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The Journal of Immunology, 2004, 172: 6988-6993.
Copyright © 2004 by The American Association of Immunologists

Volume-Sensitive Chloride Channels Do Not Mediate Activation-Induced Chloride Efflux in Human Neutrophils1

Patricia Perez-Cornejo*,{ddagger}, Jorge Arreola{dagger},§, Foon-Yee Law*, Joanne B. Schultz* and Philip A. Knauf2,*

* Department of Biochemistry and Biophysics, School of Medicine, and {dagger} Center for Oral Biology, Aab Institute of Biomedical Sciences, University of Rochester, Rochester, NY 14642; and {ddagger} Facultad de Medicina and § Instituto de Fisica Universidad Autonoma de San Luis Potosi, San Luis Potosi, Mexico


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Many agents that activate neutrophils, enabling them to adhere to venular walls at sites of inflammation, cause a rapid Cl– efflux. This Cl– efflux and the increase in the number and affinity of {beta}2 integrin surface adhesion molecules (up-regulation) are all inhibited by ethacrynic acid and certain aminomethyl phenols. The effectiveness of the latter compounds correlates with their inhibition of swelling-activated Cl– channels (IClvol), suggesting that IClvol mediates the activator-induced Cl– efflux. To test this hypothesis, we used whole-cell patch clamp in hypotonic media to examine the effects of inhibitors of up-regulation on IClvol in neutrophils and promyelocytic leukemic HL-60 cells. Both the channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid and [3-methyl-1-p-sulfophenyl-5-pyrazolone-(4)]-[1,3-dibutylbarbituric acid]-pentamethine oxonol (WW781), a nonpenetrating oxonol, inhibited IClvol at concentrations similar to those that inhibit {beta}2 integrin up-regulation. However, ethacrynic acid, at the same concentration that inhibits activator-induced Cl– efflux and up-regulation, had no effect on IClvol and swelling-activated Cl– efflux, providing evidence against the involvement of IClvol in the activator-induced Cl– efflux.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Various agents that activate human neutrophils and enable them to firmly adhere to microvascular endothelial cells, including FMLP, TNF-{alpha}, and GM-CSF, all cause the amount of {beta}2 integrins, principally CD11b/CD18 integrin (Mac-1), exposed on the cell surface to increase greatly (up-regulation) (1). Agents that increase intracellular Ca2+, such as A23187 and phorbol esters, that activate protein kinase C (PKC)3 have similar effects (2). In addition, neutrophil activators as well as phorbol esters at high concentration have been shown to cause rapid efflux of >50% of the total intracellular Cl– (3, 4). The Cl– efflux was inhibited by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and ethacrynic acid (EA) (4). Although activated neutrophils undergo shape changes and are difficult to patch, in a few cases Cl– currents have been observed in neutrophils treated with either FMLP (5) or TNF-{alpha} (6). In the latter case the currents were nearly completely blocked by the Cl– channel inhibitor, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB).

The importance of anion transport to neutrophil physiological function was first suggested by the fact that {beta}2 integrin up-regulation and the release of enzymes from intracellular vesicles (granules) to the external medium triggered by neutrophil activators were inhibited by DIDS or 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (2, 7, 8, 9, 10, 11). Moreover, EA inhibits neutrophil spreading and O2– production induced either by TNF-{alpha} on fibronectin-coated surfaces (12) or by cross-linking of {beta}2 integrins (13), in parallel with its effects on Cl– efflux. The blockade by EA and another Cl– efflux inhibitor, 2-aminomethyl-4-(1-methyl-1-phenylethyl)-6-iodophenol hydrochloride (MK-447A), of the TNF-{alpha} induced increase in mAb24 binding (14), a marker for the activator-induced high affinity conformation of {beta}2 integrins (15, 16), provides further evidence for a role of Cl– efflux in neutrophil signaling. Conversely, when the neutrophil Cl– content is lowered by incubation in low Cl– medium, there is an increase in mAb24 binding (14). These data suggest that a decrease in Cl– content and/or concentration or an increase in Cl– efflux are necessary and/or sufficient to trigger the change in {beta}2 integrin conformation and affinity as well as neutrophil functions, such as attachment, spreading, superoxide production, and release of enzymes from storage vesicles. However, the mechanism that mediates the Cl– efflux remains to be identified.

Neutrophils are reported to have a variety of Cl– transport mechanisms, including a Cl-HCO3 exchanger and several putative types of Cl– channels. Ca2+-activated (6), swelling-activated (17), and PKC-activated (5) Cl– channels have been reported electrophysiologically. In addition, mRNAs for ClC-2, ClC-3, ClC-4, and ClC-5 Cl– channels have been identified in neutrophils using RT-PCR (18). TNF-{alpha} at 11.4 nM, a concentration reported to increase intracellular Ca2+ (4, 6), appears to increase a Ca2+-activated Cl– conductance. However, GM-CSF and TNF-{alpha} also activated the Cl– efflux at concentrations at which there was no detectable increase in cytoplasmic Ca2+ (4). Furthermore, intracellular Ca2+ buffering with BAPTA-AM did not prevent the Cl– efflux caused by {beta}2 integrin cross-linking (13), arguing against a role for Ca2+-stimulated conductance in the Cl– efflux. In contrast, the PKC-activating phorbol esters, such as PMA, at high concentrations (1 µM) cause Cl– efflux and Cl– currents similar to those caused by FMLP in human neutrophils (3, 5). However, the fact that these putative channels are so strongly outwardly rectifying, permitting very little inward current (corresponding to Cl– efflux), argues against this idea. More importantly, the PKC inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) did not reduce the Cl– efflux (13).

The observation that the 2-(aminomethyl) phenol inhibitors, MK-447 and MK-447A (19), inhibited spreading and superoxide generation caused by {beta}2 integrin cross-linking (13) at concentrations similar to those that block neutrophil swelling-activated Cl– channels (IClvol) (17) suggests a possible role for IClvol in the activation-induced Cl– efflux. Moreover, MK-447A, the most potent inhibitor of the swelling-activated channels (17), inhibits the change in {beta}2 integrin conformation sensed by mAb24 in parallel with its effects on Cl– efflux (14).

Thus, the pharmacological data to date indicate that IClvol is the most likely candidate to serve as the mechanism of the activation-induced Cl– efflux in human neutrophils. To test this hypothesis, we have examined the effects of two agents known to inhibit activation-induced Cl– efflux or Cl– currents, EA and NPPB, on IClvol in human neutrophils and in promyelocytic leukemic HL-60 cells, a cell culture model for the promyelocyte, a neutrophil precursor. We have also measured the effects of [3-methyl-1-p-sulfophenyl-5-pyrazolone-(4)]-[1,3-dibutylbarbituric acid]-pentamethine oxonol (WW781), a nonpenetrating analog of the oxonol bis-(1,3-dibutylbarbituric acid)-pentamethine oxonol (diBA-(5)-C4), which is a potent inhibitor of IClvol in HL-60 cells (20). The ability of these compounds to inhibit IClvol has been compared with their effectiveness in inhibiting the activator-induced Cl– efflux and neutrophil {beta}2 integrin up-regulation. NPPB and WW781 have qualitatively similar effects on IClvol and on {beta}2 integrin up-regulation, as predicted by the hypothesis. However, EA at a concentration that inhibits the activator-induced Cl– efflux as well as {beta}2 integrin up-regulation has no effect on the IClvol in human neutrophils, providing evidence against the involvement of IClvol in neutrophil activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Neutrophil isolation

Neutrophils were isolated from heparinized human blood by the Ficoll-Hypaque method. Briefly, blood was cooled at room temperature for 30 min before its use. Then 3 ml of neutrophil isolation medium (Cardinal Associates) or Polymorphprep (Axis-Shield, Rodeløkka, Oslo, Norway) were added to an 8-ml polystyrene tube (Falcon; BD Biosciences, Franklin Lakes, NJ) and layered with 3.5 ml of blood. The tubes were centrifuged at 1500 rpm (470 x g) for 40 min at room temperature. After centrifugation the neutrophil-containing layer was collected and resuspended in 5 ml of HBSS (without Ca2+ or Mg2+) supplemented with 10 mM HEPES and 0.1% BSA (fraction V, IgG free) for washing. (For flow cytometry experiments, low endotoxin BSA was used.) Then the cells were centrifuged for 10 min at 1000 rpm. This washing step was repeated once. The cells were washed a third time in HBSS and 10 mM HEPES with 1.5 mM calcium and 1 mM magnesium and resuspended for treatment in this buffer without or with BSA for patch-clamp or flow cytometry experiments, respectively.

HL-60 cell growth and culture

HL-60 cells were obtained from American Type Culture Collection (Manassas, VA) and were grown in RPMI 1640 medium (Life Technologies, Gaithersburg, MD) containing 25 mM HEPES, 10% FBS (heat inactivated) and 1% penicillin/streptomycin in a 5% CO2 atmosphere at 37°C. All reagents were purchased from Life Technologies.

Whole-cell patch clamp

Solutions. To record swelling-activated Cl– currents from human neutrophils and HL-60 cells we generally, except for Fig. 3, used a bath solution containing 140 mM tetraethylammonium-chloride, 20 mM HEPES, and 0.5 mM CaCl2 (pH 7.2; 280 mOsmol/kg). The pipette solution included 141 mM tetraethylammonium-Cl, 10 mM EGTA, 20 mM HEPES, and 2 mM ATP-Na2 (pH 7.2; 310 mOsmol/kg). For experiments in Fig. 2 the bath solution contained 140 mM N-methyl-D-glucamine (NMDG)-Cl, 0.5 mM CaCl2, and 10 mM HEPES (pH 7.2; 278 mOsmol/kg). The composition of the corresponding pipette solution was 60 mM NMDG-Cl, 80 mM NMDG-glutamate, 10 mM NMDG-EGTA, and 20 mM HEPES (pH 7.2; 326 mOsmol/kg). The extracellular solutions used were always hypotonic relative to the pipette solution to spontaneously activate volume-sensitive channels. The stock solutions of blockers used for patch-clamp experiments were prepared in ethanol (EA) or DMSO (WW781 and NPPB).



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  		FIGURE 3. Effect of WW781 on IClvol in neutrophils. A, Representative Cl– current traces obtained from the same neutrophil without (left), with (middle), and after washing away (right) 25 µM WW781. B, Average current-voltage curves (n = 4) obtained before ({blacksquare}) and after (•) exposure to WW781.

 


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  		FIGURE 2. NPPB blocks IClvol in neutrophils. A, Whole-cell currents recorded in a hypotonic extracellular solution were elicited by a series of voltage pulses from –80 to +100 mV in 20-mV steps. The holding potential was 0 mV. B, Currents from the same cell after exposure to 100 µM NPPB. C, Average current-voltage relationships (n = 6) obtained before ({blacksquare}) and after (•) addition of NPPB. D, The fraction of unblocked channels (FNB) is plotted as a function of membrane voltage to illustrate the voltage dependence of the block by NPPB. The unblocked fraction of channels was obtained dividing the total current in the presence of NPPB by the current in the absence of NPPB.

 
Patch clamp. To record macroscopic currents the whole cell configuration of the patch-clamp technique was used, as previously described (20). Before electrophysiological recording the HL-60 cells were plated on 5-mm diameter coverslips coated with poly-L-lysine. Neutrophils were plated directly onto clean coverslips and allowed to adhere for 5–10 min. Cl– currents were recorded at room temperature with an Axopatch 200B amplifier (Axon Instruments, Foster City, CA). Glass pipettes were fabricated with borosilicate glass (Accufil; VWR, South Plainfield, NJ) to have resistance values between 6 and 8 M{Omega} when filled with the intracellular solution. No series resistance or leak compensation was performed. Data points were acquired using pCLAMP software (Axon Instruments, Union City, CA). Cells were held at –60 mV and stepped for 400 or 800 (see Fig. 5) ms to membrane voltage values from –100 to +100 mV or –80 to +120 mV for neutrophils and HL-60 cells, respectively.



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  		FIGURE 5. Lack of EA inhibition of IClvol in neutrophils and HL-60 cells. IClvol obtained from a representative cell in the absence (A) and the presence (B) of 250 µM EA. C, The average current-voltage relationships (n = 6) are shown in the absence ({blacksquare}) and the presence (•) of EA. D, Average current-voltage relationships (n = 5) of IClvol obtained from HL-60 cells are shown in the absence ({blacksquare}) and the presence (•) of EA.

 
Chloride efflux. To lyse residual RBC, the purified neutrophils were resuspended for 30 s in Dulbecco’s PBS without calcium or magnesium (Life Technologies) diluted to 14% by volume, and then 4x isotonic PBS containing BSA was added to restore tonicity and give a final BSA concentration of 0.1%. Cells were centrifuged, washed once in BSS– (146 mM NaCl, 5.4 mM KCl, 5.5 mM D-glucose, and 10 mM HEPES, pH 7.4, at 37°C) containing 0.1% BSA, and resuspended at a final concentration of 2 x 107 cells/ml. Cl– efflux measurements were performed as we previously described for HL-60 cells (21). Briefly, for each flux measurement, 0.9 ml of this suspension was incubated with 2.5 µCi/ml 36Cl– for 2 h at 37°C, then washed twice at room temperature in BSS– (pH 7.4 at room temperature) to eliminate excess isotope. Isotope-loaded cells were resuspended in 4.5 ml of low chloride isotonic or hypotonic medium at 21°C, except as indicated. Isotonic low chloride medium contained 168 mM sodium glucuronate, 1.5 mM CaSO4, 1 mM MgSO4, 5.5 mM D-glucose, 10 mM HEPES (pH 7.4 at room temperature), and 0.1% BSA (only 0.01% was used when EA was present). The hypotonic medium was made by mixing equal volumes of isotonic medium and diluent (1.5 mM CaSO4, 1 mM MgSO4, 5.5 mM glucose, and 10 mM HEPES, pH 7.4), and contained in addition 1 µM gramicidin (added from a 1 mg/ml stock solution in ethanol).

For measurements of the effect of EA on FMLP-induced Cl– efflux, isotope-loaded cells were resuspended in isotonic medium containing 0 or 0.5 mM EA, and then 10 nM FMLP was added at time zero. For measurements of the effect of EA on swelling-induced Cl– efflux, isotope-loaded cells were resuspended in hypotonic medium containing different concentrations of EA at time zero.

At each predetermined time interval, 0.5 ml of cell suspension was layered over 0.5 ml of Silicone AR 200 fluid (Serva) and mineral oil (Sigma-Aldrich, St. Louis, MO), and centrifuged at 12,800 x g for 30 s in an Eppendorf centrifuge. The silicone and mineral oils were mixed to obtain a density of 1.03 or 1.02 g/ml for isotonic or hypotonic conditions, respectively. After centrifugation, 0.3 ml of the supernatant was taken, mixed with 0.4 ml of SDS (0.1%), and added to 5 ml of scintillation fluid for 10 min. Replicate 0.3-ml samples of the cell suspension were added directly to SDS and scintillation fluid to determine the total cpm in the suspension, yinf. The rate constant for Cl– efflux was calculated by fitting the cpm (y) at various times (t) to the equation y = y0 + (yinf – y0)(1 – exp(–kt)), where y0 is the cpm at time zero. Values of y0 and k were determined from a nonlinear least squares fit using the program Origin (OriginLab, Northampton, MA), with yinf held constant at the measured value.

Immunofluorescence flow cytometry

Neutrophil samples were pretreated for 5 min at 37°C in 250 µM ethacrynic acid (EA stock was prepared in HBSS alone or in ethanol, as in the patch-clamp experiments), and then stimulated for 15 min at 37°C with 10 nM FMLP or 1.14 nM TNF-{alpha}. Control samples received no pretreatment with EA but were incubated at 37°C for 5 min before stimulation. All samples were immediately placed on ice for Ab labeling. Surface Ag levels were determined by incubating samples at 4°C for 45 min on a rotating platform with saturating levels of anti-CD18 labeled with FITC or anti-CD11b labeled with PE. Mean fluorescence in arbitrary units was then measured in a Elite flow cytometer (Beckman Coulter, Fullerton, CA).

Materials

HBSS (without calcium and magnesium; catalog no. 14175) was obtained from Life Technologies. BSA (fraction V, either low endotoxin, IgG-free or {gamma}-globulin-free), EA, NPPB, and FMLP were purchased from Sigma-Aldrich, and recombinant human TNF-{alpha} was purchased from R&D Systems (Minneapolis, MN). FITC-labeled anti-CD18 and PE-labeled anti-CD11b were obtained from Immunotech (Westbrook, ME). WW781 was purchased from Molecular Probes (Eugene, OR).

Patch-clamp data analysis

Data analysis was performed using the Clampfit software of the pCLAMP suite version 8 (Axon Instruments). Current amplitudes measured at the end of each pulse were used to construct the current-voltage relationships. All data are given as the mean ± SE, with n designating the number of cells for which measurements were averaged. Statistical analysis of current blockade was performed using a paired t test (OriginLab).

The electrical distance to the blocking site for WW781 was calculated using the Woodhull model (22), described by the following equation:

(1)
where FNB is the fraction of channels not blocked by the inhibitor concentration ([I]) used, Kd is the dissociation constant for inhibitor binding to the channel at 0 mV, z is the valence of the inhibitor, {delta} represents the fraction of the membrane electric field traversed by the external inhibitor to reach its binding site, Vm is the membrane voltage, k is Boltzmann’s constant, and T is the absolute temperature. Application of this model to our data requires the assumption that binding of WW781 has reached equilibrium.

A total of 23 neutrophils were patch-clamped under hypotonic conditions, and five were patch-clamped under hypertonic conditions. Current was recorded from all cells bathed in hypotonic medium. Six of nine neutrophils treated with EA maintained good seals and were used for data analysis. Current from all six cells exposed to NPPB was blocked. Treatment with WW781 was completed in four of seven cells. For HL-60 cell experiments we used a total of 18 cells; of those, 10 cells were treated with EA, but only five maintained good seals and therefore were used for data analysis. Of the remaining eight cells, only five treated with WW781 were used for data analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To study IClvol in both neutrophils and HL-60 cells we have used recording solutions without intracellular calcium or PKC to avoid activation of calcium- or PKC-dependent Cl– currents, respectively.

Whole-cell Cl– currents in human neutrophils

IClvol were recorded from unstimulated neutrophils after the whole-cell configuration was attained. Activation of Cl– channels was achieved using a bath solution that was hypotonic with respect to the pipette solution. Fig. 1 shows the time course of current activation ({blacksquare}) as well as the effect of high tonicity (•) on whole-cell current. The current activates following an exponential time course with a time constant of ~100 s and reaches a maximum amplitude of about +120 pA at +80 mV. In contrast, whole-cell current recorded under hypertonic conditions was only +5.3 pA at +80 mV. Therefore, almost the entire current recorded under our experimental conditions was mediated by volume-sensitive channels. As both the bath and pipette solutions contained Cl– as the singular permeable anion, it is likely that the volume-sensitive current was carried by Cl–. This is supported by the observation that the reversal potential was near 0 under symmetrical chloride conditions or –17 mV with a chloride gradient (Cli = 60 and Clo = 141 mM). In most of the experiments the Cl– concentration inside and outside of the cell was 140 mM to maximize the current amplitude recorded.



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  		FIGURE 1. Time course of IClvol activation. Whole-cell current was recorded at +80 mV using a 400-ms test pulse applied every 10 s ({blacksquare}; n = 10). Recordings started 20 s after reaching the whole cell configuration under hypotonic conditions. The continuous line is a single exponential fit with a maximum of 122 ± 4 pA and a time constant of 96 ± 9 s. For comparison, the current recorded from cells bathed in a hypertonic solution (•; n = 5) is also plotted.

 
Fig. 2A shows control raw currents obtained from a representative neutrophil. At all potentials, current traces showed little or no inactivation. This observation is in agreement with previously reported data for neutrophils (17); nevertheless, this contrasts with a strong time-dependent inactivation process reported for volume-activated channels in other cell types (23). The corresponding current-voltage relationship, shown in Fig. 2C ({blacksquare}), illustrates the outward rectifying behavior reported previously for these channels (19). Without a Cl– gradient (Cli = Clo = 140 mM), currents reversed at 0 mV as expected.

The volume-sensitive Cl– conductance recorded from many mammalian cells is sensitive to NPPB, DIDS, and tamoxifen (24). In the following section we show that the volume-sensitive currents from human neutrophils are inhibited by NPPB. Fig. 2B illustrates whole-cell Cl– currents obtained from the same cell as those shown in Fig. 2A after addition of 100 µM NPPB to the extracellular medium. Currents were strongly inhibited by this concentration of NPPB. Fig. 2C shows the corresponding average current-voltage relationships of IClvol in the presence of NPPB (•). The unblocked fraction did not change significantly with voltage (Fig. 2D). The percentages of current inhibited by NPPB at +80 and –80 mV were 75.9 ± 7.9 and 69.5 ± 2.1%, respectively.

Effect of WW781 on IClvol in neutrophils and HL-60 cells

In HL-60 cells IClvol is inhibited with high affinity by the oxonol dye diBA-(5)-C4 (20), an analog of WW781. Unlike diBA-(5)-C4, WW781 is membrane impermeable (25), and therefore its effects are expected to be restricted to sites accessible from the extracellular side of the membrane. In our experiments this was confirmed by the absence of blue color inside the cells. Additional evidence to support the idea that WW781 blocks IClvol by interacting with an external site was obtained from the lack of voltage dependence of blockade in both neutrophils and HL-60 cells. Fig. 3A shows whole-cell Cl– currents obtained from a human neutrophil before, during, and after application of 25 µM WW781 to the bath solution. The oxonol dye inhibits effectively and in a reversible manner the Cl– currents at all potentials. Fig. 3B illustrates the effect of WW781 on the average current-voltage relationship. In these experiments the macroscopic current reversed at –17 mV with Cli = 60 mM and Clo = 141 mV (ECl = –21.5 mV). Current amplitudes were diminished at all voltages without significant voltage dependence. At +80 or –80 mV, the percent inhibition of current was 72.6 ± 16 or 66.5 ± 24.5%, respectively; these two values were not significantly different (p > 0.05). The chloride gradient imposed was used to verify the Cl– dependence of volume-activated currents.

To further document the inhibitory effect of WW781 on IClvol, we recorded the IClvol from HL-60 cells, a cell culture model that can be differentiated into neutrophil-like cells. HL-60 cells express robust IClvol currents with similar properties to those displayed by human neutrophils (17, 20). Thus, these currents can be used to test unambiguously the effects of different inhibitors. Fig. 4, A and B, displays the whole-cell Cl– currents before and after treatment with 10 µM WW781, respectively. The corresponding current-voltage curves are shown in Fig. 4C. Just as in human neutrophils, WW781 inhibited IClvol in HL-60 cells, decreasing the current amplitude at all voltages. The inhibitions at +80 and –80 mV were 77 ± 10.4 and 66 ± 8.9%, respectively. These two values were significantly different (p < 0.05), indicating a slight voltage dependence of the inhibitory action of WW781 (Fig. 4D). The effects of voltage were analyzed with the Woodhull model (see Materials and Methods); the fit is shown by the line through data points in Fig. 4D. The analysis suggests that WW781 traverses 4 ± 0.3% of the electrical field to reach its binding site, which is essentially equivalent to having the binding site readily accessible from the extracellular side of the HL-60 cell plasma membrane. This very small effect of voltage is in agreement with the neutrophil data shown in Fig. 3.



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  		FIGURE 4. Effect of WW781 on IClvol in HL-60 cells. Whole-cell currents obtained from the same cell before (A) and after (B) treatment with 10 µM WW781. C, Average current-voltage relationships (n = 5) obtained under control conditions ({blacksquare}) and in the presence of WW781 (•). D, Voltage dependence of WW781 blockade shown as the fraction of unblocked channels plotted against the membrane voltage. The continuous line is the fit to data with a Woodhull model (equation 1). Kd at 0 mV = 3.8 ± 0.06 µM; electrical distance ({delta}) = 4% within the electrical field.

 
Effect of EA on Cl– efflux and IClvol in neutrophils and HL-60 cells

Previous work has reported that 250 µM EA inhibited both the Cl– efflux and mAb24 binding to an integrin activation-specific epitope in TNF-{alpha} stimulated neutrophils (14). If IClvol is the mechanism of activation-induced Cl– efflux, then EA would be expected to inhibit IClvol. To reveal the effects of EA on IClvol, whole-cell currents were recorded before and after addition of 250 µM EA to the bath. Fig. 5, A and B, shows current traces without or with EA present, respectively. Current kinetics and magnitudes were nearly identical in both conditions. Fig. 5C summarizes the effects of 250 µM EA on current magnitude at different membrane potentials. We also tested the effect of EA on IClvol in HL-60 cells. The current-voltage relationships before ({blacksquare}) and after (•) addition of 250 µM EA are depicted in Fig. 5D. Cl– current through IClvol expressed in HL-60 cells was also insensitive to EA used at the same high concentration. Thus, EA has no effect on IClvol in either human neutrophils or HL-60 cells.

To corroborate that EA under more physiological conditions does not block volume-activated currents, we obtained a dose-response curve using a population of intact neutrophils in suspension. Fig. 6 shows the contrasting sensitivity to EA of the FMLP-activated ({circ}) and swelling-activated (•, {blacktriangledown}, {blacktriangleup}, and {blacksquare}) fluxes. A concentration of 500 µM EA blocked most of the FMLP-activated Cl– efflux as expected, whereas the swelling-activated efflux was only reduced by ~17% at this concentration (based on the least squares best-fit line). The inset shows typical flux data for 2 and 4 mM EA obtained under hypotonic conditions.



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  		FIGURE 6. EA inhibition of Cl– efflux in neutrophil suspensions. IClvol was measured by loading cells with 36Cl– and then measuring the appearance of 36Cl– in a medium lacking Cl–, as described in Materials and Methods, either under hypotonic conditions with 1 µM gramicidin at 21°C (•, {blacktriangleup}, {blacktriangledown}, and {blacksquare}) or after stimulation with 10 nM FMLP ({circ}) in isotonic medium at 21°C, except for one experiment which was performed at 37°C. Data were obtained using neutrophils from four different donors, indicated by different filled symbols, and were fitted by nonlinear least squares to the equation for single site inhibition, a = 1/(1 + [I]/IC50), where a is the fractional activity, [I] is the EA concentration, and IC50 is the concentration that gives half-inhibition. For the swelling-activated flux, the IC50 was 2.5 ± 0.4 mM, and for the FMLP-stimulated flux it was 0.13 ± 0.02 mM. The inset shows plot of raw flux data obtained under hypotonic conditions with 0 (control; {circ}), 2 ({blacktriangledown}), or 4 mM (*) EA.

 
Effect of EA on neutrophil {beta}2 integrin surface expression

When neutrophils are stimulated with TNF-{alpha}, the total number of {beta}2 integrins on the cell surface increases as well as the number of binding sites for the mAb24 Ab (14). We tested the effectiveness of EA on the activator-induced increase in total {beta}2 integrins, as measured by binding of an anti-CD18 ({beta}2-chain) Ab.

Fig. 7 shows that treatment of human neutrophils with either 10 nM FMLP or 1.14 nM TNF-{alpha} caused a pronounced increase in CD18 surface expression ({blacksquare}). EA ({square}) at 250 µM (control) caused a small, but significant, increase in the CD18 level. EA also significantly inhibited the increase caused by 10 nM FMLP. The inhibition of the increase induced by 1.14 nM TNF-{alpha} was only marginally significant (p = 0.057), but was consistent in three experiments with neutrophils from different donors. When the EA stock was made in ethanol, as in the patch-clamp experiments, instead of HBSS, similar results were obtained (

). Thus EA, at the same concentration (250 µM) that has no significant effect on swelling-activated Cl– currents does have a significant effect on the FMLP-induced up-regulation of CD18, ruling out the possibility that our failure to see effects on the Cl– channels might be in some way due to the EA treatment conditions.



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  		FIGURE 7. Effect of 250 µM EA on neutrophil CD18 surface expression. Levels of surface CD18, obtained by flow cytometric analysis with FITC-labeled anti-CD18 as described in Materials and Methods, are reported as the percent increase in mean channel fluorescence intensity compared with control cells without EA or activators. Data are from a single experiment with CD18, representative of two experiments with CD18 and one with CD11b, the major {alpha}-chain partner of CD18. Error bars show the range of two determinations, except for the rightmost column for TNF-{alpha}, which represents a single determination. Analysis of the three experiments, by applying an unpaired t test to all the measured values for EA dissolved in HBSS, showed that EA alone (control) caused a significant increase (p < 0.005; five measurements in three experiments performed with cells isolated from different donors). EA also caused a significant inhibition of the increase induced by 10 nM FMLP (p < 0.005; six measurements in three experiments) and a marginally significant (p = 0.057) inhibition of the increase stimulated by 1.14 nM TNF-{alpha}. When the data for EA dissolved in ethanol were included, similar levels of significance were obtained.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Effects of NPPB and WW781

NPPB is a highly effective inhibitor of IClvol in several cell types (24). In the experiments presented in this report, 100 µM NPPB causes ~80% inhibition of IClvol in neutrophils (Fig. 2). WW781 is an even more potent inhibitor of IClvol in both neutrophils and HL-60 cells (Figs. 3 and 4). Because the dark blue WW781 does not cause any noticeable staining of the cells, it appears that it remains extracellular, probably because of its very polar sulfonic acid group (25), and thus inhibits by binding to a site that is accessible from the extracellular side of the membrane. This is consistent with the very small voltage dependence of inhibition, which suggests that the doubly negatively charged WW781 binds to a site that is readily accessible from the outside. In these experiments a chloride gradient was imposed to verify the Cl– dependence of volume-activated currents. As the WW781 block was nearly voltage independent, the Cl– gradient used for these experiments should have no effect on the percent inhibition.

In separate experiments reported previously (26), NPPB at 100 µM nearly completely inhibited FMLP-induced up-regulation of CD11b, the major {alpha} integrin ({alpha}M) associated with CD18. WW781 at 25 µM has effects on the CD18 ({beta}2 integrin) up-regulation similar to those caused by NPPB on CD11b (26). These data are consistent with the hypothesis that the FMLP-induced Cl– efflux takes place through IClvol, and that this, in turn, participates in CD11b and CD18 up-regulation. Up-regulation by two other activators, TNF-{alpha} and GM-CSF, however, was less sensitive to NPPB and WW781 (J. B. Schultz and P. A. Knauf, unpublished observations).

Effects of EA

EA is a very effective inhibitor of the activator-induced Cl– efflux in neutrophils caused by various activators, with an IC50 of <80 µM (4, 12, 13, 14). It also inhibits other neutrophil functions, such as adhesion (12), mAb24 binding (4), superoxide production (12, 13), and spreading on fibronectin-coated surfaces (12). Nevertheless, EA had no significant effect on IClvol in either neutrophils or HL-60 cells (Fig. 5). Consistent with these results on individual cells, the swelling-activated 36Cl– efflux measured in neutrophil suspensions displays a very low EA sensitivity, with an IC50 of 2.5 ± 0.4 mM (Fig. 6). Because of the scatter in the data presented in Fig. 6 at low EA concentrations, it would be difficult to rule out the possibility that there is a small component of the volume-activated Cl– efflux that is inhibited by low concentrations of EA. This possibility is rendered highly unlikely, however, by the absence of an effect of 250 µM EA in the more precise IClvol measurements (Fig. 5).

This lack of inhibitory potency could not have been due to ineffectiveness of the EA preparation or nonspecificity of the Cl– efflux measurement, because in our experiments EA caused a very significant inhibition of the FMLP-induced Cl– efflux (Fig. 6) and up-regulation of CD18 (Fig. 7). EA does not inhibit anion exchange in neutrophils (27). However, EA is not a very selective inhibitor, interfering with various Cl– transport processes and other enzymatic activities. This nonselectivity makes it difficult to identify the cause of the EA effects on Cl– efflux and other neutrophil functions stimulated by activators, but it does not weaken the evidence against involvement of IClvol. The simplest explanation for the effects of EA (4, 12, 13, 14) is that it directly inhibits activation-induced Cl– efflux. If IClvol mediates this Cl– efflux, then EA must inhibit IClvol, regardless of whether EA has other nonspecific effects. Thus, our observation that EA has no effect on IClvol provides strong evidence that IClvol is not the mediator of the activation-induced Cl– efflux.

The only alternative explanation that might rescue the hypothesis that IClvol, mediates the Cl– efflux would be that EA inhibits the activation-induced Cl– efflux indirectly by affecting the signaling process that causes the increased Cl– efflux rather than by acting directly on IClvol. However, we are aware of no evidence that external EA inhibits intracellular signaling cascades. Furthermore, EA inhibits Cl– efflux triggered by various activators, including cross-linking of {beta}2 integrins, which almost certainly use different signaling pathways, making the possibility that EA acts by inhibiting all these signaling processes unlikely.

Our data thus provide strong evidence against the former most likely candidate for the Cl– efflux mechanism, IClvol. It seems that NPPB, WW781, and EA all exert their effects on neutrophil Cl– efflux and/or neutrophil activation phenomena related to the Cl– efflux by interacting with a transporter or channel, different from IClvol, that remains to be identified.


   Acknowledgments
 
The assistance of Dr. Peter Keng with the flow cytometry experiments is greatly appreciated.


   Footnotes
 
1 This work was supported by National Heart, Lung, and Blood Institute, National Institutes of Health, Grant P01HL18208. Back

2 Address correspondence and reprint requests to Dr. Philip A. Knauf, Department of Biochemistry and Biophysics, University of Rochester, 601 Elmwood Avenue, Box 712, Rochester, NY 14642. E-mail address: philip.knauf{at}rochester.edu Back

3 Abbreviations used in this paper: PKC, protein kinase C; diBA-(5)-C4, bis-(1,3-dibutylbarbituric acid)-pentamethine oxonol; DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; {delta}, fraction of the membrane electric field traversed by the external inhibitor to reach its binding site; EA, ethacrynic acid; IClvol, swelling-activated Cl– channel; MK-447A, 2-aminomethyl-4-(1-methyl-1-phenylethyl)-6-iodophenol hydrochloride; NMDG, N-methyl-D-glucamine; NPPB, 5-nitro-2-(3-phenylpropylamino)-benzoic acid; WW781, [3-methyl-1-p-sulfophenyl-5-pyrazolone-(4)]-[1,3-dibutylbarbituric acid]-pentamethine oxonol. Back

Received for publication October 2, 2003. Accepted for publication March 18, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Carlos, T. M., J. M. Harlan. 1990. Membrane proteins involved in phagocyte adherence to endothelium. Immunol. Rev. 114:5.[Medline]
  2. Vedder, N. B., J. M. Harlan. 1988. Increased surface expression of CD11b/CD18 (Mac-1) is not required for stimulated neutrophil adherence to cultured endothelium. J. Clin. Invest. 81:676.
  3. Myers, J. B., H. F. Cantiello, J. H. Schwartz, A. I. Tauber. 1990. Phorbol ester-stimulated human neutrophil membrane depolarization is dependent on Ca2+-regulated Cl– efflux. Am. J. Physiol. 259:C531.
  4. Shimizu, Y., H. R. Daniels, M. A. Elmore, M. J. Finnen, M. E. Hill, J. M. Lackie. 1993. Agonist-stimulated Cl– efflux from human neutrophils. Biochem. Pharmacol. 45:1743.[Medline]
  5. Schumann, M. A., T. A. Raffin. 1994. Activation of a voltage-dependent chloride current in human neutrophils by phorbol 12-myristate 13-acetate and formyl-methionyl-leucyl-phenylalanine. J. Biol. Chem. 269:2389.[Abstract/Free Full Text]
  6. Schumann, M. A., P. Gardner, T. A. Raffin. 1993. Recombinant human tumor necrosis factor {alpha} induces calcium oscillation and calcium-activated chloride current in human neutrophils. J. Biol. Chem. 268:2134.[Abstract/Free Full Text]
  7. Korchak, H. M., B. A. Eisenstat, S. T. Hoffstein, P. B. Dunham, G. Weissmann. 1980. Anion channel blockers inhibit lysosomal enzyme secretion from human neutrophils without affecting generation of superoxide anion. Proc. Natl. Acad. Sci. USA 77:2721.[Abstract/Free Full Text]
  8. Korchak, H. M., B. A. Eisenstat, J. E. Smolen, L. E. Rutherford, P. B. Dunham, G. Weissmann. 1982. Stimulus-response coupling in the human neutrophil. The role of anion fluxes in degranulation. J. Biol. Chem. 257:6912.
  9. Smith, R. J., B. J. Bowman, S. S. Iden. 1984. Effects of an ion channel blocker, 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS), on human neutrophil function. Biochem. Biophys. Res. Commun. 120:964.[Medline]
  10. Smith, R. J., B. J. Bowman, S. C. Speziale. 1986. Interleukin-1 stimulates granule exocytosis from human neutrophils. Int. J. Immunopharmacol. 8:33.[Medline]
  11. Smith, R. J., S. C. Speziale, R. G. Ulrich, B. J. Bowman. 1986. Characteristics of aggregated immunoglobulin G as an immunologic phagocytic stimulus for granule enzyme release from human neutrophils. Inflammation 10:131.[Medline]
  12. Menegazzi, R., S. Busetto, P. Dri, R. Cramer, P. Patriarca. 1996. Chloride ion efflux regulates adherence, spreading, and respiratory burst of neutrophils simulated by tumor necrosis factor on biological surfaces. J. Cell Biol. 135:511.[Abstract/Free Full Text]
  13. Menegazzi, R., S. Busetto, E. Decleva, R. Cramer, P. Dri, P. Patriarca. 1999. Triggering of chloride ion efflux from human neutrophils as a novel function of leukocyte {beta}2 integrins: relationship with spreading and activation of the respiratory burst. J. Immunol. 162:423.[Abstract/Free Full Text]
  14. Menegazzi, R., S. Busetto, R. Cramer, P. Dri, P. Patriarca. 2000. Role of intracellular chloride in the reversible activation of neutrophil {beta}2 integrins: a lesson from TNF stimulation. J. Immunol. 165:4606.[Abstract/Free Full Text]
  15. Dransfield, I., N. Hogg. 1989. Regulated expression of Mg2+ binding epitope on leukocyte integrin {alpha} subunits. EMBO J. 8:3759.[Medline]
  16. Shimaoka, M., J. Takagi, T. A. Springer. 2002. Conformational regulation of integrin structure and function. Annu. Rev. Biophys. Biomol. Struct. 31:485.[Medline]
  17. Stoddard, J. S., J. H. Steinbach, L. Simchowitz. 1993. Whole cell Cl– currents in human neutrophils induced by cell swelling. Am. J. Physiol. 265:C156.
  18. Jiang, B., N. Hattori, B. Liu, K. Kitagawa, C. Inagaki. 2002. Expression of swelling- and/or pH-regulated chloride channels (ClC-2, 3, 4 and 5) in human leukemic and normal immune cells. Life Sci. 70:1383.[Medline]
  19. Simchowitz, L., J. A. Textor, E. J. Cragoe, Jr. 1993. Cell volume regulation in human neutrophils: 2-(aminomethyl)phenols as Cl– channel inhibitors. Am. J. Physiol. 265:C143.
  20. Arreola, J., K. R. Hallows, P. A. Knauf. 1995. Volume-activated chloride channels in HL-60 cells: potent inhibition by an oxonol dye. Am. J. Physiol. 269:C1063.
  21. Hallows, K. R., P. A. Knauf. 1994. Regulatory volume decrease in HL-60 cells: Importance of rapid changes in permeability of Cl– and organic solutes. Am. J. Physiol. 267:C1045.
  22. Woodhull, A. M.. 1973. Ionic blockage of sodium channels in nerve. J. Gen. Physiol. 61:687.[Abstract/Free Full Text]
  23. Nilius, B., J. Eggermont, T. Voets, G. Buyse, V. Manolopoulos, G. Droogmans. 1997. Properties of volume-regulated anion channels in mammalian cells. Prog. Biophys. Mol. Biol. 68:69.[Medline]
  24. Fürst, J., M. Gschwentner, M. Ritter, G. Bottà, M. Jakab, M. Mayer, L. Garavaglia, C. Bazzini, S. Rodighiero, G. Meyer, et al 2002. Molecular and functional aspects of anionic channels activated during regulatory volume decrease in mammalian cells. Pflügers Arch. Eur. J. Physiol. 444:1.[Medline]
  25. George, E. B., P. Nyirjesy, M. Basson, L. A. Ernst, P. R. Pratap, J. C. Freedman, A. S. Waggoner. 1988. Impermeant potential-sensitive oxonol dyes: Evidence for an "on-off" mechanism. J. Membr. Biol. 103:245.[Medline]
  26. Knauf, P., J. Schultz. 2002. Effects of chloride channel inhibitors on activator-induced CD11b up-regulation in human neutrophils. FASEB J. 16:A513.
  27. Simchowitz, L., P. DeWeer. 1986. Chloride movements in human neutrophils: diffusion, exchange, and active transport. J. Gen. Physiol. 88:167.[Abstract/Free Full Text]



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