Pathophysiology and Metabolism of the Leukocyte

Bass, David A.

Abstract

This project proposes to examine stimulus-response coupling in the human neutrophil. The basic hypothesis is that mobilization of Ca++ is an early event following membrane perturbation which then results in two sequellae: the mobilization and subsequent metabolism of arachidonic acid and activation of the respiratory burst. The role of Ca++ will be investigated by comparing Ca++ transport induced by various stimuli with arachidonate metabolism and respiratory burst activity. Specificity will be examined by the use of calcium channel blockers and the intracellular calcium chelator, TMB-8. The possible role of calmodulin in these processes will be evaluated. Lipid metabolism in the stimulated neutrophil will be examined using cells pre-labelled with 3H-labelled arachidonate and 14C-stearate. Emphasis will be placed on the source and mechanism of arachidonate release as well as its subsequent metabolism to various bioactive intermediates. The possible relationship of the lipid alterations to the respiratory burst will be examined by investigating the two phenomena in patients with chronic granulomatous disease as well as by incubating normal cells with various phospholipases and products of phospholipase metabolism. Finally, we are hypothesizing that the respiratory burst is mediated by a multi-component electron transport chain. Neutrophils (both resting and activated) will be fractionated by previously defined techniques and oxidase activity in each fraction (plasma membrane, specific granule, and azurophil granule) determined by three separate assays (O2- production, H2O2 production, and NADP formation). These activities will be correlated with the subcellular distribution of cytochrome b and ubiquinone. The kinetics of appearance of each of the above activities in the phagocytic vacuole will be measured. These experiments will be performed with both cells from normal donors and from patients with chronic granulomatous disease. Finally, reconstitution experiments will be performed in an attempt to activate the system in vitro by combining fractions containing the differing activities.

Funding Agency

• Agency: National Institute of Health (NIH)
• Institute: National Institute of Allergy and Infectious Diseases (NIAID)
• Type: Research Project (R01)
• Project #: 5R01AI010732-18
• Application #: 3124786
• Study Section: Bacteriology and Mycology Subcommittee 1 (BM)
• Project Start: 1979-03-01
• Project End: 1993-06-30
• Budget Start: 1989-07-01
• Budget End: 1990-06-30
• Support Year: 18
• Fiscal Year: 1989

Institution

• Name: Wake Forest University Health Sciences
• Type: Schools of Medicine
• DUNS #: 041418799
• City: Winston-Salem
• State: NC
• Country: United States
• Zip Code: 27106

Publications

Bauldry, S A; Nasrallah, V N; Bass, D A (1992) Activation of NADPH oxidase in human neutrophils permeabilized with Staphylococcus aureus alpha-toxin. A lower Km when the enzyme is activated in situ. J Biol Chem 267:323-30

Bauldry, S A; Elsey, K L; Bass, D A (1992) Activation of NADPH oxidase and phospholipase D in permeabilized human neutrophils. Correlation between oxidase activation and phosphatidic acid production. J Biol Chem 267:25141-52

Bauldry, S A; Wykle, R L; Bass, D A (1991) Differential actions of diacyl- and alkylacylglycerols in priming phospholipase A2, 5-lipoxygenase and acetyltransferase activation in human neutrophils. Biochim Biophys Acta 1084:178-84

Bauldry, S A; Bass, D A; Cousart, S L et al. (1991) Tumor necrosis factor alpha priming of phospholipase D in human neutrophils. Correlation between phosphatidic acid production and superoxide generation. J Biol Chem 266:4173-9

Bauldry, S A; McCall, C E; Cousart, S L et al. (1991) Tumor necrosis factor-alpha priming of phospholipase A2 activation in human neutrophils. An alternative mechanism of priming. J Immunol 146:1277-85

Bauldry, S A; Wykle, R L; Bass, D A (1988) Phospholipase A2 activation in human neutrophils. Differential actions of diacylglycerols and alkylacylglycerols in priming cells for stimulation by N-formyl-Met-Leu-Phe. J Biol Chem 263:16787-95

Caldwell, S E; McCall, C E; Hendricks, C L et al. (1988) Coregulation of NADPH oxidase activation and phosphorylation of a 48-kD protein(s) by a cytosolic factor defective in autosomal recessive chronic granulomatous disease. J Clin Invest 81:1485-96

Bass, D A; McPhail, L C; Schmitt, J D et al. (1988) Selective priming of rate and duration of the respiratory burst of neutrophils by 1,2-diacyl and 1-O-alkyl-2-acyl diglycerides. Possible relation to effects on protein kinase C. J Biol Chem 263:19610-7

Bass, D A; Gerard, C; Olbrantz, P et al. (1987) Priming of the respiratory burst of neutrophils by diacylglycerol. Independence from activation or translocation of protein kinase C. J Biol Chem 262:6643-9

Gerard, C; McPhail, L C; Marfat, A et al. (1986) Role of protein kinases in stimulation of human polymorphonuclear leukocyte oxidative metabolism by various agonists. Differential effects of a novel protein kinase inhibitor. J Clin Invest 77:61-5