CLINICAL

Vol.

IMMUNOL~Y

AND

62, No. 1, January,

IMMUNOPATHOL~Y

pp. 16-24,

1992

A Common Epitope Is Recognized by ~onoclonal Antibodies Prepared against Purified Human Neutrophil FcyRlll (CD16) HOWARD B. FLEIT,~ATHERINE ~e~~rtrnent

o~Patho~ogy,

State

D.KOBASIUK, Uniuersity

ofNew

NANCYS.PERESS, York

FcyRIII is one of two FcyR constitutively expressed by human neutrophils. We have prepared a panel of antiFcyRIII mAb following immunization of mice with FcyRIII purifmd from human neutrophih. Ten mAb which reacted with neutrophils, NK cells, and monocyte-derived macrophageswere produced. Immunohistochemical staining demonstrated that these mAb also identified macrophagesin the red pulp of spleen. Competitive cross-inhibition binding assays demonstrate that nine of the ten mAb reacted with a common epitope that is spatially associatedwith the ligand binding site. Thesenine mAb blocked the binding of immune complexes to neutrophils by 65 to 90%. In addition, two other anti-CD16 mAb, which also blocked immune complex binding to neutrophils, inhibited the binding of each of these nine mAb to neutrophils. One of the mAb produced here, 214.1, failed to block immune complex binding. In addition to immunoprecipitating the native FcyRIII glycoprotein, mAb 214.1 was capable of immunoprecipitating a 28.kDa polypeptide following deglycosylation of FcyRIII isolated from neutrophils. The results of cross-competition experiments suggest that mAb 214.1 may recognize the epitope identi~ed by mAb BW209f2. Thus mAb 214.1 identifies a polypeptide epitope distinct from the ligand binding site of FcyRIII on neutrophils. D 19% Academic PRSS. IIIC.

INTRODUCTION Three distinct classes of FcyR have been identified on human leukocytes based on affinity for ligand, molecular size, cellular distribution, and antigenic differences identified by mAb (1,2). FcyRI (CD641 is a highaffinity FcyR, binds monomeric IgG, and is expressed on mononucle~ phagocytes (3). FcyRII (CD321 is a lowaffinity receptor with broad cellular distribution (4). FcyRII is found on monocytes, neutrophils, eosinophils, B cells, platelets, and hematopoietic cell lines. FcyRIII (CD161 is also a low-affinity receptor present on neutrophils, NK cells, and mature mononuclear phagocytes (5-8). The cDNAs for each of these receptors have been molecularly cloned and demonstrate that FcyR are members of the Ig supergene family (9-15). FcyRIII exists in two alternative membrane forms, a transmembrane-anchored form expressed on NK cells and ~~-1229/92

$1.50

0 1692 by Academic Press, Inc. of reproduction in any form reserved.

Brook,

Stony

SHELLEY Brook,

New

ARRIAN York

FLEIT

11794-8691

macrophages and a phosphatidylinositol glycananchored form expressed on neutrophils (16, 17). In addition to having structurally distinct membrane-anchored forms of FcyRIII on different cell types (18, 19), heterogeneity exists in the structure of FcyRIII expressed on neutrophils. A biallelic neutrophi1 antigen system with three phenotypes has been identified and shown to be associated with FcyRIII (20). These phenotypes were defined originally by alloantisera and are homozygous (NAlINAl, NA2/NA2~ or heterozygous (NAlNA2) 121). mAb to FcyRIII have been beneficial in identifying polymorphisms in this molecule. Thus mAb CLBGRAN 11 and GRM 1 identify the NAl and NA2 phenotype, respectively (22). In the studies presented here, we have examined fur. ther the immunologic structure of FcyRIII on neutrophils. mAb were prepared from mice immunized with FcyRIII purified from neutrophils obtained from mul tiple donors (23) to determine whether polymorphisms could be identified. The results of these studies demon strated that these mAb recognized a common epitope on FcyRIII. Nine of ten mAb isolated for these studies blocked immune complex binding to neutrophils and were competitively inhibited by mAb 3G8. Another mAb isolated for these studies, 214.1, identified a polypeptide epitope distinct from the ligand binding site on FcyRIII. MATERIALSANDMETHODS Cells Peripheral blood was obtained by venipuncture from healthy adult volunteers. Heparinized blood was separated on Ficoll-H~aq~e gradients (Pharmacia Fine Chemicals, Piscataway, NJf (24). Mononuclear cells (MNL) were isolated from the Ficoll-Hypaque interface and neutrophils were recovered from the pellet by dextran sedimentation and hypotonic lysis of residual erythrocytes. Monocytes were isolated by adherence to plastic as previously described (251. Purification

of FcyRIII

from PMN

FcyRIII was purified from neutrophils which had been stored at - 30°C as described previously (23). 16

Copyright All rights

at Stony

AND

MONOCLONAL

Preparation

ANTIBODIES

TO Fc-yRIII

of mAb to FcyRIII

Competitive

A BALBic female mouse (17 g) was immunized intraperitoneally with 5 pg of FcyRIII in complete Freund’s adjuvant (GIBCO, Grand Island, NY) followed by three weekly injections of 5 pg FcyRIII in incomplete Freund’s adjuvant. The final immunization was given intravenously in saline and the spleen was removed 4 days later. Spleen cells (10’) were fused with lo7 P3X63-AG8.653 cells as described (26). The hybrids were plated in ten 96-well plates in RPM1 containing 10% FBS, 10% NCTC 109, and HAT. Detection of mAb to FqRIII

by ELISA

FcyRIII (IO p.g/ml) was boiled in 0.5% SDS in 0.1 W phosphate buffer, pH 7, for 3 min and diluted to 50 rig/ml in 0.1 &# sodium carbonate buffer, pH 9.6, containing 0.02% NaNs. Wells of Immulon I ELISA plates (Dynatech, Alexandria, VA) were coated with 5 ng of FcyRIII for 2 hr at 37”C, washed three times with 0.05% Tween 20/0.9% NaCl, and incubated for 2 hr sequentially with 100 ~1 of hybridoma supernatant, 40 pg/ml of goat anti-mouse IgG (Fc fragment specific) Ftab’):! (Cappel, Malvern, PA), and rabbit anti-goat IgG conjugated with alkaline phosphatase (Sigma Chemical Co., St. Louis, MO), diluted 1:lOO. The substrate, p-nitrophenyl phosphate (Sigma), was added at 1 mg/ml in 0.1 M glycine buffer, pH 10.4, containing 1 mM’ MgC12 and 1 m&f ZnCl,. The plates were read in a Dynatech ELISA reader at 405 nm. Anti-FcyRIII secreting hybridomas were subcloned by limiting dilution. The isotypes of all of the anti-FcyRIII mAb were determined to be IgGl, by ELISA (SBA ~lonot~ing System, Southern Biotechnology Associates, Birmingham, AL). Ascites fluids were prepared by injecting 5 x lo5 hybridoma cells into the peritoneal cavity of BALB/c mice which had been primed twice with 0.5 ml of 2,6,10,14-tetramethylpentadecane (Aldrich Chemical Co., Milwaukee, WI). Par~~~ation

and Iodi~utian

RECOGNIZE

A COMMON

Binding

EPITOPE

17

Assays

The binding of 12?labeled anti-F~~RIII was performed as described previously (5, 28). Nonspecific binding of anti-FcyRIII mAb was determined in the presence of a 20- to loo-fold excess of unlabeled antibody. For competitive binding assays, iodinated antiFcyIII mAb were used at half-maximal concentrations in the presence of increasing concentrations of unlabeled competing mAb. Eleven mAb to CD16 were obtained as ascites fluids from the Fourth International Workshop on Leukocyte Differentiation Antigens (29). Inhibitory mAb were defined as those resulting in ~25% of binding by the iodinated mAb. Flow Cytometry MNL, neutrophils (l-2 x 10” per sample), and monocyte-derived macrophages (1 x lo5 per sample) were incubated with 50 ~1 of mAb anti-FcyRIII or MOPC 21 IgG (an isotype control) for 45 min at 4°C. Cells were washed twice with PBS-5% FBS and incubated with 50 ~1 of FITC-conjugated goat anti-mouse IgG F(ab’)2 (Jackson ImmunoResearch, West Grove, PA) at 75 pg/ml for 45 min at 4°C. Cells were washed twice with PBS-5% FBS, fixed for 10 min in 100 pl of 10% phosphate-buffered formalin, and then washed with PBS0.02% NaN, prior to analysis by flow cytometry. For two-color flow cytometry MNL were incubated with phycoerythrin-conjugated Leu 19 (CD56) (BectonDickinson, Mountain View, CA) immediately after incubation with FIT&conjugated goat anti-mouse IgG F(ab’)2. Flow cytometry was performed on a FACStar (Beckton-Dickinson) equipped with a 2-W argon laser which provided exciting light at 488 nm at an incident power of 275 mW. Emitted light was filtered through a 530nm bandpass filter. The fluorescence intensity of 10,000 MNL, neutrophils, or monocyte-derived macrophages, gated for granularity and size, was collected on a logarithmic scale and presented linearly as channel numbers from 1 to 256.

of Immanog~oba~~ns

Immunoglobulin preparations were purified from ascites fluid by two precipitations with saturated ammonium sulfate followed by DE-52 chromatography. Immune complexes of rabbit anti-DNP-BSA IgG and DNP-BSA were prepared at a 7:l ratio (IgG:DNP-BSA) as previously described (5). Fifty micrograms of mAb anti-Fc~RIII was labeled with 1 mCi of 12?I using Iodobeads (Pierce Chemical Co., Rockford, IL) (27). Labeled protein was separated from free -iodide by passage over a 0.4-ml column of Dowex 1X-8Cl-. Specific activities of anti-FcyRIII mAb ranged from 2.84 x lo6 to 1.23 x lo7 cpm/Fg protein. Rabbit anti-DNP IgG was labeled to an average specific activity of 3.91 X lo6 cpm/+g protein.

Samples of normal human spleen obtained at autopsy or from fresh surgical specimens were frozen in liquid nitrogen-chilled isopentane. Immunohistochemistry was performed as described previously (30). Serial air-dried cryostat sections, with or without brief (5 min) fixation in absolute acetone, were preincubated with normal horse serum for 30 min at room temperature, followed by incubation in a humidified chamber at 4°C for 24 hr with 50 kg/ml of anti-FeyRIII mAb or MOPC 21 IgG. Sections were washed with PBS, incubated for 1 hr with a biotinylated horse anti-mouse IgG (Vector Labs, Burlingame, CA), washed again with PBS, and incubated for 1 hr with ABC-glucose oxidase (Vector). The slides were washed with PBS and incu-

18

FLEIT

60

ET AL.

1

I

I

,

0

I

,

I

,

30

I

,

I

, ’

60

CD16 FIG. 1. Binding of anti-FcyRIII mAb to MNL determined by flow cytometry. MNL were incubated sequentially with anti-FcyRIII mAb, FITC-conjugated goat anti-mouse IgG F(ab’)B, and phycoerythrin-conjugated Leu 19 (CD561 and analyzed by flow cytometry as described under Materials and Methods. To determine reactivity with NK cells in the MNL population gates were set on the lymphocyte population based on size and granularity. The nonspecific binding of the negative controls, MOPC 21 IgG, and phycoerythrinconjugated mouse IgGl was used to establish the fluorescence gates and are represented by the fluorescence in quadrant 3. This panel, which is representative of all ten anti-FcyRIII mAb, displays the staining of mAb 214.1 and phycoerythrin-conjugated Leu 19 (CD56). Ten percent of the gated cells were found in quadrant 2, indicating staining by both mAb 214.1 and Leu 19. The data are representative of two separate experiments.

bated with the substrate, nitroblue tetrazolium, for 1 hr. Slides were washed with PBS and fixed with 4% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.6.

oxidase for 30 min at 4°C (31). Labeled cells were washed four times with PBS containing 10 mM KI and lysed in 1 ml of lysis buffer containing 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, and 0.23 U/ml of aprotinin. The lysate was centrifuged at 11,500g and the supernatant was collected. The supernatant was incubated overnight with 50 ~1 of either 3G8 Fab or 214.1 conjugated to Sepharose and then washed three times with 0.6 M NaCl, 0.0125 M KPO,, 0.02% NaN,, three times with 0.05% Nonidet P-40, 0.1% SDS, 0.3 M NaCl, 10 mM Tris-HCl, pH 8.6 (MDB buffer), and three times with 0.5% deoxycholate, 0.1 M NaCl, 10 n& Tris-HCI, pH 8. Adsorbed protein was eluted with 160 ~1 of 0.5% SDS and 0.1 M P-mercaptoethanol by boiling for 10 min. Eluted protein was diluted to 0.17% SDS, 0.033 M p-mercaptoethanol, 0.2 M sodium phosphate, pH 8.6, and incubated with 10 U/ml of N-glycanase in the presence of 10 mM l,lOphenanthroline hydrate and 1.25% Nonidet P-40 at 37°C overnight (32). Digested protein was incubated with 50 ~1 of rabbit anti-FcyRIII F(ab’)B conjugated to Sepharose or with 5 ~1 of anti-FcyRIII mAb 214.1 ascites overnight. Complexes of deglycosylated FcyRIII and mAb 214.1 were precipitated with rabbit antimouse IgG F(ab’ )2 conjugated to Sepharose. The precipitates were washed twice with MDB buffer and prepared for electrophoresis as described and analyzed on 5-10% gradient gels (33). Kodak X-Omat AR film was exposed at - 70°C to the dried gels with an enhancing screen. RESULTS

Immunoprecipitation, Deglycosylation, Polyacrylamide Gel Electrophoresis

and Selection

Neutrophils (8 x lo71 were labeled with 2 mCi of lz51 using 32 pg of lactoperoxidase and 0.14 U of glucose

FLUORESCENCE

of Anti-FcyRW

Of 578 hybrid-containing rived which reacted with

mAb wells, purified

10 clones were deFcyRIII as deter-

INTENSITY

FIG. 2. Binding of anti-FcyRIII mAb to neutrophils determined by flow cytometry. and prepared for flow cytometry as described under Materials and Methods. The dotted incubation with MOPC 21 IgG. The solid line represents staining with the indicated experiments.

Neutrophils were incubated with line represents immunofluorescent mAb. The data are representative

the indicated mAb staining following of three separate

MONOCLONAL

ANTIBODIES

TO FcyRIII

RECOGNIZE

A COMMON

19

EPITOPE

~~~*~~*~~*~~I*~~ 0

100

200

0

loo

200

0

100

Fluorescence

200

0

100

200

0

loo

200

Intensity

FIG. 3. Binding of anti-FcyRIII mAb to monocyte-derived macrophages determined by flow cytometry. the indicated mAb and prepared for flow cytometry as described under Materials and Methods. The dotted staining following incubation with MOPC 21 IgG. The solid line represents staining with the indicated of three separate experiments.

Macrophages were incubated with line represents immunofluorescent mAb. The data are representative

mined by ELISA. The OD 405 nm ranged from 0.479 to 1.637, which was 10 to 33 times background. Determination of Anti-FcyRIII mAb Binding to MNL, Neutrophils, and Monocyte-Derived Macrophages by Flow Cytometry

Previous studies have demonstrated that antiFcyRIII (CD16) mAb can identify polymorphisms in FcyRIII among the different cell types expressing this molecule. Thus mAb B73.1 recognizes FcyRIII on all NK cells but only on neutrophils from 50% of the donors (34). Moreover, mAb CLBGRAN 11 and GRM 1 identify the NAl and NA2 allotypes on FcyRIII on neutrophils, respectively. (22). However, CLBGRAN 11 does not recognize CD16 on NK cells or macrophages (25, 35). Because of these observations, we examined the binding of these ten anti-FcyRIII mAb to mononuclear cells (Fig. l), to neutrophils (Fig. 21, and to monocyte-derived macrophages (Fig. 3). To characterize the population of cells in the mononuclear cell fraction which reacted with the antiFcyRIII mAb, two-color flow cytometry was performed. Cells were stained sequentially with the anti-FcyRIII mAb, FITC goat anti-mouse IgG, and phycoerythrinconjugated Leu 19 (CD56). The results of these experiments demonstrated that between 9 and 13% of the lymphocytes stained with both anti-CD16 mAb and Leu 19, indicating that they are NK cells. Figure 1 displays the staining of mAb 214.1 and Leu 19 and is representative of the staining displayed by all ten mAb. All of the anti-FcyRIII mAb reacted with neutrophils (Fig. 2). Two peaks were observed, a relatively small

FIG. 4. Immunohistochemical localization of FcyRIII in human spleen. Cryostat sections of a quick frozen surgical sample of normal human spleen were incubated as described under Materials and Methods. (a) Section of spleen stained with mAb 214.1. An intense reaction product is observed in the red pulp and around a small vessel (arrows). (b) Section of spleen stained with MOPC 21. No reaction product is observed in the red pulp which occupies the unstained area on the left two-thirds of the photograph; some reaction product is observed around the small vessel. The sections were not counterstained. Magnification is 125 x .

20

FLEIT

number of low intensity cells and a large population of much brighter cells. The low intensity peak may represent binding of the anti-CD16 mAb to eosinophils as observed previously (4). All of the anti-FcyRIII mAb bound to monocytederived macrophages (Fig. 3). However, heterogeneity was noted in the level of fluorescence intensity with which these mAb bound to macrophages. Thus, mAb 30.2 and 58.4 bound with less intensity than the other anti-FcyRIII mAb. Immunohistochemical in Spleen

Localization

of FcyRIII

In addition to being expressed in vitro on monocytederived macrophages, FcyRIII has been identified on mononuclear phagocytes from the lung, liver, and spleen (5, 7, 36). Therefore, we examined the expression of epitopes identified by mAb to FcyRIII in spleen sections derived from quick-frozen histologically normal surgical and autopsy specimens. All of the antiFcyRIII mAb we prepared reacted with sections of spleen with the surgical specimens consistently yielding a more positive result. Figure 4a demonstrates that the FcyRIII staining was observed primarily in the red pulp and marginal zone where the splenic macrophages predominate. Competitive

Binding

Assays

To determine whether these anti-FcyRIII mAb identify multiple epitopes on FcyRIII, a series of competi-

ET AL.

tive binding assays was performed. Saturation binding curves with radiolabeled mAb were performed on neutrophils. The half-maximal saturating concentration of iz51-labeled mAb was used in the competition assays together with unlabeled competing mAb ranging from equal concentrations to a 20-fold excess. This concentration range of the corresponding unlabeled mAb was found to inhibit the iodinated mAbs by >75%. Inhibitory mAb were defined by their ability to reduce binding of iodinated mAb to