Humoral and Cellular Sensitivity to Collagen in Type II Collagen-Induced Arthritis in Rats

We have recently described a new animal model of arthritis induced by intradermal injection of a distinct type of collagen found in cartilage (type II collagen). Since immunologic sensitivity to collagen could play a role in the pathogenesis of this type II collagen-induced arthritis in rats, the ability of purified types of native collagens to induce cellular and humoral responses was quantified by antigeninduced tritiated thymidine incorporation into lymphocytes by collagen and passive hemagglutination, respectively. Rats injected intradermally with native heterologous or homologous type II collagens in adjuvant developed type-specific cellular as well as humoral reactivity. Types I and III collagens were less immunogenic than was type II. The latter collagen induced brisk cellular and humoral responses that were equivalent whether complete Freund's adjuvant or incomplete Freund's adjuvant were employed. Both responses could be induced by native type II collagens modified by limited pepsin digestion, indicating that they are not attributable to determinants in the telopeptide regions of the molecule. Thus, these studies demonstrate the unique immunogenic as well as arthritogenic properties of the type II collagen molecule and indicate that both result from a helical conformation of its structurally distinct alpha-chains. Further, they suggest that type II collagen may, by humoral or cellular mechanisms, provoke or perpetuate inflammation in other arthritic diseases.     

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.     

Autoimmunity to type II collagen an experimental model of arthritis

We have found that intradermal injection of native type II collagen extracted from human, chick or rat cartilage induces an inflammatory arthritis in approximately 40% of rats of several strains whether complete Freund's adjuvant or incomplete Freund's adjuvant is used. Type I or III collagen extracted from skin, cartilage proteoglycans and alpha1(II) chains were incapable of eliciting arthritis, as was type II collagen injected without adjuvant. The disease is a chronic proliferative synovitis, resembling adjuvant arthritis in rats and rheumatoid arthritis in humans. Native type II co-lagen modified by limited pepsin digestion still produces arthritis, suggesting that type- specific determinants residing in the helical region of the molecule are responsible for the induction of disease. Since homologous type II collagen emulsified in oil without bacterial preparations regularly causes the disease, this new animal model of arthritis represents a unique example of experimentally-inducible autoimmunity to a tissue component.     

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.     

Adoptive transfer of suppression of arthritis in the mouse model of collagen-induced arthritis. Evidence for a type II collagen-specific suppressor T cell.

This study details the suppressive mechanism involved in the antigen-specific suppression of collagen-induced arthritis. Intravenous injection of 500 micrograms of soluble native type II collagen 3 d before immunization with native type II collagen emulsified in complete Freund's adjuvant resulted in animals with decreased in vitro cellular and humoral immune response to native and denatured type II collagen compared with control groups. Control groups were composed of animals preinoculated with saline and type I collagen and established the antigen-specific nature of the observed suppression. Mice with reduced immune responses to type II collagen also were observed to portray little or no erythema and edema associated with collagen-induced arthritis. Adoptive transfer experiments established the requirement of T cells for the suppression of collagen-induced arthritis. Analysis of the phenotype of responding splenic cells in chronic immunotherapeutically suppressed mice in vitro revealed that responding cells were Ly1-2+ (suppressor/cytotoxic) T cells. On the other hand, the cellular phenotype of T cells responding to type II collagen in nonsuppressed collagen-induced arthritic mice was Ly1+2- (helper/inducer T cells). The data indicate that type II collagen-specific T cells are generated on intravenous inoculation of soluble native type II collagen. These cells are observed in type II collagen-immune animals, which are nonarthritic and portray reduced humoral and in vitro cellular immune response to type II collagen. This study suggests that specific suppression of immune responses to type II collagen by T-suppressor cells can be immunotherapeutic in certain forms of arthritis.     

Synergy between adjuvant arthritis and collagen-induced arthritis in rats

Adjuvant arthritis (AA) in rats is susceptible to cell-mediated passive transfer. Collagen-induced arthritis (CIA) in rats is susceptible to passive transfer with antibody to type II collagen. We report here the development of strikingly severe arthritis in Lewis rats as the result of synergy between passively transferred antibody to type II collagen from rats with CIA and concanavalin A (Con A)-stimulated lymph node or spleen cells from syngeneic rats with AA. Similar synergy was seen in rats with AA given anticollagen antibody, in rats with CIA given Con A-stimulated adjuvant spleen cells, and in rats actively immunized with CII and complete Freund's adjuvant. The synergistic process caused a very severe polyarthritis, characterized by marked swelling and erythema in all the joints of the distal extremities, with histologic and radiographic evidence of early, extensive erosion of articular cartilage. Synergy was apparent if the lymphoid cells from AA rats were given up to 1 mo after a single injection of anticollagen antibody. No synergy was seen when normal rat immunoglobulin or anti-ovalbumin antibody was substituted for anticollagen antibody, when Con A-stimulated lymphoid cells from normal rats or donors with CIA were used, or when Con A-stimulated AA lymphoid cells were irradiated before transfer. Synergy between separate immune effector mechanisms may represent a general phenomenon in the pathogenesis of inflammatory joint disease.     

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.     

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.     

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.     

胶原诱导性关节炎大鼠滑膜组织中磷酸化p38与JNK通路及痹肿消汤的影响

背景：有研究报道滑膜细胞信号传导异常是类风湿关节炎发病的重要机制之一。目的：观察胶原诱导性关节炎大鼠中丝裂原活化蛋白激酶通路中磷酸化p38和磷酸化JNK的活化及痹肿消汤的影响。方法：将72只SD大鼠随机分为正常组、模型组、痹肿消汤组。模型组及痹肿消汤组大鼠足趾注射胶原蛋白乳剂制备胶原诱导性关节炎模型大鼠,10d后加强免疫,初次免疫后第14天开始给痹肿消汤组大鼠每天痹肿消汤灌胃,模型组蒸馏水灌胃,正常组自由饮水。结果与结论：Westernblot检测结果显示,胶原诱导性关节炎大鼠中磷酸化p38及JNK的表达较正常组显著增高（P〈0.05）。与模型组相比,痹肿消汤组能下调磷酸化p38及JNK蛋白的表达（P〈0.05）。说明痹肿消汤可抑制类风湿关节炎中磷酸化p38及JNK的高表达。     

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.     

Depletion of alpha/beta T cells by a monoclonal antibody against the alpha/beta T cell receptor suppresses established adjuvant arthritis, but not established collagen-induced arthritis in rats

The effects of treatment with a monoclonal antibody (R73 mAb) against T cell receptor alpha/beta (TCR-alpha/beta) on both established adjuvant arthritis (EAA) and established collagen-induced arthritis (ECIA) in rats have been investigated. Rats were treated with R73 mAb when arthritis reached a peak. Treatment with the anti-TCR-alpha/beta mAb markedly suppressed EAA, whereas ECIA was not affected by the mAb treatment. Histologically, R73 mAb-treated rats with EAA showed mild hyperplasia of synovial tissues, sparse infiltration of inflammatory cells, and minimal erosion of cartilage, whereas arthritic rats treated with PBS and an irrelevant control mAb against Giardia had marked hyperplasia of synovium with pannus, massive inflammatory cell infiltrate, and severe destruction of cartilage and subchondral bone. R73 mAb-treated rats with ECIA exhibited pronounced formation of pannus containing many inflammatory cells and marked cartilage and subchondral damage similar to those in arthritic rats that received the control treatments. Treatment with R73 mAb depleted markedly alpha/beta+ T cells in both peripheral blood and synovial tissues of rats with EAA and ECIA. R73 mAb treatment was associated with marked reduction in arthritogen-specific delayed-type hypersensitivity responses in both EAA and ECIA. The titers of antibodies against type II collagen produced in rats with ECIA were not affected by the mAb. Thus, alpha/beta+ T cells appear to have a central role in EAA, but not in chronic ECIA.     

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.     

Arthritis induced in rats by cloned T lymphocytes responsive to mycobacteria but not to collagen type II.

We have been studying the pathogenesis of adjuvant arthritis in rats using a long-term cell line of T lymphocytes, the A2 line, which can induce polyarthritis and can also be used to vaccinate rats against adjuvant arthritis. Although line A2 was selected for its proliferative response to mycobacteria, it also responded to collagen type II. To elucidate its role of responsiveness to collagen type II and the relationship between arthritogenicity and vaccination, we cloned A2 and selected a subline A2b. We now report that subline A2b, which bore a marker of helper/delayed hypersensitivity T lymphocytes, was strongly arthritogenic, but could not vaccinate against arthritis. Moreover, A2b showed no response to collagen type II. Therefore, reactivity to collagen type II is not a requisite for arthritogenicity, and mediation of arthritis and vaccination can be distinct properties of different populations of T lymphocytes.     

Oleuropein aglycone, an olive oil compound, ameliorates development of arthritis caused by injection of collagen type II in mice

The aim of this study was to investigate the effect of oleuropein aglycone, an olive oil compound, on the modulation of the inflammatory response in mice subjected to collagen-induced arthritis (CIA). CIA was induced in mice by an intradermal injection of 100 μl of an emulsion containing 100 μg of bovine type II collagen (CII) and complete Freund's adjuvant (CFA) at the base of the tail. On day 21, a second injection of CII in CFA was administered. Mice developed erosive hind paw arthritis when immunized with CII in CFA. Macroscopic clinical evidence of CIA first appeared as periarticular erythema and edema in the hind paws. The incidence of CIA was 100% by day 28 in the CII-challenged mice and the severity of CIA progressed over a 35-day period with resorption of bone. The histopathology of CIA included erosion of the cartilage at the joint. Treatment with oleuropein aglycone starting at the onset of arthritis (day 25) ameliorated the clinical signs at days 26 to 35 and improved histological status in the joint and paw. The degree of oxidative and nitrosative damage was also significantly reduced in oleuropein aglycone-treated mice. Plasma levels of the proinflammatory cytokines were also significantly reduced by oleuropein aglycone. In addition, we have confirmed the beneficial effects of oleuropein aglycone on an experimental model of CIA in a therapeutic regimen of post-treatment, with treatment started at day 28, demonstrating that oleuropein aglycone exerts an anti-inflammatory effect during chronic inflammation and ameliorates the tissue damage associated with CIA.     

Tibetan Medicated-Bath Therapy may Improve Adjuvant Arthritis in Rat

Tibetan medicated-bath therapy has been applied to patients with rheumatoid arthritis for centuries. However, the detailed action mechanism of Tibetan medicated-bath therapy on the morphology and function of joints remains unknown. We designed our investigation to evaluate the efficacy of Tibetan medicated-bath therapy on adjuvant arthritis (AA) of rats in comparison with water-bath and dexamethasone administration. AA was induced by intradermal injection of Mycobacterium butyricum suspended in sterile mineral oil. The control animals were similarly injected with sterile vehicle. Eight days after injection, rats were treated with fresh-water bath, Tibetan medicated-bath (40 degrees C, 15 min) or intramuscular injection with dexamethasone for 21 consecutive days after which we evaluated the severity of arthritis visually and microscopically and measured serum interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha levels. While arthritis did not significantly change after water-bath treatment, the Tibetan medicated-bath and dexamethasone groups showed diminished joint swelling and alleviation of, inflammatory cell infiltration and the destruction of bone and cartilage. Serum IL-6 and TNF-alpha levels significantly decreased. Our results demonstrated that Tibetan medicated-bath therapy exerted a reliable effect on rat adjuvant arthritis, which may be involved in the inflammatory cytokines, IL-6 and TNF-alpha. Our data provide evidence for clinical use of Tibetan-medicated bath therapy for arthritis patients.     

Protective effect of Withania somnifera root powder in relation to lipid peroxidation, antioxidant status, glycoproteins and bone collagen on adjuvant-induced arthritis in rats

The present investigation was carried out to evaluate the protective effect of Withania somnifera Linn.Dunal (family-Solanaceae), commonly known as Ashwagandha, on adjuvant-induced arthritic rats. Results were compared with those for Indomethacin, a nonsteroidal anti-inflammatory drug. Arthritis was induced by intradermal injection of complete Freund's adjuvant (0.1 mL) into the right hind paw of Wistar albino rats. Withania somnifera root powder (1000 mg/kg/day) and Indomethacin (3 mg/kg/day) were orally administered for 8 days (from 11th to 18th day) after adjuvant injection. The anti-arthritic effect of W. somnifera root powder was assessed by measuring changes in lipid peroxidation, antioxidant status, and glycoprotein levels in plasma and spleen of arthritic animals. In addition, cartilage degradation was also assessed by estimating bone collagen, and urinary constituents in arthritic animals. Results of the present investigation showed significant increase in the level of lipid peroxides, glycoproteins, and urinary constituents with the depletion of antioxidant status and bone collagen in arthritic animals. These biochemical alterations observed were ameliorated significantly by oral administration of W. somnifera root powder (1000 mg/kg body weight) in arthritic animals. The results of this study clearly indicate that W. somnifera root powder is capable of rectifying the above biochemical changes in adjuvant arthritis.     

佐剂关节炎大鼠心功能变化与细胞因子、调节T细胞和Foxp3表达的相关性

目的:观察佐剂性关节炎(adjuvant arthritis,AA)大鼠足跖肿胀度(E)、关节炎指数(AI)、心功能、细胞因子、调节T细胞及Foxp3的表达,探讨细胞因子、转录因子Foxp3与调节T细胞(Treg)在AA大鼠心功能改变中的作用机制.方法:24只Wistar雄性大鼠随机分为正常组和模型组(Model组)各...     

Spontaneous and evoked release of met-enkephalin-like material from the spinal cord of arthritic rats in vivo

Perfusion of the intrathecal space with artificial CSF was achieved in control and arthritic rats under halothane anaesthesia in order to collect the met-enkephalin-like material (MELM) released from the whole spinal cord. On the fourth week following the intradermal injection of Freund's adjuvant to induce arthritis, a marked reduction (-56%) in the spontaneous outflow of MELM was noted in arthritic rats. This effect did not involve changes in the degradation process of MELM, since it persisted when kelatorphan was added to the perfusing fluid in order to inhibit completely the peptidases acting on met-enkephalin. Raising the K+ concentration in the perfusing fluid from 2.4 to 40 mM, as well as moving the hind paws, produced a significant enhancement of MELM release which was (at least) as pronounced in arthritic as in control rats. These results suggest that the basal activity of spinal enkephalinergic neurones, but not that triggered by various stimuli, is reduced in arthritic rats.     

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.