Interleukin-35 gene therapy exacerbates experimental rheumatoid arthritis in mice.

Interleukin (IL)-35 was initially described as an immunosuppressive cytokine specifically produced by CD4(+)FoxP3(+) regulatory T cells (Treg). Since Treg play a major role in autoimmunity control and protect from inflammation, we aimed at evaluating the role of IL-35 in collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA), using a non-viral gene transfer strategy. The clinical and histological effect of IL-35 was assessed in mice with CIA receiving an injection of two distinct plasmids encoding IL-35 gene (pIGneo-mIL-35 or pORF-mIL-35) 3 and 18 days after CIA induction. Treg and Th17 were characterized by flow cytometry in the spleen and lymph nodes of treated mice. Our results showed that whatever the plasmid used, IL-35 gene transfer resulted in a statistically significant increase in clinical scores of CIA compared to results with empty plasmid. The underlying cellular mechanisms of this effect were shown to be related to an increased Th17/Treg ratio in the spleen of pORF-mIL-35 treated mice. In conclusion, we show an unexpected but clear exacerbating effect of IL-35 gene transfer in an autoimmune and inflammatory RA model, associated with a modification of the Th17/Treg balance. Altogether, these result shows that this cytokine can promote chronic inflammation.

Protection from articular damage by passive or active anti-tumour necrosis factor (TNF)-α immunotherapy in human TNF-α transgenic mice depends on anti-TNF-α antibody levels.

Active anti-tumour necrosis factor (TNF)-α immunization with the kinoid of TNF-α (TNF-K) induces polyclonal anti-TNF-α antibodies and ameliorates arthritis in human TNF-α (hTNF-α) transgenic mice (TTg). We compared the efficacy of TNF-K to that of infliximab (IFX) and of TNF-K and IFX co-administration, and evaluated whether the titres of anti-hTNF-α antibodies induced by immunization were a determinant of TNF-K efficacy. Forty-eight TTg mice received one of the following treatments: TNF-K immunization (TNF-K group); weekly IFX throughout the study duration (IFXw0-15); TNF-K plus weekly IFX for 4 weeks (TNF-K + IFX); and weekly IFX for 4 weeks (IFXw0-4); PBS. Animals were killed at week 16. Anti-hTNF-α antibody titres and clinical and histological scores were compared. All TNF-K immunized mice (TNF-K and TNF-K + IFX) produced anti-hTNF-α antibodies. Titres were higher in TNF-K versus TNF-K + IFX (P < 0·001) and correlated inversely with histological inflammation (R = -0·78; P = 0·0001) and destruction (R = -0·67; P = 0·001). TNF-K + IFX had higher histological inflammation and destruction versus TNF-K (P < 0·05). A receiver operating characteristic (ROC) analysis of anti-hTNF-α antibody titres identified the criterion cut-off value to discriminate most effectively between the TNF-K and TNF-K + IFX groups. Mice with high versus low titres had less histological inflammation and destruction (P < 0·05). In a model of TNF-α-dependent arthritis, protection from articular damage by TNF-K correlates with the titres of induced anti-hTNF-α antibodies. The co-administration of TNF-K and a short course of infliximab does not result in less articular damage versus solely TNF-K, due probably to lower anti-hTNF-α antibody production. These results are relevant for future development of active anti-TNF-α immunization in human disease.

Ultrasound and Doppler micro-imaging in a model of rheumatoid arthritis in mice.

OBJECTIVES: The evaluation of joints in arthritis using conventional ultrasonography is not really feasible in mice because of the small size of the animal. However, compared with classical analysis (clinical and histological examination) it is a non-invasive method that allows follow-up of the same animal throughout the whole experiment. Moreover, power Doppler allows the study of blood flow that reflects inflammatory activity within the synovium of arthritic joints. Our aim was to determine whether ultrasonography analysis could accurately detect arthritis lesions in a mouse model of rheumatoid arthritis, namely collagen-induced arthritis. METHODS: Collagen-induced arthritis was induced in 28 mice by immunising with collagen type II. Every week for 8 weeks, ultrasonography and Doppler analysis were performed on knees and ankles of all mice using the ultrasound biomicroscope (UBM), which is particularly dedicated to studying the mouse. Clinical and histological evaluations were performed as usual. RESULTS: We established a semiquantitative analysis by setting an UBM scoring. UBM grades were correlated to clinical and histological scores of arthritis. Vascularisation within the synovium could be estimated by power Doppler analysis and a semiquantitative vascularisation scale was established, which allowed us to show a good correlation between vascularisation scores and histological or clinical scores of arthritis. CONCLUSIONS: This is one of the first studies that shows it is possible to visualise a selected set of joints in a small animal using UBM analysis. It provides new perspectives in evaluating experimental models of rheumatoid arthritis and other joint diseases.

Early and long-lasting protection from arthritis in tumour necrosis factor alpha (TNFalpha) transgenic mice vaccinated against TNFalpha.

OBJECTIVE: To evaluate the effect in mice with arthritis of active anti-tumour necrosis factor (TNF)alpha immunotherapy based on a keyhole limpet haemocyanin-human TNFalpha heterocomplex (hTNFalpha kinoid or TNFK) adjuvanted in incomplete Freund adjuvant. Immunotherapy was evaluated also with methotrexate. METHODS: Human TNFalpha-transgenic mice received TNFK with or without methotrexate. Follow-up ranged from 6 weeks (short term) to 17 weeks (long term). Arthritis was evaluated clinically and histologically. Monitoring included titration of anti-hTNFalpha antibodies by ELISA and neutralisation assay. RESULTS: Vaccination with TNFK was associated with rapid-onset, long-lasting protection. Long-term results showed significantly milder arthritis in vaccinated animals than in control animals at the peak of the disease. Vaccination was followed by resolution of the clinical evidence of arthritis, contrasting with severe progressive arthritis in the control group. Histological improvements with decreased inflammation and destruction were noted in all immunised groups, even after the shortest follow-up (6 weeks). High titres of neutralising anti-hTNFalpha antibodies were detected as early as the fifth week post immunisation and persisted over time. Methotrexate given concomitantly with the vaccine did not influence either the effect on arthritis or the anti-hTNFalpha antibody titres. CONCLUSION: Anti-cytokine induction of autoimmune protection against chronic hTNFalpha overproduction is an efficient alternative to TNFalpha blockade in experimental arthritis and can be achieved using a TNFK vaccine.

Synoviocyte infection with adeno-associated virus (AAV) is neutralized by human synovial fluid from arthritis patients and depends on AAV serotype.

Intraarticular gene transfer with adeno-associated viral (AAV) vectors may allow efficient therapeutic transgene expression within the joint in diseases such as rheumatoid arthritis (RA), allowing high expression of the protein within the joint, preventing both systemic diffusion and side effects. However, humans demonstrate antibodies against AAV, which can influence gene transfer. To better understand critical obstacles to intraarticular gene therapy with AAV, we have previously shown that synovial fluid (SF) contains IgG to AAV that neutralizes chondrocyte infection in vitro. Our objective was therefore to compare neutralization exerted by SF from RA patients for four different AAV serotypes (AAV serotypes 1, 2, 5, and 8) on human primary synoviocytes. Serotype 2 infected synoviocytes most efficiently followed, in decreasing order, by serotypes 1, 5, and 8. SF from all patients partially inhibited infection of synoviocytes by at least one of the four serotypes. Infection with serotypes 1 and 2 was the most inhibited by SF, whereas inhibition was weak for serotypes 5 and 8. Last, we have shown that inhibition of AAV1/interleukin (IL)-4 infection of synoviocytes by SF could be reversed by increasing the number of AAV1/IL-4 particles, with a dose-dependent effect. We conclude that the most infectious AAV serotypes (1 and 2) in synoviocytes are also the serotypes most neutralized by SF. Thus, serotype 5 seems to demonstrate the best infection efficiency:immunogenicity ratio for local use in articular diseases. These data may be useful for tailoring intraarticular AAV-mediated gene therapy to individual patients.

Interleukin-10-mediated T cell apoptosis during the T helper type 2 cytokine response in murine Schistosoma mansoni parasite infection.

The pathogenesis of infection with the helminth parasite Schistosoma (S) mansoni in mice has been reported to involve a T helper (Th)1 to Th2 cytokine switch, associated with a pathogenic granulomatous response to parasite eggs and to a global defect in Th1-cell effector functions. Here we report that the Th2 cytokine response, which begins 6 weeks after infection, at the time of parasite egg laying (i) does not occur in the context of a genuine Th1 to Th2 cytokine switch, but is associated with a persistent capacity of Th1 (or Th0) cells to secrete IL-2 and IFN-gamma in response to T cell receptor (TCR) stimulation; (ii) is associated, in vitro, with spontaneous death by apoptosis of a significant fraction of the CD4 and CD8 T cells, which is greatly enhanced by TCR stimulation; and (iii) is associated, in vivo, with numerous and large clusters of apoptotic cells in the spleen and in the inflammatory infiltrates surrounding the parasite egg deposits in the liver. The in vitro addition of antibodies to the Th2 cytokine IL-10 had both a preventive effect on TCR-induced T cell apoptosis and an enhancing effect on TCR-induced T cell secretion of Th1 cytokines. Taken together, these findings suggest that the downregulation of Th1-cell-mediated effector functions in S. mansoni-infected mice may not be related to a lack of Th1 cell production, but to a process of IL-10-mediated and activation-induced premature T cell death, that include Th1 (or Th0) cells. Further identification of mechanisms involved in the regulation of T cell apoptosis has implications for the understanding of the pathogenesis of immunosuppression associated with chronic infectious diseases.

Novel pro-inflammatory interleukins: potential therapeutic targets in rheumatoid arthritis.

Among potential targets for nonspecific anti-inflammatory immunointervention, three pro-inflammatory interleukins (ILs) have recently been found to play a pivotal role in rheumatoid arthritis (RA). IL-15 has both chemoattractant and proinflammatory properties and may promote bone destruction. IL-17, a product of T lymphocytes, has proinflammatory effects and induces production of metalloproteinases such as MMP-1. IL-18 not only has proinflammatory, angiogenic, and chemoattractant effects but also promotes cartilage destruction. These cytokines are potential targets for specific or nonspecific anti-inflammatory therapy. Thus, blocking IL-15 by its receptor reduces the severity of experimental collagen-induced arthritis (CIA). In this model, IL-17 levels fall after administration of anti-inflammatory cytokines such as IL-4 or IL-13. Finally, monoclonal anti-IL-18 antibodies prevent streptococcal cell wall arthritis, and IL-18 binding protein, which is a naturally occurring IL-18 inhibitor, prevents CIA.

Therapeutic gene transfer for rheumatoid arthritis.

Rheumatoid arthritis (RA) is a severe autoimmune systemic disease. Chronic synovial inflammation results in destruction of the joints. No conventional treatment is efficient in RA. Gene therapy of RA targets mainly the players of inflammation or articular destruction: TNF-alpha or IL-1 blocking agents (such as anti-TNF-alpha monoclonal antibodies, soluble TNF-alpha receptor, type II soluble receptor of IL-1, IL-1 receptor antagonist), anti-inflammatory cytokines (such as IL-4, IL-10, IL-1), growth factors. In this polyarticular disease, the vector expressing the therapeutic protein can be administered as a local (intra articular injection) or a systemic treatment (extra articular injection). All the main vectors has been used in experimental models, including the more recent lentivirus and adeno-associated virus. Ex vivo gene transfer was done with synovial cells, fibroblasts, T cells, dendritic cells, and different cells from xenogenic origin. In vivo gene therapy is simpler, although less controlled method. Clinical trials in human RA has started with ex vivo retrovirus expressing IL-1 receptor antagonist and have demonstrated the feasibility of the strategy of gene therapy. The best target remains to be determined and extensive researches have to be conducted in pre-clinical studies.

[Gene therapy in rheumatoid polyarthritis: perspectives]. / Thérapie génique dans la polyarthrite rhumatoïde: perspectives.

THE CONCEPT OF GENE THERAPY: Gene therapy is applicable in diseases involving several genes such as rheumatoid arthritis. Gene transfer is the insertion in vivo of genetic material necessary to produce a molecule with therapeutic action. This strategy is currently in experimental stages; feasibility studies in humans are in the preliminary stage. SEVERAL TARGETS: In experimental models of rheumatoid arthritis, the most widely studied target genes are those which code for inflammation inhibitors such as IL-1 receptor antagonists or anti-inflammatory cytokines (IL-4, IL-10, IL-13). Another interesting target would concern genes coding for molecules inhibiting joint destruction (for example metalloprotease inhibitors). VECTORS: The development of high-performance vectors (both viral and nonviral vectors) will greatly improve the expected benefit/risk potential of gene therapy in general. IN RHEUMATOID ARTHRITIS: The particular problem in rheumatoid arthritis is the choice of the transfection site. An articular site would require multiple injections in the different affected joints. A systemic approach would take into account the general disseminated nature of the disease.

Interleukin-6 receptor blockade enhances CD39+ regulatory T cell development in rheumatoid arthritis and in experimental arthritis.

OBJECTIVE: The rationale for blocking interleukin-6 (IL-6) in rheumatoid arthritis (RA) lies chiefly in the proinflammatory effect of this cytokine. Few studies have evaluated the consequences of anti-IL-6 receptor (IL-6R) antibody treatment on Treg cells. This study was undertaken to elucidate the mechanism of action of anti-IL-6R antibody treatment by studying the effects on Treg cells in an experimental arthritis model and in patients with RA. METHODS: Mice with collagen-induced arthritis (CIA) were treated with a mouse anti-IL-6R antibody (MR16-1), and changes in Treg, Th1, and Th17 cells were assessed at key time points during the course of the disease. Peripheral blood from 15 RA patients was collected on day 0 and after 3 months of tocilizumab treatment for flow cytometry analysis of Th17 and Treg cells. RESULTS: In MR16-1-treated mice, Th17 cell frequencies were unchanged, whereas Treg cell frequencies were increased. The Treg cell phenotype showed marked changes, with an increase in the frequency of CD39+ Treg cells in the lymph nodes and spleen. Interestingly, similar CD39+ Treg cell expansion was observed in RA patients who were tocilizumab responders at 3 months, with no change in Th17 cell frequency. Moreover, fluorescence-activated cell-sorted CD39+ Treg cells from responder RA patients were functionally able to suppress the proliferation of conventional T cells. CONCLUSION: In both CIA and RA, the frequency of functionally suppressive CD39+ Treg cells is increased as a result of anti-IL-6R treatment, whereas Th17 cells are unaffected. The modification of Treg cell frequency and phenotype may be one of the mechanisms involved in the therapeutic effect of IL-6 blockade in RA.

Intra-articular electrotransfer of plasmid encoding soluble TNF receptor variants in normal and arthritic mice.

BACKGROUND: Anti-inflammatory gene therapy is promising in inflammatory diseases such as rheumatoid arthritis (RA). We have previously demonstrated that intra-muscular (i.m.) electrotransfer (ET) of plasmids encoding three different human tumor necrosis factor-alpha-soluble receptor I variants (hTNFR-Is) exert protective effects in an experimental RA model. However, such a systemic approach could be responsible for side effects. The present study aimed at performing an intra-articular (i.a.) gene therapy by electrotransfer using the hTNFR-Is plasmids. METHODS AND RESULTS: We evaluated targeting of mice joints by CCD optical imaging after i.a. ET of a luciferase-encoding plasmid and we showed that ET led to strongly increased transgene expression in a plasmid dose-dependent manner. Moreover, articular and seric hTNFR-Is was detectable for 2 weeks. As expected, systemic hTNFR-Is rates were lower after i.a. ET than after i.m. ET. A longer protein secretion could be achieved with several i.a. ETs. Also, we observed that hTNFR-Is expression within arthritic joints was slightly higher than in normal joints. CONCLUSIONS: In collagen-induced arthritis (CIA), a mouse model for RA, we demonstrated that hTNFR-Is/mIgG1-encoding plasmid i.a. ET decreased joint destruction in the ankles. In conclusion, our results suggest that local TNFR-Is gene therapy may play a role in decreasing joint destruction in CIA.

Immune responses to gene therapy vectors: influence on vector function and effector mechanisms.

Circumventing the immune response to the vector is a major challenge with all vector types. Viral vectors are the most likely to induce an immune response, especially those, like adenovirus and AAV, which express immunogenic epitopes within the organism. The first immune response occurring after vector transfer emerges from the innate immune system, mainly consisting in a rapid (few hours) inflammatory cytokines and chemokines secretion around the administration site. This reaction is high with adenoviral vectors and almost null with AAV. It is noteworthy that plasmid DNA vectors, because of CpG stimulatory islets, also stimulate the innate immunity via the stimulation of TLR receptors on leukocytes. Specific immune response leading to antibodies production and T lymphocytes activation also occurs within a few days after vector introduction. Capsid antigens are mostly responsible for specific immunity toward adenoviruses, and are also involved in the response against AAV. In the former case only, however, viral gene-encoded proteins can also be immunogenic. The pre-existing humoral immunity coming from early infections with wild-type AAV or adenovirus can prevent efficient gene transfer with the corresponding vectors. In all cases, some parameters like route of administration, dose, or promoter type have been extensively described as critical factors influencing vector immunity. Strategies to fight against vector-induced immunity can come from the immunology field, since tolerance induction or immunosuppression are a possibility. Alterations to vector structure have also been extensively performed to circumvent the immune system and thus enhance gene transfer efficiency and safety.

[Treatment of inflammation with anti-inflammatory cytokines: example in models of auto-immune diseases]. / Traitement de l'inflammation par des cytokines anti-inflammatoires: l'exemple des modèles de pathologies auto-immunes.

Cytokines may exert anti-inflammatory properties, allowing the hypothesis of their potential therapeutic use. Targeting the cytokines balance may modify the course of subsequent inflammatory events in an autoimmune disease. Many data are available. Interferon-gamma can be blocked by an anti-interferon-gamma monoclonal antibody: if the treatment is administered in the early phase of an autoimmune disease such as collagen-induced arthritis, the course of the disease is worsened; if the treatment is given later, the disease can be improved; these results are mirrored by treatment with high doses of the cytokine itself. Pharmacologic effects of antiinflammatory cytokines such as interleukin (IL)-4, IL-10 or IL-13 are a protection against several experimental autoimmune diseases. The very short half-life of cytokines makes them difficult and expensive to use directly; in such occurrence, high quantities have to be frequently injected. In this context, gene therapy appears as an effective alternative solution: the transfection of cells with cytokines genes (e.g. either IL-4 or IL-13) then the engraftment of these vectors in animals, permit the in vivo secretion of high levels of cytokines, and result in the protection of the animals from the development of the disease.

[Experimental gene therapy in models of inflammatory arthritis]. / Thérapie génique expérimentale dans les modèles d'arthrite inflammatoire.

Gene therapy is rheumatoid arthritis is presently in an experimental phase. Genes encoding for antiinflammatory proteins can be transfected in joint cells. Therefore gene encoding for interleukin-1 receptor antagonist has been transfected into synovial cells or into chondrocytes. Gene expression can be maintained as long as 8 weeks after cell transfer. We have reported that clinical and histopathological parameters of collagen-induced arthritis (an animal model of rheumatoid arthritis) can be reduced by engraftment of CHO cells transfected with genes encoding for antiinflammatory cytokines such as interleukine-4 or interleukine-13. Some of the questions which need to be assessed before planning clinical trial are the choice of vectors and the most efficient target cells; and the genes to be transfected have to be chosen among many candidates.

Adeno-associated virus-mediated delivery of IL-4 prevents collagen-induced arthritis.

Immunomodulation of autoimmune inflammatory diseases like rheumatoid arthritis can be achieved by anti-inflammatory T2 cytokines such as interleukin (IL)-4 administered by gene therapy. In this study we investigated the efficiency of adeno-associated viruses (AAV) vectors in collagen-induced arthritis (CIA). After injection of AAV-LacZ in the tarsus area of mice, the expression of the transgene was localized in the deep muscles cells near the bone. LacZ expression was found in liver, heart and lung after i.m. injection of AAV-LacZ, showing a spread of the vector over the body. Anti-AAV neutralizing antibodies were detected in the serum after i.m. injection of AAV-LacZ, but they did not alter the transgene expression after re-administration of AAV-LacZ. Long-term IL-4 expression persisted 129 days after intra-muscular injection of 3.7 x 10(10) or 11.2 x 10(10) AAV-IL-4 p.p. (average 7.7 or 17.5 pg IL-4/mg proteins, respectively). More importantly, the treatment of CIA with AAV-IL-4 vector in mice produced a therapeutic benefit, since we show a diminished prevalence of the disease, a significant reduction in paw swelling, attenuated histological synovitis and a 10 days delayed onset of arthritis. This is the first evidence that AAV vector-mediated gene therapy using a T2 cytokine is efficient in an animal model of rheumatoid arthritis.

Attenuation of collagen-induced arthritis in mice by treatment with vector cells engineered to secrete interleukin-13.

The anti-inflammatory effects of the recently identified cytokine interleukin (IL)-13 on collagen-induced arthritis (CIA) was explored and compared to those of IL-4 using systemic administration of these cytokines via two injections of xenogeneic vector cells transfected with a plasmid construct. CIA was induced in DBA/I mice by immunization with native bovine type II collagen (CII). Chinese hamster ovary (CHO) fibroblasts transfected with the mouse IL-13 or IL-4 genes were inoculated subcutaneously on days 10 and 25 post-priming with CII and mice were monitored for signs of arthritis by observers unaware of the status of the animal. Incidence and severity of CIA were significantly reduced in the groups of mice treated with IL-13 and IL-4 gene-transfected CHO cells compared to control groups receiving nontransfected cells. Expression of various cytokines in spleen cells from individual mice was assessed by quantitative reverse transcriptase-polymerase chain reaction at different times after immunization. Our data show that IL-13-induced suppression of CIA coincided with a decreased TNF-alpha mRNA expression in the spleen of treated animals. This may explain at least partially the anti-inflammatory effects of IL-13 in CIA. Thus, our results may have important implications for the clinical use of T helper (Th)1/TH2 modulatory cytokines as therapeutic agents in the treatment of autoimmune diseases.

Encapsulation in hollow fibres of xenogeneic cells engineered to secrete IL-4 or IL-13 ameliorates murine collagen-induced arthritis (CIA).

A strategy of gene therapy using IL-4 or IL-13 xenogeneic transfected cells encapsulated into permeable hollow fibres (HF) was used to treat CIA. Hydrogel-based hollow fibres were obtained from AN-69 copolymer, already known for its biocompatibility and tolerance in rodents. Permeability to IL-4 and lack of cell leakage from the fibres were ascertained in vitro and in vivo. Chinese hamster ovary (CHO) fibroblasts transfected with mouse IL-4 gene were encapsulated in HF (6.25 x 105 cells/HF). IL-4 was detected in vitro in the culture supernatant of filled fibres for at least 19 days. IL-4 or IL-13 transfected CHO cells encapsulated in HF were implanted in the peritoneum of mice on days 11-13 after immunization with type II collagen. Control mice were treated with fibre containing CHO cells transfected with beta-galactosidase (betagal) gene; a positive control group consisted of mice treated by subcutaneous injection of 106 cells on days 10 and 25. Mice were monitored for signs of arthritis by observers unaware of the status of animals. Results of these experiments indicate that severity of the articular disease was significantly reduced in the groups of mice treated with CHO/IL-4 or CHO/IL-13 cells encapsulated in HF, compared with control groups receiving CHO/betagal cells encapsulated in HF. Histological analysis confirmed these data and extended them to a better inhibitory effect of encapsulated cells compared with free cells on inflammatory and destructive joint disease. Moreover, such long-term treatment with HF was well tolerated; macroscopic and histological aspects of peritoneal cavity were moderately inflammatory. Thus, our results may have important implications for clinical use of gene transfected cells as therapeutic agents in the treatment of autoimmune diseases.

Biphasic effect of interferon-gamma in murine collagen-induced arthritis.

Interferon-gamma (IFN-gamma) exerts both enhancing and suppressing influences on collagen-induced arthritis (CIA), depending on the route and protocol of administration. To study the role of IFN-gamma on the autoimmune process of CIA, we treated DBA/1 mice with two different rat monoclonal antibodies (mAb) to murine IFN-gamma. Treatments, given twice weekly for 4 weeks, consisted of intraperitoneal injections of either mAb. In early treatments, starting from the day of immunization with type II collagen (CII), the severity of arthritis was reduced in both groups of anti-IFN-gamma-treated mice compared with control groups. Moreover, anti-CII antibody levels decreased in the sera of these mice. CIA was also down-regulated in mice treated from days 14 or 28 post immunization. In contrast, late treatments with anti-IFN-gamma mAb either induced aggravating effects, or did not affect the course of the disease. On the other hand, administration of high doses (8 x 10(4) U three times/week) of rat recombinant IFN-gamma exerted a transient increase of CIA severity. These findings suggest that IFN-gamma may play a critical role during both the induction and the course of CIA, first enhancing the immune response, and then regulating the arthritis process.