FcγR-Mediated Phagocytosis Stimulates
Localized Pinocytosis in Human Neutrophils
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J Immunol 2002; 169:4423-4429; ;
doi: 10.4049/jimmunol.169.8.4423
http://www.jimmunol.org/content/169/8/4423
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References
Roberto J. Botelho, Hans Tapper, Wendy Furuya, Donna
Mojdami and Sergio Grinstein
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*
P
rofessional phagocytes, comprised of monocytes, macrophages, and neutrophils, are key to the innate immune defense system and, by removing apoptotic bodies, also contribute to tissue remodeling. Neutrophils often mount the initial
response to infection because of their rapid chemotactic response
toward bacterial peptides and inflammatory cytokines. Upon
reaching the infected area, neutrophils curb pathogen activity by
ingestion of microorganisms, free radical synthesis, cytokine release, and degranulation (1, 2).
The antimicrobial responses of phagocytes are triggered by surface receptors that recognize either conserved patterns on the surface of microorganisms or opsonins that coat them. The latter receptors include Fc␥R, which are responsible for the phagocytosis
*Program in Cell Biology, Hospital for Sick Children, and Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada; and †Department of Cell and
Molecular Biology, Section for Molecular Pathogenesis, Lund University, Lund,
Sweden
Received for publication March 6, 2002. Accepted for publication August 2, 2002.
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
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by the Canadian Institutes for Health Research, the
Arthritis Society of Canada, the Arthritis Center of Excellence, the Sanatorium Association, a Canadian Institutes for Health Research Graduate Studentship (to R.J.B.),
and the Swedish Medical Research Council (Grants 12182, 12613, and 7480), The
Magnus Bergvall Foundation, The Crafoord Foundation, The Greta and Johan Kock
Foundation, The Kungliga Fysiografiska Sallskapet, and The Alfred Osterlund Foundation (to H.T.).
2
R.J.B. and H.T. contributed equally to this work.
3
Address correspondence and reprint requests to Dr. Sergio Grinstein, Program in
Cell Biology, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario,
Canada M5G 1X8. E-mail address: [email protected]
4
S.G. is a Canadian Institutes of Health Research Distinguished Scientist and the
current holder of the Pitblado Chair in Cell Biology at Hospital for Sick Children.
Cross-appointed to the Department of Biochemistry of University of Toronto.
Copyright © 2002 by The American Association of Immunologists, Inc.
of IgG-opsonized particles (1, 3, 4). Particle engulfment is triggered by Fc␥R clustering, which induces localized activation of
Src family and Syk tyrosine kinases at the phagocytic cup. These
initial events are followed by stimulation of phosphatidylinositol
3-kinase (PI3K)5 and phospholipase C␥ (4, 5), which hydrolyses
phosphatidylinositol-4,5-bisphosphate into diacylglycerol and inositol-1,4,5-trisphosphate. The latter mediator is responsible for the
rise in the free cytosolic Ca⫹2 concentration ([Ca⫹2]i) observed
during Fc␥R-mediated phagocytosis (6 – 8). Rac and Cdc42, members of the Rho family of small GTPases, are then activated and
coordinate actin remodeling at the sites of phagocytosis, culminating in the engulfment of the microbe into an intracellular vacuole
or phagosome (9 –12).
In neutrophils, Fc␥R signaling also causes degranulation. Neutrophils possess at least four types of secretory organelles: primary
(azurophilic), secondary (specific), and tertiary (gelatinase) granules and secretory vesicles (1, 13). Primary granules are enriched
in lysosomal hydrolases and myeloperoxidase, and they can be
identified by the presence of CD63 on their membrane. Secondary
granules contain lactoferrin and lysozyme and express CD66b on
their membrane. Tertiary granules contain gelatinase, while secretory vesicles are rich in albumin and alkaline phosphatase (1).
These organelles do not necessarily undergo secretion simultaneously, since the signals leading to their exocytosis differ in type
and/or activation threshold (14, 15).
Exocytosis of multiple types of secretory organelles contributes
additional surface area to the target membrane. In other systems
that undergo similar acute and vigorous secretion, such as chromaffin cells, the net area of the membrane is maintained approximately constant by the concomitant activation of endocytosis
5
Abbreviations used in this paper: PI3K, phosphatidylinositol 3-kinase; LY, Lucifer
Yellow; [Ca2⫹]i, cytosolic free Ca2⫹ concentration.
0022-1767/02/$02.00
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Engulfment of IgG-coated particles by neutrophils and macrophages is an essential component of the innate immune response.
This process, known as phagocytosis, is triggered by clustering of Fc␥R at sites where leukocytes make contact with the opsonized
particles. We found that phagocytosis is accompanied by a burst of fluid phase pinocytosis, which is largely restricted to the
immediate vicinity of the phagosomal cup. Fc␥R-induced pinocytosis preceded and appeared to be independent of phagosomal
sealing. Accordingly, fluid phase uptake was accentuated by actin depolymerization, which precludes phagocytosis. Stimulation of
pinocytosis required phosphatidylinositol 3-kinase activity and was eliminated when changes in the cytosolic free Ca2ⴙ concentration were prevented. Because stimulation of Fc␥R also induces secretion, which is similarly calcium and phosphatidylinositol
3-kinase dependent, we studied the possible relationship between these events. Neutrophil fragments devoid of secretory granules
(cytoplasts) were prepared by sedimentation through Ficoll gradients. Cytoplasts could perform Fc␥R-mediated phagocytosis,
which was not accompanied by activation of pinocytosis. This observation suggests that granule exocytosis is required for stimulation of pinocytosis. Analysis of the cytosolic Ca2ⴙ dependence of secretion and pinocytosis suggests that primary (lysosomal)
granule exocytosis is the main determinant of pinocytosis during Fc␥R stimulation. Importantly, primary granules are secreted
in a polarized fashion near forming phagosomes. Focal pinocytosis during particle engulfment may contribute to Ag processing
and presentation and/or to retrieval of components of the secretory machinery. Alternatively, it may represent an early event in
the remodeling of the phagosomal membrane, leading to phagosomal maturation. The Journal of Immunology, 2002, 169: 4423– 4429.
4424
(16 –19). Endocytosis also serves to retrieve components of the
secretory machinery to be reused in subsequent rounds of stimulation. For these reasons, endocytosis (pinocytosis) is also likely to
be activated during Fc␥R-mediated phagocytosis. It is noteworthy,
however, that, unlike chromaffin cells, phagocytes are capable of
focal secretion during particle engulfment, targeting the secreted
material to the area of the plasma membrane where phagosomes
are being generated or to the lumen of formed phagosomes (20,
21). It is therefore conceivable that localized signals may, in fact,
trigger focal pinocytosis during phagocytosis. Indeed, clathrin, dynamin, and amphiphysin were detected around the phagosomal cup
(22–24). To test these hypotheses we studied whether pinocytosis
is, in fact, activated during Fc␥R-mediated phagocytosis and, if so,
whether it occurs locally at or near nascent phagosomes. In addition, we analyzed the signals leading to membrane retrieval.
Materials and Methods
Reagents
Preparation of human neutrophils and cytoplasts
Human neutrophils were isolated from heparinized blood from healthy donors by Ficoll-Hypaque gradient centrifugation as previously described
(25, 26) or using the 1-Step Polymorph Isolation kit (Accurate Chemical
and Scientific, Westbury, NY). Contaminating RBC were removed by
NH4Cl lysis when required, and neutrophils were then counted using a
Coulter counter (model ZM; Hialeah, FL). Neutrophils were maintained in
either HEPES-buffered RPMI or complete HBSS at room temperature until
use, within 5 h of isolation. When required, cells were washed with Ca2⫹free HBSS supplemented with 1 mM MgCl2. Cytoplasts and karyoplasts
were prepared as described previously (27).
Phagocytosis and pinocytosis assays
Zymosan and latex beads were opsonized with 1–2 mg/ml human IgG for
at least 1 h and were washed three times with PBS. Particles were then
added to adherent or suspended neutrophils to initiate phagocytosis. When
in suspension, cells and particles were rapidly cosedimented by centrifugation to synchronize phagocytosis. To observe fluid phase endocytosis
(pinocytosis) during particle ingestion, phagocytosis proceeded in the presence of 1 mg/ml LY for the indicated times and was arrested by paraformaldehyde fixation. The phagocytic index was quantified by counting the
number of internalized particles per 100 cells. Pinocytosis was quantified
by flow cytometry or by measuring the endocytic index, defined as the
number of neutrophils with at least three distinct LY-labeled vesicles. During quantification of pinocytosis, early time points were employed to minimize the contribution of phagosome-derived vesicles.
Confocal microscopy and flow cytometry
Following the desired treatment, neutrophils were fixed with 4% paraformaldehyde for 15 min, and extracellular particles were identified by staining with Cy3- or Cy5-conjugated anti-human Abs for 30 min at 1/1000. To
stain for total CD63 and CD66b, cells, cytoplasts, and karyoplasts were
permeabilized with 0.1% Triton X-100 for 10 min, followed by blocking
for 1 h with 5% donkey serum and incubation for 1 h with anti-CD63 or
anti-CD66b mAbs diluted to 1/100 and 1/200, respectively. After washing,
the cells were stained using the respective secondary Abs for 1 h, washed,
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 epifluorescence microscope (model DM-IRB; Leica, Rockleigh, NJ) or a LSM 510
laser scanning confocal microscope (Zeiss, New York, NY) equipped with
a ⫻100 oil immersion objective. Images were prepared using Adobe PhotoShop 6.0 and Illustrator 10.0 (Adobe Systems, San Jose, CA).
Endocytic uptake of LY and secretion of CD63/CD66b were quantified
using a FACScan flow cytometer (BD Biosciences, Mountain View, CA).
Preparation of samples was performed as described above, but cells were
diluted in PBS and maintained in suspension. For every sample, at least
10,000 ungated cells were counted. Selection of the population of interest
was performed after the acquisition of raw data using LYSIS II analysis
software as described previously (28).
Spectrofluorometry and calcium manipulations
[Ca2⫹]i was quantified by spectrofluorometry using Indo-1 as previously
described (26, 28). Briefly, neutrophils were loaded with 1 ␮M Indo-1/AM
for 30 min at 37°C, washed, and maintained in HCO3⫺-free, Ca2⫹-free
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
added to the medium. Calibration of [Ca2⫹]i was accomplished by adding
10 ␮M ionomycin, followed by 2 mM CaCl2 to attain maximal fluorescence, and subsequently 2 mM MnCl2 to quench Indo-1 fluorescence for
determination of autofluorescence and scattering.
Intracellular calcium depletion was accomplished by pretreatment of
cells with 100 nM thapsigargin or 1 ␮M ionomycin in nominally Ca2⫹-free
medium containing 1 mM EGTA for 25 min at 37°C before stimulation.
Alternatively, cells were pretreated with 10 ␮M BAPTA/AM in Ca2⫹-free
medium containing 1 mM EGTA for 30 min at 37°C before stimulation.
Results
Fc␥R-mediated phagocytosis stimulates pinocytosis in human
neutrophils
Fc␥R-mediated phagocytosis was shown to induce secretion that is
preferentially targeted to the phagocytic cup in macrophages and
neutrophils (20, 21). We therefore analyzed whether localized retrieval of membranes also occurs during phagocytosis, using human neutrophils as a model system. When unstimulated, these
cells have a remarkably low rate of spontaneous pinocytosis, facilitating the detection of stimulation-induced events. Indeed,
when resting neutrophils were incubated with the fluid-phase
marker LY for 15 min, very few neutrophils (⬍5%) were visibly
labeled (not illustrated). Upon exposure to IgG-opsonized beads,
distinct LY-containing vesicles were noticeable in many of the
cells (Fig. 1). Note that pinocytic vesicles were present in cells
associated with beads (arrows in Fig. 1), but not in adjacent cells
that failed to bind beads. Pinocytic events were observed as early
as 30 s during synchronized phagocytosis and seemed to precede
sealing of the phagosome. As shown in Fig. 1, at the time when
pinocytosis was clearly discernible (A) the opsonized particles
were still accessible to Abs added extracellularly (B and inset),
implying that phagocytosis was still in progress. It is also noteworthy that at the early stages the endocytic vesicles accumulated
in the vicinity of nascent phagosomes, where they were probably
formed. Pinocytosis was also stimulated by IgG-opsonized and
unopsonized zymosan (not illustrated). The latter suggests that
mannose- and/or ␤-glucan receptor-mediated phagocytosis can
likewise induce pinocytosis.
As phagosomes sealed, indicated by the inaccessibility of the
particles to Abs (Fig. 1, D and inset), the number of LY-containing
vesicles increased, and they distributed more widely throughout
the cell (Fig. 1C). The continued formation of labeled vesicles may
represent ongoing pinocytic activity at the cell surface, but may
alternatively result from fission of membranes from sealed, LYcontaining phagosomes (29, 30). Accordingly, LY trapped along
with the particles disappeared gradually as the phagosomes
matured.
The cytoskeleton and Fc␥R-induced endocytosis
Phagocytosis of IgG-opsonized particles is stringently dependent
on remodeling of the actin cytoskeleton and is obliterated by treatment with cytochalasins (5). On the other hand, the secretion that
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Cytochalasin B, colchicine, PMA, thapsigargin, EGTA, fibronectin, fMLP,
and human IgG were obtained from Sigma-Aldrich (St. Louis, MO). Pefabloc 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
Molecular Probes (Eugene, OR). Latex beads were purchased from Bangs
Laboratories (Carmel, IN). Mouse anti-CD63 and anti-CD66b Abs were
obtained from Caltag (San Francisco, CA), Serotec (Oxford, U.K.), and the
Hybridoma Developmental Studies Bank (Iowa City, IA). Fluorochromeconjugated anti-human and anti-mouse Abs were purchased from Jackson
ImmunoResearch Laboratories (West Grove, PA) and Molecular Probes.
PHAGOCYTOSIS-DEPENDENT PINOCYTOSIS
The Journal of Immunology
4425
accompanies receptor activation in phagocytic cells is, in fact, potentiated by cytoskeletal disruption (21). These divergent effects of
cytoskeletal inhibitors provided a means of distinguishing whether
endocytosis was tightly linked to either the particle engulfment or
secretory processes.
Neutrophils were pretreated with cytochalasin B or D and then
exposed to IgG-opsonized beads or zymosan in the presence of
LY. As expected, capping the barbed end of filamentous actin
filaments with cytochalasin virtually eliminated phagocytosis (Fig.
2). Remarkably, the pinocytosis induced by interaction with opsonized particles not only persisted, but was, in fact, more noticeable
than in control cells (Fig. 2, A–D). Similar results were obtained
whether opsonized latex (Fig. 2, A and B) or opsonized zymosan
(Fig. 2, C and D) was used as the phagocytic target. It is noteworthy that despite the failure of the cells to ingest particles, the pinocytic events occurred preferentially in the immediate vicinity of
the adherent particles. These observations imply that closure of the
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
secretion may be associated with the increased fluid phase uptake.
Because the polarized secretion of primary granules in neutrophils
requires an intact microtubular network, we tested the effects of
colchicine on particle-induced pinocytosis (21). We found that the
efficiency of phagocytosis and pinocytosis decreased somewhat in
colchicine-treated cells (Fig. 2, G and H). More importantly, although no systematic quantitation was attempted, it was clear that
the pinocytic vesicles no longer accumulated to the same extent in
the vicinity of the adherent opsonized particles (Fig. 2, E and F).
Jointly, these observations indicate that while actin-dependent particle engulfment is not required for focal pinocytosis, secretion of
granules may play a role in the induction of pinocytosis.
FIGURE 2. Effects of cytochalasin and colchicine on Fc␥R-induced pinocytosis. Neutrophils were pretreated with 1 ␮M cytochalasin B for 5 min
(A–D) or with 10 ␮M colchicine for 20 min (E and F). Subsequently, the
cells were allowed to interact with IgG-opsonized beads (A, B, E, and F)
or IgG-opsonized zymosan (C and D) for 10 min in the presence of LY (1
mg/ml) and fixed. Extracellular particles (arrows) were then detected by
staining with Cy5-labeled anti-human Abs (not shown for beads; D for
zymosan). In C and D, the outlines of the neutrophil and zymosan particle
were traced and are illustrated. A, C, and E, Three-dimensional reconstructions of serial confocal slices of LY fluorescence. B and F, Differential
interference contrast acquisitions onto which A and E, respectively, were
overlaid. Scale bar ⫽ 5 ␮m. Phagocytosis was quantified by counting the
percentage of neutrophils containing at least one fully internalized particle
in control (Ctl), cytochalasin B-treated (CB) and colchicine-treated (colch)
cells (G). Pinocytosis was measured by counting the number of neutrophils
with at least three readily discernible LY-stained vesicles (H). Data are the
mean ⫾ SE of three separate experiments, each with at least 100 cells
counted.
Calcium-induced granule secretion promotes pinocytosis
In neutrophils, granule secretion can be elicited by artificially increasing [Ca2⫹]i, bypassing the activation of surface receptors.
This strategy was used to further explore the relationship between
secretion and pinocytosis. As shown in Fig. 3A, [Ca2⫹]i could be
readily increased beyond the resting physiological level by addition of ionomycin, a Ca2⫹ ionophore, or thapsigargin, an inhibitor
of sarco(endo)plasmic reticulum calcium ATPase-type Ca2⫹ATPases (Fig. 3A). The levels attained suffice to induce exocytosis
of all secretory granules and vesicles of human neutrophils (14, 15)
(our unpublished observations). Addition of ionomycin (Fig. 3B)
and thapsigargin (Fig. 3C) also induced a remarkable burst of fluid
phase endocytosis in ⬎90% and ⱖ85% of the cells, respectively.
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FIGURE 1. Fluid phase pinocytosis in neutrophils during Fc␥R-mediated 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 reconstructions of serial confocal slices of LY fluorescence. B and D, Differential 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 experiments. Scale bar ⫽ 10 ␮m.
4426
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 cytoplast fraction that is enriched in cytosol 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 respiratory burst (27). The distribution of primary and secondary granules
in cytoplasts and karyoplasts is compared with that of intact neutrophils in Fig. 4. As expected, both primary (CD63) and secondary granule markers (CD66b) are abundant in intact cells and inside karyoplasts. By contrast, no CD63 was detectable in
cytoplasts, and CD66b was only detectable in the limiting membrane of a fraction of the cytoplasts. The appearance of CD66b on
the membrane is indicative of some degranulation during the centrifugation procedure.
We proceeded to test the uptake of LY in cytoplasts. Like intact
cells, unstimulated cytoplasts have very low rates of fluid phase
uptake; we were unable to detect pinocytosis even after 15 min of
incubation with LY (Fig. 5, A and B). In accordance with the
results reported by Roos et al. (27), we found that cytoplasts were
capable of phagocytosis (Fig. 5D). Opsonized yeast (zymosan)
particles were used for these experiments because they are porous
and capable of trapping fluid phase markers. Indeed, when LY was
present at the time of phagocytosis but removed thereafter, the
probe was found trapped in the phagosome, confirming that sealing
of the phagocytic vacuole had occurred. Importantly, pinocytic
Of note, in both instances the LY-containing vesicles were homogeneously dispersed throughout the cell.
Role of PI3K in the induction of pinocytosis
Secretion in neutrophils is markedly inhibited by antagonists of
PI3K (31–33). 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 LYcontaining vesicles in cells stimulated with IgG-coated particles.
Wortmannin was also a powerful antagonist of ionomycin-dependent 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 necessary precursor to the stimulation of fluid phase uptake. Supporting 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 discounted, since in some systems fluid phase uptake was reportedly
inhibited by the PI3K antagonist (37–39).
Granule-deficient cytoplasts do not exhibit phagocytosisdependent pinocytosis
While suggestive of a relationship between secretion and pinocytosis, the evidence provided by wortmannin and calcium iono-
FIGURE 4. Cytoplasts are depleted of primary and secondary granules.
Epifluorescence microscopy images of fixed and permeabilized whole neutrophils (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 granules with anti-CD66b (B, D, and F). Insets show corresponding differential
interference contrast images. Scale bar ⫽ 10 ␮m.
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FIGURE 3. Calcium stimulates and wortmannin inhibits pinocytosis. A,
Neutrophils were loaded with Indo-1 and suspended in Ca2⫹-containing
medium, and [Ca2⫹] was determined by spectrofluorometry. Where indicated by the arrow, 1 ␮M ionomycin or 100 nM thapsigargin was added.
B and C, Neutrophils suspended in Ca2⫹-containing medium with LY were
incubated with either 1 ␮M ionomycin (B) or 100 nM thapsigargin (C) for
10 min before fixation. Representative fluorescence images are illustrated.
Size bars ⫽ 5 ␮m. D, Quantitation of pinocytosis in resting neutrophils (C)
or in neutrophils stimulated by thapsigargin (T) or ionomycin (I), from
experiments similar to those in B and C. E, Effect of wortmannin on FcRinduced pinocytosis. Cells were pretreated with (W) or without (C) 100 nM
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 LYstained vesicles. The data in D and E are the mean ⫾ SE of three separate
experiments, each with at least 100 cells counted.
PHAGOCYTOSIS-DEPENDENT PINOCYTOSIS
The Journal of Immunology
vesicles were not found in cytoplasts up to 10 min after
phagocytosis.
These results suggest that the pinocytosis elicited by FcR crosslinking requires the presence and probably the secretion of granules. However, it is possible that the cytoplast isolation procedure
may have directly impaired their pinocytic ability. This was tested
using PMA, an activator of protein kinase C that is a potent activator of endocytosis in a variety of cells, including phagocytes
(40 – 42). As illustrated in Fig. 5, E and F, PMA effectively induced LY uptake in cytoplasts. Together, these observations point
to an essential role of degranulation in the stimulation of pinocytosis by IgG-opsonized particles. Because LY persisted for extended periods inside the cytoplast phagosome, we conclude that
clearance of soluble phagosomal contents also requires prior fusion with secretory organelles and/or occurs by “kiss-and-run”
with such organelles (30).
Calcium dependence of FcR-induced pinocytosis
Several studies have demonstrated that elevated [Ca2⫹]i is required
for secretion during neutrophil stimulation (21, 43, 44). Moreover,
detailed analysis of the [Ca2⫹]i dependence of secretion has revealed that the threshold of activation of individual granule types
varies in the order: primary granules secondary granules tertiary
granules secretory vesicles (14, 15). We took advantage of the
known [Ca2⫹]i dependence of exocytosis to verify the relationship
between secretion and the induction of pinocytosis and to try to
identify the granule types involved.
As shown in Fig. 6A, when neutrophils suspended in Ca2⫹-free
medium were treated with ionomycin or thapsigargin, they underwent a transient increase in [Ca2⫹]i, attributable to Ca2⫹ release
FIGURE 6. Secretion of primary and secondary granules and pinocytosis in calcium-depleted cells. A, [Ca2⫹]i determinations in Indo-1-loaded
neutrophils suspended in calcium-free medium. After establishment of
basal [Ca2⫹]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 15–25 min, when
[Ca2⫹]i had returned to or below the baseline level, aggregated IgG was
added as described above (arrowhead). Traces are representative of at least
three experiments of each kind. B, Quantification of secretion of CD63 by
flow cytometry. Neutrophils were left untreated (Ctl) or were treated with
thapsigargin, ionomycin, or BAPTA-AM to deplete or buffer calcium,
respectively. The cells were then stimulated with opsonized zymosan
(OPZ ⫹) or were left unstimulated (OPZ ⫺), as indicated. C, Quantification of secretion of CD66b by flow cytometry. Conditions are as described
in B. D, Quantification of the extent of pinocytosis stimulated by phagocytosis in the presence of Ca2⫹ (control; Ctl) or after Ca2⫹ manipulation,
as described above. The endocytic index is defined as the number of neutrophils containing at least three LY-positive vesicles. The data in B–D are
the mean ⫾ SE of three experiments of each kind.
from internal stores, followed by extrusion across the plasmalemma. In both instances, [Ca2⫹]i had returned to baseline within
5 min, implying depletion of the mobilizable Ca2⫹ stores. Accordingly, subsequent stimulation of FcR (Fig. 6A, arrowhead) failed to
produce any detectable changes in [Ca2⫹]i, which contrasts with
the sharp [Ca2⫹]i peak induced by aggregation of FcR in Ca2⫹
replete cells (Fig. 6A, upper right trace).
Prior depletion of Ca2⫹ stores decreased the ability of opsonized
particles to induce primary granule secretion (Fig. 6B). Surface
exposure of CD63 in response to FcR clustering assessed by flow
cytometry was reduced by ⱖ65%. An even more pronounced inhibition was obtained in cells loaded with the Ca2⫹-buffering agent
BAPTA.
Secondary granule secretion assessed by surface exposure of
CD66b was affected by the Ca2⫹ depletion manipulations in a
different manner (Fig. 6C). First, pretreatment with thapsigargin
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FIGURE 5. Phagocytosis in cytoplasts is not accompanied by pinocytosis. 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
A, C, and E were acquired by epifluorescence microscopy. B, D, and F,
differential interference contrast images of the samples in A, C, and E,
respectively. Scale bar ⫽ 10 ␮m.
4427
4428
alone sufficed to stimulate exocytosis, and an even larger response
was elicited by ionomycin. This secretion occurred in response to
the transient [Ca2⫹]i increase triggered by the Ca2⫹-mobilizing
agents (see Fig. 6A). In accordance with this interpretation, no such
effect was induced by BAPTA. These findings are in good agreement with the lower [Ca2⫹]i threshold for activation of secondary
granules (15, 45, 46). Subsequent stimulation of the depleted cells
with opsonized particles produced an additional stimulation that,
although reduced, brought the total secretion of CD66b to levels
similar to or higher than those recorded in control cells (Fig. 6C).
Despite extensive secretion of CD66b during ionomycin- or
thapsigargin-mediated Ca2⫹ depletion (Fig. 6C), the rate of pinocytosis in such cells was virtually unaffected when particulate
stimuli were omitted (Fig. 6D). However, subsequent addition of
opsonized particles to Ca2⫹-depleted cells induced a sizable increase in pinocytosis, although the maximal rates attained were
lower than in Ca2⫹-replete cells (Fig. 6D). Together, these results
suggest that pinocytosis correlates well with the secretion of primary, but not secondary, granules.
Our results demonstrate that Fc␥R-mediated phagocytosis signals
pinocytic uptake at phagocytic sites. The stimulation of pinocytosis occurs before and independently of phagosome formation,
since 1) vesicles trapping LY were clearly discernible in cells with
unsealed phagocytic cups; and 2) pretreatment of cells with cytochalasin abolished phagocytosis, yet greatly stimulated pinosome formation. Therefore, while fission of these vesicles may be
akin to that mediating phagosome maturation, the phenomenon
reported here clearly precedes phagosome sealing and remodeling.
While not requiring completion of phagocytosis, LY-stained endosomes were nevertheless formed predominantly on or very near
the patch of membrane juxtaposed to the opsonized particle. These
results are consistent with observations that clathrin, amphiphysin
II, and dynamin-2 localize to phagocytic cups (22–24). Moreover,
when exposed to soluble immune complexes, Fc␥R undergo receptor-mediated endocytosis, a key process in Ag processing and
in the genesis of inflammation (3, 47). By analogy, it is conceivable that cross-linking of Fc␥R by the opsonized particles initiates
receptor-mediated endocytosis. However, this would require detachment of the IgG from the opsonized particle or disengagement
of the receptor-ligand complex, which entails cessation of signaling. We regard this mechanism as improbable because, unlike receptor-mediated endocytosis, pinosome formation required elevation of cytosolic calcium and was sensitive to inhibitors of PI3K
and because it was absent in cytoplasts.
Instead, our results suggest that pinocytosis was coupled to the
occurrence of exocytosis. In accordance with this idea, stimulation
of secretion with calcium ionophore promoted extensive pinosome
formation. Moreover, the enhancement of pinocytosis noted in
cells treated with cytochalasin is reminiscent of the stimulation of
secretion that this drug induces in neutrophils (48). Our results, in
addition, point to a central role of primary (lysosomal) granules in
the induction of pinocytosis. Briefly, the calcium sensitivity profile
(Fig. 6) and the preferential occurrence of pinocytosis in the immediate vicinity of the phagosomal cup (Figs. 1 and 2) closely
parallel the established behavior of primary granules (14, 21). Of
note, Fittschen and Henson (49) previously reported that endocytosis can also be triggered in neutrophils by chemotactic peptides
and that it correlates with primary granule secretion.
The coincident occurrence of fluid phase endocytosis and secretion
may reflect parallel, yet independent, events, which may share common signaling elements and are therefore similarly sensitive to pharmacological interventions. On the other hand, the events may be se-
quential and causally related. We believe that pinocytosis is at least
partly dependent on prior secretion, to the extent that degranulated
cytoplasts failed to form pinocytic vesicles. A similar consecutive and
causal relationship between secretion and endocytosis has been postulated for neurons and endocrine cells (16 –18, 50). It is currently
unclear whether soluble contents or membrane-associated components of the secretory granules are the factors that prompt endocytosis.
Transmembrane proteins of the granules may serve as nucleation sites
for the assembly of endocytic coats, such as clathrin. On the other
hand, proteases or other enzymes released from the granules may
induce pinocytosis by cleaving exofacial membrane components. In
this regard, protease inhibitors have been reported by several authors
to inhibit phagocytosis (Refs. 48 –50 and our own unpublished observations using Pefabloc).
What is the functional purpose of pinocytosis during Fc␥R-mediated phagocytosis? Pinocytosis may have a role in recycling
membrane components such as soluble N-ethylmaleimide sensitive
factor attachment receptors for use in subsequent rounds of secretion. While this may not be a critical response in neutrophils,
which have a short biological half-life, it may play an important
role in the case of macrophages. Alternatively, pinocytosis during
Fc␥R-mediated phagocytosis may participate in the initiation of
the inflammatory response or in Ag processing and presentation (3,
51). The latter is a critical aspect of macrophage function (52) and
is also observed in neutrophils treated with GM-CSF, IL-3, or
IFN-␥, which express MHC class II and can activate T cells both
in vitro and in vivo (53–57). Lastly, it is possible that the membrane fission observed during phagocytosis represents an early
stage of phagosome maturation. This premature remodeling would
be exacerbated when phagocytosis is frustrated in cytochalasintreated cells. Indeed, a proportion of the LY-containing vesicles
detected in our experiments may have originated during the maturation of sealed phagosomes. Both clathrin and coatomer protein
I, which have been shown to contribute to phagosomal recycling
(58, 59), could contribute to budding of the observed vesicles.
Possible additional mechanisms include caveolae-like structures or
other membrane coats perhaps related to sorting nexins (60, 61).
Alternatively, fluid may have exchanged during the process of
“kiss-and-run” (30), whereby secretory organelles, probably including primary granules, would transiently fuse with the phagosomal membrane.
In summary, we have described the induction of focal pinocytosis at sites of phagosome formation. Such endocytosis often precedes and is independent of phagosome sealing and correlates with
the localized secretion of primary granules. The signals that trigger
pinocytosis may be generated by cross-linking of Fc␥R, but components delivered to the membrane by exocytosis also appear to be
essential, to the extent that cytoplasts are capable of phagocytosis,
yet fail to activate pinocytosis. The functional significance of the
accelerated pinocytic uptake remains to be defined, but a role in the
early stages of phagosome maturation appears likely. In this regard, it would be of interest to monitor phagosomal maturation in
cytoplasts, where the initial fission events appear to be lacking.
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