Immunology 1996 89 301-307

Stem cell factor potentiates histamine secretion by multiple mechanisms, butdoes not affect tumour necrosis factor-7a release from rat mast cells

T. J. LIN, E. Y. BISSONNETTE, A. HIRSH & A. D. BEFUS Pulmonary Research Group, Departments of Medicineand Physiology, University of Alberta, Edmonton AB, Canada

SUMMARY

The effect of stem cell factor (SCF) on histamine and tumour necrosis factor-a (TNF-cx) releasefrom rat peritoneal mast cells (PMC) was determined and the intracellular pathways involved inthe potentiation of histamine secretion were investigated. The effects of SCF (2-100 ng/ml) wereexamined following both short-term (0 and 20 min) and long-term (up to 24 hr) preincubationswith SCF. Pretreatment of PMC with SCF for 0 min (concurrent) or 20 min did not inducehistamine secretion directly, but significantly increased antigen (Ag)-induced histamine secretion.SCF potentiated Ag-induced intracellular Ca2+ increase and calcium ionophore A23187-inducedhistamine secretion. Pertussis toxin (PT) inhibited SCF-induced potentiation of IgE-dependenthistamine secretion, indicating that PT-sensitive G-proteins are involved in the immediate effectsof SCF. In long-term incubation experiments, SCF pretreatment for 18-24 hr significantlyenhanced Ag-induced histamine secretion, but did not affect Ag-induced intracellular Ca2± levels.The effects of long-term incubation with SCF, but not the short-term effects, were blocked bycycloheximide. Interestingly, spontaneous and Ag-induced TNF-a release from rat PMC were notaffected by pretreatment with SCF (2-500ng/ml) for 1 to 24 hr. Thus, through immediate anddelayed mechanisms, SCF potentiates histamine release from PMC, but has no effect on TNF-aXrelease. The regulation of MC by SCF may be important in allergic and other inflammatorydiseases.

INTRODUCTION

Stem cell factor (SCF) is encoded by the SI locus on murinechromosome 10. It is the ligand for SCF receptor (SCFR), atyrosine kinase-type receptor that is involved in haemato-poiesis, gametogenesis and development and function of mastcells (MC) and melanocytes.1 SCF2 and soluble SCFR3 arepresent in human serum at about 3-3ng/ml and 300ng/ml,respectively, suggesting that MC are exposed to SCF ona chronic basis. Signals through SCFR can regulatethe migration,4 adhesion,5 chemotaxis,4 proliferation,6'7maturation6'7 and mediator synthesis8 and release915 of MC.

SCF can directly stimulate MC secretion or potentiate thesecretory activity of MC stimulated through the high-affinityreceptor for IgE (FcERI).9'5 The direct degranulation of MCby SCF has been observed in several MC populations, such asmouse peritoneal MC (PMC),9 human skin MC,'0 but not

Received 5 January 1996; revised 3 June 1996; accepted 9 June 1996.Abbreviations: Ag, antigen; Caf', intracellular calcium; FceRI,

high-affinity receptor for IgE; MC, mast cells; PMC, peritoneal mastcells; PT, pertussis toxin; SCF, stem cell factor; TNF-a, tumournecrosis factor-a.

Correspondence: Dr A. D. Befus, Pulmonary Research Group,Department of Medicine, Room 574 HMRC, University of Alberta,Edmonton, Alberta, T6G 2S2, Canada.

human lung MC'" and rat PMC.12 Recently, it was reportedthat freshly isolated rat PMC were relatively unresponsive toSCF, but progressively acquired responsiveness to SCF andantigen (Ag).'3 In addition, effects of SCF on the secretoryactivity of MC stimulated through FcsRI were differentafter short-term (10-30min) or long-term (up to 48hr)exposures.915 After short-term exposure, SCF enhanced IgE-dependent MC degranulation in human skin MC,'0 humanlung MC"l and mouse PMC.9 After long-term exposure to SCF(up to 48 hr), different MC varied in their responsiveness toFceRI cross-linking. IgE-mediated MC degranulation wasenhanced by SCF in mouse PMC,9 but was not affected bySCF in human skin MC.'0

The mechanisms for the differences in SCF-potentiated IgE-dependent MC secretion seen in short-term exposure asopposed to long-term exposure to SCF have not been defined.In rat PMC, we re-examined the short-term and long-termeffects of SCF on IgE-dependent MC secretion and comparedthe mechanisms that underlie these effects.MC can synthesize and release several cytokines, including

the multifunctional tumour necrosis factor-ax (TNF-cx).16 Becausethere are no reports of the effects of SCF on cytokine releaseand synthesis of MC, we compared the effects of SCF onhistamine secretion to the spontaneous and Ag-induced releaseof TNF-a.

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We demonstrate for the first time that SCF potentiatesAg-induced histamine secretion from rat PMC throughdistinct immediate (0-20min) and delayed (18-24hr)mechanisms. The immediate effects of SCF involve potentia-tion of Ag-induced intracellular Ca2+ (Ca2+)- and pertussistoxin (PT)-sensitive G-proteins, and are independent of newprotein synthesis. In contrast, the delayed effects of SCF aredependent on new protein synthesis and do not involve themodification of Ag-induced Ca2+ increase. Interestingly, SCFdid not affect spontaneous or Ag-stimulated TNF-a releasefrom PMC.

MATERIALS AND METHODS

AnimalsAdult male Sprague-Dawley rats were obtained from CharlesRiver Canada Inc. (Quebec, Canada) and maintained in filter-topped cages to minimize unwanted infection. Five to 6weeks before MC isolation, rats of 200-300 g were infectedwith 3000 third-stage larvae of Nippostrongylus brasiliensis by asingle subcutaneous (s.c.) injection to induce IgE-dependentsensitization. 17 These experimental protocols were approved bythe Health Sciences Laboratory Animal Services, University ofAlberta, in accordance with the guidelines of the CanadianCouncil on Animal Care. Animals were given food and waterad libitum and maintained on a 12-hr (07.00 hr)-12 hr (19.00 hr)light-dark cycle.

ReagentsRecombinant rat SCF (rrSCF) was a kind gift from Amgen Inc.[>95% pure by sodium dodecyl sulphate-polyacrylamide gelelectrophoresis (SDS-PAGE); Thousand Oaks, CA]. L-a-phosphatidyl-l-serine, dipalmitoyl (PS), compound 48/80,cycloheximide, calcium ionophore A23187, o-phthaldialdehyde(OPT) and ethyleneglycol-bis-(f1-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) were purchased from Sigma ChemicalCo. (St Louis, MO). Pertussis toxin (PT) was purchased fromICN Biomedical Inc. (Aurora, OH). RPMI-1640 medium,RPMI-1640 Ca2+ free medium and HEPES-buffered Tyrode'ssolution (HBTS) were purchased from Gibco BRL (GrandIsland, NY). Chelex 100 resin (Chelex) was purchase fromBioRad Laboratories Inc. (Richmond, CA).

AntigenAg used to activate in vivo-sensitized MC was a collectionof soluble excretory and secretory products of adult N.brasiliensis prepared by incubating worms in phosphate-buffered solution (PBS; pH7-2) at 370 for 4hr.'8 The Agconcentration was described as worm equivalents (We/ml andthe final protein concentration of antigen (5 We/ml) was7-1 ± 0-88g/ml. The final Ca2+ concentration in the Ag(1OWe/ml) was <10MM, as tested by the Spectro-serviceLaboratory (University of Alberta). The Ag, RPMI-1640medium and HBTS contained less than 0-01 ng/ml ofendotoxinwhen tested by the E-Toxate kit (Sigma). Endotoxin-free water(Baxter Co., Toronto, Canada) was used in all experiments.The induction of histamine secretion by Ag was specific toPMC derived from N. brasiliensis-infected rats. Histaminesecretion could not be induced by Ag in PMC from uninfectedrats, and SCF (100 ng/ml) did not alter this lack ofresponsiveness(data not shown).

MC isolation, incubation and histamine assayPMC were prepared as described previously,'9 with purity of>96%. In several experiments (long-term), PMC weresuspended in RPMI-1640 medium and incubated in thepresence or absence of rrSCF for up to 24hr, then washedand resuspended in HBTS and challenged with Ag (5 We/ml).In other experiments (short-term), PMC were suspended inHBTS and incubated in the presence or absence of rrSCF andthen challenged with one of several stimulants without firstremoving the SCF. In all experiments, MC stimulation wasconducted in a total volume of 200,Ml. Histamine was measuredin both supernatant and pellet fractions by fluorometric assay20using a CytoFluor 2350 Fluorescence Spectrometer (Millipore,Bedford, MA). Unless specified otherwise, the spontaneousrelease of histamine in the absence of stimulant was subtractedto establish histamine release specific to the secretogoguesemployed.

Caft measurementPMC (1 x 106 cells/ml) were incubated in RPMI medium for45 min with 3 gM fura-2/AM (Molecular Probes Inc., Eugene,OR) and an equal volume of Pluronic F-127 (20% w/v). Afterwashing, PMC were resuspended in RPMI medium at aconcentration of I x 106 cells/ml. Fluorescence was measuredby placing 500Ml PMC in a 370 thermostated quartz cuvettewith magnetic stirring in a spectrofluorometer (CAF-100 JascoInc, Tokyo, Japan). Fluorescence was recorded at 510 nm afterexcitation at 340 and 380 nm. Autofluorescence of the unloadedPMC was subtracted. Cytosolic-free calcium was calculatedfrom the equation: [Ca2+] = Kd[(R - Rmin)/(Rmax-R)]Fo/F. R isthe measured fluorescence. Rmax is maximum 360/380 in asaturated calcium environment after addition of 60 gM digitonin,and Rmin is the fluorescent ratio at low calcium levels afteraddition of EGTA. F0 is F380 at zero Ca2+ (10mm EGTA). F isF380 at saturated calcium concentrations. A value of 226 nM wasassumed for Kd in these calculations.2'

TNF-a bioassayThe bioactivity of TNF-a was tested as cytotoxicity of WEHI164-13 (obtained from Dr T. R. Mosmann, Dept of MedicalMicrobiology and Immunology, University of Alberta,Canada) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT; Sigma Chemical Co.) assay asdescribed previously.22 Briefly, 50 Ml/well of standards orsamples was added into flat-bottomed Linbro plate (FlowLaboratories, Inc. McLean, VA). Each sample was tested in sixdilutions (twofold serial dilutions). Cell lysates used in TNF-aassay were prepared by sonication (10 seconds sonication and10 seconds rest, three cycles, power 50 watts, with Sonicator XL2010; Heat Systems Co., Farmingdale, NY). Mouse recombi-nant TNF-a (Genzyme Co., Cambridge, MA) was used as astandard. Eight, twofold, serial dilutions starting from 100 pg/ml were used to establish the standard curve. Fifty Ml/well of1 x 105 WEHI 164 13 cells/ml in RPMI medium, supplementedwith 10% fetal bovine serum (FBS) and SOMm 2-mercaptoetha-nol, was added and incubated for 20hr. Then 10,Ml/well ofMTT (5 mg/ml) was added and further incubated for 3hr.Isopropanol-HCl (150Ml) was used to dissolve the purpleformazan precipitates. The plate was read on a Vmax kineticmicroplate reader (Molecular Devices Co., Menlo Park, CA) at570 nm.

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a)0)

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Figure 1. Short-term effects of rat rSCF on antigen-induced histaminesecretion from rat peritoneal MC. The cells were concurrentlyincubated with SCF (2, 20, 50, 100 ng/ml) and Ag (5 We/ml) for10 min. Results are means ± SEM for five experiments (*P < 0 05 bycomparison with cells not treated with SCF).

Statistical analysisAnalysis ofvariance, Scheffe multiple comparison and the pairedStudent's t-test were used for statistical evaluation of data.Results were considered significantly different when P < 0-05.Throughout the text, data are expressed as mean ± SEM.

RESULTS

SCF enhances Ag-induced histamine secretion from PMCfollowing short-term (0-20 min) incubation

The addition of SCF concurrently with Ag (5 We/ml) led to a

concentration-dependent enhancement of histamine release,but SCF itself in the absence of Ag did not induce histaminerelease (Fig. 1). Ag-induced histamine secretion was signifi-cantly enhanced by SCF at a dose of 2 ng/ml, a level similar tothat of SCF in normal human serum.2

We confirmed that SCF (100 ng/ml) alone did not directlyinduce histamine secretion even after 20-180 min incubationwith PMC. Histamine release induced by different concentra-tions of Ag (0 1-1OWe/ml) was significantly enhanced bypreincubation with SCF (50 ng/ml) for 20 min (enhancementfrom 226-9% to 382-1%; n = 3).

In contrast, in the absence ofAg stimulation SCF (100 ng/ml)induced significant histamine secretion when 50 and 100 Yug/ml PS(13 9% and 23 5% specific histamine secretion, respectively) wasused. PS is known to regulate the activities of multiple enzymesthat are important in intracellular signal transduction pathways,such as protein kinase C (PKC),23 nitric oxide synthase,24 Na+/K+-ATPase,25 diacylglycerol kinase26 and raf-1 kinase.27 Tominimize the complexity of elements influencing the interactionsbetween signals from SCFR and FceRI, the experimentsdescribed below were conducted in the absence of PS.

SCF enhances calcium ionophore A23187-induced histaminerelease and Ag-induced Ca?+ increase

To explore the hypothesis that elements of the intracellularsignalling pathways, such as Ca?', might be shared by SCFRand FceRI ligation, we investigated the effects of SCF onhistamine secretion induced by calcium ionophore, A23187.

1996 Blackwell Science Ltd, Immunology, 89, 301-307

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Figure 2. Effect of 20-min incubation of rat peritoneal MC with ratrSCF on calcium ionophore A23187-induced histamine release. Thecells were incubated with SCF (50ng/ml) for 20min, then challengedwith calcium ionophore A23187 for Omin. Results are means ± SEMfor four experiments (*P < 0 05 by comparison with cells challengedwith the same concentration of A23187 but not treated with SCF).

Pretreatment with SCF (50 ng/ml) for 20 min enhancedhistamine secretion induced by suboptimal doses (0-1 and0-2pM) of A23187 by 85 3% and 53 5% respectively (Fig. 2).Fura-2/AM was used to study the role of Caf+ in SCF-inducedenhancement of Ag-stimulated histamine secretion (Fig. 3 andTable 1). A suboptimal dose of Ag (1 We/ml) was used toinitiate Ca3+ increase (Fig. 3b). SCF itself (immediate) induceda significant Ca2+ increase (21-9 ± 5-8 nM, P < 0-05) (Fig. 3a).One minute after the addition of SCF (25 ng/ml), the Ca2+increase had reached a plateau and then PMC were stimulatedwith Ag (1 We/ml). A specific Ag-induced Caf+ increase wasrecorded. SCF significantly potentiated the Ag-induced Ca3'increase (Fig. 3c and Table 1). Given the potentiation by PS ofboth the Ag- and SCF-induced histamine secretion from ratPMC, effects of PS on Ag- and SCF-induced Ca3+ increasewere observed. Both Ag (1 We/ml)- and SCF (25 ng/ml)-induced Caf+ increases were significantly enhanced by PS(100pg/ml), 368% and 78% enhanced, respectively.

To identify the importance of extracellular Ca2+ in thepotentiation of SCF on Ag-induced histamine secretion, Ca2+-free HBTS was used to exclude the extracellular Ca2+ pool.When PMC were incubated with SCF (2, 20, 50 and 100 ng/ml)for 20 min, then challenged with Ag (1OWe/ml) for Omin inCa2+-free HBTS, Ag-induced histamine secretion in theabsence of SCF was reduced by 44-7 ± 14-2% (n = 4).However, the effects of SCF (2-100 ng/ml) on Ag-inducedhistamine secretion were not significantly reduced (4 0-17-2%reduction, n = 4). We attempted to study the roles ofintracellular Ca2+ pools in SCF effects using chelation withEGTA (1 mm for 3 hr), but unfortunately this completelyinhibited Ag-induced histamine secretion and was not a usefulapproach.

Effects of PT on the SCF-induced enhancement of PMCresponsiveness to Ag

PT-sensitive G-proteins are important components in theactivation of MC by some stimuli (eg. compound 48/80 andthrombin), but are not involved in Ag-induced histaminesecretion from MC.28 Thus we postulated that PT-sensitive

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G-protein(s) might be involved in SCF-mediated alterations in

histamine secretion from PMC. To assess the possible role ofG-proteins in the SCF-induced increase ofPMC responsiveness

to Ag, PT was used to inactivate PT-sensitive G-proteins byADP ribosylation. Our time-course and dose-response studiesconfirmed that treatment of MC with 100 ng/ml PT for 2-3 hrcaused a maximum inhibition (80-90%) of compound 48/80-induced histamine release (data not shown). Thus PMC were

incubated in RPMI-1640 medium in the presence or absence ofPT (100ng/ml) for 3hr, washed and resuspended in HBTS.Aliquots of PMC then were preincubated with SCF (2, 20, 50,OOng/ml) for 20 min and challenged with Ag (5 We/ml) foranother 1Omin. As shown in Fig. 4, PT pretreatment partiallyinhibited (20 9 ± 5.1%-28 2 + 5 4%) the effect ofSCF on IgE-dependent histamine release.

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Long-term treatment with SCF (18-24 hr) enhances Ag-inducedhistamine secretion from PMC

When MC were treated with SCF for 2, 8, 18 or 24 hr and thenwashed, it was established that 18 hr of incubation was requiredto enhance Ag-induced histamine release (Fig. 5). SCFenhanced Ag (5 We/ml)-induced histamine release in a dose-dependent manner. In contrast, this long-term treatment withSCF in the absence of Ag challenge did not induce histaminesecretion (<3%; no significant difference compared to thespontaneous release in the no-treatment group of PMC). SCF(50 ng/ml, 24 hr) enhanced histamine secretion by approxi-mately twofold (e.g. from 17% to 31% specific secretion) atdoses of Ag ranging from 01 to 10 we/ml (data not shown;n = 5). No change in number or viability of PMC was observedafter 24 hr incubation with SCF (200 ng/ml) in comparison tosham-treated cells (data not shown).

When PMC were incubated together with SCF andcycloheximide (25, 50 or 100 ng/ml) for 24 hr, the enhancementof histamine release by SCF (50 ng/ml) was blocked bycycloheximide in a dose-dependent manner (Fig. 6). Toexclude the possibility that cycloheximide directly interactswith SCF, PMC were incubated together with cycloheximide(100 ng/ml) and SCF (2, 20, 50 and 100 ng/ml) and concurrentlystimulated with Ag (5 We/ml) for 0 min. SCF (2-100 ng/ml)-potentiated histamine secretion was not affected by this short-term incubation with cycloheximide.

Given the significant change of Cal' in the immediateeffects of SCF, the possible role of Ca>2 in long-term (24hr)effects of SCF was studied. PMC were incubated with SCF (100ng/ml) for 24 hr in Ca2+-free medium supplemented with 5%FBS from which Ca2+ was removed by Chelex 100 resin. Afterwashing, PMC were resuspended in normal HBTS andchallenged with Ag (5 We/ml). Interestingly, SCF-potentiatedhistamine secretion was partially decreased (inhibition was

22-3 ± 8-5%, n = 3) when the pretreatment with SCF occurredin the absence of extracellular Ca2+. Thus, both extracellularand intracellular Ca2+ may contribute to the long-term effectsof SCF.

To determine if SCF (24hr) modified Ag-induced Ca>

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Figure 3. Direct and priming effects of SCF on intracellular Ca>+changes. Rat peritoneal MC were loaded with 3 JiM Fura-2/AM and an

equal volume of Pluronic F- 127 (20% w/v) for 45 min, then washed andchallenged with (a) SCF (25 ng/ml), (b) Ag (I We/ml), (c) SCF (25 ng/ml) and Ag (1 We/ml). Fluorescence was recorded at 510nm afterexcitation at 340 and 380 nm.

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Figure 4. Effect of PT on SCF-induced enhancement of histaminerelease from rat peritoneal MC stimulated by Ag. The cells were

incubated with PT (100ng/ml) for 3hr, then washed. Then the cellswere aliquoted and incubated with SCF (2, 20, 50, lOOng/ml) for20min, and challenged with 5We/ml Ag for 10min. Results are

means ± SEM for three experiments (*P < 0-05 by comparison withcells treated with the same concentration of SCF but not PT).

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SCF modulation ofMC histamine and TNF-c secretion

Table 1. Immediate and delayed effects of SCF on Ag-induced Ca"+ increase inrat PMC

Net Ca.+ increase (nM)by Ag stimulation

Without SCF With SCF

Immediate effect of SCF (1 min pretreatment) 18-1 ± 2-9 49-1 ± 37Delayed effect of SCF (24 hr pretreatment) 28 2 ± 2 2 28 2 i 13

In the immediate effect experiment, peritoneal MC were loaded with Fura-2/AMfor 45 min, washed and challenged with SCF (25 ng/ml) or medium for 1 min, andthen stimulated with antigen (1 We/ml). Ca.+ was measured during these processes.In the delayed effect experiment, peritoneal MC were treated with SCF (100 ng/ml)for 24 hr and then washed and loaded with Fura-2/AM before antigen (1 We/ml)challenge. Specific antigen-induced Ca.± changes were recorded. *P < 005compared to without SCF group. n = 3.

change, PMC were washed after 24 hr treatment with SCF(100 ng/ml), loaded with Fura-2/AM, and challenged with Ag(1 We/ml). In contrast to the immediate effect of SCF,pretreatment of PMC with SCF for 24 hr did not affectAg-induced Ca.+ increase (Table 1) indicating that distinctmechanisms are involved in the immediate and delayedenhancement of Ag-induced histamine secretion by SCF.

No effects of SCF on TNF-i release and synthesis of PMC

Given the marked effects of SCF on histamine secretion, we

studied whether treatment with SCF for various times wouldmodify Ag-induced TNF-a release from PMC. However, SCF(100 ng/ml) did not affect the spontaneous and Ag-stimulatedTNF-oa release from PMC (Table 2). In time-course (1, 3, 6, 12and 24hr) or dose-response studies (2, 20, 50, 100, 300 and500 ng/ml) with SCF, no effects were noted on TNF-a contentsin supernatants and cell pellets in either Ag-stimulated or

unstimulated PMC (data not shown).

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20 ng/ml

DISCUSSION

A large body of in vitro and in vivo data shows that SCF can

regulate MC development and secretory function.' Two typesof experiments have been used in the past, namely, short-termand long-term incubation with SCF. In short-term treatment(10-60min) experiments, MC were challenged with stimuli inthe presence of SCF,9-'5 while in long-term experiments (e.g.48 hr) in vitro, SCF was removed by washing and the MC were

challenged in the absence of SCF.9 In long-term experiments,hyperplasia of MC was observed in vivo following subcuta-neous administration of SCF for 21 days629 and incubation ofMC with SCF for 48 hr in vitro potentiated secretory function.9Other authors have investigated the effects of short-term (10-60min) incubation with SCF.9'5 Most of these experimentsestablished that SCF enhances mediator secretion from MCstimulated through FcERI.

We have demonstrated that the mechanisms of SCF-potentiated histamine secretion are different in these two

cnCu

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2 ng/ml

35 1SCF (50 ng/ml, 24 hr)

El No SCF

0 25 50 100

Cycloheximide (ng/ml, 24 hr)0 5 10 15 20 25

Time (hr)

Figure 5. Effect of long-term incubation (up to 24 hr) of rat peritonealMC with rat rSCF on antigen-induced histamine release. Mast cells(>99% pure) were incubated with SCF for 2, 8, 18 and 24hr, thenwashed and challenged with 5We/ml Ag for 10min. Results are

mean ± SEM for three experiments (*P < 0-05 by comparison withsham-treated cells).

Figure 6. Effect of cycloheximide on SCF-induced enhancement ofhistamine release from rat peritoneal MC stimulated by antigen. Mastcells (>99% pure) were incubated with rat rSCF (50 ng/ml) and a range

of concentrations of cycloheximide (25, 50, 100 ng/ml) for 24 hr.Thereafter the mast cells were washed and challenged with 5 We/ml Agfor Omin. Results are means + SEM histamine secretion for fiveexperiments (*P < 0-05 by comparison with cells treated with SCF butnot cycloheximide).

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Table 2. No effects of SCF (6 hr) on spontaneous andAg-induced TNF-a release from rat PMC

Treatment TNF-a (pg/million mast cells)

No treatment 56 7 ± 9.7SCF (100 ng/ml) 51 5 + 12-1Antigen (5 We/ml) 124-7 ± 40.9*SCF + antigen 123-1 ± 436*

After 6hr incubation of peritoneal MC with SCF(100 ng/ml) and Ag (5 we/ml), TNF-a contents in super-natants were tested. Results are mean ± SEM for fourexperiments. *P < 0 05 by comparison with no treatmentand SCF groups.

kinds of experimental designs (short-term and long-termincubations). The short-term (immediate) effects of SCF areassociated with enhanced levels of free Caif, partiallysusceptible to PT inactivation of G-proteins and independentof new protein synthesis (100ng/ml of cycloheximide had noeffect). In contrast, the long-term effects of SCF are dependenton new protein(s) synthesis (Fig. 6) and do not involve themodification of Ag-induced Ca2+ increase (Table 1).

To explore the hypothesis that elements of intracellularsignal transduction pathways might be shared by SCFR andFcERI, short-term (0-20 min) experiments were employed.Twenty minutes pretreatment ofPMC with SCF or concurrenttreatment of PMC with SCF and Ag significantly augmentedthe magnitude of histamine release in response to IgE-dependent activation (Fig. 1). To assess the role of Ca?+ inthe short-term effect of SCF, calcium ionophore A23 187-induced histamine secretion and Ag-Ca?+ change were used.Calcium ionophore A23187 (01, 0-2 /M)-induced histaminerelease was significantly increased by SCF (Fig. 2) andAg-induced increase of Ca 2+ was significantly enhanced bySCF (Fig. 3). These data suggest that Ca2+ may be one siteof convergence of intracellular signalling from SCFR andFceRI. To investigate the roles of stimulus-secretion couplingG-proteins in the short-term effect of SCF, PT, whichinactivates PT-sensitive G-proteins,24 was used. Optimalpretreatment of MC with PT for 3 hr only partially inhibited(23-9 ± 3 5%) the potentiation of SCF on IgE-dependenthistamine release (Fig. 4). Given the complex of intracellularsignals involving G-proteins, Ca2+, phospholipases andkinases, it is impossible to identify clearly the mechanism ofthe partial PT-induced inhibition of SCF effects. Recently,studies with various inhibitors have suggested that severalkinases are important in the action of SCF on MC.30 Takentogether, our observations suggest that multiple elements ofsignal transduction pathways are involved in the immediateSCF-induced augmentation of MC secretory function.

The long-term effects of SCF require new protein(s)synthesis, as evidenced by the time-course analysis (Fig. 5)and blockage of the SCF response in a dose-dependent mannerby the protein synthesis inhibitor cycloheximide (Fig. 6).Indeed, it has been reported elsewhere that SCF inducestranscription of c-fos, c-jun and jun B genes3' and enhancestranslation of tyrosine and serine/threonine kinases, suchas p21ras32 Raf-1, mitogen-activated protein kinase

(MAPK), 33,34 pp9orsk and pp70-S6 kinase.34 Interestingly, ourprevious work demonstrated that the down-regulatory actionof interferons (IFN) on MC also requires new proteinsynthesis. 19,35

Although it has been suggested in a review36 that SCFinduces TNF-a production and release from human MC, thereare no published data on the effect of SCF on MC TNF-arelease and synthesis. We investigated the effects of SCF onTNF-ox synthesis and release from MC and found that after24hr incubation with SCF (2-100ng/ml), TNF-a contents inMC supernatants and cell pellets were not significantly affected.Furthermore, SCF (100 ng/ml) had no significant effect inAg-induced TNF-a synthesis and release from PMC (Table 2).These experiments extend others that have shown MCdegranulation and cytokine production are regulated differ-ently by microenvironmental factors.37

The biological significance of SCF-induced enhancement ofMC responsiveness to IgE-dependent stimulation is not fullyunderstood. Our observations and those of others9-12"14'15 showthat SCF significantly potentiates the secretory activities ofMCstimulated through FceRI. Surprisingly, normal mice chroni-cally treated with SCF for 21 days exhibit fewer deaths due toIgE-dependent anaphylaxis than those treated with vehicle (1/IIversus 8/11, respectively, P < 0.01).29 Although the mechanismsthat account for MC hyperplasia and activation on one handand yet reduced severity ofanaphylaxis on the other remain to bedefined, it is likely that the biological effects of SCF on IgE-dependent MC secretion may be influenced or regulated by othermicroenvironmental factors that coregulate MC developmentand function. At least two major cellular interactions appear tobe important in regulation of MC development and function,namely T-cell and fibroblast-dependent mechanisms.38 T-helpertype-I (Thi) and Th2 lymphocytes produce several cytokinesthat modulate MC development and function, includinginterleukin-3 (IL-3), IL-4, IL-5, IL-9, IL-10, IFN-y etc.39Unfortunately, the effects of many of these cytokines on MCare incompletely known. In contrast, fibroblast-dependentmodulation of MC development and function appears to belargely mediated by SCF.36 It will be of great interest todetermine to what extent the interaction between T-cell andfibroblast-dependent mechanisms regulates MC developmentand function, and the precise mechanisms by which SCFinfluences MC in in vivo systems analogous to our short-termand long-term models of exposure to SCF.

ACKNOWLEDGMENTS

We thank Ms Jennifer Pick and Mr Arion Predika for the skilful help inNippostrongylus brasiliensis worm preparation. This work was sup-ported by Medical Research Council of Canada. Dr A. Dean Befus is aScholar of the Alberta Heritage Foundation of Medical Research. DrE. Y. Bissonnette is a Scholar of the Medical Research Council/Canadian Lung Association.

REFERENCES

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