Heat shock response inhibits NF-kappaB activation and cytokine production in murine Kupffer cells.
BACKGROUND
Kupffer cells play a crucial role in the pathogenesis of sepsis through production of proinflammatory mediators and control of systemic endotoxemia. The anti-inflammatory effects of heat shock response (HSP) have been well documented. However, the role of HSP in lipopolysaccharide (LPS) induced Kupffer cell activation has not been fully investigated. In this study, we investigated the effects of HSP on LPS induced Kupffer cell NF-kappaB activation and cytokine production.


METHODS
Kupffer cells were isolated from mice by collagenase digestion and HSP was induced by culturing Kupffer cells with sodium arsenite. Kupffer cells were stimulated in vitro by LPS. Heat shock protein (HSP)-70 expression and cytoplasmic IkappaBalpha protein was determined by Western blot. Supernatant tumor necrosis factor (TNF)-alpha, interleukin (IL)-6 and IL-10 levels were measured by ELISA. NF-kappaB activation was analyzed by electrophoresis mobility shift assay. Cytokine and IkappaBalpha mRNA expression were determined by RT-PCR. Toll-like receptor 4 expression on Kupffer cells was determined by flow cytometry.


RESULTS
HSP pre-conditioning significantly inhibited LPS-induced cytokine TNF-alpha and IL-6 production and mRNA expression. NF-kappaB activation and IkappaBalpha degradation induced by LPS were attenuated by HSP. HSP up-regulated expression of IkappaBalpha mRNA. No effect of HSP on cell surface expression of TLR4 was observed.


CONCLUSIONS
Increased IkappaBalpha stability and up-regulation of IkappaBalpha gene expression may be one of the mechanisms of the inhibition of LPS induced Kupffer cell activation by HSP. HSP also inhibited expression of the anti-inflammatory cytokine IL-10, and the mechanism and biological significance of this effect merit further investigation.
Sun
Dongxu
D
Department of Critical Care Medicine, Peking Union Medical College Hospital, Peking Union Medical College & Chinese Academy of Medical Science, Beijing, PR China. sund@uthscsa.edu
Chen
Dechang
D
Du
Bin
B
Pan
Jiaqi
J
D000818:Animals; D018053:Arsenites / Q000494:pharmacology; D002478:Cells, Cultured; D016207:Cytokines / Q000096:biosynthesis*; D024202:Electrophoretic Mobility Shift Assay; D018840:HSP70 Heat-Shock Proteins / Q000032:analysis; D018869:Heat-Shock Response / Q000502:physiology*; D020672:I-kappa B Proteins / Q000032:analysis / Q000235:genetics; D016753:Interleukin-10 / Q000032:analysis / Q000235:genetics; D015850:Interleukin-6 / Q000032:analysis / Q000235:genetics; D007728:Kupffer Cells / Q000187:drug effects / Q000502:physiology*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D051379:Mice; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017670:Sodium Compounds / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis / Q000235:genetics
D018053:Arsenites; D016207:Cytokines; D018840:HSP70 Heat-Shock Proteins; D020672:I-kappa B Proteins; D015850:Interleukin-6; D008070:Lipopolysaccharides; D016328:NF-kappa B; C000606205:Nfkbia protein, mouse; D017670:Sodium Compounds; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; D000072000:NF-KappaB Inhibitor alpha; C017947:sodium arsenite