Endothelial cell autoantigens in systemic sclerosis

Systemic sclerosis (SSc), or scleroderma, is an autoimmune connective tissue disease affecting skin and internal organs. It has long been unclear which of the three salient pathologic features, i.e. vascular abnormalities, perivascular mononuclear cell infiltration, and increased collagen deposition, is the primary pathogenic event in SSc. To study the initial stage of such a complex disease animal models are of great value. The University of California at Davis (UCD)-200 chicken line is the only spontaneous animal model displaying all key symptoms of SSc, i.e. endothelial lesions, perivascular lymphocytic infiltration, fibrosis of skin and viscera, antinuclear antibodies (ANA), and anti-endothelial cell antibodies (AECA). A parallel comparative study of skin biopsies of UCD-200 chickens and human patients revealed that endothelial cell apoptosis is a primary event in the pathogenesis of SSc. Subsequent in vitro studies showed that this microvascular endothelial cell apoptosis is induced by anti-endothelial cell antibody dependent cellular cytotoxicity (ADCC) via Fas/Fas-ligand interaction. Based on these findings, the overall goal of our investigations is the identification of the primary (auto)antigen(s) recognized by AECA that mediate endothelial cell apoptosis. In a pilot study using UCD-200 sera from the initial disease stage, a stage that is not accessible in humans, we could identify a candidate autoantigen by 2-D gel electrophoresis, western blotting, and mass spectrometry. The aims of the present study are: (a) PCR-cloning and purification of this endothelial cell membrane protein, (b) verification of the AECA specificity for the purified recombinant antigen by western blotting, (c) screening of SSc and control sera to analyze if human SSc sera are also directed against this endothelial cell antigen recognized by UCD-200 sera, (d) development of diagnostic tests if antibodies prove to be specific for SSc, and (e) the functional analyses of antibodies binding to this antigen, i.e. apoptosis induction. Should none of the SSc sera be positive for antibodies to this special protein, we will try to identify other putative autoantigens using 2-D gel electrophoresis or chromatography techniques, as apropriate, western blots, and mass spectrometry. The identification of the primary autoantigen will give insight into the pathogenesis of SSc and should also lead to the development of new diagnostic tests to help improve the classification of SSc.

Systemic sclerosis (SSc), or scleroderma, is an autoimmune connective tissue disease affecting skin and internal organs. It has long been unclear which of the three salient pathologic features, i.e. vascular abnormalities, perivascular mononuclear cell infiltration, and increased collagen deposition, is the primary pathogenic event in SSc. To study the initial stage of such a complex disease animal models are of great value. The University of California at Davis (UCD)-200 chicken line is the only spontaneous animal model displaying all key symptoms of SSc, i.e. endothelial lesions, perivascular lymphocytic infiltration, fibrosis of skin and viscera, antinuclear antibodies (ANA), and anti-endothelial cell antibodies (AECA). A parallel comparative study of skin biopsies of UCD-200 chickens and human patients revealed that endothelial cell apoptosis is a primary event in the pathogenesis of SSc. Subsequent in vitro studies showed that this microvascular endothelial cell apoptosis is induced by anti-endothelial cell antibody dependent cellular cytotoxicity (ADCC) via Fas/Fas-ligand interaction. Based on these findings, the overall goal of our investigations is the identification of the primary (auto)antigen(s) recognized by AECA that mediate endothelial cell apoptosis. In a pilot study using UCD-200 sera from the initial disease stage, a stage that is not accessible in humans, we could identify a candidate autoantigen by 2-D gel electrophoresis, western blotting, and mass spectrometry. The aims of the present study are: (a) PCR-cloning and purification of this endothelial cell membrane protein, (b) verification of the AECA specificity for the purified recombinant antigen by western blotting, (c) screening of SSc and control sera to analyze if human SSc sera are also directed against this endothelial cell antigen recognized by UCD-200 sera, (d) development of diagnostic tests if antibodies prove to be specific for SSc, and (e) the functional analyses of antibodies binding to this antigen, i.e. apoptosis induction. Should none of the SSc sera be positive for antibodies to this special protein, we will try to identify other putative autoantigens using 2-D gel electrophoresis or chromatography techniques, as apropriate, western blots, and mass spectrometry. The identification of the primary autoantigen will give insight into the pathogenesis of SSc and should also lead to the development of new diagnostic tests to help improve the classification of SSc.