Patients suffering extrapulmonary trauma or burn injury commonly suffer extensive lung injury, termed the adult respiratory distress syndrome (ARDS). This syndrome causes substantial morbidity and mortality despite successful wound closure and aggressive therapeutic support. There is convincing evidence that this syndrome is due to degranulation and oxidant production by neutrophils in the alveolar interstitium. Previous studies have shown that circulating neutrophils in these conditions are """"""""primed"""""""" by exposure to C5a for heightened responsiveness to second stimuli. Alveolar macrophages from patients suffering ARDS secrete a variety of cytokines, including TNF-alpha, IL-1, and neutrophil chemoattractant activity believed to be IL-8 and LTB4. We hypothesize that these macrophage products induce migration of C5a-primed neutrophils into the alveolar interstitium and there induce release of neutrophil-derived oxidants and enzymes. These responses are mediated through cell surface receptors. We have documented preservation of TNF-alpha receptors on the neutrophil surface on patient (C5a-activated) neutrophils, a response not mirrored by pretreatment of normal neutrophils with C5a. We will characterize receptor protection mechanisms for patient neutrophil receptors. We will then characterize cytokine oxidant and enzyme release for matrix-adherent neutrophils. We will use a novel matrix connective tissue technology developed for in vivo epithelial support. Initial reports of adherent neutrophil oxidant production in response to TNF-alpha indicate a prolonged lag phase and a prolonged secretory phase markedly different than the pattern seen with cells in suspension. We will explore the hypothesis that the oxidant response of cytokine-triggered adherent neutrophils is dependent upon protein synthesis and represents a novel mechanisms for neutrophil exocytic responses. We will then examine the effects of alveolar macrophages from normals and from patients suffering early ARDS upon neutrophil oxidant release. For these experiments, co-cultured LPS-triggered peripheral blood monocytes and then alveolar macrophages will be used to examine oxidant and enzyme release by adherent neutrophils. We believe this approach is necessary because the mixture of cytokines and other stimuli released by monocytes nd macrophages likely conditions the neutrophil response to individual cytokines. Information from this project will provide insight into mechanisms producing ARDS and will allow precise definition of potential therapeutic strategies.
