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PHAGOCYTES, GRANULOCYTES, AND MYELOPOIESIS
The GTPase-activating protein ARAP3 regulates chemotaxis and
adhesion-dependent processes in neutrophils
*Laure Gambardella,1 *Karen E. Anderson,1 Claudia Nussbaum,2 Anne Segonds-Pichon,3 Tânia Margarido,1 Laura Norton,1
Thomas Ludwig,4 Markus Sperandio,2 Phillip T. Hawkins,1 Len Stephens,1 and Sonja Vermeren1
1Inositide
Laboratory, The Babraham Institute, Cambridge, United Kingdom; 2Walter-Brendel-Zentrum, Ludwig Maximilians Universität München, Munich, Germany;
of Bioinformatics, The Babraham Institute, Cambridge, United Kingdom; and 4Institute for Cancer Genetics, Columbia University, New York, NY
3Department
Neutrophils form a vital part of the innate
immune response, but at the same time
their inappropriate activation contributes
to autoimmune diseases. Many molecular
components are involved in fine-tuning
neutrophil function. We report here the
first characterization of the role of ARAP3,
a PI3K and Rap-regulated GTPaseactivating protein for RhoA and Arf6 in
murine neutrophils. We show that neutro-
phils lacking ARAP3 are preactivated in
vitro and in vivo, exhibiting increased ␤2
integrin affinity and avidity. ARAP3-deficient neutrophils are hyperresponsive in
several adhesion-dependent situations in
vitro, including the formation of reactive
oxygen species, adhesion, spreading, and
granule release. ARAP3-deficient cells adhere more firmly under flow conditions in
vitro and to the vessel wall in vivo. Finally,
loss of ARAP3 interferes with integrindependent neutrophil chemotaxis. The results of the present study suggest an important function of ARAP3 downstream of Rap.
By modulating ␤2 integrin activity, ARAP3
guards neutrophils in their quiescent state
unless activated. (Blood. 2011;118(4):
1087-1098)
Introduction
Neutrophils are an essential component of the innate immune
responses to invading pathogens and tissue damage. In response to
inflammatory stimuli, neutrophils are recruited from the bloodstream to infected tissues, where they produce reactive oxygen
species (ROS) and phagocytose and kill pathogens.1 It is vital that
the neutrophil response to inflammatory stimuli is well controlled.
Leukocyte adhesion deficiency (LAD) patients, in whom the
recruitment process is impaired, suffer from repeated, severe
bacterial infections. Conversely, excessive neutrophil responsiveness is also detrimental, causing tissue damage and inflammation,
as exemplified in autoimmune diseases.
Work aimed at identifying the molecular machinery controlling
neutrophils has identified important regulatory players. Rap isoforms regulate adhesion-dependent processes by modulating integrin affinity and avidity.2 Loss of leukocyte Rap1 activation is a
cause of LAD-III,3,4 demonstrating the crucial role of this small
GTPase in neutrophil control. Agonist-activated PI3Ks generate on
activation the lipid second messengers phosphatidylinositol-(3,4,5)trisphosphate [PtdIns(3,4,5)P3] and PtdIns(3,4)P2. These cause plasma
membrane recruitment and activation of PI3K effectors, which regulate
multiple enzymes and pathways.5 Known PI3K effectors include
regulators of Rho and Arf family small GTPases, GTPase-activating
proteins (GAPs), and guanine nucleotide exchange factors. Rho family
small GTPases are involved in regulating the neutrophil NADPH
oxidase and chemotaxis,6,7 whereas Arf family members regulate
phagocytosis, ROS production, and chemotaxis.8,9
ARAP3 was identified from porcine neutrophils in a screen for
PtdIns(3,4,5)P3–binding proteins. In vitro, ARAP3 is a PtdIns(3,4,5)P3–
dependent Arf6 GAP and a Rap-activated RhoA GAP. In addition, in
vivo, PtdIns(3,4,5)P3 drives the recruitment of ARAP3 to the plasma
membrane, bringing it into the vicinity of its activator, Rap-GTP, and its
substrates, RhoA-GTP and Arf6-GTP.10,11 The remaining ARAP family
members (ARAP1/2) share ARAP3’s multidomain structure.12,13 We
sought to identify ARAP3’s physiologic function in neutrophils. Using
neutrophils from a conditional Arap3⫺/⫺ mouse model in biologic
assays, we demonstrate that ARAP3 regulates adhesion-dependent
processes. Loss of ARAP3 causes the preactivation of neutrophil ␤2
integrins and hyperresponsiveness in adhesion-dependent situations,
feeding into several biologic responses such as adhesion-dependent
ROS formation, granule release, and chemotaxis. Whereas Rap activity
was not affected by the loss of ARAP3, RhoA activation was increased
in an adhesion-dependent setting, suggesting a role for ARAP3 downstream of Rap in neutrophils. Our results suggest a modulatory role for
ARAP3 in neutrophils, which prevents their activation in inappropriate
situations.
Submitted October 13, 2010; accepted April 1, 2011. Prepublished online as
Blood First Edition paper, April 13, 2011; DOI 10.1182/blood-2010-10-312959.
The online version of this article contains a data supplement.
*L.G. and K.E.A. contributed equally to this work.
An Inside Blood analysis of this article appears at the front of this issue.
BLOOD, 28 JULY 2011 䡠 VOLUME 118, NUMBER 4
Methods
Unless otherwise stated, materials were obtained from Sigma-Aldrich.
Inducible Arap3ⴚ/ⴚ mouse model
Generation of the Arap3fl/fl mouse has been described previously.14 For the
inducible knockout, Arap3fl/fl mice were crossed with Rosa-ERT2Cre⫹
mice.15 Cre expression was induced by a single intraperitoneal injection
with tamoxifen, as described previously,16 which caused a complete but
transient deletion of ARAP3 in bone marrow–derived neutrophils. Mice
were housed in open racks or individually ventilated cages in small animal
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
© 2011 by The American Society of Hematology
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BLOOD, 28 JULY 2011 䡠 VOLUME 118, NUMBER 4
GAMBARDELLA et al
barrier units at the Babraham Institute or Walter-Brendel-Zentrum and used
11 or 12 days after induction. Animal work carried out at the Babraham
Institute and Walter-Brendel-Zentrum was approved by United Kingdom
Home Office Project licenses PPL80/1875 and PPL80/2335 and Regierung
Oberbayern, Germany (AZ55.2-1-54-2531-134/08), respectively.
Neutrophil purification
Neutrophils were isolated from the bone marrow of sex- and age-matched
mice 10-14 weeks of age with discontinuous Percoll gradients (Amersham),
as described previously,17,18 using endotoxin-free reagents throughout.
Analysis of peripheral blood
Tail vein blood was collected in EDTA-coated microvettes (Sarstedt) for
analysis using a Vet ABC animal blood cell counter.
ROS production assays
ROS production was measured by chemiluminescence using a luminolbased assay in polystyrene 96-well plates (Berthold Technologies), as
described previously.19 Cells (5 ⫻ 105) were incubated with luminol
(150␮M) and HRP (18.75 U/mL) for 10 minutes at 37°C. Where indicated,
neutrophils were added manually to wells precoated with fibrinogen
(150 ␮g/mL) or polyRGD (20 ␮g/mL) with or without murine TNF␣
(20ng/mL); or to immobilized immune complexes (100␮g/mL IgG-BSA).
For soluble agonist assays (fMLF [fMet Leu Phe] 10␮M final concentration), cells were primed for 1 hour at 37°C in the absence (mock) or
presence of mouse TNF␣ (1000 U/mL) and GM-CSF (100 ng/mL). For all
assays, measurements were started immediately and light emission was
recorded. Data output was in relative light units (RLUs) per second or total
RLUs integrated over the indicated measured periods of time.
Chemotaxis assays
For micropipette chemotaxis assays, neutrophils were resuspended in
HBSS, 15mM HEPES, pH 7.4, and 0.05% BSA, and allowed to attach to a
glass coverslip. Cells were stimulated at 37°C with a point source of fMLF
delivered from a microinjection pipette (Femtotip; Eppendorf) using
50 mbar of pressure, as described previously,18 and monitored by time-lapse
imaging for 30 minutes using an inverted Zeiss Axiovert microscope and
Axiovision v3.0 software. Chemotaxis assays involving Dunn chambers or
EZ-TAXIS chambers were carried out as described previously18,20 using
fMLF as the chemoattractant. Chemotaxis in transwells was assayed using
3-␮m-pore polycarbonate filter units (Millipore) as described previously.18
Chemotaxis in a 3D collagen matrix was as described previously,21,22 except
that neutrophils were prepared by centrifugation over a discontinuous
Percoll gradient without prestimulation with TNF␣. Dulbecco PBS was
used to dilute rat tail collagen (Roche). Cells were tracked using the
“manual tracking” plug-in and tracks were analyzed using the “chemotaxis
tool” plug-in (Ibidi) in ImageJ v1.37 software.
MAPK, Erk, and PKB activation assays
MAPK, Erk, and PKB activation assays were carried out essentially as
described previously,23 with cells plated onto polyRGD-coated or heatinactivated FCS (hiFCS)–blocked dishes. For details, see supplemental
Methods (available on the Blood Web site; see the Supplemental Materials
link at the top of the online article).
Figure 1. Arap3 is inducibly knocked out in neutrophils. (A) Bone marrow–derived
neutrophils were isolated from femurs and tibias of tamoxifen- and mock-induced
Arap3fl/flERT2Cre⫹ (iko), Arap3⫹/⫹ERT2Cre⫹ (iCre), and Arap3fl/fl (floxed) mice. Cells were
boiled in sample buffer and proteins were separated by SDS-PAGE, transferred to PVDF
membranes, and subjected to blotting using an anti-ARAP3 antiserum (ARAP3).10 Blots
were reprobed using ␤-COP as a loading control (␤-COP); 1 ⫻ 106 cells were loaded per
lane. (B) Peripheral blood cells from tamoxifen- and mock-induced Arap3fl/flERT2Cre⫹
mice were analyzed using a Vet ABC animal blood cell counter. Data shown are pooled
from 2 separate experiments (means ⫾ SEM; n ⫽ 18).
Reconstitutions
Cohorts of C57B/6 mice were lethally irradiated and subsequently reconstituted by tail vein injection with 4 ⫻ 106 bone marrow cells from Arap3
model mice or from sex- and age-matched controls.
Ex vivo flow chamber assay
Rectangular glass capillaries (VitroCom) with a cross-section of
40 ␮m ⫻ 400 ␮m were coated with rmE-selectin (20 ␮g/mL), rmCXCL1
(keratinocyte-derived chemokine, 15 ␮g/mL), and rmICAM-1 (15 ␮g/mL),
and served as ex vivo flow chambers, as described previously25 (see
supplemental Methods for details).
Intravital microscopy of the cremaster muscle
Degranulation assay
Intravital microscopy was performed essentially as described previously26
(see supplemental Methods for details).
Release of gelatinase granules after plating onto a fibrinogen-coated
surface, or as induced by stimulation with fMLF and cytochalasin B, was
detected by zymography, as described previously.24
Whole mount histology
Analysis of ␤2 integrins
Surface integrins were visualized by FACS analysis of stained cell
populations. Integrin clustering was analyzed by confocal microscopy of
unstimulated, stained cells and used as a readout for ␤2 integrin avidity. To
measure integrin affinity, unstimulated cells were allowed to bind to
ICAM1-Fc in solution; bound ICAM1-Fc was measured using quantitative
immunofluorescence (see supplemental Methods for details).
Cremaster muscle whole mounts were prepared as described previously.25
Leukocytes were Giemsa stained in fixed tissues and analyzed microscopically (see supplemental Methods for details).
Small GTPase activity assays
RhoA-GTP was determined by G-LISA assay (Cytoskeleton).27 Arf6-GTP
and Rap1-GTP were identified by pull-down assay, as described previously28,29 (see supplemental Methods for details).
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BLOOD, 28 JULY 2011 䡠 VOLUME 118, NUMBER 4
ARAP3 REGULATES NEUTROPHILS
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Figure 2. Increased adhesion-induced ROS production in ARAP3-deficient neutrophils. Bone marrow–derived neutrophils from tamoxifen- and mock-induced
Arap3fl/flERT2Cre⫹ mice were prepared (A-B), primed with TNF␣ and GM-CSF or mock primed, and (all) preincubated with luminol as described in “ROS assays.” Cells
(5 ⫻ 105) were plated into 96-well plates containing fMLF as a soluble stimulus (A-B) or plates that had been coated with polyRGD (C-D), fibrinogen (E-F), or a BSA–anti-BSA
immune complex (G-H), and light emission was recorded using a Berthold Microluminat Plus luminometer. Data were recorded in duplicate and at least 3 independent
experiments were performed. Data (mean ⫾ range) from a representative experiment performed with cells from tamoxifen- and mock-induced Arap3fl/fERT2Cre⫹ mice are
shown in panels A, C, E, and G. Data shown in panels B, D, F, and H represent accumulated light emission (mean ⫾ SEM) from the pooled performed experiments (n ⫽ 3)
expressed as a percentage of the response in mock-induced neutrophils of each particular experiment. Raw data were analyzed by 2-way ANOVA with Bonferroni post tests
(B,D,F) and by t tests (Mann-Whitney; H).
Results
We recently reported that deleting Arap3 in the germline causes
embryonic lethality.14 To analyze the function of ARAP3 in
neutrophils, we crossed Arap3fl/fl mice with mice expressing a
tamoxifen-inducible Cre (ERT2Cre). A single Cre induction caused
the temporary loss of ARAP3 protein from bone marrow–derived
neutrophils of Arap3fl/flERT2Cre⫹ animals (Figure 1A). ARAP3
protein expression was not affected by injecting tamoxifen into
Arap3fl/fl or into Arap3⫹/⫹ERT2Cre⫹ control mice, or by mock
inducing (vehicle control) any of these mice (Figure 1A). ARAP1
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BLOOD, 28 JULY 2011 䡠 VOLUME 118, NUMBER 4
Figure 3. Integrin-dependent events are up-regulated in ARAP3-deficient neutrophils. (A) Integrin-dependent signaling. Bone marrow–derived neutrophils were prepared from mock
(WT) and tamoxifen-induced (KO) Arap3fl/flERT2Cre⫹ mice. Cells (5⫻10⫺6) were left to adhere to hiFCS- or polyRGD-coated tissue culture plastic to induce intracellular signaling before
being scraped into lysis buffer. Lysates were subjected to SDS-PAGE and immunoblotted for phospho-PKB (Ser473), phospho-p38 (Thr180 and Tyr182), and ␤-COP as a loading control.
A representative blot is shown on the left. Blots were quantified using ImageJ v1.37 software, and pooled data from 3 independent experiments are plotted (top right, phospho-PKB; bottom
right, phospho-p38), expressed as a percentage of the response obtained with mock-induced neutrophils plated onto polyRGD (data ⫾ SEM). (B) Gelatinase granule release. Neutrophils
were prepared as in panel A, and plated in wells that had been coated with hiFCS or fibrinogen in the presence of 20 ng/mL of TNF␣. For a positive control, neutrophils were stimulated with
1␮M fMLF in the presence of 10␮M cytochalasin B (CB). Supernatants from all wells were used for zymography. Stained gels were quantified using ImageJ v1.37 software. A
representative stained zymography gel is shown on the left. The positive control samples were not loaded immediately adjacent to adhesion-induced samples. For ease of viewing, the
2 parts of the gel were pasted here, as indicated by a dotted line. Pooled data from 3 independent experiments are plotted (data ⫾ SEM) on the right. (C) Adhesion and spreading.
Neutrophils were prepared as in panel A, and 5 ⫻ 106 cells were plated into 24-well plates that had been coated with 20 ␮g/mL of polyRGD, left to adhere for 10 minutes, and fixed. In
washed plates, adherent (phase dark) and spread cells (phase light) were counted in 4 randomly chosen fields of view from each individual experiment, and the percentage of spread cells
of total numbers was determined. Example photos are shown on the left; on the right are plotted data combined from 3 independent experiments (data ⫾ SEM). Statistical analysis was by
paired t tests performed on the raw data. *P ⬍ .05; **P ⬍ .01; ***P ⬍ .001. (D) Ex vivo flow chamber studies were performed with tamoxifen-induced Arap3fl/flERT2Cre⫹ (iko) and wild-type
animals (wt) using microflow chambers (0.4 ⫻ 0.04 mm) coated with rmE-selectin (20 ␮g/mL), rmCXCL1 (15 ␮g/mL), and rmICAM-1 (15 ␮g/mL). Flow chambers were recorded for
10 minutes, and adhesion efficiency (adherent cells/mm2 ⫻ WBC) was calculated (data ⫾ SEM; n ⫽ at least 5 chambers per group).
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BLOOD, 28 JULY 2011 䡠 VOLUME 118, NUMBER 4
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Figure 4. Higher affinity and avidity of ␤2 integrins on
ARAP3-deficient neutrophils. (A) Surface Mac-1 and
LFA-1 were analyzed by FACS from flushed bone marrow
cells from mock- and tamoxifen-induced Arap3fl/
flERT2Cre⫹ mice that had or had not been prestimulated
with 20 ng/mL of TNF␣ at 37°C before being labeled with
PE-conjugated anti-GR1, APC-conjugated anti–Mac-1,
and FITC-conjugated anti–LFA-1. For FACS analysis,
GR1-positive cells were gated and Mac-1 and LFA-1
staining was measured; results were analyzed using
FlowJo v6.4.7 software. Cells from 27 tamoxifen- and
mock-injected mice were analyzed in 5 separate experiments. Representative traces are shown. Gray lines
represent cells from tamoxifen-induced and black lines
from mock-induced Arap3fl/flERT2Cre⫹ mice; broken lines
represent unstimulated samples and full lines TNF␣stimulated samples. (B) Binding of unstimulated, bone
marrow–derived neutrophils from mock-induced (WT) or
tamoxifen-induced (KO) Arap3fl/flERT2Cre⫹ mice to
ICAM1-Fc in solution as a readout of affinity was measured by quantitative immunofluorescence using an
FV1000 confocal microscope (Olympus) with a 60⫻
objective, as detailed in “Analysis of ␤2 integrins.” Mean
fluorescence intensity of 88 WT and 148 KO cells pooled
from 3 separate experiments are plotted (right) and
representative examples are shown (left). (C) Mac-1 and
LFA-1 distribution in unstimulated neutrophils in suspension was visualized microscopically. Forty cells were
analyzed for each condition in each of 3 experiments;
representative examples are shown (left). Cells were
scored for integrin clustering as a readout for high avidity
status. Integrated values obtained from 3 separate experiments are shown graphically (see also supplemental
Figure 3 for an analysis of integrin clustering using
ImageJ v1.37 software). (B-C) Statistical analysis by
paired t test. *P ⬍ .05; ***P ⬍ .001.
and ARAP2, which are expressed only weakly in murine bone
marrow–derived neutrophils, were not affected by inducibly deleting Arap3 (data not shown). Similarly, peripheral blood cell counts
from tamoxifen- or mock-induced Arap3fl/flERT2Cre⫹ animals
were not affected (Figure 1B).
ARAP3 regulates adhesion-dependent formation of ROS
Neutrophils produce ROS on appropriate stimulation. PI3Ks and
Rho, Rap, and Arf family small GTPases are involved in regulating
the phagocyte oxidase.6,9,30 We found no difference in ROS
production by neutrophils from tamoxifen- or mock-induced
Arap3fl/flERT2Cre⫹ mice or controls after stimulation with the
soluble agonist fMLF (Figure 2A-B and supplemental Figure 1A
for controls), nor with the nonphysiologic, soluble stimulus phorbol 12-myristate 13-acetate (data not shown). This indicated that
the machinery required for ROS production was intact in neutrophils lacking ARAP3.
Neutrophils attached to several surfaces generate long-lasting,
␤2 integrin–dependent ROS when costimulated with an additional
proinflammatory stimulus (eg, a cytokine or bacterial product).31,32
In contrast, the synthetic multivalent integrin ligand polyRGD does
not require any costimulation.23 We found that adhesion-dependent
ROS production after plating cells onto polyRGD-coated surfaces
was increased in tamoxifen-induced Arap3fl/flERT2Cre⫹ neutrophils compared with controls (Figure 2C-D and supplemental
Figure 1B) in the presence or absence of TNF␣. We observed the
same trend with neutrophils plated onto fibrinogen-coated surfaces,
again with or without costimulation with TNF␣ (Figure 2E-F and
supplemental Figure 1C). This suggested that ␤2 integrins in the
ARAP3-deficient neutrophils might be present in a preactivated
state, able to produce adhesion-dependent ROS even in the absence
of costimulation with a proinflammatory cytokine.
Immune complexes activate neutrophils by signaling through
cell-surface Fc receptors. This also induces ROS production,24 a
function that is critical for autoimmune inflammatory disorders. Fc
receptors and integrins share signaling intermediates.33 We assessed ROS production after plating tamoxifen-induced Arap3fl/
flERT2Cre⫹ neutrophils (and the relevant controls) in immobilized
immune complex–coated wells (BSA–anti-BSA). In agreement
with the results described in the previous paragraph, we observed a
heightened response when cells lacking ARAP3 were plated onto
immune complex–coated surfaces (Figure 2G-H and supplemental
Figure 1D). In summary, ROS production due to integrin (or
Fc-receptor) engagement, but not to stimulation with a soluble
agonist, was up-regulated in neutrophils lacking ARAP3.
Loss of ARAP3 causes increased responses in
adhesion-dependent processes in neutrophils
Prompted by the results obtained with the ROS assays, we
investigated whether ARAP3 might also be important for other
events downstream of integrin engagement in neutrophils. We
analyzed the phosphorylation status of protein kinase B (PKB, also
called Akt) and of p38 MAPK, 2 downstream components of
outside-in signaling. We plated neutrophils onto dishes that had
been blocked with hiFCS or coated with polyRGD. Both kinases
were hyperphosphorylated in tamoxifen-induced Arap3fl/flERT2Cre⫹
neutrophils, but not in controls, after plating on polyRGD (Figure
3A and supplemental Figure 2A).
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BLOOD, 28 JULY 2011 䡠 VOLUME 118, NUMBER 4
Figure 5. ARAP3 controls integrin-dependent chemotaxis. Bone marrow–derived neutrophils were prepared from mock-induced (WT) and tamoxifen-induced (KO)
Arap3fl/flERT2Cre⫹ mice as detailed in “Neutrophil purification.” (A) Cells were allowed to adhere to glass-bottomed dishes before dishes were filled with buffer and a
micropipette filled with 1␮M fMLF in buffer was inserted. A slow, constant flow of fMLF was initiated using a microinjection system, and cell movements were followed by
phase-contrast time-lapse imaging over 30 minutes, taking an exposure every 30 seconds using a Zeiss Axiovert 200 microscope with a 32⫻ objective and Axiovision v3.0
software. The needle was moved away from the cells when they threatened to migrate into the micropipette and potentially block it (WT cells only). Individual cells were tracked
using the manual tracking plug-in for ImageJ. The final images of representative videos containing the tracks are shown. For the WT cells, asterisks indicate previous positions of the
micropipette tip. (B-D) Chemotaxis in Dunn chambers. Cells were allowed to migrate toward 300nM fMLF in Dunn chambers and their movements were recorded by time-lapse imaging
using 5⫻ magnification. (B) Pooled tracks of individual cells obtained from experiments carried out on 3 separate days with separate cell preparations were plotted using the Ibidi
chemotaxis tool plug-in for ImageJ. The source of fMLF is at the bottom. The tracks were analyzed using the chemotaxis tool’s statistics feature. Accumulated and Euclidean
distances (C) as well as cell speed (D) of WT and KO cells are plotted (mean ⫾ SEM). Data were analyzed using t tests (Mann-Whitney), and differences were found to be statistically
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BLOOD, 28 JULY 2011 䡠 VOLUME 118, NUMBER 4
In neutrophils, integrin outside-in signaling leads to degranulation, firm adhesion, and spreading. To determine whether ARAP3
is involved in these functions, we plated mock- and tamoxifeninduced Arap3fl/flERT2Cre⫹ neutrophils in hiFCS-blocked and
fibrinogen-coated plastic wells and quantified gelatinase granule
release. Gelatinase granule release was increased in tamoxifeninduced Arap3fl/flERT2Cre⫹ neutrophils (Figure 3B and supplemental Figure 2B), but not in controls, upon plating the cells on
fibrinogen. Control stimulation with a powerful soluble stimulus
(fMLF in combination with cytochalasin B) did not lead to an
increased release of gelatinase granules in ARAP3-deficient cells
(Figure 3B and supplemental Figure 2B), indicating that the
cellular machinery required for granule release was unaffected.
We analyzed cell adhesion and spreading 10 minutes after
plating neutrophils onto dishes coated with integrin ligands and
found increased adhesion and spreading of tamoxifen-induced
Arap3fl/flERT2Cre⫹ neutrophils compared with controls (Figure 3C
shows adhesion and spreading after plating onto polyRGD and
supplemental Figure 2C shows the controls). The same trend was
observed when cells were plated onto fibrinogen (data not shown).
We analyzed adhesion of peripheral blood neutrophils to
immobilized E-selectin, ICAM1, and CXCL1 ex vivo in a flow
chamber system,25 a setup that represents a more accurate reflection
of the situation of neutrophils in the bloodstream because of the
constant shear forces. ARAP3-deficient cells again adhered better
to the coated chamber surface (Figure 3D). Our results show that
intracellular events downstream of integrin activation were upregulated in Arap3⫺/⫺ neutrophils (outside-in signaling).
Loss of ARAP3 regulates ␤2 integrin affinity and avidity but not
surface expression
We focused on the 2 major neutrophil ␤2 integrins, LFA-1 and
Mac-1, to identify the reason for the observed increased responses
of adhesion-dependent neutrophil functions. The total amount of
Mac-1 in neutrophils that did or did not contain ARAP3 was
unaltered (supplemental Figure 3A). Similarly, surface LFA-1 and
Mac-1 on neutrophils that had or had not been prestimulated with
TNF␣ was nearly identical on tamoxifen- and mock-induced
Arap3fl/flERT2Cre⫹ neutrophils (Figure 4A), suggesting that the
differences in biologic activities observed were not due to an
increased number of surface integrins.
Integrin ligand binding affinity and avidity are regulated by
inside-out signaling. Integrins reversibly change their tertiary
structure between low-, intermediate-, and high-affinity status, and
they reversibly cluster, modulating their binding avidity.34 To
compare ␤2 integrin affinity of neutrophils from tamoxifen- or
mock-induced Arap3fl/flERT2Cre⫹ mice in the absence of affinity
ARAP3 REGULATES NEUTROPHILS
1093
status–specific antibodies for murine ␤2 integrins, we allowed cells
to bind to ICAM1 in solution and analyzed binding using quantitative immunofluorescence on cells that had settled on glass slides
after fixation. ARAP3-deficient cells bound to ICAM1 significantly
more strongly than controls (Figure 4B), whereas cells isolated
from tamoxifen and mock-induced Arap3⫹/⫹ERT2Cre⫹ mice behaved in the same manner (supplemental Figure 3B). We next
analyzed the avidity of LFA-1 and Mac-1 by staining neutrophils
kept in solution using antibodies for these surface integrins. After
fixation, cells were allowed to settle on slides for microscopic
analysis. LFA-1 was significantly more clustered in ARAP3deficient neutrophils than in controls (Figure 4C and supplemental
Figure 3C); this trend was also observed for Mac-1, whereas cells
from tamoxifen- and mock-induced Arap3⫹/⫹ERT2Cre⫹ mice
displayed similar distributions of both LFA-1 and Mac-1 (supplemental Figure 3D). In summary, ␤2 integrin affinity and avidity
were increased in ARAP3-deficient neutrophils, indicating increased inside-out signaling.
ARAP3 regulates chemotaxis
We analyzed chemotaxis in neutrophils that did or did not express
ARAP3. We initially performed “needle chemotaxis” assays in
which a chemoattractant-filled micropipette coupled to a microinjector set to a constant, slow stream was inserted into a glassbottomed dish containing adherent neutrophils, causing the formation of a steep gradient of chemoattractant. Control neutrophils
adopted a polarized shape and migrated consistently toward the tip
of the micropipette, but neutrophils from tamoxifen-induced Arap3fl/fl
ERT2Cre⫹ mice were unable to chemotax toward fMLF in this
assay (Figure 5A and supplemental Videos 1-2; controls are shown
in supplemental Figure 4A). These neutrophils extended a pseudopod in one direction for a short time, then retracted this pseudopod
and generated another one in a different direction and so forth,
making little if any net progress in any direction in the process.
This indicated that neutrophils lacking ARAP3 had a severe
chemotaxis defect, although they clearly reacted to being stimulated with fMLF.
For a more quantifiable response, we performed chemotaxis
assays in Dunn chambers. For these assays, cells moved on a
“bridge” through a 20-␮m-deep gap between 2 glass surfaces in a
shallow gradient of chemoattractant.35 Unexpectedly, Arap3⫺/⫺
neutrophils were able to move directionally in Dunn chambers
(Figure 5B). However, tracking the chemotaxing cells and analyzing the tracks confirmed that neutrophils from tamoxifen-induced
Arap3fl/flERT2Cre⫹mice had a significantly reduced ability to
chemotax toward fMLF in terms of Euclidean distance (the shortest
distance between the start and end points) and total accumulated
Figure 5. (continued) significant (P ⬍ .001). (E-G) Neutrophils from mock-induced (WT) and tamoxifen-induced (KO) Arap3fl/flERT2Cre⫹ mice were used in chemotaxis
assays in the EZ-TAXIS chamber. Cells were lined up at the bridge of an EZ-TAXIS chamber and allowed to migrate toward 3␮M fMLF while being followed by time-lapse
imaging using a BD pathway reflection microscopy system. (E) Cell movements were tracked and analyzed as for Dunn chamber chemotaxis; cell tracks from pooled cells
tracked in 2 separate experiments are shown. (F-G) Tracks were analyzed using the statistical features of the Ibidi chemotaxis tool, and plotted results show pooled data from
3 experiments performed on separate days with separate cell preparations. Accumulated and Euclidean distances traveled by WT and KO cells (left) and their cell speeds were
significantly different, as shown by t test (P ⬍ .001). (H) Transwell chemotaxis. Flushed bone marrow cells from tamoxifen- and mock-induced Arap3fl/flERT2Cre⫹ mice were
placed in the top well of polycarbonate transwells with 3-␮m pores. The bottom chambers contained the indicated concentrations of fMLF. Cells that migrated through the filters
in 40 minutes were counted. Graph shows pooled data (mean ⫾ SEM) obtained from 3 individual experiments performed on separate days with separate cell preparations.
Stimulation with fMLF caused significant activation of chemotaxis (P ⬍ .0001), whereas there was no significant difference between WT and KO cells (P ⫽ .5098; 2-way
ANOVA with Bonferroni post test). (J-K) Chemotaxis in a 3D collagen matrix. Bone marrow neutrophils from tamoxifen-induced (KO) and mock-induced (WT)
Arap3fl/flERT2Cre⫹ mice were mixed with type I collagen in custom-made chambers. After allowing the collagen to polymerize, creating a 3 mg/mL matrix, gels were overlaid
with 0.5␮M fMLF and the chambers sealed. Gradients were allowed to develop and chemotaxing cells were followed by time-lapse imaging for 15 minutes, taking an exposure
every 30 seconds. Cells were tracked through the frames using the manual tracking and chemotaxis tools plug-ins in ImageJ v1.37. The tracks in panel J represent pooled
tracks from randomly chosen cells from 3 independent runs performed in one day. The graph in panel K represents the pooled accumulated and Euclidean distances traveled
by the randomly tracked cells from 3 independent runs of WT and KO cells in experiments performed on 3 separate days with separate cell preparations (mean ⫾ SEM). Data
were analyzed using t test (Mann-Whitney); **P ⬍ .01.
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GAMBARDELLA et al
distance covered, and also in their speed (Figure 5C-D). As
expected, there were no significant differences between neutrophils
from tamoxifen- or mock-induced Arap3⫹/⫹ERT2Cre⫹ control
mice in the Dunn chamber assays (supplemental Figure 4B).
Neutrophils from tamoxifen- and mock-induced Arap3fl/fl
ERT2Cre⫹ mice in EZ-TAXIS chambers36 in which cells were
lined up at a narrow gap (5-␮m depth) through which they
squeezed when exposed to a shallow gradient of chemoattractant
(fMLF; Figure 5E) exhibited the same trend. Interestingly, the
differences between neutrophils from tamoxifen- and mockinduced Arap3fl/flERT2Cre⫹ mice chemotaxing in EZ-TAXIS chambers were more subtle than in those measured in the Dunn chamber
assays (Figure 5F-G).
Several in vitro studies with neutrophils lacking integrins21,37,38
or in which integrin function had been inhibited using antibodies or
chelators39 have shown that integrins are required for migration on
2D but not in 3D substrates (or when “chimneying” between close
glass surfaces). To our knowledge, this issue has not been
addressed in depth in cells with increased integrin activity. We
wondered whether the different results we obtained in the different
chemotaxis assays might reflect the fact that we were measuring
different mixtures of integrin-dependent and integrin-independent
chemotaxis. We reasoned that needle chemotaxis assayed highly
integrin-dependent chemotaxis on a 2D glass coverslip, whereas
Dunn and EZ-TAXIS chambers, both of which analyze chemotaxis
of cells squeezing through more or less narrow gaps, might assay
mixtures of 2D and 3D chemotaxis. To determine whether ARAP3
might be most important for integrin-dependent chemotaxis, we
performed chemotaxis assays in integrin-independent setups in
which cells migrated in 3D substrates. We measured chemotaxis
toward fMLF in Boyden chambers (transwells), in which the cells
squeeze through 3-␮m polycarbonate pores, which has previously
been documented as being integrin independent,40 and found no
significant differences between ARAP3-deficient and control neutrophils (Figure 5H). We also analyzed neutrophil chemotaxis in a
3D collagen matrix (Figure 5J), which has previously been shown
to support chemotaxis of entirely integrin-deficient leukocytes.21
Interestingly, analysis of tracks from neutrophils from tamoxifenand mock-induced Arap3fl/flERT2Cre⫹ mice migrating in the
collagen gels indicated that there was no difference in the total
accumulated distances migrated (or in the speed, data not shown) in
the 2 groups of cells, but Euclidean distances were different (Figure
5K). Hence, ARAP3-deficient cells ended up significantly nearer to
their starting points than controls, suggesting a directionality defect
in ARAP3-deficient cells. Neutrophils from tamoxifen- or mockinduced Arap3⫹/⫹ERT2Cre⫹ mice behaved identically (supplemental Figure 4C).
In summary, our experiments show that ARAP3-deficient
neutrophils displayed a context-dependent chemotaxis defect in
vitro. Our data suggest an integrin-dependent component to
ARAP3-regulated chemotaxis, which affects the total distance
traveled by the cells in a substrate-dependent fashion. They also
suggest an additional directional chemotaxis defect in neutrophils
lacking ARAP3, which appears to be integrin independent.
ARAP3 regulates adhesion in vivo
During inflammation, the recruitment of neutrophils that circulate
in the bloodstream in a quiescent, nonadhesive state follows a
well-established cascade of adhesion and activation steps,41 starting with the capture of free-flowing neutrophils to the inflamed
endothelium, followed by rolling along the endothelium. Both
capture and rolling are mediated by selectins. During rolling,
BLOOD, 28 JULY 2011 䡠 VOLUME 118, NUMBER 4
endothelial-expressed chemokines, together with selectin-mediated
signaling events, trigger the activation of neutrophil-expressed ␤2
integrins (inside-out signaling), which leads to further slowing
down and eventually to firm neutrophil arrest on the endothelium.
This is followed by steady strengthening of the adhesive contacts
and neutrophil spreading along the endothelium, postarrest steps
that require interactions between neutrophil integrins and their
endothelial ligands (outside-in-signaling). Finally, neutrophils start
crawling along the luminal surface of the inflamed endothelium in
search of an appropriate exit point out into the tissue.
To address the relevance of our observations in vivo, we
analyzed neutrophil recruitment in mouse cremaster muscle venules
in the absence of ARAP3. Because our in vitro assays suggested a
preactivated state of ␤2 integrins in ARAP3-deficient neutrophils,
we evaluated baseline adhesion and extravasation of neutrophils in
unstimulated cremaster muscle whole mounts. To prevent traumainduced inflammatory changes, tamoxifen- and mock-induced
Arap3fl/flERT2Cre⫹ and Arap3⫹/⫹ERT2Cre⫹ mice were killed
before preparation of the cremaster muscle. In agreement with the
previous in vitro adhesion and flow chamber assays, intravascular
adhesion was increased in tamoxifen-induced Arap3fl/flERT2Cre⫹
animals compared with controls, which exhibited low numbers of
adherent intravascular neutrophils under these conditions (Figure
6A). In addition, the baseline number of perivascular neutrophils
was also found to be higher in ARAP-3–deficient animals compared with controls (Figure 6B and supplemental Figure 5). These
findings corroborate our in vitro data indicating that deletion of
ARAP3 causes preactivation of neutrophils.
To further analyze the functional relevance of ARAP3 in vivo
under inflammatory conditions and to exclude a possible effect of
ARAP3 in the endothelial compartment, we evaluated neutrophil
adhesion and extravasation in TNF␣-stimulated cremaster muscle
whole mounts of tamoxifen-induced Arap3fl/flERT2Cre⫹ and
Arap3⫹/⫹ERT2Cre⫹ bone marrow chimeras. Stimulation with
TNF␣ resulted in an increased recruitment of neutrophils in both
groups compared with baseline conditions. However, the number
of ARAP3-deficient, adherent intravascular neutrophils was significantly higher than that of controls. There were also more perivascular ARAP3-deficient cells (Figure 6C). We next directly evaluated
intravascular adhesion in postcapillary venules of the cremaster
muscle by intravital microscopy. Microvascular and hemodynamic
parameters were similar between the groups (supplemental Table
1). These experiments showed an increased adhesion efficiency
using the equation (adherent cells/mm2)/systemic WBCs in ARAP3deficient neutrophils compared with controls (Figure 6D). More
adherent ARAP3-deficient neutrophils stayed firmly attached and
showed a decreased tendency to crawl intraluminally compared
with controls (Figure 6E and supplemental Videos 3-4). In agreement with observations from chemotaxis assays in 3D matrices in
vitro, extravascular ARAP3-deficient cells were clearly able to
migrate interstitially.
ARAP3 does not modulate Rap activity, but affects RhoA
in neutrophils
Rap1 has previously been shown to modulate integrin affinity and
avidity in several settings.2 We measured Rap1 activity in neutrophils that were kept in suspension or that had been allowed to
adhere to polyRGD. We saw a robust activation of Rap1 on plating
cells on polyRGD, but there was no significant difference in Rap1
activity between cells lacking ARAP3 and controls (Figure 7A).
This indicated that Rap1 activity was not affected by the presence
or absence of ARAP3 in neutrophils.
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BLOOD, 28 JULY 2011 䡠 VOLUME 118, NUMBER 4
ARAP3 REGULATES NEUTROPHILS
1095
Figure 6. ARAP3 regulates neutrophil adhesion in vivo.
Cremaster muscle experiments were performed on tamoxifen- and mock-induced Arap3fl/flERT2Cre⫹ (iko) and Arap3⫹/⫹
ERT2Cre⫹ (iCre) bone marrow chimeras. Baseline levels
of intravascular neutrophil adhesion (A) and neutrophil
extravasation (B) were histologically evaluated in untreated, Giemsa-stained cremaster muscle whole mounts
(13-17 vessels per group). (C) TNF␣ (500 ng intrascrotal)–
stimulated neutrophil recruitment was assessed in cremaster muscle whole mounts of tamoxifen-induced
Arap3⫹/⫹ERT2Cre⫹ (22 vessels in 3 animals) and Arap3fl/
flERT2Cre⫹ mice (37 vessels in 4 mice) by quantification
of intravascular and extravascular leukocytes 3 hours
after injection. (D) Intravital microscopy of the TNF␣stimulated cremaster muscle was performed to assess
adhesion efficiency—(adherent cells/mm2)/systemic WBC
count—in postcapillary venules of tamoxifen-induced
Arap3⫹/⫹ERT2Cre⫹ (11 vessels in 3 animals) and Arap3fl/
flERT2Cre⫹ mice (18 vessels in 5 animals). (E) For
evaluation of intraluminal crawling, time-lapse intravital
microscopy of cremaster muscle venules (at least 3 mice
per group) were recorded via CCD camera (20 frames
per minute) and analyzed offline using the manual tracking plug-in of ImageJ. All data are given as means
⫾ SEM. Data were analyzed using Kruskal-Wallis 1-way
ANOVA on ranks with the Dunn post hoc test for multiple
comparison or the Mann-Whitney rank-sum test for pairwise comparison. *P ⬍ .05; ** P ⬍ .01. (Detailed information on microcirculatory parameters of intravital experiments is provided in supplemental Table 1.)
We previously showed that ARAP3 regulates the small GTPases
Arf6 and RhoA. We therefore performed activity assays with neutrophils kept in suspension or plated onto polyRGD. Measuring global
Arf6-GTP in lysates from these neutrophils, we saw a dramatic
activation of Arf6 upon adhesion to polyRGD, but we did not detect any
significant differences between the 2 genotypes (Figure 7B). When
analyzing RhoA, we saw an increased activity in cells plated onto
polyRGD. Although the extent of activation was variable between
experiments, the RhoA activation in ARAP3-deficient cells was increased every time (Figure 7C), whereas RhoA activation in neutrophils
from tamoxifen- and mock-induced Arap3⫹/⫹ERT2Cre⫹ mice was very
similar (supplemental Figure 6). In summary, our data suggest that
ARAP3 modulates RhoA in an adhesion-dependent context. These
findings are in agreement with our previous work identifying ARAP3 as
a Rap-regulated RhoA GAP.11
Discussion
ARAP3, one of 24 PtdIns(3,4,5)P3–binding proteins identified
from porcine neutrophils,10 is abundant in human and mouse
neutrophils. The present study represents the first characterization
of ARAP3 function in these highly specialized immune cells.
Because germline deletion of Arap3 is embryonically lethal,14 we
used a tamoxifen-inducible, ubiquitous Cre to delete Arap3 conditionally in the adult mouse to efficiently (although transiently)
delete Arap3 in bone marrow–derived neutrophils. Our mouse
model was unsuitable for any longer-term in vivo experiments such
as models of autoimmune diseases. In addition, we observed
variable basal peritoneal leukocyte numbers even in intraperitoneally mock-injected control mice, rendering our model unsuitable
for assessment of leukocyte recruitment after thioglycollateinduced sterile peritonitis. Given that Cre expression can in some
circumstances lead to nonspecific artifacts, tamoxifen induction
was kept to a minimum by routinely comparing tamoxifen- and
mock-induced Arap3fl/flERT2Cre⫹ mice with tamoxifen- and mockinduced Arap3⫹/⫹ERT2Cre⫹ mice, controlling for tamoxifen and
for Cre expression. None of the phenotypes described for ARAP3deficient neutrophils were due to tamoxifen or to inducing Cre.
However, in agreement with previous studies finding nonspecific
effects caused by expression of Cre,42,43 we observed a significant
loss of erythrocytes in the bone marrow of tamoxifen-induced
Arap3fl/flERT2Cre⫹ and Arap3⫹/⫹ERT2Cre⫹ mice 12 days after
tamoxifen induction (supplemental Figure 7). This side effect was
temporary, because no differences in bone marrow erythrocytes
were found 6 weeks after induction (data not shown). Because we
observed no differences in circulating blood cells, we are confident
that this temporary loss of bone marrow erythrocytes had no effect
on any of the measurements we carried out.
We noted increased responses with ARAP3-deficient neutrophils in all adhesion-dependent responses that were tested, including in vitro assays carried out with purified bone marrow–derived
neutrophils, ex vivo assays in flow chambers, and in vivo observations in the cremaster muscle system. A response was triggered
even in ARAP3-deficient, unstimulated cells both in vitro (eg,
increased ROS production in response to plating onto fibrinogen in
the absence of TNF␣ and increased gelatinase granule release in
cells plated in heat-inactivated FCS-blocked wells) and in vivo (eg,
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1096
GAMBARDELLA et al
BLOOD, 28 JULY 2011 䡠 VOLUME 118, NUMBER 4
Figure 7. Assaying activities of small GTPases. Bone
marrow–derived neutrophils were prepared from
tamoxifen-induced (KO) and mock-induced (WT) Arap3fl/
flERT2Cre⫹ mice. Neutrophils were or were not plated
onto polyRGD. Ten minutes later, cells were lysed using
ice-cold lysis buffer. (A-B) Clarified lysates were used for
pull-down assays to determine GTP-bound fractions of
small GTPases: Rap1 pull-downs using GST-Ral GDS
beads are shown in panel A and Arf6 pull-downs using
GST-MT2 beads are shown in panel B. After incubation
of baits with lysate, beads were washed, boiled in
sample buffer, and proteins were subjected to SDSPAGE and immunoblotted using anti-Rap1 (A) and antiArf6 (B) antibodies. Graphs show pooled data (means
⫾ SEM) obtained from 3 independent experiments. Photographs of representative experiments are shown. Two
different exposure lengths of the same blot are shown in
the Arf6 pull-down panel because there was a large
difference between unstimulated and stimulated samples
(dotted line). For the same reason, the 2 conditions were
quantified independently. (C) Clarified lysates were
used for RhoA G-LISA assays. To allow comparison of
4 independent experiments, suspension readings were
equalized (left graph). Statistical analyses were carried
out with the raw data using 2-way ANOVA with Bonferroni
post tests or paired t tests for pairwise comparisons.
*P ⬍ .05.
increased baseline adhesion in unstimulated cremaster muscle
venules). We measured no differences in surface Mac-1 and LFA-1,
the most abundant, constitutively expressed neutrophil integrins.
Instead, both the affinity and the avidity of these ␤2 integrins were
significantly increased in cells lacking ARAP3 (inside-out signaling). Our data support 2 conclusions: (1) both inside-out and
outside-in signaling could be altered independently of one another,
or (2) the increased responses observed in events downstream of
integrin ligation could, at least in part, be a direct consequence of
the increased inside-out signaling we measured.
In addition to regulating responses that are classically thought
of as downstream of integrin ligation, we observed a striking,
context-dependent chemotaxis defect in ARAP3-deficient neutrophils. The literature describes integrin-dependent and integrinindependent modes of cell migration. Integrin-dependent migration
involves force generation because of integrin-dependent, transient
adhesions to a typically 2D substratum, whereas integrinindependent migration involves amoeboid squeezing of cells
through narrow pores of a 3D matrix, propelled by the force of a
polarized actomyosin cytoskeleton. Relevant experiments have
been carried out either with healthy human neutrophils in which
integrin function was inhibited, using chelators or blocking antibodies,39 or with cells taken from LAD patients.37 More recently,
analysis of chemotaxis with integrin-deficient murine neutrophils
supported very convincingly the conclusions drawn in these earlier
studies.21,38 Fewer studies have addressed the integrin dependency
of chemotaxis in cells with hyperactive integrins, as is the case in
ARAP3-deficient neutrophils. We conclude that in ARAP3deficient neutrophils, integrin-dependent chemotaxis (needle chemotaxis, in which cells migrate on a glass coverslip) is defective
because the adhesive contacts formed with the glass coverslip are
too tight. Chemotaxis in Dunn or EZ-TAXIS chambers represent
intermediates between integrin-dependent and integrin-independent
chemotaxis, with an increasing amount of chimneying between the glass
coverslips forming the bridge performed by cells the tighter the gap
between the 2 coverslips. This explains why we observed a smaller
defect with ARAP3-deficient neutrophils in EZ-TAXIS chambers with
their narrow gaps (which were previously used to assay neutrophil
chemotaxis in the presence of chelating agents40) than in Dunn
chambers, in which the gap is significantly wider. Finally, chemotaxis in
integrin-independent circumstances (eg, in a 3D collagen gel or a
polycarbonate transwell in vitro or in interstitial migration in vivo) was
not affected in ARAP3-deficient cells in terms of the total accumulated
distance traveled by the neutrophils. Therefore, varying integrin dependency in different chemotaxis assays is the most likely explanation for
the reproducible yet seemingly contradictory results we obtained with
these different chemotaxis assays. We propose that ARAP3 is required
for integrin-dependent chemotaxis, but is dispensable for integrinindependent chemotaxis. In addition, our data hint that there may be an
additional defect in directionality of chemotaxis. ARAP3-deficient cells
failed to persistently polarize in the orientation of the chemoattractant in
needle chemotaxis assays. In addition, despite equal total accumulated
distances, ARAP3-deficient neutrophils migrated shorter Euclidean
distances in 3D matrices. Future work will be directed at establishing the
nature of any directionality defect in chemotaxis of ARAP3-deficient
neutrophils.
When analyzing cremaster muscle venules, we observed increased numbers of intravascular, but also perivascular, ARAP3deficient leukocytes, suggesting increased extravasation. However,
given the increased adhesion to the endothelium, it is conceivable
that transmigration of a fraction of these cells could have masked a
mild transmigration defect. Unfortunately, we were unable to
address this possibility experimentally because we could not use
the sterile peritonitis model.
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BLOOD, 28 JULY 2011 䡠 VOLUME 118, NUMBER 4
ARAP3 REGULATES NEUTROPHILS
Having identified PI3K as major regulator of ARAP3,10,14 the
phenotype of ARAP3-deficient neutrophils was somewhat surprising. We previously showed that ARAP3 is regulated by Rap-GTP in
addition to PI3K.11 Interestingly, Rap proteins have been demonstrated to be major modulators of integrin inside-out signaling in
lymphocytes, macrophages, neutrophils, and platelets, affecting
integrin affinity and avidity (although not surface expression),
ligand binding, and cell spreading.30,44-46 Rap activity was not
affected in neutrophils devoid of ARAP3, indicating (in agreement
with our previous work11) that ARAP3 is not upstream of Rap.
We assessed Arf6-GTP in neutrophils kept in solution or plated
onto polyRGD, which led to significant Arf6 activation. We did not
notice any differences between ARAP3-deficient neutrophils and
controls. Given that ARAP3 is just one of several Arf6 GAPs, and
that we analyzed global Arf6 activity, this negative result was not
very surprising. It is conceivable that an analysis of localized Arf6
activity would reveal differences that are hidden behind the robust
activation of Arf6 on plating neutrophils onto polyRGD.
When assaying RhoA activity, we noticed that the increase
measured in ARAP3-deficient cells was subtly increased compared
with controls in each experiment (on average, 1.6-fold). RhoA has
been shown to regulate leukocyte integrin activation in several
situations.47-50 In thymocytes, RhoA was shown to be required for
optimal integrin activation by Rap1,51 indicating that RhoA can be
part of Rap-dependent inside-out activation of integrins. Given that
Rap regulates ARAP3 as a Rho GAP and that ARAP3-deficient
neutrophils are characterized by heightened ␤2 integrin activity
and hyperresponsiveness in adhesion-dependent processes, we
speculate that Rap activates ARAP3 as a Rho GAP to fine-tune
integrin activity in neutrophils. Because we know that PI3K
activity is required for ARAP3 activation, it is likely to function as
a coincidence detector for PtdIns(3,4,5)P3 and Rap-GTP. It will be
1097
interesting to dissect the regulatory inputs of PI3K and Rap into
ARAP3 in the future.
Acknowledgments
The authors thank Simon Walker for help with image analysis,
Nick Ktistakis for the anti-␤COP antibody, Michelle Janas for help
with procedures, Anthony Green (Cambridge Institute for Medical
Research, Cambridge, United Kingdom) for use of a blood cell
counter, and John Ferguson, Su Kulkarni, and Michael Sixt
(Institute of Science and Technology, Klosterneuberg, Austria), and
Klaus Ley (La Jolla Institute for Allergy and Immunology, La Jolla,
CA) for helpful discussions.
This work was supported by the Biotechnology and Biological
Sciences Research Council and the Medical Research Council
(G0700740). S.V. holds a David Phillips Fellowship (BB/C520712).
Authorship
Contribution: L.G., K.E.A., C.N., M.S., and S.V. designed experiments; L.G., K.E.A., C.N., T.M., L.N., M.S., and S.V. performed
experiments; T.L. provided reagents; L.G., C.N., A.S.-P., P.T.H.,
M.S., L.S., and S.V. analyzed and/or interpreted data; and S.V. and
M.S. wrote the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Sonja Vermeren (née Krugmann), The Inositide Laboratory, The Babraham Institute, Babraham Research
Campus, Cambridge CB22 3AT, United Kingdom; e-mail:
sonja.vermeren@bbsrc.ac.uk.
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From www.bloodjournal.org by guest on March 5, 2016. For personal use only.
2011 118: 1087-1098
doi:10.1182/blood-2010-10-312959 originally published
online April 13, 2011
The GTPase-activating protein ARAP3 regulates chemotaxis and
adhesion-dependent processes in neutrophils
Laure Gambardella, Karen E. Anderson, Claudia Nussbaum, Anne Segonds-Pichon, Tânia
Margarido, Laura Norton, Thomas Ludwig, Markus Sperandio, Phillip T. Hawkins, Len Stephens and
Sonja Vermeren
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Phagocytes, Granulocytes, and Myelopoiesis (543 articles)
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