Mechanisms of Toll-Like Receptor 4 (TLR4)-Mediated... : Transplantation (original) (raw)

Basic and Experimental Research

Mechanisms of Toll-Like Receptor 4 (TLR4)-Mediated Inflammation After Cold Ischemia/Reperfusion in the Heart

Kaczorowski, David J.1,; Nakao, Atsunori2,; Vallabhaneni, Raghuveer1; Mollen, Kevin P.1; Sugimoto, Ryujiro2; Kohmoto, Junichi2; Zuckerbraun, Brian S.1; McCurry, Kenneth R.1,2; Billiar, Timothy R.1,3

1 Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA. 2 Thomas E. Starzl Transplantation Institute, University of Pittsburgh Medical Center, Pittsburgh, PA.

This work was supported by National Institutes of Health grants GM053789 (T.R.B.) and GM050441 (T.R.B.). D.J. Kaczorowski and R. Vallabhaneni are each recipients of American College of Surgeons Resident Research Scholarships.

The first two authors contributed equally to this work.

Address correspondence to: Timothy R. Billiar, M.D., Department of Surgery, University of Pittsburgh School of Medicine, 200 Lothrop Street, Presbyterian Hospital F1200, Pittsburgh, PA 15213.

E-mail: [email protected]

Received November 18, 2008

Received in revised form December 5, 2008

Accepted February 9, 2009

Abstract

Background.

Toll-Like Receptor 4 (TLR4) signaling mediates early inflammation after cold ischemia-reperfusion (I/R). We hypothesized that the TLR4 coreceptor CD14, the intracellular adaptor proteins myeloid differentiation factor 88 (MyD88) and TIR domain-containing-adaptor inducing IFNβ (TRIF) would be required for cold I/R induced inflammation. High mobility group box 1 (HMGB1) is a putative endogenous activator of TLR4. Therefore, we also assessed the contribution of HMGB1 in cold I/R induced inflammation.

Methods.

Syngeneic heart transplants were performed in mice deficient in CD14, MyD88, TRIF, or wild-type mice. In other experiments, anti-HMGB1 neutralizing antibody or control IgG was administered at reperfusion. Donor hearts were subjected to 2 hr of cold ischemia and retrieved after 3 hr of reperfusion.

Results.

After cold I/R, grafts revealed striking translocation of HMGB1 out of the nucleus in cardiac myocytes. Administration of an anti-HMGB1 neutralizing antibody resulted in reduced systemic interleukin (IL)-6, tumor necrosis factor alpha (TNFα), and intercellular adhesion molecule-1 (ICAM-1) messenger RNA (mRNA) levels (_P_≤0.05). Compared with controls, CD14 knock-out (KO) mice exhibited significantly lower (_P_≤0.05) systemic IL-6 and JE/monocyte chemotractant protein-1 levels after cold I/R. Intragraft TNFα and IL-1β mRNA levels were also significantly lower (_P_≤0.05) in CD14 KO grafts. MyD88 KO mice exhibited significantly lower (_P_≤0.05) systemic IL-6 levels compared with control mice after cold I/R. Intragraft TNFα, IL-6, and ICAM-1 mRNA levels were also significantly lower (_P_≤0.05) in MyD88 KO grafts. Significantly lower levels (_P_≤0.05) of serum IL-6, monocyte chemotractant protein-1 as well as intragraft TNFα, IL-6, IL-1β, and ICAM-1 were observed after cold I/R in TRIF deficient animals compared with controls.

Conclusions.

CD14, MyD88, TRIF, and HMGB1 contribute to the inflammatory response that occurs after cold I/R. These results provide insight into the mechanisms of TLR4-mediated inflammation after cold I/R.