The pathogenesis of CLD is complex, but may be related to age-specific characteristics of the lung microenvironment, lung leukocyte function, or an interaction between these. Persistence of lung neutrophils is a hallmark of CLD, and delayed apoptosis of lung neutrophils in neonates at risk for CLD has been observed. Neonatal neutrophils have prolonged survival compared with adult neutrophils, which is linked to decreased activity of caspase-3, a key mediator of Fas-mediated apoptosis. Important and unexplored components of CLD relate to age-specific leukocyte function, including the inflammatory and cytotoxic function of apoptotic neutrophils and those with prolonged survival, and the interactions of such neutrophils with monocytes and macrophages. OBJECTIVESThe main goals of this proposal, which focus on neutrophil survival and apoptosis, are to define the involved ontogeny-specific regulatory mechanisms, to determine their inflammatory and cytotoxic function, and to define their inflammatory interactions with monocytes and macrophages. METHODS: Neonatal neutrophils undergoing apoptosis will be studied for their expression of Fas-associated genes using microarray gene analysis, kinetic PCR determinations, Western blots, enzymatic assays and flow cytometry. To determine inflammatory and cytotoxic functions of surviving and apoptotic neutrophils, co-cultures with target cells, including lung stroma, monocytes and macrophages, will serve as the biologic read-outs. Methodologies for these studies include: proliferation and adhesion assays, multi-array protein analyses, ELISA, and flow cytometry. HEALTH RELATEDNESS: CLD is a potentially debilitating disorder with significant morbidity, mortality, and financial burden. Targeted therapies for adult diseases associated with aberrant apoptosis have met with some success, and CLD may share similar mechanisms. Thus, defining the biology of neutrophil apoptosis in the developing human is critical to future treatment modalities for CLD and other types of neonatal inflammation.
