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Dynamic Ca2+changes in neutrophil phagosomes A source for intracellular Ca2+during phagolysosome formation?
Cell Calcium (2000) 27 (6), 353–362 © 2000 Harcourt Publishers Ltd
Research
doi: 10.1054/ceca.2000.0130, available online at http://www.idealibrary.com on
Dynamic Ca2+ changes in neutrophil phagosomes A source for intracellular Ca2+ during phagolysosome formation? H. Lundqvist-Gustafsson,1 M. Gustafsson,2 C. Dahlgren3 1
Division of Pathology II, Faculty of Health Sciences, Linköping University, Sweden Department of Internal Medicine, Vrinnevi Hospital, Norrköping, Sweden 3 The Phagocyte Research Laboratory, Department of Medical Microbiology and Immunology, University of Göteborg, Sweden 2
Summary An increase in cytosolic Ca2+ concentration periphagosomally is critical for phagolysosomal formation and neutrophil elimination of microbes. The Ca2+ increase could be achieved through release of Ca2+ from mobilized intracellular stores. Alternatively, Ca2+ that passively enter the phagosome during phagocytosis could be provided by the phagosome. Intraphagosomal Ca2+ changes in single human neutrophils was measured during phagocytosis of serum opsonized Fura-2-conjugated zymosan particles, using a digital image processing system for microspectrofluorometry. A decrease in phagosomal Ca2+ down to nanomolar concentrations was seen within minutes following phagosomal closure. Blockage of plasma membrane Ca2+ channels by econazole abolished this decrease. The fluorescence properties of Fura-2 zymosan were retained after phagocytosis and stable to pH changes, reactive oxygen species, and proteolytic enzymes. We suggest that Ca2+ ions present in the phagosome enter the cell cytosol through Ca2+ channels in the phagosomal membrane, achieving a localized Ca2+ rise that is important for phagosome processing. © 2000 Harcourt Publishers Ltd
INTRODUCTION The neutrophil granulocyte is an important inflammatory cell participating in the body’s defense against invading microorganisms. The neutrophil exerts its function by phagocytosis and intracellular killing of pathogenic microorganisms. Binding of a foreign microorganism to neutrophil surface receptors generates signals that regulate the phagocytic process [1]. During this process, the plasma membrane forms an invagination that surrounds the microbe. Phagocytosis is completed when the invaginated membrane is closed; forming a new membrane enclosed intracellular vesicle, the phagosome. However, killing of microbial intruders also includes the delivery of the cellular bactericidal arsenal to the phagosome. These bactericidal weapons are stored in subcellular organelles, Received 24 February 2000 Revised 23 June 2000 Accepted 23 June 2000 Correspondence to: Helen Lundqvist-Gustafsson, Division of Pathology II, Faculty of Health Sciences, S-581 85 Linköping, Sweden. Tel: +46 13 221 525; fax: +46 13 221 529; e-mail: [email protected]
granules (reviewed in 2), of which the azurophil and the specific granules are the best characterized. The granules contain a variety of proteins including myeloperoxidase (MPO, present in the azurophil granules) and a unique b type cytochrome (present mainly in the specific granules). The b cytochrome is the electron transporting protein of the superoxide anion (O2–) and hydrogen peroxide (H2O2) generating nicotinamide adenine dinuclotide phosphate(NADPH-) oxidase [3,4]. The reduced oxygen species formed by the oxidase can be metabolized further to hypochlorous acid (HOCl) and other potent microbicidal substances [5]. Assembly and activation of the NADPHoxidase in the membrane of the phagosome [6,7] as well as phagosome-lysosome fusion are, thus, processes required for the efficient killing of an ingested microorganism. Neutrophil phagocytosis, and the activation of the bactericidal arsenal are highly regulated processes that are mediated by signals generated secondary to the binding of a particle to phagocyte membrane receptors. The complement receptor 3 (CR3; CD11b/CD18) is essential for phagocytosis of complement opsonized particles. During phagocytosis mediated by this receptor, phospholipase D 353
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(PLD) is activated which results in diacylglyceride formation and activation of protein kinase C [8], however, heterotrimeric G-proteins or inherent tyrosine kinases are not involved in the activation process [9]. The precise molecular basis of neutrophil activation through this receptor remains to be determined. In neutrophils, as well as in many other cells, changes in the cytosolic concentration of calcium ions ([Ca2+]i) play an important role in cell activation. A rise in [Ca2+]i is not required for uptake of microorganisms through phagocytosis [10], but it is a necessary signal for the killing of the ingested prey [11]. The requirement for Ca2+ in post-ingestion killing probably relates to the regulatory function of this ion in the assembly/activation of the NADPH-oxidase [11,12] as well as in the docking/fusion process leading to the formation of a phagolysosome [13,14]. Furthermore, an increase in [Ca2+]i is a prerequisite for dissolving the actin barrier around the phagosome, thus enabling fusion of granules and assembly of the NADPH-oxidase in the phagosomal membrane [15]. The formation of inositol-1,4,5-trisphosphate (Ins(-1,4,5)P3) [16,17] is the direct signal to release Ca2+ from intracellular stores probably closely related to the endoplasmic reticulum. However, these Ca2+ stores are not the only source for Ca2+, since Ca2+ ions can be transported from the extracellular fluid through plasma membrane channels and be released directly into the cytosol [18]. The local rise in [Ca2+]i in the periphagosomal area that is seen during phagocytosis [13] has been attributed to either a release of Ca2+ from intracellular stores, or an opening of Ca2+ channels present in the plasma membrane, or a combination of both these processes. It has been shown that the cytosolic Ca2+ stores translocate to the vicinity of the phagosome, and the local increase in [Ca2+]i (mediating post-ingestion processing of the phagosome) has been proposed to be a result of a Ca2+ release from these accumulated stores [19]. Based on the fact that the content of the phagosome constitutes a part of the previously extracellular milieu (that has a Ca2+ concentration above 1 mM) [16], we investigated if the release of calcium ions through the phagosomal membrane into the cytosol might contribute to the local Ca2+ signal required for post-ingestion processing of the phagosome. To investigate whether Ca2+ from the phagosome itself can enter the cytosolic compartment and, thus, facilitate phagolysosome fusion, we used serum opsonized Fura-2 conjugated zymosan particles to directly measure intraphagosomal Ca2+ levels during the phagocytosis process. MATERIALS AND METHODS Isolation of neutrophils Neutrophils were isolated from heparinized blood obtained from healthy adults. After dextran sedimentaCell Calcium (2000) 27(6), 353–362
tion at 1 g, hypotonic lysis of the remaining erythrocytes and centrifugation on a Ficoll-Pacque gradient [20], the neutrophils were washed twice and resuspended in Krebs-Ringer Glucose buffer (KRG, containing 10 mM glucose, 1 mM Ca2+ and 1.5 mM Mg2+, pH 7.3), and stored on melting ice. Samples were withdrawn and used in the experiments within 4 h of preparation. Preparation of Fura-2 conjugated zymosan particles Molecular Probes (Eugene, OR) has developed an aminereactive Fura derivative that upon request can be conjugated to proteins for site-selective targeting. In order to obtain a particle that should allow us to determine intraphagosomal Ca2+ changes, amine-reactive Fura derivative was covalently bound to zymosan particles. The freeze-dried Fura-2 conjugated zymosan particles were suspended and washed in PBS and resuspended in PBS containing 2 mM azide (1.5 × 107 particles/ml) and stored at –20°C until use. Video microscopy and ratio imaging Two different microscope systems were used in the experiments: 1. A Zeiss IM-35 (Carl Zeiss, Oberkochen, Germany) microscope equipped for epi-fluorescence with a 75 W xenon arc lamp as the excitation source was used together with a glycerol-immersed Zeiss neofluar Ph3, 100x objective. During measurements, the sample was alternatively excited at 340 nm and 380 nm, and the 510 nm emission light was recorded by an image intensifier (Hamamatsu C-2100) attached to an integrating colour CCD camera (Zeiss-ZVS-47-E, used in B/W mode). For simultaneous phase contrast microscopy, light emitting diodes emitting at 640 nm was used as a light source. In the emission path, a 580 nm dichroic mirror extracted the Fura-2 emission from the transillumination light. The latter was recorded with a Newicon B/W camera (Hamamatsu C2400–7), The resulting images were captured and digitized by a frame grabber (Innovative Vision AB, Linköping, Sweden) attached to a personal computer (Hewlett Packard Vectra U-66). One image pair and one phase contrast image, captured within 2 s from each other, were grabbed every 10 s, each image being calculated as the mean of four successive video frames (jumping average). The image sequences along with timing information were continuously stored on the hard disk for later evaluation. 2. In some experiments, a Zeiss axiovert 135 TV microscope with a 100 W mercury arc lamp as the excitation source was used together with a © Harcourt Publishers Ltd 2000
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glycerol-immersed Zeiss achrostigmat Ph3, 40x objective. A computer controlled filter wheel (Hamamatsu C-4312) was used to alternate between 340 nm and 380 nm filters, and the fluorescence was recorded by an image intensifier (Hamamatsu C-2400–80) attached to a CCD camera (Hamamatsu C-2400–77).
Phagocytosis experiments Fura-2 zymosan (1 µl containing approximately 1.5 × 104 particles) were added to glass cover slips and the drop was allowed to dry whilst being mechanically spread with a pipette tip. The slides were mounted in a chamber to which 300 µl normal human serum was added. The chambers were incubated for 5–10 min at the heated (37°C) microscope stage to opsonize the particles and allow a surface bound chemotactic gradient to be built up [21]. Prewarmed KPG (3 ml) and neutrophils (30 µl containing 3× 105 cells) were then added. The Fura-2 fluorescence (emission wavelength 510 nm) of the particles was followed, exciting the particles alternately with 340 and 380 nm light. A measurement was started when a neutrophil, crawling on the cover slip, approached a zymosan particle. Microscope fields were chosen where phagocytosed as well as non-phagocytosed zymosan particles were present and could be analyzed in parallel.
The ratiometric response of suspended particles to Ca2+, is very similar to the response of free Fura-2 sodium salt. Our Kd determination reveals a value for the particles that are very close to that for free Fura-2 salt, allowing us to use the established Kd value for the free Fura-2 salt (224 nM) in our calculations of intraphagosomal Ca2+-levels. Subcellular fractionation Neutrophils (2 × 108/ml) were resuspended in ice-cold disruption buffer (KCl, 100 mM; NaCl, 3 mM; MgCl2, 3.5 mM; adenosine trisphosphate, 1 mM; piperazine-N,Nbis(2-etanesulfonic acid), 10 mM; pH 7.4) and disrupted by nitrogen cavitation (Parr Instrument Company, Moline, IL). The post nuclear supernatant was centrifuged on a two-layer Percoll gradient as described by Borregaard et al. [25]. The gradients, containing three visible bands corresponding to the azurophil granules (α-fraction), the specific granules (β-fraction) and the plasma membranes/secretory vesicles (γ-fraction), were collected (1.5 ml fractions) by aspiration from the bottom of the centrifuge tube. Percoll was removed by centrifugation and the azurophil granule and specific granule fractions were resuspended in disruption buffer (2 ml). To disrupt the membranes, the granule fractions were freeze-thawed three times immediately before use. Reagents
Evaluation of optical data, calculation of [Ca2+]i The stored images were evaluated off line using a slightly modified computer program, previously described in detail [22]. Briefly, the ratio between the 340 nm and the 380 nm images was calculated pixel by pixel and the resulting image was presented on a monitor after calibrated pseudo colouring. This procedure allows estimation of [Ca2+] while correcting for differences in illumination intensity, photo bleaching, cell thickness and probe concentration [22–24]. Quantitative data were extracted from the images as described [22] for later plotting. For visual assessment, the phase contrast images and corresponding ratio images were sequentially displayed side by side on the monitor in an infinite loop manner. [Ca2+] was calculated using the formula: [Ca2+]=Kd × β × (R-Rmin)/(Rmax-R), where R is the ratio between the 340 nm and 380 nm fluorescence intensity values, Kd is the dissociation constant for the Fura-2/Ca2+ complex (224 nM), β is the ratio between fluorescence from free Fura-2 and Ca2+-saturated Fura-2 excited at 380 nm, Rmax is the ratio obtained when Fura-2 is saturated with Ca2+, Rmin is the ratio obtained when Fura-2 is free from Ca2+ according to Grynkiewicz et al. [23]. © Harcourt Publishers Ltd 2000
Reagents were bought from the following companies: Dextran, Ficoll-Pacque and Percoll: Pharmacia Fine Chemicals, Uppsala, Sweden. Custom synthesized Fura-2 conjugated zymosan A BioParticles: Molecular Probes Inc., Eugene, OR, USA, H2O2: Merck, Darmstadt, Germany. Econazole and adenosine trisphosphate: Sigma Chemical Co, IL, USA. MPO was a generous gift from Dr Inge Olsson, Lund, Sweden.
RESULTS Fluorescence of Fura-2 zymosan particles during phagocytosis The fluorescence changes of Fura-2 particles were followed during phagocytosis of the particles. After formation of a phagosome the 340 nm/380 nm ratio decreased, while the ratio of the non-phagocytosed Fura2 particles remained stable (Fig. 1A). The fluorescence intensity of ingested Fura-2 zymosan particles declined (faded) when excited by both at 340 and 380 nm, while the fluorescence of non-phagocytosed adjacent particles only showed a slight fading (Fig. 1A). Fura-2 zymosan particles phagocytosed in the absence of Ca2+ (the buffer Cell Calcium (2000) 27(6), 353–362
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Fig. 1 Fluorescence properties and ratio changes of Fura-2 zymosan after phagocytosis. Immediately after closure of a phagosome, the fluorescence ratio of Fura-2 excited at 340 nm and 380 nm was rapidly decreased in the presence of Ca2+ (A), while the ratio remained constant in the absence of Ca2+ (1 mM EGTA added; B). The arrows indicate the time for closure of the phagosome. Insert: free (non-phagocytosed) control particles analyzed in parallel with the phagocytosed ones. Data are from one representative measurement out of at least nine. The fluorescence values are given in arbitrary units (a.u.)
containing 2 mM EGTA) showed a similar decrease in fluorescence intensity. However, more importantly, the 340 nm/380 nm ratio of the particles was not affected in the absence of Ca2+, but remained at a constant Rmin level throughout the measurements (Fig. 1B). Furthermore, the ratio was constant in econazole-treated cells (see below) showing that the conjugated particles retain their sensitivity for Ca2+. The intraphagosomal Ca2+-concentration is lowered after phagocytosis The Ca2+-concentration in KRG (1 mM) is sufficient to saturate the Fura-2 molecules on the particles, and, thus, Cell Calcium (2000) 27(6), 353–362
Rmax of each individual particle was determined from the first pre-phagocytosis data points in each experiment. The variation of Rmax was 2.05 ± 0.54; mean ± sd, n=16. Owing to the fact that the Rmin values varied only little between different particles (0.58 ± 0.05, n=16), the same Rmin value (0.58) was used for all particles. From these Rmax and Rmin values, and from the ratio of phagocytosed Fura-2 zymosan, we calculated the corresponding Ca2+ concentration in the phagosomes during the phagocytic process. A dramatic decrease in Ca2+-concentration was seen already within seconds after closure of the phagosome, and the concentration reached nanomolar levels (583 nM ± 130 nM, mean ± sd, n=6) after a few minutes. Data from a representative measurement is shown in © Harcourt Publishers Ltd 2000
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Fig. 2 Fura-2 zymosans used as probes to determine calcium changes in the neutrophil phagosome/phagolysosome. Panel I: Phase contrast microphotographs of a neutrophil (a) and two Fura-2 zymosans (b and c). The neutrophil reaches one of the Fura-2 zymosans (b) 40 s after the start of the measurement (A) and after 80 s the ingestion is terminated and a phagosome is formed (B). Micrographs were also taken after 230 s (C) and 360 s (D). The free Fura-2 zymosan (c) is a control particle. From the fluorescence values of the phagocytosed Fura-2 zymosan (b), the corresponding Ca2+ concentration in the maturing phagosome was calculated (Panel II). (A–D) the arrows correspond to the microphotographs taken. The fluorescence and the corresponding Ca2+ values of the non-phagocytosed Fura-2 zymosan (c) is shown in panel III. The experiment was repeated with neutrophils from ten different blood donors, and we analyzed 3–13 phagocytosed and at least three non-phagocytosed particles at each occasion. Data from one representative measurement. a.u.: arbitrary units.
Figure 2. Thus, lowering of phagosomal Ca2+ down to levels corresponding to those that have been measured in the periphagosomal area [13,26,27]. seem to bee an early event in the processing of the phagosome. The role of Ca2+ channels in the efflux of Ca2+ from the phagosome In order to determine if the lowering in Ca2+ concentrations in the phagosome truly reflects an efflux of Ca2+ through Ca2+ channels in the phagosomal membrane, we blocked the opening of plasma membrane Ca2+ channels with the antimycotic imidazole derivative econazole [28]. The plasma membrane derived membrane enclosing a prey should then not be able to transport calcium from the phagosome. We found that econazole dose-depen© Harcourt Publishers Ltd 2000
dently blocked intraphagosomal Ca2+-changes [Fig. 3], further supporting the specificity of Fura-2 zymosan as tracers of phagosomal Ca2+ changes. Effects of pH changes, reactive oxygen species and granule constituents on Fura-2 zymosan fluorescence The decline in fluorescence after phagocytosis during excitation at either 340 nm or 380 nm, seen irrespectively if the Ca2+ concentration is changed or not, might reflect a functional loss of Fura-2. This could be due to a combinatory effect of oxidation, halogenation, pH changes, and other processes that occur within the phagosome. To test the stability of Fura-2, we investigated the fluorescence properties of the Fura-2 zymosan particles under various environmental conditions. Cell Calcium (2000) 27(6), 353–362
358 H Lundqvist-Gustafsson, M Gustafsson, C Dahlgren
Fig. 4 Effect of pH on the ratio of 340 nm/380 nm fluorescence of Fura-2 zymosan. Particles were spread on the bottom of a microscope chamber in buffer at different pH. The ratio of the 340 nm/380 nm fluorescence of 20 particles was measured at each pH. The numerals represent mean ± sd values. Statistically (Student’s t-test) significant changes from the value obtained at pH 7.3 are marked with **(P<0.05) and ***(P<0.01) respectively.
values in an alkaline milieu (pH~8), but there was no significant difference in ratio when the pH was lowered from physiological levels down to pH 5 (Fig. 4).
Fig. 3 The Ca2+ channel blocker econazole abolishes intraphagosomal calcium changes during phagocytosis of Fura-2 zymosan. Ca2+ measurements were performed as described in the MATERIAL AND METHODS section, with addition of 5, 10 or 50 µM econazole to the buffer (final concentration). Econazole dose-dependently abolished the lowering of Ca2+ in phagosomes (A). Ca2+ concentration in a phagosome (the arrow indicates the time for closure of the phagosome), and the Ca2+ concentration around a free Fura-2 zymosan (insert) in the presence of 50 µM econazole is shown from a representative measurement. The experiment was repeated with neutrophils from three different blood donors, and we analyzed at least six phagocytosed and six free particles at each occasion.
Influence of pH The fluorescence ratio of Fura-2 zymosan was assessed in KRG buffers with a pH-range between 5.0 and 8.6, since pH in the phagosome/phagolysosome may vary between these values during phagocytosis (29, M. G. unpublished observations). We noticed a small shift to higher ratio Cell Calcium (2000) 27(6), 353–362
Influence of H2O2 and HOCl Hydrogen peroxide (H2O2), as well as hypochlorous acid (HOCl), is released into the phagosome and subject microorganisms to oxidative attack. Myeloperoxidase, an enzyme stored in the azurophil granules of neutrophils, mediates (in the presence of chloride anions) the conversion of H2O2 to HOCl. Considering the fact that photo bleaching of Fura-2 has been shown to be related to oxidation of the molecule [30], an oxidative attack could also change the fluorescence properties of Fura-2 zymosan. As can be seen in Figure 5, exposing the particles to H2O2 or hypochlorite (by addition of H2O2 in conjunction with MPO) had no effect, either on fluorescence at the two wavelengths, or on the ratio between them. Influence of granule constituents During fusion between granules and the phagosome, a number of degradative enzymes are released into the phagosome and these bactericidal/proteolytically active factors might affect the fluorescence of the Fura-2 zymosan. A set of experiments was performed in which the particles were subjected to freeze-thawed granule material obtained from isolated azurophil and specific granules respectively. No change in fluorescence during exposure could be seen upon addition of granule © Harcourt Publishers Ltd 2000
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Fig. 5 Resistance of the Fura-2 zymosan to oxygen metabolites. Particles were exposed to H2O2 (1 mM;A), or HOCI (in a mixture of 0.1 mM H2O2 and 1.25 µg purified MPO; B), at times indicated by the arrows. Fluorescence values (a. u. = arbitrary units) of representative particles are shown to the left, and the ratios of the 340 nm/380 nm fluorescence values calculated from the data are shown to the right. Data are from one representative measurement out of four.
extracts originating from the two major granule populations of neutrophils (not shown). In conclusion, the results clearly show that Fura-2 zymosans are stable with respect to the attack from granule material that is delivered to neutrophil phagosomes during phagolysosome formation Fura-2 zymosan particles retain their Ca2+ sensitivity also after phagocytosis From the results above, we can conclude that neither changes in pH, oxidative stress or exposure to granule © Harcourt Publishers Ltd 2000
material (proteases and other destructive enzymes), influences the fluorescence properties of Fura-2 zymosan. However, to make sure that the particles retain their function as Ca2+ indicators also after phagocytosis, we recovered ingested Fura-2 zymosan by lysing cells with Triton X-100 and these particles were then investigated with respect to the response to changes in Ca2+. We found that the Fura-2 particles that have experienced the milieu in the phagolysosome preserve their ability to function as Ca2+ probes as efficiently as do free particles that have not been phagocytosed (Fig. 6).
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Fig. 6 Spectroscopic response of Fura-2 zymosan after phagocytosis. Neutrophils were allowed to phagocytose Fura-2 zymosan on glass slides for 30 min (to ensure that fully processed phagolysosomes were formed). The cells were then lysed with 0.3% Triton X-100. The 340/380 fluorescence ratios of the phagocytosed, Triton X-100-released Fura-2 zymosan were determined in the presence of Ca2+ (1 mM) and in the absence of Ca2+ (EGTA). Representative 340 nm/380 nm ratios of a phagocytosed and released particle (A) and a free control (nonphagocytosed) Fura-2 zymosan (B) are shown. The experiment was repeated with neutrophils from three different blood donors, and we analyzed at least five phagocytosed and five free particles at each occasion.
DISCUSSION Phagocytic uptake of serum opsonized particles, mediated by complement receptors 3 (CR3) on the neutrophil surface, is independent of the presence of Ca2+ in the extracellular medium and an increase in cytosolic Ca2+. However, buffering of intracellular Ca2+ combined with a Cell Calcium (2000) 27(6), 353–362
removal of extracellular Ca2+ affects the post-ingestion processing of the phagosome. This processing includes translocation to the phagosomal membrane of Ca2+ regulated phospholipid binding proteins, e.g. the annexins III and XI [31,32] and cytosolic components required for assembly of the NADPH-oxidase [11] as well as recruitment of granules [13]. Moreover, the redistribution of cytosolic proteins and granules to the phagosomal membrane is facilitated by depolymerization of the surrounding actin network, a Ca2+ dependent process [15]. Thus, intracellular Ca2+ homeostasis, probably especially so in the periphagosomal region, is of crucial importance for the antimicrobial action of neutrophils. It should be noted though, that intraphagosomal NADPH-oxidase activity [11] as well as phagolysosomal fusion [13], also can occur in Ca2+ depleted cells, provided that Ca2+ is present in the extracellular medium. A localized rise in the Ca2+ concentration in the periphagosomal area has been described by other authors [13,27]. This finding has been attributed to a release from intracellular Ca2+ stores that redistribute and accumulate around the phagosome early in the phagocytic process [19]. In this paper, we demonstrate that a decrease in Ca2+ concentration in the phagosome from millimolar (the concentration in the buffer used), down to nanomolar levels, occurs within a few minutes after closure of the plasma membrane invagination around a particle. Recent data suggest that the amount of invaginated plasma membrane is considerably larger than has been previously appreciated, thus increasing the likelihood of a significant coingestion of extracellular constituents [33]. Econazole, an antimycotic imidazole that has been shown to inhibit opening of Ca2+ channels present in the neutrophil plasma membrane [28], abolished the decrease in the intraphagosomal concentration of Ca2+ in a dose-dependent way. This strongly suggests that an opening of plasma membrane channels is the mechanism responsible for the Ca2+ dissipation from the phagosome, even though we cannot rule out that Ca2+/Na+ and/or Ca2+/H+ exchangers are participating in the transport as well [34]. Our results also suggest a mechanism by which the local rise in [Ca2+]i in the periphagosomal area that is seen during neutrophil phagocytosis could be obtained in Ca2+ depleted cells. On the basis of our results and those of others, we suggest that Ca2+ needed for post-ingestion processing of the phagosome can be provided either from mobilized intracellular stores releasing Ca2+ in the periphagosomal area, or from the phagosome itself. The phagosome could then act as an intracellular Ca2+-store and mediate a localized rise in Ca2+ around the phagosome mediated by Ca2+channels in the phagosomal membrane. In a situation that resembles the normal physiological state of a cell (Ca2+ present both intracellularly and in the extracellular © Harcourt Publishers Ltd 2000
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environment), phagocytosis is associated with a rapid, transient increase in [Ca2+]i starting immediately when a neutrophil comes into contact with the prey. The initial rise in [Ca2+]i is followed by a sustained response that ceases after a couple of minutes when [Ca2+]i returns to resting values [13]. The initial rise is dependent on release of Ca2+ from intracellular stores and the second phase is related to an opening of calcium channels in the plasma membrane resulting in an influx of Ca2+ from the extracellular milieu [35]. The plasma membrane pathway, activated by emptying the intracellular Ca2+ stores, is disabled in the presence of cytochrome P-450 inhibitors like econazole [28]. Consequently, the inhibitory effect of this drug in our study indicates that the opening of the Ca2+ channels in the plasma membrane-derived phagosomal membrane is regulated by the same mechanism. Since the concentration of Ca2+ in the extracellular fluid (1 mM) differs from that in the cytosol of a resting cell by a factor 104 [16] and the Kd of Fura-2 is in the nanomolar range, the probe is reasonably accurate for Ca2+ measurements between 50 and 1000 nM, but is rather insensitive to Ca2+ changes in the millimolar range. Resting levels in neutrophils lie typically between 80 and 200 nM, and the concentration in activated cells usually do not exceed 2000 nM [13,26,36]. Although the accuracy for Ca2+ determination in the initial phase of the phagocytic process, therefore, is low, Fura-2 zymosan seem to be useful for studies of intraphagosomal Ca2+changes. The Ca2+-sensitive fluorescence properties are retained after phagocytosis and stable with respect to the influence of reactive oxygen species, proteolytic enzymes and pH changes to which they are exposed during formation of phagolysosomes. The ratio between the 340 nm and 380 nm fluorescence intensity of Fura-2 zymosan did change slightly at an alkaline pH, but this finding was of minor importance for the interpretation of our data. If an alkalinization of the phagosome should occur [29], the decrease in apparent intraphagosomal Ca2+ as registered in our system, would be underestimated rather than overestimated. However, we have not been able to confirm the occurrence of a pH increase during phagocytosis of zymosan (unpublished observations). The Ca2+-independent decline in fluorescence of Fura-2 observed could not be shown to relate to effects induced by reactive oxygen species, pH changes, or any component present in the specific or azurophil granules. At present, we do not know why the intensity of Fura-2 fluorescence decreases after phagocytosis both when excitated with 340 nm and 380 nm light. However, the ratio technique inherently compensate for non-specific quenching of fluorescence, such as photobleaching and non-specific (Ca2+-independent) alterations in fluorescence [23] and as a consequence the Fura-2 zymosan © Harcourt Publishers Ltd 2000
could still respond to changes in the concentration of Ca2+. In a Ca2+-depleted medium as well as in econazoletreated cells in 1 mM Ca2+ medium, the 340 nm/380 nm ratio of the particles remain constant, while it rapidly falls in particles phagocytosed by untreated neutrophils in 1 mM Ca2+ medium, even though fading is seen in all cases. The most direct way to determine changes in the calcium concentration in a phagosome is to label the prey, but other approaches have been used to study Ca2+ changes in other subcellular compartments of endocytic origin. Using such a technique, Gerasimenko et al. [37] found a rapid loss of Ca2+ also from endosomes, suggesting that the rapid loss of Ca2+ may be a general property of organelles of endocytic/phagocytic pathways. In summary, we suggest that the Ca2+ needed for postingestion processing of a phagosome is provided not only from intracellular stores, but also from the phagosome, containing extracellular medium passively entering the phagosome during invagination of the plasma membrane. Since the post-ingestion processing of the phagosome is required for proper neutrophil killing of a phagocytosed prey, it is interesting that some intracellular parasites have developed strategies to inhibit this processing, and thereby escape a neutrophil attack. Our experimental model may prove useful for studies of intraphagosomal Ca2+ changes and the processing of the phagosome in general, and in studies of the mechanisms evolved by pathogens to interfere with this processing. This opens up new possibilities to improve phagolysosomal fusion, with pharmacological means, in situations in which the Ca2+ channels remain closed after phagocytosis. ACKNOWLEDGEMENTS This work was supported by grants from the Swedish Medical Research Council, King Gustaf the V 80-year foundation, The Anna-Greta Crafoord Foundation, The Lundberg Research Foundation, The Swedish Society against Rheumatism, The Swedish Medical Society, The Swedish Heart and Lung foundation, Tore Nilsons Fund for Medical Research and the Östergötland County Research Fund. REFERENCES 1. Brown EJ. Phagocytosis. Bio Essays 1995; 17: 109–117. 2. Borregaard N, Cowland JB. Granules of the human neutrophilic polymorphonuclear leukocyte. Blood 1997; 89: 3503–3521. 3. Clark RA, Leidal KG, Pearson DW, Nauseef WM. NADPH oxidase of human neutrophils. Subcellular localization and characterization of an arachidonate-activatable superoxidegenerating system. J Biol Chem 1987; 262: 4065–4074. 4. Klebanoff SJ. Oxygen metabolites from phagocytes. In: Gallin JI, Snyderman R (eds) Inflammation Basic principles and clinical correlates Raven press 1999; 721–768.
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5. DeLeo FR, Quinn MT. Assembly of the phagocyte NADPH oxidase: molecular interaction of oxidase proteins. J Leukoc Biol 1996; 60: 677–691. 6. Hampton MB, Kettle AJ, Winterbourn CG. Inside the neutrophilic phagosome. Blood 1998; 92: 3007–3017. 7. Grennberg S. Biology of phagocytosis. In: Gallin JI, Synderman R Inflammation. Basic principles and clinical correlates (eds) Baltimore: Lippincott Williams & Wilkins, 1999; 681–702. 8. Fällman M, Gullberg M, Hellberg C, Andersson T. Complement receptor-mediated phagocytosis is associated with accumulation of phosphatidylcholine-derived diglyceride in human neutrophils. J Biol Chem 1992; 267: 2656–2663. 9. Krause C-H, Clarke RA, Wymann MP. Meeting Report: European workshop on human phagocytes. J Leukoc Biol 1997; 61: 1–6. 10. Lew DP, Andersson T, Hed J, Di Virgilio F, Pozzan T, Stendahl O. Ca2+-dependent and Ca2+-independent phagocytosis in human neutrophils. Nature 1985; 315: 509–511. 11. Wilson Å, Lundqvist H, Gustafsson M, Stendahl O. Killing of phagocytosed Staphyloccocus aureus by human neutrophils requires intracellular free calcium. J Leukoc Biol 1996; 59: 902–907. 12. Lew PD, Wollheim CB, Waldvogel FA, Pozzan T. Modulation of cytosolic-free calcium transients by changes in intracellular calcium-buffering capacity: correlation with exocytosis and O2-production in human neutrophils. J Cell Biol 1984; 99: 1212–1220. 13. Jaconi MEE, Lew DP, Carpentier J-L, Magnusson KE, Sjögren M, Stendahl O. Cytosolic free calcium elevation mediates the phagosome-lysosome fusion during phagocytosis in human neutrophils. J Cell Biol 1990; 110: 1555–1564. 14. Lew PD, Monod A, Waldvogel FA, Dewald B, Baggiolini M, Pozzan T. Quantitative analysis of the cytosolic free calcium dependency of exocytosis from three subcellular compartments in intact human neutrophils. J Cell Biol 1986; 102: 2197–2204. 15. Bengtsson T, Jaconi MEE, Gustafsson M, Magnusson K-E, Theler J-M, Lew DP, Stendahl O. Actin dynamics in human neutrophils during adhesion and phagocytosis is controlled by changes in intracellular free calcium. Eur J Cell Biol 1993; 62: 49–58. 16. Uhling RL, Snyderman R. Chemoattractant stimulus-response coupling. In: Gallin JI, Synderman R (eds) Inflammation. Basic principles and clinical correlates. Raven press 1999; 607–626. 17. Berridge MJ. Inositol trisphosphate and calcium signalling. Nature 1993; 361: 315–325. 18. Davies EV, Campbell AK, Hallett MB. Dissociation of store release from transmembrane influx of calcium in human neutrophils. FEBS 1992; 313: 121–125. 19. Stendahl O, Krause K-H, Kricher J, Jerström P, Theler J-M, Clark RA, Carpentier J-L, Lew DP. Redistribution of intracellular Ca2+ stores during phagocytosis in human neutrophils. Science 1994; 265: 1439–1441. 20. Böyum A. Isolation of mononuclear cells and granulocytes from human blood. Scand J Clin Lab Invest 1968; 21(suppl 97): 77–89. 21. Dahlgren C, Hed J, Stendahl O. Chemotaxis of polymorphonuclear leukocytes in response to surface-bound
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Research
doi: 10.1054/ceca.2000.0130, available online at http://www.idealibrary.com on
Dynamic Ca2+ changes in neutrophil phagosomes A source for intracellular Ca2+ during phagolysosome formation? H. Lundqvist-Gustafsson,1 M. Gustafsson,2 C. Dahlgren3 1
Division of Pathology II, Faculty of Health Sciences, Linköping University, Sweden Department of Internal Medicine, Vrinnevi Hospital, Norrköping, Sweden 3 The Phagocyte Research Laboratory, Department of Medical Microbiology and Immunology, University of Göteborg, Sweden 2
Summary An increase in cytosolic Ca2+ concentration periphagosomally is critical for phagolysosomal formation and neutrophil elimination of microbes. The Ca2+ increase could be achieved through release of Ca2+ from mobilized intracellular stores. Alternatively, Ca2+ that passively enter the phagosome during phagocytosis could be provided by the phagosome. Intraphagosomal Ca2+ changes in single human neutrophils was measured during phagocytosis of serum opsonized Fura-2-conjugated zymosan particles, using a digital image processing system for microspectrofluorometry. A decrease in phagosomal Ca2+ down to nanomolar concentrations was seen within minutes following phagosomal closure. Blockage of plasma membrane Ca2+ channels by econazole abolished this decrease. The fluorescence properties of Fura-2 zymosan were retained after phagocytosis and stable to pH changes, reactive oxygen species, and proteolytic enzymes. We suggest that Ca2+ ions present in the phagosome enter the cell cytosol through Ca2+ channels in the phagosomal membrane, achieving a localized Ca2+ rise that is important for phagosome processing. © 2000 Harcourt Publishers Ltd
INTRODUCTION The neutrophil granulocyte is an important inflammatory cell participating in the body’s defense against invading microorganisms. The neutrophil exerts its function by phagocytosis and intracellular killing of pathogenic microorganisms. Binding of a foreign microorganism to neutrophil surface receptors generates signals that regulate the phagocytic process [1]. During this process, the plasma membrane forms an invagination that surrounds the microbe. Phagocytosis is completed when the invaginated membrane is closed; forming a new membrane enclosed intracellular vesicle, the phagosome. However, killing of microbial intruders also includes the delivery of the cellular bactericidal arsenal to the phagosome. These bactericidal weapons are stored in subcellular organelles, Received 24 February 2000 Revised 23 June 2000 Accepted 23 June 2000 Correspondence to: Helen Lundqvist-Gustafsson, Division of Pathology II, Faculty of Health Sciences, S-581 85 Linköping, Sweden. Tel: +46 13 221 525; fax: +46 13 221 529; e-mail: [email protected]
granules (reviewed in 2), of which the azurophil and the specific granules are the best characterized. The granules contain a variety of proteins including myeloperoxidase (MPO, present in the azurophil granules) and a unique b type cytochrome (present mainly in the specific granules). The b cytochrome is the electron transporting protein of the superoxide anion (O2–) and hydrogen peroxide (H2O2) generating nicotinamide adenine dinuclotide phosphate(NADPH-) oxidase [3,4]. The reduced oxygen species formed by the oxidase can be metabolized further to hypochlorous acid (HOCl) and other potent microbicidal substances [5]. Assembly and activation of the NADPHoxidase in the membrane of the phagosome [6,7] as well as phagosome-lysosome fusion are, thus, processes required for the efficient killing of an ingested microorganism. Neutrophil phagocytosis, and the activation of the bactericidal arsenal are highly regulated processes that are mediated by signals generated secondary to the binding of a particle to phagocyte membrane receptors. The complement receptor 3 (CR3; CD11b/CD18) is essential for phagocytosis of complement opsonized particles. During phagocytosis mediated by this receptor, phospholipase D 353
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(PLD) is activated which results in diacylglyceride formation and activation of protein kinase C [8], however, heterotrimeric G-proteins or inherent tyrosine kinases are not involved in the activation process [9]. The precise molecular basis of neutrophil activation through this receptor remains to be determined. In neutrophils, as well as in many other cells, changes in the cytosolic concentration of calcium ions ([Ca2+]i) play an important role in cell activation. A rise in [Ca2+]i is not required for uptake of microorganisms through phagocytosis [10], but it is a necessary signal for the killing of the ingested prey [11]. The requirement for Ca2+ in post-ingestion killing probably relates to the regulatory function of this ion in the assembly/activation of the NADPH-oxidase [11,12] as well as in the docking/fusion process leading to the formation of a phagolysosome [13,14]. Furthermore, an increase in [Ca2+]i is a prerequisite for dissolving the actin barrier around the phagosome, thus enabling fusion of granules and assembly of the NADPH-oxidase in the phagosomal membrane [15]. The formation of inositol-1,4,5-trisphosphate (Ins(-1,4,5)P3) [16,17] is the direct signal to release Ca2+ from intracellular stores probably closely related to the endoplasmic reticulum. However, these Ca2+ stores are not the only source for Ca2+, since Ca2+ ions can be transported from the extracellular fluid through plasma membrane channels and be released directly into the cytosol [18]. The local rise in [Ca2+]i in the periphagosomal area that is seen during phagocytosis [13] has been attributed to either a release of Ca2+ from intracellular stores, or an opening of Ca2+ channels present in the plasma membrane, or a combination of both these processes. It has been shown that the cytosolic Ca2+ stores translocate to the vicinity of the phagosome, and the local increase in [Ca2+]i (mediating post-ingestion processing of the phagosome) has been proposed to be a result of a Ca2+ release from these accumulated stores [19]. Based on the fact that the content of the phagosome constitutes a part of the previously extracellular milieu (that has a Ca2+ concentration above 1 mM) [16], we investigated if the release of calcium ions through the phagosomal membrane into the cytosol might contribute to the local Ca2+ signal required for post-ingestion processing of the phagosome. To investigate whether Ca2+ from the phagosome itself can enter the cytosolic compartment and, thus, facilitate phagolysosome fusion, we used serum opsonized Fura-2 conjugated zymosan particles to directly measure intraphagosomal Ca2+ levels during the phagocytosis process. MATERIALS AND METHODS Isolation of neutrophils Neutrophils were isolated from heparinized blood obtained from healthy adults. After dextran sedimentaCell Calcium (2000) 27(6), 353–362
tion at 1 g, hypotonic lysis of the remaining erythrocytes and centrifugation on a Ficoll-Pacque gradient [20], the neutrophils were washed twice and resuspended in Krebs-Ringer Glucose buffer (KRG, containing 10 mM glucose, 1 mM Ca2+ and 1.5 mM Mg2+, pH 7.3), and stored on melting ice. Samples were withdrawn and used in the experiments within 4 h of preparation. Preparation of Fura-2 conjugated zymosan particles Molecular Probes (Eugene, OR) has developed an aminereactive Fura derivative that upon request can be conjugated to proteins for site-selective targeting. In order to obtain a particle that should allow us to determine intraphagosomal Ca2+ changes, amine-reactive Fura derivative was covalently bound to zymosan particles. The freeze-dried Fura-2 conjugated zymosan particles were suspended and washed in PBS and resuspended in PBS containing 2 mM azide (1.5 × 107 particles/ml) and stored at –20°C until use. Video microscopy and ratio imaging Two different microscope systems were used in the experiments: 1. A Zeiss IM-35 (Carl Zeiss, Oberkochen, Germany) microscope equipped for epi-fluorescence with a 75 W xenon arc lamp as the excitation source was used together with a glycerol-immersed Zeiss neofluar Ph3, 100x objective. During measurements, the sample was alternatively excited at 340 nm and 380 nm, and the 510 nm emission light was recorded by an image intensifier (Hamamatsu C-2100) attached to an integrating colour CCD camera (Zeiss-ZVS-47-E, used in B/W mode). For simultaneous phase contrast microscopy, light emitting diodes emitting at 640 nm was used as a light source. In the emission path, a 580 nm dichroic mirror extracted the Fura-2 emission from the transillumination light. The latter was recorded with a Newicon B/W camera (Hamamatsu C2400–7), The resulting images were captured and digitized by a frame grabber (Innovative Vision AB, Linköping, Sweden) attached to a personal computer (Hewlett Packard Vectra U-66). One image pair and one phase contrast image, captured within 2 s from each other, were grabbed every 10 s, each image being calculated as the mean of four successive video frames (jumping average). The image sequences along with timing information were continuously stored on the hard disk for later evaluation. 2. In some experiments, a Zeiss axiovert 135 TV microscope with a 100 W mercury arc lamp as the excitation source was used together with a © Harcourt Publishers Ltd 2000
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glycerol-immersed Zeiss achrostigmat Ph3, 40x objective. A computer controlled filter wheel (Hamamatsu C-4312) was used to alternate between 340 nm and 380 nm filters, and the fluorescence was recorded by an image intensifier (Hamamatsu C-2400–80) attached to a CCD camera (Hamamatsu C-2400–77).
Phagocytosis experiments Fura-2 zymosan (1 µl containing approximately 1.5 × 104 particles) were added to glass cover slips and the drop was allowed to dry whilst being mechanically spread with a pipette tip. The slides were mounted in a chamber to which 300 µl normal human serum was added. The chambers were incubated for 5–10 min at the heated (37°C) microscope stage to opsonize the particles and allow a surface bound chemotactic gradient to be built up [21]. Prewarmed KPG (3 ml) and neutrophils (30 µl containing 3× 105 cells) were then added. The Fura-2 fluorescence (emission wavelength 510 nm) of the particles was followed, exciting the particles alternately with 340 and 380 nm light. A measurement was started when a neutrophil, crawling on the cover slip, approached a zymosan particle. Microscope fields were chosen where phagocytosed as well as non-phagocytosed zymosan particles were present and could be analyzed in parallel.
The ratiometric response of suspended particles to Ca2+, is very similar to the response of free Fura-2 sodium salt. Our Kd determination reveals a value for the particles that are very close to that for free Fura-2 salt, allowing us to use the established Kd value for the free Fura-2 salt (224 nM) in our calculations of intraphagosomal Ca2+-levels. Subcellular fractionation Neutrophils (2 × 108/ml) were resuspended in ice-cold disruption buffer (KCl, 100 mM; NaCl, 3 mM; MgCl2, 3.5 mM; adenosine trisphosphate, 1 mM; piperazine-N,Nbis(2-etanesulfonic acid), 10 mM; pH 7.4) and disrupted by nitrogen cavitation (Parr Instrument Company, Moline, IL). The post nuclear supernatant was centrifuged on a two-layer Percoll gradient as described by Borregaard et al. [25]. The gradients, containing three visible bands corresponding to the azurophil granules (α-fraction), the specific granules (β-fraction) and the plasma membranes/secretory vesicles (γ-fraction), were collected (1.5 ml fractions) by aspiration from the bottom of the centrifuge tube. Percoll was removed by centrifugation and the azurophil granule and specific granule fractions were resuspended in disruption buffer (2 ml). To disrupt the membranes, the granule fractions were freeze-thawed three times immediately before use. Reagents
Evaluation of optical data, calculation of [Ca2+]i The stored images were evaluated off line using a slightly modified computer program, previously described in detail [22]. Briefly, the ratio between the 340 nm and the 380 nm images was calculated pixel by pixel and the resulting image was presented on a monitor after calibrated pseudo colouring. This procedure allows estimation of [Ca2+] while correcting for differences in illumination intensity, photo bleaching, cell thickness and probe concentration [22–24]. Quantitative data were extracted from the images as described [22] for later plotting. For visual assessment, the phase contrast images and corresponding ratio images were sequentially displayed side by side on the monitor in an infinite loop manner. [Ca2+] was calculated using the formula: [Ca2+]=Kd × β × (R-Rmin)/(Rmax-R), where R is the ratio between the 340 nm and 380 nm fluorescence intensity values, Kd is the dissociation constant for the Fura-2/Ca2+ complex (224 nM), β is the ratio between fluorescence from free Fura-2 and Ca2+-saturated Fura-2 excited at 380 nm, Rmax is the ratio obtained when Fura-2 is saturated with Ca2+, Rmin is the ratio obtained when Fura-2 is free from Ca2+ according to Grynkiewicz et al. [23]. © Harcourt Publishers Ltd 2000
Reagents were bought from the following companies: Dextran, Ficoll-Pacque and Percoll: Pharmacia Fine Chemicals, Uppsala, Sweden. Custom synthesized Fura-2 conjugated zymosan A BioParticles: Molecular Probes Inc., Eugene, OR, USA, H2O2: Merck, Darmstadt, Germany. Econazole and adenosine trisphosphate: Sigma Chemical Co, IL, USA. MPO was a generous gift from Dr Inge Olsson, Lund, Sweden.
RESULTS Fluorescence of Fura-2 zymosan particles during phagocytosis The fluorescence changes of Fura-2 particles were followed during phagocytosis of the particles. After formation of a phagosome the 340 nm/380 nm ratio decreased, while the ratio of the non-phagocytosed Fura2 particles remained stable (Fig. 1A). The fluorescence intensity of ingested Fura-2 zymosan particles declined (faded) when excited by both at 340 and 380 nm, while the fluorescence of non-phagocytosed adjacent particles only showed a slight fading (Fig. 1A). Fura-2 zymosan particles phagocytosed in the absence of Ca2+ (the buffer Cell Calcium (2000) 27(6), 353–362
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Fig. 1 Fluorescence properties and ratio changes of Fura-2 zymosan after phagocytosis. Immediately after closure of a phagosome, the fluorescence ratio of Fura-2 excited at 340 nm and 380 nm was rapidly decreased in the presence of Ca2+ (A), while the ratio remained constant in the absence of Ca2+ (1 mM EGTA added; B). The arrows indicate the time for closure of the phagosome. Insert: free (non-phagocytosed) control particles analyzed in parallel with the phagocytosed ones. Data are from one representative measurement out of at least nine. The fluorescence values are given in arbitrary units (a.u.)
containing 2 mM EGTA) showed a similar decrease in fluorescence intensity. However, more importantly, the 340 nm/380 nm ratio of the particles was not affected in the absence of Ca2+, but remained at a constant Rmin level throughout the measurements (Fig. 1B). Furthermore, the ratio was constant in econazole-treated cells (see below) showing that the conjugated particles retain their sensitivity for Ca2+. The intraphagosomal Ca2+-concentration is lowered after phagocytosis The Ca2+-concentration in KRG (1 mM) is sufficient to saturate the Fura-2 molecules on the particles, and, thus, Cell Calcium (2000) 27(6), 353–362
Rmax of each individual particle was determined from the first pre-phagocytosis data points in each experiment. The variation of Rmax was 2.05 ± 0.54; mean ± sd, n=16. Owing to the fact that the Rmin values varied only little between different particles (0.58 ± 0.05, n=16), the same Rmin value (0.58) was used for all particles. From these Rmax and Rmin values, and from the ratio of phagocytosed Fura-2 zymosan, we calculated the corresponding Ca2+ concentration in the phagosomes during the phagocytic process. A dramatic decrease in Ca2+-concentration was seen already within seconds after closure of the phagosome, and the concentration reached nanomolar levels (583 nM ± 130 nM, mean ± sd, n=6) after a few minutes. Data from a representative measurement is shown in © Harcourt Publishers Ltd 2000
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Fig. 2 Fura-2 zymosans used as probes to determine calcium changes in the neutrophil phagosome/phagolysosome. Panel I: Phase contrast microphotographs of a neutrophil (a) and two Fura-2 zymosans (b and c). The neutrophil reaches one of the Fura-2 zymosans (b) 40 s after the start of the measurement (A) and after 80 s the ingestion is terminated and a phagosome is formed (B). Micrographs were also taken after 230 s (C) and 360 s (D). The free Fura-2 zymosan (c) is a control particle. From the fluorescence values of the phagocytosed Fura-2 zymosan (b), the corresponding Ca2+ concentration in the maturing phagosome was calculated (Panel II). (A–D) the arrows correspond to the microphotographs taken. The fluorescence and the corresponding Ca2+ values of the non-phagocytosed Fura-2 zymosan (c) is shown in panel III. The experiment was repeated with neutrophils from ten different blood donors, and we analyzed 3–13 phagocytosed and at least three non-phagocytosed particles at each occasion. Data from one representative measurement. a.u.: arbitrary units.
Figure 2. Thus, lowering of phagosomal Ca2+ down to levels corresponding to those that have been measured in the periphagosomal area [13,26,27]. seem to bee an early event in the processing of the phagosome. The role of Ca2+ channels in the efflux of Ca2+ from the phagosome In order to determine if the lowering in Ca2+ concentrations in the phagosome truly reflects an efflux of Ca2+ through Ca2+ channels in the phagosomal membrane, we blocked the opening of plasma membrane Ca2+ channels with the antimycotic imidazole derivative econazole [28]. The plasma membrane derived membrane enclosing a prey should then not be able to transport calcium from the phagosome. We found that econazole dose-depen© Harcourt Publishers Ltd 2000
dently blocked intraphagosomal Ca2+-changes [Fig. 3], further supporting the specificity of Fura-2 zymosan as tracers of phagosomal Ca2+ changes. Effects of pH changes, reactive oxygen species and granule constituents on Fura-2 zymosan fluorescence The decline in fluorescence after phagocytosis during excitation at either 340 nm or 380 nm, seen irrespectively if the Ca2+ concentration is changed or not, might reflect a functional loss of Fura-2. This could be due to a combinatory effect of oxidation, halogenation, pH changes, and other processes that occur within the phagosome. To test the stability of Fura-2, we investigated the fluorescence properties of the Fura-2 zymosan particles under various environmental conditions. Cell Calcium (2000) 27(6), 353–362
358 H Lundqvist-Gustafsson, M Gustafsson, C Dahlgren
Fig. 4 Effect of pH on the ratio of 340 nm/380 nm fluorescence of Fura-2 zymosan. Particles were spread on the bottom of a microscope chamber in buffer at different pH. The ratio of the 340 nm/380 nm fluorescence of 20 particles was measured at each pH. The numerals represent mean ± sd values. Statistically (Student’s t-test) significant changes from the value obtained at pH 7.3 are marked with **(P<0.05) and ***(P<0.01) respectively.
values in an alkaline milieu (pH~8), but there was no significant difference in ratio when the pH was lowered from physiological levels down to pH 5 (Fig. 4).
Fig. 3 The Ca2+ channel blocker econazole abolishes intraphagosomal calcium changes during phagocytosis of Fura-2 zymosan. Ca2+ measurements were performed as described in the MATERIAL AND METHODS section, with addition of 5, 10 or 50 µM econazole to the buffer (final concentration). Econazole dose-dependently abolished the lowering of Ca2+ in phagosomes (A). Ca2+ concentration in a phagosome (the arrow indicates the time for closure of the phagosome), and the Ca2+ concentration around a free Fura-2 zymosan (insert) in the presence of 50 µM econazole is shown from a representative measurement. The experiment was repeated with neutrophils from three different blood donors, and we analyzed at least six phagocytosed and six free particles at each occasion.
Influence of pH The fluorescence ratio of Fura-2 zymosan was assessed in KRG buffers with a pH-range between 5.0 and 8.6, since pH in the phagosome/phagolysosome may vary between these values during phagocytosis (29, M. G. unpublished observations). We noticed a small shift to higher ratio Cell Calcium (2000) 27(6), 353–362
Influence of H2O2 and HOCl Hydrogen peroxide (H2O2), as well as hypochlorous acid (HOCl), is released into the phagosome and subject microorganisms to oxidative attack. Myeloperoxidase, an enzyme stored in the azurophil granules of neutrophils, mediates (in the presence of chloride anions) the conversion of H2O2 to HOCl. Considering the fact that photo bleaching of Fura-2 has been shown to be related to oxidation of the molecule [30], an oxidative attack could also change the fluorescence properties of Fura-2 zymosan. As can be seen in Figure 5, exposing the particles to H2O2 or hypochlorite (by addition of H2O2 in conjunction with MPO) had no effect, either on fluorescence at the two wavelengths, or on the ratio between them. Influence of granule constituents During fusion between granules and the phagosome, a number of degradative enzymes are released into the phagosome and these bactericidal/proteolytically active factors might affect the fluorescence of the Fura-2 zymosan. A set of experiments was performed in which the particles were subjected to freeze-thawed granule material obtained from isolated azurophil and specific granules respectively. No change in fluorescence during exposure could be seen upon addition of granule © Harcourt Publishers Ltd 2000
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Fig. 5 Resistance of the Fura-2 zymosan to oxygen metabolites. Particles were exposed to H2O2 (1 mM;A), or HOCI (in a mixture of 0.1 mM H2O2 and 1.25 µg purified MPO; B), at times indicated by the arrows. Fluorescence values (a. u. = arbitrary units) of representative particles are shown to the left, and the ratios of the 340 nm/380 nm fluorescence values calculated from the data are shown to the right. Data are from one representative measurement out of four.
extracts originating from the two major granule populations of neutrophils (not shown). In conclusion, the results clearly show that Fura-2 zymosans are stable with respect to the attack from granule material that is delivered to neutrophil phagosomes during phagolysosome formation Fura-2 zymosan particles retain their Ca2+ sensitivity also after phagocytosis From the results above, we can conclude that neither changes in pH, oxidative stress or exposure to granule © Harcourt Publishers Ltd 2000
material (proteases and other destructive enzymes), influences the fluorescence properties of Fura-2 zymosan. However, to make sure that the particles retain their function as Ca2+ indicators also after phagocytosis, we recovered ingested Fura-2 zymosan by lysing cells with Triton X-100 and these particles were then investigated with respect to the response to changes in Ca2+. We found that the Fura-2 particles that have experienced the milieu in the phagolysosome preserve their ability to function as Ca2+ probes as efficiently as do free particles that have not been phagocytosed (Fig. 6).
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Fig. 6 Spectroscopic response of Fura-2 zymosan after phagocytosis. Neutrophils were allowed to phagocytose Fura-2 zymosan on glass slides for 30 min (to ensure that fully processed phagolysosomes were formed). The cells were then lysed with 0.3% Triton X-100. The 340/380 fluorescence ratios of the phagocytosed, Triton X-100-released Fura-2 zymosan were determined in the presence of Ca2+ (1 mM) and in the absence of Ca2+ (EGTA). Representative 340 nm/380 nm ratios of a phagocytosed and released particle (A) and a free control (nonphagocytosed) Fura-2 zymosan (B) are shown. The experiment was repeated with neutrophils from three different blood donors, and we analyzed at least five phagocytosed and five free particles at each occasion.
DISCUSSION Phagocytic uptake of serum opsonized particles, mediated by complement receptors 3 (CR3) on the neutrophil surface, is independent of the presence of Ca2+ in the extracellular medium and an increase in cytosolic Ca2+. However, buffering of intracellular Ca2+ combined with a Cell Calcium (2000) 27(6), 353–362
removal of extracellular Ca2+ affects the post-ingestion processing of the phagosome. This processing includes translocation to the phagosomal membrane of Ca2+ regulated phospholipid binding proteins, e.g. the annexins III and XI [31,32] and cytosolic components required for assembly of the NADPH-oxidase [11] as well as recruitment of granules [13]. Moreover, the redistribution of cytosolic proteins and granules to the phagosomal membrane is facilitated by depolymerization of the surrounding actin network, a Ca2+ dependent process [15]. Thus, intracellular Ca2+ homeostasis, probably especially so in the periphagosomal region, is of crucial importance for the antimicrobial action of neutrophils. It should be noted though, that intraphagosomal NADPH-oxidase activity [11] as well as phagolysosomal fusion [13], also can occur in Ca2+ depleted cells, provided that Ca2+ is present in the extracellular medium. A localized rise in the Ca2+ concentration in the periphagosomal area has been described by other authors [13,27]. This finding has been attributed to a release from intracellular Ca2+ stores that redistribute and accumulate around the phagosome early in the phagocytic process [19]. In this paper, we demonstrate that a decrease in Ca2+ concentration in the phagosome from millimolar (the concentration in the buffer used), down to nanomolar levels, occurs within a few minutes after closure of the plasma membrane invagination around a particle. Recent data suggest that the amount of invaginated plasma membrane is considerably larger than has been previously appreciated, thus increasing the likelihood of a significant coingestion of extracellular constituents [33]. Econazole, an antimycotic imidazole that has been shown to inhibit opening of Ca2+ channels present in the neutrophil plasma membrane [28], abolished the decrease in the intraphagosomal concentration of Ca2+ in a dose-dependent way. This strongly suggests that an opening of plasma membrane channels is the mechanism responsible for the Ca2+ dissipation from the phagosome, even though we cannot rule out that Ca2+/Na+ and/or Ca2+/H+ exchangers are participating in the transport as well [34]. Our results also suggest a mechanism by which the local rise in [Ca2+]i in the periphagosomal area that is seen during neutrophil phagocytosis could be obtained in Ca2+ depleted cells. On the basis of our results and those of others, we suggest that Ca2+ needed for post-ingestion processing of the phagosome can be provided either from mobilized intracellular stores releasing Ca2+ in the periphagosomal area, or from the phagosome itself. The phagosome could then act as an intracellular Ca2+-store and mediate a localized rise in Ca2+ around the phagosome mediated by Ca2+channels in the phagosomal membrane. In a situation that resembles the normal physiological state of a cell (Ca2+ present both intracellularly and in the extracellular © Harcourt Publishers Ltd 2000
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environment), phagocytosis is associated with a rapid, transient increase in [Ca2+]i starting immediately when a neutrophil comes into contact with the prey. The initial rise in [Ca2+]i is followed by a sustained response that ceases after a couple of minutes when [Ca2+]i returns to resting values [13]. The initial rise is dependent on release of Ca2+ from intracellular stores and the second phase is related to an opening of calcium channels in the plasma membrane resulting in an influx of Ca2+ from the extracellular milieu [35]. The plasma membrane pathway, activated by emptying the intracellular Ca2+ stores, is disabled in the presence of cytochrome P-450 inhibitors like econazole [28]. Consequently, the inhibitory effect of this drug in our study indicates that the opening of the Ca2+ channels in the plasma membrane-derived phagosomal membrane is regulated by the same mechanism. Since the concentration of Ca2+ in the extracellular fluid (1 mM) differs from that in the cytosol of a resting cell by a factor 104 [16] and the Kd of Fura-2 is in the nanomolar range, the probe is reasonably accurate for Ca2+ measurements between 50 and 1000 nM, but is rather insensitive to Ca2+ changes in the millimolar range. Resting levels in neutrophils lie typically between 80 and 200 nM, and the concentration in activated cells usually do not exceed 2000 nM [13,26,36]. Although the accuracy for Ca2+ determination in the initial phase of the phagocytic process, therefore, is low, Fura-2 zymosan seem to be useful for studies of intraphagosomal Ca2+changes. The Ca2+-sensitive fluorescence properties are retained after phagocytosis and stable with respect to the influence of reactive oxygen species, proteolytic enzymes and pH changes to which they are exposed during formation of phagolysosomes. The ratio between the 340 nm and 380 nm fluorescence intensity of Fura-2 zymosan did change slightly at an alkaline pH, but this finding was of minor importance for the interpretation of our data. If an alkalinization of the phagosome should occur [29], the decrease in apparent intraphagosomal Ca2+ as registered in our system, would be underestimated rather than overestimated. However, we have not been able to confirm the occurrence of a pH increase during phagocytosis of zymosan (unpublished observations). The Ca2+-independent decline in fluorescence of Fura-2 observed could not be shown to relate to effects induced by reactive oxygen species, pH changes, or any component present in the specific or azurophil granules. At present, we do not know why the intensity of Fura-2 fluorescence decreases after phagocytosis both when excitated with 340 nm and 380 nm light. However, the ratio technique inherently compensate for non-specific quenching of fluorescence, such as photobleaching and non-specific (Ca2+-independent) alterations in fluorescence [23] and as a consequence the Fura-2 zymosan © Harcourt Publishers Ltd 2000
could still respond to changes in the concentration of Ca2+. In a Ca2+-depleted medium as well as in econazoletreated cells in 1 mM Ca2+ medium, the 340 nm/380 nm ratio of the particles remain constant, while it rapidly falls in particles phagocytosed by untreated neutrophils in 1 mM Ca2+ medium, even though fading is seen in all cases. The most direct way to determine changes in the calcium concentration in a phagosome is to label the prey, but other approaches have been used to study Ca2+ changes in other subcellular compartments of endocytic origin. Using such a technique, Gerasimenko et al. [37] found a rapid loss of Ca2+ also from endosomes, suggesting that the rapid loss of Ca2+ may be a general property of organelles of endocytic/phagocytic pathways. In summary, we suggest that the Ca2+ needed for postingestion processing of a phagosome is provided not only from intracellular stores, but also from the phagosome, containing extracellular medium passively entering the phagosome during invagination of the plasma membrane. Since the post-ingestion processing of the phagosome is required for proper neutrophil killing of a phagocytosed prey, it is interesting that some intracellular parasites have developed strategies to inhibit this processing, and thereby escape a neutrophil attack. Our experimental model may prove useful for studies of intraphagosomal Ca2+ changes and the processing of the phagosome in general, and in studies of the mechanisms evolved by pathogens to interfere with this processing. This opens up new possibilities to improve phagolysosomal fusion, with pharmacological means, in situations in which the Ca2+ channels remain closed after phagocytosis. ACKNOWLEDGEMENTS This work was supported by grants from the Swedish Medical Research Council, King Gustaf the V 80-year foundation, The Anna-Greta Crafoord Foundation, The Lundberg Research Foundation, The Swedish Society against Rheumatism, The Swedish Medical Society, The Swedish Heart and Lung foundation, Tore Nilsons Fund for Medical Research and the Östergötland County Research Fund. REFERENCES 1. Brown EJ. Phagocytosis. Bio Essays 1995; 17: 109–117. 2. Borregaard N, Cowland JB. Granules of the human neutrophilic polymorphonuclear leukocyte. Blood 1997; 89: 3503–3521. 3. Clark RA, Leidal KG, Pearson DW, Nauseef WM. NADPH oxidase of human neutrophils. Subcellular localization and characterization of an arachidonate-activatable superoxidegenerating system. J Biol Chem 1987; 262: 4065–4074. 4. Klebanoff SJ. Oxygen metabolites from phagocytes. In: Gallin JI, Snyderman R (eds) Inflammation Basic principles and clinical correlates Raven press 1999; 721–768.
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