Several IEC lines, including the Caco-2 cell line, respond minimally to LPS because of very low TLR4 expression [20], [22], [31]. Six hours post stimulation with cPAF, done we observed a subsequent dose-dependent increase in TLR4 mRNA in Caco-2 cells using Real Time PCR analysis (Figure 2c). We addressed the functionality of the induced TLR4 by exposing these cells to LPS. We found increased IL-8 secretion by Caco-2 cells exposed to 5 mM PAF for 24 h, then stimulated for 18 h with 10 ng/ml LPS as measured by ELISA. In contrast, no increase in IL-8 secretion occurred when Caco-2 cells were treated with either PAF or LPS alone (Figure 3). These results indicate that LPS could stimulate IEC secretion of IL-8, but only if TLR4 expression was first induced by treating the cells with PAF and later stimulated with TLR4 ligand.

Figure 2 PAF-induced TLR4 expression in intestinal epithelial cells. Figure 3 PAF increases IL-8 in intestinal epithelial cells with TLR4 ligand stimulation. Interestingly, the rat (IEC-6, Fig. 2b) and human (Caco-2, Fig. 2c) cell lines used possessed very different dose responses to PAF. IEC-6 cells undergo apoptosis at higher than 2 ��M PAF concentrations, thus making it difficult to impossible to measure gene expression at those conditions. Additionally, the baseline TLR4 expression is significantly higher in IEC-6. Nevertheless, TLR4 expression in both cell lines is subject to regulation by PAF. Platelet-activating factor increases TLR4 protein expression in intestinal epithelial cells To determine whether or not the increased mRNA of IEC following PAF stimulation leads to a corresponding increase in TLR4 protein expression, immunohistochemistry was performed.

Due to extremely low expression levels in Caco-2 cells, we could not detect TLR4 and commercially available antibodies did not detect rat TLR4 in IEC-6. Upon testing various cell lines, we have found that human TLR4 is readily detected in HT29-CL19A cells. Therefore, HT29-CL19A cells were treated with or without PAF and probed with anti-TLR4 antibodies. As seen in Figure 4, there appeared to be increased TLR4 expression in cells treated with PAF as compared to unstimulated control. These data suggest that although TLR4 is weakly expressed on the surface of IEC, platelet activating factor stimulation upregulates expression of the receptor.

This could potentially result in increased recognition of TLR4 ligands at the intestinal epithelial surface and subsequently lead to production of inflammatory cytokines. Figure 4 PAF induces TLR4 expression in intestinal epithelial cells. Platelet-activating Brefeldin_A factor induces TLR4 promoter activation The PAF-induced changes in TLR4 mRNA and protein levels could be due to regulation of transcription or could be a result of posttranscriptional mechanisms.