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Gomes, Rachel N.*; Figueiredo, Rodrigo T.; Bozza, Fernando A.§; Pacheco, Patrícia*; Amâncio, Rodrigo T.*; Laranjeira, Andréa P.; Castro-Faria-Neto, Hugo C.*; Bozza, Patrícia T.*; Bozza, Marcelo T.

doi: 10.1097/01.shk.0000228801.56223.92
Basic Science Aspects

ABSTRACT: The chemokine monocyte chemoattractant protein 1/CC chemokine ligand 2 (MCP-1/CCL2) is a potent chemoattractant of mononuclear cells and a regulatory mediator involved in a variety of inflammatory diseases. In the present study, we demonstrate that mcp-1/ccl2-deficient mice are more susceptible to systemic inflammatory response syndrome induced by lipopolysaccharide and to polymicrobial sepsis induced by cecum ligation and puncture (CLP) when compared with wild-type mice. Interestingly, in the CLP model, mcp-1/ccl2-deficient mice efficiently cleared the bacteria despite an impaired recruitment of leukocytes, especially mononuclear cells. The increased lethality rate in these models correlates with an impaired production of interleukin (IL) 10 in vivo. Furthermore, macrophages from mcp-1/ccl2-deficient mice activated with lipopolysaccharide also produced lower amounts of IL-10 and similar tumor necrosis factor compared with wild-type mice. We observed a drastic increase in the amounts of macrophage migration inhibitory factor at 6 and 24 h after CLP in mcp-1/ccl2-deficient mice. These results indicate that endogenous MCP-1/CCL2 positively regulates IL-10 but negatively controls macrophage migration inhibitory factor during peritoneal sepsis, thus suggesting an important immunomodulatory role for MCP-1/CCL2 in controlling the balance between proinflammatory and anti-inflammatory factors in sepsis.

ABBREVIATIONS-CFU; colony forming unit, CLP; cecum ligation and puncture, H&E; hematoxilin/eosin, HMGB-1; high mobility group box, MCP-1/CCL2; monocyte chemoattractant protein-1/CC chemokine ligand-2, MIF; macrophage migration inhibitory factor, SIRS; systemic inflammatory response syndrome

*Laboratório de Imunofarmacologia, Departamento de Fisiologia e Farmacodinâmica, IOC, and LATEB, Biomanguinhos, Fundac¸ão Oswaldo Cruz, Rio de Janeiro, RJ, 21.045-900, Brasil; Laboratório de Inflamac¸ão e Imunidade, Departamento de Imunologia, Instituto de Microbiologia, Universidade Federal do Rio de Janeiro; §Centro de Tratamento Intensivo, Instituto de Pesquisa Clinica Evandro Chagas, Fundac¸ão Oswaldo Cruz, Rio de Janeiro, RJ, 21.045-900, Brasil

Received 18 Apr 2006; first review completed 8 May 2006; accepted in final form 11 May 2006

Address reprint requests to Dr. Marcelo T. Bozza, Laboratório de Inflamação e Imunidade, Departamento de Imunologia, Instituto de Microbiologia, Universidade Federal do Rio de Janeiro-UFRJ. Av. Brigadeiro Trompowsky s/n CCS Bloco I, Ilha do Fundão, Rio de Janeiro, RJ, 21.941-590 Brasil. E-mail: Dra. Patricia Bozza, Laboratório de Imunofarmacologia, Departamento de Fisiologia e Farmacodinâmica, IOC, Fundação Oswaldo Cruz. Av. Brasil 4365, Manguinhos, Rio de Janeiro, RJ, Brazil, CEP 21045-900. E-mail:

Disclosures: The authors declare that they have no competing financial interests.Received 18 Apr 2006; first review completed 8 May 2006; accepted in final form 11 May 2006

This work was supported in part by grants from Conselho de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Fundação José Bonifácio (FuJB), Howard Hughes Medical Institute (HHMI), and Programa de Núcleos de Excelência (Pronex).

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Activation of the immune system and the consequent inflammation during bacterial infections are not only responsible for protection but are also critically involved in pathogenesis. The immune/inflammatory responses triggered by molecules of the infectious agents are amplified by molecules of host origin including cytokines, lipid mediators, and reactive oxygen species (1). These inflammatory mediators influence the recruitment and activation of leukocytes affecting pathogen clearance at potential cost of promoting tissue damage. In fact, in experiments where high doses of bacterial products are administered, the neutralization or gene deletion of selected proinflammatory mediators significantly reduced mortality rate (2-4). However, in models of localized bacterial infection, the lack of a proinflammatory cytokine such as tumor necrosis factor (TNF) was detrimental, causing increased bacterial growth and lethality rate (5-7). The production and effects of inflammatory mediators are regulated and counterbalanced by anti-inflammatory cytokines. The lack of endogenous interleukin (IL) 10, a prototypic anti-inflammatory cytokine, resulted in increased levels of TNF and enhanced mortality in mouse models of endotoxemia, whereas in models of bacterial infection, endogenous IL-10 impairs the bacterial clearance (8-11).

The recruitment of leukocytes to tissues is an essential phenomenon of the response to infection because these emigrated cells participate in the clearance of the infectious agents. Chemokines are critically involved on leukocyte migration but also affect the biology of leukocytes in several ways (12). Monocyte chemoattractant protein 1/CC chemokine ligand 2 (MCP-1/CCL2) is a CC chemokine, with chemoattractant activity for monocytes, T cells, mast cells, and basophils (13-15). Bacterial challenge of baboons caused a time-dependent increase in plasma MCP-1/CCL2 levels (16). Monocyte chemoattractant protein 1/CC chemokine ligand 2 was also increased in patients with sepsis and septic shock (17). Treatment with recombinant MCP-1/CCL2 increased the bacterial clearance and protected the mice systemically infected with Pseudomonas aeruginosa or Salmonella typhimurium (18). The pretreatment of mice with anti-MCP-1/CCL2 increased lethality rate and was associated with impaired bacterial clearance and reduced leukocyte recruitment in a model of peritoneal sepsis (19). In this septic model, endogenous MCP-1/CCL2 influenced the systemic cytokine balance in favor of anti-inflammatory and immune-enhancing cytokines (20). Furthermore, the administration of recombinant MCP-1/CCL2 protects the mice from a lethal challenge with lipopolysaccharide (LPS) accompanied by increased levels of the anti-inflammatory cytokine IL-10, whereas neutralization of endogenous MCP-1/CCL2 caused an enhanced lethality upon sublethal LPS dose (21). Together, these findings suggest that MCP-1/CCL2 possesses important regulatory properties, protecting the host against the damage caused by the inflammatory response, in addition to the well-known effect on leukocyte recruitment and activation. However, the role of MCP-1/CCL2 in controlling the magnitude of the inflammatory response during sepsis is not fully characterized.

In the present study, we took advantage of mice genetically deficient of MCP-1/CCL2 to investigate the role of endogenous MCP-1/CCL2 in models of systemic inflammatory response syndrome induced by LPS and peritoneal sepsis induced by cecal ligation and puncture (CLP). In both models, mcp-1/ccl2-deficient mice are more susceptible than wild-type (WT) mice. Interestingly, in the CLP model, mcp-1/ccl2-deficient mice efficiently cleared bacteria. Moreover, we identified MCP-1/CCL2 as a positive regulator of IL-10 and a negative regulator of the proinflammatory cytokine macrophage migration inhibitory factor (MIF), thus suggesting the mechanism for the increased lethality rate in the absence of MCP-1/CCL2.

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Female or male wild-type and mcp-1/ccl2-deficient mice of C57Bl/6X129Sv/J genetic background (22), with 6 to 8 weeks of age were obtained from Dr. Craig Gerard (Harvard Medical School, Boston, Mass). The animals were kept at constant temperature (25°C), with free access to food and water in a room with a 12-h light/dark cycle. The experiments were approved by the Institutional Animal Welfare Committee (CEUA-Fiocruz).

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Lipopolysaccharide from Escherichia coli (serotype 0111:B4) was purchased from Sigma Chemical Company (St. Louis, Mo). Duo Set enzyme-linked immunosorbent assay (ELISA) development kits to quantify murine TNF-α, IL-6, IL-10, and KC were purchased from R&D systems (Minneapolis, Minn). The quantification of mouse MIF was performed using a kit from Chemicon International (Temecula, CA). Thiopental was from Abbott Laboratory (Abbott Park, IL), and ketamine was from Cristália (Sao Paulo, Brazil).

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Murine model of endotoxemia

A murine model of endotoxemia was performed by injecting mice intraperitoneally with 100 μg/cavity of LPS (sublethal dose) diluted in sterile saline. Animals were followed for clinical symptoms or eventual death every 12 h during 6 days.

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Induction of peritoneal sepsis

Cecum ligation and puncture was performed, as previously described (23). In brief, mice were anesthetized with a mixture of thiopental (40 mg/kg) and ketamine (80 mg/kg) diluted in sterile saline and administered intraperitoneally (0.2 mL). Laparotomy was performed, and the cecum was exposed and carefully ligated below the ileocecal junction to avoid causing bowel obstruction. The cecum was punctured once with an 18-gauge needle and was then gently squeezed to empty its contents through the puncture. The incision was closed in layers using a 3-0 nylon suture line. Immediately after the surgery, 0.5 mL of sterile saline was administered subcutaneously to the animals for volume resuscitation. Sham-operated mice were subjected to identical procedures, except that ligation and puncture of the cecum were omitted. Animals subjected to CLP developed early signs of sepsis, including lethargy, piloerection, and diarrhea. Survival rate of mice subjected to CLP or sham injury was determined daily for 7 days. In the next set of experiments, the CLP and sham-operated mice group were anesthetized at specific time points and euthanized in CO2 chamber. Each animal had its peritoneal cavity opened and washed with 3 mL of sterile saline, and the lavage fluids were collected for total and differential leukocyte numbers. In CLP group, an aliquot with 20 μL of lavage fluid were preserved for the assessment of bacteria colony forming units (CFUs). The fluids were centrifuged at 2000 rpm for 10 min at 4°C, and cell-free peritoneal wash samples were collected. The samples of lavage fluid and plasma were stored at −70°C for determination of the levels of cytokines using ELISA. In selected experiments, mice were inoculated intraperitoneally with 4 × 106 CFUs of E. coli (ATCC25922); after 6 h, the peritoneal fluid was obtained as described for quantification of CFUs.

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Leukocyte counts

Leukocyte numbers were evaluated in the peritoneal samples of sham or CLP group. Total leukocyte counts were performed in Neubawer chambers under optical microscopy after diluting the samples in Türk solution (2% acetic acid). Differential leukocyte counts in peritoneal samples were performed in cytospin smears stained by the May-Grünwald-Giemsa method.

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Determination of CFU

Ten-fold serial dilutions of peritoneal lavage fluid and blood from each mouse were plated on agar plates and incubated at 37°C. After 24 h, the number of colonies was determined.

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Cytokine and chemokines measurements

The magnitude of the inflammatory response was evaluated by measuring the levels of IL-6, MIP 2, IL-10, TNF-α, KC, and MIF in the peritoneal fluid, using ELISA. Mice were killed in a carbon dioxide chamber at designated time points, and the peritoneal cavity was opened and rinsed with Hanks' buffered salt solution without calcium or magnesium. The particulate matter was removed by centrifugation, and the supernatant fractions were used for immunoassays. All the measurements were performed in duplicate, following the manufacturer's instructions.

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Isolation of peritoneal macrophage

Peritoneal macrophages were obtained, washing the peritoneal cavity with 3 mL of saline. Two million cells were distributed to culture plates and were placed into a 37°C CO2 incubator for 2 h. All nonadherent cells were subsequently removed, and the adherent cells were stimulated with LPS (50 ng/mL). The supernatants were removed 24 h later for cytokine analysis.

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Statistical analysis

Survival curves were generated with Prism computer software (Graphpad Software, Inc, San Diego, Calif), and comparisons between curves were made using the Mantel-Cox log-rank test. All other data are expressed as mean ± SEM and compared using a 2-tailed Student t test. Data were considered statistically significant if P values were less than 0.05.

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Endogenous CCL2 has anti-inflammatory activity in an endotoxemia model

It was previously shown that treatment with recombinant MCP-1/CCL2 reduces the LPS-induced lethality and that neutralization of endogenous MCP-1/CCL2 enhances lethality (21). Initially, we tested the effect of a sublethal dose of LPS (100 μg/mice) on WT and mcp-1/ccl2-deficient mice. The lack of MCP-1/CCL2 significantly increased the susceptibility to LPS (Fig. 1). The quantification of TNF-α and CXCL1/KC in the plasma demonstrated similar high levels in WT and mcp-1/ccl2-deficient mice (Fig. 2A). On the other hand, mcp-1/ccl2-deficient mice had a marked reduction of LPS-induced plasma levels of MIP-2 and IL-10, when compared with WT mice. Macrophages from mcp-1/ccl2-deficient mice stimulated in vitro with LPS produce similar levels of TNF-α and lower amounts of IL-10 (Fig. 2B). These results suggest that endogenous MCP-1/CCL2 has anti-inflammatory effects protecting mice from LPS-induced lethality through the induction of IL-10 by macrophages.

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Increased susceptibility of mcp-1/ccl-2-deficient mice after CLP

To characterize the contribution of endogenous MCP-1/CCL2 in the host response to peritoneal infection, we performed the CLP model on WT and MCP-1/CCL2-deficient mice. The absence of MCP-1/CCL2 significantly increased the mortality rate in a period of 6 days after CLP (Fig. 3). All mcp-1/ccl2-deficient mice have died, whereas 60% of the WT mice were still alive. These findings confirmed previous studies using neutralizing antibodies (19), thus indicating that endogenous MCP-1/CCL2 has a protective role in acute septic peritonitis.

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Mice deficient in mcp-1/ccl2 gene have decreased leukocyte recruitment to the peritoneal cavity after CLP

The recruitment of leukocytes into the infectious foci is required to eliminate invading microorganisms. As expected, in WT mice, the recruitment of total leukocytes, especially neutrophils, occurred a few hours after CLP, whereas monocytes accumulated later. The lack of mcp-1/ccl2 gene caused a modest reduction of total leukocyte and neutrophil recruitment at 24 h after the CLP procedure (Fig. 4, A and B). On the other hand, the increased recruitment of monocytes observed in the WT mice after CLP was completely abolished in the absence of mcp-1/ccl2-deficient (Fig. 4C). These results indicate that MCP-1/CCL2 partially contributes to the recruitment of neutrophils but is essential to the recruitment of monocytes to the infectious foci during bacterial sepsis.

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Accelerated bacterial clearance in the absence of MCP-1/CCL2 upon septic peritonitis

To determine the involvement of endogenous MCP-1/CCL2 in antibacterial defense, we counted CFU in the peritoneal cavity and in the blood after 6 and 24 h of CLP. Six hours after CLP, reduced number of CFUs was recovered from the peritoneal fluid of mcp-1/ccl2-deficient mice compared with WT mice (Fig. 5A). To confirm the involvement of endogenous MCP-1/CCL2 impairing bacterial clearance, WT and mcp-1/ccl2-deficient mice received an intraperitoneal injection of approximately 4 × 106 CFUs of E. coli; 6 h later, the number of CFUs was determined. Similar to the CLP model, mcp-1/ccl2-deficient mice had an accelerated clearance of E. coli in the peritoneal cavity (Fig. 5B). Twenty-four hours after the surgical procedure, the number of CFUs was similar in WT and mcp-1/ccl2-deficient mice both in the peritoneal fluid and in the blood (Fig. 5, C and D). These results suggest that the increased mortality observed in mcp-1/ccl2-deficient mice is not associated with an impaired bacterial clearance.

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Mice deficient in mcp-1/ccl2 gene have reduced concentrations of IL-10 and increased MIF in a model of polymicrobial sepsis

The observation that mcp-1/ccl2-deficient mice are more susceptible to CLP and LPS-induced lethality suggest that endogenous MCP-1/CCL2 participates in the control of cytokine production and promotes tissue protection during CLP. Quantification of CXCL1/KC, TNF-α, and IL-6 revealed that mcp-1/ccl2-deficient and WT mice produce similar amounts of these cytokines at 6 h after CLP; at 24 h, CXCL1/KC and IL-6 were reduced on mcp-1/ccl2-deficient mice compared with WT mice (Fig. 6, A-C). Considering the essential role of the proinflammatory cytokine MIF in the pathogenesis of sepsis (3, 4, 24, 25), we decided to measure its levels on WT and mcp-1/ccl2-deficient mice. The mcp-1/ccl2-deficient mice presented approximately a 5-fold increase of MIF levels both at 6 and 24 h after CLP (Fig. 6D). Moreover, the lack of MCP-1/CCL2 caused an almost complete abrogation of IL-10 production at 24 h after CLP.

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In the present study, we characterized the role of endogenous MCP-1/CCL2 using mice genetically deficient of this chemokine in a model of systemic inflammatory response syndrome (SIRS) induced by LPS and in a model of polymicrobial sepsis induced by CLP. The increased lethality rate of mcp-1/ccl2-deficient mice in these models correlates with an impaired production of IL-10. Interestingly, in the CLP model, mcp-1/ccl2-deficient mice efficiently cleared bacteria and produced increased levels of the proinflammatory cytokine MIF. Our results suggest that endogenous MCP-1/CCL2 protects mice from the SIRS by regulating proinflammatory and anti-inflammatory cytokine production.

The increased lethality rate of mcp-1/ccl2-deficient mice to a sublethal dose of LPS confirmed a previous study using neutralizing antibodies (21). The absence of MCP-1/CCL2 caused a drastic impairment of IL-10 production with similar levels of TNF-α and CXCL-1/KC upon in vivo challenge with LPS. We observed that lack of endogenous MCP-1/CCL2 also caused a substantial reduction of IL-10 levels after CLP. This reduction of IL-10 might have an important role in the increased lethality rate of mcp-1/ccl2-deficient mice. In fact, IL-10-deficient mice have increased lethality rate despite the enhanced clearance of E. coli in a model of peritoneal sepsis (11). Resident macrophages from mcp-1/ccl2-deficient mice activated with LPS displayed a profile equivalent to the in vivo response, with lower IL-10 secretion and similar TNF-α production compared with WT mice. This effect might be caused by an autocrine role of MCP-1/CCL2 on IL-10 secretion by macrophages. A previous study demonstrated that administration of recombinant MCP-1/CCL2 protects mice from a lethal challenge with LPS accompanied by increased levels of the anti-inflammatory cytokine IL-10 (21). However, treatment with MCP-1/CCL2 was unable to enhance the secretion of IL-10 by resident macrophages from WT or mcp-1/ccl2-deficient mice stimulated with LPS (data not shown). The role of MCP-1/CCL2 seems to be different from other inflammatory mediators that induce IL-10 secretion on macrophages (26, 27). In fact, our result suggests a different mechanism, such as a selective defect of MCP-1/CCL2 in recruiting to noninflamed peritoneal cavity a subpopulation of mononuclear cells able to produce higher amounts of IL-10 when stimulated with LPS in vitro. CC chemokine receptor 2 (CCR2) and its principal ligand, MCP-1/CCL2, have been considered essential to the recruitment of mononuclear phagocytes from the blood to inflammatory sites, without affecting the physiological mobilization of these cells to noninflamed tissues (22, 28). A recent study proposed, however, that the axis CCR2 and MCP-1/CCL2 have a crucial role in mobilizing CD11b+Ly6Chi monocytes from the bone marrow to the circulation during infection with Listeria monocytogenes or inflammation induced by thioglycollate (29). This subpopulation produces high amounts of TNF upon activation and is critically involved in protection to L. monocytogenes infection.

The lack of endogenous MCP-1/CCL2 by genetic deficiency increased the lethality rate induced by CLP. This result recapitulates the effect of treatment with anti-MCP-1/CCL2 in this model of sepsis (19). However, the mechanisms involved in the increased lethality rate in both cases are different. Treatment with anti-MCP-1/CCL2 caused a significant impairment of bacterial clearance that correlated with a profound failure on monocytes and neutrophils recruitment (19). The mcp-1/ccl2-deficient mice were able to perform an efficient bacterial clearance, despite the reduced recruitment of monocytes. Neutrophils are considered important to bacterial clearance, and the recruitment of these leukocytes was barely affected by the absence of MCP-1/CCL2 at 6 h, when we observed a significant increase on bacterial clearance. Interestingly, a recent report demonstrated the expression of CCR2, the main receptor for MCP-1/CCL2, on neutrophils of septic animals and a direct role of MCP-1/CCL2 in recruiting these leukocytes (30). The discrepancies of the genetic deficiency and antibody neutralization of MCP-1/CCL2 on neutrophil recruitment and bacterial clearance might have different reasons, such as a compensatory effect of another inflammatory mediator or chemokine on mcp-1/ccl2-deficient mice, an incomplete neutralization of MCP-1/CCL2, or a neutralization of another chemokine by the antibody treatment. Studies demonstrating that mice deficient of mcp-1/ccl2 gene had an increased lethality rate during aspiration-induced pneumonitis likely because of an inability to restrict the inflammatory response (31) and had impaired resolution processes during acute P. aeruginosa pneumonia, without affecting the bacterial clearance (32), are consistent with our findings. It is possible that in the absence of MCP-1/CCL2, neutrophils perform a more efficient bacterial killing and tissue damage. However, future analyses are required to clarify this issue.

An increased lethality rate to CLP has also been recently shown in the absence of CCR2 signaling (33, 34). This increased lethality rate of mice treated with neutralizing CCR2 correlated with a reduced bacterial clearance and a significant increase of IL-10 production (33). Together, these results highlight the protective role of the axis MCP-1/CCL2-CCR2 during peritoneal septic episodes, despite opposite effects of MCP-1/CCL2 and CCR2 deficiencies on bacterial clearance and IL-10 production. Interestingly, the induction of MCP-1/CCL2 during SIRS causes the generation of alternative activated macrophages responsible for an impaired bactericidal response upon subsequent peritoneal sepsis (35). Differential roles of MCP-1/CCL2 and CCR2 in host defense to other infection agents have been demonstrated. In fact, MCP-1/CCL2 deficiency had no effect on coronavirus clearance or survival but impaired the recruitment of macrophages to the infectious site, although ccr2-deficient mice succumb to the infection with higher viral titers (36). On the other hand, a protective role of both endogenous MCP-1/CCL2 and CCR2 during cytomegalovirus infection has been demonstrated (37). In this infection model, the deficiency of MCP-1/CCL2 or CCR2 had a similar effect, impairing the viral clearance, recruiting macrophages and natural killer cells, and producing inflammatory mediators.

The analysis of inflammatory cytokines indicates that endogenous MCP-1/CCL2 has a minor effect on TNF-α, CXCL1/KC, and IL-6 but profoundly influences the secretion of MIF. The absence of MCP-1/CCL-2 causes a 5-fold increase in MIF levels during CLP. Considering the well-established role of MIF as an inducer of TNF secretion, the dissociation of TNF-α and MIF may be seen as unexpected. In fact, MIF regulates the expression of toll-like receptor 4, affecting the production of TNF-α induced by LPS and gram-negative bacteria (38). It is important to observe, however, that MIF is a later mediator in the inflammatory cascade triggered by bacterial infection, with peak levels occurring hours after TNF-α (24). The kinetics of MIF release is similar to that observed for high mobility group box; the treatment of mice with anti-high mobility group box hours after the onset of the septic episode also protects against lethality (39). Treatment with anti-TNF has been proven ineffective unless used prophylactically probably because TNF-α is released very early after infection. Macrophage migration inhibitory factor gene deficiency protects against LPS lethality and P. aeruginosa instilled in the lungs; MIF neutralization with antibodies later, after the septic episode, led to better survival rate in a mouse model of peritoneal sepsis (4, 24). Finally, elevated MIF levels in recently diagnosed septic patients seem to be an early indicator of poor outcome (25).

In conclusion, our results suggest that lethality of mcp-1/ccl2-deficient mice in models of SIRS and peritoneal sepsis is not caused by a bacterial overgrowth. In fact, the present study identified the proinflammatory versus anti-inflammatory imbalance as responsible for the higher lethality rate in the SIRS and septic model in mice lacking MCP-1/CCL2.

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The authors thank Dr. Barret J. Rollins from the Dana-Farber Cancer Institute and Dr. Craig Gerard from the Children's Hospital (Harvard Medical School) for kindly providing MCP-1/CCL2-deficient mice and their backcrossed controls.

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MCP-1; CCL2; CLP; endotoxin; sepsis; cytokines

©2006The Shock Society