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IL-8 inhibits cAMP-stimulated alveolar epithelial fluid transport via a GRK2/PI3K-dependent mechanism.

ABSTRACT: Patients with acute lung injury (ALI) who retain maximal alveolar fluid clearance (AFC) have better clinical outcomes. Experimental and small clinical studies have shown that ?2-adrenergic receptor (?2AR) agonists enhance AFC via a cAMP-dependent mechanism. However, two multicenter phase 3 clinical trials failed to show that ?2AR agonists provide a survival advantage in patients with ALI. We hypothesized that IL-8, an important mediator of ALI, directly antagonizes the alveolar epithelial response to ?2AR agonists. Short-circuit current and whole-cell patch-clamping experiments revealed that IL-8 or its rat analog CINC-1 decreases by 50% ?2AR agonist-stimulated vectorial Cl(-) and net fluid transport across rat and human alveolar epithelial type II cells via a reduction in the cystic fibrosis transmembrane conductance regulator activity and biosynthesis. This reduction was mediated by heterologous ?2AR desensitization and down-regulation (50%) via the G-protein-coupled receptor kinase 2 (GRK2)/PI3K signaling pathway. Inhibition of CINC-1 restored ?2AR agonist-stimulated AFC in an experimental model of ALI in rats. Finally, consistent with the experimental results, high pulmonary edema fluid levels of IL-8 (>4000 pg/ml) were associated with impaired AFC in patients with ALI. These results demonstrate a novel role for IL-8 in inhibiting ?2AR agonist-stimulated alveolar epithelial fluid transport via GRK2/PI3K-dependent mechanisms.-Roux, J., McNicholas, C. M., Carles, M., Goolaerts, A., Houseman, B. T., Dickinson, D. A., Iles, K. E., Ware, L. B., Matthay, M. A., Pittet, J.-F. IL-8 inhibits cAMP-stimulated alveolar epithelial fluid transport via a GRK2/PI3K-dependent mechanism.

SUBMITTER: Roux J

PROVIDER: S-EPMC3574281 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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IL-8 inhibits cAMP-stimulated alveolar epithelial fluid transport via a GRK2/PI3K-dependent mechanism.

Roux Jérémie J McNicholas Carmel M CM Carles Michel M Goolaerts Arnaud A Houseman Benjamin T BT Dickinson Dale A DA Iles Karen E KE Ware Lorraine B LB Matthay Michael A MA Pittet Jean-François JF

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 20121206 3

Patients with acute lung injury (ALI) who retain maximal alveolar fluid clearance (AFC) have better clinical outcomes. Experimental and small clinical studies have shown that β2-adrenergic receptor (β2AR) agonists enhance AFC via a cAMP-dependent mechanism. However, two multicenter phase 3 clinical trials failed to show that β2AR agonists provide a survival advantage in patients with ALI. We hypothesized that IL-8, an important mediator of ALI, directly antagonizes the alveolar epithelial respon ...[more]

PMID: 23221335

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Project description: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 ? cm2 vs 28 ± 4 ? 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.

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IL-8 inhibits cAMP-stimulated alveolar epithelial fluid transport via a GRK2/PI3K-dependent mechanism.

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IL-8 inhibits cAMP-stimulated alveolar epithelial fluid transport via a GRK2/PI3K-dependent mechanism.

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