Soluble Antigen-antibody Complexes Research Articles

Impact, orthodoxy and peer review

Impact, orthodoxy and peer review

Development and Proliferation of Radioimmunoassay Technology

Radioimmunoassay (RIA) developed as a fallout from investigations concerning the nature of maturity-onset diabetes. We studied the metabolism of 131I-labeled insulin in nondiabetic and diabetic subjects to determine whether rapid insulin metabolism was responsible for Type 2 diabetes. Unexpectedly, we observed that radioactive insulin disappeared more slowly from the plasma of patients who had received insulin than from the plasma of subjects never treated with insulin. While immunologists of the mid-1950s did not believe that a 6,000-dalton peptide could be immunogenic, we found that the retarded rate of insulin disappearance was due to binding of labeled insulin to insulin-binding antibodies. This first use of radioisotopic methods for detection of soluble antigen-antibody complexes introduced a revolution in theoretical immunology. It is now appreciated that very small peptides can be antigenic and that the equilibrium constants for antigen-antibody reactions can be as great as 1014 liters per mole, a value up to 108 times greater than predicted by Pauling's theory of 1940. The RIA for insulin was developed when we realized that the concentration of insulin in a solution could be determined by comparing its inhibitory effect on the binding of radioactive insulin to antibody with the inhibitory effect of known insulin standards. Our initial observations with insulin suggested that antigenicity was a more general property than had been appreciated, and since non-antibody materials with specific binding characteristics were also available, RIA and related techniques quickly proliferated for the detection and measurement of a vast array of substances in our own laboratory and around the world.

Immune complexes inhibit antimicrobial responses through interleukin-10 production. Effects in severe combined immunodeficient mice during Listeria infection.

The presence of soluble antigen-antibody complexes renders mice highly susceptible to infection with the intracellular pathogen Listeria monocytogenes. In this report we show that this inhibition is manifest at the level of the innate immune response and is mediated by IL-10. Like immuno-competent mice, mice with the severe combined immunodeficient mutation (SCID) injected with immune complexes died from a sublethal dose of L. monocytogenes. These mice were protected if pretreated with neutralizing antibodies to IL-10. In vitro, immune complexes stimulated IL-10 production by SCID splenocytes and splenic macrophages. Likewise, immune complexes inhibited TNF and IFN-gamma production by SCID splenocytes cultured with heat-killed-L. monocytogenes. This inhibition was reversed by neutralization of IL-10 but not IL-4 or TGF-beta. Immune complexes and rIL-10 inhibited cytokine production by SCID splenocytes if added before or simultaneously with heat-killed-L. monocytogenes. These data support a model in which immune complexes modulate host defense and the immune response by stimulating the production of IL-10 from macrophages.

A di-leucine motif mediates endocytosis and basolateral sorting of macrophage IgG Fc receptors in MDCK cells.

An important function of the low affinity IgG Fc receptor FcRII-B2 (FcR) on macrophages is the internalization of soluble antigen-antibody complexes for lysosomal degradation. Most endocytic receptors possess tyrosine-containing cytoplasmic determinants required for endocytosis. In many proteins, signals which overlap with the endocytosis determinant and share the same critical tyrosine residue also mediate basolateral sorting in the trans-Golgi network of epithelial cells. Despite the presence of two tyrosine residues in the FcR cytosolic domain, neither one is absolutely required for coated pit localization or basolateral targeting. Nevertheless, a short domain of 13 residues containing one of the non-critical tyrosine residues mediates endocytosis and basolateral delivery. Alanine scan mutagenesis of this region now revealed a critical role of a leucine-leucine motif in both events. These findings suggest that endocytosis and basolateral sorting can be mediated by both tyrosine- and di-leucine-based signals and confirm the close relationship between the two determinants already observed for 'classical' tyrosine-dependent motifs.

Determining molecular weight distributions of antigen–antibody complex by quasi‐elastic light scattering

Physiological properties of soluble antigen-antibody (Ag-Ab) complexes depend in part on the size of the complexes. In previous work, the size distribution and structure of model Ag-Ab complexes were determined by electron microscopy. In this study, we used constrained regularization analysis of quasi-elastic light scattering data to estimate molecular weight distributions of model Ag-Ab complexes. A conformational model was necessary to determine appropriate correlations between molecular weight and diffusion coefficient, and to estimate particle structure factors. Porod-Kratky theory proved to be an adequate conformational model for these purposes. The molecular weight distributions determined by constrained regularization compared favorably with distributions obtained either by electron microscopy or by thermodynamic modeling.

Natural cytotoxicity and serum blocking in malignant cervical neoplasia.

Peripheral blood lymphocytes from 100 patients with various stages and histological types of squamous cell carcinoma of the uterine cervix were assayed for natural killer (NK) cell activity against K562 cells using the single cell cytotoxicity assay in agarose. The effect of autologous serum on NK cell activity and a quantitation of soluble antigen-antibody complexes was also carried out. The cancer patients showed reduced NK activity compared with normal controls, the reduction increasing with tumor load. Another observation was that patients with poorly differentiated tumours showed lower levels of NK activity. Addition of autologous serum resulted in further depression of NK activity in all groups of patients. An increase in circulating immune complexes was also evident in all groups of patients. Here again increase in tumor load and poorly differentiated tumors showed the highest levels. The results point toward a possible disturbance in NK cell activity that could be further depressed by autologous serum factors.

Immunochemical characterization of juvenile hormone esterase from different species of lepidoptera

Cross-immunoreactivity of juvenile hormone esterase (JHE) from different species was tested using anti-JHE ( Trichoplusia ni ) (Noctuidae) polyclonal antibody. Partial cross-reactivity was observed between JHE from Hyphantria cunea , Isia isabella (Arctuidae) and Spodoptera exigua (Noctuidae) in immunoblot analysis. Soluble antigen-antibody complex formation was observed between anti-JHE ( T. ni ) and antigen(s) from Heliothis virescens (Noctuidae) during immunotitration of antigen(s). Using an ELISA method the highest cross-reactivity was observed for both species from Arctuidae and lower cross-reactivity for antigen(s) from H. virescens .

Fibronectin binding to C1q associated with antigen-antibody complexes in EDTA-treated plasma.

In this report we have investigated the association of fibronectin with antigen-antibody-C1q complexes incubated in fibronectin-depleted and C1q-depleted plasma. When BSA--anti-BSA immune aggregates are incubated in plasma depleted of both fibronectin and C1q to which 125I-fibronectin has been reconstituted, little radio-activity is bound to the immune complexes. However, pre-incubation of immune complexes with purified C1q prior to incubation in the plasma causes an approximately 10-fold increase in the amount of radioactivity bound. The binding of 125I-fibronectin to preformed antigen-antibody-C1q complexes is specific, since the reaction is inhibited by the addition of unlabelled fibronectin but not by ovalbumin. When antigen-antibody-C1q complexes are incubated in C1q-depleted plasma containing physiological concentrations of fibronectin, and analysed by immunoblotting, fibronectin antigens are detected on the immune complexes. Identical results are obtained using immune complexes composed of sheep erythrocyte rabbit anti-sheep erythrocyte C1q (EAC1q) cells. There is no specific requirement for preformed antigen-antibody-C1q complexes, since fibronectin can be detected on antigen-antibody complexes after incubation in normal human serum or in C1q-depleted ethylenediamineteraacetic acid (EDTA) serum reconstituted with purified C1q prior to incubation with the complexes. Finally, we also demonstrate that in the presence of C1q, 125I-fibronectin will associate with soluble antigen-antibody complexes.

Antigen-antibody complexes bound to B-lymphocyte Fcγ receptors regulate B-lymphocyte differentiation

We studied the effect of soluble antigen-antibody complexes on the responses of polyclonally activated murine B lymphocytes. For this, normal B lymphocytes were stimulated with rabbit F(ab′) 2 anti-μ and lymphokines. IgG complexes, particularly in antigen excess, inhibited the plaque-forming cell response (55–70%), while proliferation was unaffected. Maximal inhibition was obtained with small amounts (0.2–1.0 μg/ml) of complexes. Neither antigen or antibody alone was inhibitory. Inhibition was mediated via binding of the IgG complexes to Fcγ receptors of B lymphocytes: (1) neither T lymphocytes or adherent accessory cells were required; (2) IgM complexes did not inhibit; and (3) inhibition was not seen when monoclonal anti-Fcγ receptor antibodies prevented binding of the IgG complexes to these receptors. Kinetic experiments showed that B lymphocytes are susceptible to this inhibitory signal for only a short time after stimulation. We conclude that IgG complexes bound to the Fcγ receptors of B lymphocytes regulate B-lymphocyte differentiation.

Immunoconglutinin and Suppression of an Induced Immune Response by Plasma from Rats Infected with Trypanosoma brucei rhodesiense

Fresh plasma from rats infected with Trypanosoma brucei rhodesiense, incubated with splenic lymphocytes from rats previously immunized with sheep blood cells, suppressed the capacity of the splenic lymphocytes to produce antibody as was indicated by reductions in the numbers of hemolytic Jerne plaques produced by the treated cells. The effect was maximal in plasma samples drawn on the sixth to eighth day of infection when they contained elevated amounts of soluble immune complex, high titers of immunoconglutinin (IK), and reduced titers of lytic complement. We suggest that the active plasma may have affected the antibody-producing cells by one or both of two mechanisms. Soluble antigen-antibody complexes may have interacted with Fc receptors of activated lymphocytes to suppress antibody production. Alternatively, complement-fixing soluble immune complexes may have reacted with C3b receptors of the lymphocytes. These lymphocytes coated with the antigen for IK could then be injured by immunoconglutination.

Rapid, homogeneous phase, liposome-based assays for total complement activity.

A simple, rapid assay for determining total complement activity has been developed. Complement activity is quantitated spectrophotometrically by measuring the amount of liposome-encapsulated enzyme unmasked by the action of anti-Dnp antibody and complement on Dnp-tagged liposomes. The assay is homogeneous in nature and is nonisotopic. The activity of complement in guinea pig serum has been measured and shown to be proportional to complement concentration. The assay was modified to measure the complement-fixing titer of anti-Dnp antibody preparations. We have compared two monoclonal anti-Dnp antibodies (IgG1 and IgM) for their ability to fix complement. The IgM antibody preparation was 450-fold more effective than the IgG1 preparation in mediating complement-dependent damage to Dnp liposomes. In addition, the test was modified to measure complement fixation by soluble antigen-antibody complexes. This complement fixation format is capable of detecting 2 pmol Dnp antigen.

Immune Complexes and Immunoconglutinin Interactions Associated with Altered Lymphocyte Activity in Plasmodium chabaudi Infections

Fresh plasma from rats infected with Plasmodium chabaudi, incubated with splenic lymphocytes from rats immunized 5 days previously with sheep blood cells, suppressed the capacity of the spleen cells to produce antibody against the sheep cells as was indicated by reductions in the numbers of hemolytic Jerne plaques formed by the treated cells. The effect was maximal in plasma of rats drawn on the 7th day of infection at a time the rats experienced a hemolytic crisis. Serologic studies indicated that the active plasma contained elevated titers of antibody against fibrinogen products, antibody against the soluble serum antigens elaborated during blood infections and antibody against the third component of fixed complement (C3) or immunoconglutinin. Titers of lytic complement were reduced and amounts of soluble immune complex precipitated with polyethylene glycol 6000 were elevated. The active plasma may have affected the antibody producing cells by one or both of two mechanisms. Soluble antigen-antibody complexes could have interacted with Fc receptors of activated lymphocytes to alter their function. Alternatively, the complexes may have fixed complement and interacted with receptors for fixed C3 on the lymphocyte membrane. Such cells, being coated with the antigen for immunoconglutinin, could be altered by immunoconglutination. Inasmuch as the immune complexes in the active plasma were generated in vivo, it would seem unlikely that the plasma would contain significant amounts of complex that had not fixed complement. With immunoconglutinin present in the plasma, alteration of the cells by immunoconglutination seems a more likely possibility.

Dissociation of soluble antigen-antibody complexes by rheumatoid factor IgM.

Intact rheumatoid factor (RF)-active IgM dissociated soluble antigen-antibody complexes formed in the antigen excess zone, whereas trypsin-digested protein had less effect. The dissociation mechanism involved an interaction between the RF IgM and the Fc gamma of antibodies in the complexes. RF-active IgM had no demonstrable effect on antigen-antibody complexes when the antigen or the antibody had been immobilized. This was true irrespective of whether the experiments were performed in the antigen or in the antibody excess zone and despite binding between RF IgM and the immobilized proteins.

Studies on the molecular mechanisms of immune complexes triggering phagocytosis and respiratory burst in phagocytes

When guinea pig polymorphonuclear leukocytes and macrophages encounter immune complexes, they exhibit phagocytic activity and promote O-2 production. The triggering mechanisms of the complexes for inducing these activities were studied with various protease inhibitors. The sucrose density gradient fractionation of homogenized macrophages revealed that diisopropyl fluorophosphate (DFP) blocked the ingestion of soluble antigen-antibody complex, probably the formation of phagosomes since it caused a marked accumulation of the complex in a region between the plasma membrane and lysosomes. On the contrary, p-tosyl-L-lysine chloromethyl ketone accumulated the complex in the lysosomes by inhibiting the intralysosomal digestion of the complex. The enhancement of O-2 production from C3 b-zymosan-stimulated polymorphonuclear leukocytes also was inhibited by DFP. The assay of the isolated membrane fractions of the cells for NADPH oxidase demonstrated that DFP inhibited directly the activation process of the enzyme with C3 bzymosan. These results provide strong support to the involvement of a certain serine protease in the triggering mechanisms of immune complexes leading to phagocytosis as well as activation of NADPH oxidase.

8] Detection of Fc receptors

There are an almost bewildering array of assays one can use to detect Fc-receptors, but we have focused, for the most part, on relatively rapid and convenient methods--those that have become standard, e.g., EA rosetting procedures or uptake of soluble antigen-antibody complexes. For most types of Fc-receptor studies these methods are quite adequate. We have also tried to stress the newer methodology of detecting Fc receptors, i.e., the use of anti Fc-receptor antibodies, including monoclonal antibodies. The advent of monoclonal antibody technology, described in detail by Galfrè and Milstein, is changing the approach of many immunological procedures, and detection of Fc receptors is no exception. There are, it should be noted, many newer Fc-receptor methods, e.g., a solid-phase radioimmunoassay and a quantitative fluorometric assay, and methods such as these may be the most appropriate ones to choose in a particular experimental situation. In addition, we have not stressed "functional" Fc-receptor detecting assays, for example, antibody-dependent cell-mediated cytotoxicity (ADCC), which can be used to monitor the activity of Fc receptors in a whole cell population, rather than in single cells.

Study of antigenic structure and inhibition of activity of human growth hormone and chorionic somatomammotropin by monoclonal antibodies

Distinct antigenic determinants were identified on native molecules of human growth hormone (hGH) and chorionic somatomammotropin (hCS) on the basis of competitive inhibition assays with eight murine monoclonal antibodies. Effective competition for antigen binding within a pair of antibodies indicated overlapping combining site specificities whereas a lack of competition suggested binding to sterically distinct structural moieties. An antigenic determinant, specific for hGH was detected by antibodies QA68 and NA27. whilst another marginally hCS-cross-reactive site was bound by NA71. Two distinct determinants fully expressed by either hGH or hCS were bound by antibody pairs NA39/EB2 and EBI/EB3 respectively, whereas a single hCS-specific determinant was recognized by antibody EB4. An unexpected reciprocal cross-inhibition of soluble antigen-antibody complex binding was observed between antibodies reacting to distinct determinants, i.e. for NA27 towards NA39/EB2 and for NA71 towards EBI/EB3. These results were tentatively interpreted in terms of conformational changes of antigen when bound in soluble immune complexes, but an alternative explanation of steric hindrance cannot yet be excluded. The effect of monoclonal antibodies on the hormonal biological activity was investigated in a dose-response study of the hormone-dependent growth stimulation of NB2 lymphoma cells in tissue culture. Although all eight antibodies were specifically growth-inhibitory, major quantitative differences in their efficacies have been observed. At limiting hormone doses antibodies EB2/NA39 were most effective whereas QA68 and NA71 were the most potent at excess hormone input. Various mechanisms operating through inhibition of hormone binding and/or modulation of cell receptor-bound complexes have been considered.

The role of la antigens and Fc receptor-bearing T-cells in the inhibition of macrophage receptors for cytophilic antibody induced by soluble immune complexes

Soluble antigen-antibody complexes composed of 3 M KCl-extracted L1210 antigens and alloantibody to L1210 given to C3H mice caused immunosuppression in the mice. This was reflected in part by the inhibition of cytophilic antibody receptors on macrophages which could be used as a measure of the suppression. Thymocytes or splenic T cells from mice treated with immune complexes could adoptively transfer the suppression to normal syngeneic mice. These cells, which we have termed suppressor inducers, were found to be Ia positive: specifically, I-A +, I-J −. Thus, treatment of the inducers with anti-la or anti-I-A antibodies and complement in vitro abrogated their ability to transfer the suppression to normal mice. In contrast treatment with anti-I-J serum and complement had no effect. Through a similar approach, the cooperating (acceptor) T cells were found to be I-A +, I-J −. Pretreatment of mice with anti-Ia or anti-I-A serum before the administration of antigen-antibody complexes prevented the inhibition of macrophages. This was due at least in part to steric hindrance of adjacent Fc receptors on the FcR + T cells with which the complexes interacted. Early interaction of immune complexes with FcR + T cells was in fact demonstrated directly by the inability of the complexes to induce suppression when FcR + T cells were depleted. The thymocytes or splenic T cells from anti-Ia-pretreated mice failed to transfer the suppression to recipient mice. In contrast, treatment with either anti-Ia or anti-I-A after the immune complexes did not abrogate the generation of suppressor inducers. Treatment of normal recipient mice with anti-Ia serum in vivo before they received the suppressor inducer cells did not prevent cooperation between the two types of cells. By the same token, blocking of Ia antigens of the inducers in vitro with anti-Ia serum (without complement) also did not impair the cooperative interaction. These results indicate that antigen-antibody complexes generate I-A-positive, I-J-negative T-suppressor inducer cells from FcR + naive T cells. These in turn interact with Ia-positive (I-A + and I-J −) normal thymocytes or spleen T cells. This interaction most likely generates the ultimate suppressor T cells that suppress cytophilic antibody receptors on macrophages in vivo. However, the I-region determined antigens did not appear to be directly involved in the T-T interaction of suppressor inducer and acceptor cells.

Exclusive Binding of Immunoglobulin to Fcγ Receptors on Macrophages in 3-Methylcholanthrene- Induced Murine Tumors<xref ref-type="fn" rid="fn2">2</xref><xref ref-type="fn" rid="fn3">3</xref>

Immunogenic, chemically induced, murine tumors generally are infiltrated with large numbers of Fc gamma receptor (Fc gamma R)-positive cells, most of which are mononuclear phagocytes. This study demonstrates in inbred C3H/HeN mice that such immunogenic tumors contain large amounts of cell-bound immunoglobulin (Ig) and that, within the sensitivity limits of indirect immunofluorescence, the tumor-associated Ig is bound exclusively to host-derived Fc gamma R-positive cells. Depletion of Fc gamma R-positive cells from tumor cell suspensions removed all Ig-bearing cells. Specific competitive elution of the endogenous Ig with heterologous nonimmune IgG established that the endogenous Ig was bound through the Fc gamma R. Tumor-associated Fc gamma R-positive, Ig-bearing cells could be detected with soluble antigen-antibody complexes, and mononuclear phagocytes to which the exogenous complexes were bound expressed Fc gamma R at a level significantly above normal. The role of this macrophage-associated Ig in determining the level of interaction between host cells and the tumor remains to be determined.

Effects of protease inhibitors on ingestion and digestion of soluble antigen-antibody complexes by macrophages.

The inhibitory effects of diisopropyl fluorophosphate (DFP) and p-tosyl-L-lysine chloromethyl ketone (TLCK) on the ingestion and digestion of 125I-alpha-amylase [B alpha A, EC 3.2.1.1] complexed with antibody by guinea pig peritoneal macrophages were studied by sucrose density gradient fractionation. When macrophages were exposed to the complex at 37 degrees C and homogenized after washing, most of the trichloroacetic acid (TCA)-insoluble radioactivity was recovered in two fractions which behaved like the plasma membrane and lysosomes in the density gradient, and the TCA-soluble radioactivity was found in the fraction not moving into the gradient. The complex bound to Fc receptors on the cell surface, therefore, was shown to be internalized, transported to the lysosomes, and finally digested to amino acid catabolites. The inhibition with DFP caused an accumulation of the complex in a region between the plasma membrane and lysosomes. This distribution of radioactivity resembled that of cytochalasin B-inhibited macrophages. TLCK also inhibited the digestion of the complex, but the distribution of radioactivity revealed a marked accumulation of the complex in the region of lysosomes. These results provide strong support to the hypothesis that DFP blocks the ingestion of the complex, as in the case of cytochalasin B, whereas TLCK inhibits the intralysosomal proteolysis of the complex.

Interactions between lymphocyte membrane molecules. II. Characterization of an interaction between B lymphocyte surface IgD and Fc IgG receptors that differs from the surface IgM-Fc IgG receptor interaction.

The B lymphocyte surface membrane receptors IgD (sigD) and Fc IgG receptors (Fc gamma R) were evaluated for interactions by means of immunofluorescence. Ligand-(F(ab')2 anti-delta) induced capping of sIgD resulted in co-capping of Fc gamma R if the latter were occupied during the capping process by soluble antigen-antibody complexes (which themselves provided insufficient cross-linking to result in capping), but not if the Fc gamma R were occupied by monomeric IgG or unoccupied. Capping of Fc gamma R by highly cross-linked complexes did not cause co-capping of sIgD occupied by monomeric F(ab') anti-delta. The interaction between sIgD and Fc gamma R was specific in that cross-reactions between ligands were excluded and ligand-induced capping of sIgD did not cause co-capping of ligand-occupied sIgM or I-A antigens. The sIgD-Fc gamma R interaction occurred on only approximately 60% of B lymphocytes, and this B cell subpopulation did not correlate with other B cell subpopulations (CBA/N strain B cells and B cells bearing either large or small amounts of sIgD). The sIgD-Fc gamma R interaction differed from the sIgM-Fc gamma R interaction in that co-redistribution of the Fc gamma R was occupied by monomeric IgG and involved nearly all B lymphocytes. The qualitative and quantitative differences between the sIgD-Fc gamma R and sIgM-Fc gamma R interactions suggest a mechanism whereby the two antigen receptors could provide different signals to the B lymphocyte.