CHEMOKINE REGULATION IN PULMONARY OXYGEN INJURY

University Of Rochester, Rochester NY

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Abstract

DESCRIPTION (Adapted from applicant's abstract) Bronchopulmonary dysplasia (BPD) is a major morbidity in preterm infants. Production of the chemokine interleukin (IL)-8 by alveolar macrophages (AMs) is crucial to the hyperoxia-induced pulmonary injury that recapitulates some aspects of Bpd. This proposal's hypothesis is that IL-1Beta and tumor necrosis factor a (TNFalpha) are competence factors for the in vivo oxygen induction of IL-8 in neonatal rabbit /AMs. Its specific aims are to: 1. Measure the contributions of IL-1Beta, TNFalpha and oxygen to the regulation of IL-8 expression in the oxygen-stressed alveolar macrophage. Treatment of isolated rabbit AMs with exogenous IL-1Beta and TNFalpha will investigate the sufficiency of these cytokines to induce IL-8 production in response to oxygen. Interference with IL-1Beta and TNFalpha activity by the addition of IL-1 receptor antagonist (IL-1ra) and pentoxifylline, respectively, will test their necessity to the response in oxygen-exposed cells from the human U937 histiocytic cell line. 2. Determine the specific cis-and trans-activating factors responsible for the expression of IL-8 in the isolated rabbit alveolar macrophage. Isolation of a genomic clone of rabbit IL-8 will allow generation of genomic probes and a reporter construct containing the IL-8 promoter. Deletion mutagenesis, co-transfection experiments, electromobility shift analysis (EMSA) and site-directed mutagenesis will define specific cis- and trans-activating factors using the rabbit promoter in the U937 cell line and in transiently transfected rabbit Ams. 3. Determine the proximate events and trans-activating factors responsible for IL-8 expression in the rabbit alveolar macrophage exposed in vivo to oxygen stress. Adult rabbits will be exposed to >95% oxygen for 64 hours and AMs isolated. EMSA will measure differences from previous in vitro stimulation. IL-8, IL-1Beta and TNFalpha expression will be compared to in vitro stimulation. 4. Measure the differences between adult and newborn rabbit alveolar macrophages' regulation of IL-8 following oxygen stress. Newborn rabbit AMs will be co-stimulated with oxygen and exogenous IL-1Beta and/or TNFalpha. Adult and newborn AMs will be exposed to pulmonary lavage fluid from hyperoxia-exposed adult and newborn animals to assess independently possible host and cellular immaturities in the response.