Further evidence for heterogeneity of Fc gamma-receptors on guinea pig splenic B and T lymphocytes: analysis using monoclonal antibodies to two distinct types of Fc gamma-receptor

In our previous paper, we reported that guinea pig splenic lymphocytes expressed two distinct Fc-receptors for homologous IgG (Fc gamma Rs), one monospecific for IgG2 (Fc gamma 2R) and the other bispecific for IgG1 and IgG2 (Fc gamma 1/gamma 2R), when analyzed by EA-rosette assay. These Fc gamma Rs on the cells were further studied by using two monoclonal antibodies toward the Fc gamma Rs on guinea pig peritoneal macrophages (anti-Fc gamma 1/gamma 2R and anti-Fc gamma 2R antibody). The anti-Fc gamma 1/gamma 2R antibody completely inhibited the rosette formation of splenic lymphocytes with IgG1-sensitized sheep erythrocytes [EA(IgG1)]. On the other hand, EA(IgG2)-rosette formation was inhibited partially by anti-Fc gamma 2R but not by anti-Fc gamma 1/gamma 2R antibody. Complete inhibition of the EA (IgG2)-rosette formation was achieved by simultaneous additions of both anti-Fc gamma 2R and anti-Fc gamma 1/gamma 2R antibodies. The binding of IgG2 antibody complexed with ovalbumin to the cells was partially inhibited by either anti-Fc gamma R antibody, and complete inhibition occurred in the presence of both the antibodies, indicating that two types of Fc gamma R, Fc gamma 1/gamma 2R, and Fc gamma 2R, are expressed on the cells. The determination of these Fc gamma Rs on B and T lymphocytes by two-color flow cytometry showed that about 52% of B lymphocytes expressed Fc gamma 1/gamma 2R alone and 32% of the cells expressed both the Fc gamma Rs. On the other hand, about 12% of T lymphocytes was found to express Fc gamma 2R alone and the cells expressing Fc gamma 1/gamma 2R were in the minority (3.8%). T lymphocytes expressing both the Fc gamma Rs were not detected. These results show that guinea pig B lymphocytes bear two types of Fc gamma Rs and are heterogeneous with regard to their Fc gamma Rs and that T lymphocytes express Fc gamma 2R mainly.     

Insoluble immune complexes suppress mitogen-induced proliferation of human T lymphocytes bearing Fc gamma receptors.

Human peripheral blood lymphocytes were mixed with erythrocyte-antibody (EA) complexes and separated into EA-rosette forming cell (EA-RFC)-enriched and EA-RFC-depleted suspensions. Thymidine incorporation of EA-RFC-enriched population in the presence of T cell mitogens (PHA, Con A, PWM) was about half of that of EA-RFC-depleted or of unseparated cells. The dose-response curves and kinetics of proliferation were found to be very similar in the three populations. Proliferative response of EA-RFC-enriched lymphocytes was strictly T cell dependent, although non-T cells were later recruited to incorporate thymidine. The interaction of T lymphocytes bearing surface receptors for IgG (T_G) with insoluble complexes followed by a post-binding temperature sensitive event, resulted in the modulation of Fc receptors associated with an impaired proliferative response to PHA, Con A, and PWM, without significant change in metabolic cell activity as shown by cell viability, sponaneous leucine incorporation, or β₂ microglobulin release.     

Clinical evaluation of the EA-rosette test in the early detection of cervical cancer

It was previously found that a negative EA-rosette test, showing EA-rosette-forming cells in a cervical cell suspension, excluded the presence of cells of invasive carcinoma (predictive value of 99.9%). This study on 2,462 patients confirmed the applicability of the EA-rosette test in screening for precancerous as well as cancerous lesions. In 98.6% of the cases of dysplasia, carcinoma in situ and invasive carcinoma, the cervical cell suspensions contained EA-rosette forming cells (the rosette test was positive). With a negative EA-rosette test, the probability of missing a specimen with class III cytology (mild/moderate dysplasia) was 1.4%, of missing one with class IV cytology (severe dysplasia/carcinoma in situ) was 0.8% and of missing one with class V cytology (invasive carcinoma) was 0.25%. The predictive value of a negative EA-rosette test was 98.6%. The false-negative rate for negative EA-rosette tests was 3.7% for invasive carcinoma, 17.5% for carcinoma in situ and severe dysplasia and 41.4% for mild to moderate dysplasia.     

IgG Fc receptor-bearing cells during early lymphoid cell development in the chicken

Cells from intraembryonic mesenchyme, yolk sac, bursa of Fabricius, and thymus from chicken embryos at different stages of development were studied for the presence of IgG Fc receptors by EA-rosette formation and binding of heat-aggregated chicken IgG (agg IgG). Cells with Fc receptors were found in high frequency in the intraembryonic mesenchyme as early as on the third day of incubation, in the yolk sac on the 7th day, in the bursa on the 10th day, and in the thymus on the 16th day of embryonic development. In the bursa the number of agg IgG binding cells increased with the age of the embryo and remained high after hatching, whereas in the thymus the peak value (76%) was observed on the 16th embryonic day, and after hatching only about 10% of the cells expressed the agg IgG receptors. The results also suggest that the appearance of IgG Fc receptors precedes the expression of B-L (Ia-like) antigens and of cytoplasmic and surface immunoglobulins on early lymphoid cells of the chicken embryo.     

Quantitative parameters of the IgG binding by Fc receptors and the functional activity of blood lymphocytes in healthy cattle and cattle with chronic lympholeukemia

The binding of IgG to lymphocyte Fc receptors in the blood of healthy cattle and of cattle with chronic lympholeukemia has been studied by fluorometric techniques before and after the incubation of lymphocytes in a serum-free medium at 37 degrees C. The study has shown that changes in the intensity of binding of IgG to Fc receptors of normal and leukemic lymphocytes correlate with changes in the cell-mediated cytotoxic activity of the corresponding lymphocytes. Lower cell-mediated cytotoxic activity of leukemic lymphocytes in comparison with that of normal lymphocytes was parallelled by a lower association constant of IgG with leukemic lymphocytes Fc receptors.     

Interaction of blood lymphocyte Fc receptors with IgG during in vitro cultivation

Bovine blood lymphocytes taken from normal cows and those suffering from chronic lymphocytic leukemia were cultured in complete medium 199 with 10% of heat-inactivated fetal bovine serum. After a 48 hour culturing an enhanced quantity of the Fc-receptor bound fluoresceinated immunoglobulin G (IgG) was established. When lymphocyte fractions enriched with T- or B-cells were cultured, the binding capacity of Fc-receptors for IgG (48 hours after culture) increased in both the cell populations. A study of the kinetics of interactions of Fc-receptors with IgG showed that the increased number of Fc-receptors after culturing was followed by an enhanced affinity of Fc-receptors towards IgG. The affinity of Fc-receptors of blood lymphocytes of cows with chronic lymphocytic leukemia was lower than that of normal lymphocytes.     

Antigen-initiated B lymphocyte differentiation. IX. Characterization of memory AFC progenitors by buoyant density and sedimentation velocity separation.

The characteristics of memory B cell antibody-forming cell (AFC) progenitors from long-term hapten-primed CBA mice were investigated by using sedimentation velocity and buoyant density separation to isolate physically distinct B cell sub-sets. The isolated fractions were assayed by the adoptive immune response to NIP-POL antigen, under conditions where neither T cells nor other accessory cells were limiting the IgM or IgG AFC responses. The results were compared to previous studies on the IgM AFC-progenitors of unprimed adult mice. Splenic IgM and IgG memory AFC-progenitor activity was largely found among the typical B cells of slow to medium sedimentation rate, in contrast to the fastre sedimenting IgM AFC-progenitor activity of unprimed animals. Splenic IgM and IgG memory AFC-progenitor activity was found among the medium to light density cells, and so resembled by this parameter the IgM AFC-progenitor activity in unprimed animals. Thoracic duct lymphocytes from hapten-primed mice also exhibited memory IgM and IgG AFC-progenitor activity in the slow-medium sedimentation range. However, in contrast to spleen, the IgM and IgG memory AFC-progenitor activity in lymph was found among very dense B cells. Two physically distinct sub-populations of memory B cells have thus been identified, namely: i) small, medium-light density, presumably tissue-resident B lymphocytes found in spleen; and ii) small, dense, presumably recirculating B lymphocytes found in lymph. Both physical forms include IgM and IgG progenitors. Both forms are distinct from the larger, medium-light density "virgin" AFC-progenitors in the spleen of unprimed adult mice.     

Delineation of the development of T lymphocytes from leukemic null lymphocytes upon induction by conditioned medium

Null lymphocytes, lacking B- and T-lymphocyte markers, were isolated from PBL of patients with acute lymphocytic leukemia. Upon culture in the presence of medium produced from PHA-stimulated allogeneic lymphocytes, induction of these cells to T lymphocytes occurred. Within 18 hr, they acquired the capacity to form SRBC rosettes and bind complement components, IgG or IgM. This differentiation, as supported by cell cycle kinetic analysis, was accompanied by lymphocyte activation, inferable from warm SRBC rosetting. Capping of these rosettes was identified as indicative of early T lymphocytes. During T-lymphocyte development subclasses bearing complement receptors, followed by IgG and finally IgM receptors appeared. The emergence of subclasses, functionally expressed by a gain in MLC responsiveness and stimulatory capacity reflected T-lymphocyte maturation. Simultaneous with the later decrease of the subclasses was a decrease in MLC reactivity. This elucidation of T-lymphocyte development may help in dissection of the lymphoid system.     

Immunogold silver staining method for light microscopic detection of peripheral blood lymphocyte surface antigens with monoclonal antibodies.

Labelling of peripheral blood Lymphocytes surface antigens was carried out using the method of colloidal gold, enhanced with silver staining. Instead of PBS the minimal essential medium (MEM) according the Eagle, pH 7.2, was used rinsing of isolated lymphocytes. Visibility of positive reactions on lymphocytes at application of both mentioned media was the same. Positive reaction at demonstration of p24 BLV on cells acquired the from of black cap while the IgG expression was observed in the from of diffuse dispersion of colloidal gold on cells. Differences between the application of individual media were observed in the shape of peripheral lymphocytes in smears. Utilization of Eagle's MEM resulted in more uniform shapes and optically smooth surfaces when viewed under a light microscope.     

An appraisal of Fc receptors on human peripheral blood B and L lymphocytes.

Human circulating lymphocytes with easily detectable surface immunoglobulin have been divided into two populations, B cells and L cells. This second population lacks membrane-incorporated Ig, but has a receptor for membrane-labile cytophilic IgG. In this study purified B and L lymphocytes were examined for Fc receptors that bind aggregated IgG and IgG complexed to erythrocytes. Purified lymphocyte populations were prepared by nylon columns and by negative selection with rosetting techniques. L lymphocytes bound aggregated guinea pig and human IgG, and formed rosettes with human erythrocytes sensitized with Ripley IgG (EA). Treatment of L lymphocytes with trypsin had no effect on the receptors for IgG. B lymphocytes did not bind EA and attachment of aggregated IgG was variable; up to one-third of these cells fixed aggregated human IgG to the cell membrane. Trypsin treatment abolished binding of Agg-IgG to B cells in sharp contrast to its effect on L cells. Furthermore, double-label immunofluorescence studies showed that cells with both membrane-incorporated Ig and receptors for aggregated guinea pig IgG were rare. These studies indicate that human peripheral blood B lymphocytes lack a high affinity, trypsin-resistant Fc receptor that is present on L lymphocytes.     

Isolation and immunochemical characterization of immunoglobulin-binding factor from bovine peripheral blood lymphocytes

The shedding of immunoglobulin-binding factor (IBF) has been studied in peripheral blood lymphocytes during 1 h incubation at 37 degrees C. IBF was detectable in the incubating medium on the basis of their ability to bind immunoglobulin G (IgG) specifically. IBF was affinity purified on Sepharose beads coated with bovine IgG, fluoresceinated and identified by their biological activities, i.e., for binding immunoglobulin-binding factor labeled with fluoresceinizothiocyanate (FITC-IBF) to erythrocytes coated with purified antibodies (EAIgG) by fluorometric binding assay. The effect of various pH, temperatures and proteolytic enzymes on the binding properties of FITC-IBF to EAIgG was also studied. We showed that IBF are sensitive to pronase E, higher temperature and pH.     

Effects of antibodies to MHC gene products on different Fc-receptor-mediated cell functions

The effects of rabbit anti-HLA-DR and anti-β₂-microglobulin (anti-β₂m) antibodies on three different Fc-receptor-mediated cell functions of peripheral blood mononuclear leukocytes were studied. Both IgG and F(ab′)₂ fragments of anti-HLA-DR antibodies inhibited the cytotoxicity against a monolayer of antibody-sensitized sheep erythrocytes (plaque formation) whereas no effect was observed on the cytotoxicity against antibody-sensitized chicken erythrocytes in suspension (⁵¹Cr release), or on EA-rosette formation. On the other hand, all the three functions were inhibited by the IgG fraction of anti-β₂m antibodies but not by the corresponding F(ab′)₂ fragments. The results demonstrate that the plaque-forming cells (PFC) carry HLA-DR-like antigens. Furthermore, a closer association exists between the HLA-DR-like antigens and the Fc receptors than between the β₂m molecules and the Fc receptors on the PFC. The results further support our earlier investigations suggesting that the PFC are of monocytic origin.     

Determination of IgM and IgG on lymphocyte surfaces by means of registering rosette formation with anti-immunoglobulin-coated erythocytes

To identify IgM and IgG on the surface of normal bovine B-lymphocytes, the test of antiglobulin-rosette formation (anti-Ig rosettes) was used. IgM and IgG were found to exist on separate B-lymphocyte populations. The composition of receptors on the surface of lymphocytes, forming Ig rosettes, was investigated. The distribution of these lymphocytes in bovine blood and lymphoid organs was determined. IgM and IgG were detected on the surface of thymocytes, their origin remains open to question.     

Efficient production of IGG human monoclonal antibodies by lymphocytes stimulated by lipopolysaccharide,pokeweed mitogen,and interleukin 4

Summary Extensive screening of the mitogens lipopolysaccharide (LPS), pokeweed mitogen (PWM), andStaphylococcus aureus Cowan I (SAC I), alone and in combination and with and without interleukin (IL) was performed forin vitro activation of regional lymph node lymphocytes from lung cancer patients for the production of human IgG, IgM, and IgA. As assessed by electrofusion of the lymphocytes following their exposure to these agents with mouse myeloma cells and incubation of the fused hybridoma, a remarkable stimulatory effect was shown by LPS and particularly by LPS plus IL-4, which was substantially greater than that of either SAC I or PWM with or without various IL. Optimization studies indicated that the addition of PWM to LPS and IL-4 in the culture medium further stimulated the human antibody (Ab) production, and that the optimal formulation for stimulations of human IgG production was a culture medium containing 20 μg/ml of LPS, 1/500 of PWM, and 100 u/ml of IL-4.     

Immunofluorescence studies of the immunoglobulin receptors of B-lymphocytes]

The surface membrane Ig receptors of B lymphocytes in patients with atopic pollen asthma were studied by the direct immunofluorescence method. Use was made of labelled sera against human IgE, and IgG. It was shown that on the average 2.1 +/- 0.33% lymphocytes had IgE receptors, 8.4 +/- 0.63% IgG receptors and 18.7 +/- 1.16% human globulin receptors. A specific nature of B lymphocytes is suggested.     

Detection of receptors for the IgG Fc fragment as well as for the C3 component of complement in the spleen T-lymphocytes of CBA mice]

The presence of receptors to the Fc fragment of IgG and to the C3 component in the complement of T lymphocytes in the spleen of CBA mice was studied. The study was made by the method based on Coons's indirect immunofluorescence test with the use of antithymocytic globulin, in combination with EA and EAC rossette-formation. A significant percentage of T lymphocytes was found to have the receptor to the Fc fragment of IgG, while only a small part of T lymphocytes was found to have the receptor to the C3 component of the complement.     

Studies of surface immunoglobulins on human B lymphocytes. I. Dissociation of cell-bound immunoglobulins with acid pH or at 37 degrees C.

Lymphocyte preparations isolated from the human peripheral blood were exposed to different acid pH or incubated at 37 degrees C and the presence of immunoglobulin (Ig) on the cell surface was examined by immunofluorescence (IF) tests. Subsequently, such treated cells were incubated in the autologous serum or in the purified IgG, IgA or IgM proteins and their ability to bind each class of Ig was examined. The results showed that IgG molecules dissociated from large proportions of IgG-positive cells upon exposure to pH 4 at 1 degrees C for 1 min or upon incubation at 37 degrees C for 20 min. The cells from which IgG had been dissociated could again combine with IgG, whereupon the number of positive cells increased, being restored to the number of equivalent to or higher than those before acid or 37 degrees C treatment. These results indicated that the treatment could elute the cell-bound IgG present on the cell and that the receptor sites were not degraded by the treatment and could combine with IgG. These cell-bound IgG were observed not only on the monocytes, but also on the small lymphocytes. It was also found that certain proportions of mononuclear cells carried the cell-bound IgA that could be dissociated with acid pH or 37 degrees C. No cell-bound IgM was observed on any mononuclear cells. Microscopic observations before and after acid or 37 degrees C treatment revealed that the staining distribution of the cell-bound IgG and IgA on the cell was granular, appearing as a discontinuous fluorescence ring and forming multiple aggregates but no typical polar caps on warming. In contrast, IgG, IgA, and IgM stable to acid or 37 degrees C treatment were found on the lymphocytes but not on the monocytes, and their staining distribution was uniformaly diffuse, appearing as a continuous ring and forming a typical cap on warming. Exposure of the cells to pH 4 or 37 degrees C could also elute the cell-bound IgG passively adsorbed to the human lymphoid cells in a culture, but did not affect the intrinsic S.Ig on the lymphoid cells in a culture or on the lymphoma cells. These results indicate that the exposure of the cells to acid pH or to 37 degrees C may enable us to detect unfailingly S.Ig lymphocytes by removing the cell-bound IgG and IgA present on the monocytes and/or lymphocytes. Thus, an average value of approximately 10% was obtained for the S.Ig lymphocyte in the lymphocyte preparations from 11 healthy individuals. In addition, the results provided the evidence that, even in normal peripheral blood lymphocytes, there may be a population of B lymphocytes which lack the S.Ig but carry the cell-bound Ig.     

Activated T lymphocytes resist the toxic effects of the adenosine deaminase inhibitor, 2-deoxycoformycin

Tonsil lymphocytes from three adults and three children were examined for immunoglobulin (Ig) production before and after Epstein-Barr virus (EBV) transformation. T-cell depletion was required to obtain cell lines from EBV-seropositive individuals. Cytoplasmic Ig was mainly IgG in adult lymphocytes before and after transformation; IgA and IgM were more prominent after than before. IgM and IgG predominated in lymphocytes of children before and after transformation; IgA was more prominent after than before. Cytoplasmic Ig of peripheral blood lymphocytes from these individuals was mainly IgM. Secreted Ig from tonsil lymphocytes was mainly IgA or IgG; after transformation IgM predominated with adult cell lines, and IgG or IgM with cell lines from children. IgE was consistently sparse in spite of ragweed and/or grass allergies of the adults.     

Preliminary lymphocyte activation by a mitogen as a condition for demonstrating the ability of the Fab-fragment of normal IgG to inhibit lymphocyte transformation

Peculiarities attending inhibition of the PHA-induced blast-cell transformation of human lymphocytes by F(ab')2 fragment of rabbit IgG were studied. It was shown that the fragment did not affect the intensity of blast-cell transformation if the lymphocytes were preliminarily incubated with the fragment for 24 h at 37 degrees or 4 degrees C and then transferred to the fresh medium containing PHA. However, if the fragment was added to the cells 24 or 48 h following PHA it produced a significant inhibition of the blast-cell transformation. These data may indicate that F(ab')2 fragment interferes with the lymphocyte transformation only when the cells are already activated with PHA.