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The Journal of Immunology, 2003, 171: 4485-4492.
Copyright © 2003 by The American Association of Immunologists

Role of IL-12 Receptor {beta}1 in Regulation of T Cell Response by APC in Experimental Autoimmune Encephalomyelitis 1

Guang-Xian Zhang*, Shuo Yu*, Bruno Gran*, Jifen Li{dagger}, Ines Siglienti*, Xiaohan Chen{ddagger}, Divina Calida*, Elvira Ventura*, Malek Kamoun{dagger} and Abdolmohamad Rostami2,*

* Department of Neurology, Thomas Jefferson University, Philadelphia, PA 19107; and Departments of {dagger} Pathology and Laboratory Medicine and {ddagger} Neurosurgery, University of Pennsylvania School of Medicine, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IL-12 was thought to be involved in the development of experimental autoimmune encephalomyelitis (EAE), a Th1 cell-mediated autoimmune disorder of the CNS. However, we have recently found that IL-12 responsiveness, via IL-12R{beta}2, is not required in the induction of EAE. To determine the role of IL-12R{beta}1, a key subunit for the responsiveness to both IL-12 and IL-23, in the development of autoimmune diseases, we studied EAE in mice deficient in this subunit of IL-12R. IL-12R{beta}1-/- mice are completely resistant to myelin oligodendrocyte glycoprotein (MOG)-induced EAE, with an autoantigen-specific Th2 response. To study the mechanism underlying this Th2 bias, we cocultured purified CD4+ T cells and APCs of MOG-immunized mice. We demonstrate that IL-12R{beta}1-/- APCs drive CD4+ T cells of both wild-type and IL-12R{beta}1-/- mice to an Ag-induced Th2 phenotype, whereas wild-type APCs drive these CD4+ T cells toward a Th1 type. IL-12R{beta}1-/- CD4+ T cells, in turn, appear to exert an immunoregulatory effect on the capacity of wild-type APCs to produce IFN-{gamma} and TNF-{alpha}. Furthermore, decreased levels of IL-12p40, p35, and IL-23p19 mRNA expression were found in IL-12R{beta}1-/- APCs, indicating an autocrine pathway of IL-12/IL-23 via IL-12R{beta}1. IL-18 production and IL-18R{alpha} expression are also significantly decreased in IL-12R{beta}1-/- mice immunized with MOG. We conclude that in the absence of IL-12R{beta}1, APCs play a prominent regulatory role in the induction of autoantigen-specific Th2 cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interleukin-12, a heterodimeric cytokine, is a potent inducer of Th1 differentiation (1). IL-12 regulates the growth and function of T cells and especially promotes the development/propagation of Th1 cells by stimulating the production of IFN-{gamma} (2, 3). To date, two subunits of the IL-12R (3), IL-12R{beta}1 and IL-12R{beta}2, have been identified and cloned (4, 5). When individually expressed in COS-7 cells, human IL-12R{beta}1 and {beta}2 each bind IL-12 with low affinity, while coexpression of both IL-12R{beta}1 and {beta}2 results in both low and high affinity IL-12 binding and responsiveness (4). Splenocytes from IL-12R{beta}1-deficient mice fail to display IL-12-induced enhancement of NK lytic activity, suggesting that IL-12R{beta}1 is an essential component for IL-12 responsiveness in vitro and vivo (6). Further, both in humans and mice, IL-12R{beta}2 is selectively expressed on Th1 clones but not on Th2 clones, while IL-12R{beta}1 is consistently expressed on both cell types (7, 8). Gene targeting studies suggest that the absence of either IL-12R{beta}1 or {beta}2 impairs functional responses to IL-12 (6, 9). Recently, IL-23, a novel heterodimeric cytokine (p40p19), has been described (10, 11, 12, 13, 14). IL-23 shares the p40 subunit with IL-12 and has similar as well as distinct functions from IL-12. The receptor for IL-23 is a heterodimer composed of IL-12R{beta}1 and a newly cloned IL-23R (15). IL-12R{beta}1 is required for biological response to both IL-12 and IL-23.

Experimental autoimmune encephalomyelitis (EAE) 3 is a Th1 cell-mediated autoimmune disease of the CNS, and an animal model of multiple sclerosis (16). IL-12 enhances the pathogenicity of myelin-reactive T cells in adoptive transfer EAE (17, 18). However, it is not required for the development of active EAE, as demonstrated by recent studies in IL-12p35-/- mice (19, 20). Using IL-23p19-/- mice, Cua et al. (21) found that IL-23, but not IL-12, plays a critical role in the induction of EAE. Less information is available on the role of IL-12R in EAE. Previous observations from our group suggest that in vitro restimulation with Ag (and IL-2) up-regulates both IL-12{beta}1 and {beta}2 expression on splenocytes from EAE mice, and TGF-{beta} effectively down-regulates both IL-12R{beta}1 and {beta}2 (22). It has also been suggested that IL-12R{beta}2 subunit mRNA is expressed in EAE-susceptible, but not -resistant, strains (23). However, contrary to our expectation, we have recently found that IL-12R{beta}2-/- mice developed severe EAE, extensive inflammation and demyelination, and higher production of proinflammatory cytokines and NO (24). These results suggest that the involvement of IL-12R in EAE could be more complex than previously thought, and are consistent with the finding of the role of IL-23 in EAE. The role of the IL-12R{beta}1 subunit in EAE has not been studied. Furthermore, the role of IL-12R in APCs, the main source and an important autocrine target of IL-12/IL-23, has not been elucidated.

APCs could play an important regulatory role for Th differentiation (25, 26, 27, 28). It was previously reported that APCs provide Th cells not only with Ag (signal 1) and costimulatory signals (signal 2), but also with a polarizing signal (signal 3) (27, 29). Certain stimuli could induce APCs to produce a high level of IL-12 and, consequently, drive Th1 differentiation (APC1), whereas APCs producing a low level of IL-12 drive a Th2 differentiation (APC2) (27, 29). To directly address the role of IL-12R{beta}1 in the development of autoimmune diseases, we studied EAE in IL-12R{beta}1-deficient mice, focusing on the role of APC function in Th differentiation. Our results strongly suggest that IL-12R{beta}1 plays an essential role in the activation and differentiation of Th1-type myelin oligodendrocyte glycoprotein (MOG)-specific autoreactive T cells in vivo. APCs in IL-12R{beta}1-/- mice have functional properties of APC2 and play a prominent role in inducing a Th2 phenotype in Ag-reactive T cells.


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

Eight- to 10-wk-old (B6 x 129) mice homozygous for IL-12R{beta}1 mutation and their wild-type controls were purchased from The Jackson Laboratory (Bar Harbor, ME). The IL-12R{beta}1 gene mutation was created as described and screened by RT-PCR and Southern blot analysis (6). We confirmed the genotypes in our laboratory by using sequence-specific primers according to the protocol recommended by The Jackson Laboratory. To induce EAE, mice were each injected s.c. with 300 µg of MOG35–55 in CFA containing 4 mg/ml Mycobacterium tuberculosis H37Ra (Difco, Detroit, MI) over two sites at the back. Seventy-five nanograms of pertussis toxin were given i.v. on days 0 and 2 post immunization (p.i.). EAE was scored according to a 0–5 scale as follows (30): 1, limp tail or waddling gait with tail tonicity; 2, waddling gait with limp tail (ataxia); 2.5, ataxia with partial limb paralysis; 3, full paralysis of one limb; 3.5, full paralysis of one limb with partial paralysis of second limb; 4, full paralysis of two limbs; 4.5, moribund; and 5, death.

Histopathology

On weeks 3 and 6 p.i, mice were extensively perfused, and spinal cords were harvested. Sections (5 µm) were stained with H&E or Luxol fast blue (myelin stain). Slides were assessed in a blinded fashion for inflammation and demyelination was described as follows (31). For inflammation: 0, none; 1, a few inflammatory cells; 2, organization of perivascular infiltrates; and 3, increasing severity of perivascular cuffing with extension into the adjacent tissue. For demyelination: 0, none; 1, rare foci; 2, a few areas of demyelination; 3, large (confluent) areas of demyelination.

Isolation of CNS cells and flow cytometry

Mononuclear cells (MNCs) from the CNS of MOG-immunized mice were isolated by Percoll gradient centrifugation as described with modifications (32). Briefly, mice were sacrificed and transcardially perfused with ice-cold GKN solution (32), mechanically dissociated through a 100-µm cell strainer and enzymatically digested by incubation with 250 µg/ml collagenase at 37°C for 20–30 min. The digested CNS preparation was washed with GKN/BSA and the pellet was fractionated on a 70/37/30% Percoll gradient. MNCs (microglia and infiltrating MNCs) were recovered from the 37/70 interface, washed, and resuspended in RPMI 1640 with 10% FCS. After blocking FcRs with anti-CD16/CD32, 1 x 106 pooled cells were incubated with Abs to murine CD4, CD8, CD11b, CD11c, and CD45 (all from BD PharMingen, San Jose, CA). MNCs were gated and fluorescence was analyzed directly using CellQuest software (BD Biosciences, Mountain View, CA). Data represent 10,000 events.

Proliferation and production of cytokines and NO

Suspensions of MNCs from the spleen were prepared on day 21. Cells were cultured at a cell density of 2.5 x 106/ml in medium containing MOG35–55 at the final concentration of 1, 10, and 25 µg/ml, Con A at 5 µg/ml, or without Ag/mitogen. Supernatants were collected after 48 h. Quantitative ELISAs for IFN-{gamma}, IL-2, GM-CSF, IL-4, IL-5, and IL-10 were performed using paired mAbs according to the manufacturer’s recommendation (BD PharMingen). For proliferation, cells were cultured in triplicate with various doses of MOG35–55, 5 µg/ml Con A, or without Ag/mitogen. After 60 h of incubation, the cells were pulsed for 12 h with 1 µCi [3H]methylthymidine. Cells were harvested and counts read using a beta counter. The results were expressed as stimulation index, which was calculated by dividing the cpm from culture in the presence of Ag or mitogen by the cpm from culture without Ag/mitogen. Because secreted NO quickly decays with oxygen-yielding nitrite, the level of nitrite as a reflection of NO production in culture supernatants was measured using the Griess reagent.

Purification of CD4+ T cells and APCs

MNCs from spleen of MOG-immunized IL-12R{beta}1-/- and wild-type mice were harvested at day 10 p.i. and pooled in each group. Erythrocytes in the cell pellets were hemolyzed by adding NH4Cl-Tris buffer for 5 min at room temperature followed by washing. CD4+ T cells from spleen of IL-12R{beta}1-/- and wild-type mice were purified by positive selection with MACS magnetic cell sorting. Briefly, splenocytes were centrifuged over a cushion of lympholyte M (Cedarlane Laboratories, Hornby, Ontario, Canada). After blocking with CD16/CD32, cells were incubated with anti-CD4 microbeads (Miltenyi Biotec, Auburn, CA) for 30 min on ice, washed, and passed through a Type AS column following the manufacturer’s protocol (Miltenyi Biotec). The non-CD4 fraction was collected, centrifuged, and CD8+ T cells were depleted with a monoclonal anti-CD8 Ab (clone 3.155) and complement (Cedarlane Laboratories). These CD4-, CD8-depleted cells serve as APCs in the coculture system. The purity of CD4+ T cells and APCs was confirmed by FACScan. On average, this preparation yielded 97.6% CD4+ of the viable cells in the positive selection fraction and 98% of CD4-, CD8- cells in the APC fraction. This fraction was composed of macrophages (CD11b+; 57%), B cells (CD19+; 32%), and dendritic cells (DCs) (CD11c+; 15%).

Coculture system for intracellular cytokine staining

Intracellular staining of cytokines was conducted using Cytofix/Cytoperm kits (BD PharMingen) following the manufacturer’s recommendations. Briefly, purified CD4+ T cells from IL-12R{beta}1-/- or wild-type mice were mixed with purified APCs from these mice, respectively, at a ratio of 1:1. These cells were cultured at a cell density of 2.0 x 106/ml in RPMI 1640 and 10% FCS containing MOG35–55 (25 µg/ml) for 20 h. GolgiStop, a protein transport inhibitor containing brefeldin A, was added during the last 4 h of culture. Following incubation, the cells were harvested and washed. After blocking with Fc Block Abs, 1 x 106 cells/tube were incubated with Abs to murine CD11b (macrophage) and CD4 as cell surface markers. We chose macrophages as a representative of APCs because these cells play an important role in the induction of EAE (33, 34) and, in the present study, comprised the majority of APCs in the periphery (see Purification of CD4+ T cells and APCs) and in the CNS (see Fig. 2). Further, macrophages are the main producers of TNF-{alpha}, a proinflammatory cytokine regulated by IL-12. Cells were thoroughly resuspended and fixed in Cytofix/Cytoperm solution for 20 min. Cells were permeabilized with 1x Perm/Wash solution and stained for intracellular cytokines using PE-conjugated anti-mouse cytokine mAbs (BD PharMingen; IFN-{gamma} clone XMG1.2, rat IgG2a, TNF-{alpha}, clone MP6-XT22, IL-4 clone 11B11, and IL-10 clone JES5-16E3, all rat IgG1). PE-conjugated isotype Abs (rat IgG1 and IgG2a) were used in parallel as negative controls. All steps for incubation and centrifugation were at 4°C. After staining, the samples were immediately analyzed by FACS. All data were acquired on a FACSCalibur (BD Biosciences) and data were analyzed using CellQuest software (BD Biosciences). Data represent 10,000 events.



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  		FIGURE 2. Altered pattern of CNS cell infiltrates in IL-12R{beta}1-/- mice. MNCs from spinal cord cells (n = 5 in each group) were isolated from IL-12R{beta}1-/- and IL-12R{beta}1+/+ littermate controls at week 3 p.i. The percentages and absolute numbers of infiltrating CD4+ and CD8+ T cells were increased in wild-type controls (A) compared with IL-12R{beta}1-/- mice (B). In contrast to IL-12R{beta}1+/+ mice that developed severe EAE with numerous CNS CD11b+CD45high macrophages/activated microglia (C), the majority of cells isolated from the CNS of IL-12R{beta}1-/- mice were CD11b+CD45low resident microglia (D) (summarized in Table I). CD11c+ cells (DCs) in IL-12R{beta}1+/+ mice (E) were composed of both CD11b- and CD11b+ populations. Few CD11c+ cells were found in IL-12R{beta}1-/- mice (F). The differences between the two groups for CD4+, CD8+, CD11b+, and CD11c+ cells were all significant (p < 0.01 or <0.001). One representative experiment of three is shown.

 
mRNA expression of IL-12/IL-23 subunits in APCs by quantitative real-time PCR

Quantitative real-time PCR was performed as described (35) to determine mRNA expression of IL-12p40, p35, and IL-23p19. Briefly, total RNA was extracted from purified APCs (1 x 107) using TRIzol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The purity and integrity of RNA were determined by absorbance at A260/280 and gel electrophoresis. Contamination by genomic DNA was excluded by real-time PCR amplification of RNA by {beta}-actin primers. One-step RT-PCR was performed in 20 µl of Hot Start Reagent Mix (LightCycler-RNA Master SYBR Green I) according to the manufacturer’s instructions (Roche Diagnostic Systems) containing 1 µl (250 ng) of RNA template and monitored in real time with the fluorescent DNA binding dye SYBR green (Roche Diagnostic Systems, Somerville, NJ). The primer pairs used were shown, see Table I. Standard curves for these molecules were generated by performing 10-fold dilutions of known amounts of cytokine RNA (LightCycler Control Kit RNA; Roche Diagnostic Systems). The mRNA level for each sample was normalized by dividing the calculated value by the housekeeping {beta}-actin value. mRNA levels in the mutant mice were then quantified in comparison to the respective levels in wild-type mice using the relative standard curve method (Roche Diagnostic Systems).


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  		Table I. The sequence of primers for real-time PCR

 
FACS analysis of IL-18R{alpha} expression and IL-18 ELISA

Analysis of IL-18R{alpha} expression was performed as previously described (36). Briefly, isolated splenocytes from immunized mice were first cultured with MOG35–55 (10 µg/ml) for 48 h. After extensive washing, cells were incubated for 10 min with 1 µg of rat anti-mouse CD16/32 (Fc block; BD PharMingen) to block nonspecific binding of goat Ig/anti-IL-18R{alpha} to FcRs. Cells were then incubated for 30 min with either 1 µg of biotinylated goat IgG or 1 µg of biotinylated goat anti-mouse IL-18R{alpha} (R&D Systems, Minneapolis, MN) diluted in PBS-BSA, and then resuspended in streptavidin-PerCP and anti-mouse CD4-allophycocyanin. Data were acquired and analyzed as above. The concentration of IL-18 in the supernatants after 48 h of culture was determined using a sandwich ELISA kit according to the manufacturer’s recommendation (BD PharMingen).

Statistics

ANOVA was used for the comparison of clinical score, proliferative responses, and cytokine profiles between different groups. Flow cytometry data were analyzed using K-S statistics. All significance tests were two-sided.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IL-12R{beta}1-/- mice are completely resistant to clinical EAE

To study the role of IL-12R{beta}1 in the development of EAE, we immunized IL-12R{beta}1-deficient B6.129 mice (IL-12R{beta}1-/-) and their wild-type controls (IL-12R{beta}1+/+) with MOG35–55 in CFA, and scored for signs of disease. EAE developed in all wild-type mice, starting 12.5 ± 1.5 days p.i. in female mice, with an average maximal clinical score of 3.1 ± 0.9, and a progressive course (Fig. 1A). In male mice, EAE started 13.8 ± 0.7 days p.i., with an average maximal clinical score of 2.5 ± 0.2, and a monophasic course (Fig. 1B). The total incidence of EAE for wild-type mice was 100% (20/20). In contrast, neither female nor male IL-12R{beta}1-/- mice developed any signs of EAE during the entire period of observation (total incidence: 0/20; p < 0.001).



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  		FIGURE 1. IL-12R{beta}1-/- mice are resistant to the induction of EAE. Female wild-type and IL-12R{beta}1-/- mice (A; n = 5 in each group) were immunized with 300 µg of MOG35–55 peptide in CFA. Clinical EAE was scored daily by two blinded observers. Data were expressed as the mean clinical score ± SEM. Experiments were repeated in male mice (B; n = 6 in each group), and similar results were obtained. Overall, the incidence of EAE in wild-type mice was 100 vs 0% in IL-12R{beta}1-/- mice (n = 20 in each group). The overall clinical score is significantly different (p < 0.001) between two groups. At the end of the experiment, spinal cords were harvested after extensive perfusion, and 5-µm sections were stained with H&E (C, wild type; D, IL-12R{beta}1-/-) and Luxol fast blue (E, wild type; F, IL-12R{beta}1-/-). Original magnifications, x 40. The difference between two groups was significant (p < 0.001). Similar results were obtained from repeated experiments (total n = 20 in each group).

 
At histological evaluation, multiple inflammatory foci were observed in the white matter of the spinal cord and the infiltration score was 2.2 ± 0.5 in wild-type mice (exemplified in Fig. 1C) with extensive demyelination (Fig. 1D). In contrast, few inflammatory cell (exemplified in Fig. 1E) and no demyelination were detected in IL-12R{beta}1-/- mice (Fig. 1F; p < 0.001 compared with wild-type mice). Our observations indicate that IL-12R{beta}1 is crucial for the development of EAE.

Diminished recruitment/accumulation of T cells and monocytes in the CNS of IL-12R{beta}1-/- mice

To determine the pattern of infiltrating cell composition, we performed flow cytometric analysis on MNCs from the CNS at the peak of disease (week 3). In IL-12R{beta}1+/+ mice, the number of MNCs recovered from each spinal cord was approximately five times (5.7 x 104) higher than in IL-12R{beta}1-/- mice (1.1 x 104 per cord, similar to the number obtained from naive mice). The inflammatory cells were composed of CD4+ cells, CD8+ cells (Fig. 2A), infiltrating macrophages and activated microglia (CD11b+ cells; Fig. 2C) and DCs (CD11c+; Fig. 2E). Few CD4+, CD8+ T cells, or DCs were found in IL-12R{beta}1-/- mice (Fig. 2, B and F). Differential CD45 staining intensity coupled with CD11b staining was used to distinguish between infiltrating macrophages and resident microglia (37, 38). Shown in Fig. 2E and Table II are numerous CD45highCD11b+ (corresponding to infiltrating macrophages) and CD45intCD11b+ (activated microglia) isolated from IL-12R{beta}1+/+ mice. However, almost all the CD11b+ cells from the spinal cords of IL-12R{beta}1-/- mice were CD45low (Fig. 2F), corresponding to resting resident microglia, similar to those from naive mice (data not shown).


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  		Table II. CD11b/CD45 expression in the CNS in IL-12R{beta}1+/+ and IL-12R{beta}1-/- micea

 
Peripheral Ag recall immune responses

When mice were sacrificed at week 3, lymphocyte proliferation to MOG35–55 was reduced in IL-12R{beta}1-/- compared with wild-type mice (Fig. 3). When stimulated with MOG35–55, lymphocytes from IL-12R{beta}1-/- mice produced significantly lower levels of IFN-{gamma}, IL-2, GM-CSF, and NO than wild-type mice (Fig. 3). In contrast, IL-12R{beta}1-/- mice produced significantly higher levels of the Th2 cytokines IL-4, IL-5, and IL-10 than IL-12R{beta}1+/+ mice when cells were stimulated with MOG35–55. Lymphocytes from IL-12R{beta}1-/- mice also produced high levels of Th2 cytokines when stimulated with the polyclonal mitogen Con A (Fig. 3). These data indicate that lymphocytes in IL-12R{beta}1-/- mice exhibit a Th2 bias. At week 6, all Th responses to MOG35–55 were decreased, compared with those detected at week 3, indicating a decreased immune response at this time point (data not shown).



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  		FIGURE 3. MOG35–55-induced proliferative responses, cytokine, and NO production. Spleen cells (4 x 105) from IL-12R{beta}1-/- mice and IL-12R{beta}1+/+ control mice (n = 5 in each group) were cultured with MOG35–55 at 10 µg/ml, Con A at 5 µg/ml, or without Ag/mitogen. Proliferative response results were expressed as stimulation index, which was calculated by dividing the cpm from culture in the presence of Ag or mitogen by the cpm from culture without Ag/mitogen. Production of cytokines was determined from 48-h culture supernatants by sandwich ELISA. Columns indicate mean values, and bars SD. Values of p refer to comparisons between wild-type and IL-12R{beta}1-/- mice. **, p < 0.01; ***, p < 0.001. One representative experiment of three is shown.

 
Inhibition of MOG-induced Th1 responses by APCs from IL-12R{beta}1-/- mice

To dissect the role of CD4+ T cells and APCs of IL-12R{beta}1-/- mice in EAE, we purified these cells from MOG-immunized IL-12R{beta}1-/- mice and wild-type mice 10 days p.i., and used these cells in coculture studies. For Th1 response, cells producing IFN-{gamma} and TNF-{alpha} were determined by intracellular staining in both CD4+ T cells and APCs. The main findings for IFN-{gamma} (summarized in Fig. 4) are as follows. In panel 1, coculture of wild-type APCs and wild-type CD4+ T cells had high numbers of cells producing Th1 cytokines IFN-{gamma} (9.2% in CD4+ T cells and 6.0% in macrophages). When IL-12R{beta}1-/- CD4 T cells were cultured with wild-type APCs (panel 2), both the numbers of IFN-{gamma} producing CD4+ T cells and APCs were significantly reduced compared with those found in cocultures of wild-type APCs and CD4+ T cells (panel 2 compared with panel 1; all p < 0.001), indicating that a deficit in the T cell response to IL-12/IL-23 also leads to reduced activation of APCs. Importantly, when IL-12R{beta}1-/- APCs were cultured with CD4+ T cells from wild-type mice, which develop severe EAE, these encephalitogenic CD4+ T cells produced dramatically lower levels of IFN-{gamma}, compared with the number of IFN-{gamma}-producing cells when wild-type T cells were cocultured with wild-type APCs (0.8 vs 9.2%; p < 0.001; Fig. 4, panel 3 compared with panel 1).



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  		FIGURE 4. Intracellular IFN-{gamma} in CD4/APC coculture system. CD4+ T cells from spleen of MOG-immunized IL-12R{beta}1-/- and wild-type mice (n = 5 in each group) were purified by positive selection using microbeads at day 10 p.i. APCs were obtained from the non-CD4 fraction and then depleted CD8+ cells with Abs and complement. Purified CD4+ T cells and APCs were cocultured in the presence of MOG35–55. Intracellular production of IFN-{gamma} was analyzed by flow cytometry. Numbers refer to the percentage of cytokine-producing cells among CD4+ T cells or CD11b+ cells (macrophages; representing APCs). One representative experiment of two is shown.

 
The results for TNF-{alpha}, another Th1 cytokine involved in EAE pathogenesis, were consistent with the IFN-{gamma} findings. Two important findings are shown in Fig. 5: 1) when IL-12R{beta}1-/- CD4 T cells were cultured with wild-type APCs (panel 2), the number of both TNF-{alpha} producing CD4+ T cells and TNF-{alpha} producing APCs were significantly reduced (panel 2 compared with panel 1, all p < 0.001), suggesting a modulating effect of T cells derived from IL-12R{beta}1-/- mice on APCs; 2) importantly, when IL-12R{beta}1-/- APCs were cultured with wild-type CD4+ T cells, these encephalitogenic CD4+ T cells produced dramatically lower levels of TNF-{alpha} than when wild-type T cells were cocultured with wild-type APCs (0.9 vs 2.6%; p < 0.001; Fig. 5, panel 3 compared with panel 1).



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  		FIGURE 5. Intracellular staining for TNF-{alpha}. Purified CD4+ T cells and APCs from spleen of MOG-immunized IL-12R{beta}1-/- and wild-type mice (n = 5 in each group) were prepared and cultured as described in Fig. 4. Intracellular production of TNF-{alpha} was analyzed by flow cytometry. Numbers refer to the percentage of cytokine-producing cells among CD4+ T cells or CD11b+ cells. One representative experiment of two is shown.

 
APCs from IL-12R{beta}1-/- mice drive a Th2 differentiation

To further investigate the modulator effect of APCs in Th response, we determined the numbers of CD4+ T cells and macrophages producing the Th2 cytokines IL-4 and IL-10 in the coculture system. The main findings are shown in Figs. 6 and 7. When wild-type APCs were cultured with wild-type CD4+ T cells, low numbers of these CD4+ T cells produced IL-4 (0.8%) and IL-10 (0.6%) (panel 1). When cocultured with wild-type APCs, few CD4+ T cells from IL-12R{beta}1-/- mice produced IL-4 (0.7%) or IL-10 (1.0%) (panel 2). However, IL-12R{beta}1-/- APCs induced significantly higher numbers of IL-4- and IL-10-producing CD4+ T cells (p < 0.001 when compared with cultures where using wild-type APCs) (panels 3 and 4). The highest numbers of IL-4- and IL-10-producing cells were found in wild-type CD4+ T cells when cultured with IL-12R{beta}1-/- APCs (2.7% for IL-4 and 11.2% for IL-10) (panel 3). IL-10-producing wild-type CD4+ T cells, in turn, help IL-12R{beta}1-/- APCs produce significantly more IL-10 than when these APCs were cultured with IL-12R{beta}1-/- CD4+ T cells (Fig. 7, panel 3 compared with panel 4). These data indicate that 1) the T cell phenotype was plastic, based upon the influence of APCs; 2) the function of APCs can also be modulated by T cells; and 3) it is the APCs in IL-12R{beta}1-/- mice that play a predominant role in driving the differentiation of Ag-specific Th2 cells.



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  		FIGURE 6. Intracellular staining for IL-4. Purified CD4+ T cells and APCs from spleen of MOG-immunized IL-12R{beta}1-/- and wild-type mice (n = 5 in each group) were prepared and cultured as described in Fig. 4. Intracellular production of IL-4 was analyzed by flow cytometry. Numbers refer to the percentage of cytokine-producing cells among CD4+ T cells. One representative experiment of two is shown.

 


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  		FIGURE 7. Intracellular staining for IL-10. Purified CD4+ T cells and APCs from spleen of MOG-immunized IL-12R{beta}1-/- and wild-type mice (n = 5 in each group) were prepared and cultured as described in Fig. 4. Intracellular production of IL-10 was analyzed by flow cytometry. Numbers refer to the percentage of cytokine-producing cells among CD4+ T cells or CD11b+ cells. One representative experiment of two is shown.

 
IL-12p35, p40, and IL-23p19 mRNA expression in APCs of IL-12R{beta}1-/- mice

To find out the potential effect of IL-12R{beta}1 deficiency on IL-12/IL-23 expression by APCs, we analyzed mRNA expression of IL-12/IL-23 subunits p35, p40, and p19 directly in the purified APC populations of MOG35–55-immunized wild-type and IL-12R{beta}1-/- mice by quantitative real-time PCR. As shown in Fig. 8, APCs of wild-type mice expressed substantial amounts of p40, p35, and p19 mRNA, whereas these cytokine subunits were significantly decreased in IL-12R{beta}1-/- mice (p < 0.01 for p40, p < 0.001 for p35 and p19, respectively). These results indicate an autocrine pathway of IL-12/IL-23 production via IL-12R{beta}1, and provide a further mechanism for the development of APC2 in the absence of IL-12/IL-23 responsiveness.



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  		FIGURE 8. IL-12p40, p35, and IL-23p19 mRNA expression in APCs by quantitative real-time PCR. APCs from spleen of MOG-immunized IL-12R{beta}1-/- and wild-type mice (n = 5 in each group) were prepared and cultured as described in Fig. 4. mRNA expression of IL-12p40, p35, and IL-23p19 was determined as described in Materials and Methods. **, p < 0.01; ***, p < 0.001. One representative experiment of three is shown.

 
Decreased expression of IL-18R{alpha} and production of IL-18 in IL-12R{beta}1-/- mice

To study the status of the IL-18/IL-18R system in the absence of IL-12/IL-23 responsiveness in IL-12R{beta}1-/- mice, we determined IL-18 production by splenocytes and IL-18R{alpha} expression on CD4+ T cells. Production of IL-18 was not detectable in IL-12R{beta}1-/- mice but high levels were produced in wild-type mice (Fig. 9A; p < 0.001). Further, we found a 2-fold increased number of CD4+ T cells expressing IL-18R{alpha} on wild-type mice (19.2%) compared with IL-12R{beta}1-/- mice (9.2%; p < 0.001; Fig. 9B).



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  		FIGURE 9. IL-18R{alpha} expression and IL-18 production. Female IL-12R{beta}1-/- and wild-type mice (n = 5 in each group) were immunized with 300 µg of MOG35–55 peptide as in Fig. 1. Mice were sacrificed at week 3 p.i. Production of IL-18 (A) was determined with sandwich ELISA. Columns indicate mean values, and bars SD. **, p < 0.01; ***, p < 0.001. Splenocytes were stained with anti-CD4, and anti-IL-18R{alpha} Abs (B). Data presented are percentages of positive cells calculated using isotype-matched Abs as controls. The difference between two groups was significant (p < 0.001). One representative experiment of three is shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The goal of our present study was to characterize the role of IL-12R{beta}1 in the development of EAE. This is of particular interest since the recent demonstration that mice deficient in IL-12 are susceptible to EAE (19, 20), whereas mice deficient in IL-23 are resistant (21). Importantly, we have recently found increased susceptibility to the induction of EAE in IL-12R{beta}2-/- mice (24), indicating that selective block of IL-12 signaling does not impair, but indeed augments, pathogenic mechanisms in autoimmune demyelination. To better understand the role of the IL-12R system in EAE we extended our study to mice lacking IL-12R{beta}1. We report here that, unlike IL-12R{beta}2-/- mice, IL-12R{beta}1-/- mice were completely resistant to clinical and pathological EAE. This is associated with a Th2 bias and decreased IL-18/IL-18R interaction. APCs from IL-12R{beta}1-/- mice express decreased levels of IL-12/IL-23 subunits p40, p35, and p19, indicating an autocrine pathway for IL-12/23 production. Further, we provide evidence that IL-12R{beta}1-/- APCs may play a prominent role in disease resistance as APC2 by driving an Ag-specific Th2 shift.

The degree of CNS inflammation and demyelination correlated with the clinical phenotype in wild-type and IL-12R{beta}1-/- mice. Inflammatory cells in IL-12R{beta}1+/+ mice were mainly comprised of CD4+ T cells, CD8+ T cells, activated microglia (CD11b+CD45int), and infiltrating macrophages (CD11b+CD45high). Infiltrating monocytes/macrophages have been reported to function as effectors and amplifiers of CNS inflammation (39, 40). In contrast, the pathological features and cell numbers from the CNS of immunized IL-12R{beta}1-/- mice were similar to those of nonimmunized naive mice. Only few CD4 or CD8+ T cells were found in these mice. Almost all the CD11b+ cells from the spinal cords of IL-12R{beta}1-/- mice were CD45low, corresponding to resident microglia, similar to those from naive mice (data not shown). Of note is that a significant fraction of CD11b+ cells in the CNS of wild-type mice had a DC-like phenotype (CD11c+). The paucity of DCs in the CNS of IL-12R{beta}1-/- mice compared with IL-12R{beta}1+/+ mice indicates that IL-12/IL-23 pathways play a crucial role in DC infiltration from the periphery (CD11b+CD11c+ or CD11b-CD11c+) or differentiation from resident microglia (CD11b+CD11c+) (41, 42). In summary, deficiency of IL-12R{beta}1 reduced the infiltration of peripheral T cells, macrophages, and DCs to the CNS, the activation of resident microglia, and the differentiation of DC in the CNS, resulting in resistance to EAE.

Although the role of IL-12 in the regulation of T cell responses has been well-identified, the role of IL-12R subunits is more complex. In the mouse, IL-12R{beta}1 is required for high affinity IL-12 binding and IL-12R{beta}2 for IL-12 responsiveness (4). IL-12R{beta}2 contains tyrosine residues in its cytoplasmic domain and is the signaling component of the IL-12R (4), while IL-12R{beta}1 is an essential component of the functional, high affinity IL-12R in humans and mice (43). In the current study, we show that lymphocytes from IL-12R{beta}1-sufficient mice proliferate more vigorously to MOG35–55 than those from IL-12R{beta}1-/- mice. MNCs from IL-12R{beta}1-/- mice produced a significantly lower level of MOG-induced IL-2 and undetectable IFN-{gamma} and GM-CSF, a potent inducer of the differentiation of parenchymal microglia to CNS DCs (42) (see also Fig. 3E). Although the role of IFN-{gamma} and NO in the induction of EAE is controversial (44, 45, 46), our data indicate that IFN-{gamma} and NO production correlate positively with the susceptibility to EAE in wild-type and IL-12R{beta}1-/- mice. Furthermore, significantly increased levels of IL-4, IL-5, and IL-10 upon stimulation with MOG35–55 were found in IL-12R{beta}1-/- mice compared with wild-type mice (p < 0.001). These results, together with the observation that IL-12R{beta}2-/- mice (responsive to IL-23, but not to IL-12) produced high levels of proinflammatory cytokines (47), indicate that blocking IL-23, but not IL-12, is required to skew immune response toward a Th2 phenotype.

APCs have been suggested to play an important role in Th differentiation (25, 26, 27, 28). It is possible that APCs lacking IL-12/IL-23 responsiveness will be APC2 that drive a Th2 differentiation of CD4+ T cells (27, 29). To dissect the role of CD4+ T cells and APCs of IL-12R{beta}1-/- mice in EAE, we purified these cells from MOG-immunized IL-12R{beta}1-/- mice and wild-type mice, and studied the Th1/Th2 differentiation of CD4+ T cells in a coculture system. The influence of CD4+ T cells on APCs was analyzed in parallel. The main finding in this system is that wild-type APCs were able to drive both wild-type and IL-12R{beta}1-/- CD4+ T cells to produce IFN-{gamma} and TNF-{alpha}, whereas IL-12R{beta}1-/- APCs diminished the ability of CD4+ T cells to produce Th1 cytokines but strongly drove these cells to produce IL-4 and IL-10. These results indicate that it is APCs that play a prominent role in Th differentiation. Further, the finding that IL-12R{beta}1-/- CD4+ T cells inhibited the production of IFN-{gamma} and TNF-{alpha} by wild-type APCs suggests an immunoregulatory effect of IL-12R{beta}1-/- T cells on wild-type APCs.

Of note is that a significant fraction of APCs from IL-12R{beta}1-sufficient mice produce IFN-{gamma}, while this capacity is dramatically decreased in IL-12R{beta}1-/- APCs. Previous research has focused on the production of IFN-{gamma} by T cells and NK cells, but recent data has significantly broadened the spectrum of IFN-{gamma}-producing cells, including APCs (macrophages, DCs, and B cells) (for review, see Ref.48). Technological advances including intracellular staining and genetic manipulation have made these observations convincing (48, 49, 50, 51, 52). A potential role of IL-23 in the production of IFN-{gamma} by APCs has also been suggested (48). Our study provides evidence that the IL-12/IL-23 pathway via IL-12R{beta}1 is essential for the production of IFN-{gamma} by macrophages in EAE.

It is not clear why the highest numbers of IL-4- and IL-10-producing cells were found in wild-type CD4+ T cells cocultured with IL-12R{beta}1-/- APCs. One possible explanation is that, because these cells were harvested at an early stage after immunization (day 10 p.i.), a high percentage of activated MOG-reactive CD4+ T cells may have a Th0 phenotype, which is still susceptible to Th1 or Th2 skewing depending on the type of APCs (27, 29). Moreover, when IL-12R{beta}1-/- APCs drove wild-type CD4+ T cells to produce high levels of Th2 cytokines, these Th2 type CD4 T cells, in turn, help IL-12R{beta}1-/- APCs to produce higher levels of IL-10. Taken together, our data indicate that 1) the T cell phenotype at an early stage of activation (e.g., day 10 p.i.) was plastic; 2) APCs from IL-12R{beta}1-/- mice play a prominent role in driving the Ag-specific Th2 shift, even strong enough to overcome the already polarized Th1 phenotype; and 3) CD4+ T cells are able to determine the cytokine profile produced by APCs.

To understand the mechanism underlying the development of APC2 in IL-12R{beta}1-/- mice, we studied the influence of IL-12R{beta}1 deficiency on IL-12 and IL-23 mRNA expression in APCs. We and others have found both functional IL-12R{beta}1 and {beta}2 on APCs in the periphery (53) and on microglia in the CNS (54, 55, 56). A functional characterization of IL-23R complex in murine DCs and macrophages has also been provided (25, 56). IL-12 and IL-23, thus, possess the ability to promote the Th1 costimulatory function of APCs. Indeed, we found that IL-12p40, p35, and IL-23p19 mRNA expression was significantly decreased on APCs of IL-12R{beta}1-deficient mice, implying an autocrine pathway of IL-12 and IL-23 production via IL-12R{beta}1. The decreased production of IL-12/IL-23, in turn, would affect both T cells and APC activation, resulting in the development of APC2 and Th2 phenotypes (25). IL-10 has been reported to antagonize disease-promoting effects of IL-12 in autoimmune diseases. In turn, endogenous production of IL-12 suppresses IL-10 production, while anti-IL-12 treatment up-regulates the production of IL-10 (18). Therefore, manipulation of the IL-12/IL-10 immunoregulatory circuit could have a decisive effect on the incidence of autoimmune diseases (18). Our finding that IL-12R{beta}1-/- APCs produce high levels of IL-10 supports the significance of this IL-12/IL-10 immunoregulatory circuit. In addition, our data indicate that the lack of IL-12R{beta}1 interrupts an autocrine pathway for IL-12 and IL-23 production by APCs. These data also provide a molecular mechanism underlying the development of APC2 and a Th2 bias in absence of IL-12R{beta}1.

One interesting question is why IL-18, a Th1-driving cytokine, did not compensate for the lack of IL-12/IL-23 responsiveness. IL-18 is critical in the induction of IFN-{gamma}, GM-CSF, TNF-{alpha}, and IL-1 (57) and is involved in the development of EAE (58, 59). IL-18 also synergizes with IL-12 and IL-23 in the induction of Th1 responses (15, 60). In the present study, we found significantly lower expression of the IL-18R{alpha} on CD4+ cells and lower MOG-induced IL-18 production in MNCs of IL-12R{beta}1-/- mice as compared with wild-type mice. These data suggest that both IL-18 production and responsiveness were reduced in the absence of IL-12R{beta}1, and suggest a role for IL-12R{beta}1 in the activation of the IL-18/IL-18R system.

In summary, we have found that the absence of IL-12R{beta}1 not only blocks Ag-specific Th1 cells but also promotes the development of Th2 cells. It is APCs that play a key role in this process. These findings may be important for understanding basic mechanisms of cell-mediated autoimmunity, and may aid in designing novel therapeutic strategies for the treatment of autoimmune diseases such as multiple sclerosis.


   Acknowledgments
 
We thank Dr. Youhai Chen for critical discussions, and Katherine Regan for excellent secretarial assistance.


   Footnotes
 
1 This study was supported by grants from the National Institutes of Health and the National Multiple Sclerosis Society. Back

2 Address correspondence and reprint requests to Dr. Abdolmohamad Rostami, Department of Neurology, Thomas Jefferson University, 1025 Walnut Street, Suite 310, Philadelphia, PA 19107-5083. E-mail address: a.m.rostami{at}jefferson.edu Back

3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; MNC, mononuclear cell; MOG, myelin oligodendrocyte glycoprotein; p.i., postimmunization; DC, dendritic cell; int, intermediate. Back

Received for publication April 1, 2003. Accepted for publication August 20, 2003.


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 Materials and Methods
 Results
 Discussion
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