Metalloproteinases regulate CD40L shedding from platelets and pulmonary recruitment of neutrophils in abdominal sepsis.
ABSTRACT Platelets promote sepsis-induced activation of neutrophils via secretion of CD40L. However, the mechanism regulating the release of platelet-derived CD40L is not known. We hypothesized that matrix metalloproteinases (MMPs) might regulate shedding of platelet-expressed CD40L and neutrophil activation in sepsis.
Wild-type C57BL/6 mice were subjected to cecal ligation and puncture (CLP). Animals were pretreated with a broad-range MMP inhibitor, GM6001, prior to CLP induction. Edema formation, CXC chemokine and myeloperoxidase (MPO) levels and bronchoalveolar neutrophils in the lung as well as plasma levels of CD40L were quantified. Flow cytometry was used to determine expression of Mac-1 on neutrophils and CD40L on platelets. Intravital fluorescence microscopy was used to analyze leukocyte-endothelial cell interactions in the pulmonary microcirculation.
The MMP inhibitor reduced sepsis-induced release of CD40L and maintained normal levels of CD40L on platelets. Inhibition of MMP decreased CLP-induced neutrophil expression of Mac-1, formation of CXC chemokines and edema as well as neutrophil infiltration in the lung. Intravital fluorescence microscopy revealed that the MMP inhibitor attenuated leukocyte adhesion in venules whereas capillary trapping of leukocytes was not affected by MMP inhibition.
We describe a novel role of metalloproteinases in regulating platelet-dependent activation and infiltration of neutrophils in septic lung injury which might be related to controlling CD40L shedding from platelets. We conclude that targeting metalloproteinases may be a useful strategy for limiting acute lung injury in abdominal sepsis.
- SourceAvailable from: Isabel Freitas[Show abstract] [Hide abstract]
ABSTRACT: Purpose. Warm hepatic ischemia-reperfusion (I/R) injury can lead to multiorgan dysfunction. The aim of the present study was to investigate whether acute liver I/R does affect the function and/or structure of remote organs such as lung, kidney, and heart via modulation of extracellular matrix remodelling. Methods. Male Sprague-Dawley rats were subjected to 30 min partial hepatic ischemia by clamping the hepatic artery and the portal vein. After a 60 min reperfusion, liver, lung, kidney, and heart biopsies and blood samples were collected. Serum hepatic enzymes, creatinine, urea, Troponin I and TNF-alpha, and tissue matrix metalloproteinases (MMP-2, MMP-9), myeloperoxidase (MPO), malondialdehyde (MDA), and morphology were monitored. Results. Serum levels of hepatic enzymes and TNF-alpha were concomitantly increased during hepatic I/R. An increase in hepatic MMP-2 and MMP-9 activities was substantiated by tissue morphology alterations. Notably, acute hepatic I/R affect the lung inasmuch as MMP-9 activity and MPO levels were increased. No difference in MMPs and MPO was observed in kidney and heart. Conclusions. Although the underlying mechanism needs further investigation, this is the first study in which the MMP activation in a distant organ is reported; this event is probably TNF-alpha-mediated and the lung appears as the first remote organ to be involved in hepatic I/R injury.The Scientific World Journal 01/2014; 2014:867548. · 1.73 Impact Factor
Article: The role of platelets in sepsis.[Show abstract] [Hide abstract]
ABSTRACT: Platelets are small circulating anucleate cells that are of crucial importance in haemostasis. Over the last decade, it has become increasingly clear that platelets play an important role in inflammation and can influence both innate and adaptive immunity. Sepsis is a potentially lethal condition caused by detrimental host response to an invading pathogen. Dysbalanced immune response and activation of the coagulation system during sepsis are fundamental events leading to sepsis complications and organ failure. Platelets, being major effector cells in both haemostasis and inflammation, are involved in sepsis pathogenesis and contribute to sepsis complications. Platelets catalyse the development of hyperinflammation, disseminated intravascular coagulation and microthrombosis, and subsequently contribute to multiple organ failure. Inappropriate accumulation and activity of platelets are key events in the development of sepsis-related complications such as acute lung injury and acute kidney injury. Platelet activation readouts could serve as biomarkers for early sepsis recognition; inhibition of platelets in septic patients seems like an important target for immune-modulating therapy and appears promising based on animal models and retrospective human studies.Thrombosis and haemostasis. 06/2014; 112(2).
Article: Targeting neutrophils in sepsis.[Show abstract] [Hide abstract]
ABSTRACT: Sepsis continues to have a high mortality rate worldwide. The multi-step effects of this syndrome make it difficult to develop a comprehensive understanding of its pathophysiology and to identify a direct treatment. Neutrophils play a major role in controlling infection. Interestingly, the recruitment of these cells to an infection site is markedly reduced in severe sepsis. The systemic activation of Toll-like receptors and high levels of TNF-α and nitric oxide are involved in the reduction of neutrophil recruitment due to down-regulation of CXCR2 in neutrophils. By contrast, CCR2 is expressed in neutrophils after sepsis induction and contributes to their recruitment to organs far from the infection site, which contributes to organ damage. This review provides an overview of the recent advances in the understanding of the role of neutrophils in sepsis, highlighting their potential as a therapeutic target.Expert Review of Clinical Immunology 05/2014; · 2.89 Impact Factor
ORIGINAL RESEARCH PAPER
Metalloproteinases regulate CD40L shedding from platelets
and pulmonary recruitment of neutrophils in abdominal sepsis
Milladur Rahman•Jonas Roller•Su Zhang•Ingvar Syk•
Michael D. Menger•Bengt Jeppsson•Henrik Thorlacius
Received: 29 September 2011/Accepted: 28 January 2012
? Springer Basel AG 2012
neutrophils via secretion of CD40L. However, the mech-
anism regulating the release of platelet-derived CD40L is
not known. We hypothesized that matrix metalloprotein-
ases (MMPs) might regulate shedding of platelet-expressed
CD40L and neutrophil activation in sepsis.
Wild-type C57BL/6 mice were subjected to cecal
ligation and puncture (CLP). Animals were pretreated with a
Edema formation, CXC chemokine and myeloperoxidase
(MPO) levels and bronchoalveolar neutrophils in the lung as
well as plasma levels of CD40L were quantified. Flow
cytometry was used to determine expression of Mac-1 on
neutrophils and CD40L on platelets. Intravital fluorescence
microscopy was used to analyze leukocyte–endothelial cell
interactions in the pulmonary microcirculation.
The MMP inhibitor reduced sepsis-induced
release of CD40L and maintained normal levels of CD40L
on platelets. Inhibition of MMP decreased CLP-induced
neutrophil expression of Mac-1, formation of CXC che-
mokines and edema as well as neutrophil infiltration in the
lung. Intravital fluorescence microscopy revealed that the
Platelets promote sepsis-induced activation of
MMP inhibitor attenuated leukocyte adhesion in venules
whereas capillary trapping of leukocytes was not affected
by MMP inhibition.
We describe a novel role of metalloprotein-
asesin regulating platelet-dependent
infiltration of neutrophils in septic lung injury which might
conclude that targeting metalloproteinases may be a useful
strategy for limiting acute lung injury in abdominal sepsis.
Lung injury ? Sepsis
Adhesion ? Neutrophils ? Chemokines ?
Cecal ligation and puncture
Bronchoalveolar lavage fluid
Cytokine-induced neutrophil chemoattractant
Macrophage inflammatory protein-2
Abdominal sepsis remains a major cause of mortality in
intensive care units in spite of significant research efforts
[1–3]. Management of patients with sepsis is largely limited
to supportive therapies, which is partly due to an incomplete
understanding of the underlying pathophysiology. Intestinal
Responsible Editor: Artur Bauhofer.
M. Rahman ? J. Roller ? S. Zhang ? I. Syk ?
B. Jeppsson ? H. Thorlacius (&)
Department of Clinical Sciences, Malmo, Section of Surgery,
Ska ˚ne University Hospital, Lund University,
205 02 Malmo ¨, Sweden
J. Roller ? M. D. Menger
Institute for Clinical and Experimental Surgery,
University of Saarland, Homburg/Saar, Germany
perforation and contamination of the abdominal cavity with
microbes and toxins stimulate local formation of numerous
pro-inflammatory compounds, which subsequently diffuse
into the systemic circulation [4, 5]. The lung is the most
sensitive and clinically important end organ for the systemic
inflammatory response in abdominal sepsis . It is widely
held that activation of neutrophils constitutes a central
component in septic lung injury. For example, inhibition of
neutrophil recruitment by targeting adhesion molecules,
such as Mac-1 and LFA-1, effectively protects against pul-
monary damage in sepsis . CXC chemokines, including
macrophage inflammatory protein-2 (MIP-2) and cytokine-
induced neutrophil chemoattractant (KC), are primarily
responsible for coordinating accumulation of neutrophils at
sites of inflammation. However, the complex signaling
cascades triggering neutrophil activation and recruitment in
toxins are largely unknown [8, 9].
Platelets, beyond their classical role in thrombosis and
hemostasis, exert numerous effects amplifying inflamma-
tory responses and tissue injury. Recent data have
demonstrated that platelets play an important role in
abdominal sepsis  by releasing CD40L in the circula-
tion, which, in turn, provokes up-regulation of Mac-1 and
promotes pulmonary infiltration of neutrophils and tissue
damage . Indeed, a recent study demonstrated that
plasma levels of soluble CD40L are markedly elevated in
patients with sepsis . In fact, most of the soluble
CD40L in plasma is derived from platelets [11, 13].
However, the mechanisms regulating CD40L release from
(MMPs) comprise a large family of more than 25 struc-
turally and functionally related endopeptidases  with
capacity to cleave the majority of matrix proteins as well as
many non-matrix targets, such as chemokines, cytokines,
adhesion molecules and surface receptors . However,
the role of MMPs in regulating cleavage of CD40L from
platelets in abdominal sepsis remains elusive.
Based on the above, in this paper we hypothesized that
MMPs may be involved in the regulation of shedding of
platelet-expressed CD40L. More specifically, we evaluated
CLP model of abdominal sepsis in mice.
Materials and methods
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. Wild-type
male C57BL/6 (Jackson Laboratory,BarHarbor,ME,USA)
mice (20–25 g) were used in the experiments. Animals were
anaesthetized by intraperitoneal (i.p.) administration of
75 mg ketamine hydrochloride (Hoffman-La Roche, Basel,
Switzerland) and 25 mg xylazine (Janssen Pharmaceutica,
Beerse, Belgium) per kg body weight.
Experimental model of sepsis
Polymicrobial sepsis was induced by cecal ligation and
puncture (CLP) as previously described in detail . In
brief, mice were anesthetized and the abdomen was opened
to exteriorize the cecum which was filled with feces by
milking stool backwards from the ascending colon and a
ligature was placed below the ileocecal valve. Cecum was
then soaked in phosphate-buffered saline (PBS) (pH 7.4),
punctured twice with a 21-gauge needle and returned into
the peritoneal cavity. The abdominal wall was closed
with a suture and mice were resuscitated with 1 ml of
PBS subcutaneously. In order to delineate the role of
MMPs, we used a potent, broad-spectrum hydroxamic
acid inhibitor of MMPs, GM6001 (Galardin, N-[(2R)-2-
tophan methylamide; Calbiochem, Darmstadt, Germany).
GM6001 was given (40 mg/kg i.p.) 1 h before the CLP
induction. Sham mice underwent the same surgical pro-
cedures, i.e., laparotomy and resuscitation, but the cecum
was not ligated or punctured. The mice were then returned
to their cages and provided food and water ad libitum.
Animals were re-anesthetized at 6 and 24 h after CLP
induction. The left lung was ligated and excised for edema
quantification. From the right lung bronchoalveolar lavage
fluid (BALF) was collected, in which the number of neu-
trophils was determined. Next, the lung was perfused with
PBS and one part 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 ELISA and
myeloperoxidase (MPO) assays as described below.
Intravital fluorescence microscopy
In separate animals, a midline laparotomy was performed
and extended to the side along the lower border of the right
rib cage from the subxyphoidal to the midaxillary level.
Under transient lowering of the stroke volume to 100 ll,
the right diaphragm was incised to create a right-sided
pneumothorax. The diaphragm was then stepwise coagu-
lated and incised along the ventral chest wall to the
midaxillary level. A parasternal thoracotomy was per-
formed up to the level of the 4th intercostal space after
coagulating the internal mammary and the intercostal
M. Rahman et al.
vessels. By this method, the main part of the right thorax
wall could be averted to the side. During the preparation,
great care was taken not to directly manipulate the lung
tissue and the lung surface was intermittently rinsed with
saline (37?C). A micromanipulator was used to fix a cov-
erslip horizontally on the surface of the right lung.
Horizontal movements of the lung tissue could be mini-
mized by modulating a positive end-expiratory pressure
between 5 and 7 cm H2O and adjusting stroke volume
(minimum 150 ll) and stroke frequency (minimum
100 strokes/min). Immediately after surgical preparation,
the mice were put on the microscopic stage. Intravital
fluorescence microscopy was performed after retrobulbar
injection of 0.1 ml 0.1% rhodamine 6G (Sigma-Aldrich,
Taufkirchen, Germany) for direct staining of white blood
cells and 0.1 mL 5% FITC-dextran (MW 150 000, contrast
enhancement; Sigma Chemical Co., St. Louis, MO, USA).
The subpleural pulmonary microvasculature was visual-
ized by means of a modified Olympus microscope
(BX50WI, Olympus Optical Co. GmbH, Hamburg, Ger-
many) equipped with a 100 W mercury lamp and filter
sets for blue (450-490 nm excitation and [520 nm
emission wavelength) and green (530-560 nm excitation
and [580 nm emission wavelength) light epi-illumina-
tion. Microscopic images were televised by means of
a charge-coupled device video camera and recorded
digitally. By means of a 209 objective (NA 0.4) a mag-
nification of 9990 was achieved. With this setup, all parts
of the subpleural pulmonary microvasculature, i.e. arte-
rioles, venules and capillaries, could be identified. For the
measurement of 3-5 venules and capillaries, Regions of
Interest (ROIs) were selected randomly in each animal.
Leukocyte rolling was determined by counting the num-
ber of such cells passing a reference point in the venule
per 20 s. Firm adhesion was measured by counting the
number of cells adhering to the venular endothelium and
remaining stationary for 20 s.
Systemic leukocyte and platelet counts
Blood was collected from the tail vein and mixed with
Turk’s solution (0.2 mg gentian violet in 1 ml glacial
acetic acid, 6.25% v/v) in a 1:20 dilution for quantification
of polymorphonuclear leukocytes (PMNL) and monomor-
phonuclear leukocytes (MNL) or with Stromatol solution
(Mascia Brunelli SpA, Viale Monza, Milan, Italy) in a
dilution of 1:50 for identification of platelets in a Burker
The left lung was excised, washed in PBS, gently dried
using 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 an indicator
of lung edema formation.
Bronchoalveolar lavage fluid
Bronchoalveolar lavage fluid was collected by washing five
times with 1 ml of PBS containing 5 mM EDTA and was
then centrifuged; numbers of MNLs and PMNLs were
counted in a Burker chamber.
MPO in the lung tissue was assayed as described by
Asaduzamman et al. . Briefly, frozen tissue was
thawed and homogenized in 1 ml of 0.5% hexadecyl-
trimethylammonium bromide. Next, the sample was freeze-
thawed,after which the MPO activityof the supernatant was
measured. The enzyme activity was determined spectro-
540 nm, 25?C). Values were expressed as MPO units per
Plasma levels of CD40L and lung homogenate levels of
MIP-2 and KC were analyzed 6 h and 24 h after CLP,
respectively, by using commercially available ELISA kits
(R&D Systems). For soluble CD40L analysis, plasma was
collected on ice using citrate as anticoagulant and centri-
fuged for 20 min at 2,000g immediately after collection.
An additional centrifugation at 10,000g for 10 min at 4?C
was employed for complete removal of platelets and stored
at -20?C for further use. Plasma samples were then diluted
with a sterile buffer (10% fetal calf serum in PBS, pH 7.4)
and analyzed using the protocols provided.
For analysis of surface CD40L expression on platelets and
Mac-1 expression on circulating neutrophils, blood was
collected into syringes containing 1:10 acid citrate dextrose
at 6 h post CLP induction. Immediately after collection,
blood samples were incubated with an anti-CD16/CD32
antibody blocking FccIII/II receptors in order to reduce
non-specific labeling for 10 min at room temperature (RT),
and then incubated with PE-conjugated anti-Gr-1 (clone
RB6-8C5, rat IgG2b), and APC-conjugated anti-Mac-1
(clone M1/70, integrin aMchain, rat IgG2b) antibodies.
Another set of samples were stained with FITC-conjugated
anti-CD41 (clone MWReg30, integrin aIIbchain, rat IgG1)
and PE-conjugated anti-CD40L (clone MR1, hamster IgG,
MMP and abdominal sepsis
eBioscience, San Diego, CA, USA) antibodies (all anti-
bodies except where indicated were purchased from BD
Biosciences Pharmingen, San Jose, CA, USA). Cells were
fixed with 1% formaldehyde solution; erythrocytes were
lysed using FACS lysing solution (BD Biosciences
Pharmingen) and then neutrophils and platelets were
recovered following centrifugation. Flow-cytometric anal-
ysis was performed according to standard settings on a
FACScalibur flow cytometer (Becton–Dickinson, Moun-
tain View, CA, USA) and a viable gate was used to exclude
dead and fragmented cells.
Lungs samples were fixed in 4% formaldehyde phosphate
buffer overnight and then dehydrated and paraffin-embed-
ded. Six-lm sections were stained with hematoxylin and
Data are presented as mean values ± standard error 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.
MMPs regulate platelet shedding of CD40L
CLP increased plasma levels of CD40L by more than
2.5-fold, from 71 ± 6 pg/ml in the sham animals to
209 ± 23 pg/ml (Fig. 1a; P\0.05 vs. Sham, n = 5). In
parallel, surface expression of CD40L on platelets
decreased; mean fluorescent intensity (MFI) was 50 ± 4 in
sham and 29± in septic mice (Fig. 1b; P\0.05, n = 5).
Administration of GM6001, a broad-spectrum MMP inhib-
itor, reduced CLP-induced plasma levels of CD40L bymore
than 87% (Fig. 1a; P\0.05 vs. Vehicle ? CLP, n = 5).
Concomitantly, inhibition of MMP activity completely
blocked the CLP-induced reduction of surface CD40L on
platelets (Fig. 1b, c; P\0.05 vs. Vehicle ? CLP, n = 5).
Moreover, CLP caused a significant reduction in the sys-
temic number of platelets after 24 h, which was reversed to
normal levels by pretreatment with GM6001 (Table 1).
MMPs regulate Mac-1 expression on neutrophils
Induction of CLP increased surface expression of Mac-1 on
neutrophils (Fig. 2a). MFI values of Mac-1 were 734 ± 22
in Sham and 1402 ± 74 in septic mice (Fig. 2b; P\0.05
vs. Sham, n = 5). This indicates circulating neutrophils are
indeed activated in this model of sepsis. Administration of
GM6001 greatly decreased neutrophil up-regulation of
Fig. 1 MMPs regulate CLP-
induced shedding of CD40L
from platelets. Plasma levels of
soluble CD40L (a), surface
expression of CD40L on
platelets (b) and a representative
histogram (c) 6 h after CLP.
Animals were treated with
vehicle or the MMP inhibitor
GM6001 prior to CLP
animals served as negative
controls. Data represents
mean ± SEM and n = 5.
*P\0.05 versus Sham and
Vehicle ? CLP
M. Rahman et al.
Mac-1 in CLP mice (Fig. 2a). In fact, MFI values of Mac-1
on neutrophils decreased from 1402 ± 74 down to
963 ± 83 in CLP mice pretreated with the MMP inhibitor,
corresponding to a 65% reduction (Fig. 2b; P\0.05 vs.
Vehicle ? CLP, n = 5).
MMPs regulate neutrophil recruitment in the lung
Pulmonary accumulation of neutrophils was quantified by
analyzing levels of myeloperoxidase (MPO), an indicator
of neutrophils, in the lung. CLP increased MPO levels in
the lung by more than 12-fold (Fig. 3a; P\0.05 vs. Sham,
n = 5). Administration of GM6001 decreased MPO
activity in the lung by more than 56% in septic mice
(Fig. 3a; P\0.05 vs. Vehicle ? CLP, n = 5). Moreover,
cellular analysis of BALF showed that the number of
neutrophils in the bronchoalveolar space increased by
13-fold 24 h after induction of CLP (Fig. 3b; P\0.05 vs.
Sham, n = 5). Notably, inhibition of MMP activity
reduced CLP-induced recruitment of neutrophils into the
bronchoalveolar compartment from 143 ± 17 9 103down
to 50 ± 12 9 103cells, corresponding to a 70% reduction
in neutrophil accumulation (Fig. 3b; P\0.05 vs. Vehicle
? CLP, n = 5). CLP induction in mice provoked a clear-
cut leukocytopenia at 24 h. Administration of GM6001 had
no effect on the levels of circulating leukocytes in CLP
mice (Table 1).
MMPs regulate CXC chemokine formation in the lung
CXC chemokines, such as MIP-2 and KC, are known to
regulate neutrophil trafficking in the lung. Levels of MIP-2
and KC were low but detectable in sham animals (Fig. 3d).
We observed that production of CXC chemokines in the
lung was markedly increased in CLP mice (Fig. 3d;
P\0.05 vs. Sham, n = 5–6). Interestingly, it was found
that pretreatment with GM6001 reduced CLP-induced
formation of MIP-2 and KC in the lung by more than 43
and 64%, respectively (Fig. 3d; P\0.05 vs. Vehicle ?
CLP, n = 5–6).
Inhibition of MMPs protects against septic lung injury
CLP caused significant pulmonary damage, characterized
by a prominent enhancement in lung edema formation:
wet:dry ratio increased from 4.5 ± 0.1 to 5.4 ± 0.1
(Fig. 3c; P\0.05 vs. Sham, n = 5). Note that baseline
values of wet:dry ratio in sham mice represent normal
levels in healthy animals and only an increase in wet:dry
ratio represents actual edema formation. Administration of
GM6001 decreased CLP-induced lung wet:dry ratio from
5.4 ± 0.1 to 4.4 ± 0.1, corresponding to 95% reduction in
lung edema (Fig. 3c; P\0.05 vs. Vehicle ? CLP, n = 5).
Moreover, morphologic examination revealed normal
microarchitecture in lungs of sham-operated animals
(Fig. 4a), whereas CLP caused severe destruction of the
Table 1 Systemic platelet and leukocyte differential counts
Platelets MNLsPMNLs Total
Sham681 ± 53
458 ± 15a
694 ± 14b
5.2 ± 0.3
1.1 ± 0.1a
1.7 ± 0.3
0.7 ± 0.1a
6.9 ± 0.6
1.8 ± 0.2a
Vehicle ? CLP
GM6001 ? CLP1.4 ± 0.4 0.8 ± 0.12.2 ± 0.5
Blood was collected from sham-, vehicle- and GM6001-treated mice
subjected to cecal ligation and puncture (CLP) for 24 h. Cells were
identified as platelets, monomorphonuclear leukocytes (MNLs) and
polymorphonuclear leukocytes (PMNLs). Data represents mean ±
SEM, 106cells/ml and n = 5–6
aP\0.05 versus Sham
bP\0.05 versus Vehicle ? CLP
Fig. 2 MMPs regulate CLP-induced expression of Mac-1 on neu-
trophils. Mac-1 expression on neutrophils (Gr-1?cells) 6 h after
induction of CLP. A representative histogram (a) and aggregate data
(b) of Mac-1 expression on neutrophils. Animals were treated with
vehicle or the MMP inhibitor (GM6001) prior to CLP induction.
Sham-operated animals served as negative controls. Data are
representative of four other experiments (n = 5)
MMP and abdominal sepsis
pulmonary tissue structure characterized by extensive
edema of the interstitial tissue and massive infiltration of
neutrophils (Fig. 4b). Pretreatment with GM6001 markedly
reduced CLP-induced destruction of the tissue architecture
and reduced neutrophil infiltration in the lung (Fig. 4c).
MMPs regulate leukocyte adhesion in the lung
In order to study the detailed influence of MMPs on sepsis-
induced leukocyte–endothelial cell interactions in the
pulmonary microcirculation, we adopted intravital fluo-
rescence microscopy. It was found that CLP triggered a
clear-cut increase in leukocyte adhesion in capillaries and
venules of the pulmonary microvasculature (Fig. 5a, e).
Inhibition of MMP activity decreased CLP-induced leu-
kocyte adhesion in venules by 64% (Fig. 5b, d; P\0.05
vs. Vehicle ? CLP, n = 5) but had no effect on capillary
sticking of leukocytes (Fig. 5f, g). Administration of
GM6001 had no impact on leukocyte rolling in the lung
microcirculation of septic animals (Fig. 5c).
Our data show that MMPs regulate sepsis-induced lung
injury via promotion of neutrophil recruitment to the lung.
We found that MMPs reduce CD40L shedding from
platelets and up-regulation of Mac-1 on neutrophils as well
as formation of CXC chemokines in the lung. Thus, this
study demonstrates that MMPs are involved in the regu-
lation of platelet shedding of CD40L and activation of
neutrophils in septic lung damage and may be a useful
target in abdominal sepsis.
Emerging data show that platelets are not only essential
for homeostasis, thrombosis, and wound healing, but also
play an important role in inflammation and tissue injury.
We have recently demonstrated that platelets constitute a
critical component in polymicrobial sepsis by activating
and supporting neutrophil recruitment to the lung . This
platelet-dependent activation of neutrophils and subsequent
lung damage has recently been shown to be mediated by
CD40L released from platelets in abdominal sepsis . In
this context, it is also interesting to note that clinical studies
have shown that plasma levels of CD40L in the plasma are
elevated in patients with sepsis [12, 16]. However, the
mechanisms regulating sepsis-induced platelet shedding of
CD40L are not known. We focused here on the role of
MMPs and found that administration of a MMP inhibitor
reduced soluble levels of CD40L and concomitantly
increased expression of CD40L on the surface of platelets
in septic mice, suggesting that platelet shedding of CD40L
is controlled by MMPs in polymicrobial sepsis. This study
is the first to show that MMPs can regulate platelet
Fig. 3 MMPs regulate CLP-
inflammation. Lung MPO
(a) levels 6 h after CLP, number
of BALF neutrophils (b), edema
formation (c) and pulmonary
levels of CXC chemokines
(MIP-2 and KC) (d) 24 h after
CLP induction. Animals were
treated with vehicle or the MMP
inhibitor GM6001 prior to CLP
animals served as negative
controls. Data represents
mean ± SEM and n = 5–6.
*P\0.05 versus Sham and
#P\0.05 versus Vehicle?CLP
M. Rahman et al.
functions in sepsis. In this context, it should be noted that
one weakness with the present study is that we did not
study the effect of MMP inhibition on survival.
It is widely held that neutrophil recruitment causing
tissue injury and disturbed gaseous exchange is a rate-
limiting step in septic lung injury [7, 17]. In this study, it
was observed that inhibition of MMPs reduced pulmonary
infiltration of neutrophils. Additionally, we observed that
MMP inhibition not only reduced neutrophil recruitment
but also decreased sepsis-induced edema formation and
tissue destruction in the lung, suggesting that targeting
MMPs may protect against pulmonary damage in abdom-
inal sepsis. This notion is in line with most studies on MMP
inhibition in models of endotoxemia and severe infections
[18–20], although some exceptions have been reported.
Indeed, ample data show that mice challenged by different
bacteria or bacterial toxins, different doses of bacteria and
toxins and routes of administration display different phe-
notypes. Indeed, injection of single-bacteria toxins may not
represent the pathophysiology of clinical sepsis very well.
In contrast, the CLP model, in which the intestine is
punctured and the bowel contents are allowed to contam-
inate the abdominal cavity, seems to be more reminiscent
of the events and course in polymicrobial sepsis in terms of
cytokine responses and vascular and metabolic changes
[21, 22]. To study the detailed impact of MMPs on
leukocyte–endothelium interactions, we used intravital
fluorescence microscopy of the lung microcirculation. We
were able to demonstrate that MMP inhibition decreased
sepsis-induced leukocyte adhesion in venules but not cap-
illary trapping of leukocytes. Considering that venular
adhesion of leukocytes is mediated by specific adhesion
molecules and that trapping of leukocytes in capillaries is
dependent on size restrictions in the capillary lumen due to
increased stiffness of leukocytes [23–26], our data suggest
that MMPs mainly regulate the adhesion molecule-depen-
dent accumulation of leukocytes in the lung. This notion is
also supported by the observation here that inhibition of
MMPs decreased neutrophil expression of Mac-1, which is
known to mediate pulmonary recruitment of neutrophils in
abdominal sepsis . We have previously shown that
neutrophil expression of Mac-1 is dependent on platelet-
derived CD40L in polymicrobial sepsis . As mentioned
above, MMPs also abolished sepsis-triggered platelet
shedding of CD40L underling the importance of the
CD40L–Mac-1 axis in abdominal sepsis. Tissue navigation
of neutrophils is coordinated by secreted CXC chemokines
. In the present study, we observed that MMP inhibi-
tion attenuated sepsis-induced formation of MIP-2 and KC
in the lung, which may also contribute to the protective
Fig. 4 MMPs regulate CLP-induced lung damage. Representative
hematoxylin and eosin-stained sections of lung tissue. Sham-operated
animals served as negative controls (a). Animals were treated with
vehicle (b) or the MMP inhibitor GM6001 (c) prior to CLP induction
and samples were harvested 24 h later (n = 5). Scale bar indicates
MMP and abdominal sepsis
effect of MMP inhibition in septic lung damage. The
mechanism by which MMPs control CXC chemokine
formation in the lung is not known at present. However,
numerous studies have shown that MMPs can promote
Toll-like receptor (TLR)-4 activation on macrophages by
generating several different TLR4 ligands, such as soluble
CD14 , which, in turn, can trigger generation of CXC
chemokines. Thus, MMPs’ functions in sepsis are not
limited to the regulation of the CD40L–Mac-1 axis but are
most likely operating at multiple concomitant levels to
promote neutrophil accumulation at sites of inflammation.
Taken together, our results demonstrate that inhibition
of MMPs reduced platelet shedding of CD40L, Mac-1 up-
regulation on neutrophils and CXC chemokine formation
in the lung, although the relative importance of these
parameters in mediating neutrophil-dependent lung injury
remains elusive. Moreover, we found that blocking MMPs
not only decreased pulmonary infiltration of neutrophils
but also reduced lung damage in septic animals. Thus,
based on our results, we suggest that MMPs may be a
useful target for inhibiting lung damage in abdominal
Swedish Medical Research Council (2009-4872), Crafoordska stif-
telsen, Einar och Inga Nilssons stiftelse, Harald och Greta Jaenssons
stiftelse, Greta och Johan Kocks stiftelser, Fro ¨ken Agnes Nilssons
stiftelse, Franke och Margareta Bergqvists stiftelse fo ¨r fra ¨mjande av
cancerforskning, Magnus Bergvalls stiftelse, Mossfelts stiftelse,
Nanna Svartz stiftelse, Ruth och Richard Julins stiftelse, Svenska
La ¨karesa ¨llskapet, Allma ¨na sjukhusets i Malmo ¨ stiftelse fo ¨r beka ¨m-
pande av cancer, MAS fonder, Malmo ¨ University Hospital and Lund
This work was supported by grants from the
1. Cohen J. The immunopathogenesis of sepsis. Nature. 2002;420:
2. Heyland DK, Hopman W, Coo H, Tranmer J, McColl MA. Long-
term health-related quality of life in survivors of sepsis. Short
Form 36: a valid and reliable measure of health-related quality of
life. Crit Care Med. 2000;28:3599–605.
3. Martin GS, Mannino DM, Eaton S, Moss M. The epidemiology
of sepsis in the United States from 1979 through 2000. N Engl J
4. Gorbach SL, Bartlett JG. Anaerobic infections. 1. N Engl J Med.
5. Simon GL, Gorbach SL. Intestinal flora in health and disease.
Fig. 5 MMPs regulate CLP-
induced leukocyte adhesion in
the lung microvasculature.
microscopy was used to study
interactions in the pulmonary
microcirculation 6 h after CLP
induction as described in :
‘‘Materials and methods’’.
Leukocyte rolling and adhesion
in venules (a–d). The number of
trapped leukocytes was
determined in capillaries (e–g).
Animals were treated with
vehicle or the MMP inhibitor
GM6001 prior to CLP
animals served as negative
(baseline) controls. Data
represents mean ± SEM and
n = 5. *P\0.05 versus Sham
Vehicle ? CLP
M. Rahman et al.
6. Babayigit H, Kucuk C, Sozuer E, Yazici C, Kose K, Akgun H.
Protective effect of beta-glucan on lung injury after cecal ligation
and puncture in rats. Intensive Care Med. 2005;31:865–70.
7. Asaduzzaman M, Zhang S, Lavasani S, Wang Y, Thorlacius H.
LFA-1 and MAC-1 mediate pulmonary recruitment of neutrophils
and tissue damage in abdominal sepsis. Shock. 2008;30:254–9.
8. Parrillo JE. Mechanisms of disease––pathogenetic mechanisms of
septic shock. N Engl J Med. 1993;328:1471–7.
9. Remick DG. Pathophysiology of sepsis. Am J Pathol. 2007;170:
10. Asaduzzaman M, Lavasani S, Rahman M, Zhang S, Braun OO,
Jeppsson B, et al. Platelets support pulmonary recruitment of
neutrophilsinabdominal sepsis.CritCareMed. 2009;37:1389–96.
11. Rahman M, Zhang S, Chew M, Ersson A, Jeppsson B, Thorlacius
H. Platelet-derived CD40L (CD154) mediates neutrophil upreg-
ulation of Mac-1 and recruitment in septic lung injury. Ann Surg.
12. Chew M, Rahman M, Ihrman L, Erson A, Zhang S, Thorlacius H.
Soluble CD40L (CD154) is increased in patients with shock.
Inflamm Res. 2010;59:979–82.
13. Henn V, Steinbach S, Buchner K, Presek P, Kroczek RA. The
inflammatory action of CD40 ligand (CD154) expressed on
activated human platelets is temporally limited by coexpressed
CD40. Blood. 2001;98:1047–54.
14. Ra HJ, Parks WC. Control of matrix metalloproteinase catalytic
activity. Matrix Biol. 2007;26:587–96.
15. Stamenkovic I. Extracellular matrix remodelling: the role of
matrix metalloproteinases. J Pathol. 2003;200:448–64.
16. Inwald DP, Faust SN, Lister P, Peters MJ, Levin M, Heyderman
R, et al. Platelet and soluble CD40L in meningococcal sepsis.
Intensive Care Med. 2006;32:1432–7.
17. Czermak BJ, Breckwoldt M, Ravage ZB, Huber-Lang M, Schmal
H, Bless NM, et al. Mechanisms of enhanced lung injury during
sepsis. Am J Pathol. 1999;154:1057–65.
18. Cena JJ, Lalu MM, Cho WJ, Chow AK, Bagdan ML, Daniel EE,
et al. Inhibition of matrix metalloproteinase activity in vivo
protects against vascular hyporeactivity in endotoxemia. Am J
Physiol Heart Circ Physiol 298:H45–H51.
19. Maitra SR, Bhaduri S, Valane PD, Tervahartiala T, Sorsa T,
Ramamurthy N. Inhibition of matrix metalloproteinases by
chemically modified tetracyclines in sepsis. Shock. 2003;20:
20. Steinberg J, Halter J, Schiller HJ, Dasilva M, Landas S, Gatto LA,
et al. Metalloproteinase inhibition reduces lung injury and
improves survival after cecal ligation and puncture in rats. J Surg
21. Remick DG, Newcomb DE, Bolgos GL, Call DR. Comparison of
the mortality and inflammatory response of two models of sepsis:
lipopolysaccharide vs. cecal ligation and puncture. Shock.
22. Wichterman KA, Baue AE, Chaudry IH. Sepsis and septic shock–
a review of laboratory models and a proposal. J Surg Res.
23. Downey GP, Worthen GS, Henson PM, Hyde DM. Neutrophil
sequestration and migration in localized pulmonary inflamma-
tion. Capillary localization and migration across the interalveolar
septum. Am Rev Respir Dis. 1993;147:168–76.
24. Nolte D, Kuebler WM, Muller WA, Wolff KD, Messmer K.
Attenuation of leukocyte sequestration by selective blockade of
PECAM-1 or VCAM-1 in murine endotoxemia. Eur Surg Res.
25. Sikora L, Johansson AC, Rao SP, Hughes GK, Broide DH,
Sriramarao P. A murine model to study leukocyte rolling and
intravascular trafficking in lung microvessels. Am J Pathol.
26. Yoshida K, Kondo R, Wang Q, Doerschuk CM. Neutrophil
cytoskeletal rearrangements during capillary sequestration in
bacterial pneumonia in rats. Am J Respir Crit Care Med.
27. Schramm R, Thorlacius H. Staphylococcal enterotoxin B-induced
acute inflammation is inhibited by dexamethasone: important role
of CXC chemokines KC and macrophage inflammatory protein 2.
Infect Immun. 2003;71:2542–7.
28. Senft AP, Korfhagen TR, Whitsett JA, Shapiro SD, LeVine AM.
Surfactant protein-D regulates soluble CD14 through matrix
metalloproteinase-12. J Immunol. 2005;174:4953–9.
MMP and abdominal sepsis