Interaction between streptococcal IgG Fc receptors and human and rabbit IgG domains.

Groups A, C and G streptococci were tested for their ability to bind 125I-labelled fragments of human and rabbit IgG in order to localize their sites of interaction with IgG domains. Among the Group A streptococci, strains with IgG Fc receptors bound 85% of the added IgG Fc fragments in the test systems, whereas these strains showed practically no reactivity with F(ab')2, Facb (F(ab')2 + C gamma 2 domains) or pFc' (C gamma 3 domains). The Group C and Group G strains bound 48-100% of IgG Fc, but could also bind up to 36% of the added F(ab')2 in accordance with a previously described 'alternative' Fab reactivity. However, unlabelled IgG F(ab')2 or Facb showed no, or only slight, inhibitory capacity for the binding of 125I-labelled IgG Fc to the C and G strains. Collectively, these results indicate that Groups A, C and G streptococci require both the C gamma 2 and C gamma 3 domains for interaction with IgG, and most probably also bind in the interface region between the C gamma 2 and C gamma 3 domains as has been shown for staphylococcal protein A.     

Binding of IgM rheumatoid factor to group A, C and G streptococci with IgG Fc receptors

The possibility that IgM rheumatoid factors bind to streptococci was studied. Using a sequence of Sephadex G200 gel filtration, protein A-Sepharose CL-4B chromatography and preparatory electrophoresis, IgM was isolated from the sera of 2 patients with rheumatoid arthritis and then radiolabelled with 125I. There was significant binding of radiolabelled IgM to group-A streptococci types M1, M15 and M22, and to a group-C and a group-G strain, all expressing IgG Fc receptors, but none to Staphylococcus aureus, Escherichia coli or to 11 other strains of streptococci without IgG Fc receptors. The radiolabelled IgM was separated by affinity chromatography on a column containing human IgG. Types M1 and M15 bound the fraction retained on the column, whereas M22 bound both this fraction and the non-retained fraction. Commercial human IgG, even at high concentrations, did not inhibit binding. The binding reaction, which is perhaps triggered either by the IgM rheumatoid factor or by IgG complexed with rheumatoid factor, could be a useful tool for removal of anti-immunoglobulin from the blood of patients with rheumatoid arthritis.     

Fc receptors for IgG, IgM and IgE on human leukaemic lymphocytes.

Fc receptors for IgG, IgM, IgE and the cell surface immunoglobulins (SIg) were analysed on lymphocytes from seventeen patients with chronic lymphatic leukaemia (CLL), one with lympho-sarcoma cell leukaemia (LSL), two each with hairy cell leukaemia (HCL), acute lymphatic leukaemia (ALL) and Sézary syndrome. Fc receptors for IgG and IgM were detected by rosette formation with ox erythrocytes (E_O) sensitized with rabbit IgG (E_OA_G) and IgM (E_OA_M) anti-E_O antibodies, respectively. Fc receptors for IgE were analysed either with E_O coated with glutaraldehyde coupled complexes consisting of rabbit Fab'' fragments of anti-E_O antibodies and Fc fragments of an IgE myeloma protein (E_OA_E), or with aldehyde fixed E_O to which IgE was adsorbed. SIg of classes IgM, IgD, IgG and κ and λ light chain type were detected with E_O coated with complexes consisting of Fab''-anti-E_O and purified F(ab'')₂ fragments of specific goat antibodies.Lymphocytes of all patients with CLL, LSL and HCL had Fc receptors for IgG (65±15% E_OA_G⁺, normal 22·0±5·8%). Ten patients had significant numbers of cells with IgM Fc receptors (37±22% E_OA_M⁺, normal 1·2±1·5%) which were detected without overnight culturing of the lymphocytes. Lymphocytes of four patients (two CLL, one LSL, one HCL) had Fc receptors for IgE (22–88% E_OA_E⁺, normal 1·8±0·7%). The cells of three of these four patients were also E_OA_M⁺. The high numbers of rosetting cells indicated that individual lymphocytes must have carried more than one class of Fc receptors. The lymphocytes of the ALL and Sézary syndrome patients had few Fc receptor positive cells.Of the seventeen patients with CLL, twelve were SIgM⁺ and/or SIgD⁺, only four κ⁺ or λ⁺ and one had no SIg. The cells of the LSL and one of the HCL patients were SIgM⁺ and SIgD⁺, whilst the cells of the other HCL patient were SIgG, λ⁺. None of the other patients had more than 10% SIgG⁺ cells. The ALL and Sézary syndrome patients had low numbers of SIg⁺ and Fc receptor positive cells.These data indicate that lymphocytes of patients with B-cell leukaemias can carry different classes of Fc receptors simultaneously; the different classes are found in a decreasing frequency of IgG>IgM>IgE.     

Idiotypic interactions between normal human polyspecific IgG and natural IgM antibodies

Pooled normal human polyspecific IgG (IVIg) contain anti-idiotypes against a variety of autoantibodies from patients with autoimmune diseases and IgG autoantibodies present in IVIg. The present study indicates that IVIg may also react through idiotypic/anti-idiotypic interactions with human natural IgM antibodies. Sixty-four percent of IgM secreted by B lymphoid cell lines derived from B cells of healthy elderly donors and 18% of IgM secreted by cloned EBV-transformed cord B cells that were tested, bound through their variable region to F(ab')2 fragments of IVIg. The binding to 2,4,6-trinitrophenyl (TNP) of a polyreactive IgM with anti-TNP specificity, was inhibited by F(ab')2 fragments from IVIg, indicating the presence in IVIg of anti-idiotypes that may interfere with the antibody-combining site of polyreactive IgM antibodies. The ability of IgM antibodies to interact with idiotypes on IVIg was not related to the degree of polyreactivity of natural antibodies. Our observations further document that IVIg contain antibody specificities against Ig from normal individuals and suggest that IgG originating from the physiologically expressed repertoire may modulate the expression of the potential B cell repertoire. The results may be relevant to the suppressive effect of IVIg in autoimmune diseases.     

Surface markers on human activated T lymphocytes. I. Fc receptors for IgG and IgM

Expression of Fc gamma and Fc mu receptors on human peripheral blood T lymphocytes of two subsets with high (E early rosette forming presumably in vivo activated cells, TEe) and low affinity of E receptors (E late rosette forming presumable resting cells, TEl) was investigated. Different distribution pattern of T gamma and T mu cells in the both examined T cell subsets was found. Thus TEe and TEl subsets have been partially enriched in T mu and T gamma cells, respectively. Furthermore, the results obtained in the PHA-stimulated system have shown that Fc mu receptors do not function as the markers of T cell activation. However, in opposition to this finding Fc gamma receptors may be the early activation markers but only of T cells originally bearing high-affinity E receptors.     

Cytotoxicity mediated by human Fc receptors for IgG

The Fc receptors for IgG(Fc gamma R) play a major role in the removal of antibody-coated infectious agents and may be important molecules for triggering cytotoxicity of tumor cells; they may also serve as an entry for infection of Fc gamma R-bearing cells by viral (including HIV and Dengue), and perhaps other infectious agents. Although central to immune defense, an understanding of the role of these Fc gamma R in cytotoxicity has been complicated in part by the presence of several biochemically distinct types of receptor that have different distributions, specificities, affinities and modes of activation for killing. The development of monoclonal antibodies specific for Fc gamma R on human leukocytes has established the existence of three distinct Fc gamma R and furthermore has helped clarify the function of each of these receptors. In this review, Michael Fanger and colleagues discuss the use of Fc gamma R-specific mAb and the hybridoma cell lines that produce them in examining the ability of each of these unique receptors to mediate killing of tumor and red cell targets. In particular, the use of self-directed hybridoma cells as a model of tumor-cell killing and of bi-specific antibodies to link target cells to effector cells through the different Fc gamma R is discussed. The results of these studies suggest that the ability of a given Fc gamma R to trigger killing is sometimes dependent on the type of Fc gamma R, but is also markedly influenced by the type of target cell and by the nature and state of activation of the effector cell.     

Crystallization of a complex between the Fab fragment of a human immunoglobulin M (IgM) rheumatoid factor (RF-AN) and the Fc fragment of human IgG4.

Rheumatoid factors (RF) are the characteristic autoantibodies found in patients with rheumatoid arthritis. They recognize epitopes in the Fc region of immunoglobulin G (IgG) and are often of the IgM isotype. In order to analyse the nature of RF-Fc interactions, we have crystallized a complex between the Fab fragment of a human monoclonal IgM rheumatoid factor (RF-AN) and the Fc fragment of human IgG4. The stoichiometry of the complex within the crystals was found to be 2:1 Fab:Fc. The crystals diffracted X-rays to 0.3 nm resolution, and the space group was C2, with cell dimensions a = 16.03 nm, b = 8.19 nm, c = 6.42 nm, beta = 98.3 degrees. We have also determined the sequence of the variable region of the RF-AN light chain, not hitherto reported. This belongs to the V lambda III-a subgroup and is closely related to the germline gene Humlv318, from which it differs in three amino acid residues. This is the first reported crystallized complex between a human autoantibody and its autoantigen.     

Fc IgM receptors on human lymphoblastoid B cell lines.

Five human lymphoblastoid cell lines have been investigated for their ability to secrete immunoglobulins (IgG, IgA, IgM) and for the presence of different cell surface markers, with special emphasis on the Fc IgM receptor, using a rosette technique with IgM-coated bovine red blood cells (EA-IgM). Four cell lines (Hu, 8432, SB, PA3) were characterized as having as cell origin due to the presence of surface immunoglobulins, complement receptors, mouse red blood cell rosette formation, low avidity Fc IgG receptors and absence of sheep red blood cell rosette formation. Two of these B cell lines (Hu and PA3) secreted IgM, two cell lines (8432 and SB) secreted IgG, while the human T cell line Molt 4 did not secrete Ig. All four B cell lines exhibited Fc IgM receptors (17–35%) while the T cell line Molt 4 had no detectable Fc IgM receptor. The receptor was specific for IgM. Neither aggregated IgG, soluble IgG immune complexes nor EDTA could abrogate the rosette formation, IgM immune complexes, pure human IgM, as well as normal human serum had an inhibitory effect on EA-IgM rosette formation. The receptor was trypsin-sensitive and required protein synthesis for expression. There was no correlation between the expression of Fc IgM receptors and secretion of any given Ig class, indicating that B cells may express Fc IgM receptors independently of their commitment to produce IgM or IgG.     

The interference of IgM rheumatoid factor in enzyme-linked immunosorbent assays of rubella IgM and IgG antibodies

The interference of IgM rheumatoid factor (RF) in the indirect enzyme-linked immunosorbent assay (ELISA) for rubella IgM and IgG antibodies was evaluated quantitatively. In an ELISA for rubella IgM antibodies IgM RF produced false positive results in tests of sera with a rubella IgG concentration exceeding approximately 30 I.U./ml and an IgM RF concentration higher than 3.5 I.U./ml as determined by ELISA. Sera from 3 of 70 patients with recent rubella and sera from a similar proportion of blood donors contained IgM RF in a concentration exceeding this level.The false positive results were reproduced when an IgM RF preparation was added to sera containing rubella IgG. The rubella IgG values in ELISA, however, decreased after the addition of IgM RF . RF was absorbed by incubation of sera with a suspension of latex particles coated with aggregated human IgG. This method prevented the false positive results of the rubella IgM assay and increased the rubella IgM values of rubella convalescent sera. The activity in the rubella IgG assay also increased after absorption of RF. The interference of RF has been explained by a secondary binding of RF to rubella IgG-antigen complexes. The RF might subsequently be detected by the anti-IgM conjugate or compete with the anti-IgG conjugate.The common occurrence of IgM RF in concentrations sufficient to produce false positive results of the ELISA for specific IgM antibodies necessitates routine testing of IgM antibody positive sera for RF by a sensitive method and/or absorption of RF from such sera.     

IgG rheumatoid factor in subacute bacterial endocarditis: relationship to IgM rheumatoid factor and circulating immune complexes.

With recently developed radioimmunoassays, we have been able to study the levels and properties of IgG rheumatoid factor (IgG RF) and IgM rheumatoid factor (IgM RF) in patients with subacute bacterial endocarditis (SBE), as well as the relationship of these autoantibodies to circulating immune complexes. We found significantly elevated amounts of IgG RF and IgM RF in SBE sera. The IgG RF chromatographed on Sepharose 6B as an intermediate complex, indistinguishable from the pattern seen in rheumatoid arthritis. RF levels peaked later in the course of SBE than did levels of circulating immune complexes. With antibiotic treatment RF levels declined, although not as fast nor as completely as circulating immune complexes. These results suggest that both IgG RF and IgM RF in SBE may be part of a polyvalent antibody response to elevated levels of circulating immune complexes which do not themselves contain RF.     

ELISA assays for IgM and IgG rheumatoid factors

Enzyme-linked immunosorbent assays (ELISA) for the detection of IgM RF and IgG RF are described. A quantitation step was introduced into the IgM RF assay allowing expression of results as mg/ml of IgM RF. The effect of pepsin digestion of test sera on the IgG RF assay was established and the possible advantages of including pepsin digestion of sera in the assay considered. The assays have been developed to allow the screening of large numbers of sera in as rapid a time as possible.     

IgG rheumatoid factor in human and rabbit transplantation sera

Sera of human recipients of kidney allografts and rabbit recipients of skin allografts were tested by means of enzyme immunoassays (EIA) for IgG rheumatoid factor (RF) combining with human IgG (RF-H) or rabbit IgG (RF-R). In humans, the mean OD value in EIA for RF-H was significantly higher in 185 sera of allograft recipients than in 46 pretransplantation sera and 100 sera of normal subjects. Furthermore, the mean OD in patients who rejected their grafts was higher than in patients with satisfactory graft function. Interestingly, there was no difference in OD values for RF-R between sera of graft recipients and sera of normal subjects. Of 52 rabbit recipients of skin allografts, 24 were positive for both RF-R and RF-H, 4 were positive only for RF-R, another 4 only for RF-H, and 20 were negative for both. Renal allografts originating from the previous donors of skin grafts resulted, in most instances, in a significant decrease in the strength of the reaction for both RF-R and RF-H. The possible role of graft rejection in stimulation of RF formation as well as the possible role of RF in graft rejection should be considered.     

A solid-phase radioimmunoassay for IgG and IgM antigammaglobulin factor in rheumatoid arthritis sera.

Rheumatoid factors of IgG and IgM class were detected by means of a solid-phase radioimmunoassay using formalinized sheep erythrocytes sensitized with rabbit anti-sheep erythrocyte serum and 125I-labelled anti-human IgG or anti-human IgM antibody. 28 of 35 sera and all of 7 synovial fluids of patients with rheumatoid arthritis showed positive reaction for both IgG and IgM rheumatoid factor, though the activity of IgM factor was mostly higher than that of IgG factor. However, rheumatoid factor present in synovial fluids of 3 seronegative patients was mainly of IgG class. Of 14 sera of patient with other collagen diseases, 11 showed IgG and 6 showed IgM rheumatoid factor. The IgG rheumatoid factor seemed to be associated with autologous IgG to form macro-molecular complexes which could be dissociated into 7S molecules of IgG rheumatoid factor and IgG by acid treatment.     

Tissue distribution of IgG Fc receptors.

The mechanism causing deposition of circulating immune complexes is largely unknown. The possible role of tissue IgG Fc receptors in immune complex localization has been evaluated using IgG coated ox RBC (ox erythrocyte antisera [EA]) as indicator particles. Cryostat tissue sections of normal human synovium, skin, kidney, choroid plexus, lung and uveal tract were examined for the presence of IgG Fc receptors, with human spleen used as a positive control. Ox EA were shown to bind to splenic red pulp. This binding could be almost completely blocked by heat aggregated human IgG. In none of the other normal tissues examined were IgG Fc receptors demonstrated. To investigate the possibility that inflamed tissues express Fc receptors, biopsy specimens of rheumatoid synovium and skin demonstrating vasculitis were studied. No ox EA binding to these tissues was noted. We concluded that IgG Fc receptors are probably not present in tissues that are targets for immune complex deposition and are therefore unlikely to play a role in this process.     

Herpesviral Fc receptors and their relationship to the human Fc receptors

Nearly two decades ago, it was observed that cells infected with herpes simplex virus (HSV) acquired an IgG Fc binding activity. The properties of the viral Fc receptor (FcR) have now been characterized by several laboratories. The Fc binding activity appears on the surface of the infected cell prior to formation of progeny virions. The FcR induced by HSV has been identified as the HSV glycoprotein, gE. When HSV gE forms a complex with a second HSV glycoprotein, gI, the receptor binds IgG with higher affinity. Varicella-zoster virus (VZV), which is closely related to HSV, has also been shown to induce an FcR. Like the HSV FcR, the FcR specified by VZV possesses characteristics common to viral glycoproteins. VZV encodes two glycoproteins, gpI and gpIV, which are the homologs of HSV gE and gI. The VZV glycoproteins have many properties common to cell surface receptors, including O-linked glycans and phosphorylation sites. However, extensive computer-assisted analyses of the amino acid sequences of VZV gpI and gpIV did not uncover regions of homology to the human cellular Fc receptors for IgG.     

Molecular analyses of the interactions between human NK receptors and their HLA ligands

NK cell cytotoxicity is regulated by the action of multiple families of receptors. The interactions of these receptors with their ligands control different activating/inhibiting signal pathways and it is the balance of these signals which determines the behavior of the NK cell. The major described inhibitory pathways begin either with the recognition of a target cell classical class I HLA molecule by a killer cell immunologlobulin-like receptor (KIR) or the binding of the non-classical class I molecule HLA-E to the CD94/NKG2-A heterodimer. Activating counterparts to these inhibitory NK receptors have also been described and this review focuses on the molecular details of the binding of the inhibitory and activating receptors to their HLA ligands.     

Molecular definition of interaction sites on human IgG for Fc receptors (huFcγR)

Evidence from several experimental approaches allows us to conclude that the primary amino acid sequence of the lower hinge region (residues 234–237) of human IgG molecules determines recognition by human FcγRI, FcγRII and FcγRIII. Glycosylation of the CH2 domain is also essential, although the carbohydrate is not accessible for direct interaction with ligands. The role of the carbohydrate moiety may be to maintain a protein conformation that allows accessibility to amino acid side chains essential for ligand recognition and binding. It appears logical that the evolutionarily-related Fcγ R molecules should interact with overlapping non-identical sites on the IgG molecule.