Immunoregulatory properties of interferon-γ in collagen-induced arthritis : effects on CD4+ CD25+ regulatory T cell function and neutrophil infiltration

Abstract

Over a period of several years, work in our laboratory has addressed the role of interferon-g (IFN-g) in collagen-induced arthritis (CIA), a well-characterized experimental model for rheumatoid arthritis (RA) in humans. Mice lacking a functional IFN-g receptor (IFN-gR KO) were found to be more susceptible to CIA than wild-type mice: disease onset is accelerated and arthritic symptoms are more severe. CD4+CD25+ regulatory T (Treg) cells have proven to be important in the control of various autoimmune diseases. In addition, aggrevated disease in IFN-gR KO mice is associated with a high proportion of neutrophils in the joints. In this thesis we therefore investigated the possibilities that IFN-g exerts its protective effect in CIA through stimulation of Treg cells or through inhibition of neutrophil-specific chemokines. By rendering wild-type mice deficient in Treg cells and by a single transfer of Treg cells in wild-type mice, we demonstrated the importance of Treg cells in the pathogenesis of CIA. Wild-type mice treated with depleting anti-CD25 antibodies developed a significantly more severe arthritis, comparable to the disease course in IFN-gR KO mice. Thus, we proposed that the higher susceptibility of IFN-gR KO mice to CIA might be ascribed to defects in the production or function of these Treg cells. Having demonstrated that IFN-g receptor deficiency did not affect the number of CD4+CD25+ cells in the central and peripheral lymphoid tissues, nor their potential to suppress effector T (Teff) cell proliferation in vitro, we examined the effect of immunization on Treg cell numbers and function. After immunization with collagen type II (CII) in complete Freund’s adjuvant (CFA), the capacity of Treg cells to suppress TCR-triggered proliferation of Teff cells was significantly lower as compared to that of naive Treg cells. In addition, suppressive activity of Treg cells became significantly more impaired in the absence of IFN-g signaling. Accordingly, expression of Foxp3, a highly specific marker for Treg cells, was lower in Treg cells obtained from immunized IFN-gR KO mice. We completed this part of the study by showing that the effect of endogenous IFN-g, which accounts for the more suppressive activity in immunized wild-type as compared to IFN-gR KO mice, concerns both Treg cells and accessory cells. Together, these data demonstrate that the decrease in Treg cell activity in CIA is counter-regulated by endogenous IFN-g. Since a single transfer of Treg cells significantly improved clinical symptoms of arthritis without affecting humoral responses, we investigated whether the effects of Treg cells in CIA may rely on the inhibition of osteoclastogenesis, a major pathogenic process in CIA. Osteoclast differentiation was found to be significantly decreased in the presence of Treg cells. Inhibition of osteoclastogenesis was accompanied by increased expression levels of cytokines inhibiting osteoclastogenesis, including granulocyte macrophage colony-stimulating factor (GM-CSF), IL-5 and IL-10. These data provide an explanation for the beneficial effect of Treg cells in CIA and suggest that Treg cells may be used for the treatment of RA. In a subsequent part of our work we investigated the mechanism underlying the excessive proportion of neutrophils in the inflammatory lesions of IFN-gR KO mice. Neutrophils are considered important in the pathogenesis of CIA, for example by their ability to destroy cartilage. We documented significantly increased levels of myeloperoxidase and matrix metalloproteinase-9 in synovia of arthritic IFN-gR KO mice as compared to wild-type counterparts, thereby quantificating the higher infiltration of neutrophils in the joints of IFN-gR KO mice. The excessive proportion of neutrophils in synovia of arthritic IFN-gR KO mice could be explained by significantly increased levels of the CXC chemokine granulocyte chemotactic protein-2 (GCP-2), a major neutrophil-attracting chemokine in mice that is considered to be the functional equivalent of IL-8 in humans. We further demonstrated that heat-killed mycobacteria present in CFA elicit production of GCP-2 in mouse embryo fibroblast (MEF) cultures, and that this production is inhibited by IFN-g. Inhibition of GCP-2 production by IFN-g was found to be signal transducer and activator of transcription-1 (STAT-1) dependent. In addition we found that IL-17, known to be important in the pathogenesis of arthritis, synergizes with mycobacteria for the production of GCP-2. Again, IFN-g was found to inhibit this production of GCP-2. IFN-gR KO mice treated with neutralizing anti-GCP-2 antibodies were completely protected from CIA, indicating the in vivo importance of GCP-2 in the pathogenesis of CIA. These data support the notion that one of the mechanisms whereby endogenous IFN-g mitigates the manifestations of CIA consists in inhibiting production of GCP-2, thereby limiting mobilisation and infiltration of neutrophils, which are important actors in joint inflammation. To summarize our investigations, we can state that IFN-g exerts its protective effect in CIA in part by inhibition of the decrease in Treg cell activity elicited by immunization with CII in CFA. The protective effect of Treg cells in CIA could be explained by inhibition of osteoclastogenesis. In addition, IFN-g was found to limit the infiltration of neutrophils at the site of inflammation through downregulation of the production of GCP-2. Our data may provide an explanation for the protective effect of IFN-g in other autoimmune models that rely on the use of CFA.status: publishe