CRITICAL CARE II

Functional toll-like receptor 4 signaling in liver non-parenchymal cells is required for hepatic ischemia/reperfusion (I/R) injuryAllan Tsung MD, Sarah McAshon, Rosemary Hoffman PhD,David Geller MD, Timothy Billiar MD, FACSUniversity of Pittsburgh Medical Center, Pittsburgh, PA

INTRODUCTION: Recent evidence suggests that endogenous li-gands from damaged cells can activate the innate immune system viaToll-like receptor 4(TLR4) signaling. We have previously shown thatthe injury and inflammation following hepatic warm I/R requiresactivation of TLR4 using TLR4-deficient mice. To determinewhether the TLR4-dependent injury required TLR4 expression onliver parenchymal (hepatocytes) or non-parenchymal cells (Kupffercells, endothelial cells), we performed adoptive cell transfer to pro-duce chimeric mice.

METHODS: Chimeric mice were produced by adoptive transfer ofdonor bone marrow cells into irradiated recipient animals using com-binations of TLR4 wild type(HeOuj) and TLR4-deficient(HeJ) micein the following combinations: WT/WT, WT/Mutant, Mutant/Mutant, Mutant/WT. Partial warm ischemia was produced in the leftand median lobes of mice.

RESULTS: Consistent with previous results we found the TLR4-deficient mice were protected from hepatic I/R-induced damage inassociation with markedly lower TNF, IL-6, and ICAM-1 mRNAlevels. Further, we report here that hepatic JNK, but not P38 activa-tion was lower in TLR4-deficient mice. Our studies with chimericmice show that WT/Mutant(recipient/donor) chimeric mice consist-ing of recipient TLR4 WT parenchymal cells and TLR4-deficientdonor non-parenchymal cells were protected from hepatic I/R andexhibited decreased JNK activation compared to WT/WT chimericmice. In contrast, Mutant/WT chimeric mice were not protectedfrom I/R and were associated with increased JNK activation com-pared to Mutant/Mutant chimeric mice.

CONCLUSIONS: This study demonstrates that TLR4-dependenthepatic I/R injury activates JNK signaling. We also show that TLR4signaling in non-parenchymal cells and not hepatocytes is requiredfor the I/R induced injury.

Inhibition of nuclear export attenuates IkBaphosphorylationMark Walsh MD, Anirban Banerjee PhD, Christine Hamiel BS,Aaron Cheng MD, Guillermo Escobar MD, Lynn Gries MD,Robert McIntyre MDUniversity of Colorado Health Sciences Center, Denver, CO

INTRODUCTION: The NF-kB inhibitor, IkBa, is a predominantlycytoplasmic protein that undergoes basal nuclear/cytoplasmic shuttling.

Cellular stimulation results in IkBa phosphorylation and degradation,followed by resynthesis and transport to the nucleus. Nuclear IkBa bindsNF-kB and recycles it to the cytoplasm via the protein exportin-1. Wehypothesized that inhibition of exportin-1 would cause nuclear seques-tration of IkBa and attenuate IkBa phosphorylation.

METHODS: Human microvascular endothelial cells were treatedwith 20 nM Leptomycin B (LMB), a specific exportin-1 inhibitor, orvehicle for 60 minutes. Following IL-1B (100 ng/ml)stimulation,cells were fractionated; nuclear and cytoplasmic extracts were col-lected. Western blotting was performed to examine total IkBa, phos-phorylated IkBa (P-IkBa), phosphorylated IKKa/B, and phosphory-lated p38 MAP kinase. The chemokine GRO-a was measured as areadout of NF-kB activation.

RESULTS: Cytoplasmic P-IkBa was upregulated at 5 minutes andwas sustained thirty and sixty minutes after IL-1B stimulation. InLMB�IL-1B treated cells, cytoplasmic levels of P-IkBa were decreasedby 50%. Following IL-1B alone, cytoplasmic IkBa levels were markedlydecreased whereas cells exposed to LMB�IL-1B demonstrated persis-tent cytoplasmic IkBa. LMB�IL-1B treated cells demonstrated seques-tration of IkBa but not P-IkBa in the nuclear fraction. PhosphorylatedIKKa/B, the activator of IkBa, and phosphorylated p38 MAPK wereunchanged with exportin-1 inhibition. GRO-a production was inhib-ited in LMB�IL-1B treated cells by 70% (p�0.05).

CONCLUSIONS: Cellular shuttling is essential to NF-kB signal-ing. Nuclear export inhibition sequesters IkBa in the nucleus andattenuates cytoplasmic IkBa phosphorylation and degradation, lead-ing to diminished NF-kB activation.

A polymerized hemoglobin based oxygen carrierattenuates TLR4 stimulated NF-kB activation byinhibiting phosphorylation of IkB-aAaron M Cheng MD, Ernest E Moore MD, FACS,Jeffrey Johnson MD, Mark Walsh MD, Christine Hamiel BS,Anirban Banerjee PhD, Tomohiko Masuno MD,Christopher Silliman MD, PhDUniversity of Colorado Health Sciences Center/Denver Health,Denver, CO

INTRODUCTION: Previous clinical trials using polymerized hemo-globin (PolyHb) suggested that this hemoglobin-based oxygen car-rier (HBOC) also conveys anti-inflammatory effects. In this study wehypothesize that this HBOC inhibits TLR4-stimulated activation ofthe NF-kB pathway.

METHODS: Cultured human pulmonary endothelial cells werepre-incubated with PolyHb/cell media (50% v/v) mixture for 5 hrs.After pre-incubation, cells were stimulated with LPS in fresh media.NF-kB activation was determined by a p65 DNA binding assay andby p65 nuclear translocation; IkB-a phosphorylation was determinedby protein immunoblot.

RESULTS: In the LPS-treated groups, PolyHb decreased nuclearlysate p65 DNA binding by 79% compared to control (0.26 � 0.05vs. 1.23 � 0.27, p� 0.05). By digital immunofluorescence, PolyHbalso decreased LPS-induced p65 intensity in the nucleus (MFI/Cell:

Chimeric mice(Recipient/Donor) WT/WT WT/Mutant Mutant/Mutant Mutant/WT

ALT 1210 � 138 482 � 74* 303 � 36* 801 � 168**

Data are mean � SEM; n�6 per group; *indicates p�0.05 vs WT/WT; **indicatedp�0.05 vs Mutant/Mutant.

S34© 2005 by the American College of Surgeons ISSN 1072-7515/05/$30.00Published by Elsevier Inc.