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The Journal of Immunology, 2008, 181, 4208 -4218
Copyright © 2008 by The American Association of Immunologists, Inc.
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Essential Involvement of CX3CR1-Mediated Signals in the Bactericidal Host Defense during Septic Peritonitis1

Yuko Ishida2,*, Takahito Hayashi2,3,*, Takatsugu Goto4,{dagger}, Akihiko Kimura*, Shigeru Akimoto{dagger}, Naofumi Mukaida{ddagger} and Toshikazu Kondo5,*

* Department of Forensic Medicine and {dagger} Department of Microbiology, Wakayama Medical University, Wakayama, Japan; and {ddagger} Division of Molecular Bioregulation, Cancer Research Institute, Kanazawa University, Kanazawa, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Cecal ligation and puncture (CLP) caused septic peritonitis in wild-type (WT) mice, with ~33% mortality within 7 days after the procedure. Concomitantly, the protein level of intraperitoneal CX3CL1/fractalkine was increased, with infiltration by CX3CR1-expressing macrophages into the peritoneum. CLP induced 75% mortality in CX3CR1-deficient (CX3CR1–/–) mice, which, however, exhibited a similar degree of intraperitoneal leukocyte infiltration as WT mice. Despite this, CX3CR1–/– mice exhibited impairment in intraperitoneal bacterial clearance, together with a reduction in the expression of intraperitoneal inducible NO synthase (iNOS) and bactericidal proinflammatory cytokines, including IL-1β, TNF-{alpha}, IFN-{gamma}, and IL-12, compared with WT mice. Bactericidal ability of peritoneal phagocytes such as neutrophils and macrophages was consistently attenuated in CX3CR1–/– mice compared with WT mice. Moreover, when WT macrophages were stimulated in vitro with CX3CL1, their bactericidal activity was augmented in a dose-dependent manner, with enhanced iNOS gene expression and subsequent NO generation. Furthermore, CX3CL1 enhanced the gene expression of IL-1β, TNF-{alpha}, IFN-{gamma}, and IL-12 by WT macrophages with NF-{kappa}B activation. Thus, CX3CL1-CX3CR1 interaction is crucial for optimal host defense against bacterial infection by activating bacterial killing functions of phagocytes, and by augmenting iNOS-mediated NO generation and bactericidal proinflammatory cytokine production mainly through the NF-{kappa}B signal pathway, with few effects on macrophage infiltration.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Sepsis is a severe systemic bacterial infection, irrespective of the initially infected organs, and arises from complex host immune, inflammatory, and coagulation responses to infecting microbes, resulting frequently in lethal shock with coagulopathy and multiple organ failure (1, 2). In the United States alone, approximately 750,000 sepsis cases develop every year, and its frequency is increasing due to increases in the numbers of severely injured, aged, immune-suppressed, and terminal patients (3). Its overall mortality rate is still as high as 35% despite recent development in the treatment consisting of broad-spectrum antibiotics, adequate fluid replenishment, and artificial support of organ function (3).

Infection by polymicrobial flora in the intestine causes septic peritonitis with a higher mortality of 60–80% (4). Septic peritonitis is characterized by a massive infiltration of neutrophils and macrophages into the peritoneal cavity. Their infiltration is indispensable for clearance of bacteria from the peritoneum, but their exaggerated activation can further augment systemic inflammation, resulting in severe damage to multiple organs. Proinflammatory cytokines regulate the recruitment and activation of leukocytes (2, 3). Thus, TNF-{alpha} antagonists (5, 6, 7) or IL-1 receptor antagonists (8) have been administered to reduce severe inflammatory reactions frequently observed in sepsis, including septic peritonitis, but with little success.

Chemokines have potent chemotactic activity on leukocytes after binding to their specific G protein-coupled receptors with seven transmembrane portions (9, 10), and they are classified into four subgroups of CXC, CC, CX3C, and C chemokines, based on their cysteine motif. CX3CL1/fractalkine is a unique member of CX3C family and is expressed by inflamed endothelial cells, macrophages, neurons, and glial cells (11). CX3CL1 exists in soluble and membrane-bound forms (12), and it binds to its only specific receptor, CX3CR1 (13), which is expressed by monocytes, lymphocytes, and NK cells. The soluble form of CX3CL1 exhibits leukocyte chemotactic activity similar to that of other chemokines. Besides its chemotactic activity, membrane-bound CX3CL1 can act as an adhesion molecule by binding to CX3CR1 independently of selectins or integrins (11, 12, 13).

CX3CL1- and CX3CR1-deficient mice (CX3CR1–/– mice) exhibited no apparent phenotypes under unchallenged conditions (14, 15), but CX3CR1–/– mice exhibited an increased survival of allografts, with reduced NK cell infiltration, in a model of cardiac allograft rejection (16). Furuichi and his colleagues demonstrated that CX3CR1 promoted renal interstitial fibrosis after ischemia-reperfusion injury (17). Moreover, in support of the assumption that the CX3CL1-CX3CR1 axis would be essential for the development of atherosclerosis (18, 19, 20, 21, 22), the genetic disruption of CX3CR1 resulted in attenuated atherosclerotic lesions in the aorta of apolipoprotein E-deficient mice with reduced macrophage accumulation (18, 19). In humans, several mutations in CX3CR1 gene such as CX3CR1-I249 (isoleucine at position 249) or CX3CR1-M280 (methionine at position 280) can lead to lower CX3CR1 expression (23) and are associated with rapid progression of AIDS and protection from cardiovasuclar disease (23, 24).

Cecal ligation and puncture (CLP)6 has often been used as a model of septic peritonitis and causes a massive infiltration of neutrophils and macrophages (25, 26, 27, 28, 29, 30). CX3CR1 expression by infiltrating macrophages prompted us to examine the pathophysiological roles of the CX3CL1-CX3CR1 axis in CLP-induced sepsis. Herein, we provide definitive evidence that the lack of CX3CR1 reduced bactericidal activity of phagocytes by impairing macrophage-derived proinflammatory cytokine production, and inducible NO synthase (iNOS) expression followed by NO generation, and that this accounted for exaggerated susceptibility to CLP with an enhanced lethality in CX3CR1–/– mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents and antibodies

Recombinant murine CX3CL1 and LPS were obtained from R&D Systems and Difco, respectively. In the present study, the following mAbs or polyclonal Abs (pAbs) were used for immunocytochemistry and a double-color immunofluorescence analysis: rat anti-mouse F4/80 mAb (Dainippon Pharmaceutical), rabbit anti-CX3CR1 pAbs, mouse anti-iNOS mAb (Santa Cruz Biotechnology), rat anti-CD3 mAb (Serotec), rat anti-mouse pan-NK cells mAb (BD Pharmingen), goat anti-mouse CX3CL1 pAbs (R&D Systems), cyanine dye 3-conjugated donkey anti-rabbit IgG or anti-goat IgG pAbs, and FITC-conjugated donkey anti-rat IgG pAbs (Jackson Immunoresearch Laboratories). Western blot analysis was performed by the use of following Abs: rabbit anti-p38 MAPK pAbs, rabbit anti-JNK pAbs (Santa Cruz Biotechnology), rabbit anti-ERK mAb (Cell Signaling Technology), rabbit anti-phosphorylated (p)-p38 MAPK pAbs (BIOMOL), rabbit anti-p-JNK pAbs (BioVision), rabbit anti-p-ERK pAbs (Cell Signaling Technology), rabbit anti-β-actin pAbs (Sigma-Aldrich), or mouse anti-{alpha}-tubulin mAb (Santa Cruz Biotechnology).

Animals

Specific pathogen-free 8- to 10-wk-old male C57BL/6 mice were obtained from Sankyo Laboratories and designated as wild-type (WT) mice in this study. CX3CR1–/– mice were a generous gift from Drs. P. M. Murphy and J. L. Gao (National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD) (17). Age- and sex-matched CX3CR1–/– mice, backcrossed to C57BL/6 mice for 8–10 generations, were used in the following experiments. All mice were housed individually in cages under the specific pathogen-free conditions, and all animal experiments were approved by the Committee on Animal Care and Use in Wakayama Medical University.

CLP surgery and the evaluation of survival rate

CLP surgery was performed to induce sepsis as described previously (30). Briefly, mice were deeply anesthetized with an i.p. injection of pentobarbital (50 mg/kg). After making a 1-cm-long incision to the lower left abdomen, cecum was exposed, ligated tightly with 3–0 silk suture below the ileocecal valve, and punctured through once with a 20-gauge needle. Thereafter, the cecum was returned into the peritoneal cavity, and the incision was closed. Mice were then subcutaneously injected with 1 ml sterile PBS to avoid dehydration, and they were warmed on a heating pad until they recovered from the anesthesia. To evaluate the survival rates, WT and CX3CR1–/– mice were monitored for 7 days after CLP surgery. In some experiments, WT mice were administered i.p. with recombinant CX3CL1 (3 µg/mouse) or an equal amount PBS at 1 h after CLP surgery.

Blood biochemical analysis

At the indicated intervals after CLP surgery, whole blood samples were collected to determine serum levels of alanine transferase (ALT), blood urea nitrogen (BUN), and creatinine (Cr) with a Fuji Dri-Chem 3500V as instructed by the manufacturer (Fujifilm Medical Systems).

Determination of bacterial CFU

Bacterial CFU was determined on the peripheral blood and the peritoneal lavage as described previously (27). Briefly, at the indicated time intervals after CLP, mice were euthanized by exsanguination under deep anesthesia. The peritoneal cavities were washed with 2 ml of sterile PBS, and the peritoneal lavage fluids were harvested under sterile conditions. Ten microliters of peritoneal lavage fluids or peripheral blood was placed on ice and serially diluted with sterile PBS. Five microliters of each dilution was placed on trypticase soy agar (TSA) plates with 5% sheep blood (BD Biosciences) and incubated in a moist chamber overnight at 37°C, after which the numbers of aerobic bacteria colonies were counted. Data were expressed as CFU/5 µl. In another series of experiments, WT or CX3CR1–/– mice were i.p. injected with 1 x 106 CFU of Escherichia coli (strain WME1). At the indicated time intervals after the injection, bacterial CFU in the peritoneal cavity were determined as described above.

Immunocytochemistry

Smear slides from the peritoneal lavage fluid were fixed immediately in 100% methanol. The slides were immersed in 0.3% H2O2 in methanol for 30 min to eliminate endogenous peroxidase activities. After the rehydration in PBS-0.1% Tween 20, the slides were further incubated with PBS containing 1% appropriate normal serum and 1% BSA for 1 h at room temperature to reduce nonspecific reactions. The slides were incubated with anti-CX3CR1 pAbs, anti-CD3 mAb, or anti-pan NK cell mAbs at a concentration of 1 µg/ml at 4°C overnight. After the incubation of biotinylated secondary Ab, immune complexes were visualized using a catalyzed signal amplification system (DakoCytomation).

Peritoneal leukocyte counts and cell differentials

At the indicated intervals after CLP, the peritoneal lavage fluids were collected as mentioned above. The cell pellets were obtained by centrifugation at 8500 x g for 1 min at 4°C and were resuspended in PBS. The total numbers of peritoneal leukocytes were counted with a hemocytometer and are expressed as leukocytes (x106 or 105) per cavity. Differential cell analyses were conducted on Giemsa-stained smear slides prepared using the resuspensions. Moreover, T cells and NK cells were immunocytochemically detected with anti-CD3 Ab and anti-pan NK Ab, respectively. The percentage for each leukocyte population, based on a count of 300 cells from multiple high-powered fields, was multiplied by the total peritoneal cell count to determine the absolute number of each cell type.

ELISA

At the indicated time points after CLP, cell-free peritoneal lavage fluid samples were collected and used to determine the levels of murine cytokines using commercial ELISA kits (CX3CL1, IFN-{gamma}, and IL-12p70, R&D Systems; IL-1β and TNF-{alpha}, Pierce). The detection limit of each method was as follows: CX3CL1, >80 pg/ml; IL-1β, >3 pg/ml; TNF-{alpha}, >9 pg/ml; IFN-{gamma}, >2 pg/ml; and IL-12p70, >2.5 pg/ml.

Cell culture

WT or CX3CR1–/– mice were i.p. injected with 2 ml of 3% thioglycolate (Sigma-Aldrich), and intraperitoneal macrophages were harvested (macrophage purity >95%) 3 days later as described previously (31). The cells were suspended in antibiotic-free RPMI 1640 medium containing 10% FBS and incubated at 37°C in three 24-well cell culture plates. Two hours later, nonadherent cells were removed, and the medium was replaced. The cells were stimulated with LPS and/or the indicated concentrations of murine CX3CL1 for 12 h, and then applied to subsequent analyses.

In vitro phagocytosis and killing activity assays

Peritoneal macrophages were isolated and infected with 1 x 106 CFU of E. coli (strain WME1) in 24-well culture plates. After 20-min incubation at 37°C in 5% CO2, the wells were extensively washed with fresh medium to remove unphagocytosed bacteria. The cells in one plate were lysed with sterile 0.5% Triton X-100 for bacterial phagocytosis assay. Wells in the other plate were replaced with prewarmed fresh medium and incubated for an additional 20 min, and then cells were lysed with 0.5% Triton X-100 for bacterial killing assay. The serially diluted cell lysates were plated on TSA-blood plates and incubated at 37°C overnight, and the numbers of colonies were counted. In some experiments, the peritoneal macrophages (1 x 106/well) from WT mice were stimulated with the indicated concentrations of recombinant murine CX3CL1 for 12 h, and the effects of CX3CL1 on phagocytic and killing activities were evaluated (31). In another series of experiments, neutrophils were collected from WT and CX3CR1–/– mice at 6 h after the injection with 2 ml of 3% thioglycolate, and their bactericidal activity was also examined as described above. Moreover, the effects of supernatant of culture medium of CX3CL1-stimulated macrophages on killing activities of neutrophils were evaluated.

Measurement of NO generation

The peritoneal macrophages (1 x 106/well) from WT and CX3CR1–/– mice were cultured in RPMI 1640 medium containing 10% FCS at 37°C at 5% CO2 overnight. Then, after the cells were incubated with LPS (10 µg/ml) for an additional 24 h, the supernatant were obtained by centrifugation. Thereafter, NO level was determined by measuring nitrite and nitrate, stable endproducts of NO metabolism, using a commercial NO assay kit (Assay Designs). In some experiments, peritoneal macrophages (1 x 106/well) from WT mice were incubated with LPS and the indicated doses of recombinant murine CX3CL1 for 12 h before the determination of NO levels in the culture supernatants. The cell-free peritoneal lavage fluid samples were also collected from WT and CX3CR1–/– mice to determine NO levels.

Extraction of total RNAs and semiquantitative RT-PCR

Semiquantitative RT-PCR analysis was performed as described previously (32, 33). Briefly, total RNAs were extracted from the cell pellets of the peritoneal lavage fluids or from peritoneal macrophages using ISOGEN (Nippon Gene) according to the manufacturer’s instructions. Five micrograms of total RNA was reverse-transcribed at 42°C for 1 h in 20 µl reaction mixture containing mouse Moloney leukemia virus reverse transcriptase (Toyobo) with oligo(dT) primers (American-Pharmacia Biotech Japan). Thereafter, cDNA was amplified using Taq polymerase (Nippon Gene) and the specific sets with the optimal cycles consisting of 94°C for 1 min, optimal annealing temperature for 1 min, and 72°C for 1 min (Table I), followed by incubation at 72°C for 3 min. The amplified PCR products were fractionated on a 2% agarose gel and visualized by ethidium bromide staining. The band intensities were measured using image analysis software (Scion Image), and the ratios of each band to β-actin were calculated.


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  		Table I. Sequences of the primers used for RT-PCRa

 
Western blotting

The cell pellets from the peritoneal lavage fluids or isolated macrophages were homogenized with a lysis buffer (20 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA) containing Complete protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktails for serine/threonine protein phosphatases and tyrosine protein phosphatases (P2850 and P5726; Sigma-Aldrich) and were centrifuged to obtain lysates. The lysates (equivalent to 30 µg protein) were electrophoresed in a 10% SDS-polyacrylamide gel and transferred onto a nylon membrane. After the membrane was sequentially reacted with optimally diluted primary Abs and HRP-conjugated secondary Abs, the immune complexes were visualized using ECL system (Amersham Biosciences).

Measurement of NF-{kappa}B p65 DNA-binding activity

Nuclear proteins were extracted from the isolated macrophages using the NE-PER method (Pierce) as described previously (33). Subsequently, a multiwell chemiluminescent assay was performed to detect NF-{kappa}B p65 protein using the EZ-Detect transcription factor kit (Pierce) according to the manufacturer’s instructions. The p65 contents were expressed as relative light units divided by total protein contents for each sample.

Double-color immunofluorescence analysis

Smear slides from the peritoneal lavage fluid were fixed immediately in 100% methanol. After rehydration in PBS-0.1% Tween 20, the slides were incubated with PBS containing 1% normal donkey serum and 1% BSA to reduce nonspecific reactions. Thereafter, the slides were incubated overnight at 4°C with pairs of anti-CX3CR1 pAbs and anti-F4/80 mAb, or anti-CX3CL1 pAbs and anti-F4/80 mAb. After washing, the slides were incubated with fluorochrome-conjugated secondary Abs at room temperature for 1 h. The slides were then observed under a fluorescence microscopy.

Statistical analyses

The means and SEM were calculated for all parameters determined in this study. Statistical significance was evaluated using one-way ANOVA with post hoc testing with the Scheffé’s F multiple comparisons test or Mann-Whitney’s U test. The survival curves by the Kaplan-Meier procedure were analyzed by a log-rank test. p < 0.05 was accepted as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Intraperitoneal CX3CL1 and CX3CR1 expression in WT mice after CLP

CX3CL1 was barely detectable in the peritoneal lavage fluids of WT mice (Fig. 1a). Six hours after CLP procedure, CX3CL1 levels were increased significantly, and they were further enhanced by 24 h (Fig. 1a). Moreover, CX3CL1 protein was detected in F4/80-positive macrophages recruited into the peritoneal cavity (Fig. 1b–d). Similarly, CX3CR1 mRNA was barely detectable in peritoneal cells from untreated WT mice under the employed experimental conditions (Fig. 1e). CLP procedures augmented the gene expression of CX3CR1 beginning at 6 h (Fig. 1, e and f). Immunocytochemical analysis demonstrated that mononuclear cells expressed CX3CR1 protein 24 h after CLP surgery (Fig. 1g). Moreover, double-color immunofluorescence analysis revealed that most of the F4/80-positive macrophages in the peritoneum expressed CX3CR1 (Fig. 1h–j). These observations indicated that, in the course of CLP-induced sepsis, CX3CL1 is produced and can induce the accumulation and probably activation of CX3CR1-positive macrophages.


Figure 1
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  		FIGURE 1. a, Intraperitoneal CX3CL1/fractalkine levels in WT mice after CLP. At the indicated intervals, 2-ml peritoneal lavage fluids were harvested, and CX3CL1/fractalkine protein levels in the lavage fluids were measured as described in Materials and Methods. All values represent the means ± SEM (n = 6). **, p < 0.01 vs untreated WT. b–d, A double-color immunofluorescence analysis on a smear from peritoneal lavage fluid of WT mice at 24 h after CLP surgery. Representative results from six independent experiments are shown here: (b) anti-CX3CL1 pAbs and (c) anti-F4/80 mAb. Signals were digitally merged in d. Original magnification, x400. e and f, CX3CR1 gene expression in the intraperitoneal cells was examined by RT-PCR as described in Materials and Methods. Representative results from six independent experiments are shown in e. The ratios of CX3CR1 to β-actin were calculated and are shown in f. All values represent means ± SEM (n = 6). **, p < 0.01 vs untreated WT. g, Immunocytochemical analysis on smear slides from peritoneal lavage fluid of WT mice at 24 h after CLP surgery. Representative results from six independent experiments are shown here (original magnification, x400). h–j, A double-color immunofluorescence analysis on a smear from peritoneal lavage fluid of WT mice at 24 h after CLP surgery. Representative results from six independent experiments are shown here: (h) anti-CX3CR1 pAbs and (i) anti-F4/80 mAb. Signals were digitally merged in j. Original magnification, x400.

 
Increased susceptibility to CLP-induced sepsis is associated with exaggerated organ damage and systemic inflammation in CX3CR1–/– mice

To determine the pathogenic roles of the CX3CL1-CX3CR1 interactions in CLP-induced sepsis, we performed CLP procedures on WT and CX3CR1–/– mice. Twelve of 18 WT mice (66.7%) survived 7 days after CLP surgery, whereas only 25% of CX3CR1–/– mice (6 of 24 mice) survived (Fig. 2a). Since CLP-induced sepsis causes multiple organ failure and enhanced systemic inflammation (25, 26, 27, 28, 29, 30), we evaluated liver and renal injury by measuring serum ALT, BUN, and Cr levels, respectively. There were no significant differences between untreated WT and CX3CR1–/– mice in terms of serum ALT, BUN, and Cr levels (Fig. 2, b–d). Serum levels of these markers were obviously elevated in both WT and CX3CR1–/– mice later than 6 h after CLP surgery. The increases in serum chemistry levels were significantly greater at 6 h and even more so at 24 h after CLP surgery in CX3CR1–/– mice than in WT mice, indicating that liver and kidney were more damaged in CX3CR1–/– mice than in WT mice (Fig. 2, b–d). Thus, the absence of CX3CR1 increased the susceptibility to CLP-induced sepsis, accompanied with exaggerated organ damage. When WT mice were administered with CX3CL1 (3 µg/mouse) after CLP surgery, the survival rate at 7 days was significantly improved (Fig. 2e; CX3CL1 vs control, 1 death per 15 mice vs 6 deaths per 15 mice, p < 0.05). Thus, these observations implied that the CX3CL1-CX3CR1 axis was protective for CLP-induced sepsis.


Figure 2
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  		FIGURE 2. a, The survival rates in WT (n = 18) and CX3CR1–/– mice (n = 24) were evaluated for 7 days after CLP. b–d, Exaggerated organ damages in CX3CR1–/– mice. At the indicated time intervals after CLP, whole blood samples were collected, and serum levels of ALT (b), BUN (c), and Cr (d) were determined as described in Materials and Methods. All values represent means ± SEM (n = 8). **, p < 0.01 and *, p < 0.05 CX3CR1–/– vs WT. e, The effects of exogenous CX3CL1 on the survival rates of WT mice after CLP surgery.

 
Impaired bacterial clearance in CX3CR1–/– mice

CLP-induced sepsis arises from intraperitoneal dissemination of intestinal polymicrobial flora. Hence, we next examined the bacterial load in the peritoneal fluid and blood of WT and CX3CR1–/– mice after CLP surgery. No bacteria were recovered from the peritoneal cavity and whole blood of untreated WT and CX3CR1–/– mice (data not shown). The mean peritoneal CFU counts were higher in CX3CR1–/– mice at 6 and 24 h after CLP surgery than in WT mice (Fig. 3, a and b, WT vs CX3CR1–/– mice: 0.65 x 102 vs 3.2 x 102 at 6 h; 3.3 x 102 vs 4.3 x 104 at 24 h). Bacteremia was observed in 25% of WT mice (2 of 8 mice) with a mean of 4.0 CFU counts/5 µl blood 24 h after the CLP procedure (Fig. 3d). In contrast, at the same time, bacteria were recovered from 75% of CX3CR1–/– mice (6 of 8 mice) with a mean of 7.0 x 102 CFU counts/5 µl blood (Fig. 3, c and d). Moreover, when E. coli (1 x 106 CFU) was injected i.p., CX3CR1–/– mice showed significant impairment in bacterial clearance from the peritoneal cavity at the indicated intervals compared with WT mice (Fig. 3, e and f; WT vs CX3CR1–/– (n = 8 in each strain): 1.1 x 102 vs 2.7 x 103 at 6 h, p < 0.01; 1.1 x 10 vs 1.0 x 103 at 24 h, p < 0.01). Collectively, these observations indicated that, in the absence of CX3CR1, bacterial clearance from the peritoneal cavity was impaired, eventually resulting in the development of greater bacteremia and systemic inflammation.


Figure 3
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  		FIGURE 3. Impaired bacterial clearance in CX3CR1–/– mice. At the indicated intervals after CLP, peritoneal fluids (a and b) and peripheral blood (c and d) were collected from both WT and CX3CR1–/– mice. e and f, Bacterial CFU in the peritoneal cavity was also determined in WT and CX3CR1–/– mice at the indicated intervals after the i.p. injection of 1 x 106 CFU of E. coli (strain WME1). After the serial dilution, 5 µl of each dilution was plated on TSA-blood plates, and bacterial CFU in each sample were determined as described in Materials and Methods. The horizontal bar indicates the mean for each group (n = 8 per group). **, p < 0.01 and *, p < 0.05 for CX3CR1–/– vs WT.

 
CX3CR1-mediated signal pathway was not essentially involved in leukocyte recruitment during CLP-induced sepsis

The recruitment of leukocytes is presumed to be vital to bacterial clearance. Hence, we examined the effects of CX3CR1 deficiency on intraperitoneal trafficking of leukocytes. CLP caused a similar degree of massive leukocyte recruitment into the peritoneal cavity in WT and CX3CR1–/– mice (Fig. 4a). There were no significant differences in intraperitoneal neutrophil numbers between WT and CX3CR1–/– mice until 48 h after CLP (Fig. 4b). Consistent with the previous reports (25, 27, 28, 30), intraperitoneal macrophage numbers decreased transiently 6 h after CLP surgery and increased progressively thereafter in WT mice (Fig. 4c). These findings may mirror the observation (25, 27) that CLP surgery activated peritoneal macrophages to increase their capacity to adhere to peritoneum transiently. Although macrophage numbers were only marginally lower in CX3CR1–/– mice than in WT mice at 24 h, no significant differences were observed at 48 h between these two strains (Fig. 4c). Moreover, both T cells and NK cells were recruited into the peritoneal cavity to similar extents in WT and CX3CR1–/– mice after CLP surgery (Fig. 4, d and e). These observations implied that the CX3CL1-CX3CR1 axis was not indispensable for leukocyte recruitment in CLP-induced sepsis.


Figure 4
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  		FIGURE 4. Changes in intraperitoneal leukocyte numbers after CLP. At the indicated intervals after CLP, intraperitoneal cells were collected from WT and CX3CR1–/– mice, and the numbers of total leukocytes (a), neutrophils (b), macrophages (c), CD3-positive T cells (d), and NK cells (e) were counted as described in Materials and Methods. All values represent means ± SEM (n = 8).

 
Essential involvement of CX3CR1 in bacterial killing activity of macrophages and neutrophils

However, CX3CR1–/– mouse-derived peritoneal macrophages exhibited a reduced capacity to kill bacteria without any impairment in phagocytosis, compared with WT macrophages (Fig. 5, a and b). Moreover, CX3CL1 augmented bacterial killing, but not phagocytic ability, of WT macrophages in a dose-dependent manner (Fig. 5, c and d). However, CX3CL1 failed to enhance bacterial killing activity of CX3CR1–/– mouse-derived peritoneal macrophages (data not shown). Thus, these observations implied that the CX3CL1-CX3CR1 interaction has an important role in maintaining the bacterial killing capabilities of macrophages but has no effect on their trafficking and phagocytic abilities. Accumulating evidence implied i.p.-infiltrating neutrophils as a major effector cell in bacterial clearance (34, 35, 36). Indeed, the bacterial killing activity of neutrophils from CX3CR1–/– mice was significantly depressed compared with that of WT mice (Fig. 5e). Moreover, when WT macrophages were stimulated by recombinant CX3CL1, the culture supernatants enhanced bacterial killing activities of neutrophils from WT mice (Fig. 5f). However, the culture supernatant from CX3CR1–/– macrophages stimulated by CX3CL1 failed to enhance the bactericidal activities of WT neutrophils (data not shown). Thus, the CX3CL1-CX3CR1 interactions can enhance bacterial killing activities of neutrophils directly and indirectly by acting on macrophages.


Figure 5
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  		FIGURE 5. Phagocytic and bacterial killing activities of macrophages. a and b, Phagocytic (a) and bacterial killing activities (b) were determined on WT- or CX3CR1–/– mouse-derived peritoneal macrophages, as described in Materials and Methods. All values represent means ± SEM (eight independent experiments). **, p < 0.01 for CX3CR1–/– vs WT. c and d, Phagocytic (c) and bacterial killing activities (d) were determined on WT mouse-derived peritoneal macrophages after the culture in the presence of the indicated concentrations of CXC3CL1 for 12 h, as described in Materials and Methods. **, p < 0.01 vs control; #, p < 0.05 for CX3CL1 at 10 ng/ml vs CX3CL1 at 100 ng/ml. e, Bacterial killing activities of neutrophils were determined in WT and CX3CR1–/– mice, as described Materials and Methods. All values represent means ± SEM (eight independent experiments). *, p < 0.05 for CX3CR1–/– vs WT. f, The effects of the supernatant from culture medium of CX3CL1-stiumlated WT macrophages on the bacterial killing activity of WT-derived neutrophils. **, p < 0.01 and *, p < 0.05 vs control; #, p < 0.05 for CX3CL1 at 10 ng/ml vs CX3CL1 at 100 ng/ml.

 
Essential roles of the CX3CL1-CX3CR1 axis in iNOS expression and NO generation

NO, mainly produced by iNOS, has potent bactericidal activities and it therefore is reported to protect against sepsis (37). iNOS mRNA was faintly expressed in the peritoneal cells of both untreated WT and CX3CR1–/– mice to a similar extent. CLP augmented iNOS mRNA expression in WT mice, but to a markedly lesser extent in CX3CR1–/– mice (Fig. 6, a and b). Concomitantly, iNOS protein levels were more enhanced in peritoneal cell pellets of WT mice after CLP than in CX3CR1–/– mice (Fig. 6c). Moreover, nitrate (a stable byproduct of NO) concentrations in the peritoneal cavities of WT mice were markedly elevated after CLP, but were again attenuated in CX3CR1–/– mice (Fig. 6d). LPS stimulation augmented iNOS gene expression in WT mouse-derived peritoneal macrophages, but to a lesser extent in CX3CR1–/– mouse-derived macrophages (Fig. 7, a and b). Accordingly, nitrate concentration was significantly lower in the culture medium of CX3CR1–/– macrophages compared with WT macrophages (Fig. 7c). However, WT and CX3CR1–/– macrophages expressed TLR-4 mRNA to similar extents (data not shown). Exogenous CX3CL1, in a dose-dependent manner, enhanced LPS-induced iNOS mRNA expression and the resultant NO generation by WT macrophages (Fig. 7d–f) but not CX3CR1–/– macrophages (data not shown). LPS induced CX3CL1 mRNA expression to similar extents in WT and CX3CR1–/– macrophages (data not shown), consistent with the previous report (38). Thus, CX3CR1–/– macrophages exhibited lower responsiveness to LPS probably because they failed to respond to CX3CL1 produced locally upon the stimulation with LPS. These observations would indicate that the CX3CL1-CX3CR1 interaction protects against peritoneal bacterial dissemination arising from CLP by inducing iNOS gene expression and the subsequent generation of NO, a crucial bacterial killing effector molecule.


Figure 6
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  		FIGURE 6. a and b, CLP-induced gene expression for iNOS in intraperitoneal cells from WT mice (open bars) and CX3CR1–/– mice (filled bars). RT-PCR was performed as described in Materials and Methods. Representative results from six independent experiments are shown in a. The ratios of iNOS to β-actin were calculated and are shown in b. All values represent means ± SEM (n = 6). **, p < 0.01 for CX3CR1–/– vs WT. c, Western blotting analysis of iNOS expression in the intraperitoneal cells. At the indicated intervals after CLP, intraperitoneal cells were collected and Western blotting analysis was conducted to detect iNOS protein, as described in Materials and Methods. Representative results from six independent experiments are shown here. d, Nitrate concentrations in the peritoneal lavage fluids were determined on WT mice (open bars) and CX3CR1–/– mice (filled bars) at the indicated intervals after CLP. All values represent means ± SEM (n = 6). **, p < 0.01 for CX3CR1–/– vs WT.

 

Figure 7
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  		FIGURE 7. a–c, Macrophages (1 x 106) from WT and/or CX3CR1–/– mice were incubated in the presence of LPS (10 µg/ml) for 24 h. a and b, iNOS gene expression in macrophages was determined by RT-PCR as described in Materials and Methods. Representative results from six independent experiments are shown in a. The ratios of iNOS to β-actin were calculated and are shown in b. All values represent means ± SEM (six independent experiments). **, p < 0.01 for CX3CR1–/– vs WT. c, NO contents in the supernatants were determined as described in Materials and Methods. All values represent means ± SEM (six independent experiments). **, p < 0.01 for CX3CR1–/– vs WT. (d) to (f) The effects of CX3CL1 on LPS-induced iNOS gene expression and NO generation in WT macrophages. WT mouse-derived macrophages were incubated in the presence of LPS (10 µg/ml), together with the indicated concentrations of CX3CL1 for 24 h. d and e, iNOS gene expression in macrophages was determined by RT-PCR as described in Materials and Methods. Representative results from six independent experiments are shown in d. The ratios of iNOS to β-actin were calculated and are shown in e. All values represent means ± SEM (six independent experiments). **, p < 0.01 vs control; ##, p < 0.01 vs LPS alone. f, NO contents in the supernatants were determined as described in Materials and Methods. All values represent means ± SEM (six independent experiments). **, p < 0.01 vs control; ##, p < 0.01 and #, p < 0.05 vs LPS alone.

 
Impaired cytokine production in the absence of CX3CR1

Accumulating evidence indicates that antibacterial activities can be augmented by cytokines such as IL-1, TNF-{alpha}, IFN-{gamma}, and IL-12 (39, 40, 41, 42). Given the capacity of macrophages to produce these cytokines, we examined the effects of CX3CR1 deficiency on the production of these cytokines. There were no significant differences in the peritoneal contents of these cytokines of untreated WT and CX3CR1–/– mice (Fig. 8). However, CLP markedly increased the intraperitoneal contents of these cytokines in WT mice but less so in CX3CR1–/– mice (Fig. 8). CX3CR1–/– peritoneal macrophages exhibited a reduction in LPS-stimulated mRNA expression of these cytokines compared with WT mice (data not shown). Moreover, exogenous CX3CL1 augmented, in a dose-dependent manner, gene expression of these cytokines by WT peritoneal macrophages (Fig. 9) but not by CX3CR1–/– peritoneal macrophages (data not shown). These observations indicated that the CX3CL1-CX3CR1 interactions augmented both NO generation and cytokine production by macrophages, as well as their bacterial killing activity.


Figure 8
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  		FIGURE 8. Intraperitoneal levels of proinflammatory cytokines in WT and CX3CR1–/– mice. At the indicate intervals after CLP, cell-free peritoneal lavage fluids were collected to determine IL-1β (a), TNF-{alpha} (b), IFN-{gamma} (c), and IL-12p70 levels (d), as described in Materials and Methods. All values represent means ± SEM (n = 8). **, p < 0.01 and *, p < 0.05 for CX3CR1–/– vs WT.

 

Figure 9
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  		FIGURE 9. WT mouse-derived peritoneal macrophages were incubated in the presence or the absence of LPS (10 µg/ml), together with the indicated concentrations of CX3CL1 for 24 h. RT-PCR was performed on macrophages as described in Materials and Methods. Representative results from six independent experiments are shown in a. The ratios of IL-1β (b), TNF-{alpha} (c), IFN-{gamma} (d), IL-12p35 (e), and IL-12p40 (f) to β-actin were calculated and are shown. All values represent means ± SEM (six independent experiments). **, p < 0.01 vs control; ##, p < 0.01 and #, p < 0.05 vs LPS alone.

 
Effects of CX3CL1 on NF-{kappa}B, p38 MAPK, JNK, and ERK signaling pathways

Because CX3CL1 activated several signaling pathways (43, 44, 45, 46, 47), we finally examined the effects of CX3CL1 on NF-{kappa}B, p38 MAPK, JNK, and ERK signaling pathways in WT-derived macrophages. CX3CL1 significantly enhanced the DNA binding activity of NF-{kappa}B in WT macrophages in a dose-dependent manner (Fig. 10a). p38 MAPK and JNK were phosphorylated only in the presence of the highest dose of CX3CL1 that we examined (Fig. 10b–d), whereas ERK phosphorylation was not enhanced (Fig. 10, b and e). These observations implied that the CX3CL1-CX3CR1 interactions mainly activated the NF-{kappa}B signaling pathway, resulting in the enhancement of target gene expression.


Figure 10
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  		FIGURE 10. a, DNA binding activity of the nuclear NF-{kappa}B p65 in peritoneal macrophage after CX3CL1 treatment. All values represent the means ± SEM (six experiments in each group). **, p < 0.01 vs vehicle. b, Immunochemical detection of p38 MAPK, phosphorylated p38 MAPK (p-p38 MAPK), JNK, p-JNK, ERK, p-ERK, and {alpha}-tubulin on peritoneal macrophages after CX3CL1 treatment, as described in Materials and Methods. Western blotting analysis using anti-{alpha}-tubulin Ab confirmed that an equal amount of protein was loaded onto each lane. Representative results from six independent experiments are shown in b. p-p38 MAPK/p38 MAPK ratios (c), p-JNK/JNK (d), and p-ERK/ERK ratios (e) obtained by densitometry are shown here. All values represent means ± SEM (six independent experiments). **, p < 0.01 vs control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Eradication of bacteria needs the killing activity of phagocytes such as neutrophils and macrophages. CLP-induced polymicrobial infection in the peritoneum and resultant phagocyte infiltration into peritoneal cavity and their subsequent activation are crucial for clearance of microbes and the prevention of CLP-induced sepsis (25, 26, 27, 28, 29, 30). Chemokines, in collaboration with adhesion molecules, can regulate the trafficking of phagocytes. Indeed, several chemokines contribute to the pathogenesis of CLP-induced sepsis in different ways (25, 26, 27, 28, 29, 30). CXCR2-, CCR1-, or CCR8-deficient mice are more resistant to CLP-induced sepsis (25, 26, 27). The absence of CCR1 or CCR8 enhanced antibacterial responses in macrophages (26, 27). On the contrary, MIP-1{alpha}/CCL3 deficiency, or treatment with anti-MCP-1/CCL2 or anti-macrophage-derived chemokine (MDC)/CCL22 Ab, decreased survival rates following CLP-induced sepsis and was accompanied by enhanced recovery of viable bacteria from the peritoneum (48). The receptors for CCL2, CCL3, and CCL22 are expressed on macrophages. Mirroring the expression of their cognate receptors on macrophages, CCL2, CCL3, and CCL22 enhanced antibacterial responses of macrophages (29, 30, 31, 48, 49). These observations suggest that various chemokines can regulate macrophage bactericidal activities in different manners.

CX3CL1 is expressed as a membrane-bound form on endothelial cells and is processed by TNF-{alpha} converting enzyme to generate a soluble form (50). Its expression is further increased by stimulation with LPS, TNF-{alpha}, IL-1, and IFN-{gamma} (12). Because CX3CL1 contains its chemokine domain at the top of a cell-bound extended mucin-like stalk, it can also function as an adhesion molecule. Consequently, cells expressing CX3CR1, a unique receptor for CX3CL1, bind rapidly and with high affinity to CX3CL1-expressing endothelial cells under both static and physiological flow conditions (13). Thus, CX3CL1 expressed on activated endothelial cells likely mediates efficient leukocyte recruitment from blood into the interstitial tissue space. CX3CR1 expression is restricted to lymphocytes, NK cells, and monocytes. During CLP-induced peritonitis, both CX3CR1 and its specific ligand, CX3CL1, were expressed by intraperitoneal macrophages, which prompted us to evaluate CLP-induced peritonitis in CX3CR1–/– mice compared with WT mice. Herein, we observed that CX3CR1–/– mice exhibited reduced bacterial clearance from the peritoneum, with increased mortality arising from depressed bacterial killing activity of both neutrophils and macrophages. Thus, CX3CR1-expressing macrophages have a major protective role against bacterial infection, similar to CCR2- or CCR4-expressing macrophages. This protective effect is not dependent on macrophage recruitment but on their bactericidal functions.

Geissmann and colleagues (51) proposed that murine peripheral blood monocytes can be classified into two subsets based on the expression levels of CX3CR1 and CCR2: CX3CR1highCCR2– monocytes resident in tissue, and CX3CR1lowCCR2+ monocytes recruited into tissue upon inflammation. Conversely, Ancuta and colleagues reported that in humans, CX3CR1 is expressed by CD16+ monocytes, which represent 5–10% of peripheral blood monocytes under normal conditions and are dramatically expanded in several pathological conditions, including sepsis and cancer (52). In line with the latter observations, peritoneal macrophages in untreated mice expressed a low level of CX3CR1, but CLP markedly enhanced its expression by macrophages (Y. Ishida and T. Kando, unpublished data). Although gene disruption or immunoneutralization of CX3CR1 diminished macrophage recruitment in atherosclerosis and renal diseases (17, 18, 19, 53, 54), the lack of CX3CR1 had few effects on macrophage infiltration in thioglycolate-induced peritonitis (14, 15) or, in this study, on CLP-induced peritonitis. Accumulating evidence indicates that the CCL2-CCR2 interaction can crucially regulate macrophage infiltration in various disease models including CLP-induced sepsis (28, 30, 55, 56, 57, 58). Indeed, CLP increased the intraperitoneal content of CCL2 to a similar extent in WT and CX3CR1–/– mice (Y. Ishida and T. Kando, unpublished data). Thus, the CCL2-CCR2 interactions can predominantly regulate CLP-induced macrophage infiltration into the peritoneum. Most peritoneal macrophages strongly expressed both CX3CR1 and CCR2, even at 1 h and thereafter until 24 h after CLP surgery (Y. Ishida and T. Kando, unpublished data). Because the CCL2-CCR2 interactions can rapidly up-regulate CX3CR1 expression on macrophages (59), these observations may suggest that infiltrating CCR2+ inflammatory macrophages acquire CX3CR1 expression. However, rapid appearance of CCR2highCX3CR1high macrophages favors the assumption that i.p.-infiltrating macrophages may be phenotypically distinct from so-called resident and inflammatory macrophages.

Activated macrophages express iNOS, the enzyme that can synthesize NO, with inhibitory effects on pathogen replication (37). They simultaneously produce TNF-{alpha} and IFN-{gamma}, both of which also exert potent antibacterial activities (39, 40, 41, 42). CLP enhanced iNOS expression, subsequent NO generation, and TNF-{alpha} and IFN-{gamma} production in the peritoneal cavity, and these increases were depressed in CX3CR1–/– mice, with a concomitant impairment in bacterial clearance. Thus, iNOS, TNF-{alpha}, and IFN-{gamma} expression may also have protective roles in CLP-induced peritonitis. Intravenous infection with Listeria monocytogenes induced intrasplenic production of CCL2, which attracted CCR2-expressing monocytes (60). Infiltrating monocytes contained L. monocytogenes infection by expressing iNOS, TNF-{alpha}, and IFN-{gamma}, as similarly observed in CLP-induced peritonitis. However, the molecular mechanisms underlying the expression of these molecules remain to be investigated. We observed that peritoneal macrophages from uninfected WT mice expressed a low level of CX3CR1 but still retained the capacity to express iNOS, TNF-{alpha}, and IFN-{gamma} in response to CX3CL1. Thus, it is tempting to speculate that CX3CL1 may activate monocytes/macrophages infiltrating into the peritoneum under the guidance of CCL2 to express these effector molecules.

Despite lack of CX3CR1 expression on neutrophils, their bactericidal activity in CX3CR1–/– mice was significantly reduced compared WT mice. Thus, we hypothesized that the CX3CL1-CX3CR1 axis on macrophages might indirectly regulate neutrophil functions. Actually, the bacterial killing activity of WT-derived neutrophils was enhanced by the addition of the culture medium of CX3CL1-stimulated WT macrophages but not by CX3CL1-stimulated CX3CR1–/– macrophages (Y. Ishida and T. Kando, unpublished data). Supportingly, Moreno and colleagues (36) demonstrated that IL-12 enhanced the bactericidal activity of neutrophils. In line with this, the addition of anti-IL-12 to the supernatant of CX3CL1-stimulated macrophages abrogated the enhancement in the bactericidal activity of neutrophils (Y. Ishida and T. Kando, unpublished data). Thus, the impairment of bactericidal activity in CX3CR1–/– mice was at least attributable to reduced IL-12 production in CX3CR1–/– macrophages.

Upon microbial infection, macrophages recognize pathogen-associated molecular pattern and become activated to express iNOS and to produce various cytokines including IL-1β, TNF-{alpha}, IFN-{gamma}, and IL-12 (61). These cytokines act on macrophages in an autocrine and paracrine manner to augment macrophage bactericidal function (39, 40, 41, 42). Most pathogen-associated molecular pattern receptors can activate NF-{kappa}B, p38 MAPK, and/or JNK signaling pathways, and the net result is the induction of iNOS and cytokine gene expression (62, 63). CX3CR1 shares common signaling pathways with other chemokine receptors, including stimulation of intracellular calcium mobilization (13), MAPK activation (43), and PI3K activation (44). Moreover, CX3CL1 can activate NF-{kappa}B in human intestinal epithelial cells (45) and aortic smooth muscle cells (46). Actually, exogenous CX3CL1 significantly exaggerated the activation of NF-{kappa}B in peritoneal macrophages. Signaling pathways of p38 MAPK and JNK were activated by only a high dose of CX3CL1. Thus, CX3CL1 can induce iNOS and bactericidal cytokine expression by macrophages, mainly through activating NF-{kappa}B signal pathway, which is crucially involved in the expression of those genes.

Herein, we provided the first definitive evidence to indicate that CX3CR1 deficiency in mice enhanced their susceptibility to peritoneal infection. Humans with CX3CR1 M280 or I249 alleles express lower CX3CL1 binding capacity on their PBMC, and they are more prone to rapidly progress to develop AIDS upon HIV infection (23). Thus, it is probable that decreased CX3CR1 expression can enhance susceptibility to infection in general. Therefore, along with conventional treatment, manipulation of CX3CR1-mediated signals may prove to be beneficial for the treatment of microbial infection, particularly sepsis.


   Acknowledgments
 
We thank Drs. Philip M. Murphy and Ji-Liang Gao (National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD) for providing us with CX3CR1–/– mice. We appreciate Dr. Joost J. Oppenheim for his thoughtful suggestions on this work.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflicts of interest.


   Footnotes
 
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 in part from Grants-in-Aid from the Ministry of Education, Culture, Science, and Technology of the Japanese Government. T.H. is a recipient of a Research Fellowship for Young Scientists from the Japanese Society for the Promotion of Science. Back

2 Y.I. and T.H. contributed equally to this work. Back

3 Current address: Department of Legal Medicine, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544, Japan. Back

4 Current address: Division of Anaerobe Research, Life Science Research Center, Gifu University, Gifu 501-1194, Japan. Back

5 Address correspondence and reprint requests to Dr. Toshikazu Kondo, Department of Forensic Medicine, Wakayama Medical University, 811-1 Kimiidera, Wakayama 641-8509, Japan. E-mail address: kondot{at}wakayama-med.ac.jp Back

6 Abbreviations used in this paper: CLP, cecal ligation and puncture; iNOS, inducible NO synthase; pAb, polyclonal Ab; WT, wild type; ALT, alanine transferarse; BUN, blood urea nitrogen; Cr, creatinine; TSA, trypticase soy agar. Back

Received for publication April 27, 2007. Accepted for publication July 8, 2008.


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 Materials and Methods
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Y. Luo, B. M. Isaac, A. Casadevall, and D. Cox
Phagocytosis Inhibits F-Actin-Enriched Membrane Protrusions Stimulated by Fractalkine (CX3CL1) and Colony-Stimulating Factor 1
Infect. Immun., October 1, 2009; 77(10): 4487 - 4495.
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