Model Immune Complexes Research Articles

Generation, Characterization, and Quantitative Bioanalysis of Drug/Anti-drug Antibody Immune Complexes to Facilitate Dedicated In Vivo Studies

PurposeImmunogenicity against biotherapeutics can lead to the formation of drug/anti-drug-antibody (ADA) immune complexes (ICs) with potential impact on safety and drug pharmacokinetics (PK). This work aimed to generate defined drug/ADA ICs, characterized by quantitative (bio) analytical methods for dedicated determination of IC sizes and IC profile changes in serum to facilitate future in vivo studies.MethodsDefined ICs were generated and extensively characterized with chromatographic, biophysical and imaging methods. Quantification of drug fully complexed with ADAs (drug in ICs) was performed with an acid dissociation ELISA. Sequential coupling of SEC and ELISA enabled the reconstruction of IC patterns and thus analysis of IC species in serum.ResultsCharacterization of generated ICs identified cyclic dimers, tetramers, hexamers, and larger ICs of drug and ADA as main IC species. The developed acid dissociation ELISA enabled a total quantification of drug fully complexed with ADAs. Multiplexing of SEC and ELISA allowed unbiased reconstruction of IC oligomeric states in serum.ConclusionsThe developed in vitro IC model system has been properly characterized by biophysical and bioanalytical methods. The specificity of the developed methods enable discrimination between different oligomeric states of ICs and can be bench marking for future in vivo studies with ICs.

CD32 Ligation Promotes the Activation of CD4+ T Cells.

Low affinity receptors for the Fc portion of IgG (FcγRs) represent a critical link between innate and adaptive immunity. Immune complexes (ICs) are the natural ligands for low affinity FcγRs, and high levels of ICs are usually detected in both, chronic viral infections and autoimmune diseases. The expression and function of FcγRs in myeloid cells, NK cells and B cells have been well characterized. By contrast, there are controversial reports about the expression and function of FcγRs in T cells. Here, we demonstrated that ~2% of resting CD4+ T cells express cell surface FcγRII (CD32). Analysis of CD32 expression in permeabilized cells revealed an increased proportion of CD4+CD32+ T cells (~9%), indicating that CD4+ T cells store a CD32 cytoplasmic pool. Activation of CD4+ T cells markedly increased the expression of CD32 either at the cell surface or intracellularly. Analysis of CD32 mRNA transcripts in activated CD4+ T cells revealed the presence of both, the stimulatory FcγRIIa (CD32a) and the inhibitory FcγRIIb (CD32b) isoforms of CD32, being the CD32a:CD32b mRNA ratio ~5:1. Consistent with this finding, we found not only that CD4+ T cells bind aggregated IgG, used as an IC model, but also that CD32 ligation by specific mAb induced a strong calcium transient in CD4+ T cells. Moreover, we found that pretreatment of CD4+ T cells with immobilized IgG as well as cross-linking of CD32 by specific antibodies increased both, the proliferative response of CD4+ T cells and the release of a wide pattern of cytokines (IL-2, IL-5, IL-10, IL-17, IFN-γ, and TNF-α) triggered by either PHA or anti-CD3 mAb. Collectively, our results indicate that ligation of CD32 promotes the activation of CD4+ T cells. These findings suggest that ICs might contribute to the perpetuation of chronic inflammatory responses by virtue of its ability to directly interact with CD4+ T cells through CD32a, promoting the activation of T cells into different inflammatory profiles.

Immune complexes that contain HIV antigens activate peripheral blood T cells.

Uninfected donor T cells were treated in vitro by model immune complexes that contained either HIV or hepatitis C virus (HCV) antigens. Unlike HCV antigen-containing complexes, the immune complexes that contained HIV antigens have been shown to activate peripheral blood T cells of uninfected donors under in vitro conditions. Both the antiviral antibodies and HIV antigen were involved in the activation process. The unique properties of the immune complexes formed by HIV antigens and antiviral antibodies are believed to result from the virus-specific antibody properties and molecular conformation of the antigen-antibody complex.

Effect of complement on sizes of model immune complexes.

Effect of complement on sizes of model immune complexes.

Effect of medium pH and antibody Fc fragment on the size of model immune complexes

Effect of medium pH and antibody Fc fragment on the size of model immune complexes

Alkylhydroxybenzenes modify immune complex size

Alkylhydroxybenzenes (AHBs) are a large group ofbiologically active substances of microbial and plantorigin that are capable of nonspecific interaction withdifferent biopolymers [1–7]. AHB homologues differin the length of the alkyl radical, which determines thedegree of hydrophobicity of the molecules and theirreactivity [1, 2]. The functioning of AHBs as “chemical chaperones” [3, 4] determines the development ofeffects in different systems. For example, changes inthe presence of micromolar concentrations of AHBswere shown for the catalytic activity of some enzymes[3, 4], the physicochemical properties of DNA [5],and the antigenbinding ability and functional activityof antibodies [6, 7]. It was also shown that the molecular weight and sizes of proteins modified with AHBsaltered [3]. However, information about the effect ofAHB on the dimensional characteristics of immunecomplexes is missing.MATERIALS AND METHODSSuspensions of model immune complexes wereformed from purified tetanus toxoid (400 EU/mL) andhuman antitetanus γglobulin containing not less than95% IgG of the total concentration (10%) of protein inthe sample. The study was performed at antigen/antibody ratios of 1 : 32/1 : 8, 1 : 16/1 : 2, 1 : 32/1 : 2, and1 : 64/1 : 2. We used chemical analogues of microbialAHBs (99.9% purity) differing by the length of thealkyl radical: C

Contribution of Chondroitin Sulfate A to the Binding of Complement Proteins to Activated Platelets

BackgroundExposure of chondroitin sulfate A (CS-A) on the surface of activated platelets is well established. The aim of the present study was to investigate to what extent CS-A contributes to the binding of the complement recognition molecule C1q and the complement regulators C1 inhibitor (C1INH), C4b-binding protein (C4BP), and factor H to platelets.Principal FindingsHuman blood serum was passed over Sepharose conjugated with CS-A, and CS-A-specific binding proteins were identified by Western blotting and mass spectrometric analysis. C1q was shown to be the main protein that specifically bound to CS-A, but C4BP and factor H were also shown to interact. Binding of C1INH was dependent of the presence of C1q and then not bound to CS-A from C1q-depleted serum. The specific interactions observed of these proteins with CS-A were subsequently confirmed by surface plasmon resonance analysis using purified proteins. Importantly, C1q, C4BP, and factor H were also shown to bind to activated platelets and this interaction was inhibited by a CS-A-specific monoclonal antibody, thereby linking the binding of C1q, C4BP, and factor H to exposure of CS-A on activated platelets. CS-A-bound C1q was also shown to amplify the binding of model immune complexes to both microtiter plate-bound CS-A and to activated platelets.ConclusionsThis study supports the concept that CS-A contributes to the binding of C1q, C4BP, and factor H to platelets, thereby adding CS-A to the previously reported binding sites for these proteins on the platelet surface. CS-A-bound C1q also seems to amplify the binding of immune complexes to activated platelets, suggesting a role for this molecule in immune complex diseases.

Surface‐bound anti–type II collagen–containing immune complexes induce production of tumor necrosis factor α, interleukin‐1β, and interleukin‐8 from peripheral blood monocytes via fcγ receptor IIA: A potential pathophysiologic mechanism for humoral anti–type II collagen immunity in arthritis

Type II collagen (CII) is a major component of hyaline cartilage, and antibodies against CII are found in a subgroup of patients with rheumatoid arthritis. We undertook this study to investigate whether and how antibodies directed against CII can form solid-phase immune complexes (ICs) with cytokine-inducing properties in a model theoretically resembling the situation in the inflamed joint, in which CII is exposed for interaction with anti-CII antibodies during periods of inflammation. Sixty-five arthritis patients with varying levels of anti-native CII antibodies and 10 healthy controls were evaluated concerning anti-CII and cytokines induced in a solid-phase IC model. Monocytes were either depleted or enriched to define responder cells. Antibodies blocking Fc gamma receptors (Fc gammaR) were used to define the responsible T cell surface receptors. ICs containing anti-CII from arthritis patients induced the production of tumor necrosis factor alpha (TNFalpha), interleukin-1beta (IL-1beta), and IL-8. We found a close correlation between enzyme-linked immunosorbent assay optical density values and induction of TNFalpha (r = 0.862, P < 0.0001), IL-1beta (r = 0.839, P < 0.0001), and IL-8 (r = 0.547, P < 0.0001). The anti-CII-containing IC density threshold needed for cytokine induction differed among peripheral blood mononuclear cell donors. Anti-CII-containing IC-induced cytokine production was almost totally abolished (>99%) after monocyte depletion, and receptor blocking studies showed significant decreases in the production of TNFalpha, IL-1beta, and IL-8 after blocking Fc gammaRIIa, but not after blocking Fc gammaRIII. These findings represent a possible mechanism for perpetuation of joint inflammation in the subgroup of arthritis patients with high levels of anti-CII. Blockade of Fc gammaRIIa and suppression of synovial macrophages are conceivable treatment options in such patients.

The levels of immune complexes in the blood sera of calves in the neonatal period and in adult cattle

In this work immune complexes (IC) in the sera of healthy calves from birth to four months of age, heifers and cows were investigated. Polyethylen glycol (PEG) precipitation assay was applied after previous standardization. Optical density (OD) of redissolved precipitates, formed after adding PEG to serum samples, was a measure of IC level. The upper-limit values for normal IC levels were established using isolated cattle IgG in different amounts and aggregated by heat as an IC model in vitro, in the same assay. In newborn calves before colostrum intake, OD of 0.047±0.024 was registered in redissolved PEG precipitates, far below the values obtained for native monomeric IgG. Therefore, these values can not be considered to refer to immune complexes. After colostrum intake, OD values for normal IC levels were higher, with significant interindividual differences: in calves at 6 24 and 48 hours after birth the mean OD values of solubilized PEG precipitates were 0.069±0.025, 0.148±0.079, and 0.136±0.062 respectively. In the sera of calves 10 days after birth mean OD value was slightly lower 0.089±0.053, whereas in the serum of 1, 2, 3 and 4 month old calves normal levels of IC rose to 0.165±0.067, 0.157±0.080, 0,231±0,124 and 0.261±0.092 respectively. OD values for IC levels for adult animals were found to be 0,456±0,150 for heifers, and 0,183±0,031 for cows. Together, the obtained data may be included in a reference values system for normal levels of IC at different ages, in relation to which the IC levels under pathological conditions could be estimated.

Analyses of the in vivo trafficking of stoichiometric doses of an anti-complement receptor 1/2 monoclonal antibody infused intravenously in mice.

Complement plays a critical role in the immune response by opsonizing immune complexes (IC) and thymus-independent type 2 Ags with C3 breakdown product C3dg, a CR2-specific ligand. We used a C3dg-opsonized IC model, anti-CR1/2 mAb 7G6, to investigate how such substrates are processed. We used RIA, whole body imaging, flow cytometry, and fluorescence immunohistochemistry to examine the disposition of 0.1- to 2-microg quantities of mAb 7G6 infused i.v. into BALB/c mice. The mAb is rapidly taken up by the spleen and binds preferentially to marginal zone (MZ) B cells; within 24 h, the MZ B cells relocate and transfer mAb 7G6 to follicular dendritic cells (FDC). Transfer occurs coincident with loss of the extracellular portion of MZ B cell CR2, suggesting that the process may be mediated by proteolysis of CR2. Intravenous infusion of an FDC-specific mAb does not induce comparable splenic localization or cellular reorganization, emphasizing the importance of MZ B cells in intrasplenic trafficking of bound substrates. We propose the following mechanism: binding of C3dg-opsonized IC to noncognate MZ B cells promotes migration of these cells to the white pulp, followed by CR2 proteolysis, which allows transfer of the opsonized IC to FDC, thus facilitating presentation of intact Ags to cognate B cells.

The C(H)1 domain of IgG is not essential for C3 covalent binding: importance of the other constant domains as targets for C3.

The covalent binding of C3 to antigen-antibody complexes [immune complexes (IC)] plays a pivotal role in the elimination of antigens. C3 prevents the formation of large IC lattices promoting their solubilization. Subsequently, bound C3 fragments determine the efficacy of antigen presentation, and the generation of antibody responses and immunological memory. C3 binding to IgG-IC generates IgG-C3b-C3b complexes which are detected by SDS-PAGE as two major bands: C3alpha65-heavy chain and C3alpha65-C3alpha43 covalent complexes. Using human heat-aggregated IgG1 as a model of IC, a C3b binding site was localized only in the Cgamma1 domain. However, with true IC of ovalbumin and rabbit IgG anti-ovalbumin, C3b binds to both the Fab and Fc regions of IgG. To study the binding of C3b to the different domains of IgG and particularly to evaluate the involvement of the Cgamma1 domain, we have constructed recombinant single-chain antibodies without Cgamma1, which have the structure: V(H)-linker-V(L)-hinge-Cgamma2-Cgamma3 (scAb). The variable domains were from a mouse mAb anti-HSA and the constant region (hinge-C(H)2-C(H)3) from human IgG1 or rabbit IgG. C3 binds very efficiently to IC formed with human (h-scAb) or rabbit (r-scAb) recombinant antibodies (scAb-HSA) and generates also two bands on SDS-PAGE (C3alpha65-scAb and C3alpha65-C3alpha43), which are the counterparts of those of the complete antibody. In addition, IC formed with scAb activate the alternative pathway to a similar extent as IC of the entire IgG. These data indicate that the Cgamma1 domain is a dispensable region for C3b binding and that the remaining constant domains are as efficient as Cgamma1 in C3b binding. Overall these results support the view that C3 does not specifically recognize a unique site in the Cgamma1 domain. Rather it seems to be able to attach along the antibody molecule. Probably this implies an advantage for effective processing of C3b-IC and elimination of antigens in vivo.

Characterization of Elevated Neutrophil-Associated IgG in Various Autoimmune Disorders: Not Anti-Neutrophil Autoantibodies, But Possibly Immune Complexes, Bind to Neutrophils

Neutropenia is frequently observed in a variety of autoimmune disorders. As the mechanism of neutropenia in these disorders, the destruction of neutrophils by anti-neutrophil autoantibodies has been believed since elevated levels of neutrophil-associated IgG (NAIgG) have been described. However, no data exists to characterize the nature of NAIgG and show NAIgG is an anti-neutrophil autoantibodies. We investigated whether the elevated NAIgG in these patients consists of anti-neutrophil autoantibodies. The NAIgGs of 91 patients with autoimmune disorders including 50 patients with idiopathic thrombocytopenic purpura, 13 patients with systemic lupus erythematosus, 11 patients with Hashimoto's thyroiditis and 10 patients with Graves' disease were analyzed. The level of NAIgG was high in 36 of 91 patients. Elution studies were performed to determine whether NAIgG has a nature of autoantibodies. In model experiments, the ether elu-ate from neutrophils sensitized with neutrophil-specific alloantibody (anti-NA2) reacted with donor neutrophils, whereas the eluates from those with model immune complexes (ICs) failed. These data indicated that the ether elution technique is useful to determine whether NAIgG consists of anti-neutrophil autoantibodies. The NAIgG on patient's neutrophils was eluted with ether and the reactivity of the eluate with normal neutrophils was investigated. The eluates from 34 of 36 patients with various autoimmune disorders with elevated NAIgG level failed to react with donor neutrophils. These data indicated that the elevated NAIgG in the majority of these patients did not consist of anti-neutrophil autoantibodies, but possibly of ICs.

Binding of model soluble immune complexes to modified C-reactive protein.

Abstract We have identified a binding interaction between a modified form of C-reactive protein (mCRP) and model immune complexes. We have characterized these interactions in terms of their relative affinity, specificity, pH dependence, and recognition site on mCRP. First, binding isotherms were generated for the reaction of immobilized mCRP with heat-aggregated IgG (HAG) which gave an estimate for the value of the dissociation constant, Kd, of 1.6 nM. Second, competitive binding assays were performed using immobilized HAG to determine the relative affinity (IC50) for the interaction between HAG or monomeric IgG and mCRP in the fluid phase. While the binding of HAG to mCRP occurred with high affinity (IC50=0.72 nM), the binding of monomeric IgG to mCRP occurred with &amp;gt;2000-fold lower affinity (IC50&amp;gt;1.66 muM). Third, immune complex binding to immobilized mCRP was studied using defined ratios of horseradish peroxidase and rabbit anti-peroxidase Ab. The binding of these complexes to mCRP was strongly pH dependent, with a maximum at pH 5.5. At this optimal pH, preformed peroxidase-antiperoxidase immune complexes (approximately 2:3 Ab:Ag molar ratio) were shown to bind immobilized mCRP with a Kd of 111 nM. Fourth, using mCRP-specific mAbs, HAG and peroxidase- antiperoxidase binding sites were localized to CRP sequences to 130 to 138 and 200 to 206. Since inflammatory processes often cause microenvironments to become acidic, and since mCRP selectively binds immune complexes at acidic pH, we propose that mCRP mediates the specific binding of immune complexes in vivo and potentiates effector reactions for immune complex removal.

Influence of C1q on the Interaction of Model Immune Complexes with Human Platelets

Influence of C1q on the Interaction of Model Immune Complexes with Human Platelets

Purification of immune complexes clearance enhancing polysaccharide from the leaves of panax ginseng, and its biological activities

Water and alkaline-soluble crude polysaccharides, which were prepared from the roots or leaves of Panax ginseng C. A. Meyer, were tested for the enhancing activity of immune complexes binding to macrophages. Binding of glucose oxidase-anti-glucose oxidase complexes (GAG), a model immune complexes, to macrophages was enhanced by the treatment with acidic polysaccharides (GL-2 and GR-3) from the leaves and the roots, respectively. The most active polysaccharide fraction, GL-2, was purified and the active polysaccharide, GL-4IIb2, was obtained. GL-4IIb2 was an acidic polysaccharide containing 33 % uronic acid. GAG binding to macrophages by GL-4IIb2 increased in a dose dependent fashion. When mice were given GL-4IIb2 intraperitoneally, the immune complexes clearance from circulation was also significantly enhanced compared with those of the control in a dose dependent fashion. Scatchard analysis indicated enhanced expression of Fc receptor (FcR) on the surface by the action of GL-4IIb2. The enhancement of GAG binding by GL-4IIb2 was inhibited by the presence of actinomycin D or cycloheximide. The GAG binding enhancing activity of GL-4IIb2 was reduced by periodate oxidation but not by Pronase® digestion of GL-4IIb2. These results suggest that the carbohydrate moiety of GL-4IIb2 activated the mononuclear phagocytic system in vivo through an increase of FcRs expression mediated by de novo synthesis of the receptor protein, and resulted in enhanced immune complexes clearance.

Platelet Activation and Binding of Complement Components to Platelets Induced by Immune Complexes

Clinical disorders such as malignant diseases, infectious diseases or autoimmune diseases are associated with circulating immune complexes. These immune complexes can activate the complement system in the blood or interact with complement or Fc receptors on the surface of cells. Complement activation may cause cytolysis and the immune complex interaction with receptors may cause activation of cells. We have used flow cytometry and labelled chicken antibodies to study the in vitro effects of model immune complexes on platelets and show that such immune complexes activate platelets and deposit Clq, C4 and C5 on them. Either low levels or no C3 could be detected on the platelets by flow cytometry. The immune complexes also induced formation of microparticles from purified platelets. Flow cytometry might become a useful tool in estimation of risk of thrombosis or thrombocytopenia in patients with autoimmune disease. Chicken antibodies are superior to mammalian antibodies for the measurement of platelet bound plasma proteins as they do not induce complement activation or platelet activation.

An Immune Complex Selective Affinity Column Utilizing Site-Specific Attachment of Bovine Conglutinin

A simple method to isolate immune complexes by column chromatography is described. The immune complex affinity column was constructed by the site-specific attachment of bovine conglutinin to agarose. Covalent attachment of conglutinin to agarose was achieved via hydrazone chemistry, which reacts aidehydes of oxidized oligosaccharides in the collagenous domain of conglutinin with hydrazide functional groups in the solid support. The constructed conglutinin affinity column captured complement-fixed model immune complexes of heat-aggregated human IgG and more classical complexes of chicken ovalbumin-antichicken ovalbumin. Neither the nonfixed immune complexes nor their individual components were retained by the column. The captured material was specifically eluted with EDTA without the use of low pH. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot analysis, and protein sequence analysis of the eluates revealed the presence of the expected individual components, verifying that both antibody and antigen used to prepare the soluble immune complexes wore recovered from the conglutinin column. The advantages of this approach over traditional methods of immune complex isolation and characterization are discussed.

Galactose-containing epitopes on the surface of IgG model immune complexes are accessible for specific binding with the high molecular weight ligand, ricinus agglutinin, in solution—light-scattering studies

Immunoglobulin G (IgG) molecules contain covalently linked carbohydrate chains with galactose residues in their branched “antennae”. The ability of galactose-containing epitopes on the surface of IgG model immune complexes (IC) to interact with a high mol. wt ligand in solution has been elucidated. Different types of IgG model IC with pre-detennined molecular mass were mixed with Ricinus Agglutinin (RC1), which is known to bind specifically to galactose-containing oligosaccharides. The relative light-scattering increases (Δ I) in the reaction mixture were measured as a function of time. The galactose-associated epitopes of the IgG model IC were accessible for binding with RC1. The rate of the interaction between IgG model IC and RC1 was dependent on the molecular mass of the complexes; the larger the model IC molecular mass, the faster the rate of interaction. The binding of RC1 to IgG model IC was highly specific because it was completely abolished in the presence of lactose. The galactose-containing epitopes of monomeric IgG were also able to interact with RC1 but the kinetics of the interaction was much slower. We suggest than an increase in the density of the epitopes on the surface of the model IC, by close attachment of the IgG molecules, mainly determines the ability of galactose-containing epitopes to be recognized by RC1. The data presented support the importance of IgG glycans in recognition events of IgG by biologically active molecules.

A Functionally Active Complement System Is Present in Uterine Secretion of the Mouse Prior to Implantation

Sephadex beads were placed carefully in the uterus on days 2 and 3 and left for 6 to 8 h to absorb uterine secretion. The beads were then removed with volatile silicon oil and mounted on small pieces of nitrocellulose paper. Immuno-staining of these bead blots showed they contained the complement components C1q, C3, C4, and C5. We demonstrated that complement component C3 in the uterine secretion could be activated and deposited on model immune complexes, and also that antibody-coated erythrocytes were lysed in utero, that is, a membrane attack complex was produced. Thus, the mouse uterine secretion at the preimplantation stage contains a functionally active complement system.

A method to differentiate between anti-C1q antibodies and C1q-binding immune complexes using collagenase-digested solid phase C1q

A method for the detection of circulating immune complexes in the presence of autoantibodies to C1q is described. Solid phase C1q-digestion with bacterial collagenase results in the elimination of the collagen-like region of C1q. Binding of model immune complexes to this modified solid phase C1q is practically unaltered, while reactivity of anti-C1q antibodies is abolished by this procedure. In conjunction with an ELISA using the collagen-like region of C1q as antigen this modified C1q solid phase assay may be used to determine immune complexes and anti-C1q antibodies in the sera of patients with autoimmune rheumatic diseases.