Abstract 636 Poster Session III, Monday, 5/3 (poster 181)

Systemic administration of LPS (endotoxin) to rats produces intestinal injury. LPS is a known activator of NF-κB (p50-p65), a transcription factor that regulates many pro-inflammatory cytokines. We previously demonstrated that: 1) intestinal NF-κB (p50-p65 dimer) is activated by LPS (8 mg/kg IV); 2) LPS effect involves endogenous PAF and PMN activation; and 3) PAF (1.5 µg/kg) induces NF-κB (p50-p50) activation. The purpose of this study is to examine the effect of LPS on NF-κB activation in IEC cells and the role of PAF and PMNs. Subconfluent (90%) cultured IEC-6 cells (4×106 cells/flask) were treated with: 1) LPS (1 µg/ml) for 15 to 120 min; 2) LPS + rat peritoneal PMNs (1×106/ml) for 30 min; 3) LPS + WEB 2170 (0.5 µg/ml, a PAF antagonist) for 30 min; and 4) PAF (0.5 µg/ml) for 30 min. The NF-κB-DNA binding activity of cell's nuclear extracts and subunits were determined by EMSA and supershift experiments. We found that in IEC-6 cells, LPS activates NF-κB p50-p65, which reaches a peak at 30 min and remains high at 2 h. This effect was not modified by either WEB 2170 or the addition of PMNs. Further, PAF itself induced occasional minimal NF-κB activation. These facts suggest that in IEC-6 cells, unlike in vivo, 1) LPS activates NF-κB by a PAF-independent mechanism; 2) NF-κB activation is not affected by the addition of PMNs; and 3) PAF alone has no or minimal effect on NF-κB activation.

Supported by NIH grants HD 31840 and DK 34574