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.     

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.     

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.     

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.     

IFN-alpha and IFN-gamma have different regulatory effects on IL-4-induced membrane expression of Fc epsilon RIIb and release of soluble Fc epsilon RIIb by human monocytes

We used highly purified human monocytes to study the regulation of cell surface and secretion of the low affinity FcR for IgE (Fc epsilon RIIb). IL-4 induces Fc epsilon RIIb expression and soluble Fc epsilon RIIb release in a dose-dependent manner. Significant levels of Fc epsilon RIIb expression were obtained after 12 h of incubation with IL-4 and maximal expression was observed between 24 to 48 h after which the expression declined. Surface expression was followed by secretion of soluble Fc epsilon RIIb which reached maximal levels after 3 to 4 days of incubation and which remained constant throughout 7 days of culture. Induction of Fc epsilon RIIb expression by IL-4 was completely blocked by anti-IL-4 antibodies. Furthermore, IL-1 alpha, IL-2, IL-5, granulocyte-macrophage-CSF, IFN-alpha, IFN-gamma, low m.w. BCGF and also LPS all failed to induce Fc epsilon RIIb expression, demonstrating the specificity of the induction. Fc epsilon RIIb membrane expression induced by IL-4 was reduced in the presence of IFN-gamma and IFN-alpha. Strong inhibition of IL-4-induced Fc epsilon RIIb expression was observed at IFN-alpha concentrations of 450 U/ml (80%), and 100 U/ml of IFN-gamma reduced IL-4-induced Fc epsilon RIIb expression by 70%. Interestingly, soluble Fc epsilon RIIb release was strongly inhibited by IFN-alpha. In contrast, IFN-gamma did not affect soluble Fc epsilon RIIb release, suggesting that reduced membrane expression of Fc epsilon RIIb observed in the presence of IFN-gamma does not reflect inhibition of Fc epsilon RIIb expression but may represent enhanced cleavage or reduced anchoring in the membrane of Fc epsilon RIIb. Finally, IL-5 that has been shown to enhance IL-4-induced Fc epsilon RII on B cells does not enhance significantly IL-4-induced Fc epsilon RIIb membrane expression or subsequent soluble Fc epsilon RIIb release by monocytes. Taken together these results show that IFN-alpha and IFN-gamma have different regulatory effects on IL-4-induced Fc epsilon RIIb membrane expression and soluble Fc epsilon RIIb release by human monocytes.     

A novel human immunoglobulin Fc gamma Fc epsilon bifunctional fusion protein inhibits Fc epsilon RI-mediated degranulation

Human mast cells and basophils that express the high-affinity immunoglobulin E (IgE) receptor, Fc epsilon receptor 1 (Fc epsilon RI), have key roles in allergic diseases. Fc epsilon RI cross-linking stimulates the release of allergic mediators. Mast cells and basophils co-express Fc gamma RIIb, a low affinity receptor containing an immunoreceptor tyrosine-based inhibitory motif and whose co-aggregation with Fc epsilon RI can block Fc epsilon RI-mediated reactivity. Here we designed, expressed and tested the human basophil and mast-cell inhibitory function of a novel chimeric fusion protein, whose structure is gamma Hinge-CH gamma 2-CH gamma 3-15aa linker-CH epsilon 2-CH epsilon 3-CH epsilon 4. This Fc gamma Fc epsilon fusion protein was expressed as the predicted 140-kappa D dimer that reacted with anti-human epsilon- and gamma-chain specific antibodies. Fc gamma Fc epsilon bound to both human Fc epsilon RI and Fc gamma RII. It also showed dose- and time-dependent inhibition of antigen-driven IgE-mediated histamine release from fresh human basophils sensitized with IgE directed against NIP (4-hydroxy-3-iodo-5-nitrophenylacetyl). This was associated with altered Syk signaling. The fusion protein also showed increased inhibition of human anti-NP (4-hydroxy-3-nitrophenylacetyl) and anti-dansyl IgE-mediated passive cutaneous anaphylaxis in transgenic mice expressing human Fc epsilon RI alpha. Our results show that this chimeric protein is able to form complexes with both Fc epsilon RI and Fc gamma RII, and inhibit mast-cell and basophil function. This approach, using a Fc gamma Fc epsilon fusion protein to co-aggregate Fc epsilon RI with a receptor containing an immunoreceptor tyrosine-based inhibition motif, has therapeutic potential in IgE- and Fc epsilon RI-mediated diseases.     

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.     

Characterization of B-cell populations bearing Fc epsilon receptor II

We have characterized the B-cell population that expresses low affinity Fc receptors for IgE (Fc epsilon RII). Fc epsilon RII+ B cells from normal adult BALB/c mice expressed high levels of surface IgD and low/medium levels of surface IgM and constituted the majority of mature splenic B cells. Fc epsilon RII+ splenic B cells expressed high levels of class II MHC antigens and medium to high levels of B220, and consisted of approximately equal numbers of J11dhigh and J11dlow cells. CD5+ B cells did not appear to be Fc epsilon RII+, and interleukin 4 did not induce Fc epsilon RII expression on CD5+ B cells. Fc epsilon RII was not expressed by B cells that had differentiated to secrete immunoglobulin but was expressed on activated B cells. These data suggest that Fc epsilon RII is a differentiation marker expressed on mature and activated B lymphocytes in the major B-cell lineage.     

Intracellular cleavage of newly synthesized low affinity Fc epsilon receptor (Fc epsilon R2) provides a second pathway for the generation of the 28-kDa soluble Fc epsilon R2 fragment

It has been reported that the 45-kDa low affinity Fc epsilon R (Fc epsilon R2) on B cells is cleaved spontaneously from the cell surface to release a 28-kDa soluble fragment (sFc epsilon R2). This study demonstrates an additional mechanism by which B cells generate this fragment. Data from 35S methionine pulse-chase experiments with the Fc epsilon R2 bearing human B lymphoblastoid cell line, RPMI 8866, and immunoprecipitations of cell lysates and culture supernatants with an Fc epsilon R2 specific mAb, mAb 25, demonstrates the existence of a cell-associated 28-kDa Fc epsilon R2 fragment. This fragment was shown by partial amino(NH2)-terminal sequence analysis to be identical to the previously described 28-kDa sFc epsilon R2. The resistance to cell treatment with trypsin indicated that it was located intracellularly. Its appearance early in the biosynthesis of the Fc epsilon R2 (within a 10-min pulse), before the Fc epsilon R2 reached the cell surface, suggested that some of this fragment was generated intracellularly. Neutralization of acidic organelles with NH4Cl inhibited the formation of this intracellular fragment, strongly suggesting that it was a produce of intracellular cleavage of the Fc epsilon R2. Finally, this 28-kDa intracellular fragment was shown to be released into the culture supernatant, suggesting an intracellular mechanism by which the cells generate sFc epsilon R2.     

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.     

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.     

Fc receptors and immunoglobulin binding factors

Receptors for the Fc portion of Ig (Fc receptors, FcR) are found on all cell types of the immune system. Three types of FcR react with IgG: Fc gamma RI is a high-affinity receptor binding IgG monomers whereas Fc gamma RII and Fc gamma RIII are low-affinity receptors binding IgG immune complexes; the three types of Fc gamma R are members of the Ig superfamily. Two FcR react with IgE:Fc epsilon RI is a multichain receptor binding IgE with high affinity; it is composed of an IgE-binding alpha chain, homologous to Fc gamma RIII, and of gamma and beta chains that are necessary for receptor expression and signal transduction. The low-affinity Fc epsilon RII is the only FcR described so far that is not a member of the Ig superfamily but resembles animal lectins; it is composed of a transmembrane chain with an intracytoplasmic NH2 terminus. Fc alpha R has homology with Fc gamma R and is a member of the Ig superfamily. Receptors for IgM and IgD are not characterized yet. Finally, Ig transport is made by FcR-like molecules such as the poly-Ig receptor or an MHC-like receptor found on neonatal intestine. A remarkable property of most FcR is the fact that they are released in cell supernatants and circulate in biological fluids as immunoglobulin binding factors (IBF) generated either by cleavage at the cell membrane or by splicing of FcR transmembrane exon. Immunoglobulin binding factors may interfere with Ig-mediated functions and have direct immunoregulatory activities. Involvement of FcR or IBF has been postulated in several diseases, and monoclonal antibodies to FcR are beginning to be used in therapeutics, particularly to target cytotoxic effector lymphocytes and monocytes to tumor cells.     

Effect of B cell stimulatory factor-1 (interleukin 4) on Fc epsilon and Fc gamma receptor expression on murine B lymphocytes and B cell lines

Culture of murine splenic B cells with interleukin 4 (IL-4) caused the up-regulation of the lymphocyte Fc receptor for immunoglobulin E (IgE) (Fc epsilon R) over a similar dose range as required for Ia up-regulation. However, the expression level of the Fc receptor for immunoglobulin G (Fc gamma R) did not increase, rather IL-4 caused a slight but consistent decrease in the Fc gamma R level on the B cells. Fc epsilon R+ B hybridoma cells also responded to IL-4 by exhibiting increased Fc epsilon R expression; with the hybridoma cells Fc gamma R levels were unaffected. IL-4 caused an increase in the number of Fc epsilon R per cell and the highest levels of expression were obtained by having both IgE and IL-4 present in the culture. The specificity of the increase was demonstrated by blocking IL-4-mediated actions with monoclonal anti-IL-4 (11B11). Experiments following the incorporation of [35S]methionine into the Fc epsilon R demonstrated that IL-4 increased the rate of Fc epsilon R biosynthesis; this provides an explanation for the IL-4-induced increase in Fc epsilon R expression. IL-4, unlike IgE, had no effect on the rate of degradation of the Fc epsilon R. Interferon-gamma (IFN-gamma) totally abrogated IL-4-mediated Fc epsilon R up-regulation; at the same concentration of IFN-gamma Ia up-regulation is also suppressed, although not as effectively. IFN-gamma was shown to directly suppress Fc epsilon R synthesis, thereby explaining the inhibitory action on Fc epsilon R levels. Finally, it was shown that 11B11 inhibited the increased expression of Fc epsilon R on B cells obtained from mice during the early, but not the late, stages of Nippostrongylus brasiliensis infection. This latter finding suggests that the high Fc epsilon R levels seen early in parasite infections are dependent upon IL-4. The results overall provide further insight into the biologic activities of IL-4.     

Immunoglobulin E-binding site in Fc epsilon receptor (Fc epsilon RII/CD23) identified by homolog-scanning mutagenesis.

The IgE-binding site of the human low-affinity receptor for IgE (Fc epsilon RII/CD23) has previously been mapped to the extracellular domain between amino acid residues 160 and 287. We now have investigated which conformational epitope within this domain specifies the receptor-ligand interaction. The analysis of homolog-scanning mutants expressed in mammalian cells demonstrates that amino acid side chains that affect IgE binding are located in two discontinuous segments, between residues 165-190 and 224-256. The overall structure of the chimeric binding domains, as probed with 11 conformation-sensitive monoclonal antibodies, is generally not distorted, except by replacement of residues 165-183. In this region, disruption of binding function appears to be caused by global conformational constraints on the binding site. Substitution and deletion mutants demonstrate that six out of eight extracellular cysteines, Cys163, Cys174, Cys191, Cys259, Cys273, and Cys282, are necessary for IgE binding and are most likely involved in intramolecular disulfide bridges. We show that the Fc epsilon RII domain delineated by Cys163 and Cys282 encodes all the structural information required to form the IgE-binding site.     

Mechanisms of IgE homeostasis. Sequestration of IgE by murine type II IgE Fc receptor-bearing B cell hybridomas.

Among all classes of Ig, IgE exhibits the highest rate of fractional catabolism of which the site and mechanisms is not understood. We construct a panel of murine B cell hybridomas to investigate the catabolism of IgE; one of these hybridomas, 17A11, constitutively expresses high levels of type II IgE FcR (Fc epsilon RII, CD23) (Kd:1.77 nM; B max: 1.65 x 10(5], and is capable of clearing receptor-bound IgE. Receptor-mediated endocytosis of IgE ligand ensues after binding monomeric and DNP-BSA:IgE immune complexes, and the binding is inhibited by treating 17A11 with anti-CD23. IgE ligands are sequestered and are not susceptible to acid stripping from the cell surface. The internalized IgE ligands redistributed into acid hydrolase containing high density lysosomal vesicles and were degraded; metabolic inhibitors such as chloroquine and monensin that elevate intracellular pH of 17A11 also prevent entry of IgE ligand into lysosomes. These observations raise the possibility that normal Fc epsilon RII-bearing mature B cells in the circulation and lymphoid tissues may function in sequestration and catabolic turnover of IgE molecules through IgE or IL-4 up-regulated Fc epsilon RII uptake; B cell Fc epsilon RII may perform an important role in determining the short biological half-life of IgE molecules, and contributes to IgE homeostasis.     

Human Fc epsilon RIalpha-specific human single-chain Fv (scFv) antibody with antagonistic activity toward IgE/Fc epsilon RIalpha-binding

The alpha-chain of Fc epsilon RI (Fc epsilon RIalpha) plays a critical role in the binding of IgE to Fc epsilon RI. A fully human antibody interfering with this interaction may be useful for the prevention of IgE-mediated allergic diseases. Here, we describe the successful isolation of a human single-chain Fv antibody specific to human Fc epsilon RIalpha using human antibody phage display libraries. Using the non-immune phage antibody libraries constructed from peripheral blood lymphocyte cDNA from 20 healthy subjects, we isolated three phage clones (designated as FcR epsilon 27, FcR epsilon 51, and FcR epsilon 70) through two rounds of biopanning selection. The purified soluble scFv, FcR epsilon 51, inhibited the binding of IgE to recombinant Fc epsilon RIalpha, although both FcR epsilon 27 and FcR epsilon 70 showed fine binding specificity to Fc epsilon RIalpha. Since FcR epsilon 51 was determined to be a monomer by HPLC, BIAcore analysis was performed. The dissociation constant of FcR epsilon 51 to Fc epsilon RIalpha was estimated to be 20 nM, i.e., fortyfold lower than that of IgE binding to Fc epsilon RIalpha (K(d) = 0.5 nM). With these characteristics, FcR epsilon 51 exhibited inhibitory activity on the release of histamine from passively sensitized human peripheral blood mononuclear cells.