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Localized Pinocytosis in Human Neutrophils R-Mediated Phagocytosis Stimulates Γ Fc FcγR-Mediated Phagocytosis Stimulates Localized Pinocytosis in Human Neutrophils Roberto J. Botelho, Hans Tapper, Wendy Furuya, Donna Mojdami and Sergio Grinstein This information is current as of October 1, 2021. J Immunol 2002; 169:4423-4429; ; doi: 10.4049/jimmunol.169.8.4423 http://www.jimmunol.org/content/169/8/4423 Downloaded from References This article cites 61 articles, 30 of which you can access for free at: http://www.jimmunol.org/content/169/8/4423.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • 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 *average by guest on October 1, 2021 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 © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Fc␥R-Mediated Phagocytosis Stimulates Localized Pinocytosis in Human Neutrophils1 Roberto J. Botelho,2* Hans Tapper,2† Wendy Furuya,* Donna Mojdami,* and Sergio Grinstein3,4* Engulfment of IgG-coated particles by neutrophils and macrophages is an essential component of the innate immune response. This process, known as phagocytosis, is triggered by clustering of Fc␥R at sites where leukocytes make contact with the opsonized particles. We found that phagocytosis is accompanied by a burst of fluid phase pinocytosis, which is largely restricted to the immediate vicinity of the phagosomal cup. Fc␥R-induced pinocytosis preceded and appeared to be independent of phagosomal sealing. Accordingly, fluid phase uptake was accentuated by actin depolymerization, which precludes phagocytosis. Stimulation of -pinocytosis required phosphatidylinositol 3-kinase activity and was eliminated when changes in the cytosolic free Ca2؉ concen tration were prevented. Because stimulation of Fc␥R also induces secretion, which is similarly calcium and phosphatidylinositol Downloaded from 3-kinase dependent, we studied the possible relationship between these events. Neutrophil fragments devoid of secretory granules (cytoplasts) were prepared by sedimentation through Ficoll gradients. Cytoplasts could perform Fc␥R-mediated phagocytosis, which was not accompanied by activation of pinocytosis. This observation suggests that granule exocytosis is required for stim- (ulation of pinocytosis. Analysis of the cytosolic Ca2؉ dependence of secretion and pinocytosis suggests that primary (lysosomal granule exocytosis is the main determinant of pinocytosis during Fc␥R stimulation. Importantly, primary granules are secreted in a polarized fashion near forming phagosomes. Focal pinocytosis during particle engulfment may contribute to Ag processing http://www.jimmunol.org/ and presentation and/or to retrieval of components of the secretory machinery. Alternatively, it may represent an early event in the remodeling of the phagosomal membrane, leading to phagosomal maturation. The Journal of Immunology, 2002, 169: 4423–4429. rofessional phagocytes, comprised of monocytes, macro- of IgG-opsonized particles (1, 3, 4). Particle engulfment is trig- phages, and neutrophils, are key to the innate immune de- gered by Fc␥R clustering, which induces localized activation of P fense system and, by removing apoptotic bodies, also con- Src family and Syk tyrosine kinases at the phagocytic cup. These tribute to tissue remodeling. Neutrophils often mount the initial initial events are followed by stimulation of phosphatidylinositol response to infection because of their rapid chemotactic response 3-kinase (PI3K)5 and phospholipase C␥ (4, 5), which hydrolyses toward bacterial peptides and inflammatory cytokines. Upon phosphatidylinositol-4,5-bisphosphate into diacylglycerol and ino- by guest on October 1, 2021 reaching the infected area, neutrophils curb pathogen activity by sitol-1,4,5-trisphosphate. The latter mediator is responsible for the ϩ2 ϩ2 ingestion of microorganisms, free radical synthesis, cytokine re- rise in the free cytosolic Ca concentration ([Ca ]i) observed lease, and degranulation (1, 2). during Fc␥R-mediated phagocytosis (6–8). Rac and Cdc42, mem- The antimicrobial responses of phagocytes are triggered by sur- bers of the Rho family of small GTPases, are then activated and face receptors that recognize either conserved patterns on the sur- coordinate actin remodeling at the sites of phagocytosis, culminat- face of microorganisms or opsonins that coat them. The latter re- ing in the engulfment of the microbe into an intracellular vacuole ceptors include Fc␥R, which are responsible for the phagocytosis or phagosome (9–12). In neutrophils, Fc␥R signaling also causes degranulation. Neu- trophils possess at least four types of secretory organelles: primary *Program in Cell Biology, Hospital for Sick Children, and Department of Biochem- (azurophilic), secondary (specific), and tertiary (gelatinase) gran- istry, University of Toronto, Toronto, Ontario, Canada; and †Department of Cell and ules and secretory vesicles (1, 13). Primary granules are enriched Molecular Biology, Section for Molecular Pathogenesis, Lund University, Lund, in lysosomal hydrolases and myeloperoxidase, and they can be Sweden identified by the presence of CD63 on their membrane. Secondary Received for publication March 6, 2002. Accepted for publication August 2, 2002. granules contain lactoferrin and lysozyme and express CD66b on The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance their membrane. Tertiary granules contain gelatinase, while secre- with 18 U.S.C. Section 1734 solely to indicate this fact. tory vesicles are rich in albumin and alkaline phosphatase (1). 1 This work was supported by the Canadian Institutes for Health Research, the These organelles do not necessarily undergo secretion simulta- Arthritis Society of Canada, the Arthritis Center of Excellence, the Sanatorium As- neously, since the signals leading to their exocytosis differ in type sociation, a Canadian Institutes for Health Research Graduate Studentship (to R.J.B.), and the Swedish Medical Research Council (Grants 12182, 12613, and 7480), The and/or activation threshold (14, 15). Magnus Bergvall Foundation, The Crafoord Foundation, The Greta and Johan Kock Exocytosis of multiple types of secretory organelles contributes Foundation, The Kungliga Fysiografiska Sallskapet, and The Alfred Osterlund Foun- additional surface area to the target membrane. In other systems dation (to H.T.). that undergo similar acute and vigorous secretion, such as chro- 2 R.J.B. and H.T. contributed equally to this work. maffin cells, the net area of the membrane is maintained approx- 3 Address correspondence and reprint requests to Dr. Sergio Grinstein, Program in imately constant by the concomitant activation of endocytosis Cell Biology, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8. E-mail address: [email protected] 4 S.G. is a Canadian Institutes of Health Research Distinguished Scientist and the current holder of the Pitblado Chair in Cell Biology at Hospital for Sick Children. 5 Abbreviations used in this paper: PI3K, phosphatidylinositol 3-kinase; LY, Lucifer 2ϩ 2ϩ Cross-appointed to the Department of Biochemistry of University of Toronto. Yellow; [Ca ]i, cytosolic free Ca concentration. Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00 4424 PHAGOCYTOSIS-DEPENDENT PINOCYTOSIS (16–19). Endocytosis also serves to retrieve components of the Endocytic uptake of LY and secretion of CD63/CD66b were quantified secretory machinery to be reused in subsequent rounds of stimu- using a FACScan flow cytometer (BD Biosciences, Mountain View, CA). lation. For these reasons, endocytosis (pinocytosis) is also likely to Preparation of samples was performed as described above, but cells were ␥ diluted in PBS and maintained in suspension. For every sample, at least be activated during Fc R-mediated phagocytosis. It is noteworthy, 10,000 ungated cells were counted. Selection of the population of interest however, that, unlike chromaffin cells, phagocytes are capable of was performed after the acquisition of raw data using LYSIS II analysis focal secretion during particle engulfment, targeting the secreted software as described previously (28). material to the area of the plasma membrane where phagosomes are being generated or to the lumen of formed phagosomes (20, Spectrofluorometry and calcium manipulations 21). It is therefore conceivable that localized signals may, in fact, 2ϩ [Ca ]i was quantified by spectrofluorometry using Indo-1 as previously trigger focal pinocytosis during phagocytosis. Indeed, clathrin, dy- described (26, 28). Briefly, neutrophils were loaded with 1 ␮M Indo-1/AM Ϫ 2ϩ namin, and amphiphysin were detected around the phagosomal cup for 30 min at 37°C, washed, and maintained in HCO3 -free, Ca -free (22–24). To test these hypotheses we studied whether pinocytosis medium (140 mM NaCl,
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