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Proteinase 3, the Autoantigen in Granulomatosis with Polyangiitis, Associates with Calreticulin on Apoptotic Neutrophils, Impairs Macrophage Phagocytosis, and This information is current as Promotes Inflammation of September 28, 2021. Julie Gabillet, Arnaud Millet, Magali Pederzoli-Ribeil, Pascale Tacnet-Delorme, Loïc Guillevin, Luc Mouthon, Philippe Frachet and Véronique Witko-Sarsat J Immunol published online 27 July 2012 Downloaded from http://www.jimmunol.org/content/early/2012/07/27/jimmun ol.1200600 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 28, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published July 27, 2012, doi:10.4049/jimmunol.1200600 The Journal of Immunology Proteinase 3, the Autoantigen in Granulomatosis with Polyangiitis, Associates with Calreticulin on Apoptotic Neutrophils, Impairs Macrophage Phagocytosis, and Promotes Inflammation Julie Gabillet,*,†,‡,1 Arnaud Millet,*,†,‡,1 Magali Pederzoli-Ribeil,*,†,‡ Pascale Tacnet-Delorme,x,{,‖ Loı¨c Guillevin,# Luc Mouthon,*,†,‡,# Philippe Frachet,x,{,‖ and Ve´ronique Witko-Sarsat*,†,‡ Proteinase 3 (PR3) is the target of anti-neutrophil cytoplasm Abs in granulomatosis with polyangiitis, a form of systemic vasculitis. Upon neutrophil apoptosis, PR3 is coexternalized with phosphatidylserine and impaired macrophage phagocytosis. Calreticulin Downloaded from (CRT), a protein involved in apoptotic cell recognition, was found to be a new PR3 partner coexpressed with PR3 on the neutrophil plasma membrane during apoptosis, but not after degranulation. The association between PR3 and CRT was demonstrated in neutrophils by confocal microscopy and coimmunoprecipitation. Evidence for a direct interaction between PR3 and the globular domain of CRT, but not with its P domain, was provided by surface plasmon resonance spectroscopy. Phagocytosis of apoptotic neutrophils from healthy donors was decreased after blocking lipoprotein receptor-related protein (LRP), a CRT receptor on mac- rophages. In contrast, neutrophils from patients with granulomatosis with polyangiitis expressing high membrane PR3 levels http://www.jimmunol.org/ showed a lower rate of phagocytosis than those from healthy controls not affected by anti-LRP, suggesting that the LRP-CRT pathway was disturbed by PR3-CRT association. Moreover, phagocytosis of apoptotic PR3-expressing cells potentiated proinflam- matory cytokine in vitro by human monocyte-derived macrophages and in vivo by resident murine peritoneal macrophages, and diverted the anti-inflammatory response triggered by the phagocytosis of apoptotic cells after LPS challenge in thioglycolate-elicited murine macrophages. Therefore, membrane PR3 expressed on apoptotic neutrophils might amplify inflammation and promote autoimmunity by affecting the anti-inflammatory “reprogramming” of macrophages. The Journal of Immunology, 2012, 189: 000–000. by guest on September 28, 2021 ranulomatosis with polyangiitis (GPA), formerly called In vitro, ANCA bind to their cognate Ag to trigger neutrophil Wegener’s granulomatosis, is a systemic disease char- adhesion to endothelial cells and activation. Described first in G acterized by granulomatous inflammation of the upper azurophilic granules, its main storage compartment, PR3 is also and lower respiratory tract, glomerulonephritis, and necrotizing present in the secretory vesicles and on the plasma membrane (3). vasculitis (1). GPA is associated with anti-neutrophil cytoplasmic The peculiarity of PR3 membrane expression is its heterogeneity autoantibodies (ANCAs) directed against proteinase 3 (PR3) (2). (4): the percentage of membrane PR3-expressing neutrophils varies among individuals but is genetically determined. Notably, a high percentage of membrane PR3-expressing positive neu- † *INSERM U1016, Paris 75014, France; Institut Cochin, Universite´ Paris Descartes, trophils is observed in patients with chronic inflammatory diseases Paris 75014, France; ‡Centre National de la Recherche Scientifique-Unite´ Mixte de Recherche 8104, Paris 75014, France; xUniversite´ Joseph Fourier Grenoble 1, Institut such as ANCA-associated vasculitis. In addition, unlike other { de Biologie Structurale Jean-Pierre Ebel, Grenoble F-38027 Cedex 1, France; Centre neutrophil granule proteins, PR3 is coexternalized with phospha- National de la Recherche Scientifique, Grenoble F-38000, France; ‖Commissariat a` l’Energie Atomique, Institut de Biologie Structurale Jean-Pierre Ebel, Grenoble tidylserine (PS) during neutrophil apoptosis because of its asso- F-38000, France; and #Service de Me´decine Interne, Hoˆpital Cochin, Paris 75014, ciation with phospholipid scramblase 1 and impaired macrophage France phagocytosis (5). We recently reported that PR3, which is a pe- 1J.G. and A.M. contributed equally to this work. ripheral membrane protein, has a hydrophobic patch essential for Received for publication February 17, 2012. Accepted for publication June 25, 2012. its membrane anchorage and for its proinflammatory activity (6). This work was supported by the Association “Vaincre La Mucoviscidose” (to J.G.), Accordingly, a hydrophobic patch-deficient PR3 mutant was not INSERM (to A.M.), Agence Nationale de la Recherche-genopat (to V.W.-S.), the externalized on apoptosis and did not impair phagocytosis (6). Chancellerie des Universite´s de Paris (Legs Poix to V.W.-S.), Agence Nationale de la Recherche (ANR-09-PIRIbio to P.F.), and Joseph Fourier University in Grenoble Phagocytosis of apoptotic cells, also known as efferocytosis, (to P.F.). prevents the release of intracellular contents and unwanted immune Address correspondence and reprint requests to Dr. Ve´ronique Witko-Sarsat, responses to self-Ags derived from dying cells (7). During inflam- INSERM U1016, Institut Cochin, 27 Bis Rue du Faubourg St Jacques, 75014 Paris, mation, neutrophils are massively recruited at the inflammation site France. E-mail address: [email protected] where they play a fundamental role in pathogen destruction and Abbreviations used in this article: 7-AAD, 7-amino-actinomycin D; ANCA, anti- neutrophil cytoplasmic autoantibody; BFA, brefeldin A; CRT, calreticulin; GPA, then undergo apoptosis. Apoptotic neutrophils are recognized and granulomatosis with polyangiitis; LRP, lipoprotein receptor-related protein; MFI, ingested by macrophages, thereby inducing an anti-inflammatory mean fluorescence intensity; PR3, proteinase 3; PS, phosphatidylserine; RBL, rat phenotype and promoting the resolution of inflammation (7). Ap- basophilic cell line; SPR, surface plasmon resonance. optotic cell uptake involves many phagocyte receptors that bind Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 molecules expressed at the apoptotic cell surface called “eat-me” www.jimmunol.org/cgi/doi/10.4049/jimmunol.1200600 2 PROTEINASE 3 AND CALRETICULIN IN APOPTOTIC NEUTROPHILS signals. These signals include the adsorption of soluble proteins Flow cytometry analysis of PR3, CRT, CD177, and LRP, and (such as C1q and thrombospondin) from outside the cell, and the CD66b membrane expression translocation of molecules such as PS and calreticulin (CRT) from After FcR blocking, 5 3 105 cells were incubated for 30 min with one of inside the cell to the surface, as well as the suppression of “don’t- the following Abs: mouse monoclonal anti-PR3 (clone CLB12.8; 2 mg/ml; eat-me” signals such as CD47 (8). The best characterized eat-me Sanquin), rabbit polyclonal anti-CRT (clone PA3-900; 5 mg/ml; Thermo- signal is PS, exposed at the cell surface in the early stages of Scientific), mouse monoclonal anti-CRT (clone SPA 601; 30 mg/ml; Stress- m apoptosis. PS is recognized by phagocyte receptors, among them Gen), mouse monoclonal anti-hCD177 (1.2 g/ml; Serotec), mouse monoclonal anti-LRP (6 mg/ml; Becton Dickinson), or mouse monoclonal TIM4 and BAI1 (7, 8). Unlike necrotic cells, apoptotic cell recog- anti–CD66b-FITC (25 mg/ml; Immunotech), followed by incubation with nition induces the release from macrophages of anti-inflammatory fluorochrome-conjugated anti-mouse IgG or anti-rabbit IgG (Immunotech). mediators such as TGF-b,IL-10,andPGE2, and inhibits proin- flammatory cytokine secretion, such as TNF-a and IL-8 (9). Confocal microscopy analysis of immunofluorescence labeling It is now commonly accepted that CRT, originally characterized of PR3 and CRT as an endoplasmic reticulum chaperone protein (10), is also a Neutrophils were induced to adhere to poly-L-lysine–precoated coverslips, majoreat-me signal expressed at the surface of apoptotic cells, then fixed and permeabilized with streptolysin-O as described previously including neutrophils (11). Accordingly, decreased CRT mem- (5). After saturation in PBS with 1% BSA, cells were incubated for 45 min with the mouse anti-PR3 or rabbit