Using the enzyme-linked immunosorbent assay (ELISA), we detected antibodies to interstitial (type I) and basement membrane (type IV) collagens in the sera of patients with scleroderma (systemic sclerosis). Antibodies against type IV collagen were found in significant levels in these patients and correlated with the presence of abnormal pulmonary diffusion capacity. Levels of antibodies to type I collagens also correlated significantly with pulmonary diffusion capacity. Absorption of sera with type I or type IV collagens before analysis in the ELISA eliminated reactivity in an antigen-specific pattern, indicating that these antibodies reacted with determinants specific for either type I or type IV collagens. The removal of immune complexes by ultracentrifugation had no effect on serum antibody levels. Autoantibo-
The immune response to connective tissue components of basement membrane (type IV collagen and laminin) and to interstitial collagen (type I) has been examined in human and murine systems. We also examined the role that immunologic sensitization to autologous connective tissue components might play in inducing an inflammatory response resulting in pathologic sequelae. Mice receiving a single subcutaneous injection of 5 micrograms type IV or type I murine collagens, or murine laminin in complete Freund's adjuvant mount a delayed-type hypersensitivity response characterized by a mononuclear cell infiltrate when challenged in the footpad with the sensitizing antigen. Cell-mediated immunity to these connective tissue antigens can be transferred to normal syngeneic mice with sensitized T-lymphocytes. In addition, repeated immunizations with these homologous connective tissue components elicit antibody responses in mice. Our data demonstrate the immunogenic nature of types IV and I collagen, and of laminin in a syngeneic murine model. We have demonstrated autoantibodies to the basement membrane and interstitial collagens in the sera of patients with scleroderma (systemic sclerosis); ELISA ratios correlate directly with the extent of pulmonary fibrosis in these patients. Anti-type IV collagen autoantibodies were found to be primarily IgM and anti-type I collagen antibodies, primarily IgG. An antibody response to autologous connective tissue antigens could lead to complement activation, immune complex formation, and deposition of the complexes along vascular endothelium with recruitment of blood monocytes in situ, mirroring the early scleroderma lesion (perivascular mononuclear cell filtrates). In vitro we examined the role of human peripheral blood mononuclear cells in the activation of fibroblasts. Adherent human blood monocytes release mediators which stimulate fibroblast proliferation and collagen deposition. A model is presented for the induction of immunity to autologous connective tissue components, leading to mononuclear cell inflammation, fibroblast activation and fibrosis. Selective immunity to basement membrane collagens may influence the clinical expression of diffuse connective tissue syndromes such as scleroderma (systemic sclerosis).
The immune response to the noncollagenous, attachment glycoprotein of basement membrane (laminin) was studied in a syngeneic murine system. Delayed-type hypersensitivity (DTH) was assayed in C57BU6 mice by measuring footpad swelling following challenge with connective tissue antigens. Mice receiving a single sensitizing injection of laminin in Freund's complete adjuvant (FCA) developed a significant DTH response which peaked on Day 7, 24 hr after challenge with laminin or collagenase-treated laminin. Laminin-sensitized mice failed to show any significant footpad swelling when challenged with types I or IV collagen, or fibronectin throughout these experiments. Normal mice displayed no significant DTH when challenged with these collagenous or noncollagenous connective tissue antigens. Adoptive transfer of laminin-sensitized spleen cells into normal mice resulted in significant DTH responsiveness to challenge with laminin; depletion of T cells from the immune spleens abrogated this response. Twenty-four hours after challenge with laminin, histology of the footpad lesions of laminin-sensitized mice revealed a mononuclear cell infiltrate, characteristic of a DTH response. Mice receiving repeated injections of laminin in Freund's incomplete adjuvant (FIA) developed significant antibody responses as detected by the enzyme-linked immunosorbent assay (ELISA). Furthermore Pronase, but not collagenase, treatment of laminin destroyed its antigenicity. Laminin immune sera showed no reactivity when assayed on fibronectin or collagen types I-V. No crossreactivity was exhibited by murine anti-type IV or anti-type I collagen antisera with laminin. These studies demonstrate the ability of isologous laminin to induce antigen-specific cellmediated and humoral immunity in a murine model.
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