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FcγR-Mediated Stimulates Localized in Human 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

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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

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 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 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 , which is similarly calcium and phosphatidylinositol Downloaded from 3-kinase dependent, we studied the possible relationship between these events. 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 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 . 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 , 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 , free radical synthesis, cytokine re- rise in the free cytosolic Ca concentration ([Ca ]i) observed lease, and (1, 2). during Fc␥R-mediated phagocytosis (6Ð8). 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 ceptors include Fc␥R, which are responsible for the phagocytosis or (9Ð12). In neutrophils, Fc␥R signaling also causes degranulation. Neu- trophils possess at least four types of secretory organelles: primary *Program in 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 , 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

(16Ð19). 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, , 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 (22Ð24). To test these hypotheses we studied whether pinocytosis medium (140 mM NaCl, 5 mM KCl, 10 mM glucose, 1 mM MgCl2, and 10 mM HEPES, pH 7.4). Where noted, 1 mM EGTA or 1 mM CaCl2 was is, in fact, activated during Fc␥R-mediated phagocytosis and, if so, 2ϩ added to the medium. Calibration of [Ca ]i was accomplished by adding ␮ whether it occurs locally at or near nascent phagosomes. In addi- 10 M ionomycin, followed by 2 mM CaCl2 to attain maximal fluores- tion, we analyzed the signals leading to membrane retrieval. cence, and subsequently 2 mM MnCl2 to quench Indo-1 fluorescence for determination of autofluorescence and scattering. Materials and Methods Intracellular calcium depletion was accomplished by pretreatment of ␮ 2ϩ Reagents cells with 100 nM thapsigargin or 1 M ionomycin in nominally Ca -free medium containing 1 mM EGTA for 25 min at 37¡C before stimulation. Alternatively, cells were pretreated with 10 ␮M BAPTA/AM in Ca2ϩ-free

Cytochalasin B, , PMA, thapsigargin, EGTA, fibronectin, fMLP, Downloaded from and human IgG were obtained from Sigma-Aldrich (St. Louis, MO). Pe- medium containing 1 mM EGTA for 30 min at 37¡C before stimulation. fabloc SC was purchased from Roche (Indianapolis, IN). Ionomycin and wortmannin were obtained from Calbiochem (La Jolla, CA). Zymosan, Lucifer Yellow (LY), Indo-1/AM, and BAPTA-AM were obtiained from Results Molecular Probes (Eugene, OR). Latex beads were purchased from Bangs Fc␥R-mediated phagocytosis stimulates pinocytosis in human Laboratories (Carmel, IN). Mouse anti-CD63 and anti-CD66b Abs were neutrophils obtained from Caltag (San Francisco, CA), Serotec (Oxford, U.K.), and the Hybridoma Developmental Studies Bank (Iowa City, IA). Fluorochrome- Fc␥R-mediated phagocytosis was shown to induce secretion that is http://www.jimmunol.org/ conjugated anti-human and anti-mouse Abs were purchased from Jackson preferentially targeted to the phagocytic cup in macrophages and ImmunoResearch Laboratories (West Grove, PA) and Molecular Probes. neutrophils (20, 21). We therefore analyzed whether localized re- Preparation of human neutrophils and cytoplasts trieval of also occurs during phagocytosis, using hu- man neutrophils as a model system. When unstimulated, these Human neutrophils were isolated from heparinized blood from healthy do- nors by Ficoll-Hypaque gradient centrifugation as previously described cells have a remarkably low rate of spontaneous pinocytosis, fa- (25, 26) or using the 1-Step Polymorph Isolation kit (Accurate Chemical cilitating the detection of stimulation-induced events. Indeed, and Scientific, Westbury, NY). Contaminating RBC were removed by when resting neutrophils were incubated with the fluid-phase NH4Cl lysis when required, and neutrophils were then counted using a marker LY for 15 min, very few neutrophils (Ͻ5%) were visibly Coulter counter (model ZM; Hialeah, FL). Neutrophils were maintained in labeled (not illustrated). Upon exposure to IgG-opsonized beads, by guest on October 1, 2021 either HEPES-buffered RPMI or complete HBSS at room temperature until use, within5hofisolation. When required, cells were washed with Ca2ϩ- distinct LY-containing vesicles were noticeable in many of the

free HBSS supplemented with 1 mM MgCl2. Cytoplasts and karyoplasts cells (Fig. 1). Note that pinocytic vesicles were present in cells were prepared as described previously (27). associated with beads (arrows in Fig. 1), but not in adjacent cells Phagocytosis and pinocytosis assays that failed to bind beads. Pinocytic events were observed as early as 30 s during synchronized phagocytosis and seemed to precede Zymosan and latex beads were opsonized with 1Ð2 mg/ml human IgG for at least 1 h and were washed three times with PBS. Particles were then sealing of the phagosome. As shown in Fig. 1, at the time when added to adherent or suspended neutrophils to initiate phagocytosis. When pinocytosis was clearly discernible (A) the opsonized particles in suspension, cells and particles were rapidly cosedimented by centrifu- were still accessible to Abs added extracellularly (B and inset), gation to synchronize phagocytosis. To observe fluid phase endocytosis implying that phagocytosis was still in progress. It is also note- (pinocytosis) during particle ingestion, phagocytosis proceeded in the pres- worthy that at the early stages the endocytic vesicles accumulated ence of 1 mg/ml LY for the indicated times and was arrested by parafor- maldehyde fixation. The phagocytic index was quantified by counting the in the vicinity of nascent phagosomes, where they were probably number of internalized particles per 100 cells. Pinocytosis was quantified formed. Pinocytosis was also stimulated by IgG-opsonized and by flow cytometry or by measuring the endocytic index, defined as the unopsonized zymosan (not illustrated). The latter suggests that number of neutrophils with at least three distinct LY-labeled vesicles. Dur- mannose- and/or ␤-glucan -mediated phagocytosis can ing quantification of pinocytosis, early time points were employed to min- imize the contribution of phagosome-derived vesicles. likewise induce pinocytosis. As phagosomes sealed, indicated by the inaccessibility of the Confocal microscopy and flow cytometry particles to Abs (Fig. 1, D and inset), the number of LY-containing Following the desired treatment, neutrophils were fixed with 4% parafor- vesicles increased, and they distributed more widely throughout maldehyde for 15 min, and extracellular particles were identified by stain- the cell (Fig. 1C). The continued formation of labeled vesicles may ing with Cy3- or Cy5-conjugated anti-human Abs for 30 min at 1/1000. To represent ongoing pinocytic activity at the cell surface, but may stain for total CD63 and CD66b, cells, cytoplasts, and karyoplasts were permeabilized with 0.1% Triton X-100 for 10 min, followed by blocking alternatively result from fission of membranes from sealed, LY- for 1 h with 5% donkey serum and incubation for 1 h with anti-CD63 or containing phagosomes (29, 30). Accordingly, LY trapped along anti-CD66b mAbs diluted to 1/100 and 1/200, respectively. After washing, with the particles disappeared gradually as the phagosomes the cells were stained using the respective secondary Abs for 1 h, washed, matured. and mounted using mounting medium (DAKO, Carpenteria, CA). Where specified, permeabilization was omitted from the above protocol to detect exofacial CD63 or CD66b. Samples were analyzed using an epifluores- The and Fc␥R-induced endocytosis cence microscope (model DM-IRB; Leica, Rockleigh, NJ) or a LSM 510 laser scanning confocal microscope (Zeiss, New York, NY) equipped with Phagocytosis of IgG-opsonized particles is stringently dependent a ϫ100 oil immersion objective. Images were prepared using Adobe Photo- on remodeling of the actin cytoskeleton and is obliterated by treat- Shop 6.0 and Illustrator 10.0 (Adobe Systems, San Jose, CA). ment with cytochalasins (5). On the other hand, the secretion that The Journal of Immunology 4425

FIGURE 1. Fluid phase pinocytosis in neutrophils during Fc␥R-medi- Downloaded from ated phagocytosis. Human blood neutrophils were allowed to bind and internalize non-synchronously IgG-coated latex beads for 5 min (A and B) or 12 min (C and D) in the presence of LY (1 mg/ml). Cells were then fixed, and bound extracellular particles were identified by staining with Cy5-conjugated anti-human Abs (insets). A and C, Three-dimensional re- constructions of serial confocal slices of LY fluorescence. B and D, Dif- http://www.jimmunol.org/ ferential interference contrast images where the fluorescence images of A and C, respectively, were overlaid. Arrows point to partially engulfed beads, still accessible to external anti-IgG (inset). Arrowheads point to fully internalized beads. The data shown are representative of four exper- iments. Scale bar ϭ 10 ␮m.

accompanies receptor activation in phagocytic cells is, in fact, po- tentiated by cytoskeletal disruption (21). These divergent effects of FIGURE 2. Effects of cytochalasin and colchicine on Fc␥R-induced pi- ␮

cytoskeletal inhibitors provided a means of distinguishing whether nocytosis. Neutrophils were pretreated with 1 M for 5 min by guest on October 1, 2021 endocytosis was tightly linked to either the particle engulfment or (AÐD) or with 10 ␮M colchicine for 20 min (E and F). Subsequently, the secretory processes. cells were allowed to interact with IgG-opsonized beads (A, B, E, and F) Neutrophils were pretreated with cytochalasin B or D and then or IgG-opsonized zymosan (C and D) for 10 min in the presence of LY (1 exposed to IgG-opsonized beads or zymosan in the presence of mg/ml) and fixed. Extracellular particles (arrows) were then detected by staining with Cy5-labeled anti-human Abs (not shown for beads; D for LY. As expected, capping the barbed end of filamentous actin zymosan). In C and D, the outlines of the neutrophil and zymosan particle filaments with cytochalasin virtually eliminated phagocytosis (Fig. were traced and are illustrated. A, C, and E, Three-dimensional reconstruc- 2). Remarkably, the pinocytosis induced by interaction with opso- tions of serial confocal slices of LY fluorescence. B and F, Differential nized particles not only persisted, but was, in fact, more noticeable interference contrast acquisitions onto which A and E, respectively, were than in control cells (Fig. 2, AÐD). Similar results were obtained overlaid. Scale bar ϭ 5 ␮m. Phagocytosis was quantified by counting the whether opsonized latex (Fig. 2, A and B) or opsonized zymosan percentage of neutrophils containing at least one fully internalized particle (Fig. 2, C and D) was used as the phagocytic target. It is notewor- in control (Ctl), cytochalasin B-treated (CB) and colchicine-treated (colch) thy that despite the failure of the cells to ingest particles, the pi- cells (G). Pinocytosis was measured by counting the number of neutrophils nocytic events occurred preferentially in the immediate vicinity of with at least three readily discernible LY-stained vesicles (H). Data are the Ϯ the adherent particles. These observations imply that closure of the mean SE of three separate experiments, each with at least 100 cells counted. phagosomes is not essential for receptor-induced pinocytosis and that actin assembly is not involved in targeting the pinocytic events. The resistance, indeed the potentiation, of pinocytosis observed in cytochalasin-treated cells is consistent with the idea that granule Calcium-induced granule secretion promotes pinocytosis secretion may be associated with the increased fluid phase uptake. In neutrophils, granule secretion can be elicited by artificially in- 2ϩ Because the polarized secretion of primary granules in neutrophils creasing [Ca ]i, bypassing the activation of surface receptors. requires an intact microtubular network, we tested the effects of This strategy was used to further explore the relationship between 2ϩ colchicine on particle-induced pinocytosis (21). We found that the secretion and pinocytosis. As shown in Fig. 3A, [Ca ]i could be efficiency of phagocytosis and pinocytosis decreased somewhat in readily increased beyond the resting physiological level by addi- colchicine-treated cells (Fig. 2, G and H). More importantly, al- tion of ionomycin, a Ca2ϩ ionophore, or thapsigargin, an inhibitor though no systematic quantitation was attempted, it was clear that of sarco(endo)plasmic reticulum calcium ATPase-type Ca2ϩ- the pinocytic vesicles no longer accumulated to the same extent in ATPases (Fig. 3A). The levels attained suffice to induce exocytosis the vicinity of the adherent opsonized particles (Fig. 2, E and F). of all secretory granules and vesicles of human neutrophils (14, 15) Jointly, these observations indicate that while actin-dependent par- (our unpublished observations). Addition of ionomycin (Fig. 3B) ticle engulfment is not required for focal pinocytosis, secretion of and thapsigargin (Fig. 3C) also induced a remarkable burst of fluid granules may play a role in the induction of pinocytosis. phase endocytosis in Ͼ90% and Ն85% of the cells, respectively. 4426 PHAGOCYTOSIS-DEPENDENT PINOCYTOSIS

phores is circumstantial. We therefore sought an approach to more directly test the nature of the relationship between these processes. To this end, we used a preparation of enucleated and degranulated neutrophils, originally developed by Roos et al. (27). Degranulated cell fragments, called cytoplasts, can be obtained by sedimentation of cells through a discontinuous Ficoll density gradient, which induces fission of the cells in two components: a dense karyoplast that contains the nucleus and secretory granules, and a lighter cy- toplast fraction that is enriched in and light membranes, including the plasmalemma. The process is conservative, so that no cellular components are lost, and remarkably the cytoplasts retain the ability to perform phagocytosis and to mount a respira- tory burst (27). The distribution of primary and secondary granules in cytoplasts and karyoplasts is compared with that of intact neu- trophils in Fig. 4. As expected, both primary (CD63) and second- ary granule markers (CD66b) are abundant in intact cells and in- side karyoplasts. By contrast, no CD63 was detectable in cytoplasts, and CD66b was only detectable in the limiting mem- brane of a fraction of the cytoplasts. The appearance of CD66b on Downloaded from the membrane is indicative of some degranulation during the cen- trifugation procedure. We proceeded to test the uptake of LY in cytoplasts. Like intact FIGURE 3. Calcium stimulates and wortmannin inhibits pinocytosis. A, ϩ cells, unstimulated cytoplasts have very low rates of fluid phase Neutrophils were loaded with Indo-1 and suspended in Ca2 -containing medium, and [Ca2ϩ] was determined by spectrofluorometry. Where indi- uptake; we were unable to detect pinocytosis even after 15 min of cated by the arrow, 1 ␮M ionomycin or 100 nM thapsigargin was added. incubation with LY (Fig. 5, A and B). In accordance with the http://www.jimmunol.org/ B and C, Neutrophils suspended in Ca2ϩ-containing medium with LY were results reported by Roos et al. (27), we found that cytoplasts were incubated with either 1 ␮M ionomycin (B) or 100 nM thapsigargin (C) for capable of phagocytosis (Fig. 5D). Opsonized yeast (zymosan) 10 min before fixation. Representative fluorescence images are illustrated. particles were used for these experiments because they are porous Size bars ϭ 5 ␮m. D, Quantitation of pinocytosis in resting neutrophils (C) and capable of trapping fluid phase markers. Indeed, when LY was or in neutrophils stimulated by thapsigargin (T) or ionomycin (I), from present at the time of phagocytosis but removed thereafter, the experiments similar to those in B and C. E, Effect of wortmannin on FcR- probe was found trapped in the phagosome, confirming that sealing induced pinocytosis. Cells were pretreated with (W) or without (C) 100 nM of the phagocytic vacuole had occurred. Importantly, pinocytic wortmannin and then incubated with IgG-opsonized zymosan for 5 min at 37¡C before fixation and analysis by fluorescence microscopy. Ordinate in

D and E, Percentage of cells with at least three readily discernible LY- by guest on October 1, 2021 stained vesicles. The data in D and E are the mean Ϯ SE of three separate experiments, each with at least 100 cells counted.

Of note, in both instances the LY-containing vesicles were homo- geneously dispersed throughout the cell.

Role of PI3K in the induction of pinocytosis Secretion in neutrophils is markedly inhibited by antagonists of PI3K (31Ð33). On the other hand, most endocytic processes are either insensitive or only modestly affected by inhibition of PI3K (34, 35). This enabled us to test the causal relationship between these events. As illustrated in Fig. 3E, pretreatment of the cells with 100 nM wortmannin greatly depressed the formation of LY- containing vesicles in cells stimulated with IgG-coated particles. Wortmannin was also a powerful antagonist of ionomycin-depen- dent pinocytosis (not shown). These effects are unlikely to result from a direct impairment of pinocytosis and could instead be an indirect result of the inhibition of secretion, which may be a nec- essary precursor to the stimulation of fluid phase uptake. Support- ing this idea, wortmannin was shown to block ionomycin-induced exocytosis in pituitary gonadotrophs (36). Nevertheless, a direct inhibitory effect of wortmannin on pinocytosis cannot be dis- counted, since in some systems fluid phase uptake was reportedly inhibited by the PI3K antagonist (37Ð39). FIGURE 4. Cytoplasts are depleted of primary and secondary granules. Granule-deficient cytoplasts do not exhibit phagocytosis- Epifluorescence microscopy images of fixed and permeabilized whole neu- dependent pinocytosis trophils (A and B), cytoplasts (C and D), and karyoplasts (E and F) stained for primary granules with anti-CD63 (A, C, and E) or for secondary gran- While suggestive of a relationship between secretion and pinocy- ules with anti-CD66b (B, D, and F). Insets show corresponding differential tosis, the evidence provided by wortmannin and calcium iono- interference contrast images. Scale bar ϭ 10 ␮m. The Journal of Immunology 4427 Downloaded from http://www.jimmunol.org/

FIGURE 5. Phagocytosis in cytoplasts is not accompanied by pinocy- tosis. Cytoplasts were incubated with LY (1 mg/ml) in the absence of stimulus (A and B), during phagocytosis of IgG-coated zymosan (C and D), or during stimulation with 100 nM PMA for 10 min (E and F). Images in FIGURE 6. Secretion of primary and secondary granules and pinocy- A, C, and E were acquired by epifluorescence microscopy. B, D, and F, ϩ tosis in calcium-depleted cells.A, [Ca2 ] determinations in Indo-1-loaded differential interference contrast images of the samples in A, C, and E, i neutrophils suspended in calcium-free medium. After establishment of respectively. Scale bar ϭ 10 ␮m. 2ϩ basal [Ca ]i, neutrophils were stimulated with 2 mg/ml heat-aggregated IgG (arrowhead, control). Alternatively, 1 ␮M ionomycin (iono) or 100 nM thapsigargin (thaps) was added first (arrow). After 15Ð25 min, when by guest on October 1, 2021 vesicles were not found in cytoplasts up to 10 min after 2ϩ [Ca ]i had returned to or below the baseline level, aggregated IgG was phagocytosis. added as described above (arrowhead). Traces are representative of at least These results suggest that the pinocytosis elicited by FcR cross- three experiments of each kind. B, Quantification of secretion of CD63 by linking requires the presence and probably the secretion of gran- flow cytometry. Neutrophils were left untreated (Ctl) or were treated with ules. However, it is possible that the cytoplast isolation procedure thapsigargin, ionomycin, or BAPTA-AM to deplete or buffer calcium, may have directly impaired their pinocytic ability. This was tested respectively. The cells were then stimulated with opsonized zymosan using PMA, an activator of protein kinase C that is a potent acti- (OPZ ϩ) or were left unstimulated (OPZ Ϫ), as indicated. C, Quantifica- vator of endocytosis in a variety of cells, including phagocytes tion of secretion of CD66b by flow cytometry. Conditions are as described (40Ð42). As illustrated in Fig. 5, E and F, PMA effectively in- in B. D, Quantification of the extent of pinocytosis stimulated by phago- 2ϩ 2ϩ duced LY uptake in cytoplasts. Together, these observations point in the presence of Ca (control; Ctl) or after Ca manipulation, as described above. The endocytic index is defined as the number of neu- to an essential role of degranulation in the stimulation of pinocy- trophils containing at least three LY-positive vesicles. The data in BÐD are tosis by IgG-opsonized particles. Because LY persisted for ex- the mean Ϯ SE of three experiments of each kind. tended periods inside the cytoplast phagosome, we conclude that clearance of soluble phagosomal contents also requires prior fu- sion with secretory organelles and/or occurs by “kiss-and-run” from internal stores, followed by extrusion across the plasma- 2ϩ with such organelles (30). lemma. In both instances, [Ca ]i had returned to baseline within 5 min, implying depletion of the mobilizable Ca2ϩ stores. Accord- Calcium dependence of FcR-induced pinocytosis ingly, subsequent stimulation of FcR (Fig. 6A, arrowhead) failed to 2ϩ 2ϩ Several studies have demonstrated that elevated [Ca ]i is required produce any detectable changes in [Ca ]i, which contrasts with 2ϩ 2ϩ for secretion during neutrophil stimulation (21, 43, 44). Moreover, the sharp [Ca ]i peak induced by aggregation of FcR in Ca 2ϩ detailed analysis of the [Ca ]i dependence of secretion has re- replete cells (Fig. 6A, upper right trace). vealed that the threshold of activation of individual granule types Prior depletion of Ca2ϩ stores decreased the ability of opsonized varies in the order: primary granules secondary granules tertiary particles to induce primary granule secretion (Fig. 6B). Surface granules secretory vesicles (14, 15). We took advantage of the exposure of CD63 in response to FcR clustering assessed by flow 2ϩ Ն known [Ca ]i dependence of exocytosis to verify the relationship cytometry was reduced by 65%. An even more pronounced in- between secretion and the induction of pinocytosis and to try to hibition was obtained in cells loaded with the Ca2ϩ-buffering agent identify the granule types involved. BAPTA. As shown in Fig. 6A, when neutrophils suspended in Ca2ϩ-free Secondary granule secretion assessed by surface exposure of medium were treated with ionomycin or thapsigargin, they under- CD66b was affected by the Ca2ϩ depletion manipulations in a 2ϩ 2ϩ went a transient increase in [Ca ]i, attributable to Ca release different manner (Fig. 6C). First, pretreatment with thapsigargin 4428 PHAGOCYTOSIS-DEPENDENT PINOCYTOSIS alone sufficed to stimulate exocytosis, and an even larger response quential and causally related. We believe that pinocytosis is at least was elicited by ionomycin. This secretion occurred in response to partly dependent on prior secretion, to the extent that degranulated 2ϩ 2ϩ the transient [Ca ]i increase triggered by the Ca -mobilizing cytoplasts failed to form pinocytic vesicles. A similar consecutive and agents (see Fig. 6A). In accordance with this interpretation, no such causal relationship between secretion and endocytosis has been pos- effect was induced by BAPTA. These findings are in good agree- tulated for and endocrine cells (16Ð18, 50). It is currently 2ϩ ment with the lower [Ca ]i threshold for activation of secondary unclear whether soluble contents or membrane-associated compo- granules (15, 45, 46). Subsequent stimulation of the depleted cells nents of the secretory granules are the factors that prompt endocytosis. with opsonized particles produced an additional stimulation that, Transmembrane proteins of the granules may serve as nucleation sites although reduced, brought the total secretion of CD66b to levels for the assembly of endocytic coats, such as clathrin. On the other similar to or higher than those recorded in control cells (Fig. 6C). hand, proteases or other released from the granules may Despite extensive secretion of CD66b during ionomycin- or induce pinocytosis by cleaving exofacial membrane components. In thapsigargin-mediated Ca2ϩ depletion (Fig. 6C), the rate of pino- this regard, protease inhibitors have been reported by several authors cytosis in such cells was virtually unaffected when particulate to inhibit phagocytosis (Refs. 48Ð50 and our own unpublished ob- stimuli were omitted (Fig. 6D). However, subsequent addition of servations using Pefabloc). opsonized particles to Ca2ϩ-depleted cells induced a sizable in- What is the functional purpose of pinocytosis during Fc␥R-me- crease in pinocytosis, although the maximal rates attained were diated phagocytosis? Pinocytosis may have a role in recycling lower than in Ca2ϩ-replete cells (Fig. 6D). Together, these results membrane components such as soluble N-ethylmaleimide sensitive suggest that pinocytosis correlates well with the secretion of pri- factor attachment receptors for use in subsequent rounds of secre- mary, but not secondary, granules. tion. While this may not be a critical response in neutrophils, Downloaded from which have a short biological half-life, it may play an important Discussion role in the case of macrophages. Alternatively, pinocytosis during Our results demonstrate that Fc␥R-mediated phagocytosis signals Fc␥R-mediated phagocytosis may participate in the initiation of pinocytic uptake at phagocytic sites. The stimulation of pinocyto- the inflammatory response or in Ag processing and presentation (3, sis occurs before and independently of phagosome formation, 51). The latter is a critical aspect of function (52) and since 1) vesicles trapping LY were clearly discernible in cells with is also observed in neutrophils treated with GM-CSF, IL-3, or unsealed phagocytic cups; and 2) pretreatment of cells with cy- IFN-␥, which express MHC class II and can activate T cells both http://www.jimmunol.org/ tochalasin abolished phagocytosis, yet greatly stimulated pino- in vitro and in vivo (53Ð57). Lastly, it is possible that the mem- some formation. Therefore, while fission of these vesicles may be brane fission observed during phagocytosis represents an early akin to that mediating phagosome maturation, the phenomenon stage of phagosome maturation. This premature remodeling would reported here clearly precedes phagosome sealing and remodeling. be exacerbated when phagocytosis is frustrated in cytochalasin- While not requiring completion of phagocytosis, LY-stained en- treated cells. Indeed, a proportion of the LY-containing vesicles dosomes were nevertheless formed predominantly on or very near detected in our experiments may have originated during the mat- the patch of membrane juxtaposed to the opsonized particle. These uration of sealed phagosomes. Both clathrin and protein

results are consistent with observations that clathrin, amphiphysin I, which have been shown to contribute to phagosomal recycling by guest on October 1, 2021 II, and -2 localize to phagocytic cups (22Ð24). Moreover, (58, 59), could contribute to budding of the observed vesicles. when exposed to soluble immune complexes, Fc␥R undergo re- Possible additional mechanisms include -like structures or ceptor-mediated endocytosis, a key process in Ag processing and other membrane coats perhaps related to sorting nexins (60, 61). in the genesis of inflammation (3, 47). By analogy, it is conceiv- Alternatively, fluid may have exchanged during the process of able that cross-linking of Fc␥R by the opsonized particles initiates “kiss-and-run” (30), whereby secretory organelles, probably in- receptor-mediated endocytosis. However, this would require de- cluding primary granules, would transiently fuse with the phago- tachment of the IgG from the opsonized particle or disengagement somal membrane. of the receptor-ligand complex, which entails cessation of signal- In summary, we have described the induction of focal pinocy- ing. We regard this mechanism as improbable because, unlike re- tosis at sites of phagosome formation. Such endocytosis often pre- ceptor-mediated endocytosis, pinosome formation required eleva- cedes and is independent of phagosome sealing and correlates with tion of cytosolic calcium and was sensitive to inhibitors of PI3K the localized secretion of primary granules. The signals that trigger and because it was absent in cytoplasts. pinocytosis may be generated by cross-linking of Fc␥R, but com- Instead, our results suggest that pinocytosis was coupled to the ponents delivered to the membrane by exocytosis also appear to be occurrence of exocytosis. In accordance with this idea, stimulation essential, to the extent that cytoplasts are capable of phagocytosis, of secretion with calcium ionophore promoted extensive pinosome yet fail to activate pinocytosis. The functional significance of the formation. Moreover, the enhancement of pinocytosis noted in accelerated pinocytic uptake remains to be defined, but a role in the cells treated with cytochalasin is reminiscent of the stimulation of early stages of phagosome maturation appears likely. In this re- secretion that this drug induces in neutrophils (48). Our results, in gard, it would be of interest to monitor phagosomal maturation in addition, point to a central role of primary (lysosomal) granules in cytoplasts, where the initial fission events appear to be lacking. the induction of pinocytosis. Briefly, the calcium sensitivity profile (Fig. 6) and the preferential occurrence of pinocytosis in the im- References mediate vicinity of the phagosomal cup (Figs. 1 and 2) closely 1. Witko-Sarsat, V., P. Rieu, B. Descamps-Latscha, P. Lesavre, and L. Halbwachs- parallel the established behavior of primary granules (14, 21). Of Mecarelli. 2000. Neutrophils: molecules, functions and pathophysiological as- note, Fittschen and Henson (49) previously reported that endocy- pects. Lab. Invest. 80:617. tosis can also be triggered in neutrophils by chemotactic peptides 2. Zhang, P., W. R. Summer, G. J. Bagby, and S. Nelson. 2000. Innate immunity and pulmonary host defense. Immunol. Rev. 173:39. and that it correlates with primary granule secretion. 3. Sylvestre, D. L., and J. V. Ravetch. 1994. Fc receptors initiate the Arthus reac- The coincident occurrence of fluid phase endocytosis and secretion tion: redefining the inflammatory cascade. Science 265:1095. may reflect parallel, yet independent, events, which may share com- 4. Greenberg, S. 1999. Modular components of phagocytosis. J. Leukocyte Biol. 66:712. mon signaling elements and are therefore similarly sensitive to phar- 5. May, R. C., and L. M. Machesky. 2001. Phagocytosis and the actin cytoskeleton. macological interventions. On the other hand, the events may be se- J. Cell Sci. 114:1061. The Journal of Immunology 4429

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