Calcium signalling is altered in myeloid cells with a deficiency in NADPH oxidase activity

Abstract

The relation of O₂^.−-production and Ca²⁺ homeostasis was investigated in PLB-985 cell lines and neutrophilic granulocytes from peripheral blood. In differentiated wild-type PLB-985 cells, a high level of O₂^.−-production was associated with a significant decrease in the membrane potential and the inhibition of capacitative Ca²⁺ entry. These correlations were not observed in gp91^{phox –/–} cells or in cells transfected with a non-functional mutant of gp91^phox (Thr341Lys). Membrane depolarization and inhibition of Ca²⁺ entry reappeared in cells transfected with wild-type gp91^phox. These experiments demonstrate that inhibition of Ca²⁺ entry depends on the presence of a functional NADPH oxidase. The Ca²⁺ signal induced by stimulation of chemotactic receptors also showed remarkable differences: [Ca²⁺]_ic in the sustained phase was higher in gp91^phox–/– than in wild-type cells. Alteration of the Ca²⁺ signal was reproduced by treating peripheral blood neutrophils with the NADPH oxidase inhibitor diphenylene-iodonium. It is concluded that the deficiency in O₂^.−-production is accompanied by significant alterations of Ca²⁺ homeostasis in myeloid cells.

Introduction

Stimulation of chemotactic receptors of neutrophilic granulocytes initiates an increase in intracellular Ca²⁺ concentration. The Ca²⁺ signal in neutrophils consists of two components: Ca²⁺ release from intracellular stores and subsequent Ca²⁺ entry by a capacitative mechanism, i.e. via store-operated calcium channels (SOC) [1,2]. This Ca²⁺ signal is a key element in the organization of various effector responses of the cell, such as superoxide (O₂^.−) production, exocytosis of proteins from various granules, cell shape changes and chemotaxis [3]. Interestingly, both the chemotactic agent N-formyl-methionyl-leucyl-phenylalanine (fMLP) and phorbol 12-myristate 13-acetate (PMA), which are effective stimuli of superoxide production, have been shown to inhibit capacitative Ca²⁺ entry in neutrophilic granulocytes [4,5].

The O₂^.−-producing NADPH oxidase enzyme of phagocytic cells transfers electrons from intracellular NADPH to extracellular O₂, thus removing in each cycle one negative charge from the cell. This electrogenic process results in a dramatic change in the membrane potential: from approx. − 60 mV in resting cells up to + 30 to + 50 mV in stimulated cells [6–8]. In a previous study, carried out on blood neutrophils, we demonstrated a close relationship between O₂^.−-production and inhibition of Ca²⁺ entry [9]. The extensive depolarization of the plasma membrane that is associated with O₂^.−-production reduces significantly the driving force for Ca²⁺ entry. However, the possible role of additional regulatory mechanisms directly affecting Ca²⁺ entry pathway(s) such as phosphorylation by protein kinase C has been raised repeatedly [5, 10–14].

The effect of the depolarization due to O₂^.−-production is certainly not limited to Ca²⁺ entry, because it alters the driving force for all ionic components, such as Na⁺, K⁺ and Cl^–. We suggested an additional role for all these electrophysiological alterations in the pathological state developing in cases of chronic granulomatous disease (CGD) [15]. Most recently, Segal and co-workers presented experimental evidence for the critical role of depolarization-induced K⁺ efflux in bacterial killing [16]. An alteration of Ca²⁺ metabolism and [Ca²⁺]_ic could have important secondary effects on the movement of all major ions by modulation of plasma membrane transporters such as Ca²⁺ activated K⁺ channels, Ca²⁺ activated Cl^– channels or the Na⁺/Ca²⁺ exchanger.

Taken together, these findings warrant a detailed investigation of the mechanisms involved in the regulation of Ca²⁺ entry into O₂^.−-producing phagocytic cells and the possible alterations of these processes in CGD cells. The limited availability of cells from CGD patients prompted us to search for a suitable model cell line. PLB-985 cells are human promyelocytic leukaemia cells which, in differentiated form, are able to produce O₂^.− [17]. Zhen et al. [18] developed a cell line from PLB-985 cells in which the gene for gp91^phox was disrupted by homologous recombination (CGD PLB-985 cells). In the present study, we performed a detailed comparison of calcium movements in normal and CGD PLB-985 cells and found that both Ca²⁺ entry and the sustained phase of the calcium signal are significantly elevated in cells deficient in O₂^.− -production. We also demonstrate that introduction of the gp91^phox gene into CGD cells partially corrects alterations of the calcium homeostasis.

Materials and Methods

Materials

FURA-2/AM was obtained from Calbiochem (San Diego, CA, USA); Percoll from Pharmacia (Peapack, NJ, USA); fetal bovine serum from Agrobio Gmk (Gödöllő, Hungary); di-O-C₅(3) from Molecular Probes (Eugene, OR, USA); valinomycin from Fluka (Buchs, Switzerland); hygromycin from Invitrogen (Grand Island, NY, USA); l-glutamine, dimethyl-formamide, penicillin–streptomycin, NaHCO₃, RPMI-1640 medium, thapsigargin, cytochrome-c, nitroblue tetrazolium (NBT), diphenylene iodonium (DPI) and ionomycin from Sigma (St Louis, MO, USA). All other reagents were of research grade. The extracellular medium (called H-medium) contained: 145 mm NaCl, 5 mm KCl, 1 mm MgCl₂, 0,8 mm CaCl₂, 10 mm HEPES, 5 mm glucose, pH = 7·4. The Ca²⁺ free H-medium had the same composition except that 1 mm Na-EGTA was substituted for CaCl₂.

Cell lines

Wild-type and gp91^phox deficient PLB-985 cell lines were a kind gift from Dr M. Dinauer, Indianapolis, IN, USA. Thr341Lys mutant of gp91^phox was obtained by site-directed mutagenesis using the Quickchange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) according to the manufacturer's instructions. cDNA constructs encoding the wild-type (referred to hereafter as S1 cells) or mutant gp91^phox (referred to hereafter as M1 cells) were cloned into pLZRS. pLZRS constructs were expressed into a retroviral packaging cell line (φnx-α). After puromycin selection, virus was harvested and used for retroviral transduction of PLB-985 X-CGD cells (using 10 µg/ml of Dotap (Roche, Basel, Switzerland)). Transduced cells were stained with 7D5 (kind gift from Dr M. Nakamura, Nagasaki, Japan) and phycoerythrin-labelled Fab2 fragments of goat-antimouse-Ig, and sorted by means of a FacsStar (Becton Dickinson, Franklin Lakes, NJ, USA) cell sorter.

Differentiation into neutrophilic granulocytes was carried out in the presence of 0·5% dimethyl-formamide. Wild-type, PLB-985 X-CGD and M1 cell lines were differentiated for 6–7 days, and the PLB-985 S1 cells for 5 days. Differentiation was controlled on the basis of CD11b expression, using phycoerythrin-labelled anti-CD11b antibodies. Analysis of the appearance of the differentiation marker CD11b and gp91^phox on the cell surface of the different cell lines used in this study is summarized in Fig. 1.

Fig. 1.

Differentiation and expression of gp91^phox in different PLB-985 cell lines. Surface expression of the neutrophil differentiation marker CD11b (upper panels; ···, isotype control; —, anti-CD11b) and the larger membrane-bound subunit of the NADPH oxidase complex, gp91^phox recognized by antibody 7D5 (lower panels; ···, isotype control;, —7D5) on differentiated, unstimulated cells of four different PLB-985 cell lines was assessed by flow cytometry as described in Materials and methods.

Preparation of neutrophil granulocytes from human blood

Venous blood was drawn from healthy volunteers who gave written consent following the presciptions of the Ethical Committee of the Semmelweis University (permission no. 40/1998). Dextran sedimentation was followed by Ficoll-Paque gradient centrifugation according to the procedure described in [19]. Contaminating red cells were removed by hypotonic lysis. Cells were finally resuspended in H-medium and kept at room temperature until use. Preparations contained more than 95% neutrophils, viability as determined by erythrosin B dye exclusion exceeded 95%.

Measurement of Ca²⁺ entry

The cells were loaded with 4 µm FURA-2/AM at a concentration of 5 × 10⁷/ml for 30 min at 37°C in the dark. Thereafter, the cells were washed twice with H-medium (500 g, 10 min, 25°C) to remove the extracellular dye. The washed cells were resuspended in H-medium at a density of 5 × 10⁷/ml and stored at room temperature until use.

For measurement of calcium entry 3 × 10⁶ of FURA-2-loaded cells were resuspended in 3 ml of Ca²⁺ free H-medium in a methylacrylate-cuvette and incubated at 37°C for 4–5 min under stirring conditions. Changes of FURA-2 fluorescence were monitored in a Deltascan dual-wavelength spectrofluorimeter (Photon Technology International, South Brunswick, NJ, USA) using wavelengths 340 nm and 380 nm for excitation and 505 nm for emission. The speed of data collection was two measurements/ s. [Ca²⁺]_ic was calculated from the ratio of the fluorescent data at 340 and 380 nm (after the subtraction of the background values) with the use of the method described in [20]. Data were analysed with the Felix software (PTI).

Measurement of Mn²⁺ entry

Mn²⁺ influx measurements were made under the same conditions as the calcium measurements except that the excitation wavelength was 360 nm, at which the fluorescence of FURA-2 is not influenced by the calcium-binding state of the dye. Mn²⁺ ions entering the cells quench the fluorescence of the dye and thereby cause a decrease in the fluorescence. In the protocol, the cells were suspended in Ca²⁺ free H-medium and the capacitative calcium entry pathway was activated by the addition of 100 nm thapsigargin. Eight minutes after thapsigargin, 100 nm PMA or its solvent DMSO (as control) was added, and 10 min after thapsigargin 1 mm MnCl₂ was introduced into the cuvette.

Measurement of O₂^.−-production

Superoxide production of the cells was tested with the superoxide dismutase-inhibitable cytochrome-c reduction. To measure superoxide production in the extracellular medium, cells (10⁶/ml) were suspended in H-medium containing 100 µm cytochrome-c. Control probes contained 12·5 µg/ml superoxide-dismutase. Aliquots (200 µl) of the suspension were added into wells of a 96-well plate and prewarmed at 37°C for 5 min in a shaking ELISA-reader (Labsystems iEMS Reader MF). The cells were activated with the requisite stimuli by the addition of 5 µl of the stimulus solution. The changes in absorption at 550 nm were recorded for 10 min with two measurements/min at 37°C with gentle shaking. After subtracting the background values superoxide production was calculated with the use of an absorption coefficient of 21 mm⁻¹cm⁻¹ for cytochrome-c.

Measurement of membrane potential changes

Changes in the membrane potential were followed by the potential-sensitive fluorescent dye 3–3′-dipentyloxacarbocyanine, di-O-C₅(3) as described in [21]. Cells, 10⁶, were suspended in 3 ml of H-medium in a stirred cuvette at 37°C and the changes in fluorescence were followed in the spectrofluorimeter used for calcium measurements with 484 nm as excitation and 510 nm as emission wavelengths. The cells were preincubated for 5 min. Subsequently, 100 nm di-O-C₅(3) was added, then the dye was allowed to equilibrate for 6–7 min until baseline fluorescence values were stable. Thereafter, the cells were exposed to various stimuli (fMLP, PMA, ATP) and the changes in fluorescence were recorded. The fluorescence data obtained were converted to membrane potential values (mV) by a calibration curve generated by the application of 2 µg/ml valinomycin to cells suspended in medium containing a series of different external K⁺-concentrations as described in [9].

Statistical analysis

Data are presented either as representative traces of the indicated number of experiments or as the mean ± s.e.m. of the number of determinations indicated (n).

Results

Comparison of O₂^.−-production and accompanying membrane potential changes in the different PLB-985 cell lines

Superoxide production was measured by cytochrome-c reduction upon stimulation with 100 nm PMA (Fig. 2a). Wild-type PLB-985 cells produced 22·7 ± 2·07 nmol O₂^.−/10⁶ cell/10 min (n = 12), which is comparable to the PMA-induced O₂^.−-production of peripheral blood neutrophils. S1 cells with transfected wild-type gp91^phox generated five times less O₂^.− (4·32 ± 0·6 nmol O₂^.−/10⁶ cell/10 min, n = 17). As expected, neither the CGD-PLB model cell line nor M1 cells containing the non-functional mutant gp91^phox produced detectable amounts of O₂^.−.

Fig. 2.

Superoxide production (a) and membrane potential changes (b) in differentiated PLB-985 cells. (a) 10⁶ cells/ml were suspended in H-medium and the SOD-inhibitable cytochrome-c reduction was measured for 10 min at 37°C after stimulation with 100 nm PMA. The results are the mean ± s.e.m. of at least eight different experiments.(b) 3·3 × 10⁵ cells/ml were suspended in H-medium, 100 nm di-O-C₅(3) was added, the cells were stimulated with 100 nm PMA and the changes in fluorescence were measured and calibrated into membrane potential values. The results are representative of three independent experiments. X-CGD cells were transfected with wild-type gp91^phox (S1 cells) or the non-functional Thr341Lys mutant of gp91^phox (M1 cells).

Relative changes of the plasma membrane potential were followed by means of di-O-C₅(3) fluorescent dye (Fig. 2b). Addition of PMA to wild-type PLB-985 cells was followed by a lag-phase of approx. 30 s, corresponding to the typical lag-phase of PMA-induced O₂^.−-production. Thereafter, continuous depolarization ensued for over 200 s. In case of CGD-PLB cells, addition of PMA did not cause any detectable change in the membrane potential. Stimulation of S1 cells with PMA induced clearly detectable depolarization that was significantly smaller than in the wild-type cells. Similarly to CGD-PLB cells, no change of the membrane potential was detected in M1 cells.

Comparison of the effect of phorbol ester on capacitative Ca²⁺ entry in the different PLB-985 cell lines

To investigate Ca²⁺ entry via store-operated channels in the plasma membrane, cells were suspended in a Ca²⁺ free medium and treated with thapsigargin (TG). This drug is a potent inhibitor of the SERCA type Ca²⁺ ATPase of the microsomal membranes, thus preventing the reuptake of Ca²⁺ ions leaking continuously from intracellular stores [22]. The Ca²⁺ releasing effect of TG is indicated clearly by the transient increase in intracellular [Ca²⁺] following TG treatment at 120 s (indicated by * in Fig. 3a,b,c,d).

Fig. 3.

Effect of PMA on the thapsigargin-induced capacitative calcium entry in differentiated PLB-985 cells. FURA-2-loaded cells (10⁶/ml) were allowed to equilibrate for 4–5 min in Ca²⁺ free medium (a–d). Capacitative calcium entry was initiated by addition of 100 nm thapsigargin (marked by *) (T and PT) and 10 min later 1 mm CaCl₂ was added (marked by v) to the extracellular medium. Where indicated, cells have been stimulated by 100 nm PMA 2 min (marked by +) before addition of CaCl₂ (PT). In control experiments (c) basic calcium influx was measured in the presence of DMSO only. (e) Statistical analysis of the inhibitory effect of PMA on capacitative calcium entry in different PLB cells. Inhibition was calculated on the basis of fluorescence change in the initial 30 s after calcium addition. Mean ± s.e.m. of five (wild-type and X-CGD cells) or three (S1) independent experiments is represented, whereas in the case of the M1 cells the average of two measurements is presented.

Addition of Ca²⁺ at t = 720 s to resting cells caused a small increase in the fluorescent signal, due probably to reaction with extracellular dye (marked ‘C’ in Fig. 3). In contrast, addition of Ca²⁺ to TG-pretreated cells induced a rapid rise in [Ca²⁺]_ic from approx. 100 nm up to the µm range (curves ‘T’ in Fig. 3). This increase in the [Ca²⁺]_ic indicates rapid Ca²⁺ influx in the cells via transport pathways opened by store depletion, called capacitative Ca²⁺ entry channels. When the phorbol ester PMA was added (indicated by ‘+’ in Fig. 3) to the TG-pretreated wild-type PLB-985 cells 30 s prior to Ca²⁺, the rise in [Ca²⁺]_ic was significantly smaller (curve ‘PT’ in Fig. 3a). In the experiments carried out on wild-type PLB-985 cells, PMA brought about a significant inhibition of capacitative Ca²⁺ entry. In four separate cell preparations, an average of 77·1 ± 5·4% inhibition of Ca²⁺ influx was attained during the first 30 s (Fig. 3e).

Under identical experimental conditions, no inhibitory effect of PMA was detected upon capacitative Ca²⁺ entry into CGD-PLB cells (Fig. 3b). In cells transfected with wild-type gp91^phox (S1 cells), the inhibitory effect of PMA on Ca²⁺ entry reappeared (Fig. 3c). However, the inhibition observed in S1 cells was weaker than in the wild-type cells, with an average of 45·9 ± 0. 12·1% (Fig. 3e). This difference in the inhibition of Ca²⁺ entry corresponds to the lower rate of O₂^.−-production and the moderate change of the membrane potential observed in S1 cells (Fig. 2). In cells expressing the non-functional mutant of gp91^phox, no inhibitory action of PMA was detected (Fig. 3d).

Monitoring of the quenching effect of Mn²⁺ is another, widely applied method for investigation of the capacitative Ca²⁺ transport pathway. Manganese enters the cells via the same ion channels as Ca²⁺, but it is not a substrate for Ca²⁺ pumps. Reaction of Mn²⁺ with fura-2 quenches the fluorescence of the dye, thus the rate of fluorescence decrease measured at the isosbestic point represents the influx of manganese. The rapid drop in the fluorescence signal upon addition of Mn²⁺ represents the reaction with extracellular dye. In untreated cells there was no change of the fluorescence level after this initial drop (Fig. 4, curves ‘C’) whereas a gradual decrease of fluorescence occurred in thapsigargin-treated cells (Fig. 4, curves ‘T’). Addition to thapsigargin-treated cells of PMA 120 s prior to Mn²⁺ resulted in strong inhibition of fluorescence quenching in case of the wild-type cells (Fig. 4a, curve ‘PT’), whereas no change was observed in CGD-PLB cells (Fig. 4b). PMA also inhibited Mn²⁺ entry into S1 cells (Fig. 4c) but similarly to Ca²⁺ entry this effect was weaker than in wild-type cells. Similar to CGD-PLB cells, PMA was without any effect in M1 cells.

Fig. 4.

Effect of PMA on the thapsigargin-induced Mn²⁺ entry in the PLB-985 cells. FURA-2-loaded PLB-985 cells were suspended in Ca²⁺ free H-medium (10⁶/ml) and allowed to equilibrate for 4–5 min. Capacitative calcium influx was induced by the addition of 100 nm thapsigargin (T, PT), whereas in the control experiment DMSO was added (c). Ten minutes after thapsigargin, 1 mm MnCl₂ was introduced into the extracellular medium (marked by the arrow) and the quenching of the fluorescence was followed in time. Where indicated, 100 nm PMA (PT) was added to the cells 2 min before MnCl₂. The results are representative for five (wild-type and X-CGD) or four (S1) independent experiments.

Dependence of the chemoattractant-induced Ca²⁺ signal on O₂^.−-production in the different PLB-985 cell lines

Ligand binding to the various chemoattractant receptors induces a Ca²⁺ signal both in neutrophils and in promyelocytic cell lines. Comparison of the transient change of [Ca²⁺]_ic in the presence and absence of extracellular Ca²⁺ allows a clear distinction of two different phases: a first, rapid phase representing release from internal stores and a second, prolonged phase dependent on Ca²⁺ entry. Our experiments indicate that O₂^.−-production inhibits Ca²⁺ entry. Thus, the Ca²⁺ signal evoked by O₂^.−-producing chemoattractants is expected to depend on the ability of the cell to generate superoxide. Following this rationale, we compared the Ca²⁺ signal initiated by the chemotactic peptide fMLP in two cell lines, one capable, the other not capable of O₂^.−-production. FMLP-stimulated O₂^.−-production attained an average of 0·97 nmol/10 min/10⁶ cells in nine separate measurements in S1 cells, whereas it was not detectable in CGD-PLB cells.

Comparison of the fMLP-initiated Ca²⁺ signal in S1 and CGD-PLB cells is shown in Fig. 5. In the presence of extracellular Ca²⁺, the decline of the Ca²⁺ signal was significantly slower in the CGD-PLB cells, in which a clear sustained phase appeared (Fig. 5a). The difference in [Ca²⁺]_ic was greatest approx. 120 s after addition of fMLP, when it reached 133 ± 13 nm (n = 4). In the absence of extracellular Ca²⁺, there was no detectable difference between the two cell types in the decline of the Ca²⁺ signal (Fig. 5b), suggesting that release of Ca²⁺ from intracellular stores did not depend on the ability to generate O₂^.−. The amplitude of the Ca²⁺ signal showed no consistent difference between the two cell types, either in the presence or in the absence of extracellular Ca²⁺.

Fig. 5.

Comparison of the fMLP-induced calcium signals in PLB-985 X-CGD and S1 cells in Ca²⁺ containing and Ca²⁺ free medium. FURA-2-loaded, differentiated cells (10⁶/ml) were suspended in Ca²⁺ containing medium (a) or in Ca²⁺ free medium (b). The cells were allowed to equilibrate for 4–5 min and then stimulated by 1 µm fMLP (indicated by the arrow). The curves are representative for three independent experiments.

Dependence of the chemoattractant-induced Ca²⁺ signal on the ability of O₂^.−-production in neutrophilic granulocytes

Our studies on the promyelocytic cell line showed that the chemoattractant-induced Ca²⁺ signal depends on O₂^.−-production, raising the possibility that Ca²⁺ homeostasis may be altered in neutrophils of CGD patients. In earlier studies the fMLP-induced Ca²⁺ signal was investigated in normal and CGD neutrophils, and no difference could be detected [23]. However, those measurements were carried out with the fluorescent dye Quin 2, which has a lower affinity for Ca²⁺ and therefore causes a serious perturbation of intracellular Ca²⁺ handling.

In our experiments, which were carried out on neutrophilic granulocytes prepared from the peripheral blood of healthy volunteers, we abrogated O₂^.−-production by DPI. When applied in a concentration of 5 µm, DPI inhibited O₂^.−-production by 98%. Interestingly, the reduction in the depolarization of the plasma membrane was less intense. In typical experiments such as the one shown in Fig. 6b, even in the presence of 5 µm DPI, 20–30 mV depolarization occurred upon fMLP treatment. (In five experiments, 5 µm DPI inhibited plasma membrane depolarization by 53·2 ± 3·2%.) However, a clear alteration in the Ca²⁺ signal was detected: in the presence of DPI the [Ca²⁺]_ic in the sustained phase was elevated (Fig. 6a). Measured 120 s after addition of fMLP, the amplitude of the Ca²⁺ signal in the DPI-treated cells was 148·5 ± 5·9% (n = 10) of the value detected in the nontreated cells. Neither plasma membrane depolarization nor Ca²⁺ signal induced by fMLP were influenced by addition of superoxide dismutase to the extracellular medium (data not shown). Thus, generated O₂^.−per se cannot be responsible for the observed difference in the Ca²⁺ signal. In neutrophilic granulocytes, a typical Ca²⁺ signal can be also evoked by ATP. However, this agent does not initiate O₂^.−-generation and does not induce any change in the plasma membrane potential (Fig. 6d). The ATP-stimulated Ca²⁺ signal was not influenced by treatment of the cells by DPI (Fig. 6c).

Fig. 6.

Stimulus-induced calcium signals and membrane potential changes in fMLP- and ATP-treated human neutrophils. Calcium signals (a, c) and membrane potential changes (b, d) of human neutrophils stimulated with 1 µm fMLP or 10 µm ATP were compared. Where indicated, DPI was added in a concentration of 5 µm, 2 min prior to fMLP or ATP (addition of DPI is indicated by the open arrows, that of fMLP and ATP by the black arrows). The results presented are representative of 11 (a), five (b) or three (c, d) independent experiments.

Discussion

Our data show that initiation of O₂^.− production results in a significant depolarization in differentiated wild-type PLB cells similar to peripheral neutrophilic granulocytes. In contrast, stimulants of the respiratory burst have no effect on the membrane potential in the CGD-PLB cell line. Thus, PLB cells provide a useful cell line for studying the electrophysiological changes accompanying activation of the NADPH oxidase.

In the present study, inhibition of Ca²⁺ entry showed a clear correlation with the capability of O₂^.−-production and membrane depolarization. Ca²⁺ influx was decreased in the wild-type cells and in CGD cells after transfection with the wild-type gp91^phox. No inhibition of Ca²⁺ (or Mn²⁺) entry was detected either in the absence of any gp91^phox molecule (CGD-PLB cells) or in the presence of a mutant gp91^phox unable to transfer electrons (M1 cell line). Thus, in neutrophilic granulocytes, inhibition of Ca²⁺ entry by phorbol ester requires a functional oxidase. The present experiments carried out on genetically modified cell lines are in good agreement with our previous experiments based on inhibitor substances and indicate clearly that the decrease in Ca²⁺ influx is the consequence of the drop in the driving force due to the electrogenic nature of the oxidase [9]. In the absence of a functional oxidase, treatment with the PKC activator PMA had no effect at all. Thus, any direct action of PKC on the Ca²⁺ entry pathway(s) of neutrophilic granulocytes seems improbable. Our data are at variance with results described in other cell types such as mesangial cells and keratinocytes, where PKC was suggested to be involved directly in the regulation of store-operated calcium channels [12–14]. It is possible that in different cell types regulation of the capacitative influx pathway(s) involves different mechanisms. However, in none of the previous reports has the membrane potential been measured, although O₂^.−-production was also shown in mesangial cells [24].

All data summarized in the present paper suggest that there are significant alterations in the Ca²⁺ homeostasis of CGD neutrophils. In addition to differences in Ca²⁺ entry following thapsigargin-induced store depletion (Figs 3 and 4), the Ca²⁺ signal initiated by ligand binding to the fMLP receptor was also different (Figs 5 and 6). In the sustained phase, [Ca²⁺]_ic was higher both in the absence of a functional gp91^phox in the CGD-PLB cells (Fig. 5) and when electron transfer was largely inhibited by DPI in peripheral blood neutrophils (Fig. 6). The effect is specific for oxidase-activating agonists, because ATP did not induce any O₂^.−-production and no difference was observed in the ATP-induced Ca²⁺ signal of wild-type and CGD cells.

Our experiments indicate a difference of approx. 100 nm in [Ca²⁺]_ic between normal and CGD cells. In evaluating the biological significance of this difference, two conditions have to be considered. (1) The maximally effective concentration of DPI inhibited O₂^.−-production by 98% but membrane depolarization was reduced to only approx. 50%. In CGD cells, in the complete absence of gp91^phox, no change in membrane potential could be observed at all (Fig. 3 in [9]). Thus the true alteration in the Ca²⁺ signal is probably underestimated in our model experiments. (2) Our Ca²⁺ measurements carried out on cell suspensions provide a value of [Ca²⁺]_ic averaged for the entire cytosolic space. However, at certain intracellular locations, such as the subplasmalemmal space, [Ca²⁺] is significantly higher than the averaged value [25].

Abnormal Ca²⁺ influx might have several important consequences in the functioning of CGD neutrophilic granulocytes. Both spontaneous and induced apoptosis was shown to be significantly slower in CGD neutrophils than in cells from healthy individuals [26]. In several cell types calcium has a central, triggering role in the apoptotic response. Interestingly, apoptosis in neutrophils reacts opposite to other cell types: elevation of [Ca²⁺]_ic prevents apoptosis [27]. Delayed apoptosis observed in CGD neutrophils may be due to the abnormally increased influx of Ca²⁺.

Elevated [Ca²⁺]_ic has also been shown to trigger the activation of phospholipase A2 [28] and the de novo synthesis of several cytokines. In case of the chemotactic agent IL-8 it has been reported that a rise of 50–100 nm in [Ca²⁺]_ic is able to induce a significant increase in the synthesis and secretion of this peptide [29]. It is thus possible that retarded apoptosis and increased chemotactic attraction via elevated IL-8 secretion may participate in the formation of granulomas typical for CGD.