Abstract
Robust and reproducible in vitro models are required for investigating the pathways involved in fluid homeostasis in the human alveolar epithelium. We performed functional and phenotypic characterisation of ion transport in the human pulmonary epithelial cell lines NCI-H441 and A549 to determine their similarity to primary human alveolar type II cells. NCI-H441 cells exhibited high expression of junctional proteins ZO-1, and E-cadherin, seal-forming claudin-3, -4, -5 and Na+-K+-ATPase while A549 cells exhibited high expression of pore-forming claudin-2. Consistent with this phenotype NCI-H441, but not A549, cells formed a functional barrier with active ion transport characterised by higher electrical resistance (529 ± 178 Ω cm2 vs 28 ± 4 Ω cm2), lower paracellular permeability ((176 ± 42) ×10−8 cm/s vs (738 ± 190) ×10−8 cm/s) and higher transepithelial potential difference (11.9 ± 4 mV vs 0 mV). Phenotypic and functional properties of NCI-H441 cells were tuned by varying cell seeding density and supplement concentrations. The cells formed a polarised monolayer typical of in vivo epithelium at seeding densities of 100,000 cells per 12-well insert while higher densities resulted in multiple cell layers. Dexamethasone and insulin-transferrin-selenium supplements were required for the development of high levels of electrical resistance, potential difference and expression of claudin-3 and Na+-K+-ATPase. Treatment of NCI-H441 cells with inhibitors and agonists of sodium and chloride channels indicated sodium absorption through ENaC under baseline and forskolin-stimulated conditions. Chloride transport was not sensitive to inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) under either condition. Channels inhibited by 5-nitro-1-(3-phenylpropylamino) benzoic acid (NPPB) contributed to chloride secretion following forskolin stimulation, but not at baseline. These data precisely define experimental conditions for the application of NCI-H441 cells as a model for investigating ion and water transport in the human alveolar epithelium and also identify the pathways of sodium and chloride transport.
Highlights
The alveolar lining fluid is a very thin liquid layer which is essential for maintaining efficient gas exchange, surfactant homeostasis, and defence against inhaled toxins and pathogens [1]
Transepithelial electrical resistance (TEER) and transepithelial potential difference (TEPD) values of NCI-H441 cells and A549 cells were seen on day 9, this time point was selected for permeability measurements using the paracellular tracer sodium fluorescein
The concentration vs. time plots were linear for both cell lines with R2 = 0.97 for NCI-H441 and R2 = 0.99 for A549 respectively (Fig 1C), indicating that the data are consistent with the passive diffusion model used for calculating permeability
Summary
The alveolar lining fluid is a very thin liquid layer which is essential for maintaining efficient gas exchange, surfactant homeostasis, and defence against inhaled toxins and pathogens [1]. Type I and II cells express junctional proteins such as E-cadherin, claudins, occludin and zona occludens (ZO) [3,4,5] These junctions seal the paracellular clefts between neighboring cells, serving as a mechanical barrier, and a determinant for the paracellular permeability and selectivity to water and different ions. Studies in AQP knockout mice did not affect fluid clearance or edema formation suggesting that their functional significance for water transport in the alveoli is limited [9, 10]. These studies point to the ongoing evolution in our understanding of alveolar fluid transport
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