Expression of iNOS message in human buccal mucosal [keratinocytes] cell line TR146 using RT-PCR and immunocytochemistry

ABSTRACT

This study was carried out at the department of Oral Pathology, Queen Mary College of Medicine and Dentistry, Barts and The London to determine whether human oral buccal mucosal [keratinocytes] cell line, TR 146, expressed iNOS message and whether the expression of iNOS is varied when TR146 cells were exposed to different cytokines such as IL-15, IL-8, IL-1 beta, or TNF-alpha using RT-PCR and Immuno-cytochemistry and to determine the effect of the above mentioned cytokines on NO function by measuring nitrite production in TR146 cells. Immuno-cytochemistry analysis revealed that TR146 cells expressed iNOS proteins when incubated with IL-15 and IL-8 and a modest increase was seen with IL-1 beta /TNF-alpha. RT-PCR for iNOS indicated a marked increase in expression when the cells were exposed to IL-8, IL-15 or IL-1beta/ TNF-alpha. NOS activity was assessed by measuring nitrite activity. It was observed that treating the cells with cytokines caused significant increase in nitrite levels, except in the case of IL-8. These results suggest that TR146 cells expressed iNOS, the levels of which varied with various cytokines. It is clear that IL-15, IL-1beta /TNF-a and IL-8 are regulators of iNOS expression in oral keratinocytes and affect NOS activity