Amelioration of collagen II-induced arthritis in rats by the type IV phosphodiesterase inhibitor Rolipram.

The 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-alpha) and interferon-gamma (IFN-gamma) mRNA expression, whereas no effects were seen on IL-2 mRNA synthesis after in vivo challenge with native RCII emulsified in Freund'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.

Polyclonal Th1 cells transfer oil-induced arthritis.

T-cells play a critical role in oil-induced arthritis (OIA) in DA rats. The present study focuses on the involvement of CD4/CD8 T cells in OIA by using adoptive transfer. Mitogen-activated T cells from DA rats previously injected with incomplete Freund's adjuvant (IFA) were depleted of CD4+ T cells or CD8+ T cells before transfer to irradiated naive receipients. The results indicate that CD4+ T cells are essential for the induction of passively induced OIA. However, in vitro blocking experiments with monoclonal antibodies (mAb) to the CD4 molecule of the T cells before transfer did not affect the passive OIA. Neither was passive OIA inhibited by treating the CD4+ T cells with mAb to intracellular adhesion molecule-1 (ICAM-1) in order to block cell-cell interactions or migration. The arthritogenic CD4+ T cells were sensitive, however, to in vitro treatment with mAb to the interleukin-2 receptor, which inhibited the disease or delayed the onset of passive OIA in recipients. The arthritogenic CD4+ T cells were also analysed for expression of specific T-cell receptor (TCR) variable (V) beta chains, critical for recognition of autoantigen, by utilizing V beta gene-specific polymerase chain reaction (PCR). The results show a heterogeneous expression of V beta segments of the TCR, indicating a polyclonal origin of the pathogenic cells. Moreover, an investigation of the T helper (Th)1/Th2 status of the CD4+ T cells, defined by cytokine expression, was made at the mRNA level by using in situ hybridization. High numbers of interleukin-2 (IL-2) mRNA expressing cells and also interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha)-expressing cells could be identified. We conclude from this study that non-immunogenic IFA triggers polyclonal, IL-2-dependent Th1 cells which induce arthritis. The contribution of the CD4 or ICAM-1 molecules for arthritis induction seem to be of minor importance.

Altered Th1/Th2 balance associated with non-major histocompatibility complex genes in collagen-induced arthritis in resistant and non-resistant rat strains.

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.

Cytokine production in synovial tissue of mice with collagen-induced arthritis (CIA).

The 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-alpha), IL-6, IL-2, IL-4, IL-5 interferon-gamma (IFN-gamma), transforming growth factor-beta 2 (TGF-beta2) and TGF-beta3. Locally produced TNF-alpha, IL-6 and TGF-beta2 were observed within the lining layer, sublining and pannus at all stages of disease. The staining of TNF-alpha was particularly intense at the cartilage-pannus junction. In contrast to the monokines, IFN-gamma and TGF-beta3 were only expressed in scattered cells within the deeper layers of the synovia. Interestingly, IFN-gamma was not present in the late phase of CIA, despite the continued presence of TNF-alpha 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.

Dynamic expression of transforming growth factor-betas (TGF-beta) and their type I and type II receptors in the synovial tissue of arthritic rats.

A well characterized animal model that shares many characteristic features with rheumatoid arthritis (RA) is collagen-induced arthritis (CIA) in DA rats. Recent studies have demonstrated that TGF-beta, a multifunctional cytokine, is an important modulator of the immune response in CIA, and possibly also in RA. In this study we have investigated the expression of the precursor forms of TGF-beta1, TGF-beta2, TGF-beta3, as well as TGF-beta type I receptor (TGF-betaRI) and TGF-beta type II receptor (TGF-betaRII) in the synovial tissue of arthritic rats during the course of the disease. By using immunohistochemical techniques, an abundant expression of all three TGF-beta isoforms and their receptors was observed in the arthritic synovia, an expression that increased with time after onset of disease. Antibodies to TGF-beta1, TGF-beta2, TGF-betaRI and TGF-betaRII stained blood vessels intensively, already at the early onset of inflammation, whereas the synovial lining layer and chondrocytes expressed strong immunoreactivity later on in the inflammatory process. The most intense staining with these antibodies was detected in fibroblasts within fibrotic tissue, in particular at the cartilage pannus junction. Interestingly, TGF-beta3 only stained macrophage-like cells and chondrocytes in the synovia. The data suggest that the abundant expression of TGF-beta1, TGF-beta2, TGF-beta3 as well as TGF-betaRI and TGF-betaRII in the synovia, is of pathogenic importance in the development of CIA, although the question of how the different TGF-beta isoforms may enhance or counteract different arthritogenic events remains open.

Increased serum levels of cartilage oligomeric matrix protein and bone sialoprotein in rats with collagen arthritis.

The aim of this study was to investigate whether serum levels of cartilage and bone macromolecules can be used to monitor tissue destruction in experimental arthritis in rats. Serum levels of cartilage oligomeric matrix protein (COMP) and bone sialoprotein (BSP) were measured by novel immunoassays in the chronic and destructive arthritis induced in DA rats after immunization with autologous rat native collagen type II in Freund's incomplete adjuvant. Increased serum levels of COMP and BSP were seen on day 21 after immunization, and even higher levels were observed on day 28 at termination of the experiment, paralleling increases in the clinical joint score and histopathological signs of cartilage and bone erosions. Serum analyses of these markers may be a useful non-invasive way of monitoring tissue involvement in experimental arthritis and possibly the effects of therapeutic interventions aimed at retarding tissue destruction.

Cytokines in relapsing experimental autoimmune encephalomyelitis in DA rats: persistent mRNA expression of proinflammatory cytokines and absent expression of interleukin-10 and transforming growth factor-beta.

Experimental autoimmune encephalomyelitis (EAE) in rats is typically a brief and monophasic disease with sparse demyelination. However, inbred DA rats develop a demyelinating, prolonged and relapsing encephalomyelitis after immunization with rat spinal cord in incomplete Freund's adjuvant. This model enables studies of mechanisms related to chronicity and demyelination, two hallmarks of multiple sclerosis (MS). Here we have investigated, in situ, the dynamics of cytokine mRNA expression in the central nervous system (CNS) and peripheral lymphoid organs (lymph node cells and splenocytes) of diseased DA rats. We demonstrate that peripheral lymphoid cells stimulated in vitro with encephalitogenic peptides 69-87 and 87-101 of myelin basic protein responded with high mRNA expression for proinflammatory cytokines; interferon-gamma, interleukin-12 (IL-12), tumour necrosis factors alpha and beta, IL-1 beta and cytolysin. A high expression of mRNA for these proinflammatory cytokines was also observed in the CNS where it was accompanied by classical signs of inflammation such as expression of major histocompatibility complex class I and II, CD4, CD8 and IL-2 receptor. The expression of mRNA for proinflammatory cytokines was remarkably long-lasting in DA rats as compared to LEW rats which display a brief and monophasic EAE. Furthermore, mRNAs for putative immunodownmodulatory cytokines, i.e. transforming growth factor-beta (TGF-beta), IL-10 and IL-4 were almost absent in DA rats, in both the CNS and in vitro stimulated peripheral lymphoid cells, while their levels were elevated in the CNS of LEW rats during the recovery phase. We conclude that the MS-like prolonged and relapsing EAE in DA rats is associated with a prolonged production of proinflammatory cytokines and/or low or absent production of immunodownmodulatory cytokines.

Specific and long-lasting protection from collagen-induced arthritis and oil-induced arthritis in DA rats by administration of immunogens.

DA rats develop transient arthritis after subcutaneous immunization with adjuvant-oil, while chronic arthritis and collagen autoreactivity ensues when collagen is added to the oil. We show here that DA rats can be protected from oil-induced arthritis (OIA) and rat collagen-induced arthritis (rCIA) by addition of antigen to these arthritogenic inocula. We have investigated this remarkable phenomenon and demonstrate that both foreign and self antigens can be protective, apparently provided they are immunogenic; hence HSP-65kDa, ovalbumin, rat myelin basic protein, rat IgG and bovine albumin are effective while rat albumin is not. This protection is long-lasting and disease-specific because rats protected from rCIA resist a later attempt to induce arthritis, but not experimental autoimmune encephalomyelitis (EAE). Protection from rCIA depends neither on the blocking of humoral autoreactivity to collagen nor on a change in the isotype profile of anti-collagen antibodies. We demonstrate that immunogens can also be protective when injected intraperitoneally only a few days before onset of arthritis. Our results indicate that protection is mediated through bystander immune reactions towards the co-immunized antigen and that the arthritogenicity of a given provocation, be it adjuvants, microbes or autoantigens, may be a complex net result of arthritogenic and contra-arthritogenic immune reactions.

TNF-alpha dominates cytokine mRNA expression in lymphoid tissues of rats developing collagen- and oil-induced 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.

Restricted T cell receptor usage in DA rats during early collagen-induced arthritis.

We have investigated the usage of T cell receptor (TcR) V beta gene structures in DA rats undergoing homologous collagen-induced arthritis induced by immunization with homologous collagen type II emulsified in Freund's incomplete adjuvant. TcR V beta expression within freshly isolated inflamed synovial tissue (ST) and primary draining lymph nodes was analyzed in a semi-quantitative polymerase chain reaction assay. A highly restricted TcR V beta gene expression was observed in ST samples 2 days after onset of clinical disease, with V beta 6, 8.2 and 8.5 comprising more than 60% of the total V beta expression measured. The corresponding primary draining lymph nodes from the diseased animals showed some bias in V beta expression at this time but not as remarkable as that found in ST. One week after onset of disease, consistent V beta gene bias was much less evident in either site. These results indicate that T cells which infiltrate synovia early in disease are highly restricted with respect to V beta expression.