Superoxide output and expression of NADPH oxidase 1 in human colonic epithelial cells

Background

Translocation of microorganisms from the gut lumen may contribute to morbidity in sepsis. Conservation of normal barrier function should, therefore, be a goal in critical illness, but its components is only partly defined. Beyond mechanical contribution, the colonic epithelium may possess antimicrobial activity via yet undefined mediators [1]. Superoxide (O₂^-), generated by membrane-bound NADPH oxidase (Nox), is a key antimicrobial agent of phagocytes, but output from human colonic epithelial cells has not been demonstrated.

Aims

To measure O₂^- output and expression of the non-phagocytic NADPH oxidase, Nox1, in short term cultures of primary epithelial cells from normal human colon and in Caco-2 colonic epithelial cell line.

Methods

Colonic epithelial cells were isolated from biopsies of subjects with uninflamed bowel and Caco-2 cells were grown in 24-well plates [2]. Output of O₂^- was measured by the cytochrome c assay or luminol-enhanced luminescence and localised by the nitrobluetetrazolium (NBT) assay [3].Effects of 1 mM NADPH, oxidoreductase inhibition by 10 µM diphenylene iodonium (DPI) and protein kinase C-activation by 0.5 µg/ml phorbol myristate ester (PMA) were assessed. mRNA expression of Nox1 was analysed by RT-PCR.

Results

Identical results were obtained in primary epithelial cells (see Figs) and Caco-2 cells. NBT-reduction was observed at the outer cell membrane and Nox1 mRNA was detected in all cell cultures. NADPH increased output of O₂^- within seconds although cell membranes are impermeable to NADPH.

Figure

Superoxide output in cultures of primary colonic epithelial cells. O₂^- output (mean ± SEM, n = 5) was adjusted to mg protein. ^*P < 0.05 compared with basal (t-test). SOD, O₂^- dismutase.

Conclusions

These results show that cultures of human colonic epithelial cells produce extracellular O₂^- possibly through membrane-bound Nox1, which appears to independent of protein kinase C activation. O₂^- from epithelial cells may possess antimicrobial activity and contribute to colonic barrier function.