Effects of bacterial lipopolysaccharides (LPS) and tumour necrosis factor-alpha (TNF alpha) on rat tracheal epithelial cells in culture: morphology, proliferation and induction of nitric oxide (NO) synthase.

Rat tracheal epithelial cells were cultured and the effects of LPS and TNF alpha on cell morphology, rate of proliferation and NO synthase activity were studied. NO synthase activity was determined by measuring the accumulation of 3H-L-citrulline during incubation of confluent monolayer with 3H-L-arginine. In untreated cells no significant 3H-L-citrulline formation was detected, and bradykinin and the calcium ionophore A 23187 failed to stimulate 3H-L-citrulline formation excluding a constitutively expressed, calcium-dependent NO synthase activity. After culturing the cells for 18 h in the presence of LPS (10 micrograms/ml) and TNF alpha (500 U/ml) a marked formation of 3H-L-citrulline could be detected, which was largely inhibited by N(G)-monomethyl-L-arginine (L-NMMA) indicating the induction of NO synthase activity which could be prevented by dexamethasone. Exposure of confluent monolayer to LPS and TNF alpha for up to 4 days resulted in a reduction in cell density by 20% within 1 to 2 days and in additional marked changes in cell morphology. The normal honeycomb-like structure of the culture was lost and a considerable number of cells developed "dendritic' outgrowths. These morphological changes as well as the reduction in cell density was attenuated by dexamethasone, but not by the NO synthase inhibitor L-NMMA. The rate of cell proliferation was determined in non-confluent cultures 24 h after passage by determination of the incorporation of tritium into DNA during 24 h of incubation with 3H-thymidine. 3H-thymidine incorporation was reduced by about 40-45% when LPS or TNF alpha was present during exposure to 3H-thymidine, and by about 65%, when LPS and TNF alpha were present in combination. Neither L-NMMA nor dexamethasone significantly affected the 3H-thymidine incorporation nor the inhibitory effects of LPS and TNF alpha. In conclusion, airway epithelial cells are markedly affected by LPS and TNF alpha and the various responses (changes in the cell morphology, inhibition of cell proliferation and induction of NO synthase activity) appear to be caused by different (dexamethasone-sensitive and -insensitive), cellular mechanisms. An enhanced formation of endogenous NO may not be responsible for the observed morphological changes or the inhibition of cell proliferation.