The broad long-term objectives of the proposal are to understand the regulation of the neutrophil response to environmental dusts, by focusing on the cytochrome P-450LTB of human polymorphonuclear leukocytes (PMN) which regulates the metabolism of the chemotactic lipid 5S,12R-dihydroxy- 6,14-cis-8,10-trans-eicosatetraenoic acid, leukotriene (LT)B4. Exposure to the toxic environmental dusts silica and asbestos results in the recruitment of PMN to the alveolar space, with a resulting neutrophilic alveolitis followed by chronic fibrosis. The mechanism of recruitment of PMN to the alveoli, in response to these inorganic dusts, is thus a crucial factor in the pathogenesis of chronic asbestosis and silicosis. LTB4 has been suggested to play a major role in the recruitment of PMN in these disorders. P-450LTB is the cytochrome P-450 located in the microsomes of PMN which regulates the biological activity of LTB4 by catalyzing the progressive oxidation of the C:20 carbon of LTB4.
The specific aims are to purify, clone the cDNA, and study the regulation of P-450LTB in cultured cells. P-450LTB will be purified using Fast Protein Liquid Chromatography, or immunoaffinity chromatography. Purified P-450LTB will be transferred to immobilon membranes, and the N-terminal amino acid sequence determined. Tryptic digestion will also be performed on soluble purified enzyme or on enzyme transferred to nitrocellulose membranes. Tryptic fragments will be isolated by high performance liquid chromatography, and then subjected to amino acid sequencing. Oligonucleotide probes will be constructed and utilized directly or to construct polymerase chain reaction (PCR) products to probe a cDNA library constructed from mRNA obtained from human PMN of patients with chronic myelogenous leukemia, cells abundant in mRNA. Alternatively, this library will be probed with the putatively homologous cDNA of rabbit lung prostaglandin omega-hydroxylase (P-450PGomega). The factors which control the induction of P-450LTB in HL-60 cells induced to differentiate phenotypically mature neutrophils will be determined.
