Research Article Gram-Negative Bacterial …downloads.hindawi.com/journals/ije/2013/789012.pdfGram-Negative Bacterial Lipopolysaccharide Stimulates Activin A Secretion from Human Amniotic

Hindawi Publishing CorporationInternational Journal of EndocrinologyVolume 2013 Article ID 789012 6 pageshttpdxdoiorg1011552013789012

Research ArticleGram-Negative Bacterial Lipopolysaccharide StimulatesActivin A Secretion from Human Amniotic Epithelial Cells

Yumiko Abe1 Risa Marukawa2 Nami Tsuru3 Maki Sato4 Hiroko Matsuda5

Hisanobu Sadakata5 Takashi Kameda5 and Takashi Minegishi5

1 Department of Laboratory Sciences Graduate School of Health Sciences Gunma University 3-39-22 Showa MaebashiGunma 371-8514 Japan

2 Kuki General Hospital Kuki Saitama 346-0021 Japan3Miyazaki Prefectural Nobeoka Hospital Nobeoka Miyazaki 882-0835 Japan4Yokota Maternity Hospital Maebashi Gunma 371-0031 Japan5Department of Obstetrics and Gynecology Graduate School of Medicine Gunma University 3-39-22 Showa MaebashiGunma 371-8511 Japan

Correspondence should be addressed to Yumiko Abe abegunma-uacjp

Received 29 April 2013 Accepted 1 July 2013

Academic Editor Maria L Dufau

Copyright copy 2013 Yumiko Abe 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

Activin A is involved in inflammation The present study was performed to clarify if lipopolysaccharide a component of Gram-negative bacteria stimulates activin A secretion from human amniotic epithelial cells and to determine if activin A plays arole in amnionitis Fetal membranes were obtained during elective cesarean sections performed in full-term pregnancies ofpatients without systemic disease signs of premature delivery or fetal complications Amniotic epithelial cells were isolated bytrypsinization The activin A concentrations in the culture media were measured by enzyme-linked immunosorbent assay andcell proliferation was assessed by 5-bromo-21015840-deoxyuridine incorporation Amniotic epithelial cells secreted activin A in a celldensity-dependent manner and lipopolysaccharide (10120583gmL) enhanced the secretion at each cell density Lipopolysaccharide(10ndash50120583gmL) also stimulated activin A secretion in a dose-dependent manner Contrary to the effect of activin A secretionlipopolysaccharide inhibited cell proliferation in amniotic epithelial cells The present study suggests that lipopolysaccharidestimulation of activin A secretion may be a mechanism in the pathogenesis of amnionitis

1 Introduction

Activins which were first identified as stimulators of FSHsecretion are pluripotent growth factors in the TGF-betasuperfamily [1] Among the many functions of activins theinvolvement of activin A in inflammation has been noted [1ndash6] The administration of lipopolysaccharide (LPS) a Gram-negative bacterial cell wall component prominently increasesthe serum activin A level in sheep and mice [7 8] Activin Alevels in the circulation were higher in mice that died than inmice that survived after the administration of a sublethal doseof LPS [8] Furthermore cotreatment with follistatin whichneutralizes activin by binding to it increased the survival rateof LPS-treatedmice [8]The activinA release inmice depends

on a signaling cascade through Toll-like receptor 4 (TLR4) areceptor for LPS [8]

Serum activin A concentrations are elevated in patientswith septicemia [9] During pregnancy serum activin Aconcentrations increase [10] and activin A dimers and activinbeta-A subunits are detected in trophoblasts and amnioticepithelial cells (AEC) in the human placenta [11 12] Theexpression of activin beta-A subunit mRNA in fetal mem-branes increases during labor [13] Activin A concentrationsin amniotic fluid are higher in women with preterm laborthan in women without preterm labor at the same stage ofgestation [13] Activin A concentrations in amniotic fluid arealso elevated in women with intra-amniotic infection [14]

2 International Journal of Endocrinology

The notion that activin A is involved in chorioamnionitisis also supported by in vitro studies Activin A secretionfrom gestational tissues and cells is stimulated by inflamma-tory cytokines Tumor necrosis factor-120572 (TNF-120572) stimulatesactivin A production in explant cultures of human amnionand choriodecidua [15] and cultured human AEC [16]Interleukin (IL)-1120573 also stimulates activin A production inthe amnion choriodecidua [15] and AEC [16] Activin Amodulates the secretion of IL-6 IL-8 and prostaglandin E2in explant cultures of human amnion and choriodecidua [17]

Compared to the effects of TNF-120572 and IL-1120573 the effect ofLPS on activin A secretion from human AEC is inconsistentdespite the expression of functional TLR4 in human AEC[18] Rosenberg et al reported that LPS stimulated activin Arelease from cultured amniochorion explants but not fromplacental villous tissue [14] On the other hand Keelanet al reported that LPS did not affect activin A secretionfrom amnion explant cultures [15] Since the amnion is anavascular tissue andAEC are located in the innermost layer ofthe amnion secretions of AEC must be directly released intothe amniotic fluid and affect the fetus The present study wasperformed to determine if LPS stimulates activin A secretionfrom AEC and to verify the notion that activin A is involvedin amnionitis

2 Materials and Methods

21 Reagents LPS that was phenol extracted from E coliO26 was purchased from Paesel amp Lorei GmbH (HanauGermany) (catalogue no 100976 Lot 15303)

22 AEC Culture With the permission of the InstitutionalReview Board of Gunma University Hospital and the writteninformed consent of the patients we obtained fetal mem-brane samples during elective cesarean sections performedon four patients with full-term pregnancies who did nothave any systemic disease signs of premature deliveryor fetal complications AEC were prepared as previouslyreported [19] on the basis of the method established byOkita et al [20] with slight modifications Briefly thechorion was removed from amnion mechanically and theamnion was washed thoroughly with phosphate-bufferedsaline The removal of the chorion was ascertained by usinghematoxylineosin-stained paraffin sections of amnions Theamniotic membrane was cut into pieces and incubated in170mL of Krebs-Ringer solution containing 015 trypsin126mgmL sodium bicarbonate 25mM HEPES 100 120583gmLstreptomycin and 05 120583gmL amphotericin B at 37∘C in aspinner flask The liberated cells were decanted at 30minintervals and the incubationwas performed seven times withfreshly made trypsin solution Each fraction of dispersedcells was centrifuged and resuspended in DMEHamrsquos F12medium supplemented with 10 fetal bovine serum (FBS)100 120583gmL streptomycin and 05 120583gmL amphotericin BThefirst fractionwas discardedThe cell viability of the remainingfractions was determined by trypan blue exclusion and thefractions with viabilities of at least 80 were pooled Cells in

0

200

400

600

800

Activ

in A

(pg

mL)

ControlLPS

a

b

b

1250 2500 5000 10000 20000Cell number (well)

1000

1200

1400

1600 lowast

lowast

lowast

lowast

Figure 1 Effect of LPS on activin A secretion at various densities ofAEC Human AEC were seeded in 96-well microplates at densitiesof 1250 2500 5000 10000 and 20000 cells per well in 200 120583L ofDMEHamrsquos F12 medium supplemented with 10 FBS 100120583gmLstreptomycin and 05 120583gmL amphotericin B Either LPS (10 120583gmL)or vehicle (control) was added to each well After 96 h of cultureactivin A in the medium was measured Closed rectangles LPSOpen rectangles control The results from the quadruplicate assayare shown as the mean plusmn SE (119899 = 4) a

119875 lt 005 compared withthe control value at 1250 cellswell b

119875 lt 001 compared with thecontrol value at 1250 cellswell lowast119875 lt 001 comparedwith the controlvalue at each cell density

200120583L of medium were seeded in each well of 96-well platesand cultured in a humidified atmosphere containing 5CO

2-

95 air at 37∘C These cells were used for measuring activinA and cell proliferation

23 Activin A ELISA Activin A concentrations in culturemedium were measured by using an activin A assay kit(Oxford Bio-Innovations Oxfordshire UK) according to themanufacturerrsquos instructions The samples which were pre-treated with sodium dodecylsulfate and hydrogen peroxidewere added along with assay diluent to microwells coatedwith a monoclonal antibody specific for the beta-A subunitof activin After 1 h of incubation biotinylated monoclonalantibody for the beta-A subunit of activin was added andincubated overnight The following day wells were washedand streptavidin-alkaline phosphatase solution was addedAfter 1 h of incubation the wells were washed and incubatedwith substrate solution for 2 h Then amplifier solution wasadded and the absorbance at 490 nm was measured

International Journal of Endocrinology 3

0

100

200

300

400

500

600

700

800

0 1 3 10 30

Activ

in A

conc

entr

atio

n (p

gm

L)

lowast

lowast

LPS (120583gmL)

(a)

0

100

200

300

400

500

600

700

800

Activ

in A

conc

entr

atio

n (p

gm

L)

0 1 3 10 50

lowast

lowast

LPS (120583gmL)

(b)

Activ

in A

conc

entr

atio

n (p

gm

L)

LPS (120583gmL)0 1 10 30 50

350

300

250

200

150

100

50

0

lowast

lowast

(c)

Figure 2 Dose-dependent effects of LPS on activinA secretion in three independent experiments Fetalmembraneswere obtained from threepatients andAEC from each patient were cultured independently at a density of 20000 cells per well inDMEHamrsquos 12medium supplementedwith 10 FBS 100120583gmL streptomycin 05 120583gmL amphotericin B and various concentrations of LPS for 96 h Each independent result isshown in panels (a) (b) and (c) The results are shown as the mean plusmn SE (119899 = 4) 119875 lt 005 compared with the control (LPS 0 ngmL) valuelowast119875 lt 001 compared with the control (LPS 0 ngmL) value

24 Cell Proliferation Assay Cell proliferation was measuredby assessing 5-bromo-21015840-deoxyuridine (BrdU) incorporationby using a Cell Proliferation ELISA BrdU (colorimetric) kit(Roche Diagnostics Mannheim Germany) The assay wasperformed in accordance with the manufacturerrsquos instruc-tions Cells were seeded into 96-well microplates with orwithout LPS After a 96 h incubation 20120583L of 100120583MBrdU solution was added to each well containing 200120583L ofmedium and the cells were reincubated for another 24 hThe culture medium was removed the cells were fixed andthe DNA was denatured The cells were then incubatedwith mouse anti-BrdU monoclonal antibody conjugated toperoxidase at room temperature for 90min After removalof the antibody the immune complexes were detected bysubsequent reaction with tetramethylbenzidineThe reactionwas stopped by the addition of sulfuric acid and the productwas quantified by measuring the absorbance at 450 nm

25 Statistics The data from quadruplicate cultures arepresented as themeanplusmn SE Comparison between groupswasperformed by using one-way ANOVA and the significanceof the differences between the mean values was tested byusing Fisherrsquos PLSD test Comparison between two groupswas performed by using the Studentrsquos t-test 119875 values lt 005were considered statistically significant

3 Results

Variable densities of AECwere seeded into 96-well plates andincubated After 96 h of incubation activin A concentrations

in the media were measured Increased activin A concentra-tions in themedia were observed inAECwithout LPS in a celldensity-dependent manner and the activin A concentrationswere significantly higher at cell densities of 5000 cellswellor higher (119875 lt 005) (Figure 1) LPS (10 120583gmL) significantlyincreased activin A concentrations at densities of 2500cellswell and greater (119875 lt 001) Activin A concentrations ofcell lysates from either LPS-stimulated AEC or control AECwere below assay sensitivity (data not shown) Thereforethe increase of activin A concentrations in the mediumwas equivalent to the increase of activin A production andsecretion in AEC

The stimulatory effects of various concentrations of LPSon activin A secretion in AEC were confirmed by threeindependent studies of AEC from three patients In eachexperiment a dose-dependent increase in activin A secretionfrom AEC occurred after 96 h of culture with LPS (Figure 2)

The effects of LPS on AEC proliferation were also stud-ied LPS (10120583gmL) suppressed cell proliferation at eachcell density (1250ndash20000 cellswell) (Figure 3(a)) A dose-dependent inhibitory effect of LPS on AEC proliferation wasalso shown by three independent studies that used AEC fromthree patients (Figure 3(b))

4 Discussion

AEC secreted activin A in a cell density-dependent mannerin cultures of AEC prepared from the trypsinization ofamnions from women with full-term pregnancies This typeof AEC culture has been utilized in studies of the syntheses


ControlLPS

00

01

02

03

04

05

06

lowast

lowast

lowast

Cell number (well)1250 2500 5000 10000 20000

Abs4

50

nm

(a)

20

40

60

80

100

120

Abso

rban

ce (

)

00 10 100

LPS (120583gmL)

lowast

lowast

lowast

lowast

(b)

Figure 3 Effect of LPS on BrdU incorporation in AEC (a) AEC were seeded in 96-well microplates at densities of 1250 2500 500010000 and 20000 cells per well in 200 120583L of DMEHamrsquos F12 medium supplemented with 10 FBS 100120583gmL streptomycin and 05 120583gmLamphotericin B Either LPS (10 120583gmL) or vehicle (control) was added to each well After 96 h of culture BrdU incorporation was studiedThe results from the quadruplicate assay are shown as the mean plusmn SE (119899 = 4) 119875 lt 005 compared with the control value at each cell densitylowast119875 lt 001 comparedwith the control value at each cell density (b) Fetalmembranes were obtained from three patients AEC from each patientwere cultured independently in DMEHamrsquos 12 medium supplemented with 10FBS 100120583gmL streptomycin 05 120583gmL amphotericin Band various concentrations of LPSAfter 96 hof culture BrdU incorporationwas studied Closed circles closed rectangles and closed trianglesshow AEC from each patient The absorbance at AEC cultured without LPS is shown as 100 The results from the quadruplicate assay areshown as the mean plusmn SE (119899 = 4) 119875 lt 005 compared with the control (LPS 0 ngmL) value lowast119875 lt 001 compared with the control (LPS0 ngmL) value

and secretion of phospholipids and prostaglandins [20 21]matrix metalloproteinase-9 and extracellular matrix metal-loproteinase inducer [22 23] brain natriuretic peptide [24]endothelin-1 [24 25] fibronectin [26 27] albumin and glyco-gen [28] and cystic fibrosis transmembrane conductanceregulator [29] Keelan et al used this method to study activinA production by AEC [16] The cell density and incubationtime of our AEC cultures were comparable (although notidentical) to the conditions used by Keelan et al

LPS enhanced the activin A secretion at each cell density(2500ndash20000 cellswell) LPS also stimulated activin Asecretion dose dependently in three independent culturesof AEC from three patients Rosenberg et al reported thatLPS stimulated activinA release from cultured amniochorionexplants but not from placental villous tissue [14] Our studyhas clearly shown that LPS stimulates activin A secretionfrom AEC On the other hand Keelan et al reported thatLPS did not affect activin A production in amnion explantcultures [15] The components of amniotic explants otherthan AEC might inhibit the effects of LPS or the LPS doseused in their study (5 120583gmL) might not be sufficient tostimulate activin A secretion In a mouse epithelial Sertoli

cell line the secretion of activin A is enhanced through TLR4by LPS stimulation [8] Since functional TLR4 is expressedin human AEC [18] future studies must determine if similarmechanisms affect activin A secretion by LPS-stimulatedAEC

LPS stimulated activin A secretion from AEC at dosesof 10 120583gmL or higher The endotoxin concentrations inthe amniotic fluid of women with premature rupture ofmembranes were between several hundred pgmL to several120583gmL as determined by the Limulus amebocyte lysateassay with Ecoli LPS as the standard [30] Compared tothe endotoxin concentrations in the amniotic fluid the LPSdoses that stimulated activin A secretion from AEC werehigher On the other hand the LPS doses used in the presentstudy are comparable to the doses used in previous studiesof human gestational tissues and cells [31ndash35] Local LPSconcentrations in the microenvironment of AEC must behigher than the concentrations in amniotic fluid whenGram-negative bacteria invade the amnion [36]

LPS suppressed cell proliferation Therefore the increaseof activin A secretion is caused by enhanced productionand secretion of activin A in AEC rather than an increased


number of AEC LPS induces apoptosis directly or indirectlyin several types of cells [37ndash41] Apoptosis of AEC occurs infetal membranes from patients with chorioamnionitis [42]The present results are in accordance with these findingsThe tensile strength of fetal membranes is provided bycollagens in the amnion and the tensile strength is reducedin chorioamnionitis by the degradation of collagens bymatrixmetalloproteinases [43] LPS itself might weaken the strengthof membranes by suppressing AEC proliferation

In conclusion LPS stimulated activin A secretion fromhuman AEC whichmay be a mechanism in the pathogenesisof amnionitis

Acknowledgments

The authors thank Ms Junko Sakurai for her assistanceThis work was supported by a Grant-in-Aid from the JapanSociety for the Promotion of Science (15591729 18591794 and22591816)

References

[1] D M de Kretser R E OrsquoHehir C L Hardy and M P HedgerldquoThe roles of activin A and its binding protein follistatinin inflammation and tissue repairrdquo Molecular and CellularEndocrinology vol 359 no 1-2 pp 101ndash106 2012

[2] S Werner and C Alzheimer ldquoRoles of activin in tissue repairfibrosis and inflammatory diseaserdquoCytokine andGrowth FactorReviews vol 17 no 3 pp 157ndash171 2006

[3] D J Phillips D M de Kretser and M P Hedger ldquoActivinand related proteins in inflammation not just interestedbystandersrdquo Cytokine and Growth Factor Reviews vol 20 no2 pp 153ndash164 2009

[4] S Ebert R Nau and U Michel ldquoRole of activin in bacte-rial infections a potential target for immunointerventionrdquoImmunotherapy vol 2 no 5 pp 673ndash684 2010

[5] S Huber andC Schramm ldquoRole of activin A in the induction ofFoxp3+ and Foxp3minus CD4+ regulatory T cellsrdquo Critical Reviewsin Immunology vol 31 no 1 pp 53ndash60 2011

[6] M P Hedger W R Winnall D J Phillips and D M deKretser ldquoThe regulation and functions of activin and follistatinin inflammation and immunityrdquo Vitamins and Hormones vol85 pp 255ndash297 2011

[7] K L Jones J N Brauman N P Groome D M De Kretserand D J Phillips ldquoActivin A release into the circulation is anearly event in systemic inflammation and precedes the releaseof follistatinrdquo Endocrinology vol 141 no 5 pp 1905ndash1908 2000

[8] K L Jones A Mansell S Patella et al ldquoActivin A is a criticalcomponent of the inflammatory response and its binding pro-tein follistatin reduces mortality in endotoxemiardquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 104 no 41 pp 16239ndash16244 2007

[9] U Michel S Ebert D Phillips and R Nau ldquoSerum concen-trations of activin and follistatin are elevated and run in parallelin patients with septicemiardquo European Journal of Endocrinologyvol 148 no 5 pp 559ndash564 2003

[10] S Muttukrishna P A Fowler L George N P Groome and PG Knight ldquoChanges in peripheral serum levels of total activinA during the humanmenstrual cycle and pregnancyrdquo Journal ofClinical Endocrinology and Metabolism vol 81 no 9 pp 3328ndash3334 1996

[11] J Rabinovici P C Goldsmith C L Librach and R B JaffeldquoLocalization and regulation of the activin-A dimer inhuman placental cellsrdquo Journal of Clinical Endocrinology andMetabolism vol 75 no 2 pp 571ndash576 1992

[12] F Petraglia M M Anceschi L Calza et al ldquoInhibin andactivin in human fetal membranes evidence for a local effecton prostaglandin releaserdquo Journal of Clinical Endocrinology andMetabolism vol 77 no 2 pp 542ndash548 1993

[13] F Petraglia A M Di Blasio P Florio et al ldquoHigh levels offetalmembrane activin120573Aand activin receptor IIBmRNAs andaugmented concentration of amniotic fluid activin A in womenin term or preterm laborrdquo Journal of Endocrinology vol 154 no1 pp 95ndash101 1997

[14] VA Rosenberg I A Buhimschi A TDulay et al ldquoModulationof amniotic fluid activin-a and inhibin-a in women withpreterm premature rupture of the membranes and infection-induced preterm birthrdquo American Journal of ReproductiveImmunology vol 67 no 2 pp 122ndash131 2012

[15] J A Keelan R L Zhou L W Evans N P Groome and MD Mitchell ldquoRegulation of activin A inhibin A and follistatinproduction in human amnion and choriodecidual explants byinflammatory mediatorsrdquo Journal of the Society for GynecologicInvestigation vol 7 no 5 pp 291ndash296 2000

[16] J A Keelan N P Groome and M D Mitchell ldquoRegulation ofactivin-A production by human amnion decidua and placentain vitro by pro-inflammatory cytokinesrdquo Placenta vol 19 no5-6 pp 429ndash434 1998

[17] J A Keelan R-L Zhou and M D Mitchell ldquoActivin a exertsboth pro- and anti-inflammatory effects on human term gesta-tional tissuesrdquo Placenta vol 21 no 1 pp 38ndash43 2000

[18] K M Adams J Lucas R P Kapur and A M Stevens ldquoLPSinduces translocation of TLR4 in amniotic epitheliumrdquo Pla-centa vol 28 no 5-6 pp 477ndash481 2007

[19] Y Abe H Sinozaki T Takagi et al ldquoIdentification of 2378-tetrachlorodibenzo-p-dioxin (TCDD)-inducible genes inhuman amniotic epithelial cellsrdquo Reproductive Biology andEndocrinology vol 4 article no 27 2006

[20] J R Okita N Sagawa M L Casey and J M Snyder ldquoAcomparison of human amnion tissue and amnion cells inprimary culture by morphological and biochemical criteriardquo InVitro vol 19 no 2 pp 117ndash126 1983

[21] K Sun R Ma X Cui et al ldquoGlucocorticoids induce cytosolicphospholipase A2 and prostaglandin H synthase type 2 butnot microsomal prostaglandin E synthase (PGES) and cytosolicPGES expression in cultured primary human amnion cellsrdquoJournal of Clinical Endocrinology and Metabolism vol 88 no11 pp 5564ndash5571 2003

[22] W Li and J R G Challis ldquoCorticotropin-releasing hormoneand urocortin induce secretion of matrix metalloproteinase-9 (MMP-9) without change in tissue inhibitors of MMP-1 bycultured cells from human placenta and fetal membranesrdquoJournal of Clinical Endocrinology and Metabolism vol 90 no12 pp 6569ndash6574 2005

[23] W Li N Alfaidy and J R G Challis ldquoExpression of extracel-lular matrix metalloproteinase inducer in human placenta andfetal membranes at term laborrdquo Journal of Clinical Endocrinol-ogy and Metabolism vol 89 no 6 pp 2897ndash2904 2004

[24] H Itoh N Sagawa M Hasegawa et al ldquoBrain natriureticpeptide is present in the human amniotic fluid and is secretedfrom amnion cellsrdquo Journal of Clinical Endocrinology andMetabolism vol 76 no 4 pp 907ndash911 1993


[25] H Itoh N Sagawa M Hasegawa et al ldquoTransforming growthfactor-120573 stimulates and glucocorticoids and epidermal growthfactor inhibit brain natriuretic peptide secretion from culturedhuman amnion cellsrdquo Journal of Clinical Endocrinology andMetabolism vol 79 no 1 pp 176ndash182 1994

[26] S Guller L Kong RWozniak and C J Lockwood ldquoReductionof extracellular matrix protein expression in human amnionepithelial cells by glucocorticoids a potential role in pretermrupture of the fetal membranesrdquo Journal of Clinical Endocrinol-ogy and Metabolism vol 80 no 7 pp 2244ndash2250 1995

[27] Y Ma C J Lockwood A L Bunim D A Giussani P WNathanielsz and S Guller ldquoCell type-specific regulation offetal fibronectin expression in amnion conservation of gluco-corticoid responsiveness in human and nonhuman primatesrdquoBiology of Reproduction vol 62 no 6 pp 1812ndash1817 2000

[28] S Takashima H Ise P Zhao T Akaike and T NikaidoldquoHuman amniotic epithelial cells possess hepatocyte-like char-acteristic and functionsrdquoCell Structure and Function vol 29 no3 pp 73ndash84 2004

[29] S V Murphy R Lim P Heraud et al ldquoHuman amnion epi-thelial cells induced to express functional cystic fibrosis trans-membrane conductance regulatorrdquo PLoS One vol 7 no 9Article ID e46533 2012

[30] R Romero P Roslansky EOyarzun et al ldquoLabor and infectionII Bacterial endotoxin in amniotic fluid and its relationship tothe onset of preterm laborrdquo American Journal of Obstetrics andGynecology vol 158 no 5 pp 1044ndash1049 1988

[31] J A Keelan T Sato andM DMitchell ldquoInterleukin (IL)-6 andIL-8 production by human amnion regulation by cytokinesgrowth factors glucocorticoids phorbol esters and bacteriallipopolysacchariderdquo Biology of Reproduction vol 57 no 6 pp1438ndash1444 1997

[32] M Lappas M Permezel H M Georgiou and G E RiceldquoRegulation of phospholipase isozymes by nuclear factor-kappaB in human gestational tissues in vitrordquo Journal of ClinicalEndocrinology and Metabolism vol 89 no 5 pp 2365ndash23722004

[33] M D Mitchell M C Chang T Chaiworapongsa et al ldquoIdenti-fication of 911120572-prostaglandin F2 in human amniotic fluid andcharacterization of its production by human gestational tissuesrdquoJournal of Clinical Endocrinology and Metabolism vol 90 no 7pp 4244ndash4248 2005

[34] K Y Kim H K Shin J M Choi and K W Hong ldquoInhibitionof lipopolysaccharide-induced apoptosis by cilostazol in humanumbilical vein endothelial cellsrdquo Journal of Pharmacology andExperimental Therapeutics vol 300 no 2 pp 709ndash715 2002

[35] J A Keelan S Khan F Yosaatmadja and M D MitchellldquoPrevention of inflammatory activation of human gestationalmembranes in an ex vivomodel using a pharmacologicalNF-120581Binhibitorrdquo Journal of Immunology vol 183 no 8 pp 5270ndash52782009

[36] A Splichalova I Trebichavsky Y Muneta Y Mori and ISplichal ldquoEffect of bacterial virulence on IL-18 expression inthe amnion infected with Escherichia colirdquo American Journal ofReproductive Immunology vol 53 no 5 pp 255ndash260 2005

[37] S J Fortunato R Menon and S J Lombardi ldquoSupportfor an infection-induced apoptotic pathway in human fetalmembranesrdquo American Journal of Obstetrics and Gynecologyvol 184 no 7 pp 1392ndash1398 2001

[38] T Suzuki M Kobayashi K Isatsu et al ldquoMechanisms involvedin apoptosis of human macrophages induced by lipopolysac-charide from Actinobacillus actinomycetemcomitans in the

presence of cycloheximiderdquo Infection and Immunity vol 72 no4 pp 1856ndash1865 2004

[39] H Karahashi K S Michelsen and M Arditi ldquoLipopoly-saccharide-induced apoptosis in transformed bovine brainendothelial cells and human dermal microvessel endothelialcells the role of JNKrdquo Journal of Immunology vol 182 no 11pp 7280ndash7286 2009

[40] Y Zhao G Cui N Zhang Z Liu W Sun and Q PengldquoLipopolysaccharide induces endothelial cell apoptosis via acti-vation of Na

+H+exchanger 1 and calpain-dependent degrada-

tion of Bcl-2rdquo Biochemical and Biophysical Research Communi-cations vol 427 no 1 pp 125ndash132 2012

[41] J H Fan G G Feng L Huang et al ldquoRole of naofen in apop-tosis of hepatocytes induced by lipopolysaccharide throughmitochondrial signaling in ratsrdquo Hepatology Research vol 42no 7 pp 696ndash705 2012

[42] S Kataoka I Furuta H Yamada et al ldquoIncreased apoptosis ofhuman fetal membranes in rupture of the membranes andchorioamnionitisrdquo Placenta vol 23 no 2-3 pp 224ndash231 2002

[43] S Parry and J F Strauss III ldquoPremature rupture of the fetalmembranesrdquoTheNew England Journal of Medicine vol 338 no10 pp 663ndash670 1998

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


The notion that activin A is involved in chorioamnionitisis also supported by in vitro studies Activin A secretionfrom gestational tissues and cells is stimulated by inflamma-tory cytokines Tumor necrosis factor-120572 (TNF-120572) stimulatesactivin A production in explant cultures of human amnionand choriodecidua [15] and cultured human AEC [16]Interleukin (IL)-1120573 also stimulates activin A production inthe amnion choriodecidua [15] and AEC [16] Activin Amodulates the secretion of IL-6 IL-8 and prostaglandin E2in explant cultures of human amnion and choriodecidua [17]

Compared to the effects of TNF-120572 and IL-1120573 the effect ofLPS on activin A secretion from human AEC is inconsistentdespite the expression of functional TLR4 in human AEC[18] Rosenberg et al reported that LPS stimulated activin Arelease from cultured amniochorion explants but not fromplacental villous tissue [14] On the other hand Keelanet al reported that LPS did not affect activin A secretionfrom amnion explant cultures [15] Since the amnion is anavascular tissue andAEC are located in the innermost layer ofthe amnion secretions of AEC must be directly released intothe amniotic fluid and affect the fetus The present study wasperformed to determine if LPS stimulates activin A secretionfrom AEC and to verify the notion that activin A is involvedin amnionitis

2 Materials and Methods

21 Reagents LPS that was phenol extracted from E coliO26 was purchased from Paesel amp Lorei GmbH (HanauGermany) (catalogue no 100976 Lot 15303)

22 AEC Culture With the permission of the InstitutionalReview Board of Gunma University Hospital and the writteninformed consent of the patients we obtained fetal mem-brane samples during elective cesarean sections performedon four patients with full-term pregnancies who did nothave any systemic disease signs of premature deliveryor fetal complications AEC were prepared as previouslyreported [19] on the basis of the method established byOkita et al [20] with slight modifications Briefly thechorion was removed from amnion mechanically and theamnion was washed thoroughly with phosphate-bufferedsaline The removal of the chorion was ascertained by usinghematoxylineosin-stained paraffin sections of amnions Theamniotic membrane was cut into pieces and incubated in170mL of Krebs-Ringer solution containing 015 trypsin126mgmL sodium bicarbonate 25mM HEPES 100 120583gmLstreptomycin and 05 120583gmL amphotericin B at 37∘C in aspinner flask The liberated cells were decanted at 30minintervals and the incubationwas performed seven times withfreshly made trypsin solution Each fraction of dispersedcells was centrifuged and resuspended in DMEHamrsquos F12medium supplemented with 10 fetal bovine serum (FBS)100 120583gmL streptomycin and 05 120583gmL amphotericin BThefirst fractionwas discardedThe cell viability of the remainingfractions was determined by trypan blue exclusion and thefractions with viabilities of at least 80 were pooled Cells in

0

200

400

600

800

Activ

in A

(pg

mL)

ControlLPS

a

b

b

1250 2500 5000 10000 20000Cell number (well)

1000

1200

1400

1600 lowast

lowast

lowast

lowast

Figure 1 Effect of LPS on activin A secretion at various densities ofAEC Human AEC were seeded in 96-well microplates at densitiesof 1250 2500 5000 10000 and 20000 cells per well in 200 120583L ofDMEHamrsquos F12 medium supplemented with 10 FBS 100120583gmLstreptomycin and 05 120583gmL amphotericin B Either LPS (10 120583gmL)or vehicle (control) was added to each well After 96 h of cultureactivin A in the medium was measured Closed rectangles LPSOpen rectangles control The results from the quadruplicate assayare shown as the mean plusmn SE (119899 = 4) a

119875 lt 005 compared withthe control value at 1250 cellswell b

119875 lt 001 compared with thecontrol value at 1250 cellswell lowast119875 lt 001 comparedwith the controlvalue at each cell density

200120583L of medium were seeded in each well of 96-well platesand cultured in a humidified atmosphere containing 5CO

2-

95 air at 37∘C These cells were used for measuring activinA and cell proliferation

23 Activin A ELISA Activin A concentrations in culturemedium were measured by using an activin A assay kit(Oxford Bio-Innovations Oxfordshire UK) according to themanufacturerrsquos instructions The samples which were pre-treated with sodium dodecylsulfate and hydrogen peroxidewere added along with assay diluent to microwells coatedwith a monoclonal antibody specific for the beta-A subunitof activin After 1 h of incubation biotinylated monoclonalantibody for the beta-A subunit of activin was added andincubated overnight The following day wells were washedand streptavidin-alkaline phosphatase solution was addedAfter 1 h of incubation the wells were washed and incubatedwith substrate solution for 2 h Then amplifier solution wasadded and the absorbance at 490 nm was measured


0

100

200

300

400

500

600

700

800

0 1 3 10 30

Activ

in A

conc

entr

atio

n (p

gm

L)

lowast

lowast

LPS (120583gmL)

(a)

0

100

200

300

400

500

600

700

800

Activ

in A

conc

entr

atio

n (p

gm

L)

0 1 3 10 50

lowast

lowast

LPS (120583gmL)

(b)

Activ

in A

conc

entr

atio

n (p

gm

L)

LPS (120583gmL)0 1 10 30 50

350

300

250

200

150

100

50

0

lowast

lowast

(c)




3 Results





4 Discussion



ControlLPS

00

01

02

03

04

05

06

lowast

lowast

lowast

Cell number (well)1250 2500 5000 10000 20000

Abs4

50

nm

(a)

20

40

60

80

100

120

Abso

rban

ce (

)

00 10 100

LPS (120583gmL)

lowast

lowast

lowast

lowast

(b)










Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

100

200

300

400

500

600

700

800

0 1 3 10 30

Activ

in A

conc

entr

atio

n (p

gm

L)

lowast

lowast

LPS (120583gmL)

(a)

0

100

200

300

400

500

600

700

800

Activ

in A

conc

entr

atio

n (p

gm

L)

0 1 3 10 50

lowast

lowast

LPS (120583gmL)

(b)

Activ

in A

conc

entr

atio

n (p

gm

L)

LPS (120583gmL)0 1 10 30 50

350

300

250

200

150

100

50

0

lowast

lowast

(c)




3 Results





4 Discussion



ControlLPS

00

01

02

03

04

05

06

lowast

lowast

lowast

Cell number (well)1250 2500 5000 10000 20000

Abs4

50

nm

(a)

20

40

60

80

100

120

Abso

rban

ce (

)

00 10 100

LPS (120583gmL)

lowast

lowast

lowast

lowast

(b)










Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















ControlLPS

00

01

02

03

04

05

06

lowast

lowast

lowast

Cell number (well)1250 2500 5000 10000 20000

Abs4

50

nm

(a)

20

40

60

80

100

120

Abso

rban

ce (

)

00 10 100

LPS (120583gmL)

lowast

lowast

lowast

lowast

(b)










Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Acknowledgments


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article Gram-Negative Bacterial …downloads.hindawi.com/journals/ije/2013/789012.pdfGram-Negative Bacterial Lipopolysaccharide Stimulates Activin A Secretion from Human Amniotic