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Objective: To investigate the indirect effects of anti-CD4 treatment on the functions of macrophages (CD4 2 in mice) in the acute and early chronic phase of mouse antigen induced arthritis (AIA). Methods: C57BL/6 mice with AIA were treated intraperitoneally with the anti-CD4 mAb GK1.5 or control rat IgG on days 21, 0, 1, 3, 5, and 7. Proinflammatory cytokines (IL1b, IL6, and TNFa) were quantified by sandwich ELISA in joint extracts, serum, and supernatants of ex vivo stimulated spleen/lymph node cells or peritoneal macrophages (+LPS/IFNc). Nitric oxide (NO) levels in supernatants of ex vivo stimulated peritoneal macrophages were measured by the Griess reaction. Proteolytic activity in joint homogenates was analysed by gelatin, casein, and elastin zymography, and substrate assays. Results: Anti-CD4 treatment significantly reduced joint swelling in acute (days 3, 5) and early chronic AIA (day 7) and diminished inflammation and destruction scores in late chronic AIA (day 21). On day 3, anti-CD4 treatment significantly reduced IL6 levels in all compartments. IL1b was reduced in joint extracts, unaffected in serum or cells from lymphoid organs, and increased in stimulated peritoneal macrophages. TNFa was significantly increased in the joints, decreased in serum, and otherwise unchanged. NO production by stimulated peritoneal macrophages was significantly reduced by anti-CD4 treatment. Lower activity of matrix metalloproteinases and neutrophil elastase was seen in joint extracts of anti-CD4 treated animals than in IgG treated AIA controls. Conclusion: CD4+ T cell directed treatment had strong local and systemic effects on macrophages. These indirect effects may contribute to the reduction of destructive mediators/joint destruction in AIA.

Conclusion. MIF is an important Th1 and Th2 cell-derived proinflammatory cytokine that stimulates MMP expression in FLS from arthritic mice, and therefore inhibition of MIF might be a promising target for novel therapeutic strategies in human RA.Macrophage migration inhibitory factor (MIF) is a structurally unique cytokine that is a critical mediator of several acute and chronic inflammatory diseases. Of note, MIF has been prominently detected in supernatants of T cells and in macrophages (1-5). Although it was originally described as a soluble factor that inhibits the undirected migration of macrophages, an effect known to participate in delayed-type hypersensitivity reactions (1,6), it has become evident that MIF functions in a chemokine-like manner to trigger the directed migration of leukocytes, an effect that involves CXCR2 ligation and the induction of monocyte chemoattractant protein 1 release by endothelial cells (7,8).

Cartilage and bone degradation, observed in human rheumatoid arthritis (RA), are caused by aberrant expression of proteinases, resulting in an imbalance of these degrading enzymes and their inhibitors. However, the role of the individual proteinases in the pathogenesis of degradation is not yet completely understood. Murine antigen-induced arthritis (AIA) is a well-established animal model of RA. We investigated the time profiles of expression of matrix metalloproteinase (MMP), cathepsins, tissue inhibitors of matrix metalloproteinases (TIMP) and cystatins in AIA. For primary screening, we revealed the expression profile with Affymetrix oligonucleotide chips. Realtime polymerase chain reaction (PCR) analyses were performed for the validation of array results, for tests of more RNA samples and for the completion of the time profile. For the analyses at the protein level, we used an MMP fluorescence activity assay and zymography. By a combination of oligonucleotide chips, realtime PCR and zymography, we showed differential expressions of several MMPs, cathepsins and proteinase inhibitors in the course of AIA. The strongest dysregulation was observed on days 1 and 3 in the acute phase. Proteoglycan loss analysed by safranin O staining was also strongest on days 1 and 3. Expression of most of the proteinases followed the expression of pro-inflammatory cytokines. TIMP-3 showed an expression profile similar to that of anti-inflammatory interleukin-4. The present study indicates that MMPs and cathepsins are important in AIA and contribute to the degradation of cartilage and bone.

The transcription factor STAT-1 (signal transducer and activator of transcription-1) plays a pivotal role in the expression of inflammatory gene products involved in the pathogenesis of arthritis such as various cytokines and the CD40/CD40 ligand (CD40/CD40L) receptor-ligand dyad. The therapeutic efficacy of a synthetic decoy oligodeoxynucleotide (ODN) binding and neutralizing STAT-1 was tested in murine antigen-induced arthritis (AIA) as a model for human rheumatoid arthritis (RA). The STAT-1 decoy ODN was injected intra-articularly in methylated bovine serum albumin (mBSA)-immunized mice 4 h before arthritis induction. Arthritis was evaluated by joint swelling measurement and histological evaluation and compared to treatment with mutant control ODN. Serum levels of pro-inflammatory cytokines, mBSA-specific antibodies and auto-antibodies against matrix constituents were assessed by enzyme-linked immunosorbent assay (ELISA). The transcription factor neutralizing efficacy of the STAT-1 decoy ODN was verified in vitro in cultured synoviocytes and macrophages. Single administration of STAT-1 decoy ODN dose-dependently suppressed joint swelling and histological signs of acute and chronic arthritis. Delayed-type hypersensitivity (DTH) reaction, serum levels of interleukin-6 (IL-6) and anti-proteoglycan IgG titres were significantly reduced in STAT-1 decoy ODN-treated mice, whereas mBSA, collagen type I and type II specific immunoglobulins were not significantly affected. Intra-articular administration of an anti-CD40L (anti-CD154) antibody was similarly effective. Electrophoretic mobility shift analysis (EMSA) of nuclear extracts from synoviocytes incubated with the STAT-1 decoy ODN in vitro revealed an inhibitory effect on STAT-1. Furthermore, the STAT-1 decoy ODN inhibited the expression of CD40 mRNA in stimulated macrophages. The beneficial effects of the STAT-1 decoy ODN in experimental arthritis presumably mediated in part by affecting CD40 signalling in macrophages may provide the basis for a novel treatment of human RA. IndroductionHuman rheumatoid arthritis (RA) is a chronic systemic disorder of unknown aetiology, characterized by intimal lining layer hyperplasia, infiltration of the sublining area by macrophages, T and B lymphocytes, plasma cells and other inflammatory cells as well as progressive destruction of joint structures [1,2]. Despite the uncertainty about its aetiology, RA is thought to be an immune-mediated disease promoting inflammation and tissue destruction. Besides the pro-inflammatory cytokines tumour necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6, which mainly derive from macrophages, a dominant T-helper (Th)1-response is associated with the disease, which is characterized by an imbalance of interferon (IFN)-γ over 4]. AIA = antigen-induced arthritis; AP = alkaline phosphatase; bp = base pairs; CD40L = CD40 ligand (CD154); DMEM = Dulbecco's modified Eagle's medium; DTH = delayed-type hypersensitivity; ELISA = enzyme-linked immunosorbent assay; EMSA = electrophoretic mobility s...