From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
PHAGOCYTES, GRANULOCYTES, AND MYELOPOIESIS
C-C motif chemokine CCL3 and canonical neutrophil attractants promote
neutrophil extravasation through common and distinct mechanisms
Christoph A. Reichel,1 Daniel Puhr-Westerheide,1 Gabriele Zuchtriegel,1 Bernd Uhl,1 Nina Berberich,2 Stefan Zahler,2
Matthias P. Wymann,3 Bruno Luckow,4 and Fritz Krombach1
1Klinikum der Universität München, Walter Brendel Centre of Experimental Medicine, Campus Grosshadern, Ludwig-Maximilians-Universität München, Munich,
Germany; 2Department of Pharmacy, Ludwig-Maximilians-Universität München, Munich, Germany; 3Department of Biomedicine, Institute of Biochemistry and
Genetics, University of Basel, Basel, Switzerland; and 4Klinikum der Universität München, Medizinische Poliklinik, Campus Innenstadt, Arbeitsgruppe Klinische
Biochemie, Ludwig-Maximilians-Universität München, Munich, Germany
Initial observations suggested that C-C
motif chemokines exclusively mediate
chemotaxis of mononuclear cells. In addition, recent studies also implicated these
chemotactic cytokines in the recruitment
of neutrophils. The underlying mechanisms remained largely unknown. Using
in vivo microscopy on the mouse cremaster muscle, intravascular adherence and
subsequent paracellular transmigration
of neutrophils elicited by the chemokine
(C-C motif) ligand 3 (CCL3, synonym
MIP-1␣) were significantly diminished in
mice with a deficiency of the chemokine
(C-C motif) receptor 1 (Ccr1 ⴚ/ⴚ ) or
5 (Ccr5ⴚ/ⴚ). Using cell-transfer techniques, neutrophil responses required
leukocyte CCR1 and nonleukocyte
CCR5. Furthermore, neutrophil extravasation elicited by CCL3 was almost completely abolished on inhibition of
G protein–receptor coupling and PI3K␥dependent signaling, while neutrophil
recruitment induced by the canonical
neutrophil attractants chemokine (C-X-C
motif) ligand 1 (CXCL1, synonym KC) or
the lipid mediator platetelet-activating factor (PAF) was only partially reduced. More-
over, Ab blockade of ␤2 integrins, of ␣4
integrins, or of their putative counter receptors ICAM-1 and VCAM-1 significantly
attenuated CCL3-, CXCL1-, or PAF-elicited
intravascular adherence and paracellular
transmigration of neutrophils. These data
indicate that the C-C motif chemokine
CCL3 and canonical neutrophil attractants exhibit both common and distinct
mechanisms for the regulation of intravascular adherence and transmigration of
neutrophils. (Blood. 2012;120(4):880-890)
Introduction
Leukocyte recruitment from the microvasculature to the site of
inflammation is a hallmark in the inflammatory response. This
highly regulated multistep process requires the coordinated interplay of chemoattractants, signaling molecules, and adhesion receptors ultimately controlling intravascular rolling and firm adherence
as well as transmigration of leukocytes to the inflamed tissue.1-4
Although the general principles of leukocyte extravasation have
been studied in detail in the past decades, the mechanisms
underlying subtype-specific leukocyte responses remain incompletely understood.
Chemokines are small molecules (8-14 kDa) which can be
classified into C, C-C, C-X-C, and C-X3-C motif chemokines
according to the arrangement of their N-terminal cysteine residues.
Interaction of chemokines with specific G protein–coupled chemokine receptors is thought to activate intracellular signaling pathways ultimately mediating adhesion and directed migration of
leukocytes.5-7
According to our current knowledge, spatiotemporal expression
patterns of chemokines, as well as expression of specific chemokine receptors on different leukocyte subtypes, are suggested to
enable the guidance of defined leukocyte subsets to the site of
inflammation. In this context, neutrophils are thought to be
attracted by C-X-C motif chemokines (eg, CXCL1/KC, CXCL2/
MIP-2), whereas C-C motif chemokines (eg, CCL3/MIP-1␣) are
supposed to primarily mediate the migration of mononuclear
cells.3,5-7 In addition to this well-known function, however, there is
increasing evidence that C-C motif chemokines are also involved
in the migration of granulocytes including eosinophils,8,9 basophils,10,11 and, in particular, neutrophils.12-16 These reports reveal a
previously unrecognized role of these chemotactic cytokines in the
inflammatory response. The exact mechanisms underlying neutrophil recruitment by C-C motif chemokines are still unknown.
Recently, C-C motif chemokine receptors including CCR1 and
CCR5 have been identified on the surface of neutrophils.17-22
Notably, these chemokine receptors are also present on a variety of
resident cell populations known to promote extravasation of
neutrophils such as endothelial cells,23,24 smooth muscle cells,25 or
mast cells.26,27 The relative contribution of these receptors to the
recruitment of neutrophils has not yet been investigated. Moreover,
the relevant signaling and adhesion events involved in C-C motif
chemokine-dependent neutrophil responses are largely unexplored.
Here, we demonstrate that CCL3-dependent intravascular firm
adherence and subsequent paracellular transmigration of neutrophils require both C-C motif chemokine receptor CCR1 and CCR5.
In this context, leukocyte CCR1 and nonleukocyte CCR5 are
engaged regulating C-C motif chemokine-dependent neutrophil
responses via G protein–receptor coupling as well as PI3K␥dependent signaling pathways. As a consequence, firm adherence
Submitted January 2, 2012; accepted May 26, 2012. Prepublished online as
Blood First Edition paper, June 6, 2012; DOI 10.1182/blood-2012-01-402164.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
© 2012 by The American Society of Hematology
880
BLOOD, 26 JULY 2012 䡠 VOLUME 120, NUMBER 4
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
BLOOD, 26 JULY 2012 䡠 VOLUME 120, NUMBER 4
to the vascular endothelium is particularly mediated by the ␤2
integrins lymphocyte function-associated Ag-1 (CD11a/LFA-1)
and macrophage-1 Ag (CD11b/Mac-1), to a lesser degree by ␣4
integrins (␣4␤1 integrin/VLA-4 and/or ␣4␤7 integrin) as well as by
their (putative) counter receptors intercellular adhesion molecule-1
(CD54/ICAM-1) and vascular cell adhesion molecule-1 (CD106/
VCAM-1), whereas subsequent paracellular transmigration of
neutrophils is facilitated through platelet/endothelial cell adhesion
molecule-1 (CD31/PECAM-1) and intercellular adhesion
molecule-2 (CD102/ICAM-2).
Methods
Animals
BALB/cAnNCrl (briefly BALB/c) mice were obtained from Charles River.
Ccr1-deficient mice (Ccr1tm1Gao) and Ccr5-deficient mice (Ccr5tm1Blck) have
been generated as described28,29 and backcrossed for 10 (Ccr1) or
13 generations (Ccr5) to the BALB/c background. Experiments were
performed exclusively with male mice at the age of 20 ⫾ 5 weeks. Mice
were raised in individually ventilated cages under specific pathogen-free
conditions and housed during the experiments under conventional conditions with free access to food and water. All experiments were performed in
compliance with the German legislation for the protection of animals and
were approved by the Regierung von Oberbayern (government of Upper
Bavaria).
Musculus cremaster assay
The surgical preparation of the cremaster muscle was performed as
originally described by Baez with minor modifications.30,31 Mice were
anesthetized using a ketamine/xylazine mixture (100 mg/kg ketamine and
10 mg/kg xylazine), administrated by IP injection. The left femoral artery
was cannulated in a retrograde manner for administration of microspheres
and drugs (see “Quantification of leukocyte kinetics and microhemodynamic parameters”). The right cremaster muscle was exposed through a
ventral incision of the scrotum. The muscle was opened ventrally in an
avascular zone, using careful electrocautery to stop any bleeding, and
spread over the transparent pedestal of a custom-made microscopy stage.
Epididymis and testicle were detached from the cremaster muscle and
placed into the abdominal cavity. Throughout the procedure as well as after
surgical preparation during in vivo microscopy, the muscle was superfused
with warm-buffered saline.
In vivo microscopy
The setup for in vivo microscopy was centered around an Olympus BX
50 upright microscope (Olympus Microscopy), equipped for stroboscopic
fluorescence epiillumination microscopy. Light from a 75-W xenon source
was narrowed to a near-monochromatic beam of a wavelength of 700 nm by
a galvanometric scanner (Polychrome II; TILL Photonics) and directed onto
the specimen via a FITC filter cube equipped with dichroic and emission
filters (DCLP 500, LP515; Olympus). Microscopy images were obtained
with Olympus water immersion lenses (20⫻/numerical aperture [NA]
0.5 and 10⫻/NA 0.3) and recorded with an analog black-and-white
charge-coupled device video camera (Cohu) and an analog video recorder
(Panasonic). Oblique illumination was obtained by positioning a mirroring
surface (reflector) directly below the specimen and tilting its angle relative
to the horizontal plane. The reflector consisted of a round cover glass
(thickness, 0.19-0.22 mm; diameter, 11.8 mm), which was coated with
aluminum vapor (Freichel) and brought into direct contact with the
overlying specimen as described previously.31 For measurement of centerline blood flow velocity, green fluorescent microspheres (2-␮m diameter;
Molecular Probes) were injected via the femoral artery catheter, and their
passage through the vessels of interest was recorded using the FITC filter
cube under appropriate stroboscopic illumination (exposure, 1 ms; cycle
time, 10 ms; ␭ ⫽ 488 nm), integrating video images for sufficient time
CCR1 AND CCR5 PROMOTE NEUTROPHIL RECRUITMENT
881
(⬎ 80 ms) to allow for the recording of several images of the same bead on
one frame. Beads that were flowing freely along the centerline of the vessels
were used to determine blood flow velocity (see next section).
Quantification of leukocyte kinetics and microhemodynamic
parameters
For offline analysis of parameters describing the sequential steps of
leukocyte extravasation, we used the Cap-Image image analysis software
(Dr Zeintl). Rolling leukocytes were defined as those moving slower than
the associated blood flow and quantified as described previously. Firmly
adherent cells were determined as those resting in the associated blood flow
for ⬎ 30 seconds and related to the luminal surface per 100-␮m vessel
length. Transmigrated cells were counted in regions of interest (ROI),
covering 75 ␮m on both sides of a vessel over 100-␮m vessel length. By
measuring the distance between several images of one fluorescent bead
under stroboscopic illumination, centerline blood flow velocity was determined. From measured vessel diameters and centerline blood flow velocity,
apparent wall shear rates were calculated, assuming a parabolic flow
velocity profile over the vessel cross-section.
Quantification of fluorescent leukocyte responses
To investigate the contribution of leukocyte and nonleukocyte target
proteins to CCL3-elicited leukocyte responses, a cell-transfer technique
was used as described previously.32,33 Briefly, bone marrow leukocytes
were isolated from donor mice by flushing the femur and tibia bones with
PBS. Cells were then sieved and counted, resuspended in PBS containing
BSA (0.25%), and incubated with calcein-AM (Molecular Probes; 10␮M
final concentration at 37°C for 30 minutes) as well as in separate
experiments also with pertussis toxin (PTx), wortmannin, or drug vehicle.
After 2 washes, the cells were injected intravenously into recipient mice via
the right jugular vein (107 cells/mouse) 120 minutes before the surgical
preparation. Fluorescent cells were counted in 175 high-power fields (HPF)
per animal, this being equivalent to the total quantifiable area of an
exteriorized cremaster muscle in the present studies. Results are shown as
the number of adherent or transmigrated calcein-labeled cells/HPF.
Reagents
A nonblocking Alexa Fluor 488–conjugated anti–PECAM-1 mAb (clone
390; 40 ␮g/mL, applied into the superfusion solution; BioLegend) was used
to delineate endothelial junctions. Clodronate liposomes (injection into the
tail vein 48 hours [200 ␮L], 24 hours [100 ␮L], and 6 hours [100 ␮L]
before the experiment; VUmc FdG) were prepared as described elsewhere
and used to deplete monocytes and macrophages according to previous
protocols.34,35 Recombinant murine CCL3, CXCL1 (300 ng in 0.4 mL of
PBS; intrascrotally; R&D Systems), or platelet-activating factor (PAF;
0.4 mL of 10⫺6M solution, intrascrotally; Sigma-Aldrich) were used to
induce leukocyte extravasation. Blocking anti–LFA-1 mAb (clone M17/4),
anti–Mac-1 mAb (clone M1/70), anti-␣4 integrin mAbs (blocking ␣4␤1
integrin/VLA-4 and ␣4␤7 integrin; clone R1-2 and clone PS/2), anti–
PECAM-1 mAb (clone MEC 13.3), anti–ICAM-1 mAb (clone YN1),
anti–ICAM-2 mAb (clone 3C4), and anti–VCAM-1 mAb (clone 429; 50 ␮g
in 150 ␮L of saline, i.a.; BioLegend) were used to inhibit interactions with
the respective adhesion molecules, and the nonblocking anti–PECAM-1
mAb (clone 390; 50 ␮g in 150 ␮L of saline, intra-arterially; BioLegend)
was used as additional control. PTx (4 ␮g in 150 ␮L of saline, intraarterially; Sigma-Aldrich) was used to inhibit G protein–receptor coupling.
Wortmannin (irreversible pan-PI3K inhibitor; 1 mg/kg BW, intra-arterially;
Sigma-Aldrich), PI103 (reversible pan-PI3K inhibitor; 3 ⫻ 5 mg/kg BW
intraperitoneally), AS605240 (reversible PI3K␥ inhibitor; 3 ⫻ 5 mg/kg
BW IP), and IC87114 (reversible PI3K␦ inhibitor; 3 ⫻ 5 mg/kg BW
intraperitoneally) were used to inhibit PI3K activity.
Experimental groups
Animals were assigned randomly to the following groups: PBS-treated
wild-type (WT) control mice as well as WT, Ccr1⫺/⫺, and Ccr5⫺/⫺ mice
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
882
BLOOD, 26 JULY 2012 䡠 VOLUME 120, NUMBER 4
REICHEL et al
Figure 1. Role of CCR1 and CCR5 for CCL3-elicited
leukocyte responses. (A) Representative RLOT in vivo
microscopy images of postcapillary venules in WT, Ccr1-,
and Ccr5-deficient mice after stimulation with CCL3 (scale
bar: 20 ␮m). Leukocyte intravascular firm adherence and
transmigration were quantified in postcapillary venules of the
cremaster muscle as detailed in “Quantification of leukocyte
kinetics and microhemodynamic parameters.” (B-C) Results
for PBS-treated WT control mice as well as for WT, Ccr1-,
and Ccr5-deficient mice after stimulation with CCL3
(mean ⫾ SEM for n ⫽ 6 per group; #P ⬍ .05, vs unstimulated; *P ⬍ .05, vs WT). (D) Intravascular adherence and (E)
transmigration of fluorescence-labeled bone marrow leukocytes were quantified in the cremaster muscle using in vivo
fluorescence microscopy as detailed in “Quantification of
florescent leukocyte responses.” Panels show results for WT
mice receiving leukocytes from WT, Ccr1-, or Ccr5-deficient
donors as well as for Ccr1- and Ccr5-deficient mice receiving
leukocytes from WT donors after stimulation with CCL3
(mean ⫾ SEM for n ⫽ 7 per group; #P ⬍ .05, vs unstimulated; *P ⬍ .05, vs WT3WT).
undergoing intrascrotal stimulation with murine recombinant CCL3
(180 minutes; n ⫽ 6). Moreover, leukocyte responses were analyzed in the
cremaster muscle of WT mice receiving fluorescent leukocytes from WT,
Ccr1⫺/⫺, or Ccr5⫺/⫺ donors as well of Ccr1⫺/⫺ and of Ccr5⫺/⫺ mice
receiving fluorescent leukocytes from WT donors undergoing intrascrotal
stimulation with CCL3 (180 minutes; n ⫽ 7 each group). Leukocyte
responses were also analyzed in the cremaster muscle of WT mice treated
with PTx, wortmannin, or drug vehicle receiving fluorescent leukocytes from
WT mice coincubated with PTx, wortmannin, or drug vehicle undergoing
intrascrotal stimulation with CCL3 (180 minutes; n ⫽ 4 each group).
In addition, experiments were conducted in PBS-treated WT control
mice as well as in WT mice treated with PTx, with PI3K inhibitors PI103,
AS605240, IC87114, with an anti–LFA-1 mAb, an anti–Mac-1 mAb,
anti-␣4 integrin mAbs (clone R1-2 and clone PS/2), anti–PECAM-1 mAbs
(clone Mec13.3 and clone 390), an anti–ICAM-1 mAb, an anti–ICAM-2
mAb, an anti–VCAM-1 mAb, or with a respective isotype control Ab/drug
vehicle undergoing intrascrotal stimulation with CCL3, CXCL1, or PAF
(180 minutes; n ⫽ 4 each group). Furthermore, experiments were performed in WT animals receiving clodronate liposomes or control PBS
liposomes undergoing intrascrotal stimulation with CCL3, CXCL1, or PAF
(180 minutes; n ⫽ 4 each group). Finally, leukocyte transmigration routes
were analyzed in the cremaster muscle of WT animals receiving a
nonblocking Alexa Fluor 488–conjugated anti–PECAM-1 mAb (clone 390)
undergoing intrascrotal stimulation with CCL3, CXCL1, or PAF (180 minutes; n ⫽ 3 each group).
Experimental protocols
the spread-out cremaster muscle among those that were at least 150 ␮m
away from neighboring postcapillary venules and did not branch over a
distance of at least 150 ␮m. Abs/inhibitors were applied 5 minutes before
the intrascrotal injection of inflammatory mediators (see “Reagents”). After
having obtained recordings of migration parameters, blood flow velocity
was determined as described in “Quantification of leukocyte kinetics.”
After in vivo microscopy, tissue samples of the cremaster muscle were
taken for immunohistochemistry (see supplemental Methods, available on
the Blood Web site; see the Supplemental Materials link at the top of the
online article). Blood samples were collected by cardiac puncture for the
determination of systemic leukocyte counts using a Coulter ACT counter
(Coulter Corp). Anesthetized animals were then killed by bleeding to death.
Statistics
Data analysis was performed with a statistical software package (SigmaStat
for Windows; Jandel Scientific). The ANOVA on ranks test followed by the
Student-Newman-Keuls test was used for the estimation of stochastic
probability in intergroup comparisons. Mean values and SEM are given.
P values ⬍ .05 were considered significant.
Mouse gene nomenclature
The officially approved gene symbols from the Mouse Genome Informatics
Database are used throughout this report.
Results
Role of CCR1 and CCR5 in CCL3-elicited leukocyte responses
For the analysis of CCL3-, CXCL1-, and PAF-dependent leukocyte
responses, leukocyte recruitment to the cremaster muscle was induced by
intrascrotal injection of recombinant murine CCL3, CXCL1, or PAF. After
180 minutes, 5 vessel segments were randomly chosen in a central area of
Using near-infrared transillumination in vivo microscopy, the role
of the chemokine receptors CCR1 and CCR5 for chemokinedependent rolling, firm adherence, and transmigration of leukocytes
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
BLOOD, 26 JULY 2012 䡠 VOLUME 120, NUMBER 4
CCR1 AND CCR5 PROMOTE NEUTROPHIL RECRUITMENT
B
30
25
Leukocyte transmigration
4
2
[n/10 µm ]
Leukocyte firm adherence
4
2
[n/10 µm ]
A
unstimuated
vehicle
PTx
#
20
15
10
*
5
0
30
25
#
20
15
*
10
5
0
CCL3
CCL3
D
30
25
20
vehicle
pan-PI3K inhibitor
PI3KJ inhibitor
PI3KG inhibitor
#
#
15
10
5
*
*
0
CCL3
Leukocyte transmigration
4
[n/10 µm²]
C
A
Leukocyte firm adherence
4
[n/10 µm²]
883
40
30
#
#
20
10
*
*
0
CCL3
E
Figure 2. Role of G protein–receptor coupling and PI3K for CCL3-elicited leukocyte responses. (A,C) Leukocyte firm adherence, and (B,D) transmigration were
quantified in postcapillary venules of the cremaster muscle using RLOT in vivo microscopy as detailed in “Quantification of leukocyte kinetics and microhemodynamic
parameters.” Panels show results for PBS-treated WT control mice as well as for WT mice receiving PTx, the PI3K inhibitors PI103 (pan-PI3K), AS605240 (PI3K␥), and
IC87114 (PI3K␦), or respective drug vehicle after stimulation with CCL3 (mean ⫾ SEM for n ⫽ 4 per group; #P ⬍ .05, vs unstimulated; *P ⬍ .05, vs vehicle). (E) Intravascular
adherence and transmigration of fluorescence-labeled bone marrow leukocytes were quantified in the cremaster muscle using in vivo fluorescence microscopy as detailed in
“Quantification of florescent leukocyte responses.” Panels show results for WT mice treated with PTx, the irreversible PI3K inhibitor wortmannin, or drug vehicle receiving
leukocytes from WT donors pretreated with PTx, wortmannin, or drug vehicle after stimulation with CCL3 (mean ⫾ SEM for n ⫽ 4 per group; #P ⬍ .05, vs unstimulated;
*P ⬍ .05, vs WT3WT ⫹ drug vehicle).
were analyzed in the mouse cremaster muscle 3 hours after intrascrotal
injection of CCL3 (Figure 1A).
It is well known that surgical preparation of the cremaster
muscle induced leukocyte rolling in postcapillary venules. No
significant differences were observed in numbers of rolling leukocytes among all experimental groups (data not shown).
In contrast, the number of firmly adherent leukocytes
(11.6 ⫾ 1.9 ␮m2) was significantly increased on stimulation with
CCL3 compared with PBS-treated control animals (4.3 ⫾ 0.9 ␮m2).
This increase was almost completely abolished in animals lacking
the chemokine receptor Ccr1 (6.1 ⫾ 1.4 ␮m2) or Ccr5
(6.3 ⫾ 1.5 ␮m2; Figure 1B).
Moreover, there was a significant elevation in numbers of
transmigrated leukocytes (22.5 ⫾ 2.9 ␮m2) in response to CCL3
compared with controls (4.9 ⫾ 0.8 ␮m2). This elevation was
significantly attenuated in Ccr1⫺/⫺ (11.5 ⫾ 2.3 ␮m2) and Ccr5⫺/⫺
(9.6 ⫾ 1.3 ␮m2) animals (Figure 1C).
Role of leukocyte versus nonleukocyte CCR1 and CCR5 in
CCL3-elicited leukocyte responses
To enable investigations into the contributions of leukocyte and
nonleukocyte chemokine receptors in CCL3-elicited leukocyte
responses, bone marrow leukocytes were isolated from WT,
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
A
100
Leukocyte rolling flux
[n/30 s]
BLOOD, 26 JULY 2012 䡠 VOLUME 120, NUMBER 4
REICHEL et al
80
60
unstimulated
isotype control
anti-ICAM-1 mAb
anti-Mac-1 mAb
anti-D4 integrin mAb (clone R1-2)
anti-D4 integrin mAb (clone PS/2)
#
40
*
20
B
100
Leukocyte rolling flux
[n/30 s]
884
80
60
unstimulated
isotype control
anti-LFA-1 mAb
anti-PECAM-1 mAb (clone Mec13.3)
anti-PECAM-1 mAb (clone 390)
anti-ICAM-2 mAb
anti-VCAM-1 mAb
40
20
0
0
CCL3
CCL3
D30
30
25
20
15
#
*
10
*
5
*
*
0
Leukocyte firm adherence
4
[n/10 µm²]
Leukocyte firm adherence
4
2
[n/10 µm ]
C
25
20
#
#
#
#
15
*
10
*
5
0
CCL3
CCL3
E
F
40
30
#
20
10
0
#
*
*
*
#
*
Leukocyte transmigration
4
[n/10 µm²]
Leukocyte transmigration
4
2
[n/10 µm ]
Figure 3. Role of ␤2 and ␣4 integrins as well as
PECAM-1, ICAM-1, ICAM-2, and VCAM-1 for CCL3elicited leukocyte responses. (A-B) Leukocyte rolling,
(C-D) firm adherence, and (E-F) transmigration were
quantified in postcapillary venules of the cremaster muscle
using RLOT in vivo microscopy as detailed in “Quantification of leukocyte kinetics and microhemodynamic parameters.” Panels show results for PBS-treated WT control
mice as well as for WT mice receiving blocking mAbs
directed against LFA-1, Mac-1, ␣4 integrins, PECAM-1
(clone Mec13.3), ICAM-1, ICAM-2, and VCAM-1 or isotype control and a nonblocking anti-PECAM-1 mAb (clone
390) after stimulation with CCL3 (mean ⫾ SEM for
n ⫽ 4 per group; #P ⬍ .05, vs unstimulated; *P ⬍ .05, vs
isotype control).
40
30
#
#
20
#
10
*
#
*
*
*
0
Ccr1⫺/⫺, or Ccr5⫺/⫺ donor mice, fluorescently labeled with calcein
AM, and injected intravenously into WT, Ccr1⫺/⫺, or Ccr5⫺/⫺
recipient mice. Three hours after intrascrotal injection of CCL3, the
number of fluorescent cells adherent or transmigrated was quantified in the cremaster muscle by in vivo fluorescence microscopy.
In unstimulated control mice, only a few fluorescent cells were
found attached to the wall of the postcapillary venules (0.14 ⫾ 0.03/
HPF) or within the perivascular tissue (0.12 ⫾ 0.02/HPF). In
contrast, stimulation with CCL3 caused a significant elevation in
numbers of adherent (0.84 ⫾ 0.11/HPF; Figure 1D) and transmigrated (0.86 ⫾ 0.17/HPF; Figure 1E) fluorescent cells. This elevation in the number of adherent and transmigrated fluorescent cells
was significantly diminished in WT animals receiving Ccr1⫺/⫺
cells (0.42 ⫾ 0.09/HPF; 0.31 ⫾ 0.06/HPF) as well as in Ccr5⫺/⫺
animals receiving WT cells (0.44 ⫾ 0.06/HPF; 0.45 ⫾ 0.10/HPF),
respectively.
Role of G protein–receptor coupling and PI3K activation for
CCL3-elicited leukocyte responses
Interaction of chemokines to their cognate receptors is supposed to
activate intracellular G proteins. To explore the functional relevance of
these processes for CCL3-elicited leukocyte responses, another series of
experiments was performed. Using reflected light oblique transillumination (RLOT) in vivo microscopy, CCL3-elicited intravascular firm
adherence and (subsequent) transmigration of leukocytes were nearly
abolished in animals treated with PTx (a compound blocking G protein–
receptor coupling; Figure 2A-B), whereas leukocyte rolling was not
significantly altered (data not shown). Furthermore, treatment with a
reversible pan-PI3K inhibitor or a specific PI3K␥ inhibitor, but not with
a PI3K␦ inhibitor, almost completely abrogated CCL3-elicited leuko-
cyte responses (Figure 2C-D). Using a cell transfer technique, we found
that stimulation with CCL3 caused a significant increase in the numbers
of adherent and transmigrated fluorescent cells compared with controls
(Figure 2E). This increase was almost completely abolished in animals
treated systemically with PTx or the irreversible pan-PI3K inhibitor
wortmannin as well as in animals receiving WT donor cells pretreated
with PTx or wortmannin.
Role of ␤2 and ␣4 integrins as well as of ICAM-1, ICAM-2,
VCAM-1, and PECAM-1 in CCL3-elicited leukocyte responses
In a next step, candidate adhesion molecules involved in the
leukocyte recruitment process were analyzed for their functional
relevance in CCL3-dependent leukocyte responses. In our experiments, intravascular firm adherence (Figure 3C-D) and subsequent
transmigration of leukocytes (Figure 3E-F) elicited by CCL3 were
almost completely abolished on Ab blockade of the ␤2 integrins
LFA-1 or Mac-1 compared with animals receiving isotype control
or the nonblocking anti–PECAM-1 mAb “clone390,” whereas
leukocyte rolling remained unaffected (Figure 3A-B). It is noteworthy that blockade of their putative counter receptor ICAM-1 nearly
abolished intravascular firm adherence and transmigration of
leukocytes and significantly enhanced the number of rolling
leukocytes. In contrast, blockade of ␣4 integrins only slightly
attenuated CCL3-elicited leukocyte responses, while blockade of
its putative counter receptor VCAM-1 significantly diminished
intravascular adherence and subsequent transmigration of leukocytes. Finally, blockade of PECAM-1 or of ICAM-2 selectively
reduced transmigration of leukocytes on stimulation with CCL3
without affecting intravascular rolling or firm adherence of
leukocytes.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
BLOOD, 26 JULY 2012 䡠 VOLUME 120, NUMBER 4
CCR1 AND CCR5 PROMOTE NEUTROPHIL RECRUITMENT
40
20
0
CXCL1
80
60
#
25
20
#
E
unstimulated
vehicle
pan-PI3K inhibitor
PI3KJ inhibitor
PI3KG inhibitor
40
20
*
10
5
0
30
#
20
#
60
40
20
0
Leukocyte firm adherence
4
[n/10 µm²]
Leukocyte rolling flux
[n/30 s]
80
40
#
*
#*
10
5
0
25
20
#
10
#
*
5
0
PAF
PAF
#
#
*
0
PAF
80
#
#
60
#
#
40
20
0
CXCL1
I
30
15
*
20
CXCL1
H
unstimulated
vehicle
pan-PI3K inhibitor
PI3KJ inhibitor
PI3KG inhibitor
#
60
CXCL1
F
25
15
80
PAF
#
CXCL1
100
#
CXCL1
0
G
*
15
PAF
Leukocyte firm adherence
4
[n/10 µm²]
Leukocyte rolling flux
[n/30 s]
100
Leukocyte transmigration
4
[n/10 µm²]
60
30
Leukocyte transmigration
4
[n/10 µm²]
unstimulated
vehicle
PTx
#
Leukocyte transmigration
4
[n/10 µm²]
80
Leukocyte firm adherence
4
[n/10 µm²]
Leukocyte rolling flux
[n/30 s]
100
D
C
B
A
885
40
30
20
10
#
#
#
*
0
PAF
Figure 4. Role of G protein–receptor coupling and PI3K for CXCL1- and PAF-elicited leukocyte responses. (A,D,G) Leukocyte rolling, (B,E,H) firm adherence, and
(C,F,I) transmigration were quantified in postcapillary venules of the cremaster muscle using RLOT in vivo microscopy as detailed in “Quantification of leukocyte kinetics and
microhemodynamic parameters.” Panels show results for PBS-treated WT control mice as well as for WT mice receiving PTx, the PI3K inhibitors PI103 (pan-PI3K), AS605240
(PI3K␥), and IC87114 (PI3K␦), or respective vehicle after stimulation with CXCL1 or PAF (mean ⫾ SEM for n ⫽ 4 per group; #P ⬍ .05, vs unstimulated; *P ⬍ .05, vs vehicle).
Role of G protein–receptor coupling and PI3K activation for
CXCL1- or PAF-elicited leukocyte responses
In further experiments, the mechanisms underlying neutrophil
responses elicited by canonical neutrophil attractants were analyzed. On stimulation with the C-X-C motif chemokine CXCL1 or
with the lipid mediator PAF, application of PTx only partially
reduced intravascular adherence (Figure 4B) and (subsequent)
transmigration of leukocytes (Figure 4C), while leukocyte rolling
remained unaltered (Figure 4A). In addition, CXCL1-elicited
intravascular adherence of leukocytes was only slightly attenuated
in animals treated with the pan-PI3K inhibitor or the specific
PI3K␥ inhibitor, but not with the PI3K␦ inhibitor, while leukocyte
rolling and transmigration in response to CXCL1 were not
significantly altered on treatment with the PI3K inhibitors (Figure
4D-F). Moreover, treatment with the pan-PI3K inhibitor, but not
with the PI3K␥ or PI3K␦ inhibitor significantly reduced PAFelicited leukocyte responses (Figure 4G-I).
Role of ␤2 and ␣4 integrins as well as of ICAM-1, ICAM-2,
VCAM-1, and PECAM-1 in CXCL1- or PAF-elicited leukocyte
responses
On stimulation with CXCL1 (Figures 5A,C,E and 6A,C,E) or PAF
(Figures 5B,D,F and 6B,D,F), intravascular firm adherence and
subsequent transmigration of leukocytes were almost completely
abolished on Ab blockade of the ␤2 integrins Mac-1 or LFA-1
compared with animals receiving isotype control or the nonblocking anti–PECAM-1 mAb “clone390,” whereas leukocyte rolling
remained unchanged in these experiments. Interestingly, on stimu-
lation with CXCL1 or PAF, blockade of ICAM-1 also almost
completely abrogated firm adherence and transmigration of leukocytes, while only in PAF-elicited inflammation the number of
rolling leukocytes was additionally enhanced. Furthermore, blockade of ␣4 integrins (␣4␤1 integrin/VLA-4 and/or ␣4␤7 integrin) or
of their putative counter receptor VCAM-1 attenuated CXCL1- and
PAF-elicited leukocyte responses. Finally, blockade of PECAM-1
partially reduced intravascular adherence and transmigration of
leukocytes on stimulation with CXCL1, but not in response to PAF.
Interestingly, blockade of ICAM-2 significantly diminished intravascular firm adherence of leukocytes elicited by CXCL1 or PAF,
but did not significantly alter leukocyte transmigration to the
inflamed tissue.
Transmigration routes of leukocytes in response to CCL3,
CXCL1, or PAF
Transmigration of leukocytes occurs via the paracellular or the
transcellular transmigration route. To characterize the transmigration route of extravasating leukocytes in CCL3-, CXCL1-, or
PAF-elicited inflammation, endothelial boundaries in postcapillary
venules of the cremaster muscle were visualized by fluorescence in
vivo microscopy and, concomitantly, the localization of firmly
arrested leukocytes was traced by using near-infrared RLOT in vivo
microscopy (Figure 7A). Three hours after intrascrotal injection of
CCL3 (90.8% ⫾ 5.7%), CXCL1 (93.3% ⫾ 4.2%), or PAF
(94.2% ⫾ 1.8%), firmly adherent leukocytes predominantly colocalized
with PECAM-1–immunoreactive endothelial junctions (Figure 7B).
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
BLOOD, 26 JULY 2012 䡠 VOLUME 120, NUMBER 4
REICHEL et al
A
Leukocyte rolling flux
[n/30 s]
100
80
60
unstimulated
isotype control
anti-ICAM-1 mAb
anti-Mac-1 mAb
anti-D4 integrin mAb (clone R1-2)
anti-D4 integrin mAb (clone PS/2)
40
20
B
100
Leukocyte rolling flux
[n/30 s]
886
80
60
unstimulated
isotype control
anti-ICAM-1 mAb
anti-Mac-1 mAb
anti-D4 integrin mAb (clone R1-2)
anti-D4 integrin mAb (clone PS/2)
#
40
*
20
0
0
PAF
CXCL1
25
#
20
15
*
10
*
5
*
*
0
E
#
40
20
*
*
25
20
#
15
*
10
*
5
*
*
0
F
80
60
30
PAF
CXCL1
*
*
0
Leukocyte transmigration
4
2
[n/10 µm ]
Leukocyte firm adherence
4
2
[n/10 µm ]
30
Leukocyte firm adherence
4
2
[n/10 µm ]
D
C
Leukocyte transmigration
4
2
[n/10 µm ]
Figure 5. Role of Mac-1, ␣4 integrins, and ICAM-1 for
CXCL1- or PAF-elicited leukocyte responses.
(A-B) Leukocyte rolling, (C-D) firm adherence, and
(E-F) transmigration were quantified in postcapillary
venules of the cremaster muscle using RLOT in vivo
microscopy as detailed in “Quantification of leukocyte
kinetics and microhemodynamic parameters.” Panels show
results for PBS-treated WT control mice as well as for
WT mice receiving blocking mAbs directed against Mac-1,
␣4 integrins, ICAM-1, or isotype control after stimulation
with CXCL1 or PAF (mean ⫾ SEM for n ⫽ 4 per group;
#P ⬍ .05, vs unstimulated; *P ⬍ .05, vs isotype control).
40
30
#
20
#*
10
*
*
#*
0
CXCL1
Phenotyping of transmigrated leukocytes
Phenotyping of transmigrated leukocytes was performed by immunostaining of paraffin-embedded tissue sections of the cremaster
muscle. In response to CCL3, CXCL1, or PAF, ⬎ 80% of
transmigrated CD45⫹ leukocytes were Ly-6G⫹ neutrophils. The
remaining 10%-20% were F4/80⫹ monocytes/macrophages.
Effect of monocyte/macrophage depletion on CCL3-, CXCL1-,
or PAF-elicited neutrophil responses
To characterize the role of monocytes/macrophages for CCL3-,
CXCL1-, or PAF-elicited neutrophil responses, experiments were
performed in animals receiving monocyte/macrophage-depleting
clodronate liposomes. On stimulation with CCL3, CXCL1, or PAF,
no significant differences in numbers of rolling, firmly adherent, or
transmigrated leukocytes were detected among animals receiving
clodronate liposomes or control PBS liposomes (supplemental
Figure 1).
Effect of CCL3, CXCL1, or PAF on activation of endothelial cells
To further dissect the mechanisms underlying CCL3-, CXCL1-, or
PAF-elicited neutrophil responses, the effect of inflammatory
mediators on activation of endothelial cells was characterized. To
measure endothelial cell activation, RNA expression of E-selectin
was determined in cremaster muscle samples by using RT-PCR. On
stimulation with CCL3, there was a strong increase in RNA
expression of E-selectin (149.4% ⫾ 15.4%) compared with PBStreated controls. By contrast, RNA levels of E-selectin were not
PAF
significantly altered on stimulation with CXCL1 (⫺4.3% ⫾ 33.6%)
and only slightly enhanced in response to PAF (10.0% ⫾ 1.3%;
supplemental Figure 2).
Systemic leukocyte counts and microhemodynamic
parameters
To assure intergroup comparability, systemic leukocyte counts and
microhemodynamic parameters including inner vessel diameter,
blood flow velocity, and wall shear rate were analyzed in each
experiment. No significant differences were detected among all
experimental groups (supplemental Table 1).
Discussion
Leukocyte migration to the site of inflammation is a key event in
innate and adaptive immunity.1-4 In this context, it is widely
accepted that C-C motif chemokines, which represent 1 of the
4 major classes of chemokines, primarily govern the migration of
mononuclear cells.3,5-7 It is noteworthy that this assumption appears
to be no longer valid as recent studies have clearly indicated that
chemokines belonging to the C-C motif subfamily do also play a
crucial role for the recruitment of eosinophils,8,9 basophils,10,11 and,
in particular, neutrophils.12-16 CCL3 binds to 2 closely related
chemokine receptors designated CCR1 and CCR5. Recently, the
chemokine receptors CCR1 and CCR5 have been detected on
neutrophils.17,19,21,22 The relative contribution of both chemokine
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
BLOOD, 26 JULY 2012 䡠 VOLUME 120, NUMBER 4
100
80
60
unstimulated
isotype control
anti-LFA-1 mAb
anti-PECAM-1 mAb (clone Mec13.3)
anti-PECAM-1 mAb (clone 390)
anti-ICAM-2 mAb
anti-VCAM-1 mAb
40
20
B
100
Leukocyte rolling flux
[n/30 s]
Leukocyte rolling flux
[n/30 s]
A
80
60
unstimulated
isotype control
LFA-1 mAb
anti-PECAM-1 mAb (clone Mec13.3)
anti-PECAM-1 mAb (clone 390)
anti-ICAM-2 mAb
anti-VCAM-1 mAb
#
40
PAF
D
Leukocyte firm adherence
4
[n/10 µm²]
30
#
#
25
20
#*
15
#*
*
*
10
5
0
30
25
20
#
#
#
15
*
10
*
*
5
0
CXCL1
PAF
E
F
60
50
40
#
#
30
#
#
20
10
*
*
0
CXCL1
receptors to discrete steps of the CCL3-mediated extravasation of
neutrophils is still unclear.
In a first approach, we therefore sought to evaluate the
functional relevance of CCR1 and CCR5 for CCL3-dependent
neutrophil responses. Using in vivo microscopy on the mouse
cremaster muscle, we observed that CCL3-induced intravascular
firm adherence followed by transmigration of neutrophils were
significantly diminished in Ccr1- or Ccr5-deficient animals, whereas
leukocyte rolling remained unaltered. Our findings extend previous
studies demonstrating that deficiency/blockade of CCR1 or CCR5
was associated with reduced neutrophil recruitment in animal
models of ischemia-reperfusion injury13,14,16 or in experimental
aspergillosis,29 and convincingly demonstrate that both chemokine
receptors play a significant role during the extravasation of neutrophils.
Interestingly, CCR1 and CCR5 are not only expressed on
neutrophils and mononuclear cells as these chemokine receptors
have previously been detected on the surface of resident cells (eg,
endothelial cells,23,24 smooth muscle cells,25 or mast cells26,27).
Binding of C-C motif chemokines to leukocyte chemokine receptors is supposed to elicit immediate affinity changes of surfaceexpressed integrins,3,5-7 while in resident cells these chemotactic
cytokines are known to primarily induce the generation of further
leukocyte-attracting mediators.36,37 To which extent leukocyte and
nonleukocyte chemokine receptors contribute to CCL3-mediated
neutrophil recruitment remained to be determined. Using a celltransfer technique, we found that leukocyte CCR1 and nonleukocyte CCR5 mediate CCL3-elicited intravascular adherence and
subsequent transmigration of neutrophils to inflamed tissue. Moreover, we show that monocytes and macrophages are not involved in
#
*
Leukocyte transmigration
4
[n/10 µm²]
Leukocyte transmigration
4
[n/10 µm²]
*
20
CXCL1
C
887
0
0
Leukocyte firm adherence
4
2
[n/10 µm ]
Figure 6. Role of LFA, PECAM-1, ICAM-2, and VCAM-1
for CXCL1- or PAF-elicited leukocyte responses.
(A-B) Leukocyte rolling, (C-D) firm adherence, and
(E-F) transmigration were quantified in postcapillary
venules of the cremaster muscle using RLOT in vivo
microscopy as detailed in “Quantification of leukocyte
kinetics and microhemodynamic parameters.” Panels show
results for PBS-treated WT control mice as well as for WT
mice receiving blocking mAbs directed against LFA-1,
PECAM-1 (clone Mec13.3), ICAM-2, and VCAM-1 or
isotype control and a nonblocking anti–PECAM-1 mAb
(clone 390) after stimulation with CXCL1 or PAF
(mean ⫾ SEM for n ⫽ 4 per group; #P ⬍ .05, vs unstimulated; *P ⬍ .05, vs isotype control).
CCR1 AND CCR5 PROMOTE NEUTROPHIL RECRUITMENT
40
30
#
#
#
20
10
#
#*
*
0
PAF
CCL3-dependent neutrophil responses. Collectively, our observations point to a complex interplay between chemokine receptors
CCR1 in neutrophils and CCR5 in resident cells for the regulation
of CCL3-dependent neutrophil recruitment.
Inflammatory stimuli individually use specific mechanisms for
the control of the single steps of the leukocyte recruitment process.
Which signaling and adhesion molecules C-C motif chemokines
engage for the single steps of the neutrophil recruitment process,
however, has not yet been investigated. Previous in vitro data
suggest that ligand binding to a chemokine receptor on the cell
surface causes allosteric changes of the receptor which, in turn,
activates intracellular proteins of the G-protein family. These
processes subsequently allow interaction of G proteins with
different target proteins.3,5-7 Here, we show that CCL3-induced
intravascular adherence and subsequent transmigration of neutrophils strictly require the activation of PTx-sensitive G proteins. Of
note, on stimulation with canonical neutrophil attractants such as
the C-X-C motif chemokine CXCL1 or the lipid mediator PAF,
treatment with PTx only partially inhibited neutrophil extravasation.
Activated G proteins interact with intracellular target proteins
such as the catalytic subunit isoforms p110␥ and p110␦ of PI3K.
These lipid kinases, in turn, produce phosphatidylinositol-3,4,5trisphosphate (PIP3), an intracellular second messenger which is
involved in a variety of cellular functions.38,39 In our experiments,
we found that signaling through the catalytic subunit isoform
p110␥, but not through the catalytic subunit isoform p110␦ of
PI3K controls CCL3-induced intravascular firm adherence and
subsequent transmigration of neutrophils. Interestingly, neutrophil extravasation elicited by CXCL1 did not require PI3K, and
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
888
BLOOD, 26 JULY 2012 䡠 VOLUME 120, NUMBER 4
REICHEL et al
A
B
CCL3
CXCL1
PAF
Non-junctional
Junctional
0
20
40
60
80
100
localization of firmly adherent
leukocytes [%]
Figure 7. Transmigration routes in CCL3-, CXCL1-, and PAF-elicited leukocyte
responses. (A) Representative in vivo microscopy image of a postcapillary venule in
the inflamed cremaster muscle, immunostained for PECAM-1 (scale bars: 20 ␮m).
Arrow indicates a firmly adherent leukocyte colocalizing with PECAM-1–immunoreactive endothelial junctions (green). (B) Results for the relative localization of firmly
adherent leukocytes to PECAM-1–immunoreactive endothelial junctions in postcapillary venules of WT mice undergoing stimulation with CCL3, CXCL1, or PAF
(mean ⫾ SEM for n ⫽ 3 per group).
might therefore be regulated via different signaling pathways. In
contrast, PAF-elicited neutrophil recruitment was significantly
attenuated on broad-spectrum blockade of PI3K, but not after
specific inhibition of the catalytic subunit isoforms p110␥ or
p110␦. Consequently, neutrophil responses induced by PAF
might primarily be regulated via PI3K catalytic subunit isoforms
different from PI3K␥ or PI3K␦ (eg, by PI3K␣ or PI3K␤), which
are supposed to be activated independently of G proteins.38,39
Collectively, these data lead us to the conclusion that receptor
coupling of PTx-sensitive G proteins and activation of PI3K␥
are essential events for the initiation of the CCL3-elicited
inflammatory response, while this signaling pathway is not
critical for neutrophil recruitment mediated by the canonical
neutrophil attractants CXCL1 or PAF.
Activation of neutrophils results in immediate affinity changes
of surface-expressed integrins thereby regulating adhesion and
migration of these inflammatory cells.1-3 Which integrins C-C
motif chemokines use for the recruitment of neutrophils is still
unknown. In our experiments, we observed that, particularly, the ␤2
integrins Mac-1 and LFA-1 as well as their putative counter
receptor ICAM-1 are critically involved in the regulation of
CCL3-dependent intravascular adherence and subsequent transmi-
gration of neutrophils. Moreover, neutrophil responses elicited by
CXCL1 or PAF were also found to require Mac-1, LFA-1, and
ICAM-1 underlining the elementary role of these proteins for the
neutrophil recruitment process. Interestingly, blockade of ICAM-1
significantly enhanced leukocyte rolling on stimulation with CCL3
or PAF, while in CXCL1-elicited inflammation, leukocyte rolling
was not significantly altered on blockade of Mac-1. These observations point to a stimulus-specific control of the initial steps in the
leukocyte recruitment cascade. In this context, it should be noted
that at present we have no clear answer to the question of whether
ICAM-1 serves as a binding partner of Mac-1 and LFA-1 under
these inflammatory conditions or whether these promiscuous
integrins interact with one of their multiple other ligands (eg,
ICAM-2, RAGE, or components of the complement system as well
as of the fibrinolytic system for Mac-1; ICAM-2, ICAM-3, or
JAM-A for LFA-1) in CCL3-, CXCL1-, or PAF-elicited neutrophil
responses.
Astonishingly, we found that ␣4 integrins (␣4␤1 integrin/VLA-4
and/or ␣4␤7 integrin, which are highly expressed in lymphocytes,
but only weakly in neutrophils) participate in CCL3-, CXCL1-, and
PAF-induced neutrophil extravasation. Moreover, it is interesting that blockade of their putative counter receptor VCAM-1
was also associated with significantly diminished intravascular
adherence and subsequent transmigration of neutrophils elicited
by CCL3, CXCL1, or PAF. Our results extend previously
published observations. VLA-4 has been shown to mediate
neutrophil recruitment in experimental peritonitis40 or vasculitis41 and VCAM-1 has been reported to control neutrophil
infiltration in a model of myocardial ischemia reperfusion
(I/R).42 These data highlight a formerly neglected role of these
molecules in the innate immune response.
After their adhesion to endothelial cells, leukocytes either
squeeze between adjacent endothelial cells or directly penetrate
endothelial cells (paracellular vs transcellular transmigration
route) to enter the perivascular space.1,2 In this report, we
demonstrate that on stimulation with CCL3, CXCL1, or PAF,
⬎ 90% of firmly adherent leukocytes colocalized with endothelial junctions. These observations strongly suggest that under
these inflammatory conditions (CCL3-, CXCL1-, or PAFinduced) neutrophils predominantly take the paracellular transmigration route to overcome the endothelial barrier. Our data
agree with previous reports as the leukocyte transmigration
route was not dependent on the inflammatory stimulus applied.43,44 Moreover, we found that PECAM-1 and ICAM-2
selectively mediate CCL3-elicited transmigration of neutrophils
to the inflamed tissue extending previous results under different
inflammatory conditions. In this context, ICAM-2 is suggested
to act in an early stage of leukocyte transmigration whereas
PECAM-1 (and other molecules such as JAM-A) are thought to
be involved later in this sequential process.45 Interestingly, we
found that PECAM-1 was engaged in the extravasation process
of neutrophils already on the level of intravascular adherence on
stimulation with CXCL1 while this molecule was not required
for PAF-elicited neutrophil recruitment. Furthermore, ICAM-2
was dispensable for leukocyte transmigration in response to
CXCL1 or PAF. These results are in line with previous
observations demonstrating that PECAM-1 and ICAM-2 facilitate leukocyte transmigration elicited by the cytokine IL-1␤, but
not by TNF-␣.45 A possible explanation for the stimulus-specific
control of leukocyte recruitment might be that individual
inflammatory mediators differentially activate distinct target
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
BLOOD, 26 JULY 2012 䡠 VOLUME 120, NUMBER 4
CCR1 AND CCR5 PROMOTE NEUTROPHIL RECRUITMENT
cell populations involved in the leukocyte extravasation process
thereby engaging different molecular mechanisms: while nonleukocyte cells (eg, endothelial cells, mast cells) strongly respond
to stimulation with C-C motif chemokines, canonical neutrophil
attractants such as C-X-C motif chemokines and lipid mediators
serve as potent activators of neutrophils.37 From this perspective, the entirety of our experimental data also reveals that C-C
motif chemokines, C-X-C motif chemokines, and lipid mediators exhibit both common and distinct mechanisms for the
regulation of the neutrophil recruitment process.
In conclusion, our in vivo findings demonstrate that both
CCR1 and CCR5 are critically involved in CCL3-dependent
intravascular firm adherence and subsequent paracellular transmigration of neutrophils. In this context, leukocyte CCR1 and
nonleukocyte CCR5 are engaged, initiating intracellular signal
transduction through G protein–receptor coupling and activation
of PI3K␥. Subsequently, intravascular adherence is particularly
mediated by the ␤2 integrins LFA-1 and Mac-1, to a lesser
degree by ␣4 integrins as well as by their putative counter
receptors ICAM-1 and VCAM-1, whereas paracellular transmigration of neutrophils is facilitated through PECAM-1 and
ICAM-2. These findings corroborate the significance of C-C
motif chemokines for neutrophil recruitment, provide a rationale
of how these inflammatory mediators exert their effects, and
identify these chemotactic cytokines as well as their receptors as
potential therapeutic targets for the treatment of neutrophildriven inflammatory diseases.
889
Acknowledgments
The authors thank A. Schropp and G. Adams for technical
assistance. Data presented in this manuscript are part of the
doctoral thesis of D.P.-W.
This work was supported by Deutsche Forschungsgemeinschaft
(DFG; RE 2885-1/1 [C.A.R.]; LU612/4-3 [B.L.]; SFB 914 [C.A.R.,
F.K.]) and Friedrich-Baur-Stiftung (C.A.R.).
Authorship
Contribution: C.A.R. designed and performed experiments, and
contributed to data analysis, interpretation, and the writing of the
manuscript; D.P.-W., G.Z., B.U., N.B., and S.Z. performed experiments and contributed to data analysis, interpretation, and the
writing of the manuscript; M.P.W. and B.L. provided key reagents
and contributed to data analysis, interpretation, and the writing of
the manuscript; and F.K. contributed to data analysis, interpretation, and the writing of the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Dr Christoph A. Reichel, Walter Brendel Centre of
Experimental Medicine, Marchioninistr 15, D-81366 München, Germany; e-mail: [email protected].
References
1. Nourshargh S, Hordijk PL, Sixt M. Breaching multiple barriers: leukocyte motility through venular
walls and the interstitium. Nat Rev Mol Cell Biol.
2010;11(5):366-378.
2. Ley K, Laudanna C, Cybulsky MI, Nourshargh S.
Getting to the site of inflammation: the leukocyte
adhesion cascade updated. Nat Rev Immunol.
2007;7(9):678-689.
3. Weber C. Novel mechanistic concepts for the
control of leukocyte transmigration: specialization
of integrins, chemokines, and junctional molecules. J Mol Med. 2003;81(1):4-19.
4. Phillipson M, Kubes P. The neutrophil in vascular
inflammation. Nat Med. 2011;17(11):1381-1390.
5. Viola A, Luster AD. Chemokines and their receptors: drug targets in immunity and inflammation.
Annu Rev Pharmacol Toxicol. 2008;48:171-197.
Weber C, Soehnlein O. Hyperlipidemia-triggered
neutrophilia promotes early atherosclerosis. Circulation. 2010;122(18):1837-1845.
13. Reichel CA, Khandoga A, Anders HJ, et al.
Chemokine receptors Ccr1, Ccr2, and Ccr5 mediate neutrophil migration to postischemic tissue.
J Leukoc Biol. 2006;79(1):114-122.
14. Krishnadasan B, Farivar AS, Naidu BV, et al.
Beta-chemokine function in experimental lung
ischemia-reperfusion injury. Ann Thorac Surg.
2004;77(3):1056-1062.
15. Standiford TJ, Kunkel SL, Lukacs NW, et al. Macrophage inflammatory protein-1 alpha mediates
lung leukocyte recruitment, lung capillary leak,
and early mortality in murine endotoxemia. J Immunol. 1995;155(3):1515-1524.
6. Charo IF, Ransohoff RM. The many roles of
chemokines and chemokine receptors in inflammation. N Engl J Med. 2006;354(6):610-621.
16. Furuichi K, Gao JL, Horuk R, et al. Chemokine
receptor CCR1 regulates inflammatory cell infiltration after renal ischemia-reperfusion injury.
J Immunol. 2008;181(12):8670-8676.
7. Rot A, von Andrian UH. Chemokines in innate
and adaptive host defense: basic chemokinese
grammar for immune cells. Annu Rev Immunol.
2004;22:891-928.
17. Cheng SS, Lai JJ, Lukacs NW, Kunkel SL.
Granulocyte-macrophage colony stimulating factor up-regulates CCR1 in human neutrophils.
J Immunol. 2001;166(2):1178-1184.
8. Lukacs NW, Strieter RM, Warmington K, et al.
Differential recruitment of leukocyte populations
and alteration of airway hyperreactivity by C-C
family chemokines in allergic airway inflammation. J Immunol. 1997;158(9):4398-4404.
18. Iida S, Kohro T, Kodama T, Nagata S,
Fukunaga R. Identification of CCR2, flotillin, and
gp49B genes as new G-CSF targets during neutrophilic differentiation. J Leukoc Biol. 2005;78(2):
481-490.
9. Rot A, Krieger M, Brunner T, et al. RANTES and
macrophage inflammatory protein 1 alpha induce
the migration and activation of normal human eosinophil granulocytes. J Exp Med. 1992;176(6):
1489-1495.
19. Ottonello L, Montecucco F, Bertolotto M, et al.
CCL3 (MIP-1alpha) induces in vitro migration of
GM-CSF-primed human neutrophils via CCR5dependent activation of ERK 1/2. Cell Signal.
2005;17(3):355-363.
10. Iikura M, Ebisawa M, Yamaguchi M, et al. Transendothelial migration of human basophils. J Immunol. 2004;173(8):5189-5195.
11. Heinemann A, Hartnell A, Stubbs VE, et al. Basophil responses to chemokines are regulated by
both sequential and cooperative receptor signaling. J Immunol. 2000;165(12):7224-7233.
12. Drechsler M, Megens RT, van Zandvoort M,
20. Johnston B, Burns AR, Suematsu M, et al.
Chronic inflammation upregulates chemokine receptors and induces neutrophil migration to
monocyte chemoattractant protein-1. J Clin Invest. 1999;103(9):1269-1276.
21. Hartl D, Krauss-Etschmann S, Koller B, et al. Infiltrated neutrophils acquire novel chemokine receptor expression and chemokine responsive-
ness in chronic inflammatory lung diseases.
J Immunol. 2008;181(11):8053-8067.
22. Speyer CL, Gao H, Rancilio NJ, et al. Novel
chemokine responsiveness and mobilization of
neutrophils during sepsis. Am J Pathol. 2004;
165(6):2187-2196.
23. Edinger AL, Mankowski JL, Doranz BJ, et al.
CD4-independent, CCR5-dependent infection of
brain capillary endothelial cells by a neurovirulent
simian immunodeficiency virus strain. Proc Natl
Acad Sci U S A. 1997;94(26):14742-14747.
24. Hwang J, Son KN, Kim CW, et al. Human CC
chemokine CCL23, a ligand for CCR1, induces
endothelial cell migration and promotes angiogenesis. Cytokine. 2005;30(5):254-263.
25. Edinger AL, Amedee A, Miller K, et al. Differential
utilization of CCR5 by macrophage and T cell
tropic simian immunodeficiency virus strains.
Proc Natl Acad Sci U S A. 1997;94(8):4005-4010.
26. Alam R, Kumar D, Anderson-Walters D,
Forsythe PA. Macrophage inflammatory protein-1
alpha and monocyte chemoattractant peptide-1
elicit immediate and late cutaneous reactions and
activate murine mast cells in vivo. J Immunol.
1994;152(3):1298-1303.
27. Oliveira SH, Lukacs NW. Stem cell factor and
igE-stimulated murine mast cells produce chemokines (CCL2, CCL17, CCL22) and express
chemokine receptors. Inflamm Res. 2001;50(3):
168-174.
28. Luckow B, Joergensen J, Chilla S, et al. Reduced
intragraft mRNA expression of matrix metalloproteinases Mmp3, Mmp12, Mmp13 and Adam8,
and diminished transplant arteriosclerosis in
Ccr5-deficient mice. Eur J Immunol. 2004;34(9):
2568-2578.
29. Gao JL, Wynn TA, Chang Y, et al. Impaired host
defense, hematopoiesis, granulomatous inflammation and type 1-type 2 cytokine balance in
mice lacking CC chemokine receptor 1. J Exp
Med. 1997;185(11):1959-1968.
30. Baez S. An open cremaster muscle preparation
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
890
BLOOD, 26 JULY 2012 䡠 VOLUME 120, NUMBER 4
REICHEL et al
for the study of blood vessels by in vivo microscopy. Microvasc Res. 1973;5(3):384-394.
31. Mempel TR, Moser C, Hutter J, et al. Visualization of leukocyte transendothelial and interstitial
migration using reflected light oblique transillumination in intravital video microscopy. J Vasc Res.
2003;40(5):435-441.
32. Reichel CA, Uhl B, Lerchenberger M, et al.
Urokinase-type plasminogen activator promotes
paracellular transmigration of neutrophils via
Mac-1, but independently of urokinase-type plasminogen activator receptor. Circulation. 2011;
124(17):1848-1859.
33. Woodfin A, Reichel CA, Khandoga A, et al.
JAM-A mediates neutrophil transmigration in a
stimulus-specific manner in vivo: evidence for
sequential roles for JAM-A and PECAM-1 in neutrophil transmigration. Blood. 2007;110(6):18481856.
34. Kreisel D, Nava RG, Li W, et al. In vivo twophoton imaging reveals monocyte-dependent
neutrophil extravasation during pulmonary inflammation. Proc Natl Acad Sci U S A. 2010;107(42):
18073-18078.
35. Van RN, Sanders A. Liposome mediated deple-
tion of macrophages: mechanism of action,
preparation of liposomes and applications. J Immunol Methods. 1994;174:83-93.
36. Ramos CD, Canetti C, Souto JT, et al. MIP1alpha[CCL3] acting on the CCR1 receptor mediates neutrophil migration in immune inflammation
via sequential release of TNF-alpha and LTB4.
J Leukoc Biol. 2005;78(1):167-177.
37. Reichel CA, Rehberg M, Lerchenberger M, et al.
Ccl2 and Ccl3 mediate neutrophil recruitment via
induction of protein synthesis and generation of
lipid mediators. Arterioscler Thromb Vasc Biol.
2009;29(11):1787-1793.
38. Marone R, Cmiljanovic V, Giese B, et al. Targeting phosphoinositide 3-kinase: moving towards
therapy. Biochim Biophys Acta. 2008;1784(1):
159-185.
39. Morello F, Perino A, Hirsch E. Phosphoinositide
3-kinase signalling in the vascular system. Cardiovasc Res. 2009;82(2):261-271.
40. Henderson RB, Lim LH, Tessier PA, et al. The
use of lymphocyte function-associated antigen
(LFA)-1-deficient mice to determine the role of
LFA-1, Mac-1, and alpha4 integrin in the inflam-
matory response of neutrophils. J Exp Med.
2001;194(2):219-226.
41. Kuligowski MP, Kwan RY, Lo C, et al. Antimyeloperoxidase antibodies rapidly induce alpha-4integrin-dependent glomerular neutrophil adhesion. Blood. 2009;113(25):6485-6494.
42. Bowden RA, Ding ZM, Donnachie EM, et al. Role
of alpha4 integrin and VCAM-1 in CD18independent neutrophil migration across mouse
cardiac endothelium. Circ Res. 2002;90(5):562569.
43. Schulte D, Kuppers V, Dartsch N, et al. Stabilizing
the VE-cadherin-catenin complex blocks leukocyte extravasation and vascular permeability.
EMBO J. 2011;30(20):4157-4170.
44. Woodfin A, Voisin MB, Beyrau M, et al. The junctional adhesion molecule JAM-C regulates polarized transendothelial migration of neutrophils in
vivo. Nat Immunol. 2011;12(8):761-769.
45. Woodfin A, Voisin MB, Imhof BA, et al. Endothelial cell activation leads to neutrophil transmigration as supported by the sequential roles of
ICAM-2, JAM-A, and PECAM-1. Blood. 2009;
113(24):6246-6257.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2012 120: 880-890
doi:10.1182/blood-2012-01-402164 originally published
online June 6, 2012
C-C motif chemokine CCL3 and canonical neutrophil attractants
promote neutrophil extravasation through common and distinct
mechanisms
Christoph A. Reichel, Daniel Puhr-Westerheide, Gabriele Zuchtriegel, Bernd Uhl, Nina Berberich,
Stefan Zahler, Matthias P. Wymann, Bruno Luckow and Fritz Krombach
Updated information and services can be found at:
http://www.bloodjournal.org/content/120/4/880.full.html
Articles on similar topics can be found in the following Blood collections
Phagocytes, Granulocytes, and Myelopoiesis (616 articles)
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society
of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.