Am J Physiol Cell Physiol 288: C863–C871, 2005.
First published November 24, 2004; doi:10.1152/ajpcell.00358.2004.
Control of neutrophil pseudopods by fluid shear: role of Rho family GTPases
Ayako Makino,1 Michael Glogauer,2 Gary M. Bokoch,3 Shu Chien,1 and Geert W. Schmid-Schönbein1
1
Department of Bioengineering, The Whitaker Institute of Biomedical Engineering, University of California, San Diego,
La Jolla, California; 2Canadian Institutes of Health Research Group in Matrix Dynamics, University of Toronto,
Toronto, Ontario, Canada; and 3Department of Immunology, The Scripps Research Institute, La Jolla, California
Submitted 26 July 2004; accepted in final form 19 November 2004
microcirculation; mechanotransduction; actin polymerization; transgenic mouse; leukocyte
SHEAR STRESS due to blood flow greatly influences vascular
structure and the inflammatory response. These phenotypic
alterations resulting from shear stress have been examined
mostly in vascular endothelial and smooth muscle cells and
involve a coordinated sequence of events with rapid activation
of flow-sensitive K⫹ and Cl⫺ channels (34, 40) and GTPbinding proteins (G proteins) (18, 29, 53), changes in cell
membrane fluidity (5, 19) and intracellular pH (58), and mobilization of intracellular calcium (2, 25, 47). The early re-
Address for reprint requests and other correspondence: G. W. SchmidSchönbein, Dept. of Bioengineering, The Whitaker Institute of Biomedical
Engineering, University of California, San Diego, 9500 Gilman Dr., La Jolla,
CA 92093.
http://www.ajpcell.org
sponses are followed by activation of a host of gene and protein
regulatory responses (32, 39, 42) and by cytoskeletal remodeling (11, 35, 53).
We have obtained increasing evidence suggesting that fluid
shear stress also affects circulating cells, such as leukocytes.
The leukocyte-endothelial cell adhesion is a critical step during
immune responses against bacterial infection and the inflammatory cascade. The binding of circulating leukocytes to the
endothelium is regulated by fluid shear (8, 24, 30). Fluid shear
controls the morphology of neutrophils, but different from that
of endothelial cells or smooth muscle cells, and it controls the
cytoplasmic properties as well as the distribution of membrane
adhesion molecules (12, 37, 48).
The molecular details of cytoskeletal reorganization induced
by fluid shear stress in leukocytes, however, are unexplored.
This is the focus of the current report. The Rho family small
GTPases, which are members of the Ras superfamily of small
GTP-binding proteins, have been shown to play an essential
role in a variety of cellular functions, including the regulation
of actin cytoskeleton, membrane trafficking, gene transcription, oxidant generation, cell growth, chemotaxis, and cell
adhesion (20). Neutrophils have the ability to project thin
membrane-covered cytoplasm regions called pseudopods.
There are several general shapes, including veil-like lamellipodia, finger-like projections, filopodia, and uropodia during
pseudopod retraction. Pseudopod formation is mediated by
several signaling pathways, including the Rho family GTPases.
Activated cdc42 and Rac stimulate the Arp2/3 complex via
Wiskott-Aldrich syndrome protein (WASP) and WASP-family
verprolin protein, respectively (23, 51). The activated Arp2/3
complex serves as an actin-nucleating factor. Cdc42 forms
actin-rich filopodia and Rac stimulates the formation of lamellipodia and membrane ruffles (20). Although stress fibers or
focal adhesion sites are not formed in leukocytes, it has been
reported that RhoA regulates uropod detachment by stimulating actomyosin filaments (1, 54).
Thus we examine here the small GTPase activity in neutrophils during shear-induced pseudopod retraction. We use fresh
leukocytes and human promyelocytic HL-60 cells differentiated by dimethyl sulfoxide (DMSO) into neutrophil-like cells
(9, 22). Because appropriate expression vectors and transfection methods have been developed that allow expression of
exogenous proteins in HL-60 cell line (41), we use these cells
as part of the in vitro experiments.
We show that Rac1 and Rac2 make an important contribution to the fluid shear response as well as to the adhesion to
endothelium in postcapillary venules.
The 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.
0363-6143/05 $8.00 Copyright © 2005 the American Physiological Society
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Makino, Ayako, Michael Glogauer, Gary M. Bokoch, Shu
Chien, and Geert W. Schmid-Schönbein. Control of neutrophil
pseudopods by fluid shear: role of Rho family GTPases. Am J Physiol
Cell Physiol 288: C863–C871, 2005. First published November 24,
2004; doi:10.1152/ajpcell.00358.2004.—Blood vessels and blood
cells are under continuous fluid shear. Studies on vascular endothelium and smooth muscle cells have shown the importance of this
mechanical stress in cell signal transduction, gene expression, vascular remodeling, and cell survival. However, in circulating leukocytes,
shear-induced signal transduction has not been investigated. Here we
examine in vivo and in vitro the control of pseudopods in leukocytes
under the influence of fluid shear stress and the role of the Rho family
small GTPases. We used a combination of HL-60 cells differentiated
into neutrophils (1.4% dimethyl sulfoxide for 5 days) and fresh
leukocytes from Rac knockout mice. The cells responded to shear
stress (5 dyn/cm2) with retraction of pseudopods and reduction of
their projected cell area. The Rac1 and Rac2 activities were decreased
by fluid shear in a time- and magnitude-dependent manner, whereas
the Cdc42 activity remained unchanged (up to 5 dyn/cm2). The Rho
activity was transiently increased and recovered to static levels after
10 min of shear exposure (5 dyn/cm2). Inhibition of either Rac1 or
Rac2 slightly but significantly diminished the fluid shear response.
Transfection with Rac1-positive mutant enhanced the pseudopod
formation during shear. Leukocytes from Rac1-null and Rac2-null
mice had an ability to form pseudopods in response to plateletactivating factor but did not respond to fluid shear in vitro. Leukocytes
in wild-type mice retracted pseudopods after physiological shear
exposure, whereas cells in Rac1-null mice showed no retraction
during equal shear. On leukocytes from Rac2-null mice, however,
fluid shear exerted a biphasic effect. Leukocytes with extended pseudopods slightly decreased in length, whereas initially round cells
increased in length after shear application. The disruption of Rac
activity made leukocytes nonresponsive to fluid shear, induced cell
adhesion and microvascular stasis, and decreased microvascular density. These results suggest that deactivation of Rac activity by fluid
shear plays an important role in stable circulation of leukocytes.
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MATERIALS AND METHODS
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Biological materials and reagents. Human promyelocytic leukemic
HL-60 cells from the American Type Culture Collection were cultured in RPMI 1640 (Invitrogen) supplemented with 10% heatinactivated fetal bovine serum (Omega Scientific) in 5% CO2 at 37°C.
HL-60 cell differentiation was induced by treatment with 1.4%
DMSO (American Type Culture Collection) for 5 days. The rabbit
anti-Rac2 polyclonal antibody, GST-PBD, GFP-tagged T17NRac1,
T17NRac2, Q16LRac1, and Q16LRac2 have been described previously (4). The mouse anti-Rac1 monoclonal antibody was from Santa
Cruz Biotechnology (Santa Cruz, CA), and the Rho Activation Assay
Kit and mouse anti-cdc42 monoclonal antibody were obtained from
Upstate Biotechnology (Waltham, MA). The protease inhibitors,
PMSF, aprotinin, and leupeptin, and the chemoattractant, formylmethionyl-leucyl-phenylalanine (fMLP), and platelet-activating factor
(PAF) were from Sigma (St. Louis, MO).
Transfection of HL-60 cells. HL-60 cells were cultured in the
presence of 1.4% DMSO for 4 days to initiate differentiation before
transfection. On day 4, the cells were washed and suspended in
Opti-MEM reduced serum medium (Invitrogen) at a cell concentration of 2 ⫻ 106 cells/well. cDNA (4 ␮g) were suspended in OptiMEM and mixed with the cationic lipid 1,2-dimyristyloxypropyl-3dimethylhydroxyethyl ammonium bromide/cholesterol (Invitrogen, 4
␮l/well). The cationic lipid/cDNA mixture was added to the cells and
incubated at 37°C for 4 h. An equal volume of RPMI 1640 medium
containing 20% heat-inactivated fetal bovine serum plus DMSO
(1.4% final concentration) was then added, and the cells were cultured
for 20 h. On day 5, the cells were washed and suspended in fresh
RPMI that contained 10% FBS and were cultured for an additional
24 h before the experiment.
Animals. Conditional Rac1 knockout (Rac1c/⫺LysMcre/⫹) mice had
previously been generated by targeted disruption of the rac1 gene in
neutrophils (16). All experiments were compared in neutrophils isolated from Rac1c/⫺LysMcre/⫹ mice and Rac1⫹/⫹LyseMcre/⫹. Rac2⫺/⫺
mice (45) were backcrosses (⬎12 generations) into C57BL/6J mice
(Jackson Laboratory, Bar Harbor, ME). The mice used in these
experiments were 9 –17 wk of age. All animal procedures were
approved by the University of California, San Diego, Animal Subject
Committee.
Murine leukocyte isolation. Whole blood leukocytes and enriched
neutrophil populations were isolated from whole blood of anesthetized mice (50 mg/kg ip pentobarbital sodium). Anticoagulated blood
samples (10 U/ml ammonium heparin) were collected by cardiac
puncture using a 1-ml syringe with a 23-G needle. For whole blood
leukocytes, a leukocyte-rich buffy coat was obtained by centrifugation
at 200 g for 5 min at 4°C. Sporadic red blood cells were removed via
hypotonic lysis performed at 4°C. The mixture of platelets and
leukocytes was immediately resuspended with RPMI 1640 containing
10% FBS.
Human leukocyte isolation. Fresh leukocytes from finger prick
blood of volunteers were collected in capillary hematocrit tubes
(ammonium heparin prefilled; Clay Adams). The red blood cells were
allowed to sediment at room temperature for 1 h. The supernatant
mixture of platelets and leukocytes was resuspended in RPMI 1640
containing 10% FBS.
Fluid shear application in a flow chamber. The differentiated
HL-60 cells or mouse leukocytes were subjected to laminar shear flow
in a rectangular flow channel sandwiched with a gasket (230 ␮m
thick) between a coverslip and a glass plate (15). The chamber was
mounted on the stage of an inverted phase-contrast microscope
(model IX70, Olympus, Japan). All flow experiments were carried out
at 24°C. Cells were suspended in RPMI 1640 containing 10% FBS
and loaded into the flow chamber at a slow flow rate with wall shear
stress lower than ⬃1 dyn/cm2. Laminar shear stress was generated by
perfusion of HBSS (pH 7.4 adjusted with 100 ␮mol/l HEPES, containing Ca2⫹ and Mg2⫹ at 1 mmol/l each, Life Technologies) with an
automated syringe pump (Harvard Apparatus) attached to the inlet
side of the flow chamber.
Images of the cells were recorded via a ⫻60 objective and a ⫻1.6
eyepiece with a charge-coupled device camera (model VI-470,
Optronics) and stored digitally (Scion Image Software). Images were
recorded during 10 min of fluid shearing and for 10 min during the
recovery phase. Each cell contour was outlined manually and the area
was computed (Scion Image Software) (Fig. 1A). This measurement
will be referred to as projected cell area. Some of the results are shown
as normalized cell area (projected cell area divided by the average of
the projected cell area during the control period before shear).
Fluid shear application in a cone-and-plate device. We used the
cone-and-plate device to collect sufficient concentrations of protein
for the pull-down assay to measure small GTPase activity. This
approach permits the use of suspended cells. But the flow and shear
Fig. 1. Response of differentiated human leukemia (HL-60) cells and human
neutrophils to fluid shear. A: differentiated HL-60 cells before and after the
application of fluid shear stress (5 dyn/cm2). The perimeter of the cell was
outlined by hand, and the cell area was digitally computed with appropriate
length calibration. The length of the bar equals 10 ␮m. B and C: projected cell
area of differentiated HL-60 cells (B) and human neutrophils (C) during fluid
shear application at 5 dyn/cm2 for 10 min in the flow chamber. No fluid shear
was applied in the static control cells. Bar shows 10 ␮m. Static control cells:
open symbols, n ⫽ 6; sheared cells: closed symbols, n ⫽ 13. These numbers
refer to the total number of cells measured. *P ⬍ 0.05 compared with sheared
and static control cells at the same time point.
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NEUTROPHIL RESPONSE TO FLUID SHEAR
AJP-Cell Physiol • VOL
venules (25– 40 ␮m in diameter). Platelet-activating factor (PAF;
⬃10⫺9–10⫺8 M) was superfused to allow cells to project pseudopods.
Flow stasis with near zero shear stress was induced for 3–5 min by
obstruction at upstream locations of venular segments with a micropipette. The micropipette was then removed and the venule was returned
to physiological shear stress for 5 min. Leukocytes that remained on
the venular endothelium after shear restoration but did not adhere to
the wall for ⬎30-s period were used to determine the cell length. L
refers to maximum length across the cell (cell body plus pseudopod),
and L0 refers to the diameter of the cells in its spherical state without
pseudopods.
Another set of mice was used for determining the number of
adherent cells. A leukocyte was considered adherent to the venular
endothelium if it remained stationary within a preselected area of
venule for 30 s. After the experiments, cremaster muscle samples
were stained with FITC-labeled Bandeirea simplicifolia lectin
(Sigma) to outline the microvascular bed. Fluorescence images were
obtained using confocal microscopy (excitation/emission ⫽ 488/515;
model MRC-1024UV, Bio-Rad), and they were digitized and quantified using an automated computer vessel-counting method previously
described (43).
To measure the wall shear stress in the postcapillary venule, 0.5 ml
of fluorescently labeled erythrocytes from donor mouse (DiIC16,
Molecular Probes) was intravenously injected. The velocities of 75
labeled erythrocytes in each venule were measured using an intravital
fluorescence microscope with a silicone-intensified target camera
(DAGE-MTI). The wall shear stress on the endothelium in each
venule was estimated as 4 ⫻ mean velocity ⫻ plasma viscosity/
internal radius of the vessel, according to flow at a low Reynolds
number in a cylindrical blood vessel.
Statistical analysis. Measurements are presented as means ⫾ SE.
For statistical comparison, two-way ANOVA with repeated measurements was carried out, and the Bonferroni (Dunn) test was used as a
post hoc test. Comparisons between groups of the Western blot data
were carried out by unpaired Student’s t-test.
RESULTS
Differentiated HL-60 cell and human neutrophil response to
fluid shear. To examine how differentiated HL-60 cells respond to fluid shear, we applied a shear stress (5 dyn/cm2 for
10 min) to the cells while they adhered to the wall of the flow
chamber. Before shear, the cells were migrating on the coverslip and spreading their cytoplasm. Fluid shear stress induced
a rapid and significant reduction of projected cell area in the
differentiated HL-60 cells. During shear, the cells had a smaller
projected area with reduced cytoplasmic spreading. After cessation of fluid shear, there was no significant recovery of cell
spreading over a 10-min period (Fig. 1B). In line with HL-60
cells, application of fluid shear to adherent human neutrophils
caused significant retraction of projected pseudopods and also
the recovery of cell area was not seen during 10 min after shear
application (Fig. 1C).
Shear stress controls small GTPase activity in a time- and
magnitude-dependent manner. To assess whether physiological shear stress in a buffer solution (5.2 dyn/cm2 at a shear rate
of 450 s⫺1) affects the activity of the Rho family small
GTPases, we applied fluid shear to differentiated HL-60 cells
in the cone-and-plate device. The sheared cells were immediately lysed and subjected to a pull-down assay to detect the
active forms of small GTPases. Rac1 and Rac2 activities were
significantly decreased by fluid shear stress applied for 5 min,
whereas cdc42 exhibited no significant change at any time
point up to 10 min (Fig. 2A). RhoA activity was transiently
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stress pattern on the leukocyte membrane is different from that in the
parallel plate flow chamber (49). One main difference between the
two situations is that leukocytes in free suspension experience on their
membrane higher rates of change of shear stress with respect to time
compared with cells adhering to a substrate surface, e.g., in the flow
chamber. Leukocytes in the circulation obviously experience both
situations as they are carried in the free circulation to a position where
they adhere to the endothelium.
We suspended 107 cells with 0.5 ml of HBSS and subjected to
shear stress in the cone-and-plate device (model DV II, Brookfield,
Middleboro, MA). The device consists of a rotating 0.8° cone placed
over a stationary plate. The gap between the cone and plate at the
center is 12.5 ␮m. The experimental apparatus was maintained at
24 –25°C. To apply fluid shear stress, the cell suspensions were placed
in the gap between the cone and plate. The cone turned at a constant
rotational speed, generating a uniform fluid shear field to the entire
cell suspension.
Rac, cdc42, and Rho pull-down assays. Rac and cdc42 activation
assays were performed as described (4). Immediately after the cessation of shear in the cone-and-plate device, an equal volume of lysate
buffer (0.5 ml; 100 mmol/l Tris 䡠 HCl, pH 7.4, 1 mol/l NaCl, 2 mmol/l
MgCl2, 1% Igepal, 20% glycerol, 2 mmol/l EGTA, 20 ␮g/ml leupeptin, 20 ␮g/ml aprotinin and 2 mmol/l PMSF) was added to arrest cell
activation. The cell lysate was centrifuged and 600 ␮l of the supernatant was incubated with PAK1-PBD agarose (10 ␮g) for 1 h at 4°C,
washed three times, and eluted with SDS sample buffer. Total Rac or
cdc42 proteins were measured using 10 ␮l of lysate. Rac1, 2, and
cdc42 proteins were detected with Western blot analysis using antiRac1 mouse monoclonal antibody (dilution 1:2,000), anti-Rac2 rabbit
polyclonal antibody (1:2,000), and anti-cdc42 mouse monoclonal
antibody (1:1,000), respectively. To measure the activation of Rho,
the cells were lysed in 500 ␮l of lysate buffer and 800 ␮l of the lysate
were incubated with Rhotekin RBD agarose (20 ␮g, Upstate Biotechnology) following the manufacturer’s recommendations. The total
Rho present in 10 ␮l of the lysate was measured before incubation
with Rhotekin RBD. Total Rho and activated Rho bound to RhotekinRBD were detected by Western blot analysis with the use of antiRhoA mouse polyclonal antibody (1:2,000). The immunoblots were
detected with the ECL Western blotting detection reagents (Amersham Biosciences). Densitometric analysis was performed using
Scion Image software. The densitometric quantification was attained
from the ratio of the active form and the total GTPase protein.
Surgical procedure for intravital microscopy. Male mice were
anesthetized with pentobarbital sodium (50 mg/kg ip). Anesthesia was
maintained with supplemental doses of pentobarbital sodium (5 mg/kg
iv) as needed. The left femoral vein was cannulated for the administration of supplemental anesthesia. Mice were placed in a dorsal
recumbent position on a microscope stage, maintained at 37°C with an
optically clear viewing pedestal, and surgically prepared. Briefly, an
incision was made in the scrotal skin to expose the left cremaster
muscle, which was then carefully removed from the associated fascia.
A lengthwise incision was made on the ventral surface of the cremaster muscle using cautery. The testicle and the epididymis were
separated from the underlying muscle and moved into the abdominal
cavity. The muscle was then spread over an optically clear view
pedestal and secured along the edges with 6-0 thread. The exposed
tissue was superfused with warm Krebs-Henseleit solution (KHS, pH
7.4, 35–37°C) that was bubbled with a mixture of 5% CO2 and 95%
N2. The cremaster postcapillary venules were observed through an
intravital microscope (Leitz) with a ⫻60 water-immersion objective
lens (Olympus). The images were recorded at various time points
during the experiment with the use of a color charge-coupled device
television camera (model DEI-470, Optronics, Goleta, CA) and stored
on a videocassette recorder (VHS model BR-S601MU; JVC).
Experimental protocol for intravital microscopy. Measurements
were obtained after a 30-min stabilization period with constant KHS
suffusion. Leukocytes were observed in the unbranched cremaster
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NEUTROPHIL RESPONSE TO FLUID SHEAR
Fig. 2. Effects of fluid shear stress on GTPase activities in differentiated
HL-60 cells. A: time course of each activity after shear stress (5.2 dyn/cm2) for
10 min. B: activities in response to different shear stress magnitudes. The
differentiated HL-60 cells were subjected to fluid shear for 10 min before
measurements of Rac1, Rac2, and cdc42 activities, or 2 min before Rho
activity measurement. The fluid shear was applied to the cells in the coneand-plate device, and the activity was measured by pull-down assay. The
activity of each GTPase was calculated as the amount of GTP-bound form
relative to the total GTPase and normalized by the static control value. Values
are means ⫾ SE from three to four independent experiments. *P ⬍ 0.05
compared with static control.
increased after 2 min of shear and returned to its control level
at 10 min (Fig. 2A). On the basis of these results, we examined
the effect of the shear stress magnitude on the small GTPase
activities. We applied fluid shear at different magnitudes (0,
1.2, 2.5, and 5.2 dyn/cm2) to the differentiated HL-60 cells for
10 min (in the cases of Rac and cdc42 activities) or for 2 min
(in the case of Rho activity) (Fig. 2B). A shear stress at 5.2
dyn/cm2 caused a significant reduction in Rac1 and Rac2
activities, no significant change in cdc42 activity, and a marked
enhancement of the RhoA activity.
Shear stress-induced reduction in projected cell area is
associated with deactivation of Rac1 and Rac2. To investigate
the role of Rac in fluid shear-induced reduction of the projected
area of differentiated HL-60 cells, we tested whether positive
or negative Rac mutant affects the cellular response to shear
stress (Fig. 3). GFP fused to dominant negative (T17N) or
positive (Q61L) Rac was transiently transfected into the differentiated HL-60 cells. cDNA encoding for only GFP was
transfected into the cells as a control. T17N Rac1 weakened the
shear stress-induced reduction in projected cell area, whereas
AJP-Cell Physiol • VOL
Fig. 3. Effect of fluid shear on HL-60 cells transfected with negative or
positive mutant of Rac. Open symbols show the response of control cells
transfected with green fluorescent protein (GFP) only. Closed symbols show
the response of the cells transfected either with negative (T17N) or positive
(Q61L) mutants of Rac1 or Rac2. The values for the projected cell area were
normalized by those under static condition. Values are means ⫾ SE. Shear
stress (5 dyn/cm2) was applied for 10 min in the flow chamber, followed by 10
min of stasis. *P ⬍ 0.05 compared with control cells.
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Q61L Rac1 caused an increase in the projected cell area after
shear application. Rac2 mutants blocked the shear-induced
reduction in cell area.
fMLP increased small GTPase activities, inhibited shear
stress-induced deactivation of Rac1 and Rac2 and blocked the
cell response to fluid shear. fMLP, which increases small
GTPase activities and enhances the migration of human neutrophils (4, 7, 57), was used to test whether it may affect the
shear stress-induced small GTPase activities (Fig. 4). The
differentiated HL-60 cells were suspended in HBSS with fMLP
(10⫺9 M) and sheared in the cone-and-plate device. Each
GTPase activity was measured in unsheared (static) and
sheared (5.2 dyn/cm2 for 10 min) cells in the absence or
presence of fMLP. Under static conditions, fMLP markedly
increased the activities of all three GTPases. The shear stressinduced downregulation of Rac activity (Fig. 2) was abolished
by fMLP pretreatment, and actually caused a significant increase in Rac2 activity above that under static condition (Fig.
4A). fMLP pretreatment also caused significant increases in
cell spreading as measured by the projected cell area of
differentiated HL-60 cells. Real-time images of the cells suspended in culture medium with fMLP (10⫺9 M) also confirm
NEUTROPHIL RESPONSE TO FLUID SHEAR
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Fig. 4. Effect of fMLP on the shear-induced change in small GTPase activities. A: differentiated HL-60 cells were subjected to shear stress at 5.2 dyn/cm2
for 10 min in the cone-and-plate device. The activity of each GTPase was
calculated as the amount of GTP-bound form relative to the total GTPase, and
the data were normalized by the static control value in presence of fMLP.
Values are means ⫾ SE from 4 to 6 independent experiments. *P ⬍ 0.05
comparison with fMLP-treated static cells. B: effect of fMLP on the change in
projected cell area in response to fluid shear. After 1 h of equilibration in the
presence or absence of fMLP, the fluid shear was applied at 5 dyn/cm2 in the
flow chamber. Typical photomicrographs show the response of fMLP-treated
cell to the fluid shear. Bar shows 10 ␮m. Open symbols represent control cells
(n ⫽ 13); closed symbols represent fMLP-treated cells (n ⫽ 8). *P ⬍ 0.05,
compared with fMLP-treated and control cells.
that the projected cell area does not decrease during fluid shear
(Fig. 4B).
Leukocytes from both Rac1-null mice and Rac2-null mice do
not exhibit shear-induced pseudopod retraction in vitro. Leukocytes from Rac-deficient mice exhibited enhanced spreading
activity in the resting state (spreading cells: wild type, 2.38 ⫾
1.8%; Rac1-null, 7.46 ⫾ 1.8%; Rac2-null, 7.18 ⫾ 3.9%), and
over half of the cells from each group maintained the ability to
form pseudopods in response to 5 ⫻ 10⫺8 M PAF (cells with
small pseudopods ⫹ spreading cells: wild type, 59.9 ⫾ 3.4%;
Rac1-null, 51.6 ⫾ 5.3%; Rac2-null, 59.8 ⫾ 5.1%) (Fig. 5A).
The response of leukocytes, which had pseudopods without
stimulation, to fluid shear was examined in the flow chamber.
Fluid shear (5 dyn/cm2) induced a reduction of cell area in
wild-type leukocytes, whereas cells taken from either Rac1null or Rac2-null mice not only lost their response to the fluid
shear but also markedly increased their cell area during shear
exposure (Fig. 5B).
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Fig. 5. Phenotype of Rac-deficient neutrophils in vitro. A: the response to the
5 ⫻ 10⫺8 M platelet-activating factor (PAF) of each cell. Each group has 4
mice and includes ⬎50 cells per mouse. B: effect of Rac deficiency on the
shear-induced pseudopod retraction. The fluid shear was applied at 5 dyn/cm2
in the flow chamber. Open symbols show the response of wild-type neutrophils
(n ⫽ 5), and closed symbols show the response of Rac-deficient neutrophils
(Rac1-null; n ⫽ 5, Rac2-null; n ⫽ 11). *P ⬍ 0.05, between wild-type and
Rac-deficient cells at the same time point.
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In vivo leukocyte response of Rac-deficient mice to physiological shear. Selected postcapillary venules were occluded by
a micropipette for 3–5 min, during which time the leukocytes
were allowed to form pseudopods. After occlusion release, the
cells were exposed to physiological shear stress (12.6 ⫾ 0.7
dyn/cm2). We encountered a wide variety of cell length responses and classified them into two groups according to their
initial values, i.e., initially more extended (L/L0 ⬎ 1.4) and less
extended (L/L0 ⱕ 1.4) cells. This approach is optimal to
summarize the different cell responses we encountered.
Both more and less extended cells in wild-type mice reduced
their length during fluid shear. In contrast, cells from Rac1-null
mice exhibited no change of their cell shape during shear. In
Rac2-null mice, cells that were initially more extended decreased their length but did not reach a round shape. In
contrast, cells that were initially less extended increased their
length in response to fluid shear (Fig. 6).
Rac-deficient mice exhibited vascular stasis in response to
PAF and had lower microvascular density than wild-type mice.
Compared with the wild-type mice, Rac1-null and Rac2-null
mice had significantly lower body weight. After treatment with
PAF, both forms of Rac-deficient mice significantly increased
the number of leukocytes adhering to, and readily induced
stasis in, the postcapillary venules (Table 1). Rac1-null mice
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NEUTROPHIL RESPONSE TO FLUID SHEAR
had a lower microvascular density in their cremaster vascular
bed compared with wild-type mice.
DISCUSSION
Previous in vitro studies suggest that human neutrophils in
an activated state respond to fluid shear by pseudopod retraction (14, 37, 48), whereas spherical leukocytes without pseudopods may actually project pseudopods when exposed to fluid
shear (10). Trigger signals to induce pseudopod retraction or
projection may involve calcium influx and nitric oxide production (14, 37). We demonstrate here that the shear-induced
signaling pathway is mediated by Rho family small GTPases.
Specifically, Rac controls shape adjustment of leukocytes in
Table 1. Phenotype of knockout mice
Body weight, g
Microvascular density,
mm/mm3
Adherent cells, cells/
80 ␮m
Control
PAF (10⫺9 M)
PAF (10⫺8 M)
Time to stasis in
postcapillary
venules, min
PAF (10⫺9 M)
PAF (10⫺8 M)
Wild Type
Rac1-Null
Rac2-Null
28.2⫾0.7 (12)
25.3⫾0.6*(11)
26.1⫾0.5*(10)
1951⫾232 (5)
1411⫾97*(6)
1641⫾146 (5)
0⫾0 [12]
1⫾1 [9]
1⫾0 [4]
⬎120 (5)
⬎120 (4)
2⫾1 [18]
6⫾1*[7]
26⫾5*[10]
1⫾0 [13]
12⫾2*[5]
16⫾2*[9]
⬎120 (4)
93⫾16*(4)
⬎120 (4)
96⫾9*(5)
Values are means ⫾ SE. PAF, platelet-activating factor. Parentheses show
the number of mice. Brackets show the number of observed areas. Adherent
cells were counted in an area of 30 ␮m (width) ⫻ 80 ␮m (length). *P ⬍ 0.05,
significant difference between wild-type and knockout mice.
AJP-Cell Physiol • VOL
response to fluid shear in vitro and in vivo. Physiological fluid
shear induces retraction of lamellipodia with a reduction of the
projected cell area in human neutrophils as well as in differentiated HL-60 (Fig. 1). The cell responses we see here are all
evoked by physiological but small fluid shear stresses (5
dyn/cm2 is ⬃5/981 cmH2O) far below the values needed to
achieve a passive viscoelastic response (⬃100 to 1,000 dyn/
cm2), representing a short-term reaction compared with the
majority of longer-term shear responses reported for endothelial cells or smooth muscle cells. The current studies show the
response for a period of ⬍10 min, a short period compared
with the typical 1 to 24 h required to establish steady state
elongation of endothelial cells (in the direction of fluid shear)
or smooth muscle cells (perpendicular to fluid shear) (6, 31,
52, 53).
A key concern was the use of different measures to quantify
the rather complex shape changes encountered in leukocytes
forming pseudopods. In pilot studies, we experimented with
three different morphological parameters to describe the cell
response to shear stress; viz., projected cell area, longest length
across the cell, and a cell shape index S ([cell area]2/[perimeter
length]/4␲) (data not shown). Because the cells have small
pseudopods, the longest length across the cell or the shape
index were not particularly sensitive indicators. Instead, the
projected cell area was found to be the most sensitive index for
detecting the morphological response to shear in vitro, and it
was used in this analysis.
Neutrophil motility depends on the cycling of actin protein
subunits between monomeric and polymeric pools and the
reversible cross-linking of the polymeric actin into threedimensional networks. There are multiple mechanisms for the
control of the actin state. These mechanisms are not mutually
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Fig. 6. Response of neutrophils to fluid shear in
vivo. A: photomicrographs of wild-type neutrophils during shear application. L refers to maximum length across the cell and L0 refers to the
diameter of the cells in its spherical state without
pseudopods. W indicates leukocyte, and EC indicates endothelial cell. The length of the bar equals
10 ␮m. B: after 3- to 5-min occlusion of postcapillary venule, the cells were again exposed to
physiological levels of fluid shear. The exact neutrophil length in a high red cell population during
stasis cannot be exactly recognized due to light
scatter by the surrounding red cells. We therefore
determined the neutrophil length at the instant
(⬃60 –90 ms) after restoration of blood flow,
which preceded any shape change of leukocytes
under these experimental conditions. Open symbols represent the cells with initial L/L0 ⱕ 1.4, and
closed symbols represent those with initial L/L0 ⬎
1.4. The numbers of those two types of cells in
each panel are, respectively, 12 and 11 for the
wild-type, 5 and 6 for Rac1-null, and 11 and 14 for
Rac2-null.
NEUTROPHIL RESPONSE TO FLUID SHEAR
AJP-Cell Physiol • VOL
fMLP is an effective chemoattractant for human neutrophils
(4, 46). fMLP (10⫺9 M) also markedly increases the activities
of all three Rho family small GTPases in differentiated HL-60
cells. In the presence of fMLP, the shear stress-induced Rac
deactivation was completely abolished (Fig. 4A). In line with
these results, fluid shear did not induce a reduction of the
projected cell area after pretreatment with fMLP (Fig. 4B),
suggesting again that the deactivation of Rac plays a major role
in the retraction of pseudopod by physiological fluid shear
stress.
Neutrophils from Rac1-null or Rac2-null mice maintain the
ability to form pseudopods in response to PAF (Fig. 5A). In
contrast, the response of neutrophils from both types of null
mice to fMLP was weakened compared with neutrophils from
the wild-type mice (16, 17, 45). Although both fMLP and PAF
bind to the receptor and initiate cell migration, their signaling
pathways are slightly different (3, 26, 38).
When examined in the living microcirculation, neutrophils
reduced their cell length in response to fluid flow. The response
was faster than the one we observed in vitro (Fig. 6), in part
because the wall shear stress in vivo was higher than the one
used in vitro (12.6 ⫾ 0.7 vs. 5 dyn/cm2). Most of the leukocytes adhering to postcapillary endothelium are neutrophils
with occasional monocytes (⬍3%), but no detectable lymphocytes, eosinophils, or basophils (37). Rac1-null neutrophils
showed no change of cell length after fluid shear exposure.
Fluid shear, however, produces a biphasic response in Rac2null leukocytes. Cells that had longer pseudopods at the time of
fluid shear stress application exhibited a slight decrease in cell
length, whereas initially round cells showed an increase in
length after shear application. The shear-induced cell response
was affected by Rac1 deletion more than Rac2 deletion, an
effect that might occur because mouse neutrophils have higher
Rac1 than Rac2 expression, contrary to the dominant Rac2
expression in human neutrophils (28).
Fluid shear-induced retraction of neutrophil pseudopods
plays an important role in the microcirculation. The retraction
of pseudopods serves to minimize leukocyte entrapment in
capillaries, and the circulation of leukocytes with projected
pseudopods may cause significant flow reduction in capillaries
(13, 50).
Pseudopods also facilitate leukocyte spreading on the endothelium and may induce microvascular entrapment and capillary stasis (21, 44). We observed that Rac1-null neutrophils
exhibited enhanced membrane adhesion compared with wildtype neutrophils (Table 1). The density of a microvascular
network depends on a balance between angiogenesis and cell
death. Adherence of neutrophils to endothelial cells is the first
step of diapedesis and infiltration into the medium, where they
affect the smooth muscle cells and the connective tissue with
cell death and tissue alterations. Rac1-null mice showed fewer
microvessels than the wild-type mice. Ti et al. (17) reported
that Rac1-deficient hematopoietic stem/progenitor cells
showed a significantly reduced ability to form cells. The high
adherence of leukocytes and low ability for cell formation by
hematopoietic stem/progenitor cells might cause less microvascular density in cremaster muscle of Rac1-null mice.
It is not clear how neutrophils decrease Rac activation in
response to fluid shear. We recently reported that fluid shear
serves to cleave CD18 binding sites (12), one of the integrins
that can induce Rac activation. It is possible that the cleavage
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exclusive, but small GTPases of the Rho family, the bestknown members of which are Rac1, Rac2, cdc42, and RhoA,
are prominent membrane-targeted proteins that function as
critical regulators of actin cytoskeletal remodeling in neutrophils (57). Mechanical fluid stresses may also regulate the state
of actin in a number of ways. Rho family GTPases are involved
in cell motility induced by different mechanical stresses in
endothelial and smooth muscle cells. In endothelial cells, fluid
shear stress induces cytoskeletal reorganization mediated by
Rac1 (52) or Rac1 and RhoA (53). The inhibition of RhoA
activity with the cell-permeable C3 toxin reduces the stretchdependent actin synthesis of vascular smooth muscle (56). The
early effect of the stretch, which is associated with reduced cell
polarization and directionality of smooth muscle cells, is mediated by Rac1 activity (27). In human neutrophils, osmotic
pressure induces actin polymerization and cytoskeleton remodeling via activation of Rac and cdc42 (33). When differentiated
HL-60 cells are subjected to physiological levels of shear
stress, Rac1 and Rac2 activities are significantly decreased by
shear stress in a time- and magnitude-dependent manner (Fig.
2). Because Rac induces lamellipodia formation (20), these
results agree with the morphological picture of differentiated
HL-60 cells under shear stress in the flow chamber because the
cell area change was induced by the lamellipodia retraction.
RhoA activity was transiently increased after shear stress
application and returned to control level by 10 min, whereas
cdc42 activity exhibited no change at any time or magnitude of
shear stress (up to 5.2 dyn/cm2). Interestingly, this finding is
similar to the response of endothelial cells to shear stress,
although neutrophils do not make actin stress fibers. RhoA
might facilitate cell detachment from the glass surface in the
early stage as a regulator of uropod retraction, possibly by
stimulating actomyosin filament contraction or actin depolymerization (1, 54). Cdc42 induces extension of filopodia,
finger-like protrusions in the form of parallel arrays of actin
filaments (20), but filopodia were rarely observed compared
with lamellipodia in the flow chamber. In this study, neither a
shift in cdc42 activity nor filopodia formation was observed.
GTPases function as molecular switches by cycling between
an inactive GDP-bound form and an active GTP-bound form.
Several proteins, such as guanine exchange factors and
GTPase-activating proteins, regulate the ratio of GTP/GDPbound forms, which determines the activities of Rho GTPases
(20). Mutations that disrupt the cycling of the two forms of
GTPases have been identified in the guanine nucleotide binding domain or the effector domain. Mutations at positions 12
(G12V) and 61 (Q61L) inhibit GTP hydrolysis to produce
constitutively active mutants (55), and a mutation at position
17 (T17N) destabilizes the guanine nucleotide-binding site to
result in a dominant negative mutant (36). Because there is no
selective chemical inhibitor or activator for Rac only, these
mutants are used to test the role of Rac in the signaling
pathway. Both T17NRac1 and T17NRac2 diminished the shear
stress-induced reduction of the projected cell area in the flow
chamber (Fig. 3). Together with the ability of Q61LRac1 and
Q61LRac2 to block the shear stress-induced reduction of cell
motility, these data suggest that deactivation of Rac is required
for the reduction of projected cell area in response to fluid
shear stress. This result was also supported by the data with the
cells taken from Rac-deficient mice (Fig. 5B).
C869
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NEUTROPHIL RESPONSE TO FLUID SHEAR
of CD18 induced by fluid shear decreases spontaneous Rac
activity. Further studies are required to clarify the signaling
mechanisms that cause cell retraction by fluid shear stress.
Rac has two major functions: 1) It regulates the organization
of the actin cytoskeleton, and 2) it controls the activity of the
NADPH oxidase enzyme complex, which is capable of producing superoxide. The lack of Rac activity may represent a
risk factor for various cardiovascular complications because of
the deficient leukocyte retraction in response to fluid shear in
addition to unbalanced superoxide generation.
17.
18.
19.
ACKNOWLEDGMENTS
20.
21.
The authors thank Daria Theodora for assistance with the tissue cultures and
Gerard Norwich for technical assistance with the microscopy.
22.
This work was supported by National Institute of Health Program Project
Grant HL-43026 (to G. W. Schmid-Schönbein) and Grant GM-39434 (to G. M.
Bokoch).
23.
24.
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