CB2 cannabinoid receptors contribute to bacterial invasion and mortality in polymicrobial sepsis.
ABSTRACT Sepsis is a major healthcare problem and current estimates suggest that the incidence of sepsis is approximately 750,000 annually. Sepsis is caused by an inability of the immune system to eliminate invading pathogens. It was recently proposed that endogenous mediators produced during sepsis can contribute to the immune dysfunction that is observed in sepsis. Endocannabinoids that are produced excessively in sepsis are potential factors leading to immune dysfunction, because they suppress immune cell function by binding to G-protein-coupled CB(2) receptors on immune cells. Here we examined the role of CB(2) receptors in regulating the host's response to sepsis.
The role of CB(2) receptors was studied by subjecting CB(2) receptor wild-type and knockout mice to bacterial sepsis induced by cecal ligation and puncture. We report that CB(2) receptor inactivation by knockout decreases sepsis-induced mortality, and bacterial translocation into the bloodstream of septic animals. Furthermore, CB(2) receptor inactivation decreases kidney and muscle injury, suppresses splenic nuclear factor (NF)-kappaB activation, and diminishes the production of IL-10, IL-6 and MIP-2. Finally, CB(2) receptor deficiency prevents apoptosis in lymphoid organs and augments the number of CD11b(+) and CD19(+) cells during CLP.
Taken together, our results establish for the first time that CB(2) receptors are important contributors to septic immune dysfunction and mortality, indicating that CB(2) receptors may be therapeutically targeted for the benefit of patients suffering from sepsis.
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ABSTRACT: The purpose of this review is to illustrate the expanding view of the endocannabinoid system (ECS) in relation to its roles in inflammation. According to the formal classification, the ECS consists of two cannabinoid receptors, their endogenous fatty acid-derived ligands, and a number of enzymes involved in their synthesis and breakdown. However, many endogenous congeners of classical endocannabinoids have now been discovered, together with a set of receptors structurally or functionally related to the cannabinoid receptors. Endocannabinoids per se behave 'promiscuously' with regard to their receptor interactions. It is increasingly recognized how tightly this expanded ECS is intertwined with key processes involved in inflammation. A continuous dynamic exchange of substrates and metabolites exists between ECS and eicosanoid pathways. Endocannabinoids can also be oxygenated by cyclooxygenase and other enzymes to biologically active 'hybrid' structures. Diet is among the main factors determining synthesis and release of endocannabinoids and related mediators. The complexity of what may be called the 'endocannabinoidome' requires approaches that take into account its dynamics and interconnections with other regulatory systems. This endocannabinoidome continues to offer possibilities for prevention and intervention, but multiple target approaches will probably provide the only keys to success.Current opinion in clinical nutrition and metabolic care. 01/2014;
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ABSTRACT: The systemic inflammatory response syndrome can be self-limited or can progress to severe sepsis and septic shock. Despite significant advances in the understanding of the molecular and cellular mechanisms of septic shock, it is still one of the most frequent and serious problems confronting clinicians in the treatments. And the effects of cannabinoid receptor 2 (CB2R) on the sepsis still remain undefined. The present study was aimed to explore the role and mechanism of CB2R in acute sepsis model of mice. Here, we found that mice were more vulnerable for lipopolysaccharide- (LPS-) induced death and inflammation after CB2R deletion (CB2R(-/-)). CB2R agonist, GW405833, could significantly extend the survival rate and decrease serum proinflammatory cytokines in LPS-treated mice. GW405833 dose-dependently inhibits proinflammatory cytokines release in splenocytes and peritoneal macrophages as well as splenocytes proliferation, and these effects were partly abolished in CB2R(-/-) splenocytes but completely abolished in CB2R(-/-) peritoneal macrophages. Further studies showed that GW405833 inhibits LPS-induced phosphorylation of ERK1/2 and STAT3 and blocks I κ B α degradation and NF- κ B p65 nuclear translocation in macrophages. All data together showed that CB2R provides a protection and is a potential therapeutic target for the sepsis.Mediators of Inflammation 01/2013; 2013:741303. · 2.42 Impact Factor
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ABSTRACT: Sepsis is a complex condition that results from a dysregulated immune system in response to a systemic infection. Current treatments lack effectiveness in reducing the incidence and mortality associated with this disease. The endocannabinoid system offers great promise in managing sepsis pathogenesis due to its unique characteristics. The present study explored the effect of modulating the CB2 receptor pathway in an acute sepsis mouse model. Endotoxemia was induced by intravenous injection of lipopolysaccharide (LPS) in mice and intestinal microcirculation was assessed through intravital microscopy. We found that HU308 (CB2 receptor agonist) reduced the number of adherent leukocytes in submucosal venules but did not restore muscular and mucosal villi FCD in endotoxemic mice. AM630 (CB2 receptor antagonist) maintained the level of adherent leukocytes induced by LPS but further reduced muscular and mucosal villi FCD. URB597 (FAAH inhibitor) and JZL184 (MAGL inhibitor) both reduced the number of adherent leukocytes in submucosal venules but did not restore the mucosal villi FCD. Using various compounds we have shown different mechanisms of activating CB2 receptors to reduce leukocyte endothelial interactions in order to prevent further inflammatory damage during sepsis.Mediators of Inflammation 01/2014; 2014:978678. · 2.42 Impact Factor
CB2Cannabinoid Receptors Contribute to Bacterial
Invasion and Mortality in Polymicrobial Sepsis
Bala ´zs Cso ´ka1, Zolta ´n H. Ne ´meth1,2, Partha Mukhopadhyay3, Zolta ´n Spolarics1, Mohanraj Rajesh3,
Stephanie Federici1, Edwin A. Deitch1, Sa ´ndor Ba ´tkai3, Pa ´l Pacher3*, Gyo ¨rgy Hasko ´1*
1Department of Surgery, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, New Jersey, United States of America, 2Department of
Surgery, Morristown Memorial Hospital, Morristown, New Jersey, United States of America, 3National Institute on Alcohol Abuse and Alcoholism, Bethesda, Maryland,
United States of America
Background: Sepsis is a major healthcare problem and current estimates suggest that the incidence of sepsis is
approximately 750,000 annually. Sepsis is caused by an inability of the immune system to eliminate invading pathogens. It
was recently proposed that endogenous mediators produced during sepsis can contribute to the immune dysfunction that
is observed in sepsis. Endocannabinoids that are produced excessively in sepsis are potential factors leading to immune
dysfunction, because they suppress immune cell function by binding to G-protein-coupled CB2receptors on immune cells.
Here we examined the role of CB2receptors in regulating the host’s response to sepsis.
Methods and Findings: The role of CB2receptors was studied by subjecting CB2receptor wild-type and knockout mice to
bacterial sepsis induced by cecal ligation and puncture. We report that CB2receptor inactivation by knockout decreases
sepsis-induced mortality, and bacterial translocation into the bloodstream of septic animals. Furthermore, CB2receptor
inactivation decreases kidney and muscle injury, suppresses splenic nuclear factor (NF)-kB activation, and diminishes the
production of IL-10, IL-6 and MIP-2. Finally, CB2receptor deficiency prevents apoptosis in lymphoid organs and augments
the number of CD11b+and CD19+cells during CLP.
Conclusions: Taken together, our results establish for the first time that CB2receptors are important contributors to septic
immune dysfunction and mortality, indicating that CB2receptors may be therapeutically targeted for the benefit of patients
suffering from sepsis.
Citation: Cso ´ka B, Ne ´meth ZH, Mukhopadhyay P, Spolarics Z, Rajesh M, et al. (2009) CB2Cannabinoid Receptors Contribute to Bacterial Invasion and Mortality in
Polymicrobial Sepsis. PLoS ONE 4(7): e6409. doi:10.1371/journal.pone.0006409
Editor: Neeraj Vij, Johns Hopkins School of Medicine, United States of America
Received June 2, 2009; Accepted June 22, 2009; Published July 29, 2009
This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public
domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
Funding: This work was supported by the National Institutes of Health (NIH) Grant GM066189-05A2 and the Intramural Research Program of NIH, National
Institute on Alcohol Abuse and Alcoholism. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org (PP); email@example.com (GH)
Sepsis is defined as systemic illness caused by microbial invasion
of normally sterile parts of the body. Sepsis is a major healthcare
problem because its incidence is in the order of sepsis is in the
order of 750,000 annually, and sepsis causes more than 200,000
deaths each year in the United States alone [1,2]. Currently,
therapeutic management of sepsis is limited mostly to supportive
measures, in a large part due to a failure to fully establish the
pathophysiology of this complex and heterogeneous syndrome.
Multiple organ dysfunction syndrome and death in sepsis are
consequences of an inability to kill invading pathogens effectively
due to immunosuppression [3,4]. Potentially contributing to
immune suppression after a septic insult are immune cell
apoptosis, inefficient phagocytosis of microbial pathogens by
neutrophils and macrophages, decreased ability of antigen-
presenting cells to present antigens, as well as decreased
responsiveness of macrophages and T cells to release proinflam-
matory cytokines in conjunction with overzealous production of
the anti-inflammatory cytokine IL-10 [5–7].
One view holds that immune dysfunction during sepsis is a
result of autocrine/paracrine immunoregulatory mediators that
are produced primarily at the site of infection/injury (including the
bloodstream) and suppress immune cell function via acting on G-
protein-coupled receptors. One group of these mediators are
endocannabinoids, which elicit their cellular effects by binding to
two subtypes of G-protein-coupled cannabinoid receptor proteins
on the cell surface, termed CB1 and CB2 receptors [8–10].
Endocannabinoids are released from macrophages, dendritic cells,
platelets, and parenchimal cells in response to inflammatory
stimuli and oxidative stress [11–15] and are present at elevated
concentrations in the sera of patients and animals suffering from
septic, hemorrhagic or cardiogenic shock [16–20]. CB2receptors
are the dominant cannabinoid receptors on macrophages,
neutrophils, and lymphocytes, and triggering CB2receptors has
an overall anti-inflammatory and immunosuppressive effect .
CB2 receptor activation augments the production of the anti-
inflammatory cytokine, IL-10, by murine macrophages , and
disrupts antigen processing by these cells, which leads to
incomplete antigen-presentation to T cells [23,24].
PLoS ONE | www.plosone.org1July 2009 | Volume 4 | Issue 7 | e6409
Despite the recent enormous advances in our knowledge of how
CB2receptors regulate immune function, the role of CB2receptors
in regulating bacterial sepsis is unknown. In the present study,
using a genetic approach we examined the role of CB2receptors in
regulating the host’s response to polymicrobial sepsis.
Materials and Methods
CB2knockout (KO) mice and their wild-type (WT) littermates
were developed as described previously and had been backcrossed
to a C57Bl/6J background . All mice were maintained in
accordance with the recommendations of the ‘‘Guide for the Care
and Use of Laboratory Animals’’, and the experiments were
approved by the New Jersey Medical School Animal Care
Cecal ligation and puncture (CLP)
Polymicrobial sepsis was induced by subjecting mice to CLP, as
we have described previously [25,26]. Eight- to twelve-week-old
male CB2KO or WT mice were anesthetized with Pentobarbital
(50 mg/kg), given intraperitoneally (i.p.). Under aseptic condi-
tions, a 2-cm midline laparotomy was performed to allow exposure
of the cecum with adjoining intestine. Approximately two-thirds of
the cecum was tightly ligated with a 3.0 silk suture, and the ligated
part of the cecum was perforated twice (through and through) with
a 20-gauge needle (BD Biosciences). The cecum was then gently
squeezed to extrude a small amount of feces from the perforation
sites. The cecum was then returned to the peritoneal cavity and
the laparotomy was closed in two layers with 4.0 silk sutures. The
mice were resuscitated with 1 ml of physiological saline injected
subcutaneously (s.c.) and returned to their cages with free access to
food and water. One group of mice was monitored daily and
survival was recorded for 7 days. Another group of mice was
reanesthetized with Pentobarbital (80 mg/kg i.p.) 16 hour after
the operation, and blood, peritoneal lavage fluid, and various
organs were harvested.
Collection of blood, peritoneal lavage fluid, and organs
Blood samples were obtained aseptically by cardiac puncture
using heparinized syringes after opening the chest and placed on
ice into heparinized Eppendorf tubes until further processing for
bacteriological analysis. After serial dilutions for bacteriological
analysis were made (see below), the blood was centrifuged at
2,0006g for 10 min and the recovered plasma was stored at
270uC until further use. For peritoneal lavage, the abdominal skin
was cleansed with 70% ethanol and the abdominal wall was
exposed by opening the skin. Two milliliters of sterile physiological
saline were then installed into the peritoneal cavity via an 18-
gauge needle. The abdomen was massaged gently for 1 min while
keeping the tip of the needle in the peritoneum, after which
peritoneal fluid was recovered through the needle. Recovered
peritoneal lavage fluid was placed on ice until processed for
bacteriological examination. After serially diluting the peritoneal
lavage fluid to determine colony forming unit (CFU) numbers (see
below), the peritoneal lavage fluid was centrifuged at 5,0006g for
10 min and the supernatant was stored at 270uC until further
analysis. Samples from spleen and thymus, were excised and
immediately frozen in liquid nitrogen.
Quantification of bacterial CFUs from peritoneal lavage
fluid and blood
100 ml of blood or peritoneal lavage fluid was diluted serially in
sterile physiological saline. 50 ml of each dilution was aseptically
plated and cultured on trypticase blood agar plates (BD
Biosciences) at 37uC. After 24 hours, the number of bacterial
colonies was counted. Quantitative cultures are expressed as CFUs
per milliliter of blood or peritoneal lavage fluid.
Flow cytometric analysis of leukocyte subsets
Flow cytometric detection of leukocyte subsets was performed as
previously described . In brief, the percent distribution of
leukocyte subsets in blood was analyzed by specifically staining
CD3+T-cells, CD19+B-cells and CD11b+myeloid cells using
antibodies against CD markers conjugated with FITC, PERCP or
PE (BD Biosciences) in three-color incubations. Aliquots of 0.1 ml
whole blood were incubated with the respective markers for
15 min followed by incubation with BD FACS lysing solution (BD
Biosciences) for 7 min at 37uC. Cells were washed twice with BD
FACS wash buffer and then fixed with 1% methanol free
formaldehyde. FACS acquisitions were performed in a centralized
flow cytometry facility. At least 30,000 events were collected for
Protein extraction and Western blot analysis
Frozen organs were homogenized in a Dounce homogenizer in
modified radioimmunoprecipitation assay buffer (50 mM Tris
HCl, 150 mM NaCl, 1 mM EDTA, 0.25% sodium deoxycho-
late, 1% Nonidet P-40, 1 mg/ml pepstatin, 1 mg/ml leupeptin,
1 mM PMSF, 1 mM Na3VO4). The lysates were centrifuged at
15,0006g for 15 min, and the supernatant was recovered.
Protein concentrations were determined using the Bio-Rad
protein assay kit. A total of 40 mg of sample was separated on
8–12% Tris-glycine gel (Invitrogen Life Technologies) and
transferred to nitrocellulose membrane. The membranes were
probed with polyclonal rabbit anti-cleaved caspase-3, polyclonal
rabbit anti-cleaved poly(ADP-ribose) polymerase (PARP), and
polyclonal rabbit anti- inhibitory subunit of nuclear factor (NF)-
kB (IkBa), (all from Cell Signaling Technology). Thereafter, the
membranes were incubated with a secondary HRP-conjugated
anti-rabbit antibody (Santa Cruz Biotechnology). HRP-conju-
gated polyclonal goat anti-b actin antibody to assess equal
loading was used from Santa Cruz Biotechnology. Bands were
detected using ECL Western Blotting Detection Reagent
Determination of lactate dehydrogenase (LDH), aspartate
aminotransferase (AST), alanine aminotransferase (ALT),
blood urea nitrogen (BUN), and creatine phosphokinase
Plasma concentrations of LDH, AST, ALT, BUN, and CK were
analyzed using a clinical chemistry analyzer system (VetTest8008,
Determination of cytokine and chemokine levels
Concentrations of IL-10, IL-6, and MIP-2, were determined
using commercially available ELISA kits (R&D Systems) according
to the manufacturer’s instructions. The lower detection limit for all
these cytokines was 10 pg/ml.
Survival statistics were performed using Kaplan-Meier curve
and log rank test. Two-tailed t testing was used to compare
cytokine concentrations, CFUs, and other laboratory parame-
ters. Statistical significance was assigned to p values smaller than
CB2Receptors in Sepsis
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CB2receptors contribute to sepsis-induced mortality and
To begin to study the role of CB2receptors, we first investigated
the effect of CB2deficiency in CLP-induced septic peritonitis by
monitoring the survival of CB2 WT and KO mice. As
demonstrated in Figure 1a, CB2 receptor KO mice had
significantly lower mortality rates compared with WT mice,
which became apparent on the 2nd day of observation. On the 7th
day following CLP, the mortality rate of CB2 KO mice was
markedly (by more than 40%) lower than that of CB2WT mice.
No additional changes in mortality were detected when the mice
were monitored for up to 10 days (data not shown). Because
persistence of local bacterial infection and bloodstream invasion
play important roles in mortality in the CLP model, we next
assessed the impact of CB2receptor inactivation on bacterial levels
at the primary peritoneal site of infection and in the blood stream.
We found markedly decreased numbers of bacteria in the blood
but not peritoneal lavage fluid of CB2receptor KO mice when
compared to WT animals at 16 hours after CLP (Figure 1b, c).
Taken together, these studies document that CB2 receptors
contribute to bacterial translocation into the bloodstream and
mortality in polymicrobial sepsis.
CB2receptor inactivation diminishes the production of
IL-10, IL-6 and MIP-2 in CLP-induced sepsis
Because IL-10 overproduction contributes to the impairment of
host antibacterial defenses seen in mice undergoing CLP [28–31],
CB2KO and WT mice subjected to CLP. CB2KO mice exhibited
markedly lower levels of IL-10 at 16 h after CLP (Figure 2a, b).
Figure 1. CB2deficiency decreases mortality and bacterial burden in polymicrobial sepsis elicited by CLP. (a) Surviving CB2KO and WT
mice were counted every day for 7 days after inducing polymicrobial sepsis by way of cecal ligation and puncture (CLP). p,0.001 versus WT. (b)
Blood and (c) peritoneal lavage fluid obtained from CB2KO or WT mice 16 hour after CLP were cultured on soy-trypticase agar plates, and the
number of bacterial colonies was counted. Data are the mean6SEM of n=6–9 mice per group. *p,0.05.
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Figure 2. CB2receptor deficiency decreases IL-10, IL-6, and MIP-2 levels in the plasma and peritoneal lavage fluid of mice subjected
to CLP. IL-10 (a,b), IL-6 (c,d) and MIP-2 (e,f) concentrations were measured at 16 hours after surgery using ELISA. Data are the mean6SEM of n=6–9
mice per group. ***p,0.001; **p,0.01.
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Because IL-6 blockade with neutralizing Abs has been shown to
be protective in CLP-induced sepsis , we next assessed the role
of CB2 receptors in regulating IL-6 production during sepsis.
Plasma and peritoneal lavage fluid had lower levels of IL-6 in CLP-
induced CB2KO mice than their WT littermates (Figure 2c,d).
We then determined the levels of macrophage-inflammatory
protein-2 (MIP-2), a crucial chemokine that mediates inflamma-
tory responses, in the plasma and peritoneal lavage fluid of CB2
KO and WT mice subjected to CLP, and we found that CLP-
induced concentrations of MIP-2 were diminished in CB2KO
Figure 3. CB2KO mice have less tissue damage, kidney and muscle injury in sepsis than their WT counterparts. LDH (a), AST (b), ALT
(c), and CK (d) activity, and BUN (e) levels were measured in plasma samples 16 hour after CLP using a clinical chemistry analyzer system
(VetTest8008, IDEXX Laboratories). Data are the mean6SEM of n=6–9 mice per group. **p,0.01; * p,0.05.
CB2Receptors in Sepsis
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mice as compared with their WT counterparts when measured at
16 h after CLP (Figure 2e, f).
Mice deficient in CB2receptor show decreased level of
markers of tissue damage, and kidney and muscle injury
We next measured markers of disease severity and organ
damage in an attempt to provide further explanation for the
improved survival of CB2 receptor KO mice. Levels of LDH
(Figure 3a), were lower in CB2KO mice, indicating less tissue
damage in general. Markers of liver (AST and ALT) function were
not different between the WT and KO groups (Figure 3b, c). In
addition, we could not detect any lung inflammation in CLP-
challenged mice as assessed by histological analysis of lung
sections, and detecting myeloperoxidase activity in lung tissue
homogenates after 16 hour of CLP (data not shown). Finally, CK
activity and BUN levels were lower in CB2KO mice indicating
preserved kidney function and lessened muscle (both heart and
skeletal) damage, respectively (Figure 3d,e).
CB2receptor deficiency decreases NF-kB activation in
Microbial components and endogenous danger signals trigger
the activation of signaling cascades leading to induction of the NF-
kB system during sepsis. Persistent activation of NF-kB may cause
excessive inflammatory responses culminating in tissue injury,
organ dysfunction, and death. We, therefore, studied the
activation of NF-kB by measuring levels of the IkBa in spleen of
septic animals. As Figure 4 shows, the levels of IkBa were
increased in the spleen of CB2KO as compared to WT mice,
indicating decreased NF-kB activation in KO mice.
Genetic deletion of the CB2receptor diminishes
apoptosis in lymphoid organs
Sepsis provokes extensive immune cell apoptosis that contrib-
utes to immune dysregulation and mortality. This was borne out
by studies demonstrating that transfer of apoptotic splenocytes
worsens survival in CLP-induced sepsis . Because proteolytic
cleavage of caspase-3 and PARP is a good indicator of apoptosis,
we tested whether CB2 receptor deficiency would affect the
cleavage of caspase-3 and PARP in the spleen and thymus of mice
subjected to CLP. Figure 5 shows that 16 hours after the onset of
sepsis, the cleavage of both caspase-3 and PARP was markedly
decreased in thymus and spleen of CB2receptor KO mice.
Lack of CB2receptors augments the number of CD11b+
and CD19+cells during CLP
CLP-challenged mice exhibit a decrease in white blood cell
numbers,which includes CD11b+cells (mostly neutrophils), CD3+T
KO mice have cell counts comparable to their WT counterparts
. Flow-cytometric analysis of cell counts revealed that CLP-
challenged CB2KO mice have increased numbers of total white
blood cells (Figure 6a), CD11b+(Figure 6b) cells and CD19+B
lymphocytes (Figure 6c) in comparison with CB2WT mice, whereas
the number of CD3+T cells (Figure 6d) was comparable between
WT and KO mice undergoing CLP. We propose that preserved
Figure 4. CB2receptor deficiency is associated with augmented
IkBa levels in CLP-induced sepsis. IkBa degradation was assessed
using Western blotting of spleen protein extracts of CB2WT and KO
mice. Protein extracts were generated from spleen taken 16 hours after
sepsis induction. Bands were detected by enchanced chemilumines-
cence (ECL). Results (mean6SEM) shown are representative of 3
experiments. **p,0.01 versus WT.
Figure 5. CB2receptor deficiency decreases apoptosis in septic thymus and spleen. Proteolytic cleavage of caspase-3 and PARP in thymus
and spleen protein extracts from CB2WT and KO mice was examined using Western blotting. Bands were detected by enchanced chemiluminescence
(ECL). Results shown are representative of 3 experiments.
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white blood cell numbers in KO animals contribute to the decreased
bacterial growth and mortality of these mice.
Current concepts suggest that sepsis is the consequence of the
inability of the immune system to ward off infecting pathogens due
to immune system dysfunction. The mechanisms underlying these
immune functional abnormalities are largely unknown. Recent
studies have expanded the list of potential mediators to molecules
that are produced locally by infected and inflamed tissues and act
on specific G protein-coupled receptors expressed on immune cells
to inhibit their function. These molecules include adenosine
[37,38], which bind to and trigger their receptors on lymphocytes,
macrophages, and neutrophils, thereby diminishing anti-bacterial
defenses. In these studies we have focused on a new type of
immunosuppressive G protein-coupled receptor, the CB2canna-
binoid receptor, which is expressed primarily by immune cells and
is activated by locally released endocannabinoids. Using the CLP
model of sepsis, we found that CB2 receptor activation by
endogenously released cannabinoids contributes to mortality,
bacterial invasion, IL-10 production, and immune cell death in
Studies utilizing antibiotic therapy have shown that systemic
bacterial dissemination is a major factor contributing to the
mortality of both experimental animals and humans during sepsis
[39–41]. Our studies showed that bacterial burden was decreased
in CB2receptor KO mice suggesting that CB2receptor activation
contributes mortality by increasing systemic bacterial burden
during sepsis. One potential explanation for the decreased
bacterial load in mice lacking CB2receptors is a decrease in the
levels of the immunosuppressive IL-10 leading to a better
preserved phagocytic response. IL-10 is an immunoregulatory
cytokine that is released primarily by macrophages during sepsis.
IL-10 is an important contributor to the dysregulated immune
system that is observed in sepsis [28–31,42]. Recent studies have
shown that CB2 receptor activation can upregulate IL-10
production across a number of experimental systems that utilize
macrophages [22,43]. Given the relevance of IL-10 in sepsis, the
effect of CB2receptor activation on IL-10 release is likely to be a
major determinant of the immunomodulatory action of CB2
receptor activation in sepsis.
Sepsis instigates widespread immune cell apoptosis, and
mortality in this illness is thought to be, at least in part, a
consequence of dysregulated immune cell death [44–46]. CB2
receptor triggering induces both T and B lymphocyte apoptosis in
Figure 6. Effect of CB2receptor inactivation on the leukocyte cell subsets in the blood during CLP. Numbers of total white cell (a), as
well as CD11b+(b), CD19+(c), and CD3+(d) cells were monitored in blood by flow cytometric analysis after 16 hours of sepsis induced by CLP. Data
are the mean6SEM of n=6–9 mice per group. *p,0.05; **p,0.01.
CB2Receptors in Sepsis
PLoS ONE | www.plosone.org7July 2009 | Volume 4 | Issue 7 | e6409
vitro [47–52]. Our data showing decreased levels of caspase-3
cleavage as well as PARP cleavage in CB2KO mice following
sepsis indicate that CB2receptors are essential contributors to
apoptotic processes also in vivo. Moreover, we found increased
numbers of CD11b+and CD19+cells in blood of CB2KO mice
suggesting that CB2receptors contribute to bacterial invasion by
CD11b+and B cell depletion in the bloodstream. The immuno-
suppressive role of CB2receptors has been confirmed recently in
vivo by inducing immune-mediated inflammatory disease in CB2
receptor KO and WT mice. CB2receptor KO mice develop a
more severe form of experimental allergic encephalomyelitis in
comparison with WT mice, which is a consequence of increased
T-cell activation and decreased T-cell apoptosis in KO vs. WT
mice . In another model, CB2receptor KO mice displayed
increased allergic responses in the skin, which was secondary to
increased production of proinflammatory cytokines . More-
over, we have recently demonstrated that CB2receptor KO mice
exhibit exacerbated liver injury following hepatic ischemia/
reperfusion, which is associated with increased production of
pro-inflammatory cytokines and neutrophil infiltration into the
liver . Our results that CB2receptor inactivation decreases
mortality during CLP-induced bacterial (non-sterile) sepsis might
seem contradictory to the observations in experimental allergic
encephalomyelitis, allergic skin inflammation, and ischemia/
reperfusion-induced inflammation in that CB2receptor inactiva-
tion is injurious in these sterile inflammation models. But we
believe the data seen as a whole suggests that differences in
outcome in the two types of model are due to immunosuppression
being beneficial in sterile inflammation/ischemia but detrimental
in clinically relevant models of infection-induced sepsis where
mortality depends more upon the inability to mount an immune
response leading to a loss of control of bacterial growth.
In summary, we found that CB2 receptor activation by
endogenously released cannabinoids contributes to mortality,
bacterial invasion, IL-10 production, and immune cell death in
sepsis. Based on these observations, we suggest that CB2receptor
triggering contributes to the development of immune system
dysfunction that leads to mortality in sepsis.
Conceived and designed the experiments: BC. Performed the experiments:
BC ZHN PM MR SF. Analyzed the data: BC ZS PP GH. Contributed
reagents/materials/analysis tools: ED SB PP. Wrote the paper: BC PP
1. Angus DC, Linde-Zwirble WT, Lidicker J, Clermont G, Carcillo J, et al. (2001)
Epidemiology of severe sepsis in the united states: Analysis of incidence,
outcome, and associated costs of care. Crit Care Med 29(7): 1303–1310.
2. Martin GS, Mannino DM, Eaton S, Moss M (2003) The epidemiology of sepsis
in the united states from 1979 through 2000. N Engl J Med 348(16): 1546–1554.
3. Benjamim CF, Hogaboam CM, Kunkel SL (2004) The chronic consequences of
severe sepsis. J Leukoc Biol 75(3): 408–412.
4. Oberholzer A, Oberholzer C, Moldawer LL (2001) Sepsis syndromes:
Understanding the role of innate and acquired immunity. Shock 16(2): 83–96.
5. Hotchkiss RS, Karl IE (2003) The pathophysiology and treatment of sepsis.
N Engl J Med 348(2): 138–150.
6. Riedemann NC, Guo RF, Bernacki KD, Reuben JS, Laudes IJ, et al. (2003)
Regulationby C5a ofneutrophil activation during sepsis. Immunity19(2): 193–202.
7. Ayala A, Chaudry IH (1996) Immune dysfunction in murine polymicrobial
sepsis: Mediators, macrophages, lymphocytes and apoptosis. Shock 6 Suppl 1:
8. Pacher P, Batkai S, Kunos G (2006) The endocannabinoid system as an
emerging target of pharmacotherapy. Pharmacol Rev 58(3): 389–462.
9. Di Marzo V (2008) Targeting the endocannabinoid system: To enhance or
reduce? Nat Rev Drug Discov 7(5): 438–455.
10. Howlett AC, Barth F, Bonner TI, Cabral G, Casellas P, et al. (2002)
International union of pharmacology. XXVII. classification of cannabinoid
receptors. Pharmacol Rev 54(2): 161–202.
11. Di Marzo V, De Petrocellis L, Sepe N, Buono A (1996) Biosynthesis of
anandamide and related acylethanolamides in mouse J774 macrophages and
N18 neuroblastoma cells. Biochem J 316 ( Pt 3)(Pt 3): 977–984.
12. Pacher P, Hasko G (2008) Endocannabinoids and cannabinoid receptors in
ischaemia-reperfusion injury and preconditioning. Br J Pharmacol 153(2): 252–262.
13. Matias I, Pochard P, Orlando P, Salzet M, Pestel J, et al. (2002) Presence and
regulation of the endocannabinoid system in human dendritic cells.
Eur J Biochem 269(15): 3771–3778.
14. Batkai S, Osei-Hyiaman D, Pan H, El-Assal O, Rajesh M, et al. (2007)
Cannabinoid-2 receptor mediates protection against hepatic ischemia/reperfu-
sion injury. FASEB J 21(8): 1788–1800.
15. Mukhopadhyay P, Batkai S, Rajesh M, Czifra N, Harvey-White J, et al. (2007)
Pharmacological inhibition of CB1 cannabinoid receptor protects against
doxorubicin-induced cardiotoxicity. J Am Coll Cardiol 50(6): 528–536.
16. Wang Y, Liu Y, Ito Y, Hashiguchi T, Kitajima I, et al. (2001) Simultaneous
measurement of anandamide and 2-arachidonoylglycerol by polymyxin B-
selective adsorption and subsequent high-performance liquid chromatography
analysis: Increase in endogenous cannabinoids in the sera of patients with
endotoxic shock. Anal Biochem 294(1): 73–82.
17. Varga K, Wagner JA, Bridgen DT, Kunos G (1998) Platelet- and macrophage-
derived endogenous cannabinoids are involved in endotoxin-induced hypoten-
sion. FASEB J 12(11): 1035–1044.
18. Wagner JA, Varga K, Kunos G (1998) Cardiovascular actions of cannabinoids
and their generation during shock. J Mol Med 76(12): 824–836.
19. Wagner JA, Varga K, Ellis EF, Rzigalinski BA, Martin BR, et al. (1997)
Activation of peripheral CB1 cannabinoid receptors in haemorrhagic shock.
Nature 390(6659): 518–521.
20. Wagner JA, Hu K, Bauersachs J, Karcher J, Wiesler M, et al. (2001)
Endogenous cannabinoids mediate hypotension after experimental myocardial
infarction. J Am Coll Cardiol 38(7): 2048–2054.
21. Klein TW (2005) Cannabinoid-based drugs as anti-inflammatory therapeutics.
Nat Rev Immunol 5(5): 400–411.
22. Correa F, Mestre L, Docagne F, Guaza C (2005) Activation of cannabinoid CB2
receptor negatively regulates IL-12p40 production in murine macrophages: Role
of IL-10 and ERK1/2 kinase signaling. Br J Pharmacol 145(4): 441–448.
23. McCoy KL, Matveyeva M, Carlisle SJ, Cabral GA (1999) Cannabinoid
inhibition of the processing of intact lysozyme by macrophages: Evidence for
CB2 receptor participation. J Pharmacol Exp Ther 289(3): 1620–1625.
24. Matveyeva M, Hartmann CB, Harrison MT, Cabral GA, McCoy KL (2000)
Delta(9)-tetrahydrocannabinol selectively increases aspartyl cathepsin D proteo-
lytic activity and impairs lysozyme processing by macrophages. Int
J Immunopharmacol 22(5): 373–381.
25. Nemeth ZH, Csoka B, Wilmanski J, Xu D, Lu Q, et al. (2006) Adenosine A2A
receptor inactivation increases survival in polymicrobial sepsis. J Immunol
26. Csoka B, Nemeth ZH, Selmeczy Z, Koscso B, Pacher P, et al. (2007) Role of
A(2A) adenosine receptors in regulation of opsonized E. coli-induced
macrophage function. Purinergic Signal 3(4): 447–452.
27. Chandra R, Villanueva E, Feketova E, Machiedo GW, Hasko G, et al. (2008)
Endotoxemia down-regulates bone marrow lymphopoiesis but stimulates
myelopoiesis: The effect of G6PD deficiency. J Leukoc Biol 83(6): 1541–1550.
increased production of interleukin-10 by cells of the immune system with a negative
impact on resistance to infection. Ann Surg 226(4): 450–8; discussion 458–60.
29. Steinhauser ML, Hogaboam CM, Kunkel SL, Lukacs NW, Strieter RM, et al.
(1999) IL-10 is a major mediator of sepsis-induced impairment in lung
antibacterial host defense. J Immunol 162(1): 392–399.
30. Song GY, Chung CS, Chaudry IH, Ayala A (2000) Immune suppression in
polymicrobial sepsis: Differential regulation of Th1 and Th2 responses by p38
MAPK. J Surg Res 91(2): 141–146.
31. Kalechman Y, Gafter U, Gal R, Rushkin G, Yan D, et al. (2002) Anti-IL-10
therapeutic strategy using the immunomodulator AS101 in protecting mice from
sepsis-induced death: Dependence on timing of immunomodulating interven-
tion. J Immunol 169(1): 384–392.
32. Riedemann NC, Neff TA, Guo RF, Bernacki KD, Laudes IJ, et al. (2003)
Protective effects of IL-6 blockade in sepsis are linked to reduced C5a receptor
expression. J Immunol 170(1): 503–507.
33. Hotchkiss RS, Chang KC, Grayson MH, Tinsley KW, Dunne BS, et al. (2003)
Adoptive transfer of apoptotic splenocytes worsens survival, whereas adoptive
transfer of necrotic splenocytes improves survival in sepsis. Proc Natl Acad
Sci U S A 100(11): 6724–6729.
34. Hotchkiss RS, Swanson PE, Cobb JP, Jacobson A, Buchman TG, et al. (1997)
Apoptosis in lymphoid and parenchymal cells during sepsis: Findings in normal
and T- and B-cell-deficient mice. Crit Care Med 25(8): 1298–1307.
35. Shelley O, Murphy T, Paterson H, Mannick JA, Lederer JA (2003) Interaction
between the innate and adaptive immune systems is required to survive sepsis
and control inflammation after injury. Shock 20(2): 123–129.
CB2Receptors in Sepsis
PLoS ONE | www.plosone.org8 July 2009 | Volume 4 | Issue 7 | e6409
36. Buckley NE, McCoy KL, Mezey E, Bonner T, Zimmer A, et al. (2000)
Immunomodulation by cannabinoids is absent in mice deficient for the
cannabinoid CB(2) receptor. Eur J Pharmacol 396(2–3): 141–149.
37. Hasko G, Cronstein BN (2004) Adenosine: An endogenous regulator of innate
immunity. Trends Immunol 25(1): 33–39.
38. Hasko G, Linden J, Cronstein B, Pacher P (2008) Adenosine receptors:
Therapeutic aspects for inflammatory and immune diseases. Nat Rev Drug
Discov 7(9): 759–770.
39. Enoh VT, Fairchild CD, Lin CY, Varma TK, Sherwood ER (2006) Differential
effect of imipenem treatment on wild-type and NK cell-deficient CD8 knockout
mice during acute intra-abdominal injury. Am J Physiol Regul Integr Comp
Physiol 290(3): R685–93.
40. Enoh VT, Lin CY, Varma TK, Sherwood ER (2006) Differential effect of
imipenem treatment on injury caused by cecal ligation and puncture in wild-type
and NK cell-deficient beta(2)-microgloblin knockout mice. Am J Physiol
Gastrointest Liver Physiol 290(2): G277–84.
41. Leibovici L, Drucker M, Konigsberger H, Samra Z, Harrari S, et al. (1997)
Septic shock in bacteremic patients: Risk factors, features and prognosis.
Scand J Infect Dis 29(1): 71–75.
42. Oberholzer A, Oberholzer C, Moldawer LL (2002) Interleukin-10: A complex
role in the pathogenesis of sepsis syndromes and its potential as an anti-
inflammatory drug. Crit Care Med 30(1 Supp): S58–S63.
43. Zhu LX, Sharma S, Stolina M, Gardner B, Roth MD, et al. (2000) Delta-9-
tetrahydrocannabinol inhibits antitumor immunity by a CB2 receptor-mediated,
cytokine-dependent pathway. J Immunol 165(1): 373–380.
44. Chung CS, Xu YX, Wang W, Chaudry IH, Ayala A (1998) Is fas ligand or
endotoxin responsible for mucosal lymphocyte apoptosis in sepsis? Arch Surg
45. Hotchkiss RS, Tinsley KW, Swanson PE, Chang KC, Cobb JP, et al. (1999)
Prevention of lymphocyte cell death in sepsis improves survival in mice. Proc
Natl Acad Sci U S A 96(25): 14541–14546.
46. Hotchkiss RS, Tinsley KW, Swanson PE, Schmieg RE Jr, Hui JJ, et al. (2001)
Sepsis-induced apoptosis causes progressive profound depletion of B and CD4+
T lymphocytes in humans. J Immunol 166(11): 6952–6963.
47. Lombard C, Nagarkatti M, Nagarkatti P (2007) CB2 cannabinoid receptor
agonist, JWH-015, triggers apoptosis in immune cells: Potential role for CB2-
selective ligands as immunosuppressive agents. Clin Immunol 122(3): 259–270.
48. Rieder SA, Chauhan A, Singh U, Nagarkatti M, Nagarkatti P (2009)
Cannabinoid-induced apoptosis in immune cells as a pathway to immunosup-
49. Jia W, Hegde VL, Singh NP, Sisco D, Grant S, et al. (2006) Delta9-
tetrahydrocannabinol-induced apoptosis in jurkat leukemia T cells is regulated
by translocation of bad to mitochondria. Mol Cancer Res 4(8): 549–562.
50. McKallip RJ, Jia W, Schlomer J, Warren JW, Nagarkatti PS, et al. (2006)
Cannabidiol-induced apoptosis in human leukemia cells: A novel role of
cannabidiol in the regulation of p22phox and Nox4 expression. Mol Pharmacol
51. McKallip RJ, Lombard C, Martin BR, Nagarkatti M, Nagarkatti PS (2002)
Delta(9)-tetrahydrocannabinol-induced apoptosis in the thymus and spleen as a
mechanism of immunosuppression in vitro and in vivo. J Pharmacol Exp Ther
52. Do Y, McKallip RJ, Nagarkatti M, Nagarkatti PS (2004) Activation through
cannabinoid receptors 1 and 2 on dendritic cells triggers NF-kappaB-dependent
apoptosis: Novel role for endogenous and exogenous cannabinoids in
immunoregulation. J Immunol 173(4): 2373–2382.
53. Maresz K, Pryce G, Ponomarev ED, Marsicano G, Croxford JL, et al. (2007)
Direct suppression of CNS autoimmune inflammation via the cannabinoid
receptor CB1 on neurons and CB2 on autoreactive T cells. Nat Med 13(4):
54. Karsak M, Gaffal E, Date R, Wang-Eckhardt L, Rehnelt J, et al. (2007)
Attenuation of allergic contact dermatitis through the endocannabinoid system.
Science 316(5830): 1494–1497.
CB2Receptors in Sepsis
PLoS ONE | www.plosone.org9 July 2009 | Volume 4 | Issue 7 | e6409