The Mechanism of Action and Interaction of Leukotriene B4 and Platelet-Activating Factor as Mediators of Neutrophil Activation

Rossi, Adriano Giorgio (1987) The Mechanism of Action and Interaction of Leukotriene B4 and Platelet-Activating Factor as Mediators of Neutrophil Activation. PhD thesis, University of Glasgow.

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Human neutrophils can be stimulated by a plethora of soluble and particulate stimuli, the majority of which interact with specific recognition sites (receptors) located on the plasma membrane. These agonists evoke a series of cellular responses including chemotaxis, aggregation, degranulation, superoxide generation and the formation of numerous lipid products, for example LTB4 and PAF, which are derived from membrane phospholipids. LTB4 and PAF interact with specific receptors on, and are themselves potent activators of, human neutrophils. Therefore, these lipids have the potential to act as endogenous mediators or amplifiers of neutrophil activation. The mechanisms by which agonist receptor occupancy lead to such cellular activation remain to be fully established. It remains possible that in neutrophils, as in some other cells, reactivity may be regulated by the production of at least two second messenger molecules, 1,2-diacylglycerol (DAG) and [Ca2+]i that are produced as a consequence of phospholipase C catalysed phosphoinositide hydrolysis. The precise nature and role of this receptor mediated sequence of events in the human neutrophil, particularly concerning LTB4 and PAF, still remains to be fully elucidated and is frequently controversial. In this study I attempted to investigate the mechanism of action and interaction of the arachidonic acid metabolite, LTB4, and the ether lipid, PAP, as mediators of neutrophil activation by comparing the responses elicited by these lipids to those evoked by other neutrophil stimuli, namely the chemotactic tripeptide, FMLP, the calcium ionophore, ionomycin and the phorbol eater, PMA. I also examined the possible involvement of G proteins and the role of protein kinase C activation as stimulatory and regulatory mechanisms in the neutrophil. In addition, I explored the role of PAF and LTB4 as endogenous mediators or amplifiers of neutrophil activation induced by other agonists. Neutrophil reactivity was assessed by: a. Examining ultrastructural features using electron microscopy. b. Monitoring aggregation photometrically using a standard platelet aggregometer. c. Determining NAG and lysozyme release using a fluorimetric and a spectrophotometric technique respectively. d. Measuring LTB4 generation by specific radioimmunoassay and the authenticity confirmed using reverse phase HPLC. e. Observing changes in [Ca2+]i using the fluorescent calcium indicator dye, quin2. f. Following inositol phospholipid metabolism in cells prelabelled with [32P]-orthophosphate and monitoring changes in the levels of [32P]-PtdA, [32P]-PtdIns, [32P]-PIP and [32P]-PIP2. Using these in vitro techniques, the major observations and conclusions are listed below: 1. The ultrastructural features associated with neutrophils activated by FMLP, PAF and LTB4 were similar. Control, unstimulated cells were generally rounded with smooth contours and the occasional extension of fine projections. Exposure of cells to agonists caused a marked plasma membrane ruffling followed by cell polarization and the formation of large lamellipodia. Pretreatment of neutrophils with the fungal metabolite, cytochalasin B, caused a marked vacuolation and subsequent additions of agonists resulted in the formation of blebs giving the cells a bizarre appearance. Caution ought to be a priority when using or interpreting data generated by cytochalasin B. 2. LTB4, PAF and EMLP all caused a reversible, concentration-dependent neutrophil aggregation. The aggregatory response produced by ionomycin and PMA was also concentration-dependent but was slower in onset and irreversible. 3. The receptor directed agonists caused a cytochalasin B dependent release of NAG (an azurophil granule marker) and lysozyme (an azurophil and specific granule marker). The calcium ionophore induced the release of both markers independent of pretreatment with cytochalasin B. PMA could release lysozyrae in the absence of the fungal metabolite but the phorbol ester only induced a limited cytochalasin B dependent release of NAG. 4. Ionomycin elicited a concentration-dependent generation and release of LTB4. FMLP and PAP only released barely detectable levels of the arachidonic acid metabolite. 5. All agonists except PMA caused a rapid concentration-dependent elevation of neutrophil [Ca2+]i. Ionomycin induced a greater maximal increase than the receptor directed agonists. (Abstract shortened by ProQuest.).

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Pharmacology
Date of Award: 1987
Depositing User: Enlighten Team
Unique ID: glathesis:1987-77581
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 14 Jan 2020 11:53
Last Modified: 14 Jan 2020 11:53

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