The effects of mesoporphyrin on experimental arthritis in mice

The effects of mesoporphyrin, a novel porphyrin derivative, on type II collagen-induced arthritis in mice were studied. Mesoporphyrin (10–30 mg/kg) and prednisolone (5 mg/kg; reference drug) reduced the incidence and severity of type II collagen-induced arthritis in mice, as assayed by clinical observation and histopathological studies. Although both agents inhibited type II collagen-induced delayed type hypersensitivity in arthritic mice, only prednisolone inhibited humoral immunity to type II collagen. The effects of mesoporphyrin on T cell dependent allergic inflammation were examined, in order to study the mechanism by which it inhibits arthritis. Staphylococcal enterotoxin B (SEB; superantigen)-potentiated collagen-induced arthritis and sheep red blood cell-induced delayed type hypersensitivity reaction were clearly inhibited by mesoporphyrin. Moreover, the superantigen-induced CD-25 expression on T cells was inhibited by mesoporphyrin. These results indicate that mesoporphyrin inhibits type II collagen-induced arthritis by inhibiting the activation of T cells.accepted by M. Katori     

Humokal and Cellulaa Immunologic Aspects of Adjuvant and Collagfn Arthritis in Rats

Systemic and local immunological responses of rats sensitized with either M. butyricumor native type II collagen have been evaloatod. In rats exhibiting adjuvant-induced arthritis no antibodies to collagen could be detected. In animals exhibiting collagen-induced arthritis, high antibody titsrs developed by day 14, and could be correlated with the severity of the arthritis.

Delayed type hypersensitivity (DTH) responses were measured by a 5-iodo-2'-deoxyuridine 125-I (125-IUdR) uptake assay. Arthritic scores in rats immunized with collagen were not accompanied by a positive DTFt response, where as adjuvant arthritic rats showed a positive response. T-lymphocyte cellular responses in both adjuvant- and collagen-induced arthritic rats were measured. In neither syndrome were major alterations observed in T-lymphocytz subpopulations. These results provide evidence that adjuvant-induced arthritis and type II collagen-induced arthritis are distinct entities, and that they may be discriminated by the nature of the humoral response.     

Effect of clodronate on established collagen-induced arthritis in rats

The collagen-induced arthritis model in rats was used to study the effect of disodium clodronate on inflammation and destruction of tarsal, metatarsal, and interphalangeal bones and joints. Female DA rats were immunized with heterologous type II collagen. Fourteen days after immunization, rats with similar scores were assigned to the different experimental groups. They were treated subcutaneously either with saline (controls) or with clodronate at doses of 12.5 and 25 mg/kg/day five times a week for 2 weeks. Clinical signs of arthritis including the severity of paw swelling were assessed weekly. At the time of killing, histological features of the non-decalcified tarsus with tarsal, tarsometatarsal and interphalangeal joints were assessed for inflammatory soft-tissue, articular, and bone changes. All the arthritic control rats developed severe arthritis as shown by the total histological scores of the hindpaw. The treatment with clodronate (25 mg/kg) decreased clinical signs of arthritis, the activity of the collagen-degrading lysosomal enzyme,-N-acetylglucosaminidase, in inflamed hindpaw tissue, serum osteocalcin level and serum cross-linked telopeptide of type I collagen level. Histological evaluation indicated moderate arthritis in 29% of the rats and severe arthritis in 71%. The results show that clodronate given therapeutically to arthritic rats, induced with type II collagen, suppresses the intensity of inflammation and bone lesions in the tibiotarsal and tarsometatarsal regions.     

Methotrexate,combined with cyclophosphamide attenuates murine collagen induced arthritis by modulating the expression level of Breg and DCs

To explore the mechanism of methotrexate (MTX) and its combination with cyclophosphamide (CTX) in collagen-induced arthritis (CIA), we investigated the levels of several immune cells and cytokines in mice with different treatments. CIA was induced in DBA/1 mice at the age of 7 weeks by primary immunization with 100 μl emulsion containing 2 mg/ml bovine type II collagen which was mixed with complete Freund's adjuvant (CFA). The booster immunization was performed with 50–100 μl emulsion containing 2 mg/ml bovine type II collagen (CII) mixed with incomplete Freund's adjuvant (IFA). MTX, CTX or both were administrated after the booster immunization. Therapeutic effect was evaluated by arthritic scores, X-rays and assessment of histopathological joint destruction. The expression of TNF-α, IL-6, IL-23, IL-10 were also measured. The frequencies of different immune cell subsets in the lymph node, spleen and bone marrow were determined by flow cytometry analysis. Our results showed that CTX and MTX treatment attenuated the severity of arthritis of CIA mice and reduced the levels of several cytokines. CTX and MTX treated mice showed a lower frequency of B cells in bone marrow. Also, when treated the CIA mice with MTX, alone or together with CTX, the lymph nodes and spleen exhibited a decrease in regulatory B cells (Breg) and dendritic cells (DCs). Notably, the combination of MTX and CTX had a more pronounced effect. By measuring the levels of different immune cells those participated in the development of rheumatoid arthritis (RA), our experiment may help to evaluate the therapeutic effects and prognosis of arthritic diseases.     

Comparison of inflammatory changes in established type II collagen- and adjuvant-induced arthritis using outbred Wistar rats

Type II collagen- and adjuvant-induced arthritis in outbred Wistar rats were compared using parameters that measured the inflammatory response, cellular and humoral immunity, blood protein changes, drug metabolism and histopathological and bony changes of the inflamed paws. There was a lesser incidence (40-70%) and severity of collagen disease than the adjuvant model (incidence approximately 100%). The use of MDP increased the incidence and severity of collagen arthritis. The acute phase protein response (plasma fibrinogen) was similar in both models during the peak of inflammatory response. Drug metabolism was inhibited in both type II collagen boosted with MDP or M. butyricum sensitized rats with arthritis; however, arthritic rats sensitized with collagen alone produced no inhibition. Only collagen arthritic rats produced type II collagen antibody and exhibited delayed hypersensitivity to type II collagen. Bony changes as assessed by radiographic evaluation were more severe in adjuvant arthritic rats than in the collagen arthritic model; histopathological findings from these animals confirmed this observation. The primary lesions in both models were periosteal reaction of the bone and ankylosis. Several classes of antiarthritic drugs were compared in both models using paw edema measurements and bony changes by radiographic evaluation. Drugs with inhibitory activity in both models were indomethacin, methylprednisolone, D-penicillamine and gold sodium thiomalate. Levamisole, chloroquine and auranofin were inactive in both models.     

Anti-inflammatory properties of zinc protoporphyrin disodium (Zn−PP−2Na)

Anti-inflammatory properties of zinc protoporphyrin disodium (Zn–PP–2Na) were studied. Zn–PP–2Na exhibits anti-allergic action against type III and IV reactions (passive Arthus reaction in rats and tuberculin-induced footpad reaction in mice), but does not affect type I and II reactions (homologous passive cutaneous anaphylaxis in mice and reversed cutaneous anaphylaxis in rats). Zn–PP–2Na also clearly inhibits type II collagen-induced arthritis in mice. The agent inhibits general arthritis symptoms, anti-type-II collagen antibody production and type II collagen-induced delayed type hypersensitivity (DTH) in arthritic mice. Zn–PP–2Na, however, did not affect carrageenin-induced paw edema and histamine- and serotonin-induced skin reactions in rats. Zn–PP–2Na inhibits IL-1-induced mouse lymphocyte proliferation, but does not affect PMA-induced O ₂ ^– generation from guinea-pig neutrophil. These results indicate that Zn–PP–2Na inhibits type II collagen-induced arthritis in mice due to the antagonism of IL-1 activity and the inhibition of DTH against type II collagen.     

Effects of methotrexate on collagen-induced arthritis in male Wistar rats

To evaluate the effect of methotrexate on collagen-induced arthritis, micro-computed tomography (micro-CT) and histopathological analyses were used in male Wistar rats. Rats were divided randomly into three groups. Group 1 was treated with 0.9% saline, and groups 2 and 3 were boosted with type Ⅱ collagen. From day 21 to 42, groups 1 and 2 were orally treated with 0.9% saline and group 3 was orally treated with 1.5 mg/kg methotrexate. All rats were sacrificed at day 42 after the first collagen treatment. Micro-CT analyses showed bony parameters, such as bone volume and trabecular number, were decreased in group 2 compared to group 1, and these parameters were recovered in group 3. Histopathological examination and pathological parameter scoring showed that the knee joints of rats in group 2 had severe joint destruction, showing cartilage and bone erosion, enlarged cavities with inflammatory cell infiltration and activation of synovial fibroblasts. By contrast, these changes were reduced in group 3. Taken together, methotrexate treatment showed therapeutic potential in male rat collagen-induced arthritis model, and micro-CT analysis and histopathological tools could be integrated to assess the quantification/qualification of arthritic lesions.     

Anti-IL-12 and anti-TNF antibodies synergistically suppress the progression of murine collagen-induced arthritis.

The co-ordinate role of the Th1 cytokine IL-12 and the proinflammatory cytokine TNF in arthritis was explored using the DBA/1 mouse model, collagen-induced arthritis (CIA). In this study, mice with established arthritis were treated with anti-IL-12 and/or anti-TNF antibodies for 10 days from the onset of disease. Clinical assessment showed that the combined antibody treatment ameliorated disease severity to a greater extent than anti-TNF alone. Supporting these observations, histological analysis revealed that there was a reduced joint damage in the mice that received combined anti-IL-12 and anti-TNF treatment, compared to the other treatment groups. Anti-IL-12 had no statistically significant effect on the clinical outcome of disease. The combination of anti-IL-12 and anti-TNF treatment was found to reduce collagen type II (CII)-specific lymph node cell IFN-gamma production and proliferation, as well as decrease the anti-CII IgG2a:IgG1 ratio more effectively than either treatment alone. When the antibodies were added to synovial cells from arthritic mice and bone marrow macrophages in vitro, anti-TNF diminished IL-12 production, but anti-IL-12 had no effect on TNF production. These data suggest that, through the partial regulation of IL-12, TNF modulates the immune response in arthritis, as well as the inflammatory response. The synergistic action of anti-TNF and anti-IL-12 on CIA may provide a new therapeutic approach for treating rheumatoid arthritis.     

Immunomodulating effects of the new anti-rheumatic drug tenidap on collagen-induced arthritis

We investigated the in vivo action of the newly developed anti-rheumatic agent tenidap, CP-66,248 (Pfizer Inc., New York), on arthritis in collagen-induced arthritic mice. The inhibitory effect of tenidap on the development of arthritis was statistically more significant than piroxicam. The serum anti-type II collagen antibody titer was markedly inhibited in the mice treated by tenidap. These results suggest that, unlike NSAIDs, tenidap inhibits the progress of collagen-induced arthritis through its immunomodulating effect.     

PGE2受体EP2和EP4调节CIA小鼠脾B细胞表面分子和细胞因子表达

目的: 探讨前列腺素E₂(PGE₂)受体EP2和EP4在胶原诱导性关节炎(CIA)小鼠脾B细胞免疫调节中的作用。方法: 建立CIA小鼠模型,用CD19⁺ 免疫磁珠分选脾B细胞,流式细胞术检测MHCⅡ、CD80和CD86的表达,实时荧光定量PCR技术检测EPs 和细胞因子IFN-γ、TNF-α、IL-6、IL-4、IL-10和TGF-β的表达。结果: 小鼠脾B细胞表达EP的4个亚型,CIA模型小鼠EP2和EP4表达增加;EP2阻断剂可以降低MHCⅡ、CD80和CD86的表达,而EP4阻断剂对CD80没有明显影响;EP2和EP4阻断剂均可以降低IFN-γ、TNF-α 和IL-6 的表达(P<0.05或P<0.01),促进IL-10的表达(P<0.01或P<0.05),并可以分别促进IL-4和TGF-β的表达(P<0.01)。结论: PGE₂可通过EP2/EP4调节B细胞表面分子和细胞因子参与CIA发病,EP2/EP4有可能成为类风湿关节炎治疗的新靶点。     

A fish oil diet reduces the severity of collagen induced arthritis after onset of the disease.

We have previously reported that compared to a corn oil diet a fish oil diet (5% by weight) fed to B10R.III mice before the induction of collagen induced arthritis markedly reduced disease severity. In this study we determine whether a fish oil diet could reduce the severity of collagen induced arthritis if begun after the arthritis was clinically apparent. Mice were initially fed either a fish oil or corn oil diet and immunized with bovine type II collagen 4 weeks later. At the onset of collagen-induced arthritis, half of the corn oil fed mice were switched to fish oil and arthritis assessed on a weekly basis. Four weeks after the diet change until killing 5 weeks later, the mice switched to fish oil developed much less severe arthritis than the corn oil fed controls. Thus the severity index of corn oil fed mice ranged between 9.4 and 7.1; the severity index of fish oil fed mice was between 6.8 and 4.3 while the mice switched to fish oil ranged between 7.2 and 5.6. Analysis of peritoneal macrophages 13 weeks after immunization showed that macrophages from fish oil fed mice incorporated eicosapentaenoic acid into phospholipids and produced less arachidonate products than corn oil fed mice. There was no difference between macrophages obtained from mice switched from corn oil to fish oil and those maintained on fish oil with respect to fatty acid composition of membrane phospholipids or prostaglandin profile. These results suggest that arthritis severity may be modulated after the onset of CIA by altering the PG profile of macrophages present at inflammatory sites.     

Estrogen induced suppression of collagen arthritis. V: Physiological level of estrogen in DBA/1 mice is therapeutic on established arthritis, suppresses anti-type II collagen T-cell dependent immunity and stimulates polyclonal B-cell activity

Immunization of castrated female DBA/1 mice with rat type II collagen (CII) induces severe polyarthritis with an onset 3-5 weeks after immunization and with 80-100% incidence. Estrogen treatment, inducing physiological 17 beta-estradiol (E2) levels, during a limited period before and after the immunization, or during another period before the expected onset of arthritis, delayed the arthritic onset by approximately 10 days but did not affect the incidence of severity of arthritis. Treatment with physiological doses of E2 after onset of arthritis decreased severity and duration of disease. The T-cell dependent anti-CII autoantibody response was suppressed if the E2 treatment was given immediately before and after CII immunization and was not significantly affected if E2 treatment was given after CII immunization. Neither the total anti-CII Ig levels nor the anti-CII IgG2a levels correlated with development of arthritis. We also titrated the serum levels of estrogen and recorded the vaginal smear response after injections of various doses of E2. This enabled us to work in a physiological range of estrogen levels, spanning the levels found at the end of pregnancy and those found during the normal estrous cycle. These levels were found to suppress antigen-specific T-cell functions but enhance certain B-cell activities since the delayed type hypersensitivity (DTH) reaction against CII was suppressed while the total number of splenic Ig-secreting cells increased. These findings suggest that estrogen in physiological doses is therapeutic for the development of collagen-induced arthritis and that estrogen exerts dualistic effects on the immune system by suppressing T-cell functions and stimulating certain B-cell activities. The suppressive effect on arthritis could not be explained by suppression of anti-CII autoantibody response and must therefore depend on other T-cell-mediated functions.     

Involvement of interleukin-1-like factor(s) in type II collagen-induced arthritis in mice

To explore the role of interleukins in development of arthritis, we induced collagen-induced arthritis in mice and examined interleukin activities in the inflamed joints. Arthritis developed in 90% of mice 4-5 weeks after primary immunization with type II collagen. Joint extracts from mice with collagen-induced arthritis contained high levels of interleukin 1 (IL-1)-like activity but not interleukin 2 (IL-2) or interleukin 4 (IL-4) activity. IL-1-like activities in the lesions were correlated with development of arthritis assessed by joint swelling and erythema. These results suggest that IL-1-like factor(s) may participate in the etiopathogenesis of collagen-induced arthritis in mice.     

The anti-arthritic and immunosuppressive effects of cyclosporine on arthritis induced in the rat by type II collagen

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-induced arthritis in rats. Evidence that autoimmunity to homologous collagens types I, II, IX and XI is not involved in the pathogenesis of arthritis.

We examined the sera of arthritic outbred Wistar and Sprague-Dawley rats and inbred Fisher 344 and Wistar-Lewis rats for autoantibodies to rat type I, II, IX and XI collagens following the induction of arthritis with mycobacteria (MTB). Although many sera collected over an extended time were assayed in addition to acid eluates of arthritic joints, convincing evidence for autoimmunity to collagen could not be demonstrated. Instead, modest non-specific reactions were observed to collagen, irrelevant proteins, and buffer-treated plastic microtitre wells. In contrast, antibodies to purified protein derivative (PPD) were detected in the sera of rats developing adjuvant-induced arthritis, and antibodies to type II collagen, in the sera and joint eluate of rats developing experimental collagen-induced arthritis. Lastly, delayed-type hypersensitivity responses to collagen could not be detected, nor could adjuvant-induced arthritis be attenuated by soluble collagen injected intravenously before challenge with MTB. We conclude that adjuvant-induced arthritis and experimental collagen-induced arthritis are distinct models of rheumatic disease and that autoimmunity to collagen is neither prevalent in adjuvant-induced arthritis nor necessary for its pathogenesis.     

Effects of ageing and exercise on soleus and extensor digitorum longus muscles of female rats.

The effects of ageing and of exercise on muscle mass, fiber cross-sectional area, and fiber type composition of a weight-bearing muscle, the soleus and a non-weight-bearing muscle, the extensor digitorum longus (EDL) were investigated in female Long-Evans rats. The animals were exercised by means of voluntary wheel running beginning at 4 months. Runners and sedentary controls were studied at 9 months and 27 months of age. In sedentary rats, the soleus muscle weighed 26% less, and the EDL weighed 19% less at age 27 months, than at 9 months. This decline in muscle mass was accounted for by a similar decrease in muscle fiber cross-sectional area. The wheel running resulted in significant hypertrophy of the soleus in both 9- and 27-month-old rats; as a consequence the 27-month-old runners had larger soleus muscles than the 9-month-old sedentary rats. The running did not prevent atrophy of the EDL in the old rats, but did increase the proportion of type IIa fibers. The exercise also increased the number of capillaries per fiber in the soleus muscles of both young and old rats. In conclusion, the finding that wheel running prevented atrophy with ageing of the weight-bearing soleus but not of the non-weight-bearing EDL emphasizes the specificity of exercise, and shows that exercise-induced muscle hypertrophy can be maintained in old age by appropriate exercise.     

Differing roles for urokinase and tissue-type plasminogen activator in collagen-induced arthritis

The plasminogen activators, urokinase PA (u-PA) and tissue-type PA (t-PA), are believed to play important roles in inflammatory cell infiltration, fibrin deposition, and joint destruction associated with rheumatoid arthritis; however, their precise roles in such processes, particularly u-PA, have yet to be defined. Using gene-deficient mice we examined the relative contribution of the PAs to the chronic systemic collagen-induced arthritis model. Based on clinical and histological assessments, u-PA-/- mice developed significantly milder disease and t-PA-/- mice more severe disease compared with the relevant wild-type mice. Fibrin deposition within joints paralleled disease severity and was particularly pronounced in t-PA-/- mice. Likewise, cytokine levels in the synovium reflected the severity of disease, with interleukin-1beta levels in particular being lower in u-PA-/- mice and increased in t-PA-/- mice. The antibody response to type II collagen was normal in both knockouts; however, T cells from u-PA-/- mice had a reduced proliferative response and produced less interferon-gamma on antigen stimulation in vitro. These results indicate that the major effect of u-PA in the collagen-induced arthritis model is deleterious, whereas that of t-PA is protective. Our data highlight the complexities of PA function, and suggest that approaches either to target u-PA or to enhance local t-PA activity in joints may be of therapeutic benefit in rheumatoid arthritis.     

Pinealectomy ameliorates collagen II-induced arthritis in mice.

To extend our previous findings that exposure to constant darkness (stimulation of endogenous melatonin release) as well as treatment with exogenous melatonin magnifies the severity of collagen-induced arthritis in mice, we have examined the effects of melatonin cutback by removing the pineal gland. Two strains of mice, DBA/1 and NFR/N, were subjected to surgical pinealectomy. The melatonin levels in sera were reduced by approximately 70% by the pinealectomy compared with the corresponding sham-operated controls. After 3-4 weeks of rest the mice were immunized with rat type II collagen to induce autoimmune arthritis, and the animals were kept in constant darkness during the experiments. In comparison with the controls, all groups of pinealectomized mice showed reduced severity of the arthritis by means of (i) a slower onset of the disease, (ii) a less severe course of the disease (reduced clinical scores), and (iii) reduced serum levels of anti-collagen II antibodies. These effects were not significant in all experiments, but the trends were always the same. Thus, the present result strengthen the hypothesis that high physiological levels of melatonin (which can be induced by exposure to darkness) stimulate the immune system and cause exacerbation of autoimmune collagen II arthritis, while inhibition of melatonin release (pinealectomy or exposure to light) has a beneficial effect.     

Cyclosporin A induces a selective, reversible suppression of T-helper lymphocyte regeneration after syngeneic bone marrow transplantation: association with syngeneic graft-versus-host disease in rats.

The presence of species-specific and species-non-specific (common) epitopes has been demonstrated on type II collagen (CII) using monoclonal antibodies. In this study, we investigated the role of antibody response to some species-specific and common epitopes in mice immunized with human CII for the induction of collagen-induced arthritis (CIA). Antibody responses to species-specific epitopes in arthritic mice appeared significantly higher than that in non-arthritic mice. However, no significant difference of antibody responses to common epitopes was found between arthritic and non-arthritic mice. Monoclonal antibody reactive with one of the common epitopes exhibited the ability to induce arthritis in mice previously given the primary injection of CII, indicating the involvement of this epitope in the induction of CIA. Finally, we investigated the epitope specificity of anti-human CII antibody present in serum samples of patients with rheumatoid arthritis and relapsing polychondritis, and found antibodies to some common epitopes.     

Characterization of monoclonal antibody specific for human type II collagen: possible implication in collagen-induced arthritis

We obtained monoclonal antibodies specific for human type II collagen and characterized them using human collagen type I, II, III and V and tropocollagen A (3/4) (TCA) and tropocollagen B (1/4) (TCB) fragments of type II collagen which were obtained by digestion with tadpole collagenase. These antibodies were of the IgG2a class and specific for the conformational determinant of TCA fragment of type II collagen. When injected intravenously into DBA/1J mice, one of the monoclonal antibodies induced arthritis, which was characterized by early onset, mildness in severity and preferential localization mainly in the peripheral joints of the lower extremities. These results suggest that, at least, one of the arthritogenic determinants of type II collagen for collagen-induced arthritis of mice exists in the three quarter region from the N-terminus of type II collagen.