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SUMMARYBiomarker quantification in disease tissues from animal models of rheumatoid arthritis (RA) can help to provide insights into the mechanisms of action of novel therapeutic agents. In this study we validated the kinetics of IL-1 b , TNF-a and IL-6 mRNA and protein expression levels in joints from DBA/ 1OlaHsd murine collagen-induced arthritis (CIA) and Lewis rat Streptococcal cell wall (SCW)-induced arthritis by real-time polymerase chain reaction (PCR) TaqMan® and Enzyme-linked immunosorbent assay (ELISA). Prednisolone was used as a reference to investigate any correlation between clinical response and cytokine levels at selected time-points. To our knowledge this is the first report showing a close pattern of expression between mRNA and protein for IL-1 b and IL-6, but not for TNF-a , in these two models of RA. The kinetics of expression for these biomarkers suggested that the optimal sampling time-points to study the effect of compounds on both inflammation and cytokine levels were day 4 postonset in CIA and day 3 after i.v challenge in SCW-induced arthritis. Prednisolone reduced joint swelling through a mechanism associated with a reduction in IL-1 b and IL-6 protein and mRNA expression levels. At the investigated time points, protein levels for TNF-a in arthritic joints were lower than the lower limit of detection of the ELISA, whereas mRNA levels for this cytokine were reliably detected. These observations suggest that RT-PCR TaqMan® is a sensitive technique that can be successfully applied to the quantification of mRNA levels in rodent joints from experimental arthritis models providing insights into mechanisms of action of novel anti-inflammatory drugs.Keywords animal models mice rats collagen-induced arthritis SCW-induced arthritis cytokines interleukins prednisolone

Objective. To characterize chronic murine pristane-induced arthritis (PIA) with regard to the response to antirheumatic agents, expression levels of proinflammatory cytokines, and immunopathologic features.Methods. Male DBA/1 mice were injected intraperitoneally with pristane oil to induce a chronic polyarthritis, which was monitored by visual scoring. Serum antibody and splenocyte responses to a panel of putative joint-derived autoantigens were measured. Whole paws were evaluated postmortem for changes in the levels of proinflammatory cytokines tumor necrosis factor ␣ (TNF␣), interleukin-1␤ (IL-1␤), and IL-6 by enzymelinked immunosorbent assay, and standard histopathology techniques were used to determine joint structural changes. Therapeutic studies were performed for up to 8 weeks of dosing with prednisolone, methotrexate, 3 nonsteroidal antiinflammatory drugs (celecoxib, diclofenac, and indomethacin), a p38 MAPK inhibitor, SB242235, and human soluble TNF receptor (sTNFR; etanercept) and murine sTNFR fusion proteins.Results. Antibody and cellular responses to the putative joint autoantigens revealed a broad extent of autoimmunity in PIA. TNF␣, IL-1␤, and IL-6 were all persistently up-regulated in PIA joints. Prednisolone, methotrexate, celecoxib, indomethacin, and SB242235 all significantly reduced the arthritis scores. Etanercept was ineffective in reducing the arthritis scores, whereas murine sTNFR produced a significant, but nonsustained, benefit. Only prednisolone significantly reduced the expression of TNF␣, IL-1␤, and IL-6 in the joints. Prednisolone and methotrexate demonstrated the most effective joint protection.Conclusion. We have markedly extended the characterization of PIA as a murine model of chronic inflammatory arthritis by demonstrating cellular and humoral autoantigenicity, elevation of clinically precedented joint cytokines, and variation in the response to several antirheumatic therapies. PIA offers significant potential for the long-term study of immunopathologic mechanisms and novel therapies in rheumatoid arthritis.

SUMMARYThe aim of this study was to understand the immune processes controlling the initiation and spontaneous resolution of adjuvant arthritis (AA). We investigated synovial T-cell recruitment and mRNA expression of IL-17 and other important disease related cytokines, IFN-g , IL-2, IL-4, TNF and TGF-b in inguinal lymph node (ILN) and synovial membrane (SM). Arthritis severity was assessed by a numerical rating score and rats were sacrificed every 3±4 days postadjuvant induction. Further assessment involved quantitative radiology and histology of the ankle joints on each day, and the ILN and SM were removed for RNA extraction. Cytokine mRNA expression was measured using RT-PCR and densitometry. Paraffin sections of rat ankle joints were stained for T-cells (CD3) by immunohistochemistry. In the ILN, there was an increase in IL-17, TNF and IFN-g expression in the early stages of disease, with a secondary sustained increase in IFN-g expression. In the SM, there was expression of T-cell cytokines in early arthritis (day 13), and prolonged TNF and TGF-b expression, which reflected disease progression. IL-4 mRNA expression increased in the later stages of AA. Synovial T-cell numbers transiently increased at day 6, and remained high from days 13±28. Increased pro-inflammatory cytokine expression, including IL-17, in the ILN reflects the initiating events in the early stage of disease. IL-17 may therefore play an important role in the pathogenesis of AA. The increase in IL-4 (an antiinflammatory cytokine) in the SM in the later stages of AA suggests that IL-4 is involved in the spontaneous resolution of AA. The initial increase in IFN-g in the ILN may reflect a pro-inflammatory response, while the prolonged secondary increase may indicate activation of regulatory T-cells.

SUMMARY Anti‐TNF therapy is effective in rheumatoid arthritis (RA); however, its mechanisms of action are incompletely understood. T cell‐driven mechanisms are thought to play an important role in RA and the effects of TNF blockade on these mechanisms are unclear. Adjuvant arthritis (AA) is a T cell dependent model of inflammatory arthritis. The aims of this study were to investigate the effects of TNF blockade on in vivo T cell cytokine expression and to clarify the role of TNF in the inguinal lymph nodes (ILN) in early arthritis. AA was induced in male DA rats. Rats received either 3 mg/kg or 10 mg/kg PEG sTNF‐RI at days 0, 2 and 4 postinduction or 10 mg/kg anti‐TNF antibody on day of arthritis induction. Control rats received either saline or normal sheep serum. Paw volume was assessed every 3–4 days. Rats were sacrificed on days 0, 6, 13 and 21 postinduction. Ankles were removed for quantitative radiology and histology. Synovium and ILN were removed for cell culture and to determine mRNA expression of cytokines using semiquantitative RT‐PCR. TNF and IFN‐γ protein production was measured using a bioassay and an ELISA. TNF blockade did not suppress mRNA expression of T cell cytokines in the ILN of rats in the early phase of AA, suggesting ongoing T cell activity. TNF protein production by ILN cells in culture was reduced in PEG sTNF‐RI treated rats, although mRNA expression was increased in the ILN prior to culture. Early administration of PEG sTNF‐RI did not attenuate AA, in contrast to an anti‐TNF antibody, which suppressed disease. A shorter half‐life for the PEG sTNF‐RI compared with the anti‐TNF antibody or the development of anti‐PEG sTNF‐RI antibodies may account for these results.