The effects of surfactant on neutrophil apoptosis in lipopolysaccharide induced acute lung injury in rat

Abstract

Background: The therapeutic effects of surfactant on acute lung injury derive not only from its recruiting action on collapsed alveoli but also from its anti-inflammatory effects. Pro-apoptotic action on alveolar neutrophils represents one of the important anti-inflammatory mechanisms of surfactant. In the present study, we evaluated the effects of surfactant on the apoptosis of human peripheral and rat alveolar neutrophils. Methods: In the (Ed- the article is not definitely needed but it helps to separate the two prepositions 'in') in vitro study, human neutrophils were collected from healthy volunteers. An equal number of neutrophils (1×106) (Ed-confirm) was treated with LPS (10, 100, 1000ng/ml), surfactant (10, 100, 1000μg /ml), or a combination of LPS (1000ng/ml) and surfactant (10, 100, 1000μg/ml). After incubation for 24 hours, the apoptosis of neutrophils was evaluated by Annexin V method. In the in vivo study, induction of acute lung injury in SD rats by intra-tracheal instillation of LPS (5mg/kg) was followed by intra-tracheal administration of either surfactant (30mg/kg) or normal saline (5ml/kg). Twenty-four hours after LPS instillation, alveolar neutrophils were collected and the apoptotic rate was evaluated by Annexin V method. In addition, changes of the respiratory mechanics of rats (respiratory rate, tidal volume, and airway resistance) were evaluated with one chamber body plethysmography before, and 23 hours after, LPS instillation. Results: In the in vitro study, LPS treatment decreased the apoptosis of human peripheral blood neutrophils (control; 47.4±5.0%, LPS 10ng/ml; 30.6±10.8%, LPS 100ng/ml; 27.5±9.5%, LPS 1000ng/ml; 24.4±7.7%). The combination of low to moderate doses of surfactant with LPS promoted apoptosis (LPS 1000ng/ml + Surf 10μg/ml; 36.6±11.3%, LPS 1000ng/ml + Surf 100μg/ml; 41.3±11.2%). The high dose of surfactant (1000μg/ml) decreased apoptosis (24.4±7.7%) and augmented the anti-apoptotic effect of LPS (LPS 1000μg/ml + Surf 1000μg/ml; 19.8±5.4%). In the in vivo study, the apoptotic rate of alveolar neutrophils of surfactant-treated rats was higher than that of normal saline-treated rats (6.03± 3.36% vs. 2.95±0.58%). The airway resistance (represented by Penh) of surfactant-treated rats was lower than that of normal saline-treated rats at 23 hours after LPS injury (2.64±0.69 vs. 4,51±2.24, p<0.05). Conclusions: Surfactant promotes the apoptosis of human peripheral blood and rat alveolar neutrophils. Pro-apoptotic action on neutrophils represents one of the important anti-inflammatory mechanisms of surfactant.