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Phagocytosis (and endocytosis) is an unusual cellular process that results in the formation of a novel subcellular organelle, the phagosome. This phagosome contains not only the internalised target of phagocytosis but also the external medium, creating a new border between extracellular and intracellular environments. The boundary at the plasma membrane is, of course, tightly controlled and exploited in ionic cell signalling events. Although there has been much work on the control of phagocytosis by ions, notably, Ca 2+ ions influxing across the plasma membrane, increasing our understanding of the mechanism enormously, very little work has been done exploring the phagosome/cytosol boundary. In this paper, we explored the changes in the intra-phagosomal Ca 2+ ion content that occur during phagocytosis and phagosome formation in human neutrophils. Measuring Ca 2+ ion concentration in the phagosome is potentially prone to artefacts as the intra-phagosomal environment experiences changes in pH and oxidation. However, by excluding such artefacts, we conclude that there are open Ca 2+ channels on the phagosome that allow Ca 2+ ions to "drain" into the surrounding cytosol. This conclusion was confirmed by monitoring the translocation of the intracellularly expressed YFP-tagged C2 domain of PKC-γ. This approach marked regions of membrane at which Ca 2+ influx occurred, the earliest being the phagocytic cup, and then the whole cell. This paper therefore presents data that have novel implications for understanding phagocytic Ca 2+ signalling events, such as peri-phagosomal Ca 2+ hotspots, and other phenomena.

Neutrophils exhibit rapid cell spreading and phagocytosis, both requiring a large apparent increase in the cell surface area. The wrinkled surface topography of these cells may provide the membrane reservoir for this. Here, the effects of manipulation of the neutrophil cell surface topography on phagocytosis and cell spreading were established. Chemical expansion of the plasma membrane or osmotic swelling had no effects. However, osmotic shrinking of neutrophils inhibited both cell spreading and phagocytosis. Triggering a Ca 2+ signal in osmotically shrunk cells (by IP 3 uncaging) evoked tubular blebs instead of full cell spreading. Phagocytosis was halted at the phagocytic cup stage by osmotic shrinking induced after the phagocytic Ca 2+ signalling. Restoration of isotonicity was able to restore complete phagocytosis. These data thus provide evidence that the wrinkled neutrophil surface topography provides the membrane reservoir to increase the available cell surface area for phagocytosis and spreading by neutrophils., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The measurement and manipulation of cytosolic free Ca 2+ of neutrophils is crucial for investigating the mechanisms within living neutrophils which generate Ca 2+ signals and the cellular responses triggered by them. Optical methods for this are the most applicable for neutrophils, and are discussed here, especially the use of fluorescent indicators of Ca 2+ and photoactivation of reagents involved in Ca 2+ signaling. Both of these synthetic agents can be loaded into neutrophils as lipid-soluble esters or can be microinjected into the cell. In this chapter, we outline some of the techniques that have been used to monitor, visualize, and manipulate Ca 2+ in neutrophils.

The measurement and manipulation of cytosolic free Ca(2+) of neutrophils is crucial for investigating the mechanisms within living neutrophils which generate Ca(2+) signals and the cellular responses triggered by them. Optical methods for this are the most applicable for neutrophils and are discussed here, especially the use of fluorescent indicators of Ca(2+) and photoactivation of reagents involved in Ca(2+) signaling. Both of these synthetic agents can be loaded into neutrophils as lipid-soluble esters or can be microinjected into the cell. In this chapter, we will outline some of the techniques that have been used to monitor, visualize, and manipulate Ca(2+) in neutrophils.

In order to establish whether non-mitochondrial oxidase activity in human neutrophils is tightly related to cytosolic Ca(2+) concentration, we simultaneously measured Ca(2+) oscillations induced by ATP and oxidant production in single adherent neutrophils using confocal microscopy. ATP induced fast damped Ca(2+) spikes with a period of 15s and slower irregular spikes with a period greater than 50s. Spikes in Ca(2+) occurred in the absence of Ca(2+) influx, but the amplitude was damped by inhibition of Ca(2+) influx. Using the oxidation of hydroethidine as a cytosolic marker of oxidant production, we show that the generation of reactive oxygen species by neutrophils adherent to glass was accelerated by ATP. The step-up in NADPH oxidase activity followed the first elevation of cytosolic Ca(2+) but, despite subsequent spikes in Ca(2+) concentration, no oscillations in oxidase activity could be detected. ATP induced spikes in Ca(2+) in a very reproducible way and we propose that the Ca(2+) signal is an on-switch for oxidase activity, but the activity is apparently not directly correlated with spiking activity in cytosolic Ca(2+)., (2011 Elsevier B.V. All rights reserved.)

The first report that high speed Ca(2+) waves travel in a restricted zone around the cell periphery in a clockwise manner and which can only be revealed by very high speed imaging has been quietly retracted. In the original report, a single small region of high Ca(2+) was seen to spin around the cell periphery and around phagosomes formed within phagocytes in a manner unlike anything reported previously. This consequently caused a lot of interest. Several other papers reporting a similar phenomenon in a variety of cells were also published by the same lab and the phenomenon has been included in high profile reviews such as in Science. However, several groups have failed to reproduce this work and attempts to detect the Ca(2+) waves or the mechanism for their generation have failed. Now, the original report has been quietly retracted. In this short commentary, we give a brief account of the sorry story of the non-existent circulating Ca(2+) waves (z-waves) in the hope that the literature can be rectified and that future cell calcium biologists are not misled by these fantastic and spurious Ca(2+) reports.

Many cell signalling pathways are driven by changes in cytosolic calcium. We studied the effects of a range of inhibitors of connexin channels on calcium signalling in cardiac cells and HeLa cells expressing connexins. Gap 26 and 27, peptides that mimic short sequences in each of the extracellular loops of connexin 43, and anti-peptide antibodies generated to extracellular loop sequences of connexins, inhibited calcium oscillations in neonatal cardiac myocytes, as well as calcium transients induced by ATP in HL-1 cells originating from cardiac atrium and HeLa cells expressing connexin 43 or 26. Comparison of single with confluent cells showed that intracellular calcium responses were suppressed by interaction of connexin mimetic peptides and antibodies with hemichannels present on unapposed regions of the plasma membrane. To investigate how inhibition of hemichannels in the plasma membrane by the applied reagents was communicated to calcium store operation in the endoplasmic reticulum, we studied the effect of Gap 26 on calcium entry into cells and on intracellular IP3 release; both were inhibited by Gap 26. Calcium transients in both connexin 43- and connexin 26-expressing HeLa cells were inhibited by the peptides suggesting that the extended cytoplasmic carboxyl tail domain of larger connexins and their interactions with intracellular scaffolding/auxiliary proteins were unlikely to feature in transmitting peptide-induced perturbations at hemichannels in the plasma membrane to IP3 receptor channel central to calcium signalling. The results suggest that calcium levels in a microenvironment functionally connecting plasma membrane connexin hemichannels to downstream IP3-dependent calcium release channels in the endoplasmic reticulum were disrupted by the connexin mimetic peptide, although implication of other candidate hemichannels cannot be entirely discounted. Since calcium signalling is fundamental to the maintenance of cellular homeostasis, connexin hemichannels emerge as therapeutic targets open to manipulation by reagents interacting with external regions of these channels.

Patients with sepsis commonly develop leukocytosis, which is presumed to reflect a host response to infection. Effective phagocytosis by neutrophils is crucial in the clearance of invading microbes. However, efficacy of phagocytosis in sepsis is controversial. We hypothesized that host phagocytic capacity in sepsis can be affected by immature neutrophils that are released into the circulation. Circulating neutrophils were evaluated in 16 patients with severe sepsis and 5 healthy donors. Immature neutrophils were identified by the cell morphology. Phagocytosis was evaluated by micromanipulation technique and simultaneous cytosolic-free Ca2+ imaging. Leukocytosis was present in 12 of 16 patients. Nine of the 12 patients with leukocytosis and 3 of 4 patients with normal white blood cell counts had increased circulating immature neutrophils (mean, 39.3% +/- 20.7%; normal

Ultra-localised and peripherally restricted zones of elevated Ca2+ (z-waves) have been reported to cycle around the periphery of neutrophils at low frequency (1/20s) in the absence of conventional localised Ca2+ (puffs) and global Ca2+ (waves) signals. However, we report here that fast confocal laser scanning of human neutrophils loaded with either cytosolic fluo4 or its membrane associated analogue, MOMO reports both "conventional" stationary Ca2+ "puffs" (diameter c.3 microm) and global Ca2+ waves that sweep across the cell. The Ca2+ puff size and frequency of detection suggests that each neutrophil contained only a single release site and that its detection was limited by the location of the confocal plane relative to the event. Both formylated peptide receptor stimulation and cytosolic IP3 uncaging generated Ca2+ puffs (c.6% of cells) and global Ca2+ signals (c.75% of cells). The Ca2+ puffs peaked at approx. 250 nM and had a duration of approx. 235 msec and remained at a single locus. This was similar to other Ca2+ events in other cell types but in direct contrast to the reported z-waves. It was concluded that the micro-events which underlie Ca2+ signalling in neutrophils are conventional and that the existence of novel Ca2+z-waves is doubtful.

Several events accompany integrin-mediated phagocytosis by myeloid cells. These include local pseudopod and phagocytic cup formation followed by Ca(2+) signalling. However, there is also a role for localised phosphatidylinositol (3,4,5) trisphosphate [PtdIns(3,4,5)P(3)] production. Here we report that in neutrophilic HL-60 cells expressing PH-Akt-GFP, binding of iC3b-coated zymosan particles (2 microm in diameter) via beta2 integrin induces an incomplete phagocytic cup to form before either PtdIns(3,4,5)P(3) or phosphatidylinositol (3,4) bisphosphate [PtdIns(3,4)P(2)] production or Ca(2+) signalling. These phosphoinositides then accumulated locally at the site of the phagocytic cup and Ca(2+) signalling and phagosome closure follows immediately. Although photobleaching showed that PH-Akt-GFP was freely diffusible in the cytosol and able to dissociate from the phagocytic cup, it was restricted to the plasma membrane of the formed but open phagosome and failed to diffuse into the surrounding plasma membrane or neighbouring phagocytic cups even if connected. Inhibition of phosphoinositide (PI) 3-kinase or depletion of membrane cholesterol inhibited both Ca(2+) signalling and phagosome closure, but had no effect on particle binding or phagocytic cup formation. We therefore conclude that PtdIns(3,4,5)P(3) or PtdIns(3,4)P(2) generation was not required for the events that initiate the formation of the phagocytic cup, but that anchoring of PtdIns(3,4,5)P(3) at the phagocytic cup is an essential step for phagosome closure and Ca(2+) signalling.

In neutrophils, as in most other cell types, Ca(2+) signalling is important for a number of cellular activities. Although inositol(1,4,5)trisphosphate-mediated release of Ca(2+) from intracellular stores is a necessary prelude, it is the Ca(2+) influx that is responsible for many of the neutrophil responses. We report here that although elevations of cytosolic Ca(2+) do not accompany Fas-mediated apoptosis in neutrophils, the Ca(2+) influx component of the response to N-formyl-methionyl-leucyl-phenylalanine (FMLP) becomes selectively inactived as the neutrophils progress towards accelerated apoptosis induced by Fas (CD95) cross-linking. After 4 hr incubation at 37 degrees, untreated neutrophils display an exaggerated Ca(2+) influx phase in response to FMLP. This was absent in neutrophils that had been Fas-activated at the same time. No Ca(2+) influx component was demonstrable by the removal of extracellular Ca(2+) or by Ca(2+) channel blockade with Ni(2+) and no Mn(2+) influx was detectable. The defect could not be attributed to a decrease in receptor sensitivity, receptor coupling or receptor number because the release of stored Ca(2+) remained constant during incubation and was unaffected by Fas activation. Ca(2+) influx became uncoupled from store release before detectable gross morphological changes or phosphatidyl serine externalization and was also insensitive to caspase 3 and 8 inhibitors. These results suggest a mechanism other than caspase-mediated proteolytic damage to components important for Ca(2+) influx.

The chemical signals within neutrophils that control their behaviour are complex and these signals control the complex activity of neutrophils with precision. Failure of neutrophils to reform their antibacterial activity would lead to infection, while over-activity of neutrophils may lead to tissue damage and inflammatory disease. The identity of some of the intracellular signals is becoming clear and insights into the potential for interplay between them are being sought. Although it is well established that cytosolic free Ca(2+) plays a role, it is only recently that the importance of intracellular protease, calpain, and the 3-position phosphorylated phosphatidyl inositides is becoming recognised. In this review these three key signals are discussed as potential therapeutic targets for the modulation of neutrophil activity.

Cytosolic free Ca(2+)and intranuclear Ca(2+)behave similarly in human neutrophils. However, conventional laser scanning at 350 ms/frame resolution at lower than physiological temperatures demonstrates that (i) the nuclear fluo3-Ca(2+)signal persists longer than the cytosolic signal in some (but not all) nuclear lobes, (ii) the neutrophil nuclear membrane and fine inter-lobe filaments present barriers to diffusion of fluo3-Ca(2+)and lucifer yellow, and (iii) the diffusion barrier correlates with condensed chromosomal material on the nuclear envelope and blockage of the movement of fluo3-Ca(2+)into individual nuclear lobes.

Glycosyl-phosphatidylinositol (GPI)-anchored proteins are unique in that they penetrate only the outer leaflet of the plasma membrane but are still able to mediate intracellular signalling events following antibody-induced ligation. Detergent solubilisation studies suggest that microdomains exist at the cell surface within which are sequestered GPI-linked proteins. Here we report the construction and expression of a fluorescent GPI anchor on the surface of CHO, EL4, and U937 cells by fusing green fluorescent protein (GFP) to the GPI-attachment site of CD59. The resultant GFP-GPI has properties comparable to that of endogenously expressed GPI-anchored molecules as shown by Triton X-114 partitioning. However, sucrose gradient floatation showed that GFP-GPI was only partially resistant to detergent solubilisation. Furthermore confocal scanning laser microscopy revealed a homogeneous distribution of GFP-GPI at the cell surface, which only became clustered following cross-linking of the GPI anchor via an anti-GFP antibody. Surprisingly, GFP-GPI signalled Ca2+ change upon cross-linking demonstrating its signalling competence. Our results suggest that the GPI-anchor itself does not confer a clustered distribution to molecules but that clustering occurs following ligation with antibody, which allows the protein to become Ca2+ signalling competent., (Copyright 2002 Elsevier Science (USA).)