Characterization of the IgE Fc receptors on monocytes and macrophages

Subpopulations of human monocytes (15%) and alveolar macrophages (AM phi, 8%) and rat and mouse AM phi (89%) and peritoneal M phi (57%) bear Fc receptors for IgE (Fc epsilon R) as shown by IgE-specific rosette formation. Cells from M phi-like cell lines of human, rat, and mouse origins also express Fc epsilon R. Monomeric IgE binds to Fc epsilon R on M phi with an equilibrium association constant Ka congruent to 10(7) M-1. The Fc epsilon R on human monocytes and M phi are antigenically similar to Fc epsilon R on lymphocytes but differ from Fc epsilon R on basophilic granulocytes. The Fc epsilon R on human and mouse M phi promote phagocytosis and lysis of IgE-coated erythrocytes. Patients with active IgE-mediated allergic diseases have elevated percentages of Fc epsilon R(+) monocytes (56%) that show allergic increased lytic activity against IgE-coated erythrocytes as compared to monocytes from normal humans. M phi from rats infested with Nippostrongylus brasiliensis parasites express more Fc epsilon R than normal M phi. The data indicate that Fc epsilon R expressed on M phi differ from those on mast cells and basophils, increase in number during IgE immune responses, and are likely to play an important role in the host's defense against parasites and in the pathogenesis of allergic diseases.     

Lymphocytes bearing Fc receptors for IgE. II. Induction of Fcepsilon-receptor bearing rat lymphocytes by IgE.

The proportion of lymphocytes bearing receptors for IgE (FcepsilonR) markedly increased after infection of rats with Nippostrongylus brasiliensis (Nb). The FcepsilonR-bearing lymphocytes from the infected animals bound more IgE-coated erythrocytes in rosette assay than FcepsilonR-bearing cells from normal rats, suggesting that the number of FcepsilonR per cell may also increase following the infection. In contrast, the number of IgE-receptors on peritoneal mast cells did not change after Nb infection. The increase in the proportion of FcepsilonR-bearing lymphocytes in Nb-infected rats is probably due to an increased concentration of IgE in the environment. The proportion of FcepsilonR-bearing cells in normal rat lymphocyte suspensions increased by culture of the cells with rat IgE of 1 microgram/ml or higher concentration. Other immunoglobulins such as rat IgG, human IgE, or rabbit IgG failed to induce either FcepsilonR-bearing cells or FcgammaR-bearing cells. It was also found that induction of Fc receptors by rat IgE is confined to FcepsilonR. Kinetic studies on the induction of FcepsilonR-bearing lymphocytes in vitro showed that the proportion of these cells in lymphocyte suspensions increased within 8 hr incubation with rat IgE but not within 4 hr. Evidence was obtained that both RNA synthesis and protein synthesis, but no DNA synthesis, are required for the induction of FcepsilonR-bearing cells or the expression of the receptors on the cell surface.     

Human monocytes, B lymphocytes, and non-B lymphocytes each have structurally unique Fc gamma receptors

Human Fc gamma-binding macromolecules were isolated from subpopulations of mononuclear cells by repetitive affinity chromatography. Mononuclear cells, nylon wool-filtered cells, plastic-nonadherent cells, and plastic-adherent cells from normal donors were radiolabeled by using 125I and lactoperoxidase. Washed cells were solubilized in 1% NP-40 buffer containing proteinase inhibitors at 0 degrees C. Fc gamma receptors were purified on human IgG-Sepharose columns by use of the repetitive affinity chromatography procedure. Analysis by SDS-polyacrylamide gel electrophoresis demonstrated only a 52,000 to 58,000 Mr Fc gamma receptor from nonadherent cell populations. Both rosetting and nonrosetting subpopulations of non-B lymphocytes expressed the 52,000 to 58,000 Mr receptor. The predominant Fc gamma receptor isolated from plastic-adherent cells was a 60,000 to 68,000 Mr macromolecule. Cell preparations enriched in B lymphocytes yielded prominent 43,000 Mr Fc gamma receptors. Thus human monocytes, B lymphocytes, and non-B lymphocytes each appear to have structurally distinct and unique Fc gamma receptors.     

Action of pertussigen (pertussis toxin) on serum IgE and on Fc epsilon receptors on lymphocytes

Pertussigen (pertussis toxin (PT] is one of the most effective stimulators of IgE production in mice and rats. Employing flow microfluorimetric analysis (FMF), we showed that PT increases the percentage of blood and spleen lymphocytes with IgE on their surface. The percentage of IgE-bearing cells in the spleen of normal untreated C57Bl/10SCN mice of various ages varied from 2.2 to 12.2%, with an average value of 6.1 +/- 5.4%. In mice treated with 400 ng of PT and 1 mg of chicken egg albumin (EA), the percentage of these cells increased, 14 days after immunization, to an average value of 31.1 +/- 2.2%. Immunization of mice with PT alone increase the percentage of IgE-bearing cells only slightly (13.1 +/- 2.2% of the splenic lymphocytes) while injection of 1 mg of EA alone did not have any detectable action. As little as 6 ng of PT, when given simultaneously with 1 mg of EA, increased the percentage of IgE-bearing lymphocytes. A booster dose of 10 micrograms of EA given on Day 14 induced a further increase in the percentage of these cells even when as little as 0.039 ng of PT had been given at the time of initial immunization. PT was effective when given 4 days before or 5 days after EA. EA was effective when given 4 days before or 4 days after PT, but not 8 days after. The increase in IgE-bearing cells was mainly due to cytophilic binding of IgE to receptors for the epsilon chain of IgE (Fc epsilon) on the surface of lymphocytes rather than to a greater number of IgE-producing cells. This was shown by removing the IgE from Fc epsilon receptors by acid treatment which reduced the percentage of IgE-bearing cells to nearly normal values. The antibodies of IgE class with specificity to EA were increased dramatically, while antibodies with specificity to PT were not detected.     

Fc receptors for IgA on human B, and human non-B, non-T lymphocytes.

Recently, receptors for IgA were demonstrated on subpopulations of human T lymphocytes. In this report, TNP-modified ox erythrocytes coated with the IgA myeloma MOPC-315 were used to detect IgA receptor-bearing lymphocytes within the human non T cell lymphocyte population. A mean of 5.3% (range 2.9 to 12.4%) of E-rosette negative human lymphocytes bound IgA-coated indicator cells. Blocking studies with soluble IgA, IgG, and IgM demonstrated that the IgA receptors on the non-T cell populations were separate and distinct from the Fc-receptors for IgG and IgM. Fractionation of the non-T lymphocytes on anti-human (Fab)2 columns into sIg+ and sIg- populations or by rosetting with EAC to provide CRL+ and CRL- populations demonstrated that Fc-IgA receptors were present on a subpopulation of sIg+, CRL+ lymphocytes, and also on sIg- (non-T, non-B) lymphocytes.     

Studies of Fc gamma receptors of human B lymphocytes: phospholipase A2 activity of Fc gamma receptors

The presence of phospholipase A2 activity within human B cell Fc gamma receptors was investigated. Lysate produced by detergent treatment of chronic lymphocytic leukemia cells that had 1% of the cells surface radioiodinated was subjected to affinity chromatography by using either rac-1-(9-carboxynonyl)-2-hexadecylglycero-3-phosphorylcholine-Sepharose (PC-Sepharose) or heat-aggregated human IgG-Sepharose 4B conjugate (IgG-Sepharose). The materials eluted from both adsorbants by ethylenediaminetetraacetate- or urea-containing buffer were further purified by gel filtration and isoelectric focusing in the presence of 6 M urea. Both isolated PC- and IgG-binding materials were homogeneous, when judged by gel filtration and isoelectric focusing, and had identical isoelectric points (pI = 6.5), peptide maps, and amino acid compositions. Furthermore, both preparations catalyzed equally the hydrolysis of phosphatidylcholine to release fatty acid from the 2 position. Optimal enzymatic activity depended on the presence of Ca2+, was maximal at pH 9.5, and was augmented by Fc gamma fragments. Both preparations specifically bound to the Fc portion of IgG and inhibited human antibody-coated erythrocyte rosette formation by peripheral mononuclear cells. Our data thus demonstrate the identity of PC- and IgG-binding materials and suggest that a functional activity of the human B cell Fc gamma receptor is the generation of phospholipase A2 activity within the plasma membrane.     

Fc receptors for IgE (Fc epsilon R) on human lymphoid cells: inducible expression of Fc epsilon RII (CD23) on lymphocytes and detection by monoclonal anti-Fc epsilon RII antibody

The studies presented herein describe (1) a sensitive, quantitative, and objective assay for detecting cell membrane-bound form of Fc receptors for IgE displayed on human lymphoid cells based on measuring unlabeled Fc epsilon R-bound IgE by a solid-phase RIA of cell lysate fluids; (2) the development and characterization of an IgM monoclonal antibody, termed 7E4, which is specific for human lymphocyte Fc epsilon RII (CD23) molecules; and (3) a system for reproducibly inducing de novo synthesis and expression of Fc epsilon RII proteins on human lymphocytes following exposure to the mitogenic lectin, pokeweed mitogen. The Fc epsilon RII molecules induced by exposure to PWM were proven to be present on lymphocytes, and not on other cell types in several ways, including (1) documenting sensitivity of such proteins to both acid pH and trypsin treatment, the latter manipulation being ineffective in removing Fc epsilon RII molecules on basophils and mast cells; (2) demonstrating specific reactivity of the expressed Fc epsilon RII molecules with the 7E4 monoclonal antibody, which is specific for human lymphocyte Fc epsilon RII molecules and does not react with Fc epsilon R molecules on other cell types; and (3) observing the required concomitant presence of both T and B lymphocytes during the induction process and proving that the induced Fc epsilon R+ cells are indeed B cells of the Leu-12+ phenotype by fluorescence analysis. The ability to induce expression of Fc epsilon RII molecules on human lymphocytes exposed to a mitogen such as PWM requires special technical attention to the method of preparation and isolation of human lymphoid cells from peripheral blood. This in vitro system for up-regulating Fc epsilon RII expression on human lymphocytes should provide us with an important new tool to analyze the participation of such cells in the regulatory mechanisms controlling the human IgE antibody system.     

Fc receptors and human T lymphocytes in frozen-thawed peripheral blood cells

The present study was carried out to investigate the influence of cryopreservation on human T-cell subsets defined by their membrane receptors for Fc IgM (TM) and Fc IgG (TG) and by their membrane antigens. For this purpose isolated T cells, obtained by neuraminidase-treated sheep erythrocyte (E-N) rosetting, and enriched mononuclear cells were cryopreserved using a programmed freezing procedure. A significant decrease of the TM and TG cells was found whereas the proportion of T cells and their subsets determined by monoclonal antibodies seemed not to be influenced. The effectiveness of T-cell separation by E-N rosetting of frozen lymphocytes demonstrated no impairment of the E-receptor binding capacity of T cells. The PHA reactivity of separated T cells was maintained after cryopreservation; however, the spontaneous blastogenesis was reduced significantly. The selective loss of the TM and TG cells seemed to be dependent on the length of the phase transition time; over 90 sec the capacity of the expression of Fc receptors was profoundly affected. Neither an additional 20 hr incubation after hypotonic shock prior to cryopreservation nor incubation after thawing could repair this function of T cells. The data suggest irreversible damage of the Fc receptor expression capacity on the cell membrane as a result of a disturbance of metabolic pathways rather than a preferentially greater sensitivity of these cells to cryopreservation.     

Murine B cell hybridomas bearing ligand-inducible Fc receptors for IgE

Interest in the regulation of IgE synthesis has generated investigation of low-affinity Fc receptors for IgE (Fc epsilon R) and the related immunoregulatory IgE-binding factors. In an effort to facilitate biochemical analysis of the B lymphocyte Fc epsilon R, hybridoma technology has been used to create stable cell lines that maintain Fc epsilon R in high numbers. Fusion of the HAT-sensitive B lymphoma, M12.4.5, with murine B cells from Nippostrongylus brasiliensis infected BALB/c mice led to the formation of hybrid cells of B cell phenotype, all of which were Fc epsilon R+, including several that had greater than 50,000 Fc epsilon R/cell. The Fc epsilon R on these cells were biochemically identical to the Fc epsilon R on normal B cells with respect to binding affinity (approximately equal to 10(8) M-1), m.w. (49,000), and tryptic peptides. Each hybridoma cell line specifically increased its Fc epsilon R level between twofold and fourfold when cultured with rat or mouse IgE. Additional studies demonstrated that the increased IgE binding ability was due to an increase in receptor number rather than an affinity change, and the Fc epsilon R increase was seen on the entire cell population. Dose studies indicated that oligomeric IgE was 10-fold more effective than monomeric IgE in causing upregulation, and the effective concentrations required indicated that induction occurred only if IgE was present in saturating concentrations. Upon addition of IgE, peak Fc epsilon R levels were reached after 15 to 20 hr of culture; blocking protein synthesis with cycloheximide largely blocked the increase in Fc epsilon R levels. Additionally, the inductive signal IgE must constantly be present to maintain upregulated Fc epsilon R levels in that its removal from the culture resulted in a rapid decline of Fc epsilon R from induced to normal levels. Because Fc receptor upregulation is important to several systems describing Ig isotype-specific regulation, the ability to examine such receptor upregulation at a clonal level should aid in discerning the role of the Fc epsilon R in the regulation of IgE antibody synthesis.     

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.     

Fc receptors on monocytes cause OKT3-treated lymphocytes to internalize T3 and to secrete IL-2

Monocytes cause OKT3-treated T cells to secrete IL-2 and to lose cell surface T3. We have studied the fate of the "lost" T3. Immunofluorescence microscopy on permeabilized cells showed that monocytes induce T cells to internalize T3. Furthermore, experiments with radioiodinated T cells showed that the internalized T3 was not degraded and exhibited an unaltered polypeptide composition for at least 16 hr. The role of Fc receptors in inducing internalization was also investigated. Fc receptors were depleted from monocytes by allowing the phagocytes to spread on immune complexes. Such depleted monocytes exhibit a fourfold reduction in their ability to promote both internalization of T3 and production of IL-2. A comparable reduction is seen if F(ab')2 fragments of OKT3 were employed in place of intact IgG. Furthermore, monocyte Fc receptors that have been blocked by heat-aggregated human IgG also show much reduced capability for induction of OKT3-mediated T-cell proliferation. We therefore conclude that Fc receptors bind to the Fc domain of OKT3 and thereby cause OKT3-treated T cells to internalize T3 and become activated.     

Murine follicular dendritic cells and low affinity Fc receptors for IgE (Fc epsilon RII).

The present study was undertaken to determine whether mouse follicular dendritic cells (FDC) bear Fc epsilon RII (CD23) and whether IgE-immune complexes are retained by FDC. Mouse Fc epsilon RII was localized by both L and electron microscopy using the mAb B3B4. In lymph nodes of normal mice, Fc epsilon RII was low but detectable on FDC. By 14 days after Nippostrongylus brasiliensis infection, the level of Fc epsilon RII increased on B lymphocytes located in the cortex of draining mesenteric lymph nodes. However, the Fc epsilon RII level on FDC remained low. Although numerous IgE-producing plasma cells were seen at day 14, very little IgE was associated with FDC. By 26 days after infection, Fc epsilon RII was observed on FDC in increased levels and IgE binding was clearly associated with FDC. Unexpectedly, FDC of control mice immunized with albumin in CFA to elicit an IgG response showed intense labeling for Fc epsilon RII. In contrast, the B cells exhibited very little Fc epsilon RII. IgE immune complexes were observed in association with FDC in the CFA-immunized mice. When mice were given a hapten-specific monoclonal of the IgE isotype, hapten carrier complexes were trapped and retained on Fc epsilon RII-bearing FDC. In conclusion, FDC were clearly one of the major murine cell types bearing Fc epsilon RII. IgE immune complexes were found in association with FDC and Fc epsilon RII appeared to play a major role in trapping and retaining IgE immune complexes. FDC Fc epsilon RII was subject to regulatory control, but the Fc epsilon RII level on FDC was regulated very differently from the Fc epsilon RII level on B cells.     

Human monocytes and U937 cells bear two distinct Fc receptors for IgG

Several convergent lines of evidence have led us to propose that human monocytes and the related cell line U937 possess a second class of IgG Fc receptor (FcR) in addition to the 72-Kd high affinity FcR previously described. IgG affinity purification from detergent lysates of surface radiolabeled U937 cells has yielded both a 40-Kd IgG-binding membrane protein (p40) and the 72-Kd FcR protein. By the same procedure, only the p40 was isolated from the erythroblast cell line K562 and from the B cell lines, Daudi and Raji. Serologic cross-reactivity between the 40-Kd FcR on U937 and Daudi cells was demonstrated using a goat anti-FcR antiserum. A murine (m) monoclonal antibody, raised against the FcR of K562 cells, precipitated the 40-Kd FcR from lysates of U937 and K562 cells but not from Daudi or Raji cells. This antibody, referred to as anti-p40 (IV.3), selectively inhibited the binding of murine IgG1-coated erythrocytes to U937 cells, whereas monomeric human IgG selectively inhibited binding of human anti-Rh(D)-coated erythrocytes to U937 cells. Both Daudi and U937 cells mediated mIgG1 anti-T3 (Leu-4)-induced stimulation of T lymphocytes. In contrast, mIgG2a anti-T3 (OKT3)-induced stimulation was supported effectively by U937 cells but only modestly by Daudi cells. Intact IgG or Fab fragments of anti-p40 (IV.3) blocked mIgG1 anti-T3 (Leu-4) stimulation but not mIgG2a anti-T3 (OKT3) stimulation of T cells; monomeric human IgG blocked only OKT3-induced stimulation. The simplest interpretation of these results is that human monocytes and U937 cells bear two classes of IgG FcR, one of 72 Kd and the other, as described above, of 40 Kd. We propose that the 72-Kd FcR mediates rosette formation with red cells coated by human anti-Rh IgG as well as T cell stimulation by mIgG2a anti-T3 (OKT3) and that the 40-Kd FcR mediates rosette formation with erythrocytes bearing mIgG1 as well as T cell stimulation by mIgG1 anti-T3 (Leu-4). Furthermore, we suggest that these two FcR are the human homologues of the murine macrophage FcRI (binding mIgG2a) and FcRII (binding mIgG2b/1).     

Differential modulation of stimulatory and inhibitory Fc gamma receptors on human monocytes by Th1 and Th2 cytokines

Immune complex-mediated inflammatory responses are initiated by Fc gamma R on phagocytes. We report in this study that an inhibitory receptor, Fc gamma RIIb2, is expressed on circulating human monocytes, and when co-cross-linked with stimulatory Fc gamma R it down-regulates effector function. Fc gamma RIIb2 expression is increased by IL-4 and decreased by IFN-gamma, in contrast to the activating receptor, Fc gamma RIIa, which is increased by IFN-gamma and decreased by IL-4. Thus, Th1 and Th2 cytokines differentially regulate the opposing Fc gamma R systems, altering the balance of activating and inhibiting Fc gamma R. The detection and cytokine modulation of Fc gamma RIIb2 in human myeloid cells provide evidence of a negative regulator of immune complex-mediated responses in human phagocytes and offer a new approach to limit Ab-triggered inflammation in autoimmune disease.     

Monoclonal antibody (H107) inhibiting IgE binding to Fc epsilon R(+) human lymphocytes

A hybridoma-producing monoclonal antibody blocking the binding of human IgE to lymphocytes Fc receptor (Fc epsilon R) was established by the fusion of murine myeloma cells. P3X63.653.Ag8, with BALB/c spleen cells immunized with Fc epsilon R(+) human B lymphoblastoid cell line cells, RPMI1788. A clone of the hybridoma (H107) produced a monoclonal IgG2b antibody that inhibited the rosette formation of Fc epsilon R(+) human B lymphoblastoid cell line cells (RPMI1788, RPMI8866, CESS, Dakiki, and IM9) with fixed ox red blood cells (ORBC) conjugated with human IgE (IgE-ORBC). In contrast, the rosette formation with IgG-conjugated ORBC (IgG-ORBC) on Fc gamma R(+), Fc epsilon R(-) Daudi cells were not affected by the H107 antibodies. A close association of Fc epsilon R and the antigenic determinant recognized by H107 antibody was suggested by the following results. First, the bindings of 125I-labeled IgE (125I-IgE) or 125I-labeled H107 IgG2b antibody (125I-H107) to RPMI8866 cells were inhibited by cold human IgE and H107 IgG2b but not by other classes of human Ig (IgA and IgG), MPC11 IgG2b, or unrelated monoclonal antibodies. Second, H107 antibody reacted with Fc epsilon R(+) B cell lines but not with Fc epsilon R(-) B cell lines as determined by an indirect immunofluorescence. Third, Fc epsilon R(+) cells were depleted by the incubation in the dish coated with H107 antibody or IgE but not in the dish coated with unrelated antibodies. Finally, there was a correlation between the increase of Fc epsilon R(+) cells and that of H107(+) cells in the peripheral blood lymphocytes of the patients with atopic dermatitis. The surface antigens on Fc epsilon R(+) RPMI8866 cells recognized by H107 antibodies had the molecular size of 45,000 as determined by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis.     

Molecular basis for a polymorphism involving Fc receptor II on human monocytes

IgG Fc receptor II (Fc gamma RII) on human monocytes is polymorphic with respect to its appearance on gels after isoelectric focusing and with respect to its ability to mediate T lymphocyte proliferation induced by murine anti-CD3 mAb of the IgG1 isotype (i.e., its ability to bind murine IgG1). To determine the molecular basis for this polymorphism, we isolated total cellular RNA from PBMC of responders and nonresponders (defined by Leu-4-induced [3H] thymidine incorporation) and synthesized corresponding cDNA. Sequences encoding the extracellular domain of Fc gamma RII were then amplified using the Taq polymerase chain reaction. Amplified DNA fragments were cloned into pUC vectors, and sequenced. Analysis of clones from two nonresponders revealed a single base change (G for A) at position 519, which would result in the substitution of a histidine for an arginine at residue 133 in the mature Fc gamma RII protein. These findings suggest that the polymorphism involving human monocyte Fc gamma RII results from allelic variation of a single gene.     

IgA-induced avidity maturation of IgA Fc receptors on murine T lymphocytes

The analysis of 30 well characterized murine T lymphocyte populations using a cytofluorometric IgA binding assay has identified many populations that are constitutive and/or inducible for IgA receptor expression, and has identified two distinct mechanisms by which IgA up-regulates the IgA-binding properties of murine T cells. Studies with lymphomas, hybridomas, Ag-specific clones and activated normal splenic T cells identified many examples of CD4 and CD8 lineage cells that constitutively expressed IgA receptors. T cell populations that constitutively expressed IgA receptors exhibited enhanced IgA binding after incubation with oligomeric IgA for 18 h. The IgA-induced up-regulation of IgA binding resulted from two distinct processes: 1) an increase in the number of surface membrane IgA binding sites and 2) an increase in the avidity of IgA binding without a change in the number of binding sites. The IgA-induced avidity increase was reflected by a 5- to 10-fold decrease in the apparent Kd. Depending on the T cell population examined the enhanced binding of IgA involved one or both of these mechanisms. T cell populations that did not constitutively express IgA receptors failed to bind IgA after prolonged incubation with oligomeric IgA suggesting that if such cells can express IgA receptors they require other signals to induce their expression. Consistent with this possibility is the finding that resting splenic T cells did not bind IgA but their activation with Con A or mAb anti-T3 resulted in high level expression of IgA receptors. These studies have identified multiple distinct mechanisms that alter the IgA-binding properties of murine T cells and are discussed in terms of their possible physiologic significance.