Streptococcal M1 protein-provoked CXC chemokine formation, neutrophil recruitment and lung damage are regulated by Rho-kinase signaling.

Abstract

Streptococcal toxic shock syndrome is frequently caused by Streptococcus pyogenes of the M1 serotype. The aim of this study was to determine the role of Ras-homologous (Rho)-kinase signaling in M1 protein-provoked lung damage. Male C57BL/6 mice received the Rho-kinase-specific inhibitor Y-27632 before administration of M1 protein. Edema, neutrophil accumulation and CXC chemokines were quantified in the lung 4 h after M1 protein challenge. Flow cytometry was used to determine Mac-1 expression. Quantitative RT-PCR was used to determine gene expression of CXC chemokine mRNA in alveolar macrophages. M1 protein increased neutrophil accumulation, edema and CXC chemokine formation in the lung as well as enhanced Mac-1 expression on neutrophils. Inhibition of Rho-kinase signaling significantly reduced M1 protein-provoked neutrophil accumulation and edema formation in the lung. M1 protein-triggered pulmonary production of CXC chemokine and gene expression of CXC chemokines in alveolar macrophages was decreased by Y-27632. Moreover, Rho-kinase inhibition attenuated M1 protein-induced Mac-1 expression on neutrophils. We conclude that Rho-kinase-dependent neutrophil infiltration controls pulmonary tissue damage in response to streptococcal M1 protein and that Rho-kinase signaling regulates M1 protein-induced lung recruitment of neutrophils via the formation of CXC chemokines and Mac-1 expression.

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http://dx.doi.org/10.1159/000336182
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Streptococcal M1 protein-provoked CXC chemokine formation, neutrophil recruitment,
and lung damage are regulated by Rho-kinase signaling
Songen Zhang1, Milladur Rahman1, Su Zhang1, Heiko Herwald2, Zhongquan Qi1, Bengt Jeppsson1 ,
and Henrik Thorlacius1*
Department of Clinical Sciences, 1Section for Surgery, 20502 Malmö, 2Section for Clinical and
Experimental Infection Medicine, 22184 Lund, Lund University, Sweden

Key Words: Chemokines · Kinases · Leukocytes · Lung · Sepsis





*Correspondence and Request for Reprints to:
Henrik Thorlacius, MD, PhD
Department of Clinical Sciences, Surgery
Lund University
205 02 Malmö, SWEDEN
Telephone: Int+46-40-331000
Telefax: Int+46-40-336207
E-mail: henrik.thorlacius@med.lu.se

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Abstract
Streptococcal toxic shock syndrome is frequently caused by S. pyogenes of the M1 serotype. The
aim of this study was to determine the role of Rho-kinase signaling in M1 protein-provoked lung
damage. Male C57BL/6 mice received the Rho-kinase specific inhibitor Y-27632 before
administration of M1 protein. Edema, neutrophil accumulation and CXC chemokines were
quantified in the lung 4 h after M1 protein challenge. Flow cytometry was used to determine
Mac-1 expression. Quantitative RT-PCR was used to determine gene expression of CXC
chemokine mRNA in alveolar macrophages. M1 protein increased neutrophil accumulation,
edema and CXC chemokine formation in the lung as well as enhanced Mac-1 expression on
neutrophils. Inhibition of Rho-kinase signaling significantly reduced M1 protein-provoked
neutrophil accumulation and edema formation in the lung. M1 protein-triggered pulmonary
production of CXC chemokine and gene expression of CXC chemokines in alveolar
macrophages was decreased by Y-27632. Moreover, Rho-kinase inhibition attenuated M1
protein-induced Mac-1 expression on neutrophils. We conclude that Rho-kinase-dependent
neutrophil infiltration controls pulmonary tissue damage in response to streptococcal M1 protein
and that Rho-kinase signaling regulates M1 protein-induced lung recruitment of neutrophils via
formation of CXC chemokines and Mac-1 expression.

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Introduction
The microbial etiology of septic shock has traditionally been dominated by gram-negative
bacteria. However, a recent resurgence of Gram-positive bacterial infections has markedly
changed the microbial etiology in septic patients [1,2,3]. Streptococcus pyogenes is a common
cause of Gram-positive infections presenting as uncomplicated cases of pharyngitis to severe and
fatal conditions, such as streptococcal toxic shock syndrome (STSS). STSS is an insidious
condition associated with a mortality rate surpassing 50% [4,5,6]. S. pyogenes express a versatile
spectrum of virulence factors, such as M proteins. Up to now, more than 80 different M
serotypes have been described in S. pyogenes. Importantly, convincing data have shown that the
M1 serotype is most commonly associated with STSS [4]. M1 protein is a potent stimulator of
the innate immunity triggering neutrophil [5] and monocyte activation [7]. M1 protein forms
complexes with fibrinogen, which activate neutrophils by binding to beta2 integrins [5].
Neutrophils constitute the first line of defense against invading microorganisms but excessive
activation and infiltration of neutrophils is also known to be a rate-limiting step in acute lung
injury [8,7]. It is widely considered that the lung is the most critical organ involved in STSS
patients [9,10]. Extravascular accumulation of neutrophils at sites of inflammation is regulated
by adhesion molecules, including P-selectin and Mac-1 as well as CXC chemokines, such as
macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant
(KC) [11,12,13]. A recent study demonstrated that M1 protein-induced pulmonary infiltration of
neutrophils is critically dependent on the formation and action of CXC chemokines [14]. Thus,
the chemokine-mediated mechanisms behind accumulation of neutrophils in the lung are
relatively well known, but the signaling pathways controlling M1 protein-provoked
accumulation of neutrophils and lung injury remain elusive.

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Extracellular stress situations, such as ischemia and infection, trigger intracellular signaling
cascades converging on specific transcription factors regulating gene expression of inflammatory
mediators [15,16]. This signal transmission is largely regulated by intracellular kinases
phosphorylating down-stream targets [17]. For example, small (~21 kDa) guanosine
triphosphatases of the Ras-homologous (Rho) family and one of their effectors, Rho-kinase, are
known to act as molecular switches regulating numerous important cellular functions, such as
cytoskeleton organization, cell adhesion, reactive oxygen species formation and oncogenic
transformation [17,18]. Moreover, a previous study demonstrated that Rho-kinase is an important
regulator of chemoattractant-induced neutrophil migration in vitro [19]. Notably, Rho-kinase
inhibitors have been demonstrated to ameliorate reperfusion and endotoxemic injury in the liver
[20] as well as protecting against tissue fibrosis [21], obstructive cholestasis [22], intestinal
ischemia [23] and pulmonary hypertension [24]. Previous studies have shown that CXC
chemokine formation in acute pancreatitis [25], colonic ischemia-reperfusion [23] as well as
cholestatic [22] and endotoxemic [20] liver injury is regulated by Rho-kinase. However, the role
of Rho-kinase signaling in regulating CXC chemokine formation, neutrophil recruitment and
tissue edema in M1 protein-induced acute lung injury is not known. Another group of significant
kinases are mitogen-activated protein kinases (MAPKs), including p38 MAPK, extracellular
signal-regulated protein kinases (ERK1/2), and c-Jun NH2-terminal protein kinases (JNKs) [26].
Signal transduction through MAPKs has been shown to control production of inflammatory
cytokines and chemokines [27,28]. Notably, we have recently observed that p38 MAPK is a key
molecule in regulating M1 protein-induced neutrophil infiltration and lung damage [29].
Based on these considerations, the aim of the present study was to define the functional
significance of Rho-kinase signaling in regulating CXC chemokine production, neutrophil

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activation and recruitment as well as edema formation and p38 MAPK activity in acute lung
injury provoked by streptococcal M1 protein.

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Materials and Methods
Animals. 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. Male C57BL/6 mice weighing 23 to 25 g were
used for experiments and kept under standard laboratory conditions, maintained on a 12-12 hour
light dark cycle and fed a laboratory diet and water ad libitum. Animals were anesthetized with
7.5 mg of ketamine hydrochloride (Hoffman-La Roche, Basel, Switzerland) and 2.5 mg of
xylazine (Janssen Pharmaceutica, Beerse, Belgium) per 100 g body weight.

Experimental model. M1 protein was purified from the isogenic mutant MC25 strain (derived
from the AP1 S. pyogenes strain 40/58 from the WHO Collaborating Centre for references and
Research on Streptococci, Institute of Hygiene and Epidemiology, Prague, Czech Republic) as
described previously [5]. Mice were intravenously injected with 15 μg of M1 protein in
phosphate-buffered saline (PBS). M1 protein was purified from a mutated S. pyogenes strain [8]
making the likelihood of endotoxin contamination close to zero. Nevertheless we also measured
the endotoxin content in the M1 protein samples and confirmed that endotoxin levels were below
the detection limit. Sham mice received PBS intravenously (i.v.) only. Vehicle or the Rho-kinase
inhibitor, Y-27632 (Calbiochem, San Diego, USA), was given (0.5 or 5 mg/kg) intraperitoneally
(i.p.) 10 min prior to M1 protein challenge. Animals were re-anesthetized 4 h after M1 protein
challenge. The left lung was ligated and excised for edema measurement. The right lung was
used for collecting bronchoalveolar lavage fluid (BALF) to quantify neutrophils. Then, the lung
was excised and one lobe was fixed in formaldehyde for histology and the remaining lung tissue
was snap-frozen in liquid nitrogen and stored at -80 C for later myeloperoxidase (MPO) assays

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and enzyme-linked immunosorbent assay (ELISA) as described subsequently.

Systemic leukocyte counts. Blood was collected from the tail vein and mixed with Turks solution
(0.2 mg gentian violet in 1 ml glacial acetic acid, 6.25% v/v) in a 1:20 dilution. Leukocytes were
identified as monomorphonuclear (MNL) and polymorphonuclear (PMNL) cells in a Burker
chamber.

Lung edema. The left lung was excised, washed in PBS, gently dried using a blotting paper and
weighed. The tissue was then dried at 60 C for 72 h and re-weighed. The change in the ratio of
wet weight to dry weight was used as indicator of lung edema formation.

MPO activity. Lung tissue was thawed and homogenized in 1 ml of 0.5%
hexadecyltrimethylammonium bromide. Samples were freeze-thawed, after which the MPO
activity of the supernatant was determined spectrophotometrically as the MPO-catalysed change
in absorbance in the redox reaction of H2O2 (450 nm, with a reference filter 540 nm, 25C) as
previously described [30]. Values were expressed as MPO unit per g tissue.

ELISA. Levels of MIP-2 and KC in lung homogenates were analyzed by using double antibody
Quantikine ELISA kits (R & D Systems, Europe, Abingdon, Oxon, UK) using recombinant
murine MIP-2 and KC as standards. The lower limit of the assay was 0.5 pg/ml.

Flow cytometry. For analysis of surface molecules expression on circulating neutrophils, blood
was collected (1:10 acid citrate dextrose) 4 h after M1 protein challenge and incubated (10 min,

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RT) with an anti-CD16/CD32 antibody for blocking Fcγ III/II receptors to reduce non-specific
labeling and then incubated with PE-conjugated anti-Gr-1 (clone RB6-8C5, rat IgG2b,
eBioscience, San Diego, CA, USA), and FITC-conjugated anti-Mac-1 (clone M1/70, integrin αM
china, rat IgG2b). The mean fluorescence intensity (MFI) was determined by comparisons to an
isotype control antibody (FITC-conjugated rat IgG2b). All antibodies were purchased from BD
Biosciences Pharmingen, San Jose, CA, USA except indicated. Cells were fixed and erythrocytes
were lysed by BD lysis buffer (BD Biosciences, USA) and then neutrophils were recovered
following centrifugation. Flow cytometric analysis was performed by first gating the neutrophil
population of cells based on forward and side scatter characteristics and then Mac-1 expression
was determined on Gr-1+ cells in these gate on a FACSCalibur flow cytometer (Becton
Dickinson, Mountain View, CA, USA). A viable gate was used to exclude dead and fragmented
cells.

Histology. Lung samples were fixed in 4% formaldehyde phosphate buffer overnight and then
dehydrated and paraffin-embedded. Six m sections were stained with haematoxylin and eosin.
Lung injury was quantified in a blinded manner by adoption of a pre-existing scoring system as
described [31], including size of alveolar spaces, thickness of alveolar septas, alveolar fibrin
deposition and neutrophil infiltration graded on a 0 (absent) to 4 (extensive) scale.

In vitro activation of neutrophils. Blood was collected from healthy animals containing 1:10 acid
citrate dextrose. Whole blood was incubated with M1 protein (1 µg/ml) and Y-27632 (10 μM,
Sigma Chemical, St. Louis, MO) or vehicle at 37C for 20 min. Cells were stained for flow
cytometric analysis of Mac-1 expression on neutrophils (Gr-1+) as described above.

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Western blot. Lung sections were weighed and homogenized in lysing buffer. The samples were
centrifuged at 14000 rpm for 5 min and 25 µl of the supernatants were loaded onto SDS-
polyacrylamide gel electrophoresis and transferred onto immunoblot membranes. The
membranes were blocked with non-fat milk for 2 h and incubated with an anti-phospho-p38
MAPK monoclonal antibody (Thr180/Tyr182) or an anti-p38 MAPK antibody (Cell Signaling
Technology, Beverly, MA, USA). The membranes were then washed three times and incubated
with a horseradish peroxidase-coupled secondary antibody (Santa Cruz Biotechnology, Santa
Cruz, CA, USA) for 2 h. Blots were again washed three times and developed by the ECL®
detection system (Santa Cruz Biotechnology). The resultant signal was quantified by using
densitometer (GS-800™ Calibrated Densitometer, BIO-RAD) and the value obtained from the
sham animals were set as 100.

Quantitative RT-PCR. Alveolar macrophages were isolated as previously described [32] 30 min
after challenge with M1 protein. Total RNA was then isolated from alveolar macrophages by use
of RNeasy Mini Kit (Qiagen, West Sussex, UK) and treated with RNase-free DNase (DNase I;
Amersham Pharmacia Biotech, Sollentuna, Sweden) to remove potential genomic DNA
contaminants. RNA concentrations were determined by measuring the absorbance at 260 nm.
Each cDNA was synthesized by reverse transcription from 10 µg of total RNA by use of
StrataScript First-Strand Synthesis System and random hexamers primers (Stratagene, AH
diagnostics, Stockholm, Sweden). Real-time PCR was performed using a Brilliant SYBRgreen
QPCR master mix and MX 3000P detection system (Stratagene). The primers sequences of MIP-
2, KC and β-actin were as follows: MIP-2 (f) 5´-GCT TCC TCG GGC ACT CCA GAC-3´, MIP-

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2 (r) 5´-TTA GCC TTG CCT TTG TTC AGT AT-3´; KC (f) 5´-GCC AAT GAG CTG CGC
TGT CAA TGC-3´, KC (r) 5´-CTT GGG GAC ACC TTT TAG CAT CTT-3´; β-actin (f) 5´-
ATG TTT GAG ACC TTC AAC ACC-3´, β-actin (r) 5´-TCT CCA GGG AGG AAG AGG AT-
3´. Standard PCR curves were generated for each PCR product to establish linearity of the RT-
PCR reaction. PCR amplifications were performed in a total volume of 50 µl, containing 25 µl of
SYBRgreen PCR 2 x master mix, 2 µl of 0.15 µM each primer, 0.75 µl of reference dye, and one
1 µl cDNA as a template adjusted up to 50 µl with water. PCR reactions were started with 10 min
denaturing temperature of 95°C, followed by a total of 40 cycles (95°C for 30 s and 55°C for 1
min) and 1 min of elongation at 72°C. The relative differences in expression between groups
were expressed by using cycling time values. Cycling time values for the specific target genes
were first normalized with that of β-actin in the same sample, and then relative differences
between groups were expressed as percentage of control.

Statistics. Data are presented as mean values + standard errors of the means (SEM). Statistical
evaluations were performed using Kruskal-Wallis one-way analysis of variance on ranks
followed by multiple comparisons versus control group (Dunnett’s method). P < 0.05 was
considered significant and n represents the number of animals in each group.

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Results
Pulmanry edema and damage. Challenge with M1 protein induced clear-cut lung injury,
indicated by the significant increase in lung edema formation (Fig. 1). Thus, lung wet:dry ratio
increased in M1 protein-treated animals from 4.6  0.1 to 5.3  0.06 (Fig. 1). Administration of 5
mg/kg of the Rho-kinase inhibitor Y-27632 reduced lung wet:dry ratio to 4.9  0.05 in mice
challenged with M1 protein (Fig. 1). Although not significant, lung edema tended to be higher in
mice receiving with 5 mg/kg of Y27632 alone. Thus, inhibition of Rho-kinase signaling
decreased M1 protein-provoked lung edema by 53%. Moreover, morphologic examination
revealed normal lung microarchitecture in sham-operated mice (Fig. 2A), whereas M1 protein
caused clear-cut destruction of the lung tissue structure characterized by interstitial edema,
capillary congestion and neutrophil accumulation (Fig. 2B). It was observed that inhibition of
Rho-kinase activity reduced M1 protein-provoked changes of the microarchitecture and
neutrophil accumulation in the lung (Fig. 2C and 2D). Quantification of the morphological
changes revealed that M1 protein increased the lung injury score and that administration of the
Rho-kinase inhibitor significantly decreased the lung injury score in animals challenged with M1
protein (Fig. 2E).

Neutrophil infiltration. Injection of M1 protein increased lung levels of MPO by more than 13-
fold (Fig. 3A). Inhibition of Rho-kinase signaling reduced the M1 protein-provoked increase in
pulmonary MPO activity by 54% (Fig. 3A). Quantification of BALF neutrophils revealed a
massive enhancement in the number of alveolar neutrophils 4 h after administration of M1
protein (Fig. 3B). We observed that treatment with 5 mg/kg of Y-27632 reduced number of
pulmonary neutrophils from 96.0  6.2 x 103 to 49.6  3.7 x 103 in the lung, corresponding to a

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66% reduction, 4 h after M1 protein challenge (Fig. 3B). Moreover, it was found that
administration of M1 protein reduced the number of PMNLs and MNLs in the blood (Table 1).
Inhibition of Rho-kinase signaling significantly reduced this M1 protein-provoked leukocopenia
(Table 1).

Mac-1 expression and CXC chemokine formation. Challenge with M1 protein greatly increased
neutrophil expression of Mac-1 compared to PBS-treated control mice (Fig. 4). We found that
inhibition of Rho-kinase activity abolished M1 protein induced increases of Mac-1 expression on
the surface of neutrophils (Fig. 4), suggesting that Rho-kinase signaling controls M1 protein-
provoked expression of Mac-1 on neutrophils in vivo. In order to determine whether this
inhibitory impact of Rho-kinase inhibition is a direct or indirect effect, neutrophils were
incubated with M1 protein with or without Y-27632 in vitro. We observed that M1 protein
enhanced expression of Mac-1 on neutrophils in vitro although this increase was lower than that
observed in vivo (Fig. 5). Co-incubation with the Rho-kinase inhibitor had no impact on M1
protein-induced Mac-1 expression on neutrophils in vitro (Fig. 5), indicating that the inhibitory
effect of Y-27632 is an indirect effect in vivo. In vivo, it has recently been shown that the
CXCL2-CXCR2 axis regulates neutrophil expression of Mac-1 in M1 protein-induced
inflammation [33]. Thus, we next analyzed the role of Rho-kinase signaling in regulating
pulmonary formation of CXC chemokines in vivo. Lung levels of MIP-2 and KC were low in
sham-operated animals whereas administration of M1 protein caused a more than 116-fold
increase in CXC chemokine production in the lung (Fig. 6a). We observed that treatment with Y-
27632 dose-dependently reduced M1 protein-provoked production of MIP-2 and KC in the lung
(Fig. 6a). We next isolated alveolar macrophages from the BALF in animals challenged with M1

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protein and/or Y-27632. We observed that Y-27632 markedly reduced mRNA levels of MIP-2
and KC in the alveolar macrophages in M1 protein-treated animals (Fig. 6b).

Phosphorylation of p38 MAPK. We have recently observed that p38 MAPK activity plays a
central role in M1 protein-induced neutrophil infiltration and lung injury [27]. Next, we asked
whether Rho-kinase signaling and p38 MAPK phosphorylation might be related in M1 protein-
induced lung inflammation. Herein, it was found that administration of M1 protein enhanced p38
MAPK phosphorylation in the lung (Fig. 7). Administration of 5 mg/kg of the Rho-kinase
inhibitor significantly decreased M1 protein-induced phosphorylation of p38 MAPK (Fig. 7).

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Discussion
The present study documents that Rho-kinase signaling plays constitute a key feature in
streptococcal M1 protein-induced acute lung injury. The findings show that inhibition of Rho-
kinase decrease M1 protein-provoked production of CXC chemokines, neutrophil activation and
recruitment in the lung. In fact, it was observed that Rho-kinase inhibition not only decreased
M1 protein-induced neutrophil infiltration but also abolished edema formation and tissue damage
in the lung. Moreover, our data also indicate that phosphorylation of p38 MAPK is regulated by
Rho-kinase signaling. Thus, these novel results indicate that targeting Rho-kinase signaling
pathways may be an effective strategy to protect against acute lung damage in systemic
streptococcal infections.
Potentially fatal streptococcal infections, such as STSS, are commonly triggered by S.
pyogenes of the M1 serotype [34]. During bacterial invasion the M1 protein is shed from the
surface of S. pyogenes into the blood circulation causing widespread activation of the host innate
immune cells. Activated neutrophils and monocytes secrete massive levels of cytokines and
chemokines [6,35], provoking a systemic inflammatory response, which may cause acute lung
damage and compromised blood oxygenation, which is a feared complication in STSS.
Recruitment of neutrophils to extravascular sites of inflammation is known to be a rate-limiting
step in septic lung damage [36,37]. For example, ample data have demonstrated that
immunoneutralization of specific adhesion molecules, such as ICAM-1, Mac-1, LFA-1 and
PSGL-1 not only decreases neutrophil infiltration but also ameliorates acute lung damage in
endotoxemia and abdominal sepsis [8,38]. Herein, we found that Rho-kinase inhibition of
reduced lung MPO activity and the number of neutrophils in the bronchoalveolar space in
animals exposed to M1 protein, suggesting that Rho-kinase signaling is a significant regulator of