Fc epsilon RI-mediated hydrolysis of phosphoinositides in ghosts derived from rat basophilic leukemia cells.

Aggregation of the high affinity receptor for IgE (Fc epsilon RI) on mast cells by a polyvalent Ag leads to hydrolysis of phosphoinositides (PI) catalyzed by phospholipase C (PI-PLC). To understand this phenomenon in molecular terms, it is important to obtain active, cell-free preparations. In extensive preliminary studies, we could not demonstrate Fc epsilon RI-mediated activation of PI-PLC in plasma membranes prepared by conventional methods from rat basophilic leukemia cells. We now report a stepwise approach involving preparation of cytoplasts from such cells and then hypotonic lysis of the cytoplasts to obtain active membrane vesicles. These membranes, best described as "ghosts," appear to reseal after losing greater than 90% of their soluble, cytoplasmic components and contain receptors that when aggregated, activate PI-PLC to hydrolyze endogenous phospholipids. Per unit of plasma membrane, the ghosts retain approximately 25% of Fc epsilon RI-mediated stimulation of PI-PLC relative to the cells. This activity requires ATP, magnesium, phosphoenolpyruvate, and, to a limited degree, calcium. Although an adequate amount of phosphatidylinositol biphosphate is present, the predicted spike of (1,4,5)-inositol trisphosphate is not seen, and the predominant inositol phosphate isomer is (1,4)-inositol bisphosphate. This is the first report of Fc epsilon RI-mediated activation of PI-PLC in a cytoplasm-depleted system that demonstrates activation of endogenous enzyme acting on endogenous substrate. In addition, it is the first such report for any receptor of the Ig superfamily.     

Signal transduction by the cytoplasmic domains of Fc epsilon RI-gamma and TCR-zeta in rat basophilic leukemia cells.

The gamma subunit of the high affinity IgE receptor, Fc epsilon RI, is a member of a family of proteins which form disulfide-linked dimers. This family also includes the zeta- and eta-chains of the T cell receptor. Engagement of Fc epsilon RI activates src-related protein tyrosine kinases in basophils and mast cells. However, the role of individual subunits of Fc epsilon RI in this activation is still not known. In an effort to determine the function of Fc epsilon RI-gamma, we used chimeric proteins containing the extracellular and transmembrane domains of the alpha chain of the human interleukin 2 receptor (Tac) and the cytoplasmic domains of either T cell receptor-zeta or Fc epsilon RI-gamma. We show that while cross-linking of the Tac chimeras in the rat basophilic leukemia cell line RBL-2H3 resulted in the tyrosine phosphorylation of a subset of proteins and a portion of the degranulation normally observed after Fc epsilon RI-mediated stimulation, no detectable activation of p56lyn or pp60c-src was observed. In contrast, an apparent transient deactivation of these two kinases was observed after Tac chimera cross-linking. These observations suggest that Fc epsilon RI-gamma is responsible for some, but not all, of the signaling that occurs after engagement of its receptor, and that other receptor subunits may also play important roles in this signaling process.     

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.     

The fate of IgE bound to rat basophilic leukemia cells.

The present study investigates the fate of the cell-bound IgE by using a well-characterized rat basophilic leukemia cell line and a purifed IgE myeloma protein. Both histamine-releasing and nonreleasing cell lines were examined. In both cases, no evidence for cell-mediated IgE catabolism could be elicited. Both the dissociated IgE and the receptors remained intact for prolonged periods of time, as demonstrated by binding assays. Internalization and/or recycling of membrane-bound IgE could not be demonstrated by E. M. autoradiography. We found only limited time-dependent changes in accessibility to anti-IgE antibody, trypsin, or elution at low pH (2.9 to 3.1). A biphasic dissociation of cell-bound 125I-IgE during incubation in the presence of excess unlabeled IgE was reproducibly observed; the more slowly dissociated IgE was also less readily dissociated at pH 3.4. These studies lead us to conclude that, in vitro, IgE resides in a functional orientation on the surface of RBL-1 cells, for prolonged periods of time.     

The fate of IgE bound to rat basophilic leukemia cells. IV. Functional association between the receptors for IgE

Rat basophilic leukemia cells (RBL-2H3) have receptors for immunoglobulin E (IgE) and immunoglobulin G (IgG). These receptors for IgE mediate the endocytosis of chemically or immunochemically cross-linked IgE but not monomeric IgE. However, unoccupied receptors were endocytosed with cross-linked IgE. To further assess the degree and specificity of the observed coendocytosis, we exposed cells carrying monomeric rat IgE and monomeric mouse IgE anti-DNP to a DNP-protein conjugate. We found that up to 30% of the surface-bound monomeric rat IgE redistributed at 0 to 4 degrees C and was then internalized at 37 degrees C with the immunochemically cross-linked mouse IgE. To assess the specificity of the coendocytosis, we exposed cells carrying monomeric rat IgE to immunochemically cross-linked mouse IgG. We found that the binding, patching, and endocytosis of cross-linked mouse IgG had no effect on the monomerically bound rat IgE. The rate of coendocytosis was the same as the rate of endocytosis (t 1/2 3 to 5 min). The extent of coendocytosis depended on the extent of endocytosis but was relatively insensitive to changes in the ratio between mouse and rat IgE over a broad range. These results indicate that some of the receptors for IgE are associated in a specific fashion.     

Small oligomers of immunoglobulin E (IgE) cause large-scale clustering of IgE receptors on the surface of rat basophilic leukemia cells

We examined the distribution of small oligomers of IgE bound to rat basophilic leukemia cells using fluorescence microscopy. The oligomers were seen to cluster into visible patches on the cell surface at 4 degrees C; at higher temperatures internalization also was observed. In contrast, cells labeled with IgE monomers remained predominantly ring-stained. Evidence is provided that the observed clustering of IgE oligomers is a cell-induced phenomenon, and the possible significance of this clustering is discussed in the context of the oligomer-triggered degranulation of rat basophilic leukemia cells.     

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.     

IgE receptor-mediated hydrolysis of phosphoinositides by cytoplasts from rat basophilic leukemia cells

Cytoplasts (plasma membrane sacs containing cytoplasm, endoplasmic reticulum, and few organelles) were prepared from rat basophilic leukemia cells by treatment with cytochalasin B and centrifugation at 33 degrees C through stepwise gradients of Ficoll. To compare the relative ability of cytoplasts and cells to generate second-messengers (inositol phosphates, Ca2+) in response to stimulation of the high affinity receptor for IgE, we normalized our results per recovered receptor by using the tightly bound IgE as a marker. This marker correlated well with other estimates of plasma membrane recovery. Furthermore, data normalized on this basis correlated well with data expressed as percentage of phosphoinositides hydrolyzed. The purest fraction of cytoplasts (containing about 6% of the receptors) was satisfactorily devoid of organelles and, at early times, generated about 50% as much inositol phosphates per receptor as did the intact, untreated cells. This response of the cytoplasts, like that of the cells, was totally dependent upon aggregation of the receptors. The response by the cytoplasts (in the 5-min time frame which we examined), unlike that of the cells, was not enhanced by the presence of extracellular Ca2+. Furthermore, unlike the cells, the cytoplasts failed to raise their intracellular free Ca2+ levels after addition of polyvalent Ag. This result suggests that aggregation of the receptors may be insufficient, by itself, to open the normal Ca2+ channels.     

Membrane-bound IgE receptor complexes fused with rat basophilic leukemia cells mediate degranulation

The high affinity receptor for IgE on rat basophilic leukemia (RBL) cells mediates antigen-triggered cellular degranulation. Polyethylene glycol-induced membrane fusion methods were used to introduce exogenous IgE receptors into living RBL cells, and these were tested for normal activities. In cell-cell fusion experiments, RBL cells with fluorescein-labeled rat IgE bound to receptors and containing [5-1,2-3H(N)]hydroxytryptamine binoxalate ([3H]5HT) in their secretory granules were fused to cells with receptors occupied by rhodamine-labeled anti-dinitrophenyl mouse IgE. The fused cells showed a uniform surface distribution of both types of IgE, which could be patched independently by anti-IgE or dinitrophenylated bovine gamma globulin (DNP16BGG). [3H]5HT release could be triggered specifically by DNP16BGG. In vesicle-cell fusion experiments, plasma membrane vesicles, with receptors occupied by fluorescein- and 125I-labeled anti-DNP mouse IgE, were fused to RBL cells containing [3H]5HT. The cells showed substantial associated fluorescein fluorescence and 125I counts, and [3H]5HT release could be triggered specifically by DNP16BGG. These experiments indicate that IgE receptors can be dissociated from their natural cellular interactions and retain the ability to reassociate with another cell's components to deliver the transmembrane signal for degranulation.     

Temperature effect on IgE binding to CD23 versus Fc epsilon RI

A chimeric soluble CD23, consisting of the extracellular domain of mouse CD23 and a modified leucine zipper (lz-CD23), has been shown to inhibit IgE binding to the FcepsilonRI. A similar human CD23 construct was also shown to inhibit binding of human IgE to human FcepsilonRI. In both systems, the inhibition was found to be temperature dependent; a 10-fold molar excess of lz-CD23 gave 90-98% inhibition at 4 degrees C, dropping to 20-30% inhibition at 37 degrees C. Surface plasmon resonance analysis of lz-CD23 binding to an IgE-coated sensor chip suggested that the effective concentration of lz-CD23 was lower at the higher temperatures. Analysis of (125)I-IgE binding to CD23(+)-Chinese hamster ovary cells also indicated that increased temperature resulted in a lower percentage of IgE capable of interacting with CD23. In contrast, IgE interacts more effectively with FcepsilonRI(+)-rat basophilic leukemia cells at 37 degrees C compared with 4 degrees C. The results support the concept that the open and closed IgE structures found by crystallography interact differently with the two IgE receptors and suggest that temperature influences the relative percentage of IgE in the respective structural forms. Changes in CD23 oligomerization also plays a role in the decreased binding seen at physiological temperatures.     

Rotational dynamics of the Fc receptor for immunoglobulin E on histamine-releasing rat basophilic leukemia cells

The rotational diffusion of immunoglobulin E (IgE) bound to its specific Fc receptor on the surface of living rat basophilic leukemia cells was determined from time-resolved phosphorescence emission and anisotropy measurements. The IgE-receptor complexes are mobile throughout the range of temperatures of 5-38 degrees C. The residual anisotropy does not reach zero, indicating that the rotational diffusion is hindered. The values of rotational correlation times for each temperature are consistent with dispersed receptors rotating freely in the cell membrane and rule out any significant aggregation of occupied receptors before cross-linking by antigen or anti-IgE antibodies. The rotational correlation times decrease with increasing temperature from 65 microseconds at 5.5 degrees C to 23 microseconds at 38 degrees C. However, the degree of orientational constraint experienced by the probe is unchanged. Thus, the temperature dependence can be attributed primarily to a change in the effective viscosity of the cellular plasma membrane. The phosphorescence depolarization technique is very sensitive (our probe concentrations were 10-100 nM) and thus generally applicable to studies of surface receptors and antigens on living cells.     

A cell surface glycoprotein of rat basophilic leukemia cells close to the high affinity IgE receptor (Fc epsilon RI). Similarity to human melanoma differentiation antigen ME491.

A monoclonal antibody (mAb), AD1, was isolated that recognized a cell surface protein on rat basophilic leukemia cells (RBL-2H3). At high concentration, this antibody inhibited IgE-mediated but not calcium ionophore-induced histamine release (49% inhibition at 100 micrograms/ml). The mAb AD1 did not inhibit the binding of IgE or of several antibodies directed to the high affinity IgE receptor (Fc epsilon RI). Likewise, IgE did not inhibit mAb AD1 binding. However, several anti-Fc epsilon RI antibodies did inhibit mAb AD1 binding as intact molecules but not as Fab fragments. Therefore, the sites on the cell surface to which mAb AD1 binds are close to Fc epsilon RI. The mAb AD1 immunoprecipitated a broad, 50-60-kDa band from 125I-surface-labeled RBL-2H3 cells that upon peptide N-glycosidase F treatment was transformed into a sharp 27-kDa band. A similar 27-kDa protein was immunoprecipitated from surface-radiolabeled cells after culture with tunicamycin. Thus, the protein recognized by mAb AD1 is highly glycosylated with predominantly N-linked oligosaccharides. The N-terminal sequence of 43 amino acids was found to be different from any subunit of Fc epsilon RI but nearly identical to that of the human melanoma-associated antigen ME491. Therefore, mAb AD1 binds to a surface glycoprotein on RBL-2H3 cells sterically close to the Fc epsilon RI but distinct from the recognized subunits of the receptor.     

IgE receptor-mediated depolarization of rat basophilic leukemia cells measured with the fluorescent probe bis-oxonol

Receptor-mediated changes in plasma membrane potential were recorded in rat basophilic leukemia (RBL) cells with the potential-sensitive fluorescent indicator bis-oxonol. Depolarization of the mitochondria with metabolic inhibitors was not detected by bis-oxonol, suggesting that only potential changes across the plasma membrane were being measured. The resting membrane potential of RBL cells was largely generated by the equilibrium distribution of K+ and not through electrogenic activity of the sodium pump. Depolarization was maintained as long as IgE receptors remained aggregated. We believe that at physiologic calcium concentrations a large portion of the measured potential change may be due to calcium influx across the plasma membrane. Prevention of calcium influx by lanthanum, disruption of aggregated receptors, or prior depolarization in a high K+ saline solution completely inhibited the antigen-induced depolarization. The time course of the antigen-stimulated increase in bis-oxonol fluorescence was similar, but not identical, to the antigen-stimulated rise in cytoplasmic free ionized calcium measured with fura-2. Antigen-stimulated depolarization was inhibited by removing both calcium and sodium and could be restored by the addition of either ion. Reduction of total cellular adenosine triphosphate inhibited depolarization in response to antigen stimulation.     

Binding of monoclonal antibody AA4 to gangliosides on rat basophilic leukemia cells produces changes similar to those seen with Fc epsilon receptor activation.

The mAb AA4 binds to novel derivatives of the ganglioside Gd1b on rat basophilic leukemia (RBL-2H3) cells. Some of the gangliosides are located close to the high affinity IgE receptor (Fc epsilon RI), and binding of mAb AA4 inhibits Fc epsilon RI-mediated histamine release. In the present study, mAb AA4 was found to bind exclusively to mast cells in all rat tissues examined. In vitro, within 1 min of mAb AA4 binding, the cells underwent striking morphologic changes. They lost their normal spindle shaped appearance, increased their ruffling, and spread over the surface of the culture dish. These changes were accompanied by a redistribution of the cytoskeletal elements, actin, tubulin, and vimentin, but only the actin was associated with the membrane ruffles. Binding of mAb AA4 also induces a rise in intracellular calcium, stimulates phosphatidyl inositol breakdown, and activates PKC. However, the extent of these changes was less than that observed when the cells were stimulated with antigen or antibody directed against the Fc epsilon RI. None of these changes associated with mAb AA4 binding were seen when the cells were exposed to nonspecific IgG, IgE, or four other anti-cell surface antibodies, nor were the changes induced by binding mAb AA4 at 4 degrees C or in the absence of extracellular calcium. Although mAb AA4 does not stimulate histamine release, it enhances the effect of the calcium ionophore A23187 mediated release. The morphological and biochemical effects produced by mAb AA4 are similar to those seen following activation of the cell through the IgE receptor. Therefore, the surface gangliosides which bind mAb AA4 may function in modulating secretory events.     

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.     

Peroxisome proliferator-activated receptor ligands negatively regulate the expression of the high-affinity IgE receptor Fc epsilon RI in human basophilic KU812 cells

The high-affinity IgE receptor Fc epsilon RI is expressed on the cell surface of mast cells and basophils, and plays a central role in IgE-mediated inflammatory reactions. Recently, peroxisome proliferator-activated receptors (PPARs) have been implicated in the anti-inflammatory response. To investigate a possible role for PPAR in human basophils, the effect of PPAR ligands on Fc epsilon RI expression in human basophilic KU812 cells was studied. The PPARalpha ligand, leukotriene B(4), did not affect the cell surface expression of Fc epsilon RI. However, prostaglandin (PG) A(1) and 15-deoxy-Delta(12,14) PGJ(2) (15d-PGJ(2)), which are PPARbeta and gamma ligands, respectively, were both able to decrease Fc epsilon RI expression. Treatment with PGA(1) or 15d-PGJ(2) separately also reduced histamine release from KU812 cells in response to cross-linkage of Fc epsilon RI. In addition, RT-PCR analysis showed that KU812 cells expressed the mRNA for PPARalpha, beta, and gamma, indicating that PPARbeta or gamma may negatively regulate the cell activation via Fc epsilon RI. Cells treated with 15d-PGJ(2) expressed lower levels of Fc epsilon RI alpha and gamma mRNA, and PGA(1) treatment decreased the level of Fc epsilon RI gamma mRNA. These results suggest that the suppression of Fc epsilon RI expression by PPARs may be due to the down-regulation of Fc epsilon RI alpha or gamma mRNA.     

Phospholipase C-gamma 1 is translocated to the membrane of rat basophilic leukemia cells in response to aggregation of IgE receptors.

Aggregation of the high affinity receptor for IgE (Fc epsilon RI) on the surface of mast cells results in the rapid hydrolysis of membrane inositol phospholipids by phospholipase C (PLC). Although at least seven isoenzymes of PLC have been characterized in different mammalian cells, the isoenzyme involved in Fc epsilon RI-mediated signal transduction and the mechanism of its activation have not been demonstrated. We now report that PLC-gamma 1 is translocated to the membrane of mast cells after aggregation of Fc epsilon RI. Activation of rat basophilic leukemia cells, a rat mast cell line, with oligomeric IgE resulted in an increase in PLC activity in washed membrane preparations in a cell free assay containing exogenous [3H]phosphatidylinositol (PI). The increase in PLC activity has the same dose-response to oligomeric IgE as receptor mediated hydrolysis of inositol lipids (PI hydrolysis) in intact cells. Analysis by Western blot probed with anti-PLC-gamma 1 antibody revealed that there is a three- to fourfold increase in PLC-gamma 1 in membranes from activated cells. The increase in PLC activity is augmented a further 20% by the addition of orthovanadate to the incubation medium suggesting that a tyrosine phosphatase is involved in the down-regulation of this phenomenon. These findings demonstrate translocation of PLC-gamma 1 to the membrane following activation of a receptor which does not contain intrinsic tyrosine kinase activity. Activation of PLC-gamma 1 by this pathway may account for Fc epsilon RI-mediated PI hydrolysis.