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.     

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.     

Monoclonal antibody specific for T cell-derived human IgE binding factors

A B cell hybridoma secreting monoclonal antibody against human IgE binding factors was obtained by immunization of BALB/c mice with partially purified IgE binding factors, and fusion of their spleen cells with SP-2/0-AG14 cells. The monoclonal antibody bound all of the 60,000, 30,000, and 15,000 dalton IgE binding factors from two T cell hybridomas and those from activated T cells of a normal individual. The antibody bound both IgE-potentiating factors, which had affinity for lentil lectin, and IgE-suppressive factors, which had affinity for peanut agglutinin. However, the monoclonal anti-IgE-binding factor bound neither Fc epsilon R on RPMI 8866 cells nor IgE binding factors from the B lymphoblastoid cells. A monoclonal antibody against Fc epsilon R on B cells (H107) bound the 60,000 and 30,000 dalton IgE binding factors from both T cell hybridomas and RPMI 8866 cells but did not bind the 15,000 dalton IgE binding factors from either T cells or B cells. The results indicate that T cell-derived IgE binding factors have a unique antigenic determinant that is lacking in both Fc epsilon R on B cells and B cell-derived IgE binding factors. The anti-IgE binding factor and anti-Fc epsilon R monoclonal antibodies both failed to stain cell surface components of IgE binding factor-producing T cell hybridomas. However, both antibodies induced the T cell hybridoma to form IgE binding factors. The results suggest that the T cell hybridomas bear low numbers of Fc epsilon R that share antigenic determinants with IgE binding factors secreted from the cells.     

The binding of IgE to murine Fc epsilon RII is calcium-dependent but not inhibited by carbohydrate

Despite extensive study, little is known about the functions of the moderate affinity IgE receptors (Fc epsilon RII) on B cells. Recent cDNA and genomic cloning studies have demonstrated that, in contrast to other FcR, Fc epsilon RII is not a member of the Ig gene superfamily. Moreover, it uniquely expresses a region that is highly homologous with a membrane-associated, calcium-dependent binding lectin, the asialoglycoprotein receptor. We now report that the interaction between IgE and the Fc epsilon RII of murine B cells and macrophages requires calcium. Furthermore, as might be expected of asialoglycoprotein lectins, this binding was pH-dependent and resulted in ligand internalization. However, although 125I-Fc epsilon RII bound in a calcium-dependent manner to monosaccharide-agarose beads, high concentrations of mono- and disaccharides did not inhibit the interaction between either 125I-IgE and intact B cells or 125I-Fc epsilon RII (from surface-labeled B cells) and IgE-Sepharose. These results suggest that although murine Fc epsilon RII is a lectin, it is not strictly dependent upon IgE oligosaccharides for its binding to IgE.     

Isolation, characterization, and expression of cDNA clones encoding the mouse Fc receptor for IgE (Fc epsilon RII)1

We have isolated cDNA clones encoding a mouse low affinity receptor for IgE (Fc epsilon RII) from a cDNA library of BALB/c splenic B cells activated with LPS and IL-4. The 2.2-kb cDNA clone encodes a 331 amino acid membrane glycoprotein that is homologous to human Fc epsilon RII (CD23) and a family of carbohydrate-binding proteins. COS7 cells transfected with the cDNA clones expressed a 45,000 m.w. protein that bound IgE and the anti-Fc epsilon RII mAb, B3B4. Fc epsilon RII mRNA was up-regulated in mouse B cells by culture with IL-4, but not in B cells cultured with IgE. Fc epsilon RII mRNA was detected in IgM+/IgD+ B cell lines, but not in pre-B cell lines or in B cell lines which have undergone differentiation to secrete Ig. The monocyte line P388D1, mast cell lines MC/9 and PT18, and peritoneal macrophages stimulated with IL-4 lacked detectable Fc epsilon RII mRNA, as did Thy-1.2+, CD4+, and CD8+ normal T cells and Thy-1.2+ T cells from Nippostrongylus brasiliensis-infected mice.     

Recombinant soluble Fc epsilon receptor II (Fc epsilon RII/CD23) has IgE binding activity but no B cell growth promoting activity

We have undertaken the production of recombinant soluble Fc epsilon receptor II (Fc epsilon RII) as a secretory protein, but not as a cleavage product of membrane-bound receptor. Several plasmid constructs containing soluble receptor sequence were prepared. Only a chimeric gene containing the sequences encoding IL-6 signal peptide and the soluble moiety of Fc epsilon RII could be expressed in Xenopus laevis oocytes and CHO cells, resulting in the secretion of soluble Fc epsilon RII. The recombinant soluble Fc epsilon RII was also produced in the yeast expression system. The NH2-terminal sequence analysis of the chimeric gene product generated by oocytes demonstrated the correct cleavage of IL-6 leader sequence by a signal peptidase. Moreover, most of CHO cell and all of the yeast-derived recombinant molecules were products identical with the native B cell-derived soluble Fc epsilon RII. These recombinant products as well as the natural soluble receptor derived from a human B cell line could bind both human IgE and two different anti-Fc epsilon RII mAb and could competitively inhibit the binding of IgE to Fc epsilon RII-expressing cells. However, the recombinant soluble Fc epsilon RII and highly purified native molecules did not display any B cell growth-promoting activity.     

Superinduction of the murine B cell Fc epsilon RII by T helper cell clones. Role of IL-4

Th cell clones are known to induce an IL-4 dependent polyclonal IgE synthesis. Because IL-4 can induce the expression of the low affinity FcR for IgE (Fc epsilon RII) the ability of Th cell clones to induce Fc epsilon RII on purified splenic B cells was analyzed. It was found that a TH2 clone could cause a 50- to 100-fold superinduction of Fc epsilon RII after 2 days in culture; after 3 days, the Fc epsilon RII levels had almost returned to base line. The superinduction was inhibited by an anti-IL-4 antibody, 11B11, indicating its dependence on IL-4. A TH1 clone could cause a modest (four fold) induction of Fc epsilon RII, and this induction was not influenced by 11B11. A similar Fc epsilon RII induction was seen when using the supernatant from activated TH1 cells. The component(s) causing this relatively low level Fc epsilon RII induction is not known; a variety of known lymphokines were tested, and only IL-4 demonstrated any capacity for Fc epsilon RII induction on LPS-activated B cells. Addition of rIL-4 at concentrations of 400 U/ml or greater to the TH1 culture was sufficient to cause a Fc epsilon RII superinduction similar to that seen with the TH2 clone, while 40 U/ml was not. In order to determine a potential role for the Fc epsilon RII or its soluble fragment on the IgE synthesis mediated by TH2, a monoclonal anti-Fc epsilon RII, B3B4, was added to the culture. The addition of B3B4 did not have an influence on IgE levels in this system.     

Induction of Fc epsilon RII/CD23 on phytohemagglutinin-activated human peripheral blood T lymphocytes. I. Enhancement by IL-2 and IL-4.

Despite evidence for the expression of low affinity Fc receptor for IgE (Fc epsilon RII)/CD23 in T cell lines and pathologic T cells, Fc epsilon RII/CD23 in normal human T cells is still unclear. We studied the expression of Fc epsilon RII/CD23 on T cells in short-term culture of normal human PBMC stimulated with 15 micrograms/ml PHA. PHA stimulation also resulted in the release of soluble Fc epsilon RII/CD23 (IgE binding factor). Using two-dimensional flow cytometry, more than 10% of the Fc epsilon RII/CD23+ cells were found to co-express CD3 Ag. Both CD4+ and CD8+ T cells expressed Fc epsilon RII/CD23. The induction of Fc epsilon RII/CD23 on PHA-activated T cells was enhanced by IL-2 as well as IL-4. Both IL-2 and IL-4 also augmented PHA-induced production of soluble Fc epsilon RII/CD23. The enhanced expression of Fc epsilon RII/CD23 on T cells by both lymphokines was suppressed by rabbit anti-IL-4 antiserum, suggesting the involvement of an IL-4-dependent process even in the IL-2-dependent Fc epsilon RII/CD23 expression on T cells. The expression of mRNA for Fc epsilon RII/CD23 on PHA and IL-4-stimulated PBMC was examined by Northern blot analysis. Fc epsilon RII/CD23 mRNA was detected in RNA prepared from the T cell fraction depleted of B cells and macrophages (Fc epsilon RII+CD3+ = 6.2%, Fc epsilon RII+CD3- = 0.8%). The expression of the mRNA for Fc epsilon RII/CD23 on CD3+ T cells was also confirmed by in situ hybridization with Fc epsilon RII/CD23 cDNA combined with CD3 rosette formation at the single cell level.     

The murine lymphocyte receptor for IgE. IV. The mechanism of ligand-specific receptor upregulation on B cells

Rodent B cells respond to culture with IgE by increasing their IgE-specific Fc receptors (Fc epsilon R). The mechanism of this upregulation was characterized on Fc epsilon R+ murine B cell hybridoma lines. Measurements of [35S]methionine incorporated into the Fc epsilon R over time indicated that IgE did not appreciably increase the rate of Fc epsilon R synthesis. In contrast analysis of Fc epsilon R decay from surface radioiodinated B hybridoma cells demonstrated that IgE acted to slow the rate of Fc epsilon R degradation. Very little endocytosis of monomeric IgE was seen; this, combined with the observation that lysomotropic agents failed to inhibit Fc epsilon R degradation suggested that decay occurs at the cell surface. A soluble receptor immunoassay was developed, using monoclonal anti-Fc epsilon R, and this assay demonstrated that cell-bound IgE inhibited the release into the culture media of soluble immunoreactive Fc epsilon R. Examination of the soluble Fc epsilon R by SDS-PAGE after isolation with monoclonal anti-Fc epsilon R demonstrated that it was 10,000 m.w. smaller than the cell-associated Fc epsilon R. IgE affinity columns failed to bind the Fc epsilon R fragment, indicating that the ligand binding activity was largely lost. Thus this study demonstrated that IgE-dependent Fc epsilon R induction on B cells occurs because IgE upon binding to the B cell surface, inhibits the proteolytic cleavage and release of the Fc epsilon R into the surrounding medium, and it is this inhibition of degradation that causes the higher Fc epsilon R levels.     

In vivo and in vitro regulation of IgE production in murine hybridomas

Normal BALB/c mice injected i.p. with the IgE-secreting hybridomas B53 (epsilon, kappa anti-DNP), SE1.3 (epsilon, kappa, anti-arsonate) or A3B1 (epsilon, kappa, anti-TNP) were monitored for serum IgE concentrations and frequencies of splenic T lymphocytes with surface membrane receptors for the Fc portion of IgE (Fc epsilon R+ T lymphocytes). Mice with B53 or SE1.3 hybridomas initially developed high concentrations of IgE and CD8+ Fc epsilon R+ T lymphocytes, followed by a progressive decline in both serum IgE and expression of cytoplasmic epsilon-chains in the hybridoma cells. Serum IgE concentrations in mice with A3B1 hybridomas progressively increased without development of Fc epsilon R+ T lymphocytes nor a subsequent decline in IgE or change in cytoplasmic epsilon-chain expression in the A3B1 cells. An in vitro system in which the IgE-secreting hybridoma cells were cocultured with spleen cells harvested from mice with established B53 tumors was used to investigate the mechanisms involved in the inhibition of IgE production by the hybridoma cells. The results of these studies indicate that: 1) the induction/upregulation of Fc epsilon R on CD8+ T lymphocytes in vivo requires factors in addition to high serum IgE concentrations; 2) in addition to CD8+ Fc epsilon R+ T lymphocytes and monocytes, another, as yet unidentified, splenic cell component appears to contribute to the process by which epsilon-chain expression in IgE-secreting hybridoma cells is suppressed, and 3) a hybridoma (A3B1) that fails to induce CD8+, Fc epsilon R+ T lymphocytes in vivo and is not inhibited in IgE expression in vivo, nonetheless is inhibited in IgE expression in vitro when cocultured with spleen cells from mice with B53 tumors.     

Superinduction of low affinity IgE receptors on murine B lymphocytes by lipopolysaccharide and IL-4

Recent work in both the human and murine systems has demonstrated that IL-4 is capable of specifically inducing the synthesis of the low affinity receptor for IgE (Fc epsilon RII). In addition, in conjunction with LPS, IL-4 will induce IgG1 and IgE synthesis. To analyze the correlation between Fc epsilon RII induction and IgE secretion, Fc epsilon RII and IgE levels were measured by RIA on murine splenic B cells stimulated with LPS and IL-4 over 7 days of culture. Treatment with LPS and IL-4 gave a 20- to 50-fold (day 3) "superinduction" of Fc epsilon RII levels compared with a 3- to 5-fold induction with IL-4 alone; removal of IL-4 resulted in a rapid decline in Fc epsilon RII levels. The cells expressing high Fc epsilon RII levels were determined to be blasts. Superinduction of Fc epsilon RII occurs at 10 U/ml IL-4 and remains relatively constant in the range of 10 to 1000 U/ml. In contrast, with increasing IL-4, IgE levels increase, reaching microgram levels at day 7 with 300 U/ml IL-4. Triggering the cells with anti-Ig, as expected, gave no Ig secretion, and in addition, Fc epsilon RII superinduction by IL-4 and anti-Ig was not seen. PMA is known to block Ig secretion induced by LPS. Concentrations of PMA that totally abrogated IgE secretion had no effect on Fc epsilon RII superinduction, indicating that the latter phenomena can be separated from IL-4-induced Ig secretion. Superinduction also results in higher levels of Fc epsilon RII fragment release into the media. Thus, attempts were made to influence IgE secretion by adding additional purified Fc epsilon RII fragment to the culture. The purified fragment did not have a significant influence on IgE levels in this system.     

Characterization of a monoclonal antibody directed against the murine B lymphocyte receptor for IgE

A rat hybridoma producing a high-affinity IgG2a monoclonal antibody (B3B4) directed against against the murine lymphocyte IgE receptor (Fc epsilon R) was established by using purified Fc epsilon R from Fc epsilon R+ murine hybridoma B cells as immunogen. The monoclonal and polyclonal anti-Fc epsilon R inhibited the binding of IgE to the murine lymphocyte Fc epsilon R and were also used to isolate the Fc epsilon R. B3B4 specifically recognized only the 49-Kd Fc epsilon R on murine B lymphocyte as determined by immunoprecipitation and SDS-PAGE analysis. In addition to its reaction with intact Fc epsilon R, B3B4 also recognized Fc epsilon R fragments that were present in the culture media of Fc epsilon R+ hybridoma cells. The predominant fragments isolated were 38 Kd and 28 Kd by SDS-PAGE analysis. When tested for reactivity with other cell types, B3B4 was highly specific for murine B lineage cells in that it did not significantly react with Fc epsilon R on macrophages and T cells and, in addition, did not react with the high affinity mast cell Fc epsilon R. B3B4 completely blocked IgE rosetting, and a reciprocal inhibition of binding was seen in a dose-dependent fashion between IgE and B3B4, indicating a close proximity of the IgE and B3B4 binding sites. Saturation binding analysis indicated that the Fab' fragment of B3B4 bound to twice as many sites/cell as IgE, suggesting that there are two identical B3B4 determinants per 49-Kd Fc epsilon R or that the IgE binding site is formed by the association of at least two 49-Kd Fc epsilon R. However, unlike IgE, neither B3B4 nor F(ab')2-B3B4 nor Fab'-B3B4 were very effective in causing Fc epsilon R upregulation on murine hybridoma B cells; in fact, B3B4 prevented this upregulation when added in combination with IgE. These results suggest that a site-specific interaction provided only by IgE may be essential for ligand-specific upregulation. Both polyclonal and monoclonal antibodies will be useful in further studies concerning the functional relationship between the membrane Fc epsilon R and the soluble Fc epsilon R fragments.     

Characterization of the murine T cell receptor for IgE (Fc epsilon RII). Demonstration of shared and unshared epitopes with the B cell Fc epsilon RII

Antigenic relationships between the low affinity Fc epsilon R present on murine B and T lymphocytes were studied. A rat mAb (B3B4) and two polyclonal antisera produced by immunizing with the murine B lymphocyte Fc epsilon RII were examined for their ability to inhibit binding of IgE to murine B or T lymphocytes, using an IgE-specific rosette assay. One polyclonal antiserum (goat-anti-mouse Fc epsilon R) inhibited binding of IgE to both B and T lymphocytes, whereas another polyclonal antiserum (rabbit-anti-mouse Fc epsilon R) and the rat mAb inhibited the binding of IgE to B lymphocytes but did not influence the binding of IgE to T lymphocytes. When lymphocytes were surface labeled with 125I, 49-kDa and 38-kDa IgE-binding proteins were immunoprecipitated from B lymphocyte lysates by B3B4 and from B and T lymphocyte lysates by the goat antiserum. Taken together, these results suggest that the Fc epsilon R present on murine B and T lymphocytes are structurally related receptors that share some, but not all, epitopes.     

Lack of Fc epsilon RII expression by murine B cells after in vivo immunization is directly associated with Ig secretion and not Ig isotype switching.

The "low affinity" Fc receptor for IgE (Fc epsilon RII) has been reported to be absent from normal murine and human B cells that express a membrane (m)Ig isotype other than mIgM or mIgD in vivo. This would suggest that Fc epsilon RII expression is specifically lost after in vivo Ig isotype switching. We demonstrate that during a murine immune response to the bacterium Brucella abortus, to goat anti-mouse IgD (G alpha M delta) antibody, or to infection with the nematode parasites Nippostrongylus brasilienis or Heligmosomoides polygyrus, Fc epsilon RII expression is low or absent on virtually all B cells secreting IgM, IgG1, IgG2a, and IgE. However, up to 50% of B cells that express mIgG1 after G alpha M delta injection continue to express Fc epsilon RII. These mIgG1 + Fc epsilon RII+ cells secrete little, if any, IgG1 when placed in vitro, in contrast to their mIgG1 + Fc epsilon RII- counterparts. The mIgG1 + Fc epsilon RII+ cells may be a transitional cell population, because they undergo substantial loss of Fc epsilon RII in culture, unlike mIgM+ Fc epsilon RII+ cells, which maintain constant levels of Fc epsilon RII throughout a comparable culture period. Thus, low or absent expression of Fc epsilon RII after immunization in vivo is directly associated with B cell differentiation to Ig production in the presence or absence of Ig isotype switching. However, all post-switched B cells may eventually lack Fc epsilon RII expression, independently of their differentiative state.     

Production and characterization of a monoclonal antibody specific for the human lymphocyte low affinity receptor for IgE: CD 23 is a low affinity receptor for IgE

In the present study a gamma 1 kappa monoclonal antibody, Mab 25, specific for the receptor for the Fc fragment of IgE on lymphocytes (Fc epsilon RL) was established. This antibody was generated after fusion of spleen cells from mice immunized with the EBV-transformed lymphoblastoid cell line RPMI 8866, which is known to express Fc epsilon RL at high density. Mab 25 inhibits strongly the binding of IgE to RPMI 8866 cells and to other Fc epsilon RL-positive EBV-transformed lymphoblastoid cell lines. A 50% inhibition of IgE binding was observed at a Mab 25 concentration of 10 ng/ml. The binding of IgE was also inhibited by Fab fragments of Mab 25, suggesting that the inhibition is not simply due to steric hindrance or to an eventual binding through its Fc portion. Mab 25 only binds to cell lines expressing Fc epsilon RL. Mab 25 immunoprecipitated a single polypeptide with an apparent m.w. of 42 Kd, pI 4.9. The membrane molecule bound to and eluted from a Mab 25 immunoabsorbent had the same apparent m.w. and pI as the Fc epsilon RL purified from an IgE immunoabsorbent. Additionally, when RPMI 8866 cell lysates were cleared with Mab 25, no Fc epsilon RL could be bound to or eluted from an IgE immunoabsorbent. Mab 25 was found to weakly bind to a minor proportion of blood (1 to 4%), tonsil (2 to 9%) and spleen (4 to 5%) mononuclear cells with a low intensity. By double fluorescence analysis, most of the Fc epsilon RL-positive cells were found to be CD 20-positive B lymphocytes. The staining pattern of Mab 25 and the biochemical characteristics of the antigen detected by Mab 25 were comparable to those of the CD 23 Mab. The four CD 23 Mab MHM 6, PL 13, HD 50, and Tü 1 were found to inhibit the binding of IgE. PL 13 was found to totally inhibit the binding of Mab 25 to RPMI 8866 cells, whereas Tü 1 and MHM 6 only partially inhibited Mab 25 binding. HD 50 was unable to block the binding of Mab 25. The finding that different CD 23/Fc epsilon RL-specific monoclonal antibodies recognizing distinct epitopes have in common the capacity of inhibiting the binding of IgE suggests that upon binding they induce a conformational alteration of the Fc epsilon RL resulting in a loss of the IgE binding capacity. In conclusion, our data demonstrate that the CD 23 antigen is a low affinity receptor for IgE on lymphocytes.     

Human neutrophils express the high-affinity receptor for immunoglobulin E (Fc epsilon RI): role in asthma.

Polymorphonuclear neutrophils (PMNs) are important effector cells in host defense and the inflammatory response to antigen. The involvement of PMNs in inflammation is mediated mainly by the Fc receptor family, including IgE receptors. Recently, PMNs were shown to express two IgE receptors (CD23/Fc epsilon RII and galectin-3). In allergic diseases, the dominant role of IgE has been mainly ascribed to its high-affinity receptor, Fc epsilon RI. We have examined the expression of Fc epsilon RI by PMNS: mRNA and cell surface expression of Fc epsilon RI alpha chain was identified on PMNs from asthmatic subjects. Furthermore, preincubation with human IgE Fc fragment blocks completely the binding of anti-Fc epsilon RI alpha chain (mAb15--1) to human PMNS: Conversely, preincubation of PMNs with mAb15--1 inhibits significantly the binding of IgE Fc fragment to PMNs, indicating that IgE bound to the cell surface of PMNs mainly via the Fc epsilon RI. Peripheral blood and bronchoalveolar lavage (BAL) PMNs from asthmatic subjects also express intracellular Fc epsilon RI alpha and beta chain immunoreactivity. Engagement of Fc epsilon RI induces the release of IL-8 by PMNS: Collectively, these observations provide new evidence that PMNs express the Fc epsilon RI and suggest that these cells may play a role in allergic inflammation through an IgE-dependent activation mechanism.     

B cell activator. Effects on B cell expression of CD23, proliferation, and antibody secretion.

The studies herein describe a B cell hybridoma-derived, low m.w. (less than 1000 Da), hydrophilic mediator denoted B cell activator (BCA). BCA stimulates B cell expression of IgE-specific FcR (Fc epsilon RII or CD23) in a manner similar to IL-4. However, BCA can be readily distinguished from IL-4 because it does not 1) enhance B cell Ia expression; 2) bind 11B11 anti-IL-4 mAb; or 3) elicit superinduction of Fc epsilon RII expression or IgE production in cultures of LPS-activated B cells. Moreover, BCA is considerably more mitogenic than IL-4 for LPS-activated B cells and, in contrast to IL-4, lacks mitogenicity for anti-mu-activated B cells. BCA can enhance IgG2b and IgG3 production by LPS-activated B cells, responses that are suppressed by IL-4. BCA alone did not stimulate IgE and IgG1 production by LPS-activated B cells, but exerted synergistic activity when combined with IL-4 in stimulating secretion of these antibody isotypes. Finally, secondary Ag-driven IgG1, IgE, and IgA antibody responses can be stimulated by BCA in vitro. Thus, BCA appears to be a novel mediator with broad B cell activation properties.     

Mapping of the high affinity Fc epsilon receptor binding site to the third constant region domain of IgE.

Identification of the precise region(s) on the IgE molecule that take part in the binding of IgE to its high affinity receptor (Fc epsilon RI) may lead to the design of IgE analogues able to block the allergic response. To localize the Fc epsilon RI-binding domain of mouse IgE, we attempted to confer on human IgE, which normally does not bind to the rodent receptor, the ability to bind to the rat Fc epsilon RI. Employing exon shuffling, we have expressed chimeric epsilon-heavy chain genes composed of a mouse (4-hydroxy-3-nitrophenyl)acetic acid (NP)-binding VH domain, and human C epsilon in which various domains were replaced by their murine counterparts. This has enabled us to test the Fc epsilon RI-binding of each mouse IgE domain while maintaining the overall conformation of the molecule. All of the chimeric IgE molecules which contain the murine C epsilon 3, bound equally to both the rodent and human receptor, as well as to monoclonal antibodies recognizing a site on IgE which is identical or very close to the Fc epsilon RI binding site. Deletion of the second constant region domain did not impair either the binding capacity of the mutated IgE or its ability to mediate mast cell degradation. These results assign the third epsilon domain of IgE as the principal region involved in the interaction with the Fc epsilon RI.