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 (CCR1؊/؊) mice were significantly protected from the lethal effects of sepsis when compared with wild-type (WT) controls. The peritoneal and systemic cytokine profile in CCR1؊/؊ 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 CCR1؊/؊ resident or recruited peri toneal cells during sepsis. Peritoneal macrophages isolated from naive CCR1؊/؊ 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. epsis is the most common cause of death in noncoronary mune response to sepsis (7, 8). Although differences in critical care units in the U.S. with Ͼ751,000 cases per year chemokine receptor expression in septic patients have been re- S at an estimated overall cost of $16.7 billion (increasing by ported (9), the significance of these differences is not under- 1.5% per year) (1). Due to the paucity of effective therapeutic stood. Almost nothing is known about the role these receptors treatments (2), the care of septic patients is predominantly sup- play in the septic response (10, 11). portive and mortality rates remain high. Although recombinant ac- CCR1 is expressed by a broad spectrum of leukocytes, including tivated protein C has shown some promise (3), investigators con- neutrophils, monocytes, eosinophils, and lymphocytes (12). Stud- tinue to search for novel therapies. ies of CCR1-deficient (CCR1Ϫ/Ϫ) mice have implicated CCR1 in During a normal infection, the immune system of an immuno- the modulation of leukocyte trafficking (13), parasite and viral competent host works to contain and destroy the pathogen. A sep- clearance (14, 15), and the balance of type 1 and type 2 cytokines tic response occurs when a pathogen circumvents the innate and (16); however, little is known about its role in the innate immune acquired immune defenses, resulting in systemic spread of the in- response during sepsis and multiorgan failure. In vitro, CCR1 has fection. In a continued attempt to eliminate the infection, the host been shown to bind several ligands, including CCL3, CCL5–9, enhances production of several proinflammatory cytokines and CCL14–16, and CCL23, although CCL3, CCL5, and CCL6 are the chemokines that act to increase the infiltration and activation of major agonists identified in vivo (17, 18). Serum levels of CCL3 inflammatory leukocytes. These factors have been shown to play a and CCL5 are elevated in septic patients (19), and peritoneal CCL3 significant role in the resulting tissue damage preceding sepsis- (20) and CCL6 (7) concentrations are increased in the cecal liga- associated multiple organ failure (4, 5) and may serve as potential tion and puncture (CLP) mouse model of sepsis. Both CCL3 (21) targets for immunotherapy. and CCL6 (7) have been demonstrated to play protective roles Chemokines are a family of small, primarily secreted proteins against sepsis-induced lethality and injury in a murine CLP model, that are responsible for modulating multiple aspects of inflam- while the role of CCL5 has yet to be explored. matory responses and host defense (6). Many studies have dem- The purpose of this study was to investigate the role of CCR1 in the onstrated several of these to be key mediators in the host im- innate immune response to experimental sepsis. CCR1Ϫ/Ϫ mice were significantly protected against CLP-induced lethality. Although CCR1 deficiency had no effect on the inflammatory cell recruitment *Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109; and †Children’s Hospital, Harvard Medical School, Boston, MA 02115 to the peritoneal cavity, loss of the receptor promoted accelerated cytokine expression and enhanced macrophage activity, both of Received for publication June 15, 2004. Accepted for publication September 22, 2004. which contributed to a more efficient and regulated antibacterial The costs of publication of this article were defrayed in part by the payment of page response. CCL5 was identified as a key modulator of the host charges. This article must therefore be hereby marked advertisement in accordance response that acted in a CCR1-dependent manner to trigger the with 18 U.S.C. Section 1734 solely to indicate this fact. unregulated, exaggerated expression of proinflammatory cyto- 1 This work was supported by Grants HL031237 and P50HL074024 from the Na- kines, resulting in increased injury and mortality following sepsis. 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: 3 Abbreviations used in this paper: CLP, cecal ligation and puncture; TSA, thymic- [email protected] 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 of 300 cells from multiple high-powered fields, was multiplied by the total Mice peritoneal cell count to determine the number for each cell type. Three independent experiments (three to five mice per group) showed similar Specific pathogen-free wild-type (WT) BALB/c mice (6–8 wk of age) results, and data were pooled. were purchased from The Jackson Laboratory (Bar Harbor, ME). CCR1- deficient (CCR1Ϫ/Ϫ) mice were generated, as previously described, and Measurement of cytokines and chemokines by ELISA were backcrossed onto a BALB/c genetic background (22). Mice were bred Concentrations of murine TNF-␣, IFN-␥, IL-12 (p70), IL-10, MIP-2, KC, and housed in the animal care facility (University Laboratory of Animal CCL2, CCL3, CCL5, CCL6, CCL17, CXCL9, and CXCL10 were mea- Medicine) at the University of Michigan. The Animal Use Committee at sured in cell-free peritoneal lavage fluid, serum, and cell culture superna- the University of Michigan approved all experimental procedures tants using a standardized sandwich ELISA previously described in detail involving mice. (25). Briefly, flat-bottom 96-well microtiter plates (Nunc, Roskilde, Den- Cecal ligation and puncture 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, CLP was used to induce acute septic peritonitis, as previously described Rochester, MN). Specific Ab for capture and detection of KC was obtained (23). Mice were anesthetized with a combination of 2.25 mg of ketamine from PeproTech. Plates were coated with 0.4 ␮g/ml (KC), 0.5 ␮g/ml HCl (Abbott Laboratories, Chicago, IL) and 150 ␮g of xylazine (Lloyd (IFN-␥, IL-12, CCL2, and CCL5), or 1.0 ␮g/ml (TNF-␣, IL-10, MIP-2, Laboratories, Shenandoah, IA) administered i.p. A 1-cm incision was made CCL3, CCL6, CCL17, CXCL9, and CXCL10) appropriate capture Abs. to the lower left abdomen of the mouse, and the cecum was exposed. The Plates were washed with PBS-Tween 20 (0.05%) and blocked with 2% cecum was ligated distally with 3.0 silk suture and punctured through and BSA in PBS for 90 min at 37°C. Plates were rinsed four times, and samples through with a 21- or 26-gauge needle. The cecum was returned to the were loaded and incubated at 37°C for 1 h. After washing, a biotinylated peritoneal cavity, and surgical staples were used to close the incision. Mice secondary polyclonal Ab specific for the cytokine being measured (R&D immediately received 1 ml of saline s.c. for fluid resuscitation and were Systems; PeproTech for KC) was added at 0.5 ␮g/ml (or 0.25 ␮g/ml for warmed on a heating pad to facilitate their revival from the anesthetic. CCL5) for 30 min at 37°C. The plate was washed, and streptavidin-per- 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 ϳ50 pg/ml. ELISA spec- on male mice. Survival of CLP groups (n ϭ 8–10 mice per group) was ificity was confirmed for each assay.