CCR1 and CC Chemokine Ligand 5

Interactions Exacerbate Innate Immune

This information is current as
of October 26, 2022.
Responses during Sepsis
Traci L. Ness, Kristin J. Carpenter, Jillian L. Ewing, Craig J.
Gerard, Cory M. Hogaboam and Steven L. Kunkel
J Immunol 2004; 173:6938-6948; ;
doi: 10.4049/jimmunol.173.11.6938
http://www.jimmunol.org/content/173/11/6938

Downloaded from http://www.jimmunol.org/ by guest on October 26, 2022

References This article cites 44 articles, 23 of which you can access for free at:
http://www.jimmunol.org/content/173/11/6938.full#ref-list-1

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision
• No Triage! Every submission reviewed by practicing scientists
• Fast Publication! 4 weeks from acceptance to publication
*average

Subscription Information about subscribing to The Journal of Immunology is online at:

http://jimmunol.org/subscription
Permissions Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Email Alerts Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by

The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2004 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
 The Journal of Immunology

CCR1 and CC Chemokine Ligand 5 Interactions Exacerbate

Innate Immune Responses during Sepsis1

Traci L. Ness,* Kristin J. Carpenter,* Jillian L. Ewing,* Craig J. Gerard,†

Cory M. Hogaboam,2* and Steven L. Kunkel*
CCR1 has previously been shown to play important roles in leukocyte trafficking, pathogen clearance, and the type 1/type
2 cytokine balance, although very little is known about its role in the host response during sepsis. In a cecal ligation and
puncture model of septic peritonitis, CCR1-deficient (CCR1ⴚ/ⴚ) mice were significantly protected from the lethal effects of
sepsis when compared with wild-type (WT) controls. The peritoneal and systemic cytokine profile in CCR1ⴚ/ⴚ mice was
characterized by a robust, but short-lived and regulated antibacterial response. CCR1 expression was not required for leu-
kocyte recruitment, suggesting critical differences extant in the activation of WT and CCR1ⴚ/ⴚ resident or recruited peri-
toneal cells during sepsis. Peritoneal macrophages isolated from naive CCR1ⴚ/ⴚ mice clearly demonstrated enhanced

Downloaded from http://www.jimmunol.org/ by guest on October 26, 2022

cytokine/chemokine generation and antibacterial responses compared with similarly treated WT macrophages. CCR1 and
CCL5 interactions markedly altered the inflammatory response in vivo and in vitro. Administration of CCL5 increased
sepsis-induced lethality in WT mice, whereas neutralization of CCL5 improved survival. CCL5 acted in a CCR1-dependent
manner to augment production of IFN-␥ and MIP-2 to damaging levels. These data illustrate that the interaction between
CCR1 and CCL5 modulates the innate immune response during sepsis, and both represent potential targets for therapeutic
intervention. The Journal of Immunology, 2004, 173: 6938 – 6948.

S epsis is the most common cause of death in noncoronary
critical care units in the U.S. with ⬎751,000 cases per year
at an estimated overall cost of $16.7 billion (increasing by
1.5% per year) (1). Due to the paucity of effective therapeutic
treatments (2), the care of septic patients is predominantly sup-
portive and mortality rates remain high. Although recombinant ac-
tivated protein C has shown some promise (3), investigators con-
tinue to search for novel therapies.
During a normal infection, the immune system of an immuno-
competent host works to contain and destroy the pathogen. A sep-
tic response occurs when a pathogen circumvents the innate and
acquired immune defenses, resulting in systemic spread of the in-
fection. In a continued attempt to eliminate the infection, the host
enhances production of several proinflammatory cytokines and
chemokines that act to increase the infiltration and activation of
inflammatory leukocytes. These factors have been shown to play a
significant role in the resulting tissue damage preceding sepsis-
associated multiple organ failure (4, 5) and may serve as potential
targets for immunotherapy.
Chemokines are a family of small, primarily secreted proteins
that are responsible for modulating multiple aspects of inflam-
matory responses and host defense (6). Many studies have dem-
onstrated several of these to be key mediators in the host im-
*Department of Pathology, University of Michigan Medical School, Ann Arbor, MI
48109; and †Children’s Hospital, Harvard Medical School, Boston, MA 02115
Received for publication June 15, 2004. Accepted for publication September
22, 2004.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by Grants HL031237 and P50HL074024 from the Na-
tional Institutes of Health to S.L.K.
2
Address correspondence and reprint requests to Dr. Cory M. Hogaboam, Depart-
ment of Pathology, University of Michigan Medical School, 1301 Catherine Road,
Room 5214, Medical Sciences I, Ann Arbor, MI 48109-0602. E-mail address:
hogaboam@med.umich.edu
mune response to sepsis (7, 8). Although differences in
chemokine receptor expression in septic patients have been re-
ported (9), the significance of these differences is not under-
stood. Almost nothing is known about the role these receptors
play in the septic response (10, 11).
CCR1 is expressed by a broad spectrum of leukocytes, including
neutrophils, monocytes, eosinophils, and lymphocytes (12). Stud-
ies of CCR1-deficient (CCR1⫺/⫺) mice have implicated CCR1 in
the modulation of leukocyte trafficking (13), parasite and viral
clearance (14, 15), and the balance of type 1 and type 2 cytokines
(16); however, little is known about its role in the innate immune
response during sepsis and multiorgan failure. In vitro, CCR1 has
been shown to bind several ligands, including CCL3, CCL5–9,
CCL14 –16, and CCL23, although CCL3, CCL5, and CCL6 are the
major agonists identified in vivo (17, 18). Serum levels of CCL3
and CCL5 are elevated in septic patients (19), and peritoneal CCL3
(20) and CCL6 (7) concentrations are increased in the cecal liga-
tion and puncture (CLP) mouse model of sepsis. Both CCL3 (21)
and CCL6 (7) have been demonstrated to play protective roles
against sepsis-induced lethality and injury in a murine CLP model,
while the role of CCL5 has yet to be explored.
The purpose of this study was to investigate the role of CCR1 in the
innate immune response to experimental sepsis. CCR1⫺/⫺ mice were
significantly protected against CLP-induced lethality. Although
CCR1 deficiency had no effect on the inflammatory cell recruitment
to the peritoneal cavity, loss of the receptor promoted accelerated
cytokine expression and enhanced macrophage activity, both of
which contributed to a more efficient and regulated antibacterial
response. CCL5 was identified as a key modulator of the host
response that acted in a CCR1-dependent manner to trigger the
unregulated, exaggerated expression of proinflammatory cyto-
kines, resulting in increased injury and mortality following sepsis.
3
Abbreviations used in this paper: CLP, cecal ligation and puncture; TSA, thymic-
shared Ag; WT, wild type.

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00

The Journal of Immunology 6939

Materials and Methods
Mice
Specific pathogen-free wild-type (WT) BALB/c mice (6 – 8 wk of age)
were purchased from The Jackson Laboratory (Bar Harbor, ME). CCR1-
deficient (CCR1⫺/⫺) mice were generated, as previously described, and
were backcrossed onto a BALB/c genetic background (22). Mice were bred
and housed in the animal care facility (University Laboratory of Animal
Medicine) at the University of Michigan. The Animal Use Committee at
the University of Michigan approved all experimental procedures
involving mice.
Cecal ligation and puncture
CLP was used to induce acute septic peritonitis, as previously described
(23). Mice were anesthetized with a combination of 2.25 mg of ketamine
HCl (Abbott Laboratories, Chicago, IL) and 150 ␮g of xylazine (Lloyd
Laboratories, Shenandoah, IA) administered i.p. A 1-cm incision was made
to the lower left abdomen of the mouse, and the cecum was exposed. The
cecum was ligated distally with 3.0 silk suture and punctured through and
through with a 21- or 26-gauge needle. The cecum was returned to the
peritoneal cavity, and surgical staples were used to close the incision. Mice
immediately received 1 ml of saline s.c. for fluid resuscitation and were
warmed on a heating pad to facilitate their revival from the anesthetic.
of 300 cells from multiple high-powered fields, was multiplied by the total
peritoneal cell count to determine the number for each cell type. Three
independent experiments (three to five mice per group) showed similar
results, and data were pooled.
Measurement of cytokines and chemokines by ELISA
Concentrations of murine TNF-␣, IFN-␥, IL-12 (p70), IL-10, MIP-2, KC,
CCL2, CCL3, CCL5, CCL6, CCL17, CXCL9, and CXCL10 were mea-
sured in cell-free peritoneal lavage fluid, serum, and cell culture superna-
tants using a standardized sandwich ELISA previously described in detail
(25). Briefly, flat-bottom 96-well microtiter plates (Nunc, Roskilde, Den-
mark) were coated overnight at 4°C with mAb (for IL-10) or affinity-
purified polyclonal Abs for the specific cytokine of interest (R&D Systems,
Rochester, MN). Specific Ab for capture and detection of KC was obtained
from PeproTech. Plates were coated with 0.4 ␮g/ml (KC), 0.5 ␮g/ml
(IFN-␥, IL-12, CCL2, and CCL5), or 1.0 ␮g/ml (TNF-␣, IL-10, MIP-2,
CCL3, CCL6, CCL17, CXCL9, and CXCL10) appropriate capture Abs.
Plates were washed with PBS-Tween 20 (0.05%) and blocked with 2%
BSA in PBS for 90 min at 37°C. Plates were rinsed four times, and samples
were loaded and incubated at 37°C for 1 h. After washing, a biotinylated
secondary polyclonal Ab specific for the cytokine being measured (R&D
Systems; PeproTech for KC) was added at 0.5 ␮g/ml (or 0.25 ␮g/ml for
CCL5) for 30 min at 37°C. The plate was washed, and streptavidin-per-

Downloaded from http://www.jimmunol.org/ by guest on October 26, 2022

oxidase conjugate (Bio-Rad, Richmond, CA) was added to the wells for 30
Experimental protocols min at 37°C. This was followed by another set of washes before the ad-
dition of a chromogenic substrate (Bio-Rad). After full development oc-
The first set of survival studies was performed to determine the effect of the
curred, the reaction was stopped and the plate was read in an ELISA plate
presence of CCR1 on survival following induction of acute septic perito-
reader at 490 nm. Recombinant murine cytokines/chemokines (R&D Sys-
nitis in female and male mice. Due to differential susceptibility, female
tems) were used to generate standard curves, and concentrations were ex-
mice were subjected to 21-gauge CLP, while 26-gauge CLP was performed
pressed as ng or pg/ml. Limits of detection were ⬃50 pg/ml. ELISA spec-
on male mice. Survival of CLP groups (n ⫽ 8 –10 mice per group) was
ificity was confirmed for each assay. Experimental groups consisted of
monitored for 7 days following surgery. Duplicate survival studies for the
three to five mice or triplicate samples in vitro. ELISAs were run on sam-
female mice had similar results, and, therefore, the data were pooled. For
ples from three independent experiments, and representative data are
the remaining in vivo studies, female mice were used in the 21-gauge CLP
shown.
model, while peritoneal cells were isolated from both male and female
mice for in vitro analyses, as described later.
A second set of survival studies focused on the role of exogenous and
endogenous CCL5 (RANTES) in the context of sepsis induced by 21-
gauge CLP. Two hours following CLP, 1 ␮g of murine rCCL5 (PeproTech,
Rocky Hill, NJ) or 0.1 ml of saline was administered i.p. (n ⫽ 5–10 per
group). Endogenous CCL5 was blocked in mice using a previously de-
scribed neutralizing goat polyclonal Ab against CCL5 (24) kindly provided
by R. Strieter (University of California, Los Angeles, CA). CCL5-specific
IgG was purified from antiserum using a protein G column (Pierce, Rock-
ford, IL). Mice were pretreated with 0.1 ml containing 0.5 mg of CCL5 IgG
or control goat IgG (Sigma-Aldrich, St. Louis, MO) i.p. 2 h before CLP and
every 2 days following surgery. In these studies, survival was followed for
4 days after surgery. CCL5 administration (three studies) and neutralization
(two studies) experiments (n ⫽ 5–10 per group) produced similar survival
curves, and, therefore, data were pooled for each treatment group. Female
CCR1⫺/⫺ mice were similarly treated with either saline or 1 ␮g of rCCL5
i.p. 2 h after 21-gauge CLP (n ⫽ 9 per group), after which survival was
monitored for 4 days.
In other studies, naive and CLP mice (4, 8, and 24 h after surgery; n ⫽
3– 6 per group) were anesthetized and bled. The mice were euthanized, and
peritoneal lavages were performed with 2 ml of sterile saline. Lavage fluid
was collected and saved for bacteria, cell, and protein analyses.

Determination of CFU
Blood samples obtained for the purpose of determining CFU were imme-
diately mixed with EDTA (final 4 mM) to prevent coagulation. Peritoneal
lavage fluid and EDTA-treated blood from 8- or 24-h post-CLP were
placed on ice and serially diluted in sterile saline. A 10-␮l aliquot of each
dilution was spread on thymic-shared Ag (TSA) agar plates (Difco, Detroit,
MI) and incubated at 37°C overnight. Colonies were counted and ex-
pressed as CFU/10 ␮l. Groups contained four to six mice, and the exper-
iment was repeated on one to four different occasions. Results were similar
for each experiment and were subsequently pooled. The mean for each
group was calculated and indicated by a horizontal bar.

Peritoneal leukocyte counts and cell differentials
The total number of peritoneal leukocytes per lavage sample was deter-
mined by diluting 10 ␮l of lavage fluid with trypan blue and counting in a
hemocytometer. The total was expressed as leukocytes ⫻ 106 per cavity.
Differential cell analyses were performed on Diff-Quik-stained cytospin
preparations (Dade Behring, Düdingen, Switzerland) from peritoneal la-
vage fluid. The percentage for each leukocyte population, based on a count
FIGURE 1. CCR1-deficient mice were less susceptible to CLP-induced
lethality. A, Female WT BALB/c (CCR1⫹/⫹) and CCR1⫺/⫺ mice were
subjected to 21-gauge CLP. The graph represents pooled survival data from
duplicate studies showing similar results. B, Due to their increased sus-
ceptibility to sepsis, 26-gauge CLP was used to induce a less severe form
of sepsis in the male WT and CCR1⫺/⫺ mice. Experiments contained 8 –10
mice per group, and survival was followed for 7 days after surgery.
6940 CCR1 IN SEPSIS

Peritoneal macrophage isolation
Naive mice were euthanized and subjected to peritoneal lavages with 10 ml
of sterile saline containing 5 mM EDTA. Lavages were pooled for mice in
the same group (i.e., WT or CCR1⫺/⫺). RBC were lysed in ammonium
chloride buffer (150 mM NH4Cl, 10 mM NaHCO3, 1 mM EDTA-tetraso-
dium salt), and the remaining cells were thoroughly washed with saline.
Cells were counted and subjected to Diff-Quik staining, as described above,
to determine the number of peritoneal macrophages. Cells were resus-
pended in complete DMEM (BioWhittaker, Walkersville, MD) containing
5% FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 U/ml strepto-
mycin. Cells were plated in plastic plates and incubated 1–2 h at 37°C in
5% CO2. Nonadherent cells were removed, and adherent cells were washed
with complete DMEM. Cells were either treated immediately or rested
overnight, depending on the assay.
Phagocytosis assay
Escherichia coli strain O86a:K61 (American Type Culture Collection, Ma-
nassas, VA) was grown to mid-log phase in tryptic soy broth (Difco, De-
troit, MI) and used to infect freshly isolated naive macrophages (106 mac-
rophages per well) in 24-well cell culture plates (Corning Glass, Corning,
NY). Cells were infected in antibiotic-free DMEM containing 5% FCS and
2 mM L-glutamine at a 1:1 ratio of macrophages to bacteria. After 1 h at
remove extracellular bacteria. The cells were lysed with 0.5 ml of sterile
0.5% Triton X-100 (Sigma-Aldrich). Serial 10-fold dilutions of the lysates
were plated on TSA agar plates, and CFU were enumerated, as described
above.
Nitrite production
NO, one of the major antimicrobial effector molecules generated by mac-
rophages, is difficult to measure directly due to its very short t1/2. In vitro,
concentrations of nitrite (NO2⫺), a stable, oxidative end product of NO, are
assessed from the medium of activated macrophages as an indirect indi-
cator of NO production by these cells (26). Naive macrophages were plated
at 5 ⫻ 105/well of a 96-well cell culture plate (Corning Glass) and rested
overnight. Fresh DMEM (complete with 5% FCS) was added to each well,
and cells were treated with medium alone, 20 U/ml IFN-␥ (PeproTech),
100 ng/ml LPS O55:B5 (Sigma-Aldrich), or both IFN-␥ and LPS (n ⫽ 3– 6
per treatment group). After 48 h at 37°C in 5% CO2, 50 ␮l of cell-free
supernatants was transferred to a flat-bottom 96-well plate and treated with
100 ␮l of 0.5% sulfanilamide (Sigma-Aldrich) and 0.05% naphthylethyl-
enediamine dihydrochloride (Sigma-Aldrich) in 2.5% phosphoric acid
(H3PO4). The absorbance was read at 550 nm in a microplate reader. A
standard curve was generated using known concentrations of sodium nitrite
(NaNO2; Sigma-Aldrich) in DMEM. Similar results were shown in at least

Downloaded from http://www.jimmunol.org/ by guest on October 26, 2022

37°C in 5% CO2, cells were washed four to five times with medium to three independent experiments. In vivo, NO2⫺ can be further oxidized to

FIGURE 2. CCR1⫺/⫺ mice had significantly en-

hanced bacterial clearance and reduced bacteremia after
CLP. At 8 or 24 h after CLP, mice (n ⫽ 3– 6 per group)
were bled and subjected to 2-ml peritoneal washes with
sterile saline. Serial 10-fold dilutions of peritoneal la-
vage fluid (A) and EDTA-treated blood (B) were spread
on TSA plates and incubated overnight at 37°C. Levels
of bacteria were expressed as CFU per 10 ␮l. The
graphs depict data pooled from one to four independent
studies showing similar results. The horizontal bar in-
dicates the mean for each group. C, Peritoneal lavage
fluid was collected from WT (f) and CCR1⫺/⫺ (䡺)
mice at 0, 4, 8, or 24 h post-CLP (n ⫽ 3–5 per group).
Nitrate (NO3⫺) was converted into nitrite (NO2⫺), and
then total nitrite concentration was assessed. The graph
depicts data from three pooled experiments. ⴱ, p ⬍ 0.05
compared with WT-CLP mice.
The Journal of Immunology 6941

FIGURE 3. CCR1 had no significant effect on leu-

kocyte recruitment to the peritoneal cavity after CLP.
Mice were euthanized at 0, 4, 8, or 24 h post-CLP, and
peritoneal lavages (2 ml) were collected (n ⫽ 4 – 6 per
group). A, The total number of peritoneal cells was
counted for each individual mouse. Data were collected
from three independent experiments. B, Differential counts
of mononuclear cell and neutrophil populations were per-
formed from Diff-Quik-stained cytospins prepared from
each lavage. Data were analyzed from two different ex-
periments. The results for total counts and differentials
were similar in all experiments and were pooled. The
graphs depict averages for each group ⫾ SEM.

Downloaded from http://www.jimmunol.org/ by guest on October 26, 2022

form nitrate (NO3⫺). To measure total in vivo NO activity in samples,
NO3⫺ was first converted to NO2⫺, and then total NO2⫺ concentration was
measured. Peritoneal lavage fluid from naive and CLP mice was mixed
with an equal volume of nitrate reductase mixture (1 mg/ml NADPH and
0.4 U/ml nitrate reductase (Sigma-Aldrich) in 0.2 M KH2PO4, pH ⫽ 7.4)
and incubated overnight at 37°C/5%CO2. The next day, 40 ␮l of 0.5%
sulfanilamide and 0.05% napthylethylenediamine dihydrochloride in 2.5%
H3PO4 were added to each standard and sample, and the absorbance was
read at 550 nm. Total NO2⫺ in peritoneal lavage fluid was measured in
three different experiments. Each experiment exhibited similar results and
was consequently pooled.
In vitro LPS stimulation
tration. Twenty micrograms of each extract was analyzed according to the
manufacturer’s instructions for the ELISA kit. The absorbance at 450 nm
was read, and background absorbance (negative control) was subtracted
from each sample.
Statistics
In the case of survival studies, the log rank test was used to evaluate
significance. All other data are shown as the mean ⫾ SEM. Bacterial CFU
data were analyzed with the nonparametric Mann-Whitney U test, while all
other data were subjected to the unpaired Student’s t test. A p value ⬍0.05
(ⴱ) was considered statistically significant. Highly significant p values
⬍0.005 (ⴱⴱ) and ⬍0.0005 (ⴱⴱⴱ) were also indicated.

Naive peritoneal macrophages were plated at 5 ⫻ 105/well of a 24-well cell Results

culture plate (Corning Glass). After resting overnight, macrophages were
treated with medium alone or 1 ␮g/ml LPS O55:B5 (Sigma-Aldrich). Cells
were incubated 24 h at 37°C/5% CO2, after which cell-free supernatants
were collected. Cytokine/chemokine production was measured by ELISA
(as described above) in at least three experiments. All experiments had
similar results.
NF-␬B p65 ELISA
Four WT and CCR1⫺/⫺ mice were injected with 3 ml of sterile 4% thio-
glycolate i.p. After 5 days, peritoneal cells were isolated, as described
above, and 107 macrophages were plated per 100-mm cell culture plate
(Corning Glass). Macrophages were rested overnight and then treated with
10 –100 ng/ml CCL5, 1 ␮g/ml LPS, or both CCL5 and LPS. Untreated
cells were included as a control. Four hours later, nuclear extracts were
prepared according to manufacturer’s instructions and analyzed using the
TransAM NF-␬B p65 transcription factor assay kit (Active Motif, Carls-
bad, CA). Briefly, cells were washed and scraped into ice-cold PBS con-
taining 6.25 mM NaF, 12.5 mM ␤-glycerophosphate, 12.5 mM para-nitro-
phenyl phosphate, and 1.25 mM NaV03 (Sigma-Aldrich). Cells were
pelleted and resuspended in ice-cold hypotonic buffer (20 mM HEPES, pH
7.5, 5 mM NaF, 10 ␮M Na2MoO4, and 1 mM EDTA; Sigma-Aldrich).
After 15 min on ice, cells were lysed with 0.5% Nonidet P-40 (Sigma-
Aldrich). The nuclear fraction was pelleted at 15,000 ⫻ g for 15 min and
resuspended in complete lysis buffer. After 30 min of agitation at 4°C,
debris was removed by pelleting, and nuclear extracts were snap frozen in
a dry ice/ethanol bath. Extracts were stored at ⫺80°C. Before the ELISA,
extracts were subjected to Bradford analysis to determine protein concen-
CCR1-deficient mice were significantly less susceptible to
CLP-induced lethality
To determine the role of CCR1 in a murine model of experimental
sepsis, both WT BALB/c (CCR1⫹/⫹) and CCR1⫺/⫺ mice were
subjected to CLP. In female WT mice, 21-gauge CLP was almost
100% lethal (19 of 20 mice) as early as 5 days after CLP (Fig. 1A).
However, female CCR1⫺/⫺ mice were significantly protected
against CLP-induced lethality. They experienced only 41% mor-
tality (7 of 17 mice) at day 5 and 59% mortality (10 of 17 mice)
at day 7, demonstrating 54 and 36% increased survival, respec-
tively. Due to enhanced susceptibility of male mice to CLP, a
26-gauge needle was used to induce sepsis in these mice. As
shown in Fig. 1B, even in this less severe form of sepsis, complete
mortality was observed in WT mice at the end of the experiment.
Once again, male CCR1⫺/⫺ mice were more resistant to these
lethal effects. None of the CCR1⫺/⫺ mice died until 3 days post-
CLP, at which time 90% of the WT mice (9 of 10 mice) were dead.
At day 7 post-CLP, 40% of the CCR1⫺/⫺ mice survived (4 of 10
mice) compared with 0% of the WT mice. The rest of this inves-
tigation focused on identifying the immunomodulatory mechanism
in CCR1⫺/⫺ mice that resulted in decreased CLP susceptibility
and increased survival.
6942 CCR1 IN SEPSIS

FIGURE 4. Cytokine and che-

Downloaded from http://www.jimmunol.org/ by guest on October 26, 2022

mokine expression were signifi-
cantly altered in CCR1⫺/⫺ mice
following CLP. At 0, 4, 8, or 24 h
post-CLP, mice were sacrificed
and bled, and 2-ml peritoneal la-
vages were performed. Concen-
trations of cytokines/chemokines
in serum (A) and lavage fluid (B)
from WT BALB/c mice (f) and
CCR1⫺/⫺ mice (䡺) were mea-
sured using specific sandwich
ELISAs. The data from three in-
dependent experiments were sim-
ilar, and representative data are
shown (n ⫽ 3–5 per group). ⴱ,
p ⬍ 0.05; ⴱⴱ, p ⬍ 0.005; ⴱⴱⴱ,
p ⬍ 0.0005, compared with sim-
ilarly treated WT mice.

CCR1⫺/⫺ mice displayed increased bacterial clearance
subsequent to accelerated production of cytotoxic mediators
following CLP
The process of CLP causes leakage of the polymicrobial flora into
the peritoneal cavity. Peritoneal lavages were performed on mice
to assess the degree of local infection over time, while blood was
collected to measure systemic spread of the infection. The amount
of bacteria recovered from the peritoneal cavities of CCR1⫺/⫺
mice was significantly lower than WT mice (Fig. 2A). At 8 h
post-CLP, CCR1⫺/⫺ mice had an average of 0.11 ⫻ 102 CFU/10
␮l peritoneal lavage fluid, while WT levels were significantly el-
evated (2.14 ⫻ 103 CFU/10 ␮l). At 24 h post-CLP, mean bacterial
burdens were 28-fold higher in WT mice (2.66 ⫻ 106 CFU/10 ␮l)
compared with CCR1⫺/⫺ mice (9.52 ⫻ 104 CFU/10 ␮l). In WT
mice, decreased clearance and containment of bacteria at the local
site of infection (peritoneal cavity) contributed to exacerbated
The Journal of Immunology
spread throughout the animal (Fig. 2B). WT mice had dramatically
higher systemic levels of bacteria at both 8 and 24 h post-CLP
(1.25 ⫻ 103 and 5.25 ⫻ 103 CFU/10 ␮l, respectively), when com-
pared with CCR1⫺/⫺ mice (0.10 ⫻ 102 and 2.80 ⫻ 102 CFU/10
␮l, respectively).
Both macrophages and neutrophils produce a variety of nitrogen
and oxygen radicals that contribute to their repertoire of antimi-
crobial activity. Peritoneal lavage fluid was collected from mice at
various times after CLP and evaluated for nitrite, a stable byprod-
uct of NO (Fig. 2C). After CLP, peritoneal nitrite concentrations
steadily increased in WT mice and were highest at 24 h. In con-
trast, nitrite concentrations in CCR1⫺/⫺ mice peaked and were
higher than in WT mice at 4 h after CLP (12.49 ⫾ 2.80 vs 6.24 ⫾
0.66 ␮M in CCR1⫺/⫺ and WT mice, respectively). Nitrite con-
centrations in CCR1⫺/⫺ mice remained steady without further in-
crease over the 24-h period. At 24 h post-CLP, these levels were
significantly lower than the amount of nitrite produced in WT mice
(11.90 ⫾ 2.84 vs 44.51 ⫾ 11.38 ␮M in WT), directly correlating
with the decreased bacterial load, and, therefore, decreased stim-
ulus, present in CCR1⫺/⫺ mice at that time.
CCR1 was nonessential to the chemotaxis of leukocytes to the
peritoneal cavity in response to sepsis
One obvious explanation for the increased bacterial clearance and
accelerated nitrite production observed in CCR1⫺/⫺ mice was po-
tential differences in the leukocyte populations recruited to the
peritoneal cavity following CLP. Therefore, peritoneal lavages
were examined from WT and CCR1⫺/⫺ mice at various times after
CLP to assess leukocyte infiltration (Fig. 3). In these studies, CLP
was a strong stimulus for recruitment of cells into the peritoneal
cavity; however, no differences were observed in total numbers of
leukocytes between the two groups (Fig. 3A). As expected, a ro-
bust neutrophilic response occurred quickly after CLP in WT mice
(peaking at 8 –24 h; Fig. 3B). No discrepancies were noted in
mononuclear cell and neutrophil recruitment between WT and
CCR1⫺/⫺ mice after CLP, indicating that enhanced bacterial clear-
ance in the CCR1⫺/⫺ mice was not due to changes in cellular
recruitment.
Temporal changes in expression and an altered
cytokine/chemokine profile were observed in CCR1⫺/⫺ mice
following CLP
Studies have shown that following CLP, the local and systemic
expression of many cytokines and chemokines is augmented.
Therefore, the cytokine profiles of naive and CLP-treated WT and
CCR1⫺/⫺ mice were measured in blood and peritoneal lavage fluid
at various times after surgery. CCR1⫺/⫺ mice produced signifi-
cantly higher levels of systemic IFN-␥ and CCL2 than WT mice
4 h after CLP (Fig. 4A). Lower levels of these cytokines, as well
as MIP-2 and KC, were observed later after CLP, compared with
WT. In general, peritoneal cytokine and chemokine expression
was either unchanged or decreased at later times (8 and 24 h) in
CCR1⫺/⫺ mice when compared with WT mice (Fig. 4B). In WT
mice, peritoneal TNF-␣ levels were induced at 4 h post-CLP and
were significantly amplified throughout the 24-h period after CLP.
Conversely, TNF-␣ peaked at 4 h in CCR1⫺/⫺ mice and was sig-
nificantly reduced in comparison with WT levels at 8 and 24 h
post-CLP. Several inflammatory chemokines (MIP-2, KC, CCL2,
and CXCL9) were significantly higher in WT mice, compared with
CCR1⫺/⫺ mice at 8 and/or 24 h post-CLP. Conversely, peritoneal
CXCL10, CCL6, and CCL17 expression in CCR1⫺/⫺ mice was
significantly increased over WT expression at 8 h post-CLP.
6943
CCR1⫺/⫺ macrophages had enhanced innate immune responses
Although there were no apparent differences in leukocyte recruit-
ment to the peritoneal cavity, key differences were observed in
bacterial clearance and cytokine/chemokine expression in
CCR1⫺/⫺ mice. These data implied that intrinsic differences ex-
isted in the cellular activation of cells within the peritoneal cavity
of these animals. The next series of experiments were designed to
assess the specific effects of CCR1 deficiency on resident perito-
neal macrophages.
Peritoneal macrophages were isolated from naive WT and
CCR1⫺/⫺ mice and infected in vitro with E. coli. Phagocytosis
was 7.3-fold higher in CCR1⫺/⫺ macrophages (mean ⫽ 3.7 ⫻ 104
CFU/well) than WT macrophages (mean ⫽ 5.1 ⫻ 103 CFU/well)
after 1 h of infection (Fig. 5A). The combined stimulus of IFN-␥
and LPS was required to stimulate production and release of nitrite
from WT macrophages (15.34 ⫾ 0.63 ␮M) in culture (Fig. 5B).
However, CCR1⫺/⫺ macrophages were activated by IFN-␥ alone
(14.86 ⫾ 0.56 ␮M) and demonstrated a greatly amplified response
to the combination of IFN-␥ and LPS (36.9 ⫾ 1.1 ␮M).
Downloaded from http://www.jimmunol.org/ by guest on October 26, 2022
In other experiments, naive peritoneal macrophages were stim-
ulated in vitro with LPS. Supernatants were collected 24 h later
and assayed by ELISA. CCR1⫺/⫺ macrophages expressed signif-
icantly higher constitutive levels of MIP-2, CCL2, CCL5, CCL6,
and CCL22 (Fig. 6). Although LPS stimulated substantial produc-
tion of multiple cytokines and chemokines from WT macrophages,
it provoked significantly higher levels from CCR1⫺/⫺ macro-
phages (Fig. 6).
FIGURE 5. Macrophages deficient in CCR1 displayed enhanced phago-
cytic and cytotoxic activity. A, Peritoneal cells were isolated from naive
WT (f) and CCR1⫺/⫺ mice (䡺) and plated at 106 macrophages per well
(24-well plate). Nonadherent cells were removed, and macrophages were
infected with 106 E. coli for 1 h at 37°C. Extracellular bacteria were re-
moved, cells were lysed, and concentrations of CFU were determined.
Results were similar from two independent studies, and representative data
are shown. Horizontal bars represent the mean for each group (n ⫽ 5– 6).
B, Naive macrophages were plated (5 ⫻ 105/well of 96-well plate) and
rested overnight. WT (f) and CCR1⫺/⫺ (䡺) macrophages were untreated
or stimulated with 100 ng/ml LPS, 20 U/ml IFN-␥, or LPS ⫹ IFN-␥ for
48 h. Nitrite (NO2⫺) concentrations were measured in the supernatants
(n ⱖ 3 per group). Similar results were obtained from three independent
studies, and representative data are shown. ⴱⴱⴱ, p ⬍ 0.0005 compared with
similarly treated WT macrophages.
6944
FIGURE 6. Cytokine/chemokine production was
significantly increased in CCR1⫺/⫺ macrophages. Na-
ive macrophages from WT (f) and CCR1⫺/⫺ mice (䡺)
were treated with medium or 1 ␮g/ml LPS for 24 h.
Supernatants were collected and clarified, and cytokine/
chemokine levels were measured by ELISA (n ⫽ 3 per
group). Results are presented as average for the
group ⫾ SEM. Representative data are shown for four
independent experiments. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.005;
ⴱⴱⴱ, p ⬍ 0.0005 compared with similarly treated WT
macrophages.
CCL5 increased lethality in CLP-induced sepsis
CCR1 ⫺/⫺
mice were clearly protected against the lethal effects of
sepsis, and their macrophages demonstrated significantly enhanced
innate immune responses. Because CCL3 and CCL6 were previ-
ously recognized to play protective roles in this model of sepsis (7,
21), we investigated the role of CCL5 in this model. CCL5 ex-
pression was strongly up-regulated following CLP in WT and
CCR1⫺/⫺ mice (Fig. 7). It was detected as early as 4 h in the
peritoneal cavity; however, peak expression was not observed until
at least 24 h after CLP in the peritoneal cavity and the serum of
both mouse strains (Fig. 7). WT-CLP mice expressed significantly
higher systemic levels of CCL5 at 24 h post-CLP than CCR1⫺/⫺
mice. When rCCL5 was administered 2 h post-CLP, day 4 mor-
tality was significantly higher (88%) than in CLP-saline-treated
mice (59% mortality; p ⫽ 0.03; Fig. 8A). In contrast, when en-
dogenous CCL5 was blocked with a specific goat anti-murine
CCL5 IgG 2 h prior and 48 h after CLP, survival was significantly
improved (Fig. 8B). Two days after CLP, only 30% of control
IgG-treated mice were alive, whereas 80% of mice treated with
anti-CCL5 IgG were remaining ( p ⫽ 0.0036). When CCR1⫺/⫺
mice were treated with rCCL5 2 h following CLP (Fig. 8C), no
significant effect was seen on the 4-day mortality (1 of 9 mice)
when compared with saline-treated CCR1⫺/⫺-CLP mice (3 of 9
mice; p ⫽ 0.32).
CCR1 IN SEPSIS
Downloaded from http://www.jimmunol.org/ by guest on October 26, 2022
CCL5 acted through CCR1 to increase NF-␬B activation and
CLP-induced inflammatory cytokine production
When thioglycolate-elicited peritoneal macrophages were treated
with CCL5 in vitro, no significant differences were observed in the
NF-␬B activation of WT or CCR1⫺/⫺ cells; however, CCR1⫺/⫺
macrophages had slightly higher constitutive levels of activation
(Fig. 9A). LPS induced similar levels of NF-␬B activation in WT
and CCR1⫺/⫺ macrophages. In the context of LPS stimulation,
CCL5 caused a dose-dependent increase in NF-␬B activation in
WT, but not CCR1⫺/⫺, macrophages. These data provide evidence
that CCL5 interacts primarily with CCR1 in vitro.
When rCCL5 was administered 2 h after CLP, peritoneal ex-
pression of IFN-␥ and MIP-2 was amplified 264 and 125%, re-
spectively, in WT mice (Fig. 9B), while CCR1⫺/⫺ mice were un-
responsive to the effects of this ligand. WT production of
peritoneal KC, CCL22, and CXCL9 (53, 51, and 72%, respec-
tively) as well as systemic IL-12, IFN-␥, and KC (81, 69, and
104%, respectively) was also augmented to a lesser degree by
CCL5 treatment after CLP (data not shown). No changes were
observed in CCL3 expression following CCL5 administration in
naive or CLP-treated WT or CCR1⫺/⫺ mice (data not shown).
Cytokine/Chemokine expression in CCR1⫺/⫺-CLP mice remained
unchanged. In contrast, CCL5 appeared to have no effect on the
expression of protective chemokines, such as CXCL10 or CCL6,
The Journal of Immunology
FIGURE 7. CLP induced local and systemic expression of CCL5 in WT
and CCR1⫺/⫺ mice. At 0, 4, 8, or 24 h post-CLP, mice were sacrificed and
bled, and 2-ml peritoneal lavages were performed. CCL5 was measured in
peritoneal wash (PW) fluid (A) and serum (B) from WT (f) and CCR1⫺/⫺
mice (䡺) using a specific ELISA. The data are representative of two in-
dependent studies (n ⫽ 3–5 per group). ⴱ, p ⬍ 0.05 compared with sim-
ilarly treated WT mice.
which continued to be expressed at significantly higher levels in
CCR1⫺/⫺ mice. These data independently verified the interaction
of CCL5 with CCR1 in a relevant sepsis model. Most likely, the
exaggerated inflammatory cytokine expression observed after CLP
in WT CCL5-treated mice was subsequent to the previously ob-
served augmentation of CCR1-dependent NF-␬B activation.
Discussion
Mounting evidence emphasizes the importance of both activated
neutrophils and macrophages for an effective host response against
pathogen infections, including sepsis (8). CCR1, a widely ex-
pressed chemokine receptor found on both of these cell types, has
proven to be fundamental in cellular trafficking and activation (13)
as well as performing a central role in modulating the cytokine
balance in several immunological models (16). In light of the
widespread distribution of this receptor, especially on phagocytic
cells, we were interested in the role that CCR1 plays in the host
response to sepsis. This study clearly indicated that CCR1, while
dispensable for leukocyte recruitment, was a pivotal focus of the
immune response to sepsis in which the balance of pro- and anti-
inflammatory cytokines rested. Additionally, CCR1 regulated
macrophage responses to bacterial infection and LPS stimulation.
CCL5 functioned in a CCR1-dependent manner to detrimentally
amplify proinflammatory cytokine production and increase sepsis-
related mortality.
Cytokines are often described as double-edged swords in that they
are required for recruitment and activation of cells necessary for re-
sponding to a foreign insult; however, when produced in excess pre-
cipitate damage against the host for which they were produced to
protect. A successful response to infection, as in sepsis, is comprised
of a complex balance of pro- and anti-inflammatory cytokines. A local
type 1 cytokine-driven response (dominated by IFN-␥ and IL-12) is
crucial to contain and eliminate the source of the infection (23), while
a systemic type 2 cytokine response (i.e., IL-10) is essential to mod-
ulate the inflammatory response, protecting the host from tissue and
organ injury (27). Several CC and CXC chemokines have also been
6945
Downloaded from http://www.jimmunol.org/ by guest on October 26, 2022
FIGURE 8. CCL5 significantly increased mortality of mice following
CLP. A, Female WT BALB/c mice received saline (f) or 1 ␮g of CCL5
(䡺) i.p. 2 h post-CLP. B, WT mice received 0.5 mg of control (f) or
anti-murine CCL5 IgG (䡺) i.p. 2 h before and 48 h after CLP. Similar
results were observed in replicate studies; therefore, data were pooled. C,
Female CCR1⫺/⫺ mice received saline (f) or 1 ␮g of CCL5 (䡺) i.p. 2 h
post-CLP. Survival was followed for 4 days after surgery (n ⫽ 5–10 per
group).
shown to exert pro- and anti-inflammatory effects during the septic
response (7, 8, 28).
IFN-␥ is a powerful inducer of NO, and, therefore, promotes
bacterial clearance and host survival (29). CCR1-deficient mice
responded more rapidly to CLP and attained earlier peak systemic
expression of IFN-␥ (4 vs 24 h in WT). Simultaneously, increased
concentrations of nitrite were measured in the peritoneal cavities
of CCR1⫺/⫺ CLP mice. Consequently, significantly lower local
and systemic levels of bacteria were found in these mice, com-
pared with WT-CLP mice, indicating that an accelerated antimi-
crobial response was highly effective. Despite radically amplified
IFN-␥ and nitrite production at later times after CLP (8 and 24 h),
WT mice were not able to effectively control the infection, once
again emphasizing the importance of an early response.
CLP also induced quicker systemic expression of CCL2 in
CCR1⫺/⫺ mice (4 h), compared with 24-h peak expression in
WT-CLP mice. CCL2 has previously been shown to increase CLP
survival by promoting activation of macrophages and production
of anti-inflammatory cytokines, protecting the host from excessive
inflammation and resulting tissue damage (28, 30). This immuno-
modulation was observed in the CCR1⫺/⫺ mice at 8 and 24 h
post-CLP when systemic expression of inflammatory cytokines/
6946
FIGURE 9. CCL5 increased NF-␬B activation by
LPS in vitro and CLP-induced inflammatory cytokine
expression in vivo. A, Thioglycolate-elicited macro-
phages from WT (f) and CCR1⫺/⫺ mice (䡺) were
treated in culture with 0, 10, or 100 ng/ml CCL5 ⫾ LPS
for 4 h. Nuclear extracts were prepared and assayed by
ELISA for the p65 subunit of NF-␬B. Absorbance was
read at 450 nm. B, WT (f) and CCR1⫺/⫺ mice (䡺)
were subjected to 21-gauge CLP, followed 2 h later by
i.p. injection with saline or 1 ␮g of CCL5. Additional
mice received saline or CCL5 with no surgery. Four
hours after administration of saline/CCL5 (6 h post-
CLP), mice were sacrificed and peritoneal lavages were
performed. Cytokine/chemokine concentrations were
measured in lavage fluid using specific ELISAs. ⴱ, p ⬍
0.05; ⴱⴱ, p ⬍ 0.005; ⴱⴱⴱ, p ⬍ 0.0005 compared with
similarly treated WT mice (n ⫽ 5 per group); #, p ⬍
0.05 compared with CLP (without CCL5).
chemokines and nitrite production were significantly lower than in
WT-CLP mice. Similar regulation occurred in the peritoneal cav-
ity, where inflammatory mediators peaked at 4 h post-CLP in
CCR1⫺/⫺ mice, but continued to rise throughout the 24-h period
after CLP in WT mice. TNF-␣, while critical to the immune re-
sponse, is able to solely initiate many of the immunopathological
features of septic shock (31), and, therefore, must be rapidly reg-
ulated. Its continued expression may serve to propagate the ex-
tended inflammatory response observed after CLP in WT mice.
Interestingly, CCR1⫺/⫺ mice showed significantly increased
peritoneal expression of CXCL10, CCL6, and CCL17 when com-
pared with WT mice at 8 –24 h post-CLP. Studies from our labo-
ratory previously demonstrated major protective roles for both
CXCL10 and CCL6 against CLP-induced lethality (7, 11).
CXCL10 acted through an unidentified mechanism, while CCL6
augmented peritoneal macrophage activity and reduced bacterial
leak from the gut. The role of CCL17, a predominantly recognized
Th2 lymphocyte chemoattractant, in sepsis is unknown. Its expres-
sion may contribute to the regulation of the proinflammatory type
1 response in CCR1⫺/⫺ mice after CLP.
Peritoneal macrophages are the principal resident cells, and are
known to play an essential role in infection and inflammation by
producing a large variety of mediators, including cytokines/che-
mokines, in response to infectious bacteria or bacterial components
such as LPS (32). Previous work from our laboratory has illus-
trated the importance of these cells in the CLP model of sepsis (7,
CCR1 IN SEPSIS
Downloaded from http://www.jimmunol.org/ by guest on October 26, 2022
8, 11). In the absence of altered leukocyte recruitment after CLP,
changes in bacterial clearance and cytokine expression suggested
intrinsic differences in the activation of WT and CCR1⫺/⫺ peri-
toneal cells. In vitro analyses of CCR1⫺/⫺ macrophages demon-
strated overall enhanced innate responses, including increased
phagocytosis, cytotoxicity, and cytokine/chemokine production.
These data support the accelerated responses seen in CCR1⫺/⫺
mice after CLP and emphasize the central function that peritoneal
macrophages execute in the innate response following sepsis.
In this study, we confirmed that the CCR1 ligands, CCL3 and
CCL6, were produced in WT mice following CLP (7, 20). How-
ever, this study was the first to explore the expression and role of
the CCR1 ligand, namely CCL5, in a CLP model of sepsis. Pre-
viously, it was shown that CCL5 was elevated in septic patients as
well as volunteers treated with LPS (19, 33). In a colon ascendens
stent peritonitis model of sepsis, CCL5 induction was present dur-
ing renal failure (34). In the CLP model, local and systemic ex-
pression of CCL5 was strongly provoked in WT mice. CCR1⫺/⫺
expression of CCL3 and CCL5 was equal to or less than WT
expression at all times after CLP. In contrast, CCL6 expression
was much higher in CCR1⫺/⫺ than WT mice at 8 and 24 h after
CLP, supporting the idea that CCL6 has only one receptor in vivo
(18). The elevations in CCL6 presumably reflected a continued
accumulation of ligand in the absence of functional receptor to
bind and regulate its production. Both CCL3 and CCL5 are known
to interact with multiple receptors, principally CCR1 and CCR5
The Journal of Immunology
(12). In the absence of CCR1, mice were protected against the
deleterious effects of sepsis, indicating that CCR1 is a central
player in this injurious response. Its ligand counterpart was pre-
dicted to have a similar detrimental effect. Considering the previ-
ously established protective roles of CCL3 (21) and CCL6 (7) in
this model, CCL5 was the obvious ligand to consider. In our
model, CCL5 demonstrated a negative effect on CLP-associated
survival in wild-type, but not CCR1⫺/⫺ mice, implicating its in-
volvement as another key mediator of the deleterious host
response.
CCR1⫺/⫺ mice and macrophages were nonresponsive to CCL5,
indicating that CCL5 interacted directly with CCR1. Although
CCL3 is generally thought to be the principal in vivo ligand
for CCR1, other studies have demonstrated CCL5 action through
CCR1. CCL5 was identified as a potent agonist that stimulated
CCR1-dependent chemotaxis of mast cells (35). CCR1 was also
recognized as the major receptor for CCL5 binding in memory and
naive CD4⫹ T cells, as well as acting in CCL5-induced chemo-
taxis of memory T cells (36). Although CCL5 has been predom-
inantly studied as an important mediator of type 2-driven diseases,
it has become apparent that the regulatory function of CCL5 has
broader implications than previously recognized. CCL5 is a potent
stimulus for macrophage recruitment into the lung following en-
dotoxemia (37) as well as into synovial tissue during rheumatoid
arthritis, propagating the chronic inflammation that is characteris-
tic of the disease (38). Intradermal injection of rCCL5 results in
pathology characterized by monocytic and eosinophilic inflamma-
tory responses (39). CCL5 acts not only as a chemoattractant, but
operates synergistically with IFN-␥ to activate macrophages, NK
cells, and T cells, alluding to its central role in modulating type 1
immune responses (40).
In the sepsis model, CCL5 acted to amplify the expression of
inflammatory cytokines in WT mice without affecting the produc-
tion of protective mediators such as CXCL10 and CCL6. CCL5
triggered an overwhelming IFN-␥ response within the peritoneal
cavity after CLP in WT mice. Although the protective function of
IFN-␥ has been demonstrated in a number of infection models (41)
as well as in the CCR1⫺/⫺ mice following CLP, overproduction or
abundance of this cytokine directly after CLP significantly in-
creases lethality (42, 43). In our model, CCL5 also up-regulated
MIP-2 production, as well as marginally affecting the expression
of other inflammatory cytokines/chemokines. It is well-established
that MIP-2 contributes to neutrophil recruitment, resulting in host
injury and CLP mortality (20). CCL5 may augment type 1 cyto-
kine production directly or indirectly by decreasing anti-
inflammatory type 2 cytokine expression. Given that the levels of
type 2 cytokines are very low during the acute phase after CLP, it
is more likely that CCL5 acts directly on the expression of type 1
cytokines. Locati et al. (44) have previously shown that CCL5
stimulation of human monocytes in vitro induces RNA expression
of key proinflammatory mediators IL-1␤ and IL-8 (human homo-
logue of MIP-2) as well as other inflammatory chemokines such as
CCL2 and CCL3. Additionally, in vitro LPS activation of the in-
flammatory p65 subunit of NF-␬B was amplified in a dose-depen-
dent manner by CCL5, lending further credence to a direct effect
of CCL5 on the inflammatory response.
In summary, we have shown that genetic deletion of CCR1 in-
creased survival in septic mice. Although no effect on leukocyte
recruitment was observed, CCR1⫺/⫺ mice exhibited an acceler-
ated, effective antimicrobial response that was quickly down-reg-
ulated following bacterial clearance. Peritoneal macrophages from
CCR1⫺/⫺ mice displayed enhanced innate immune responses that
were also differentially regulated. We demonstrated that CCL5
production after CLP had a negative effect on survival, in contrast
6947
to CCL3 and CCL6, other previously identified CCR1 ligands.
Exogenous CCL5 administered after CLP induced a hyperinflam-
matory response dominated by IFN-␥ in WT, but not in CCR1⫺/⫺
mice, indicating that the deleterious actions of CCL5 were CCR1
dependent. Thus, these data suggested that CCR1 and CCL5 are
both key mediators in modulating the innate inflammatory re-
sponse generated during a septic insult and are potential targets for
immunotherapy in septic patients.
References
1. Angus, D. C., W. T. Linde-Zwirble, J. Lidicker, G. Clermont, J. Carcillo, and
M. R. Pinsky. 2001. Epidemiology of severe sepsis in the United States: analysis
of incidence, outcome, and associated costs of care. Crit. Care Med. 29:1303.
2. Zanotti, S., and A. Kumar. 2002. Cytokine modulation in sepsis and septic shock.
Exp. Opin. Investig. Drugs 11:1061.
3. Esmon, C. T. 2002. Protein C pathway in sepsis. Ann. Med. 34:598.
4. Bhatia, M., and S. Moochhala. 2004. Role of inflammatory mediators in the
pathophysiology of acute respiratory distress syndrome. J. Pathol. 202:145.
5. Makhija, R., and A. N. Kingsnorth. 2002. Cytokine storm in acute pancreatitis.
J. Hepatobiliary Pancreat. Surg. 9:401.
6. Baggiolini, M., B. Dewald, and B. Moser. 1994. Interleukin-8 and related che-
motactic cytokines: CXC and CC chemokines. Adv. Immunol. 55:97.
Downloaded from http://www.jimmunol.org/ by guest on October 26, 2022
7. Steinhauser, M. L., C. M. Hogaboam, A. Matsukawa, N. W. Lukacs,
R. M. Strieter, and S. L. Kunkel. 2000. Chemokine C10 promotes disease reso-
lution and survival in an experimental model of bacterial sepsis. Infect. Immun.
68:6108.
8. Matsukawa, A., C. M. Hogaboam, N. W. Lukacs, P. M. Lincoln, H. L. Evanoff,
and S. L. Kunkel. 2000. Pivotal role of the CC chemokine, macrophage-derived
chemokine, in the innate immune response. J. Immunol. 164:5362.
9. Cummings, C. J., T. R. Martin, C. W. Frevert, J. M. Quan, V. A. Wong,
S. M. Mongovin, T. R. Hagen, K. P. Steinberg, and R. B. Goodman. 1999.
Expression and function of the chemokine receptors CXCR1 and CXCR2 in
sepsis. J. Immunol. 162:2341.
10. Chvatchko, Y., A. J. Hoogewerf, A. Meyer, S. Alouani, P. Juillard, R. Buser,
F. Conquet, A. E. Proudfoot, T. N. Wells, and C. A. Power. 2000. A key role for
CC chemokine receptor 4 in lipopolysaccharide-induced endotoxic shock. J. Exp.
Med. 191:1755.
11. Ness, T. L., C. M. Hogaboam, R. M. Strieter, and S. L. Kunkel. 2003. Immu-
nomodulatory role of CXCR2 during experimental septic peritonitis. J. Immunol.
171:3775.
12. Matsukawa, A., N. W. Lukacs, C. M. Hogaboam, S. W. Chensue, and
S. L. Kunkel. 2001. III. Chemokines and other mediators. 8. Chemokines and
their receptors in cell-mediated immune responses in the lung. Microsc. Res.
Tech. 53:298.
13. Shang, X., B. Qiu, K. A. Frait, J. S. Hu, J. Sonstein, J. L. Curtis, B. Lu, C. Gerard,
and S. W. Chensue. 2000. Chemokine receptor 1 knockout abrogates natural
killer cell recruitment and impairs type-1 cytokines in lymphoid tissue during
pulmonary granuloma formation. Am. J. Pathol. 157:2055.
14. Rodriguez-Sosa, M., L. E. Rosas, L. I. Terrazas, B. Lu, C. Gerard, and
A. R. Satoskar. 2003. CC chemokine receptor 1 enhances susceptibility to Leish-
mania major during early phase of infection. Immunol. Cell Biol. 81:114.
15. Domachowske, J. B., C. A. Bonville, J. L. Gao, P. M. Murphy, A. J. Easton, and
H. F. Rosenberg. 2000. The chemokine macrophage-inflammatory protein-1␣
and its receptor CCR1 control pulmonary inflammation and antiviral host defense
in paramyxovirus infection. J. Immunol. 165:2677.
16. Gao, J. L., T. A. Wynn, Y. Chang, E. J. Lee, H. E. Broxmeyer, S. Cooper,
H. L. Tiffany, H. Westphal, J. Kwon-Chung, and P. M. Murphy. 1997. Impaired
host defense, hematopoiesis, granulomatous inflammation and type 1-type 2 cy-
tokine balance in mice lacking CC chemokine receptor 1. J. Exp. Med. 185:1959.
17. Lukacs, N. W., S. H. Oliveira, and C. M. Hogaboam. 1999. Chemokines and
asthma: redundancy of function or a coordinated effort? J. Clin. Invest. 104:995.
18. Ma, B., Z. Zhu, R. J. Homer, C. Gerard, R. Strieter, and J. A. Elias. 2004. The
C10/CCL6 chemokine and CCR1 play critical roles in the pathogenesis of IL-
13-induced inflammation and remodeling. J. Immunol. 172:1872.
19. Cavaillon, J. M., M. Adib-Conquy, C. Fitting, C. Adrie, and D. Payen. 2003.
Cytokine cascade in sepsis. Scand. J. Infect. Dis. 35:535.
20. Walley, K. R., N. W. Lukacs, T. J. Standiford, R. M. Strieter, and S. L. Kunkel.
1997. Elevated levels of macrophage inflammatory protein 2 in severe murine
peritonitis increase neutrophil recruitment and mortality. Infect. Immun. 65:3847.
21. Takahashi, H., T. Tashiro, M. Miyazaki, M. Kobayashi, R. B. Pollard, and
F. Suzuki. 2002. An essential role of macrophage inflammatory protein 1␣/CCL3
on the expression of host’s innate immunities against infectious complications.
J. Leukocyte Biol. 72:1190.
22. Gerard, C., J. L. Frossard, M. Bhatia, A. Saluja, N. P. Gerard, B. Lu, and
M. Steer. 1997. Targeted disruption of the ␤-chemokine receptor CCR1 protects
against pancreatitis-associated lung injury. J. Clin. Invest. 100:2022.
23. Steinhauser, M. L., C. M. Hogaboam, N. W. Lukacs, R. M. Strieter, and
S. L. Kunkel. 1999. Multiple roles for IL-12 in a model of acute septic peritonitis.
J. Immunol. 162:5437.
24. Belperio, J. A., M. D. Burdick, M. P. Keane, Y. Y. Xue, J. P. Lynch III,
B. L. Daugherty, S. L. Kunkel, and R. M. Strieter. 2000. The role of the CC
chemokine, RANTES, in acute lung allograft rejection. J. Immunol. 165:461.
6948
25. Evanoff, H. L., M. D. Burdick, S. A. Moore, S. L. Kunkel, and R. M. Strieter.
1992. A sensitive ELISA for the detection of human monocyte chemoattractant
protein-1 (MCP-1). Immunol. Invest. 21:39.
26. Green, S. J., C. A. Nacy, R. D. Schreiber, D. L. Granger, R. M. Crawford,
M. S. Meltzer, and A. H. Fortier. 1993. Neutralization of ␥ interferon and tumor
necrosis factor ␣ blocks in vivo synthesis of nitrogen oxides from L-arginine and
protection against Francisella tularensis infection in Mycobacterium bovis BCG-
treated mice. Infect. Immun. 61:689.
27. Sewnath, M. E., D. P. Olszyna, R. Birjmohun, F. J. ten Kate, D. J. Gouma, and
T. van Der Poll. 2001. IL-10-deficient mice demonstrate multiple organ failure
and increased mortality during Escherichia coli peritonitis despite an accelerated
bacterial clearance. J. Immunol. 166:6323.
28. Matsukawa, A., C. M. Hogaboam, N. W. Lukacs, P. M. Lincoln, R. M. Strieter,
and S. L. Kunkel. 1999. Endogenous monocyte chemoattractant protein-1
(MCP-1) protects mice in a model of acute septic peritonitis: cross-talk between
MCP-1 and leukotriene B4. J. Immunol. 163:6148.
29. Moreira, A. L., L. Tsenova, P. J. Murray, S. Freeman, A. Bergtold, L. Chiriboga,
and G. Kaplan. 2000. Aerosol infection of mice with recombinant BCG secreting
murine IFN-␥ partially reconstitutes local protective immunity. Microb. Pathog.
29:175.
30. Matsukawa, A., C. M. Hogaboam, N. W. Lukacs, P. M. Lincoln, R. M. Strieter,
and S. L. Kunkel. 2000. Endogenous MCP-1 influences systemic cytokine bal-
ance in a murine model of acute septic peritonitis. Exp. Mol. Pathol. 68:77.
31. Cohen, J. 2002. The immunopathogenesis of sepsis. Nature 420:885.
32. Suzuki, T., S. Hashimoto, N. Toyoda, S. Nagai, N. Yamazaki, H. Y. Dong,
J. Sakai, T. Yamashita, T. Nukiwa, and K. Matsushima. 2000. Comprehensive
gene expression profile of LPS-stimulated human monocytes by SAGE. Blood
96:2584.
33. Kopydlowski, K. M., C. A. Salkowski, M. J. Cody, N. van Rooijen, J. Major,
T. A. Hamilton, and S. N. Vogel. 1999. Regulation of macrophage chemokine
expression by lipopolysaccharide in vitro and in vivo. J. Immunol. 163:1537.
34. Maier, S., K. Emmanuilidis, M. Entleutner, N. Zantl, M. Werner, K. Pfeffer, and
C. D. Heidecke. 2000. Massive chemokine transcription in acute renal failure due
to polymicrobial sepsis. Shock 14:187.
35. Juremalm, M., N. Olsson, and G. Nilsson. 2002. Selective CCL5/RANTES-in-
duced mast cell migration through interactions with chemokine receptors CCR1
and CCR4. Biochem. Biophys. Res. Commun. 297:480.
CCR1 IN SEPSIS
36. Sato, K., H. Kawasaki, C. Morimoto, N. Yamashima, and T. Matsuyama. 2002.
An abortive ligand-induced activation of CCR1-mediated downstream signaling
event and a deficiency of CCR5 expression are associated with the hyporespon-
siveness of human naive CD4⫹ T cells to CCL3 and CCL5. J. Immunol.
168:6263.
37. VanOtteren, G. M., R. M. Strieter, S. L. Kunkel, R. Paine III, M. J. Greenberger,
J. M. Danforth, M. D. Burdick, and T. J. Standiford. 1995. Compartmentalized
expression of RANTES in a murine model of endotoxemia. J. Immunol.
154:1900.
38. Shadidi, K. R., T. Aarvak, J. E. Henriksen, J. B. Natvig, and K. M. Thompson.
2003. The chemokines CCL5, CCL2 and CXCL12 play significant roles in the
migration of Th1 cells into rheumatoid synovial tissue. Scand. J. Immunol.
57:192.
39. Meurer, R., G. Van Riper, W. Feeney, P. Cunningham, D. Hora, Jr.,
M. S. Springer, D. E. MacIntyre, and H. Rosen. 1993. Formation of eosinophilic
and monocytic intradermal inflammatory sites in the dog by injection of human
RANTES but not human monocyte chemoattractant protein 1, human macro-
phage inflammatory protein 1␣, or human interleukin 8. J. Exp. Med. 178:1913.
40. Dorner, B. G., A. Scheffold, M. S. Rolph, M. B. Huser, S. H. Kaufmann,
A. Radbruch, I. E. Flesch, and R. A. Kroczek. 2002. MIP-1␣, MIP-1␤, RANTES,
and ATAC/lymphotactin function together with IFN-␥ as type 1 cytokines. Proc.
Natl. Acad. Sci. USA 99:6181.
41. Bohn, E., and I. B. Autenrieth. 1996. IL-12 is essential for resistance against
Yersinia enterocolitica by triggering IFN-␥ production in NK cells and CD4⫹ T
Downloaded from http://www.jimmunol.org/ by guest on October 26, 2022
cells. J. Immunol. 156:1458.
42. Echtenacher, B., M. A. Freudenberg, R. S. Jack, and D. N. Mannel. 2001. Dif-
ferences in innate defense mechanisms in endotoxemia and polymicrobial septic
peritonitis. Infect. Immun. 69:7271.
43. Miles, R. H., T. P. Paxton, D. J. Dries, and R. L. Gamelli. 1994. Interferon-␥
increases mortality following cecal ligation and puncture. J. Trauma 36:607.
44. Locati, M., U. Deuschle, M. L. Massardi, F. O. Martinez, M. Sironi, S. Sozzani,
T. Bartfai, and A. Mantovani. 2002. Analysis of the gene expression profile
activated by the CC chemokine ligand 5/RANTES and by lipopolysaccharide in
human monocytes. J. Immunol. 168:3557.