Toll-Like Receptor 4 Mediates the Early Inflammatory Response After Cold Ischemia/Reperfusion

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Abstract
Ischemia/reperfusion (I/R) injury leads to graft dysfunction and may contribute to alloimmune responses posttransplantation. The molecular mechanisms of cold I/R injury are only partially characterized but may involve toll-like receptor (TLR)-4 activation by endogenous ligands. We tested the hypothesis that TLR4 mediates the early inflammatory response in the setting of cold I/R in a murine cardiac transplant model. Syngeneic heart transplants were performed in mutant mice deficient in TLR4 signaling (C3H/HeJ) and wild-type mice (C3H/HeOuJ). Transplants were also performed between the strains (mutant hearts into wild-type recipients and the converse). Donor hearts were subjected to 2 hr of cold ischemia. The grafts were retrieved at 3 and 24 hr after reperfusion. Serum samples were collected for cytokine analysis. Reverse-transcription polymerase chain reaction and histologic analysis were used to assess intra-graft inflammation. After transplant, serum tumor necrosis factor (TNF), interleukin (IL)-6, JE/monocyte chemotractant protein (MCP)-1, IL-1beta, and troponin I levels, as well as intragraft TNF, IL-1beta, IL-6, early growth response (EGR)-1, intercellular adhesion molecule (ICAM)-1, and inducible nitric oxide synthase (iNOS) mRNA levels, were significantly lower in the mutant-->mutant group compared to the wild-type-->wild-type group (P< or =0.05). Intermediate levels of serum IL-6, JE/MCP-1, as well as intragraft TNF, IL-1beta, IL-6, and ICAM-1 mRNA were observed after transplants in the mutant-->wild-type and wild-type-->mutant groups. Immunohistochemistry revealed less myocardial nuclear factor-kappaB nuclear translocation at and less neutrophil infiltration in the mutant-->mutant group compared to the wild-type-->wild-type group. These findings demonstrate that TLR4 signaling is central to both the systemic and intragraft inflammatory responses that occur after cold I/R in the setting of organ transplantation and that TLR4 signaling on both donor and recipient cells contributes to this response.