Regulation of autoimmune arthritis by the pro-inflammatory cytokine interferon-gamma

The pathogenesis of T cell-mediated diseases like rheumatoid arthritis (RA) has typically been explained in the context of the Th1-Th2 paradigm: the initiation/propagation by pro-inflammatory cytokines, and downregulation by Th2 cytokines. However, in our study based on the adjuvant-induced arthritis (AA) model of RA, we observed that Lewis (LEW) (RT.1(l)) rats at the recovery phase of AA showed the highest level of IFN-gamma in recall response to mycobacterial heat-shock protein 65 (Bhsp65), whereas AA-resistant Wistar-Kyoto (WKY) (RT.1(l)) rats secreted high levels of IFN-gamma much earlier following disease induction. However, no significant secretion of IL-10 or TGF-beta was observed in either strain. Furthermore, pre-treatment of LEW rats with a peptide of self (rat) hsp65 (R465), which induced T cells secreting predominantly IFN-gamma, afforded protection against AA and decreased IL-17 expression by the arthritogenic epitope-restimulated T cells. These results provide a novel perspective on the pathogenesis of autoimmune arthritis.     

Diversification of response to hsp65 during the course of autoimmune arthritis is regulatory rather than pathogenic

Summary: Determinant spreading has been implicated in the pathogenesis of certain autoimmune diseases in animal models. We have observed that during the course of adjuvant arthritis (AA) in the Lewis rat, there is 'diversification' of response to the bacterial 65-kDa heat shock protein (Bhsp65) towards its carboxy-terminal determinants (BCTD). Strikingly, pretreatment of naive Lewis rats with BCTD affords significant protection from AA. Our preliminary studies indicate that the diversification of response to BCTD in the Lewis rat is probably triggered in vivo by the induction and enhanced processing of self(rat) hsp65. Thus, the self hsp65-directed T-cell responses appear to be involved in mediating natural remission from acute inflammatory arthritis induced by a foreign antigen, Myco-bacterium tuberculosis. This the first report describing that the new T-cell specificities arising during the course of an autoimmune disease are regulatory/protective rather than pathogenic. Moreover, our results suggest that a final common mechanism involving BCTD might be recruited by other rac strains which either are resistant to AA (WKY rats) or whose susceptibility to AA is modulated significantly by microbial flora (Fisher rats). The results of this study would contribute significantly to understanding of the pathogenesis of human rheumatoid arthritis, and in devising new therapeutic strategies for this disease.     

Responses of the rat immune system to arthritogenic adjuvant oil

T-cell mediated inflammatory joint diseases with similarities to rheumatoid arthritis (RA) can be triggered in arthritis-prone rat strains by intradermal injection of adjuvant oils. The pathogenesis of oil-induced arthritis (OIA) remains elusive, and a largely unresolved question is how the rat immune system responds to arthritogenic oils such as incomplete Freund's adjuvant (IFA). Here we report that IFA already induces increased plasma levels of the acute-phase reactants (APR) fibrinogen and alpha1-acid glycoprotein at day 4 postinjection (p.i). In contrast, no early responses were detected in the joints before infiltration of the T cells, which coincided with arthritis onset at 11-14 days post injection (d.p.i.) The infiltrating cells were possibly derived from draining lymph nodes (LN), which were hyperplastic and contained increased cell numbers from 4 days p.i. and onwards. The magnitude of the early increase in cell numbers and APR was regulated by non-major histocompatibility complex (MHC) genes, as determined by comparison between arthritis-susceptible DA rats and arthritis-resistant but MHC-identical LEW.lAV1 and PVG.1AV1 rats. Arthritisprone DA rats developed a weak acute-phase response, suggesting that this systemic response may be counteracting disease. The DA rats also had the largest early increase in LN-cell numbers, suggesting that the LN hyperplasia is part of a disease pathway. The analysis of hyperplastic LN after in vivo labelling with bromodeoxyuridine (BrdU) revealed increased numbers and proportions of proliferating lymphocytes, including T cells. Furthermore, polymerase chain reaction (PCR)-analysis of LN cytokine mRNA revealed upregulation of interleukin (IL)-1beta at 4 d.p.i. We conclude that adjuvant oil exposure triggers both systemic acute phase reactions and local activation of the peripheral lymphoid system. These responses are genetically regulated and may determine arthritis development and susceptibility.     

Measurement of streptococcal cell wall in tissues of rats resistant or susceptible to cell wall-induced chronic erosive arthritis.

The quantity of streptococcal cell wall localized in the joints of rats of strains which are either susceptible (Sprague-Dawley, LEW/N, M520/N) or resistant (Buffalo, WKY/N, F344/N) to cell wall-induced chronic erosive arthritis was measured after intraperitoneal injection of group A streptococcal cell wall fragments. Susceptibility or resistance was not associated with a difference in the amount of cell wall localized in limbs or other tissues. It is concluded that although localization of cell wall in joint tissue is essential for development of arthritis, the relative resistance of certain rat strains reflects genetic regulation of inflammatory response rather than a quantitative difference in localization of cell wall in joints.     

In the rat, citrullinated autologous fibrinogen is immunogenic but the induced autoimmune response is not arthritogenic

Conversion of arginyl to citrullyl residues (citrullination) is essential for the formation of the epitopes recognized by rheumatoid arthritis (RA)-associated autoantibodies to citrullinated proteins (ACPA). ACPA are secreted by plasma cells of the rheumatoid synovial tissue where their major target, citrullinated fibrin, is abundant. Although numerous arguments suggest that ACPA play an important role in RA, their pathological relevance remains to be established. In the present study, we assessed the immunogenicity and arthritogenicity of complete Freund's adjuvant-emulsified autologous citrullinated (C-rFBG) or non-citrullinated (NC-rFBG) fibrinogen in Lewis (LEW) and Brown-Norway rats, which exhibit drastic differences in their susceptibility to induced autoimmune diseases. NC-rFBG induced no antibody response. In contrast, a single injection of C-rFBG induced an IgG response directed mainly to citrullinated determinants of rFBG. However, all rat strains remained devoid of clinical and histological signs of arthritis up to 3 months after C-rFBG inoculation. Next, in LEW rats, we tested whether autoimmunity to C-rFBG could aggravate acute ankle arthritis triggered by intra-articular injection of incomplete Freund's adjuvant (IFA). However, such arthritis evolved identically in the presence or absence of anti-C-rFBG autoantibodies. However, IFA-injected joints were devoid of citrullinated fibrin deposits. Therefore, citrullination allows breakdown of immunological tolerance but the autoimmune response developed is not spontaneously arthritogenic. Whether or not it can aggravate arthritis with citrullinated fibrin deposits remains to be evaluated.     

Collagen arthritis in rats, arthritogenic lymphokines and other aspects

This review will mainly highlight data from selected, independent studies which collectively implicate a primary role for T cells in the pathogenesis of collagen arthritis in rats. Conferring insusceptibility to this experimental disease with the use of polyclonal, T cell specific antiserum provided direct initial evidence for this conclusion. Substantiation for the theory of a dominant T cell role in collagen arthritis was afforded by T cell line vaccination; scrutiny showed that the mechanism accounting for this protection was a specific down-regulation of the cellular response to collagen. Additional support came from experiments which showed that as few as 10(3) type II collagen specific T line cells were capable of provoking a sustained proliferative synovitis when instilled into the knee joint cavity of syngeneic naive rats. Further analysis of this phenomenon revealed that the arthritogenic capacity of various collagen-reactive line cells correlated with their ability to release a 65-Kd, collagen-binding lymphokine. This antigen-specific lymphokine was designated arthritogenic factor, based on an arthritogenic activity in the knee joint bioassay similar to that of the cells. A functional and physicochemically identical rat arthritogenic factor has also been identified in the adjuvant model of arthritis. These data support the premise that a major effector mechanism in experimental rat arthritis is the release of arthritogenic factor by expanded clones of autoreactive T cells; they also indicate that substantive efforts should be undertaken to seek to identify arthritogenic factor-like lymphokines in patients with chronic inflammatory synovial disease. As an equally plausible alternative hypothesis, the review will close with a brief discussion of recent findings supporting the possible involvement of cartilage-binding, complement-fixing anti-type II collagen antibodies in the pathogenesis of rheumatoid arthritis.     

Balance of Th1/Th2 Cytokines Associated with the Preventive Effect of Incomplete Freund's Adjuvant on the Development of Adjuvant Arthritis in LEW Rats

Complete Freund's adjuvant (CFA) could induce adjuvant arthritis (AA) in LEW rats and incomplete Freund's adjuvant (IFA) could induce oil induced arthritis (OIA) in DA but not in LEW rats. Lymph node cells (LNCs) from these AA and OIA rats showed increased mRNA expression of IFN-gamma, IL-2 and TNF-alpha but not IL-4. LNCs from IFA immunized LEW rats showed increased expression of IL-4, reduced expression of IFN-gamma and TNF-alpha and no IL-2, in contrast to IFA immunized DA rats. The pretreatment of IFA before CFA challenge could completely prevent AA in LEW rats and their LNCs showed increased expression of IL-4 and IFN-gamma but not IL-2 and TNF-alpha. In F1 (LEW x DA) rats, IFA could not induce OIA but the pretreatment of IFA before CFA challenge could induce very mild AA with 80% incidence, LNCs showing an elevated expression of all the above cytokines. These findings suggest that increased Th1 cytokine expression is associated with disease development and that increased IL-4 expression or the balance of Th2 over Th1 cytokine expression plays an important regulatory role in disease development.     

Collagen Arthritis in Rats,Arthritogenic Lymphokines and Other Aspects

This review will mainly highlight data from selected, independent studies which collectively implicate a primary role for T cells in the pathogenesis of collagen arthritis in rats. Conferring insusceptibility to this experimental disease with the use of polyclonal, T cell specific antiserum provided direct initial evidence for this conclusion. Substantiation for the theory of a dominant T cell role in collagen arthritis was afforded by T cell line vaccination; scrutiny showed that the mechanism accounting for this protection was a specific down-regulation of the cellular response to collagen. Additional support came from experiments which showed that as few as 10³ type II collagen specific T line cells were capable of provoking a sustained proliferative synovitis when instilled into the knee joint cavity of syngeneic naive rats. Further analysis of this phenomenon revealed that the arthritogenic capacity of various collagen-reactive line cells correlated with their ability to release a 65-Kd, collagen-binding lymphokine. This antigen-specific lymphokine was designated arthritogenic factor, based on an arthritogenic activity in the knee joint bioassay similar to that of the cells. A functional and physicochemically identical rat arthritogenic factor has also been identified in the adjuvant model of arthritis. These data support the premise that a major effector mechanism in experimental rat arthritis is the release of arthritogenic factor by expanded clones of autoreactive T cells; they also indicate that substantive efforts should be undertaken to seek to identify arthritogenic factor-like lymphokines in patients with chronic inflammatory synovial disease. As an equally plausible alternative hypothesis, the review will close with a brief discussion of recent findings supporting the possible involvement of cartilage-binding, complement-fixing anti-type II collagen antibodies in the pathogenesis of rheumatoid arthritis.     

Cytokine expression and synovial pathology in the initiation and spontaneous resolution phases of adjuvant arthritis: interleukin-17 expression is upregulated in early disease

The 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-gamma, IL-2, IL-4, TNF and TGF-beta 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-gamma expression in the early stages of disease, with a secondary sustained increase in IFN-gamma expression. In the SM, there was expression of T-cell cytokines in early arthritis (day 13), and prolonged TNF and TGF-beta 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 anti-inflammatory 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-gamma in the ILN may reflect a pro-inflammatory response, while the prolonged secondary increase may indicate activation of regulatory T-cells.     

Spontaneous recovery from early glomerular inflammation is associated with resistance to anti-GBM glomerulonephritis: tolerance and autoimmune tissue injury

Different susceptibility to anti-GBM glomerulonephritis (GN) among animal strains has been reported. Using our rat model for T cell-mediated anti-GBM GN, this study initiated an investigation on the mechanism related with GN susceptibility. Anti-GBM GN was induced either through immunization with the nephritogenic T cell epitope pCol(28-40) from Col4alpha3NC1 or through the transfer of specific T cells. WKY rats were highly susceptible to GN while immuno-compatible LEW rats were GN-resistant. GN-resistance in LEW rats was not associated to the immune response to pCol(28-40). First, both strains mounted a Th1 T cell response to pCol(28-40) with identical specificities; transfer of T cells from LEW to WKY rats induced glomerular injury. Second, co-transfer of antibody from WKY to LEW failed to induce GN. Time-course studies revealed that LEW rats did develop T cell-mediated inflammation in glomeruli at early stages similar to WKY rats, as evidenced by histopathology, proteinuria, CD4(+) T cell infiltration in glomeruli, and glomerular expression of inflammatory molecules. However, glomerular inflammation in LEW rats was transient followed by a full recovery. Thus, GN-resistance in LEW rats was due to its ability to contain early T cell-mediated autoimmune glomerular damage. Our model may reveal a potential tolerance mechanism after autoimmune tissue damage has been initiated.     

Arthritogenicity of collagen type II is increased by chlorination

During inflammation, activated neutrophils, monocytes and macrophages produce and release myeloperoxidase (MPO). MPO converts hydrogen peroxide to hypochlorous acid, a highly reactive and oxidizing agent. Proteins subjected to hypochlorous acid become chlorinated. We analysed how chlorination of the cartilage antigen collagen type II (CII) affects its immunogenic and arthritogenic properties by studying immune responses to chlorinated CII in comparison to immune responses to CII and by studying the development of arthritis in rats immunized with CII-Cl. CII-Cl immunization of LEW.1AV1 rats caused a 100% incidence of arthritis with a mean maximum score of 9.2 (maximal score possible 16). The same dose of non-chlorinated CII did not induce arthritis at all. Rats immunized with CII-Cl developed high anti-CII-Cl IgG titres and also developed IgG antibodies recognizing the non-chlorinated form of CII. Analysis of cytokine mRNA expression in lymph nodes 10 days after immunzation revealed an increased expression of interferon (IFN)-gamma mRNA and interleukin (IL)-1beta mRNA in CII-Cl-immunized rats compared to CII-immunized rats. Thus, chlorination of CII increased its immunogenicity as well as its arthritogenicity. As neutrophils, monocytes and macrophages are abundant cells in arthritic joints of patients with rheumatoid arthritis, chlorination might be a mechanism by which immunoreactivity to CII is induced and by which chronic joint inflammation is supported.     

A Monoclonal Antibody to the Mycobacterial 65kDa Heat Shock Protein (ML 30) Binds to Cells in Normal and Arthritic Joints of Rats

A monoclonal antibody reactive with the mycobacterial 65 kDa heat shock protein (ML 30) was investigated for reactivity with biopsies from normal rat joints and with inflamed joints due to adjuvant arthritis (AA) or collagen induced arthritis (CIA). Immunohistochemical stainings with the anti-hsp 65 antibody on paraffin sections from normal rat joints revealed a weak but exclusive staining of cells within the synovial lining. Also normal chondrocytes and bone marrow cells showed occasional staining. In biopsies from inflamed joints obtained from rats suffering from AA or CIA, an intense staining with ML 30 was seen within the cartilage-pannus junction as well as sites of bone erosion. An increased staining, compared with the normal, was also seen in chondrocytes of the eroded cartilage and in some bone marrow cells. No staining with ML 30 was seen in biopsies from inflammatory lesions due to delayed type hypersensitivity reactions in the skin of rats. Reactivity of ML 30 was also seen in a Western blot assay performed on lysates from inflamed synovia from rats with CIA, preferentially with a component slightly below 60 kDa in molecular weight. The demonstration of epitopes cross-reactive with hsp 65 of mycobacteria in normal and, in higher quantity, in arthritic rat joints, suggests, together with our preliminary biochemical findings, that a recently identified mammalian counterpart to bacterial hsp 65 is both preferentially expressed in normal joints and subject to increased expression in arthritis of different aetiologies.     

Association of Pepsin with Type II Collagen (CII) Breaks Control of CII Autoimmunity and Triggers Development of Arthritis in Rats

Lewis rats develop arthritis after immunization with heterologous but not homologous rat type II collagen (CII). We have observed that if the rat CII is prepared by pepsin digestion without subsequent extensive purification, it is arthritogenic in Lewis rats. To address whether pepsin in the CII preparations contributed to the development of arthritis and whether this was associated with the induction of an immune response to CII, Lewis rats were immunized with rat CII of various degrees of purity and with various pepsin contents. After immunization with a crude preparation of CII, containing relatively large amounts of pepsin, Lewis rats developed arthritis with an incidence of 80% together with a strong anti-CII autoantibody production. Further purification of the CII on DEAE-Sepharose, which removes pepsin, eliminated the arthritogenic properties and the capacity to activate CH-specific B cells. Likewise, lathyritic CII, prepared without pepsin, induced neither a CII-specific immune response nor arthritis. If, however, pepsin was added to non-arthritogenic batches of rat CII, arthritis appeared at an incidence of 40%. By using an ELISPOT technique to detect antigen-specific interferon-γ-producing T cells and antibody-producing B cells, the immune response to CII and pepsin can be evaluated. Eleven days after immunization with lathyritic CII and pepsin, a B-eell response towards both CII and pepsin was seen. Pepsin-specific T cells were also seen at day 11, but CII-specific T cells did not appear until day 14 after immunization. In addition, a weak CII -specific proliferative response of the T cells could be demonstrated at day 14 but not at day 11 or 12. These data show that pepsin plays an important role in the triggering of a CII-specific immune response. We suggest a carrier-hapten mechanism where pepsin acts as a carrier and CII as a ‘hapten’ which will activate CII-specific B cells. Subsequently these CII-specific B cells will break the T-cell tolerance and evoke a T-cell-mediated immune response towards CII.     

Heat-shock protein T-cell epitopes trigger a spreading regulatory control in a diversified arthritogenic T-cell response

Summary: Adjuvant arthritis (AA) in Lewis rats is T-cell mediated and seems to depend on T cells recognising the 180–188 epitope of mycobacterial heat-shock protein (hsp) 60. Analysis of arthritogenic T-cell clone A2b has revealed a mimicry of this particular epitope with an articular cartilage-associated target T-cell epitope. Nasal administration of synthetic peptides covering this 180–188 sequence led to epitope-.specific tolerance and resistance to AA. Since this tolerisation protocol also inhibited avridine arthritis, one may conclude that this form of epitope-specific tolerance had effectuated a spreading tolerisation at the level of target antigens that included a diverse set of possible arthritis -associated antigens. In vitro anergised T cells exhibited suppressive activity in a co-culture system. As in this case - depending on the presence of the antigen of the anergic T cell – such T cells suppressed responder T cells of a different antigenic specificity, we postulated that anergic T cells may be responsible for a spreading of tolerance. It seemed that such spreading of tolerance was channelled through the antigen-presenting cells (APC) and was dependent on direct cell-cell contact. This and additional forms of spreading of tolerance could be responsible for specific nasal tolerance, causing inhibition of the development of an arthritogenic inflammatory response. This can be similarly che case for the arthritis protection that resulted from immunisation with hsps. Analysis of T-cell responses following hsp immunisations revealed that the arthritis inhibitory activity resided in T cells with specificity for a conserved part of microbial hsp60. The same T cells cross-responded to rat self-hsp60. Low level expression of the latter molecule on non-professional APC could possibly have induced a suppressive anergic state in these autoreactive cells. Thus, immunisation with microbial hsp would have led to an expansion of such T cells, leading to raised disease-suppressive potential when selectively trapped and activated in the inflamed self-hsp-overexpressing joint. Alternatively the cross-recognised self-hsp epitope could have the regulatory qualities of an altered peptide ligand or a partial agonist for T cells that see the microbial homologue as the full agonist.     

The arthritogenic adjuvant squalene does not accumulate in joints, but gives rise to pathogenic cells in both draining and non-draining lymph nodes

A single intradermal injection of the adjuvant‐oil squalene induces T cell‐mediated arthritis in DA rats. The chain of events leading from non‐specific provocation of the immune system to arthritis, with clinical similarities to rheumatoid arthritis, is largely undetermined. Here, we combined in vivo tracking of tritium‐labelled squalene with lymph node (LN) cell transfer experiments to determine where critical activation events may take place. The majority of squalene remained at the injection site (79%). The amounts recovered in peripheral joints (<1%) were equal to that recovered in other organs that can be targets in autoimmune diseases. This argues that arthritis does not develop as a consequence of adjuvant accumulation in joints. In contrast, substantial amounts of squalene were recovered in hyperplastic LN draining the injection site (1–13%). The adjuvant was deposited to a larger extent in cells than in extracellular matrix. The draining LN cells could transfer arthritis to naïve irradiated DA rats following in vitro stimulation with conA. Interestingly, non‐draining LN were also hyperplastic and harboured arthritogenic cells, although they contained low amounts of squalene (<1%). Consequently, the amount of arthritogenic adjuvant in a particular LN is not closely linked to the development of pathogenic cells. The distribution pattern of squalene was similar in MHC‐identical but arthritis‐resistant PVG.1AV1 and LEW.1AV1 rats, and it was unaffected by T cell depletion with a monoclonal antibody (R73). Thus, T cells and non‐MHC genes do not regulate dissemination of squalene, but rather determine arthritis development at the level of adjuvant response.     

Immunogenicity of Mycobacterium tuberculosis RD1 region gene products in infected cattle

Rheumatoid arthritis (RA) is a chronic inflammatory disease primarily affecting cartilaginous joints but also extra-articular tissues such as the nose and upper respiratory tract. We have investigated extra-articular cartilage involvement in two commonly used animal models for RA, collagen-induced and pristane-induced arthritis, by immunizing rats with different susceptibility to disease (LEW.1 A, LEW.1F and DA rats). We found that nasal and tracheolaryngeal cartilage is affected in LEW.1 A and DA rats to varying degrees in collagen-induced arthritis but not in any strain in the pristane-induced model. Antibodies to matrilin-1, a cartilage-specific protein expressed mainly in tracheolaryngeal and nasal cartilage but not in joints, were positively associated with the presence of inflammation in nasal cartilage. In contrast, no antibody response to matrilin-1 could be detected in pristane-induced arthritis. In addition, nasal vaccination with collagen type II prior to immunization in DA rats significantly decreased the antibody response to matrilin-1 at day 56, but not at earlier time points, indicating a late protective effect on extra-articular cartilage. We conclude that pristane-induced arthritis is a joint-specific model whereas collagen-induced arthritis affect joints as well as extra-articular cartilage. Furthermore, collagen immunization induces an antibody response to matrilin-1.     

Strain differences in sensitivity of rats to Mycoplasma arthritidis ISR 1 infection are under multiple gene control.

At least 5 female rats from each of 24 inbred (ACI, AS, BDIX, BH, BN, BS, BUF, DA, LE, LEW, MWF, OM, SPRD-Cu3, W-Krypt, and WKY), RT1 congenic [BH.1L(LEW), LEW.1A(AVN), LEW.1C(WIST), LEW.1LV3(BH), LEW.1K(SHR), and LEW.1N(BN)], and F1 hybrid [(LEW x BN)F1, (LEW.1W x LEW.1A)F1, and (LEW x LEW.1W)F1] strains, representing eight independent major histocompatibility complex (MHC) haplotypes (a, b, c, dv1, k, l, n, and u) and five related RT1 haplotypes (av1, lv1, lv3, uv2, and uv3), were inoculated intravenously with Mycoplasma arthritidis, and the severity of the polyarthritis that developed was determined by estimating arthritis scores and weight reductions. The 24 inbred, congenic, and F1 hybrid rat strains differed considerably in their sensitivity to infection with M. arthritidis and in the severity of the polyarthritis that they developed. Statistical evaluation showed that in the acute phase (days 1 to 42 after infection) as well as in the chronic phase (days 39 to 121 after infection) of the disease, the means of the arthritis scores for the strains form a continuous variation without significant interruptions, with the very sensitive LEW rats, the RT1 congenic rats on LEW background, the F1 hybrids with LEW, and the MWF, BS, BH, and DA rats on one end and the resistant WKY, BUF, W-Krypt, LE, and OM rats on the other end. A continuous variation was also observed for the means of the growth rates. There were, however, no significant differences between the sensitive and the resistant rat strains in the antibody titers determined by complement fixation test and enzyme immunoassay. Heritabilities of arthritis scores were calculated for all strains (h2 = 0.39 to 0.62), for the RT1 congenic strains (h2 = 0.04 to 0.14), and for several strains with identical MHC genes (h2 = 0.61 to 0.93). The results show that non-MHC genes are probably responsible for the sensitivity of rats to infection with M. arthritidis.     

Streptococcal cell wall-induced polyarthritis in the rat. Mechanisms for chronicity and regulation of susceptibility

Streptococcal cell wall (SCW)-induced arthritis is a chronic, erosive polyarthritis that can be induced in euthymic, susceptible Lewis rats by a single i.p. injection of a sterile, aqueous suspension of SCW. Nude Lewis rats and most other rats strains, including histocompatible F344 rats, are resistant to chronic disease. To study the mechanisms of chronicity and susceptibility to bacterium-induced arthritis, we compared immunological parameters in Lewis and F344 rats. A first observation was that Lewis rats mounted T-cell proliferative responses to SCW after immunisation with SCW or arthritis induction, while F344 rats were completely unable to do so. Depletion of OX8+ cells partially restored this defective response in F344 rats; it did not make them susceptible to polyarthritis, however. As SCW are present throughout the body and the disease manifests itself mainly, and sometimes uniquely as a joint inflammation, a reason for localisation had to be found. One explanation is the crossreactivity of SCW-primed T cells to cartilage components which can be demonstrated in Lewis but not in F344 rats, in vitro and in vivo. We considered this T-cell unresponsiveness in F344 rats as tolerance to threatening antigens or epitopes, so we changed the state of tolerance in both Lewis and F344 rats followed by induction of arthritis. Tolerance to bacteria was prevented in F344 rats by using them as germfree (GF) animals and was induced in Lewis rats by pretreatment with a bacterial common antigen, the 65 kD mycobacterial heat shock protein. The changed state of tolerance coincided with a reversal of the susceptibility to SCW-induced arthritis in both strains. We suggest that in arthritis-prone individuals (Lewis) tolerance to arthritogenic epitopes is defective, while in normal individuals (F344) tolerance and thus arthritis-resistance is induced and/or maintained by exogenous bacteria or gut flora. Another point to be considered is the involvement of T cells in the chronicity of joint inflammation. We demonstrated that a subsiding arthritis can be reactivated by systemic administration of a small amount of bacteria. This so called flare up is dependent on specific T cells and can therefore be induced in Lewis, but not in F344 rats. Of importance is the observation that even unrelated bacteria are able to reactivate and thus to maintain arthritis induced by streptococci.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of arthritogenic adjuvants of self and foreign origin

The lack of defined triggers for human inflammatory joint diseases warrants efforts to identify candidate molecules. For this task, it may be an important lead that nonspecific activation of the immune system can precipitate arthritis in rats. Consequently, arthritis-prone rat strains were used to search for disease-triggering factors among molecules which initially induce innate defence reactions rather than specific immune responses. A variety of immunological adjuvants were investigated by intradermal injection into DA and LEW.1AV1 rats and monitoring of clinical signs for 30 days. Several arthritogenic cell-wall structures from yeast and bacteria were identified, such as beta-glucan, lipopolysaccharide and trehalosedimycolate. The test procedures also revealed arthritogens of chemical origin, such as dioctadecyldiammoniumbromide (DDA = C38H80NBr) and heptadecane (C17H36). Furthermore, it allowed the precise definition of arthritogenic determinants of lipids, since C16H34 induced arthritis, whereas the closely related linear hydrocarbons C16H32, C16H33Br and C15H32 did not. The observed pathogenicity of organic lipids raised the question of whether endogenous lipids can also precipitate arthritis. Indeed, this was true for the cholesterol precursor squalene (C30H50). In conclusion, this article describes the rational use of arthritis-prone rat strains to identify arthritogenic factors of both foreign and self origin. Although structurally unrelated, the pathogenic molecules defined here share the feature of being nonspecific triggers of the immune system. This consolidates a general principle for the induction of adjuvant arthritis which may provide clues to the aetiology of human arthritides, including rheumatoid arthritis.     

TNF-α 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 FLA. 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-α, IL-2, IFN-γ and IL-4 was determined. The results show a rapid and pronounced accumulation of TNF-α mRNA expression, in RCII/FIA and FIA immunized rats. This pronounced expression of TNF-α 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-γ 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-α mRNA after restimulation with RCII, whereas restimulation with the mitogen Con A generated a cytokine mRNA response dominated by IL-2 and IFN-γ.
These and other results indicate that a strong local expression of TNF-α, 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.