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We previously reported that joint swelling, synovial thickening, and cartilage matrix depletion induced by the injection of anti-collagen monoclonal antibodies and lipopolysaccharide (LPS) in BALB/c mice are increased in the absence of inhibitory leukocyte immunoglobulin (Ig)-like receptor B4 (LILRB4; formerly gp49B1) in a neutrophil-dependent manner. Because both mast cells and neutrophils express LILRB4, we sought a mast cell requirement with mast cell-deficient mouse strains, but unexpectedly obtained full arthritis in Kit(W-sh) mice and full resistance in Kit(W/KitW-v) mice. Kit(W-sh) mice were indeed mast cell deficient as assessed by histology and the absence of IgE/mast cell-dependent passive cutaneous anaphylaxis in the ear and joint as well as passive systemic anaphylaxis. Deletion of LILRB4 in Kit(W-sh) mice exacerbated anti-collagen/LPS-induced joint swelling that was abolished by neutrophil depletion, establishing a counterregulatory role for LILRB4 in the absence of mast cells. Whereas blood neutrophil levels and LPS-elicited tissue neutrophilia were equal in Kit(W-sh) and Kit+ mice, both were impaired in Kit(W/KitW-v) mice. Although both strains are mast cell deficient and protected from IgE-mediated anaphylactic reactions, their dramatically different responses to autoantibody-mediated, neutrophil-dependent immune complex arthritis suggest that other host differences determine the extent of mast cell involvement. Thus, a conclusion for an absolute mast cell role in a pathobiologic process requires evidence from both strains.

Collagens are the most abundant proteins in the human body, important in maintenance of tissue structure and hemostasis. Here we report that collagens are high affinity ligands for the broadly expressed inhibitory leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1). The interaction is dependent on the conserved Gly-Pro-Hyp collagen repeats. Antibody cross-linking of LAIR-1 is known to inhibit immune cell function in vitro. We now show that collagens are functional ligands for LAIR-1 and directly inhibit immune cell activation in vitro. Thus far, all documented ligands for immune inhibitory receptors are membrane molecules, implying a regulatory role in cell-cell interaction. Our data reveal a novel mechanism of peripheral immune regulation by inhibitory immune receptors binding to extracellular matrix collagens.

Pathogenic mechanisms relevant to rheumatoid arthritis occur in the mouse model of collagen-induced arthritis (CIA). Cytosolic phospholipase A2alpha (cPLA2alpha) releases arachidonic acid from cell membranes to initiate the production of prostaglandins and leukotrienes. These inflammatory mediators have been implicated in the development of CIA. To test the hypothesis that cPLA2alpha plays a key role in the development of CIA, we backcrossed cPLA2alpha-deficient mice on the DBA/1LacJ background that is susceptible to CIA. The disease severity scores and the incidence of disease were markedly reduced in cPLA2alpha-deficient mice compared with wild-type littermates. At completion of the study, >90% of the wild-type mice had developed disease whereas none of the cPLA2alpha-deficient mice had more than one digit inflamed. Furthermore, visual disease scores correlated with severity of disease determined histologically. Pannus formation, articular fibrillation, and ankylosis were all dramatically reduced in the cPLA2alpha-deficient mice. Although the disease scores differed significantly between cPLA2alpha mutant and wild-type mice, anti-collagen antibody levels were similar in the wild-type mice and mutant littermates. These data demonstrate the critical role of cPLA2alpha in the pathogenesis of CIA.

A characteristic feature of rheumatoid arthritis is the abundance of inflammatory cells in the diseased joint. Two major components of this infiltrate are neutrophils in the synovial fluid and macrophages in the synovial tissue. These cells produce cytokines including tumor necrosis factor alpha and other proinflammatory mediators that likely drive the disease through its effector phases. To investigate what mechanisms underlie the recruitment of these cells into the synovial fluid and tissue, we performed expression analyses of chemoattractant receptors in a related family that includes the anaphylatoxin receptors and the formyl-MetLeuPhe receptor. We then examined the effect of targeted disruption of two abundantly expressed chemoattractant receptors, the receptors for C3a and C5a, on arthritogenesis in a mouse model of disease. We report that genetic ablation of C5a receptor expression completely protects mice from arthritis.

Removal of apoptotic cells is essential for maintenance of tissue homeostasis, organogenesis, remodeling, development, and maintenance of the immune system, protection against neoplasia, and resolution of inflammation. The mechanisms of this removal involve recognition of the apoptotic cell surface and initiation of phagocytic uptake into a variety of cell types. Here we provide evidence that C1q and mannose binding lectin (MBL), a member of the collectin family of proteins, bind to apoptotic cells and stimulate ingestion of these by ligation on the phagocyte surface of the multifunctional protein, calreticulin (also known as the cC1qR), which in turn is bound to the endocytic receptor protein CD91, also known as the alpha-2-macroglobulin receptor. Use of these proteins provides another example of apoptotic cell clearance mediated by pattern recognition molecules of the innate immune system. Ingestion of the apoptotic cells through calreticulin/CD91 stimulation is further shown to involve the process of macropinocytosis, implicated as a primitive and relatively nonselective uptake mechanism for C1q- and MBL-enhanced engulfment of whole, intact apoptotic cells, as well as cell debris and foreign organisms to which these molecules may bind.

The combination of hemorrhagic pneumonitis and rapidly progressive glomerulonephritis is a characteristic feature of Goodpasture's syndrome (GPS), an autoimmune disease resulting from the interaction of pathogenic anti-collagen type IV (C-IV) antibodies with alveolar and glomerular basement membranes. Lack of a suitable animal model for this fatal disease has hampered both a basic understanding of its etiology and the development of therapeutic strategies. We now report a novel model for GPS using mice deficient in a central regulatory receptor for immunoglobulin (Ig)G antibody expression and function, the type IIB Fc receptor for IgG (FcgammaRIIB). Mutant mice immunized with bovine C-IV reproducibly develop massive pulmonary hemorrhage with neutrophil and macrophage infiltration and crescentic glomerulonephritis. The distinctive linear, ribbon-like deposition of IgG immune complex seen in GPS was observed along the glomerular and tubulointerstitial membranes of diseased animals. These results highlight the role of FcgammaRIIB in maintaining tolerance and suggest that it may play a role in the pathogenesis of human GPS.

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.

Autoimmune diseases, like rheumatoid arthritis, result from a dysregulation of the immune response culminating in hyperactivation of effector cells leading to immune-mediated injury. To maintain an appropriate immune response and prevent the emergence of autoimmune disease, activation signals must be regulated by inhibitory pathways. Biochemical and genetic studies indicate that the type IIB low-affinity receptor for immunoglobulin (Ig)G (FcgammaRIIB) inhibits cellular activation triggered through antibody or immune complexes and may be an important component in preventing the emergence of autoimmunity. To investigate the role of FcgammaRIIB in the development of type II collagen (CII)-induced arthritis (CIA), a model for rheumatoid arthritis in humans, we have examined its contribution in determining the susceptibility to CIA in the nonpermissive H-2(b) haplotype. H-2(b) mice immunized with bovine CII do not develop appreciable disease. In contrast, immunization of the FcgammaRIIB-deficient, H-2(b) mice with bovine CII induced CIA at an incidence of 42.2%. The maximal arthritis index of the FcgammaRIIB-deficient mice developing CIA (6.9 +/- 3.6) was comparable to that of DBA/1 mice (8.6 +/- 1.9), an H-2(q) strain susceptible for CIA induction. IgG1, IgG2a, and IgG2b antibody responses against CII were elevated in the FcgammaRIIB-deficient animals, especially in those mice showing arthritis, but less pronounced than DBA/1 mice. Histological examinations of the arthritic paws from FcgammaRIIB-deficient mice revealed that cartilage was destroyed and bone was focally eroded in association with marked lymphocyte and monocyte/macrophage infiltration, very similar to the pathologic findings observed in DBA/1 mice. These results indicate that a nonpermissive H-2(b) haplotype can be rendered permissive to CIA induction through deletion of FcgammaRIIB, suggesting that FcgammaRIIB plays a critical role in suppressing the induction of CIA.

Rheumatoid arthritis (RA) is an autoimmune disease that is strongly associated with the expression of several HLA-DR haplotypes, including DR1 (DRB1*0101). Although the antigen that initiates RA remains elusive, it has been shown that many patients have autoimmunity directed to type II collagen (CII). To test the hypothesis that HLA-DR1 is capable of mediating an immune response to CII, we have generated transgenic mice expressing chimeric (human/mouse) HLA-DR1. When the DR1 transgenic mice were immunized with human CII (hCII), they developed a severe autoimmune arthritis, evidenced by severe swelling and erythema of the limbs and marked inflammation and erosion of articular joints. The development of the autoimmune arthritis was accompanied by strong DR1-restricted T and B cell responses to hCII. The T cell response was focused on a dominant determinant contained within CII(259-273) from which an eight amino acid core was defined. The B cell response was characterized by high titers of antibody specific for hCII, and a high degree of cross-reactivity with murine type II collagen. These data demonstrate that HLA-DR1 is capable of presenting peptides derived from hCII, and suggest that this DR1 transgenic model will be useful in the development of DR1-specific therapies for RA.

Collagen-induced arthritis in the DBA/1 mouse is an experimental model of human rheumatoid arthritis. To examine the role of leukotrienes in the pathogenesis of this disease, we have developed embryonic stem (ES) cells from this mouse strain. Here, we report that DBA/1 mice made deficient in 5-lipoxygenase-activating protein (FLAP) by gene targeting in ES cells develop and grow normally. Zymosan-stimulated leukotriene production in the peritoneal cavity of these mice is undetectable, whereas they produce substantial amounts of prostaglandins. The inflammatory response to zymosan is reduced in FLAP-deficient mice. The severity of collagen-induced arthritis in the FLAP-deficient mice was substantially reduced when compared with wild-type or heterozygous animals. This was not due to an immunosuppressive effect, because anti-collagen antibody levels were similar in wild-type and FLAP-deficient mice. These data demonstrate that leukotrienes play an essential role in both the acute and chronic inflammatory response in mice.

Genetic studies have indicated that susceptibility to rheumatoid arthritis (RA) maps to the HLA-DR locus of the major histocompatibility complex. Strong linkage disequilibrium between certain HLA-DQ genes and HLA-DR genes associated with RA, however, suggests that HLA-DQ molecules may also play a role in RA susceptibility. To examine the role of HLA-DQ molecules in arthritis, we generated transgenic mice expressing the DQA1*0301 and DQB1*0302 genes from an RA predisposing haplotype (DQ8/DR4Dw4). The transgenes were introduced into mouse class II-deficient H-2Ab0 mice, and their susceptibility to experimental collagen-induced arthritis was evaluated. The HLA-DQ8+,H-2Ab0 mice displayed good expression of the DQ8 molecule, while no surface expression of endogenous murine class II molecules could be detected. The DQ8 molecule also induced the selection of CD4+ T cells expressing a normal repertoire of V beta T cell receptors. Immunization of HLA-DQ8+,H-2Ab0 mice with bovine type II collagen (CII) induced a strong antibody response that was cross-reactive to homologous mouse CII. Also, in vitro proliferative responses against bovine CII, which were blocked in the presence of an antibody specific for HLA-DQ and mouse CD4, were detected. Finally, a severe polyarthritis developed in a majority of HLA-DQ8+,H-2Ab0 mice, which was indistinguishable from the disease observed in arthritis susceptible B10.T(6R) (H-2Aq) controls. In contrast, HLA-DQ8-,H-2Ab0 fullsibs did not generate CII antibody and were completely resistant to arthritis. Therefore, these results strongly suggest that HLA-DQ8 molecules contribute to genetic susceptibility to arthritis and also establish a novel animal model for the study of human arthritis.

Antigen priming of naive CD4 T cells can generate effector CD4 T cells that produce interleukin 4 (T helper [Th]2-like) or interferon-gamma (Th1-like). Using a system in which priming leads to responses dominated by one or the other of these cell types, we show that varying either the antigenic peptide or the major histocompatibility complex class II molecule can determine whether Th1-like or Th2-like responses are obtained. Our results show that peptide/major histocompatibility complex class II complexes that interact strongly with the T cell receptor favor generation of Th1-like cells, while those that bind weakly favor priming of Th2-like T cells. Thus, signals from the T cell receptor can influence the differentiation of CD4 T cells into specific types of effector cells.

Collagen-induced arthritis (CIA) is an animal model of autoimmune inflammatory polyarthritis that has features similar to rheumatoid arthritis (RA). Much like RA, susceptibility to mouse CIA is influenced by the major histocompatibility complex (MHC), H-2, and restricted to the H-2q and H-2r haplotypes. Whereas the role of the H-2A molecule in susceptibility to CIA is well established, little is known about the role of H-2E molecule in the disease. In this study, we analyzed the effect of a transgenic E beta d molecule on CIA susceptibility in a recombinant mouse B10.RQB3, which expresses the CIA susceptible Aq genes and an Eak gene, but does not produce an E molecule since Ebq is nonfunctional. In the presence of an Ebd transgene, a viable E molecule is generated. Whereas B10.RQB3 were susceptible to CIA, B10.RQB3-E beta d+ showed a dramatic reduction in the incidence of arthritis as well as a decrease in the level of anti-mouse and anti-bovine CII antibodies in their serum. No clear cut differences in the expression of T cell receptor (TCR) V beta was observed between E beta d+ and E beta d- transgenic mice. Mechanisms underlying the protective effect of E beta d transgenic molecule on CIA may shed light on how HLA-DR molecules influence human RA.

A critical event in an immune response is the T cell recognition of peptides bound to major histocompatibility complex (MHC) molecules on the surface of an antigen presenting cell (APC). Although the majority of eukaryotic proteins are glycosylated, it has not yet been shown that T cell recognition of such proteins involves recognition of the bound carbohydrates. Type II collagen (CII), the major protein constituent of joint cartilage, is posttranslationally modified by hydroxylation and glycosylation of lysines. In this report we show that posttranslational modifications of the immunodominant peptide CII(256-270) generate a structural determinant that is distinct from the determinant represented by the corresponding synthetic peptide. Elimination of carbohydrates, present on CII, by two different biochemical methods revealed that the carbohydrates, O-linked to the hydroxylysines within the CII(256-270) determinant, were crucial for the reactivity towards the posttranslationally modified peptide. Furthermore, a T cell hybridoma specific for the glycosylated determinant was stimulated by tryptic CII-peptides presented by fixed APCs, thus showing that the carbohydrates are involved in the trimolecular complex T cell receptor/peptide/MHC. Finally, the importance of the bound carbohydrates for the arthritogenicity of CII was investigated by comparing the development of arthritis after immunization with carbohydrate-depleted and glycosylated CII, respectively. Incidence, time of onset, and severity of the disease were significantly affected by the elimination of carbohydrates, whereas no significant difference in anti-CII antibody titers was seen.

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.

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.

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.

Neovascularization is observed in a spectrum of diseases such as solid tumors, diabetic retinopathy, and rheumatoid arthritis. It is also evident in rat collage-induced arthritis (CIA), an animal model with histologic, clinical, and radiographic manifestations resembling rheumatoid arthritis. To evaluate the effects of angioinhibition in CIA, Louvain rats were immunized with type II collagen to induce arthritis and then administered an angiogenesis inhibitor, AGM-1470, in an attempt to either prevent arthritis or suppress established disease. Using clinical and radiographic criteria, AGM-1470 prevented CIA and significantly suppressed established disease without evidence of immunosuppression. Histologic sections from control ankle joints manifested pannus and neovascularization, which were absent in experimental animals. This is the first study to investigate this novel agent in an autoimmune disease, and additional evaluation of this promising compound in other diseases that are potentially angiogenesis dependent, such as rheumatoid arthritis, might be warranted.

Collagen arthritis (CA), an autoimmune model of rheumatoid arthritis (RA), has been studied in various animals. However, it has not been studied in an animal with a genetic background relevant to RA. We selected rats from a diabetic-resistant (DR) subline of the diabetic BB rat because they have an autoimmune disease-prone background, but not the immunodeficiencies of the diabetic BB rat, and the third hypervariable region (HVRIII) of the BB RT1.D beta gene appeared to encode a nucleotide sequence of the human HLA DR beta gene, which has been reported to be associated with susceptibility to RA. We synthesized oligonucleotide primers flanking the RT1.D beta HVRIII, cloned polymerase chain reaction-amplified DNA into M13mp18, and confirmed the presence of the susceptibility sequence (SS) (RRRAA) by the dideoxy sequencing method in a colony of DR BB/Wor-UTM rats. When immunized with human type II collagen (CII) in incomplete Freunds adjuvant (IFA), arthritis developed rapidly by day 10 with 100% incidence. Light and electron microscopy revealed an unusually severe and aggressive, bidirectional pattern of cartilage resorption by synovial and subchondral mononuclear and multinucleated inflammatory cells. These findings coincided with a predominant humoral response to the cyanogen bromide (CB) 11 fragment of the human CII molecule by the pathogenic IgG2a isotype. This study provides further support to the role of CA as a relevant RA model, the specific roles of the CB11 fragment as a major site of arthritogenic epitopes, and of antibody mechanisms in the pathogenesis of CA. Furthermore, the identification of an RA SS in an immune response gene of the DR BB rat presents a novel opportunity to determine with an animal model the role of other antigens as well as this SS in RA.

Purified chick type II collagen was cleaved with cyanogen bromide (CB), and the resulting peptides isolated and renatured. Sera from arthritic DBA/1 mice, immunized with chick type II collagen, were tested for reactivity with each peptide. The sera preferentially recognized peptides 11, 10, and 8, in that order. Some reactivity was also detected to peptides 9, 7, and 12. Because arthritis depends upon binding of antibody to autologous type II collagen in the joint, sera were also tested for reactivity with mouse type II collagen. There was a strong positive correlation between reactivity with peptide 11 and reactivity with mouse collagen, but no correlation was found with any of the other peptides. Peptides 11, 10, and 8 were also used for immunization. Antibodies were detected in response to each of these peptides, but arthritis developed only in mice immunized with peptide 11. We conclude that a major immunogenic and arthritogenic epitope on type II collagen resides in the region of the molecule represented by CB peptide 11.

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.

Studies were performed to determine if cultured human endothelial cells synthesized basement membrane collagen. In culture, endothelial cells were attached to grossly visible membranous structures which on light microscopy were composed of ribbons of dense, amorphous material. On transmission electron microscopy, these membranous structures consisted of amorphous basement membrane, and material morphologically similar to microfibrils and elastic fibers. By immunofluorescence microscopy, these membranous structures stained brightly with antisera to human glomerular basement membrane. Cultured endothelial cells incorporated [3H]proline into protein; 18% of the incorporated [3H]proline was solubilized by purified collagenase. When endothelial cells were cultured with [14C]proline, 7.1% of the incorporated counts were present as [14C]hydroxyproline. Cultured endothelial cells were labeled with [3H]glycine and [3H]proline and digested with pepsin. The resulting fractions on analysis by SDS-polyacrylamide gel electrophoresis contained two radioactive protein peaks of mol wt 94,200 and 120,500. Both these peaks disappeared after digestion with purified collagenase. The peak of mol wt 120,500 corresponds to that of alpha1 (IV) collagen; the peak of the mol wt 94,200 probably corresponds to that of alpha1 (III) collagen. Thus, cultured human endothelial cells synthesize material which is morphologically and immunologically like amorphous basement membrane and biochemically like basement membrane collagen. Cultured endothelial cells probably also synthesize material which is morphologically similar to microfibrils and elastic fibers.

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.

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.

Immunization of two cynomolugus and three rhesus monkeys with purified type II collagen resulted in the development of polyarthritis. Arthritis first became clinically apparent 7 wk after primary immunization and persisted for 16 mo. Radiologic examination of the limbs demonstrated soft tissue swelling with severe joint destruction including loss of cartilage and bone. Involved joints eventually became ankylosed with permanent loss of some motion. All of the monkeys developed a response to the immunizing collagen as determined by ELISA of serum for antibodies. Arthritis was associated with weight loss and constitutional symptoms, including lethargy and refusal to eat. One monkey became so debilitated that it was necessary to euthanize it. Histologic examination of the joints showed synovial hypertrophy with pannus formation. A control monkey immunized with type I collagen suffered no apparent ill effects.

University of California, Davis (UCD) line 200 White Leghorn Chickens spontaneously develop a syndrome that has many analogous features to human progressive systemic sclerosis. This syndrome is characterized by progressive involution of comb, dermal fibrosis, and distal polyarthritis. These three features occur within 6 wk after hatching, and are accompanied by a 40% mortality as a result of vaso-occlusive disease, with development of secondary infection of peripheral gangrenous lesions. Birds that survive greater than 2 mo after hatching progressively develop fibrosis of the esophagous and mononuclear infiltration of heart and kidney, with prominent occlusion of small and medium sized blood vessels. In addition, line 200 chickens develop rheumatoid factors, antinuclear antibodies, and antibodies to collagen, but do not have antibodies to thymocytes, DNA, or extractable nuclear antigens. Moreover, antinuclear antibodies when studied using HEp-2 cells as substrate demonstrate predominantly a speckled pattern. This syndrome of line 200 chickens is not detectable in F1 crosses to several UCD inbred lines. F1 X parental line BC1 backcrosses have an approximately 50% incidence of disease, suggesting that this syndrome is inherited as autosomal recessive. However, only 4% of F2 generation birds show abnormal symptoms, suggesting the presence of modifying genes. There is no appearance of IgG deposition, as determined by immunofluorescence, in either skin, blood vessels, esophagus, or heart. However, approximately 20% of chickens have a glomerulonephritis; this feature appears to be a terminal event and does not appear clinically significant. Although this syndrome of line 200 chickens has several features that are in sharp distinction to human scleroderma, the presence of common immunologic and pathologic denominators suggest that this spontaneous disease may be an appropriate model to develop a better understanding of autoimmune connective tissue diseases.

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.

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.

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

Analysis of the IgG autoantibody subclass response in the collagen II autoimmune arthritis (CII AIA)-susceptible D1 strain mice revealed that the onset of disease was associated with a predominance of IgG2a autoantibody. In a comparative study, resistance in the B6 strain was associated with a deficient IgG2a autoantibody response. B6 IgG1, 2b, and 3 autoantibody responses generally overlapped those of arthritic D1 mice, and estimates of antibody crossreactivity and affinity were similar for both strains. In crosses between D1 and B6, arthritis developed only in those F1 mice with IgG2a autoantibody responses approximating or exceeding those in arthritic D1 mice. Additional studies with B6 and B10 strains suggested an alternate role for the IgG2b autoantibody response. In inbred strains with known genetic backgrounds, a dissociation between the magnitude of the total IgG autoantibody response and the percent of total as IgG2a was demonstrated. The H-2q, Ig-1c D1 strain was a high-total and high-percent IgG2a responder, while the H-2d, Ig-1c D2 strain was a low-total but high-percent IgG2a responder. The H-2b, Ig-1b B6 strain was a low-total and low-percent IgG2a responder, while the H-2b/q, Ig-1b/c (B6D1)F1 hybrid was a low-total but high-percent IgG2a responder. A further dissociation between high-percent IgG2a autoantibody responsiveness and the H-2 haplotype was demonstrated by the H-2 congenic B10.D2/n (H-2d, Ig-1b) strain, in which a low-percent IgG2a response was observed to differ from the D2 strain. High-percent IgG2a autoantibody responsiveness also appeared to be inherited as a dominant trait based upon high responses in all (B6D1)F1 hybrids and backcrosses to D1. These findings suggest that the H-2 haplotype is involved in the total IgG autoantibody response but that the relative fraction of the total response as IgG2a is independent of the H-2 haplotype and possibly related to Igh-C genes. C5-deficient SWR (H-2q, Ig-1c) mice were found to have a high total autoantibody response to mouse CII and IgG2a comparable to arthritic D1 mice, but these mice did not develop arthritis. Based upon these observations, we conclude that susceptibility to CII AIA requires the interaction of multiple genes, both major histocompatibility complex (MHC) and non-MHC, which influence the magnitude (total IgG) and the quality (IgG subclass) of the autoimmune response and the availability of appropriate mediators (C5) to initiate the inflammatory reaction.

In the present study, we demonstrate delayed-type hypersensitivity (DTH) to homologous type I collagen that cross-reacts with type IV collagen. Mice immunized with native or denatured type I collagens and challenged with these same antigens or native type IV collagen develop a peak DTH response on day 7. Challenge with denatured type IV collagen or collagenase-treated type IV collagen failed to elicit DTH in type I collagen-sensitized mice. Type I collagen-sensitized spleen cells adoptively transferred DTH to types IV and I collagen to normal recipients; T cell-depleted spleen cells failed to transfer immunity. Periodate-treated type IV collagen did not elicit DTH in mice sensitized to type I collagen; however, mice sensitized with type IV collagen displayed significant DTH when challenged with periodate-treated type IV collagen. Furthermore, treatment of type IV collagen with a mixed glycosidase or alpha-glucosidase before challenge eliminated the DTH response in type I collagen-sensitized mice; beta-galactosidase treatment of type IV collagen had no effect on this response. Mice sensitized with type IV collagen, however, displayed significant DTH when challenged with these glycosidase-treated antigens. Antibodies produced to types I and IV collagen by repeated immunizations were specific for the sensitizing antigen and did not react with other connective tissue antigens. These studies indicate that a CMI response to type I collagen recognizes similar antigenic determinants on the type IV collagen molecule. These cross-reacting determinants are dependent on conformation and contain carbohydrates, particularly glucose residues.

The level of antibody produced in response to calf skin collagen in mice is influenced by genes which are closely linked to the I region of the H-2 major histocompatibility complex. This influence is shown to be expressed during lymphoid maturation by testing the antibody responsiveness to collagen in two types of chimeric mice. First, high responder and low responder parental strain mice were lethally irradiated and restored with fetal liver cells from (high X low responder) F1 mice. These F1 leads to parent chimeras exhibited an immune response phenotype characteristic of the irradiated parental strain animals, establishing that H-2 determinants of the host affect antigen responsiveness. Second, (high X low responder) F1 congenitally athymic (nude) mice were restored with fetal thymus transplants from either high or low responder parental strain mice. After a period of maturation these mice were shown to be competent for a T-dependent IgG response to SRBC. The responsiveness to collagen in these mice was characteristic of the parental strain thymus donors, indicating that the expression of H-2 determinants in thymic tissue during lymphoid maturation influences the antibody response phenotype expressed by mice.

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.

The ability of collagen and collagen-derived peptides to act as chemotactic stimuli was investigated by in vitro chemotaxis assays. Native human and chick skin collagen (type I) and alpha-chains obtained from purified chick skin collagen were each chemotactic for human peripheral blood monocytes. In addition, smaller peptides obtained either by digesting native collagen with bacterial collagenase or by degrading purified alpha-chains with cyanogen bromide or pepsin were also chemotactic for monocytes. In contrast, native collagen, alpha-chains, and smaller collagen-derived peptides were not chemotactic for human neutrophils. Since collagen is degraded at sites of tissue damage and inflammation, our findings suggest the possibility that such collagen-derived degradation products might directly serve as chemotactic stimuli for human peripheral blood monocytes in vivo.

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.

Sera from patients with poststreptococcal glomerulonephritis (PSGN) known to have antibodies to proteoglycans were studied for the presence of antibodies against other basement membrane (BM) components. BM collagen (type IV) was isolated in the native state by extracting bovine anterior lens capsule (ALC) with 0.5 M acetic acid. The 7-S (collagenous) domain and the NC-1 (noncollagenous) domain of type IV collagen were obtained after bacterial collagenase digestion of ALC followed by gel filtration. Laminin was isolated from the mouse EHS tumor and fibronectin from human plasma. Immunologic studies, using an ELISA and electroimmunoblot, revealed the presence of antibodies that reacted with intact, native type IV collagen and the 7-S collagenous domain of this molecule. Reaction with the NC-1 (noncollagenous) domain was minimal, and not higher than that obtained with control sera. Laminin reaction strongly with the patients' sera, but fibronectin did not. Unlike sera from patients with Goodpasture syndrome, which contain antibodies primarily against the NC-1 (noncollagenous) domain of type IV collagen, sera from patients with acute PSGN contain antibodies against all the major macromolecular components of BM. This difference in immunologic reactivity may account for the observed differences in the pathologic picture at the glomerular level.

A model of arthritis was established by the injection of type II collagen into mice. Only mice bearing the H-2q haplotype were susceptible to the disease. Susceptibility was further mapped by the use of recombinant strains on the Iq locus. Type II collagen arthritis was observed in the (resistant X susceptible) F1 cross. Mice strains were designated high, intermediate, or low responders with respect to the anti-type II antibody levels measured by radioimmunoassay. Arthritis-susceptible strains were all classified as high antibody responders. The clinical and histological appearance of type II collagen arthritis in the mouse indicates that it may be a good animal model for the investigation of various immunogenetic traits in rheumatoid arthritis.

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.

Collagen, the major extracellular matrix protein, is also a membrane protein. Two types of collagen are detected on the normal human fibroblast membrane in culture, type I collagen and a new immunologically and chemically distinct collagen, type M (membrane) collagen. Antibodies to type M collagen elicited complement-mediated cytotoxicity, which could be blocked by pretreatment of the cells with bacterial collagenase or the antibody with type M collagen. Pretreatment of the cells with other proteolytic enzymes or the antibody with type I collagen or type III collagen had no effect on this complement-mediated cytotoxicity. Although type I collagen is the major collagen synthesized by normal human fibroblasts type M collagen may be the major cell membrane collagen and may be a major cell membrane component.