Intraphagosomal Free Ca2+ Changes during Phagocytosis.

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, Ca2+ 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 Ca2+ ion content that occur during phagocytosis and phagosome formation in human neutrophils. Measuring Ca2+ 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 Ca2+ channels on the phagosome that allow Ca2+ 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 Ca2+ 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 Ca2+ signalling events, such as peri-phagosomal Ca2+ hotspots, and other phenomena.

Microinjection and Micropipette-Controlled Phagocytosis Methods for Neutrophils.

The ability to microinject substances into the cytosol of living neutrophils opens the possibility of manipulating the chemistry within the cell and also of monitoring changes using indicators which otherwise cannot be introduced into the cell. However, neutrophils cannot be microinjected by the conventional glass pipette insertion method. Here we outline two techniques which work well with neutrophils, namely, SLAM (Simple Lipid-Assisted Microinjection) and electromicroinjection. As these methods utilize micropipettes, we also include a simple method which uses a micropipette to deliver a phagocytic stimulus to a specific cell at a defined time, enable detailed study of the phagocytic process from particle contact to particle internalization.

Minimal impact electro-injection of cells undergoing dynamic shape change reveals calpain activation.

The ability of neutrophils to rapidly change shape underlies their physiological functions of phagocytosis and spreading. A major problem in establishing the mechanism is that conventional microinjection of substances and indicators interferes with this dynamic cell behaviour. Here we show that electroinjection, a "no-touch" point-and-shoot means of introducing material into the cell, is sufficiently gentle to allow neutrophils to be injected whilst undergoing chemokinesis and spreading without disturbing cell shape change behaviour. Using this approach, a fluorogenic calpain-1 selective peptide substrate was introduced into the cytosol of individual neutrophils undergoing shape changes. These data showed that (i) physiologically elevated cytosolic Ca(2+) concentrations were sufficient to trigger calpain-1 activation, blockade of Ca(2+) influx preventing calpain activation and (ii) calpain-1 activity was elevated in spreading neutrophil. These findings provide the first direct demonstration of a physiological role for Ca(2+) elevation in calpain-1 activation and rapid cell spreading. Electroinjection of cells undergoing dynamic shape changes thus opens new avenues of investigation for defining the molecular mechanism underlying dynamic cell shape changes.

Microinjection methods for neutrophils.

The ability to microinject substances into the cytosol of living neutrophils opens the possibility of manipulating the chemistry within the cell and also of monitoring changes using indicators which otherwise cannot be introduced into the cell. However, neutrophils cannot be microinjected by the conventional glass pipette insertion method. Here, we outline two techniques which work well with neutrophils, namely, SLAM (simple lipid-assisted microinjection) and electroinjection.

Fibroblast dysfunction is a key factor in the non-healing of chronic venous leg ulcers.

Chronic age-related degenerative disorders, including the formation of chronic leg wounds, may occur due to aging of the stromal tissues and ensuing dysfunctional cellular responses. This study investigated the impact of environmental-driven cellular aging on wound healing by conducting a comprehensive analysis of chronic wound fibroblast (CWF) behavior in comparison with patient-matched healthy skin normal fibroblasts (NF). The dysfunctional wound healing abilities of CWF correlated with a significantly reduced proliferative life span and early onset of senescence compared with NF. However, pair-wise comparisons of telomere dynamics between NF and CWF indicated that the induction of senescence in CWF was telomere-independent. Microarray and functional analysis suggested that CWFs have a decreased ability to withstand oxidative stress, which may explain why these cells prematurely senescence. Microarray analysis revealed lower expression levels of several CXC chemokine genes (CXCL-1, -2, -3, -5, -6, -12) in CWF compared with NF (confirmed by ELISA). Functionally, this was related to impaired neutrophil chemotaxis in response to CWF-conditioned medium. Although the persistence of non-healing wounds is, in part, due to prolonged chronic inflammation and bacterial infection, our investigations show that premature fibroblast aging and an inability to correctly express a stromal address code are also implicated in the disease chronicity.

Lipid-protein cargo transfer: a mode of direct cell-to-cell communication for lipids and their associated proteins.

Cells in tissues or in experimental cell colonies respond to stimuli in a co-ordinated manner when they are electrically and chemically coupled by gap junctions. These junctions permit the cell-to-cell passage of small molecules, such as inositol tris phosphate (IP(3)) within the colony and are important in co-ordinating tissue activity. This is the only recognised mechanism of direct chemical signalling that does not involve the release of an extracellular messenger between cells. However, the data in this article demonstrates a new mode of intercellular communication. Two potentially important signalling lipids, PIP(2) and ganglioside G-M1 were shown to move between cells in colonies by tracking (i) fluorescent lipids loaded into the plasma membranes of individual cells in a cell colony using a novel micropipette technique and (ii) movement of fluorescent lipids after localised photobleaching. Furthermore, a large protein molecule, cholera toxin B subunit bound to extracellularly facing ganglioside G-M1 was also shown to transfer between cells. The transfer was inhibited by pre-treatment with poly-L-lysine and polyethylenimine, suggesting a role for tight junctions, perhaps by permitting diffusion of lipids and their protein "cargo" across these cell-to-cell contact points. This is a hitherto unsuspected form of molecular signalling within cell colonies and tissues which may have implications for understanding co-ordinated cell colony behaviour.

Ca influx shutdown in neutrophils induced by Fas (CD95) cross-linking.

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.

Ca(2+), calpain and 3-phosphorylated phosphatidyl inositides; decision-making signals in neutrophils as potential targets for therapeutics.

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.

Exclusion of exogenous phosphatidylinositol-3,4,5-trisphosphate from neutrophil-polarizing pseudopodia: stabilization of the uropod and cell polarity.

Although there is accumulating evidence that the generation and localization of phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) have important functions in neutrophil polarization and chemotaxis, the mechanism of this linkage has yet to be established. Here, using exogenous fluorescent PtdIns(3,4,5)P(3) introduced into the inner leaflet of the neutrophil plasma membrane by a cationic carrier, we show that: first, PtdIns(3,4,5)P(3) uniformly delivered to the neutrophil plasma membrane is excluded from newly forming pseudopodia; second, PtdIns(3,4,5)P(3) translocates to and is immobilized at the pole opposite a stable polarizing pseudopod; third, asymmetric delivery of PtdIns(3,4,5)P(3) to the neutrophil triggers the generation of polarizing pseudopodia at the opposite pole; and finally, PtdIns(3,4,5)P(3) triggers repetitive Ca(2+) signals, the onset of which precedes morphological polarization. These data suggest that translocation and immobilization of PtdIns(3,4,5)P(3) or a 3,x-phosphorylated metabolite in the uropod functions as an important polarization cue that defines neutrophil polarity and stabilizes the generation of pseudopodia at the opposite pole.

Intranuclear Ca2+ signals within individual nuclear lobes of neutrophils.

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.

Phagosomal oxidative activity during beta2 integrin (CR3)-mediated phagocytosis by neutrophils is triggered by a non-restricted Ca2+ signal: Ca2+ controls time not space.

The temporal and spatial relationship between particle binding to the neutrophil by beta2 integrin (CR3), the Ca2+ elevation and subsequent oxidase activation has been unclear. This is because of the difficulty in studying the time course of individual phagocytic events in individual neutrophils. Here, we have used a micromanipulation technique to present C3bi-opsonised zymosan particles to the neutrophil under observation. In this way, the moment of particle contact, pseudopod formation and internalisation has been established and cytosolic free Ca2+ and oxidation of dichlorodihydrofluorescein (DCDHF)-labelled particles determined simultaneously. Using this approach, we have found that the Ca2+ signal, which is triggered by CR3-mediated phagocytosis, can be resolved into two temporally separated components. The first Ca2+ signal occurs during beta2 integrin engagement as the phagocytic cup forms but does not trigger oxidation of the particle. The second global Ca2+ signal, which is triggered about the time of phagosomal closure, causes an abrupt activation of the oxidase. This second Ca2+ signal was not restricted to the region of the phagosome yet only triggered the oxidase activation locally in the phagosome, with no evidence of activation at other sites in the neutrophil. This points to a dual control of oxidase activation, with Ca2+ controlling the timing of oxidase activation but slower and more localised molecular events, perhaps involving oxidase assembly and phosphatidylinositol 3-phosphate generation, determining the site of oxidase activation.