SUMMARYThe kinetics of cytokine production in arthritic limbs of mice with CIA was determined by using modified immunohistochemical techniques. Tissue cryostat sections of undecalcified whole paws were analysed for the presence of tumour necrosis factor-alpha (TNF-a), IL-6, IL-2, IL-4, IL-5 interferongamma (IFN-g), transforming growth factor-beta 2 (TGF-b2) and TGF-b3. Locally produced TNF-a, IL-6 and TGF-b2 were observed within the lining layer, sublining and pannus at all stages of disease. The staining of TNF-a was particularly intense at the cartilage-pannus junction. In contrast to the monokines, IFN-g and TGF-b3 were only expressed in scattered cells within the deeper layers of the synovia. Interestingly, IFN-g was not present in the late phase of CIA, despite the continued presence of TNF-a and IL-6 in the pannus. Production of IL-2, IL-4 or IL-5 was not detected in any joint. The observed pattern of a relative paucity of T cell-derived cytokines and an abundance of monokines during the late phase of T cell-dependent CIA indicates that the synovial cytokine pattern previously described in rheumatoid arthritis (RA) is fully compatible with a pathogenic role of T cells. The temporal as well as spatial dissociation between expression of T cell-derived cytokines and monokines indicates that T cell-independent mechanisms may also be of importance in the triggering of monokine production during arthritis.

Experimental arthritis can be induced in the DA rat strain with rat type II collagen (RCII) administered in Freund's incomplete adjuvant oil (FIA) or with only FIA. If ovalbumin (Ova), is added to these arthritogens the development of arthritis is blocked. To investigate the mechanisms responsible for induction of arthritis, as well as inhibition of arthritis, a kinetic study of the local cytokine expression in lymph nodes has been performed after immunization with the above mentioned agents. By using in situ hybridization techniques, mRNA expression of TNF-alpha, IL-2, IFN-gamma and IL-4 was determined. The results show a rapid and pronounced accumulation of TNF-alpha mRNA expression, in RCII/FIA and FIA immunized rats. This pronounced expression of TNF-alpha mRNA was not recorded in the Ova/FIA immunized animals, which instead were the only animals in which the IL-4 gene was expressed. The expression of IFN-gamma mRNA was limited in RCII/FIA- and FIA-immunized rats, whereas IL-2 mRNA expression was detected only after RCII/FIA injection. Lymph node cells from RCII-immunized animals generated a high amount of TNF-alpha mRNA after restimulation with RCII, whereas restimulation with the mitogen Con A generated a cytokine mRNA response dominated by IL-2 and IFN-gamma. These and other results indicate that a strong local expression of TNF-alpha, induced by arthritogenic stimuli, may be important for the induction of arthritis. Moreover, the elicitation of an immune reaction against Ova, may inhibit arthritis development by contributing to a shift in the initial arthritogenic cytokine response.

SUMMARYThe effect of Rolipram, a selective inhibitor of the cyclic AMP specific phosphodiesterase (PDE IV) was evaluated in the rat collagen type II (RCII)-induced arthritis model in the DA rat. Rolipram was given either shortly before expected onset of disease (days 10-14) or shortly after the onset of clinically evident arthritis (days 15-19 after immunization). Administration at days 10-14 delayed the onset of arthritis for approximately 5 days, but the severity of arthritis was thereafter comparable to that seen in a non-treated control group. Rolipram treatment of animals with manifest arthritis inhibited further arthritis development and also tended to diminish its severity at a phase of disease where non-treated control animals showed a rapidly progressing disease development. Serum levels of antibodies to RCII were in all experiments similar between Rolipram-treated and control animals. An in situ hybridization method for determining cytokine mRNA synthesis in regional lymph nodes, after administration of Rolipram (at days 2-7), demonstrated a strong inhibitory effect on tumour necrosis factor-alpha (TNF-a) and interferon-gamma (IFN-g) mRNA expression, whereas no effects were seen on IL-2 mRNA synthesis after in vivo challenge with native RCII emulsified in Freund 0 s incomplete adjuvant. The results thus demonstrate strong preventive as well as therapeutic effects of Rolipram in a model for arthritis that is very similar to human rheumatoid arthritis with respect to cytokine regulation, and suggest that Rolipram has its major effects in the effector stage of the arthritogenic immune response.

Collagen-induced arthritis (CIA) is a T cell-dependent disease in which susceptibility is controlled by genes both within and outside the major histocompatibility complex (MHC). In the present study, we compared the humoral responses and kinetics of cytokine secretion patterns in the draining lymph nodes of arthritis-susceptible DA rats and arthritis-resistant F344 and DA MHC congenic PVG.1AV1 rats immunized with rat type II collagen (RCII) in incomplete Freund's adjuvant. The results demonstrate a marked humoral RCII response and a Th1 cytokine profile, with expression of interferon-gamma and interleukin (IL)-2 mRNA in DA rats; a limited humoral RCII response and a Th2 cytokine profile, with expression of IL-4 mRNA in arthritis-resistant F344 rats; and a marked humoral RCII response in arthritis-resistant PVG.1AV1 rats. However, in contrast to DA rats, PVG.1AV1 rats produce IgG1 autoantibodies which, together with strong expression of IL-4 mRNA, indicates the involvement of Th2 subsets. From these data, we conclude that non-MHC gene(s) determines the direction of the anti-RCII response towards a Th1 disease-promoting, or a Th2 disease-limiting response.

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