Toll-like receptor 4 mediates the early inflammatory response after cold ischemia/reperfusion.
BACKGROUND
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA 15213, USA.
David J
DJ
Kaczorowski
Atsunori
A
Nakao
Kevin P
KP
Mollen
Raghuveer
R
Vallabhaneni
Ryujiro
R
Sugimoto
Junichi
J
Kohmoto
Kimimasa
K
Tobita
Brian S
BS
Zuckerbraun
Kenneth R
KR
McCurry
Noriko
N
Murase
Timothy R
TR
Billiar
D000818:Animals; D003080:Cold Temperature; D016027:Heart Transplantation / Q000009:adverse effects*; D007249:Inflammation / Q000503:physiopathology* / Q000517:prevention & control; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D018345:Mice, Knockout; D011183:Postoperative Complications / Q000517:prevention & control; D015427:Reperfusion Injury / Q000503:physiopathology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D051197:Toll-Like Receptor 4 / Q000172:deficiency / Q000235:genetics / Q000502:physiology*
C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4