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Guinea-pig peritoneal macrophage receptor for IgG—II. Purification of the receptor and its partial characterization
MoiecufarImmwrofogy,Vol. 20, No.
0161-5890/83%3.00 + 0.00 Q 1983 Pergamon Press Ltd
11, pp. 1149-l 155, 1983
Printed in Great Britain
GUINEA-PIG PERITONEAL MACROPHAGE RECEPTOR FOR IgG-II. PURIFICATION OF THE RECEPTOR AND ITS PARTIAL CHARACTERIZATION* MARIA
Department
JANUSZ,~ KRYSTYNA STAROSCIK, WOJCIECH GORCZYCA, ZBICNIEW WIECZOREK$ and J~ZEF LISOWSKI of Immunochemistry and SLaboratory of Immunobiology, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrodaw, Poland (First received 11 January
1983; accepted in revised form 11April 1983)
Abstract-The Fey receptor of guinea-pig peritoneal macrophages was purified by affinity chromatography by using rabbit IgG or guinea-pig IgG2 coupled to Sepharose. Lysates prepared by treatment of L251-labeledmacrophages with NP-40 were first applied to BSA-Sepharose and then to IgG-Sepharose and eluted with 0.5 M acetic acid containing 1% NP-40. The specific binding was determined by interaction of the ‘251-labeledreceptor with IgG-Sepharose in the presence and absence of soluble IgG. The specific binding of the purified receptor was 41-82x. Interactions of the purified receptor with IgG-Sepharose were equally we11inhibited by soluble rabbit IgG or guinea-pig IgG2, but not by F(ab’)? fragments. Inclusion of NP-40 in the buffer used in the assay reduced nonspecific binding of the receptor to the affinity gels. The purified receptor can be stored for 20 days at 4°C without a significant loss of the specific binding activity. Analysis of the receptor by SD~polyacrylamide gel electrophoresis, under nonreducing and reducing conditions, revealed two major peaks of radioactivity corresponding to mol. wts of about 50,000 and 25,000, and one very minor peak corresponding to a mol. wt of about 30,000. The results obtained suggest that the protein of the second major peak is a product of the dissociation of the protein of the first major peak rather than a product of its reduction by 2-mercaptoethanol.
INTRODIJCTION
Macrophages possess receptors for the Fc portion of immunoglobulins (FcR). They play an important role in antibody-dependent cytotoxicity, phagocytosis and regulation of the immune response [for reviews see Dickler (1976), Dorrington (1977), Gergely et at. (1982), Leslie (1982), Lovchik and Hong (1977) and Lydyard and Fanger (1982)]. Fey receptors from rabbit and murine macrophages and macrophage-like cells were isolated and partially characterized in several laboratories (Bourgois et al., 1977; Kulczycki et ai., 1980; Loube and Dorrington, 1980; Schneider et al., 1981; Suzuki et al., 1982; Unkeless, 1977; Urso-Coward and Cone, 1978). Although the studies on properties of the guinea-pig peritoneal macrophage Fc receptors started in 1967 (Davey and Asherson, 1967), very little is still known about properties of the receptor. Studies on interactions of the guinea-pig peritoneal macrophage Fc receptor with various classes and subclasses of immu*This work was supported by Polish Academy of Sciences, Grant No. 10.5. tTo whom correspondence should be addressed. ~Abbre~ations: BSA, bovine serum albumin; BBS, 0.2 M borate buffer containing 0.15 M NaCl. oH 8: NP-40. Nonidet P-40; PBS, 0.02-M phosphate b&er cdntaining 0.15 M NaCl, pH 7.2; PMSF, phenylmethylsulfonyl fluoride; SDS-PAGE, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate.
noglobulins have been carried out in several laboratories (Alexander et al., 1978; Liew, 1971; Leslie and Cohen, 1976). However, no info~ation is available concerning the isolation, purification and molecular properties of the receptor. Recently, two papers on properties of the solubilized but not purified guineapig peritoneal macrophage Fey receptor have been published. In the first, Yagawa et al. (1979) presented results of studies on binding properties, sensitivity to enzymatic treatment, and solubilization of the Fey receptor. In the second paper, Sugiyama et al. (198 1) described studies on solubilization of the receptor and its interaction with immune complexes containing guinea-pig IgGl or IgG2 antibodies. The authors
suggested the existence of two Fey receptors on guinea-pig macrophages; one interacting equally well with IgGl and IgG2 and the other primarily interacting with IgG2. However, the solubilized receptors are indistinguishable from one another by means of gel filtration on Sepharose-6B in the presence or absence of detergent [Nonidet P-40 (NP-401.4 Results of our studies on properties of the guineapig Fey receptor, interactions of the receptor with immunoglobulins, and e&cts of chemicals and enzymes on the activity of the receptor have been published in our former paper (Janusz et al., 1980). In this report we present results of studies on solubilization and isolation of the guinea-pig peritoneal macrophage Fey receptor in an active form.
1149
MARIA JANUSZ PI ui.
I 150 MATERIALS AND METHODS
Preparation
and radiolubeling
of’ macrophuge
mem -
branes
DEAE-~eiIulose (DE-32) was obtained from Whatman, England. Sephadex G-200 and Sepharose-4B were products of Pharmacia, Sweden. Bromocyan, PMSF, sodium dodecyl sulfate and acrylamide were purchased from Serva, F.R.G. Lactoperoxidase, glucose oxidase and BSA were obtained from Sigma, U.S.A., glucose from Polfa, Poland, potassium iodide from Merck, F.R.G., NP-40 from Fluka, Switzerland, BioBeads SM-2 from BioRad, U.K. Sucrose was a product of B.D.H., U.K. Na”? was purchased from the Institute of Nuclear Research, Poland. All reagents were of analytical grade. To prevent any bacterial growth, all buffer and protein soiutions contained 0.03% sodium azide. Animuls
Guinea-pigs and rabbits were obtained Animal Farm, Trestno, Poland.
from the
Randomly bred guinea-pigs weighing 30&400 g were used as donors of peritoneal macrophages. The animals were injected intraperitoneally with 30 ml of sterilized liquid paraffin and 4 days after the injection macrophages were harvested and isolated as described in our former paper (Janus2 et al., 1980). lmmunogiohulins
Rabbit IgG was prepared by chromatography of gamma-globulins on DE-32 cellulose in 0.01 M phosphate (K) buffer, pH 7.5, containing 0.045 M NaCI. Guinea-pig IgG2 was obtained according to the procedure of Leslie and Cohen (1970). IgG monomers were prepared by gel filtration on Sephadex G-200 in PBS. Aggregated IgG was prepared by incubation of 2% immunoglobulin solutions in PBS at 63°C for 20min. The aggregates were filtered through Sephadex G-200 in PBS and the ascending limb of the first peak was pooled for tests. Digestion
of itntnunoglobulins
with pepsin
F(ab’), fragments of IgG were prepared according to the method of Nisonoff ef 01. (1960) and purified by gel filtration on Sephadex G-200, Radiolabeling
and lysis of macrophuges
Macrophages were labeled with “‘1 in the presence of lactoperoxidase and glucose oxidase, as described by Kulczycki et al. (1980). The labeled macrophages were lysed by treatment with NP-40 (1 x IO8 cells suspended in 1 ml of PBS containing 1% NP-40 and 2 mM PMSF) for 5 min at 4°C. The lysed cells were centrifuged for 20 min at 30,OOOg (4°C). The supernatant obtained was used for isolation of the Fey receptor.
Macrophages, 2 x IO’ cells in 2 ml of PBS, were disrupted by ultrasonication (21 cycles, ultrasonicator MSE, U.K.; 3 x 1 min in an ice-water bath) and centrifuged at 20,OOOg for 30 min at 4 C. The supernatant obtained was then centrifuged at 100,OOOg for 60min at 4°C. The membranes were labeled with ‘251(Kulczycki et ul., 1980). Membrane pellets obtained from 1 x 10’ macrophages were suspended in 0.4 ml of PBS, ultrasonicated for 1 min and treated with 12~1 of lactoperoxidase (I mg;ml), 1.2~1 of KI (1 x 10m4M), 12~11of glucose oxidase (0.15 mg/ml), and 0.5 mCi of Na’*‘I. The reaction was carried out for 15 min at room temp with constant stirring. The radioiodinated membranes were washed twice by centrifugation (lO~,OOO~, 1 hr, 4.C) with PBS containing 2 mM PMSF and 3 x 10 ’ M KI, and finally dialysed against PBS to remove unbound iodine. The dialysed membranes were centrifuged at 100,OOOg for 1 hr (4°C) and resuspended in PBS containing 2 mM PMSF, and uftrasonicated for 1min. The suspension of membranes was mixed with an equal volume of PBS containing 2 mM PMSF and 2% NP-40, left for 30 min at 4“C, and centrifuged for 20min at 30,OOOg (4°C). The supernatant obtained was used for isolation of the Fey receptor.
CNBr-activated Sepharose-4B (Cuatrecasas, 1970) was mixed with an equal volume of BSA or IgG (l&15 mg/ml) and rotated end-over-end overnight at 4°C. On average, 13 mg of BSA or 8.5 mg of IgG were coupled to I ml of packed gel. Before use, immunoadsorbents were incubated for 3Omin at room temp with BBS containing 1% BSA to minimize nonspecific binding. Then, the gels were extensively washed with BBS containing BSA (0.3 mg/ml), sodium azide (0.03%) and 1% NP-40. Puri~c~t~on
qf‘ Fq
receptor
The method of purification described here is an adaptation of the procedure of Kulczycki et al. (1980) used for purification of the Fey receptor from rabbit alveolar macrophages. All steps in the receptor purification were carried out at 4°C. The material obtained after lysis of cells or cell membranes (1 mi in PBS containing 1% NP-40 and 2 mM PMSF) was preincubated with BSA-Sepharose (5mg of BSA coupled to Sepharose per lysate obtained from 1 x 10’ cells) for 30 min with constant rotation. The supernatant containing material unbound to BSA-Sepharose was then incubated with IgG-Sepharose (5-l 0 mg of rabbit IgG coupled to Sepharose per lysate obtained from 1 x lo8 macrophages) overnight on a rotator. The incubation mixture was centrifuged at 400g for 5 min. The supernatant was removed, the gel was suspended in BBS with 2mlEP PMSF and 1% NP-40, and transferred
Guinea-pig macrophage Fey receptor into 1 x 6 cm columns. The immunoadsorbents in columns were first washed with BBS containing 1% NP-40 to remove material unbound to IgG. The Fey receptor containing fraction was eluted either with 0.5 M acetic acid containing 1% NP-40, 0.5 N HCl containing 1% NP-40, or 6 M guanidine hydrochloride containing 1% NP-40. The elution was carried out with l-ml portions of the eluting solutions. The eluates were collected into tubes with 0.375 ml of 2 M Tri-HCl buffer, pH 8.6, containing 1% NP-40 (elution with acetic acid) or 2 M Tris containing 1% NP-40 (elution with HCl). IgG-binding activity of the receptor fractions eluted from the immunoadsorbent was measured after dialysis of the fractions against buffers used in the assay.
When necessary, NP-40 was removed by incubating the receptor fractions with BioBeads SM-2 (300 mg of BioBeads per 1 ml of the receptor fraction) with continuous rotation for 1.5 hr and the beads were removed by centrifugation. Lysis of erythrocytes was used as a control for the removal of the detergent. Polyacrylamide gel electrophoresis
The electrophoresis was performed in 8, 10 or 15% gel in the presence of SDS as described by Ugel et al. (1971). After separation, gels were cut into 2-mm segments and the radioactivity was measured in each segment in a gamma counter. As standards, IgG, BSA, ovalbumin and chymotrypsinogen labeled with ‘25I(Doran and Spar, 1980) were used. RESULTS
Immunoadsorbent assay for Fey receptor activity
To determine the activity of the labeled receptor, its binding to IgG coupled to Sepharose was studied in the presence (nonspecific binding) and absence (total binding) of soluble IgG. Binding of constant amounts of the receptor to various amounts of IgG-Sepharose was measured to find optimal conditions for the assay. The addition of 98Opg of IgG coupled to Sepharose was enough for binding the receptor prepared from 6 x lo6 macrophages. The presence of a three-fold excess of soluble IgG with respect to the amount of IgG coupled to Sepharose was sufficient for determination of the nonspecific binding. The assay was performed in plastic tubes in BBS with BSA (0.3 mg/ml), NP-40 (1%) and sodium azide (0.03’4). The final volume of the reaction mixture was equal to 1 ml. The samples were incubated for 1.5 hr at 4°C on an orbital shaker, centrifuged at 400g for 1 min, and the immunoadsorbent beads were washed twice with 1 ml of cold BBS containing NP-40, BSA and sodium azide. Washed beads were counted in a gamma counter for “‘1 (Gamma counter A-22M, Polon, Poland). The amount of the labeled receptor specifically bound was calculated by subtracting the radioactivity bound in the presence of soluble IgG (nonspecifically bound receptor) from that bound in the absence of soluble IgG (total binding). The activity of the purified receptor fractions was also measured using the inhibition assay. Constant amounts of the receptor (equivalent to 6 x lo6 macrophages) were first preincubated for 1 hr at 4°C with immunoglobulins or immunoglobulin fragments (three-fold excess in respect to IgG coupled to Sepharose) and then the binding experiments were performed as described earlier. The radioactivity measured in control experiments (immunoglobulins replaced with buffer) was assumed to be 100%. The percentage of inhibition was calculated as the difference between the radioactivity measured in the control experiments and the radioactivity of samples preincubated with immunoglobulins.
1151
PuriJication of the Fq
receptor
Macrophages were labeled with ‘25Iand solubilized in buffers containing NP-40, as described in Materials and Methods. Because of the reported susceptibility of solubilized Fey receptors [e.g. Bourgois et al. (1977)] to proteolysis, we used PMSF throughout the isolation procedure. The solubilized material was first applied to BSA-Sepharose which bound about 26% of the radioactivity applied. The unretarded material was then applied to IgG-Sepharose and the unbound fraction (88% of the radioactivity applied, on average) was eluted with BBS containing 1% NP-40. Elution of the putative Fey receptor was accomplished with one of the following reagents: 1% NP-40 containing 0.5 M acetic acid, 0.5 N HCI or 6 M guanidine hydrochloride. Assuming the radioactivity initially applied to IgG-Sepharose as loo%, the reagents eluted 0.059-0.126% of the radioactivity applied (Table 1). Although significant amounts of labeled membrane proteins were bound to IgG-Sepharose-on average, 8.45% of the radioactivity applied--elution of these proteins from the beads was difficult even under strong dissociating conditions. Only 1.64% (the average value) of the radioactivity bound was eluted with the reagents applied. Prolonged washing of the immunoadsorbent with 1% NP-40 containing 6 M guanidine hydrochloride did not improve the recovery of the material bound to IgG-Sepharose. The material eluted from the immunoadsorbent with each of the eluting agents used showed a similar activity. However, 0.5 M acetic acid was routinely used as a less drastic reagent. No improvement in the yield of the receptor fraction was obtained when the receptor was purified from labeled and lysed macrophage membranes. Similar yields of the receptor were obtained irrespective of the use of immunoadsorbents prepared by coupling to Sepharose homologous guinea-pig IgG2 or heterologous rabbit IgG, in a monomeric or aggregated form. The degree of purification of the receptor was evaluated after each step of purification by deter-
MARIA JANIJSZ et cd. Table
I. Affinity
chromatography of IgG-binding material guinea-pig peritoneal macrophages Radloactivity recovered ,n the receptor fraction”
Eluting
agent
0.5 M acetic acid 0.5 M HCI 6 M guanidine hvdrochloride
obtamed
from lysates of
(““)
IgG-binding activity (specific bmding)b (:“)
0.109 0.059
41-82 43-70
0. I26
19-69
“Radioactivity recovered in the receptor fraction is expressed as a percentage of the radioactivity applied to IgG-Sepharose immunoadsorbents. The values presented are the average of eight determinations. bThe specific binding IS described as the difference between the total binding assumed as 100% and the binding m the presence of an excess of soluble IgG (nonspecific bmding). The binding varied from one preparation to another. so the range of binding activities is presented. All eluting agents contained 1% NP-40.
Table 2. Binding activity of preparations obtained after various steps of purification of the guinea-pig peritoneal macrophage Fey receptor
Preparation Cell lysate Supernatant after absorption of the cell lysate with BSA-Sepharose Material bound to IgG-Sepharose and eluted as purified receptor Material unbound to IgGSepharose
Radioactwity bound to IgG-Sepharose (total binding) (“,“)
Specific binding” (%)
0.06-2.5
lo-16
0.1 -2
12-13
2 I --49
41-82
O.l&l.3
O-8
“The specific binding is defined as described under Table I. In each experiment, amounts of the receptor fraction used for the assay corresponded to 6 x IO” macrophages.
mination of the total and specific binding of fractions containing the receptor for IgG-Sepharose (Table 2). The purified active receptor prepared from 1.6 x 1O8 cells was applied to fresh IgG-Sepharose (16 mg of IgG coupled to Sepharose) and incubated overnight at 4°C on a rotatory shaker. It was found that 56% of the radioactivity applied was retained by Table 3. Inhibition
Preparation I 2
of binding Inhibitor protein Guinea-pig IgG2 Rabbit IgG
4
Guinea-pig IgG2 Rabbit IgG
5
BSA
6
Rabbit IgG--F(ab’),
3
the immunoadsorbent. The bound radioactivity was eluted with 0.5 M acetic acid with a 68% yield. The radiolabeled purified receptor could also bind soluble IgG. Preincubation of the receptor with soluble IgG, in three-fold excess of the amount of IgG coupled to Sepharose, inhibited adsorption of the receptor on the immunoadsorbent. Inhibition of the binding (46-63”/,) was observed after preincubation of the receptor both with homologous and heterologous immunoglobulins (Table 3). BSA showed only a weak inhibition, whereas F(ab’), fragments did not inhibit the binding. Removal of NP-40 from the purified receptor decreased its specific binding to IgG-Sepharose by 15-20x. Addition of NP-40 did not restore the binding to the initial value. The presence of NP-40 in the buffer reduced nonspecific binding of the receptor to the affinity gels. The purified receptor could be stored at 4°C in the presence or absence of NP-40 without an appreciable loss of the specific binding activity. The specific binding of the purified receptor preparations stored for 20 days was 25-357: lower than the binding of fresh preparations. The total binding was lower by 40%.
of the isolated
Fey receptor
Immunoadsorbent Guinea-pig IgC2Xepharose Rabbit IgG Sepharose Rabbit IgGSepharose Guinea-pig IgG2XSepharose Rabbit IgGSepharose Rabbit IgG Sepharose
to immunoadsorbents” Inhibition of specific bindingb (%) 46.1-62.9 48.5-5X.6 47.2-63.1 46.9-61.4 I l--13.2 0
dThe receptor was purified on guinea-pig IgG2 coupled to Sepharose (preparations I and 4) or on rabbit IgG coupled to Sepharose (preparations 2, 3, 5 and 6). The purified receptor was preincubated with indicated proteins and adsorption on IgGZ- or IgG-Sepharose was assayed. hRadioactivity of receptor samples preincubated with BBS and bound to IgG-Sepharose (control experiments) was taken to be 100%. The percentage of inhibition is expressed as the difference between specific binding of control samples and samples preincubated with proteins.
1153
Guinea-pig macrophage Fey receptor Molecular size of the purified receptor
The purified receptor, eluted from the immunoadsorbent with 0.5 M acetic acid was analysed using SDS-PAGE (Fig. I). Two major peaks of radioactivity correspond to mol. wts of 56,000 (range 40,00&70,000) and 25,000 (range 21,000-29,000) and a very minor peak (shoulder) of mol. wt about 30,000 were found under nonreducing conditions. The relative mobilities of these peaks did not change on reduction. Electrophoresis under reducing conditions again revealed two major peaks, with mol. wts of 45,000 (range 37,00&52,000) and 23,000 (range 19,00&27,000). Under nonreducing conditions the first peak appeared as a rather broad band. The electrophoretic patterns obtained were very reproducible. Similar electrophoretic patterns were obtained when the electrophoresis of the receptor purified by using repetitive affinity chromatography on IgG-Sepharose was performed. Similar patterns were also obtained after electrophoresis of receptor preparations eluted with HCI or guanidine hydrochloride. Although the relative mobilities of the two peaks were the same under nonreducing and reducing conditions, the relative proportions of radioactivity in the first and the second peak did change. It was found that the radioactivity of the second peak was higher under reducing conditions than that under nonreducing conditions. Radioactivity of the minor peak did not change.
Gel slice
number
r (
4
DISCUSSION
The positive aspect of our isolation procedure is that the purified receptor retains its specific ligandbinding activity. The binding specificity of the isolated receptor for the Fc portion of IgG was proven by the observation that this membrane component could be bound and rebound to IgG-Sepharose. In addition, binding of the purified guinea-pig peritoneal macrophage Fey receptor to IgG-Sepharose could be blocked by preincubation of the receptor with IgG, but not by BSA or the F(ab’), fragment. The IgG binding activity of the isolated receptor and inhibition of the binding by IgG was similar or better than activities described for other Fey receptors purified from lymphocytes or macrophages (Cooper et al., 1977; Kulczycki et al., 1980; Schneider et al., 1981; Suzuki et al., 1980; Yagawa et al., 1979). It is worth mentioning here that our direct assay to determine the binding activity of the isolated receptor allows us to measure reversible and specific binding and not only the total binding of the receptor to IgG. The isolated receptor showed surprisingly high stability. During 20 days storage of the isolated receptor at 4°C the specific activity was only about 30% lower. This is in agreement with our earlier observation that macrophages stored in PBS for 14 days at 4°C lost only 15% of their binding activity (Janusz et al., 1980).
3 : P ; 4 +! 5
i
I
IO
20
30
Gel slice
number
I
,
40
50
Fig. 1. SDS-polyacrylamide gel electrophoresis of the isolated guinea-pig peritoneal macrophage Fey receptor (fraction eluted from IgG-Sepharose with 0.5 M acetic acid containing 1% NP-40). Electrophoresis was performed in 10% gel, under nonreducing (a) and reducing (b) conditions. After electrophoresis, the gels were sliced into 2-mm segments and the radioactivity of each segment was measured
in a gamma counter.
1154
MAKIA JANUSZct crl
There are numerous reports on the isolation and characterization of IgG-Fc-binding proteins. Proteins with apparent mol. wts varying from 25,000 to 120,000 displaying Fc-binding properties have been isolated from various cells (Cooper rt ul., 1977; Cunningham-Rundles et al., 1978; Frade and Kourilsky, 1977; Kulczycki et al., 1980; Loube and Dorrington, 1980; Rask et al., 1975; Schneider et al., 1981; Suzuki et al., 1982; Urso-Coward and Cone, 1978). The purified guinea-pig peritoneal macrophage Fey receptor was resolved on SDSPAGE into two major (mol. wts about 50,000 and 25,000) fractions and one very minor fraction (mol. wt about 30,000). The electrophoretic patterns were the same under nonreducing and reducing conditions. Under nonreducing conditions the first peak appeared as a rather broad band. The facts that: (I) the purification of the receptor was performed in the absence of reducing agents, and (2) that the presence of the second peak was also observed after electrophoresis under nonreducing conditions argues against a possibility that the protein of the second peak was a product of reduction of the protein of the first peak. In our opinion, the protein of the second peak was a product of dissociation of the protein of the first peak. This suggests that interactions of 25,000 mol. wt subunits were rather strong and resistant to complete dissociation by SDS. Reduction of intramolecular disulfide bonds might facilitate the dissociation of the 50,000 mol. wt protein into 25,000 mol. wt subunits. The detergent-solubilized guineapig peritoneal macrophage Fey receptor filtered on a column of Sepharose6B in the presence of NP-40 emerges somewhat earlier than guinea-pig IgG (Sugiyama et al., 1981; Yagawa et al., 1979; our unpublished results). When gel filtration is performed in the absence of NP-40, the receptor appears in the void volume. Because of the difficulty in eluting tightly bound Fey receptor from the affinity gels, we cannot be certain that the radiolabeled proteins represent the sole membrane receptor. Thus, other Fey receptors may exist, which were either not eluted from the affinity gels or were not radioiodinated under conditions employed. The presence of proteins with mol. wts of 50,00&57,000 and 25,00&28,000 is also observed after SDS-PAGE of receptors isolated from macrophage-like cells (Loube and Dorrington, 1980; Suzuki et al., 1982) or murine leukemic Ll210 cells (Cooper et al., 1977). Kulczycki et al. (1980) found an apparent mol. wt of 50,00~57,000 in 5.6”/, gels and of 35,000-55,000 in 9% gels for the receptor isolated from rabbit alveolar macrophages. Size heterogeneity revealed by SDS-PAGE of isolated Fey receptors from other sources has been noted by other investigators (Cooper et al., 1977; Loube and Dorrington, 1980; Rask et al., 1975; Suzuki et al., 1982; Urso-Coward and Cone, 1978). The heterogeneity of the isolated Fey receptors may represent
degradation products of larger receptors (Bourgois (II ul., 1977). To prevent this, we used PMSF throughout the isolation procedure. However, even the presence of PMSF cannot completely exclude the possibility of proteolysis. In conclusion, we have successfully isolated an Fey-binding protein from the surface of radiolabeled guinea-pig peritoneal macrophages that is a membrane Fcl; receptor and retains its ligand-binding activity. It appears to bind equally well to rabbit IgG and guinea-pig IgG2.
acknowledge the excellent technical assistance of MS Helena Kostecka.
Acknowledgement-We
REFERENCES
Alexander M. D., Andrews .I. A. and Leslie R. G. Q. (1978) The binding of human and guinea pig IgG subclasses to homologous macrophage and monocyte Fc receptors. Immunology 35, 115-123. Bourgois A., Abney E. R. and Parkhouse R. M. E. (1977) Structure of mouse Fc receptor. Eur. J. Immun. 7, 691-695. Cooper S. M., Sambray Y. and Friou G. J. (1977) Isolation of separate Fc receptors for IgG complexed to antigen and native IgG from a murine leukemia. Nuture, Lond. 270, 253-255. Cuatrecasas P. (1970) Protein purification by affinity chromatography. Derivatizations of agarose and polyacrylamide beads. J. hiol. Chem. 2451 3059-3065. Cunningham-Rundles C., Seiaal F. P. and Good R. A. (19785 Isolation and charac&zation of a human mononuclear cell Fc receptor. Immunochemistry, 15, 365-370. Davey M. J. and Asherson G. L. (1967) Cytophilic antibody I. Nature of the macrophage receptor. Immunology 12, 13-20. Dickler H. B. (1976) Lymphocyte receptors for immunoglobulins. Adv. Immun. 24, 1677214. Doran D. M. and Spar J. L. (1980) Oxidative iodine monochloride iodination technique. J. Immun. Me/h. 39, 155-163. Dorrington K. J. (1977) Properties of the Fc receptor on macrophages and monocytes. In Immunology of Receptors (Edited by Cinader B.), pp. 183-200. Marcel Dekker. New York. Frade R. and Kourilsky F. M. (I 977) Preliminary characterization of a glycoprotein having Fc receptor properties extracted from a T cell lymphoma L-5 17X-Y. Eur. J. Immun. 7, 230-235. Gergely J., Erdei A., Sandor M., Sarmay G. and Uher F. (1982) The Fc receptor model of membrane cytoplasmic signalling. Molec. Immun. 19, 1223-I 228. Janusz M., Ugorski M., Mikulska J., Staroscik K., Wieczorek Z. and Lisowski J. (1980) Studies on properties of guinea pig peritoneal macrophage Fc receptor. Arch. Immun. Ther. Exp. 28, 275-285. Kulczycki A., Jr., Krause V., Killion C. Ch. and Atkinson J. P. (1980) Purification of Fey receptor from rabbit alveolar macrophages that retains ligand-binding activity. J. Immun. 124, 2772-2779. Leslie R. G. Q. (1982) Macrophage interactions with antibodies and soluble immune complexes. Immunohiology 161, 322-333. Leslie R. G. Q. and Cohen S. (I 970) Chemical properties of guinea pig immunoglobulins IgGI, IgG2 and IgM. Biothem. J. 120, 787-795. Leslie R. G. Q. and Cohen S. (1976) Comparison of the
Guinea-pig macrophage Fey receptor cytophilic activities of guinea pig IgGl and IgG2 antibodies. Eur. J. Immun. 6, 848-855. Liew F. Y. (1971) The binding of the Fc fragment of guinea pig cytophilic antibody to peritoneal macrophages. Immunology 20, 8 17-829. Loube S. R. and Dorrington K. _I. (1980) Isolation of biosynthetically-labeled Fc-binding proteins from detergent lysates and spent culture fluid of a macrophagelike cell line P388Dl. J. Immun. 125. 970-975. Lovchik J. C. and Hong R. (1977) Antibody-dependent cell-mediated cytolysis (ADCC): analyses and projections. Prog. Allergy 22, 144. Lydyard P. M. and Fanger M. W. (1982) Characteristics and function of Fc receptors on human lymphocytes. Immunology 47, l-17.
Nisonoff A., Wissler F. C., Lipman L. N. and Woennley W. L. (1960) Separation of univalent fragments from the bivalent rabbit antibody molecule by reduction of disulfide bonds. Archs Biochem. Biophys. 89, 23&244. Rask L., Klareskog L., Gstberg L. and Peterson P. A. (1975) Isolation and properties of a murine spleen cell Fc receptor. Nature, iond. 257, 231-233. _ Schneider R. J., Atkinson J. P.. Krause V. and Kulczvcki A. (1981) Characterization of hgand-binding activity-of isolated murine Fey receptor. J. Immun. 126, 935-940. Sugiyama N., Tamoto K. and Koyama J. (1981) Evidence
MlMM20/l l-e
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for two distinct Fc receptors on guinea pig peritoneal macrophages. Molec. Immun. 18, 999-1005. Suzuki T., Sadisavan R., Wood G. and Bayer W. L. (1980) Isolation and characterization of biologically active Fc receptors of human B lymphocytes. Molec. Immun. 17, 491-503. Suzuki T., Saito-Taki T., Sadisavan R. and Nitta T. (1982) Biochemical signal transmitted by Fey receptors: phospholipase A, activity of Fcy2b receptor of murine macrophage cell line P388 D,. Proc. n&n. Acud. Sci. U.S.A. 79, 591-595. Ugel A. R., Chrambach A. and Rodbard D. (1971) Fractionation and characterization of an oligomeric series of bovine keratohyalin by polyacrylamide gel electrophoresis. Analyt. Biochem. 43, 41W26. Unkeless J. (1977) The presence of two Fc receptors on mouse macrophages: evidence from a variant cell line and differential trypsin sensitivity. J. exp. Med. 145, 931-947. Urso-Coward M. d’ and Cone R. E. (1978) Membrane proteins of the P388Dl macrophage cell line: isolation of membrane polypeptides that bind to the Fc portion of aggregated IgG. J. Immun. 121, 1973-1978. Yagawa K., Onoue K. and Aida Y. (1979) Structural studies of Fc receptors. I. Binding properties, solubilization, and partial characterization of Fc receptors of macrophages. J. Immun. 122, 366373.
0161-5890/83%3.00 + 0.00 Q 1983 Pergamon Press Ltd
11, pp. 1149-l 155, 1983
Printed in Great Britain
GUINEA-PIG PERITONEAL MACROPHAGE RECEPTOR FOR IgG-II. PURIFICATION OF THE RECEPTOR AND ITS PARTIAL CHARACTERIZATION* MARIA
Department
JANUSZ,~ KRYSTYNA STAROSCIK, WOJCIECH GORCZYCA, ZBICNIEW WIECZOREK$ and J~ZEF LISOWSKI of Immunochemistry and SLaboratory of Immunobiology, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrodaw, Poland (First received 11 January
1983; accepted in revised form 11April 1983)
Abstract-The Fey receptor of guinea-pig peritoneal macrophages was purified by affinity chromatography by using rabbit IgG or guinea-pig IgG2 coupled to Sepharose. Lysates prepared by treatment of L251-labeledmacrophages with NP-40 were first applied to BSA-Sepharose and then to IgG-Sepharose and eluted with 0.5 M acetic acid containing 1% NP-40. The specific binding was determined by interaction of the ‘251-labeledreceptor with IgG-Sepharose in the presence and absence of soluble IgG. The specific binding of the purified receptor was 41-82x. Interactions of the purified receptor with IgG-Sepharose were equally we11inhibited by soluble rabbit IgG or guinea-pig IgG2, but not by F(ab’)? fragments. Inclusion of NP-40 in the buffer used in the assay reduced nonspecific binding of the receptor to the affinity gels. The purified receptor can be stored for 20 days at 4°C without a significant loss of the specific binding activity. Analysis of the receptor by SD~polyacrylamide gel electrophoresis, under nonreducing and reducing conditions, revealed two major peaks of radioactivity corresponding to mol. wts of about 50,000 and 25,000, and one very minor peak corresponding to a mol. wt of about 30,000. The results obtained suggest that the protein of the second major peak is a product of the dissociation of the protein of the first major peak rather than a product of its reduction by 2-mercaptoethanol.
INTRODIJCTION
Macrophages possess receptors for the Fc portion of immunoglobulins (FcR). They play an important role in antibody-dependent cytotoxicity, phagocytosis and regulation of the immune response [for reviews see Dickler (1976), Dorrington (1977), Gergely et at. (1982), Leslie (1982), Lovchik and Hong (1977) and Lydyard and Fanger (1982)]. Fey receptors from rabbit and murine macrophages and macrophage-like cells were isolated and partially characterized in several laboratories (Bourgois et al., 1977; Kulczycki et ai., 1980; Loube and Dorrington, 1980; Schneider et al., 1981; Suzuki et al., 1982; Unkeless, 1977; Urso-Coward and Cone, 1978). Although the studies on properties of the guinea-pig peritoneal macrophage Fc receptors started in 1967 (Davey and Asherson, 1967), very little is still known about properties of the receptor. Studies on interactions of the guinea-pig peritoneal macrophage Fc receptor with various classes and subclasses of immu*This work was supported by Polish Academy of Sciences, Grant No. 10.5. tTo whom correspondence should be addressed. ~Abbre~ations: BSA, bovine serum albumin; BBS, 0.2 M borate buffer containing 0.15 M NaCl. oH 8: NP-40. Nonidet P-40; PBS, 0.02-M phosphate b&er cdntaining 0.15 M NaCl, pH 7.2; PMSF, phenylmethylsulfonyl fluoride; SDS-PAGE, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate.
noglobulins have been carried out in several laboratories (Alexander et al., 1978; Liew, 1971; Leslie and Cohen, 1976). However, no info~ation is available concerning the isolation, purification and molecular properties of the receptor. Recently, two papers on properties of the solubilized but not purified guineapig peritoneal macrophage Fey receptor have been published. In the first, Yagawa et al. (1979) presented results of studies on binding properties, sensitivity to enzymatic treatment, and solubilization of the Fey receptor. In the second paper, Sugiyama et al. (198 1) described studies on solubilization of the receptor and its interaction with immune complexes containing guinea-pig IgGl or IgG2 antibodies. The authors
suggested the existence of two Fey receptors on guinea-pig macrophages; one interacting equally well with IgGl and IgG2 and the other primarily interacting with IgG2. However, the solubilized receptors are indistinguishable from one another by means of gel filtration on Sepharose-6B in the presence or absence of detergent [Nonidet P-40 (NP-401.4 Results of our studies on properties of the guineapig Fey receptor, interactions of the receptor with immunoglobulins, and e&cts of chemicals and enzymes on the activity of the receptor have been published in our former paper (Janusz et al., 1980). In this report we present results of studies on solubilization and isolation of the guinea-pig peritoneal macrophage Fey receptor in an active form.
1149
MARIA JANUSZ PI ui.
I 150 MATERIALS AND METHODS
Preparation
and radiolubeling
of’ macrophuge
mem -
branes
DEAE-~eiIulose (DE-32) was obtained from Whatman, England. Sephadex G-200 and Sepharose-4B were products of Pharmacia, Sweden. Bromocyan, PMSF, sodium dodecyl sulfate and acrylamide were purchased from Serva, F.R.G. Lactoperoxidase, glucose oxidase and BSA were obtained from Sigma, U.S.A., glucose from Polfa, Poland, potassium iodide from Merck, F.R.G., NP-40 from Fluka, Switzerland, BioBeads SM-2 from BioRad, U.K. Sucrose was a product of B.D.H., U.K. Na”? was purchased from the Institute of Nuclear Research, Poland. All reagents were of analytical grade. To prevent any bacterial growth, all buffer and protein soiutions contained 0.03% sodium azide. Animuls
Guinea-pigs and rabbits were obtained Animal Farm, Trestno, Poland.
from the
Randomly bred guinea-pigs weighing 30&400 g were used as donors of peritoneal macrophages. The animals were injected intraperitoneally with 30 ml of sterilized liquid paraffin and 4 days after the injection macrophages were harvested and isolated as described in our former paper (Janus2 et al., 1980). lmmunogiohulins
Rabbit IgG was prepared by chromatography of gamma-globulins on DE-32 cellulose in 0.01 M phosphate (K) buffer, pH 7.5, containing 0.045 M NaCI. Guinea-pig IgG2 was obtained according to the procedure of Leslie and Cohen (1970). IgG monomers were prepared by gel filtration on Sephadex G-200 in PBS. Aggregated IgG was prepared by incubation of 2% immunoglobulin solutions in PBS at 63°C for 20min. The aggregates were filtered through Sephadex G-200 in PBS and the ascending limb of the first peak was pooled for tests. Digestion
of itntnunoglobulins
with pepsin
F(ab’), fragments of IgG were prepared according to the method of Nisonoff ef 01. (1960) and purified by gel filtration on Sephadex G-200, Radiolabeling
and lysis of macrophuges
Macrophages were labeled with “‘1 in the presence of lactoperoxidase and glucose oxidase, as described by Kulczycki et al. (1980). The labeled macrophages were lysed by treatment with NP-40 (1 x IO8 cells suspended in 1 ml of PBS containing 1% NP-40 and 2 mM PMSF) for 5 min at 4°C. The lysed cells were centrifuged for 20 min at 30,OOOg (4°C). The supernatant obtained was used for isolation of the Fey receptor.
Macrophages, 2 x IO’ cells in 2 ml of PBS, were disrupted by ultrasonication (21 cycles, ultrasonicator MSE, U.K.; 3 x 1 min in an ice-water bath) and centrifuged at 20,OOOg for 30 min at 4 C. The supernatant obtained was then centrifuged at 100,OOOg for 60min at 4°C. The membranes were labeled with ‘251(Kulczycki et ul., 1980). Membrane pellets obtained from 1 x 10’ macrophages were suspended in 0.4 ml of PBS, ultrasonicated for 1 min and treated with 12~1 of lactoperoxidase (I mg;ml), 1.2~1 of KI (1 x 10m4M), 12~11of glucose oxidase (0.15 mg/ml), and 0.5 mCi of Na’*‘I. The reaction was carried out for 15 min at room temp with constant stirring. The radioiodinated membranes were washed twice by centrifugation (lO~,OOO~, 1 hr, 4.C) with PBS containing 2 mM PMSF and 3 x 10 ’ M KI, and finally dialysed against PBS to remove unbound iodine. The dialysed membranes were centrifuged at 100,OOOg for 1 hr (4°C) and resuspended in PBS containing 2 mM PMSF, and uftrasonicated for 1min. The suspension of membranes was mixed with an equal volume of PBS containing 2 mM PMSF and 2% NP-40, left for 30 min at 4“C, and centrifuged for 20min at 30,OOOg (4°C). The supernatant obtained was used for isolation of the Fey receptor.
CNBr-activated Sepharose-4B (Cuatrecasas, 1970) was mixed with an equal volume of BSA or IgG (l&15 mg/ml) and rotated end-over-end overnight at 4°C. On average, 13 mg of BSA or 8.5 mg of IgG were coupled to I ml of packed gel. Before use, immunoadsorbents were incubated for 3Omin at room temp with BBS containing 1% BSA to minimize nonspecific binding. Then, the gels were extensively washed with BBS containing BSA (0.3 mg/ml), sodium azide (0.03%) and 1% NP-40. Puri~c~t~on
qf‘ Fq
receptor
The method of purification described here is an adaptation of the procedure of Kulczycki et al. (1980) used for purification of the Fey receptor from rabbit alveolar macrophages. All steps in the receptor purification were carried out at 4°C. The material obtained after lysis of cells or cell membranes (1 mi in PBS containing 1% NP-40 and 2 mM PMSF) was preincubated with BSA-Sepharose (5mg of BSA coupled to Sepharose per lysate obtained from 1 x 10’ cells) for 30 min with constant rotation. The supernatant containing material unbound to BSA-Sepharose was then incubated with IgG-Sepharose (5-l 0 mg of rabbit IgG coupled to Sepharose per lysate obtained from 1 x lo8 macrophages) overnight on a rotator. The incubation mixture was centrifuged at 400g for 5 min. The supernatant was removed, the gel was suspended in BBS with 2mlEP PMSF and 1% NP-40, and transferred
Guinea-pig macrophage Fey receptor into 1 x 6 cm columns. The immunoadsorbents in columns were first washed with BBS containing 1% NP-40 to remove material unbound to IgG. The Fey receptor containing fraction was eluted either with 0.5 M acetic acid containing 1% NP-40, 0.5 N HCl containing 1% NP-40, or 6 M guanidine hydrochloride containing 1% NP-40. The elution was carried out with l-ml portions of the eluting solutions. The eluates were collected into tubes with 0.375 ml of 2 M Tri-HCl buffer, pH 8.6, containing 1% NP-40 (elution with acetic acid) or 2 M Tris containing 1% NP-40 (elution with HCl). IgG-binding activity of the receptor fractions eluted from the immunoadsorbent was measured after dialysis of the fractions against buffers used in the assay.
When necessary, NP-40 was removed by incubating the receptor fractions with BioBeads SM-2 (300 mg of BioBeads per 1 ml of the receptor fraction) with continuous rotation for 1.5 hr and the beads were removed by centrifugation. Lysis of erythrocytes was used as a control for the removal of the detergent. Polyacrylamide gel electrophoresis
The electrophoresis was performed in 8, 10 or 15% gel in the presence of SDS as described by Ugel et al. (1971). After separation, gels were cut into 2-mm segments and the radioactivity was measured in each segment in a gamma counter. As standards, IgG, BSA, ovalbumin and chymotrypsinogen labeled with ‘25I(Doran and Spar, 1980) were used. RESULTS
Immunoadsorbent assay for Fey receptor activity
To determine the activity of the labeled receptor, its binding to IgG coupled to Sepharose was studied in the presence (nonspecific binding) and absence (total binding) of soluble IgG. Binding of constant amounts of the receptor to various amounts of IgG-Sepharose was measured to find optimal conditions for the assay. The addition of 98Opg of IgG coupled to Sepharose was enough for binding the receptor prepared from 6 x lo6 macrophages. The presence of a three-fold excess of soluble IgG with respect to the amount of IgG coupled to Sepharose was sufficient for determination of the nonspecific binding. The assay was performed in plastic tubes in BBS with BSA (0.3 mg/ml), NP-40 (1%) and sodium azide (0.03’4). The final volume of the reaction mixture was equal to 1 ml. The samples were incubated for 1.5 hr at 4°C on an orbital shaker, centrifuged at 400g for 1 min, and the immunoadsorbent beads were washed twice with 1 ml of cold BBS containing NP-40, BSA and sodium azide. Washed beads were counted in a gamma counter for “‘1 (Gamma counter A-22M, Polon, Poland). The amount of the labeled receptor specifically bound was calculated by subtracting the radioactivity bound in the presence of soluble IgG (nonspecifically bound receptor) from that bound in the absence of soluble IgG (total binding). The activity of the purified receptor fractions was also measured using the inhibition assay. Constant amounts of the receptor (equivalent to 6 x lo6 macrophages) were first preincubated for 1 hr at 4°C with immunoglobulins or immunoglobulin fragments (three-fold excess in respect to IgG coupled to Sepharose) and then the binding experiments were performed as described earlier. The radioactivity measured in control experiments (immunoglobulins replaced with buffer) was assumed to be 100%. The percentage of inhibition was calculated as the difference between the radioactivity measured in the control experiments and the radioactivity of samples preincubated with immunoglobulins.
1151
PuriJication of the Fq
receptor
Macrophages were labeled with ‘25Iand solubilized in buffers containing NP-40, as described in Materials and Methods. Because of the reported susceptibility of solubilized Fey receptors [e.g. Bourgois et al. (1977)] to proteolysis, we used PMSF throughout the isolation procedure. The solubilized material was first applied to BSA-Sepharose which bound about 26% of the radioactivity applied. The unretarded material was then applied to IgG-Sepharose and the unbound fraction (88% of the radioactivity applied, on average) was eluted with BBS containing 1% NP-40. Elution of the putative Fey receptor was accomplished with one of the following reagents: 1% NP-40 containing 0.5 M acetic acid, 0.5 N HCI or 6 M guanidine hydrochloride. Assuming the radioactivity initially applied to IgG-Sepharose as loo%, the reagents eluted 0.059-0.126% of the radioactivity applied (Table 1). Although significant amounts of labeled membrane proteins were bound to IgG-Sepharose-on average, 8.45% of the radioactivity applied--elution of these proteins from the beads was difficult even under strong dissociating conditions. Only 1.64% (the average value) of the radioactivity bound was eluted with the reagents applied. Prolonged washing of the immunoadsorbent with 1% NP-40 containing 6 M guanidine hydrochloride did not improve the recovery of the material bound to IgG-Sepharose. The material eluted from the immunoadsorbent with each of the eluting agents used showed a similar activity. However, 0.5 M acetic acid was routinely used as a less drastic reagent. No improvement in the yield of the receptor fraction was obtained when the receptor was purified from labeled and lysed macrophage membranes. Similar yields of the receptor were obtained irrespective of the use of immunoadsorbents prepared by coupling to Sepharose homologous guinea-pig IgG2 or heterologous rabbit IgG, in a monomeric or aggregated form. The degree of purification of the receptor was evaluated after each step of purification by deter-
MARIA JANIJSZ et cd. Table
I. Affinity
chromatography of IgG-binding material guinea-pig peritoneal macrophages Radloactivity recovered ,n the receptor fraction”
Eluting
agent
0.5 M acetic acid 0.5 M HCI 6 M guanidine hvdrochloride
obtamed
from lysates of
(““)
IgG-binding activity (specific bmding)b (:“)
0.109 0.059
41-82 43-70
0. I26
19-69
“Radioactivity recovered in the receptor fraction is expressed as a percentage of the radioactivity applied to IgG-Sepharose immunoadsorbents. The values presented are the average of eight determinations. bThe specific binding IS described as the difference between the total binding assumed as 100% and the binding m the presence of an excess of soluble IgG (nonspecific bmding). The binding varied from one preparation to another. so the range of binding activities is presented. All eluting agents contained 1% NP-40.
Table 2. Binding activity of preparations obtained after various steps of purification of the guinea-pig peritoneal macrophage Fey receptor
Preparation Cell lysate Supernatant after absorption of the cell lysate with BSA-Sepharose Material bound to IgG-Sepharose and eluted as purified receptor Material unbound to IgGSepharose
Radioactwity bound to IgG-Sepharose (total binding) (“,“)
Specific binding” (%)
0.06-2.5
lo-16
0.1 -2
12-13
2 I --49
41-82
O.l&l.3
O-8
“The specific binding is defined as described under Table I. In each experiment, amounts of the receptor fraction used for the assay corresponded to 6 x IO” macrophages.
mination of the total and specific binding of fractions containing the receptor for IgG-Sepharose (Table 2). The purified active receptor prepared from 1.6 x 1O8 cells was applied to fresh IgG-Sepharose (16 mg of IgG coupled to Sepharose) and incubated overnight at 4°C on a rotatory shaker. It was found that 56% of the radioactivity applied was retained by Table 3. Inhibition
Preparation I 2
of binding Inhibitor protein Guinea-pig IgG2 Rabbit IgG
4
Guinea-pig IgG2 Rabbit IgG
5
BSA
6
Rabbit IgG--F(ab’),
3
the immunoadsorbent. The bound radioactivity was eluted with 0.5 M acetic acid with a 68% yield. The radiolabeled purified receptor could also bind soluble IgG. Preincubation of the receptor with soluble IgG, in three-fold excess of the amount of IgG coupled to Sepharose, inhibited adsorption of the receptor on the immunoadsorbent. Inhibition of the binding (46-63”/,) was observed after preincubation of the receptor both with homologous and heterologous immunoglobulins (Table 3). BSA showed only a weak inhibition, whereas F(ab’), fragments did not inhibit the binding. Removal of NP-40 from the purified receptor decreased its specific binding to IgG-Sepharose by 15-20x. Addition of NP-40 did not restore the binding to the initial value. The presence of NP-40 in the buffer reduced nonspecific binding of the receptor to the affinity gels. The purified receptor could be stored at 4°C in the presence or absence of NP-40 without an appreciable loss of the specific binding activity. The specific binding of the purified receptor preparations stored for 20 days was 25-357: lower than the binding of fresh preparations. The total binding was lower by 40%.
of the isolated
Fey receptor
Immunoadsorbent Guinea-pig IgC2Xepharose Rabbit IgG Sepharose Rabbit IgGSepharose Guinea-pig IgG2XSepharose Rabbit IgGSepharose Rabbit IgG Sepharose
to immunoadsorbents” Inhibition of specific bindingb (%) 46.1-62.9 48.5-5X.6 47.2-63.1 46.9-61.4 I l--13.2 0
dThe receptor was purified on guinea-pig IgG2 coupled to Sepharose (preparations I and 4) or on rabbit IgG coupled to Sepharose (preparations 2, 3, 5 and 6). The purified receptor was preincubated with indicated proteins and adsorption on IgGZ- or IgG-Sepharose was assayed. hRadioactivity of receptor samples preincubated with BBS and bound to IgG-Sepharose (control experiments) was taken to be 100%. The percentage of inhibition is expressed as the difference between specific binding of control samples and samples preincubated with proteins.
1153
Guinea-pig macrophage Fey receptor Molecular size of the purified receptor
The purified receptor, eluted from the immunoadsorbent with 0.5 M acetic acid was analysed using SDS-PAGE (Fig. I). Two major peaks of radioactivity correspond to mol. wts of 56,000 (range 40,00&70,000) and 25,000 (range 21,000-29,000) and a very minor peak (shoulder) of mol. wt about 30,000 were found under nonreducing conditions. The relative mobilities of these peaks did not change on reduction. Electrophoresis under reducing conditions again revealed two major peaks, with mol. wts of 45,000 (range 37,00&52,000) and 23,000 (range 19,00&27,000). Under nonreducing conditions the first peak appeared as a rather broad band. The electrophoretic patterns obtained were very reproducible. Similar electrophoretic patterns were obtained when the electrophoresis of the receptor purified by using repetitive affinity chromatography on IgG-Sepharose was performed. Similar patterns were also obtained after electrophoresis of receptor preparations eluted with HCI or guanidine hydrochloride. Although the relative mobilities of the two peaks were the same under nonreducing and reducing conditions, the relative proportions of radioactivity in the first and the second peak did change. It was found that the radioactivity of the second peak was higher under reducing conditions than that under nonreducing conditions. Radioactivity of the minor peak did not change.
Gel slice
number
r (
4
DISCUSSION
The positive aspect of our isolation procedure is that the purified receptor retains its specific ligandbinding activity. The binding specificity of the isolated receptor for the Fc portion of IgG was proven by the observation that this membrane component could be bound and rebound to IgG-Sepharose. In addition, binding of the purified guinea-pig peritoneal macrophage Fey receptor to IgG-Sepharose could be blocked by preincubation of the receptor with IgG, but not by BSA or the F(ab’), fragment. The IgG binding activity of the isolated receptor and inhibition of the binding by IgG was similar or better than activities described for other Fey receptors purified from lymphocytes or macrophages (Cooper et al., 1977; Kulczycki et al., 1980; Schneider et al., 1981; Suzuki et al., 1980; Yagawa et al., 1979). It is worth mentioning here that our direct assay to determine the binding activity of the isolated receptor allows us to measure reversible and specific binding and not only the total binding of the receptor to IgG. The isolated receptor showed surprisingly high stability. During 20 days storage of the isolated receptor at 4°C the specific activity was only about 30% lower. This is in agreement with our earlier observation that macrophages stored in PBS for 14 days at 4°C lost only 15% of their binding activity (Janusz et al., 1980).
3 : P ; 4 +! 5
i
I
IO
20
30
Gel slice
number
I
,
40
50
Fig. 1. SDS-polyacrylamide gel electrophoresis of the isolated guinea-pig peritoneal macrophage Fey receptor (fraction eluted from IgG-Sepharose with 0.5 M acetic acid containing 1% NP-40). Electrophoresis was performed in 10% gel, under nonreducing (a) and reducing (b) conditions. After electrophoresis, the gels were sliced into 2-mm segments and the radioactivity of each segment was measured
in a gamma counter.
1154
MAKIA JANUSZct crl
There are numerous reports on the isolation and characterization of IgG-Fc-binding proteins. Proteins with apparent mol. wts varying from 25,000 to 120,000 displaying Fc-binding properties have been isolated from various cells (Cooper rt ul., 1977; Cunningham-Rundles et al., 1978; Frade and Kourilsky, 1977; Kulczycki et al., 1980; Loube and Dorrington, 1980; Rask et al., 1975; Schneider et al., 1981; Suzuki et al., 1982; Urso-Coward and Cone, 1978). The purified guinea-pig peritoneal macrophage Fey receptor was resolved on SDSPAGE into two major (mol. wts about 50,000 and 25,000) fractions and one very minor fraction (mol. wt about 30,000). The electrophoretic patterns were the same under nonreducing and reducing conditions. Under nonreducing conditions the first peak appeared as a rather broad band. The facts that: (I) the purification of the receptor was performed in the absence of reducing agents, and (2) that the presence of the second peak was also observed after electrophoresis under nonreducing conditions argues against a possibility that the protein of the second peak was a product of reduction of the protein of the first peak. In our opinion, the protein of the second peak was a product of dissociation of the protein of the first peak. This suggests that interactions of 25,000 mol. wt subunits were rather strong and resistant to complete dissociation by SDS. Reduction of intramolecular disulfide bonds might facilitate the dissociation of the 50,000 mol. wt protein into 25,000 mol. wt subunits. The detergent-solubilized guineapig peritoneal macrophage Fey receptor filtered on a column of Sepharose6B in the presence of NP-40 emerges somewhat earlier than guinea-pig IgG (Sugiyama et al., 1981; Yagawa et al., 1979; our unpublished results). When gel filtration is performed in the absence of NP-40, the receptor appears in the void volume. Because of the difficulty in eluting tightly bound Fey receptor from the affinity gels, we cannot be certain that the radiolabeled proteins represent the sole membrane receptor. Thus, other Fey receptors may exist, which were either not eluted from the affinity gels or were not radioiodinated under conditions employed. The presence of proteins with mol. wts of 50,00&57,000 and 25,00&28,000 is also observed after SDS-PAGE of receptors isolated from macrophage-like cells (Loube and Dorrington, 1980; Suzuki et al., 1982) or murine leukemic Ll210 cells (Cooper et al., 1977). Kulczycki et al. (1980) found an apparent mol. wt of 50,00~57,000 in 5.6”/, gels and of 35,000-55,000 in 9% gels for the receptor isolated from rabbit alveolar macrophages. Size heterogeneity revealed by SDS-PAGE of isolated Fey receptors from other sources has been noted by other investigators (Cooper et al., 1977; Loube and Dorrington, 1980; Rask et al., 1975; Suzuki et al., 1982; Urso-Coward and Cone, 1978). The heterogeneity of the isolated Fey receptors may represent
degradation products of larger receptors (Bourgois (II ul., 1977). To prevent this, we used PMSF throughout the isolation procedure. However, even the presence of PMSF cannot completely exclude the possibility of proteolysis. In conclusion, we have successfully isolated an Fey-binding protein from the surface of radiolabeled guinea-pig peritoneal macrophages that is a membrane Fcl; receptor and retains its ligand-binding activity. It appears to bind equally well to rabbit IgG and guinea-pig IgG2.
acknowledge the excellent technical assistance of MS Helena Kostecka.
Acknowledgement-We
REFERENCES
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for two distinct Fc receptors on guinea pig peritoneal macrophages. Molec. Immun. 18, 999-1005. Suzuki T., Sadisavan R., Wood G. and Bayer W. L. (1980) Isolation and characterization of biologically active Fc receptors of human B lymphocytes. Molec. Immun. 17, 491-503. Suzuki T., Saito-Taki T., Sadisavan R. and Nitta T. (1982) Biochemical signal transmitted by Fey receptors: phospholipase A, activity of Fcy2b receptor of murine macrophage cell line P388 D,. Proc. n&n. Acud. Sci. U.S.A. 79, 591-595. Ugel A. R., Chrambach A. and Rodbard D. (1971) Fractionation and characterization of an oligomeric series of bovine keratohyalin by polyacrylamide gel electrophoresis. Analyt. Biochem. 43, 41W26. Unkeless J. (1977) The presence of two Fc receptors on mouse macrophages: evidence from a variant cell line and differential trypsin sensitivity. J. exp. Med. 145, 931-947. Urso-Coward M. d’ and Cone R. E. (1978) Membrane proteins of the P388Dl macrophage cell line: isolation of membrane polypeptides that bind to the Fc portion of aggregated IgG. J. Immun. 121, 1973-1978. Yagawa K., Onoue K. and Aida Y. (1979) Structural studies of Fc receptors. I. Binding properties, solubilization, and partial characterization of Fc receptors of macrophages. J. Immun. 122, 366373.