Macrophage migration inhibitory factor is involved in the pathogenesis of collagen type II-induced arthritis in mice
Macrophage migration inhibitory factor is involved in the pathogenesis of collagen type II-induced arthritis in mice. was significantly inhibited by anti-MIF MoAb treatment ( 0001). AIA was also significantly inhibited by anti-MIF MoAb ( Trigonelline 002). DEX treatment induced a dose-dependent inhibition of AIA, which was significant at 02 mg/kg ( 005). MIF treatment reversed the effect of therapeutic DEX on AIA ( 0001). DEX also significantly inhibited DTH reactions ( 005) but rMIF had no effect on this effect of DEX. DTH and AIA are MIF-dependent models of inflammation and arthritis. The reversal of glucocorticoid suppression of AIA by MIF supports the concept that MIF is a counter-regulator of glucocorticoid control of synovial inflammation. Although DTH was observed to be MIF-dependent and glucocorticoid-sensitive, rMIF had no reversing effect on the suppression of DTH by glucocorticoids. This suggests that inflammatory processes in specific tissues may respond differently to MIF in the presence of glucocorticoids. model of arthritis. We initially established that the model of interest was dependent upon MIF and was inhibited by glucocorticoids. The chief finding of this study is that MIF does indeed reverse the effects of therapeutic glucocorticoids on a model of arthritis. This finding is consistent with the hypothesis that MIF is a key counter-regulator of glucocorticoid actions, and implicates it in steroid resistance. MATERIALS AND METHODS Antigen-induced arthritis (AIA) was induced in male C57Bl6 mice (9C12 weeks). Groups of at least six animals were used for all studies. Methylated bovine serum albumin (mBSA; 100 g) (Sigma, Sydney, Australia) in 100 l Freund’s complete adjuvant (Sigma) was injected subcutaneously on each flank and (04 109 organisms/400 l saline per mouse) (Bioscientific, Sydney, Australia) was injected intraperitoneally. Antigen challenge was performed 8 days later with 60 g mBSA/10 l saline administered by intra-articular injection into the left knee joint. As a control, an equal volume of saline was injected into the right knee joint. All reagents were screened for endotoxin using the assay (Sigma). Cutaneous DTH was induced by intradermal injection of 50 g mBSA/20 l saline (right footpad) or saline alone in the control left footpad on day 4 post intra-articular antigen challenge. Skin thickness was measured on day 5 using calibrated skin fold callipers, and results expressed as the difference in skin thickness (mm) between mBSA- and saline-injected sites. Treatment of mice with dexamethasone (DEX; Sigma) 002C02 mg/kg and rMIF (1 mg/kg) was performed by daily i.p. injection on days 3 and 4 post-antigen challenge. Treatment with anti-MIF MoAb, which recognizes murine MIF [8,14], was performed on days 0, 2 and 4 after antigen challenge. Control mice were treated with isotype-matched IgG1. Mice were killed on day 5 post-antigen challenge. Serum was obtained from tail vein puncture prior to killing. Synovium from Trigonelline knee joints was collected for frozen section and routine haematoxylin and eosin (HCE) staining as described . Briefly, specimens Trigonelline of whole mouse knee joint were fixed in perodate-lysine-paraformaldehyde (PLP) and decalcified in a solution of 75% polyvinylpyrrolidone (PVP; Sigma) and 10% ethylenediamine tetra acetic acid (EDTA; BDH Chemicals, Sydney, Australia). Decalcified joints were embedded in OCT (Tissue Tek, Westhaven, CT) and frozen. Frozen tissue was cut into 7- m sections using a cryostat (Reichardt-Jung Cryocut 1800; Nussloch, Germany). Histological counts were obtained by counting cells in two to four 02-mm2 areas of the synovium using a graticule. Counts were averaged and results expressed as mean s.e.m. cells/02 mm2. RESULTS Compared with non-pre-immunized animals (313 34 cells/mm2) or to contralateral saline-injected joints in Antxr2 pre-immunized animals (323 13 cells/mm2), intra-articular injection of mBSA in pre-immunized animals was associated with a significant increase in synovial cellularity (694 55 cells/mm2, 0005) (Figs 1 and ?and2).2). Anti-MIF MoAb 30 mg/kg treatment completely prevented AIA (368 24 cells/mm2, 002) (Figs 1 and ?and2),2), while a weaker inhibitory effect of anti-MIF MoAb 15 mg/kg was non-significant (653 70 cells/mm2, NS). All control animals received isotype-matched control mouse IgG in matched concentrations. Administration of rMIF 1 mg/kg induced a trend towards increased severity of AIA (791 69 Trigonelline cells/mm2) which did not reach statistical significance (= 10) (Fig. 2). Open in a separate window Fig. 1 Mice received were pre-immunized with methylated bovine serum albumin (mBSA) 8 days prior to intra-articular injection into the knee of (a) saline or (b,c) mBSA. Arthritis on day 5 after intra-articular injection was analysed.