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The Role of IL-12 in the Induction of Intravenous Tolerance in Experimental Autoimmune Encephalomyelitis¹

Department of Neurology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Intravenous administration of autoantigen is an effective method to induce Ag-specific tolerance against experimental autoimmune encephalomyelitis (EAE). IL-12 is a potent Th1 stimulator and an essential cytokine in the induction of EAE. The role of IL-12 in the induction of i.v. tolerance is not clear. In this study, we induced tolerance by i.v. administering myelin basic protein (MBP) peptide Ac1–11 (MBP1–11) in EAE. We observed significant suppression of IL-12 production by the lymph node cells of MBP1–11-injected mice. To see whether the low level of IL-12 is the cause or effect of tolerance, we administered IL-12 to the EAE mice at the time of i.v. MBP1–11 injection. Exogenous IL-12 abrogated the suppression of clinical and pathological EAE by i.v. tolerance. IL-12 blocked the suppressive effect of i.v. tolerance on the proliferative response to MBP1–11 and MBP1–11-induced production of IL-12 and IFN-. Furthermore, IL-12 completely blocked the i.v. tolerance-induced type 1 T regulatory cell response. These data suggest that i.v. administration of autoantigen results in the suppression of endogenous IL-12 and the consequent switching of the immune response from an immunogenic to a tolerogenic form.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Immune tolerance is a process that eliminates or neutralizes autoreactive cells. Breakdown of this system can give rise to autoimmune disease (1). Experimental autoimmune encephalomyelitis (EAE),³ an animal model of multiple sclerosis, is a T cell-mediated autoimmune disease of the CNS. Autoreactive T cells directed against myelin Ags, including myelin basic protein (MBP), produce proinflammatory cytokines and promote cell-mediated immunity. These T cells produce high levels of IFN-, TNF-, and TNF- (2, 3). Conversely, immunoregulatory cytokines (e.g., IL-10) may be protective (4, 5).

Induction of i.v. tolerance with specific autoantigens has been accomplished by administration of the Ag in a variety of tolerogenic forms, including soluble protein/peptide and Ag-coupled splenocytes (6). Different mechanisms are involved in the induction of i.v. tolerance, including selective suppression of Ag-specific Th1 cytokines and induction of regulatory cytokines (7, 8). We have shown that when soluble Ag is administered after disease onset in EAE, clonal deletion of Ag-reactive T cells by apoptosis is a major mechanism for the reversal of EAE (9). In contrast, when soluble Ag is administered before or immediately after immunization for the EAE induction, an Ag-specific regulatory mechanism, but not deletion, prevails and appears to be the main mechanism for tolerance induction (10).

IL-12 is a heterodimeric cytokine, composed of an H chain or p40 and an L chain or p35. IL-12 is a potent, and obligatory, inducer of Th1 differentiation (11, 12). IL-12 regulates the growth and function of T cells and especially promotes the development of Th1 cells by stimulating the production of IFN- (11, 12). There is increasing evidence that implicates IL-12 in the pathogenesis of multiple sclerosis and EAE (13, 14, 15, 16, 17). Because i.v. tolerance against EAE preferentially involves suppression of Th1 response (6, 18) and IL-12 is important in Th1 differentiation and maintenance, we hypothesized that regulation of IL-12 was involved in the induction of i.v. tolerance in EAE. Our data show that i.v. administration of MBP peptide Ac1–11 (MBP1–11) significantly suppresses clinical EAE, as well as IFN- and IL-12 production. The role of IL-12 in the induction of tolerance was confirmed by in vivo administration of IL-12 to MBP1–11 i.v. injected mice, resulting in abrogation of tolerance. The effect of IL-12 on a suggested IL-12/IL-10 circuit controlling the regulation of autoimmunity/tolerance was also determined.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice, Ag, and reagents

Female (SJL/J x PL/J)F₁ mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All mice were housed in University of Pennsylvania Medical School (Philadelphia, PA) animal care facilities. MBP peptide Ac1–11 (AcASQKRPSQRHG) was synthesized at the Protein Chemistry Laboratory of the University of Pennsylvania. Bordetella pertussis toxin was purchased from List Biological Laboratories (Campbell, CA). Recombinant murine (rm)IL-12 was a generous gift of the Genetics Institute (Cambridge, MA).

Induction of EAE and i.v. tolerance

Sixteen mice at 8–10 wk of age were each injected s.c. with 400 µg of MBP1–11 in CFA containing 4 mg/ml Mycobacterium tuberculosis H37Ra (Difco, Detroit, MI) over two sites at the back. A total of 500 ng of pertussis toxin was given i.p. on days 0 and 2 postimmunization (p.i.). To induce tolerance, MBP1–11 at the dose of 200 µg/mouse was i.v. injected in eight mice from day 0 p.i. and then every 3 days for five times (MBP1–11-i.v. mice). Same volumes (200 µl) of PBS were i.v. injected in eight mice in parallel as control (PBS-i.v. mice). EAE was scored as follows (19): level 1, limp tail; level 2, partial hind leg paralysis; level 3, total hind leg or partial hind and front leg paralysis; level 4, total hind leg and partial front leg paralysis; level 5, moribund or dead. Mice were examined daily for signs of EAE in a blind fashion and sacrificed on day 28 p.i. All work was performed in accordance with the University of Pennsylvania guidelines for animal use and care.

Administration of rmIL-12

To determine the role of IL-12 in the induction of i.v. tolerance, we repeated the i.v. tolerance induction and administered rmIL-12 to the MBP1–11-i.v. mice. Briefly, 12 mice were immunized with MBP1–11 plus CFA to induce EAE as above, and then randomly divided into three groups. At the time points when MBP1–11 was i.v. injected, four MBP1–11-i.v. mice were injected i.p. with 100 ng of rmIL-12 in PBS with 1% mouse serum (MBP1–11-i.v. + IL-12-i.p. mice), four MBP1–11-injected mice were injected with 1% mouse serum in PBS (MBP1–11-i.v. + PBS-i.p. mice), and four mice were injected i.v. with PBS and i.p. with 1% mouse serum in PBS (PBS-i.v. + PBS-i.p. mice). Clinical scores were monitored as described above.

Histopathological assessment of EAE

Mice (n = 4 in each group) were perfused through the left ventricle with 100 ml of physiological saline containing 2 U/ml heparin, followed by 50 ml of 10% buffered formalin phosphate (Fisher Chemicals, Fair Lawn, NJ). Spinal cords were removed and fixed in the same fixative for 48 h, after which the tissue was removed and stored in PBS. Tissue was dehydrated in a graded ethanol series, infiltrated with toluene, embedded in paraffin, sectioned (6 µm) and stained with H&E, counterstained with cresyl violet, and scored for infiltrate mononuclear cells (MNCs). Two investigators unaware of the experimental groups to which the tissues belonged assessed inflammation as follows (19): 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.

Cell preparation from lymphoid organs

Suspensions of MNCs from the inguinal and popliteal lymph nodes were prepared and pooled in each group, respectively. The cells were suspended in complete RPMI 1640 culture medium containing 1% (v/v) MEM (Life Technologies, Rockville, MD), 2 mM glutamine (Life Technologies), 50 IU/ml penicillin, 50 µg/ml streptomycin, and 10% (v/v) FCS (Life Technologies). The cells were washed three times and then diluted to a cell concentration of 2.5 x 10⁶/ml.

Proliferative responses

Triplicate aliquots (200 µl) of MNC suspensions were applied to 96-well round-bottom microtiter plates (Nunc, Naperville, IL) at a cell density of 2.5 x 10⁶/ml. Ten-microliter aliquots of either MBP1–11 or Con A was added to appropriate wells at final concentrations of 10 µg/ml (MBP1–11) or 5 µg/ml (Con A), and without Ag/mitogen. To study the in vitro effect of IL-12 on the proliferative responses of cells from MBP1–11-i.v. and PBS-i.v. mice, 5 ng/ml rmIL-12 was added to certain cultures. After 60 h of incubation, the cells were pulsed for 12 h with 1 µCi of [³H]methylthymidine (sp. act., 42 Ci/mmol). Cells were harvested on fiberglass filters, and thymidine incorporation was measured at a scintillation counter. The results were expressed as the cpm from culture in the presence of Ag, mitogen, and without Ag/mitogen.

Cytokine profiles

Lymph node cells were cultured in medium without Ag or containing MBP1–11 at a final concentration of 10 µg/ml. Supernatants were collected after 48 h and kept at -70°C. Quantitative ELISA for IFN-, IL-4, IL-5, IL-10, and IL-12 were performed using paired mAbs according to the manufacturer’s recommendations (BD PharMingen, San Diego, CA). Briefly, microtiter plates (Costar, Cambridge, MA) were coated with 100 µl/well capture Ab at 4°C overnight. Uncoated sites were blocked with 10% FCS. Supernatants were added and incubated for 2 h at room temperature. Then, plates were incubated for 2 h with biotinylated detection Abs, followed by HRP-streptavidin (Genzyme, Cambridge, MA). The color was developed with tetramethylbenzidine microwell peroxidase substrate (1-C; Kirkegaard & Perry Laboratories, Gaithersburg, MD), and OD values were detected at 450 nM reader. The concentrations of the cytokines detected were automatically calculated by computer software based on the standard curves obtained from known concentrations.

Statistics

ANOVA was used for the comparison of average clinical scores, proliferative responses, and cytokine profiles among different groups. All tests were two sided.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
i.v. MBP1–11 suppresses clinical EAE and inhibits the production of autoantigen-induced IL-12

To evaluate the involvement of IL-12 in the induction of i.v. tolerance against EAE, we induced i.v. tolerance in mice immunized with MBP1–11 plus CFA. All PBS-i.v. mice (eight of eight) developed severe EAE. The disease onset in PBS-i.v. mice was on day 11 ± 0.5 p.i. The mean maximum score was 3, and the disease was progressive. In contrast, only three of eight MBP1–11-i.v. mice exhibited clinical disease, but much less severe. The mean onset was on day 15 ± 0.7 p.i. (p < 0.01 compared with PBS-i.v. mice). The mean maximum score was 0.4 (p < 0.01 compared with PBS-i.v. mice), and mice recovered in few days (Fig. 1).

View larger version (16K): [in this window] [in a new window]   FIGURE 1. Effect of i.v. administration of MBP1–11 on clinical EAE. Mice were randomly assigned into two groups and immunized with 400 µg of MBP1–11 in CFA. To induce i.v. tolerance, MBP1–11 at a dose of 200 µg/mouse was i.v. injected from day 0 p.i. and every 3 days for five times (MBP1–11-i.v.). Mice receiving PBS served as controls (PBS-i.v.). Data were expressed as the mean clinical score (n = 8 in each group) every other day for all the animals in the groups.

View larger version (36K): [in this window] [in a new window]   FIGURE 2. i.v. MBP1–11 inhibited production of Ag-induced IL-12 p70 and IFN- but increased IL-5 and IL-10. Draining lymph node cells from PBS-i.v. mice and MBP1–11-i.v. mice (n = 8 in each group) were cultured in the presence and absence of MBP1–11 for 48 h. Supernatants were analyzed for the production of IL-12 p70, IFN-, IL-4, IL-5, and IL-10 by sandwich ELISA. IL-4 was not detectable. Values of p were <0.01 for IL-5 and <0.001 for all other comparisons between the two groups.

Because IL-12 has a strong ability to stimulate Th1 cells, we determined whether in vitro IL-12 could reverse the established i.v. MBP1–11-induced tolerance. In vitro proliferative response to autoantigen MBP1–11, polyclonal immune stimulator Con A, and without Ag/mitogen was studied in the presence or absence of rmIL-12. Low proliferative responses were observed in both MBP1–11-i.v. and PBS-i.v. mice without Ag stimulation. Cells from PBS-i.v. mice strongly responded to rmIL-12 in the presence and absence of Ag. This observation is consistent with previous studies, which showed that IL-12 could induce high proliferative response in already activated lymphocytes (20, 21). When the cells were stimulated with MBP1–11, a strong proliferative response was observed in PBS-i.v. mice but not in MBP1–11-i.v. mice. This state of hyporesponsiveness could not be reversed by rmIL-12 (Fig. 3).

View larger version (48K): [in this window] [in a new window]   FIGURE 3. IL-12 in vitro failed to reverse the suppressed proliferative response induced by i.v. tolerance. A total of 4 x 105 of lymph node cells from PBS-i.v. mice or MBP1–11-i.v. mice (n = 8 in each group) was cultured with 10 µg/ml MBP1–11, 5 µg/ml Con A, or without Ag/mitogen. To study the in vitro effect of IL-12 on the proliferative response, 5 ng/ml rmIL-12 was added to certain cultures. After 60 h of incubation, the cells were pulsed for 12 h with 1 µCi of [3H]methylthymidine. Columns refer to mean values, and bars refer to SD. Significant differences were found between PBS-i.v. mice and MBP-i.v. mice (p < 0.01), and between wells with and without rmIL-12 in PBS-i.v. mice (p < 0.01), respectively.

IL-12 in vivo abrogates tolerance induced by i.v. MBP in EAE

To confirm the role of decreased IL-12 production in the induction of i.v. tolerance, we repeated the tolerance induction with i.v. injection of MBP1–11, and i.p. injected rmIL-12 concurrently (MBP1–11-i.v. + IL-12-i.p. mice). The same volume of PBS was injected i.p. to another MBP1–11-i.v. group as control (MBP1–11-i.v. + PBS-i.p. mice). Clinical EAE and pathological signs were monitored. PBS-i.v. + PBS-i.p. mice exhibited severe clinical EAE, while MBP1–11-i.v. + PBS-i.p. mice exhibited much later and less severe clinical disease (p < 0.01). In contrast, administration of rmIL-12 (MBP1–11-i.v. + IL-12-i.p.) blocked the suppression by i.v. tolerance (Fig. 4). Upon receiving rmIL-12, MBP1–11-i.v. mice developed as early and as severe clinical EAE as PBS-i.v. + PBS-i.p. mice, and no significant difference was observed between those two groups. The difference between MBP1–11-i.v. + PBS-i.p. mice and MBP1–11-i.v. + IL-12-i.p. mice was highly significant (p < 0.01).

View larger version (18K): [in this window] [in a new window]   FIGURE 4. Effect of rmIL-12 on the induction of i.v. tolerance in vivo. Twelve mice were randomly assigned into three groups. EAE was induced, and i.v. administration of PBS or MBP1–11 was performed as described in Fig. 1. At the indicated time points, four MBP1–11-i.v. mice were injected i.p. with 100 ng of rmIL-12 in PBS with 1% mouse serum (MBP1–11-i.v. + IL-12-i.p. mice), four MBP1–11-i.v. mice were injected i.p. with 1% mouse serum in PBS (MBP1–11-i.v. + PBS-i.p. mice), and four PBS-i.v. mice were injected i.p. with 1% mouse serum in PBS (PBS-i.v. + PBS-i.p. mice). Clinical scores were monitored as described. Data were expressed as the mean clinical score every other day for all the animals in the groups. Significant differences were observed between MBP1–11-i.v. + PBS-i.p. mice compared with the other two groups (p < 0.01 for all comparisons).

Consistent with the clinical finding, histological examination of the CNS tissue 28 days p.i. revealed a dramatic difference between MBP1–11-i.v. + PBS-i.p. mice and PBS-i.v. + PBS-i.p. mice or MBP1–11-i.v. + IL-12-i.p. mice. In PBS-i.v. + PBS-i.p. mice, multiple inflammatory foci were observed in the white matter of the spinal cord. Fig. 5, A and B, shows representative sections of the spinal cord of these mice. By contrast, few inflammatory cells were detected in MBP1–11-i.v. + PBS-i.p. mice, as shown in Fig. 5, C and D. After administration of IL-12, MBP-11-i.v. mice showed multiple inflammatory foci in the white matter of their spinal cords, as severe as in PBS-i.v. + PBS-i.p. mice. Significant differences were shown between MBP1–11-i.v. + PBS-i.p. mice and other two groups (p < 0.01). The difference was not significant between PBS-i.v. + PBS-i.p. mice and MBP1–11-i.v. + IL-12-i.p. mice (Table I).

View larger version (126K): [in this window] [in a new window]   FIGURE 5. Histopathology of the spinal cord. Spinal cord sections of PBS-i.v. + PBS-i.p. mice (A and B), MBP1–11-i.v. + PBS-i.p. mice (C and D), and MBP1–11-i.v. + IL-12-i.p. mice (E and F) were stained as described in Materials and Methods (n = 4 in each group). Original magnification, x40 in A, C, and E; x100 in B, D, and F.

Because in vitro IL-12 failed to reverse i.v. tolerance-induced suppression of proliferative response to autoantigen MBP1–11, while in vivo IL-12 significantly blocked the i.v. tolerance induction, we were interested in testing whether IL-12 can block the effect of i.v. tolerance on Ag-induced proliferative response in vivo. As shown in Fig. 6, lymph node cells from all three groups had a similar proliferative response without Ag stimulation. When the cells were stimulated with autoantigen MBP1–11, a strong proliferative response was observed in PBS-i.v. mice, while the level of proliferative response in MBP1–11-i.v. mice remained similar to the level without Ag stimulation. In contrast, cells from mice injected with MBP1–11 and IL-12 strongly responded to MBP1–11. The differences between MBP1–11-i.v. + PBS-i.p. mice and the other two groups were all highly significant (p < 0.001).

View larger version (52K): [in this window] [in a new window]   FIGURE 6. IL-12 in vivo blocked i.v. tolerance-induced suppression of proliferative responses. A total of 4 x 105 of MNC suspensions of lymph node cells from PBS-i.v. + PBS-i.p. mice, MBP1–11-i.v. + PBS-i.p. mice, and MBP1–11-i.v. + IL-12-i.p. mice (n = 4 in each group) was cultured with or without 10 µg/ml MBP1–11. After 60 h of incubation, the cells were pulsed for 12 h with 1 µCi of [3H]methylthymidine, and thymidine incorporation was measured with a beta counter. Columns refer to mean values, and bars refer to SD. Significant differences were found between MBP1–11-i.v. + PBS-i.p. mice and the other two groups (all p < 0.01), respectively.

We determined the effect of IL-12 injection on the production of IL-12, IFN-, IL-4, IL-5, and IL-10. When lymph node cells were cultured in the absence of Ag, which represents spontaneous secretion of cytokines, there were no differences in the levels of these cytokines among all three groups. When stimulated with MBP1–11, draining lymph node cells from MBP1–11-i.v. + PBS-i.p. mice produced a significantly lower level of IL-12 compared with PBS-i.v. + PBS-i.p. mice (7.2 ± 1.3 pg/ml; p < 0.001), and MBP1–11- i.v. + IL-12-i.p. mice (7.6 ± 0.1 pg/ml; p < 0.001). Decreased IFN- was also found in MBP1–11-i.v. + PBS-i.p. mice compared with PBS-i.v. + PBS-i.p. mice (p < 0.001), and PBS-i.v. + IL-12-i.p. mice (p < 0.001). No IL-4 production was detected in all three groups. Lymph nodes from MBP1–11-i.v. + PBS-i.p. mice, compared with PBS-i.v. + PBS-i.p. mice, produced higher level of IL-5 (p < 0.01) and IL-10 (p < 0.001). In MBP1–11-i.v. + IL-12-i.p. mice, the up-regulation of IL-5 and IL-10 induced by i.v. tolerance was significantly blocked (all p < 0.001 compared with MBP1–11-i.v. + PBS-i.p. mice) (Fig. 7).

View larger version (31K): [in this window] [in a new window]   FIGURE 7. IL-12 blocked the effect of tolerance on cytokine production. Draining lymph node cells from PBS-i.v. + PBS-i.p. mice, MBP1–11-i.v. + PBS-i.p. mice, and MBP1–11-i.v. + IL-12-i.p. mice (n = 4 in each group) were cultured for 48 h, in the presence and absence of MBP1–11. The production of IL-12 p70, IFN-, IL-4, IL-5, and IL-10 was detected in triplicate by sandwich ELISA. IL-4 was undetectable in all three groups. Significant differences were observed between MBP1–11-i.v. + PBS-i.p. mice compared with the other two groups (p < 0.001 for all comparisons). Error bars represent SD.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Tolerance can be induced in EAE by administration of specific encephalitogenic Ags in a variety of tolerogenic forms and by various routes, including mucosal, i.v., i.p., and s.c. (22). Although soluble Ags including proteins and naive/altered peptides have been used, Ag has also been coupled to splenocytes (6, 23). The mechanisms behind tolerance induction might include physical elimination (clonal deletion), functional inactivation (anergy) of autoantigen-reactive T cells (9, 24, 25, 26, 27, 28), and subpopulation dysregulation by the production of Th2 cells and immunoregulatory cells (10, 29). Using a mAb (19G) (30) to trace MBP TCR transgenic T cells, we showed no clonal deletion when i.v. tolerance was induced concurrent with the induction of active EAE. Furthermore, decreased Ag-specific proliferative response could not be reversed by the presence of IL-2 (G. X. Zhang and A. M. Rostami, unpublished data). When the same protocol was used to induce i.v. tolerance against EAE in Lewis rats, an Ag-specific regulatory mechanism prevailed, and appeared to be the main mechanism for tolerance induction (10, 31).

Recently, the role of IL-12, a potent inducer and promoter of Th1 response, in tolerance induction has been studied in certain animal models. IL-12 blocked/reversed UV radiation-induced immunosuppression (32, 33, 34), experimental autoimmune thyroiditis (35), neonatal tolerance in proteolipid protein-induced EAE (36) and in transplantation (37), and OVA-induced tolerance (38, 39), by promoting Th1 response and/or inhibiting Th2 polarization. IL-12 injection mimicked the adjuvant effects of CFA with respect to phenotype, clonal expansion, effector function, and establishment of memory of CD8⁺ T cells (40). Although IL-12 may play an important role in the pathogenesis of multiple sclerosis (41, 42, 43) and EAE (13, 44, 45, 46, 47), the role of IL-12 in i.v. tolerance induction in EAE is not clear. It has been shown that i.v. injection of proteolipid protein peptide-coupled splenocytes induced resistance to EAE in SJL mice, and, in certain conditions, injection of IL-2 or IL-12 reversed the suppression (48). However, the mechanism for this phenomenon has not been studied. In this study, we further addressed the effect of IL-12 on i.v. tolerance-induced cytokine regulation upon Ag stimulation. Administration of IL-12 significantly blocked the suppressive effect of i.v. tolerance on MBP1–11-induced proliferation and the production of IFN- and IL-12. Although the role of IFN- in the induction of EAE is controversial (49, 50, 51, 52), our data showed that IFN- was positively correlated with the severity of EAE in all groups, implying that IFN- plays a proinflammatory role in EAE. Because the primary effect of IL-12 is the induction of Th1 response, especially IFN- production, the blockade of i.v. tolerance-induced down-regulation of IFN- by IL-12 provides an explanation for the enhancement of EAE.

The mechanism for down-regulation of IL-12 production by i.v. MBP peptide is not clear. Our study suggests the following possibilities: 1) Tolerance induction may inhibit T cell-dependent and IFN--induced IL-12 production, as suggested by several studies (53, 54). Our ex vivo data (Fig. 7A) showed that, after stimulation with autoantigen, lymph node cells from EAE mice or IL-12-i.p. mice produce high levels of IL-12, indicating a T cell-dependent IL-12 production pathway. T cell- and autoantigen-dependent IL-12 production was also reported by a neonatal tolerance study (55). Furthermore, this study showed that the expression of CD40 ligand on tolerized splenic T cells is defective, leading to ineffective cooperation between T cells and APCs, and the lack of IL-12 production (55). 2) The suppression of IL-12 production may result indirectly from a tolerance-induced high level of antiinflammatory molecules, e.g., Th2 cytokines (27, 29) and immunoregulatory chemokines. When oral tolerance was induced in EAE, a decrease of IL-12 production in the mucosal tissue was observed, accompanied by increased monocyte chemoattractant protein-1 expression. Antimonocyte chemoattractant protein-1 abrogated oral tolerance induction and resulted in restoration of mucosal IL-12 expression, as well as peripheral Ag-specific Th1 response (56). Recent data suggest that T regulatory cells (Tr) play a critical role in the induction and maintenance of tolerance. Among them, type 1 T regulatory cells (Tr1) have been well defined based on their unique profile of cytokine production: high level of IL-10, normal level of TGF-, moderate amounts of IFN- and IL-5, low level of IL-2, and no IL-4 (57). This cytokine profile is distinct from the profiles of classical Th1 and Th2 cells. In our present study, cells from MBP1–11-i.v. mice produce undetectable IL-4, low level IFN-, moderate level of IL-5, and high level of IL-10 upon MBP1–11 stimulation. This phenotype indicates an i.v. MBP1–11-driven Tr1-like response.

The importance of IL-10, the hallmark of Tr1 response, has been addressed in the induction of tolerance against T cell-mediated autoimmune diseases, including EAE (4, 58, 59, 60). Mucosal administration of IL-10 enhances tolerance induction in EAE (61, 62). IL-10 can down-regulate costimulatory signals by macrophages, dendritic cell-driven IFN- production by T cells, and T cell responses to Ag through inhibition of IL-12 production and IL-12R expression (63). Adoptive transfer of autoreactive T cells genetically designed to secrete IL-10 delayed the onset of EAE (64). T cells from IL-10^-/- mice exhibit a stronger Ag-specific proliferation, produce more proinflammatory cytokines (IFN- and TNF-) when stimulated with an encephalitogenic peptide, and induce severe EAE upon transfer into wild-type mice (5). It has recently been suggested that, in the innate immune system, there may be an IL-10/IL-12 immunoregulatory circuit controlling susceptibility to autoimmune disease (46). In this circuit, the disease-promoting effects of IL-12 are antagonized by IL-10. In turn, endogenous production of IL-12 could suppress IL-10 production, while anti-IL-12 treatment up-regulates the production of IL-10 (46). Therefore, manipulation of the cytokine milieu and the IL-12/IL-10 balance could have a decisive effect on the incidence of autoimmune diseases (46, 65). Such IL-12/IL-10 immunoregulatory circuit hypothesis, along with other studies, provides a convincing explanation for the interaction between IL-12 and the induction of i.v. tolerance. First of all, i.v. tolerance may inhibit IL-12 production indirectly as a result of the enhancement of IL-10, as suggested by this IL-12/IL-10 immunoregulatory circuit hypothesis. Our data support the existence of this IL-12/IL-10 immunoregulatory circuit. They also suggest that exogenous IL-12 could block i.v. tolerance-induced high production of IL-10, thus promoting the production of endogenous IL-12 and IFN-. Furthermore, a high level of exogenous IL-12, along with endogenous IL-12, will commit and expand an Ag-specific Th1 population (66) that has been suppressed by i.v. tolerance. These mechanisms, as a result, could contribute to blocking the induction of i.v. tolerance in EAE.

Of note in our study is that IL-12 in vitro failed to restore impaired Ag-specific proliferative response after the i.v. tolerance had already been established (Fig. 2), while IL-12 in vivo strongly blocked tolerance-induced suppression of autoantigen-specific proliferative response (Fig. 6). These results indicate that IL-12 plays distinct roles at different stages of i.v. tolerance induction. In addition to our finding, several studies have also suggested that the effect of IL-12 on blocking/reversing tolerance-induced suppression of T cell proliferation is a complex process. In a model of i.v. tolerance against contact hypersensitivity, Ushio et al. (67) showed that IL-12 reversed established tolerance in vitro and in vivo. However, Van Parijs et al. (39) showed that the administration of IL-12 at the time of tolerance induction stimulated Th1 differentiation, but did not promote Ag-specific T cell proliferation. By contrast, the combination of IL-12 and anti-CTLA-4 completely reversed the tolerogen-induced suppression of both T cell proliferation and Th1 differentiation by tolerogen. It is not clear why i.v. tolerance-induced suppression of Ag-specific proliferation could be blocked in vivo, but failed to do so in the established i.v. tolerance in vitro. One possibility is that cells from mice that have been tolerized produced a high level of IL-10, which can overcome the effect of in vitro IL-12, while when IL-12 was administered in vivo before the induction of IL-10 by i.v. tolerance, it could block the IL-10 induction. The effect of i.v. tolerance on costimulatory signals, as pointed out by Van Parijs et al. (39), might also play a different part in affecting the function of IL-12 in vivo or in vitro.

In summary, we observed significant suppression of IL-12 production in lymph node cells of EAE mice tolerized by i.v. MBP1–11. Exogenous IL-12 resulted in reversal of this suppression and abrogation of tolerance. i.v. tolerance suppressed MBP1–11-induced IFN- production and proliferative response; IL-12 blocked these effects. Furthermore, IL-12 completely blocked the i.v. tolerance-induced Tr1 response. These data suggest that i.v. administration of autoantigen results in the suppression of endogenous IL-12 and the consequent switching of immune response from an immunogenic to a tolerogenic form.

	Acknowledgments

 
We thank Dr. Bruno Gran for critical discussion, Katherine Regan for excellent secretarial assistance, Elvira Ventura for technical assistance, and the Bioanalytical Science Department of the Genetics Institute for rmIL-12.

	Footnotes

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

² Address correspondence and reprint requests to Dr. Abdolmohamad Rostami, Department of Neurology, University of Pennsylvania Medical Center, 3400 Spruce Street, Philadelphia, PA 19104. E-mail address: rostamia{at}mail.med.upenn.edu

³ Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; MBP, myelin basic protein; MNC, mononuclear cell; p.i., postimmunization; rm, recombinant murine; Tr1, type 1 T regulatory cell.

Received for publication September 4, 2001. Accepted for publication December 19, 2001.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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