Neutrophil Signalling Group, University Department of Surgery, University of Wales College of Medicine, Heath Park, Cardiff, CF14 4XN, UK
* Author for correspondence (e-mail: hallettmb{at}cf.ac.uk)
Accepted 21 March 2003
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Summary |
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Key words: Neutrophils, Phagocytosis, Cytosolic free Ca2+, Oxidase activation, ß2 integrin
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Introduction |
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Materials and Methods |
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Preparation of C3bi-opsonised oxidant indicator zymosan
Zymosan particles (10 mg/ml) were opsonised either by incubation with human
serum (50% diluted, 30 minutes, 37°C) or with purified human C3bi (1
mg/ml; 30 minutes, 4°C). The particles were then washed by centrifugation
and resuspension to remove unfixed C3bi and either labelled immediately or
stored at -20°C. Opsonised zymosan labelling was achieved in bicarbonate
buffer (0.1 M, pH 8.3, 10 mg/ml) (NaHCO3 and HEPES buffer-titrate).
Succimidyl ester of 2'7'dichlorodihydrofluorescein diacetate (1
mg, Molecular Probes, Eugene, Oregon) was dissolved in 100 µl DMSO
(anhydrous), and added dropwise to zymosan (1 ml), mixing between each drop.
The solution was then drawn into a foil-coated syringe, capped to exclude air
and allowed to react overnight at 4°C. This resulted in coupling between
the acetylated, oxidant-insensitive probe and the zymosan. In order to
de-acetylate the product to generate the oxidant-sensitive
dichlorodihydrofluorescein (DCDHF) for use, 100 µl hydroxylamine (105
mg/ml) was added to the suspension, which was then withdrawn into a syringe
and capped for a further 3 hours. The DCDHF-labelled zymosan suspension was
washed several times by centrifugation and resuspension with BSS (balanced
salt solution), aliquotted and stored at -20°C. Fluorescein isothiocyanate
(FITC) was conjugated to zymosan by over-night incubation, at 4°C at pH
9.0 as previously described (Morris et
al., 2003).
Micromanipulated delivery of particles
Neutrophils were allowed to adhere to glass coverslips for 1-2 minutes.
Zymosan particles were allowed to sediment among cells and a micropipette (tip
diameter 1 µm) was brought into the field of view. A particle was drawn
into the mouth of the micropipette by applying slight negative pressure, and
was lightly placed in contact with a neutrophil. After adherence between cell
and particle was established, the zymosan was released by removing the
negative pressure and phagocytosis allowed to proceed
(Dewitt and Hallett, 2002).
Simultaneous cytosolic free Ca2+, oxidase and phase
contrast imaging
Neutrophils were loaded with the Ca2+ indicator, fura-2-AM as
previously described (Hallett et al.,
1996) or fura2-dextran by micro-injection and allowed to adhere to
glass coverslips maintained at 37°C with a temperature-controlled
microscope stage heater. Light was transmitted to an inverted microscope
(Nikon Eclipse) with an oil immersion 100x objective using a rapid
monochromator (Delta RAM, PTI, Surbiton, UK) at three excitation wavelengths -
340 nm, 380 nm (for fura2) and 490 nm (for DCDHF) - delivered sequentially
(see below). Phase contrast images were simultaneously taken under far red
illumination (690 nm) using an appropriate dichroic mirror and a red-sensitive
CCD camera. The fluorescent images were collected using a CCD camera (IC100,
Photon Technology International, Surbiton, UK), and 340/380 nm ratio images
and 490 nm intensity images were calculated and captured using ImageMaster
(PTI, UK) software. The sequence of the excitation wavelengths was 340 nm, 490
nm and 380 nm, thus the two data sets for a single Ca2+ ratio image
and the DCDHF images were overlapping temporally. Images were acquired through
the same emission filter with maximum transmission at 505 nm, with 16-frame
averaging, threshold background subtracted and ratio or intensity calculated
and pseudocoloured. The sum intensity (SI) (rather than average intensity) of
the zymosan particles was used to monitor oxidase activity to avoid errors
associated with the periphery of the zymosan particle where the nonoxidised
indicator often had subthreshold fluorescence. The SI was therefore calculated
as SI=Nø.Iav, where Nø is the number of non-zero pixels and Iav
is the average intensity of the pixels.
Micro-injection of fura2-dextran
The large molecular weight conjugate of fura2, fura2-dextran (molecular
mass 10 kDa), was used to measure cytosolic free Ca2+ concentration
with reduced diffusion of the fura2-Ca2+ complex within the cell.
The fura2-dextran (Molecular Probes) was micro-injected into neutrophils by
the simple lipid assisted micro-injection technique (SLAM) as previously
described (Laffafian and Hallett,
1998). The probe was dissolved in intracellular medium (KCl, 150
mM, HEPES, 25 mM, pH 7.0) to give a final concentration of 500 µM and
either loaded into a micropipette (tip diameter 0.5 µm), before tip lipid
coating, as described previously
(Laffafian and Hallett, 1998
),
or loaded into a pre-lipid-coated micropipette (Cell Engineering, Swansea,
UK). On contact between the micropipette and the neutrophil, transfer of
fura2-dextran into the cell was monitored by an increase in fluorescence at
360 nm to give intracellular concentrations of fura2-dextran of between 10-50
µM. After successful micro-injection, the neutrophils remained fully
functional and able to undergo phagocytosis in response to challenge
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Results |
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Temporal relationship between Ca2+ signalling and
phagosomal oxidation
DCDHF-labelled zymosan particles triggered the same typical sequence of
phagocytic events with particle binding, cup formation and local
Ca2+ signalling, giving rise to a global Ca2+ rise due
to influx (and inhibitable by Ni2+) and then phagosome closure
(Fig. 2). The global
Ca2+ change was also observed as a single transient increase, with
a shoulder or double peak. The DCDHF-labelled zymosan particles were therefore
a useful model for studying phagocytosis by neutrophils.
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The simple addition of hydrogen peroxide (H2O2) to a suspension of DCDHF-labelled particles produced an increase in particle-associated fluorescence excited at 490 nm (Fig. 3a). The rate of H2O2-induced increase in intensity was greatly enhanced by peroxidase activity (Fig. 3c). After phagocytosis, C3bi-opsonised DCDHF-labelled zymosan particles that had been engulfed by adherent neutrophils were significantly brighter than those which had not (Fig. 3b). This was not a general effect of the phagosomal environment on the 'parent molecule', fluorescein, as fluorescein-labelled particles, which were insensitive to oxidation, did not increase in intensity during phagocytosis (4/4 cells) and often decreased in intensity (3/4 cells; Fig. 3d). Thus, the increase in fluorescence intensity of DCDHF-labelled zymosan during phagocytosis was used as a monitor of oxidative activity, particularly in the presence of peroxidase activity.
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In order to establish the time sequence of oxidase activation and Ca2+ signalling during phagocytosis, phase contrast images, the DCDHF signal from the particle and Ca2+ signals from the cell were recorded simultaneously. Using this simultaneous imaging approach, it was immediately evident that the Ca2+ signal in the neutrophil began many seconds before the onset of oxidase activation detectable by increased DCDHF fluorescence (42/47 cells). To determine whether the onset correlated with any of the phases of the complex Ca2+ signal, neutrophils that displayed distinct double Ca2+ peaks were chosen for analysis because the separate Ca2+ phases could be more easily distinguished and timed. In the majority of the cells studied (18/25 cells), the onset of DCDHF oxidation (increased fluorescence) was shown to correlate with the second of the two Ca2+ peaks (Fig. 4a). It was difficult to time the onset of oxidation accurately in the other cells tested (7/25), either because the particle was moving in and out of focus during phagocytosis or because there was little oxidative increase after phagocytosis. The relationship between oxidation and the later Ca2+ signal was not unique to these 'twin peak' cells, as it was also evident in cells with less obvious separation of the two signals, such as those giving a Ca2+ shouldered response (Fig. 4b).
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The second Ca2+ event usually occurred around the same time as phagosomal closure. In the majority of cells, phagosomal closure occurred just after the second peak in the Ca2+ signal (21/33); in some cells (8/33) phagosomal closure occurred while the second Ca2+ signal was subsiding, but in one cell, phagosomal closure occurred just before the second peak, and in the remainder (3/33), phagosome closure occurred after the return of the second Ca2+ peak to base-line. As oxidative activation also occurred near the time of phagosome closure and the second Ca2+ peak (see Fig. 4a,b), we considered the possibility that the oxidase was actually activated and peroxidase secreted before phagosome closure, but that the local oxidant and peroxidase concentrations were too low (as a result of diffusional dilution before phagosome closure) to be effective in causing oxidation of DCDHF on the zymosan until the phagosome closed, preventing dilution. However, this explanation seemed unlikely to account for the abrupt increase in oxidation within the phagosome because first, this mechanism would be expected to cause a gradual increase in DCDHF fluorescence, in contrast to the abrupt changes observed (Fig. 4a,b, Figs 5 and 6); second, there was no evidence that the base of the zymosan particle deepest in the phagocytic cup, where the diffusion path to the outside was longest, became oxidised earlier or before phagosome closure (e.g. Fig. 4a,b, Fig. 6); and third, in some cells (e.g. Fig. 4a,b), phagosome closure occurred several seconds before the onset of detectable oxidation, there being no sudden effect of oxidation at the time of phagosomal closure. It was therefore concluded that during phagocytosis, activation of the oxidase and triggering of the release of peroxidase into the phagosome did not occur during the first global change in Ca2+ signal but correlated temporally with the second phase of the Ca2+ signal.
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To establish the role for the phagocytic-induced Ca2+ signal in
oxidase activation, two pharmacological inhibitors of the Ca2+
signal were used. Ni2+ (2 mM), which blocked Ca2+
influx, totally prevented the global Ca2+ signal and slowed
phagocytosis (Dewitt and Hallett,
2002). Blocking Ca2+ influx in this way also reduced
the increase in DCDHF-zymosan fluorescence, the oxidative response being
significantly reduced by Ni2+ (P<0.05, n=6,
Fig. 5). The
phosphatidylinositol 3-kinase [PtdIns(3) kinase] inhibitor, LY294002 (50
µM), also inhibited the Ca2+ signal and also slowed phagocytosis
(Dewitt and Hallett, 2002
).
Again, there was a significant decrease in the oxidative response
(Fig. 5). It was therefore
concluded that during phagocytosis, the global Ca2+ signal was
obligatory for maximum phagosomal oxidation (during the second Ca2+
phase), but it was not sufficient alone to trigger oxidative activation (in
the first Ca2+ phase).
Contribution of myeloperoxidase degranulation to oxidation
signal
As myeloperoxidase (MPO), which is responsible for the generation of HOCl
from H2O2 in neutrophil phagosomes, is delivered to the
phagosome by granule-phagosome fusion, and has a major effect on DCDHF-zymosan
oxidation (Fig. 3), it was
important to consider the role of this enzyme in the oxidation signal
observed. DCDHF-labelled zymosan was therefore delivered to neutrophils in the
presence of the potent MPO inhibitor, azide (10 mM N3-).
Under these conditions, Ca2+ signalling was normal, but the
fluorescent signal from internalised particles was significantly reduced
(P<0.01), although it could not be totally inhibited
(n=6) (Fig. 5). This
may have resulted from an inability of azide to cause total peroxidase
inhibition, as the residual response could be inhibited further by the
Ca2+ channel blocker, Ni2+ (P<0.001,
n=2, Fig. 5). This
reduced the response to that observed in MPO-deficient neutrophils
(P<0.001, Fig. 5).
In these latter cells, the oxidative response was restored to normality by
addition of extracellular MPO (5.5 nM, by haem content) or horseradish
peroxidase (0.25 units/ml) before phagocytosis. It was therefore concluded
that delivery of MPO to the phagosome played an important role in the
DCDHF-oxidation signal.
In the absence of MPO-activity (MPO-deficiency), although the DCDHF signal was reduced and slowed as with N3- treatment, a detectable response was observed which commenced at the second phase of the Ca2+ signal. As this suggested that the second phase of the Ca2+ signal was also important in activating the oxidase (in the absence of peroxidase degranulation), this proposal was tested by measuring oxidation of DCDHF-zymosan during phagocytosis in the presence of extracellular MPO or HRP (0.25 units/ml). This strategy, which would eliminate the dependence of degranulation of peroxidase into the phagosome, had no effect on the timing of the DCDHF response. It was therefore concluded that the oxidase (or delivery of preformed oxidant) was not activated earlier than the second phase of the Ca2+ signal and that oxidase activation occurred with the second Ca2+ signal.
Detection of oxidative activity was restricted to the phagosome
As the second Ca2+ signal triggered oxidative activity but was
not restricted within the cytosol, the possibility that oxidase and peroxidase
release were also triggered at nonphagosomal sites was investigated.
Neutrophils were chosen that had oxidant-detecting zymosan particles close by
or touching, which would act as a sentinel reporting extracellular oxidation
during phagocytosis. In these experiments, no oxidants were detected at
extracellular sites around the cell (47/47) even if the sentinel zymosan
particles were near the open mouth of the phagosome (e.g.
Fig. 6a, particle 3). Sentinel
particles near or touching the outer face of the phagocytic cup
(Fig. 6a, particle 2) also
failed to become oxidised. Thus, it appeared that the second phase
Ca2+ rise triggered oxidase assembly and granule fusion restricted
to the inner face of the forming phagosome. However, the concentration of
oxidants near extracellular zymosan particles (i.e. not within phagosomes) was
expected to be less as a result of dilution and so detection of oxidation of
DCDHF might have been preferential within the phagosome. This was confirmed in
neutrophils in which pseudopodia formation and internalisation of particles
was prevented by pretreatment with cytochalasin B (5 µg/ml). Although this
treatment did not inhibit the Ca2+ signal
(Dewitt and Hallett, 2002), no
oxidation of the extracellular particle was detected
(Fig. 6b). Cytochalasin B does
not inhibit but enhances oxidase-mediated oxygen consumption
(Al-Mohanna and Hallett, 1987
)
and causes nondirected MPO release, which was consistent with extracellular
dilution of oxidants preventing the detection of extracellular oxidation by
this approach. This conclusion was also supported by the failure of phorbol
myristate acetate (PMA), a non-phagocytic stimulus of the oxidase, but not of
MPO release, to oxidise extracellular DCDHF-zymosan. A second indicator of
oxidase activity, independent of MPO, was therefore used. Nitroblue
teterazolium (NBT) is reduced by O2- (and may also
accept electrons directly from the oxidase) to form insoluble formazan. As the
precipitate cannot diffuse, it provides spatial information of oxidase
activity. This indicator reported strong oxidation within the phagosome,
beginning on closure of the phagosome (n=3), but produced no evidence
of oxidase activity at adjacent plasma membrane sites
(Fig. 6c). There was evidence
of oxidation of NBT within the cytosol near the phagosome, perhaps as a result
of diffusion of oxidants from localised oxidase activity within the phagosome
(Fig. 6c) (see also
Sullivan, 2003
). There was,
however, no clear formazan precipitation at membranes other than within the
phagosome (Fig. 6c).
Whether or not activation of the oxidase was restricted absolutely to the
inner phagosomal membrane, formazan precipitation clearly showed it was
localised at least to that part of the cell undergoing phagocytosis. As the
activation of the oxidase appeared to be spatially restricted, yet
Ca2+ signalling was unrestricted throughout the cytosol, we
examined the possibility that the apparent global distribution of elevated
Ca2+ was generated artefactually as a result of the rapid diffusion
of the fura2-Ca2+ complex away from a more restricted region of the
cytosol. This possibility was considered because the initial small localised
Ca2+ event, which occurs on initial contact between an opsonised
particle and a neutrophil, is best observed when the geometry of the
phagocytic cup includes a narrowing before the main body of the cell
(Dewitt and Hallett, 2002).
This would be explained if the narrowing at the base of some phagocytic cups
imposed a 'diffusion resistance' to the fura2-Ca2+ complex and so
more accurately reported the site of elevated Ca2+. The possibility
that the oxidase-triggering second Ca2+ signal was also restricted
to a zone around the phagosome was therefore tested by using a larger
molecular weight dextran conjugate of fura2 (molecular mass 10 kDa) as a more
slowly diffusible Ca2+ reporter. After micro-injection with
fura2-dextran (50-100 µM), the phagocytic event was identical to that seen
in noninjected neutrophils or those micro-injected with an irrelevant molecule
(lucifer yellow). Furthermore, apart from the initial Ca2+ event,
which was observed as localised in 1/3 phagocytotic events, fura2-dextran also
reported synchronised global Ca2+ signals (3/3), with no evidence
of a time delay between the periphagosomal region and elsewhere
(Fig. 7). Also, fast confocal
imaging (33 mseconds resolution) failed to reveal a wave of Ca2+
originating from the phagosome (data not shown). There was thus no evidence
that the global Ca2+ changes originated from a localised source of
Ca2+ in the phagocytic region or that the cytosolic free
Ca2+ concentration was higher there (in Ca2+ ratio
images, the DCDHF signal contaminates the Ca2+ image as it gets
brighter after phagocytosis and give an artefactually higher ratio value at
that location). It was therefore concluded that the Ca2+ signal
that triggered the oxidation response was the result of influx of
Ca2+ across the plasma membrane around the whole cell perimeter.
However, as this elevated Ca2+ alone was insufficient to trigger
oxidase activity in non-phagosomal locations in the cell, it was concluded
that other key factors required for activation of the oxidase and peroxidase
release by Ca2+ were limited to the phagosomal membrane.
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Discussion |
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This study raises some important questions. The first set of questions
concern the mechanism underlying the complexity of the phagocytic
Ca2+ signal. We have recently shown that the initial ß2
integrin-mediated Ca2+ signal causes an increase in mobility of
ß2 integrin molecules distant from the contact site, which results in an
acceleration of phagocytosis (Dewitt and
Hallett, 2002). The second phase of ß2 integrin binding may
be responsible for the second Ca2+ peak observed in some
neutrophils. It is probable that the same events exist in all neutrophils, but
appeared as a single Ca2+ peak, a Ca2+ peak with
shoulder or a double Ca2+ peak, depending on the some critical
values for key kinetic parameters of integrin mobilisation or Ca2+
homeostasis. A recent mathematical model suggests that splitting of
Ca2+ signals may arise in individual cells as a result of
differences in the number of their Ca2+ storage proteins
(Baker et al., 2002
) or in
Ca2+ reuptake rates. The question also arises about the mechanism
by which the global Ca2+ signal is generated. Clearly, with the
technique used here, the stimulus delivered to the cell was very localised,
yet no evidence for localisation of the 'global' Ca2+ signal was
found (Fig. 4a,b,
Fig. 7). As the global
Ca2+ signal was driven by Ca2+ influx, three
possibilities exist: first, a factor was released into the medium that
diffused extracellularly to trigger remote Ca2+ influx; second, a
factor was released into the cytosol that diffused intracellularly to trigger
remote Ca2+ influx; or third, a factor was released into the plasma
membrane that diffused around the cell membrane to trigger remote
Ca2+ influx. There is some evidence in support of either of the
latter two possibilities, as a water-soluble factor (Ca2+ influx
factor) has been described (Randriamampita
and Tsein, 1993
) and can be isolated from neutrophils
(Davies and Hallett, 1995
), and
inhibitors of PI(3) kinase inhibit Ca2+ signals triggered by
ß2 integrin (Dewitt and Hallett,
2002
) and phosphatidylinositol-3,4,5-trisphosphate
[PtdIns(3,4,5)P3] itself can induce Ca2+
signals in neutrophils. For any of the three mechanisms, the Ca2+
concentration may be elevated nearer the generation site of the
'Ca2+ influx signal' as it diffuses down a concentration gradient.
In an earlier study, a wave of Ca2+ (17 µm/s) originating near
the phagosome (Schwab et al.,
1992
) was indeed reported. However, we and others
(Theler et al., 1995
) have not
been able to show such a Ca2+ wave.
The Ca2+ influx signal was responsible for abrupt oxidation of
internalised zymosan (Fig. 5).
This may be attributed to the abrupt delivery to the phagosome of MPO by
granulephagosome fusion, the abrupt activation of the oxidase in the phagosome
membrane, the delivery of long-lived oxidants preformed in a granule (that
perhaps also contain the MPO) or a combination of these. It has previously
been shown that high Ca2+ levels are required for
MPO-granule-phagosome fusion, with K50 values estimated by quin2
buffering as 2.6 µM (Lew et al.,
1986; Jaconi et al.,
1990
) or by internal perfusion at near 100 µM
(Nüsse et al., 1998
). As
these levels of Ca2+ are higher than the bulk Ca2+ level
(approximately 700 nM), either ß2 integrin engagement increased the
efficacy of Ca2+ for degranulation or Ca2+ just under
the phagosomal membrane reached much higher levels. Although two early papers
reported a long-lived localised Ca2+ signal around the phagosome
(Sawyer et al., 1985
;
Murata et al., 1987
), this
could not be shown here. However, Ca2+ influx in neutrophils has
been shown to cause high submembrane changes in Ca2+, to give a
Ca2+ concentration of at least 50 µM
(Davies and Hallett, 1998
),
which has been suggested to mediate subplasma membrane calpain activation
(Kd 30 µM) (Dewitt
and Hallett, 2002
). As the concentration of Ca2+ within
the phagosome decreases after phagocytosis
(Lundqvist-Gustafsson et al.,
2000
), the possibility exists that Ca2+ channel opening
in the phagosomal membrane may elevate Ca2+ concentrations to very
high levels immediately around the phagosome. However, it is clear that this
Ca2+ signal alone is insufficient to activate the oxidase as its
activity was not triggered by the initial Ca2+ change. The
possibility exists that between the first and second Ca2+ event,
the oxidase was assembled in a form awaiting the Ca2+ signal for
full activation. It is known that components of the oxidase p40phox and
p47phox must be assembled (Segal,
1996
) and that PtdIns(3)P binding and generation occurs
(Ellson et al., 2001a
;
Kanai et al., 2001
). If
PtdIns(3)P is localised to the phagosomal membrane in neutrophils, as
it is in RAW 264.7 cells (Ellson et al.,
2001b
), the effect of the global Ca2+ signal would only
be evident locally within the phagosome. Although the oxidase complex may be
partially active without the Ca2+ signal, and thus account for the
inability to completely inhibit oxidative activity by blocking the
Ca2+ signal (Fig.
5), the Ca2+ signal was associated with a massive
increase in oxidative activity in the phagosome. As it is well documented that
activators and inhibitors of protein kinase C (PKC) activity also activate and
inhibit oxidase activation (e.g. Cooke and
Hallett, 1985
), and PKC associates with the oxidase component
p40phox (Reeves et al., 1999
)
the possibility exists that the action of Ca2+ on the oxidase may
be mediated by PKC activity.
An issue important for pathology raised in this study is whether leakage of oxidants into the extracellular medium can occur from the activated oxidase. No leakage was detected by zymosan particles near by or even at the mouth of the phagosome. However, this may have arisen artefactually as a result of dilution of oxidants in the extracellular medium (an event which cannot occur within the enclosed phagosome). In some cells, efflux of oxidants from the forming phagosome may thus remain a possibility, because although there was a good correlation between the second phase on the Ca2+ signal and the activation of oxidative activity, this was not tightly coupled to the time of phagosomal closure. This means that in some cells, the oxidase was activated before complete closure of the phagosome and that, theoretically, extracellular oxidation would be possible.
The work here has therefore provided a role for the second of the two Ca2+ signal phases triggered during ß2 integrin-mediated phagocytosis - namely, in activation of the oxidase and triggering granule-phagosome fusion. Previously, we have shown that the first phase was required for the liberation of immobile ß2 integrin to cause acceleration of phagocytosis. However, it still remains to be established whether the first localised Ca2+ signal fulfils any role and how the Ca2+ signal interacts with other events before a full explanation of the roles of the complex Ca2+ signalling of phagocytosis by neutrophils can be given.
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Acknowledgments |
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Footnotes |
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References |
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