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.

Phagocytosis is one of the most polarised of all cellular activities. Both the stimulus (the target for phagocytosis) and the response (its internalisation) are focussed at just one part of the cell. At the locus, and this locus alone, pseudopodia form a phagocytic cup around the particle, the cytoskeleton is rearranged, the plasma membrane is reorganised, and a new internal organelle, the phagosome, is formed. The effect of signals from the stimulus must, thus, both be complex and yet be restricted in space and time to enable an effective focussed response. While many aspects of phagocytosis are being uncovered, the mechanism for the restriction of signalling or the effects of signalling remains obscure. In this review, the details of the problem of restricting chemical intracellular signalling are presented, with a focus on diffusion into the cytosol and of signalling lipids along the plasma membrane. The possible ways in which simple diffusion is overcome so that the restriction of signalling and effective phagocytosis can be achieved are discussed in the light of recent advances in imaging, biophysics, and cell biochemistry which together are providing new insights into this area.

Although we know a wealth of detail about the molecular and cell biology of phagocytosis, there are many unsolved mysteries remaining. In this final chapter, some important may be tangential) questions are raised, that the bulk of researchers are not really addressing. In this chapter, some suggestions are given for this type of "blue skies" future work. These include new approaches to understanding phagocytosis and the possibility that this new knowledge may provide a solution to anti-microbial resistance. This future phagocytosis research would have an impact, not only on our understanding of phagocytosis, but potentially on the future of human health.

The construction of a low affinity Ca2+-probe that locates to the cell cortex and cell surface wrinkles, is described called. EPIC3 (ezrin-protein indicator of Ca2+). The novel probe is a fusion of CEPIA3 with ezrin, and is used in combination with a Ca2+-insensitive probe, ezrin-mCherry, both of which locate at the cell cortex. EPIC3 was used to monitor the effect of Ca2+ influx on intra-wrinkle Ca2+ in the macrophage cell line, RAW 264.7. During experimentally-induced Ca(2+)influx, EPIC3 reported Ca2+ concentrations at the cell cortex in the region of 30-50 mu M, with peak locations towards the tips of wrinkles reaching 80 mu M. These concentrations were asso-ciated with cleavage of ezrin (a substrate for the Ca2+ activated protease calpain-1) and released the C-terminal fluors. The cortical Ca2+ levels, restricted to near the site of phagocytic cup formation and pseudopodia extension during phagocytosis also reached high levels (50-80 mu M) during phagocytosis. As phagocytosis was completed, hotspots of Ca2+ near the phagosome were also observed.

Phagocytosis is usually defined as the cellular process by which cells internalise particulate matter larger than about 0.5 mu m in diameter. It is an endocytic process, distinct from pinocytosis and macropinocytosis. These latter processes may internalise small particles suspended the extracellular fluid, but this is a by-product of internalising the fluid, and is not phagocytosis per se. In contrast, phagocytosis is targeted at solid particulates, usually microbes, which are internalised and "digested" either to provide food, or as part of the immune system of higher animals. The mechanism of phagocytosis may have, at its core, many primitive elements, but it is a highly complex and coordinated series of cell biological and molecular events which together result in the uptake of a particle. In this introduction, the basis of phagocytosis and some ideas of its origin are discussed.

During phagocytosis, there is an apparent expansion of the plasma membrane to accommodate the target within a phagosome. This is accompanied (or driven by) a change in membrane tension. It is proposed that the wrinkled topography of the phagocyte surface, by un-wrinkling, provides the additional available membrane and that this explains the changes in membrane tension. There is no agreement as to the mechanism by which unfolding of cell surface wrinkles occurs during phagocytosis, but there is a good case building for the involvement of the actin-plasma membrane crosslinking protein ezrin. Not only have direct measurements of membrane tension strongly implicated ezrin as the key component in establishing membrane tension, but the cortical location of ezrin changes at the phagocytic cup, suggesting that it is locally signalled. This chapter therefore attempts to synthesise our current state of knowledge about ezrin and membrane tension with phagocytosis to provide a coherent hypothesis.

Phagocytosis is usually defined as the cellular process by which cells internalise particulate matter larger than about 0.5 μm in diameter. It is an endocytic process, distinct from pinocytosis and macropinocytosis. These latter processes may internalise small particles suspended the extracellular fluid, but this is a by-product of internalising the fluid, and is not phagocytosis per se. In contrast, phagocytosis is targeted at solid particulates, usually microbes, which are internalised and “digested” either to provide food, or as part of the immune system of higher animals. The mechanism of phagocytosis may have, at its core, many primitive elements, but it is a highly complex and coordinated series of cell biological and molecular events which together result in the uptake of a particle. In this introduction, the basis of phagocytosis and some ideas of its origin are discussed.

This chapter outlines some of the more significant steps in our understanding of the phenomenon and mechanism of phagocytosis. These are mainly historical, ranging from near the advent of microscopy in the seventeenth and eighteenth century up to the period before the Second World War (1930s). During this time, science itself moved from being the domain of the wealthy enthusiast to the professional and funded university scientist. Not surprisingly progress was slow of the first two centuries of phagocytic research, but accelerated around the late nineteenth century and the turn of the twentieth century. Since then progress has accelerated still further. This chapter however aims to put our current progress into a historical context and to explore some of the interesting personalities who have set the ground work for our current understanding of the subject of this book, namely phagocytosis.

Although the cytosolic Ca2+ signalling event in phagocytosis is well established, and the mechanism for generating such signals also understood, the target for the Ca2+ signal and how this relates to the phagocytic outcome is less clear. In this chapter, we present the evidence for a role of the Ca2+ activated protease, calpain, in phagocytosis. The abundant evidence for Ca2+ changes and calpain activation during cell shape changes is extended to include the specific cell shape change which accompanies phagocytosis. The discussion therefore includes a brief description of the domain structure of calpain and their functions. Also the mechanism by which calpain activation is limited at the cell periphery subdomains, and how this would allow phagocytic pseudopodia to form locally.

The measurement and manipulation of cytosolic free Ca2+ of neutrophils is crucial for investigating the mechanisms within living neutrophils which generate Ca2+ 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 Ca2+ and photoactivation of reagents involved in Ca2+ 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 Ca2+ in neutrophils.

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 Ca2+ signal in osmotically shrunk cells (by IP3 uncaging) evoked tubular blebs instead of full cell spreading. Phagocytosis was halted at the phagocytic cup stage by osmotic shrinking induced after the phagocytic Ca2+ 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.

The intracellular events underlying phagocytosis, a crucial event for innate immunity, are still unresolved. In order to test whether the reservoir of membrane required for the formation of the phagocytic pseudopodia is maintained by cortical ezrin, and that its cleavage is a key step in releasing this membrane, the cleavage of cortical ezrin was monitored within living phagocytes (the phagocytically competent cell line RAW264.7) through expressing two ezrin constructs with fluorescent protein tags located either inside the FERM or at the actin-binding domains. When ezrin is cleaved in the linker region by the Ca2+-activated protease calpain, separation of the two fluorophores would result. Experimentally induced Ca2+ influx triggered cleavage of peripherally located ezrin, which was temporally associated with cell expansion. Ezrin cleavage was also observed in the phagocytic pseudopodia during phagocytosis. Thus, our data demonstrates that peripheral ezrin is cleaved during Ca2+-influx-induced membrane expansion and locally within the extending pseudopodia during phagocytosis. This is consistent with a role for intact ezrin in maintaining folded membrane on the cell surface, which then becomes available for cell spreading and phagocytosis.

Perhaps the most important feature of neutrophils is their ability to rapidly change shape. In the bloodstream, the neutrophils circulate as almost spherical cells, with the ability to deform in order to pass along narrower capillaries. Upon receiving the signal to extravasate, they are able to transform their morphology and flatten onto the endothelium surface. This transition, from a spherical to a flattened morphology, is the first key step which neutrophils undergo before moving out of the blood and into the extravascular tissue space. Once they have migrated through tissues towards sites of infection, neutrophils carry out their primary role-killing infecting microbes by performing phagocytosis and producing toxic reactive oxygen species within the microbe-containing phagosome. Phagocytosis involves the second key morphology change that neutrophils undergo, with the formation of pseudopodia which capture the microbe within an internal vesicle. Both the spherical to flattened stage and the phagocytic capture stage are rapid, each being completed within 100 s. Knowing how these rapid cell shape changes occur in neutrophils is thus fundamental to understanding neutrophil behaviour. This article will discuss advances in our current knowledge of this process, and also identify an important regulated molecular event which may represent an important target for anti-inflammatory therapy.

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.

It has been proposed that Ca2+ activation of calpain-1 is important for the rapid cell shape changes which accompany phagocytosis. In this paper, we use a fluorogenic calpain substrate, (CBZ-Ala Ala)2 R110, and find that there was a low calpain activity measureable in resting (ie without intentional activation) neutrophils, but that it was accelerated by an elevation of cytosolic free Ca2+ (ionomycin -induced) and inhibited by calpeptin (an established calpain-1 inhibitor). The fluorescence signal was sufficiently bright for detection in individual neutrophils that enabled the quantification of dynamic changes in calpain activity to be related to elevations in cytosolic Ca2+ within individual neutrophils. It was found that during phagocytosis of C3bi-opsonised zymosan particles, calpain activity was elevated incrementally, each step increase corresponding to the phagocytosis of an individual particle. The sub-cellular source of the fluorescent product of calpain activity was the phagocytic site itself and originated at the phagocytic cup. It was thus concluded that calpain was activated locally during the formation of the phagocytic cup. These data were consistent with central role of Ca2+ activated calpain activation in controlling phagocytosis.

The interest in the signal transduction mechanisms of the neutrophil stems from a desire to understand not only general cell biology, but also the specific role of these mechanisms in pathological processes. As Metchnikoff had clearly demonstrated, the physiological function of these cells is the prevention of pathology by combatting infection. If probabilistic phenomena underlie critical steps in the activation of neutrophils, the possibility exists that as with the neuron, the coupling of stimulation to response may be unreliable. There are features of stimulus-response coupling in the neutrophil that may have pathogenic significance. There is growing evidence that neutrophils can unleash their destructive power not only against the infecting microorganism but also against the surrounding extracellular matrix and neighboring cells. One important difference between the neutrophil and the muscle cell is in the type of cellular responses that are triggered. All the cell types belonging to the neutrophil family group can produce number of cellular responses, which are often coordinated.

A synthesis and collation of the recent material regarding the role of the neutrophil in basic pathological processes is presented in this volume. The mechanisms of chemotaxis, secretion, phagocytosis, intracellular killing, oxygen radical production and arachidonate production are comprehensively reviewed. Stimulus response coupling in the neutrophil, with chapters on intracellular Ca2+, C-Kinase, phospholipid metabolism, microfilaments and membrane electrophysiology are extensively discussed. Each chapter provides a critical review by experts with over 1,000 cited references. Invaluable to graduate students and medical and scientific researchers, this book provides a unique, up-to-date account of cellular biochemistry and physiology of the neutrophil.