The role of the NFKB signalling pathway in the inflamed intestine

Abstract

The nuclear factor kappa B (NFKB) signalling pathway is essential in the establishment and propagation of inflammation in the intestine. An increased number of cells, predominantly of the macrophage and intestinal epithelial cell (IEC) type, are known to contain the active form of the NF1d3-p65 subunit in inflamed and noninflamed intestinal tissue from Crahn's disease (CD) patients, though this remains to be confirmed. However the stimuli that induce NFKB activation in IECs and the mechanism of NFKB activation in macrophages, are only poorly understood. As such, this thesis has investigated the NFKB signalling pathway and its role in intestinal inflammation. Increased levels of NFKB DNA-binding activity and inhibitor kappa B alpha (IKBa) protein levels were found in both inflamed and non-inflamed intestinal tissue from CD patients. However, Bcl-3 levels did not significantly change. In HeLa Ohio cells, a human mucosal epithelial cell line, interleukin-l ~ (IL-l ~), lipopolysaccharide (LPS) and Phorbol 12-myritate I3-acetate (PMA) were shown to induce NFKB activation. However, when these same stimuli were used in another human IEC line, Caco-2, little NFKB-mediated gene expression was observed unless a combination of stimuli, IL-l~, LPS and tumour necrosis factor alpha (TNFa), was used. In RAW 264.7 cells, a murine macrophage cell line, LPS-stimulated NFKBmediated NO production was shown to involve protein kinase C epsilon (PKCc). Subsequently, PKC€ protein levels were also shown to be up-regulated in inflamed intestinal tissue from TNBS-treated rats. This was associated with increased NFlcB activation and IKBa protein levels, increases that were absent in non~inflamed tissue from TNBS-treated rats. In addition, IKB~ and Bcl-3 protein levels did not differ between inflamed and non-inflamed tissues, although they did vary with intestinal region. In conclusion, this study shows that abnormal NFKB activation and IKBa expression occurs in CD, and also suggests increased NFKB activation IKBa expression can coexist within inflamed intestinal tissue. In addition, the IEC line Caco-2 is shown to be relatively unresponsive to NFKB activation. In the macrophage cell line, RAW 264.7, PKC£ is involved in NFKB-mediated gene expression, and PKC£ protein levels are increased in the inflamed, TNBS-treated, intestine.