Apical, but not basolateral, endotoxin preincubation protects alveolar epithelial cells against hydrogen peroxide-induced loss of barrier function: The role of nitric oxide synthesis

The influence of LPS preincubation on hydrogen peroxide (H₂O₂)-induced loss of epithelial barrier function was investigated in rat alveolar epithelial type II cells (ATII). Both apical and basolateral H₂O₂ administration caused a manyfold increase in transepithelial [³H]mannitol passage. Apical but not basolateral preincubation of ATII with LPS did not influence control barrier properties but fully abrogated the H₂O₂-induced leakage response. The effect of apical LPS was CD14 dependent and was accompanied by a strong up-regulation of NO synthase II mRNA and protein and NO release. Inhibition of NO by N^G-mono-methyl-L-arginine suppressed the LPS effect, whereas it was reproduced by exogenous application of gaseous NO or NO donor agents. Manipulation of the glutathione homeostasis (buthionine-(S,R)-sulfoximine) and the cGMP pathway (1H-(1,2,4)oxadiazolo[4,3-α]quinoxaline-1-one; zaprinast) did not interfere with the protective effect of LPS. Superoxide (O₂) generation by ATII cells was reduced by exogenous NO and LPS preincubation. O₂^.-scavenging with exogenous superoxide dismutase, the intracellular superoxide dismutase analog Mn(III)tetrakis(4-benzoic acid) porphyrin, and the superoxide scavenger nitroblue tetrazolium and, in particular, hydroxyl radical scavenging with hydroxyl radical scavenger 1,3-dimethyl-thiourea inhibited the H₂O₂-induced epithelial leakage response. In conclusion, apical but not basolateral LPS preincubation of ATII cells provides strong protection against H₂O₂-induced transepithelial leakage, attributable to an up-regulation of epithelial NO synthesis. It is suggested that the LPS-induced NO formation is effective via interaction with reactive oxygen species, including superoxide and hydroxyl radicals. The polarized epithelial response to LPS may be part of the lung innate immune system, activated by inhaled endotoxin or under conditions of pneumonia.