Modulation of neutrophil apoptosis by murine pulmonary microvascular endothelial cell inducible nitric oxide synthase

Neutrophils contribute significantly to ALI (acute lung injury) through adhesion to pulmonary microvascular endothelial cells (PMEC), trans-PMEC migration and alveolar infiltration. Trans-PMEC migration delays expression of neutrophil apoptosis, which promotes intra-alveolar neutrophil survival and neutrophil mediated ALI. We assessed the role of neutrophil vs PMEC inducible nitric oxide (NO) synthase (iNOS) in modulating neutrophil apoptosis. Apoptosis of wild-type vs iNOS−/− neutrophils was quantified by microscopy and FACS annexin-V binding. In a murine model of ALI, neutrophils isolated by BAL(broncho-alveolar lavage) from iNOS−/− mice had increased expression of apoptosis after 24 h culture ex vivo than wild-type neutrophils (15.2 ± 3.3 vs 3.0 ± 0.4%, mean ± sd, p < 0.01). Apoptosis rates of isolated bone marrow iNOS+/+ vs iNOS−/− neutrophils were similar under basal and LPS/IFN-γ stimulation, and following LPS/IFN-γ-stimulated trans-PMEC migration. Apoptosis of both iNOS+/+ and iNOS−/− neutrophils was inhibited by trans-PMEC migration only across iNOS+/+ PMEC (1.6 ± 0.3 and 1.5 ± 0.3%, respectively; p < 0.05 for each vs non-migrated neutrophils) but not across iNOS−/− PMEC (4.3 ± 1 and 3.1 ± 0.6%, respectively). PMEC iNOS-dependent inhibition of neutrophil apoptosis was independent of changes in neutrophil caspase-3 activity. We conclude that PMEC iNOS, but not neutrophil iNOS, has an important inhibitory effect on neutrophil apoptosis during trans-PMEC neutrophil migration, which is independent of caspase-3 activity. Further studies will define the mechanism of PMEC iNOS-dependent inhibition of neutrophil apoptosis and assess the potential relevance of this phenomenon in human neutrophils and ALI.

Research highlights
► Neutrophils isolated by BAL in iNOS−/− mice have increased expression of apoptosis.
► Neutrophil iNOS does not have an autocrine effect on murine neutrophil apoptosis.
► PMEC iNOS has an inhibitory effect on neutrophil apoptosis during transmigration.
► Modulation of neutrophil apoptosis by iNOS is independent of caspase-3 activity.