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Research ArticleProminent Levels of the Profibrotic ChemokineCCL18 during Peritonitis In Vitro Downregulation byVitamin D Receptor Agonists
Marta Ossorio1 Virginia Martiacutenez 2 Maria-Auxiliadora Bajo1 Gloria Del Peso1
Maria-Joseacute Castro 1 Sara Romero1 Rafael Selgas12 and Teresa Belloacuten 2
1Nephrology Service Hospital La Paz Institute for Health Research (IdiPAZ) La Paz University Hospital and IRSIN Madrid Spain2Hospital La Paz Institute for Health Research (IdiPAZ) Madrid Spain
Correspondence should be addressed to Teresa Bellon teresabellonsaludmadridorg
Received 13 November 2017 Revised 29 January 2018 Accepted 27 February 2018 Published 4 April 2018
Academic Editor Gernot Zissel
Copyright copy 2018 Marta Ossorio et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Peritoneal dialysis (PD) is used as a renal replacement therapy which can be limited by peritoneal membrane ultrafiltration failure(UFF) secondary to fibrotic processes Peritonitis a frequent complication of PD is a major risk factor for peritoneal membranefibrosis and UFF Low peritoneal levels of the chemokine CCL18 are associated with preservation of peritoneal membrane functionin PD Given that CCL18 is involved in fibrotic processes and recurrent peritonitis it is a risk factor for peritoneal membrane failurethus we evaluated CCL18 concentrations in peritoneal effluents from patients undergoing peritonitis episodes Pharmacologicalinterventions aimed at diminishing the production of CCL18 were also explored Fivefold higher CCL18 peritoneal concentrationswere found during acute bacterial peritonitis in parallel with the increased infiltration of macrophages Unexpectedly CCL18 wasalso highly (50-fold) increased during sterile eosinophilic peritonitis and peritoneal eosinophils were found to express CCL18 Invitro treatment of peritoneal macrophages with the vitamin D receptor agonist paricalcitol was able to reduce the secretion andthe expression of CCL18 in isolated peritoneal macrophages In conclusion our study suggests that the chemokine CCL18 can bea mediator of peritoneal membrane failure associated with peritonitis episodes as well as providing a new potential therapeutictarget
1 Introduction
Peritoneal dialysis (PD) is a form of renal replacement usedas an alternative to hemodialysis for the treatment of end-stage renal disease It is based on the use of the peritoneumas a semipermeable membrane across which ultrafiltrationand diffusion take place upon exposing it to bioincompat-ible glucose-containing hyperosmotic solutions Howevercontinuous exposure to bioincompatible PD solutions causesacute and chronic inflammation and injury to the peritonealmembrane which progressively leads to ultrafiltration failure(UFF) and irreversible structural fibrosis compromisingtreatment efficacy and worsening patient outcomes [1 2] Insome cases PD-associated chronic peritoneal inflammationcan result in encapsulating peritoneal sclerosis (EPS) [3]a fatal form of peritoneal inflammation characterized by a
fibrous thickening of the peritoneum Treatment for morethan 4 years with bioincompatible PD solutions is knownto present the highest risk for EPS [4 5] Additional riskfactors for peritoneal membrane fibrosis are high glucosecontent and recurrent peritonitis episodes which are afrequent complication in PD [4 6ndash8] Although bioincom-patible solutions can themselves induce sterile peritonealinflammation [9 10] PD is affected by episodes of bacterialinfection leading to localized inflammation evidenced asperitonitis a potentially destructive disease that exacer-bates local peritoneal inflammation Both acute and chronicperitoneal inflammation can lead to UFF [11] Peritonealinflammation is characterized by local upregulation of severalcytokines and collagen synthesis by mesothelial cells andfibroblasts leading to the loss of membrane integrity andfibrosis
HindawiBioMed Research InternationalVolume 2018 Article ID 6415892 12 pageshttpsdoiorg10115520186415892
2 BioMed Research International
The successful repair of injured tissue requires a tightlycontrolled response to limit the structural alteration Under-standing the role of the various players involved can helpin designing strategies aimed at limiting inflammation-mediated tissue injury
The pathophysiological mechanisms that are involved inperitoneal functional impairment and UFF are not com-pletely understood although local conversion of mesothelialcells (MCs) by epithelial-to-mesenchymal transition (EMT)during the inflammatory and repair responses that areinduced by PD might play a key role [12] MCs and residentor infiltrating immune cells including macrophages mono-cytes lymphocytes and neutrophils can produce a largenumber of cytokines growth factors and chemokines leadingto membrane deterioration and fibrosis [10 13]
Among leukocytes alternatively activated macrophages(M2) are important players in tissue repair and the devel-opment of fibrosis [14] and might contribute to the fibroticprocess of the peritoneum under PD [15]
The human chemokine CCL18 which has been involvedin various progressive fibrotic disorders [16ndash19] is notablypresent at easily measurable levels in the effluent of patientstreated with PD [15 20] CCL18 is also a hallmark of M2macrophages in humans [14 21] High peritoneal CCL18effluent levels in PD patients in relation to UFF and topredicting the future development of peritoneal sclerosishave recently been reported [15 20] Moreover CD163+ M2expressing CCL18 infiltrate the peritoneum in PD patientstheir number increases during peritonitis episodes andperitoneal macrophages isolated from patients with activeinfectious peritonitis synthesize high amounts of CCL18when cultured ex vivo [15] In a recent study we reported thatPD patients with sustained low levels of peritoneal CCL18 areat lower risk of developing peritoneal membrane dysfunction[22]
Given that CCL18 is involved in fibrotic processes andthat recurrent peritonitis is a major risk factor for devel-oping peritoneal membrane failure and peritoneal fibrosiswe sought to evaluate CCL18 protein concentrations inperitoneal effluents from patients experiencing peritonitisepisodes Pharmacological interventions aimed at diminish-ing the production of CCL18were also explored Among drugcandidates paricalcitol a vitamin D receptor (VDR) agonisthas been shown to ameliorate fibrosis in a murine modelof PD [23] In humans a preliminary study suggests thatparicalcitol could increase the ultrafiltration capacity with adecrease of peritoneal protein losses in PD [24] The absenceof CCL18 in rodents precludes the study of paricalcitol effectson CCL18 peritoneal levels in the murine model We hereinanalyzed the effect of this drug on CCL18 production byperitoneal macrophages
CCL18 peritoneal concentrations were found to increaseduring the course of infectious bacterial peritonitis in parallelwith the increased infiltration ofmacrophages UnexpectedlyCCL18 effluent levels were also highly increased duringthe course of sterile eosinophilic peritonitis and peritonealeosinophils were found to express CCL18
In addition in vitro treatment of peritoneal macrophageswith the VDR agonist paricalcitol was able to reduce the
secretion and the expression of CCL18 in isolated peritonealmacrophages (pMΦs)
In conclusion our study suggests that the chemokineCCL18 can be a mediator of peritoneal membrane failureassociated with peritonitis episodes as well as providing anew potential therapeutic target
2 Patients and Methods
21 Patients Patients were recruited from the PeritonealDialysis Unit of La Paz University Hospital in Madrid Thediagnosis of PD-associated peritonitis was based on the pres-ence of abdominal pain cloudy PD effluent with a leukocytecount above 100 cells120583L and a positive microbiologicalculture The diagnosis of eosinophilic peritonitis (EP) wasbased on the presence of cloudy effluent (white blood cellcount above 100 cells120583L) with no infectious agent andeosinophils above 10
The study was approved by the Research Ethics Com-mittee of La Paz University Hospital (PI12 0024) All clin-ical investigations were conducted according to the princi-ples expressed in the declaration of Helsinki and writteninformed consent was obtained from all the patients
22 Cytokine Determinations CCL18 was quantified using aDuoSet ELISA Development System (RampD Systems EuropeUK) IL-6 concentrations were measured by flow cytometryusing the corresponding Cytometric Bead Array Flex Set kit(BD Biosciences)
23 Cytospin and Immunostaining For cytospin prepara-tions peritoneal effluent cells were applied to slides at 105cellsslide The cells were then fixed in 4 paraformalde-hyde at room temperature Sequential blocking was thenperformed with 003 hydrogen peroxide in methanol andin 10 normal goat serum1 bovine serum albumin01saponin in Tris-buffered saline Biotinylated goat anti-humanCCL18 (RampD Systems) was added and incubated overnightat 4∘C Nonimmune serum was used as a negative back-ground control staining The sections were washed threetimes with phosphate-buffered saline and the signal wasdeveloped with peroxidase-conjugated streptavidin (RampD)and 3-31015840-diaminobenzidine Slides were counterstained withhematoxylin prior to visualization
24 Cell Isolation and Treatment Cells were isolated fromPD effluents by centrifugation at 500119892 for 15 minutes at4∘C Peripheral blood mononuclear cells (PBMCs) fromcontrol donors were prepared by Ficoll-Hypaque (AmershamPharmacia Biotech Sweden) density gradient centrifugationpMΦs were obtained from PD effluent concentrates Positiveselection was performed by magnetic isolation using CD14microbeads For isolation of polymorphonuclear leukocytesFITC-conjugated CD66b-specific antibodies followed byanti-FITC microbeads (Miltenyi Biotec GmbH Germany)were used as described previously [15] Purity of macrophagefractions was at least 95 as assessed by flow cytome-try CD14-APC CD66b-FITC and CD16-FITC antibodies
BioMed Research International 3
(Miltenyi Biotec) were used for cytometry analysis of peri-toneal cells
Monocytes were isolated from PBMCs by the cell cul-ture adherence method For monocyte isolation by adher-ence PBMCs were seeded onto cell culture plates andallowed to adhere in a 5 CO
2incubator at 37∘C for
1 hour in serum-free medium Nonadherent cells wereremoved and the adherent cells were carefully washed twicewith medium before culturing in RPMI medium plus 10heat-inactivated fetal calf serum (FCS) Peripheral bloodmonocyte-derivedM2 cells were generated as described [25]Briefly monocytes were incubated in RPMI with 10 heat-inactivated FCS and 50 UmL IL-4 (Peprotech UK) for 72hours
Cells were seeded at 200000 cellswell in 96-well platesand incubated with various doses of paricalcitol In someexperiments the commercial form (Zemplar) was used asthe source of drug and cultures in the presence of theequivalent concentration of vehicle (30 50 20 propyleneglycol water ethanol)were established in parallel as negativecontrols Stock solutions of pure paricalcitol (Abbott) andvitamin D3 [125(OH)
2D3] (Roche) were prepared in EtOH
at 10minus3MCell viability was analyzed by exclusion of propidium
iodide staining and flow cytometry
25 Quantitative Reverse Transcription-Polymerase ChainReaction (qRT-PCR) Total RNA was isolated using theHigh Pure RNA Isolation Kit (Roche Germany) and 1 120583gwas reverse transcribed with random hexanucleotides andavian myeloblastosis virus (AMV) reverse transcriptase(Promega) in a final volume of 20120583L for 1 hour at 42∘CFor quantitative analysis of mRNA content 1120583L aliquotsof the resulting cDNAs were PCR amplified in a LightCycler (Roche) with FastStart DNA Master SYBR Green I(Roche) in duplicate Primers used for PCR amplificationwere previously described [15] CCL18 sense 51015840-ACA AAGAGC TCT GCT GCC TC-31015840 CCL18 antisense 51015840-CCCACT TCT TAT GGG GTC A-31015840 B2M sense 51015840-CCA GCAGAG AAT GGA AAG TC-31015840 B2M antisense 51015840-GAT GCTGCT TAC ATG TCT CG-31015840 Standard curves for targetmRNAexpressionwere generated by amplifying 10-fold serialdilutions of known quantities of the specific PCR productsQuantification of target gene expression was obtained usingLight Cycler system software Relative units (RU) estimatedfrom the quantification represent the ratio between CCL18mRNA molecules and B2M mRNA molecules in each sam-ple
26 Statistical Analysis The statistical analysis was per-formed using GraphPad Prism software (San Diego CA)Parametric or nonparametric tests were used according tothe distribution of the data Data are shown as mean plusmnSEM mean plusmn SD values or median and interquartile ranges(IQR) as indicated in each figure Data were analyzed withparametric or nonparametric 119905-tests for unpaired or pairedsamples Correlation was assessed using Spearman correla-tion tests with 119901 values 119901 values less than or equal to 05 wereconsidered statistically significant
Gram +0
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CCL1
8 (n
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(N = 13) (N = 26)
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Figure 1The concentration of CCL18was evaluated in 39 peritonealeffluents from patients diagnosed with infectious peritonitis Efflu-entswere collected on the first day of cloudy effluentHorizontal barsrepresent mean plusmn SEM values No significant differences were foundbetween Gramminus (119873 = 13) and Gram+ bacteria (119873 = 26) infectedcases (unpaired 119905-test)
3 Results
31 CCL18 Measurements in Peritonitis Effluent SamplesPrevious studies propose CCL18 as a marker and mediatorof peritoneal fibrosis and UFF in patients treated with PD[15 16] Given recurrent peritonitis is a risk factor forperitoneal fibrosis and UFF the concentration of CCL18was evaluated in a group of 39 PD patients with infectiousperitonitis Effluent samples were obtained at diagnosis andthe concentration of CCL18 was measured by enzyme-linkedimmunosorbent assay (ELISA) The mean concentration ofCCL18 in peritonitis samples was 1502 plusmn 787 ngml whereasmean values previously reported ranged between 1 and6 ngml No significant differences were found in patientsinfected with Gram+ or Gramminus bacteria Mean observedvalues were 133 plusmn 243 ngml in the Gramminus versus 1586 plusmn145 ngml in the Gram+ samples (Figure 1)
We evaluated CCL18 concentrations in serial samplesfrom four of the PD patients with infectious peritonitis(IP) (Figure 2(a)) CCL18 effluent concentrations showedthe highest values during the first days of peritonitis anddecreased slowly following a kinetics similar to absolutemacrophage counts in these samples (Figure 2(c)) and inagreement with previous reports describing macrophages asthe primary source of this chemokine [15]
Serial samples were also analyzed in one additionalcase of sterile EP associated with an accidental exposureof the peritoneum to chlorhexidine Surprisingly whereasthe mean CCL18 concentrations on day 1 and on days2ndash4 in the IP samples were 155 plusmn 61 ngml and 117 plusmn69 ngml respectively in the EP samples the concentrationof CCL18 was 19812 ngml on day 1 and 20465 ngml on day3 (Figure 3 and Supplemental Figure 1(A)) IL-6 was alsoevaluated for comparative purposes as the hallmark cytokinein inflammatory processes and in PD-associated peritonitis[26] Elevated levels of IL-6were found in spent effluents fromall patients on the first day of peritonitis with similar values
4 BioMed Research International
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Figure 2 Serial cytokine concentrations and cell counts in peritoneal effluent during peritonitis episodes (a) Evolution of CCL18 effluentlevels in serial samples from four patients with infectious peritonitis (IP) shown as IP-1 to IP-4 (b) Evolution of IL-6 effluent levels inserial samples from the same patients (c) Comparison of neutrophils (PMN) and macrophage (MΦ) cell counts in serial effluent samplesDifferential cell counts on D26 (IP-2) D22 (IP-3) and D13 (IP-4) were not available given that the total cell counts were lt50 cells120583l in thosesamples
in IP samples and in the EP sample (Supplemental Figure1(B)) A sharp decline in IL-6 occurred in all the IP cases aspreviously described [26 27] (Figure 2(b)) and in contrast toCCL18 levels which remained elevated during the followingdays and declined more slowly (Figure 2(a))
32 Peritoneal Eosinophils Are a Source of CCL18 in Eosin-ophilic Peritonitis Cytokine concentrations were comparedwith absolute and differential cell counts in effluent samplesfrom the patient with EP (Figure 3) A significant correlationwas found between the kinetics of CCL18 concentration and
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Figure 3 Kinetics of effluent cell counts and cytokine concentrations during EP (andashd) CCL18 effluent levels and cell counts in serial effluentsamples (endashh) IL-6 effluent levels and cell counts in serial effluent samples
6 BioMed Research International
(a)
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Figure 4 Cytospin preparations showing effluent cell content in EP (a) Hematoxylin and eosin staining (b) Immunocytochemistry showingCCL18 staining of eosinophils (c) Background staining with nonimmune serum
the macrophage counts in the EP samples (119903 = 0889119901 = 043) (Figure 3(c)) However the strongest correlationwas found between CCL18 effluent levels and eosinophils(119903 = 0949 119901 = 013) with peak CCL18 concentrationsdetected in effluents with the highest absolute eosinophilcount (Figure 3(d)) In contrast IL-6 effluent levels paralleledpolymorphonuclear neutrophils (PMNs) and absolute cellnumbers in effluent samples from EP (Figures 3(e) and3(f)) and no correlation was observed between effluent IL-6 concentrations and eosinophil cell count (Figure 3(h))
Although CCL18 is thought to be primarily produced byM2 macrophages involved in tissue repair and fibrosis andby dendritic cells [28] eosinophils have occasionally beenreported to produce CCL18 during inflammatory conditions[29 30] The kinetics of CCL18 effluent concentrations in EPand eosinophil counts suggested that not only macrophagesbut also eosinophils might contribute to the production ofCCL18 in this case To test this hypothesis immunostainingof cytospin preparations from eosinophilic effluents wasperformed Specific staining was detected in the cytoplasmof typical binucleated cells supporting the role of eosinophilsas a source of CCL18 in this case (Figure 4)
In a previous report we found that only peritonealmacrophages were able to produce CCL18 during infectiousperitonitis given that the cytokine was not detected in cul-ture supernatants from neutrophils (PMNs) or lymphocytes[15] To further analyze the putative role of eosinophils asproducers of CCL18 in the patient with EP CD66b+ polymor-phonuclear leukocytes (among which 85 were eosinophils
as assessed by CD16 negative staining of eosinophils versusCD16 positive neutrophils) were isolated from peritonealeffluent cells on D6 as well as CD14+ macrophages forcomparative purposes (Figures 5(a)ndash5(c)) CCL18 transcriptswere detected in both the CD66b+ and CD14+ fractions(Figure 5(d)) Quantitative PCR analysis confirmed CCL18mRNA transcripts in polymorphonuclear leukocytes in thiscase (Figure 5(e)) Altogether the data strongly suggest thateosinophils are producers of CCL18 in EP
33 Paricalcitol Downregulates the Production of CCL18 byPeritoneal Macrophages Paricalcitol a VDR agonist hasbeen shown to ameliorate peritoneal fibrosis in a murinemodel of PD [23] Given that CCL18 has been involvedin peritoneal fibrosis in PD patients [15 20 22] we testedthe effect of paricalcitol in the production of CCL18 byhuman pMΦs isolated fromPD effluents drawn frompatientssuffering from peritonitis and cultured with various dosesof nontoxic concentrations of Zemplar as a source of par-icalcitol or vehicle (supplementary Figure 2) As shown inFigures 6(a)ndash6(c) pharmacological treatment was able tosignificantly downregulate the production of CCL18 in thesecells at concentrations equivalent to 10minus8M and 10minus9M ofparicalcitol This effect was obtained as early as 24 hours andit was also observed at 48 hours and 72 hours of cell cultureSimilar results were obtained with the pure substance andwith equivalent doses of vitamin D3 (Figure 6(d))
It has been suggested that the regulation of CCL18 proteinsynthesis and secretion can be a complex process with some
BioMed Research International 7
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Figure 5 (a) Flow cytometry analysis of peritoneal effluent leukocytes on D6 according to morphological parameters LΦ lymphocytesMΦ macrophages (b) Flow cytometry analysis of CD16 expression in peritoneal effluent cells on D6 The cutoff level for positive stainingwas set according to isotype control background staining PMN polymorphonuclear neutrophils (c) Flow cytometry analysis of CD66b+polymorphonuclear cells isolated from peritoneal effluent cells (d) RT-PCR of CCL18 gene expression in CD66+ and CD14+ purifiedperitoneal leukocytes (e) Quantitative RT-PCR analysis of CCL18 gene expression in CD66+ and CD14+ purified peritoneal leukocytes
degree of posttranscriptional regulation [28] To analyzewhether VDR agonists regulate CCL18 mRNA levels quan-titative RT-PCR assays were performed in isolated pMΦstreated with paricalcitol CCL18 mRNA levels were downreg-ulated in parallel with the diminished production of CCL18protein in pMΦs from two different donors representinginfected and uninfected peritoneal samples (Figures 7(a) and7(b)) Given that IL-4 is one of the main inducers of CCL18[31] we tested if VDR agonists couldmodulate the expressionof the chemokine in M2 macrophages generated in vitroupon treatment of peripheral blood monocytes with IL-4 Asshown in Figure 7(c) the expression of CCL18 was highlyupregulated in monocyte-derived macrophages treated for 3days with IL-4 and pretreatment with paricalcitol or vitaminD was able to prevent the upregulation of CCL18 in thesecells
4 Discussion
Peritonitis episodes constitute a frequent complication in PDpatients and recurrent peritonitis is a well-known clinicalrisk factor for peritoneal membrane dysfunction and EPS
There is an unmet requirement for biomarkers as toolsto identify patients who are at higher risk of techniquefailure to guide interventions that could improve clinicaloutcomes [32] Soluble factors released during inflammatoryprocesses such as IL-6 and IL-17 have been proposed asbiomarkers and mediators of peritoneal membrane deteri-oration (reviewed in [32 33]) However none are used inthe clinical routine and some cytokines such as IL-17 arenot easily measured in PD effluents in humans We proposeCCL18 as a biomarker of peritoneal membrane outcome inPD [22]
8 BioMed Research International
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(d)
Figure 6 (andashc) Peritoneal macrophages isolated from patients experiencing peritonitis episodes were treated with vehicle or Zemplar indoses equivalent to 10minus8M and 10minus9M of paricalcitol during 24 h (a) 48 h (b) or 72 h (c) Equivalent cell cultures were established in theabsence of any treatment The concentration of CCL18 was evaluated in cell-free supernatants and the production of CCL18 in the presenceof vehicle or drug referred to the production in cell culture media alone Differences between cultures in the presence of paricalcitol or vehiclewere analyzed by theWilcoxon matched pairs rank test (d) Peritoneal macrophages were treated with paricalcitol as pure substance vitaminD or vehicle (ethanol) for 48 h
Given that high levels of the human chemokine CCL18have been shown to be related to peritoneal membranefailure and fibrosis [15 20 22] whereas sustained low CCL18peritoneal concentrations appear to be protective [22] weevaluated CCL18 levels in the peritoneum of patients under-going peritonitis episodes We found that the peritoneal con-centration of CCL18 was increased approximately 5-fold ineffluents from patients with infectious peritonitis comparedwith reported values in uninfected samples In a previousstudy involving a longitudinal analysis of 43 patients CCL18effluent concentrations ranged from 28 plusmn 16 ngml at thestart of dialysis to 34plusmn 18 ngml after 3 years of PD treatment[22] In a cross-sectional analysis of 61 patients treated formore than 3 years with PDmean effluent CCL18 values were395 plusmn 26 ngml [22] The high concentrations during IPare consistent with a previous finding showing that pMΦsfrom patients undergoing peritonitis or who recently had
peritonitis were able to secrete higher amounts of CCL18compared with pMΦs from uninfected patients [15]
Unexpectedly in a patient identified with sterileeosinophilic peritonitis the peritoneal effluent was extremelyrich in CCL18 with levels approximately 10-fold higher thanthose found in patients with IP and 50-fold more thanthose found in uninfected samples whereas effluent levelsof IL-6 which were evaluated in the same samples forcomparative purposes were similar on day 1 in both theIP and EP samples The differences between a peritonealinflammatory recognized marker IL-6 and CCL18 duringperitonitis episodes should be remarked It is of note thatthe peritoneal concentrations of CCL18 remained elevatedfor several days whereas there was a sharp decrease in IL-6Both IL-6 and CCL18 have been reported to increase indifferent inflammatory conditions some leading to tissuefibrosis [34ndash37] As a main difference although CCL18 is
BioMed Research International 9
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Pari Vit D3Vmdash
(c)
Figure 7 (a) Peritoneal macrophages from a patient with recent infectious peritonitis were treated with paricalcitol or vehicle for 24 h Theprotein concentration of CCL18 was evaluated in cell culture supernatants by ELISA and CCL18 mRNA levels were estimated by quantitativeRT-PCR in the same cultures (b) Peritoneal macrophages from an uninfected peritoneum were isolated from the peritoneal lavage uponcatheter insertion and treated as in (a) (c) Peripheral bloodmonocytes were pretreated with vehicle (V) 10minus7Mparicalcitol (Pari) or vitaminD3 for 24 h The cells were treated thereafter with IL-4 for 3 days before RNA extraction and evaluation expression of CCL18 mRNA levelsby quantitative RT-PCR Mean plusmn SD values from four independent experiments are shown
primarily produced by cells of myeloid origin multiplecell types are producers of IL-6 with endothelial cellsbeing an important source of this cytokine Infiltratingneutrophils as well as mesothelial cells could also be animportant source of IL-6 during bacterial peritonitis [37]The analysis of serial samples in four patients with IP showedthat the concentrations of CCL18 paralleled the numbers ofmacrophages infiltrating the peritoneum and are consistentwith previous descriptions of macrophages andmononuclearphagocytes as the primary producers of CCL18 [21] Cellcounts in the serial samples from our case of EP suggest that
the peritoneal eosinophilic infiltrate was likely contributingto the peritoneal production of CCL18 Evidences of CCL18production by activated peritoneal eosinophils in this caseare cytospin analysis of CCL18 cytoplasmic staining ineosinophils and gene expression data in polymorphonuclearleukocytes among which 85 were eosinophils
Although macrophages are typically reported as themajor source of this chemokine [21] CCL18 protein pro-duction by eosinophils has occasionally been described asbeing related to the activation status of these cells given thatCCL18 was found only in eosinophil culture supernatants
10 BioMed Research International
from eosinophilic donors but not from individuals withnonactivated eosinophils [29] Our data support the activatedstatus of eosinophils in EP given that strong staining ofCC18 was detected in peritoneal eosinophils Eosinophilshave also been related to tissue fibrosis in several diseasesand are a source of several profibrotic mediators that alterfibroblast activity and tissue remodeling [38 39] Some ofthesemediators could also stimulate the profibrotic activity ofpMΦTheproduction ofCCL18 by eosinophils in EPprovidesa new additional explanation for the association of recurrentEP during PD with UFF and potentially peritoneal fibrosis[40]
As part of its profibrotic activity CCL18 has been shownto stimulate collagen deposition [41] and fibroblast prolif-eration [42] In consonance with these actions circulatingCCL18 levels have been demonstrated to be a biomarker fordisease activity and outcome in various fibrotic diseases [1943 44] as well as in some eosinophilic disorders [30 45 46]Moreover CCL18 has been shown to promote EMT in variouscell systems [47ndash50] It is thus possible that it could alsocontribute to the EMT of mesothelial cells which has beenproposed as one of the mechanisms leading to peritonealfibrosis and membrane failure [12]
In terms of peritoneal fibrosis CCL18 has been demon-strated to herald the development of peritoneal functionaldeterioration in PD patients [15 20] in whom the abilityof peritoneal macrophages to stimulate fibroblast prolifer-ation is correlated with CCL18 mRNA levels Moreoverrecombinant CCL18 was able to stimulate the proliferation ofperitoneal fibroblasts [15]
Several molecules have been tested for their ability toprevent peritoneal fibrosis in vitro and in animal models(reviewed in [8]) Among them paricalcitol is a selectiveVDR agonist VDR is a nuclear hormone receptor expressedin various cell types including those of the immune sys-tem VDR agonists have been shown to modulate immuneresponses inflammation and fibrosis [51]
Considering the role played by inflammation in peri-toneal fibrosis during PDwe studied the effects of paricalcitolin the production of CCL18 We found that the release andgene expression of CCL18 were downregulated in pMΦsand in in vitro-differentiated human M2 cells which are theprimary producers of CCL18These results appear to contrastwith previous data showing upregulation of CCL18 by vita-min D in monocyte-derived immature dendritic cells [52]However recent reports demonstrate that the modulationof immune signaling by vitamin D in human mononuclearphagocytes is cell-type-specific with differential responses inmonocytes macrophages and dendritic cells [53] It is thuspossible that opposite effects are found on the regulation ofCCL18 by VDR agonists in different cellular systems
On the other hand in a mouse model of PD intraperi-toneal administration of paricalcitol was shown to reduceperitoneal fibrosis and ultrafiltration failure [23] In thatmodel the beneficial effects appeared to be dependent ona reduction of intraperitoneal concentrations of IL-17 Thelack of rodent orthologues of human CCL18 [28] precludesthe investigation of CCL18 and its involvement in fibrosis inmurine models On the other hand IL-17 is highly diluted
in human peritoneal effluents The need to concentrate thesamples for measurement of this cytokine hampers accuratedeterminations in human samples
A preliminary study involving 23 patients on PD suggeststhat paricalcitol could improve the ultrafiltration capacitywith less peritoneal protein loss [24] The authors suggestan effect of VDR upon the renin-angiotensin-aldosteronesystem but it is tempting to speculate that lower peritonealconcentrations of CCL18 might be present in paricalcitol-treated patients In this sense a slight reduction in theperitoneal concentration of CCL18 was observed in sixpatients who were more than 3 years on PD and treated withparicalcitol compared with a set of 31 patients of the samevintage (data not shown)
5 Conclusion
In conclusion our study suggests that the chemokine CCL18can be a mediator of peritoneal membrane failure associatedwith peritonitis episodes and might provide a new potentialtherapeutic target
Further clinical studies are needed to confirm the useful-ness of VDR agonists to preserve the peritoneal membraneand to analyze the putative mechanisms involved
Conflicts of Interest
All the authors have no potential conflicts of interest todeclare
Authorsrsquo Contributions
Marta Ossorio and Virginia Martınez contributed equally tothis work
Acknowledgments
This study has been partially supported by ISCIII (ldquoInsti-tuto de Investigacion Sanitaria Carlos IIIrdquo) Grants FIS PI1200204 PI1301768 and PI 1500120 FEDER (Funds fromthe European Union) REDinREN 20 FEDER and ReinaSofıa Institute of Nephrology Research (IRSIN) monetaryfunds The authors would like to thank Juliette Siegfriedand her team at ServingMedcom for their editing of themanuscript
Supplementary Materials
Supplemental Figure 1 serial cytokine concentrations andcell counts in peritoneal effluent during infectious peritoni-tis and eosinophilic peritonitis (A) Comparison of CCL18concentration in serial effluent samples from infectiousperitonitis (IP) and eosinophilic peritonitis (EP) (B) IL-6effluent levels in serial samples from IP and EP D1ndashD26indicate days of evolution Supplemental Figure 2 dose-dependent effects of VDR agonists on cell viability Cellviability of human monocytes was assessed by exclusion ofpropidium iodide staining and analyzed by flow cytometry
BioMed Research International 11
Results are shown as mean plusmn SD values of triplicates(A)ndash(C) Viability of monocytes upon culture during 24 48or 72 hours with decreasing concentrations of Zemplar orequivalent vehicle (305020 propylene glycolwaterethanol)(D)ndash(F) Viability of monocytes upon culture during 2448 or 72 hours with different concentrations of paricalcitol(pure substance) vitamin D or equivalent vehicle (EtOH)(Supplementary Materials)
References
[1] R T Krediet ldquoThe peritoneal membrane in chronic peritonealdialysisrdquo Kidney International vol 55 no 1 pp 341ndash356 1999
[2] R T Krediet ldquoPeritoneal physiology - Impact on solute andfluid clearancerdquo Advances in Chronic Kidney Disease vol 7 no4 pp 271ndash279 2000
[3] M Merkle and M Wrnle ldquoSclerosing peritonitis A rare butfatal complication of peritoneal inflammationrdquo Mediators ofInflammation vol 2012 Article ID 709673 p 4 2012
[4] D W Johnson Y Cho B E R Livingston et al ldquoEncapsulat-ing peritoneal sclerosis incidence predictors and outcomesrdquoKidney International vol 77 no 10 pp 904ndash912 2010
[5] G Balasubramaniam E A Brown and A Davenport ldquoThePan-Thames EPS study treatment and outcomes of encapsulat-ing peritoneal sclerosisrdquo Nephrology Dialysis Transplantation vol 24 no 10 pp 3209ndash3215 2009
[6] E D Sousa-Amorim G D Peso M A Bajo et al ldquoCan EPSdevelopment be avoided with early interventionsThe potentialrole of tamoxifenmdasha single-center studyrdquo Peritoneal DialysisInternational vol 34 no 6 pp 582ndash593 2014
[7] M R Korte D E SampimonM GH Betjes and R T KredietldquoEncapsulating peritoneal sclerosis the state of affairsrdquo NatureReviews Nephrology vol 7 no 9 pp 528ndash538 2011
[8] M A Bajo G del Peso and I Teitelbaum ldquoPeritoneal Mem-brane Preservationrdquo Seminars in Nephrology vol 37 no 1 pp77ndash92 2017
[9] M F Flessner ldquoInflammation from sterile dialysis solutions andthe longevity of the peritoneal barrierrdquo Clinical Nephrology vol68 no 6 pp 341ndash348 2007
[10] K N Lai and J C K Leung ldquoInflammation in peritonealdialysisrdquo Nephron Clinical Practice vol 116 no 1 pp c11ndashc182010
[11] S Yung and T M Chan ldquoPathophysiological changes to theperitoneal membrane during PD-related peritonitis the role ofmesothelial cellsrdquo Mediators of Inflammation vol 2012 ArticleID 484167 21 pages 2012
[12] L S Aroeira A Aguilera J A Sanchez-Tomero et al ldquoEpithe-lial tomesenchymal transition and peritonealmembrane failurein peritoneal dialysis patients pathologic significance andpotential therapeutic interventionsrdquo Journal of the AmericanSociety of Nephrology vol 18 no 7 pp 2004ndash2013 2007
[13] N Topley T Liberek A Davenport F-K Li H Fear andJ D Williams ldquoActivation of inflammation and leukocyterecruitment into the peritoneal cavityrdquo Kidney InternationalSupplements vol 50 no 56 pp S17ndashS21 1996
[14] F O Martınez and S Gordon ldquoThe M1 and M2 paradigmof macrophage activation time for reassessmentrdquo F1000PrimeReports vol 6 article 13 2014
[15] T Bellon V Martınez B Lucendo et al ldquoAlternative activa-tion of macrophages in human peritoneum implications for
peritoneal fibrosisrdquo Nephrology Dialysis Transplantation vol26 no 9 pp 2995ndash3005 2011
[16] A Prasse D V Pechkovsky G B Toews et al ldquoA vicious circleof alveolar macrophages and fibroblasts perpetuates pulmonaryfibrosis via CCL18rdquoAmerican Journal of Respiratory and CriticalCare Medicine vol 173 no 7 pp 781ndash792 2006
[17] J M Estep A Baranova N Hossain et al ldquoExpression ofcytokine signaling genes in morbidly obese patients with non-alcoholic steatohepatitis and hepatic fibrosisrdquo Obesity Surgeryvol 19 no 5 pp 617ndash624 2009
[18] F Kollert M Binder C Probst et al ldquoCCL18 - Potentialbiomarker of fibroinflammatory activity in chronic periaortitisrdquoThe Journal of Rheumatology vol 39 no 7 pp 1407ndash1412 2012
[19] A Prasse D V Pechkovsky G B Toews et al ldquoCCL18 as anindicator of pulmonary fibrotic activity in idiopathic interstitialpneumonias and systemic sclerosisrdquo Arthritis amp Rheumatologyvol 56 no 5 pp 1685ndash1693 2007
[20] S Ahmad B V North A Qureshi et al ldquoCCL18 in peritonealdialysis patients and encapsulating peritoneal sclerosisrdquo Euro-pean Journal of Clinical Investigation vol 40 no 12 pp 1067ndash1073 2010
[21] V Kodelja C Muller O Politz N Hakij C E Orfanos andS Goerdt ldquoAlternative macrophage activation-associated CC-chemokine-1 a novel structural homologue of macrophageinflammatory protein-1120572 with a Th2- associated expressionpatternrdquo The Journal of Immunology vol 160 no 3 pp 1411ndash1418 1998
[22] M Ossorio M A Bajo G Del Peso et al ldquoSustained low peri-toneal effluent CCL18 levels are associated with preservationof peritoneal membrane function in peritoneal dialysisrdquo PLoSONE vol 12 no 4 Article ID e0175835 2017
[23] G T Gonzalez-Mateo V Fernandez-Mıllara T Bellon etal ldquoParicalcitol reduces peritoneal fibrosis in mice throughthe activation of regulatory T cells and reduction in IL-17productionrdquo PLoS ONE vol 9 no 10 Article ID e108477 2014
[24] F Coronel S Cigarran A Gomis et al ldquoChanges in peritonealmembrane permeability and proteinuria in patients on peri-toneal dialysis after treatment with paricalcitolmdasha preliminarystudyrdquo Clinical Nephrology vol 78 no 2 pp 93ndash99 2012
[25] E Song N Ouyang M Horbelt B Antus M Wang and MS Exton ldquoInfluence of alternatively and classically activatedmacrophages on fibrogenic activities of human fibroblastsrdquoCellular Immunology vol 204 no 1 pp 19ndash28 2000
[26] S M Hurst T S Wilkinson R M McLoughlin et al ldquoIL-6 andits soluble receptor orchestrate a temporal switch in the patternof leukocyte recruitment seen during acute inflammationrdquoImmunity vol 14 no 6 pp 705ndash714 2001
[27] M Kozioł-Montewka A Ksiazek L Janicka and I BaranowiczldquoSerial cytokine changes in peritoneal effluent and plasma dur-ing peritonitis in patients on continuous ambulatory peritonealdialysis (CAPD)rdquo Inflammation Research vol 46 no 4 pp 132ndash136 1997
[28] E Schutyser A Richmond and J Van Damme ldquoInvolvementof CC chemokine ligand 18 (CCL18) in normal and pathologicalprocessesrdquo Journal of Leukocyte Biology vol 78 no 1 pp 14ndash262005
[29] I Schraufstatter H Takamori L Sikora P Sriramarao and RG DiScipio ldquoEosinophils and monocytes produce pulmonaryand activation-regulated chemokine which activates culturedmonocytesmacrophagesrdquoAmerican Journal of Physiology-LungCellular and Molecular Physiology vol 286 no 3 pp L494ndashL501 2004
12 BioMed Research International
[30] C Gunther N Carballido-Perrig T Kopp J M Carballidoand C Pfeiffer ldquoCCL18 is expressed in patients with bullouspemphigoid and parallels disease courserdquo British Journal ofDermatology vol 160 no 4 pp 747ndash755 2009
[31] AMantovani A Sica S Sozzani P Allavena A Vecchi andMLocati ldquoThe chemokine system in diverse forms of macrophageactivation and polarizationrdquo Trends in Immunology vol 25 no12 pp 677ndash686 2004
[32] C Aufricht R Beelen M Eberl et al ldquoBiomarker researchto improve clinical outcomes of peritoneal dialysis consensusof the European Training and Research in Peritoneal Dialysis(EuTRiPD) networkrdquo Kidney International vol 92 no 4 pp824ndash835 2017
[33] D Lopes Barreto andR T Krediet ldquoCurrent status and practicaluse of effluent biomarkers in peritoneal dialysis patientsrdquoAmerican Journal of Kidney Diseases vol 62 no 4 pp 823ndash8332013
[34] L A Boven M van Meurs R G Boot et al ldquoGaucher cellsdemonstrate a distinct macrophage phenotype and resemblealternatively activated macrophagesrdquo American Journal of Clin-ical Pathology vol 122 no 3 pp 359ndash369 2004
[35] L Cocho I Fernandez M Calonge et al ldquoGene expression-based predictive models of graft versus host disease-associateddry eyerdquo Investigative Ophtalmology Visual Science vol 56 no8 pp 4570ndash4581 2015
[36] MHasegawa ldquoBiomarkers in systemic sclerosisTheir potentialto predict clinical coursesrdquoThe Journal of Dermatology vol 43no 1 pp 29ndash38 2016
[37] S A Jones D J Fraser C A Fielding and G W JonesldquoInterleukin-6 in renal disease and therapyrdquoNephrologyDialysisTransplantation vol 30 no 4 pp 564ndash574 2015
[38] A Munitz and F Levi-Schaffer ldquoEosinophils rsquoNewrsquo roles forrsquooldrsquo cellsrdquo Allergy European Journal of Allergy and ClinicalImmunology vol 59 no 3 pp 268ndash275 2004
[39] S S Aceves ldquoRemodeling and fibrosis in chronic eosinophilinflammationrdquo Digestive Diseases vol 32 no 1-2 pp 15ndash212014
[40] Y Nakamura H Okada A Yasui T Koh and T YamaneldquoSclerosing encapsulating peritonitis associated with recurrenteosinophilic peritonitisrdquo Nephrology Dialysis Transplantation vol 14 no 3 pp 768ndash770 1999
[41] S P Atamas I G Luzina J Choi et al ldquoPulmonary andactivation-regulated chemokine stimulates collagen productionin lung fibroblastsrdquo American Journal of Respiratory Cell andMolecular Biology vol 29 no 6 pp 743ndash749 2003
[42] A Wimmer S K Khaldoyanidi M Judex N Serobyan RG DiScipio and I U Schraufstatter ldquoCCL18PARC stimulateshematopoiesis in long-term bone marrow cultures indirectlythrough its effect on monocytesrdquo Blood vol 108 no 12 pp3722ndash3729 2006
[43] A Prasse C Probst E Bargagli et al ldquoSerum CC-chemokineligand 18 concentration predicts outcome in idiopathic pul-monary fibrosisrdquo American Journal of Respiratory and CriticalCare Medicine vol 179 no 8 pp 717ndash723 2009
[44] J Chen Y Yao C Gong et al ldquoCCL18 from tumor-associatedmacrophages promotes breast cancermetastasis via PITPNM3rdquoCancer Cell vol 19 no 4 pp 541ndash555 2011
[45] K L Hon G K Ching P C Ng and T F Leung ldquoExplor-ing CCL18 eczema severity and atopyrdquo Pediatric Allergy andImmunology vol 22 no 7 pp 704ndash707 2011
[46] A J Lucendo A Arias L C De Rezende et al ldquoSubepithelialcollagen deposition profibrogenic cytokine gene expressionand changes after prolonged fluticasone propionate treatment inadult eosinophilic esophagitis a prospective studyrdquoThe Journalof Allergy and Clinical Immunology vol 128 no 5 pp 1037ndash1046 2011
[47] H Li D Zhang J Yu et al ldquoCCL18-dependent translocationof AMAP1 is critical for epithelial to mesenchymal transition inbreast cancerrdquo Journal of Cellular Physiology vol 233 no 4 pp3207ndash3217 2017
[48] Z Lin W Li H Zhang et al ldquoCCL18PITPNM3 enhancesmigration invasion and EMT through the NF-120581B signalingpathway in hepatocellular carcinomardquo Tumor Biology vol 37no 3 pp 3461ndash3468 2016
[49] F Meng W Li C Li Z Gao K Guo and S SongldquoCCL18 promotes epithelial-mesenchymal transition invasionand migration of pancreatic cancer cells in pancreatic ductaladenocarcinomardquo International Journal of Oncology vol 46 no3 pp 1109ndash1120 2015
[50] T Ploenes B Scholtes A Krohn et al ldquoCC-Chemokine Ligand18 Induces Epithelial to Mesenchymal Transition in LungCancer A549 Cells and Elevates the Invasive Potentialrdquo PLoSONE vol 8 no 1 Article ID e53068 2013
[51] J Rojas-Rivera C De La Piedra A Ramos A Ortiz andJ Egido ldquoThe expanding spectrum of biological actions ofvitamin Drdquo Nephrology Dialysis Transplantation vol 25 no 9pp 2850ndash2865 2010
[52] M Vulcano S Struyf P Scapini et al ldquoUnique regulation ofCCL18 production by maturing dendritic cellsrdquo The Journal ofImmunology vol 170 no 7 pp 3843ndash3849 2003
[53] R Kundu A Theodoraki C T Haas et al ldquoCell-type-specificmodulation of innate immune signalling by vitamin D inhuman mononuclear phagocytesrdquo The Journal of Immunologyvol 150 no 1 pp 55ndash63 2017
Stem Cells International
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Disease Markers
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Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
2 BioMed Research International
The successful repair of injured tissue requires a tightlycontrolled response to limit the structural alteration Under-standing the role of the various players involved can helpin designing strategies aimed at limiting inflammation-mediated tissue injury
The pathophysiological mechanisms that are involved inperitoneal functional impairment and UFF are not com-pletely understood although local conversion of mesothelialcells (MCs) by epithelial-to-mesenchymal transition (EMT)during the inflammatory and repair responses that areinduced by PD might play a key role [12] MCs and residentor infiltrating immune cells including macrophages mono-cytes lymphocytes and neutrophils can produce a largenumber of cytokines growth factors and chemokines leadingto membrane deterioration and fibrosis [10 13]
Among leukocytes alternatively activated macrophages(M2) are important players in tissue repair and the devel-opment of fibrosis [14] and might contribute to the fibroticprocess of the peritoneum under PD [15]
The human chemokine CCL18 which has been involvedin various progressive fibrotic disorders [16ndash19] is notablypresent at easily measurable levels in the effluent of patientstreated with PD [15 20] CCL18 is also a hallmark of M2macrophages in humans [14 21] High peritoneal CCL18effluent levels in PD patients in relation to UFF and topredicting the future development of peritoneal sclerosishave recently been reported [15 20] Moreover CD163+ M2expressing CCL18 infiltrate the peritoneum in PD patientstheir number increases during peritonitis episodes andperitoneal macrophages isolated from patients with activeinfectious peritonitis synthesize high amounts of CCL18when cultured ex vivo [15] In a recent study we reported thatPD patients with sustained low levels of peritoneal CCL18 areat lower risk of developing peritoneal membrane dysfunction[22]
Given that CCL18 is involved in fibrotic processes andthat recurrent peritonitis is a major risk factor for devel-oping peritoneal membrane failure and peritoneal fibrosiswe sought to evaluate CCL18 protein concentrations inperitoneal effluents from patients experiencing peritonitisepisodes Pharmacological interventions aimed at diminish-ing the production of CCL18were also explored Among drugcandidates paricalcitol a vitamin D receptor (VDR) agonisthas been shown to ameliorate fibrosis in a murine modelof PD [23] In humans a preliminary study suggests thatparicalcitol could increase the ultrafiltration capacity with adecrease of peritoneal protein losses in PD [24] The absenceof CCL18 in rodents precludes the study of paricalcitol effectson CCL18 peritoneal levels in the murine model We hereinanalyzed the effect of this drug on CCL18 production byperitoneal macrophages
CCL18 peritoneal concentrations were found to increaseduring the course of infectious bacterial peritonitis in parallelwith the increased infiltration ofmacrophages UnexpectedlyCCL18 effluent levels were also highly increased duringthe course of sterile eosinophilic peritonitis and peritonealeosinophils were found to express CCL18
In addition in vitro treatment of peritoneal macrophageswith the VDR agonist paricalcitol was able to reduce the
secretion and the expression of CCL18 in isolated peritonealmacrophages (pMΦs)
In conclusion our study suggests that the chemokineCCL18 can be a mediator of peritoneal membrane failureassociated with peritonitis episodes as well as providing anew potential therapeutic target
2 Patients and Methods
21 Patients Patients were recruited from the PeritonealDialysis Unit of La Paz University Hospital in Madrid Thediagnosis of PD-associated peritonitis was based on the pres-ence of abdominal pain cloudy PD effluent with a leukocytecount above 100 cells120583L and a positive microbiologicalculture The diagnosis of eosinophilic peritonitis (EP) wasbased on the presence of cloudy effluent (white blood cellcount above 100 cells120583L) with no infectious agent andeosinophils above 10
The study was approved by the Research Ethics Com-mittee of La Paz University Hospital (PI12 0024) All clin-ical investigations were conducted according to the princi-ples expressed in the declaration of Helsinki and writteninformed consent was obtained from all the patients
22 Cytokine Determinations CCL18 was quantified using aDuoSet ELISA Development System (RampD Systems EuropeUK) IL-6 concentrations were measured by flow cytometryusing the corresponding Cytometric Bead Array Flex Set kit(BD Biosciences)
23 Cytospin and Immunostaining For cytospin prepara-tions peritoneal effluent cells were applied to slides at 105cellsslide The cells were then fixed in 4 paraformalde-hyde at room temperature Sequential blocking was thenperformed with 003 hydrogen peroxide in methanol andin 10 normal goat serum1 bovine serum albumin01saponin in Tris-buffered saline Biotinylated goat anti-humanCCL18 (RampD Systems) was added and incubated overnightat 4∘C Nonimmune serum was used as a negative back-ground control staining The sections were washed threetimes with phosphate-buffered saline and the signal wasdeveloped with peroxidase-conjugated streptavidin (RampD)and 3-31015840-diaminobenzidine Slides were counterstained withhematoxylin prior to visualization
24 Cell Isolation and Treatment Cells were isolated fromPD effluents by centrifugation at 500119892 for 15 minutes at4∘C Peripheral blood mononuclear cells (PBMCs) fromcontrol donors were prepared by Ficoll-Hypaque (AmershamPharmacia Biotech Sweden) density gradient centrifugationpMΦs were obtained from PD effluent concentrates Positiveselection was performed by magnetic isolation using CD14microbeads For isolation of polymorphonuclear leukocytesFITC-conjugated CD66b-specific antibodies followed byanti-FITC microbeads (Miltenyi Biotec GmbH Germany)were used as described previously [15] Purity of macrophagefractions was at least 95 as assessed by flow cytome-try CD14-APC CD66b-FITC and CD16-FITC antibodies
BioMed Research International 3
(Miltenyi Biotec) were used for cytometry analysis of peri-toneal cells
Monocytes were isolated from PBMCs by the cell cul-ture adherence method For monocyte isolation by adher-ence PBMCs were seeded onto cell culture plates andallowed to adhere in a 5 CO
2incubator at 37∘C for
1 hour in serum-free medium Nonadherent cells wereremoved and the adherent cells were carefully washed twicewith medium before culturing in RPMI medium plus 10heat-inactivated fetal calf serum (FCS) Peripheral bloodmonocyte-derivedM2 cells were generated as described [25]Briefly monocytes were incubated in RPMI with 10 heat-inactivated FCS and 50 UmL IL-4 (Peprotech UK) for 72hours
Cells were seeded at 200000 cellswell in 96-well platesand incubated with various doses of paricalcitol In someexperiments the commercial form (Zemplar) was used asthe source of drug and cultures in the presence of theequivalent concentration of vehicle (30 50 20 propyleneglycol water ethanol)were established in parallel as negativecontrols Stock solutions of pure paricalcitol (Abbott) andvitamin D3 [125(OH)
2D3] (Roche) were prepared in EtOH
at 10minus3MCell viability was analyzed by exclusion of propidium
iodide staining and flow cytometry
25 Quantitative Reverse Transcription-Polymerase ChainReaction (qRT-PCR) Total RNA was isolated using theHigh Pure RNA Isolation Kit (Roche Germany) and 1 120583gwas reverse transcribed with random hexanucleotides andavian myeloblastosis virus (AMV) reverse transcriptase(Promega) in a final volume of 20120583L for 1 hour at 42∘CFor quantitative analysis of mRNA content 1120583L aliquotsof the resulting cDNAs were PCR amplified in a LightCycler (Roche) with FastStart DNA Master SYBR Green I(Roche) in duplicate Primers used for PCR amplificationwere previously described [15] CCL18 sense 51015840-ACA AAGAGC TCT GCT GCC TC-31015840 CCL18 antisense 51015840-CCCACT TCT TAT GGG GTC A-31015840 B2M sense 51015840-CCA GCAGAG AAT GGA AAG TC-31015840 B2M antisense 51015840-GAT GCTGCT TAC ATG TCT CG-31015840 Standard curves for targetmRNAexpressionwere generated by amplifying 10-fold serialdilutions of known quantities of the specific PCR productsQuantification of target gene expression was obtained usingLight Cycler system software Relative units (RU) estimatedfrom the quantification represent the ratio between CCL18mRNA molecules and B2M mRNA molecules in each sam-ple
26 Statistical Analysis The statistical analysis was per-formed using GraphPad Prism software (San Diego CA)Parametric or nonparametric tests were used according tothe distribution of the data Data are shown as mean plusmnSEM mean plusmn SD values or median and interquartile ranges(IQR) as indicated in each figure Data were analyzed withparametric or nonparametric 119905-tests for unpaired or pairedsamples Correlation was assessed using Spearman correla-tion tests with 119901 values 119901 values less than or equal to 05 wereconsidered statistically significant
Gram +0
10
20
30
40
CCL1
8 (n
gm
l)
(N = 13) (N = 26)
ns
Gram minus
Figure 1The concentration of CCL18was evaluated in 39 peritonealeffluents from patients diagnosed with infectious peritonitis Efflu-entswere collected on the first day of cloudy effluentHorizontal barsrepresent mean plusmn SEM values No significant differences were foundbetween Gramminus (119873 = 13) and Gram+ bacteria (119873 = 26) infectedcases (unpaired 119905-test)
3 Results
31 CCL18 Measurements in Peritonitis Effluent SamplesPrevious studies propose CCL18 as a marker and mediatorof peritoneal fibrosis and UFF in patients treated with PD[15 16] Given recurrent peritonitis is a risk factor forperitoneal fibrosis and UFF the concentration of CCL18was evaluated in a group of 39 PD patients with infectiousperitonitis Effluent samples were obtained at diagnosis andthe concentration of CCL18 was measured by enzyme-linkedimmunosorbent assay (ELISA) The mean concentration ofCCL18 in peritonitis samples was 1502 plusmn 787 ngml whereasmean values previously reported ranged between 1 and6 ngml No significant differences were found in patientsinfected with Gram+ or Gramminus bacteria Mean observedvalues were 133 plusmn 243 ngml in the Gramminus versus 1586 plusmn145 ngml in the Gram+ samples (Figure 1)
We evaluated CCL18 concentrations in serial samplesfrom four of the PD patients with infectious peritonitis(IP) (Figure 2(a)) CCL18 effluent concentrations showedthe highest values during the first days of peritonitis anddecreased slowly following a kinetics similar to absolutemacrophage counts in these samples (Figure 2(c)) and inagreement with previous reports describing macrophages asthe primary source of this chemokine [15]
Serial samples were also analyzed in one additionalcase of sterile EP associated with an accidental exposureof the peritoneum to chlorhexidine Surprisingly whereasthe mean CCL18 concentrations on day 1 and on days2ndash4 in the IP samples were 155 plusmn 61 ngml and 117 plusmn69 ngml respectively in the EP samples the concentrationof CCL18 was 19812 ngml on day 1 and 20465 ngml on day3 (Figure 3 and Supplemental Figure 1(A)) IL-6 was alsoevaluated for comparative purposes as the hallmark cytokinein inflammatory processes and in PD-associated peritonitis[26] Elevated levels of IL-6were found in spent effluents fromall patients on the first day of peritonitis with similar values
4 BioMed Research International
CCL1
8 (n
gm
l)CC
L18
(ng
ml)
CCL1
8 (n
gm
l)
Days of evolution
CCL1
8 (n
gm
l)
D1 D3 D120
2
4
6
8
10IP-1
0
5
10
15
20
25
D1 D2 D5 D26
IP-2
0
5
10
15
20
25
D1 D4 D8 D13
IP-4
02468
10121416
D1 D3 D5 D22
IP-3
(a)
0
1
1
2
2
3
3
4
D1 D3 D12
IP-1
0
5
10
15
20
D1 D2 D5 D26
IP-2
0
5
10
15
D1 D3 D5 D22
IP-3
0
1
2
3
4
5
6
D1 D4 D8 D13
IL-6
(ng
ml)
IL-6
(ng
ml)
IL-6
(ng
ml)
IL-6
(ng
ml)
IP-4
(b)
Days of evolution
1
10
100
1000
10000
100000
D1 D3 D12
1
10
100
1000
10000
100000
D1 D2 D4 D5
1
10
100
1000
10000
D1 D3 D5
1
10
100
1000
10000
D1 D4 D8
IP-1
IP-2
IP-3
IP-4
(Cel
ls
l)(C
ells
l)
(Cel
ls
l)(C
ells
l)
MΦlPMNl
(c)
Figure 2 Serial cytokine concentrations and cell counts in peritoneal effluent during peritonitis episodes (a) Evolution of CCL18 effluentlevels in serial samples from four patients with infectious peritonitis (IP) shown as IP-1 to IP-4 (b) Evolution of IL-6 effluent levels inserial samples from the same patients (c) Comparison of neutrophils (PMN) and macrophage (MΦ) cell counts in serial effluent samplesDifferential cell counts on D26 (IP-2) D22 (IP-3) and D13 (IP-4) were not available given that the total cell counts were lt50 cells120583l in thosesamples
in IP samples and in the EP sample (Supplemental Figure1(B)) A sharp decline in IL-6 occurred in all the IP cases aspreviously described [26 27] (Figure 2(b)) and in contrast toCCL18 levels which remained elevated during the followingdays and declined more slowly (Figure 2(a))
32 Peritoneal Eosinophils Are a Source of CCL18 in Eosin-ophilic Peritonitis Cytokine concentrations were comparedwith absolute and differential cell counts in effluent samplesfrom the patient with EP (Figure 3) A significant correlationwas found between the kinetics of CCL18 concentration and
BioMed Research International 5
000
5000
10000
15000
20000
25000
D1 D3 D6 D17 D190
1000
2000
3000
4000
5000
CCL1
8 (n
gm
L)
CCL18 Total cells
Cel
ls
L
(a)
000
5000
10000
15000
20000
25000
D1 D3 D6 D17 D190
500
1000
1500
2000
2500
CCL1
8 (n
gm
L)
CCL18PMN
PMN
L
(b)
000
5000
10000
15000
20000
25000
D1 D3 D6 D17 D190
200
400
600
800
CCL1
8 (n
gm
L)
CCL18
MΦ
L
MΦ
(c)
000
5000
10000
15000
20000
25000
D1 D3 D6 D17 D19
CCL1
8 (n
gm
L)
0
100
200
300
400
500
600
700
CCL18 Eos
Eos
L
(d)
IL-6Total cells
0
2000
4000
6000
8000
10000
D1 D3 D6 D17 D190
1000
2000
3000
4000
5000
IL-6
(pg
mL)
Cel
ls
L
(e)
0
2000
4000
6000
8000
10000
D1 D3 D6 D17 D190
500
1000
1500
2000
2500
IL-6PMN
IL-6
(pg
mL)
PMN
L
(f)
0
2000
4000
6000
8000
10000
D1 D3 D6 D17 D190
200
400
600
800
IL-6
IL-6
(pg
mL)
MΦ
L
MΦ
(g)
0
2000
4000
6000
8000
10000
D1 D3 D6 D17 D190
100
200
300
400
500
600
700
IL-6
(pg
mL)
IL-6Eos
Eos
L
(h)
Figure 3 Kinetics of effluent cell counts and cytokine concentrations during EP (andashd) CCL18 effluent levels and cell counts in serial effluentsamples (endashh) IL-6 effluent levels and cell counts in serial effluent samples
6 BioMed Research International
(a)
(b) (c)
Figure 4 Cytospin preparations showing effluent cell content in EP (a) Hematoxylin and eosin staining (b) Immunocytochemistry showingCCL18 staining of eosinophils (c) Background staining with nonimmune serum
the macrophage counts in the EP samples (119903 = 0889119901 = 043) (Figure 3(c)) However the strongest correlationwas found between CCL18 effluent levels and eosinophils(119903 = 0949 119901 = 013) with peak CCL18 concentrationsdetected in effluents with the highest absolute eosinophilcount (Figure 3(d)) In contrast IL-6 effluent levels paralleledpolymorphonuclear neutrophils (PMNs) and absolute cellnumbers in effluent samples from EP (Figures 3(e) and3(f)) and no correlation was observed between effluent IL-6 concentrations and eosinophil cell count (Figure 3(h))
Although CCL18 is thought to be primarily produced byM2 macrophages involved in tissue repair and fibrosis andby dendritic cells [28] eosinophils have occasionally beenreported to produce CCL18 during inflammatory conditions[29 30] The kinetics of CCL18 effluent concentrations in EPand eosinophil counts suggested that not only macrophagesbut also eosinophils might contribute to the production ofCCL18 in this case To test this hypothesis immunostainingof cytospin preparations from eosinophilic effluents wasperformed Specific staining was detected in the cytoplasmof typical binucleated cells supporting the role of eosinophilsas a source of CCL18 in this case (Figure 4)
In a previous report we found that only peritonealmacrophages were able to produce CCL18 during infectiousperitonitis given that the cytokine was not detected in cul-ture supernatants from neutrophils (PMNs) or lymphocytes[15] To further analyze the putative role of eosinophils asproducers of CCL18 in the patient with EP CD66b+ polymor-phonuclear leukocytes (among which 85 were eosinophils
as assessed by CD16 negative staining of eosinophils versusCD16 positive neutrophils) were isolated from peritonealeffluent cells on D6 as well as CD14+ macrophages forcomparative purposes (Figures 5(a)ndash5(c)) CCL18 transcriptswere detected in both the CD66b+ and CD14+ fractions(Figure 5(d)) Quantitative PCR analysis confirmed CCL18mRNA transcripts in polymorphonuclear leukocytes in thiscase (Figure 5(e)) Altogether the data strongly suggest thateosinophils are producers of CCL18 in EP
33 Paricalcitol Downregulates the Production of CCL18 byPeritoneal Macrophages Paricalcitol a VDR agonist hasbeen shown to ameliorate peritoneal fibrosis in a murinemodel of PD [23] Given that CCL18 has been involvedin peritoneal fibrosis in PD patients [15 20 22] we testedthe effect of paricalcitol in the production of CCL18 byhuman pMΦs isolated fromPD effluents drawn frompatientssuffering from peritonitis and cultured with various dosesof nontoxic concentrations of Zemplar as a source of par-icalcitol or vehicle (supplementary Figure 2) As shown inFigures 6(a)ndash6(c) pharmacological treatment was able tosignificantly downregulate the production of CCL18 in thesecells at concentrations equivalent to 10minus8M and 10minus9M ofparicalcitol This effect was obtained as early as 24 hours andit was also observed at 48 hours and 72 hours of cell cultureSimilar results were obtained with the pure substance andwith equivalent doses of vitamin D3 (Figure 6(d))
It has been suggested that the regulation of CCL18 proteinsynthesis and secretion can be a complex process with some
BioMed Research International 7
0 200 400 600 800 1000FSC-H
PolymorphonuclearLeukocytes
0
200
400
600
800
1000
SSC-
HR1 = 715 LΦR2 = 24 MΦR3 = 6885
(a)CD16-FITC
767PMN
0
200
400
600
800
1000
SSC-
H
100 101 102 103 104
(b)
CD14-APC
999
CD66
b-FI
TC
100
100
101
101
102
102
103 104
103
104
(c)
500
300
400
200
300
200 CCL18
B2M
100
bp la
dder
NEG
CD66<+
(Eos
)
CD14+
(MΦ
)
(d)
CD14+ CD66b+
CCL1
8B2
M m
RNA
(RU
)
25E minus 05
20E minus 05
15E minus 05
10E minus 05
50E minus 06
00E + 00
(e)
Figure 5 (a) Flow cytometry analysis of peritoneal effluent leukocytes on D6 according to morphological parameters LΦ lymphocytesMΦ macrophages (b) Flow cytometry analysis of CD16 expression in peritoneal effluent cells on D6 The cutoff level for positive stainingwas set according to isotype control background staining PMN polymorphonuclear neutrophils (c) Flow cytometry analysis of CD66b+polymorphonuclear cells isolated from peritoneal effluent cells (d) RT-PCR of CCL18 gene expression in CD66+ and CD14+ purifiedperitoneal leukocytes (e) Quantitative RT-PCR analysis of CCL18 gene expression in CD66+ and CD14+ purified peritoneal leukocytes
degree of posttranscriptional regulation [28] To analyzewhether VDR agonists regulate CCL18 mRNA levels quan-titative RT-PCR assays were performed in isolated pMΦstreated with paricalcitol CCL18 mRNA levels were downreg-ulated in parallel with the diminished production of CCL18protein in pMΦs from two different donors representinginfected and uninfected peritoneal samples (Figures 7(a) and7(b)) Given that IL-4 is one of the main inducers of CCL18[31] we tested if VDR agonists couldmodulate the expressionof the chemokine in M2 macrophages generated in vitroupon treatment of peripheral blood monocytes with IL-4 Asshown in Figure 7(c) the expression of CCL18 was highlyupregulated in monocyte-derived macrophages treated for 3days with IL-4 and pretreatment with paricalcitol or vitaminD was able to prevent the upregulation of CCL18 in thesecells
4 Discussion
Peritonitis episodes constitute a frequent complication in PDpatients and recurrent peritonitis is a well-known clinicalrisk factor for peritoneal membrane dysfunction and EPS
There is an unmet requirement for biomarkers as toolsto identify patients who are at higher risk of techniquefailure to guide interventions that could improve clinicaloutcomes [32] Soluble factors released during inflammatoryprocesses such as IL-6 and IL-17 have been proposed asbiomarkers and mediators of peritoneal membrane deteri-oration (reviewed in [32 33]) However none are used inthe clinical routine and some cytokines such as IL-17 arenot easily measured in PD effluents in humans We proposeCCL18 as a biomarker of peritoneal membrane outcome inPD [22]
8 BioMed Research International
0
20
40
60
80
100
120
C
CL18
pro
duct
ion
Pari VV Pari(10minus8 M) (10minus9 M)
lowastp = 0156 lowastp = 0156
(a)
0
20
40
60
80
100
120
C
CL18
pro
duct
ion
Pari VV Pari(10minus8 M) (10minus9 M)
p = 0625
(b)
0
20
40
60
80
100
120
C
CL18
pro
duct
ion
Pari VV Pari(10minus8 M) (10minus9 M)
lowastp = 0313lowastlowastp = 0039
(c)
0
20
40
60
80
100
ParicalcitolVehicle Vit D
120
C
CL18
pro
duct
ion
(10minus8 M) (10minus8 M)
(d)
Figure 6 (andashc) Peritoneal macrophages isolated from patients experiencing peritonitis episodes were treated with vehicle or Zemplar indoses equivalent to 10minus8M and 10minus9M of paricalcitol during 24 h (a) 48 h (b) or 72 h (c) Equivalent cell cultures were established in theabsence of any treatment The concentration of CCL18 was evaluated in cell-free supernatants and the production of CCL18 in the presenceof vehicle or drug referred to the production in cell culture media alone Differences between cultures in the presence of paricalcitol or vehiclewere analyzed by theWilcoxon matched pairs rank test (d) Peritoneal macrophages were treated with paricalcitol as pure substance vitaminD or vehicle (ethanol) for 48 h
Given that high levels of the human chemokine CCL18have been shown to be related to peritoneal membranefailure and fibrosis [15 20 22] whereas sustained low CCL18peritoneal concentrations appear to be protective [22] weevaluated CCL18 levels in the peritoneum of patients under-going peritonitis episodes We found that the peritoneal con-centration of CCL18 was increased approximately 5-fold ineffluents from patients with infectious peritonitis comparedwith reported values in uninfected samples In a previousstudy involving a longitudinal analysis of 43 patients CCL18effluent concentrations ranged from 28 plusmn 16 ngml at thestart of dialysis to 34plusmn 18 ngml after 3 years of PD treatment[22] In a cross-sectional analysis of 61 patients treated formore than 3 years with PDmean effluent CCL18 values were395 plusmn 26 ngml [22] The high concentrations during IPare consistent with a previous finding showing that pMΦsfrom patients undergoing peritonitis or who recently had
peritonitis were able to secrete higher amounts of CCL18compared with pMΦs from uninfected patients [15]
Unexpectedly in a patient identified with sterileeosinophilic peritonitis the peritoneal effluent was extremelyrich in CCL18 with levels approximately 10-fold higher thanthose found in patients with IP and 50-fold more thanthose found in uninfected samples whereas effluent levelsof IL-6 which were evaluated in the same samples forcomparative purposes were similar on day 1 in both theIP and EP samples The differences between a peritonealinflammatory recognized marker IL-6 and CCL18 duringperitonitis episodes should be remarked It is of note thatthe peritoneal concentrations of CCL18 remained elevatedfor several days whereas there was a sharp decrease in IL-6Both IL-6 and CCL18 have been reported to increase indifferent inflammatory conditions some leading to tissuefibrosis [34ndash37] As a main difference although CCL18 is
BioMed Research International 9
NEG
CCL1
8 (p
gm
l)
0
200
400
600
800
1000
12001057
522
Pari (10minus7 M) NEG000
020
040
060
080
100
120100
025Fold
mRN
A C
CL18
Pari (10minus7 M)
(a)
NEG
408
279
0
100
200
300
400
500
CCL1
8 (p
gm
l)
Pari (10minus7 M)
100
045
000
050
100
150
NEG
Fold
mRN
A C
CL18
Pari (10minus7 M)
(b)
000
025
050
075
100
125
Fold
mRN
A C
CL18
NEG
Mo + IL-4
Pari Vit D3Vmdash
(c)
Figure 7 (a) Peritoneal macrophages from a patient with recent infectious peritonitis were treated with paricalcitol or vehicle for 24 h Theprotein concentration of CCL18 was evaluated in cell culture supernatants by ELISA and CCL18 mRNA levels were estimated by quantitativeRT-PCR in the same cultures (b) Peritoneal macrophages from an uninfected peritoneum were isolated from the peritoneal lavage uponcatheter insertion and treated as in (a) (c) Peripheral bloodmonocytes were pretreated with vehicle (V) 10minus7Mparicalcitol (Pari) or vitaminD3 for 24 h The cells were treated thereafter with IL-4 for 3 days before RNA extraction and evaluation expression of CCL18 mRNA levelsby quantitative RT-PCR Mean plusmn SD values from four independent experiments are shown
primarily produced by cells of myeloid origin multiplecell types are producers of IL-6 with endothelial cellsbeing an important source of this cytokine Infiltratingneutrophils as well as mesothelial cells could also be animportant source of IL-6 during bacterial peritonitis [37]The analysis of serial samples in four patients with IP showedthat the concentrations of CCL18 paralleled the numbers ofmacrophages infiltrating the peritoneum and are consistentwith previous descriptions of macrophages andmononuclearphagocytes as the primary producers of CCL18 [21] Cellcounts in the serial samples from our case of EP suggest that
the peritoneal eosinophilic infiltrate was likely contributingto the peritoneal production of CCL18 Evidences of CCL18production by activated peritoneal eosinophils in this caseare cytospin analysis of CCL18 cytoplasmic staining ineosinophils and gene expression data in polymorphonuclearleukocytes among which 85 were eosinophils
Although macrophages are typically reported as themajor source of this chemokine [21] CCL18 protein pro-duction by eosinophils has occasionally been described asbeing related to the activation status of these cells given thatCCL18 was found only in eosinophil culture supernatants
10 BioMed Research International
from eosinophilic donors but not from individuals withnonactivated eosinophils [29] Our data support the activatedstatus of eosinophils in EP given that strong staining ofCC18 was detected in peritoneal eosinophils Eosinophilshave also been related to tissue fibrosis in several diseasesand are a source of several profibrotic mediators that alterfibroblast activity and tissue remodeling [38 39] Some ofthesemediators could also stimulate the profibrotic activity ofpMΦTheproduction ofCCL18 by eosinophils in EPprovidesa new additional explanation for the association of recurrentEP during PD with UFF and potentially peritoneal fibrosis[40]
As part of its profibrotic activity CCL18 has been shownto stimulate collagen deposition [41] and fibroblast prolif-eration [42] In consonance with these actions circulatingCCL18 levels have been demonstrated to be a biomarker fordisease activity and outcome in various fibrotic diseases [1943 44] as well as in some eosinophilic disorders [30 45 46]Moreover CCL18 has been shown to promote EMT in variouscell systems [47ndash50] It is thus possible that it could alsocontribute to the EMT of mesothelial cells which has beenproposed as one of the mechanisms leading to peritonealfibrosis and membrane failure [12]
In terms of peritoneal fibrosis CCL18 has been demon-strated to herald the development of peritoneal functionaldeterioration in PD patients [15 20] in whom the abilityof peritoneal macrophages to stimulate fibroblast prolifer-ation is correlated with CCL18 mRNA levels Moreoverrecombinant CCL18 was able to stimulate the proliferation ofperitoneal fibroblasts [15]
Several molecules have been tested for their ability toprevent peritoneal fibrosis in vitro and in animal models(reviewed in [8]) Among them paricalcitol is a selectiveVDR agonist VDR is a nuclear hormone receptor expressedin various cell types including those of the immune sys-tem VDR agonists have been shown to modulate immuneresponses inflammation and fibrosis [51]
Considering the role played by inflammation in peri-toneal fibrosis during PDwe studied the effects of paricalcitolin the production of CCL18 We found that the release andgene expression of CCL18 were downregulated in pMΦsand in in vitro-differentiated human M2 cells which are theprimary producers of CCL18These results appear to contrastwith previous data showing upregulation of CCL18 by vita-min D in monocyte-derived immature dendritic cells [52]However recent reports demonstrate that the modulationof immune signaling by vitamin D in human mononuclearphagocytes is cell-type-specific with differential responses inmonocytes macrophages and dendritic cells [53] It is thuspossible that opposite effects are found on the regulation ofCCL18 by VDR agonists in different cellular systems
On the other hand in a mouse model of PD intraperi-toneal administration of paricalcitol was shown to reduceperitoneal fibrosis and ultrafiltration failure [23] In thatmodel the beneficial effects appeared to be dependent ona reduction of intraperitoneal concentrations of IL-17 Thelack of rodent orthologues of human CCL18 [28] precludesthe investigation of CCL18 and its involvement in fibrosis inmurine models On the other hand IL-17 is highly diluted
in human peritoneal effluents The need to concentrate thesamples for measurement of this cytokine hampers accuratedeterminations in human samples
A preliminary study involving 23 patients on PD suggeststhat paricalcitol could improve the ultrafiltration capacitywith less peritoneal protein loss [24] The authors suggestan effect of VDR upon the renin-angiotensin-aldosteronesystem but it is tempting to speculate that lower peritonealconcentrations of CCL18 might be present in paricalcitol-treated patients In this sense a slight reduction in theperitoneal concentration of CCL18 was observed in sixpatients who were more than 3 years on PD and treated withparicalcitol compared with a set of 31 patients of the samevintage (data not shown)
5 Conclusion
In conclusion our study suggests that the chemokine CCL18can be a mediator of peritoneal membrane failure associatedwith peritonitis episodes and might provide a new potentialtherapeutic target
Further clinical studies are needed to confirm the useful-ness of VDR agonists to preserve the peritoneal membraneand to analyze the putative mechanisms involved
Conflicts of Interest
All the authors have no potential conflicts of interest todeclare
Authorsrsquo Contributions
Marta Ossorio and Virginia Martınez contributed equally tothis work
Acknowledgments
This study has been partially supported by ISCIII (ldquoInsti-tuto de Investigacion Sanitaria Carlos IIIrdquo) Grants FIS PI1200204 PI1301768 and PI 1500120 FEDER (Funds fromthe European Union) REDinREN 20 FEDER and ReinaSofıa Institute of Nephrology Research (IRSIN) monetaryfunds The authors would like to thank Juliette Siegfriedand her team at ServingMedcom for their editing of themanuscript
Supplementary Materials
Supplemental Figure 1 serial cytokine concentrations andcell counts in peritoneal effluent during infectious peritoni-tis and eosinophilic peritonitis (A) Comparison of CCL18concentration in serial effluent samples from infectiousperitonitis (IP) and eosinophilic peritonitis (EP) (B) IL-6effluent levels in serial samples from IP and EP D1ndashD26indicate days of evolution Supplemental Figure 2 dose-dependent effects of VDR agonists on cell viability Cellviability of human monocytes was assessed by exclusion ofpropidium iodide staining and analyzed by flow cytometry
BioMed Research International 11
Results are shown as mean plusmn SD values of triplicates(A)ndash(C) Viability of monocytes upon culture during 24 48or 72 hours with decreasing concentrations of Zemplar orequivalent vehicle (305020 propylene glycolwaterethanol)(D)ndash(F) Viability of monocytes upon culture during 2448 or 72 hours with different concentrations of paricalcitol(pure substance) vitamin D or equivalent vehicle (EtOH)(Supplementary Materials)
References
[1] R T Krediet ldquoThe peritoneal membrane in chronic peritonealdialysisrdquo Kidney International vol 55 no 1 pp 341ndash356 1999
[2] R T Krediet ldquoPeritoneal physiology - Impact on solute andfluid clearancerdquo Advances in Chronic Kidney Disease vol 7 no4 pp 271ndash279 2000
[3] M Merkle and M Wrnle ldquoSclerosing peritonitis A rare butfatal complication of peritoneal inflammationrdquo Mediators ofInflammation vol 2012 Article ID 709673 p 4 2012
[4] D W Johnson Y Cho B E R Livingston et al ldquoEncapsulat-ing peritoneal sclerosis incidence predictors and outcomesrdquoKidney International vol 77 no 10 pp 904ndash912 2010
[5] G Balasubramaniam E A Brown and A Davenport ldquoThePan-Thames EPS study treatment and outcomes of encapsulat-ing peritoneal sclerosisrdquo Nephrology Dialysis Transplantation vol 24 no 10 pp 3209ndash3215 2009
[6] E D Sousa-Amorim G D Peso M A Bajo et al ldquoCan EPSdevelopment be avoided with early interventionsThe potentialrole of tamoxifenmdasha single-center studyrdquo Peritoneal DialysisInternational vol 34 no 6 pp 582ndash593 2014
[7] M R Korte D E SampimonM GH Betjes and R T KredietldquoEncapsulating peritoneal sclerosis the state of affairsrdquo NatureReviews Nephrology vol 7 no 9 pp 528ndash538 2011
[8] M A Bajo G del Peso and I Teitelbaum ldquoPeritoneal Mem-brane Preservationrdquo Seminars in Nephrology vol 37 no 1 pp77ndash92 2017
[9] M F Flessner ldquoInflammation from sterile dialysis solutions andthe longevity of the peritoneal barrierrdquo Clinical Nephrology vol68 no 6 pp 341ndash348 2007
[10] K N Lai and J C K Leung ldquoInflammation in peritonealdialysisrdquo Nephron Clinical Practice vol 116 no 1 pp c11ndashc182010
[11] S Yung and T M Chan ldquoPathophysiological changes to theperitoneal membrane during PD-related peritonitis the role ofmesothelial cellsrdquo Mediators of Inflammation vol 2012 ArticleID 484167 21 pages 2012
[12] L S Aroeira A Aguilera J A Sanchez-Tomero et al ldquoEpithe-lial tomesenchymal transition and peritonealmembrane failurein peritoneal dialysis patients pathologic significance andpotential therapeutic interventionsrdquo Journal of the AmericanSociety of Nephrology vol 18 no 7 pp 2004ndash2013 2007
[13] N Topley T Liberek A Davenport F-K Li H Fear andJ D Williams ldquoActivation of inflammation and leukocyterecruitment into the peritoneal cavityrdquo Kidney InternationalSupplements vol 50 no 56 pp S17ndashS21 1996
[14] F O Martınez and S Gordon ldquoThe M1 and M2 paradigmof macrophage activation time for reassessmentrdquo F1000PrimeReports vol 6 article 13 2014
[15] T Bellon V Martınez B Lucendo et al ldquoAlternative activa-tion of macrophages in human peritoneum implications for
peritoneal fibrosisrdquo Nephrology Dialysis Transplantation vol26 no 9 pp 2995ndash3005 2011
[16] A Prasse D V Pechkovsky G B Toews et al ldquoA vicious circleof alveolar macrophages and fibroblasts perpetuates pulmonaryfibrosis via CCL18rdquoAmerican Journal of Respiratory and CriticalCare Medicine vol 173 no 7 pp 781ndash792 2006
[17] J M Estep A Baranova N Hossain et al ldquoExpression ofcytokine signaling genes in morbidly obese patients with non-alcoholic steatohepatitis and hepatic fibrosisrdquo Obesity Surgeryvol 19 no 5 pp 617ndash624 2009
[18] F Kollert M Binder C Probst et al ldquoCCL18 - Potentialbiomarker of fibroinflammatory activity in chronic periaortitisrdquoThe Journal of Rheumatology vol 39 no 7 pp 1407ndash1412 2012
[19] A Prasse D V Pechkovsky G B Toews et al ldquoCCL18 as anindicator of pulmonary fibrotic activity in idiopathic interstitialpneumonias and systemic sclerosisrdquo Arthritis amp Rheumatologyvol 56 no 5 pp 1685ndash1693 2007
[20] S Ahmad B V North A Qureshi et al ldquoCCL18 in peritonealdialysis patients and encapsulating peritoneal sclerosisrdquo Euro-pean Journal of Clinical Investigation vol 40 no 12 pp 1067ndash1073 2010
[21] V Kodelja C Muller O Politz N Hakij C E Orfanos andS Goerdt ldquoAlternative macrophage activation-associated CC-chemokine-1 a novel structural homologue of macrophageinflammatory protein-1120572 with a Th2- associated expressionpatternrdquo The Journal of Immunology vol 160 no 3 pp 1411ndash1418 1998
[22] M Ossorio M A Bajo G Del Peso et al ldquoSustained low peri-toneal effluent CCL18 levels are associated with preservationof peritoneal membrane function in peritoneal dialysisrdquo PLoSONE vol 12 no 4 Article ID e0175835 2017
[23] G T Gonzalez-Mateo V Fernandez-Mıllara T Bellon etal ldquoParicalcitol reduces peritoneal fibrosis in mice throughthe activation of regulatory T cells and reduction in IL-17productionrdquo PLoS ONE vol 9 no 10 Article ID e108477 2014
[24] F Coronel S Cigarran A Gomis et al ldquoChanges in peritonealmembrane permeability and proteinuria in patients on peri-toneal dialysis after treatment with paricalcitolmdasha preliminarystudyrdquo Clinical Nephrology vol 78 no 2 pp 93ndash99 2012
[25] E Song N Ouyang M Horbelt B Antus M Wang and MS Exton ldquoInfluence of alternatively and classically activatedmacrophages on fibrogenic activities of human fibroblastsrdquoCellular Immunology vol 204 no 1 pp 19ndash28 2000
[26] S M Hurst T S Wilkinson R M McLoughlin et al ldquoIL-6 andits soluble receptor orchestrate a temporal switch in the patternof leukocyte recruitment seen during acute inflammationrdquoImmunity vol 14 no 6 pp 705ndash714 2001
[27] M Kozioł-Montewka A Ksiazek L Janicka and I BaranowiczldquoSerial cytokine changes in peritoneal effluent and plasma dur-ing peritonitis in patients on continuous ambulatory peritonealdialysis (CAPD)rdquo Inflammation Research vol 46 no 4 pp 132ndash136 1997
[28] E Schutyser A Richmond and J Van Damme ldquoInvolvementof CC chemokine ligand 18 (CCL18) in normal and pathologicalprocessesrdquo Journal of Leukocyte Biology vol 78 no 1 pp 14ndash262005
[29] I Schraufstatter H Takamori L Sikora P Sriramarao and RG DiScipio ldquoEosinophils and monocytes produce pulmonaryand activation-regulated chemokine which activates culturedmonocytesmacrophagesrdquoAmerican Journal of Physiology-LungCellular and Molecular Physiology vol 286 no 3 pp L494ndashL501 2004
12 BioMed Research International
[30] C Gunther N Carballido-Perrig T Kopp J M Carballidoand C Pfeiffer ldquoCCL18 is expressed in patients with bullouspemphigoid and parallels disease courserdquo British Journal ofDermatology vol 160 no 4 pp 747ndash755 2009
[31] AMantovani A Sica S Sozzani P Allavena A Vecchi andMLocati ldquoThe chemokine system in diverse forms of macrophageactivation and polarizationrdquo Trends in Immunology vol 25 no12 pp 677ndash686 2004
[32] C Aufricht R Beelen M Eberl et al ldquoBiomarker researchto improve clinical outcomes of peritoneal dialysis consensusof the European Training and Research in Peritoneal Dialysis(EuTRiPD) networkrdquo Kidney International vol 92 no 4 pp824ndash835 2017
[33] D Lopes Barreto andR T Krediet ldquoCurrent status and practicaluse of effluent biomarkers in peritoneal dialysis patientsrdquoAmerican Journal of Kidney Diseases vol 62 no 4 pp 823ndash8332013
[34] L A Boven M van Meurs R G Boot et al ldquoGaucher cellsdemonstrate a distinct macrophage phenotype and resemblealternatively activated macrophagesrdquo American Journal of Clin-ical Pathology vol 122 no 3 pp 359ndash369 2004
[35] L Cocho I Fernandez M Calonge et al ldquoGene expression-based predictive models of graft versus host disease-associateddry eyerdquo Investigative Ophtalmology Visual Science vol 56 no8 pp 4570ndash4581 2015
[36] MHasegawa ldquoBiomarkers in systemic sclerosisTheir potentialto predict clinical coursesrdquoThe Journal of Dermatology vol 43no 1 pp 29ndash38 2016
[37] S A Jones D J Fraser C A Fielding and G W JonesldquoInterleukin-6 in renal disease and therapyrdquoNephrologyDialysisTransplantation vol 30 no 4 pp 564ndash574 2015
[38] A Munitz and F Levi-Schaffer ldquoEosinophils rsquoNewrsquo roles forrsquooldrsquo cellsrdquo Allergy European Journal of Allergy and ClinicalImmunology vol 59 no 3 pp 268ndash275 2004
[39] S S Aceves ldquoRemodeling and fibrosis in chronic eosinophilinflammationrdquo Digestive Diseases vol 32 no 1-2 pp 15ndash212014
[40] Y Nakamura H Okada A Yasui T Koh and T YamaneldquoSclerosing encapsulating peritonitis associated with recurrenteosinophilic peritonitisrdquo Nephrology Dialysis Transplantation vol 14 no 3 pp 768ndash770 1999
[41] S P Atamas I G Luzina J Choi et al ldquoPulmonary andactivation-regulated chemokine stimulates collagen productionin lung fibroblastsrdquo American Journal of Respiratory Cell andMolecular Biology vol 29 no 6 pp 743ndash749 2003
[42] A Wimmer S K Khaldoyanidi M Judex N Serobyan RG DiScipio and I U Schraufstatter ldquoCCL18PARC stimulateshematopoiesis in long-term bone marrow cultures indirectlythrough its effect on monocytesrdquo Blood vol 108 no 12 pp3722ndash3729 2006
[43] A Prasse C Probst E Bargagli et al ldquoSerum CC-chemokineligand 18 concentration predicts outcome in idiopathic pul-monary fibrosisrdquo American Journal of Respiratory and CriticalCare Medicine vol 179 no 8 pp 717ndash723 2009
[44] J Chen Y Yao C Gong et al ldquoCCL18 from tumor-associatedmacrophages promotes breast cancermetastasis via PITPNM3rdquoCancer Cell vol 19 no 4 pp 541ndash555 2011
[45] K L Hon G K Ching P C Ng and T F Leung ldquoExplor-ing CCL18 eczema severity and atopyrdquo Pediatric Allergy andImmunology vol 22 no 7 pp 704ndash707 2011
[46] A J Lucendo A Arias L C De Rezende et al ldquoSubepithelialcollagen deposition profibrogenic cytokine gene expressionand changes after prolonged fluticasone propionate treatment inadult eosinophilic esophagitis a prospective studyrdquoThe Journalof Allergy and Clinical Immunology vol 128 no 5 pp 1037ndash1046 2011
[47] H Li D Zhang J Yu et al ldquoCCL18-dependent translocationof AMAP1 is critical for epithelial to mesenchymal transition inbreast cancerrdquo Journal of Cellular Physiology vol 233 no 4 pp3207ndash3217 2017
[48] Z Lin W Li H Zhang et al ldquoCCL18PITPNM3 enhancesmigration invasion and EMT through the NF-120581B signalingpathway in hepatocellular carcinomardquo Tumor Biology vol 37no 3 pp 3461ndash3468 2016
[49] F Meng W Li C Li Z Gao K Guo and S SongldquoCCL18 promotes epithelial-mesenchymal transition invasionand migration of pancreatic cancer cells in pancreatic ductaladenocarcinomardquo International Journal of Oncology vol 46 no3 pp 1109ndash1120 2015
[50] T Ploenes B Scholtes A Krohn et al ldquoCC-Chemokine Ligand18 Induces Epithelial to Mesenchymal Transition in LungCancer A549 Cells and Elevates the Invasive Potentialrdquo PLoSONE vol 8 no 1 Article ID e53068 2013
[51] J Rojas-Rivera C De La Piedra A Ramos A Ortiz andJ Egido ldquoThe expanding spectrum of biological actions ofvitamin Drdquo Nephrology Dialysis Transplantation vol 25 no 9pp 2850ndash2865 2010
[52] M Vulcano S Struyf P Scapini et al ldquoUnique regulation ofCCL18 production by maturing dendritic cellsrdquo The Journal ofImmunology vol 170 no 7 pp 3843ndash3849 2003
[53] R Kundu A Theodoraki C T Haas et al ldquoCell-type-specificmodulation of innate immune signalling by vitamin D inhuman mononuclear phagocytesrdquo The Journal of Immunologyvol 150 no 1 pp 55ndash63 2017
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BioMed Research International 3
(Miltenyi Biotec) were used for cytometry analysis of peri-toneal cells
Monocytes were isolated from PBMCs by the cell cul-ture adherence method For monocyte isolation by adher-ence PBMCs were seeded onto cell culture plates andallowed to adhere in a 5 CO
2incubator at 37∘C for
1 hour in serum-free medium Nonadherent cells wereremoved and the adherent cells were carefully washed twicewith medium before culturing in RPMI medium plus 10heat-inactivated fetal calf serum (FCS) Peripheral bloodmonocyte-derivedM2 cells were generated as described [25]Briefly monocytes were incubated in RPMI with 10 heat-inactivated FCS and 50 UmL IL-4 (Peprotech UK) for 72hours
Cells were seeded at 200000 cellswell in 96-well platesand incubated with various doses of paricalcitol In someexperiments the commercial form (Zemplar) was used asthe source of drug and cultures in the presence of theequivalent concentration of vehicle (30 50 20 propyleneglycol water ethanol)were established in parallel as negativecontrols Stock solutions of pure paricalcitol (Abbott) andvitamin D3 [125(OH)
2D3] (Roche) were prepared in EtOH
at 10minus3MCell viability was analyzed by exclusion of propidium
iodide staining and flow cytometry
25 Quantitative Reverse Transcription-Polymerase ChainReaction (qRT-PCR) Total RNA was isolated using theHigh Pure RNA Isolation Kit (Roche Germany) and 1 120583gwas reverse transcribed with random hexanucleotides andavian myeloblastosis virus (AMV) reverse transcriptase(Promega) in a final volume of 20120583L for 1 hour at 42∘CFor quantitative analysis of mRNA content 1120583L aliquotsof the resulting cDNAs were PCR amplified in a LightCycler (Roche) with FastStart DNA Master SYBR Green I(Roche) in duplicate Primers used for PCR amplificationwere previously described [15] CCL18 sense 51015840-ACA AAGAGC TCT GCT GCC TC-31015840 CCL18 antisense 51015840-CCCACT TCT TAT GGG GTC A-31015840 B2M sense 51015840-CCA GCAGAG AAT GGA AAG TC-31015840 B2M antisense 51015840-GAT GCTGCT TAC ATG TCT CG-31015840 Standard curves for targetmRNAexpressionwere generated by amplifying 10-fold serialdilutions of known quantities of the specific PCR productsQuantification of target gene expression was obtained usingLight Cycler system software Relative units (RU) estimatedfrom the quantification represent the ratio between CCL18mRNA molecules and B2M mRNA molecules in each sam-ple
26 Statistical Analysis The statistical analysis was per-formed using GraphPad Prism software (San Diego CA)Parametric or nonparametric tests were used according tothe distribution of the data Data are shown as mean plusmnSEM mean plusmn SD values or median and interquartile ranges(IQR) as indicated in each figure Data were analyzed withparametric or nonparametric 119905-tests for unpaired or pairedsamples Correlation was assessed using Spearman correla-tion tests with 119901 values 119901 values less than or equal to 05 wereconsidered statistically significant
Gram +0
10
20
30
40
CCL1
8 (n
gm
l)
(N = 13) (N = 26)
ns
Gram minus
Figure 1The concentration of CCL18was evaluated in 39 peritonealeffluents from patients diagnosed with infectious peritonitis Efflu-entswere collected on the first day of cloudy effluentHorizontal barsrepresent mean plusmn SEM values No significant differences were foundbetween Gramminus (119873 = 13) and Gram+ bacteria (119873 = 26) infectedcases (unpaired 119905-test)
3 Results
31 CCL18 Measurements in Peritonitis Effluent SamplesPrevious studies propose CCL18 as a marker and mediatorof peritoneal fibrosis and UFF in patients treated with PD[15 16] Given recurrent peritonitis is a risk factor forperitoneal fibrosis and UFF the concentration of CCL18was evaluated in a group of 39 PD patients with infectiousperitonitis Effluent samples were obtained at diagnosis andthe concentration of CCL18 was measured by enzyme-linkedimmunosorbent assay (ELISA) The mean concentration ofCCL18 in peritonitis samples was 1502 plusmn 787 ngml whereasmean values previously reported ranged between 1 and6 ngml No significant differences were found in patientsinfected with Gram+ or Gramminus bacteria Mean observedvalues were 133 plusmn 243 ngml in the Gramminus versus 1586 plusmn145 ngml in the Gram+ samples (Figure 1)
We evaluated CCL18 concentrations in serial samplesfrom four of the PD patients with infectious peritonitis(IP) (Figure 2(a)) CCL18 effluent concentrations showedthe highest values during the first days of peritonitis anddecreased slowly following a kinetics similar to absolutemacrophage counts in these samples (Figure 2(c)) and inagreement with previous reports describing macrophages asthe primary source of this chemokine [15]
Serial samples were also analyzed in one additionalcase of sterile EP associated with an accidental exposureof the peritoneum to chlorhexidine Surprisingly whereasthe mean CCL18 concentrations on day 1 and on days2ndash4 in the IP samples were 155 plusmn 61 ngml and 117 plusmn69 ngml respectively in the EP samples the concentrationof CCL18 was 19812 ngml on day 1 and 20465 ngml on day3 (Figure 3 and Supplemental Figure 1(A)) IL-6 was alsoevaluated for comparative purposes as the hallmark cytokinein inflammatory processes and in PD-associated peritonitis[26] Elevated levels of IL-6were found in spent effluents fromall patients on the first day of peritonitis with similar values
4 BioMed Research International
CCL1
8 (n
gm
l)CC
L18
(ng
ml)
CCL1
8 (n
gm
l)
Days of evolution
CCL1
8 (n
gm
l)
D1 D3 D120
2
4
6
8
10IP-1
0
5
10
15
20
25
D1 D2 D5 D26
IP-2
0
5
10
15
20
25
D1 D4 D8 D13
IP-4
02468
10121416
D1 D3 D5 D22
IP-3
(a)
0
1
1
2
2
3
3
4
D1 D3 D12
IP-1
0
5
10
15
20
D1 D2 D5 D26
IP-2
0
5
10
15
D1 D3 D5 D22
IP-3
0
1
2
3
4
5
6
D1 D4 D8 D13
IL-6
(ng
ml)
IL-6
(ng
ml)
IL-6
(ng
ml)
IL-6
(ng
ml)
IP-4
(b)
Days of evolution
1
10
100
1000
10000
100000
D1 D3 D12
1
10
100
1000
10000
100000
D1 D2 D4 D5
1
10
100
1000
10000
D1 D3 D5
1
10
100
1000
10000
D1 D4 D8
IP-1
IP-2
IP-3
IP-4
(Cel
ls
l)(C
ells
l)
(Cel
ls
l)(C
ells
l)
MΦlPMNl
(c)
Figure 2 Serial cytokine concentrations and cell counts in peritoneal effluent during peritonitis episodes (a) Evolution of CCL18 effluentlevels in serial samples from four patients with infectious peritonitis (IP) shown as IP-1 to IP-4 (b) Evolution of IL-6 effluent levels inserial samples from the same patients (c) Comparison of neutrophils (PMN) and macrophage (MΦ) cell counts in serial effluent samplesDifferential cell counts on D26 (IP-2) D22 (IP-3) and D13 (IP-4) were not available given that the total cell counts were lt50 cells120583l in thosesamples
in IP samples and in the EP sample (Supplemental Figure1(B)) A sharp decline in IL-6 occurred in all the IP cases aspreviously described [26 27] (Figure 2(b)) and in contrast toCCL18 levels which remained elevated during the followingdays and declined more slowly (Figure 2(a))
32 Peritoneal Eosinophils Are a Source of CCL18 in Eosin-ophilic Peritonitis Cytokine concentrations were comparedwith absolute and differential cell counts in effluent samplesfrom the patient with EP (Figure 3) A significant correlationwas found between the kinetics of CCL18 concentration and
BioMed Research International 5
000
5000
10000
15000
20000
25000
D1 D3 D6 D17 D190
1000
2000
3000
4000
5000
CCL1
8 (n
gm
L)
CCL18 Total cells
Cel
ls
L
(a)
000
5000
10000
15000
20000
25000
D1 D3 D6 D17 D190
500
1000
1500
2000
2500
CCL1
8 (n
gm
L)
CCL18PMN
PMN
L
(b)
000
5000
10000
15000
20000
25000
D1 D3 D6 D17 D190
200
400
600
800
CCL1
8 (n
gm
L)
CCL18
MΦ
L
MΦ
(c)
000
5000
10000
15000
20000
25000
D1 D3 D6 D17 D19
CCL1
8 (n
gm
L)
0
100
200
300
400
500
600
700
CCL18 Eos
Eos
L
(d)
IL-6Total cells
0
2000
4000
6000
8000
10000
D1 D3 D6 D17 D190
1000
2000
3000
4000
5000
IL-6
(pg
mL)
Cel
ls
L
(e)
0
2000
4000
6000
8000
10000
D1 D3 D6 D17 D190
500
1000
1500
2000
2500
IL-6PMN
IL-6
(pg
mL)
PMN
L
(f)
0
2000
4000
6000
8000
10000
D1 D3 D6 D17 D190
200
400
600
800
IL-6
IL-6
(pg
mL)
MΦ
L
MΦ
(g)
0
2000
4000
6000
8000
10000
D1 D3 D6 D17 D190
100
200
300
400
500
600
700
IL-6
(pg
mL)
IL-6Eos
Eos
L
(h)
Figure 3 Kinetics of effluent cell counts and cytokine concentrations during EP (andashd) CCL18 effluent levels and cell counts in serial effluentsamples (endashh) IL-6 effluent levels and cell counts in serial effluent samples
6 BioMed Research International
(a)
(b) (c)
Figure 4 Cytospin preparations showing effluent cell content in EP (a) Hematoxylin and eosin staining (b) Immunocytochemistry showingCCL18 staining of eosinophils (c) Background staining with nonimmune serum
the macrophage counts in the EP samples (119903 = 0889119901 = 043) (Figure 3(c)) However the strongest correlationwas found between CCL18 effluent levels and eosinophils(119903 = 0949 119901 = 013) with peak CCL18 concentrationsdetected in effluents with the highest absolute eosinophilcount (Figure 3(d)) In contrast IL-6 effluent levels paralleledpolymorphonuclear neutrophils (PMNs) and absolute cellnumbers in effluent samples from EP (Figures 3(e) and3(f)) and no correlation was observed between effluent IL-6 concentrations and eosinophil cell count (Figure 3(h))
Although CCL18 is thought to be primarily produced byM2 macrophages involved in tissue repair and fibrosis andby dendritic cells [28] eosinophils have occasionally beenreported to produce CCL18 during inflammatory conditions[29 30] The kinetics of CCL18 effluent concentrations in EPand eosinophil counts suggested that not only macrophagesbut also eosinophils might contribute to the production ofCCL18 in this case To test this hypothesis immunostainingof cytospin preparations from eosinophilic effluents wasperformed Specific staining was detected in the cytoplasmof typical binucleated cells supporting the role of eosinophilsas a source of CCL18 in this case (Figure 4)
In a previous report we found that only peritonealmacrophages were able to produce CCL18 during infectiousperitonitis given that the cytokine was not detected in cul-ture supernatants from neutrophils (PMNs) or lymphocytes[15] To further analyze the putative role of eosinophils asproducers of CCL18 in the patient with EP CD66b+ polymor-phonuclear leukocytes (among which 85 were eosinophils
as assessed by CD16 negative staining of eosinophils versusCD16 positive neutrophils) were isolated from peritonealeffluent cells on D6 as well as CD14+ macrophages forcomparative purposes (Figures 5(a)ndash5(c)) CCL18 transcriptswere detected in both the CD66b+ and CD14+ fractions(Figure 5(d)) Quantitative PCR analysis confirmed CCL18mRNA transcripts in polymorphonuclear leukocytes in thiscase (Figure 5(e)) Altogether the data strongly suggest thateosinophils are producers of CCL18 in EP
33 Paricalcitol Downregulates the Production of CCL18 byPeritoneal Macrophages Paricalcitol a VDR agonist hasbeen shown to ameliorate peritoneal fibrosis in a murinemodel of PD [23] Given that CCL18 has been involvedin peritoneal fibrosis in PD patients [15 20 22] we testedthe effect of paricalcitol in the production of CCL18 byhuman pMΦs isolated fromPD effluents drawn frompatientssuffering from peritonitis and cultured with various dosesof nontoxic concentrations of Zemplar as a source of par-icalcitol or vehicle (supplementary Figure 2) As shown inFigures 6(a)ndash6(c) pharmacological treatment was able tosignificantly downregulate the production of CCL18 in thesecells at concentrations equivalent to 10minus8M and 10minus9M ofparicalcitol This effect was obtained as early as 24 hours andit was also observed at 48 hours and 72 hours of cell cultureSimilar results were obtained with the pure substance andwith equivalent doses of vitamin D3 (Figure 6(d))
It has been suggested that the regulation of CCL18 proteinsynthesis and secretion can be a complex process with some
BioMed Research International 7
0 200 400 600 800 1000FSC-H
PolymorphonuclearLeukocytes
0
200
400
600
800
1000
SSC-
HR1 = 715 LΦR2 = 24 MΦR3 = 6885
(a)CD16-FITC
767PMN
0
200
400
600
800
1000
SSC-
H
100 101 102 103 104
(b)
CD14-APC
999
CD66
b-FI
TC
100
100
101
101
102
102
103 104
103
104
(c)
500
300
400
200
300
200 CCL18
B2M
100
bp la
dder
NEG
CD66<+
(Eos
)
CD14+
(MΦ
)
(d)
CD14+ CD66b+
CCL1
8B2
M m
RNA
(RU
)
25E minus 05
20E minus 05
15E minus 05
10E minus 05
50E minus 06
00E + 00
(e)
Figure 5 (a) Flow cytometry analysis of peritoneal effluent leukocytes on D6 according to morphological parameters LΦ lymphocytesMΦ macrophages (b) Flow cytometry analysis of CD16 expression in peritoneal effluent cells on D6 The cutoff level for positive stainingwas set according to isotype control background staining PMN polymorphonuclear neutrophils (c) Flow cytometry analysis of CD66b+polymorphonuclear cells isolated from peritoneal effluent cells (d) RT-PCR of CCL18 gene expression in CD66+ and CD14+ purifiedperitoneal leukocytes (e) Quantitative RT-PCR analysis of CCL18 gene expression in CD66+ and CD14+ purified peritoneal leukocytes
degree of posttranscriptional regulation [28] To analyzewhether VDR agonists regulate CCL18 mRNA levels quan-titative RT-PCR assays were performed in isolated pMΦstreated with paricalcitol CCL18 mRNA levels were downreg-ulated in parallel with the diminished production of CCL18protein in pMΦs from two different donors representinginfected and uninfected peritoneal samples (Figures 7(a) and7(b)) Given that IL-4 is one of the main inducers of CCL18[31] we tested if VDR agonists couldmodulate the expressionof the chemokine in M2 macrophages generated in vitroupon treatment of peripheral blood monocytes with IL-4 Asshown in Figure 7(c) the expression of CCL18 was highlyupregulated in monocyte-derived macrophages treated for 3days with IL-4 and pretreatment with paricalcitol or vitaminD was able to prevent the upregulation of CCL18 in thesecells
4 Discussion
Peritonitis episodes constitute a frequent complication in PDpatients and recurrent peritonitis is a well-known clinicalrisk factor for peritoneal membrane dysfunction and EPS
There is an unmet requirement for biomarkers as toolsto identify patients who are at higher risk of techniquefailure to guide interventions that could improve clinicaloutcomes [32] Soluble factors released during inflammatoryprocesses such as IL-6 and IL-17 have been proposed asbiomarkers and mediators of peritoneal membrane deteri-oration (reviewed in [32 33]) However none are used inthe clinical routine and some cytokines such as IL-17 arenot easily measured in PD effluents in humans We proposeCCL18 as a biomarker of peritoneal membrane outcome inPD [22]
8 BioMed Research International
0
20
40
60
80
100
120
C
CL18
pro
duct
ion
Pari VV Pari(10minus8 M) (10minus9 M)
lowastp = 0156 lowastp = 0156
(a)
0
20
40
60
80
100
120
C
CL18
pro
duct
ion
Pari VV Pari(10minus8 M) (10minus9 M)
p = 0625
(b)
0
20
40
60
80
100
120
C
CL18
pro
duct
ion
Pari VV Pari(10minus8 M) (10minus9 M)
lowastp = 0313lowastlowastp = 0039
(c)
0
20
40
60
80
100
ParicalcitolVehicle Vit D
120
C
CL18
pro
duct
ion
(10minus8 M) (10minus8 M)
(d)
Figure 6 (andashc) Peritoneal macrophages isolated from patients experiencing peritonitis episodes were treated with vehicle or Zemplar indoses equivalent to 10minus8M and 10minus9M of paricalcitol during 24 h (a) 48 h (b) or 72 h (c) Equivalent cell cultures were established in theabsence of any treatment The concentration of CCL18 was evaluated in cell-free supernatants and the production of CCL18 in the presenceof vehicle or drug referred to the production in cell culture media alone Differences between cultures in the presence of paricalcitol or vehiclewere analyzed by theWilcoxon matched pairs rank test (d) Peritoneal macrophages were treated with paricalcitol as pure substance vitaminD or vehicle (ethanol) for 48 h
Given that high levels of the human chemokine CCL18have been shown to be related to peritoneal membranefailure and fibrosis [15 20 22] whereas sustained low CCL18peritoneal concentrations appear to be protective [22] weevaluated CCL18 levels in the peritoneum of patients under-going peritonitis episodes We found that the peritoneal con-centration of CCL18 was increased approximately 5-fold ineffluents from patients with infectious peritonitis comparedwith reported values in uninfected samples In a previousstudy involving a longitudinal analysis of 43 patients CCL18effluent concentrations ranged from 28 plusmn 16 ngml at thestart of dialysis to 34plusmn 18 ngml after 3 years of PD treatment[22] In a cross-sectional analysis of 61 patients treated formore than 3 years with PDmean effluent CCL18 values were395 plusmn 26 ngml [22] The high concentrations during IPare consistent with a previous finding showing that pMΦsfrom patients undergoing peritonitis or who recently had
peritonitis were able to secrete higher amounts of CCL18compared with pMΦs from uninfected patients [15]
Unexpectedly in a patient identified with sterileeosinophilic peritonitis the peritoneal effluent was extremelyrich in CCL18 with levels approximately 10-fold higher thanthose found in patients with IP and 50-fold more thanthose found in uninfected samples whereas effluent levelsof IL-6 which were evaluated in the same samples forcomparative purposes were similar on day 1 in both theIP and EP samples The differences between a peritonealinflammatory recognized marker IL-6 and CCL18 duringperitonitis episodes should be remarked It is of note thatthe peritoneal concentrations of CCL18 remained elevatedfor several days whereas there was a sharp decrease in IL-6Both IL-6 and CCL18 have been reported to increase indifferent inflammatory conditions some leading to tissuefibrosis [34ndash37] As a main difference although CCL18 is
BioMed Research International 9
NEG
CCL1
8 (p
gm
l)
0
200
400
600
800
1000
12001057
522
Pari (10minus7 M) NEG000
020
040
060
080
100
120100
025Fold
mRN
A C
CL18
Pari (10minus7 M)
(a)
NEG
408
279
0
100
200
300
400
500
CCL1
8 (p
gm
l)
Pari (10minus7 M)
100
045
000
050
100
150
NEG
Fold
mRN
A C
CL18
Pari (10minus7 M)
(b)
000
025
050
075
100
125
Fold
mRN
A C
CL18
NEG
Mo + IL-4
Pari Vit D3Vmdash
(c)
Figure 7 (a) Peritoneal macrophages from a patient with recent infectious peritonitis were treated with paricalcitol or vehicle for 24 h Theprotein concentration of CCL18 was evaluated in cell culture supernatants by ELISA and CCL18 mRNA levels were estimated by quantitativeRT-PCR in the same cultures (b) Peritoneal macrophages from an uninfected peritoneum were isolated from the peritoneal lavage uponcatheter insertion and treated as in (a) (c) Peripheral bloodmonocytes were pretreated with vehicle (V) 10minus7Mparicalcitol (Pari) or vitaminD3 for 24 h The cells were treated thereafter with IL-4 for 3 days before RNA extraction and evaluation expression of CCL18 mRNA levelsby quantitative RT-PCR Mean plusmn SD values from four independent experiments are shown
primarily produced by cells of myeloid origin multiplecell types are producers of IL-6 with endothelial cellsbeing an important source of this cytokine Infiltratingneutrophils as well as mesothelial cells could also be animportant source of IL-6 during bacterial peritonitis [37]The analysis of serial samples in four patients with IP showedthat the concentrations of CCL18 paralleled the numbers ofmacrophages infiltrating the peritoneum and are consistentwith previous descriptions of macrophages andmononuclearphagocytes as the primary producers of CCL18 [21] Cellcounts in the serial samples from our case of EP suggest that
the peritoneal eosinophilic infiltrate was likely contributingto the peritoneal production of CCL18 Evidences of CCL18production by activated peritoneal eosinophils in this caseare cytospin analysis of CCL18 cytoplasmic staining ineosinophils and gene expression data in polymorphonuclearleukocytes among which 85 were eosinophils
Although macrophages are typically reported as themajor source of this chemokine [21] CCL18 protein pro-duction by eosinophils has occasionally been described asbeing related to the activation status of these cells given thatCCL18 was found only in eosinophil culture supernatants
10 BioMed Research International
from eosinophilic donors but not from individuals withnonactivated eosinophils [29] Our data support the activatedstatus of eosinophils in EP given that strong staining ofCC18 was detected in peritoneal eosinophils Eosinophilshave also been related to tissue fibrosis in several diseasesand are a source of several profibrotic mediators that alterfibroblast activity and tissue remodeling [38 39] Some ofthesemediators could also stimulate the profibrotic activity ofpMΦTheproduction ofCCL18 by eosinophils in EPprovidesa new additional explanation for the association of recurrentEP during PD with UFF and potentially peritoneal fibrosis[40]
As part of its profibrotic activity CCL18 has been shownto stimulate collagen deposition [41] and fibroblast prolif-eration [42] In consonance with these actions circulatingCCL18 levels have been demonstrated to be a biomarker fordisease activity and outcome in various fibrotic diseases [1943 44] as well as in some eosinophilic disorders [30 45 46]Moreover CCL18 has been shown to promote EMT in variouscell systems [47ndash50] It is thus possible that it could alsocontribute to the EMT of mesothelial cells which has beenproposed as one of the mechanisms leading to peritonealfibrosis and membrane failure [12]
In terms of peritoneal fibrosis CCL18 has been demon-strated to herald the development of peritoneal functionaldeterioration in PD patients [15 20] in whom the abilityof peritoneal macrophages to stimulate fibroblast prolifer-ation is correlated with CCL18 mRNA levels Moreoverrecombinant CCL18 was able to stimulate the proliferation ofperitoneal fibroblasts [15]
Several molecules have been tested for their ability toprevent peritoneal fibrosis in vitro and in animal models(reviewed in [8]) Among them paricalcitol is a selectiveVDR agonist VDR is a nuclear hormone receptor expressedin various cell types including those of the immune sys-tem VDR agonists have been shown to modulate immuneresponses inflammation and fibrosis [51]
Considering the role played by inflammation in peri-toneal fibrosis during PDwe studied the effects of paricalcitolin the production of CCL18 We found that the release andgene expression of CCL18 were downregulated in pMΦsand in in vitro-differentiated human M2 cells which are theprimary producers of CCL18These results appear to contrastwith previous data showing upregulation of CCL18 by vita-min D in monocyte-derived immature dendritic cells [52]However recent reports demonstrate that the modulationof immune signaling by vitamin D in human mononuclearphagocytes is cell-type-specific with differential responses inmonocytes macrophages and dendritic cells [53] It is thuspossible that opposite effects are found on the regulation ofCCL18 by VDR agonists in different cellular systems
On the other hand in a mouse model of PD intraperi-toneal administration of paricalcitol was shown to reduceperitoneal fibrosis and ultrafiltration failure [23] In thatmodel the beneficial effects appeared to be dependent ona reduction of intraperitoneal concentrations of IL-17 Thelack of rodent orthologues of human CCL18 [28] precludesthe investigation of CCL18 and its involvement in fibrosis inmurine models On the other hand IL-17 is highly diluted
in human peritoneal effluents The need to concentrate thesamples for measurement of this cytokine hampers accuratedeterminations in human samples
A preliminary study involving 23 patients on PD suggeststhat paricalcitol could improve the ultrafiltration capacitywith less peritoneal protein loss [24] The authors suggestan effect of VDR upon the renin-angiotensin-aldosteronesystem but it is tempting to speculate that lower peritonealconcentrations of CCL18 might be present in paricalcitol-treated patients In this sense a slight reduction in theperitoneal concentration of CCL18 was observed in sixpatients who were more than 3 years on PD and treated withparicalcitol compared with a set of 31 patients of the samevintage (data not shown)
5 Conclusion
In conclusion our study suggests that the chemokine CCL18can be a mediator of peritoneal membrane failure associatedwith peritonitis episodes and might provide a new potentialtherapeutic target
Further clinical studies are needed to confirm the useful-ness of VDR agonists to preserve the peritoneal membraneand to analyze the putative mechanisms involved
Conflicts of Interest
All the authors have no potential conflicts of interest todeclare
Authorsrsquo Contributions
Marta Ossorio and Virginia Martınez contributed equally tothis work
Acknowledgments
This study has been partially supported by ISCIII (ldquoInsti-tuto de Investigacion Sanitaria Carlos IIIrdquo) Grants FIS PI1200204 PI1301768 and PI 1500120 FEDER (Funds fromthe European Union) REDinREN 20 FEDER and ReinaSofıa Institute of Nephrology Research (IRSIN) monetaryfunds The authors would like to thank Juliette Siegfriedand her team at ServingMedcom for their editing of themanuscript
Supplementary Materials
Supplemental Figure 1 serial cytokine concentrations andcell counts in peritoneal effluent during infectious peritoni-tis and eosinophilic peritonitis (A) Comparison of CCL18concentration in serial effluent samples from infectiousperitonitis (IP) and eosinophilic peritonitis (EP) (B) IL-6effluent levels in serial samples from IP and EP D1ndashD26indicate days of evolution Supplemental Figure 2 dose-dependent effects of VDR agonists on cell viability Cellviability of human monocytes was assessed by exclusion ofpropidium iodide staining and analyzed by flow cytometry
BioMed Research International 11
Results are shown as mean plusmn SD values of triplicates(A)ndash(C) Viability of monocytes upon culture during 24 48or 72 hours with decreasing concentrations of Zemplar orequivalent vehicle (305020 propylene glycolwaterethanol)(D)ndash(F) Viability of monocytes upon culture during 2448 or 72 hours with different concentrations of paricalcitol(pure substance) vitamin D or equivalent vehicle (EtOH)(Supplementary Materials)
References
[1] R T Krediet ldquoThe peritoneal membrane in chronic peritonealdialysisrdquo Kidney International vol 55 no 1 pp 341ndash356 1999
[2] R T Krediet ldquoPeritoneal physiology - Impact on solute andfluid clearancerdquo Advances in Chronic Kidney Disease vol 7 no4 pp 271ndash279 2000
[3] M Merkle and M Wrnle ldquoSclerosing peritonitis A rare butfatal complication of peritoneal inflammationrdquo Mediators ofInflammation vol 2012 Article ID 709673 p 4 2012
[4] D W Johnson Y Cho B E R Livingston et al ldquoEncapsulat-ing peritoneal sclerosis incidence predictors and outcomesrdquoKidney International vol 77 no 10 pp 904ndash912 2010
[5] G Balasubramaniam E A Brown and A Davenport ldquoThePan-Thames EPS study treatment and outcomes of encapsulat-ing peritoneal sclerosisrdquo Nephrology Dialysis Transplantation vol 24 no 10 pp 3209ndash3215 2009
[6] E D Sousa-Amorim G D Peso M A Bajo et al ldquoCan EPSdevelopment be avoided with early interventionsThe potentialrole of tamoxifenmdasha single-center studyrdquo Peritoneal DialysisInternational vol 34 no 6 pp 582ndash593 2014
[7] M R Korte D E SampimonM GH Betjes and R T KredietldquoEncapsulating peritoneal sclerosis the state of affairsrdquo NatureReviews Nephrology vol 7 no 9 pp 528ndash538 2011
[8] M A Bajo G del Peso and I Teitelbaum ldquoPeritoneal Mem-brane Preservationrdquo Seminars in Nephrology vol 37 no 1 pp77ndash92 2017
[9] M F Flessner ldquoInflammation from sterile dialysis solutions andthe longevity of the peritoneal barrierrdquo Clinical Nephrology vol68 no 6 pp 341ndash348 2007
[10] K N Lai and J C K Leung ldquoInflammation in peritonealdialysisrdquo Nephron Clinical Practice vol 116 no 1 pp c11ndashc182010
[11] S Yung and T M Chan ldquoPathophysiological changes to theperitoneal membrane during PD-related peritonitis the role ofmesothelial cellsrdquo Mediators of Inflammation vol 2012 ArticleID 484167 21 pages 2012
[12] L S Aroeira A Aguilera J A Sanchez-Tomero et al ldquoEpithe-lial tomesenchymal transition and peritonealmembrane failurein peritoneal dialysis patients pathologic significance andpotential therapeutic interventionsrdquo Journal of the AmericanSociety of Nephrology vol 18 no 7 pp 2004ndash2013 2007
[13] N Topley T Liberek A Davenport F-K Li H Fear andJ D Williams ldquoActivation of inflammation and leukocyterecruitment into the peritoneal cavityrdquo Kidney InternationalSupplements vol 50 no 56 pp S17ndashS21 1996
[14] F O Martınez and S Gordon ldquoThe M1 and M2 paradigmof macrophage activation time for reassessmentrdquo F1000PrimeReports vol 6 article 13 2014
[15] T Bellon V Martınez B Lucendo et al ldquoAlternative activa-tion of macrophages in human peritoneum implications for
peritoneal fibrosisrdquo Nephrology Dialysis Transplantation vol26 no 9 pp 2995ndash3005 2011
[16] A Prasse D V Pechkovsky G B Toews et al ldquoA vicious circleof alveolar macrophages and fibroblasts perpetuates pulmonaryfibrosis via CCL18rdquoAmerican Journal of Respiratory and CriticalCare Medicine vol 173 no 7 pp 781ndash792 2006
[17] J M Estep A Baranova N Hossain et al ldquoExpression ofcytokine signaling genes in morbidly obese patients with non-alcoholic steatohepatitis and hepatic fibrosisrdquo Obesity Surgeryvol 19 no 5 pp 617ndash624 2009
[18] F Kollert M Binder C Probst et al ldquoCCL18 - Potentialbiomarker of fibroinflammatory activity in chronic periaortitisrdquoThe Journal of Rheumatology vol 39 no 7 pp 1407ndash1412 2012
[19] A Prasse D V Pechkovsky G B Toews et al ldquoCCL18 as anindicator of pulmonary fibrotic activity in idiopathic interstitialpneumonias and systemic sclerosisrdquo Arthritis amp Rheumatologyvol 56 no 5 pp 1685ndash1693 2007
[20] S Ahmad B V North A Qureshi et al ldquoCCL18 in peritonealdialysis patients and encapsulating peritoneal sclerosisrdquo Euro-pean Journal of Clinical Investigation vol 40 no 12 pp 1067ndash1073 2010
[21] V Kodelja C Muller O Politz N Hakij C E Orfanos andS Goerdt ldquoAlternative macrophage activation-associated CC-chemokine-1 a novel structural homologue of macrophageinflammatory protein-1120572 with a Th2- associated expressionpatternrdquo The Journal of Immunology vol 160 no 3 pp 1411ndash1418 1998
[22] M Ossorio M A Bajo G Del Peso et al ldquoSustained low peri-toneal effluent CCL18 levels are associated with preservationof peritoneal membrane function in peritoneal dialysisrdquo PLoSONE vol 12 no 4 Article ID e0175835 2017
[23] G T Gonzalez-Mateo V Fernandez-Mıllara T Bellon etal ldquoParicalcitol reduces peritoneal fibrosis in mice throughthe activation of regulatory T cells and reduction in IL-17productionrdquo PLoS ONE vol 9 no 10 Article ID e108477 2014
[24] F Coronel S Cigarran A Gomis et al ldquoChanges in peritonealmembrane permeability and proteinuria in patients on peri-toneal dialysis after treatment with paricalcitolmdasha preliminarystudyrdquo Clinical Nephrology vol 78 no 2 pp 93ndash99 2012
[25] E Song N Ouyang M Horbelt B Antus M Wang and MS Exton ldquoInfluence of alternatively and classically activatedmacrophages on fibrogenic activities of human fibroblastsrdquoCellular Immunology vol 204 no 1 pp 19ndash28 2000
[26] S M Hurst T S Wilkinson R M McLoughlin et al ldquoIL-6 andits soluble receptor orchestrate a temporal switch in the patternof leukocyte recruitment seen during acute inflammationrdquoImmunity vol 14 no 6 pp 705ndash714 2001
[27] M Kozioł-Montewka A Ksiazek L Janicka and I BaranowiczldquoSerial cytokine changes in peritoneal effluent and plasma dur-ing peritonitis in patients on continuous ambulatory peritonealdialysis (CAPD)rdquo Inflammation Research vol 46 no 4 pp 132ndash136 1997
[28] E Schutyser A Richmond and J Van Damme ldquoInvolvementof CC chemokine ligand 18 (CCL18) in normal and pathologicalprocessesrdquo Journal of Leukocyte Biology vol 78 no 1 pp 14ndash262005
[29] I Schraufstatter H Takamori L Sikora P Sriramarao and RG DiScipio ldquoEosinophils and monocytes produce pulmonaryand activation-regulated chemokine which activates culturedmonocytesmacrophagesrdquoAmerican Journal of Physiology-LungCellular and Molecular Physiology vol 286 no 3 pp L494ndashL501 2004
12 BioMed Research International
[30] C Gunther N Carballido-Perrig T Kopp J M Carballidoand C Pfeiffer ldquoCCL18 is expressed in patients with bullouspemphigoid and parallels disease courserdquo British Journal ofDermatology vol 160 no 4 pp 747ndash755 2009
[31] AMantovani A Sica S Sozzani P Allavena A Vecchi andMLocati ldquoThe chemokine system in diverse forms of macrophageactivation and polarizationrdquo Trends in Immunology vol 25 no12 pp 677ndash686 2004
[32] C Aufricht R Beelen M Eberl et al ldquoBiomarker researchto improve clinical outcomes of peritoneal dialysis consensusof the European Training and Research in Peritoneal Dialysis(EuTRiPD) networkrdquo Kidney International vol 92 no 4 pp824ndash835 2017
[33] D Lopes Barreto andR T Krediet ldquoCurrent status and practicaluse of effluent biomarkers in peritoneal dialysis patientsrdquoAmerican Journal of Kidney Diseases vol 62 no 4 pp 823ndash8332013
[34] L A Boven M van Meurs R G Boot et al ldquoGaucher cellsdemonstrate a distinct macrophage phenotype and resemblealternatively activated macrophagesrdquo American Journal of Clin-ical Pathology vol 122 no 3 pp 359ndash369 2004
[35] L Cocho I Fernandez M Calonge et al ldquoGene expression-based predictive models of graft versus host disease-associateddry eyerdquo Investigative Ophtalmology Visual Science vol 56 no8 pp 4570ndash4581 2015
[36] MHasegawa ldquoBiomarkers in systemic sclerosisTheir potentialto predict clinical coursesrdquoThe Journal of Dermatology vol 43no 1 pp 29ndash38 2016
[37] S A Jones D J Fraser C A Fielding and G W JonesldquoInterleukin-6 in renal disease and therapyrdquoNephrologyDialysisTransplantation vol 30 no 4 pp 564ndash574 2015
[38] A Munitz and F Levi-Schaffer ldquoEosinophils rsquoNewrsquo roles forrsquooldrsquo cellsrdquo Allergy European Journal of Allergy and ClinicalImmunology vol 59 no 3 pp 268ndash275 2004
[39] S S Aceves ldquoRemodeling and fibrosis in chronic eosinophilinflammationrdquo Digestive Diseases vol 32 no 1-2 pp 15ndash212014
[40] Y Nakamura H Okada A Yasui T Koh and T YamaneldquoSclerosing encapsulating peritonitis associated with recurrenteosinophilic peritonitisrdquo Nephrology Dialysis Transplantation vol 14 no 3 pp 768ndash770 1999
[41] S P Atamas I G Luzina J Choi et al ldquoPulmonary andactivation-regulated chemokine stimulates collagen productionin lung fibroblastsrdquo American Journal of Respiratory Cell andMolecular Biology vol 29 no 6 pp 743ndash749 2003
[42] A Wimmer S K Khaldoyanidi M Judex N Serobyan RG DiScipio and I U Schraufstatter ldquoCCL18PARC stimulateshematopoiesis in long-term bone marrow cultures indirectlythrough its effect on monocytesrdquo Blood vol 108 no 12 pp3722ndash3729 2006
[43] A Prasse C Probst E Bargagli et al ldquoSerum CC-chemokineligand 18 concentration predicts outcome in idiopathic pul-monary fibrosisrdquo American Journal of Respiratory and CriticalCare Medicine vol 179 no 8 pp 717ndash723 2009
[44] J Chen Y Yao C Gong et al ldquoCCL18 from tumor-associatedmacrophages promotes breast cancermetastasis via PITPNM3rdquoCancer Cell vol 19 no 4 pp 541ndash555 2011
[45] K L Hon G K Ching P C Ng and T F Leung ldquoExplor-ing CCL18 eczema severity and atopyrdquo Pediatric Allergy andImmunology vol 22 no 7 pp 704ndash707 2011
[46] A J Lucendo A Arias L C De Rezende et al ldquoSubepithelialcollagen deposition profibrogenic cytokine gene expressionand changes after prolonged fluticasone propionate treatment inadult eosinophilic esophagitis a prospective studyrdquoThe Journalof Allergy and Clinical Immunology vol 128 no 5 pp 1037ndash1046 2011
[47] H Li D Zhang J Yu et al ldquoCCL18-dependent translocationof AMAP1 is critical for epithelial to mesenchymal transition inbreast cancerrdquo Journal of Cellular Physiology vol 233 no 4 pp3207ndash3217 2017
[48] Z Lin W Li H Zhang et al ldquoCCL18PITPNM3 enhancesmigration invasion and EMT through the NF-120581B signalingpathway in hepatocellular carcinomardquo Tumor Biology vol 37no 3 pp 3461ndash3468 2016
[49] F Meng W Li C Li Z Gao K Guo and S SongldquoCCL18 promotes epithelial-mesenchymal transition invasionand migration of pancreatic cancer cells in pancreatic ductaladenocarcinomardquo International Journal of Oncology vol 46 no3 pp 1109ndash1120 2015
[50] T Ploenes B Scholtes A Krohn et al ldquoCC-Chemokine Ligand18 Induces Epithelial to Mesenchymal Transition in LungCancer A549 Cells and Elevates the Invasive Potentialrdquo PLoSONE vol 8 no 1 Article ID e53068 2013
[51] J Rojas-Rivera C De La Piedra A Ramos A Ortiz andJ Egido ldquoThe expanding spectrum of biological actions ofvitamin Drdquo Nephrology Dialysis Transplantation vol 25 no 9pp 2850ndash2865 2010
[52] M Vulcano S Struyf P Scapini et al ldquoUnique regulation ofCCL18 production by maturing dendritic cellsrdquo The Journal ofImmunology vol 170 no 7 pp 3843ndash3849 2003
[53] R Kundu A Theodoraki C T Haas et al ldquoCell-type-specificmodulation of innate immune signalling by vitamin D inhuman mononuclear phagocytesrdquo The Journal of Immunologyvol 150 no 1 pp 55ndash63 2017
Stem Cells International
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Disease Markers
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Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
4 BioMed Research International
CCL1
8 (n
gm
l)CC
L18
(ng
ml)
CCL1
8 (n
gm
l)
Days of evolution
CCL1
8 (n
gm
l)
D1 D3 D120
2
4
6
8
10IP-1
0
5
10
15
20
25
D1 D2 D5 D26
IP-2
0
5
10
15
20
25
D1 D4 D8 D13
IP-4
02468
10121416
D1 D3 D5 D22
IP-3
(a)
0
1
1
2
2
3
3
4
D1 D3 D12
IP-1
0
5
10
15
20
D1 D2 D5 D26
IP-2
0
5
10
15
D1 D3 D5 D22
IP-3
0
1
2
3
4
5
6
D1 D4 D8 D13
IL-6
(ng
ml)
IL-6
(ng
ml)
IL-6
(ng
ml)
IL-6
(ng
ml)
IP-4
(b)
Days of evolution
1
10
100
1000
10000
100000
D1 D3 D12
1
10
100
1000
10000
100000
D1 D2 D4 D5
1
10
100
1000
10000
D1 D3 D5
1
10
100
1000
10000
D1 D4 D8
IP-1
IP-2
IP-3
IP-4
(Cel
ls
l)(C
ells
l)
(Cel
ls
l)(C
ells
l)
MΦlPMNl
(c)
Figure 2 Serial cytokine concentrations and cell counts in peritoneal effluent during peritonitis episodes (a) Evolution of CCL18 effluentlevels in serial samples from four patients with infectious peritonitis (IP) shown as IP-1 to IP-4 (b) Evolution of IL-6 effluent levels inserial samples from the same patients (c) Comparison of neutrophils (PMN) and macrophage (MΦ) cell counts in serial effluent samplesDifferential cell counts on D26 (IP-2) D22 (IP-3) and D13 (IP-4) were not available given that the total cell counts were lt50 cells120583l in thosesamples
in IP samples and in the EP sample (Supplemental Figure1(B)) A sharp decline in IL-6 occurred in all the IP cases aspreviously described [26 27] (Figure 2(b)) and in contrast toCCL18 levels which remained elevated during the followingdays and declined more slowly (Figure 2(a))
32 Peritoneal Eosinophils Are a Source of CCL18 in Eosin-ophilic Peritonitis Cytokine concentrations were comparedwith absolute and differential cell counts in effluent samplesfrom the patient with EP (Figure 3) A significant correlationwas found between the kinetics of CCL18 concentration and
BioMed Research International 5
000
5000
10000
15000
20000
25000
D1 D3 D6 D17 D190
1000
2000
3000
4000
5000
CCL1
8 (n
gm
L)
CCL18 Total cells
Cel
ls
L
(a)
000
5000
10000
15000
20000
25000
D1 D3 D6 D17 D190
500
1000
1500
2000
2500
CCL1
8 (n
gm
L)
CCL18PMN
PMN
L
(b)
000
5000
10000
15000
20000
25000
D1 D3 D6 D17 D190
200
400
600
800
CCL1
8 (n
gm
L)
CCL18
MΦ
L
MΦ
(c)
000
5000
10000
15000
20000
25000
D1 D3 D6 D17 D19
CCL1
8 (n
gm
L)
0
100
200
300
400
500
600
700
CCL18 Eos
Eos
L
(d)
IL-6Total cells
0
2000
4000
6000
8000
10000
D1 D3 D6 D17 D190
1000
2000
3000
4000
5000
IL-6
(pg
mL)
Cel
ls
L
(e)
0
2000
4000
6000
8000
10000
D1 D3 D6 D17 D190
500
1000
1500
2000
2500
IL-6PMN
IL-6
(pg
mL)
PMN
L
(f)
0
2000
4000
6000
8000
10000
D1 D3 D6 D17 D190
200
400
600
800
IL-6
IL-6
(pg
mL)
MΦ
L
MΦ
(g)
0
2000
4000
6000
8000
10000
D1 D3 D6 D17 D190
100
200
300
400
500
600
700
IL-6
(pg
mL)
IL-6Eos
Eos
L
(h)
Figure 3 Kinetics of effluent cell counts and cytokine concentrations during EP (andashd) CCL18 effluent levels and cell counts in serial effluentsamples (endashh) IL-6 effluent levels and cell counts in serial effluent samples
6 BioMed Research International
(a)
(b) (c)
Figure 4 Cytospin preparations showing effluent cell content in EP (a) Hematoxylin and eosin staining (b) Immunocytochemistry showingCCL18 staining of eosinophils (c) Background staining with nonimmune serum
the macrophage counts in the EP samples (119903 = 0889119901 = 043) (Figure 3(c)) However the strongest correlationwas found between CCL18 effluent levels and eosinophils(119903 = 0949 119901 = 013) with peak CCL18 concentrationsdetected in effluents with the highest absolute eosinophilcount (Figure 3(d)) In contrast IL-6 effluent levels paralleledpolymorphonuclear neutrophils (PMNs) and absolute cellnumbers in effluent samples from EP (Figures 3(e) and3(f)) and no correlation was observed between effluent IL-6 concentrations and eosinophil cell count (Figure 3(h))
Although CCL18 is thought to be primarily produced byM2 macrophages involved in tissue repair and fibrosis andby dendritic cells [28] eosinophils have occasionally beenreported to produce CCL18 during inflammatory conditions[29 30] The kinetics of CCL18 effluent concentrations in EPand eosinophil counts suggested that not only macrophagesbut also eosinophils might contribute to the production ofCCL18 in this case To test this hypothesis immunostainingof cytospin preparations from eosinophilic effluents wasperformed Specific staining was detected in the cytoplasmof typical binucleated cells supporting the role of eosinophilsas a source of CCL18 in this case (Figure 4)
In a previous report we found that only peritonealmacrophages were able to produce CCL18 during infectiousperitonitis given that the cytokine was not detected in cul-ture supernatants from neutrophils (PMNs) or lymphocytes[15] To further analyze the putative role of eosinophils asproducers of CCL18 in the patient with EP CD66b+ polymor-phonuclear leukocytes (among which 85 were eosinophils
as assessed by CD16 negative staining of eosinophils versusCD16 positive neutrophils) were isolated from peritonealeffluent cells on D6 as well as CD14+ macrophages forcomparative purposes (Figures 5(a)ndash5(c)) CCL18 transcriptswere detected in both the CD66b+ and CD14+ fractions(Figure 5(d)) Quantitative PCR analysis confirmed CCL18mRNA transcripts in polymorphonuclear leukocytes in thiscase (Figure 5(e)) Altogether the data strongly suggest thateosinophils are producers of CCL18 in EP
33 Paricalcitol Downregulates the Production of CCL18 byPeritoneal Macrophages Paricalcitol a VDR agonist hasbeen shown to ameliorate peritoneal fibrosis in a murinemodel of PD [23] Given that CCL18 has been involvedin peritoneal fibrosis in PD patients [15 20 22] we testedthe effect of paricalcitol in the production of CCL18 byhuman pMΦs isolated fromPD effluents drawn frompatientssuffering from peritonitis and cultured with various dosesof nontoxic concentrations of Zemplar as a source of par-icalcitol or vehicle (supplementary Figure 2) As shown inFigures 6(a)ndash6(c) pharmacological treatment was able tosignificantly downregulate the production of CCL18 in thesecells at concentrations equivalent to 10minus8M and 10minus9M ofparicalcitol This effect was obtained as early as 24 hours andit was also observed at 48 hours and 72 hours of cell cultureSimilar results were obtained with the pure substance andwith equivalent doses of vitamin D3 (Figure 6(d))
It has been suggested that the regulation of CCL18 proteinsynthesis and secretion can be a complex process with some
BioMed Research International 7
0 200 400 600 800 1000FSC-H
PolymorphonuclearLeukocytes
0
200
400
600
800
1000
SSC-
HR1 = 715 LΦR2 = 24 MΦR3 = 6885
(a)CD16-FITC
767PMN
0
200
400
600
800
1000
SSC-
H
100 101 102 103 104
(b)
CD14-APC
999
CD66
b-FI
TC
100
100
101
101
102
102
103 104
103
104
(c)
500
300
400
200
300
200 CCL18
B2M
100
bp la
dder
NEG
CD66<+
(Eos
)
CD14+
(MΦ
)
(d)
CD14+ CD66b+
CCL1
8B2
M m
RNA
(RU
)
25E minus 05
20E minus 05
15E minus 05
10E minus 05
50E minus 06
00E + 00
(e)
Figure 5 (a) Flow cytometry analysis of peritoneal effluent leukocytes on D6 according to morphological parameters LΦ lymphocytesMΦ macrophages (b) Flow cytometry analysis of CD16 expression in peritoneal effluent cells on D6 The cutoff level for positive stainingwas set according to isotype control background staining PMN polymorphonuclear neutrophils (c) Flow cytometry analysis of CD66b+polymorphonuclear cells isolated from peritoneal effluent cells (d) RT-PCR of CCL18 gene expression in CD66+ and CD14+ purifiedperitoneal leukocytes (e) Quantitative RT-PCR analysis of CCL18 gene expression in CD66+ and CD14+ purified peritoneal leukocytes
degree of posttranscriptional regulation [28] To analyzewhether VDR agonists regulate CCL18 mRNA levels quan-titative RT-PCR assays were performed in isolated pMΦstreated with paricalcitol CCL18 mRNA levels were downreg-ulated in parallel with the diminished production of CCL18protein in pMΦs from two different donors representinginfected and uninfected peritoneal samples (Figures 7(a) and7(b)) Given that IL-4 is one of the main inducers of CCL18[31] we tested if VDR agonists couldmodulate the expressionof the chemokine in M2 macrophages generated in vitroupon treatment of peripheral blood monocytes with IL-4 Asshown in Figure 7(c) the expression of CCL18 was highlyupregulated in monocyte-derived macrophages treated for 3days with IL-4 and pretreatment with paricalcitol or vitaminD was able to prevent the upregulation of CCL18 in thesecells
4 Discussion
Peritonitis episodes constitute a frequent complication in PDpatients and recurrent peritonitis is a well-known clinicalrisk factor for peritoneal membrane dysfunction and EPS
There is an unmet requirement for biomarkers as toolsto identify patients who are at higher risk of techniquefailure to guide interventions that could improve clinicaloutcomes [32] Soluble factors released during inflammatoryprocesses such as IL-6 and IL-17 have been proposed asbiomarkers and mediators of peritoneal membrane deteri-oration (reviewed in [32 33]) However none are used inthe clinical routine and some cytokines such as IL-17 arenot easily measured in PD effluents in humans We proposeCCL18 as a biomarker of peritoneal membrane outcome inPD [22]
8 BioMed Research International
0
20
40
60
80
100
120
C
CL18
pro
duct
ion
Pari VV Pari(10minus8 M) (10minus9 M)
lowastp = 0156 lowastp = 0156
(a)
0
20
40
60
80
100
120
C
CL18
pro
duct
ion
Pari VV Pari(10minus8 M) (10minus9 M)
p = 0625
(b)
0
20
40
60
80
100
120
C
CL18
pro
duct
ion
Pari VV Pari(10minus8 M) (10minus9 M)
lowastp = 0313lowastlowastp = 0039
(c)
0
20
40
60
80
100
ParicalcitolVehicle Vit D
120
C
CL18
pro
duct
ion
(10minus8 M) (10minus8 M)
(d)
Figure 6 (andashc) Peritoneal macrophages isolated from patients experiencing peritonitis episodes were treated with vehicle or Zemplar indoses equivalent to 10minus8M and 10minus9M of paricalcitol during 24 h (a) 48 h (b) or 72 h (c) Equivalent cell cultures were established in theabsence of any treatment The concentration of CCL18 was evaluated in cell-free supernatants and the production of CCL18 in the presenceof vehicle or drug referred to the production in cell culture media alone Differences between cultures in the presence of paricalcitol or vehiclewere analyzed by theWilcoxon matched pairs rank test (d) Peritoneal macrophages were treated with paricalcitol as pure substance vitaminD or vehicle (ethanol) for 48 h
Given that high levels of the human chemokine CCL18have been shown to be related to peritoneal membranefailure and fibrosis [15 20 22] whereas sustained low CCL18peritoneal concentrations appear to be protective [22] weevaluated CCL18 levels in the peritoneum of patients under-going peritonitis episodes We found that the peritoneal con-centration of CCL18 was increased approximately 5-fold ineffluents from patients with infectious peritonitis comparedwith reported values in uninfected samples In a previousstudy involving a longitudinal analysis of 43 patients CCL18effluent concentrations ranged from 28 plusmn 16 ngml at thestart of dialysis to 34plusmn 18 ngml after 3 years of PD treatment[22] In a cross-sectional analysis of 61 patients treated formore than 3 years with PDmean effluent CCL18 values were395 plusmn 26 ngml [22] The high concentrations during IPare consistent with a previous finding showing that pMΦsfrom patients undergoing peritonitis or who recently had
peritonitis were able to secrete higher amounts of CCL18compared with pMΦs from uninfected patients [15]
Unexpectedly in a patient identified with sterileeosinophilic peritonitis the peritoneal effluent was extremelyrich in CCL18 with levels approximately 10-fold higher thanthose found in patients with IP and 50-fold more thanthose found in uninfected samples whereas effluent levelsof IL-6 which were evaluated in the same samples forcomparative purposes were similar on day 1 in both theIP and EP samples The differences between a peritonealinflammatory recognized marker IL-6 and CCL18 duringperitonitis episodes should be remarked It is of note thatthe peritoneal concentrations of CCL18 remained elevatedfor several days whereas there was a sharp decrease in IL-6Both IL-6 and CCL18 have been reported to increase indifferent inflammatory conditions some leading to tissuefibrosis [34ndash37] As a main difference although CCL18 is
BioMed Research International 9
NEG
CCL1
8 (p
gm
l)
0
200
400
600
800
1000
12001057
522
Pari (10minus7 M) NEG000
020
040
060
080
100
120100
025Fold
mRN
A C
CL18
Pari (10minus7 M)
(a)
NEG
408
279
0
100
200
300
400
500
CCL1
8 (p
gm
l)
Pari (10minus7 M)
100
045
000
050
100
150
NEG
Fold
mRN
A C
CL18
Pari (10minus7 M)
(b)
000
025
050
075
100
125
Fold
mRN
A C
CL18
NEG
Mo + IL-4
Pari Vit D3Vmdash
(c)
Figure 7 (a) Peritoneal macrophages from a patient with recent infectious peritonitis were treated with paricalcitol or vehicle for 24 h Theprotein concentration of CCL18 was evaluated in cell culture supernatants by ELISA and CCL18 mRNA levels were estimated by quantitativeRT-PCR in the same cultures (b) Peritoneal macrophages from an uninfected peritoneum were isolated from the peritoneal lavage uponcatheter insertion and treated as in (a) (c) Peripheral bloodmonocytes were pretreated with vehicle (V) 10minus7Mparicalcitol (Pari) or vitaminD3 for 24 h The cells were treated thereafter with IL-4 for 3 days before RNA extraction and evaluation expression of CCL18 mRNA levelsby quantitative RT-PCR Mean plusmn SD values from four independent experiments are shown
primarily produced by cells of myeloid origin multiplecell types are producers of IL-6 with endothelial cellsbeing an important source of this cytokine Infiltratingneutrophils as well as mesothelial cells could also be animportant source of IL-6 during bacterial peritonitis [37]The analysis of serial samples in four patients with IP showedthat the concentrations of CCL18 paralleled the numbers ofmacrophages infiltrating the peritoneum and are consistentwith previous descriptions of macrophages andmononuclearphagocytes as the primary producers of CCL18 [21] Cellcounts in the serial samples from our case of EP suggest that
the peritoneal eosinophilic infiltrate was likely contributingto the peritoneal production of CCL18 Evidences of CCL18production by activated peritoneal eosinophils in this caseare cytospin analysis of CCL18 cytoplasmic staining ineosinophils and gene expression data in polymorphonuclearleukocytes among which 85 were eosinophils
Although macrophages are typically reported as themajor source of this chemokine [21] CCL18 protein pro-duction by eosinophils has occasionally been described asbeing related to the activation status of these cells given thatCCL18 was found only in eosinophil culture supernatants
10 BioMed Research International
from eosinophilic donors but not from individuals withnonactivated eosinophils [29] Our data support the activatedstatus of eosinophils in EP given that strong staining ofCC18 was detected in peritoneal eosinophils Eosinophilshave also been related to tissue fibrosis in several diseasesand are a source of several profibrotic mediators that alterfibroblast activity and tissue remodeling [38 39] Some ofthesemediators could also stimulate the profibrotic activity ofpMΦTheproduction ofCCL18 by eosinophils in EPprovidesa new additional explanation for the association of recurrentEP during PD with UFF and potentially peritoneal fibrosis[40]
As part of its profibrotic activity CCL18 has been shownto stimulate collagen deposition [41] and fibroblast prolif-eration [42] In consonance with these actions circulatingCCL18 levels have been demonstrated to be a biomarker fordisease activity and outcome in various fibrotic diseases [1943 44] as well as in some eosinophilic disorders [30 45 46]Moreover CCL18 has been shown to promote EMT in variouscell systems [47ndash50] It is thus possible that it could alsocontribute to the EMT of mesothelial cells which has beenproposed as one of the mechanisms leading to peritonealfibrosis and membrane failure [12]
In terms of peritoneal fibrosis CCL18 has been demon-strated to herald the development of peritoneal functionaldeterioration in PD patients [15 20] in whom the abilityof peritoneal macrophages to stimulate fibroblast prolifer-ation is correlated with CCL18 mRNA levels Moreoverrecombinant CCL18 was able to stimulate the proliferation ofperitoneal fibroblasts [15]
Several molecules have been tested for their ability toprevent peritoneal fibrosis in vitro and in animal models(reviewed in [8]) Among them paricalcitol is a selectiveVDR agonist VDR is a nuclear hormone receptor expressedin various cell types including those of the immune sys-tem VDR agonists have been shown to modulate immuneresponses inflammation and fibrosis [51]
Considering the role played by inflammation in peri-toneal fibrosis during PDwe studied the effects of paricalcitolin the production of CCL18 We found that the release andgene expression of CCL18 were downregulated in pMΦsand in in vitro-differentiated human M2 cells which are theprimary producers of CCL18These results appear to contrastwith previous data showing upregulation of CCL18 by vita-min D in monocyte-derived immature dendritic cells [52]However recent reports demonstrate that the modulationof immune signaling by vitamin D in human mononuclearphagocytes is cell-type-specific with differential responses inmonocytes macrophages and dendritic cells [53] It is thuspossible that opposite effects are found on the regulation ofCCL18 by VDR agonists in different cellular systems
On the other hand in a mouse model of PD intraperi-toneal administration of paricalcitol was shown to reduceperitoneal fibrosis and ultrafiltration failure [23] In thatmodel the beneficial effects appeared to be dependent ona reduction of intraperitoneal concentrations of IL-17 Thelack of rodent orthologues of human CCL18 [28] precludesthe investigation of CCL18 and its involvement in fibrosis inmurine models On the other hand IL-17 is highly diluted
in human peritoneal effluents The need to concentrate thesamples for measurement of this cytokine hampers accuratedeterminations in human samples
A preliminary study involving 23 patients on PD suggeststhat paricalcitol could improve the ultrafiltration capacitywith less peritoneal protein loss [24] The authors suggestan effect of VDR upon the renin-angiotensin-aldosteronesystem but it is tempting to speculate that lower peritonealconcentrations of CCL18 might be present in paricalcitol-treated patients In this sense a slight reduction in theperitoneal concentration of CCL18 was observed in sixpatients who were more than 3 years on PD and treated withparicalcitol compared with a set of 31 patients of the samevintage (data not shown)
5 Conclusion
In conclusion our study suggests that the chemokine CCL18can be a mediator of peritoneal membrane failure associatedwith peritonitis episodes and might provide a new potentialtherapeutic target
Further clinical studies are needed to confirm the useful-ness of VDR agonists to preserve the peritoneal membraneand to analyze the putative mechanisms involved
Conflicts of Interest
All the authors have no potential conflicts of interest todeclare
Authorsrsquo Contributions
Marta Ossorio and Virginia Martınez contributed equally tothis work
Acknowledgments
This study has been partially supported by ISCIII (ldquoInsti-tuto de Investigacion Sanitaria Carlos IIIrdquo) Grants FIS PI1200204 PI1301768 and PI 1500120 FEDER (Funds fromthe European Union) REDinREN 20 FEDER and ReinaSofıa Institute of Nephrology Research (IRSIN) monetaryfunds The authors would like to thank Juliette Siegfriedand her team at ServingMedcom for their editing of themanuscript
Supplementary Materials
Supplemental Figure 1 serial cytokine concentrations andcell counts in peritoneal effluent during infectious peritoni-tis and eosinophilic peritonitis (A) Comparison of CCL18concentration in serial effluent samples from infectiousperitonitis (IP) and eosinophilic peritonitis (EP) (B) IL-6effluent levels in serial samples from IP and EP D1ndashD26indicate days of evolution Supplemental Figure 2 dose-dependent effects of VDR agonists on cell viability Cellviability of human monocytes was assessed by exclusion ofpropidium iodide staining and analyzed by flow cytometry
BioMed Research International 11
Results are shown as mean plusmn SD values of triplicates(A)ndash(C) Viability of monocytes upon culture during 24 48or 72 hours with decreasing concentrations of Zemplar orequivalent vehicle (305020 propylene glycolwaterethanol)(D)ndash(F) Viability of monocytes upon culture during 2448 or 72 hours with different concentrations of paricalcitol(pure substance) vitamin D or equivalent vehicle (EtOH)(Supplementary Materials)
References
[1] R T Krediet ldquoThe peritoneal membrane in chronic peritonealdialysisrdquo Kidney International vol 55 no 1 pp 341ndash356 1999
[2] R T Krediet ldquoPeritoneal physiology - Impact on solute andfluid clearancerdquo Advances in Chronic Kidney Disease vol 7 no4 pp 271ndash279 2000
[3] M Merkle and M Wrnle ldquoSclerosing peritonitis A rare butfatal complication of peritoneal inflammationrdquo Mediators ofInflammation vol 2012 Article ID 709673 p 4 2012
[4] D W Johnson Y Cho B E R Livingston et al ldquoEncapsulat-ing peritoneal sclerosis incidence predictors and outcomesrdquoKidney International vol 77 no 10 pp 904ndash912 2010
[5] G Balasubramaniam E A Brown and A Davenport ldquoThePan-Thames EPS study treatment and outcomes of encapsulat-ing peritoneal sclerosisrdquo Nephrology Dialysis Transplantation vol 24 no 10 pp 3209ndash3215 2009
[6] E D Sousa-Amorim G D Peso M A Bajo et al ldquoCan EPSdevelopment be avoided with early interventionsThe potentialrole of tamoxifenmdasha single-center studyrdquo Peritoneal DialysisInternational vol 34 no 6 pp 582ndash593 2014
[7] M R Korte D E SampimonM GH Betjes and R T KredietldquoEncapsulating peritoneal sclerosis the state of affairsrdquo NatureReviews Nephrology vol 7 no 9 pp 528ndash538 2011
[8] M A Bajo G del Peso and I Teitelbaum ldquoPeritoneal Mem-brane Preservationrdquo Seminars in Nephrology vol 37 no 1 pp77ndash92 2017
[9] M F Flessner ldquoInflammation from sterile dialysis solutions andthe longevity of the peritoneal barrierrdquo Clinical Nephrology vol68 no 6 pp 341ndash348 2007
[10] K N Lai and J C K Leung ldquoInflammation in peritonealdialysisrdquo Nephron Clinical Practice vol 116 no 1 pp c11ndashc182010
[11] S Yung and T M Chan ldquoPathophysiological changes to theperitoneal membrane during PD-related peritonitis the role ofmesothelial cellsrdquo Mediators of Inflammation vol 2012 ArticleID 484167 21 pages 2012
[12] L S Aroeira A Aguilera J A Sanchez-Tomero et al ldquoEpithe-lial tomesenchymal transition and peritonealmembrane failurein peritoneal dialysis patients pathologic significance andpotential therapeutic interventionsrdquo Journal of the AmericanSociety of Nephrology vol 18 no 7 pp 2004ndash2013 2007
[13] N Topley T Liberek A Davenport F-K Li H Fear andJ D Williams ldquoActivation of inflammation and leukocyterecruitment into the peritoneal cavityrdquo Kidney InternationalSupplements vol 50 no 56 pp S17ndashS21 1996
[14] F O Martınez and S Gordon ldquoThe M1 and M2 paradigmof macrophage activation time for reassessmentrdquo F1000PrimeReports vol 6 article 13 2014
[15] T Bellon V Martınez B Lucendo et al ldquoAlternative activa-tion of macrophages in human peritoneum implications for
peritoneal fibrosisrdquo Nephrology Dialysis Transplantation vol26 no 9 pp 2995ndash3005 2011
[16] A Prasse D V Pechkovsky G B Toews et al ldquoA vicious circleof alveolar macrophages and fibroblasts perpetuates pulmonaryfibrosis via CCL18rdquoAmerican Journal of Respiratory and CriticalCare Medicine vol 173 no 7 pp 781ndash792 2006
[17] J M Estep A Baranova N Hossain et al ldquoExpression ofcytokine signaling genes in morbidly obese patients with non-alcoholic steatohepatitis and hepatic fibrosisrdquo Obesity Surgeryvol 19 no 5 pp 617ndash624 2009
[18] F Kollert M Binder C Probst et al ldquoCCL18 - Potentialbiomarker of fibroinflammatory activity in chronic periaortitisrdquoThe Journal of Rheumatology vol 39 no 7 pp 1407ndash1412 2012
[19] A Prasse D V Pechkovsky G B Toews et al ldquoCCL18 as anindicator of pulmonary fibrotic activity in idiopathic interstitialpneumonias and systemic sclerosisrdquo Arthritis amp Rheumatologyvol 56 no 5 pp 1685ndash1693 2007
[20] S Ahmad B V North A Qureshi et al ldquoCCL18 in peritonealdialysis patients and encapsulating peritoneal sclerosisrdquo Euro-pean Journal of Clinical Investigation vol 40 no 12 pp 1067ndash1073 2010
[21] V Kodelja C Muller O Politz N Hakij C E Orfanos andS Goerdt ldquoAlternative macrophage activation-associated CC-chemokine-1 a novel structural homologue of macrophageinflammatory protein-1120572 with a Th2- associated expressionpatternrdquo The Journal of Immunology vol 160 no 3 pp 1411ndash1418 1998
[22] M Ossorio M A Bajo G Del Peso et al ldquoSustained low peri-toneal effluent CCL18 levels are associated with preservationof peritoneal membrane function in peritoneal dialysisrdquo PLoSONE vol 12 no 4 Article ID e0175835 2017
[23] G T Gonzalez-Mateo V Fernandez-Mıllara T Bellon etal ldquoParicalcitol reduces peritoneal fibrosis in mice throughthe activation of regulatory T cells and reduction in IL-17productionrdquo PLoS ONE vol 9 no 10 Article ID e108477 2014
[24] F Coronel S Cigarran A Gomis et al ldquoChanges in peritonealmembrane permeability and proteinuria in patients on peri-toneal dialysis after treatment with paricalcitolmdasha preliminarystudyrdquo Clinical Nephrology vol 78 no 2 pp 93ndash99 2012
[25] E Song N Ouyang M Horbelt B Antus M Wang and MS Exton ldquoInfluence of alternatively and classically activatedmacrophages on fibrogenic activities of human fibroblastsrdquoCellular Immunology vol 204 no 1 pp 19ndash28 2000
[26] S M Hurst T S Wilkinson R M McLoughlin et al ldquoIL-6 andits soluble receptor orchestrate a temporal switch in the patternof leukocyte recruitment seen during acute inflammationrdquoImmunity vol 14 no 6 pp 705ndash714 2001
[27] M Kozioł-Montewka A Ksiazek L Janicka and I BaranowiczldquoSerial cytokine changes in peritoneal effluent and plasma dur-ing peritonitis in patients on continuous ambulatory peritonealdialysis (CAPD)rdquo Inflammation Research vol 46 no 4 pp 132ndash136 1997
[28] E Schutyser A Richmond and J Van Damme ldquoInvolvementof CC chemokine ligand 18 (CCL18) in normal and pathologicalprocessesrdquo Journal of Leukocyte Biology vol 78 no 1 pp 14ndash262005
[29] I Schraufstatter H Takamori L Sikora P Sriramarao and RG DiScipio ldquoEosinophils and monocytes produce pulmonaryand activation-regulated chemokine which activates culturedmonocytesmacrophagesrdquoAmerican Journal of Physiology-LungCellular and Molecular Physiology vol 286 no 3 pp L494ndashL501 2004
12 BioMed Research International
[30] C Gunther N Carballido-Perrig T Kopp J M Carballidoand C Pfeiffer ldquoCCL18 is expressed in patients with bullouspemphigoid and parallels disease courserdquo British Journal ofDermatology vol 160 no 4 pp 747ndash755 2009
[31] AMantovani A Sica S Sozzani P Allavena A Vecchi andMLocati ldquoThe chemokine system in diverse forms of macrophageactivation and polarizationrdquo Trends in Immunology vol 25 no12 pp 677ndash686 2004
[32] C Aufricht R Beelen M Eberl et al ldquoBiomarker researchto improve clinical outcomes of peritoneal dialysis consensusof the European Training and Research in Peritoneal Dialysis(EuTRiPD) networkrdquo Kidney International vol 92 no 4 pp824ndash835 2017
[33] D Lopes Barreto andR T Krediet ldquoCurrent status and practicaluse of effluent biomarkers in peritoneal dialysis patientsrdquoAmerican Journal of Kidney Diseases vol 62 no 4 pp 823ndash8332013
[34] L A Boven M van Meurs R G Boot et al ldquoGaucher cellsdemonstrate a distinct macrophage phenotype and resemblealternatively activated macrophagesrdquo American Journal of Clin-ical Pathology vol 122 no 3 pp 359ndash369 2004
[35] L Cocho I Fernandez M Calonge et al ldquoGene expression-based predictive models of graft versus host disease-associateddry eyerdquo Investigative Ophtalmology Visual Science vol 56 no8 pp 4570ndash4581 2015
[36] MHasegawa ldquoBiomarkers in systemic sclerosisTheir potentialto predict clinical coursesrdquoThe Journal of Dermatology vol 43no 1 pp 29ndash38 2016
[37] S A Jones D J Fraser C A Fielding and G W JonesldquoInterleukin-6 in renal disease and therapyrdquoNephrologyDialysisTransplantation vol 30 no 4 pp 564ndash574 2015
[38] A Munitz and F Levi-Schaffer ldquoEosinophils rsquoNewrsquo roles forrsquooldrsquo cellsrdquo Allergy European Journal of Allergy and ClinicalImmunology vol 59 no 3 pp 268ndash275 2004
[39] S S Aceves ldquoRemodeling and fibrosis in chronic eosinophilinflammationrdquo Digestive Diseases vol 32 no 1-2 pp 15ndash212014
[40] Y Nakamura H Okada A Yasui T Koh and T YamaneldquoSclerosing encapsulating peritonitis associated with recurrenteosinophilic peritonitisrdquo Nephrology Dialysis Transplantation vol 14 no 3 pp 768ndash770 1999
[41] S P Atamas I G Luzina J Choi et al ldquoPulmonary andactivation-regulated chemokine stimulates collagen productionin lung fibroblastsrdquo American Journal of Respiratory Cell andMolecular Biology vol 29 no 6 pp 743ndash749 2003
[42] A Wimmer S K Khaldoyanidi M Judex N Serobyan RG DiScipio and I U Schraufstatter ldquoCCL18PARC stimulateshematopoiesis in long-term bone marrow cultures indirectlythrough its effect on monocytesrdquo Blood vol 108 no 12 pp3722ndash3729 2006
[43] A Prasse C Probst E Bargagli et al ldquoSerum CC-chemokineligand 18 concentration predicts outcome in idiopathic pul-monary fibrosisrdquo American Journal of Respiratory and CriticalCare Medicine vol 179 no 8 pp 717ndash723 2009
[44] J Chen Y Yao C Gong et al ldquoCCL18 from tumor-associatedmacrophages promotes breast cancermetastasis via PITPNM3rdquoCancer Cell vol 19 no 4 pp 541ndash555 2011
[45] K L Hon G K Ching P C Ng and T F Leung ldquoExplor-ing CCL18 eczema severity and atopyrdquo Pediatric Allergy andImmunology vol 22 no 7 pp 704ndash707 2011
[46] A J Lucendo A Arias L C De Rezende et al ldquoSubepithelialcollagen deposition profibrogenic cytokine gene expressionand changes after prolonged fluticasone propionate treatment inadult eosinophilic esophagitis a prospective studyrdquoThe Journalof Allergy and Clinical Immunology vol 128 no 5 pp 1037ndash1046 2011
[47] H Li D Zhang J Yu et al ldquoCCL18-dependent translocationof AMAP1 is critical for epithelial to mesenchymal transition inbreast cancerrdquo Journal of Cellular Physiology vol 233 no 4 pp3207ndash3217 2017
[48] Z Lin W Li H Zhang et al ldquoCCL18PITPNM3 enhancesmigration invasion and EMT through the NF-120581B signalingpathway in hepatocellular carcinomardquo Tumor Biology vol 37no 3 pp 3461ndash3468 2016
[49] F Meng W Li C Li Z Gao K Guo and S SongldquoCCL18 promotes epithelial-mesenchymal transition invasionand migration of pancreatic cancer cells in pancreatic ductaladenocarcinomardquo International Journal of Oncology vol 46 no3 pp 1109ndash1120 2015
[50] T Ploenes B Scholtes A Krohn et al ldquoCC-Chemokine Ligand18 Induces Epithelial to Mesenchymal Transition in LungCancer A549 Cells and Elevates the Invasive Potentialrdquo PLoSONE vol 8 no 1 Article ID e53068 2013
[51] J Rojas-Rivera C De La Piedra A Ramos A Ortiz andJ Egido ldquoThe expanding spectrum of biological actions ofvitamin Drdquo Nephrology Dialysis Transplantation vol 25 no 9pp 2850ndash2865 2010
[52] M Vulcano S Struyf P Scapini et al ldquoUnique regulation ofCCL18 production by maturing dendritic cellsrdquo The Journal ofImmunology vol 170 no 7 pp 3843ndash3849 2003
[53] R Kundu A Theodoraki C T Haas et al ldquoCell-type-specificmodulation of innate immune signalling by vitamin D inhuman mononuclear phagocytesrdquo The Journal of Immunologyvol 150 no 1 pp 55ndash63 2017
Stem Cells International
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Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
BioMed Research International 5
000
5000
10000
15000
20000
25000
D1 D3 D6 D17 D190
1000
2000
3000
4000
5000
CCL1
8 (n
gm
L)
CCL18 Total cells
Cel
ls
L
(a)
000
5000
10000
15000
20000
25000
D1 D3 D6 D17 D190
500
1000
1500
2000
2500
CCL1
8 (n
gm
L)
CCL18PMN
PMN
L
(b)
000
5000
10000
15000
20000
25000
D1 D3 D6 D17 D190
200
400
600
800
CCL1
8 (n
gm
L)
CCL18
MΦ
L
MΦ
(c)
000
5000
10000
15000
20000
25000
D1 D3 D6 D17 D19
CCL1
8 (n
gm
L)
0
100
200
300
400
500
600
700
CCL18 Eos
Eos
L
(d)
IL-6Total cells
0
2000
4000
6000
8000
10000
D1 D3 D6 D17 D190
1000
2000
3000
4000
5000
IL-6
(pg
mL)
Cel
ls
L
(e)
0
2000
4000
6000
8000
10000
D1 D3 D6 D17 D190
500
1000
1500
2000
2500
IL-6PMN
IL-6
(pg
mL)
PMN
L
(f)
0
2000
4000
6000
8000
10000
D1 D3 D6 D17 D190
200
400
600
800
IL-6
IL-6
(pg
mL)
MΦ
L
MΦ
(g)
0
2000
4000
6000
8000
10000
D1 D3 D6 D17 D190
100
200
300
400
500
600
700
IL-6
(pg
mL)
IL-6Eos
Eos
L
(h)
Figure 3 Kinetics of effluent cell counts and cytokine concentrations during EP (andashd) CCL18 effluent levels and cell counts in serial effluentsamples (endashh) IL-6 effluent levels and cell counts in serial effluent samples
6 BioMed Research International
(a)
(b) (c)
Figure 4 Cytospin preparations showing effluent cell content in EP (a) Hematoxylin and eosin staining (b) Immunocytochemistry showingCCL18 staining of eosinophils (c) Background staining with nonimmune serum
the macrophage counts in the EP samples (119903 = 0889119901 = 043) (Figure 3(c)) However the strongest correlationwas found between CCL18 effluent levels and eosinophils(119903 = 0949 119901 = 013) with peak CCL18 concentrationsdetected in effluents with the highest absolute eosinophilcount (Figure 3(d)) In contrast IL-6 effluent levels paralleledpolymorphonuclear neutrophils (PMNs) and absolute cellnumbers in effluent samples from EP (Figures 3(e) and3(f)) and no correlation was observed between effluent IL-6 concentrations and eosinophil cell count (Figure 3(h))
Although CCL18 is thought to be primarily produced byM2 macrophages involved in tissue repair and fibrosis andby dendritic cells [28] eosinophils have occasionally beenreported to produce CCL18 during inflammatory conditions[29 30] The kinetics of CCL18 effluent concentrations in EPand eosinophil counts suggested that not only macrophagesbut also eosinophils might contribute to the production ofCCL18 in this case To test this hypothesis immunostainingof cytospin preparations from eosinophilic effluents wasperformed Specific staining was detected in the cytoplasmof typical binucleated cells supporting the role of eosinophilsas a source of CCL18 in this case (Figure 4)
In a previous report we found that only peritonealmacrophages were able to produce CCL18 during infectiousperitonitis given that the cytokine was not detected in cul-ture supernatants from neutrophils (PMNs) or lymphocytes[15] To further analyze the putative role of eosinophils asproducers of CCL18 in the patient with EP CD66b+ polymor-phonuclear leukocytes (among which 85 were eosinophils
as assessed by CD16 negative staining of eosinophils versusCD16 positive neutrophils) were isolated from peritonealeffluent cells on D6 as well as CD14+ macrophages forcomparative purposes (Figures 5(a)ndash5(c)) CCL18 transcriptswere detected in both the CD66b+ and CD14+ fractions(Figure 5(d)) Quantitative PCR analysis confirmed CCL18mRNA transcripts in polymorphonuclear leukocytes in thiscase (Figure 5(e)) Altogether the data strongly suggest thateosinophils are producers of CCL18 in EP
33 Paricalcitol Downregulates the Production of CCL18 byPeritoneal Macrophages Paricalcitol a VDR agonist hasbeen shown to ameliorate peritoneal fibrosis in a murinemodel of PD [23] Given that CCL18 has been involvedin peritoneal fibrosis in PD patients [15 20 22] we testedthe effect of paricalcitol in the production of CCL18 byhuman pMΦs isolated fromPD effluents drawn frompatientssuffering from peritonitis and cultured with various dosesof nontoxic concentrations of Zemplar as a source of par-icalcitol or vehicle (supplementary Figure 2) As shown inFigures 6(a)ndash6(c) pharmacological treatment was able tosignificantly downregulate the production of CCL18 in thesecells at concentrations equivalent to 10minus8M and 10minus9M ofparicalcitol This effect was obtained as early as 24 hours andit was also observed at 48 hours and 72 hours of cell cultureSimilar results were obtained with the pure substance andwith equivalent doses of vitamin D3 (Figure 6(d))
It has been suggested that the regulation of CCL18 proteinsynthesis and secretion can be a complex process with some
BioMed Research International 7
0 200 400 600 800 1000FSC-H
PolymorphonuclearLeukocytes
0
200
400
600
800
1000
SSC-
HR1 = 715 LΦR2 = 24 MΦR3 = 6885
(a)CD16-FITC
767PMN
0
200
400
600
800
1000
SSC-
H
100 101 102 103 104
(b)
CD14-APC
999
CD66
b-FI
TC
100
100
101
101
102
102
103 104
103
104
(c)
500
300
400
200
300
200 CCL18
B2M
100
bp la
dder
NEG
CD66<+
(Eos
)
CD14+
(MΦ
)
(d)
CD14+ CD66b+
CCL1
8B2
M m
RNA
(RU
)
25E minus 05
20E minus 05
15E minus 05
10E minus 05
50E minus 06
00E + 00
(e)
Figure 5 (a) Flow cytometry analysis of peritoneal effluent leukocytes on D6 according to morphological parameters LΦ lymphocytesMΦ macrophages (b) Flow cytometry analysis of CD16 expression in peritoneal effluent cells on D6 The cutoff level for positive stainingwas set according to isotype control background staining PMN polymorphonuclear neutrophils (c) Flow cytometry analysis of CD66b+polymorphonuclear cells isolated from peritoneal effluent cells (d) RT-PCR of CCL18 gene expression in CD66+ and CD14+ purifiedperitoneal leukocytes (e) Quantitative RT-PCR analysis of CCL18 gene expression in CD66+ and CD14+ purified peritoneal leukocytes
degree of posttranscriptional regulation [28] To analyzewhether VDR agonists regulate CCL18 mRNA levels quan-titative RT-PCR assays were performed in isolated pMΦstreated with paricalcitol CCL18 mRNA levels were downreg-ulated in parallel with the diminished production of CCL18protein in pMΦs from two different donors representinginfected and uninfected peritoneal samples (Figures 7(a) and7(b)) Given that IL-4 is one of the main inducers of CCL18[31] we tested if VDR agonists couldmodulate the expressionof the chemokine in M2 macrophages generated in vitroupon treatment of peripheral blood monocytes with IL-4 Asshown in Figure 7(c) the expression of CCL18 was highlyupregulated in monocyte-derived macrophages treated for 3days with IL-4 and pretreatment with paricalcitol or vitaminD was able to prevent the upregulation of CCL18 in thesecells
4 Discussion
Peritonitis episodes constitute a frequent complication in PDpatients and recurrent peritonitis is a well-known clinicalrisk factor for peritoneal membrane dysfunction and EPS
There is an unmet requirement for biomarkers as toolsto identify patients who are at higher risk of techniquefailure to guide interventions that could improve clinicaloutcomes [32] Soluble factors released during inflammatoryprocesses such as IL-6 and IL-17 have been proposed asbiomarkers and mediators of peritoneal membrane deteri-oration (reviewed in [32 33]) However none are used inthe clinical routine and some cytokines such as IL-17 arenot easily measured in PD effluents in humans We proposeCCL18 as a biomarker of peritoneal membrane outcome inPD [22]
8 BioMed Research International
0
20
40
60
80
100
120
C
CL18
pro
duct
ion
Pari VV Pari(10minus8 M) (10minus9 M)
lowastp = 0156 lowastp = 0156
(a)
0
20
40
60
80
100
120
C
CL18
pro
duct
ion
Pari VV Pari(10minus8 M) (10minus9 M)
p = 0625
(b)
0
20
40
60
80
100
120
C
CL18
pro
duct
ion
Pari VV Pari(10minus8 M) (10minus9 M)
lowastp = 0313lowastlowastp = 0039
(c)
0
20
40
60
80
100
ParicalcitolVehicle Vit D
120
C
CL18
pro
duct
ion
(10minus8 M) (10minus8 M)
(d)
Figure 6 (andashc) Peritoneal macrophages isolated from patients experiencing peritonitis episodes were treated with vehicle or Zemplar indoses equivalent to 10minus8M and 10minus9M of paricalcitol during 24 h (a) 48 h (b) or 72 h (c) Equivalent cell cultures were established in theabsence of any treatment The concentration of CCL18 was evaluated in cell-free supernatants and the production of CCL18 in the presenceof vehicle or drug referred to the production in cell culture media alone Differences between cultures in the presence of paricalcitol or vehiclewere analyzed by theWilcoxon matched pairs rank test (d) Peritoneal macrophages were treated with paricalcitol as pure substance vitaminD or vehicle (ethanol) for 48 h
Given that high levels of the human chemokine CCL18have been shown to be related to peritoneal membranefailure and fibrosis [15 20 22] whereas sustained low CCL18peritoneal concentrations appear to be protective [22] weevaluated CCL18 levels in the peritoneum of patients under-going peritonitis episodes We found that the peritoneal con-centration of CCL18 was increased approximately 5-fold ineffluents from patients with infectious peritonitis comparedwith reported values in uninfected samples In a previousstudy involving a longitudinal analysis of 43 patients CCL18effluent concentrations ranged from 28 plusmn 16 ngml at thestart of dialysis to 34plusmn 18 ngml after 3 years of PD treatment[22] In a cross-sectional analysis of 61 patients treated formore than 3 years with PDmean effluent CCL18 values were395 plusmn 26 ngml [22] The high concentrations during IPare consistent with a previous finding showing that pMΦsfrom patients undergoing peritonitis or who recently had
peritonitis were able to secrete higher amounts of CCL18compared with pMΦs from uninfected patients [15]
Unexpectedly in a patient identified with sterileeosinophilic peritonitis the peritoneal effluent was extremelyrich in CCL18 with levels approximately 10-fold higher thanthose found in patients with IP and 50-fold more thanthose found in uninfected samples whereas effluent levelsof IL-6 which were evaluated in the same samples forcomparative purposes were similar on day 1 in both theIP and EP samples The differences between a peritonealinflammatory recognized marker IL-6 and CCL18 duringperitonitis episodes should be remarked It is of note thatthe peritoneal concentrations of CCL18 remained elevatedfor several days whereas there was a sharp decrease in IL-6Both IL-6 and CCL18 have been reported to increase indifferent inflammatory conditions some leading to tissuefibrosis [34ndash37] As a main difference although CCL18 is
BioMed Research International 9
NEG
CCL1
8 (p
gm
l)
0
200
400
600
800
1000
12001057
522
Pari (10minus7 M) NEG000
020
040
060
080
100
120100
025Fold
mRN
A C
CL18
Pari (10minus7 M)
(a)
NEG
408
279
0
100
200
300
400
500
CCL1
8 (p
gm
l)
Pari (10minus7 M)
100
045
000
050
100
150
NEG
Fold
mRN
A C
CL18
Pari (10minus7 M)
(b)
000
025
050
075
100
125
Fold
mRN
A C
CL18
NEG
Mo + IL-4
Pari Vit D3Vmdash
(c)
Figure 7 (a) Peritoneal macrophages from a patient with recent infectious peritonitis were treated with paricalcitol or vehicle for 24 h Theprotein concentration of CCL18 was evaluated in cell culture supernatants by ELISA and CCL18 mRNA levels were estimated by quantitativeRT-PCR in the same cultures (b) Peritoneal macrophages from an uninfected peritoneum were isolated from the peritoneal lavage uponcatheter insertion and treated as in (a) (c) Peripheral bloodmonocytes were pretreated with vehicle (V) 10minus7Mparicalcitol (Pari) or vitaminD3 for 24 h The cells were treated thereafter with IL-4 for 3 days before RNA extraction and evaluation expression of CCL18 mRNA levelsby quantitative RT-PCR Mean plusmn SD values from four independent experiments are shown
primarily produced by cells of myeloid origin multiplecell types are producers of IL-6 with endothelial cellsbeing an important source of this cytokine Infiltratingneutrophils as well as mesothelial cells could also be animportant source of IL-6 during bacterial peritonitis [37]The analysis of serial samples in four patients with IP showedthat the concentrations of CCL18 paralleled the numbers ofmacrophages infiltrating the peritoneum and are consistentwith previous descriptions of macrophages andmononuclearphagocytes as the primary producers of CCL18 [21] Cellcounts in the serial samples from our case of EP suggest that
the peritoneal eosinophilic infiltrate was likely contributingto the peritoneal production of CCL18 Evidences of CCL18production by activated peritoneal eosinophils in this caseare cytospin analysis of CCL18 cytoplasmic staining ineosinophils and gene expression data in polymorphonuclearleukocytes among which 85 were eosinophils
Although macrophages are typically reported as themajor source of this chemokine [21] CCL18 protein pro-duction by eosinophils has occasionally been described asbeing related to the activation status of these cells given thatCCL18 was found only in eosinophil culture supernatants
10 BioMed Research International
from eosinophilic donors but not from individuals withnonactivated eosinophils [29] Our data support the activatedstatus of eosinophils in EP given that strong staining ofCC18 was detected in peritoneal eosinophils Eosinophilshave also been related to tissue fibrosis in several diseasesand are a source of several profibrotic mediators that alterfibroblast activity and tissue remodeling [38 39] Some ofthesemediators could also stimulate the profibrotic activity ofpMΦTheproduction ofCCL18 by eosinophils in EPprovidesa new additional explanation for the association of recurrentEP during PD with UFF and potentially peritoneal fibrosis[40]
As part of its profibrotic activity CCL18 has been shownto stimulate collagen deposition [41] and fibroblast prolif-eration [42] In consonance with these actions circulatingCCL18 levels have been demonstrated to be a biomarker fordisease activity and outcome in various fibrotic diseases [1943 44] as well as in some eosinophilic disorders [30 45 46]Moreover CCL18 has been shown to promote EMT in variouscell systems [47ndash50] It is thus possible that it could alsocontribute to the EMT of mesothelial cells which has beenproposed as one of the mechanisms leading to peritonealfibrosis and membrane failure [12]
In terms of peritoneal fibrosis CCL18 has been demon-strated to herald the development of peritoneal functionaldeterioration in PD patients [15 20] in whom the abilityof peritoneal macrophages to stimulate fibroblast prolifer-ation is correlated with CCL18 mRNA levels Moreoverrecombinant CCL18 was able to stimulate the proliferation ofperitoneal fibroblasts [15]
Several molecules have been tested for their ability toprevent peritoneal fibrosis in vitro and in animal models(reviewed in [8]) Among them paricalcitol is a selectiveVDR agonist VDR is a nuclear hormone receptor expressedin various cell types including those of the immune sys-tem VDR agonists have been shown to modulate immuneresponses inflammation and fibrosis [51]
Considering the role played by inflammation in peri-toneal fibrosis during PDwe studied the effects of paricalcitolin the production of CCL18 We found that the release andgene expression of CCL18 were downregulated in pMΦsand in in vitro-differentiated human M2 cells which are theprimary producers of CCL18These results appear to contrastwith previous data showing upregulation of CCL18 by vita-min D in monocyte-derived immature dendritic cells [52]However recent reports demonstrate that the modulationof immune signaling by vitamin D in human mononuclearphagocytes is cell-type-specific with differential responses inmonocytes macrophages and dendritic cells [53] It is thuspossible that opposite effects are found on the regulation ofCCL18 by VDR agonists in different cellular systems
On the other hand in a mouse model of PD intraperi-toneal administration of paricalcitol was shown to reduceperitoneal fibrosis and ultrafiltration failure [23] In thatmodel the beneficial effects appeared to be dependent ona reduction of intraperitoneal concentrations of IL-17 Thelack of rodent orthologues of human CCL18 [28] precludesthe investigation of CCL18 and its involvement in fibrosis inmurine models On the other hand IL-17 is highly diluted
in human peritoneal effluents The need to concentrate thesamples for measurement of this cytokine hampers accuratedeterminations in human samples
A preliminary study involving 23 patients on PD suggeststhat paricalcitol could improve the ultrafiltration capacitywith less peritoneal protein loss [24] The authors suggestan effect of VDR upon the renin-angiotensin-aldosteronesystem but it is tempting to speculate that lower peritonealconcentrations of CCL18 might be present in paricalcitol-treated patients In this sense a slight reduction in theperitoneal concentration of CCL18 was observed in sixpatients who were more than 3 years on PD and treated withparicalcitol compared with a set of 31 patients of the samevintage (data not shown)
5 Conclusion
In conclusion our study suggests that the chemokine CCL18can be a mediator of peritoneal membrane failure associatedwith peritonitis episodes and might provide a new potentialtherapeutic target
Further clinical studies are needed to confirm the useful-ness of VDR agonists to preserve the peritoneal membraneand to analyze the putative mechanisms involved
Conflicts of Interest
All the authors have no potential conflicts of interest todeclare
Authorsrsquo Contributions
Marta Ossorio and Virginia Martınez contributed equally tothis work
Acknowledgments
This study has been partially supported by ISCIII (ldquoInsti-tuto de Investigacion Sanitaria Carlos IIIrdquo) Grants FIS PI1200204 PI1301768 and PI 1500120 FEDER (Funds fromthe European Union) REDinREN 20 FEDER and ReinaSofıa Institute of Nephrology Research (IRSIN) monetaryfunds The authors would like to thank Juliette Siegfriedand her team at ServingMedcom for their editing of themanuscript
Supplementary Materials
Supplemental Figure 1 serial cytokine concentrations andcell counts in peritoneal effluent during infectious peritoni-tis and eosinophilic peritonitis (A) Comparison of CCL18concentration in serial effluent samples from infectiousperitonitis (IP) and eosinophilic peritonitis (EP) (B) IL-6effluent levels in serial samples from IP and EP D1ndashD26indicate days of evolution Supplemental Figure 2 dose-dependent effects of VDR agonists on cell viability Cellviability of human monocytes was assessed by exclusion ofpropidium iodide staining and analyzed by flow cytometry
BioMed Research International 11
Results are shown as mean plusmn SD values of triplicates(A)ndash(C) Viability of monocytes upon culture during 24 48or 72 hours with decreasing concentrations of Zemplar orequivalent vehicle (305020 propylene glycolwaterethanol)(D)ndash(F) Viability of monocytes upon culture during 2448 or 72 hours with different concentrations of paricalcitol(pure substance) vitamin D or equivalent vehicle (EtOH)(Supplementary Materials)
References
[1] R T Krediet ldquoThe peritoneal membrane in chronic peritonealdialysisrdquo Kidney International vol 55 no 1 pp 341ndash356 1999
[2] R T Krediet ldquoPeritoneal physiology - Impact on solute andfluid clearancerdquo Advances in Chronic Kidney Disease vol 7 no4 pp 271ndash279 2000
[3] M Merkle and M Wrnle ldquoSclerosing peritonitis A rare butfatal complication of peritoneal inflammationrdquo Mediators ofInflammation vol 2012 Article ID 709673 p 4 2012
[4] D W Johnson Y Cho B E R Livingston et al ldquoEncapsulat-ing peritoneal sclerosis incidence predictors and outcomesrdquoKidney International vol 77 no 10 pp 904ndash912 2010
[5] G Balasubramaniam E A Brown and A Davenport ldquoThePan-Thames EPS study treatment and outcomes of encapsulat-ing peritoneal sclerosisrdquo Nephrology Dialysis Transplantation vol 24 no 10 pp 3209ndash3215 2009
[6] E D Sousa-Amorim G D Peso M A Bajo et al ldquoCan EPSdevelopment be avoided with early interventionsThe potentialrole of tamoxifenmdasha single-center studyrdquo Peritoneal DialysisInternational vol 34 no 6 pp 582ndash593 2014
[7] M R Korte D E SampimonM GH Betjes and R T KredietldquoEncapsulating peritoneal sclerosis the state of affairsrdquo NatureReviews Nephrology vol 7 no 9 pp 528ndash538 2011
[8] M A Bajo G del Peso and I Teitelbaum ldquoPeritoneal Mem-brane Preservationrdquo Seminars in Nephrology vol 37 no 1 pp77ndash92 2017
[9] M F Flessner ldquoInflammation from sterile dialysis solutions andthe longevity of the peritoneal barrierrdquo Clinical Nephrology vol68 no 6 pp 341ndash348 2007
[10] K N Lai and J C K Leung ldquoInflammation in peritonealdialysisrdquo Nephron Clinical Practice vol 116 no 1 pp c11ndashc182010
[11] S Yung and T M Chan ldquoPathophysiological changes to theperitoneal membrane during PD-related peritonitis the role ofmesothelial cellsrdquo Mediators of Inflammation vol 2012 ArticleID 484167 21 pages 2012
[12] L S Aroeira A Aguilera J A Sanchez-Tomero et al ldquoEpithe-lial tomesenchymal transition and peritonealmembrane failurein peritoneal dialysis patients pathologic significance andpotential therapeutic interventionsrdquo Journal of the AmericanSociety of Nephrology vol 18 no 7 pp 2004ndash2013 2007
[13] N Topley T Liberek A Davenport F-K Li H Fear andJ D Williams ldquoActivation of inflammation and leukocyterecruitment into the peritoneal cavityrdquo Kidney InternationalSupplements vol 50 no 56 pp S17ndashS21 1996
[14] F O Martınez and S Gordon ldquoThe M1 and M2 paradigmof macrophage activation time for reassessmentrdquo F1000PrimeReports vol 6 article 13 2014
[15] T Bellon V Martınez B Lucendo et al ldquoAlternative activa-tion of macrophages in human peritoneum implications for
peritoneal fibrosisrdquo Nephrology Dialysis Transplantation vol26 no 9 pp 2995ndash3005 2011
[16] A Prasse D V Pechkovsky G B Toews et al ldquoA vicious circleof alveolar macrophages and fibroblasts perpetuates pulmonaryfibrosis via CCL18rdquoAmerican Journal of Respiratory and CriticalCare Medicine vol 173 no 7 pp 781ndash792 2006
[17] J M Estep A Baranova N Hossain et al ldquoExpression ofcytokine signaling genes in morbidly obese patients with non-alcoholic steatohepatitis and hepatic fibrosisrdquo Obesity Surgeryvol 19 no 5 pp 617ndash624 2009
[18] F Kollert M Binder C Probst et al ldquoCCL18 - Potentialbiomarker of fibroinflammatory activity in chronic periaortitisrdquoThe Journal of Rheumatology vol 39 no 7 pp 1407ndash1412 2012
[19] A Prasse D V Pechkovsky G B Toews et al ldquoCCL18 as anindicator of pulmonary fibrotic activity in idiopathic interstitialpneumonias and systemic sclerosisrdquo Arthritis amp Rheumatologyvol 56 no 5 pp 1685ndash1693 2007
[20] S Ahmad B V North A Qureshi et al ldquoCCL18 in peritonealdialysis patients and encapsulating peritoneal sclerosisrdquo Euro-pean Journal of Clinical Investigation vol 40 no 12 pp 1067ndash1073 2010
[21] V Kodelja C Muller O Politz N Hakij C E Orfanos andS Goerdt ldquoAlternative macrophage activation-associated CC-chemokine-1 a novel structural homologue of macrophageinflammatory protein-1120572 with a Th2- associated expressionpatternrdquo The Journal of Immunology vol 160 no 3 pp 1411ndash1418 1998
[22] M Ossorio M A Bajo G Del Peso et al ldquoSustained low peri-toneal effluent CCL18 levels are associated with preservationof peritoneal membrane function in peritoneal dialysisrdquo PLoSONE vol 12 no 4 Article ID e0175835 2017
[23] G T Gonzalez-Mateo V Fernandez-Mıllara T Bellon etal ldquoParicalcitol reduces peritoneal fibrosis in mice throughthe activation of regulatory T cells and reduction in IL-17productionrdquo PLoS ONE vol 9 no 10 Article ID e108477 2014
[24] F Coronel S Cigarran A Gomis et al ldquoChanges in peritonealmembrane permeability and proteinuria in patients on peri-toneal dialysis after treatment with paricalcitolmdasha preliminarystudyrdquo Clinical Nephrology vol 78 no 2 pp 93ndash99 2012
[25] E Song N Ouyang M Horbelt B Antus M Wang and MS Exton ldquoInfluence of alternatively and classically activatedmacrophages on fibrogenic activities of human fibroblastsrdquoCellular Immunology vol 204 no 1 pp 19ndash28 2000
[26] S M Hurst T S Wilkinson R M McLoughlin et al ldquoIL-6 andits soluble receptor orchestrate a temporal switch in the patternof leukocyte recruitment seen during acute inflammationrdquoImmunity vol 14 no 6 pp 705ndash714 2001
[27] M Kozioł-Montewka A Ksiazek L Janicka and I BaranowiczldquoSerial cytokine changes in peritoneal effluent and plasma dur-ing peritonitis in patients on continuous ambulatory peritonealdialysis (CAPD)rdquo Inflammation Research vol 46 no 4 pp 132ndash136 1997
[28] E Schutyser A Richmond and J Van Damme ldquoInvolvementof CC chemokine ligand 18 (CCL18) in normal and pathologicalprocessesrdquo Journal of Leukocyte Biology vol 78 no 1 pp 14ndash262005
[29] I Schraufstatter H Takamori L Sikora P Sriramarao and RG DiScipio ldquoEosinophils and monocytes produce pulmonaryand activation-regulated chemokine which activates culturedmonocytesmacrophagesrdquoAmerican Journal of Physiology-LungCellular and Molecular Physiology vol 286 no 3 pp L494ndashL501 2004
12 BioMed Research International
[30] C Gunther N Carballido-Perrig T Kopp J M Carballidoand C Pfeiffer ldquoCCL18 is expressed in patients with bullouspemphigoid and parallels disease courserdquo British Journal ofDermatology vol 160 no 4 pp 747ndash755 2009
[31] AMantovani A Sica S Sozzani P Allavena A Vecchi andMLocati ldquoThe chemokine system in diverse forms of macrophageactivation and polarizationrdquo Trends in Immunology vol 25 no12 pp 677ndash686 2004
[32] C Aufricht R Beelen M Eberl et al ldquoBiomarker researchto improve clinical outcomes of peritoneal dialysis consensusof the European Training and Research in Peritoneal Dialysis(EuTRiPD) networkrdquo Kidney International vol 92 no 4 pp824ndash835 2017
[33] D Lopes Barreto andR T Krediet ldquoCurrent status and practicaluse of effluent biomarkers in peritoneal dialysis patientsrdquoAmerican Journal of Kidney Diseases vol 62 no 4 pp 823ndash8332013
[34] L A Boven M van Meurs R G Boot et al ldquoGaucher cellsdemonstrate a distinct macrophage phenotype and resemblealternatively activated macrophagesrdquo American Journal of Clin-ical Pathology vol 122 no 3 pp 359ndash369 2004
[35] L Cocho I Fernandez M Calonge et al ldquoGene expression-based predictive models of graft versus host disease-associateddry eyerdquo Investigative Ophtalmology Visual Science vol 56 no8 pp 4570ndash4581 2015
[36] MHasegawa ldquoBiomarkers in systemic sclerosisTheir potentialto predict clinical coursesrdquoThe Journal of Dermatology vol 43no 1 pp 29ndash38 2016
[37] S A Jones D J Fraser C A Fielding and G W JonesldquoInterleukin-6 in renal disease and therapyrdquoNephrologyDialysisTransplantation vol 30 no 4 pp 564ndash574 2015
[38] A Munitz and F Levi-Schaffer ldquoEosinophils rsquoNewrsquo roles forrsquooldrsquo cellsrdquo Allergy European Journal of Allergy and ClinicalImmunology vol 59 no 3 pp 268ndash275 2004
[39] S S Aceves ldquoRemodeling and fibrosis in chronic eosinophilinflammationrdquo Digestive Diseases vol 32 no 1-2 pp 15ndash212014
[40] Y Nakamura H Okada A Yasui T Koh and T YamaneldquoSclerosing encapsulating peritonitis associated with recurrenteosinophilic peritonitisrdquo Nephrology Dialysis Transplantation vol 14 no 3 pp 768ndash770 1999
[41] S P Atamas I G Luzina J Choi et al ldquoPulmonary andactivation-regulated chemokine stimulates collagen productionin lung fibroblastsrdquo American Journal of Respiratory Cell andMolecular Biology vol 29 no 6 pp 743ndash749 2003
[42] A Wimmer S K Khaldoyanidi M Judex N Serobyan RG DiScipio and I U Schraufstatter ldquoCCL18PARC stimulateshematopoiesis in long-term bone marrow cultures indirectlythrough its effect on monocytesrdquo Blood vol 108 no 12 pp3722ndash3729 2006
[43] A Prasse C Probst E Bargagli et al ldquoSerum CC-chemokineligand 18 concentration predicts outcome in idiopathic pul-monary fibrosisrdquo American Journal of Respiratory and CriticalCare Medicine vol 179 no 8 pp 717ndash723 2009
[44] J Chen Y Yao C Gong et al ldquoCCL18 from tumor-associatedmacrophages promotes breast cancermetastasis via PITPNM3rdquoCancer Cell vol 19 no 4 pp 541ndash555 2011
[45] K L Hon G K Ching P C Ng and T F Leung ldquoExplor-ing CCL18 eczema severity and atopyrdquo Pediatric Allergy andImmunology vol 22 no 7 pp 704ndash707 2011
[46] A J Lucendo A Arias L C De Rezende et al ldquoSubepithelialcollagen deposition profibrogenic cytokine gene expressionand changes after prolonged fluticasone propionate treatment inadult eosinophilic esophagitis a prospective studyrdquoThe Journalof Allergy and Clinical Immunology vol 128 no 5 pp 1037ndash1046 2011
[47] H Li D Zhang J Yu et al ldquoCCL18-dependent translocationof AMAP1 is critical for epithelial to mesenchymal transition inbreast cancerrdquo Journal of Cellular Physiology vol 233 no 4 pp3207ndash3217 2017
[48] Z Lin W Li H Zhang et al ldquoCCL18PITPNM3 enhancesmigration invasion and EMT through the NF-120581B signalingpathway in hepatocellular carcinomardquo Tumor Biology vol 37no 3 pp 3461ndash3468 2016
[49] F Meng W Li C Li Z Gao K Guo and S SongldquoCCL18 promotes epithelial-mesenchymal transition invasionand migration of pancreatic cancer cells in pancreatic ductaladenocarcinomardquo International Journal of Oncology vol 46 no3 pp 1109ndash1120 2015
[50] T Ploenes B Scholtes A Krohn et al ldquoCC-Chemokine Ligand18 Induces Epithelial to Mesenchymal Transition in LungCancer A549 Cells and Elevates the Invasive Potentialrdquo PLoSONE vol 8 no 1 Article ID e53068 2013
[51] J Rojas-Rivera C De La Piedra A Ramos A Ortiz andJ Egido ldquoThe expanding spectrum of biological actions ofvitamin Drdquo Nephrology Dialysis Transplantation vol 25 no 9pp 2850ndash2865 2010
[52] M Vulcano S Struyf P Scapini et al ldquoUnique regulation ofCCL18 production by maturing dendritic cellsrdquo The Journal ofImmunology vol 170 no 7 pp 3843ndash3849 2003
[53] R Kundu A Theodoraki C T Haas et al ldquoCell-type-specificmodulation of innate immune signalling by vitamin D inhuman mononuclear phagocytesrdquo The Journal of Immunologyvol 150 no 1 pp 55ndash63 2017
Stem Cells International
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MEDIATORSINFLAMMATION
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Disease Markers
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Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
6 BioMed Research International
(a)
(b) (c)
Figure 4 Cytospin preparations showing effluent cell content in EP (a) Hematoxylin and eosin staining (b) Immunocytochemistry showingCCL18 staining of eosinophils (c) Background staining with nonimmune serum
the macrophage counts in the EP samples (119903 = 0889119901 = 043) (Figure 3(c)) However the strongest correlationwas found between CCL18 effluent levels and eosinophils(119903 = 0949 119901 = 013) with peak CCL18 concentrationsdetected in effluents with the highest absolute eosinophilcount (Figure 3(d)) In contrast IL-6 effluent levels paralleledpolymorphonuclear neutrophils (PMNs) and absolute cellnumbers in effluent samples from EP (Figures 3(e) and3(f)) and no correlation was observed between effluent IL-6 concentrations and eosinophil cell count (Figure 3(h))
Although CCL18 is thought to be primarily produced byM2 macrophages involved in tissue repair and fibrosis andby dendritic cells [28] eosinophils have occasionally beenreported to produce CCL18 during inflammatory conditions[29 30] The kinetics of CCL18 effluent concentrations in EPand eosinophil counts suggested that not only macrophagesbut also eosinophils might contribute to the production ofCCL18 in this case To test this hypothesis immunostainingof cytospin preparations from eosinophilic effluents wasperformed Specific staining was detected in the cytoplasmof typical binucleated cells supporting the role of eosinophilsas a source of CCL18 in this case (Figure 4)
In a previous report we found that only peritonealmacrophages were able to produce CCL18 during infectiousperitonitis given that the cytokine was not detected in cul-ture supernatants from neutrophils (PMNs) or lymphocytes[15] To further analyze the putative role of eosinophils asproducers of CCL18 in the patient with EP CD66b+ polymor-phonuclear leukocytes (among which 85 were eosinophils
as assessed by CD16 negative staining of eosinophils versusCD16 positive neutrophils) were isolated from peritonealeffluent cells on D6 as well as CD14+ macrophages forcomparative purposes (Figures 5(a)ndash5(c)) CCL18 transcriptswere detected in both the CD66b+ and CD14+ fractions(Figure 5(d)) Quantitative PCR analysis confirmed CCL18mRNA transcripts in polymorphonuclear leukocytes in thiscase (Figure 5(e)) Altogether the data strongly suggest thateosinophils are producers of CCL18 in EP
33 Paricalcitol Downregulates the Production of CCL18 byPeritoneal Macrophages Paricalcitol a VDR agonist hasbeen shown to ameliorate peritoneal fibrosis in a murinemodel of PD [23] Given that CCL18 has been involvedin peritoneal fibrosis in PD patients [15 20 22] we testedthe effect of paricalcitol in the production of CCL18 byhuman pMΦs isolated fromPD effluents drawn frompatientssuffering from peritonitis and cultured with various dosesof nontoxic concentrations of Zemplar as a source of par-icalcitol or vehicle (supplementary Figure 2) As shown inFigures 6(a)ndash6(c) pharmacological treatment was able tosignificantly downregulate the production of CCL18 in thesecells at concentrations equivalent to 10minus8M and 10minus9M ofparicalcitol This effect was obtained as early as 24 hours andit was also observed at 48 hours and 72 hours of cell cultureSimilar results were obtained with the pure substance andwith equivalent doses of vitamin D3 (Figure 6(d))
It has been suggested that the regulation of CCL18 proteinsynthesis and secretion can be a complex process with some
BioMed Research International 7
0 200 400 600 800 1000FSC-H
PolymorphonuclearLeukocytes
0
200
400
600
800
1000
SSC-
HR1 = 715 LΦR2 = 24 MΦR3 = 6885
(a)CD16-FITC
767PMN
0
200
400
600
800
1000
SSC-
H
100 101 102 103 104
(b)
CD14-APC
999
CD66
b-FI
TC
100
100
101
101
102
102
103 104
103
104
(c)
500
300
400
200
300
200 CCL18
B2M
100
bp la
dder
NEG
CD66<+
(Eos
)
CD14+
(MΦ
)
(d)
CD14+ CD66b+
CCL1
8B2
M m
RNA
(RU
)
25E minus 05
20E minus 05
15E minus 05
10E minus 05
50E minus 06
00E + 00
(e)
Figure 5 (a) Flow cytometry analysis of peritoneal effluent leukocytes on D6 according to morphological parameters LΦ lymphocytesMΦ macrophages (b) Flow cytometry analysis of CD16 expression in peritoneal effluent cells on D6 The cutoff level for positive stainingwas set according to isotype control background staining PMN polymorphonuclear neutrophils (c) Flow cytometry analysis of CD66b+polymorphonuclear cells isolated from peritoneal effluent cells (d) RT-PCR of CCL18 gene expression in CD66+ and CD14+ purifiedperitoneal leukocytes (e) Quantitative RT-PCR analysis of CCL18 gene expression in CD66+ and CD14+ purified peritoneal leukocytes
degree of posttranscriptional regulation [28] To analyzewhether VDR agonists regulate CCL18 mRNA levels quan-titative RT-PCR assays were performed in isolated pMΦstreated with paricalcitol CCL18 mRNA levels were downreg-ulated in parallel with the diminished production of CCL18protein in pMΦs from two different donors representinginfected and uninfected peritoneal samples (Figures 7(a) and7(b)) Given that IL-4 is one of the main inducers of CCL18[31] we tested if VDR agonists couldmodulate the expressionof the chemokine in M2 macrophages generated in vitroupon treatment of peripheral blood monocytes with IL-4 Asshown in Figure 7(c) the expression of CCL18 was highlyupregulated in monocyte-derived macrophages treated for 3days with IL-4 and pretreatment with paricalcitol or vitaminD was able to prevent the upregulation of CCL18 in thesecells
4 Discussion
Peritonitis episodes constitute a frequent complication in PDpatients and recurrent peritonitis is a well-known clinicalrisk factor for peritoneal membrane dysfunction and EPS
There is an unmet requirement for biomarkers as toolsto identify patients who are at higher risk of techniquefailure to guide interventions that could improve clinicaloutcomes [32] Soluble factors released during inflammatoryprocesses such as IL-6 and IL-17 have been proposed asbiomarkers and mediators of peritoneal membrane deteri-oration (reviewed in [32 33]) However none are used inthe clinical routine and some cytokines such as IL-17 arenot easily measured in PD effluents in humans We proposeCCL18 as a biomarker of peritoneal membrane outcome inPD [22]
8 BioMed Research International
0
20
40
60
80
100
120
C
CL18
pro
duct
ion
Pari VV Pari(10minus8 M) (10minus9 M)
lowastp = 0156 lowastp = 0156
(a)
0
20
40
60
80
100
120
C
CL18
pro
duct
ion
Pari VV Pari(10minus8 M) (10minus9 M)
p = 0625
(b)
0
20
40
60
80
100
120
C
CL18
pro
duct
ion
Pari VV Pari(10minus8 M) (10minus9 M)
lowastp = 0313lowastlowastp = 0039
(c)
0
20
40
60
80
100
ParicalcitolVehicle Vit D
120
C
CL18
pro
duct
ion
(10minus8 M) (10minus8 M)
(d)
Figure 6 (andashc) Peritoneal macrophages isolated from patients experiencing peritonitis episodes were treated with vehicle or Zemplar indoses equivalent to 10minus8M and 10minus9M of paricalcitol during 24 h (a) 48 h (b) or 72 h (c) Equivalent cell cultures were established in theabsence of any treatment The concentration of CCL18 was evaluated in cell-free supernatants and the production of CCL18 in the presenceof vehicle or drug referred to the production in cell culture media alone Differences between cultures in the presence of paricalcitol or vehiclewere analyzed by theWilcoxon matched pairs rank test (d) Peritoneal macrophages were treated with paricalcitol as pure substance vitaminD or vehicle (ethanol) for 48 h
Given that high levels of the human chemokine CCL18have been shown to be related to peritoneal membranefailure and fibrosis [15 20 22] whereas sustained low CCL18peritoneal concentrations appear to be protective [22] weevaluated CCL18 levels in the peritoneum of patients under-going peritonitis episodes We found that the peritoneal con-centration of CCL18 was increased approximately 5-fold ineffluents from patients with infectious peritonitis comparedwith reported values in uninfected samples In a previousstudy involving a longitudinal analysis of 43 patients CCL18effluent concentrations ranged from 28 plusmn 16 ngml at thestart of dialysis to 34plusmn 18 ngml after 3 years of PD treatment[22] In a cross-sectional analysis of 61 patients treated formore than 3 years with PDmean effluent CCL18 values were395 plusmn 26 ngml [22] The high concentrations during IPare consistent with a previous finding showing that pMΦsfrom patients undergoing peritonitis or who recently had
peritonitis were able to secrete higher amounts of CCL18compared with pMΦs from uninfected patients [15]
Unexpectedly in a patient identified with sterileeosinophilic peritonitis the peritoneal effluent was extremelyrich in CCL18 with levels approximately 10-fold higher thanthose found in patients with IP and 50-fold more thanthose found in uninfected samples whereas effluent levelsof IL-6 which were evaluated in the same samples forcomparative purposes were similar on day 1 in both theIP and EP samples The differences between a peritonealinflammatory recognized marker IL-6 and CCL18 duringperitonitis episodes should be remarked It is of note thatthe peritoneal concentrations of CCL18 remained elevatedfor several days whereas there was a sharp decrease in IL-6Both IL-6 and CCL18 have been reported to increase indifferent inflammatory conditions some leading to tissuefibrosis [34ndash37] As a main difference although CCL18 is
BioMed Research International 9
NEG
CCL1
8 (p
gm
l)
0
200
400
600
800
1000
12001057
522
Pari (10minus7 M) NEG000
020
040
060
080
100
120100
025Fold
mRN
A C
CL18
Pari (10minus7 M)
(a)
NEG
408
279
0
100
200
300
400
500
CCL1
8 (p
gm
l)
Pari (10minus7 M)
100
045
000
050
100
150
NEG
Fold
mRN
A C
CL18
Pari (10minus7 M)
(b)
000
025
050
075
100
125
Fold
mRN
A C
CL18
NEG
Mo + IL-4
Pari Vit D3Vmdash
(c)
Figure 7 (a) Peritoneal macrophages from a patient with recent infectious peritonitis were treated with paricalcitol or vehicle for 24 h Theprotein concentration of CCL18 was evaluated in cell culture supernatants by ELISA and CCL18 mRNA levels were estimated by quantitativeRT-PCR in the same cultures (b) Peritoneal macrophages from an uninfected peritoneum were isolated from the peritoneal lavage uponcatheter insertion and treated as in (a) (c) Peripheral bloodmonocytes were pretreated with vehicle (V) 10minus7Mparicalcitol (Pari) or vitaminD3 for 24 h The cells were treated thereafter with IL-4 for 3 days before RNA extraction and evaluation expression of CCL18 mRNA levelsby quantitative RT-PCR Mean plusmn SD values from four independent experiments are shown
primarily produced by cells of myeloid origin multiplecell types are producers of IL-6 with endothelial cellsbeing an important source of this cytokine Infiltratingneutrophils as well as mesothelial cells could also be animportant source of IL-6 during bacterial peritonitis [37]The analysis of serial samples in four patients with IP showedthat the concentrations of CCL18 paralleled the numbers ofmacrophages infiltrating the peritoneum and are consistentwith previous descriptions of macrophages andmononuclearphagocytes as the primary producers of CCL18 [21] Cellcounts in the serial samples from our case of EP suggest that
the peritoneal eosinophilic infiltrate was likely contributingto the peritoneal production of CCL18 Evidences of CCL18production by activated peritoneal eosinophils in this caseare cytospin analysis of CCL18 cytoplasmic staining ineosinophils and gene expression data in polymorphonuclearleukocytes among which 85 were eosinophils
Although macrophages are typically reported as themajor source of this chemokine [21] CCL18 protein pro-duction by eosinophils has occasionally been described asbeing related to the activation status of these cells given thatCCL18 was found only in eosinophil culture supernatants
10 BioMed Research International
from eosinophilic donors but not from individuals withnonactivated eosinophils [29] Our data support the activatedstatus of eosinophils in EP given that strong staining ofCC18 was detected in peritoneal eosinophils Eosinophilshave also been related to tissue fibrosis in several diseasesand are a source of several profibrotic mediators that alterfibroblast activity and tissue remodeling [38 39] Some ofthesemediators could also stimulate the profibrotic activity ofpMΦTheproduction ofCCL18 by eosinophils in EPprovidesa new additional explanation for the association of recurrentEP during PD with UFF and potentially peritoneal fibrosis[40]
As part of its profibrotic activity CCL18 has been shownto stimulate collagen deposition [41] and fibroblast prolif-eration [42] In consonance with these actions circulatingCCL18 levels have been demonstrated to be a biomarker fordisease activity and outcome in various fibrotic diseases [1943 44] as well as in some eosinophilic disorders [30 45 46]Moreover CCL18 has been shown to promote EMT in variouscell systems [47ndash50] It is thus possible that it could alsocontribute to the EMT of mesothelial cells which has beenproposed as one of the mechanisms leading to peritonealfibrosis and membrane failure [12]
In terms of peritoneal fibrosis CCL18 has been demon-strated to herald the development of peritoneal functionaldeterioration in PD patients [15 20] in whom the abilityof peritoneal macrophages to stimulate fibroblast prolifer-ation is correlated with CCL18 mRNA levels Moreoverrecombinant CCL18 was able to stimulate the proliferation ofperitoneal fibroblasts [15]
Several molecules have been tested for their ability toprevent peritoneal fibrosis in vitro and in animal models(reviewed in [8]) Among them paricalcitol is a selectiveVDR agonist VDR is a nuclear hormone receptor expressedin various cell types including those of the immune sys-tem VDR agonists have been shown to modulate immuneresponses inflammation and fibrosis [51]
Considering the role played by inflammation in peri-toneal fibrosis during PDwe studied the effects of paricalcitolin the production of CCL18 We found that the release andgene expression of CCL18 were downregulated in pMΦsand in in vitro-differentiated human M2 cells which are theprimary producers of CCL18These results appear to contrastwith previous data showing upregulation of CCL18 by vita-min D in monocyte-derived immature dendritic cells [52]However recent reports demonstrate that the modulationof immune signaling by vitamin D in human mononuclearphagocytes is cell-type-specific with differential responses inmonocytes macrophages and dendritic cells [53] It is thuspossible that opposite effects are found on the regulation ofCCL18 by VDR agonists in different cellular systems
On the other hand in a mouse model of PD intraperi-toneal administration of paricalcitol was shown to reduceperitoneal fibrosis and ultrafiltration failure [23] In thatmodel the beneficial effects appeared to be dependent ona reduction of intraperitoneal concentrations of IL-17 Thelack of rodent orthologues of human CCL18 [28] precludesthe investigation of CCL18 and its involvement in fibrosis inmurine models On the other hand IL-17 is highly diluted
in human peritoneal effluents The need to concentrate thesamples for measurement of this cytokine hampers accuratedeterminations in human samples
A preliminary study involving 23 patients on PD suggeststhat paricalcitol could improve the ultrafiltration capacitywith less peritoneal protein loss [24] The authors suggestan effect of VDR upon the renin-angiotensin-aldosteronesystem but it is tempting to speculate that lower peritonealconcentrations of CCL18 might be present in paricalcitol-treated patients In this sense a slight reduction in theperitoneal concentration of CCL18 was observed in sixpatients who were more than 3 years on PD and treated withparicalcitol compared with a set of 31 patients of the samevintage (data not shown)
5 Conclusion
In conclusion our study suggests that the chemokine CCL18can be a mediator of peritoneal membrane failure associatedwith peritonitis episodes and might provide a new potentialtherapeutic target
Further clinical studies are needed to confirm the useful-ness of VDR agonists to preserve the peritoneal membraneand to analyze the putative mechanisms involved
Conflicts of Interest
All the authors have no potential conflicts of interest todeclare
Authorsrsquo Contributions
Marta Ossorio and Virginia Martınez contributed equally tothis work
Acknowledgments
This study has been partially supported by ISCIII (ldquoInsti-tuto de Investigacion Sanitaria Carlos IIIrdquo) Grants FIS PI1200204 PI1301768 and PI 1500120 FEDER (Funds fromthe European Union) REDinREN 20 FEDER and ReinaSofıa Institute of Nephrology Research (IRSIN) monetaryfunds The authors would like to thank Juliette Siegfriedand her team at ServingMedcom for their editing of themanuscript
Supplementary Materials
Supplemental Figure 1 serial cytokine concentrations andcell counts in peritoneal effluent during infectious peritoni-tis and eosinophilic peritonitis (A) Comparison of CCL18concentration in serial effluent samples from infectiousperitonitis (IP) and eosinophilic peritonitis (EP) (B) IL-6effluent levels in serial samples from IP and EP D1ndashD26indicate days of evolution Supplemental Figure 2 dose-dependent effects of VDR agonists on cell viability Cellviability of human monocytes was assessed by exclusion ofpropidium iodide staining and analyzed by flow cytometry
BioMed Research International 11
Results are shown as mean plusmn SD values of triplicates(A)ndash(C) Viability of monocytes upon culture during 24 48or 72 hours with decreasing concentrations of Zemplar orequivalent vehicle (305020 propylene glycolwaterethanol)(D)ndash(F) Viability of monocytes upon culture during 2448 or 72 hours with different concentrations of paricalcitol(pure substance) vitamin D or equivalent vehicle (EtOH)(Supplementary Materials)
References
[1] R T Krediet ldquoThe peritoneal membrane in chronic peritonealdialysisrdquo Kidney International vol 55 no 1 pp 341ndash356 1999
[2] R T Krediet ldquoPeritoneal physiology - Impact on solute andfluid clearancerdquo Advances in Chronic Kidney Disease vol 7 no4 pp 271ndash279 2000
[3] M Merkle and M Wrnle ldquoSclerosing peritonitis A rare butfatal complication of peritoneal inflammationrdquo Mediators ofInflammation vol 2012 Article ID 709673 p 4 2012
[4] D W Johnson Y Cho B E R Livingston et al ldquoEncapsulat-ing peritoneal sclerosis incidence predictors and outcomesrdquoKidney International vol 77 no 10 pp 904ndash912 2010
[5] G Balasubramaniam E A Brown and A Davenport ldquoThePan-Thames EPS study treatment and outcomes of encapsulat-ing peritoneal sclerosisrdquo Nephrology Dialysis Transplantation vol 24 no 10 pp 3209ndash3215 2009
[6] E D Sousa-Amorim G D Peso M A Bajo et al ldquoCan EPSdevelopment be avoided with early interventionsThe potentialrole of tamoxifenmdasha single-center studyrdquo Peritoneal DialysisInternational vol 34 no 6 pp 582ndash593 2014
[7] M R Korte D E SampimonM GH Betjes and R T KredietldquoEncapsulating peritoneal sclerosis the state of affairsrdquo NatureReviews Nephrology vol 7 no 9 pp 528ndash538 2011
[8] M A Bajo G del Peso and I Teitelbaum ldquoPeritoneal Mem-brane Preservationrdquo Seminars in Nephrology vol 37 no 1 pp77ndash92 2017
[9] M F Flessner ldquoInflammation from sterile dialysis solutions andthe longevity of the peritoneal barrierrdquo Clinical Nephrology vol68 no 6 pp 341ndash348 2007
[10] K N Lai and J C K Leung ldquoInflammation in peritonealdialysisrdquo Nephron Clinical Practice vol 116 no 1 pp c11ndashc182010
[11] S Yung and T M Chan ldquoPathophysiological changes to theperitoneal membrane during PD-related peritonitis the role ofmesothelial cellsrdquo Mediators of Inflammation vol 2012 ArticleID 484167 21 pages 2012
[12] L S Aroeira A Aguilera J A Sanchez-Tomero et al ldquoEpithe-lial tomesenchymal transition and peritonealmembrane failurein peritoneal dialysis patients pathologic significance andpotential therapeutic interventionsrdquo Journal of the AmericanSociety of Nephrology vol 18 no 7 pp 2004ndash2013 2007
[13] N Topley T Liberek A Davenport F-K Li H Fear andJ D Williams ldquoActivation of inflammation and leukocyterecruitment into the peritoneal cavityrdquo Kidney InternationalSupplements vol 50 no 56 pp S17ndashS21 1996
[14] F O Martınez and S Gordon ldquoThe M1 and M2 paradigmof macrophage activation time for reassessmentrdquo F1000PrimeReports vol 6 article 13 2014
[15] T Bellon V Martınez B Lucendo et al ldquoAlternative activa-tion of macrophages in human peritoneum implications for
peritoneal fibrosisrdquo Nephrology Dialysis Transplantation vol26 no 9 pp 2995ndash3005 2011
[16] A Prasse D V Pechkovsky G B Toews et al ldquoA vicious circleof alveolar macrophages and fibroblasts perpetuates pulmonaryfibrosis via CCL18rdquoAmerican Journal of Respiratory and CriticalCare Medicine vol 173 no 7 pp 781ndash792 2006
[17] J M Estep A Baranova N Hossain et al ldquoExpression ofcytokine signaling genes in morbidly obese patients with non-alcoholic steatohepatitis and hepatic fibrosisrdquo Obesity Surgeryvol 19 no 5 pp 617ndash624 2009
[18] F Kollert M Binder C Probst et al ldquoCCL18 - Potentialbiomarker of fibroinflammatory activity in chronic periaortitisrdquoThe Journal of Rheumatology vol 39 no 7 pp 1407ndash1412 2012
[19] A Prasse D V Pechkovsky G B Toews et al ldquoCCL18 as anindicator of pulmonary fibrotic activity in idiopathic interstitialpneumonias and systemic sclerosisrdquo Arthritis amp Rheumatologyvol 56 no 5 pp 1685ndash1693 2007
[20] S Ahmad B V North A Qureshi et al ldquoCCL18 in peritonealdialysis patients and encapsulating peritoneal sclerosisrdquo Euro-pean Journal of Clinical Investigation vol 40 no 12 pp 1067ndash1073 2010
[21] V Kodelja C Muller O Politz N Hakij C E Orfanos andS Goerdt ldquoAlternative macrophage activation-associated CC-chemokine-1 a novel structural homologue of macrophageinflammatory protein-1120572 with a Th2- associated expressionpatternrdquo The Journal of Immunology vol 160 no 3 pp 1411ndash1418 1998
[22] M Ossorio M A Bajo G Del Peso et al ldquoSustained low peri-toneal effluent CCL18 levels are associated with preservationof peritoneal membrane function in peritoneal dialysisrdquo PLoSONE vol 12 no 4 Article ID e0175835 2017
[23] G T Gonzalez-Mateo V Fernandez-Mıllara T Bellon etal ldquoParicalcitol reduces peritoneal fibrosis in mice throughthe activation of regulatory T cells and reduction in IL-17productionrdquo PLoS ONE vol 9 no 10 Article ID e108477 2014
[24] F Coronel S Cigarran A Gomis et al ldquoChanges in peritonealmembrane permeability and proteinuria in patients on peri-toneal dialysis after treatment with paricalcitolmdasha preliminarystudyrdquo Clinical Nephrology vol 78 no 2 pp 93ndash99 2012
[25] E Song N Ouyang M Horbelt B Antus M Wang and MS Exton ldquoInfluence of alternatively and classically activatedmacrophages on fibrogenic activities of human fibroblastsrdquoCellular Immunology vol 204 no 1 pp 19ndash28 2000
[26] S M Hurst T S Wilkinson R M McLoughlin et al ldquoIL-6 andits soluble receptor orchestrate a temporal switch in the patternof leukocyte recruitment seen during acute inflammationrdquoImmunity vol 14 no 6 pp 705ndash714 2001
[27] M Kozioł-Montewka A Ksiazek L Janicka and I BaranowiczldquoSerial cytokine changes in peritoneal effluent and plasma dur-ing peritonitis in patients on continuous ambulatory peritonealdialysis (CAPD)rdquo Inflammation Research vol 46 no 4 pp 132ndash136 1997
[28] E Schutyser A Richmond and J Van Damme ldquoInvolvementof CC chemokine ligand 18 (CCL18) in normal and pathologicalprocessesrdquo Journal of Leukocyte Biology vol 78 no 1 pp 14ndash262005
[29] I Schraufstatter H Takamori L Sikora P Sriramarao and RG DiScipio ldquoEosinophils and monocytes produce pulmonaryand activation-regulated chemokine which activates culturedmonocytesmacrophagesrdquoAmerican Journal of Physiology-LungCellular and Molecular Physiology vol 286 no 3 pp L494ndashL501 2004
12 BioMed Research International
[30] C Gunther N Carballido-Perrig T Kopp J M Carballidoand C Pfeiffer ldquoCCL18 is expressed in patients with bullouspemphigoid and parallels disease courserdquo British Journal ofDermatology vol 160 no 4 pp 747ndash755 2009
[31] AMantovani A Sica S Sozzani P Allavena A Vecchi andMLocati ldquoThe chemokine system in diverse forms of macrophageactivation and polarizationrdquo Trends in Immunology vol 25 no12 pp 677ndash686 2004
[32] C Aufricht R Beelen M Eberl et al ldquoBiomarker researchto improve clinical outcomes of peritoneal dialysis consensusof the European Training and Research in Peritoneal Dialysis(EuTRiPD) networkrdquo Kidney International vol 92 no 4 pp824ndash835 2017
[33] D Lopes Barreto andR T Krediet ldquoCurrent status and practicaluse of effluent biomarkers in peritoneal dialysis patientsrdquoAmerican Journal of Kidney Diseases vol 62 no 4 pp 823ndash8332013
[34] L A Boven M van Meurs R G Boot et al ldquoGaucher cellsdemonstrate a distinct macrophage phenotype and resemblealternatively activated macrophagesrdquo American Journal of Clin-ical Pathology vol 122 no 3 pp 359ndash369 2004
[35] L Cocho I Fernandez M Calonge et al ldquoGene expression-based predictive models of graft versus host disease-associateddry eyerdquo Investigative Ophtalmology Visual Science vol 56 no8 pp 4570ndash4581 2015
[36] MHasegawa ldquoBiomarkers in systemic sclerosisTheir potentialto predict clinical coursesrdquoThe Journal of Dermatology vol 43no 1 pp 29ndash38 2016
[37] S A Jones D J Fraser C A Fielding and G W JonesldquoInterleukin-6 in renal disease and therapyrdquoNephrologyDialysisTransplantation vol 30 no 4 pp 564ndash574 2015
[38] A Munitz and F Levi-Schaffer ldquoEosinophils rsquoNewrsquo roles forrsquooldrsquo cellsrdquo Allergy European Journal of Allergy and ClinicalImmunology vol 59 no 3 pp 268ndash275 2004
[39] S S Aceves ldquoRemodeling and fibrosis in chronic eosinophilinflammationrdquo Digestive Diseases vol 32 no 1-2 pp 15ndash212014
[40] Y Nakamura H Okada A Yasui T Koh and T YamaneldquoSclerosing encapsulating peritonitis associated with recurrenteosinophilic peritonitisrdquo Nephrology Dialysis Transplantation vol 14 no 3 pp 768ndash770 1999
[41] S P Atamas I G Luzina J Choi et al ldquoPulmonary andactivation-regulated chemokine stimulates collagen productionin lung fibroblastsrdquo American Journal of Respiratory Cell andMolecular Biology vol 29 no 6 pp 743ndash749 2003
[42] A Wimmer S K Khaldoyanidi M Judex N Serobyan RG DiScipio and I U Schraufstatter ldquoCCL18PARC stimulateshematopoiesis in long-term bone marrow cultures indirectlythrough its effect on monocytesrdquo Blood vol 108 no 12 pp3722ndash3729 2006
[43] A Prasse C Probst E Bargagli et al ldquoSerum CC-chemokineligand 18 concentration predicts outcome in idiopathic pul-monary fibrosisrdquo American Journal of Respiratory and CriticalCare Medicine vol 179 no 8 pp 717ndash723 2009
[44] J Chen Y Yao C Gong et al ldquoCCL18 from tumor-associatedmacrophages promotes breast cancermetastasis via PITPNM3rdquoCancer Cell vol 19 no 4 pp 541ndash555 2011
[45] K L Hon G K Ching P C Ng and T F Leung ldquoExplor-ing CCL18 eczema severity and atopyrdquo Pediatric Allergy andImmunology vol 22 no 7 pp 704ndash707 2011
[46] A J Lucendo A Arias L C De Rezende et al ldquoSubepithelialcollagen deposition profibrogenic cytokine gene expressionand changes after prolonged fluticasone propionate treatment inadult eosinophilic esophagitis a prospective studyrdquoThe Journalof Allergy and Clinical Immunology vol 128 no 5 pp 1037ndash1046 2011
[47] H Li D Zhang J Yu et al ldquoCCL18-dependent translocationof AMAP1 is critical for epithelial to mesenchymal transition inbreast cancerrdquo Journal of Cellular Physiology vol 233 no 4 pp3207ndash3217 2017
[48] Z Lin W Li H Zhang et al ldquoCCL18PITPNM3 enhancesmigration invasion and EMT through the NF-120581B signalingpathway in hepatocellular carcinomardquo Tumor Biology vol 37no 3 pp 3461ndash3468 2016
[49] F Meng W Li C Li Z Gao K Guo and S SongldquoCCL18 promotes epithelial-mesenchymal transition invasionand migration of pancreatic cancer cells in pancreatic ductaladenocarcinomardquo International Journal of Oncology vol 46 no3 pp 1109ndash1120 2015
[50] T Ploenes B Scholtes A Krohn et al ldquoCC-Chemokine Ligand18 Induces Epithelial to Mesenchymal Transition in LungCancer A549 Cells and Elevates the Invasive Potentialrdquo PLoSONE vol 8 no 1 Article ID e53068 2013
[51] J Rojas-Rivera C De La Piedra A Ramos A Ortiz andJ Egido ldquoThe expanding spectrum of biological actions ofvitamin Drdquo Nephrology Dialysis Transplantation vol 25 no 9pp 2850ndash2865 2010
[52] M Vulcano S Struyf P Scapini et al ldquoUnique regulation ofCCL18 production by maturing dendritic cellsrdquo The Journal ofImmunology vol 170 no 7 pp 3843ndash3849 2003
[53] R Kundu A Theodoraki C T Haas et al ldquoCell-type-specificmodulation of innate immune signalling by vitamin D inhuman mononuclear phagocytesrdquo The Journal of Immunologyvol 150 no 1 pp 55ndash63 2017
Stem Cells International
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Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
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Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
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Immunology ResearchHindawiwwwhindawicom Volume 2018
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Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
BioMed Research International 7
0 200 400 600 800 1000FSC-H
PolymorphonuclearLeukocytes
0
200
400
600
800
1000
SSC-
HR1 = 715 LΦR2 = 24 MΦR3 = 6885
(a)CD16-FITC
767PMN
0
200
400
600
800
1000
SSC-
H
100 101 102 103 104
(b)
CD14-APC
999
CD66
b-FI
TC
100
100
101
101
102
102
103 104
103
104
(c)
500
300
400
200
300
200 CCL18
B2M
100
bp la
dder
NEG
CD66<+
(Eos
)
CD14+
(MΦ
)
(d)
CD14+ CD66b+
CCL1
8B2
M m
RNA
(RU
)
25E minus 05
20E minus 05
15E minus 05
10E minus 05
50E minus 06
00E + 00
(e)
Figure 5 (a) Flow cytometry analysis of peritoneal effluent leukocytes on D6 according to morphological parameters LΦ lymphocytesMΦ macrophages (b) Flow cytometry analysis of CD16 expression in peritoneal effluent cells on D6 The cutoff level for positive stainingwas set according to isotype control background staining PMN polymorphonuclear neutrophils (c) Flow cytometry analysis of CD66b+polymorphonuclear cells isolated from peritoneal effluent cells (d) RT-PCR of CCL18 gene expression in CD66+ and CD14+ purifiedperitoneal leukocytes (e) Quantitative RT-PCR analysis of CCL18 gene expression in CD66+ and CD14+ purified peritoneal leukocytes
degree of posttranscriptional regulation [28] To analyzewhether VDR agonists regulate CCL18 mRNA levels quan-titative RT-PCR assays were performed in isolated pMΦstreated with paricalcitol CCL18 mRNA levels were downreg-ulated in parallel with the diminished production of CCL18protein in pMΦs from two different donors representinginfected and uninfected peritoneal samples (Figures 7(a) and7(b)) Given that IL-4 is one of the main inducers of CCL18[31] we tested if VDR agonists couldmodulate the expressionof the chemokine in M2 macrophages generated in vitroupon treatment of peripheral blood monocytes with IL-4 Asshown in Figure 7(c) the expression of CCL18 was highlyupregulated in monocyte-derived macrophages treated for 3days with IL-4 and pretreatment with paricalcitol or vitaminD was able to prevent the upregulation of CCL18 in thesecells
4 Discussion
Peritonitis episodes constitute a frequent complication in PDpatients and recurrent peritonitis is a well-known clinicalrisk factor for peritoneal membrane dysfunction and EPS
There is an unmet requirement for biomarkers as toolsto identify patients who are at higher risk of techniquefailure to guide interventions that could improve clinicaloutcomes [32] Soluble factors released during inflammatoryprocesses such as IL-6 and IL-17 have been proposed asbiomarkers and mediators of peritoneal membrane deteri-oration (reviewed in [32 33]) However none are used inthe clinical routine and some cytokines such as IL-17 arenot easily measured in PD effluents in humans We proposeCCL18 as a biomarker of peritoneal membrane outcome inPD [22]
8 BioMed Research International
0
20
40
60
80
100
120
C
CL18
pro
duct
ion
Pari VV Pari(10minus8 M) (10minus9 M)
lowastp = 0156 lowastp = 0156
(a)
0
20
40
60
80
100
120
C
CL18
pro
duct
ion
Pari VV Pari(10minus8 M) (10minus9 M)
p = 0625
(b)
0
20
40
60
80
100
120
C
CL18
pro
duct
ion
Pari VV Pari(10minus8 M) (10minus9 M)
lowastp = 0313lowastlowastp = 0039
(c)
0
20
40
60
80
100
ParicalcitolVehicle Vit D
120
C
CL18
pro
duct
ion
(10minus8 M) (10minus8 M)
(d)
Figure 6 (andashc) Peritoneal macrophages isolated from patients experiencing peritonitis episodes were treated with vehicle or Zemplar indoses equivalent to 10minus8M and 10minus9M of paricalcitol during 24 h (a) 48 h (b) or 72 h (c) Equivalent cell cultures were established in theabsence of any treatment The concentration of CCL18 was evaluated in cell-free supernatants and the production of CCL18 in the presenceof vehicle or drug referred to the production in cell culture media alone Differences between cultures in the presence of paricalcitol or vehiclewere analyzed by theWilcoxon matched pairs rank test (d) Peritoneal macrophages were treated with paricalcitol as pure substance vitaminD or vehicle (ethanol) for 48 h
Given that high levels of the human chemokine CCL18have been shown to be related to peritoneal membranefailure and fibrosis [15 20 22] whereas sustained low CCL18peritoneal concentrations appear to be protective [22] weevaluated CCL18 levels in the peritoneum of patients under-going peritonitis episodes We found that the peritoneal con-centration of CCL18 was increased approximately 5-fold ineffluents from patients with infectious peritonitis comparedwith reported values in uninfected samples In a previousstudy involving a longitudinal analysis of 43 patients CCL18effluent concentrations ranged from 28 plusmn 16 ngml at thestart of dialysis to 34plusmn 18 ngml after 3 years of PD treatment[22] In a cross-sectional analysis of 61 patients treated formore than 3 years with PDmean effluent CCL18 values were395 plusmn 26 ngml [22] The high concentrations during IPare consistent with a previous finding showing that pMΦsfrom patients undergoing peritonitis or who recently had
peritonitis were able to secrete higher amounts of CCL18compared with pMΦs from uninfected patients [15]
Unexpectedly in a patient identified with sterileeosinophilic peritonitis the peritoneal effluent was extremelyrich in CCL18 with levels approximately 10-fold higher thanthose found in patients with IP and 50-fold more thanthose found in uninfected samples whereas effluent levelsof IL-6 which were evaluated in the same samples forcomparative purposes were similar on day 1 in both theIP and EP samples The differences between a peritonealinflammatory recognized marker IL-6 and CCL18 duringperitonitis episodes should be remarked It is of note thatthe peritoneal concentrations of CCL18 remained elevatedfor several days whereas there was a sharp decrease in IL-6Both IL-6 and CCL18 have been reported to increase indifferent inflammatory conditions some leading to tissuefibrosis [34ndash37] As a main difference although CCL18 is
BioMed Research International 9
NEG
CCL1
8 (p
gm
l)
0
200
400
600
800
1000
12001057
522
Pari (10minus7 M) NEG000
020
040
060
080
100
120100
025Fold
mRN
A C
CL18
Pari (10minus7 M)
(a)
NEG
408
279
0
100
200
300
400
500
CCL1
8 (p
gm
l)
Pari (10minus7 M)
100
045
000
050
100
150
NEG
Fold
mRN
A C
CL18
Pari (10minus7 M)
(b)
000
025
050
075
100
125
Fold
mRN
A C
CL18
NEG
Mo + IL-4
Pari Vit D3Vmdash
(c)
Figure 7 (a) Peritoneal macrophages from a patient with recent infectious peritonitis were treated with paricalcitol or vehicle for 24 h Theprotein concentration of CCL18 was evaluated in cell culture supernatants by ELISA and CCL18 mRNA levels were estimated by quantitativeRT-PCR in the same cultures (b) Peritoneal macrophages from an uninfected peritoneum were isolated from the peritoneal lavage uponcatheter insertion and treated as in (a) (c) Peripheral bloodmonocytes were pretreated with vehicle (V) 10minus7Mparicalcitol (Pari) or vitaminD3 for 24 h The cells were treated thereafter with IL-4 for 3 days before RNA extraction and evaluation expression of CCL18 mRNA levelsby quantitative RT-PCR Mean plusmn SD values from four independent experiments are shown
primarily produced by cells of myeloid origin multiplecell types are producers of IL-6 with endothelial cellsbeing an important source of this cytokine Infiltratingneutrophils as well as mesothelial cells could also be animportant source of IL-6 during bacterial peritonitis [37]The analysis of serial samples in four patients with IP showedthat the concentrations of CCL18 paralleled the numbers ofmacrophages infiltrating the peritoneum and are consistentwith previous descriptions of macrophages andmononuclearphagocytes as the primary producers of CCL18 [21] Cellcounts in the serial samples from our case of EP suggest that
the peritoneal eosinophilic infiltrate was likely contributingto the peritoneal production of CCL18 Evidences of CCL18production by activated peritoneal eosinophils in this caseare cytospin analysis of CCL18 cytoplasmic staining ineosinophils and gene expression data in polymorphonuclearleukocytes among which 85 were eosinophils
Although macrophages are typically reported as themajor source of this chemokine [21] CCL18 protein pro-duction by eosinophils has occasionally been described asbeing related to the activation status of these cells given thatCCL18 was found only in eosinophil culture supernatants
10 BioMed Research International
from eosinophilic donors but not from individuals withnonactivated eosinophils [29] Our data support the activatedstatus of eosinophils in EP given that strong staining ofCC18 was detected in peritoneal eosinophils Eosinophilshave also been related to tissue fibrosis in several diseasesand are a source of several profibrotic mediators that alterfibroblast activity and tissue remodeling [38 39] Some ofthesemediators could also stimulate the profibrotic activity ofpMΦTheproduction ofCCL18 by eosinophils in EPprovidesa new additional explanation for the association of recurrentEP during PD with UFF and potentially peritoneal fibrosis[40]
As part of its profibrotic activity CCL18 has been shownto stimulate collagen deposition [41] and fibroblast prolif-eration [42] In consonance with these actions circulatingCCL18 levels have been demonstrated to be a biomarker fordisease activity and outcome in various fibrotic diseases [1943 44] as well as in some eosinophilic disorders [30 45 46]Moreover CCL18 has been shown to promote EMT in variouscell systems [47ndash50] It is thus possible that it could alsocontribute to the EMT of mesothelial cells which has beenproposed as one of the mechanisms leading to peritonealfibrosis and membrane failure [12]
In terms of peritoneal fibrosis CCL18 has been demon-strated to herald the development of peritoneal functionaldeterioration in PD patients [15 20] in whom the abilityof peritoneal macrophages to stimulate fibroblast prolifer-ation is correlated with CCL18 mRNA levels Moreoverrecombinant CCL18 was able to stimulate the proliferation ofperitoneal fibroblasts [15]
Several molecules have been tested for their ability toprevent peritoneal fibrosis in vitro and in animal models(reviewed in [8]) Among them paricalcitol is a selectiveVDR agonist VDR is a nuclear hormone receptor expressedin various cell types including those of the immune sys-tem VDR agonists have been shown to modulate immuneresponses inflammation and fibrosis [51]
Considering the role played by inflammation in peri-toneal fibrosis during PDwe studied the effects of paricalcitolin the production of CCL18 We found that the release andgene expression of CCL18 were downregulated in pMΦsand in in vitro-differentiated human M2 cells which are theprimary producers of CCL18These results appear to contrastwith previous data showing upregulation of CCL18 by vita-min D in monocyte-derived immature dendritic cells [52]However recent reports demonstrate that the modulationof immune signaling by vitamin D in human mononuclearphagocytes is cell-type-specific with differential responses inmonocytes macrophages and dendritic cells [53] It is thuspossible that opposite effects are found on the regulation ofCCL18 by VDR agonists in different cellular systems
On the other hand in a mouse model of PD intraperi-toneal administration of paricalcitol was shown to reduceperitoneal fibrosis and ultrafiltration failure [23] In thatmodel the beneficial effects appeared to be dependent ona reduction of intraperitoneal concentrations of IL-17 Thelack of rodent orthologues of human CCL18 [28] precludesthe investigation of CCL18 and its involvement in fibrosis inmurine models On the other hand IL-17 is highly diluted
in human peritoneal effluents The need to concentrate thesamples for measurement of this cytokine hampers accuratedeterminations in human samples
A preliminary study involving 23 patients on PD suggeststhat paricalcitol could improve the ultrafiltration capacitywith less peritoneal protein loss [24] The authors suggestan effect of VDR upon the renin-angiotensin-aldosteronesystem but it is tempting to speculate that lower peritonealconcentrations of CCL18 might be present in paricalcitol-treated patients In this sense a slight reduction in theperitoneal concentration of CCL18 was observed in sixpatients who were more than 3 years on PD and treated withparicalcitol compared with a set of 31 patients of the samevintage (data not shown)
5 Conclusion
In conclusion our study suggests that the chemokine CCL18can be a mediator of peritoneal membrane failure associatedwith peritonitis episodes and might provide a new potentialtherapeutic target
Further clinical studies are needed to confirm the useful-ness of VDR agonists to preserve the peritoneal membraneand to analyze the putative mechanisms involved
Conflicts of Interest
All the authors have no potential conflicts of interest todeclare
Authorsrsquo Contributions
Marta Ossorio and Virginia Martınez contributed equally tothis work
Acknowledgments
This study has been partially supported by ISCIII (ldquoInsti-tuto de Investigacion Sanitaria Carlos IIIrdquo) Grants FIS PI1200204 PI1301768 and PI 1500120 FEDER (Funds fromthe European Union) REDinREN 20 FEDER and ReinaSofıa Institute of Nephrology Research (IRSIN) monetaryfunds The authors would like to thank Juliette Siegfriedand her team at ServingMedcom for their editing of themanuscript
Supplementary Materials
Supplemental Figure 1 serial cytokine concentrations andcell counts in peritoneal effluent during infectious peritoni-tis and eosinophilic peritonitis (A) Comparison of CCL18concentration in serial effluent samples from infectiousperitonitis (IP) and eosinophilic peritonitis (EP) (B) IL-6effluent levels in serial samples from IP and EP D1ndashD26indicate days of evolution Supplemental Figure 2 dose-dependent effects of VDR agonists on cell viability Cellviability of human monocytes was assessed by exclusion ofpropidium iodide staining and analyzed by flow cytometry
BioMed Research International 11
Results are shown as mean plusmn SD values of triplicates(A)ndash(C) Viability of monocytes upon culture during 24 48or 72 hours with decreasing concentrations of Zemplar orequivalent vehicle (305020 propylene glycolwaterethanol)(D)ndash(F) Viability of monocytes upon culture during 2448 or 72 hours with different concentrations of paricalcitol(pure substance) vitamin D or equivalent vehicle (EtOH)(Supplementary Materials)
References
[1] R T Krediet ldquoThe peritoneal membrane in chronic peritonealdialysisrdquo Kidney International vol 55 no 1 pp 341ndash356 1999
[2] R T Krediet ldquoPeritoneal physiology - Impact on solute andfluid clearancerdquo Advances in Chronic Kidney Disease vol 7 no4 pp 271ndash279 2000
[3] M Merkle and M Wrnle ldquoSclerosing peritonitis A rare butfatal complication of peritoneal inflammationrdquo Mediators ofInflammation vol 2012 Article ID 709673 p 4 2012
[4] D W Johnson Y Cho B E R Livingston et al ldquoEncapsulat-ing peritoneal sclerosis incidence predictors and outcomesrdquoKidney International vol 77 no 10 pp 904ndash912 2010
[5] G Balasubramaniam E A Brown and A Davenport ldquoThePan-Thames EPS study treatment and outcomes of encapsulat-ing peritoneal sclerosisrdquo Nephrology Dialysis Transplantation vol 24 no 10 pp 3209ndash3215 2009
[6] E D Sousa-Amorim G D Peso M A Bajo et al ldquoCan EPSdevelopment be avoided with early interventionsThe potentialrole of tamoxifenmdasha single-center studyrdquo Peritoneal DialysisInternational vol 34 no 6 pp 582ndash593 2014
[7] M R Korte D E SampimonM GH Betjes and R T KredietldquoEncapsulating peritoneal sclerosis the state of affairsrdquo NatureReviews Nephrology vol 7 no 9 pp 528ndash538 2011
[8] M A Bajo G del Peso and I Teitelbaum ldquoPeritoneal Mem-brane Preservationrdquo Seminars in Nephrology vol 37 no 1 pp77ndash92 2017
[9] M F Flessner ldquoInflammation from sterile dialysis solutions andthe longevity of the peritoneal barrierrdquo Clinical Nephrology vol68 no 6 pp 341ndash348 2007
[10] K N Lai and J C K Leung ldquoInflammation in peritonealdialysisrdquo Nephron Clinical Practice vol 116 no 1 pp c11ndashc182010
[11] S Yung and T M Chan ldquoPathophysiological changes to theperitoneal membrane during PD-related peritonitis the role ofmesothelial cellsrdquo Mediators of Inflammation vol 2012 ArticleID 484167 21 pages 2012
[12] L S Aroeira A Aguilera J A Sanchez-Tomero et al ldquoEpithe-lial tomesenchymal transition and peritonealmembrane failurein peritoneal dialysis patients pathologic significance andpotential therapeutic interventionsrdquo Journal of the AmericanSociety of Nephrology vol 18 no 7 pp 2004ndash2013 2007
[13] N Topley T Liberek A Davenport F-K Li H Fear andJ D Williams ldquoActivation of inflammation and leukocyterecruitment into the peritoneal cavityrdquo Kidney InternationalSupplements vol 50 no 56 pp S17ndashS21 1996
[14] F O Martınez and S Gordon ldquoThe M1 and M2 paradigmof macrophage activation time for reassessmentrdquo F1000PrimeReports vol 6 article 13 2014
[15] T Bellon V Martınez B Lucendo et al ldquoAlternative activa-tion of macrophages in human peritoneum implications for
peritoneal fibrosisrdquo Nephrology Dialysis Transplantation vol26 no 9 pp 2995ndash3005 2011
[16] A Prasse D V Pechkovsky G B Toews et al ldquoA vicious circleof alveolar macrophages and fibroblasts perpetuates pulmonaryfibrosis via CCL18rdquoAmerican Journal of Respiratory and CriticalCare Medicine vol 173 no 7 pp 781ndash792 2006
[17] J M Estep A Baranova N Hossain et al ldquoExpression ofcytokine signaling genes in morbidly obese patients with non-alcoholic steatohepatitis and hepatic fibrosisrdquo Obesity Surgeryvol 19 no 5 pp 617ndash624 2009
[18] F Kollert M Binder C Probst et al ldquoCCL18 - Potentialbiomarker of fibroinflammatory activity in chronic periaortitisrdquoThe Journal of Rheumatology vol 39 no 7 pp 1407ndash1412 2012
[19] A Prasse D V Pechkovsky G B Toews et al ldquoCCL18 as anindicator of pulmonary fibrotic activity in idiopathic interstitialpneumonias and systemic sclerosisrdquo Arthritis amp Rheumatologyvol 56 no 5 pp 1685ndash1693 2007
[20] S Ahmad B V North A Qureshi et al ldquoCCL18 in peritonealdialysis patients and encapsulating peritoneal sclerosisrdquo Euro-pean Journal of Clinical Investigation vol 40 no 12 pp 1067ndash1073 2010
[21] V Kodelja C Muller O Politz N Hakij C E Orfanos andS Goerdt ldquoAlternative macrophage activation-associated CC-chemokine-1 a novel structural homologue of macrophageinflammatory protein-1120572 with a Th2- associated expressionpatternrdquo The Journal of Immunology vol 160 no 3 pp 1411ndash1418 1998
[22] M Ossorio M A Bajo G Del Peso et al ldquoSustained low peri-toneal effluent CCL18 levels are associated with preservationof peritoneal membrane function in peritoneal dialysisrdquo PLoSONE vol 12 no 4 Article ID e0175835 2017
[23] G T Gonzalez-Mateo V Fernandez-Mıllara T Bellon etal ldquoParicalcitol reduces peritoneal fibrosis in mice throughthe activation of regulatory T cells and reduction in IL-17productionrdquo PLoS ONE vol 9 no 10 Article ID e108477 2014
[24] F Coronel S Cigarran A Gomis et al ldquoChanges in peritonealmembrane permeability and proteinuria in patients on peri-toneal dialysis after treatment with paricalcitolmdasha preliminarystudyrdquo Clinical Nephrology vol 78 no 2 pp 93ndash99 2012
[25] E Song N Ouyang M Horbelt B Antus M Wang and MS Exton ldquoInfluence of alternatively and classically activatedmacrophages on fibrogenic activities of human fibroblastsrdquoCellular Immunology vol 204 no 1 pp 19ndash28 2000
[26] S M Hurst T S Wilkinson R M McLoughlin et al ldquoIL-6 andits soluble receptor orchestrate a temporal switch in the patternof leukocyte recruitment seen during acute inflammationrdquoImmunity vol 14 no 6 pp 705ndash714 2001
[27] M Kozioł-Montewka A Ksiazek L Janicka and I BaranowiczldquoSerial cytokine changes in peritoneal effluent and plasma dur-ing peritonitis in patients on continuous ambulatory peritonealdialysis (CAPD)rdquo Inflammation Research vol 46 no 4 pp 132ndash136 1997
[28] E Schutyser A Richmond and J Van Damme ldquoInvolvementof CC chemokine ligand 18 (CCL18) in normal and pathologicalprocessesrdquo Journal of Leukocyte Biology vol 78 no 1 pp 14ndash262005
[29] I Schraufstatter H Takamori L Sikora P Sriramarao and RG DiScipio ldquoEosinophils and monocytes produce pulmonaryand activation-regulated chemokine which activates culturedmonocytesmacrophagesrdquoAmerican Journal of Physiology-LungCellular and Molecular Physiology vol 286 no 3 pp L494ndashL501 2004
12 BioMed Research International
[30] C Gunther N Carballido-Perrig T Kopp J M Carballidoand C Pfeiffer ldquoCCL18 is expressed in patients with bullouspemphigoid and parallels disease courserdquo British Journal ofDermatology vol 160 no 4 pp 747ndash755 2009
[31] AMantovani A Sica S Sozzani P Allavena A Vecchi andMLocati ldquoThe chemokine system in diverse forms of macrophageactivation and polarizationrdquo Trends in Immunology vol 25 no12 pp 677ndash686 2004
[32] C Aufricht R Beelen M Eberl et al ldquoBiomarker researchto improve clinical outcomes of peritoneal dialysis consensusof the European Training and Research in Peritoneal Dialysis(EuTRiPD) networkrdquo Kidney International vol 92 no 4 pp824ndash835 2017
[33] D Lopes Barreto andR T Krediet ldquoCurrent status and practicaluse of effluent biomarkers in peritoneal dialysis patientsrdquoAmerican Journal of Kidney Diseases vol 62 no 4 pp 823ndash8332013
[34] L A Boven M van Meurs R G Boot et al ldquoGaucher cellsdemonstrate a distinct macrophage phenotype and resemblealternatively activated macrophagesrdquo American Journal of Clin-ical Pathology vol 122 no 3 pp 359ndash369 2004
[35] L Cocho I Fernandez M Calonge et al ldquoGene expression-based predictive models of graft versus host disease-associateddry eyerdquo Investigative Ophtalmology Visual Science vol 56 no8 pp 4570ndash4581 2015
[36] MHasegawa ldquoBiomarkers in systemic sclerosisTheir potentialto predict clinical coursesrdquoThe Journal of Dermatology vol 43no 1 pp 29ndash38 2016
[37] S A Jones D J Fraser C A Fielding and G W JonesldquoInterleukin-6 in renal disease and therapyrdquoNephrologyDialysisTransplantation vol 30 no 4 pp 564ndash574 2015
[38] A Munitz and F Levi-Schaffer ldquoEosinophils rsquoNewrsquo roles forrsquooldrsquo cellsrdquo Allergy European Journal of Allergy and ClinicalImmunology vol 59 no 3 pp 268ndash275 2004
[39] S S Aceves ldquoRemodeling and fibrosis in chronic eosinophilinflammationrdquo Digestive Diseases vol 32 no 1-2 pp 15ndash212014
[40] Y Nakamura H Okada A Yasui T Koh and T YamaneldquoSclerosing encapsulating peritonitis associated with recurrenteosinophilic peritonitisrdquo Nephrology Dialysis Transplantation vol 14 no 3 pp 768ndash770 1999
[41] S P Atamas I G Luzina J Choi et al ldquoPulmonary andactivation-regulated chemokine stimulates collagen productionin lung fibroblastsrdquo American Journal of Respiratory Cell andMolecular Biology vol 29 no 6 pp 743ndash749 2003
[42] A Wimmer S K Khaldoyanidi M Judex N Serobyan RG DiScipio and I U Schraufstatter ldquoCCL18PARC stimulateshematopoiesis in long-term bone marrow cultures indirectlythrough its effect on monocytesrdquo Blood vol 108 no 12 pp3722ndash3729 2006
[43] A Prasse C Probst E Bargagli et al ldquoSerum CC-chemokineligand 18 concentration predicts outcome in idiopathic pul-monary fibrosisrdquo American Journal of Respiratory and CriticalCare Medicine vol 179 no 8 pp 717ndash723 2009
[44] J Chen Y Yao C Gong et al ldquoCCL18 from tumor-associatedmacrophages promotes breast cancermetastasis via PITPNM3rdquoCancer Cell vol 19 no 4 pp 541ndash555 2011
[45] K L Hon G K Ching P C Ng and T F Leung ldquoExplor-ing CCL18 eczema severity and atopyrdquo Pediatric Allergy andImmunology vol 22 no 7 pp 704ndash707 2011
[46] A J Lucendo A Arias L C De Rezende et al ldquoSubepithelialcollagen deposition profibrogenic cytokine gene expressionand changes after prolonged fluticasone propionate treatment inadult eosinophilic esophagitis a prospective studyrdquoThe Journalof Allergy and Clinical Immunology vol 128 no 5 pp 1037ndash1046 2011
[47] H Li D Zhang J Yu et al ldquoCCL18-dependent translocationof AMAP1 is critical for epithelial to mesenchymal transition inbreast cancerrdquo Journal of Cellular Physiology vol 233 no 4 pp3207ndash3217 2017
[48] Z Lin W Li H Zhang et al ldquoCCL18PITPNM3 enhancesmigration invasion and EMT through the NF-120581B signalingpathway in hepatocellular carcinomardquo Tumor Biology vol 37no 3 pp 3461ndash3468 2016
[49] F Meng W Li C Li Z Gao K Guo and S SongldquoCCL18 promotes epithelial-mesenchymal transition invasionand migration of pancreatic cancer cells in pancreatic ductaladenocarcinomardquo International Journal of Oncology vol 46 no3 pp 1109ndash1120 2015
[50] T Ploenes B Scholtes A Krohn et al ldquoCC-Chemokine Ligand18 Induces Epithelial to Mesenchymal Transition in LungCancer A549 Cells and Elevates the Invasive Potentialrdquo PLoSONE vol 8 no 1 Article ID e53068 2013
[51] J Rojas-Rivera C De La Piedra A Ramos A Ortiz andJ Egido ldquoThe expanding spectrum of biological actions ofvitamin Drdquo Nephrology Dialysis Transplantation vol 25 no 9pp 2850ndash2865 2010
[52] M Vulcano S Struyf P Scapini et al ldquoUnique regulation ofCCL18 production by maturing dendritic cellsrdquo The Journal ofImmunology vol 170 no 7 pp 3843ndash3849 2003
[53] R Kundu A Theodoraki C T Haas et al ldquoCell-type-specificmodulation of innate immune signalling by vitamin D inhuman mononuclear phagocytesrdquo The Journal of Immunologyvol 150 no 1 pp 55ndash63 2017
Stem Cells International
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Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
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Hindawiwwwhindawicom Volume 2018
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Disease Markers
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BioMed Research International
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Oxidative Medicine and Cellular Longevity
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Hindawiwwwhindawicom Volume 2018
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Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
8 BioMed Research International
0
20
40
60
80
100
120
C
CL18
pro
duct
ion
Pari VV Pari(10minus8 M) (10minus9 M)
lowastp = 0156 lowastp = 0156
(a)
0
20
40
60
80
100
120
C
CL18
pro
duct
ion
Pari VV Pari(10minus8 M) (10minus9 M)
p = 0625
(b)
0
20
40
60
80
100
120
C
CL18
pro
duct
ion
Pari VV Pari(10minus8 M) (10minus9 M)
lowastp = 0313lowastlowastp = 0039
(c)
0
20
40
60
80
100
ParicalcitolVehicle Vit D
120
C
CL18
pro
duct
ion
(10minus8 M) (10minus8 M)
(d)
Figure 6 (andashc) Peritoneal macrophages isolated from patients experiencing peritonitis episodes were treated with vehicle or Zemplar indoses equivalent to 10minus8M and 10minus9M of paricalcitol during 24 h (a) 48 h (b) or 72 h (c) Equivalent cell cultures were established in theabsence of any treatment The concentration of CCL18 was evaluated in cell-free supernatants and the production of CCL18 in the presenceof vehicle or drug referred to the production in cell culture media alone Differences between cultures in the presence of paricalcitol or vehiclewere analyzed by theWilcoxon matched pairs rank test (d) Peritoneal macrophages were treated with paricalcitol as pure substance vitaminD or vehicle (ethanol) for 48 h
Given that high levels of the human chemokine CCL18have been shown to be related to peritoneal membranefailure and fibrosis [15 20 22] whereas sustained low CCL18peritoneal concentrations appear to be protective [22] weevaluated CCL18 levels in the peritoneum of patients under-going peritonitis episodes We found that the peritoneal con-centration of CCL18 was increased approximately 5-fold ineffluents from patients with infectious peritonitis comparedwith reported values in uninfected samples In a previousstudy involving a longitudinal analysis of 43 patients CCL18effluent concentrations ranged from 28 plusmn 16 ngml at thestart of dialysis to 34plusmn 18 ngml after 3 years of PD treatment[22] In a cross-sectional analysis of 61 patients treated formore than 3 years with PDmean effluent CCL18 values were395 plusmn 26 ngml [22] The high concentrations during IPare consistent with a previous finding showing that pMΦsfrom patients undergoing peritonitis or who recently had
peritonitis were able to secrete higher amounts of CCL18compared with pMΦs from uninfected patients [15]
Unexpectedly in a patient identified with sterileeosinophilic peritonitis the peritoneal effluent was extremelyrich in CCL18 with levels approximately 10-fold higher thanthose found in patients with IP and 50-fold more thanthose found in uninfected samples whereas effluent levelsof IL-6 which were evaluated in the same samples forcomparative purposes were similar on day 1 in both theIP and EP samples The differences between a peritonealinflammatory recognized marker IL-6 and CCL18 duringperitonitis episodes should be remarked It is of note thatthe peritoneal concentrations of CCL18 remained elevatedfor several days whereas there was a sharp decrease in IL-6Both IL-6 and CCL18 have been reported to increase indifferent inflammatory conditions some leading to tissuefibrosis [34ndash37] As a main difference although CCL18 is
BioMed Research International 9
NEG
CCL1
8 (p
gm
l)
0
200
400
600
800
1000
12001057
522
Pari (10minus7 M) NEG000
020
040
060
080
100
120100
025Fold
mRN
A C
CL18
Pari (10minus7 M)
(a)
NEG
408
279
0
100
200
300
400
500
CCL1
8 (p
gm
l)
Pari (10minus7 M)
100
045
000
050
100
150
NEG
Fold
mRN
A C
CL18
Pari (10minus7 M)
(b)
000
025
050
075
100
125
Fold
mRN
A C
CL18
NEG
Mo + IL-4
Pari Vit D3Vmdash
(c)
Figure 7 (a) Peritoneal macrophages from a patient with recent infectious peritonitis were treated with paricalcitol or vehicle for 24 h Theprotein concentration of CCL18 was evaluated in cell culture supernatants by ELISA and CCL18 mRNA levels were estimated by quantitativeRT-PCR in the same cultures (b) Peritoneal macrophages from an uninfected peritoneum were isolated from the peritoneal lavage uponcatheter insertion and treated as in (a) (c) Peripheral bloodmonocytes were pretreated with vehicle (V) 10minus7Mparicalcitol (Pari) or vitaminD3 for 24 h The cells were treated thereafter with IL-4 for 3 days before RNA extraction and evaluation expression of CCL18 mRNA levelsby quantitative RT-PCR Mean plusmn SD values from four independent experiments are shown
primarily produced by cells of myeloid origin multiplecell types are producers of IL-6 with endothelial cellsbeing an important source of this cytokine Infiltratingneutrophils as well as mesothelial cells could also be animportant source of IL-6 during bacterial peritonitis [37]The analysis of serial samples in four patients with IP showedthat the concentrations of CCL18 paralleled the numbers ofmacrophages infiltrating the peritoneum and are consistentwith previous descriptions of macrophages andmononuclearphagocytes as the primary producers of CCL18 [21] Cellcounts in the serial samples from our case of EP suggest that
the peritoneal eosinophilic infiltrate was likely contributingto the peritoneal production of CCL18 Evidences of CCL18production by activated peritoneal eosinophils in this caseare cytospin analysis of CCL18 cytoplasmic staining ineosinophils and gene expression data in polymorphonuclearleukocytes among which 85 were eosinophils
Although macrophages are typically reported as themajor source of this chemokine [21] CCL18 protein pro-duction by eosinophils has occasionally been described asbeing related to the activation status of these cells given thatCCL18 was found only in eosinophil culture supernatants
10 BioMed Research International
from eosinophilic donors but not from individuals withnonactivated eosinophils [29] Our data support the activatedstatus of eosinophils in EP given that strong staining ofCC18 was detected in peritoneal eosinophils Eosinophilshave also been related to tissue fibrosis in several diseasesand are a source of several profibrotic mediators that alterfibroblast activity and tissue remodeling [38 39] Some ofthesemediators could also stimulate the profibrotic activity ofpMΦTheproduction ofCCL18 by eosinophils in EPprovidesa new additional explanation for the association of recurrentEP during PD with UFF and potentially peritoneal fibrosis[40]
As part of its profibrotic activity CCL18 has been shownto stimulate collagen deposition [41] and fibroblast prolif-eration [42] In consonance with these actions circulatingCCL18 levels have been demonstrated to be a biomarker fordisease activity and outcome in various fibrotic diseases [1943 44] as well as in some eosinophilic disorders [30 45 46]Moreover CCL18 has been shown to promote EMT in variouscell systems [47ndash50] It is thus possible that it could alsocontribute to the EMT of mesothelial cells which has beenproposed as one of the mechanisms leading to peritonealfibrosis and membrane failure [12]
In terms of peritoneal fibrosis CCL18 has been demon-strated to herald the development of peritoneal functionaldeterioration in PD patients [15 20] in whom the abilityof peritoneal macrophages to stimulate fibroblast prolifer-ation is correlated with CCL18 mRNA levels Moreoverrecombinant CCL18 was able to stimulate the proliferation ofperitoneal fibroblasts [15]
Several molecules have been tested for their ability toprevent peritoneal fibrosis in vitro and in animal models(reviewed in [8]) Among them paricalcitol is a selectiveVDR agonist VDR is a nuclear hormone receptor expressedin various cell types including those of the immune sys-tem VDR agonists have been shown to modulate immuneresponses inflammation and fibrosis [51]
Considering the role played by inflammation in peri-toneal fibrosis during PDwe studied the effects of paricalcitolin the production of CCL18 We found that the release andgene expression of CCL18 were downregulated in pMΦsand in in vitro-differentiated human M2 cells which are theprimary producers of CCL18These results appear to contrastwith previous data showing upregulation of CCL18 by vita-min D in monocyte-derived immature dendritic cells [52]However recent reports demonstrate that the modulationof immune signaling by vitamin D in human mononuclearphagocytes is cell-type-specific with differential responses inmonocytes macrophages and dendritic cells [53] It is thuspossible that opposite effects are found on the regulation ofCCL18 by VDR agonists in different cellular systems
On the other hand in a mouse model of PD intraperi-toneal administration of paricalcitol was shown to reduceperitoneal fibrosis and ultrafiltration failure [23] In thatmodel the beneficial effects appeared to be dependent ona reduction of intraperitoneal concentrations of IL-17 Thelack of rodent orthologues of human CCL18 [28] precludesthe investigation of CCL18 and its involvement in fibrosis inmurine models On the other hand IL-17 is highly diluted
in human peritoneal effluents The need to concentrate thesamples for measurement of this cytokine hampers accuratedeterminations in human samples
A preliminary study involving 23 patients on PD suggeststhat paricalcitol could improve the ultrafiltration capacitywith less peritoneal protein loss [24] The authors suggestan effect of VDR upon the renin-angiotensin-aldosteronesystem but it is tempting to speculate that lower peritonealconcentrations of CCL18 might be present in paricalcitol-treated patients In this sense a slight reduction in theperitoneal concentration of CCL18 was observed in sixpatients who were more than 3 years on PD and treated withparicalcitol compared with a set of 31 patients of the samevintage (data not shown)
5 Conclusion
In conclusion our study suggests that the chemokine CCL18can be a mediator of peritoneal membrane failure associatedwith peritonitis episodes and might provide a new potentialtherapeutic target
Further clinical studies are needed to confirm the useful-ness of VDR agonists to preserve the peritoneal membraneand to analyze the putative mechanisms involved
Conflicts of Interest
All the authors have no potential conflicts of interest todeclare
Authorsrsquo Contributions
Marta Ossorio and Virginia Martınez contributed equally tothis work
Acknowledgments
This study has been partially supported by ISCIII (ldquoInsti-tuto de Investigacion Sanitaria Carlos IIIrdquo) Grants FIS PI1200204 PI1301768 and PI 1500120 FEDER (Funds fromthe European Union) REDinREN 20 FEDER and ReinaSofıa Institute of Nephrology Research (IRSIN) monetaryfunds The authors would like to thank Juliette Siegfriedand her team at ServingMedcom for their editing of themanuscript
Supplementary Materials
Supplemental Figure 1 serial cytokine concentrations andcell counts in peritoneal effluent during infectious peritoni-tis and eosinophilic peritonitis (A) Comparison of CCL18concentration in serial effluent samples from infectiousperitonitis (IP) and eosinophilic peritonitis (EP) (B) IL-6effluent levels in serial samples from IP and EP D1ndashD26indicate days of evolution Supplemental Figure 2 dose-dependent effects of VDR agonists on cell viability Cellviability of human monocytes was assessed by exclusion ofpropidium iodide staining and analyzed by flow cytometry
BioMed Research International 11
Results are shown as mean plusmn SD values of triplicates(A)ndash(C) Viability of monocytes upon culture during 24 48or 72 hours with decreasing concentrations of Zemplar orequivalent vehicle (305020 propylene glycolwaterethanol)(D)ndash(F) Viability of monocytes upon culture during 2448 or 72 hours with different concentrations of paricalcitol(pure substance) vitamin D or equivalent vehicle (EtOH)(Supplementary Materials)
References
[1] R T Krediet ldquoThe peritoneal membrane in chronic peritonealdialysisrdquo Kidney International vol 55 no 1 pp 341ndash356 1999
[2] R T Krediet ldquoPeritoneal physiology - Impact on solute andfluid clearancerdquo Advances in Chronic Kidney Disease vol 7 no4 pp 271ndash279 2000
[3] M Merkle and M Wrnle ldquoSclerosing peritonitis A rare butfatal complication of peritoneal inflammationrdquo Mediators ofInflammation vol 2012 Article ID 709673 p 4 2012
[4] D W Johnson Y Cho B E R Livingston et al ldquoEncapsulat-ing peritoneal sclerosis incidence predictors and outcomesrdquoKidney International vol 77 no 10 pp 904ndash912 2010
[5] G Balasubramaniam E A Brown and A Davenport ldquoThePan-Thames EPS study treatment and outcomes of encapsulat-ing peritoneal sclerosisrdquo Nephrology Dialysis Transplantation vol 24 no 10 pp 3209ndash3215 2009
[6] E D Sousa-Amorim G D Peso M A Bajo et al ldquoCan EPSdevelopment be avoided with early interventionsThe potentialrole of tamoxifenmdasha single-center studyrdquo Peritoneal DialysisInternational vol 34 no 6 pp 582ndash593 2014
[7] M R Korte D E SampimonM GH Betjes and R T KredietldquoEncapsulating peritoneal sclerosis the state of affairsrdquo NatureReviews Nephrology vol 7 no 9 pp 528ndash538 2011
[8] M A Bajo G del Peso and I Teitelbaum ldquoPeritoneal Mem-brane Preservationrdquo Seminars in Nephrology vol 37 no 1 pp77ndash92 2017
[9] M F Flessner ldquoInflammation from sterile dialysis solutions andthe longevity of the peritoneal barrierrdquo Clinical Nephrology vol68 no 6 pp 341ndash348 2007
[10] K N Lai and J C K Leung ldquoInflammation in peritonealdialysisrdquo Nephron Clinical Practice vol 116 no 1 pp c11ndashc182010
[11] S Yung and T M Chan ldquoPathophysiological changes to theperitoneal membrane during PD-related peritonitis the role ofmesothelial cellsrdquo Mediators of Inflammation vol 2012 ArticleID 484167 21 pages 2012
[12] L S Aroeira A Aguilera J A Sanchez-Tomero et al ldquoEpithe-lial tomesenchymal transition and peritonealmembrane failurein peritoneal dialysis patients pathologic significance andpotential therapeutic interventionsrdquo Journal of the AmericanSociety of Nephrology vol 18 no 7 pp 2004ndash2013 2007
[13] N Topley T Liberek A Davenport F-K Li H Fear andJ D Williams ldquoActivation of inflammation and leukocyterecruitment into the peritoneal cavityrdquo Kidney InternationalSupplements vol 50 no 56 pp S17ndashS21 1996
[14] F O Martınez and S Gordon ldquoThe M1 and M2 paradigmof macrophage activation time for reassessmentrdquo F1000PrimeReports vol 6 article 13 2014
[15] T Bellon V Martınez B Lucendo et al ldquoAlternative activa-tion of macrophages in human peritoneum implications for
peritoneal fibrosisrdquo Nephrology Dialysis Transplantation vol26 no 9 pp 2995ndash3005 2011
[16] A Prasse D V Pechkovsky G B Toews et al ldquoA vicious circleof alveolar macrophages and fibroblasts perpetuates pulmonaryfibrosis via CCL18rdquoAmerican Journal of Respiratory and CriticalCare Medicine vol 173 no 7 pp 781ndash792 2006
[17] J M Estep A Baranova N Hossain et al ldquoExpression ofcytokine signaling genes in morbidly obese patients with non-alcoholic steatohepatitis and hepatic fibrosisrdquo Obesity Surgeryvol 19 no 5 pp 617ndash624 2009
[18] F Kollert M Binder C Probst et al ldquoCCL18 - Potentialbiomarker of fibroinflammatory activity in chronic periaortitisrdquoThe Journal of Rheumatology vol 39 no 7 pp 1407ndash1412 2012
[19] A Prasse D V Pechkovsky G B Toews et al ldquoCCL18 as anindicator of pulmonary fibrotic activity in idiopathic interstitialpneumonias and systemic sclerosisrdquo Arthritis amp Rheumatologyvol 56 no 5 pp 1685ndash1693 2007
[20] S Ahmad B V North A Qureshi et al ldquoCCL18 in peritonealdialysis patients and encapsulating peritoneal sclerosisrdquo Euro-pean Journal of Clinical Investigation vol 40 no 12 pp 1067ndash1073 2010
[21] V Kodelja C Muller O Politz N Hakij C E Orfanos andS Goerdt ldquoAlternative macrophage activation-associated CC-chemokine-1 a novel structural homologue of macrophageinflammatory protein-1120572 with a Th2- associated expressionpatternrdquo The Journal of Immunology vol 160 no 3 pp 1411ndash1418 1998
[22] M Ossorio M A Bajo G Del Peso et al ldquoSustained low peri-toneal effluent CCL18 levels are associated with preservationof peritoneal membrane function in peritoneal dialysisrdquo PLoSONE vol 12 no 4 Article ID e0175835 2017
[23] G T Gonzalez-Mateo V Fernandez-Mıllara T Bellon etal ldquoParicalcitol reduces peritoneal fibrosis in mice throughthe activation of regulatory T cells and reduction in IL-17productionrdquo PLoS ONE vol 9 no 10 Article ID e108477 2014
[24] F Coronel S Cigarran A Gomis et al ldquoChanges in peritonealmembrane permeability and proteinuria in patients on peri-toneal dialysis after treatment with paricalcitolmdasha preliminarystudyrdquo Clinical Nephrology vol 78 no 2 pp 93ndash99 2012
[25] E Song N Ouyang M Horbelt B Antus M Wang and MS Exton ldquoInfluence of alternatively and classically activatedmacrophages on fibrogenic activities of human fibroblastsrdquoCellular Immunology vol 204 no 1 pp 19ndash28 2000
[26] S M Hurst T S Wilkinson R M McLoughlin et al ldquoIL-6 andits soluble receptor orchestrate a temporal switch in the patternof leukocyte recruitment seen during acute inflammationrdquoImmunity vol 14 no 6 pp 705ndash714 2001
[27] M Kozioł-Montewka A Ksiazek L Janicka and I BaranowiczldquoSerial cytokine changes in peritoneal effluent and plasma dur-ing peritonitis in patients on continuous ambulatory peritonealdialysis (CAPD)rdquo Inflammation Research vol 46 no 4 pp 132ndash136 1997
[28] E Schutyser A Richmond and J Van Damme ldquoInvolvementof CC chemokine ligand 18 (CCL18) in normal and pathologicalprocessesrdquo Journal of Leukocyte Biology vol 78 no 1 pp 14ndash262005
[29] I Schraufstatter H Takamori L Sikora P Sriramarao and RG DiScipio ldquoEosinophils and monocytes produce pulmonaryand activation-regulated chemokine which activates culturedmonocytesmacrophagesrdquoAmerican Journal of Physiology-LungCellular and Molecular Physiology vol 286 no 3 pp L494ndashL501 2004
12 BioMed Research International
[30] C Gunther N Carballido-Perrig T Kopp J M Carballidoand C Pfeiffer ldquoCCL18 is expressed in patients with bullouspemphigoid and parallels disease courserdquo British Journal ofDermatology vol 160 no 4 pp 747ndash755 2009
[31] AMantovani A Sica S Sozzani P Allavena A Vecchi andMLocati ldquoThe chemokine system in diverse forms of macrophageactivation and polarizationrdquo Trends in Immunology vol 25 no12 pp 677ndash686 2004
[32] C Aufricht R Beelen M Eberl et al ldquoBiomarker researchto improve clinical outcomes of peritoneal dialysis consensusof the European Training and Research in Peritoneal Dialysis(EuTRiPD) networkrdquo Kidney International vol 92 no 4 pp824ndash835 2017
[33] D Lopes Barreto andR T Krediet ldquoCurrent status and practicaluse of effluent biomarkers in peritoneal dialysis patientsrdquoAmerican Journal of Kidney Diseases vol 62 no 4 pp 823ndash8332013
[34] L A Boven M van Meurs R G Boot et al ldquoGaucher cellsdemonstrate a distinct macrophage phenotype and resemblealternatively activated macrophagesrdquo American Journal of Clin-ical Pathology vol 122 no 3 pp 359ndash369 2004
[35] L Cocho I Fernandez M Calonge et al ldquoGene expression-based predictive models of graft versus host disease-associateddry eyerdquo Investigative Ophtalmology Visual Science vol 56 no8 pp 4570ndash4581 2015
[36] MHasegawa ldquoBiomarkers in systemic sclerosisTheir potentialto predict clinical coursesrdquoThe Journal of Dermatology vol 43no 1 pp 29ndash38 2016
[37] S A Jones D J Fraser C A Fielding and G W JonesldquoInterleukin-6 in renal disease and therapyrdquoNephrologyDialysisTransplantation vol 30 no 4 pp 564ndash574 2015
[38] A Munitz and F Levi-Schaffer ldquoEosinophils rsquoNewrsquo roles forrsquooldrsquo cellsrdquo Allergy European Journal of Allergy and ClinicalImmunology vol 59 no 3 pp 268ndash275 2004
[39] S S Aceves ldquoRemodeling and fibrosis in chronic eosinophilinflammationrdquo Digestive Diseases vol 32 no 1-2 pp 15ndash212014
[40] Y Nakamura H Okada A Yasui T Koh and T YamaneldquoSclerosing encapsulating peritonitis associated with recurrenteosinophilic peritonitisrdquo Nephrology Dialysis Transplantation vol 14 no 3 pp 768ndash770 1999
[41] S P Atamas I G Luzina J Choi et al ldquoPulmonary andactivation-regulated chemokine stimulates collagen productionin lung fibroblastsrdquo American Journal of Respiratory Cell andMolecular Biology vol 29 no 6 pp 743ndash749 2003
[42] A Wimmer S K Khaldoyanidi M Judex N Serobyan RG DiScipio and I U Schraufstatter ldquoCCL18PARC stimulateshematopoiesis in long-term bone marrow cultures indirectlythrough its effect on monocytesrdquo Blood vol 108 no 12 pp3722ndash3729 2006
[43] A Prasse C Probst E Bargagli et al ldquoSerum CC-chemokineligand 18 concentration predicts outcome in idiopathic pul-monary fibrosisrdquo American Journal of Respiratory and CriticalCare Medicine vol 179 no 8 pp 717ndash723 2009
[44] J Chen Y Yao C Gong et al ldquoCCL18 from tumor-associatedmacrophages promotes breast cancermetastasis via PITPNM3rdquoCancer Cell vol 19 no 4 pp 541ndash555 2011
[45] K L Hon G K Ching P C Ng and T F Leung ldquoExplor-ing CCL18 eczema severity and atopyrdquo Pediatric Allergy andImmunology vol 22 no 7 pp 704ndash707 2011
[46] A J Lucendo A Arias L C De Rezende et al ldquoSubepithelialcollagen deposition profibrogenic cytokine gene expressionand changes after prolonged fluticasone propionate treatment inadult eosinophilic esophagitis a prospective studyrdquoThe Journalof Allergy and Clinical Immunology vol 128 no 5 pp 1037ndash1046 2011
[47] H Li D Zhang J Yu et al ldquoCCL18-dependent translocationof AMAP1 is critical for epithelial to mesenchymal transition inbreast cancerrdquo Journal of Cellular Physiology vol 233 no 4 pp3207ndash3217 2017
[48] Z Lin W Li H Zhang et al ldquoCCL18PITPNM3 enhancesmigration invasion and EMT through the NF-120581B signalingpathway in hepatocellular carcinomardquo Tumor Biology vol 37no 3 pp 3461ndash3468 2016
[49] F Meng W Li C Li Z Gao K Guo and S SongldquoCCL18 promotes epithelial-mesenchymal transition invasionand migration of pancreatic cancer cells in pancreatic ductaladenocarcinomardquo International Journal of Oncology vol 46 no3 pp 1109ndash1120 2015
[50] T Ploenes B Scholtes A Krohn et al ldquoCC-Chemokine Ligand18 Induces Epithelial to Mesenchymal Transition in LungCancer A549 Cells and Elevates the Invasive Potentialrdquo PLoSONE vol 8 no 1 Article ID e53068 2013
[51] J Rojas-Rivera C De La Piedra A Ramos A Ortiz andJ Egido ldquoThe expanding spectrum of biological actions ofvitamin Drdquo Nephrology Dialysis Transplantation vol 25 no 9pp 2850ndash2865 2010
[52] M Vulcano S Struyf P Scapini et al ldquoUnique regulation ofCCL18 production by maturing dendritic cellsrdquo The Journal ofImmunology vol 170 no 7 pp 3843ndash3849 2003
[53] R Kundu A Theodoraki C T Haas et al ldquoCell-type-specificmodulation of innate immune signalling by vitamin D inhuman mononuclear phagocytesrdquo The Journal of Immunologyvol 150 no 1 pp 55ndash63 2017
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
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Diabetes ResearchJournal of
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Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
BioMed Research International 9
NEG
CCL1
8 (p
gm
l)
0
200
400
600
800
1000
12001057
522
Pari (10minus7 M) NEG000
020
040
060
080
100
120100
025Fold
mRN
A C
CL18
Pari (10minus7 M)
(a)
NEG
408
279
0
100
200
300
400
500
CCL1
8 (p
gm
l)
Pari (10minus7 M)
100
045
000
050
100
150
NEG
Fold
mRN
A C
CL18
Pari (10minus7 M)
(b)
000
025
050
075
100
125
Fold
mRN
A C
CL18
NEG
Mo + IL-4
Pari Vit D3Vmdash
(c)
Figure 7 (a) Peritoneal macrophages from a patient with recent infectious peritonitis were treated with paricalcitol or vehicle for 24 h Theprotein concentration of CCL18 was evaluated in cell culture supernatants by ELISA and CCL18 mRNA levels were estimated by quantitativeRT-PCR in the same cultures (b) Peritoneal macrophages from an uninfected peritoneum were isolated from the peritoneal lavage uponcatheter insertion and treated as in (a) (c) Peripheral bloodmonocytes were pretreated with vehicle (V) 10minus7Mparicalcitol (Pari) or vitaminD3 for 24 h The cells were treated thereafter with IL-4 for 3 days before RNA extraction and evaluation expression of CCL18 mRNA levelsby quantitative RT-PCR Mean plusmn SD values from four independent experiments are shown
primarily produced by cells of myeloid origin multiplecell types are producers of IL-6 with endothelial cellsbeing an important source of this cytokine Infiltratingneutrophils as well as mesothelial cells could also be animportant source of IL-6 during bacterial peritonitis [37]The analysis of serial samples in four patients with IP showedthat the concentrations of CCL18 paralleled the numbers ofmacrophages infiltrating the peritoneum and are consistentwith previous descriptions of macrophages andmononuclearphagocytes as the primary producers of CCL18 [21] Cellcounts in the serial samples from our case of EP suggest that
the peritoneal eosinophilic infiltrate was likely contributingto the peritoneal production of CCL18 Evidences of CCL18production by activated peritoneal eosinophils in this caseare cytospin analysis of CCL18 cytoplasmic staining ineosinophils and gene expression data in polymorphonuclearleukocytes among which 85 were eosinophils
Although macrophages are typically reported as themajor source of this chemokine [21] CCL18 protein pro-duction by eosinophils has occasionally been described asbeing related to the activation status of these cells given thatCCL18 was found only in eosinophil culture supernatants
10 BioMed Research International
from eosinophilic donors but not from individuals withnonactivated eosinophils [29] Our data support the activatedstatus of eosinophils in EP given that strong staining ofCC18 was detected in peritoneal eosinophils Eosinophilshave also been related to tissue fibrosis in several diseasesand are a source of several profibrotic mediators that alterfibroblast activity and tissue remodeling [38 39] Some ofthesemediators could also stimulate the profibrotic activity ofpMΦTheproduction ofCCL18 by eosinophils in EPprovidesa new additional explanation for the association of recurrentEP during PD with UFF and potentially peritoneal fibrosis[40]
As part of its profibrotic activity CCL18 has been shownto stimulate collagen deposition [41] and fibroblast prolif-eration [42] In consonance with these actions circulatingCCL18 levels have been demonstrated to be a biomarker fordisease activity and outcome in various fibrotic diseases [1943 44] as well as in some eosinophilic disorders [30 45 46]Moreover CCL18 has been shown to promote EMT in variouscell systems [47ndash50] It is thus possible that it could alsocontribute to the EMT of mesothelial cells which has beenproposed as one of the mechanisms leading to peritonealfibrosis and membrane failure [12]
In terms of peritoneal fibrosis CCL18 has been demon-strated to herald the development of peritoneal functionaldeterioration in PD patients [15 20] in whom the abilityof peritoneal macrophages to stimulate fibroblast prolifer-ation is correlated with CCL18 mRNA levels Moreoverrecombinant CCL18 was able to stimulate the proliferation ofperitoneal fibroblasts [15]
Several molecules have been tested for their ability toprevent peritoneal fibrosis in vitro and in animal models(reviewed in [8]) Among them paricalcitol is a selectiveVDR agonist VDR is a nuclear hormone receptor expressedin various cell types including those of the immune sys-tem VDR agonists have been shown to modulate immuneresponses inflammation and fibrosis [51]
Considering the role played by inflammation in peri-toneal fibrosis during PDwe studied the effects of paricalcitolin the production of CCL18 We found that the release andgene expression of CCL18 were downregulated in pMΦsand in in vitro-differentiated human M2 cells which are theprimary producers of CCL18These results appear to contrastwith previous data showing upregulation of CCL18 by vita-min D in monocyte-derived immature dendritic cells [52]However recent reports demonstrate that the modulationof immune signaling by vitamin D in human mononuclearphagocytes is cell-type-specific with differential responses inmonocytes macrophages and dendritic cells [53] It is thuspossible that opposite effects are found on the regulation ofCCL18 by VDR agonists in different cellular systems
On the other hand in a mouse model of PD intraperi-toneal administration of paricalcitol was shown to reduceperitoneal fibrosis and ultrafiltration failure [23] In thatmodel the beneficial effects appeared to be dependent ona reduction of intraperitoneal concentrations of IL-17 Thelack of rodent orthologues of human CCL18 [28] precludesthe investigation of CCL18 and its involvement in fibrosis inmurine models On the other hand IL-17 is highly diluted
in human peritoneal effluents The need to concentrate thesamples for measurement of this cytokine hampers accuratedeterminations in human samples
A preliminary study involving 23 patients on PD suggeststhat paricalcitol could improve the ultrafiltration capacitywith less peritoneal protein loss [24] The authors suggestan effect of VDR upon the renin-angiotensin-aldosteronesystem but it is tempting to speculate that lower peritonealconcentrations of CCL18 might be present in paricalcitol-treated patients In this sense a slight reduction in theperitoneal concentration of CCL18 was observed in sixpatients who were more than 3 years on PD and treated withparicalcitol compared with a set of 31 patients of the samevintage (data not shown)
5 Conclusion
In conclusion our study suggests that the chemokine CCL18can be a mediator of peritoneal membrane failure associatedwith peritonitis episodes and might provide a new potentialtherapeutic target
Further clinical studies are needed to confirm the useful-ness of VDR agonists to preserve the peritoneal membraneand to analyze the putative mechanisms involved
Conflicts of Interest
All the authors have no potential conflicts of interest todeclare
Authorsrsquo Contributions
Marta Ossorio and Virginia Martınez contributed equally tothis work
Acknowledgments
This study has been partially supported by ISCIII (ldquoInsti-tuto de Investigacion Sanitaria Carlos IIIrdquo) Grants FIS PI1200204 PI1301768 and PI 1500120 FEDER (Funds fromthe European Union) REDinREN 20 FEDER and ReinaSofıa Institute of Nephrology Research (IRSIN) monetaryfunds The authors would like to thank Juliette Siegfriedand her team at ServingMedcom for their editing of themanuscript
Supplementary Materials
Supplemental Figure 1 serial cytokine concentrations andcell counts in peritoneal effluent during infectious peritoni-tis and eosinophilic peritonitis (A) Comparison of CCL18concentration in serial effluent samples from infectiousperitonitis (IP) and eosinophilic peritonitis (EP) (B) IL-6effluent levels in serial samples from IP and EP D1ndashD26indicate days of evolution Supplemental Figure 2 dose-dependent effects of VDR agonists on cell viability Cellviability of human monocytes was assessed by exclusion ofpropidium iodide staining and analyzed by flow cytometry
BioMed Research International 11
Results are shown as mean plusmn SD values of triplicates(A)ndash(C) Viability of monocytes upon culture during 24 48or 72 hours with decreasing concentrations of Zemplar orequivalent vehicle (305020 propylene glycolwaterethanol)(D)ndash(F) Viability of monocytes upon culture during 2448 or 72 hours with different concentrations of paricalcitol(pure substance) vitamin D or equivalent vehicle (EtOH)(Supplementary Materials)
References
[1] R T Krediet ldquoThe peritoneal membrane in chronic peritonealdialysisrdquo Kidney International vol 55 no 1 pp 341ndash356 1999
[2] R T Krediet ldquoPeritoneal physiology - Impact on solute andfluid clearancerdquo Advances in Chronic Kidney Disease vol 7 no4 pp 271ndash279 2000
[3] M Merkle and M Wrnle ldquoSclerosing peritonitis A rare butfatal complication of peritoneal inflammationrdquo Mediators ofInflammation vol 2012 Article ID 709673 p 4 2012
[4] D W Johnson Y Cho B E R Livingston et al ldquoEncapsulat-ing peritoneal sclerosis incidence predictors and outcomesrdquoKidney International vol 77 no 10 pp 904ndash912 2010
[5] G Balasubramaniam E A Brown and A Davenport ldquoThePan-Thames EPS study treatment and outcomes of encapsulat-ing peritoneal sclerosisrdquo Nephrology Dialysis Transplantation vol 24 no 10 pp 3209ndash3215 2009
[6] E D Sousa-Amorim G D Peso M A Bajo et al ldquoCan EPSdevelopment be avoided with early interventionsThe potentialrole of tamoxifenmdasha single-center studyrdquo Peritoneal DialysisInternational vol 34 no 6 pp 582ndash593 2014
[7] M R Korte D E SampimonM GH Betjes and R T KredietldquoEncapsulating peritoneal sclerosis the state of affairsrdquo NatureReviews Nephrology vol 7 no 9 pp 528ndash538 2011
[8] M A Bajo G del Peso and I Teitelbaum ldquoPeritoneal Mem-brane Preservationrdquo Seminars in Nephrology vol 37 no 1 pp77ndash92 2017
[9] M F Flessner ldquoInflammation from sterile dialysis solutions andthe longevity of the peritoneal barrierrdquo Clinical Nephrology vol68 no 6 pp 341ndash348 2007
[10] K N Lai and J C K Leung ldquoInflammation in peritonealdialysisrdquo Nephron Clinical Practice vol 116 no 1 pp c11ndashc182010
[11] S Yung and T M Chan ldquoPathophysiological changes to theperitoneal membrane during PD-related peritonitis the role ofmesothelial cellsrdquo Mediators of Inflammation vol 2012 ArticleID 484167 21 pages 2012
[12] L S Aroeira A Aguilera J A Sanchez-Tomero et al ldquoEpithe-lial tomesenchymal transition and peritonealmembrane failurein peritoneal dialysis patients pathologic significance andpotential therapeutic interventionsrdquo Journal of the AmericanSociety of Nephrology vol 18 no 7 pp 2004ndash2013 2007
[13] N Topley T Liberek A Davenport F-K Li H Fear andJ D Williams ldquoActivation of inflammation and leukocyterecruitment into the peritoneal cavityrdquo Kidney InternationalSupplements vol 50 no 56 pp S17ndashS21 1996
[14] F O Martınez and S Gordon ldquoThe M1 and M2 paradigmof macrophage activation time for reassessmentrdquo F1000PrimeReports vol 6 article 13 2014
[15] T Bellon V Martınez B Lucendo et al ldquoAlternative activa-tion of macrophages in human peritoneum implications for
peritoneal fibrosisrdquo Nephrology Dialysis Transplantation vol26 no 9 pp 2995ndash3005 2011
[16] A Prasse D V Pechkovsky G B Toews et al ldquoA vicious circleof alveolar macrophages and fibroblasts perpetuates pulmonaryfibrosis via CCL18rdquoAmerican Journal of Respiratory and CriticalCare Medicine vol 173 no 7 pp 781ndash792 2006
[17] J M Estep A Baranova N Hossain et al ldquoExpression ofcytokine signaling genes in morbidly obese patients with non-alcoholic steatohepatitis and hepatic fibrosisrdquo Obesity Surgeryvol 19 no 5 pp 617ndash624 2009
[18] F Kollert M Binder C Probst et al ldquoCCL18 - Potentialbiomarker of fibroinflammatory activity in chronic periaortitisrdquoThe Journal of Rheumatology vol 39 no 7 pp 1407ndash1412 2012
[19] A Prasse D V Pechkovsky G B Toews et al ldquoCCL18 as anindicator of pulmonary fibrotic activity in idiopathic interstitialpneumonias and systemic sclerosisrdquo Arthritis amp Rheumatologyvol 56 no 5 pp 1685ndash1693 2007
[20] S Ahmad B V North A Qureshi et al ldquoCCL18 in peritonealdialysis patients and encapsulating peritoneal sclerosisrdquo Euro-pean Journal of Clinical Investigation vol 40 no 12 pp 1067ndash1073 2010
[21] V Kodelja C Muller O Politz N Hakij C E Orfanos andS Goerdt ldquoAlternative macrophage activation-associated CC-chemokine-1 a novel structural homologue of macrophageinflammatory protein-1120572 with a Th2- associated expressionpatternrdquo The Journal of Immunology vol 160 no 3 pp 1411ndash1418 1998
[22] M Ossorio M A Bajo G Del Peso et al ldquoSustained low peri-toneal effluent CCL18 levels are associated with preservationof peritoneal membrane function in peritoneal dialysisrdquo PLoSONE vol 12 no 4 Article ID e0175835 2017
[23] G T Gonzalez-Mateo V Fernandez-Mıllara T Bellon etal ldquoParicalcitol reduces peritoneal fibrosis in mice throughthe activation of regulatory T cells and reduction in IL-17productionrdquo PLoS ONE vol 9 no 10 Article ID e108477 2014
[24] F Coronel S Cigarran A Gomis et al ldquoChanges in peritonealmembrane permeability and proteinuria in patients on peri-toneal dialysis after treatment with paricalcitolmdasha preliminarystudyrdquo Clinical Nephrology vol 78 no 2 pp 93ndash99 2012
[25] E Song N Ouyang M Horbelt B Antus M Wang and MS Exton ldquoInfluence of alternatively and classically activatedmacrophages on fibrogenic activities of human fibroblastsrdquoCellular Immunology vol 204 no 1 pp 19ndash28 2000
[26] S M Hurst T S Wilkinson R M McLoughlin et al ldquoIL-6 andits soluble receptor orchestrate a temporal switch in the patternof leukocyte recruitment seen during acute inflammationrdquoImmunity vol 14 no 6 pp 705ndash714 2001
[27] M Kozioł-Montewka A Ksiazek L Janicka and I BaranowiczldquoSerial cytokine changes in peritoneal effluent and plasma dur-ing peritonitis in patients on continuous ambulatory peritonealdialysis (CAPD)rdquo Inflammation Research vol 46 no 4 pp 132ndash136 1997
[28] E Schutyser A Richmond and J Van Damme ldquoInvolvementof CC chemokine ligand 18 (CCL18) in normal and pathologicalprocessesrdquo Journal of Leukocyte Biology vol 78 no 1 pp 14ndash262005
[29] I Schraufstatter H Takamori L Sikora P Sriramarao and RG DiScipio ldquoEosinophils and monocytes produce pulmonaryand activation-regulated chemokine which activates culturedmonocytesmacrophagesrdquoAmerican Journal of Physiology-LungCellular and Molecular Physiology vol 286 no 3 pp L494ndashL501 2004
12 BioMed Research International
[30] C Gunther N Carballido-Perrig T Kopp J M Carballidoand C Pfeiffer ldquoCCL18 is expressed in patients with bullouspemphigoid and parallels disease courserdquo British Journal ofDermatology vol 160 no 4 pp 747ndash755 2009
[31] AMantovani A Sica S Sozzani P Allavena A Vecchi andMLocati ldquoThe chemokine system in diverse forms of macrophageactivation and polarizationrdquo Trends in Immunology vol 25 no12 pp 677ndash686 2004
[32] C Aufricht R Beelen M Eberl et al ldquoBiomarker researchto improve clinical outcomes of peritoneal dialysis consensusof the European Training and Research in Peritoneal Dialysis(EuTRiPD) networkrdquo Kidney International vol 92 no 4 pp824ndash835 2017
[33] D Lopes Barreto andR T Krediet ldquoCurrent status and practicaluse of effluent biomarkers in peritoneal dialysis patientsrdquoAmerican Journal of Kidney Diseases vol 62 no 4 pp 823ndash8332013
[34] L A Boven M van Meurs R G Boot et al ldquoGaucher cellsdemonstrate a distinct macrophage phenotype and resemblealternatively activated macrophagesrdquo American Journal of Clin-ical Pathology vol 122 no 3 pp 359ndash369 2004
[35] L Cocho I Fernandez M Calonge et al ldquoGene expression-based predictive models of graft versus host disease-associateddry eyerdquo Investigative Ophtalmology Visual Science vol 56 no8 pp 4570ndash4581 2015
[36] MHasegawa ldquoBiomarkers in systemic sclerosisTheir potentialto predict clinical coursesrdquoThe Journal of Dermatology vol 43no 1 pp 29ndash38 2016
[37] S A Jones D J Fraser C A Fielding and G W JonesldquoInterleukin-6 in renal disease and therapyrdquoNephrologyDialysisTransplantation vol 30 no 4 pp 564ndash574 2015
[38] A Munitz and F Levi-Schaffer ldquoEosinophils rsquoNewrsquo roles forrsquooldrsquo cellsrdquo Allergy European Journal of Allergy and ClinicalImmunology vol 59 no 3 pp 268ndash275 2004
[39] S S Aceves ldquoRemodeling and fibrosis in chronic eosinophilinflammationrdquo Digestive Diseases vol 32 no 1-2 pp 15ndash212014
[40] Y Nakamura H Okada A Yasui T Koh and T YamaneldquoSclerosing encapsulating peritonitis associated with recurrenteosinophilic peritonitisrdquo Nephrology Dialysis Transplantation vol 14 no 3 pp 768ndash770 1999
[41] S P Atamas I G Luzina J Choi et al ldquoPulmonary andactivation-regulated chemokine stimulates collagen productionin lung fibroblastsrdquo American Journal of Respiratory Cell andMolecular Biology vol 29 no 6 pp 743ndash749 2003
[42] A Wimmer S K Khaldoyanidi M Judex N Serobyan RG DiScipio and I U Schraufstatter ldquoCCL18PARC stimulateshematopoiesis in long-term bone marrow cultures indirectlythrough its effect on monocytesrdquo Blood vol 108 no 12 pp3722ndash3729 2006
[43] A Prasse C Probst E Bargagli et al ldquoSerum CC-chemokineligand 18 concentration predicts outcome in idiopathic pul-monary fibrosisrdquo American Journal of Respiratory and CriticalCare Medicine vol 179 no 8 pp 717ndash723 2009
[44] J Chen Y Yao C Gong et al ldquoCCL18 from tumor-associatedmacrophages promotes breast cancermetastasis via PITPNM3rdquoCancer Cell vol 19 no 4 pp 541ndash555 2011
[45] K L Hon G K Ching P C Ng and T F Leung ldquoExplor-ing CCL18 eczema severity and atopyrdquo Pediatric Allergy andImmunology vol 22 no 7 pp 704ndash707 2011
[46] A J Lucendo A Arias L C De Rezende et al ldquoSubepithelialcollagen deposition profibrogenic cytokine gene expressionand changes after prolonged fluticasone propionate treatment inadult eosinophilic esophagitis a prospective studyrdquoThe Journalof Allergy and Clinical Immunology vol 128 no 5 pp 1037ndash1046 2011
[47] H Li D Zhang J Yu et al ldquoCCL18-dependent translocationof AMAP1 is critical for epithelial to mesenchymal transition inbreast cancerrdquo Journal of Cellular Physiology vol 233 no 4 pp3207ndash3217 2017
[48] Z Lin W Li H Zhang et al ldquoCCL18PITPNM3 enhancesmigration invasion and EMT through the NF-120581B signalingpathway in hepatocellular carcinomardquo Tumor Biology vol 37no 3 pp 3461ndash3468 2016
[49] F Meng W Li C Li Z Gao K Guo and S SongldquoCCL18 promotes epithelial-mesenchymal transition invasionand migration of pancreatic cancer cells in pancreatic ductaladenocarcinomardquo International Journal of Oncology vol 46 no3 pp 1109ndash1120 2015
[50] T Ploenes B Scholtes A Krohn et al ldquoCC-Chemokine Ligand18 Induces Epithelial to Mesenchymal Transition in LungCancer A549 Cells and Elevates the Invasive Potentialrdquo PLoSONE vol 8 no 1 Article ID e53068 2013
[51] J Rojas-Rivera C De La Piedra A Ramos A Ortiz andJ Egido ldquoThe expanding spectrum of biological actions ofvitamin Drdquo Nephrology Dialysis Transplantation vol 25 no 9pp 2850ndash2865 2010
[52] M Vulcano S Struyf P Scapini et al ldquoUnique regulation ofCCL18 production by maturing dendritic cellsrdquo The Journal ofImmunology vol 170 no 7 pp 3843ndash3849 2003
[53] R Kundu A Theodoraki C T Haas et al ldquoCell-type-specificmodulation of innate immune signalling by vitamin D inhuman mononuclear phagocytesrdquo The Journal of Immunologyvol 150 no 1 pp 55ndash63 2017
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
10 BioMed Research International
from eosinophilic donors but not from individuals withnonactivated eosinophils [29] Our data support the activatedstatus of eosinophils in EP given that strong staining ofCC18 was detected in peritoneal eosinophils Eosinophilshave also been related to tissue fibrosis in several diseasesand are a source of several profibrotic mediators that alterfibroblast activity and tissue remodeling [38 39] Some ofthesemediators could also stimulate the profibrotic activity ofpMΦTheproduction ofCCL18 by eosinophils in EPprovidesa new additional explanation for the association of recurrentEP during PD with UFF and potentially peritoneal fibrosis[40]
As part of its profibrotic activity CCL18 has been shownto stimulate collagen deposition [41] and fibroblast prolif-eration [42] In consonance with these actions circulatingCCL18 levels have been demonstrated to be a biomarker fordisease activity and outcome in various fibrotic diseases [1943 44] as well as in some eosinophilic disorders [30 45 46]Moreover CCL18 has been shown to promote EMT in variouscell systems [47ndash50] It is thus possible that it could alsocontribute to the EMT of mesothelial cells which has beenproposed as one of the mechanisms leading to peritonealfibrosis and membrane failure [12]
In terms of peritoneal fibrosis CCL18 has been demon-strated to herald the development of peritoneal functionaldeterioration in PD patients [15 20] in whom the abilityof peritoneal macrophages to stimulate fibroblast prolifer-ation is correlated with CCL18 mRNA levels Moreoverrecombinant CCL18 was able to stimulate the proliferation ofperitoneal fibroblasts [15]
Several molecules have been tested for their ability toprevent peritoneal fibrosis in vitro and in animal models(reviewed in [8]) Among them paricalcitol is a selectiveVDR agonist VDR is a nuclear hormone receptor expressedin various cell types including those of the immune sys-tem VDR agonists have been shown to modulate immuneresponses inflammation and fibrosis [51]
Considering the role played by inflammation in peri-toneal fibrosis during PDwe studied the effects of paricalcitolin the production of CCL18 We found that the release andgene expression of CCL18 were downregulated in pMΦsand in in vitro-differentiated human M2 cells which are theprimary producers of CCL18These results appear to contrastwith previous data showing upregulation of CCL18 by vita-min D in monocyte-derived immature dendritic cells [52]However recent reports demonstrate that the modulationof immune signaling by vitamin D in human mononuclearphagocytes is cell-type-specific with differential responses inmonocytes macrophages and dendritic cells [53] It is thuspossible that opposite effects are found on the regulation ofCCL18 by VDR agonists in different cellular systems
On the other hand in a mouse model of PD intraperi-toneal administration of paricalcitol was shown to reduceperitoneal fibrosis and ultrafiltration failure [23] In thatmodel the beneficial effects appeared to be dependent ona reduction of intraperitoneal concentrations of IL-17 Thelack of rodent orthologues of human CCL18 [28] precludesthe investigation of CCL18 and its involvement in fibrosis inmurine models On the other hand IL-17 is highly diluted
in human peritoneal effluents The need to concentrate thesamples for measurement of this cytokine hampers accuratedeterminations in human samples
A preliminary study involving 23 patients on PD suggeststhat paricalcitol could improve the ultrafiltration capacitywith less peritoneal protein loss [24] The authors suggestan effect of VDR upon the renin-angiotensin-aldosteronesystem but it is tempting to speculate that lower peritonealconcentrations of CCL18 might be present in paricalcitol-treated patients In this sense a slight reduction in theperitoneal concentration of CCL18 was observed in sixpatients who were more than 3 years on PD and treated withparicalcitol compared with a set of 31 patients of the samevintage (data not shown)
5 Conclusion
In conclusion our study suggests that the chemokine CCL18can be a mediator of peritoneal membrane failure associatedwith peritonitis episodes and might provide a new potentialtherapeutic target
Further clinical studies are needed to confirm the useful-ness of VDR agonists to preserve the peritoneal membraneand to analyze the putative mechanisms involved
Conflicts of Interest
All the authors have no potential conflicts of interest todeclare
Authorsrsquo Contributions
Marta Ossorio and Virginia Martınez contributed equally tothis work
Acknowledgments
This study has been partially supported by ISCIII (ldquoInsti-tuto de Investigacion Sanitaria Carlos IIIrdquo) Grants FIS PI1200204 PI1301768 and PI 1500120 FEDER (Funds fromthe European Union) REDinREN 20 FEDER and ReinaSofıa Institute of Nephrology Research (IRSIN) monetaryfunds The authors would like to thank Juliette Siegfriedand her team at ServingMedcom for their editing of themanuscript
Supplementary Materials
Supplemental Figure 1 serial cytokine concentrations andcell counts in peritoneal effluent during infectious peritoni-tis and eosinophilic peritonitis (A) Comparison of CCL18concentration in serial effluent samples from infectiousperitonitis (IP) and eosinophilic peritonitis (EP) (B) IL-6effluent levels in serial samples from IP and EP D1ndashD26indicate days of evolution Supplemental Figure 2 dose-dependent effects of VDR agonists on cell viability Cellviability of human monocytes was assessed by exclusion ofpropidium iodide staining and analyzed by flow cytometry
BioMed Research International 11
Results are shown as mean plusmn SD values of triplicates(A)ndash(C) Viability of monocytes upon culture during 24 48or 72 hours with decreasing concentrations of Zemplar orequivalent vehicle (305020 propylene glycolwaterethanol)(D)ndash(F) Viability of monocytes upon culture during 2448 or 72 hours with different concentrations of paricalcitol(pure substance) vitamin D or equivalent vehicle (EtOH)(Supplementary Materials)
References
[1] R T Krediet ldquoThe peritoneal membrane in chronic peritonealdialysisrdquo Kidney International vol 55 no 1 pp 341ndash356 1999
[2] R T Krediet ldquoPeritoneal physiology - Impact on solute andfluid clearancerdquo Advances in Chronic Kidney Disease vol 7 no4 pp 271ndash279 2000
[3] M Merkle and M Wrnle ldquoSclerosing peritonitis A rare butfatal complication of peritoneal inflammationrdquo Mediators ofInflammation vol 2012 Article ID 709673 p 4 2012
[4] D W Johnson Y Cho B E R Livingston et al ldquoEncapsulat-ing peritoneal sclerosis incidence predictors and outcomesrdquoKidney International vol 77 no 10 pp 904ndash912 2010
[5] G Balasubramaniam E A Brown and A Davenport ldquoThePan-Thames EPS study treatment and outcomes of encapsulat-ing peritoneal sclerosisrdquo Nephrology Dialysis Transplantation vol 24 no 10 pp 3209ndash3215 2009
[6] E D Sousa-Amorim G D Peso M A Bajo et al ldquoCan EPSdevelopment be avoided with early interventionsThe potentialrole of tamoxifenmdasha single-center studyrdquo Peritoneal DialysisInternational vol 34 no 6 pp 582ndash593 2014
[7] M R Korte D E SampimonM GH Betjes and R T KredietldquoEncapsulating peritoneal sclerosis the state of affairsrdquo NatureReviews Nephrology vol 7 no 9 pp 528ndash538 2011
[8] M A Bajo G del Peso and I Teitelbaum ldquoPeritoneal Mem-brane Preservationrdquo Seminars in Nephrology vol 37 no 1 pp77ndash92 2017
[9] M F Flessner ldquoInflammation from sterile dialysis solutions andthe longevity of the peritoneal barrierrdquo Clinical Nephrology vol68 no 6 pp 341ndash348 2007
[10] K N Lai and J C K Leung ldquoInflammation in peritonealdialysisrdquo Nephron Clinical Practice vol 116 no 1 pp c11ndashc182010
[11] S Yung and T M Chan ldquoPathophysiological changes to theperitoneal membrane during PD-related peritonitis the role ofmesothelial cellsrdquo Mediators of Inflammation vol 2012 ArticleID 484167 21 pages 2012
[12] L S Aroeira A Aguilera J A Sanchez-Tomero et al ldquoEpithe-lial tomesenchymal transition and peritonealmembrane failurein peritoneal dialysis patients pathologic significance andpotential therapeutic interventionsrdquo Journal of the AmericanSociety of Nephrology vol 18 no 7 pp 2004ndash2013 2007
[13] N Topley T Liberek A Davenport F-K Li H Fear andJ D Williams ldquoActivation of inflammation and leukocyterecruitment into the peritoneal cavityrdquo Kidney InternationalSupplements vol 50 no 56 pp S17ndashS21 1996
[14] F O Martınez and S Gordon ldquoThe M1 and M2 paradigmof macrophage activation time for reassessmentrdquo F1000PrimeReports vol 6 article 13 2014
[15] T Bellon V Martınez B Lucendo et al ldquoAlternative activa-tion of macrophages in human peritoneum implications for
peritoneal fibrosisrdquo Nephrology Dialysis Transplantation vol26 no 9 pp 2995ndash3005 2011
[16] A Prasse D V Pechkovsky G B Toews et al ldquoA vicious circleof alveolar macrophages and fibroblasts perpetuates pulmonaryfibrosis via CCL18rdquoAmerican Journal of Respiratory and CriticalCare Medicine vol 173 no 7 pp 781ndash792 2006
[17] J M Estep A Baranova N Hossain et al ldquoExpression ofcytokine signaling genes in morbidly obese patients with non-alcoholic steatohepatitis and hepatic fibrosisrdquo Obesity Surgeryvol 19 no 5 pp 617ndash624 2009
[18] F Kollert M Binder C Probst et al ldquoCCL18 - Potentialbiomarker of fibroinflammatory activity in chronic periaortitisrdquoThe Journal of Rheumatology vol 39 no 7 pp 1407ndash1412 2012
[19] A Prasse D V Pechkovsky G B Toews et al ldquoCCL18 as anindicator of pulmonary fibrotic activity in idiopathic interstitialpneumonias and systemic sclerosisrdquo Arthritis amp Rheumatologyvol 56 no 5 pp 1685ndash1693 2007
[20] S Ahmad B V North A Qureshi et al ldquoCCL18 in peritonealdialysis patients and encapsulating peritoneal sclerosisrdquo Euro-pean Journal of Clinical Investigation vol 40 no 12 pp 1067ndash1073 2010
[21] V Kodelja C Muller O Politz N Hakij C E Orfanos andS Goerdt ldquoAlternative macrophage activation-associated CC-chemokine-1 a novel structural homologue of macrophageinflammatory protein-1120572 with a Th2- associated expressionpatternrdquo The Journal of Immunology vol 160 no 3 pp 1411ndash1418 1998
[22] M Ossorio M A Bajo G Del Peso et al ldquoSustained low peri-toneal effluent CCL18 levels are associated with preservationof peritoneal membrane function in peritoneal dialysisrdquo PLoSONE vol 12 no 4 Article ID e0175835 2017
[23] G T Gonzalez-Mateo V Fernandez-Mıllara T Bellon etal ldquoParicalcitol reduces peritoneal fibrosis in mice throughthe activation of regulatory T cells and reduction in IL-17productionrdquo PLoS ONE vol 9 no 10 Article ID e108477 2014
[24] F Coronel S Cigarran A Gomis et al ldquoChanges in peritonealmembrane permeability and proteinuria in patients on peri-toneal dialysis after treatment with paricalcitolmdasha preliminarystudyrdquo Clinical Nephrology vol 78 no 2 pp 93ndash99 2012
[25] E Song N Ouyang M Horbelt B Antus M Wang and MS Exton ldquoInfluence of alternatively and classically activatedmacrophages on fibrogenic activities of human fibroblastsrdquoCellular Immunology vol 204 no 1 pp 19ndash28 2000
[26] S M Hurst T S Wilkinson R M McLoughlin et al ldquoIL-6 andits soluble receptor orchestrate a temporal switch in the patternof leukocyte recruitment seen during acute inflammationrdquoImmunity vol 14 no 6 pp 705ndash714 2001
[27] M Kozioł-Montewka A Ksiazek L Janicka and I BaranowiczldquoSerial cytokine changes in peritoneal effluent and plasma dur-ing peritonitis in patients on continuous ambulatory peritonealdialysis (CAPD)rdquo Inflammation Research vol 46 no 4 pp 132ndash136 1997
[28] E Schutyser A Richmond and J Van Damme ldquoInvolvementof CC chemokine ligand 18 (CCL18) in normal and pathologicalprocessesrdquo Journal of Leukocyte Biology vol 78 no 1 pp 14ndash262005
[29] I Schraufstatter H Takamori L Sikora P Sriramarao and RG DiScipio ldquoEosinophils and monocytes produce pulmonaryand activation-regulated chemokine which activates culturedmonocytesmacrophagesrdquoAmerican Journal of Physiology-LungCellular and Molecular Physiology vol 286 no 3 pp L494ndashL501 2004
12 BioMed Research International
[30] C Gunther N Carballido-Perrig T Kopp J M Carballidoand C Pfeiffer ldquoCCL18 is expressed in patients with bullouspemphigoid and parallels disease courserdquo British Journal ofDermatology vol 160 no 4 pp 747ndash755 2009
[31] AMantovani A Sica S Sozzani P Allavena A Vecchi andMLocati ldquoThe chemokine system in diverse forms of macrophageactivation and polarizationrdquo Trends in Immunology vol 25 no12 pp 677ndash686 2004
[32] C Aufricht R Beelen M Eberl et al ldquoBiomarker researchto improve clinical outcomes of peritoneal dialysis consensusof the European Training and Research in Peritoneal Dialysis(EuTRiPD) networkrdquo Kidney International vol 92 no 4 pp824ndash835 2017
[33] D Lopes Barreto andR T Krediet ldquoCurrent status and practicaluse of effluent biomarkers in peritoneal dialysis patientsrdquoAmerican Journal of Kidney Diseases vol 62 no 4 pp 823ndash8332013
[34] L A Boven M van Meurs R G Boot et al ldquoGaucher cellsdemonstrate a distinct macrophage phenotype and resemblealternatively activated macrophagesrdquo American Journal of Clin-ical Pathology vol 122 no 3 pp 359ndash369 2004
[35] L Cocho I Fernandez M Calonge et al ldquoGene expression-based predictive models of graft versus host disease-associateddry eyerdquo Investigative Ophtalmology Visual Science vol 56 no8 pp 4570ndash4581 2015
[36] MHasegawa ldquoBiomarkers in systemic sclerosisTheir potentialto predict clinical coursesrdquoThe Journal of Dermatology vol 43no 1 pp 29ndash38 2016
[37] S A Jones D J Fraser C A Fielding and G W JonesldquoInterleukin-6 in renal disease and therapyrdquoNephrologyDialysisTransplantation vol 30 no 4 pp 564ndash574 2015
[38] A Munitz and F Levi-Schaffer ldquoEosinophils rsquoNewrsquo roles forrsquooldrsquo cellsrdquo Allergy European Journal of Allergy and ClinicalImmunology vol 59 no 3 pp 268ndash275 2004
[39] S S Aceves ldquoRemodeling and fibrosis in chronic eosinophilinflammationrdquo Digestive Diseases vol 32 no 1-2 pp 15ndash212014
[40] Y Nakamura H Okada A Yasui T Koh and T YamaneldquoSclerosing encapsulating peritonitis associated with recurrenteosinophilic peritonitisrdquo Nephrology Dialysis Transplantation vol 14 no 3 pp 768ndash770 1999
[41] S P Atamas I G Luzina J Choi et al ldquoPulmonary andactivation-regulated chemokine stimulates collagen productionin lung fibroblastsrdquo American Journal of Respiratory Cell andMolecular Biology vol 29 no 6 pp 743ndash749 2003
[42] A Wimmer S K Khaldoyanidi M Judex N Serobyan RG DiScipio and I U Schraufstatter ldquoCCL18PARC stimulateshematopoiesis in long-term bone marrow cultures indirectlythrough its effect on monocytesrdquo Blood vol 108 no 12 pp3722ndash3729 2006
[43] A Prasse C Probst E Bargagli et al ldquoSerum CC-chemokineligand 18 concentration predicts outcome in idiopathic pul-monary fibrosisrdquo American Journal of Respiratory and CriticalCare Medicine vol 179 no 8 pp 717ndash723 2009
[44] J Chen Y Yao C Gong et al ldquoCCL18 from tumor-associatedmacrophages promotes breast cancermetastasis via PITPNM3rdquoCancer Cell vol 19 no 4 pp 541ndash555 2011
[45] K L Hon G K Ching P C Ng and T F Leung ldquoExplor-ing CCL18 eczema severity and atopyrdquo Pediatric Allergy andImmunology vol 22 no 7 pp 704ndash707 2011
[46] A J Lucendo A Arias L C De Rezende et al ldquoSubepithelialcollagen deposition profibrogenic cytokine gene expressionand changes after prolonged fluticasone propionate treatment inadult eosinophilic esophagitis a prospective studyrdquoThe Journalof Allergy and Clinical Immunology vol 128 no 5 pp 1037ndash1046 2011
[47] H Li D Zhang J Yu et al ldquoCCL18-dependent translocationof AMAP1 is critical for epithelial to mesenchymal transition inbreast cancerrdquo Journal of Cellular Physiology vol 233 no 4 pp3207ndash3217 2017
[48] Z Lin W Li H Zhang et al ldquoCCL18PITPNM3 enhancesmigration invasion and EMT through the NF-120581B signalingpathway in hepatocellular carcinomardquo Tumor Biology vol 37no 3 pp 3461ndash3468 2016
[49] F Meng W Li C Li Z Gao K Guo and S SongldquoCCL18 promotes epithelial-mesenchymal transition invasionand migration of pancreatic cancer cells in pancreatic ductaladenocarcinomardquo International Journal of Oncology vol 46 no3 pp 1109ndash1120 2015
[50] T Ploenes B Scholtes A Krohn et al ldquoCC-Chemokine Ligand18 Induces Epithelial to Mesenchymal Transition in LungCancer A549 Cells and Elevates the Invasive Potentialrdquo PLoSONE vol 8 no 1 Article ID e53068 2013
[51] J Rojas-Rivera C De La Piedra A Ramos A Ortiz andJ Egido ldquoThe expanding spectrum of biological actions ofvitamin Drdquo Nephrology Dialysis Transplantation vol 25 no 9pp 2850ndash2865 2010
[52] M Vulcano S Struyf P Scapini et al ldquoUnique regulation ofCCL18 production by maturing dendritic cellsrdquo The Journal ofImmunology vol 170 no 7 pp 3843ndash3849 2003
[53] R Kundu A Theodoraki C T Haas et al ldquoCell-type-specificmodulation of innate immune signalling by vitamin D inhuman mononuclear phagocytesrdquo The Journal of Immunologyvol 150 no 1 pp 55ndash63 2017
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
BioMed Research International 11
Results are shown as mean plusmn SD values of triplicates(A)ndash(C) Viability of monocytes upon culture during 24 48or 72 hours with decreasing concentrations of Zemplar orequivalent vehicle (305020 propylene glycolwaterethanol)(D)ndash(F) Viability of monocytes upon culture during 2448 or 72 hours with different concentrations of paricalcitol(pure substance) vitamin D or equivalent vehicle (EtOH)(Supplementary Materials)
References
[1] R T Krediet ldquoThe peritoneal membrane in chronic peritonealdialysisrdquo Kidney International vol 55 no 1 pp 341ndash356 1999
[2] R T Krediet ldquoPeritoneal physiology - Impact on solute andfluid clearancerdquo Advances in Chronic Kidney Disease vol 7 no4 pp 271ndash279 2000
[3] M Merkle and M Wrnle ldquoSclerosing peritonitis A rare butfatal complication of peritoneal inflammationrdquo Mediators ofInflammation vol 2012 Article ID 709673 p 4 2012
[4] D W Johnson Y Cho B E R Livingston et al ldquoEncapsulat-ing peritoneal sclerosis incidence predictors and outcomesrdquoKidney International vol 77 no 10 pp 904ndash912 2010
[5] G Balasubramaniam E A Brown and A Davenport ldquoThePan-Thames EPS study treatment and outcomes of encapsulat-ing peritoneal sclerosisrdquo Nephrology Dialysis Transplantation vol 24 no 10 pp 3209ndash3215 2009
[6] E D Sousa-Amorim G D Peso M A Bajo et al ldquoCan EPSdevelopment be avoided with early interventionsThe potentialrole of tamoxifenmdasha single-center studyrdquo Peritoneal DialysisInternational vol 34 no 6 pp 582ndash593 2014
[7] M R Korte D E SampimonM GH Betjes and R T KredietldquoEncapsulating peritoneal sclerosis the state of affairsrdquo NatureReviews Nephrology vol 7 no 9 pp 528ndash538 2011
[8] M A Bajo G del Peso and I Teitelbaum ldquoPeritoneal Mem-brane Preservationrdquo Seminars in Nephrology vol 37 no 1 pp77ndash92 2017
[9] M F Flessner ldquoInflammation from sterile dialysis solutions andthe longevity of the peritoneal barrierrdquo Clinical Nephrology vol68 no 6 pp 341ndash348 2007
[10] K N Lai and J C K Leung ldquoInflammation in peritonealdialysisrdquo Nephron Clinical Practice vol 116 no 1 pp c11ndashc182010
[11] S Yung and T M Chan ldquoPathophysiological changes to theperitoneal membrane during PD-related peritonitis the role ofmesothelial cellsrdquo Mediators of Inflammation vol 2012 ArticleID 484167 21 pages 2012
[12] L S Aroeira A Aguilera J A Sanchez-Tomero et al ldquoEpithe-lial tomesenchymal transition and peritonealmembrane failurein peritoneal dialysis patients pathologic significance andpotential therapeutic interventionsrdquo Journal of the AmericanSociety of Nephrology vol 18 no 7 pp 2004ndash2013 2007
[13] N Topley T Liberek A Davenport F-K Li H Fear andJ D Williams ldquoActivation of inflammation and leukocyterecruitment into the peritoneal cavityrdquo Kidney InternationalSupplements vol 50 no 56 pp S17ndashS21 1996
[14] F O Martınez and S Gordon ldquoThe M1 and M2 paradigmof macrophage activation time for reassessmentrdquo F1000PrimeReports vol 6 article 13 2014
[15] T Bellon V Martınez B Lucendo et al ldquoAlternative activa-tion of macrophages in human peritoneum implications for
peritoneal fibrosisrdquo Nephrology Dialysis Transplantation vol26 no 9 pp 2995ndash3005 2011
[16] A Prasse D V Pechkovsky G B Toews et al ldquoA vicious circleof alveolar macrophages and fibroblasts perpetuates pulmonaryfibrosis via CCL18rdquoAmerican Journal of Respiratory and CriticalCare Medicine vol 173 no 7 pp 781ndash792 2006
[17] J M Estep A Baranova N Hossain et al ldquoExpression ofcytokine signaling genes in morbidly obese patients with non-alcoholic steatohepatitis and hepatic fibrosisrdquo Obesity Surgeryvol 19 no 5 pp 617ndash624 2009
[18] F Kollert M Binder C Probst et al ldquoCCL18 - Potentialbiomarker of fibroinflammatory activity in chronic periaortitisrdquoThe Journal of Rheumatology vol 39 no 7 pp 1407ndash1412 2012
[19] A Prasse D V Pechkovsky G B Toews et al ldquoCCL18 as anindicator of pulmonary fibrotic activity in idiopathic interstitialpneumonias and systemic sclerosisrdquo Arthritis amp Rheumatologyvol 56 no 5 pp 1685ndash1693 2007
[20] S Ahmad B V North A Qureshi et al ldquoCCL18 in peritonealdialysis patients and encapsulating peritoneal sclerosisrdquo Euro-pean Journal of Clinical Investigation vol 40 no 12 pp 1067ndash1073 2010
[21] V Kodelja C Muller O Politz N Hakij C E Orfanos andS Goerdt ldquoAlternative macrophage activation-associated CC-chemokine-1 a novel structural homologue of macrophageinflammatory protein-1120572 with a Th2- associated expressionpatternrdquo The Journal of Immunology vol 160 no 3 pp 1411ndash1418 1998
[22] M Ossorio M A Bajo G Del Peso et al ldquoSustained low peri-toneal effluent CCL18 levels are associated with preservationof peritoneal membrane function in peritoneal dialysisrdquo PLoSONE vol 12 no 4 Article ID e0175835 2017
[23] G T Gonzalez-Mateo V Fernandez-Mıllara T Bellon etal ldquoParicalcitol reduces peritoneal fibrosis in mice throughthe activation of regulatory T cells and reduction in IL-17productionrdquo PLoS ONE vol 9 no 10 Article ID e108477 2014
[24] F Coronel S Cigarran A Gomis et al ldquoChanges in peritonealmembrane permeability and proteinuria in patients on peri-toneal dialysis after treatment with paricalcitolmdasha preliminarystudyrdquo Clinical Nephrology vol 78 no 2 pp 93ndash99 2012
[25] E Song N Ouyang M Horbelt B Antus M Wang and MS Exton ldquoInfluence of alternatively and classically activatedmacrophages on fibrogenic activities of human fibroblastsrdquoCellular Immunology vol 204 no 1 pp 19ndash28 2000
[26] S M Hurst T S Wilkinson R M McLoughlin et al ldquoIL-6 andits soluble receptor orchestrate a temporal switch in the patternof leukocyte recruitment seen during acute inflammationrdquoImmunity vol 14 no 6 pp 705ndash714 2001
[27] M Kozioł-Montewka A Ksiazek L Janicka and I BaranowiczldquoSerial cytokine changes in peritoneal effluent and plasma dur-ing peritonitis in patients on continuous ambulatory peritonealdialysis (CAPD)rdquo Inflammation Research vol 46 no 4 pp 132ndash136 1997
[28] E Schutyser A Richmond and J Van Damme ldquoInvolvementof CC chemokine ligand 18 (CCL18) in normal and pathologicalprocessesrdquo Journal of Leukocyte Biology vol 78 no 1 pp 14ndash262005
[29] I Schraufstatter H Takamori L Sikora P Sriramarao and RG DiScipio ldquoEosinophils and monocytes produce pulmonaryand activation-regulated chemokine which activates culturedmonocytesmacrophagesrdquoAmerican Journal of Physiology-LungCellular and Molecular Physiology vol 286 no 3 pp L494ndashL501 2004
12 BioMed Research International
[30] C Gunther N Carballido-Perrig T Kopp J M Carballidoand C Pfeiffer ldquoCCL18 is expressed in patients with bullouspemphigoid and parallels disease courserdquo British Journal ofDermatology vol 160 no 4 pp 747ndash755 2009
[31] AMantovani A Sica S Sozzani P Allavena A Vecchi andMLocati ldquoThe chemokine system in diverse forms of macrophageactivation and polarizationrdquo Trends in Immunology vol 25 no12 pp 677ndash686 2004
[32] C Aufricht R Beelen M Eberl et al ldquoBiomarker researchto improve clinical outcomes of peritoneal dialysis consensusof the European Training and Research in Peritoneal Dialysis(EuTRiPD) networkrdquo Kidney International vol 92 no 4 pp824ndash835 2017
[33] D Lopes Barreto andR T Krediet ldquoCurrent status and practicaluse of effluent biomarkers in peritoneal dialysis patientsrdquoAmerican Journal of Kidney Diseases vol 62 no 4 pp 823ndash8332013
[34] L A Boven M van Meurs R G Boot et al ldquoGaucher cellsdemonstrate a distinct macrophage phenotype and resemblealternatively activated macrophagesrdquo American Journal of Clin-ical Pathology vol 122 no 3 pp 359ndash369 2004
[35] L Cocho I Fernandez M Calonge et al ldquoGene expression-based predictive models of graft versus host disease-associateddry eyerdquo Investigative Ophtalmology Visual Science vol 56 no8 pp 4570ndash4581 2015
[36] MHasegawa ldquoBiomarkers in systemic sclerosisTheir potentialto predict clinical coursesrdquoThe Journal of Dermatology vol 43no 1 pp 29ndash38 2016
[37] S A Jones D J Fraser C A Fielding and G W JonesldquoInterleukin-6 in renal disease and therapyrdquoNephrologyDialysisTransplantation vol 30 no 4 pp 564ndash574 2015
[38] A Munitz and F Levi-Schaffer ldquoEosinophils rsquoNewrsquo roles forrsquooldrsquo cellsrdquo Allergy European Journal of Allergy and ClinicalImmunology vol 59 no 3 pp 268ndash275 2004
[39] S S Aceves ldquoRemodeling and fibrosis in chronic eosinophilinflammationrdquo Digestive Diseases vol 32 no 1-2 pp 15ndash212014
[40] Y Nakamura H Okada A Yasui T Koh and T YamaneldquoSclerosing encapsulating peritonitis associated with recurrenteosinophilic peritonitisrdquo Nephrology Dialysis Transplantation vol 14 no 3 pp 768ndash770 1999
[41] S P Atamas I G Luzina J Choi et al ldquoPulmonary andactivation-regulated chemokine stimulates collagen productionin lung fibroblastsrdquo American Journal of Respiratory Cell andMolecular Biology vol 29 no 6 pp 743ndash749 2003
[42] A Wimmer S K Khaldoyanidi M Judex N Serobyan RG DiScipio and I U Schraufstatter ldquoCCL18PARC stimulateshematopoiesis in long-term bone marrow cultures indirectlythrough its effect on monocytesrdquo Blood vol 108 no 12 pp3722ndash3729 2006
[43] A Prasse C Probst E Bargagli et al ldquoSerum CC-chemokineligand 18 concentration predicts outcome in idiopathic pul-monary fibrosisrdquo American Journal of Respiratory and CriticalCare Medicine vol 179 no 8 pp 717ndash723 2009
[44] J Chen Y Yao C Gong et al ldquoCCL18 from tumor-associatedmacrophages promotes breast cancermetastasis via PITPNM3rdquoCancer Cell vol 19 no 4 pp 541ndash555 2011
[45] K L Hon G K Ching P C Ng and T F Leung ldquoExplor-ing CCL18 eczema severity and atopyrdquo Pediatric Allergy andImmunology vol 22 no 7 pp 704ndash707 2011
[46] A J Lucendo A Arias L C De Rezende et al ldquoSubepithelialcollagen deposition profibrogenic cytokine gene expressionand changes after prolonged fluticasone propionate treatment inadult eosinophilic esophagitis a prospective studyrdquoThe Journalof Allergy and Clinical Immunology vol 128 no 5 pp 1037ndash1046 2011
[47] H Li D Zhang J Yu et al ldquoCCL18-dependent translocationof AMAP1 is critical for epithelial to mesenchymal transition inbreast cancerrdquo Journal of Cellular Physiology vol 233 no 4 pp3207ndash3217 2017
[48] Z Lin W Li H Zhang et al ldquoCCL18PITPNM3 enhancesmigration invasion and EMT through the NF-120581B signalingpathway in hepatocellular carcinomardquo Tumor Biology vol 37no 3 pp 3461ndash3468 2016
[49] F Meng W Li C Li Z Gao K Guo and S SongldquoCCL18 promotes epithelial-mesenchymal transition invasionand migration of pancreatic cancer cells in pancreatic ductaladenocarcinomardquo International Journal of Oncology vol 46 no3 pp 1109ndash1120 2015
[50] T Ploenes B Scholtes A Krohn et al ldquoCC-Chemokine Ligand18 Induces Epithelial to Mesenchymal Transition in LungCancer A549 Cells and Elevates the Invasive Potentialrdquo PLoSONE vol 8 no 1 Article ID e53068 2013
[51] J Rojas-Rivera C De La Piedra A Ramos A Ortiz andJ Egido ldquoThe expanding spectrum of biological actions ofvitamin Drdquo Nephrology Dialysis Transplantation vol 25 no 9pp 2850ndash2865 2010
[52] M Vulcano S Struyf P Scapini et al ldquoUnique regulation ofCCL18 production by maturing dendritic cellsrdquo The Journal ofImmunology vol 170 no 7 pp 3843ndash3849 2003
[53] R Kundu A Theodoraki C T Haas et al ldquoCell-type-specificmodulation of innate immune signalling by vitamin D inhuman mononuclear phagocytesrdquo The Journal of Immunologyvol 150 no 1 pp 55ndash63 2017
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
12 BioMed Research International
[30] C Gunther N Carballido-Perrig T Kopp J M Carballidoand C Pfeiffer ldquoCCL18 is expressed in patients with bullouspemphigoid and parallels disease courserdquo British Journal ofDermatology vol 160 no 4 pp 747ndash755 2009
[31] AMantovani A Sica S Sozzani P Allavena A Vecchi andMLocati ldquoThe chemokine system in diverse forms of macrophageactivation and polarizationrdquo Trends in Immunology vol 25 no12 pp 677ndash686 2004
[32] C Aufricht R Beelen M Eberl et al ldquoBiomarker researchto improve clinical outcomes of peritoneal dialysis consensusof the European Training and Research in Peritoneal Dialysis(EuTRiPD) networkrdquo Kidney International vol 92 no 4 pp824ndash835 2017
[33] D Lopes Barreto andR T Krediet ldquoCurrent status and practicaluse of effluent biomarkers in peritoneal dialysis patientsrdquoAmerican Journal of Kidney Diseases vol 62 no 4 pp 823ndash8332013
[34] L A Boven M van Meurs R G Boot et al ldquoGaucher cellsdemonstrate a distinct macrophage phenotype and resemblealternatively activated macrophagesrdquo American Journal of Clin-ical Pathology vol 122 no 3 pp 359ndash369 2004
[35] L Cocho I Fernandez M Calonge et al ldquoGene expression-based predictive models of graft versus host disease-associateddry eyerdquo Investigative Ophtalmology Visual Science vol 56 no8 pp 4570ndash4581 2015
[36] MHasegawa ldquoBiomarkers in systemic sclerosisTheir potentialto predict clinical coursesrdquoThe Journal of Dermatology vol 43no 1 pp 29ndash38 2016
[37] S A Jones D J Fraser C A Fielding and G W JonesldquoInterleukin-6 in renal disease and therapyrdquoNephrologyDialysisTransplantation vol 30 no 4 pp 564ndash574 2015
[38] A Munitz and F Levi-Schaffer ldquoEosinophils rsquoNewrsquo roles forrsquooldrsquo cellsrdquo Allergy European Journal of Allergy and ClinicalImmunology vol 59 no 3 pp 268ndash275 2004
[39] S S Aceves ldquoRemodeling and fibrosis in chronic eosinophilinflammationrdquo Digestive Diseases vol 32 no 1-2 pp 15ndash212014
[40] Y Nakamura H Okada A Yasui T Koh and T YamaneldquoSclerosing encapsulating peritonitis associated with recurrenteosinophilic peritonitisrdquo Nephrology Dialysis Transplantation vol 14 no 3 pp 768ndash770 1999
[41] S P Atamas I G Luzina J Choi et al ldquoPulmonary andactivation-regulated chemokine stimulates collagen productionin lung fibroblastsrdquo American Journal of Respiratory Cell andMolecular Biology vol 29 no 6 pp 743ndash749 2003
[42] A Wimmer S K Khaldoyanidi M Judex N Serobyan RG DiScipio and I U Schraufstatter ldquoCCL18PARC stimulateshematopoiesis in long-term bone marrow cultures indirectlythrough its effect on monocytesrdquo Blood vol 108 no 12 pp3722ndash3729 2006
[43] A Prasse C Probst E Bargagli et al ldquoSerum CC-chemokineligand 18 concentration predicts outcome in idiopathic pul-monary fibrosisrdquo American Journal of Respiratory and CriticalCare Medicine vol 179 no 8 pp 717ndash723 2009
[44] J Chen Y Yao C Gong et al ldquoCCL18 from tumor-associatedmacrophages promotes breast cancermetastasis via PITPNM3rdquoCancer Cell vol 19 no 4 pp 541ndash555 2011
[45] K L Hon G K Ching P C Ng and T F Leung ldquoExplor-ing CCL18 eczema severity and atopyrdquo Pediatric Allergy andImmunology vol 22 no 7 pp 704ndash707 2011
[46] A J Lucendo A Arias L C De Rezende et al ldquoSubepithelialcollagen deposition profibrogenic cytokine gene expressionand changes after prolonged fluticasone propionate treatment inadult eosinophilic esophagitis a prospective studyrdquoThe Journalof Allergy and Clinical Immunology vol 128 no 5 pp 1037ndash1046 2011
[47] H Li D Zhang J Yu et al ldquoCCL18-dependent translocationof AMAP1 is critical for epithelial to mesenchymal transition inbreast cancerrdquo Journal of Cellular Physiology vol 233 no 4 pp3207ndash3217 2017
[48] Z Lin W Li H Zhang et al ldquoCCL18PITPNM3 enhancesmigration invasion and EMT through the NF-120581B signalingpathway in hepatocellular carcinomardquo Tumor Biology vol 37no 3 pp 3461ndash3468 2016
[49] F Meng W Li C Li Z Gao K Guo and S SongldquoCCL18 promotes epithelial-mesenchymal transition invasionand migration of pancreatic cancer cells in pancreatic ductaladenocarcinomardquo International Journal of Oncology vol 46 no3 pp 1109ndash1120 2015
[50] T Ploenes B Scholtes A Krohn et al ldquoCC-Chemokine Ligand18 Induces Epithelial to Mesenchymal Transition in LungCancer A549 Cells and Elevates the Invasive Potentialrdquo PLoSONE vol 8 no 1 Article ID e53068 2013
[51] J Rojas-Rivera C De La Piedra A Ramos A Ortiz andJ Egido ldquoThe expanding spectrum of biological actions ofvitamin Drdquo Nephrology Dialysis Transplantation vol 25 no 9pp 2850ndash2865 2010
[52] M Vulcano S Struyf P Scapini et al ldquoUnique regulation ofCCL18 production by maturing dendritic cellsrdquo The Journal ofImmunology vol 170 no 7 pp 3843ndash3849 2003
[53] R Kundu A Theodoraki C T Haas et al ldquoCell-type-specificmodulation of innate immune signalling by vitamin D inhuman mononuclear phagocytesrdquo The Journal of Immunologyvol 150 no 1 pp 55ndash63 2017
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom