IMMUNOLOGIC MECHANISMS IN LUPUS NEPHRITIS

The long term goals of this project are to determine the structural properties and renal antigen (Ag) ligands of immune deposit forming lupus autoantibodies (autoAb). The experimental hypotheses are: i) specific V gene sequences of autoAb, by determining their Ag binding properties, confer the capacity of Ig to form immune deposits by direct interactions with glomerular Ag, and ii) the site of immune deposit formation is influenced by the location of the Ag target within the glomerulus. These phenomena, per se, are critical for and contribute to the observed variations in the phenotypic expression of lupus nephritis, and they emphasize the disease relevance of both the Ag binding regions of pathogenic autoAb and the glomerular Ag. To further examine the molecular basis of these interactions an existing panel of over 2000 B cell hybridomas and renal cell lines derived from lupus prone MRL-lpr/lpr mice will be used to pursue the following Specific Aims: 1. To examine structural properties of immune deposit forming lupus autoAb. The experimental approach is facilitated by previous identification, characterization and V gene sequence analysis of subgroups of m anti-DNA Ab that produce distinguishable patterns of glomerular immune deposits after passive transfer to normal mice, including the following prototypes: a) anti-DNA-SE/MES (mesangial and subendothelial deposits); b) anti-DNA-BM (basement membrane and mesangial deposits; and c) anti-DNA IL/IM (intraluminal and intramembranous deposits). Oligonucleotide probes specific for CDR1 and CDR2 of the V genes used to encode pathogenic Ig will be utilized to select Ig from our existing panel of hybridomas that are encoded by closely related V gene sequences. The capacity of individual Ig to transfer disease will then be compared to prototypic m anti-DNA Ab. Determination of critical a.a. residues will be evaluated by comparison of immune deposit forming autoAb to mIg that do not form deposits using the primary sequence data and computer generated models of the Ag binding grooves. If necessary, in second order experiments, site-directed mutagenesis of V genes encoding immune deposit forming Ab will be utilized to examine the influence of specific a.a. residues. 2. To identify relevant glomerular Ag targets of nephritogenic lupus autoAb. Previous work demonstrated that immune deposit forming autoAb bind to: a protein extract derived from normal glomeruli; cell surface Ag present on normal mesangial cells; and matrix proteins that normally reside within the glomerulus. The studies described in this proposal focus on more precise identification of the Ag targets of immune deposit forming m autoAb. The three mAb groups described in the first Specific Aim will serve as prototypes. Initial experiments will be directed at purification and sequence analysis of relevant proteins. Degenerate oligonucleotide probes, based on partial a.a. sequence of these purified protein(s), will then be used to screen a murine kidney cDNA library. Relevant clones will be sequenced to determine the identity of the protein(s). Concurrently, the anti-DNA mAb will be used to directly screen murine renal cell cDNA expression libraries; relevant clones will be evaluated in a similar manner.