Kupffer Cell-derived CCL2 Recruits Neutrophils To Promote Hepatic Ischemia/reperfusion Injury Through TLR4/CCR2 Signaling

Background:Liver ischemia/reperfusion (I/R) injury, which affects patient's life and prognosis severely, is a kind of pathological process characterized by liver immune disorder and systemic inflammatory response resulting from hemorrhagic shock, liver transplantation, hepatic resection, sepsis.As an early infiltrating immune cell population, neutrophils are recruited egress from bone marrow into injured liver under the chemokine concentration, eliciting significant tissue damage during hepatic I/R injury. Multiple chemokines participate in this process, CXCL1 and CXCL2 are most important chemokines involved in neutrophil recruitment. Furthermore, inflammatory monocytes as cytokine pool, which mobilization and infiltrating are slightly later than neutrophils, can secrete a variety of chemokines and cytokines to expand the immune response during hepatic I/R injury. This process is CCL2-CCR2 signaling dependent. However, the precise mechanism for their recruitment and infiltration and crosstalk between immune cells remains to be fully characterized.Objective:1. Explore the mechanisms of the recruitment of neutrophils during hepatic I/R injury.2. Clarify the crosstalk between innate immune cells and between major receptor molecules during hepatic I/R injury.Methods:A hepatic partial I/R model was reproduced in wild-type, CCL2-/-, CCR2-/- and TLR4-/- mice. Tissue damage was evaluated by serum enzyme analysis, hematoxylin-eosin staining and cytokine production measurement. Mobilization of neutrophils from the bone marrow and subsequent infiltration into the liver were measured by flow cytometry. CCR2 expression on neutrophils and CCL2 chemotaxis were measured using flow cytometry. The cellular source of CCL2 in the liver was determined by deleting specific cell groups and performing intracellular staining. The crosstalk between Kupffer cells and neutrophils was determined by the depletion of Kupffer cells using clodronate liposomes.Results: Liver damage was ameliorated and neutrophil recruitment and accumulation were decreased in CCL2-/-, CCR2-/- and TLR4-/- mice compared with wild-type mice. Neutrophils displayed activated expression of CCR2 during I/R, and these cells were required for CCL2-induced chemotaxis. Depletion of Kupffer cells protected the liver from I/R injury. Furthermore, genetic ablation of CCL2 reduced liver injury, as demonstrated by decreases in the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and subsequent reductions in neutrophil recruitment and accumulation. Inflammatory monocytes are recruited to liver in a CCL2/CCR2-dependent manner. Neutrophils activate CCR2 through TLR4 receptor.Conclusions:Kupffer cells secrete CCL2 to promote inflammatory monocytes and CCR2-expressing neutrophil recruitment from the bone marrow and subsequent infiltration into the liver through TLR4 during I/R. These findings reveal a novel pro-inflammatory role of cell-mediated CCL2-CCR2 interactions during this sterile insult.