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The Journal of Immunology, 2003, 170: 5491-5501.
Copyright © 2003 by The American Association of Immunologists

IL-12 Administration Accelerates Autoimmune Diabetes in Both Wild-Type and IFN-{gamma}-Deficient Nonobese Diabetic Mice, Revealing Pathogenic and Protective Effects of IL-12-Induced IFN-{gamma}1

Sylvie Trembleau2, Giuseppe Penna, Silvia Gregori, Nadia Giarratana and Luciano Adorini3

BioXell, Milan, Italy


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IL-12 administration to nonobese diabetic (NOD) mice induces IFN-{gamma}-secreting type 1 T cells and high circulating IFN-{gamma} levels and accelerates insulin-dependent diabetes mellitus (IDDM). Here we show that IL-12-induced IFN-{gamma} production is dispensable for diabetes acceleration, because exogenous IL-12 could enhance IDDM development in IFN-{gamma}-deficient as well as in IFN-{gamma}-sufficient NOD mice. Both in IFN-{gamma}+/- and IFN-{gamma}-/- NOD mice, IL-12 administration generates a massive and destructive insulitis characterized by T cells, macrophages, and CD11c+ dendritic cells, and increases the number of pancreatic CD4+ cells secreting IL-2 and TNF-{alpha}. Surprisingly, IL-12-induced IFN-{gamma} hinders pancreatic B cell infiltration and inhibits the capacity of APCs to activate T cells. Although pancreatic CD4+ T cells from IL-12-treated IFN-{gamma}-/- mice fail to up-regulate the P-selectin ligand, suggesting that their entry into the pancreas may be impaired, T cell expansion is favored in these mice compared with IL-12-treated IFN-{gamma}+/- mice because IL-12 administration in the absence of IFN-{gamma} leads to enhanced cell proliferation and reduced T cell apoptosis. NO, an effector molecule in {beta} cell destruction, is produced ex vivo in high quantity by pancreas-infiltrating cells through a mechanism involving IL-12-induced IFN-{gamma}. Conversely, in IL-12-treated IFN-{gamma}-deficient mice, other pathways of {beta} cell death appear to be increased, as indicated by the up-regulated expression of Fas ligand on Th1 cells in the absence of IFN-{gamma}. These data demonstrate that IFN-{gamma} has a dual role, pathogenic and protective, in IDDM development, and its deletion allows IL-12 to establish alternative pathways leading to diabetes acceleration.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The key feature of insulin-dependent diabetes mellitus (IDDM)4 is a cell-mediated destruction of the insulin-secreting {beta} cells of the pancreatic islets (1), and the nonobese diabetic (NOD) mouse has been instrumental in providing a better understanding of diabetes pathogenesis (2, 3). T lymphocytes cause IDDM (4), but the events that initiate the disease, the relative importance of the different cell types that progressively infiltrate the pancreatic islets, and the precise mechanism(s) of {beta} cell destruction are still incompletely understood.

IDDM, like most autoimmune diseases, is characterized by overproduction of type-1 cytokines such as IFN-{gamma}, IL-2, and TNF-{alpha}. Interestingly, macrophages and dendritic cells (DCs) of NOD mice present several abnormalities compared with non-autoimmune-prone mouse strains, including the capacity to secrete elevated levels of IL-12 upon in vitro stimulation (5, 6). In addition, IL-12 mRNA increases in the islets in parallel to {beta} cell destruction (7). The IL-12p40 chain of NOD and other autoimmune disease-prone mice carries two amino acid substitutions compared with NOR and other mouse strains, and IL-12p40 levels, both basal and induced, are reduced in the NOD mouse (8). Reduced levels of the potentially inhibitory IL-12p40 chain coupled with enhanced production of the IL-12p75 heterodimer could explain the enhanced Th1 cell development observed in the NOD mouse (8, 9, 10). Administration of IL-12p40 homodimer to NOD mice before the onset of insulitis antagonizes IL-12p75 and leads to reduced IDDM development (11). Thus, IL-12 produced by APCs of NOD mice has a primary role in inducing pathogenic Th1/Tc1 cells, and thereby IDDM. Consistent with the pathogenic role of Th1 cells, administration of IL-12 leads to premature IDDM onset, characterized by high numbers of pancreas-infiltrating DCs as well as Th1/Tc1 cells (12). Interestingly, stimulation of NOD DCs with IL-12 itself induces them to secrete high levels of IL-12 (6), and IL-12 contributes to the enhanced ability of NOD DCs to stimulate Ag-specific CD4+ and CD8+ T cells (13). Thus, injection of IL-12 into NOD mice may amplify the APC function of macrophages and DCs by inducing them to produce more IL-12, resulting in enhanced autoantigen-specific Th1/Tc1 cell induction. In addition, IL-12 treatment can directly stimulate T cells to produce higher levels of proinflammatory cytokines and notably IFN-{gamma}.

Several studies have implicated IFN-{gamma} in IDDM development. Blockade of IFN-{gamma} reduces significantly the incidence of diabetes (14, 15, 16), likely by disrupting different mechanisms from homing of diabetogenic T cells to the pancreas (17) to {beta} cell death (18). Surprisingly, diabetes develops in IFN-{gamma}-deficient NOD mice, although with a slightly delayed onset (19). IDDM also develops in IFN-{gamma}R {beta}-chain-deficient mice (20). The absence of the IFN-{gamma}R{alpha} (21) has been reported to lead to disease protection, likely because non-NOD genes were inherited during the backcrosses (22). Thus, IFN-{gamma} and the IFN-{gamma}R appear dispensable for IDDM development in the NOD mouse. IL-12-deficient NOD mice also develop IDDM with a similar incidence and tempo as controls (23). The genetic absence of IL-12 or IFN-{gamma} may favor the early development of compensatory pathways, which are not induced in genetically unmanipulated NOD mice. At least 20 susceptibility loci contribute to IDDM (24), and the NOD mouse presents multiple gene defects favoring the preferential development of Th1 cells and disrupting regulatory mechanisms that might overcome the absence of IFN-{gamma} or IL-12.

IFN-{gamma} is a major proinflammatory cytokine and, in addition, it synergizes with IL-12 in Th1 development (25). Thus, IFN-{gamma} would be expected to be an important component of IDDM acceleration induced by IL-12 administration. In the present study, we have analyzed IDDM development after IL-12 injection in IFN-{gamma}-/-, IFN-{gamma}+/-, and wild-type NOD mice. Surprisingly, IL-12 could accelerate IDDM to the same extent in the presence or absence of IFN-{gamma}. In both cases, IL-12 increased dramatically the number of T cells and APCs infiltrating the pancreas, resulting in higher production of inflammatory cytokines. Intriguingly, the acceleration of IDDM induced by IL-12 administration to IFN-{gamma}-deficient or IFN-{gamma}-sufficient NOD mice was mediated by a different combination of positive and negative effects on the inflammatory process, which nevertheless always resulted in IDDM acceleration.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

BALB/c, C3H, and NOD/Lt mice were purchased from Charles River Breeding Laboratories (Calco, Italy). NOD mice with a targeted deletion of the ifg gene were obtained from T. Stewart (Genentech, South San Francisco, CA) at the N9 generation. NOD males, heterozygous for the IFN-{gamma} mutation (IFN-{gamma}+/-), were further backcrossed to NOD/Lt female mice in our animal facility for three additional generations, and the heterozygous (IFN-{gamma}+/-) mice generated were then intercrossed. Subsequently, the colony was maintained by brother/sister mating of IFN-{gamma}-/- and IFN-{gamma}+/- mice to generate IFN-{gamma}-/- and IFN-{gamma}+/- littermate NOD mice. Experiments were conducted with female mice only. Blood glucose levels in tail venous blood were quantified using a Glucometer Elite (Bayer, Wuppertal, Germany). A diagnosis of diabetes was made after two sequential measurements higher than 200 mg/dl.

Antigens

Hen egg-white lysozyme (HEL) was purchased from Sigma-Aldrich (St. Louis, MO). The peptide HEL10-23, AAMKRHGLDNYRGY, was synthesized by standard Fmoc/tBu chemistry. The crude peptide was purified by reverse-phase HPLC, and the sequence was confirmed by amino acid analysis and fast atom bombardment mass spectrometry.

Recombinant mouse IL-12

Recombinant mouse IL-12 was produced in serum-free medium by transfected Chinese hamster ovary cells and purified by sequential chromatography, as previously described (26). The IL-12 used in this study was >95% pure, as assessed by SDS-PAGE analysis, and the endotoxin content was <5 U/mg IL-12, as determined by the Limulus amebocyte assay. IL-12 was diluted in PBS containing 100 µg/ml mouse serum albumin (Sigma-Aldrich) and injected into mice i.p. at 7.5 µg/kg daily for 10 days or for 4–8 wk, as indicated.

T cell hybridomas

The T cell hybridoma 2D12.1, specific for the core sequence HEL12-21 (MKRHGLDNYR), has been previously described (27). T hybridoma cells (5 x 104 cells/well) were cultured in 96-well plates with various concentrations of HEL protein or HEL10-23 peptide and with splenocytes depleted of erythrocytes and T cells from IL-12-treated or vehicle-treated IFN-{gamma}-/-, IFN-{gamma}+/-, and wild-type NOD mice (2.5 x 105 cells/well). Culture medium was RPMI 1640 supplemented with 2 mM L-glutamine, 50 µg/ml gentamicin, 50 µM 2-ME, and 10% FCS. After 24 h of culture, the supernatant was collected from each well and the IL-2 concentration measured by ELISA as previously described (28).

Flow cytometric analysis

Pancreas-infiltrating and spleen cells were isolated as previously described (11). Pancreatic CD45+ mononuclear cells or CD4+ plus CD8+ cells, purified by positive selection using microbeads (Miltenyi Biotech, Bergish Gladbach, Germany), were stained with the following mAbs: anti-CD4 (RM-4-4), anti-CD8 (53-6.7), anti-B220 (RA3-6B2), and anti-CD11b (Mac1) (all from BD PharMingen, San Diego, CA) and anti-TCR (H57-597), anti-Thy 1.2 (HO-13-4), anti-I-Ag7 (10.3.62), and anti-CD11c (N418) (all from American Type Culture Collection, Manassas, VA). Alternatively, purified pancreas-infiltrating CD4+ plus CD8+ cells were stained with PE-labeled anti-CD4 (RM4-4) and P-selectin IgG chimeric protein (a kind gift from D. Westweber, University of Muenster, Germany), followed by FITC-labeled rabbit F(ab')2 anti-human IgG. Pancreatic T cells were stained intracellularly for the detection of IFN-{gamma}, TNF-{alpha}, IL-4, IL-2, and IL-10 using the method previously described (29). Analysis was performed with a FACScan flow cytometer (BD Biosciences, Mountain View, CA) equipped with CellQuest software.

Immunohistology

Pancreata were snap-frozen in Tissue Tek (Miles Laboratories, Elkhart, IN), and 5-µm-thick sections were stained with biotinylated GK1.5 anti-CD4, 53-6.7 anti-CD8, RA3-6B2 anti-B220, and Mac1 anti-CD11b mAbs (all from BD PharMingen) or with N418 anti-CD11c mAb (American Type Culture Collection), followed by streptavidin-peroxidase conjugate. 3-Amino-9-ethylcarbazole (DAKO, Carpenteria, CA) was used as chromogen, and hematoxylin was used as a counterstain.

T cell activation by TCR ligation

To obtain purified T cells, lymph node cells (LNCs) were depleted of adherent cells by incubation at 37°C for 1 h in RPMI 1640 containing 10% FCS in petri dishes, and they were depleted of Ig+ cells with anti-mIg coated Dynabeads for 30 min at 4°C. The fraction obtained yielded 96–97% T cells as determined by flow cytofluorometry. Total LNCs or purified T cells were cultured in 200 µl of HL-1 medium (BioWhittaker Europe, Verviers, Belgium) supplemented with 2 mM glutamine and 50 µg/ml gentamicin in 96-well round bottom plates previously coated with the indicated concentrations of anti-TCR mAb (H57-597). After a 48-h incubation, cell proliferation was assessed by 16-h [3H]thymidine (1 µCi) incorporation, and IFN-{gamma} production was measured by ELISA as previously described (28).

Detection of apoptotic cells

Spleen cells were stained with Annexin-V FITC (BD PharMingen) and propidium iodide (50 µg/ml; Sigma-Aldrich) in the presence of rat anti-mouse CD4 conjugated to allophycocyanin (BD PharMingen) for 15 min at room temperature. Cells were analyzed with a FACScan flow cytometer using CellQuest software.

Inducible NO synthase (iNOS) expression and NO production

For the detection of iNOS protein, pancreas-infiltrating CD45+ cells and peritoneal exudate cells (PECs) were permeabilized with PBS, 5% FCS, 0.5% saponin, and 0.1% sodium azide (PBS/FCS/saponin) for 10 min and then were stained with FITC-conjugated anti-iNOS mAb (BD Transduction Laboratories, Lexington, KY) or with an isotype control. The cells were washed and the cell surface was stained with PE-conjugated anti-CD14 mAb (BD PharMingen). Analysis was performed on CD14+ gated cells with a FACScan flow cytometer. For the detection of NO, 3 x 105 cells/well were cultured for 24 h in flat-bottom 96-well plates in complete RPMI 1640 medium alone or supplemented with 10 µg/ml LPS with or without 500 U/ml IFN-{gamma}. For quantification of NO, 100 µl of titrated culture supernatants were incubated with 100 µl of Griess reagent, and absorbance was read at 550 nm.

Fas ligand (FasL) expression on Th1 cells

CD4+ T cells were enriched from lymph nodes of normal NOD mice by positive selection using CD4 beads (Miltenyi Biotec). CD4+ T cells were stimulated for 6 days on anti-TCR mAb coated plates in the presence of 100 pg/ml IL-12 and 10 µg/ml anti-IL-4 mAb (11B11). Th1 cell lines (<0.1% IL-4+ cells in lines from IFN-{gamma}-/- and NOD; 78% IFN-{gamma}+ cells in lines from NOD mice) were expanded in IL-2 and restimulated with anti-TCR for 4 h before staining with anti-FasL mAb (K10) from BD PharMingen.

Statistical analysis

The kinetics of progression to IDDM in paired groups of mice were compared by a two-tailed Gehan’s test (a procedure comparing two survival curves). The proportions of mice that eventually became diabetic were compared by a two-tailed Fisher’s exact test. When indicated, differences between groups were evaluated using a two-tailed Mann-Whitney U test or unpaired two-tailed Student’s t test. Differences were considered to be statistically significant at p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IL-12 administration accelerates IDDM in IFN-{gamma}-/- NOD mice

Vehicle-treated IFN-{gamma}-/- NOD mice developed IDDM with a significant delay compared with vehicle-treated NOD mice (p = 0.0062; Fig. 1) in agreement with a previous report (19). However, IL-12 administration clearly accelerated IDDM onset in IFN-{gamma}-/- NOD mice (p = 0.0132) and in NOD mice (p = 0.00006), compared with vehicle-treated control littermates (Fig. 1), demonstrating that this acceleration is IFN-{gamma} independent. Although IL-12 accelerated IDDM onset, it did not modify, compared with vehicle-injected controls, the proportion of IFN-{gamma}-/- NOD (60%; p = 0.95) and NOD mice (86%; p = 0.99) that eventually became diabetic. Time to IDDM onset appeared slightly delayed in IL-12-treated IFN-{gamma}+/- NOD mice vs IL-12-treated NOD mice, but the difference did not reach statistical significance (p = 0.059).



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  		FIGURE 1. IL-12 administration accelerates IDDM in IFN-{gamma}-deficient NOD mice. IFN-{gamma}-/-, IFN-{gamma}+/-, and wild-type NOD mice were injected i.p. daily with 7.5 µg/kg recombinant mouse IL-12 or with vehicle (PBS containing 100 µg/ml mouse albumin) starting at 11 wk of age. The treatment was continued for 32 days for NOD mice and for 56 days for IFN-{gamma}-/- and IFN-{gamma}+/- NOD mice, because the latter mice develop IDDM more slowly than do NOD mice. The time to IDDM was significantly faster in IL-12-treated IFN-{gamma}-/- mice compared with vehicle-treated IFN-{gamma}-/- NOD mice (p = 0.0132), in IL-12-treated wild-type NOD mice compared with vehicle-treated wild-type NOD mice (p < 0.00006), in vehicle-treated wild-type compared with vehicle-treated IFN-{gamma}-/- NOD mice (p = 0.0062), and in IL-12-treated wild-type NOD mice compared with IL-12-treated IFN-{gamma}-/- NOD mice (p = 0.02), as determined by a two-tailed Gehan’s test. In contrast, there was no statistical difference in the time to IDDM in IL-12-treated IFN-{gamma}+/- vs IL-12-treated NOD mice (p = 0.059).

 
IL-12 administration induces severe destructive insulitis in IFN-{gamma}-/- NOD and control mice

IDDM is the direct consequence of a selective {beta} cell destruction after mononuclear cell infiltration of the pancreatic islets. To clarify the mechanisms underlying IL-12-induced IDDM acceleration in IFN-{gamma}-/- NOD mice, we first characterized the cell populations infiltrating the pancreas. CD45+ leukocytes were purified from the pancreas of 11-wk-old IFN-{gamma}-/- and IFN-{gamma}+/- NOD mice, treated or not with IL-12, and the lymphoid and myeloid cell populations were assessed by flow cytometry. The overall number of pancreas-infiltrating CD45+ cells was slightly higher in vehicle-treated IFN-{gamma}+/- compared with IFN-{gamma}-/- NOD mice (Fig. 2A). However, IL-12 treatment induced a fivefold and eightfold increase in the total quantity of pancreatic leukocytes in IFN-{gamma}+/- mice and IFN-{gamma}-/- NOD mice, respectively. The increment of CD8+ was more prominent than that of CD4+ T cells in IFN-{gamma}+/-, as previously reported in NOD mice (12), whereas this was not the case in IFN-{gamma}-/- NOD mice (Fig. 2A). This result indicates that IL-12-induced IFN-{gamma} may favor the expansion of CD8+ T cells. IL-12 administration induced a limited increase (twofold) of B220+ cells in IFN-{gamma}+/- mice, but a prominent increase (13-fold) of these cells in IFN-{gamma}-/- NOD mice. Thus, the infiltration and/or expansion of B220+ cells, mostly B lymphocytes, in the pancreas of IL-12-treated mice, seems to be negatively regulated by IFN-{gamma}. Finally, IL-12 treatment markedly increased the number of pancreatic CD11c+ CD11b+ cells in both IFN-{gamma}+/- and IFN-{gamma}-/- NOD mice, suggesting an IFN-{gamma}-independent process (Fig. 2A). Double cell surface staining with CD11c and CD11b mAb allows differentiation of DC and macrophage populations, and CD11c+ CD11b+ cells represent a subset of myeloid DCs (30). Thus, IL-12 administration enhances, in IFN-{gamma}-deficient NOD mice, the pancreatic infiltration not only of T cells but also of APCs.



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  		FIGURE 2. Modification of pancreatic leukocyte infiltrate after IL-12 administration in IFN-{gamma}-deficient and IFN-{gamma}-sufficient NOD mice. A, Pancreatic CD45+ cells were purified from vehicle or IL-12-treated mice (10 injections) and were double stained for the indicated cell surface markers. The proportions of positive cells were determined by flow cytometry and the cell numbers were calculated. The number of CD45+ cells/pancreas obtained in two experiments for vehicle-treated IFN-{gamma}+/-, IL-12-treated IFN-{gamma}+/-, vehicle-treated IFN-{gamma}-/-, and IL-12-treated IFN-{gamma}-/- NOD mice were 96–29, 390–218, 32–28, and 275–236 x 104, respectively. The numbers adjacent to the bars indicate the fold increase for each cell subset in IL-12-treated compared with vehicle-treated groups. The filled bars indicate the number of cells expressing I-Ag7. Results are the mean of two experiments. B, Insulitis in IL-12-treated, IFN-{gamma}-deficient and IFN-{gamma}-sufficient NOD mice. Pancreatic sections from IFN-{gamma}+/- (left panels) and IFN-{gamma}-/- (right panels) NOD mice injected with IL-12 for 10 consecutive days were stained with GK1.5 anti-CD4, 53.6.7 anti-CD8, RA3-6B2 anti-B220, 10.3.62 anti-A{beta}g7, Mac1 anti-CD11b, or N418 anti-CD11c mAb, as indicated. Consecutive sections of a representative islet are shown. The arrows indicate the presence of mononuclear cells in the exocrine pancreas. Some cells with a triple positive staining for I-Ag7 CD11b and CD11c molecules are visible in the exocrine pancreas of IFN-{gamma}+/- mice (arrows on the left). A CD11b+ CD11c+ cell is shown in the exocrine pancreas of IFN-{gamma}-/- mice (arrows on the right). Magnification, x250.

 
Next, to determine whether these cells infiltrated the islets, histological analysis of pancreata was performed. IFN-{gamma}-/- NOD mice injected with IL-12 for 10 days displayed a severe destructive insulitis composed of CD4+ and CD8+ T cells, B cells, and DCs, similar to the one observed in IFN-{gamma}+/- mice (Fig. 2B) and wild-type NOD mice treated with IL-12 (12). The only difference was a higher number of islet-infiltrating B220+ cells in IFN-{gamma}-/- vs IFN-{gamma}+/- NOD mice (Fig. 2B), as quantified in Fig. 2A. In all mice, I-Ag7 class II was found on both B cells and DCs and was highly expressed in the infiltrate (Fig. 2B). We have previously explained that IL-12 administration to NOD mice induces high class II expression in the exocrine pancreas, mostly on DCs, as revealed by CD11c expression, but perhaps also on other cells such as vessel endothelial cells (12). However, class II expression was modest in the exocrine pancreas of IL-12-treated IFN-{gamma}-/- and IFN-{gamma}+/- NOD mice (Fig. 2B), suggesting that in the exocrine pancreas the induction by IL-12 of high class II expression on particular cell types may require double allelic expression of IFN-{gamma}.

In conclusion, IL-12 administration enhances destructive insulitis compared with vehicle-treated controls, both in IFN-{gamma}-deficient and in IFN-{gamma}-sufficient NOD mice. However, this is associated with a high increase (7-fold) of CD11c+ CD11b+ and a minor increase (2-fold) of B220+ cells in IFN-{gamma}+/- NOD mice, in contrast with the major increase of both CD11c+ CD11b+ (12-fold) and B220+ (13 fold) cells in IFN-{gamma}-/- NOD mice.

IL-12 administration alters the frequency of lymphoid and myeloid cell subsets in peripheral lymphoid organs

Because the priming of diabetogenic cells is considered to occur in peripheral lymphoid organs (31), we also analyzed cell subsets in spleen and lymph nodes. IL-12 administration to IFN-{gamma}-sufficient NOD mice induced a major decrease of T and B cells in the spleen and peripheral blood, but not in mesenteric lymph nodes, indicating that lymphoid organs are differently affected by IL-12-induced IFN-{gamma} (data not shown). Conversely, in IL-12-treated compared with untreated IFN-{gamma}-deficient NOD mice, T cells were only slightly decreased and B cell numbers remained unchanged. The decrease of splenic lymphoid cells was accompanied by an elevation of cells expressing high levels of J11d (data not shown), a marker for early hematopoietic cells (32). These progenitors were induced by IL-12 mostly in IFN-{gamma}-sufficient NOD mice (data not shown), in concordance with results obtained in other mouse strains (33, 34). Interestingly, the number of CD11c+ DCs was increased 2-fold in the spleen of IL-12-treated mice, independently of IFN-{gamma} (data not shown). Moreover, IL-12 up-regulated the proportion of splenic CD11b+ cells by 3-fold and 5-fold, respectively, in IFN-{gamma}-sufficient and IFN-{gamma}-deficient mice (data not shown). Thus, IL-12 leads to increased macrophages and DC counts, not only in the pancreas but also in the spleen. We also checked whether in vivo IL-12 could modulate class II and costimulatory molecule expression on professional APCs. Splenic DCs from IL-12-treated or untreated IFN-{gamma}+/- and IFN-{gamma}-/- NOD mice displayed similar levels of I-Ag7 molecules, whereas the proportion of CD86+ DCs was only slightly increased in all IL-12-treated mice. Conversely, pancreatic DCs from IL-12-treated IFN-{gamma}+/- and IFN-{gamma}-/- NOD mice presented a slight down-modulation of their class II expression, as shown by the decreased fluorescence intensity compared with vehicle-treated mice, but the proportion of CD86+ pancreatic DCs remained constant, between 5 and 7% in all groups (data not shown).

IL-12 treatment decreases the APC capacity to activate T cells, mostly due to the presence of IFN-{gamma}

Next, to assess whether the increased number of professional APCs might have favored T cell stimulation, we evaluated the in vivo effect of IL-12 on APC function. We used T cell-depleted splenocytes from IL-12-treated or untreated mice and cultured them with Ag and with the T cell hybridoma 2D12.1, which recognizes the HEL10-23 peptide presented by I-Ag7 molecules (27). This T cell hybridoma was similarly activated by splenic APCs from vehicle-treated IFN-{gamma}+/-, IFN-{gamma}-/-, and wild-type NOD mice in the presence of HEL10-23 or HEL protein (Fig. 3, left panels). However, quite unexpectedly, splenic APCs from IL-12-treated IFN-{gamma}+/- and wild-type NOD mice displayed a significant reduction in their capacity to stimulate the 2D12.1 hybridoma (Fig. 3, right panels). The APCs were still able to process HEL, because both the core peptide 10-23 and the protein could activate the T cell hybridoma similarly (Fig. 3, right panels). Thus, this APC dysfunction does not appear to be due to a strong APC maturation induced by IL-12 preventing HEL uptake. Interestingly, splenic APCs from IL-12-treated IFN-{gamma}-/- NOD mice were more efficient than cells from IL-12-treated wild-type NOD or IFN-{gamma}+/- NOD mice in stimulating the 2D12.1 hybridoma (Fig. 3). This suggests that the APC dysfunction associated with IL-12 administration is mostly due to the presence of IFN-{gamma}. In addition, we obtained the same results using T cell-depleted LNCs (data not shown). Therefore, the APC defect is unlikely to reflect the lower number of B cells or higher number of progenitor cells because lymph nodes do not present these cellular alterations. Our data reveal that IL-12 can induce unexpected immunosuppressive effects. This is consistent with reports indicating that APC defects in the NOD mouse are associated with the development of autoimmunity (35, 36), but it does not support the possibility that IL-12 may increase the APC function, as recently suggested (13). In conclusion, IL-12 administration to NOD mice considerably reduces the APC capacity, largely via IFN-{gamma}. Therefore, the priming of diabetogenic T cells may be favored in IL-12-treated, IFN-{gamma}-deficient mice compared with IFN-{gamma}-sufficient mice, and this could represent an important mechanism enhancing IDDM development in IL-12-treated, IFN-{gamma}-deficient mice.



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  		FIGURE 3. Effect of in vivo IL-12 treatment on the capacity of APCs from NOD, IFN-{gamma}+/-, and IFN-{gamma}-/- mice to process and present Ag. The I-Ag7-restricted, HEL-specific T cell hybridoma 2D12.1 (5 x 104 cells/well) was incubated with the indicated concentrations of HEL protein or HEL peptide 10-23 and 2.5 x 105 T cell-depleted splenocytes/well from NOD, IFN-{gamma}+/-, and IFN-{gamma}-/- mice injected with vehicle (PBS containing 100 µg/ml serum albumin) or IL-12 for 10 consecutive days. After 24 h of culture, supernatants were collected and the IL-2 content was measured by ELISA. Each curve represents the mean (±SE) IL-2 produced by cells from three individual mice.

 
Pancreas-infiltrating T cells of IFN-{gamma}+/- and IFN-{gamma}-/- NOD mice secrete proinflammatory cytokines

Pancreas-infiltrating CD4+ T cells of adult NOD mice belong mostly to the Th1 phenotype (9, 37). They are characterized by high production of IFN-{gamma} and are crucial in the progression to IDDM. IL-12 administration markedly increases the number of pancreas-infiltrating T cells that produce IFN-{gamma}, and this is linked to an earlier and more rapid disease (12). We characterized the cytokine profile of IFN-{gamma}-/- NOD mice by measuring the intracytoplasmic production of the proinflammatory cytokines IL-2 and TNF-{alpha} and the Th2/T regulatory cytokines IL-4 and IL-10. In both IL-12-treated IFN-{gamma}+/- and IFN-{gamma}-/- NOD mice compared with vehicle-treated mice, the increased number of CD4+ cells infiltrating the pancreas (Fig. 2A) was associated with an increased number of cytokine-producing CD4+ cells (Fig. 4A, upper panels). In IL-12-treated IFN-{gamma}+/- mice, IFN-{gamma}-producing CD4+ cells showed the highest increase, followed by TNF-{alpha}-, IL-2-, and IL-10-producing cells (Fig. 4A, upper panels). The pattern of cytokine-producing cells was similar in IL-12-treated IFN-{gamma}-/- and IL-12-treated IFN-{gamma}+/- mice, except for the lack of IFN-{gamma}-producing cells in IFN-{gamma}-/- mice. IL-4-producing cells remained nearly undetectable in all groups (Fig. 4). The in vivo effect of IL-12 was also clearly evidenced by the variation in the proportion of cytokine-producing cells in mice treated with IL-12 compared with vehicle. Only the proportion of IFN-{gamma}+ CD4+ cells increased significantly in IL-12-treated IFN-{gamma}+/- mice over controls (Fig. 4A, lower panels). In both IFN-{gamma}+/- and IFN-{gamma}-/- mice injected with IL-12, compared with controls, the percentage of cells producing IL-2 was slightly lower, and of those producing IL-10 it was slightly higher, whereas the proportion of TNF-{alpha}-producing cells remained unchanged (Fig. 4A, lower panels). In IL-12-treated IFN-{gamma}+/- mice, most IL-2-producing cells acquired the capacity to also synthesize IFN-{gamma} (Fig. 4B), suggesting the induction of Th1 effector cells. Interestingly, we observed a 3-fold increase of cells that secreted IL-10 without coproducing TNF-{alpha} in both IFN-{gamma}+/- and IFN-{gamma}-/- mice after IL-12 administration (Fig. 4B).



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  		FIGURE 4. Pancreas-infiltrating CD4+ T cells secrete proinflammatory cytokines. Pancreatic T cells from IFN-{gamma}+/- and IFN-{gamma}-/- mice injected with vehicle (PBS containing 100 µg/ml serum albumin) or IL-12 for 10 consecutive days were purified and labeled with Cy-Chrome anti-CD4 mAb. The analysis was performed on gated CD4+ cells. In addition, cells were double stained intracellularly with cytokine-specific mAbs from BD PharMingen. The double stainings were performed with PE-IL-4/FITC-IFN-{gamma}, PE-IL-2/FITC-IFN-{gamma}, PE-IL-10/FITC-IFN-{gamma}, and PE-IL-10/FITC-TNF-{alpha}. A, For clarity, the mean number of CD4+ cells secreting an individual cytokine only is reported (upper panels). The proportion of CD4+ cells secreting individual cytokines is shown in the lower panels. Circles indicate different experiments performed with pooled cells from two to five mice/group. B, Representative stainings of cytokine-producing CD4+ T cells are shown.

 
In conclusion, the main effect of IL-12 administration is to enhance the frequency of IFN-{gamma}-producing CD4+ cells, as we previously observed (9, 12). IL-12 treatment does not significantly modify the proportion of CD4+ cells secreting the proinflammatory cytokines IL-2 and TNF-{alpha}. Nevertheless, the number of CD4+ cells secreting these cytokines increases because of the IL-12-mediated entry/expansion of mononuclear cells into the pancreas of IFN-{gamma}-/- as well as IFN-{gamma}+/- mice. Thus, the overall number of potential effector CD4+ T cells increases in the pancreas of both IFN-{gamma}-deficient and IFN-{gamma}-sufficient mice. In addition, IL-12 treatment slightly favors the induction of IL-10-producing cells, suggesting that it may also induce regulatory T cells.

The expression of P-selectin ligand on pancreas-infiltrating CD4+ T cells is regulated by IL-12-induced IFN-{gamma}

The ligand for P-selectin is absent on naive T cells, but becomes preferentially expressed on Th1 cells and contributes to T cell recruitment into inflammatory sites (38, 39, 40). IL-12 is known to enhance the expression of functional P-selectin ligand on T cells, but the role of IFN-{gamma} in this process has not been fully evaluated (41). Thus, we investigated the expression of P-selectin ligand on pancreas-infiltrating T cells and found that almost 50% of pancreatic CD4+ T cells isolated from IL-12-treated IFN-{gamma}+/- NOD mice could bind P-selectin IgG fusion protein, compared with 10% in vehicle-treated mice (Fig. 5). In contrast, no significant increase of P-selectin ligand expression on CD4+ T cells was observed after injection of IL-12 in IFN-{gamma}-/- NOD mice (Fig. 5). These data demonstrate, for the first time, that IFN-{gamma} is required for the increase of P-selectin ligand expression on CD4+ T cells induced by IL-12 treatment. Although the number of pancreatic CD4+ T cells was always slightly lower in IFN-{gamma}-/- compared with IFN-{gamma}+/- NOD mice, IL-12 administration resulted in 8-fold and 4-fold increases of these cells in IFN-{gamma}-/- and IFN-{gamma}+/- NOD mice, respectively (Fig. 2A). Thus, the large accumulation of CD4+ T cells observed in the pancreatic islets of IL-12-treated IFN-{gamma}-/- mice proceeds in the absence of enhanced P-selectin ligand expression on CD4+ cells, likely through mechanisms different from those operating in IFN-{gamma}+/- NOD mice.



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  		FIGURE 5. IL-12-induced IFN-{gamma} up-regulates P-selectin ligand expression on pancreas-infiltrating CD4+ T cells. Pancreatic T cells from IFN-{gamma}+/- and IFN-{gamma}-/- NOD mice injected with vehicle (PBS containing 100 µg/ml serum albumin) or IL-12 for 10 consecutive days were double stained with P-selectin IgG chimeric protein and anti-CD4 mAb. The bars indicate the mean percent (±SE) of pancreatic CD4+ cells binding P-selectin from two to four experiments. The asterisk indicates a statistical difference (p < 0.05 by two-tailed Mann-Whitney U test) with the vehicle-treated controls.

 
IL-12-induced IFN-{gamma} reduces T cell proliferation in response to TCR ligation

Previous reports have shown that thymic and splenic T cells from NOD mice proliferate poorly due to a defect in TCR-mediated signaling (42, 43). We could confirm that LNCs from NOD mice exhibit a slightly lower proliferation in response to TCR ligation compared with cells from BALB/c or C3H mice (data not shown). However, strikingly, T cells from IL-12-treated NOD mice showed, compared with controls, an even more pronounced defect in anti-TCR-induced T cell proliferation, and a progressive decrease of T cell proliferation paralleled the increasing number of IL-12 injections (Fig. 6A). Nevertheless, T cells, even if proliferating poorly, secreted abundant amounts of IFN-{gamma} (Fig. 6A). IFN-{gamma} is known to have antiproliferative effects on a number of cells, including T and B cells (44, 45). Therefore, we compared the proliferative capacity of T cells from IL-12-treated IFN-{gamma}-/- and IFN-{gamma}+/- NOD mice. Strikingly, LNCs from IL-12-treated IFN-{gamma}-/- NOD mice showed enhanced proliferation after stimulation with plate-bound anti-TCR Ab, whereas the proliferation of cells from IFN-{gamma}+/- NOD mice was profoundly inhibited compared with controls (Fig. 6B). Similarly, we found that purified pancreatic T cells (5 x 104 cells/well) stimulated with 3 µg/ml insolubilized anti-TCR mAb proliferate less when obtained from IL-12-treated IFN-{gamma}+/- (4.8 x 103 cpm) compared with IFN-{gamma}-/- NOD mice (12.5 x 103 cpm). These results indicate that T cells from IFN-{gamma}-/- mice, in contrast with IFN-{gamma}+/- NOD mice, undergo an enhanced proliferation upon TCR ligation.



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  		FIGURE 6. IL-12-induced IFN-{gamma} inhibits T cell proliferation. A, T cells from IL-12-injected NOD mice show a progressively reduced proliferation in response to TCR ligation. Purified T cells (2 x 105/well) from NOD mice injected with vehicle (PBS containing 1% NOD serum) or IL-12 for different lengths of time, as indicated, were cultured in plates precoated with anti-TCR mAb. After 48-h incubation, the proliferation was assessed by 16-h [3H]thymidine incorporation. Alternatively, purified T cells (2 x 105/well) were cultured for 48 h in plates precoated with 10 µg/ml anti-TCR mAb, supernatants were collected, and IFN-{gamma} production was quantified by ELISA. Open and filled symbols indicate values of vehicle-treated and IL-12-treated mice, respectively. Values shown represent the mean (±SE) from two mice/group. B, Total LNCs from IFN-{gamma}+/- and IFN-{gamma}-/- NOD mice injected with vehicle (PBS containing 100 µg/ml serum albumin) or IL-12 for 9 consecutive days were cultured in plates precoated with anti-TCR mAb. After a 48-h incubation, the proliferation was assessed by 16-h [3H]thymidine incorporation. The curves represent the mean (±SE) from three individual mice/group, except for the IL-12-treated IFN-{gamma}-/- mice (mean of two mice).

 
IL-12 increases the number of apoptotic CD4+ cells via an IFN-{gamma}-dependent mechanism

Next, we examined the role of IFN-{gamma} in CD4+ T cell apoptosis. Apoptosis of CD4+ T cells from mice injected for 10 days with IL-12 or vehicle was quantified by double staining with Annexin V-FITC and propidium iodide. Increased proportions of splenic CD4+ cells from IFN-{gamma}+/- (40%) and wild-type NOD mice (27%) were apoptotic after IL-12 administration, compared with 13% in vehicle-treated controls (Fig. 7). In contrast, there was no significant increase of apoptotic CD4+ cells in the spleen from IL-12-treated IFN-{gamma}-/- mice compared with vehicle-treated controls (p = 0.3; Fig. 7). Thus, IL-12-induced IFN-{gamma} leads to an increased proportion of apoptotic CD4+ cells. This result indicates that survival of activated CD4+ cells after IL-12 administration is favored in IFN-{gamma}-/- compared with IFN-{gamma}+/- and wild-type NOD mice.



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  		FIGURE 7. The increased apoptosis of CD4+ T cells after in vivo IL-12 treatment is IFN-{gamma} dependent. A, Representative staining for Annexin V-FITC and propidium iodide of gated splenic CD4+ lymphocytes, obtained from individual mice injected with vehicle (PBS with 100 µg/ml serum albumin) or IL-12 for 10 consecutive days. Percentages of apoptotic and late apoptotic/necrotic cells are indicated in the lower and upper quadrants, respectively. B, Mean percentage (±SE) of apoptotic CD4+ lymphocytes obtained from 5–11 individual mice, injected as indicated in A. The statistical difference between IL-12-treated and vehicle-treated groups was evaluated as follows: **, p < 0.001; *, p = 0.016; ns, not significant; p = 0.3 by two-tailed Mann-Whitney U test.

 
Regulation of {beta} cell-targeting molecules by IFN-{gamma}: IL-12 treatment increases NO in IFN-{gamma}-sufficient mice, but selectively up-regulates FasL on Th1 cells from IFN-{gamma}-deficient mice

Finally, we asked which pathways induced by IL-12 treatment could provoke {beta} cell destruction during the late phase of IDDM development. In NOD mice, {beta} cell death is mediated by several mechanisms, including direct cell/cell contact via FasL expressed on activated lymphocytes with Fas on {beta} cells and soluble effector molecules such as cytokines and iNOS-induced NO (46). Because we found a prominent increase of CD11b+ macrophages, a possible source of NO, in IL-12-treated NOD mice, we quantified this effector molecule in the presence or absence of IL-12-induced IFN-{gamma}. In untreated IFN-{gamma}-sufficient and -deficient NOD mice, the amounts of iNOS protein detected in peritoneal or pancreatic CD14+ cells were similar (Fig. 8, A and B), resulting in a similar production of NO (Fig. 8C). Thus, the absence of IFN-{gamma} does not influence iNOS and NO production in untreated mice. In contrast, IL-12 injection induced a substantial (5-fold) increase of iNOS only in PECs from IFN-{gamma}-sufficient NOD mice (Fig. 8A), paralleled by a 9-fold increase of NO (Fig. 8C). The difference in iNOS between IFN-{gamma}-/- and NOD mice after IL-12 treatment was less pronounced in pancreatic macrophages compared with PECs (Fig. 8B). Nevertheless, high production of NO in the pancreatic infiltrate ex vivo was only measured in IL-12-treated IFN-{gamma}-sufficient NOD mice, reflecting in this case the same pattern seen in PECs (Fig. 8C). To further clarify the role of IFN-{gamma}, we stimulated peritoneal and pancreas-infiltrating cells with LPS. LPS induced a 9-fold increase of NO production by PECs independently of IFN-{gamma} (Fig. 8, C and D). Conversely, LPS induced little modification of NO production by PECs from IL-12-treated IFN-{gamma}-/- mice, and this could only be increased by the addition of IFN-{gamma} (Fig. 8, C–E), revealing that IL-12 treatment induces an IFN-{gamma}-dependent regulation of NO secretion. Interestingly, in IL-12-treated NOD mice, the pancreatic infiltrate stimulated with exogenous IFN-{gamma} did not up-regulate its secretion of NO, which was already at maximal levels (Fig. 8, C–E). In contrast, in IL-12-treated IFN-{gamma}-deficient NOD mice, the pancreatic infiltrate produced NO optimally only when IFN-{gamma} was added to the culture (Fig. 8, C–E). Thus, IL-12 treatment of NOD mice results in a strong IFN-{gamma}-dependent increase of NO. Because high levels of NO in the islets kill the {beta} cells in vivo (47), in IFN-{gamma}-sufficient NOD mice NO is likely to represent a major component of {beta} cell death induced by IL-12 treatment, and thereby would be an important factor in IDDM acceleration. In contrast, because NO secretion was unchanged in IL-12-treated compared with untreated IFN-{gamma}-deficient NOD mice (Fig. 8C), we conclude it has no impact on IL-12-mediated IDDM acceleration. In the search for another mechanism leading to {beta} cell death in IL-12-treated IFN-{gamma}-deficient NOD mice, we measured FasL expression on Th1 cells. Because FasL expression is transient, we quantified it on Th1 lines stimulated with anti-TCR and found it preferentially expressed on Th1 cells from IFN-{gamma}-deficient NOD mice. Thus, in IL-12-treated IFN-{gamma}-deficient mice, FasL may represent a preferential pathway of {beta} cell death. Indeed, in the absence of IFN-{gamma}, Th1 cells are greatly expanded by IL-12 treatment (Figs. 2 and 4), are not subject to apoptosis (Fig. 7), and express FasL (Fig. 8).



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  		FIGURE 8. Regulation of {beta} cell-targeting molecules by IFN-{gamma}. A and B, NOD and IFN-{gamma}-deficient NOD mice were injected for 10 consecutive days with IL-12 or were left untreated. PECs and pancreas-infiltrating CD45+ cells isolated from these mice were stained intracellularly with FITC-conjugated anti-iNOS mAb and on the cell surface with PE-labeled anti-CD14 mAb, as described in Materials and Methods. Analysis was performed on CD14+ gated cells with a FACScan flow cytometer. The values indicate the difference of fluorescence intensity ({Delta} Geo mean) between the staining with anti-iNOS mAb and the isotype control. The bars represent the mean (±SE) from three to four individual mice/group (A) or the value from three to four pooled mice/group (B). C–E, For the detection of NO, 3 x 105 cells/well, either PECs or pancreatic CD45+ cells from the same mice described in A and B, were cultured for 24 h in flat-bottom 96-well plates in complete RPMI 1640 medium alone or supplemented with 10 µg/ml LPS with or without 500 U/ml IFN-{gamma}. NO was quantified in culture supernatants as described in Materials and Methods. For PECs, the bars represent the mean (±SE) from three to four individual mice/group, and for pancreatic CD45+ cells, they represent the value from three to four pooled mice/group. The asterisks indicate a statistical difference between IL-12-treated and the corresponding untreated mice: *, p < 0.05; and **, p < 0.005; by unpaired two-tailed Student’s t test. F–G, Th1 lines, generated as described in Materials and Methods, were stimulated with anti-TCR mAb for 4 h and stained with anti-FasL mAb. The percentage of positive cells is indicated.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we show that IL-12 administration accelerates IDDM in IFN-{gamma}-deficient as well as in IFN-{gamma}-sufficient NOD mice, indicating that IL-12-induced IFN-{gamma} is dispensable for diabetes provocation. In both groups of mice, daily injections of IL-12 led to aggressive insulitis composed predominantly of APCs and T cells. Apart from the lack of IFN-{gamma} secretion, the typical cytokine profile induced by IL-12 is conserved in CD4+ T cells from IFN-{gamma}-/- NOD mice, and it could be concluded that IL-12 administration induces pathogenic Th1-like cells in the absence of IFN-{gamma}. Administration of rIL-12 was also shown to trigger a Th1-mediated autoimmune arthritis in collagen-primed DBA/1 mice, but in this case the effect was mediated by IFN-{gamma}, as shown by the abrogation of arthritis in IL-12-treated mice receiving anti-IFN-{gamma} mAb (48).

The requirement for IFN-{gamma} in Th1 cell development has been controversial, the issue revolving around the local balance between IL-4 and IFN-{gamma} (49). In the complete absence of IL-4, IFN-{gamma} is notrequired and T cells become committed to the Th1 pathway in the presence of IL-12 only (50). IL-12 administration to IFN-{gamma}-deficient NOD mice did not induce IL-4-secreting cells, consistent with the observation that NOD mice have a defective IL-4 production (42) and explaining why IL-12 can induce Th1 cells independently of IFN-{gamma} in NOD mice.

IL-12, a proinflammatory cytokine, induces Th1 cell differentiation, CD8+ T cell cytotoxicity, and NK cell activation, enhancing protective (51, 52) or detrimental (12, 48) type 1 T cell responses. Therefore, it was rather surprising to observe that administration of IL-12 to NOD mice lowered the capacity of lymph node and splenic APCs to activate T cells. These results are reminiscent of the immunosuppressive effects induced by IL-12 during lymphocytic choriomeningitis virus infection in mice, leading to decreased anti-viral CTL activity and a poorer outcome caused by IFN-{gamma} induction of iNOS activity (53, 54).

How could APC dysfunction and low T cell activation be reconciled with IDDM acceleration in IFN-{gamma}-sufficient NOD mice? We have previously shown that injection of IL-12 in adult (12) as well as in 12-day-old NOD mice (28), before the beginning of insulitis, induces autoantigen-specific cells and enhances IDDM development. Thus, effector cells are induced and diabetes is accelerated even if, as shown in the present study, IL-12 administration to IFN-{gamma}-sufficient NOD mice reduces T cell activation. Interestingly, suboptimal T cell activation due to defects in APC differentiation and function was described as favoring the development of diabetogenic T cells in NOD mice, possibly by preventing efficient negative selection (36). In addition to blunting T cell activation, IL-12 administration to IFN-{gamma}-sufficient NOD mice increases apoptosis of CD4+ T cells. Similarly, intrathecal delivery of IFN-{gamma} has been shown to increase apoptosis of encephalitogenic T cells, but in this case the development of experimental autoimmune encephalomyelitis was inhibited (55). Moreover, IL-12 administration protects from experimental autoimmune uveitis, likely by hyperinduction of systemic IFN-{gamma}, which causes apoptotic deletion of uveitogenic effector cells as they are primed (56). In this study, the mice were immunized with retinal autoantigen emulsified in CFA and treated for 5 days with IL-12. Because CFA is an IL-12 inducer, the authors argued that an excess of IL-12 during priming could curtail the development of effector Th1 cells. Likewise, mice immunized with type II collagen emulsified in CFA and treated with high doses of IL-12 are protected from arthritis (57). In contrast with these models, hyperinduction of IFN-{gamma} in IL-12-treated IFN-{gamma}-sufficient NOD mice does not prevent disease, possibly because the reduced T cell activation curtails the premature apoptosis of diabetogenic cells. In addition, other IFN-{gamma}-dependent mechanisms, such as up-regulation of P-selectin ligand, could also explain IDDM acceleration. In IL-12-treated IFN-{gamma}-deficient NOD mice, the lower APC dysfunction, enhanced T cell proliferation, and absence of IFN-{gamma}-driven apoptotic effects could compensate for the lack of P-selectin ligand up-regulation and contribute to IL-12-mediated IDDM acceleration.

All types of APCs, DCs, macrophages, and B cells have been implicated in NOD IDDM, although each may be required at a specific stage of the disease process. Interestingly, we found that B cells represent a prominent population in the islet infiltrate induced by IL-12 administration in IFN-{gamma}-/- but not IFN-{gamma}+/- NOD mice. In addition, B cell numbers were decreased in the spleen and peripheral blood of IL-12-treated IFN-{gamma}-sufficient mice. Thus, IL-12-induced IFN-{gamma} appears to inhibit B cell development, which usually occurs in the bone marrow, and/or B cell migration to the spleen and pancreas. The latter would be consistent with the observation that the autocrine secretion of IFN-{gamma} can prevent immature B cells from homing into nonsplenic secondary lymphoid organs (58) by down-regulating their integrin-mediated adhesion to fibronectin (59). Therefore, IFN-{gamma}-deficient mice exhibit significantly higher levels of immature B cells in peripheral lymph nodes than do their wild-type counterparts (58). In addition, IL-12 can directly bind to some B cell subsets (60, 61), especially to B-1 cells which are present in increased numbers in autoimmune conditions (62, 63), and can down-regulate them, likely through the induction of cytokines such as IFN-{gamma} (64, 65). Thus, the absence of IFN-{gamma} in IL-12-treated NOD mice may favor the activation of autoimmunity-associated B cell subsets and facilitate their recirculation to inflammatory sites, such as the pancreas. Moreover, several studies have shown that B cells are required APCs in the activation of diabetogenic T cells (66, 67, 68, 69) and that B cell-deficient NOD mice fail to develop insulitis and diabetes (70). Islet-reactive T cells display impaired activation in the absence of B cells, which are needed to provide sufficient costimulation (71). B lymphocytes also appear necessary to amplify as well as to diversify the T cell response to epitopes from glutamic acid decarboxylase 65, a major autoantigen candidate in IDDM (68, 69). Thus, our results suggest a role for B cells in enhancing IDDM development in IL-12-treated IFN-{gamma}-deficient mice. However, this does not exclude an important participation of other APCs such as CD11c+ CD11b+ DCs, which are equally increased in these mice.

In NOD mice, a few DCs and macrophages are constitutively present in the pancreas, but they progressively accumulate as IDDM progresses in the perivascular areas and around the islets, even before any sign of lymphocyte infiltration (72). An important characteristic of DCs is their capacity to endocytose Ag in tissues and to migrate to the draining lymph nodes where they can prime naive T cells. This likely occurs also in the NOD mouse, because islet-reactive T cells are initially activated in pancreatic lymph nodes and afterward enter the pancreas (31). NOD DCs, due to their marked NF-{kappa}B activation, can stimulate Ag-specific CD4+ and CD8+ T cells to a greater extent compared with DCs from other mouse strains (13), and neutralization of IL-12p75 has been shown to block the capacity of DCs to activate T cells (13). In contrast, our data show that IL-12 administration hinders the capacity of splenic and lymph node APCs to activate T cells and that this effect is due in part to IFN-{gamma}. However, it is conceivable that macrophages and not DCs are responsible for this APC dysfunction. Indeed, we have shown that macrophages from IL-12-treated IFN-{gamma}-sufficient mice up-regulate iNOS, leading to high amounts of NO, an effector molecule that has been associated with inhibition of Ag-specific T cell proliferation (56). Thus, the negative effect of macrophages on T cell priming could predominate over the immunogenic capacity of DCs.

Macrophages appear to be key players during the late phases of diabetes development (72), and we could isolate high numbers of CD11b+ CD11c- macrophages from the pancreas of IL-12-treated IFN-{gamma}+/- and IFN-{gamma}-/- NOD mice, in part due to their abundance in the exocrine pancreas. Macrophages are known to enhance the islet-destructive function of T cells through NO-dependent pathways (73) or to kill {beta} cells directly via NO (46). Our results show that IL-12-induced IFN-{gamma} results in hyperinduction of NO by the pancreatic infiltrate, suggesting a role for this effector molecule in the IDDM acceleration observed in IL-12-treated IFN-{gamma}-sufficient NOD mice. In contrast, in IL-12-treated IFN-{gamma}-deficient mice, which do not show enhanced levels of NO, other pathways of {beta} cell death must operate. A likely one is via Fas/FasL interaction, although we cannot rule out {beta} cell damage through perforin release by cytotoxic T cells or TNF-{alpha} production by macrophages and T cells.

In conclusion, IDDM acceleration in IL-12-treated IFN-{gamma}-sufficient NOD mice is not an obvious event. Indeed, IL-12-induced IFN-{gamma} blunts the inflammatory process via different mechanisms, by inducing APC dysfunction, reducing T cell proliferation, and increasing T cell apoptosis. In contrast, IL-12-mediated IFN-{gamma} up-regulates P-selectin ligand expression, thus favoring T cell migration, and it enhances NO production, a toxic molecule for {beta} cells. Conversely, acceleration of IDDM in IL-12-treated IFN-{gamma}-deficient NOD mice involves an increased entry of APCs (in particular B cells) in the insulitic lesion, a higher Ag-presenting capacity of APCs, a selective enhancement of Th1-type cell development, and the expansion of T cells not controlled by apoptosis. In these mice, Th1 cells might kill pancreatic {beta} cells mainly through FasL/Fas interaction. These pathogenic effects are counterbalanced by lack of P-selectin ligand up-regulation and absence of enhanced NO production. Thus, the regulation of the inflammatory process in IL-12-treated IFN-{gamma}-sufficient and IFN-{gamma}-deficient NOD mice appears quite different, although some IFN-{gamma}-independent pathways required for disease acceleration may be similar, such as DC induction. These data highlight the dual role of IFN-{gamma}, pathogenic and protective, in IDDM development and show how its deletion may allow IL-12 to establish alternative pathways leading to disease provocation.


   Acknowledgments
 
We are grateful to Dr. T. Stewart (Genentech) for providing breeding pairs of IFN-{gamma}-deficient NOD mice.


   Footnotes
 
1 This work was supported in part by European Union Contract QLK2-CT-2001-02103. Back

2 Current address: Department of Internal Medicine III, AKH, University of Vienna, Waehringer Guertel 18, A-1090 Vienna, Austria. Back

3 Address correspondence and reprint requests to Dr. Luciano Adorini, BioXell, Via Olgettina 58, I-20132 Milan, Italy. E-mail address: luciano.adorini{at}bioxell.com Back

4 Abbreviations used in this paper: IDDM, insulin-dependent diabetes mellitus; NOD, nonobese diabetic; DC dendritic cell; HEL, hen egg-white lysozyme; LNC, lymph node cell; iNOS, inducible NO synthase; PEC, peritoneal exudate cell; FasL, Fas ligand. Back

Received for publication September 3, 2002. Accepted for publication March 20, 2003.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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