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Published on: May 21, 2021
Membrane proteinase 3 expression and ANCA-induced neutrophil activation
Adrian Schreiber1, Friedrich C Luft, Ralph Kettritz
1HELIOS Klinikum-Berlin, Franz Volhard Clinic, and Max Delbrück Center for Molecular Medicine, Medical Faculty of the Charité, Humboldt University of Berlin, Germany.
Background:
Proteinase 3 is the major autoantigen in Wegener's granulomatosis (WG). Membrane PR3 expression is bimodal; low expressing cells (mPR3(low)) can be distinguished from cells with high expression (mPR3(high)) within a given individual. High mPR3 expression is a WG risk factor and is associated with relapse. However, no mechanisms for this important clinical observation have been provided. We tested the hypothesis that mPR3 expression, rather than the expression of other membrane molecules implicated in anti-neutrophil cytoplasmic autoantibodies (ANCA) activation, determines the robustness of the PR3-ANCA-mediated response.
Methods:
mPR3(low) and mPR3(high) neutrophils from a given individual were separated by magnetic cell sorting. Superoxide was measured by the ferricytochrome assay, and Akt phosphorylation by Western blotting. Double staining and flow cytometry were used to assay Fc gamma-receptor and beta 2-integrin expression with respect to the mPR3 phenotype. Degranulation was measured via beta-glucuronidase activity, migration with fibronectin-coated transwells, and cell quantification by the myeloperoxidase (MPO) assay.
Results:
PR3-ANCA-treated mPR3(high) versus mPR3(low) neutrophils showed more superoxide generation (33.7 +/- 15.2 nmol O(2) (-) to 14.6 +/- 8.4, P < 0.01), more degranulation (29%+/- 5 to 22%+/- 3, P < 0.05), and more PI3-K/Akt activation. In contrast, all responses in both mPR3 subsets were similar after other stimuli. We observed no differences in the beta 2-integrin, Fc gamma R IIa, and III expression with respect to the mPR3 subtype. Furthermore, we found no differences in the mobilization of PR3-containing granules and no differences in migration through fibronectin.
Conclusion:
The degree of neutrophil mPR3 expression has definitive functional consequences.
Insights
High membrane Proteinase 3 (mPR3) expression on neutrophils amplifies anti-neutrophil cytoplasmic autoantibody (ANCA)-mediated responses, including superoxide generation and degranulation. This finding explains the clinical observation linking high mPR3 to Wegener's granulomatosis relapse.
Area of Science:
- Immunology
- Cell Biology
Background:
- Proteinase 3 (PR3) is a key autoantigen in Wegener's granulomatosis (WG).
- Neutrophil membrane PR3 (mPR3) expression is bimodal, with low (mPR3(low)) and high (mPR3(high)) expressing cells.
- High mPR3 expression is a risk factor for WG and relapse, but underlying mechanisms remain unclear.
Purpose of the Study:
- To investigate the functional consequences of differential mPR3 expression on neutrophil activation by PR3-ANCA.
- To determine if mPR3 expression level, rather than other membrane molecules, dictates the PR3-ANCA-mediated response.
Main Methods:
- Neutrophils from individuals were separated into mPR3(low) and mPR3(high) subsets using magnetic cell sorting.
- Functional assays included superoxide generation, Akt phosphorylation, degranulation, and migration.
- Flow cytometry assessed Fc gamma-receptor and beta 2-integrin expression.
Main Results:
- PR3-ANCA-stimulated mPR3(high) neutrophils exhibited significantly higher superoxide generation and degranulation compared to mPR3(low) neutrophils.
- PI3-K/Akt activation was also elevated in mPR3(high) neutrophils following PR3-ANCA stimulation.
- No differences in beta 2-integrin or Fc gamma-receptor expression were observed between the subsets.
Conclusions:
- The level of neutrophil mPR3 expression directly influences functional responses to PR3-ANCA.
- This differential expression provides a mechanistic link between high mPR3 and WG pathogenesis/relapse.
