The Mammalian Actin-Binding Protein 1 Is Critical for Spreading and

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This information is current as
of January 15, 2017.
The Mammalian Actin-Binding Protein 1 Is
Critical for Spreading and Intraluminal
Crawling of Neutrophils under Flow
Conditions
Ingrid Hepper, Jürgen Schymeinsky, Ludwig T. Weckbach,
Sascha M. Jakob, David Frommhold, Michael Sixt, Melanie
Laschinger, Markus Sperandio and Barbara Walzog
Supplementary
Material
http://www.jimmunol.org/content/suppl/2012/03/26/jimmunol.110087
8.DC2.html
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J Immunol published online 26 March 2012
http://www.jimmunol.org/content/early/2012/03/26/jimmun
ol.1100878
Published March 26, 2012, doi:10.4049/jimmunol.1100878
The Journal of Immunology
The Mammalian Actin-Binding Protein 1 Is Critical for
Spreading and Intraluminal Crawling of Neutrophils under
Flow Conditions
Ingrid Hepper,* Jürgen Schymeinsky,* Ludwig T. Weckbach,* Sascha M. Jakob,*
David Frommhold,† Michael Sixt,‡ Melanie Laschinger,x Markus Sperandio,* and
Barbara Walzog*
R
ecruitment of polymorphonuclear neutrophils (PMN) to
the site of inflammation follows a well-defined multistep
adhesion cascade. It includes leukocyte capturing and
rolling along the activated endothelium, firm adhesion, adhesion
strengthening, spreading, and intraluminal crawling to the emigration sites where PMN eventually extravasate. Whereas selectins
are the key molecules that allow PMN capture and rolling, adhesion
is mainly dependent on adhesion molecules of the b2 integrin
family (CD11/CD18) (1, 2). During rolling of PMN, chemokines
*Walter Brendel Centre of Experimental Medicine, Ludwig Maximilians University
Munich, 80336 Munich, Germany; †Department of Neonatology, Children’s Hospital,
University of Heidelberg, 69120 Heidelberg, Germany; ‡Department of Molecular
Medicine, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany; and
x
Department of Surgery, Technische Universität München, 81675 Munich, Germany
Received for publication April 4, 2011. Accepted for publication March 6, 2012.
This work was supported by Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 914) Grants A2 (to B.W.), B1 (to M. Sperandio), and A4 (to M.L.).
Address correspondence and reprint requests to Prof. Barbara Walzog, Department
of Cardiovascular Physiology and Pathophysiology, Walter Brendel Centre of Experimental Medicine, Ludwig-Maximilians-Universität München, Schillerstr. 44,
D-80336 München, Germany. E-mail address: walzog@lrz.uni-muenchen.de
The online version of this article contains supplemental material.
Abbreviations used in this article: Arp2/3, actin-related protein 2 and 3; dHL-60,
neutrophil-like differentiated HL-60; EGFP, enhanced GFP; Hem-1, hematopoietic
protein 1; mAbp1, mammalian actin-binding protein 1; N-WASP, neural WASP;
PLL, poly(L-lysine); PMN, polymorphonuclear neutrophil; RNAi, RNA interference;
shRNA, short hairpin RNA; TIRF, total internal reflection fluorescence; WASP, Wiskott–Aldrich syndrome protein; WAVE, WASP family Verprolin-homologous protein; WIP, WASP-interacting protein.
Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100878
presented by the endothelium as well as selectin-ligand interactions can activate the b2 integrins (inside-out signaling) by
a shift of the conformation of the b2 integrin (3, 4). The b2
integrins exist in three different conformations, the low-affinity
conformation that is mainly expressed on nonactivated PMN, as
well as the intermediate-affinity and the high-affinity state that can
be primarily found upon cellular activation (5).
Activation of b2 integrins is indispensable for different steps of
the recruitment cascade. Whereas b2 integrins in the intermediateaffinity state contribute to slow rolling of PMN (4), firm adhesion under flow conditions requires the high-affinity conformation of the b2 integrins enabling PMN to remain adherent and
withstand shear forces (1, 6). In addition, there is increasing evidence for different molecular functions of the b2 integrins LFA-1
(CD11a/CD18, aLb2) and Mac-1 (CD11b/CD18, aMb2) in the
recruitment process. In vivo studies by Phillipson et al. (7–9) on
adhesion and crawling of PMN in venules of the inflamed
cremaster muscle in mice revealed that initial LFA-1–mediated
adhesion was followed by crawling via Mac-1. In this study,
crawling of the PMN to endothelial junctions was almost exclusively dependent on interaction between Mac-1 and endothelial
ICAM-1 that eventually allowed efficient emigration out of the
blood vessel.
Ligand binding of b2 integrins leads to integrin clustering and
subsequent outside-in signaling, which contributes to adhesion
strengthening and cell motility by controlling cytoskeletal rearrangements and cell polarization (10–13). Similar to classical
immunoreceptor signaling, for example, via the TCR or the BCR,
b2 integrin-mediated outside-in signaling causes phosphorylation
Recently, the mammalian actin-binding protein 1 (mAbp1; Hip-55, SH3P7, debrin-like protein) was identified as a novel component
of the b2 integrin-mediated signaling cascade during complement-mediated phagocytosis and firm adhesion of polymorphonuclear
neutrophils (PMN) under physiological shear stress conditions. In this study, we found that the genetic ablation of mAbp1 severely
compromised not only the induction of adhesion, but also subsequent spreading of leukocytes to the endothelium as assessed by
intravital microscopy of inflamed vessels of the cremaster muscle of mice. In vitro studies using murine PMN confirmed that
mAbp1 was required for b2 integrin-mediated spreading under shear stress conditions, whereas mAbp1 was dispensable for
spreading under static conditions. Upon b2 integrin-mediated adhesion and chemotactic migration of human neutrophil-like
differentiated HL-60 cells, mAbp1 was enriched at the leading edge of the polarized cell. Total internal reflection fluorescence
microscopy revealed that mAbp1 formed propagating waves toward the front of the lamellipodium, which are characteristic for
dynamic reorganization of the cytoskeleton. Accordingly, binding of mAbp1 to actin was increased upon b2 integrin-mediated
adhesion, as shown by coimmunoprecipitation experiments. However, chemotactic migration under static conditions was unaffected in the absence of mAbp1. In contrast, the downregulation of mAbp1 by RNA interference technique in neutrophil-like
differentiated HL-60 cells or the genetic ablation of mAbp1 in leukocytes led to defective migration under flow conditions in vitro
and in inflamed cremaster muscle venules in the situation in vivo. In conclusion, mAbp1 is of fundamental importance for
spreading and migration under shear stress conditions, which are critical prerequisites for efficient PMN extravasation during
inflammation. The Journal of Immunology, 2012, 188: 000–000.
2
Materials and Methods
Mice
Mice carrying the Dbnltm1Jwnd allele (mAbp12) and wild-type control mice
maintained on the BALB/c background were provided by J. Wienands
(Göttingen, Germany) (41). LFA-12/2 mice and wild-type control mice
were maintained on the C57BL/6 background. LFA-12/2 mice were provided by N. Hogg (London, U.K.) (42). Animal experiments were institutionally approved.
Reagents and Abs
BSA, diisopropylfluorophosphate, DMSO, DTT, fibrinogen from human or
mouse plasma, fMLP, HEPES, Percoll, piceatannol, PMSF, p-nitrophenyl
phosphate, protease inhibitor mixture, sodium chloride, sodium deoxycholate, sodium metavanadate, sodium orthovanadate, Triton X-100, trypsin
inhibitor, and Tween 20 were obtained from Sigma-Aldrich (Deisenhofen,
Germany). Sodium fluoride and Tris were delivered by AppliChem (Darmstadt, Germany). RPMI 1640 medium, FCS, HBSS, penicillin/streptomycin, poly(L-lysine) (PLL), and PBS were purchased from Biochrom
(Berlin, Germany). GFP-Trap-A beads were delivered by Chromotek
(Munich, Germany). Human and murine ICAM-1 and murine P-selectin
were obtained from R&D Systems (Minneapolis, MN). Keratinocytederived chemokine (KC; CXCL1) was purchased from PeproTech (Hamburg, Germany). Murine TNF-a was ordered from Cell Signaling Technology (Danvers, MA). The mouse anti-Syk mAb (IgG2a, clone 4D10,
sc1240), the mouse anti-Wiskott–Aldrich syndrome protein (WASP) mAb
(IgG2a, clone B-9, sc13139), the goat anti-actin Ab (I-19, sc-1616), and
the rabbit anti–WASP-interacting protein (WIP) Ab (H-224, sc-25533)
were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The
goat anti-mAbp1 Ab (ab2836) was obtained from Abcam (Cambridge,
U.K.). The mouse anti-mAbp1 mAb (IgG1, 612614) was purchased from
BD Biosciences (Heidelberg, Germany). The HRP-conjugated goat antimouse IgG L chain Ab was obtained from Jackson ImmunoResearch
Laboratories (West Grove, PA). The function-blocking mouse anti-CD18
mAb (IgG2a, clone IB4) and the HRP-conjugated rabbit anti-goat IgG Ab
were purchased from Calbiochem (Darmstadt, Germany). Alexa 488conjugated goat anti-mouse IgG, Alexa 546-conjugated donkey anti-goat
IgG, Alexa 488-conjugated donkey anti-rabbit IgG Abs, and Alexa 555conjugated phalloidin were ordered from Invitrogen (Karlsruhe, Germany).
Isolation of bone marrow PMN and cell culture
Murine PMN were isolated, as described previously (28). The human
myeloid HL-60 cells (ACC 3) and the murine WEHI-3B cells (ACC 26)
were obtained from the German Resource Centre for Biological Material
(Braunschweig, Germany). Differentiation of neutrophil-like HL-60 cells
was induced in culture using RPMI 1640 medium supplemented with
1.3% DMSO for 6 d. For pharmacological inhibition of Syk, cells were
treated with 30 mM piceatannol for 30 min at 37˚C or vehicle (DMSO) for
control.
Stable transduction of HL-60 cells
The mAbp1-EGFP expression vector was a gift of M. Deckert (INSERM,
Université de Nice-Sophia-Antipolis, Nice, France). For viral transduction,
the coding region of mAbp1-EGFP was subcloned into the FuVal lentiviral
vector (Fu-mAbp1-EGFP). To generate virus-containing supernatant, HEK
293T cells (American Type Culture Collection; CRL-11268) were plated
on 6-well tissue culture plates in RPMI 1640 medium (supplemented with
10% FCS and 25 mM HEPES) and transfected with the transducing vector
Fu-mAbp1-EGFP, the packaging vector pCMVΔR8.9, and the envelope
vector pVSV-G (43) using Lipofectamine 2000 (Invitrogen, Karlsruhe,
Germany), according to the manufacturer’s instructions. Subsequently,
HL-60 cells were incubated overnight in RPMI 1640 medium supplemented with 33% virus-containing supernatant. After 2 wk, transduced
cells were sorted for fluorescence intensity using a FACSAria system (BD
Biosciences, Heidelberg, Germany) and subcloned. Fluorescence intensity
of the mAbp1-EGFP expressing HL-60 cell clones was measured using
a FACScan flow cytometer (BD Biosciences). The expression of the 85kDa mAbp1-EGFP fusion protein was detected by Western blot technique
(data not shown).
For the generation of the cell line that stably expressed a short hairpin (sh)
RNA to downregulate mAbp1 by the RNA interference (RNAi) technique,
we cloned the specific DNA oligonucleotide (59-TCG AAA GTC CCC
GAC CGA CTG GGC TTT CAA GAG AAG CCC AGT CGG TCG GGG
ACT TTT TTT-39; Biomers.net, Ulm, Germany) or a scrambled DNA
oligonucleotide as control into the shRNA vector pSuppressorRetro
of ITAMs in the adaptor proteins DAP12 and Fc receptor g-chain
by Src family kinases (14, 15). The phosphorylation of ITAMs
allows binding and activation of the nonreceptor tyrosine kinase
Syk (16, 17). Previous studies have demonstrated a crucial role of
Syk for different b2 integrin-mediated PMN functions, including
slow leukocyte rolling and firm adhesion during inflammation
in vivo, as well as the respiratory burst, cell spreading, migration, and phagocytosis (4, 16, 18–24). Signaling downstream of
Syk involves several molecules, including phospholipase C-g, the
guanine nucleotide exchange factor Vav, the Src homology 2
domain-containing leukocyte phosphoprotein of 76 kDa, the
adaptor protein Cbl, and the regulatory subunit p85 of the PI3K
class IA (18, 22, 25–30). In addition, the mammalian actin-binding
protein 1 (mAbp1; HIP-55, SH3P7, debrin-like protein) has been
shown to become phosphorylated by Src and Syk family kinases
in fibroblasts and in lymphocytes (31–33).
The 55-kDa intracellular adaptor protein mAbp1 binds to
actin via its N-terminal–located evolutionary conserved actindepolymerizing factor homology domain and the neighboring
helical domain (32, 34–36). At its C terminus, mAbp1 contains a
Src homology 3 domain, as well as a proline-rich domain, which
mediate protein-protein interactions (37, 38). In lymphocytes,
mAbp1 has been shown to be critical for TCR-mediated signaling
and Ag internalization, processing, and presentation in B cells
(39, 40). Recently, we were able to demonstrate that mAbp1
was indispensable for Mac-1–mediated phagocytosis in PMN
(21). Moreover, the genetic absence of mAbp1 caused a severe
defect of PMN adhesion in TNF-a–stimulated cremaster muscle
venules and in a microflow chamber under physiologic shear
stress conditions in vitro, whereas adhesion was not affected
under static conditions (21). The analysis of the underlying
molecular mechanism revealed that mAbp1 was required for
stabilization of CD18 clusters in the high-affinity conformation
under flow conditions. Thus, mAbp1 seems to be involved in
the reinforcement of the high-affinity conformation of the b2
integrins under shear stress conditions, thereby allowing adhesion
strengthening, which is critical to withstand shear forces during
adhesion (21).
As the high-affinity conformation of b2 integrins is known to be
critical not only for the reinforcement of adhesion, that is, adhesion strengthening, but also plays an important role for other steps
of the recruitment cascade (3), we studied whether mAbp1 has
a functional impact on the induction of firm adhesion, spreading,
and intraluminal crawling. We found that enhanced GFP (EGFP)tagged mAbp1 (mAbp1-EGFP) was translocated to the lamellipodium of polarized neutrophil-like differentiated HL-60 (dHL60) cells during b2 integrin-mediated adhesion and migration. In
this study, mAbp1-EGFP formed wavelike structures that are
known to be characteristic for actin reorganization. Accordingly,
b2 integrin binding specifically increased the interaction of
mAbp1 with actin. However, chemotactic migration and spreading
of murine PMN under static conditions were not affected by the
genetic absence of mAbp1. This was in sharp contrast to physiological shear stress conditions in which the induction of firm
adhesion, spreading, and intraluminal crawling of leukocytes was
significantly impaired in mAbp12/2 mice in vivo compared with
mAbp1+/+ control mice. Mechanotactic crawling on immobilized
ICAM-1 was found to depend on Mac-1, as the crawling ability of
isolated mAbp1+/+ PMN was significantly reduced on truncated
ICAM-1, which lacked the binding site for Mac-1. In summary,
our data provide evidence that mAbp1 was critical for the induction of adhesion, spreading, and intraluminal crawling of PMN
under flow conditions.
mAbp1 IS CRITICAL FOR INTRALUMINAL CRAWLING
(Imgenex, San Diego, CA). After subcloning, the efficiency of mAbp1
downregulation was measured by Western blot analysis (compare Fig. 6F).
Transient transfection of HL-60 cells
To generate dHL-60 cells transiently expressing mAbp1-EGFP, a 400-ml
aliquot of dHL-60 cells (2 3 107 cells/ml) in RPMI 1640 medium was
transferred to a Gene Pulser cuvette with an 0.4-cm electrode (Bio Rad,
Hercules, CA) and mixed with 30 mg endotoxin-free mAbp1-EGFP expression vector. Samples were subjected to an electroporation pulse of 290
V and 1050 mF (EasyJect T+ electroporator; Equibio, Kent, U.K.).
Expression of ICAM-1(D1-2)-Fc fusion protein
The coding region of domains D1-D2 of ICAM-1 derived from cDNA of
murine spleen was cloned into the expression vector pcDNA3.1 IgG (44).
HEK 293T cells (American Type Culture Collection; CRL-11268) were
transfected with the expression vector, and the fusion protein ICAM-1(D12)-Fc containing the human Fcg1 fragment was purified from supernatant
(compare Fig. 5C).
Scanning confocal microscopy and total internal reflection
fluorescence microscopy
Spreading and migration under flow conditions
To analyze spreading of isolated murine PMN, IBIDI m-slides VI 0.4
(IBIDI GmbH, Martinsried, Germany) were coated overnight with murine
fibrinogen (50 mg/ml) or murine ICAM-1 (12.5 mg/ml). After rinsing with
PBS, the chambers were filled with 3 3 105 murine PMN/sample. After
incubation of PMN for 10 min at 37˚C with fMLP (1 mM) in adhesion
medium (HBSS; Biochrom, Berlin, Germany) supplemented with 20 mM
HEPES (pH 7.2), 0.25% (w/v) BSA, 0.1% (w/v) glucose, 1.2 mM Ca2+,
and 1.0 mM Mg2+, shear stress (1 dyne/cm2) was applied for 10 min using
a high-precision syringe pump (model KDS-232; KD Scientific, Holliston,
MA). Samples were fixed with 3.7% formaldehyde, and microscopy was
performed using a Zeiss Axiovert 200M microscope with a PlanApochromat original magnification 363/1.4 oil objective, an AxioCam
HR digital camera, and the AxioVision 4 software (Zeiss). Spreading of
PMN was assessed by measuring the cell area using Image J software
provided by the National Institutes of Health.
Crawling of dHL-60 cells or murine PMN under flow conditions was
analyzed in IBIDI m-slides VI 0.4 (IBIDI GmbH) or flow chambers (0.2 3
2-mm cross-section; VitroCom, Mountain Lake, NJ) coated with murine
ICAM-1 (12.5 mg/ml) or human ICAM-1 (12.5 mg/ml) alone or with
a combination of murine P-selectin (20 mg/ml), murine ICAM-1 (12.5 mg/
ml), and KC (15 mg/ml), as indicated. A total of 3 3 105 cells/sample was
allowed to adhere for 10 min at 37˚C. Crawling was recorded under shear
stress (1 dyne/cm2) for 10 min at 37˚C using the system mentioned above.
Crawling was analyzed offline using Image J software plugin Manual
Tracking and the Chemotaxis and Migration tool.
Spreading and migration under static conditions
Isolated murine PMN were seeded on coverslips coated with murine fibrinogen (50 mg/ml) and were stimulated for 30 min at 37˚C with 100 ng/
ml TNF-a or 1 mM Mn2+. Microscopic images of cells fixed with 3.7%
formaldehyde were recorded using a Zeiss Axiovert 200M microscope
with a Plan-Apochromat original magnification 363/1.4 oil objective, an
AxioCam HR digital camera, and the AxioVision 4 software (Zeiss). Cell
area of spreaded cells was measured offline using Image J software.
Chemotactic migration of isolated murine PMN in response to a gradient
of 10 mM fMLP was analyzed on immobilized murine fibrinogen (50 mg/
ml) or murine ICAM-1 (12.5 mg/ml) in Zigmond chambers (45) using
a Zeiss Axiovert 200M microscope equipped with Plan-Apochromat
original magnification 310/0.25 or Plan-Apochromat original magnification 320/0.75 objectives, an AxioCam HR digital camera, and the AxioVision 4 software (Zeiss), as described previously (28). Tracking of PMN
migration was carried out, as described above.
For time-lapse video microscopy of migrating dHL-60 cells transiently
expressing mAbp1-EGFP, cells were seeded onto fibrinogen-coated IBIDI
m-slides Chemotaxis (IBIDI GmbH). Chemotactic migration in response to
a gradient of 10 nM fMLP was recorded using a Plan-Apochromat 203/
0.75 objective (Zeiss) on the microscope system mentioned above.
Intravital microscopy
Intravital microscopy and recording of the cremaster muscle were conducted, as reported previously, using an upright microscope (model 512815/
20; Leitz, Wetzlar, Germany) with a saline immersion objective (SW 40/
0.75 NA) (19). Microvascular parameters were measured using a digital
image processing system (46). For assessment of induction of adhesion and
spreading of leukocytes, the cremaster muscle was superfused with 1 mM
fMLP in bicarbonate-buffered saline (19). The number of adherent leukocytes in inflamed cremaster muscle venules was assessed, as described
(47). Leukocytes were considered to be adherent when they were attached
at the same position for .30 s. Rolling leukocyte flux fraction was defined
as the percentage of rolling leukocytes to all leukocytes passing the same
vessel in 1 min (47). Spreading of adherent leukocytes against the vessel
wall was measured as a decrease in perpendicular diameter (19). To study
intraluminal leukocyte crawling, murine TNF-a (500 ng per mouse) was
injected intrascrotally 2–4 h prior to intravital microscopy. Leukocyte
crawling was defined as displacement of the cell by at least one cell diameter within the observation period of 10 min, and was analyzed offline
using Image J software plugin Manual Tracking and the Chemotaxis and
Migration tool (48). Percentage of cells migrating in the direction of blood
flow was defined as the number of leukocytes migrating within an angle of
60˚ in the direction of blood flow in the venule compared with the number
of all migrating leukocytes in the given vessel segment.
Coimmunoprecipitation
For coimmunoprecipitation experiments, dHL-60 cells stably expressing
mAbp1-EGFP (8 3 106/sample) or dHL-60 control cells suspended in
medium (0.9% NaCl, 20 mM HEPES, 0.1% glucose, 1.2 mM Ca2+, 1 mM
Mg2+) were seeded onto dishes coated with fibrinogen (250 mg/ml),
ICAM-1 (12.5 mg/ml), truncated ICAM-1 (D1-2) (12.5 mg/ml), or PLL
(100 mg/ml) and incubated at 37˚C for 10 min. To inhibit b2 integrinmediated adhesion, cells were treated with a function-blocking Ab
against CD18 (clone IB4, 50 mg/ml) for 20 min at room temperature prior
to the onset of the experiment. Adhesion of cells was induced by addition
of 1 mM Mn2+. After 15 min, cells were lysed using a modified radioimmunoprecipitation assay buffer without SDS (25 mM Tris [pH 7.5], 150
mM NaCl, 1% [v/v] Triton X-100, 1% [w/v] sodium deoxycholate, 1 mM
DTT, 1 mM diisopropyl fluorophosphate, 100 mM sodium orthovanadate,
100 mM NaF, 1 mM PMSF, 0.25 mM sodium metavanadate, protease inhibitor mixture [Sigma-Aldrich; 1:1000], 50 mg/ml trypsin inhibitor, 10
mM p-nitrophenyl phosphate) for 30 min at 4˚C. GFP-Trap beads (Chromotek, Munich, Germany) were added and immunoprecipitation was
performed, according to manufacturer’s protocol. Finally, beads were
resuspended in SDS sample buffer and boiled for 10 min at 95˚C. Western
blot was performed using the mouse anti-mAbp1 mAb (BD Biosciences,
Heidelberg, Germany) or the goat anti-actin Ab (sc-1616; Santa Cruz
Biotechnology) and secondary HRP-conjugated goat anti-mouse IgG L
chain (Jackson ImmunoResearch Laboratories) or rabbit anti-goat IgG
(Calbiochem, Darmstadt, Germany) Abs, respectively.
Statistical analysis
Data shown represent means 6 SD or means 6 SEM, respectively. Statistical significance was determined using Student t test, the Mann–
Whitney rank sum test, or Kruskal–Wallis ANOVA test, respectively. A
p value , 0.05 was considered statistically significant.
Results
Localization of mAbp1 during leukocyte adhesion and
migration
During b2 integrin-mediated phagocytosis of complementopsonized Escherichia coli, mAbp1 has been shown to be dynamically enriched at the phagocytic cup of dHL-60 cells and
primary murine PMN. This localization of mAbp1 at the phago-
Scanning confocal microscopy of cells fixed with 3.7% formaldehyde was
conducted with a Zeiss LSM 410/Axiovert 135 microscope using a Zeiss
633/1.2 water or a Zeiss 633/1.4 oil objective (Zeiss, Göttingen, Germany). The quantitative analysis of the subcellular distribution of mAbp1
was performed offline by two different investigators in a blinded manner
using confocal images.
For total internal reflection fluorescence (TIRF) microscopy, dHL-60
cells expressing mAbp1-EGFP (1–2 3 105) were suspended in medium
(PBS supplemented with 0.25% BSA, 1.2 mM Ca2+, 1 mM Mg2+, and
0.1% glucose), plated onto fibrinogen (250 mg/ml)-coated coverslips (Saur,
Reutlingen, Germany), and incubated for 5 min at 37˚C. Subsequently,
cells were stimulated with 100 nM fMLP and analyzed at 37˚C using an
Axiovert 200 (Zeiss) microscope with a TIRF 488/561-nm laser system
(Visitron Systems), Cool-Snap-HQ camera (Roper Scientific), and Metamorph software (Molecular Devices).
3
4
cytic cup depended on active Syk (21). In this work, we studied
the subcellular localization of mAbp1 during b2 integrin-mediated
adhesion and migration. By means of confocal microscopy, we
found that upon induction of adhesion to immobilized fibrinogen
by the bacteria-derived tripeptide fMLP (100 nM), EGFP-tagged
mAbp1 (mAbp1-EGFP) was markedly enriched at the leading
edge of polarized dHL-60 cells and colocalized with F-actin at the
lamellipodium (Fig. 1A). The enrichment of mAbp1 at the leading
edge was compromised when Syk was pharmacologically inhibited by piceatannol (Fig. 1B). The quantitative analysis revealed
that the percentage of polarized cells with a localization of mAbp1
at the cell front was significantly reduced from 65.7% of control
FIGURE 1. Localization of mAbp1, WASP, and WIP at the leading edge was dependent on active Syk. (A) Confocal microscopy image of adherent and
polarized dHL-60 cells transiently transfected with mAbp1-EGFP (mAbp1-EGFP) and stimulated with fMLP (100 nM). F-actin was stained with Alexa
555-conjugated phalloidin (red). mAbp1-EGFP (green) was enriched at the leading edge (arrows) and colocalized with F-actin (yellow, merged image). (B,
D, and E) Confocal microscopy images of dHL-60 cells incubated with 30 mM piceatannol (PIC) or vehicle for control (control). Adhesion and polarization
of dHL-60 cells on immobilized fibrinogen were induced by fMLP (100 nM) for 30 min. (B) Indirect fluorescence staining of Syk was performed using
a mouse anti-Syk mAb and a secondary Alexa 488-conjugated anti-mouse IgG Ab (green). mAbp1 was assessed using a goat anti-mAbp1 Ab and
a secondary Alexa 546-conjugated anti-goat IgG Ab (red). Upon stimulation, Syk and mAbp1 were enriched at the leading edge (arrows) of polarized dHL60 cells. The merged images demonstrate colocalization (yellow). Piceatannol treatment induced the formation of multiple lamellipodia (arrowheads), and
dHL-60 cells showed a homogenous distribution of Syk and mAbp1. Images are representative for three independent experiments. Scale bar, 10 mm. (C)
Quantitative analysis of subcellular mAbp1 distribution. Polarized cells that formed at least one lamellipodium upon fMLP stimulation were counted
according to the subcellular distribution of mAbp1. n = 239 (control); n = 275 (PIC) in three independent experiments. Means 6 SD; hom = homogeneous;
*p , 0.05. (D and E) Indirect fluorescence staining of WASP was performed using a mouse anti-WASP mAb and a secondary Alexa 488-conjugated antimouse IgG Ab (D, green). WIP was assessed using a rabbit anti-WIP Ab and a secondary Alexa 488-conjugated anti-rabbit IgG Ab (E, green). Upon
stimulation, WASP and WIP were enriched at the leading edge (arrows) of polarized dHL-60 cells and colocalized with F-actin (yellow, merged images).
Piceatannol treatment induced the formation of multiple lamellipodia (arrowheads), and proper localization of WASP and WIP at the lamellipodia was
diminished. Images are representative for four independent experiments. Scale bar, 10 mm. (F) Box-whisker plot of Pearson’s correlation coefficient of
F-actin and WASP or F-actin and WIP, respectively. Median numbers are indicated. n = 73 (WASP; control), n = 78 (WASP; PIC) of four independent
experiments. n = 108 (WIP; control), n = 135 (WIP; PIC) of four independent experiments. *p , 0.05.
expressing dHL-60 cells, we investigated the mAbp1 dynamics
at the plasma membrane with high resolution. Using this approach, we identified wavelike structures formed by dynamic
accumulation of mAbp1 toward the leading edge of the cell (Fig.
2B, Supplemental Video 1). Such propagating waves have been
described previously for actin and other actin-associated proteins
such as myosin-IB, the Arp2/3 complex, and coronin in Dictyostelium, and for hematopoietic protein 1 (Hem-1) in dHL-60 cells
(53–56). Therefore, we studied whether mAbp1 may link the b2
integrin function to the actin cytoskeleton. dHL-60 cells expressing
mAbp1-EGFP were left unstimulated in suspension for control or
were seeded on immobilized fibrinogen or ICAM-1. Adhesion was
induced by addition of Mn2+ (1 mM), which is known to stabilize
the high-affinity conformation of b2 integrins (57). Coimmunoprecipitation technique revealed that the mAbp1-actin interaction
was markedly induced upon b2 integrin-mediated adhesion on
fibrinogen or ICAM-1 compared with cells in suspension or cells
exposed to PLL for control (Fig. 2C). An induction of the mAbp1actin interaction was also observed in cells adherent to truncated
ICAM-1 (ICAM-1 D1-2), which lacked the binding domains of
the b2 integrin Mac-1 (44). In this study, adhesion was mediated
by an interaction of the b2 integrin LFA-1 and ICAM-1 (D1-2).
The mAbp1-actin interaction was markedly decreased when the
cells were exposed to ICAM-1 in the presence of a function-
FIGURE 2. Subcellular dynamics of mAbp1. (A) Time-lapse microscopy of live dHL-60 cells transiently transfected with mAbp1-EGFP (mAbp1EGFP). Migration on immobilized fibrinogen in response to a gradient of 10 nM fMLP is shown (◢). (1)–(5) were recorded at intervals of 30 s. mAbp1EGFP was enriched at the leading edge (arrows, upper panel) of the migrating cell during the observed time period. The lamellipodium of the corresponding cells (arrows, lower panel) is shown in transmission. Images are representative for three independent experiments. Scale bar, 10 mm. (B) TIRF
microscopy image of an adherent and polarized dHL-60 cell stably expressing mAbp1-EGFP (mAbp1-EGFP) taken from a time-lapse recording. For better
visualization, mAbp1-EGFP fluorescence intensitiy was displayed in pseudocolors. mAbp1-EGFP was translocated to the lamellipodium in a wavelike
manner. Waves are depicted in the schematic. Scale bar, 10 mm. (C) Western blot of coimmunoprecipitates of mAbp1-EGFP by GFP-Trap beads in mAbp1EGFP expressing dHL-60 cells (mAbp1-EGFP) after stimulation with Mn2+ on immobilized PLL, human fibrinogen (Fbg; 250 mg/ml), human ICAM-1
(12.5 mg/ml), or truncated ICAM-1 lacking domains 3–5 (ICAM-1 D1-2; 12.5 mg/ml), or left unstimulated in suspension. b2 integrins were functionally
blocked by the mouse anti-CD18 mAb (clone IB4; a-CD18). mAbp1-EGFP was detected by the mouse anti-mAbp1 mAb; actin was detected by the goat
anti-actin Ab. (D) Semiquantitative analysis of protein levels of coimmunoprecipitated actin. Protein levels of actin were normalized to amount of precipitated mAbp1-EGFP. Amount of actin interacting with mAbp1 in unstimulated mAbp1-EGFP expressing dHL-60 cells in suspension or in cells adherent
to fibrinogen, ICAM-1, and ICAM-1 D1-2 is shown relative to levels of coimmunoprecipitated actin on the nonintegrin ligand PLL. The level of coimmunoprecipitated actin was specifically increased after induction of b2 integrin-mediated cell adhesion. n = 4, mean 6 SD, *p , 0.05.
cells to 36.7% of cells treated with piceatannol (Fig. 1C), suggesting that active Syk was required for the proper localization of
mAbp1. Similarly, the inhibition of Syk interfered with the proper
localization of two additional cytoskeleton-associated proteins,
namely the WASP and WIP at the lamellipodium of polarized
dHL-60 cells (Fig. 1D, 1E). WASP is known to activate the actinrelated protein 2 and 3 (Arp2/3) complex, which is indispensable
for generation and reorganization of actin. Activity and stability of
WASP are regulated by WIP (49–52). Quantitative analysis of the
colocalization between WASP and WIP with F-actin by calculating the Pearson’s correlation coefficient revealed that inhibition
of Syk resulted in a diminished colocalization of these proteins
with F-actin (Fig. 1F). Thus, the three proteins, WASP, WIP, and
mAbp1, seem to represent cytoskeleton-associated components of
the b2 integrin-mediated signaling complex downstream of Syk.
To study the subcellular dynamics of mAbp1 during migration in
detail, we performed time-lapse video microscopy of live dHL-60
cells expressing EGFP-tagged mAbp1 (mAbp1-EGFP). We found
that mAbp1-EGFP was enriched at the leading edge of cells migrating toward a gradient of the chemoattractant fMLP (10 nM) on
immobilized fibrinogen (Fig. 2A). For further analysis, we used
a lentivirally transduced dHL-60 cell clone that stably expressed
EGFP-tagged mAbp1 comparable to the endogenous mAbp1
level (21). Using TIRF microscopy of polarized mAbp1-EGFP
5
6
Table I. Hemodynamic parameters in fMLP-stimulated cremaster muscle venules
Abp1+/+
Abp12/2
Mice (n)
Venules (n)
7
6
26
19
Diameter
(mm)
Centerline Velocity
(mm/s)
Wall Shear Rate
(s21)
Systemic Leukocyte Counts
(ml21)
29 6 1
31 6 1
NS
1750 6 120
1700 6 120
NS
1500 6 90
1400 6 100
NS
5100 6 300
7300 6 200
p , 0.05
All values are means 6 SEM.
Cytoskeletal rearrangements are indispensable for leukocyte recruitment, allowing firm adhesion, spreading, and migration on the
endothelium under flow conditions (58). To elucidate the functional relevance of our findings for leukocyte recruitment, we
studied leukocyte rolling, adhesion, and spreading in unstimulated and fMLP-stimulated cremaster muscle venules in mAbp12/2
and mAbp1+/+ mice using intravital microscopy. Microvascular
parameters are presented in Table I and were comparable between
mAbp12/2 and mAbp1+/+ mice in diameter of the venules, centerline velocity of the blood flow, and wall shear rate. Systemic
leukocyte counts per microliter as assessed in whole blood
samples were significantly higher in mAbp12/2 mice compared
with mAbp1+/+ mice. In unstimulated cremaster muscle venules,
rolling flux fraction observed within the first 30 min after cremaster muscle exteriorization was similar between the two groups
(Fig. 3A, 0.32 6 0.02 in mAbp1+/+ mice and 0.33 6 0.03 in
mAbp12/2 mice), suggesting that mAbp1 was dispensable for
leukocyte rolling at least in this setting, where rolling was induced by surgical trauma and dependent on P-selectin (48). Next,
we analyzed leukocyte adhesion in unstimulated cremaster
muscle venules (pre) and found no significant difference in the
number of adherent leukocytes between mAbp1+/+ mice (200 6
24/mm2) and mAbp12/2 mice (236 6 28/mm2) (Fig. 3C, pre).
Because leukocyte adhesion greatly depends on systemic leukocyte counts, we calculated the adhesion efficiency (adherent
leukocytes per mm2/systemic leukocyte count per ml), which
nevertheless was not affected in mAbp12/2 mice (0.032 6 0.004)
compared with mAbp1+/+ mice (0.039 6 0.005) (Fig. 3B).
However, upon local stimulation of the cremaster muscle by
fMLP (1 mM) superfusion, the number of adherent leukocytes
increased in mAbp1+/+ mice within 15 min (Fig. 3C, 476 6 62/
mm2) accompanied by a concomitant decrease in rolling flux
fraction (Fig. 3D, 215.8 6 2.1%). This response was almost
completely absent in mAbp12/2 mice (251 6 44 adherent leukocytes/mm2 and 27.1 6 2.3% rolling flux fraction after 15
min), suggesting that fMLP-induced leukocyte adhesion was
dependent on mAbp1. Next, we studied leukocyte spreading
measured as cell flattening against the vessel wall in vivo, that is,
the decrease of the perpendicular leukocyte diameter (Fig. 4A).
During fMLP stimulation, the cell diameter of adherent leukocytes
in mAbp1+/+ mice continuously decreased from 6.44 6 0.13 mm
to 5.15 6 0.11 mm after 15 min, representing cell spreading, as
expected. In contrast, there was no spreading detectable in response to fMLP in mAbp12/2 mice from 6.41 6 0.10 mm to 6.53
6 0.13 mm after 15 min. These findings clearly indicated a crucial
role for mAbp1 in PMN recruitment, namely induction of adhesion and spreading in vivo.
To further characterize the impact of mAbp1 on spreading, we
performed in vitro flow chamber assays using bone marrow-derived
PMN from mAbp12/2 mice and mAbp1+/+ mice (Fig. 4B). In this
setting, an increase of the cell area indicates cell spreading on
the substrate. Compared with mAbp1+/+ PMN, the cell area of
mAbp12/2 PMN adherent to immobilized fibrinogen or ICAM-1
was significantly lower, confirming the spreading defect under
physiological shear stress conditions (1 dyne/cm2) in response to
FIGURE 3. Leukocyte rolling and adhesion in unstimulated and fMLPstimulated cremaster muscle venules in vivo. Initial rolling flux fraction
(A) and adhesion effiency (B) of leukocytes before local superfusion of
cremaster muscle. Adhesion (C) and rolling flux fraction (D) of leukocytes
before (pre) and during local stimulation of cremaster muscle venules of
mAbp12/2 and mAbp1+/+ mice with fMLP (1 mM). Initial rolling flux
fraction (A) and adhesion effiency (B) of leukocytes were not altered in the
genetic absence of mAbp1 compared with mAbp1+/+ mice. (C) Induction
of leukocyte adhesion upon stimulation with fMLP was impaired in
mAbp12/2 mice, resulting in a (D) decreased change of rolling flux
fraction. (A–D) n = 7 mAbp1+/+ mice, n = 6 mAbp12/2 mice, means 6
SEM, *p , 0.05.
blocking anti-CD18 mAb (Fig. 2C). Semiquantitative analysis
showed a significant 1.8-fold increase of the interaction between
mAbp1 and actin upon induction of adhesion on b2 integrin
substrates fibrinogen, ICAM-1, and ICAM-1 (D1-2) compared
with control cells exposed to PLL in the presence of 1 mM Mn2+
(Fig. 2D). Functional blockade of CD18 abrogated the effect,
indicating that b2 integrin engagement was specifically required to
induce the interaction between mAbp1 and actin. Of note, both b2
integrins, Mac-1 (via binding to fibrinogen) as well as LFA-1 (via
binding to ICAM-1 D1-2), seemed to be able to induce interaction
between mAbp1 and actin.
Role of mAbp1 for leukocyte adhesion and spreading in vitro
and in vivo
7
fMLP (1 mM) in vitro. In contrast, spreading of mAbp12/2 PMN
under static conditions upon stimulation by TNF-a (100 ng/ml) or
Mn2+ (1 mM) was completely normal compared with mAbp1+/+
PMN (Fig. 4C, 4D). Thus, mAbp1 was only indispensable for
spreading under shear stress conditions, but not required under
static conditions.
Impact of mAbp1 for leukocyte migration and crawling under
shear stress conditions
Next, we studied crawling of isolated murine PMN from mAbp12/2
mice and mAbp1+/+ mice using an in vitro flow chamber assay.
Analysis of mechanotactic migration of murine PMN on immobilized ICAM-1 under physiological shear stress conditions (1
dyne/cm2) revealed that mAbp1+/+ PMN migrated under flow
conditions. In contrast, the majority of mAbp12/2 PMN showed
some motility, but no significant net movement (Fig. 5A, Supplemental Video 2). Quantitative analysis showed that the majority
of mAbp1+/+ PMN performed substantial migration under flow
conditions, whereas this percentage was significantly reduced
from 53.4 6 22.8% in mAbp1+/+ PMN to 31.5 6 12.9% in
mAbp12/2 PMN (Fig. 5B, left panel). As shown in Fig. 5B (right
panel), the ability of adherent mAbp1+/+ PMN to migrate under
shear stress conditions was severely compromised on truncated
ICAM-1 (ICAM-1 D1-2, Fig. 5C) lacking the binding domains of
the b2 integrin Mac-1 (44). In this setting, cellular interactions
were only possible via binding of LFA-1 to ICAM-1. Subsequently, the percentage of migrating mAbp1+/+ PMN was significantly reduced to similar levels, as seen for mAbp12/2 PMN on
immobilized full-length ICAM-1, indicating that mechanotactic
crawling was dependent on Mac-1, as expected. To confirm this
finding, we studied mechanotactic crawling on full-length ICAM1 using LFA-12/2 PMN. The percentage of migrating LFA-12/2
PMN was similar compared with LFA-1+/+ PMN (Fig. 5D), indicating that crawling of PMN under shear stress conditions was
independent of LFA-1.
Detailed analysis of mechanotactic migration of isolated murine PMN on immobilized ICAM-1 and KC revealed that not only
the percentage of migrating cells was diminished. Also, the migration velocity of the remaining mAbp12/2 PMN that crawled
during 10 min of physiological shear stress of 1 dyne/cm2 was
significantly reduced from 1.9 6 0.8 mm/min in mAbp1+/+ PMN
to 1.4 6 0.8 mm/min in mAbp12/2 PMN (Fig. 6A). Moreover,
calculation of the frequency distribution of the migration velocity
revealed that there were two groups of crawling mAbp1+/+ PMN.
One group crawled at a low migration velocity (,2 mm/min), and
the second group migrated at a higher migration velocity of .2
mm/min. In contrast, the majority of mAbp12/2 PMN crawled at
the low migration velocity of ,2 mm/min.
To study intraluminal crawling of leukocytes in vivo, we performed intravital microscopy of TNF-a–stimulated cremaster
muscle venules of mAbp12/2 mice and mAbp1+/+ mice as control.
The analysis of the migration tracks of leukocytes that crawled on
the inflamed endothelium showed that wild-type leukocytes of
mAbp1+/+ mice preferentially migrated perpendicular or against
the direction of blood flow, as described by Phillipson et al. (59),
for mechanotactic crawling of murine PMN under flow conditions
in vitro and in vivo (Fig. 6B, Supplemental Video 3; WT). In
contrast, a high number of mAbp12/2 leukocytes crawled in the
direction of blood flow (Fig. 6B, Supplemental Video 3; KO).
Quantitative analysis revealed that the percentage of cells that
were not able to crawl perpendicular or against the direction of
blood flow, but crawled in the direction of blood flow, was significantly increased in mAbp12/2 mice to 46.3 6 6.2% compared
with 26.0 6 6.4% in mAbp1+/+ mice (Fig. 6C).
FIGURE 4. Leukocyte spreading was impaired in absence of mAbp1 under flow conditions. (A) Diameters of adherent leukocytes from mAbp12/2 and
mAbp1+/+ mice were measured perpendicular to the vessel at indicated time points during superfusion with fMLP (1 mM). Adherent leukocytes from
mAbp12/2 mice failed to spread against the vessel wall, resulting in a constant cell diameter. n = 345 cells from seven mAbp1+/+ mice, n = 201 cells from
six mAbp12/2 mice, means 6 SEM, *p , 0.05. (B–D) Measurement of cell area and microscopic images of adherent PMN isolated from mAbp12/2 and
mAbp1+/+ mice in an in vitro flow chamber assay (B) and under static conditions (C, D). (B) Upon stimulation with 1 mM fMLP for 10 min, spreading on
murine fibrinogen or ICAM-1 was diminished in mAbp12/2 PMN under physiological flow conditions (1 dyne/cm2) compared with mAbp1+/+ PMN. (C
and D) In contrast, under static conditions, spreading of mAbp12/2 PMN on fibrinogen was similar to that of mAbp1+/+ PMN after stimulation for 30 min
with murine TNF-a (100 ng/ml) or Mn2+ (1 mM), respectively. (B) n = 400 from four mice, means 6 SEM. (C) Images are representative for four independent experiments. Scale bar, 10 mm. (D) n = 400 from four mice, means 6 SD. *p , 0.05.
8
Discussion
FIGURE 5. mAbp1 was critical for Mac-1–mediated crawling of PMN
under flow conditions. (A) Microscopic images and single-cell tracks of
migrating murine mAbp1+/+ and mAbp12/2 PMN in a flow chamber
coated with murine ICAM-1 (12.5 mg/ml) upon stimulation with fMLP (1
mM) at physiological shear stress (1 dyne/cm2, direction of flow is indicated) applied for 10 min. Microscopic images were taken from the time
point 600 s. Dotted lines indicate position of cells at time point 0 s. Microscopic images and single-cell tracks are representative for seven independent experiments. Scale bar, 10 mm. (B, D) Quantitative analysis of the
percentage of migrating murine mAbp1+/+ and mAbp12/2 PMN (B) or
murine LFA-1+/+ and LFA-12/2 PMN (D) in a flow chamber coated with
murine ICAM-1 [12.5 mg/ml, (B) left panel, and (D)] or truncated murine
ICAM-1 lacking domains 3–5 [ICAM-1 D1-2, 12.5 mg/ml, (B) right panel]
upon shear stress (1 dyne/cm2) for 10 min. PMN were considered to migrate, if they moved at least one cell diameter within the observation period of 10 min. n = 7 (B, ICAM-1), n = 5 (B, ICAM-1 D1-2), n = 4 (D),
means 6 SD, *p , 0.05. (C) Schematics of the Fc-tagged fusion proteins
of full-length murine ICAM-1 and its truncated form (ICAM-1 D1-2) and
silver staining obtained from SDS-PAGE of 50 ng protein each. (A and B,
left panel) The genetic absence of mAbp1 diminished the ability of PMN
to migrate under flow conditions on immobilized ICAM-1. (B, right panel)
Crawling of mAbp1+/+ was abrogated when truncated murine ICAM-1 was
used as substrate. (D) LFA-1 was dispensable for migration on ICAM-1
under flow conditions.
To analyze the impact of mAbp1 in the human system, we
studied crawling of dHL-60 cells under shear stress conditions at 1
dyne/cm2 on immobilized ICAM-1 (Fig. 6D). In accordance with
the results obtained from murine PMN, dHL-60 cells downregulating mAbp1 expression by RNAi technique (shAbp1) to 19 6
7% compared with control cells expressing a scrambled RNA
sequence (shControl, 100%) crawled at a significantly lower migration velocity, resulting in decreased accumulated migration
distance compared with control cells (Fig. 6E, 6F). This was also
The present study indicates a role of mAbp1 for induction of b2
integrin-mediated adhesion, spreading, and intraluminal crawling
of PMN under flow conditions. In dHL-60 cells, mAbp1 was
translocated to the leading edge during Mac-1–mediated adhesion
and migration on immobilized fibrinogen. This enrichment was
dependent on active Syk, indicating that mAbp1 is part of the
Syk-controlled signaling complex, which stabilizes the leading edge
of migrating PMN (22, 28, 60). Also, WASP and WIP showed
compromised enrichment at the leading edge and impaired
colocalization with actin upon inhibition of Syk. Thus, mAbp1,
WASP, and WIP may represent cytoskeleton-associated components of the signaling cascade downstream of Syk, which is known
to become activated upon ligand binding of b2 integrins controlling postadhesion events in PMN (14–16, 18, 21). Similar to
WASP and WIP, mAbp1 colocalized with F-actin at the lamellipodium. This finding is in accordance with a previous study
showing polarized recruitment of mAbp1 to the cell periphery of
migrating fibroblasts, where it colocalized with F-actin. In this
study, the accumulation of mAbp1 occurred predominantly at sites
of high F-actin dynamics at the leading edge (35).
By means of TIRF microscopy, we identified an even more complex pattern of mAbp1 distribution forming propagating waves of
mAbp1-EGFP toward the lamellipodium of polarized cells. These
waves of mAbp1 resembled the discrete nonuniformal propagating
waves of Hem-1 in fMLP-simulated HL-60 cells found by Weiner
et al. (56, 61). Hem-1 is a hematopoietic-specific component of the
Arp2/3 complex-activating WASP family Verprolin-homologous
protein (WAVE) complex. It was found to accumulate at the
leading edge of fMLP-treated HL-60 cells, and TIRF microscopy
revealed that Hem-1 forms sharp actin-based wavefronts at the
plasma membrane of the lamellipodium, probably resulting from
rapid Hem-1 recruitment and release (56, 61). The Arp2/3 complex, which catalyzes nucleation and branching of actin filaments,
is controlled by nucleation-promoting factors like WASP and
WAVE family proteins (52). In contrast to its yeast homolog
true for the calculated Euclidean distance. Further analysis of the
single migration tracks and frequency distribution of migration
angles (rose diagram) showed that the downregulation of mAbp1
in dHL-60 cells prevented the ability of the cells to migrate
against or perpendicular to the direction of flow almost completely, whereas the majority of the control cells again crawled
perpendicular or against the direction of flow (Fig. 6D), confirming the in vivo findings in the human system. These results
indicated a crucial role of mAbp1 in mechanotactic crawling of
PMN under shear stress conditions in vitro and in the situation
in vivo.
To examine the role of mAbp1 in chemotactic migration, we
used Zigmond chambers with fMLP (10 mM) as chemoattractant.
We found that the genetic absence of mAbp1 in PMN did not
affect chemotactic migration toward the gradient of fMLP on
immobilized fibrinogen when compared with mAbp1+/+ control
PMN (Fig. 7A). This was also true for migration velocity and
mean accumulated and Eucledian distance (Fig. 7C, upper panel),
suggesting that chemotactic migration under static in vitro conditions did not require mAbp1. Similar results were obtained on
immobilized ICAM-1. In this study, chemotactic migration and
migratory parameters of PMN were again not affected in the absence of mAbp1 (Fig. 7B, 7C, lower panel). Taken together, our
results demonstrate that mAbp1 was indispensable for spreading
and Mac-1–dependent intraluminal crawling under shear stress
conditions, but not required for spreading or chemotactic migration under static conditions.
9
Abp1p, mammalian Abp1 exerts no nucleation-promoting function, and mAbp1 cannot activate the Arp2/3 complex directly.
However, Pinyol et al. (34, 52, 62) showed that mAbp1 directly
interacted with the Arp2/3 complex activator neural WASP (NWASP) in neuronal cells and thereby promoted actin polymerization together with N-WASP. Furthermore, mAbp1 was shown to
cooperate with the small GTPase Cdc42, which in turn is known
to mediate WASP/N-WASP activation. Because mAbp1 is part of
the N-WASP/Arp2/3 complex, waves of mAbp1 might reflect
dynamics of the WASP/N-WASP complex at the lamellipodium
similar to Hem-1 in the WAVE complex.
Functional analysis revealed a substantial impact of mAbp1 for
the induction of leukocyte adhesion to the endothelium in fMLPstimulated cremaster muscle venules and for subsequent spreading
against the vessel wall, as both responses were almost completely
absent in mAbp12/2 mice. In contrast, the genetic absence of
mAbp1 did not interfere with P-selectin–mediated rolling, as the
initial rolling flux fraction was not different between mAbp1+/+
FIGURE 6. Crawling of PMN was affected by the absence or downregulation of mAbp1 under flow conditions. (A) Crawling velocity of isolated murine
mAbp1+/+ and mAbp12/2 PMN in a flow chamber coated with murine P-selectin (20 mg/ml), murine ICAM-1 (12.5 mg/ml), and KC (15 mg/ml) at
a physiological flow of 1 dyne/cm2. Genetic absence of mAbp1 diminished the crawling velocity under flow conditions in vitro. Left, Box-whisker plot of
velocities 6 SD; median numbers are indicated. n = 3, *p , 0.05. Right, Frequency distribution of velocities. n = 60 mAbp1+/+ PMN and n = 90 mAbp12/2
PMN from three independent experiments. (B) Microscopic images and single-cell tracks of leukocytes from mAbp1+/+ and mAbp12/2 mice that crawled
on the endothelium assessed from intravital microscopy of inflamed venules of the cremaster muscle. Arrows resemble migration tracks of leukocytes.
Dotted arrows indicate the direction of blood flow in the venule. Microscopic images and single-cell tracks are representative for four (mAbp1+/+) or five
(mAbp12/2) independent experiments. Scale bar, 50 mm. (C) Quantitative analysis of the percentage of leukocytes from mAbp1+/+ and mAbp12/2 mice
crawling on the inflamed endothelium in the direction of the blood flow. Leukocytes were considered to migrate, when they moved at least one cell diameter
within the observation period of 10 min. The genetic absence of mAbp1 diminished the ability of leukocytes to migrate perpendicular or against the
direction of blood flow. n = 157 cells in 13 venules from four mAbp1+/+ mice, n = 189 cells in 15 venules from five mAbp12/2 mice, means 6 SEM, *p ,
0.05. (D and E) Trajectories and migratory parameters of crawling dHL-60 cells on human ICAM-1 (12.5 mg/ml) stimulated with fMLP (100 nM) after 10
min under flow conditions (1 dyne/cm2, direction of flow is indicated). The final positions relative to their original starting point are indicated by a filled
circle (D, left panel). In the rose diagram (D, right panel), the area of each sector is proportional to the frequency of the migration vectors of the tracked
cells pointed in the respective direction. Under physiological flow conditions, the downregulation of mAbp1 by RNAi technique (shAbp1) diminished the
migration velocity and migration distance of crawling cells and the frequency of the migration vectors pointing against direction of the applied flow
compared with control dHL-60 cells (shControl). n = 4 (shControl), n = 5 (shAbp1). (F) Representative Western blot of dHL-60 cell clones stably
expressing a shRNA against mAbp1 (shAbp1) or a scrambled control (shControl) and wild-type dHL-60 cells (WT). mAbp1 was detected by the mouse
anti-mAbp1 mAb; actin was detected by the goat anti-actin Ab. Protein levels of mAbp1 were normalized to amount of actin. Numbers indicate relative
level of mAbp1 expression of shAbp1 cells compared with shControl (100%). n = 3, mean 6 SD.
10
and mAbp12/2 mice, providing evidence that the initial steps of
leukocyte recruitment were not disturbed by the genetic absence
of mAbp1. The observed defect in leukocyte spreading in the
inflamed cremaster of mAbp12/2 mice resembles the spreading
defect of Syk2/2 leukocytes in vivo, as reported earlier (19). Interestingly, Syk was found to be indispensable for adhesion and
spreading even under static in vitro conditions, whereas mAbp1
was not required for adhesion or spreading under these conditions
(19, 21). Thus, unlike Syk, mAbp1 seems to be only required
when mechanical force is applied on the b2 integrins.
Intraluminal crawling of neutrophils is a postarrest step of the
recruitment cascade, which has been shown to be mediated by
interaction of Mac-1 and endothelial ICAM-1 in mice in vivo (8,
48, 59). This was also true in our experimental setting using
isolated murine PMN migrating on immobilized ICAM-1 under
shear stress conditions. In this study, the percentage of migrating
cells was severely compromised in mAbp12/2 PMN, suggesting
an impact of mAbp1 for Mac-1–mediated postarrest steps in
neutrophil recruitment. Further analysis revealed a similar defect
in the human system when mAbp1 was downregulated by RNAi
technique. In addition to the overall migration capacity, the genetic ablation of mAbp1 in mice or the downregulation of mAbp1
by RNAi technique in dHL-60 cells interfered with the ability of
the cells to migrate against and perpendicular to the direction of
flow. In contrast, this behavior was typical for mAbp1+/+ leuko-
cytes in the inflamed cremaster muscle model in vivo as well as in
control dHL-60 cells in an in vitro flow chamber assay. Phillipson
et al. (8, 59) described perpendicular intraluminal crawling of
PMN as a mechanism to find emigration sites. In Vav2/2 mice,
PMN were not able to migrate perpendicular to the direction of
blood flow, but crawled with the flow. Our findings on the impact
of mAbp1 on migration under shear stress resembled the observation on Vav2/2 PMN. Similar to mAbp1, Vav is known to be
important for neutrophil migration and was found to be part of the
signaling complex downstream of Syk (28), arguing for an important impact of b2 integrin-mediated signaling via Syk for Mac1–dependent intraluminal crawling.
Analysis of spreading and chemotactic migration of mAbp12/2
PMN under static conditions revealed no differences compared with
control cells. Similarly, the downregulation of the mAbp1 ortholog
Dabp1 in Dictyostelium discoideum by RNAi technique (siDabp1)
did not affect cell migration (63). In contrast, the overexpression
of Dabp1 led to an impaired migration, suggesting a functional
compensation by other signaling molecules in the siDabp1 cells
(63). A compensatory mechanism could also work in mAbp12/2
PMN, allowing adhesion, spreading, and migration at least under
static conditions. In yeast, Abp1p has redundant functions with the
protein Sla2p (34). Less is known about the mammalian ortholog
of Sla2p, the Huntingtin-interacting protein-1–related protein
(Hip1R). However, it has been shown to bind F-actin, to associate
FIGURE 7. mAbp1 was dispensable for chemotactic migration of PMN under static conditions. (A–C) Trajectories and migratory parameters of murine
PMN isolated from mAbp1+/+ and mAbp12/2 mice migrating in response to a gradient of 10 mM fMLP (◢) on immobilized fibrinogen (A, C, upper panel)
or murine ICAM-1 (B, C, lower panel), respectively. The final positions relative to their original starting point are indicated by a filled circle (A, B, left
panels). In the rose diagram (A, B, right panels), the area of each sector is proportional to the frequency of the migration vectors of the tracked cells pointed
in the respective direction. Under static conditions, chemotactic migration on immobilized fibrinogen or ICAM-1 was similar between the two groups. n = 4
on immobilized fibrinogen, n = 3 on immobilized ICAM-1. (C) Means 6 SD.
Acknowledgments
We thank Katy Niedung, Melitta Weissinger, and Susanne Bierschenk for
excellent technical assistance.
Disclosures
The authors have no financial conflicts of interest.
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with clathrin-coated vesicles, and to form a complex with cortactin, which acts as a negative regulator of actin polymerization
in HeLa cells, suggesting that Hip1R may compensate for the
absence of mAbp1 during chemotactic migration (64).
Growing evidence supports the concept that mechanical force
may be required for the development of adhesion sites by inducing
recruitment of additional proteins. Force can be generated either
internally, for example, by actin polymerization, or externally, for
example, by shear stress (65). In this study, we demonstrated that
actin binding of mAbp1 was enhanced upon induction of adhesion
via b2 integrins. It may be reasonable to speculate that mAbp1
might be a novel component of the adhesion protein network that
is necessary to connect the ligand-bound b2 integrins to the cytoskeleton, which in turn is indispensable to resist shear forces
during adhesion, spreading, and migration under flow conditions.
Accordingly, our data implicate that mAbp1 exerts its function
rather under the influence of high external forces like exposure to
shear stress than upon generation of moderate internal forces like
actin polymerization during spreading and migration under static
conditions. Under flow conditions, adhesion as well as crawling
depends on the affinity regulation of b2 integrins (1, 3, 6). We
have previously shown that mAbp1 was critical for the establishment and/or maintenance of the high-affinity conformation of
b2 integrins under flow conditions (21). Accordingly, similar to
the adhesion defect under shear stress conditions, also the impairment of mechanotactic crawling may be caused by the inability of the cells to tightly control affinity of the b2 integrins in
the absence of mAbp1 under flow conditions.
In addition to mAbp1, an essential role for firm adhesion and
migration under shear stress has been reported for WASP, whereas
adhesion and migration under static conditions were largely not
affected in the absence of WASP. In this study, WASP has been
associated with integrin clustering to promote adhesion strengthening under shear stress (66). A functional impact on the reinforcement of the high-affinity conformation of b2 integrins under
flow conditions was demonstrated for mAbp1 (21). As the genetic
absence or downregulation of mAbp1 by RNAi technique did not
affect the subcellular localization of WASP under static and flow
conditions, mAbp1 was obviously not required for the proper localization of WASP (21). However, the localization of mAbp1 as
well as WASP and WIP was dependent of active Syk. This finding
suggests that mAbp1, WASP, and WIP may represent components
of the same b2 integrin-mediated signaling complex downstream
of Syk, leading to cytoskeletal reorganization, which in turn is
thought to be essential for control of the affinity regulation of b2
integrins during PMN recruitment.
Taken together, we found that mAbp1 was critical for b2
integrin-mediated postarrest functions of PMN, namely spreading
and crawling under flow conditions. However, mAbp1 was only
indispensable when external forces were applied to the integrin,
whereas spreading and migration were normal under static conditions. Accordingly, mAbp1 seems to be critical for the transmission of mechanical force from the ligand-bound b2 integrin to
the actin cytoskeleton, which is not only crucial for induction of
adhesion under flow conditions, but also for intraluminal spreading and crawling of neutrophils during their recruitment to sites of
inflammation.
11
12
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The Mammalian Actin-Binding Protein 1 Is Critical for Spreading and

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