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

We examined the effect of lipopolysaccharide (LPS), a cell wall constituent of Gram negative bacteria, on nuclear factor κB (NF-κB) activation in the intestine and the roles of endogenous platelet-activating factor (PAF), tumour necrosis factor-α (TNF) and neutrophils. We also compared the time course of NF-κB activation in response to PAF and LPS. Ileal nuclear extracts from LPS (8 mg kg−1, IV)-injected rats were assayed for NF-κB-DNA-binding activity and identification of the subunits. Some rats were pretreated with WEB2170 (a PAF receptor antagonist), anti-TNF antibody, or anti-neutrophil antiserum. NF-κB p65 was localized by immunohistochemistry. An additional group was challenged with PAF (2 μg kg−1, IV) for comparison. LPS activates intestinal NF-κB, both as p50-p50 and p50-p65 dimers within 15 min, and the effect peaks at 2 h. The effect is slower and more sustained than that of PAF, which peaks at 30 min. Activated NF-κB was immunolocalized within epithelial and lamina propria cells. LPS effect was reduced by 41, 37 and 44%, respectively, in animals pretreated with WEB2170, anti-TNF antibody, or anti-neutrophil antiserum (P<0.05). LPS activates intestinal NF-κB in vivo and neutrophil activation is involved in its action. The LPS effect is mediated by both endogenous PAF and TNF. Keywords: Lipopolysaccharide, inflammation, transcription factors, in vivo Introduction NF-κB is a key transcription factor in the inflammatory response due to its regulatory role on pro-inflammatory cytokines, enzymes and adhesion molecules (Baeuerle & Henkel, 1994). In many cells, NF-κB exists in the cytoplasm in an inactive state, linked to a potent inhibitor, inhibitor protein κB (IκB), and becomes active only upon stimulation by agents such as LPS, reactive oxygen species (ROS), cytokines, and phorbol esters (Baeuerle & Henkel, 1994; Kopp & Ghosh, 1995). These agents trigger NF-κB/IκB dissociation and nuclear translocation of NF-κB, which regulates the gene transcription of several cytokines and adhesion molecules (Baeuerle & Henkel, 1994; Kopp & Ghosh, 1995). LPS, a cell wall constituent of gram-negative bacteria, plays a major role in the pathogenesis of septic shock. Administration of LPS in vivo produces shock and intestinal injury (Hsueh et al., 1987) and stimulates the production of IL1, IL6, chemokines (Baeuerle & Henkel, 1994; Kopp & Ghosh, 1995; Libermann & Baltimore, 1990; Couturier et al., 1992; Geng et al., 1993), adhesion molecules (Henninger et al., 1997) and TNF, a pivotal mediator in septic shock (Heumann et al., 1992; Tracey et al., 1986), which is known to activate NF-κB (Shakhov et al., 1990; Schutze et al., 1995; Sun et al., 1993). The transcription of many of these mediators is modulated by NF-κB. LPS also induces the production of PAF, a phospholipid mediator involved in shock (Benveniste, 1988; Handley, 1990) and bowel inflammation (Gonzalez-Crussi & Hsueh, 1983). PAF, when administered systemically, causes bowel necrosis (Gonzalez-Crussi & Hsueh, 1983) and leukocyte-endothelium adhesion (Sun et al., 1996; Kubes et al., 1990). Many of the in vivo effects of LPS are abrogated by pretreatment of the animal with anti-TNF antibody (Tracey et al., 1987) or PAF antagonists (Hsueh et al., 1987; Toth, 1990). These observations suggest that endogenous TNF and PAF mediate the adverse effects of LPS. Several protein subunits (p50, p65, p52, cRel, RelB) compose the NF-κB complex, forming either a homo or a heterodimer. It is known that LPS activates p50-p65 in endothelial cells in vivo (Essani et al., 1996) and haematopoietic cells in vitro (Ziegler-Heithbrock et al., 1994; Sanceau et al., 1995), but has no effect on the intestinal T84 epithelial cell line (Savkovic et al., 1997). Very few in vivo studies on NF-κB activation have been done. LPS has been shown to induce p50-p65 in rat lung tissue (Blackwell et al., 1996). We recently showed that PAF upregulates the gene expression of NF-κB (Tan, et al., 1994) and activates NF-κB in the rat intestine 30 min after its injection, mainly as p50 homodimers (De Plaen, et al., 1998). Furthermore, we found that neutrophils were necessary for its activation. However, the in vivo effect of LPS on NF-κB activation in the intestine has not been examined to date. The purpose of this study is to: (a) determine if LPS activates NF-κB in vivo in rat intestine; (b) examine the subunit composition and the time course of LPS-induced NF-κB activation; (c) determine if endogenous PAF and TNF mediate LPS effect on NF-κB activation; and (d) examine the role of polymorphonuclear leukocytes (PMN) in the process.