IL-12 and IL-12 antagonist administration to nonobese diabetic (NOD) mice accelerates and prevents insulin-dependent diabetes mellitus (IDDM), respectively. To further define the role of endogenous IL-12 in the development of diabetogenic Th1 cells, IL-12-deficient NOD mice were generated and analyzed. Th1 responses to exogenous Ags were reduced by ∼80% in draining lymph nodes of these mice, and addition of IL-12, but not IL-18, restored Th1 development in vitro, indicating a nonredundant role of IL-12. Moreover, spontaneous Th1 responses to a self Ag, the tyrosine phosphatase-like IA-2, were undetectable in lymphoid organs from IL-12-deficient, in contrast to wild-type, NOD mice. Nevertheless, wild-type and IL-12-deficient NOD mice developed similar insulitis and IDDM. Both in wild-type and IL-12-deficient NOD mice, ∼20% of pancreas-infiltrating CD4+ T cells produced IFN-γ, whereas very few produced IL-10 or IL-4, indicating that IDDM was associated with a type 1 T cell infiltrate in the target organ. T cell recruitment in the pancreas seemed favored in IL-12-deficient NOD mice, as revealed by increased P-selectin ligand expression on pancreas-infiltrating T cells, and this could, at least in part, compensate for the defective Th1 cell pool recruitable from peripheral lymphoid organs. Residual Th1 cells could also accumulate in the pancreas of IL-12-deficient NOD mice because Th2 cells were not induced, in contrast to wild-type NOD mice treated with an IL-12 antagonist. Thus, a regulatory pathway seems necessary to counteract the pathogenic Th1 cells that develop in the absence of IL-12 in a spontaneous chronic progressive autoimmune disease under polygenic control, such as IDDM.

Interleukin-12 is an inflammatory cytokine secreted by neutrophils, macrophages, microglia, and dendritic cells in response to different stimuli, including bacteria, bacterial products, and interaction with T cells (1). IL-12 and IL-4 secreted during the initiation of an Ag-specific immune response play a dominant role in directing the differentiation of CD4+ T cell precursors toward the Th1 and Th2 phenotype, respectively (2, 3). Th1 cells secrete IFN-γ, as well as IL-2 and TNF-β, whereas Th2 cells are characterized by secretion of IL-4, IL-5, and IL-10 (4).

The important and nonredundant role of IL-12 in the induction of Th1 responses has been demonstrated in mice deficient for IL-12 (5), IL-12Rβ1 (6), or Stat4 (7). IL-12-deficient mice fail to control mycobacterial infections due to a decreased ability to develop Th1-mediated protective immunity (8). Similarly, humans with genetic deficiency of IL-12 or the IL-12 receptor demonstrate systemic dissemination of otherwise poorly pathogenic bacteria (9). In contrast, IL-12 deficiency does not alter the control of viral infections, indicating that alternative pathways for the generation of type 1 responses may be induced (10, 11).

A pathogenic role of Th1 cells is documented in several autoimmune diseases, such as experimental allergic encephalomyelitis (EAE),4 collagen-induced arthritis (CIA), experimental autoimmune uveitis (EAU), and experimental autoimmune myasthenia gravis (EAMG) (reviewed in Refs. 12, 13). In all of these models, IL-12-deficient mice are protected from disease, either completely (14, 15) or partially (16, 17).

In contrast to induced autoimmune disease models, insulin-dependent diabetes mellitus (IDDM) develops spontaneously in nonobese diabetic (NOD) mice (18). The disease is characterized by a progressive mononuclear cell infiltration in the pancreatic islets of NOD mice, which starts at about 4 wk of age and leads to β cell destruction and hyperglycemia (18, 19). Pancreas-infiltrating CD4+ and CD8+ T cells have a type 1 phenotype, as demonstrated by intracytoplasmic staining for cytokine production (13).

We have previously shown that daily administration of IL-12 accelerated IDDM, and this acceleration was associated with increased type 1 cytokine production by pancreas-infiltrating CD4+ and CD8+ T cells and selective elimination of islet β cells (20). These data, consistent with a dominant role of Th1 cells in the pathogenesis of IDDM, did not determine whether IL-12 is obligatory for the development of diabetogenic Th1 cells. We addressed this question by targeting endogenous IL-12 in NOD mice with the IL-12 antagonist (p40)2 (21). Treatment with (p40)2 started at 3 wk of age, resulted in the deviation of pancreas-infiltrating CD4+ cells to the Th2 phenotype at the expense of Th1-type cells, as well as in delay and reduction of IDDM incidence. Reduction of IDDM incidence by IL-12 antagonist administration to NOD mice was also obtained by Rothe et al. (22). Nevertheless, it remained unclear whether Th2 cells exerted a direct protective role, inhibiting pathogenic Th1 cells, or whether the decrease in Th1 development could by itself account for IDDM protection. The role of Th2 cells in IDDM is still controversial. Th2 lymphocytes may be neutral (23, 24), may actively inhibit pathogenic Th1 activity (25), and, in immunodeficient hosts, they may even be pathogenic (26).

We have generated IL-12-deficient NOD mice to clarify the requirement for IL-12 in the development of diabetogenic Th1 cells, as well as to provide further insight into the role of Th1/Th2 regulation in IDDM pathogenesis. Unexpectedly, our findings indicate that the development of diabetogenic type 1 T cells and their accumulation in pancreatic islets can occur via IL-12-independent mechanisms. In addition, they suggest that a regulatory pathway is required to counteract the pathogenic type 1 T cells that develop in the absence of IL-12.

A functionally inactivated IL-12p40 allele originally generated on the 129/SvEv genetic background (5) was backcrossed to NOD/Lt mice obtained from The Jackson Laboratory (Bar Harbor, ME) for a total of eight generations. Progeny were screened for the presence of the IL-12p40 mutation by Southern blot analysis, as described previously (5). The IL-12p40 gene maps to a region of chromosome 11 close to the Idd4 locus. Thus, mice used for experimental analyses were all derived from a single seventh generation backcross female that was shown by the previously described PCR-typing protocols (27) to be homozygous for NOD allelic variants at the Idd4 microsatellite linkage markers D11 Mit115, D11 Mit140, and D11 Mit320. At the eighth backcross generation, IL-12p40+/− heterozygotes were then intercrossed to generate two types of mice on the NOD background: mice homozygous for the IL-12p40 mutation (IL-12-deficient NOD mice) and mice homozygous for the wild-type IL-12p40 allele (wild-type NOD mice). All mice were bred and maintained under specific pathogen-free conditions. A diagnosis of diabetes was made after two sequential measurements of blood glucose levels higher than 200 mg/dl.

Mouse IL-12(p40)2 [(p40)2] was produced by Chinese hamster ovary cells stably transfected with the mouse IL-12p40 cDNA and purified as previously described (28). The endotoxin contamination of the purified (p40)2 was <2 e.u./mg (p40)2, as assayed by the Limulus amebocyte assay. For in vivo administration, (p40)2 was diluted in Dulbecco’s PBS (Life Technologies, Grand Island, NY) containing 100 μg/ml mouse albumin (Sigma, St. Louis, MO). NOD/Lt mice were injected i.p. daily, from 3 to 10 wk of age, with 3 mg/kg recombinant mouse (p40)2 or with vehicle, as previously described (21).

Mice were injected i.p. with 400 μg of LPS from Salmonellaabortus equi (Sigma). The mice were bled 3 and 6 h later, and serum IL-12p40 and IFN-γ, respectively, were measured by ELISA.

NOD mice were immunized into the hind footpads with either CFA containing H37Ra mycobacteria (Difco, Detroit, MI) or with 3.5 nmoles/mouse HEL emulsified in IFA. Draining lymph nodes were removed 9 days after immunization, and 6 × 105 cells/well were cultured in 96-well culture plates with Ag in synthetic HL-1 medium (Ventrex Laboratories, Portland, ME) supplemented with 2 mM l-glutamine and 50 μg/ml gentamicin (Sigma). The recall Ag was either 30 μg/ml PPD (Statens Seruminstitut, Copenhagen, Denmark), for the mice immunized with CFA, or 10 μM HEL. In addition, cells were incubated with or without 0.5 ng/ml recombinant mouse IL-12 (kindly provided by Dr. M. K. Gately, Hoffmann-La Roche, Nutley, NJ) or 12 ng/ml IL-18 (Research Diagnostic, Flanders, NJ). After 72 h of culture, supernatants were collected for quantification of secreted cytokines by ELISA. The cells were harvested, washed, and recultured for an additional 72 h in RPMI 1640 medium (Life Technologies) supplemented with 50 μM 2-ME (Fluka Biochemica, Buchs, Switzerland), 2 mM l-glutamine, 50 μg/ml gentamicin, and 10% FCS (complete medium) for the detection of intracellular production of cytokines. Afterwards, the cells were stimulated by either PPD or PMA/ionomycin. In the first case, 2.5 × 105 cells/well were incubated in 96-well plates for 24 h at 37°C with PPD and 106 T cell-depleted mitomycin C-treated splenocytes from either IL-12-deficient or wild-type NOD mice in the presence of 10 μg/ml brefeldin A (BFA; Novartis, Basel, Switzerland). Cells were resuspended in PBS containing 10 μg/ml BFA before adding an equal volume of 4% paraformaldehyde. After fixing for 20 min, cells were stained for intracytoplasmic cytokines. Otherwise, 2.5 × 105 cells/well were stimulated with 50 ng/ml PMA and 750 ng/ml ionomycin (all from Sigma) for 4 h at 37°C, and 10 μg/ml BFA was added for the last 2 h. Cells were resuspended in PBS containing 10 μg/ml BFA, fixed as above, and stained for intracytoplasmic cytokines.

Total spleen cells (106 cells/well) from 10-wk-old nonimmunized NOD mice were cultured in 96-well plates in complete medium containing 2.5% horse serum (Life Technologies) with or without 0.3 μM recombinant mouse IA-2. After 48 h of incubation at 37°C, IFN-γ and IL-10 secretion were determined in culture supernatants by ELISA. Alternatively, splenic or mesenteric lymph node CD4+ cells were sorted by positive selection on MiniMACS (Miltenyi Biotec, Auburn, CA). These CD4+ cells (2 × 105/well) were cultured with T cell-depleted and mitomycin C-treated spleen cells (106 cells/well) in 96-well plates with the indicated concentrations of IA-2. After 72 h, IFN-γ and IL-10 were determined in culture supernatants. Purified recombinant mouse IA-2 protein (protein tyrosine phosphatase-like, PTP 35) (29) was a kind gift of Dr. Antonella Isacchi (Pharmacia-Upjohn, Milan, Italy).

Cytokines were quantified by two-sites ELISA. To detect IFN-γ, polyvinyl microtiter plates (Falcon 3012) were coated with 100 μl of AN-18.17.24 mAb (30) in carbonate buffer. After blocking, samples (50 μl/well) diluted in test solution (PBS containing 5% FCS and 1 g/L phenol) were incubated together with 50 μl peroxidase-conjugated XMG1.2 mAb (31). After overnight incubation at room temperature, bound peroxidase was detected by 3,3′,5,5′-tetramethylbenzidine (Fluka Chemical, Ronkonkoma, NY), and adsorbance read at 450 nm with an automated microplate ELISA reader (MR5000; Dynatech Laboratories, Chantilly, VA). To detect IL-12p40, the mAb used for capture was 10F6 anti-IL-12p40 followed by biotin-conjugated goat anti-IL-12, as described (32). Anti-IL-12 Abs were kindly provided by Dr. M. K. Gately (Hoffmann-LaRoche). IL-4 and IL-10 were determined using paired mAb from PharMingen (San Diego, CA). For capture, the mAb were BVD4-1D11 or 11B11 (anti-IL-4) and JESS-2A5 (anti-IL-10). Samples were titrated in test solution and incubated overnight at 4°C. To detect bound cytokines, plates were then incubated with the biotinylated mAb BVD6-24G2 (anti-IL-4) or SXC-1 (anti-IL-10) in PBS containing 0.1% Tween 20 and 1% BSA (PBSA-Tw). After washing, the bound biotinylated Abs were revealed by an additional 30-min incubation with alkaline phosphatase-conjugated streptavidin (Jackson ImmunoResearch Laboratories, Avondale, PA) diluted 1/5000. The plates were washed again and incubated with the developing substrate p-nitrophenylphosphate disodium (Sigma) in diethanolamine buffer (pH 9.6; 100 μl/well). The reaction was stopped by adding 50 μl/well NaOH 3N, and absorbance was read at 405 nm. Cytokines were quantified from two to three titration points using standard curves generated by purified recombinant mouse cytokines and results expressed as cytokine concentration in ng or pg/ml. Detection limits were 15 pg/ml for all cytokines, unless otherwise stated.

After removal of all visible pancreatic lymph nodes, pancreata were individually digested in 3 ml HBSS containing 1 mg/ml collagenase IV (Sigma), by shaking (200 rpm) at 37°C for 15 min. Single cell suspensions were collected after diluting the enzyme with ice-cold HBSS containing 5% FCS and removal of the aggregates by settling for 2 min on ice. Aggregates were further digested with collagenase IV at 0.5 mg/ml for 10 min, and at 0.25 mg/ml for 6 min. Single cell suspensions were washed three times, and CD4+ and CD8+ cells were sorted by positive selection on MiniMACS. Pancreas-infiltrating CD4+ and CD8+ cells were double stained by incubating them with optimal concentrations of PE-labeled rat anti-mouse CD4 (RM4-4) or CD8 (53-6.7) in the presence of rat anti-mouse FcR (2.4G2) (all from PharMingen) and P-selectin-IgG chimeric protein (kind gift of Dr. D. Vestweber, University of Muenster, Muenster, Germany) followed by FITC-labeled rabbit F(ab′)2 anti-human IgG. Alternatively, pancreas-infiltrating CD4+ and CD8+ cells were directly stimulated with PMA and ionomycin in complete medium, fixed, and stained the next day for intracellular production of cytokines.

Cells were stained for IFN-γ, IL-4, and IL-10 using the method described by Openshaw (33) and mAbs obtained from PharMingen. Reagents for intracytoplasmic staining contained 1% FCS, 0.5% saponin (Sigma), and 0.1% sodium azide, and all incubations were performed at room temperature. Cells were washed, preincubated for 10 min with PBS/FCS/saponin, and then incubated with FITC-labeled rat anti-mouse IFN-γ (XMG1.2) and PE-labeled rat anti-mouse IL-4 (11B11), or PE-labeled rat anti-mouse IL-10 (JES5-16E3). Isotype controls were FITC- and PE-labeled rat IgG1 (R3-34). After 30 min, cells were washed twice with PBS/FCS/saponin and then with PBS containing 5% of FCS without saponin to allow membrane closure. The cell surface was then stained with CyChrome-labeled anti-CD4 (L3T4) for 15 min at room temperature. Analysis was performed with a FACScan flow cytometer (Becton Dickinson, Mountain View, CA) equipped with CellQuest software, and ∼10,000 events were acquired.

Pancreata were snap-frozen in Tissue Tek (Miles Laboratories, Elkhart, IN), and 5-μm-thick sections stained with hematoxilin and eosin. Insulitis score was quantified as follows: 0, no insulitis; 1, peri-insulitis; 2, insulitis in <50% of the islet; 3, insulitis in >50% of the islet. A mean score was calculated from 40–50 islets per individual pancreas. In addition, pancreas cryostat sections were stained with biotinylated mAb directed against CD4, CD8, B220, CD11b (all purchased from PharMingen), or CD11c (N418; American Type Culture Collection, Manassas, VA), followed by streptavidin-peroxidase conjugate. 3-amino-9-ethylcarbazole (Dako, Carpenteria, CA) was used as chromogen and hematoxylin as a counterstain.

LPS-induced serum IFN-γ levels in IL-12-deficient B6 or BALB/c mice are reduced by ∼80%, as compared with wild-type littermates (5). We analyzed LPS-induced serum levels of IL-12p40 and IFN-γ in IL-12-deficient and wild-type NOD mice. Results shown in Fig. 1 confirmed the absence of IL-12p40 in IL-12-deficient NOD mice and the reduction of serum IFN-γ levels by ∼80%. Therefore, IFN-γ production in response to endotoxin is strongly, although not completely, inhibited in IL-12-deficient NOD mice, consistent with results obtained in other mouse strains.

FIGURE 1.

LPS-induced IL-12p40 and IFN-γ production in vivo in IL-12-deficient and wild-type NOD mice. IL-12-deficient and wild-type NOD mice (four females and two males/group) were injected i.p. with LPS and bled 3 and 6 h later for serum IL-12p40 and IFN-γ quantification, respectively. Individual serum cytokine levels and their means (horizontal bars) are shown. Similar results were obtained in a separate experiment.

FIGURE 1.

LPS-induced IL-12p40 and IFN-γ production in vivo in IL-12-deficient and wild-type NOD mice. IL-12-deficient and wild-type NOD mice (four females and two males/group) were injected i.p. with LPS and bled 3 and 6 h later for serum IL-12p40 and IFN-γ quantification, respectively. Individual serum cytokine levels and their means (horizontal bars) are shown. Similar results were obtained in a separate experiment.

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To test the role of endogenous IL-12 in the development of Th1 responses in NOD mice, we examined cytokine production in response to two different Ags, HEL and PPD. PPD was chosen for its capacity to preferentially induce IL-12-dependent Th1 responses (34). IL-12-deficient and wild-type NOD mice were immunized with either HEL emulsified in IFA or with CFA alone. Nine days later, draining lymph node cells were restimulated in vitro with HEL or PPD, respectively. The immune cells from NOD mice secreted high levels of IFN-γ, 15 and 23 ng/ml, respectively (Fig. 2, A and B). In contrast, the amount of IL-4 secreted by HEL or PPD-specific cells was 120 pg/ml and <15 pg/ml, respectively. As compared with wild-type mice, IL-12-deficient NOD mice showed near abrogation of Ag-specific IFN-γ secretion, whereas IL-4 was increased and IL-10 secretion was slightly enhanced (Fig. 2, A and B). The phenotype of PPD-specific CD4+ was characterized at the single cell level by intracytoplasmic staining for IFN-γ and IL-4 production. Immune lymph node cells, after culture for 72 h with PPD, were rested for an additional 72 h without Ag and restimulated with either PPD or PMA/ionomycin before staining for intracytoplasmic cytokines and surface CD4 expression. With both stimuli, IL-4-producing CD4+ cells were undetectable either in wild-type or IL-12-deficient NOD mice (Fig. 2,C). In contrast, 10% and 25% of CD4+ lymph node cells from wild-type NOD mice produced IFN-γ when restimulated with PPD or PMA/ionomycin, respectively (Fig. 2 C). These proportions were reduced by 80% and 64%, respectively, in CD4+ cells from IL-12-deficient NOD mice. Thus, a qualitatively similar pattern of intracytoplasmic cytokine production was induced by restimulation with PPD or PMA/ionomycin. Altogether, the results show that Th1 development is impaired in IL-12-deficient NOD mice, without an appreciable induction of Th2 cells.

FIGURE 2.

Ag-induced cytokine production by immune lymph node cells from IL-12-deficient and wild-type NOD mice. A, HEL-specific cytokine secretion. IL-12-deficient and wild-type NOD female mice were immunized with HEL in IFA, and lymph nodes were harvested 9 days later. Lymph node cells were cultured in the presence of 10 μM HEL with or without IL-12 or IL-18, and secreted IFN-γ, IL-4, and IL-10 in culture supernatants were quantified by ELISA. Bars represent mean values ± SE from two separate experiments. B, PPD-specific cytokine secretion. The experiment was performed as in A, but mice were immunized with CFA only and lymph node cells were cultured with 30 μg/ml PPD. Bars represent mean values ± SE from two separate experiments. C, Intracytoplasmic cytokine production by PPD-specific CD4+ T cells. After collecting the supernatants, cells from the experiment described in B were washed and recultured without Ag for 72 h. The cells were then stimulated by either PPD or PMA/ionomycin as indicated in Materials and Methods. Cells were stained for intracytoplasmic IL-4 and IFN-γ as well as cell surface CD4 expression, and analyzed by flow cytometry. Acquisition was performed on CD4+ cells. Percentages of cytokine-positive cells, set according to the isotype controls, are shown in the top right-hand corner of each quadrant.

FIGURE 2.

Ag-induced cytokine production by immune lymph node cells from IL-12-deficient and wild-type NOD mice. A, HEL-specific cytokine secretion. IL-12-deficient and wild-type NOD female mice were immunized with HEL in IFA, and lymph nodes were harvested 9 days later. Lymph node cells were cultured in the presence of 10 μM HEL with or without IL-12 or IL-18, and secreted IFN-γ, IL-4, and IL-10 in culture supernatants were quantified by ELISA. Bars represent mean values ± SE from two separate experiments. B, PPD-specific cytokine secretion. The experiment was performed as in A, but mice were immunized with CFA only and lymph node cells were cultured with 30 μg/ml PPD. Bars represent mean values ± SE from two separate experiments. C, Intracytoplasmic cytokine production by PPD-specific CD4+ T cells. After collecting the supernatants, cells from the experiment described in B were washed and recultured without Ag for 72 h. The cells were then stimulated by either PPD or PMA/ionomycin as indicated in Materials and Methods. Cells were stained for intracytoplasmic IL-4 and IFN-γ as well as cell surface CD4 expression, and analyzed by flow cytometry. Acquisition was performed on CD4+ cells. Percentages of cytokine-positive cells, set according to the isotype controls, are shown in the top right-hand corner of each quadrant.

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We next examined whether IL-12 could restore in vitro Th1 cell development in IL-12-deficient NOD mice. Addition of exogenous IL-12 and PPD to immune lymph node cells from wild-type NOD mice recruited new cells to secrete IFN-γ, increasing the frequency of Th1 cells by 2- to 3-fold (Fig. 2,C), and the amount of secreted IFN-γ increased accordingly (Fig. 2,B). Addition of IL-12, together with PPD, to lymph node cells from IL-12-deficient NOD mice yielded a similar proportion of IFN-γ-producing CD4+ cells and similar levels of IFN-γ secretion, as in controls. In addition, it inhibited the limited secretion of IL-4 (Fig. 2 B).

Since IL-18 shares some biological functions with IL-12 (35), we also tested the capacity of IL-18 to induce differentiation of Th1 cells. Addition of IL-18 to the cell culture from wild-type NOD mice did not modify the proportion of cells secreting IFN-γ, in contrast to the results obtained with exogenous IL-12 (Fig. 2,C). However, the total amount of IFN-γ in the culture supernatant increased substantially, indicating that IL-18 stimulated individual Th1 cells to produce more IFN-γ. This effect was IL-12-dependent, since it was abrogated in cells from IL-12-deficient NOD mice (Fig. 2,B). Addition of IL-18 to the cell culture from IL-12-deficient NOD mice induced a slight increase, ∼2-fold, in the number of IFN-γ-producing CD4+ cells (Fig. 2,C) and, consequently, in the level of secreted IFN-γ (Fig. 2 B). This suggests that IL-18 may have a minor role in the development of Th1 cells in IL-12-deficient NOD mice.

In conclusion, IL-12-deficient NOD mice have greatly reduced Th1 responses to exogenous Ags, confirming the important role of IL-12 for Th1 development in NOD mice. However, as in other mouse strains, a residual low level of IL-12-independent Th1 development does occur, which could be mediated in part by IL-18.

A defect in peripheral Th1 responses was expected to reduce the progression to IDDM in IL-12-deficient NOD mice. Strikingly, the onset and cumulative incidence of diabetes were similar in wild-type and IL-12-deficient NOD mice. About 70% female and 60% male wild-type or IL-12-deficient NOD mice developed spontaneous IDDM by 45 wk of age (Fig. 3). No statistically significant difference in the insulitis score could be detected between either male or female wild-type and IL-12-deficient NOD mice (Fig. 4). Histologic examination of the pancreata from 12-wk-old wild-type and IL-12-deficient NOD mice revealed in both a severe insulitis composed of CD4+ and CD8+ T cells, B cells, macrophages, and dendritic cells, as determined by CD4, CD8, B220, Mac-1, and CD11c expression (Fig. 4).

FIGURE 3.

Similar IDDM incidence in IL-12-deficient and wild-type NOD mice. Blood glucose levels from 33 IL-12-deficient and 38 wild-type NOD mice were measured every 2 wk, from 10 to 45 wk of age. There was no statistical difference in the time to IDDM (two-tailed Gehan’s test) nor in the proportion of mice becoming diabetic (two-tailed Fisher’s exact test) between IL-12-deficient and wild-type NOD mice.

FIGURE 3.

Similar IDDM incidence in IL-12-deficient and wild-type NOD mice. Blood glucose levels from 33 IL-12-deficient and 38 wild-type NOD mice were measured every 2 wk, from 10 to 45 wk of age. There was no statistical difference in the time to IDDM (two-tailed Gehan’s test) nor in the proportion of mice becoming diabetic (two-tailed Fisher’s exact test) between IL-12-deficient and wild-type NOD mice.

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FIGURE 4.

Insulitis in IL-12-deficient and wild-type NOD mice. Histological scoring of insulitis (left panels) was performed on hematoxylin/eosin-stained pancreatic sections from 12-wk-old IL-12-deficient and wild-type NOD female mice, as detailed in Materials and Methods. Each bar represents the mean score of ∼40–50 islets from an individual mouse. In the right panels, representative pancreatic sections from 12-wk-old wild-type (upper panels) and IL-12-deficient NOD female mice (lower panels) are shown. Consecutive sections of the same islet were stained with anti-CD4, anti-CD8, anti-B220, anti-CD11b (Mac-1), or anti-CD11c (N418) mAb.

FIGURE 4.

Insulitis in IL-12-deficient and wild-type NOD mice. Histological scoring of insulitis (left panels) was performed on hematoxylin/eosin-stained pancreatic sections from 12-wk-old IL-12-deficient and wild-type NOD female mice, as detailed in Materials and Methods. Each bar represents the mean score of ∼40–50 islets from an individual mouse. In the right panels, representative pancreatic sections from 12-wk-old wild-type (upper panels) and IL-12-deficient NOD female mice (lower panels) are shown. Consecutive sections of the same islet were stained with anti-CD4, anti-CD8, anti-B220, anti-CD11b (Mac-1), or anti-CD11c (N418) mAb.

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The lack of difference in IDDM progression between wild-type and IL-12-deficient NOD mice was rather unexpected, since administration of the IL-12 antagonist (p40)2 to NOD mice could inhibit disease development (21). In an attempt to determine the reason for this discrepancy, we analyzed the CD4+ T cell response to a candidate autoantigen in IDDM, the tyrosine phosphatase-like IA-2. Like glutamic acid decarboxylase (GAD)65, another major autoantigen in IDDM, IA-2 induces a spontaneous cytokine response in spleen cells from unprimed NOD, but not other mouse strains (S. Trembleau et al., manuscript in preparation). Spleen cells from NOD mice treated with the IL-12-antagonist (p40)2 from 3 wk of age for 50 consecutive days, IL-12-deficient, and control NOD mice were stimulated in vitro with recombinant mouse IA-2. IFN-γ and IL-10 secretion were measured after 48 h of culture. The IFN-γ response to IA-2 was inhibited by ∼50% in spleen cells from (p40)2-treated NOD mice and was abrogated in spleen cells from IL-12-deficient NOD mice (Fig. 5,A). In contrast, IA-2-specific IL-10 secretion was similar in spleen cells from IL-12 antagonist-treated, IL-12-deficient, and control NOD mice (Fig. 5,A). To determine whether these cytokines were secreted by IA-2-specific CD4+ T cells, CD4+ cells were purified from peripheral lymphoid organs and stimulated with IA-2 presented by T cell-depleted mitomycin C-treated NOD spleen cells. Splenic and mesenteric lymph node CD4+ T cells from control NOD mice secreted dose-dependent high levels of IFN-γ in response to IA-2, whereas CD4+ cells from IL-12-deficient NOD mice did not secrete any detectable IFN-γ (Fig. 5,B). IA-2-induced IL-10 secretion by CD4+ T cells was similar in both groups (Fig. 5 B). Thus, Th1 responses to IA-2 are decreased in IL-12 antagonist-treated NOD mice and undetectable in IL-12-deficient NOD mice.

FIGURE 5.

Comparison of spontaneous cytokine responses in peripheral lymphoid organs and in the pancreas of IL-12 antagonist-treated, IL-12-deficient, and control NOD mice. A, IA-2-induced IFN-γ and IL-10 secretion by spleen cells. A total of 106 total spleen cells from 10-wk-old nonimmunized female mice (three mice/group) were cultured in complete medium with or without 0.3 μM recombinant mouse IA-2. After 48 h of incubation at 37°C, IFN-γ and IL-10 secretion were determined in culture supernatants by ELISA. IFN-γ and IL-10 were undetectable in the absence of IA-2, the detection limits being 45 pg and 15 pg/ml, respectively. The upper panels show mean and SEM of cytokine production from three individual mice, whereas data in the lower panels are from pooled cells from three mice/group. ∗, Indicates a significant decrease in IFN-γ secretion in (p40)2-vs vehicle-treated mice (p = 0.022 by one-tailed paired t test). B, IA-2-specific CD4+ T cell responses in peripheral lymphoid organs. Splenic or mesenteric lymph node CD4+ cells from nonimmunized IL-12-deficient or wild-type NOD female mice (pool of three mice/group) were sorted by positive selection on MiniMACS. A total of 2 × 105 CD4+ cells were cultured with 106 T cell-depleted and mitomycin C-treated spleen cells, and with 0.1, 0.3, or 1 μM IA-2. After 72 h, IFN-γ and IL-10 were determined in culture supernatants. IFN-γ and IL-10 were undetectable in cultures without IA-2, the detection limits being 45 pg and 15 pg/ml, respectively. C, IFN-γ and IL-10 production by pancreas-infiltrating CD4+ cells. Pancreas-infiltrating CD4+ plus CD8+ cells were positively selected on MiniMACS from pools (three mice/group) of IL-12-deficient, wild-type IL-12 antagonist-treated and vehicle-treated NOD female mice. The latter mice were injected daily with 3 mg/kg (p40)2 or vehicle from 3 to 10 wk of age before collecting the pancreas-infiltrating T cells. Pancreas-infiltrating T cells were stimulated with PMA/ionomycin for 4 h and analyzed by flow cytometry for IFN-γ (abscissa) and IL-10 (ordinate) production. During acquisition, cells were gated in CD4+ and CD4 (CD8+) populations, using CyChrome-labeled anti-CD4 mAb. Cytokine production by CD4+ cells only is shown. Percentages of cytokine-positive cells, set according to the isotype controls, are shown in the top right-hand corner of each quadrant.

FIGURE 5.

Comparison of spontaneous cytokine responses in peripheral lymphoid organs and in the pancreas of IL-12 antagonist-treated, IL-12-deficient, and control NOD mice. A, IA-2-induced IFN-γ and IL-10 secretion by spleen cells. A total of 106 total spleen cells from 10-wk-old nonimmunized female mice (three mice/group) were cultured in complete medium with or without 0.3 μM recombinant mouse IA-2. After 48 h of incubation at 37°C, IFN-γ and IL-10 secretion were determined in culture supernatants by ELISA. IFN-γ and IL-10 were undetectable in the absence of IA-2, the detection limits being 45 pg and 15 pg/ml, respectively. The upper panels show mean and SEM of cytokine production from three individual mice, whereas data in the lower panels are from pooled cells from three mice/group. ∗, Indicates a significant decrease in IFN-γ secretion in (p40)2-vs vehicle-treated mice (p = 0.022 by one-tailed paired t test). B, IA-2-specific CD4+ T cell responses in peripheral lymphoid organs. Splenic or mesenteric lymph node CD4+ cells from nonimmunized IL-12-deficient or wild-type NOD female mice (pool of three mice/group) were sorted by positive selection on MiniMACS. A total of 2 × 105 CD4+ cells were cultured with 106 T cell-depleted and mitomycin C-treated spleen cells, and with 0.1, 0.3, or 1 μM IA-2. After 72 h, IFN-γ and IL-10 were determined in culture supernatants. IFN-γ and IL-10 were undetectable in cultures without IA-2, the detection limits being 45 pg and 15 pg/ml, respectively. C, IFN-γ and IL-10 production by pancreas-infiltrating CD4+ cells. Pancreas-infiltrating CD4+ plus CD8+ cells were positively selected on MiniMACS from pools (three mice/group) of IL-12-deficient, wild-type IL-12 antagonist-treated and vehicle-treated NOD female mice. The latter mice were injected daily with 3 mg/kg (p40)2 or vehicle from 3 to 10 wk of age before collecting the pancreas-infiltrating T cells. Pancreas-infiltrating T cells were stimulated with PMA/ionomycin for 4 h and analyzed by flow cytometry for IFN-γ (abscissa) and IL-10 (ordinate) production. During acquisition, cells were gated in CD4+ and CD4 (CD8+) populations, using CyChrome-labeled anti-CD4 mAb. Cytokine production by CD4+ cells only is shown. Percentages of cytokine-positive cells, set according to the isotype controls, are shown in the top right-hand corner of each quadrant.

Close modal

Th1-mediated autoimmune diseases can develop, in particular conditions, in the context of a deviant Th2-like effector response (26, 36, 37). To test whether this was the case, we characterized the phenotype of pancreas-infiltrating T cells in IL-12-deficient and IL-12 antagonist-treated NOD mice. Pancreatic CD4+ and CD8+ T cells were isolated, stimulated with PMA/ionomycin, and stained for intracytoplasmic IFN-γ and IL-4, as well as IFN-γ and IL-10 production. As shown in Fig. 2, a short restimulation with PMA/ionomycin is qualitatively comparable to Ag in revealing the cytokine profile of previously activated cells. Surprisingly, both in wild-type and IL-12-deficient NOD mice, ∼20% of pancreatic CD4+ cells produced IFN-γ only, 6% IFN-γ and IL-10, and 3% IL-10 only (Fig. 5,C). No IL-4-producing cells were detected (data not shown). Therefore, a similar percentage of Th1-type pancreas-infiltrating CD4+ cells was present both in wild-type and IL-12-deficient NOD mice. In contrast, the proportion of IL-10-producing CD4+ cells increased by 3-fold in NOD mice treated with the IL-12 antagonist (p40)2 (Fig. 5,C), consistent with previous results (21). The proportion of IFN-γ-producing CD4+ cells remained similar in vehicle and IL-12(p40)2-treated NOD mice (Fig. 5 C). However, when IL-12(p40)2-treated mice were tested individually at a later age and stratified in diabetic and still normoglycemic, the increase of pancreas-infiltrating Th2-type cells was found associated with decreased Th1 and with protection from IDDM (21). Pancreas-infiltrating CD8+ cells were also tested for their cytokine profile. CD8+ cells from IL-12 antagonist-treated NOD, IL-12-deficient NOD, and control NOD mice produced IFN-γ, but not IL-4 nor IL-10, with a similar percentage of IFN-γ-producing cells in all groups (data not shown). These results are in agreement with the lack of deviation to the type 2 phenotype in pancreas-infiltrating CD8+ cells from IL-12(p40)2-treated NOD mice (21).

In conclusion, in IL-12-deficient NOD mice, the development of Th1 cells was strongly impaired in peripheral lymphoid organs and nevertheless they did accumulate and/or expand in the pancreas leading to IDDM development. Only Th1 and not Th2 cells were found in the pancreas of IL-12-deficient NOD mice. In contrast, administration of the IL-12 antagonist (p40)2 induced Th2 cells in the pancreas of NOD mice. This immune deviation was found to be associated with protection from IDDM (21). Thus, the lack of Th2 cells may leave the pathogenic Th1 cells that develop in the absence of IL-12 unchecked.

Leukocyte extravasation into inflamed tissues requires P- and E-selectin expression on endothelial cells, and the ligand for P-selectin is expressed by chronically stimulated, but not resting, CD4+ T lymphocytes (38) and by Th1 but not Th2 cells (39, 40). High numbers of P-selectin-binding CD4+ T cells were found at inflammatory sites in delayed-type hypersensitivity and autoimmune colitis, two Th1-mediated conditions (41, 42). Therefore, it was of interest to compare the expression of P-selectin ligand on pancreas-infiltrating T cells from IL-12-deficient and wild-type NOD mice. Unexpectedly, a larger fraction of ex vivo labeled CD4+ and CD8+ pancreas-infiltrating cells from IL-12-deficient compared with control mice bound P-selectin/Ig fusion protein (Fig. 6). The increase in P-selectin ligand-positive CD4+ and CD8+ cells was ∼2- and 3-fold, respectively. These results suggest a compensatory mechanism for type 1 T cell recruitment in the pancreas of IL-12-deficient NOD mice.

FIGURE 6.

Cell surface expression of P-selectin ligand by pancreas-infiltrating CD4+ and CD8+ cells from prediabetic IL-12-deficient and wild-type NOD mice. Pancreas-infiltrating T cells from IL-12-deficient and wild-type NOD female mice were double-stained for the detection of P-selectin ligand and either CD4 or CD8. The bars in the top panel indicate the mean percent (±SD) of pancreas-infiltrating CD4+ and CD8+ cells binding P-selectin from three separate experiments. Double stainings from a representative experiment are shown below.

FIGURE 6.

Cell surface expression of P-selectin ligand by pancreas-infiltrating CD4+ and CD8+ cells from prediabetic IL-12-deficient and wild-type NOD mice. Pancreas-infiltrating T cells from IL-12-deficient and wild-type NOD female mice were double-stained for the detection of P-selectin ligand and either CD4 or CD8. The bars in the top panel indicate the mean percent (±SD) of pancreas-infiltrating CD4+ and CD8+ cells binding P-selectin from three separate experiments. Double stainings from a representative experiment are shown below.

Close modal

The present study demonstrates that Th1 responses to exogenous Ags are reduced by ∼80% in lymph nodes of IL-12-deficient NOD mice, confirming results obtained in other IL-12-deficient mouse strains (5). In addition, Th1 responses to the self-Ag IA-2 are undetectable in IL-12-deficient NOD in contrast to control mice. However, rather unexpectedly, IDDM develops equally well in IL-12-deficient and wild-type NOD mice, and in both it is associated with pancreas-infiltrating type 1 T cells only. Thus, Th1 development is impaired in peripheral lymphoid organs of IL-12-deficient NOD mice, but IL-12 appears dispensable for pancreatic infiltration of IFN-γ-producing cells and IDDM development. In contrast, pancreas-infiltrating CD4+ cells from IL-12 antagonist-treated NOD mice are skewed to a Th2 phenotype, which was found to be associated with protection from IDDM (21). These findings suggest that the development of pathogenic Th1 cells can be inhibited when a Th2-type regulation is induced and that the incapacity to generate this regulatory pathway may contribute to IDDM development in IL-12-deficient NOD mice.

The impaired Th1 development in IL-12-deficient mice is usually associated with a propensity to develop Th2 responses (5). For example, wild-type 129/Sv/Ev mice develop a Th1-dominated immune response and are resistant to L. major, whereas mice lacking IL-12 mount a polarized Th2 response and become susceptible to the infection (43). However, IL-12 deficiency is not always paralleled by an expansion of Th2 cells (8). Inhibition of Th1 favors the establishment of Th2-promoting conditions (44), but if these conditions are intrinsically defective, development of Th2 cells will be precluded. Th2-promoting factors include the predisposition to produce IL-4. Since NOD mice have a defect in IL-4 production (45), this could explain why IL-12-deficient NOD mice fail to demonstrate an appreciable induction of Th2 responses.

The contrasting results obtained in IL-12(p40)2-treated and IL-12-deficient NOD mice suggest that IL-12 or (p40)2 may be involved in Th2 cell generation. IL-12p75 has been shown to contribute to Th2 cell development (46). In addition, a direct or indirect role of (p40)2 itself could also explain the enhancement of Th2 responses observed after (p40)2 administration in NOD mice. IL-12 is composed of two covalently associated chains, p40 and p35. Upon activation, cells secreting the biologically active IL-12p75 heterodimer also secrete a large excess of monomeric p40 as well as (p40)2 (47, 48). IL-12(p40)2 strongly inhibits IL-12 activities, and thus represents a natural antagonist (49), although an agonist role has also been hypothesized (50). Analysis of IDDM development in IL-12p40- and in IL-12p35-deficient NOD mice could have been useful to distinguish between the role of endogenous IL-12p75 and monomeric or homodimeric p40. Unfortunately, the backcross of the IL-12p35-deficient mice to the NOD background is uninformative due to the close linkage of several Idd loci on chromosome 3, where the IL-12p35 gene is located (51).

Spontaneous IDDM in NOD mice is unique among the autoimmune disease models so far examined because of its capacity to develop as efficiently in IL-12-deficient and in control mice. IL-12 deficiency consistently leads to decreased autoantigen-specific Th1 responses in induced autoimmune diseases, such as CIA, EAMG, EAU, and EAE. However, the concomitant induction of Th2-type responses or other immunoregulatory pathways is variable (14, 15, 16, 17). Interestingly, IL-12-deficient mice are only partially protected from CIA and EAMG, whereas they appear to be completely protected from EAE and EAU. In these cases, complete protection from autoimmunity seems associated with an immunoregulatory circuit involving IL-10 (14, 15). Thus, an impaired development of Th1 cells may not be sufficient for complete inhibition of an autoimmune disease, and the induction of an immunoregulatory pathway could be necessary. This regulation could depend more on IL-10 than IL-4, as indicated by the observation that IL-4 transgenic mice do develop EAE, but IL-10 transgenic are completely protected (52), and by the capacity of IL-10-producing Tr1 cells to inhibit autoimmune colitis (53). IL-12-deficient NOD mice show a major reduction of Ag-specific IFN-γ, but little enhancement of IL-4 and IL-10 secretion. Likewise, very few pancreas-infiltrating T cells produce IL-4 or IL-10. A defective IL-4 production by NOD CD4+ cells has been implicated in IDDM development (45), possibly through impairment of NK1.1+CD4+ cells that could be involved in early IL-4 production (54), and it is possible that immunoregulatory pathways involving IL-10 are impaired as well. Consistent with this assumption, administration of a noncytolytic IL-10-fusion protein completely protects NOD mice from IDDM (55). In addition, IL-10-transduced islet-specific Th1 cells prevent IDDM transfer in NOD mice (56).

The absence of Th2-type regulation could, in part, explain the accumulation of residual Th1 cells into the pancreas of IL-12-deficient NOD mice. Could other cytokines, such as IFN-γ or IL-18, compensate for the lack of IL-12 and induce diabetogenic Th1 cells? In the mouse, IFN-γ synergizes with IL-12 for Th1 development (57). Similar to the situation observed with IL-12, inhibition of endogenous IFN-γ protects from diabetes (58, 59), but IDDM develops in IFN-γ-deficient NOD mice (60). In contrast to the latter result, insulitis does not develop in IFN-γRα-deficient NOD mice (61). While there is no clear explanation at present for the discrepancy between IFN-γ- and IFN-γRα-deficient mice, this has been observed also in other models (62). Recently, IFN-γRβ-deficient mice were found to differ from IFN-γRα-deficient mice in their ability to develop Th1 responses (63). More detailed understanding of the IFN-γ signaling pathway may explain these seemingly conflicting results. In any case, the genetic absence of IL-12 or IFN-γ allows the development of compensatory mechanisms not available in unmanipulated NOD mice, in which IDDM can be prevented by treatment with cytokine antagonists.

A cytokine potentially able to replace IL-12 could be the IFN-γ-inducing factor IL-18 (64). A rise in both IL-18 and IL-12p40 mRNA levels has been detected in the adherent spleen cell population of cyclophosphamide-treated NOD mice (65). Our results demonstrate that IL-18 synergizes with IL-12, but is not able to restore the production of IFN-γ by HEL or PPD-specific T cells from IL-12-deficient NOD mice. Thus, IL-18 only acts on IL-12-primed Th1-developing cells, stimulating them to produce more IFN-γ, but in the absence of IL-12 is inefficient in inducing the differentiation of Th1 cells. These data are consistent with results indicating that IL-12 is sufficient for normal Th1 development in the absence of IL-18 (35), and that IL-18 by itself does not induce Th1 cell development (66, 67). However, mice deficient in both IL-12 and IL-18 display a more profound impairment in the bacillus Calmette-Guerin-induced Th1 response, as compared with IL-12-deficient mice, suggesting that IL-12-independent Th1 development could be induced by the cooperative action of IL-18 and other factor(s), yet unidentified (35). However, this pathway might only account for the residual Th1 development in IL-12-deficient NOD mice. Although IL-18 could substitute in part for the lack of IL-12, accumulation of diabetogenic Th1 cells in the pancreas of IL-12-deficient NOD mice is likely to depend on alternative mechanisms. In addition to Th1-promoting cytokines, the nature of the autoantigen(s) and the chronicity of IDDM combined with a genetic deficiency in immunoregulation could lead, even in the absence of IL-12, to diabetogenic Th1 cell development in the NOD mouse.

Both CD4+ and CD8+ T cells are necessary for IDDM development in unmanipulated NOD mice. However, under some circumstances, either CD4+ or CD8+ T cells alone are able to induce the disease (68, 69). Interestingly, the cytotoxic activity of CD8+ T cells is unaffected in IL-12-deficient mice (5), or even increased in IFN-γ-deficient mice (70). Thus, it is possible that IL-12-deficient NOD mice develop IDDM via a mechanism(s) involving predominantly CD8+ T cells, as compared with their wild-type littermates. A similar number of pancreas infiltrating CD4+ and CD8+ cells was visualized in histology or counted after their purification in IL-12-deficient and wild-type NOD mice. However, the proportion of pancreas infiltrating cells expressing P-selectin ligand is 2-fold higher in CD4+ and 3-fold higher in CD8+ T cells from IL-12-deficient as compared with wild-type NOD mice. P-selectin ligand expression has been associated with subsets of skin or mucosa-seeking memory/effector T cells that produce proinflammatory cytokines (41, 42). Therefore, T cells and, in particular, CD8+ cells from IL-12-deficient NOD mice may have a higher diabetogenic potential.

In conclusion, spontaneous IDDM in NOD mice is unique among autoimmune disease models for its capacity to develop in IL-12-deficient mice. Nevertheless, administration of the IL-12 antagonist (p40)2 does prevent IDDM in NOD mice (21). The following scenario could be envisioned to explain why IL-12 is dispensable for IDDM development. Th1 cell development in NOD mice, as in other mouse strains, is impaired, although not completely prevented, in the absence of IL-12. The residual Th1 development may be due in part to the cooperative action of IL-18 and other factor(s) (35). Th1 impairment is not sufficient for IDDM prevention, and the induction of a regulatory pathway is necessary for protection against Th1-mediated autoimmunity. Neutralization of endogenous IL-12 in normal NOD mice, when started at 3 wk of age, results in high numbers of pancreas-infiltrating IL-4-producing CD4+ cells and in increased IL-10-producing CD4+ cells, associated with protection from IDDM (21). Intriguingly, these Th2-type cells are not present in IL-12-deficient NOD mice, suggesting that IL-12 or (p40)2 may be involved in their generation. The lack of immune deviation to the Th2 pathway is the most obvious difference between IL-12-deficient NOD mice and wild-type NOD mice in which endogenous IL-12 has been targeted by IL-12 antagonist administration. Thus, IL-12-deficient NOD mice appear to have a genetic deficiency in developing a regulatory pathway able to counteract diabetogenic Th1 cells. In the absence of a regulatory pathway controlling Th1 responses, the residual Th1 cells accumulate in the pancreas of IL-12-deficient NOD mice to levels quantitatively and qualitatively similar to controls. T cell infiltration and accumulation may also be favored in IL-12-deficient NOD mice by increased P-selectin ligand expression on CD4+ and CD8+ cells, and this could further contribute to compensate for the defective Th1 cell pool recruitable from peripheral lymphoid organs.

4

Abbreviations used in this paper: EAE, experimental allergic encephalomyelitis; CIA, collagen-induced arthritis; EAU, experimental autoimmune uveitis; EAMG, experimental autoimmune myasthenia gravis; HEL, hen egg-white lysozyme; IDDM, insulin-dependent diabetes mellitus; NOD, nonobese diabetic; PPD, protein purified derivative.

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