Localized Exocytosis of Primary (Lysosomal) Granules During Phagocytosis: Role of Ca 2+ -Dependent Tyrosine Phosphorylation and Microtubules This information is current as of September 24, 2021. Hans Tapper, Wendy Furuya and Sergio Grinstein J Immunol 2002; 168:5287-5296; ; doi: 10.4049/jimmunol.168.10.5287 http://www.jimmunol.org/content/168/10/5287 Downloaded from References This article cites 52 articles, 26 of which you can access for free at: http://www.jimmunol.org/content/168/10/5287.full#ref-list-1 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 24, 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 © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Localized Exocytosis of Primary (Lysosomal) Granules During Phagocytosis: Role of Ca2؉-Dependent Tyrosine Phosphorylation and Microtubules1 Hans Tapper,2* Wendy Furuya,† and Sergio Grinstein† The uptake and killing of bacteria by human neutrophils are dependent on the fusion of secretory granules with forming phago- somes. The earliest component of exocytosis was found to precede phagosome closure, so that granular membrane constituents were detectable on the plasmalemma. We show that during phagocytosis of IgG-opsonized particles, this early secretory response is highly polarized in the case of primary granules, but less so for specific granules. The vectorial discharge of primary granules was dependent on calcium, but no evidence was found that calcium is involved in determining the polarity of exocytosis. In particular, a redistribution of endomembrane calcium stores toward forming phagosomes could not be detected. Polarized granule Downloaded from exocytosis was accompanied by focal tyrosine phosphorylation and actin polymerization, although the latter was not required for the response. Instead, microtubules seemed to contribute to the vectorial nature of the response. During particle ingestion, the microtubule-organizing center relocated toward forming phagosomes, and colchicine treatment altered the pattern of exocytosis, reducing its directionality. We hypothesize that the focal activation of tyrosine kinases generates localized signals that induce exocytosis in a calcium-dependent manner, and that reorientation of microtubules facilitates preferential delivery of granules toward the forming phagosome. The Journal of Immunology, 2002, 168: 5287–5296. http://www.jimmunol.org/ rofessional phagocytes, e.g., macrophages and neutrophils, lated to secondary granules, and share some of their contents, but play an important role in our innate defense against invad- are distinct in that they contain gelatinase and lack CD66b. Finally, P ing pathogens, in the resolution of inflammation, and in neutrophils possess small secretory vesicles, which are filled with the general maintenance of tissue homeostasis (1, 2). These func- albumin and express on their membranes latent alkaline phospha- tions are highly dependent on the ability of these cells to internal- tase and CD35. All four types of granules coexist in each cell. ize particles and on other events that are coupled to or triggered by They are, however, secreted under different conditions and with phagocytosis (e.g., secretion, oxidative burst). Unlike most other distinct sensitivity toward stimuli (3, 4). Secretory vesicles are the cells, neutrophils are endowed with several distinct types of se- most responsive and are secreted first, even before the neutrophil by guest on September 24, 2021 cretory organelles. They are intended to secrete content proteins, reaches the site of infection. Tertiary granules are more sensitive as well as deliver integral proteins to the cell surface and phago- than secondary granules, which are in turn more responsive than somal membranes. At least four distinct types of secretory or- primary granules. In permeabilized cells or in cells treated with ganelles are currently recognized (1, 3). Primary or azurophilic ionophores, this progression can be mimicked in vitro as the con- granules are lysosomal in nature. They contain proteases, myelo- centration of Ca2ϩ is increased (5, 6). Clearly, the function of the peroxidase, etc. Their membranes express CD63, which is a useful individual granule types is distinct, as are the signal transduction marker for immunofluorescence and flow cytometry. Secondary pathways leading to their release. granules contain lactoferrin, vitamin B12-binding protein, ly- As part of the microbial killing process, the contents of secretory sozyme, etc. They are smaller and less dense than primary granules granules are emptied into the phagosomal space. Concurrently, and possess CD66b on their membranes. Tertiary granules are re- granular membrane proteins are inserted into the phagosomal membrane, where they play an essential role in killing (e.g., the Hϩ-ATPase and some membrane-bound forms of cathepsin). *Department of Cell and Molecular Biology, Section for Molecular Pathogenesis, Clearly, such delivery of granular components must be targeted: Lund University, Lund, Sweden; and †Program in Cell Biology, Hospital for Sick Children and Department of Biochemistry, University of Toronto, Toronto, Ontario, random insertion throughout the cell membrane would not only be Canada wasteful, but potentially deleterious. Indeed, uncontrolled release Received for publication July 27, 2001. Accepted for publication March 6, 2002. of elastase and other proteases is among the major causes of lung The costs of publication of this article were defrayed in part by the payment of page damage in cystic fibrosis and of joint pathogenesis in arthritis as charges. This article must therefore be hereby marked advertisement in accordance well (1, 7). In the present study, studies have been performed to with 18 U.S.C. Section 1734 solely to indicate this fact. spatially locate secretion of primary and secondary granules during 1 This work was supported by grants from the Canadian Institutes for Health Re- phagocytosis of yeast (zymosan) coated with Ig to engage the FcR. search, The Sanatorium Association, and the Arthritis Society of Canada. H.T. re- ceived financial support from the Swedish Medical Research Council (Grants 12182 Early studies (8–10) have described exocytosis occurring during and 12613), the Magnus Bergvall Foundation, the Crafoord Foundation, the Greta and neutrophil phagocytosis, before closure of the phagosome. Using a Johan Kock Foundation, the Kungliga Fysiografiska Sa¨llskapet, the Alfred O¨ sterlund Foundation, and the Wenner-Gren Center Foundation. S.G. is an International Scholar combination of flow cytometry and dual-wavelength confocal im- of the Howard Hughes Medical Institute and is the current holder of the Pitblado munofluorescence, we found that exocytosis of both primary and Chair in Cell Biology at The Hospital for Sick Children. secondary granules precedes sealing of the phagosomal space. 2 Address correspondence and reprint requests to Dr. Hans Tapper, Department of More importantly, CD63 was found to be highly localized to the Cell and Molecular Biology, Section for Molecular Pathogenesis, BMC, B14, Lund University, Tornava¨gen 10, SE-221 84 Lund, Sweden. E-mail address: prephagosomal cup, while CD66b was more randomly distributed. [email protected] Because the phagosomal membrane is formed by invagination of Copyright © 2002 by The American Association of Immunologists 0022-1767/02/$02.00 5288 LOCALIZED EXOCYTOSIS DURING PHAGOCYTOSIS the plasma membrane, it is not immediately apparent how exocy- rosine were obtained from Zymed Laboratories (clones PY20, PY-7E1, tosis can be selectively targeted to the phagosome. Polarized exo- PY-Plus-cocktail; San Francisco, CA) and from Upstate Biotechnology cytosis exists in other cell types (11). Neurons have well-defined (clone 4G10; Lake Placid, NY). mAbs to tyrosine tubulin (clone TUB- 1A2) and ␣-tubulin (clone DM 1A) were obtained from Sigma-Aldrich. presynaptic densities in which vesicles accumulate and fuse, at the Donkey serum and the secondary Abs used for immunofluorescence (Cy3- exclusion of other areas of the surface membrane. Similarly, acinar conjugated donkey anti-rabbit and FITC- and Cy3-conjugated donkey anti- cells of exocrine tissues deliver their secretory contents to the lu- mouse) were from Jackson ImmunoResearch (West Grove, PA). men of the duct via the apical membrane, without occurrence of Experimental media basolateral secretion. Yet these systems differ from the neutrophil Nominally bicarbonate-free solution RPMI 1640 was buffered to pH 7.3 in that polarization is a permanent, preestablished feature of these ϩ cells. By contrast, neutrophils are seemingly symmetric cells be- with 25 mM HEPES. Na -based solution (Na medium) was also buffered to pH 7.3 and contained (in mM): 127 NaCl, 1.2 KH2PO4, 5.4 KCl, 0.8 fore their acute