Regulation of High-affinity IgE Receptor-mediated Mast Cell Activationby Murine Low-affinity IgG ReceptorsMarc Daeron, Odile Malbec, Sylvain Latour, Michel Arock,* and Wolf H. FridmanLaboratoire d'Immunologie Cellulaire et Clinique, INSERMU255, Institut Curie, Paris;and *Laboratoire d'Hematologie, Faculte de Pharmacie de l'Universite' Paris V

Abstract

Allergic symptoms result from the release of granular andlipidic mediators and of cytokines by inflammatory cells.The whole process is initiated by the aggregation of mastcell and basophil high-affinity IgE receptors (FceRI) by IgEand antigen. We report here that IgE-induced release ofmediator and cytokine can be inhibited by cross-linkingFceRI to low-affinity IgG receptors (FcyRH) which are con-stitutively expressed on mast cells and basophils. Using amodel of stable transfectants in RBL-2H3 cells expressingendogeneous rat FceRI and recombinant murine FcVyRH,we showed that inhibition requires that FcERI be cross-linked to FcyRII by the same multivalent ligand. Inhibitionof cross-linked receptors left non-cross-linked FceRI capa-ble of triggering mediator release and was reversible upondisengagement. Both isoforms of wild-type FcyRll wereequally capable of inhibiting FceRI-mediated mast cell acti-vation provided they had an intact intracytoplasmic domain.Our results demonstrate that mast cell secretory responsestriggered by high-affinity receptors for IgE may be con-trolled by low-affinity receptors for IgG. This regulation ofFceRI-mediated mast cell activation is of potential interestin mast cell physiology and in allergic pathology. (J. Clin.Invest. 1995. 95:577-585.) Key words: Fcy receptors II FcLreceptors I * immune complexes * mediators and cytokines.allergy

Introduction

Allergic manifestations are initiated by the aggregation of high-affinity IgE receptors (FceRI)' expressed on mast cells andbasophils, when FceRI-bound IgE antibodies bind multivalentallergens (1). FceRI are multichain-receptors composed of an

Address correspondence to Dr. Marc Daeron, Laboratoire d'Immunolo-gie Cellulaire & Clinique, INSERMU.255, Institut Curie, 26, rue d'Ulm,75005 Paris, France. Phone: 1-4432-4220; FAX: 1-4051-0420.

Receivedfor publication 19 May 1994 and in revisedform 23 August1994.

1. Abbreviations used in this paper: BCR, B cell receptor; BMMC,bone marrow-derived mast cells; FceRI. high-affinity receptors for theFc portion of IgE; FcyRII and III, murine type H and III low-affinityreceptors for the Fc portion of IgG; IC, intracytoplasmic; MAR, mouseanti-rat immunoglobulins; RAM, rat anti-mouse immunoglobulins; rIgE,rat IgE.

IgE-binding a subunit, associated with a four-transmembranedomain /3 subunit and a homodimeric y subunit (2). A consen-sus tyrosine-containing activation motif, in intracytoplasmic(IC) domains of FceRI/3 and y (3), is thought to account forthe cell-activating properties of FceRI (4).

Mast cells (5, 6) and basophils (7, 8) also express low-affinity IgG receptors (FcyR). Depending on their subtype,mouse mast cells express FcyRII and FcyRIII in variable pro-portions (6). FcyRIII are multichain-receptors composed ofa IgG-binding a subunit (9), associated in mast cells with thesame 3 (10) and y ( 11) subunits as FceRIa. Whenaggregatedby multivalent IgG immune complexes, FcyRIII also triggerthe release of inflammatory mediators (12) and cytokines(13). Under the same conditions, FcyRII do not (12, 13).FcyRII are single-chain receptors which exist as two isoformsgenerated by alternative splicing of sequences of the first ICexon, FcyRIIbl and FcyRIIb2, which differ by a 47-aminoacid insertion in the IC domain of FcyRIIbl (9). Humanbasophils express FcyRII (8), but no FcyRIII, and they failedto release histamine when challenged with anti-FcyRII (C.Lawrence and M. Daeron, unpublished data). Ancient experi-ments aiming at demonstrating the ability of IgG immune com-plexes to trigger histamine release in human basophils werecontradictory (14, 15). Human FcyRII are of two types:FcyRIIA/C and FcyRIIB. FcyRIIA/C are single-chain recep-tors the IC domain of which possesses a tyrosine-containingactivation motif (9) with cell-triggering capability (16).FcyRIIB resemble closely murine FcyRII and they share acommon IC aminoacid sequence (9).

Evidence that FcR aggregation is a necessary and sufficientsignal for triggering a cellular response (17-20) was based onstudies using multivalent ligands specific of a single FcR. Mostcells, however, express several types of FcR and antibodies ofmore than one isotype are usually produced together against agiven antigen. Whenantigen reaches FcERI-bound IgE antibod-ies on mast cells, it is therefore probably complexed to IgGantibodies, if IgG antibodies were generated against the sameantigen. This is likely to occur during immunotherapy in allergicpatients, when high titers of anti-allergen IgG antibodies havebeen raised (21), but also during a normal immune response.Under these conditions, FceRI are therefore not only aggregated,they are also cross-linked to adjacent FcyR. In the present work,we examined the consequences of cross-linking FceRI toFcyRII on IgE-induced mast cell activation.

We found that, when cross-linked to FceRI, both FcyRIIisoforms inhibit mediator and cytokine release triggered byFceRI aggregation and that inhibition depends on FcyRII intra-cytoplasmic sequences common to FcyRIIbl and b2. Weshowed that inhibition requires FceRI-FcyRII cross-linking, af-fects only crosslinked FceRI and is reversible. Our results pro-vide a new hypothesis for the development of allergic manifes-tations and offer a possible mechanism for desensitization in

Regulation of IgE-dependent Mast Cell Activation by IgG Receptors 577

J. Clin. Invest.© The American Society for Clinical Investigation, Inc.0021-9738/95/02/0577/09 $2.00Volume 95, February 1995, 577-585

allergic patients. Because mediator release could be abrogatedin mast cells previously sensitized with IgE, they suggest newtherapeutic approaches of allergic diseases.

Methods

Antibodies, antibody fragments, and antigens. The rat monoclonal IgELO-DNP-30 and IR162 were from IMEX (Universit6 de Louvain, Brux-elles, Belgium). The mouse IgE anti-DNP 2682-I mAb was a gift ofDr. Ulrich Blank (Institut Pasteur, Paris). It was purified from culturesupernatant of a subclone of DNP-Hl-e-26 hybridoma cells (22). Themouse IgE 14.205 Al mAb (23) was purified by immunoaffinity chro-matography from ascitic fluid, generously offered by Dr. Colette Kanel-lopoulos (Institut Jacques Monod, Paris). IgE-rich anti-ovalbulin serumraised in the Copacabana strain of Swiss mice was kindly provided byDr. Annie Prouvost-Danon (Fundacao Oswaldo Cruz, Rio de Janeiro,Brazil). Rat and mouse IgE bound equally well to rat and mouse mastcell FceRI. Mouse IgGI anti-DNP mAb, purified on Protein G-sepha-rose, was a gift of Dr. Jacques Couderc (Institut Curie, Paris). Unlabeledand FITC-labeled polyclonal mouse anti-rat Ig (MAR) F(ab')2 frag-ments, as well as rat anti-mouse Ig (RAM) F(ab')2 fragments werefrom Jackson Immunoresearch Laboratories (West Grove, PA). MARF(ab') 2 reacted with rat Ig, but not with mouse Ig, and RAMF(ab')2reacted with mouse Ig, but not with rat Ig. This was checked both byindirect immunofluorescence and by serotonin release on RBL cellssensitized with mouse and rat IgE respectively. The anti-FcyRII/III2.4G2 mAb (24) was purified by affinity-chromatography on ProteinG-sepharose from ascitic fluid of nude mice inoculated with 2.4G2hybridoma cells intraperitoneally. F(ab')2 fragments were obtained bypepsin digestion for 48h. The purity of 2.4G2 IgG and F(ab')2 frag-ments was assessed by SDS-PAGEanalysis. 2.4G2 reacted with mouseFcyR, but not with rat FcyR.

Heteroconjugates made by chemically crosslinking Fab fragmentsof 2.4G2 and affinity-purified rabbit polyclonal anti-DNP antibodies(referred to as [2.4G2 x a-DNP] Fab2) were described previously(25). (2.4G2 x a-DNP) Fab2 bind to FcyRII via the 2.4G2 Fab moietyand to dinitrophenyl conjugates via the anti-DNP Fab moiety. Theywere a generous gift of Dr. David M. Segal (NIH, Bethesda, MD).

Mouse IgE 14.205 Al, BSA (Sigma Chemical Co., St. Louis, MO)and ovalbumin (Sigma Chemical Co.) were dinitrophenylated using2-,4-dinitrobenzene sulfonic acid (Eastman Kodak Co., Rochester, NY).After passage over Sephadex G25 (Pharmacia, Uppsala, Sweden), theaverage substitution numbers of dinitrophenylated proteins were 7.5moles DNP/mole IgE, 13 moles DNP/mole BSA and 4 moles DNP/mole ovalbumin. Rat IgE IR162 was biotinylated using the kit fromSigma Chemical Co. Streptavidin was from Pierce (Rockford, IL), L-lysine and N-e-DNP-L-lysine from Sigma Chemical Co.

Cells. BMMCwere prepared from BALB/c bone marrow cells asdescribed (26). They were maintained in culture in RPMI medium(Seromed, Biochrom A. G., Berlin, Germany) supplemented with 10%FCS, 100 IU/ml penicillin, 100 pLg/ml streptomycin, and 50% condi-tioned medium from WEHI-3 cells. P815 mouse mastocytoma cells andL929 cells were cultured in RPMI supplemented with 10% FCS, 100IU/ml penicillin and 100,tg/ml streptomycin. RBL-2H3 cells (27) werecultured in DMEMsupplemented with 10% FCS, 100 IU/ml penicillinand 100 ,ug/ml streptomycin. Only adherent RBL cells were used. Theywere recovered with trypsin-EDTA. Except RPMI, all culture reagentswere from GIBCO BRL (Paisley, Scotland, UK).

Immunoprecipitation of iodine-labeled mast cells. BMMCor P815cells were 1251I-labeled using lactoperoxydase. Membrane FcyR wereprecipitated with 2.4G2 and analyzed by SDS-PAGE under reducingconditions, before and after endoglycosidase-F (Bohringer Biochimica,Mannheim, Germany) treatment as previously described (6).

cDNA constructions and transfectants. RBL-2H3 cells stablytransfected with cDNAs encoding murine FcyRIIbl (28) and FcyRIIb2(29), IC domain-deleted FcyRIIb2(IC1) (30), or chimeric FcyRII-ICIIIa ( 12) were described previously ( 12). All transfectants used were

cloned as described ( 12). They were maintained in culture in the pres-ence of 250 ,g/ml G418 (Geneticin; GIBCOBRL) and the expressionof recombinant receptors on clones remained stable over the durationof experiments.

Immunofluorescence. Nontransfected and transfected RBL-2H3 cellswere incubated for 1 h at 0C with 10 ,Lg/ml rat monoclonal IgE LO-DNP-30 or 2.4G2 mAbin HBSScontaining 5% FCS, or with mediumalone. Cells were washed and stained by being incubated for 30 min at0C with 50 Mig/ml FITC-labeled MARF(ab')2. Fluorescence wasanalyzed by flow cytometry using a FACScan (Becton-Dickinson,Mountain View, CA).

Serotonin release. Transfected or nontransfected RBL-2H3 cells, re-suspended in RPMI medium supplemented with 10% FCS at 1 X 106cells/ml, were incubated at 370C for 1 h. with 2 ,uCi/ml [3H] serotonin(Amersham, Les Ulis, France), washed, resuspended in RPMI-FCS,incubated for another hour at 370C, washed again, resuspended in thesame medium and distributed in 96-well microculture plates at 2 x 105cells/well and incubated for 1 h at 37TC with IgE in a final volume of50 jyd. Adherent cells were washed four times with 200 Ad HBSS(GIBCO BRL), 25 Mtl culture medium were added to each well andcells were warmed at 37°C for 15 min before challenge. Cells werechallenged for 30 min at 37°C with 25 I-L of MARF(ab')2, RAMF(ab')2, DNP-BSA or streptavidin, previously warmed at 37°C for 15min. Reactions were stopped by adding 50 Ad ice-cold medium and byplacing plates on ice. 50 Ald of supernatants were mixed with 200 pIAqualuma-Plus scintillation fluid (Lumac, The Netherlands) andcounted in a /-plate counter (Pharmacia, Uppsala, Sweden). The per-centage of serotonin release was calculated using as 100% cpm con-tained in 50 41 harvested from wells containing the same number ofcells that were lysed in 100,il of 0.5% SDS and 0.5% NP40.

BMMCwere sensitized by an overnight incubation at 37°C with IgE-rich anti-ovalbumin immune serum before being loaded with [3H]-serotonin under the same conditions as RBL cells. After washings andwarming, cells were challenged for 10 min at 37°C with DNP-ovalbuminin Eppendorf tubes.

TNF release and assay. Aliquots of 7 X 105 RBL cells, previouslysensitized by a 1-h incubation at 37°C with indicated concentrations ofIgE, were resuspended in 150 j41 culture medium and incubated for 3h at 37°C with 50 ,g/ml MARF(ab')2 fragments. Cell-free superna-tants were harvested and assayed for TNFa. TNFa was measured by acytotoxic assay on the TNFa-sensitive L929 cells as described (13).Briefly, 3 x 104 L929 cells were plated in each well of 96-well tissueculture plates. After a 20-h incubation at 37°C, culture medium wasreplaced by serial twofold dilutions of cell-free supernatants from stimu-lated cells. Supernatants were diluted in complete medium containing1.5 ,ug/ml actinomycin D (Boehringer). After a 20-h incubation at 37°C,medium was removed, adherent cells were stained with 0.2% crystal-violet (Sigma Chemical Co.) in 1% ethanol for 15 min, wells werewashed, dried, and the absorbance was measured at 570 nm using aTiterteck spectrophotometer (Labsystems, Helsinki, Finland).

Results

Inhibition of IgE-induced secretory responses by complexationof antigen to IgG antibodies in FcyRII-expressing mast cells.Bone marrow-derived mast cells (BMMC), sensitized withpolyclonal IgE anti-ovalbumin (ova), released serotonin uponchallenge with DNP-ova (Fig. 1 a). Nonsensitized BMMCdidnot release serotonin when challenged with DNP-ova or withDNP-ova complexed to monoclonal IgGl anti-DNP (Fig. 1a, open circles). DNP-ova-induced IgE-dependent serotoninrelease was found to decrease progressively if DNP-ova wascomplexed to increasing concentrations of IgGI anti-DNP (Fig.1 a, closed symbols). Inhibition was also observed when DNP-ova was complexed with dilutions of ascitic fluid of the sameIgGI anti-DNP hybridoma (not shown). The same was ob-

578 Daeron et al.

P815 BMMC-+ - +

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Figure 1. Inhibition of IgE-induced bone marrow-derived mast cell(BMMC) activation by IgG immune complexes and FcyR expressedby BMMC. (a) BMMCwere sensitized or not with polyclonal mouseIgE anti-ova by an overnight incubation at 370C with IgE-rich anti-ovalbumin sernm diluted 1 / 1000 (squares) or 1/3000 (closed circles),or with culture medium only (open circles). Cells were challenged for10 min at 370C with 20 sg/ml DNP-ova, not complexed, or complexedwith indicated concentrations of purified IgGl anti-DNP mAb. Thefigure shows the percentage of total radioactivity released in superna-tants. (b) Membrane FcyR were precipitated from '25I-labeled BMMCand P815 cells with 2.4G2 and examined by SDS-PAGEunder reducingconditions, before and after deglycosylation with endoglycosidase F.

served in three other experiments of the same design, on BMMCsensitized with monoclonal IgE anti-DNP and challenged withDNP-BSAcomplexed to IgG1 anti-DNP (not shown). Antigencomplexation to IgG antibodies therefore decreased IgE-medi-ated serotonin release by mouse mast cells.

Low-affinity IgG receptors expressed by BMMCwere immu-noprecipitated by the rat anti-mouse FcyRII/HIl mAb 2.4G2and examined by SDS-PAGEanalysis before and after endogly-cosidase-F treatment. Deglycosylated polypeptides with an ap-parent m.w. of 37, 32, and 28 kD were precipitated from thecontrol mouse mastocytoma cells P815. They were previouslyidentified as FcyRIIbl, FcyRIIb2, and FcyRIlla respectively(6, 31 ). Confirming our previous results (6), large amounts ofFcyRI~bl were precipitated from BMMC,but only traces ofFcyRIIb2 and Fc-yRllIa (Fig. 1 b). These results altogetherraised the possibility that the crosslinking of FcyRLIbl to FceRIby immune complexes could inhibit IgE-induced mast cell acti-vation and the subsequent secretory response.

Inhibition of IgE-induced secretory responses by cross-link-ing FceRI and FcyRII in RBL-2H3 transfectants. The latterhypothesis was tested using Rat Basophilic Leukemia cells

100 101 102 1 0 l01 102 103 104Fluorescence intensity

Figure 2. Structure and expression of wild-type and mutant murineFcyRII in RBL-2H3 cells. (a) The figure schematizes the structure ofrecombinant murine FcyRIIbl, FcyRllb2, IC domain-deleted FcyRII(ICI), or chimeric Fc-yRll-ICIfla encoded by the cDNAs transfectedinto RBL-2H3 cells. EC, extracellular; TM, transmembrane; and IC,intracytoplasmic domains. (b) Clones of stable transfectants were exam-ined by immunofluorescence for the expression of FceRI and murineFcyR. The binding of the rat monoclonal IgE LO-DNP-30 (open grayhistograms) and of 2.4G2 (open black histograms) to nontransfectedRBL-2H3 cells (nt) and to RBL-2H3 cells transfected with cDNAen-coding wild-type FcyRIIbl and Fc-yRIIb2, deleted Fc-yRll(ICl) orchimeric FcyRll-ICIIa was examined by indirect immunofluorescencewith FITC-labeled (MAR) F(ab')2. Shaded histograms represent thefluorescence of cells incubated with FITC-MAR F(ab')2 only.

(RBL-2H3) stably transfected with cDNAs encoding wild-type,deleted or chimeric murine FcyRII (Fig. 2 a). The surfaceexpression of murine FcyRlI and of endogenous FceRI wasassessed by indirect immunofluorescence with 2.4G2 and withrat monoclonal IgE respectively (Fig. 2 b). The expression ofFcyRII was checked again, systematically, in all subsequentexperiments. When sensitized with rat IgE, all transfectantsreleased serotonin upon challenge with MARF(ab')2 (Fig.3). When preincubated with 2.4G2 F(ab')2 fragments, notransfectant released serotonin above background upon chal-lenge with MARF(ab')2 (Fig. 3, open circles). The effect ofcross-linking FcyRII to FceRI was searched for by studyingserotonin release triggered by MARF(ab') 2 in RBL transfec-tants sensitized with rat IgE, and incubated with increasingconcentrations of F(ab')2 fragments of the rat mAb 2.4G2(Fig. 3, closed symbols). IgE-induced serotonin release wasinhibited by crosslinking FceRI to FcyRIIbl (Fig. 3 a) orFcyRIIb2 (Fig. 3 b), but not to FcyRII deleted of all butone intracytoplasmic aminoacids (FcyRII [IC 1], Fig. 3 c) or tochimeric receptors having the extracellular and transmembrane

Regulation of IgE-dependent Mast Cell Activation by IgG Receptors 579

Endo FkD

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Figure 3. Inhibition of IgE-inducedserotonin release by wild-type FcyRllin RBL transfectants. RBL transfec-tants expressing wild-type FcyRIIbl(a) or FcyRllb2 (b), deleted FcyRll-(ICI) (c) or chimeric FcyRll-ICHla(d) were incubated for 1 h at 37TCwith 0 (open circles), 0.1 (closed cir-cles), or 0.3 (squares) ug/ml rat IgELO-DNP-30 and with indicated con-centrations of 2.4G2 F(ab')2. Afterbeing washed, cells were challengedfor 30 min at 37TC with 50 pg/mlMARF(ab')2, and serotonin re-leased in supemnatants was measured.

domains of Fc-yRII and the IC domain of FcyRIIIa (FcyRII-ICHla, Fig. 3 d). The same results were obtained in at leastthree experiments for each transfectant. They were repeatedseveral times, with comparable results, in FcyRIIbl-,Fc-yRIIb2-, and Fc-yRII(ICl)-expressing cells obtained in atleast one other independent transfection. Inhibition dependstherefore on specific IC sequences common to FcyRIIbl andFcyRllb2.

FcyRIIb2-expressing transfectants, sensitized with rat IgEand challenged with MARF(ab')2 for 3 h at 37°C, producedTNFa that could be quantitated by assaying cytotoxicity of cell-free supernatants on L929 cells. Cytotoxicity depended on theconcentration of IgE used for sensitization (Fig. 4, open cir-cles). It was markedly reduced if cells were incubated with2.4G2 F(ab')2 before challenge with MARF(ab')2 (Fig. 4,closed symbols). Like inhibition of serotonin release, inhibitionof TNF production was proportional to the dilution of IgE usedfor sensitization. IgE-induced mediator release and cytokineproduction were therefore both inhibited by crosslinking FceRIto FcyRII.

Inhibition of IgE-induced mediator release requires FceRI-FcyRII cross-linking. In order to determine whether the de-creased serotonin release might result from a change in thekinetics of the release process, FcyRIIb2-expressing cells were

sensitized with rat IgE in the presence or absence of 2.4G2

rat IgE 0.3 lg/mI100

80

6000

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F(ab')2 and challenged with MARF(ab')2 for 15, 30, 45, or

60 min. Inhibition of serotonin release was of the same magni-tude whatever the duration of challenge, when cells had beenpreincubated with 2.4G2 F(ab' )2 (data not shown). The cross-

linking of FceRI to FcyRHI therefore did not slow down secre-

tion but indeed decreased the amount of mediator released.In order to determine whether FcyRII occupancy would af-

fect IgE-induced serotonin release, FcyRIIb2-expressingtransfectants were incubated with or without 2.4G2 F(ab')2,sensitized with mouse IgE anti-DNP, and challenged with DNP-BSA. Serotonin release was of the same magnitude in cellspreincubated or not with 2.4G2 F(ab')2 (data not shown).Ligand binding to FcyRII (binding of 2.4G2 was checked byindirect immunofluorescence) therefore had no inhibitory ef-fect. To determine whether an independent FcyRII aggregationwould affect IgE-induced serotonin release, FcyRIIb2-express-ing transfectants were incubated with 2.4G2 F(ab ' )2 and sensi-tized with mouse IgE anti-DNP. They were washed, preincu-bated for various periods of time at 370C with MARF(ab ')2,and finally challenged with DNP-BSA (Fig. 5). Compared tocells not preincubated with MARF(ab')2 (open symbols),serotonin release induced by DNP-BSA was unaffected in the

presence of MARF (ab' ) 2, whatever the duration of the prein-cubation with MARF(ab')2 (closed symbols). The aggrega-tion of FcyRH therefore had no effect on IgE-induced serotonin

rat IgE 1 yg/ml100

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Figure 4. Inhibition of IgE-induced TNFa production by RBL transfectants expressing FcyRIIb2. FcyRllb2-expressing transfectants were sensitizedwith 0.3, 1, or 3 Ag/ml rat IgE LO-DNP-30, in the presence of 0 (open circles), 1 (closed squares), or 10 (closed triangles) Ag/ml 2.4G2 F(ab')2.They were challenged with 50 ,g/ml MARF(ab')2 for 3 h at 37°C. Cell-free supernatants were harvested and twofold dilutions were tested forcytotoxicity on L929 cells. The figure represents the percentage of cytotoxicity as a function of the dilution of supernatants.

580 Daeron et al.

Fc)RIIbl40r a

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50 A -- requirement for50 FcyRMb2-mediated inhi-g40 ° bition of IgE-induced

2.1 serotonin release.*C 30 - FcyRIIb2-expressing

2 20 transfectants were sensi-tized with 0 (circles),

810 0.03 (squares), or 0.1o * * * * (triangles) Ag/ml mouse

0 L IgE anti-DNP 2682-I in0 0 10 20 30 the presence of 3 jg/ml

Duration of preincubation 2 4G2 F(ab ' )2. Cellswith MARF(ab'J2 (mmn)

were then preincubatedwith 0 (open symbols) or 50 (closed symbols) jig/ml MARF(ab')2for indicated times at 370C before they were washed and challengedwith 3 jig/ml DNP-BSA for 30 min.

release, even if achieved 30 min before challenge with antigen.No effect on IgE-induced serotonin release was seen followingan independent FcyRII aggregation in two other experiments.

In above experiments, FceRI were cross-linked to FcyRII bythe same reagent that was used also for challenge (DNP-BSA orMARF [ ab' ] 2, depending on the IgE). Under these conditions,FceRI-FcyRII cross-linking and FceRI aggregation were simul-taneous. To dissociate the two events, FcyRIIb2-expressingtransfectants were sensitized with a dinitrophenylated mousemonoclonal IgE, not directed against DNP, 14.205 Al, (DNP-mIgE) that could be subsequently cross-linked to FcyRII withheteroaggregates made of 2.4G2 Fab fragments and rabbit anti-DNPFab fragments (referred to as [2.4G2 X a-DNP] Fab2),without being aggregated. Cells were then challenged with ratanti-mouse Ig (RAM) F(ab')2 to aggregate FceRI. Serotoninrelease induced by RAMF (ab' ) 2 was abolished, whatever theconcentration of DNP-mIgE used for sensitization, if cells werepreincubated with as little as 0.3 jig/ml (2.4G2 x a-DNP) Fab2(Fig. 6 a).

Nontransfected cells and transfectants expressing FcyRIIblor FcyRII(ICl ) were sensitized by the same DNP-mIgE, incu-bated with or without (2.4G2 x a-DNP) Fab2, and challengedwith RAMF(ab')2. When not exposed to (2.4G2 X a-DNP)Fab2, all three cells released serotonin upon challenge. Seroto-nin release was abolished if FcyRI~bl-expressing transfectants,but not FcyRII(ICI )-expressing transfectants or nontransfectedcells, were preincubated with (2.4G2 X a-DNP) Fab2. Underthe same conditions, (2.4G2 X a-DNP) Fab2 did not inhibitserotonin release by FcyRIIbl-expressing transfectants sensi-tized with the same IgE, but not dinitrophenylated (Fig. 6 b).Identical results were seen in at least three experiments of thesame design performed with each transfectant. FceRI-FcyRllcrosslinking, prior to FcERI aggregation, therefore abrogatedsubsequent serotonin release.

Inhibition reversibly affects only FceRI cross-linked toFcyRII. The possibility to dissociate FceRI-FcyRII crosslinkingfrom FcERI aggregation enabled to examine whether inhibitionwould affect only crosslinked FceRI or whether it would affectalso other FceRI. This was achieved by sensitizing mast cellswith two types of IgE which could be cross-linked to FcyRIIor aggregated independently. When sensitized with dinitrophe-nylated mouse IgE 14.205 Al (DNP-mIgE), FcyRIIb2-ex-pressing transfectants released serotonin upon challenge withRAMF(ab' )2 (Fig. 7 a, open circles), but not with streptavidin

(not shown). Serotonin release was inhibited if cells were prein-cubated with (2.4G2 X a-DNP) Fab2 (Fig. 7 a, closed circles).When sensitized with biotinylated rat IgE IR162 (Biot-rIgE),FcyRIIb2-expressing transfectants released serotonin uponchallenge with streptavidin (Fig. 7 b, open circles), but notwith RAMF(ab')2 (not shown). Serotonin release was notaffected if cells were preincubated with (2.4G2 X a-DNP) Fab2(Fig. 7 b, closed circles). When sensitized with a mixture ofDNP-mIgE and Biot-rIgE, FcyRIlb2-expressing transfectantsreleased serotonin upon challenge with either RAMF(ab') 2or streptavidin, and a dose-dependent competition between thetwo IgE used for sensitization could be seen (Fig. 7 c -f opencircles). Serotonin release induced by RAMF(ab')2 (Fig. 7c and e, closed circles), but not serotonin release induced bystreptavidin (Fig. 7 d, and f closed circles), was abolished ifcells were incubated with (2.4G2 X a-DNP) Fab2 prior tochallenge. It follows that inhibition was restricted to FcERIcrosslinked to FcyRll and left non-cross-linked FceRI fullycapable of triggering mediator release upon aggregation. Thesame conclusion could be drawn from two additional experi-ments of the same design performed with the same reagents.

Finally, we wondered whether inhibition observed uponFceRI-FcyRll cross-linking would be reversed upon cross-linking disengagement in the presence of an excess of monova-lent hapten. FcyRIIb2-expressing transfectants were sensitizedwith dinitrophenylated mouse IgE 14.205 Al (DNP-mIgE) thatwas subsequently cross-linked or not cross-linked to FcyRllb2with (2.4G2 X a-DNP) Fab2, as in above experiments. Cellswere then incubated for increasing periods of time at 37°C withan excess of DNP-lysine or, as a negative control, of lysine,before they were challenged with RAMF(ab')2. As expected,serotonin release was inhibited when cells not exposed to haptenwere incubated with (2.4G2 X a-DNP) Fab2 (Fig. 8, opencircles). DNP-lysine (black circles) had no effect on serotoninrelease induced by the aggregation of DNP-mIgE not cross-linked to FcyRHb2 (broken line), but it reduced by 50% inhibi-tion of serotonin release induced by aggregating DNP-mIgEcrosslinked to FcyRIIb2 (solid line). The same molar concen-tration of lysine (grey circles) had no effect on inhibition (solidline). Comparable results were obtained in two other experi-ments. This indicates that FcERI whose triggering ability waspreviously abolished recovered from inhibition upon cross-link-ing disengagement. Inhibition therefore is reversible and re-quires that FceRI-FcyRH cross-linking be maintained.

Discussion

Our results demonstrate that mast cell activation via high-affin-ity receptors for IgE may be controlled by low-affinity receptorsfor IgG. This control was effective whenever FceRI were cross-linked to FcyRII with an intact IC domain, irrespectively of thecross-linking ligand. It required FceRI-FcyRII cross-linking,affected only FceRI crosslinked to FcyRll and was reversibleupon cross-linking disengagement. Such a regulation might beof physiological and of clinical relevance.

Inhibition was observed in RBL transfectants, when endogen-eous rat FceRI were cross-linked to recombinant murineFcyRIIbl or to FcyRIb2. Inhibition was not due to a competi-tion between IgE and 2.4G2 for MARF(ab' )2 or to extracellu-lar steric hindrance for the following two reasons: (a) the MARF(ab')2 concentration used for crosslinking IgE and 2.4G2F(ab')2 was 25-fold higher than the concentration sufficient

Regulation of IgE-dependent Mast Cell Activation by IgG Receptors 581

RBL-FCRiib2

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40 1

30 -

20 -

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u 'I'-L-II--2-1 0 -2 -1 0

0 10 10 10 0 10 10 10

0

0-

0 10 10 10

(2.4G2 x a-DNP) Fab2 (ig/ml)

Figure 6. Inhibition of IgE-induced serotonin release by cross-linking FceRI to FcyRll before challenge. (a) FcyRllb2-expressing transfectantswere sensitized with 0 (open circles), 1 (closed circles), 3 (closed squares), or 10 (closed triangles) ,ug/ml dinitrophenylated mouse IgE 14.205Al (DNP-mIgE). After being washed, cells were incubated for 1 h at 37°C with indicated concentrations of (2.4G2 x a-DNP) Fab2, washed again,and challenged with 50 gg/ml RAMF(ab')2 for 30 min. (b) FcyRIIbl- or FcyRll(ICl)-expressing transfectants and nontransfected RBL-2H3cells were sensitized with 0 (open circles) or 3 j.g/ml IgE 14.205 Al, dinitrophenylated (DNP-mIgE, closed circles) or not (mIgE, closed squares).After being washed, cells were incubated for 1 h at 37°C with indicated concentrations of (2.4G2 x a-DNP) Fab2, washed again, and challengedwith 50 pHg/ml RAMF(ab')2 for 30 min.

for triggering an optimal serotonin release upon aggregation ofIgE, and comparable inhibitions were seen when 50, 25 or

12.5 ig/ml MARF(ab')2 were used for challenge (data notshown); (b) Inhibition was not seen in cells expressing FcyRIIdeletants lacking IC domain, which bound comparable amountsof 2.4G2 F(ab')2 fragments as wild-type FcyRII as judgedby immunofluorescence with FITC-conjugated MARF(ab')2.Steric hindrance of IC domains could neither account for inhibi-tion because it was not seen in transfectants expressing chimericreceptors having the extracellular and transmembrane domainsof FcyRII and the IC domain of FcyRIlla. Inhibition thereforedepends on specific IC sequences shared by FcyRIIbl andFczyRIIb2. Murine FcyRII have been known for long to inhibitB cell activation triggered by B cell receptor (BCR) aggrega-

tion, and IC sequences required for FcyRII to inhibit BCR-mediated B cell activation have recently been identified (32,33). Whether the same FcyRII IC sequences are responsiblefor FcyRII-mediated inhibition of IgE-induced mast cell activa-tion is currently under study. BCR, like FceRI and FcyRIII,is composed of a ligand-binding subunit, associated with twosubunits whose IC domains possess consensus tyrosine-con-

taining activation motifs similar to those of FceRIy and p (34),and it may be a general property of FcyRII to regulate tyrosine-containing activation motif-dependent cell activation.

Inhibition affected secretory responses of mast cells as differ-ent as the release of preformed mediators and the de novo

synthesis of cytokines. It is therefore susceptible to control boththe initiation of allergic reactions and the late phase reaction inwhich cytokines play a major role (35). Inhibition, howeverwas not the consequence of a general desensitization of mastcells since non-cross-linked receptors could function normallyand triggered serotonin release upon aggregation while cross-

linked receptors could not. Inhibition was not an all-or-nothingphenomenon. Experiments on FcyRIIb2-expressing transfec-tants incubated with lower concentrations of 2.4G2 F(ab')2than those used in Fig. 3 showed a clearcut dose-dependentinhibition (not shown). Inhibition required the crosslinking ofFceRI to FcyRll. MARF(ab') 2 could indeed crosslink rat IgELO-DNP-30 and rat 2.4G2 F(ab')2 inasmuch as (a) the same

MARF(ab')2 conjugated to FITC bound to both rat reagentsin indirect immunofluorescence experiments (see Fig. 2), and(b) MARF(ab')2 were functionally divalent since they trig-

582 Daeron et al.

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Figure 7. Inhibition of IgE-induced serotonin release affects only FceRI crosslinked to FcyRII. FcyRllb2-expressing transfectants were sensitizedwith dinitrophenylated mouse IgE 14.205 Al (DNP-mIgE) and/or biotinylated rat IgE IR162 (Biot-rIgE) at concentrations indicated in abscissa,and preincubated for 1 h at 370C with (closed circles) or without (open circles) 1 jig/ml (2.4G2 x a-DNP) Fab2. Cells were washed and challengedwith either 50 jig/ml RAMF(ab')2 (upper panel) or 10 tig/ml streptavidin (lower panel) for 30 min at 370C.

gered serotonin release and TNF secretion by RBL cells sensi-tized with rat IgE (this manuscript) and by transfectants ex-pressing FcyRHI preincubated with 2.4G2 F(ab')2 (12, 13).Neither FcyRLI occupancy by 2.4G2 F(ab')2 nor FcyRll ag-gregation by 2.4G2 F(ab ' )2 and MARF(ab ')2, by themselves,affected serotonin release. This was confirmed with (2.4G2 X a-DNP) Fab2 which had no effect when bound only to FcyRII (inFcyRIIb 1-expressing cells sensitized with nondinitrophenylatedIgE), or to IgE only (in nontransfected cells sensitized withDNP-IgE), but abolished serotonin release when cross-linkingIgE to FcyRll with an intact IC domain (in FcyRIIbl- andFcyRIIb2-, but not in FcyRII(ICI )-expressing cells, sensitizedwith DNP-IgE). Moreover, inhibition could be reversed whenDNP-mIgE previously cross-linked to FcyRII by (2.4G2 X a-DNP) Fab2 were disengaged from cross-linking with monova-lent DNP-Lysine. One may notice that a prolonged exposure toDNP-Lysine was required for inhibition to be reduced. Whether

this reflects a low dissociation rate of multiple interactions be-tween (2.4G2 x a-DNP) Fab2 and DNP7.5-mIgE or whetherFcERI required time to recover from cross-linking is not known.We are currently investigating the intracellular consequencesof receptor cross-linking.

Inhibition was not seen only in RBL transfectants, whenrecombinant murine FcyRII were artificially cross-linked to ratFceRI with monoclonal anti-FcyR antibodies and MARF(ab ')2 or with (2.4G2 x a-DNP) Fab2 heteroaggregates. IgE-induced serotonin release was also inhibited in nontransformedmast cells, which express constitutive FcyRII, sensitized withpolyclonal serum IgE, when receptor cross-linking wasachieved by IgG immune complexes. To rule out a possiblecompetition of antibodies for the same epitope, we used IgEand IgG antibodies of different specificities. The complexationof DNP-BSA to IgGI anti-DNP mAbalso inhibited the releaseof serotonin by BMMCsensitized with mouse monoclonal IgE

Regulation of IgE-dependent Mast Cell Activation by IgG Receptors 583

0-

0 30 60 90 120

Duration of incubation with hapten (min)

Figure 8. Reversal of in-hibition of IgE-inducedserotonin release upon

disengagement ofFceRI-FcyRII cross-

linking. FcyRIb2-ex-pressing transfectantswere sensitized with 10j.g/ml dinitrophenylatedmouse IgE 14.205 Al,washed, and preincu-bated with (solid lines)or without (broken line)1 ttg/ml (2.4G2 x a-

DNP) Fab2 for 1 h at 370C. They were washed and incubated at 370Cfor indicated periods of time with 100 /tM DNP-Lysine (black circles)or lysine (gray circles), added at various times (all cells were incubatedfor a total of 2 h). Cells were washed and challenged with 50 jsg/mlRAMF(ab')2 for 30 min at 370C.

anti-DNP, as well as the production of TNFa by the murinemastocytoma cells MC9sensitized with IgE anti-DNP (data notshown). Under these conditions, competition between IgG andIgE antibodies for the same epitopes cannot be excluded. Aninhibitory effect of FcyRII, rather than a competition for antigenor an extracellular steric hindrance, however, was strongly sug-

gested by the finding that inhibition of the TNF response was

abolished if MC9 cells were preincubated with with 2.4G2F(ab') 2 before challenge with immune complexes (data notshown). 2.4G2 F(ab')2, indeed, block the Fc-binding site ofFcyRII (24). Immune complex-induced inhibition mimicsphysiologic conditions, when IgG antibodies directed againstidentical or different epitopes of the same antigen as IgE anti-bodies interact with FcyRll by their Fc portions, while antigenbinds FceRI-bound IgE on the same cell. A reasonable hypothe-sis would be that, during the course of a normal immune re-

sponse, physiologic IgE-mediated mast cell activation mightoccur in the presence of antigen, which would be normallymaintained at low levels by IgG antibodies bound to the same

antigen, so that no sign of hypersensitivity is detectable. If so,

one can speculate that allergic manifestations might arise when,for any reason, this control is not efficient enough.

This also reminds the situation when allergic patients are

treated by the injection of increasing amounts of allergen. Suchan immunotherapy is usually successful after high titers of IgGantibodies have been induced against allergen (21), but no

satisfactory mechanism was yet proposed. HumanFcyRIIB are

the human equivalent of murine FcyRII, and they were recentlyreported to inhibit murine B cell activation (36). One mighttherefore expect that FcyRIIB could be capable of regulatingFceRI in human basophils. If so, inhibition described here mightnot only explain the success of desensitization, it might alsoexplain its relative inefficacy. When FceRI aggregation andcrosslinking to FcyRII were simultaneously produced by MARF(ab')2 reacting with FcyRII-bound 2.4G2 F(ab')2 andFceRI-bound rat IgE, or by antigen-IgG immune complexes,inhibition was partial and inversely proportional to the concen-

tration of IgE used for sensitization. Under these conditions, one

cannot prevent adjacent IgE molecules from being aggregatedwithout being cross-linked to 2.4G2, and chances of IgE aggre-

gation can be expected to increase with the density of IgE atthe cell surface.

It was recently reported that asthmatic patients injected withIgG immune complexes made with allergen and affinity-purifiedanti-allergen IgG antibodies had markedly reduced symptoms(37, 38). The mechanism of this effect was not investigatedand we propose the inhibition described here as a likely explana-tion. This suggests that manoeuvres aiming at crosslinkingFceRI to FcyRI might be successful for controlling hypersensi-tivity manifestations in allergic patients. One advantage ofFcyRll-mediated inhibition of IgE-induced mast cell activation,in terms of therapeutics, is that it can operate after mast cellswere already sensitized with IgE, as it is the case in allergicpatients. Our model where DNP-IgE are crosslinked to FcyRIIby (2.4G2 X a-DNP) Fab2, simulates such a therapeutic ap-proach. Under these conditions, the inhibitory effect was deepenough for enabling less than 1 jLg/ml (2.4G2 X a-DNP) Fab2to abolish serotonin release triggered by 50 ,ig/ml MARF(ab ' )2 in RBL cells sensitized with 10 jtg/ml IgE. The higherefficacy of bispecific heteroaggregates in inhibiting IgE-inducedserotonin release, compared with that of 2.4G2 F(ab')2 andMARF(ab' )2, may be explained because the preincubation oftransfectants with (2.4G2 x a-DNP) Fab2 enabled FceRI to becross-linked to FcyRII before FceRI-bound IgE were aggre-gated by RAMF(ab' )2. Appropriate ligands capable to repro-duce the same cross-linking in humans are currently under in-vestigation.

Acknowledgments

Weare grateful to: Drs. P. M. Hogarth (Melbourne University, Parkville,Victoria), J. V. Ravetch (Memorial Sloan Kettering Cancer Center,New York, NY) and I. Mellman (Yale University School of Medicine,New Haven, CT) for their generous gifts of cDNAs, to Dr. J. Kanello-poulos (Institut Pasteur, Paris) for RBL-2H3 cells, to Dr. D. M. Segal(NIH, Bethesda, MD) for (2.4G2 x a-DNP) Fab2, and to Drs. U. Blank(Institut Pasteur, Paris), A. Prouvost-Danon (Fundacfao Oswaldo Cruz,Rio de Janeiro), J. Couderc (Institut Curie, Paris), and C. Kanellopoulos(Institut Jacques Monod, Paris) for antibodies. We acknowledge theexpert technical assistance of Ms. Fabienne Vozy, Annie Galinha, Ma-rie-Annick Provost-Marloie and Genevieve Averlant.

This work was supported in part by the INSERM, Roussel-UCLAFand the Institut Curie.

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Regulation of IgE-dependent Mast Cell Activation by IgG Receptors 585