Nonuniform distribution of Ca(2+) uptake sites within human neutrophils.

The rate at which Ca(2+) returns towards the basal concentration is controlled by the action of Ca(2+) pumps, both on the plasma membrane and on organelles within the cytosol. The distribution of Ca(2+) uptake sites within the cytosol was investigated using rapid confocal imaging (55 ms/frame) of fluo3-loaded human neutrophils. In some zones within the cell, the uptake of Ca(2+) from the cytosol followed a single exponential time course, whereas in others, there was accelerated kinetics after about 3 s. Using the full array of data, to produce a cell-map of Ca(2+) uptake rates a clear nonuniformity of Ca(2+) uptake sites throughout the neutrophil cytosol was observed. The location of the Ca(2+) uptake sites did not correlate with the granules or the main body of the nucleus, but Ca(2+) uptake was highest near the vestigial Golgi/ER, the edges of the nuclear lobes and at the leading cell edge. The possibility exists that the nonuniform distribution of Ca(2+) uptake sites plays a role in restricting Ca(2+) signals with the neutrophil cytosol.

Techniques for measuring and manipulating free Ca2+ in the cytosol and organelles of neutrophils.

Ca(2+) signalling in neutrophils is important for triggering and coordinating the behaviour of neutrophils. Fluorescent probes for cytosolic free Ca(2+) concentration, e.g., fura2 and fluo3, have been widely used in neutrophils. These probes can be used to monitor Ca(2+) in the cytosol, the nucleus, near the plasma membrane and theoretically within Ca(2+) storage organelles. The longer wavelength indicators, e.g., fluo3 and calcium green, can be used confocally to monitor subcellular Ca(2+) changes in the cytosol of neutrophils and in the nucleus. Confocal techniques also permit "impossible views" imaging of Ca(2+) and newer scanning techniques promise very fast temporal resolution. Techniques using chlortetracycline (CTC) and DiOC(6)(3) are also described for monitoring the position of Ca(2+) storage sites in neutrophils and for manipulating their activity. Thus, in this review, a spectrum of new (and older) optical techniques are presented which are useful for measuring, monitoring and manipulating cytosolic free Ca(2+) concentration and Ca(2+) storage in neutrophils. With these techniques, it is hoped that more insight will be gained into both the mechanism of and the consequences of Ca(2+) signalling in neutrophils.

Cytosolic free calcium and the cytoskeleton in the control of leukocyte chemotaxis.

In response to a chemotactic gradient, leukocytes extravasate and chemotax toward the site of pathogen invasion. Although fundamental in the control of many leukocyte functions, the role of cytosolic free Ca2+ in chemotaxis is unclear and has been the subject of debate. Before becoming motile, the cell assumes a polarized morphology, as a result of modulation of the cytoskeleton by G protein and kinase activation. This morphology may be reinforced during chemotaxis by the intracellular redistribution of Ca2+ stores, cytoskeletal constituents, and chemoattractant receptors. Restricted subcellular distributions of signaling molecules, such as Ca2+, Ca2+/calmodulin, diacylglycerol, and protein kinase C, may also play a role in some types of leukocyte. Chemotaxis is an essential function of most cells at some stage during their development, and a deeper understanding of the molecular signaling and structural components involved will enable rational design of therapeutic strategies in a wide variety of diseases.

Release of 'caged' cytosolic Ca2+ triggers rapid spreading of human neutrophils adherent via integrin engagement.

The role of the transient rise in cytosolic free Ca2+ which occurs during neutrophil adhesion and cell spreading is unclear. In order to establish whether such a Ca2+ signal triggers neutrophil shape change, neutrophils co-loaded with fluo3 and Nitr5 ('caged' Ca2+) were used with rapid-time confocal laser scanning microscopy. Here we show that the photolytic generation of a Ca2+ rise in neutrophils which were adherent to an integrin-engaging surface, triggered a rapid change in cell morphology, with increases in cell diameter of approximately 175% occurring within 90 seconds of the Ca2+ signal. In non-adhered neutrophils or neutrophils on plain glass, no acceleration of the rate of spreading occurring in response to the release of 'caged Ca2+' could be demonstrated. It was concluded that although a rise in cytosolic free Ca2+ was not the sole trigger for neutrophil shape change, with other signals generated by integrin engagement, a rise in cytosolic free Ca2+ accelerated the rate of neutrophil spreading.

Ca2+ signalling delays in neutrophils: effects of prior exposure to platelet activating factor or formyl-met-leu-phe.

Rapid-time confocal scanning of fluo3-loaded neutrophils revealed that in individual cells there were grossly heterogeneous time intervals between stimulation with either f-met-leu-phe or platelet activating factor (PAF) and the initiation of Ca2+ influx, ranging from 75 msec to several seconds. The distribution of lag times after stimulation with f-met-leu-phe (100 nM) was influenced by prior stimulation with either f-met-leu-phe or PAF. However, whereas prior stimulation with f-met-leu-phe (50 nM) caused the subsequent cytosolic free Ca2+ response to second challenge with f-met-leu-phe to be delayed, prior stimulation with PAF (100 nM) caused an increase in the rapidity of the onset of the second response to f-met-leu-phe. With both stimuli, the cytosolic free Ca2+ in some neutrophils (non-Ca2+ responders) in the population did not increase significantly. However, some of these cells responded to the subsequent challenge. However, with both pre-treatment stimuli, those cells in which a significant Ca2+ response was provoked by the first stimulus, responded significantly faster than the initial 'non-Ca2+ responders.' However, the reduced lag time provoked by pre-stimulation was not inhibited in neutrophils in which cytosolic free Ca2+ changes were dampened by intracellular BAPTA. These data point to post-receptor events, other than prior cytosolic free Ca2+ elevation, being important in determining the response Ca2+ delay to subsequent stimulation.

Two distinct Ca2+ storage and release sites in human neutrophils.

It is well established that changes in cytosolic free Ca2+ play a role in a number of neutrophil responses such as cell shape change and oxidase activation. The release of intracellular stored Ca2+ occurs with stimuli that either act by occupancy of seven membrane-spanning domain receptors or those which act by receptor cross-linking. Here, two distinct Ca2+ storage and release sites have been identified in neutrophils. Using chlortetracycline fluorescence as an indicator of Ca2+ storage, two separate Ca2+ storage sites have been identified. One site was located peripherally under the plasma membrane and the other was in the juxtanuclear space. Confocal imaging of Fluo3-loaded neutrophils demonstrated that the central Ca2+ storage site released Ca2+ in response to N-formyl-methionyl-leucyl-phenylalanine, whereas engagement and clustering of CD11b/CD18 integrins causes Ca2+ release from the peripheral stores. The release sites also correlated with organelles that stained with DiOC6(3). Localized phototoxicity generated by DiOC6(3) excitation resulted in inhibition of the release of stored Ca2+, which was selective for the stimulus used. The presence of two distinct cellular locations for these Ca2+ stores and their independent release raises the possibility that separate intracellular messengers for their release are generated.

Does actin polymerization status modulate Ca2+ storage in human neutrophils? Release and coalescence of Ca2+ stores by cytochalasins.

The aim of this paper was to establish whether actin polymerization modulated cytosolic Ca2+ storage in human neutrophils. Over the concentration ranges which inhibit actin polymerization, cytochalasins A, B, and D liberated Ca2+ from membrane-bound stores within neutrophils. Two Ca2+ storage sites were identified in neutrophils by the accumulation of the Ca2+ binding probe, chlortetracycline: one at the center of the cell and the other at the cell periphery. Confocal imaging demonstrated that cytochalasins released Ca2+ from the neutrophil periphery, but not from the central Ca2+ store. Ca2+ store release was coupled to Ca2+ influx, suggesting that the peripheral site may be a physiological store containing a Ca2+ influx factor. 3,3'-Dihexyloxacarbocyanine iodide staining organelles, which correlate with Ca2+ release sites, coalesced in neutrophils after treatment with cytochalasins. We propose that peripheral Ca2+ storage sites are restricted from coalescence by cortical polymerized actin and that Ca2+ store coalescence and Ca2+ release are coupled events.

Stochastic events underlie Ca2+ signalling in neutrophils.

In order to probe the events which couple receptor occupancy to elevation of cytosolic free Ca2+ fast laser scanning of fluo3-loaded neutrophils was used to determine the timing of the initial phase of the Ca2+ response. This approach demonstrated that there was a measurable delay between the addition of stimulus and the onset of the cytosolic free Ca2+ signal which varied from cell to cell variable from 75 ms to greater than 1.5 s. The distribution of lag times was similar to that expected if the delay resulted from a series of obligatory stochastic processes. From the Poisson equation, a probability density function for the delays was generated which depended on n, the number of stochastic events in the series, and lambda, the stochastic rate for the events. The best fit between the data and theory was found to be for six stochastic steps in the series occurring with a stochastic rate similar to that expected for diffusion of known small molecular weight signalling molecules within the cell. We, therefore, propose that the delays in onset of the Ca2+ response in neutrophils were the result of sequence of diffusion steps, each with sufficiently small numbers of intracellular messenger molecules to generate stochastic behaviour.

The microanatomy of calcium stores in human neutrophils: relationship of structure to function.

As changes in cytosolic free Ca2+ play key roles in coupling responses in neutrophils, it is important to locate and identify Ca2+ storage sites within these cells. Here, recent data is presented which highlights the functional link between microanatomical structure and cell signalling function. Fluorescent optical probes for cytosolic free Ca2+ have been used, together with organelle specific markers. We present evidence from conventional fluorescence microscopy, together with ratiometric and confocal laser scanning fluorescence microscopy, which pin-points two cellular locations for Ca2+ within the neutrophil; one within the nuclear lobes, and the other towards the cell periphery. Knowledge of these two locations provides a clear insight into how signalling in this cell type is regulated and provides a framework for explaining how specific stimuli act to produce specific responses.

The timing and magnitude of Ca2+ signaling by CD32 depends on its redistribution on the cell surface.

Ca2+ signaling was correlated with microaggregation and capping of CD32 molecules on the myeloid cell line, U937. The cytosolic free Ca2+ signal was related to the extent of CD32 cross-linking and arose asymmetrically within individual cells. Both the magnitude and the delay before Ca2+ signaling via CD32 on U937 cells was dependent on the extent of CD32 cross-linking. The delay time was extended in cells in which lateral diffusion in the membrane was reduced by covalently cross-linking of surface proteins. Under these conditions, capping but not surface microaggregation of CD32 molecules was prevented. The delay time before Ca2+ signaling but not the magnitude was also affected. At a higher density of covalent cross-linking of surface proteins, the magnitude of the Ca2+ signal by CD32 was also reduced and could be completely inhibited. This evidence therefore shows that the formation of a CD32 "cap" was not required for Ca2+ signaling by this route. However, the signaling delay time was a consequence of lateral diffusion of CD32 molecules in the membrane to form signaling-competent microaggregates, and the redistribution of CD32 molecules on the cell surface was required for Ca2+ signal generation.

Pulsatile Ca2+ influx in human neutrophils undergoing CD11b/CD18 integrin engagement.

Rapid-time confocal scanning has demonstrated that neutrophils undergoing CDllb/CD18 integrin-dependent adhesion show repeated elevations in cytosolic free Ca2+ concentration, due to Ca2+ influx. The magnitude of each individual influx, compared to the previous one, depended upon the time between the two, and not upon the cytosolic Ca2+ concentration at the start of the influx. Influx events occurring less than 100 seconds after the previous Ca2+ rise were observed not to reach the concentration of the preceding peak, whilst events occurring later usually exceeded the magnitude of the previous Ca2+ concentration. This suggested temporary inactivation, followed by recovery, of the Ca2+ influx mechanism. In addition, Ca2+ influx was most likely to occur immediately following this recovery. The involvement of Ca2+ store release at the site of integrin engagement suggested that the cytoskeletal connection between the peripheral store and membrane was facilitating the signalling of Ca2+ influx.

Temporal and spatial resolution of Ca2+ release and influx in human neutrophils using a novel confocal laser scanning mode.

Confocal laser scanning demonstrated that stimulation of neutrophils with the surface receptor agonist, f-met-leu-phe, resulted in the release of stored Ca2+ from a single site. From reconstructions of neutrophils stained with acridine orange, it was shown that the central Ca2+ release site was always close to the nucleus, and correlated with a site which stained with DiOC(6)3. Elevated Ca2+ was locally restricted to within 1 micron of this site. Release of Ca2+ by this pathway was accompanied by the influx of Ca2+, less than 1 second after store release. In each neutrophil, the Ca2+ store release component preceded the Ca2+ influx, and their spatial separation suggested communication between the Ca2+ store and the plasma membrane by a messenger. The (distance)2 of the release site from the plasma membrane was correlated to the time between Ca2+ release and influx, consistent with the diffusion of a messenger from the storage site signalling Ca2+ influx.

Localised and global cytosolic Ca2+ changes in neutrophils during engagement of Cd11b/CD18 integrin visualised using confocal laser scanning reconstruction.

A confocal laser scanning technique was used to provide optical sections in the vertical plane through living neutrophils during engagement of integrin and shape change. This has permitted the visualisation of cytosolic free Ca2+ rises localised to the sites of integrin immobilisation. These localised Ca2+ changes resulted from the release of Ca2+ from intracellular stores, and were not inhibited by removal of extracellular Ca2+ or by the Ca2+ channel blocking nickel ions. After integrin engagement and initial cell shape changes, a second rapid phase of cell spreading occurred, which was accompanied by global Ca2+ signalling bursts that continued sporadically after maximum spreading. This global signalling was driven mainly by Ca2+ influx from the extracellular medium. We propose that the localised and global Ca2+ signalling triggered by integrin engagement results from a common underlying mechanism and these signals are important for neutrophil shape change and extravasation.

Early Ca2+ signalling events in neutrophils detected by rapid confocal laser scanning.

Characterization of early Ca2+ signalling events is crucial for understanding the mechanisms which lead to cell signalling. Rapid confocal laser scanning of Fluo3-loaded neutrophils was used to provide spatially resolved cytosolic free Ca2+ measurements from neutrophils stimulated with formyl-Met-Leu-Phe with a time resolution of 12.5 ms. Heterogeneity of the magnitude and timing of the response was observed between individual neutrophils. There was always a delay between contact with the stimulus and onset of the Ca2+ signal, with a minimum delay of 75 ms and a mean delay of 530 ms (n = 150). There was no delay in the cytosolic free Ca2+ rise induced by ionomycin. The earliest Ca2+ event detected following stimulation with formyl-Met-Leu-Phe (1 microM) was a localized rise in cytosolic free Ca2+ from a location within the cell, pointing to Ca2+ store release as the initial event for triggering whole-cell Ca2+ changes in these cells.