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Originally published online as doi:10.1189/jlb.1203645 on May 3, 2004

Published online before print May 3, 2004
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(Journal of Leukocyte Biology. 2004;76:368-373.)
© 2004 by Society for Leukocyte Biology

CCL2, a product of mice early after systemic inflammatory response syndrome (SIRS), induces alternatively activated macrophages capable of impairing antibacterial resistance of SIRS mice

Yasuhiro Tsuda*, Hitoshi Takahashi*, Makiko Kobayashi*,{dagger}, Toshiaki Hanafusa{ddagger}, David N. Herndon{dagger} and Fujio Suzuki*,{dagger},1

* Department of Internal Medicine, The University of Texas Medical Branch, Galveston;
{dagger} Shriners Hospitals for Children, Galveston, Texas; and
{ddagger} First Department of Internal Medicine, Osaka Medical College, Takatsuki, Japan

1Correspondence: The University of Texas Medical Branch, Department of Internal Medicine, 301 University Boulevard, Galveston, TX 77555-0435. E-mail: fsuzuki{at}utmb.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Infection associated with systemic inflammatory response syndrome (SIRS) is a major cause of morbidity and mortality in patients with major surgery, polytrauma, and severe burn injury. In previous studies, mice with severe pancreatitis (a mouse model of SIRS, SIRS mice) have been shown to be greatly susceptible to various infections. In the present study, a mechanism involved in the impaired resistance of SIRS mice to infectious complications was investigated. Sera from SIRS mice impaired the resistance of normal mice to infectious complications induced by cecal ligation and puncture (CLP). CC chemokine ligand 2 (CCL2) was detected in sera of SIRS mice. Resident macrophages (RM{phi}) cultured with SIRS mouse sera converted to alternatively activated macrophages (AAM{phi}), which were also demonstrated in mice treated with recombinant murine CCL2. However, AAM{phi} were not demonstrated in mice injected with SIRS mouse sera and anti-CCL2 monoclonal antibody (mAb) in combination. Furthermore, normal mice that received SIRS mouse sera and anti-CCL2 mAb resisted CLP-induced infectious complications. These results indicate that the resistance of SIRS mice to infectious complications is impaired by AAM{phi} generated from RM{phi} in response to SIRS-associated CCL2 production.

Key Words: SIRS • CCL2 • neutrophils • infectious complications


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mortality rates in critically ill patients (patients with major surgery, polytrauma, and severe burn injury) have not decreased despite significant advances in the antibiotic arsenal and in intensive care technology [1 2 3 4 5 ]. Sepsis is one of the leading causes of death in these patients [2 , 3 ]. In the recent decade, the cascade of events leading from local infection or initial injuries to systemic inflammatory response syndrome (SIRS), sepsis, multiple organ dysfunction syndrome, multiple organ failure, and death has been clearly described [1 ]. In some SIRS patients, persistent and overwhelming inflammation with elevated levels of proinflammatory mediators is associated with an increased risk of death [2 ]. Therefore, the clinical course of the above patients appears strongly in association with the manifestation of SIRS [6 ]. A number of strategies with inflammatory modulating therapies have been tested to curb the progression of SIRS [7 ]. However, none of these interventions has resulted in the significant improvement of survival rates [7 ]. Activated protein C has only been approved for the treatment of severe cases of sepsis [7 ]. For immunological intervention, further studies for the underlying relationship between SIRS and host antibacterial innate immunities are required.

Innate immunity typically serves as the rapid, first-line defense against invading pathogens and foreign antigens [8 ]. Macrophages (M{phi}) are one of the critical participants in innate immune responses [8 ]. Recently, five different procedures for M{phi} activation have been described [9 ], and three different types of activated M{phi} have been reported [10 ]. In general, however, classically activated M{phi} (CAM{phi}) and alternatively activated M{phi} (AAM{phi}) have been widely recognized as activated M{phi} populations [11 , 12 ]. CAM{phi} are known as antibacterial effector cells [11 , 12 ]. AAM{phi} do not kill intracellular pathogens and cells infected with these pathogens [11 ]. CAM{phi} appear when resident M{phi} (RM{phi}) are exposed to interferons (IFNs), lipopolysaccharide (LPS), CpG DNA, and double-stranded RNA [11 12 13 14 ]. In contrast, AAM{phi} are commonly found in patients with burn injuries, psychological stress, human immunodeficiency virus infection, or malignancies [11 12 13 14 15 16 ].

Previously, we have examined the susceptibility of SIRS mice to various infections [17 ]. In these experiments, mice with severe, acute pancreatitis [17 ] were used as SIRS mice, as these mice were well recognized as hosts with typical SIRS [18 19 20 ]. In the results obtained, SIRS mice were shown to be susceptible to infections with Enterococcus faecalis and Staphylococcus aureus and infectious complications induced by cecal ligation and puncture (CLP) [17 ]. Therefore, in a murine SIRS model, we investigated why SIRS mice are greatly susceptible to infections. In the results obtained, M{phi}-related, antibacterial innate immunities were not developed in severe SIRS mice, which were shown to be hosts with a predominance of inhibitory cells (AAM{phi}) on the antibacterial innate immunities. Also, CC chemokine ligand 2 (CCL2) produced in response to SIRS development was shown to be a trigger on AAM{phi} generation. The susceptibility of SIRS mice to infections might be directly influenced by AAM{phi} generated from RM{phi} in response to SIRS-associated CCL2 production.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Eight- to 9-week-old BALB/c mice (The Jackson Laboratory, Bar Harbor, ME) were used in these experiments. The Institutional Animal Care and Use Committee of the University of Texas Medical Branch at Galveston (IACUC Approval Number 01-04-010) approved experimental protocols for animal studies.

Reagents, media, and cells
Recombinant murine CCL2 (rCCL2), anti-CCL2 neutralizing monoclonal antibody (mAb) and isotype control antibody were purchased from BD PharMingen (San Diego, CA). rCCL17 and mAb for CCL17 were purchased from R&D Systems (Minneapolis, MN). Cerulein was purchased from Bachem (Torrance, CA), and Escherichia coli LPS was obtained from Difco (Detroit, MI). Peritoneal M{phi} prepared from freshly isolated peritoneal exudates of normal mice were used as RM{phi}. For the isolation of M{phi}, 2 x 108 peritoneal exudate cells in 10 ml RPMI-1640 medium supplemented with 2% heat-inactivated fetal bovine serum (FBS; maintenance medium) were cultured in fibronectin-coated petri dishes for 15 min at 37°C [17 ]. At the end of cultivation, dishes were washed twice with warm maintenance medium (37°C). M{phi} were recovered from the dishes using a rubber policeman. As required, obtained cell preparations were treated with magnetic beads coupled with anti-CD3, anti-B220, and anti-CD11c mAb (Dynal, Norway) to deplete T cells, B cells, or dendritic cells (DCs). The purity of M{phi} obtained was routinely more than 92% when it was analyzed using phycoerythrin-conjugated anti-CD11b mAb (BD PharMingen) and a FACSVantage flow cytometer (Becton Dickinson, San Jose, CA). In some experiments, peritoneal M{phi} were obtained from mice injected with normal mouse sera or SIRS mouse sera [250 µl/mouse, intravenously (i.v.)] in combination with or without anti-CCL2 mAb [10 µg/mouse, subcutaneously (s.c.)] or isotype antibody (10 µg/mouse, s.c.). For cultivation, M{phi} were resuspended in RPMI-1640 medium supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin; complete medium).

A mouse model of pancreatitis
Acute pancreatitis was produced in mice, according to a previously reported protocol [17 18 19 20 ]. This model has been recognized as a carrier of typical SIRS [18 19 20 ]. To create pancreatitis mice, mice were treated with cerulein [50 µg/kg, intraperitoneally (i.p.)] hourly for 6 h in combination with LPS (1.6 mg/kg, i.p.), 5 h after the first injection of cerulein. Markedly enhancing damages (edema, inflammatory cell infiltration, hemorrhage, and necrosis) were demonstrated histologically in the pancreas of SIRS mice. Indicators of multiple organ dysfunction (amylase, glutamic pyruvic transaminase, glutamic oxaloacetic transaminase) were found in the sera of these mice. They also had a decrease in body temperature (<35.5°C) and white blood cell count (<2000/mm3).

Infection experiments
A well-controlled CLP technique was used in this study, as infectious complications induced by CLP have been described as similar to the sepsis developed in various patients [21 ]. This laboratory developed a modified procedure to perform a well-controlled cecal ligation and 26-gauge puncture [22 , 23 ]. To perform CLP, mice were anesthetized with pentobarbital (50 mg/kg, i.p.). To ensure the consistent severity of CLP, a minimum-sized incision (less than 1.0 cm) to the lower-left quadrant of the abdomen was made, and the cecum was drawn out. To avoid dehydration, the exposure of the cecum to air was kept to a minimum. The distal one-third section was ligated with silk suture, and two punctures were made on the ligated cecum with a 26-gauge needle. Then, the cecum was returned and placed away from the incision. The peritoneal incision was closed using sutures (not surgical glue). All mice were treated with 2 ml sterile saline (s.c.) for fluid resuscitation during the postoperative period. From our accumulated data, 37% lethality rate in normal BALB/c mice (24/65) resulted from the CLP with two punctures in the above procedure. In some experiments, serum specimens (250 µl, i.v.) from normal mice or mice 3 h after SIRS induction were injected into these mice 0.5 h before CLP. Additionally, in some experiments, anti-CCL2 mAb (10 µg/mouse, s.c.) or isotype antibody (10 µg/mouse, s.c.) was injected into these mice 6 and 0.5 h before and 24 and 48 h after CLP. All of these mice were observed daily to determine their mortalities (percent survival, 7 days after CLP). The survival percent of tested groups was compared with that of appropriate controls. All experiments were performed two or three times, and figures show data from the results of repeated experiments.

Detection of circulating CCL2
To determine circulating CCL2 levels, serum specimens obtained from normal mice or mice various hours after SIRS induction were assayed using enzyme-linked immunosorbent assay (ELISA). The detection limit for this chemokine was 15 pg/ml in our assay systems. Each assay was performed three times.

Induction and assay for AAM{phi} generation
RM{phi} (1x106 cells/ml) were cultured with media supplemented with sera (15%, v/v) of mice 3 h after SIRS induction, in the presence or absence of anti-CCL2 mAb (10 µg/ml). RM{phi} cultured with the same amount of sera from normal mice served as a control. Twenty-four hours after cultivation, these M{phi} were washed three times with complete medium, and M{phi} harvested were examined for their AAM{phi} properties. Also, in combination with anti-CCL2 mAb (10 µg/mouse, s.c.) or isotype antibody (10 µg/mouse, s.c.), SIRS mouse sera (250 µl/mouse, i.v.) were injected two times into mice 30 and 24 h before they were killed. M{phi} from mice injected with the same amount of normal mouse sera (250 µl/mouse, i.v.) served as a control. In some experiments, mice were injected with rCCL2 (100 ng/mouse, s.c.) instead of SIRS mouse sera, 30 and 24 h before they were killed. Peritoneal M{phi} (1x106 cells/ml) were obtained from these mice and cultured for 24 h to examine their AAM{phi} properties. M{phi} were evaluated as AAM{phi} when they produced CCL17 and expressed mannose receptor mRNA. These parameters have been shown to be typical properties of AAM{phi} [11 , 12 ]. For the production of CCL17, culture fluids harvested were assayed for CCL17 using ELISA, according to the manufacturer’s protocols. In our assay system, the detection limit for these chemokines was 22 pg/ml. Each assay was performed three times. For the analysis of mRNA expression, M{phi} (1x106 cells) were sorted into a 1-ml RNA isolater. First-strand cDNA was synthesized using random hexamer primers and murine leukemia virus reverse transcriptase. Polymerase chain reaction was performed using synthesized oligonucleotide primers (Sigma-Genosys, Woodlands, TX): mannose receptor, 5'-CCATCGAGACTGCTGCTGAG-3' (forward) and 5'-AGCCCTTGGGTTGAGGATCC-3' (reverse). The predicted products were analyzed by electrophoresis in 2% agarose gel containing ethidium bromide.

Statistical analysis
The results obtained were analyzed statistically using ANOVA test. Survival curves were analyzed using the Kaplan-Meier test. All calculations were performed on a computer using the Statview 4.5 program from Brain Power (Calabasas, CA). The result was considered significant if the probability value was lower than 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of SIRS mouse sera on AAM{phi} generation
In many previously published papers, various proinflammatory soluble factors have been demonstrated in sera of mice early after SIRS induction [18 19 20 ]. Therefore, the effect of soluble factors contained in SIRS mouse sera on AAM{phi} generation was examined. AAM{phi} have been characterized as effector cells on the impaired resistance of the host’s antibacterial innate immunities [11 , 12 ]. Serum specimens (250 µl/mouse) prepared from mice 3 h after SIRS induction were transferred (i.v.) to normal mice just before CLP. As a control, normal mouse sera or saline were injected to normal mice exposed to the same CLP. When 27–33% of normal mice treated with saline or normal mouse sera died after exposure to CLP, the same CLP killed 100% of normal mice treated with SIRS mouse sera (Fig. 1 ). This pattern on the survival curve of normal mice treated with SIRS mouse sera was shown to be similar to that of the survival curve demonstrated in SIRS mice exposed to the same CLP (Fig. 1) . These results suggest that a soluble factor(s) contained in SIRS mouse sera has the activity to impair the resistance of SIRS mice to CLP-induced, infectious complications.



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  		Figure 1. Susceptibility of SIRS mice or normal mice injected with SIRS mouse sera to CLP-induced, infectious complications. Normal mice exposed to CLP were treated with 250 µl/mouse (i.v.) of sera from normal mice (12 mice, {circ}) or mice 3 h after SIRS induction (12 mice, •). As controls, normal mice (11 mice, {square}) and mice 3 h after SIRS induction (SIRS mice, 13 mice, {blacktriangleup}) were treated with saline and exposed to the same CLP. Mice were monitored for a 7-day experimental period. The survival percent obtained in each day postinfection is plotted in the figure. *, P < 0.01, compared with normal mice and mice injected with normal mouse sera.

 
Therefore, in the next experiments, the activity of SIRS mouse sera on the generation of AAM{phi} was examined. RM{phi} (peritoneal M{phi} from normal mice) were cultured with media supplemented with 15% (v/v) of SIRS mouse sera for 24 h. Cells harvested were examined for their AAM{phi} properties by their abilities to produce CCL17 and to express mannose receptor mRNA. These cellular properties have been described as typical parameters of AAM{phi} [11 , 12 ]. RM{phi} cultured with normal mouse sera did not produce any amounts of CCL17 into their culture fluids (Fig. 2A ). However, CCL17 was detected in culture fluids of RM{phi} that were cultured with media supplemented with SIRS mouse sera (15%, v/v; Fig. 2A ). Also, mannose receptor mRNA was expressed by RM{phi} after cultivation with SIRS mouse sera (Fig. 2B) , and RM{phi} cultured with normal mouse sera did not express this mRNA. These results indicate that a soluble factor(s) contained in SIRS mouse sera acts as an effector molecule on the AAM{phi} generation.



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  		Figure 2. Effect of SIRS mouse sera on the generation of AAM{phi}. RM{phi} (1x106 cells/ml) were cultured for 24 h with media supplemented with 15% (v/v) of normal mouse sera or SIRS mouse sera. Cells harvested were washed three times with media and cultured for an additional 24 h. (A) The culture fluids obtained were examined for CCL17 by ELISA. Also, the amount of CCL17 in SIRS mouse sera was measured. Data are displayed as mean ± SD. (B) The cells obtained were assayed for their mRNA expression of the mannose receptor, as described in text. Data are representative of three independent experiments.

 
The role of CCL2 detected in SIRS mouse sera on AAM{phi} generation
In preliminary studies about the active substance for AAM{phi} generation, SIRS mouse sera were tested for their chemokine and cytokine contents. As a result, high levels of CCL2 were detected in SIRS mouse sera, and this chemokine was not demonstrated in normal mouse sera. CCL2 was first detected in sera of mice 1 h after SIRS induction. The CCL2 production peaked at 3 h after SIRS induction. Within 24 h after SIRS induction, CCL2 in sera was gradually declined (Fig. 3 ). In the next series of experiments, the effect of CCL2 in SIRS mouse sera on AAM{phi} induction was tested in mice after an injection of SIRS mouse sera and anti-CCL2 mAb in combination. As shown in Figure 4A , peritoneal M{phi} from mice injected with SIRS mouse sera (250 µl/mouse, i.v.) produced CCL17, and this typical parameter of AAM{phi} was not produced by peritoneal M{phi} from mice that were injected with a combination of SIRS mouse sera and anti-CCL2 mAb (10 µg/mouse, s.c.). These results strongly suggested that an active molecule in SIRS mouse sera for AAM{phi} generation was a chemokine, CCL2.



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  		Figure 3. CCL2 production in mice various hours after SIRS induction. Serum specimens from normal mice and mice various hours after SIRS induction were assayed for CCL2 using ELISA. Each point is displayed as mean ± SD (n=5). Data shown in the figure are the representative result within three independent experiments.

 


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  		Figure 4. CCL2 as an effector molecule on the SIRS mouse sera-associated AAM{phi} generation. (A) Peritoneal M{phi} (1x106 cells/ml) from mice treated with SIRS mouse sera and anti-CCL2 mAb in combination were cultured for 24 h without any stimulation. As controls, peritoneal M{phi} from mice treated with normal mouse sera or SIRS mouse sera and isotype antibody were similarly cultured. Culture fluids harvested were assayed for CCL17. Data are displayed as mean ± SD. *, Statistical difference (P<0.001) of the results compared with the control (isotype antibody-treated). (B) Survival of CLP mice injected with SIRS mouse sera and anti-CCL2 mAb in combination. SIRS mouse sera were injected to normal mice 0.5 h before CLP in combination with anti-CCL2 mAb (11 mice, {circ}) or isotype antibody (10 mice, •). As a control, normal mice were treated with saline and exposed to the same CLP (11 mice, {square}). *, Statistical differences (P<0.01) of the results compared with the control (isotype antibody-treated).

 
Further, after being treated with or without anti-CCL2 mAb in vivo, SIRS mouse sera were examined for their abilities to impair the resistance of normal mice against CLP-induced sepsis. SIRS mouse sera (250 µl/mouse, i.v.) were injected into mice 0.5 h before CLP. Anti-CCL2 mAb (10 µg/mouse, s.c.) or isotype antibody (10 µg/mouse, s.c.) was injected into these mice 6 and 0.5 h before and 24 and 48 h after CLP. As a control, normal mice treated with saline were exposed to the same CLP. The results obtained are shown in Figure 4B . All of the mice that were treated with SIRS mouse sera and isotype antibody died within 5 days of CLP. However, 63% of CLP mice (seven of 11) treated with SIRS mouse sera and anti-CCL2 mAb in combination survived (P<0.01). These results suggest that the resistance of mice to CLP-induced sepsis is impaired by AAM{phi}, which are induced by CCL2, appearing in response to the SIRS development.

To confirm the AAM{phi}-inducing activity of CCL2, mice were injected (s.c.) with a 100-ng/mouse dose of rCCL2 instead of SIRS mouse sera at 30 and 24 h before they were killed. Peritoneal M{phi} were obtained from these mice and cultured for 24 h without any stimulation. Culture fluids harvested were assayed for CCL17 as a parameter of AAM{phi}. The results obtained are shown in Table 1 . M{phi} from mice treated with rCCL2 produced 820 pg/ml CCL17 into their culture fluids. These results indicated that rCCL2 has the ability to induce AAM{phi}. These results shown in Figures 3 and 4 A and B , and Table 1 indicate that CCL2 is a pivotal factor when SIRS mouse sera impair the resistance of mice to CLP-induced sepsis.


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  		Table 1. Effect of rCCL2 on AAM{phi} Generation in Normal Mice

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In our previous studies, mice with severe SIRS (SIRS mice) have been shown to be susceptible to infections with E. faecalis, S. aureus, and CLP-induced sepsis [17 ]. SIRS mice have been predominated by AAM{phi} with an ability to inhibit antibacterial effector M{phi} (CAM{phi}) [17 ]. SIRS-associated AAM{phi} have been identified as the cells responsible for the increased susceptibility of SIRS mice to CLP-induced sepsis, as the resistance of normal mice to CLP-induced sepsis has been impaired by the inoculation of AAM{phi} to the level observed in SIRS mice [17 ]. In this study, therefore, the mechanism involved in the SIRS-associated generation of AAM{phi} was investigated. Serum specimens from mice 3 h after SIRS induction (SIRS mouse sera) were shown to have a capacity to impair the resistance of normal mice against CLP-induced infectious complications. CCL2 was detected in sera of SIRS mice but not in normal mice. RM{phi} cultured with SIRS mouse sera and peritoneal M{phi} from mice treated with rCCL2 produced CCL17, a typical parameter of AAM{phi}, in their culture fluids. However, this chemokine was not produced by peritoneal M{phi} from mice that were injected with SIRS mouse sera and anti-CCL2 mAb in combination. Also, normal mice exposed to CLP survived after an injection of SIRS mouse sera and anti-CCL2 mAb. These results indicate that CCL2 produced in association with the SIRS development is a key chemokine on the increased susceptibility of SIRS mice (or on the stimulation of RM{phi} to generate AAM{phi}).

Many papers have described that CAM{phi} are important effector cells for the host’s innate immunities against various infections [10 11 12 13 14 ]. CAM{phi} were generated from RM{phi} in response to the engagement of Toll-like receptors or binding of IFN receptors by IFN-{alpha}/ß or IFN-{gamma} [24 25 26 27 ]. CAM{phi} exhibit high oxygen consumption and the strong killing activity against intracellular pathogens. In fact, CAM{phi} have been shown to eradicate infections with Mycobacterium avium complex, Salmonella typhimurium, Trypanosoma cruzi, or lymphocytic choriomeningitis virus [28 29 30 31 ]. In our previous studies, mild SIRS mice (mice with mild pancreatitis) were shown to be resistant to various bacterial infections, as compared with normal mice [17 ]. The same antibacterial resistance was replicated in normal mice inoculated with peritoneal M{phi} from mild SIRS mice. In following studies, these M{phi} from mild SIRS mice were shown to exhibit typical properties for CAM{phi} [17 ]. These results indicate that mild SIRS enhances antibacterial innate immunities through the generation of CAM{phi}. In contrast, AAM{phi} have been shown to play a role on the negative regulation of CAM{phi} [32 33 34 ]. Recently, the inhibitory effect of AAM{phi} on CAM{phi} generation was demonstrated in our laboratory [33 ]. RM{phi} did not polarize to CAM{phi} after cultivation with AAM{phi} in a dual-chamber transwell supplemented with CpG DNA, while CAM{phi} were easily generated from RM{phi} stimulated with CpG DNA, a typical CAM{phi} inducer. In addition, RM{phi} stimulated with CpG DNA did not convert to CAM{phi} when they were cultured with the culture fluids of AAM{phi}. IL-10 and CCL17 released from AAM{phi} were identified as inhibitory molecules on CAM{phi} generation [33 ]. It has been well recognized that AAM{phi} are generated from RM{phi} after exposure to T helper cell type 2 (Th2) cytokines [interleukin (IL)-4, IL-10, and/or IL-13] or glucocorticoids [11 , 12 ]. So far, the effect of CCL2 on AAM{phi} generation has not been described. The results shown in this paper indicate that CCL2 is a key chemokine in the stimulation of RM{phi} to generate AAM{phi}. In fact, AAM{phi} were generated in normal mice treated with rCCL2. These data may help to explain why the susceptibility to various infections increases in hosts whose AAM{phi} predominate.

Next, questions concerning the cellular source of CCL2 arose. In our recent studies, CCL2 production was demonstrated in cultures of a mixture of peripheral blood monocytes (PBM) and neutrophils (PMN) from mice 3 h after severe SIRS induction [35 ]. When PBM and PMN from these mice were individually cultured at a cell density of 1 x 106 cells/ml, the CCL2 production was not demonstrated. However, PMN produced CCL2 after stimulation with S. aureus Cowan I bacteria (0.0075%). Under the same condition, CCL2 was not produced by PBM. These results indicate that PMN might be a major source of CCL2 in mice early after severe SIRS induction.

Conversely, CCR2 (the receptor for CCL2) is highly expressed on monocytes and activated T cells, including memory T cells, Th1 cells, and Th2 cells [36 ]. Previously, CCL2 has been described as a key chemokine in the development of Th2 responses [37 ]. However, Th1 responses have been not developed in CCR2 knockout mice that are susceptible to infections with intracellular pathogens [38 , 39 ]. These facts suggest that additional ligands of CCR2 may function differently from CCL2. CCL8 and CCL12 are known to be ligands of murine CCR2 [36 , 40 ]. These chemokines are known to be potent chemoattractants for monocytes, macrophages, immature DCs, natural killer cells, and activated T cells [36 , 40 , 41 ]. The role of these ligands on AAM{phi} generation remains unknown.

Compensatory anti-inflammatory response syndrome (CARS) has been described as an important regulator of SIRS [42 , 43 ]. CARS usually appeared in response to SIRS development [42 ]. However, some of the important host defenses against infections are down-regulated by CARS through the excessive production of anti-inflammatory cytokines (IL-4, IL-10, and IL-13) [43 ]. Recently, the necessity of CCL2 for the generation of CARS effector cells has been demonstrated [35 ]. Notably, CCL2-deficient mice exhibited resistance to infection with Leishmania major [37 ]. Also, CCL2 overexpressing transgenic mice were shown to be greatly susceptible to Listeria monocytogenes or Mycobacterium tuberculosis [44 ]. Our earlier papers have described that CCL2 has a function to escalate the development of herpes encephalomyelitis [45 ] and cryptococcal encephalitis [46 ]. Our results presented herein suggest that if CCL2 production in the early phase of severe SIRS induction could be regulated, the subsequent AAM{phi} generation or the increased susceptibility of hosts as a result of AAM{phi} generation would be minimized. As the majority of patients with major surgery, trauma, and burn injury is diagnosed as SIRS, and infection-associated mortality of these patients is extremely high, CCL2 may be a possible, therapeutical target to control opportunistic infections immunologically.


   ACKNOWLEDGEMENTS
 
Grant #8690 from Shriners of North America supported this work.

Received December 19, 2003; revised March 24, 2004; accepted April 6, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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