A cytosolic calcium transient is not necessary for degranulation or oxidative burst in immune complex-stimulated neutrophils

Seetoo, Kurt F.; Schonhorn, Jeremy E.; Gewirtz, Andrew T.; Zhou, Ming; McMenamin, Mary E.; Delva, Luisette; Simons, Elizabeth R.

doi:10.1002/jlb.62.3.329

Cited by 25 publications

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“…Activation of PLD appears to be necessary but not sufficient for Fc␥ receptormediated degranulation of PMN [2]. In contrast, changes in cytoplasmic [Ca 2ϩ ] are neither necessary nor sufficient to elicit degranulation, although they can, to a limited extent, modulate this effector function [3]. Consistent with this view, modulation of degranulation by pH in appeared to be mediated via PLD, but not by ⌬[Ca 2ϩ ] in .…”

Section: Discussionmentioning

confidence: 83%

“…Although PLD activation is also required for PMN superoxide secretion [27][28][29], our results suggest that, at least in response to maximal Fc␥ receptor stimulation, neither PLD activation nor acidification is limiting. Perhaps for that secretory response, ⌬[Ca 2ϩ ] in plays a more important regulatory role because preventing such changes potently (Ͼ75%) inhibits PMN superoxide release [3].…”

Section: Discussionmentioning

confidence: 99%

“…Cytoplasmic pH was measured by spectrofluorimetry using the intracellular pH probe biscarboxyethylcyanineflourescein (BCECF) as previously described [3].…”

Section: Ph Measurementsmentioning

confidence: 99%

“…Our laboratory has investigated the mechanism by which activation of Fc␥ receptors leads to degranulation in PMN. These studies revealed that phospholipase D (PLD) is probably an essential controlling signaling event for Fc receptor-mediated degranulation [2] whereas, in contrast, changes in cytoplasmic [Ca 2ϩ ] play a modulatory role but are not absolutely required [3]. In addition, other signaling events may be required for neutrophil function and its regulation; the identification of these events is the goal of this study.…”

Section: Introductionmentioning

confidence: 99%

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Neutrophil degranulation and phospholipase D activation are enhanced if the Na+/H+ antiport is blocked

Gewirtz

Seetoo

Simons

1998

Journal of Leukocyte Biology

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To investigate the role of the pH transient in controlling degranulation, the Na ؉ /H ؉ antiport was inhibited either with 100 M dimethylamiloride (DMA) or by substituting N-methyl-glucamine for extracellular sodium. Blocking the antiport with DMA led to hyperacidified PMN, which exhibited an increase in degranulation, but did not affect generation of superoxide. DMA did not alter the ability of neutrophils to phagocytose and oxidize dichlorodihydrofluoresceinated HIC, suggesting the increase in degranulation was not the result of failed phagocytosis. Investigation into whether the observed increase in degranulation when the antiport was blocked was mediated by PLD or ⌬[Ca 2؉ ] in revealed that blocking the antiport increased HICinduced PLD activity but had no effect on HICinduced ⌬[Ca 2؉ ] in . Blocking the Na ؉ /H ؉ antiport by ion substitution caused similar effects on PMN signaling and secretion as was seen with DMA. These results indicate that Na ؉ /H ؉ antiport activity is not necessary for degranulation or superoxide release in HICstimulated PMN and that hyperacidification of the cytoplasm can modulate degranulation. Therefore, pH in , via its effect on PLD, may be a control point of degranulation and may represent one way that neutrophils achieve differential control of their antibacterial products. J. Leukoc. Biol. 64: 98-103; 1998.

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Section: Discussionmentioning

confidence: 83%

Section: Discussionmentioning

confidence: 99%

“…Cytoplasmic pH was measured by spectrofluorimetry using the intracellular pH probe biscarboxyethylcyanineflourescein (BCECF) as previously described [3].…”

Section: Ph Measurementsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Neutrophil degranulation and phospholipase D activation are enhanced if the Na+/H+ antiport is blocked

Gewirtz

Seetoo

Simons

1998

Journal of Leukocyte Biology

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“…They were loaded with the Ca 21 indicator Indo-1, as previously described (15,16,35,36), and then were kept shaking gently in PBS 1 glucose (0.5 mM) at 48C until needed (never more than 6 h from blood drawing to use). Serum from each donor was saved and used for opsonization of Se, on that day, either as is (normal) or after incubation at 568C for 30 min to inactivate complement components (DH).…”

Section: Methodsmentioning

confidence: 99%

Initial cytoplasmic and phagosomal consequences of human neutrophil exposure to Staphylococcus epidermidis

2009

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Microorganisms are recognized by specific phagocyte surface receptors. Liganded receptors then signal a series of events leading to phagocytosis and destruction of the organism by oxidative, lytic, and associated processes. Some organisms, such as Mycobacterium tuberculosis (Mtb), Cryptococcus neoformans (Cf), and others, evade such destruction, surviving and sometimes multiplying within the phagosome to later cause disease. To study such evasion, we developed protocols which permit simultaneous kinetic measurement of early cytoplasmic signaling and of phagosomal pH (pH p ) and oxidative burst, on a cell-by-cell basis, of polymorphonuclear (PMN) leukocytes exposed to fluorescently labeled, nonpathogenic Staphylococcus epidermidis (Se). The availability of a new, highly sensitive pH probe, pHrodo TM , permits observation of increasing pH p . Simultaneous labeling of the organism, applicable to any phagocyte target, with a probe insensitive to pH and oxidative species, such as AlexaFluor350 TM , permits distinction between binding and functional responses to it by ratioing fluorescences. Addition of an extracellular-specific quencher (Trypan blue) permits distinction between bound and phagosome-enclosed targets, so that conditions within the closed phagosome can be studied. We found that opsonization is required for functional activation of PMN by Se, that the organism causes early alkalinization of the phagosome (in contrast to Cf which hyperacidifies it), and that extracellular Ca 21 is not required for cytoplasmic Ca 21 signaling but contributes markedly to binding of Se to PMN and to ensuant bactericidal functions. These findings lead to a new approach to the study of select organisms, like Cf and Mtb, which evade killing by manipulating the phagosomal environment. ' International Society for Advancement of CytometryKey terms calcium; pH; reactive oxygen species PHAGOCYTIC cells, such as polymorphonuclear (PMN) neutrophils and tissue macrophages (MP), are essential components of the human innate defense against infection. Phagocyte surface receptors recognize invading bacteria by a variety of mechanisms, including complement-dependent opsonization, antibody/antigen binding via Fc receptors, or pattern recognition (e.g., mannose-, scavenger-, or Toll-) receptor liganding. They then signal, via changes in intracellular cation concentrations, in transmembrane potentials, and in secondary signaling (e.g., via activation of specific phospholipases) so as to initiate a series of processes that result in phagocytosis with subsequent intraphagosomal killing and clearance of the organism [for some very recent reviews see (1-7)].Many of these signaling events occur early, within seconds of ligand-receptor binding, and have been shown to vary with the specific receptors involved (8)(9)(10)(11)(12)(13)(14). Yet, the changes initiated by these signals to generate effector functions of the cell do not become measurable by conventional methods until at least minutes later. Furthermore, responses to such stimulation...

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Measurement of Phagocytosis and of the Phagosomal Environment in Polymorphonuclear Phagocytes by Flow Cytometry

Simons

2010

CP Cytometry

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Phagocytes are the most important early components of the immune response, programmed to recognize, engulf, and destroy immune complexes (formed when antibodies recognize their specific antigens), foreign particles, bacteria, mycobacteria, apoptotic cells, etc. Neutrophils, monocytes, macrophages, and dendritic cells all participate in this process. Flow cytometry permits observation of phagocytes that have responded and, with the appropriate fluorescent probes, of the environment in the phagosome that has enclosed the foreign matter. This unit gives the background and the protocols for performing such studies. Curr. Protoc. Cytom. 51:9.31.1‐9.31.10. © 2010 by John Wiley & Sons, Inc.

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A cytosolic calcium transient is not necessary for degranulation or oxidative burst in immune complex-stimulated neutrophils

Abstract: Abstract:Receptor-mediated activation of neutrophils

Cited by 25 publications

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Neutrophil degranulation and phospholipase D activation are enhanced if the Na+/H+ antiport is blocked

Neutrophil degranulation and phospholipase D activation are enhanced if the Na+/H+ antiport is blocked

Initial cytoplasmic and phagosomal consequences of human neutrophil exposure to Staphylococcus epidermidis

Measurement of Phagocytosis and of the Phagosomal Environment in Polymorphonuclear Phagocytes by Flow Cytometry

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