Proteinase 3 on apoptotic cells disrupts immune silencing in autoimmune vasculitis
Arnaud Millet, … , Sylvain Perruche, Véronique Witko-Sarsat
Arnaud Millet, … , Sylvain Perruche, Véronique Witko-Sarsat
Published October 5, 2015
Citation Information: J Clin Invest. 2015;125(11):4107-4121. https://doi.org/10.1172/JCI78182.
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Research Article Immunology

Proteinase 3 on apoptotic cells disrupts immune silencing in autoimmune vasculitis

Abstract

Granulomatosis with polyangiitis (GPA) is a systemic necrotizing vasculitis that is associated with granulomatous inflammation and the presence of anti-neutrophil cytoplasmic antibodies (ANCAs) directed against proteinase 3 (PR3). We previously determined that PR3 on the surface of apoptotic neutrophils interferes with induction of antiinflammatory mechanisms following phagocytosis of these cells by macrophages. Here, we demonstrate that enzymatically active membrane-associated PR3 on apoptotic cells triggered secretion of inflammatory cytokines, including granulocyte CSF (G-CSF) and chemokines. This response required the IL-1R1/MyD88 signaling pathway and was dependent on the synthesis of NO, as macrophages from animals lacking these pathways did not exhibit a PR3-associated proinflammatory response. The PR3-induced microenvironment facilitated recruitment of inflammatory cells, such as macrophages, plasmacytoid DCs (pDCs), and neutrophils, which were observed in close proximity within granulomatous lesions in the lungs of GPA patients. In different murine models of apoptotic cell injection, the PR3-induced microenvironment instructed pDC-driven Th9/Th2 cell generation. Concomitant injection of anti-PR3 ANCAs with PR3-expressing apoptotic cells induced a Th17 response, revealing a GPA-specific mechanism of immune polarization. Accordingly, circulating CD4+ T cells from GPA patients had a skewed distribution of Th9/Th2/Th17. These results reveal that PR3 disrupts immune silencing associated with clearance of apoptotic neutrophils and provide insight into how PR3 and PR3-targeting ANCAs promote GPA pathophysiology.

Authors

Arnaud Millet, Katherine R. Martin, Francis Bonnefoy, Philippe Saas, Julie Mocek, Manal Alkan, Benjamin Terrier, Anja Kerstein, Nicola Tamassia, Senthil Kumaran Satyanarayanan, Amiram Ariel, Jean-Antoine Ribeil, Loïc Guillevin, Marco A. Cassatella, Antje Mueller, Nathalie Thieblemont, Peter Lamprecht, Luc Mouthon, Sylvain Perruche, Véronique Witko-Sarsat

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Figure 1

PR3 membrane expression on apoptotic neutrophils was increased in GPA.

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PR3 membrane expression on apoptotic neutrophils was increased in GPA. (...
(A and B) Membrane expression of PR3 and CD11b on apoptotic neutrophils (CD15+Annexin V+7AAD–) from healthy controls (HC) (n = 8) and GPA patients (n = 10) was assessed in whole blood maintained at 37°C for 20 hours. Values are presented as mean ± SEM. **P < 0.01. (C) PR3 expression on unstimulated neutrophils (basal) correlated with PR3 expression on TNF-α–stimulated neutrophils. (D) No correlation in membrane expression of PR3 (mean fluorescence intensity [MFI]) on basal and apoptotic neutrophils was observed. Data are presented for n = 19 healthy controls (white circles) and n = 15 GPA (black squares) with a mean age of 47.6 ± 3.2 versus 51.7 ± 4.4 years, respectively, and with a similar sex ratio, provided in Supplemental Table 1. (E) Within granulomatous inflammation in GPA, PR3+ (green, upper right panel) and cleaved caspase 3+ (red, lower left panel) cells detected by immunofluorescence analyses using confocal microscopy colocalized within the same cell (merge, lower right panel). Nuclei visualized using DAPI are depicted in gray (upper left panel). Scale bars: 5 μm. Significant differences between groups were determined by Mann-Whitney U test (A and B), and correlations were assessed using Pearson’s tests (C and D).