Neutrophils are phagocytic white blood cells which act as the first line of defence against entry of foreign microorganisms. Neutrophils are recruited to their target site through the process of spreading, extravasation and phagocytosis involving complex signal transduction within the cells, which might include the activation of the cytosolic Ca2+ activated protease, calpain-1. The work described here investigates the role of calpain-1 in regulating neutrophil functions such as spreading, trans-endothelial migration, chemotaxis, phagocytosis and Ca2+ signalling. Through the work done at European Mutant Mouse Archive (EMMA), Oxford, and by using intracellular sperm injection (ICSI) of calpain-1 deleted gene from mice generated in the USA, and with a selective genotype breeding programme, a colony of homozygous calpain-1 KO mouse has been generated in Cardiff. Homozygous calpain-1 KO neutrophils appeared to have a smaller surface spreading area and their recruitment into the peritoneal cavity of the mouse in vivo was disrupted. In vitro experiments showed significant defects in their ability to cross the ICAM-1 expressing endothelial cells in trans-endothelial migration assay. Disruption in this transmigration was only evident with ICAM-1 upregulated (TNF-treated) endothelial cells, suggesting a specific defect in the β2 integrin-ICAM-1 signalling process. Calpain-1 absence did not affect signal transduction as neutrophils were able to signal cytosolic Ca2+ in response to β2 integrin engagement (C3bi-opsonised zymosan) and also to release intracellular Ca2+ store upon IP3 uncaging. This showed that the IP3 pathway in the cells was not affected by knocking-out calpain-1 and continued to be functional. The key signalling mechanisms from β2 integrin also remained intact and this is consistent with calpain-1 activation by Ca2+ being an important event in trans-endothelial migration. In conclusion, calpain-1 absence has significantly affected the ability of neutrophils to undergo trans-endothelial migration and this effect is directed towards the event which happens downstream to the increase in cytosolic free Ca2+ concentration.

Our understanding of the dynamic chemical changes within living cells has increased enormously as a direct result of imaging and manipulating techniques which rely on the use of light to penetrate the cell. These optical techniques are at the intersection of the three sciences: physics, chemistry and biology. However, the understanding of the physics of illumination (e. g. lasers, confocal microscopy) and the chemistry of fluors (synthetic and protein) is way ahead of the understanding of the biological interface posed by the cell itself. In this critical review we will show that ignoring the optical properties of living cells can lead to serious errors of interpretation and that even seemingly compelling images can result from a ''trick of the light''.

The ability to manipulate the intracellular environment within living cells and to monitor the cytosolic chemical changes which occur during cell stimulation has lead to major advances in our understanding of how cells read and respond to their environment. Perhaps the most powerful suite of techniques for achieving these dual objectives is based on the use of light (photons). Because cells are ‘transparent’, light has been used to both interrogate and manipulate the chemistry inside living cells, exploiting technical advances in both the physical and biochemical sciences. However, cells are neither transparent nor homogeneous with respect to their optical properties. The interface between light and the living cell cytoplasm thus represent an important, yet largely ignored, interface. There has been no review of the optical properties of cytoplasm and little discussion about how the optical properties of living cytoplasm influence the outcome of such measurements and manipulations. In this short review, we discuss the importance of understanding the optical properties of cytoplasm for such techniques and how imperfections in experimental interpretation can arise.

Several events accompany integrin-mediated phagocytosis by myeloid cells. These include local pseudopod and phagocytic cup formation followed by Ca2+ signalling. However, there is also a role for localised phosphatidylinositol (3,4,5) trisphosphate [PtdIns(3,4,5)P3] production. Here we report that in neutrophilic HL-60 cells expressing PH-Akt-GFP, binding of iC3b-coated zymosan particles (2 μm in diameter) via β2 integrin induces an incomplete phagocytic cup to form before either PtdIns(3,4,5)P3 or phosphatidylinositol (3,4) bisphosphate [PtdIns(3,4)P2] production or Ca2+ signalling. These phosphoinositides then accumulated locally at the site of the phagocytic cup and Ca2+ 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 Ca2+ signalling and phagosome closure, but had no effect on particle binding or phagocytic cup formation. We therefore conclude that PtdIns(3,4,5)P3 or PtdIns(3,4)P2 generation was not required for the events that initiate the formation of the phagocytic cup, but that anchoring of PtdIns(3,4,5)P3 at the phagocytic cup is an essential step for phagosome closure and Ca2+ signalling.

Cystic fibrosis (CF) is characterized by a neutrophil-dominated chronic inflammation of the airways with persistent infections. In order to investigate whether neutrophils contribute to an inadequacy in the pulmonary defence mechanism, the phagocytic activity of pulmonary and peripheral blood neutrophils from CF and non-CF respiratory patients were compared. Neutrophils were isolated from both the blood and bronchoalveolar lavage fluid of 21 patients with CF (12 male, 9 female; mean age 7.5 years, range 0.25-16.4 years) and 17 non-CF subjects (9 male, 8 female; mean age 5.4 years, range 0.2-13.1 years). The ex vivo phagocytic rate of normal pulmonary neutrophils to internalize zymosan particles opsonized with iC3b was faster than that of circulating neutrophils (P < 0.05), but the maximum capacity (9 particles/cell) was similar. In contrast, pulmonary neutrophils from patients with CF had a lower phagocytic capacity than circulating neutrophils either from the same patients or from normal subjects. This deficiency could not be attributed to (i) the cell surface density of CR3 (CD18/CD11b) receptors, which were not significantly different between the other groups (ii) the signalling ability of the CR3 receptors, using cytosolic free Ca(2+) signalling as the receptor activity read-out or (iii) a decrease in cellular ATP concentration. As CFTR was not detectable on neutrophils from any source by either histochemistry or Western blotting, it was concluded that the reduced phagocytic capacity was not the direct result of a CFTR mutation, but was attributed to a failure of neutrophil phagocytic priming during translocation into the CF lung.

Exosomes are nanometer-sized vesicles secreted by various cells, with potentially diverse roles in physiology. Although emphasis has been placed on their involvement in immune modulation, their potential for more wide-ranging biological effects has not been appreciated. A common exosome feature is the expression of adhesion molecules, which include the integrin family. We have for the first time addressed the possible function of B cell-derived exosome-integrins by examining adhesive interactions of exosomes (immobilized onto beads) with extracellular matrix (ECM) components and cytokine-treated fibroblasts. Integrin (beta1 and beta2) expression was demonstrated by Western blotting and flow cytometry. Binding studies (with blocking antibodies) demonstrated their function in adhesion to collagen-I, fibronectin, and tumor necrosis factor (TNF)-alpha-activated fibroblasts. Exosome adhesion to TNF-alpha-activated fibroblasts also triggered integrin-dependent changes in cytosolic calcium, measured by single cell imaging. Thus, B cell-derived exosomes express functional integrins, which are capable of mediating anchorage to ECM and cell-surface adhesion molecules, and may be a novel mode of delivering adhesion signals at distances beyond that of direct cell-cell contact during inflammation.

In neutrophils, as in most other cell types, Ca2+ signalling is important for a number of cellular activities. Although inositol(1,4,5)trisphosphate-mediated release of Ca2+ from intracellular stores is a necessary prelude, it is the Ca2+ influx that is responsible for many of the neutrophil responses. We report here that although elevations of cytosolic Ca2+ do not accompany Fas-mediated apoptosis in neutrophils, the Ca2+ 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 37degrees, untreated neutrophils display an exaggerated Ca2+ influx phase in response to FMLP. This was absent in neutrophils that had been Fas-activated at the same time. No Ca2+ influx component was demonstrable by the removal of extracellular Ca2+ or by Ca2+ channel blockade with Ni2+ and no Mn2+ 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 Ca2+ remained constant during incubation and was unaffected by Fas activation. Ca2+ 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 Ca2+ 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.

Although there is accumulating evidence that the generation and localization of phosphatidylinositol‐3,4,5‐trisphosphate (PtdIns(3,4,5)P3) 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)P3 introduced into the inner leaflet of the neutrophil plasma membrane by a cationic carrier, we show that: first, PtdIns(3,4,5)P3 uniformly delivered to the neutrophil plasma membrane is excluded from newly forming pseudopodia; second, PtdIns(3,4,5)P3 translocates to and is immobilized at the pole opposite a stable polarizing pseudopod; third, asymmetric delivery of PtdIns(3,4,5)P3 to the neutrophil triggers the generation of polarizing pseudopodia at the opposite pole; and finally, PtdIns(3,4,5)P3 triggers repetitive Ca2+ signals, the onset of which precedes morphological polarization. These data suggest that translocation and immobilization of PtdIns(3,4,5)P3 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.

The temporal and spatial relationship between particle binding to the neutrophil by β2 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 β2 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.

Phagocytosis of microbes coated with opsonins such as the complement component C3bi is the key activity of neutrophils. However, the mechanism by which opsonins enhance the rate of phagocytosis by these cells is unknown and has been difficult to study, partly because of the problem of observing and quantifying the events associated with phagocytosis. In this study, C3bi-opsonized particles were presented to neutrophils with a micromanipulator, so that the events of binding, pseudopod cup formation, engulfment, and completion of phagocytosis were clearly defined and distinguished from those involved with chemotaxis. Using this approach in combination with simultaneous phase contrast and Ca2+ imaging, the temporal relationship between changes in cytosolic free Ca2+ concentration and phagocytosis were correlated. Here we show that whereas small, localized Ca2+ changes occur at the site of particle attachment and cup formation as a result of store release, rapid engulfment of the particle required a global change in cytosolic free Ca2+ which resulted from Ca2+ influx. This latter rise in cytosolic free Ca2+ concentration also liberated a fraction of β2 integrin receptors which were initially immobile on the neutrophil surface, as demonstrable by both fluorescence recovery after laser bleaching and by visualization of localized β2 integrin labelling. Inhibitors of calpain activation prevented both the Ca2+-induced liberation of β2 integrin and the rapid stage of phagocytosis, despite the persistence of the global Ca2+ signal. Therefore, we propose that Ca2+ activation of calpain causes β2 integrin liberation, and that this signal plays a key role in the acceleration of β2 integrin–mediated phagocytosis.