Impaired apoptotic death signaling in inflammatory lung neutrophils is associated with decreased expression of interleukin-1beta converting enzyme family proteases (caspases)

Abstract

Background. Fas and tumor necrosis factor receptor 1 (TNFR1) are membrane proteins that signal for apoptotic cell death by downstream activation of proteins of the interleukin-1β converting enzyme (ICE) family. Spontaneous apoptosis is delayed in neutrophils activated by transmigration into an inflammatory focus. In this study we evaluated the effects of transmigration on Fas and TNFR1-induced apoptosis and apoptotic gene expression. Methods. Sprague-Dawley rats were killed 4 hours after intratracheal challenge with 500 μg lipopolysaccharide (LPS). Neutrophils isolated from the systemic circulation (circulation) or bronchoalveolar lavage fluid (lung) were incubated with or without an agonistic antibody to Fas (clone CH-11, 100 ng/ml) or TNF (10 ng/ml) for 24 hours. Apoptosis and Fas expression were assessed by flow cytometry. Expression of the antiapoptotic protein Bcl-2 and proapoptotic proteins ICE and CPP32 were measured by Western blots. Results. Neutrophils transmigrating into the lung in response to LPS showed delayed apoptosis compared with circulating neutrophils and failed to undergo apoptosis in response to anti-Fas antibody or TNF-α. Fas expression was unaltered; however, TNFR1 expression was reduced. Bcl-2 was not detected in either group; both the pro- and active forms of ICE and active CPP32 were significantly decreased in lung neutrophils. The specific ICE inhibitor, YVAD-CMK, partially blocked the increased rates of apoptosis resulting from engagement of Fas or TNFR1. Conclusions. Neutrophil transmigration retards apoptosis through engagement of the death receptors Fas and TNFR1. This refractory state is associated with reduced levels of proapoptotic proteins. Blunted responsiveness to physiologic apoptotic stimuli prolongs neutrophil functional survival during acute inflammation and may contribute to the tissue injury associated with acute respiratory distress syndrome.