Immunology 1976 30 267
In vitro uptake and digestion of immune complexes containing guinea-pig IgG1 and IgG2 antibodies by macrophages
T. SHINO MI YA & J. KOYA MA Department of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan
Received 13 June 1975; acceptedfor publication 14 August 1975
body alone can be demonstrated (Uhr, 1965; Phillips-Quagliata, Levine, Quagliata and Uhr, 1971; Arend and Mannik, 1972; Leslie and Cohen, 1974b). For example, the formation of immune complex with homologous antigen increases the strength of binding of guinea-pig IgG2 antibody, known as a cytophilic antibody, to macrophages, although the antibody alone is capable of binding to the cells weakly (Leslie and Cohen, 1974a, b). The binding is supposed to be mediated by the Fc parts of antibody molecules, since it is inhibited by the Fc fragments, but not by the F(ab')2 fragments. On the other hand, there is another subclass, IgG1, in guinea-pig IgG, which is not cytophilic but homocytotropic (Ovary, Benacerraf and Bloch, 1963; Sandberg, Oliveira and Osler, 1971; Berken and Benacerraf, 1966). The IgG1 antibody is distinguished from the IgG2 antibody by both structure and biological function of the Fc part. In order to elucidate the mechanism of elimination of antigen by the IgG1 antibody and also of ingestion of immune complexes by macrophages, we have studied the in vitro uptake and digestion of the IgG1 INTRODUCTION antibody by guinea-pig peritoneal macrophages using 1251-labelled IgG1 antibody to hen ovalbumin Certain immune complexes bind to the surfaces of (OA). The present paper reports the enhanced macrophages even when little or no binding of antiingestion of the IgG1 antibody by the complex formation with OA. In particular, the mode of Correspondence: Professor J. Koyama, Department of uptake and digestion of IgGI complex was compared Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, 060, Japan. with that of IgG2 complex. 267
Summary. The uptake and digestion of immune complexes by peritoneal macrophages from oilstimulated guinea-pigs were studied using l 25I1 labelled guinea-pig IgG1 and IgG2 antibodies to hen ovalbumin. When the IgGi or IgG2 antibody was incubated with homologous antigen at antigenantibody ratios ranging from 0-01 to 10, the complexes produced were preferentially taken up by macrophages in the absence of complement and digested by the intracellular enzymes. The uptake and digestion of complexes reached a maximum at an antigen-antibody ratio of 0-1-05. In this respect, the IgG1 antibody was indistinguishable from the IgG2 antibody having a cytophilic activity. These observations suggest that some conformational changes in both the IgG1 and IgG2 antibodies or specific molecular arrangement in the lattice work of both the complexes may increase the strength of binding of complexes to macrophages, independently of the difference in biological activities between these two antibodies.
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MATERIALS AND METHODS Preparation of guinea-pig antibodies to OA Guinea-pig IgGI and IgG2 anti-OA antibodies were specifically purified from the hyperimmune serum by immunoadsorbent techniques previously described (Nakamura and Koyama, 1975). Elution of the bound antibodies from a column of agarose gel coupled with OA by the CNBr method (Porath, Axen and Ernback, 1967) was performed using either 01 M glycine buffer, pH 3-5, or 3 M NaSCN (Carrel and Barandun, 1971). The IgGI and IgG2 antibodies thus purified were separated by zone electrophoresis in a starch block (Nakamura, Ofune, Tamoto and Koyama, 1972). Preparation of F(ab')2 fragments of guinea-pig IgG1 and IgG2 antibodies The F(ab')2 fragments of guinea-pig IgGI and IgG2 anti-OA antibodies were prepared from the antibody preparations specifically purified from the hyperimmune serum. The digestion with pepsin was performed according to the method of Nisonoff, Wissler, Lipman and Woernley (1960). The F(ab')2 fragments were purified by gel filtration with Sephadex G-200. Isotopic labelling and further purification of proteins Labelling of proteins with radioactive iodine was performed by the ICL method (Grossberg, Radzimski and Pressman, 1962). Iodine was incorporated at less than 1 atom of iodine per molecule of protein. Some aggregated proteins forming during the purification and labelling were removed by gel filtration with Sephadex G-200. Monomeric proteins isolated were used in experiments. The l25l-labelled IgG1 and IgG2 antibodies and the F(ab')h fragments thus obtained were highly pure on immunoelectrophoresis using rabbit antiserum to guinea-pig serum. On the basis of amounts of the radioactivity adsorbed on the immunoadsorbent, more than 95 per cent of each protein was found to be anti-OA antibody or the F(ab')2 fragments capable of binding to OA.
Isolation ofperitoneal macrophages Guinea-pigs of the Hartley strain weighing 400-600 g were used as donors of peritoneal macrophages. The animals were injected intraperitoneally with 20 ml of sterilized liquid paraffin. Four days after the injection, macrophages were harvested from the
peritoneal cavity using the Dulbecco's balanced salt solution containing 1 u of heparin/ml (Dulbecco and Vogt, 1954). The cells were centrifuged at 500 g for 10 min and washed twice at 40 with 50 ml of the MEM medium (minimal essential medium) containing 20 mm HEPES. Culture of macrophages Culture of macrophages was performed essentially according to the method described by Calderon and Unanue (1974). The cells isolated were suspended in the MEM medium containing 5 per cent calf serum. 107 cells in 1 ml of the medium were planted in plastic dishes (Falcon, 35 x 10 mm) and incubated at 370 for 20 h in a C02-air incubator. After incubation, the dishes containing the adherent macrophages were washed twice with the MEM medium supplemented with 20 mm HEPES, which had been added to MEM in order to maintain a stable pH outside the incubator. At this time, the dishes contained about 40 per cent of the original number of cells planted, when estimated on the basis of the determination of cellular proteins with the Folin reagent. More than 98 per cent of the cells were typical, well-spread macrophages forming an interrupted monolayer. After washes, the dishes were incubated with I251-labelled antibody and antigen in the MEM medium supplemented with 0 1 per cent bovine serum albumin which had been added to prevent non-specific adsorption of proteins to the dishes or to the cells. After incubation for appropriate times, the ingestion of immune complexes were investigated. Each experiment was run in duplicate and repeated two or three times. Analysis of 1 25I-labelled products 25I-labelled materials were examined from culture supernatants or cells after the incubation of complexes containing ' 25I-labelled antibodies with macrophages. Culture supernatants were centrifuged at 500 g for 5 min in order to remove any possible cells and then precipitated by the addition of an equal volume of 10 per cent trichloroacetic acid. On occasion, 5 mg of bovine serum albumin was added to the culture supernatants as a carrier to obtain a sufficient amount of precipitates. The precipitates and supernatant fluids thus obtained were assumed to represent protein-bound and non-
protein-bound 1251. The radioactivity associated with the cells (cellassociated 1251) was obtained by the treatment of the
269
Uptake of immune complexes by macrophages twice washed cell monolayers with 0-5 ml of 01 N NaOH. The cellular materials were extracted after gently scraping the cells from the dishes with a rubber policeman and then added to 5 mg of bovine serum albumin. The cell-associated I 25I-labelled materials were fractionated to protein-bound and non-protein-bound 1 251 by the addition of trichloroacetic acid at 5 per cent. The measurements of radioactivity were performed in a Packard automatic well-type gamma counter.
Estimation ofproteins The absorbancy at 280 nm was used for calculation of protein concentration. The values of A' Per Cent were taken as 13-5 for IgG as well as F(ab')2 fragment and 7-35 for OA, respectively. The following molecular weights were assumed: 150,000 for IgG, 100,000 for F(ab')2 fragment and 45,000 for OA. Materials Crystalline OA was prepared in our laboratory (Nakamura et al., 1972). Bovine serum albumin and HEPES [4-(2-hydroxyethyl)-1-piperazine ethanesulphonic acid] were obtained from Sigma Chemical Company. The MEM medium was 10 |H
obtained from Nissui Seiyaku Company, Ltd, Japan. RESULTS
Uptake and digestion of IgG1 and IgG2 antibodies depending on the complex formation with antigen The uptake and digestion of '25l-labelled IgGi and IgG2 anti-OA antibodies by macrophages were studied in the presence of OA. Representative results are shown in Fig. 1. Incubation of macrophages with 125I-labelled IgGi or IgG2 anti-OA antibody alone resulted in little binding of radioactivity, detectable after two washes. However, a marked increase in the uptake of IgGi and IgG2 antibodies occurred when a constant amount of the IgG1 or IgG2 antibody was added to the cells after incubation with varying the amounts of OA at 370 for 1 h. In particular, the uptake and digestion of both IgG1 and IgG2 antibodies reached a maximum when macrophages were incubated with each complex having an antigen-antibody ratio between 0-1 and 0 5, corresponding to an equivalence point of precipitin reactions of these two antibodies (Nakamura et al., 1972).
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Figure 1. Effect of complex formation with OA on the uptakes of IgGI and IgG2 anti-OA antibodies by macrophages. Soluble complexes were prepared by incubation of 20 ,ug of (a) '251-labelled IgG 1 or (b) IgG2 antibody with varying the amounts of OA in the range of molar ratio of antigen to antibody from 0 to 10. The complexes were reacted with macrophages at 370 for 4 h. The amounts of protein-bound "25I (0 0) and non-protein-bound "25I (--- -0) associated with cells and non-proteinbound 125I released into the culture fluids (0- --0) were determined and expressed as percentages against total radioactivity added. H
270
T. Shinomiya & J. Koyama
The analysis of ' 251-labelled materials in the culture fluids revealed that the cell-bound antibodies were digested during the incubation and the digestive products were released into the culture fluids. The amount of released 1251 was in proportion to that of cell-bound antibody; the maximum release again occurred with the complexes having an antigenantibody ratio between 0-1 and 0 5. On the other hand, the cell-bound 1251 was mainly of proteinbound nature, since the amount of non-proteinbound 1251 remained remarkably small, indicating that the digested 1251-labelled compounds were removed rapidly out of the cells. With regard to the uptake of complexes, there seems to be the possibility that the protein-bound radioactivity did not represent the cell-associated 125I but 1251-labelled antibody precipitated with OA during the incubation. However, when 20 ug of 1251-labelled IgGi or IgG2 anti-OA antibody were incubated with 3 ug of OA in empty dishes, no visible precipitate formed and radioactivity which adhered to the dishes and was recovered with 01 N NaOH was less than 0 5 per cent of the added activity. Therefore, the radioactivity of cells, detectable after two washes which were sufficient to remove the unbound complexes, could be assumed to represent the cell-associated 1251. It was very probable that the concentration of antigen-antibody complexes used was too low to make the complexes precipitable. Effect of co-existing IgG2 antibody on the uptake of IgG1 complex The results shown in Fig. 1 demonstrated that the
IgGi antibody, known to be non-cytophilic, was indistinguishable from the cytophilic IgG2 antibody by susceptibility to the ingestion by macrophages, when reacted in a form of soluble complex. However, this might not be caused by a property of the IgGI antibody itself, but by a helper function of a trace amount of the IgG2 antibody existing as a contaminant in the IgGi antibody preparation used, though immunoelectrophoretic analysis could not demonstrate any contaminating IgG2 antibody, as shown in Fig. 2. In order to investigate further this point, the effect of coexisting IgG2 antibody on the uptake of 1251. labelled IgGl antibody by macrophages was studied using some mixed complexes composed of 1251_ labelled IgGi and non-labelled IgG2 anti-OA antibodies. The complexes used were prepared by incubation of a constant amount of OA (6 7 x 10- 11 moles) with the antibody mixtures (1 3x10-10 moles) containing the IgGI and IgG2 antibodies at various ratios. In control experiments, other mixed complexes were prepared in the same manner, using 125I-labelled F(ab')2 fragments of IgG1 and IgG2 anti-OA antibodies in the place of 1 251_ labelled IgG1 antibody, since the F(ab')2 fragments are known to have no ability to adhere to the surfaces of macrophages even when bound to homologous antigen (Arend and Mannik, 1972). After incubation of these complexes with macrophages at 370 for 4 h, the cells and culture fluids were analysed for the cell-associated 1251 and the released 1 2 5I-labelled compounds. As shown in Fig. 3, both the uptake of 125I-labelled IgG1 antibody and the release of digested 1251-labelled compounds were reduced with decreasing ratios of
IgG1
IgG2
Figure 2. Immunoelectrophoresis of purified IgG1 and IgG2 anti-OA antibodies. Electrophoresis of purified IgG1 and IgG2 antibodies was performed as previously described by Nakamura et al. (1972). Precipitin arcs were developed using rabbit antiserum to guinea-pig serum.
271
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'251-labelled IgG1 antibody to non-labelled IgG2 antibody in the mixed complexes. This result demonstrated clearly that the coexistence of IgG2 antibody rather inhibited competitively the uptake of IgG1 antibody and the IgG1 complex alone was capable of binding to the surfaces of macrophages similarly to the IgG2 complex. On the contrary, the uptake of '125-labelled F(ab')2 fragments and the release of digested '251-labelled compounds occurred when the fragments formed the mixed antigen-antibody complexes in the presence of IgG2 antibody. The amounts of cell-associated 125I and 125I-labelled compounds released increased with increasing the ratios of 1251_ labelled F(ab')2 fragments to IgG2 antibody in the mixed complexes. This result indicated that the F(ab')2 fragments lacking the Fc part could bind to macrophages only when reacted in a form of mixed complexes with IgG2 antibody, probably the binding being mediated by the Fc part of IgG2 antibody. Effect of incubation time In order to investigate effects of incubation time on the binding of complexes and the release of digested products, macrophages were cultured in the presence of IgG1 or IgG2 complex with an antigen-antibody ratio of 0-5. As shown in Fig. 4, the release of digested '25I-labelled compounds into the culture fluids continued to increase during the course of the (b)
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Incubation time (h) Figure 4. Effect of incubation time on the uptake of IgGl and IgG2 complexes. (a) The IgG1 and (b) IgG2 complexes were prepared by incubation of 20 pg of each '25I-labelled antibody with 3 pg of OA. After varying the incubation times, the amounts of protein-bound 125I (0 *) and non-protein-bound 125I (-- - -o) associated with cells and non-protein-bound 125i (o- - -o) released into the culture fluids were determined and expressed as percentages against total radioactivity added.
T. Shinomiya & J. Koyama
272
experiment. Furthermore, both the IgGi and IgG2 complexes were found to be digested in an identical manner. On the contrary, the amounts of protein-bound , 25I associated with the cells remained at a constant level of 3 per cent of the added complexes throughout the entire course of the experiment, although the amounts of non-protein-bound 125I associated with the cells increased slightly. The persistence of constant amounts of the protein-bound 1251 suggests that a rate-limited step of the digestion of complexes may be either ingestion of complexes into cells or digestion by the intracellular enzymes. The binding of complexes to the surfaces of macrophages appears to proceed relatively so fast that the protein-bound radioactivity associated with cells reaches the maximum level within 30 min. This view may be supported by the fact that, at 30 min, the cellassociated 1251 was found to be exclusively of macromolecular form. Amounts of IgG1 and IgG2 complexes taken up by macrophages The abilities of IgGi and IgG2 complexes to bind to macrophages were compared using varying amounts of both the complexes. On the basis of the 3 2
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results shown in Fig. 4, the incubation was performed for 30 min, since all the cell-associated 125I was of macromolecular form and no appreciable amount of 12I1 was released into the culture fluids at 30 min. The amounts of IgGI and IgG2 complexes taken up by macrophages increased with increasing amounts of complexes, as shown in Fig. 5. The IgG1 and IgG2 complexes again were not significantly different in their abilities to bind to macrophages.
Digestion of macrophage-associated complexes The digestive processes of IgG1 and IgG2 complexes taken up by macrophages were studied by the measurements of disappearance of the cell-associated 125I. For this purpose, macrophages were cultured at 370 for 1 h in the presence of 1 25I-labelled antibodyantigen complexes and then washed twice to remove the soluble complexes which had not bound to the cells. The washed cells were cultured further for varying the incubation time. As shown in Fig. 6, there was a progressive loss of the radioactivity from the cells and a concomitant increase in the culture fluids, indicating that the original cell-associated complexes were digested constantly. In both the cases of IgG1 and IgG2 complexes, the rate of disappearance of protein-bound 1 25I associated with the cells was found to be equal to that of appearance of digested I25I-labelled compounds into the culture fluids. This result also seems to indicate that the rate-limited step of digestion is either ingestion of complexes or proteolysis of the ingested complexes to small peptides, but not release of digested 1251_ labelled compounds. Comparison of the results obtained with the IgGI and IgG2 complexes revealed that they were not appreciably different in both the rates of disappearance of cell-associated complexes and of release of digested 125I-labelled compounds, showing similar susceptibilities to ingestion by macrophages and also to digestion with the intracellular enzymes. During the incubation, some trichloroacetic acid-insoluble materials were found to be released into the culture fluids, though their amounts were markedly small. This fact may be caused by lysis of some macrophages containing the complexes, since about 10 per cent of the macrophages was found to lyse during the incubation for 6 h, as estimated by the determination of lactic dehydrogenase activity released into the culture fluids using the method described by Wroblewski and LaDue
273
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Incubation time (h) Figure 6. Digestion of cell-associated IgG 1 and IgG2 complexes. The IgG and IgG2 complexes were prepared by incubation of 20 pg of either '25I-labelled IgGi or IgG2 antibody with 3 0 pg of OA. These complexes were reacted with macrophages at 370 for I h. The macrophages which had taken up (a) the IgGI or (b) the IgG2 complexes were washed twice and then incubated at 370 for varying incubation times. The digestion of macrophage-bound complexes was followed by the measurements of amounts of protein-bound 125I (e *) and non-protein-bound 12"J (- -- --) associated with the cells. The appearance of digested products also was determined by the measurements of amounts of protein-bound 125I (0 0) and non-proteinbound I25I (0 -0) released into the culture fluids. Each amount was expressed as a percentage against total radioactivity initially bound to the cells.
(1955). On the other hand, any possible proteolytic enzymes released by the lysis of macrophages did not seem to affect the results obtained; the extracts obtained by repeated freeze-thawing of the same number of macrophages as that used for the experiments could not produce any appreciable amount of trichloroacetic acid-soluble materials when incubated with the IgG1 and IgG2 complexes.
DISCUSSION It is well known that certain immune complexes bind to the surfaces of macrophages even when little or no binding of antibody alone can be observed (Uhr, 1965; Phillips-Quagliata et al., 1971; Arend and Mannik, 1972; Leslie and Cohen, 1974b). However, the immunoglobulin class or subclass specificity for the attachment to macrophages has remained uncertain to some extent. In the case of guinea-pig IgG, Leslie and Cohen (1974b) have reported that the IgG2 antibody bind to macrophages when combined with antigen. However, whether the IgGl complex can bind to macrophages remained to be solved. The results reported in this paper clarified this problem and demonstrated that the IgG1 complex was easily taken up by macro-
phages and subsequently digested by the intracellular system in the same manner as that of IgG2 complex. Recently, Arend and Mannik (1972) have observed that immune complexes of human serum albumin and rabbit antibody containing more than two molecules of antibody per molecule of antigen preferentially adhered to rabbit macrophages in the absence of complement. In this respect, both the IgG1 and IgG2 complexes seem to behave in the same manner as does rabbit IgG complex, since maximum amounts of complexes taken up as well as maximum rates of release of digested products were obtained with each complex with an antigenantibody ratio between 01 and 0-5, which may correspond to an equivalence point of the precipitin reactions of IgG1 and IgG2 anti-OA antibodies (Ag/Ab =04) (Nakamura et al., 1972). This result might indicate that the reaction of complexes with macrophages observed in the present experiments is not concerned with the direct interaction of soluble complexes with macrophages, but the interaction of complexes which either precipitated or adhered to the dish walls during the incubation. However, control experiments may support the direct mechanism, since no visible antigen-antibody complex formed during the incubation in the enzyme
274
T. Shinomiya & J. Koyama
absence of macrophages and only less than 0 5 per cent of complexes added was recovered by elution of some possible complexes adhering to the dish walls. On the other hand, the facts that the enhancement of uptake of IgG1 as well as IgG2 complexes were maximum at antigen-antibody equivalence, that was under conditions which favoured lattice formation, and were markedly diminished in antigen excess appear to support the view that the enhancement of binding in the presence of antigen may be due to some conformational changes in the antibody molecules or specific molecular arrangement in the lattice work of complexes, as discussed by others (Phillips-Quagliata et al., 1971; Arend and Mannik, 1972). The IgG1 antibody seems to be indistinguishable from the IgG2 antibody by these properties. In this respect, it seems to be of interest to speculate on the identical behaviour of IgG1 and IgG2 complexes against macrophages. The Fc parts of these two antibodies are definitely different in both structure and function. The IgG2 antibody is capable of fixing complement by the classical pathway but incapable of eliciting passive cutaneous anaphylaxis (Bloch, Kourilsky, Ovary and Benacerraf, 1963; Ovary et al., 1963). On the contrary, the IgGI antibody is capable of eliciting passive cutaneous anaphylaxis and also of fixing complement, but by the alternative pathway (Ovary et al., 1963; Sandberg et al., 1971). However, these differences did not affect the binding of complexes to macrophages. A similar phenomenon has been observed with the interaction of neutrophils with various heat-aggregated immunoglobulins including four subclasses of human IgG and two subclasses of human IgA; these aggregates interact with neutrophils and induce the release of some lysosomal enzymes in an identical manner (Henson, 1971). Therefore, the adherence of certain immune complexes to phagocytes may not require such strict class or subclass specificity as those for complement fixation and passive cutaneous anaphylaxis. With regard to the mechanism of adherence of immune complexes to macrophages, two pathways have been proposed by others. One involves the attachments of complexes to the surfaces of cells via surface receptors for the Fc parts of antibodies (Berken and Benacerraf, 1966; Messner and Jelinek, 1970). The other depends on the union of the fixed C3 of complement with receptors also present on the surfaces of macrophages (Lay and Nussenzweig,
1968; Huber and Fudenberg, 1970; Mantovani, Rabinovitch and Nussenzweig, 1972). All the experiments presented in this paper were performed in the absence of complement. Therefore, the binding reaction of complexes seems to be concerned directly with the interaction via the surface receptors for the Fc parts of antibodies. The species specificity of rabbit macrophage receptor for IgG has been observed by inhibition experiments using rabbit, human and sheep IgG preparations (Arend and Mannik, 1972). Our preliminary experiments using the IgGI and IgG2 isolated from guinea-pig hyperimmune serum to Bacillus subtilis a-amylase indicated that the presence of free IgG1 inhibited the uptakes of IgGI and IgG2 complexes in an identical manner. Furthermore, the replacement of the IgGI by IgG2 did not vary the inhibition of uptakes of IgGI and IgG2 complexes. These results suggest that both the complexes may adhere to macrophages via the union with the same receptor, but not with different receptors, each specific for either IgG I or IgG2 complex. At present, we have no further evidence for the subclass specificity of receptors present on the surfaces of macrophages. Further studies are required for elucidation of this problem as well as of a possible effect of fixed complement components on the uptake of complexes.
ACKNOWLEDGMENTS This work was supported in part by a grant from the Ministry of Education, Japan. The authors are deeply indebted to Dr Y. Kikuchi, Faculty of Medicine, Hokkaido University, for her valuable discussion on isolation of macrophages. REFERENCES AREND W.P. & MANNIK M. (1972) In vitro adherence of soluble immune complexes to macrophages. J. exp. Med. 136, 514. BERKEN A. & BENACERRAF B. (1966) Properties of antibodies cytophilic for macrophages. J. exp. Med. 123, 119. BLOCH K.J., KoURILSKY F.M., OVARY Z. & BENACERRAF B. (1963) Properties of guinea-pig 7S antibodies. III. Identification of antibodies involved in complement fixation and haemolysis. J. exp. Med. 117, 965. CALDERON J. & UNANUE E.R. (1974) The release of antigen molecules from macrophages: characterization of the phenomena. J. Immunol. 112, 1804.
Uptake of immune complexes by macrophages CARREL S. & BARANDUN S. (1971) Protein-containing polyacrylamide gels: Their use as immunoadsorbents of high capacity. Immunochemistry, 8, 39. DULBECCO R. & VOGT M. (1954) Plaque formation and isolation of pure lines with poliomyelitis viruses. J. exp. Med. 99, 167. GROSSBERG A.L., RADZIMSKI G. & PRESSMAN D. (1962) Effect of iodination on the active site of several antihapten antibodies. Biochemistry, 1, 391. HENSON P.M. (1971) Interaction of cells with immune complexes: Adherence, release of constituents, and tissue injury. J. exp. Med. 134, 114. HUBER H. & FUDENBERG H.H. (1970) The interaction of monocytes and macrophages with immunoglobulins and complement. Ser. Haematol. 2, 160. LAY W.H. & NUSSENZWEIG V. (1968) Receptors for complement on leucocytes. J. exp. Med. 128, 991. LESLIE R.G.O. & COHEN S. (1 974a) Cytophilic activity of IgG2 from sera of unimmunized guinea-pigs. Immunology, 27, 577. LESLIE R.G.Q. & COHEN S. (1974b) Cytophilic activity of IgG2 from sera of guinea-pigs immunized with bovine y-globulin. Immunology, 27, 589. MANTOVANI B., RABINOVITCH M. & NUSSENZWEIG V. (1972) Phagocytosis of immune complexes by macrophages. J. exp. Med. 135, 780. MESSNER R.P. & JELINEK J.G. (1970) Receptors for human yG globulin on human neutrophils. J. cdin. Invest. 49, 2165.
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NAKAMURA T. & KOYAMA J. (1975) Variability among guinea pig antibody populations produced by varying the immunizing dose of Bacillus subtilis a-amylase. Immunochemistry. (In press.) NAKAMURA T., OFUNE K., TAMOTO K. & KOYAMA J. (1972) Immunochemical studies on guinea pig non-precipitating antibodies to ovalbumin. J. Biochem. 71, 245. NISONOFF A., WISSLER F.C., LIPMAN L.N. & WOERNLEY F.L. (1960) Separation of univalent fragments from the bivalent rabbit antibody molecule by reduction of disulfide bonds. Arch. Biochem. Biophys. 89, 230. OVARY Z., BENACERRAF B. & BLOCH K.J. (1963) Properties of guinea-pig 7S antibodies. II. Identification of antibodies involved in passive cutaneous and systemic anaphylaxis. J. exp. Med. 117, 951 PHILLIPS-QUAGLIATA J.M., LEVINE B.B., QUAGLIATA F. & UHR J.W. (1971) Mechanisms underlying binding of immune complexes to macrophages. J. exp. Med. 133,589. PORATH J., AXEN R. & ERNBACK S. (1967) Chemical coupling of proteins to agarose. Nature (Lond.), 215, 1491. SANDBERG A.L., OLIVEIRA B. & OSLER A.G. (1971) Two complement interaction sites in guinea-pig immunoglobulins. J. Immunol. 106, 282. UHR J.W. (1965) Passive sensitization of lymphocytes and macrophages by antigen-antibody complexes. Proc. nat. Acad. Sci. (Wash.), 54, 1599. WR6BLEWSKI F. & LADUE J.S. (1955) Lactic dehydrogenase activity in blood. Proc. Soc. exp. Biol. (N. Y.), 90, 210.
In vitro uptake and digestion of immune complexes containing guinea-pig IgG1 and IgG2 antibodies by macrophages
T. SHINO MI YA & J. KOYA MA Department of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan
Received 13 June 1975; acceptedfor publication 14 August 1975
body alone can be demonstrated (Uhr, 1965; Phillips-Quagliata, Levine, Quagliata and Uhr, 1971; Arend and Mannik, 1972; Leslie and Cohen, 1974b). For example, the formation of immune complex with homologous antigen increases the strength of binding of guinea-pig IgG2 antibody, known as a cytophilic antibody, to macrophages, although the antibody alone is capable of binding to the cells weakly (Leslie and Cohen, 1974a, b). The binding is supposed to be mediated by the Fc parts of antibody molecules, since it is inhibited by the Fc fragments, but not by the F(ab')2 fragments. On the other hand, there is another subclass, IgG1, in guinea-pig IgG, which is not cytophilic but homocytotropic (Ovary, Benacerraf and Bloch, 1963; Sandberg, Oliveira and Osler, 1971; Berken and Benacerraf, 1966). The IgG1 antibody is distinguished from the IgG2 antibody by both structure and biological function of the Fc part. In order to elucidate the mechanism of elimination of antigen by the IgG1 antibody and also of ingestion of immune complexes by macrophages, we have studied the in vitro uptake and digestion of the IgG1 INTRODUCTION antibody by guinea-pig peritoneal macrophages using 1251-labelled IgG1 antibody to hen ovalbumin Certain immune complexes bind to the surfaces of (OA). The present paper reports the enhanced macrophages even when little or no binding of antiingestion of the IgG1 antibody by the complex formation with OA. In particular, the mode of Correspondence: Professor J. Koyama, Department of uptake and digestion of IgGI complex was compared Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, 060, Japan. with that of IgG2 complex. 267
Summary. The uptake and digestion of immune complexes by peritoneal macrophages from oilstimulated guinea-pigs were studied using l 25I1 labelled guinea-pig IgG1 and IgG2 antibodies to hen ovalbumin. When the IgGi or IgG2 antibody was incubated with homologous antigen at antigenantibody ratios ranging from 0-01 to 10, the complexes produced were preferentially taken up by macrophages in the absence of complement and digested by the intracellular enzymes. The uptake and digestion of complexes reached a maximum at an antigen-antibody ratio of 0-1-05. In this respect, the IgG1 antibody was indistinguishable from the IgG2 antibody having a cytophilic activity. These observations suggest that some conformational changes in both the IgG1 and IgG2 antibodies or specific molecular arrangement in the lattice work of both the complexes may increase the strength of binding of complexes to macrophages, independently of the difference in biological activities between these two antibodies.
268
T. Shinomiya & J. Koyama
MATERIALS AND METHODS Preparation of guinea-pig antibodies to OA Guinea-pig IgGI and IgG2 anti-OA antibodies were specifically purified from the hyperimmune serum by immunoadsorbent techniques previously described (Nakamura and Koyama, 1975). Elution of the bound antibodies from a column of agarose gel coupled with OA by the CNBr method (Porath, Axen and Ernback, 1967) was performed using either 01 M glycine buffer, pH 3-5, or 3 M NaSCN (Carrel and Barandun, 1971). The IgGI and IgG2 antibodies thus purified were separated by zone electrophoresis in a starch block (Nakamura, Ofune, Tamoto and Koyama, 1972). Preparation of F(ab')2 fragments of guinea-pig IgG1 and IgG2 antibodies The F(ab')2 fragments of guinea-pig IgGI and IgG2 anti-OA antibodies were prepared from the antibody preparations specifically purified from the hyperimmune serum. The digestion with pepsin was performed according to the method of Nisonoff, Wissler, Lipman and Woernley (1960). The F(ab')2 fragments were purified by gel filtration with Sephadex G-200. Isotopic labelling and further purification of proteins Labelling of proteins with radioactive iodine was performed by the ICL method (Grossberg, Radzimski and Pressman, 1962). Iodine was incorporated at less than 1 atom of iodine per molecule of protein. Some aggregated proteins forming during the purification and labelling were removed by gel filtration with Sephadex G-200. Monomeric proteins isolated were used in experiments. The l25l-labelled IgG1 and IgG2 antibodies and the F(ab')h fragments thus obtained were highly pure on immunoelectrophoresis using rabbit antiserum to guinea-pig serum. On the basis of amounts of the radioactivity adsorbed on the immunoadsorbent, more than 95 per cent of each protein was found to be anti-OA antibody or the F(ab')2 fragments capable of binding to OA.
Isolation ofperitoneal macrophages Guinea-pigs of the Hartley strain weighing 400-600 g were used as donors of peritoneal macrophages. The animals were injected intraperitoneally with 20 ml of sterilized liquid paraffin. Four days after the injection, macrophages were harvested from the
peritoneal cavity using the Dulbecco's balanced salt solution containing 1 u of heparin/ml (Dulbecco and Vogt, 1954). The cells were centrifuged at 500 g for 10 min and washed twice at 40 with 50 ml of the MEM medium (minimal essential medium) containing 20 mm HEPES. Culture of macrophages Culture of macrophages was performed essentially according to the method described by Calderon and Unanue (1974). The cells isolated were suspended in the MEM medium containing 5 per cent calf serum. 107 cells in 1 ml of the medium were planted in plastic dishes (Falcon, 35 x 10 mm) and incubated at 370 for 20 h in a C02-air incubator. After incubation, the dishes containing the adherent macrophages were washed twice with the MEM medium supplemented with 20 mm HEPES, which had been added to MEM in order to maintain a stable pH outside the incubator. At this time, the dishes contained about 40 per cent of the original number of cells planted, when estimated on the basis of the determination of cellular proteins with the Folin reagent. More than 98 per cent of the cells were typical, well-spread macrophages forming an interrupted monolayer. After washes, the dishes were incubated with I251-labelled antibody and antigen in the MEM medium supplemented with 0 1 per cent bovine serum albumin which had been added to prevent non-specific adsorption of proteins to the dishes or to the cells. After incubation for appropriate times, the ingestion of immune complexes were investigated. Each experiment was run in duplicate and repeated two or three times. Analysis of 1 25I-labelled products 25I-labelled materials were examined from culture supernatants or cells after the incubation of complexes containing ' 25I-labelled antibodies with macrophages. Culture supernatants were centrifuged at 500 g for 5 min in order to remove any possible cells and then precipitated by the addition of an equal volume of 10 per cent trichloroacetic acid. On occasion, 5 mg of bovine serum albumin was added to the culture supernatants as a carrier to obtain a sufficient amount of precipitates. The precipitates and supernatant fluids thus obtained were assumed to represent protein-bound and non-
protein-bound 1251. The radioactivity associated with the cells (cellassociated 1251) was obtained by the treatment of the
269
Uptake of immune complexes by macrophages twice washed cell monolayers with 0-5 ml of 01 N NaOH. The cellular materials were extracted after gently scraping the cells from the dishes with a rubber policeman and then added to 5 mg of bovine serum albumin. The cell-associated I 25I-labelled materials were fractionated to protein-bound and non-protein-bound 1 251 by the addition of trichloroacetic acid at 5 per cent. The measurements of radioactivity were performed in a Packard automatic well-type gamma counter.
Estimation ofproteins The absorbancy at 280 nm was used for calculation of protein concentration. The values of A' Per Cent were taken as 13-5 for IgG as well as F(ab')2 fragment and 7-35 for OA, respectively. The following molecular weights were assumed: 150,000 for IgG, 100,000 for F(ab')2 fragment and 45,000 for OA. Materials Crystalline OA was prepared in our laboratory (Nakamura et al., 1972). Bovine serum albumin and HEPES [4-(2-hydroxyethyl)-1-piperazine ethanesulphonic acid] were obtained from Sigma Chemical Company. The MEM medium was 10 |H
obtained from Nissui Seiyaku Company, Ltd, Japan. RESULTS
Uptake and digestion of IgG1 and IgG2 antibodies depending on the complex formation with antigen The uptake and digestion of '25l-labelled IgGi and IgG2 anti-OA antibodies by macrophages were studied in the presence of OA. Representative results are shown in Fig. 1. Incubation of macrophages with 125I-labelled IgGi or IgG2 anti-OA antibody alone resulted in little binding of radioactivity, detectable after two washes. However, a marked increase in the uptake of IgGi and IgG2 antibodies occurred when a constant amount of the IgG1 or IgG2 antibody was added to the cells after incubation with varying the amounts of OA at 370 for 1 h. In particular, the uptake and digestion of both IgG1 and IgG2 antibodies reached a maximum when macrophages were incubated with each complex having an antigen-antibody ratio between 0-1 and 0 5, corresponding to an equivalence point of precipitin reactions of these two antibodies (Nakamura et al., 1972).
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Figure 1. Effect of complex formation with OA on the uptakes of IgGI and IgG2 anti-OA antibodies by macrophages. Soluble complexes were prepared by incubation of 20 ,ug of (a) '251-labelled IgG 1 or (b) IgG2 antibody with varying the amounts of OA in the range of molar ratio of antigen to antibody from 0 to 10. The complexes were reacted with macrophages at 370 for 4 h. The amounts of protein-bound "25I (0 0) and non-protein-bound "25I (--- -0) associated with cells and non-proteinbound 125I released into the culture fluids (0- --0) were determined and expressed as percentages against total radioactivity added. H
270
T. Shinomiya & J. Koyama
The analysis of ' 251-labelled materials in the culture fluids revealed that the cell-bound antibodies were digested during the incubation and the digestive products were released into the culture fluids. The amount of released 1251 was in proportion to that of cell-bound antibody; the maximum release again occurred with the complexes having an antigenantibody ratio between 0-1 and 0 5. On the other hand, the cell-bound 1251 was mainly of proteinbound nature, since the amount of non-proteinbound 1251 remained remarkably small, indicating that the digested 1251-labelled compounds were removed rapidly out of the cells. With regard to the uptake of complexes, there seems to be the possibility that the protein-bound radioactivity did not represent the cell-associated 125I but 1251-labelled antibody precipitated with OA during the incubation. However, when 20 ug of 1251-labelled IgGi or IgG2 anti-OA antibody were incubated with 3 ug of OA in empty dishes, no visible precipitate formed and radioactivity which adhered to the dishes and was recovered with 01 N NaOH was less than 0 5 per cent of the added activity. Therefore, the radioactivity of cells, detectable after two washes which were sufficient to remove the unbound complexes, could be assumed to represent the cell-associated 1251. It was very probable that the concentration of antigen-antibody complexes used was too low to make the complexes precipitable. Effect of co-existing IgG2 antibody on the uptake of IgG1 complex The results shown in Fig. 1 demonstrated that the
IgGi antibody, known to be non-cytophilic, was indistinguishable from the cytophilic IgG2 antibody by susceptibility to the ingestion by macrophages, when reacted in a form of soluble complex. However, this might not be caused by a property of the IgGI antibody itself, but by a helper function of a trace amount of the IgG2 antibody existing as a contaminant in the IgGi antibody preparation used, though immunoelectrophoretic analysis could not demonstrate any contaminating IgG2 antibody, as shown in Fig. 2. In order to investigate further this point, the effect of coexisting IgG2 antibody on the uptake of 1251. labelled IgGl antibody by macrophages was studied using some mixed complexes composed of 1251_ labelled IgGi and non-labelled IgG2 anti-OA antibodies. The complexes used were prepared by incubation of a constant amount of OA (6 7 x 10- 11 moles) with the antibody mixtures (1 3x10-10 moles) containing the IgGI and IgG2 antibodies at various ratios. In control experiments, other mixed complexes were prepared in the same manner, using 125I-labelled F(ab')2 fragments of IgG1 and IgG2 anti-OA antibodies in the place of 1 251_ labelled IgG1 antibody, since the F(ab')2 fragments are known to have no ability to adhere to the surfaces of macrophages even when bound to homologous antigen (Arend and Mannik, 1972). After incubation of these complexes with macrophages at 370 for 4 h, the cells and culture fluids were analysed for the cell-associated 1251 and the released 1 2 5I-labelled compounds. As shown in Fig. 3, both the uptake of 125I-labelled IgG1 antibody and the release of digested 1251-labelled compounds were reduced with decreasing ratios of
IgG1
IgG2
Figure 2. Immunoelectrophoresis of purified IgG1 and IgG2 anti-OA antibodies. Electrophoresis of purified IgG1 and IgG2 antibodies was performed as previously described by Nakamura et al. (1972). Precipitin arcs were developed using rabbit antiserum to guinea-pig serum.
271
Uptake of immune complexes by macrophages
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IgG 2 (molar ratio) Figure 3. Effect of co-existing IgG2 antibody on the uptakes of IgGl antibody or the F(ab')2 fragments by macrophages. Soluble complexes (0) were prepared by incubation of a constant amount of OA with antibody mixtures containing 1251-labelled IgGI and non-labelled IgG2 antibodies at various ratios, as described in text. Other mixed complexes (0) were prepared in the same manner, using '251-labelled F(ab')2 fragments in the place of 1251-labelled IgGI antibody. These complexes were reacted with macrophages at 370 ) and for 4 h. The amounts of cell-associated 1251 ( non-protein-bound 12 51 released into the culture fluids (--- ) were determined and expressed as percentages against total radioactivity added. 5-
'251-labelled IgG1 antibody to non-labelled IgG2 antibody in the mixed complexes. This result demonstrated clearly that the coexistence of IgG2 antibody rather inhibited competitively the uptake of IgG1 antibody and the IgG1 complex alone was capable of binding to the surfaces of macrophages similarly to the IgG2 complex. On the contrary, the uptake of '125-labelled F(ab')2 fragments and the release of digested '251-labelled compounds occurred when the fragments formed the mixed antigen-antibody complexes in the presence of IgG2 antibody. The amounts of cell-associated 125I and 125I-labelled compounds released increased with increasing the ratios of 1251_ labelled F(ab')2 fragments to IgG2 antibody in the mixed complexes. This result indicated that the F(ab')2 fragments lacking the Fc part could bind to macrophages only when reacted in a form of mixed complexes with IgG2 antibody, probably the binding being mediated by the Fc part of IgG2 antibody. Effect of incubation time In order to investigate effects of incubation time on the binding of complexes and the release of digested products, macrophages were cultured in the presence of IgG1 or IgG2 complex with an antigen-antibody ratio of 0-5. As shown in Fig. 4, the release of digested '25I-labelled compounds into the culture fluids continued to increase during the course of the (b)
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Incubation time (h) Figure 4. Effect of incubation time on the uptake of IgGl and IgG2 complexes. (a) The IgG1 and (b) IgG2 complexes were prepared by incubation of 20 pg of each '25I-labelled antibody with 3 pg of OA. After varying the incubation times, the amounts of protein-bound 125I (0 *) and non-protein-bound 125I (-- - -o) associated with cells and non-protein-bound 125i (o- - -o) released into the culture fluids were determined and expressed as percentages against total radioactivity added.
T. Shinomiya & J. Koyama
272
experiment. Furthermore, both the IgGi and IgG2 complexes were found to be digested in an identical manner. On the contrary, the amounts of protein-bound , 25I associated with the cells remained at a constant level of 3 per cent of the added complexes throughout the entire course of the experiment, although the amounts of non-protein-bound 125I associated with the cells increased slightly. The persistence of constant amounts of the protein-bound 1251 suggests that a rate-limited step of the digestion of complexes may be either ingestion of complexes into cells or digestion by the intracellular enzymes. The binding of complexes to the surfaces of macrophages appears to proceed relatively so fast that the protein-bound radioactivity associated with cells reaches the maximum level within 30 min. This view may be supported by the fact that, at 30 min, the cellassociated 1251 was found to be exclusively of macromolecular form. Amounts of IgG1 and IgG2 complexes taken up by macrophages The abilities of IgGi and IgG2 complexes to bind to macrophages were compared using varying amounts of both the complexes. On the basis of the 3 2
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results shown in Fig. 4, the incubation was performed for 30 min, since all the cell-associated 125I was of macromolecular form and no appreciable amount of 12I1 was released into the culture fluids at 30 min. The amounts of IgGI and IgG2 complexes taken up by macrophages increased with increasing amounts of complexes, as shown in Fig. 5. The IgG1 and IgG2 complexes again were not significantly different in their abilities to bind to macrophages.
Digestion of macrophage-associated complexes The digestive processes of IgG1 and IgG2 complexes taken up by macrophages were studied by the measurements of disappearance of the cell-associated 125I. For this purpose, macrophages were cultured at 370 for 1 h in the presence of 1 25I-labelled antibodyantigen complexes and then washed twice to remove the soluble complexes which had not bound to the cells. The washed cells were cultured further for varying the incubation time. As shown in Fig. 6, there was a progressive loss of the radioactivity from the cells and a concomitant increase in the culture fluids, indicating that the original cell-associated complexes were digested constantly. In both the cases of IgG1 and IgG2 complexes, the rate of disappearance of protein-bound 1 25I associated with the cells was found to be equal to that of appearance of digested I25I-labelled compounds into the culture fluids. This result also seems to indicate that the rate-limited step of digestion is either ingestion of complexes or proteolysis of the ingested complexes to small peptides, but not release of digested 1251_ labelled compounds. Comparison of the results obtained with the IgGI and IgG2 complexes revealed that they were not appreciably different in both the rates of disappearance of cell-associated complexes and of release of digested 125I-labelled compounds, showing similar susceptibilities to ingestion by macrophages and also to digestion with the intracellular enzymes. During the incubation, some trichloroacetic acid-insoluble materials were found to be released into the culture fluids, though their amounts were markedly small. This fact may be caused by lysis of some macrophages containing the complexes, since about 10 per cent of the macrophages was found to lyse during the incubation for 6 h, as estimated by the determination of lactic dehydrogenase activity released into the culture fluids using the method described by Wroblewski and LaDue
273
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Incubation time (h) Figure 6. Digestion of cell-associated IgG 1 and IgG2 complexes. The IgG and IgG2 complexes were prepared by incubation of 20 pg of either '25I-labelled IgGi or IgG2 antibody with 3 0 pg of OA. These complexes were reacted with macrophages at 370 for I h. The macrophages which had taken up (a) the IgGI or (b) the IgG2 complexes were washed twice and then incubated at 370 for varying incubation times. The digestion of macrophage-bound complexes was followed by the measurements of amounts of protein-bound 125I (e *) and non-protein-bound 12"J (- -- --) associated with the cells. The appearance of digested products also was determined by the measurements of amounts of protein-bound 125I (0 0) and non-proteinbound I25I (0 -0) released into the culture fluids. Each amount was expressed as a percentage against total radioactivity initially bound to the cells.
(1955). On the other hand, any possible proteolytic enzymes released by the lysis of macrophages did not seem to affect the results obtained; the extracts obtained by repeated freeze-thawing of the same number of macrophages as that used for the experiments could not produce any appreciable amount of trichloroacetic acid-soluble materials when incubated with the IgG1 and IgG2 complexes.
DISCUSSION It is well known that certain immune complexes bind to the surfaces of macrophages even when little or no binding of antibody alone can be observed (Uhr, 1965; Phillips-Quagliata et al., 1971; Arend and Mannik, 1972; Leslie and Cohen, 1974b). However, the immunoglobulin class or subclass specificity for the attachment to macrophages has remained uncertain to some extent. In the case of guinea-pig IgG, Leslie and Cohen (1974b) have reported that the IgG2 antibody bind to macrophages when combined with antigen. However, whether the IgGl complex can bind to macrophages remained to be solved. The results reported in this paper clarified this problem and demonstrated that the IgG1 complex was easily taken up by macro-
phages and subsequently digested by the intracellular system in the same manner as that of IgG2 complex. Recently, Arend and Mannik (1972) have observed that immune complexes of human serum albumin and rabbit antibody containing more than two molecules of antibody per molecule of antigen preferentially adhered to rabbit macrophages in the absence of complement. In this respect, both the IgG1 and IgG2 complexes seem to behave in the same manner as does rabbit IgG complex, since maximum amounts of complexes taken up as well as maximum rates of release of digested products were obtained with each complex with an antigenantibody ratio between 01 and 0-5, which may correspond to an equivalence point of the precipitin reactions of IgG1 and IgG2 anti-OA antibodies (Ag/Ab =04) (Nakamura et al., 1972). This result might indicate that the reaction of complexes with macrophages observed in the present experiments is not concerned with the direct interaction of soluble complexes with macrophages, but the interaction of complexes which either precipitated or adhered to the dish walls during the incubation. However, control experiments may support the direct mechanism, since no visible antigen-antibody complex formed during the incubation in the enzyme
274
T. Shinomiya & J. Koyama
absence of macrophages and only less than 0 5 per cent of complexes added was recovered by elution of some possible complexes adhering to the dish walls. On the other hand, the facts that the enhancement of uptake of IgG1 as well as IgG2 complexes were maximum at antigen-antibody equivalence, that was under conditions which favoured lattice formation, and were markedly diminished in antigen excess appear to support the view that the enhancement of binding in the presence of antigen may be due to some conformational changes in the antibody molecules or specific molecular arrangement in the lattice work of complexes, as discussed by others (Phillips-Quagliata et al., 1971; Arend and Mannik, 1972). The IgG1 antibody seems to be indistinguishable from the IgG2 antibody by these properties. In this respect, it seems to be of interest to speculate on the identical behaviour of IgG1 and IgG2 complexes against macrophages. The Fc parts of these two antibodies are definitely different in both structure and function. The IgG2 antibody is capable of fixing complement by the classical pathway but incapable of eliciting passive cutaneous anaphylaxis (Bloch, Kourilsky, Ovary and Benacerraf, 1963; Ovary et al., 1963). On the contrary, the IgGI antibody is capable of eliciting passive cutaneous anaphylaxis and also of fixing complement, but by the alternative pathway (Ovary et al., 1963; Sandberg et al., 1971). However, these differences did not affect the binding of complexes to macrophages. A similar phenomenon has been observed with the interaction of neutrophils with various heat-aggregated immunoglobulins including four subclasses of human IgG and two subclasses of human IgA; these aggregates interact with neutrophils and induce the release of some lysosomal enzymes in an identical manner (Henson, 1971). Therefore, the adherence of certain immune complexes to phagocytes may not require such strict class or subclass specificity as those for complement fixation and passive cutaneous anaphylaxis. With regard to the mechanism of adherence of immune complexes to macrophages, two pathways have been proposed by others. One involves the attachments of complexes to the surfaces of cells via surface receptors for the Fc parts of antibodies (Berken and Benacerraf, 1966; Messner and Jelinek, 1970). The other depends on the union of the fixed C3 of complement with receptors also present on the surfaces of macrophages (Lay and Nussenzweig,
1968; Huber and Fudenberg, 1970; Mantovani, Rabinovitch and Nussenzweig, 1972). All the experiments presented in this paper were performed in the absence of complement. Therefore, the binding reaction of complexes seems to be concerned directly with the interaction via the surface receptors for the Fc parts of antibodies. The species specificity of rabbit macrophage receptor for IgG has been observed by inhibition experiments using rabbit, human and sheep IgG preparations (Arend and Mannik, 1972). Our preliminary experiments using the IgGI and IgG2 isolated from guinea-pig hyperimmune serum to Bacillus subtilis a-amylase indicated that the presence of free IgG1 inhibited the uptakes of IgGI and IgG2 complexes in an identical manner. Furthermore, the replacement of the IgGI by IgG2 did not vary the inhibition of uptakes of IgGI and IgG2 complexes. These results suggest that both the complexes may adhere to macrophages via the union with the same receptor, but not with different receptors, each specific for either IgG I or IgG2 complex. At present, we have no further evidence for the subclass specificity of receptors present on the surfaces of macrophages. Further studies are required for elucidation of this problem as well as of a possible effect of fixed complement components on the uptake of complexes.
ACKNOWLEDGMENTS This work was supported in part by a grant from the Ministry of Education, Japan. The authors are deeply indebted to Dr Y. Kikuchi, Faculty of Medicine, Hokkaido University, for her valuable discussion on isolation of macrophages. REFERENCES AREND W.P. & MANNIK M. (1972) In vitro adherence of soluble immune complexes to macrophages. J. exp. Med. 136, 514. BERKEN A. & BENACERRAF B. (1966) Properties of antibodies cytophilic for macrophages. J. exp. Med. 123, 119. BLOCH K.J., KoURILSKY F.M., OVARY Z. & BENACERRAF B. (1963) Properties of guinea-pig 7S antibodies. III. Identification of antibodies involved in complement fixation and haemolysis. J. exp. Med. 117, 965. CALDERON J. & UNANUE E.R. (1974) The release of antigen molecules from macrophages: characterization of the phenomena. J. Immunol. 112, 1804.
Uptake of immune complexes by macrophages CARREL S. & BARANDUN S. (1971) Protein-containing polyacrylamide gels: Their use as immunoadsorbents of high capacity. Immunochemistry, 8, 39. DULBECCO R. & VOGT M. (1954) Plaque formation and isolation of pure lines with poliomyelitis viruses. J. exp. Med. 99, 167. GROSSBERG A.L., RADZIMSKI G. & PRESSMAN D. (1962) Effect of iodination on the active site of several antihapten antibodies. Biochemistry, 1, 391. HENSON P.M. (1971) Interaction of cells with immune complexes: Adherence, release of constituents, and tissue injury. J. exp. Med. 134, 114. HUBER H. & FUDENBERG H.H. (1970) The interaction of monocytes and macrophages with immunoglobulins and complement. Ser. Haematol. 2, 160. LAY W.H. & NUSSENZWEIG V. (1968) Receptors for complement on leucocytes. J. exp. Med. 128, 991. LESLIE R.G.O. & COHEN S. (1 974a) Cytophilic activity of IgG2 from sera of unimmunized guinea-pigs. Immunology, 27, 577. LESLIE R.G.Q. & COHEN S. (1974b) Cytophilic activity of IgG2 from sera of guinea-pigs immunized with bovine y-globulin. Immunology, 27, 589. MANTOVANI B., RABINOVITCH M. & NUSSENZWEIG V. (1972) Phagocytosis of immune complexes by macrophages. J. exp. Med. 135, 780. MESSNER R.P. & JELINEK J.G. (1970) Receptors for human yG globulin on human neutrophils. J. cdin. Invest. 49, 2165.
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NAKAMURA T. & KOYAMA J. (1975) Variability among guinea pig antibody populations produced by varying the immunizing dose of Bacillus subtilis a-amylase. Immunochemistry. (In press.) NAKAMURA T., OFUNE K., TAMOTO K. & KOYAMA J. (1972) Immunochemical studies on guinea pig non-precipitating antibodies to ovalbumin. J. Biochem. 71, 245. NISONOFF A., WISSLER F.C., LIPMAN L.N. & WOERNLEY F.L. (1960) Separation of univalent fragments from the bivalent rabbit antibody molecule by reduction of disulfide bonds. Arch. Biochem. Biophys. 89, 230. OVARY Z., BENACERRAF B. & BLOCH K.J. (1963) Properties of guinea-pig 7S antibodies. II. Identification of antibodies involved in passive cutaneous and systemic anaphylaxis. J. exp. Med. 117, 951 PHILLIPS-QUAGLIATA J.M., LEVINE B.B., QUAGLIATA F. & UHR J.W. (1971) Mechanisms underlying binding of immune complexes to macrophages. J. exp. Med. 133,589. PORATH J., AXEN R. & ERNBACK S. (1967) Chemical coupling of proteins to agarose. Nature (Lond.), 215, 1491. SANDBERG A.L., OLIVEIRA B. & OSLER A.G. (1971) Two complement interaction sites in guinea-pig immunoglobulins. J. Immunol. 106, 282. UHR J.W. (1965) Passive sensitization of lymphocytes and macrophages by antigen-antibody complexes. Proc. nat. Acad. Sci. (Wash.), 54, 1599. WR6BLEWSKI F. & LADUE J.S. (1955) Lactic dehydrogenase activity in blood. Proc. Soc. exp. Biol. (N. Y.), 90, 210.
