Characterization of interactions involving anti-metatype antibodies and immune complexes.

Immunizations of high affinity anti-fluorescein monoclonal antibody 4-4-20 affinity labeled with fluorescein 5-isothiocyanate into a rabbit elicited antibodies specific for the liganded conformation of 4-4-20 (termed "anti-metatype" antibodies). Reaction of liganded 4-4-20 with anti-metatype antibodies caused significant delay (up to 23-fold) in the rate of dissociation of fluorescein ligand from the active site. In this study, structural analogues of fluorescein, including fluorescein 5-isothiocyanate, fluorescein 6-isothiocyanate, 5-dichlorotriazinyl aminofluorescein and 5-carboxyfluorescein, were bound by monoclonal antibody 4-4-20 and anti-metatype antibody reactivity was observed through delay in the dissociation rate of ligand from Mab 4-4-20. Significant delays (ranging from 5- to 242-fold) were observed for all structural analogues examined indicating that 4-4-20 maintained similar but not necessarily identical conformations upon binding fluorescein structural analogues. Additionally, fluorescein 5-isothiocyanate and fluorescein 6-isothiocyanate were conjugated to carrier molecules of increasing mol. wt (ranging from 225 to 14,600 D) in an attempt to sterically interfere with "metatopes" at the mouth of the active site and localize regions of anti-metatype antibody binding. These fluorescein-conjugated compounds were reacted with 4-4-20, and binding of anti-metatype antibodies delayed dissociation rates from 24- to greater than 1500-fold. These results indicated that the mechanism whereby anti-metatype antibodies delay the release of fluorescyl ligands from the active site probably does not solely involve steric hindrance of the ligand due to binding of anti-metatype antibodies at the mouth of the active site. Studies with 4-4-20 Fab fragments and a single chain derivative of 4-4-20 (consisting of the variable regions tethered by a 14 amino acid linker) indicated that anti-metatype reactivity was specific for the immunoglobulin variable region.     

The glomerular deposition of soluble immune complexes prepared with reduced and alkylated antibodies and with intact antibodies in mice.

The kidney localization and glomerular deposition of soluble immune complexes in mice were greater and more persistent following the intravenous administration of complexes prepared with reduced and alkylated antibodies than following the administration of complexes prepared with intact antibodies. The increased glomerular deposition following the administration of complexes prepared with reduced and alkylated antibodies was associated with the persistence of circulating complexes composed of more than two antigen and two antibody molecules (Haakenstad AO, Mannik M:Lab Invest 35:283, 1976). The deposition of immune complexes in glomeruli, as detected by immunofluorescence, appeared to precede the detection of mouse C3 in glomerular deposits following the administration of both preparations of complexes. The deposition of mouse C3 was more intense and persisted longer in mice receiving complexes containing reduced and alkylated antibodies than in mice receiving complexes containing intact antibodies. The ultrastructural studies indicated that both preparations of complexes initially localized as electron-dense material in endothelial cell fenestrae and in the subendothelial space of the glomerular capillary loops and subsequently accumulated in the mesangial matrix between mesangial cells. The material persisted in the mesangium of mice receiving complexes with reduced and alkylated antibodies, whereas it was removed from the mesangium of mice receiving complexes with intact antibodies. The mechanism for removal of complexes from the mesangial matrix was not defined, but it did not appear to occur through phagocytosis by the mesangial cell.     

The disappearance kinetics of soluble immune complexes prepared with reduced and alkylated antibodies and with intact antibodies in mice.

Soluble immune complexes prepared with reduced and alkylated antibodies persisted longer in the circulation than complexes prepared with intact antibodies, when these were administered intravenously to mice. The disappearance of complexes with reduced and alkylated antibodies was delayed in part because the initial phase of vascular permeability was considerably less than that seen following the administration of complexes with intact antibodies. In addition, large complexes with lattice structure of more than two antigen and two antibody molecules persisted longer in the circulation after administration of complexes with reduced and alkylated antibodies than after administration of complexes with intact antibodies. Thus, the concentration of large latticed complexes with reduced and alkylated antibodies was significantly greater than the concentrations of large latticed complexes with intact antobodies at all observed times through 96 hours. The persistence of large latticed complexes with reduced and alkylated antibodies was associated with significantly decreased hepatic localization of complexes with reduced and alkylated antibodies compared to the hepatic localization of complexes with intact antibodies at 1, 4, 12, and 24 hours. The observations indicated that the removal of large latticed complexes from the circulation by the hepatic mononuclear phagocyte system was decreased when reduced and alkylated antibodies were used for the preparation of immune complexes. The persistence of large latticed complexes with reduced and alkylated antibodies in the circulation was associated with enhanced and prolonged presence of glomerular deposits of immune complexes, as reported in the accompanying article (Haakenstad AO, Striker GE, Mannik M: Lab Invest 35:293, 1976.     

Human sera with precipitating antibodies to human soluble immune complexes. A brief communication

INTRODUCTION: Previous numerous papers by the senior author dealt with the human serum factor referred to as anti-antibody which is specifically directed against IgG antibodies that underwent molecular transformation in the course of the reactions with their corresponding antigens. The reactions of this serum factor could be conveniently detected by means of agglutination of Rh-positive erythrocytes sensitized by anti Rh antibodies. No precipitation tests could be developed. MATERIAL AND METHODS: Most studies were conducted by means of double diffusion in gel precipitation. RESULTS: A rheumatoid arthritis serum, G, was noted that produced a strong reaction of double diffusion in gel precipitation with serum samples of a renal graft recipient, T. Further screening detected one more rheumatoid arthritis serum reacting with T; of 28 sera from renal graft recipients, 6 reacted in a similar way to T, but the reactions were considerably weaker and poorly reproducible. Evidence was presented that the precipitin in the two rheumatoid arthritis sera under study had properties of previously described anti-antibody. CONCLUSIONS: Sera with precipitating anti-antibodies may serve as exquisite reagents for detection of soluble immune complexes in human sera.     

Macrophage handling of soluble immune complexes: Evaluation of mechanisms involved in the selective clearance of complexes from the circulation

The binding and release of soluble guinea pig IgG2-containing DNPBSA-anti-DNP complexes and antigen-free, covalently-linked anti-DNP IgG2 oligomers of similar size, by guinea pig peritoneal macrophages, has been examined in the absence and presence of monomeric IgG2, of unrelated antibody specificity, or the monovalent hapten, DNP lysine. Complex binding was found to differ from the binding of the oligomers in that it was about twice as efficient and was essentially irreversible even in the presence of an inhibitor of ingestion, cytochalasin B. On the other hand, quantitative complex release could be achieved, in the presence of the ingestion inhibitor, by including 1.5mM DNP lysine in the medium. Complex handling by macrophages at 37°C was also examined in the presence of monomeric IgG2, at its serum concn, and in the absence and presence of cytochalasin B. Inhibiting ingestion did not impair the capacity of the macrophages to take up complexes under these conditions. On the basis of these findings and previous reports that complexes bound to a receptor-bearing membrane undergo additional antibody-antigen bond formation [Dower et al, Biochemistry20, 6326–6334 (1981a) and Leslie, Protides biol. Fluids29, 431–434 (1982)] it is proposed that complex aggregation at the phagocyte surface may constitute the critical irreversible event required for the selective clearance of complexes in vivo. Other biological implications of receptor-mediated complex aggregation are also discussed.     

Macrophage handling of soluble immune complexes ingestion and digestion of surface-bound complexes at 4, 20 and 37 °C

Radioassays have been developed to measure, as separate events, the ingestion and degradation of macrophage-bound guinea pig IgG anti-DNP (2,4-dinitrophenyl)- DNP-BSA (bovine serum albumin) complexes of defined size and subclass. Complex ingestion was observed to be temperature-dependent and was effectively blocked only by a combination of inhibitors of respiration and glycolysis indicating that the process is under the same metabolic control as fluid phase pinocytosis. On the other hand, the half-life of membrane-bound complexes at 37 °C (t_1/2 = 5.6 ± 1.7 min) was considerably shorter than the value expected from membrane turnover studies (t_1/2 = 22.4 min) suggesting that complexes are selectively cleared from the cell surface. The rate of ingestion at 20°C was independent of complex size and of the IgG subclass used in complex formation, but was affected by in vivo stimulation of the macrophages before assay. Complex digestion was shown to be highly temperature-dependent and, at 37 °C, proceeded at a rate (t_1/2 = 1.5-5 h) which was 20-60-fold slower than the rate of ingestion indicating that the latter is unlikely to influence digestion kinetics. On the other hand, the selective action of 2-deoxyglucose in blocking digestion, but not ingestion, suggests that pinosome-lysosome fusion may play a part in determining the overall catabolic rate. A 2 to 3-fold difference in digestion rates was observed between the proteins employed as antibody (guinea pig IgG₁ or IgG₂) and as antigen (DNP-BSA). This finding suggests that the intrinsic susceptibility of ingested proteins to enzymatic hydrolysis may be the prime determinant of digestion rate. As with ingestion, no discrimination was observed in the degradation of complexes of different size or IgG antibody subclass. The observations in this and the preceding study (Eur. J. Immunol. 1980. 10: 317) indicate that complex size is important in determining the level of uptake by phagocytes but not subsequent events associated with catabolism.     

Clearance kinetics of soluble pre-formed immune complexes containing IgM antibodies in normal and decomplemented rabbits.

The clearance kinetics of homologous soluble pre-formed 125I-BSA/IgM anti-BSA immune complexes (IgM . IC) injected into the blood was studied in the rabbit. IgM . IC close to equivalence were cleared with a greater velocity than those in x 5 or x 10 antigen excess. Clearance of BSA specifically bound to antibody, was measured by the Farr assay and found to exhibit an exponential mode of elimination suggesting a selective removal of large over small lattice complexes or free antigen. The role of C3 in the clearance of IgM . IC was examined by treating rabbits with cobra venom factor (CVF). While more variation was noted in CVF-treated rabbits, no statistically significant differences could be detected between C3-depleted and control animals. It is, therefore, concluded that soluble preformed IgM . IC are cleared from the rabbit circulation by a C3b-independent mechanism. This finding for soluble IC is clearly different from the clearance of IgM-coated red cells from the circulation, which has previously shown to be highly C3b-dependent.     

Interaction between soluble immune complexes and glass-fiber filters.

The two components of soluble antigen--antibody complexes, at the antigen excess, exhibit an increase in their binding ability to glass-fiber filters. This is demonstrated in two systems. In the 125I-BSA--anti-BSA system the proportion of 125I-BSA bound to the filter is markedly increased in the presence of anti-BSA antibodies. More than 80% of the antibody bound BSA can be removed by passage through the filter. In the other system, mouse gamma globulin (MGG)--125I-anti-MGG, the proportion of antibody bound to the filter increases with the increase in antigen concentration, whilst the presence of another, non-related, gamma globulin has little effect on the binding. The possible mechanisms for the binding of soluble complexes to the glass fibers are suggested and discussed.     

In vivo generation and clearance of soluble immune complexes containing IgM antibodies in normal and decomplemented rabbits.

Soluble immune complexes containing IgM antibodies (IgM.IC) were generated in vivo utilizing a passive induction model, whereby purified antibodies were injected into rabbits with circulating radiolabelled bovine serum albumin (BSA) as antigen. A triphasic response was obtained consisting of an initial rapid elimination of TCA-precipitable antigen in the first 30 min, followed by a progressive diminution in the clearance velocity as antigen from the tissues moved back into the circulation to re-equilibrate, and subsequent elimination of the antigen at a rate close to that of free BSA. The dynamics of IC formation and disappearance were studied by a combination of Farr assay and solid-phase C1q binding. The results show that the rate of clearance decreased as the complexes progressively moved into antigen excess, and that the decrease in the proportion of complexed antigen was mirrored by a similar decrease in the ability of the complexes to bind C1q. Depletion of complement by treatment with cobra venom factor did not inhibit the clearance of the antigen, but may have inhibited solubilization of the complexes in vivo. Tissue localization experiments indicated that the liver is the organ predominantly involved in the uptake and catabolism of in vivo-generated IgM.IC. These results show that the clearance velocity of soluble IgM.IC is critically dependent on the antigen/antibody ratio, and that clearance is mediated via a C3b-independent mechanism in the RES.     

Demonstration of soluble immune complexes in biopsy tissues

The percentage of immune complexes revealed in the intercellular adhesive substance of the epidermis in acantholytic pemphigus was raised to 100% after cryostat sections of biopsy tissues were pretreated with 30–50% ethanol. Immune complexes were also found in all cases of the benign familial chronic pemphigus (Hailey-Hailey disease). It is suggested that 30–50% ethanol can gently denature the proteins and stabilize the bonds between antibody and antigen in the case of a weak affinity of antibodies, and can cause soluble immune complexes to aggregate into larger protein conglomerates, which prevents them from being flushed out of the tissues. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 120, № 11, pp. 545–547, November, 1995 Presented by V. I. Shumakov, Member of the Russian Academy of Medical Sciences     

The relationship of soluble immune complexes, insulin antibodies and insulin-anti-insulin complexes to platelet and coagulation factors in type 1 diabetic patients with and without proliferative retinopathy.

The possible correlation between soluble immune factors and platelet and coagulation factors has been evaluated in Type 1 diabetic patients with and without proliferative retinopathy, and in non-diabetic controls. Soluble immune complexes, platelet factor IV (PF4), beta-thromboglobulin, fibrinogen, factor VIII related antigen and anti-thrombin III were significantly increased in Type 1 diabetic patients with retinopathy as compared to non-diabetic controls. Fibrinogen and anti-thrombin III were also higher in those patients with retinopathy compared to those without retinopathy. A significant correlation was found between positive values of soluble immune complexes and increased levels of PF4 and beta-thromboglobulin in diabetic patients with retinopathy. The presence of soluble immune complexes and insulin-anti-insulin complexes was associated with a significantly greater number of elevated haemostatic factors in retinopathic patients. Our findings suggest that the interaction of platelets and soluble immune complexes or insulin-anti-insulin complexes may be pathologically relevant to the development of diabetic retinopathy.     

Chemiluminescence of guinea pig peritoneal macrophages stimulated by immune precipitates and soluble immune complexes

The chemiluminescence (CL) response of guinea pig peritoneal macrophages to immune precipitates and soluble immune complexes has been investigated. The rapid burst of intense light emission observed in response to both stimuli, was inhibited by superoxide dismutase (SOD). With soluble immune complexes, this was followed by prolonged CL of lower intensity susceptible to both SOD and catalase inhibition. The magnitude of the CL response was directly related to the size of the soluble complexes reacting with the macrophages. These findings suggest that circulating, as distinct from deposited immune complexes, may play a role in the pathogenesis of complex-mediated diseases.     

Quantitative determination of anti-dsDNA antibodies and antibody/dsDNA stoichiometries in prepared,soluble complement-fixing antibody/dsDNA immune complexes

We have investigated quantitatively the complement-mediated binding of prepared, soluble ¹²⁵I-7S IgG antibody/³H-dsDNA immune complexes to human red blood cells (RBCs). We have performed these studies by using a detailed modification of the RBC-CF assay [Pedersen et al., J. Immun. Meth. 38, 2692–2280 (1980)] which now allows for the simultaneous measurement of both ³H-DNA and ¹²⁵I-binding to the cells. Our results indicate that, in the case of three SLE patients, their anti-dsDNA antibody titers are sufficiently high that a small fraction of their ¹²⁵I-7S IgG antibodies (ca 0.1–0.2%) can be identified as specifically anti-dsDNA. We have also used an indirect method (with ¹²⁵I-labelled rabbit anti-human IgG) for the determination of IgG anti-dsDNA antibodies in complement-fixing antibody/dsDNA immune complexes that bind to RBCs, and the results of these measurements are in reasonable agreement with the direct binding experiments. These studies have also allowed us to estimate the antibody/DNA stoichiometries in complement-fixing immune complexes. The results of these experiments may provide a useful standard for the analysis of monoclonal anti-dsDNA antibodies.     

Detection of circulating soluble immune complexes in patients with various renal diseases.

Sera from patients with various types of glomerulonephritis (GN) as well as sera from rabbits with acute serum sickness were studied for the presence of circulating immune complexes (IC). The method used is based on the observation that IC inhibit the uptake of IgG aggregates by guinea-pig peritoneal macrophages. Inhibition significantly greater than with normal human sera was found with sera of patients with membranous GN, membranoproliferative GN, focal glomerular sclerosis, minimal change nephrotic syndrome, acute septicaemic glomerular diseases and systemic lupus erythematous. IC were also detected in rabbits with acute serum sickness during the period of immune elimination.     

Antigen-specific detection of soluble immune complexes in conglutinin-binding assays.

A modification of the conglutinin-binding assay has been used for antigen identification in circulating immune complexes (IC). The complexed antigen was identified, in conglutinin-bound complexes, by antibodies or antibody fragments, specifically directed against the antigen. These antibody preparations were either radiolabelled or detected with 125I-protein A. This system was found efficient using in vitro-formed bovine serum albumin--anti-bovine serum albumin and tetanus toxoid--anti-tetanus toxoid soluble immune complexes at equivalence or at slight antigen or antibody excess. In addition, with 125I-IgG--anti-HBs and with 125I-F(ab')2--anti-HBs, we examined 44 sera from 41 patients with viral type B hepatitis and the presence of HBs was observed in 18 (40.9%) IC containing tested samples. Therefore, this method appears applicable to clinical investigation.     

Neutrophil Fcγ and complement receptors involved in binding soluble IgG immune complexes and in specific granule release induced by soluble IgG immune complexes

We examined the effect of soluble IgG immune complex (IC) characteristics on the binding of IC to human neutrophils and IC-induced specific granule release of neutrophils via Fcγ receptors (CD16 and CD32) and complement receptors (CR1 and CR3). A set of soluble IgG IC varying in size, IgG subclass, antigen epitope density and complement (C) incorporation were formed between 5-iodo-4-hydroxy-3-nitrophenacetyl (NIP) coupled to bovine serum albumin (BSA) and chimeric mouse-human anti-NIP monoclonal antibodies (mAb) of all four IgG subclasses. High and low epitope density IC of all four IgG subclasses induced specific granule release with C, but in the absence of C only IgG1 and IgG3 IC were functionally active. The Fcγ and C receptors responsible for IgG IC-induced specific granule release and IC binding were determined using mAb specific for the ligand binding sites of CD16, CD32 and CR3, and recombinant soluble CR1. Each defined IC displayed a unique pattern of receptor preference, dependent upon subclass and antigenic epitope density. IC binding and IC-induced specific granule release was not mediated by the same receptor, or combination of receptors. High and low epitope density IgG3 IC binding and induction of specific granule release was mediated predominantly via CD16. Other IC subclasses bound differently, i.e. IgG1 IC used CD16 and CR3; IgG2 and IgG4 predominantly used complement receptors; but all three induced specific granule release via CD32. In vivo these results may translate into differential activation of neutrophils by soluble IC dependent upon their characteristics, leading to subtle nuances in the etiology, pathology and control of the immune response in IC-related diseases.