Ag-Ab Complexes Research Articles

Macrophage metabolism during phagocytosis and digestion of normal and IgG antibody-coated sheep erythrocytes.

The respiration rate, aerobic glycolysis and glycolytic enzyme activities of purified mouse peritoneal macrophages increase during phagocytosis of either free or antibody-coated SRBC. These changes, however, seem to be roughly parallel to the amount of phagocytized antigen. In the digestive phase, Ag-Ab complexes induce more marked changes in macrophage metabolism than a comparable amount of free antigen. Lactate production, activity of several glycolytic enzymes and alanine-aminotransferase as well as glycogen content are greatly affected. Since in cell transfer experiments macrophage-associated SRBC can prime normal recipients for antibody response, whereas cell-associated SRBC-antibody complexes cannot, it is concluded that the changed macrophage metabolism drives antibody-coated antigen into metabolic channels allowing its rapid and total degradation.

In vitro and in vivo studies on uptake of antigen-antibody complexes by eosinophils.

<i>In vitro </i>and <i>in vivo </i>uptake of soluble Ag-Ab complexes by eosinophilic leukocytes was studied. In the <i>in vitro </i>experiments, soluble complexes containing ragweed antigen and human IgM or IgG antibodies were much more frequently taken up by eosinophils than complexes containing IgA antibodies. Complexes containing IgE antibodies (purified) prepared in extreme and moderate antigen (purified fraction of ragweed extract) excess were taken up more frequently by the eosinophils (4.8 or 4.9%) than complexes containing purified IgG antibodies (1.7 and 0.9%). <i>In vivo </i>experiments were performed on rats using soluble Ag-Ab complexes of rat system (homologous) consisting of ragweed antigen and rat antibodies and complexes of rabbit and human systems (heterologous) consisting of ragweed and corresponding rabbit or human antibodies. Rats injected with homologous complexes showed a definitely higher percentage of eosinophils taking up complexes (14.1%) than rats injected with heterologous complexes of rabbit (2.3%) or human (1.3%) system.

The Biologic Significance of Fc Receptor on Mouse B-Lymphocytes

Abstract Evidence exists that antigen is trapped in lymphoid follicles complexed with antibody (1–6) and that antigen-antibody complexes (Ag-Ab) prepared in vitro localize in these same areas (7). The localization of the complexes depends on the presence of the Fc of the antibody since complexes of the antigen with Fab fragment do not localize in the follicles (8). These studies consider that the binding of Ag-Ab complexes and Fc fragment occurs by an Fc receptor present on reticular cells. We have previously described the presence of a receptor for antibody Fc on the surface of mouse spleen lymphocytes (9–11). This receptor could bind isolated Fc fragments as well as the Fc of antibody in Ag-Ab complexes. The binding was independent of complement. In the present communication we present some evidence for the possible biologic role of this receptor during the primary response and during the interaction of rabbit anti-mouse thymocyte serum (ALS) with lymphocytes both in vitro and in vivo.

Modulation of in vitro lymphocyte transformation by antibodies: Enhancement by antigen-antibody complexes and inhibition by antibody excess

The in vitro lymphoproliferative responses to diphtheria and tetanus toxoids by lymph node lymphocytes from guinea pigs immunized to manifest both antibody production and delayed hypersensitivity were inhibited by antisera to these antigens. These inhibitory effects were greater in the presence of fresh sera containing complement than in heat inactivated sera. The antitoxins interfered to a lesser extent with the lymphocyte response to phytohemagglutinin, purified protein derivative, bovine serum albumin, and dinitrophenol guinea pig albumin than with tetanus toxoid and were therefore not nonspecifically cytotoxic. After 60 min of exposure the inhibitory effects of the antisera could still be reversed by washing the lymphocytes free of antibodies and restimulating them with either of the toxoids. Conversely, partial inhibition by antitetanus was achieved even when the antiserum was added 18 hr after the tetanus toxoid. This suggests that even after initiation of the lymphocyte response to antigen it could be reversed by the addition of antibodies. Antigen-antibody (Ag-Ab) complexes were prepared and the precipitates separated from the supernatants. The supernatants of the Ag-Ab complexes in antibody excess often stimulated lymphocytes to a limited extent and concomitantly inhibited the lymphocyte from responding to any additional antigen. Supernatants of Ag-Ab complexes at equivalence and in antigen excess stimulated the lymphocytes more effectively. In contrast all the precipitated Ag-Ab complexes including those prepared in antibody excess stimulated the lymphocytes significantly and often up to twice as effectively as the uncomplexed antigen. The presence of fresh sera containing complement was not a prerequisite for obtaining lymphocyte stimulation with the precipitated complexes. The precipitated Ag-Ab complexes did not stimulate lymphocytes from unsensitized guinea pigs. These findings indicate that an excess of soluble antibodies can block the response of sensitized lymphocytes to specific antigens, whereas antigen aggregated by antibody could enhance the proliferative response. These in vitro studies suggest that antibodies can affect the cellular immune response by modulating the lymphoproliferative reaction to antigens.

The macrophage in the antibody-mediated suppression of the humoral response.

Inbred Balb/c mice were used throughout the experiments. Highly purified IgM-type and IgG-type anti-sheep red blood cell (SRBC) antibodies were used. <sup>51</sup>Cr-labelled SRBC were incubated with antibodies at different antigen-antibody ratios. Thioglycollate-induced peritoneal macrophages were incubated with SRBC or antibody-complexed SRBC and injected into syngeneic animals. The number of macrophages varied within groups, the radioactivity being, however, constant. The antibody response was quantitated by the Jerne plaque technique at different intervals after transfer. Macrophage-associated antigen triggers a significant immune response. IgG-complexed SRBC given in macrophages evoke only a marginal response even when only a minority of available antigenic determinants are antibody-complexed. The immune response after transfer of IgM-complexed macrophage-associated antigen is suppressed when tested on the 4th day; but on the 7th day, a moderate reaction is discernible. Suppression was observed also when antigen was given in macrophages and antibody intravenously. Suppression was dependent on the antibody dose injected or used for complexing with antigen, particularly in the IgM system. The degradation of engulfed Ag-Ab complexes proceeds in macrophages more efficiently than in free antigen. Two possible mechanisms of antibody-mediated suppression are discussed, i.e., blocking of antigen in macrophages by antibody or increased decay of Ab-Ag complexes in the lysosomal system.

Separation of soluble antigen-antibody complexes by acrylamide gel electrophoresis and ultracentrifugation

Soluble antigen-antibody complexes between ovotransferrin and its antibody were separated by rate zonal centrifugation and by acrylamide gel electrophoresis in 5.0% gel or in a discontinuous gel gradient. The isolation of centrifuged fractions was facilitated by photopolymerizing the tube content in acrylamide gel. Both methods gave identical types of antigen-antibody complexes, characterized by electrophoretic mobility, antigen-antibody ration, and sedimentation constant. The acrylamide gel electrophoresis in 5.0% gel is particularly suitable for the identification and the quantitative determination of various Ag Ab complexes. Small quantities of concentrated Ag Ab complexes can be obtained easily in the “pore limit” electrophoresis, larger quantities by zonal centrifugation in acrylamide containing sucrose gradients.

Polymer-induced precipitation of antigen-antibody complexes: ‘Precipiplex’ reactions

Incorporating high molecular weight polyethylene glycol into mixtures of antigen (Ag) and antibody (Ab) accelerated greatly Ag-Ab precipitation, increased amounts of specific precipitates, and permitted recovery of Ag-Ab complexes not obtainable by conventional procedures. The contracted adjectival term ‘precipiplex’ is introduced to describe the induced precipitation of Ag-Ab complexes by certain watersoluble polymers and to distinguish such reactions from classical precipitin reactions. The concept of induced precipitation may be applied to a variety of immunoassays dependent upon precipitin reactions.

Studies on antigen-antibody complexes. I. Elimination of soluble complexes from rabbit circulation.

Solid phase immunoadsorbents were prepared by coupling antigens to agarose. With this technique specific antibodies were easily isolated in large amounts. The gammaG-globulin class of antibodies isolated in this manner were not denatured as judged by their normal biological half-life in rabbits. Soluble immune complexes at fivefold antigen excess were prepared from isolated specific antibodies and HSA, human lambda-chains, human lambdaG-globulins, and a Waldenström's macroglobulin as antigens. In all these preparations a characteristic immune complex was encountered that represented the smallest stable antigen-antibody union. In the HSA-anti-HSA system they were found to be AgAb(2) complexes, and Ag(2)Ab complexes in the gammaG-anti-gammaG system. These stable complexes fixed complement ineffectively. Also, a spectrum of larger complexes was present in each system, and these complexes fixed complement effectively. With intact antibodies the disappearance curves of immune complexes from the circulation were composed of three exponential components. The immune complexes larger than AgAb(2) were quickly removed from the circulation with half-lives of 0.09-0.37 hr. Their clearance was not dependent on complement components, in that depletion of complement by cobra venom factor and aggregated gammaG-globulin did not alter the pattern of their removal from the circulation. However, when the interchain disulfide bonds of antibodies were reduced and alkylated, the removal of the lambda-anti-lambda, HSA-anti-HSA, and gammaG-anti-gammaG complexes was altered. In these experiments the disappearance curves were composed of two exponential components and the rapid removal of the greater than AgAb(2) complexes did not occur. The immune complexes prepared from reduced and alkylated antibodies fixed complement ineffectively. The presented data indicate that the rapid removal of circulating immune complexes, containing gammaG-globulin molecules as antibodies, depends primarily on the number of antibodies involved. Furthermore, complement fixation is not involved in the rapid removal of such complexes. Nevertheless, the rapid removal of immune complexes and their ability to fix complement have similarities for optimal function in that both processes require intact interchain disulfide bonds of antibodies and complexes that exceed the AgAb(2) combination.

Passive Anaphylaxis in Mice with γG Antibodies

Summary Anaphylactic release of histamine from mouse mast cells passively sensitized by purified homologous anti-hapten antibodies was demonstrated. The reaction was critically dependent on the Ag-Ab proportions used for sensitization and challenge and could be provoked by addition of freshly prepared Ag-Ab complexes. No fixation of the antibodies to the cells prior to the Ag-Ab reactions could be demonstrated. The release of histamine was complete 2 min after challenge.

Equilibrium and kinetic studies of the reaction between conalbumin and anticonalbumin

Methods are presented by which the two phases of the primary AgAb reaction can be studied independently. It is experimentally justified that the first phase of the reaction can be treated as an equilibrium reaction. In one special example, in the FCA-AFCA and FITC-AFCA systems, experimental data are presented for the calculation of the association constant of the reaction from equilibrium fluorescence measurements. The usefulness of fluorescence quenching measurements for both equilibrium and kinetic measurements is demonstrated in the FCA-AFCA system. Our data suggest that the fluorescence quenching of labeled protein antigens would be generally applicable for measuring the rate of formation of the primary AgAb complex between the soluble protein Ag and its Ab. The presence of the fluorescent label does not change significantly the combining or precipitating properties of the Ag, or the rate of the primary combination of Ag with Ab, but changes significantly the rate of growth of the primary AgAb complex. These data suggest that different combining sites may be involved in the primary union of Ag with Ab than in the growth of the AgAb complex.

Termination of immunologic tolerance in mice by antigen-antibody complexes.

Eisen and Karush(l) proposed a model for the extracellular control of immunity and tolerance based on the assumption that bimolecular antigen-antibody (AgAb) complexes stimulate antibody formation, while trimolecular Ag2Ab complexes induce immunologic tolerance because they are not taken up by the immunologically competent cells. The model predicts that it should be possible to initiate an immune response in cells from tolerant animals provided they are stimulated with the appropriate AgAb complexes. Newborn NIH white mice were made tolerant to human serum albumin (HSA). At 7 weeks of age, single-cell suspensions were prepared from the individual spleens of the tolerant animals and exposed in vitro to HSA and rabbit anti-HSA mixed in 1:1 or 10:1 molecular ratios, to HSA only, or to a salt solution. The spleen cells were then injected intraperitoneally in the same mouse from which they had been removed. After challenge with HSA in incomplete Freund's adjuvant, the mice were tested by the tanned-cell hemagglutination procedure and by measurement of the rate of HSA elimination. Within 2 weeks following challenge, tolerance was broken in 55% of the mice whose cells had been exposed to AgAb complexes. In contrast, almost 90% of the mice whose cells had been treated with antigen only or with saline remained tolerant.

Liver Localizing Antibodies—The Limited Contribution of Ag-Ab Complexes

Summary The contribution of antigen-antibody complexes in the liver localization of purified anti-rat-liver antibodies was studied by the use of Sephadex G-200 and the paired label technique and found to be unimportant. The 7 S antibody fraction eluted from the Sephadex column (Peak II) which is free of complexes, localized in the liver to a high extent, indicating the liver localization of anti-liver serum is due to specific antibody and not to nonspecific uptake of antigen-antibody complexes. The contribution of anti-rat-plasma antibody present in the anti-liver serum (which forms complexes upon injection into assay rats) is negligible since it was largely eliminated during the purification process as shown by the paired label experiments. The macroglobulin antibody may participate also in the liver localization of the purified anti-liver antibody preparation. The soluble complexes formed by the anti-liver globulin and a large excess of soluble antigens from liver connective tissue, localized poorly in the liver and was eliminated much more rapidly than the localizing antibody.

BRONCHOCONSTRICTOR EFFECTS OF SOLUBLE ANTIGEN-ANTIBODY COMPLEXES IN ISOLATED LUNG, AND THEIR INHIBITION BY NORMAL SERUM.

RECENT investigations in this laboratory with the perfused guinea pig lung have shown that a broncho-constriction, accompanied by histamine release, follows injection into the perfusate of soluble antigen–antibody (Ag–Ab) complexes prepared in antigen excess. It is known that soluble Ag–Ab complexes produce an increase in capillary permeability when injected subcutaneously in unsensitized animals1, and an anaphylactic syndrome when given intravenously2. They have also been shown to induce a contraction of guinea pig ileum in vitro3.

Labelling of antibodies by 5-dimethylamino-1-naphthalene sulfonyl chloride, its effect on antigen-antibody reactions.

Summary and conclusionProperties of the fluorescent dye DNS for labelling proteins have been described. Investigations of conditions of DNS binding to serum proteins revealed ratios of 3-4 M DNS/M gamma-globulin and about 7 M DNS/M albumin. Dealing with the use of DNS in immunochemistry, experiments have been carried out with the ADH-rabbit antiserum system, measuring enzyme inhibition by the Ab. Labelling of Ab with DNS did not affect rate of formation of Ag-Ab complex and had no influence on quantity of formed precipitate. The reported data suggest that because DNS does not affect the Ag-Ab reaction, it can be successfully applied in histochemical studies.