Localized of Primary (Lysosomal) Granules During : Role of Ca 2+ -Dependent Tyrosine Phosphorylation and 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

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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 by human 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 Downloaded from exocytosis was accompanied by focal tyrosine phosphorylation and 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 -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 , e.g., 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 , 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 . 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 by guest on September 24, 2021 cretory . 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 , Hospital for Sick Children and Department of Biochemistry, University of Toronto, Toronto, Ontario, random insertion throughout the 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 (8Ð10) 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 activation. MgSO , 1.8 CaCl , 5.6 glucose, and 10 HEPES. In calcium-free Na me- 2ϩ 4 2 Increased cytosolic Ca is essential for exocytosis of primary dium, CaCl2 was replaced by EGTA (1 mM). All media were adjusted to and secondary granules in neutrophils (3, 6). Thus, removal of 290 Ϯ 5 mOsm with the major salt. external Ca2ϩ and preloading with bis(2-aminophenoxy)ethane- Coating of coverslips N,N,NЈ,NЈ-tetraacetate (BAPTA)3 eliminate exocytosis. It is well established that release of internal Ca2ϩ stores suffices for the se- Glass coverslips were washed with methanol and overlaid with 0.25 ml cretory response and that omission of external Ca2ϩ has little ef- poly-L-lysine (0.2 mg/ml in water). After evaporating the added fluid at 2ϩ 50Ð65¡C, the poly-L-lysine-coated coverslips were washed twice with dis- fect. There is some discrepancy regarding the pattern of Ca re- tilled water. Coverslips were used within 1 day of coating. lease from stores, but some authors have pointed out that cytosolic Downloaded from 2ϩ Neutrophil isolation, preparation of zymosan, and protocol for free calcium concentration ([Ca ]i) is preferentially elevated in the vicinity of the phagosome (12, 13). This could readily account stimulation of cells for the occurrence of localized exocytosis near the phagosomal Human neutrophils (Ͼ98% pure) were isolated from fresh heparinized ϩ cup. It has been also suggested that the Ca2 stores themselves blood of healthy volunteers by dextran sedimentation, followed by Ficoll- rearrange, concentrating around the phagosome (13). Obviously, Hypaque gradient centrifugation. Contaminating red cells were removed by 2ϩ NH Cl lysis. Neutrophils were counted using a model ZM Coulter counter this could account for the observed [Ca ] localization. It is not 4 http://www.jimmunol.org/ i (Hialeah, FL), resuspended in HEPES-buffered RPMI 1640 medium at 107 clear whether this repositioning of the stores is sufficiently fast to cells/ml, and maintained in this medium at room temperature until use. The account for the exocytosis that precedes closure of the phagosome. viability of the cells was greater than 97%, as judged by trypan blue ex- Another early response to phagocytic stimuli is cascades of ty- clusion. All experiments were performed within3hofneutrophil isolation. rosine phosphorylation of multiple cellular targets (14Ð16). In the Zymosan particles are baker’s yeast (Saccharomyces cerevisiae) that has 2ϩ been subjected to boiling and extensive trypsin treatment (19). The zymo- present work, we will present data on the role of Ca and phos- san particles used were relatively homogeneous in size with an average phorylation on tyrosine in the targeting of secretory responses to- particle diameter of 3 ␮m. Zymosan particles were dispersed in PBS by ward early phagosomes. vortexing and gentle sonication. After one wash, the particles were opso- Regulated changes in the actin and microtubule cytoskeletal net- nized by incubation for1hat37¡C with 2 mg human IgG/mg zymosan. works are required for phagocytosis to proceed normally, and After two subsequent washes, the opsonized particles were counted using by guest on September 24, 2021 the Coulter counter. That the particle suspension was well dispersed was might be necessary also for focal targeting of secretory responses. routinely verified by phase contrast microscopy, and freshly prepared par- Conditions have been reported that produce either centripetal or ticles were used in all experiments. centrifugal movement of lysosomal organelles along microtubules Synchronization of the interaction between neutrophils and zymosan in phagocytes and other cells (17, 18). These include acute treat- was achieved by rapidly sedimenting the cells together with zymosan in a microcentrifuge tube, followed by resuspension. This process was com- ment with cAMP, changes in pH, and others. Such vectorial dis- plete within 30 s. placement, if directed to one pole of the cell, could also contribute to focal exocytosis induced by phagocytic stimuli. Fluorescence microscopy Fixation of neutrophils with 1.6% paraformaldehyde in PBS was initiated Materials and Methods at 4¡C for 30 min and continued at room temperature for 1 h. In some Materials experiments, fixed cells were permeabilized by treatment with a buffer containing 0.01% Triton X-100, 100 mM PIPES (pH 6.8), 5 mM EGTA,

Unlabeled and Texas Red-conjugated zymosan particles, rhodamine phal- 100 mM KOH, and 2 mM MgCl2 for 5 min. Similar results were obtained loidin, and the acetoxymethyl esters of Indo-1 and BAPTA were obtained using higher concentrations of Triton X-100 (up to 0.1%). Blocking was from Molecular Probes (Eugene, OR). RPMI 1640, HEPES, human IgG, performed with 5% donkey serum in PBS for 2 h. After washing with PBS, cytochalasin B, colchicine, fibrinogen, and poly-L-lysine were from Sigma- cells were incubated at room temperature with the indicated primary Ab for Aldrich (St. Louis, MO). Ionomycin was from Calbiochem (La Jolla, CA). 2Ð4 h in PBS containing 1% BSA. Following washing, incubation with Erbstatin analog was obtained from Biomol Research Laboratories (Ply- secondary Ab was for 1 h, also in PBS containing 1% BSA. Cells were mouth Meeting, PA). Powdered PBS was obtained from Pierce (Rockford, adhered to poly-L-lysine-coated coverslips after staining with secondary IL). BSA was from Boehringer Mannheim (Mannheim, Germany). mAbs Ab. After washing, the samples were overlaid with Slow Fade or with to both CD63 (CLB-CD63) and CD66b (CLB-CD66b) were the generous ProLong (Molecular Probes) before mounting. gift of A. J. Verhoeven (Red Cross Blood Transfusion Center, Central To label F-actin, rhodamine phalloidin (6.6 ␮M stock solution in meth- Laboratory of The Netherlands). Rabbit polyclonal anti-peptide Abs raised anol) was evaporated and redissolved in PBS, according to the instructions against calreticulin and SERCA2 were the generous gift of K. H. Krause of the manufacturer (final concentration 165 nM). This staining solution (Division of Infectious Diseases, University Hospital, Geneva, Switzer- was applied to fixed cells for 20 min at room temperature, after which the land). A rabbit polyclonal Ab to calnexin was the generous gift of D. coverslips were washed twice with PBS and mounted for fluorescence Williams (University of Toronto). For some experiments, rabbit polyclonal microscopy. Samples stained with phalloidin or with fluorescent Abs were Abs were precleared of zymosan-reactive Abs by an incubation with zy- analyzed using a Leica (Deerfield, IL) TCS 4D laser confocal microscope. mosan particles for1hat37¡C. mAb to SERCA2 (clone IID8) was ob- At least 100 cells were examined in each experiment. tained from Affinity Bioreagents (Golden, CO). mAbs against phosphoty- Flow cytometry

3 Abbreviations used in this paper: BAPTA, bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-tet- Neutrophils were fixed and stained, as described above for immunofluo- 2ϩ rescence microscopy. After washing, the cells were diluted in PBS and raacetate; [Ca ]i, cytosolic free calcium concentration; MTOC, microtubule- organizing center; PYK2, proline-rich tyrosine kinase 2; OZ, IgG-opsonized zymosan analyzed using a FACScan flow cytometer (BD Biosciences, Mountain particle. View, CA). For every sample, at least 10,000 ungated cells were counted. The Journal of Immunology 5289

Selection of cell populations of interest was done after the acquisition of analyzing single confocal sections (Fig. 1, cf A and D), as well as raw data, using the Lysis II analysis software, as described previously (20). after reconstructing serial sections of the entire cell (Fig. 1, cf B Ͼ Measurement of cytosolic Ca2ϩ concentration and E). Similar results were obtained in 10 preparations using blood from different donors. 2ϩ [Ca ]i was measured using Indo-1 and a Hitachi Model F-4000 spec- trofluorometer, set at 37¡C and equipped with magnetic stirring. Neutro- Role of calcium in the polarized exocytosis of CD63: phils were loaded with Indo-1 by incubation with 2 ␮M of the acetoxy- extracellular vs intracellular sources methyl ester form of the dye for 15 min at 37¡C. After washing twice, 106 cells were allowed to interact with IgG-opsonized zymosan particles (OZ) Calcium is widely accepted to have an essential role in neutrophil by cosedimentation at room temperature. The cells were rapidly resus- granule secretion (3, 6). It is therefore conceivable that the polar- pended in a phosphate-free Na medium at 37¡C, and recording of Indo-1 ized exocytosis of CD63 is generated by a localized increase in fluorescence was initiated within 30 s of cosedimentation. Indo-1 fluores- 2ϩ 2ϩ cence was monitored using excitation and emission wavelengths of 331 Ca . In fact, cytosolic Ca gradients have been reported to form and 410 nm, respectively, and was calibrated using ionomycin and Mn2ϩ, in the vicinity of the phagosome (12). To determine the role of as described (21). A dissociation constant of 250 nM for the indo-1-Ca2ϩ cytosolic Ca2ϩ in the targeting of CD63 toward the forming 2ϩ complex was used to calculate [Ca ]i (22). phagosome, we first defined the source of the divalent cation uti- lized during exocytosis induced by OZ. As illustrated in Fig. Results 2A–C, addition of a calcium ionophore in the absence of OZ suf- Exocytosis of primary and secondary granules during ficed to induce exocytosis, which was substantially inhibited by phagocytosis: detection by confocal immunomicroscopy omission of external Ca2ϩ (Fig. 2G). Importantly, the distribution Exocytosis during the course of FcR-mediated phagocytosis was of CD63 on the surface of ionomycin-activated cells was random, Downloaded from studied using immunocytochemical detection of luminal epitopes indicating that there are no preexisting regions of higher fusogenic on membrane proteins of primary and secondary granules (CD63 activity. Also, when cells were stimulated by phorbol ester ϩ iono- and CD66b, respectively). These epitopes, which are inaccessible mycin or with cytochalasin B ϩ fMLP, was the induced exocytosis in intact, unstimulated cells, become exposed to extracellular Abs nonlocalized (not shown). In contrast to the effects of the iono- upon insertion of the granule membrane into the plasmalemma (5). phore, exocytosis of CD63 in response to OZ was not only re-

Using this approach, we had earlier shown that the secretory re- stricted to the membrane of the forming phagosome, but was http://www.jimmunol.org/ sponse induced by IgG-opsonized particles precedes the formation largely independent of the presence of extracellular Ca2ϩ (Fig. of sealed phagosomes (20). We now extend these studies, using 2D–G). This suggests that endomembrane stores may be the pri- confocal microscopy, to define the site of exocytosis with respect mary source of the Ca2ϩ required for OZ-induced exocytosis to the forming phagosome. By immunostaining of permeabilized of CD63. neutrophils during various stages of phagocytosis, we could not The increased Ca2ϩ found in the vicinity of the phagosome has demonstrate an obvious accumulation of either primary or second- been attributed to a redistribution of intracellular Ca2ϩ stores, ary granules in the part of the cell closer to the zymosan (not which ostensibly accumulate near the phagosomal membrane (13). shown). However, a rapid fusion of granules with the phagosome Preferential Ca2ϩ release from endomembranes attracted to the might preclude such an accumulation. Next, human neutrophils region of the forming phagosome could readily account for the by guest on September 24, 2021 were exposed for short (1-min) periods to OZ particles, fixed, and observed focalized exocytosis of CD63. We therefore sought to subjected to immunostaining without permeabilization. As shown confirm the redistribution of Ca2ϩ stores following phagocytosis, in Fig. 1C, the site of attachment of OZ, where phagosomes are by analyzing the localization of calnexin, calreticulin, and being formed, can be defined readily by differential interference SERCA2b, acknowledged as resident proteins of the endoplasmic contrast microscopy (Fig. 1, C and F). Shortly after induction of reticulum, the primary endomembrane Ca2ϩ store. Unlike Sten- phagocytosis, CD63 was found to accumulate preferentially in the dahl et al. (13), we repeatedly failed to see accumulation of the vicinity of the opsonized particles (Fig. 1, A and B), while CD66b markers in the vicinity of the phagosome (Fig. 3, A–H). Concen- distributed more homogeneously on the neutrophil surface (Fig. 1, tration of fluorescence around the opsonized particles was only D–F). These differential distribution patterns were evident when seen when crude polyclonal IgG preparations were used (insets in

FIGURE 1. OZ induces differentially localized expression of CD63 and CD66b at the surface mem- brane of human neutrophils. Suspended human neu- trophils were mixed with Texas Red-labeled OZ at a particle-cell ratio of 2:1 and rapidly cosedimented. Cells and particles were resuspended and incubated at 37¡C for 1 min before cooling on ice. This was followed by fixation, immunostaining, and attach- ment of the stained cells onto poly-L-lysine-coated coverslips. Cells were stained with Abs against CD63 (A–C) or CD66b (D–F), followed by FITC- labeled secondary Ab. Single confocal xy-sections are shown in A and D, while composites of serial sections (slices spaced ϳ0.5 ␮m apart) are shown in B and E. The corresponding Nomarski images are shown in C and F. Arrowheads identify OZ in con- tact with cells. No labeling of cells or particles was seen with secondary Ab alone. Bars ϭ 10 ␮m. Sim- ilar results were obtained in at least 10 separate experiments. 5290 LOCALIZED EXOCYTOSIS DURING PHAGOCYTOSIS

FIGURE 2. OZ-triggered primary granule exocyto- sis is not dependent on calcium influx. A–C, Sus- pended human neutrophils were stimulated with iono- mycin (0.5 ␮M, 5 min at 37¡C in calcium-containing Na medium). D–F, Cells were rapidly sedimented with Texas Red-labeled OZ, resuspended, and incu- bated for 1 min at 37¡C in calcium-free Na medium (containing 1 mM EGTA). Stimulation was followed by cooling on ice, fixation, immunostaining, and at- tachment of the stained cells onto poly-L-lysine-coated coverslips. Cells were stained with Abs against CD63, followed by FITC-labeled secondary Ab. Single con- focal xy-sections are shown in A and D, while com- Downloaded from posites of serial sections (slices spaced ϳ0.5 ␮m apart) are shown in B and E. The corresponding No- marski images are shown in C and F. Arrowheads identify OZ in contact with the cells. No labeling of cells or particles was seen with secondary Ab alone. Images are representative of at least five separate ex- http://www.jimmunol.org/ periments. G, Cells were stimulated as above (in Na medium with or without calcium, as indicated) and immunostained for CD63, and their fluorescence was quantified using flow cytometric analysis, as described in Materials and Methods. IgG-Z, IgG-zymosan. Ex- perimental data are expressed as percentage of the highest fluorescence observed in each experiment, and are presented as means of eight separate experiments. by guest on September 24, 2021

Fig. 3, A–F), but not when using mAbs. However, perusal of the of OZ is associated with a large, transient elevation of cytosolic preparations revealed that such staining represented binding of IgG Ca2ϩ (Fig. 4A, upper trace). This increase could be completely to the zymosan particles, and was unrelated to the endoplasmic prevented by prior loading of the cells with the Ca2ϩ-buffering reticulum (notice staining of OZ not associated with cells in the agent BAPTA (Fig. 4B, lower trace). While OZ was ineffective insets of Fig. 3, A–F). Indeed, such staining was eliminated when under these conditions, subsequent addition of ionomycin pro- zymosan was used to preclear the sera, yielding a more purified Ab duced a concentration-dependent, sustained increase in Ca2ϩ (Fig. that produced staining pattern resembling that obtained with the 4B), as anticipated from the finite buffering power of BAPTA. mAb. In view of these data, we failed to demonstrate that the Because the ionophore is likely to distribute homogeneously in the polarized exocytosis of CD63 during phagocytosis is coupled to a ϩ plasmalemma, the Ca2 change recorded should have occurred redistribution of endomembrane Ca2ϩ stores to the vicinity of the diffusely throughout the cell, i.e., without preferential accumula- phagosome. tion of the cation near the forming phagosome. OZ-induced exocytosis in the absence of Ca2ϩ gradients In parallel flow cytometric experiments, pretreatment of the 2ϩ cells with BAPTA was shown to inhibit OZ-triggered expression Even though the redistribution of endomembrane Ca stores can- 2ϩ ϩ of CD63 at the cell surface, even when extracellular Ca was not be detected, localized Ca2 release may nevertheless occur if second messengers such as inositol 1,4,5-trisphosphate are gener- reintroduced (Fig. 4C; see also Fig. 5A). This is consistent with the 2ϩ ated in the vicinity of the forming phagosome. We therefore de- notion that Ca is required for primary granule exocytosis (3, 6). vised an experimental protocol that enabled us to assess the exo- Accordingly, the addition of a low concentration of ionomycin (0.5 cytosis of CD63 in the absence of localized Ca2ϩ release. To this ␮M) to BAPTA-loaded cells restored their ability to up-regulate ϩ end, we obliterated the endogenous changes in Ca2ϩ elicited by CD63 in response to OZ in Ca2 -containing medium (Fig. 4C). OZ, yet allowed exocytosis to occur by artificially elevating Ca2ϩ Importantly, this concentration of the ionophore was a relatively in a diffuse manner, using an ionophore. The data justifying this poor inducer of CD63 exocytosis in cells not exposed to OZ, most approach are compiled in Fig. 4. As reported earlier, phagocytosis likely due to the attenuated magnitude and retarded kinetics of the The Journal of Immunology 5291

FIGURE 3. Calcium stores are not redistributed toward forming phagosomes. Human neutrophils and unlabeled OZ (particle-cell ratio 2:1) were mixed and immediately cosedimented to favor interactions. Af- ter resuspension and incubation for 1 min at 37¡C, the cells were put on ice. This was followed by fix- ation, permeabilization, immunostaining, and attach- ment of the stained cells onto poly-L-lysine-coated coverslips. Cells were stained with rabbit polyclonal Abs against calnexin (A and B), calreticulin (C and D), SERCA (E and F), or a mouse mAb against

SERCA (G and H), followed by the appropriate Cy3- Downloaded from labeled secondary Ab. The leftmost images in A–F show staining obtained with Ab purified against zy- mosan, as described in Materials and Methods, while the smaller images show the staining obtained with the nonpurified Abs. Single confocal xy-sections from the midregion of the cells, and the correspond- ing Nomarski images are shown. Arrowheads iden- http://www.jimmunol.org/ tify OZ. Discrimination of particles from nuclei was aided by additional Nomarski images (not shown). No labeling of cells or particles was seen with sec- ondary Ab alone. Bars ϭ 10 ␮m. Similar results were obtained in at least five separate experiments. by guest on September 24, 2021

Ca2ϩ increase. It therefore appears that the phagocytic event fa- tyrosine accumulation was also clearly observed adjacent to form- cilitated exocytosis of primary granules at lower, otherwise sub- ing OZ phagosomes in neutrophils. The specificity of the immu- threshold Ca2ϩ levels. nostaining is indicated by the inhibitory effects of erbstatin, a As shown in Fig. 5, while little surface CD63 was detectable by tyrosine kinase antagonist (Fig. 6, C and D). immunofluorescence in BAPTA-treated cells exposed to OZ (Fig. Removal of extracellular Ca2ϩ had no discernible effect on the 5, A and B), surface epitope was readily detectable when 0.5 ␮M extent or localization of the phosphotyrosines (Fig. 6, E and F), but ionomycin was also added. Strikingly, the fluorescence was again chelation of intracellular Ca2ϩ with BAPTA greatly reduced the concentrated in the region of the forming phagosomes (Fig. 5C). staining (Fig. 7, A and B). This reduction in phosphotyrosine la- Similar observations were made in three separate experiments. Un- beling was due to the divalent cation-binding properties of BAPTA der the conditions used, any calcium released locally by OZ was and is not due to nonspecific effects of the chelator or the loading neutralized by BAPTA (Fig. 4A). We therefore conclude that ad- process. This was concluded from the observation that staining ditional signals, unrelated to Ca2ϩ, are generated locally by the reappeared upon addition of ionomycin, to impose an elevated FcR, inducing polarized exocytosis of primary granules. Although level of Ca2ϩ (Fig. 7, C and D). Restoration of tyrosine the process is Ca2ϩ dependent, localized calcium gradients may phosphorylation by the ionophore was also detectable by flow cy- not be essential to polarization. However, such gradients could tometry (Fig. 7E). As for CD63 exocytosis, the concentration of certainly contribute to the response in a more physiological setting. ionomycin used was not a potent inducer of tyrosine phosphory- lation in the absence of OZ (Fig. 7E). Remarkably, the reappear- Accumulation of phosphotyrosine is spatially restricted and ance of phosphotyrosine accumulation was restricted to the nas- calcium dependent cent phagosome (Fig. 7C), despite the diffuse influx in Ca2ϩ Phosphotyrosine accumulation near phagosomes has been demon- caused by ionomycin. This observation parallels the effects de- strated previously (14). Because tyrosine phosphorylation is scribed for CD63 exocytosis and suggests that the focal accumu- thought to be an important early event in FcR-mediated signaling lation of phosphotyrosine is an important step in dictating the po- (15, 16), it could be a determinant of the spatial localization of larization of exocytosis. It also implies that a localized signal exocytosis in neutrophils. As shown in Fig. 6, A and B, phospho- promotes the Ca2ϩ-dependent accumulation of phosphotyrosine 5292 LOCALIZED EXOCYTOSIS DURING PHAGOCYTOSIS

FIGURE 5. Calcium influx is permissive for localized primary granule Downloaded from exocytosis triggered by OZ. BAPTA-loaded neutrophils were cosedi- mented with Texas Red-labeled OZ at a particle-cell ratio of 2:1, resus- pended, and incubated at 37¡C for 1 min before cooling on ice. A and B, FIGURE 4. Azurophilic granule exocytosis can be prevented by the Calcium-containing Na medium was added 5 min before cosedimentation buffering of intracellular calcium. [Ca2ϩ] was measured in neutrophils i of cells and particles. C and D, Calcium-containing Na medium and iono- loaded with Indo-1. The break in the traces indicates the removal of cells mycin (0.5 ␮M) were added 5 min before cosedimentation. After fixation, from the cuvette for mixing with OZ. After rapid cosedimentation, the immunostaining for CD63, and attachment of the stained cells onto poly- http://www.jimmunol.org/ pellet was immediately resuspended and placed in the cuvette, and record- L-lysine-coated coverslips, analysis by confocal microscopy was per- ing was resumed. A, Upper trace, cells stimulated by OZ (particle-cell formed. The xy-sections of cells and their corresponding Nomarski images ratio, 5:1); lower trace, untreated cells. B, Both traces, BAPTA-pretreated are shown in A and C and in B and D, respectively. Arrowheads identify ␮ cells (10 M BAPTA-acetoxymethyl ester, 20 min at 37¡C in calcium-free OZ in contact with cells. No labeling of cells or particles was seen with Na medium containing 1 mM EGTA) were stimulated with OZ as in A. secondary Ab alone. Confocal xy-sections from the midplane of the cells Calcium-containing Na medium (without BAPTA) was added at time of are shown, and these are representative of three separate experiments. stimulation. Arrow and arrowheads denote the addition of 0.5 ␮M(upper trace in B) and 1 ␮M ionomycin, respectively. The traces shown are rep- resentative of three separate experiments. C, Primary granule exocytosis in a characteristic cup. However, failure of actin to polymerize in the BAPTA-loaded cells was quantified after stimulation with either Texas presence of cytochalasin did not preclude the accumulation of by guest on September 24, 2021 Red-labeled OZ (1 min at 37¡C after sedimentation and resuspension of a phosphotyrosine in the region of contact with the particle (Fig. 8, particle-cell mixture, ratio 2:1) and/or ionomycin (0.5 ␮M, 5 min at 37¡C). E and F). More importantly, exocytosis of primary granules per- Stimulation was either in calcium-free Na medium (with 1 mM EGTA) or sisted in cells that associated with particles (Fig. 8, G and H). in calcium-containing Na medium, as indicated. After cooling on ice, cells Remarkably, CD63 was found to accumulate preferentially in the were immunostained for CD63, and their fluorescence was quantified using area of contact with the OZ. Thus, actin polymerization appears flow cytometric analysis, as described in Materials and Methods. IgG-Z, not to be critically required for the phagosomal targeting of pri- IgG-zymosan. Experimental data were expressed as percentage of the high- est fluorescence observed in each experiment, and are presented as means mary granules. of eight separate experiments. Role of microtubules in polarization during exocytosis Microtubules are involved in the intracellular transport and local- near the nascent phagosome. This signal could be a low, barely ization of a variety of organelles, including secretory granules ϩ detectable level of Ca2 -independent tyrosine phosphorylation or (25). Recently, it was demonstrated that polarized exocytosis of another, as yet undefined response. secretory by dendritic cells involves microtubule-me- diated recruitment of granules (26). It was therefore conceivable Role of OZ-triggered actin polymerization in targeting that polarized exocytosis of primary granules during phagocytosis exocytosis is similarly mediated by microtubules. In suspended (nonadherent) Completion of phagocytosis requires the integrity of the actin cy- neutrophils, well-defined microtubules are very difficult to discern toskeleton (23, 24). Because actin polymerizes actively in the re- by immunostaining. However, the localization of the microtubule- gion of the nascent phagosome, we considered the possibility that organizing center (MTOC) can be readily defined. As shown in the may be involved in targeting secretory granules to Fig. 9, A and B, the MTOC was consistently localized very close this region. In cells stained with rhodamine phalloidin, typical ac- to nascent phagosomes in neutrophils. To quantify this phenome- tin cups can be observed during the early stages of OZ internal- non, the cells were arbitrarily subdivided into three regions: the ization, as illustrated in Fig. 8, A and B. As reported, we found that third closest to the forming phagosome, the middle third, and the cytochalasin inhibited phagocytosis and reduced the number of OZ distal third (see Fig. 9G). After staining for tubulin, the location of particles associated with the surface of neutrophils. Nevertheless, the MTOC with respect to the phagosome was defined in 500 cells, because of the cosedimentation protocol utilized, sufficient num- from three separate experiments. The compiled data, shown in Fig. bers of interacting particles could be found for systematic analysis 9G, demonstrate that the MTOC preferentially locates in the im- by confocal immunofluorescence microscopy. Fig. 8, C and D, mediate vicinity of the phagocytic cup. shows that, despite intimate contact between the cell and particle, We next investigated whether the focal accumulation of phos- F-actin staining was reduced and not preferentially concentrated in photyrosine and the localized exocytosis of primary granules were The Journal of Immunology 5293 Downloaded from http://www.jimmunol.org/

FIGURE 6. Phosphotyrosine accumulation close to nascent phago- somes is not caused by calcium influx. Suspended human neutrophils and unlabeled OZ (at a particle-cell ratio of 2:1) were rapidly cosedimented. Cells and particles were resuspended and incubated at 37¡C for 1 min before cooling on ice. The cells were subsequently fixed, permeabilized, and stained with anti-phosphotyrosine Ab and Cy3-labeled secondary Ab. Stained cells were then sedimented onto poly-L-lysine-coated coverslips for analysis by confocal microscopy. Stimulation with particles was per- FIGURE 7. Calcium influx is permissive for localized phosphotyrosine by guest on September 24, 2021 formed in Na medium (A–D) or in calcium-free Na medium containing 1 accumulation. BAPTA-loaded human neutrophils and unlabeled OZ (at a mM EGTA (E and F). C and D, Cells were preincubated with erbstatin (5 particle-cell ratio of 2:1) were rapidly cosedimented. A and B, Calcium- ␮g/ml for1hat37¡C) before stimulation with OZ. No labeling of cells or containing Na medium was added 5 min before cosedimentation of cells particles was seen with secondary Ab alone. Confocal xy-sections from the and particles; C and D, calcium-containing Na medium and ionomycin (0.5 midplane of the cells are shown, together with their corresponding Nomar- ␮M) were added 5 min before cosedimentation. Cells and particles were ski images. Representative of three separate experiments. resuspended and incubated at 37¡C for 1 min before cooling on ice. The cells were subsequently fixed, permeabilized, and stained with anti- phosphotyrosine Ab and Cy3-labeled secondary Ab. Stained cells were microtubule-dependent processes. Initial experiments revealed that then sedimented onto poly-L-lysine-coated coverslips for analysis by con- focal microscopy. The xy-sections of cells and their corresponding Nomar- microtubule-disrupting agents such as colchicine induced a relo- ski images are shown in A and C and in B and D, respectively. Arrowheads cation of the MTOC, which was less frequently associated with identify OZ. No labeling of cells or particles was seen with secondary Ab nascent phagosomes. In fact, the MTOC was often seen on the alone. Confocal xy-sections from the midplane of the cells are shown, and opposite side of the cells (not shown). Despite the disruption of these are representative of three separate experiments. E, Phosphotyrosine microtubules, tyrosine-phosphorylated proteins still accumulated staining of BAPTA-loaded cells was quantified by flow cytometry after close to phagosomes, as determined by immunostaining (Fig. 9, C stimulation with Texas Red-labeled OZ (1 min at 37¡C; particle-cell ratio, and D). However, primary granule exocytosis was much less lo- 2:1) and/or ionomycin (0.5 ␮M, 5 min at 37¡C). Stimulation was either in calized in cells treated with colchicine (Fig. 9, E and F), implying calcium-free Na medium with 1 mM EGTA added, or in calcium-contain- that microtubules may be responsible, at least in part, for targeting ing Na medium, as indicated. After cooling on ice, cells were immuno- the secretory response to the area of the phagocytic cup. stained for phosphotyrosine, and their fluorescence was quantified using flow cytometric analysis, as described in Materials and Methods. Phos- photyrosine fluorescence was normalized, relative to the sample showing Discussion maximal staining within each experimental series. IgG-Z, IgG-zymosan. Role of calcium in targeting of exocytosis in neutrophils Data are means Ϯ SEM of four separate experiments. It is well established that Ca2ϩ triggers exocytosis in neurons and neuroendocrine cells (27), and neutrophil degranulation is gener- ally accepted to be also a Ca2ϩ-dependent process (6). However, at least four types of secretory granules and vesicles exist in neu- ry) granules, specific (secondary) granules, and -like (pri- trophils, and these display distinct profiles of Ca2ϩ sensitivity (3, mary) granules. The subcellular Ca2ϩ gradients reported to appear 6). Secretory vesicles have the lowest Ca2ϩ threshold and are also during the course of phagocytosis in neutrophils (12) could in prin- most readily mobilized in vivo (3, 4). In decreasing order of Ca2ϩ ciple dictate the preferential exocytosis of granules near the phago- sensitivity, neutrophils also possess gelatinase-containing (tertia- some. Moreover, those granules with the lowest Ca2ϩ sensitivity 5294 LOCALIZED EXOCYTOSIS DURING PHAGOCYTOSIS Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 8. Cytochalasin does not prevent localization of either phos- photyrosine accumulation or exocytosis of CD63. Suspended human neu- trophils were mixed with unlabeled OZ at a particle-cell ratio of 2:1 and rapidly cosedimented. Immediately hereafter, cells and particles were re- suspended and incubated at 37¡C for 1 min before cooling on ice. C–H, FIGURE 9. Microtubules/MTOC are required for localized primary Neutrophils were preincubated with cytochalasin B (5 ␮M, 10 min at granule exocytosis, but not for localized phosphotyrosine accumulation. 37¡C), and stimulation was in the continued presence of this drug. Cells Suspended human neutrophils were rapidly cosedimented with unlabeled were subsequently fixed and stained for rhodamine phalloidin (A–D), phos- OZ (particle-cell ratio of 2:1). Immediately hereafter, cells and particles photyrosine (E and F), or CD63 (G and H). Single xy-planes through the were resuspended and incubated at 37¡C for 1 min before cooling on ice. ␮ midplane of the stained cells were obtained by confocal microscopy, and C–F, Cells were preincubated with colchicine (20 M, 10 min at 37¡C), these are shown together with corresponding Nomarski images. Arrow- and stimulation was in the continued presence of this drug. Subsequently, heads identify OZ in contact with cells. Images shown are representative of cells were fixed, permeabilized (A–F), and sedimented onto poly-L-lysine- ␣ three separate experiments. coated coverslips for immunostaining for tyrosine-tubulin (A and B), -tu- bulin (insets in A and B), phosphotyrosine (C and D), or CD63 (E and F). Single xy-planes through the midplane of the stained cells were obtained by would be expected to polarize most effectively, since other gran- confocal microscopy, and these are shown together with corresponding ϩ Nomarski images. Arrowheads identify OZ. Representative of three sepa- ules would be more likely to reach the secretory Ca2 threshold rate experiments. G, Cells stained with ␣-tubulin were divided into three not only near the phagosome, but elsewhere as well. zones, as indicated, and 500 cells were examined by fluorescence micros- 2ϩ The involvement of Ca in polarized exocytosis was tested copy to determine the localization of their MTOC. using BAPTA. As reported before for specific granules (28), exo- cytosis of primary granules induced by opsonized particles was inhibited by chelation of Ca2ϩ. This stresses the differences be- served only after formation of a phagosome (12). Such delayed tween neutrophils and macrophages, in which phagosome-lyso- gradient formation could not account for the rapid polarization of some fusion was recently demonstrated to be a Ca2ϩ-independent primary granule exocytosis reported in this work. Also, migration process (29). Although essential for secretion, Ca2ϩ does not ap- of human neutrophils, a process that is ostensibly dependent on pear to be the main determinant of the polarized exocytosis oc- polarized exocytosis of endomembranes (30), appears to proceed curring during neutrophil phagocytosis of IgG-opsonized zymo- normally when Ca2ϩ gradients are eliminated (31, 32). Jointly, our san, as shown in the present study. It has earlier been shown that results suggest that, while Ca2ϩ plays a permissive role in exocy- the initial Ca2ϩ elevation observed upon cell-particle contact is not tosis, it appears not to be the sole determinant of focal exocytosis localized, and that a nonuniform distribution of Ca2ϩ can be ob- of primary granules in neutrophils. The Journal of Immunology 5295

Role of tyrosine phosphorylation in the targeting of exocytosis supported by the following observations: 1) kinesin, a motor pro- An early event likely to partake in signaling the spatial restriction tein that displaces organelles toward the plus end of microtubules, of primary granule exocytosis is a localized tyrosine phosphory- is found associated with secretory granules of neutrophils (45); 2) lation. Human neutrophils express Fc␥RII (CD32) and Fc␥RIII the MTOC appears to relocate during phagocytosis to the vicinity (CD16), and ligation of these receptors leads to protein tyrosine of the nascent phagosome; and 3) disruption of microtubules with phosphorylation. Kinases of the src family are thought to phos- colchicine, which alters the relocation of the MTOC, is associated phorylate the receptors, which in turn promotes the recruitment with a more disperse pattern of exocytosis. Thus, coalescence of and activation of p72syk. Clustering of this kinase has been shown phagosomes with lysosomes may be facilitated by two concomi- to be a sufficient signal to trigger phagocytosis (15). In accordance tant microtubule-mediated events: centrifugal movement of gran- with an important role for tyrosine phosphorylation in phagocyto- ules and centripetal displacement of sealed phagosomes. Colchi- sis, it has been demonstrated that the tyrosine phosphatase YopH cine, which had been reported earlier to partially inhibit secretion of Yersinia can inhibit its own phagocytic uptake, as well as Fc␥R- in neutrophils (42), may decrease the efficiency of the exocytic mediated phagocytosis of other phagocytic prey (33). process by reducing the rate at which the granules collide with the The initial round of tyrosine phosphorylation triggered by FcR plasma membrane, where they presumably dock and fuse via a leads to the activation of and the subsequent Ca2ϩ soluble N-ethylmaleimide-sensitive factor attachment protein re- release from inositol trisphosphate-sensitive stores. Our experi- ceptor-mediated process. ments revealed that a sizeable fraction of the tyrosine phosphory- Experimental data from yeast and mammalian cell systems (46, lation depended on the elevation of Ca2ϩ. This finding is not nec- 47) suggest that a large protein complex known as the exocyst, Downloaded from essarily incompatible with the sequence of events postulated which includes Sec6/8, plays a role in the polarization of secretory above, but instead reflects the existence of two components of pathways through the recruitment of vesicles to specific domains tyrosine phosphorylation: an early phase that activates phospho- on the plasma membrane. We therefore investigated the localiza- lipase C and is presumably Ca2ϩ independent, and a secondary tion of Sec6/8 in phagocytosing human neutrophils by immuno- phase that requires prior elevation of Ca2ϩ. A component of Ca2ϩ- blotting of plasma membrane and early phagosomal fractions. In dependent tyrosine phosphorylation has been reported earlier in preliminary experiments, no accumulation of Sec6/8 was observed neutrophils (34, 35) and also in platelets (36Ð39). Recently, a in early (2- to 5-min) phagosomal membranes (not shown), sug- http://www.jimmunol.org/ Ca2ϩ-dependent tyrosine kinase, known as proline-rich tyrosine gesting that factors other than the exocyst dictate the targeting of kinase 2 (PYK2), cell adhesion kinase ␤, or related adhesion focal azurophil granules to phagosomes. tyrosine kinase, was described to exist in a variety of cell types, Rapid repositioning of the MTOC upon stimulation has been including neutrophils (31Ð33). This type of nonreceptor tyrosine reported in a few cell types, including macrophages challenged kinase may well be responsible for the secondary, Ca2ϩ-dependent with immobilized immune complexes (48). At present, compara- wave of phosphorylation during phagocytosis and may partake in tively little is known about the underlying mechanism. In T cells, the displacement of the MTOC was found to require extracellular the activation of primary granule exocytosis. This would account 2ϩ 2ϩ calcium and most likely involves elevated cytosolic Ca (49). for the Ca dependence of exocytosis, while some process other by guest on September 24, 2021 than Ca2ϩ changes would accumulate PYK2 near the phagosome. This displacement is of likely relevance for the formation of re- In this regard, it is noteworthy that PYK2 is related to focal ad- ceptor signaling complexes and the activation of tyrosine phos- hesion kinase, a tyrosine kinase that clusters in focal adhesions in phorylation events (50). Also, during ingestion of Chlamydiae, aCa2ϩ-independent manner (40). tyrosine phosphorylation of host cell proteins is required to trigger a microtubule-dependent redistribution of the (51). Of note, phosphorylation of tyrosine residues on tubulin (52), as The role of the cytoskeleton in the targeting of exocytosis well as tubulin tyrosination (53), are felt to be important in mi- Actin polymerization is an absolute requirement for completion of crotubule remodelling. The linkage between FcR engagement and phagocytosis, but the exocytosis induced by opsonized particles the activation of these processes, and their precise role in directed persists when polymerization at the barbed end is prevented by exocytosis remain to be defined. cytochalasin (41). Remarkably, while actin is thought to be a cen- In summary, our findings indicate that in neutrophils, fusion of tral element in the establishment of neutrophil polarity, focal exo- lysosomal granules with the phagosome is an early, vectorial cytosis of CD63 was observed in the presence of cytochalasin, at event. While exocytosis requires calcium, this cation appears not doses that obliterated phagocytosis. Moreover, tyrosine phosphor- to be directly responsible for the polarization of exocytosis, nor is ylation remained concentrated in the area of contact between the de novo actin polymerization required. Instead, the localized acti- OZ particle and the leukocyte. Thus, while actin polymerization vation of tyrosine kinases may promote relocation of the MTOC, and/or cross-linking may be needed for optimal stimulation by FcR possibly directing microtubules toward the phagosome. Kinesin- and for formation of phagosomal cups, the actin cytoskeleton does mediated movement of granules toward the phagosome would then not appear to mediate the targeting of exocytosis to the area of the favor preferential exocytosis at or near the forming phagosome. phagosome. Microtubules had been implicated earlier in the process of pha- Acknowledgments gosomal maturation. Late phagosomes in neutrophils were found We thank Dr. A. J. Verhoeven (Red Cross, The Netherlands) for gener- to localize adjacent to the (42), and movement of phago- ously providing the Abs against CD63 and CD66b. We are also grateful to somes along microtubules was documented directly (43). In addi- Dr. Karl-Heinz Krause (Division of Infectious Diseases, University Hos- tion, fusion of phagosomes with lamp 2-containing vesicles was pital) for generously providing Abs against calreticulin and SERCA2, and shown to be a microtubule-dependent process in J774 cells (44). to Dr. David Williams (University of Toronto) for generously providing Ab Jointly, these findings can be interpreted to mean that fusion of against calnexin. phagosomes with lysosomes is promoted by the centripetal move- ment of the phagosome. Our data suggest that an additional mech- References 1. Witko-Sarsat, V., P. Rieu, B. Descamps-Latscha, P. Lesavre, and anism may be involved, namely the directed movement of secre- L. Halbwachs-Mecarelli. 2000. 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