OK COBRE: NEUTROPHIL PROTEINASE-3 AUTOANTIGEN EXPRESSION AND REGULATION

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Pro-inflammatory cytokines, such as tumor necrosis factor (TNF), play an important role in the pathophysiology of diseases characterized by tissue destruction. Recently, the TNF precursor was found to be a substrate for neutrophil proteinase-3 (PR3). PR3 is a serine proteinase expressed on the surface of neutrophils and is the autoantigen of pro-inflammatory PR3-ANCA antibodies in patients with Wegener's granulomatosis, a severe autoimmune vasculitis. We showed that the soluble endothelial protein C receptor (sEPCR), a member of the protein C anticoagulant and anti-inflammatory pathway, binds to activated neutrophils via PR3 and PR3-ANCA from some, but not all, Wegener's patients reduced sEPCR binding to neutrophils. Our current data include the novel observation that PR3 activity toward the TNF precursor is down-modulated by sEPCR. These observations lead to our hypothesis that expression of neutrophil PR3 provides for both an immediate host response to inflammation, as well as a template for sEPCR binding and subsequent down-modulation of the response. Common or unique PR3 epitopes recognized by ANCA may alter this response pathway and contribute to the disease manifestations. Aim 1 studies use mutagenesis techniques and functional assays to identify the structural requirements governing sEPCR's interaction with PR3. Aim 2 studies evaluate how these molecular interactions are subsequently influenced by PR3-ANCA with respect to epitope specificity and disease manifestations. Aim 3 studies determine the importance of laminar shear stress forces on neutrophil PR3 expression, reasoning that in vivo rheological forces lead to neutrophil PR3 exposure and access to Wegener's autoantibodies under flow conditions found in small-to-medium sized vessels. Aim 4 studies determine relationships between PR3 DNA sequence and PR3 expression levels to identify mechanisms that result in the observed stable variability in PR3 expression phenotype between individuals. Unique polymorphisms or promoter cytosine methylation levels may influence PR3 expression levels or alter regions important for binding sEPCR, potentially contributing to risk of Wegener's vasculitis or disease severity. Correlation of the structural and functional data with the patient clinical condition will contribute to a better understanding of the underlying processes that result in endothelial damage in these patients. These studies will also impact our understanding of how PR3 contributes to cytokine generation and the role of sEPCR in modulating neutrophil inflammatory responses in auto-immune-mediated vasculitis. This regulatory mechanism may contribute to localizing the response to ANCA and preventing more systemic, deleterious effects by the cytokine.