Immunology 1995 86 434-440

Monocyte chemotactic protein-i provokes mast cell aggregationand [3H15HT release

P. CONTI,* W. BOUCHERt R. LETOURNEAU,t C. FELICIANI,4 M. REALE,* R. C. BARBACANE,*P. VLAGOPOULOS,t G. BRUNEAU,§ J. THIBAULT§ & T. C. THEOHARIDESt *ImmunologyDivision,

Institute of Experimental Medicine, and tDermatology Department, University of Chieti, Chieti, Italy,tDepartment of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, Boston,

Massachusetts, USA and §Laboratoire de Biochimie Cellulaire, College de France, Paris, France

SUMMARY

Monocyte chemotactic protein-l (MCP-1) and MCP-3, the most active and representativecompounds of the CC chemokine family, are proinflammatory cytokines that attract and activatespecific types ofleucocytes. We have used highly purified isolated rat peritoneal mast cells (RPMC)cultured for different lengths of time with and without MCP-1 (200, 100, 50 and 25 nM). Our dataclearly show that MCP-1 (200 nM) causes a marked release of [3H]serotonin ([3H]5HT) andhistamine, which reach a peak at 40 min of incubation (56 6 ± 5-3 and 34-7 ± 6 above the control,respectively). In dose-response experiments, MCP-1 (200, 100, 50, 25, 12 5, 6-25 and 312nM)provoked a dose-dependent release of [3H]5HT and histamine from RPMC, which was maximumat 200 nm. After preparation of the histidine decarboxylase (HDC) probe, a Northern blot analysiswas determined for HDC mRNA. After 4 hr, steady-state levels of HDC mRNA were induced in a

dose-dependent manner by MCP-1 (200-25 nM), compared to the controls. However, MCP-lfailed to prime RPMC in [3H]5HT and histamine release when C48/80 (0 05 mg/ml) or anti-IgE wasused. In contrast, murine interleukin-3 (IL-3) in combination with MCP-1 (200 and 100nM)provoked a greater release of histamine and [3H]5HT than the compounds alone. Moreover,RPMC treated with MCP-1 (200nM) showed, under light microscopy (20x), greater clumpformation, a phenomenon absent in the controls (untreated cells). The electron microscope studiesrevealed that treatment with MCP-l (200 nM) promoted binding of RPMC and clearlydemonstrated a communication between the cytoplasms of adjacent mast cells. Our reportdescribes additional biological activities for MCP-1, suggesting for the first time that this humanmonocyte chemoattractant plays a fundamental role in histamine and serotonin release and cellaggregation in rat peritoneal mast cells.

INTRODUCTION

Chemokines are small proteins inducible in a number ofpathophysiological processes. -3 Two subfamilies have beendiscovered, according to the position of the first two cysteinesin the conserved motif: CXC chemokines (separated by oneamino acid) and CC chemokines (with adjacent cysteines).Monocyte chemoattractant protein-l (MCP-1) is the prototypeof the CC chemokine subfamily, purified from different sourceswith chemoattractant and activator properties.4 5 Recently, ithas been reported that MCP-1 is a potent proinflammatoryprotein, as effective as the complement component C5a in

Received 31 December 1994; revised 11 July 1995; accepted 12 July1995.

Correspondence: Professor P. Conti, Immunology Division,Institute of Experimental Medicine, University of Chieti, Via deiVestini, 66100 Chieti, Italy.

allergic diseases and much more potent than interleukin-8(IL-8).6'7 MCP-l induced high levels of histamine release fromhuman basophils, and stimulated exocytosis.67 It is highlyeffective as an inducer of mediator release but has onlymoderate chemotactic activity, whereas CXC chemokineshave an opposite effect.2'3

Mast cells located in connective tissues are unique fortheir metachromasia with toluidine blue. Basophils and mastcells are a potent source of vasoactive and inflammatorymediators. They accumulate in the tissues in a variety ofdiseases, where their function in most cases is unclear.8-" Theaggregation of IgE bound to high-affinity receptors (FceRI) onthe surfaces of basophils and mast cells results in degranulationand the release of mediators such as histamine, proteases,chemotactic factors, arachidonic acid metabolites and cyto-kines. MCP-l is a novel cytokine and acts on human basophils,eliciting histamine and leukotriene release.67"2 These effectscan occur without priming with IL-3 or other cytokines.'2"13

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MCPJ-provokes most cell aggregation and [3H]5HT release

Recently, some cell-derived agonists eliciting basophil mediatorrelease have been defined; however, little is known about mastcells and the factors that induce their migration, activation andmediator release at the site of inflammation. With theseobservations in mind, it was pertinent to study the effect ofMCP-l on histamine and [3H]serotonin ([3H]5HT) release frommast cells and on mast cell aggregation.

In this study, we have endeavoured to determine the effectof MCP-l on [3H]5HT and histamine release from ratperitoneal mast cells (RPMC), using anti-IgE and the classicpolycationic secretagogue, compound 48/80 (C48/80), asagonists. Because histidine decarboxylase (HDC) is the soleenzyme responsible for the production of histamine fromhistidine, we also studied the effect of MCP-l on HDC mRNAproduction by RPMC. In addition, we determined the effect ofMCP-l on RPMC aggregation. We report that MCP-l (itself)causes histamine and 3H-5HT release and provokes aggre-gation in cultured RPMC.

MATERIALS AND METHODS

Animals and materialsSprague-Dawley rats, obtained from Tacomic (Germantown,NY), were kept in virus-free sections of a modem animalfacility and were allowed access to food and water ad libitum.The animals were killed by inhalation of CO2 followed bydecapitation. Recombinant human MCP- I was purchasedfrom R&D Systems (no. 279-MC; Minneapolis, MN, USA).C48/80 and recombinant murine IL-3 was purchased fromSigma (St Louis, MO). The scintillation cocktail (Ready-solvCP) was from Beckman Instruments Inc. (Fullertown, CA).Metrizamide was from Accurate Chemical and Scientific Corp.(Westbury, NY); histamine methyltransferase was a gift fromthe laboratory of Dr D. Cochrane (Medford, MA); and anti-IgE antibody (anti-DNP-IgE) and dinitrophenyl-bovineserum albumin (DNP-BSA) were provided by Dr F.-T. Liu(Scripps Clinic, La Jolla, CA). The anti-IgE antibody is amouse anti-rat IgE (E-chain specific) that reacts strongly withrat IgE and will not react with other rat isotypes.

Isolation ofRPMCRPMC were obtained by peritoneal lavage from maleSprague-Dawley rats, approximately 350 g in weight, usingHEPES-buffered Locke's solution, pH 7-2 (150 mm NaCl, 5mMKCl, 5mM HEPES, 2mm CaCl2, 1 g dextrose/l, 1 g BSA,pH 7-2. Cells were purified (> 90% purity) over 22-5%metrizamide23 and resuspended in the same buffer. The cellswere loaded with [3H]5HT (15-30Ci/mmol; New EnglandNuclear, Boston, MA) for 1 hr at 370, washed twice and thenresuspended in the indicated buffer (4 x 105/ml) in tubes(Falcon 2063) with a total sample volume of 05 ml/tube.They were then preincubated for 15 min at 370 with thecompound to be tested, followed by C48/80 for 10min and/orMCP- I for 40 min. At the end of the incubation, the cells werepelleted by centrifugation at 100 x g. The supernatant wasremoved, 2% Triton-X-100 added to the pellet to lyse the cells,and both supernatant and pellet radioactivity were quantifiedby a fl-counter. The release was expressed as the percentage oftotal [3H]5HT present in the supernatant relative to the total inthe pellet and the supernatant combined.

In other experiments, histamine content was determined

fluorometrically as described previously.'4"15 For radioimmuno-assay; an aliquot of the sample to be analysed for histaminerelease was incubated with the methyl donor, S-adenosyl-L-(methyl-[17]C) methionine and the enzyme histamine-N-methyltransferase (HMT), to convert histamine to radio-labelled N-methylhistamine. The source of HMT was apartially purified enzyme preparation, obtained from ratkidneys as follows. The kidneys were homogenized, centrifugedat 40 000g, and then the soluble HMT in the supernatant wassubjected to ammonium sulphate precipitation. The HMT,which precipitates between 45% and 70% saturation, wasdialysed and the dialysate (volume 14-16 ml; 20 ml protein/ml)was stored at - 200. The final enzyme preparation was diluted1: 50 prior to use in the enzyme assay. For this histamine assaythe incubation mixture consisted of 50 ul HMT preparation,100 M1 perfusion fraction or histamine standard, 2 nm S-adenosy-L-(methyl-[17]C)methionine (54mCi/mmol; New EnglandNuclear) and 0 1 M sodium phosphate buffer, pH 7 9, to makea total volume of 400 M1. After incubation at 370 for 2 hr, thereaction was halted by the addition of 0-2 ml of ice-cold 0-4Nperchloric acid. Then 0 2 ml of 5N NaOH was added and thelabelled methylhistamine extracted into 4ml of chloroform.The chloroform extract was washed once with 1-0 ml 3 3 NNaOH,then transferred to scintillation vials, evaporated to dryness(during 30min incubation at 550 using an Evaporac, YaleUniversity, New Haven, CT) and assayed for radioactivity.

Aliquots of 50yl of various dilutions of anti-IgE antibody(mouse anti-rat IgE) were incubated with 450 yA RPMCsuspensions for different lengths of time in a shaking waterbath at 37°. Each experiment was done in triplicate. In controlsamples, 50 sl of Lock's buffer was added at the same time asanti-IgE. The optimal concentration of anti-IgE for inducinghistamine release was a 1:1000 dilution of antibody. Thehistamine content of the supernatants was measured using aradioenzymatic method.'6

The results are expressed as the mean counts per minute(c.p.m.) ± standard deviation (SD).

Preparation of the histidine decarboxylase (HDC) probeA probe made from a reverse-transcribed rat brain poly(A)+mRNA was used. Total cellular RNA was extracted from NewEngland Deaconess Hospital (Boston, MA) rat brain. Poly(A)mRNA was purified by one-step chromatography on an oligo-dT column. A sample of 2 mg poly(A)+ mRNA was reversetranscribed at 420 for 40 min in a 20-ml mixture containing 4 mlof 5 x reverse transcriptase buffer (250mm Tris-HCl, pH 8-3 at420, 50mM MgCl2, 250mm KCl, 15mm DTT, 10U placentalRNase inhibitor, 0 5mm each dNTP, 50 pM oligo-dT primerand 20U avian myeloblastosis virus reverse transcriptase).After reverse transcription, the HDC cDNA was amplified bypolymerase chain reaction (PCR) using two specific primerssynthesized on a gene assembler plus (Pharmacia, Uppsala,Sweden): 5' primer, 5'-ATGATGG-AGCCCAGTGAATACCand 3' primer, 5'-CCAGAATTCGCATGT-CTGAGG-TAG.Four millilitres of single-stranded cDNA mixture was supple-mented with 50pM of each 5' and 3' primers in a volume of50 ml denatured for 2 min in a boiling bath and added to a 50 mlmix prewarmed at 720 containing 0-25 mm each dNTP, 10 ml10x Taq polymerase buffer (500mm KC1, 100mm Tris-HCl,pH 8-3 at 25°, 15mM MgCl2 and 0-1% gelatin) and 1-5 U ofTaqpolymerase (Perkin-Elmer Cetus, Branchberg, NJ). The PCR

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programme consisted of one cycle of 1 min at 940 and 15 min at72', followed by 35 cycles of 30 seconds at 940, 30 seconds at 550and 4 min at 720, and was completed by an additional annealingat 550 for 30 seconds and a final elongation at 72° for 15min.PCR was performed in a Techne PHC-2 programmable heatingblock. Amplified products were purified by glass-milk proce-dure (Geneclean BIO 101, Boston, MA), blunt-ended with theKlenow fragment and cut by EcoRI. The resulting blunt end-EcoRI fragments were cloned in the p-MAL vector (Biolabs,Boston, MA) cut by both StuI and EcoRI restriction enzymes.The resulting recombinant plasmid was amplified in the TB 1Escherichia coli strain. Plasmid DNA was sequenced by thedouble-stranded protocol of the sequenase kit (USB Cleveland,OH). Plasmid-containing amplified HDC cDNA was preparedaccording to the alkaline lysis method and purified on a CL4Bcolumn.

Northern blot analysisTotal RNA was isolated by guanidine hydrochloride asdescribed previously.'7"18 Total RNA (10 mg/lane) was frac-tioned by electrophoresis on a 2% agarose gel, transferred tonylon membranes (Hybond N; Amersham Int., Amersham,UK) and hybridized with 32p (2 x 108 c.p.m./mg). It was thenwashed four times at room temperature for 15 min in 2 x salinesodium citrate (SSC) and 0-1% sodium dodecyl sulphate (SDS),heated to 48° for 30 min, then washed twice in 0 1 x SSC and0-1% SDS. Membranes were finally exposed to Kodak XAR5for 3 days at - 70°. Signals were compared with ribosomalRNA to evaluate an equal quantity of RNA for each lane.

Electron microscopy studies ofRPMCElectron microscopy preparation and examination on RPMCwas performed as described previously.'9 Briefly, after culture,the cells were washed at 40 with a solution containing 10mMHEPES, 135 mm NaCl, 5mM KCl, mg/ml BSA, pH 70, andfixed for 1 hr at room temperature with 3% glutaraldehyde in0-1 M cacodylate-HCl buffer (pH 7 2). At the end of thisfixation period, cells were scraped and pelleted at 550g in aSorval Clinical Table Top centrifuge (Boston, MA).

Cells were then washed twice with cacodylate buffer. Theywere then post-fixed with 1% OS04 in cacodylate buffer for 1 hrat room temperature, washed with cacodylate buffer andpelleted. The pellets were then infiltrated with two 10-minchanges of 100% propylene oxide followed by overnightexposure to a 1: 1 mixture of propylene oxide and DMP-30.The next day cells were embedded in Epon (Fluka, Switzerland)with DMP-30. Embedded cells were placed in a 56° oven topolymerize for 48.hr.

Thick and thin sections were cut on Sorvall MT-I and MT-2B Ultramicrotomes (Boston, MA) equipped with glass anddiamond knives, respectively. Sections (1000 A) were picked upon 300-mesh copper grids and stained with both uranium andlead salts. The sections were then examined and photographedusing a JEOL JEM-100 s transmission electron microscope(Philips, the Netherlands) operated at an accelerating voltageof 80 kV.

Stainingfor light microscopyThe cells were stained with 0 25% toluidine blue, pH 2-5, for5 min. Peritoneal mast cells were observed in a high power field(x 10, x20, x40, x 100). Some samples were photographed and

all observations were performed using a Nikon Diaphote-THDmicroscope.

Determination of cell viabilityCell viability was evaluated by measuring lactic dehydrogenase(LDH) release spectrophotometrically20 and by trypan blueexclusion, using a 0 1% trypan blue solution. Viability isexpressed as the percentage of cells that did not take up trypanblue.

Statistical analysesData from different experiments were combined and reportedas the mean ± SD. Student's t-test for independent means wasused to provide a statistical analyses (P > 0 05 was consideredas not significant).

RESULTS

MCP-1 causes 13HI5HT and histamine release from RPMC

MCP-1 has been presented as a potent histamine-releasingfactor for basophils.6'7 To evaluate this effect more fully, wecultured RPMC in the presence of MCP-1 (200 nM) for 0, 5, 10,20 and 40 min at 370 in a shaking water bath (Fig. 1). Theaddition of MCP-1 produced a marked release of [3H]5HT andhistamine, which was maximal between 40 and 80 min(56 6 ± 5 3% and 34-7 + 6 0%, respectively).

The effect of doses of MCP-1 on [3H]5HT and histaminerelease from RPMC was evaluated at molar concentrations of200, 100, 50, 25, 12 5, 6 5 and 3 12nM after 40min incubationtime (Table 1). A dose-dependent release was seen and themaximal effect was obtained with 200 nM MCP-l: 62 35 ± 12 4[3H]5HT and 42 8 ± 8 1 histamine. Doses higher than 200 nmdid not prove to be more effective.

Table 2 records the percentage total histamine and[3H]5HT release from RPMC cultured for 10min with C48/80 (0-2-05j5g/ml), or for 40min with MCP-l (200-6-25nM)with C48/80 (0 05 jig/ml) added for the last 10 min.

All concentrations of C48/80 provoked histamine release ina dose-dependent manner. In order to evaluate the primingeffect of MCP- 1, we used suboptimal concentrations of C48/80(0 05 jg/ml) in combination with scaled doses of MCP-1. Nosignificant increase of histamine and [3H]5HT release was

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Figure 1. The kinetics of MCP- 1 (200 rlM)-induced [3H]5HT andhistamine release from RPMC (4 x 105cells/ml). The values ± SD arerepresentative of three experiments in triplicate cultures incubated at370.

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MCPI-provokes most cell aggregation and [3H]5HT release

Table 1. Percentage histamine release after treatment with MCP-l

Treatment [3H]5HT Histamine P <

MCP-l 200nM 62-35 + 12 4 42-8 ± 8-1 0 01MCP-1 lOOnM 477 +10-7 3582 ± 93 0.01MCP-1 50nM 37-4 10-0 3375 ± 89 0.05MCP-1 25nM 30 3 i 115 25-1 + 7-8 0 05MCP-1 1250nM 215 ±112 19-4 75 005MCP-1 625nM 90 + 8-5 102 + 89 NSMCP-1 3 12 nM 7-3 ± 7-0 5-5 ± 5 3 NS

Percentage release of [3H]5HT and histamine (± SD), compared tothe control, by RPMC (4 x 105 cells/ml) following the addition ofincreasing concentrations of MCP-1 for 40 min.

P-values were calculated comparing treated cells with untreatedcells.

NS, not significant.

found when the cells were pretreated with MCP-1 (625-200 nM) in combination with C48/80 (005 Mg/ml), comparedwith C48/80 (0 05 pg/ml) alone.

In humans there is evidence that IgE antibodies have theability to mediate immediate-type hypersensitivity reactions.IgE antibodies exhibit a capacity to bind with high affinity toreceptors on basophils and mast cells, and elicit mediatorrelease. In these studies we evaluated the effect of 40 minincubation with MCP-1 (200-50nM) on the release of totalhistamine and [3H]5HT release from RPMC in response toanti-IgE antibodies (Fig. 2). Exposure of cells to MCP-1 (50-200nM) plus anti-IgE (1:1000 dilution) caused no significantincrease in release of [3H]5HT and histamine above thatobserved from RPMC treated with anti-IgE alone.

Stimulation of HDC mRNA levels by MCP-1 in RPMC

A probe was prepared to detect mRNA encoding the HDCgene. Oligonucleotide primers specific for HDC sequences

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* Histamine* [3H]5HT

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Figure 2. [3H]5HT and total histamine release by RPMC (4 x 105 cells/ml) + SD following the addition of increasing concentrations ofMCP- 1 (50-200 nM) for 40 min and after, anti-IgE for 10 min. Student'st-tests were performed and all the MCP-1 data compared with thecontrol were not significant (P > 0-05). Cell viability was evaluated bymeasuring lactic dehydrogenase release spectrophotometrically'5 andby trypan blue exclusion, using 0 1% trypan blue solution. (a) Control;(b) anti-IgE; (c) MCP-l (200 nM) + anti-IgE; (d) MCP-l (100mM) +anti-IgE; (e) MCP- 1 (50 nM) + anti-IgE.

were used to amplify a product from rat brain cDNA, andthis yielded a 1019-bp fragment of DNA following digestionwith EcoRI restriction enzyme. After cloning into thep-Mal plasmid, this was used to detect HDC mRNA byNorthern blot hybridization.'7 Since MCP-1 has been pre-sented as a stimulator of histamine synthesis, in this study wecultured RPMC in the presence of either MCP-1 (200-25 nM)or anti-IgE (1: 1000 dilution). Northern blot analysis was usedto determine HDC message. After 4 hr in culture, steady-statelevels of HDC mRNA in control unstimulated RPMC werelow (Fig. 3, lane 1). In the presence of MCP-1, HDC mRNAwas induced in a dose-dependent manner compared to thecontrols. HDC mRNA reached maximum levels when the cellswere treated with anti-IgE (1:1000) (positive control) or with200nm MCP-1 (lanes 6 and 5, respectively). When the cells

Table 2. Percentage histamine and [3H]5HT release after treatment with C48/80 + MCP-1

Histamine releaseTreatment (% of total histamine) P < [3H]5HT P <

Control 9-8 ± 5 4 4-2 ± 1-6C48/80 0-2 pg/ml 39 4 + 5-1 43-6 + 7 0C48/80 01lug/ml 23-0 ± 4-4 38-5 ± 4-1C48/80 005 pg/ml 211 ± 3-2 * 201 ±33 *MCP-1 200 + C48/80 005 ug/ml 23-8 + 4 0 NS 22-0 ± 4 0 NSMCP-l 100 + C48/80 0 05 pg/ml 25-5 ± 3-7 NS 23 9 ± 3 0 NSMCP- 1 50 + C48/80 005 pg/ml 24-8 ± 3-5 NS 21 8 ± 2 5 NSMCP-1 25 + C48/80 005 pg/ml 22 5 ± 38 NS 20 7 ± 4-3 NSMCP-1 125 + C48/80 005 pg/ml 216 ±2-6 NS 20-3 38 NSMCP-1 6 25 + C48/80 0-05 pg/ml 20 7 3-0 NS 21 2 ± 2-6 NS

Percentage release of total histamine and [3H]5HT by RPMC (4 x 105 cell/ml) ± SD, following theaddition of increasing concentrations of C48/80 (0-2-0 05 pg/ml) for 10 min, or MCP-1 (200-6-25 nM)for 40 min and then C48/80 005 pg/ml for 10 min.

P-values were calculated comparing C48/80 (0 05 ug/ml) treated cells * with MCP-1 plus C48/80-treated cells.

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P. Conti et al.

1 2 3 4 5 6

U U SU" "* (a)

'Win (b)

Figure 3. Effect of MCP-1 (25-200nM) and anti-IgE on histidinedecarboxylase mRNA expression in normal rat peritoneal mast cells,RPMC were exposed to MCP-1 for 4hr and then Northern blotanalysis was determined for histidine decarboxylase mRNA. In lane 1the cells were not treated and received only the medium at the samevolume as the treated cells (control); lane 2 to lane 5 with MCP-1 (25-200nM); and lane 6 the cells were treated with anti-IgE (positivecontrol). This is a representative experiment of four. Cell viability wasevaluated by measuring lactic dehydrogenase release spectrophoto-metrically'5 and by trypan blue exclusion, using 0 1% trypan bluesolution. Lane 1, control; lane 2, MCP-1 25nM; lane 3, MCP-1 50nM;lane 4, MCP-l lOOnM; lane 5, MCP-1 200nM; lane 6, anti-IgE (1:1000dilution).

were treated with 100 nm MCP- 1, there was a small increase inHDC mRNA (lane 4), while the addition of MCP-1 at 50 and25 nm had minimal effects (lanes 3 and 2) compared to thecontrol.

Activation of RPMC by IL-3 in combination with MCP-1

It has been reported that IL-3, in combination with othercompounds, can influence, histamine release.'2"3,21,22 Inanother set of experiments, we studied the effect of IL-3 incombination with MCP-1 (Table 3). Release of histamine fromRPMC incubated for 40 min with MCP-1 in combination withIL-3 (20 ng/ml) was significantly greater (P < 0 05) than theeffect of MCP-1 (200 and 100 nM) alone. The release obtainedwhen the cells were incubated with MCP- 1 (50 nM) incombination with IL-3 (20 ng/ml) was not significantly different

Table 3. Percentage histamine release after treatment withIL-3 + MCP-l

% release P <

IL-3 20ng/ml 12 2 ± 6-9MCP-1 200nM 33-0 ± 37 *MCP-1 100nM 309 34 **MCP-1 50nM 20 7 + 2-2IL-3 20ng/ml + MCP-l 200nM 48 0 ± 3 5 0-05IL-3 20ng/ml + MCP-1 lOOnM 40 2 + 4 1 0 05IL-3 20ng/ml + MCP-1 50nM 24-4 + 3 0 NS

Percentage release ± SD of histamine, compared tocontrol values, following the addition of IL-3 (20 ng/ml) for40 min or increasing concentrations of MCP- 1 (50-200 nM)alone and in combination with IL-3 (20 ng/ml) for 40 min.The data are representative of four experiments in triplicateand the values are expressed as the mean ± SD.

P-values are calculated comparing MCP-1 200 nm ,lOOnM **, and 50nM *** treated cells with IL-3 + MCP-l-treated cells, respectively.

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Figure 4. (a) Light micrographs (x20) of purified mast cells fromSprague-Dawley rats. The cells were untreated (control) and culturedfor 40 min. No clumps were found. (b) Light micrographs (x20) ofpurified mast cells from Sprague-Dawley rats. The cells were treatedwith MCP-1 (200 nM) for 40 min. Mast cells were stained with Giemsa,and over 20 different fields were examined from each condition and onerepresentative field is shown below. Great clumps were found.

from RPMC incubated with MCP- 1 (50 nM) alone. IL-3 alonecaused a little increase of histamine release.

Morphology of RPMC cultured in the presence and absence ofMCP-1

Pure RPMC (4 x 105 cells/ml) was cultured for 40min in thepresence or absence of MCP-1 (200 nM). After culture, the cellswere stained with toluidine blue and observed by lightmicroscopy (Fig. 4). In untreated cells (control) the RPMCappeared morphologically normal and there was no clumpformation (Fig. 4a). In the presence of 200nM MCP- 1, greatclumps were formed (Fig. 4b), while at lower concentrations(50-100 nM) there was a decrease in the number of clumpsobserved (data not shown).

To analyse the clumping effect better, RPMC was culturedfor 40 min in the presence of 200 nM MCP-l and the cellsobserved by electron microscopy (Fig. 5). In untreated cells theRPMC were close together and their cytoplasmic membranesdid not communicate (Fig. Sa), while in cells treated withMCP- 1 (200 nM), the RPMC stuck together and there was cyto-plasmic communication between the two mast cells (Fig. 5b).

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MCP1-provokes most cell aggregation and [3H]5HT release

Figure 5. (a) Transmission electromicrogaphy of two representativenormal peritoneal mast cells (control, nil) from Sprague-Dawley rats(magnified x 21 700). The cells were cultured for 40 min. (b) Trans-mission electromicrogaphy of two representative peritoneal mast cellsfrom Sprague-Dawley rats treated with MCP-1 (200nM) (magnifiedx61 000). The cells were cultured for 40 min.

DISCUSSION

MCP-1 is a recently cloned cytokine that causes chemotaxisand stimulation of histamine release from human baso-phils.68"2 In these studies highly purified RPMC cultureswere used to follow the behaviour of the cells during a

continuous exposure to MCP-1 at various concentrations. Wehave demonstrated that MCP- 1, a proinflammatory chemokineof the CC subfamily, is a potent histamine and [3H]5HTreleasing factor for mast cells. The inclusion of MCP-1 incultures of RPMC promoted a dose-dependent increase inHDC mRNA synthesis. The induction of HDC transcriptsoccurred within 4 hr. The stimulatory effect of MCP- 1 on HDCmRNA expression was compared with the effect of anti-IgEused as positive control. The enhancement of HDC mRNA can

be explained by de novo synthesis of this enzyme induced byMCP-l and anti-IgE. This effect was abolished in the presenceof cycloheximide or actinomycin D (data not shown), whichinhibit, respectively, protein synthesis and mRNA transcrip-tion. As it has been reported that histamine synthesis can beinduced by other compounds, such as IL-3, granulocyte-macrophage colony-stimulating factor and prostaglandin E2,all of which are agents that increase cAMP levels,'13 it is possiblethat MCP- I also acts upon HDC activity by a cAMP-dependent intracellular mechanism.

1995 Blackwell Science Ltd, Immunology, 86, 434-440

These results extend previous observations that MCP-1 is astimulus of basophil mediator release.3 4'6'7 The effect of MCP-1 appeared to be direct and dose-dependent, acting as anagonist on RPMC. Moreover, MCP-1 (200-100nM) did nothave a priming effect on mast cells activated with C48/80 (theeffect of MCP-1 + C48/80 was not additive, probably becausethe effect ofMCP- I was masked by C48/80) or by anti-IgE. Ourdata suggest that murine IL-3 has a role in MCP-1-dependentstimulation of [3H]5HT and histamine release from RPMC.Heat-inactivated murine IL-3 was used as a control and had noeffect (data not shown). The magnitude of [3H]5HT andhistamine release by MCP-l can be augmented if the cells areprimed with IL-3. The mechanism by which IL-3 interacts oncell targets for activation and proliferation is still unknown.However, there are at least two mechanisms involved in IL-3cell mediation. One involves a ligand-receptor-mediatedmechanism, based on the ability of the cell target to absorbIL-3 activity; a second non-specific mechanism does not involveligand-receptor interactions, and is based on the lack ofabsorption of IL-3 activity, which is associated with ATPconcentrations and provokes a rapid and transient redistribu-tion of protein kinase C from the cytosol to the plasmamembrane, an effect similar to the phorbol myristate acetate(PMA).23 Taken together, the above observations demonstratethat, even if there is only low amino acid identity between themurine and rat IL-3 proteins, the murine IL-3 might be activeon mast cells in accordance with the second mechanism.

As reported in the literature,24 cells pretreated with IL-3(20 ng/ml) in combination with MCP- 1 (200 or 100 nM) showedincreased mediator release compared to the effect of MCP-1alone. However, MCP- 1 at 50 nm had no effect. The reason whythe effects of recombinant MCP-1 worked only at higher(200nm-100nM) concentrations is not known. However, itcould depend on the source. Purified MCP- 1 from somelaboratories are more active at lower concentrations ( < 100 nM)(J. J. Oppenheim, personal communication).

In aggregation experiments21'25 we have provided newevidence that MCP-1 is important in homotypic adhesion ofmast cells. Increased aggregation of mast cells is usuallyassociated with increased cellular motility, adherence andchemotaxis, characteristics which have been recognized inmany pathological conditions involving inflammation andimmune reactions in vivo. The involvement of MCP- 1 inmast cell aggregation indicates that this cytokine may play arole in augmenting various inflammatory responses of baso-phils and mast cells. The mechanism by which MCP-l inducescell aggregation is not known. However, the effect may bedue to the expression of adhesion-promoting glycoproteinson the surface of the mast cells.26 The dose-dependent activityof MCP-1 on mast cells leads us to hypothesize a unique modeof action, such as a novel mast cell activation signaltransduction pathway that may be different from those ofother cytokines.

Histamine production itself seems to be markedly influ-enced by the cytokine network, acting both as a modulator ofcytokine-receptor interactions and as a target of cytokineaction.22'24'27 The production of MCP-1 by macrophagesrepresents an important pathogenetic interaction betweenthese cells and connective tissue mast cells in a wide varietyof immunological disorders.28'29 The proinflammatory effectsof MCP-l on mast cell histamine and [3H]5HT release, and cell

439

440 P. Conti et al.

aggregation responses in vitro, suggest a complex role played byMCP-l in many in vivo pathological states involving leucocyteinfiltration. Our data are in accordance with others,6"2'30 whofound that MCP-1 is a releasing factor for basophils. However,since we used recombinant MCP-l instead of purified MCP-1,our active concentrations were higher than the concentrationsused by the other authors.6,7,30-32

Further elucidation of the role of MCP-1 on the specificityof stimulation of the inflammatory response in vitro and in vivois needed.

ACKNOWLEDGMENTS

We wish to thank Harry W. Maskell, Chief Medical Photographer,Tufts University, Boston, MA for the realization of the photosprovided for this manuscript.

These studies were supported by a grant (1993) from the Ministry ofUniversity, Scientific and Technological Research, Italy.

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© 1995 Blackwell Science Ltd, Immunology, 86, 434-440