John M. Stuart

Autoimmunity due to molecular mimicry as a cause of neurological disease

One hypothesis that couples infection with autoimmune disease is molecular mimicry. Molecular mimicry is characterized by an immune response to an environmental agent that cross-reacts with a host antigen, resulting in disease. This hypothesis has been implicated in the pathogenesis of diabetes, lupus and multiple sclerosis (MS). There is limited direct evidence linking causative agents with pathogenic immune reactions in these diseases. Our study establishes a clear link between viral infection, autoimmunity and neurological disease in humans. As a model for molecular mimicry, we studied patients with human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a disease that can be indistinguishable from MS (refs. 5,6,7). HAM/TSP patients develop antibodies to neurons. We hypothesized these antibodies would identify a central nervous system (CNS) autoantigen. Immunoglobulin G isolated from HAM/TSP patients identified heterogeneous nuclear ribonuclear protein-A1 (hnRNP-A1) as the autoantigen. Antibodies to hnRNP-A1 cross-reacted with HTLV-1-tax, the immune response to which is associated with HAM/TSP (refs. 5,9). Immunoglobulin G specifically stained human Betz cells, whose axons are preferentially damaged. Infusion of autoantibodies in brain sections inhibited neuronal firing, indicative of their pathogenic nature. These data demonstrate the importance of molecular mimicry between an infecting agent and hnRNP-A1 in autoimmune disease of the CNS.

Nature and specificity of the immune response to collagen in type II collagen-induced arthritis in mice.

To determine the role of collagen-immunity in the development of collagen-induced arthritis, DBA/1 mice were immunized with type II collagen and observed for the development of polyarthritis. 96% of the mice immunized with native type II collagen developed inflammatory arthritis between 4 and 5 wk after primary immunization. Immunization with denatured type II collagen in exactly the same manner was not effective in inducing arthritis. Cell-mediated immunity in arthritic mice was assessed by measuring [3H]thymidine incorporation by mononuclear cells cultured in the presence of collagen. The maximal proliferative response to collagen occurred at 2 wk after immunization. Equally good incorporation of label occurred when cells were cultured with native or denatured type II collagen or type I collagen. The cellular response of nonarthritic mice immunized with denatured collagen was indistinguishable from that seen in arthritic mice. Humoral immunity was assessed by an ELISA assay for antibodies to collagen. The immunoglobulin M (IgM) response peaked at 2 wk and the IgG response at 5 wk after immunization. Antisera from arthritic mice immunized with native type II collagen were relatively specific for conformational determinants on the native type II molecule although some reactivity with denatured collagen was noted. Antisera from nonarthritic mice immunized with denatured collagen primarily recognized covalent structural determinants. It was concluded that native type II collagen was essential for the induction of arthritis and that an antibody response specific for native type II collagen may be important for the development of arthritis.

Type II collagen-induced autoimmune sensorineural hearing loss and vestibular dysfunction in rats.

Autoimmune sensorineural hearing loss was induced in rats by immunizing them with native bovine type II collagen. Type I and denatured type II collagen, administered by an identical immunization procedure, would not induce disease. Evidence of sensorineural hearing loss was obtained by measuring the brainstem evoked potential and by observing histopathologic changes consisting of cochlear nerve degeneration and perineural vasculitis in affected animals. Immunized animals had high levels of antibodies to native type II collagen.

Cell-mediated immunity to collagen and collagen α chains in rheumatoid arthritis and other rheumatic diseases

Peripheral blood mononuclear cells from patients with rheumatoid arthritis, gout, ankylosing spondylitis and degenerative joint disease were cultured in the presence of native types I, II and III collagens and alpha chains from each of these types of collagen. The culture supernatant fluids were harvested and assayed for lymphocyte-derived chemotatic factor for monocytes. Reactions to one or more of the native collagens was found in 50 per cent (10 of 20) of the patients with rheumatoid arthritis, 20 per cent (two of 10) of the patients with gout and ankylosing spondylitis but in none of the 10 patients with degenerative joint disease or in normal subjects. Reaction to one or more alpha chains was found in 90 per cent (18 of 20) of the patients with rheumatoid arthritis, 60 per cent (six of 10) of the patients with gout, 50 per cent (five of 10) of the patients with ankylosing spondylitis, 30 per cent (three of 10) of the patients with degenerative joint disease and in 10 per cent of the normal subjects (one of 10). All the reactions were quantiatively stronger in patients with rheumatoid arthritis. These results indicate that patients with rheumatoid arthritis have cell-mediated immunity to homologous native and denatured collagens but that the reaction is not specific for rheumatoid arthritis. Some patients with gout, ankylosing spondylitis and degenerative joint disease also have low levels of immunity.

Type II Collagen-Induced Autoimmune Otospongiosis A Preliminary Report

Otospongiosis-like lesions were induced in rats by immunizing them with native type II collagen. Immunized rats had antibody responses specific for native type II collagen and developed otospongiosis-like lesions. The spongiotic lesion was characterized by numerous osteocytes and osteoblasts in the vascular spaces and by dark staining probably due to the chemical alteration of ground substances. Bone resorption and new bone formation were clearly visible. Immunofluorescence studies demonstrated deposition of immunoglobulin and complement on the bone matrix and wall within the area of spongiosis. An antibody-mediated etiopathogenesis was suspected.

Collagen autoimmunity and arthritis.

Collagen‐induced arthritis in animals is an example of polyarthritis that sufficiently resembles human rheumatoid arthritis to be used as a model. It is caused by immunizing susceptible animals with type II collagen isolated from articular cartilage. Susceptibility is genetically determined and linked to the major histocompatibility locus. It is important because some human arthritis is also associated with major histocompatibility genes and may be caused or aggravated by the presence of autoimmunity to normal cartilage components. Collagen‐induced arthritis is also important because it is an example of immunologically mediated joint destruction, which may share some of the mechanisms present in human disease. Although it is caused by autoimmunity to collagen, susceptibility and responsiveness to type II collagen are not completely correlated, and there are examples of animals with high levels of collagen immunity who do not develop arthritis. The initial lesion appears to be the deposition of an antibody on the surface of articular cartilage, which precedes development of overt arthritis by several days. Disease can be readily transferred with specific antibody. Arthritogenic antibodies appear to have restricted epitope specificity, which may partially explain the disparities between responsiveness to immunization with collagen and susceptibility to arthritis, but precise delineation of the epitopes involved has not yet been accomplished. Complement activation also appears to be intimately involved since the disease correlates with the presence of high levels of complement‐binding IgG isotypes, and passive transfer is possible only into complement‐sufficient recipients. Inflammation progresses rapidly so that cartilage destruction and marginal erosion develop over a period of a few days. Collagen‐induced arthritis offers a unique opportunity to study autoimmune‐mediated arthritis in which the inducing antigen is well characterized and readily available. Analysis of the disease has permitted the proposal of a schema for its pathogenesis.— Stuart, J. M.; Watson, W. C.; Kang, A. H. Collagen autoimmunity and arthritis. FASEB J. 2: 2950‐2956; 1988.

Cross-Reactivity between Immunodominant Human T Lymphotropic Virus Type I tax and Neurons: Implications for Molecular Mimicry

Human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is associated with immunoreactivity to HTLV-I tax. Antibodies isolated from patients with HAM/TSP and monoclonal antibodies (MAbs) to HTLV-I tax stained neurons. In neuronal extracts, HAM/TSP immunoglobulin G identified heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) as the autoantigen. Importantly, tax MAbs reacted with hnRNP A1. To identify the epitope recognized by the tax MAbs, the fine epitope specificity of the antibodies was determined using overlapping peptides. This analysis identified an epitope at the C-terminus (tax(346-353)), which overlaps a human immunodominant domain. Preincubation of this peptide with tax MAbs inhibited antibody binding to tax, hnRNP A1, and neurons. This indicates that a cross-reactive immune response between HTLV-I tax and neuronal hnRNP A1 is contained within the human immunodominant epitope of tax and suggests that molecular mimicry plays a role in the pathogenesis of HAM/TSP.

Detection of early changes in autoimmune T cell phenotype and function following intravenous administration of type II collagen in a TCR-transgenic model.

To study the phenotypic and functional changes in naive type II collagen (CII)-specific autoimmune T cells following a tolerogenic signal, a TCR-transgenic (Tg) mouse model of collagen-induced arthritis was developed. These Tg mice express an I-Aq-restricted CII (260–267)-specific TCR that confers severe accelerated autoimmune arthritis following immunization with CII. Despite the fact that >90% of the αβ T cells express the Tg, these mice can be rendered completely tolerant to the induction of arthritis by i.v. administration of 200 μg of CII. As early as 24 h after CII administration, CII-specific T cells demonstrated a decreased ability to proliferate in response to the CII immunodominant peptide and phenotypically altered the expression of L-selectin to CD62Llow and of phagocytic glycoprotein-1 to CD44high, expression levels consistent with the phenotype of memory T cells. In addition, they up-regulated the expression of the activation markers CD71 and CD69. Functionally, following tolerogenic stimulation, the CII-specific T cells produced similar levels of IL-2 in comparison to controls when challenged with CII peptide, however, by 48 h after exposure to tolerogen, IL-2 production dropped and was replaced by high levels of IL-10 and IL-4. Based on their production of Th2 cytokines, these data suggest that T regulatory cells expressing activation and memory markers are induced by the tolerogen and may exert their influence via cytokines to protect the animals from the induction of arthritis.

Relevance of Posttranslational Modifications for the Arthritogenicity of Type II Collagen

To establish the role of posttranslational modification in modulating the immune response to collagen, recombinant human type II collagen (rCII) was produced using a yeast expression system (rCIIpic) and a baculovirus expression system (rCIIbac). The biosynthesis of CII requires extensive posttranslational modification including the hydroxylation of prolyl and lysyl residues and glycosylation of selected hydroxylysyl residues. Amino acid analyses indicated that the rCIIbac was adequately hydroxylated at prolyl residues but underhydroxylated at lysyl residues and underglycosylated compared with tissue-derived CII, whereas rCIIpic was adequately hydroxylated at prolyl residues but unhydroxylated at lysyl residues and had no glycosylation. When DBA/1 mice were immunized with rCII, rCIIpic induced a lower incidence of arthritis than tissue-derived CII, whereas rCIIbac induced an intermediate level of arthritis. The severity of the arthritis was significantly lower in mice immunized with rCIIpic compared with mice immunized with tissue-derived CII, whereas that of rCIIbac was intermediate. These data indicate that the degree of lysine hydroxylation and glycosylation plays a role in the induction of arthritis. The recombinant collagens were then compared with tissue-derived CII when given as i.v. or oral tolerogens to suppress arthritis. Both recombinant collagens were less potent than tissue-derived CII, and this decrease in arthritis was associated with a decrease in Ab response to CII. These data suggest that the degree of glysosylation affects the immune response to CII, so that underglycosylated CII is less effective in the induction of arthritis and in its ability to suppress collagen-induced arthritis.

Analog peptides of type II collagen can suppress arthritis in HLA-DR4 (DRB1*0401) transgenic mice

Rheumatoid arthritis (RA) is an autoimmune disease associated with the recognition of self proteins secluded in diarthrodial joints. We have previously established that mice transgenic for the human DR genes associated with RA are susceptible to collagen-induced arthritis (CIA) and we have identified a determinant of type II collagen (CII263–270) that triggers T-cell immune responses in these mice. We have also determined that an analog of CII263–270 would suppress disease in DR1 transgenic mice. Because the immunodominant determinant is the same for both DR1 transgenic and DR4 transgenic mice, we attempted to determine whether the analog peptide that was suppressive in DR1 transgenic mice would also be effective in suppressing CIA in DR4 transgenic mice. We treated DR4 transgenic mice with two analog peptides of CII that contained substitutions in the core of the immunodominant determinant: CII256–276 (F263N, E266D) and CII256–270 (F263N, E266A). Mice were observed for CIA, and T-cell proliferative responses were determined. Either peptide administered at the time of immunization with CII significantly downregulated arthritis. Binding studies demonstrated that replacement of the phenylalanine residue in position 263 of the CII peptide with asparagine significantly decreased the affinity of the peptide for the DR4 molecule. In contrast, replacement of the glutamic acid residue in position 266 with aspartic acid or with alanine had differing results. Aspartic acid reduced the affinity (35-fold) whereas alanine did not. Both peptides were capable of suppressing CIA. With the use of either peptide, CII256–276 (F263N, E266D) or CII256–270 (F263N, E266A), the modulation of CIA was associated with an increase in T-cell secretion of IL-4 together with a decrease in IFN-γ. We have identified two analog peptides that are potent suppressors of CIA in DR4 transgenic mice. These experiments represent the first description of an analog peptide of CII recognized by T cells in the context of HLA-DR4 that can suppress autoimmune arthritis.