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of June 14, 2017.
Focal Adhesion Kinase Regulates
Pathogen-Killing Capability and Life Span of
Neutrophils via Mediating Both
Adhesion-Dependent and -Independent
Cellular Signals
Anongnard Kasorn, Pilar Alcaide, Yonghui Jia, Kulandayan
K. Subramanian, Bara Sarraj, Yitang Li, Fabien Loison,
Hidenori Hattori, Leslie E. Silberstein, William F.
Luscinskas and Hongbo R. Luo
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The Journal of Immunology is published twice each month by
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Copyright © 2009 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2009; 183:1032-1043; Prepublished online 26
June 2009;
doi: 10.4049/jimmunol.0802984
http://www.jimmunol.org/content/183/2/1032
The Journal of Immunology
Focal Adhesion Kinase Regulates Pathogen-Killing Capability
and Life Span of Neutrophils via Mediating Both
Adhesion-Dependent and -Independent Cellular Signals1
Anongnard Kasorn,* Pilar Alcaide,‡ Yonghui Jia,† Kulandayan K. Subramanian,† Bara Sarraj,§
Yitang Li,† Fabien Loison,† Hidenori Hattori,† Leslie E. Silberstein,† William F. Luscinskas,‡
and Hongbo R. Luo2†
N
eutrophils are the most abundant cell type among circulating white blood cells and constitute the first line of
host defense against invading microorganisms. In response to inflammatory stimuli, neutrophils migrate from the circulating blood to infected tissues, where they protect their host by
phagocytosing, killing, and digesting bacterial and fungal pathogens (1–3). A variety of neutrophil functions are mediated by integrin, a superfamily of transmembrane heterodimeric glycoproteins composed of one ␣ and one ␤ subunit. Neutrophil recruitment
to sites of infection or inflammation involves sequential interactions with vascular endothelial and extravascular compartments.
Integrin receptors expressed on neutrophils play a central role in
these interactions, mediating linkages between the cellular cytoskeleton and the external environment. The migrating neutro-
*Department of Community Medicine, Faculty of Medicine, Srinakharinwirot University, Bangkok, Thailand; †Department of Pathology, Harvard Medical School,
Dana-Farber/Harvard Cancer Center, and Department of Lab Medicine, Children’s
Hospital Boston, Boston, MA 02115; ‡Center for Excellence in Vascular Biology,
Department of Pathology, Brigham and Women’s Hospital and Harvard Medical
School, Boston, MA 02115; and §Division of Organ Transplant, Department of Surgery, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611
Received for publication September 10, 2008. Accepted for publication May
12, 2009.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
B.S. is supported by National Institutes of Health Training Grant HL066987, and
H.L. is supported by National Institutes of Health Grants HL085100, AI076471, and
GM076084 and a Research Scholar Grant from American Cancer Society.
2
Address correspondence and reprint requests to Dr. Hongbo R. Luo, Karp Family
Research Building, Room 10214, Boston, MA 02115. E-mail address: Hongbo.Luo@
childrens.harvard.edu
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0802984
phils must establish transient and dynamic adhesive contacts with
extracellular matrix proteins, and the integrin activity should be
tightly regulated during neutrophil locomotion (4 – 8). Lack of
functional ␤2 integrin is associated with leukocyte adhesion deficiency type 1 immunodeficiency in which neutrophils fail to migrate to sites of inflammation.
In resting neutrophils, integrins are in a conformationally inactive state, unable to bind their extracellular ligands. Upon stimulation with cytokines, chemokines, or anti-integrin Abs, the integrins are primed to an active form, which can be activated by
ligand binding and subsequently initiates downstream signaling
(5– 8). Integrin ligation, either by cell-matrix or cell-cell interaction, elicits outside-in signaling in neutrophils that leads to cellular
responses such as cell spreading, firm adhesion, cell survival,
degranulation, and reactive oxygen species (ROS)3 production.
Some neutrophil responses, particularly NADPH oxidase-mediated ROS production, can be elicited solely by integrin ligation in
the absence of any other inflammatory stimuli (9 –11).
The ability of neutrophils to generate ROS after adherence to
extracellular matrices varies depending on the nature of the substrate. For example, adherence to fibronectin results in a more
pronounced ROS production and shorter lag period compared with
adherence to laminin or HUVEC surface (12). Although engagement of integrin receptor can induce ROS production by itself, it
3
Abbreviations used in this paper: ROS, reactive oxygen species; CR3, complement
receptor 3; FAK, focal adhesion kinase; PtdIns(3,4,5)P3, phosphatidylinositol (3,4,5)trisphosphate; LB, Luria-Bertani; PLC, phospholipase C; PS, phosphatidylserine;
SH2, Src homology 2; RIP, receptor-interacting protein.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
Various neutrophil functions such as phagocytosis, superoxide production, and survival are regulated by integrin signaling.
Despite the essential role of focal adhesion kinase (FAK) in mediating this signaling pathway, its exact function in neutrophils is
ill defined. In this study, we investigated the role of FAK in neutrophils using a myeloid-specific conditional FAK knockout mouse.
As reported in many other cell types, FAK is required for regulation of focal adhesion dynamics when neutrophils adhere to
fibronectin or ICAM-1. Adhesion on VCAM-1-coated surfaces and chemotaxis after adhesion were not altered in FAK null
neutrophils. In addition, we observed significant reduction in NADPH oxidase-mediated superoxide production and complementmediated phagocytosis in FAK null neutrophils. As a result, these neutrophils displayed decreased pathogen killing capability both
in vitro and in vivo in a mouse peritonitis model. In adherent cells, the defects associated with FAK deficiency are likely due to
suppression of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) signaling and chemoattractant-elicited calcium signaling. Disruption of FAK also reduced chemoattractant-elicited superoxide production in suspended neutrophils in the absence of
cell adhesion. This may be solely caused by suppression of PtdIns(3,4,5)P3 signaling in these cells, because the fMLP-elicited
calcium signal was not altered. Consistent with decreased PtdIns(3,4,5)P3/Akt signaling in FAK null neutrophils, we also observed
accelerated spontaneous death in these cells. Taken together, our results revealed previously unrecognized roles of FAK in
neutrophil function and provided a potential therapeutic target for treatment of a variety of infectious and inflammatory
diseases. The Journal of Immunology, 2009, 183: 1032–1043.
The Journal of Immunology
1033
trophils in RPMI 1640 containing 0.1% BSA (1 ␮l of 3 ⫻ 106 cells/ml)
were placed into the single hole at the bottom of a 4-␮m depth ⫻ 260-nm
width microchip. One microliter of medium with or without 1 ␮M fMLP
was loaded into the contrahole. Cells were allowed to adhere for 3 min
before starting experiment. Chemotaxis was recorded at 37°C for 20 min
with a 30-s interval using a charge-coupled device camera. Outlines of
migrating cells were traced using DIAS software (Solltech), and tracks
were generated using centroid-based methods. Chemotactic parameters
were then analyzed from the cell tracks using an in-house Matlab program
as previously described. A description of chemotactic parameters is described in supplemental Fig. 5.4
Random migration
Freshly prepared bone marrow-derived wild-type and FAK⫺/⫺ mouse neutrophils in RPMI plus 2% FCS were plated onto fibronectin (10 ␮g/ml)
precoated glass-bottom dishes (MatTek) and allowed to adhere for 3 min.
Cells were then uniformly stimulated with 100 nM fMLP, and images were
captured from multiple fields every 10 s for 20 min using a ⫻60 objective
on an inverted microscope (Olympus IX17).
Flow-based adhesion assay
The flow-based adhesion assay was conducted essentially as previously
described for leukocyte interactions with adhesion molecules (28). Briefly,
Dia glass coverslips (25 mm; Carolina Biological Supply) were coated with
fibronectin (5 ␮g/ml; Sigma-Aldrich), BSA (20 ␮g/ml; Sigma-Aldrich),
murine ICAM-1 (20 ␮g/ml; R&D Systems), or murine VCAM-1 (20 ␮g/
ml; R&D Systems). Mouse neutrophils were pretreated with 1 ␮M fMLP
for 1 min or PMA for 15 min before being loaded. Neutrophil adhesion was
examined under conditions of fluid shear stress in a parallel-plate flow
chamber. Neutrophils (0.5 ⫻ 106/ml) suspended in flow buffer (Dulbecco
PBS-0.1% human serum albumin) were drawn through the chamber at
decreasing flow rates corresponding to an estimated shear stress of 1 dyne/
cm2, 0.8 dyne/cm2, and 0.5 dyne/cm2. Neutrophil accumulation was determined after the initial minute of each flow rate by counting the number
of cells in four different fields. Polymorphonuclear neutrophil interactions
with the coated surfaces were recorded using a ⫻20 phase contrast objective, a videomicroscopy, and VideoLab software. Data are means ⫾ SEM
of three experiments. A value of p ⬍ 0.05 was considered statistically
significant using the paired t test or one-way ANOVA for multiple groups.
Cremaster muscle preparation
The surgical procedure to expose the cremaster muscle was performed as
described previously (29) with slight modification. Mice were anesthetized
i.p. with a mixture of 200 ␮g of ketamine and 20 ␮g of xylazine per mouse.
The hair covering the right testes area was shaved with an electric razor
followed by application of hair removal lotion. A diagonal incision was
made starting at the tip of the testis, which was exposed on a Plexiglas
custom-built stage. A longitudinal incision was made at the ventral side of
the muscle using heat cauterizer, and the muscle was spread using 6-0
sutures. The muscle was continuously superfused with 35°C-thermostable
0.09% saline. Temperature at the muscle surface was frequently monitored
by a digital thermometer probe. The surgical procedure takes ⬃13 min.
Intravital video microscopy acquisition
Materials and Methods
Mice
loxP/loxP
The conditional FAK knockout mice (FAK
) were gifts from Dr.
Louis F. Reichardt (University of California, San Francisco, CA; Ref. 26),
and the myeloid-specific Cre mice (B6.129P2-Lyz2tm1(cre)Ifo/J) were purchased from The Jackson Laboratory. In all experiments, wild-type littermate (FAKwt/wtCre⫹/⫹) mice were used as experimental controls. All procedures involving mice were conducted in accordance with the Animal
Welfare Guidelines of Children’s Hospital (Boston, MA) and were approved and monitored by the Children’s Hospital Animal Care and Use
Committee. Bone marrow-derived mouse neutrophils were isolated from
femurs and tibias as previously described (27). We routinely obtain 4 – 8
million cells per mouse, and ⬎90% of them are morphologically mature
neutrophils (27).
Chemotaxis assay
The EZ-TAXIScan MIC-1000 (Hirata Corp. of America) was used to investigate real-time horizontal chemotaxis of mouse neutrophils. The EZTAXIScan consists of an etched silicon substrate and a flat glass plate, both
of which form two compartments. Glass coverslips (Corning) with or without fibronectin coating were placed on the glass plate at the bottom of the
compartment. Purified bone marrow-derived wild-type and FAK⫺/⫺ neu-
The muscle preparation was transferred to an intravital microscope (IV500; Micron Instruments) with epifluorescence capability. Digital recording of videos was acquired by QED Imaging software (MediaCybernetics).
At least three vessels with diameters of 20 – 40 ␮m were recorded per time
point. A waiting period of 15 min was observed to stabilize blood flow
before any recording was taken. The mouse was injected intrascrotally with
300 ␮l of sterile saline alone (control) or saline with 1 ␮g of MIP-2, 2 h
before video acquisition. To measure the blood flow centerline velocity,
100 ␮l of rhodamine 6G (2 ␮g/ml) were injected i.v. Videos of 30 – 60 s
were recorded under a ⫻40 water immersion objective with a frame rate of
10 frames per second (fps), and 1 or 2 binning. Epifluorescent videos were
taken at 96 fps/4 ⫻ 4 binning (for rhodamine 6G).
Intravital video microscopy analysis
Offline analysis of recorded videos was performed by frame-by-frame playback using Quicktime software. Rolling cells were defined as the cells
moving over a 100-␮m-long vessel with a velocity below the velocity of
RBCs. Adherent cells were defined as cells staying still for at least 30 s.
Emigrating cells were calculated as the number of cells that left the blood
4
The online version of this article contains supplemental material.
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suppresses ROS production induced by soluble cytokine stimuli,
e.g., fMLP or TNF-␣ (12–14). Integrin also plays an essential role
in phagocytosis of pathogens by neutrophils (15, 16). To be recognized and engulfed by neutrophils, foreign particles (e.g., invading pathogens) must be coated with either IgG or complement
fragment C3bi, a process known as opsonization. IgG and C3bi
then bind to Fc␥R and complement receptor 3 (CR3), respectively,
and initiate a complex series of cellular events including actin
polymerization, membrane remodeling, extension of pseudopods,
phagosome closure, and particle engulfment (17). CR3 (CD11b/
CD18, ␣M␤2, Mac-1) is one of the members of ␤2 integrin subfamily and is necessary for productive phagocytic signals. Blockade of CR3 receptor using mAbs greatly impairs the ability of
neutrophils to engulf complement-opsonized bacteria (18). ␤1 integrin is also involved in the phagocytosis of certain bacteria via
regulating phagosome maturation through Rac expression (19).
Although it is well accepted that integrins transmit various activating signals in neutrophils, the underlying pathways are still not
completely understood. At sites of integrin adhesion to the extracellular matrix, a multiprotein focal adhesion complex is formed.
This complex contains clustered integrins and numerous cytoplasmic proteins, such as talin, paxillin, vinculin, focal adhesion kinase
(FAK), and zyxin. FAK was first identified in 1992 as a 125-kDa,
highly tyrosine-phosphorylated protein associated with the v-Src
oncogene and localized within integrin-enriched focal adhesion
contact sites (20 –23). It is an important mediator functioning between cells and the extracellular matrix and has been implicated in
controlling several integrin-dependent biological processes, including cell spreading, migration, and regulation of cell survival
(24, 25).
Despite the essential role of FAK in mediating integrin signaling, the exact function of FAK in neutrophils has not yet been
clearly defined. This is partially due to the embryonic lethality of
FAK knockout mice. In this study, we have investigated the role of
FAK in the regulation of neutrophil function by using a myeloidspecific FAK knockout mouse. We found that FAK is required for
fibronectin- and ICAM-1-, but not VCAM-1-mediated neutrophil
adhesion. It also plays a crucial role in NADPH oxidase-mediated
ROS production and complement-mediated phagocytosis. Disruption of FAK leads to reduced pathogen killing capability by neutrophils. In addition, the phosphatidylinositol (3,4,5)-trisphosphate
(PtdIns(3,4,5)P3)/Akt prosurvival pathway in FAK null neutrophils was significantly reduced, leading to elevated death of these
cells.
1034
vessel and still within a distance of 50 ␮m on both sides, thus in an area
of 100 ␮m ⫻ 50 ␮m ⫻ 2 ⫽ 104 ␮m2. Rolling velocity (10 cells/vessel) was
measured as the distance (micrometers) between two points over time (seconds). Centerline blood velocity (Vrbc) was measured for 20 cells/vessel
under a high recording rate of 96 fps. Mean blood flow velocity was calculated as centerline velocity ⫻ 0.625. Wall shear rate was calculated as
8 ⫻ 2.12 (mean blood velocity/diameter) (30).
Superoxide production
Determination of superoxide production was performed as previously described (31). Briefly, reaction mixture in HBSS containing 0.4 ⫻ 106
mouse neutrophils, 4 U/ml HRP (type XII; Sigma-Aldrich), and 5.5 ␮M
luminol was allowed to equilibrate to 37°C for 4 min in a 1420 Wallac
Victor multilabel counter. fMLP (20 ␮l of 10⫻ concentrated) was then
injected into the mixture via the injection port of the luminometer and
luminescence was recorded (for 2 s) at the fixed time intervals.
In vitro bacterial killing assay
Mouse peritonitis model
Wild-type and FAK⫺/⫺ mice were injected i.p. with 200 ␮l of 1 ⫻ 107
E. coli (strain 19138; American Type Culture Collection) in 0.9% saline.
After 4 h, mice were sacrificed, and the peritoneal cavity was flushed with
10 ml of ice-cold PBS. Dilutions of peritoneal lavage were plated onto LB
agar, and the number of bacterial colonies was enumerated to present the
in vivo killing capability. The number of cells in peritoneal cavity was
quantified by using a hemocytometer, and the differential cell count was
obtained by microscopic analysis of Wright-Giemsa-stained cytospins. The
total number of neutrophils was determined as previously described (32).
IgG- and complement-mediated phagocytosis
FITC-labeled E. coli particles (K-12 strain; Molecular Probes) were
opsonized with either 50% mouse serum or 20 ␮g/ml purified mouse
IgG (Sigma-Aldrich) at 37°C for 30 min. For C3 complement-mediated
phagocytosis, E. coli particles were incubated with purified mouse IgM
(10 ␮g/ml; Accurate Chemical and Scientific) for 1 h at 37°C, followed
by an incubation with 10% C5-deficient serum (Sigma-Aldrich) for another 30 min. The opsonized E. coli particles were washed twice and
resuspended in PBS. Mouse neutrophils (4 ⫻ 106 cells) were incubated
with either serum-opsonized or IgG-opsonized E. coli (1.7 ⫻ 108) at
37°C for the indicated time points. For C3 complement-mediated
phagocytosis, neutrophils were treated with 100 ng/ml PMA (SigmaAldrich) for 15 min at 37°C before E. coli addition. At each time point,
phagocytosis was terminated by putting the tubes on ice and quantified
using an inverted fluorescence microscope (Olympus IX17; ⫻100 objective); ⬎100 cells were counted from random fields per coverslip.
Results are presented as the phagocytosis index which is defined as the
total number of internalized particles per 100 neutrophils.
Measurement of calcium flux in nonadherent cells
Mouse neutrophils (5 ⫻ 106/ml) were loaded with 5 ␮M fura 2-AM (Invitrogen) for 45 min at room temperature, then washed twice with warm
PBS to remove the extracellular dye, and resuspended with HBSS buffer
containing Ca2⫹ and Mg2⫹ at a density of 5 ⫻ 106/ml. Neutrophils were
left at room temperature for at least 15 min before the start of the experiment. Fura-2-loaded neutrophils were transferred to a cuvet, resuspended
in 3.6 ml of HBSS buffer, and allowed to equilibrate for at least 10 min
before the addition of 1 ␮M fMLP. Fluorescence changes were recorded
for 3 min in QuantaMaster Spectrofluorometer QM-8/2005 (Photon Technology International) using 340 and 380 nm for excitation and 510 nm for
emission wavelengths. All experiments were performed at room temperature with continuous stirring. The mean of the first 10 points of fluorescence ratios was taken as a baseline for each experiment and subtracted
from subsequent fluorescence ratios to allow generation of changes in flu-
orescence ratio (⌬F340/380) values. The maximum increase of intracellular
calcium induced by fMLP was calculated as the peak of the response.
Calcium flux in adherent cells
Mouse neutrophils (5 ⫻ 106/ml) were loaded with 2 ␮M fura 2-AM (Invitrogen) as described above. The final pellets were resuspended in HBSS
buffer containing Ca2⫹, Mg2⫹, 20 mM HEPES, and 1% BSA at a density
of 5 ⫻ 106/ml. Fura-2-loaded neutrophils (100 ␮l) were plated onto fibronectin (10 ␮g/ml)-precoated glass-bottom dishes (MatTek) and allowed
to loosely adhere for 1.5 min. Cells were then uniformly stimulated with 1
␮M fMLP. Emitted fluorescences were collected simultaneously at 340 and
380 nm excitation and 510 nm emission for 6 min with a 2-s interval using
an inverted fluorescence microscope (Olympus IX17; ⫻40 oil immersion
objective). The changes in fluorescence ratio in each cell were analyzed by
IPLab software (Scanalytics).
FACS analysis of neutrophil spontaneous death
Bone marrow-derived mouse neutrophils were cultured in RPMI 1640 with
10% FCS for the indicated time and stained with annexin V-FITC and 7-aminoactinomycin D-PE staining. FACS analysis was performed by using a
FACScan flow cytometer (BD Biosciences) equipped with a 488-nm argon
laser. Ten thousand cells were collected and analyzed by FlowJo software.
Results
Myeloid-specific FAK knockout mice
Conventional FAK knockout mice die in the early stage of embryonic development (E8.5) due to defective gastrulation events
(33). To investigate the role of FAK in neutrophils, we generated myeloid-specific conditional FAK knockout mice by crossing a FAK-floxed mouse with a mouse expressing Cre recombinase under the control of lysozyme M promoter which is
activated only in the myeloid-specific linage including mature
macrophages, monocytes and neutrophils (supplemental Fig. 1).
Western blotting analysis showed that FAK protein expression
levels were significantly reduced in bone marrow neutrophils
isolated from Cre⫹/⫺FAKloxP/loxP or Cre⫹/⫹FAKloxP/loxP mice
compared with Cre⫺/⫺FAKloxP/loxP littermates (supplemental
Fig. 2 and data not shown). Homozygous mice (Cre⫹/⫺;
FAKloxP/loxP or Cre⫹/⫹; FAKloxP/loxP) were viable, fertile, and
normal in size and displayed no physical or behavioral abnormalities. The number of neutrophils in peripheral blood was
also normal in these mice (supplemental Fig. 3). In addition,
microscopic examination of blood smears did not show any morphological abnormality in these neutrophils (data not shown).
Disruption of FAK impairs fibronectin and ICAM-1-, but not
VCAM-1-mediated neutrophil adhesion
FAK is a crucial component of focal adhesion complex and has
been implicated in integrin-mediated cellular signaling (34 –36).
FAK null fibroblasts show enhanced focal-contact formation. It
has been hypothesized that FAK signaling is associated with the
disassembly of integrin-based adhesion sites (33). Accordingly, we
first addressed whether FAK is involved in the regulation of integrin-mediated adhesion in neutrophils. To mimic the physiological
flow condition and to investigate whether there is a change in the
dynamics of integrin activation, we examined neutrophil adhesion
using a flow-based adhesion assay (Fig. 1). Bone marrow-derived
neutrophils were stimulated or unstimulated with chemoattractant
and then plated on fibronectin (ligand for ␣9␤1, ␣4␤1/VLA-4,
␣M␤2/Mac-1), VCAM-1 (another ligand for ␣4␤1/VLA-4)- or
ICAM-1 (ligand for ␣L␤2/LFA-1 and ␣M␤2/Mac-1)-coated surface. Neutrophil adhesion was examined under conditions of fluid
shear stress in a parallel plate flow chamber. In the absence of any
extracellular stimuli, only a small percentage of cells were adherent (data not shown). Upon stimulation with peptide fMLP (100
nM and 1 ␮M), a tripeptide widely used as a model chemoattractant in studies of neutrophil chemotaxis (37), neutrophil adhesion
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Bacteria (Escherichia coli strain 19138; American Type Culture Collection) was subcultured at 37°C to logarithmic growth from an overnight
culture; 2.5 ⫻ 107 bacteria were opsonized with 12.5% mouse serum at
37°C for 30 min. Opsonized bacteria (12.5 ⫻ 105 E. coli) were added to
2.5 ⫻ 105 purified wild-type or FAK null neutrophils and incubated at
37°C for 30 and 120 min. After each time point, 900 ␮l of ice-cold distilled
water were added to the sample to lyse neutrophils. Viable bacterial counts
from each mixture were measured by plating serial dilutions of bacteria
onto Luria-Bertani (LB) plates in triplicate. Bacteria survival was assessed
by comparing colony number (CFU) to identically handled and plated untreated bacteria (no neutrophils).
ROLE OF FAK IN NEUTROPHILS
The Journal of Immunology
1035
Disruption of FAK did not affect in vivo neutrophil trafficking
FIGURE 1. FAK⫺/⫺ neutrophils exhibit impaired adhesion to fibronectin
and ICAM-1, but not VCAM-1, under flow conditions. Wild-type (WT) and
FAK⫺/⫺ (KO) neutrophils (PMN; 0.5 ⫻ 106/ml) suspended in flow buffer
(PBS, 0.1% BSA) were drawn through the flow chamber at decreasing flow
rates corresponding to an estimated shear stress of 1, 0.8, and 0.5 dyne/cm2.
Neutrophils were pretreated with (w/) 1 ␮M fMLP for 1 min (A–D and F–H),
or PMA for 15 min (E) before being loaded. Data represent the accumulation
of wild-type and FAK-deficient (FAK⫺/⫺) neutrophils on glass coverslips
coated with BSA (A), fibronectin (B and F), recombinant VCAM-1 (C and G),
or ICAM-1 (D and H) under the indicated shear stresses. Data are means ⫾
SEM of three experiments. A value of p ⬍ 0.05 was considered statistically
significant using the paired t test or one-way ANOVA for multiple groups.
A–E, No extra fMLP was added during the assay. F–G, The assay was conducted in the presence of fMLP (100 nM).
was much enhanced compared with cells that were not stimulated
at all. A significant reduction was observed in the FAK null neutrophils when cells were plated on fibronectin or ICAM-1-coated
surface, no matter whether the assay was conducted in the absence
(Fig. 1, B and D) or presence of chemoattractant fMLP (Fig. 1, F
and H). Interestingly and unexpectedly, FAK⫺/⫺ neutrophils
showed an adhesion efficiency similar to that of wild-type cells
when the surface was coated with VCAM-1, suggesting that the
effect of FAK on cell adhesion is ligand specific (Fig. 1, C and G).
In addition, disruption of FAK did not impair cell adhesion induced by PMA, indicating that FAK might not directly regulate
cell adhesion; instead, it may be involved in chemoattractant-elicited signaling pathways leading to firm adhesion (Fig. 1E).
Disruption of FAK did not affect neutrophil migration
FAK also plays an essential role in regulating adhesion turnover in
migrating cells. Disruption of FAK in fibroblasts and macrophages
Circulating neutrophils initiate tissue entry through a complex series of interactions with the endothelial cells that line local postcapillary venules. The process can be divided into at least four
discrete phases: capture and rolling; activation; arrest or firm adhesion; and diapedesis or transmigration from circulation across
endothelium into tissues (39 – 41). We investigated the role of
FAK in neutrophil transendothelial migration in live animals using
a cremaster muscle model and intravital microscopy. Wild-type
and FAK⫺/⫺ mice were intrascrotally injected with either saline
(control) or MIP-2, 2 h before microscopy analysis. We detected a
similar number of rolling neutrophils in MIP-2-challenged
FAK⫺/⫺ and wild-type mice (Fig. 3A). In addition, similar rolling
velocity, adhesion, and emigration were also observed in these
mice (Fig. 3, B–D and supplemental Movies 8 and 9). Consistently, in a mouse peritonitis inflammation model, we observed
similar degree of recruitment of neutrophils to inflamed peritoneal
cavity in FAK⫺/⫺ and wild-type mice (Fig. 3E). Collectively,
these results suggest that FAK is not essential for neutrophil transendothelial migration in vivo.
Disruption of FAK led to reduced complement-mediated
phagocytosis
Phagocytosis of invading pathogens is mediated by either IgG or
complement fragment C3bi. Integrin plays an essential role in
complement-mediated phagocytosis by neutrophils (15, 16). CR3
is one of the members of ␤2 integrin subfamily, and blockade of
CR3 receptor greatly impairs the ability of neutrophils to engulf
complement opsonized bacteria (18). Consistently, FAK has been
suggested to be involved in the process of E. coli phagocytosis by
insect hemocytes (42). However, the role of FAK in pathogen
phagocytosis by mammalian neutrophils has not been fully investigated. Therefore, we examined whether deletion of FAK affects
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lead to defects in cell motility (33, 36, 38). We therefore investigated whether deletion of FAK leads to migration defect in neutrophils. We first performed a chemotaxis assay using an EZTaxiscan chemotaxis device in which a stable chemoattractant
gradient was formed in a 260-␮m-wide channel (supplemental Fig.
4). In the absence of fMLP gradient, chemotaxis was barely observed in neutrophils isolated from wild-type and FAK⫺/⫺ bone
marrow (data not shown). In the presence of fMLP gradient, migration of FAK⫺/⫺ neutrophils on either uncoated or fibronectincoated coverslips was similar in speed and directionality index to
that of wild-type neutrophils (Fig. 2, A and B; supplemental Fig. 4;
and supplemental Movies 1 and 2). Similar results were observed
when higher concentrations (25, 50, and 100 ␮g/ml) of fibronectin
were used (supplemental Movies 3–5). We next examined the
speed and morphology of neutrophils during chemoattractant-elicited random migration (chemokinesis). Neutrophils were plated on
fibronectin (10 ␮g/ml)-coated glass-bottom dishes and uniformly
stimulated with 100 nM fMLP. Random migrations were monitored by time lapse imaging for a period of 10 min at 10 s intervals.
Wild-type and FAK⫺/⫺ neutrophils were predominantly round before fMLP stimulation (Fig. 2C). Upon stimulation, both wild-type
and FAK⫺/⫺ neutrophils polarized, form distinct pseudopods and
uropods, and move randomly (Fig. 2C and supplemental Movie 6).
No detectable impairment in polarization and random migration
speed was observed in FAK⫺/⫺ neutrophils (Fig. 2, C and D).
Similar results were obtained when higher concentration of fibronectin (50 ␮g/ml) was used (supplemental Movie 7). We therefore concluded that FAK is not essential for chemotaxis and chemokinesis in neutrophils.
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ROLE OF FAK IN NEUTROPHILS
phagocytosis in neutrophils. Wild-type and FAK⫺/⫺ bone marrow-derived neutrophils were incubated with serum-opsonized
FITC-labeled E. coli for either 30 or 120 min. In both cases, the
efficiency of phagocytosis was significantly decreased in FAK⫺/⫺
neutrophils (Fig. 4A). The phagocytosis index in FAK⫺/⫺ neutrophils (number of ingested particles per 100 neutrophils) was reduced by 45% at 30 min and ⬃35% at 120 min, compared with
wild-type neutrophils. The augmented phagocytosis was likely a
result of enhanced engulfment, given that there was essentially no
difference in the initial bacteria-binding capability between wildtype and FAK null neutrophils (Fig. 4B). To investigate whether
complement- or IgG-mediated phagocytosis was affected, we opsonized bioparticles using either purified IgG or IgG-depleted
C3i-rich serum. As expected, FAK⫺/⫺ neutrophils showed a clear
defect in C3 complement-mediated phagocytosis, but not in IgGmediated phagocytosis (Fig. 4, C and D). These results demonstrated that FAK plays an essential role in complement-mediated
phagocytic signaling pathway.
⫺/⫺
FIGURE 3. The in vivo neutrophil trafficking is normal in FAK
mice. Wild-type (WT) and FAK⫺/⫺ (KO) mice were intrascrotally injected
with MIP-2 (1 ␮g/mouse). The contralateral cremaster muscle was externalized for observation. The indicated parameters were assessed by intravital video microscope. A, The number of cells rolling per minute; B, rolling velocity; C, number of adherent cells per minute; D, transmigration.
Results are means ⫾ SD from two to three experiments. Two videos of the
experiment described in this figure are included in supplemental Movie 9).
E, Wild-type and FAK⫺/⫺ mice were injected i.p. with 2 ⫻ 106 E. coli cells
in 0.9% NaCl. After 4 h, mice were killed, and the peritoneal lavage fluid
was collected. The total number of neutrophils in peritoneal cavity was
determined using microscopic examination of Wright-Giemsa-stained cytospins. Results are means ⫾ SD of four mice. f, Wild type; 䡺, FAK⫺/⫺.
Disruption of FAK reduced both adhesion and
chemoattractant-elicited superoxide production
Neutrophil adhesion to extracellular matrix or the surface of endothelial cells elicits integrin-mediated signaling pathway leading
to NADPH oxidase activation and subsequent release of superoxide. Although FAK was identified as a key component in integrin
signaling, whether it is essential for adhesion-elicited superoxide
production is still largely unknown. To test this, we conducted an
in vitro luminal chemiluminescence assay using bone marrow-derived neutrophils. Cell adhesion-induced superoxide production in
FAK null neutrophils was significantly reduced at each time points
examined, whereas the time course for the increase and subsequent
decrease in superoxide production was not altered (Fig. 5A). When
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FIGURE 2. FAK⫺/⫺ neutrophils exhibit normal chemotaxis and chemokinesis upon fMLP stimulation. A and B, Wild-type (WT) and FAK⫺/⫺ (KO)
neutrophils were plated onto uncoated or fibronectin-coated coverslips. Chemotaxis was visualized using an EZ-TAXIScan device (supplemental Fig. 4).
Chemotaxis speed and directionality were quantified using DIAS imaging software as described in supplemental Fig. 5. Results are presented as means ⫾
SD of ⬎20 neutrophils. C, Bone marrow-derived wild-type and FAK⫺/⫺ neutrophils were plated onto fibronectin (10 ␮g/ml)-coated MatTek dish and
uniformly stimulated with 100 nM fMLP. Shown are representative images of neutrophils before (left) and 10 min after fMLP stimulation (right). Two
videos of the experiment described in this figure are included in supplemental Movie 6). Images were captured using a ⫻40 optical lens (recorded at 1
frame/10 s). D, Migration speeds of wild-type and FAK⫺/⫺ neutrophils during chemokinesis were calculated as total distance of migration from origin
divided by total time. Results are means ⫾ SD of 10 neutrophils. f, Wild type; 䡺, FAK⫺/⫺.
The Journal of Immunology
adherent neutrophils were stimulated with chemoattractant
fMLP, a much higher level of superoxide was produced. Under
this condition, similar inhibition was observed in FAK null neutrophils (⬎70% reduction compared with wild-type neutrophils;
Fig. 5B).
We next examined whether this effect occurred only in adherent
neutrophils. We suppressed neutrophil adhesion by preblocking
the assay plate with a high concentration of BSA. Reduced adhesion was confirmed by microscopy examination (supplemental Fig.
6). Wild-type and FAK⫺/⫺ neutrophils were then stimulated with
fMLP, and superoxide products were measured using a luminal
chemiluminescence assay as described in Materials and Methods.
Surprisingly, we could still detect a pronounced reduction of superoxide production in the FAK⫺/⫺ neutrophils, suggesting that
FAK might also play a role in chemoattractant-elicited NADPH
oxidase activation independent of cell adhesion (Fig. 5C). To confirm this unexpected result, we examined superoxide production in
neutrophils treated with latrunculin B, which rapidly and specifically disrupted the actin cytoskeleton and completely prevented
cell adhesion (supplemental Fig. 7). Disruption of actin cystoskeleton with latrunculin B markedly enhanced fMLP-stimulated superoxide production in neutrophils (Fig. 5D) as previously reported (43). Consistent with what was observed in Fig. 5C,
disruption of FAK in these neutrophils decreased the fMLP-elicited superoxide production by ⬎60% compared with wild-type
neutrophils (Fig. 5D). Although many cellular processes can be
affected by latrunculin, in this study we examined ROS production
by wild-type and FAK KO neutrophils under exactly the same
condition, and thus the comparison of interest should most likely
be the effect of genetic deficiency rather than effects of latrunculin
treatment. Altogether, these results suggest that FAK is involved
FIGURE 5. Superoxide production is reduced in FAK⫺/⫺ neutrophils.
Bone marrow-derived wild-type (WT) and FAK⫺/⫺ (KO) neutrophils were
used in a luminol-dependent chemiluminescence assay to investigate superoxide productions. Superoxide production was monitored in a luminometer at 37°C. Chemiluminescence (arbitrary unit lights) was recorded (for
2 s) at indicated time points. A, Adhesion-mediated superoxide production.
B, fMLP-induced superoxide production. C, fMLP-induced superoxide
production in plate preblocked with 5% BSA. D, fMLP-induced superoxide production in latrunculin B-treated neutrophils. Results are means ⫾
SD of duplicated samples from one experiment representative of three. f,
Wild-type; 䡺, FAK⫺/⫺.
not only in integrin-mediated but also in chemoattractant receptormediated NADPH oxidase activation independent of cell adhesion.
FAK is a component in chemoattractant-elicited cellular signal
pathway leading to NADPH oxidase activation in both adherent
and suspended neutrophils
FAK activation and autophosphorylation creates the binding sites
for p85 subunit of PI3K and phosphatidylinositol-specific phospholipase C (PLC; Refs. 44 – 46). Association of FAK with these
proteins leads to activation of the corresponding enzymes and the
subsequent generation of signal molecules PtdIns(3,4,5)P3, IP3,
and calcium, which have all been implicated in the activation of
NADPH oxidase. Nevertheless, FAK role and relative contribution to chemoattractant-elicited NADPH oxidase activation
signaling have not been fully investigated. To examine this, we
explored whether loss of FAK can lead to down-regulation of
fMLP-induced productions of PtdIns(3,4,5)P3 and intracellular
calcium transient. Phosphorylation of Akt was used as an indicator of the PtdIns(3,4,5)P3 signaling pathway. Akt contains a
PtdIns(3,4,5)P3-specific pleckstrin homology domain and can be
recruited onto the plasma membrane via its specific binding to
PtdIns(3,4,5)P3. Only the Akt molecules on the plasma membrane
can be phosphorylated by two phosphatidylinositol-dependent protein kinases and become activated (47, 48). Akt phosphorylation
was monitored by Western blot analysis using a phospho-Akt-specific Ab. In both adherent (Fig. 6, A and B) and suspended (Fig. 6,
C and D) neutrophils, Akt phosphorylation was hardly detected
before challenging with chemoattractant. Levels of phospho-Akt
increased substantially after fMLP stimulation and reached the
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FIGURE 4. FAK⫺/⫺ neutrophils show decreased complement-mediated
phagocytosis. FITC-labeled E. coli bioparticles were opsonized with
mouse serum (A and B), IgG (20 ␮g/ml; C), or C3 complement (D). Bone
marrow-derived wild-type (WT) and FAK⫺/⫺ neutrophils were incubated
with opsonized E. coli for 30 and 120 min (approximate E. coli-neutrophil
ratio, 46:1). Phagocytosis of E. coli particles was determined under the
inverted fluorescence microscope. The phagocytosis index was expressed
as the number of bioparticles engulfed by 100 neutrophils (A, C, and D).
Binding index was expressed as the number of bioparticles bound to 100
neutrophils (B). More than 200 neutrophils were counted in each group.
Results are means ⫾ SD from at least three independent experiments.
ⴱ, p ⬍ 0.05, ⴱⴱ, p ⬍ 0.01 vs wild-type neutrophils by Student’s t test.
f, Wild type; 䡺, FAK⫺/⫺.
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1038
ROLE OF FAK IN NEUTROPHILS
maximum value between 1 and 3 min. Disruption of FAK significantly decreases the level of Akt phosphorylation after fMLP
stimulation in adherent and suspended neutrophils. These results
demonstrated that FAK is involved in chemoattractant-elicited
PtdIns(3,4,5)P3 signaling pathway activation independent of cell
adhesion.
The increase of intracellular calcium concentration was measured in wild-type and FAK⫺/⫺ neutrophils using fura-2 AM ratiometric fluorescence indicator. When neutrophils loosely adhered
on a 10 ␮g/ml fibronectin-coated dish, fMLP stimulation-elicited
inside-out signal initiated further integrin engagement, resulting in
activation of downstream signals such as calcium. FAK⫺/⫺ neutrophils displayed a reduced response to fMLP compared with
wild-type neutrophils, indicating a crucial role of FAK in this signaling process (Fig. 6E, supplemental Fig. 8, and supplemental
Movie 10). Different from what was observed in adherent neutrophils, the level of fMLP-induced increase in intracellular Ca2⫹ was
comparable in wild-type and FAK⫺/⫺ neutrophils in suspension
with continuously stirring (Fig. 6, F and G). The maximum increases of intracellular calcium in these cells were comparable
(Fig. 6G). These results suggest that FAK plays a role in chemoat-
tractant-elicited calcium signaling in adherent neutrophils, but not
in suspended neutrophils. It is likely that the engagement of integrin receptors on the cell surface is necessary for the involvement
of FAK in chemoattractant-elicited calcium signaling.
The bacterial killing capability was reduced in FAK null
neutrophils
Both phagocytosis and NADPH oxidase-mediated superoxide production play an important role in bacterial killing by neutrophils.
In chronic granulomatous disease, impairment of NADPH oxidase
function leads to elevated susceptibility of patients to microbial
infection (49). Similarly, the defects of phagocytosis and superoxide production in FAK null neutrophils may lead to impaired antimicrobial mechanism in FAK knockout mice. To test this, we
first conducted an in vitro bacterial killing assay. Serum-opsonized
E. coli particles were incubated with either wild-type or FAK⫺/⫺
neutrophils at a ratio of 5 E. coli to 1 neutrophil at 37°C for 30 and
120 min. The numbers of surviving bacteria were quantified using
a colony-forming assay. Although the bacterial killing ability of
wild-type and FAK⫺/⫺ neutrophils was still comparable at 30 min,
FAK⫺/⫺ neutrophils showed a significant defect in killing E. coli
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FIGURE 6. fMLP-mediated signaling pathways are reduced in FAK⫺/⫺ neutrophils. A and B, fMLP-induced Akt phosphorylation in adherent neutrophils. Bone marrow-derived wild-type (WT) and FAK⫺/⫺ (KO) neutrophils were allowed to adhere at 37°C for 4 min and then stimulated with 1 ␮M fMLP
for the indicated time (0 –90 s). A, Western blotting analysis. B, Results of densitometry. C and D, fMLP-induced Akt phosphorylation in nonadherent
neutrophils. Wild-type and FAK⫺/⫺ neutrophils in suspension were stimulated with 1 mM fMLP for the indicated time (0 –5 min). C, Western blotting
analysis. D, Results of densitometry. Expression levels of phosphorylated Akt and total Akt were detected by Western blotting analysis using antiphosphorylated Akt (Ser473; 1/1000) and anti-Akt (1/1000) Abs (Cell Signaling), respectively. Results are presented as ratio of phospho-Akt to total Akt with
actin normalization. ⴱ, p ⬎ 0.05 vs wild-type neutrophils by Student’s t test. E and F, fMLP-induced calcium signaling in neutrophils. Wild-type and
FAK⫺/⫺ neutrophils were loaded with fura 2-AM for 45 min at 37°C. E, fura 2-loaded wild-type and FAK⫺/⫺ neutrophils were allowed to loosely adhere
on a fibronectin (10 ␮g/ml)-coated MatTek dish for 1.5 min. Cells were uniformly stimulated with 1 ␮M fMLP, and changes of [Ca2⫹]i were monitored
every 2 s for 10 min using a ⫻40 objective. Results are a single cell from wild-type and FAK⫺/⫺ neutrophils representative of at least 40 cells from two
independent experiments. Red line, wild type; blue line, FAK⫺/⫺. Two videos of the experiment described in this figure are included in supplement data
(Movie 10). F, Neutrophils in suspension were stimulated with 1 ␮M fMLP. The changes in intracellular calcium ([Ca2⫹]i) were monitored for 3 min after
stimulation. Results are means ⫾ SD from two independent experiments. Red line, wild type; blue line, FAK⫺/⫺. Arrow, addition of fMLP. G, Peaks of
changes in [Ca2⫹]i of wild-type and FAK⫺/⫺ neutrophils after fMLP stimulation. Results are means ⫾ SD from two independent experiments. f, Wild
type; 䡺, FAK⫺/⫺.
The Journal of Immunology
(⬃60% reduction compared with wild-type) at 2 h (Fig. 7A). We
also investigated the function of FAK in in vivo bacterial killing
using the peritonitis model. Live E. coli cells were injected into the
peritoneum of wild-type and FAK⫺/⫺ mice. Peritoneal lavage was
collected from these mice 4 h after the injection. Serial dilutions of
this lavage (1/500, 1/1000, and 1/5000) were plated onto LB agar
to measure the number of surviving bacteria (Fig. 7B). Three times
more live E. coli cells were detected in the inflamed peritoneal
cavity of FAK⫺/⫺ mice than in the wild-type littermates (Fig. 7C).
Because the number of neutrophils accumulated in the peritoneal
cavity after E. coli injection was comparable in the wild-type and
FAK⫺/⫺ mice (Fig. 3E), the elevated bacteria number in FAK
knockout mice is most likely due to a bacterial killing defect in
neutrophils.
The spontaneous death was accelerated in FAK null neutrophils
Neutrophils are terminally differentiated cells. They normally have
a very short life span (6 –7 h in blood and 1– 4 days in tissue) and
readily undergo spontaneous programmed cell death (apoptosis;
FIGURE 8. FAK deletion enhances spontaneous neutrophil death. A,
Bone marrow-derived wild-type (WT) and FAK⫺/⫺ (KO) neutrophils were
cultured in RPMI 1640 containing 10% heat-inactivated FBS at a density
of 5 ⫻ 105 cells/ml. Apoptotic cells were detected by annexin V-FITC
staining and 7-aminoactinomycin D (7-AAD) staining. Ten thousand cells
were collected at the indicated time points and analyzed by FlowJo software. Region R1, viable cell; Region R2, early apoptotic cells; Regions R3
and R4, late apoptotic cells and necrotic cells. B, Time course of neutrophil
spontaneous death. Results are means ⫾ SD of two separate experiments.
Ref. 50). Neutrophil death shares many features of classical apoptosis, such as cell body shrinkage, exteriorization of phosphatidylserine (PS) from the inner to the outer leaflet of plasma membrane, mitochondria depolarization, nuclear condensation, and
DNA fragmentation (51). Recently, we established PtdIns(3,4,5)P3/
Akt deactivation as one of the causal mediators of apoptosis in
neutrophils. PtdIns(3,4,5)P3/Akt signal is strongly deactivated
during neutrophil death. Inhibition of this signal pathway promotes
neutrophil spontaneous death, whereas augmentation of this signal
prevents neutrophil death (27). Because the PtdIns(3,4,5)P3/Akt
pathway was significantly suppressed in both adherent and suspended FAK null neutrophils, we next explored whether the programmed death of these neutrophils was altered. The number of
neutrophil undergoing spontaneous death was quantified using
FACS analysis. In the FACS assays, we use Annexin V, an anticoagulant protein that has high affinity and selectivity for PS, to
detect PS exteriorization and 7-aminoactinomycin D, a nucleic
acid dye which intercalates into the double-stranded nucleic acid
and penetrates cell membranes of dying or dead cells (Fig. 8A).
The PS exposure was evident in neutrophils by 16 h, and the level
increased to 35 ⫾ 4% at 24 h. We detected a concomitant increase
in the apoptotic (R2, lower right quadrant) and necrotic (R3 plus
R4) populations at 24 h, suggesting that at later time points many
of the apoptotic cells have proceeded into secondary necrosis (Fig.
8A). As expected, FAK null neutrophils displayed a much higher
level of cell death than did wild-type neutrophils, and this effect
was observed at all time points examined. Only 34% of FAK null
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FIGURE 7. FAK⫺/⫺ neutrophils are defective in killing of E. coli in
vitro and in vivo. A, Wild-type (WT) and FAK⫺/⫺ (KO) neutrophils were
incubated with serum-opsonized E. coli at 37°C for 30 and 120 min, and
the numbers of surviving bacteria were determined and presented as CFUs.
B and C, Wild-type and FAK⫺/⫺ mice were injected i.p. with 2 ⫻ 106 E.
coli in 0.9% NaCl. After 4 h, mice were killed, and the peritoneal lavage
fluid was collected. Surviving bacteria were enumerated by plating several
dilutions (1/500, 1/1000, and 1/5000) onto LB agar (B) and total numbers
of surviving bacteria per animal were calculated (C). Results are means ⫾
SD of four mice. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01 vs wild-type neutrophils by
Student’s t test. f, Wild type; 䡺, FAK⫺/⫺.
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1040
neutrophils could live longer than 24 h in the culture, whereas
⬃64% wild-type neutrophils were detected healthy under the same
condition (Fig. 8B). These results demonstrated that FAK also
plays an essential role in modulating the life span of neutrophils,
probably through the Akt/PIP3 pathway.
Discussion
flammation model, we observed a similar degree of recruitment of
neutrophils to an inflamed peritoneal cavity in FAK⫺/⫺ and wildtype mice, suggesting that FAK is not essential for neutrophil
transendothelial migration in vivo. These results are somewhat surprising, considering that FAK is essential for fibronectin and
ICAM-1-mediated adhesion. In the myeloid specific FAK knockout mice, FAK expression is also ablated in monocytes and macrophages, and maybe some dendritic cells. However, in the peritonitis model and the cremaster muscle model described above,
macrophages migrated to the sites of inflammation after neutrophil
recruitment. Thus, it is unlikely that macrophages are involved in
the recruitment of neutrophils. Neutrophil recruitment to the peritoneum in response to bacteria has previously been shown not to
require ␤2 integrins (56). However, it seems that other types of
integrins (e.g., ␤1 and ␤7) are involved in this model, given that it
was recently reported that extravasation of integrin⫺/⫺ leukocytes
(all integrins were depleted in these mice) from the blood stream
into the inflamed sites was completely abolished, suggesting that
integrins are essential mediators in neutrophil transmigration (57).
In addition, it was well known that in the cremaster muscle model
used above, ␤2 integrins are critical for chemokine-induced neutrophil transmigration (58). Thus, the most logical explanation for
the observed normal neutrophil adhesion/trafficking in the FAK
knockout mice is that neutrophil adhesion on endothelial cells in
these models might be mainly mediated by VCAM-1 or other factors that are not regulated by FAK.
The pathogen-killing capability of neutrophils is mediated by a
network of intracellular signaling pathways. Our data have defined
FAK as a key regulator in neutrophil phagocytosis and NADPH
oxidase-mediated superoxide production. Its involvement in
phagocytosis is not surprising because it is well known that integrin plays an essential role in phagocytosis of pathogens by neutrophils (15, 16). Phagocytosis is mediated by either Fc␥R or CR3
(17). CR3 (CD11b/CD18, ␣M␤2, Mac-1) is one of the members of
the ␤2 integrin subfamily and is necessary for productive phagocytic signaling. Impairment of CR3 receptor leads to defect in
engulfment of complement-opsonized bacteria by neutrophils (18).
Additionally, ␤1 integrin has been implicated in the phagocytosis
of certain bacteria via regulating phagosome maturation through
Rac expression (19). Our findings underscored the essential role of
FAK in mediating integrin signaling during complement-mediated
phagocytosis. In FAK null neutrophils, no abnormality was detected in integrin-independent IgG-mediated phagocytosis. It was
previously reported that complement-mediated phagocytosis is dependent on Rho activation, whereas IgG-mediated is dependent on
Cdc42 and Rac (59). We investigated whether Rho activation is
diminished in FAK⫺/⫺ neutrophils during complement-mediated
phagocytosis. No significant difference in the cellular level of activate form of Rho (Rho-GTP) was detected between the wild-type
and FAK knockout neutrophils (supplemental Fig. 10). This suggests that phagocytosis-induced Rho activation might not be a
downstream event of phosphatidylinositol 4,5-trisphosphate signaling, which was significantly reduced in the FAK knockout neutrophils. Or at least phosphatidylinositol 4,5-trisphosphate is not
the only signal that controls Rho activation.
Neutrophil adhesion to extracellular matrix or the surface of
endothelial cells elicits NADPH oxidase-mediated superoxide production. We have found that adhesion-induced superoxide production and fMLP-induced superoxide production in adherent neutrophils are much reduced in FAK-deficient neutrophils. This is
consistent with the involvement of integrin in these adhesion-mediated cellular events. FAK is localized within integrin-enriched
focal adhesion contact sites, and the FAK-mediated signaling pathway leads to NADPH oxidase activation and subsequent release of
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In current study, using myeloid-specific conditional FAK knockout
mice, we demonstrated that FAK, a non-receptor protein tyrosine
kinase which is mostly known as a regulator of cell adhesion and
migration, plays a crucial role in regulating various neutrophil
functions. These results revealed previously unrecognized roles of
FAK in neutrophil function and provided a potential target for
treatment of a variety of infectious and inflammatory diseases.
FAK is a ubiquitously expressed protein tyrosine kinase, the
function of which has been well studied in fibroblasts, where it
plays an important regulatory function in cell matrix/integrin-dependent adhesion and motility (24, 25). Following integrin-mediated binding to extracellular matrix proteins such as fibronectin,
FAK is catalytically activated and undergoes autophosphorylation
at Tyr397, which serves as a binding site for Src homology 2 (SH2)
domain containing Src family kinases. The FAK-Src kinase complex leads to further phosphorylation at additional FAK sites and
to the recruitment and activation of multiple downstream signaling
proteins, including PI3K and PLC␥. It has been hypothesized that
FAK signaling is associated with the disassembly of integrin-based
adhesion sites and thus coordinates lamellipodial formation and
regulates adhesion turnover in migrating cells. FAK null fibroblasts show excess focal contact formation and defect in cell
movement (33). FAK is also expressed in hemopoietic cells. Owen
et al. (38) recently reported that FAK null macrophages exhibited
elevated protrusive activity at the cell periphery, reduced adhesion
turnover, and a marked inability to form the stable lamellipodia
necessary for directional movement. As a result, these macrophages failed to migrate to sites of infection and inflammation in
vivo. FAK also plays a role in megakaryopoiesis and platelet function. FAK null platelets exhibit diminished spreading on immobilized fibrinogen (52). Stimuli-induced FAK phosphorylation was
also observed in neutrophils. Ryu et al. reported that pituitary
growth hormone (GH) triggered tyrosine phosphorylation of FAK
and focal localization of phosphorylated FAK into the membrane
rufflings and uropods of human neutrophils (53). FAK phosphorylation was also detected in neutrophils stimulated with disintegrin
peptides (54) and after integrin activation (55). Consistently, in
current study, we demonstrated that FAK is required for regulation of focal adhesion dynamics when neutrophils adhere to
fibronectin or ICAM-1. Interestingly and unexpectedly, FAK⫺/⫺ neutrophils showed an adhesion efficiency similar to that of wild-type
cells when the surface was coated with VCAM-1, suggesting that the
effect of FAK on cell adhesion might be ligand specific. In addition,
FAK⫺/⫺ neutrophils also exhibited normal chemoattractant-elicited
chemotaxis and chemokinesis, indicating that FAK is also not essential for neutrophil migration. An alternative explanation involves the
FAK homolog pyk2 which is highly expressed in neutrophils. It is
possible that pyk2 is able to compensate in the absence of FAK to
maintain normal neutrophil migration, despite the fact that pyk2 protein expression and phosphorylation were not altered in the FAK null
neutrophils (supplemental Fig. 9). It will be intriguing to examine
whether any neutrophil migration defect can be detected in FAK/pyk2
double-knockout mice.
We also examined neutrophil trafficking in live animals. We
detected similar rolling influx, rolling velocity, adhesion, and emigration in MIP-2-challenged FAK⫺/⫺ and wild-type mice in a
cremaster muscle model. Consistently, in a mouse peritonitis in-
ROLE OF FAK IN NEUTROPHILS
The Journal of Immunology
superoxide. However, the fact that FAK is also involved in fMLPinduced superoxide production in suspended neutrophils is somewhat surprising. This suggests that FAK is also regulated by G
protein-coupled chemoattractant receptor, independent of cell adhesion-elicited integrin activation. A similar effect was recently
observed in chemokine CXCL12-stimulated REH pro-B cells (60)
and hemopoietic progenitor cells (61). Upon CXCL12 stimulation,
activated CXCR4 receptors form clusters in lipid raft domains and
subsequently trigger signaling molecules, such as Gi protein, Src
family proteins, and FAK. This event can happen in suspension
without cell adhesion. It provides an inside-out signaling that activates progenitor B cell surface integrins, such as VLA-4, and
subsequently promotes cell adhesion (60).
To delineate the mechanisms underlying the cellular defects observed in FAK-deficient neutrophils, we examined the involvement of several downstream targets in FAK-mediated neutrophil
functions. Phosphorylation and activation of FAK provides a binding site for SH2 domain-containing proteins such as Src, p85 subunit of PI3K, and PLC␥ (24, 25, 62). The FAK-Src kinase complex
leads to further phosphorylation at additional FAK sites and to the
recruitment and activation of other downstream signaling proteins.
Associations of FAK with p85 leads to activation of PI3K and the
subsequent generation of signal molecule PtdIns(3,4,5)P3. As another important signaling molecule, Ins(1,4,5)P3, is formed from
the hydrolysis of phosphatidylinositol 4,5-bisphosphate by PLC
(63). In mammals, multiple PLC genes were identified and divided
into four major types, ␤, ␥, ␦, and ␧. They differ in their domain
structure, regulation, and tissue distribution. PLC␥ contains an
SH2 domain and can be activated by FAK and responsible for cell
adhesion-mediated calcium signaling. In agreement with these previous reports, we revealed that in adherent cells, disruption of FAK
suppressed both PtdIns(3,4,5)P3 signaling and chemoattractantelicited calcium signaling. Interestingly, in suspended neutrophils,
only PtdIns(3,4,5)P3 signaling was reduced upon FAK disruption,
whereas the fMLP-elicited calcium signal was not altered, indicating that engagement of fMLP receptor can initiate calcium signaling required for NADPH oxidase activation independent of FAK
(Fig. 9). The PLC␤ family, which consists of four isoforms, ␤1–␤4,
can be directly regulated by G protein. It was reported that PLC␤
is activated by fMLP in neutrophils and that the activation is mediated by the G protein ␤␥ subunits that are released from the Gi
protein upon chemoattractant receptor activation (64, 65). The exact pathway leading to G protein-coupled chemoattractant receptor-induced FAK activation in the absence of cell adhesion is still
largely unknown. It appears that Src plays a role in adhesioninduced FAK activation, because NADPH oxidase activity was
inhibited by Src inhibitors (data not shown). In contrast, Src inhibitors had no effect on fMLP-induced FAK activation (Fig. 9). A
recent study demonstrated that RhoG is important in superoxide
release in neutrophils (66). However, because the level of activated
Rho is the same in the FAK null neutrophils (supplemental Fig.
10), it is unlikely that the NADPH oxidase defect seen in FAK⫺/⫺
neutrophils is caused by impaired Rho activation.
The reduced PtdIns(3,4,5)P3/Akt signaling in FAK null neutrophils parallels and may account for the accelerated spontaneous
death observed in these cells. PtdIns(3,4,5)P3/Akt signaling pathway possesses prosurvival and antiapoptotic activities in a variety
of cell types. Akt contains a pleckstrin homology domain which
specifically binds PtdIns(3,4,5)P3. The PtdIns(3,4,5)P3-mediated
membrane translocation of Akt is essential for its phosphorylation
and activation. Activated Akt, in turn, phosphorylates a variety of
proteins, including several associated with cell survival/death pathways such as BAD, Forkhead, ASK1, and NF-␬B, leading to diminished apoptotic cell death (67, 68). We recently established
Akt deactivation as a causal mediator in neutrophil spontaneous
death. Augmentation of PtdIns(3,4,5)P3/Akt signal prevents neutrophil spontaneous death, whereas inhibition of PtdIns(3,4,5)P3/
Akt signal further promotes neutrophil death. Thus, the elevated
death in FAK null neutrophils is likely due to the reduced
PtdIns(3,4,5)P3/Akt signaling in these cells, although we cannot
completely rule out the involvement of other death/survival pathways downstream of FAK. It has been reported that activated FAK
can augment the activity of Ras-MAPK pathway (25, 62), resulting
in elevated cell survival (24, 25). FAK also binds to the death
domain kinase receptor-interacting protein (RIP), a major component of the death receptor complex that has been shown to interact
with Fas and TNFR1 through its binding to adapter proteins. The
proapoptotic activity of RIP is suppressed by its binding to FAK
(69). Nevertheless, the involvement of RIP and Ras in FAK-mediated neutrophil survival/death remains unknown and should be
further investigated. Neutrophil spontaneous death might play a
role in neutrophil homeostasis. We examined whether disruption
of FAK can increase peripheral blood neutrophil count due to delayed spontaneous death. However, we did not detect any alteration in the peripheral blood neutrophil count (supplemental Fig.
3) or the percentage of apoptotic neutrophils in the peripheral
blood (supplemental Fig. 11). The peripheral blood neutrophil
count is decided by multiple cellular processes, such as cytokineelicited mobilization from bone marrow, spontaneous death, transmigration from blood to tissues, as well as clearance by phagocytic
cells. Conceivably, depletion of FAK can affect processes other
than spontaneous death and these effects are able to overcome the
effect elicited by delayed neutrophil death, leading to unaltered
peripheral blood neutrophil count. Alternatively, neutrophil spontaneous death might not be a deciding factor for regulating peripheral blood neutrophil count in mouse.
Acknowledgments
We thank John Manis, Li Chai, and people in Joint Program in Transfusion
Medicine for helpful discussions.
Disclosures
The authors have no financial conflict of interest.
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
FIGURE 9. FAK mediates NADPH oxidase activation in mouse neutrophils. Cell adhesion-mediated integrin aggregation leads to activation of
FAK and its downstream signaling molecules including Src family kinases,
PtdIns(3,4,5)P3, and Ca2⫹. Elevation of PtdIns(3,4,5)P3 and Ca2⫹ results
in activation of NADPH oxidase. In nonadherent neutrophils, binding of
fMLP to its receptor(s) triggers activation of FAK and downstream
PtdIns(3,4,5)P3 signaling. Ca2⫹ signaling is directly regulated by fMLP
receptor independent of FAK. GPCR, G protein-coupled chemoattractant
receptor.
1041
1042
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Supplemental Figure 1
A
FAK line
FAK exon 2
Wild-type (FAK wt)
LoxP
LoxP
Conditional allele (FAK loxP)
Cre recombinase
FAK mutant allele (FAK -)
Cre line
B
Lys M gene promoter (activated in myeloid linage)
M lysozyme gene
Wild-type (Cre -)
Lys M gene promoter (activated in myeloid linage)
Cre transgene (Cre +)
Cre recombinase gene
C
X
Cre +/+; FAK wt/wt
X
Cre +/-; FAK wt/loxP
Cre +/+; FAK wt/loxP
Cre +/+; FAK wt/wt
Wild-type FAK
Cre +/+; FAK loxP/loxP
Homozygous FAK mutant
Heterozygous FAK mutant
Cre -/-; FAK loxP/loxP
Cre +/-; FAK wt/loxP
Cre -/-; FAK wt/loxP
Cre +/-; FAK wt/loxP
Cre +/-; FAK wt/wt
Cre +/-; FAK loxP/loxP
Wild-type FAK
Homozygous FAK mutant
Heterozygous FAK mutant
Cre -/-; FAK wt/wt
Cre -/-; FAK loxP/loxP
Wild-type FAK
(In the absence of Cre, mutant FAK can not be generated
from FAK-loxP allele)
Supplemental Figure 1. Myeloid specific FAK-/- mice. (A) The conditional FAK knockout mouse line in which two loxP sequences were
inserted on either side of the exon 2 of FAK encoding the phosphatase domain. (B) The myeloid-specific Cre mouse line. In these mice, the
Cre recombinase gene was inserted into the endogenous M lysozyme locus, and therefore is under the control of lysozyme promoter which
is activated only in myeloid linage, including monocytes, mature macrophages, and neutrophils. (C) The strategy for producing myeloidspecific FAK knockout mice. Only in myeloid linage, the loxP site will be cut by Cre recombinase, leading to the disruption of FAK. Cre–
mediated deletion of loxP–flanked FAK gene in myeloid cells is both specific and highly efficient. The amount of Cre recombinase
expressed from only one copy of the Cre gene is enough to initiate FAK deletion. The expression of wild-type FAK protein is completely
abolished in neutrophils isolated from either Cre+/-;FAK loxP/loxP or Cre+/+; FAK loxP/loxP mice.
Supplemental Figure 2
FAK
WT
KO
A
FAK
Actin
B
FAK Protein (normalized)
1.2
1.0
0.8
0.6
0.4
0.2
0.0
WT
FAK KO
Supplemental Figure 2. The expression of FAK protein is completely abolished in FAK-/- neutrophils. (A) Bone
marrow-derived neutrophils were isolated from wild type and FAK-/- mice by centrifugation over a three-layer Percoll
gradient (76%/64%/52%). Protein extracts were resolved on SDS-PAGE and immunoblotted for FAK using anti-FAK
monoclonal antibody (Upstate, USA). Actin was used as the loading control. (B) Relative amounts of FAK protein in (A)
were quantified using a NIH Image software and normalized with wild-type samples. Black bar, wild type; white bar,
FAK-/-.
Supplemental Figure 3
15
5
12
4
x 103 Neutrophils/µ l
x 103 White blood cells/µ l
A
9
6
3
0
WT
FAK KO
3
2
1
0
WT
FAK KO
B
Parameters
WT Mice (n=28)
FAK-/- Mice (n=32)
p Value
White blood cells (x 103 cells/µl)
8.52 ± 0.477
7.49 ± 0.368
0.0876
Neutrophils (x 103 cells/µl)
1.61 ± 0.135
1.66 ± 0.161
0.8222
Supplemental Figure 3. Blood parameter of wild type and FAK-/- mice. Peripheral blood collected from eye of each
mouse was analyzed. (A) Scatter plot of white blood cells and neutrophils per ml of blood from n = 28-32 wild type or
FAK-/- mice. (B) Blood parameters presented as mean ± (SD) from 28-32 mice.
Supplemental Figure 4
Before
After (10 min)
fMLP gradient
A
WT
FAK KO
B
WT
FAK KO
Uncoated
Fibronectin
(10 mg/ml)
Supplemental Figure 4. (A) Wild type and FAK-/- neutrophils were plated onto uncoated or fibronectin (10 µg/ml) coated
coverslip. One microliter of neutrophils were added to the bottom well and fMLP was added to the top well of EZ-TAXIScan
device. This creates a shallow chemoattractant gradient between the wells which drives cells to migrate toward the fMLP
containing well. Images of migrating cells in each channel were captured in parallel using a 10X lens coupled to a CCD camera.
Shown are representative images of before and 10 min after fMLP stimulation. Four videos of the experiment described in this
figure are included in Supplement Data (Movie 1-4). (B) Cell tracks analysis of wild type and FAK-/- neutrophils during
chemotaxis using DIAS imaging software (Solltech, Oakdale, IA).
Supplemental Figure 5
y
(x0, yf)
(µm)
(xf, yf, tf)
Chemoattractant Gradient
df
(xc, yc, tc)
dS
dc
db
da
(xb, yb, tb)
(xa, ya, ta)
(x0, y0, t0)
(0, 0)
(µm)
x
Supplemental Figure 5. Chemotaxis parameters for neutrophil migration in response to a chemoattractant
gradient. If n is the number successive frames analyzed and (xn, yn, tn) denotes the position of the neutrophil
(xn,yn) at any time tn the chemotaxis parameters can be calculated as follows: Directionality (0 to1) is dS / (da + db
…+df), Speed (µm/min) is (da/(ta-t0) + db/(tb-ta) + …+df/(tf-tc)) / n. Here, ‘f’ denotes the final position of the cell,
‘0’ denotes the initial position, dn is the distance migrated between two successive frames (xn,yn) and (xn-1,yn-1),
dS is the straight line migration distance.
Supplemental Figure 6
A
B
Supplemental Figure 6. Mouse neutrophils in 5 % BSA. Bone marrow-derived mouse neutrophils were
resuspended in HBSS buffer. (A) Neutrophils were able to spread and adhere on an un-preblocked surface. (B)
Adhesion of neutrophils was abolished by preblocking the glass bottom dish with 5 % BSA at 37ºC for 1 h.
Supplemental Figure 7
Control neutrophils
Latrunculin B-treated neutrophils
Supplemental Figure 7. Latrunculin B-treated mouse neutrophils. Bone marrow-derived neutrophils were
treated with latrunculin B (1 µM) at 37ºC for 1 h. Cells were then resuspended in HBSS buffer containing 1 µM
latrunculin B. (A) Control neutrophil (no latrunculin B) were able to adhere and spread on a glass bottom dish.
(B) Latrunculin B-treated neutrophils were rounded and not able to adhere or spread on a glass bottom dish.
Supplemental Figure 8
A
2.0
[Ca2+] i
( ∆ F340/380 ratio)
WT1
WT2
1.5
WT3
WT4
1.0
0.5
0.0
0
50
100
150
200
250
300
350
400
Time (sec)
B
fMLP
2.0
[Ca2+]i
(∆ F340/380 ratio)
FAK KO1
FAK KO2
1.5
FAK KO3
FAK KO4
1.0
0.5
0.0
0
50
fMLP
100
150
200
250
300
350
400
Time (sec)
Supplemental Figure 8. fMLP-mediated changes in [Ca2+]i in adherent neutrophils. Fura-2 AM loaded wild type (A) and FAK-/(B) neutrophils were plated on 10 µg/ml fibronectin coated MatTek dish and allowed to adhere for 1.5 min prior to 1 µM fMLP uniform
stimulation. Shown are graphs of 4 wild type and 4 FAK KO neutrophils.
Supplemental Figure 9
A
WT
0
1
3
KO
5
0
1
3
5
fMLP (min)
P-Pyk 2
Pyk 2
Actin
Total-Pyk2 / Actin
P-Pyk2 / Total-Pyk2
B
Time (min)
Time (min)
Supplement Figure 9. FAK disruption does not affect Pyk2 expression and fMLP-elicited Pyk2 phosphorylation in
neutrophils. (A) Bone marrow-derived wild type and FAK-/- neutrophils were stimulated with 1 µM fMLP for indicated
time (0-5 min). The levels of phosphorylated Pyk2 and total Pyk2 were detected by western blotting analysis using antiphosphorylated Pyk2 (1:1000) and anti-Pyk2 (1:1000) antibodies (Cell Signaling, MA) respectively. (B) Relative
amounts of phosphorylated Pyk2 and total Pyk2 in (A) were quantified using NIH Image software as previously
described (Jia et al., 2007). fMLP-elicited Pyk2 phosphorylation was presented as ratio of phospho-Pyk2 to total Pyk2.
The Pyk2 expression levels were normalized to actin levels in the same sample. Results are the means (± SD) of 3
independent experiments. No significant difference (P<0.05% versus wild-type neutrophils by Student’s t test) was
detected between the WT and FAK KO neutrophils.
Supplemental Figure 10
WT
WT
FAK-/-
FAK-/-
Long exposure
Short exposure
Total RhoA
Rho-GTP
Supplemental Figure 10. WT and FAK-/- neutrophils (107 cells) were primed with 100nM PMA and then
mixed with 5x108 E-coli bioparticles that were opsonized with 12.5% mouse serum. After 2 hours incubation at
37°C, cells were lysed and probed for active Rho using a GST-RBD-Rhotekin pulldown assay. Immunoblot
shows active Rho (Rho-GTP) (right) and total Rho (left) in WT and FAK-/- lysates. Methods:
Fluorescein-conjugated E. coli (Molecular Probes) was reconstituted in HBSS and opsonized with 12.5% mouse
serum at 37°C for 30min. Mouse bone marrow neutrophils from WT and FAK-/- (107 cells) were primed with
100ng/mL PMA (sigma) at 37°C for 15 min, then added to the serum-opsonized bioparticles at a ratio of 1:50
(neutrophils : bioparticles), and incubated at 37°C for 2 hours. Cells were then spun down at 1500xg for 1 min
and lysed with 500 µl ice-cold lysis buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1% NP-40,
1 mM DTT and 5% glycerol, supplemented with Roche protease inhibitor cocktail and 1mM PMSF). Precleared
lysates were then probed for active Rho using the GST-RBD-Rhotekin pulldown assay kit (Pierce
Biotechnology), as per the manufacturer’s instructions.
Supplemental Figure 11
n>=6
Supplemental Figure 11. The percentage of apoptotic neutrophils in peripheral blood. Peripheral blood cells
from wild type and FAK-/- mice were isolated by dextran sedimentation followed by red blood cell lysis. Cells were
then stained with an anti- Gr-1 antibody, Annexin V-FITC, and Propidium Iodide, and analyzed by FACS. PMN
were characterized by the FCS/SSC pattern and the Gr-1 high staining. The percentage of Annexin V negative cells
(live cells) and the percentage of Annexin V positive cells (apoptotic cells) were reported on the histograms. Results
were obtained from n = 6 wild type and FAK-/- mice.
Supplemental Movie 1. Bone marrow derived wild-type (left) and FAK-/- (right) neutrophils were
plated on uncoated coverslip in the presence of fMLP gradient in the EZ-TAXScan device. Images
were acquired every 30 sec for 20 min.
Supplemental Movie 2. Bone marrow derived wild-type (left) and FAK-/- (right) neutrophils were
plated on 10 µg/ml fibronectin coated coverslip in the presence of fMLP gradient in the EZ-TAXScan
device. Images were acquired every 30 sec for 20 min.
Supplemental Movie 3. Bone marrow derived wild-type (left) and FAK-/- (right) neutrophils were
plated on 25 µg/ml fibronectin coated coverslip in the presence of fMLP gradient in the EZ-TAXScan
device. Images were acquired every 30 sec for 20 min.
Supplemental Movie 4. Bone marrow derived wild-type (left) and FAK-/- (right) neutrophils were
plated on 50 µg/ml fibronectin coated coverslip in the presence of fMLP gradient in the EZ-TAXScan
device. Images were acquired every 30 sec for 20 min.
Supplemental Movie 5. Bone marrow derived wild-type (left) and FAK-/- (right) neutrophils were
plated on 100 µg/ml fibronectin coated coverslip in the presence of fMLP gradient in the EZ-TAXScan
device. Images were acquired every 30 sec for 20 min.
Supplemental Movie 6. Bone marrow derived wild-type (left) and FAK-/- (right) neutrophils were
plated on 10 µg/ml fibronectin coated MatTek dish and uniformly stimulated with 100 nM fMLP.
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Image sequences were captured from multiple fields every 2 sec for 6 min. fMLP was added after the
fifth frame.
Supplemental Movie 7. Bone marrow derived wild-type (left) and FAK-/- (right) neutrophils were
plated on 50 µg/ml fibronectin coated MatTek dish and uniformly stimulated with 100 nM fMLP.
Image sequences were captured from multiple fields at 10 sec/frame for 6 min. fMLP was added after
the fifth frame.
Supplemental Movie 8. Wild type (left) and FAK-/- (right) mice were intrascrotally injected with
saline. Leukocyte trafficking was monitored at 2 hour post injection. Frame rate is 5fps. The video was
accelerated 20 times.
Supplemental Movie 9. Wild type (left) and FAK-/- (right) mice were intrascrotally injected with 1 µg
MIP-2. Leukocyte trafficking was monitored at 2 hour post injection. Frame rate is 5fps. The video
was accelerated 20 times.
Supplemental Movie 10. Fura-2 AM loaded bone marrow derived wild-type (left) and FAK-/- (right)
neutrophils were plated onto 10 µg/ml fibronectin coated MatTek dish and uniformly stimulated with 1
µM fMLP at the fifth frame. Images were captured every 2 sec for 6 min. Calcium concentration was
indicated as fluorescence ratios F340/F380. The videos show pseudocolor representations of the ratio
between the pixel intensities of the 340 and 360 images. Red represents high calcium, and green
represents low calcium. There is a brief flash of red in both WT and FAK null cells right after the
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addition of fMLP. However, this event is independent of chemoattractant stimulation. The flash was
observed when HBSS alone was added.
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