The overall aim of this project is to develop a better understanding of the immunologic events leading to glomerular immune deposit formation in individuals with Systemic Lupus Erythematosus. In previous studies, murine and human monoclonal anti-DNA antibodies (Ab) were identified that produced glomerulonephritis following transfer to normal mice. Of particular relevance, the location of immune deposit formation and disease phenotype varied with the mAb. Furthermore, these individual pathogenic Ab bound directly to glomerular cell surface antigens, however each monoclonal anti-DNA Ab recognized a different cell surface proteins. Based on these observations, it was postulated that different autoantibody-glomerular antigen interactions, in vivo, contributes to the phenotypic diversity observed both among the monoclonal Ab and among individuals with lupus. A primary goal of this project is to fully identify the glomerular cell surface antigens for three nephritogenic lupus autoantibodies: anti-DNA MES and anti-DNA SE, derived from MRL-lpr/lpr mice; and RH-14, a human anti-DNA Ab. Anti-DNA MES produces mesangial deposits and binds to mesangial cells, whereas anti-DNA SE produces subendothelial deposits and binds to glomerular endothelial cells. RH14 produces massive subendothelial deposits on transfer to SCID mice, and it binds to glomerular endothelial cells. Candidate cell surface protein antigens were isolated for the autoantibodies. Peptides derived from the isolated proteins will be sequenced and then used to generate both degenerate oligonucleotides and anti-peptide antibodies to screen cDNA libraries, in order to define the full sequence and identity of the immunoreactive proteins. Another primary goal of the project is to further determine the pathogenic relevance of these autoantibody-glomerular cell interactions by examining: i) the immune response to the purified cell surface proteins, ii) other spontaneously produced autoantibodies with anti-cell surface protein activity; and iii) the cellular and functional consequences of Ab ligation of the cell surface proteins. Studies will be performed to begin to determine the overall relevance of direct binding of human lupus autoantibodies to glomerular antigens, in general, using: human lupus sera from the Lupus Collaborative Study and controls, the purified cell surface antigens, and individual glomerular cells. Collectively, the results should identify disease-relevant glomerular antigens for pathogenic lupus autoantibodies and provide insights into the overall pathogenic relevance of autoantibody-glomerular cell surface antigen interactions in lupus nephritis.
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