Sophie Guinard, Anne Olland, Jérémie Reeb, Mélanie Burban, Fatiha Zobairi, Ferhat Meziani, Pierre-Emmanuel Falcoz, Valérie Schini-Kerth, Laurence Kessler, Florence Toti, Gilbert Massard
Institution : Service de chirurgie thoracique, nouvel hôpital civil, Strasbourg
Objectives : In vitro study of paracrine effect of alveolar microparticles (MPs) generated during lung ischemia reperfusion, on alveolar epithelial cells (AEC) type II.
Methods : The bronchoalveolar lavage (BAL) of rat lungs is collected after 20h of cold ischemia and 2h of reperfusion. MPs are isolated, concentrated and their cellular origin determined after capture on annexin-5 or on specific antibodies. Washed and concentrated MPs are applied, during 20 hours on rat AEC type II (RLE-6TN) at 70% of confluence. Cellular integration of MPs is evaluated after labeling with a fluorescent probe PKH26. Induced apoptosis is assessed by the percentage of cells carrying hypodiploide DNA, the cellular function by the presence of surfactant protein B and inflammation by the rate of interleukin 8 (IL8).
Results : The yield of MPs gathered in BAL is 80% after 2h of ultracentrifugation. The microparticles are endothelial and leukocyte origin. More than 90% of MPs are integrated in cells after 24h of stimulation. The percentage of apoptosis induced by alvolar MPs at concentrations of 5, 10, 15 nMPhSer is 3±1.4%, 3±0.9%, 4.4±2.2% respectively, vs 3.4±1.4% for untreated cells and 9.9±4.5% for cells sitmulated by 0.1 µg/ml of actinomycin. Stimulation by alveolar MPs at concentrations of 10 and 15 nMPhSer significantly increase the concentration of SpB in cell supernatant compared to untreated cells (p<0.001). We have not highlighted IL8 in cell surpernatant after stimulation by MPs.
Conclusion : Alveolar microparticles are integrated in alveolar epithelial cell type II and have a cell activator effect by the stimulation of the production of lung surfactant.
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