Reversible and irreversible loss of Fc receptor function of human monocytes as a consequence of interaction with immunoglobulin G

Reversible and irreversible loss of Fc receptor

function of human monocytes as a

consequence of interaction with

immunoglobulin G.

R J Kurlander

J Clin Invest. 1980;66(4):773-781. https://doi.org/10.1172/JCI109915.

The effects of IgG in different configurations on the Fc receptor function of human monocytes were studied. Receptor function was assessed by quantitating immune adherence and/or ingestion of human erythrocytes coated with IgG anti-D antibody.

Monomeric IgGl in solution inhibited the Fc receptor function of monocytes, but this function was restored completely after washing. In contrast, monomeric IgG that was adsorbed nonspecifically to a plastic surface inhibited the Fc receptor function of monocytes even after washing away unbound IgGl. This loss of function could be blocked by sodium azide and was reversed when the IgG adsorbed to plastic was degraded by trypsin, suggesting that loss of function was the reversible consequence of localized binding of most of the monocyte's receptors at the point of contact with immobilized IgGl. Fluid-phase aggregates of IgGl also reduced the Fc receptor function of monocytes as a consequence of direct binding to the monocyte surface. High concentrations of purified aggregates rapidly reduced Fc receptor function but function was reversed by trypsin even after incubation for 18 h. Lower concentrations of aggregates reduced Fc receptor function more slowly, but after 18 h of incubation, lost function was not restored by trypsin treatment. Because the transition from reversible to irreversible loss was blocked by sodium azide, an energy-dependent process of ingestion, shedding or denaturation of receptors is […]

Research Article

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Reversible and Irreversible

Loss

of

Fc Receptor

Function of Human Monocytes

as a

Consequence of Interaction with Immunoglobulin

G

R.J. KURLANDER, Myrtle Bell Lane Laboratory, Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710

A B S T R A C T The effects of IgG in different con-figurations ontheFc receptor function of human mono-cytes were studied.Receptorfunctionwas assessedby

(juantitating

immune adherence and/or ingestion of human erythrocytes coated with IgG anti-D antibody. Monomeric IgGl in solutioninhibited the Fc receptor

function of monocytes, but this function was restored completely after washing. In contrast, monomeric IgG

that was adsorbed nonspecifically to a plastic surfaice

inhibited the Fc receptor function of monocytes even

after washingawayunbound IgGl. This loss of function

could be blocked by sodium azide and was reversed

when the IgG adsorbed to plastic was degraded by trypsin,suggesting that loss of function was the

revers-ible consequence of localized binding of most of the

monocyte's receptors at the point of contact with

immobilized IgGI.

Fluid-phase aggregates ofIgGI also reducedthe Fe

receptor function of monocytes as a consequence of directbindingto themonocyte surface. High concen-trations of purified aggregates rapidly reduced Fc receptorfunctionbutfunction wasreversed by trypsin evenafterincubationfor18h.Lowerconcentrations of aggregates reduced Fcreceptor function more slowly,

butafter 18h ofincubation, lostfunction was not

re-storedbytrypsinitreatment.Becausethe transitionfrom

reversible to irreversible loss was blockedby sodium azide,anenergy-dependentprocessofingestion, shed-dingordenaturation ofreceptors isresponsiblefor this

irreversible loss ofFc receptor function. Rabbit IgG

anti-human IgGboundto IgG adsorbedtothe surface

ofmonocytes alsomediated aloss ofFc receptor

func-tion as aresult of the bindingofFc receptors tothe Fc

portionoftherabbitIgGmolecule,aprocessanalogous

to the binding of aggregated IgG. After irreversible depletion ofFe receptor

fuinction

by anti-IgG, partial

Portions of this work werepresentecl tothe American

So-ciety ofHematology,

Phoeniix,

Ariz., December 1979.

Receivedfor publication 7March1980andinrevisedform

30April 1980.

recovery of

fuinction

wasdetectablewithin 12-24 h of

incubation in vitro, and this recovery was blocked by cycloheximide, suggesting that new receptor

syn-thesis wasre(quired forrestoration offtunction.

INTRODUCTION

The destruction oferythrocytes coatedwith IgG

anti-body by reticuloendothelial cells in vitro (1, 2)and in

vivo (3, 4) is mediatedbyreceptors for the Fe portionof

IgG.Variations in thequantity(2, 5),configuration (5),

and subclass(6, 7) oftheantibodycoatingof the

erythro-cyteshave been showntoinfluence

profoundly

therate

of leukocyte-mediated erythrocyte

destruietion.

In

addition, recent studies suggest that variations in

reticuloendothelialfunctionalsoinfluenceFc

receptor-mediated immune clearance. Patients with systemic lupus erythematosus (8), nephritis (9), vasculitis (9), or Sj6gren's syndrome (10) sequester and

destroy

erythrocytes coated with IgG antibodyin theirspleen

more slowly than normal volunteers. This reduction in Fcreceptorfunctionmay beaprimarydisorder con-tributing to tissuie damage in these (liseases (8, 10) or

maybe

duie

tointerference with Fc receptor function by immune complexes

containinig

IgG (9, 11). In the

fol-lowingstudiesinvitro, wedemonstratethatcomplexes

containing IgG can reversibly reduce Fc receptor

function of human monocytes, and that this process

proceeds by an energy-dependent step(s) to the

irre-versible

loss of receptorfunction.Thisprocess of recep-tor degradation may be of importance in the patho-physiology of a variety ofdisease states.

METHODS

Dulbecco's phosphate-buffered satline (PBS)' (pH 7.4), New-man-Tytell sertumless medlitum (N-T), RPMI 1640, fetal calf

'Abbreviationtsused in thispajper:EA,htumanerythrocytes coated with IgGanti-Dantibody; EApapain, papain-treated EA; FCS, fetal calf sertum; N-T, Newmani-Tytell serumless

medliumiii; PBS, Dulbecco's pIhosphate-bufferedl salinie; P+S, penicillini and streptomycin soltutioni.

serum (FCS), and penicillin and streptomycin solution (P

+ S)(10,000 U/ml and 10,000Zg/ml, respectively) were

ob-tainedfromGibco Laboratories,Grand IslandBiological Co.,

Grand Island, N. Y. Latex beads (0.8 mM), cycloheximide,

pepsin (1:10,000), papain (two times crystallized in 0.05 M

sodium acetate), glutaraldehyde (25%), and pancreatic trypsin type II were obtained from Sigma Chemical Co.,

St.Louis, Mo.Sodium heparinwasobtained from UpjohnCo.

(Kalamazoo, Mich.).

Monoclonalhuman IgG wasobtained from the serumofa

singlepatientwithmultiple myeloma. The IgGlwas

precipi-tatedina 50%saturatedammoniumsulfatesolution,dialyzed free of ammonium sulfate, and purified by ion exchange

chromatographyusing DEAE cellulose and a 0.005 M phos-phate buffer at pH 8.0. The IgG was collected,

concen-trated by vacuum dialysis, and stored at 4°C with 0.01% sodium azide. Azidewas removedby dialysis against PBSbeforeuse.Theconcentrationof IgGIinthese fractions

wascalculated based on theabsorbance oflight at280 ,Lm, assuming an extinctioncoefficient (El% cm) of13.3 for IgG. Plastic microtiterwellscoated with IgGl wereprepared by incubation witha10-mg/ml solution ofmonomeric IgGlfor18

hat4°C.Coated surfaceswerethen washedwith PBS 10 times toremoveunbound IgGl.

Preparation of IgGI aggregates. Heat-treated IgGl was

prepared by incubation ofanIgGlsolution (20mg/ml)at63°C for20 min (12). Purified IgGlaggregates wereprepared by

passing heat-treated IgGI through a 1 x 80-cm column of

Sepharose4B(PharmaciaFineChemicals,Div.ofPharmacia,

Inc., Piscataway, N.J.)asdescribedby others (13). Fractions

withan apparentmolecularweight larger thanthatof

mono-meric IgG were collected, pooled, and concentrated by

vacuumdialysis.

Preparation of anti-IgG. Anti-IgG was prepared as de-scribed previously (14). Apurified IgGfraction of this anti-serum was prepared by passage ofserum dialyzed against a 0.05-M buffer through a 2.0 x20.0-cm column of DEAE

Affigel Blue (Bio-Rad Laboratories, Richmond, Calif.)using

the method recommended by the manufacturer. Fab2 frag-mentswereprepared byincubation of IgG with 1% by weight

ofpepsin in sodium acetate bufferat pH 4.4 for 36 h. Fab2 fragments were thenisolatedby passage through a 100 x 2-cmSephadex G-150column(15) (PharmaciaFineChemicals).

Preparation ofEA. Human O+ erythrocytes weredrawn and storedinAlsever'ssolutionat4°Cforup to 2wk.

Erythro-cytes tobesensitizedwith anti-Dantibodywerewashedthree timeswithPBSand50,ulofwashed, packed erythrocyteswere

incubated with20,ulof IgGanti-Dantibody(RhoGam,Ortho

Diagnostics, Inc., Raritan, N. J.) in 1ml ofPBS at 37°Cfor1h.

EAthenwerewashed fourtimesandsuspendedin12.5 mlof

RPMI 1640plus 10% FCS.

Preparation of EA using papain-treated erythrocytes

(EApapaiJd.

Papain-treated erythrocytes were prepared by incubating50,ul of packed erythrocytes suspendedin 0.4ml

of0.15 MNaCl with20,ulofpapainsolutionatroom tempera-ture for 30 min. The erythrocytes were then washed four times with PBS, suspended in 1 ml ofPBS, and incubated with 5_,lA ofanti-Dantibodyfor 1 h, washed,andsuspended

in 12.5 mlofRPMI 1640plus 10% FCS.

PreparationofEC43wascarried out using methods

previ-ouslydescribed (16). Human O+erythrocyteswereincubated with an IgM coldagglutinin antibodyand fresh human serum

for10minat4°C and10min at37°C,andthen werewashed

thoroughlytoremoveremainingantibodyand unbound

com-plement components. Erythrocytes prepared in this fashion

have been showninthe past tobe coatedwith C3 and C4. Preparationofhuman serum. 50 mlofblood were drawn

sterilely,cooled to40C,andcentrifugedat12,000 g for1min.

The plasma thenwasremoved, incubatedat37°Cfor 30 min,

andserum wasthenseparated mechanically from the clot.

Preparation of adherent monocytes. The mixture of erythrocytes, platelets, and leukocytes remaining after the removal of plasma above was mixed rapidly with 20cm3 of RPMI 1640 containing 10U/ml of sodium heparin. Thecell suspension was thenwashed threetimeswithRPMI 1640and

layeredover 20 mlof Ficoll-Hypaque (17) (Pharmacia Fine

Chemicals). The tubewascentrifugedat 500 gfor10minand

1,000 g for20 min. The mixed mononuclear cells obtained from the interface werewashedthreetimes with RPMI and wereresuspendedin RPMI1640plus 20% FCSat a concen-trationof5-10x 106leukocytes/ml.50-,ul portionsof this sus-pension were addedtowells in sterile, 96-well, flat bottom microtiterplates (Falcon Labware,Div., Becton,Dickinson& Co., Oxnard, Calif.), and incubatedat 37°Cfor30 min.Each

well was then washed six times with PBS to remove

non-adherent lymphocytes, erythrocytes, and platelets. Greater

than 90% of adherent leukocyteswere monocytes, asjudged by morphologic appearance and the capacity to ingest latex beads. Monocyte monolayers to be maintained in vitro for

<24 h were overlaid with RPMI 1640 with 10% FCS and

P +S, but formore prolonged incubations monocytes were

incubatedin N-Tmediumwith 20%autologoushuman serum

andP+S in amodification ofthe method ofJohnsonetal.(18) at37°C in amoisturized5%CO2 incubator.

Incubation of monocytes with IgGi. Unless otherwise stated, incubations were performed by suspending mixed

mononuclear cells (5x 106/ml) in medium containing IgGl in 12 x 75-mmplastic tubesat37°C.After incubation, the cells

werewashed fourtimes withPBS,resuspendedin RPMI1640

plus 20% FCS, and monocytes were allowed to adhere to microtiterplates,asdescribed above, beforeassessmentof Fc

receptor (orother) function.

Incubation of monocytes with anti-IgG. Unless

other-wisenoted, incubationwasperformed by adding50plof anti-IgG diluted 1:250 in RPMI 1640 to wells containing

ad-herent, purified monocytes prepared as described above. After incubation, eachwell waswashedatotal ofeight times

withPBS to removeunboundanti-IgGbefore theassessment of Fc receptorfunction.

Trypsin treatment of adherent monocytes. Wells

con-taining adherent monocytes werewashed fourtimeswith PBS toremoveunboundIgGl orotherprotein,and100 ,ulofRPMI 1640containing trypsin(1 mg/ml)wasaddedtoeach well. The ad-herent cells were incubated at 37°C for 30 min and were

washedagain with PBSfourtimes.

Assessmentof Fc receptorfunction. Adherent monocytes with or withoutprior treatment with trypsin were overlaid

with 50 ,ulofa0.4%suspensionofEAinRPMI 1640with10% FCS and erythrocytes werepermitted to sedimentonto the

adherentmonocytes at37°C. After incubation for60min, 50pul

of0.5%glutaraldehyde insalinewas addedto each well for

10minat roomtemperature andthen each wellwaswashed throughly with PBS, dried, and stained with Giemsa stain. Receptor activity was assessed in each well of the inverted microtiterplate at x200. Monocytes with three or more ad-herent erythrocytes or with one or more ingested, or partially

ingested, erythrocyteswereconsidered to possess Fc receptor function. The percentage of monocytes with Fc receptor func-tion was quantitated in each of three microtiter wells by

scoring at least 100 cells for evidence of interaction with EA. The mean and standard deviation was calculated for each set

of samples.Less than 1%ofmonocytesinteractedwith human

erythrocytesnotcoated with IgGantibody.

Complementreceptor function was measuredbyadding50

pl

of a 0.4% suspensionofEC43 tomicrotiter wells contain-ing adherent monocytes that had been washed asdescribed

above. Cells to be evaluated for complement receptor func-tion were nottreated withtrypsinwhichisknowntodestroy

complement receptor function (19). The cells in microtiter

wells wereincubated at370Cfor1h,treated with glutaralde-hyde, and stained as described above. The adherence of three or more erythrocytes to the monocyte membrane was considered evidence ofcomplement receptor function. The percentage of monocytes possessing complement receptor function was measured intriplicate.

Phagocytic capacity of adherent monocytes was assessed by adding50,ulofa suspension(1 x 108particles/cm3) oflatex

beads to microtiter wells containing adherent cells.

Unin-gested particleswerewashedawayaftera1-hincubationat37°C and 100cellsin each wellwerescored for evidence oflatex

ingestion underan inverted phase microscope at x 100.

RESULTS

The effectof IgGl adsorbed to plastic upon the Fc

receptorfunction of monocytes. When freshly pre-pared monocytes were permitted to adhere in

micro-titerwells, most (50- 100%) cells had demonstrable Fc receptor function as evidenced by the binding or in-gestion ofEA. However, monocytes permitted to

ad-here to microtiterwells coated withmonoclonal IgGl monomerhadmarkedly inhibited Fc receptor function.

Inhibitionwasmediatedby theFcportionofIgGI

be-cause monocyte adherence to microtiter wells coated

with Fab2fragmentsof IgGl did not alter monocyte re-ceptor function. The loss of Fc rere-ceptor function could

be prevented if IgG-coated wells were treated with trypsinbefore the adherence ofmonocytes tothewells, andcould be reversed bytrypsin evenafter the

mono-cytes had adhered to the IgGI-coated wells (Table I, experimentA). Similar inhibition of Fc receptor func-tion, which also could be reversed bytreatment with trypsin, wasproduced when IgGl (10mg/ml) was added to wells already containing monolayers ofmonocytes. Sincethe Fcreceptorfunction ofmonocytesincubated

with IgGIintesttubes andwashedtoremoveunbound

IgG before adherence of cells to wells was undimin-ished,this inhibition mustbedependent in part upon

TABLE I

Effect of IgGI MonomeronFC Receptor

FunctionofMonocytes

Percent monocytes with Fc receptor function Before trypsin After trypsin

treatment treatment

Expt. A Fc receptor function of monocytesadherent to plastic wells:

coated with IgGI monomer, coated with IgG monomer and

then treated with trypsin be-fore addition of monocytes,

coated with Fab2 fragments of IgGl,

withoutcontact with IgGI Expt. B Fc receptorfunctionof

ad-herent monocytes:

after incubation for3h in micto-titerwellswith medium contain-ing IgGl monomer 10mg/ml, after incubation with IgGI (10

mg/ml)monomer in a testtube for 3 h and washing before

transfertoplasticwells, withmedium without IgGl

mon-omer

0.3±0.6*4 97.0±2.1

95.3+2.5

95.0±+2.5

90.3+2.1

ND

ND 97.0+2.1

50.3+3.14 96.0+1.7

95.0±2.5 94.0±1.7

94.7±2.5 94.0± 1.7

* +SD.

4P<0.001.

ND, notdone.

theadherenceof IgGI to the wallsofthe microtiter well

(Table II).

Theeffect of aggregated IgGl upon the Fc receptor function of monocytes. To prevent artifactual

inhibi-tion ofFc receptor function due to adsorption of free IgG to microtiter wells, in the remaining experiments

TABLE II

Effect of PurifiedAggregatesofIgGi Upon Fc Receptor Function

% MonocyteswithFcreceptorfunction after incubation

1 h 18h

Monocytes incubatedin Without Followed by Without Followed by

mediumcontainingpurified trypsin trypsin trypsin trypsin aggregatesof IgGI treatment treatment treatment treatment

6mg/ml _22.0±30*t 87.7+2.5 4.3+2.54 84.3+ 10.6 0.6mg/ml 34.3±2.5* 61.7+5.0 4.6+1.74 9.3+0.64

Medium alone 87.3+2.6 90.0+2.6 91.7+3.8 89.3+3.5

monocytes were treated with IgGI by incubating mixed mononuclear cells with IgGl in test tubes and washing away IgGlbeforethe adherence ofmonocytes to micro-titer wells.

Fc receptorfunction ofmonocytes was markedly

re-duced by incubationwithsolutionscontaining IgGI ag-gregates. High concentrations of purified aggregates (6 mg/ml) rapidly reduced Fc receptor function, how-ever, evenafter incubation for 18 hthisinhibition was

reversed bytreatmentofmonocyteswithtrypsin(Table II).Pretreatment of monocytes with trypsin (to uncover receptor sitesblocked withcytophilic IgG)beforethe

additionofaggregatesdidnotpreventeithertheloss of

receptor function or the restoration of function

pro-duced bya secondincubationwith trypsinaftercontact withpurified aggregates (data not shown).

Monocytes incubated with a more dilute solution of

purified aggregates (0.6 mg/ml)lost Fc receptor

func-tion moreslowly, but after18 hreceptorlosswasnearly complete and was notreversed by trypsin (Table II). A 10-mg/ml solution of heat-aggregated IgG (which contained -30%aggregated IgGI and70% monomeric IgGl) reduced Fc receptor function minimally even

after incubation for 18h; however, if after incubation with this solution for 1 h monocyteswere washed and incubated inmedium alone, Fc receptorfunction was lost over thefollowing 17-hperiod (Table III).During

the first12hof incubation after washing,thislossofFc receptor functioncould be partially reversed bytrypsin

but afterlonger intervals, loss offunctioncouldnotbe reversed(Fig. 1).The failure of crude heat-aggregated IgGItoreduce Fcreceptorfunctionappearstobe due

TABLE III

Effecton Fc Receptor Function ofIncubation with IgGI Followed by Washing and IncubationinMedium

%Monocyteswith

receptorfunction Beforetrypsin Aftertrypsin

treatment treatment

Monocytes incubated for 18 h in

mediumcontainingheat-treated

IgGl, 10mg/ml 90.7±5.9* 93.7±4.6 Monocytes washed and incubated

in medium for 17 h after incu-bationfor1 hwith

monomericIgGI,10mg/ml, 99.0± 1.7 96.3±2.5

heat-treatedIgGl F(ab)2,

10mglml, 98.3±2.9 NDt

heat-treated IgGl, 10mg/ml; 1.0±1.7§ 5.0±3.6§

medium 95.3±3.1 99.0±1.0

*

±SD.

4Notdone.

§ Difference significantwithP <0.001.

O2 sU

,, 60

\ \

40

-20

-4 8 12 24

DURATIONOFINCUBATION IN MEDIUM FREE OFIg6i (hours)

FIGURE 1 Time-course ofthe loss ofFc receptor function

ofmonocytessuspendedinmedium free ofIgGafter

incuba-tionfor1 hinmedium containing heat-treated IgGl (10 mg/

ml).Monocytes Fc receptorfunction assayed: *,without tryp-sintreatment; 0, aftertreatment of monocytes with trypsin.

tothepresenceofmonomeric IgG since theloss of Fc receptor function of monocytes after a 1-h incubation

with heat-aggregated IgGI and washing could be blockedby the re-addition ofmonomeric IgG tothe

me-dium during the subsequent 17-h incubation (Fig. 2).

Heat-aggregated Fab2 fragments ofIgGl did not alter Fcreceptorfunction (Table III).

The effect of anti-IgGon Fc receptorfunction. Fc receptor function ofmonocytes was markedly

dimin-ishedby

incubation

withrabbitanti-IgG antiserum (at

dilutions of1:250to 10,000) for1-2h. Treatmentwith trypsinafter10-30minofincubation

partially

reversed the loss ofFcreceptorfunction but after

longer

incuba-tionthe loss ofFc receptorfunction wasnotreversible

(Fig.3).A

purified

IgG fraction of

anti-IgG

also had

ac-tivity,butFab2 fragments of IgG anti-IgG didnotcause alossof receptor functionand,in fact, blockedthe

re-duction of receptor function

produced

by unaltered

anti-IgG(TableIV). Normal nonimmune

rabbit

serum at similar dilutions didnotalter monocyte Fcreceptor function. Inhibition ofFc receptorfunction

produced

byanti-IgGwasblockedbytheadditionof 1 mg/mlof monomeric IgG to the incubation medium (data

notshown).

Theeffect ofFcreceptor alteration upon

phagocyto-sis and complement receptor function. Monocytes

depletedofFcreceptorfunctionasaconsequence of

TABLE IV

EffectofAn2ti-IgG Upon FcReceptorFtunictioni

%Nionocvteswith NMonoCvtesinclul)atedwith receptorftunction1

An1ti-IgG antiseruIm1 6.7+3.2*

IgGanti-IgG 3.7±+1.1l4

IgG

F(ab),

anti-IgG 91.0±5.3

IgG F(ab)2anti-IgG, 82.8+2.3

followeled by aniti-IgG

Medium 89.3±+-5.0

* +SD.

4P<0.001. Concentration of Monomeric IgGi(mg/ml)

FIGURE2 FC receptor function ofmonocytes incubated in

medium containing varying amounts ofmonomericIgGI for 17hafter incubationwithheat-treated IgGl(10mg/ml) for1h.

cul)ation with IgGl, or anti-IgG, still phagoeytosed latex beads and rosetted EC43 normally (data not shown).

The effect of sodiuim azide on receptor depletion. The lossof Fereceptorfunction associated with the

ad-herence ofmonocytes toplastic coatedwithmonomeric

IgGIwasmarkedly inhibitedbypriorincubationofthe

cells witlh sodliuimazide. Sodium azide didnot prevent

the loss ofreceptorftunction produced byincubation of

monocytes with heat-treated IgGl or anti-IgG. How-ever,itdidpartiallyblockthetransformationbywhich

IOOi

90

80

70

60

50-40

30

20-I0

10 20 30 40 50 60

MINUTES OF INCUBATION WITH ANTI-Ig6

FIGURE 3 Fcreceptorfunction ofmonocytesincubatedwith anti-IgG (1:500 dilution) for 1 h. Fc receptor function was eitherassayed directly (0)oraftertreatmentofmonocyteswith

trypsin(0).

receptorlossprodlueed by these reagentsbecame

irre-versible despitetreatmentwith trypsin (Table V). The effect of incubation in vitro upon Fc receptor

function. Fe receptor fuinction of adherent

mono-cytes was assessed after incubation in N-T with 20% autologouis human serum for 1-4 d. Because

mono-meric IgG present in serum could alter Fc receptor functionby nonspecificadherencetothe walls of micro-titerwells (see al)ove),monocytes werealwaystreated

withtrypsin justbeforeassayforFcreceptorfunction.

Fc receptor fuinction of adherent monocytes

incti-blated in vitro decreased considerably dturingthe first

2 dof culture (Fig.40) although nearlyall monocytes

stillpossessedFcreceptorsdetectable after incubation

with EApapain (Fig. 4, 0). IfFc receptor function was depleted by treatment with anti-IgG, it was restored very slowly in vitroand did notreturn to the level of undepletedmonocytessimilarlyculturedevenafter4d (Fig. 5, 0), but significant restoration ofFc receptor

functioncould be detected usingEApapainwithin 6-12h of incubation(Fig. 5, 0).Thisrestorationwasinhibited by the addition of cycloheximide (2 ,ug/ml (Fig. 6,

0) or by the deletion of serum from the incubation medium (Fig. 6, A).

DISCUSSION

Inthese experimentswe demonstrated thattheFe

re-ceptor function of human monocytes may be reduiced markedly byincubation with IgG in vitro. Loss of

re-ceptor function may result either from the reversible

saturationofsurface FcreceptorswithIgGorfrom irre-versible loss of receptor function via an energy-de-pendentprocess. MonomericIgGl insolution inhibits

interactionofmonocyteswithEApresumably by

com-peting with IgG antibody bound to erythrocytes for

alimited number ofreceptorsites,butsincebindingis

of lowaffinityandrapidly reversible, Fcreceptor

func-tion is restoredas soon as unbound IgGI monomer is washed away. However, when monocytes are per-mittedtoadheretoaplastic surface coated with mono-0

a) Cie

u

0

LL

TABLE V

Effect of Sodium AzideonDepletion ofMonocyte FcReceptor FunctionProduced byIgGI and Anti-IgG

Percentmonocytes withFcreceptor functionafter incubation in medium

Withoutsodium azide Withsodium azide Without trypsin With trypsin Without trypsin With trypsin

treatment treatment treatment treatment

Monocytes permitted to adhere to wells

coated with IgGl for1 h 11.7±2.4* ND 90.0+6.9t ND

Monocytesincubated with purified

aggre-gates, 100,ug/ml for3h 1.7±1.5 57.0±5.3 0.3±0.6§ 90.9±5.0"

Monocytesincubated with anti-IgG for2h 13.0±8.2 17.3±3.6 17.0±3.6§ 58.3±2.3" *SD.

Medium contained sodium azide(15.0

mM).

5Medium contained sodiumazide(0.15 mM).

"Fc receptorfunction significantly increased (P<0.01) compared with comparably treated cells incubated in

the absence of sodium azide.

meric IgG, thebinding ofFcreceptors toIgGl on the

lower surface ofthemonocyteresults in thepersistent loss of Fc receptor function even after unbound IgGlis

washed away.AsimilardiminutioninFcreceptor func-tion, designated "modulation" (20), has been noted

when murine macrophage (20-22) or human

mono-cytes (23)are permitted to adhere to a plastic surface coated with immunecomplexes containing IgG.

100

90

80

70,

DAYS OFINCUBATION INVITRO

FIGURE4 Fc receptor function ofmonocytes incubated in

N-Twith 20%autologousserumfor 1-4d. Fcreceptor func-tion was assessed either with "standard" EA (-) or with EApapain (0).

Ourstudies extend priorobservations in several

re-spects: (a) "modulation" is probably an

energy-de-pendent process since it isinhibitedby sodium azide, (b) modulation of human monocytes can be initiated byadsorbed monomericIgGaswell asby immune com-plexes, despite the low avidity of binding of soluble IgG monomer to the Fc receptor (24). We presume eitherthat theconcurrentadherence to the monocytes

toplastic mechanicallystabilizesthe interaction of the

Fc receptor with adsorbed IgGI or, alternatively, that

100

90 /

80

z 70

E 60

-50

40

-4

m

30-2 3

DAYS OFINCUBATIONAFTERTREATMENT WITH ANTI-Ig6

FIGURE 5 Evaluationof the rate of recovery ofFc receptor

function after treatment with anti-IgG (1:250) and incuba-tionin N-Twith20%autologousserum.FCreceptorfunction

wasassessed either with standardEA(0)orwithEApapain (0).

80

'L, 60

40-20

2

DAYSOF INCUBATION AFTER TREATMENT WITH ANTI-Ig6

FIGURE6 Evaluation ofthe rate ofrecovery of Fc receptor

function aftertreatment with anti-IgG usingEApapain to

de-tect Fereceptorfunction. Cellswereincubated inN-Twith

20% autologousserumwith(0)orwithout(0)2 ug/ml ofcyclo-heximide,or withmedium withoutserum (A).

binding ofmonomer to plastic alters the conformation ofthe IgG, permitting firm IgG-Fc receptor binding

in the absence ofantibody-antigen interaction or ag-gregation ofthe IgG and, (c) that modulation can be reversed completely bytrypsin,whichdegrades IgGl,

but doesnotdestroy theFc receptor(25).

Ourfindings supportthehypothesis (20, 21, 23) that

modulation results from the formation ofstable

com-plexes between adsorbed IgG and Fc receptors that

migrate tothe siteofcontactwith IgGfrom other parts

ofthe cell surface (Fig. 7A). Because receptor loss is

reversible, it is unlikely that receptor denaturation or

internalizationisimportant in the loss of function as has

beensuggested by others(22).Of practicalimportance,

since similaradsorption ofIgG may occur whenever concentrated solutions of IgG are incubated with monocytes adherenttoplastic, Fcreceptorfunction of

monocytes that have been incubated in medium con-taining IgG (such as autologous serum) cannot be

as-sessed accurately unless the cells are transferred from the IgG-coated surface or are treated with trypsin to

degrade adsorbed IgG.

Because aggregates of IgG are known to bind to the Fc receptor of mononuclear cells more avidly than

A

B

- Rabbitanti-HumanIgG

HumanIgG,

1FMonocyteFcReceptor

FIGURE7 Proposed mechanisms forreduction of Fcreceptor

functionof(A)monocytes incubatedin asurfaicecoatedwith IgGl monomer, (B)formonocytesincubated withaggregated IgGI and(C) formonocytestreatedwithanti-IgG.

monomeric IgG (24) with a much slower rate of (is-sociation (26), we presume that the rapid loss of Fc

re-ceptor functionfollowing incubation of monocytes with concentrated solutions of purified aggregates results from the saturation of available receptors with IgGI ag-gregates. We attribute the return of function after trypsin treatment to the degradation of IgGI aggregates stillbound to the receptors on the cell membrane. Since function was restored by trypsin, even after incuba-tion withconcentrated aggregates for 18 h, IgG-Fc re-ceptorcomplexes apparently can remain on the

mem-branesurfacewithout ingestion,shedding,or denatura-tion for prolonged periods.

When monocytes wereincuibatedwithlower concen-trationsofpurified aggregates or with heat-treated IgGl and then washed to remove contaminating monomeric IgGI, Fc receptor function gradually was lost over a 12-18-h period and could notberestored with trypsin. Weproposethatdelayed loss of Fc receptorfunction oc-curred because smaller amounts of multivalent aggre-gates bound to the monocyte membrane initially by onlyafew of the total Fc fragments available could

sub-sequently bind additional receptors (Fig. 7B). The eventual irreversibilityof receptor loss in this instance suggests that these complexes are ingested by mono-cytes as are other receptor-ligand complexes (27).

Alternatively, receptors may bedenaturedorshed,

al-thoughthere is no data as yet to support these

mecha-nisms.

It is unclear why irreversible receptor loss did not

degree of Fc receptor binding per aggregate is insuffi-cient to initiate ingestion.

Human monocytes isolated and washed in vitro have

been shown to be coated with surface IgG (28). Our findings suggest that binding of anti-IgG to this

mem-brane-bound IgG may initiate binding of Fc receptors with the subsequent ingestion oftheIgG-anti-IgG

com-plexes. Though the antibody-antigen interaction (rather than IgG-Fc receptor interaction) is responsible

for the binding ofanti-IgG to the surface of monocytes, receptor loss mediated by anti-IgG appears to proceed bythe same mechanism as described above (Fig. 7C),

since the loss of Fc receptor activity is dependent uponmembrane binding ofIgG-anti-IgG with an intact Fc fragment. The ability to deplete selectively Fc receptor function in vitro permits the quantitative study of the rate of regrowth of receptors in vitro. Such re-growth has beenevaluatedpreviously, but the methods used for depleting Fc receptorfunction have had

sub-stantial limitations (21, 29). The methods of depletion

described here are simple, specific, andwithout major

morphologic consequences to the treated cells and, thus, appear better suited to the study of membrane

receptor dynamics.

The perceivedlevelofFcreceptor function of mono-cytes incubated in vitro in these experiments varied depending upon the characteristics of the

antibody-coatederythrocytes usedtodetectreceptorfunction.Fc receptor function detected with erythrocytes coated with IgG anti-D antibody diminished substantially

during incubation invitro,andreceptorregrowth after depletion was incomplete after incubation in vitro for

4 d. In contrast, Fc receptor function detected using

erythrocytes treatedwith papainbeforeantibody

sensi-tization waspresentin >90% ofmonocytesafter2d of incubationinvitro.Afterdepletion with anti-IgG,

func-tion was largely restored within 12-24 h,arate of re-covery close tothat reported previously (21, 29). This recovery was blocked by cycloheximide and, thus, probably required the synthesis ofnew Fc receptors

in vitro.

We are unable to determine whether these dif-ferencesin

perceived

receptorfunctionreflectthe

pres-ence of two separate populations of Fc receptors

analogoustothosereportedinthemouse(30),onlyone

of which is detected by standard EA or whether the differences noted reflect only a greater propensity of

papain-treated EA for interaction with monocytes possessingalow level ofFcreceptor function.

Which-ever is the case, it is clear thatreceptor function is at

least partially restored within 24 h but that receptor function of monocytes incubatedinvitrounder the

con-ditionsdescribed,whether treated withanti-IgGornot,

is diminishedcompared with unculturedmonocytes.

Ourstudies suggest that thebindingand clearance of immune complexes containing IgG by the

reticulo-endothelial system may result in the selective deple-tion of Fc receptor function. Such a mechanism might account for the decreased clearance of IgG anti-D-coated erythrocytes noted in patients with a variety

ofdiseases sharing in common the presence of circulat-ingimmune complexes. If Fc receptor function is

selec-tivelydepleted as a consequence of immune clearance in vivo, the rate at which the reticuloendothelial sys-tem can regenerate receptor function may be an im-portantdeterminant both of the rate ofimmune

clear-ance and of the severity of immune-mediated

de-struction.

ACKNOWLEDGMENTS

We are deeply indebted to Ms. Janet Batker for technical as-sistance in these experiments and to Ms. Norma Martell for help in preparation of the manuscript.

Roger Kurlander is supported by the Medical Research Service of the Veterans Administration. This research was supported in part by National Institutes of Health grant

RO1 CA-10267-11.

REFERENCES

1. LoBuglio, A.F.,R. S. Cotran,andJ. H.Jandl. 1967. Red

cells coated with immunoglobulin G: binding and

sphering by mononuclear cells in man. Science (Wash.

D. C.). 158: 1582-1584.

2. Huber, H., S. D. Douglas, and H. H. Fudenberg. 1969. The IgG receptor: an immunologic marker for the

characterization ofmononuclear cells. Immunology. 17:

7-21.

3. Mollison,P.L.,andN.C.Hughes-Jones. 1967.Clearance of Rh positive redcellsbylow concentrations of Rh

anti-body. Immunology. 12: 63-73.

4. Schreiber,A.D., andM. M.Frank. 1972. Role ofantibody and complementin theimmune clearance and destruc-tion oferythrocytes. I. In vivo effects of IgG and IgM

complement-fixing sites.J. Clin. Invest. 51: 575-582. 5. Kurlander, R. J., W. F. Rosse, and G. L. Logue. 1977.

Qualitative influence ofantibodyand complement

coat-ing of redcells onmonocyte-mediatedcell lysis. J. Clin. Invest. 61: 1309-1319.

6. Abramson,N.,E. W.Gelfand,J.H.Jandl,andF. S. Rosen.

1970. The interaction between human monocytes and red cells. Specificity for IgG subclasses and IgG

frag-ments.J. Exp. Med. 132: 1207-1215.

7. vanLoghem,J. J.,E. W. vanderMeulen,A.Fleer,M.I.A. van der Hart, A. E. G. Kr von dem Borne, and C. P.

Engelfriet. 1977. The importance of the Fc receptor for

red cell destruction under the influence ofnoncomplement

binding antibodies inhuman blood groups. 5th

Internia-tionalConvocationofImmunology, Buffalo,N.Y., Karger AG, Basel.75-84.

8. Frank, M. M., M. I. Hamburger, T. J. Lawley, R. P.

Kimberly,andP. H.Plotz. 1979. Defective reticuloendo-thelial system Fc receptor function in systemic lupus erythematosus.N.Engl.J. Med. 300: 518-523.

9. Lockwood, C. M., S.Worrledge, A.Nicholas, C. Cotton,

andD. K.Peters. 1979.Reversal ofimpaired splenic

func-tion inpatients with nephritis orvasculitis (orboth) by

plasma exchange.N. Engl. J. Med. 300: 524-530. 10. Hamburger,M.I.,H. M.Moutsopoulos,T.J.Lawley,andl

M. M. Frank. 1979. Sjogren's syndrome. A defect in

reticuloendothelial system Fe receptor specific clear-ance. Ann. Intern. Med. 91: 534-538.

11. Cooper, S. M. 1979. N.Engl. J. Med. 300: 1486 (Letter). 12. Dickler, H. B., and H. G. Kunkel. 1972. Interaction of

aggregated y-globulin with B lymphocytes. J. Exp. Med. 136: 191-196.

13. Anderson, C. L., and H. M. Grey. 1974. Receptors for ag-gregated IgG on mouse lymphocytes: their presence on thymocytes, thymus-derived, and bone marrow-derived lymphocytes. J. Exp.Med. 139: 1175-1188.

14. Dixon, R. H., W. F. Rosse, and L. Ebbert. 1975. Quanti-tativedetermination of antibody in idiopathic thrombo-cytopenic purpura. N.Engl.J. Med.292: 230-236. 15. Stanworth,D.R.,andM. W.Turner.1978.

Immunochemi-cal analysis of immunoglobulins and their subunits. In

Handbook ofExperimental Immunology. D. M. Weir, editor. Blackwell Scientific Publications, Oxford, 3rd edition. 6.20-6.21.

16. Rosse, W. F., G. L. Logue,J. Adams, and J. H. Crookston. 1974. Mechanisms of immune lysis of the red cells in hereditaryerythroblastic multinuclearity with a positive acidified serum test and paroxysmal nocturnal

hemo-globinuria.J. Clin.Invest. 53: 31-43.

17. Boyum, A. 1968. Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Lab Invest. 21(Suppl. 97): 77-89.

18. Johnson, W. D., B. Mei,andZ.Cohen. 1977.The separa-tion, longterm cultivation,and maturation of the human monocyte.J. Exp. Med. 146: 1613-1626.

19. Henson, P. M. 1969. The adherence ofleucocytes and platelets induced byfixed IgGantibodyorcomplement. Immunology. 16: 107-121.

20. MIichl, J., NI. M. Pieezonka, J. C. Unkeless, and S. C.

Silverstein. 1979. Effects of immobilized immune

com-plexes on Fc- and complement-receptor function in

resident and thioglycollate-elicited mouse peritoneal macrophage. J. Exp. Med. 150: 607-621.

21. Rabinovitch, M., R. E. Manesias, and V. Nussenzweig. 1975. Selective phagocytic paralysis induced by im-mobilized immunecomplexes.J. Exp. Med. 142:827-838. 22. Ragsdale, C. G., and W. P. Arend. 1980. Loss of Fc re-ceptor activity after culture of human monocytes on surface bound immune complexes. Mediation by cyclic nucleotides.J. Exp. Med. 151: 32-44.

23. Douglas,S. D. 1976. Human monocytespreadinginvitro inducers and effects on Fc and C3 receptors. Cell.

Immunol.21: 344-349.

24. Segal, D. NI., and E. Hurwitz. 1977. Binding ofaffinity

cross-linkedoligomersofIgGtocellbearingFc receptors. J.Immunol. 118: 1338-1347.

25. Davey, M. J., and G. L. Asherton. 1967.Cytophilic

anti-bodyI. Natureof themacrophagereceptor.Immunology.

12: 13-20.

26. Knutson, D. W., A.Kijlstra,and L. A. van Es. 1977. Asso-ciation and dissoAsso-ciation ofaggregatedIgG fromrat

peri-toneal macrophages.J. Exp.Med. 145: 1368-1381. 27. Kaplan, J., and M. L. Nielson. 1979. Analysis of

macro-phage surface receptors. II. Internalization of

a-macro-globulin-trypsin complexes by rabbit alveolar

macro-phages.J. Biol. Chem.254: 7329-7335.

28. Slease, R. B., R. Wistar, Jr., and I. Scher. 1979. Surface

immunoglobulin density on human peripheral blood

mononuclear cells.Blood.54: 72-87.

29. Schmidt, M. E., and S. D. Douglas. 1972. Disappearance and recovery of human monocyte IgG receptor activity

after phagocytosis.J.Immunol. 109: 914-917.

30. Walker, W. S. 1976. Separate Fc-receptors for