William F. Pendergraft III, Ph.D.

Dissertation research performed under the guidence of Dr. Ronald J. Falk

ABSTRACT
    This dissertation consists of 3 projects that examine proteinase 3 (PR3) from several perspectives. These studies unveil novel mechanisms underlying autoimmunity and inflammation. The first project, described in Chapter 1, proposes a hypothesis that autoimmune disease could stem from an immune response directed against a protein of endogenous or exogenous origin that is complementary in nature to self-protein(s). The culmination of these studies resulted in a novel theory, termed the theory of autoantigen complementarity. Experiments pertaining to proteinase 3-anti-neutrophil cytoplasmic autoantibodies (PR3-ANCA) support this theory. The second project, investigated in Chapter 2, addresses the controversy of expression of the ANCA autoantigens by the human endothelium. If endothelial cells expressed PR3 and myeloperoxidase (MPO), then autoantibodies directed against these proteins could interact directly with the endothelium thereby inducing vascular inflammation. We provide extensive data that endothelial cells do not express PR3 and MPO. The onset of a certain type of an autoimmune response is typically a harbinger for acute and subsequently chronic inflammation of vessels. Given the prominent role of neutrophil and monocyte granule constituents in acute and chronic inflammation, respectively, it is critical to understand the link between granule proteins and vascular injury. The third project (Chapter 3) demonstrates that PR3 rapidly enters endothelial cells and cleaves intracellular substrates that are also cleaved by caspases. Here we describe substrates of PR3, namely NF-kappaB and p21Cip1/WAF1. This ability to cleave downstream death substrates constitutes an apoptotic effector mechanism of immune cell proteases that is independent of that of the endogenous endothelial apoptotic cascade. A number of preliminary studies stemming from the major aims described above (see Chapter 4) provides data for the identification of an apoptotic death domain within PR3, suggesting that PR3 is capable of inducing apoptosis even when inactive as a serine protease. A recombinant form of PR3 (rPR3) was produced for affinity purification of PR3-ANCA. This material is antigenically similar to native PR3 resulting in rPR3 that could be used as a surrogate for native PR3 in clinical assays for the presence of PR3-ANCA. Together, these projects shed light on mechanisms of autoimmunity and inflammation.