Journal of Leukocyte Biology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published online as doi:10.1189/jlb.0406248 on January 29, 2007

Published online before print January 29, 2007
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0406248v1
81/4/1054    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Steinckwich, N.
Right arrow Articles by Nüße, O.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Steinckwich, N.
Right arrow Articles by Nüße, O.
(Journal of Leukocyte Biology. 2007;81:1054-1064.)
© 2007 by Society for Leukocyte Biology

Potent inhibition of store-operated Ca2+ influx and superoxide production in HL60 cells and polymorphonuclear neutrophils by the pyrazole derivative BTP2

Natacha Steinckwich*,1, Jean-Pol Frippiat*, Marie-José Stasia{dagger}, Marie Erard{ddagger},§, Rachel Boxio*,2, Christiane Tankosic*, Isabelle Doignon§,|| and Oliver Nüße§,||

* University Henri Poincaré Nancy 1, EA3442, Vandoeuvre-les-Nancy, France;
{dagger} University Joseph Fourier, GREPI EA2938, Grenoble, France;
{ddagger} CNRS, UMR8000, Orsay, France;
§ University Paris-Sud, Orsay, France; and
|| INSERM, U757, Orsay, France

1 Correspondence: EA3442, Université Henri Poincaré Nancy 1, Faculté des Sciences, BP239, 54506 Vandoeuvre-les-Nancy, France. E-mail: natacha.steinckwich{at}scbiol.uhp-nancy.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Store-operated calcium entry (SOCE) is a key regulator in the activation of leukocytes. 3,5-Bistrifluoromethyl pyrazole (BTP) derivatives have been identified recently as inhibitors of T lymphocyte activation. The inhibitory effect of one of these compounds, N-(4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl)-4-methyl-1,2,3-thiadiazole-5-carboxamide (BTP2), appears to be a result of inhibition of SOC influx. Polymorphonuclear neutrophils provide effective protection against bacterial infection, but they are also involved in tissue damage during chronic inflammation. As for T lymphocytes, their activation relies on SOCE. We therefore investigated the effect of BTP2 on calcium homeostasis and functional responses of human neutrophils. BTP2 significantly inhibited the calcium influx after stimulation with thapsigargin or fMLF. This inhibition was seen after 5 min of incubation with 10 µM BTP2 and after 24 h with lower concentrations. With 24 h incubation, the effect appeared irreversible, as the removal of BTP2 3 h before the experiment did not reduce this inhibition in granulocyte-differentiated HL60 cells. In human neutrophils, BTP2 reduced superoxide anion production by 82% after 24 h of incubation. On the contrary, phagocytosis, intraphagosomal radical production, and bacterial killing by neutrophils were not reduced significantly, even after 24 h treatment with 10 µM BTP2. This work suggests that BTP2 could become an important tool to characterize calcium signaling in neutrophils. Furthermore, BTP2 or related compounds could constitute a new approach to the down-regulation of neutrophils in chronic inflammatory disease without compromising antibacterial host defense.

Key Words: human cells • signal transduction • calcium channel inhibitor • phagocytosis • bacterial killing


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Intracellular calcium is a key regulator in all cells of the immune system. Engagement of TCRs causes long-lasting calcium oscillations, which are essential for cytokine production and proliferation [1 ]. Activation of polymorphonuclear neutrophils (PMN) involves rapid and transient elevations in the intracellular free calcium concentration [Ca2+] triggering bactericidal functions. Calcium is released from intracellular stores or enters the cell through ion channels in the plasma membrane. The exact nature and the mechanism of activation of these channels are not yet known. However, ion channels are excellent drug targets, and the prospect of modulating the immune system by selective ion channel inhibitors is quite attractive.

In a search for safer immunosuppressive drugs, a new family of calcium influx inhibitors in lymphocytes has been described recently: The 3,5-bistrifluoromethyl pyrazole (BTP) derivatives inhibit cytokine gene transcription in Th1 and Th2 lymphocytes by suppressing the calcium-dependent translocation of the transcription factor NFAT [2 , 3 ]. One of these compounds, N-(4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl)-4-methyl-1,2,3-thiadiazole-5-carboxamide (BTP2; also called YM-58483) turned out to inhibit store-operated calcium entry (SOCE) in T lymphocytes [4 ]. This calcium entry mechanism is based on the depletion of endoplasmic reticulum calcium stores and subsequent activation of plasma membrane ion channels [5 ]. In mast cells and lymphocytes, SOCE generates a significant ionic current, calcium release-activated current (ICRAC). It has been suggested that BTP2 could inhibit ICRAC directly [4 ] or activate a nonselective cation channel, which causes membrane depolarization and thereby reduces the driving force for calcium entry [6 ]. The responsible ion channel may belong to the transient receptor potential (TRP) protein family [7 ]. A direct, inhibitory effect of BTP2 on two similar TRP channels, TRPC3 and TRPC5, was shown by expressing these proteins in several cell lines [8 ]. A third member of the TRP family, TRPV6, remained unaffected by BTP2 [8 , 9 ].

PMN are the most abundant circulating cells of the human immune system. They are essential for the phagocytosis and killing of pathogenic bacteria. However, they are also involved in tissue damage during chronic inflammation or ischemia-reperfusion injury. Neutrophil action against bacteria relies on the production of reactive oxygen intermediates by the phagocyte NADPH oxidase and on the release of proteases and pore-forming bactericidal peptides. The same effector mechanisms cause tissue destruction in the case of inappropriate neutrophil activation. The control of the critical balance between antimicrobial defense and inflammatory tissue damage needs to be understood to identify new drug targets that reduce inflammation and preserve host defense. As intracellular calcium and in particular, SOCE play a central role in neutrophil activation, they are potential drug targets [10 ]. Several mechanisms involving multiple ion channels appear to contribute to SOCE in neutrophils [11 ].

Electrophysiological and pharmacological data suggest that different immune cells have different calcium entry mechanisms [10 ]. However, they all have SOC influx, which is central for the control of their cellular functions. Therefore, we wanted to determine whether BTP2 acts on neutrophils and whether it affects neutrophil functions. We show here that BTP2 is a potent inhibitor of SOC influx in human neutrophils and granulocyte-differentiated HL60 cells and causes significant inhibition of certain cellular functions without impairing bacterial killing.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
HL60 cells (American Type Culture Collection, Manassas, VA, USA) were cultured at 37°C with 5% CO2 in RPMI-1640 medium supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, and 100 U/ml penicillin-streptomycin. To differentiate HL60 cells into the neutrophil lineage, cells were grown in the presence of 1.3% DMSO for 3 days. Afterwards, the medium was replaced by fresh medium containing 0.65% DMSO, and the culture continued for 3 more days.

Neutrophil preparation
PMN were purified from fresh, healthy human blood samples recovered on EDTA by the French blood bank (EFS, Vandoeuvre-les-Nancy, France). The appropriate review committee approved the protocol. Whole blood was diluted in HBSS, loaded onto Polymorphprep® (Axis-Shield PoC AS, Oslo, Norway), and centrifuged for 45 min at 450 g. The neutrophil layer was collected, washed two times in PBS, and resuspended in 2 ml 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM Hepes, pH 7.2, 9 mM glucose (ES buffer). PMN purity using this method was above 95%, as determined by nuclear staining with Türk’s solution. Some of our experiments used PMN after 24 h of culture. In this case, purified neutrophils were cultured in the presence or absence of BTP2 at 3.5 x 105 cells/ml in RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, and 100 U/ml penicillin-streptomycin (except for killing experiments) at 37°C with 5% CO2. After 24 h of incubation, the viability of the cells was controlled by flow cytometry (EPICS® XL-MCL, Beckman Coulter, Fullerton, CA, USA) with the Vybrant® apotosis assay kit (Molecular ProbesTM, Invitrogen, Carlsbad, CA, USA). Necrotic cells were labeled by propidium iodide, and annexin V (Alexa Fluor® 488 conjugate) binding indicated the beginning of apoptosis.

Intracellular calcium
Neutrophils and differentiated HL60 cells (5x106 cells/ml) were incubated in 500 µl loading buffer (ES buffer with 4 µM indo-1 AM, 250 µM sulfinpyrazone, 0.02% pluronic acid, and 0.1% BSA) for 1 h at room temperature. Then, cells were washed and resuspended in 500 µl ES buffer with 250 µM sulfinpyrazone and maintained in this buffer until use. Just before the measurement, cells were washed and resuspended at 2.5 x 106 cells/ml in ES buffer, with or without calcium. Fluorescence of indo-1 was monitored in black 96-well plates (Costar® 3916, Corning Inc., Corning, NY, USA) at 37°C with a multilabel plate reader Wallac 1420 (Perkin Elmer, Courtaboeuf, France) at 405 and 485 nm with excitation at 355 nm every 9.5 or 12 s. Calibration was obtained by measuring the signal of cells incubated for 1 h with 10 µM ionomycin in ES buffer (maximal calcium signal) or with 10 µM ionomycin and 30 mM EGTA in calcium-deprived ES buffer (minimal calcium signal). The intracellular calcium was calculated with the following equation [12 ]: [Ca2+]i = Kd x Q x (R–Rmin)/(Rmax–R), where Kd = 230 nM, Q = F485min/F485max, and R = F405/F485. Intracellular Ca2+ response was stimulated with 1 µM fMLF or with 200 nM thapsigargin (TG). A control experiment was performed with DMSO, the solvent of fMLF and TG, to check that DMSO did not stimulate calcium influx.

Manganese influx
Neutrophils or HL60 cells were loaded with fura-2 as described above for indo-1. Quenching of fura-2 fluorescence in 106 cells in 1.7 ml was recorded 10 times/s at 360 nm excitation and 505 nm emission wavelengths at 37°C under constant stirring (spectrofluorimeter, Varian Eclipse, Les Ulis, France, or FL200, Safas, Monaco, Monaco). Where indicated, cells were incubated with 1 or 10 µM BTP2 or solvent (DMSO) for 8 min and 200 nM TG for 4 min prior to addition of 0.5 mM MnCl2. At the end of each experiment, 5 mM EGTA was added to dequench extracellular fura ({Delta}Fe). Mn2+ influx (Mi) was analyzed in the linear portion of the fluorescence decay between the point of Mn2+ addition (F0) and 40 s (HL60) or 120 s (PMN) later (F1), according to Demaurex et al. [13 ]. In addition, the Mi values were normalized to the fluorescence immediately before Mn2+ addition: Mi = (F0–F1–{Delta}Fe)/F0.

Secretion of ß-glucosaminidase by HL60 cells
The protocol for granule release was adapted to microplate format [14 , 15 ]. Briefly, 2 x 105 cells in ES buffer were loaded per well of a 96-well culture plate (353072 Microtest®, Falcon®) with 5 µg/ml cytochalasin B and the compound to be tested (BTP2, Calbiochem, VWR, France). After an incubation of 10 min at 37°C, 1 µM fMLF was added. After 15 min of incubation at 37°C, 100 µl ice-cooled ES buffer was added to stop the reaction. The plate was centrifuged at 400 g, 4°C, for 5 min. Two samples of 50 µl supernatant were collected and placed in a black plate with 50 µl reaction buffer (0.1 M acetate sodium, pH 4 buffer, 0.02% Triton X-100, 5 mM 4-methylumbelliferyl-N-acetyl-ß-D-glucosamine). After 1 h of incubation at 37°C in the dark, the reaction was stopped with 50 µl stop solution (0.1 M EGTA, 3 M glycine, pH 10.4). The fluorescence was monitored with the multilabel plate reader Wallac 1420 (excitation 355 nm; emission 460 nm). Results were expressed as a percentage of the maximal fluorescence produced with HL60 cells lysed with 0.02% Triton X-100. Background release of unstimulated cells was subtracted. DMSO, the solvent of BTP2, did not stimulate ß-glucosaminidase release.

Production of superoxide anions by neutrophils and differentiated HL60 cells
Reactive oxygen species (ROS) were quantified by measuring luminol-dependent chemiluminescence every 6 s at 37°C with a multilabel plate reader Wallac 1420 using white BSA-coated polypropylene 96-well plates (Nunc, Roskilde, Denmark) [15 ]. Human neutrophils or HL60 cells (5x104 cells) were resuspended in ES buffer or calcium-deprived ES buffer with 10 µg/ml luminol and 4 U/ml HRP in a final volume of 200 µl per well. Cells were stimulated with 1 µM fMLF or 100 nM PMA, and superoxide anion production was measured for 15–60 min. For quantitative comparisons, total ROS production was assessed by integrating photon counts over the entire measurement period. The solvent control, DMSO, did not stimulate ROS production.

Phagocytosis of bioparticles by human neutrophils
The phagocytic capacity of PMN was evaluated by monitoring the internalization of fluorescent bioparticles (Molecular Probes, Eugene, OR, USA) following the supplier’s instructions. Fluorescein-labeled, killed Escherichia coli (K-12 strain) or zymosan (Saccharomyces cerevisiae) were opsonized by rabbit polyclonal IgG antibodies against E. coli or zymosan for 1 h at 37°C. After three washings with ice-cold PBS buffer, PMN were resuspended in RPMI-1640 medium without phenol red (Gibco/Invitrogen, UK), supplemented with 10% heat-inactivated FBS and 2 mM L-glutamine. Neutrophils (3x106/ml) were coincubated with 10 E. coli particles or five zymosan particles for one PMN in 96-well culture plates at 37°C with 5% CO2. After an incubation of 30 min for E. coli and 90 min for zymosan, the phagocytosis was stopped by transfer into ice-cold PBS buffer. The addition of ice-cold trypan blue quenched the fluorescence of extracellular particles to visualize only internalized particles. The percentage of phagocytosing neutrophils was quantified by flow cytometry (EPICS® XL-MCL) after gating with neutrophils incubated without bioparticles.

Measurement of intraphagosomal NADPH oxidase activity
Heat-killed S. cerevisiae were labeled with an oxidant-sensitive probe, dichlorodihydrofluorescein (DCFH2), which is poorly fluorescent before oxidation by ROS and becomes significantly brighter when the yeast is engulfed in a phagosome. These labeled yeasts were shown to be a tool to measure the oxidase activity directly in the phagosome [16 ]. For DCFH2 labeling, yeasts were washed two times in a carbonate buffer (100 mM, pH 8.3) and resuspended at a concentration of 10 mg/ml. Succimidyl ester of 2'7' DCFH2 diacetate (Molecular Probes) was dissolved in anhydrous DMSO (100 µl, 1 mg/ml) and added drop-wise to the yeast (1 ml). The solution was allowed to react 1 h at room temperature with a continuous stirring under a nitrogen flow in the dark. To deacylate the product and to generate the oxidant-sensitive probe, 100 µl of a 1.5 M solution of hydroxylamine, pH 8.5, was added. After 1 h with a continuous stirring under a nitrogen flow in the dark, yeasts were washed two times with degassed PBS, resuspended at a density of 108 yeasts/ml in PBS, and stored in sealed vials at –20°C for several months. Before use, yeast was opsonized with a polyclonal rabbit antiyeast antibody (1/100, 1 h, 37°C), washed two times with PBS, and resuspended in ES buffer supplemented with 5% heat-inactivated FBS.

For the measurement of the oxidase activity level, opsonized, DCFH2-labeled yeasts were added to the neutrophil suspension with a yeast:neutrophil ratio of 5:1 for 1 h at 37°C. The phagocytosis was stopped by addition of cold buffer (4°C). The fluorescence of neutrophils that had engulfed DCFH2 yeasts was analyzed by flow cytometry, with or without a previous, 24 h incubation of neutrophils with 10 µM BTP2. The fluorescence of these neutrophils reflected the number of ingested particles and their oxidation state, which increased with phagosomal ROS production. To control the degree of phagocytosis, the same yeast particles were labeled with FITC instead of DCFH2, oponized, and phagocytosed under the same conditions as DCFH2-labeled yeasts. No effect of BTP2 on the number of ingested particles was observed by flow cytometry (data not shown). Thus, any changes in fluorescence of DCFH2-labeled yeast reflect their oxidation state.

Killing of Pseudomonas aeruginosa by human neutrophils
The capacity of PMN to kill P. aeruginosa strain PAO1 was evaluated by monitoring bacterial plate counts during and after coincubation [17 ]. Bacteria were cultured overnight in Luria-Bertani (LB) liquid medium, collected by centrifugation, and washed once in modified HEPES buffer (15 mM HEPES, 8 mM glucose, 4 mM KCl, 140 mM NaCl, 0.5 mM MgCl2, 0.9 mM CaCl2). Then, bacteria were opsonized for 5 min with pooled normal human serum (NHS). After purification, 5 x 106 PMN were mixed with 5 x 107 CFU of opsonized P. aeruginosa (multiplicity of infection of 10) in 1 ml culture medium containing 10% NHS. This mixture was shaken for 1 h at 37°C. During and after the incubation period, 50 µl samples of the coincubation mixture were collected, serially diluted in LB medium, and plated on Pseudomonas isolation agar plates. Bacterial colonies were counted after 24 h of incubation at 37°C.

Data analyses and statistics
To evaluate the impact of each treatment, data were subjected to ANOVA analyses followed by the post hoc test of Newmann-Keuls with a threshold lower than 5%. This statistical treatment allowed multiple comparisons of several means calculated after three to five repetitions of each experiment. Data are shown as mean ± SEM.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
We analyzed the effect of BTP2 on two types of granulocytes: neutrophils extracted from human peripheral blood and HL60 cells differentiated into granulocytes. This second cell type was used mainly to confirm results observed with PMN but also to study the effect of a long-term incubation with BTP2.

Impact of BTP2 on calcium influx of neutrophils
The effect of BTP2 on calcium influx was investigated on three types of responses. First, we analyzed the impact of BTP2 on SOC influx induced by TG and readdition of calcium. Second, we investigated the action of BTP2 on the entry of Mn2+ by SOC channels after a TG stimulation. Finally, we studied the impact of BTP2 on the calcium response induced by fMLF. The concentrations of BTP2 and incubation times tested were similar to those efficient in T lymphocytes [4 ].

The SOCE in human neutrophils was induced with TG in a calcium-deprived buffer followed by the addition of CaCl2 (Fig. 1A ). Indeed, TG induces a depletion of intracellular calcium stores and an activation of store-operated channels. Therefore, when calcium is added in the buffer, there is an entry of extracellular calcium into the cells. The intracellular Ca2+ concentration ([Ca2+]i) was determined from the fluorescence ratio, F405:F485, of the indo-1 probe. Fluorescence data were calibrated, and the intracellular-free calcium concentration was calculated. The basal calcium level was subtracted to analyze the increase as a result of TG and calcium readdition treatment ({Delta} [Ca2+]i, Fig. 1B ). These figures demonstrate clearly that BTP2, when used at 10 µM, reduced the calcium entry into neutrophils by 81%.


Figure 1
View larger version (29K):
[in this window]
[in a new window]

  		Figure 1. Effect of BTP2 on calcium and manganese influx of neutrophils stimulated by TG. Calcium and manganese entry was monitored with indo-1 (A, B) and fura-2 (C, D), respectively. The [Ca2+]i was calculated with the ratio of fluorescence at 405 and 485 nm:F405/F485 (A, B). Loaded cells were placed in a calcium-free buffer, and calcium store depletion was induced by adding 200 nM TG. About 4 min later, 5 mM CaCl2 was added. After this external calcium addition, the [Ca2+]i reached a plateau before slowly returning to basal levels. The difference of [Ca2+]i {Delta} [Ca2+]i, was defined as the average value at the beginning of the plateau minus the basal level prior to TG addition. Cells were incubated with various concentrations of BTP2 before adding TG. To follow manganese influx (Mi) (C, D), neutrophils were placed in a calcium-free buffer. Four minutes after the beginning of the recording, 200 nM TG was added to induce the opening of store-operated channels. Four minutes later, 2 mM MnCl2 was added. Fura-2 fluorescence appears as a line whose slope is proportional to the rate of Mi. (A, C) Representative of three independent experiments. (B, D) Mean variations ± SEM of the [Ca2+]i or manganese entry. *, Statistically significant differences; #, differences not statistically significant.

 
To confirm these results, we investigated the impact of BTP2 on the manganese entry. Mn2+ also enters through store-operated channels [13 ] and can be detected by fura-2 fluorescence quenching at the calcium-independent excitation wavelength of 360 nm. As described before [13 ], the beginning of the quenching response was linear, allowing a quantitative assessment of the rate of manganese entry. In neutrophils, we demonstrated that 10 µM BTP2 reduced by 74% the TG-induced manganese entry, and 1 µM BTP2 reduced this entry by 38% (Fig. 1C and 1D) .

Chemoattractants, such as the bacterial peptide fMLF, induce a transient rise in [Ca2+]i. This calcium peak can be divided in three steps: 1) an important and abrupt increase that corresponds to the depletion of calcium stores, 2) a slow decrease, followed by 3) a stabilization of the signal at a concentration higher than the basal level [18 ]. This plateau is a result of the opening of store-operated channels that maintain the elevated intracellular calcium concentration. We wanted to determine whether the complex calcium response to chemoattractant stimulation, under physiological calcium concentrations, was affected by BTP2. To this end, we stimulated the calcium response of neutrophils with 1 µM fMLF (Fig. 2A ). It is interesting that when BTP2 was added to the medium, the plateau of the calcium response (evaluated 3 min after the peak) was situated at a lower concentration, and the height of the peak was not affected significantly (Fig. 2B and 2C) . Indeed, if neutrophils were incubated for 4 min with 10 µM BTP2, we noticed that the intracellular calcium concentration, 3 min after the peak, was reduced by 60% in neutrophils (Fig. 2C) . However, these values are different from the control condition, where cells were placed in calcium-deprived medium, which reduced calcium influx by 97% (Fig. 2C) , suggesting that calcium entry was not inhibited completely.


Figure 2
View larger version (13K):
[in this window]
[in a new window]

  		Figure 2. Effect of BTP2 on calcium influx of neutrophils stimulated by fMLF. Neutrophils were placed in a buffer, with or without 2 mM calcium, and stimulated with 1 µM fMLF. Cells were incubated with various concentrations of BTP2 for 4 min before the addition of fMLF. Changes of indo-1 fluorescence were monitored to follow the [Ca2+]i. (A) Results are representative of three independent experiments. Variations of [Ca2+]i were calculated at the maximum of the peak (B) and 3 min later (C). The calcium concentration corresponding to the basal level was subtracted to determine {Delta} [Ca2+]i. (B, C) Mean ± SEM of three experiments. *, Statistically significant differences; #, differences not statistically significant. Ext. Ca2+, Extracellular calcium.

 
BTP2 had similar effects in differentiated HL60 cells. Indeed, 1 µM BTP2 was able to reduce by 79% the calcium entry induced by TG, and 10 µM BTP2 reduced this entry by 94% (Fig. 3A ). Furthermore, we found that 1 µM BTP2 inhibited manganese entry by 61% and 10 µM BTP2 by 83% (Fig. 3B) . These results confirmed that BTP2 is able to inhibit store-operated divalent cation entry. In addition, the peak of the fMLF-stimulated calcium response was not affected significantly by 10 µM BTP2 (Fig. 3C) . However, at this concentration, the compound reduced the plateau significantly 3 min after the peak (Fig. 3D) . The calcium responses of neutrophils and HL60 cells have a similar sensibility to BTP2 (Figs. 2C and 3D) . However, these two cell types have a different sensibility to calcium-free conditions (Figs. 2B and 3C) . Indeed, the peak of the fMLF response is reduced by 41% in neutrophils and by 96% in HL60 cells.


Figure 3
View larger version (19K):
[in this window]
[in a new window]

  		Figure 3. Effect of BTP2 on calcium and manganese influx of differentiated HL60 cells stimulated by TG (A, B) or by fMLF (C, D). The SOC influx was monitored with indo-1-loaded HL60 cells placed in a calcium-free buffer. Cells were stimulated with 200 nM TG and the readdition of 5 mM CaCl2. (A) The {Delta} [Ca2+]i. (B) Results show the mean slope, which corresponds to manganese influx following the addition of 2 mM Mn2+ on fura-2-loaded HL60 cells stimulated with 200 nM TG in a calcium-low buffer (100 µM CaCl2). (C, D) Mean values of {Delta} [Ca2+]i obtained by stimulating indo-1-loaded HL60 cells with 1 µM fMLF in a buffer, with or without 2 mM CaCl2. Variations of [Ca2+]i were calculated at the maximum of the peak (C) and 3 min later (D). (A–D) Mean ± SEM of at least three experiments. *, Statistically significant differences; #, differences not statistically significant.

 
Long-term effect and reversibility of the action of BTP2 on calcium influx of differentiated HL60 cells
In previous studies [4 , 8 ], it has been shown that BTP2 could have an impact on calcium entry at a much lower concentration if the incubation time were extended to 24 h. We used HL60 cells differentiated into granulocytes to study the long-term impact of BTP2. Twenty-four-hour incubation with 0.2 µM BTP2 inhibited the calcium influx by 97% after TG and CaCl2 addition (Fig. 4B ) and by 68% after fMLF addition (Fig. 4D) .


Figure 4
View larger version (20K):
[in this window]
[in a new window]

  		Figure 4. Long-term effect of BTP2 on calcium signaling in neutrophils and differentiated HL60 cells. Neutrophils (A) or HL60 cells (B–D) were incubated without BTP2 or with 0.2, 1, or 10 µM BTP2 for 24 or 21 h and washed 3 h before monitoring the fluorescence variations. Cells were stimulated by TG in calcium-free buffer and calcium addition 5 min later (A, B) or by fMLF in a buffer, with or without calcium (C, D). Kinetics of the calcium responses had the same profiles as in Figures 1A and 2A . {Delta} [Ca2+]i was obtained after subtraction of the basal level. Values were read at the plateau after calcium addition (A, B) or at the peak (C) and at the plateau (D) induced by fMLF. Data represent the mean ± SEM of three independent experiments. *, Statistically significant differences; #, differences not statistically significant.

 
During previous studies, a constant BTP2 concentration had been maintained throughout the entire experiment, including washing steps and periods of dye-loading [4 ]. We wondered if the action of this compound was reversible. To address this issue, we cultured HL60 cells with 0.2 µM BTP2 for only 21 h; afterward, BTP2 was removed by centrifugation and cells placed in dye-loading buffer without BTP2 for 3 h. It is interesting that this treatment produced an inhibition of the calcium influx similar to that observed when cells were incubated for 24 h with 0.2 µM BTP2 (Fig. 4B 4C 4D) , thereby indicating that the action of BTP2 was irreversible for at least 3 h.

Impact of BTP2 on bactericidal functions of human PMN
Having shown that BTP2 is able to substantially reduce the capacitive calcium entry in neutrophils, we investigated if BTP2 could have an impact on microbicidal functions of neutrophils.

At first, we looked at the impact of 10 µM BTP2 after a short incubation of 5 min. In these conditions, removal of extracellular calcium did not reduce ROS production after stimulation with fMLF. Consequently, a short incubation of 5 min with 10 µM BTP2 was insufficient to significantly inhibit the superoxide anion secretion by NADPH oxidase after stimulation with fMLF (Fig. 5A and 5B ). Then, we investigated the impact of BTP2 on E. coli-FITC phagocytosis (Fig. 5C) and the capacity of neutrophils to kill P. aeruginosa bacteria (data not shown). The percentage of phagocytosis was evaluated by flow cytometry. The effect of BTP2 on bacterial killing was determined by calculating the percentage of bacteria that survived when they were coincubated with PMNs in the presence or absence of BTP2. Addition of BTP2 did not have a significant effect on bacteria phagocytosis and killing.


Figure 5
View larger version (15K):
[in this window]
[in a new window]

  		Figure 5. Short-term effect of BTP2 on ROS production and E. coli phagocytosis by neutrophils. The dynamics of ROS production were studied by following the variations of luminescence of the luminol that reacts with ROS. Neutrophil responses were induced by 1 µM fMLF, with or without 10 µM of BTP2 added 5 min before the fMLF addition. (A) Kinetics of the responses shown are representative of four independent experiments. (B) Mean values ± SEM of the total production during 15 min after stimulation. (C) The mean percentage ± SEM of neutrophils, which ingested E. coli bioparticles labeled with fluorescein and opsonized with polyclonal antibodies against E. coli. The phagocytosis was stopped after 30 min of coincubation of cells with bioparticles at 37°C. The fluorescence of extracellular particles was quenched with trypan blue, and the percentage of PMN having internalized bioparticles was evaluated by flow cytometry. #, Differences not statistically significant.

 
These results raised the question whether longer incubation with BTP2 would affect neutrophil functions. We tested the effect of BTP2 on neutrophil calcium signaling after 24 h of incubation (Fig. 4A) . Our results show that in these conditions, BTP2 was able to inhibit the capacitive calcium entry induced by TG stimulation and calcium readdition. Moreover, after 24 h, the inhibition induced by 1 µM BTP2 is as strong as the one induced by 10 µM BTP2 (Fig. 4A) . Increasing the incubation time with BTP2 could also act on the ROS production by human neutrophils. Whyte et al. [19 ] have shown that the functional ability of neutrophils after 24 h in culture was reduced strongly among the apoptotic cells, whereas nonapoptotic cells were capable of ROS production and phagocytosis. We used propidium iodide staining to detect signs of necrosis and annexin V staining to reveal early signs of apoptosis on neutrophils after 24 h in tissue-culture medium. We found less than 0.5% of cells labeled by propidium iodide, and annexin V binding was detected on 58.5 ± 0.7% and 61.8 ± 0.7% of control neutrophils and BTP2-treated neutrophils, respectively. Thus, any functional differences in these cells were not a result of differential apoptosis and most likely reflect the effect of BTP2 on the remaining 40% nonapoptotic cells.

Consequently, we also tested the impact of BTP2 on fMLF-stimulated NADPH oxidase activity after 24 h of incubation. Under these conditions, ROS production was inhibited by BTP2 to the same level as in the absence of extracellular calcium, corresponding to a decrease of 74% in the total amount of ROS secretion (Fig. 6A and 6B ). The total ROS production in cultured neutrophils was lower than in freshly isolated neutrophils (Fig. 5B) , presumably as a result of the loss of function in apoptotic cells. To know if the effect of BTP2 was specific for the calcium dependence of oxidase activation, we tested the impact of BTP2 on ROS production stimulated by PMA on neutrophils incubated for 24 h in the presence of 10 µM BTP2. Our results demonstrate that BTP2 had no significant impact on PMA-stimulated ROS production (Fig. 6C and 6D) .


Figure 6
View larger version (32K):
[in this window]
[in a new window]

  		Figure 6. Long-term effect of BTP2 on ROS production of human neutrophils. The dynamics of ROS production were studied by following the variations of luminescence of the luminol. BTP2 was added 24 h before the addition of fMLF (A, B) or PMA (C, D), where indicated. Neutrophil responses were induced by 1 µM fMLF (A, B) in a buffer, with or without 2 mM CaCl2. (C, D) Results obtained by stimulating with 100 nM PMA. (A, C) The kinetics of the responses are representative of three and four independent experiments, respectively. (B, D) Mean values ± SEM of the total ROS production during 15 min (B) or 60 min (D) after stimulation.

 
Would BTP2 also compromise the phagocytosis of antibody-opsonized E. coli or zymosan particles? No significant inhibition of phagocytosis was observed after 24 h of incubation with 10 µM BTP2 (Fig. 7A and 7B ). Similarly, when PMN were cultured for 24 h in the presence of 10 µM BTP2, bacterial killing was not reduced significantly (Fig. 7D) . This is in apparent contrast to the observation that longer incubation with BTP2 inhibited chemoattractant-induced ROS production in the absence of phagocytosis (Fig. 6B) , which may be similar to the over-reaction of PMN in chronic inflammation. ROS production is known to be essential to efficient bacterial killing by PMN. However, our previous experiments on ROS production did not determine the phagosomal ROS production. However, it is the phagosomal production that is important for the antibactericidal functions of neutrophils. Dewitt et al. [16 ] recently demonstrated a method for microscopic detection of intraphagosomal ROS production. The technique is based on yeast particles labeled with an oxidant-sensitive probe, DCFH2. Using this technique with flow cytometry, we found no substantial difference of intraphagosomal ROS production as a result of 24 h BTP2 treatment (Fig. 7C) . Thus, 24 h treatment with BTP2 had a substantial impact on SOC influx and global ROS production, and phagocytosis, intraphagosomal ROS production, and bacterial killing were not impaired.


Figure 7
View larger version (12K):
[in this window]
[in a new window]

  		Figure 7. Long-term effect of BTP2 on phagocytosis (A, B), intraphagosomal ROS production (C), and bacterial killing activity (D) of human neutrophils, which were preincubated, with or without 10 µM BTP2, for 24 h before being coincubated at 37°C with E. coli or zymosan bioparticles labeled with fluorescein and opsonized with polyclonal antibodies against E. coli (A) or zymosan (B). Phagocytosis was stopped after 30 min (E. coli) or 90 min (zymosan) of coincubation, and trypan blue was added to quench the fluorescence of unphagocytozed particles. The number of PMN having internalized bioparticles was evaluated by flow cytometry. Results are expressed as percentage of phagocytosing neutrophils (A, B). Intraphagosomal NADPH oxidase activity was evaluated with ROS-sensitive, DCFH2-labeled yeast (C). The mean fluorescence of PMNs, which had engulfed bioparticles after an incubation of 1 h, was measured by flow cytometry and compared with the mean fluorescence of bioparticles before internalization. Killing activity of human neutrophils was tested on P. aeruginosa strain PAO-1 after 24 h treatment with 10 µM BTP2 (D). This activity was evaluated by following the decrease of colony numbers of P. aeruginosa 24 h after incubation of PMNs with bacteria for 0–45 min. Mean values ± SEM of three (B–D) or four (A) independent experiments. #, Differences not statistically significant.

 
Impact of BTP2 on bactericidal functions of differentiated HL60 cells
BTP2 had similar effects on calcium signaling in human neutrophils and HL60 cells. Although neutrophils undergo apoptosis during prolonged incubation periods, cell lines such as HL60 can be incubated for days, an advantage in the investigation of potential drugs. Therefore, we investigated the impact of BTP2 on calcium-dependent functions of these cells, namely, ROS production and exocytosis of the primary granule marker ß-glucosaminidase. Our data show that 10 µM BTP2 inhibited fMLF-induced activity of the NADPH oxidase by 87%, similar to the lack of extracellular calcium, which reduced this production by 75% (Fig. 8A ). The kinetics of the two reduced responses were identical (data not shown). Moreover, the effect of BTP2 was specific for the calcium dependence of oxidase activation, as it had no impact on ROS production induced by the addition of PMA (Fig. 8B) . Concerning ß-glucosaminidase exocytosis, after stimulation by 1 µM fMLF, 10 µM BTP2 inhibited exocytosis by 32%, and the lack of extracellular calcium reduced this function by 94% (Fig. 8C) . These experiments indicated that BTP2 is able to reduce two major functions of differentiated HL60 cells.


Figure 8
View larger version (19K):
[in this window]
[in a new window]

  		Figure 8. Effect of BTP2 on the NADPH oxidase activity (A, B) and ß-glucosaminidase secretion (C) of differentiated HL60 cells. ROS production was followed by monitoring the variations of luminol luminescence. Cells were incubated with different concentrations of BTP2 for 5 min and then stimulated by the addition of 1 µM fMLF (A) or 100 nM PMA (B). (A, B) Total ROS production during 15 min (A) or 30 min (B) after stimulation. (C) The percentage of ß-glucosaminidase secretion induced by the addition of 1 µM fMLF to the medium after 5 min of incubation with various concentrations of BTP2. Values were expressed as a percentage of the maximal response induced by Triton X-100 addition. Results represent the mean ± SEM of three independent experiments. *, Statistically significant differences; #, differences not statistically significant.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
SOCE plays a crucial role in lymphocytes, neutrophils, and other hematopoietic cells. SOC channels are potential yet unexploited, anti-inflammatory drug targets [10 ]. Exploration of the calcium entry pathway would be facilitated substantially by specific inhibitors. We report here that the 3,5-bis(trifluoromethyl)-pyrazol derivative BTP2 inhibited TG-induced SOC and manganese influx in human neutrophils and HL60 cells completely, as shown before for T lymphocytes (Figs. 1 and 3) .

With short incubation times, micromolar levels of BTP2 were required. In HL60 cells, complete inhibition could also be achieved with 50 times less BTP2, when the drug was given for 24 h instead of 5 min. Moreover, washing away the drug after 21 h of treatment did not abolish the effect. Similarly, in human neutrophils, 24 h incubation markedly increased the inhibitor efficiency of 1 µM BTP2 (Fig. 4A) .

Currently, it is not clear why the long-term inhibition is much more potent. Previous studies about lymphocytes and other cells also reported a slow action of BTP2, although the time course and the required concentration differed between experimental systems [4 , 8 ]. BTP2 seems to act from the outside of the cell, most likely on the channel itself and does not require potentially slow entry through the membrane [4 , 8 ]. In T lymphocytes, the CRAC channels themselves seem to be the target of BTP2 [4 ]. Their identity is not known, but members of the TRP protein family are likely to be involved in SOCE. He et al. [8 ] suggested that BTP2 acts on TRPC channels, in particular, on TRPC3. Recently, Takezawa et al. [6 ] reported that BTP2 activates the nonselective cation channel TRPM4, which may counteract SOCE. However, this model required low nanomolar concentrations of BTP2 and short incubation periods [6 ], which had no effect in PMN or HL60 cells. Our data are consistent with the hypothesis that BTP2 acts directly on SOCE, potentially via TRP channels. However, other targets for BTP2 may be relevant for the observed effects.

A further clue to the mechanism of action of BTP2 came from the question of whether its action is readily reversible. We show here and for the first time that the long-term inhibition is irreversible for at least 3 h (Fig. 4) . This may suggest a covalent interaction between BTP2 and its target. Alternatively, the compound may induce degradation of the channel itself or of a partner necessary to its function. If BTP2 were to be used as a pharmaceutical drug, the apparent irreversibility of its effect may be a major advantage to maintain the effect between two administrations. However, it would also slow down recovery of the immune response after treatment. In one preclinical study of BTP in primates, the animals showed less than 50% recovery in 4 days [20 ]. It should be pointed out that this study used plasma concentration of 2–5 µM for 14 days to achieve strong and persistent suppression of IL-2 production. Moreover, serum components in the animal as well as in cell culture experiments may bind BTP2 and thereby, alter the concentration of free BTP2.

It is generally accepted that SOC influx represents the main pathway of calcium entry stimulated by G-protein-coupled receptors of chemoattractants such as fMLF. However, recent data suggest that fMLF stimulates more than one calcium entry pathway, and some calcium entry may be store-independent [21 , 22 ]. In neutrophils, BTP2 completely inhibited SOC influx induced by TG, whereas fMLF-stimulated calcium entry was not inhibited completely (Fig. 2) . This raises the possibility that BTP2 inhibits only the store-operated channels. Their inhibition by BTP2 could be compensated partially by store-independent pathways, allowing some calcium entry after fMLF stimulation.

The role of calcium signaling in the immune response has been emphasized often. It has been demonstrated that degranulation and superoxide production by neutrophils and HL60 cells are strongly supported by changes of intracellular calcium concentration [14 , 23 , 24 ]. The effects of BTP2 described above let us think that this molecule could be useful to determine the implication of this signaling pathway in some microbicidal functions of neutrophils.

We first showed that BTP2 had no impact on ROS production by human neutrophils when it was used at 10 µM during 5 min (Fig. 5) . The removal of extracellular calcium had only minor effects on the fMLF-induced ROS production, in agreement with previous publications [25 , 26 ]. However, intracellular calcium buffering with chelators reduced ROS production strongly [25 ], suggesting that calcium release rather than calcium influx is needed [27 ]. Short incubation in calcium-free buffer affected the peak calcium response after stimulation with fMLF in HL60 cells much more than in neutrophils (Figs. 3C vs. 2B ), suggesting that the intracellular calcium stores are depleted more readily in HL60 cells. This may explain why HL60 needed extracellular calcium for ROS production and was sensitive to short treatment with BTP2, and freshly isolated neutrophils did not (Figs. 8A vs. 5B ). In neutrophils, 24 h treatment with BTP2 presumably prevents the refilling of intracellular stores for which a calcium entry from extracellular medium is necessary. Consequently, the ROS production was blocked in 24 h-cultured neutrophils. In support of this hypothesis, we observed in calcium-free buffer a decrease in TG-induced calcium release in neutrophils cultured in the presence of BTP2 for 24 h. This effect was not observed on freshly purified neutrophils (data not shown). Moreover, it appears that BTP2 does not act directly on the NADPH oxidase itself or its assembly, as PMA-induced ROS production was not affected significantly by 24 h treatment with BTP2 (Fig. 6D) . This is a first example, which shows that BTP2 can act as an immediate inhibitor of immune function, in contrast to slower effects on gene expression in lymphocytes shown previously [1 , 2 , 4 ].

Similarly, Han et al. [28 ] also noted that the ROS production could be affected by an inhibitor of TNF-induced calcium signaling, "neucalcin". The mechanism of TNF-induced calcium increase is not well understood; therefore, it is unclear whether neucalcin could act on the same target as BTP2. Alternatively, different stimuli could be linked to ROS production by multiple calcium entry pathways [22 ]. If neucalcin and BTP2 turned out to be specific inhibitors for different pathways, they would be useful to analyze each pathway separately.

IgG-dependent and complement-dependent phagocytosis in PMN is thought to have different calcium requirements [29 ]. Our data indicate that phagocytosis of IgG-opsonized E. coli or zymosan particles is insensitive to BTP2, even at high concentration and after prolonged incubation (Fig. 7A and 7B) . Furthermore, BTP2 did not affect intraphagosomal ROS production (Fig. 7C) . In contrast, fMLF induces mainly extracellular ROS production [30 ], which seemed to require BTP2-sensitive calcium entry under conditions where intracellular calcium release was insufficient. Apparently, intraphagosomal ROS production does not depend on the same calcium entry mechanism as extracellular ROS production. Finally, BTP2 had no significant effect on bacterial killing (Fig. 7D) , which involves phagocytosis, ROS production, and granule release into the phagosome. These events are calcium-dependent, but the source of calcium is not certain. Stendahl et al. [31 ] have shown accumulation of intracellular calcium stores around the phagosome. Lundqvist-Gustafsson et al. [32 ] have suggested that calcium release from the phagosome could also contribute to the increase in periphagosomal calcium. Our data suggest that these pathways are insensitive to BTP2 and that SOCE was unnecessary for these bactericidal functions under our experimental conditions. A much larger screen will be required to determine whether phagocytosis via other receptors and destruction of other particles, in particular, pathogens, are also insensitive to BTP2.

In conclusion, neutrophils play a key role in the host defense against bacterial infections. However, excessive neutrophil activation contributes to tissue damage in acute and chronic inflammatory diseases such as inflammatory bowel disease or cystic fibrosis. The destructive role of neutrophils involves extracellular ROS production. The ideal modulator of neutrophil activation would inhibit these extracellular actions and preserve the phagocytic capacity and bactericidal properties of neutrophils. As phagocytosis appears to be less-dependent on calcium influx than degranulation and ROS production, inhibitors of calcium influx are excellent drug candidates [10 ]. In this study, we showed that BTP2 inhibits SOC influx in human neutrophils and HL60 cells and several calcium-dependent neutrophil functions. However, this inhibition is incomplete, presumably as a result of parallel calcium entry mechanisms. This new molecule may help to distinguish these pathways and identify their respective role. Moreover, BTP2 did not reduce the phagocytosis and killing functions, thereby suggesting that it could potentially reduce damaging neutrophil reactions without impairing their bactericidal activity.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the French Ministry of Science (Programs ACI Microbiology and DRAB) and the University Nancy 1 (BQR). We thank Jean-Claude Sulpice for his help with the human neutrophils and Elisabeth Maquet for her technical assistance with bactericidal killing experiments. We are grateful to Christine Mathieu and Cindy Granjenette for help with flow cytometry analysis and Marie Christine Bene for critical reading of the manuscript (University Nancy 1, EA3442).


   FOOTNOTES
 
2 Current address: INSERM, U514, Reims, France. Back

Received April 5, 2006; revised December 15, 2006; accepted December 15, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Lewis, R. S. (2001) Calcium signaling mechanisms in T lymphocytes Annu. Rev. Immunol. 19,497-521[CrossRef][Medline]
  2. Djuric, S. W., BaMaung, N. Y., Basha, A., Liu, H., Luly, J. R., Madar, D. J., Sciotti, R. J., Tu, N. P., Wagenaar, F. L., Wiedeman, P. E., et al (2000) 3,5-Bis(trifluoromethyl)pyrazoles: a novel class of NFAT transcription factor regulator J. Med. Chem. 43,2975-2981[CrossRef][Medline]
  3. Trevillyan, J. M., Chiou, X. G., Chen, Y. W., Ballaron, S. J., Sheets, M. P., Smith, M. L., Wiedeman, P. E., Warrior, U., Wilkins, J., Gubbins, E. J., et al (2001) Potent inhibition of NFAT activation and T cell cytokine production by novel low molecular weight pyrazole compounds J. Biol. Chem. 276,48118-48126[Abstract/Free Full Text]
  4. Zitt, C., Strauss, B., Schwarz, E. C., Spaeth, N., Rast, G., Hatzelmann, A., Hoth, M. (2004) Potent inhibition of Ca2+ release-activated Ca2+ channels and T-lymphocyte activation by the pyrazole derivative BTP2 J. Biol. Chem. 279,12427-12437[Abstract/Free Full Text]
  5. Parekh, A. B., Putney, J. W., Jr (2005) Store-operated calcium channels Physiol. Rev. 85,757-810[Abstract/Free Full Text]
  6. Takezawa, R., Cheng, H., Beck, A., Ishikawa, J., Launay, P., Kubota, H., Kinet, J. P., Fleig, A., Yamada, T., Penner, R. (2006) A pyrazole derivative potently inhibits lymphocyte Ca2+ influx and cytokine production by facilitating TRPM4 channel activity Mol. Pharmacol. 69,1413-1420[Abstract/Free Full Text]
  7. Nilius, B., Voets, T. (2005) TRP channels: a TR(I)P through a world of multifunctional cation channels Pflugers Arch. 451,1-10[CrossRef][Medline]
  8. He, L. P., Hewavitharana, T., Soboloff, J., Spassova, M. A., Gill, D. L. (2005) A functional link between store-operated and TRPC channels revealed by the 3,5-bis(trifluoromethyl)pyrazole derivative, BTP2 J. Biol. Chem. 280,10997-11006[Abstract/Free Full Text]
  9. Schwarz, E. C., Wissenbach, U., Niemeyer, B. A., Strauss, B., Philipp, S. E., Flockerzi, V., Hoth, M. (2006) TRPV6 potentiates calcium-dependent cell proliferation Cell Calcium 39,163-173[CrossRef][Medline]
  10. Li, S. W., Westwick, J., Poll, C. T. (2002) Receptor-operated Ca2+ influx channels in leukocytes: a therapeutic target? Trends Pharmacol. Sci. 23,63-70[CrossRef][Medline]
  11. Itagaki, K., Kannan, K. B., Singh, B. B., Hauser, C. J. (2004) Cytoskeletal reorganization internalizes multiple transient receptor potential channels and blocks calcium entry into human neutrophils J. Immunol. 172,601-607[Abstract/Free Full Text]
  12. Grynkiewicz, G., Poenie, M., Tsien, R. Y. (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties J. Biol. Chem. 260,3440-3450[Abstract/Free Full Text]
  13. Demaurex, N., Monod, A., Lew, D. P., Krause, K. H. (1994) Characterization of receptor-mediated and store-regulated Ca2+ influx in human neutrophils Biochem. J. 297,595-601
  14. Nusse, O., Serrander, L., Foyouzi-Youssefi, R., Monod, A., Lew, D. P., Krause, K. H. (1997) Store-operated Ca2+ influx and stimulation of exocytosis in HL-60 granulocytes J. Biol. Chem. 272,28360-28367[Abstract/Free Full Text]
  15. Boxio, R., Bossenmeyer-Pourie, C., Steinckwich, N., Dournon, C., Nusse, O. (2004) Mouse bone marrow contains large numbers of functionally competent neutrophils J. Leukoc. Biol. 75,604-611[Abstract/Free Full Text]
  16. Dewitt, S., Laffafian, I., Hallett, M. B. (2003) Phagosomal oxidative activity during ß2 integrin (CR3)-mediated phagocytosis by neutrophils is triggered by a non-restricted Ca2+ signal: Ca2+ controls time not space J. Cell Sci. 116,2857-2865[Abstract/Free Full Text]
  17. Dacheux, D., Attree, I., Schneider, C., Toussaint, B. (1999) Cell death of human polymorphonuclear neutrophils induced by a Pseudomonas aeruginosa cystic fibrosis isolate requires a functional type III secretion system Infect. Immun. 67,6164-6167[Abstract/Free Full Text]
  18. Mandeville, J. T., Maxfield, F. R. (1996) Calcium and signal transduction in granulocytes Curr. Opin. Hematol. 3,63-70[Medline]
  19. Whyte, M. K., Meagher, L. C., MacDermot, J., Haslett, C. (1993) Impairment of function in aging neutrophils is associated with apoptosis J. Immunol. 150,5124-5134[Abstract]
  20. Birsan, T., Dambrin, C., Marsh, K. C., Jacobsen, W., Djuric, S. W., Mollison, K. W., Christians, U., Carter, G. W., Morris, R. E. (2004) Preliminary in vivo pharmacokinetic and pharmacodynamic evaluation of a novel calcineurin-independent inhibitor of NFAT Transpl. Int. 17,145-150[Medline]
  21. Itagaki, K., Kannan, K. B., Livingston, D. H., Deitch, E. A., Fekete, Z., Hauser, C. J. (2002) Store-operated calcium entry in human neutrophils reflects multiple contributions from independently regulated pathways J. Immunol. 168,4063-4069[Abstract/Free Full Text]
  22. Itagaki, K., Kannan, K. B., Hauser, C. J. (2005) Lysophosphatidic acid triggers calcium entry through a non-store-operated pathway in human neutrophils J. Leukoc. Biol. 77,181-189[Abstract/Free Full Text]
  23. Foyouzi-Youssefi, R., Petersson, F., Lew, D. P., Krause, K. H., Nusse, O. (1997) Chemoattractant-induced respiratory burst: increases in cytosolic Ca2+ concentrations are essential and synergize with a kinetically distinct second signal Biochem. J. 322,709-718
  24. Hallett, M. B., Davies, E. V., Campbell, A. K. (1990) Oxidase activation in individual neutrophils is dependent on the onset and magnitude of the Ca2+ signal Cell Calcium 11,655-663[CrossRef][Medline]
  25. Lew, P. D., Wollheim, C. B., Waldvogel, F. A., Pozzan, T. (1984) 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. 99,1212-1220[Abstract/Free Full Text]
  26. Kessels, G. C., Roos, D., Verhoeven, A. J. (1991) fMet-Leu-Phe-induced activation of phospholipase D in human neutrophils. Dependence on changes in cytosolic free Ca2+ concentration and relation with respiratory burst activation J. Biol. Chem. 266,23152-23156[Abstract/Free Full Text]
  27. Geiszt, M., Szeberenyi, J. B., Kaldi, K., Ligeti, E. (1999) Role of different Ca2+ sources in the superoxide production of human neutrophil granulocytes Free Radic. Biol. Med. 26,1092-1099[CrossRef][Medline]
  28. Han, H., Stessin, A., Roberts, J., Hess, K., Gautam, N., Kamenetsky, M., Lou, O., Hyde, E., Nathan, N., Muller, W. A., Buck, J., Levin, L. L., Nathan, C. (2005) Calcium-sensing soluble adenylyl cyclase mediates TNF signal transduction in human neutrophils J. Exp. Med. 202,353-361[Abstract/Free Full Text]
  29. Lew, D. P., Andersson, T., Hed, J., Di Virgilio, F., Pozzan, T., Stendahl, O. (1985) Ca2+-dependent and Ca2+-independent phagocytosis in human neutrophils Nature 315,509-511[CrossRef][Medline]
  30. Dahlgren, C., Karlsson, A. (1999) Respiratory burst in human neutrophils J. Immunol. Methods 232,3-14[CrossRef][Medline]
  31. Stendahl, O., Krause, K. H., Krischer, J., Jerstrom, P., Theler, J. M., Clark, R. A., Carpentier, J. L., Lew, D. P. (1994) Redistribution of intracellular Ca2+ stores during phagocytosis in human neutrophils Science 265,1439-1441[Abstract/Free Full Text]
  32. Lundqvist-Gustafsson, H., Gustafsson, M., Dahlgren, C. (2000) Dynamic Ca2+ changes in neutrophil phagosomes. A source for intracellular Ca2+ during phagolysosome formation? Cell Calcium 27,353-362[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J. Leukoc. Biol.Home page
S. Brechard and E. J. Tschirhart
Regulation of superoxide production in neutrophils: role of calcium influx
J. Leukoc. Biol., November 1, 2008; 84(5): 1223 - 1237.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
I. Torii, S. Oka, M. Hotomi, W. H. Benjamin Jr, T. Takai, J. F. Kearney, D. E. Briles, and H. Kubagawa
PIR-B-Deficient Mice Are Susceptible to Salmonella Infection
J. Immunol., September 15, 2008; 181(6): 4229 - 4239.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
A. J. Sandoval, J. P. Riquelme, M. D. Carretta, J. L. Hancke, M. A. Hidalgo, and R. A. Burgos
Store-operated calcium entry mediates intracellular alkalinization, ERK1/2, and Akt/PKB phosphorylation in bovine neutrophils
J. Leukoc. Biol., November 1, 2007; 82(5): 1266 - 1277.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0406248v1
81/4/1054    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Steinckwich, N.
Right arrow Articles by Nüße, O.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Steinckwich, N.
Right arrow Articles by Nüße, O.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS