On the interaction of IgG subclasses with the low affinity Fc gamma RIIa (CD32) on human monocytes, neutrophils, and platelets Analysis of a functional polymorphism to human IgG2

On the interaction of IgG subclasses with the

low affinity Fc gamma RIIa (CD32) on human

monocytes, neutrophils, and platelets. Analysis

of a functional polymorphism to human IgG2.

P W Parren, … , L A Aarden, J G van de Winkel

J Clin Invest. 1992;90(4):1537-1546. https://doi.org/10.1172/JCI116022.

An allotypic form of the low affinity IgG Fc receptor Fc gamma RIIa (CD32), termed low responder (LR) because of its weak reactivity with mouse (m) IgG1, interacts efficiently with human (h) IgG2. Fc gamma RIIaLR is the first known human FcR that binds this IgG

subclass. In this study, we analyzed the role of Fc gamma RIIa in binding of stable hIgG-subclass dimers, and in induction of T cell mitogenesis using chimeric anti-CD3 mAb. We demonstrate that the functional polymorphism to hIgG2 is expressed on the majority of Fc gamma R-bearing peripheral blood cells: monocytes, neutrophils, and platelets. We were able to assess Fc gamma RII-mediated IgG-binding without interference of other Fc gamma R-classes, by blockade of Fc gamma RI on monocytes, and by using neutrophils of an individual deficient for the Fc gamma RIIIB gene. This study indicates as subclass

specificity: hIgG3 >hIgG1,hIgG2 >> hIgG4 for Fc gamma RIIaLR and hIgG3,hIgG1 >> hIgG2 > hIgG4 for Fc gamma RIIaHR. Comparing the serum hIgG levels of individuals

homozygous for the two fc gamma RIIa allotypic forms, we observed significantly lower hIgG2 serum levels in individuals expressing the hIgG2-binding LR allotypic form. This observation may implicate that Fc gamma RIIa regulates hIgG subclass production or turnover in man.

Research Article

Find the latest version:

On

the Interaction of

IgG

Subclasses with the Low

Affinity

FcyRlla

(CD32)

on

Human

Monocytes, Neutrophils,

and

Platelets

Analysis ofa Functional Polymorphismto Human IgG2

Paul W. H. 1. Parren, *Petra A. M.Warmerdam,t LeonieC.M.Boeije,* JanineArts,*Nomdo A.C. Westerdaal,*

Arjen Vlug,* PeterJ. A.Capel,t Lucien A.Aarden,*andJan G. J. vandeWinkel8

*Central Laboratory ofthe Netherlands Red CrossBloodTransfusion Service and_{Laboratory of Experimental}and Clinical

Immunology, University ofAmsterdam, andtDepartment ofImmunology, University Hospital Utrecht, TheNetherlands

Abstract

Anallotypic form of thelowaffinity IgGFcreceptorFcyRIIa

(CD32), termed low responder (LR) because of its weak

reac-tivity withmouse (m)IgG1, interacts efficientlywith human (h) IgG2.

Fc-yRIIaIA

isthefirstknownhuman FcR that binds this IgG subclass. In this study, we analyzed the role of

Fc~yRIIa

inbinding of stable hIgG-subclass dimers,andin in-duction ofT cell mitogenesis using chimeric anti-CD3 mAb.

Wedemonstratethat thefunctional polymorphismtohIgG2 is expressed on the majorityofFc-yR-bearing peripheral blood

cells: monocytes, neutrophils, and platelets. Wewereableto assess FcyRHI-mediated IgG-binding without interference of

otherFcyR-classes, by blockade ofFcyRI onmonocytes, and byusing neutrophilsofanindividual deficient fortheFcyRIIIB

gene. This study indicates as subclass specificity: hIgG3

>hIgGl,hIgG2 > hIgG4 for

Fc'yRIla't

and hIgG3,hIgGl

>hIgG2>hIgG4 forFc7RIIaHR.Comparing theserumhIgG

levelsof individuals homozygous for thetwoFcyRIIaallotypic

forms,weobserved significantly lowerhIgG2 serumlevelsin

individuals expressing the hIgG2-binding LRallotypic form. This observation mayimplicate thatFcyRIIaregulates hIgG

subclassproductionor turnover in man.(J. Clin.Invest. 1992.

90:1537-1546.) Keywords:chimeric anti-CD3* T cell activa-tion* serumIgG*CD16*CD64

Introduction

Receptorsspecific for the Fc moiety of IgG(FcyR),expressed

on abroadrangeof hematopoietic cells, create an important linkage between humoral and cellular defence mechanisms.

Address correspondencetoDr.JanG.J. vandeWinkel, Departmentof Immunology, University Hospital Utrecht, RmG.04.614,P.O. Box

85500,3508 GA Utrecht, The Netherlands.

Paul Parren's present address is Baxter Healthcare, Biology Skill

Center, 10555 West Flagler St., Miami,FL33174.

Received for publication 29 January 1992 and in revised form 7

April1992.

1.Abbreviationsused in thispaper:

Fc-yR,

IgG Fc receptor; GPI,

glyco-syl-phosphatidylinositol;GPI-PLC, GPI-specific phospholipaseC; h, human; HR, high responder(tomIgGl); IMDM, Iscove's modified Dulbecco's medium;LR, lowresponder(to mIgG1);m, mouse; PLC, phospholipaseC; PNH,paroxysmalnocturnalhemoglobinuria;TcR, Tcell receptor.

J.Clin.Invest.

©TheAmerican Society for Clinical Investigation,Inc.

0021-9738/92/10/1537/10 $2.00 Volume 90, October 1992, 1537-1546

On human_leukocytes,threedistinctclasses ofIgGFcreceptors

(FcyR)

arecurrently recognized.

FcyRI

(CD64) is a 72-kD

high

affinityreceptor(Ka- ₁₀₈- 109

M-')

that is

expressed

constitutively

on_monocytesandmacrophages,and is

IFN-y-andG-CSF-inducibleon neutrophils (1, 2).

FcyRII (CD32)

andFc-yRIII (CD16) displaylower affinities(Ka< 107

M-')

and primarily bind immune complexes. Both FcyRII and

Fc'yRIII

have beendemonstratedon neutrophils,eosinophils,

andmacrophages.

FcyRII

is, furthermore,expressedon

mono-cytes,B

cells, basophils,

andplatelets (3,

4).

ThesubclassspecificityofFc~yRII (on platelets)was

previ-ouslydeterminedashuman(h)IgGl =hIgG3>

hIgG2,hIgG4

(5).

This viewmaybeincomplete; however,sincecDNA

clon-ingof

FcyRII

revealedmultipleFc'yRII-isoformsand

allotypes

(6-8),

whichmay express differencesinIgGsubclass

specific-ity.Three distinct genestermedhFcyRIIA, B,andC have been

described,which encodehighly homologous proteins

regarding

theextracellular parts,but whichdisplay strongstructural

di-vergenceincytoplasmic domains (6, 7, 9, 10).Thisindicates

that differences in subclass specificity between the various

forms, anddifferential expression ondistinctcells mayhave

major consequencesforantibody effector functions.FcyRIIis knowntobepolymorphicwithrespecttoitsinteraction with mouse(m) IgGIcomplexes (4, 1 1).It wasrecentlyshown that this functional polymorphism resides in a single

Arg-His

amino acidchangeatposition 131 inFczyRIIa ( 12-14).

Excit-ingly,it was found that thischangealsohasimplicationsfor the

interaction with hIgG: transfected fibroblasts expressing

FcyRIIa

(131-His), which was termed low responder

(Fc-yRIIaLR)

because ofits weakreactivity with mIgGl, ap-peared to bind hIgG2 dimers efficiently, in contrast to the

Fc-yRIIahigh responder(FcyRIIaHR)allotypic form( 13).

Recently, we developed a matched setof mouse/human chimericanti-CD3 mAb( 15).mAbdirected against the

multi-molecularTcR/CD3 complex on the cell surface of humanT

cells,arecapableofinducingT cellactivation, bothinvitro and

in vivo( 16). Accessory cellssuchas monocytes, however, are required to induce fullactivation and proliferation of the T cells and provide crosslinking of the anti-CD3 mAb via Fc receptors, aswell asproduction of lymphokinessuch as IL- 1 (11, 17). Using these antibodies, we demonstrated thatthis

Fc-yRII polymorphism is functionally expressedinanti-CD3

mAb induced T cell activation, both with monocytes and

FcyRIIaHR_ or

FcyRIIaLR

transfected fibroblastsasaccessory

cells( 18).

Acomplication in linkingtheabove observationstoother

cellsexpressingFc-yRIIisthepotential coexpression of

multi-ple isoforms ofFcyRII, suchas FcyRIIb, which expresses a

distinct subclass specificity (Warmerdam, P. A. M., I. E.

Oudyk,P.W. H.I.Parren,N. A.C. Westerdaal, J. G. J.vande

Winkel, and P.J.A.Capel. Submitted for publication),and

coexpression of members of different FchyR classes onmost

blood cells (4). Furthermore, studies with hybrid antibodies have suggested thatoneIgG moleculemaysimultaneously

in-teract withtwodistinct Fc'yR molecules ( 19), this might imply that potential cooperation between different classes ofFc-YR

moleculesmayaffect IgG binding avidity.

We now demonstrate the functional Fc'yRIIa polymor-phism for hIgG2 complexes onthemajorityof FcyR bearing

peripheralblood cells: monocytes, neutrophils, and platelets. Comparing IgG serum levelsbetween groups of individuals homozygous for either FcyRIIa allotype, we found signifi-cantly lower serum hIgG2 levels in

FcyRIIaLR

individuals. This observation mayimplicate thatFcyRIIaLR, which is the

firstFcR known to bind hIgG2 efficiently,maybe involved in modulation of hIgGserumlevels.

Methods

Monoclonal antibodies. Hybridoma cell line CLB-T3/4.1, producing a

mIgGlK mAbreactive withthee chain oftheTcR/CD3 complex(20)

and itsheavy chain isotype variants have been described in detail (15, 21).

Anti-Fc'yRI mAb 197 (y2asK) and mAb 22 ('Y1,K; FITC-conju-gated), anti-Fc'yRII mAb IV.3 ('y2bK)andanti-FcyRIII mAb 3G8

('y1,K)werekindly providedbyMedarex, Inc. (W. Lebanon, NH).

Anti-FcyRIImAb41H16('y2asK)wasprovidedby Dr T. Zipf(Univer-sity ofTexas, Houston, TX), and anti-FcyRIII mAb CLB GranI

('y2aK)wasfromtheCLB.Anti-CD28 mAbCLB-CD28/1('y1,K)was

providedby Dr R. van Lier (CLB,Amsterdam).

PurificationofPBC. T cells were isolated by sheepE-rosette sedi-mentation from PBMC, obtained by Percoll density centrifugation from buffycoats, asdescribedin( 15 ). Residual monocyteswere

re-moved byplasticadherence. T cellpuritywas checked bymeasuring LPS-induced monocyticIL-6production (22),which was found < 5

pg/ml IL-6in anovernight incubation of 200,000cells/ml, whereas

unseparatedPBMCproduced 2,000to 10,000 pg/ml.

Monocytes wereisolated from PBMC bycounterflow

centrifuga-tion,asdescribed elsewhere (23),andwere >95% pure,asdetermined incytocentrifuge preparationsafterstaining for nonspecificesterase,

and May Grtinwald-Giemsa.

Granulocyteswereseparated fromPBMC andplatelets by

centrifu-gationof trisodium citrate (14 mM) anticoagulatedbloodon 1.076 g/mlPercoll.Erythrocyteswere removedfromthegranulocyte pellet bylysisinisotonic NH4C1onice,resultinginaPMNpreparation

con-taining>95%neutrophils(24). Induction of PMN-FcyRIwas

ana-lyzed after incubation ofcells in culture medium(see below)

supple-mented with 300 U / mlIFN-y(Genentech,SanFrancisco, CA), inhibi-tion analyses of IFN-'y induced effects were performed by

supplementinginhibitoryanti-IFN-'ymAbMD-1 ( 10 tg/ml)(25).

Platelet-rich plasmawasobtained bycentrifugation(15min,250g) ofEDTA(5mM) anticoagulatedblood.Plateletswerethen

concen-trated bycentrifugation(7 min, 1,200 g)and three washeswere per-formed withPBS/5mMEDTA/0.5%FCS(26).

PMNexpressingreduced levels ofFcyRIIIwere isolatedfromtwo

patientswithdiagnosed paroxysmalnocturnalhemoglobinuria (PNH) (27, 28).Theneutrophil preparationsfrom thesepatients contained

- 80% and - 100% of(GPI-linked) FcyRIIIb deficient cells,

respec-tively, and expressed - 10% ofnormal Fc'YRIIIb levels.

FcyRIII-deficient(Fc-yRIIIOe)PMNwereisolated fromanFcyRIIIB-deficient

donor. The absence of theFcyRIIIBgene,Fc'yRIIIbmRNA, and solu-bleFcyRIIIbin serumwaspreviously demonstratedinastudy by Hui-zingaetal.(29).PMN from thisFcyRIIIb"*_{donor showedacomplete}

absence ofFcyRIII expressioninimmunofluorescencestudies(see

Re-sults; Fig.4B).

GPI-PLCtreatment.FcoyRIIIbwasremoved from normalPMNby

incubationwithGPI-specific phospholipaseC(GPI-PLC).PMNwere

washedandincubatedfor 1 h at370CinIscove's modified Dulbecco's medium (IMDM) supplementedwith a 1:1,000 dilutionofGPI-PLC. GPI-PLC isolated from Bacillus thuringiensis was kindly provided by

Dr. T.Schumacher (NetherlandsCancer Institute,Amsterdam). Tcellproliferation assay. Highly purified T cells were stimulated

with graded amounts ofanti-CD3 mAb in 200

_I1

IMDM, supple-mented with 5%heat-inactivatedpooled human serum,

_50MgM

2-mer-captoethanol, 100IU/ml penicillin,and 100

_,g/ml

streptomycin.All

incubations were performed in the presence of 1 Mg/ml anti-CD28

mAb as a T cellcostimulus (18). Withoutaccessory cells, no T cell

proliferationwas observed by theanti-CD28/anti-CD3mAb

combina-tion alone. As accessory cells, either 60,000 PMN or 2 x

I0'

platelets

were added per well. To generate optimalT cell triggering,we first

titrated PMN inanti-CD3-inducedT cellproliferation experiments

using an optimal amount ofanti-CD3mAb (40ng/ml). Remarkably,

the optimum formIgGl anti-CD3induced proliferationwas reached by supplementing - 30,000 PMN per well,whereasthe potencyof

hIgG2 anti-CD3 induced T cell proliferation still increased signifi-cantly up to PMNconcentrationsof- 60,000 cells per well(observed

in fiveexperiments using cells fromdifferentdonors). Since this

in-crease ofPMN-density did not affect the mIgGl anti-CD3 induced mitogenicity, further experiments were performed using - 60,000

PMN per well.

Titrationofplatelet concentration in thepresenceof an optimal amountsof anti-CD3 mAb, revealed an optimal T cellproliferationat a_densityof 2X

₁₀₅

_plateletsper well.

Inhibitionbyanti-Fc-yRmAb was assessed byincubationof T cells with anoptimalamount of anti-CD3 mAb (40 ng/ml) in thepresence

ofeithermAb 197

_(20,gg/

ml), mAbIV.3 (5Mg/ml),mAb CLB Gran I (10,ug/ml),CLBGranl F(ab')2fragments(20

_,ug/ml),

or mAb 3G8 (20 ,ug/ml). Theseinhibitory mAb werepreincubated for 1 h with

accessorycells before purified T cells were added to thecultures.A 4-h

[3H1thymidine

(0.2_,Ci/well)incorporationassay wasperformedon day 4. Allincubationswereperformedintriplicate,and SD were al-ways < 10%.

Weperformed control experimentsto check for(nonspecific) ef-fects ofanti-FcyRmAb on anti-CD3inducedT cellproliferation,(via potential activation andsubsequent degranulation of PMN) during culture.Anti-FcyR mAb were added to anFcyR-independentT cell

activation system (30).In this assay, mIgAanti-CD3mAb werecoated

at highdensitytoculture wells;acondition inducingpotent

prolifera-tion of purified T cells (this mIgA mAb is a heavy chain isotype switch

variant(21) of the anti-CD3 mAb used in rest of ourstudies). No

inhibitoryeffects of anyanti-FcyRmAb wereobservedin thissystem.

Purification of IgG paraproteins. Purified hIgG subclass proteins

with kappa lightchainswereisolatedfrom sera ofpatientswith multi-plemyelomaby(NH4)2S04precipitationandDEAE-Sephadex (Phar-macia, Uppsala, Sweden)ion-exchange chromatography.Ifnecessary,

proteins were furtherpurified on protein A oranti-hIgGsubclass Sepha-rose. All preparations werecheckedforimpurities and IgG subclass

contentinimmunoelectrophoresis,radialimmunodiffusion,and

hem-agglutinationassays (31, 32). The hIgGparaproteinsused in this study were > 98% pure, asevaluatedwith thesemethods. PurifiedhIgG was either used in amonomericform, afterremovalofaggregatesby

ultra-centrifugation( 1 h at 150,000 g), or as stable dimericcomplexes(see below).

Dimer binding studies. Purified IgG paraproteins were incubated overnightwithF(ab')2fragmentsof a mouseanti-humanK-lightchain mAb K35, in a molar ratio of 1:1, whichresultedin stabletetrameric complexes,as detailed in (24). Humanmonocytes(3X I0 in100

_;l

PBS/1%BSA) wereincubatedwith 25Mlof

anti-Fc'yR

mAb 197 (7 Mg/ml)and 25MlofanoptimalamountofhIgGdimers( 25,g/ml) for 45min at40C.The amount of mAb 197 wasoptimizedto result in complete blockade of bothmonomeric IgG binding to FcyRI,and

Fc-yRI-mediated EA-rosetting(seebelow). Afterwashing,cells were

subsequently incubatedwithFITC-conjugated F(ab')2 fragments of

goat

anti-hMg

antiserum(Cappel, Cochranville, PA),and

FITC-conju-gated

F(ab')2

fragmentsof rabbitanti-goatantiserum(Cappel),each

for45 minat40C. Cell-boundhIgGdimersweredetected flow cytomet-rically by aFACScanO (Becton Dickinson Immunocytometry Sys., Mountain View, CA).Toremovecrossreactiveantibodies,all

FITC-conjugateswereabsorbed withmIgG-coated Sepharosebeadspriorto use.Statisticalanalysiswasperformed usingaStudent'st test.

Dimer binding to PMN was essentially performed as detailed

above,butwithoutaddition ofmAb 197.Since hIgG4dimers didnot bindtoFcyRIIof either monocytesorPMN of any donortested,we checked theintegrityofthisbindingreagentby assessingdimerbinding

toU937cells, whichwereincubated withIFN-y (300 U/ml overnight) beforeuse toenhanceFc-yRI expression (1).

EA-rosetting;specificityof inhibitory anti-FcyRI andanti-Fc-yRII

mAb.Tocheck forspecificinhibition byanti-FcyRIandanti-FcyRII

mAb inourassays,wetested these mAb inEA-rosetting experiments.

Two types of indicatorerythrocyteswereused:rhesus-Dpositive hu-manerythrocyteswereoptimally sensitized eitherwithahuman

anti-serum directed against rhesus-D (EA-hIgG; Merz and Dade,

Dudingen, Switzerland), orwitha mIgGi anti-glycophorinA mAb

(EA-mIgGl) (23, 33). Rosettingwasscored microscopically as

de-scribed before (33).

Inhibition of FcyRI- orFcyRII-mediated IgG-bindingwas

per-formed by addingmAb 197 (7;g/ml)ormAb IV.3 (2.5 ug/ml), respectively. Bothantibodieswereusedatconcentrations that inhib-itedspecific EA-rosettingviaFcyRIorFcyRII using purified mono-cytes andEA-hIgGorEA-mIgG1,respectively (23, 33).Rosetteswere

readilyobservedafter incubation of monocytes withEA-hIgG:70±3% of cellswerescoredpositive (n = 3). Formationofrosettes was not inhibitedbyadditionofmAb IV.3(73±4%rosetting cells;n=3), in contrast to mAb 197, which blocked EA-hIgG rosetting efficiently (2±2%rosettes;n= _3).FcyRII-mediatedrosettingwasmeasuredby adding EA-mIgGItoHR monocytes,whichresulted in58±3%rosettes

(n = 3). Addition of identical concentrations oftheblocking

anti-Fc'yRmAbasabove,resulted inefficient inhibition of EA-mIgGI

ro-setting bymAbIV.3(3±1%rosettes;n=3),whereas mAb 197didnot

significantly impederosetteformation (56±2%rosettes;n=3).

Wenextevaluated whether FcyRIwasefficientlyblockedby

prein-cubation with mAb 197and,therefore,could be usedtostudyspecific bindingtoFc'yRIIaonmonocytes.Monocyteswerefirstincubated for 30 minat37°Cin RPMI 1640 medium(withoutserum)to remove

cytophilicIgG.Cellswerewashedtwice in medium andincubated for

15 minat4°CinPBS/0.5%BSA/0.1% NaN3 alone, or supplemented with eitheranti-FcyRImAb 197 (7jg/ml) or mAb IV.3(2.5,ug/ml).

Subsequently, 10 _gg/mlof monomeric hIgGwasadded, andaftera 45-minincubation at4°C, cells were washed. Cell-bound hIgG was detectedflow cytometrically byaFACScanO, using FITC-conjugated F(ab')2 fragmentsof goatanti-hIgGantiserum (Cappel). Addition of monomeric hIgG tomonocytesresulted in 79±3% positive staining cells(n= _2),whichwaseffectivelyblocked bypreincubatingthe cells with mAb 197 (4±2%positivecells; n =2), but notwith mAb IV.3

(70±4%positivecells;n=2).

MeasurementofserumIgG.Sera andcitrated blood sampleswere collectedby venipuncturefrom 69randomlyselectedvolunteering

col-leagues,43 males and 26females(mean age30±6,range 21-57yr), and sera were stored at -20°C until used. hIgGl, hIgG2, hIgG3, hIgG4,andtotalhIgGconcentrationsweremeasured

nephelometri-cally (Vlug, A., E. Nieuwenhuis, I. Knop, and R. V. W. van Eijk,

manuscriptin preparation). Statisticalanalysiswasperformedby a

Mann-WhitneyUtest.

FcyRIIphenotyping.TheFcyRIIaphenotype of cells from differ-ent donorswasanalyzed by comparingthe potencyof mIgG1 and

hIgG2 anti-CD3-mAbinducedTcellproliferation in PBMC isolated from citrated blood,asdetailed in (18). Briefly, 40,000 PBMC were culturedfor4din culturemedium (see above) after addition of either

mIgGl,orhIgG2anti-CD3 mAb inanoptimal concentration (40 ng/

ml).Asdescribed before (18), three groups ofdonors could be discrim-inated:twogroupswith PBMConly responsivetoeithermIgGl, or hIgG2anti-CD3mAb,andone_groupwitha_comparablereactivityto

bothanti-CD3mAb,indicating homozygous

Fc-yRIIa"',

homozygous

Fc'yRIIa'L,andheterozygousFczyRIIaLR.HRindividuals,respectively. Inaddition,FcyRIIaphenotypesof donorswereassessedby using

immuno fluorescenceanalysison aFACScanusinganti-FcyRIImAb 41H16(which selectivelyreactswithFcyRIIaHR[34])and mAbIV.3

(recognizingboth FcyRIIa allotypic forms), followed by detection withFITC-conjugatedF(ab')2fragmentsofgoat anti-mouse antiserum

(G26M17F, CLB)asdescribedpreviously ( 18, 34). To avoid

cyto-philic binding ofthemIgG2amAb 41H16tothehigh-affinity FcyRI,

allmAbincubationswereperformedin the presence of 25% heat-inac-tivated humanserum.

Allexperimentsinthis studywereperformed usingcells isolated from eitherFcyRIIa'L orFcyRIIaHRhomozygous donors.

Results

HumanIgGdimerbindingtomonocytes. Humanmonocytes

express both FcyRI and FcyRIIa constitutively (4). When

usedasaccessory cellsinanti-CD3induced T cellproliferation,

mitogenicitytochimerichIgGl, hIgG3,andhIgG4anti-CD3

wasfoundtobeFcyRI-mediated( 18). The hIgG2 anti-CD3

mAb, however, induced

FcyRIIa-mediated

T cell prolifera-tion,whichwasonlyfound with PBMC of individuals

express-ingthe

FcyRIIaLR

allotypicform(bothhomo- and heterozy-gotes) (18). We now studiedbinding of well-defined stable

.0 E z

C,

0

0

a)

cc

af

I 0

0)

0

LR-Monocyles

A

HR-Monocytes

B

101 102 103 104 101 102 103 104

LogFluorescenceIntensity (ArbitraryUnits)

higG1 hIgG2 hIgG3

LR HR LR HR LR HR

Figure 1.BindingofdimerichIgGsubclasscomplexesto

FcyRI-blockedpurified monocytes.Thehigh affinityFcyRIonmonocytes

wasblockedbyaddition ofasaturatingamountofinhibitory

anti-FcyRI mAb 197. Stable dimerichIgGl, hIgG2,orhIgG3 complexes

wereadded,andbindingwasanalyzed cytofluorometrically.In(A

andB), representative experimentsareshownillustratingthe

immu-nofluorescenceobservedafter additionofhIgG2dimericcomplexes

tomonocytesfromFcyRIIl' andFcyRIIHR-homozygous donors,

re-spectively. Bindingwasassessedafter additionofhIgG2-dimersalone (solid line),orinthepresenceof mAbIV.3(dashed line). Back-ground fluorescence,observed intheabsence of dimericcomplexes,

is indicatedby shading. (C-E)showthebindingofhIgG1, hIgG2,

andhIgG3dimericcomplexestomonocytesfromFcyRIIa'L-(n

=_9),andFcyRIIaHR-typed (n=_{5) homozygous donors, represented}

asthepercentageof cells withfluorescenceintensities above back-groundlevels.

Functional_{Fc-yRII Polymorphism}on_PeripheralBlood Cells 1539

C D * E

me o

20~~~~~~~~~~~~~~~~~0

20

-A:~~~~~~~~20

dimerichIgG complexes of the four hIgG subclasses to these cells. To allowexclusive FcyRIIa-mediated binding of hIgG dimers, we blocked the high affinity IgG Fc receptor specifi-callyusinganti-FcyRImAb 197 (see Methods).

Purified monocytes isolated from homozygous FcyRIIa-typed donors,wereincubated with dimeric complexes of de-fined hIgG subclasses. hIgG2 dimers were bound differentially by cells from

FcyRIIa"'

andFcyRIIaHRindividuals, as illus-tratedin Fig. 1A, andB,respectively. Significant binding of hIgG2dimeric complexes was only apparent to cells bearing the

FcyRIIa'L

allotypic form (Fig. 1A), whereas the homozy-gousFc.yRIIaHR-expressingcellsremained negative (Fig. 1B). The same pattern of binding was observed with monocytes from nine LR and five HR donors (Fig. 1 D), and with four different hIgG2 myeloma proteins (data not shown). Parallel experiments were performed using hIgGI, hIgG3, and hIgG4 dimeric complexes. Both hIgGl and hIgG3 dimers bound

effec-tivelytomonocytesfromthe two typesofdonors, asshown in Fig. 1 C,and 1 E,respectively. Nodifference was observed in hIgGl dimerbindingbetween the homozygousFcyRIIaI _and

Fc'yRIIa'H

monocytes (Fig. 1 C). Cells expressing the

FcyRIIaLR allotypic form, however, exhibited a significantly highercapacitytobindhIgG3dimers (P<0.001;Fig. 1E). In none oftheexperiments performed we observed binding of hIgG4dimerstoFcyRI-blockedmonocytes. The percentageof

cells with fluorescence above background was always <2%, both with monocytes from FcyRIIaLR (n = 15), and

FcyRIIa'

(n=8)individuals.Toappraisethevalidity ofthe

hIgG4dimeric complexesasbindingreagents, weperformed binding experimentswith U937 cells (expressing FcyRIand

FcyRIIaHR

[8]). Upon incubation of these cells withhIgG4 dimers,99% of the U937 cells stainedpositive,which could be

inhibited bymAb 197(6%positive cells),butnotbymAb IV.3

(99%positive cells) (n=2).This

susceptibility

ofhIgG4

bind-ingtoinhibitionsolely byanti-FcyRImAb 197indicates, fur-thermore, that FcyRIIaon U937 is

incapable

of

interacting

with hIgG4dimers(as observedwith monocytes).

Thefunctional FcyRIIa polymorphism to

hIgG2

is

ex-pressedonplatelets.AsubstantialportionofFcyRIIpresenton

PBC is expressed onplatelets. Platelets donotexpress other classesof FcyR (35), andseemthereforea

superb

model

sys-tem tostudyFcyRII-IgG interactions.

Consequently,

we stud-iedanti-CD3-inducedTcellactivationin the presence

ofplate-letsasaccessory cells.

Fig. 2AandBrepresentsimmunofluorescence analyses of

platelets used in the Tcellmitogenesis experimentsshown in

panels C andD,

respectively.

After

staining

with mAbIV.3, platelets derived from both donors

displayed

a

comparable

fluo-rescenceintensity,indicating

comparable

FcyRIIa

expression

levels.Incubation of platelets with mAb 41H16 revealed simi-larstainingasmAb IV.3 for the

FcyRIIa

' _{homozygousdonor}

andbackground stainingfor the

FcyRIIa'L

homozygous

do-nor.Thissuggestedanantigenicallysimilar

Fc'yRIIa

polymor-phismasfoundonmonocytes andPMN.

Incontrast toTcellmitogenesisstudies with monocytesas

accessory cells, addition ofanti-CD3 mAbtoplatelets alone

wasinsufficienttoinduceTcellproliferation.Platelets appar-ently lacked theabilitytoprovideanaccessorysignalfor full activation ofT cells. Wecircumvented this requirement by addingananti-CD28 mAbascostimulatortotheincubations.

Addition of anti-CD28 mAb aloneor anti-CD28 combined with theanti-CD3mAbCLB-T3/4didnotresultin

prolifera-.0

E-z

1._

z

LR-Platelets

41H161

AI

It~

I I

I-A

HR-Platelets

41H16

IV3

~-

--I

B

101 102 103 104 101 102 103 104

LogFluorescence Intensity (Arbitrary Units)

c

n

CL 0O

._2

II

90 /

-80-LR-Platelets

70- v

60- V/

50-

40-

30-

20-

10-IC IA-,D

HR-Platelets _e*

/

"I -rI 91q

*mlgG_Il

vhIgG2

0 100 10' 10 103° 0 10° lo' lo, 103

Anti-CD3 mAb concentration (ng/ml)

Figure 2. CharacterizationofplateletFcyRexpression, and anti-CD3-induced T cellproliferationin the presence of platelets as ac-cessory cells. Platelets,isolatedfromFcyRIIa"'-(A) or FcyRIIaHR-(B)homozygousdonors, wereincubated either with buffer alone

(control;shaded), anti-FcyRIImAb IV.3(dashed line), or

anti-FcyRIIa'

mAb 41H16(solid line), followed by FITC-conjugated F(ab')2fragments ofgoatanti-mouseIgGantiserum. Platelets from these donors weresubsequentlyused asaccessory cells inmIgGl and

hIgG2anti-CD3-inducedTcellproliferation.(C and D) represent theanti-CD3-induced mitogenesis of highlypurified T cells using Fc'yRIIa'L-typed,andFcyRIIaHR-typed platelets, respectively. [3H]thymidineincorporationis plotted against the anti-CD3 mAb

concentration. Experiments performedwith platelets derived from twoFc'yRJIa'-,and oneFcyRIIa'H-homozygousdonors,

respec-tively, yielded similarresults.

tion ofpurifiedT cells.Platelets, isolated fromhomozygous

FcoyRIIaHRor

FcyRIIaLR

individualswere used to study T cell proliferation induced by quantitated amounts of mIgGl or hIgG2 anti-CD3 mAb (shown in Fig. 2 C and D). Platelets derived froman

FcyRIIa'L

individual supported hIgG2

anti-CD3-induced proliferation efficiently, whereas mIgGl

in-ducedproliferationwashardlydetectable(Fig.2C).Platelets isolated from

FcyRIIa"'

individualswereobservedtosupport mIgGl anti-CD3-inducedTcell mitogenesis, but not hIgG2 anti-CD3 induced activation. Addition of mAb IV.3tothese cultures resulted in complete inhibition of both hIgG2 and

mIgGl anti-CD3-induced Tcell mitogenesis(> 99%

inhibi-tion;n=2).Plateletsexpressingthe

FcyRIIa'L

allotypic form,

furthermore supported induction ofT cell proliferation by

hIgGl anti-CD3 efficiently,butnotwith hIgG3,orhIgG4 (n

=2).

Interaction between(chimeric) anti-CD3mAb and FcyR on PMN. Human neutrophils constitutively express both FcyRIIa and aGPI-linked form ofFcyRIII; i.e., FcyRIIIb.

Fc'yRIII

hasbeenimplicatedinmodulation of FcyRIIaffinity

and triggering (36-38). To examine this in moredetail, we

studied antibody-FcyR interactions on normal PMN and PMNimpairedinexpressionofFcyRIIIb.

PMN wereisolated from healthydonorshomozygousfor

either FcyRIIa phenotype, and they were used asaccessory

cells in anti-CD3-induced T cell activation experiments, as

shown in Fig. 3. In these experiments, we also added anti-CD28 mAbas aTcellcostimulus, since anti-CD3 mAb

com-bined withPMNalonewereincapable ofinducingTcell prolif-eration. A dose-dependent mIgGl anti-CD3 mAb induced T cell activation was only observed using PMN expressing FcYRIIaHR, whereashIgG2 anti-CD3 mAbpreferentially in-ducedproliferation in thepresenceofFcyRIIaLR PMN. Cells from both types of individuals supported proliferation with hIgGl anti-CD3, but not hIgG3 or hIgG4 subclass variants (Fig. 3).Evaluationofthe roleof FcyRIIin theseresponsesis

complicatedby expression of bothFcyRIIaand

FcyRIIIb

on

thesecells. Inhibition analyseswere, therefore, performedon normalandonFcyRIIIbnegative

(FcyRIII¶eg)

PMN.

PhenotypesofPMNusedfor these inhibition experiments

wereanalyzedcytofluorometricallyas showninFig.4 AandB. The PMN analyzed in Fig. 4 B, isolated from a donorwho showedacomplete lack of PMN-FcyRIIIb expression caused byagenomicdeletionin theFcyRIIIBlocus(29), stained

posi-tive with

anti-FcyRII

mAb(IV.3),butnotwitha

FcyRIIa'-specific (41H116) or

anti-FcyRIII

mAb (CLB Granl ). This

indicated expression ofthe

FcyRIIa'L

allotypic form

exclu-sively onthese cells. Fig. 4Ashows theanalysis ofanormal donor matched forexpression of thisFcyRIIaallotypicform.

PMN ofboth these donors, furthermore, did not express

Fc-yRI,asindicated by absent staining withanti-FcyRImAb 197, and22(datanotshown).

Toevaluate whichFcyRclasses wereinvolved inanti-CD3 inducedTcell mitogenesis in thepresenceofPMN,we

ana-lyzed T cell proliferation in this system, in the presence or

absenceofblocking

anti-FcyRI

(197),

anti-FcyRII

(IV.3),or

anti-FcyRIII(CLBGranl) mAb. Inhibitionpatternsof anti-CD3inducedTcellproliferation bythese

anti-FcyR

mAbare

shown inFig.4C andD.Fig.4C shows thatPMN-dependent

hIgG2 anti-CD3-inducedTcellproliferationwascompletely

inhibiteduponaddition of mAb IV.3.PMNderived from the

r 60

-00

o 40

.6~30-V

'

20

A

10 /

0

0 100 101 102 103 0 100 10' 102 103

Anti-CD3 mAb concentration (ng/ml)

Figure 3. Induction of T cellmitogenesisbyanti-CD3 isotype variants

usingPMN asaccessory cells.Highly purifiedTcells wereincubated withaquantitated amount ofmIgGI, hIgGI,hIgG2, hIgG3, and hIgG4anti-CD3 mAb in the presence of PMN expressing either the

FcyRIIa'L

(A),ortheFcyRIIaHRallotypic form (B). Anti-CD28 mAb(1 ,ug/ml)wasadded as acostimulatory signal.[3H]thymidine

incorporationwasplotted against the concentration of anti-CD3 mAb. Theexperiment shown is representative of two separate

experi-mentswith cellsfrom the same donors. Similar results were obtained withPMNderivedfrom 5Fc-yRIIaL'-and 2FcyRIIaHR-expressing

donors. *,mIgGI;v,hIgGI; v, hIgG2; o, hIgG3; *, hIgG4.

n

z

aC

-a')

A CLBGranI B

41H16

I _IV

Log Fluorescene 104nt r01 10t2 3 104 LogFluorescenceIntensity(Arbitrary Units)

--D

LR-PMN LR-PMN_FCyR1lneg

_ Za

higrO2 hIaS 1 m,cG'

Figure4.CharacterizationofFc-yR-phenotypeandhIgG1,andhIgG2 anti-CD3-induced T cellproliferationin thepresenceof normaland

FcyRIIIne*PMN.Cytofluorometricanalysisof PMN isolated from normal,andFcyRIIIbnh',FcyRIIa'L-homozygousindividualsare

shown in(AandB), respectively.PMNfrom both donorswere

incu-bated either with bufferalone(control; shaded),anti-FcyRIImAb IV.3(whichbinds bothallotypicformsofFc'yRIIa;dashedline), anti-Fc-yRIIaHR-specificmAb 41H16(solid line),oranti-Fc'YRIII

mAb CLBGranI(dotted line),followed byFITC-conjugatedF(ab')2

fragmentsofgoat-anti-mouse IgGassecondary antibody. (CandD) showananalysisofhIgG I,andhIgG2anti-CD3 inducedproliferation ofhighly purifiedTcells in thepresenceof normalandFcyRIIIa'*

PMN, respectively. Tcellswerestimulated withanoptimalamount of anti-CD3 mAb in medium alone(black bars),orinthepresence of eitheranti-FcyRImAb 197,anti-Fc-yRIImAbIV.3,anti-FcyRIII mAbCLB Grani,or acombination of mAb IV.3 and mAb 197.(C) showsanexperiment representativefor PMNfrom fiveFcyRIIa' homozygousdonors,(D)showsanexperiment representativefor two separateexperiments usingcells fromasingleFcyRIII"*donor. .,

-;n,IV.3; , 197;m,CLBGranl; IV.3+ 197.

FcyRIIIn`

donor (analyzed in Fig.4D), also showedcomplete

susceptibility ofhIgG2anti-CD3-inducedTcellmitogenicity

tomAbIV.3,similartoFcyRIIaLR-bearingPMNfromall

nor-mal donors evaluated (n= _12).Incontrast,hIgGl

anti-CD3-inducedTcell proliferationwasstronglyinhibitedafter

block-adeof FcyRIIIb (Fig.4C).Surprisingly,however, the absence

of FcyRIIIb didnotaffecttheability ofFcyRIIIbn" PMN to support Tcellmitogenesis inducedbythis hIgG subclass.The

data shown inFig.4 Dsuggestedthis proliferation-inductionto

bepartly FcyRIImediated, supportedby a decreaseinT cell

responsiveness after addition ofmAb IV.3. Remarkably, a

syn-ergistically increased inhibition was observed by combining

mAb IV.3withmAb 197. Indeed, a complication in these cul-tureexperimentsmaybeinductionofFcyRIon PMN by

IFN-'y

(1), producedbyproliferatingTcells, which may then medi-atebindingofhIgGlmAb.We testedthishypothesisby

analyz-ing cells from the cultures cytofluorometrically, using a

FHTC-conjugate

ofanti-FcyRImAb 22, as shown in Table I.

AftergatingonPMN, wefoundasignificantincreaseofFcyRI

expression after culturingthe cells overnight, provided

anti-CD3-stimulatedTcells were presentinthe culture. The level of FchyRIexpressiononthesePMNwassimilarto the

expres-Functional Fc-yRII PolymorphismonPeripheral Blood Cells 1541 LR-PMN_FCN-RiIlneg

sion level induced after culturing overnight in medium

supple-mented with IFN-'y(n=4). In boththese experiments, Fc'yRI-induction couldbecompletely inhibited by addition of anti-IFN-,y mAb.

Turningourattentionto

FcyRIIIb,

weobservedadramatic

decrease in the expression level of this FcyR on PMN

mea-sured duringthe first 2 d of culture (n = 3), as indicated in

TableII.Thepresenceof solubleFcyRIIIin thesupernatants

(datanotshown)indicated thatFcyRIIIbis shed fromthecell membrane during culture (39).As acontrol,westained cells

witha(granulocyte-specific)CD66mAb, CLB Gran 10,which

revealed allgated cellstobepositive, without reduced

fluores-cence.The observationthatFcyRIisrapidlyinducedduring culture, while FcyRIIIb expression on the cell surface is de-creased, might implicatea minorrole forFcyRIIIb inhIgGl anti-CD3 induced T cell mitogenesis. Expression ofFc'yRI,

furthermore, poseda practical difficulty inthatanti-FcyRIII

mAb CLB Gran1 (mIgG2a subclass)may inhibit binding to

Fc'yRI via its Fc-moiety. We, therefore, used CLB Granl F(ab')2 fragments and mAb 3G8 (mIgGl subclass) in

inhibi-tion analyses, which didnot(CLB Granl [Fab']2)orweakly (mAb3G8) affecthIgG1 anti-CD3-inducedTcellactivation.

Human IgG dimer binding by normal and

FcyRIII"9

PMN. We analyzed binding of dimeric hIgG subclass

com-plexes to PMN, with normal, and absent expression of

FchyRIIIb. Cellsofa normal

FcyRIIa'u

homozygous donor and the Fc'yRIIIbn*donor described above were incubated withoptimalamountsof dimers(Fig. 5).5C and 5Dindicate that PMN of both donors were capable ofbinding hIgG2 dimers,whichwascompletelyinhibitableby addingmAbIV.3. PMNofbothtypesofdonors,furthermore,boundhIgGl and hIgG3 dimersefficiently(Fig. 5A, B, E, and F,respectively),

although the binding capacityof normalPMN fordimers of theseisotypesweresignificantly higher.On normalPMN,the

majorcomponentof thisbindingwasFcyRIIIb-mediated,

in-dicated by susceptibility toinhibition by anti-FcyRIII mAb

3G8(Fig.5AandE).The lowamountofhIgGl /hIgG3

bind-ingobserved in the presence ofmAb3G8, was decreasedto background level when this mAb was combined with mAb

IV.3 (data not shown; n = _2). The experiment with

FcyRIIlbnl*

PMN showed that, even in the absence of

TableI.InductionofFc-yRIonCulturedPMN

Stimuli* %CD64-positivePMN5'

None 7.1

IFN-Ty 68.4

hIgGIanti-CD3 7.3

hIgGI anti-CD3+Tcells 60.5

IFN-,y+anti-IFN-'y 11.4

hIgG1 anti-CD3+Tcells+Anti-IFN-'y 9.5

*_{Freshly isolated PMNwereculturedovernightinculture medium}

supplementedwith the indicated agents in the presenceorabsence ofhighly purifiedTcells. $PMNwerewashed and incubated witha

FITC-conjugateof anti-FcyRI mAb 22. AftergatingonPMN,the percentage of cellsdisplayingfluorescence intensities above back-ground level (PMN incubated in buffer without mAb22-FITC)was assessed. § As acontrol, the cellswerestained with

granulocyte-spe-cific CD66 mAb CLB Gran1O,which showed allgatedcellstobe

positive.

TableII.Expression ofFcyRIIIb on Cultured PMN

Days of culture* % CD I 6-positive cells(mean)50

0 100 (1,038)

1 74(311)

2 66 (161)

* FreshlyisolatedPMN were incubated in culture medium for the numberofdays indicated. t PMN were washed and subsequently incubated withanti-FcyRIIImAb 3G8 and FITC-conjugatedF(ab)2

fragmentsof a goat anti-mouse antiserum. The percentage of cells, displaying fluorescence intensities above background level, and mean

fluorescenceintensities of the cell-population (between parentheses) areindicated. § As a control, PMN were stained with CD66 mAb CLB Gran10,which showed all cells to be positive.

Fc'yRIIIb,

PMN were still capable of binding significant

amountsofhlgGl and hIgG3. Thisbinding,however,was now

completely susceptibletoblockade by mAb IV.3 (Fig. 5Band F), and, hence,

FcyRII-mediated.

Binding of hIgG4 dimeric complexestothese cellswasalsostudied,butnoneofthe PMN

preparationsevaluated inourlaboratory(n= 15),was capable

ofsignificantly bindingthis hIgG subclass (Fig. 5 G andH).

hIgG2

C &!03

.~~ ~ ~ ~ ~~~~~

3

iv:3 S /

j~~~~~~~~I 3138

E

z L)

4:

hlgGd

G H

i IV

LA

101 102 ₁₀₃ 104 10 102 103 10'

LogFluorescenceIntensity(Arbitrary Units)

Figure5.BindingofdimerichIgGsubclass complexestoPMN froma

normalandanFcnyRIII"g individual. Highly purifiedPMN froma normal(A, C, E,andG), andaFcyRIIIn*g(B, D, F, and H)

FcYRIIa'L-homozygous donorwereincubated with dimerichIgGl (A, B),hIgG2 (C, D), hIgG3 (E, F),and hIgG4(G, H)complexes. Afterwashing, bound dimerswerequantitated cytofluorometrically. BindingtoPMNwasassessed after addition of hIgG dimers either

alone(solid line),orinthepresenceofanti-FcyRIImAbIV.3 (dashed line),oranti-Fc'yRIIImAb3G8(dotted line).Background fluorescence isindicatedby shading.Theexperimentshownwas

re-peatedseveral times with normal(n=5),andFcyRIIIb"*PMN(n

ComparinghIgG2 dimer bindingtonormal PMN,

homo-zygously expressingthe LRorHRallotypicform ofFcyRIIa,

weobservedasimilarpattern as on monocytes.Efficient

bind-ing was only apparent on PMN expressing FcyRIIaLR

(31±15%positive cells;n= 12), whereas

FcyRIIaHR

homozy-gous PMNboundhIgG2poorly (7±4% positive cells;n = 12).

Incubation ofPMNwith GPI-PLC, which resulted in removal

of - _90% ofthe GPI-linked

FcyRIIIb,

did not significantly affecttheabilityof these cellsto bindhIgG2 (n = 3). hIgG2 dimer binding studies were, furthermore, performed with

PMNisolated fromtwopatients sufferingfrom PNH, which is

anacquireddisorderresulting in strongly diminished

expres-sion of GPI-linkedmembraneproteins,such asFcyRIIIb(27, 28). PMN of both these patients were found to express

FcayRIIaHR

only, and interacted weakly with hIgG2 dimers (re-sulting in4and 8%positive stainingcells,respectively).

Correlation between Fc-yRIIa polymorphism and donor

hIgG2 serum levels. The FcyRIIaLR allotypic form seems

uniquein itsabilitytobind hIgG2,sincenootherhFcYRhas beenreportedtointeractefficientlywith this isotype ([4, 10], this study). It has been postulated that so-called Igbinding factors (most likely soluble FcyR) mayaffectIgproduction

andproliferation ofBcells (40). This promptedus toanalyze

hIgGsubclass levels in seraof healthydonors. We randomly

analyzed 69 donors, ofwhom 10werefoundhomozygous for

FczyRIIaHR,

and 22 for

FcyRIIaLR.

Fig. 6A shows the total

hIgGcontents measured in theseraofthese 32homozygous donors.Comparingthetwogroupsof individuals,wefoundno

significantdifferences in total hIgG concentrations. Remark-ably,astriking differencewasfound for hIgG2subclass levels,

whichwerecalculated relative to the hIgG1 concentration, to correctfor variations in total hIgGserum levels between

differ-entindividuals (indicated in Fig. 6A). Asshown in Fig.6B,

Fc~yRIIaLR

individuals had significantly lower hIgG2 serum levelsthan

FcyRIIaHR

individuals (P=0.02). Significant

dif-C 20

15

oS

LR HR

*.2

20.8_

p0.6_, *

CV

>,0.4_ .

E

e 0.2

.2

0.6

HR

Figure6.hIgG serum

levels inFcyRIIaLRand

FcyRIIaHR homozy-gous donors. Levels of A hIgGI, and hIgG2

sub-classes, and totalhIgG

were analyzed in sera of 69randomly chosen volunteers. The serum levels of the 22

FcYRIIaLR, and 10

FcayRIIaHRhomozygous individuals in this group areshown.(A) repre-sentsthe totalhIgG

concentration measured

B inthese sera.(B) shows thehIgG2 serumlevels, calculatedrelativeto thehIgG1 concentra-tion inindividuals,to correctfor variations in the absoluteserum hIgG levels. Themean values±SEMare indi-catedfor each donor group.

ferencesbetweenthetwogroupswerealso foundby

calculating

hIgG2(P=0.05)andhIgG1(P=0.008) levels relativetototal

hIgGconcentration.Inthese analyses,hIgG2levelswerefound

decreased while hIgGIlevelsappearedincreased in

FcyRIIaLR

versus

FcyRIIaHR

donors. Absolute hIgGl and hIgG2 levels,

however,didnotdiffersignificantly.Nosignificant differences

were observed in hIgG3 or hIgG4 serum levels. IfhIgG2 is

moreefficientlycleared by theLRcomparedtotheHR

allo-typic form of FcyRIIa, then also heterozygous individuals

mightbeexpectedtohavelowerrelativehIgG2subclass levels

compared with homozygous

FcyRIIaHR

donors.

Comparing

the hIgG2/hIgGl ratio between the three groups, we found thatthis ratio (mean±SEM) ofheterozygotes(0.54±0.04)was intermediate between the ratio ofHR (0.67±0.05) and LR

homozygotes(0.50±0.04). Thedifferencebetween the

hetero-zygotes and HR homozygoteswas statistically significant (P

=0.04), whereas thedifferencebetweenheterozygotesandLR

homozygoteswasnot.

Discussion

Recently, it was shown that the classical polymorphism of

hFcyRIIa,asoriginally definedinmIgG1anti-CD3-inducedT

cellmitogenesis(4, l 1), hasprofound implicationsforhIgG2 binding.TransfectedfibroblastsexpressingtheFcyRIIaLR

allo-typic form were found to interact efficiently with this hIgG

subclass, incontrast to

FcyRIIaHR-transfected

cells( 13).Tcell

mitogenesisexperimentsusing hIgG2 anti-CD3,furthermore,

indicated thatdifferential binding ofhIgG2 was inherent to

normal monocytes,and wascorrelated withFcyRIIa

pheno-type(18).

In thepresent studywe demonstrate that the functional

FcyRIIapolymorphismisexpressedonmonocytes,PMN, and platelets. We studied this intwosystems:first,bybinding

analy-seswith stabledimeric hIgG complexes,andsecondly, in

anti-CD3-inducedTcellproliferation in which large,heavily

op-sonized complexes were presented to FcyR. Both in hIgG

dimerbindingandanti-CD3-induced T cell mitogenesis,we

wereabletodisplace hIgG2 from cells with mAb IV.3,

support-ing FcyRIIa-mediated bindsupport-ing.FcyRIIa, furthermore, bound antibodiesofthehIgGlandhIgG3, butnothIgG4 isotypes,as

shownby dimerbindingtoFcyRI-blockedmonocytes(Fig. 1).

Inthese analyses, hIgG3 dimerswereboundsignificantly better by

FcyRIIa'L

than by FcyRIIaHR. Sincecross-talk between

FcoyRI and II has been observed in signalling in U937 cells ( 19), itseemspossiblethatligation ofFcyRI on monocytesby mAb 197mayaffect binding viaFcyRII. In our opinion, this is notsupportedby ourstudies with

FcyRIIa-transfected

fibro-blasts( 13), which showed identical binding characteristicsof

IgG dimers, as shownwith theFcyRI-blockedmonocytes in

thepresentstudy.Blocking of FcyRIbyintactmAb 197 might

causeproblems resulting from cross-reactivityof its Fc region

withother FcyR classes. All available FcyRImAb, however,

bindto an epitope outsidethe ligand-binding site (41 ), and

onlyoneofthem (mAb 197, as used in the present study) is

inhibitory by an interaction of its mIgG2a Fc region to the

Fc-yRI-bindingsite("Kurlander phenomenon"[4]). Interfer-encein IgG dimer bindingto FcyRIIa, however, seems

improb-able since

mIgG2a

has only minor affinity for this class of FcyR(4), indicatedfurthermore by the inability ofFcyRIIa transfected fibroblaststo support mIgG2aanti-CD3-induced

FunctionalFcyRII PolymorphismonPeripheral Blood Cells 1543

Tcellactivation( 18).Binding of dimerstoFcyRII on PMN

exhibited similar characteristics as on monocytes (Fig. 5).

After blockingFcyRIIIbonthesePMNbymAb 3G8,hIgGl andhIgG3 dimerswerestillbinding weaklytothe cells,which could beabrogatedby combining mAb 3G8 with mAb IV.3. Thefluorescence intensities observed with hIgGl,hIgG2, and

hIgG3 dimers to PMN on which FcyRIIIbwas blocked by

mAb3G8 (Figs.5 A, C, andE), werecomparableto the

stain-inglevels observed with

FcyRIII

"*_{PMN(Figs.5B,D,and F).}

These data were in agreement with data from the

anti-CD3-induced T cell proliferation studies. hIgG2 anti-CD3 mAb actedas apotent Tcell mitogen in thepresenceof mono-cytes(18), platelets(Fig.2), and neutrophils (Fig. 3), provid-ing the

FcyRIIa'

allotypic formwasexpressed. hIgGl anti-CD3mAb, incontrast, induced proliferation ofTcells in the

presence of accessory cells from all donors, regardless of

Fc'yRIIa-phenotype. This mitogenic activity wasblocked

al-mostcompletely by

anti-FcyRI

mAb197 (18)oranti-FcyRIII

mAb CLB Granl (Fig.4 C)whenmonocytes orneutrophils

were used as accessorycells, respectively. Using

FcyRIIa'/

FchyRIIIb"*PMN as accessorycells, wealsoobservedan

in-duction ofTcell mitogenesisby hIgGl anti-CD3 mAb. Our

observations indicatethatthismitogenicity isbothFcyRI-and

FcyRII-mediated,

suggested by low susceptibility to either mAb 197 ormAb IV.3 alone, butasynergistic inhibitionby

combiningtheseanti-FcyRmAb withPMNfrombothtypesof

donors.Cytofluorometric analyses ofPMNduringTcell mito-genesis experiments showed, furthermore,inductionof Fc'YRI-expressiononPMN,whichwasmediatedby

IFN-'y,

produced

byactivatedTcells. This issupported byinhibitionof

Fc'yRI-inductionin thepresence ofaneutralizinganti-IFN-ymAb.

Culturing of PMN, furthermore,affectedthe level of FcyRIIIb

expression,whichwasreduceddramatically.This

observation,

andtheabsentorweakinhibition bymAb CLBGranlF(ab')2 fragmentsand mAb3G8, respectively,suggestaminor rolefor

Fc'yRIIIb

inanti-CD3-induced Tcell activation. Thestrong

inhibitionby

anti-FcyRIII

mAb CLBGranl ofhIgGl anti-CD3-inducedTcell

mitogenesis (Fig.

4

C) is, therefore,

most likely mediatedby theFc-moiety ofthis (mIgG2a)mAb

(Kur-landerphenomenon

[41]).

Apossible explanation fortheinhibitionpatternofhIgGl anti-CD3-inducedTcell

mitogenesis (shown

inFig.4)maybe the following: hIgGl, in first instance, most likely interacts with FcyRIIIb and FcyRIIa, supported by susceptibility of

hIgGl dimer bindingonnormalPMNtoinhibitionbymAb

3G8and mAbIV.3, whichresults in activation ofTcells.

Pro-duction of

IFN-'y

bytheseactivated Tcells leadstoinduction

of FcyRI. In

parallel,

FcyRIIIb is shed from the cell

mem-brane. Thisimplicates that theseFcyR

might

be involved in hIgG1-inducedTcellmitogenicityatdifferent time

points

dur-ingthe culture.

Anti-CD3-inducedTcell

proliferation

with

platelets

as

ac-cessory cells indicated that the FcyRIIa

polymorphism

to hIgG2 was also expressed on

platelets. hIgG2

anti-CD3

in-ducedTcell

proliferation

was

only

observed in thepresenceof

Fc'yRIIal

expressingcells

(Fig. 2).

The pattern of

subclass-specificT cell

mitogenesis

inthese

experiments,

in

fact,

ap-peared identical to T cell

mitogenesis

in the presence of

FcyRIIa-transfected murine fibroblasts(18). Rosenfeldetal. (42) have shown stable interdonor variation in FcyRIIa

ex-pressionlevels. Differences

resulting

from this

polymorphism

werecontrolledfor in thisstudy,

by matching

different donors

(two

FcyRIIa'L-

and one FcoyRIIaHR-homozygous) for

Fc'yRIIa

expressionlevels ontheirplatelets (Fig. 2 A and B).

Comparing thedataobtained withhIgG3 indimer-binding and T-cell mitogenicity studies, an inconsistency was found:

hIgG3- andhIgGl-dimersbothboundFcyRpositivecells

effi-ciently, whereas the hIgGl chimeric mAb was superior to

hIgG3 in inducingTcellmitogenesis.Inthe present and

pre-vious studies,wehaveobserved that hIgG3 anti-CD3 mAb, in contrast tohIgGI anti-CD3,were poor T cell mitogens when accessorycellsfrommosthealthydonors were used ( 15, 18).

Thiswasobserved fortwo different allotypic forms of hIgG3

(15). Strikingly,we observed no differences in cytophilic

bind-ing ofhIgGland hIgG3anti-CD3mAb to

IFN-'y

treated U937

cells (P. Parren, unpublished results). Similar observations

were reported byBruggemannet al. (43) and Steplewski et al.

(44),who compared theefficacyofhIgGl andhIgG3 chimeric

mAbin antibody-dependentcellular cytotoxicity, and found a greater potencyof hIgGl. Consistent with ourobservations, the latter study also reported this remarkable difference, in

spite ofasimilarbinding of chimeric mAb of these two

sub-classestoFcyRon IFN-,y treated U937 cells (44).

Summarizingtheresults obtained withmonocytes, PMN,

and platelets, thepresentstudysuggests as subclass specificity

hIgG3 > hIgGl,hIgG2 > hIgG4 forthe

FcyRIIa'L

allotypic

form,andhIgG3,hIgGl >hIgG2>hIgG4 forthe

FcyRIIa'R

allotypicform.

UsingPMNof normal andan

_FcyRIII"'g

donor, we

ana-lyzed whether cooperation between FcyRIIa and FcyRIII is

taking place. Such cooperation has been described in

func-tional studies of FcyRII-mediated phagocytosis (37),

im-mune-complex-induced PMN actin assembly (36), and

re-leaseofhydrolyticenzymesinduced byIgManti-FcyR

autoan-tibodies (38). Thequestion ofpotential cooperation between

Fc-yR

classesseemsrelevant in that inPNHpatients whohave

strongly diminished expression of GPI-linked proteins, the

Fc'yRIIIb

expression is preserved (albeit at reduced level), likely because ofa preferential assembly of GPI-anchors to

FcyRIIIb comparedtootherGPI-linked membrane proteins (28).ThissuggeststhatcompleteFcyRIIIb deficiencymaybe harmful.Apartfromadiseased FcyRIIIb11* individual

suffer-ing from systemic lupus erythematosus (45); however, two

healthy

_FcyRIII"*

individuals have recently been described

who were deficient for PMN-FcyRIIIb expression, probably because ofadisruptionof the structural FcyRIIIBgene(29).

We have nowstudied hIgG dimer interactions withPMNof

oneofthese donors indetail. Effects of

FcyRIIIb

on IgG-bind-ingby FcyRII,however,were notevidentfromourstudy(Figs.

4and 5). After blockade ofFcyRIIIb onnormal PMN, no

differences were observed between normal and

FcyRIII"*

PMNintheirabilitytobind

hIgGl,

hIgG2,orhIgG3 dimers,

whichwasfound

FcyRIIa'u-mediated.

Interestingly,the

effi-cacyofhIgGl anti-CD3-inducedTcell

proliferation

wasnot

affected when normal PMN were substituted by

FcyRIII"9

cells and with both PMN

populations,

FcyRI and Fc'yRII seemed involved in

sustaining

Tcell

mitogenicity.

Thesedata raisequestionsabout the in vivo role ofthe third class ofFcyR,

and maypointto astructurallyredundant system.

An importantquestion regardingthefunctional FcyRIIa

polymorphismremains:Is thereaninvivo relevance?A

study

byWeeetal.(46)suggestsarelevancein

immunotherapy,

in

that differences in the

efficacy

ofmIgG1 anti-CD8 induced

clearingofTcellsin

kidney

transplant

recipients

wereobserved

betweenHRand LR individuals. In thepresentstudy,we

ob-served a significant correlation between hIgGi and hIgG2

serumlevels, and theFcyRIIa-phenotype in healthydonors. To correct for differences in total hIgG serumlevels, which varyconsiderably between adults (47), we calculated hIgGl and hIgG2 relative to hIgG serum levels in each individual.

This seems correctsince theproportionof each subclass rela-tiveto the total hIgGcontentis maintained withina narrow range(reviewed in [48]). Our datasuggestthat FcyRIIamay

insome wayregulate hIgG subclassproduction/turnover.The

mechanism behind thisregulation,however, remainstobe

elu-cidated. It seems unlikely that regulationoccurs viaFcyRII

expressedonBcells, sinceBcellsareequippedwithFcyRIIb isoforms,which donot expresstheHR-LRpolymorphism(7, 10, 34). Inthe murine system, modulation of Ig production andproliferationofBcells has been describedbysoluble FcR,

collectively termed"IgG bindingfactors." Such factorswere

alsodemonstrated in humanserum(39). Recently,molecular cloning of humanFcyRIIahasgeneratedcDNA clones lacking

atransmembranedomain(8,49),theseFcyRmay represent

putative soluble FcyRIla-molecules expressing the polymor-phism.Itwill,therefore,beinterestingtoevaluate the possible roleof solubleFcyR furtherwith respecttotheobserved

differ-encesin hIgG2serumlevelsfound in vivo.Interestingly,hIgG2 is the mainisotypeinduced in the humoralresponseafter chal-lengewith bacterialpolysaccharides,andit mediatesan

impor-tantrole inimmuneresponses tocertaincapsulatedbacteria, such as Streptococcus pneumoniae andHaemophilus

influen-zae typeb. This is indicated byanincreased susceptibilityto

thesepathogensinpatientswith selectivehIgG2subclass

defi-ciency(48). On theconditionthat theseprotective properties of hIgG2antibodiesareofcell-mediatednature,suggestedbya

low activity in Clq-binding and complement-mediated lysis (43) (although hIgG2maybeeffectiveathighantigendensities

[50]), it is important to investigate whether the

FcyRIIa-phenotypeislinked with susceptibilitytobacterialinfections.

Acknowledgments

We aregratefultoDr E. van der Schoot for help with obtaining blood from donors with decreasedFc-yRIIIbexpression levels. We thank M. de Haas and M.KleijerformeasuringsolubleFcyRIII,Dr. D. Roos for

critically readingthemanuscript, and W. Schaasberg for help with

sta-tistical analyses.

References

1.Perussia,B., E. T. Dayton, R. Lazarus, V. Fanning, and G. Trinchieri. 1983. Immuneinterferoninduces the receptor for monomeric IgGI on human

monocyticandmyeloid cells. J. Exp.Med. 158:1092-1113.

2. Repp, R., Th. Valerius, A. Sendler, M.Gramatzki,H. Iro, J. R. Kalden, and E.Platzer. 1991.Neutrophils express the high affinity receptor for IgG(FcyRI, CD64)after in vivo application ofrecombinant human granulocyte

colony-stim-ulatingfactor. Blood. 78:885-889.

3. Unkeless, J. C. 1989. Function and heterogeneity ofhuman Fc receptors for

immunoglobulinG.J. Clin. Invest. 83:355-361.

4. Van de Winkel, J. G. J. and C. L. Anderson. 1991. Biology of human

immunoglobulinGFc receptors. J. LeukocyteBiol.49:511-524.

5.Karas, S. P., W. F. Rosse, and R. J. Kurlander. 1982. Characterization of theIgG-Fc receptor on human platelets. Blood. 60:1277-1282.

6.Stuart,S._G.,E.Simister,S.B.Clarkson,B.M._Kacinski,M._Shapiro,and I.

Mellman.1989. Human_IgGFc_receptor

_(hFcRH;

_CD32)existsas_multiple iso-forms in_{macrophages, lymphocytes}and_{IgG-transportingplacentalepithelium.} EMBO(Eur.

_Mol.

Biol.Organ.) J.8:3657-3666.

7.Brooks,D.G.,W.Q. Qiu,A. D.Luster,andJ. V. Ravetch. 1989. Structure

andexpressionofhumanIgGFcRII(CD32).Functionalheterogeneityis

en-codedbythealternatively spliced productsofmultiplegenes. J. Exp. Med. 170:1369-1385.

8.Warmerdam,P. A.M.,J. G. J. Van deWinkel,E. J.Gosselin,and P. J. A. Capel. 1990. Molecular basis forapolymorphismof humanFcy receptorII (CD32).J.Exp.Med. 172:19-25.

9._Qiu,W.Q.,D. DeBruin,B.H.Brownstein,R.Pearse,andJ. V. Ravetch. 1990.Organizationofthe human andmouselow-affinity FcyRgenes: duplica-tion and recombinaduplica-tion. Science(Wash. DC).248:732-735.

10.Ravetch,J.V.,and J-P. Kinet. 1991. Fcreceptors.Annu. Rev. Immunol. 9:457-492.

11.Tax,W. J.M.,F.F.Hermes,R. W.Willems,P. J. A.Capel,andR. A. P. Koene. 1984. Fc receptors formouseIgGIonhuman monocytes:polymorphism androle inantibody-inducedT cellproliferation.J.Immunol.133:1185-1189.

12.Clark,M.R.,S.B.Clarkson,P. A.Ory,N.Stollman,and I. M. Goldstein. 1989.Molecular basis forapolymorphism involvingFc receptor IIonhuman monocytes.J.Immunol. 143:1731-1734.

13.Warmerdam,P. A.M.,J. G. J. Van deWinkel,A.Vlug,N. A. C.

Wester-daal,and P.J. A.Capel.1991. Asingleamino acid in the secondIg-likedomain of the humanFcy receptorII is critical for humanIgG2 binding. J. Immunol. 147:1338-1343.

14.Clark,M.R.,S.G.Stuart,R.P.Kimberly,P. A.Ory,and I. M. Goldstein. 1991. Asingleamino aciddistinguishesthehigh-responderfrom the low-re-sponderform of FcreceptorIIonhumanmonocytes.Eur. J. Immunol. 21:191

1-1916.

15.Parren,P.W. H.I.,M. E. J.Geerts,L.C. M.Boeije,andL. A. Aarden. 1991. Induction of T-cellproliferationbyrecombinant mouseand chimeric

mouse/humananti-CD3 monoclonal antibodies. Res. Immunol. 142:749-763. 16.Clevers, H.,B.Alarcon,T.Wileman,and C. Terhorst. 1988. The T cell

receptor/CD3 complex: adynamic protein ensemble. Annu. Rev. Immunol. 6:629-662.

17.Palacios, R. 1985. Mechanismsbywhich accessory cells contribute in growthofrestingTlymphocytesinitiatedbyOKT3antibody.Eur. J. Immunol.

15:645-651.

18.Parren,P.W. H.I.,P.A. M.Warmerdam,L. C. M.Boeije,P. J. A.Capel, J. G. J. Van deWinkel,and L.A. Aarden. 1992. Characterization ofIgG FcR-me-diatedproliferationofhuman T-cells inducedbymouseandhuman anti-CD3 monoclonalantibodies; identificationofafunctionalpolymorphismtohuman IgG2anti-CD3. J. Immunol. 148:695-701.

19.Koolwijk, P.,J.G. J. Van deWinkel,L. C.Pfefferkorn,C.W. M.Jacobs,I.

Otten,G. T.Spierenburg,and B. J. E. G. Bast. 1991. Induction of intracellular

Ca2" mobilization andcytotoxicity by hybridmousemonoclonal antibodies. Fc-yRII regulation ofFc-yRI-triggered functions or signaling? J. Immunol.

147:595-602.

20.Tunnacliffe, A.,C.Olsson,A.Traunecker,G. W. Krissansen, K. Karja-lainen,and A. De la Hera. 1989. Themajorityof CD3epitopesareconferredby theepsilonchain. InLeucocyte TypingIV: White Cell DifferentiationAntigens. W.Knapp,W. R.Gilks,E. P.Rieber,R. E.Schmidt,H.Stein,and A. E.G. Kr. Von demBorne,editors. Oxford University Press, Eynsham, Oxon,England.

295-296.

21. VanLier,R. A.W.,J.H. A. Boot, E. R. DeGroot, and L. A. Aarden. 1987. Induction of T-cellproliferationwith CD3 switch-variant monoclonal anti-bodies: Effects ofheavy-chain isotypein monocyte-dependent systems. Eur.J.

Immunol. 17:1599-1604.

22. Aarden, L. A., E. R. De Groot, 0. L. Schaap, and P. M. Lansdorp. 1987. Production of HGFbyhuman monocytes. Eur. J. Immunol. 17:1411-1416.

23. Van de Winkel, J. G. J., J. L. P. Van Duijnhoven, R. Van Ommen, P. J.A. Capel,andW. J. M. Tax. 1988. Selective modulation oftwo human monocyte Fc receptors forIgG byimmobilized immune complexes. J. Immunol.

140:3515-3521.

24.Huizinga,T. W.J.,M.Kerst,J. H.Nuijens,A.Vlug, A. E. G. Kr. Von dem

Borne,D.Roos,and P.A. T. Tetteroo. 1989.Bindingcharacteristics ofdimeric IgGsubclass complexes to human neutrophils. J. Immunol.142:2359-2364.

25. Van der Meide, P. H., M. Dubbeld, and H. Schellekens. 1985. Monoclo-nal antibodies to human immune interferon and their use in a sensitive

solid-phase elisa. J. Immunol. Methods.79:293-305.

26. Fijnheer,R.,R.N. I. Pieterz, D. De Korte, and D. Roos. 1989.Monitoring

ofplatelet morphology duringstorage of platelet concentrates. Transfusion (Paris).29:36-40.

27. Van der Schoot, C. E., T. W. J. Huizinga, E. T. Van 't Veer-Korthof, R. Wijmans, J. Pinkster, and A. E. G. Kr. Von dem Borne. 1990. Deficiency of

glycosyl-phosphatidylinositol-linkedmembrane glycoproteins of leukocytes in paroxysmal nocturnal hemoglobinuria, description of a new diagnostic

cytofluo-rometric assay. Blood. 76:1853-1859.

28.Edberg, J. C., J. E. Salmon, M. Whiltow, and R. P. Kimberly. 1991. Preferential expression of humanFc-yRIII-PMN(CD16) in paroxysmal noctur-nal hemoglobinuria. Discordant expression of glycosyl phosphatidylinisitol-linked proteins. J. Clin. Invest. 87:58-67.

29.Huizinga,T.W.J.,R. W. A. M._Kuijpers,M.Kleijer,T. W. J.Schulpen,

H. T. M.Cuypers,D.Roos,and A. E.G. Kr.VondemBorne. 1990. Maternal

genomic neutrophilFcRIII deficiency leading to neonatal isoimmune neutro-penia. Blood. 76:1927-1932.

30. Van Kooten, C., M. H. J. Van Oers, and L. A. Aarden. 1990. IL-6 en-hances humanIgproduction, but not as a terminal differentiation factor for B lymphocytes. Res.Immunol. 141:341-356.

31. Vlug, A., A. J.de Bot, H. Brouwer, G. G. De Lange, A. M. van Leeuwen, E. A. de Ron,and R. V. W. van Eijk. 1989. Monoclonal antibodies against IgG subclasses. InProtidesof theBiologicalFluids,William BeaumontHospital

Se-ries.H.Peeters, M. D. Poulik, J. R. Hobbs, J. Mestecky,andN.E.Miller, editors.

Pergamon Press, Inc.,Oxford.pp.39-47.

32. Goosen, P. C. M. 1980.Isolation ofsubclassofhuman IgGwith affinity chromatography. J. Immunol.Methods.37:89-93.

33. Van deWinkel,J. G. J., W. J. M. Tax, M. C. J. Van Bruggen, C. E. P. Roozendaal, H. W.Willems,A.Vlug, P. J. A.Capel, and R. A. P. Koene. 1987.

Characterization oftwo Fcreceptorsformouseimmunoglobulinsonhuman

monocytesand celllines.Scand.J. Immunol.26:663-672.

34.Gosselin,E.J.,M. F.Brown, C.L.Anderson,T. F.Zipf,and P. M.Guyre.

1990. The monoclonalantibody 41H16 detectsthe Leu 4responderform of humanFc'yRII.J.Immunol. 144:1817-1822.

35.Rosenfeld, S.I., andC.L.Anderson. 1989.Fc receptorsofhuman

plate-lets. InPlateletImmunobiology, MolecularandClinicalaspects. T. J.Kunicki

and J. N.George, editors.J.B.Lippincott Company, Philadelphia.337-353.

36. Brennan,P.J.,S. H.Zigmond,A.D.Schreiber,E. R.Smith,and F.S.

Southwick. 1991.Binding ofIgGcontaining immune complexestohuman neu-trophilFc'yRIIandFc-yRIII induces actin polymerizationby apertussis toxin-in-sensitive transduction pathway.J. Immunol.146:4282-4288.

37. Salmon, J. E., N. L.Brogle,J. C.Edberg,and R. P.Kimberley.1991.Fcy

receptorImlinduces actin polymerization in humanneutrophils andprimes phagocytosis mediated by Fcyreceptor II. J.Immunol. 146:997-1004.

38. Boros, P.,J. A.Odin,T.Muryoi, S.K.Masur, C. Bona, andJ.C. Unkeless. 1991.IgManti-Fc-yRautoantibodies triggerneutrophil degranulation. J.Exp. Med. 173:1473-1482.

39.Huizinga,T.W.J.,M.deHaas,M.Kleijer,J. H.Nuijens,D.Roos, and

A. E.G.Kr. VondemBorne. 1990.SolubleFc-yreceptor III in humanplasma originates fromreleasebyneutrophils.J.Clin.Invest.86:416-423.

40.Daeron, M., C. Sautes, C. Bonnerot,U.Blank,N.Varin,J.Even,P.M.

Hogarth,and W. H.Fridman. 1989. Murinetype IIFc-yreceptors and

IgG-bind-ing factors. In Structures and Functions of Low Affinity Fc Receptors. W. H. Fridman, editor. Karger, Basel. 21-78.

41. Guyre, P. M., R. F.Graziano, B. A. Vance, P. M. Morganelli, and M. W. Fanger.1989.Monoclonalantibodiesthatbind to distinct epitopes onFcyRIare

abletotriggerreceptorfunction.J.Immunol. 143:1650-1655.

42.Rosenfeld,S. I., D. H. Ryan, R. J. Looney, C. L. Anderson, G. N. Abra-ham, and J. P. Leddy. 1987. HumanFcyreceptors: stableinter-donor variation inquantitative expressiononplatelets correlates with functional responses. J.

Immunol. 138:2869-2873.

43.Bruggemann, M., G. T. Williams, C. I. Bindon, M. R. Clark, M. R. Walker, R.Jefferis,H. Waldmann, and M. S. Neuberger. 1987. Comparison of theeffector functions ofhumanimmunoglobulins usingamatched set of

chi-meric antibodies.J. Exp.Med. 166:1351-1361.

44.Steplewski,Z., L. K. Sun, C. W. Shearman, J. Ghrayeb, P. E. Daddona,

andH.Koprowski. 1988. Biological activity ofhuman-mouseIgGl,IgG2, IgG3, andIgG4 chimeric monoclonal antibodies with antitumor specificity.Proc.Natl.

Acad.Sci. USA.85:4852-4856.

45.Clark,M.R.,L.Liu,S. B.Clarkson,P. A.Ory, andI. M.Goldstein. 1990.

Anabnormality ofthe gene that encodesneutrophilFc receptorIII in a patient with systemic lupus erythematosus. J. Clin. Invest. 86:341-346.

46. Wee, S.L.,R. B.Colvin,J. M.Phelan, F. I.Preffer,T. A.Reichert,D. Berd, and A. B.Cosimi. 1989.Fc receptorformouseIgGI(FcyRII)and

anti-body-mediatedcell clearance inpatientstreatedwithLeu2aantibody.

Transplan-tation.48:1012-1017.

47.French,M. A.H., and G.Harrison.1984. SerumIgG subclass

concentra-tionin healthy adults: a studyusingmonoclonalantisera.Clin.Exp.Immunol. 56:473-475.

48.Jefferis,R., and D. S. Kumararatne. 1990.Selective IgGsubclass defi-ciency:quantificationandclinicalrelevance.Clin. Exp. Immunol. 81:357-367.

49.Rappaport,E. F., D. L.Cassel,D.0.Walterhouse, R. P.Meister,A.D.

Schreiber,and E.Schwartz. 1989.Isolationandcharacterization ofahuman

cDNAclonecoding forapotentiallysolubleform oftheFcyreceptor,FcyRII. Blood. 74:169a. (Abstr.)

50.Michaelsen,T.E.,P.Garred,and A. Aase.1991.HumanIgGsubclass patternofinducingcomplement-mediated cytolysis dependsonantigen

concen-trationandto alesserextent onepitopepatchiness, antibodyaffinityand