Edward F. Rosloniec

Collagen-induced arthritis

The collagen-induced arthritis (CIA) mouse model is the most commonly studied autoimmune model of rheumatoid arthritis. Autoimmune arthritis is induced in this model by immunization with an emulsion of complete Freunds adjuvant and type II collagen (CII). This protocol describes the steps necessary for acquisition, handling and preparation of CII, as well as selection of mouse strains, proper immunization technique and evaluation of the arthritis incidence and severity. Typically, the first signs of arthritis appear in this model 21–28 days after immunization, and identification of the arthritic limbs is not difficult. Using the protocol described, the investigator should be able to reproducibly induce a high incidence of CIA in various strains of genetically susceptible mice as well as learn how to critically evaluate the pathology of the disease. The total time for the preparation of reagents and the immunization of ten mice is about 1.5 h.

Impaired Invariant Chain Degradation and Antigen Presentation and Diminished Collagen-Induced Arthritis in Cathepsin S Null Mice

Cathepsins have been implicated in the degradation of proteins destined for the MHC class II processing pathway and in the proteolytic removal of invariant chain (Ii), a critical regulator of MHC class II function. Mice lacking the lysosomal cysteine proteinase cathepsin S (catS) demonstrated a profound inhibition of Ii degradation in professional APC in vivo. A marked variation in the generation of MHC class II-bound Ii fragments and presentation of exogenous proteins was observed between B cells, dendritic cells, and macrophages lacking catS. CatS-deficient mice showed diminished susceptibility to collagen-induced arthritis, suggesting a potential therapeutic target for regulation of immune responsiveness.

Collagen-induced arthritis, an animal model of autoimmunity

Collagen induced arthritis (CIA) is an autoimmune model that in many ways resembles rheumatoid arthritis (RA). Immunization of genetically susceptible strains of rodents and primates with type II collagen (CII) leads to the development of a severe polyarticular arthritis that is mediated by an autoimmune response. Like RA, synovitis and erosions of cartilage and bone are hallmarks of CIA, and susceptibility to both RA and CIA is linked to the expression of specific MHC class II molecules. Although not identical to RA, CIA clearly establishes the biological plausibility that an autoimmune reaction to a cartilage component can lead to a chronic, destructive, polyarthritis. Although it is induced in susceptible animals by immunization with heterologous CII, it is the autoreactive component of the immune response that leads to disease. A wealth of evidence indicates that synovitis is initiated by the production of pathogenic autoreactive antibodies capable of fixing and activating complement. The elucidation of the specific amino acid sequences of collagen that are recognized by the MHC molecules has enabled at least two approaches to specific immunotherapy to be considered. Firstly, small synthetic peptides representing dominant epitopes have been used as effectively as the original antigen as a tolerogen. The rather fastidious physicochemical properties of collagen that make it difficult for its routine use in therapy are thereby circumvented by the use of oligopeptides. Secondly, analysis of the specific amino acid side chains that are involved in MHC contact and TCR recognition enables analog peptides to be devised which can specifically and exquisitely inhibit the response to CII, preventing the onset of arthritis. Further investigations involving this model may contribute to the development of specific immunotherapies in the human disorder.

Immunopathogenesis of collagen arthritis.

Abstract. Collagen-induced arthritis (CIA) is an animal model of autoimmunity that has been studied extensively because of its similarities to rheumatoid arthritis (RA). CIA is induced in genetically susceptible strains of mice by immunization with type II collagen (CII), and both T cell and B cell immunity to CII are required for disease manifestation. Like RA, CIA is primarily an autoimmune disease of articular joints and susceptibility to CIA is linked to specific class II molecules of the major histocompatibility complex (H-2r and H-2q). Recently, it was demonstrated that transgenic expression of HLA-DR1 (*0101) or DR4 (*0401) molecules associated with susceptibility to RA also conferred susceptibility to CIA in the mouse model. The T cell response to CII has been extensively characterized in both the DR transgenic and naturally susceptible mouse strains, including the antigenic determinants recognized, the role of post transcriptional modifications of these determinants in the pathogenic T cell response, and the cytokines produced. Like most class II-mediated autoimmune diseases, the cytokine production of CII-specific T cells reflects a Th1 phenotype of the autoimmune response. While the direct role of T cells in the pathogenesis of CIA is unclear, the B cell response in terms of anti-CII immunoglobulin is critical to the development of the disease. This response, predominated by the IgG2 isotype, requires the activation of the complement cascade for the development of CIA. In recent years, the pathogenesis of this model has been studied extensively and the CIA model is proving to be a valuable asset for the design of new immunotherapeutics for the potential treatment of RA and other autoimmune diseases.

Ceramide signalling and the immune response

Ceramide, produced through either the induction of SM hydrolysis or synthesized de novo transduces signals mediating differentiation, growth, growth arrest, apoptosis, cytokine biosynthesis and secretion, and a variety of other cellular functions. A generalized ceramide signal transduction scheme is shown in Fig. 2 in which ceramide is generated through the activation of distinct SMases residing in separate subcellular compartments in response to specific stimuli. Clearly, specificity of cellular responses to ceramide depends upon many factors which include the nature of the stimulus, co-stimulatory signals and the cell type involved. Ceramide derived from neutral SMase activation is thought to be involved in modulating CAPK and MAP kinases, PLA2 (arachidonic acid mobilization), and CAPP while ceramide generated through acid SMase activation appears to be primarily involved in NF-kappa B activation. While there is no apparent cross-talk between these two ceramide-mediated signalling pathways, there is likely to be significant cross-talk between ceramide signalling and other signal transduction pathways (e.g., the PKC and MAP kinase pathways). Other downstream targets for ceramide action include Cox, IL-6 and IL-2 gene expression, PKC zeta, Vav, Rb, c-Myc, c-Fos, c-Jun and other transcriptional regulators. Many, if not all, of these ceramide-mediated signalling events have been identified in the various cells comprising the immune system and are integral to the optimal functioning of the immune system. Although the role of the SM pathway and the generation of ceramide in T and B lymphocytes have only recently been recognized, it is clear from these studies that signal transduction through SM and ceramide can strongly affect the immune response, either directly through cell signalling events, or indirectly through cytokines produced by other cells as the result of signalling through the SM pathway. An overview of the signalling mechanisms coupling ceramide to the modulation of the immune response is depicted in Fig. 3 and shows how ceramide may play pivotal roles in regulating a number of complex processes. The SM pathway represents a potentially valuable focal point for therapeutic control of immune responses, perhaps for either enhancement of the activity of T cells in the elimination of tumors, or the down-regulation of lymphocyte function in instances of autoimmune disease. The recent explosion of knowledge regarding ceramide signalling notwithstanding, a number of critical questions need to be answered before a comprehensive, mechanistic understanding can be formulated relative to the incredibly varied effects of ceramide on cell function. For example, (i) how is a structurally simple molecule like ceramide able to mediate so many different, and sometimes paradoxical, physiological responses ranging from cell proliferation and differentiation to inhibition of cell growth and apoptosis, (ii) what are the molecular identities and modes of activation of the various SMase isoforms, (iii) what determines the distribution of the unique isoforms of SMase in cells of different lineages or at different stages of differentiation, (iv) what is the relative contribution of ceramide generated through SM hydrolysis versus de novo synthesis, and (v) by what means does ceramide interact with specific intracellular targets? Although a number of ceramide-activatable kinases, phosphatases, and their protein substrates have been identified, a more extensive search for additional cellular targets will be indispensable in determining the phosphorylation cascades linking the activation of the SM pathway to the regulation of nuclear events. Clearly, cross-talk between ceramide-induced signal transduction cascades and other signalling pathways adds to the inherent difficulty in distinguishing the specific effects of complex, intertwining signalling pathways.

The cartilage collagens: a review of their structure, organization, and role in the pathogenesis of experimental arthritis in animals and in human rheumatic disease

Abstract This contribution reviews the structure and organization of collagen molecules found in cartilage and the roles that they may play in rheumatic diseases. Cartilage is unique in its physical properties and molecular composition, and contains sufficient amounts of types II, IX, X, and XI collagen to deem these molecules as ”cartilage-specific.” The vitreous body of the eye, a ”cartilagelike” tissue is also rich in the same collagens but is type X deficient. Types VI and XII collagen are present in cartilage as well as noncartilaginous tissues. Types II, IX, and XI collagen are organized into matrix fibrils, where type II constitutes the bulk of the fibril, type XI regulates fibril size, and type IX facilitates fibril interaction with proteoglycan macromolecules. Genetic defects in these collagens can produce mild to severe developmental abnormalities, including spondyloepiphyseal dysplasia often accompanied by an accelerated form of osteoarthritis. Sensitization with collagen can produce experimental rheumatic diseases. Type II collagen induces an erosive polyarthritis in certain strains of rats, mice, and higher primates which can resemble rheumatoid arthritis and relapsing polychondritis. Type XI collagen is arthritogenic in rats but not mice; type IX induces autoimmunity in both species but not arthritis. Arthritis is initiated by complement fixing antibodies that bind to type II collagen in autologous cartilage, and the production of these antibodies is MHC restricted and T cell dependent. It is unclear whether T cells alone can induce arthritis, although they probably help sustain it. Mapping and characterizing the of T cell epitopes on type II collagen has resulted in the synthesis of small homolog and substituted peptides of type II collagen which suppress arthritis in an antigen-specific manner by a variety of routes, including mucosal. Moreover, collagen-induced arthritis has proven a valuable model to study the contribution of cytokines and other biological agents in the pathogenesis of joint injury and how they might be used to develop new therapies. Collagen autoimmunity has been implicated in the pathogenesis rheumatoid arthritis and polychondritis. Circulating antibodies to type II collagen are found in both diseases. Antibodies to types IX and XI collagen are also present in rheumatoid sera but are less prevalent. Rheumatoid cartilage and synovium contain antibodies to type II collagen at a prevalence far greater than serum, suggesting an intra-articular antigen-driven immune process. Although effective in animal models, attempts to treat rheumatoid arthritis with orally administered type II collagen have proven elusive. Different approaches using newer formulations and selected or modified oligopeptides remain to be tested and could prove effective in the treatment of the human rheumatic diseases.

Genetic ablation of interferon-gamma up-regulates interleukin-1beta expression and enables the elicitation of collagen-induced arthritis in a nonsusceptible mouse strain.

OBJECTIVE To determine whether the lack of interferon-gamma (IFNgamma) alters resistance to collagen-induced arthritis (CIA) in a nonsusceptible mouse strain, and if so, to identify changes in the antibody, cellular type II collagen (CII)-specific immune responses, and cytokine gene expression that might account for the altered susceptibility. METHODS CIA-resistant C57BL/6 and C57BL/6 IFNgamma-/- mice were immunized with bovine CII in Freunds complete adjuvant (CFA) or in CFA alone. Animals were monitored for signs of arthritis for up to 80 days; arthritis severity was assessed visually and histologically. Sera were collected at various time points after immunization for measurement of anti-CII antibody levels. T cell responses to bovine CII were assessed in proliferation assays. Cytokine messenger RNA (mRNA) expression in lymph node cells and in synovial cells from arthritic paws was measured by RNase protection assays, and levels of cytokine protein production were determined by enzyme-linked immunosorbent assay. RESULTS IFNgamma-/- mice developed a severe autoimmune arthritis that was dependent on immunization with CII. IFNgamma-/- mice produced significantly higher amounts of IgG1 and IgG2b antibody to the autoantigen, murine CII, compared with wild-type C57BL/6 mice and had an enhanced T cell proliferative response to bovine CII. Enhanced production of mature interleukin-1/beta (IL-1beta) protein was observed, but no significant changes in Th1 or Th2 cytokines. Although IL-6 and tumor necrosis factor alpha transcripts were clearly evident in the synovial cells from the arthritic paws of IFNgamma-/- mice, neither message was elevated to the levels measured for IL-1beta expression. Treatment of IFNgamma-/- mice with anti-IL-1beta significantly reduced the incidence and severity of the inflammation. CONCLUSION Endogenous IFNgamma plays a role in the regulation of IL-1beta, in this model of autoimmune arthritis.

Estradiol treatment redirects the isotype of the autoantibody response and prevents the development of autoimmune arthritis

A number of clinical and experimental observations have been made relating elevated estrogen levels with the amelioration of autoimmune diseases, yet questions remain about the levels required for efficacy as well as the mechanism of disease inhibition. Using the collagen-induced arthritis (CIA) model, we have studied the effects of physiological, sustained levels of 17β-estradiol in preventing the development of autoimmune arthritis and analyzed the changes in the autoimmune response. Using time-release pellets of 17β-estradiol, arthritis development was significantly inhibited in three different strains of CIA-susceptible mice compared with the effect of placebo treatment, and serum estradiol levels similar to those of mice in estrus were found to be equally effective as higher estradiol concentrations. Analysis of the autoimmune response in the estradiol-treated mice indicated that T cell production of IFN-γ was markedly decreased, and significant decreases were also observed in levels of IL-10 and GM-CSF produced by lymph nodes cells from estradiol-treated mice. Although the total IgG anti-CII response was only minimally affected by estrogen treatment, a significant reduction in the levels of IgG2a anti-CII Abs and an increase in the levels of IgG1 anti-CII Abs were observed in estradiol-treated mice. These data indicate that estradiol treatment altered the Th profile of the autoimmune T cell response, which, in turn, altered the production of IgG Abs to an isotype that is poor at fixing complement, an important component in the immunopathogenesis of CIA.

Collagen-induced arthritis.

The mouse model collagen‐induced arthritis (CIA) is a widely studied autoimmune model of rheumatoid arthritis. In this model, autoimmune arthritis is induced by immunization with type II collagen (CII) emulsified in complete Freunds adjuvant. This unit describes the steps necessary for the acquisition, handling, and preparation of CII, in addition to the selection of mouse strains, proper immunization technique, and methods for evaluation of the incidence and severity of arthritis. In this model, the first signs of arthritis appear approximately 21 to 28 days after immunization. The protocols in this unit should provide the investigator with all the necessary information required to reproducibly induce a high incidence of CIA in genetically susceptible strains of mice, and to critically evaluate the pathology of the disease. Curr. Protoc. Immunol. 89:15.5.1‐15.5.25.

HLA-DR1 (DRB1*0101) and DR4 (DRB1*0401) Use the Same Anchor Residues for Binding an Immunodominant Peptide Derived from Human Type II Collagen

Rheumatoid arthritis is an autoimmune disease in which susceptibility is strongly associated with the expression of specific HLA-DR haplotypes, including DR1 (DRB1*0101) and DR4 (DRB1*0401). As transgenes, both of these class II molecules mediate susceptibility to an autoimmune arthritis induced by immunization with human type II collagen (hCII). The dominant T cell response of both the DR1 and DR4 transgenic mice to hCII is focused on the same determinant core, CII(263–270). Peptide binding studies revealed that the affinity of DR1 and DR4 for CII(263–270) was at least 10 times less than that of the model Ag HA(307–319), and that the affinity of DR4 for the CII peptide is 3-fold less than that of DR1. As predicted based on the crystal structures, the majority of the CII-peptide binding affinity for DR1 and DR4 is controlled by the Phe263; however, unexpectedly the adjacent Lys264 also contributed significantly to the binding affinity of the peptide. Only these two CII amino acids were found to provide binding anchors. Amino acid substitutions at the remaining positions had either no effect or significantly increased the affinity of the hCII peptide. Affinity-enhancing substitutions frequently involved replacement of a negative charge, or Gly or Pro, hallmark amino acids of CII structure. These data indicate that DR1 and DR4 bind this CII peptide in a nearly identical manner and that the primary structure of CII may dictate a different binding motif for DR1 and DR4 than has been described for other peptides that bind to these alleles.