CD18 ICAM-1-DEPENDENT OXIDATIVE NF-KAPPA-B ACTIVATION LEADING TO NITRIC-OXIDE PRODUCTION IN RAT KUPFFER CELLS COCULTURED WITH SYNGENEIC HEPATOMA-CELLS/

Previous studies have indicated that nitric oxide (NO) released from K upffer cells modulates biological viability of cocultured hepatoma cel ls. This study was designed to evaluate the mechanisms by which Kupffe r cells synthesize and release NO in reponse to cocultured hepatoma ce lls. Kupffer cells isolated from male Wistar rats were cocultured with rat hepatoma cell line, AH70 cells. The sum of nitrite and nitrate le vels increased in the culture medium of Kupffer cells with AH70 cells as compared with those of Kupffer cells or AH70 cells alone. Increased expressions of iNOS and iNOS mRNA in Kupffer cells cocultured with AH 70 cells were detected by an immunofluorescence staining and a fluores cence in situ hybridization study, respectively. A fluorescence in sit u DNA-protein binding assay revealed that NF-kappa B activation occurs in Kupffer cells and activated NF-kappa B moved into the nuclei prece ding to an increased production of NO. Oxidative stress indicated by d ichlorofluorescein fluorescence was observed in Kupffer cells cocultur ed with AH70 cells. An increased calcium mobilization indicated as inc reased fluo-3-associated fluorescence was also induced in Kupffer cell s after coculture with AH70 cells. Monoclonal antibodies directed agai nst rat CD18 and ICAM-1, as well as TMB-8, a calcium inhibitor, preven ted the calcium mobilization, active oxygen production, and NF-kappa B activation in addition to the increased production of NO. Pyrrolidine dithiocarbamate, an inhibitor of oxidative NF-kappa B activation, dip henylene iodonium, an NADPH oxidase inhibitor, and quinacrine, a phosp holipase A(2) inhibitor, significantly attenuated the increase in dich lorofluorescein fluorescence, NF-kappa B activation, and NO production . Therefore, this study suggests that CD18/ICAM-1-dependent cell-to-ce ll interaction with hepatoma cells causes calcium mobilization and oxi dative activation of NF-kappa B, which may lead to the increased produ ction of NO in Kupffer cells.