Sheep macrophages express at least two distinct receptors for IgG which have similar affinity for homologous IgG1 and IgG2

"etermarv Immunology and Immunopathologv, 33 ( 1 9 9 2 ) 321-337

321

Elsevier Science P u b h s h e r s B V , A m s t e r d a m

Sheep macrophages express at least two distinct receptors for IgG which have similar affinity for homologous IgG 1 and IgG2 Thomas W Jungl, Thlerry Francey, Marlja Brclc, Burkhard Pohl and Ernst Peterhans lnstttute of Veterinary Virology, Umversttvof Berne, Langgass-Strasse 122, CH-3012Berne, Swztzerland (Accepted 15 November 1991 )

ABSTRACT Jung~, T W , Francey, T , Brcic, M , Pohl, B and Peterhans, E , 1992 Sheep macrophages express at least two distinct receptors for IgG which have similar affinity for homologous IgG 1 and IgG2 Iet Immunol hmnunopathol 33 321-337 Ovine bone marrow-derived macrophages ( B M M ) may express several IgG receptor ( F% receptor, FcR) subsets To study this, model particles (opsonlzed erythrocytes, EA), which are selectively handled by certain FcR subsets of human macrophages were used in cross-inhibition studies and found to react in a similar manner with FcR subsets of sheep macrophages In experiments with monoclonal antibodies against subsets of human FcR, human erythrocytes (E) treated with human anti-D-IgG (antl-D-EA hu ) and sheep E treated with bovine IgG 1 (Bo 1-EA~) were handled selectively by human macrophage FcRI and FcRII, respectively Rabblt-IgG-coated sheep E (Rb-EA s) were recognized by FcRI, FcRII and possibly also by FcRIII of human macrophages Anti-D-EA h", BoI-EA s and Rb-EA ~ were also ingested by sheep BMM Competitive inhibition tests, using various homologous and heterologous IgG isotypes as fluid phase lnhlbltors and the particles used as FcR-specific tools in man (antl-D-EA hu and Bol-EAS), revealed a heterogeneity of FcR also in sheep BMM Thus, ingestion of antl-D-EA hu by ovine BMM was inhibited by low concentrations of competitor IgG from rabbit or man In the fluid phase, but not at all by bovine IgG1, whereas ingestion of Bol-EA s was inhibited by bovine lgG1 This suggested that ant~-D-EA hu were recogmzed by a FcR subset distinct from that recognizing bovlne-lgG1 It was concluded that sheep BMM express functional analogs of human macrophage FcRI and FcRII and that Bol-EA S and antl-D-EA hu are handled by distinct subsets of BMM FcR All EA hu tested (EA hu treated with anti-D, sheep IgG 1 or sheep IgG2 ) were Ingested to a lower degree than EA s This inefficient phagocytosis could be enhanced by treatment of E A hu with antlglobuhn from the rabbit, suggesting that it is caused by a low degree of activity of opsonlzIng antibodies rather than special properties of the erythrocytes themselves Several lines of evidence suggested that both FcR subsets of ovine BMM recognize both ovine IgG1 and IgG2 In contrast, bovine lgG1 reacts with one FcR subset and bovine IgG2 interacts inefficiently with all FcR ofovlne BMM ABBREVIATIONS Anti-D-EA hu, h u m a n antl-D-IgG-coated h u m a n erythrocytes, BMM, bone marrow-derived macrophages, Bo 1-EA S, b o v i n e IgG 1-coated sheep erythrocytes, d, days, E, erytbrocytes, EA, C o r r e s p o n d e n c e to D r T W Jungl, Institute o f Veterinary Virology, University o f Berne, Langgass-Str 122, CH-3012 Berne, Switzerland © 1992 Elsevier Science P u b h s h e r s B V All rights reserved 0 1 6 5 - 2 4 2 7 / 9 2 / $ 0 5 00

322

T W JUNGIET A.L

opsomzed erythrocytes, FcR, Fc~ receptor, IDso, dose causing 50% inhibition of phagoc;ytosls PB, phosphate buffer, PBS, phosphate buffered saline, PI, phagocytic index, Rb-EAs rabbit IgG-coated sheep erythrocytes

INTRODUCTION

IgG antibodies interact with leukocytes by means of receptors specific for the Fc portion of IgG (Fc) receptors, FcR) Their activation triggers an array of effector functions, Including phagocytosis, cytotoxlclty, discharge of an oxidative burst, release of lysosomal enzymes and generation of lipid and cytoklne-type mediators of inflammation Recent studies suggest that man and mouse express three types of FcR distinct with respect to structure, antigenic characteristics, binding affinity and protease sensitivity (reviewed by Unkeless, 1986, Anderson and Looney, 1986, Klnet, 1989, Ravetch and Anderson, 1990 ) Despite these differences, all FcR types are members of the IgG superfamily (Kmet, 1989), suggesting that hgands and their receptors evolved in parallel Different cells of the defense system have disparate patterns of FcR membrane expression In particular, the expression of FcRI, a high-affinity Fc~R type, is restricted to mononuclear phagocytes and lnterferon-7-treated neutrophlls (Perussla et al • 1983, Unkeless, 1986, Looney et al, 1986a) In contrast, Fc,,RII, a low-affinity FcR type, is much more widely expressed and is found on platelets, B cells and cells of non-hematopoletlc origin (Rosenfeld et al, 1985, Looney et al, 1986b, Stuart et al, 1989) Fc~,RIII is expressed on neutrophils, natural killer cells, a subset of monocytes, and on mature macrophages (Flelt et al, 1982, Fleit and Kuhnle, 1988, Klaassen et al, 1990, Trezzlnl et al, 1990) Different FcR subtypes are up- and down-regulated by cytokines in a distinct manner As an example, FcRI is upregulated by interferon-7 (Guyre et al, 1983, Perussla et al, 1983, Yoshie et al, 1985, Jungx et al, 1988), FcRII is rather upregulated by granulocyte-macrophage colony stimulating factor (Llesveld et al, 1988 ) and FcRIII by transforming growth factor type/~ (Welch et al, 1990) In contrast to mouse and man, little is known regarding heterogeneity of Fc receptors in domestic animals Various approaches have previously been used to assess FcR heterogeneity These include the use of subtype-specific monoclonal antibodies or genetic probes, differential protease sensitivity and the differential reactivity of heterologous or homologous IgG subclasses The development of monoclonal antibodies and DNA probes specific for ruminant FcR and the application of human or murlne reagents to the study of ruminant FcR have not been reported In the present study, FcR heterogeneity of

SHEEP M ~CROPHAGE Fc RECEPTORS

323

sheep bone marrow-derived macrophages (BMM) was addressed by exploiting the subset-specificity of appropriately opsonlzed erythrocytes Erythrocytes opsonlzed in a particular manner are shown to be specific for either FcRI or FcRII of human monocyte-derlved macrophages by using FcR subset-specific monoclonal antibodies as competitive lnhlbltors Ovlne BMM are then shown to be capable of Ingesting erythrocytes opsonlzed with heterologous and homologous IgG ISOtypes in a similar manner to human macrophages Using IgG lsotypes of various sources and determining the 50% Inhibitory-dose (IDso) we show that bovine IgG1 in the fluid phase does not cross-compete the phagocytosls of human antl-D-opsonized human erythrocytes This points to an FcR type selectively recognizing the latter particle and another one selectively recognizing bovine IgG1 This is in close analogy to our previous findings with human macrophages and permitted the preparation of two model particles, sheep erythrocytes opsonlzed with bovine IgG 1 ( B o 1 -EA S) and human erythrocytes ( E ) opsomzed with human antl-D (antiD-EAhU), that serve as selective tools for two distinct FcR subsets on sheep macrophages The two subsets defined in this work are functional analogs of human FcR type I (FcRI) and type II (FcRII) The studies suggest that ovxne macrophages resemble their human and murlne counterparts by expressing more than one FcR subtype which can be distinguished by their affinity for heterologous IgG The model particles used to discriminate between the two FcR subsets may prove helpful in the further structural and functional characterization of ovme FcR There IS evidence from cattle that ruminant IgG subclasses are specific for certain FcR subsets, which are differentially expressed on various cells (McGuire et al, 1979, Howard et al., 1980) Here we provide evidence that, despite significant physlcochemlcal differences, the affinity of sheep IgG1 and IgG2 lsotypes is similar for two distract FcR expressed on BMM This points to an important functional difference between bovine and ovine IgG lsotypes MATERIALS AND METHODS Collectton and culture o f ovme bone marrow cells

Bone marrow was isolated from sterna of freshly slaughtered sheep aged 49 months from the local abattoir (Francey et al, 1992a,b) The manubrlum sterni and the adjacent three or four sternebrae were sequentially cut across with large sterile scissors, followed by scraping the red bone marrow out of the opened spongiosa with a bone curette (oval, L7) The bone marrow fragments were minced into small pieces with forceps, put in an Erlenmeyer flask and stirred for 10-15 mln Iscove's medium containing EDTA (1 mM), HEPES (25 mM) and heparin (500 U ml -~) was added to the sponglosa fragments, the suspension was filtered through sterile gauze to remove the

324

T W JUNG1 ET AL

bone fragments, centrifuged ( 10 mln, 300 × g, 25 ° C ) and washed three times with phosphate buffered saline ( 10 mM, pH 7 4, PBS) containing EDTA ( 1 m M ) at 25°C After a hypotonlc shock to lyse any erythrocytes, cells were washed three more times with EDTA-free PBS at 4°C Cell viability, as assessed by trypan blue dye exclusion, was 81% _+6% (n = 16 ) The bone marrow cell suspension (2 × 105, 5 × 105 or 10 × 105 viable cells ml - 1) was sealed into hydrophobic, gas permeable teflon bags of appropriate size (5-40 ml) and Incubated for 18-20 days (d) at 37 °C in a humidified atmosphere of 5% CO, in air, without changing the m e d i u m Iscove's m e d i u m supplemented with penicillin ( 100 IU ml - 1 ) , streptomycin ( 100 #g ml - 1), funglzone (25 /~g ml - 1 ) , glutamlne (2 m M ), nonessential amino acids for m i n i m u m essential m e d i u m (Gibco, 1%), m i n i m u m essential m e d i u m vitamin solution (Glbco, 1% ), sodium pyruvate ( 1 m M ), fl-mercaptoethanol ( 50 #M ), HEPES ( 10 m M ) , 20% fetal calf serum (FCS) and 20% autologous serum was used Teflon bags were made in our laboratory by cutting and sealing teflon foil type 100 A (Angst & Pfister, Zurich, Switzerland) to the desired size and presterllizlng with ethylene oxide Ninety-four percent of cells harvested at Day 18 or 20 showed macrophage morphology in cytospin preparations stained with Dlff-Qulk (Merz & Dade, Dudlngen, Switzerland ) and were positive for nonspecific esterase, using o~-naphthyl butyrate as a substrate (Koskl et al, 1976 ) and a 60-90 mln staining period

Culture of human monocytes Mononuclear cells were isolated by a modified Flcoll-Hypaque procedure from buffy coats of whole blood donations (Jungl and Hafner, 1986, Kuhnert et al, 1990) Monocytes were purified by selective adherence to polystyrene flasks and were dislodged after an overnight culture by vortexing Purified monocytes were sealed in teflon bags in RPMI 1640 (Jungl and Hafner, 1986 ) and cultured for up to 10 days

Antzbody and IgG preparations Rabbit affinity-purified anti-sheep E IgG was obtained from Diamedix (Miami, FL) Bovine anti-sheep E IgG was obtained from serum of a healthy cow i m m u n i z e d repeatedly with sheep erythrocytes Ovxne anti-human E IgG was isolated from serum of an ewe i m m u n i z e d with h u m a n (blood group AB/ Rh + ) erythrocytes The antl-E titer was determined by direct hemagglutlnation in round-bottom microtlter plates Bovine IgG containing antl-E and IgG from sera or colostrum of n o n l m m u n e cows, was isolated by a m m o n i u m sulfate precipitation at 40%, followed by anion exchange chromatography (Fey et al, 1976) IgG was applied to a DEAE cellulose column in 0 01 M phosphate buffer (PB), pH 7 0 The u n b o u n d material was IgG2 Using a discon-

SHEEP MACROPHAGE Fc RECEPTORS

325

tinuous NaC1 gradient of ascending molarlty, IgG 1-enriched fractions were eluted at 0 05 M and at 0 l M The latter fraction was used for the further experiments after its purity and that of the IgG2 was verified by immunoelectrophoresls, in which IgG1 moves faster than IgG2, and by Ouchterlony, using lsotype-speclfiC antIsera With both methods, no contamination of IgG1 was detected in the IgG2 fraction and vice versa Sheep IgG was prepared in a similar manner, but using 0 02 M PB, pH 7 5 Purity of the ovine IgG1 and IgG2 was verified by lmmunoelectrophoresis and assays indicating established functions of the two subclasses Thus, IgG2 binds to both protein A and protein G, whereas IgG 1 binds to protein G only The isolated IgG 1 fraction was strongly stained by enzyme-labeled protein A, whereas the IgG 1 fraction was not, both subclasses were stained by enzyme-labeled protein G Other evidence for resolution of IgG into its major two subclasses was the distinct Interaction with heterologous complement, erythrocytes opsonlzed with sheep IgG1, but not those opsonized with sheep IgG2, were lysed by diluted rabbit complement Both bovine and sheep IgG2 and IgG1 were stored frozen in aliquots at - 20 ° C Monoclonal antibodies IV 3, 3G8 and 3G8 F(ab' )2 were purchased from Medarex, Lebanon IV 3 is a blocking anti-human FcRII antibody of the mouse IgG lsotype and 3G8 is a neutrahzing antl-FcRIII of the IgG2b lsotype CE7 2a, a murlne IgG2a switch variant of CE7, which is specific for neuroblastoma cells (Schonmann et al, 1986), was kindly provided by Drs K Blaser and C d'Usclo, Swiss Institute of Allergy Research, Davos, Switzerland As monomerlc IgG2a has a high affinity for human FcRI, this antibody was used to selectively block an interaction with FcRI Human IgG (standard gammaglobuhn, 16%) and human antI-D ( 16% IgG) were obtained from the Central Laboratory of the Swiss Red Cross Blood Transfusion Service, Berne, Switzerland

Opsonlzatlon of erythrocytes IgG-coated erythrocytes (EA) were prepared by exposure of washed E to IgG antibody preparations at various subagglutinating concentrations ( 30 rain at 37°C, followed by 30 mln at 4°C), followed by repeated washing with phosphate-buffered saline (PBS) (Jungi and Barandun, 1985) EA were stored in Alsever's solution at 4 °C for up to 4 weeks Immediately before use, they were washed and resuspended in a 1 1 mixture of PBS and phagocytosls medium [minimum essential medium buffered with 30 mM HEPES, pH 7 4, containing 0 5% human serum albumin (Behrlngwerke, Marburg, Germany) ] EA coated with bovine IgG1 were always freshly prepared immediately before use

Spectrometric erythrophagocytosls assay Phagocytosls was determined by a spectrometric method (Jungl and Hal-

326

T W JUNGI ET NL

ner, 1986, Jungl, 1988) Cells were washed once with ice-cold PBS, assessed for trypan blue exclusion and resuspended in phagocytosls medium Briefly, BMM or human macrophages were allowed to adhere to mlcrotlter plates for 2 h and were then offered EA After a 1 h ingestion phase at 37°C in a humi&fled chamber and in the absence of added complement, nonlngested EA were removed by rinsing and hypotonic lysis Monolayers containing macrophages with ingested EA were detergent-lysed and a peroxldase substrate was added Color conversion catalysed by hemoglobin-associated pseudoperoxldase was proportional to the number of ingested EA and was determined in an ELISA-reader The number of phagocytosed EA was calculated by means of a calibration curve constructed from wells containing BMM and known numbers of EA added at the end of the assay Phagocytosls was expressed as phagocytic index (PI), which represented the number of erythrocytes ingested per macrophage recovered at the end of the experiment The number of macrophages per well was determined by counting the macrophage nuclei recovered from control wells in a hematocytometer, after lysmg monolayers with cetavlon-amidoblack (Nakagawara and Nathan, 1983) Each determination was performed in trlphcate and the coefficient of variation was usually < 15% When results from several experiments with cells from different donors were averaged, the coefficient of variation was normally < 45% Phagocytosis inhibition by either monoclonal antibodies or by fluid-phase IgG was tested over a broad concentratmn range with serial 3 16-fold dflutmns of antibodies Percentage mhlbmon was expressed by the formula [1

(PI ofopsonlzed E in presence ol lrlhlbltor-Pl ofnonopsonlzed E~] - \ PI ol opsonlzed E in absence of inhibitor- PI of nonopsonlz~d E ,/J× 100

Inhibitions between - 2 5 % and +25% were not regarded as significant Inhibition of less than - 5 0 % (1 e actual enhancement) was assumed to be caused by natural ant~-E antibodies and results with such IgG preparations were disregarded The 50% inhibitory dose (ID5o) was calculated from the linear regression obtained from the data points within the quasl-hnear portion of the dose-inhibition curves (usually between 10% and 90% inhibition ), using logarithmic values for the abscissa (IgG concentrations ) RESULTS

Antl-D-coated EA and bovme IgG l-coated EA as tools fop probing FcR ~ubO'pe-~pectfiCltV m human maoophages Freshly Isolated human monocytes were reported to ingest bovine IgGlcoated EA (Bol-EA ~) by FcRII only (Jungl et al, 1989, 1990), based on complete inhibition of ingestion by blocking antl-FcRII antibody Th~s was

SHEEP MACROPHAGEFc RECEPTORS

32 7

confirmed and extended in the present study to mature human macrophages (Fig 1 ) Thus, ingestion of Bo 1-EA s was strongly inhibited by monoclonal anti-FcRII at concentrations as low as 0 32 #g ml-1, but not by antl-FcRIII Murlne IgG2a, which interacts with high affinity with FcRI, did not inhibit uptake of Bo 1-EAs either In contrast, ingestion of anti-D-coated human EA (anti-D-EA hu ) was inhibited by murine IgG2a m a dose-dependent manner, an indication of FcRI involvement Antl-FcRIII antibodtes dose-dependently inhibited phagocytosls of antl-D-EA hu, an effect probably mediated by the Fc portion Thus, F(ab')2 fragments of the same anti-FcRIII did not block ingestion of anti-D-EA hu The same F(ab' )2 preparation bound to human macrophages and to a subset of human monocytes in similar amounts, as did intact antl-FcRIII, as revealed by flow cytometry (data not shown) Phagocytosis of rabbit IgG-coated EA (Rb-EA) was partially inhibited by antiFcRII, by murlne IgG2a and by intact anti-FcRIII, suggesting that more than one FcR subset (possibly all three) were involved These experiments established the use of antl-D-EA hu and Bo 1-EA Sas tools for probing selective interaction with human FcRI or FcRII, respectively

Phagocytosls of lgG-coated erythrocytes by owne BMM BMM from sheep were tested for ingestion of IgG-coated erythrocytes Erythrocytes sensitized by various subagglutlnatlng concentrattons of IgG of heterologous or homologous origin were offered, including those used as FcRselective tools in the human model (see above) The efficiency with which erythrocytes were taken up by BMM was variable (Fig 2 ) As sheep EA were generally ingested m approximately ten-fold higher numbers than human EA, Bol-EA

100

anti-D-EA

Rb-EA

A~ A

~ 75 x: 50

~ 25

0 ..C n

1

10

100

1

10

[Monoclonal antibody]

100

i

i

i

1

10

100

(/~g/ml)

Fig 1 The inhibition of human macrophage erythrophagocytosis by monoclonal antibodies selectively interacting with FcR subtypes Antibody IV 3 ( • ) is specific for FcRII, CE7 2a, a murlne IgG2a (11) interacts with its Fc portion with FcRI, 3G8 ( • ) and 3G8 F ( a b ' )2 ( A ) are specific for FcRIII Test EA were bovine IgG-l-coated sheep erythrocytes ( B o l - E A ) , rabbit ]gG-coated sheep erythrocytes (Rb-EA) and ant~-D-treated human erythrocTytes (antl-D-E a,) Data shown are means of a representative experiment performed in tnphcate

328

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X3

"13

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30

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0

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Dllubon of anhserum F~g 2 Phagoeytosls by BMM of erythrocytes from man (E h~, open symbols) or sheep (EL closed symbols) opsonlzed with various lgO anti-erythrocyte (E) preparaUons @-@, affinitTypurified rabblt-anU-EL I1-11, bowne IgGl-antl-EL A - A , bovine IgG2-anta-E~, O - O , anu-Dtreated E"", ~-~>, sheep lgGl-anU-E h~, V - V , sheep IgG2-anU-E "u Data are expressed as phagocytic radices (E& ingested per macrophage) and are the means of three experiments all performed m replicate

the efficiency of opsonlzatlon could be compared only within groups of EA types sheep or human Sheep EA coated with bovine IgG1 or with rabbit IgG were phagocytosed avidly, whereas a low degree of ingestion was observed when they were coated wtth bovine IgG2 (Fig 2) Human EA optimally opsonlzed with human IgG (anti-D) or with sheep lgG1 or IgG2 were all ingested to a similar degree The relatively low number of human EA, which had twice the volume of sheep EA, could be enhanced by a second opsonlzatlon with antl-IgG Thus, EA sensmzed first with homologous IgG followed by treatment with a second antibody directed against the first one (antiglobulin) were phagocytosed by BMM in higher numbers than control cells covered with the first antibody only (Table 1 ) This pointed to a relaUvely low efficiency of all three types of opsomzing antibodies (anti-D, sheep IgG 1, sheep IgG2 ) rather than any special properties of erythrocytes of human origin as a reason for the low PI achieved with human EA Evidence for at least two dlstmct Fc receptors expressed by ovlne B M M The question whether ovlne BMM express more than one FcR type with distinct specificity for heterologous isotypes was investigated next To this end, phagocytosls of optimally sensitized EA was allowed to proceed in the presence of varying concentrations of fluid phase IgG serving as a competitive inhibitor and IDso values were calculated The results showed that phagocytosls of anti-D-EA "u was not inhibited by any lsotype of bovine lgG, but

SHEEP MACROPHAGEFc RECEPTORS

329

TABLE 1 Phagocytosls by BMM of human erythrocytes sensitized with sheep IgG and subsequently with antlglobuhn Ddutmn of first antibody 1 160

1 320

1 640

1 1280

nd2 nd nd

423 133 77

09 89 52

02 58 24

0 96 44 19

05 43 17

01 20 08

nd nd nd

Ftrst antibody sheep IgG l Second antibody none rabbit anti-sheep IgG 1 1600 rabbit anti-sheep IgG 1 3200

Ftrst anubodv sheep IgG2 Second antibody none rabbit anti-sheep IgG 1 3200 rabbit anti-sheep IgG 1 6400

IE were opsomzed with the first antibody, washed, subsequently opsomzed w~th the second antibody and washed again 2n d not done ~Phagocytlc index (mean of three experiments ), expressed as number of EA ingested per macrophage TABLE2 Inhlb~tmn of BMM erythrophagocytosls by fluid-phase IgG Inhibitor IgG

Human IgG Bovine IgGl Bovine IgG2 O~lne IgG1 Ovme IgG2

Particles offered to BMM Antl-D-EAhu

Bovine IgG 1-EAS

9 81 (3) 1 > 10000 (4) > 10000 (3) 111 (2) 160 (3)

1082 2380 > 10000 2317 2317

(4) (7) (4) (4) (4)

1The ID~o the concentration causing 50% lnhlbltmn of phagocytos~s, expressed in/tg m l - 1 Numbers in parentheses refer to the number of experiments from which the geometric mean has been determined

was inhibited by low concentrations of human IgG (Table 2) Conversely, ingestion of Bo 1-EA s was inhibited by bovine IgG1, human IgG and by both ovlne IgG lsotypes in a similar concentration range This is consistent w~th the hypothesis that anti-D-EA hu are handled by a FcR type not recognizing bovine IgG 1 It follows that Bo 1-EA S are recognized by an o w n e macrophage FcR type distract from that recognizing antl-D-EA hu and it was assumed that ovlne BMM ingest antl-D-EA h° and Bo 1-EA Svia functional analogs of human FcRI and FcRI1, respectively (Anderson and Looney, 1986, Ruegg and Jungl, 1988, Jungl et a l , 1989) Henceforth FcRI and FcRII are used as operational terms for describing the two distinct FcR subsets of ovlne BMM An IDso in the range of 10/zg m l - 1 for FcRI is consistent with human FcRI being a highaffinity FcR and an IDso in the range of 2 mg m l - ~ for FcRII corresponds to the low-affinity for IgG of human FcRII

330

T W JUNGI ET AL

Lack of distraction by ovlne FcRI and FcRI1 between homologous IgG1 and IgG 2 The above evidence, for two FcR types with distinct affinity for particles covered with heterologous IgG isotypes, raised the question whether FcRI and II also distinguish between sheep IgG 1 and IgG2, which occur at different concentrations in serum, milk and inflammatory lesions (Butler, 1973, 1983, Johnson et al, 1983 ) This was addressed in several ways Firstly, ovine IgG1 and IgG2 were used as competitive lnhlbltors in phagocytosls assays with either FcRI-speclfiC or FcRII-specific particles Secondly, inhibition of ingestion of erythrocytes coated with either ovlne IgG 1 or IgG2 by IgG1 and IgG2 was evaluated Ingestion of anti-D-treated E ha was inhibited by both ovlne IgG1 and ovlne IgG2, IDs0 values being approximately 20-fold higher than that of human IgG (Table 2 ) The inhibitory activity of IgG1 was better than that of IgG2 Likewise, inhibition of Bo 1-EASuptake was achieved by IgG 1 and by IgG2 at similar concentrations, IDso values being in the range of that of bovine IgG1 Cross-competition experiments showed that ovlne IgG 1 and IgG2 both inhibited the phagocytosis of E hu opsonlzed by ovlne IgG 1 and of E hu opsonlzed by IgG2 at similar concentrations (Table 3) In this purely homologous system, IgGl was 20 times more active than IgG2 These results are consistent with the hypothesis that both FcRI and FcRII recognize either ovlne IgG lsotype Still another approach to probe FcRII specificity for ovine IgG subclasses was to inhibit the uptake of particles, sensitized with either ovlne IgG1 or ovlne IgG2, with bovine IgG1 in the fluid phase The concentration range to be tested was limited by the presence of opsonlzing, natural antibodies against human erythrocytes in bovine IgG 1, becoming manifest at concentrations > 50 pg ml-1 In the testable concentration range, phagocytosis of erythrocytes coated with either ovlne subclass was clearly inhibited, whereas uptake of antiD-EA h~ was unaffected (Fig 3) Using sheep EA coated with either bovine IgG1 or rabbit IgG, the former particles were completely prevented from upTABLE3 Inhibition o f B M M erythrophagocytosls by o v m e IgG in the fluid-phase Inhibitor IgG

O v m e IgG 1 O v l n e IgG2

Particles offered to B M M O v m e IgG 1-E hu

O v l n e IgG2-E hu

247 ~ 3162

145 5288

~The IDso the c o n c e n t r a t i o n causing 50% inhibition o f p h a g o c y t o s l s , expressed m p g m l - 1 (geometric m e a n of three i n d e p e n d e n t e x p e r i m e n t s )

SHEEPMACROPH4,GEFc RECEPTORS

IO0

331

~ ~ 0 ~ _ ~ _ ~ 0 / 0

N

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o

, none

~/

'

'

'

1

3 2

10

i

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31 6

[Bov,ne IgG1] (/zg/ml) Fig 3 Phagocytosls of various opsomzed EA in the presence of fluid-phase bovine IgG 1 Er~throc)tes were antx-D-EAhu ( O ) and human erythrocytes coated with sheep IgG1 ( & ) or sheep lgG2 (x7) Results of a representative experiment performed in triplicate are shown

take at high bovine IgG1 concentrations, whereas the latter could not be blocked completely (data not shown) DISCUSSION

Investigation of the phagocytes of mouse and man suggested that they express up to three antlgenlcally and structurally &stlnct receptors for the Fc port~on of IgG (Unkeless, 1986, Anderson and Looney, 1986, Ravetch and Anderson, 1990) This report provides the first evidence that sheep bone marrow-derived macrophages (BMM) also express at least two Fc receptors which share certain properties with those of human macrophages Both FcR subsets defined in this work interact with homologous IgGl and IgG2 The &stmctlon between two FcR subsets was made possible by the use of opsomzed particles shown to be recogmzed solely by one subset of human Fc receptors, demonstrated by the use of monoclonal antibodies which selectively blocked the function of the FcR subsets Thus, uptake of anU-D-treated human erythrocytes was inhibited by murlne IgG2a, the Fc portion of which has a high affimty for FcRI, but not by an antl-FcRII monoclonal antibody or by F9 (ab')2 fragments blocking FcRIII This proves the selectivity of antlD-EA hu for FcRI Conversely, Bo 1-EA s were found to interact only with FcRII, as monoclonal ant~-FcRII blocked phagocytosls of Bo 1-EAs at low concentrations, whereas antl-FcRIII and murlne IgG2a did not lnh~blt The behavior of erythrocyte-bound bovine IgG 1 was reflected by that of fluid-phase IgG 1, which selectively interfered with the uptake of particles handled by FcRII On the other hand, human IgG, corresponding in subclass composition to the

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preparation used to coat antl-D-EA, interacts with both FcRI and FcRII in fluid-phase inhibition assays The F c R subset specificity ofanti-D-EA hu is the result of the particular arrangement of these antibodies on the erythrocyte surface and is probably not caused by species or subclass restriction Notably, D-specific antibodies are non-hemagglutinatlng and fall to activate the classical complement pathway when b o u n d to erythrocytes, although the antibody lsotypes involved (IgG3, IgG1, IgM) are able to interact in principle, with C l q (Nydegger and Kazatchklne, 1983, Issatt, 1985) The observed h u m a n F c R subtype specificity of anti-D-EA, B o l - E A S and fluid-phase bovine IgG1 indicated the existence of more than one F c R on sheep macrophages Thus, in competitive experiments, inhibition of antl-DEA uptake was found to follow a different pattern to that of BoI-EA S It was intriguing that, despite their phylogenetic distance, both human and sheep macrophages showed an entirely analogous behavior in their interaction with Bo 1-EA s and anti-D-EA hu This raises the posslblhty that these particles m~ght serve as F c R subtype-selective agents in other species as well Murlne IgG2a and IgG3 have a high affinity for h u m a n FcRI and a much lower affinity for FcRII and FcRIII, whereas murlne IgG1 and 2b preferentially interact with human FcRII (Lubeck et al, 1985, Anderson and Looney, 1986, Ravetch and Anderson, 1990) These agents of murlne origin have been used to address FcR heterogeneity in man (Van de Wlnkel et a l , 1988 ) but not yet in sheep As both bovine IgG1 and h u m a n antl-D IgG can be isolated in large quantities much more readily than murine IgG subclasses, Bol-EA ~ and anti-DEA hu may serve as alternatives to m u r m e IgG lsotypes to probe F c R subtype specificity Several other methods lend themselves to addressing F c R heterogeneity FcR-subtype-spec~fic genetic probes and monoclonal antibodies are not yet available in sheep A method to distinguish between different FcR is to induce F c R modulation by surface-bound IgG interacting with one FcR type only This has been used successfully in mouse and man (Michl et a l , 1979, Ezekowltz et al, 1983, Sung, 1985, Van de Winkel et al, 1988) Consistent with our findings, goat IgG more readily modulated FcRII than FcRI, whereas rabbit IgG modulated FcRI rather than FcRII (Ezekowitz et al, 1983 ) However, selective modulation was not absolute and F c R comodulatlon has also been reported to occur (Sung, 1985 ) Surface-bound IgG modulates both Bo lEAS-specific F c R ( F c R I I ) and antl-D-EAhU-speclfiC F c R ( F c R I ) of h u m a n monocytes (M B r o c and T W Jungi, unpublished observation, 1990) and bovine IgG loses F c R subtype specificity when b o u n d to polystyrene (L Schalch, unpublished observation, 1991 ) Yet another posslbdlty for FcR subtype d i s t i n c t i o n is the variation In protease sensitivity, m u r m e FcRI is sensitive to trypslnlsation but murlne FcRII IS not (Unkeless, 1977, Pontzer and Russell, 1990) However, human FcRI appears to be more trypsin-resist-

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ant In sheep macrophages, there do not appear to be any differences m trypsin sensitivity of phagocytosls via FcRI and via FcRII (T W Jungl, unpublished observation, 1991 ) Ruminants express at least two physlcochemlcally easily distinguishable IgG isotypes, which appear at different concentrations in serum, milk and inflammatory effusions (Butler, 1973. 1983, Johnson et al, 1983) IgG2, but not IgG1, from cattle, goat and sheep is cytophlllc for homologous PMN (Watson, 1976, McGulre et al, 1979, Fleit et al, 1986) In cattle, evidence for two PMN FcR types with lsotype preference has been provided (Mossmann et al, 1981 ) Thus, PMN-rosetting IgGl-erythrocytes are inhibited more readlly by IgG 1 than by IgG2a and vice versa One possible interpretation of these findings is that bovine PMN rosette IgG 1-erythrocytes via a FcRII analog and IgG2a-erythrocytes via another FcR (FcRIII analogg) However, ruminant lgGl~ but not IgG2, is cytophihc for homologous alveolar macrophages and monocyte-derlved macrophages (McGulre et al, 1979, Howard et al, 1980, Flelt et al. 1986) This would suggest that ruminant macrophages lack a receptor expressed on PMN although this is at variance with results obtained in man and mouse (Ravetch and Anderson, 1990) The differences between PMN and macrophage FcR in sheep have not been addressed here However, the lack of macrophage cytophlllc properties of IgG2 does not exclude the possibility that this subclass interacts with FcR at a lower affimty Our data are consistent with the view that sheep IgG2 is recognized by homologous macrophages In cross-competition experiments, fluid-phase IgG2 was 20 t~mes less effective in inhibiting IgG 1- or IgG2-opsonlzed particles than fluidphase IgG 1 (Table 3) In contrast, when tested with particles interacting with one FcR subset only (antl-D-EA hu and Bo 1-EAs), the inhibitory power of IgG 1 and IgG2 was virtually the same (Table 2) This affinity difference may be caused by the contribution of a further FcR subset which could not be analysed in isolation, but which does interact with homologous IgG 1 Ovlne FcR appear to interact more efficiently w~th heterologous IgG (e g from rabbit or man) than with homologous IgG, a conclusion based on two pieces of evidence Firstly, the uptake of EA hu opsonlzed with ovlne IgG was inefficient, but could be enhanced by treatment with heterologous antxglobuhn (Table 1 ) Secondly, the IDs0 value of fluid-phase ovlne IgG was higher than that for rabbit or human IgG This observation may be restricted to FcRI and FcRII and may not be vahd for the other Fc receptors which have not been probed here The possibility that ovlne IgG might be more susceptible to the isolation procedures used than bovine IgG and have been partially inactivated has been ruled out by comparing the inhibitory power, on a molar bas~s, of ~solated sheep IgG with that of whole, heat-reactivated serum (data not shown) Consistent w~th our findings, an earher lmmunofluorescence study suggested that sheep alveolar macrophages bind little monomerlc IgG1 and no IgG2 (FleIt et al, 1986)

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The observation that ovine FcRII mediates phagocytosis of particles opsomzed by bovine IgG1 is also of practical importance as IgGl is the major antibody tsotype in bovine colostrum (Butler. 1973, 1983) IgG 1 is often used to protect small rummants that for some reason fad to receive homologous colostrum For example, a program to eradicate Caprme Arthritls-Encephahtls in Switzerland is based on the removal of kids from their mothers prior to uptake of colostrum, thereby preventing a verttcal transmission of the virus (Peterhans et al, 1988 ) These kids are given bovine colostrum, or commercial IgG made therefrom If data from sheep can be extrapolated to goat, one would predict an efficient interaction of the heterologous IgG w~th cellular Fc receptors of the recipient In conclusion, evidence for the expresston of two dtstlnct FcR types by ovlne macrophages has been presented The &stlnCtlon is based on different avidity for particles covered by heterologous IgG These FcR subtype-selective parttcles (antl-D-EA hu and Bo 1-EAs) may prove useful in the further characterlzatlon of structure, signal transductton and function of ovlne FcR Both FcR types investigated mediate phagocytosls Contrary to expectattons based on earlier reports (Watson, 1976, Yasmeen, 1981, Flelt et al, 1986), the two major sheep IgG isotypes do not have a preference for one of the two described FcR subsets of macrophages ACKNOWLEDGMENTS

This work was supported by the Swiss National Science Fund (grant no 31-26248 89 to TWJ and 31-288 l0 90 to EP) The generous g i f t of bovine anti-sheep red blood cell antiserum by Dr R Zwahlen, Department of Veterlnary Pathology, University of Berne, is gratefully acknowledged REFERENCES Anderson, C L and Looney, R J , 1986 Human leukocyte IgG Fc receptors Immunol Toda) 7 264-266 Butler J E, 1973 Synthesis and distribution of bowne lmmunoglobullns J Am Vet Med Ass, 163 795-798 Butler, J E, 1983 Bovine lmmunoglobuhns, an augmented rewew Vet Immunol Immunopathol, 4 43-152 Ezekowltz, R A B Bampton, M and Gordon, S, 1983 Macrophage activation selectively enhances expression of Fc receptors for IgG2a J Exp Med, 157 807-812 Fey, H , Pfister H , Messerh, J , Sturzenegger N and Grohmund, F , 1976 Methods for isolation, purification and quantltatlon of bovine immunoglobuhns Zbl Vet Med B 23 2693O0 Flelt H B and Kuhnle, M 1988 Biochemical characterization of an Fc gamma receptor purLfled from human neutrophds J Immunol, 140 3120-3125 Fleet H B, Wright, S D and Unkeless J C , 1982 Human neutrophll Fc gamma receptor distribution and structure Proc Natl Acad Scl U S A 79 3275-3279

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Van de Wlnkel, J G , van Duonhoven H L , van Ommen, R and Capel, P J , 1988 Selective modulation of two human monocyte Fc receptors for IgG by immobilized ~mmune complexes J Immunol, 140 3515-3521 Watson, D L 1976 The effect ofcytophilic IgG2 on phagocytosls by ovlne polymorphonuclear leukocytes Immunology, 31 159-165 Welch G R , Wong, H L and Wahl, S M , 1990 Selective reduction of Fc gamma RIll on human monocytes by transforming growth factor-beta J Immunol, 144 3444-3448 Yasmeen D 1981 Antigen-specific cytophilic activity of sheep IgG1 and IgG2 antibodies ~kust J Exp Blol Med Scl, 59 297-302 Yoshle, O 4.so, H , Sakaklbara, A and Ishlda, N , 1985 Differential effects of recombinant human interferon-alpha A / D on expression of three types of Fc receptors on murlne macrophages in VlVOand in vitro J Interferon Res, 5 531-540