Although inflammation of the fetal membranes often results in premature delivery, recent studies have revealed that elements of the lung immune response can advance crucial aspects of fetal lung maturation. In a previous study, we demonstrated that exposure of fetal rat lung explants to the bacterial endotoxin, lipopolysaccharide (LPS), evoked a spontaneous increase in airway branching morphogenesis over a permissive concentration range (Land and Darakhshan, 2003). Although LPS stimulates a diverse range of cellular responses, recent studies show a positive correlation between the administration of a pharmacological NO-donor ((Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]-diazen-1-ium-1, 2-diolate-NO) and improved airway bifurcation (Young et al. 2002), therefore, we hypothesised that the increase in airway branching evoked by this toxin requires the activation of inducible nitric oxide synthase (iNOS) and endogenous NO-release.

Gestation day 14 rats and their fetuses were killed humanely and fetal rat lung explants were cultured for 96 h at fetal (23 mmHg) or ambient (142 mmHg) PO₂. Low concentration of LPS (2 µg/ml) increased the airway surface complexity (ASC = perimeter/square root of area), from 1.66 ± 0.09 to 1.99 ± 0.14 (P < 0.05) and 1.54 ± 0.11 to 2.01 ± 0.14 (P < 0.05, all values are Mean ± S.E.M.; ANOVA, post hoc Tukey’s HSD) at fetal and ambient PO₂, respectively (n = 5). This concentration of LPS also increased the release of nitrate (a measure of NO release, determined by the Griess assay), from 3.63 ± 0.66µM nitrate/µg protein to 22.07 ± 2.68 (P < 0.01) and 11.22 ± 1.89 to 44.25 ± 7.4 (P < 0.01) at fetal and ambient PO₂, respectively (n = 7). High concentration of LPS (50µg/ml) evoked widespread necrosis in explants and no increase in ASC was observed. Whereas 2 µg/ml LPS increased iNOS protein in the explants, selective inhibition of iNOS by 100 µM L-N6-(1-Iminoethyl)lysine hydrochloride (L-NIL) abrogated LPS-induced nitrate release (n = 9) and the resultant increase in ASC (n = 6).

LPS stimulates the synthesis and release of transforming growth factor-β (TGF-β), a potent inhibitor of branching morphogenesis and respiratory epithelial cell differentiation in fetal lung (Zeng et al. 2001). The dose dependent morphogenic effect we observed with LPS may therefore arise through the TGF-β-mediated suppression of iNOS activity, as noted in other studies (Vicencio et al. 2002; Vodovotz et al. 1999). LPS (0.5-50 µg/ml) had no effect on the expression of the 3 TGF-β receptor isoforms (TβRI, II and III) at either fetal or ambient PO₂, raising the possibility that LPS may act directly to modulate the release of TGF-β. We conclude that LPS-mediated iNOS activation and endogenous NO release is a potent inducer of branching morphogenesis in fetal rat lung explants. Constitutive expression of all three TGF-β receptor isoforms in the fetal lung highlights the possibility that TGF-β may negatively regulate lung morphogenesis via this pathway.

This work was supported by the Medical Research Council, Tenovus (Scotland) and the Anonymous Trust