Cross-linked IgG Research Articles

Inhibition of murine fibrocyte differentiation by cross-linked IgG is dependent on FcγRI.

Monocyte-derived, fibroblast-like cells, called fibrocytes, participate in wound-healing and the formation of fibrotic lesions. Aggregated or cross-linked IgG are key effectors in infections, autoimmune diseases, anaphylaxis, and immunotherapy. Cells, including monocytes and fibrocytes, bind IgG using FcγRs, and aggregated or cross-linked IgG inhibits fibrocyte differentiation. Mice have four different FcγRs, and which of these, if any, mediate the cross-linked IgG effect on fibrocyte differentiation is unknown. We find that in mice, deletion of FcγRI or the common signaling protein FcRγ significantly reduces the ability of cross-linked IgG or IgG2a to inhibit fibrocyte differentiation. Cells from FcγRIIb/III/IV KO mice are still sensitive to cross-linked IgG, whereas cells from FcγRI/IIb/III/IV KO mice are insensitive to cross-linked IgG. These observations suggest that IgG-mediated inhibition of fibrocyte differentiation is mediated by FcγRs, with FcγRI mediating most of the signaling.

Improved serum-free culture conditions for spleen-derived murine fibrocytes

Both wound repair and fibrosing diseases involve circulating monocytes entering a tissue and differentiating into fibroblast-like cells called fibrocytes. Fibrocyte biology has been extensively studied in both humans and mice. However, current in vitro techniques to culture murine fibrocytes can take up to two weeks and can require multiple mice to obtain enough circulating monocytes for a single experiment. An alternative source of fibrocytes is the splenic reservoir of monocytes, where one can obtain significantly more cells compared to the peripheral blood. We found that in serum-free medium, fibrocytes differentiate from murine spleen cells within 5 days. To maximize fibrocyte yield, we found the optimal purification technique was to digest the spleen with a collagenase/DNase cocktail, pass the cells through a cell strainer, and lyse the red blood cells. We found that IL-13 and M-CSF significantly enhanced fibrocyte differentiation and that the optimal cell density to promote differentiation was 1.75 × 10 6 cells/ml. Serum amyloid P (SAP) and cross-linked IgG are two factors known to inhibit the differentiation of human monocytes into fibrocytes. We found that SAP and cross-linked IgG also inhibited the differentiation of murine spleen cells into fibrocytes. These results suggest that culturing murine spleen cells in serum-free medium is a rapid and efficient system to study factors that can affect fibrocyte differentiation.

C-reactive protein-mediated phagocytosis and phospholipase D signalling through the high-affinity receptor for immunoglobulin G (FcgammaRI).

C-reactive protein (CRP) is the prototypic acute-phase protein in man which performs innate immune functions. CRP-mediated phagocytosis may be indirect, through activation of complement and complement receptors, or direct, through receptors for the Fc portion of immunoglobulin G (IgG; FcgammaRs) or even a putative CRP-specific receptor. No strong evidence has been shown to indicate which receptors may be responsible for phagocytosis or signalling responses. Using BIAcore technology, we confirm that CRP binds directly to the extracellular portion of FcgammaRI with a threefold higher affinity than IgG (KD = 0.81 x 10-9 m). Binding is Ca2+ dependent and is inhibited by IgG1 but not by phosphorylcholine (PC). CRP opsonization (using CRP concentrations within the normal human serum range) of PC-conjugated sheep erythrocytes increased phagocytosis of these particles by COS-7 cells transfected with FcgammaRI-II chimaera or FcgammaRI/gamma-chain. Interferon-gamma-treated U937 cells, which signal through FcgammaRI to activate phospholipase D (PLD) in response to cross-linked IgG, were also activated by CRP without any requirement for further cross-linking. These studies indicate that CRP is capable of binding to and cross-linking FcgammaRI thereby resulting in PLD activation and increased phagocytosis. Uptake by FcgammaRI has been reported to promote various acquired immune responses suggesting that CRP could act in a similar way.

The receptor with high affinity for IgE on rat mast cells is a functional receptor for rat IgG2a

Rat mast cells express high-affinity receptors for IgE (Fcϵ RI) and low-affinity receptors for IgG (Fcγ R). In this study, the capacity of IgG to activate the rat basophilic leukemia (RBL-2H3) and rat peritoneal mast cells was investigated. Immune complexes formed with purified rat IgG and antigen as well as chemically cross-linked rat IgG induced histamine release from RBL-H3 cells. This stimulation was inhibited by pre-incubation of the cells with saturating concentrations of monomeric IgE. With chemically cross-linked rat IgG of each subclass, only IgG2a stimulated histamine release from RBL-2H3 cells and this release was also inhibited by prior saturation of the Fcϵ RI with monomeric IgE. Identical results were obtained with rat peritoneal mast cells. In binding experiments, IgE and cross-linked rat IgG2a bound to rat Fcϵ RI transfected into CHO cells. Monomeric rat IgG2a, cross-linked rat IgG1, IgG2b, IgG2c and rabbit IgG did not bind to Fcϵ RI. Stimulation of RBL-2H3 cells with aggregated IgG2a induced phosphorylation of tyrosines in the β and γ subunits of the Fcϵ RI. Thus, although RBL-2H3 and rat peritoneal mast cells have Fcγ R, the IgG-mediated stimulation of these cells for histamine release was by the Fcϵ RI. Altogether, these data demonstrate that the rat Fcϵ RI is a functional receptor with low affinity for rat IgG2a.

Extensive CD4 cross-linking inhibits T cell activation by anti-receptor antibody but not by antigen.

Anti-CD4 mAbs have been shown to inhibit T cell activation in a variety of ways. We have tested a panel of IgG and IgM anti-CD4 mAbs for their effects on the activation of a cloned T cell line by antigen presented by syngeneic accessory cells, by soluble anti-T cell receptor antibodies, and by mitogenic lectins. Both IgM and IgG mAbs to CD4 inhibit responses to mitogenic lectins. However, IgM, but not IgG, anti-CD4 antibodies inhibit T cell activation by mAbs specific for the TCR. This inhibitory activity appears to be due to the signaling effects of IgM mAbs, as cross-linked IgG antibodies mimic the behavior of the IgM anti-CD4 antibodies. Inhibition of T cell activation correlates with the ability of IgM and of cross-linked IgG anti-CD4 antibodies to induce tyrosine phosphorylation of the CD4-associated tyrosine kinase p56lck and an unknown substrate, pp32. Surprisingly, we find that the IgM anti-CD4 mAbs tested had no effect on the specific antigen recognition, despite their potent inhibitory effects on the other responses of the same cloned T cell line. These results suggest that multivalent CD4 interactions with ligands such as MHC class II molecules are inhibitory of T cell activation, but that this inhibition can be reversed when CD4 and the TCR bind the same ligand. We discuss the possible implications for positive intrathymic selection of these findings on signaling through CD4.

Reversible and irreversible cross-linking of immunoglobulin heavy chains through their carbohydrate residues

After periodate oxidation and incubation with a dihydrazide, cross-linking of the two heavy chains of immunoglobulins G from several species proceeds specifically through their oligosaccharides. We have used malonic acid dihydrazide, adipic acid dihydrazide and dithiodipropionic acid dihydrazide. The last compound is introduced in this work as a cleavable-carbohydrate-specific cross-linker. It was found that in rabbit and human immunoglobulins the degree of cross-linking was strongly dependent on the oxidation conditions but only very weakly dependent on the concentration and size of the dihydrazides. Papain cleavage of the cross-linked rabbit IgG indicated that the cross-linking occurred predominantly, if not exclusively, in the Fc region, probably through the two glycans linked to Asn-297 in the CH2 domain of each of the two heavy chains. The immunoglobulins from sheep, pig, goat and guinea pig show a comparable cross-linking pattern, indicating that the sugar chains from these immunoglobulins have a spatial structure closely related to that of rabbit and human IgG. When dithiodipropionic acid dihydrazide was used as the cross-linker, the cross-link could be cleaved by mercaptoethanol.

Immobilized albumin-immunoglobulin G for improved hemocompatibility of biopolymers.

Efforts to render blood contacting surfaces less thrombogenic have included chemical modification of the polymer, as well as surface modifications with proteins, anticoagulants, or antiplatelet agents. Studies reported from this laboratory in the past have shown that certain surfaces pretreated with albumin-Immunoglobulin G (alb-IgG) become relatively more hemocompatible (reduced platelet adhesion, minimal thrombus). However, absorbed proteins, including alb-IgG, desorb rapidly when exposed to circulating blood. Therefore, efforts were made to immobilize alb-IgG on glass or Biomer as follows: a 2 mg/ml solution of IgG was crosslinked with glutaraldehyde on the test surface. Crosslinked IgG was then treated with mercaptoethanol to reduce the disulfide bonds, followed by incubation with albumin previously reduced with mercaptoethanol under conditions that allowed reassociation of disulfide bridges between albumin and crosslinked IgG. Alb-IgG immobilized at the test surface following this method was found to remain at the interface when the surface was exposed to blood in vitro for up to 7 days under static conditions. Glass or Biomer surfaces with crosslinked Alb-IgG showed considerable reduction in the adhesion of 111In-platelets and adsorption of 125I-fibrinogen when test surfaces were analyzed in a flow chamber. Under identical experimental conditions, surfaces with crosslinked albumin alone were not effective in reducing the adhesion of platelets. Crosslinked IgG or immobilization of IgG on crosslinked albumin following the above methods rendered the surfaces more thrombogenic. It was noted, however, that crosslinked proteins on Biomer were dislodged rapidly in areas with turbulent flow.(ABSTRACT TRUNCATED AT 250 WORDS)

High avidity periventricular IgG-Fc receptor activity in human and rabbit brain

Periventricular tissues from human and rabbit brains were examined for receptors for the Fc domain of immunoglobulin G (Fc receptors). Fc receptors were demonstrated using antibody-coated erythrocytes (EIgG) and covalently cross-linked rabbit IgG. EIgG adhered specifically to nonneuronal periventricular glial areas and was inhibited by IgG and Fc fragments, but not by Fab fragments or albumin. EIgG adherence was 50% inhibited with 6.7 × 10 −9 M IgG. This is 1 1000 the amount of IgG required to produce 50% inhibition of EIgG adherence to arachnoid FcR. Studies with covalently cross-linked IgG demonstrated a linear relationship between ligand size and inhibition of EIgG adherence. Uncoated erythrocytes, IgM-coated erythrocytes, or F(ab′) 2-coated erythrocytes failed to bind to periventricular tissue. Many nonspecific esterase-positive cells were found in the area of periventricular EIgG adherence; this esterase activity was sensitive to fluoride. These results provide, for the first time, evidence of inherent IgG receptor activity in the periventricular tissues, and suggest that the periventricular tissues play a protective role in the removal of IgG or IgG-antigen complexes which have entered the cerebral spinal fluid.

Endocytosis of aggregated immunoglobulin G by rat basophilic leukemia cells; rate, extent, and effects on the endocytosis of immunoglobulin E.

Abstract Rat basophilic leukemia (RBL) cells have distinct receptors for IgE and IgG. We assessed the endocytosis of chemically and immunochemically cross-linked mouse-IgG and its influence on the simultaneous endocytosis of IgE. We found that at 37 degrees C, aggregates of IgG and IgE were endocytosed at about the same rate with one-half of the maximal endocytosis occurring in 5 to 13 min, and the efficiency of endocytosis for both ligands ranging from 40 to 70%. We also found that endocytosis of cross-linked IgE and IgG occurred simultaneously and neither ligand significantly affected the rate or extent of endocytosis of the other. The cells accumulated the cross-linked IgG, and then released it to the extracellular environment, at a rate (less than 3%/hr) slower than the released endocytosed IgE (greater than 10%/hr). Using an assay that discriminates between unbound and receptor-bound oligomeric IgG, we found that oligomeric IgG is endocytosed with its receptor, and that the bulk of the ligand remains bound to its receptor for greater than 120 min after endocytosis. The differences in the rate of release of endocytosed IgG vs IgE suggests that the intracellular fate or pathway of these two oligomeric ligands may differ.

The production of small IgG aggregates by glutaraldehyde cross-linking

Reaction conditions have been determined for the production of soluble IgG polymers in the size range 10 S to 30 S by covalent cross-linking with glutaraldehyde. This size range is comparable with that of the immune complexes which are frequently found in the circulation of patients with certain autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. The yield of IgG aggregates in this size range is far greater than has been reported for cross-linking by other bifunctional reagents or for aggregation by heating. Glutaraldehyde cross-linked IgG polymers are stable and biologically reactive. They can also be labelled with fluorescein and freeze-dried with minimal loss of integrity or reactivity.

Failure of covalently cross-linked human IgG myeloma subclass protein to release histamine from human leukocytes.

We have examined the ability of IgG subclass antibodies to release histamine from human leukocytes using covalently cross-linked oligomers of human myeloma proteins. Purified IgG1, G2, G3, G4, (or IgE) was incubated with dimethyl suberimidate to induce cross-linking. The resulting dimers, trimers, and higher molecular weight oligomers were isolated using gel filtration columns (Sephadex G200 and Ultrogel AcA 22) connected in tandem. None of the oligomers of IgG1, G2, G3, or G4 released histamine from leukocytes of donors whose basophils released histamine when challenged with IgE dimer. Furthermore, preincubation with subclass specific oligomers did not desensitize cells to challenge with IgE dimer or to anti-IgE. We conclude that, under our experimental conditions, oligomers of human IgG myeloma subclass antibodies do not trigger histamine release nor modulate IgE-mediated reactions.

Mechanism of binding of multivalent immune complexes to Fc receptors. 2. Kinetics of binding.

The kinetics of association and dissociation of affinity cross-linked IgG oligomers with Fc receptors on P388D1 cells have been studied at 0 degrees C. For dimers prepared with two different affinity cross-linking reagents (DIBADL and BDPE) and also trimers and a heavy oligomer fraction prepared with BDPE, monomeric IgG accelerates the rate of dissociation of bound oligomer from the cells. This observation is consistent with a binding mechanism in which monovalently, and multivalently bound species are in rapid equilibrium with one another on the cell surface and in which the rate of formation of the divalently bound species is faster than the dissociation of the monovalent species from the cells. As predicted, the rates of dissociation of BDPE cross-linked oligomers decrease with size, and dimers (cross-linked with DIBADL) which can self-aggregate on the cell surface dissociate more slowly than nonaggregating (BDPE) dimers. The association and dissociation of oligomers with P388D1 cells proceed by parallel fast and slow processes, even with cells that bind dimers with a single affinity. The origin of this heterogeneity is not known but could arise from a fraction of receptors in environments with limited accessibility to bulk solution. The fast component of the association reaction is second order, and the rate-limiting step of this process is the formation of the monovalently bound intermediate from solution-phase oligomer.

Fc (IgG) receptors on rat basophilic leukemia cells.

Receptors for IgG and IgE on rat basophilic leukemia cells (the 2H3 subline of RBL-IV) were detected by their ability bind IgE and IgG in fluorescence or radioactive assays. The 2H3 cells have approximately two-thirds as many receptors for IgG as for IgE. The IgE receptors bind monomeric IgE with high affinity and IgE binding is uninhibited by high concentrations of monomeric or oligomeric IgG. In contrast, the IgG receptors bind both (rabbit and rat) IgG and (rat) IgE. The affinity of IgG receptors for monomeric IgG and IgE is much lower than that of the IgE receptor for its ligand; binding of radiolabeled IgG or IgE to IgG receptors can be detected only with cross-linked oligomers. Although both receptors are sensitive to tryptic digestion than IgE receptors. Dual laser flow microfluorometric studies revealed that the numbers of IgG and IgE receptors per cell varied considerably among 2H3 cells and that the distributions of the 2 types of receptors were independent of each other. 2H3 cells released histamine through an IgE-mediated system, but cross-linked IgG, even in saturating amounts, did not elicit histamine release, nor have any effect upon IgE-induced release. Moreover, 2H3 cells would not mediate ADCC of IgG-coated chicken red blood cells.

Human Leukocyte Fc (IgG) Receptors: Quantitation and Affinity with Radiolabeled Affinity Cross-Linked Rabbit IgG

Quantitative studies have been made of Fc receptors on human leukocytes derived from peripheral blood, thymus, tonsil, and spleen. The relative affinities and average numbers of receptors per cell were determined by measuring the binding of 125I-labeled, affinity cross-linked trimers of rabbit IgG to various populations of cells. In parallel, the sizes of receptor-bearing populations were determined by fluorescence microscopy. Fc receptors could be detected on leukocytes from peripheral blood and spleen, but not from tonsil or thymus. In the peripheral blood, the highest density of receptors was found on polymorphonuclear leukocytes; a subpopulation of lymphocytes had somewhat fewer receptors per cell, and circulating monocytes had the lowest receptor density. Among splenocytes, most of the receptors were found on myeloid cells and monocytes. In all populations, the affinity of Fc receptors for the trimer was about the same. At 0 degrees C the average value for the association constant was 5 x 10(7) M-1.

Serum anti-immunoglobulins in psoriatic arthritis as compared with rheumatoid arthritis.

AbstractSerum anti‐immunoglobulins of IgG, IgA and IgM types were measured in patients with rheumatoid arthritis, patients with psoriatic arthritis, and normal subjects by absorption on cross‐linked IgG, with subsequent elution. Relatively elevated values of all three types of serum anti‐immunoglobulins were found in seropositive rheumatoids, and elevated values of anti‐immunoglobulins of the IgG type were found in seronegative rheumatoids. Values in patients with psoriatic arthritis fell within the normal range.