Identification of an Fc receptor for IgG1 and IgG2 on guinea-pig polymorphonuclear leukocytes

Mokcular Immunolog.~, Vol. 24, No. 8, pp. 831-837, Printedin Great Britain.

1987

6

0161-5890/87 $3.00 f0.00 1987 Pergamon JournalsLtd

IDENTIFICATION OF AN Fc RECEPTOR FOR IgGl AND IgG2 ON GUINEA-PIG POLYMORPHONUCLEAR LEUKOCYTES TOHORU NAKAMURA,

KOICHI TAMOTO, JWN-ICHI MAEYAMA, HIDEKI SATO, TOSHIRO SHIMAMURAand JIRO KOYAMA Department of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo, 060, Japan

(First received 24 October 1986; accepted in revised form 30 January 1987) Abstract--Ovalbumin (OA)-complexed guinea-pig IgGl and IgG2 antibodies were found to bind to homologous polymorphonuclear leukocytes (PMNs). As these bindings are assumed to be mediated by certain Fc receptors (FcRs) for IgGl and IgG2, the variety and properties of the FcRs on the cells were investigated by the use of two monoclonal antibodies to guinea-pig macrophage FcRs which were prepared by Shimamura T. et al., 1987 (Molec. Zmmun. 24,67-74): VI A2 IgGl to the FcR for IgGl and IgG2 (FcR,,*) and VII Al IgGl to the FcR for IgG2 (FcR,). PMNs were shown to bind the Fab’ of VI A2 IgGl (VI A2 Fab’) by flow cytofluorometry, suggesting that the cells possess a certain FcR which cross-reacts antigenically with macrophage FcR,,, In fact, VI A2 Fab’ inhibited completely the binding of OA-complexed IgGl antibody to the cells. When the FcR was isolated by affinity chromatography on the F(ab’), of VI A2 IgGl coupled to Sepharose, it gave a 55,000 mol. wt band on sodium dodecylsulfate-polyacrylamide gel electrophoresis, as in the case of macrophage FcR, *. The number of the FcR molecules per PMN cell was estimated to be 2 x lo4 by measuring the binding of ‘**I-VI A2 Fab’. The binding of OA-complexed IgG2 antibody to PMNs was also inhibited with VI A2 Fab’, but partially. This finding indicates that the FcR bound by VI A2 Fab may be an FcR,,, which is able to bind both OA-complexed IgGl and IgG2 antibodies, and also that PMNs possess another FcR, namely FcR, which binds IgG2 antibody alone. The Fab’ of VII Al IgGl (VII Al Fab’), on the other hand, did not exhibit any inhibitory activity on the bindings of OA-complexed IgGl and IgG2 antibodies to PMNs. Since no evidence indicating the binding of VII Al Fab’ to PMN cells was obtained by flow cytofluorometry, the FcR, of PMNs may be antigenically different from its macrophage counterpart. In conclusion, these results indicate that two distinct types of FcR for IgG isotypes exist on guinea-pig PMN cells: FcR,,, similar to macrophage FcR,,,, and FcR, distinct from macrophage FcR,.

INTRODUCTION As in the case of macrophages, polymorphonuclear leukoctyes (PMNs) express membranous Fc receptors (FcRs) for IgG isotypes which play an important role in some defense mechanisms, i.e. phagocytosis, 0; production and lysosomal enzyme release (Kudoh et al., 1985; Washida et al., 1980; Baxter et al., 1983; Starkey, 1977). The available information, however, suggests that PMNs differ from homologous macrophages with regard to variety and properties of FcRs. For instance, both human neutrophils and macrophages possess the 40,000 mol. wt FcR, but the 51,00&73,000 mol. wt FcR of PMNs seems distinct antigenically and immunochemically from its monocyte counterpart (the 72,000 FcR) (Looney et al., 1986qb; Fleit et al., 1982; Kulczycki, 1984). In addition, Coupland and Leslie (1983) reported that guinea-pig peritoneal neutrophils do not exhibit any FcR activity for homologous IgGl, though guineapig peritoneal macrophages possess FcRs for both homologous IgGl and IgG2. As reported in previous papers (Shimamura et al., 1986, 1987), we prepared mouse monoclonal antibodies to FcRs of guinea-pig peritoneal macro-

phages; one (VI A2 IgGl) is an antibody to FcR,,* for IgGl and IgG2, and another (VII Al IgGl) is an antibody to FcR, for IgG2 alone. To clarify the variety and biochemical properties of FcRs on guinea-pig peritoneal PMNs, we investigated the effects of these monoclonal antibodies on the bindings of hen ovalbumin (OA)-complexed IgGl and IgG2 antibodies to the cells. The existence of two types of FcRs on the cells and the isolation of an FcR for both IgGl and IgG2 by using VI A2 IgGl are reported in this paper. MATERIALS AND METHODS

Isolation

of

PMNs

Guinea-pig peritoneal exudate cells containing PMNs were harvested after elicitation with casein, and fractionated with Percoll density gradient centrifugation, as described by Kudo et al. (1985). In brief, guinea-pigs were intraperitoneally injected with 15 ml of 3% casein, 16 hr before harvest of peritoneal cells. After removal of the contaminating tissue debris and erythrocytes by sedimentation in 2.47% gelatin-containing saline and by hypotonic treatment 831

TOHORU NAKAMURA et

832

with 0.2% NaCl, the cells were washed with Krebs-Ringer phosphate buffer, pH 7.4 (KRPB), and suspended in a Percoll solution (sp. gravity: 1.040) containing Dulbecco’s phosphate-buffered saline without Ca” and Mg2+ (D-PBS). The cell suspension was layered on another Percoll solution (sp. gravity: 1.080) in a centrifuge tube. After addition of D-PBS as a top layer, the cells were centrifuged at 3000 rpm for 15 min. The cells at the bottom and at the lower interphase were collected and again fractionated in the same manner as in the first Percoll density fractionation. The cells at the lower interphase were washed with D-PBS and used as PMN. More than 95% of the cells thus purified were identified to be PMNs by Wright staining. The bindings of antigen-antibody

Fab’ or F(ab’), of mouse normal IgG in 0.1 ml of 10mM phosphate-buffered saline at pH 7.2 (PBS) supplemented with 0.1% BSA, 0.05% NaN, and 5 units/ml of heparin (BSA-PBS) at 4°C for 20 min. After washing with BSA-PBS, the cells were reacted with fluorescein isothiocyanate 2.5/lg of (FITC)-conjugated F(ab’), of rabbit IgG anti-mouse IgG antibody in 0.05 ml of BSA-PBS at 4°C for 15 min. The cells thus treated were washed 3 times with BSA-PBS and suspended in 2 ml of BSA-PBS. The cells were analyzed on an Ortho Spectrum III flow cytometer (Ortho Diagnostic Systems). The green fluorescence of PMNs (2500 cells) gated for low angle light scatter and 90°C angle light scatter to exclude some contaminating macrophages, lymphocytes, dead cells and cell debris, was measured.

complexes to PMNs

The bindings of OA-complexed IgG antibodies to PMNs were determined, as described in a previous paper (Shimamura et al., 1986). The OA-complexed IgGl and IgG2 antibodies were prepared by incubating ‘2SI-IgG1 and IgG2 anti-OA antibodies with OA at a molar antibody to antigen ratio of 1: 10 in KRPB. PMNs (1 x 10’ cells) were incubated with the OA-complexed antibodies in 1.O ml of KRPB supplemented with 0.2% NaN, and 0.5% bovine serum albumin (BSA) for 2 hr at 4°C. After incubation, the cells were washed 3 times with the same buffer. The amounts of OA-complexed antibodies bound on the cells were determined by measuring the radioactivity bound. When the inhibition of bindings of OAcomplexed antibodies by monoclonal anti-FcR antibodies were assessed, PMNs (1 x 10’ cells) were preincubated with the Fab’ of each antibody for I hr at 4°C and then the bindings of OA-complexed antibodies to the cells were measured. Preparation and jiagmentation FcR antibodies

al.

qf monoclonal

anti-

Monoclonal anti-FcR antibodies, VI A2 IgGl for macrophage FcR,., and VII Al IgGl for macrophage FcR, were the products of each hybridoma (VI A2 and VII Al) prepared by fusion of splenic cells of mice immunized with guinea-pig peritoneal macrophages with a myeloma cell line, as described by Shimamura et al. (1986, 1987). These antibodies were purified from ascites fluid of mice intraperitoneally injected with the hybridomas secreting anti-FcR antibodies, by ammonium sulfate fractionation and DEAE-cellulose column chromatography. The F(ab’)z of each monoclonal antibody was obtained by digestion with pepsin, and the Fab’ fragments were prepared from these F(ab’), by reduction with 2-mercaptoethanol, as described previously (Shimamura et al., 1986. 1987). Analysis of’ the bindings of monoclonal PMNs by jlow cytojluorometry

antibodies

to

PMNs (I x lo6 cells) were incubated in the presence or absence of 0.5 pg of VI A2 Fab’, VII Al

Analysis of the binding reactions qf monoclonal FcR antibodies to PMNs

anti-

The binding reactions of the Fab’ of monoclonal anti-FcR antibodies to PMNs were performed as in the case of macrophages (Shimamura et al., 1986). PMNs (I x IO’ cells) were reacted with varying amounts of “‘I-VI A2 or 1251-VII Al Fab’ in 1.0 ml of KRPB supplemented with 0.02% NaN, and 0.5% BSA for 2 hr at 4-C with gentle shaking. After centrifugation, the cells were washed 3 times with the same buffer and the amounts of the Fab’ bound to the cells were determined by measuring the radioactivity bound. The binding constant and the number of antigen molecules per PMN cell were estimated by Scatchard plotting of the data obtained. Isolation of the untigen of VI A 2 IgG I ,from the PMN membrane PMNs (1 x lo8 cells) radioiodinated by the method with lactoperoxidase (Cone and Marchalonis, 1974) were solubilized with Nonidet P-40, and the antigen of VI A2 IgGl was purified by affinity chromatography on a VI A2 F(ab’),-Sepharose 6B column as in the case of isolation of the antigen on macrophages (Shimamura et al., 1986, 1987). In brief, the solubilized ‘251-PMNs were centrifuged, and the supernatant was adsorbed with OA-Sepharose 6B to remove ‘*‘I-proteins nonspecifically adsorbed on the affinity carrier. For this purpose, the supernatant was incubated with 100~1 of OA-Sepharose 6B suspended in 0.5% Nonidet P-40 dissolved in PBS (NP-PBS) for 1 hr at 4-C. After centrifugation, the unbound fraction was reacted with 100 ~1 of VI A2 F(ab’),-Sepharose 6B in 1.O ml of NP-PBS for 1 hr at 4’C. The Sepharose beads were washed more than 4 times with NP-PBS, and the antigen was dissociated from the beads by suspending the beads in 0.05 ml of 0.125 M Tris-HCl buffer, pH 6.8, containing 8M urea and 4% SDS with subsequent heating in a boiling water bath for IOmin. For identification of the antigen, sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the “‘I-antigen isolated was performed with 10% gel slab with the

Identification of an Fc receptor for IgGl and IgG2 on guinea-pig PMNs discontinuous buffer system of Laemmli (1970). The ‘2SI-antigen in the dried gel was visualized by autoradiography. Preparation of guinea-pig and rabbit antibodies Guinea-pig IgGl and IgG2 antibodies to OA were highly purified from hyperimmune serum by fractionation with ammonium sulfate, DEAE-cellulose chromatography, and affinity chromatography with OASepharose 6B. To remove the contaminating antibody of another IgG isotype, the IgGl or IgG2 antibody to OA thus purified was further passed through a column of Sepharose 6B coupled to rabbit antibody specific to IgG2 or IgGl isotype (Tamoto and Koyama, 1976; Sugiyama et al., 1981; Yamashita and Koyama, 1984). No contamination by another IgG isotype was detected for each antibody preparation by Ouchterlony double diffusion with rabbit antiserum to guinea-pig IgG. Rabbit IgG antibodies were also purified by fractionation with ammonium sulfate and DEAEcellulose chromatography. Anti-guinea-pig IgG isotype antibodies were further purified by passing through a column of Sepharose 6B coupled to another guinea-pig IgG isotype, to remove some crossreactive antibodies. Anti-mouse IgG antibody was purified by affinity chromatography with mouse IgGSepharose 6B. The purified antibody was digested with pepsin (Nisonoff et al., 1960) and the F(ab’), was reacted with FITC, as described by Goding (1976). The molar ratio of FITC to F(ab’)z reacted was 2. The FITC-F(ab’), obtained was purified by gel filtration on a Sephadex G-100 column. Labeling of antibodies with radioactive iodine Proteins (Grossberg

were labeled with “‘1 by an ICI method et al., 1962).

Preparation of Sepharose beads coupled to proteins Proteins were coupled to cyanogen bromideactivated Sepharose 6B to prepare affinity columns, as described in a previous paper (Shimamura et al., 1986). Protein determination The absorbance at 280 nm was used for calculation of protein concn. The values of absorbance of 1% protein at 280 nm were taken as 7.35, 13.5 and 13.5 for OA, IgG and Fab’, and the mol. wt values as 45,000, 150,000 and 50,000, respectively. Materials The following materials were obtained from the indicated sources; lactoperoxidase from bovine milk from Sigma, St. Louis, MO; carrier-free Na ‘25I from New England Nuclear, Boston, MA; Nonidet P-40 from Nakarai Chemicals, Kyoto, Japan; Percoll and Sepharose 6B from Pharmacia Fine Chemicals AB, Uppsala, Sweden.

833

RESULTS The bindings of IgG 1 and IgG2 antibodies to PMNs To estimate the FcR activities of PMNs for IgGl and IgG2, the bindings of OA-complexed IgGl and IgG2 antibodies to the cells were examined. PMNs (1 x 1O’cells) were reacted with various amounts of 1251-IgG1 and lZSI-IgG2 anti-OA antibodies complexed with OA at a molar ratio of 1: 10. The binding of the F(ab’), of IgG antibody complexed with OA to the cells was also measured for the assessment of nonspecific binding of OA-complexed IgG antibodies. The amount of OA-complexed F(ab’), bound was found to markedly increase on increasing that of added complex beyond 5 pg (data not shown). Therefore, the binding curves obtained by addition of (r5 pg of OA-complexed IgG antibodies are presented in Fig. 1, where the amounts of bound complexes on the cells are plotted against those of added complexes. The bindings of both the IgG antibodies occurred dose-dependently. The amounts of OA-complexed IgGl antibody specifically bound were markedly smaller than those of OA-complexed IgG2 antibody. The comparison of the binding curve of OA-complexed IgGl antibody with that of OAcomplexed F(ab’), , however, demonstrates clearly that the OA-complexed IgGl antibody is able to bind to PMN cells through a certain FcR. The specific bindings of OA-complexed antibodies were estimated by subtracting the amounts of OAcomplexed F(ab’), bound to the cells from those of OA-complexed antibodies. Based on Scatchard plotting of these binding data, the apparent association constants (K,) and numbers of FcRs per cell were calculated. The number of FcRs for IgGl antibody was estimated to be 1.7 x lo4 molecules per PMN cell. This value was about one-fifth that of FcRs for IgG2 antibody (1.0 x lo5 molecules). The affinity of IgGl antibody to its FcR (K,: 3.4 x 10’ M -‘), on the other hand, seems to be of the same order of magnitude as that of IgG2 antibody (K,: 4.0 x 10’ Mm’). The bindings of anti-Fc receptor antibodies to PMN As the bindings of OA-complexed IgGl and IgG2 antibodies to PMNs were demonstrated, the FcRs involved in the reactions were assessed by the use of mouse monoclonal antibodies to macrophage FcRs. VI A2 and VII Al IgGl, which are antibodies specific for macrophage FcR,,, and FcR,, respectively, were used for this purpose, and the bindings of VI A2 Fab and VII Al Fab’ to PMN cells were examined by flow cytofluorometry. PMNs (1 x lo6 cells) were incubated with VI A2 Fab’ or VII Al Fab’, and then reacted with FITC-F(ab’), of rabbit anti-mouse IgG antibody. When the fluorescent cells (2500 cells) were analyzed on a flow cytofluorometer, almost all of the VI A2 Fab’-incubated cells were stained, indicating that the PMN cells used possess an FCR similar to macrophage FcR,., (Fig. 2). On the contrary, only a

TOHORU NAKAMURA et al

834

I =

_ OO Antibody

added (JJCJ:

\

.

Bound hntibody’F)M

x IO”!”

Fig. 1. The bindings of OA-complexed IgG antibodies to PMNs. Various amounts of OA-complexed IgGl (0) and IgG2 antibodies (a), and OA-complexed F(ab’)* of IgG2 antibody (A) were incubated with PMNs (1 x 1O’cells) at 4°C for 2 hr. After washing the cells, the amounts of immune complexes bound were determined (a). The specific binding reactions of OA-complexed IgGl (0) and IgG2 antibodies (0)

are analyzed by Scatchard plotting (b).

small part of the cells incubated with VII Al Fab’ or F(ab’), of normal mouse IgG were stained, as in the case of the cells incubated without any Fab’ or F(ab’),. This finding shows that the PMN cells do not possess any antigen specifically bound by VII Al IgGl, suggesting that the FcR for IgG2 on the PMN cells, if present, is antigenically distinguishable from macrophage FcR,

As VI A2 Fab’ was found to bind to PMNs, the number of the antigen molecules specific to VI A2 IgGl on a PMN cell was estimated by measuring the binding of 1251-labeled VI A2 Fab’ to PMNs (Fig. 3). Analysis of the binding data by Scatchard plotting gave a value of 2 x lo4 for the number of the antigen molecules per cell. This value is consistent with the number of IgGl-binding sites per PMN cell estimated

b

c

* d

2 125’ c 3 100. 75,. 50. 25..

‘0

50

lo0 150 200 2500 Log(fluorescence

50 100 intensity)

150

200

b 250

Fig. 2. Flow cytofluorometry of the PMNs bound with VI A2 Fab’ and VII Al Fab’. PMNs (1 x 10” cells) were incubated without (a) or with VI A2 Fab’ (b), VII Al Fab’ (c) and F(ab’), of normal mouse IgG (d), and then reacted with FITC-F(ab’), of anti-mouse IgG antibody. The PMNs (2500 cells) were analyzed on an Ortho Spectrum III flow cytometer.

Identification

of an Fc receptor

for IgGl

and lgG2

-no.2z [L IY .A

20- a ;;i = 2 r. 0-z

on guinea-pig

835

PMNs

b

5 .

:L -k -0 0.1. z n II_” .!. 5

Oi

A

0 Anti-FcR

T 0.5 Fad added (pg)

44 I .o

7 : mo-. 0 Bound anti-FZR Fab’ ?t+l~lO~~l

Fig. 3. The binding of VI A2 Fab’ to PMNs. PMNs (1 x 10’ cells) were incubated with various amounts of ‘25I-VI A2 Fab’ (0) and VII Al Fab’ (A) at 4°C for 2 hr. After washing the cells, the bound

radioactivities were measured (a). Scatchard plotting of the data is shown in (b).

from the binding of OA-complexed ‘ZSI-IgGl antibody (Fig. 1). The affinity of VI A2 Fab’ to the antigen on PMN cells was found to be 8 x lo8 M-l. Inhibition of the bindings of antigen-complexed bodies to PMN by VI A2 Fab’

anti-

The demonstration of the binding of VIA2 Fab’ to PMN cells suggests strongly that a certain FcR antigenically similar to macrophage FcR,,, also exists on the PMN cells. To further confirm whether the antigen bound by the monoclonal antibody is an FcR for both IgGl and IgG2 or not, the effects of VI A2 Fab’ on the bindings of OA-complexed IgGl and IgG2 antibodies to PMNs were investigated. When the inhibitory effect of VI A2 Fab’ on the binding of OA-complexed IgGl antibody to PMNs was examined, more than 0.1 pg of the Fab’ was found to inhibit strongly the binding reaction (Fig. 4). That of OA-complexed IgG2 antibody was also inhibited with VI A2 Fab’, but the inhibition was only 50% even in the presence of an excess amount of the Fab’. These results demonstrated that the antigen of PMN cells specifically bound by VI A2 IgGl is an FcR (FcR,,,) for both IgGl and IgG2. In addition, the finding that the binding of OA-complexed IgG2 antibody is only partially inhibited by VI A2 Fab’, indicates that another FcR, probably FcR, capable of binding IgG2 but not IgGl antibody, may exist on PMN cells. The failure of VII Al Fab’ to bind to PMNs may be explained by the fact that the FcR, is antigenically different from its macrophage counterpart. Mol. wt of FcR,,, As found from those

the antigen of VI A2 IgGl on to be FcR,,,, we attempted to PMN cells and to compare its of macrophage FcR,,, For this

PMN cells was isolate the FcR properties with purpose, PMNs

were labeled with ‘*‘I, using a lactoperoxidase method, solubilized with 1% Nonidet P-40, and applied to a VI A2 F(ab’),-Sepharose 6B column. When the ‘251-protein adsorbed on the column was eluted and applied to SDS-PAGE, it gave a single band with a mol. wt of 55,000 (Fig. 5). The FcR,,, of PMN again was found to be similar to macrophage FcR,,, with regard to mol. wt, since the mol. size of et al. is macrophage FcR,,, isolated by Shimamura 55,000 (Shimamura et al., 1986). DISCUSSION

Coupland and Leslie (1983) reported that guineapig IgGl antibody complexed with soluble antigen could not substantially bind to homologous PMNs, though its IgG2 antibody counterpart could bind to the cells. Based on these results, they suggested that the PMNs possessed no FcR for IgGl.

s m”

-5

0 cod’of

-1.

VlA2’?ad

-3

,o-2

(rng)Lt)

Fig. 4. Inhibition of the bindings of OA-complexed IgGl and IgG2 antibodies to PMNs with VI A2 Fab’. PMNs (1 x 10’cells) were preincubated with various amounts of VI A2 Fab’ for 1 hr at 4”C, and then reacted with OAcomplexed IgGl (0) and IgG2 antibodies (A) (Ab: 5 pg), and OA-complexed F(ab’), of IgG2 antibody [F(ab’),: 5 pg] (W), for 2 hr at 4°C. After reaction, the amounts of complexes bound were determined.

836

TOHORUNAKAMIJRAet al.

--IS0 KD

-4SKD

Fig. 5. Autoradiographic profile of ‘2SI-FcR,,, isolated from PMNs with VI A2 F(ab’),-Sepharose 6B. The ‘251-F~R,,, purified by affinity chromatography on a VI A2 F(ab’),Sepharose 6B column was applied to SDS-PAGE. IgG, BSA and OA were used as standard proteins with known mol. wts.

The results described in this paper, however, showed that not only IgG2 antibody but also IgGl antibody bound to homologous PMNs, when complexed with OA; OA-complexed IgGl antibody was also found to bind dose-dependently to PMN cells in larger quantities than OA-complexed F(ab’)* of IgG2 antibody. In this respect, there might be the possibility that the apparent binding of IgGl antibody to the cells does not reflect the real binding of IgGl antibody, but the binding of the contaminating IgG2 antibody. This, however, may be excluded by the fact that no contaminating IgG2 antibody could be detected by the Ouchterlony double diffusion test, even when the IgGl antibody preparation at a high concn of 6 mg/ml was examined. There might be another possibility that the binding of OA-complexed IgGl antibody observed does not occur on PMN cells but on some contaminating macrophage cells in the PMN preparation used. This possibility may also be excluded, since flow cytofluorometry showed that almost all of the PMN cells therein bound VI A2 Fab’ which was able to inhibit completely the binding of OA-complexed IgGl antibody to the cells. The results so far obtained really indicate that PMN cells possess not only FcR for IgG2 but also FcR for IgGl, as is the case of homologous macrophage cells. The FcR for IgGl, thus detected on PMN cells, resembles the FcR,,, on macrophage cells with regard to antigenicity and mol. wt. In addition, the in-

hibition of binding of OA-complexed IgG2 antibody by VI A2 Fab’ indicates that the FcR for IgGl on PMN cells also operates as FcR for IgG2. The FcR, therefore, may be FcR,,, for both IgGl and IgG2, and identical to the FcR,,~ on macrophage cells with regard to immunochemical and functional properties. The number of the FcR,,, molecules per PMN cell was estimated to be 1.7 x lo4 by the measurement of binding of OA-complexed IgGl antibody. This value is substantially equal to that estimated by the measurement of binding of VI A2 Fab’ (2 x lo4 molecules per PMN cell). Since homologous macrophages possess 2 x lo5 FcR,,, molecules per cell, the number of FcR,,, molecules per PMN cell is about one-tenth of that of macrophages (Shimamura et al., 1986). On the other hand, the affinity of PMN FcR,,* for OAcomplexed IgGl antibody (&: 3.4 x 10’ M-l) seems higher than that of macrophage FcR,,, (3.8 x 106M-I). The affinity of VI A2 Fab’ to the FcR,,, on PMN cells was found to be 8 x 10’ Mm’, which is of the same order of magnitude as that to the FcR,,, on macrophage cells (4 x lOEM-i) (Shimamura et al., 1986). The number of the FcR,,, molelcules per PMN cell is markedly smaller than that of FcR estimated by measuring the binding of OA-complexed IgG2 antibody; the number of FcR,,, molecules was about one-fifth that of FcR for IgG2 (1.0 x 10’ molecules). Coupland and Leslie (1983) reported a value of 1.3-2.6 x lo4 molecules for the number of FcR molecules per PMN cell through the measurement of the binding of 2,4_dinitrophenylated BSA-complexed IgG2 antibody. The value of Coupland and Leslie (1983) is smaller than that obtained by us, using OA-complexed IgG2 antibody. The difference between numbers of FcRs involved in the bindings of OA-complexed IgG2 and IgGl antibodies suggests that guinea-pig PMNs should possess at least two types of FcR for IgG isotypes, as in the case of human PMNs and mouse macrophages (Kulczycki, 1984; Looney et al., 1986~; Unkeless, 1979). the results so far obtained by us suggest that guinea-pig PMNs possess FcR,,, for both IgGl and IgG2 antibody and FcR, for IgG2 antibody alone. This assumption is supported by the following observation; differing from the binding of OA-complexed IgGl antibody to PMN cells that of OA-complexed IgG2 antibody was incompletely inhibited by VI A2 Fab’. The binding of OA-complexed IgG2 antibody was only 50% inhibited even in the presence of excessive amounts of the Fab’ fragment. This incomplete inhibition may be explained by the existence of FcR,, the binding activity of which is not affected by VI A2 Fab’. The FcR,, its presence being thus demonstrated, differs antigenically from macrophage FcR,, since the Fab’ of a monoclonal antibody to macrophage FcR, (VII Al) does not inhibit at all the binding of OA-complexed IgG2 antibody to PMN cells. Flow cytofluorometry, furthermore, showed that PMN

Identification

of an Fc receptor

for IgGl

cells do not bind VII Al F(ab’),, differing from macrophage cells. Scatchard plotting of the binding of OAcomplexed IgG2 antibody to PMNs, on the other hand, did not reflect the existence of two distinct FcRs, as in the case of homologous macrophages also having two types of FcR (Sugiyama et al., 1981); the plotting gave a straight line with a K,,, value of 4.0 x 10’ Mm’ (Fig. 1). This suggests that the affinities of FcR, and FcR,,, on PMNs for the complex are of the same order of magnitude. However, if more excessive amounts of the complex than those used in the experiment are reacted with the cells, Scatchard plotting of the binding data might become nonlinear, being made up of two parts having different slopes. Since the amount of complex nonspecifically bound to the cells increased markedly on increasing that of added complex beyond 5 pg, we could not exactly estimate the specific binding reaction in the complex-excess zone. Recently, some immunochemical diversity of FcRs for IgG isotypes was observed with human and mouse phagocytes (Looney et al., 1986a, b; Unkeless, 1979). For instance, human neutrophils have two a 40,OOOmol. wt FcR and a types of FcRs, 5 1,00&73,000 mol. wt FcR, though human monocytes have a 40,000 FcR and a 72,000 FcR. Fleit et al. (1982) demonstrated, using a monoclonal antibody to the 51,000-73,000 FcR on human neutrophils, 3G8, that the 72,000 FcR on human monocytes was antigenically distinguishable from the 5 l,OOO-73,000 mol. wt FcR on the neutrophils. Anderson and his coworkers, on the other hand, observed that the 40,000 mol. wt FcR on the neutrophils was cross-reactive with a monoclonal antibody to the 40,000 FcR on the neutrophils, IV 3 (Looney et al.,

1986a, b). Based on the analogy with human phagocytes, it appears that the FcR, on guinea-pig PMNs differs not only antigenically but also in mol. size from the homologue on homologous macrophages. The results in this paper, furthermore, indicate that even the FcR with the same specificity for IgG isotypes differs immunochemically, depending on either PMN or macrophage. At present, the immunochemical properties of FcR, on guinea-pig PMNs such as mol. wt remains unknown. To clarify precise characters of the FcR,, further investigation is now in progress. Acknowledgements--We would especially like to thank Mr Takashi Fujii for performing flow cytofluorometry and Kishimoto Clinical Laboratory for allowing us to use an Ortho Sepctrum III flow cytdmeter. This work was supported in part by Grants-in-Aid for the Fugaku Trust for Medical Research, and Scientific Research from the Ministry of Education, Science and Culture of Japan, REFERENCES

Baxter M. A., Leslie R. G. Q. and Reeves W. G. (1983) The stimulation of superoxide anion production in guinea-pig peritoneal macrophages and neutrophils by phorbol myristate acetate, opsonized zymosan and IgGZ-containing soluble immune complexes. Immunology 48, 657-665.

and IgG2 on guinea-pig

PMNs

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Cone R. E. and Marchalonis J. J. (1974) Surface. proteins of thymus-derived lymphocytes and bone-marrow-derived lymphocytes. Selective isolation and immunoglobulin and the O-antigen by non-ionic detergents. Biochem. J. 140, 3455354. Coupland K. and Leslie R. G. Q. (1983) The expression of Fc receptors on guinea-pig peritoneal macrophages and neutrophils. Immunology 48, 6477656. Fleit B. F., Wright S. D. and Unkeless J. C. (1982) Human neutrophil Fey receptor distribution and structure. Proc. natn. &ad. Sci. U.S.A. 79, 3275-3279. Godinp. J. W. (1976) Coniuaation of antibodies with fluorochromes:‘ modification-to the standard methods. J. Immun. Meth. 13, 215-226. Grossbcrg A. L., Radzimski G. and Pressman D. (1962) Effect of iodination on the active site of several antihapten antibodies. Eiochemisfry 1, 391401. Kudo M., Nakamura T. and Koyama J. (1985) Isolation of a protein labeled with diisopropyl fluorophosphate on stimulation of polymorphonuclear leukocytes with immune complexes. Molec. Immun. 22, 1099-l 105. Kudo M., Nakamura T. and Koyama J. (1985) Purification and some properties of a membrane-bound dipeptidyl peptidase IV of guinea pig casein-induced intraperitoneal leukocytes. J. Biochem. 97, 121 lL1218. Kulczycki A. Jr (1984) Human neutrophils and eosinophils have structurally distinct Fey receptors, J. Immun. 133, 849-854. Laemmii U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680. Looney R. J., Ryan D. H., Takahashi K., Fleit H. B., Cohen H. J., Abraham G. H. and Anderson C. L. (1986a) Identification of a second class of IgG Fc receptors on human neutrophils. A 40 kilodalton molecule also found on eosinophils. J. exp. Med. 163, 826836. Looney R. J., Abraham G. N. and Anderson C. L. (19866) Human monocytes and U937 cells bear two distinct Fc receptors for IgG. J. Immun. 136, 164~1647. NisonolI A., Wissler F. C., Lipman L. N. and Woernley D. L. (1960) Studies of univalent fragments from the bivalent rabbit antibody molecules by reduction of disulfite bonds. Archs Biochem. Biophys. 89, 23G244. Shimamura T., Nakamura T. and Koyama J. (1986) Analysis of Fc receptors for IgG on guinea pig peritoneal

macrophages by the use of a monoclonal antibody to one

_ of the_Fc receptors. J. Biochem. 99, 227-235. Shimamura T.. Nakamura T. and Kovama J. (1987) Demonstration of the existence of two distinct Fc recep: tors for IgG isotypes on guinea-pig macrophages by the use of monoclonal antibodies. Molec. Immun. 24, 67-74. Starkey P. M. (1977) Elastase and cathepsin G; the serine proteinases of human neutrophil leucocytes and spleen. In Proteinases in Mammalian Cells and Tissues (Edited by Barrett A. J.), pp. 57-89. North-Holland, Amsterdam. Sugiyama N., Tamoto K. and Koyama J. (1981) Evidence for two distinct Fc receptors on guinea pig peritoneal macrophages. Molec. Immun. 18, 999-1005. Tamoto K. and Koyama J. (1976) Non-precipitating guinea-pig antibodies as products of limited recognition of antigenic sites on the ovalbumin molecule. Immunology 31, 67-7’7. Unkeless J. C. (1979) Characterization of a monoclonal antibody directed against mouse macrophage and lymphocyte Fc receptors. J. exp. Med. 150, 58G-596. Washida N., Sagawa A., Tamoto K. and Koyama J. (1980) Comparative studies on superoxide anion production by polymorphonuclear leukocytes stimulated with various agents. Biochim. biophys. Acta 631, 371-379. Yamashita R. and Koyama J. (1984) Isotype specificity and heterogeneity of Fc-receptors on guinea pig splenic B and T lymphocytes. Microbial. Immun. 28, 1137-I 148.