Effect of inflammatory factors on air liquid interface epithelia

Inflammatory factors are known to disturb the transepithelial water transport in lung by altering the sodium transport. The epithelial sodium channel (ENaC) is regarded as a limiting factor for transepithelial water resorption. We investigated the effect of inflammatory factors on transepithelial transport across lung epithelia in vitro and addressed the question whether ENaC mediated mechanisms are involved.

All experiments were performed on NCl-H441 cells cultivated at air-liquid interface (ALI) conditions as a well established model of the distal lung epithelium. IL13 (10 ng/ml), TNFα (10 ng/ml), LPS (200 ng/ml), Histamin (100µM), C5a (100 ng/ml) and C3a (1000 ng/ml) were added to the basal medium to mimic inflammatory conditions. Ussing chamber experiments were performed to examine the effect on transepithelial ion transport and transepithelial electrical resistance (TEER). The novel deuterium-oxide dilution method enabled us to measure the water transport rate directly. Expression levels of Claudin 8 (cldn8), α-ENaC, β-ENaC and γ-ENaC were measured by semi quantitative real time RT-PCR.

None of the investigated factors affected the expression levels of α-ENaC subunit. However, expression levels of β- and γ-ENaC subunits were reduced by IL13, Histamin, LPS, C5a and C3a but not by TNFα. Furthermore, all inflammatory factors except TNFα reduced the ENaC-mediated transcellular sodium transport. Although most factors showed decreased ENaC activity and reduced expression levels of β- and γ-ENaC subunits only IL-13 was capable to decrease the water resorption. Even though the TEER as a parameter of paracellular permeability remained unaffected by all tested factors, we observed a down-regulation of the tight junction protein cldn8 by IL-13 and TNFα. Since cldn8 is known to support resorptive transepithelial transport by mediating paracellular permselectivity for chloride, IL-13 and TNFα potentially affect paracellular transport.

We showed that inflammatory factors have different but distinct effects on NCI-H441 epithelia. Regarding the fact that only IL-13 alters cldn8 expression as well as ENaC mediated transport, we suggest that the perturbation of both, the para- and transcellular pathway, is needed to impact the water transport in lung epithelia.