absence other fc receptors, fcyriiia transmits phagocytic...

In the Absence of Other Fc Receptors, FcyRIIIA Transmits a PhagocyticSignal That Requires the Cytoplasmic Domain of Its t Subunit

J.-G. Park, R. E. Isaacs, P. Chien, and A. D. SchreiberUniversity of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

Abstract

The transmembrane isoform of FcyRIII, FcyRIIIA, is found on

NKcells, cultured monocytes, and tissue macrophages in associ-ation with a dimer of an accessory subunit, either -y or t. Func-tions of individual Fc receptors have been difficult to analyzedue to coexpression of the receptors on hematopoietic cells andpermanent cell lines expressing Fc receptors. cDNAs for the a

and y subunits of FcyRIIIA were cotransfected into COS-1cells, which lack endogenous Fc receptors, to evaluate receptor-mediated phagocytosis and changes in ICa2+I,. Transfectantsboth bound and phagocytosed IgG-sensitized erythrocytes and,following activation of Fc-yRIIIA, increased ICa2+j I. The sub-unit was essential both for the surface expression of the recep-

tor and for transduction of the phagocytic signal. Truncation ofthe y subunit cytoplasmic domain (amino acids 65-80) elimi-nated phagocytic function. Phorbol ester inhibited phagocyto-sis in a concentration-dependent manner, but did not affectIgG-sensitized erythrocytes binding, suggesting that a proteinkinase C-dependent pathway inhibits phagocytosis. The dataindicate that a tyrosine containing cytoplasmic domain withinthe y subunit is required for phagocytosis by FcyRIIIA. (J.Clin. Invest. 1993. 92:1967-1973.) Key words: receptorsphagocytosis - signaling * immunoglobulin * macrophages

Introduction

Receptors for the Fc portion of IgG are expressed on many cellsof hematopoietic lineage. These receptors are important in hostdefense and in the ingestion of IgG-coated cells ( 1, 2). Thereare three classes of human Fcy receptors (FcyRI, FcyRII, andFc7yRIII), distinguished by IgG subclass affinity, cellular distri-bution, and reactivity with monoclonal antibodies ( 1). Thegenes for these receptors have homology in the coding regionsfor their extracellular domains, but are divergent in their trans-membrane and cytoplasmic regions, suggesting functional spec-ificity for the individual receptors. FcyRIII (CD1 6), a low af-finity Fc receptor, has two isoforms that are encoded by twohighly homologous genes (3, 4). FcyRIIIB is found only on

neutrophils as a glycosyl phosphatidylinositol-linked protein(5). FcyRIIIA is found on NKcells, cultured monocytes andmacrophages (6, 7). The receptor is a multichain complex com-

posed of a ligand-binding polypeptide, a, and a disulfide-linkeddimer of an accessory subunit, y or D. The y and r subunits

Address correspondence to Dr. Alan D. Schreiber, University of Penn-sylvania Cancer Center, 7 Silverstein Building, Philadelphia, PA19104.

Receivedfor publication 12 February 1992 and in revisedform 25February 1993.

were originally identified as components of other multimericreceptors, the high affinity receptor for IgE (FcERI)' and the Tcell receptor (TCR / CD33), respectively (8- 10 ).

Examination of the functions ascribed to the individualFcy receptors and their signal transduction pathways hasproven difficult due to coexpression of more than one class ofreceptor on hematopoietic cells that express Fc receptors. Fur-ther complexity is added by various isoforms of FcyRIIIA. TheFcyRIIIA-a polypeptide associates with a dimer of y-y, tR, or,ys that likely interacts with different second messenger path-ways and subserve different functions upon immune complexbinding ( 1).

A previous report indicated that the fibroblast-derivedCOS- 1 cell line possessed the cellular machinery for phagocyticcell function following Fc receptor ( 11 ) and mannose receptortransfection ( 12). Weused COS- 1 cells to evaluate the phago-cytic function of the Fc'yRIIIA complex, composed of an a anda homodimer of y. We observed that COS-1 transfectantsphagocytosed IgG-sensitized cells and that the cytoplasmic do-main of its y subunit is required. In addition, we undertookstudies with a known inducer of protein kinase C (PKC),PMA, to begin to dissect the signaling pathway involved inphagocytosis by this FcyRIIIA isoform.

Methods

Cell culture. COS- I cells were maintained in DMEM-high glucose (4.5mg/ml), glutamine (25 mg/ml), penicillin ( 100 U/ml), streptomycin(100 gg/ml) and 10% heat-inactivated FCS at 37°C with 5% CO2.Cells were seeded onto 3.5-cm plates or six-well plates (Falcon Lab-ware, Oxnard, CA) at a concentration of 3 x I05 cells/well, 24 h beforetransfection. The cells reached approximately 80% confluence fortransfection. All tissue culture reagents were obtained from the Univer-sity of Pennsylvania Tissue Culture Facility.

Construction of recombinant plasmids. The coding sequence of theFc-yRIIIA-a cDNA (kindly provided by Dr. Bice Perussia, JeffersonCollege of Medicine, Philadelphia, PA) was ligated into the SV40-driven eukaryotic expression vector pSVL (Pharmacia LKB, Piscata-way, NJ) digested with XbaI and Sacd. The cDNAfor the murine FcERIy-subunit (kindly provided by Dr. Jean-Pierre Kinet, National Insti-tutes of Health, Bethesda, MD) was expressed from the same vector.Truncation mutants of the 'y subunit were constructed by using thetwo-step overlap-extension PCR( 13). GAMdel- 1, a mutant with thetruncation at Lys8', was generated by changing the codon AAGfor Lysto TAGfor a termination. GAMdel-2, a mutant with the truncation atTyr65, was generated by changing the codon TACfor Tyr to TAGfor a

termination.Transient transfection of COS-J cells. Cotransfections of cDNAs

were carried out with a modified DEAE-dextran method. Briefly,300,000 COS-1 cells were seeded on 35-mm well plates, 24 h beforetransfection. Plates of 70-80% confluency were washed twice and incu-bated for 40 min with DMEM(GIBCO BRL, Grand Island, NY) with-

1. Abbreviations used in this paper: ADCC, antibody-dependent cellu-lar cytotoxicity; EA, IgG opsonized sheep erythrocyte; FcyR, Fc recep-tor for IgG; FcER, Fc receptor for IgE; PI, phagocytic index; PKC,protein kinase C; RBC, red blood cells.

Fc-yRIIIA Transmits a Phagocytic Signal 1967

J. Clin. Invest.©The American Society for Clinical Investigation, Inc.0021-9738/93/10/1722/07 $2.00Volume 92, October 1993, 1967-1973

out serum before transfection. 4 ,gg of plasmid DNA(0.5 ,g/,4l) was

slowly added to 1 ml of a transfection buffer containing Nu medium(DMEMwith 10% NuSerum [Collaborative Biomedical, Bedford,MA]), 1 mg/ml of DEAE-dextran and 100 juM chloroquine. Thetransfection buffer containing DNAwas added to COS-1 cells withincubation for 4 h at 370C. Cells were then shocked with 10%DMSOinPBS for 2 min, washed twice with DMEM,and grown in Nu medium.Cells were studied 48 h following transfection.

Antibodies. Anti-FcyRIII monoclonal antibody 3G8 (14) was iso-lated from ascites fluid (the 3G8 hybridoma cell line was kindly pro-vided by Dr. J. Unkeless, Mt. Sinai Medical School, NewYork, NY)using protein A (Affi-Gel Protein A Maps II; Bio-Rad Laboratories,Richmond, CA). The ascites fluid ( 1.5 ml) was applied to a 5-ml pro-tein A agarose column. The column was washed with 15 vol of bindingbuffer, and the IgG was eluted according to manufacturer's instruc-tions. The eluted protein was dialyzed against PBSand concentrated to7 mg/ml.

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Figure 1. (A) Fluorescence histograms of COS-1 cells expressingFcyRIIIA-a and -y (wild type and mutants). COS-1 cells were trans-fected with FcyRIIIA-a and -ey cDNAs and assayed 48 h later. Thedotted lines represent cells stained with an IgG isotype control P3,while the solid lines represent cells stained with anti-FcyRIIIA mAb3G8. Transfection with: (a) FcyRIIIA a and y wild type, (b)Fc'yRIIIA a and GAMdel-1, (c) Fc7RIIIA a and GAMdel-2, and (d)FcyRIIIA-a and pSVL vector DNAwithout -y insert are shown. (B)Schematic representation of y wild type and mutants. Shown with theschematic diagram of the y chain are signal sequence (S), externalpeptides (E), transmembrane domain (TM), and cytoplasmic do-main (CY). The expanded area shows an area of the nucleotide se-

quence of the y chain containing a conserved motif. The conservedamino acids are denoted by the bold face. The two thick horizontalbars shown at the bottom of the figure represent y mutants with atermination codon at respective 3' termini but 5' ends of the hori-zontal bars do not represent a termination (their 5' ends are identicalto that of wild type 'y).

Table I. FcyRIIIA Expression and Phagocytosis by COS-I CellsTransfected with FcyRIIIA-a and --y (Wild Type or Mutants)

FcyRIIIA MFI* Pit Phagocytosis Rosetting

%Cells + % Cells +

a + pSVL (Sham) 15 0 0 0.61a + y (Wild Type) 254 30±7.1 8.8±1.5 36±3.0a + y (GAMdel-l) 259 25±3.5 6.1±0.7 35±1.7a + -y (GAMdel-2) 303 0 0 37±5.2

COS-l cells (7 x 10' cells/well) transfected with Fc'yRIIIA-a and -'ysubunit cDNAs were incubated at 370C with EA for 40 min. AfterWright-Giemsa staining, phagocytosed RBCswere microscopicallyscored (x 1000). Results are expressed as the mean±SEMfor phago-cytic index, phagocytosis, and EA binding (rosetting). At least threeseparate experiments were performed for each clone. For each exper-iment, 300 cells were counted at each of three randomly selected sites.Unsensitized RBCsdid not bind to transfectants. * MFI, meanfluorescence intensity; § PI, phagocytic index (number of RBCsin-ternalized per 100 COS-l cells).

Flow cytometry. Anti-FcyRIII mAb3G8 was used to assess proteinexpression on the cell surface of transfected COS-1 cells. Cells weregently removed from plates, counted, and resuspended in 50 z1 stainingbuffer ( lx PBS without Ca2"/Mg2", containing 0.02% sodium azide,0.1% BSA) with 5 ,d of primary antibody and placed on ice for 45 min.After two washes, FITC-labeled F(ab')2 goat anti-mouse IgG (GAM)was added and incubation continued at 4°C for 30 min. Isotype con-trols were used for all reactions. Fluorescencewao measured on a FAC-Star® (Becton Dickinson &Co., Mountain View, CA). For all samples,10,000 events were recorded on a log fluorescence scale and meanfluorescence intensity data and contour maps were generated.

Electron microscopy. COS- 1 cells were removed from plates by pi-petting and washed to remove media. The pelleted specimens werefixed in 2%glutaraldehyde in 0.1 Msodium cacodylate buffer, pH 7.4.After washing three times in 0.1 Mcacodylate buffer, pH 7.4, the speci-mens were fixed in 1% OS04 for 30 min at room temperature. Follow-ing dehydration in graded alcohol, the specimens were embedded inEpon-EM-Bed-812 (Electron Microscopy Science, Fort Washington,PA). Thin sections were cut using an LKB Ultratome III microtome(Pharmacia Fine Chemicals, Piscataway, NJ). Pictures were takenwith a 300 electron microscope (Philips Electronic Instruments, Inc.,Mahwah, NJ).

Preparation ofIgG-sensitized red blood cells (EA). Sheep red bloodcells (RBC) were sensitized with rabbit anti-sheep RBCIgG as previ-ously described (14). Briefly, 109 sterile sheep RBC(Rockland Inc.,Gilbertsville, PA) suspended in 1 ml of calcium- and magnesium-freePBS was sensitized by incubation with an equal volume of PBS con-taining the highest subagglutinating titer of rabbit anti-sheep RBCan-tibody (Cappel Laboratories, Cochranville, PA) at 37°C for 30 min.The IgG-sensitized sheep RBC(EA) were washed twice with PBSandresuspended in a final concentration of 109 cells/ml for overlaying ontransfected COS-1 cells.

Binding and phagocytosis of IgG-sensitized RBCs. COS-l cellswere incubated with EA at 37°C for 45 min. Unbound EA was re-moved by extensive washing and stained with Wright-Giemsa to deter-mine the number of cells with bound EA. For the analysis of phagocyto-sis, COS- I cells bound with EA (after three washings) were subjected toa brief hypotonic shock (35 s) with hypotonic PBS to remove surface-bound EA before staining. Results were expressed as phagocytic index(PI), the number of intracellular EA/ 100 COS-1 cells. Phagocytosiswas also assessed by electron microscopy (with the assistance of Dr.Steven Douglas, Children's Hospital of Philadelphia, PA). Statisticalanalysis was carried out by one-way analysis of variance and the Stu-

1968 J.-G. Park, R. E. Isaacs, P. Chien, and A. D. Schreiber

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Effect of PMA on phagocytosis in COS-1 transfectants. PMA(Sigma Chemical Co., St. Louis, MO)was dissolved in DMSO.COS-ltransfectants were incubated with increasing concentrations of PMAfor 30 min followed by incubation with EA for 40 min. COS-1 cellswere washed extensively, extracellular EA removed, and the cells ana-lyzed for internalized EA as described above.

Measurement of [Ca2]i following FcyRIIIA activation. 48 h fol-lowing transfection with the FcyRIIIA-a and -y cDNAs, cells adherentto 2.5-cm glass cover slips were taken for single-cell measurement of[Ca2+]i following cross-linking of FcyRIIIA (15). Media were aspi-rated and replaced by HBSSwith Fura-2/AM (Molecular Probes, Inc.,Eugene, OR). Cells were incubated for 30 min at 370C, washed, andpreincubated for an additional 30 min with biotinylated anti-Fc'YRIIImAb 3G8. The cover slip was transfered to a Leidem cell chamber(Medical Systems, Greenvale, NY) and individual cells were analyzedfor a rise in [Ca2+]i following delivery by a micropipette of 50 Ag/mlstreptavidin. Fluorescence was imaged with a 100 Fluor objective and aDiaphot epifluorescence microscope (Nikon Inc., Melville, NY).Emission was collected at 520 nmby a photomultiplier tube connectedto a Johnson Foundation Spinning Wheel Fluorimeter (University ofPennsylvania Biomedical Instrumentation Group, Philadelphia, PA)and digitally analyzed by an IBM AT clone-based system (Indec Sys-tems, Sunnyvale, CA). In control experiments only streptavidin orbiotinylated mAbanti-FcyRIII was added to the above cells and mocktransfectants.

Results

Cell-surface expression of transfected Fc-yRIIIA. Expression ofFc-yRIIIA complexes 48 h after transfection was determined byflow cytometry using anti-FcyRIII mAb (Fig. 1 A). Surfaceexpression of the FcyRIIIA-a protein required interaction withthe accessory subunit y, as previously noted (16-18). Twodeletion mutants of the -y chain were constructed by introduc-ing stop codons at amino acids 80 (GAMdel-l mutant) or 65(GAMdel-2 mutant) (Fig. 1 B). The cDNAs of these two mu-tants or wild type y along with the cDNA of a were cotrans-fected into COS-1 cells and examined for cell-surface expres-sion of transfected receptor complexes. COS-1 transfectantscontaining wild type or mutant y exhibited comparable levelsof FcyRIIIA assessed by mean fluorescence intensity and thepercentage cells binding EA. Approximately 36% of the COS- Itransfectants containing wild type or mutant y avidly boundEA (Table I).

Phagocytosis and binding of EA mediated by FcyRIIIA.Weexamined COS-1 transfectants containing FcyRIIIA fortheir ability to bind and phagocytose IgG-sensitized cells. Weand others have demonstrated phagocytosis of EA in COS-1cells and other cell lines derived from fibroblasts, following Fcreceptor transfection ( 1 1, 19). In this study, we employed elec-tron microscopy to examine phagocytosis in COS-1 cells co-transfected with the FcyRIIIA a and y subunits. The thin-sec-tion electron micrograph demonstrates that COS-1 cells thatlack endogenous expression of Fc receptors are able to phagocy-tose EAwhen transfected with FcyRIIIA (Fig. 2 A). Light mi-croscopic visualization of Wright-Giemsa-stained COS- 1transfectants also revealed phagocytosis of EA. The ingestedEA have been found to be enclosed in distinct vesicles, withsome RBCs showing partial degradation. Transfectants ex-pressing both the a and y chains showed a substantial degree ofEA binding (- 36% of cells) and phagocytosis (-9% of cells)

(Table I, Fig. 2 B). Control transfectants, transfected withFc-yRIIIA-a and pSVL vector without the y chain cDNA, didnot exhibit binding of EA (< 1% of cells).

Internalization of EA was a time-dependent process andincreased significantly from 15 to 60 min of incubation. Therewas approximately a fourfold increase in the phagocytic indexat 60 min in comparison to 15 min (data not shown).

-y Chain is essentialfor phagocytic signal transduction. Al-though the surface expression of the FcyRIIIA a chain requiresthe y chain, it is not known whether -y is also involved in thesignal transduction events required for phagocytosis. To exam-ine its role in transduction of a phagocytic signal, y wild type ormutants were cotransfected into COS-l cells along withFc-yRIIIA a. The GAMdel- 1 mutant, containing the COOHterminus 7 amino acid deletion, exhibited a 16% decrease inphagocytic activity. However, the GAMdel-2 mutant with theCOOHterminus 22 amino acid deletion completely lost itsability to transduce a phagocytic signal (Table I), suggestingthat the cytoplasmic region spanning Tyr65 to Leu79 is criticalfor the phagocytic signal. These results demonstrate that the Ychain is essential not only for the surface expression of the achain but also for the phagocytic function of the receptor.

The effect of PMAon phagocytosis. Previous studies haveshown that treatment of PMAincreases FcyRIIA mediatedphagocytosis in neutrophils and HEL cells (20, 21). In thisstudy, addition of PMA (10-6_10-'0 M) progressively de-creased phagocytosis in transfectants expressing Fc7RIIIA,with more than a fivefold decrease observed at l0-7 M. Incontrast, transfectants expressing FcyRIIA exhibited an in-crease in phagocytic function at lower concentrations (10`1and I0 -9 M) followed by a modest decrease at higher concen-trations (10-8 and 10-6 M) (Table II). The effect of PMAdepended upon its presence during EA incubation and wasrapidly reversible. This effect of PMAon phagocytosis suggeststhat FcyRIIIA and FcyRIIA may mediate their phagocytic sig-nals through different pathways. PMAhad no effect on thepercentage of COS-l cells binding EA by either receptor.

[Ca2]i stimulation following cross-linking of FcyRIIIA.To assess changes in [Ca2+ JI, weperformed single-cell measure-ments. Upon cross-linking of FcyRIIIA with biotinylated anti-Fc-yRIII and streptavidin, [Ca2]i rose immediately to 2.5- to7-fold over baseline in 6 out of 24 cells randomly tested (Fig.3). However, none of 23 mock-transfected cells exhibited anincrease of [Ca2+ ] i flux when incubated with biotinylated anti-Fc-yRIII and streptavidin (data not shown). Streptavidin alone

Table IL PMAEffect on FcyR-mediated Phagocytosisin COS-J Transfectants

Control 10'-M 10-9M 1o-4M 10-7M 10'M

Fc-yRIIA 61±13 88±19 80±29 46±6 47±11 22±4Fc-yRIIIA 34±10 33±10 20±6 10±3 6±2 1±1

PMAwas added to cells 30 min before incubation with EA and waspresent during EA incubation. EA was allowed to bind to transfec-tants for 40 min before staining. Results are expressed as the meanPI±SEM, n = 5, P < 0.05. For experiments with Fc-yRIIA, 2 gg ofpSVL containing Fc-yRIIA and equal amounts of the vector DNAwithout insert were transfected into COS- cells. For FcYRIIIA, 2 Ogeach of pSVL containing cDNAs of a and y cDNAwere used for co-transfection.

FcyRIIIA Transmits a Phagocytic Signal 1969

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'igure 2. Phagocytosis and EA binding by COS- I cells expressing Fc-yRIIIA. COS- I transfectants were incubated with EA for 30 min. Unboundervthrocvtes were removed by washing and examined for EA binding. For phagocytic analysis. washed COS-I cells were treated with a hypotonicsolution for 35 s to remove bound EA. (A ) Thin-section electron micrograph of COS- I transfectants demonstrates phagocytosis of EA denotedby arrows. (Picture shows an image magnified by 8000.) (B) The COS- I cells were stained with Wright-Giemsa and visualized by a light micro-scope (x1000). (a) Control cells from mock-transfection incubated with EA. (h) EA binding (rosetting) in COS-I cells expressing Fc-yRIIIA.(c) Phagocvtosis of EA by COS- I transfectants.

did not stimulate a rise in [Ca2+Ji. Epinephrine (10 IAM),which evokes a Ca2" signal in COScells (22) also stimulated arise in [Ca2+]i (Fig. 3).

Discussion

Human FcyRIIIA is a receptor complex composed of threeseparate polypeptide chains, the ligand-binding subunitFc'yRIIIA-a, and its associated -y and r chains found on NKcells, activated monocytes, and macrophages (23). These asso-ciated chains belong to a family of polypeptides that dimerize,interact in a noncovalent manner with other polypeptides inmultimeric receptors, and are critical for signal transduction bythe individual receptors (24, 25). The y and r chains are re-quired for surface receptor expression of FceRI and TCR/CD3, respectively (8-10). It has recently been shown that they or r subunit significantly increases FcyRIIIA-a surface ex-pression in cotransfection experiments (16, 18). Without theinteraction of the y or r molecule, the a chain is targeted fordegradation in the endoplasmic reticulum (23). FcyRIIIA canexist in three molecular forms with the a chain associated witheither yy, Id, or -ye. The FcyRIIIA-a-,yy isoform is found inmacrophages, while all three forms occur in NKcells that ex-press both the y and r subunits (26, 27). Wechose to evaluatethe role of one specific isoform, Fc-yRIIIA-a--yy, in signalingfollowing binding of Fc-yRIIIA by IgG-coated erythrocytes(EA) or anti-FcyRIII mAbin a transient transfection system.

Prior studies evaluating individual Fc receptor functionshave relied upon the use of cells expressing more than one classof Fc receptor and receptor-specific IgG subclasses or mAbstosimulate ligand binding. Under these conditions, the relativebinding specificity, displacement of mAbs by immune com-plexes, or cooperativity between the cytoplasmic domains ofthe different Fc receptor classes may influence the results. Fcreceptor-negative COS-1 cells have previously been shown to

be an effective transfection system for evaluating mannose re-ceptor ( 12) and for individual Fc receptor function (1 1, 19).Thus, analysis of Fc receptor signaling in this system can occurwithout the potential interpretation that the cytoplasmic do-mains of different receptors interact following ligand binding.Our results obtained with thin-section electron microscopy fur-ther demonstrate that COS-1 cells are an attractive model sys-tem to study the phagocytic signal transduction pathway.

COS- 1 cells cotransfected with a and y (wild type and mu-tants) subunits of FcyIIIA demonstrated comparable levels ofcell-surface expression of the multimeric FcyRIIIA (Table I).Transfection of cells with FcyRIIIA-a chain alone did not in-duce its cell-surface expression. Thus, our data indicate that the-y chain is necessary for both the surface expression ofFc'yRIIIA and its transmission of a phagocytic signal. Duringthe preparation of our manuscript, it was shown by others thatthe y subunit of FcyRIIIA plays a critical role in antigen presen-tation in transformed B cells (28) and in Ca2l signaling intransfected T and mast cells (29). Our results further demon-strate that the -y subunit of FcyRIIIA is essential for the signaltransduction ability of this receptor. It is of note that the regiondeleted in the GAMdel-2 mutant contains two conserved tyro-sines and leucines present in several signaling molecules of theimmunoglobulin gene superfamily (30, 31 ).

An increase in [Ca2+]i was demonstrated with COS-l cellsexpressing wild type FcyRIIIA a and y following receptorcross-linking, suggesting that the FcyRIIIA-a--y isoform caninitiate events involved in Ca2+ mobilization. The cytoplasmicdomain required for a phagocytic signal, described above, alsoappears to play an important role for intracellular Ca2+ mobili-zation, as mutants of the Sy chain in which tyrosine was re-placed by phenylalanine exhibited significant reductions in in-tracellular Ca2+ mobilization (31a). Intracellular Ca2+ in-creases have been demonstrated for all three Fcy receptorclasses (2, 32, 33). Similarly, it has been observed that both


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The FcyRIIIA-a-,yy isoform mediated both binding andphagocytosis of IgG-sensitized erythrocytes in COS- 1 transfec-tants without the presence of other Fc receptors. SinceFcyRIIIA is an abundant Fcy receptor on macrophages andthe -y chain is the only member of its family expressed in thesecells, these experiments suggest that the FcyRIIIA-a-y'y iso-form in macrophages plays an important role in phagocytosis.This process may involve increases in intracellular Ca2" (34).In NKcells, different Fc7RIIIA signaling pathways involved inADCCand NK-cell activation may be mediated by oy or ¢isoforms.

Through stimulation of the second messenger pathway in-volving PKC, PMAcan affect events in the phagocytic process(20, 21 ). Weevaluated the effects of PMAon phagocytosis inCOS-1 transfectants expressing either FcyRIIIA or Fc-yRIIA.PMA( l0-7 M) has been known to elicit superoxide anionproduction in U937 cells (35) and to stimulate both comple-ment receptor-mediated phagocytosis in cultured monocytes(36) and FcyRII-mediated phagocytosis in neutrophils andHEL cells (20, 21 ). However, the same concentration of PMA(10-7 M) caused an 82% reduction of phagocytosis in COS-1transfectants expressing FcyRIIIA, while phagocytosis was re-duced only by 13%in FcyRIIA transfected cells. That FcyRIIAtransfectants consistently show moderate increases at 10-1o-10-9 Mof PMAsuggests that there are differences betweenthese two Fcy receptors in intracellular signaling. These recep-tors may mediate phagocytosis in macrophages and stimulatemonocytes by coupling with different second messenger path-ways. PKCupregulation may be able to stimulate the FcyRII-mediated pathway, while inhibiting phagocytosis by FcaRIIIA.

Wehave used a model system to study the role of individualFc receptors, in transmission of a phagocytic signal. Weob-served that macrophage FcyRIIIA is able to mediate phagocyto-sis and that the y subunit of the receptor is essential both forsurface expression and for signal transduction mediated by thereceptor following activation. Future studies will examine the

Figure 3. [Ca2"]i increase on a single celllevel following cross-linking of FcyRIIIA.COS- transfectants plated on glass cover-slips were loaded with Fura-2/AM. Afterobtaining baseline [Ca2+]i, bound biotiny-lated anti-FcyRIII was cross-linked by theaddition of streptavidin. In control experi-ments, only streptavidin or biotinylatedanti-FcyRIII was added with no increasein [Ca2+ ]i over baseline. The arrows fromleft to right (the thickest to the thinnest)represent three time points when streptavi-

4 5 din (following preincubation with biotiny-lated anti-Fc-yRIII), epinephrine, and ion-omycin are added, respectively.

role of different isomers of FcyRIIIA and map the area andindividual amino acids critical for transmission of the phago-cytic signal.

Acknowledgments

The authors wish to thank Dr. John Williamson (University of Penn-sylvania) for the use of his Leidem cell chamber and fluorescent micro-scope/computerized analysis system for evaluation of calcium signal-ing. EMwork was made possible by Mr. Natali Thomassini and Dr.Steven Douglas (Children's Hospital of Philadelphia, PA).

This work was supported by National Institutes of Health grantsAI-22193 and HL-27068.

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4. Ravetch, J. V., and B. Perussia. 1989. Alternative membrane forms ofFc~yRIII (CD16) on human NKcells and neutrophils: Cell-type specific expres-sion of two genes which differ in single nucleotide substitutions. J. Exp. Med.170:481-497.

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6. Darby, C., P. Chien, M. D. Rossman, and A. D. Schreiber. 1990. Mono-cyte / macrophage FcyRIII, unlike FcyRIII on neutrophils, is not a phosphatylino-sitol-linked protein. Blood. 75:2396-2400.

7. Perussia, B., and J. V. Ravetch. 1991. Fc-yRIII (CD1 6) on human macro-phages is a functional product of the Fc-yRIII-2 gene. Eur. J. Immunol. 21:425-429.

8. Ra, C., M.-H. E. Jouvin, and J.-P. Kinet. 1989. Complete structure of themouse mast cell receptor for IgE (FcERI) and surface expression of chimericreceptors (rat-mouse-human) on transfected cells. J. Biol. Chem. 264:15323-15327.

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absence other fc receptors, fcyriiia transmits phagocytic...

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