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.     

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.     

Resistance to collagen-induced arthritis in a nonhuman primate species maps to the major histocompatibility complex class I region.

Type II collagen-induced arthritis (CIA) is an experimentally inducible autoimmune disorder that is, just like several forms of human arthritis, influenced by a genetic background. Immunization of young rhesus monkeys (Macaca mulatta) with type II collagen (CII) induced CIA in about 70% of the animals. One major histocompatibility complex (MHC) class I allele was present only in young animals resistant to CIA and absent in arthritic animals. This strong association suggests that the MHC class I allele itself, or a closely linked gene, determines resistance to CIA. The mechanism controlling the resistance to CIA becomes less efficient in aged animals since older rhesus monkeys, which were positive for the resistance marker, developed a mild form of arthritis. At the cellular level it is demonstrated that resistance to CIA is reflected by a low responsiveness of T cells to CII. This association between a specified MHC class I allele and resistance to an autoimmune disease points at the importance of the MHC class I region in the regulation of the immune response to an autoantigen.     

Identification of an immunosuppressive epitope of type II collagen that confers protection against collagen-induced arthritis

We have previously reported that collagen-induced arthritis can be suppressed by intravenous injection of native type II (CII) but not type I collagen. We have now identified denatured fragments of CII capable of suppressing collagen-induced arthritis and inducing tolerance. Purified CII was cleaved with cyanogen bromide (CB), and the major resulting peptides were isolated. Female DBA/1 mice were administered OVA, native CII, or one of the CB peptides, intravenously, before immunization with native CII, 6 wk after immunization, mice tolerized with CII and CB11 had a markedly lower incidence of arthritis compared with controls. There was a correlation between the overall antibody response and the incidence of arthritis. In addition, animals tolerized with either CII or CB11 had a decreased antibody response not only to CII, but also to each of the other CB peptides tested. To identify the epitope involved in suppression of arthritis, five synthetic peptides, 21-26 amino acids in length, corresponding to selected regions of CB11, were generated. Each of the peptides was injected intravenously into mice before immunization. Only one of these, CB11 122-147, was capable of suppressing arthritis. In addition, mice given the synthetic peptide CB11 122-147 neonatally were suppressed for arthritis and antibody responsiveness when immunized with CII at 8 wk of age. Thus, we have identified CB11 122-147 as an epitope of CII important in induction of tolerance and suppression of disease. Further experiments narrowing down the pivotal amino acids for the immunogenicity of this epitope and the role this epitope plays in induction and regulation of disease will enhance our understanding of how the immune response to collagen affects autoimmune arthritis.     

Induction of specific immune unresponsiveness with purified mixed leukocyte culture-activated T lymphoblasts as autoimmunogen. III. Proof for the existence of autoanti-idiotypic killer T cells and transfer of suppression to normal syngeneic recipients by T or B lymphocytes

Specific immune unresponsiveness against a given set of histocompatibility antigens can be induced by immunization with autologous, antigen-specific T lymphoblasts. Such unresponsiveness can be transferred by lymphoid cells from autoblast-immunized donors to normal syngeneic recipients. The cells being most efficient in transferring the selective suppression are T lymphocytes from the spleen, especially if of Ly 1-2+3+ phenotype. By using such T lymphocytes we deem it likely that the actual underlying mechanism is one of actual transfer of autoanti-idiotypic killer T cells. In support for this view is the fact that such T cells endowed with exquisite specific, cytolytic reactivity towards autologous idiotype-positive T target cells exist in autoblast immune animals. Significant suppression may also be transferred with T cells of Ly 1+2-3- phenotype or with B cells. Here, we consider the suppressive mechanism to be one of production of autoanti-idiotypic antibodies. By using affinity fraction procedures, it was finally possible to prove that all T-cell suppressive activity resides in a population with true antigen-binding-specific receptors for the relevant idiotypes.     

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.     

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.     

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.     

Mechanisms of suppression in the transfer of contact sensitivity. Analysis of an I-J+ molecule required for Ly2 suppressor cell activity

The passive transfer of contact sensitivity (CS) by immune cells can be inhibited with an antigen-specific T suppressor factor. This factor is composed of two subfactors: an antigen-specific subfactor made by an Ly1+ cell (PC1-F) and a antigen nonspecific subfactor made by an Ly2+ T cell (TNBSA-F). The suppressive activity of the complete factor can be eliminated by depleting the assay population of Ly2+ cells, even though it is the Ly1+ cell in the population that transfers the adoptive immunity. This suggests that the Ly2+ cell in the assay population is needed to transduce the suppressive signal to the Ly1+ effector cell of DTH. We found that an Ly2+ cell from immune animals could be induced to produce a cell free subfactor that overcame the requirement for this Ttrans cell in the suppression of CS by TsF. The induction required only PC1-F, TNP-coupled spleen cells, and resulted in the production of an antigen-nonspecific I-J+ subfactor by immune Ly2+, I-J+ cells. The need for the Ly2+ transducer cell could also be overcome by addition of an I-J+ molecule secreted by Ly1 T cells hyperimmunized to SRBC. A suppressor complex made from mixing the I-J+ molecule with TNBSA-F could directly suppress the functional activity of immune T cells not only to transfer CS, but also to deliver help to B cells in an in vitro PFC response. This suppressive complex is antigen-nonspecific and does not require Ly2+ T cells in the assay population for suppressive activity. These results indicate that effector factors of the suppressor circuit require two molecules; one that contains the functional suppressor material and one that serves as a "schlepper," a molecule needed to deliver the suppression to the appropriate target cell. The ability to construct a functional suppressor complex from two subfactors raised against different antigens, using different immunization procedures, which were isolated from factors exhibiting different functional activities suggests that certain cells of the immune system may play a universal role in "transducing" the suppressive signal.     

Inducible costimulator is essential for collagen-induced arthritis

CD4(+) helper Th cells play a major role in the pathogenesis of rheumatoid arthritis. Th cell activation, differentiation, and immune function are regulated by costimulatory molecules. Inducible costimulator (ICOS) is a novel costimulatory receptor expressed on activated T cells. We, as well as others, recently demonstrated its importance in Th2 cytokine expression and Ab class switching by B cells. In this study, we examined the role of ICOS in rheumatoid arthritis using a collagen-induced arthritis model. We found that ICOS knockout mice on the DBA/1 background were completely resistant to collagen-induced arthritis and exhibited absence of joint tissue inflammation. These mice, when immunized with collagen, exhibited reduced anti-collagen IgM Ab's in the initial stage and IgG2a Ab's at the effector phase of collagen-induced arthritis. Furthermore, ICOS regulates the in vitro and in vivo expression of IL-17, a proinflammatory cytokine implicated in rheumatoid arthritis. These data indicate that ICOS is essential for collagen-induced arthritis and may suggest novel means for treating patients with rheumatoid 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.     

Paradoxical effects of cyclosporin A on collagen arthritis in rats

The effect of the immunosuppressive agent cyclosporin A (CS-A) on collagen arthritis in Sprague-Dawley rats is investigated. A 14-d course of CS-A treatment at doses of 15 mg/kg per day or more, begun on the same day as type II collagen immunization, suppressed the development of arthritis as well as humoral and delayed-type hypersensitivity (DTH) skin test responses to type II collagen, possibly by interfering with helper T cells. Additional studies demonstrated that CS-A treatment only during the induction phase of immunity proved to be successful. When CS-A treatment was started only during the immediately preclinical phase of arthritis or after the disease onset, a significant enhancement of the disease was obtained in a dose-dependent manner. This enhancement was accompanied by an augmentation of DTH skin reactions, while antibody responses were either suppressed or unaffected. These results appear to be attributable at least in part to a suppressive effect of CS-A on a population of suppressor T cells, thus resulting in a T cell-mediated helper effect. It is therefore reasonable to assume that the paradoxical effects of CS-A on collagen arthritis in rats might be caused by an altering of the sensitive balance of the two regulatory subpopulations of T cells. It is also possible that cell-mediated immune responses may play an important role in influencing the course of the disease.     

Recombinant galectin-1 and its genetic delivery suppress collagen-induced arthritis via T cell apoptosis.

Galectin-1 (GAL-1), a member of a family of conserved beta-galactoside-binding proteins, has been shown to induce in vitro apoptosis of activated T cells and immature thymocytes. We assessed the therapeutic effects and mechanisms of action of delivery of GAL-1 in a collagen-induced arthritis model. A single injection of syngeneic DBA/1 fibroblasts engineered to secrete GAL-1 at the day of disease onset was able to abrogate clinical and histopathological manifestations of arthritis. This effect was reproduced by daily administration of recombinant GAL-1. GAL-1 treatment resulted in reduction in anticollagen immunoglobulin (Ig)G levels. The cytokine profile in draining lymph node cells and the anticollagen IgG isotypes in mice sera at the end of the treatment clearly showed inhibition of the proinflammatory response and skewing towards a type 2-polarized immune reaction. Lymph node cells from mice engaged in the gene therapy protocol increased their susceptibility to antigen-induced apoptosis. Moreover, GAL-1-expressing fibroblasts and recombinant GAL-1 revealed a specific dose-dependent inhibitory effect in vitro in antigen-dependent interleukin 2 production to an A(q)-restricted, collagen type 2-specific T cell hybridoma clone. Thus, a correlation between the apoptotic properties of GAL-1 in vitro and its immunomodulatory properties in vivo supports its therapeutic potential in the treatment of T helper cell type 1-mediated autoimmune disorders.     

Cellular and genetic restrictions in the immunoregulatory activity of alpha-fetoprotein. I. Selective inhibition of anti-Ia-associated proliferative reactions

Alpha-fetoprotein (AFP), a major alpha-globulin component of fetal and newborn sera, has earlier been shown to exert significant immunosuppressive activity in vitro on T-dependent-immune responses. In the present investigation we have examined the effects of AFP on the recognition and proliferation of T lymphocytes responding in mixed leukocyte culture against histocompatibility-associated alloantigens. Fetal-derived AFP could be shown to exert differential effects on both primary and secondary responses ranging from strong inhibition to occasional enhancement, depending on the stimulating antigens. Proliferative responses against major histocompatibility complex (MHC) I region determinants, mediated predominantly by Ly 1 + cells, were markedly suppressed. Suppression was also observed in responses against Mls locus products, an antigenic system whose recognition requires concomitant recognition of I region gene products on the stimulating cells. In contrast, responses against MHC K or D region determinants, mediated predominantly by Ly 2 + cells, were generally unaffected by AFP. Similarly, non-MHC loci alloantigens distinct from Mls locus products also induced T-cell proliferation which was refractive to suppression by AFP. Because neither Ly 1 + nor Ly 2 + cells responding in this latter situation could be inhibited by AFP, we concluded that the mere fact that a T cell expresses a particular Ly phenotype does not predetermine sensitivity to AFP-induced suppression. In any case, AFP exerts a highly selective suppressive activity on I region-associated immune responses. These data may help to resolve the present controversy over the possibility that AFP has an in vivo relevance as an immunosuppressive agent by pointing out the importance of selecting proper genetic situations for study.     

Interleukin-1 potentiates the development of collagen-induced arthritis in mice

1. The subcutaneous administration of recombinant human interleukin-1 beta (rhIL-1 beta) was found to induce an increased incidence and earlier onset of collagen-induced arthritis in mice. 2. The rhIL-1 beta had different effects, depending on when it was administered after collagen-immunization. 3. The effect of rhIL-1 beta may be due, in part, to augmentation of the immune response to type II collagen. 4. Interleukin-1-accelerated, collagen-induced arthritis will provide a useful model for investigating the role of interleukin-1 in the regulation of arthritic diseases, and the development of anti-arthritic therapeutics.     

Macrophages suppress T cell responses and arthritis development in mice by producing reactive oxygen species

Reduced capacity to produce ROS increases the severity of T cell-dependent arthritis in both mice and rats with polymorphisms in neutrophil cytosolic factor 1 (Ncf1) (p47phox). Since T cells cannot exert oxidative burst, we hypothesized that T cell responsiveness is downregulated by ROS produced by APCs. Macrophages have the highest burst capacity among APCs, so to study the effect of macrophage ROS on T cell activation, we developed transgenic mice expressing functional Ncf1 restricted to macrophages. Macrophage-restricted expression of functional Ncf1 restored arthritis resistance to the level of that of wild-type mice in a collagen-induced arthritis model but not in a T cell-independent anti-collagen antibody-induced arthritis model. T cell activation was downregulated and skewed toward Th2 in transgenic mice. In vitro, IL-2 production and T cell proliferation were suppressed by macrophage ROS, irrespective of T cell origin. IFN-gamma production, however, was independent of macrophage ROS but dependent on T cell origin. These effects were antigen dependent but not restricted to collagen type II. In conclusion, macrophage-derived ROS play a role in T cell selection, maturation, and differentiation, and also a suppressive role in T cell activation, and thereby mediate protection against autoimmune diseases like arthritis.     

MHC control of CD4+ T cell subset activation

The present results demonstrate that CD4+ T cells activated in the primary in vivo response to antigen produce distinct patterns of cytokines depending upon the MHC class II haplotype of the responding mice. I-As mice were found to selectively activate IL-2/IFN-gamma-producing CD4+ T cells, whereas I-Ab mice exhibited selective activation of IL-4-producing CD4+ T cells in response to collagen IV. The effector response phenotype was found to correlate with the cytokine phenotype of CD4+ T cells activated in vivo; IL-2/IFN-gamma-producing cells giving rise to proliferative (cell-mediated) responses, IL-4-producing cells leading to secondary IgG (humoral) responses. Together the data support the notion that the outcome of a given immune response (e.g., protection vs. onset, tolerance vs. autoimmunity) may be determined in part by the type of CD4+ T cells initially activated by antigen. Moreover, the present experiments demonstrate for the first time that polymorphism in class II MHC can determine such selective activation of different cytokine-producing CD4+ T cell phenotypes.     

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.     

Localized expression of an anti-TNF single-chain antibody prevents development of collagen-induced arthritis

Although systemic administration of neutralizing anti-TNF antibodies has been used successfully in treating rheumatoid arthritis, there is a potential for side effects. We transduced a collagen reactive T-cell hybridoma with tissue-specific homing properties to assess therapeutic effects of local delivery to inflamed joints of anti-TNF single-chain antibodies (scFv) by adoptive cellular gene therapy. Cell culture medium conditioned with 1 x 10(6) scFv producer cells/ml had TNF neutralizing capacity in vitro equivalent to 50 ng/ml anti-TNF monoclonal antibody. Adding a kappa chain constant domain to the basic scFv (construct TN3-Ckappa) gave increased in vitro stability and in vivo therapeutic effect. TN3-Ckappa blocked development of collagen-induced arthritis in DBA/1LacJ mice for >60 days. Transgene expression was detected in the paws but not the spleen of treated animals for up to 55 days postinjection. No significant variations in cell proliferation or cytokine secretion were found in splenocytes or peripheral lymphocytes. IL-6 expression was blocked in the diseased paws of mice in the scFv treatment groups compared to controls. In conclusion, we have shown that local expression of an anti-inflammatory agent blocks disease development without causing demonstrable systemic immune function changes. This is encouraging for the potential development of safe adoptive cellular therapies to treat autoimmunity.     

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.