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Receptor-1, and TNF Receptor-2 MoleculesIndependent of Fas, Perforin, TNFDiabetes in Nonobese Diabetic Mice Islet-Specific Expression of IL-10 Promotes

Itoh and Nora SarvetnickBalakrishna, Pere Santamaria, Toshiaki Hanafusa, Naoto Balaji Balasa, Kurt Van Gunst, Nadja Jung, Deepika

http://www.jimmunol.org/content/165/5/2841doi: 10.4049/jimmunol.165.5.2841

2000; 165:2841-2849; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/165/5/2841.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2000 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Islet-Specific Expression of IL-10 Promotes Diabetes inNonobese Diabetic Mice Independent of Fas, Perforin,TNF Receptor-1, and TNF Receptor-2 Molecules1

Balaji Balasa,2* Kurt Van Gunst,* Nadja Jung,* Deepika Balakrishna,* Pere Santamaria,†

Toshiaki Hanafusa,‡ Naoto Itoh,§ and Nora Sarvetnick3*

Several death-signaling or death-inducing molecules have been implicated inb cell destruction, including Fas, perforin, andTNFR-1. In this study, we examined the role of each death-signaling molecule in the IL-10-accelerated diabetes of nonobesediabetic (NOD) mice. Groups of IL-10-NOD mice, each deficient in either Fas, perforin, or TNFR-1 molecules, readily developedinsulitis, and subsequently succumbed to diabetes with an accelerated kinetics and incidence similar to that observed in theirwild-type or heterozygous IL-10-NOD littermates. Similarly, a TNFR-2 deficiency did not block accelerated diabetes in IL-10-NODmice and spontaneous diabetes in NOD mice. These results demonstrate that pancreatic IL-10 promotes diabetes independent ofFas, perforin, TNFR-1, and TNFR-2 molecules. Subsequently, when cyclophosphamide, a diabetes-inducing agent, was injectedinto insulitis-free NOD.lpr/lpr mice, none of these mice developed insulitis or diabetes. Our data suggest that cyclophosphamide-but not IL-10-induced diabetes is Fas dependent. Overall, these findings provide evidence that pancreatic expression of IL-10promotes diabetes independent of the major death pathways and provide impetus for identification of novel death pathwaysprecipitating autoimmune destruction of insulin-producing b cells. The Journal of Immunology,2000, 165: 2841–2849.

I nsulin-dependent diabetes mellitus (IDDM)4 is an autoim-mune disease caused by the T cell-mediated destruction ofinsulin-producingb cells in the pancreatic islets of Langer-

hans (1, 2). The nonobese diabetic (NOD) mouse spontaneouslydevelops IDDM and has been used as an animal model of humanIDDM. In the NOD mouse, insulitis begins at 3–4 wk of age anddiabetes by 14 wk of age (3).

Cytokines produced by the mononuclear cell infiltrate (T cellsand APC) itself are clearly involved in the propagation of insulitis.Treatment of young NOD mice with anti-IL-10 mAb prevented thedevelopment of insulitis (4). Elsewhere, the expression of IL-10 inpancreaticb cells correlated with the insulitis of NOD mice (5).BALB/c mice expressing the IL-10 transgene (tg) in their insulin-producingb cells (IL-10-BALB/c mice) of the pancreas did notdevelop diabetes, but their offspring (IL-10-NOD mice) frombackcrosses (N2-N3) to NOD mice became diabetic at an accel-

erated rate (6). Similarly, NOD mice expressing the IL-10 trans-gene in glucagon-producinga cells of the pancreas developed di-abetes at an accelerated rate (5). These cumulative findings impliedthat IL-10 is an immunostimulatory factor in the IDDM of NODmice. Our recent studies have demonstrated that the promotion ofdiabetes by IL-10 in NOD mice requires the participation of anautoreactive T cell repertoire (7). Depending upon the circum-stances, pancreatic IL-10 promoted autoimmune diabetes via CD4(8) or CD8 T cells (7).

Several studies have shown that a homozygous deficiency of theFas, perforin, or TNFR-1 molecules dramatically affects the inci-dence of spontaneous insulitis and diabetes in NOD mice. Fas-deficient NOD.lpr/lprmice were free from insulitis and diabetes(9, 10), and a perforin deficiency reduced the incidence of diabetesby a dramatic 80–85%, despite the mild insulitis in pancreata (11)of these mice, as confirmed by others (12). Additionally, TNFR-1-deficient NOD mice exhibited mild insulitis, but were com-pletely resistant to spontaneous diabetes (13). Consequently, eachof these pathways participates in either the initiation and/or effec-tor phases of autoimmune diabetes.

In this study, we examined the roles of these death-signalingmolecules (Fas, perforin, and TNFR-1), and of TNFR-2 as well, inthe IL-10-accelerated autoimmune diabetes of NOD mice. TheFas-Fas ligand (FasL) pathway was also explored in cyclophosph-amide (CYP)-induced diabetes of NOD mice. Determining howthese molecules function, if at all, in the inception and accelerationof autoimmune diabetes is important to understanding how inflam-matory stimuli such as cytokines in the target organ predisposemice to autoimmune diabetes and to devising appropriate thera-peutic interventions.

Our results demonstrate that IL-10 promoted diabetes in NODmice independent of the Fas, perforin, TNFR-1, and TNFR-2 mol-ecules. A homozygous deficiency at perforin gene locus in IL-10-NOD mice slightly delayed the onset, but did not decrease theincidence of their diabetes. Although IL-10-NOD.lpr/lpr mice

*Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037;†Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada;‡Osaka Uni-versity Medical School, Suita, Osaka, Japan; and§Toyonaka Municipal Hospital,Toyonaka, Osaka, Japan

Received for publication March 22, 2000. Accepted for publication June 13, 2000.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 B.B. is supported successively by postdoctoral fellowships from the Juvenile Dia-betes Foundation International (JDFI) and the Myasthenia Gravis Foundation ofAmerica. This work was supported by a Diabetes Interdisciplinary Research Programgrant from the JDFI (to N.S.), Scientific Research Fund from the Ministry of Edu-cation, Science, and Culture of Japan (to N.I.), and the Medical Research Council ofCanada (to P.S.).2 Current address: Protein Design Labs, Inc., 34801 Campus Drive, Fremont, CA94555.3 Address correspondence and reprint requests to Dr. Nora Sarvetnick, Mail code:IMM-23, Department of Immunology, The Scripps Research Institute, 10550 NorthTorrey Pines Road, La Jolla, CA 92037. E-mail address: noras@scripps.edu4 Abbreviations used in this paper: IDDM, insulin-dependent diabetes mellitus; BG,blood glucose; CYP, cyclophosphamide; H&E, hematoxylin and eosin; KO, knock-out; NOD, nonobese diabetic; tg, transgenic; FasL, Fas ligand.

Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00

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readily developed diabetes, CYP-injected NOD.lpr/lpr mice didnot. This outcome suggests that IL-10-accelerated diabetes is Fasindependent, whereas CYP-induced diabetes is Fas dependent. Fi-nally, current findings may provide impetus for the delineation ofadditional death pathways, under the influence of cytokine-inducedinflammation, precipitating autoimmune destruction of insulin-producingb cells.

Materials and MethodsMice

NOD/shi mice were part of the rodent breeding colony at The ScrippsResearch Institute (La Jolla, CA). IL-10-BALB/c mice expressing theIL-10 transgene in their islets under control of the human insulin promoterwere backcrossed to NOD/shi mice for 10–11 generations to produce IL-10-NOD mice (7). The presence of the transgene was verified by PCR.Mice were housed under specific pathogen-free conditions.

Generation of Fas-deficient NOD.lpr/lpr mice

Initially, we backcrossed B6.MRL.lpr/lprmice to NOD mice for two gen-erations. The heterozygous offspring were intercrossed to get NOD.lpr/lprmice. IL-10-NOD mice were then backcrossed to these mice to generateN3 to N4 mice of appropriate combinations.

Subsequently, to generate a later generation of IL-10-NOD.lpr/lpr mice,IL-10-NOD mice were backcrossed to NOD.wt/lpror NOD.lpr/lpr mice ofan N9 backcross generation. The Fas mutation (lpr) was verified in tailDNA by using two pairs of primer sets (10). The first pair was composedof NIL-1, 59-CAG CAG GAA TCC TAT GAG GT-39and NIL-2, 59-CTCGCA ACG TGA ACG GTT CG-39, yielding a band of 381 bp for themutated allele. The second pair was composed of NIL-1, 59-CAG CAGGAA TCC TAT GAG GT-39 and NIL-4, 59-GCA GAG ATG CTA AGCAGC AG-39, yielding a band of 265 bp for the wild-type allele and a bandof 5.7 kb for the mutated allele.

Generation of perforin-deficient NOD mice

Perforin-deficient BALB/c mice were backcrossed onto NOD mice for twoto three generations, and the resulting heterozygous mice of generation N2or N3 were then intercrossed to generate perforin-deficient NOD mice and,subsequently, perforin-deficient IL-10-NOD mice. The perforin genotypewas determined with PCR using three primers on DNA prepared from tailbiopsies (perforin 12 primer, 59-TGG CCT AGG GTT CAC ATC CAG-39;perforin 17 primer, 59-CGT GAG AGG TCA GCA TCC TTC-39; perforin26 primer, 59-ATA TTG GCT GCA GGG TCG CTC-39). The PCR yieldeda 500-bp fragment for wild-type mice, a 350-bp fragment for KO mice, and350- and 500-bp fragments for heterozygous mice.

Generation of TNFR-1-deficient NOD mice

TNFR-1-deficient C57BL/6j mice (14) were purchased from The JacksonLaboratory (Bar Harbor, ME) and backcrossed onto NOD mice for three tofour generations. NOD.TNFR-1 heterozygous mice of N3 or N4 genera-tions were intercrossed to generate TNFR-1-deficient NOD mice, whichwere used to introduce the TNFR-1 gene deficiency into IL-10-NOD mice.TNFR-1 genotyping was determined by PCR using three primers on DNAprepared from tail biopsies in one PCR. o1MR448, 59-TGT GAA AAGGGC ACC TTT ACG GC-39(TNFR-1 wild-type primer); o1MR449, 59-GGC TGC AGT CCA GCG ACT GG-39(TNFR-1 common primer);o1MR450, 59-ATT CGC CAA TGA CAA GAC GCT GG-39(HSV-thy-midine kinase primer). The o1MR448 and o1MR449 primer set yielded470 bp for1/1 mice, The o1MR449 and o1MR450 primer set yielded300-bp fragment for2/2 mice.

Generation of TNFR-2-deficient NOD mice

TNFR-2-deficient C57BL/6j mice (15) were purchased from The JacksonLaboratory and were backcrossed onto NOD mice for three to four gen-erations. NOD.TNFR-2 heterozygous mice of N3 or N4 generations wereintercrossed to generate TNFR-2-deficient NOD mice, which were used tointroduce TNFR-2 gene deficiency into IL-10-NOD mice. The TNFR-2genotyping was determined by PCR on DNA prepared from tail biopsiesusing two primer sets in two separate reactions. o1MR338, 59-CCT CTCATG CTG TCC CGG AAT-39(wild-type primer) (forward); o1MR338,59-AGC TCC AGG CAC AAG GGC GGG-39(wild-type primer) (re-verse); o1MR340, 59-CGG TTC TTT TTG TCA AGA C-39(neo primer)(forward); o1MR341, 59-ATC CTC GCC GGG CAT GC-39(neo primer)(reverse). The o1MR338 and o1MR338 primer set yielded a 200-bp frag-ment, whereas o1MR340 and o1MR34 yielded a 400-bp fragment.

MHC typing of mice

All the second backcross mice were tested by PCR for NOD, C57BL/6, andBALB/c MHC. The presence of I-Abd was determined on tail DNA byPCR using the following primer set: forward primer, 59-GAT ACA TCTACA ACC GGG AGG AG-39, and reverse primer, 59-CTG TTC CAGTAC TCG GCG TCT G-39. PCR amplification yielded a103-bp productfrom BALB/c, but not NOD mice. The presence of I-Ead was tested in tailDNA by PCR using the following primer set: forward primer, 59-ATGAGC TCC CAG AAG TCA TGG G-39, and reverse primer, 59-GGA GAGACA GCA GCT CTC AGC-39. PCR amplification yielded a 277-bp prod-uct from BALB/c, but not NOD mice. Mice were also tested for MHC classI molecules at Kd with anti-Kd mAb (clone SF1-1.1), Kb with anti-Kb mAb(clone AF6-88.5), Dd with anti-Dd mAb (clone 34-2-12), Db with anti-H-2Db mAb (clone 28-14-8), and class II I-Ab molecule with anti-I-Ab mAb(clone AF6-120.1) (PharMingen, La Jolla, CA) by flow cytometry.

Adoptive transfers

Donor splenocytes from the mice indicated were prepared as single cellsuspensions in sterile PBS. These cells were injected (at 13 107/mouse or3 3 107/mouse) i.v. into 16-wk-old IL-10-NOD-scid/scid or NOD.scid/scid mice. For adoptive transfer of perforin-deficient splenocytes, we usedperforin-deficient (2/2) NOD mice of N10 backcross generation (12).

Assessment of diabetes

Starting at 4–5 wk of age, mice were tested for diabetes by weekly mea-surements of blood glucose (BG) levels using a one-step Bayer GlucometerElite (Bayer Corporation, Elkhart, IN). Animals were considered diabeticwhen BG levels were.300 mg/dl. In most instances, the BG levels ex-ceeded 500 mg/dl. Mice of both sexes were included in all the IL-10-NODexperiments, and female mice were employed for monitoring of diabetes inTNFR-1 KO and TNFR-2 KO mice.

Histological analysis

Lymphocytic infiltration of the islets was evaluated on hematoxylin andeosin (H&E)-stained paraffin sections of the pancreas. For insulin staining,paraffin-embedded sections of the pancreata were stained with an immu-noperoxidase method using polyclonal Abs to porcine insulin, followed bya biotinylated secondary Ab and an avidin-biotin complex, as describedearlier (16).

ResultsPancreatic IL-10 promotes autoimmune insulitis and diabetes inNOD.lpr/lpr mice

Autoimmune destruction of insulin-producingb cells involvesFas-FasL interaction, as evident because Fas-deficient NOD.lpr/lpr mice are free from spontaneous insulitis and diabetes (9, 10).Previous studies from our group demonstrated that IL-10-NODmice rapidly develop insulitis and diabetes compared with theircounterpart NOD mice (7). Because a Fas1 mononuclear cell in-filtrate in the pancreatic islets of 5-wk-old diabetic IL-10-NODmice (not shown) was observed, we tested the requirement forFas-FasL interaction in the accelerated diabetes of IL-10-NODmice. After introducing the Fas deficiency into IL-10-NOD miceby breeding them with diabetes-resistant Fas-deficient NOD.lpr/lpr mice, we monitored their offspring for diabetes at weekly in-tervals. Surprisingly, IL-10-NOD.lpr/lprmice of the N3-N4 back-cross generations (n5 8; 88%) (Fig. 1A) and N8-N9 backcrossmice (n5 13; 100%) (Fig. 1B) developed the accelerated diabetes.The kinetics and incidence of the disease in these mice duplicatedthat of the wild-type (Fas/Fas) (N3-N4 backcrossn 5 5; 100%; orN8-N9 backcross micen 5 12; 100%) as well as of the heterozy-gous littermates (Fas/lpr) (N3-N4 backcrossn 5 9; 89%; orN8-N9 backcross micen 5 12; 83%). Statistical values (p values)for N3-N4 backcross mice were as follows: IL-10-NOD.lpr/lpr vsIL-10-NOD.Fas/lpr 0.6145; IL-10-NOD.lpr/lpr vs IL-10-NOD-.Fas/Fas 0.850. Statistical values for N8-N9 backcross mice wereas follows: IL-10-NOD.lpr/lprvs IL-10-NOD.Fas/lpr0.5370; IL-10-NOD.lpr/lpr vs IL-10-NOD.Fas/Fas 0.0751. However, duringthe same period of time, none of the non-tg littermates (Fas/Fas or

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Fas/lpror lpr/lpr) became diabetic. The NOD.Fas/Fas and NOD.Fas/lprmice developed diabetes after 14 wk of age. In agreementwith the published literature, NOD.lpr/lprmice did not developdiabetes over a 24-wk period (not shown). Therefore, the Fas/FasLinteraction was not necessary for IL-10-accelerated diabetes in

NOD mice. Analysis of pancreata from IL-10-NOD.lpr/lpr (2/2)mice, by H&E staining, showed extensive lymphocytic infiltrationof the islets similar to that observed in IL-10-NOD.Fas/lpr (1/2)littermates used as controls (Fig. 1C). The insulin-positive cells inthese infiltrated islets were completely destroyed (data not shown).

FIGURE 1. A and B, Incidence of autoimmune diabetes in IL-10-NOD.lpr/lprmice and their littermate controls.C, H&E staining of the paraffin-embedded pancreata from Fas-deficient IL-10-NOD.lpr/lpr(2/2) (N9 backcross) shows severe insulitis compared with that in Fas-sufficient IL-10-NOD.Fas/Fas (1/1) littermates. Note that pancreata from NOD.lpr/lprare free from insulitis (3200).

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As expected, islets from pancreata of NOD.lpr/lpr mice were freefrom insulitis. Our results demonstrate that IL-10 promotes auto-immune insulitis and diabetes independent of the Fas/FasLpathway.

Splenocytes from diabetic IL-10-NOD.lpr/lpr mice transferdisease into NOD.scid/scid mice

Since, as Fig. 1 shows, IL-10-NOD.lpr/lpr mice readily developeddiabetes, but the non-tg NOD.lpr/lprmice did not, we testedwhether splenocytes from diabetic 8-wk-old IL-10-NOD.lpr/lprmice would transfer disease into NOD-scid/scid mice. As expected(Fig. 2A), splenocytes from littermate non-tg NOD.lpr/lpr mice didnot cause diabetes in NOD-scid/scid mice (n5 4). Conversely,splenocytes from diabetic IL-10-NOD.lpr/lpr mice provoked dia-betes in recipient NOD-scid/scid mice beginning at 16 wk post-transfer (n5 4) (p 5 0.0082). Staining of pancreata by H&Erevealed mononuclear cell infiltrates within islets from NOD-scid/scid recipients of splenocytes from diabetic IL-10-NOD.lpr/lprmice, whereas islets in NOD-scid/scid recipients of splenocytesfrom NOD.lpr/lpr mice were free from insulitis (Fig. 2B). In adifferent set of experiments, splenocytes from 5-wk-old diabeticIL-10-NOD mice (Fas/lpr) transferred disease into NOD.scid/scidmice with same kinetics that was observed with 8-wk-old diabeticIL-10-NOD.lpr/lpr mice.5 Additionally, the kinetics of diseasetransfer observed with splenocytes from 5-wk-old diabetic IL-10-NOD mice is far different from that observed with splenocytesfrom 18-wk-old diabetic NOD mice.5 The results again indicatethat the Fas/FasL pathway is not required for autoimmune diabetesin IL-10-NOD mice.

IL-10 promotes accelerated diabetes in perforin-deficientNOD mice

Next, we examined the role of perforin in autoimmune destructionof b cells of IL-10-NOD mice by introducing a perforin gene de-ficiency into IL-10-NOD mice. As Fig. 3Adepicts, IL-10-NODmice of the wild-type (1/1) (n 5 8; 88% incidence) or heterozy-gous (1/2) (n 5 15; 87% incidence) or KO (2/2) (n 5 9; 89%)for the perforin gene developed the anticipated diabetes. Interest-ingly, a gene dose effect on the incidence of accelerated diabetes at5 wk of age was noticed. As compared with 50% incidence ofdiabetes at 5 wk of age in IL-10-NOD.wild-type (1/1) mice, only7% of heterozygous and 11% of KO mice developed diabetes.However, the cumulative percentage of incidence of diabetes at 12wk was similar among all the groups (p 5 0.8185 for IL-10-NOD.perforin (2/2) vs IL-10-NOD.perforin (1/2);p 5 0.3244for IL-10-NOD.perforin (2/2) vs IL-10-NOD.perforin (1/1)).During the same interval, none of the non-tg perforin-deficient(2/2) NOD mice developed diabetes (n5 10). The pancreaticislets from diabetic IL-10-NOD mice that are heterozygous (1/2)or deficient (2/2) for perforin were completely infiltrated withmononuclear cells (Fig. 3B) and their insulin-producingb cellswere destroyed (not shown).

To further confirm that perforin is not required for accelerateddiabetes of IL-10-NOD mice, we performed adoptive transfer ex-periments. To this end, splenocytes from nondiabetic 32-wk-oldperforin-deficient (2/2) NOD mice of the N10 backcross gener-ation (12) were injected into tg IL-10-NOD-scid/scid mice (7) ornon-tg NOD.scid/scid mice. When injected, these splenocytesreadily caused diabetes in 13- to 14-wk-old IL-10-NOD-scid/scidmice (n 5 4; 100% incidence) at 4 wk posttransfer. However,NOD-scid/scid mice (n5 4; 0% incidence) that received spleno-cytes from NOD.perforin-deficient (2/2) mice did not develop

diabetes (Fig. 3C) (p 5 0.0082). Additionally, IL-10-NOD-scid/scid mice not given splenocytes from NOD.perforin-deficient(2/2) mice did not develop diabetes (n5 5) (not shown). Theseresults confirm that perforin is not essential for the accelerateddiabetes of IL-10-NOD mice.

Analysis by H&E staining (Fig. 3D) revealed that the pancreaticislets from IL-10-NOD-scid/scid mice that were injected withNOD-perforin deficient (2/2) splenocytes were completely infil-trated with mononuclear cells, causing destruction of most ofbcells, leaving few in place. Conversely, pancreatic islets fromNOD-scid/scid mice that were injected with NOD.perorin KO5 B. Balasa et al.Submitted for publication.

FIGURE 2. A, Adoptive transfer of splenocytes from female 8-wk-olddiabetic IL-10-NOD.lpr/lprmice causes disease in 8-wk-old female NOD.scid/scid mice, whereas splenocytes from littermate 8-wk-old NOD.lpr/lprmice do not. Each recipient mouse was injected i.v. with 23 107 spleno-cytes in PBS.B, Pancreata from NOD.scid/scid recipients that were in-jected with splenocytes from diabetic IL-10-NOD.lpr/lprmice show mono-nuclear cell infiltration of the islets and the loss of insulin-producingbcells, whereas splenocytes from NOD.lpr/lprmice did not cause insulitis(3200).

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FIGURE 3. A, Incidence of autoimmune diabetes in N2-N3 backcross IL-10-NOD.peforin-deficient (2/2) mice and their littermate controls.B, H&E staining of theparaffin-embedded pancreata from IL-10-NOD.peforin-deficient (2/2) shows severe insulitis compared with that in perforin-sufficient (1/2) IL-10-NOD littermates(magnification,3400).C, Adoptive transfer of splenocytes from female 32-wk-old nondiabetic perforin-deficient (2/2) NOD mice provokes disease in 8-wk-old femaleIL-10-NOD.scid/scid, but not NOD.scid/scid mice. Each recipient mouse was injected i.v. of 13 107 splenocytes in PBS.D, H&E staining of pancreata from IL-10-NOD.scid/scid and NOD.scid/scid recipient mice injected with splenocytes from 32-wk-old nondiabetic perforin-deficient (2/2) NOD mice (3200).

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(2/2) splenocytes exhibited only periinsulitis and contained intactinsulin-producingb cells. These findings further confirm the dataof Fig. 3A that IL-10 promotes diabetes independent of perforinpathway.

TNFR-1-deficient NOD mice are susceptible toIL-10-accelerated diabetes

Since neither Fas nor perforin molecules were essential for thediabetic state of IL-10-NOD mice, we questioned whether theTNFR-1 gene would fill that role. TNFR-1 gene function was dis-rupted in IL-10-NOD mice, after which their tg progeny andnon-tg littermates were monitored for diabetes beginning at 5 wkof age. The results shown in Fig. 4Aare from N3-N4 backcrossgenerations. The findings show that IL-10-NOD mice that are wildtype (1/1) (n 5 9; 89%) or heterozygous (1/2) (n 5 12; 83%)for TNFR-1 gene readily developed diabetes. Similarly, IL-10-NOD-TNFR-1-deficient (2/2) mice (n5 10; 90%) developed

diabetes with an accelerated kinetics and incidence like that in thetg littermate controls (p 5 0.6790 vs IL-10-NOD.TNFR-11/1mice; p 5 0.3359 vs IL-10-NOD.TNFR-11/2 mice). In agree-ment with previous observations (13), NOD-TNFR-1-deficientmice (2/2) did not develop diabetes (n5 7) over a period of 24wk. When pancreatic tissues from diabetic IL-10-NOD-TNFR-11/2 and IL-10-NOD-TNFR-1-deficient (2/2) mice were thenstained with H&E, islets from both groups were completely infil-trated with mononuclear cells (Fig. 4B), and their insulin-produc-ing b cells had been destroyed (not shown). Considering that the

FIGURE 5. A, Incidence of autoimmune diabetes in IL-10-NOD micethat are deficient (2/2) and sufficient (1/2) for TNFR-2 molecules. Theirnon-tg TNFR-1-deficient (2/2) littermate controls are also included. Themice used were of N3-N4 backcross generation.B, H&E staining of par-affin-embedded pancreatic sections from diabetic IL-10-NOD. TNFR-2-deficient (2/2) and sufficient (1/2) mice, respectively (3400).

FIGURE 4. A, Incidence of autoimmune diabetes in IL-10-NOD.TNFR-1-deficient (2/2) and sufficient (1/2,1/1) mice. Their non-tgTNFR-1-deficient (2/2) littermate controls are also included. The miceused were of the N3-N4 backcross generations.B, H&E staining of par-affin-embedded pancreatic sections from diabetic IL-10-NOD. TNFR-1-deficient (2/2) and sufficient (1/2) mice, respectively (3200).

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pancreatic islets from age-matched non-tg TNFR-1-deficient lit-termates were free from insulitis (data not shown), and their in-ability to develop diabetes even at 24 wk of age (n 5 8), clearlyTNFR-1 signaling plays a role in spontaneous autoimmune diabetesof NOD mice, but not in the accelerated diabetes of IL-10-NOD mice.

TNFR-2-deficient NOD mice are susceptible to spontaneous andIL-10-accelerated diabetes

Subsequently, we introduced the disrupted TNFR-2 gene into IL-10-NOD mice. As shown in Fig. 5A, IL-10-NOD mice (generationN4 backcross) that were either heterozygous (1/2) (n 5 8; 100%)or deficient (n5 6; 100%) for TNFR-2 gene developed diabetesbeginning at 4–5 wk of age. There was no statistical significancebetween these two groups (p 5 0.2059). However, their non-tgKO (n 5 5) littermates were diabetes free for that 10-wk period.

Furthermore, the pancreatic islets from both groups of micewere extensively infiltrated with autoreactive lymphocytes (Fig.5B), and their insulin-producingb cells were destroyed (notshown). The islets from pancreata of 5-wk-old littermate NOD-TNFR-2-deficient (2/2) mice exhibited periinsulitis. These micesubsequently progressed to diabetes beginning at 18 wk of ageand showed 60% incidence of diabetes by 24 wk of age (n 5 5).These findings imply that TNFR-2 signaling is irrelevant for spon-taneous and accelerated diabetes of NOD and IL-10-NOD mice,respectively.

CYP fails to provoke autoimmune insulitis and diabetes in theabsence of pancreatic inflammation

Finally, to test whether CYP would induce diabetes in a Fas-de-pendent manner, we injected CYP into Fas-deficient NOD.lpr/lpr

FIGURE 6. A, CYP injection fails to induce diabetes in insulitis-free NOD.lpr/lprmice. NOD.lpr/lpr(2/2) (n 5 10) and NOD.Fas/lpr(1/2) (n 5 10)mice were injected i.p. with CYP (200 mg/kg body weight) on days 0 and 14, then monitored for diabetes at weekly intervals. Mice were considered diabeticwhen the BG values were.300 mg/dl.B, The insulitis index for CYP-treated NOD.lpr/lpr(n 5 10) and NOD.Fas/lpr(n 5 8) mice is shown. The valuesin the parentheses represent the number of islets scored for each category.C, Representative H&E-stained paraffin-embedded pancreatic sections fromCYP-treated NOD.lpr/lprand NOD.Fas/lprmice (3400).

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(2/2) mice and theirFas-sufficient NOD.Fas/lpr(1/2) littermatecontrols on days 0 and 14. This protocol has been shown earlier toevoke or accelerate diabetes in NOD mice (17, 18). We found that,over a period of 8 wk, NOD.lpr/lprmice were completely resistantto CYP-induced diabetes. As expected, heterozygous mice (Fas/lpr) ( p 5 0.004 vslpr/lpr group) and wild-type (Fas/Fas) mice( p 5 0.0019 vslpr/lpr) rapidly developed diabetes beginning at 2wk after the first CYP injection (Fig. 6A). Additionally, most of theheterozygous NOD mice (Fas/lpr) (1/2) became diabetic within2–4 wk of receiving the first inoculation of CYP.

Although approximately 80% of the islets from CYP-injectedheterozygous (Fas/lpr) mice (n5 10) showed severe insulitis,none of the islets from CYP-treated NOD.lpr/lprmice (n5 8) hadany sign of lymphocytic infiltration in or near the islets (Fig. 6B).Occasionally, a perivascular infiltrate occupied the pancreatic tis-sue of CYP-treated NOD.lpr/lprmice, as pictured in representativeH&E-stained sections from NOD.Fas/lpr (1/2) and NOD.lpr/lpr(2/2) (Fig. 6C). Since the pancreatic islets of CYP-treatedNOD.lpr/lpr mice were free from lymphocytic infiltration andstained positively for insulin, yet those of CYP-treated heterozy-gous NOD.Fas/lprmice were filled with mononuclear cells andstained only weakly for insulin-positive cells, Fas expression is aprerequisite for CYP-induced diabetes in NOD mice.

DiscussionThe results from this study demonstrate that the expression ofIL-10 in pancreatic islets bypasses the requirement for Fas, per-forin, TNFR-1, and TNFR-2 molecules and that IL-10 can other-wise precipitate the diabetic process. In contrast to IL-10-acceler-ated diabetes in NOD.lpr/lprmice, CYP fails to provoke diabetesin NOD.lpr/lpr mice. Therefore, our findings demonstrate that IL-10-accelerated diabetes is Fas independent and that CYP-induceddiabetes is Fas dependent. This current study also demonstratesthat for the first time, in contrast to the role of TNFR-1 in diabetesof the NOD mouse, deficiency of TNFR-2 failed to block sponta-neous diabetes of NOD mice.

Destruction ofb cells in the spontaneous diabetes of NOD micerequired Fas-FasL interaction. The Fas-FasL pathway appeared tobe required for the initiation and/or effector phases of spontaneousautoimmune diabetes in former experiments with NOD mice (9,10, 19), and with TCR tg NOD mice expressing islet-specific Tcells (12, 20). The current study with CYP-induced diabetes ofNOD mice further highlights a role for Fas-FasL pathway in de-struction of b cells. However, islet transplantation experimentsprovided differing results. That is, the Fas-FasL pathway did notparticipate in the effector stages of diabetes, since NOD.lpr/lprislets transplanted into recently diabetic NOD mice were com-pletely destroyed by an autoimmune attack (21, 22) or followingCYP injection (22). Apparently different mechanisms participatein the destruction of ectopically transplanted islet grafts and ofbcells in situ. Our current findings demonstrated that expression ofthe IL-10 transgene in the pancreatic islets promoted accelerateddiabetes of NOD mice in situ without a requirement for the Fassignaling. Additionally, we showed that splenocytes from diabeticIL-10-NOD.lpr/lpr mice transferred disease into NOD.scid/scidmice, reinforcing the implication that the Fas-FasL pathway is notrequired throughout this autoimmune process of IL-10-NOD mice.

If, as seems evident, the Fas-FasL pathway does not participatein this accelerated diabetes, presumably expression of the IL-10transgene in the islets of NOD mice could awaken other deathpathways such as those that use perforin or TNFR-1 or TNFR-2molecules. However, our findings exclude that possibility. There-fore, the results described in this work contrast with earlier con-clusions that perforin is required for spontaneous diabetes (11, 12)

and CYP-induced diabetes (11). TNFR-1 molecules were also con-sidered a requirement for the spontaneous and CYP-induced dia-betes of NOD mice (13). Since TNFR-2-deficient NOD mice de-veloped spontaneous diabetes, we did not study the effect ofTNFR-2 deficiency on CYP-induced diabetes. In addition, our cur-rent findings demonstrate that Fas is also required for CYP-in-duced diabetes of NOD mice. However, the actual cause, for theabsence of disease, may have been the lack of intense inflamma-tion in the pancreatic environment that is necessary for efficientAPC activation, Ag presentation, and T cell activation, leading tothe production of inflammatory mediators.

For example, we have shown that CD40-CD40L pathway isessential for the spontaneous autoimmune insulitis and diabetes ofNOD mice, as demonstrated by Ab-blocking studies. This pathwayappears to play a role in the initiation but not the effector phase ofthis disease process (16). The requirement for this pathway inspontaneous diabetes was confirmed by Green and coworkers (23)using CD40L-deficient NOD mice. However, this pathway wasfound dispensable for the accelerated diabetes of tg IL-10-NOD(7) and TNF-a-NOD (23) mice. These findings and the resultspresented in the current study suggest that cytokine-induced in-flammation in the pancreatic environment circumvented the re-quirement for the well-established costimulation pathways,thereby short-circuiting the onset of disease. This hypothesis isfurther supported by two additional observations: 1) Expression ofthe IL-10 transgene in diabetes-free BDC2.5 NOD mice leads tothe development of diabetes. 2) CYP injection fails to provokediabetes in insulitis-free NOD.lpr/lprmice. We are of the opinionthat the failure to observe diabetes in CYP-treated NOD.lpr/lprmice is unrelated to thelpr-induced lymphoproliferative effect,because IL-10-NOD.lpr/lprmice do develop diabetes, and theirsplenocytes can transfer diabetes into NOD.scid/scid mice withdelayed kinetics.

It is well established that IL-10 promotes pathogenic cell-me-diated and humoral autoimmunity. In fact, tg IL-10-C57BL/6 miceexpressing IL-10 under control of the salivary amylase promoterdeveloped a Sjogren’s-like syndrome via a Fas-FasL pathway (24).Depending on the circumstances, then, IL-10 could exhibit its au-toimmunostimulatory effect via Fas-dependent and Fas-indepen-dent pathways. IL-10 also seems to act as an immunostimulator inhumoral autoimmunity through B cells (25). However, our previ-ous findings demonstrate that, in T cell-mediated autoimmunity,IL-10 readily promotes autoimmune diabetes independent of Bcells, because B cell-deficient IL-10-NOD mice also developedaccelerated diabetes similar to that in their wild-type counterparts(7). Continual administration of IL-10 to NZB/W F1 mice accel-erated autoimmunity, whereas treatment with anti-IL-10 Ab de-layed its onset (26). Apart from its pathogenic role in autoimmu-nity, IL-10 further exerted its immunostimulatory capacity withrespect to tumor suppression, since IL-10 expressed under a classII promoter of C57BL/6 mice limited the growth of immunogenictumors (27).

From the foregoing results, we conclude that accelerated diabe-tes in IL-10-NOD mice does not involve the classical death sig-naling molecules, Fas, perforin, and TNFR-1. Nor is the TNFR-2molecule required for diabetes to arise in IL-10-NOD mice. There-fore, pancreatic IL-10 may promote diabetes via unique deathpathway(s) involving TRAIL, TWEAK, and LIGHT molecules(28–30), or its expression may promote diabetes via compensatorydeath pathways. To prevent such disease, these novel death path-ways used by autoimmune responses to destroy insulin-producingb cells must be uncovered along with molecules that interfere withtheir destruction.

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AcknowledgmentsWe thank Antonio Lacava and Shyam Pakala for helpful discussions on thedata presented in this article.

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