Cytosolic free Ca2+ concentration was measured and imaged in human neutrophils moving towards a source of formylated peptide in a micropipette held close to the cells. Under these conditions, neutrophils changed shape and displayed chemotaxis without significant or persistent global or localised elevations in cytosolic free Ca2+. A rearto-front persistent Ca2+ gradient of less than 0.5 nM/μm was present in the migrating neutrophils, until they reached the zone of higher peptide concentration, when an abrupt rise in cytosolic free Ca2+ concentration was triggered and chemotaxis stopped. Small localised rises in cytosolic free Ca2+, which were occasionally observed during neutrophil manoeuvring, were attributed to the effect of local deformation of the neutrophil membrane, since deformation of the membrane with a blunt micropipette caused similar Ca2+ changes. These data suggest that neutrophil chemotaxis towards a source of formylated peptide occurs without significant changes in Ca2+ signalling.

Neutrophils play an important part in combating infection by phagocytosing and killing infecting bacteria. These cells leave the blood stream and migrate to the site of infection towards a source of chemotactic agents, including formylated peptides released from bacteria (Schiffmann et al., 1975). The neutrophil formylated peptide receptor is a classic seven-transmembrane spanning G-protein linked receptor (Boulay et al., 1990; Murphy, 1994), occupancy of which can cause a major increase in cytosolic free Ca2+ concentration (Hallett and Campbell, 1982; White et al., 1983; Pozzan et al., 1983) both by release of Ca2+ from an intracellular store and by inducing Ca2+ influx (Hallett et al., 1990; Thelen et al., 1993). However, the role of cytosolic free Ca2+ in chemotaxis induced by agonists of this receptor, such as fMet-Leu-Phe, remains unresolved (Maxfield, 1993; Hendey and Maxfield, 1993).

Evidence for a role for cytosolic free Ca2+ concentration in neutrophil chemotaxis is inconsistent. Although in populations of neutrophils in suspension, cytosolic free Ca2+ changes occur during chemotactic stimulation (Hallett and Campbell, 1982; White et al., 1983; Pozzan et al., 1983), within individual migrating neutrophils, changes in cytosolic free Ca2+ have been reported to occur either only at the leading edge (Sawyer et al., 1985), transiently, but globally during turning (Marks and Maxfield, 1990a,b), or not at all (Perez et al., 1989). Also chemotaxis has been shown to be reduced by blocking Ca2+ influx with lanthanum (Boucek and Snyderman, 1976), whereas buffering cytosolic free Ca2+ concentration with

BAPTA or quin2 has been reported to have no effect (Meshulam et al., 1986; Zigmond et al., 1988; Perez et al., 1989). One problem in interpreting the results of these studies arises from the role played by the surface. Recent studies have shown that some underlying substrata, which engage neutrophil integrin receptors, can provide signals to the cells (Ng-Sikorski et al., 1991; Petersen et al., 1993). It has been shown that neutrophils moving over such a surface are continually signalled by the engagement and disengagement of integrin (Marks and Maxfield, 1990a,b; Marks et al., 1991).

More recently, elegant work with giant newt eosinophils (Brundage et al., 1991, 1993; Gilbert et al., 1994), which are approx 100 μm long, has demonstrated persistent elevations in Ca2+ at the rear of the cell during chemotaxis, whether directed to a chemotactic source (newt serum) or randomly. This is similar to another large migrating non-mammalian cell, amoeba, in which aequorin was used to demonstrate that cytosolic free Ca2+ concentration was elevated at the rear (Taylor et al., 1980). We have therefore re-examined whether such a gradient can be demonstrated in human neutrophils.

In the present study, the problems of surface interaction have been avoided by measuring the cytosolic free Ca2+ within individual neutrophils during chemotaxis towards a source of fMet-Leu-Phe on plain glass. We demonstrate here that, under these conditions, neutrophils move rapidly up a concentration gradient of formylated peptide without global changes in cytosolic free Ca2+, until reaching a zone of high peptide concentration, when a Ca2+ signal is triggered. Furthermore, unlike the giant newt eosinophil (Gilbert et al., 1994), a rear-to-front

Ca2+ gradient of less than 0.5 nM/μm existed. Small, localised changes in cytosolic free Ca2+ concentration were occasionally observed when neutrophils executed sharp turning manoeuvres, which could be replicated by local membrane deformation.

Materials

fura-2/AM, pluronic F-127 and formylNle-Leu-Phe-Tyr-Lys-fluorescein derivative were purchased from Molecular Probes, Oregon, USA. fMet-Leu-Phe was from Sigma Chemicals, Poole, Dorset.

Neutrophil isolation

Neutrophils were isolated from heparinized blood of healthy volunteers as described previously (Davies et al., 1991). Following dextran sedimentation, centrifugation through Ficoll-Hypaque (Pharmacia) and hypotonic lysis of red cells, neutrophils were washed and resuspended in Krebs buffer (120 mM NaCl, 4.8 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 1.3 mM CaCl2, 25 mM HEPES and 0.1% bovine serum albumin, adjusted to pH 7.4 with NaOH).

Measurement of cytosolic free Ca2+

Neutrophils (2×107 cells/ml) were loaded with fura-2 as previously described (Al-Mohanna and Hallett, 1988). Ratio fluorescence measurements and ratio imaging of fura-2-loaded neutrophils adherent to glass coverslips were performed as previously described (Hallett et al., 1990, 1991; Davies et al., 1991). Excitation wavelengths were achieved using a Spex Fluorolog dual wavelength fluorimeter (Glen Spectra, Stanmore, UK), and ratio images acquired by an ISIS-M intensified CCD camera (Photonic Science, Tunbridge, UK) coupled to a Spex IM101 analysis system. Background subtraction, set at the autofluorescence level, avoided excluding the extreme cell periphery from subsequent ratio calculations. In our hands, this procedure generates artefactually low excitation ratios in a region 1-2 μm within the cell image (Hallett et al., 1991), but permits measurement of cytosolic free Ca2+ changes up to the apparent cell edge. Although this artefact can be removed by increasing the subtraction above the autofluorescence level, this was not done as it simply excluded visualisation of the periphery of the cell and may have masked localised Ca2+ changes near the front or trailing edges of the migrating neutrophil. Images were collected at a rate of 0.25/second. Ratio pixel values were converted to cytosolic free Ca2+ concentrations using the standard equation (Hallett et al., 1990). This approach would provide significantly different data for two regions of the image when the actual difference in cytosolic free Ca2+ concentration was greater than δ, where:

where n is the number of pixels in each of the two areas of the image, σ is the standard deviation of the distribution of Ca2+ values in the pixel arrays, Zx is the standard normal deviate exceeded with probability x, α is the significance level for the test and (1−β) is the power of the test (Armitage and Berry, 1987). Areas of interest in which the number of pixels exceed n were, therefore, chosen, in order to statistically detect a Ca2+ difference of δ, where:

This analysis sought to determine the presence of a Ca2+ gradient across the cell by taking test areas at defined distances apart. However, as seen from equations for n and δ, the ability to detect small and localised changes in Ca2+, depends on both the magnitude of the Ca2+ change and the area its occupies. Therefore, the possibility of smaller and more localised Ca2+ changes within a test area cannot be excluded by the approach.

Stimulation of chemotaxis

Neutrophils were prepared from healthy human blood as previously described and allowed to adhere to glass coverslips in Krebs medium containing bovine serum albumin (0.1% w/v). A micro-pipette with tip diameter of 1-2 μm (Sterile Femtotips, Eppendorf, Hamburg, Germany) was filled with fMet-Leu-Phe (1 μM) in Krebs medium, previously filtered through a 0.2 μm sterile filter (Millipore). After placing the micropipette tip within the microscopic field, a constant pressure (40-50 mbar) was applied using a micro-injector pressure system (Micro-injector 5242, Eppendorf, Hamburg, Germany) to slowly eject the chemotactic peptide.

Does global cytosolic free Ca2+ concentration in neutrophils change during chemotaxis?

In order to visualise and measure cytosolic free Ca2+ changes in neutrophils undergoing chemotaxis, a micropipette containing fMet-Leu-Phe (1 μM) was positioned in a field of fura2-loaded neutrophils. By including fluorescein or fluoresceinated-formylated peptide (formylNle-Leu-Phe-Tyr-Lys-fluorescein derivative), it was established that a local steady state gradient formed around the tip within 2 minutes, by applying a constant pressure (40-50 mbar). The concentration near the tip was at least 100 times greater than at a point 100 μm distant, giving gradients of approximate 0.1 nM/μm (Fig. 1a). After delays of 0.5-2 minutes, neutrophils within the 100 μm field changed shape, moved towards the micropipette tip and clustered around it (Fig. 1b). Visualisation of cytosolic free Ca2+ concentration within the neutrophils under these conditions failed to demonstrate any changes in cytosolic free Ca2+ concentration in neutrophils undergoing shape change or rapid chemotaxis (Fig. 2). However, the cytosolic free Ca2+ concentration in neutrophils within 5-10 μm of the tip rose abruptly to 500-800 nM (Figs 2 and 3). At this point, neutrophils became stationary and often rounded. Apart from the abrupt Ca2+ change as neutrophils reached the zone of relatively high peptide concentration, global changes in cytosolic free Ca2+ concentration were not detected during chemotaxis in any of the more than 50 individual neutrophils examined.

Fig. 1.

Formation of a formylated peptide gradient to stimulate neutrophil chemotaxis. (a) The leakage of fluorescein-labelled formylated peptide from a micropipette is shown after the times indicated. The images show pseudo-coloured images of the fluorescence intensity given by the colour scale. By 60 seconds, the gradient was formed which remained constant. (b) A brightfield view of human neutrophils: (i) immediately after placing the micropipette containing fMet-Leu-Phe (1 μM) in position; and (ii) 2 minutes later, showing both the accumulation of neutrophils at the pipette tip and the effect on other neutrophils in the field.

Fig. 1.

Formation of a formylated peptide gradient to stimulate neutrophil chemotaxis. (a) The leakage of fluorescein-labelled formylated peptide from a micropipette is shown after the times indicated. The images show pseudo-coloured images of the fluorescence intensity given by the colour scale. By 60 seconds, the gradient was formed which remained constant. (b) A brightfield view of human neutrophils: (i) immediately after placing the micropipette containing fMet-Leu-Phe (1 μM) in position; and (ii) 2 minutes later, showing both the accumulation of neutrophils at the pipette tip and the effect on other neutrophils in the field.

Fig. 2.

Cytosolic free Ca2+ concentration in neutrophils undergoing chemotaxis. The cytosolic free Ca2+ concentration in a group of neutrophils whilst migrating towards a micropipette containing fMet-Leu-Phe (1 μM) is shown, according to the pseudo-colour scale indicated. The position of the micropipette, which contained no fluorescent material and so was not visible, is marked by the white shape. Images shown are for the times indicated, with an acquisition time of 4 seconds. The Ca2+ change in neutrophils close to the pipette tip is observed at 150 seconds.

Fig. 2.

Cytosolic free Ca2+ concentration in neutrophils undergoing chemotaxis. The cytosolic free Ca2+ concentration in a group of neutrophils whilst migrating towards a micropipette containing fMet-Leu-Phe (1 μM) is shown, according to the pseudo-colour scale indicated. The position of the micropipette, which contained no fluorescent material and so was not visible, is marked by the white shape. Images shown are for the times indicated, with an acquisition time of 4 seconds. The Ca2+ change in neutrophils close to the pipette tip is observed at 150 seconds.

Fig. 3.

Cytosolic free Ca2+ concentration in an individual neutrophil undergoing chemotaxis. The cytosolic free Ca2+ concentration in an individual neutrophil whilst migrating towards a micropipette containing fMet-Leu-Phe (1 μM) is shown, according to the pseudo-colour scale (in Fig. 2). The position of the micropipette, which contained no fluorescent material and so was not visible, is marked by the white shape. Images shown are for the times indicated, with an acquisition time of 4 seconds. On the image for 60 seconds, a small localised Ca2+ increase may be seen at the site indicated by the arrowhead. The lower part of the figure also shows the complete the time course, with the cytosolic free Ca2+ concentration plotted (y axis) at the position of the centre of the cell (x axis), for time steps of 4 seconds (z axis).

Fig. 3.

Cytosolic free Ca2+ concentration in an individual neutrophil undergoing chemotaxis. The cytosolic free Ca2+ concentration in an individual neutrophil whilst migrating towards a micropipette containing fMet-Leu-Phe (1 μM) is shown, according to the pseudo-colour scale (in Fig. 2). The position of the micropipette, which contained no fluorescent material and so was not visible, is marked by the white shape. Images shown are for the times indicated, with an acquisition time of 4 seconds. On the image for 60 seconds, a small localised Ca2+ increase may be seen at the site indicated by the arrowhead. The lower part of the figure also shows the complete the time course, with the cytosolic free Ca2+ concentration plotted (y axis) at the position of the centre of the cell (x axis), for time steps of 4 seconds (z axis).

Was a persistent Ca2+ gradient present in migrating neutrophils?

The question arose as to whether a persistent rear-to-front Ca2+ gradient was formed in neutrophils during chemotaxis, as found in giant newt eosinophils (Brundage et al., 1991), where the concentration at the rear of the cell was approximately 100 nM higher than the front. Although no difference of this magnitude was detected, a rear-to-front Ca2+ gradient of 0.92 nM/μm (Brundage et al., 1991) would produce a difference of only 9.2 nM in a 10 μm cell. Attempts were therefore made to detect differences of this smaller magnitude.

As the detection of a difference in cytosolic free Ca2+ concentration between given areas by imaging is essentially statistical, high magnification (×1,000) of the migrating neutrophil was used to provide more sampling points per area of interest. The detection of differences between areas is limited by both the accuracy of pixel to pixel measurement (variance) and the number of pixels covering the areas of interest (see Materials and Methods). Regions of the cell corresponding to the front and rear of the cell were chosen, which occupied sufficient pixel numbers to give a sensitivity of greater than 1 nM/μm (for the variance in the pixel values in those areas). Using these criteria, no significant difference (with 80% power at 0.05 significance level) between the leading edge and the rear was detected (Fig. 4). From the measured standard deviations and pixel numbers, it was calculated that a difference between rear and front as low as 2.7 nM (gradient = 0.17 nM/μm) would have produced significantly different mean pixel values. Similar analysis of five other cells also failed to detect statistically significant Ca2+ gradients across the cell. The minimum detectable range for such gradients in all six cells was 0.17-0.5 nM/μm (Table 1). It was therefore concluded that although fluctuations in the mean cytosolic free Ca2+ concentration in the three regions were sometimes apparent, no consistent rear-to-front Ca2+ gradient greater than 0.5 nM/μm existed in migrating neutrophils.

Table 1.

The distribution of cytosolic free Ca2+ concentration in neutrophils during chemotaxis determined in the leading segement (front) and rear segment (tail)

The distribution of cytosolic free Ca2+ concentration in neutrophils during chemotaxis determined in the leading segement (front) and rear segment (tail)
The distribution of cytosolic free Ca2+ concentration in neutrophils during chemotaxis determined in the leading segement (front) and rear segment (tail)
Fig. 4.

Analysis of Ca2+ within a neutrophil during chemotaxis. The image (right) shows the cytosolic free Ca2+ concentration in an individual neutrophil during chemotaxis (acquisition time 2 seconds, magnification ×1,000), coloured according to the pseudo-colour scale (Fig. 2). The Ca2+ values for a single line of pixels through the cell from rear to front (together with smoothed data) is shown for the line indicated on the cell outline (left). Histograms of pixel frequency for sample areas from the three regions indicated are shown (middle). The values on the histograms show the mean Ca2+ for that area with standard deviation and total number of pixels. The standard errors for the mean Ca2+ values in the tail, body and front were 1.5 nM, 0.67 nM and 0.98 nM, respectively. A similar analysis failed to detect statistically significant Ca2+ gradients across five other neutrophils.

Fig. 4.

Analysis of Ca2+ within a neutrophil during chemotaxis. The image (right) shows the cytosolic free Ca2+ concentration in an individual neutrophil during chemotaxis (acquisition time 2 seconds, magnification ×1,000), coloured according to the pseudo-colour scale (Fig. 2). The Ca2+ values for a single line of pixels through the cell from rear to front (together with smoothed data) is shown for the line indicated on the cell outline (left). Histograms of pixel frequency for sample areas from the three regions indicated are shown (middle). The values on the histograms show the mean Ca2+ for that area with standard deviation and total number of pixels. The standard errors for the mean Ca2+ values in the tail, body and front were 1.5 nM, 0.67 nM and 0.98 nM, respectively. A similar analysis failed to detect statistically significant Ca2+ gradients across five other neutrophils.

Do transient cytosolic free Ca2+ increases occur during cell redirecting?

As the formylated peptide gradient developed, the transition of a round stationary neutrophil into a polarized and chemotactic cell occurred without any change in cytosolic free Ca2+ concentration (Fig. 3). Also, no global changes in cytosolic free Ca2+ concentration or persistent Ca2+ gradients were detected during neutrophil chemotaxis. However, transient, localised cytosolic free Ca2+ changes were occasionally observed during abrupt neutrophil shape changes (see for example Fig. 3, arrowhead). In order to establish whether this localised change in cytosolic free Ca2+ was a cause or a con-sequence of cell shape change, an empty blunt micropipette was used to produce an abrupt stretching of non-migrating neutrophils, in order to mimic membrane ‘stretching’, which occurs when cells turn sharply (Fig. 5). Deformation of the neutrophil plasma membrane by advancing the pipette abruptly by 5 μm provoked a transient localised change in cytosolic free Ca2+ (Fig. 5) comparable to that seen occasionally during chemotaxis. The possibility thus existed that the occasional change in cytosolic free Ca2+ concentration observed during manoeuvring of neutrophils was a result of membrane deformation rather than the reason for the cell to change its direction.

Fig. 5.

Cytosolic free Ca2+ concentration in an individual neutrophil undergoing membrane deformation by a blunt micropipette is shown, according to the pseudo-colour scale (Fig. 2). The position of the micropipette, which contained no fluorescent material and so was not visible, is marked by the black bar. The top image shows the neutrophil with the blunt pipette near the cell; the middle image shows the image taken 4 seconds after advancing the pipette 5 μm, when a localised cytosolic free Ca2+ change was evoked; and the lower image shows the result of advancing the pipette a further 5 μm (4 second acquisition time).

Fig. 5.

Cytosolic free Ca2+ concentration in an individual neutrophil undergoing membrane deformation by a blunt micropipette is shown, according to the pseudo-colour scale (Fig. 2). The position of the micropipette, which contained no fluorescent material and so was not visible, is marked by the black bar. The top image shows the neutrophil with the blunt pipette near the cell; the middle image shows the image taken 4 seconds after advancing the pipette 5 μm, when a localised cytosolic free Ca2+ change was evoked; and the lower image shows the result of advancing the pipette a further 5 μm (4 second acquisition time).

The results presented here show that neutrophils migrate towards a source of fMet-Leu-Phe chemotaxis, on glass, without detectable global elevations or persistent gradients in the cytosolic free Ca2+ of greater than 0.5 μM/μm. Occasional transient localised changes were observed during cell manoeuvring, which could similarly be provoked by deformation of the neutrophil membrane. These data question the relevance of Ca2+ gradients, which are present during chemotaxis of giant non-mammalian cells, to human neutrophil chemotaxis.

An early study on individual neutrophils reported that chemotaxis was associated with elevated Ca2+ at the front of the neutrophil (Sawyer et al., 1985). These data from non-ratiometric imaging of quin2 were probably artefactual. In recent studies, using ratiometric imaging of fura2-loaded neutrophils, both local and global changes in cytosolic free Ca2+ concentration were detected (Marks and Maxfield, 1990a,b). However, the possibility existed that Ca2+ signalling was the result of neutrophil integrin engagement (Ng-Sikorski et al., 1991; Petersen et al., 1993) rather than chemotaxis (Marks et al., 1991). In larger migrating cells, such as amoeba or newt eosinophils, the cytosolic free Ca2+ concentration becomes elevated at the rear (Taylor et al., 1980; Brundage et al., 1991, 1993; Gilbert et al., 1994), the Ca2+ gradient from rear to front in migrating newt eosinophils being 0.92 nM/μm (Brundage et al., 1991). In some recent elegant studies by Fay and colleagues (Gilbert et al., 1994) a model, based on spatial separation between diacylglycerol causing Ca2+ pump activation and InsP3 releasing Ca2+, has been proposed to explain the mechanism for these gradients (Gilbert et al., 1994). If the spatial elements and diffusion constants for this model in large newt eosinophils apply to human neutrophils, and so give rise to a similar Ca2+ gradient, then the expected difference in Ca2+ concentration between the front and the rear of the human neutrophil (diam., 10 μm) would be only 9.2 nM. The data presented here do not support the hypothesis that a persistent Ca2+ gradient of this magnitude exists in migrating human neutrophils, but they suggest that if any gradient exists it must be less than 0.5 nM/μm. A similar Ca2+ gradient in kinetoplasts (diam., 2 μm), small motile neutrophil fragments (Keller and Bessis, 1975; Malawista and Chevance, 1982), would generate a rear-to-front Ca2+ difference of only 1 nM (1% of the resting level).

Although the biochemical events accompanying chemotaxis are not resolved, the movement of neutrophils is associated with an increase in the polymerization of actin (Sheterline and Rickards, 1989). This biochemical event can be dissociated from the rise in cytosolic free Ca2+ when measured in neutrophil populations (Al-Mohanna and Hallett, 1990; Downey et al., 1990; Howard et al., 1990). The possibility exists that high-affinity receptors for fMet-Leu-Phe are coupled to events other than a rise in cytosolic free Ca2+ (Meshulam et al., 1986). For example, lower concentrations of fMet-Leu-Phe (Huang et al., 1990) or other chemotactic agents (Lloyds et al., 1995) signal tyrosine phosphorylation.

In some previous studies with human neutrophils and in migrating newt eosinophils, globally elevated Ca2+ levels during sharp turns have been reported (Marks and Maxfield, 1990a,b; Brundage et al., 1991, 1993; Gilbert et al., 1994). In the study reported here, small changes in cytosolic free Ca2+ concentration were also sometimes observed during neutrophil manoeuvring (Fig. 3). These changes in cytosolic free Ca2+ concentration are unlikely to trigger the manoeuvring, since procedures that inhibit changes in cytosolic free Ca2+ concentration do not prevent cell manoeuvring or chemotaxis (Meshulam et al., 1986; Zigmond et al., 1988; Perez et al., 1989). As membrane deformation alone was sufficient to elicit similar cytosolic free Ca2+ concentration changes (Fig. 5), the possibility exists that, rather than triggering cell shape change, these changes were a consequence of the shape change. The mechanism of Ca2+ change in neutrophils in response to membrane deformation may be related to the stretch-activated channels observed in some other cells, such as endothelial cells (Lansman et al., 1983; Goligorsky, 1988) and epithelial cells (Boitano et al., 1992). In macrophages, membrane deformation induces hyperpolarization (Reis et al., 1979) as a result of Ca2+ influx and activation of Ca2+-activated K+ channels. Alternatively, experimentally ‘prodding’ the cell could have elicited temporary and local membrane ‘damage’ that generated the Ca2+ signal. Whatever the mechanism for the membrane deformation-induced Ca2+ signal, the possibility exists that such signalling occurs during neutrophil migration and manoeuvring.

The data presented here thus question whether cytosolic free Ca2+ plays a role in signalling or controlling chemotaxis in human neutrophils.

We are grateful to the Arthritis and Rheumatism Council (UK) for support.

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