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 Ca2+ signalling in inflammatory neutrophils: implications for rheumatoid arthritis (Review).

Recognition of the ways in which neutrophil behaviour is regulated may be crucial for a full understanding of their role in inflammation and in rheumatoid arthritis. Although it is well established that changes in cytosolic free Ca2+ play a central role in triggering neutrophil responses, only recently has evidence accumulated which points strongly to the existence of two distinct Ca2+ pathways in neutrophils. One pathway is mediated by conventional agonists, such as formylated peptides, IL-8, C5a and PAF, and the other by cross-linking and immobilisation of surface receptors, such as integrins, and the Fc receptors, CD32 and CD16. In this review, we give evidence for these two signalling pathways in neutrophils, highlighting the roles of two Ca2+ storage and release organelles, one centrally located and stationary, and the other peripheral and mobile. We point out the significance of these two routes of Ca2+ signalling for the correct sequence of neutrophil responses, and suggest that aberration of this sequence could result in pathogenic neutrophil activation.

High micromolar Ca2+ beneath the plasma membrane in stimulated neutrophils.

Ca2+ near the inner face of the plasma membrane, as reported by the membrane associated fluorescent Ca2+ probe FFP-18, was higher than the bulk cytosolic free Ca2+ concentration both in resting neutrophils and in response to f-met-leu-phe. Influx caused Ca2+ close to the plasma membrane to rise more rapidly than the bulk cytosolic free Ca2+ and to reach a peak concentration of at least 30 microM. This zone of high Ca2+ was localised to just beneath the plasma membrane and did not extend more than 0.1 micron into the cell, as it was undetected by the bulk cytosolic free Ca2+ probes magfura2 and fura2. From these data, reconstruction of the distribution of Ca2+ within the neutrophil showed that the high Ca2+ signal at the cell cortex rapidly subsided to give a uniform free Ca2+ across the cell.

Spatial and temporal separation of calcium signals in myeloid cells stimulated by immune complexes.

Stimulation of large (100 microns) human myeloid cells with immune complexes resulted in Ca2+ spiking. Both global and regional changes in the intracellular cytosolic free Ca2+ concentration were detected in response to immune complex stimulation. The regional changes were mediated by release of Ca2+ from stores, whereas global changes were mediated by Ca2+ influx. They occurred independently of each other, with release of Ca2+ from intracellular Ca2+ stores being separated from transmembrane influx of Ca2+. Bromophenacyl bromide, an 1-plastin binding agent, inhibited store release without preventing transmembrane influx of Ca2+. The large size of the myeloid cells used here allowed the visualisation of the spatial and temporal separation of store release from transmembrane influx of Ca2+, providing further evidence for the existence of independent Ca2+ store release and Ca2+ influx mechanisms in these cells.

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.

Use of fluorescent dyes for measurement and localization of organelles associated with Ca2+ store release in human neutrophils.

Fura-2 and its lipid analogue, FFP-18, were used to measure changes in cytosolic free Ca2+ concentration within human neutrophils. Whereas fura-2 was employed to monitor cytosolic Ca2+ increases throughout the cytosol, FFP-18 was used to monitor Ca2+ changes only near the membrane. This latter probe was incorporated into the plasma membrane as its acetoxymethyl ester (FFP-18-AM) but as de-esterification was catalysed by cytosolic esterases, the Ca(2+)-sensing probe (FFP-18 acid) accumulated on the inner face of membrane. The fluorescence of esterified probe on the extracellularly facing membrane leaflet was quenched by the membrane-impermeant ion Ni2+. Under these conditions, near membrane Ca2+ changes which resulted from the release of Ca2+ from intracellular stores was possible by conventional ratio fluorescence measurement of FFP-18. From the timing of arrival of Ca2+ at the plasma membrane, it was proposed that there were two Ca2+ storage sites, liberated by different stimuli, one close to the plasma membrane and the other more distant. In order to discover whether organelles within the neutrophil had distributions which correlate with the Ca2+ release sites, fluorescent dyes for structures within the cytosol were employed. We have previously shown that the location of the intracellular membrane stain, DiOC6 (3) corresponds to the distant Ca2+ release site. Here a second stain, BODIPY-C5 ceramide, has also been used and is shown to stain a peripheral region of the neutrophil, in a similar pattern to the near membrane Ca2+ storage site. These data therefore raise the question of whether these stains mark the organelles in neutrophils which are the two Ca2+ storage and release sites.

Tyrosine phosphorylation in neutrophils from synovial fluid of patients with rheumatoid arthritis.

The priming of neutrophils is associated with an increase in the level of tyrosine phosphorylation of cytosolic proteins, specifically of proteins with mol. wts of 42 and 74 kDa. We show here, using dot blots and Western blotting, that neutrophils isolated from rheumatoid synovial fluid have increased tyrosine phosphorylation of these target proteins. The level of tyrosine phosphorylation within neutrophils in the synovial fluid was increased when compared with neutrophils from the blood of the same patients, normal blood or neutrophils from the synovial fluid of patients without rheumatoid arthritis. Neutrophils from the rheumatoid synovial fluid were also more active and were unable to be further primed by exogenous primers. These data suggest that this elevation of tyrosine phosphorylation was the result of the action of local priming agents within the rheumatoid synovial fluid.

Near membrane Ca2+ changes resulting from store release in neutrophils: detection by FFP-18.

FFP-18 was incorporated into the inner face of the plasma membrane of human neutrophils by incubation with its acetoxymethyl ester. Conversion to the Ca2+ sensitive intracellular indicator was monitored by the change in excitation spectra. The fluorescence from extracellularly facing FFP-18 was quenched by the membrane impermeant ion Ni2+. Ratio fluorescence measurement of FFP-18 under these conditions permitted the detection of near membrane Ca2+ changes resulting from the release of Ca2+ from intracellular stores. Near membrane and cytosolic Ca2+ changes were measured under conditions in which store release and Ca2+ influx were triggered by FMLP, thapsigargin or immune complexes. There were significant differences in the timing and magnitude of Ca2+ changes near the plasma membrane and bulk cytosolic Ca2+ concentration, which were consistent with a Ca2+ storage site deep within the neutrophil released by thapsigargin and FMLP, but Ca2+ release sites with immune complex stimulation being close to the membrane. The use of FFP-18 to monitor Ca2+ near the inner face of the plasma membrane thus provides evidence for the existence of two distinct Ca2+ storage locations in neutrophils.

A novel pathway for Ca2+ signalling in neutrophils by immune complexes.

The data presented here demonstrates that immune complexes use novel routes for stimulating a two-phase rise in cytosolic-free Ca2+ concentration. The initial transient Ca2+ rise resulted from release of Ca2+ from intracellular stores, by a route which, unlike f-met-leu-phe, was inhibited by bromophenacyl bromide. The second phase resulted from transmembrane influx and occurred in the absence of store release and by Ca2+ channels that were inhibited by Ni2+ but not SKF 96365 or econazole. Stimulation by immune complexes therefore involves novel routes for both the release of stored Ca2+ and the opening of Ca2+ channels in the plasma membrane.

Slow Ca2+ waves in large myeloid cells as a result of a diffusible cytosolic factor.

In the work reported here evidence is provided that shows the slow wave of Ca2+ large neonatal myeloid cells provoked by formyl-Met-Leu-Phe was generated by spatially delayed Ca2+ influx. Evidence is provided that the delay in Ca2+ influx was the result of diffusion of a factor from the Ca2+ storage site, which is responsible for Ca2+ channel opening. The location of the Ca2+ release site was correlated with a region near the nucleus, probably a specialized region of endoplasmic reticulum. It is proposed that similar mechanisms of Ca2+ signaling occur in mature myeloid cells, such as neutrophils, but on a shorter time scale as a consequence of their smaller size.

Complement component C9-dependent cytosolic free Ca2+ rise and recovery in neutrophils.

The cytosolic free Ca2+ concentration in rat neutrophils was determined by ratiometric fluorometry and imaging of Fura-2. Transient elevations of cytosolic free Ca2+ concentration were provoked by addition of C9 to neutrophils pre-coated with C5b-8. The rate of rise of the cytosolic free Ca2+ concentration was dependent upon the concentration of C9. These changes in cytosolic free Ca2+ concentration occurred in the absence of cell lysis, since there was no release of Fura-2, and were the result of increased permeability to extracellular Ca2+. More than 96% of the rise in cytosolic free Ca2+ generation by C9 was dependent upon the presence of extracellular Ca2+, but did not occur via channels which were inhibited by the Ca2+ channel blocker SKF96365. The decrease in the permeability of the membrane to Ca2+ after C9 was not triggered by the rise in cytosolic free Ca2+. After attack by C9, individual neutrophils remained responsive to f-met-leu-phe, or further attack by C9, both producing Ca2+ transients. The recovery of the Ca2+ signal was consistent with the complement membrane attack complex generating a series of permeability thresholds in the plasma membrane. These data have implications for the understanding of the mechanisms underlying the inappropriate responsiveness of neutrophils at inflammatory sites.

A soluble cellular factor directly stimulates Ca2+ entry in neutrophils.

A soluble factor, extracted from neutrophils and P388D1 cells, stimulated a transient rise in cytosolic free Ca2+ and a small increase in the permeability to Mn2+ in fura2-loaded neutrophils. These effects were not prevented by blockade of formylated peptide receptors by t-boc-met-leu-phe. The rise in cytosolic free Ca2+ was partly attributed to transmembrane influx and partly due to store release. Ca2+ store release but not transmembrane influx was inhibited by the PLC inhibitor, U73122, demonstrating a direct effect of the factor on channel opening. It was concluded that the soluble cellular factor directly stimulated Ca2+ entry in neutrophils.

Altered Ca2+ signalling in human neutrophils from inflammatory sites.

OBJECTIVES: To determine whether the intracellular store release of Ca2+ in neutrophils from patients with rheumatoid arthritis, other joint disease and active leg ulceration was different from normal neutrophils. METHODS: The release into the cytosol of Ca2+ from stores within individual neutrophils was determined using ratiometric imaging of fura2. The size of the elevated Ca2+ 'cloud' and its concentration were quantified in neutrophils from the circulation of patients with rheumatoid arthritis, other joint diseases, and leg ulcers and from the joints of those with joint disease. RESULTS: In neutrophils isolated from both the synovial fluid of patients with rheumatoid arthritis and other joint conditions, and also arising from leg ulcers, the amount of the cell cytosol occupied by elevated Ca2+ was significantly increased compared with neutrophils from healthy subjects; for neutrophils from rheumatoid, non-rheumatoid joints and leg ulcers p values were 0.0006, < 0.0001, 0.016 respectively (Student's t test). There was also a significant increase in Ca2+ release from circulating neutrophils from patients with rheumatoid arthritis (p = 0.09), but not in circulating neutrophils from patients with leg ulcers or non-rheumatoid joint conditions. CONCLUSIONS: It is proposed that the increased release of free Ca2+ into the cytosol of neutrophil at inflammatory sites results in increased oxidase activation.

Inappropriate neutrophil activation in venous disease.

Neutrophil oxygen radical production was studied in 18 limbs with class 2 or 3 venous disease and compared with that of nine normal limbs. Neutrophils were isolated from arm and leg venous samples. Free radical production was determined using chemiluminescence after stimulation with the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (FMLP) or the ester phorbol myristate acetate (PMA). The ratio of leg to arm luminescence was greater after FMLP stimulation in patients with venous disease (median 1.52 (95 per cent confidence interval (c.i.) 1.27-2.60)) than in controls (median 0.97 (95 per cent c.i. 0.70-1.12); P < 0.01). These changes were not observed with PMA (venous disease 1.16 (95 per cent c.i. 1.05-1.40); controls 0.95 (95 per cent c.i. 0.78-1.24)). There were fewer FMLP receptors on activated leg neutrophils (median 20.19 (95 per cent c.i. 3.58-51.42) fluorescence units) than arm neutrophils (median 36.03 (95 per cent c.i. 13.00-65.28) fluorescence units; P < 0.05), indicating an amplification of signal transduction. Intracellular calcium imaging demonstrated a larger release of calcium after stimulation of leg neutrophils (median 25.0 per cent (95 per cent c.i. 15.7-43.9 per cent)) compared with neutrophils from the arm (median 8.0 per cent (95 per cent c.i. 5.6-16.1 per cent); P = 0.04), demonstrating calcium-dependent activation. Neutrophils in patients with chronic venous disease inappropriately produce more oxygen free radical as a result of amplification of a calcium-dependent signal pathway.

Ca2+ oscillations in neutrophils triggered by immune complexes result from Ca2+ influx.

Although the mechanisms of Ca2+ signalling in neutrophils by certain chemotactic agents have been well characterized, the signalling by immune complexes is poorly understood. Here we demonstrate that immune complex stimulation, acting via Fc receptors, leads to repetitive Ca2+ spiking in neutrophils. Although the initial Ca2+ rise was the result of release of Ca2+ from intracellular stores, subsequent repetitive Ca2+ spikes resulted from transmembrane influx, as they were prevented by removal of extracellular Ca2+ and were accompanied by Mn2+ influx. The transmembrane Ca2+ spikes induced dramatic neutrophil cell shape changes. The Ca2+ spiking phase was inhibited by a phospholipase C (PLC) inhibitor, U73122, and removal of immune complex, but not by cytochalasin B. It was concluded that Ca2+ spiking was dependent upon the initial release of Ca2+ from an intracellular Ca2+ store, and driven by continued binding of immune complex, which triggered pulsatile changes in transmembrane influx.

Slow calcium waves imaged in myeloid cells derived from neonatal cord blood.

Myeloid cells were derived from neonatal cord blood by culture with granulocyte-macrophage colony-stimulating factor for approximately 8 days. The resultant cell population contained large adherent cells (diameter > or = microns), expressing formylated peptide receptors that were functionally coupled to cytosolic free Ca2+ signaling. Imaging of the cytosolic free Ca2+ changes in these cells revealed initial focal release of Ca2+ from a site from within the cell, with elevated Ca2+ also near the cell edge. Increased cytosolic free Ca2+ moved as a slow oscillating wave across the cell (velocity 1 microns/s). As similar events may occur in mature neutrophils and monocytes but be difficult to resolve because of the small size of these cells, it was concluded that neonatal myeloid cells may provide a useful model system for the investigation of Ca2+ signaling in myeloid cells.