Down-Regulation of Th1-Mediated Pathology in Experimental Arthritis by Stimulation of the Th2 Arm of the Immune Response

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1 ARTHRITIS & RHEUMATISM Vol. 48, No. 3, March 2003, pp DOI /art , American College of Rheumatology Down-Regulation of Th1-Mediated Pathology in Experimental Arthritis by Stimulation of the Th2 Arm of the Immune Response Claudia Mauri, 1 Marc Feldmann, 2 and Richard O. Williams 2 Supported by the Arthritis Research Campaign of Great Britain. 1 Claudia Mauri, PhD: Center for Rheumatology Research, Windeyer Institute of Medical Sciences, University College London, London, UK, and The Kennedy Institute of Rheumatology, Imperial College of Science, Technology and Medicine, London, UK; 2 Marc Feldmann, MB, PhD, FRCPath, Richard O. Williams, PhD: The Kennedy Institute of Rheumatology, Imperial College of Science, Technology and Medicine, London, UK. Address correspondence and reprint requests to Claudia Mauri, PhD, Centre for Rheumatology Research, Department of Medicine, Windeyer Institute of Medical Sciences, University College London, 46 Cleveland Street, London, UK. c.mauri@ucl.ac.uk. Submitted for publication July 17, 2002; accepted in revised form November 22, Objective. To assess whether systemic administration of recombinant interferon- (rifn ), a proinflammatory cytokine that influences the differentiation of naive T cells into Th1 cells, promotes the induction of arthritis in DBA/1 mice immunized with type II collagen (CII) in Freund s incomplete adjuvant (IFA) and to determine the antiarthritic effect of treatment with CII in IFA. Methods. DBA/1 mice were immunized with CII in IFA and injected intraperitoneally with rifn (8,000 units/mouse/day) or with recombinant interleukin-12 (ril-12; 100 ng/mouse/day). In another experiment, mice were immunized with CII in Freund s complete adjuvant (CFA) and treated with a single dose of CII in IFA on the day of immunization or on the day of disease onset. Mice were monitored to assess the effect of the treatment on the severity of disease. Th1/Th2 cytokines and anti-cii antibodies were measured by enzymelinked immunosorbent assay. Results. The administration of ril-12 or rifn to mice immunized with CII in IFA restored the Th1 response and resulted in the development of arthritis. We then determined whether immunization with CII in IFA had the capacity to prevent and/or ameliorate collagen-induced arthritis. A single intraperitoneal injection of CII in IFA prevented arthritis when given at the time of immunization with CII in CFA and reduced disease severity when given at the time of arthritis onset. The administration of CII in IFA resulted in a profound down-regulation of IFN production and an up-regulation of IL-10 in cultures of draining lymph node cells. Conclusion. These findings demonstrate that it is possible to deflect an ongoing pathogenic Th1 response to an antigen by reimmunization of the same antigen with a Th2-polarizing adjuvant. Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic synovitis and progressive destruction of the joint. Although many questions concerning the etiology of RA remain unanswered, cumulative evidence suggests that CD4 T cells, which exhibit a predominantly Th1 pattern of cytokine expression, play an important role in the pathogenesis of the disease (1). Collagen-induced arthritis is a model of RA induced in genetically susceptible strains of mice by immunization with type II collagen (CII) in Freund s complete adjuvant (CFA). We and other investigators have previously demonstrated that the induction phase of collagen-induced arthritis is characterized by a Th1- like cytokine response, while Th2 cytokines assume a more prominent role during the remission phase of the disease (2,3). During the induction phase of collageninduced arthritis, neutralization of interleukin-12 (IL- 12) or administration of IL-4 protects against disease, thus indicating an important role of Th1 cells in the induction of joint inflammation (4,5). However, these approaches were not successful in established arthritis, indicating that modification of an already established Th1 response requires a more radical approach than the temporary manipulation of cytokine levels. Indeed, the failure of this strategy is predictable, given the relatively stable nature of Th1/Th2 phenotypes and the difficulty 839

2 840 MAURI ET AL in reprogramming cytokine expression patterns in differentiated T cells (6). What is needed, therefore, is a therapeutic approach that involves the generation of significant numbers of Th2 cells from naive precursors, so as to counteract the effects of the existing pathogenic Th1 cells. In the present study, we set out to test the hypothesis that it is possible to suppress an ongoing pathologic Th1 response by actively reimmunizing mice with CII plus a Th2-inducing adjuvant. We have previously shown that immunization of DBA/1 mice with CII in Freund s incomplete adjuvant (IFA) produces a strong Th2 response (2). Similarly, immunization with myelin basic protein derived peptides in IFA was shown to induce a polarized Th2 response and to protect against experimental allergic encephalomyelitis (7), whereas administration of a complex of alum and CII protected against the development of collagen-induced arthritis in rats (8). We show here that immunization with CII in IFA protects against collagen-induced arthritis in mice and, more importantly, is effective in modifying an ongoing Th1 response and in ameliorating arthritis even after disease onset. Thus, administration of autoantigen in a vehicle designed to promote Th2 responses may provide an alternative strategy for reversing established autoimmune diseases such as RA. MATERIALS AND METHODS Preparation of type II collagen. Bovine CII was purified and prepared as previously described (9). Bovine CII was solubilized by stirring overnight at 4 C in 0.1M acetic acid for immunization or in 0.05M Tris, containing 0.2M NaCl, ph 7.4, for stimulation of splenocytes in vitro. Immunization of mice. Male DBA/1 mice were immunized intradermally at 8 12 weeks of age with bovine CII (200 g/mouse) emulsified in CFA or IFA (both purchased from Difco, West Moseley, UK). Beginning on day 14 after immunization, mice were examined daily for signs of arthritis. Other groups were immunized intradermally with CII in IFA and then injected intraperitoneally for 10 days with recombinant interferon- (rifn ; 8,000 units/mouse/day) or with recombinant IL-12 (ril-12; 100 ng/mouse/day), beginning on the day of immunization. This research was approved by the local Ethical Review Process Committee and by the Home Office of the UK. Prevention and treatment of arthritis. First, we assessed the ability of immunization with CII in IFA to prevent the arthritis induced by immunization with CII in CFA. Next, we assessed the ability of immunization with CII in IFA to treat existing arthritis. For the prevention of arthritis, DBA/1 mice were injected intradermally or intraperitoneally with 200 g of CII emulsified in IFA on the same day of immunization with CII in CFA. For the treatment of existing arthritis, mice were injected intradermally or intraperitoneally with a single dose of 200 g of CII in IFA on the day of arthritis onset. Antibodies. The coating/detection antibody pairings used for enzyme-linked immunosorbent assay (ELISA) were as follows: for IL-10, rat monoclonal antibodies 2A5/SXC-1, and for IFN, R46A2/XMG1.2 (obtained from the American Tissue Type Collection, Rockville, MD). Assessment of arthritis. The development of arthritis was assessed daily for the duration of the experiment. The clinical severity of arthritis was graded as follows: 0 normal, 1 slight swelling and/or erythema, 2 pronounced edematous swelling, and 3 joint rigidity. Each limb was evaluated and graded (maximum clinical score 12 per animal). Swelling of hind paws was measured with a pair of calipers. All clinical evaluations were performed in a blinded manner. Quantitation of cytokine production. Inguinal draining lymph nodes were teased apart to make a single-cell suspension, then washed and cultured in RPMI 1640 containing 10% (volume/volume) heat-inactivated fetal calf serum, 100 units/ml of penicillin, 100 g/ml of streptomycin, M 2-mercaptoethanol, and 20 mm L-glutamine. Lymph node cells were cultured in 96-well plates (Nunc, Uxbridge, UK) at a density of cells/ml (200 l/well) in medium alone or with 50 g/ml of CII for 72 hours. Supernatants were collected and analyzed for IL-10 and IFN production by a sandwich ELISA as previously described (2). T cell proliferation. For in vitro proliferation studies, lymph nodes were removed 30 days after CII immunization and cells/ml were cultured in 200 l of complete medium with CII (50 g/ml) for 72 hours in a round-bottomed 96-well plate (Nunc). During the last 9 hours of culture, cells were pulsed with 1 Ci of tritiated thymidine (Amersham, Little Chalfont, UK), then harvested and counted in a scintillation counter (LKB, Croydon, UK). All experiments were performed in triplicate. Serum anticollagen antibody levels. Levels of anti-cii antibodies were determined as previously described. Briefly, microplates (Nunc) were coated with 2 g/ml of bovine CII dissolved overnight in Tris buffer (0.05M Tris, containing 0.2M NaCl, ph 7.4), blocked with 2% bovine serum albumin (Sigma, St. Louis, MO), and then incubated with serial dilutions of test sera. A reference sample was included on each plate. Bound total IgG, IgG1, or IgG2a was detected by incubation with alkaline phosphatase conjugated sheep anti-mouse IgG, IgG1, or IgG2a, respectively (The Binding Site, Birmingham, UK), followed by dinitrophenyl phosphate. Plates were washed at least 3 times between each step with 0.01% Tween phosphate buffered saline. Optical density was measured at 405 nm. Statistical analysis. For the statistical analysis of clinical scores, the nonparametric Mann-Whitney U test was used. The analysis of arthritis incidence was performed using Fisher s exact test. The unpaired t-test was used to analyze the results of cytokine quantification experiments and serum antibody levels. A P value of less than 5% was considered statistically significant. RESULTS Induction of a nonarthritogenic Th2 response by immunization with CII in IFA. Immunization of DBA/1 mice with CII in CFA induces a destructive polyarthritis

3 DOWN-REGULATION OF TH1-MEDIATED PATHOLOGY IN ARTHRITIS 841 Table 1. Treatment Role of IFN and IL-12 in the induction of arthritis in DBA/1 mice* No. of mice Incidence of arthritis Clinical score on day 5, mean SEM Anti-CII IgG2a, mean SEM AU Anti-CII IgG1, mean SEM AU CII in CFA , CII in IFA alone , , CII in IFA rifn CII in IFA ril ,350 1, * Mice were immunized with type II collagen (CII) in Freund s complete adjuvant (CFA), CII in Freund s incomplete adjuvant (IFA) alone, or CII in IFA plus either recombinant interferon- (rifn ; 8,000 units) or recombinant interleukin-12 (ril-12; 100 ng). Recombinant cytokines were injected intraperitoneally each day for 10 days, starting on the day of immunization. The incidence of arthritis (percentage of mice) was determined 6 weeks after immunization. Clinical scores were determined 5 days after the onset of clinical arthritis (see Materials and Methods for details). Data are representative of 3 independent experiments. AU arbitrary units. P 0.05 versus CII in IFA alone. P versus CII in CFA, versus CII in IFA rifn, and versus CII in IFA ril-12. P 0.01 versus CII in IFA alone. characterized by a predominantly Th1-like response (2,3). The omission of Mycobacterium from the adjuvant not only fails to induce arthritis, but it leads to the development of a predominantly Th2-like response (2). We therefore set out to investigate the relationship between the absence of Mycobacterium, the induction of Th2 responses, and the development of arthritis. Mice were immunized with CII in IFA on day 0 and were then given daily intraperitoneal injections of rifn or ril-12 for 10 days. Table 1 shows that treatment with either rifn or ril-12 increased the incidence of clinical arthritis from 20% to %. Serologic analysis of anti-cii IgG levels in mice given CII in IFA alone revealed high levels of anti-cii IgG1 and lower levels of anti-cii IgG2a (Table 1). However, in mice given CII in IFA plus ril-12, there was a dramatic increase in the anti-cii IgG2a levels. No significant differences in the levels of total anti-cii IgG antibodies were found between the study groups (data not shown). These findings confirmed that immunization with CII in IFA leads to a nonarthritogenic and Th2-biased immune response that can be converted into an arthritogenic response by supplementation with rifn or ril-12. Prevention of collagen-induced arthritis by immunization with CII in IFA. We next addressed the question of whether immunization with CII in IFA could prevent the development of arthritis in DBA/1 mice immunized with CII in CFA. On day 0, DBA/1 mice were immunized intradermally with CII in CFA and then injected intraperitoneally or intradermally (on the same day) with CII in IFA. Controls were given CII in phosphate buffered saline. The group injected intraperitoneally with CII in IFA showed a statistically significant reduction in the incidence of clinical arthritis. In addition, in the small proportion of mice that developed clinical arthritis (30%), there were significant reductions in both the time to arthritis onset (data not shown) and the severity of arthritis (Figure 1). Treatment with CII in IFA by intradermal injection was less effective in protecting against arthritis, with 80% of mice developing clinical arthritis. However, the severity of arthritis was significantly reduced at both the clinical and histologic levels in mice treated intradermally with CII in IFA compared with the control group. We concluded from this experiment that treatment with CII in IFA around the time of immunization with CII in CFA protected against the development of arthritis. Figure 1. Protection against collagen-induced arthritis by immunization with type II collagen (CII) in Freund s incomplete adjuvant (IFA). DBA/1 mice were immunized with CII in Freund s complete adjuvant, and on the same day, they were injected with a single dose of CII in IFA (intraperitoneally) (}), CII in IFA (intradermally) (F), or CII in phosphate buffered saline alone (intraperitoneally) ( ). Clinical scores were determined as described in Materials and Methods. Values are the mean and SEM clinical scores and are representative of 3 experiments.

4 842 MAURI ET AL High levels of IFN and low levels of IL-10 were found in the lymph node cell cultures from control mice, which is indicative of a differentiated Th1 response. In contrast, treatment with CII in IFA significantly decreased Figure 2. Protection against collagen-induced arthritis by immunization with CII in IFA, even when performed after disease onset. DBA/1 mice were immunized with CII in Freund s complete adjuvant, and on the day of onset of clinical arthritis, mice were treated with CII in IFA (intraperitoneally) (}), CII in IFA (intradermally) (F), or CII in phosphate buffered saline alone (intraperitoneally) ( ). Values are the mean and SEM clinical scores and are representative of 3 experiments, each containing 10 mice per group. See Figure 1 for definitions. Amelioration of established arthritis after a single injection of CII in IFA. In the next experiment, the ability of CII in IFA treatment to reverse an established Th1-driven arthritis was tested. DBA/1 mice were immunized with CII in CFA, and on the day of arthritis onset, a single injection of CII in IFA was given intraperitoneally. The progression of arthritis was significantly reduced after administration of CII in IFA compared with the control group, which had been treated with CII in phosphate buffered saline (Figure 2). We also observed that intradermal administration of CII in IFA, although less effective than intraperitoneal administration, significantly reduced the progression of clinical arthritis compared with the control group. These findings confirmed the therapeutic potential of CII in IFA administration, even in the face of an ongoing immune response. Protective effect of CII in IFA administration associated with a reduction in Th1 cell activity and an increase in IL-10 production. We next tested whether treatment with CII in IFA had any effect on the Th1 response induced by immunization with CII in CFA. DBA/1 mice were immunized with CII in CFA on day 0 and then treated with a single intraperitoneal injection of CII in IFA or CII in phosphate buffered saline on the day of onset of arthritis. Five days after the onset of clinical arthritis, lymph node cells were obtained and cultured alone or in the presence of CII (50 g/ml). Levels of IFN and IL-10 were measured by ELISA. IL-4 was not detected in these lymph node cell cultures. Figure 3. a and b, Inhibition of T cell proliferation and interferon- (IFN ) production and increase in interleukin-10 (IL-10) production by treatment with CII in IFA. Arthritis was induced in DBA/1 mice by immunization with CII in Freund s complete adjuvant, then mice were injected intraperitoneally with CII in IFA or with CII alone. Five days after the onset of arthritis, mice (n 5) were killed and inguinal lymph nodes were excised. Lymph node cells were cultured with 50 g/ml of CII (solid bars) or with medium alone (open bars). c, The T cell proliferative response was measured as the incorporation of tritiated thymidine. Supernatants were also tested for the presence of IFN and IL-10 by enzyme-linked immunosorbent assay. Values are the mean and SEM and are representative of the results from 1 of 3 experiments. PBS phosphate buffered saline (see Figure 1 for other definitions).

5 DOWN-REGULATION OF TH1-MEDIATED PATHOLOGY IN ARTHRITIS 843 cell activity (as judged by IFN production), and an increase in the production of the antiinflammatory cytokine IL-10. Inhibition of the production of complementfixing IgG2a and anti-cii antibodies by immunization with CII in IFA. Our findings so far strongly suggest that the amelioration of arthritis observed after immunization with CII in IFA is due mainly to inhibition of Th1 responses. The expression of IgG1 and IgG2a subclasses is known to be controlled by Th2 and Th1 cytokines, respectively. We therefore investigated whether the beneficial effect of CII in IFA treatment on arthritis in DBA/1 mice was associated with a change in the anti-cii IgG isotype profile. Serum levels of anti-cii IgG1 and IgG2a were measured 30 days after mice were immunized with CII in CFA. Treatment with CII in IFA administered intraperitoneally significantly inhibited levels of anti-cii IgG2a but had no significant effect on levels of IgG1 (Figures 4a and b). It was concluded that one of the consequences of administration of CII in IFA is modulation of the anti-cii antibody response, resulting in a decrease in the ratio of IgG2a to IgG1 and a reduction in the levels of complement-fixing antibodies. Figure 4. Selective down-regulation of IgG2a anti-cii antibodies caused by treatment with CII in IFA. DBA/1 mice that had been immunized with CII in Freund s complete adjuvant (CFA) were given a single intraperitoneal injection of CII in IFA or of CII in phosphate buffered saline (PBS) on the day of onset of arthritis. Serum levels of a, IgG2a and b, IgG1 were measured by enzyme-linked immunosorbent assay on day 30 after immunization with CII in CFA. See Figure 1 for other definitions. the levels of IFN and increased the levels of IL-10 (Figures 3a and b), supporting the hypothesis of a switch from the pathogenic Th1 response to a more protective Th2-like response. Next, we tested the effect of treatment with CII in IFA on the T cell proliferative response to CII, as determined by tritiated thymidine incorporation. Lymph node cells from the control group displayed strong proliferative responses to CII. In contrast, DBA/1 mice treated intraperitoneally with CII in IFA displayed a significantly reduced proliferative response to CII (Figure 3c). Thus, treatment with CII in IFA had 3 effects on the T cell response: an overall reduction in the level of T cell proliferation, a reduction in proinflammatory Th1 DISCUSSION It is now well established that T helper cells can be grouped as Th1 cells or Th2 cells based on their cytokine secretion patterns and effector functions. There is also general agreement that Th1 cells are important mediators of pathology in organ-specific autoimmune diseases. Although RA has a number of important systemic features, it is the chronic inflammation in the joints that is the most consistently destructive feature of the disease, and a number of studies have shown that T cells in RA joints are predominantly of a Th1 phenotype (1). Immunohistochemical analysis of arthritic joints reveals abundant expression of monocyte-derived proinflammatory cytokines, which is also consistent with Th1-mediated disease, and on this basis, it has been proposed that down-regulation of the Th1 response would be beneficial in the treatment of RA. A number of groups of investigators have therefore used animal models of arthritis in which it is possible to demonstrate a clear skewing of Th1 cells to investigate the possibility of skewing the immune response away from a polarized Th1 response toward a Th2 response, with the aim of reducing pathologic changes. In view of the mutual antagonism of Th1 and Th2 subsets, it is hoped that the beneficial effects of

6 844 MAURI ET AL promoting the Th2 response would be amplified because the newly generated Th2 cells would further inhibit the Th1 response through the release of IL-4, IL-10, and IL-13. This approach, however, has met with 2 fundamental problems. First, the administration of Th2 cytokines or the blocking of antibodies to Th1 cytokines appears to be insufficient to modify a fully established Th1 response. For example, administration of IL-4 (5) or anti IL-12 monoclonal antibody (4,10) was shown to exert a protective effect on the development of arthritis if administered during the induction phase of the disease, but both were less effective or were completely ineffective if administered during established disease. Second, while it may be possible to temporarily downregulate the Th1 response using Th2 cytokines, the response reverts back to a Th1 response as soon as treatment is stopped (5). This may be attributed to the fact that the cytokine expression patterns of differentiated Th1 and Th2 cells are relatively stable over time, with little evidence to suggest that Th1 cells can be induced to assume a Th2 phenotype or vice versa. These factors prompted us to consider a different strategy for treating Th1-mediated arthritis, that of active reimmunization with antigen plus a Th2-inducing adjuvant. There are a number of potential benefits of this approach. First, administration of a Th2 adjuvant would lead to a broader range of Th2 cytokine expression than would administration of a single recombinant Th2 cytokine. Second, by reimmunizing the mice, we expected to induce the differentiation of Th2 cells from naive precursors, rather than to alter the phenotype of differentiated Th1 cells. Finally, by reimmunizing with type II collagen plus a Th2 adjuvant, Th2 cells that are specific for type II collagen would be generated, and this would promote the selective accumulation of antigenspecific Th2 cells at the site of disease activity. Based on what is known about bystander suppression in animal models (11), we also predicted that immunization with CII plus a Th2 adjuvant would suppress a pathogenic response to other antigens in the joint. This point needs particular emphasis because there is little evidence to suggest that CII is the major autoantigen in RA. However, it should be emphasized that it has not been possible to test the principle of bystander suppression in humans. We previously observed that a sustained Th2- type response was generated by immunization of DBA/1 mice with CII in IFA, with a corresponding failure to induce full-blown arthritis (2). In the present study, the absence of arthritis in mice immunized with CII in IFA could be overcome by administering ril-12 or rifn during the induction phase of collagen-induced arthritis, thereby confirming that Th1 cytokines are required for the induction of collagen-induced arthritis. Indeed, these findings are consistent with a previous study by Germann et al (12), which showed that coadministration of CII in IFA plus IL-12 led to a strongly arthritogenic response, accompanied by high levels of IFN production. However, it should be noted that IFN is a highly pleiotropic cytokine and that both disease-promoting and disease-limiting properties have been described for this cytokine in autoimmune disease models (for review, see ref. 13). Next, we took advantage of the Th2-inducing properties of IFA to evaluate the protective effect on arthritis of skewing the immune response toward a more Th2-like response. We observed that administration of CII in IFA around the time of immunization with CII in CFA was highly effective at suppressing the clinical and histologic severity of collagen-induced arthritis (Figure 1). It was also confirmed that the administration of CII in IFA during the induction of collagen-induced arthritis provoked a dramatic reduction in the intensity of the Th1 response, as shown by the decreased production of IFN by lymph node cells, decreased serum levels of anti-cii IgG2a, and increased levels of IgG1 (data not shown). In the next phase of the study, we analyzed the effect of reimmunization with CII in IFA in mice that had already developed a full-blown Th1 response. We found that even after the onset of collagen-induced arthritis, it was possible to alter the Th1/Th2 balance by administration of CII in IFA. Interestingly, neither CII alone nor IFA alone was able to suppress the Th1 response or reduce the severity of arthritis (data not shown). It was also observed that the protective effect of CII in IFA was more pronounced when administered intraperitoneally rather than intradermally. Although the reasons for this are unclear, it is known that the skin contains a large population of dendritic cells, which are known to be effective inducers of Th1 responses, and this may have reduced the protective capacity of the administration of CII in IFA. In the present study, we succeeded in validating two hypotheses. The first is that stimulation of the Th2 arm of the immune response is beneficial in terms of the outcome of arthritis. The second is that a fully established Th1 response can be down-regulated by an opposing Th2 response, even after the onset of clinical arthritis. There are clear dangers in the extrapolation of data from animal models to human disease. Further-

7 DOWN-REGULATION OF TH1-MEDIATED PATHOLOGY IN ARTHRITIS 845 more, we would not advocate the use of IFA in RA in humans because of its toxicity. However, alum is licensed for human use as an adjuvant, and when conjugated to type II collagen, it has been shown to protect against collagen-induced arthritis in rats (8). Moreover, administration of an unrelated antigen (DR4/1 peptide) in alum adjuvant was found to be safe in humans with RA (14). Oral administration of type II collagen has also been shown to be safe in RA and to produce a modest degree of benefit (15). Against this background, our findings provide a powerful argument for the testing of immunization with type II collagen/alum in RA in humans. REFERENCES 1. Miossec P, van den Berg W. Th1/Th2 cytokine balance in arthritis. Arthritis Rheum 1997;40: Mauri C, Williams RO, Walmsley M, Feldmann M. Relationship between Th1/Th2 cytokine patterns and the arthritogenic response in collagen-induced arthritis. Eur J Immunol 1996;26: Doncarli A, Stasiuk LM, Fournier C, Abehsira-Amar O. Conversion in vivo from an early dominant Th0/Th1 response to a Th2 phenotype during the development of collagen-induced arthritis. Eur J Immunol 1997;27: Joosten LA, Lubberts E, Helsen MM, van den Berg WB. Dual role of IL-12 in early and late stages of murine collagen type II arthritis. J Immunol 1997;159: Horsfall AC, Butler DM, Marinova L, Warden PJ, Williams RO, Maini RN, et al. Suppression of collagen-induced arthritis by continuous administration of IL-4. J Immunol 1997;159: Murphy E, Shibuya K, Hosken N, Openshaw P, Maino V, Davis K, et al. Reversibility of T helper 1 and 2 populations is lost after long-term stimulation. J Exp Med 1996;183: Heeger PS, Forsthuber T, Shive C, Biekert E, Genain C, Hofstetter HH, et al. Revisiting tolerance induced by autoantigen in incomplete Freund s adjuvant. J Immunol 2000;164: Mattsson L, Lorentzen JC, Svelander L, Bucht A, Nyman U, Klareskog L, et al. Immunization with alum-collagen II complex suppresses the development of collagen-induced arthritis in rats by deviating the immune response. Scand J Immunol 1997;46: Miller EJ. Structural studies on cartilage collagen employing limited cleavage and solubilization with pepsin. Biochemistry 1972;11: Malfait A-M, Butler DM, Presky DH, Maini RN, Brennan FM, Feldmann M. Blockade of IL-12 during the induction of collageninduced arthritis (CIA) markedly attenuates the severity of the arthritis. Clin Exp Immunol 1998;111: Bayrak S, Mitchison NA. Bystander suppression of murine collagen-induced arthritis by long-term nasal administration of a self type II collagen peptide. Clin Exp Immunol 1998;113: Germann T, Szeliga J, Hess H, Storkel S, Podlaski FJ, Gately MK, et al. Administration of interleukin 12 in combination with type II collagen induces severe arthritis in DBA/1 mice. Proc Natl Acad SciUSA1995;92: Matthys P, Vermeire K, Billiau A. Mac-1 myelopoiesis induced by CFA: a clue to the paradoxical effects of IFN- in autoimmune disease models. Trends Immunol 2001;22: St. Clair EW, Cohen SB, Lee ML, Fleischmann RM, Lee SH, Moreland LW, et al. Treatment of rheumatoid arthritis with a DR4/1 peptide. J Rheumatol 2000;27: Choy EH, Scott DL, Kingsley GH, Thomas S, Murphy AG, Staines N, et al. Control of rheumatoid arthritis by oral tolerance. Arthritis Rheum 2001;44: