Reactivity of bacterial Fc receptors with bovine immunoglobulins: implications and limitations for quantitative immunochemistry

Summary The interaction of bovine immunoglobulins with staphylococcal Protein A and a group C streptococcal bacterial Fc receptor (FcRc) were compared. The isolated group C streptococcal receptor was reactive with both bovine IgG₁ and IgG₂. The reactivity of the streptococcal FcRc with IgG₂ was approximately 40 fold greater than that observed with IgG₁. By contrast, protein A reacted only poorly with bovine IgG₂ and no detectable reactivity was observed with IgG₁. A two stage competitive binding assay to measure bovine IgG in serum and secretions using ¹²⁵I-labeled protein A as tracer was developed. This assay was found to be sensitive and reproducible and was used to measure serum IgG levels in cattle of differing ages and breeds.     

The interaction of macrophage receptors with bacterial ligands

Innate immune receptors play a key role in the early recognition of invading bacterial pathogens and initiate the crucial innate immune response. The diverse macrophage receptors recognise Gram-positive and Gram-negative bacteria via conserved structures on the bacterial surface and facilitate phagocytosis and/or signalling, providing the trigger for the adaptive immune response. These receptors include scavenger receptors, C-type lectins, integrins, Toll-like receptors and siglecs. The bacterial ligands generally recognised by these receptors range from lipopolysaccharides on Gram-negative bacteria to peptidoglycan and lipoteichoic acid on Gram-positive bacteria. However, emerging evidence indicates that bacterial proteins are also important ligands; for example, surface proteins from Neisseria meningitidis have been shown to be ligands for class A scavenger receptors. In addition, a group of cytosolic receptors, the NBS-LRR proteins, have been implicated in recognition of bacterial breakdown products. It is becoming increasingly apparent that macrophage receptors can act in conjunction with one another to deliver an appropriate response.     

Fc receptors and surface immunoglobulins in cells of hairy cell leukemia]

Using 125I-labelled aggregated IgG in a quantitative assay a strong expression of Fc-receptors was found on the leukemic cells of a patient with hairy cell leukemia. The Fc-receptor activity on these cells was much higher than that on monocytes and B-lymphocytes from normal blood. Surface immunoglobulins were detected by radioautography using radioactively labelled (Fab')2-fragments of monospecific antibodies directed against immunoglobulin heavy chains. Prior to radioautography the cells were stained for the tartrate resistant acid phosphatase. It was found that all cells containing this enzyme bore sigma-chains on their surface. On more than 90% of these cells a simultaneous expression of mu-chains was detected. gamma-Chains could only be demonstrated on cells which were negative for the tartrate resistant acid phosphatase; part of these cells, however, were hairy cells by morphological criteria.     

The interaction of murine IgG subclass proteins with human monocyte Fc receptors

The use of murine monoclonal antibodies in the immunotherapy of human disease has prompted interest in the interactions of murine IgG with Fc receptors (FcR) expressed on human effector cells. We examined the heterocytophilic interactions between monomeric murine IgG subclass proteins and the FcR expressed on human monocytic cells (peripheral blood monocytes and interferon (IFN)-gamma-induced U937 cells). All four murine IgG2a antibodies and both murine IgG3 antibodies that were tested bound to human monocyte FcR with high affinity (10(8) to 10(9) M-1). By contrast, the affinities of four murine IgG1 and four IgG2b monomers were 100-fold to 1000-fold lower than the affinity of the human IgG1-FcR interaction. A 68,000 to 72,000 dalton protein was isolated by affinity chromatography from blood monocytes and from IFN-gamma-induced U937 cells on murine IgG2a, IgG3, and human IgG immunoadsorbents. In binding assays with IFN-stimulated U937 cells, murine IgG2a and IgG3 antibodies showed complete cross-blocking with a human IgG1 myeloma protein, indicating that murine and human IgG interact with the same population of Fc-binding proteins. No evidence for heterogeneity of cross-reactive FcR was observed. The ability of murine IgG2a and IgG3 monomers to compete with human IgG1 monomers for binding to human monocyte FcR suggests the potential usefulness of antibodies of these isotypes in the immunotherapy of diseases in which monocyte- or macrophage-mediated, antibody-dependent cellular cytotoxicity may play a role in the modification or remission of disease.     

Plasma cells and Fc receptors in human adipose tissue--lipogenic and anti-inflammatory effects of immunoglobulins on adipocytes

We have previously reported high immunoglobulin expression in human omental adipose tissue. The aim of this work was to investigate plasma cell density and Fc receptor (FcR) expression in human adipose tissue depots and in vitro effects of immunoglobulins on adipocyte function. Plasma cell density was higher in the visceral compared to the subcutaneous depot (10.0+/-1.56% and 5.2+/-0.98%, respectively, n=20, p<0.05). Microarray analysis revealed expression of four FcR genes in adipose tissue; FCGR2A, FCGR2B, FCER1G, and FCGRT. FCGR2A was highly expressed in adipocytes in both depots and this was verified by immunohistochemistry. Expression of IL-1beta and IL-6 was markedly reduced in adipocytes after incubation with the Fc moiety of immunoglobulin G (Fc) (p<0.01). Furthermore, Fc stimulated adipocyte lipogenesis as potently as insulin (p<0.05), but did not influence lipolysis. In conclusion, immunoglobulins produced by plasma cells in human adipose tissue could influence adipocyte metabolism and cytokine production.     

An immunoblotting technique for the detection of bound and secreted bacterial Fc receptors

A rapid semiquantitative procedure that enables bacteria to be screened for surface or secreted receptors for the Fc region of human IgG is described. Surface Fc receptors were detected by direct transfer of bacterial colonies to nitrocellulose by electroblotting and then probing with ¹²⁵I-labeled human IgG in the presence of a two fold molar excess of unlabeled F(ab′)₂fragments. The blots were exposed to X-ray film and the intensity of the resulting autoradiograph was a measure of surface Fc receptors expression. This procedure reliably distinguished Staphylococcus aureus strains which expressed different levels of surface Fc receptors. When applied to the study of group A streptococci, a number of Fc receptor-positive strains were identified. Unlike the homogeneous Fc receptor expression on individual colonies of the staphylococcal strains, a wide variation in the level of Fc receptor expression was observed within a given streptococcal strain. Group A streptococcal substrains which expressed high and low levels of surface Fc receptors could be isolated from replica plates.Secreted Fc receptors were measured by a simple modification of the blotting procedure in which the nitrocellulose was placed on the opposite side of the agar from the bacterial colonies. Secreted Fc receptors was electroblotted through the agar onto nitrocellulose and probed as described above. This approach readily detected nanogram quantities of secreted type I Fc receptor (protein A) from the Staphylococcus aureus Cowan strain. None of the group A streptococcal strains tested were found to secrete detectable quantities of Fc receptors.     

A common theme in interaction of bacterial immunoglobulin-binding proteins with immunoglobulins illustrated in the equine system

The M protein of Streptococcus equi subsp. equi known as fibrinogen-binding protein (FgBP) is a cell wall-associated protein with antiphagocytic activity that binds IgG. Recombinant versions of the seven equine IgG subclasses were used to investigate the subclass specificity of FgBP. FgBP bound predominantly to equine IgG4 and IgG7, with little or no binding to the other subclasses. Competitive binding experiments revealed that FgBP could inhibit the binding of staphylococcal protein A and streptococcal protein G to both IgG4 and IgG7, implicating the Fc interdomain region in binding to FgBP. To identify which of the two IgG Fc domains contributed to the interaction with FgBP, we tested two human IgG1/IgA1 domain swap mutants and found that both domains are required for full binding, with the CH3 domain playing a critical role. The binding site for FgBP was further localized using recombinant equine IgG7 antibodies with single or double point mutations to residues lying at the CH2-CH3 interface. We found that interaction of FgBP with equine IgG4 and IgG7 was able to disrupt C1q binding and antibody-mediated activation of the classical complement pathway, demonstrating an effective means by which S. equi may evade the immune response. The mode of interaction of FgBP with IgG fits a common theme for bacterial Ig-binding proteins. Remarkably, for those interactions studied in detail, it emerges that all the Ig-binding proteins target the CH2-CH3 domain interface, regardless of specificity for IgG or IgA, streptococcal or staphylococcal origin, or host species (equine or human).     

Selective colony blotting to expand bacterial surface receptors: applications to receptors for rat immunoglobulins

Many bacterial surface receptors demonstrate a heterogeneous expression pattern among individual colonies. Methods have been developed to select bacteria expressing high levels of a stable surface receptor. This process is illustrated using a Streptococcus zooepidemicus isolate demonstrating a high level of Fc receptors for rat immunoglobulins. This strain was selected and expanded to obtain a bacterial isolate demonstrating approximately 100 fold greater reactivity with rat immunoglobulins than protein A positive Staphylococcus aureus or 30-40 fold higher reactivity for rat IgG than type III Fc receptor positive streptococcal group G strains. The optimal pH for rat IgG binding and the reactivity with rat IgG subclasses and certain rat monoclonal antibodies is described. The potential application and limitations of the selected rat Fc receptor positive bacterial strain to immunoassays based on the specificity of rat monoclonal and polyclonal antibodies is discussed.     

Lack of Fc receptors on osteoclasts

Summary Fc and C₃ receptors, which are characteristically present on macrophages, could not be demonstrated on osteoclasts maintained in situ on their normal substrates when assayed for by use of sheep red blood cells coated with immmoglobulin (Shapiro et al. 1979). The present study tested the hypotheses that Fc receptors are present only on the osteoclast surface adjacent to bone and that Fc receptors on osteoclasts can be uncovered by enzymes or stimulated to appear. Freeze-dried, inverted osteoclasts (and osteoblasts) obtained from the endocranium of newborn rats were tested for Fc receptors using the rosette assay and examined by scanning electron microscopy. No rosettes were observed on the surfaces of the osteoclasts that had been approximal to the bone. Bone specimens were cultured for 30 min at 37° C in control medium, or in medium with the addition of 10, 50 or 100 gmg/ml trypsin, 0.5 U/ml parathyroid extract (PTE), or 0.5 or 1U/ml parathyroid hormone 1–34 (PTH). Additionally, two week-old rats were injected intraperitoneally with PTE (1.5 U/g body weight or 1USP/g body weight) or with PTH (1U/g body weight) or with vehicle alone, 6 h before sacrifice. The specimens were assayed for Fc receptors and examined by scanning electron microscopy. Macrophages were always used as controls for the assay. No rosettes were present on osteoclasts subjected to any of these treatments. Accordingly, the hypotheses were not supported.     

Characterization of the family of dimers associated with Fc receptors (Fc epsilon RI and Fc gamma RIII).

The receptor for IgE (Fc epsilon RI) is a multimeric complex containing one alpha chain, one beta chain with four transmembrane domains and one homodimer of disulfide-linked gamma-chains. The Fc epsilon RI gamma-chains form additional disulfide-linked dimers with the homologous zeta- and eta-chains, as part of the TCR complex. The low affinity receptor for IgG (Fc gamma RIII)2 on NK cells is also associated with zeta-chains. Here we show that the gamma-chain is expressed in NK cells both as a group of heterogenous gamma gamma homodimers and also as a heterodimer bound to zeta. Fc gamma RIIIA is associated with three types of dimers zeta zeta, gamma zeta, and notably gamma gamma as well. In fact, gamma gamma appears to be the predominant species associating with Fc gamma RIIIA. The surface expressed Fc epsilon RI also associates with the same group of heterogenous gamma gamma homodimers. We also show that there is no C-terminal posttranslational cleavage of gamma occurring before its insertion into the plasma membrane as previously suggested. Thus, like the TCR, Fc gamma RIIIA may form a variety of receptor isoforms, though at present we do not understand the functional implications of these structures.     

Cloning, expression and interaction of human T-cell receptors with the bacterial superantigen SSA.

Superantigens (SAgs) are a class of disease-causing and immunostimulatory proteins of bacterial or viral origin that activate a large number of T-cells through interaction with the Vbeta domain of T-cell receptors (TCRs). In this study, recombinant TCR beta chains were constructed with human variable domains Vbeta5.2, Vbeta1 and Vbeta2.1, expressed as inclusion bodies, refolded and purified. The Streptococcus pyogenes SAg SSA-1 was cloned and expressed as a soluble periplasmic protein. SSA-1 was obtained both as a monomer and a dimer that has an intermolecular disulfide bond. We analyzed the biological activity of the recombinant SAgs by proliferation assays. The results suggest that SSA dimerization occludes the TCR interaction site. Naturally occurring SSA dimerization was also observed in supernatants of S. pyogenes isolates. An SSA mutant [SSA(C26S)] was produced to eliminate the Cys responsible for dimerization. Affinity assays using a resonant biosensor showed that both the mutant and monomeric wild type SSA have affinity for human Vbeta5.2 and Vbeta1 with Kd of 9-11 microm with a fast kass and a moderately fast kdiss. In spite of the reported stimulation of Vbeta2.1 bearing T-cells by SSA, we observed no measurable interaction.     

Immunoreactivity of cytomegalovirus-induced Fc receptors

The present studies were undertaken to characterize the affinity of CMV-induced Fc receptors for each of the subclasses of human IgG and to define the specific region of the IgG Fc fragment interacting with such receptors. To do this, we infected confluent human embryonic lung (HEL) cell monolayers with CMV (strain AD169) and then used a double radiolabel assay to measure adherence of antibody-coated E. coli 06 to such monolayers. Preincubating monolayers with each of the 4 subclasses of human IgG (but not IgA, IgM, or human or bovine albumin) abrogated the enhancing effect of CMV infection on adherence of antibody-coated E. coli 06 to HEL monolayers. Pepsin-derived, purified Fc fragments of human IgG had a similar abrogative effect. Preincubating these with staphylococcal protein A did not reduce their capacity to interfere with binding of antibody-coated E. coli to CMV-induced Fc receptors. These observations establish a broad range of immunoreactivity for CMV-induced Fc receptors, that encompasses all 4 subclasses of human IgG. They also provide indirect evidence that the reaction site of CMV-induced Fc receptors is in the CH2 domain of the Fc fragment.     

Design and characterization of novel dual Fc antibody with enhanced avidity for Fc receptors

Monoclonal antibodies (mAbs) have become an important class of therapeutics, particularly in the realm of anticancer immunotherapy. While the two antigen-binding fragments (Fabs) of an mAb allow for high-avidity binding to molecular targets, the crystallizable fragment (Fc) engages immune effector elements. mAbs of the IgG class are used for the treatment of autoimmune diseases and can elicit antitumor immune functions not only by several mechanisms including direct antigen engagement via their Fab arms but also by Fab binding to tumors combined with Fc engagement of complement component C1q and Fcγ receptors. Additionally, IgG binding to the neonatal Fc receptor (FcRn) allows for endosomal recycling and prolonged serum half-life. To augment the effector functions or half-life of an IgG1 mAb, we constructed a novel “2Fc” mAb containing two Fc domains in addition to the normal two Fab domains. Structural and functional characterization of this 2Fc mAb demonstrated that it exists in a tetrahedral-like geometry and retains binding capacity via the Fab domains. Furthermore, duplication of the Fc region significantly enhanced avidity for Fc receptors FcγRI, FcγRIIIa, and FcRn, which manifested as a decrease in complex dissociation rate that was more pronounced at higher densities of receptor. At intermediate receptor density, the dissociation rate for Fc receptors was decreased 6- to 130-fold, resulting in apparent affinity increases of 7- to 42-fold. Stoichiometric analysis confirmed that each 2Fc mAb may simultaneously bind two molecules of FcγRI or four molecules of FcRn, which is double the stoichiometry of a wild-type mAb. In summary, duplication of the IgG Fc region allows for increased avidity to Fc receptors that could translate into clinically relevant enhancement of effector functions or pharmacokinetics.     

Rabbit T cells with and without Fc receptors

Earlier, indirect evidence for rabbit subpopulations differing in Fc receptors and in response to mitogen has been directly tested. T cells were purified from spleen suspension by removal of adherent cells, followed by removal of Ig-bearing cells on petri dishes coated with antibody, directed against the light chain allotype of Ig receptors. The purified cells were further fractionated by formation of EA rosettes and separation on Ficoll-Hypaque. T cells which lacked Fc receptors had a larger response when stimulated with Con A or PHA than did T cells which possessed Fc receptors. Both subpopulations responded more when irradiated nonadherent B cells were added to the mixture, but the extent of help was the same for both cell populations. T cells which contained both Fc receptor-bearing cells and cells which lacked the receptor had a response which was intermediate between that of the two separated subpopulations.     

The involvement of a preformed cytoplasmic receptor pool in the reexpression of Fc receptors following their interaction with various antibodies

The mechanisms of Fc receptor blocking by anti-Ig, anti-Ia, and anti-B2 microglobulin (anti-B2Mi) antibodies were compared on human peripheral blood mononuclear cells. The results suggest that Fc receptors were “co-endocytosed” with sIg/anti-Ig and la/anti-Ia complexes, while they were “co-shed” with B2Mi/anti-B2Mi complexes during the incubation with the antibodies at 37 °C. Fc receptors recovered after the blocking by anti-B2Mi or anti-Ig, but not after anti-Ia or heat-aggregated human IgG(Agg Hg) treatment. Lymphocytes were able to replace their endocytosed or shed Fc receptors presumably from a preformed cytoplasmic receptor pool. Anti-Ia antibodies and AggHg probably exhausted this Fc receptor pool or inhibited its transport to the membrane causing an irreversible inhibition of Fc receptor expression on the appropriate cells.     

Influence of dipeptides on precipitated morphine withdrawal in the mouse

Effects of various dipeptides on naloxone-precipitated morphine withdrawal were studied in the mouse. Mice were rendered dependent on morphine by implantation of morphine pellets and the withdrawal syndrome was measured by the latency of the onset of stereotyped jumpings. In accordance with previous data, subcutaneous injection of Z-prolyl-D-leucine significantly delayed the onset of morphine withdrawal. The all-L enantiomer of the dipeptide (Z-L-prolyl-L-leucine) did not affect morphine withdrawal in the dose studied. Replacement of L-proline by L-glutamate or L-pyroglutamate (Z-L-glutamyl-L-leucine and L-pyroglutamyl-L-leucine) resulted in dipeptides which were more potent towards morphine withdrawal than Z-prolyl-D-leucine. Z-L-glycyl-L-proline attenuated the morphine withdrawal syndrome more effectively than Z-L-prolyl-D-leucine, but Z-L-leucyl-L-glycine was ineffective in this respect. The data reveal that certain dipeptides—which in their nonprotected forms are normal sequences of endogenous peptides—affect morphine withdrawal more potently than Z-prolyl-D-leucine, a synthetic dipeptide known to attenuate morphine dependence.     

Dynamics of the interaction of polyreactive immunoglobulins with immobilized antigens]

The kinetic of polyreactive immunoglobulins (PRIG) and immobilized antigen interaction was examined at different temperatures. It was shown that this process can be described by the so-called "competitive" model, and the relatively simple method for the rate constant determination for this process was developed. According to the "competitive" model PRIG molecule could be either in "active" or in "inactive" state and dynamic equilibrium exist between "active" and "inactive" molecules which strongly depend on incubation temperature. Only "active" PRIG can interact with antigens, and this is the reason of strong temperature dependence of PRIG-antigen interaction. The data also show that the mechanism of PRIG-antigen interaction differ from that of antibody-antigen interaction.