Surfactant modulates calcium response of neutrophils to physiologic stimulation via cell membrane depolarization

ABSTRACT Pulmonary surfactant (PS) reduces inflammation in the lung by poorly understood mechanisms. We have observed that surfactant-associated proteins (SAP) insert monovalent cation

channels in artificial membranes. Neutrophils are primary mediators of acute pulmonary inflammation, and their functions are activated by increases in cytosolic ionized calcium concentration

([Ca2+]) and by changes in membrane potential. We hypothesize that PS inserts SAP-dependent cation channels in neutrophils, causing membrane depolarization, altered [Ca2+] response, and

depressed activation. Human neutrophils were isolated, exposed to PS+SAP (1% Survanta), PS−SAP (1% Exosurf), or buffer, and washed before activating with selected stimulants. PS+SAP reduced

phorbol ester- and formyl peptide–stimulated adherence and aggregation by 38% (_p_ < 0.05) and 54% (_p_ < 0.02), respectively. PS+SAP also inhibited the formyl peptide–induced [Ca2+]

response of neutrophils (_p_ < 0.01), but only in the presence of external Ca2+. Further characterization of this inhibition demonstrated that PS+SAP blocked formyl peptide–induced influx

of both Ca2+ and Mn2+, and that this inhibition was present during activation by other neutrophil stimulants (IL-8, immune complexes). Prior depolarization of neutrophils with gramicidin-D

similarly inhibited the [Ca2+] response of neutrophils to formyl peptide, and analysis of neutrophil membrane potential by 3,3′-dipentyloxaearbocyanine iodide (diOC5(3)) fluorescence

revealed that PS+SAP induced rapid neutrophil depolarization. In contrast, PS−SAP exhibited little effect on neutrophil function, [Ca2+], or membrane potential. We conclude that PS+SAP

decreases neutrophil adherence and aggregation responses, blocks Ca2+ influx after physiologic stimulation, and decreases membrane potential. We speculate that these effects are caused by

membrane depolarization via SAP-dependent cation channel insertion, and that all of these effects contribute to the antiinflammatory properties of PS+SAP. SIMILAR CONTENT BEING VIEWED BY

OTHERS THE MITOCHONDRIAL CALCIUM UNIPORTER OF PULMONARY TYPE 2 CELLS DETERMINES SEVERITY OF ACUTE LUNG INJURY Article Open access 03 October 2022 MICROVESICLES RELEASED FROM

PNEUMOLYSIN-STIMULATED LUNG EPITHELIAL CELLS CARRY MITOCHONDRIAL CARGO AND SUPPRESS NEUTROPHIL OXIDATIVE BURST Article Open access 05 May 2021 OPTIMIZING EXOGENOUS SURFACTANT AS A PULMONARY

DELIVERY VEHICLE FOR CHICKEN CATHELICIDIN-2 Article Open access 10 June 2020 MAIN PS is a complex mixture of several phospholipids and four SAP (1). Reduction of alveolar surface tension is

a primary function of surfactant to which both phospholipids and SAP contribute. The presence of SAP contributes to secondary functions directed at recycling of surfactant and immune

modulation (1–4). Two of the four SAPs, SAP-B and SAP-C, are hydrophobic proteolipids present in all of the commercially available natural surfactants. Synthetic surfactants lack SAP (1). In

clinical trials comparing efficacy of natural surfactant (PS+SAP) with artificial surfactant (PS−SAP), natural surfactants promote lower oxygen requirements, fewer pulmonary air leaks, and

better survival, suggesting protective effects attributable to the secondary functions of SAP-B and SAP-C, _i.e._ surfactant recycling and immune modulation (5). Neutrophils are the most

important mediators of acute inflammation in the lung and are implicated in a variety of infectious and noninfectious pulmonary diseases, including pneumonia, idiopathic pulmonary fibrosis,

asthma, emphysema, and acute respiratory distress syndrome (6, 7). Prior studies have demonstrated that treatment of neutrophils with natural surfactant decreases neutrophil adherence,

chemotaxis, respiratory burst, and elastase production after physiologic stimulation (8–11), whereas treatment with synthetic surfactant does not (9, 10). We have previously observed that a

natural surfactant preparation, Survanta, but not a synthetic surfactant preparation, Exosurf, inserts monovalent cation channels in artificial black membranes (12). More recent experiments

using SAP isolated from Survanta confirm that SAP-B and/or SAP-C account for the observed channel activities in the artificial membranes (13). Comparable electrophysiologic investigations

have not been performed in living cells. We hypothesized that PS+SAP inserts SAP-dependent cation channels in neutrophils, causing depolarization of cell membranes, alteration of [Ca2+]

response, and depression of neutrophil activation. We tested the latter part of this hypothesis by exposing human neutrophils to PS+SAP or PS−SAP, removing the surfactant by washing and

activating the cells with physiologic stimulants. In the ensuing investigations, we observed that 1% PS+SAP, but not PS−SAP, depolarizes cell membranes of neutrophils and alters [Ca2+]

responses to physiologic stimulation through blockade of agonist-stimulated Ca2+ influx. These electrochemical effects of PS+SAP on neutrophils are associated with decreased proinflammatory

neutrophil responses to physiologic stimulation. METHODS REAGENTS AND CHEMICALS. EGTA, dextran, Ficoll, NaCl, KCl, diOC5(3), O-dianisidine, HTAB, and ƒMLP were purchased from Sigma Chemical

Co. (St. Louis, MO). PS+SAP preparation (Survanta) was available from Ross Products Division, Abbott Labs (Columbus, OH) and PS−SAP preparation (Exosurf), from Glaxo-Wellcome (Research

Triangle Park, NC). Recombinant human lactoferrin was the kind gift of Denis Headon, Ph.D. (Agennix Corp, Houston, TX). Fura2-AM and gramicidin-D were purchased from Molecular Probes

(Eugene, OR). MnCl2, CaCl2 and potassium phosphate buffer were available from Fisher Scientific Company (Fair Lawn, NJ); IL-8 from R&D Systems (Minneapolis, MN); Tris+ (base), hydrogen

peroxide, and monobasic and dibasic sodium phosphate buffers from JT Baker Chemical Company (Phillipsburg, NJ); Hanks' balanced salt solution with (HBSSw) or without Ca2+ and Mg2+

(HBSSw/o) from Bio-Whitaker (Walkersville, MD); HCl from Mallinckrodt-Chemical (Paris, KY); Hypaque 76 from Sanofi Winthrop Pharmaceuticals (New York, NY); and heparin from Elfing-Sinn

(Cherry Hill, NJ). NEUTROPHIL ISOLATION. Heparinized venous blood obtained from healthy adult volunteers of either sex was separated by Hypaque-Ficoll step-gradient centrifugation, dextran

sedimentation, and hypotonic lysis as reported (14). The cell preparations resulting from this method were typically >95% neutrophils on the basis of modified Wright-Giemsa stain

morphology. BUFFERS. Cells were suspended in either HBSSw or HBSSw/o in all experiments unless otherwise noted. In some experiments, 30 μM EGTA was added to HBSSw/o to create Ca2+-free HBSS.

In control experiments, addition of 200–300 nM Ca2+ to this Ca2+-free HBSS resulted in detectable Ca2+. PS±SAP EXPOSURES. For all [Ca2+], membrane potential, and aggregation experiments,

neutrophils were previously exposed to 1% PS±SAP or purified phospholipids for 3 min at room temperature, washed once, and resuspended in HBSS ± Ca2+. For experiments looking at aggregation

responses, neutrophils were exposed to 1% PS±SAP for 3 min without washing. PREPARATION OF LACTOFERRIN/ANTI-LACTOFERRIN IMMUNE COMPLEXES. A commercially available anti-human lactoferrin IgG

fraction (ICN Pharmaceuticals, Costa Mesa, CA) was reconstituted to the manufacturer's specifications. This antibody was combined with recombinant human lactoferrin (Agennix Corp) to

achieve a 3:1 ratio (wt/wt) of antibody to lactoferrin protein, incubated at room temperature for 30 min, and then combined (1% final concentration) with fura2-labeled neutrophils as a

stimulus (15). [CA2+] MEASUREMENTS. [Ca2+] was measured using the dual-wavelength fluorescent Ca2+ probe fura2. Neutrophils, 5 × 106 in HBSSw/o, were exposed to 2 μM fura2-AM (the methyl

ester form of fura2) for 45 min at 37°C, 5% CO2, in darkness, allowing the membrane permeant methyl ester to enter the cell and be cleaved by cytosolic esterase activity to produce cytosolic

free acid fura2. After labeling, the cells were sedimented, washed twice in 15-mL volumes of HBSSw/o, and resuspended to 10 × 106 cells/mL with HBSSw. After incubation with either 1% PS+SAP

or 1% PS−SAP, the cells were washed once with HBSSw, resuspended in the same buffer, and examined for fluorescence in an LS50B spectrofluorometer (Perkin Elmer Cetus, Norwalk, CT) at

excitation wavelengths of 340 nm and 380 nm and emission wavelength of 510 nm. After baseline readings were recorded, the agonist (either ƒMLP, 1 μM final; IL-8, 150 ng/mL final; or immune

complexes at 1% final) was added to the cells, and changes in fluorescence were recorded continuously over time. In all studies, fura2-labeled cells were examined in the experimental

conditions described using four approaches:_1_) baseline fluorescence of cells without additions;_2_) fluorescence of cells after additions (agonists);_3_) cells + 0.1% Triton X-100; and

_4_) cells + 0.1% Triton X-100 + 20 mM EGTA. These experimental approaches permitted measurements of basal neutrophil [Ca2+], stimulated neutrophil [Ca2+], maximum fura2 fluorescence ratio

(Rmax), and minimum fura2 fluorescence ratio (Rmin). Neutrophil [Ca2+] values were calculated from the equation (16) MATH 1 where R is the ratio of the 340/380 nm fluorescence, Rmin is the

minimal ratio of 340/380 nm fluorescence, Rmax is the maximal ratio of the 340/380 nm fluorescence, β is the ratio of 380 nm fluorescences under Ca2+-free/Ca2+-saturated conditions, and Kd

is the dissociation constant for Ca2+ binding to fura2 (Kd = 145 nM from calibration curves). After agonist stimulation, the calculated values of [Ca2+] were used to produce a curve of

[Ca2+]_versus_ time. [Ca2+] responses to agonist stimulation were quantitated by the area under this curve, and expressed as nanomolar·minute. In experiments examining PS effects on

ƒMLP-stimulated [Ca2+] responses in zero external Ca2+ conditions, neutrophils were exposed to 1% PS+SAP, 1% PS−SAP, or control buffer and washed. Neutrophils were then resuspended in

HBSSw/o containing 30 μM EGTA and placed in the spectrofluorometer. After a stable baseline was achieved, ƒMLP (1 μM final) was added, and the fluorescence was monitored. After 210 s, 5 mM

CaCl2 was added, and the fluorescence was monitored for an additional 240 s. MANGANESE QUENCHING ASSAY. Fura2 fluorescence is lost when it binds manganese, thus permitting assessment of

divalent cation (Mn2+) entry into fura2-labeled cells (17). For these assays, cells were loaded with fura2, exposed (3 min, 24°C) to 1% PS+SAP, 1% PS−SAP, or control buffer; washed,

resuspended in HBSSw/o, and placed in the spectrofluorometer. After stabilization of the fluorescence reading, 1 mM MnCl2 was added before stimulating neutrophils with 1 μM _f_ MLP.

Fluorescence was recorded for 5 min at excitation wavelength of 360 nm (wavelength insensitive to Ca2+ but sensitive to Mn2+) and emission wavelength of 510 nm. MEASUREMENT OF NEUTROPHIL

MEMBRANE POTENTIAL. Membrane potentials (ΔΨ) were assessed through the dye response of a potential-sensitive dye, diOC5(3) (18). Neutrophils were buffered at pH 7.4 in decreasing external

[NaCl] and increasing external [KCl] to maintain the total external cation concentration equal to 150 mM in a stirred, temperature-controlled cuvette (24°C) at excitation wavelength of 460

nM and emission wavelength of 507 nM. Dye response (ΔF%) of diOC5(3) (225 nM) in the presence of the K+ ionophore valinomycin (10 μM) was calibrated to ΔΨ by the equation MATH 2 where R =

ideal gas constant, T = temperature (Kelvin), and F = faraday's constant. Pilot experiments performed using direct addition of PS+SAP to neutrophils labeled with diOC5(3) showed that

artifactual increases in fluorescence resulted from this approach, presumably through interaction between PS+SAP and PS−SAP with the lipophilic diOC5(3). Alternatively, neutrophils were

exposed to 1% PS+SAP, 1% PS−SAP, or buffer for 3 min, washed, resuspended in HBSSw, and labeled with 225 nM diOC5(3) directly in the spectrofluorometer cuvette. When a stable fluorescence

baseline was achieved, this was considered to reflect the membrane potential status of the cells; a depolarizing agent (either 50 mM KCl or 1 μM gramicidin-D) was added, and the drop in

diOC5(3) fluorescence was measured. ΔΨ from each individual experiment was expressed as millivolts from the calibration of ΔF% to ΔΨ. NEUTROPHIL ADHERENCE ASSAY. As described (19), freshly

purified neutrophils were suspended in 500 μL of HBSSw and exposed to 1% PS+SAP or 1% PS−SAP for 3 min, and aliquots of cells (2.5 × 106/well) were transferred to 24-well tissue culture

plates (Becton Dickinson, Lincoln Park, NJ). Stimulated adherence was initiated by addition of PMA (20 ng/mL final). After incubation (15 min, 37°C), each well was gently rinsed twice with

HBSSw (to remove nonadherent cells), and the remaining adherent cells were solubilized in 0.5 mL of HTAB buffer (0.5% HTAB in 50 mM potassium phosphate, pH 6.0). The MPO activity of the

solubilized cells was measured spectrophotometrically by combining 0.1 mL of HTAB extract with 2.9 mL of MPO buffer (0.167 mg/mL O-dianisodine HCl and 0.0005% H2O2 in 50 mM potassium

phosphate buffer, pH 6.0). The change in absorbance at 460 nm (ΔOD460) was continuously monitored in a Lambda-6 spectrophotometer (Perkin Elmer Cetus). The ΔOD460 per minute correlated

directly with the number of adherent neutrophils, and the percentage of adhered cells was calculated from 100% MPO activity found in 2.5 × 106 solubilized neutrophils not placed into the

tissue culture plates. NEUTROPHIL AGGREGATION ASSAY. Freshly purified neutrophils were suspended to 10 × 106/mL in HBSSw and exposed to either 1% PS+SAP or 1% PS−SAP for 3 min, washed,

resuspended in 0.6 mL of HBSSw, and placed in a preheated (37°C) Chrono-Log model 560 platelet aggregometer (Chrom-Log Corporation, Havertown, PA) with continuous stirring at 500 rpm. During

temperature equilibration, the upper and lower limits of light transmission were set at 8 × 106 and 10 × 106 cells/mL, respectively. _f_ MLP (1 μM) was added, and the light transmission of

each cell suspension was recorded for 3 min. Hard-copy aggregation curve tracings were scanned into electronic files, and the area under the aggregation curve for 3 min was measured

(SigmaScan Pro, Jandel Scientific, San Rafael, CA). The average area under the curve was expressed in pixels2 to compare aggregation responses among different conditions (19). STATISTICAL

ANALYSIS. All data were expressed as mean ± SEM. Paired-sample _t_ tests were conducted with _p_ < 0.05 indicating significance. Unless otherwise noted, replicate experiments used

neutrophils from different donors. RESULTS EFFECT OF PS+SAP ON NEUTROPHIL ADHERENCE AND AGGREGATION. Exposure of neutrophils to PS+SAP both decreased adherence to plastic induced by PMA and

decreased aggregation induced by _f_ MLP. When neutrophils were exposed to 1% PS+SAP, but not 1% PS−SAP, adherence to plastic induced by PMA decreased 39% (control, 77 ± 3% adherence;

PS−SAP, 77 ± 9% adherence; PS+SAP, 47 ± 5% adherence;_p_ < 0.05 PS+SAP _versus_ control or PS−SAP;Fig. 1_A_). In addition to reducing adherence, exposure to PS+SAP decreased aggregation

of neutrophils induced by _f_ MLP 53% (control, 40 688 ± 2 744 pixels2; PS−SAP, 64 024 ± 3 024 pixels2; PS+SAP, 19 308 ± 3 476 pixels2;_p_ < 0.05 PS+SAP _versus_ control or PS−SAP;Fig.

1_B_). PS−SAP–exposed neutrophils had a significantly increased aggregation response to _f_ MLP—an effect that was not further investigated. On confirming previously reported observations of

the inhibitory effects of PS+SAP on neutrophil proinflammatory responses, all subsequent experiments were designed to investigate the underlying mechanism(s) of these inhibitory effects.

EFFECT OF PS+SAP ON NEUTROPHIL [CA2+] RESPONSE TO _F_ MLP ACTIVATION. PS+SAP depressed the [Ca2+] response of neutrophils to ƒMLP stimulation. Under control conditions, stimulation of

neutrophils with ƒMLP (1 μM) induced a rapid increase in [Ca2+], followed by slow decay (_closed circles,_Fig. 2). Preexposure to 1% PS+SAP followed by _f_ MLP stimulation had limited effect

on the early [Ca2+] peaking response at 30 s (_open circles,_Fig. 2), but a more rapid fall in [Ca2+] was observed after peaking occurred. From the area under the [Ca2+] response curve,

neutrophil [Ca2+] responses to _f_ MLP were suppressed 49% by PS+SAP (control, 511 ± 40 nM·min; PS−SAP, 405 ± 28 nM·min; PS+SAP, 264 ± 22 nM·min;_p_ < 0.05 PS+SAP _versus_ control or

PS−SAP;Fig. 2). EFFECT OF PS+SAP ON DIVALENT CATION (CA2+ ANDMN2+) ENTRY INTO _F_ MLP-STIMULATEDNEUTROPHILS. The _f_ MLP-stimulated [Ca2+] response of neutrophils occurs in at least two

phases. Because initial release of internal Ca2+ stores (producing the early [Ca2+] peak) is accompanied by subsequent Ca2+ influx across the neutrophil plasma membrane (20–23), and because

the early [Ca2+] peak appeared unaffected by PS+SAP in Figure 2, subsequent experiments were designed to determine whether PS+SAP exposure modified Ca2+ influx into activated neutrophils.

Accordingly, fura2-loaded neutrophils were exposed to PS+SAP, PS−SAP, or buffer, washed, and resuspended in HBSSw/o plus 30 μM EGTA (to provide a Ca2+-free buffer with minimal excess EGTA).

In the presence of the Ca2+-free buffer, the peak [Ca2+] response to _f_ MLP stimulation was not significantly altered by either of the experimental conditions including PS+SAP or PS−SAP

exposure (Fig. 3_A_). However, by comparison with the [Ca2+] rise and decay after _f_ MLP stimulation in the presence of external Ca2+ (Fig. 2), decays of [Ca2+] in the absence of external

Ca2+ for 60–210 s were equally accelerated under all three of the experimental conditions (Fig. 3_A_). The accelerated decays of neutrophil [Ca2+] under all conditions were consistent with

expected decreases of Ca2+ influx in the absence of extracellular Ca2+. Subsequently, CaCl2 (5 mM) was returned to all three of the suspending external buffers at 210 s (Fig. 3_A_). As

expected, neutrophil [Ca2+] rose promptly in all three conditions, consistent with Ca2+ influx. However, the magnitude of [Ca2+] increase in PS+SAP–exposed neutrophils (_open circles,_Fig.

3_A_), as determined by the area under the curve, was 39% less than those of control and PS−SAP–exposed cells (control, 275 ± 11 nM·min; PS−SAP, 243 ± 19 nM·min; PS+SAP, 167 ± 19 nM·min;_p_

< 0.05 PS+SAP _versus_ control or PS−SAP). To determine whether PS+SAP exposure altered other divalent cation entry into neutrophils, fura2-loaded neutrophils were exposed to PS+SAP,

PS−SAP, or buffer, washed, and resuspended in HBSSw/o plus 1 mM MnCl2. Fluorescence was recorded continuously at 360 nm (a wavelength at which fura2 fluorescence is insensitive to Ca2+ but

sensitive to Mn2+). Under these conditions, Mn2+ entry into the cell resulted in fluorescence quenching, providing quantitation of divalent cation entry (17). Both control and PS−SAP–exposed

neutrophils exhibited similar rates of fluorescence loss during the 300 s after _f_ MLP stimulation (_closed circles_ and _triangles,_Fig. 3_B_), but the loss of fluorescence in

PS+SAP–exposed cells (_open circles,_Fig. 3_B_) was significantly lower 300 s after _f_ MLP stimulation (control, 69.7 ± 0.9%; PS−SAP, 70.3 ± 0.9%; PS+SAP, 80.7 ± 2.2%;_p_ < 0.05 PS+SAP

_versus_ control and PS−SAP). Both experimental approaches, using either removal and return of external Ca2+ or substitution of Mn2+ for Ca2+, yielded results consistent with

PS+SAP–dependent blockade of divalent cation entry into neutrophils after _f_ MLP stimulation. EFFECT OF PS+SAP ON [CA2+] RESPONSE STIMULATED BYOTHER AGONISTS. The effects of PS+SAP exposure

on _f_ MLP-stimulated neutrophil [Ca2+] responses raised the question of whether similar effects occurred after non-_f_ MLP agonists. Therefore, IL-8 and lactoferrin/anti-lactoferrin immune

complexes were examined in additional experiments. Fura2-labeled neutrophils were exposed to PS+SAP, PS−SAP, or buffer, washed, resuspended in HBSSw, and stimulated with IL-8 (150 ng/mL

final). Control and PS−SAP–exposed cells exhibited similar [Ca2+] responses (_closed circles_ and _triangles,_Fig. 4), whereas PS+SAP–exposed cells (_open circles,_Fig. 4) exhibited more

rapid decay of [Ca2+] at 30 s after a comparable [Ca2+] peak. The more rapid [Ca2+] decay in PS+SAP–exposed neutrophils decreased the area under the [Ca2+] response curve by 57% (control,

225 ± 20 nM·min; PS−SAP, 192 ± 20 nM·min; PS+SAP, 98 ± 4 nM·min;_p_ < 0.05 PS+SAP _versus_ control or PS−SAP). In similar experiments substituting 1% immune complex for IL-8, [Ca2+]

responses observed for control and PS−SAP–exposed cells were similar (Fig. 5, _A_ and _C_), whereas the [Ca2+] response of PS+SAP–exposed cells (Fig. 5_B_) was significantly decreased by 87%

as calculated from measurements of areas under [Ca2+] response curves (control, 158 ± 17 nM·min; PS−SAP, 129 ± 4 nM·min; PS+SAP, 21 ± 11 nM·min;_p_ < 0.05 PS+SAP _versus_ control or

PS−SAP). EFFECT OF PURIFIED PHOSPHOLIPIDS ON NEUTROPHIL [CA2+]RESPONSE TO _F_ MLP ACTIVATION. Although we hypothesized that different effects on neutrophil [Ca2+] responses caused by PS+SAP

and PS−SAP were attributable to SAP-dependent events, the possibility of phospholipid-dependent events could not be excluded because PS+SAP and PS−SAP differ in phospholipid composition and

concentration. Accordingly, a purified preparation of dipalmitoylphosphatidylcholine, phosphatidylglycerol, and phosphatidic acid (7:2:1, 10 mg/mL) was used to mimic the phospholipid

component of PS+SAP (24). We exposed neutrophils to 1% of this preparation for 3 min, washed them, and stimulated them with 1 μM _f_ MLP. Purified phospholipids did not significantly alter

the _f_ MLP-stimulated [Ca2+] response in neutrophils (control, 315 ± 54 nM·min; PS+SAP, 195 ± 33 nM·min; purified phospholipids, 305 ± 46 nM·min; purified phospholipids not significantly

different from control; all _n_ = 3). These experiments confirmed that the major phospholipid components of PS are not responsible for the [Ca2+] effects found in neutrophils. EFFECT OF

GRAMICIDIN-D ON NEUTROPHIL [CA2+] RESPONSETO _F_ MLP ACTIVATION. We propose that PS+SAP decreases Ca2+ influx into activated neutrophils by inserting cation channels into the plasma

membrane, thereby inducing cell membrane depolarization. If cell membrane depolarization via cation channel insertion is the underlying electrochemical event producing PS+SAP inhibition of

Ca2+ influx into _f_ MLP-stimulated neutrophils, then neutrophils exposed to a cation channel inserter, gramicidin-D, should also exhibit similar inhibition (25–27). Accordingly, neutrophils

were exposed to 90 μM gramicidin-D for 1 min, washed, and resuspended in HBSSw before being exposed to 1 μM _f_ MLP. Cation channel insertion by gramicidin-D decreased _f_ MLP-stimulated

[Ca2+] response by neutrophils (control, 518 ± 46 nM·min; gramicidin-D-treated cells, 284 ± 50 nM·min;_p_ < 0.05), similar to that observed after PS+SAP exposure. EFFECT OF CELL MEMBRANE

DEPOLARIZATION BY KCL ON NEUTROPHIL[CA2+] RESPONSE TO _F_ MLP ACTIVATION. To extend the findings obtained from gramicidin-D depolarization experiments, we sought to determine whether prior

cell membrane depolarization, caused by suspending neutrophils in 150 mM KCl, would alter the inhibitory effect of PS+SAP on the _f_ MLP-stimulated [Ca2+] response. Neutrophils were

suspended in either 150 mM NaCl or 150 mM KCl ± 1% PS+SAP for 3 min, washed, resuspended in the same buffers, and stimulated with _f_ MLP. As shown in Figure 6, the _f_ MLP-stimulated [Ca2+]

response in the absence of PS+SAP was decreased 50% solely by cell membrane depolarization with 150 mM KCl. In the presence of PS+SAP, the _f_ MLP-stimulated [Ca2+] response was inhibited

to the same extent as by KCl-induced cell membrane depolarization alone (control in 150 mM NaCl, 398 ± 42 nM·min; PS+SAP in 150 mM NaCl, 209 ± 45 nM·min; control in 150 mM KCl, 194 ± 27

nM·min; PS+SAP in 150 mM KCl, 203 ± 40 nM·min;_p_ < 0.05 control in 150 mM NaCl when compared with all other conditions; all _n_ = 3), thereby suggesting that PS+SAP inhibition of [Ca2+]

responses to physiologic stimuli is dependent on cell membrane potential. EFFECT OF PS+SAP ON NEUTROPHIL MEMBRANE POTENTIAL. To examine the effect of PS+SAP on neutrophil membrane potential

independent of _f_ MLP activation, neutrophils were exposed to PS+SAP, PS−SAP, or buffer, washed, and resuspended in HBSSw. Suspended neutrophils were further diluted with HBSSw containing

diOC5(3) (225 nM final) in the spectrofluorometer. When fluorescence had stabilized, depolarization with either 50 mM KCl or 1 μM gramicidin-D was induced. Prior exposure of neutrophils to

PS+SAP decreased membrane depolarization after 50 mM KCl and 1 μM gramicidin-D exposures 28 and 32 mV, respectively (KCl-exposed, control, 30.3 ± 2.4 mV; KCl-exposed, PS−SAP, 27.2 ± 4.2 mV;

KCl-exposed, PS+SAP, 2.2 ± 4.2 mV;_p_ < 0.05 PS+SAP _versus_ control and PS−SAP; gramicidin-D-exposed, control, 83.1 ± 4.4 mV; gramicidin-D-exposed, PS−SAP, 84.7 ± 5.8 mV;

gramicidin-D-exposed, PS+SAP, 50.8 ± 2.9 mV;_p_ < 0.05 PS+SAP _versus_ control and PS−SAP;Fig. 7). These results confirm neutrophil membrane depolarization by 1% PS+SAP exposure

independent of agonist-induced neutrophil activation. DISCUSSION PS, a complex mixture of phospholipids, neutral lipids, and SAPs, is essential for normal pulmonary function (28, 29). Its

role in decreasing alveolar surface tension is widely appreciated, and it is clinically effective for the treatment of respiratory distress syndrome in preterm newborn infants (1, 29).

Although it is investigational in ongoing clinical trials, surfactant is also considered a therapeutic option for term infants, children, and adults with other life-threatening causes of

acute respiratory failure (30–37). Although previous studies have demonstrated that surfactant preparations in clinical use vary greatly with regard to _in vitro_ biophysical properties and

_in vivo_ physiologic effects (38–40), clinical trials comparing natural surfactant preparations (containing SAP) with artificial surfactant preparations (lacking SAP) demonstrate superior

outcomes for natural surfactants (1, 5). These results suggest that the nonphospholipid components (SAP) of PS contribute to improved clinical outcomes (1, 5, 38). Neutrophils are the

primary cellular component of acute inflammation and potent inducers of lung damage in both infectious and noninfectious pulmonary inflammatory diseases (6, 7). Although data are limited,

investigators have suggested that additional benefits of natural surfactants may relate to suppression of proinflammatory neutrophil functions. Finck _et al._ (11) observed that guinea pig

neutrophils treated with low concentrations of Infasurf (a natural surfactant preparation) led to decreased endotoxin-stimulated neutrophil chemotaxis. Suwabe _et al._ (8) reported that

exposure of human neutrophils to Surfactant TA (Surfacten, another natural surfactant preparation) decreased neutrophil adherence to plastic after _f_ MLP, PMA, and tumor necrosis factor-α

stimulation. By contrast, synthetic surfactants lack the antiinflammatory properties of natural surfactants. Ahuja _et al._ (9) reported that native porcine surfactant, but not a

dipalmitoylphosphatidylcholine preparation (lacking SAP), inhibits human neutrophil superoxide production induced by either _f_ MLP or PMA. Tegtmeyer _et al._ (10) observed that elastase

release induced by activated human neutrophils was decreased by natural surfactants, Survanta and Curosurf, whereas synthetic surfactant, Exosurf, exhibited only modest effects. In the

present investigation, our first experiments supported previous observations that preexposure to PS+SAP, but not PS−SAP, decreases neutrophil proinflammatory responses of adherence and

aggregation to stimulation by PMA or _f_ MLP. Collectively, the breadth of observed effects by us and others suggested to us that alteration of an intracellular signaling pathway—common to

many activated neutrophil functions—might be occurring. For neutrophils, changes in [Ca2+] are closely linked with cell response to chemoattractants, secretagogues, and activating agonists

(15, 20–23, 41–48). Accordingly, intracellular Ca2+ balance is critical to appropriate and effective neutrophil functioning. This central importance of Ca2+ metabolism to neutrophil function

prompted us to examine whether PS+SAP exposure alters neutrophil [Ca2+] responses to selected agonists, and how changes in [Ca2+] response might occur. Experiments monitoring [Ca2+]

response to agonist stimulation after transient exposure to PS+SAP, but not PS−SAP or purified phospholipids, clearly demonstrated increased decay of the [Ca2+] response after achieving an

unaffected peak of neutrophil [Ca2+] by _f_ MLP activation (Fig. 2). As it was known that the initial phase of _f_ MLP-stimulated [Ca2+] response by neutrophils results from internal

mobilization of Ca2+ from intracellular sources (20–23), the pattern of increased [Ca2+] decay after PS+SAP exposure suggested either that Ca2+ influx was decreased, Ca2+ efflux was

increased, replenishment of Ca2+ into internal stores was increased, or all three events were occurring. The probability of either increased Ca2+ efflux or internal Ca2+ store replenishment

was rendered less likely by the loss of a PS+SAP effect on neutrophil [Ca2+] response in the absence of extracellular Ca2+ (Fig. 3_A_). To address the possibility of decreased Ca2+ influx,

two approaches were taken. In the first approach, Ca2+ influx after _f_ MLP stimulation was quantitated by repletion of extracellular Ca2+ (Fig. 3_A_). The second approach was to monitor

quenching of internal fura2 fluorescence by extracellular Mn2+ (Fig. 3_B_). Both approaches demonstrated that PS+SAP–exposed neutrophils experienced decreased _f_ MLP-stimulated influx of

divalent cations, consistent with blockade of Ca2+ influx by PS+SAP exposure. Although _f_ MLP stimulation is a well-studied, soluble stimulus for neutrophil activation, its physiologic

relevance is not fully established. It was of interest, therefore, to determine whether exposure to PS+SAP modified neutrophil [Ca2+] responses to more physiologically and clinically

relevant stimuli (IL-8 and immune complexes). IL-8 is a member of the “C-X-C” chemokine subfamily, which has been highly implicated in the inflammatory response of many pulmonary diseases

(48–51). For these agonists, PS+SAP exposure significantly suppressed [Ca2+] responses relative to PS−SAP and control (Figs. 4 and 5). These results suggest a generalized inhibition of

agonist-induced neutrophil activation by blockage of Ca2+ influx. In this regard, other laboratories have observed that inhibition of Ca2+ influx by Ca2+ channel blockers or by removal of

external Ca2+ inhibits proinflammatory functions of activated neutrophils similar to that observed after PS+SAP exposure (52–55). In light of previous observations from our laboratory that

monovalent cation channel insertion into artificial black membranes occurs with PS+SAP or isolated SAP-B and SAP-C (12, 13), we investigated whether neutrophil depolarization by an

alternative route of cation channel insertion would decrease the neutrophil [Ca2+] response to _f_ MLP. Gramicidin-D, a well-characterized peptide causing depolarization of other cell types

by insertion of transmembrane channels for monovalent cations, was used to mimic the PS+SAP effect (25–27). As predicted, the effect of gramicidin-D on the _f_ MLP-induced [Ca2+] response

was nearly identical to that observed with PS+SAP. In addition, prior cell membrane depolarization by 150 mM KCl decreased _f_ MLP-stimulated [Ca2+] responses and prevented further

inhibition of [Ca2+] responses by PS+SAP (Fig. 6). Accordingly, we investigated whether PS+SAP exposure independent of neutrophil activation induced depolarization. These studies, using two

different models of cell membrane depolarization (Fig. 7), were consistent with the _in vitro_ findings in artificial membranes. PS+SAP, but not PS−SAP, caused cell membrane depolarization.

Moreover, the magnitude of depolarization by PS+SAP before depolarization by either KCl or gramicidin-D approximated 30 mV. Although these studies do not prove insertion of SAP-dependent,

monovalent cation channels in neutrophils by PS+SAP, our prior observations of channel insertion in artificial membranes by PS+SAP strongly support the possibility. The current investigation

suggests, but does not establish, a cause and effect relationship between PS+SAP–induced membrane depolarization and blockade of Ca2+ influx during physiologic stimulation. Even though

prior cell membrane depolarization with KCl did not alter the PS+SAP inhibitory effect on _f_ MLP-stimulated [Ca2+] responses, the effects of external Ca2+ on membrane potential and of

PS+SAP on other cation fluxes across neutrophil membranes were not determined. Based on our results, and supported by previous reports consistent with Na+ influx as a significant contributor

of cell membrane depolarization after _f_ MLP stimulation (56–59), we hypothesize that on insertion of SAP-dependent, monovalent cation channels, PS+SAP induces sustained neutrophil

depolarization by influx of external Na+. Neutrophil depolarization reduces the inside negative charge gradient normally present to electrically support influx of external cations, such as

Ca2+, by favorable channel conductances or electrogenic transport activities. We further speculate that whereas activation of neutrophils induces early release of internal Ca2+ stores

followed subsequently by opening of Ca2+ channels operated by plasma membrane stores, sustained neutrophil depolarization by SAP-dependent cation channels limits, or possibly prevents, Ca2+

influx. Neither prior nor current observations suggest that the SAP-dependent, monovalent cation channels support conductance of divalent cations. In summary, neutrophil exposure to PS+SAP

inhibits functional responses of activated neutrophils, induces membrane depolarization, and blocks agonist-stimulated Ca2+ influx in neutrophils. We speculate that these effects of PS+SAP

on human neutrophils result in alterations of intracellular signaling pathways dependent on membrane potential and Ca2+. In addition, such alterations of intracellular signaling pathways may

contribute to the suppression of multiple neutrophil functions and decreased inflammation observed after PS+SAP exposure. ABBREVIATIONS * [Ca2+]: ionized cytosolic calcium concentration *

_f_ MLP: formyl peptide * PMA: phorbol ester * HBSS: Hanks' balanced salt solution * SAP: surfactant-associated proteins * PS: pulmonary surfactant * HTAB: hexadecyltrimethylammonium

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Stephen J. Beebe, Kristi Mantych, and Paula Fox for their support and assistance in designing and performing these experiments. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Center for

Pediatric Research, Children's Hospital of The King's Daughters and Eastern Virginia Medical School, Norfolk, Virginia, U.S.A. Enrique Chacon-Cruz, E Stephen Buescher & David G

Oelberg Authors * Enrique Chacon-Cruz View author publications You can also search for this author inPubMed Google Scholar * E Stephen Buescher View author publications You can also search

for this author inPubMed Google Scholar * David G Oelberg View author publications You can also search for this author inPubMed Google Scholar RIGHTS AND PERMISSIONS Reprints and permissions

ABOUT THIS ARTICLE CITE THIS ARTICLE Chacon-Cruz, E., Buescher, E. & Oelberg, D. Surfactant Modulates Calcium Response of Neutrophils to Physiologic Stimulation via Cell Membrane

Depolarization. _Pediatr Res_ 47, 405–413 (2000). https://doi.org/10.1203/00006450-200003000-00020 Download citation * Received: 09 April 1999 * Accepted: 01 November 1999 * Issue Date: 01

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