Fc Receptor-Bearing Lymphoid Cells in the Chicken I. Characterization of the Fc(IgG) Receptor

The receptor for Fc(IgG) on chicken lymphoid cells was investigated by EA rosette techniques using sheep erythrocytes sensitized with a subagglutinating dose of anti-sheep erythrocyte (SRBC) chicken serum. Chicken lymphocytes did not form rosettes with SRBC coated with rabbit antibody, and human and mouse lymphocytes did not bind SRBC sensitized with chicken antibody. Only avian sera were effective in blocking the Fc receptor. Similarities between chicken and mammalian Fc receptors were demonstrated as both are pronase sensitive, trypsin resistant, and are distinct from surface immunoglobulin. Fc receptors were also distinguished from avian bursa-and thymus-specific antigens. Additional Fc receptor-bearing cells were revealed in bursa, spleen and bone marrow lymphocytes after neura-minidase treatment.     

Fc Receptor-Bearing Lymphoid Cells in the Chicken II. Relative Increase of Fc(IgG) Receptor Bearing Cells in Obese Strain Chickens

An EA rosette technique is used to study ontological development and organ distribution of Fc(IgG) receptor-bearing lymphoid cells in normal CS White Leghorn chickens, and in OS chickens with spontaneous autoimmune thyroiditis. During the embryonic period, no difference was seen between CS and OS in the tissue distribution of cells with Fc receptors. At the time of hatching and subsequently, the OS chickens possessed relatively more Fc receptor-bearing lymphoid cells than did CS chickens. The increase of Fc receptor-bearing lymphoid cells was most prominent among spleen cells. No difference in the affinity of Fc receptors between lymphocytes of the OS and CS chickens was demonstrated. The possible role of Fc receptor-carrying cells in the development of autoimmune thyroiditis is discussed.     

Biological Significance of Fc Receptor-Bearing Cells Among Activated T Lymphocytes

Lethally irradiated mice injected with syngeneic thymocytes and immunized with protein antigens develop specific helper T cells. If injected with semiallogeneic thymocytes, such mice generate H-2 antigen-specific cytotoxic T cells. Most spleen cells from these chimeric mice possess Fc receptors The present results demonstrate that the development of Fc-rcceptor-bearing cells in thymocyte injected irradiation chimeras seemingly is due to the physiological conditions in the mice rather than to the specific immunization. As a corollary, both helper T cells and cytotoxic T tells did not have Fc receptors, at least not in their effector state. Thus, Fc receptors on T cells would seem irrelevant to their immune function.     

Transient Increase of IgG Fc Receptor-Bearing T Lymphocytes following Positive PPD Skin Testing

In tuberculin-sensitive individuals, IgG Fc receptor (FcR)-bearing lymphocytes in the peripheral blood increased transiently following PPD-tuberculin skin test. This rise in circulating FcR-bearing cells appeared to peak about 36--48 h after the intradermal inoculation of PPD and seemed to occur largely in the T cell population. Skin test-negative individuals showed no significant changes in their circulating FcR-bearing cells following PPD inoculation. Peripheral blood lymphocytes from PPD-sensitive individuals were fractionated into non-T cell and T cell-enriched populations by E rosette sedimentation technique. FcR-bearing cells in the T cell-enriched population were eliminated by EA rosette sedimentation: i.e. FcR-negative T cells. Then, equal numbers (1 X 10(5) cells each) of non-T cells and unfractionated or FcR-negative T cells were recombined in culture. Prior to PPD inoculation, there was no significant difference between these two cell mixtures in the in vitro cellular response to PPD or mitogens. When these cell populations were obtained 36--48 h after PPD inoculation, however, the combination of non-T cells and FcR-negative T cells responded to PPD much better than the combination of non-T cells and unfractionated T cells, whereas the mitogen-induced cellular proliferation of these two cell mixtures did not differ from each other.     

Suppressor Activity of T Cells Bearing Fc Receptors for IgG on Lymphoid Colony Formation

Human T lymphocytes, upon phytohaemagglutinin stimulation, are able to form colonies in semisolid media. Peripheral T cells bearing Fc receptors (T_G) were studied for their possible regulatory activity on lymphoid colony development. Although no substantial differences were observed between the cloning efficiency of unfractionated T cells (thus including T_G cells) and T cells depleted of T_G, a sharp suppression of colony formation occurred when positively selected T_G cells were readded to T_G-depleted suspensions. Therefore, T_G suppressor activity seems to be strictly dependent upon cell interaction with ox IgG immune complexes used for T_G cell isolation. Different experimental approaches failed to demonstrate, although did not exclude, that suppression in this system is mediated by soluble factors, γ-irradiation of T_G cells abrogated their suppressor capacity.     

Characterization of the Fc(IgG) receptor on the pluripotential leukaemia cell K-562.

K-562 cells express an Fc receptor that is murine isotype IgG specific. The receptor was defined by rosette formation using sheep erythrocytes sensitized with murine monoclonal haemagglutinin (EA) of known isotype. Optimal rosette formation occurred at 4 degrees C or ambient temperature; however, the number of rosettes formed at ambient temperature decreased after 8 h whereas formed rosettes were stable at 4 degrees C. EA in which IgG2b was the immunoglobulin isotype gave high numbers of rosettes while IgG2a gave lower but significant numbers. Aggregated human IgG inhibited rosette formation of EA(IgG2a) more easily than those of EA(IgG2b), indicating a higher affinity of the Fc receptor for IgG2b.     

Lymphoid Stem Cells in the Intraembryonic Mesenchyme of the Chicken

Chromosomally marked cells from the 7-day intraembryonic mesenchyme were transplanted into 14-day-old irradiated chick embryos. At the age of 6 weeks donor-derived T and B lymphocytes were shown to be present in the thymus, spleen and bone marrow, indicating that cells in the 7-day intraembryonic mesenchyme are capable of developing into functional T and B lymphocytes. In addition to the sex chromosome marker, IgG allotype was used as a marker; the results demonstrate that cells from intraembryonic haemopoietic sites develop into mature IgG-producing cells. In similar experiments, the 7-day yolk sac also proved to contain lymphoid stem cells. Since lymphoid cell progenitors are not present in the 2-day yolk sac, as has been shown previously in the yolk sac-embryo chimaeras and since circulation is established from day 2 of incubation onwards, lymphoid stem cells present in the 7-day yolk sac are most likely secondary immigrants originating in the intraembryonic mesenchyme.     

Interaction Between a Synthetic Polypeptide, TIGAL, and Fc Receptors on Non-Bursa-Derived Chicken Lymphoid Cells

Autoradiographic studies have shown that radioiodinated TIGAL binds in vitro to a small but varying fraction of lymphoid cells from bursectomized, agammaglobulinemic chickens, whereas no binding of radioiodinated TGAL or a variety of other radioiodinated antigens can be observed. The binding of [125I]TIGAL is inhibited by antigen-antibody complexes. Radioiodinated antigen-antibody complexes are bound to a similar proportion of the lymphoid cells from bursectomized chickens, and this binding is inhibited by preincubation of the cells with unlabeled TIGAL but not with TGAL. These results indicate a cross-reaction at the level of Fc receptors between determinants on TIGAL and on IgG.     

Intraembryonic Origin of Lymphoid Stem Cells in the Chicken: Studies with Sex Chromosome and IgG Allotype Markers in Histocompatible Yolk Sac-Embryo Chimaeras

Two-day-old chick embryonic bodies were transplanted onto the area vasculosa of age-matched histocompatible blastoderms, resulting in the development of yolk sac-embryo chimaeras. Eighteen of these succeeded in hatching and became adults. Differences in the sex chromosomes and in IgG allotype between the embryo and the yolk sac were used to study the contribution of these two components to the lymphoid cell development. At 5-7 weeks of age the chimaeras proved to be completely normal in the IgM and IgG antibody production against human gamma globulin and Brucella abortus and in the lymphocyte responses to phytohaemagglutinin and concanavalin A. For the sex chromosome analyses bursa cells and specifically stimulated B and T lymphocytes were used. The latter was achieved by stimulating thymus, spleen, and bone marrow cells in vitro with anti-Ig and Con A. Only four out of 1498 mitoses analysed belonged to the sex opposite to that of the bird. Among the chimaeras eleven were marked through IgG allotypes. At the age of 3-20 weeks all eleven chimaeras showed serum IgG of the embryo allotype and none of the yolk sac type. These results, based on the use of two different markers, indicate that lymphoid stem cells in the chicken are originally derived from an intraembryonic source and not from the yolk sac.     

Effect of rIFN-γ on Antibody-Mediated Cytotoxicity via Human Monocyte IgG Fc Receptor II (CD32)

Human monocytes and macrophages express an isoform of IgG Fe receptor II (FcγRII), FcγRII. Two allotypic variants of this receptor could be distinguished with respect to their ability to bind murine (m)IgG1 complexes either strongly or weakly, defined as high-responder (HR) and low-responder (LR). respectively. We investigated the effect of recombinant (r)IFN-γ on the ability of freshly isolated monocytes, and those cultured for 40 h and 9 days, to mediate antibody-dependent cell-mediated cytotoxicity (ADCC), Using human erythrocytes (E) sensitized with mlgGl as target cells, FcγRII was studied selectively. Cells which had been cultured for 40 h exhibit a significantly decreased FcγRII expression, and FcγRll-mediated ADCC activity as compared with freshly isolated monocytes. Co-culture with rlFN-γ (40 h) reversed this decrease. Short-term rlFN-γ-cultured cells, and fresh cells express similar numbers of FcγRII, and exhibit comparable FcγRII-mediated ADCC activity. Phagocytic activity was not affected. Prolonged culture of monocytes for 9 days, co-cultured with rlFN-γ either from day 0 or from day 7, did not affect expression or functional activity of FcγRII. Furthermore, the effects were observed in both HR and LR individuals.
Our results show that rlFN-γ has strong effects on FcγRII-mediated responses specifically during the early stages of monocyte maturation, most likely by affecting receptor expression levels.     

IgG on Infants'' B Lymphocytes: Enhanced Binding of IgG by IgM-Bearing Lymphoid Cells in Early Childhood

IgM/IgD-bearing lymphocytes (B cells) from children in the first few weeks of life were found to also have surface IgG, unlike most normal adult B cells. The IgG was loosely bound to the lymphocyte surface and was partially or completely removed by incubation at 37 degrees C or by trypsinization. When F(ab')2 antisera were employed, very few infant B cells had surface IgG, although the IgM staining was similar to that obtained with the native antisera. IgM/IgD-positive cells bound IgG anti-Rh-coated (Ripley) erythrocytes, unlike most adult B lymphocytes. Capping experiments suggested that an Fc receptor on the cells could be redistributed by the anti-IgM-surface IgM complex. These data indicate that, during infancy, B-lymphocyte receptors for IgG are of altered affinity, frequency, or availability compared with adult B-lymphocyte Fc receptors and resemble the Fc receptors found on "third population" (Fc + Ig-) mononuclear cells and monocytes.     

Molecular recognition of antibody (IgG) by cellular Fc receptor (FcRI)

Earlier studies from this and other laboratories have provided indirect evidence for the involvement of the C gamma 2 domain of human IgG in the binding of IgG to the high affinity monocyte Fc receptor (FcRI). Two approaches have been used to extend these studies and to further localize the site of interaction on human IgG. Firstly, monoclonal antibodies (MAbs) directed against different epitopes on IgG were assayed for their capacity to inhibit the binding of radiolabelled IgG to human monocytes or U937 cells. The capacity of the MAbs to interact with their respective epitopes on FcR-bound IgG was also studied using indirect radiobinding and immunofluorescence assays. Secondly, a number of IgGs from several different species and fragments of human IgGs were assayed for their ability to inhibit the binding of radiolabelled IgG to human monocytes. The amino acid sequences of those IgGs exhibiting relatively tight, intermediate or weak binding to monocyte FcRs were compared. On the basis of these studies a possible monocyte FcR-binding site on human IgG is postulated, involving the lower hinge region of IgG (residues Leu 234-Ser 239) with possible involvement of the nearby N-proximal bend and two beta-strands (Gly 316-Lys 338).     

Direct demonstration of binding of aggregated mouse IgG1 to the 40-kDa Fc receptor for IgG (Fc gamma RII) in both high and low responders.

Several directly fluorochrome-conjugated murine monoclonal antibodies (mAb) of the IgG1 subclass and directed against B or T cell antigens were found to bind to monocytes via the 40-kDa Fc receptor for IgG (Fc gamma RII). As expected from the established polymorphism of Fc gamma RII, strong staining was observed in about 75% of individuals. In the remaining 25% staining was clearly weaker, but could be definitely demonstrated with a mAb against the B cell-specific CD19 differentiation antigen. Specificity of binding to Fc gamma RII was confirmed by the ability to block the binding of the CD19 mAb by pre-incubation with aggregated IgG1 or with mAb against Fc gamma RII. The extent of T cell proliferation induced with a CD3 mAb of the IgG1 isotype (a-Leu 4), which is dependent on the interaction of monocyte Fc gamma RII with the Fc portion of the CD3 mAb, exactly correlated with the amount of binding to Fc gamma RII in all individuals. Proliferation was dose-dependent for both high and low responders; cells of low responders did not proliferate at concentrations below 16 ng/ml of mAb, whereas there was a small but unequivocal proliferation at higher concentrations. These results confirm that monocytes from previously characterized "non-responders" are able to bind aggregated murine IgG1 via Fc gamma RII. They also demonstrate that directly labeled mAb can cause extensive nonspecific staining which may not be excluded by the use of control antibodies of the same isotype.     

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.     

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.     

Characterization of herpes simplex virus type 1-induced Fc receptor in its interaction with rabbit immunoglobulin G (IgG)

Herpes simplex virus (HSV) induces a receptor on infected cells that is able to bind the Fc part of immunoglobulin G (IgG). We have examined some basic physicochemical and binding properties of the Fc receptor induced on HSV-1 infected green monkey kidney (GMK) cells in its interaction with rabbit IgG. Fixation of HSV-1 infected cells with glutaraldehyde, formaldehyde, acetone or ethanol did not inhibit the Fc binding ability. The binding specificity of the receptor was not affected by ethanol treatment and all subsequent binding studies were performed with cells treated with ethanol. The receptor was detected within 4 hours of infection and the binding increased until 16 hours post infection. The interaction between ligand and receptor was dependent on pH with a binding optimum around pH 8.0 and 8.5. EDTA, but not EGTA, inhibited receptor binding, suggesting participation of divalent cations in the receptor-ligand interaction. Inhibition of binding was also seen when cells were preincubated for 30 min at 56 degrees, 60 degrees and 100 degrees C in contrast with cells incubated at 37 degrees and 45 degrees C. The number of binding sites on ethanol-treated GMK cells 18 hours after infection was estimated to be around 4 x 10(6)/cell and the affinity constant at approximately 2 x 10(7) M-1.     

Lymphocyte receptors. I. Receptors for Fc of IgG and complement (C3b) on immunoglobulin-bearing, antigen-binding and antibody-secreting cells.

Immunoglobulin (Ig) bearing, antigen-binding and antibody-secreting cells were investigated for the presence of membrane receptors for the Fc part of IgG and for C3b on their surface. Fc and C3b receptors were detected on the surface of most Ig-bearing and of most antigen-binding cells. Fc receptors were also detected on IgM antibody-secreting cells but not on IgG-secreting cells. C3b receptors were not detected on any antibody-secreting cells. These receptors are either lost from the B cell surface as they differentiate into antibody-secreting cells or are blocked in vivo by immune complexes or C3b.     

A Method for the Detection and Quantitation of Fc Receptor Sites on Cells

A new rosette technique for identification of Fc-receptor-bearing cells is based on the ability of sheep erythrocyte coated with protein A of Staphylococcus aureus (ES) to form rosettes with cell treated with monomeric IgG or aggregated IgG. The IgG is attached to lymphocytes through its Fc region and to a trypsin-resistant but pronase sensitive receptor (considered an Fc receptor) The ES rosette technique facilitates studies of the interaction of IgG with Fc receptor sites; the binding of any IgG preparation reacting with protein A of Staphylococcus aureus (SpA) can be studied by the technique. Inhibition of rosette formation by SpA was used for quantitation of IgG fixed to the cell surface (that is, the number of Fc reception/cell). The sensitivity of the method permits quantitation of less than 10⁵ IgG molecules bound to the Fc receptors. The relative affinity constant between Fc receptors and IgG ligands was estimated by plotting the percentage of ES rosettes as a function of IgG concentration and calculating the reciprocal of the IgG concentration giving half the maximal number of ES rosettes.