Abstract
IntroductionAnti-endothelial cell antibodies (AECAs) are thought to be critical for vasculitides in collagen diseases, but most were directed against molecules localized within the cell and not expressed on the cell surface. To clarify the pathogenic roles of AECAs, we constructed a retroviral vector system for identification of autoantigens expressed on the endothelial cell surface.MethodsAECA activity in sera from patients with collagen diseases was measured with flow cytometry by using human umbilical vein endothelial cells (HUVECs). A cDNA library of HUVECs was retrovirally transfected into a rat myeloma cell line, from which AECA-positive clones were sorted with flow cytometry. cDNA of the cells was analyzed to identify an autoantigen, and then the clinical characteristics and the functional significance of the autoantibody were evaluated.ResultsTwo distinct AECA-positive clones were isolated by using serum immunoglobulin G (IgG) from a patient with systemic lupus erythematosus (SLE). Both clones were identical to cDNA of fibronectin leucine-rich transmembrane protein 2 (FLRT2). HUVECs expressed FLRT2 and the prototype AECA IgG bound specifically to FLRT2-transfected cells. Anti-FLRT2 antibody activity accounted for 21.4% of AECAs in SLE. Furthermore, anti-FLRT2 antibody induced complement-dependent cytotoxicity against FLRT2-expressing cells.ConclusionsWe identified the membrane protein FLRT2 as a novel autoantigen of AECAs in SLE patients by using the retroviral vector system. Anti-FLRT2 antibody has the potential to induce direct endothelial cell cytotoxicity in about 10% of SLE patients and could be a novel molecular target for intervention. Identification of such a cell-surface target for AECAs may reveal a comprehensive mechanism of vascular injury in collagen diseases.
Highlights
Anti-endothelial cell antibodies (AECAs) are thought to be critical for vasculitides in collagen diseases, but most were directed against molecules localized within the cell and not expressed on the cell surface
The prevalence of AECAs was significantly higher in patients with systemic lupus erythematosus (SLE) (50.5%) and other collagen diseases compared with normal controls (2.9%)
As these data indicated the presence of autoantigens on the endothelial cells (ECs) surface, we constructed a retroviral vector system to identify cell-surface target molecules of AECAs with flow cytometry
Summary
Anti-endothelial cell antibodies (AECAs) are thought to be critical for vasculitides in collagen diseases, but most were directed against molecules localized within the cell and not expressed on the cell surface. To clarify the pathogenic roles of AECAs, we constructed a retroviral vector system for identification of autoantigens expressed on the endothelial cell surface. AECAs have the potential to induce vascular lesions directly because their targets are expressed on ECs, which are always in contact with these circulating antibodies. AECAs are considered to play roles in the development of pathologic lesions by EC cytotoxicity (complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC)), activation of EC (proinflammatory cytokine secretion and expression of adhesion molecules), induction of coagulation, and induction of apoptosis [6,7,8,9]. AECAs may be good targets for such interventions, and identification of cellsurface targets of AECAs is required
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