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A new Fc receptor on mouse macrophages binding IgG3

A new Fc receptor on mouse macrophages binding IgG3 A NEW Fc RECEPTOR ON MOUSE MACROPHAGES BINDING IgG3* BY BETTY DIAMOND AND DALE E. YELTON:[: From the Departments of Microbiology and Immunology and Medicine, and of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461 Among the effector functions exhibited by immunoglobulins is the ability to bind to Fc receptors on macrophages, lymphocytes, and granulocytes (1-10). It has recently become evident that there are different types of Fc receptors on different types of cells (11) and, moreover, that the same cell may express more than one type of Fc receptor (1, 2, 5). For example, mouse macrophage Fc receptors do not bind IgM, whereas some lymphocyte Fc receptors do (7). Mouse macrophages are known to express at least two different Fc receptors, one specifically binding mouse IgGt and IgG2b, the other binding IgG2a (12, 13). The Fc receptors are distinguishable by certain other properties as well. The IgG2a receptor is sensitive to trypsin and shows decreased binding at 4°C and in the presence of cytochalasin B; the IgGl-IgGzb receptor is trypsin resistant and unaltered at 4°C by cytochalasin B (1, 13). The IgGa class of mouse immunoglobulins has not been studied extensively with respect to its binding to Fc receptors. IgGs has an Fc fragment that is serologically and structurally different from IgG1 and IgG2 (14). Functionally, it has been shown to have an increased ability to cross the placenta 04). Earlier experiments have shown that monomeric IgG3 does not inhibit the binding of monomeric IgG2a to its Fc receptor (14). These and other early results have led to the assumption that IgG3 does not bind to Fc receptors. In light of the evidence for multiple Fc receptors, we felt it necessary to examine again the binding of IgGs to Fc receptors. We generated a monoclonal IgGs anti-sheep erythrocyte (SRBC) 1 antibody from a mouse spleen-mouse myeloma fusion in order to study directly the binding of IgGa to macrophage Fc receptors. Monoclonal antibodies are especially useful in such studies because they are homogeneous, they are the only mouse immunoglobulin in culture medium from the hybridoma cultures, and when reacted with their antigen, they form antigen-antibody complexes more natural for binding studies than artificial aggregates. Using this monoclonal IgG3 anti-SRBC antibody, we found that IgG3 does bind to macrophages through a third, independent IgG Fc receptor. Materials and Methods Cells. J774 is a reticulum cell sarcoma from a BALB/c mouse with macrophagelike properties that has been adapted to culture 05). J774.2 is a clone from the tissue culture line. * Supported by grants AI 161166, A110702, AI 1581 l, A1523 l, and CA24300 from t he National Institutes of Health, grant PCM77-25635 from the National Science Foundation, and grant IM-216 from the American Cancer Society. :~ Medical scientist trainee supported by grant 5T32GMF288 from the National Institutes of Health. J Abbreviations used in this paper: BDB, bis-diazotized benzidine; PBS, phosphate-buffered saline; SRBC, sheep erythrocytes. 514 J. ExP. MEo. ©The Rockefeller University Press • 0022-1007/81/03/0514/06 $1.00 Volume 153 March 1981 514-519 515 BETTY DIAMOND AND DALE E. YELTON A series of subclones ofJ774.2 were picked and screened for their ability to bind IgGs. J774.2.1 was found to lack IgGs receptors. It has grown to mass culture and recloned to produce J774.2.1.4. P388D1 is a macrophagelike cell line with Fc receptors and phagocytic ability. FCI.4 and FC1.6 are subclones of the FC1 cell line which arose during a fusion of MPC 11 myeloma ceils to spleen cells from a BALB/e mouse immunized with SRBC. FC 1.4 and FC 1.6 were selected for their inability to phagocytize IgG~-SRBC. Primary macrophages were obtained from resident peritoneal cells and from peritoneal cells 4 d after an intraperitoneal injection of thioglycolate broth (Difco Laboratories, Detroit, Mich.). The method for isolating adherent cells has been described previously (1). Myeloma Proteins. Myeloma proteins were obtained by injecting 107 MOPC21 (IgGa), MPCll (IgG~b), MOPC173 (IgG2a), or J606 (IgGa) cells into the peritoneal cavity of pristine- primed BALB/c mice (16). The aseites fluid was precipitated with 50% saturated ammonium sulfate. A purified -/-globulin fraction was obtained by DEAE chromatography (1). It was determined by agarose gel electrophoresis and by Ouchterlony analysis that no fraction had any contaminating protein of another subclass. Flope 21 was purchased from Bionetics, Kensington, Md. Monomeric protein was prepared by centrifuging the protein for 30 min at 150,000 g. Preparation ofIg-coated SRBC. The methods of Bianco et al. (17) were used. SRBC were incubated with antibody for 30 min at 37°C, washed, and resuspended to 0.5%. The antibodies were obtained from cloned hybridoma lines making anti-SRBC antibody, 5 #1 of ascites fluid was incubated with 1 ml of a 5% solution of SRBC. The IgGs-producing line was derived from a fusion of drug-marked P3 cells with spleen cells from a BALB/c mouse immunized with SRBC. Segregants no longer making the myeloma IgG1 heavy chain were selected. The IgGz antibody was identified by Ouchterlony analysis with commercial anti-IgG3 (Meioy Laboratories, Inc., Springfield, Va.) and with antiserum raised in the laboratory of Dr. John Cebra, University of Pennsylvania, against J606 protein and absorbed with IgGa, Ig~a, and IgG2b. Fc Rosettes and Fc-mediated Phagocytosis. This was done as previously described (1). Cells adhered to glass coverslips were incubated for 30 min at 37 ° or 4°C, washed, and assayed for rosettes. Attachment of three or more SRBC signified a rosette. For phagoeytosis, the cells were incubated for 1 h at 37°C, free SRBC were lysed in hypotonic solution, and intracellular SRBC were assayed. A phagocytic cell was any cell with three or more ingested SRBC. Controls were run with SRBC incubated in normal mouse serum, and <5% of cells rosetted or phagocytized. To study the effect of trypsin and cytochalasin, macrophages were incubated in 1 mg/ml of crystallized trypsin in phosphate-buffered saline (PBS), or 10 pg/ml of eytochalasin B (Aldrich Chemical Co., Inc., Milwaukee, Wis.) in PBS for 30 rain at 37°C, washed, and assayed for rosetting ability. Inhibition of Binding by Myeloma Proteins. MPCll (IgG2b), MOPC173 (IgG~), MOPC21 (IgGl), J606 (IgGs), or Flopc 21 (IgGs) protein was aggregated with bis-diazotized benzidine (BDB) (18). Macrophages were pretreated for 5 rain with the myeloma protein in PBS and then Ig- SRBC were added. Rosettes were assayed at 20 rain. Inhibitwn of Binding by Protein A. 20 ffl of a 5% solution of SRBC were incubated with 50/~1 of protein A at 5 mg/ml and 200 ffl of either IgGx, IgG~, IgG2b, or IgGz anti-SRBC antibody. The titer of the anti-SRBC antibody in each case was 1:500 using indirect hemagglutination with an anti-x antibody. The SRBC were washed and assayed for rosette formation. Results IgGa-coated SRBC formed rosettes with primary resident and thioglycolate-induced peritoneal macrophages, and with J774.2 and P388DI cell lines (Table I). In competitive inhibition experiments (Tables I and II), aggregates of IgG1, IgG2a, and IgG2b all failed to inhibit rosetting of IgGs-coated SRBC, suggesting a separate receptor for IgGa. In contrast, two IgGa myeloma proteins, Flopc 21 and J606, did inhibit rosetting when aggregated chemically with BDB. These results imply that the Fc RECEPTOR ON MOUSE MACROPHAGES BINDING IgG3 TABLE I Competition with Other IgG Subdasses for Fc Rosetting Primary Primary Inhibitor J774.2 P388DI resident TG induced IgGa-SRBC -- 97 93 87 89 IgGl aggregated 96 93 89 89 IgG2a aggregated 97 91 85 9 l IgG2b aggregated 98 91 86 84 IgG3 aggregated 21 22 19 22 TABLE II Rosettmg of IgGa-SRBC to Macrophages Primary Primary Inhibitor* J774.2 P388D 1 J774.2.1.4 resident TG in- duced -- 98 98 2 87 93 IgGa-SRBC J606 aggregated 17 21 2 17 21 Flopc 21 aggregated 19 20 4 22 19 Flopc monomer 95 91 3 84 97 * Inhibiting proteins were used at a concentration of 100/~g/ml. TABLE III Protein A Inhibition of Fc-mediated Rosettes J774.2 IgGa alone 98 IgGa + protein A 21 IgGl alone 97 IgGj + protein A 88 IgG~ alone 99 IgG2,, + protein A 18 IgG2b alone 99 IgG2b + protein A 21 IgG3 antibody is binding to the macrophage through its Fc terminal. Monomeric IgG3 did not inhibit rosetting. The rosetting of IgG3-SRBC was unaffected at 4°C, by cytochalasin B, or by trypsinization of the macrophages. In this respect, the IgGa receptor behaves like the receptor for IgG1 and IgG2b and unlike the receptor for IgG2a, which is altered at 4°C, and by both cytochalasin and trypsin. Although inhibition of rosetting by aggregated IgGa suggested that the binding of antibody-coated SRBC was Fc mediated, we wanted to show by a second method that IgGa was bound to receptors on macrophages through its Fc portion. We therefore examined whether protein A could inhibit the binding of IgGa. Protein A will bind to the Fc portion of several immunoglobulins, including IgG3 and IgG2. It binds poorly to IgG1 (19). It has been further shown that protein A will compete with Fc receptors for Fc binding (20). When protein A was incubated with IgG3-coated SRBC, it inhibited rosette formation. As expected, it also inhibited rosetting of IgG2-coated SRBC and not of IgGrcoated SRBC (Table III). J774.2.1.4 is a randomly picked subclone ofJ774.2. It did not rosette IgGa-SRBC BETTY DIAMOND AND DALE E. YELTON 517 TABLE IV Phagocytosis of lgG-SRBC Primary J774.2 P388D1 J774.2.1.1 Primary TG in- resident duced IgGa 89 87 2 83 91 IgGl 91 90 88 85 89 IgG2a 93 88 91 91 92 IgG2b 92 87 94 85 92 TABLE V Phagocytosis by Variant Lines FC 1.4 FC 1.6 IgGl 89 92 IgG2a 14 16 IgGab 91 93 IgG3 90 90 at all. It did, however, rosette IgGl-, IgG2a-, and IgG2b-coated SRBC in a manner indistinguishable from the parent cell line (Tables II and IV). These data again suggest that the receptor for IgG3 is independent of the other IgG receptors. We have previously reported variants from the FC 1 macrophage cell line that have altered phagocytosis through the IgG~ receptor (1). These variant lines with a selective defect in phagocytosis were both able to phagocytize IgGa-coated SRBC, suggesting again that the IgGa receptor is independent of the IgG2a receptor (Table V). Discussion It has previously been thought that IgG3 does not bind to Fc receptors on mouse macrophages. This was based on data showing the inability of IgG3 to compete successfully with IgG2a for Fc binding (14). It has since become clear that macrophages possess more than one Fc receptor and that each receptor has a restricted specificity (1, 5, 12). Two receptors have been clearly defined: one for IgG1 and IgG2b, and the other for IgG2a (4, 13). The studies reported here indicate that there is also a separate Fc receptor on mouse macrophages for IgG3. This Fc receptor is present on resident peritoneal macrophages as well as on thioglycolate-induced macrophages. In addition, the J774.2 cell line and the P388Dl cell line exhibit this receptor. This receptor is independent from the two previously identified receptors because (a) the other IgG subclasses do not compete with IgGs for binding; (b) J774.2.1.4, a subclone of J774.2, is unable to bind or phagocytize IgGa complexes but is unaffected in its IgG2~ and IgGa-IgG2b receptors; and, (c) variants of the FC1 line that are altered in their ability to phagocytize through the IgG2a receptor have an intact ability to phagocytize IgGa-SRBC. The IgGs Fc receptor is similar to the receptor for IgGa and IgG2b in that it is not affected at 4°C or by cytochalasin B or trypsin. It resembles the other IgG receptors in its ability to mediate phagocytosis. In addition, we have reported that IgG3 can also mediate antibody-dependent cell-mediated cytolysis of SRBC. Fc RECEPTOR ON MOUSE MACROPHAGES BINDING IgGs That IgGs should bind through a separate receptor is not surprising, because structurally and serologically it is the most dissimilar of the I.gG subclasses (14). The identification of this third receptor, however, raises again the question of why several independent Fc receptors are present on macrophages. On macrophages, each receptor is capable of mediating both phagocytosis and antibody-dependent cell-mediated cytolysis (21). Other cell types that bear Fc receptors may possess only one or two of these receptors in order to restrict their response to antibody or immune complexes (8, 22, 23). If Fc receptors on other cell types have the same specificity as on macrophages, perhaps the multiplicity of receptors allows for antibody-specific re- sponsiveness in other cell types. The multiplicity may be a consequence of the amount of information that must be carried in the constant region of the immunoglobulin molecule; the constraints of subclass specificity may make separate Fc receptors necessary. Further studies on both the biologically active sites of the IgG molecules and the nature of Fc receptors on other cell types are necessary to resolve these questions. Summary Monoclonal antibodies to sheep erythrocytes (SRBC) have proved useful in iden- tifying two Fc receptors on mouse macrophages, one for IgG2a, and one for IgG1 and IgG2b. We have used monoclonal IgG3 anti-SRBC to identify a third Fc receptor on mouse macrophages which binds IgG3 uniquely. This receptor is present on primary resident and thioglycolate-induced peritoneal macrophages and on some macrophage cell lines. The binding of IgGa-coated SRBC is inhibited by aggregated but not monomeric IgGa, and not by IgG1, IgG2., and IgGeb aggregates. It is unaffected by treating the macrophages with trypsin or cytochalasin B and occurs at both 4 ° and 37°C. IgGs, like all other IgG subclasses, mediates phagocytosis. We have also generated a variant macrophage line which bears the receptors for IgGa and IgG2b and for IgG2,, but not for IgGs. We would like to acknowledge the help of Dr. Peter Ralph in initiating these studies and in giving advice throughout. Received for publication 29 September 1980. References I. Diamond, B., B. R. Bloom, and M. D. Scharff. 1978. The Fc receptors of primary and cultured phagocytic cells studied with homogeneous antibodies.,]. ]mmunol. 121:1329. 2. Heusser, C. H., C. L. Anderson, and H. M. Grey. 1977. Receptors for IgG: subclass specificity of receptors on different mouse cell types and the definition of two distinct receptors on a macrophage cell line.,]. Exp. Med. 145:1316. 3. Segal, D. M., and J. A. Titus. 1978. The subclass of specificity for the binding of murine myeloma proteins to macrophage and lymphocyte cell lines and to normal spleen cell. jr. Immunol. 120:1395. 4. Unkeless, J. C. 1979. Characterization of a monoclonal antibody directed against mouse macrophage and lymphocyte Fc receptors.,]. Exp. Med. 150:.580. 5. Walker, W. S. 1976. Separate Fc-receptors for immunoglobulins IgG2, and IgG2b on an established cell line of mouse macrophages. J. Immunol. 116:911. 6. Paraskevas, F., and S. T. Lee. 1976. The helper cell function of primed T-cells. I. Marked BETTY DIAMOND AND DALE E. YELTON amplification of antibody formation by antigen-educated T-cells carrying surface Ig 6 h after priming. Eur. J. lmmunoL 6:856. 7. Andersson, B., A.-C. Skoglund, and A. Rosen. 1979. Functional characterization of mouse T lymphocytes with IgM-Fc receptors.J, lmrnunoL 123:1936. 8. Strober, W., N. E. Hague, L. G. Lure, and P. A. Henkart. 1978. IgA-Fc receptors on mouse lymphoid cells.J, lmmunoL 121:2440. 9. Scribner, D. J., and D. Farhney. 1976. Neutrophil receptors of IgG and complement: their roles in the attachment and ingestion phases of phagocytosis. J. Immunol. 116:892. 10. Mantovani, B. 1975. Different roles of IgG and complement receptors in phagocytosis by polymorphonuclear leukocytes. J. lmrnunoL 115:15. 11. McNabb, T., T. Y. Koh, K. Y. Dorrington, and R. H. Painter. 1976. Structure and function of immunoglobulin domains. V. Binding of immunoglobulin G and fragments to placental membrane preparations. J. lrarnunol. 117:882. 12. Unkeless, J. C. 1977. The presence of two Fc receptors on mouse macrophages: evidence from a variant cell line and differential trypsin sensitivity.J. Exp. Med. 142:931. 13. Diamond, B., and M. D. Scharff. 1980. IgG1 and IgG2b share the Fc receptor on mouse macrophages. J. lmmunoL 125:631. 14. Grey, H. M.,J. W. Hirst, and M. Cohn. 1971. A new mouse immunoglobulin IgG3.J. Exp. Med. 133:289. 15. Ralph, P., J. Prichard, and M. Cohn. 1975. Reticulum cell sarcoma: an effector cell in antibody-dependent cell-mediated immunity.J, lmmunoL 114:898. 16. Potter, M., J. G. Pumphrey, and J. L. Waiters. 1972. Growth of primary plasmacytomas in the mineral oil conditioned peritoneal environment.J. Natl. Cancer lnst. 49:305. 17. Bianco, C., F. M. Griffin, and S. C. Silverstein. 1975. Studies of the macrophage comple- ment receptor. Alteration of receptor function upon macrophage activation. J. Exp. Med. 141:1278. 18. Pressman, D., D. H. Campbell, and L. Pauling. 1942. The agglutination of intact azoerythrocytes by antisera homologous to the attached groups.J, lmmunol. 44:101. 19. Kronvall, G., H. M. Grey, and R. C. Williams. 1970. Protein A reactivity with mouse immunoglobulins.J, lmmunol. 105:1116. 20. Dossett, J. W., G. Kronvall, R. C. Williams, Jr., and P. G. Quie. 1969. Antiphagocytic effects of staphylococcal protein A.J. ImmunoL 103:1405. 21. Ralph, P., I. Nakoinz, B. Diamond, and D. Yelton. 1980. All classes ofmurine IgG antibody mediate macrophage phagocytosis and lysis of erythrocytes. J. lmmunoL 125:1885. 22. Gordon, J., and R. A. Murgita. 1975. Suppression and augmentation of the primary in vitro response by different classes of antibody. Cell lmmunoL 15:392. 23. Paraskevas, F., and S. T. Lee. 1976. Helper cell function of primed T cells. II. T-T cell synergism between Ig ÷ and Ig- subpopulations of primed thymocytes: a mechanism for amplification of helper cell function. Eur. J. lmmunoL 6:862. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Experimental Medicine Pubmed Central

A new Fc receptor on mouse macrophages binding IgG3

The Journal of Experimental Medicine , Volume 153 (3) – Mar 1, 1981

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Abstract

A NEW Fc RECEPTOR ON MOUSE MACROPHAGES BINDING IgG3* BY BETTY DIAMOND AND DALE E. YELTON:[: From the Departments of Microbiology and Immunology and Medicine, and of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461 Among the effector functions exhibited by immunoglobulins is the ability to bind to Fc receptors on macrophages, lymphocytes, and granulocytes (1-10). It has recently become evident that there are different types of Fc receptors on different types of cells (11) and, moreover, that the same cell may express more than one type of Fc receptor (1, 2, 5). For example, mouse macrophage Fc receptors do not bind IgM, whereas some lymphocyte Fc receptors do (7). Mouse macrophages are known to express at least two different Fc receptors, one specifically binding mouse IgGt and IgG2b, the other binding IgG2a (12, 13). The Fc receptors are distinguishable by certain other properties as well. The IgG2a receptor is sensitive to trypsin and shows decreased binding at 4°C and in the presence of cytochalasin B; the IgGl-IgGzb receptor is trypsin resistant and unaltered at 4°C by cytochalasin B (1, 13). The IgGa class of mouse immunoglobulins has not been studied extensively with respect to its binding to Fc receptors. IgGs has an Fc fragment that is serologically and structurally different from IgG1 and IgG2 (14). Functionally, it has been shown to have an increased ability to cross the placenta 04). Earlier experiments have shown that monomeric IgG3 does not inhibit the binding of monomeric IgG2a to its Fc receptor (14). These and other early results have led to the assumption that IgG3 does not bind to Fc receptors. In light of the evidence for multiple Fc receptors, we felt it necessary to examine again the binding of IgGs to Fc receptors. We generated a monoclonal IgGs anti-sheep erythrocyte (SRBC) 1 antibody from a mouse spleen-mouse myeloma fusion in order to study directly the binding of IgGa to macrophage Fc receptors. Monoclonal antibodies are especially useful in such studies because they are homogeneous, they are the only mouse immunoglobulin in culture medium from the hybridoma cultures, and when reacted with their antigen, they form antigen-antibody complexes more natural for binding studies than artificial aggregates. Using this monoclonal IgG3 anti-SRBC antibody, we found that IgG3 does bind to macrophages through a third, independent IgG Fc receptor. Materials and Methods Cells. J774 is a reticulum cell sarcoma from a BALB/c mouse with macrophagelike properties that has been adapted to culture 05). J774.2 is a clone from the tissue culture line. * Supported by grants AI 161166, A110702, AI 1581 l, A1523 l, and CA24300 from t he National Institutes of Health, grant PCM77-25635 from the National Science Foundation, and grant IM-216 from the American Cancer Society. :~ Medical scientist trainee supported by grant 5T32GMF288 from the National Institutes of Health. J Abbreviations used in this paper: BDB, bis-diazotized benzidine; PBS, phosphate-buffered saline; SRBC, sheep erythrocytes. 514 J. ExP. MEo. ©The Rockefeller University Press • 0022-1007/81/03/0514/06 $1.00 Volume 153 March 1981 514-519 515 BETTY DIAMOND AND DALE E. YELTON A series of subclones ofJ774.2 were picked and screened for their ability to bind IgGs. J774.2.1 was found to lack IgGs receptors. It has grown to mass culture and recloned to produce J774.2.1.4. P388D1 is a macrophagelike cell line with Fc receptors and phagocytic ability. FCI.4 and FC1.6 are subclones of the FC1 cell line which arose during a fusion of MPC 11 myeloma ceils to spleen cells from a BALB/e mouse immunized with SRBC. FC 1.4 and FC 1.6 were selected for their inability to phagocytize IgG~-SRBC. Primary macrophages were obtained from resident peritoneal cells and from peritoneal cells 4 d after an intraperitoneal injection of thioglycolate broth (Difco Laboratories, Detroit, Mich.). The method for isolating adherent cells has been described previously (1). Myeloma Proteins. Myeloma proteins were obtained by injecting 107 MOPC21 (IgGa), MPCll (IgG~b), MOPC173 (IgG2a), or J606 (IgGa) cells into the peritoneal cavity of pristine- primed BALB/c mice (16). The aseites fluid was precipitated with 50% saturated ammonium sulfate. A purified -/-globulin fraction was obtained by DEAE chromatography (1). It was determined by agarose gel electrophoresis and by Ouchterlony analysis that no fraction had any contaminating protein of another subclass. Flope 21 was purchased from Bionetics, Kensington, Md. Monomeric protein was prepared by centrifuging the protein for 30 min at 150,000 g. Preparation ofIg-coated SRBC. The methods of Bianco et al. (17) were used. SRBC were incubated with antibody for 30 min at 37°C, washed, and resuspended to 0.5%. The antibodies were obtained from cloned hybridoma lines making anti-SRBC antibody, 5 #1 of ascites fluid was incubated with 1 ml of a 5% solution of SRBC. The IgGs-producing line was derived from a fusion of drug-marked P3 cells with spleen cells from a BALB/c mouse immunized with SRBC. Segregants no longer making the myeloma IgG1 heavy chain were selected. The IgGz antibody was identified by Ouchterlony analysis with commercial anti-IgG3 (Meioy Laboratories, Inc., Springfield, Va.) and with antiserum raised in the laboratory of Dr. John Cebra, University of Pennsylvania, against J606 protein and absorbed with IgGa, Ig~a, and IgG2b. Fc Rosettes and Fc-mediated Phagocytosis. This was done as previously described (1). Cells adhered to glass coverslips were incubated for 30 min at 37 ° or 4°C, washed, and assayed for rosettes. Attachment of three or more SRBC signified a rosette. For phagoeytosis, the cells were incubated for 1 h at 37°C, free SRBC were lysed in hypotonic solution, and intracellular SRBC were assayed. A phagocytic cell was any cell with three or more ingested SRBC. Controls were run with SRBC incubated in normal mouse serum, and <5% of cells rosetted or phagocytized. To study the effect of trypsin and cytochalasin, macrophages were incubated in 1 mg/ml of crystallized trypsin in phosphate-buffered saline (PBS), or 10 pg/ml of eytochalasin B (Aldrich Chemical Co., Inc., Milwaukee, Wis.) in PBS for 30 rain at 37°C, washed, and assayed for rosetting ability. Inhibition of Binding by Myeloma Proteins. MPCll (IgG2b), MOPC173 (IgG~), MOPC21 (IgGl), J606 (IgGs), or Flopc 21 (IgGs) protein was aggregated with bis-diazotized benzidine (BDB) (18). Macrophages were pretreated for 5 rain with the myeloma protein in PBS and then Ig- SRBC were added. Rosettes were assayed at 20 rain. Inhibitwn of Binding by Protein A. 20 ffl of a 5% solution of SRBC were incubated with 50/~1 of protein A at 5 mg/ml and 200 ffl of either IgGx, IgG~, IgG2b, or IgGz anti-SRBC antibody. The titer of the anti-SRBC antibody in each case was 1:500 using indirect hemagglutination with an anti-x antibody. The SRBC were washed and assayed for rosette formation. Results IgGa-coated SRBC formed rosettes with primary resident and thioglycolate-induced peritoneal macrophages, and with J774.2 and P388DI cell lines (Table I). In competitive inhibition experiments (Tables I and II), aggregates of IgG1, IgG2a, and IgG2b all failed to inhibit rosetting of IgGs-coated SRBC, suggesting a separate receptor for IgGa. In contrast, two IgGa myeloma proteins, Flopc 21 and J606, did inhibit rosetting when aggregated chemically with BDB. These results imply that the Fc RECEPTOR ON MOUSE MACROPHAGES BINDING IgG3 TABLE I Competition with Other IgG Subdasses for Fc Rosetting Primary Primary Inhibitor J774.2 P388DI resident TG induced IgGa-SRBC -- 97 93 87 89 IgGl aggregated 96 93 89 89 IgG2a aggregated 97 91 85 9 l IgG2b aggregated 98 91 86 84 IgG3 aggregated 21 22 19 22 TABLE II Rosettmg of IgGa-SRBC to Macrophages Primary Primary Inhibitor* J774.2 P388D 1 J774.2.1.4 resident TG in- duced -- 98 98 2 87 93 IgGa-SRBC J606 aggregated 17 21 2 17 21 Flopc 21 aggregated 19 20 4 22 19 Flopc monomer 95 91 3 84 97 * Inhibiting proteins were used at a concentration of 100/~g/ml. TABLE III Protein A Inhibition of Fc-mediated Rosettes J774.2 IgGa alone 98 IgGa + protein A 21 IgGl alone 97 IgGj + protein A 88 IgG~ alone 99 IgG2,, + protein A 18 IgG2b alone 99 IgG2b + protein A 21 IgG3 antibody is binding to the macrophage through its Fc terminal. Monomeric IgG3 did not inhibit rosetting. The rosetting of IgG3-SRBC was unaffected at 4°C, by cytochalasin B, or by trypsinization of the macrophages. In this respect, the IgGa receptor behaves like the receptor for IgG1 and IgG2b and unlike the receptor for IgG2a, which is altered at 4°C, and by both cytochalasin and trypsin. Although inhibition of rosetting by aggregated IgGa suggested that the binding of antibody-coated SRBC was Fc mediated, we wanted to show by a second method that IgGa was bound to receptors on macrophages through its Fc portion. We therefore examined whether protein A could inhibit the binding of IgGa. Protein A will bind to the Fc portion of several immunoglobulins, including IgG3 and IgG2. It binds poorly to IgG1 (19). It has been further shown that protein A will compete with Fc receptors for Fc binding (20). When protein A was incubated with IgG3-coated SRBC, it inhibited rosette formation. As expected, it also inhibited rosetting of IgG2-coated SRBC and not of IgGrcoated SRBC (Table III). J774.2.1.4 is a randomly picked subclone ofJ774.2. It did not rosette IgGa-SRBC BETTY DIAMOND AND DALE E. YELTON 517 TABLE IV Phagocytosis of lgG-SRBC Primary J774.2 P388D1 J774.2.1.1 Primary TG in- resident duced IgGa 89 87 2 83 91 IgGl 91 90 88 85 89 IgG2a 93 88 91 91 92 IgG2b 92 87 94 85 92 TABLE V Phagocytosis by Variant Lines FC 1.4 FC 1.6 IgGl 89 92 IgG2a 14 16 IgGab 91 93 IgG3 90 90 at all. It did, however, rosette IgGl-, IgG2a-, and IgG2b-coated SRBC in a manner indistinguishable from the parent cell line (Tables II and IV). These data again suggest that the receptor for IgG3 is independent of the other IgG receptors. We have previously reported variants from the FC 1 macrophage cell line that have altered phagocytosis through the IgG~ receptor (1). These variant lines with a selective defect in phagocytosis were both able to phagocytize IgGa-coated SRBC, suggesting again that the IgGa receptor is independent of the IgG2a receptor (Table V). Discussion It has previously been thought that IgG3 does not bind to Fc receptors on mouse macrophages. This was based on data showing the inability of IgG3 to compete successfully with IgG2a for Fc binding (14). It has since become clear that macrophages possess more than one Fc receptor and that each receptor has a restricted specificity (1, 5, 12). Two receptors have been clearly defined: one for IgG1 and IgG2b, and the other for IgG2a (4, 13). The studies reported here indicate that there is also a separate Fc receptor on mouse macrophages for IgG3. This Fc receptor is present on resident peritoneal macrophages as well as on thioglycolate-induced macrophages. In addition, the J774.2 cell line and the P388Dl cell line exhibit this receptor. This receptor is independent from the two previously identified receptors because (a) the other IgG subclasses do not compete with IgGs for binding; (b) J774.2.1.4, a subclone of J774.2, is unable to bind or phagocytize IgGa complexes but is unaffected in its IgG2~ and IgGa-IgG2b receptors; and, (c) variants of the FC1 line that are altered in their ability to phagocytize through the IgG2a receptor have an intact ability to phagocytize IgGa-SRBC. The IgGs Fc receptor is similar to the receptor for IgGa and IgG2b in that it is not affected at 4°C or by cytochalasin B or trypsin. It resembles the other IgG receptors in its ability to mediate phagocytosis. In addition, we have reported that IgG3 can also mediate antibody-dependent cell-mediated cytolysis of SRBC. Fc RECEPTOR ON MOUSE MACROPHAGES BINDING IgGs That IgGs should bind through a separate receptor is not surprising, because structurally and serologically it is the most dissimilar of the I.gG subclasses (14). The identification of this third receptor, however, raises again the question of why several independent Fc receptors are present on macrophages. On macrophages, each receptor is capable of mediating both phagocytosis and antibody-dependent cell-mediated cytolysis (21). Other cell types that bear Fc receptors may possess only one or two of these receptors in order to restrict their response to antibody or immune complexes (8, 22, 23). If Fc receptors on other cell types have the same specificity as on macrophages, perhaps the multiplicity of receptors allows for antibody-specific re- sponsiveness in other cell types. The multiplicity may be a consequence of the amount of information that must be carried in the constant region of the immunoglobulin molecule; the constraints of subclass specificity may make separate Fc receptors necessary. Further studies on both the biologically active sites of the IgG molecules and the nature of Fc receptors on other cell types are necessary to resolve these questions. Summary Monoclonal antibodies to sheep erythrocytes (SRBC) have proved useful in iden- tifying two Fc receptors on mouse macrophages, one for IgG2a, and one for IgG1 and IgG2b. We have used monoclonal IgG3 anti-SRBC to identify a third Fc receptor on mouse macrophages which binds IgG3 uniquely. This receptor is present on primary resident and thioglycolate-induced peritoneal macrophages and on some macrophage cell lines. The binding of IgGa-coated SRBC is inhibited by aggregated but not monomeric IgGa, and not by IgG1, IgG2., and IgGeb aggregates. It is unaffected by treating the macrophages with trypsin or cytochalasin B and occurs at both 4 ° and 37°C. IgGs, like all other IgG subclasses, mediates phagocytosis. We have also generated a variant macrophage line which bears the receptors for IgGa and IgG2b and for IgG2,, but not for IgGs. We would like to acknowledge the help of Dr. Peter Ralph in initiating these studies and in giving advice throughout. Received for publication 29 September 1980. References I. Diamond, B., B. R. Bloom, and M. D. Scharff. 1978. The Fc receptors of primary and cultured phagocytic cells studied with homogeneous antibodies.,]. ]mmunol. 121:1329. 2. Heusser, C. H., C. L. Anderson, and H. M. Grey. 1977. Receptors for IgG: subclass specificity of receptors on different mouse cell types and the definition of two distinct receptors on a macrophage cell line.,]. Exp. Med. 145:1316. 3. Segal, D. M., and J. A. Titus. 1978. The subclass of specificity for the binding of murine myeloma proteins to macrophage and lymphocyte cell lines and to normal spleen cell. jr. Immunol. 120:1395. 4. Unkeless, J. C. 1979. Characterization of a monoclonal antibody directed against mouse macrophage and lymphocyte Fc receptors.,]. Exp. Med. 150:.580. 5. Walker, W. S. 1976. Separate Fc-receptors for immunoglobulins IgG2, and IgG2b on an established cell line of mouse macrophages. J. Immunol. 116:911. 6. Paraskevas, F., and S. T. Lee. 1976. The helper cell function of primed T-cells. I. Marked BETTY DIAMOND AND DALE E. YELTON amplification of antibody formation by antigen-educated T-cells carrying surface Ig 6 h after priming. Eur. J. lmmunoL 6:856. 7. Andersson, B., A.-C. Skoglund, and A. Rosen. 1979. Functional characterization of mouse T lymphocytes with IgM-Fc receptors.J, lmrnunoL 123:1936. 8. Strober, W., N. E. Hague, L. G. Lure, and P. A. Henkart. 1978. IgA-Fc receptors on mouse lymphoid cells.J, lmmunoL 121:2440. 9. Scribner, D. J., and D. Farhney. 1976. Neutrophil receptors of IgG and complement: their roles in the attachment and ingestion phases of phagocytosis. J. Immunol. 116:892. 10. Mantovani, B. 1975. Different roles of IgG and complement receptors in phagocytosis by polymorphonuclear leukocytes. J. lmrnunoL 115:15. 11. McNabb, T., T. Y. Koh, K. Y. Dorrington, and R. H. Painter. 1976. Structure and function of immunoglobulin domains. V. Binding of immunoglobulin G and fragments to placental membrane preparations. J. lrarnunol. 117:882. 12. Unkeless, J. C. 1977. The presence of two Fc receptors on mouse macrophages: evidence from a variant cell line and differential trypsin sensitivity.J. Exp. Med. 142:931. 13. Diamond, B., and M. D. Scharff. 1980. IgG1 and IgG2b share the Fc receptor on mouse macrophages. J. lmmunoL 125:631. 14. Grey, H. M.,J. W. Hirst, and M. Cohn. 1971. A new mouse immunoglobulin IgG3.J. Exp. Med. 133:289. 15. Ralph, P., J. Prichard, and M. Cohn. 1975. Reticulum cell sarcoma: an effector cell in antibody-dependent cell-mediated immunity.J, lmmunoL 114:898. 16. Potter, M., J. G. Pumphrey, and J. L. Waiters. 1972. Growth of primary plasmacytomas in the mineral oil conditioned peritoneal environment.J. Natl. Cancer lnst. 49:305. 17. Bianco, C., F. M. Griffin, and S. C. Silverstein. 1975. Studies of the macrophage comple- ment receptor. Alteration of receptor function upon macrophage activation. J. Exp. Med. 141:1278. 18. Pressman, D., D. H. Campbell, and L. Pauling. 1942. The agglutination of intact azoerythrocytes by antisera homologous to the attached groups.J, lmmunol. 44:101. 19. Kronvall, G., H. M. Grey, and R. C. Williams. 1970. Protein A reactivity with mouse immunoglobulins.J, lmmunol. 105:1116. 20. Dossett, J. W., G. Kronvall, R. C. Williams, Jr., and P. G. Quie. 1969. Antiphagocytic effects of staphylococcal protein A.J. ImmunoL 103:1405. 21. Ralph, P., I. Nakoinz, B. Diamond, and D. Yelton. 1980. All classes ofmurine IgG antibody mediate macrophage phagocytosis and lysis of erythrocytes. J. lmmunoL 125:1885. 22. Gordon, J., and R. A. Murgita. 1975. Suppression and augmentation of the primary in vitro response by different classes of antibody. Cell lmmunoL 15:392. 23. Paraskevas, F., and S. T. Lee. 1976. Helper cell function of primed T cells. II. T-T cell synergism between Ig ÷ and Ig- subpopulations of primed thymocytes: a mechanism for amplification of helper cell function. Eur. J. lmmunoL 6:862.

Journal

The Journal of Experimental MedicinePubmed Central

Published: Mar 1, 1981

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