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E Kitchen

Publications and source records attributed to E Kitchen.

5 recordsLinked to original sources
PubMed1998

Neutrophil priming: pathophysiological consequences and underlying mechanisms.

1. Neutrophil priming by agents such as tumour necrosis factor-alpha, granulocyte/macrophage colony-stimulating factor and lipopolysaccharide causes a dramatic increase in the response of these cells to an activating agent; this process has been shown to be critical for neutrophil-mediated tissue injury both in vitro and in vivo. 2. The principle consequence of priming, aside from a direct effect on cell polarization, deformability and integrin/selectin expression, is to permit secretagogue-induced superoxide anion generation, degranulation and lipid mediator (e.g. leukotriene B4 and arachidonic acid) release. It is now recognized that most priming agents also serve an additional function of delaying apoptosis and hence increasing the functional longevity of these cells at the inflamed site. 3. The potential mechanisms underlying priming are discussed; current data suggest a dissociation between priming and changes in receptor number and/or affinity, G-protein expression, phospholipase C and phospholipase A2 activation and changes in intracellular Ca2+ concentration. However, more recent studies support a key role for protein tyrosine phosphorylation and enhanced phospholipase D and phosphoinositide 3-kinase activity in neutrophil priming. 4. Recent work has also revealed the potential for neutrophils to spontaneously and fully 'de-prime' after an initial challenge with platelet-activating factor. This ability of neutrophils to undergo a complete cycle of priming-de-priming (and re-priming) reveals a previously unrecognized flexibility in the control of neutrophil behaviour at an inflamed site.

Enzyme Activation↗
PubMed1996

Demonstration of reversible priming of human neutrophils using platelet-activating factor.

Exposure of neutrophils to agents such as lipopolysaccharide, tumor necrosis factor-alpha (TNF-alpha), and the granulocyte-macrophage colony-stimulating factor causes a major upregulation of subsequent agonist-induced NADPH oxidase activation. This priming effect is a prerequisite for neutrophil-mediated tissue damage and has been widely considered to be an irreversible process. We have investigated the potential for neutrophils to recover from a priming stimulus by studying the effects of platelet-activating factor (PAF). PAF did not stimulate respiratory burst activity directly, but caused a rapid (maximal at 10 minutes) and concentration-dependent (EC50 50.2 nmol/L) increase in N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated superoxide anion release. At time-points > 10 minutes, this priming effect spontaneously declined, with return to basal levels of fMLP-stimulated superoxide anion generation by 120 minutes. An identical priming time-course was observed with N-methyl carbamyl PAF, a nonmetabolizable analogue of PAF, indicating that the transient nature of PAF-induced priming was not secondary to PAF metabolism. Two structurally diverse PAF receptor antagonists (UK-74,505 and WEB 2086), added 10 minutes after PAF addition, increased the rate of decay of the priming effect. In contrast, TNF-alpha-induced priming, which was of a similar magnitude to that observed for PAF, was slower to evolve (maximal at 30 minutes) and remained constant for at least 120 minutes. The reversible nature of PAF-induced priming was confirmed by demonstrating that PAF-, but not TNF-alpha-, induced cell polarization (shape change) and CD11b-dependent neutrophil binding of albumin-coated latex beads was also transient, with return to basal, unstimulated levels by 120 minutes. Furthermore, cells that had spontaneously deprimed following PAF exposure retained their capacity to be fully reprimed by a subsequent addition of either PAF or TNF-alpha. These data imply that neutrophil priming is not an irreversible event: the demonstration of a cycle of complete priming, depriming, and repriming offers the potential for functional recycling of neutrophils at sites of inflammation.

Adult↗
PubMed1996

Characterization of inositol hexakisphosphate (InsP6)-mediated priming in human neutrophils: lack of extracellular [3H]-InsP6 receptors.

1. Inositol hexakisphosphate (InsP6) is a ubiquitous and abundant cytosolic inositol phosphate that has been reported to prime human neutrophils for enhanced agonist-stimulated superoxide anion generation. This led to the proposal that the release of InsP6 from necrotic cells may augment the functional responsiveness of neutrophils at an inflammatory focus. The aim of this study was to examine whether the functional effects of InsP6 in neutrophils are receptor-mediated and establish the magnitude of this priming effect relative to other better characterized priming agents. 2. Analysis of [3H]-InsP6 binding to human neutrophil membranes in 20 mM Tris, 20 mM NaCl, 100 mM KCl, 5 mM EDTA (pH 7.7) buffer using 0.1 mg ml-1 membrane protein and 2.5 nM [3H]-InsP6 (90 min, 4 degrees C), demonstrated specific low affinity [3H]-InsP6 binding that was non-saturable up to a radioligand concentration of 10 nM. 3. [3H]-InsP6 displacement by InsP6 gave a Hill coefficient of 0.55 and best fitted a two-site logistic model (53% KD 150 nM, 47% KD 5 microM). [3H]-InsP6 binding also displayed low (3 fold) selectivity for InsP6 over Ins(1,3,4,5,6)P5. 4. The specific [3H]-InsP6 binding displayed a pH optimum of 8, was abolished by pre-boiling the membranes, and was enhanced by Ca2+, Mg2+ and Na+. 5. In incubations with intact neutrophils, where high levels of specific [3H]-LTB4 binding was observed, no [3H]-InsP6 binding could be identified. 6. Preincubation of neutrophils with 100 microM InsP6 had no effect on resting cell morphology, but caused a minor and transient (maximal at 30 s) enhancement of (0.1 nM) fMLP-induced shape change (% cells shape changed: fMLP 53 +/- 3%, fMLP+InsP6 66 +/- 4%). Similarly, InsP6 (100 microM, 30 s) had no effect on basal superoxide anion generation and, compared to lipopolysaccharide (LPS, 100 ng ml-1, 60 min), tumour necrosis factor-alpha (TNF alpha, 200 u ml-1, 30 min) or platelet-activating factor (PAF, 100 nM, 5 min) caused only a small enhancement of 100 nM fMLP-stimulated superoxide anion generation (fold-increase in superoxide anion generation over fMLP alone: InsP6 1.8 +/- 0.3, LPS 6.8 +/- 0.6, TNF alpha 5.2 +/- 0.7, PAF 5.8 +/- 0.6). 7. While these data support the presence of a specific, albeit low affinity, [3H]-InsP6 binding site in human neutrophil membrane preparations, the lack of binding to intact cells implies that the functional effects of InsP6 (ie. enhanced fMLP-stimulated superoxide anion generation and shape change) are not receptor-mediated.

Adult↗