Abstract
The process of leukocyte recruitment to the airways in real time has not been extensively studied, yet airway inflammation persists as a major contributor to lung pathology. We showed previously in vivo, that neutrophils are recruited acutely to the large airways after periods of airway distension imposed by the application of positive end-expiratory pressure (PEEP). Given extensive literature implicating products of nitric oxide synthase (NOS) in lung injury after ventilatory over-distension, we questioned whether similar mechanisms exist in airway post-capillary venules. Yet, endothelial nitric oxide has been shown to be largely anti-inflammatory in other systemic venules. Using intravital microscopy to visualize post-capillary tracheal venules in anesthetized, ventilated mice, the number of adherent leukocytes was significantly decreased in eNOS-/- mice under baseline conditions (2±1 cell/60 min observation) vs wild type (WT) C57BL/6 mice (7±2 cells). After exposure to PEEP (8 cmH2O for 1 min; 5 times), adherent cells increased significantly (29±5 cells) in WT mice while eNOS-/- mice demonstrated a significantly decreased number of adherent cells (11±4 cells) after PEEP. A similar response was seen when thrombin was used as the pro-inflammatory stimulus. In addition, mouse tracheal venular endothelial cells studied in vitro after exposure to cyclic stretch (18% elongation) or thrombin both demonstrated increased p-selectin expression that was significantly attenuated by NG-nitro-L-arginine methyl ester, N-acetylcysteine amide (NACA) and excess BH4. In vivo treatment with the ROS inhibitor NACA or co-factor BH4 abolished completely the PEEP-induced leukocyte adherence. These results suggest that pro-inflammatory stimuli cause leukocyte recruitment to tracheal endothelium in part due to eNOS uncoupling.
Highlights
Lung parenchymal injury induced by ventilatory over-distension has been studied for many years
As previously shown [15] and demonstrated in Figure 1B, distension induced by the application of intermittent positive end-expiratory pressure (PEEP) caused significant leukocyte recruitment as measured by the sum of adherent cells compared to wild type (WT) controls (p = 0.002)
The main finding of the current study is that endothelial nitric oxide synthase contributes to leukocyte recruitment to the airway when pro-inflammatory stimuli are imposed in vivo
Summary
Lung parenchymal injury induced by ventilatory over-distension has been studied for many years. Shearinduced release of endothelial-derived nitric oxide (NO) has been shown to limit Weibel-Palade body release of p-selectin [13]. In the present study we questioned whether the products of endothelial NOS (eNOS, NOS-3) contribute to or attenuate neutrophil recruitment during excessive airway distension imposed through the application of PEEP and compared it to the response of the proinflammatory protein thrombin. Because NO is known to attenuate neutrophil recruitment in systemic vascular beds, we hypothesized that neutrophil recruitment would be increased in eNOS deficient mice. Contrary to this hypothesis, results demonstrated that products of nitric oxide synthase in postcapillary venules of airways are pro-inflammatory
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