Endotoxin Increases Type II-Phospholipase A₂ Gene Expression in Rat Intestinal Epithelial Cells

Abstract 626 Poster Session III, Monday, 5/3 (poster 180)

Platelet activating factor (PAF), a potent phospholipid inflammatory mediator, is implicated in the pathogenesis of necrotizing enterocolitis (NEC). Epidemiological studies have demonstrated bacterial colonization of the gut with endotoxin (LPS) production is a risk factor for NEC. PAF production is regulated by the enzyme Phospholipase A₂ and the biological effects of PAF are mediated through the PAF Receptor (PAF-R), the gene of which has recently been isolated and sequenced. This experiment measured Type II-Phospholipase A₂ and PAF-R gene expression over time in rat intestinal crypt epithelial (IEC-6) cells following exposure to LPS. We hypothesized that IEC-6 cells exposed to LPS would increase PLA₂-II and PAF-R mRNA levels over time. Post-confluent IEC-6 cells, cultured in DMEM, were exposed to LPS at doses of 2.5, 5, 10 and 20 ug/ml. Cell cultures were harvested at 2, 4, 8, 24 and 48 hours following LPS exposure, total RNA extracted and PLA₂-II and PAF-R transcripts measured by RT-PCR amplification. The reaction products were separated by electrophoresis and transcript bands were quantified by phosphorimaging. LPS effects on PLA₂-II and PAF-R gene expression were normalized using amplification products of β-actin gene transcripts as an internal control. We found that PLA₂-II is constituitively expressed in IEC-6 cells and that following LPS exposure, PLA₂-II transcript levels increase in a time and dose-dependent manner. Maximal transcript levels were observed at 48 hours at all LPS concentrations - ranging from a 2-fold to 5.75-fold increase over baseline at LPS concentrations of 2.5 ug/ml and 20 ug/ml, respectively. In contrast, PAF-R transcript levels showed no change over baseline with increasing LPS exposure over the study period. These data indicate LPS exposure increases PLA₂-II gene expression in cultured rat intestinal epithelial cells. Although a similar increase in PAF-R gene expression was not seen in these preliminary data, PAF-R gene up-regulation may require greater LPS concentrations or longer sampling periods than those used in this study. These data suggest that LPS exposure, a known risk factor for NEC in humans, may also up-regulate PLA₂-II gene expression in vivo, with consequent increased local intestinal PAF production. Increased PLA₂-II gene expression following exposure to LPS further supports the role of PAF in the pathogenesis of NEC.

Supported in part by a grant from The March of Dimes.