Macrophage Interactions with Antibodies and Soluble Immune Complexes

Immunobiol., vol. 161, pp. 322-333 (1982)

Department of Immunology, University Hospital, Queen's Medical Centre, Nottingham, United Kingdom

Macrophage Interactions with Antibodies and Soluble Immune Complexes R. G. Q. LESLIE Abstract In vitro studies aimed at characterising (1) the binding of monomeric immunoglobulins from a variety of animal species to homologous mononuclear phagocytes, (2) the enhancement in phagocyte binding when antibodies are reacted with soluble antigens to form complexes of defined size, (3) the kinetics of complex ingestion and catabolism by macrophages and the biochemical mechanisms involved, (4) the role of complement in soluble complex catabolism and (5) the stimulatory effects of soluble complexes on phagocyte activity are reviewed. Insights gained from these studies into the in vivo clearance of soluble complexes and into the part played by circulating immune complexes in disease are discussed.

Introduction The study of macrophage interactions with antibodies and soluble immune complexes gains its significance from two in vivo observations. The first concerns the clearance of soluble complexes from the circulation after experimental administration of free or complexed antigen, or during an acute or chronic infection. Clearance of immune complexes is a selective process mediated by cells of the mononuclear phagocyte series, particularly Kupffer cells (1), and its rate is dependent both on the ability of the constituent antibodies to bind to specific macrophage membrane receptors (2) and on the size of the complexes (3). Secondly, there is the observed association of circulating immune complexes with the more aggressive forms of complex-mediated diseases such as rheumatoid arthritis or sys­ temic lupus erythematosus, where tissue damage arises primarily from the deposition of complexes at specific sites. The basis of the persistence of complexes in the blood and the part played by the circulating, as opposed to deposited, complexes in exacerbating these diseases are aspects in which the clearance capacity and responsiveness to stimulation of the mononuclear phagocytes may be crucially important. Studies in vitro provide an essential basis for evaluating these observations 1. by defining the manner in which macrophages recognise cytophilic antibodies, 2. by giving insight into the mechanism of the binding enhancement which follows antibody combina­ tion with antigen, 3. by characterising the kinetics of complex ingestion and degradation by phagocytes and the biochemical mechanisms involved and 4. by identifying the regulatory effects that soluble complexes exert on various macrophage activities.

Macrophages, Antibodies and Immune Complexes . 323

Macrophage interaction with monomeric immunoglobulins A necessary preliminary to the investigation of co~plex handling by macrophages has been the characterisation of free immunoglobulin binding to the cells. This interaction was first defined in terms of an association constant and the number of receptor sites per cell by AREND and MANNIK (4) who measured the equilibrium binding of rabbit IgG to homologous alveolar macrophages and analysed the data using the Scatchard plot, ric = - Kar + Kan, where rand c are the concentrations of bound and free IgG, respectively, n is the total concentration of immunoglobulin receptors and Ka is the binding constant (Fig. 1). This approach has subsequently been applied to homologous IgG/macrophage interactions in guinea pig, mice and man (reviewed in 5). Variation between species, both in the affinity of IgG for the macrophages or monocytes and in the number of receptor sites per cell is considerable (Table 1). The mononuclear phagocytes in guinea pig, mice, and, possibly, humans express two types of receptor disting­ uished by their IgG subclass specificity, their affinity for immunoglobulin and their abundance at the cell sudace. In mice, the existence of two receptors has been confirmed on the basis of differing susceptibility to proteolysis (6), independent variation in their expression on macrophage­ like cell lines (7) and antigenic disparity (8). Investigations of the mouse and guinea pig systems (5, 6) have established that IgG binding to macrophages is an exothermic reaction displaying a decline in association constant (Ka) with increase in temperature. At 37°C the Ka falls to about one third of the value recorded at 20°C to give a range of between 5 X 105 and 2.3 X 107 M- 1 in the species examined (5, 6). Binding constants of this magnitude have considerable in vivo significance, since they indicate that in the circulation, where the IgG concentration is between 10- 5 and 10- 4 M, more than 95 % of the immunoglobulin recep0.010

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Macrophages, Antibodies and Immune Complexes . 325

tors on macrophages are occupied by their monomeric ligands. This in turn implies that the uptake of circulating immune complexes by phagocytes occurs through competition with monomer IgG for the receptors rather than by attachment to sites which were hitherto vacant. Macrophage interaction with immune complexes Immune complex attachment to macrophages in vitro has been examined in terms of both equilibrium binding and the kinetics of association and dissociation, using immune complex mixtures of defined average size (9) purified dimers and trimers of antibodies covalently cross-linked by diva­ lent affinity-labelling reagents (10, 11) and heat aggregated IgG (12). Equilibrium studies with complex mixtures (9) or isolated hapten-carry­ ing antibody oligomers (10) have established that at 4°C each complex binds with a uniform avidity which increases with complex size (Fig. 2, Table 2). Analysis of the binding data in thermodynamic terms indicated that the enhanced avidity of complexes for the phagocyte receptors could be accounted for by simple multivalent attachment of antigen-cross-linked antibodies in their native conformation (9, 10). Recent studies by DOWER et al. (11) employing antibody oligomers from which the hapten has been released in the course of cross-linking, suggest, however, that the basis of complex binding may be more complicated. The antigen-free dimer and trimer ligands differ from conventional complexes in that they display non­ linear plots where the association constant declines with increase in the Ab :Ag

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326 . R. G. Q. LESLIE Table 2. Binding of immune complexes of IgG2 and DNP 19 BSA to guinea pig peritoneal macrophages l Ab : AG ratio in incubation mixture

Mean composition of complex

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± ± ± ±

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From Leslie, 1980 (9)

occupancy of the macrophage receptors, reflecting a decrease in the valency of attachment as the availability of vacant binding sites drops. Similar curves were obtained when hapten-carrying oligomers were reacted with the cells in high concentrations of free hapten. The linear plots normally observed with complexes are consequently viewed as arising from a combination of events in which the reduction in valency of binding at high membrane concentrations of complex is compensated by additional antibody-antigen bridging between the cell-bound complexes to form larger, more adherent aggregates. Kinetic studies using either heat-aggregated (12) or affinity cross-linked (13) IgG oligomers have established that the size-related changes in com­ plex avidity arise from differences in the rate of dissociation, rather than association. DOWER et al. (13), in their study, showed that complex association with the macrophages involved two rate components; an initial univalent attachment of the oligomer which is rate determining and is unrelated to complex size, followed by rapid secondary binding to neigh­ bouring receptors on the cell surface. The dissociation of the oligomers 100

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Macrophages, Antibodies and Immune Complexes . 327

requires synchronous release of the cross-linked antibodies from their receptor sites and consequently declines in rate with increasing ligand valency. A non-allosteric multivalent attachment model, to account for the enhanced binding of complexes to macrophages, is supported by the observation that monomeric IgG, of unrelated antibody specificity, is an effective competitor in the process. Equilibrium studies (9) have shown that the capacity of monomer to inhibit complex binding is inversely related to complex size (Fig. 3) leading to greater phagocyte discrimination between complexes of different valency in the presence of high concentrations of free IgG. Kinetic studies have confirmed that monomeric IgG competes both in the primary and secondary stages of complex binding (13). From an in vivo standpoint, the most significant aspect of these inhibition studies lies in the finding that IgG at concentrations found in serum (5-15 mg/ml) virtually abolishes the equilibrium binding of the smallest complexes (dimers and trimers) and markedly reduces the uptake of higher oligomers (11, 13). The selective clearance of small complexes in vivo cannot, therefore, be ascribed simply to their high avidity for the macrophage receptors, but must depend on additional, irreversible, events which result in their retention by the phagocyte.

Complex ingestion and degradation by macrophages The kinetics of complex ingestion have been examined by attaching soluble DNPBSA-antiDNP IgG2 complexes to guinea pig macrophages at 4°C, incubating the washed, complex-laden cells at elevated temperatures for various times and then detecting complexed antigen remaining at the cell surface with 125I-Iabelled (Fab')2 fragments of the anti-DNP antibody (14). Ingestion was found to obey first order kinetics at 20°C and 37°C (Fig. 4) and to proceed at a rate (12.5 % per minute at 37°C) which was fourfold faster than the rate of membrane intake (3 % per minute) which accom­ panies pinocytosis by unstimulated macrophages (15). Complex ingestion was thus regarded as a selective process, though it was not clear from this study whether the selectivity was simply a consequence of complex localisa­ tion 'at the sites of forming pinosomes or whether the complexes initiated pinosome formation de novo. Variation in complex size, on the other hand, was not found to influence the ingestion rate indicating either that cross­ linking of as few as two immunoglobulin receptors on the macrophage provided the necessary signal for endocytosis or that rapid rearrangement of the membrane-bound complexes into large aggregates preceded inges­ tion. These alternatives were assessed by examining microscopically the events initiated when macrophages were loaded with fluorescein labelled soluble complexes at 4°C and were subsequently raised to 37°C on a temperature-controlled stage (16). Reorganisation of the complexes from a

328 . R. G. Q. LESLIE

uniform distribution at 4°C to initially diffuse and then discrete aggregates was observed within minutes of the temperature shift and this was followed over the next half hour by progressive ingestion of all the membrane-bound material. Cytochalasin B failed to block the rearrangement, indicating that the process was not under microfilament control. Estimation of the con­ centration increase achieved at the macrophage surface when complexes on the cell and in solution are at equilibrium (approximately 600-fold) led to the conclusion that aggregation could be accounted for by additional antibody-antigen bond formation between the membrane-bound com­ plexes (16). Intracellular digestion of immune complexes by macrophages has been examined by measuring the release of TCA-soluble catabolites after loading the cells with iodine radiolabelled complexes at 4 or 20°C (14). Kinetically, catabolism was expressed as the decline, with time, in the proportion of radio-label that remained undigested, and was found to be a temperature­ dependent pseudo-first order reaction (Fig. 5) with a rate (0.3-0.6 % per minute at 37°C) that was 20- to 40-fold slower than the rate of ingestion. No variation in digestion kinetics was observed with complexes of different size, but the rates of degradation of antigen and antibody in the same complex were found to differ markedly (14) indicating that the susceptibi­ lity of the target proteins to enzymic hydrolysis, rather than the kinetics of secondary lysosome formation or catabolite excretion, was the rate deter­ mining factor. 0

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Preliminary characterisation of the individual intra-cellular events involved in complex catabolism has been achieved by observing the effect of metabolic inhibitors (KCN and 2 deoxy-glucose), disruptors of cellular organisation (colchicine, and cytochalasin B), the local anaesthetic lidocaine and a protease inhibitor (tosyllysyl chloromethyl ketone, TLCK) upon the rates of complex ingestion and catabolite excretion (14, 17). Ingestion of membrane-bound complexes was partially blocked by KCN (Table 3), was unaffected by 2 deoxy-glucose and was substantially reduced by a combination of both inhibitors (14) indicating that the endocytic process was under the same metabolic control as fluid-phase pinocytosis (18). Support for this mechanism was provided by the observation that complex internalisation is also reduced to 16 % of the normal rate by microfilament disruption with cytochalasin B (17), whilst remaining unaf­ fected by colchicine or lidocaine. Complex digestion was blocked by a Table 3. The effects of inhibitors on the rates of ingestion and digestion of soluble immune complexes by macrophages Inhibitor

Concentration

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+

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% Inhibition Ingestion Digestion

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29 -7 69 24 20 76 60

330 . R. G. Q.

LESLIE

combination of the metabolic inhibitors, but differed from ingestion in showing greater sensitivity to the action of 2 deoxy-glucose than KCN (14). Selective inhibition of catabolism was also observed with 5 mM lidocaine (19) and TLCK (17), while cytochalasin B and colchicine exerted relatively minor effects. Confirmation of selective action of cytochalasin B on ingestion and identification of the intra-cellular events blocked by lidocaine and TLCK were obtained by preincubating the complex-laden macrophages for diffe­ rent times at 3rC before adding the inhibitors. The partial blockade of digestion (ca. 24 % ) observed with cytochalasin B was completely abolished by 15 minutes preincubation (Fig. 6) indicating that its sole effect was on the rapid ingestion step and not on subsequent events. The action of lidocaine was affected to a lesser extent, but increasing the preincubation period to 45 minutes resulted in a marked depression of its inhibitory activity, suggesting that the local anaesthetic acts upon a relatively early intracellular event; presumably by preventing pinosome-Iysosome fusion rather than by blocking proteolysis within the secondary lysosomes or catabolite excretion. Inhibition of complex degradation by TLCK, on the other hand, was unaffected by preincubation and its inhibitory action upon the lysosomal proteases, rather than upon the excretion of complex diges­ tion products, was confirmed by the absence of catabolite accumulation within the inhibited cells (17). The role of complement in complex catabolism by guinea pig mac­ rophages in vitro has been examined using heat aggregates of IgG 1 and IgG2 and soluble bovine thyroglobulin (BTG)-anti BTG complexes (20-22). Enhanced degradation was observed with IgG oligomers which were large enough to activate either the classical or alternative pathways efficiently; that is, by aggregates containing twenty or more IgG molecules

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Macrophages, Antibodies and Immune Complexes . 331

(20, 21) or complexes containing four or more antibodies (22). Binding studies indicated that the enhanced digestion could be attributed to more efficient attachment of the aggregates to the cells (20) which was mediated by membrane receptors for the aggregate bound C3 fragments (22). Macrophage stimulation by soluble immune complexes The destruction of soluble complexes by macrophages, though selective at the levels of uptake and ingestion, is not necessarily a process that involves specific triggering, since phagocytes constantly pinocytose and process their fluid environment in the absence of stimuli. Triggering is most readily measured in terms of functions which are not normally expressed by the cells, such as directed movement, the release of lysosomal enzymes and neutral proteases or the production of cytotoxic oxygen compounds. CONNEL and coworkers (23) have examined the oxidative response of guinea pig macrophages upon incubation with soluble complexes of defined size. They demonstrated that complexes containing two to four antibody molecules of the IgG2 subclass produced a rapid two-phase response, comprising of a brief burst of high chemiluminescent activity which was inhibitable by superoxide dis mutase but not catalase, followed by a persis­ tent lower level of response susceptible to both enzymes. These observa­ tions indicated that soluble immune complexes were capable of stimulating oxidative metabolism in macrophages both upon attachment to the cell surface and following ingestion and transport to secondary lysosomes. A similar study with IgG 1 and IgG2 subclass antibodies has established that complexes of both subclasses stimulate superoxide production by guinea pig macrophages at rates proportional to the relative avidities of these complexes for their respective receptors (24). Other recent reports have established that the release of lysosomal hydrolyses or neutral proteinases (25) and the synthesis of the C2 complement component by human monocytes (26) are also stimulated following in vitro exposure of the phagocytes to small aggregates and soluble complexes, respectively. Concluding remarks The in vitro studies described in this review raise several points for consideration regarding the in vivo fate and pathogenetic activity of cir­ culating immune complexes. In terms of clearance, it is apparent that soluble complexes have to compete with serum IgG for phagocyte receptors but carry some binding advantage by virtue of their capacity for multipoint attachment. The cell surface receptors constitute a mechanism for concen­ trating ligands and, where complexes are uniform in terms of their antigenic component, this may lead to aggregation which enhances the binding still further. Once bound, the complexes are selectively and irreversibly ingested and may thus avoid re-release induced by the competing monomer. It may

332 . R. G. Q. LESLIE

be argued, therefore, that the clearance of complexes from the circulation depends not simply on attachment per se but also on the subsequent steps of rearrangement and ingestion. Complement may enhance the rate of complex clearance by providing a second ligand for the phagocytes in the form of complex-bound C3b, C3bi or C3d. However, its role is limited by the efficiency with which complexes activate the complement cascade and is probably restricted to aiding the rapid elimination of the larger complexes. Soluble complexes have been demonstrated to stimulate macrophage function in vitro at two levels. They are capable of inducing short term responses, such as an increase in oxidative metabolism or the release of lysosomal hydrolyses and neutral proteinases, as well as promoting more protracted changes in protein synthesis. The effects on triggering of serum concentrations of IgG or complex cross-linking at the cell surface have not yet been investigated so the in vivo relevance of these observations remains uncertain. However, recent reports that the monocytes from rheumatoid arthritis patients synthesise more C2 (27) and express higher levels of IgG receptor activity (28) than normal individuals, and the correlation of the latter change with the presence of circulating complexes emphasises the need for further examination of the stimulatory effects of soluble complexes on macrophages. References 1. BENACERRAF, B., M. SEBESTYEN, and N. S. COOPER. 1959. The clearance of antibody­ antigen complexes from the blood by the reticulo-endothelial system. J. Immuno!. 82: 131. 2. VAN Es, L. A., M. R. DAHA, and A. KIJLSTRA. 1979. Clearance of soluble immune complexes and aggregates. Protides of the Biological Fluids 26: 159. 3. MANNIK, M., A. O. HAAKENSTAD, and W. P. AREND. 1974. The fate and detection of circulating immune complexes. Progress in Immunology II 5: 91. 4. AREND, W. P., and M. MANNIK. 1973. The macrophage receptor for IgG: number and affinity of binding sites. J. Immuno!. 110: 1455. 5. LESLIE, R. G. Q., and M. D. ALEXANDER. 1979. Cytophilic antibodies. Curro Top. Microbio!. Immuno!. 88: 25. 6. UNKELESS, J. C., and H. N. EISEN. 1975. Binding of monomeric immunoglobulins to the Fe receptors of mouse maerophages. J. Exp. Med. 142: 1520. 7. UNKELESS, J. C. 1972. The presence of two Fe receptors on mouse macrophages: Evidence from a variant cell line and differential trypsin sensitivity. J. Exp. Med. 145: 931. 8. UNKELESS, J. C. 1979. Characterisation of a monoclonal antibody directed against mouse macrophage and lymphocyte Fe receptors. J. Exp. Med. 150: 580. 9. LESLIE, R. G. Q. 1980. The binding of soluble immune complexes of guinea pig IgG2 to homologous peritoneal maerophages. Determination of the avidity constants at 4 °C. Eur. J. Immuno!. 10: 317. 10. SEGAL, D. M., and E. HURWITZ. 1977. Binding of affinity cross-linked oligomers of IgG to cells bearing Fc receptors. J. Immuno!. 118: 1338. 11. DOWER, S. K., C. DELISI, J. A. TITUS, and D. M. SEGAL. 1981. The mechanism of binding of multivalent immune complexes to Fe receptors. 1. Equilibrium binding. Biochemistry, 20: 6326. 12. KNUTSON, D. W., A. KIJLSTRA, and L. A. VAN Es. 1977. Association and dissociation of aggregated IgG from rat peritoneal macrophages. J. Exp. Med. 145: 1368.

Macrophages, Antibodies and Immune Complexes . 333 13. DOWER, S. K., J. A. TITUS, C. DELISI, and D. M. SEGAL. 1981. The mechanism of binding of multivalent immune complexes to Fc receptors. II. Kinetics of binding. Biochemistry, 20: 6335. 14. LESLIE, R. G. Q. 1980. Macrophage handling of soluble immune complexes. Ingestion and digestion of surface-bound complexes at 4, 20 and 37°C. Eur. J. Immunoi. 10: 323. 15. MULLER, W. A., R. M. STEINMAN, and Z. A. COHN. 1980. Membrane flow during endocytosis. In: Mononuclear Phagocytes - Functional Aspects. Martinus Nijhoff Medi­ cal Division, The Hague, Pt. 1, pp. 595. 16. LESLIE, R. G. Q. 1982. Events initiated by soluble immune complex interaction with immunoglobulin receptors on macrophages. Protides of the Biological Fluids, 29, in press. 17. LESLIE, R. G. Q. 1980. Macrophage handling of soluble immune complexes. Use of specific inhibitors to study the biochemical events involved in complex catabolism. Eur. J. Immunoi. 10: 799. 18. KIJLSTRA, A., W. V. DoRP, M. R. DAHA, and R. G. Q. LESLIE. 1980. The effect of Lidocaine on the processing of soluble immune aggregates and immune complexes by peritoneal macrophages. Immunology 41: 237. 19. STEINMAN, R. M., J. M. SILVER, and Z. A. COHN. 1974. Pinocytosis in fibroblasts. Quantitative studies in vitro. J. Cell. BioI. 63: 949. 20. KIJLSTRA, A., L. A. v. Es, and M. R. DAHA. 1979. The role of complement in the binding and degradation of immunoglobulin aggregates by macrophages. J. Immunoi. 123: 2488. 21. DAHA, M. R., and L. A. v. Es. 1981. Enhanced alternative complement pathway­ dependent degradation of soluble immunoglobulin aggregates by macrophages. Immuno­ logy 43: 513. 22. KIJLSTRA, A., L. A. v. Es, and M. R. DAHA. 1981. Enhanced degradation of soluble immune complexes by guinea pig peritoneal macrophages in the presence of complement. Immunology 43: 345. 23. CONNELL, P. A., M. S. SEEHRA, R. G. Q. LESLIE, and W. G. REEVES. 1980. Chemiluminescence of guinea pig peritoneal macrophages stimulated by immune precipi­ tates and soluble immune complexes. Eur. J. Immunoi. 10: 966. 24. TAMATO, K., and J. KOYAMA. 1980. Superoxide anion production from guinea pig macrophages stimulated with immune complexes of different IgG subclasses. J. Biochem. 87: 1649. 25. PESTEL, J., M. JOSEPH, J.-P. DESSAINT, and A. CAPRON. 1981. Macrophage triggering by aggregated Igs. 1. Delayed effect of IgG aggregates or immune complexes. J. Immunoi. 125: 1987. 26. MACPHADEN, A. K., and K. WHALEY. 1981. Modulation of C2 biosynthesis by antigen­ antibody complexes. J. Clin. Lab. Immunol., in press. 27. DE CEULAER, C., S. PAPAZOGLOUS, and K. WHALEY. 1980. Increased biosynthesis of complement components by cultured monocytes, synovial fluid macrophages and syno­ vial membrane cells from patients with rheumatoid anhritis. Immunology 41: 37. 28. KATAYAMA, S., D. CHIA, H. NASU, and D. W. KNUTSON. 1981. Increased Fc receptor activity in monocytes from patients with rheumatoid arthritis: A study of monocyte binding and catabolism of soluble aggregates of IgG in vitro. J. Immunoi. 127: 643. 29. LESLIE, R. G. Q., and S. COHEN. 1976. Comparison of the cytophilic activities of guinea pig IgG1 and IgG2. Eur. J. Immunoi. 6: 848. 30. LESLIE, R. G. Q., and S. COHEN. 1974. Cytophilic activity of IgG2 from sera of unimmunised guinea pigs. Immunology 27: 577. 31. GANCZAKOWSKI, M., and R. G. Q. LESLIE. 1979. The binding of rabbit IgG and its enzymatically derived fragments to homologous peritoneal macrophages. Immunology 36: 487. 32. ALEXANDER, M. D., J. A. ANDREWS, R. G. Q. LESLIE, and N. J. WOOD. 1978. The binding of human and guinea pig IgG subclasses to homologous macrophage and monocyte receptors. Immunology 35: 125. Dr. R. G. Q. LESLIE, Depanment of Immunology, University Hospital, Queen's Medical Centre, Nottingham, NG7 2UH, United Kingdom