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1,25-Dihydroxyvitamin D3 Modulates Expression ofChemokines and Cytokines in Pancreatic Islets:

Implications for Prevention of Diabetes in NonobeseDiabetic Mice

Conny A. Gysemans, Alessandra K. Cardozo, Hanne Callewaert, Annapaula Giulietti, Leen Hulshagen,Roger Bouillon, Decio L. Eizirik, and Chantal Mathieu

Laboratory of Experimental Medicine and Endocrinology (C.A.G., H.C., A.G., R.B., C.M.) and Laboratory for ClinicalChemistry (L.H.), Katholieke Universiteit Leuven, 3000 Leuven, Belgium; and Laboratory of Experimental Medicine (A.K.C.,D.L.E.), Universite Libre de Bruxelles, 1070 Brussels, Belgium

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) is an immune mod-ulator that prevents experimental autoimmune diseases. Re-

ceptors for 1,25-(OH)2D3 are present in pancreatic -cells, thetarget of an autoimmune assault in nonobese diabetic (NOD)mice. The aim of this study was to investigate the in vivo andin vitro effects of 1,25-(OH)2D3 on -cell gene expression anddeath and correlate these findings to in vivo diabetes devel-opment in NOD mice. When female NOD mice were treatedwith 1,25-(OH)2D3 (5 g/kg per 2 d), there was a decrease inislet cytokine and chemokine expression, which was accom-panied by less insulitis. Complementing these findings, weobserved that exposure to 1,25-(OH)2D3 in three cell systemsINS-1E cell line, fluorescence-activated cell sorting purifiedrat-cells, and NOD-severe combined immunodeficient islets)suppressed IP-10 and IL-15 expression in the -cell itself but

did not prevent cytokine-induced -cell death. This 1,25-(OH)2D3-induced inhibition of chemokine expression in

-cells was associated with a decreased diabetes incidence insome treatment windows targeting early insulitis. Thus, al-though a short and early intervention with 1,25-(OH)2D3 (314wk of age) reduced diabetes incidence (35 vs. 58%, P < 0.05), alate intervention (from 14 wk of age, when insulitis is present)failed to prevent disease. Of note, only early and long-termtreatment(328wk of age) preventeddisease to a major extent(more than 30% decrease in diabetes incidence). We concludethat 1,25-(OH)2D3monotherapy is most effective in preventingdiabetes in NOD mice when applied early. This beneficial ef-fect of 1,25-(OH)2D3 is associated with decreased chemokineand cytokine expression by the pancreatic islets. (Endocri-nology 146: 19561964, 2005)

THE PREVALENCE OF type 1 diabetes (affecting 5.341.6 per 100,000 subjects in Europe and the UnitedStates) is estimated to rise each year by 35%, with a largerincrease in some central and eastern European countries (1,2). Against this background, prediction, prevention, and cureof type 1 diabetes are major health care issues. An increasingamount of data points toward a close relationship betweentype 1 diabetes and vitamin D. Thus, there is a higher prev-alence of type 1 diabetes in regions with low vitamin Dsupplies, whereas dietary supplementation with vitamin Dduring infancy reduces risk of developing type 1 diabetes ingenetically at-risk subjects later in life (35). In nonobesediabetic (NOD) mice, the hormonally active form of vitamin

D, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] prevents dia-betes when given throughout their life span, whereas vita-min D deficiency in early life accelerates the onset and in-creases the incidence of the disease. This is probably related

to deficient macrophage function and/or higher -cell vul-nerability (68).

1,25-(OH)2D3 exerts its function by engaging with a spe-cific nuclear receptor [vitamin D receptor (VDR)] present inmost tissues and cells, including lymphocytes, macrophages,and pancreatic -cells (9, 10). Of note, the vitamin D-depen-dent calcium binding protein (calbindin-D28K), which isthought to serve as a cytosolic calcium buffer, is present in-cells (11). Besides its well-known effects on calcium and

bone metabolism, 1,25-(OH)2D3 inhibits cell growth, inducescell differentiation, and modulates the immune system (12).In NOD mice, 1,25-(OH)2D3 prevents insulitis and diabetesthrough modulation of the immune system (6, 7). 1,25-

(OH)2D3 inhibits antigen-induced T-cell proliferation as wellas proinflammation-related cytokine production, enhancesT-cell sensitivity to apoptosis-inducing signals, reduces an-tigen-presenting and costimulating properties of dendriticcells, and induces pancreatic lymph-node CD4CD25 Tregulator cells (13, 14). Recently several studies suggestedthat 1,25-(OH)2D3 has a direct protective effect on -cells,contributing to prevention of diabetes in NOD mice. Thisissue remains, however, controversial. Whereas normal lev-els of 1,25-(OH)2D3 are necessary for secretion and de novo

biosynthesis of insulin by -cells (1517), studies on the ef-fects of 1,25-(OH)2D3 treatment on -cell vulnerability toimmune mediators provided conflicting results. Whereas

First Published Online January 6, 2005Abbreviations: FACS, Fluorescence-activated cell sorting; IFN, inter-

feron; HO,Hoechst 342; IP, inducible protein; MCP, monocyte chemoat-tractant protein; MIP, macrophage inflammatory protein; NOD, nono-

bese diabetic; 1,25-(OH)2D3, 1,25-dihydroxyvitamin D3; PI, propidiumiodide; VDR, vitamin D receptor.

Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving theendocrine community.

0013-7227/05/$15.00/0 Endocrinology 146(4):1956 1964Printed in U.S.A. Copyright 2005 by The Endocrine Society

doi: 10.1210/en.2004-1322

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Mauricio et al. (18) did not observe a protective effect of1,25-(OH)2D3 against cytokine-induced -cell death in vitro,Sandler et al. (19) and Riachy et al. (20, 21) described clearprotection.

The aim of the present study was to investigate whetherin vivo and in vitro effects of 1,25-(OH)2D3 on -cell geneexpression and death were associated with in vivo protectionagainst diabetes in NOD mice.

Materials and Methods

Animals

NOD mice, originally obtained from Professor C. Y. Wu (Departmentof Endocrinology, Peking Union Medical College Hospital, Beijing,China), were housed and inbred in our animal facility of the KatholiekeUniversiteit Leuven since 1989. Housing of NOD mice occurred undersemibarrier conditions, and animals were fed sterile chow and water adlibitum. NOD mice were screened for the onset of diabetes by evaluatingglucose levels in urine(Clinistix; Bayer Diagnostics, Tarrytown, NY) andvenous blood (Glucocard; Menarini, Florence, Italy). The cumulativeincidence of diabetes of NOD mice in our breeding stock was 85% infemales and 61% in males by 28wk at the timeof the present study. Mice

were diagnosed as diabetic when having positive glucosuria and twoconsecutive blood glucose measurements exceeding 200 mg/dl. NOD/LtSz-scid/scid [NOD-severe combined immunodeficient (SCID)] micewere bred from stocks purchased from the Jackson Laboratory (BarHarbor, ME), whereas BALB/c mice were purchased from Harlan Ned-erland (Horst, The Netherlands). Wistar male WI rats were purchasedfrom Charles River Belgium (Brussels, Belgium) and maintained in theanimal facility of the Universite Libre de Bruxelles. Animals were main-tained in accordance with the National Institutes of Health Guide fortheCare and Use of Laboratory Animals, and all experimental procedureswere approved and performed in accordance with the Ethics Commit-tees of the Katholieke Universiteit Leuven (Leuven, Belgium) and theUniversite Libre de Bruxelles (Brussels, Belgium).

Cell culture

The insulin-producing insulinoma (INS)-1E cell clone was kindlyprovided by Prof. C. Wollheim (Center Medical Universitaire, Geneva,Switzerland) and represents an extremely stable and valuable -cellmodel as reflected by a constant doubling time and balanced glucoseoxidation/use (Ortis, F., J. Rasschaert, and D. L. Eizirik, unpublishedobservations) (22). Cells (i.e. passages 6070) were seeded (250,000 cellsin 35 mm dishes) in RPMI 1640 medium (with Glutamax-I) supple-mented with 10% heat-inactivated fetal calf serum, 10 mm HEPES, 100U/ml penicillin, 100 g/ml streptomycin, 1 mm sodium pyruvate, and50 m 2-mercaptoethanol (23, 24). The next day cells were incubatedwith vehicle (ethanol) or 108 m 1,25-(OH)2D3 and 24 h later exposed toIL-1 and/or interferon (IFN)-for 8 h for mRNA extraction (seebelow)or 24 h for assessment of islet viability (see below). Recombinant humanIL-1 (10 U/ml; 38 IU/ng) was a kind gift from Dr. C. W. Reynolds(National Cancer Institute, Bethesda, MD) and mouse IFN(100 U/ml;10 IU/ng) was purchased from Invitrogen (Carlsbad, CA). The choice of

cytokine concentrations was determined by a dose-response curve as thelowest concentration giving the maximal apoptosis rate after 24 h (25).1,25-(OH)2D3 wasobtained from J. P. vande Velde (Duphar, Weesp,TheNetherlands). 1,25-(OH)2D3 was added to the culture medium at a finalconcentration of 108 m 24 h before induction of cytokine-mediated celldeath and refreshed every other day.

Islet isolation and culture

Pancreatic islets were isolated from Wistar rats (10 wk old) and NOD(4, 8, and 10 wk old) and NOD-SCID mice (10 wk old) by collagenasedigestion as previously described (26). Islets were directly frozen inliquid nitrogen and stored at 80 C (for mRNA extraction, see below)or cultured as whole islets (for assessment of islet viability, see below).Unless otherwise indicated, islet cells were cultured free floating inRPMI 1640 medium containing 10% heat-inactivated fetal calf serum, 2

mm glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin andmaintained at 37 C in a humidified incubator (95% air-5% CO2).

Purification of-cells by autofluorescence-activated cell

sorting (FACS)

Rat islet -cells were purified by FACS as described (27). These

preparations contain single

-cells that are 9095% pure (Rasschaert, J.,L. Ladriere and D. L. Eizirik, unpublished data) (28). Purified -cellswere cultured single and attached to polylysine-coated microtiterdishesat 37 C in Hams F-10 medium (Invitrogen) as previously described (28).After overnight culture, primary -cells were incubated for 24 h withvehicle (ethanol) or 108m 1,25-(OH)2D3 and thereafter exposed for 48 hto IL-1 (50 U/ml) and/or IFN (1000 U/ml) for assessment of cellviability (see below). The choice of cytokine concentrations was basedon previous studies by our group (29, 30).

Islet viability

Percentages of viable, apoptotic, and necrotic cells were assessed inINS-1E cells [in vitro treated with or without 1,25-(OH)2D3 at concen-tration of 108 m, 6000 cells/condition], rat FACS-purified -cells [invitro treated with or without 1,25-(OH)2D3 at concentration of 10

8m,

10,000 cells/condition], and NOD-SCID islets [in vivo treated with orwithout 1,25-(OH)2D3 at a dose of 5 g/kg per 2 d from weaning till 10wk of age, 15 islets/condition]. Briefly, single cells or whole islets wereincubated for 15 min with the DNA binding dyes Hoechst (HO) 342 (20g/ml) and propidium iodide (10 g/ml) and the cells examined on aninverted fluorescence microscopy as previously described for rodentand human -cells (3133). In each well a minimum of 500 cells werecounted by two independent individuals, one of them unaware of sam-ple identity. For whole islets, discrimination between necrosis andapoptosis is difficult due to superposition between cells; therefore, anapproximate estimate of the percentage of living/dead cells was per-formed by two observers, one of them unaware of sample identity, asdescribed (33).

RNA isolation, reverse transcription, and quantitative PCR

Total RNA was extracted from INS-1E

cells and whole islets using theHigh pure RNA extraction kit (Roche Diagnostics Corp., Indianapolis,IN). Either 0.5 or 1 g RNA was reverse transcribed using 100 U Su-perscript II reverse transcriptase (Invitrogen) at 42 C for 80 min in thepresence of 5 m oligodT16. Real-time PCR was performed using the7700 SDS(Applied Biosystems, FosterCity, CA)and theTaqMan system,as described previously (3436). Primers and probes for calbindin-D28K,IL-1, IL-15, IFN, monocyte chemoattractant protein (MCP)-1 (CCL-2),macrophage inflammatory protein (MIP)-3 (CCL-20), interferon in-ducible protein (IP)-10 (CXCL-10), VDR, and -actin as housekeepinggene were as described (3437). Cytokines as well as 1,25-(OH)2D3 didnot modify -actin expression (data not shown).

In vivo experimental design

For in vivo administration, 1,25-(OH)2D3 was suspended in peanut oil

and administered at a dose of 5 g/kg ip every other day. Control micereceived the treatment vehicle only (50 l). For in vivo experimentsdealing with insulitis development, islet viability and mRNA extractionstudies, NODand NOD-SCID mice were treated from weaning until theend of the follow-up period (10 wk of age).

For in vivo experiments dealing with prevention of diabetes, NODmice were randomly assigned to the control group or three treatmentgroups: treatment from 3 to 14 wk of age, 14 28 wk of age, or 328 wkof age. All mice were followed up until 28 wk of age.

Calcium and bone parameters

At the end of the in vivo experiments, blood was collected by heartpuncture, and a femur was removed. Serum and femur were stored at20 C until determinations were performed. Calcium content of thefemur, measured on HCl dissolved bone ashes, and the serum was

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Effects of in vitro treatment with 1,25-(OH)2D3 on islet cell

death and gene expression after combined

cytokine treatment

To assess whether the observed in vivo effects of 1,25-(OH)2D3 on the -cell phenotype of NOD mice was associ-ated with direct effects on the -cell, we tested in a first setof experiments whether 1,25-(OH)2D3 was able to preserveviability of the clonal rat insulinoma cell line (INS-1E cells)when exposed to IL-1 and IFN. The cells were pretreatedwith vehicle or 108 m 1,25-(OH)2D3 for 24 h. Neither IL-1nor IFNalone induced cell death in INS-1E cells (data notshown), whereas a combined cytokine treatment led to ex-tensive cell death already at 24 h of cytokine exposure (67.0

1.9 vs. 94.0

0.7% living cells in medium control, P

0.05).1,25-(OH)2D3 (108m) per se was not toxic toward islet cells

(90.0 1.2% living cells, PNS vs. medium control) but didnot protect the INS-1E cells against cytokine-induced -celldeath (63.0 1.2% living cells) (Fig. 3A). Discrimination

between apoptosis and necrosis rates revealed no differencesbetween vehicle and 1,25-(OH)2D3 conditions, and cytokine-induced accumulation of medium nitrite [an indicator ofnitric oxide production] was also unaffected by 1,25-(OH)2D3(data not shown).

Cytokine-induced cell death of FACS-purified rat -cellswas studied after a 48-h instead of a 24-h cytokine culture

because cytokine-induced cell death was shown to appearlater in primary -cells (25). Again no difference in the total

number of viable islet

-cells was observed after cytokinetreatment on addition of 1,25-(OH)2D3 (68.0 2.0, comparedwith 61.0 4.6%, living cells in cytokine control, P NS)(Fig. 3B).

Although treatment with 1,25-(OH)2D3 was unable to pro-tect against cytokine-induced islet damage in both experi-mental models, 1,25-(OH)2D3 was able to alter the gene ex-pression profile of islets (INS-1E cells) after combinedcytokine treatment. In line with previous observations fromgene array analyses (25, 30), mRNA levels of IL-1, MCP-1,IP-10, and the cytokine IL-15 were significantly up-regulatedin INS-1E cells after exposure to inflammatory cytokines (Fig.4). No MIP-3 and IFN mRNA expression could be ob-served in the INS-1E cells under basal as well as cytokine

conditions (datanot shown). 1,25-(OH)2D3 reduced cytokine-induced INS-1E IL-1 expression (Fig. 4A) and clearly de-creased cytokine-triggered MCP-1, IP-10, and IL-15 gene ex-pression during the period of incubation (Fig. 4, BD). Ofnote, calbindin-D28K and VDR mRNA levels in INS-1E cellswere slightly up-regulated by 1,25-(OH)2D3 under both basaland cytokine stimulated conditions (Fig. 4, E and F).

Effects of in vivo treatment with 1,25-(OH)2D3 in NOD-

SCID mice on islet cell death and gene expression after

combined cytokine treatment

To evaluate whether in vivo treatment with 1,25-(OH)2D3also resulted in less chemokine expression in the islet cells,

FIG. 2. Effect of in vivo treatment with 1,25-(OH)2D3 on time course of IL-1 (A), MCP-1 (B), MIP-3 (C), IFN (D), IL-15 (E), IP-10 (F),calbindin-D28K (G), and VDR (H) mRNA expression in islets of diabetes-prone NOD mice. mRNA was isolated from islets of female NOD mice;treated with either vehicle (peanut oil, white bars) or 1,25-(OH)2D3 (5 g/kg body weight,gray bars) by ip injection everyother dayfrom weaninguntil 4, 8, and 10 wk of age; and analyzed by real-time PCR. Four-week-old female BALB/c islets were used as nondiabetes-prone controls. Theresults obtained are means SEM for three similar experiments and are expressed as cytokine or chemokine copies per -actin copies.Significance (symbol) is expressed compared with mRNA expression levels in islets of control mice. *, P 0.05 vs. 4-wk-old BALB/c islets; $,

P 0.05 vs. age-related vehicle-treated NOD mice.


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we treated NOD-SCID mice, lacking T and B lymphocytesand natural killer cells, with 1,25-(OH)2D3 (5 g/kg per 2 d)until 10 wk of age. Exposing control islets to the combinedcytokines (IL-1 IFN) killed already 26.8% of islet cellsafter 48 h of culture (islet viability 73.2 14.8 vs. 95.8 3.1%living cells in medium control, P 0.05). A prolonged ex-posure of 5 d further increased cell death as reflected bydecreased general islet viability (36.0 16.0 vs. 97.4 1.9%

living cells in medium control, P

0.05) (Fig. 5). After treat-ment with 1,25-(OH)2D3 in vivo, 97.4% of cells remainedviable in cytokine-free medium (PNS vs. medium control),

but again no protection against cytokine-induced cell deathwas observed (30.0 7.6% living cells, P NS vs. cytokinecontrol) (Fig. 5).

IL-1 IFNup-regulated levels of IL-1, MCP-1, MIP-3, IP-10, and IL-15 in islet cells from control mice (Fig. 6).No IFNmRNA expression could be observed in the NOD-SCID islets under basal as well as cytokine conditions (datanot shown). In vivo treatment of NOD-SCID mice with 1,25-(OH)2D3 led to a small, but significant, decrease in IP-10 andespecially IL-15 levels in islet cells after cytokine exposure(P 0.05 vs. cytokine control) (Fig. 6E).

Early and long-term treatment with 1,25-(OH)2D3 prevents

diabetes in NOD mice

Different treatment windows with 1,25-(OH)2D3 weretested in female NOD mice to study the impact of 1,25-(OH)2D3 on diabetes development when administered dur-ing early insulitis development (314 wk of age), after insu-

litis development (1428 wk of age), and throughout thewhole process (328 wk of age). As anticipated, at 28 wk ofage, diabetes was clearly prevented [20% (18 of 90)] in thelong-term-treated group, compared with 58% (40 of 69) in thecontrol group (P 0.0001) (Fig. 7). This was accompanied bya delay in diabetes onset (mean age 147 17 vs. 139 18 din controls, P 0.0005). When therapy was initiated only at14 wk of age (when more than 75% of control NOD micealready have islet infiltration) 40% (eight of 20) of these mice

became diabetic by wk 28 (PNS vs. controls). Themean ageat onset was again delayed, compared with controls (153 24 d vs. controls, P 0.0005). When mice were treated from3 until 14 wk of age, a small delay of diabetes onset was seen(142 23 d, P 0.0005 vs. controls), and a reduction inincidence by 28 wk was observed (35 vs. 58% in controls, P0.05).

None of the different treatment regimens had an effect onbody weight at the end of the experiment, and total serumcalcium levels remained in the normal range [9.1 1.7 (314),9.4 0.6 (1428), 9.8 2.4 (328) vs. 9.6 2.0 mg/dl invehicle-treated control, PNS, respectively]. Effects of 1,25-(OH)2D3 on bone metabolism appeared from serum concen-trations of osteocalcin, a sensitive marker of osteoblast ac-tivity and bone turnover, and bone calcium contentmeasured at the end of the experiment. Osteocalcin levels inthe group treated during their entire life span was increased(102.1 11.2 vs. 37.8 3.9g/liter in vehicle-treated control,

P 0.001), whereas the bone turnover in the mice from theshorter treatment duration was in the normal range (62.6 14.7 g/liter, P 0.05 vs. vehicle-treated control). Bone cal-cium content as indicated by the calcium content of the rightfemur reflected again the impact of 1,25-(OH)2D3 treatmentduring the whole life span because the bone calcium contentof these animals was decreased (3.9 0.4 vs. 7.9 1.1 mg/femur in vehicle-treated control, P 0.001). On the otherhand, when treatment was either ended or initiated at 14 wkof age, no bone loss under 1,25-(OH)2D3 treatment was seen(6.7 0.3 and 5.1 0.4 mg/femur, P NS, respectively, vs.vehicle-treated control).

Discussion

The search for innovative therapies to prevent type 1 di-abetes in genetically at-risk children and adolescents and/orarrest further loss of islet -cells in recent-onset diabeticsstands high on the priority list of researchers in the diabetesfield. At present no treatment can prevent type 1 diabetes inhumans, although many interventions are successful in theNOD mouse (reviewed in Ref. 39). Keeping this argument inmind, we point out that this animal model is not a perfectsurrogate for the human disease but provides us with im-portant lessons and insights into the possible heterogeneityunderlying the complex pathogenesis of human diabetes.Based on data in the NOD mouse and also recent epidemi-

FIG. 3. Effect of in vitro treatment with 1,25-(OH)2D3 on cytokine-induced cell death in INS-1E cells (A) and primary rat -cells (B).INS-1E cells (6,000 cells/condition) and FACS-purified rat -cells(10,000 cells/condition) were exposed for 24 and 48 h, respectively, toIL-1 (10 U/ml) plus IFN (100 U/ml) (CYTK, dashed bars) as indi-cated in the figure. Cultures were either untreated (white bars) orpretreated with 108 M 1,25-(OH)2D3 for 24 h (gray bars). Cell via-bility was determined with the nuclear dyes HO 342 and PI. Nucleiof viable cells are stained in blue, whereas the nuclei of dead cells are

stained in red. Isletcelldeadis expressed asmeans SEM (percentageof living cells) from three independent experiments. *, P 0.05 vs.medium control (control cells, without cytokines); $, P 0.05 vs.CYTK control (control cells, with cytokines); , P 0.05 vs. medium1,25-(OH)2D3 [cells treated with 1,25-(OH)2D3, without cytokines].

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ological data of the European Community Concerted ActionProgramme in Diabetes study and observations made in aFinnish study in humans, treatment with analogs of 1,25-(OH)2D3, the active form of vitamin D, or interventions withsupplements of regular vitamin D are presently being con-sidered inpatients at risk for type1 diabetes (5, 7, 8,40). Many

studies have demonstrated that inflammation is orchestratedby cytokines and chemokines secreted by activated T cellsand macrophages during the autoimmune response (re-viewed in Ref. 41). Recently we observed that expression ofthese cytokines and chemokines were also detectable in-cells on exposure to combinations of IL-1 and IFN invitro and in pancreatic islet cells during insulitis progressionin the NOD mice in vivo (29, 42), suggesting an active role ofthe target organ it its own destruction.

In the present study, we combined in vivo and in vitroapproaches to evaluate whether 1,25-(OH)2D3 inhibits insu-litis and autoimmune diabetes in NOD mice and the mech-anisms involved in these putativebeneficialeffects at theisletlevel.

We observed that in vivo treatment of diabetes-prone NODmice with 1,25-(OH)2D3, initiated before insulitis onset, notonly diminished the severity of islet infiltration and pre-served -cell functionality during the course of insulitis, aswaspreviously observedby our group (6), but also decreasedthe levels of IL-1, IL-15, and IP-10 mRNA levels in the isletcells from NOD mice at 8 and 10 wk of age. Complementingthese findings, we found that in vivo and in vitro treatmentwith 1,25-(OH)2D3 decreased the cytokine-induced expres-sion of IL-1 and IL-15 as well as the chemokine IP-10 in islet-cells during cell culture. Our data in INS-1E cells and isletcells of immune-deficient NOD-SCID mice confirm thatthose cytokines and chemokines are expressed by the -cellsthemselves. However, in contrast to NOD-SCID mice, in thecase of NOD mice, these cytokines and chemokines may beproduced by not only-cells but also theinfiltrating cells. We

FIG. 4. Effect of in vitro treatment with 1,25-

(OH)2D3 on differential expression of cytokine-inducedinflammation-related molecule IL-1 (A),MCP-1 (B), IL-15 (C), IP-10 (D), calbindin-D28K(E), and VDR (F) mRNA in INS-1E cells. INS-1E

cells were exposed for 8 h to IL-1 (10 U/ml) plusIFN(100 U/ml) (CYTK, dashed bars) with (graybars) or without (white bars) pretreatment of1,25-(OH)2D3 (10

8M) for 24h, asindicatedin the

figure. The cells were then harvested, mRNA ex-tracted, and measured by real-time PCR with theuse of gene-specific oligonucleotide primers andfluorogenic probes. mRNA levels (the ratio be-tween the gene of interest and -actin) are ex-pressed as means SEM from three independentexperiments. *, P 0.05 vs. medium control (con-trolcells,without cytokines); $,P0.05 vs. CYTK

control (control cells, with cytokines); , P 0.05vs. medium 1,25-(OH)2D3 [cells treated with1,25-(OH)2D3, without cytokines].

FIG. 5. Effect of in vivo treatment with 1,25-(OH)2D3 on cytokine-induced cell death in NOD-SCID islets. Islets (15 per condition) iso-lated from NOD-SCID mice were exposed for 5 d to IL-1 (50 U/ml)plus IFN (1000 IU/ml) (CYTK, dashed bars), as indicated in thefigure. Cultures were from either 10-wk-old vehicle- (white bars) or1,25-(OH)2D3-treated NOD-SCID mice (5 g/kg body weight ip fromweaning per 2 d, gray bars). Cell viability was determined with thenuclear dyes HO 342 and PI. Nuclei of viable cellsare stained in blue,whereas the nuclei of dead cells are stained in red. Islet cell dead isan estimation of the percentage of livingvs. dead cells. *, P 0.05 vs.medium control (control cells, without cytokines); $, P 0.05 vs.CYTK control (control cells, with cytokines); , P 0.05 vs. medium1,25-(OH)

2D3

[cells treated with 1,25-(OH)2

D3, without cytokines].


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hypothesize that the reduced expression of inflammatory

cytokines, as well as inhibition of chemotaxis in the islet cellsby 1,25-(OH)2D3, might limit in vivo the development ofinsulitis by affecting the migration and recruitment of effec-tor T cells and macrophages to the islets. While this manu-script was in preparation, Giarratana et al. (43) showed thatin vivo treatment with an analog of 1,25-(OH)2D3, given from8 until 16 wk of age to NOD-SCID mice, inhibited basal isletexpression of the chemokines [MCP-1, IP-10, and RANTES(regulated upon activation, normal T cell expressed, andsecreted)] and delayed diabetes development after transfer

experiments with diabetogenic T cells. Finally, although oth-

ers have reported protective effects of calbindin-D28K-over-expression in TC-3 cells incubated with cytokines (44), wedid not find any clear involvement of calbindin-D28K andVDR in the 1,25-(OH)2D3-mediated disease prevention.

On the other hand, in vitro and in vivo treatment with1,25-(OH)2D3 alone (prior to islet isolation) did not protectrodent insulinoma INS-1E cells, rat FACS-purified single-cells, and whole NOD-SCID mouse islets against cytokine-induced cell death in vitro. This confirms observations ofMauricio et al. (18) but contrasts with the findings of Riachy

FIG. 6. Effect ofin vivo treatment with1,25-(OH)2D3 on differential expression

of cytokine-induced inflammation-re-lated molecule IL-1 (A), MCP-1 (B),MIP-3 (C), IL-15 (D), and IP-10 (E)mRNA in NOD-SCID islet cells. NOD-SCID islets were exposed for 24 h toIL-1 (50 U/ml) plus IFN(1000 U/ml),as indicated in the figure. mRNA wasisolated from islets of female NOD-SCID mice; treated with either vehicle(peanut oil, white bars) or 1,25-(OH)2D3(5 g/kg body weight, gray bars) by ipinjection every other day from weaninguntil 10 wkof age; and analyzed byreal-time PCR. mRNA levels (the ratio be-tween the gene of interest and -actin)are expressed as means SEM from

three independent experiments. *,P 0.05 vs. medium control (controlcells, without cytokines); $, P 0.05vs. CYTK control (control cells, withcytokines); , P 0.05 vs. medium1,25-(OH)2D3 [cells treated with 1,25-(OH)2D3, without cytokines].

FIG. 7. Effect of in vivo treatment with 1,25-(OH)2D3, applied in different time windows, on diabetes incidence in diabetes-prone NOD mice.Female NODmicewere treated witheither vehicle(peanut oil) until28 wk ofage (, n90) or 1,25-(OH)2D3 (5 g/kg body weight) by ip injectionevery other day from weaning until 28 wk of age (F, n 69) or 14 wk of age (, n 43) or from 14 until 28 wk of age (, n 20). Mice withblood glucose levels of more than 200 mg/dl at 28 wk of age were scored as having diabetes. Significance is expressed compared with

vehicle-treated NOD mice.


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et al. (20, 21), who demonstrated that in vitro treatment with1,25-(OH)2D3 protects rat insulin-producing RINm5F cellsand human islets against cytokine-induced cell death. Dif-ferences between these studies and our own experimentsinclude different exposure periods to 1,25-(OH)2D3 (rangingfrom 48 h to 6 d) and different islet cell systems used, whichmight have modified sensitivity to inflammatory cytokinesand/or response to treatment with 1,25-(OH)2D3. Of note, inour hands, 1,25-(OH)2D3 does not provide protection againstin vitro cytokine-induced -cell death even when using vari-able incubation times and multiple cellular islet systems.

Taking our previous and present data together, these ob-servations suggest that 1,25-(OH)2D3 acts on both immunecells and -cells, affecting the expression of key proinflam-matory genes expressed in the islet cells and thus reshapingthem into less inflammation-prone and chemoattractive tar-gets. Indeed, we previously observed that 1,25-(OH)2D3 is astrong immune modulator, inhibiting phytohemagglutininA-induced lymphocyte proliferation (45), stimulating mac-rophage phagocytosis of bacteria and suppressing the anti-gen-presenting capacity of these cells and dendritic cells invitro (46). In vivo, we observed that administration of 1,25-(OH)2D3 to NOD mice induced suppressor cells (7), mainlyCD4CD25 cells (14), and eliminated effector cells (47),inducing an immune shift with lower Th1 cytokine levelslocally in the inflamed islets of the native pancreas (48).Therefore, protection against insulitis and diabetes in NODmice by 1,25-(OH)2D3 may be due to a combination of effectson both effector (e.g. immune cells) and victim (e.g. -cells)of the autoimmune process culminating in type 1 diabetesmellitus.

Departing from this hypothesis, we tested different treat-ment windows for 1,25-(OH)2D3 as preventive agent againstdiabetes in NOD mice. We confirmed that protection againstdevelopment of autoimmune diabetes was observed whenmice were treated from weaning until the end of life. How-ever, also, a small but consistent protection was seen whentreatment was initiated early and terminated at 14 wk of age.On theotherhand, no protection was observedwhen therapywas initiated at 14 wk of age, a time point when most of theislets of the subjects were already heavily infiltrated andthere remained few insulin-producing -cells. From thesedata it is clear that early-life treatment (before the onset ofinsulitis) had long-lasting effects on the development of di-abetes and that interfering with an already ongoing immuneattack against the -cells is more difficult than interfering atthestart of autoimmunity. These observations arein line withprevious papers (49), in which we have shown that long-termtreatment withanalogs of 1,25-(OH)2D3, initiated early in life,was able to prevent disease. However, when treatment wasinitiated in mice with already ongoing insulitis, the analogscould not prevent progression to overt disease, unless theywere combined with a short course of an anti-T-cell agent(e.g. cyclosporine A) (50). Thus, inhibiting homing of mono-nuclear cells into the islets, by decreasing chemokine expres-sion, might be an attractive approach to prevent insulitis anddiabetes. In line with this hypothesis, Christen et al. (51)demonstrated that blocking of IP-10 by monoclonal antibodytherapy significantly reduced the incidence and onset of

diabetes by decreasing clonal expansion and migration intothe pancreas of CD8 T cells.

In conclusion, this study showed that in vitro and in vivotreatment with 1,25-(OH)2D3 did not protect -cells againstcell death by direct immuneattack but was able to modify theinflammation-induced gene expression in islet cells, shapingthem into less inflamedand less chemoattractive targets. This-cell effect, together with the previously described immuneeffects of the treatment, leads to reduced severity of isletinfiltration and preserved -cell functionality during thecourse of insulitis, eventually inhibiting the development oftype 1 diabetes,especially when therapy is initiated early andgiven during the entire life span. These observations thusdelimit the place for 1,25-(OH)2D3 and its analogs and pro-vide a rationale for its use as potential tools in the preventionof type 1 diabetes in humans.

Acknowledgments

The authors thank W. Cockx, J. Depovere, J. Laureys, D. Valckx, M.Neef, J. Schoonheydt, and M. Urbain for their excellent technicalassistance.

Received October 7, 2004. Accepted December 16, 2004.Address all correspondence and requests for reprints to: Chantal

Mathieu, M.D., Ph.D., Laboratory of Experimental Medicine and En-docrinology, Katholieke Universiteit Leuven, Herestraat 49, 3000 Leu-ven, Belgium. E-mail: [email protected].

This work was supported by the Juvenile Diabetes Research Foun-dation (JDRF) Center for Prevention of -Cell Destruction in EuropeGrant 4-2002-457 and the European Community concerted Sixth Frame-work Program Grant TONECA (LSHM-CT-2004-503245). Additionalgrants were from the Belgium Program on Interuniversity Poles ofAttraction initiated by the Belgian State (IUAP P5/17), the KatholiekeUniversiteit Leuven (KUL) (Geconcerteerde Onderzoeksacties, 2004/10), the Flemish Research Foundation [Fonds Voor WetenschappelijkOnderzoek (FWO)Vlaanderen, G.0084.02 and G.0233.04], and the Na-tional Research Foundation (Fonds National de la Recherche Scienti-fique). C.M. has a clinical research fellowship (FWO); C.A.G. has apostdoctoral FWO fellowship; A.K.C. has a postdoctoral JDRF fellow-ship; and A.G. has a doctoral scholarship from the KUL (InterfacultyCouncil for Development Co-operation).

References

1. Kyvik KO, Nystrom L, Gorus F, Songini M, Oestman J, Castell C, Green A,Guyrus E, Ionescu-Tirgoviste C, McKinney PA, Michalkova D, OstrauskasR, Raymond NT 2004 The epidemiology of type 1 diabetes mellitus is not thesame in young adults as in children. Diabetologia 47:377384

2. EURODIAB ACE Study Group 2000 Variation and trends in incidence ofchildhood diabetes in Europe. Lancet 355:873876

3. Akerblom HK, Vaarala O, Hyoty H, Ilonen J, Knip M 2002 Environmentalfactors in the etiology of type 1 diabetes. Am J Med Genet 115:1829

4. NorrisJM 2001 Canthe sunshine vitamin shed lighton type 1 diabetes?Lancet358:14761478

5. Hypponen E, Laara E, Reunanen A, Jarvelin MR, Virtanen SM 2001 Intakeof vitamin D and risk of type 1 diabetes: a birth-cohort study. Lancet 358:15001503

6. Mathieu C, Laureys J, Sobis H, Vandeputte M, Waer M, Bouillon R 19921,25-Dihydroxyvitamin D3 prevents insulitis in NOD mice. Diabetes 41:14911495

7. Mathieu C, Waer M, Laureys J, Rutgeerts O, Bouillon R 1994 Prevention ofautoimmune diabetes in NOD mice by 1,25 dihydroxyvitamin D3. Diabeto-logia 37:552558

8. Giulietti A, Gysemans C, Stoffels K, Van Etten E, Decallonne B, OverberghL, Bouillon R, Mathieu C 2004 Vitamin D deficiency in early life acceleratestype 1 diabetes in non-obese diabetic mice. Diabetologia 47:451462

9. Deluca HF, Cantorna MT 2001 Vitamin D: its role and uses in immunology.FASEB J 15:25792585

10. Holick MF 2003 Evolution and function of vitamin D. Recent Results CancerRes 164:328

11. Sooy K, Schermerhorn T, Noda M, Surana M, Rhoten WB, Meyer M, Fleis-


7/28/2019 1956.full_2

9/9

cher N, Sharp GW, Christakos S 1999 Calbindin-D(28k) controls [Ca(2)](i)and insulin release. Evidence obtained from calbindin-D(28k) knockout miceand cell lines. J Biol Chem 274:3434334349

12. Van Etten E, Decallonne B, Mathieu C 2002 1,25-Dihydroxycholecalciferol:endocrinology meets the immune system. Proc Nutr Soc 61:375380

13. Mathieu C, Adorini L 2002 The coming of age of 1,25-dihydroxyvitamin D(3)analogs as immunomodulatory agents. Trends Mol Med 8:174179

14. Adorini L 2003 Tolerogenic dendritic cells induced by vitamin D receptorligands enhance regulatory T cells inhibiting autoimmune diabetes. Ann NY

Acad Sci 987:25826115. Billaudel BJ, Faure AG, Sutter BC 1990 Effect of 1,25 dihydroxyvitamin D3on isolated islets from vitamin D3-deprived rats. Am J Physiol 258:E643E648

16. Bourlon PM, Faure-Dussert A, Billaudel B 1997 Modulatory role of 1,25dihydroxyvitamin D3 on pancreatic islet insulin release via the cyclic AMPpathway in the rat. Br J Pharmacol 121:751758

17. Bourlon PM, Faure-Dussert A, Billaudel B 1999 The de novo synthesis ofnumerous proteins is decreased during vitamin D3 deficiencyand is graduallyrestored by 1, 25-dihydroxyvitamin D3 repletion in the islets of Langerhans ofrats. J Endocrinol 162:101109

18. Mauricio D, Andersen HU, Larsen CM, Karlsen AE, Mandrup-Poulsen T,Nerup J 1997 Dexamethasone prevents interleukin-1-mediated inhibition ofrat isletinsulin secretion without decreasing nitric oxide production. Cytokine9:563569

19. Sandler S, Buschard K, Bendtzen K 1994 Effects of 1,25-dihydroxyvitamin D3and the analogues MC903 and KH1060 on interleukin-1-induced inhibitionof rat pancreatic islet -cell function in vitro. Immunol Lett 41:7377

20. Riachy R, Vandewalle B, Belaich S, Kerr-Conte J, Gmyr V, Zerimech F,

dHerbomez M,LefebvreJ, PattouF 2001 Beneficialeffect of 1,25dihydroxyvi-tamin D3 on cytokine-treated human pancreatic islets. J Endocrinol 169:161168

21. Riachy R, Vandewalle B, Kerr CJ, Moerman E, Sacchetti P, Lukowiak B,Gmyr V, Bouckenooghe T, Dubois M, Pattou F 2002 1,25-DihydroxyvitaminD3 protects RINm5F and human isletcells against cytokine-induced apoptosis:implication of the antiapoptotic protein A20. Endocrinology 143:48094819

22. Merglen A, Theander S, Rubi B, Chaffard G, WollheimCB, Maechler P 2004Glucose sensitivity and metabolism-secretion coupling studied during two-year continuous culture in INS-1E insulinoma cells. Endocrinology 145:667678

23. Asfari M, Janjic D, Meda P, Li G, Halban PA, Wollheim CB 1992 Establish-ment of 2-mercaptoethanol-dependent differentiated insulin-secreting celllines. Endocrinology 130:167178

24. JanjicD, Maechler P, SekineN, Bartley C, Annen AS,WolheimCB 1999 Freeradical modulation of insulin release in INS-1cells exposed to alloxan.BiochemPharmacol 57:639 648

25. Kutlu B, Cardozo AK, Darville MI, Kruhoffer M, Magnusson N, Orntoft T,Eizirik DL 2003 Discovery of gene networks regulating cytokine-induceddysfunction and apoptosis in insulin-producing INS-1 cells. Diabetes 52:27012719

26. Gysemans CA, Waer M, Valckx D, Laureys JM, Mihkalsky D, Bouillon R,Mathieu C 2000 Early graft failure of xenogeneic islets in NOD mice is ac-companied by high levels of interleukin-1 and low levels of transforminggrowth factor- mRNA in the grafts. Diabetes 49:19921997

27. Pipeleers DG, int Veld PA, Van de WM, Maes E, Schuit FC, Gepts W 1985A new in vitro model for the study of pancreatic A and B cells. Endocrinology117:806816

28. Ling Z, Hannaert JC, Pipeleers D 1994 Effect of nutrients, hormones andserum on survival of rat islet cells in culture. Diabetologia 37:1521

29. Chen MC, Proost P, Gysemans C, Mathieu C, Eizirik DL 2001 Monocytechemoattractant protein-1 is expressed in pancreatic islets from prediabeticNOD mice and in interleukin-1 -exposed human and rat islet cells. Diabe-tologia 44:325332

30. Cardozo AK, Kruhoffer M, Leeman R, Orntoft T, Eizirik DL 2001 Identifi-cation of novel cytokine-induced genes in pancreatic -cells by high-densityoligonucleotide arrays. Diabetes 50:909920

31. Hoorens A, Van de CM, Kloppel G, Pipeleers D 1996 Glucose promotessurvival of rat pancreatic cells by activating synthesis of proteins whichsuppress a constitutive apoptotic program. J Clin Invest 98:15681574

32. Delaney CA, Pavlovic D, Hoorens A, Pipeleers DG, Eizirik DL 1997 Cyto-

kines induce deoxyribonucleic acid strand breaks and apoptosis in humanpancreatic islet cells. Endocrinology 138:26102614

33. Liu D, Pavlovic D, Chen MC, Flodstrom M, Sandler S, Eizirik DL 2000Cytokines induce apoptosis in -cells isolated from mice lacking the inducibleisoform of nitric oxide synthase (iNOS/). Diabetes 49:11161122

34. Overbergh L, Valckx D, Waer M, Mathieu C 1999 Quantification of murinecytokine mRNAs using real time quantitative reverse transcriptase PCR. Cy-tokine 11:305312

35. Giulietti A, Overbergh L, Valckx D, Decallonne B, Bouillon R, Mathieu C

2001 An overview of real-time quantitative PCR: applications to quantifycytokine gene expression. Methods 25:386401

36. Overbergh L, Giulietti A, Valckx D, Decallonne R, Bouillon R, Mathieu C2003 The use of real-time reverse transcriptase PCR for the quantification ofcytokine gene expression. J Biomol Tech 14:3343

37. Van Cromphaut SJ, Rummens K, Stockmans I, Van Herck E, Dijcks FA,Ederveen AG, Carmeliet P, Verhaeghe J, Bouillon R, Carmeliet G 2003Intestinal calcium transporter genes are upregulated by estrogens and thereproductive cycle through vitamin D receptor-independent mechanisms.

J Bone Miner Res 18:1725173638. Verhaeghe J, Van Herck E, Van Bree R, Van Assche FA, Bouillon R 1989

Osteocalcin during the reproductive cycle in normal and diabetic rats. J En-docrinol 120:143151

39. Bach JF 2002 Immunotherapy of type1 diabetes:lessons for other autoimmunediseases. Arthritis Res 4:S3S15

40. DahlquistGG, PattersonC, Soltesz G 1999 Perinatalrisk factors for childhoodtype 1 diabetes in Europe. The EURODIAB Substudy 2 Study Group. DiabetesCare 22:16981702

41. Yoon JW, Jun HS 2001 Cellular and molecular pathogenic mechanisms ofinsulin-dependent diabetes mellitus. Ann NY Acad Sci 928:200211

42. Cardozo AK, Proost P, Gysemans C, Chen MC, Mathieu C, Eizirik DL 2003IL-1 and IFN- induce the expression of diverse chemokines and IL-15 inhuman and rat pancreatic islet cells, and in islets from pre-diabetic NOD mice.Diabetologia 46:255266

43. Giarratana N, Penna G, Amuchastegui S, Mariani R, Daniel KC, Adorini L2004 A vitamin D analog down-regulates proinflammatory chemokine pro-duction by pancreatic islets inhibiting T cell recruitment and type 1 diabetesdevelopment. J Immunol 173:22802287

44. Rabinovitch A, Suarez-Pinzon WL, Sooy K, Strynadka K, Christakos S 2001Expression of calbindin-D(28k) in a pancreatic islet -cell line protects againstcytokine-induced apoptosis and necrosis. Endocrinology 142:36493655

45. Van Etten E, Branisteanu DD, Verstuyf A, Waer M, Bouillon R, Mathieu C2000 Analogs of 1,25-dihydroxyvitamin D3 as dose-reducing agents for clas-sical immunosuppressants. Transplantation 69:19321942

46. Van Etten E, Decallonne B, Bouillon R, Mathieu C 2004 NOD bone marrow-derived dendritic cells are modulated by analogs of 1,25-dihydroxyvitamin

D(3). J Steroid Biochem Mol Biol 8990:45745947. Casteels K, Waer M, Bouillon R, Depovere J, Valckx D, Laureys J, Mathieu

C 1998 1,25-Dihydroxyvitamin D3 restores sensitivity to cyclophosphamide-induced apoptosis in non-obese diabetic (NOD) mice and protects againstdiabetes. Clin Exp Immunol 112:181187

48. Overbergh L, Decallonne B, Waer M, Rutgeerts O, Valckx D, Casteels KM,Laureys J, Bouillon R, Mathieu C 2000 1,25-dihydroxyvitamin D3 inducesan autoantigen-specific T-helper 1/T-helper 2 immune shift in NOD miceimmunized with GAD65 (p524543). Diabetes 49:13011307

49. Mathieu C,WaerM, CasteelsK, Laureys J,BouillonR 1995Prevention of typeI diabetes in NOD mice by nonhypercalcemic doses of a new structural analogof 1,25-dihydroxyvitamin D3, KH1060. Endocrinology 136:866872

50. Casteels KM, Mathieu C, Waer M, Valckx D, Overbergh L, Laureys JM,Bouillon R 1998 Prevention of type I diabetes innonobesediabeticmiceby lateintervention with nonhypercalcemic analogs of 1,25-dihydroxyvitamin D3 incombination with a short induction course of cyclosporin A. Endocrinology139:95102

51. Christen U, McGavern DB, Luster AD, von Herrath MG, Oldstone MB 2003

Among CXCR3 chemokines, IFN--inducible protein of 10 kDa (CXC chemo-kine ligand (CXCL) 10) but not monokine induced by IFN-(CXCL9) imprintsa pattern for the subsequent development of autoimmune disease. J Immunol171:68386845

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