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Adjuvant arthritis in Lewis rats is a model of T cell-mediated autoimmune arthritis resembling human rheumatoid arthritis. A nonapeptide from the 65-kD heat-shock protein of Mycobacterium bovis BCG, amino acid sequence 180-188, has been described to carry the dominant immunogenic epitope(s) for both arthritis-protective and arthritogenic T cell clones. Here we demonstrate that immunizations with the synthetic nonapeptide completely protected rats against adjuvant arthritis induced by M. tuberculosis. Interestingly, deletion of the N-terminal threonine of the nonapeptide resulted in loss of the protective activity. Pretreatments with the nonapeptide resulted in an immune response to the nonapeptide and to M. tuberculosis. After immunizations with the synthetic nonapeptide, only low titres of nonapeptide-specific antibodies were produced, whereas a significant cellular immune response to the nonapeptide was observed. In addition, the protection was transferable to naive rats by spleen T cells. These findings document the requirement of a T cell-specific immune response to the dominant epitope of the 65-kD mycobacterial heat-shock protein for the protection against adjuvant arthritis and suggest the feasibility of immune intervention in autoimmune arthritis through the use of synthetic peptides.

Previous studies from this laboratory have described Sch 24937 as a potent immunosuppressive agent that is particularly effective in suppressing humoral immune responses in mice. These findings prompted an evaluation of the effects of Sch 24937 in type II collagen-induced arthritis in mice where disease manifestations include the development of a strong humoral response to the collagen antigen. Sch 24937 reduced the incidence and severity of arthritis in collagen sensitized mice which appeared to be directly related to the immunosuppressive properties of the drug. However in contrast to the steroid betamethasone which also exhibited immunosuppressive activity, Sch 24937 did not prevent the changes occurring in the lymphocyte population of the draining lymph nodes of mice immunized with type II collagen. While the exact mechanism of the immunosuppressive activity of Sch 24937 remains to be elucidated, its mode of action in suppressing arthritis differs at least to some extent from that of a steroid.

We attempted to elucidate the mechanism of action of T cell growth factor (TCGF)-expanded cells after stimulation with concanavalin A (Con A), a process which reduces the severity of adjuvant arthritis (AA), as seen in transferred syngeneic rats. TCGF-expanded cells were fractionated by Percoll density gradient or by the panning method, before the transfer. Transfer of cells with a density of between 1070 and 1079 suppressed AA most effectively, compared with other fractions with a density of less than 1070. A large number of the cells with density 1070-1079 were of the W3/25 phenotype. The transfer of W3/25 positive cells obtained by the panning method from TCGF-expanded cells reduced AA but not W3/25 negative cells. The suppressor function of TCGF-expanded cells was examined in an assay system in vitro. The addition of Con A-stimulated cells to the assay culture (but not expanded with TCGF), suppressed both the proliferation of spleen cells stimulated with Con A and the IgG production stimulated with lipopolysaccharide. The addition of TCGF-expanded cells to the assay culture had no effect on these responses. Thus, the W3/25 cells in the TCGF-expanded cells seem to function as suppressor-inducer cells, affect the presuppressor cells of the recipient rat and differentiate to suppressor effector cells.

Adjuvant arthritis in rats was induced by a single subcutaneous injection of Freund adjuvant into a hind paw. The injected paws' mean volume increased continuously by 80 +/- 26% after 6 h (acute local response). The injected paw displayed a biphasic response peaking at day 1-2 decreasing to day 5, then increased to day 14 by 123% while the contralateral non-injected paw showed little increase until day 10 and increased by 42% at day 14. The lateral diameter of both knee-joints also increased biphasically, peaking at day 4, decreasing to day 6, then increasing to day 14 (early and late systemic phase). Mepyramine maleate, 1 mg.kg-1 (i.p.) daily, suppressed only the early systemic phase by 50%. Daily cimetidine hydrochloride, 1 mg.kg -1.day-1, almost completely abolished the inflammatory response in the knee joints up to day 12, while at day 14 the suppression was 71%. The increase in mean paw volume was also suppressed by orally administered cimetidine. Intraperitoneal doses of cimetidine (3.5 mumol.kg-1) totally suppressed paw oedema produced by subplantar injection of histamine (1.8 mM) but had no action on equiactive dose of the specific H1-agonist, 2-pyridylethylamine. Mepyramine at 3.5 mumol.kg-1 or greater produced no more than 50% suppression of the histamine response. A component of both adjuvant and histamine-induced responses thus appears to be histamine H2-receptor mediated.

On the basis of experiences proving inhibition of some immunological reactions with the ion gallium, we have demonstrated a protecting effect on adjuvant experimental arthritis of the Rat. Consequently, gallium and some metals of the same group should be proposed to treat human arthritis with immunological dysfunction.

Adjuvant arthritis in rat is inhibited by an oxonate diet, which increases uricemia. The inhibition is proportional to blood uric acid level and not to oxonate concentration in the diet. Oxonate alone does not exert an inhibitory effect.

Fisher rats from a inbred colony, when fed on a salt-free high-protein diet, developed only a mild arthritis after adjuvant injection. Their spleen cells failed to respond in vitro to concanavalin A (a T-cell mitogen), although they possessed a B-cell function of plaque formation to sheep red blood cells. When a full salt supplement was included in the diet, or magnesium or copper or zinc was included in the drinking water, adjuvant-induced arthritis was severe and the response to the T-cell mitogen was restored. The above results suggest that these trace elements may stabilize or activate certain cell populations needed for some immune responses in rats.

The muralytic enzyme mutanolysin can act in vivo to eliminate chronic erosive arthritis induced in rats by polymers of peptidoglycan-polysaccharide isolated from group A streptococci (PG-APS). The amounts of PG-APS in the livers and spleens of rats treated with mutanolysin were significantly reduced compared with the amounts in control rats treated with phosphate-buffered saline. However, the amounts of PG-APS in the limbs of mutanolysin- and phosphate-buffered saline-treated rats were comparable. PG-APS polymers extracted from the livers, spleens, and limbs of mutanolysin-treated rats were extensively degraded, whereas PG-APS extracted from phosphate-buffered saline-treated rats had a high molecular weight. We propose that mutanolysin abrogates arthritis in rats by degrading PG-APS polymers to a size which is no longer able to induce chronic erosive arthritis, even though the polymers are still present in the limbs.

Daily intraperitoneal administration from day 3 to day 10 post-adjuvant of crystalline silica in doses 1.5-3.1 mg/kg per day or of amorphous silica in doses of 12.5 mg/kg per day inhibited the development of adjuvant arthritis mostly suppressing the swelling of the non-injected paw. These doses of silica did not impair the carbon clearance. Higher doses of silica (25 mg/kg per day of the crystalline form and 50 mg/kg of the amorphous form) administered from day 17 to day 24 post-adjuvant had no effect on the already established disease.

Peptidoglycan, the substance in mycobacteria thought to be responsible for inducing adjuvant arthritis, and endotoxin (lipopolysaccharide or LPS) share many inflammatory properties. Since repeated administration of LPS produces tolerance, i.e., resistance to the toxic and inflammatory effects of LPS, we tested whether LPS and/or LPS tolerance might influence inflammation due to mycobacterial adjuvant. Male Sprague-Dawley rats were injected with Escherichia coli LPS or saline intraperitoneally and then challenged with 100 micrograms killed Mycobacteria butyricum (adjuvant) in the footpad. A single dose of 100 micrograms LPS three or 24 hours before adjuvant markedly, but transiently, reduced the local footpad swelling that begins within hours of the adjuvant injection and histologically resembles a sterile abscess. Animals that received multiple doses of LPS and were therefore tolerant or animals that received LPS 72 hours before adjuvant demonstrated adjuvant-induced footpad swelling nearly equal to controls. The anti-inflammatory effect of LPS was transient since footpad swelling in all groups was nearly comparable six days after the adjuvant injection and LPS failed to inhibit consistently the arthritis that develops two or more weeks after adjuvant injection. These studies establish that LPS can markedly inhibit the prodrome of adjuvant arthritis (footpad swelling due to M. butyricum), that inhibition of this prodrome does not prevent the subsequent development of arthritis, and that LPS tolerance diminishes this anti-inflammatory effect of LPS.

Daily oral administration of 6-mercaptopurine suppressed the development of the secondary (immune) lesions of adjuvant arthritis in a dose-related manner. The degrees of arthritis suppression corresponded closely to suppressions of concurrent development of the humoral and the cellular immune response to El4 cells. A short course of therapy during the sensitization period (day - 1 to day 5 when the day of adjuvant injection is designated as day 0) appeared to be almost as effective as continued daily dosing (day - 1 to day 15). The drug did not influence the development of the primary (nonimmune) lesions of adjuvant arthritis at all dosage levels investigated. Chronic pretreatment with 10 mg/kg/day PO for 17 days had no effect on the development of carrageenin-induced acute inflammation in the rat. The suppression of adjuvant arthritis by 6-mercaptopurine, unlike suppression by cyclophosphamide, appears to result primarily from its suppressive action on the immune response.

Catalase, peroxidase and superoxide dismutase were found to inhibit significantly carrageenin edema and the primary phase of adjuvant arthritis in rats after i.v. injection. Heat-inactivated enzymes were as effective as the native enzymes. None of 10 scavengers of oxygen radicals inhibited the adjuvant arthritis at any time. Accordingly, no evidence for a participation of oxygen radicals in the secondary arthritis phase could be found, whereas a role of oxygen radicals in the primary arthritis phase and in carrageenin edema cannot be ruled out.

Histamine, injected subcutaneously (0.3-10 mg/kg), produced a dose-related inhibition of the primary and secondary inflammation, and the development of the secondary lesions of rat adjuvant arthritis. Histamine was effective when given for short periods around the time of adjuvant administration and could also delay and possibly reverse an established arthritic response. The histamine H1-agonist, 2-(2-pyridyl)-ethylamine, inhibited rat adjuvant arthritis, whereas the histamine H2-agonists, impromidine and dimaprit, failed to affect the response. Metiamide, a histamine H2-antagonist (5 mg/kg), reduced the inflammation in the uninjected hind-paw and the development of secondary lesions. Histamine may have two effects on rat adjuvant arthritis, inhibiting the response via stimulation of H1-receptors and augmenting the response via stimulation of H2-receptors. Since histamine is known to bind to and to alter the reactivity of cells which are involved in the regulation of immune responsiveness, it is suggested that interactions with these cells are responsible for the observed effects of histamine.

HWA 486 was investigated for its ability to modify the development of adjuvant-induced polyarthritis in Lewis rats. HWA 486 (20 mg/kg/day p.o.), dosed for 8 or 16 days beginning with the day of adjuvant administration, significantly reduced edema, fibrinogen levels, and erythrocyte sedimentation rates (ESR) 42 days later. When HWA 486 (20 mg/kg/day, p.o.) and cyclosporin A (CsA 15 mg/kg/day, p.o.) were tested in the 8-day treatment regimen, the antiarthritic effects of HWA 486 were more sustained. Both compounds reduced the delayed hypersensitivity (DTH) response on day 9 followed by a rebound to an enhanced DTH response on day 21. The PHA-induced mitogenic response of splenocytes from arthritic rats was suppressed on day 9. Treatment with HWA 486 but not CsA restored the splenocyte response to the level of the negative controls.

The influence of cyclosporine (CsA) on the induction and pathogenesis of type II collagen-induced arthritis has been investigated in inbred and outbred Wistar rats. The proportion of animals developing disease and the severity of disease they developed were both diminished by treatment with CsA. These effects were accompanied by a marked suppression of the antibody response to both the immunizing collagen and also to rat type II collagen. CsA treatment also resulted in a decreased accumulation of lymphocytes in arthritic joints. The results indicate that the anti-arthritic and immunosuppressive effects of CsA probably result from a modification of both systemic antibody-mediated and local cell-mediated immunity.

Adjuvant arthritis in rats is a T-cell dependent "autoimmune" disease with close similarities to several forms of human arthritis. Injection of mycobacterial adjuvant leads to T-cell activation and proliferation, processes in which the de novo expression of the interleukin 2 (IL-2) receptor plays a pivotal role. The subsequent massive mononuclear cell infiltration of the joints ultimately results in complete joint destruction. Because activation of the helper/inducer subset of T lymphocytes is critical to the establishment of disease, we reasoned that IL2-PE40, a cytotoxic IL-2-Pseudomonas exotoxin fusion protein that targets the membrane-penetration and ADP-ribosylation domains of the toxin to cells bearing the IL-2 receptor, would be an effective and specific therapy. Adjuvant-injected rats were randomized to treatment with IL2-PE40, phosphate-buffered saline, or either of two control proteins related to IL2-PE40 but lacking either the receptor-binding moiety or an enzymatically active toxin domain and previously demonstrated to lack cytotoxicity in vitro. Intraperitoneal IL2-PE40 given before the establishment of overt clinical disease proved an effective and specific modifier of adjuvant arthritis by clinical, histological, and radiographic criteria. Our data suggest that IL2-PE40 may be effective in those diseases in which activated T-cells play an important role.

B10.RIII and B10.G mice were transferred from a diet of laboratory rodent chow to a standard diet in which all the fat (5% by weight) was supplied as either fish oil (17% eicosapentaenoic acid [EPA], 12% docosahexaenoic acid [DHA], 0% arachidonic acid [AA], and 2% linoleic acid) or corn oil (0% EPA, 0% DHA, 0% AA, and 65% linoleic acid). The fatty acid composition of the macrophage phospholipids from mice on the chow diet was similar to that of mice on a corn oil diet. Mice fed the fish oil diet for only 1 wk showed substantial increases in macrophage phospholipid levels of the omega-3 fatty acids (of total fatty acid 4% was EPA, 10% docosapentaenoic acid [DPA], and 10% DHA), and decreases in omega-6 fatty acids (12% was AA, 2% docosatetraenoic acid [DTA], and 4% linoleic acid) compared to corn oil-fed mice (0% EPA, 0% DPA, 6% DHA, 20% AA, 9% DTA, and 8% linoleic acid). After 5 wk this difference between the fish oil-fed and corn oil-fed mice was even more pronounced. Further small changes occurred at 5-9 wk. We studied the prostaglandin (PG) and thromboxane (TX) profile of macrophages prepared from mice fed the two diets just before being immunized with collagen. Irrespective of diet, macrophages prepared from female mice and incubated for 24 h had significantly more PG and TX in the medium than similarly prepared macrophages from male mice. The increased percentage of EPA and decreased percentage of AA in the phospholipids of the macrophages prepared from the fish oil-fed mice was reflected in a reduction in the amount of PGE2 and PGI2 in the medium relative to identically incubated macrophages prepared from corn oil-fed mice. When this same fish oil diet was fed to B10.RIII mice for 26 d before immunization with type II collagen, the time of onset of arthritis was increased, and the incidence and severity of arthritis was reduced compared to arthritis induced in corn oil-fed mice. The females, especially those on the fish oil diet, tended to have less arthritis than the males. These alterations in the fatty acid pool available for PG and leukotriene synthesis suggest a pivotal role for the macrophage and PG in the immune and/or inflammatory response to type II collagen.

1. The effects of FUT-175 on the development of adjuvant arthritis in rats were studied and compared with those of indomethacin. FUT-175 inhibited both primary and secondary paw lesions in the adjuvant arthritic rats when it was administered orally on a daily basis from the day before through 18th day after adjuvant injection. 2. In addition, FUT-175 inhibited the increase in hemolytic complement in adjuvant arthritic rats in a dose-dependent manner. 3. Indomethacin also showed an inhibitory effect on the development of arthritic lesion, but had no effect on the increase in hemolytic complement in the adjuvant arthritis in rats. 4. Furthermore, FUT-175 inhibited the activities of various proteases in vitro, and then strongly inhibited complement-mediated hemolysis via the classical and alternative pathways, while indomethacin had no effect on them. 5. These results suggest that the anti-inflammatory activity of FUT-175 may differ from indomethacin in the mechanisms of action and, at least in part, due to the anti-complement activity.

Alterations in the progression of the inflammatory disease in mice with established collagen-induced arthritis (CIA) by in vivo treatments with either anti-T cell antibodies or an immunosuppressive drug, whose main targets of action are T cells, were studied. It was demonstrated that the in vivo administration of either monoclonal anti-L3T4, anti-Ly-2 or the combination of anti-L3T4 and anti-Ly-2 failed to modify the inflammatory disease after the arthritis had been initiated. Nevertheless, the progression of disease was decreased by treatments with rabbit anti-mouse lymphocyte sera (ALS) in mice with established CIA. While there was a substantial reduction in the proliferative responses to the T cell mitogen concanavalin A (Con A) in these ALS-treated mice, proliferation to a B cell mitogen, lipopolysaccharide, was unaffected. Furthermore, treatments with anti-Thy-1.2 monoclonal antibody (mAb) by itself did not alter the progression of the ongoing inflammatory disease, but combined treatments with both anti-Thy-1.2 and anti-L3T4 mAb prevented the further advancement of the arthritic disease. Although mice receiving either anti-Thy-1.2 alone or both anti-Thy-1.2 and anti-L3T4 exhibited complete absence of Thy-1+ cells within their inguinal lymph nodes, the proliferative responses to Con A by LN cells from mice treated with the combination of anti-Thy-1.2 and anti-L3T4 were drastically reduced compared to the responses of either the anti-Thy-1.2 or anti-L3T4-treated groups. Finally, daily treatments with cyclosporin A, an immunosuppressive drug which preferentially acts on T cells, were also effective in modifying the clinical course of the arthritic disease in mice with established CIA. These results suggest that immunocompetent cells which express the Thy-1 surface marker, most likely Thy-1+ T cells, may play a role in maintaining and perpetuating the inflammatory disease of established CIA.

It has been established that adaptation of Wistar rats to high-altitude hypoxia led to a reduction of inflammatory lesions occurring in adjuvant arthritis. It has been shown that the mechanisms associated with a decrease in the content of mediators (serotonin, histamine) of the chemical and pathophysiological stages of allergic reactions formation in the adapted animals underlie the protective effect of adaptation. Adaptation to hypoxia appreciably lowered the sensitivity of the sensitized rats to serotonin.

The antiinflammatory action of dexamethasone in both phases of rat adjuvant arthritis was significantly reduced by injection of the RNA synthesis inhibitor actinomycin D into the inflamed paw. This effect is apparently caused by inhibition of RNA/protein synthesis and not by actinomycin D irritant activity. Thus, the present data support the view of indirect glucocorticoid activity via gene expression also in subacute and chronic adjuvant arthritis. The dexamethasone-induced antiinflammatory protein seems to be synthetized at the site of inflammation.

Collagen type II (CII)-induced arthritis (CIA) can be induced in 78% of B10.RIII mice (H2r) by intradermal (id) immunization with CII of bovine origin in complete Freund's adjuvant (CFA), whereas immunization with CII of chick origin induces arthritis in less than 5% of these mice. Nevertheless, tolerization of B10.RIII mice with intravenously injected chick CII renders the animals resistant to induction of CIA by immunization with bovine CII. Such tolerization can be achieved either by intravenous injection of 500 micrograms chick CII 1 week prior to immunization with bovine CII in CFA or by such an intravenous injection of chick CII 2 weeks after immunization with bovine CII in CFA. Postimmunization treatment results in a significant decrease in the concentration of antibody to bovine CII. Preimmunization administration of chick CII causes a marked decrease in the antibody reactive with chick CII without a significant effect on the anti-bovine CII antibody concentration. In DBA/1 mice, a strain in which both bovine CII and chick CII can induce a high incidence of the disease, intravenous injection of bovine CII can also prevent arthritis induced by chick CII, even when given 7 or 14 days after immunization. The fact that chick CII as tolerogen is quite effective in preventing arthritis in B10.RIII mice, while as immunogen it is very ineffective in inducing arthritis in this strain, may be interpreted as evidence for interaction between different epitopes on CII in the pathogenesis of CIA.

Interleukin production and the in vitro mitogenic responses from honey bee venom treated normal rat splenocytes were reduced considerably compared to controls. Addition of interleukin-1 (IL-1) or interleukin-2 (IL-2) supernatants to these cultures in vitro resulted in an increase of their responses to normal levels. These results suggest that in vivo honey bee venom treatment affects the production of IL-1 by macrophages directly. Honey bee venom treatment affects adjuvant induced disease development by inhibiting certain macrophage functions and thus indirectly inhibiting the activation of T and B cells, and possibly the activation of an endogenous virus which might be involved in adjuvant induced disease induction.

Bis(5-amidino-2-benzimidazolyl)methane, a powerful synthetic trypsin inhibitor, proved to be highly effective in suppressing the arthritis induced by streptococcal cell wall fragments in Lewis rats. It reduced not only the degree of synovitis, osteitis, and hematopoietic hyperplasia in the distal extremities, but also the degree of associated granulomatous inflammation in the liver. The results suggest that trypsin-like proteases play an important role in this arthritis model and that inhibitors may be useful in the treatment of similar arthritic conditions in humans.