Immunopharmacology and Inflammation
Rho-kinase regulates adhesive and mechanical mechanisms of pulmonary
recruitment of neutrophils in abdominal sepsis
Karzan Palania, Milladur Rahmana, Zirak Hasana, Su Zhanga, Zhongquan Qib,
Bengt Jeppssona, Henrik Thorlaciusa,⁎
aDepartment of Clinical Sciences, Section of Surgery, Malmö, Lund University, 20502 Malmö, Sweden
bOrgan Transplantation Institute of Xiamen University, Xiamen City, Fujian Province, China
a b s t r a c ta r t i c l ei n f o
Received 16 November 2011
Received in revised form 3 February 2012
Accepted 8 February 2012
Available online 21 February 2012
We hypothesized that Rho-kinase signaling plays a role in mechanical and adhesive mechanisms of neutro-
phil accumulation in lung. Male C57BL/6 mice were treated with the Rho-kinase inhibitor Y-27632 prior to
cecal ligation and puncture (CLP). Lung levels of myeloperoxidase (MPO) and histological tissue damage
were determined 6 h and 24 h after CLP. Expression of Mac-1 and F-actin formation in neutrophils were
quantified by using flow cytometry 6 h after CLP. Mac-1 expression and F-actin formation were also deter-
mined in isolated neutrophils up to 3 h after stimulation with CXCL2. Labeled and activated neutrophils co-
incubated with Y-27632, an anti-Mac-1 antibody and cytochalasin B were adoptively transferred to CLP
mice. Y-27632 reduced the CLP-induced pulmonary injury and MPO activity as well as Mac-1 on neutrophils.
Neutrophil F-actin formation peaked at 6 h and returned to baseline levels 24 h after CLP induction. Rho-
kinase inhibition decreased CLP-provoked F-actin formation in neutrophils. CXCL2 rapidly increased Mac-1
expression and F-actin formation in neutrophils. Co-incubation with Y-27632 abolished CXCL2-induced
Mac-1 up-regulation and formation of F-actin in neutrophils. Notably, co-incubation with cytochalasin B
inhibited formation of F-actin but did not reduce Mac-1 expression on activated neutrophils. Adoptive trans-
fer experiments revealed that co-incubation of neutrophils with the anti-Mac-1 antibody or cytochalasin B
significantly decreased pulmonary accumulation of neutrophils in septic mice. Our data show that targeting
Rho-kinase effectively reduces neutrophil recruitment and tissue damage in abdominal sepsis. Moreover,
these findings demonstrate that Rho-kinase-dependent neutrophil accumulation in septic lung injury is
regulated by both adhesive and mechanical mechanisms.
© 2012 Elsevier B.V. All rights reserved.
Intestinal perforation is a major cause of mortality in intensive care
units and poses a significant challenge for clinicians (Cohen, 2002;
Heyland et al., 2000; Martin et al., 2003). Dissemination of bacteria
and their toxins in the abdominal cavity provokes local formation of
various pro-inflammatory compounds, which subsequently leak into
the circulation, where these mediators activate circulating neutrophils
causing a systemic inflammatory response (Aird, 2003). The lung is
the most sensitive and clinically important end organ in abdominal
sepsis. It is widely held that pulmonary recruitment of neutrophils is a
rate-limiting step in septic lung injury. For example, neutrophil deple-
tion or targeting specific adhesion molecules, such as PSGL-1 and
(Asaduzzaman et al., 2009; Zhanget al., 2011).The recruitmentprocess
of neutrophils in the lung is more complex and far less studied than in
other organs. Under homeostatic conditions, most neutrophils, which
have a diameter larger than that of pulmonary capillaries, must deform
in order to pass through the pulmonary microcirculation (Motosugi
et al., 1996). Thus, any reduction in their deformability would
promote mechanicalsequestrationof neutrophils in thelungcapillaries
(Worthen et al., 1989). Considered together, neutrophil accumulation
may depend on both adhesive and mechanical factors in the lung. On
one hand, adhesion molecules, such as selectins and integrins may
support leukocyte–endothelium interactions in the pulmonary micro-
vasculature. On the other hand, activated leukocytes may trigger
cytoskeletal changes, including polymerization of F-actin, resulting in
cell stiffening and mechanical trapping in the narrow capillaries in the
lung. Whether adhesive and mechanical mechanisms of neutrophil
accumulation operate in parallel and/or sequentially are not known.
lar signaling cascades converging on specific transcription factors regu-
lating gene expression of inflammatory mediators. This intracellular
signal transmission is largely regulated by kinases phosphorylating
down-stream targets (Itoh et al., 1999). For example, small (~21 kDa)
European Journal of Pharmacology 682 (2012) 181–187
⁎ Corresponding author at: Department of Clinical Sciences, Section of Surgery, Malmö,
Lund University, S-20502 Malmö, Sweden. Tel.: +46 40 331000(Int); fax: +46 40
E-mail address: firstname.lastname@example.org (H. Thorlacius).
0014-2999/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
Contents lists available at SciVerse ScienceDirect
European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar
guanosine triphosphatases of the Ras-homologus (Rho) family and one
of their effectors, Rho-kinase, are known to act as molecular switches
controlling several critical functions, including, cell adhesion and
ic transformation (Alblas et al., 2001; Itoh et al., 1999; Slotta et al.,
2006).Interestingly,Rho-kinaseinhibitorshave been reportedto atten-
uate ischemia/reperfusion and endotoxemic injury in the liver (Slotta
et al., 2008) as well as protecting against tissue fibrosis (Kitamura
et al.,2007),cholestasis(Laschke etal., 2010), cerebraland intestinalis-
chemia (Santen et al., 2010; Shin et al., 2007). However, the role of the
Rho-kinase signaling in regulating pulmonary recruitment of neutro-
phils and tissue damage in abdominal sepsis is not known. Moreover,
the influence of Rho-kinase inhibition on adhesive and mechanical
aspect of sepsis-induced neutrophil accumulation in the lung remains
Based on these considerations, the aim of the present study was to
define the functional role of Rho-kinase signaling in regulating F-actin
polymerization and Mac-1 expression in neutrophils as well as their
role in the regulation of pulmonary recruitment of neutrophils and
tissue damage in sepsis. For this purpose, we used a model of polymi-
crobial sepsis based on intestinal perforation in mice.
2. Materials and methods
in filter-top cages under standardized laboratory conditions. Mice were
witha12 hlight/12 hdarkdiurnalcyclewithfoodandwateradlibitum.
All experimental procedures were performed in accordance with the
legislation on the protection of animals and were approved by the
Regional Ethical Committee for Animal Experimentation at Lund
University, Sweden. Animals were anesthetized by administration of
7.5 mg (i.p.) ketamine hydrochloride (Hoffman-La Roche, Basel,
Switzerland) and 2.5 mg(i.p.) xylazine(Janssen Pharmaceutica,Beerse,
Belgium) per 100 g body weight.
2.2. Experimental protocols
Polymicrobial sepsis was induced by cecal ligation and puncture
(CLP) as described previously (Asaduzzaman et al., 2008). Shortly,
through laparotomy incision, the exposed cecum was filled with feces
by milking stool backward from the ascending colon, and a ligature
was placed below the ileocecal valve. The cecum was soaked with
phosphate-buffered saline (PBS; pH 7.4) and punctured twice with a
21-gauge needle. The cecum was then pushed back into the abdominal
cavity and the incision was sutured. To determine the role of
Rho-kinase, vehicle (PBS) or the Rho-kinase inhibitor, Y-27632
(Calbiochem,SanDiego,USA), wasgiven(5.0 mg/kg) i.p. 30 min before
CLP induction. The dose of 5 mg/kg was chosen based on a previous
study (Awla,etal.,2011).Shammice underwentthe samesurgicalpro-
cedures, but the cecum was neither ligated nor punctured. The mice
were then returned to their cages and provided food and water.
Animals were re-anesthetized 3, 6, 12 and 24 h after CLP induction.
The lung was perfused with PBS, left lung was fixed in formaldehyde
for histology, and the remaining lung tissue was weighed, snap-frozen
in liquid nitrogen, and stored at −80°C for later myeloperoxidase
(MPO) assays as described below.
2.3. Systemic leukocyte count
Blood was collected from tail vein and was mixed with Turk's
solution (0.2 mggentian violet in 1 ml glacial acetic acid; 6.25% vol/vol)
in a 1:20 dilution. Leukocytes were identified and counted as
monomorphonuclear (MNL) and polymorphonuclear (PMNL) leuko-
cyte cells in a Burker chamber.
2.4. MPO activity
The enzyme MPO is abundant in neutrophils and has been used as
reliable marker for detection of neutrophil accumulation in inflamed
tissue and was quantified as described previously (Bradley et al.,
1982). In brief, frozen lung tissue was thawed and homogenized in
1 ml of 0.5% hexadecyltrimethylammonium bromide. Next, the sample
was freeze-thawed, after which the MPO activity of the supernatant
was measured. The enzyme activity was determined spectrophotomet-
of H2O2(450 nm, with a reference filter of 540 nm; 25 °C). Values were
expressed as MPO units per gram of tissue.
night and then dehydrated and paraffin-embedded. Six-micrometer
sections were stained with hematoxylin and eosin. Lung injury was
quantified in a blinded manner by adoption of a pre-existing scoring
system as described (Borzone et al., 2007), including size of alveolar
spaces , thickness of alveolar septas, alveolar fibrin deposition and
neutrophil infiltration, graded on a 1 (absent) to 4 (extensive) scale.
The total injury score was expressed as the sum of the four scores.
2.6. Flow cytometry
For detection and analysis of Mac-1 expression on circulating neu-
trophils, blood was collected into syringes containing 1:10 acid citrate
dextrose (22.0 g/L sodium citrate; 7.3 g/L citric acid, anhydrous; and
24.5 g/L dextrose) 3, 6, 12 and 24 h after CLP induction. Blood samples
were incubated with an anti-CD16/CD32 antibody (10 min at room
temperature) blocking Fcγ III/II receptors to reduce non-specific la-
beling and then incubated with PE-conjugated anti-Gr-1 (Clone
RB6-8C5, rat IgG2b, eBioscience, San Diego, USA), and FITC-
conjugated anti-Mac-1 (Clone M1/70, integrin αMchain, rat IgG2b)
antibodies. To determine the distribution of F-actin content within
neutrophils, another set of samples was permeabilized by L-
lysophosphatidylcholine (LPC, lysolecithin, 1-O-acyl-sn-glycero-3-
phophocholine, L-lysophosphatidylcholine-gamma-Oacyl; Sigma, St.
Louis , MO, USA) and then stained with BODIPY FL phallicidin (Molec-
ular Probes, Invitrogen, Eugene, USA) and APC-conjugated anti-Gr-1
(Clone RB6-8 C5, rat anti-mouse Ly-6G and Ly-6C) antibodies (all an-
tibodies except those indicated were purchased from BD Biosciences
Pharmingen, San Jose, CA, USA). Cells were fixed and erythrocytes
were lysed using FACS lysing solution (BD Biosciences Pharmingen,
San Jose, CA, USA) and then neutrophils were recovered following
centrifugation. Flow cytometric analysis was performed according to
standard settings on a FACSCalibur flow cytometer (Becton Dickinson,
Mountain View, CA, USA) and a viable gate was used to exclude dead
and fragmented cells.
2.7. In vitro studies
Neutrophils were freshly isolated from healthy mice by aseptically
flushing the bone marrow of femurs and tibias from healthy mice by
using Ficoll-Paque™ Research Grade (Amersham Pharmacia Biotech,
Uppsala, Sweden). The purity of the isolated neutrophils was more
than 70% as assessed in a haematocytometer. Leukocytes were then
re-suspended in PBS to 10×106/ml and co-incubated with 300 ng/
ml recombinant mouse CXCL2/MIP2 (R&D Systems, Inc., Minneapolis,
USA) for different time points. Two set of samples were pre-incubated
with Y-27632 (100 μM/100 μl) 20 min before activation by CXCL2 for
measuring F-actin content within neutrophils and Mac-1 expression.
K. Palani et al. / European Journal of Pharmacology 682 (2012) 181–187
Cells are permeabilized, stained and fixed as described above. Finally,
cells were analyzed by flow cytometry (FACSCalibur). Mac-1 expres-
sion on neutrophils and F-actin content within neutrophils were
measured in separate experiments. Lastly effects of cytochalasin B
10 μM (cytochalasin B isolated from Drechslera dematioidea; Sigma,
Sweden) on F-actin and Mac-1 were checked in two different sets of
2.8. Adoptive transfer of neutrophils
Again bone marrow leukocytes were freshly extracted from healthy
mice by using Ficoll-Paque™ as described above. Leukocytes were then
re-suspended in PBS to 10×106/ml and co-incubated with 300 ng/ml
recombinantmouse CXCL2for 10 min and 180 min separately; further-
more leukocytes were pre-incubated with cytochalasin B 10 μM 30 min
and anti-Mac-1 (Purified anti-mouse CD11b, NA/LE, from BD Biosci-
ences) for 15–20 min at room temperature before challenge with
CXCL2 co-incubation. Samples were stained with 20 μM CFDA-SE
(carboxyfluorescein diacetate-succinimidyl ester, Invitrogen, Paisley,
UK) and for 1 h at 37 °C. CFDA-SE passively diffuses into cells and is
non-fluorescent until its acetate groups are cleaved by intracellular
phils were injected intravenously into mice immediately prior to CLP,
then 4 h after CLP induced, lungs were harvested, minced, and digested
for 1 h at 37 °C in buffer containing 20 U/ml collagenase A (Sigma).
Single-cell suspensions were obtained by straining the digested tissue
through a 40-μm mesh. Cells were labeled with an APC-labeled anti-
Gr-1 antibody and fixed as described above. Finally, cells were analyzed
by flow cytometry (FACSCalibur). Lung recruitment of transferred
neutrophils were quantified by dividing the number of CFDA+/Gr-1+
cells by the number of CFDA−/Gr-1+cells in the lung extracts.
Data are presented as mean values±S.E.M. (Standard error of the
mean). Statistical evaluations were performed by using Kruskal–Wallis
one-way analysis of variance on ranks followed by multiple compari-
sons versus control group (Dunnett's method). Pb0.05 was considered
significant and n represents the number of animals in each group.
3.1. Rho-kinase activity regulates lung tissue damage
CLP caused significant pulmonary damage, characterized by severe
destruction of pulmonary tissue microstructure, extensive edema of
interstitial tissue, necrosis and massive infiltration of neutrophils (not
shown). Quantification of the morphological damage showed that CLP
enhanced the lung injury score and that pretreatment with the
Rho-kinase inhibitor Y-27632 significantly reduced the lung injury
score in CLP mice (Fig. 1A; Pb0.008 vs. vehicle+CLP, n=5). Moreover,
leukocytopenia was observed 24 h after CLP induction (Table 1). For
example, the number of neutrophils decreased by 53% 24 h after CLP
(Table 1; Pb0.05 vs. sham, n=5). Notably, this CLP-induced neutrope-
nia was significantly decreased in mice pretreated with Y-27632
(Table 1; Pb0.05 vs. vehicle+CLP, n=5).
3.2. Mac-1 expression and neutrophil accumulation are regulated by
levels of myeloperoxidase (MPO), an indicator of neutrophils in the
lung. It was found that CLP increased MPO activity in the lung by
11-fold (Fig. 1B; Pb0.008 vs. sham, n=5). Notably, Rho-kinase inhibi-
tion reduced MPO activity in the lung by 54% in septic mice (Fig. 1B;
Pb0.008 vs. vehicle+CLP, n=5). Mac-1 expression was increased on
Fig. 1. (A) Evaluation of lung injury at 24 h as described in Materials and methods.
(B) Lung MPO activity, a marker of neutrophils in the lung 6 h post-CLP. (C) Rho-kinase
activity regulates Mac-1 expression on neutrophils. Sham animals served as a negative
control and separate groups of micewere pretreated with vehicle and Y-27632before op-
eration. Data represents mean±S.E.M. and n=5. *Pb0.05 vs. sham and
Systemic leukocyte differential counts.
Blood was collected from vehicle and Y-27632 treated mice exposed to cecal ligation
and puncture (CLP) for 24 h as well as sham-operated animals. Cells were identified
as monomorphonuclear leukocytes (MNLs) and polymorphonuclear leukocytes
(PMNLs). Data represents mean ± S.E.M. x 106cells/ml and n=5.
aPb0.05 vs. sham.
bPb0.05 vs. vehicle+CLP.
K. Palani et al. / European Journal of Pharmacology 682 (2012) 181–187
the surface of neutrophils in CLP animals, indicating that circulating
neutrophils were activated in this sepsis model. Inhibition of Rho-
kinase activity markedly antagonized CLP-induced up-regulation of
Mac-1 on the surface of neutrophils. MFI (mean fluorescence intensity)
values of Mac-1 on neutrophils decreased from 285±16 down to
138±15 in CLP mice pretreated with Y-27632, corresponding to a
63% reduction (Fig. 1C; Pb0.029 vs. vehicle+CLP, n=5).
3.3. Rho-kinase activity regulates neutrophil formation of F-actin
We first analyzed the F-actin content of neutrophils at different
time points after CLP induction. F-actin formation in neutrophils in-
creased rapidly in septic animals, i.e. MFI values of F-actin increased
from 122±3 at baseline to 264±24 3 h after CLP (Fig. 2A; Pb0.008
vs. sham, n=5). Maximum levels of F-actin were observed 6 h after in-
duction of CLP when MFI values of F-actin reached 270±26 (Fig. 2A;
Pb0.008 vs. sham, n=5). F-actin formation in neutrophils returned to
baselinelevels24 hafterCLPinduction(Fig. 2A).Nextweasked wheth-
er inhibition of Rho-kinase activity mightinfluenceF-actin formation in
neutrophils. Administration of Y-27632 decreased the CLP-provoked
formation of F-actin in neutrophils (Fig. 2B). Indeed, inhibition of Rho-
kinase activity reduced neutrophil formation of F-actin by 68% in septic
mice (Fig. 2C; Pb0.001 vs. vehicle+CLP, n=5).
3.4. CXCL2-induced F-actin formation and Mac-1 expression in vitro
F-actin formation and Mac-1 expression were determined in isolat-
ed neutrophils after stimulation withCXCL2, whichis a potent activator
of neutrophils. Challenge with CXCL2 caused a rapid increase in F-actin
formation in neutrophils peaking at 10 min and returning to baseline
levels after 180 min (Fig. 3A). Neutrophil expression of Mac-1 also
peaked at 10 min but remained elevated compared to baseline values
180 min after stimulation with CXCL2 (Fig. 3B). It was found that
co-incubation of neutrophils with Y-27632 abolished CXCL2-induced
formation of F-actin and expression of Mac-1 in neutrophils (Table 2).
Interestingly, co-incubation of neutrophils with cytochalasin B, a well-
known inhibitor of F-actin polymerization, also abolished CXCL2 trig-
gered F-actin formation (Table 2; Pb0.008 vs. vehicle+CXCL2, n=5)
but had no effect on surface expression of Mac-1 (Table 2) on
3.5. Adhesive and mechanical mechanisms of neutrophil accumulation
In order to discriminate the influence of adhesive and mechanical
mechanisms in the accumulation process of neutrophils in septic lung
damage, we performed adoptive transfer of CFDA-labeled and CXCL2
activated neutrophils co-incubated with or without the anti-Mac-1
antibodyandcytochalasinB.Inlinewiththekinetic experiments show-
ing maximal expression of Mac-1 and F-actin formation in neutrophils
10 min after challenge with CXCL2, we observed that homing of adop-
tively transferred neutrophils was maximal when the cells had been
stimulated with CXCL2 for 10 min (Fig. 4A; Pb0.010 vs. sham, n=6).
It was found that homing of CFDA-labeled and activated neutrophils
to the lung in CLP mice were markedly reduced when neutrophils
were co-incubated with the anti-Mac-1 antibody (Fig. 4B). Notably,
co-incubation of labeled and activated neutrophils with cytochalasin
pulmonary accumulationof thesecells in CLP animals(Fig. 4C; Pb0.001
vs. vehicle+CXCL2, n=5).
It is widely held that systemic activation and pulmonary accumula-
tion of neutrophils are key features in sepsis. However, the signaling
pathways regulating neutrophil activation remain elusive. In the pre-
sent study, we show that the Rho-kinase inhibitor Y-27632 greatly
reduced pulmonary levels of MPO, an indicator of neutrophils, suggest-
ing that Rho-kinase controls neutrophil accumulation in septic lung
injury. Moreover, inhibition of Rho-kinase activity not only decreased
pulmonary neutrophilia but also attenuated lung tissue damage in
abdominal sepsis. Considering the close relationship between neutro-
phil recruitment and tissue damage in septic pulmonary injury
accumulation of neutrophils in the lung. These findings are in line with
sue injury in the liver and lung triggered by endotoxin (Lomas-Neira
et al., 2006; Thorlacius et al., 2006). Although toxin-based models are
the complex events and course in polymicrobial sepsis in terms of
Fig. 2. CLP rapidly increased F-actin content of neutrophils and reached maximum levels
6 h after CLP. F-actin content (MFI) of the neutrophils shown on the Y-axis against time
after CLP induction on the X-axis (a). Pre-treatment with Y-27632 significantly reduced
F-actin content at 6 h CLP (b) and (c). Data represents mean±S.E.M. and n=5. *Pb0.05
vs. sham and#Pb0.05 vs. vehicle+CLP.
K. Palani et al. / European Journal of Pharmacology 682 (2012) 181–187
cytokine responses as well as vascular and metabolic changes
(Klintman et al., 2004b; Remick et al., 2000; Wichterman et al., 1980).
Moreover, different toxins activate the host immune system in a
distinctly different manner. For example, LPS has been shown to be a
potent activator of macrophages and stimulates TNF-α production
(Ulevitch et al., 1990; Wright et al., 1990) whereas superantigens do
not provoke clear-cut TNF-α formation and activates primarily
T-lymphocytes causing FasL-dependent apoptosis (Klintman et al.,
2004a). Nonetheless, considered together with our present findings, it
may be forwarded that Rho-kinase signalling is a key feature in acute
Leukocyte recruitment in the lung is far less studied and appears to
be much more complex than that in other organs. One reason for this is
that the spherical diameter of the capillaries in the lung is smaller
(6 μm) than that of neutrophils (7 μm) which forces neutrophils to
deform and make them prone to mechanical trapping when passing
through the narrow lung capillaries (Motosugi et al., 1996). Thus,
cytoskeletal rearrangements causing any reduction in neutrophil
deformability will increase theirsequestration in thepulmonary micro-
trapping of neutrophils in the lung has been confirmed in a number of
studies (Downey et al., 1991; Frank, 1990; Saito et al., 2002). Therefore,
it was of great interest to study F-actin formation in circulating neutro-
phils in septic mice herein. We observed that F-actin markedly in-
creased in circulating neutrophils in mice with ongoing abdominal
sepsis. Moreover, this sespis-induced neutrophil F-actin formation
was abolished by administration of Y-27632, indicating that polymeri-
zation of F-actin in neutrophils is regulated by Rho-kinase signaling in
abdominal sepsis. In contrast, Tasaka et al. (2005) reported that
neutrophil formation of F-actin triggered by fMLP, a peptide from
Escherichai coli bacteria, was insensitive to treatment with Y-27632.
The reason behind these apparent discrepancies is not known.
sis in vivo is likely different from that induced by bacterial proteins in
vitro. For instance, it is well known that mechanisms of neutrophil stiff-
et al., 2002) and LPS (Erzurum et al., 1992). In addition, our findings are
ing pathway controls polymerization of F-actin in neutrophils
flammation is a marked decrease in the number of circulating neutro-
phils due to mechanical sequestration in the lung (Andonegui et al.,
Fig. 3. In vitro kinetics of F-actin content (MFI) and Mac-1 expression (MFI). Shown on
the Y-axis against time after CXCL2 activation on the X-axis. F-actin peaked at 10 min
and reduced at 180 min (a). Mac-1 expression peaked at 10 min again but remained
relatively high at 180 min (b). Data represents mean±S.E.M. and n=5.
Fig. 4. (a) Adoptive transfer of CFDA-labeled neutrophils. Bone marrow neutrophils were
labeled with CFDA and 2×106neutrophils and incubated with CXCL2 for 10 min and
180 min and then adoptively transferred to CLP mice, Data represents mean±S.E.M.
and n=6. *Pb0.05 vs. control and#Pb0.05 vs. 10 min+CLP. CFDA-labeled neutrophils
activated with CXCL2 for 10 min were co-incubated with an anti-Mac-1 (b) or cytochala-
sin B (c) prior to injection. Accumulation of transferred neutrophils was determined by
quantifying the number of neutrophils (Gr-1+ cells) in lung labeled with CFDA divided
by the total number of neutrophils (Gr-1+ cells). Data represents mean±S.E.M. and
n=5. *Pb0.05 vs. control and#Pb0.05 vs. vehicle+CXCL2.
K. Palani et al. / European Journal of Pharmacology 682 (2012) 181–187
2003). Indeed, we also found that systemic neutrophil counts were sig-
nificantly reduced in CLP mice. It is therefore interesting to note that ad-
ministrationof Y-27632 reduced
circulating neutrophils, which further lends support to the concept that
Rho-kinase signaling regulates neutrophil stiffening in abdominal sepsis.
Besides mechanical trapping, leukocytes are also recruited in the
lung byspecific adhesion molecules expressed on leukocytesand endo-
thelial cells. For example, it has been shown that PSGL-1, LFA-1 and
Mac-1 on neutrophils and ICAM-1 on endothelial cells support pulmo-
nary accumulation of neutrophils in abdominal sepsis (Asaduzzaman
et al., 2008, 2009; Hildebrand et al., 2005). Herein, we found that
expression of Mac-1 was markedly increased on the surface of circulat-
ing neutrophils in septic mice. Moreover, administration of Y-27632
abolished sepsis-induced Mac-1 expression on neutrophils, indicating
that Rho-kinase regulates Mac-1 up-regulation on neutrophils in ab-
dominal sepsis. Considered together, our findings suggest that Rho-
kinase may control sepsis-evoked neutrophil recruitment in the lung
the relative role of adhesive and mechanical mechanisms in this Rho-
kinase-dependent neutrophil recruitment in the lung, we stimulated
isolated neutrophils with CXCL2 in vitro. We observed that CXCL2
increased Mac-1 expression and F-actin formation in neutrophils in a
time-dependent manner peaking 10 min after challenge. It was found
that Y-27632 reduced CXCL2-induced neutrophil up-regulation of
Mac-1 as well as F-actin formation. In contrast, cytochalasin B, a well-
known inhibitor of F-actin polymerization (Cooper, 1987), abolished
F-actin formation but had noeffectonMac-1 expression on neutrophils
in response to CXCL2 stimulation. Next, we adoptively transferred iso-
lated and labeled neutrophils activated with CXCL2 for 10 min to mice
with ongoing abdominal sepsis and determined their accumulation in
the lung. We found that co-incubation of isolated neutrophils with an
antibody directed against Mac-1 markedly reduced pulmonary recruit-
1 expression but abolished F-actin formation,alsodecreasedneutrophil
accumulation in the lungs of septic mice. Taken together, our results
show for the first time that both adhesive and mechanical mechanisms
mediate neutrophil recruitment in the lung. Considering that Y-27632
decreased both Mac-1 up-regulation and F-actin formation in neutro-
phils, it may be suggested that Rho-kinase signaling regulates both ad-
hesive and mechanical aspects of pulmonary accumulation of
neutrophils in abdominal sepsis
Our results show that Rho-kinase signaling plays an important role
in septic lung damage. Moreover, these findings demonstrate that
Rho-kinase-dependent accumulation of neutrophils in lung is com-
posed of both adhesive (Mac-1) and mechanical (F-actin) components
ing the Rho-kinase signaling pathway may be a useful strategy to
protect against sepsis-induced lung injury.
This study was supported by the Swedish Medical Research Council
(2009-4872), Crafoordska stiftelsen, Einar och Inga Nilssons stiftelse,
Greta och Johan Kocks stiftelser, Fröken Agnes Nilssons stiftelse, Magnus
Bergvalls stiftelse, Mossfelts stiftelse, Nanna Svartz stiftelse, Ruth och
Richard Julins stiftelse, Dir. A. Påhlsson's Foundation, Swedish Cancer
Foundation, Malmö University Hospital Cancer Foundation, Lundgren's
Foundation, Gunnar Nilsson's Foundation and Apotekaren Hedberg's
Fond, Malmö University Hospital and Lund University. K.P. and Z.H. are
supported by a fellowship from Ministry of Higher Education from the
Kurdistan regional government.
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