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.     

Fine specificity of a rabbit antibody interacting with human IgG Fc receptor-like molecules

A polyclonal rabbit antibody raised against an Fc receptor (FcR)-like membrane glycoprotein fraction of chronic leukaemic lymphocytes has previously been prepared and partially characterized. This antibody, called AbA, was found to precipitate a 70-kDa and a 45-kDa fraction of the detergent lysate of U937 cells and to inhibit ligand binding to Fc gamma R on the P388D1 murine macrophage cell line. In the present work we have characterised this antibody further. All Fc gamma RII-positive B lymphoblastoid cell lines, as well as resting human B lymphocytes, were positively stained with the AbA antibody. U937 cells were found to be negative, but after stimulation with phorbol ester (PMA), 50% of the cells became positive. AbA antibody did not react with human T cell lines or with the T + 0 cell subset of peripheral blood. Monocytes were also negative. On the other hand, AbA antibody exhibited a dose-dependent inhibition of antibody-mediated cytotoxic reaction (ADCC) of monocytes, while not affecting K cell-mediated ADCC. It had an inhibitory effect of EA rosette formation of B cells and stimulated U937 cells. Furthermore, it interacted with the soluble form of Fc gamma RII released by activated B lymphocytes, and--similarly to IgG--precipitated a 33 kDa fraction from the supernatant of B cells.     

Receptors for IgG (Fc receptors) on human lymphocytes: re-evaluation by multiple techniques

This study examines whether receptors for IgG (Fc receptors), as identified by different methods, are found on identical human lymphocyte subpopulations, and the relationship of Fc receptors to surface immunoglobulin (SIg) and receptor for complement (C'). Fc receptors were identified by two rosetting techniques (antibody-sensitized human erythrocytes, HuEA, or sheep erythrocytes, ShEA) and two immunofluorescent techniques (heat-aggregated IgG of human origin, HuAgg, or of guinea-pig origin, GPAgg).

When lymphocytes depleted of cells rosetting with ShEA were compared to HuEA-depleted lymphocytes, the two subpopulations appeared to be significantly different. However, when lymphocytes were depleted of cells rosetting with ShEA which had been sensitized with lower concentrations of antibody, the subpopulation so depleted appeared to be virtually identical to HuEA-depleted lymphocytes. These studies suggest that more than one lymphocyte subpopulation has Fc receptors and that each subpopulation can, in part, be identified and distinguished from the other by the capacity to bind IgG at differing concentrations.

In particular, these experiments may serve to resolve the controversy concerning the presence of Fc receptors on lymphocytes bearing surface immunoglobulin (SIg). Depletion of lymphocytes rosetting with ShEA removed most of the SIg-bearing lymphocytes; depletion of cells rosetting with ShEA which had been sensitized with lower concentrations of IgG antibody, however, failed to deplete SIg-bearing lymphocytes even though other Fc-bearing populations were completely depleted. These results suggest that SIg-bearing lymphocytes (B lymphocytes) do have Fc receptors but that high concentrations of IgG are needed to demonstrate them.

     

Fc receptors on rabbit lymphocytes. Identification and organ distribution of rosette-forming cells; cocapping with surface immunoglobulin

Fc receptor (FcR)-bearing cells were demonstrated using ox erythrocytes coated with homologous IgG-type antibodies (EAY) in rabbit peripheral blood leukocytes (PBL) and in various lymphoid organs. Discrimination of the rosette-forming cells (RFC) is carried out after prior ingestion of tetramethylrhodamine isothiocyanate-labeled latex particles and in transmission electron microscopic studies. Most of the nonlymphoid cells (5-10%) in PBL and spleen cell suspensions expose FcR. These nonlymphoid cells are almost absent in other lymphoid organs, except in bone marrow. The average percentage of cells rosetting with IgG-sensitized erythrocytes (EAγRFC) in lymphoid cell preparations of the various tissues was as follows: PBL 25%, bone marrow 65%, appendix 37%, spleen 40%, Peyer's patches 44%, thymus 2% and peripheral lymph node 27%. The nature of FcR-bearing PBL was further studied using F(ab′)₂ anti-IgM, anti-IgA or anti-T cell conjugates. About half of the population of B cells, bearing IgM or IgA, express FcR. Moreover, about 80% of the RFC are found within the B cell population. Only a few T cells were found rosetting with EAγ suggesting that most of the non-B lymphoid RFC are “null” cells. In different lymphoid organs, the percentages of EAγRFC and B cells are comparable but not identical. A greater part of the EAγRFC also expresses the receptor for the third component of complement. After capping of membrane IgM determinants, FcR is located in the same cap on the majority (60%) of the FcR-positive IgM-capped cells.     

Fc receptors for IgG and antigen presentation on MHC class I and class II molecules

Antigens internalized through specific membrane receptors are presented to helper CD4(+) T cells at antigen concentrations 10(3) to 10(4) fold lower than antigens internalized by fluid phase. B lymphocyte antigen receptors, mannose receptors and receptors for the Fc region of immunoglobulins, promote both internalization and efficient presentation at low antigen concentrations. Thus, binding to specific membrane receptors concentrate antigens on antigen presenting cells and mediates efficient uptake. Is this 'quantitative' concentration of antigens on antigen presenting cells the end of the story? Or may 'quality', i.e. selective intracellular antigen targeting, somehow influence the efficiency or specificity of MHC class I and class II-restricted antigen presentation?     

Cell populations in leucocyte adherence inhibition: requirement for T lymphocytes with IgG Fc receptors.

The subset identity of T lymphocytes participating in the leucocyte adherence inhibition (LAI) reaction was investigated. Humans were immunized with keyhole limpet haemocyanin (KLH) and their cellular immunity was tested by means of the haemocytometer variant of the LAI method. Their lymphocytes were fractionated by rosetting methods employing neuraminidase-treated sheep erythrocytes and IgG- or IgM-coated ox erythrocytes. The T lymphocytes rosetted by IgG-coated ox cells (T gamma) reacted with KLH to give specific LAI reactions. Non-T gamma cells failed to react. The T gamma cells released a lymphokine which caused an LAI reaction of T lymphocytes from non-immunized donors. Immune non-T gamma cells, when incubated with KLH yielded inactive supernates. The normal cells which gave positive LAI responses to the lymphokine also proved to belong exclusively to the T gamma subclass. Cells positively selected with IgM-coated ox cells (T micro) were inactive while the non-T micro lymphocytes behaved like the T gamma cells. It was shown that the activity was confined to the T gamma subset throughout the time course of a primary immune response. Thus, LAI reactivity appears to be a property of a very small subclass of lymphocytes which communicate with each other by means of a soluble factor.     

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.     

ELISA for a complexed antigen with a monoclonal antibody blocking reaction with the free antigen-assay-specific for complexed prostate-specific antigen

An increased level of complexity will be encountered when developing protocols for intracellular markers. Protocols for surface markers have been successfully standardized, however it is understood that no single method is appropriate for all intracellular staining. A systematic approach should be followed, including knowledge of antigen location and functional state, selection of cell fixative and cell permeabilizer, antibody specificity and class/subclass, fluorochrome, fluorochrome to protein ratio (F:P), and use of adequate controls, including isotype-matched negative controls and positive and negative cell controls. Even though it is impossible to recommend a single technique to stain all intracellular antigens, the authors present a logical approach to follow when developing a staining protocol.     

Protein A as a molecular probe for the detection of antigen induced conformational change in Fc region of rabbit antibody

Rabbit IgG antibody which reacts with protein A of Staphylococcus aureus (SpA) and forms a soluble complex with molar composition (IgG₂-SpA₁)₂ is not able to further bind SpA or to attach to SpA-Sepharose 4B thus proving that the unengaged SpA reactive sites of IgG are shielded by the already bound SpA.On the contrary the (IgG₂-SpA₁)₂ complex was able to bind a small fragment of SpA (fSpA) clearly showing that SpA-reactive sites are present in the rabbit IgG molecules of the complex but that they are not available for the intact SpA molecule.Immune complexes contaning (IgGaFER₂-SpA₁)₂ and ferritin attach to an SpA-Sepharose 4B column showing that SpA binding sites exist on the IgG molecules and became exposed after the conformational change of the Fcγ region induced by the antigen. Therefore SpA can be used for the direct detection of conformational changes induced by antigen in the Fcγ region of rabbit antibody.     

Detection of receptors for the Fc region of IgG on streptococci

A semi-quantitative absorption test to measure Fc-reactive proteins on the surface of streptococci is described. The ability of bacteria to remove intact IgG in the presence of an equimolar amount of F(ab')2 fragments was used to identify streptococci with Fc-reactive proteins on their surface. This method was found to be more objective, reproducible and quantitative than the hemagglutination and 125I-labeled IgG binding methods currently in use. Methods for detection of secreted Fc-reactive materials with protein A-like reactivity are also described.     

The Fc receptor for IgG (Fc gamma RII; CD32) on human neonatal B lymphocytes.

B cells express an Fc receptor for IgG (FcgammaRII; CD32) which is involved in feedback inhibition of antibody production. Engagement of FcgammaRII during ligation of the antigen receptor provides an inhibitory signal. FcgammaRII exists as several isoforms, with FcgammaRIIb (which carries an immunoreceptor tyrosine-based inhibition motif; ITIM) being predominant form on adult B cells. The inhibitory role of FcgammaRIIb may be unhelpful to the infant, since primary exposure to infectious agents is likely to be in the presence of maternal IgG. We hypothesized that neonatal B cells would be less susceptible to feedback inhibition by antibody, either through the expression of activation-competent FcgammaRII isoforms (FcgammaRIIa and FcgammaRIIc) or through reduced expression of the inhibitory FcgammaRIIb isoforms. Cord and adult B cells were examined for expression of FcgammaRII isoforms using monoclonal antibodies and RT-PCR. In vitro assays were performed to assess susceptibility of cord and adult cells to FcgammaRII-mediated suppression. Although there is no phenotypic difference in FcgammaRII expression (FcgammaRIIb predominating on both adult and cord B cells), FcgammaRIIb is expressed at lower levels on cord cells. This quantitative difference in FcgammaRIIb expression may explain the reduced susceptibility of cord B cells to antibody-mediated inhibition observed in these experiments.     

Sensitive detection of two IgG Fc receptors of mouse macrophages by chemiluminescence analysis

Luminol-enhanced chemiluminescence assay was used to detect the surface expression and the consequent activation of receptors (FcRI and FcRII) of murine macrophages (M phi s). When murine IgG2a was used for the specific detection of FcRI and IgG2b for FcRII, a newly established procedure enabled us to detect the activation of each receptor with as few as 3 X 10(5) M phi s. Briefly, TNP-SRBC coated with monoclonal IgG2a or IgG2b antibodies directed to TNP (sensitized SRBC) were used as reagent, in the presence of 1 X 10(-5) M luminol, and the emission was measured with a liquid scintillation counter. When results obtained by chemiluminescence counting were compared to the results obtained by the rosette formation by adding the same SRBC reagent to peritoneal M phi s obtained after ip injection of Listeria, fortified chemiluminescence counting allowed us to obtain a more definite answer about the activation of each receptor. Under the conditions established, the specific activation of FcRI was obtained by the addition of rIFN alpha A/D to the resident M phi s in vitro and the specific activation of spleen M phi FcRII by iv injection of IAP (Immunosuppressive acidic protein) into mice. These two results supported the independence of the two receptors detected by the assay.     

Absence of IgG Fc receptors on varicella-zoster virus-infected cells

Herpesvirus-infected cells usually express receptors for the Fc portion of immunoglobulin G. Varicella-zoster virus has so far been the sole exception in the family. Both immunehemadsorption and immunofluorescence techniques failed to detect the expression of such receptors. This observation excludes the possibility of false-positive results in serological tests for antibodies to this virus. It is possible that the function of these receptors early in infection is not needed in the subsequent reactivation(s) of the virus. No variation has been shown to occur with different isolates.     

Regulation of in vivo expression of Fc receptors for IgE (Fc epsilon R) on murine lymphocytes. II. Induction of Fc epsilon R and its inhibition in mice immunized with antigen

The induction of Fc receptors for IgE (Fc epsilon R) and its regulation were studied in BALB/c, SJL/J, and nude mice by a flow cytometric assay with the use of homologous monomeric IgE. Immunization of BALB/c mice with alum-absorbed antigen induced a remarkable increase in the expression of Fc epsilon R on spleen cells, whereas no enhancement of the Fc epsilon R expression was observed in SJL/J and nude mice after immunization. This increase was correlated with the elevation of serum IgE levels. However, the IgG antibody response, which is inducible even in SJL/J mice, was not associated with the induction of Fc epsilon R. The enhanced expression of Fc epsilon R in BALB/c mice observed in the primary or secondary IgE antibody response was detected in B cells with B220, surface IgM, and IgD, but not in T cells. The induction of Fc epsilon R in immunized BALB/c mice was inhibited by suppressive factor of allergy isolated from ascites fluids of SJL/J mice inoculated with complete Freund's adjuvant. In addition, both cyclophosphamide and prednisolone had an inhibitory effect on the induction of Fc epsilon R. These results suggest that the Fc epsilon R induction is inhibited not only by suppressive factor of allergy, which is effective in inhibiting the IgE antibody response selectively, but also by some immunosuppressive agents which are capable of suppressing all isotypes.     

New method for detection of Borrelia burgdorferi antigen complexed to antibody in seronegative Lyme disease

Serologic tests for Lyme disease are problematic. Because of cross-reactive antigens Borrelia burgdorferi (Bb) shares with other organisms, Lyme disease can be overdiagnosed. However, in addition to specificity problems, serologic tests for early Lyme disease can be falsely negative due to lack of sensitivity of ELISAs and Western blots. Most routine antibody tests are designed to detect free antibodies, and in early, active disease, circulating antibodies may not be free in serum but sequestered in complexes with the antigens which originally triggered their production. This difficulty may be overcome by first isolating immune complexes (IC) from the serum and using this fraction for testing. Free Borrelia-specific antibodies can then be liberated from the immune complexes which may enhance test sensitivity in patients with active disease. We developed a technique that captures the antibody component of IC on immunobeads, and subsequently releases the antigen component of IC. Immunoblotting with monoclonal antibody detected at least one antigen to be OspA, thus definitively demonstrating a Borrelia-specific antigen in circulating IC in early Lyme disease. This test is also useful in demonstrating Bb antigen in otherwise seronegative Lyme disease patients.     

Binding and endocytosis of erythrocytes sensitized with rabbit IgG via Fc gamma receptors of human monocytes.

The properties of the monocyte Fc gamma receptors (FcR) were investigated with monoclonal antibodies (mAb) against FcRI (10.1) and FcRII (IV3). mAb against FcRI inhibited partially the binding of sheep red blood cells (SRBC) sensitized with anti-SRBC rabbit IgG (EA) at 37 degrees to monocytes pretreated with N-ethyl maleimide, which inhibits the EA ingestion. The erythrocytes (E) were sensitized with varying concentrations of anti-E rabbit IgG. The EA binding to different FcR depends on the concentration of specific antibody used to sensitize the erythrocytes. At high levels of sensitization a high proportion of rosettes form via FcRII which can be inhibited with mAb IV3. As sensitization decreases it is more difficult for FcRII to form rosettes, so an increased percentage of them is mediated via FcRI. Sensitization of SRBC with 1-1.5 x 10(3) anti-SRBC rabbit IgG molecules per erythrocyte is the threshold to allow FcRII to mediate rosettes. At the lowest levels of sensitization the total number of rosettes is even lower and all rosettes are mediated via FcRI, hence mAb 10.1 is fully inhibitory. In addition, our data strongly support the view that the ingestion of EA takes place mainly via FcRII. We show in this study that while binding of slightly sensitized erythrocytes was blocked efficiently by mAb 10.1, the ingestion of the equivalent EA was hardly inhibited by it.     

Binding and uptake of rabbit IgG complexes by diploid fibroblasts via plasma membrane Fc receptors

The existence of IgG receptors on the plasma membrane of diploid human fibroblasts is demonstrated. The receptors specifically bind heat-aggregated rabbit IgG as well as rabbit IgG within antigen-antibody complexes. Monomeric rabbit IgG were only poor ligands of the receptor. Competition experiments with Fab and Fc fragments of IgG revealed that the receptor specifically recognizes the Fc domain of the IgG molecule. Heat-aggregated IgG or antigen-antibody IgG complexes are specifically bound to the receptors, endocytosed and subsequently degraded. The receptors do not seem to be recycled because protein synthesis is a prerequisite for further cycles of endocytosis.     

Co-cross-linking of surface immunoglobulin Fc gamma receptors on B lymphocytes uncouples the antigen receptors from their associated G protein

Cross-linking of surface Ig receptors (sIg) by mitogenic forms of anti-Ig antibodies (e.g. F(ab')2 fragments of rabbit anti-Ig) causes the rapid, and prolonged breakdown of phosphatidylinositol 4,5-bisphosphate. This response involves an unidentified guanine nucleotide regulatory protein (termed Gp), which couples sIg to the polyphosphoinositide-specific phosphodiesterase. Intact (IgG) rabbit anti-Ig antibodies, which co-cross-link sIg and Fc gamma receptors on B cells, only induce short-lived inositol phospholipid breakdown and abortive B cell activation. We show here that in permeabilized B cells intact anti-Ig inhibits the reconstituted breakdown of inositol phospholipids given by a combination of F(ab')2 anti-Ig and the non-hydrolyzable GTP analogue guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S), but not the basal stimulation of Gp induced by GTP gamma S alone. These results therefore indicate that co-cross-linkage of sIg and Fc gamma receptors on B cells uncouples the antigen receptors from the associated G protein, but does not affect coupling between Gp and the phosphodiesterase. These observations therefore provide further insight into the mechanisms whereby engaging Fc receptors on B cells, by antigen-antibody complexes for example, could modulate antigen-induced B cell activation.     

Electron microscopy of Fc receptors on human lymphocytes.

The membrane receptor for Fc portions of IgG (FcR) was localized on the cell surface of humans lymphocytes by electron microscopy. The electron microscopic markers for FcR were soluble ferritin 7S anti-ferritin immune complexes prepared in forty times antigen excess than needed at equivalence. Fc receptors on the lymphocytes labelled at 0 degree in the presence of sodium azide were seen as discontinuous patches on the cell surface. In control experiments, no labelling was observed, which included lymphocytes treated with ferritin only or with F(ab')2 immune complexes as well as glutaraldehyde-fixed lymphocytes treated with 7S anti-ferritin immune complexes. The findings are discussed with relation to the widely accepted membrane fluidity model.