Serum transfer of collagen-induced arthritis in mice

Immunization of DBA/1 mice with native chick type II collagen resulted in development of polyarthritis 4-5 wk later. Sera of these mice contained high levels of anticollagen antibodies, and immunoglobulin concentrates of their sera transferred arthritis to unimmunized recipients. Histopathologically, this passively transferred arthritis resembled the early disease of immunized donors. Immunofluorescence studies demonstrated the deposition of IgG and C3 on the articular surface but not in synovial tissue of arthritic joints. Transferred, isotopically labeled anticollagen antibodies rapidly localized to the limbs and to other cartilage-containing tissues. When transfer concentrate was administered to arthritis-resistant strains, they also developed arthritis. Indeed, immunoglobulin concentrates from rats with collagen-induced arthritis transferred arthritis to naive mice. The amount of concentrate required for transfer to B10.D2 resistant mice was reduced by immunizing them with collagen 4 wk before transfer. Although susceptibility to arthritis from immunization is H-2 linked, these studies clearly demonstrate that passive transfer of arthritis depends upon injection of specific antibody and not on other host factors.     

Prevention of type II collagen-induced arthritis by in vivo treatment with anti-L3T4

The effect of in vivo administration of monoclonal anti-L3T4 antibody on the development of murine collagen-induced arthritis (CIA) was assessed. Treatment with anti-L3T4 resulted in a greater than 90% depletion of L3T4+ T cells in lymph nodes and spleen, an effect that appears entirely reversed 30 d after treatment. Administration of anti-L3T4 before immunization with type II collagen resulted in a significant decrease in arthritis incidence and delayed onset of the disease while treatment begun after a strong anticollagen IgG humoral response was underway was not effective in altering disease expression. These results suggest a prominent role for L3T4+ T cells in the pathogenesis of CIA.     

Type II collagen-induced arthritis in rats. Passive transfer with serum and evidence that IgG anticollagen antibodies can cause arthritis

We have found that serum from rats with type II collagen-induced arthritis, when fractionated with 50% ammonium sulfate and concentrated, would transfer arthritis to nonimmunized recipients. The arthritis in recipients developed within 18-72 h and displayed all of the major histopathologic characteristics of the early lesion in immunized animals but was transient and less severe. Although consideration was given to the possibility that a circulating immune complex was involved, no evidence of such a complex was detected. Further fractionation of the serum yielded an IgG anticollagen antibody that was fully active in transferring disease. The antibody's reaction was inhibited by the native bovine type II collagen used for immunization of donors and the antibody strongly cross-reacted with homologous type II collage but not with denatured collagen. These studies demonstrate that arthritis in rats can be induced with anti- type II collagen antibodies and suggest that an autoimmune process is involved. Because antibodies to collagen have also been detected in human rheumatic diseases, further investigation of the characteristics of collagen antibodies capable of inducing arthritis seems warranted.     

Passive Transfer by Cells of Type II Collagen-Induced Arthritis in Rats

To investigate the role of immunologic hypersensitivity to collagen in the causation of type II collagen-induced arthritis in rats, passive transfer experiments were performed. Wistar/Lewis rats used in these experiments were demonstrated to be histocompatible by prolonged skin graft survival and mixed lymphocyte cultures. Popliteal lymph node weight assays excluded a potential for graft-vs.-host reactivity in this strain. 9 of 32 naive rats developed arthritis after intravenous receipt of pooled spleen and lymph node cells from donors that had been injected intradermally with type II collagen emulsified in incomplete Freund's adjuvant. This passively transferred synovitis was evident clinically as well as histologically. In control cell transfer experiments involving a total of 97 recipients, transfer of arthritis was shown to require viable cells sensitized to type II collagen. These controls included 17 rats receiving cells from unimmunized donors, 20 recipients of cells from donors injected with incomplete Freund's adjuvant alone, and 24 recipients of cells from rats injected with type I collagen in adjuvant. Deliberate addition of solubilized type II collagen to unsensitized cells at the time of transfer or injection of heat-killed sensitized cells also did not cause arthritis in a total of 36 recipients. These latter two control groups indicate that disease transfer was not the result of antigen carry-over. Intravenous injection of sera from arthritic donors was incapable of passively transferring clinical or histologic synovitis in 30 recipients. Thus, these studies directly implicate immunologic sensitivity to the cartilage type of collagen in the etiology of this autoimmune disease.     

β-内啡肽对胶原诱导关节炎大鼠的免疫调节作用

目的研究β-内啡肽(β-END)对胶原诱导性关节炎(CIA)大鼠的免疫调节作用,为探索β-END治疗类风湿关节炎(RA)提供实验依据。方法采用尾根部皮内多点注射天然Ⅱ型胶原(CⅡ)的方法免疫雌性Wistar大鼠(60只),建立CIA模型。随机取CIA成模大鼠(5只/组)于初次免疫后第14~35天,给予不同浓度的β-END腹腔内注射,定期进行临床、实验室、影像学及病理指标评估。结果不同剂量β-END(0.1、1、5 nmol隔日1次共2周)治疗后CIA大鼠临床、实验室、影像学及病理指标明显缓解;正常鼠β-END给药5 nmol×2周后重要脏器功能、组织学未见明显异常。结论生理浓度的β-END体内可缓解CIA鼠关节局部及全身免疫炎性反应,这使β-END成为有潜力的治疗RA的制剂。     

Autoimmunity to collagen in adjuvant arthritis of rats.

Arthritis can be induced in rats by intradermal injection of oil containing bacterial derivatives (adjuvant-induced arthritis) or cartilage collagen (type II collagen-induced arthritis). It was of interest, therefore, to determine whether collagen functions as an autoantigen in rats with adjuvant arthritis. Blood mononuclear cells from the majority of rats with adjuvant arthritis exhibited enhanced thymidine incorporation to homologous types I and II collagens, as well as to purified protein derivative of tuberculin. In contrast, cells from rats remaining nonarthritic after injection of adjuvant did not respond to collagen, although they did react to tuberculin. Similar results were obtained with a radiometric ear assay used to quantify intradermal delayed-type hypersensitivity in vivo. Using passive hemagglutination, autoantibodies to these collagens and their denatured alpha-chains were frequently detected in the sera of rats late in the course of adjuvant arthritis. Rats with inflammation of a hindlimb induced by turpentine did not acquire sensitivity to collagen. These data indicate that autoimmunity to collagen is a common feature of adjuvant- and collagen-induced arthritis, both of which are considered to be mediated by immunologic mechanisms.     

Lymphocyte subsets and mitogen induced proliferation in adjuvant arthritis. Part II. Changes in function and numbers of lymph nodes T lymphocytes.

To evaluate the effects of lymphocytes on the pathogenesis of adjuvant arthritis (AA), the lymphocytic responses to phytohemagglutinin (PHA) and concanavalin A (Con A) and the percentages of T cell subpopulations were investigated in the inguinal lymph nodes of Long-Evans (LE) rats and Sprague-Dawley (SD) rats after a single injection of complete Freund's adjuvant. The response of LE to PHA increased to its maximum on the 14th day and correlated with the severity of AA. The responses of LE and SD rats to Con A were decreased on the 7th, 11th, and 14th days. Additionally, percentage of suppressor T cells of LE rats was lower than that of SD rats. Significant increases of percentage of helper T cells and of helper/suppressor T cell ratio were observed in LE rats, indicating a decrease or defect in the suppressor T cells in LE rats. In LE, we postulated that helper T cells are involved in the induction of AA, and a defect in the suppressor T cell function or numbers would subject LE to the development of AA. SD rats, on the other hand, with higher suppressor T cell numbers, were less susceptible to AA.     

Passive transfer of arthritis to mice by injection of human anti-type II collagen antibody

The serum IgG fraction from a patient with seronegative rheumatoid-like arthritis which contained a high anti-type II collagen antibody titer was injected intravenously into mice susceptible to type II collagen-induced arthritis. A mild, transient, inflammatory arthritis was observed in 20 to 25% of the animals, whereas histologic signs of disease were evident in most of the injected mice. Purified human anti-type II collagen immunoglobulin injected into the knee joints of mice was also shown to induce a transient, inflammatory arthritis. Radiolabeled human anti-type II collagen IgG was shown to accumulate in the peripheral joints of mice, and the specificity of the antibody was shown to be similar to the specificity of anticollagen antibody eluted from the joints of mice with collagen-induced arthritis.     

Lymphocyte subsets and mitogen induced proliferation in adjuvant arthritis. Part I. Migration and function of the peripheral T lymphocytes.

Adjuvant arthritis (AA) was used as an animal model of rheumatoid arthritis in this study. Seventy 10-week-old inbred female rats of both Long-Evans rats (LE, high responder of adjuvant arthritis) and Sprague-Dawley rats (SD, low responder of adjuvant arthritis) were inoculated with adjuvant. Using monoclonal antibodies, we demonstrated that redistribution, migration, or an imbalance of T lymphocytes rather than the change of total T cells are involved in the pathogenesis of AA. After the adjuvant injection, the lymphocytic proliferative responses of LE and SD rats to phytohemagglutin (PHA) had no significant difference, whereas the response of LE rats to concanavalin A (ConA) was significantly lower than that of SD rats. In addition, a highly significant positive correlation was found between the absolute numbers of helper T cells and the lymphocytic proliferative response to PHA, and between absolute numbers of suppressor T cells and the lymphocytic proliferative response to Con A. We postulated high responder strain LE would have a defect in suppressor T lymphocytes leading to severe arthritis, while in low responder strain SD, adjuvant-activated suppressor T lymphocytes might reduce the severity of arthritis.     

Dendritic cells genetically engineered to express IL-4 inhibit murine collagen-induced arthritis

Dendritic cells (DCs) are specialized antigen-presenting cells that migrate from the periphery to lymphoid tissues, where they activate and regulate T cells. Genetic modification of DCs to express immunoregulatory molecules would provide a new immunotherapeutic strategy for autoimmune and other diseases. We have engineered bone marrow-derived DCs that express IL-4 and tested the ability of these cells to control murine collagen-induced arthritis (CIA), a model for rheumatoid arthritis in which Th1 cells play a critical role. IL-4-transduced DCs inhibited Th1 responses to collagen type II in vitro. A single injection of IL-4-transduced DCs reduced the incidence and severity of CIA and suppressed established Th1 responses and associated humoral responses, despite only transient persistence of injected DCs in the spleen. In contrast, control DCs and IL-4-transduced T cells or fibroblastic cells failed to alter the course of the disease. The functional effects correlated well with the differential efficiency of DC migration from various sites of injection to lymphoid organs, especially the spleen. The ability of splenic T cells to produce IL-4 in response to anti-CD3 was enhanced after the administration of IL-4-transduced DCS: These results support the feasibility of using genetically modified DCs for the treatment of autoimmune disease.     

Enhancement of collagen-induced arthritis in mice by diesel exhaust particles.

The present study was undertaken to investigate the effect of diesel exhaust particles (DEP) on collagen-induced arthritis (CIA), which is an experimental model of autoimmune disease, in mice. CIA was induced by s.c. injection of type II collagen (CII) emulsified with complete Freund's adjuvant into the base of the tail (day 0) followed by a booster injection on day 21. Varying doses of DEP were intranasally administered every 2 days from days 0 to 20. The results showed that administration of DEP enhanced both the incidence and the severity of CIA. The enhancement of the disease was associated with pronounced production of anti-CII IgG and IgG2a antibodies. Treatment with DEP also augmented proliferative responses of spleen cells to CII. There was marked secretion of interferon-gamma, interleukin (IL)-2, and IL-4 from the lymphoid cells in DEP-treated mice. Administration of DEP after onset of CIA was also effective in enhancing the severity of the disease as well as production of anti-CII IgG and IgG2a antibodies and secretion of interferon-gamma, IL-2, and IL-4. These results suggest that exposure to DEP may influence autoimmune disease.     

Passive transfer of diabetes from BB/W to Wistar-Furth rats.

Autoimmune diabetes can be transferred to young, diabetes prone BB/W rats by injecting them intravenously with concanavalin A (Con A)-treated spleen cells from acute diabetic BB/W donors. This study describes the transfer of diabetes to the normal Wistar-Furth strain of rats using a similar procedure. For the successful transfer of diabetes it was necessary to immunosuppress recipient animals with a single intraperitoneal injection of cyclophosphamide 24-48 h before administering Con A-stimulated spleen cells from acute diabetic BB/W rats. Of 68 Wistar-Furth rats in immunosuppressed with a dose of 100-150 mg cyclophosphamide/kg body wt, 10 (15%) became diabetic. None of the control rats receiving either Con A-stimulated Wistar-Furth spleen cells (n = 28), freshly isolated BB/W spleen cells (n = 14), or fresh RPMI medium (n = 11) became diabetic. These data indicate that diabetes can be transferred from BB/W to Wistar-Furth rats. In addition, they support the hypothesis that cell-mediated immune processes are involved in the development of insulin-dependent diabetes and rule out any absolute requirement for BB-derived genes in the target pancreatic beta cells.     

Lymphocyte subsets and mitogen induced proliferation in adjuvant arthritis. Part III. Numbers and functions of T lymphocytes in spleen.

The changes and the possible roles of splenic T lymphocyte subpopulations in high-responder Long-Evans (LE) rats and low-responder Sprague-Dawley (SD) rats with induced adjuvant arthritis (AA) were investigated. Total splenic T lymphocytes of both strains did not change significantly after the adjuvant injection. However, LE rats exhibited an increase in percentage of helper T cells and helper/suppressor T cell ratio, whereas SD rats expressed a decrease in the ratio with concomitant increase of suppressor T cells. This observation suggested that an imbalance of T lymphocyte ratio not only existed in the peripheral blood as reported by other investigators, but also existed in the spleen of rats with AA. In vitro measurement of phytohemagglutinin (PHA) responses of splenic T lymphocytes revealed an increased PHA response in LE but a markedly decreased PHA response in SD. Both LE and SD rats showed decreased response to concanavalin A (Con A) stimulation. We concluded that PHA response, as an index of helper T cell function, coincided with the development of AA and may be responsible for the immunoregulation of the disease. The increased proliferation of suppressor T cell in SD rats may also be significant in regulating the immune response to AA.     

Expression of a transgenic T cell receptor beta chain enhances collagen- induced arthritis

SWR/J transgenic (tg) mice were generated expressing the TCR beta chain derived from an anticollagen type II (CII) arthritogenic T cell clone. The SWR/J strain was selected because it is resistant to collagen-induced arthritis (CIA) and lacks the V beta gene segment used by the T cell clone. Expression of the tg beta chain on all thymocytes and peripheral lymph node T cells led to a more efficient anti-CII immune response, but did not confer CIA susceptibility to SWR/J mice. Nevertheless, this tg beta chain enhanced predisposition to CIA as (DBA/1 x SWR) F1 beta tg mice were more susceptible than normal F1 littermates. Our results demonstrate that the expression of the tg beta chain contributes to CIA susceptibility, but by itself it is not sufficient to overcome CIA resistance in the SWR/J strain.     

Pathogenetic difference between collagen arthritis and adjuvant arthritis

Daily treatment with cyclosporin at a dose of 25 mg/kg for 14 d gave complete suppression of the development of collagen arthritis and adjuvant arthritis in Sprague-Dawley rats during an observation period of 45 d. To study whether the immunologic unresponsiveness produced by cyclosporin is antigen specific, we rechallenged the cyclosporin- protected rats with either type II collagen or complete Freund's adjuvant (CFA) after discontinuation of cyclosporin treatment. Type II collagen-immunized, cyclosporin-protected rats did not develop arthritis in response to reimmunization with type II collagen, but, they did develop arthritis in response to a subsequent injection of CFA. Similarly, CFA-injected, cyclosporin-protected rats showed a suppressed arthritogenic reaction in response to reinjection of CFA, whereas their response to a subsequent immunization with type II collagen was unaffected. On the other hand, the rats that were treated with cyclosporin without any prior antigenic challenge could develop arthritis in response to a subsequent injection of CFA or type II collagen after cessation of cyclosporin treatment. These results indicate that specific immunologic unresponsiveness can be induced by cyclosporin in the two experimental models of polyarthritis, collagen arthritis and adjuvant arthritis, and that there is no cross-reactivity between type II collagen and the mycobacterial cell wall components. The results further indicate that immunity to type II collagen plays a critical role in the pathogenesis of collagen arthritis but that its pathogenetic role in adjuvant arthritis is insignificant.     

Characterization of the T cell receptor repertoire causing collagen arthritis in mice

Collagen type II-induced arthritis (CIA) is generated in susceptible rodent strains by intradermal injections of homologous or heterologous native type II collagen in complete Freund's adjuvant. Symptoms of CIA are analogous to those of the human autoimmune disease, rheumatoid arthritis. CIA is a model system for T cell-mediated autoimmune disease. To study the T cell receptor (TCR) repertoire of bovine type II-specific T cells that may be involved in the pathogenesis of CIA in DBA/1Lac.J (H-2q) mice, 13 clonally distinct T cell hybridomas specific for bovine type II collagen have been established and the alpha and beta chains of their TCRs have been analyzed. These T cell hybridomas recognize epitopes that are shared by type II collagens from distinct species and not by type I collagens, and exhibit a highly restricted TCR-alpha/beta repertoire. The alpha chains of the TCRs employ three V alpha gene subfamilies (V alpha 11, V alpha 8, and V alpha 22) and four J alpha gene segments (J alpha 42, J alpha 24, J alpha 37, and J alpha 32). The V alpha 22 is a newly identified subfamily consisting of approximately four to six members, and exhibits a high degree of polymorphism among four mouse strains of distinct V alpha haplotypes. In addition, the beta chains of the TCRs employ three V beta gene subfamilies (V beta 8, V beta 1, and V beta 6), however the V beta 8.2 gene segment is preferentially utilized (58.3%). In contrast, the J beta gene segment usage is more heterogeneous. On the basis of the highly limited TCR-alpha/beta repertoire of the TCRs of the panel of bovine type II-specific T cell hybrid clones, a significant reduction (60%) of the incidence of arthritis in DBA/1Lac.J mice is accomplished by the use of anti-V beta 8.2 antibody therapy.     

Possible role of V beta T cell receptor genes in susceptibility to collagen-induced arthritis in mice

Arthritis was induced by immunization of type II collagen in adjuvant in mice from H-2q-bearing crosses between SWR (H-2q/q) and B10 (H-2b/b mice), two strains known to be resistant to collagen-induced arthritis (CIA). The resistance of B10 is known to be due to its MHC haplotype, but it was postulated that the resistance of SWR mice which expresses the susceptible MHC haplotype could be due to the deletion of close to 50% of the V beta genes of the T cell receptor (TCR) in them. 17% of the F1 hybrids, 33% of the SWR backcrosses, 68% of the B10 backcrosses, and 52% of the F2 hybrids developed arthritis on follow-up to 5 mo after primary immunization with collagen. There was no significant difference in anti-type II collagen antibody titers between the arthritic and nonarthritic mice in each of these crosses. The segregation of the TCR genes with arthritis was determined in the F2 population by typing with F23.1 mAb that reacts with T cells using V beta 8 subfamily genes in their TCRs. SWR mice are F23.1- as V beta 8 genes are deleted in them. All six of arthritic mice homozygous for H-2q, and thus with an H-2 haplotype similar to SWR mice, expressed the F23.1 marker. These studies indicate that for complete susceptibility to collagen-induced arthritis, not only is a susceptible MHC haplotype (H-2q) important, but possibly also the presence of a subset of T cells using certain specific V beta genes in their TCRs. Other background genes may, however, modulate the severity of arthritis.     

Inhibition of lymphoproliferative responses by SK&F 105685, a novel anti-arthritic agent.

SK&F 105685 (N,N-Dimethyl-8,8-dipropyl-2-azaspiro[4.5]decane-2-propanamine+ ++ dihydrochloride) is a novel azaspirane with beneficial activity in animal models of autoimmune diseases such as adjuvant-induced arthritis and experimental autoimmune encephalomyelitis in the Lewis rat and lupus-like disease in the MRL mouse. The effect of SK&F 105685 on the proliferation of rat lymphoid cells was examined in vitro. The compound inhibited the proliferative response of spleen, thymus and lymph node cells to the mitogen concanavalin A (Con A) in a dose-dependent manner but had little or no effect on the mitogenic response of peripheral blood lymphocytes. Although less potent than cyclosporin A, SK&F 105685 was able to inhibit the proliferation of spleen cells stimulated with PMA and ionomycin or the mitogens phytohemagglutinin (PHA), Con A and pokeweed mitogen (PWM). Relatively early event(s) in cell proliferation were affected by SK&F 105685 since delaying addition of the drug by 24 to 48 hours after Con A stimulation of rat spleen cells resulted in reduced levels of suppression. The mode of action of SK&F 105685 appeared to differ from that of cyclosporin A or rapamycin. Unlike cyclosporin A, SK&F 105685 did not affect IL-2 production by Con A-stimulated spleen cells or the IL-2-producing Jurkat cell line, but, like rapamycin, the compound significantly reduced the IL-2-induced proliferation of rat ConA blasts. These results suggest that inhibition of lymphocyte proliferation by SK&F 105685 may require the activity of an intermediate effector cell(s) present in susceptible populations such as cells from the spleen, thymus, lymph nodes and Con A blast preparations but absent or present in low numbers in resistant populations such as peripheral blood cells. Indomethacin and NG-monomethyl-L-arginine (NGMMA), a competitive inhibitor of nitric oxide synthase, were both unable to relieve SK&F 105685-induced suppression of splenic Con A responses thereby ruling out a role for the production of prostaglandins or nitric oxide by macrophages as an intermediate in drug-mediated suppression. In summary, SK&F 105685 was unable to inhibit lymphoproliferative responses by a mechanism distinct from that of cyclosporin A or rapamycin and which appears to involve regulation of cellular interactions rather than a direct effect on responding lymphocytes.     

薯蓣皂苷对胶原性关节炎大鼠的免疫调节

目的研究薯蓣皂苷(Dioscin)对大鼠胶原性关节炎(CIA)的免疫调节作用。方法将SD大鼠随机分为正常组、CIA模型组、阳性药物组和实验组。连续灌胃给药2周,停药后处死大鼠,足容积法测量继发侧足肿胀度,观察该药对CIA大鼠免疫器官的影响,MTT法检测ConA诱导的小鼠脾淋巴细胞增殖转化,中性红实验测定小鼠腹腔巨噬细胞吞噬功能,酶联免疫吸附测定法(ELISA)测定大鼠血清细胞因子IL-1、和TNF-α的水平。结果从第24天起,薯蓣皂苷(60、120 mg/kg)对CIA大鼠继发性炎症反应有明显的治疗作用。薯蓣皂苷可剂量依赖性降低CIA大鼠胸腺指数,提高淋巴细胞增殖反应及腹腔巨噬细胞吞噬能力,高、中、低剂量组给药大鼠的血清内炎性细胞因子IL-1、TNF-α水平较CIA模型组明显降低(P<0.01)。结论薯蓣皂苷有良好的抗炎及免疫调节作用,对CIA大鼠继发性炎症有治疗作用,其作用机制可能与调节机能依赖性的双向免疫及抑制炎性细胞因子产生有关。     

Induction and suppression of collagen-induced arthritis is dependent on distinct fcgamma receptors

Receptors for immunoglobulin (Ig)G (FcgammaRs) are important for the antibody-mediated effector functions of the immune system. FcgammaRI and FcgammaRIII trigger cell activation through a common gamma chain, whereas FcgammaRII acts as a negative regulator of antibody production and immune complex-triggered activation. Here we describe the in vivo consequences of FcgammaR deficiency in a mouse model of human rheumatoid arthritis. FcRgamma chain-deficient mice on arthritis-susceptible DBA/1 background were immunized with collagen for induction of collagen-induced arthritis. The DBA/1 mice lacking FcRgamma chain were protected from collagen-induced arthritis in contrast to wild-type mice, although both groups produced similar levels of IgG anticollagen antibodies. In comparison, DBA/1 mice lacking FcgammaRII developed an augmented IgG anticollagen response and arthritis. These observations suggest a crucial role of FcgammaRI and FcgammaRIII in triggering autoimmune arthritis.