Lipopolysaccharide activates nuclear factor κB in rat intestine: role of endogenous platelet‐activating factor and tumour necrosis factor

Abstract

1 We examined the eect of lipopolysaccharide (LPS), a cell wall constituent of Gram negative bacteria, on nuclear factor kB (NF-kB) activation in the intestine and the roles of endogenous platelet-activating factor (PAF), tumour necrosis factor-a (TNF) and neutrophils. We also compared the time course of NF-kB activation in response to PAF and LPS. 2 Ileal nuclear extracts from LPS (8 mg kg 71, IV)-injected rats were assayed for NF-kB-DNAbinding activity and identi®cation of the subunits. Some rats were pretreated with WEB2170 (a PAF receptor antagonist), anti-TNF antibody, or anti-neutrophil antiserum. NF-kB p65 was localized by immunohistochemistry. An additional group was challenged with PAF (2 mg kg 71 , IV) for comparison. 3 LPS activates intestinal NF-kB, both as p50-p50 and p50-p65 dimers within 15 min, and the eect peaks at 2 h. The eect is slower and more sustained than that of PAF, which peaks at 30 min. Activated NF-kB was immunolocalized within epithelial and lamina propria cells. LPS eect was reduced by 41, 37 and 44%, respectively, in animals pretreated with WEB2170, anti-TNF antibody, or anti-neutrophil antiserum (P50.05). 4 LPS activates intestinal NF-kB in vivo and neutrophil activation is involved in its action. The LPS eect is mediated by both endogenous PAF and TNF.

Lipopolysaccharide induces CXCL2/macrophage inflammatory protein‐2 gene expression in enterocytes via NF‐κB activation: independence from endogenous TNF‐α and platelet‐activating factor

“…Protein concentration was measured by Bradford's method. NF-jB was identified by EMSA using a kit from Promega (Madison, WI), as previously described 21 after gel loading of equivalent amounts of nuclear extracts. Supershift experiments for p50 and p65, the two subunits identified in our previous studies 5 were performed with anti-p50 (sc-114X) and anti-p65 (sc-109X) antibodies from Santa Cruz Biothechnology.…”

“…20 PAF was also found to mediate LPS-induced NF-jB activation. 21 It is therefore possible that PAF could play a role in mediating LPS-induced CXCL2 gene expression in enterocytes in vitro in an autocrine loop. However, in this study, we found that PAF did not mediate CXCL2 gene expression by LPS in enterocytes.…”

“…21 Here we examined whether PAF mediates LPS-induced CXCL2 gene expression in vitro in enterocytes. Pre-treatment for 30-60 min with WEB2170 (1-5)10 lg/ml), a PAF receptor antagonist, did not decrease CXCL2 gene expression induced by LPS after 2 hr.…”

“…21 Other investigators have found that LPS stimulates intestinal epithelial cells to produce TNF 22,23 and PAF. 24 Thus, it is possible that LPS may directly stimulate enterocytes to express CXCL2 via the activation of NFjB, and this effect may be mediated via endogenous production of TNF and PAF.…”

Hydroxyethyl Starch Inhibits Intestinal Production of Cytokines and Activation of Transcription Factors in Endotoxaemic Rats

“…16 Several studies have also demonstrated that LPS may activate NF-κB in rat intestine. 17,18 Based on these findings, we hypothesized that HES might exert its anti-inflammatory effects in gut during endotoxaemia and down-regulate pro-inflammatory mediators, TNF-α, IL-1β, IL-6 and IL-8, through the NF-κB signalling pathway. Our results demonstrated that HES reduced NF-κB activity and decreased TNF-α, IL-1β, IL-6 and IL-8 levels in the small intestine, thus supporting our hypothesis.…”