Background/Purpose: Autoantibodies which recognize integral membrane proteins are generally accepted as pathogenic. However, it had been technically difficult to identify plasma membrane proteins as autoantigens by conventional methods including proteomics. Anti-endothelial cell antibodies (AECAs) are autoantibodies against cell surface molecules on the endothelium, and play important roles on promoting vascular inflammation. Most of the reported autoantigens were intracellular molecules, and their target antigens on endothelium were poorly understood. In order to identify cell-surface autoantigens, we constructed a Serological identification system for Autoantigens using a Retroviral vector and Flow cytometry (SARF).

Methods: cDNA library of human umbilical vein endothelial cells (HUVECs) were generated and inserted into the retroviral vector. Retroviruses possessing the cDNA library were generated and infected into the rat myeloma cells. Rat myeloma cells which have different HUVEC cDNA were stained with serum IgG from patients with AECA-positive collagen diseases, and AECA-positive fraction was sorted by flow cytometry. After cloning of an AECA-positive cell, cDNA inserted into each clone was identified by DNA sequencing and microarray.

Results: The identified molecules included fibronectin leucine rich transmembrane protein 2 (FLRT2), ephrin type-B receptor 2 (EphB2), and Pk antigen for systemic lupus erythematosus, and intercellular adhesion molecule-1 (ICAM-1) for rheumatoid arthritis. Expressions of these molecules on ECs were confirmed by flow cytometry and AECA IgGs bound specifically to the cells which were transfected with each of the molecules. Importantly, anti-FLRT2 antibody induced complement-dependent cytotoxicity against FLRT2 expressing cells including HUVECs. Clinical manifestations of each autoantibody-positive patient were associated with the distribution of autoantigens in different cell types, indicating that AECAs can act not only on ECs but also on other target cells. We further identified autoantigens in Takayasu arteritis, in which no definite autoantigens were reported. The autoantigens in Takayasu arteritis play important roles for the coagulation and lipid metabolism, suggesting that AECAs can directly modify vascular inflammation in Takayasu arteritis.

Conclusion: We successfully identified membrane proteins as autoantigens against AECAs by using SARF and confirmed the usefulness of SARF. Moreover, identified molecules were associated with the clinical manifestations of patients, suggesting direct contributions of AECAs for promoting pathological conditions. Using this system, it is possible to achieve a comprehensive analysis of autoantibody-mediated injury in inflammatory diseases and develop more specific interventions.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/establishment-of-a-powerful-method-to-identify-autoantigens-expressed-on-the-cell-surface/