Delayed polymorphonuclear leukocyte (PMN) apoptosis exacerbates acute lung injury. To reach the alveolar spaces, PMNs must migrate across both pulmonary endothelial and epithelial cell layers. We hypothesized that transmigration across the endothelium-epithelium bilayer suppresses PMN apoptosis and sought to elucidate the underlying mechanisms. PMNs freshly isolated from normal volunteers were allowed to migrate across polycarbonate membranes alone or membranes coated with a bilayer of human lung endothelial and epithelial cells. After migration towards different chemoattractants (IL-8, fMLP or LTB₄), PMN apoptosis and caspase activities were assessed by Annexin V, histology and enzymatic assays, respectively. Messenger RNA and specific protein expression in three receptor-ligand mediated apoptosis inducing pathways (Fas, TNF- and TRAIL) were further examined by gene array, RT-PCR, flow cytometry and Western blot analyses. The data demonstrated that transbilayer migration suppressed PMN apoptosis and this effect was not chemoattractant type-specific. Kinetic analyses further showed that the delay of apoptosis was sustained to at least 18 h. Transbilayer migration caused significant decreases in caspase (-3, -8 and -9) activities. The changes in apoptosis-related gene expression support the survival role of transbilayer migration. Furthermore, the reduced apoptosis was correlated with downregulation of Fas ligand and TNF-R1 expression. Our data reveal that migration across a lung endothelium-epithelium bilayer suppresses PMN apoptosis. The decreased activity and/or expression of pro-apoptotic proteins may provide possible targets for the regulation of inappropriate delay in PMN apoptosis during lung inflammation and injury.