Intestinal epithelial cells release CXCL‐2 in response to lipopolysaccharide via NF‐KB and IKK activation

We had previously shown that CXCL-2, a critical chemokine for neutrophils, mediates neutrophil infiltration and intestinal injury induced by PAF. Intestinal epithelium is the first line of defense against microorganisms. Here, we examined the role of NF-KB in the induction of CXCL2 in enterocytes, in response to LPS and TNF. IEC-6 cells were incubated with 3 NF-KB inhibitors: PDTC 200 μM, helenalin 50 μM, NBD (specific inhibitor of IKK activation) or control peptide, 200 μM, for 30 min prior to LPS or TNF. Cells were collected after different times for whole cell extracts and total RNA extraction. CXCL2 gene expression was quantified by real-time PCR and CXCL2 protein release by ELISA. We found that LPS markedly induced CXCL2 expression after 1 h, peaking at 2h and remaining elevated at 24 h. In contrast, the CXCL2 expression induced by TNF was more rapid, significant within 30 min with a peak reached at 45 min. LPS induced CXCL2 protein release within 2 h while TNF more rapidly within 1 h. CXCL2 gene expression and protein release induced by both LPS and TNF were completely blocked by PDTC and helenalin. NBD peptide significantly blocked CXCL2 gene expression and protein release induced by LPS. In conclusion, LPS and TNF induce enterocytes to produce CXCL2 via NF-KB activation. The specific disruption of the NEMO:IKK interaction inhibits LPS-induced CXCL2 gene expression (supported by IDPH and NIH-5KO8HD044558 grants)