Abstract
Proteinase 3 is a serine protease found in neutrophil granules and on the extracellular neutrophil membrane (mPR3). mPR3 is a major antigen for anti-neutrophil cytoplasmic antibodies (PR3-ANCAs), autoantibodies causing fatal autoimmune diseases. In most individuals, a subpopulation of neutrophils also produce CD177, proposed to present additional PR3 on the surface, resulting in CD177neg/mPR3low and CD177pos/mPR3high neutrophil subsets. A positive correlation has been shown between mPR3 abundance, disease incidence, and clinical outcome. We present here a detailed investigation of the PR3:CD177 complex, verifying the interaction, demonstrating the effect of binding on PR3 proteolytic activity and explaining the accessibility of major PR3-ANCA epitopes. We observed high affinity PR3:CD177 complex formation by surface plasmon resonance. Using flow cytometry and a PR3-specific FRET assay, we found that CD177 binding reduced the proteolytic activity of PR3 in vitro using purified proteins, in neutrophil degranulation supernatants containing wtPR3 and directly on mPR3high neutrophils and PR3-loaded HEK cells. Finally, CD177pos/mPR3high neutrophils showed no migration advantage in vitro or in vivo when migrating from the blood into the oral cavity. We illuminate details of the PR3:CD177 interaction explaining mPR3 membrane orientation and proteolytic activity with relevance to ANCA activation of the distinct mPR3 neutrophil populations.
Highlights
Proteinase 3 is a serine protease found in neutrophil granules and on the extracellular neutrophil membrane. membrane PR3 (mPR3) is a major antigen for anti-neutrophil cytoplasmic antibodies (PR3-ANCAs), autoantibodies causing fatal autoimmune diseases
In addition to direct membrane interaction[20,21], we have proposed that high mPR3 levels are achieved by PR3 binding to CD177, a GPI-anchored neutrophil-specific membrane receptor present in neutrophil granules and released into the extracellular space after neutrophil activation[22]
PR3 belongs to the neutrophil serine protease (NSP) family, but is the only member that serves as a major autoantigen in associated vasculitis (AAV)
Summary
Proteinase 3 is a serine protease found in neutrophil granules and on the extracellular neutrophil membrane (mPR3). mPR3 is a major antigen for anti-neutrophil cytoplasmic antibodies (PR3-ANCAs), autoantibodies causing fatal autoimmune diseases. PR3 serves as a major autoantigen in ANCA-associated vasculitis (AAV)[8,9,10] These anti-PR3 antibodies (PR3-ANCA) bind and cross-link membrane PR3 (mPR3) causing neutrophil activation[11,12] which contributes to necrotizing vasculitis. While all neutrophils contain PR3 intracellularly, the mPR3 pattern is remarkable in that two distinct subsets exist, one displaying little (mPR3low) and one abundant (mPR3high) mPR313 The percentage of the latter ranges from 0–100%, is genetically determined and stable in a given individual, is significantly higher in AAV patients than in healthy controls, responds stronger to PR3-ANCA in vitro, and is associated with worse disease outcome[14,15,16,17,18,19]. Complex that help to clarify the presentation of mPR3 to anti-PR3 ANCA, the effects of complex formation on the proteolytic activity of PR3 and its consequences for neutrophil migration
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