Activation of hepatocytes by extracellular heat shock protein 72.
Heat shock protein (HSP) 72 is released by cells during stress and injury. HSP-72 also stimulates the release of cytokines in macrophages by binding to Toll-like receptors (TLR) 2 and 4. Circulating levels of HSP-72 increase during hepatic ischemia-reperfusion injury. The role of extracellular HSP-72 (eHSP-72) in the injury response to ischemia-reperfusion is unknown. Therefore, the objective of the present study was to determine whether eHSP-72 has any direct effects on hepatocytes. Primary mouse hepatocytes were treated with purified human recombinant HSP-72. Conditioned media were evaluated by ELISA for the cytokines, TNF-alpha, IL-6, and macrophage inflammatory protein 2 (MIP-2). Stimulation of hepatocytes with eHSP-72 did not induce production of TNFalpha or IL-6 but resulted in dose-dependent increases in MIP-2 production. To evaluate the pathway responsible for this response, expression of TLR2 and TLR4 was confirmed on hepatocytes by immunohistochemistry. Hepatocyte production of MIP-2 was significantly decreased in hepatocytes obtained from TLR2 or TLR4 knockout mice. MIP-2 production was found to be partially dependent on NF-kappaB because inhibition of NF-kappaB with Bay 11-7085 significantly decreased eHSP-72-induced MIP-2 production. Inhibitors of p38 mitogen-activated protein kinase or c-Jun NH(2)-terminal kinase had no effect on production of MIP-2 induced by eHSP-72. The data suggest that eHSP-72 binds to TLR2 and TLR4 on hepatocytes and signals through NF-kappaB to increase MIP-2 production. The fact that eHSP-72 did not increase TNF-alpha or IL-6 production may be indicative of a highly regulated signaling pathway downstream from TLR.
The Laboratory of Trauma, Sepsis and Inflammation Research, Department of Surgery, University of Cincinnati, Cincinnati, OH 45267-0558, USA.
Elizabeth
E
Galloway
Thomas
T
Shin
Nadine
N
Huber
Thorsten
T
Eismann
Satoshi
S
Kuboki
Rebecca
R
Schuster
John
J
Blanchard
Hector R
HR
Wong
Alex B
AB
Lentsch
D000818:Animals; D002478:Cells, Cultured; D054426:Chemokine CXCL2 / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D050884:HSP72 Heat-Shock Proteins / Q000235:genetics / Q000494:pharmacology*; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007376:Interleukin-2 / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors; D008099:Liver / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D009154:Mutation; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism; D009570:Nitriles / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D011994:Recombinant Proteins / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects; D013450:Sulfones / Q000494:pharmacology; D051195:Toll-Like Receptor 2 / Q000235:genetics / Q000378:metabolism; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors
C416282:BAY 11-7085; D054426:Chemokine CXCL2; C404915:Cxcl2 protein, mouse; D004791:Enzyme Inhibitors; D050884:HSP72 Heat-Shock Proteins; D007376:Interleukin-2; D016328:NF-kappa B; D009570:Nitriles; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013450:Sulfones; C493486:Tlr2 protein, mouse; C493487:Tlr4 protein, mouse; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D014409:Tumor Necrosis Factor-alpha; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases