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.

Localisation of Intracellular Signals and Responses during Phagocytosis.

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.

Ca2+-activated cleavage of ezrin visualised dynamically in living myeloid cells during cell surface area expansion.

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.

An Introduction to Phagocytosis.

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.

A Brief History of Phagocytosis.

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.

Conclusions and the Futures of Phagocytosis.

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.

Calpain Activation by Ca2+ and Its Role in 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.

Membrane Tension and the Role of Ezrin During Phagocytosis.

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.

Single cell measurement of calpain activity in neutrophils reveals link to cytosolic Ca2+ elevation and individual phagocytotic events.

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.

Neutrophil Cell Shape Change: Mechanism and Signalling during 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.

Defective rapid cell shape and transendothelial migration by calpain-1 null neutrophils.

It has been proposed that Ca2+ activation of calpain-1 is an important trigger for rapid cell spreading by neutrophils. In this paper, we have investigated this by assessing the ex vivo functioning of neutrophils from calpain-1 null mice, Calpain-1 null neutrophils failed to migrate through TNF-activated endothelial monolayers. The failure to transmigrate through endothelial monolayers was therefore unlikely to be due to a failure of chemotaxis as chemotaxis by adherent calpain-1 null neutrophils towards fMLP was unpaired. In contrast, the capacity of calpian-1 neutrophils to spontaneously spread was limited to smaller diameters than for wild type cells. Photolytic uncaging of IP3 with Individual wild type neutrophils resulted in a large Ca2+ signal and rapid cell spreading. In contrast, calpain-1 neutrophils failed to spread in response to the IP3-induced Ca2+ signal. This work has therefore demonstrated that the presence of calpain-1 was required for effective rapid cell spreading by neutrophils.

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.

Ca²âº and calpain control membrane expansion during the rapid cell spreading of neutrophils.

Following adherence of neutrophils to the endothelium, neutrophils undergo a major morphological change that is a necessary prelude to their extravasation. We show here that this shape change is triggered by an elevation of cytosolic inositol (1,4,5)-trisphosphate (IP3), to provoke physiological Ca(2+) influx through a store-operated mechanism. This transition from a spherical to 'flattened' neutrophil morphology is rapid (∼100 seconds) and is accompanied by an apparent rapid expansion of the area of the plasma membrane. However, no new membrane is added into the plasma membrane. Pharmacological inhibition of calpain-activation, which is triggered by Ca(2+) influx during neutrophil spreading, prevents normal cell flattening. In calpain-suppressed cells, an aberrant form of cell spreading can occur where an uncoordinated and localised expansion of the plasma membrane is evident. These data show that rapid neutrophil spreading is triggered by Ca(2+) influx, which causes activation of calpain and release of furled plasma membrane to allow its apparent 'expansion'.

Active calpain in phagocytically competent human neutrophils: electroinjection of fluorogenic calpain substrate.

Calpain has been implicated in the apparent expansion of neutrophil plasma membrane that accompanies cell spreading and phagocytosis. In order to test this hypothesis, an internally quenched fluorescent peptide substrate of calpain-1 which increased in fluorescence on cleavage, was micro-electroinjected into neutrophils. The fluorescence intensity increased in a significant number of neutrophils, including those which appeared to be in a morphologically resting (spherical) state. In order to test whether calpain was activated by an elevation of cytosolic Ca(2+) during the injection, Ca(2+) chelators were added to the injectate and cytosolic free Ca(2+) in the receiving neutrophil was simultaneously monitored. It was shown that this approach could be used without raising Ca(2+) within the injected cell. Despite this, approximately 75% of individual neutrophils had calpain activity which consumed the substrate within approx. 100 s. It was found that all neutrophils had elevated calpain activity were phagocytically competent; whereas neutrophils with low or undetectable calpain activity failed to undergo phagocytosis. This association was consistent with the hypothesis that calpain activity within neutrophils was necessary for them to undergo efficient phagocytosis.

The structural basis of differential inhibition of human calpain by indole and phenyl α-mercaptoacrylic acids.

Excessive activity of neutrophils has been linked to many pathological conditions, including rheumatoid arthritis, cancer and Alzheimer's disease. Calpain-I is a Ca(2+)-dependent protease that plays a key role in the extravasation of neutrophils from the blood stream prior to causing damage within affected tissues. Inhibition of calpain-I with small molecule mercaptoacrylic acid derivatives slows the cell spreading process of live neutrophils and so these compounds represent promising drug leads. Here we present the 2.05 and 2.03 Å co-crystal X-ray structures of the pentaEF hand region, PEF(S), from human calpain with (Z)-3-(4-chlorophenyl)-2-mercaptoacrylic acid and (Z)-3-(5-bromoindol-3-yl)-2-mercaptoacrylic acid. In both structures, the α-mercaptoacrylic acid derivatives bind between two α-helices in a hydrophobic pocket that is also exploited by a leucine residue of the endogenous regulatory calpain inhibitor calpastatin. Hydrophobic interactions between the aromatic rings of both inhibitors and the aliphatic residues of the pocket are integral for tight binding. In the case of (Z)-3-(5-bromoindol-3-yl)-2-mercaptoacrylic acid, hydrogen bonds form between the mercaptoacrylic acid substituent lying outside the pocket and the protein and the carboxylate group is coplanar with the aromatic ring system. Multiple conformations of (Z)-3-(5-bromoindol-3-yl)-2-mercaptoacrylic acid were found within the pocket. The increased potency of (Z)-3-(5-bromoindol-3-yl)-2-mercaptoacrylic acid relative to (Z)-3-(4-chlorophenyl)-2-mercaptoacrylic acid may be a consequence of the indole group binding more deeply in the hydrophobic pocket of PEF(S) than the phenyl ring.

Transforming growth factor-ß1 (TGF-ß1)-stimulated fibroblast to myofibroblast differentiation is mediated by hyaluronan (HA)-facilitated epidermal growth factor receptor (EGFR) and CD44 co-localization in lipid rafts.

Fibroblast to myofibroblast differentiation drives effective wound healing and is largely regulated by the cytokine transforming growth factor-ß1 (TGF-ß1). Myofibroblasts express α-smooth muscle actin and are present in granulation tissue, where they are responsible for wound contraction. Our previous studies show that fibroblast differentiation in response to TGF-ß1 is dependent on and mediated by the linear polysaccharide hyaluronan (HA). Both the HA receptor, CD44, and the epidermal growth factor receptor (EGFR) are involved in this differentiation response. The aim of this study was to understand the mechanisms linking HA-, CD44-, and EGFR-regulated TGF-ß1-dependent differentiation. CD44 and EGFR co-localization within membrane-bound lipid rafts was necessary for differentiation, and this triggered downstream mitogen-activated protein kinase (MAPK/ERK) and Ca(2+)/calmodulin kinase II (CaMKII) activation. We also found that ERK phosphorylation was upstream of CaMKII phosphorylation, that ERK activation was necessary for CaMKII signaling, and that both kinases were essential for differentiation. In addition, HA synthase-2 (HAS2) siRNA attenuated both ERK and CaMKII signaling and sequestration of CD44 into lipid rafts, preventing differentiation. In summary, the data suggest that HAS2-dependent production of HA facilitates TGF-ß1-dependent fibroblast differentiation through promoting CD44 interaction with EGFR held within membrane-bound lipid rafts. This induces MAPK/ERK, followed by CaMKII activation, leading to differentiation. This pathway is synergistic with the classical TGF-ß1-dependent SMAD-signaling pathway and may provide a novel opportunity for intervention in wound healing.

Extracellular ATP induces spikes in cytosolic free Ca(2+) but not in NADPH oxidase activity 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+).

In vivo functional analysis and genetic modification of in vitro-derived mouse neutrophils.

Mature neutrophils are notoriously short-lived immune cells that cannot be genetically manipulated. Analysis of gene function therefore requires genetically modified animals, which is expensive, time-consuming, and costly in animal life. Analysis of gene function in neutrophils in a physiologically relevant context thus represents a significant problem in the field. We sought to overcome this obstruction in the field by developing a strategy for the analysis of gene function in neutrophils in a physiologically relevant context. Here, we demonstrate the functional relevance of in vitro conditional-Hoxb8 immortalized precursor-derived neutrophils. In vitro-derived neutrophils functionally resembled primary neutrophils, but critically, neutrophils generated in this way can be adoptively transferred into live animals and tracked during inflammatory responses using single-cell analysis to define functional attributes. We have validated this approach using CD11b-deficient neutrophils and replicated the key findings observed in gene-targeted animals and in naturally CD11b-deficient humans. Furthermore, we show that by retroviral transduction, one can generate stable alterations in the precursor cell lines and thus a continuous supply of functionally altered neutrophils. This novel technological advance offers for the first time the possibility of applying higher-throughput genetic modification and in vivo functional analysis to the neutrophil-lineage.