Human neutrophil–pulmonary microvascular endothelial cell interactions in vitro: Differential effects of nitric oxide vs. peroxynitrite

Sepsis-induced acute lung injury is characterized by activation and injury of pulmonary microvascular endothelial cells (PMVEC), increased neutrophil–PMVEC adhesion and migration, and trans-PMVEC high-protein edema. Inducible NO synthase (iNOS) inhibits septic murine neutrophil migration in vivo and in vitro. The effects of NO in human neutrophil–PMVEC interactions are not known. We isolated human PMVEC using magnetic bead-bound anti-PECAM antibody. Confluent PMVEC at passage 3–4 were co-cultured with human neutrophils for assessment of neutrophil–PMVEC adhesion, and trans-PMVEC neutrophil migration and Evans-Blue dye-labeled albumin leak. Two NO donors (spermine-NONOate, S-nitroso-N-acetylpenicillamine) attenuated both cytomix-enhanced neutrophil–PMVEC adhesion by 64 ± 14% (p < 0.01) and 32 ± 3% (p < 0.05), respectively, and cytomix-induced trans-PMVEC neutrophil migration by 85 ± 16% (p < 0.01) and 43 ± 5% (p < 0.01), respectively. Correspondingly, iNOS inhibition with 1400W enhanced cytomix-stimulated neutrophil migration by 52 ± 3% (p < 0.01), but had no effect on neutrophil–PMVEC adhesion. Conversely, a peroxynitrite donor (SIN-1) increased both neutrophil–PMVEC adhesion (38 ± 2% vs. 14 ± 1% control, p < 0.01) and trans-PMVEC neutrophil migration; with both effects were completely inhibited by scavenging of NO, superoxide, or peroxynitrite (p < 0.05 for each). Scavenging of peroxynitrite also eliminated cytomix-induced neutrophil adhesion and migration. Blocking CD18-dependent neutrophil adhesion prevented cytomix-stimulated trans-PMVEC EB-albumin leak (p < 0.05), while inhibiting neutrophil migration paradoxically enhanced cytomix-stimulated EB-albumin leak (11 ± 1% vs. 7 ± 0.5%, p < 0.01). FMLP-induced neutrophil migration had no effect on trans-PMVEC EB-albumin leak. In summary, we report differential effects, including the inhibitory action of NO and stimulatory effect of ONOO− on human neutrophil–PMVEC adhesion and trans-PMVEC migration under cytomix stimulation. Moreover, neutrophil–PMVEC adhesion, but not trans-PMVEC migration, contributes to human PMVEC barrier dysfunction.