Neutrophil phagocytosis and secretion : The role of calcium and the cytoskeleton

University dissertation from Linköping : Linköping University Electronic Press

Abstract: Neutrophils assure rapid removal of bacteria by a variety of processes. They crawl out of the vessels, phagocytose the bacteria and kill them by secretion of bactericidal substances and production of oxidative metabolites. The aims of this study were to investigate the signalling pathways during these processes, in particular i) how complement receptors mediate phagocytosis and NADPH~oxidase activity ii) the role of Ca2+ in secretion and the role of a Ca2+-dependent, actin-binding protein, _gelsolin, in neutrophil phagocytosis and secretion. Conventional biochemical methods, capacitance measurements of secretion with the patchclamp technique and visualisation with fluorescence microscopy techniques were used. We found that phospholipase D (PLD) is an early Ca2+-independent signal in complementmediated phagocytosis, preceding cytoskeletal rearrangements. We also demonstrated'that the NADPH-oxidase could be activated in situ to generate oxidative metabolites intracellularly after particle stimulation of complement receptors in the absence of phagocytosis. This permits cells to use oxidative metabolites for signalling and not only to kill bacteria. This activation involved the cytoskeleton and PLD. Whereas signalling during pha~ocytosis can occur independently of Ca2+, other neutrophil functions are highly Ca +-dependent. Investigating the relative importance of Ca2+-release from intracellular stores versus Ca2+ influx over the plasma membrane, we found that secretion of primary granules induced by fMLP is dependent on Ca2+ -influx. ci+-influx alone is not sufficient to induce secretion in neutrophils. A second synergistic signal is required. This missing signal was not PLD, PLC or tyrosine kinases, but involved a pertussis-sensitive 0-protein and PI3-kinase. When further investigating the role of Ca2+ in secretion, we found that secretion of different granules is regulated by different [Ca2+:J. Primary granules are secreted at 100 ~ Ca2+ whereas the other granules are secreted at 1.5-5 ~ ci+, suggesting two mechanisms involving different Ca2+ activated systems/sensor proteins. One sensor protein could be the Ca2+-dependent, actinbinding protein, gelsolin, which has earlier been shown to stimulate secretion in different celltypes. Secretion from gelsolin-deficient mouse neutrophils was nevertheless nonnal. Gelsolin was however found to be essential for !gO-mediated-, but not complement-induced phagocytosis. Activation of the oxidase and phagolysosorne-fusion was unaffected in gelsolin-deficient neutrophils. This suggests gelsolin to be a Ca2+ -sensor specifically for !gOmediated phagocytosis. !gO-mediated phagocytosis often leads to more efficient killing than complement-mediated phagocytosis. Gelsolin seems to be part of the machinery that distinguishes the two pathways of phagocytosis. The present data show that receptor mediated activation of neutrophil functions involves several signalling pathways. This allows selective modulation of the inflammatory response.

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