The expression of IgE Fc receptors on peripheral blood monocytes in asthmatic patients]

We measured the expression of the IgE Fc receptor, Fc epsilon RII, on peripheral blood monocytes isolated from asthmatic patients and normal subjects by laser flow cytometry. After peripheral blood mononuclear cells were incubated with monoclonal antibodies, H107, which recognize Fc epsilon RII, FITC-labeled second antibodies were reacted with them. The cells were then incubated with PE-labeled Leu M3 monoclonal antibodies and the ratios of H107 positive monocytes were measured by two color analysis. The ratio of H107 positive monocytes in atopic asthmatic patients was significantly greater than the ratio in normal subjects. But the ratio was not higher in 9 out of the 14 atopic asthmatic patients. This indicated that atopic asthmatic patients are divided into two groups by the expression of Fc epsilon RII on monocytes. Total serum IgE level was not related to the ratio of H107 positive monocytes.     

Decoding IgE Fc receptors

Immunoglobulin E (IgE) plays a central role in the pathogenesis of allergic diseases by interacting with two membrane receptors: high-affinity FcεRI and low-affinity FcεRII (CD23). Allergen-induced IgE-occupied EcεRI aggregation on the mast cell or basophil cell surface leads to the activation of intracellular signaling events and eventually the release of pre-formed and de novo synthesized inflammatory mediators. The role of FcεRII in allergic diseases has been proposed to include regulation of IgE synthesis, enhanced histamine release from basophils, and a contribution to Ag-IgE complex presentation but the exact function of CD23 remains poorly understood. This review summarizes some new developments in IgE Fc-receptor studies with an emphasis on regulation of FcεRI expression and signal transduction, including monomeric IgE, lipid raft segregation, and some recently identified negative regulators. A better understanding of signaling events following IGE FcR aggregation will shed new light on how allergy patients might be treated more safely and effectively.     

IgE class-specific regulatory factor(s) and Fc epsilon receptors on lymphocytes

The IgE isotype-specific regulatory factor(s) of rodents as well as humans was shown to have an affinity to IgE molecules, suggesting that the factor(s) are Fc epsilon receptors (Fc epsilon R) on lymphocytes or include a fragment of Fc epsilon R. In order to test this possibility, the monoclonal anti-Fc epsilon R antibodies with different epitope specificities were prepared. FACS analysis showed that approximately 50 percent of B cells from normal individuals expressed Fc epsilon R and the augmentation of the expression was observed by the incubation with T cell factors and IgE. However, the Fc epsilon R expression on T cells was not detected even after induction. The result suggests that T cells may secrete Fc epsilon R but not express it on the surface or the IgE-binding factor(s) from T cells may not be antigenically cross-reactive with Fc epsilon R on B cells.     

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.     

Cloning of cDNA coding for low-affinity Fc receptors for IgE on human T lymphocytes

Using a cDNA probe corresponding to the membrane-bound formof the B cell receptor for IgE, we have isolated, sequenced,and expressed a cDNA clone which codes for a human T lymphocyteFcR from HUT-78 cells. This T cell FcR cDNA codes for 320 aminoacid residues, and shows high homology to the B cell FcR sequence.The major differences between this T cell and the B cell FcRcDNA sequences are (i) a limited stretch of nucleotides at the5' segment of the coding region which encodes a putative cytoplasmicregion of the FcR molecule and the untranslated 5' end; and(ii) an additional 64 bp segment in the untranslated 3' endcontaining two repeats in tandem with three existing repeatsin the same region. The expression of FcR on T lymphocytes mayreflect involvement of the FcR in regulation of IgE-mediatedresponses. The cytoplasmic difference implies functional activityof the FcR in T lymphocytes that is mechanistically differentfrom the FcR of B lymphocytes.     

Regulation of in vivo expression of Fc receptors for IgE (Fc epsilon R) on murine lymphocytes. I. Detection of Fc epsilon R+ lymphocytes by flow cytometry

Homologous monomeric IgE was employed in a flow cytometric assay for the detection of IgE Fc receptors (Fc epsilon R) on mouse lymphocytes. The expression of Fc epsilon R in normal BALB/c mice was detected on splenic and circulating lymphocytes, but not on bone marrow cells. The Fc epsilon R expression was observed in B cells with B220, surface IgM, and IgD, but not in T cells. Infection of mice with Nippostrongylus brasiliensis resulted in a marked increase in the expression of Fc epsilon R on splenic B cells. T cells, however, did not express Fc epsilon R even after N. brasiliensis infection. On the other hand, the Fc epsilon R expression on normal B cells decreased after a simple incubation at 37 degrees C for 24 h, while in the presence of IgE this decrease was inhibited. In contrast, B cells stimulated with interleukin 4 display Fc epsilon R with high densities. Interestingly, IgE enhanced the Fc epsilon R expression induced by interleukin 4, suggesting that both interleukin 4 and IgE may be responsible for an increase in the expression of Fc epsilon R on B cells of N. brasiliensis infected mice.     

Induction of IgE receptors on human lymphocytes

A mouse myeloma IgE protein was cross-linked with dimethylsuberimidate to induce polymerization and incubated with normal human peripheral blood T lymphocytes in tissue culture. After 24 hours at 37 degrees C approximately 30% of the cells formed rosettes with IgE sensitized red cells as compared with much lower levels in the controls. A variety of controls established that the cellular receptors involved in rosette formation were specific for IgE. Monomeric mouse IgE failed to induce a response.     

Allergen-directed expression of Fc receptors for IgE (CD23) on human T lymphocytes is modulated by interleukin 4 and interferon-gamma

T lymphocytes bearing Fc receptors (FcR) for immunoglobulins are known to have immunoglobulin class-specific regulatory functions. Here we report that expression on T cells of the low-affinity FcR for IgE (Fc epsilon RII/CD23) is preferentially induced by stimulation with antigens that cause an IgE response. T cells from eight patients allergic to the hemoglobin of Chironomus thummi thummi mosquito larvae (CHIT I) were analyzed for reactivity with the anti-FcERII/CD23 monoclonal antibody (mAb) M-L25 under various conditions. No Fc epsilon RII/CD23+ T cells were observed among freshly isolated, resting peripheral blood mononuclear cells (PBMC). Stimulation of PBMC with CHIT I, however, induced a marked although transient Fc epsilon RII/CD23 expression on a large portion of the allergen-activated T lymphocytes. It reached a maximum of 37.2 +/- 4.6% Fc epsilon RII/CD23+ T cell blasts on day 5 of culture. The selectivity of this expression became evident when compared to non-allergenic control antigens: after stimulation of PBMC with tetanus toxoid or purified protein derivative from tuberculin a maximum of 4.6% +/- 1.4% and 4.2% +/- 1.1% T cell blasts was found to express Fc epsilon RII/CD23, respectively. Activation by an anti-CD3 mAb was insufficient to induce Fc epsilon RII/CD23 on T cells. The allergen-stimulated Fc epsilon RII/CD23+ T cells exclusively belonged to the CD4+CD29+ helper inducer T cell subset. Using a cDNA probe coding for the B cell Fc epsilon RII/CD23, Northern blot analysis revealed a 1.7-kb Fc epsilon RII/CD23 mRNA in extracts of highly purified allergen-stimulated T cells. It was of the same size as Fc epsilon RII/CD23 mRNA of the lymphoblastoid B cell line WI-L2. Of several cytokines tested [interleukin (IL) 1 to IL 6, interferon-gamma (IFN-gamma), tumor necrosis factor-alpha] only IL 4 and IFN-gamma significantly modified allergen-induced Fc epsilon RII/CD23 expression on T cells. The latter was enhanced nearly twofold in the presence of IL 4, and was almost completely abrogated by IFN-gamma. IL 4, however, could not increase the number of Fc epsilon RII/CD23+ T lymphocytes either alone or in combination with an anti-CD3 mAb. Taken together, the selective induction of Fc epsilon RII/CD23 on T cells by allergen and its inclusion in the regulatory network of cytokines point to an important role of Fc epsilon RII/CD23+ T lymphocytes in the human IgE response.     

IgE Fc receptor positive T and B lymphocytes in patients with the hyper IgE syndrome.

The percentages of peripheral blood lymphocytes (PBL), bearing Fc receptors for IgE (Fc epsilon R) and IgG (Fc gamma R) were determined in four patients with the hyper IgE syndrome by a rosette assay employing IgE and IgG coated fixed ox erythrocytes. The patients had 8 +/- 3% Fc epsilon R+ and 13 +/- 8% Fc gamma R+ PBL, compared to 1.2 +/- 1% Fc epsilon R+ and 17 +/- 4% Fc gamma R+ PBL for control donors. T cells were isolated by rosetting with neuraminidase treated sheep erythrocytes (EN). Indirect immunofluorescence with Lyt 3 monoclonal antibody (MoAb) to the sheep erythrocyte receptor, followed by rosetting for Fc epsilon R and Fc gamma R showed that the patients' T cells contained less than 0.1% Fc epsilon R+ and 1.4 +/- 0.2% Fc gamma R+ cells; T cells from the control subjects contained less than 0.1% Fc epsilon R+ and 11 +/- 4% Fc gamma R+ cells. The non-T (EN rosette depleted) cells of the patients included 56 +/- 18% sIgM+/sIgD+, 45 +/- 9% Fc epsilon R+ and 35 +/- 27% Fc gamma R+ cells. Indirect immunofluorescence with MoAb to IgM, IgD, and NK cells (antibody B73.1) followed by rosetting for Fc epsilon R and Fc gamma R, indicated that 92 +/- 2% of the Fc epsilon R+ cells and 9 +/- 7% of the Fc gamma R+ cells were B cells (mu+/delta+), while 3 +/- 4% of the Fc epsilon R+ and 30 +/- 23% of the Fc gamma R+ cells were NK cells (B73.1+). Thus, most of the Fc epsilon R+ non-T cells were B cells, and only a small fraction appeared to be NK cells. On the other hand, Fc gamma R+ B cells were outnumbered by Fc gamma R+ NK cells (B73.1+) by three to one. The data indicate that patients with the hyper IgE syndrome have increased numbers of Fc gamma R+ PBL, most of them being B cells, whereas their T cells contain less than 0.1% Fc epsilon R+ cells.     

Fc receptors as determinants of allergic reactions

Activation of the high-affinity receptor for IgE (FcepsilonRI) on allergic effector cells induces a multitude of positive signals via immunoreceptor tyrosine-based activation motifs, which leads to the rapid manifestation of allergic inflammatory reactions. As a counterbalance, the coaggregation of the IgG receptor FcgammaRIIB mediates inhibitory signals via immunoreceptor tyrosine-based inhibition motifs. Advances in the positive and negative regulation of Fc receptor expression and signaling have shed light on the role of Fc receptors in our immune system, indicating them to be bifunctional, inhibitory and activating structures. Based on these findings, exciting new therapeutic strategies have been developed, such as the use of chimeric fusion proteins, which concomitantly activate FcepsilonRI and FcgammaRIIB. These new approaches successfully take advantage of the bivalent character of Fc receptors and pave the way for innovative strategies to modulate allergic immune reactions.     

Fc epsilon receptors on human cell lines and peripheral blood lymphocytes detected by binding of IgE immune complexes

To identify Fc epsilon receptors on human cell lines and peripheral blood lymphocytes, we developed a new method which relies on the binding of constructed immune complexes to Fc epsilon receptor-positive cells. Cell suspensions from either cell lines or peripheral blood lymphocytes were incubated with complexes of human myeloma IgE and murine monoclonal anti-human IgE at various ratios prior to cytocentrifugation. The complexes bound to the cells were subsequently visualized by immunoperoxidase staining. The specificity of this assay to detect cell surface Fc epsilon receptors was shown by the ability of human myeloma IgE to block the binding of the IgE complexes, resulting in unstained cells, whereas IgM, IgG, and IgA were unable to block the binding of the complexes (stained cells). This method is reproducible, allows quantification of a single sample at different times, and provides a record of the results. It can also be adapted to identify any cell surface receptor for which the ligand is known.     

Fc receptors on human blood B lymphocytes.

The frequency of Fc-receptor positive B lymphocytes in human blood was investigated. Under the conditions used heat-aggregated gammaglobulin binding and EA(ox)-rosette formation labelled the same lymphocyte populations. Using various techniques, double marking and cell separations the proportion of Fc-receptor positive cells within the surface Ig carrying population was estimated to be between 11-8 and 36-2%. The proportion of SIg carrying cells within the population forming EA-rosettes was between 11 and 26-4%. This represents extreme values due to known technical circumstances.     

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.     

IgE receptors on human lymphocytes III. Expression of IgE receptors on mitogen-stimulated human mononuclear cells

Human peripheral blood mononuclear cells (PBMC) were tested for the expression of Fee-receptor (FcεR) after stimulation with various mitogens in the absence of IgE. FcεR were found on virtually all the cells from 19 Epstein-Barr virus-transformed B cell lines including those derived from cord blood, from one agamma-globulinemic patient and VDS-0 pre-B cells. Hence, the data clearly indicate that FcεR may be expressed on very immature B cells. PBMC cultures stimulated with either pokeweed mitogen (PWM), phytohemagglutinin (PHA) or concanavalin A displayed an early increase of their content in FcεR-bearing cells followed by a decrease to levels below those of the control cultures. After fractionation of the PWM-stimulated cultures into T and B cell-enriched preparations, most of the FcεR⁺ cells were in the B cell fractions and the same low levels of FceR⁺ cells were found in the T cell fractions isolated from the PWM-stimulated and from the control cultures. Double-labeling experiments, employing biotinylated F(ab')₂ monoclonal antibody to FcR and either fluorescein isothiocyanate-conjugated B1 or Mo2 monoclonal antibodies, indicated that PWM mainly exerted its effect on B cells and on monocytes. This effect was T cell dependent and it was mediated by soluble factors of T cell origin. At the peak of the PHA or concanavalin A response, most of the FceR-bearing cells were found in the B cell fraction but the T cells isolated from mitogen-stimulated cultures contained significantly more FcεR⁺ cells than those from the control cultures, suggesting that T cell mitogens had increased the expression of FcεR on some T cells. This view was supported by the finding of a higher proportion of FceR⁺ cells in PHA-stimulated than in control cultures of highly purified T cells with a maximum response at the end of the culture period. Double-labeling experiments at the peak (day 2) of the peripheral blood mononuclear cell response indicated that the expression of FcεR was increased on B cells (Bl⁺) and on monocytes (Mo2⁺). By using the same approach at the peak of the T cell response (day 7), it was found that T cells isolated from PHA-stimulated cultures expressed more FcεR than those isolated from control cultures. Moreover, in unstimulated cultures, FcεR was mainly expressed on T helper cells (Leu 3⁺) whereas in PHA-stimulated cultures FcεR was expressed on both T helper and T suppressor/cytotoxic cells (Leu 2⁺).     

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.     

Characterization of Fc receptors for IgE on human alveolar macrophages.

Human alveolar macrophages (aM phi) isolated from lung lavages performed during bronchoscopy and after surgical removal of pulmonary lobes were analysed for Fc receptors for IgE (Fc epsilon R) and IgG (Fc gamma R) by rosette assays. A mean+/-s.d. of 8.0+/-2.6% of aM phi formed rosettes with fixed ox erythrocytes coated with an IgE myeloma protein (Eo''-IgE). The Eo''-IgE rosettes were inhibited by two IgE myeloma proteins and by IgE Fc fragments but not by myeloma proteins of the other Ig classes or by IgE denatured by heating or reduction and alkylation. Fresh ox erythrocytes sensitized with rabbit IgG antibodies (EoA) formed rosettes with 64.1+/-20.3% of the aM phi. Peripheral blood monocytes formed 10.6+/-1.2% Eo''-IgE and 90.2+/-6.0% EoA rosettes. Incubation of the aM phi with a goat antiserum to human lymphocyte Fc epsilon R inhibited Eo''-IgE rosette formation on aM phi by 80% but did not affect the percentage of EoA rosettes. The antiserum also inhibited Eo''-IgE rosettes formed by peripheral blood monocytes and cultured macrophage-like U937 cells but not those formed by basophilic granulocytes obtained from a patient with chronic myelogenous leukaemia. There was no relationship between age, sex, diagnosis or smoking history of the patients and the percentage of aM phi forming Eo''-IgE rosettes. These studies demonstrate that a subpopulation of human aM phi bear Fc epsilon R that share antigenic determinants with Fc epsilon R on lymphocytes and monocytes. Fc epsilon R(+) aM phi may play an important role in allergic and inflammatory pulmonary diseases by inducing the release of mediators of inflammation after interaction with IgE immune complexes.     

Expression of Fc epsilon receptors on activated human T lymphocytes

Our results clearly demonstrate that the low-affinity receptor for IgE (Fc epsilon R) is an activation antigen transiently expressed on a subpopulation of human T lymphocytes. It can be selectively induced by stimulation with certain antigens or lectins, but it is not found on resting T cells. The increased numbers of activated Fc epsilon R+ T cells observed after stimulation of peripheral blood mononuclear cells (PBMC) from bee venom allergic patients with the specific allergen phospholipase A2 (PLA2) suggest that Fc epsilon R+ T cells might very well be involved in the regulation of the human IgE response against the respective antigen. These results were obtained by the use of two monoclonal antibodies, M-L25 and M-L47, which were raised against the human low-affinity Fc epsilon R in our laboratory. After stimulation of PBMC with phytohemagglutinin a peak of 7.6 +/- 6% Fc epsilon R+ T cells was observed on day 3, with pokeweed mitogen of 0.8 +/- 0.8% on days 2 and 3, and with concanavalin A of 0.6 +/- 0.7% Fc epsilon R+ T cells on day 2. Stimulation of PBMC with tetanus toxoid (TT) induced Fc epsilon R on maximally 0.6 +/- 0.8% of the total T cells (day 4), stimulation with purified protein derivative from tuberculin (PPD) on 0.2 +/- 0.6% of the T cells (day 2). In contrast to these antigens, stimulation of PBMC from bee venom allergic patients with PLA2 induced as a peak 2.5 +/- 2.5% of the total T cells to express Fc epsilon R (day 5), although the stimulated T cell population was much smaller than with TT or PPD, as was shown by their stimulation indices. The allergen-stimulated Fc epsilon R+ T cells were exclusively T4+. The Fc epsilon R-expression index was determined, which for a specific antigen or lectin correlates the percentage of Fc epsilon R+ T cells to the stimulated T cell population, respectively.     

Presence of antigenic determinants common to Fc IgE receptors on human macrophages, T and B lymphocytes and IgE-binding factors

The present study indicates that two Mab specific to Fc epsilon R (MabER) on human B lymphocytes also react with Fc epsilon R on macrophage and T-cell lines. More importantly, it is also shown that MabER cross-react with IgE-BFs derived from B, T and macrophage cell lines. This conclusion is supported by the following observations: the binding of MabER to Fc epsilon R-bearing cells is blocked by the CSN of T, B and macrophage Fc epsilon R-bearing cell lines, known to contain IgE-BFs as shown by their inhibition of rosette formation between IgE-coated erythrocytes and Fc epsilon R-bearing cells; the material purified from the CSN of each Fc epsilon R(+) cell lines by affinity chromatography on MabER-Affi-gel blocks the rosetting of U937 cells with IgE but not with IgG-coated erythrocytes; the same affinity-purified material inhibits the binding of 125I-IgE to a selected anti-IgE Mab (Mab 75); and the CSN of Fc epsilon R(+) cells but not of Fc epsilon R(-) cells reacts in a solid-phase sandwich radioimmunoassay with two MabER (135-176), and their reactivity is significantly retained on IgE-Affi-gel from which it may be recovered by glycine elution. This RIA is not influenced by any class of Ig, including IgE, employed at a final concentration of 100 micrograms/ml. Human serum also reacts in the RIA, and parallel dilution curves are obtained with different CSN and human sera. The RIA proved to be a reproducible and sensitive method to quantify human IgE-BFs. The expression of the same antigenic determinants on Fc epsilon R from T, B and macrophages as well as on the IgE-BFs secreted by these cells indicates structural homology between IgE receptors and IgE-FBs and suggests that they are encoded by the same gene.