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A4653 - Alveolar-Type I Like Cells and Alveolar Type II Cells Release ATP Differentially After Trauma Induction in an Alveolar-Capillary Barrier Model
Author Block: V. E. Winkelmann1, G. Fois1, M. Fauler1, N. Hobi2, M. Huber-Lang3, P. Dietl1, M. Frick1; 1General Physiology, Ulm University, Ulm, Germany, 2ARTORG Center for Biomedical Engineering Research, University of Bern, Bern, Switzerland, 3Institute of Clinical and Experimental Trauma Immunology (ITI), Ulm University Hospital, Ulm, Germany.
Rationale: Acute respiratory distress syndrome (ARDS) is a life-threatening condition, in which the alveolar-capillary barrier is severely compromised. Despite this patho-physiological importance, the molecular and cellular mechanisms of alveolar barrier damage are hardly elaborated. It is well established that ATP is a key danger-associated molecular pattern (DAMP) molecule modulating and amplifying the inflammatory response contributing to barrier breakdown. Controversially, ATP also stimulates fluid resorption and surfactant secretion/activation via activation of purinergic receptors - potentially counteracting alveolar flooding and surfactant inactivation observed in ARDS. To delineate the role of ATP we have established a novel in vitro alveolar model reconstituting the cellular and biophysical properties of the alveolar-capillary barrier. Methods: Co-cultures of pulmonary microvascular endothelial cells (HPMEC-ST1.6R) and a mixture of primary isolated alveolar type II and/or type I cell lines (TT1 or hAELVi) on opposing sites of a transwell filter were maintained at air-liquid conditions. These cultures actively resorb alveolar fluid, maintain a high TEER and secret surfactant in response to agonists. The cultures were challenged with a polytrauma cocktail and/or NB4 cells, as neutrophil model, mimicking pulmonary and systemic disturbances from either the basolateral or apical site, respectively. Results: Triple co-cultures released ATP after a 4h basolateral challenge with polytrauma cocktail, which was accompanied with a slight TEER reduction. This ATP release could be attributed to the cytokines IL-1beta and IL-8. In co-cultures using either ATI-like or alveolar type II (ATII) cells with endothelial cells, ATP release after 4 h basolateral challenge was mainly due to ATI-like cells. ATII cells only secreted comparable amounts of ATP when stimulated with NB4 cells from the apical side. Conclusions: Increased (apical) alveolar ATP levels were observed in an in vitro alveolar-capillary barrier model following treatment with a polytrauma cocktail and/or NB4 cells from the apical or basolateral side. However, ATI-like cells mainly released ATP levels after basolateral challenge, while ATII cells only released ATP when stimulated with NB4 cells from the apical side. The mechanisms triggering this differential ATP release are currently under investigation.