The injurious effect of neutrophils on pneumocytes in vitro

Neutrophils have been implicated in the pathogenesis of pulmonary injury in many clinical entities, but in vitro studies of neutrophil-mediated pneumocyte damage are limited. To study the role of neutrophils in mediating pulmonary injury, we cocultured these cells with monolayers of human A549 pneumocytes and rat type II alveolar cells. As indexes of injury, we measured cell detachment from monolayers, frank cytolysis, and the effect on pneumocyte protein and DNA synthesis. Unstimulated neutrophils effected minimal lysis or detachment of both pneumocyte targets, but neutrophils stimulated with phorbal myristate acetate and other secretogogues produced marked target cell detachment without lysis, which was time- and dose-dependent. Supernatants of activated neutrophils were similarly effective, suggesting that the mediator was a stable, soluble substance. Catalase and superoxide dismutase were minimally inhibitory to neutrophil-mediated detachment, and neutrophils from patients with chronic granulomatous disease produced detachment comparable to that produced by normal neutrophils. Furthermore, target cells were quite resistant to reagent H2O2 and non-neutrophil-derived toxic oxygen species, further suggesting that oxidative injury was not a major factor in causing detachment. Target cells were susceptible to detachment by the neutral proteases, elastase and collagenase, whereas neutrophil-mediated detachment was markedly inhibited by neutral protease and elastase inhibitors. Human and bovine serum were also inhibitory, but not albumin or pepstatin A, an acid protease inhibitor. Furthermore, we found that activated neutrophils inhibited protein and DNA synthesis of pneumocyte targets, providing additional evidence that neutrophils cause nonlytic injury to pneumocytes. These studies indicate that stimulated neutrophils cause nonlethal injury to pneumocytes, as measured by detachment from monolayers, and inhibition of vital intracellular synthetic functions. The mechanism of detachment is through the action of granule neutral proteases, rather than toxic oxygen metabolites, and is probably due to degradation of the extracellular matrix of the pneumocytes. In vivo, detachment could lead to desquamation of alveolar cells and increased permeability of the epithelial barrier of the lung. Similarly, inhibition of protein and DNA synthesis could have profound effects on the normal function and replication of alveolar epithelium.