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.
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.
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.
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 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).
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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