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Shape F-actin and surface morphology changes during chemotactic peptide-induced polarity in human neutrophils.

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THE ANATOMICAL RECORD 241519-528 (1995)
Shape, F-Actin, and Surface Morphology Changes During
Chemotactic Peptide-Induced Polarity in Human Neutrophi Is
Departamento de Biologia Celular e Histologia, Facultad de Medicina, Uniuersidad de
Granada (E.F.S., J.M.G., A.C.), Seruicio de Inmunologia, Hospital Clinic0 Universitario
(J.L.S.), Granada, Spain
Background: The exposure of human neutrophils to uniform concentrations of chemoattractants, such as N-formyl peptides, induces morphological cell polarization. In this study we report the temporal
sequence of changes in cell shape, F-actin, and cell surface morphology
during cellular polarization induced by N-formylmethionyl-leucyl-phenylalanine (fMLP) in human neutrophils in suspension.
Methods: Neutrophil shape changes induced by lo-' M fMLP were observed with DIC microscopy. Size and cellular granularity were analyzed
by flow cytometry measuring their forward and side scattered light. To
visualize F-actin distribution, neutrophils were labeled with the fluorescence probe FITC-phalloidin, and were examined with fluorescence and
confocal laser scanning microscopy. Cell surface morphology was assessed
with scanning electron microscopy (SEM).
Results: The stimulation of round-smooth neutrophils with nanomolar
concentrations (lo-' M) of fMLP in suspension induced a temporal sequence of morphological changes during cell polarization, characterized
by 1)increase in size as determined by forward angle scattered light, 2)
rapid redistribution of F-actin from a diffuse cytoplasmic localization to the
cell periphery, and 3) rapid reorganization of cell surface morphological
features, with accumulation of plasma membrane in the front of polar cells.
Four cell shapes were identified with SEM after stimulation of roundsmooth neutrophils: round-ridged, round-ruffled, nonpolar ruffled, and polar cells. These cell shapes were correlated with a cortical localization,
focal aggregates, and multipolar distribution of F-actin. In polar neutrophils, F-actin became concentrated in the front of the cell.
Conclusions: These findings show the relation between reorganization of
the microfilamentous cytoskeleton and modifications in cell shape and surface features during cell polarization induced after fMLP activation in neutrophils. This approach offers a powerful tool for further analysis of receptor distribution in polarized, motile neutrophils. o 1995 Wiley-Liss, Inc.
Key words: Neutrophils, N-formyl peptide, Cell polarity, Flow cytometry,
F-actin, Confocal microscopy, Scanning E M
Polymorphonuclear neutrophils are recruited to inflammatory sites by a variety of soluble mediators
(chemoattractants) that stimulate neutrophil migration (chemotaxis). Two types of chemoattractants can
be distinguished: exogenous (i.e., N-formyl peptides),
and endogenous (C5a, LTB,, PAF, IL-8) (Harvath,
1991). The N-formyl peptides have been extensively
studied as probes of neutrophil activation because
these peptides stimulate a variety of biochemical and
physiological responses in addition to chemotaxis. The
cellular response begins when this signal molecule interacts with its specific receptor, formyl peptide receptor (FPR), on the plasma membrane of neutrophils.
This induces a series of responses coordinated a t the
biochemical and cellular levels: activation of pertussis
toxin-sensitive G protein and increased activity of
phospholipase C, which induces the formation of inosito1 1,4,5-trisphosphate and diacylglycerol. Inositol
1,4,5-trisphosphate increases intracellular free Ca2+,
and diacylglycerol activates protein kinase C (PKC)
(for a review, see Omann et al., 1987). These signaling
Received February 17, 1994; accepted September 16, 1994.
Address reprint requests to Prof. Antonio Campos, Departamento
de Biologia Celular e Histologia, Facultad de Medicina, Universidad
de Granada, E-18071 Granada, Spain.
pathways interact with each other to initiate or enhance a wide range of neutrophil activities, including
initiation of the respiratory burst leading to superoxide
production, degranulation, up-regulation of receptors,
and cell shape changes with projections of membrane
ruffles and pseudopodia. These cell shape changes are
correlated with dynamic changes in the organization of
the cytoskeleton, mainly in relation with microfilamentous actin (F-actin), the major cytoskeletal element in neutrophils. Exposure of neutrophils to
N-formyl peptides generates transient actin nucleation
activity, rapid polymerization of F-actin followed by
slower depolymerization (Howard and Meyer, 1984;
Howard and Oresajo, 1985; Howard and Wang, 1987;
Watts et al., 1991), F-actin cross-linking activity, and
incorporation of actin-binding proteins (Niggli and
Jenni, 1989; Fujimoto and Ogawa, 1990). These processes give rise to morphological and functional polarization of the cytoplasm and plasma membrane of neutrophils. Previous reports have indicated the spatial
reorientation of microtubules (Anderson et al., 1982),
centrosomes (Schliwa et al., 1982), and membrane proteins such as FPR (Sullivan e t al., 1984), concanavalin
A-binding sites (Weinbaumn et al., 1980), and Fc and
C3b receptors (Davis et al., 1982; Petty et al., 1989;
Pytowski et al., 1990; Walter et al., 1980). However,
conflicting results were reported when cells adherent
to a substrate were studied.
Previous studies have shown that the same morphological cell polarization process occurs in suspension,
after the exposure of human neutrophils to a n isotropic
concentration of N-formyl peptides in the absence of a
gradient or attachment to a substrate (Davis et al.,
1982; Shields and Haston, 1985). Although most studies have examined the kinetics of chemotactic peptideinduced changes in the content and distribution of
F-actin (Coates et al., 1992; Howard and Meyer, 1984;
Howard and Oresajo, 1985; Howard and Wang, 1987;
Lepedi et al., 1992; Wallace et al., 1984; Watts et al.,
1991), little is known about the relationship between
temporal changes in the cell shape, F-actin and cell
surface morphology. Moreover, detailed morphologic
analyses were not reported in any of these investigations. Consequently, the purpose of the present study
was to analyze the temporal sequence of changes in cell
shape, F-actin, and cell surface morphology during the
cellular polarization induced by fMLP in human polymorphonuclear leukocytes. To examine the relation between F-actin reorganization and changes in cell shape
and surface morphology, we used spherical-smooth
neutrophils stimulated in suspension.
DMSO, and stored frozen at -20°C in aliquots until
use. FITC-phalloidin stock solution was prepared as 3.3
x lop6 M in methanol and was stored at -20°C. The
HBSS used in polarization assays was supplemented
with 25 mM Hepes, 1.2 mM calcium chloride, and 1.0
mM magnesium chloride.
Cell Preparation
Human peripheral blood was obtained from healthy
volunteer donors by venipuncture and collected into
heparin (20 U/ml). Heparinized blood was mixed with
HBSS without Ca2+ and Mg2+,pH 7.2, and was sedimented in 6% Dextran 70 in 0.9% sodium chloride for 1
h. The leukocyte-rich plasma was layered on a FicollPaque density gradient and centrifuged at 400g for 30
min. Cells were washed (3 times, 400g, 5 min) and
resuspended in HBSS without Ca2+ and Mg2+ at a
concentration of 1 x lo6 cell/ml. The contaminating
red blood cells were lysed with distilled water for
20-30 sec. Isolated neutrophils were washed and resuspended in HBSS/Hepes buffer with Ca2+ and Mg2+.
All the above procedures were carried out under sterile
conditions. Cell preparations contained >95% neutrophils as detected by Giemsa staining and immunofluorescence flow cytometry. Neutrophil viability was
>95% by trypan blue exclusion. Cells were used for
polarization assays within 1h after isolation. To avoid
the effects of cell purification on neutrophil response to
fMLP stimulation, we used total leukocyte suspensions
for polarization assays. Briefly, the blood was immediately treated twice for 30 sec with distilled water to
lyse the erythrocytes. The leukocytes were washed
twice in HBSS without Ca2+ and Mg2+, and resuspended in HBSS/Hepes buffer with Ca and Mg2+ a t
a final concentration of 1 x lo6 cell/mI.
Neutrophil Polarization Assays
Neutrophils (5 x lo5 cell/ml) were incubated in suspension with
M fMLP in HBSS/Hepes buffer with
Ca2+ and Mg2+ in a water bath at 37°C. After fMLP
had been added, 250 pl of cell suspension was removed
and fixed with 2.5% glutaraldehyde in 0.1 M buffer
phosphate, pH 7.2, a t room temperature, at the times
indicated. Cells were fixed overnight at 4"C, then
washed (3 times) with PBS, pH 7.2. Control cells were
incubated with 0.01% DMSO in HBSS/Hepes buffer
with Ca2+ and Mg2+.
Shape Analysis
Glutaraldehyde-fixed neutrophils were mounted on
slides and coverslips and cell shape changes were deMATERIAL AND METHODS
termined with differential interference contrast miChemicals
croscopy (DIC, Nomarski optics, Zeiss Axiphot). Also,
Hanks' balanced salt solution (HBSS), Hepes, Dex- cell shape was defined by morphological parameters,
tran T-70, N-formylmethionyl-leucyl-phenylalanine such as perimeter, area, and shape factor (SF). The SF
(fMLP), poly-L-lysine (PLL), Triton X-100, and fluores- is a measure of the deviation from a circle of the shape
cein (F1TC)-phalloidin were obtained from Sigma of the cell, with a prefect circle resulting in a SF of 1.0,
Chemicals Company (St. Louis, MO). Ficoll-Paque was whereas elongation results in a decrease in SF. The
obtained from Pharmacia Fine Chemicals (Uppsala, value of SF was determined according to the equation
Sweden). Phosphate buffered saline (PBS) was pur- SF = 4 x 7~ x area/(perimeter)2.Cell outlines were
chased from Flow Laboratories (Irvine, Scotland). Glu- traced using a camera lucida drawing tube fitted to a
taraldehyde EM grade and dimethylsulfoxide (DMSO) Leitz microscope with a x 100 oil immersion objective
were obtained from Merck (Darmstadt, Germany). A on a digitizing tablet. The tracings were analyzed on a
stock solution of fMLP was prepared as
M in personal computer with a n image analysis program.
Each morphological parameter was measured in a t
least 50 cells a t the times indicated.
Flow Cytometry
Single cell suspensions of fixed neutrophils, stimulated with chemotactic peptide, were analyzed according to their size and cellular granularity by measuring
their forward (narrow angle) and side (right angle)
scattered light, respectively. Steady state analysis was
performed on a n Ortho Cytoron Absolute flow cytometer (Ortho Diagnostics Systems, Raritan, NJ) with excitation at 488 nm using a 5 W argon ion laser. In total
leukocyte suspensions, neutrophils were easily discriminated from lymphocytes and monocytes by their
characteristics forward and side scattered light. Frequency histograms were obtained by analyzing 2,000
cells at the times indicated. Data are expressed as the
mean channel number of the histograms.
Fluorescence and Confocal Microscopy
FITC-phalloidin was used to stain F-actin in neutrophils. After stimulation with lo-' M fMLP, cell suspensions were fixed by adding equal volumes of 0.2%
glutaraldehyde EM grade (final concentration 0.1%) in
PBS, pH 7.2, for 10 min at room temperature. After
fixation, neutrophils were washed with 0.05 M glycine
in PBS, pH 7.2, for 15 min at 4°C in order to quench
aldehyde groups. The cells were washed, and then permeabilized by incubation with 0.1% buffered Triton
X-100 for 5 min at room temperature, followed by
washing with PBS, pH 7.2, prior to incubation with
1.65 x
M FITC-phalloidin for 20 min at room
temperature in the dark. Neutrophils were then
washed with PBS, pH 7.2, dropped onto PLL-precoated
glass coverslips, and mounted on slides with PBS/glycerol. For conventional fluorescence microscopy, the
samples were visualized in a Leitz Laborlux 12 epifluorescence microscope. For confocal microscopy, the
samples were observed in a Zeiss 310 confocal laser
scanning microscope (Zeiss, Oberkochen, Germany).
Photographs were taken using Kodak Tri-X 400 film.
Scanning Electron Microscopy
Glutaraldehyde-fixed cells were washed, and then
dropped onto PLL-precoated coverslips overnight a t
4°C. The cells were dehydrated in a graded series of
alcohols, critical-point dried (CPD30, Balzers Union,
Liechtenstein) with COs, and gold-sputter coated
(E5000, Polaron, UK). The samples were visualized in
a Philips SEM 505 scanning electron microscope (Philips, Eindhoven, Holland) at 20-30 kV. Micrographs
were made using Polaroid type 52 film.
Shape of fMLP Stimulated Neutrophils
Human isolated neutrophils incubated with lo-' M
fMLP showed rapid, time-dependent shape changes
(Fig. 1A). Cellular perimeter and area increased, and
SF decreased significantly as a result of polarization
(Fig. lB,C). Examination with DIC microscopy showed
that >90% of the unstimulated (time 0) neutrophils
were round-smooth (Fig. 2A). Polar neutrophils were
not observed. After 30 sec to 2 min of stimulation, the
cells were rounded with small surface projections (Fig.
2B). After 5 min of chemotactic peptide stimulation,
the neutrophils were irregularly shaped, with large
surface projections. Some cells were elongated, with
clear signs of polarization. After incubation with fMLP
for 10 min, most neutrophils displayed a polar shape
with front-tail polarity (Fig. 2C). Control cells showed
no morphological changes.
We also analyzed the size and cellular granularity of
chemotactic peptide-stimulated neutrophils by cytometric measurements of forward (size) and side scattered light (cellular granularity). Stimulation with
lo-' M fMLP increased the size of neutrophils in a
time-dependent manner, in comparison with control
cells (Fig. 3). The isotropic concentration of chemoattractants induced little change in the cellular granularity of stimulated neutrophils as compared with control cells. Figure 4 illustrates the response of
neutrophils in total leukocyte suspensions after stimulation with lo-' M fMLP. The changes were similar
to those seen with purified neutrophils.
fMLP Induces F-actin Reorganization in Neutrophils
F-actin was stained with FITC-phalloidin in glutaraldehyde-fixed cells. Fixation with paraformaldehyde
induced the formation of blebs on the plasma membrane, and cell lysis of chemotactic peptide-stimulated
neutrophils (not shown). Exposure of neutrophils in
suspension to lo-' M fMLP induced a dramatic, timedependent rearrangement of F-actin, a s seen with conventional fluorescence and confocal microscopy. The
use of confocal microscopy made it possible to observe
optical sections of individual cells and thus improved
the resolution of fluorescence imaging. In unstimulated neutrophils, F-actin was distributed diffusely and
homogeneously throughout the cytoplasm (Fig. 5A).
Within 30 sec after the addition of fMLP, F-actin was
redistributed to form a peripheral submembranous
ring (Fig. 5B). After 2 min of exposure to chemotactic
peptide, actin microfilaments were redistributed as focal aggregates at the cell periphery (Fig. 5C). Five minutes after the addition of chemotactic peptide, F-actin
was asymmetrically redistributed near the pseudopods
(Fig. 5D). After 10 min of stimulation, FITC-phalloidin
stained neutrophils displayed a strongly fluorescent
ruffled lamellipodium and a weakly fluorescent tail
(Fig. 5E).
fMLP Induces Changes in Cell Surface
Morphology of Neutrophils
In an attempt to identify and correlate the time-dependent reorganization of cell surface morphology of
neutrophils after fMLP stimulation with changes in
cell shape and F-actin reorganization observed by flow
cytometry and confocal microscopy, we used SEM. The
morphological polarization of neutrophils in suspension was accompanied by a time-dependent redistribution of the cell surface. Neutrophils of five different
shapes were identified by SEM: round-smooth, roundridged, round-ruff led, nonpolar ruff led, and polarized
cells. Round-smooth cells displayed a spherical smooth
surface with few short microvilli and microridges (Fig.
6A). Round-ridged cells were spherical with ridges and
short microvilli on cell surface (Fig. 6B). Round-ruffled
cells were characterized by a rough surface with welldeveloped ridges and folds (Fig. 6C). Nonpolar ruffled
cells displayed a n asymmetric rough surface with ruf-
Time (min)
Fig. 1. A: Time-course of shape changes of neutrophils in suspension
after stimulation with lo-’ M fMLP. Cell shape was determined in
cells fixed with 2% glutaraldehyde and observed using Nomarsky
optics. Non-spherical, irregular shaped cells with surface projections
plus front-tail polarized cells, 0 ; front-tail polarized cells, a. Results
are given as percentages of cells that changed in shape (means 2 SD
of ten separate experiments). B Time-course of changes in the area (m,
0 ) and perimeter ( 0 , 0 ) of neutrophils stimuIated with lo-’ M fMLP
(closed symbols) and control cells incubated with 0.01% DMSO (open
symbols). C: Time-course of changes in shape factor (SF),determined
according to the equation SF = 4 X TI x areaiperimeter’. Neutrophils
stimulated with [email protected] fMLP, control cells incubated with 0.01%
DMSO, 0. Results are means i SD of three separate experiments with
cells isolated from different donors.
Fig. 2. Differential interference contrast (DIC) micrographs of the
effect of stimulation with lo-’ M fMLP on neutrophil shape. After
stimulation, neutrophils were fixed with 2% glutaraldehyde in suspension and then dropped onto PLL-coated coverslips. A, unstimuM fMLP for 2
lated neutrophils; B, Neutrophils stimulated with
min; and C, for 10 min. Scale bar: 10 pm.
.. .
Fig. 3. Flow cytometry frequency histograms of changes in forward
(FW-SC) and side (RT-SC) scatter parameters of chemotactic peptidestimulated neutrophils in suspension. Frequency histograms were obtained by analyzing 2,000 cells at the time indicated. Cells after the
addition of lo-' M fMLP for 30 see (A,E), 2 rnin (B,F), 5 rnin (C,G),
and 10 min (D,H). Unstimulated neutrophils (-);
stimulated with lo-' M fMLP (--).
Histograms are representative
of five separate experiments.
Fig. 4. Dot mapping of changes in forward (narrow angle) and side
(right angle) scatter parameters in neutrophils after 10 min of stimulation with lo-* M M L P in total leukocyte suspensions. Neutrophils
were gated by their characteristic forward (FW-SC) and side (RT-SC)
scattered light. Dot maps were obtained by analyzing 2,000 cells. A,
unstimulated cells; B, cells stimulated with lo-' M fMLP. Note the
increase in forward scatter in neutrophils (N). Lymphocytes (L) and
monocytes (MI show no changes in scatter parameters. Plots are representative of three separate experiments.
fles and folds (Fig. 6D). These cellular projections arose
from two or three sites, giving the cell a multipolar
appearance. Polar cells were elongated with front-tail
polarity, i.e., ruffles and folds at the frontal pole, and a
contracted rear end with a distinct tail knob at the
posterior pole (Fig. 6E). The exposure of total leukocyte
M fMLP during 10 min induced a
suspensions to
polar morphology in neutrophils, similar to that seen
in purified neutrophils. Lymphocytes and monocytes
showed no changes in cell shape (not show).
Figure 7 illustrates the sequential modifications in
the cell surface morphology of lo-' M fMLP stimulated
neutrophils. Before stimulation, neutrophils were
round-smooth (83%).Round-ridged neutrophils developed after 30 sec of chemotactic peptide stimulation
(64.2%). Cells with ridges and folds were observed after
2 min (47.8%). The formation of nonpolar ruffled cells
reached its peak at 5 min (56.8%), decreasing after 10
min of fMLP stimulation, whereas the proportion of
polar cells increased after 2 min, and reached its peak
after 10 rnin of exposure to fMLP (57.6%). Control cells,
incubated with 0.01% DMSO, showed no changes in
cell surface morphology.
In this report we analyzed the changes in cell shape,
F-actin, and surface morphology that occur during cell
polarization induced by nanomolar concentration (lo-'
M) of fMLP in human neutrophils in suspension. Our
results not only confirm earlier findings, but also provide new evidence of the relationship between the pattern of F-actin distribution and the different cell
Fig. 5. Confocal laser scanning microscopic images of the reorganization of F-actin in neutrophils stimulated with lo-' M fMLP in
suspension. Neutrophils were fixed with 0.1%glutaraldehyde, permeabilized, stained with FITC-phalloidin, and dropped onto PLL-coated
coverslips. F-actin distribution in unstimulated neutrophils (A) and
in cells after the addition of lo-' M fMLP for 30 sec (B), 2 min ( C ) , 5
min (D), and 10 min (E). Each micrograph is representative of five
separate experiments with cells isolated from different donors. Scale
bar: 10 pm.
shapes and surface features observed during the phases
of polarization. These modifications are discussed below with reference to the signal transduction pathways
in neutrophils t h a t may come into play as a result of
chemotactic peptide stimulation. This model should
provide a n appropriate morphological substrate to establish, with immunogold-labeling techniques and
SEM, the topological distribution of antigenic receptor
sites on cell surface domains throughout the polarization process (Fernandez-Segura et al., 1994)
Purified neutrophils are able to reversibly bind
chemoattractants, and are capable of responding to
their continued presence after warming to physiological temperatures (English and Graves, 1992). Nevertheless, the different isolation procedures used to purify neutrophils may have caused cellular activation
(Kuijpers e t al., 1991). Although the reasons for activation are unknown, possible factors are the exposure
of cells to endotoxin and/or centrifugation on density
gradients during cell preparation (Howard et al., 1990).
Activation may influence cell surface morphology
(Watts et al., 1991) and neutrophil response in vitro
(Haslett et al., 1985), and hence may modulate the expression of surface molecules, such as Fc receptors,
LFA-1, Mac-1, p150, CR1, or FPR (Kuipjers et al.,
1991; Norgauer et al., 1991). However, our purification
procedure did not affect cellular response, as shown by
the findings with neutrophils in total leukocyte suspensions: after stimulation with lo-' M fMLP, the
changes in cell shape were similar to those found with
purified neutrophils. Our cell preparations of purified
neutrophils contained no polarized cells, and more than
90% of the neutrophils were round as determined by
morphometric analysis (e.g., shape factor) and flow cytometry; F-actin was diffusely distributed as determined by confocal microscopy. Previous studies with
purified neutrophils under endotoxin-free conditions
reported similar results (Howard et al., 1990).
The neutrophil response to chemotactic peptide is
sensitive to different isotropic concentrations (Shields
and Haston, 1985). At low concentrations of fMLP, neutrophils rapidly acquire a polarized appearance upon
activation. However, at high concentrations, neutrophils display a multipolar morphology (McKay et al.,
1991). Isotropic concentrations of chemotactic peptide
(lo-' M fMLP) induced the sequential development of
polar cell shape, as we demonstrated by light microscopy and flow cytometry. We also found that stimulation with fMLP of neutrophils in suspension induced a
sequence of cell shape changes, cytoskeletal F-actin reorganization, and topographic redistribution of cell
surface features, which was different from the changes
observed in neutrophils stimulated on substrate (Davis
et al., 1982).
Neutrophil adhesion to a substrate may alter the cellular response, leading to cellular activation, characterized by a rise in free intracellular Ca2' (Jaconi et
al., 1991; Southwick et al., 1989), sustained burst of
superoxide production (Ginis and Tauber, 19901, increased cytoskeleton-associated actin (F-actin) (Ginis
et al., 1992), F-actin reorganization, and membrane
ruffling (Boyles and Bainton, 1979). These adhesiondependent events are mediated by heterodimer membrane proteins-integrins-which
link the cytoskeleton to the extracellular environment (Hynes, 1987). It
therefore seems logical to assume that all modifications that occur in cell shape, F-actin, and cell surface
morphology in adherent neutrophils after fMLP stimulation may be caused by a two-pronged effect: 1)activation in response to stimulation by the chemotactic
peptide itself, and/or 2) activation a s a result of a transitory, cyclic process of adhesion-detachment on the
substrate during cell polarization. However, the stimulation of neutrophils in suspension makes i t possible
to observe changes in cell shape and cell surface morphology in the absence of other stimuli. These changes
reflect only the effects of cytoskeletal F-actin reorganization during morphological polarization (Coates et
al., 1992). SEM observations of neutrophils in suspension allowed us identify different time-dependent transitional cell shapes. Our results show that cell shape
changes from round-smooth to polar after chemotacticpeptide stimulation in suspension progresses through
three different intermediate types: round-ridged,
round-ruffled, and non-polar ruffled cells. Each cell
Fig. 6. Scanning electron micrographs of changes in cell surface
morphology of neutrophils in suspension stimulated with lo-' M
fMLP. A Cell surface morphology of unstimulated neutrophils. B: 30
sec after fMLP stimulation, round-ridged cell. C: 2 min after chemoattractant stimulation, round-ruffled cell. D 5 min after the addition of
chemotactic peptide, nonpolar cell with ruffles and folds. E: Polarized
front-tail cell. Each micrograph is representative of neutrophils from
five separate experiments with cells isolated from different donors.
Scale Bar: 1 km.
shape showed a distinctive subcellular localization of
Under our isolation conditions, unstimulated neutrophils displayed a regular round-smooth appearance.
Watts et al. (1991) reported basal, endotoxin-free neutrophils with a similar cell surface appearance in scan-
ning EM observations. These results differ from earlier
studies that showed typical unstimulated neutrophils
after Ficoll-dextran sedimentation purification, bearing short ruffles and membrane folds (Davis et al.,
1982; Hoffstein et al., 1982). This morphological phenotype corresponds to cells that were preactivated dur-
Smooth cell
Ridged cell
Ruffled cell
Nonpolar c e l l
Polar c e l l
Time (min)
Fig. 7. Kinetic analysis of neutrophil surface changes observed with
scanning EM. Neutrophils were stimulated with
M fMLP in
suspension during the indicated times, fixed with 2% glutaraldehyde,
and then dropped onto PLL-coated coverslips. Data are given as percentages of cells that changed in shape (means ? SD of five separate
ing isolation procedures (Haslett et al., 1985; Howard
et al., 1990; Watts et al., 1991). Two different F-actin
pools have been described in unstimulated neutrophils:
1)a stable Triton-insoluble, gelsolin-poor F-actin pool
representing 35-40% of total cellular F-actin, and 2) a
labile, Triton-soluble, gelsolin-rich F-actin pool representing the 60-65% of total cellular F-actin (Watts and
Howard, 1992). The spatial distribution of the two
F-actin pools in the cytoplasm is different (Zaffran et
al., 1993). Watts and Howard (1992) reported t h a t in
formaldehyde-prefixed neutrophils, which retain both
stable and labile actin pools, F-actin was diffusely distributed throughout the cytoplasm. Our results showed
a similar subcellular distribution of F-actin labeled
with FITC-phalloidin after glutaraldehyde fixation.
These findings suggest that our basal cell preparations
retained both F-actin pools.
The first phase (<30 sec) of neutrophil stimulation
by fMLP is characterized by a rapid, transient rise in
free intracellular Ca2+, increased F-actin content
(Howard and Meyer, 1984; Howard and Oresajo, 1985;
Howard and Wang, 1987; Wallace et al., 1984), and
spatial redistribution of F-actin (Coates et al., 1992;
Haston, 1987; Lepedi et al., 1992). At this time of stimulation, we observed a subcortical localization of F-actin with both fluorescence and confocal microscopy.
This reorganization of F-actin coincided with a rapid
redistribution of actin-associated proteins to the cell
surface upon cell activation. In ultrastructural studies
with immunogold staining, Hartwig et al. (1989) reported a submembranous localization of gelsolin, and
suggested that this actin-binding protein translocated
short actin oligomers to the membrane surface. Furthermore, other authors have noted the rapid incorporation into the cytoskeleton of specific actin cross-linking proteins, such as a-actinin (Niggli and Jenni,
1989), which are implicated in the extension of surface
projections. During the early phase of stimulation, we
found that neutrophils displayed a rough-ridged cell
surface under SEM. The development of ridges on the
cell surface may be related with the rearrangement of
actin cytoskeleton, i.e., F-actin and actin-associated
proteins move to the cell periphery after exposure to
chemoattractants. The transductional pathway involved in actin polymerization, F-actin distribution,
and subsequent cell shape changes remains unclear,
specifically with regard to the role of intracellular calcium (Zaffran et al., 1993). Recent studies showed that
depletion and chelation of cytosolic free calcium have
no effect on actin assembly (Howard and Wang, 1987),
F-actin distribution, or membrane ruff ling in neutrophils stimulated with fMLP (Zaffran et al., 1993).However, free intracellular Ca2 may modulate the physical organization of F-actin (Omann et al., 1987).
Round-ruff led and non-polar ruff led neutro hils, cell
shapes that appear within 2-5 min after 10- M fMLP
stimulation, showed a reorganization of F-actin in peripheral focal aggregates. Kinetic studies with flow cytometry and video image analysis reported that this
asymmetric redistribution of F-actin coincides with the
depolymerization phase of F-actin, and probably precedes the development of polar shape in neutrophils
stimulated with fMLP in suspension (Coates et al.,
1992; Lepedi et al., 1992; Watts e t al., 1991). The signal
transduction pathways responsible for these various
shapes are unknown. Previous studies showed that exposure of neutrophils to PKC activators (e.g., diacylglycerols or phorbol esters) induced reorganization of
F-actin and membrane ruffling (Roos et al., 1987; Zimmermann et al., 1988; Downey et al., 1992) similar to
the changes described in the present study. However,
these chemotactic peptide-induced effects cannot be
prevented by PKC inhibitors, such as H7, staurosporine, or calphostine C; thus neither the reorganization
of F-actin nor pseudopod extension induced by fMLP
appears to depend on PKC activation. Protein kinase C
has been implicated in the maintenance of cell polarity,
i.e., pseudopod extension and tail contraction (Niggli
and Keller, 1993). In our study, after 10 min of stimulation with chemotactic peptide, most neutrophils displayed the typical polarized shape, with a broad, ruffled lamellipodium and a contracted, granular tail.
Like other workers (Coates et al., 1992; Lepedi et al.,
1992), we found that F-actin was distributed in two
foci: a main focus in the anterior pole, and a less intense, secondary focus in the posterior end. Keller and
Niggli (1993)reported the accumulation of a-actinin in
the anterior pole. In contrast, fodrin (Fujimoto and
Ogawa, 1990) and myosin (Keller and Niggli, 1993)
were preferentially concentrated at the rear end of polarized neutrophils. The co-localization of F-actin and
myosin may be related with the development of the
contracted, granular tail.
The reorganization of actin cytoskeleton and the cell
surface morphological features after fMLP stimulation
induced a n asymmetrical distribution of molecules on
the plasma membrane. The causes of this differential
reorganization of surface molecules are under debate.
Some authors have suggested that the differences are
caused by a n increase in the density of the molecules a t
the anterior pole (Haston and Maggs, 1990; McKay et
al., 1991; Sullivan et al., 1984; Walter et al., 1980;
Weinbaum et al., 1980). Alternatively, they may result
from the accumulation of plasma membrane in the
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morphology, neutrophils, induced, change, activ, surface, chemotactic, shape, polarity, human, peptide
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