Am J Physiol Cell Physiol 296: C857–C867, 2009.
First published January 28, 2009; doi:10.1152/ajpcell.00454.2008.
Counterregulation of clathrin-mediated endocytosis by the actin and
microtubular cytoskeleton in human neutrophils
Silvia M. Uriarte,1* Neelakshi R. Jog,2* Gregory C. Luerman,3 Samrath Bhimani,1 Richard A. Ward,1
and Kenneth R. McLeish1,3,4
Departments of 1Medicine, 2Microbiology and Immunology, and 3Biochemistry and Molecular Biology, University
of Louisville, and 4Veterans Affairs Medical Center, Louisville, Kentucky
Submitted 2 September 2008; accepted in final form 24 January 2009
exocytosis; signal transduction; intracellular granules
ENDOCYTOSIS is a cell-type and cargo-specific mechanism by
which the plasma membrane and material from the external
environment are internalized (9, 46). There are two broad
mechanisms of endocytosis: phagocytosis and pinocytosis.
Phagocytosis is used by specialized cells, including macrophages, monocytes, and neutrophils, to clear mircoorganisms
and other large opsonized particles. Four mechanisms of pinocytosis have been described: macropinocytosis, clathrin-mediated endocytosis, caveolae-mediated endocytosis, and clathrinand caveolae-independent endocytosis (9). Macropinocytosis
is a mechanism by which relatively large amounts of extracellular fluid and solutes are nonspecifically internalized. Consti-
* S. M. Uriarte and N. R. Jog contributed equally to this work.
Address for reprint requests and other correspondence: S. M. Uriarte,
Kidney Disease Program, Baxter I Research Bldg., 570 S. Preston St., Louisville, KY 40202 (e-mail: [email protected]).
http://www.ajpcell.org
tutive and ligand-stimulated clathrin-mediated endocytosis occurs in all mammalian cells and is essential for the uptake of
key nutrients, such as iron-laden transferrin (9). Ligand-stimulated clathrin-mediated endocytosis results in the localization
of transmembrane receptors and their bound ligand in areas of
the plasma membrane known as “coated pits,” the main constituent of which is clathrin. These coated pits are internalized
followed by scission from the plasma membrane and release of
clathrin-coated vesicles into the cytoplasm (9, 23, 53). Caveolae are plasma membrane invaginations formed from cholesterol and sphingolipid-rich microdomains, termed lipid rafts,
that are morphologically different from clathrin-coated pits.
The mechanisms of clathrin- and caveolin-independent endocytosis are unknown (25, 26). These diverse endocytic pathways function to control a large number of cell processes,
including immune surveillance and innate immunity, antigen
presentation, nutrient uptake, neurotransmitter release, signal
transduction, and protein degradation and trafficking (9, 45,
46, 48).
Actin reorganization has been shown to be required for both
phagocytosis and pinocytosis. When particles or opsonized
pathogens are engulfed by phagocytosis, pseudopod extensions
of the plasma membrane are driven by actin polymerization to
encircle the phagocytic target (29, 32). Similarly, actin reorganization is required to form the membrane protrusions leading to macropinocytosis (9). Reorganization of the actin cytoskeleton is postulated to be required at multiple steps of
clathrin-mediated endocytosis, including providing the spatial
organization of the endocytic machinery, inducing the curvature of the plasma membrane at coated pits, driving the separation of early clathrin-coated endosomes from the plasma
membrane, inducing scission of the endosome, and propelling
endosome internalization (9, 16, 23, 31). Most, but not all,
studies (6, 14, 41) have reported that pharmacological inhibition of actin reorganization prevented endocytosis.
Neutrophils are the primary cellular components of innate
immunity. These cells perform multiple forms of endocytosis,
including phagocytosis of pathogens and pinocytosis via clathrin-dependent and -independent mechanisms. We (22) have
recently reported that disruption of the actin cytoskeleton
enhanced the rate and extent of granule exocytosis after stimulation of neutrophils with N-formylmethionyl-leucyl-phenylalanine (fMLP). On the other hand, plasma membrane expression of a marker of secretory vesicles, complement receptor 1
(CR1), was reduced. The present study was initiated to deterThe costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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Uriarte SM, Jog NR, Luerman GC, Bhimani S, Ward RA,
McLeish KR. Counterregulation of clathrin-mediated endocytosis by
the actin and microtubular cytoskeleton in human neutrophils. Am J
Physiol Cell Physiol 296: C857–C867, 2009. First published January
28, 2009; doi:10.1152/ajpcell.00454.2008.—We have recently reported that disruption of the actin cytoskeleton enhanced N-formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated granule exocytosis in human neutrophils but decreased plasma membrane expression
of complement receptor 1 (CR1), a marker of secretory vesicles. The
present study was initiated to determine if reduced CR1 expression
was due to fMLP-stimulated endocytosis, to determine the mechanism
of this endocytosis, and to examine its impact on neutrophil functional
responses. Stimulation of neutrophils with fMLP or ionomycin in the
presence of latrunculin A resulted in the uptake of Alexa fluor
488-labeled albumin and transferrin and reduced plasma membrane
expression of CR1. These effects were prevented by preincubation of
the cells with sucrose, chlorpromazine, or monodansylcadaverine
(MDC), inhibitors of clathrin-mediated endocytosis. Sucrose, chlorpromazine, and MDC also significantly inhibited fMLP- and ionomycin-stimulated specific and azurophil granule exocytosis. Disruption
of microtubules with nocodazole inhibited endocytosis and azurophil
granule exocytosis stimulated by fMLP in the presence of latrunculin
A. Pharmacological inhibition of phosphatidylinositol 3-kinase, ERK1/2, and
PKC significantly reduced fMLP-stimulated transferrin uptake in the
presence of latrunculin A. Blockade of clathrin-mediated endocytosis
had no significant effect on fMLP-stimulated phosphorylation of
ERK1/2 in neutrophils pretreated with latrunculin A. From these data,
we conclude that the actin cytoskeleton functions to limit microtubule-dependent, clathrin-mediated endocytosis in stimulated human
neutrophils. The limitation of clathrin-mediated endocytosis by actin
regulates the extent of both specific and azurophilic granule exocytosis.
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CLATHRIN-MEDIATED ENDOCYTOSIS IN HUMAN NEUTROPHILS
mine if reduced CR1 expression was due to enhanced fMLPstimulated endocytosis after disruption of the actin cytoskeleton, to determine the mechanism of this endocytosis, and to
examine its impact on neutrophil functional responses. The
results show that disruption of the actin cytoskeleton allows
microtubule-dependent, clathrin-mediated endocytosis in stimulated human neutrophils and that this endocytosis participates
in exocytosis of specific and azurophilic granules.
MATERIALS AND METHODS
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RESULTS
Disruption of the actin cytoskeleton enhances clathrin-mediated endocytosis. To determine if disruption of the actin
cytoskeleton resulted in enhanced endocytosis in fMLP-stimulated neutrophils, Alexa fluor-488-conjugated BSA uptake
was measured by flow cytometry. As shown in Fig. 1A,
pretreatment of neutrophils with 1 ␮M latrunculin A, a concentration that disrupts basal and fMLP-stimulated F-actin
(22), resulted in a significant increase in Alexa fluor-488conjugated BSA uptake after stimulation with 300 nM fMLP
compared with untreated cells. Neither fMLP nor latrunculin A
alone induced significant BSA uptake, indicating that both
disruption of the actin cytoskeleton and a subsequent stimulus
were necessary to observe endocytosis of BSA by this method.
Transferrin binding to its receptor induces receptor internalization through a clathrin-mediated mechanism (9, 54), and
endocytosis of ligand-bound chemoattractant receptors in human neutrophils is clathrin dependent (30, 50, 59). Clathrinmediated endocytosis has been reported to be inhibited by
hypertonicity (10, 21), MDC (1, 7, 42, 47), and chlorpromazine
(1, 56). To determine if the combination of disruption of the
actin cytoskeleton and fMLP stimulation induced clathrinmediated endocytosis, transferrin uptake was measured in
neutrophils incubated with or without hypertonic sucrose,
chlorpromazine, or MDC before latrunculin A treatment and
fMLP stimulation. As shown in Fig. 1B, latrunculin A pretreatment, followed by stimulation with fMLP, resulted in a signif-
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Reagents. Alexa fluor 488-conjugated BSA, Alexa fluor 488conjugated transferrin, and latrunculin A were from Molecular Probes
(Eugene, OR). Cytochalasin D, sucrose, chlorpromazine, monodansylcadaverine (MDC), nystatin, nocodazole, calphostin C, genistein,
PP2, ionomycin, and fMLP were from Sigma (St. Louis, MO). The
p38 MAPK inhibitor SB-203580 and the MEK1/2 inhibitor PD098059 were from Calbiochem (La Jolla, CA). The phosphatidylinositol 3-kinase (PI3K) inhibitor LY-294002 was from BioMol (Plymouth Meeting, PA). Phospho-ERK1/2 and total ERK1/2 antibodies
were from Santa Cruz Biotechnology (Santa Cruz, CA). FITC-conjugated monoclonal anti-CR1 (CD35) and mouse IgG1 were from
Pharmingen (San Diego, CA). FITC-conjugated monoclonal antiCD66b was from Accurate Chemical (Westbury, NY). FITC-conjugated monoclonal anti-CD63 was from Ancell (Bayport, MN).
Neutrophil isolation. Neutrophils were isolated from the blood of
healthy donors using plasma-Percoll gradients as previously described
(20). After isolation, neutrophils were washed and resuspended in
bacterial LPS-free Krebs-Ringer phosphate buffer (pH 7.2) containing
0.2% dextrose (Krebs). Microscopic evaluation of isolated cells
showed that ⬎97% of cells were neutrophils. Trypan blue exclusion
indicated that ⬎97% cells were viable. The Institutional Review
Board of the University of Louisville approved the use of human
donors.
Endocytosis. To quantify the internalization of BSA or transferrin
receptors, neutrophils were incubated with 2 ␮g/ml Alexa fluor
488-conjugated BSA or 5 ␮g/ml Alexa fluor 488-conjugated transferrin in the presence and absence of 1 ␮M latrunculin A or 30 ␮M
cytochalasin D for 30 min at 37°C followed by stimulation with or
without 300 nM fMLP for 3 min or 250 nM ionomycin for 15 min.
Cells were then washed with 1 mM sodium azide, fixed in paraformaldehyde, and analyzed for internalized Alexa fluor 488 by flow cytometry using mean channel fluorescence intensity as a quantitative measure
of uptake. Aliquots of cells were also imaged by a Zeiss Axiovert 100 M
confocal microscope with a Zeiss Plan-Neofluar ⫻100/1.3 oil-immersion
lens using LSM510 (version 3.2) software. The excitation wavelength
from an argon laser was set at 488 nm and emission was set at 505 nm.
Exocytosis. Exocytosis of secretory vesicles, specific granules, and
azurophil granules was determined by measuring an increase in
plasma membrane expression of CR1 (also known as CD35), CD66b,
and CD63, respectively, using flow cytometry as previously described
(22, 58). Specific granule exocytosis was also determined by measuring lactoferrin release into neutrophil supernatants using an in-house
ELISA. NUNC immunoplates were coated with anti-lactoferrin antibody (1:1,000 dilution of a 4 mg/ml preparation, MP Biomedicals,
Aurora, OH) at 37°C. The plate was washed and then blocked with
1% BSA in BBL FTA hemagglutination buffer (BD, Sparks, MD) at
4°C. Lactoferrin standards (Sigma) were made by serial dilution in 1%
BSA. Diluted samples and standards were assayed in triplicate. Wells
were loaded and incubated at 37°C for 1 h, washed, and then
incubated for 1 h at 37°C with peroxidase-conjugated anti-lactoferrin
(ICN, Aurora, OH). Wells were washed again and exposed to peroxidase substrate for 3 min. Reactions were stopped with 8 N H2SO4.
Plates were read on a Spectramax plate reader at 490 nm using a
log-logit standard curve. Azurophil granule exocytosis was also determined by measuring myeloperoxidase (MPO) release into neutro-
phil supernatants using an ELISA (Assay Designs, Ann Arbor, MI)
per the manufacturer’s instructions.
Colocalization of neutrophil granule markers and transferrin.
Neutrophils (5 ⫻ 106 cells/ml) were incubated with 5 ␮g/ml Alexa
fluor 488-conjugated transferrin in the presence of 1 ␮M latrunculin A
for 30 min at 37°C followed by stimulation with 300 nM fMLP for 3
min. Cells were washed with Krebs solution, fixed in 3.7% paraformaldehyde for 15 min at room temperature, permeabilized with 2% saponin
for 15 min at room temperature, and then incubated with phycoerythrinconjugated CR1, CD66b, or CD63 (final concentration: 8 ␮g/ml). Cells
were imaged by a Zeiss Axiovert 100 M confocal microscope with a
Zeiss Plan-Neofluar ⫻100/1.3 oil-immersion lens using LSM510 (version 3.2) software.
Western blot analysis. Neutrophils (1 ⫻ 107 cells/ml) were incubated for 30 min at 37°C in the presence or absence of 225 mM
sucrose, 40 ␮M chlorpromazine, or 350 ␮M MDC and 1 ␮M latrunculin A, followed by stimulation with 300 nM fMLP for 1 min.
Stimulation was ended by centrifugation at 2,500 g for 20 s, and
pelleted cells were lysed in 100 ␮l of ice-cold lysis buffer [20 mM
Tris 䡠 HCl (pH 7.5), 150 mM NaCl, 1% (vol/vol) Triton X-100, 0.5%
(vol/vol) Nonidet P-40, 20 mM NaF, 20 mM NaVO3, 1 mM EDTA,
1 mM EGTA, 5 mM PMSF, 21 ␮g/ml aprotinin, and 5 ␮g/ml
leupeptin]. Samples were centrifuged at 15,000 g for 15 min at 4°C.
Equal amounts of protein (50 ␮g) were separated by 10% SDS-PAGE
and subjected to standard immunoblot procedures. Blots were probed
with phospho-ERK1/2 (1:1,000) or total ERK1/2 (1:1,000) in 1% milk
and Tris-buffered saline-Tween 20. The appropriate secondary antibodies were used at 1:2,000. Protein signals were visualized by
enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ) as directed by the manufacturer. Densitometry analysis of
the Western blot bands was performed with ImageQuant software
(Molecular Dynamics, Sunnyvale, CA).
Statistical analysis. All data are expressed as means ⫾ SE. Statistical analysis was performed using ANOVA with the Tukey-Kramer
multiple-comparison test. Differences were considered statistically
significant when P ⱕ 0.05.
CLATHRIN-MEDIATED ENDOCYTOSIS IN HUMAN NEUTROPHILS
C859
icant increase in Alexa fluor-488-conjugated transferrin uptake, which was blocked by incubation with hypertonic sucrose, chlorpromazine, or MDC for 30 min. These data
demonstrate that clathrin-mediated endocytosis was enhanced
by disruption of the actin cytoskeleton and that all three
inhibitors (hypertonic sucrose, chlorpromazine, and MDC)
effectively inhibited clathrin-mediated endocytosis in human
neutrophils. To determine if clathrin-mediated endocytosis was
also responsible for the previously observed albumin uptake,
neutrophils were incubated with hypertonic sucrose, chlorpromazine, or MDC before latrunculin A treatment and fMLP
stimulation. As shown in Fig. 1A, incubation with hypertonic
sucrose, chlorpromazine, or MDC for 30 min completely
inhibited fMLP-stimulated internalization of albumin in latrunculin A-treated cells. Additionally, albumin and transferrin
uptake were observed by confocal microscopy. These observations showed that labeled albumin and transferrin were
internalized, not associated with the plasma membrane, when
neutrophils pretreated with latrunculin A were stimulated with
fMLP (see Fig. 3, for example). Moreover, incubation of
neutrophils with hypertonic sucrose, chlorpromazine, or MDC
significantly prevented internalization of labeled albumin and
transferrin, consistent with the flow cytometric data (data not
shown).
Nystatin, a cholesterol-aggregating drug that blocks clathrinindependent endocytosis (44, 50), failed to alter fMLP-stimulated albumin or transferrin uptake (data not shown). Neutrophils pretreated with 30 ␮M cytochalasin D, which disrupts the
actin cytoskeleton by a mechanism different from that of
latrunculin A, also induced a significant increase in Alexa
fluor-488-conjugated transferrin uptake after stimulation with
fMLP (data not shown).
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Clathrin-mediated endocytosis regulates neutrophil granule
exocytosis. To determine if endocytosis plays a role in fMLPstimulated exocytosis, neutrophils were incubated with or
without latrunculin A before stimulation with fMLP in the
presence or absence of hypertonic sucrose, chlorpromazine, or
MDC. As reported previously (22), fMLP or latrunculin A
alone stimulated a significant increase in plasma membrane
expression of CR1, a marker of secretory vesicles, whereas
fMLP stimulation of neutrophils pretreated with latrunculin A
resulted in decreased CR1 expression (Fig. 2A). To determine
if that reduction in CR1 expression was due to clathrinmediated endocytosis, we examined CR1 expression in neutrophils stimulated for 10 min by fMLP with or without
latrunculin A pretreatment in the presence or absence of
hypertonic sucrose, chlorpromazine, or MDC. Pretreatment
with hypertonic sucrose, chlorpromazine, or MDC significantly
reduced CR1 expression stimulated by fMLP alone (P ⬍ 0.05),
and chlorpromazine and MDC significantly reduced latrunculin A-stimulated CR1 expression (P ⬍ 0.001 and P ⬍
0.05, respectively). The decrease in CR1 expression stimulated by fMLP plus latrunculin A was prevented by pretreatment with hypertonic sucrose (P ⬍ 0.001), chlorpromazine
(P ⬍ 0.01), and MDC (P ⬍ 0.05). Nystatin had no effect on
CR1 expression under those same conditions (data not
shown). These data suggest that fMLP normally stimulates
secretory vesicle exocytosis, resulting in increased CR1
expression, whereas reuptake of CR1 by clathrin-mediated
endocytosis in stimulated neutrophils is inhibited by an
intact actin cytoskeleton.
Stimulation of neutrophils with fMLP, latrunculin A, or the
combination of the two induced a significant increase in the
expression of CD66b, a marker of specific granules (Fig. 2B).
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Fig. 1. Endocytosis after actin disruption is
clathrin mediated. Neutrophils (4 ⫻ 106
cells/ml) were incubated with or without 225
mM sucrose, 40 ␮M chlorpromazine, 350
␮M monodansylcadaverine (MDC), and/or 1
␮M latrunculin A (Lat) for 30 min before
stimulation with 300 nM N-formylmethionyl-leucyl-phenylalanine (fMLP) for 3 min
in the presence of 2 ␮g/ml of Alexa fluor488-conjugated BSA or 5 ␮g/ml of Alexa
fluor-488-conjugated transferrin. A: results
of flow cytometric measurement of Alexa
fluor-488-conjugated BSA uptake. B: results
of flow cytometric measurement of Alexa
fluor-488-conjugated transferrin uptake. Data are
expressed as means ⫾ SE of the mean channel
fluorescence intensity (mcf) for 3 separate experiments. *P ⬍ 0.05 compared with basal (untreated) conditions; §P ⬍ 0.05 compared with
Lat ⫹ fMLP in control cells.
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CLATHRIN-MEDIATED ENDOCYTOSIS IN HUMAN NEUTROPHILS
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Fig. 2. Endocytosis is required for complement receptor 1 (CR1) internalization and
for specific and azurophil granule exocytosis. Neutrophils (4 ⫻ 106 cells/ml) were
incubated for 30 min in the presence or
absence of 225 mM sucrose, 40 ␮M chlorpromazine, or 350 ␮M MDC, and/or 1 ␮M
Lat followed by stimulation with 300 nM
fMLP for 10 min. Exocytosis of secretory
vesicles, specific granules, and azurophil
granules was determined using flow cytometry to measure the mean channel of fluorescence intensity for CR1, CD66b, and CD63,
respectively, and using ELISA to measure
the release of lactoferrin and myeloperoxidase (MPO) for specific granules and azurophil granules, respectively. A: CR1 expression in the presence or absence of sucrose,
chlorpromazine, or MDC. B: CD66b expression in the presence or absence of sucrose,
chlorpromazine, or MDC. C: lactoferrin release (in ␮g/ml) in the presence or absence
of chlorpromazine or MDC. D: CD63 expression in the presence or absence of sucrose, chlorpromazine, or MDC. E: MPO
release (in ng/ml) in the presence or absence
of chlorpromazine or MDC. Flow cytometric
data shown in A, B, and D are expressed as
means ⫾ SE of mean channel of fluorescence intensity for 5 separate experiments.
Lactoferrin and MPO data are expressed as
means ⫾ SE for 4 separate experiments.
CME, clathrin-mediated endocytosis. *P ⬍
0.05 compared with basal conditions; #P ⬍
0.05 compared with control.
Both chlorpromazine and MDC significantly reduced CD66b
expression stimulated by fMLP, latruculin A, or the two
together, whereas hypertonic sucrose pretreatment had no effect on CD66b expression under any of those conditions (Fig.
2B). To further evaluate specific granule exocytosis, lactoferrin
release was measured in neutrophils stimulated with fMLP,
latrunculin A, or the combination of the two. As shown in Fig.
2C, lactoferrin release was significantly increased in the presence of latrunculin A followed by fMLP stimulation, and both
chlorpromazine and MDC significantly reduced lactoferrin
release. The partial inhibition by chlorpromazine and MDC
suggests that endocytosis contributes to optimal specific granule exocytosis, but is not a necessary component for this
process.
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As previously reported (22), neither fMLP nor latrunculin A
alone increased the expression of CD63, a marker of azurophil
granules (Fig. 2D). Pretreatment with latrunculin A followed
by stimulation with fMLP resulted in a significant increase in
CD63 expression (P ⬍ 0.001). Pretreatment with hypertonic
sucrose or MDC significantly inhibited azurophil granule exocytosis under those conditions. No differences in CD63 expression were observed with chlorpromazine pretreatment. MPO
release from azurophil granules exhibited a pattern similar to
that of CD63 expression (Fig. 2E). A significant increase in
MPO release occurred when neutrophils were pretreated with
latrunculin A before fMLP stimulation. MPO release was
significantly reduced by pretreatment with either chlorpromazine or MDC (Fig. 2E). These results suggest that clathrin-
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CLATHRIN-MEDIATED ENDOCYTOSIS IN HUMAN NEUTROPHILS
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mediated endocytosis is required for optimal azurophil granule
exocytosis.
Colocalization of markers of clathrin-mediated endocytosis
and neutrophil granules. Our flow cytometric data showed that
pretreatment of human neutrophils with latrunculin A followed by
fMLP stimulation enhanced transferrin uptake and reduced CR1
expression. To determine if these events were connected, neutrophils were incubated with latrunculin A before stimulation with
fMLP in the presence of Alexa fluor-488-conjugated transferrin,
stained for CR1, and then examined by confocal microscopy.
Although CR1 was present in the cytosol independent of transferrin, transferrin always colocalized with CR1 (Fig. 3A),
supporting the conclusion that CR1 is internalized by clathrin-mediated endocytosis. To determine if endosomes and
specific or azurophil granules interact during exocytosis,
colocalization of transferrin with CD66b or CD63 was
determined by confocal microscopy after fMLP stimulation
in the presence of latrunculin A. Both CD66b (a marker of
specific granules) and CD63 (a marker of azurophil granules) were detected in the cytosol and at the plasma membrane (Fig. 3, B and C, in red); however, neither granule
marker colocalized with transferrin (Fig. 3, B and C). These
findings do not support endosome-granule fusion as the
mechanism by which endocytosis contributes to exocytosis.
Fig. 3. Actin disruption results in the colocalization of CR1 and transferrin uptake after fMLP stimulation. Neutrophils (5 ⫻ 106 cells/ml) were incubated with
1 ␮M Lat for 30 min, stimulated for 5 min with 300 nM fMLP in the presence of Alexa fluor-488-conjugated transferrin, and then stained with
phycoerythin-conjugated anti-CR1, anti-CD66b, or anti-CD63 as described in MATERIALS AND METHODS. Stained cells were visualized by confocal microscopy.
A: merged confocal image of anti-CR1, shown in red, and transferrrin uptake, shown in green. Colocalization of CR1 and transferrin is shown in yellow.
B: merged confocal image of anti-CD66b staining, shown in red, and transferrin uptake, shown in green. C: merged confocal image of anti-CD63 staining, shown
in red, and transferrin uptake, shown in green.
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Fig. 2.—Continued
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CLATHRIN-MEDIATED ENDOCYTOSIS IN HUMAN NEUTROPHILS
these conditions (Fig. 5, A and C). Inhibition of clathrinmediated endocytosis by chlorpromazine or MDC significantly
reduced CD66b expression stimulated by ionomycin, latrunculin A, or the two together (Fig. 5B). Whereas hypertonic
sucrose significantly reduced ionomycin-induced CD66b expression, it had no effect after the disruption of the actin
cytoskeleton with latrunculin A (Fig. 5B). These data suggest
that the relationship between clathrin-mediated endocytosis
and specific and azurophil granule exocytosis is independent of
endocytosis of ligand-bound receptors.
Clathrin-mediated endocytosis after disruption of the actin
cytoskeleton is dependent on microtubules. Previous studies
(23, 41) concluded that the actin cytoskeleton participated in
endocytosis by providing the force necessary for the deformation of the plasma membrane, for the internalization and
scission of clathrin-coated vesicles, and/or the intracellular
mobility of endocytic vesicles. As our results showed enhanced
clathrin-mediated endocytosis with disruption of the actin
cytoskeleton, we evaluated the role of the microtubule cytoskeleton in neutrophil clathrin-mediated endocytosis. Neutrophils were pretreated with latrunculin A, in the presence or
absence of nocodazole, before stimulation with fMLP in the
presence of Alexa fluor-488-conjugated transferrin. As shown
in Fig. 6, nocodazole significantly inhibited transferrin uptake,
suggesting that microtubules participate in clathrin-mediated
endocytosis in human neutrophils.
The effect of disruption of microtubules with nocodazole on
exocytosis of the different granule subsets is shown in Fig. 7.
Similar to the results with inhibition of endocytosis, pretreatment with nocodazole significantly restored the increase in
CR1expression and blunted the decrease in CD63 expression
Fig. 4. Actin disruption induces Alexa fluor488-conjugated BSA and transferrin uptake
after ionomycin (Iono) stimulation. Neutrophils (4 ⫻ 106 cells/ml) were incubated for
30 min in the presence or absence of 225
mM sucrose, 40 ␮M chlorpromazine, or 350
␮M MDC, and/or 1 ␮M Lat followed by
stimulation with 250 nM Iono for 15 min.
A: uptake of Alexa fluor-488-conjugated
BSA as determined by flow cytometry and
expressed as means ⫾ SE of mean channel
fluorescence intensity for 3 separate experiments. B: uptake of Alexa fluor-488-conjugated transferrin as determined by flow cytometry and expressed as means ⫾ SE of
mean channel fluorescence intensity for 3
separate experiments. *P ⬍ 0.05 compared
with basal conditions; §P ⬍ 0.05 compared
with Lat ⫹ fMLP in control cells.
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Ionomycin-stimulated exocytosis and endocytosis. Previous
reports have shown that formyl peptide receptors undergo
clathrin-mediated endocytosis after ligand binding (50), and
clathrin-mediated endocytosis of G protein-coupled receptors
results in the activation of some signal transduction pathways
(27, 28, 51, 59). To determine if the effects of hypertonic
sucrose, chlorpromazine, and MDC were due to inhibition of
endocytosis of ligand-bound formyl peptide receptors, we
examined the effect of disruption of the actin cytoskeleton on
endocytosis and exocytosis stimulated by a receptor-independent mechanism. Similar to the results with fMLP, pretreatment of neutrophils with latrunculin A significantly enhanced
endocytosis of Alexa fluor-488-conjugated BSA (Fig. 4A) and
Alexa fluor-488-conjugated transferrin (Fig. 4B) stimulated by
the calcium ionophore ionomycin. That enhanced endocytosis
was blocked by pretreatment with hypertonic sucrose, chlorpromazine, and MDC.
The ability of ionomycin to stimulate exocytosis of secretory
vesicles, specific granules, and azurophil granules in the presence and absence of latrunculin A and clathrin-mediated endocytosis inhibitors is shown in Fig. 5, A–C. Incubation for 15
min with ionomycin stimulated increased plasma membrane
expression of CR1 and CD66b but not CD63. Pretreatment
with latrunculin A, followed by stimulation with ionomycin,
resulted in a significant decrease in CR1 expression and a
significant increase in CD63 expression. CD66b expression did
not differ between treatments with ionomycin alone and ionomycin plus latrunculin A. Similar to the results with fMLP,
blockade of clathrin-mediated endocytosis with hypertonic
sucrose, chlorpromazine, or MDC prevented the reduction in
CR1 expression and the increase in CD63 expression under
CLATHRIN-MEDIATED ENDOCYTOSIS IN HUMAN NEUTROPHILS
C863
after stimulation by fMLP in the presence of latrunculin A.
However, disruption of microtubules had no effect on fMLPstimulated CD66b expression. These data suggest that microtubule reorganization is necessary for clathrin-mediated endocytosis, CR1 internalization, and azurophil granule exocytosis
in human neutrophils.
Signal transduction pathways mediating endocytosis. A
number of signaling pathways, including those containing p38
MAPK, PI3K, and nonreceptor tyrosine kinases, regulate exocytosis in human neutrophils (17, 37, 55, 58). To evaluate the
role of these and other kinases in clathrin-mediated endocytosis, neutrophils were pretreated with pharmacological inhibitors of various kinases before measurements of fMLP-stimulated transferrin uptake. As shown in Table 1, pretreatment
with PD-098059 (an ERK inhibitor), LY-294002 (a PI3K
inhibitor), genistein (a nonspecific tyrosine kinase inhibitor), or
AJP-Cell Physiol • VOL
calphostin C (a PKC inhibitor) significantly reduced transferrin
uptake in human neutrophils treated with a combination of
latrunculin A and fMLP. Inhibition of p38 MAPK with SB203580 and Src tyrosine kinases with PP2 had no effect. The
concentrations of PP2 and SB-203580 used in these experiments have been shown to inhibit substrate phosphorylation by
Src tyrosine kinases and p38 MAPK (data not shown). Thus,
despite the relationship between endocytosis and exocytosis,
distinctly different patterns of signal transduction pathways
mediate fMLP-stimulated endocytosis and exocytosis in human neutrophils.
The relationship between ERK1/2 activity and endocytosis
was of particular interest as the activation of ERK through
␤-arrestins has been reported to be dependent on clathrinmediated endocytosis of G protein-coupled receptors (27, 28,
51). Thus, we evaluated ERK1/2 phosphorylation stimulated
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Fig. 5. Effect of actin cytoskeleton disruption
on Iono-stimulated exocytosis. Neutrophils (4 ⫻
106 cells/ml) were incubated for 30 min in the
presence or absence of 225 mM sucrose, 40 ␮M
chlorpromazine, or 350 ␮M MDC, and/or 1 ␮M
Lat followed by stimulation with 250 nM Iono
for 15 min. Exocytosis of secretory vesicles,
specific granules, and azurophil granules was
determined using flow cytometry to measure the
plasma membrane expression of CR1, CD66b,
and CD63, respectively. A: CR1 expression in
the presence or absence of sucrose, chlorpromazine, or MDC. B: CD66b expression in the presence or absence of sucrose, chlorpromazine, or
MDC. C: CD63 expression in the presence or
absence of sucrose, chlorpromazine, or MDC.
Data are expressed as means ⫾ SE of mean
channel of fluorescence intensity for 4 separate
experiments. *P ⬍ 0.05 compared with basal
conditions; #P ⬍ 0.05 compared with control.
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CLATHRIN-MEDIATED ENDOCYTOSIS IN HUMAN NEUTROPHILS
Table 1. Inhibition of phosphatidylinositol 3-kinase, ERK1/2,
and PKC reduced transferrin uptake
Change in Transferrin Uptake
by fMLP under conditions of enhanced and inhibited endocytosis. As shown in Fig. 8, fMLP-stimulated ERK1/2 phosphorylation was modestly enhanced by pretreatment with latrunculin A. Treatment with hypertonic sucrose, chlorpromazine, or
MDC had no effect on fMLP-stimulated ERK1/2 phosphorylation in either the presence or absence of latrunculin A. These
data suggest that receptor endocytosis plays no role in fMLPstimulated ERK1/2 activation, and disruption of the actin
cytoskeleton enhances ERK1/2 activation independently of the
effect on endocytosis.
DISCUSSION
We (22) have previously reported that the actin cytoskeleton
acts as a barrier to stimulated exocytosis of neutrophil granules. The results of the present study demonstrate that the actin
cytoskeleton also functions to limit clathrin-mediated endocytosis after stimulation of human neutrophils. This conclusion is
supported by data showing that disruption of the actin cytoskeleton by latrunculin A or cytochalasin D, followed by stimulation with fMLP or ionomycin, induced a significant increase in
the uptake of albumin and transferrin. Endocytosis of transferrin is clathrin mediated (9, 54), and endocytosis of both
(Lat ⫹ fMLP) ⫺ Lat
关(Lat ⫹ fMLP) ⫺ Lat兴 ⫹ Inhibitor
LY-294002
PD-98059
Calphostin C
Genistein
SB-203580
PP2
591⫾146
645⫾54
503⫾36
703⫾81
344⫾85
557⫾104
91⫾7†
284⫾55*
299⫾76†
65⫾32‡
453⫾81
416⫾119
Data are expressed as means ⫾ SE and are represented as the subtraction of
关latrunculin A (Lat) ⫹ N-formylmethionyl-leucyl-phenylalanine (fMLP)兴 ⫺
Lat; n ⫽ 3 independent experiments. Neutrophils were incubated with 1 ␮M
Lat for 30 min with or without 3 ␮M SB-203580 (p38 MAPK inhibitor), 50
␮M PD-98059 (ERK1/2 inhibitor), 30 ␮M genistein (tyrosine kinase inhibitor), or 1 ␮M calphostin C (PKC inhibitor), or for 15 min with or without 50
␮M LY-294002 (phosphatidylinositol 3-kinase inhibitor), or for 10 min with or
without 10 ␮M PP2 (Src kinase inhibitor), followed by 300 nM fMLP
stimulation for 3 min (Lat ⫹ fMLP). Alexa fluor-488-conjugated transferrin (5
␮g/ml) was added with fMLP, and transferrin uptake was measured by flow
cytometry. *P ⬍ 0.01, †P ⬍ 0.05, and ‡P ⬍ 0.001 compared with control.
albumin and transferrin was inhibited by hypertonic sucrose,
chlorpromazine, and MDC, agents that block clathrin-mediated
endocytosis (1, 7, 10, 21, 42, 47, 56). Although neutrophils
lack caveolin (49), they still form lipid raft domains. Additionally, endocytosis of IL-2 receptors in lymphocytes lacking
caveolin 1 occurred through a clathrin-independent, cholesterolsensitive pathway (25, 26). Our data showed that nystatin, a
cholesterol-aggregating drug, did not alter albumin or transferrin uptake, ruling out a caveolin-like mechanism of endocytosis. Previous studies (6, 9, 14, 23, 31, 41) have indicated
that actin reorganization was required for, or in some circumstances played no role in, clathrin-mediated endocytosis. However, Puthenveedu and von Zastrow (40) reported that some
endocytic cargo interacted with the actin cytoskeleton, and
they postulated that this interaction delayed endocytosis. Our
data indicate that the actin cytoskeleton can impair endocytosis
in some cells, although the mechanism for that inhibition was
not determined. Thus, our results identify a new role for the
actin cytoskeleton in clathrin-mediated endocytosis.
Our previous study (22) showed that disruption of the actin
cytoskeleton enhanced the rate and extent of fMLP-stimulated
exocytosis of neutrophil gelatinase, specific, and azurophil
granules. Whereas stimulation with fMLP alone increased
Fig. 7. Effect of microtubule disruption on
fMLP-stimulated exocytosis. Neutrophils (4 ⫻
106 cells/ml) were incubated in the presence or
absence of 10 ␮M nocodazole, to disrupt microtubule formation, and 1 ␮M Lat, to disrupt
the actin cytoskeleton, for 30 min and then
stimulated for 5 min with 300 nM fMLP or
buffer. Exocytosis of secretory vesicles, specific granules, and azurophil granules was
measured by determining plasma membrane
expression of CR1, CD66b, and CD63, respectively, using flow cytometry. Data are expressed as means ⫾ SE of mean channel of
fluorescence intensity for 3 separate experiments. *P ⬍ 0.05 compared with basal conditions; #P ⬍ 0.05 compared with control.
AJP-Cell Physiol • VOL
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Fig. 6. Actin disruption results in microtubule-dependent transferrin uptake
after fMLP stimulation. Neutrophils (4 ⫻ 106 cells/ml) were incubated for 30
min in the presence or absence of 10 ␮M nocodazole (to disrupt microtubule
formation) and 1 ␮M Lat followed by stimulation with 300 nM fMLP for 3
min. Endocytosis was measured as the uptake of Alexa fluor-488-conjugated
transferrin by flow cytometry. Data are expressed as means ⫾ SE of mean
channel of fluorescence intensity for 3 separate experiments. *P ⬍ 0.05
compared with basal conditions; §P ⬍ 0.05 compared with Lat ⫹ fMLP in
control cells.
Kinase Inhibitor
CLATHRIN-MEDIATED ENDOCYTOSIS IN HUMAN NEUTROPHILS
C865
plasma membrane expression of the secretory vesicle marker
CR1, pharmacological disruption of the actin cytoskeleton
resulted in a time-dependent reduction in CR1 expression after
fMLP stimulation. The ability of hypertonic sucrose, chlorpromazine, and MDC to prevent the loss of CR1 expression
under these conditions suggests that the reduced CR1 expression observed previously was due to clathrin-mediated endocytosis of CR1 in the absence of interaction with its ligand. The
colocalization of CR1 and transferrin-containing endosomes by
confocal microscopy is consistent with this conclusion. Using
immunogold labeling and electron microscopy, Berger and
colleagues (3, 4, 52) demonstrated that fMLP and ionomycin
stimulation of human neutrophils with an intact actin cytoskeleton induced a 6- to 10-fold increase in plasma membrane CR1
expression due to exocytosis of secretory vesicles. This translocation was rapidly followed by ligand-independent endocytosis and subsequent degradation of CR1. However, we were
unable to detect endocytosis of CR1, albumin, or transferrin
without disruption of the actin cytoskeleton by confocal microscopy.
Three agents that inhibited clathrin-mediated endocytosis by
different mechanisms were used to determine the relationship
between endocytosis and exocytosis of specific and azurophil
granules. Both chlorpromazine and MDC significantly inhibited specific granule exocytosis stimulated by fMLP or ionomycin with or without latrunculin A pretreatment. On the other
hand, hypertonic sucrose pretreatment inhibited specific granule exocytosis stimulated by fMLP or ionomycin alone but
failed to alter exocytosis stimulated in the presence of latrunculin A. This observation may be explained by the ability of
osmotic stress to induce the rigid subcortical actin polymerization that would be expected to inhibit exocytosis (43). As we
have previously reported (22), azurophil granule exocytosis
AJP-Cell Physiol • VOL
could only be stimulated after disruption of the actin cytoskeleton. All three inhibitors of clathrin-mediated endocytosis
dramatically reduced azurophil granule exocytosis stimulated
by ionomycin, whereas sucrose and MDC significantly inhibited azurophil granule exocytosis stimulated by fMLP. The
reason for the failure of chlorpromazine to inhibit exocytosis of
this granule subset is not clear. Taken together, these data
suggest that the extent of clathrin-mediated endocytosis is one
determinant of the level of specific and azurophil granule
exocytosis. The data also suggest that the regulation of exocytosis by the actin cytoskeleton may be through control of
endocytosis rather than a direct effect on granule access to the
plasma membrane. Although the mechanism by which endocytosis regulates exocytosis was not examined in the present
study, the ability of inhibition of endocytosis to block both
fMLP- and ionomycin-stimulated azurophil granule exocytosis
indicates that the effect of endocytosis is not mediated by
internalization of ligand-bound receptors. Fittschen and Henson (13) previously proposed a model for the interaction
between endosomes and azurophil granules. By ultrastructural
analysis, they showed that endosomes fused with azurophil
granules and suggested that endosomal membranes provided
the contact sites for recognition and fusion with the plasma
membrane. The failure of transferrin-containing endosomes to
colocalize with a marker of azurophil granules in the present
study, however, does not support this hypothesis.
Our observation that endocytosis regulates azurophil granule
exocytosis provides one explanation for the observation that
disruption of the actin cytoskeleton is required for stimulated
exocytosis of azurophil granules (22). Botelho and colleagues
(5) described pinocytosis occurring in the vicinity of Fc␥Rmediated phagocytosis and reported that disruption of the actin
cytoskeleton blocked phagocytosis but enhanced pinocytosis.
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Fig. 8. Effect of blockade of CME on the
phosphorylation of ERK1/2. Neutrophils
(1 ⫻ 107 cells/ml) were incubated for 30 min
in the presence or absence of 225 mM sucrose, 40 ␮M chlorpromazine, or 350 ␮M
MDC, and/or 1 ␮M Lat followed by stimulation with 300 nM fMLP for 1 min. Proteins
from cell lysates were separated by 10%
SDS-PAGE, immunoblotted for phosphorylated (p)ERK1/2, stripped, and then reblotted for total ERK1/2 as a loading control. A
representative immunoblot (IB) of 4 experiments is shown. A: representative Western
blot analysis of pERK1/2. B: average densitometric analysis of 4 immunoblots. Values
were normalized to the total amount of
ERK1/2 and are expressed as percentages.
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CLATHRIN-MEDIATED ENDOCYTOSIS IN HUMAN NEUTROPHILS
AJP-Cell Physiol • VOL
family tyrosine kinases are not involved. Previous studies
(33–35, 57, 58) have shown that p38 MAPK and Src tyrosine
kinases, but not ERK1/2, play a significant role in granule
exocytosis in neutrophils. Although a more detailed analysis of
the signal transduction pathways regulating endocytosis and
exocytosis is required, our data suggest that distinctly different
signal transduction pathways regulate exocytosis and endocytosis in human neutrophils.
ACKNOWLEDGMENTS
We thank Karen Brinkley and Terri Manning for the excellent technical
assistance.
Present address of N. R. Jog: Sect. of Rheumatology, Dept. of Medicine,
Temple Univ., Philadelphia, PA 19140.
GRANTS
This work was supported by a Department of Veterans Affairs Merit
Review Grant (to K.R. McLeish), by National Institute of Diabetes and
Digestive and Kidney Diseases Grant R21-DK-62389 (to R. A. Ward and K. R.
McLeish), and American Heart Association Ohio Valley Affiliate Predoctoral
Fellowship Grant 0415191B (to N. R. Jog) and Beginning Grant-In-Aid
0765387B (to S. M. Uriarte).
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