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Splice variants of CD74 differentially modulate the activity of cathepsin L (CTSL). As CD74 and CTSL participate in the pathogenesis of inflammatory diseases such as rheumatoid arthritis (RA), we determined whether splice variants of CD74 could be biomarkers of disease activity. Gene expression was measured in mice with collagen-induced arthritis using quantitative PCR (qPCR). In vitro studies using murine macrophage/DC-lineage cells determined the relative influence of macrophage phenotype on isoform expression and the potential to produce CTSL in response to TNF. CD74 splice variants were measured in human RA synovium and RA patients’ monocytes. In arthritic mice, the expression of the p41 CD74 isoform was significantly higher in severely affected paws compared with unaffected paws or the paws of naïve mice; the p41 isoform significantly correlated with the expression of TNF in arthritic paws. Compared with M2-like macrophages, M1-like macrophages expressed increased levels of CD74 and had higher expression, secretion and activity of CTSL. RA patients that responded to TNF blockade had significantly higher expression levels of CD74 in circulating monocytes after treatment, compared with non-responders. The expression of the human CD74 isoform a was significantly higher in RA synovia, compared with osteoarthritis synovia, and was associated with CSTL enzymatic activity. This study is the first to demonstrate differential expression of the CD74 p41 isoform in an auto-immune disorder and in response to therapy. The differential expression of CD74 splice variants indicates an association, and potentially a mechanistic role, in the pathogenesis of RA.

OBJECTIVE: Tumor necrosis factor (TNF) signals via 2 receptors, TNFR type I (TNFRI) and TNFRII, with distinct cellular distribution and signaling functions. In rheumatoid arthritis (RA), the net effect of TNFR signaling favors inflammatory responses while inhibiting the activity of regulatory T cells. TNFRII signaling has been shown to promote Treg cell function. To assess the relative contributions of TNFRI and TNFRII signaling to inflammatory and regulatory responses in vivo, we compared the effect of TNF blockade, hence TNFRI/II, versus TNFRI alone in collagen-induced arthritis (CIA) as a model of RA. METHODS: Mice with established arthritis were treated for 10 days with anti-mouse TNFRI domain antibody (dAb; DMS5540), an isotype control dAb (DMS5538), or murine TNFRII genetically fused with mouse IgG1 Fc domain (mTNFRII-Fc) beginning on the day of arthritis onset, and disease progression was monitored. Systemic cytokine concentrations and numbers of T cell subsets in lymph nodes and spleens were measured, and intrinsic Treg cell function was determined by ex vivo suppression assays. RESULTS: Progression of CIA was suppressed similarly by TNFRI (DMS5540) and TNFRI/II (mTNFRII-Fc) blockade. However, blockade of TNFRI/II led to increased effector T cell activity, which was not observed after selective TNFRI blockade, suggesting an immunoregulatory role of TNFRII. In support of this, TNFRI blockade, but not TNFRI/II blockade, expanded and activated Treg cells. Furthermore, a dramatic increase in expression of the Treg cell signature genes FoxP3 and TNFRII was observed in joints undergoing remission, which supports the notion that these molecules have a physiologic role in the resolution of inflammation. CONCLUSION: We propose that a therapeutic strategy that targets TNFRI while sparing TNFRII has the potential to both inhibit inflammation and promote Treg cell activity, which might be superior to TNF blockade.

There is increasing interest in the use of combination therapy for rheumatoid arthritis and in the possibility of combining the conventional drug approach with newer biological therapies. Animal models of arthritis provide important tools for evaluating novel forms of therapy and for eludicating mechanisms of drug action. In this paper, we review the results of our own research into combination therapy in collagen-induced arthritis using biological therapies such as anti-tumor necrosis factor alpha, anti-CD4, and anti-interleukin 12 monoclonal antibodies, and small molecular weight compounds such as cyclosporin and the phosphodiesterase IV (PDE IV) inhibitor rolipram.

Type II collagen-induced arthritis (CIA) is an animal model of rheumatoid arthritis that has been used extensively to address questions of disease pathogenesis and to validate novel therapeutic targets. Susceptibility to CIA is strongly associated with major histocompatibility complex class II genes, and the development of arthritis is accompanied by a robust T- and B-cell response to type II collagen. The main pathological features of CIA include proliferative synovitis with infiltration of inflammatory cells, pannus formation, cartilage degradation, erosion of bone and fibrosis. Pro-inflammatory cytokines, such as tumour necrosis factor alpha and interleukin-1beta, are expressed in the arthritic joints in both murine CIA and human rheumatoid arthritis, and blockade of these molecules results in amelioration of disease. Hence, there is a great deal of interest in the development of small-molecular-weight inhibitors of pro-inflammatory cytokines. There is also interest in the development and testing of drugs with the capacity to modulate the immune pathways involved in driving the inflammatory response in arthritis. For these reasons, there is a need to monitor the effect of novel treatments on cytokine expression in vivo. In this review, we outline the various techniques used to detect cytokines in experimental arthritis and describe how these techniques have been used to quantify changes in cytokine expression following therapeutic intervention.

OBJECTIVE: Functionally impaired Treg cells expressing abnormally low levels of CTLA-4 have been well documented in rheumatoid arthritis (RA). However, the molecular defect underlying this reduced expression is unknown. The aims of this study were to assess the role of DNA methylation in regulating CTLA-4 expression in Treg cells isolated from RA patients and to elucidate the mechanism of their reduced suppressor function. METHODS: CTLA-4 expression in Treg cells from RA patients and healthy controls was measured by quantitative polymerase chain reaction (PCR) and flow cytometry. Methylation of the CTLA-4 gene promoter was analyzed by bisulfite-specific PCR, followed by sequencing. Methylation-dependent transcriptional activity of the CTLA-4 gene promoter was measured by luciferase assay, and NF-AT binding to the CTLA-4 gene promoter was determined by chromatin immunoprecipitation. The role of CTLA-4 expression in controlling Teff cells was analyzed using an autologous mixed lymphocyte reaction. RESULTS: Down-regulation of CTLA-4 expression in Treg cells from RA patients was caused by methylation of a previously unidentified NF-AT binding site within the CTLA-4 gene promoter. As a consequence, Treg cells were unable to induce expression and activation of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO), which in turn resulted in a failure to activate the immunomodulatory kynurenine pathway. CONCLUSION: We show for the first time that epigenetic modifications contribute to defective Treg cell function in RA through an inability to activate the IDO pathway. Therefore, this study sets a precedent for investigating potential therapeutic strategies aimed at reinforcing the IDO pathway in RA patients.

Kennedy Institute of Rheumatology, London, United Kingdom. Collagen-induced arthritis (CIA) is an experimental model of arthritis widely used to dissect the pathogenesis of human rheumatoid arthritis and to identify potential therapeutic targets. Among these, TNF-alpha has been recognized to play an important role. Here we investigate the feasibility and therapeutic efficacy of prolonged blockade of TNF-alpha activity through the adenovirus-mediated gene delivery of a dimeric chimeric human p55 TNFR-IgG fusion protein and compare it to protein therapy in established CIA. A single i.v. administration of the replication-deficient adenovirus yielded microgram serum levels of the chimeric fusion protein and ameliorated CIA for 10 days. Subsequently, benefit was lost and a rebound to greater inflammatory activity was observed despite the continual presence of bioactive TNFR fusion protein. A similar trend was also observed in mice injected directly with comparable amounts of a human TNFR-IgG fusion protein, whereas the administration of a control adenovirus-encoding beta-galactosidase or of a control human IgG1 protein did not significantly affect the disease course. The mechanisms of the rebound of CIA were investigated, and augmented Ab response to collagen type II and TNFR were identified as potential causes. Our results confirm the feasibility of adenovirus-mediated gene delivery of cytokine inhibitors in animal models of autoimmune diseases for investigational purposes and highlight the importance of prolonged studies. Further investigations are needed to optimize ways of exploiting the potential of adenoviral gene therapy in RA.

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