CCR1 and CC Chemokine Ligand 5 Interactions Exacerbate Innate ...

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 leukocyte recruitment, suggesting critical differences extant in the activation of WT and CCR1ⴚ/ⴚ resident or recruited peritoneal cells during sepsis. Peritoneal macrophages isolated from naive CCR1ⴚ/ⴚ mice clearly demonstrated enhanced 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.

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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 supportive and mortality rates remain high. Although recombinant activated protein C has shown some promise (3), investigators continue to search for novel therapies. During a normal infection, the immune system of an immunocompetent host works to contain and destroy the pathogen. A septic response occurs when a pathogen circumvents the innate and acquired immune defenses, resulting in systemic spread of the infection. 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 sepsisassociated 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 inflammatory responses and host defense (6). Many studies have demonstrated 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 National Institutes of Health to S.L.K. 2 Address correspondence and reprint requests to Dr. Cory M. Hogaboam, Department of Pathology, University of Michigan Medical School, 1301 Catherine Road, Room 5214, Medical Sciences I, Ann Arbor, MI 48109-0602. E-mail address: [email protected]

Copyright © 2004 by The American Association of Immunologists, Inc.

mune response to sepsis (7, 8). Although differences in chemokine receptor expression in septic patients have been reported (9), the significance of these differences is not understood. 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). Studies 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 ligation 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 cytokines, resulting in increased injury and mortality following sepsis.

3 Abbreviations used in this paper: CLP, cecal ligation and puncture; TSA, thymicshared Ag; WT, wild type.

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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). CCR1deficient (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.

Experimental protocols The first set of survival studies was performed to determine the effect of the presence of CCR1 on survival following induction of acute septic peritonitis in female and male mice. Due to differential susceptibility, female mice were subjected to 21-gauge CLP, while 26-gauge CLP was performed on male mice. Survival of CLP groups (n ⫽ 8 –10 mice per group) was monitored for 7 days following surgery. Duplicate survival studies for the female mice had similar results, and, therefore, the data were pooled. For the remaining in vivo studies, female mice were used in the 21-gauge CLP 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 21gauge 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 described 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, Rockford, 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.

6939 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 measured in cell-free peritoneal lavage fluid, serum, and cell culture supernatants using a standardized sandwich ELISA previously described in detail (25). Briefly, flat-bottom 96-well microtiter plates (Nunc, Roskilde, Denmark) were coated overnight at 4°C with mAb (for IL-10) or affinitypurified 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-peroxidase conjugate (Bio-Rad, Richmond, CA) was added to the wells for 30 min at 37°C. This was followed by another set of washes before the addition of a chromogenic substrate (Bio-Rad). After full development occurred, the reaction was stopped and the plate was read in an ELISA plate reader at 490 nm. Recombinant murine cytokines/chemokines (R&D Systems) were used to generate standard curves, and concentrations were expressed as ng or pg/ml. Limits of detection were ⬃50 pg/ml. ELISA specificity was confirmed for each assay. Experimental groups consisted of three to five mice or triplicate samples in vitro. ELISAs were run on samples from three independent experiments, and representative data are shown.

Determination of CFU Blood samples obtained for the purpose of determining CFU were immediately 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 expressed as CFU/10 ␮l. Groups contained four to six mice, and the experiment 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 determined 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, Du¨dingen, Switzerland) from peritoneal lavage 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 susceptibility 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 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-tetrasodium 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 resuspended in complete DMEM (BioWhittaker, Walkersville, MD) containing 5% FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin. 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, Manassas, VA) was grown to mid-log phase in tryptic soy broth (Difco, Detroit, MI) and used to infect freshly isolated naive macrophages (106 macrophages 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 37°C in 5% CO2, cells were washed four to five times with medium to

FIGURE 2. CCR1⫺/⫺ mice had significantly enhanced 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 lavage 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 indicates 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.

CCR1 IN SEPSIS 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 macrophages, 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 indicator 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% naphthylethylenediamine 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 three independent experiments. In vivo, NO2⫺ can be further oxidized to


6941

FIGURE 3. CCR1 had no significant effect on leukocyte 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 performed from Diff-Quik-stained cytospins prepared from each lavage. Data were analyzed from two different experiments. The results for total counts and differentials were similar in all experiments and were pooled. The graphs depict averages for each group ⫾ SEM.

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 Naive peritoneal macrophages were plated at 5 ⫻ 105/well of a 24-well cell 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% thioglycolate 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, Carlsbad, CA). Briefly, cells were washed and scraped into ice-cold PBS containing 6.25 mM NaF, 12.5 mM ␤-glycerophosphate, 12.5 mM para-nitrophenyl 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 (SigmaAldrich). 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-

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.

Results 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% mortality (7 of 17 mice) at day 5 and 59% mortality (10 of 17 mice) at day 7, demonstrating 54 and 36% increased survival, respectively. 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 postCLP, 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 investigation focused on identifying the immunomodulatory mechanism in CCR1⫺/⫺ mice that resulted in decreased CLP susceptibility and increased survival.

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FIGURE 4. Cytokine and chemokine expression were significantly altered in CCR1⫺/⫺ mice following CLP. At 0, 4, 8, or 24 h post-CLP, mice were sacrificed and bled, and 2-ml peritoneal lavages were performed. Concentrations of cytokines/chemokines in serum (A) and lavage fluid (B) from WT BALB/c mice (f) and CCR1⫺/⫺ mice (䡺) were measured using specific sandwich ELISAs. The data from three independent experiments were similar, and representative data are shown (n ⫽ 3–5 per group). ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.005; ⴱⴱⴱ, p ⬍ 0.0005, compared with similarly 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 elevated (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 compared 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 antimicrobial activity. Peritoneal lavage fluid was collected from mice at various times after CLP and evaluated for nitrite, a stable byproduct of NO (Fig. 2C). After CLP, peritoneal nitrite concentrations steadily increased in WT mice and were highest at 24 h. In contrast, 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 concentrations in CCR1⫺/⫺ mice remained steady without further increase 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 stimulus, 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 potential 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 robust 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 clearance in the CCR1⫺/⫺ mice was not due to changes in cellular recruitment.

6943 CCR1⫺/⫺ macrophages had enhanced innate immune responses Although there were no apparent differences in leukocyte recruitment to the peritoneal cavity, key differences were observed in bacterial clearance and cytokine/chemokine expression in CCR1⫺/⫺ mice. These data implied that intrinsic differences existed 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 peritoneal 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). In other experiments, naive peritoneal macrophages were stimulated in vitro with LPS. Supernatants were collected 24 h later and assayed by ELISA. CCR1⫺/⫺ macrophages expressed significantly higher constitutive levels of MIP-2, CCL2, CCL5, CCL6, and CCL22 (Fig. 6). Although LPS stimulated substantial production of multiple cytokines and chemokines from WT macrophages, it provoked significantly higher levels from CCR1⫺/⫺ macrophages (Fig. 6).

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 significantly 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 significantly 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.

FIGURE 5. Macrophages deficient in CCR1 displayed enhanced phagocytic 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 removed, 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.

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FIGURE 6. Cytokine/chemokine production was significantly increased in CCR1⫺/⫺ macrophages. Naive 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 previously recognized to play protective roles in this model of sepsis (7, 21), we investigated the role of CCL5 in this model. CCL5 expression 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 mortality was significantly higher (88%) than in CLP-saline-treated mice (59% mortality; p ⫽ 0.03; Fig. 8A). In contrast, when endogenous 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).

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 expression of IFN-␥ and MIP-2 was amplified 264 and 125%, respectively, in WT mice (Fig. 9B), while CCR1⫺/⫺ mice were unresponsive to the effects of this ligand. WT production of peritoneal KC, CCL22, and CXCL9 (53, 51, and 72%, respectively) 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,


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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 independent studies (n ⫽ 3–5 per group). ⴱ, p ⬍ 0.05 compared with similarly 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 observed 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 expressed 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 antiinflammatory 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 sepsisrelated mortality. Cytokines are often described as double-edged swords in that they are required for recruitment and activation of cells necessary for responding to a foreign insult; however, when produced in excess precipitate 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 modulate the inflammatory response, protecting the host from tissue and organ injury (27). Several CC and CXC chemokines have also been

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, compared with WT-CLP mice, indicating that an accelerated antimicrobial 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 immunomodulation was observed in the CCR1⫺/⫺ mice at 8 and 24 h post-CLP when systemic expression of inflammatory cytokines/

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CCR1 IN SEPSIS

FIGURE 9. CCL5 increased NF-␬B activation by LPS in vitro and CLP-induced inflammatory cytokine expression in vivo. A, Thioglycolate-elicited macrophages 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 postCLP), 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 cavity, 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 response, is able to solely initiate many of the immunopathological features of septic shock (31), and, therefore, must be rapidly regulated. Its continued expression may serve to propagate the extended inflammatory response observed after CLP in WT mice. Interestingly, CCR1⫺/⫺ mice showed significantly increased peritoneal expression of CXCL10, CCL6, and CCL17 when compared with WT mice at 8 –24 h post-CLP. Studies from our laboratory 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 expression 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/chemokines, in response to infectious bacteria or bacterial components such as LPS (32). Previous work from our laboratory has illustrated the importance of these cells in the CLP model of sepsis (7,

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⫺/⫺ peritoneal cells. In vitro analyses of CCR1⫺/⫺ macrophages demonstrated 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). However, this study was the first to explore the expression and role of the CCR1 ligand, namely CCL5, in a CLP model of sepsis. Previously, 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 during renal failure (34). In the CLP model, local and systemic expression 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 predicted to have a similar detrimental effect. Considering the previously 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 involvement 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 chemotaxis of memory T cells (36). Although CCL5 has been predominantly 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 endotoxemia (37) as well as into synovial tissue during rheumatoid arthritis, propagating the chronic inflammation that is characteristic of the disease (38). Intradermal injection of rCCL5 results in pathology characterized by monocytic and eosinophilic inflammatory 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 production 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 increases 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 cytokine production directly or indirectly by decreasing antiinflammatory 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 homologue of MIP-2) as well as other inflammatory chemokines such as CCL2 and CCL3. Additionally, in vitro LPS activation of the inflammatory p65 subunit of NF-␬B was amplified in a dose-dependent 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 increased survival in septic mice. Although no effect on leukocyte recruitment was observed, CCR1⫺/⫺ mice exhibited an accelerated, effective antimicrobial response that was quickly down-regulated 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 hyperinflammatory 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 response generated during a septic insult and are potential targets for immunotherapy in septic patients.

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