Stimulation by Leukotriene D4 of Increases in the Cytosolic Concentration ofCalcium in Dimethylsulfoxide-differentiated HL-60 CellsLaurent Baud, Edward J. Goetzl, and Catherine H. KooThe Howard Hughes Medical Institute and Departments of Medicine, Pharmacology, and Microbiology-Immunology,University of California Medical Center, San Francisco, California 94143-0724

Abstract

The C6-sulfidopeptide leukotrienes C4 (LTC4) and D4 (LTD4)evoked increases in the cytosolic concentration of intracellularcalcium (QCa`2Ij) in dimethylsulfoxide-differentiated HL-60cells, as assessed by the fluorescence of quin-2. The increasesin 1Ca"2Ji reached a peak within 15-90 s, attained 50% of themaximum level at 1.2 nM LTD4 and 60 nM LTC4, weregreater in maximal magnitude for LTD4 than LTC4, and sub-sided in 5-7 min. Flow cytometric evaluation of the LTD4-in-duced increases in ICa+2i,, reflected in increases in the fluores-cence of intracellular indo-1, revealed that a mean of 77% ofdifferentiated HL-60 cells responded, as contrasted with lesserincreases in only 50% of undifferentiated HL-60 cells. Thecapacity of pretreatment of HL-60 cells with LTD4 to preventsubsequent responses of [Ca"2I to LTC4 and LTD4, and thefinding that the serine-borate inhibitor of conversion of LTC4to LTD4 suppressed concurrently both LTC4-induced rises in[Ca+2h and increases in adherence to Sephadex G-25 indicatedthat the responses of HL-60 cells to LTC4 required conversionto LTD4. That pertussis toxin and a chemical antagonist ofLTD4 reduced the [Ca+2lI response suggested a dependence onLTD4 receptors. The LTD4-induced increases in [Ca+21I weredependent on extracellular calcium and diminished by lantha-num, but not affected by nifedipine nor associated with changesin membrane potential, as measured with the fluorescent probe3,3'-dipentyloxacarbocyanine. Thus, the increase in [Ca+2t inHL-60 cells, which is coupled to an increase in adherence,appears to involve LTD4 receptor-specific and voltage-inde-pendent calcium channels in the plasma membrane.

Introduction

Structurally diverse factors exhibiting chemotatic activity forpolymorphonuclear (PMN)' leukocytes, including N-formyl-

Address reprint requests to Dr. C. H. Koo, University of California,533 Parnassus, U-426, San Francisco, CA94143-0724.

Receivedfor publication 29 September 1986 and in revisedform 1June 1987.

1. Abbreviations used in this paper: [Cai2],, cytosolic concentration ofintracellular calcium; di-O-C5(3), 3,3'-dipentyloxacarbocyanine;DMSO,dimethylsulfoxide; EGTA, ethylene glycol-bis (,B aminoethylether) N,N,N',N'-tetraacetic acid; fMLP, N-formyl-methionyl-leucyl-phenylalanine; HBSS, Hanks' balanced salt solution without phenolred; LTB4, LTC4, LTD4, LTE4, leukotrienes B4, C4, D4, E4; [3H]-LTC4, [3H]LTD4, 14,15(N)[3H]leukotrienes C4, D4; NIF, nifedipine.

methionyl-leucyl-phenylalanine (fMLP) (1, 2) and leukotrieneB4 (LTB4) (3-5), evoke rapid increases in the cytosolic con-centration of intracellular calcium ([Ca`]i) in PMNleuko-cytes (1-5) and myelocytes derived from HL-60 cells (5, 6),which may be a critical event in the mediation of functionalresponses to the factors (7). The C6-sulfidopeptide leukotrieneconstituents of the slow-reacting substance of anaphylaxis,leukotrienes C4 (LTC4) and D4 (LTD4), lack leukocyte chemo-tactic activity, but enhance the adherence of PMNleukocytesto synthetic surfaces in vitro (8) and to vascular endothelium invivo (9). Whereas the activation of PMNleukocytes by LTC4and LTD4 is dependent in part on stimulation of the cycloox-ygenation of arachidonic acid in target cells, the major bio-chemical prerequisites for this selective recruitment of func-tion have not been elucidated definitively. The present studywas designed to evaluate the effects of the C6-sulfidopeptideleukotrienes on [Ca+2J, in myelocytic leukocytes derived fromHL-60 cells by incubation with dimethylsulfoxide (DMSO),and to examine the dependence of such effects on specificcalcium channels and the metabolism of leukotrienes by theleukocytes.

MethodsHanks' balanced salt solution without phenol red (HBSS), RPMI 1640medium, glutamine, a mixture of penicillin Gand streptomycin, andfetal bovine serum (FBS) were obtained from University of CaliforniaCell Culture Facility, San Francisco, CA; sodium dodecyl sulfate(SDS), fMLP, DMSO, lanthanum, nifedipine (NIF), and gramicidinD, from Sigma Chemical Co., St. Louis, MO; 14,15(N)[PH]LTC4([3H]LTC4) (39.0 Ci/mmol), from NewEngland Nuclear, Boston, MA;quin-2 AM, from Amersham Corp., Arlington Heights, IL; and indo- IAMand 3,3'-dipentyloxacarbocyanine (di-O-C5(3)), from MolecularProbes Inc., Junction City, OR. Synthetic LTB4, LTC4, LTD4, andLTE4 were supplied by Dr. J. Rokach of Merck Frosst Canada, Ltd.(Dorval, Canada). The dithioacetal LTD4 receptor antagonist SKF102922 was a gift of Dr. J. G. Gleason (Smith Kline & French Labora-tories, Philadelphia, PA). Purified pertussis toxin was a gift by Dr.R. D. Selcura of the National Institute of Child Health and HumanDevelopment, Bethesda, MD.

Culture and differentiation of HL-60 cells. HL-60 cells were sup-plied by Dr. Z. Bar-Shavit (Washington University Medical Center, St.Louis, MO) (10), and suspensions were cultured in 75-ml plastic flasksin RPMI 1640 medium supplemented with 10%FBS(vol/vol), 1 g/100ml of glutamine, 100 U/ml of penicillin, and 100 ,g/ml of streptomy-cin in a humidified atmosphere of 5% CO2and 95% air at 37°C. Thedensity of HL-60 cells was maintained between 5 X 105 and 2 X 106/ml. The HL-60 cells were cultured in the same medium containing 1%(vol/vol) DMSOfor 4 d to achieve differentiation into myeloid cellsresembling PMNleukocytes (1 1).

Quantification of [Ca'1i by quin-2 fluorescence. Cultured HL-60cells were loaded with quin-2 by a modification of the method of Tsienet al. (5, 12). Suspensions of 1.2 X 107 HL-60 cells per ml of HBSScontaining 0.1 g/100 ml of ovalbumin and 10 mMHEPESat pH 7.4were incubated with 20 ;M diacetomethoxy ester of quin-2 in the darkat 37°C for 10 min and at room temperature for 20 min. The quin-2-

Leukotriene D4 Stimulation of HL-60 Cells 983

J. Clin. Invest.©The American Society for Clinical Investigation, Inc.002 1-9738/87/10/0983/09 $2.00Volume 80, October 1987, 983-991

loaded HL-60 cells were washed three times with HBSS, resuspendedat 6 x 106 per ml of HBSScontaining 0.1 g/I00 ml of ovalbumin and10 mMHEPES, and kept on ice until used for the fluorescence mea-surements.

Fluorescence studies were performed as described (5) with a spec-trofluorimeter (model 650-40; Perkin-Elmer Corp., Norwalk, CT)equipped with temperature-controlled cuvettes and a magnetic stirrerunder the cuvetter holder. 3 X 106 quin-2-loaded HL-60 cells werepelleted at 8,000 g for 15 s in a microfuge (model B; Beckman Instru-ments, Inc., Palo Alto, CA), resuspended in 2 ml of HBSSat 370C, andtransferred to a cuvette held at 370C. Fluorescence was recorded con-tinuously at excitation and emission wavelengths of 339 and 492 nm,respectively. Maximum and minimum fluorescence were determinedsequentially by the addition of 20 MAl of 10% (vol/vol) Triton X- 100 indistilled water, followed by 40 Ml of 0.5 Methylene glycol-bis (ft-amino-ethyl ether) N,N,N',N'-tetraacetic acid (EGTA) (pH 10); the nanomo-lar values of [Ca+2], were calculated as before (5, 12). In some experi-ments, the standardization of quin-2 fluorescence was confirmed bythe addition to replicate suspensions of ionomycin and MnCI2 formaximum and minimum fluorescence, respectively (13). In each ex-periment, the autofluorescence value was determined under the sameconditions with HL-60 cells lacking quin-2 and was subtracted fromthe fluorescence value of quin-2-loaded HL-60 cells.

Flow cytometric assessment of [Ca+1i by indo-1 fluorescence. Rep-licate suspensions of 2 X 107 HL-60 cells per ml of RPMI 1640 wereincubated with 3 uM acetoxy-methyl ester of indo-l for 20 min at37°C in the dark, diluted 10-fold in RPMI 1640, and incubated for anadditional 20 min at 37°C as described (14). The indo- I -loaded HL-60cells were washed twice and resuspended in RPMI 1640 at 2.5 X 106per ml and kept on ice. The effects of stimulation on indo-l-loadedHL-60 cells were analyzed in a cell sorter (FACS IV; Beckton Dickin-son & Co., Sunnyvale, CA) equipped with an argon ion laser emitting600 mWat 351-364 nm (Spectra-Physics Inc., Mountain View, CA).Blue (486/4 nm) and violet (404/4 nm) band pass filters were used tocollect the fluorescence emitted by indo- 1. The ratio of violet-to-bluefluorescence, which is directly related to the [Ca+]j, was expressed on alogarithmic scale (14).

Flow cytometric evaluation of the membrane potential of HL-60cells. Replicate suspensions of 106 HL-60 cells per ml of RPMI 1640were preincubated at 37°C with 10 nM di-O-C5(3) as described (15).The fluorescence of the di-O-C5(3) in HL-60 cells was analyzed in a cellsorter (FACS IV; Beckton Dickinson & Co.) using an argon ion laseremitting 600 mWat 457 nm. Forward and 900 light scatters werecollected for all cells to resolve viable from nonviable cells. Greenfluorescence was collected through a band pass 530/30 nm filter for aminimum of 10,000 cells in each specimen and the values were ex-pressed on a logarithmic scale.

Measurement of the metabolism of [3H]LTC4 and [3H]LTD4 byHL-60 cells. To assess the extent of conversion of LTC4 to LTD4 andLTD4 to LTE4 and more polar oxidative products, DMSO-differen-tiated HL-60 cells were incubated with 4.5 mM[3H]LTC4 and 100 or300 nMnonradioactive LTC4 or with 4.5 nM [3H]LTD4 and 100 nMnonradioactive LTD4 under the same conditions as for the measure-ment of [Ca"2Ji by quin-2 fluorescence. At defined times, 200 Ml sam-ples of each suspension were removed and added to 300 Ml of methanoland 25 Ml of 0.5 N acetic acid at -20°C, followed by centrifugation at8,000 g for 2 min in a microfuge (model B; Beckman Instruments,Inc.). Each supernatant then was dried under a stream of nitrogen;redissolved in 80 Ml of methanol and 3.1 mMphosphoric acid in water(65:35 vol/vol, pH 5.5) containing 50 ng each of LTC4, LTD4, andLTE4; and analyzed by high performance liquid chromatography(HPLC) with a dual pump system (Beckman Instruments, Inc.), whichutilizes a 4.6-mm by 25-cm Ultrasil 10-Mm octadecylsilane particlereverse-phase column (Beckman Instuments, Inc.) that was developedisocratically with methanol and 3.1 mMphosphoric acid (65:35 vol/vol, pH 5.5) at I ml/min. Absorbance of the eluate at 280 nm wasmonitored continuously,and 1 ml fractions were collected and assessedfor radioactivity in a liquid scintillation counter (Beckman Instru-ments, Inc.).

Measurement of adherence of HL-60 cells to Sephadex G-25.DMSO-differentiated HL-60 cells were washed once in HBSS andresuspended in HBSScontaining 0.1 g/100 ml of ovalbumin. Replicate0.2-ml portions of suspensions of 107 differentiated HL-60 cells/mlwere incubated with stimuli for 2 min at 370C. The cells then wereapplied to 0.4-ml columns of Sephadex G-25 that had been prewashedwith 3 ml of HBSScontaining 0.1 g/100 ml of ovalbumin. The col-umns were washed twice with 2 ml of HBSScontaining 0. 1 g/ 100 ml ofovalbumin, and the effluent and washes containing the nonadherentHL,60 cells were pooled. The pools of nonadherent HL-60 cells werewashed twice with 2 ml of HBSSlacking protein and then dissolved in3 g/100 ml of SDS in distilled water. The absorbancy at 280 nmof eachof the dissolved pools and of two washed and dissolved aliquots of thesuspension applied to the columns were quantified with a spectropho-tometer (model 240; Gilford Instrument Laboratories, Inc., Oberlin,OH). Percentage adherence to Sephadex G-25 was calculated accord-ing to the formula: I - [(absorbance of nonadherent pool in the pres-ence of stimuli)/(absorbance of nonadherent pool in buffer)] X 100.

Results

LTC4- and LTD4-induced changes in the [Ca+]i of DMSO-differentiated HL-60 cells. The [Ca+2]i in unstimulatedDMSO-differentiated HL-60 cells was 142.2±12.7 nM(mean±SD, n = 16), as assessed by quin-2 fluorescence. LTC4increased the [Ca+]i in a concentration-dependent manner,with the elicitation of changes that were just reproducibly de-tectable, half-maximal, and maximal by - 12.5, 60, and 200nMLTC4, respectively (Fig. 1). At 200 nM, the response of the[Ca+2]i reached a peak within 15-45 s and thereafter returnedto the resting level over a period of 7-10 min (Fig. 2). Incontrast, the lowest effective concentration of LTC4 evoked aresponse in [Cai2], that developed and subsided more slowlythan that attributable to optimal concentrations. LTD4 alsoinduced rapid and concentration-dependent increases in[Cai2], (Fig. 1). However, the potency of LTD4 was 50-foldgreater than that of LTC4, with a 50% effective concentrationof 1.2 nM, and LTD4 elicited a maximal increase in [Ca+21Jnearly twice that attained by the optimal concentration ofLTC4 (Fig. 1). Also, even the lowest effective concentration ofLTD4 achieved the peak response in the [Ca+2], within 60 s(Fig. 3). Finally, LTE4 at concentrations up to 300 nMdid notelicit an increase in [Cai2],.

Development of responsiveness of [Ca+2]i to LTC4 andLTD4 as a function of the differentiation of HL-60 cells. LTD4and LTC4 elicited increases in [Ca12]i of undifferentiatedHL-60 cells that were significantly smaller than the corre-sponding responses observed in HL-60 cells pretreated withDMSO. The [Ca+2], of undifferentiated HL-60 cells was153±25 nM(mean±SD, n = 7) and rose to 196±7 and 351±55nMafter the introduction of 100 nMLTC4 and LTD4, respec-tively. After DMSO-induced differentiation was complete, thebasal [Ca+2]J of 143±21 nM(mean±SD, n = 7) was elevated to368±33 and 892± 143 nM, respectively, by 100 nMLTC4 andLTD4 (P < 0.001 and P < 0.005 relative to undifferentiatedHL-60 cells).

The response of [Ca+2], in the undifferentiated HL-60 cellsalso was dependent on the concentration of LTD4, but a half-maximal response occurred at 10 nM, as contrasted with 1.2nM for DMSO-differentiated HL-60 cells. The magnitude ofthe maximal response of [Ca+2Ji elicited by 100 nMLTD4 alsowas significantly lower in undifferentiated than in DMSO-dif-ferentiated HL-60 cells.

984 L. Baud, E. J. Goetzl, and C. H. Koo

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The development of greater net responsiveness to the leu-kotrienes with differentiation might be attributable to the ap-pearance of a higher percentage of responsive HL-60 cells, ascontrasted with an increase in the level of responsiveness of allof the HL-60 cells. To distinguish between these possibilities,flow cytometric assays of the responses of individual cells wereperformed with indo-1 as the fluorescent indicator of [Ca2]i(Fig. 4). After exposure to 200 nM LTD4, DMSO-differen-tiated HL-60 cells demonstrated typically homogeneous in-

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Figure 1. Concentration-dependence of theeffects of LTC4 (.) and LTD4 (o) on the[Ca+2]J in DMSO-differentiated HL-60cells. Results are expressed as the maximalvalue of the [Ca+]i attained after the intro-duction of a stimulus. Each point is the

07 l0o6 mean±SDof three to four determinationsperformed in separate experiments.

creases in the [Ca+]i (Fig. 4 A). Maximal fluorescence wasdetected in 77.1% of all of the HL-60 cells at 15 s and returnedto the prechallenge level by 3 min. In contrast, only 50.5% ofthe undifferentiated HL-60 cells exhibited any increase in fluo-rescence (Fig. 4 B), and the modal level of the increases influorescence was lower than that observed in differentiatedHL-60 cells. Also, the increases in [Ca2i evoked by LTD4 inundifferentiated HL-60 cells subsided to prechallenge levels by30 s. Thus, DMSO-induced differentiation transforms most of

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Figure 2. Time courses of the increases in the [Ca+]i of DMSO-dif-ferentiated HL-60 cells induced by exposure to three different con-centrations of LTC4. Each panel shows a typical tracing of the resultsof a representative experiment.

Figure 3. Time courses of the increases in the [Ca+2]i of DMSO-dif-ferentiated HL-60 cells induced by exposure to three different con-centrations of LTD4. Each panel shows a typical tracing of the re-sults of a representative experiment.

Leukotriene D4 Stimulation of HL-60 Cells 985

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the HL-60 cells into an LTD4-responsive state, characterizedby responses of the [Ca+2]i to LTD4 that are longer and greaterin magnitude than those of undifferentiated HL-60 cells.

Dependence of the effect of LTC4 on conversion to LTD4.The effect of prior exposure to one leukotriene on the responseof DMSO-differentiated HL-60 cells to that stimulus and otherleukotrienes was examined to further define the pathways ofactivation (Fig. 5). Prior exposure of the HL-60 cells to an

optimally active concentration of LTD4 eliminated completelyany response of the [Ca+2]i to further challenges with LTD4(Fig. 5 A) or LTC4 (Fig. 5 C). Pretreatment with a functionallysimilar concentration of LTC4 suppressed substantially the re-

sponse to subsequent challenge with LTD4 (Fig. 5 B). In con-trast, pretreatment with a maximally active concentration offMLP (100 nM) had no apparent effect on the subsequentresponse of the [Ca+2]J to LTD4 (Fig. 5 D).

The implication that LTC4 stimulated increases in the[Ca+12] only after conversion to LTD4 was supported by the100-fold greater potency of LTD4 than LTC4 (Fig. 1) and theslower rate of action of LTC4 (Fig. 2). To assess the depen-dence of the activity of LTC4 on conversion to LTD4, theeffect of the serine-borate complex inhibitor of y-glutamyltranspeptidase activity was examined (16). The presence of amixture of 5 mML-serine and 10 mMsodium borate did not

Figure 4. Effect of LTD4 on the fluorescencehistograms of indo- I -loaded HL-60 cells after(A) or before (B) 4 d of exposure to 1%DMSO.The patterns of fluorescence distribu-tion were obtained before (-) or 15 s (top),30 s (middle), and 3 min (bottom) after (- )

stimulation by 200 nMLTD4.

prevent the induction of a [Ca2J]i signal by LTD4, but totallyabolished the response of [Ca+2]i to LTC4 (Fig. 6). To delineatethe role of the target cells in metabolism of LTC4, DMSO-dif-ferentiated HL-60 cells were incubated for 1 or 3 min with 4.5nM [3H]LTC4 and 100 or 300 nMnonradioactive LTC4 in theabsence and presence of the serine-borate complex. The radio-activity was extracted from the suspensions and analyzed byHPLCin three different experiments (Table I). In the absenceof serine-borate complex, a mean of 1.7% and 9.1% of 300 and100 nM [3H]LTC4, respectively, was converted to [3H]LTD4,without a significant change in the generation of polar impuri-ties and other products. After 3 min of incubation, the con-centration of [3H]LTC4 declined further and the conversion to[3H]LTD4 reached a mean of 2.9% and 13.7% for 300 and 100nM [3HJLTC4, respectively. No LTE4 was detected. In theexperiments conducted in the presence of serine-borate com-plex, the peptidolysis of 100 and 300 nM[3H]LTC4 was inhib-ited by - 50% and over 99%, respectively (Table I). The en-

dogenous capacity of DMSO-differentiated HL-60 cells to gen-erate 5-15 nM LTD4 from LTC4 in the absence ofserine-borate complex is sufficient to evoke the observed in-creases in the [Ca+2Ji through actions of LTD4 alone (Fig. 1).The increase in the response of [Ca+2]i to LTD4 associated withDMSO-induced differentiation of HL-60 cells was not due to a

986 L. Baud, E. J. Goetzl, and C. H. Koo

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Figure 5. Effect of stimulus pretreatment on the increases in the[Ca"2Ji of DMSO-differentiated HL-60 cells elicited by leukotrienes.HL-60 cells were incubated first with LTD4 (A, C), LTC4 (B), orfMLP (D) and subsequently stimulated with maximally effectiveconcentrations of LTD4 (A, B, D) or LTC4 (C). Each panel shows atypical tracing of the results of one representative experiment from atotal of four.

modification in the capacity of the HL-60 cells to generate ormetabolize LTD4. There was < 15% change in the rate ofconversion of [3H]LTC4 to [3H]LTD4 in DMSO-differentiated

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HL-60 cells, as compared with undifferentiated HL-60 (n = 3).After a 3-min incubation at 370C with 100 nMnonradioactiveLTD4 and 4.5 nM [3H]LTD4, the radioactivity recovered was87.5±4.2% and 85.8±3.7% (mean±SD, n = 4) LTD4 for un-differentiated and DMSO-differentiated HL-60 cells, respec-tively.

That the increase in [Ca+2]i evoked by LTD4 was a recep-tor-mediated event was evidenced by significant inhibition ofthe effect of LTD4 by the SKF 102922 dithioacetal antagonistof LTD4 binding to receptors in other tissues (17). SKF 102922inhibited in a concentration-dependent relationship the in-crease in [Ca+2]i elicited by LTD4 in DMSO-differentiatedHL-60 cells (Fig. 7). Complete inhibition was achieved at 1 ;MSKF 102922, which by itself demonstrated no detectable ago-nist activity. The response of [Ca2]i1 to 100 nMfMLP was notmodified by 1 gMSKF 102922.

Cellular mechanisms of stimulation of the [Ca+]i by LTD4.To investigate a requirement for extracellular Ca+2 in the ob-served increases in the [Ca+2],, DMSO-differentiated HL-60cells were incubated in a medium without Ca3+2 and with 1mMEGTA(Fig. 8). In the presence of EGTA, LTD4 did notelicit any rise in the [Ca+2]i above the baseline concentration of143.51±2.48 nM(n = 3). Also, a concentration of lanthanumsufficient to block Ca+2 channels also prevented almost com-pletely the rise in the [Ca+2]i recorded in the complete medium(Fig. 8). NIF, a blocker of voltage-dependent calcium channelsdid not affect the maximal response of the [Ca+2], to 100 nMLTD4 in three different experiments (Fig. 9). To confirm thatthe Ca+2 channels activated by LTD4 are not of the voltage-dependent type, the membrane potential of the cells wasquantified with a membrane potential-sensitive cyanine dye,di-O-C5(3) (15). The fluorescence of di-O-C5(3) in unstimu-lated HL-60 cells, which reflects the resting membrane poten-tial, was not modified by the addition of 100 nM LTD4,whereas the membrane-depolarizing compound gramidicin at0.5 ,uM (18) decreased fluorescence (Fig. 10). Depolarizationof the HL-60 cells with 60 mMKC1 or 0.5 ,uM gramicidin didnot elicit any change in quin-2 fluorescence.

Recent findings support an association of LTD4 receptorsin guinea pig lung with a guanine nucleotide-binding regula-tory protein, Ni. Pretreatment of guinea pig lung tissue with

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_ 800 differentiated HL-60 cells to maximal con-

400 centrations of LTD4 (top) or LTC4 (bot-tom). HL-60 cells were preincubated with-out (A) or with (B) a mixture of 5 mM

20° L-serine and 10 mMsodium borate for 10min at 37°C before the addition of the leu-

_100 kotrienes. Each panel shows a tracing ofsI the results obtained in the same experiment.

Leukotriene D4 Stimulation of HL-60 Cells 987

Table I. Metabolism of [3H]LTC4 by DMSO-differentiated HL-60 Cells

Percentage of original [3H]LTC4

Without serine-borate complex With serine-borate complex

Concentration of Polar PolarTime of incubation nonradioactive LTC4 LTC4 LTD4 products LTC4 LTD4 products

min nM % % % % % %

1 300 86.5±1.9 1.7±0.4 11.8±2.2 91.4±3.4 0 8.6±3.43 300 84.3±1.1 2.9±0.8 12.7±1.2 85.7±10.8 0 14.3±10.81 100 77.0±3.0 9.1±3.8 14.2±1.0 82.3±1.5 4.7±2.1 13.2±0.83 100 69.3±3.1 13.7±1.5 17.0±1.7 77.3±2.5 7.2±3.3 15.7±2.3

DMSO-differentiated HL-60 cells were incubated at 370C in the absence or presence of a mixture of 5 mML-serine and 10 mMsodium borateand with 4.5 nM [3H]LTC4 and 100 or 300 nMunlabeled LTC4. Each value represents the mean±SDof the quantities of each factor recoveredin three different experiments.

pertussis toxin at concentrations that inactivate Ni partiallysuppressed the LTD4-induced synthesis and release of prosta-noids (17). To investigate a possible requirement for the gua-nine nucleotide-binding regulatory protein in the observedincreases in [Ca+2]i, DMSO-differentiated HL-60 cells werepreincubated for 2 h with 100 ng/ml of pertussis toxin. At thisconcentration, pertussis toxin inhibited LTD4-induced in-creases in [Cai by 85.5+6.5% (mean±SD, n = 3) (Fig. 11).

The induction by DMSOof differentiation of HL-60 cellsinto mature granulocytes with receptors for fMLP and LTB4has been well-documented (6, 11, 19-21). LTC4 and LTD4have been shown to enhance human PMNleukocyte adher-ence to synthetic surfaces in vitro (8) and to microvascularendothelium in vivo (9). To evaluate a possible functional re-sponse of DMSO-differentiated HL-60 cells to LTC4 andLTD4, the effects of the leukotrienes on the adherence of thecells to Sephadex G-25 was examined with a previously stan-dardized technique (Table II). Both LTC4 and LTD4 enhancedsignificantly the adherence of DMSO-differentiated HL-60cells to Sephadex G-25. The preincubation of the cells withserine-borate complex partially inhibited the effect of LTC4,but not LTD4, LTB4, or fMLP on adherence.

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Discussion

The C6-sulfidopeptide leukotrienes LTC4 and LTD4 have thecapacity to activate broadly or with functional selectivitysmooth muscle cells (22, 23), endothelial cells (24), glomerularepithelial (25) and mesangial cells (26), keratinocytes (27),neutrophils (8), and monocytes (28). As the pattern of func-tional responses of human leukocytes to LTC4 and LTD4 issubstantially more restricted than that elicited by other stim-uli, it was anticipated that the receptors for leukotrienes andthe transductional sequence of biochemical events might alsohave unique characteristics. DMSO-differentiated HL-60 cellsprovide a convenient model for studies of such cellular eventssince the parent line is stable and differentiates homogen-eously into myelocytic leukocytes bearing receptors for LTB4and peptide mediators, which are coupled to increases inJCa+2]j and other biochemical and functional concomitants ofactivation (5, 6). Also, the continuous suspension cultures ofdifferentiated HL-60 cells permit more reliable application ofbiochemical techniques than monolayer cultures (13).

Functionally relevant concentrations of the sulfidopeptideleukotrienes elicted a transient rise in [Ca2]i that attained a

Figure 7. Inhibition of the increases in[Ca+2]i induced by LTD4 in DMSO-differ-entiated HL-60 cells by the receptor antag-onist SKF 102922. Results are expressed asmean percentage inhibition of the maximalincrease in [Ca+2]i observed after the addi-tion of 100 nMLTD4 (n = 3).

988 L. Baud, E. J. Goetzl, and C. H. Koo

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Figure 8. Dependence of the responses of the[Ca+2]i on extracellular [Ca"]. DMSO-differen-

1600 tiated HL-60 cells were incubated in buffer with800 (A) or without (B) 1 mMCaCL2 (top). Other sus-400 pensions of HL-60 cells were maintained in a me-

dium containing 1 mMCaCl2, but no phosphatesalts, without (A) or with (B) 20 uM lanthanum

100 (bottom). Each panel shows the tracing of the re-sults of one representative experiment from atotal of three.

peak more rapidly for LTD4 than for LTC4 (Figs. 1-3). Thatthe DMSO-induced differentiation of HL-60 cells was accom-panied by an increase in the sensitivity and maximal responseof the [Cal2Ji to LTD4, relative to that of undifferentiatedHL-60 cells, linked the alterations in cellular responsiveness todifferentiation-associated changes in cellular receptors forLTD4 or postreceptor biochemical events. The results of flowcytometric assays of [Ca+2]i indicated that a subset of undiffer-entiated HL-60 cells react suboptimally to LTD4 and thatDMSOrecruits the majority of the cells into an optimallyLTD4-responsive state (Fig. 4). DMSO-induced differentiationdid not alter the rate of degradation of LTD4 by HL-60 cells.Thus, the results of analyses of the specificity and mechanismsof the increases in [Ca+2]i elicited by LTC4 and LTD4 arereferable to a relatively homogeneous population of myelo-cytic leukocytes.

Although the response of [Ca+2]i to LTC4 attained one-halfof the maximum level at - 60 nM, a concentration that corre-lates with the equilibrium dissociation constant of 50 nMfound for the binding of [3H]LTC4 to DMSO-differentiatedHL-60 cells and of 34 nM for human PMNleukocytes (29);LTC4 was not a direct agonist. The binding sites on humanPMNleukocytes were specific for the sulfido-peptide leuko-trienes since only C6-peptide leukotrienes and their structuralanalogs exhibit binding activity (29). The longer time requiredto reach a maximal [Ca+2], with LTC4 than LTD4 suggestedinitially the LTC4 was not acting directly through binding to a

separate set of receptors, but rather was being converted to anactive metabolite. The observation that high concentrations ofLTD4 were able to deactivate HL-60 cells to the subsequenteffects of LTC4, as well as LTD4, on the [Ca+]i implied thatthe two leukotrienes shared a commonpathway of activation(Fig. 5). The hypothesis that LTC4 acted only after conversionto LTD4 was confirmed by the findings that the serine-borateinhibitor of leukocyte y-glutamyl transpeptidase suppressedthe response of [Ca+2]I to LTC4, but not to LTD4, and reducedconcomitantly the LTC4-induced increases in adherence ofHL-60 cells to Sephadex G-25 (Fig. 6, Table II). Under theconditions used for determination of [Ca+2]i by quin-2 fluores-cence, the serine-borate complex inhibited the metabolic con-version of LTC4 to LTD4 by 50-100% (Table I). This rate ofconversion of LTC4 to LTD4 is sufficient to explain the in-creases in [Ca+2]i and adherence to surfaces documented forLTC4 in terms of the LTD4 generated from LTC4. In contrastto these results, previous studies have shown that the spasmo-genic activity of LTC4 in guinea pig ileum does not depend onthe bioconversion of LTC4 to LTD4 (30), and that serine-bo-rate complex enhances the contractile activity of LTC4 on

guinea pig trachea (31).That the effect of LTD4 on the [Ca+2], of HL-60 cells is

receptor-mediated was suggested by the potency and suscepti-bility to stimulus-specific deactivation of LTD4 activity. Thispossibility is supported by two other independent lines of evi-dence. First, the stimulation of increases in [Ca+2]i by LTD4

100

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2HL-60 cells. The HL-60 cells were chal-

200 lenged with 100 nMLTD4 without (A)and with (B) 1 gMNIF. The results are

'100 representative of a total of four identi-cal experiments.

Leukotriene D4 Stimulation of HL-60 Cells 989

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Figure 10. Effect of LTD4 on the fluorescence histograms of di-O-C5(3)-loaded HL-60 cells. The patterns of distribution of fluores-cence were obtained before any challenge (-), 1 min after stimula-tion by 200 nMLTD4 (-), and 7 min after addition of 0.5 ,Mgramicidin (- --).

was prevented by equimolar concentrations of a specific LTD4receptor antagonist (Fig. 7). Second, pretreatment with con-centrations of pertussis toxin that inactivate Nprotein, inhib-ited the LTD4-induced increases in [Ca+2]i in HL-60 cells (Fig.11), as has been demonstrated recently for receptor-mediatedeffects of LTD4 in other systems (17, 32).

The responses of the [Ca+2]i of DMSO-differentiatedHL-60 cells to LTD4 depend on extracellular calcium, as evi-denced by the finding that the addition of either EGTAor thecalcium entry blocker lanthanum abolishes almost completelythe [Ca2J]i rises elicited by LTD4 (Fig. 7). It is also apparentthat LTD4 stimulates calcium entry through a voltage-inde-pendent calcium channel in the plasma membrane. First, thespecific calcium channel blocker NIF does not alter the re-sponse of the [Ca+]i to LTD4 (Fig. 8). Second, the LTD4-in-duced increase in the [(a+2J] is not accompanied by changes inmembrane potential (Fig. 9). In guinea pig trachea and lungparenchyma, LTD4 also triggers cellular responses through amechanism that is inhibited by a lack of extracellular calciumand only slightly affected by NIF (33). LTD4 in both cells andtissues thus seems to open a voltage-independent calcium

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Figure 11. Prevention by pertussis toxin of the increase in [Ca'2]i in-duced by LTD4. HL-60 cells were incubated without (A) and with(B) 100 ng/ml of pertussis toxin for 2 h at 37°C before the additionof 100 nMLTD4. The tracings in A and B are from a single illustra-tive experiment from a total of three.

Table I. Effect of LTC4 and LTD4 on the Adherence of DMSO-differentiated HL-60 Cells to Sephadex G-25

Percentage increase in adherenceto Sephadex G-25*

Stimulust Without serine-borate With serine-borate

Buffer 0fMLP (10-6 M) 42.1 43.4LTB4 (10-7 M) 24.0 22.9LTC4 (10-9 M) 1.6LTC4 (10-8 M) 11.3 5.4LTC4 (10-7 M) 20.5 12.1LTC4(10-6M) 26.7LTD4 (10-9 M) 1.4LTD4 (10-8 M) 7.5 8.2LTD4 (10-7 M) 20.4 21.8LTD4 (10-6 M) 41.2

* The results presented are the mean percentage increase of HL-60cells adhering to Sephadex G-25 in two experiments performed induplicate, except for LTD4 in the presence of serine-borate and LTB4.

The HL-60 cells were preincubated for 5 min at 370C without orwith 5 mMserine-lO mMsodium borate and then for 2 min withthe stimulus or buffer alone.

channel similar to that described for the effect of angiotensin IIon smooth muscle cells (13) and of the epidermal growth fac-tor on A43 1 cells (34). These changes in [Ca1]i also are asso-ciated with a functional response in HL-60 cells, where LTC4and LTD4 enhanced their adherence to the surface of Sepha-dex G-25 particles (Table II). It is clear from these studies thatthe increased adherence of HL-60 cells in response to LTC4stimulation is primarily due to the generation of LTD4. Sinceboth LTC4 and LTD4 are able to compete with [3H]LTC4 forbinding to human PMN!eukocytes (29), the enhanced adher-ence could possibly be mediated by this population of recep-tors. In vitro studies of the responses to sulfidopeptide leuko-trienes have been limited previously to those of tissues andcellular monolayers. The results of the present studies demon-strate for the first time in suspensions of individual cells in-creases in the [Cal2Ji, which may represent a critical earlybiochemical event in the selective activation of leukocyte ad-herence to surfaces.

Acknowledgments

This work was supported in part by grants A119784 and HL31809from the National Institutes of Health. Dr. Baud was supported in partby the Ministere de la Recherche et de la Technologie.

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Leukotriene D4 Stimulation of HL-60 Cells 991