Mast cells cultured from the peripheral blood of normal donors and patients with mastocytosis originate from a CD34+/Fc epsilon RI- cell population

Mast cells may be cultured from human peripheral blood in the presence of recombinant human stem cell factor (rhSCF). The characteristics of the cells in peripheral blood that give rise to mast cells are unknown. Because mast cell precursors in human marrow are CD34+, human peripheral blood mononuclear cells from patients with mastocytosis and normal controls were sorted on the basis of CD34 expression and the positive and negative cell populations were cultured in rhSCF, recombinant human interleukin-3 (rhIL-3), or both for 6 weeks. Cell cultures were examined every 2 weeks for total and mast cell number and cell differential using Wright Giemsa and acid toluidine blue stains and antibodies to mast cell tryptase and chymase, cell-associated histamine, and expression of CD34, c-kit, Fc epsilon RI, and Fc gamma RII using flow cytometric analysis. The ultrastructural anatomy of mast cells was examined by electron microscopy. Peripheral blood CD34+ cells cultured in rhSCF with or without rhIL-3 gave rise to cell cultures consisting of greater than 80% mast cells by 6 weeks. CD34+ cells cultured in rhIL-3 alone did not give rise to mast cells, whereas rhIL- 3 plus rhSCF increased the final mast cell number eightfold when compared with cells cultured in rhSCF alone. Mast cells increased concomitantly with a decrease in large undifferentiated mononuclear cells. CD34- cells did not give rise to mast cells. Histamine content per cell at 6 weeks was approximately 5 pg. Electron microscopy of 4- week cultures showed immature mast cells containing predominantly tryptase-positive granules that were either homogeneous or contained lattice structures, partial scroll patterns, or central dense cores and mixtures of vesicles, fine granular material, and particles. The CD34+ population at day 0 expressed Kit (65%) and Fc gamma RII (95%), but not Fc epsilon RI, by fluorescence-activated cell sorter analysis. At 6 weeks, CD34+-derived mast cells exhibited Fc epsilon RI in addition to Kit and Fc gamma RII, and were negative for CD34 antigen. Patients with mastocytosis showed a higher number of mast cells per CD34+ cell cultured compared with normal controls. Thus, the mast cell precursor in human peripheral blood is CD34+/Fc epsilon RI- and gives rise to mast cells in the presence of rhSCF with or without rhIL-3, and the number of mast cells arising per CD34+ cell in culture is greater when the CD34+ cells are obtained from patients with mastocytosis compared with normal subjects.     

Human skin-derived mast cells can proliferate while retaining their characteristic functional and protease phenotypes

Human mast cells in adult tissues have been thought to have limited, if any, proliferative potential. The current study examined mast cells obtained from adult skin and cultured in serum-free medium with recombinant human stem cell factor. During the first 4 weeks of culture, the percentages of mast cells increased from 10 to almost 100. After 8 weeks, a 150-fold increase in the number of mast cells was observed. When freshly dispersed mast cells were individually sorted onto human fibroblast monolayers and cultured for 3 weeks, one or more mast cells were detected in about two thirds of the wells, and in about two thirds of these wells the surviving mast cells showed evidence of proliferation, indicating most mast cells in skin can proliferate. Such mast cells all expressed high surface levels of Kit and Fc epsilon RI, each of which were functional, indicating IgE was not required for Fc epsilon RI expression on mast cells. Such mast cells also retained the MC(TC) protease phenotype of mast cells that normally reside in the dermis. After 4 to 8 weeks of culture these mast cells degranulated in response to substance P and compound 48/80, characteristics of skin-derived mast cells that persist outside of the cutaneous microenvironment. (Blood. 2001;97:2045-2052)     

Tyrosine phosphorylation coupled to IgE receptor-mediated signal transduction and histamine release.

Antigen-induced cross-linking of IgE bound to its receptors at the surface of basophils or mast cells initiates a number of biochemical events culminating in the release of histamine-containing granules. In the present study, we investigated the possible involvement of tyrosine phosphorylation in signaling by the high-affinity IgE receptor (Fc epsilon RI). Cross-linking of Fc epsilon RI in rat basophilic leukemia cells (RBL-2H3) led to the phosphorylation of several proteins on tyrosine, the most prominent having a mass of 72 kDa. Tyrosine phosphorylation was rapid, detectable 1 min after stimulation, and correlated with both the time course and antigen dose for histamine release. Reversal of Fc epsilon RI cross-linking prevented continuation of the degranulation process and resulted in rapid loss of tyrosine phosphorylation. The receptor-mediated tyrosine phosphorylation was still induced in the absence of calcium in the medium. Depletion of protein kinase C with phorbol 12-myristate 13-acetate did not dramatically affect the tyrosine phosphorylation signal or the release of histamine. In contrast, the calcium ionophore A23187 induced histamine release in the absence of a perceptible increase in protein tyrosine phosphorylation. Thus, tyrosine phosphorylation is an early signal following Fc epsilon RI aggregation, independent of the exocytotic process itself. Taken together, our findings functionally link protein phosphorylation on tyrosine residues to Fc epsilon RI-mediated signal transduction leading to histamine release.     

Stem cell factor and interleukin-4 induce murine bone marrow cells to develop into mast cells with connective tissue type characteristics in vitro

In this study, we have developed a method to obtain mast cells with connective tissue type mast cell (CTMC) characteristics directly from mouse bone marrow (BM) cells. BM cells were grown for 3 weeks in presence of interleukin-4 (IL-4) plus stem cell factor (SCF). SCF alone poorly supported growth and development of mast cells. IL-4 dose-dependently enhanced the expression of c-kit and high-affinity receptor for IgE (Fc(epsilon)RI) on the cell surface of SCF-cultured BM cells. Furthermore, cytoplasmic granulation and histamine synthesis of BM-derived mast cells were increased in presence of IL-4 and SCF. Histochemical staining demonstrated that granules were safranin positive. BM-derived mast cells could be activated for granule exocytosis (beta-hexosaminidase release) and lipid mediator generation (LTC4 production) via Fc(epsilon)RI after sensitization with IgE and subsequent crosslinking with multivalent antigen. In addition, mast cells derived from BM cells cultured with SCF plus IL-4 could be activated by substance P, a nonimmunologic stimulus, to release beta-hexosaminidase. The results presented indicate that IL-4 and SCF both have a prominent role in the development of mast cells from murine BM cells in vitro. Mast cells can directly be derived from BM cells in presence of SCF and IL-4 and the cultured cells show typical hallmarks of CTMC, indicating that precursor cells for CTMC may be present in BM. The described culture procedure may be useful to investigate the molecular aspects of the development of committed mast cell lineages.     

Transmembrane signaling by the high-affinity IgE receptor on membrane preparations.

Aggregating the receptor with high affinity for IgE (Fc epsilon RI) stimulates a variety of phenomena in mast cells. Previous efforts to reproduce some of these events in broken-cell preparations such as isolated membranes have had limited success, possibly because the phenomena being monitored were too distal from the initial events. One of the earliest responses is now known to be the phosphorylation of tyrosine residues on several proteins, including the beta and gamma subunits of Fc epsilon RI. We show that in cell sonicates or on partially purified membranes derived from tumor mast cells, aggregating Fc epsilon RI stimulates phosphorylation of receptor tyrosine residues. As in the intact cells, receptor-mediated phosphorylation occurs only on receptors that are themselves aggregated. Because even in the unfractionated sonicates the phosphorylation of other cellular components was not detectably enhanced, and because the evidence is against the receptor itself being a kinase, our results suggest that phosphorylation of Fc epsilon RI is one of the earliest events stimulated by the receptor--an event that can now be investigated on simpler biological preparations than previously available.     

Interleukin-6 and mast cells.

Interleukin (IL) 6 is a pleiotropic cytokine (26 kDa) that originally was named interferon beta 2 or B cell-stimulating factor or differentiating B cell factor inducing immunoglobulin production. IL-6 is produced in many diseases. After secretion, IL-6 binds to its receptor IL-6R alpha (gp 80), the IL-6R alpha complex then recruits the signal-transducing beta-subunit (gp 130), which is the functional complex for signal transduction. In addition, activation of Th2 cells or mast cells also produce IL-6, which mediates immune responses, inflammation, acute phase responses, hematopoiesis, cancer, inflammatory bowel disease, etc. IL-6 also is a crucial cytokine for mast cell maturation. Human cord blood CD34+ cells differentiate and grow into mast cells in the presence of stem cell factor (SCF) and IL-6, causing increases in cell size, frequency of chymase positive cells, and intracellular histamine levels when compared with cells treated with SCF alone. Activated mast cells increase IL-6 mRNA associated with protein kinase C (PKC) activity. IL-6 also up-regulates histamine production rather than increases its storage and is an important inducing factor for the expression of immunoglobulin E (IgE) Fc epsilon RI.     

Association of protein-tyrosine kinase with phospholipase C-gamma 1 in bone marrow-derived mouse mast cells.

Bone marrow-derived mouse mast cells contain phospholipase C-gamma 1 (PLC-gamma 1), which is phosphorylated at tyrosine residues upon cross-linking of cell-bound IgE antibodies with multivalent antigen. It was found that immune complexes formed from digitonin lysates of the mast cells by monoclonal anti-PLC-gamma 1 antibodies contained protein-tyrosine kinase (PTK), which phosphorylated PLC-gamma 1 in vitro. The tyrosine kinase activity coprecipitated with PLC-gamma 1-anti-PLC-gamma 1 complexes markedly increased when the cell lysates were obtained immediately after antigen challenge. The results indicate that PTK is associated with PLC-gamma 1 in the mast cells and that the kinase is activated upon cross-linking of Fc epsilon RI. Neither beta nor gamma subunit of Fc epsilon RI nor src family PTK was coprecipitated with the PLC-gamma 1-anti-PLC-gamma 1 complexes. In situ denaturation/renaturation experiments, which detect autophosphorylated kinases, indicated that the PTK associated with PLC-gamma 1 was a 44-kDa protein.     

Aggregation of the high-affinity IgE receptor and enhanced activity of p53/56lyn protein-tyrosine kinase.

Aggregation of the receptor with high affinity for IgE (Fc epsilon RI) on the surface of mast cells and basophils stimulates phosphorylation of protein tyrosines, a process in which p53/56lyn kinase has been implicated. We measured the association between Fc epsilon RI and the kinase, using chemical crosslinking to stabilize their interaction. In the rat basophilic leukemia mast cell line, 3-4%, and at most 20%, of Fc epsilon RI appear to be associated with the kinase prior to aggregation, even though there is an excess of total cell lyn kinase. Aggregating the Fc epsilon RI causes three to four times more of the kinase to associate with receptors, a process requiring a prior phosphorylation step. In an in vitro assay, the lyn associated with the aggregated receptors becomes disproportionately more phosphorylated than would be predicted from the amount of lyn associated with the receptors. These and other data are consistent with a model in which aggregation of the receptor leads to its transphosphorylation by constitutively associated lyn kinase. We propose that additional molecules of this kinase are thereby recruited and that this markedly enhances transphosphorylation of tyrosine on the receptor and associated proteins, thereby initiating a cascade of further biochemical changes. This model is also consistent with data on receptors such as the clonotypic receptors on B and T lymphocytes, which share structural and functional features with Fc epsilon RI.     

Mouse splenic and bone marrow cell populations that express high-affinity Fc epsilon receptors and produce interleukin 4 are highly enriched in basophils.

Splenic and bone marrow cells from normal mice, and from mice that have been polyclonally activated by injection of anti-IgD antibody, contain cells that produce interleukin 4 (IL-4) in response to crosslinkage of Fc epsilon receptors (Fc epsilon R) or Fc gamma R or to ionomycin. Isolated Fc epsilon R+ cells have recently been shown to contain all of the IL-4-producing capacity of the nonlymphoid compartment of spleen and bone marrow. Here, purified Fc epsilon R+ cells are shown to be enriched in cells that contain histamine and express alcian blue-positive cytoplasmic granules. By electron microscopy, the vast majority of cytoplasmic granule-containing cells are basophils; they constitute approximately 25% and approximately 50%, respectively, of Fc epsilon R+ spleen and bone marrow cells from anti-IgD-injected mice. The Fc epsilon R- populations contain cells that form colonies typical of mast cells. The Fc epsilon R+ populations also contain cells that, upon culture with IL-3, form colonies of alcian blue-positive cells, but (in contrast to colonies derived from Fc epsilon R- populations) the colonies are small, and all the cells die within 2-3 weeks. The Fc epsilon R+ cells synthesize histamine during a 60-hr culture with IL-3, while the Fc epsilon R- cells do not. These results indicate that IL-4-producing Fc epsilon R+ cells are highly enriched in basophils.     

A novel insight to the functional role of Stathmin 1 in IgE-mediated activation of RBL-2H3 cells

IgE-mediated cell signaling, induced by cross-linking of high affinity receptor for IgE (FcεRI) in the presence of antigen (Ag), is a well known mechanism described for mast cell activation in allergy and hypersensitivity reactions, which induces a spectrum of cellular responses such as secretion and up-regulation of cell surface FcεRI. Although for several years IgE binding to FcεRI was considered to be a passive sensitization process, the outcomes of several recent studies have revealed a variety of different cellular responses to IgE binding compared to IgE plus Antigen binding. The present study applied a functional proteomics-based approach to investigate mast cell signaling events and provided new insights to FcεRI-mediated cell signaling in RBL-2H3.1 cells, and may point to the activation of alternative signaling pathways in response to IgE or IgE plus Ag. Comparative analysis by 2-D PAGE of RBL cells activated with IgE plus Ag for three and four hours compared to non-activated cells was followed by mass spectrometric protein identification and provided evidence for the induction of Stathmin 1 (STMN1) gene expression in response to IgE plus Ag activation.Complementary SDS-PAGE analysis showed a distinct up-regulation of STMN1 induction in response to challenge with IgE plus Ag compared to sensitization with IgE only. Phosphoproteomics analysis gave evidence for significant increase at phosphorylation of STMN1 on ser16 after 1min, though a slight rise at 5 min, and on ser38 after 1 and 5min sensitization with IgE and a similar result was observed for 1min IgE plus Ag-activation. IgE plus Ag-activation was also found to induce the phosphorylation of ser38 to a greater extent than sensitization with IgE. In contrast, IgE alone was more effective than IgE plus Ag at inducing phosphorylation of ser16. Collectively this study provides further insights into the role of stathmin 1 in FcRI-mediated activation of cells of mast cell lineage and might shed light on the diverse response of these cells to IgE or IgE plus Ag.     

Clinical trials results of a unique monoclonal antibody in asthma

IgE binds to high affinity receptors (Fc epsilon RI) on mast cells, basophils, alveolar macrophages, and antigen-presenting cells. This results in the degranulation of effector cells and the release of mediators, such as histamine, leukotrienes, cytokines, that are causally linked to the pathophysiology of allergic asthma. It made for an attractive target for the development of a unique humanized monoclonal antibody, rhuMAb-E25 (E25). E25 offers a novel strategic approach to allergy treatment, it is expected that using E25 to significantly decrease IgE levels will result in improved control of allergic asthma.     

Transphosphorylation as the mechanism by which the high-affinity receptor for IgE is phosphorylated upon aggregation.

When aggregated, the high-affinity receptors for IgE on mast cells (Fc epsilon RI) launch a series of phosphorylations, particularly of protein tyrosines. We have analyzed how aggregation initiates this cascade. We examined Fc epsilon RI from unstimulated cells and from cells exposed to a polyvalent hapten conjugate that aggregates the Fc epsilon RI via the receptor-bound anti-hapten IgE. We also examined the latter receptors after they had been disaggregated in vitro with monovalent hapten. By an in vitro kinase assay: (i) Unaggregated and disaggregated receptors are associated with a kinase that phosphorylates an exogenous (peptide) substrate but minimally, or not at all, the subunits of Fc epsilon RI or associated proteins (endogenous substrates). After aggregation, phosphorylation of the exogenous substrate is linear with time, but the modification of the endogenous substrates reaches a plateau, presumably because only those endogenous substrates that are adjacent to the kinase are phosphorylated. (ii) Aggregated receptors and disaggregated receptors have enhanced kinase activity toward exogenous substrate. The state of phosphorylation of the receptor correlates strongly with the yield of enhanced kinase activity. We propose that upon aggregation of Fc epsilon RI, a constitutively associated kinase phosphorylates endogenous substrates by transphosphorylation. As a result, additional kinase activity becomes manifest and this promotes further transphosphorylation. In view of the homology between Fc epsilon RI and other receptors central to the immune response, the latter receptors likely utilize a similar transphosphorylation mechanism.     

CD3 zeta subunit can substitute for the gamma subunit of Fc epsilon receptor type I in assembly and functional expression of the high-affinity IgE receptor: evidence for interreceptor complementation.

The high-affinity receptor for IgE (Fc epsilon RI) is a four-subunit structure consisting of three distinct polypeptides: the IgE-binding alpha chain, the four-fold membrane-spanning beta chain, and the disulfide-linked gamma-gamma homodimer. cDNAs encoding each subunit have previously been isolated. Here we show that microinjection of Xenopus oocytes with a mixture of in vitro transcribed RNAs encoding each subunit results in expression of IgE receptors at the oocyte surface as detected by binding of IgE or anti-Fc epsilon RI alpha subunit monoclonal antibody to intact oocytes. Surface expression of Fc epsilon RI requires injection of all three subunits (alpha, beta, and gamma) RNAs. In particular, omission of Fc epsilon RI gamma RNA from the mixtures abolishes surface binding of either IgE or anti-Fc epsilon RI alpha monoclonal antibody to microinjected oocytes. However, addition of CD3 zeta RNA to Fc epsilon RI alpha and Fc epsilon RI beta RNAs restores IgE receptor surface expression when this combination is microinjected into oocytes. Metabolic labeling and immunoprecipitation of oocyte microinjected with a mixture of CD3 zeta plus Fc epsilon RI alpha and Fc epsilon RI beta RNAs reveals a noncovalent association between the CD3 zeta-zeta disulfide-linked homodimer and Fc epsilon RI alpha-beta. These results provide direct evidence for the functional relatedness of CD3 zeta and Fc epsilon RI gamma.     

NTAL phosphorylation is a pivotal link between the signaling cascades leading to human mast cell degranulation following Kit activation and Fc epsilon RI aggregation

Aggregation of high-affinity receptors for immunoglobulin E (Fc epsilon RI) on the surface of mast cells results in degranulation, a response that is potentiated by binding of stem cell factor (SCF) to its receptor Kit. We observed that one of the major initial signaling events associated with Fc epsilon RI-mediated activation of human mast cells (HuMCs) is the rapid tyrosine phosphorylation of a protein of 25 to 30 kDa. The phosphorylation of this protein was also observed in response to SCF. This protein was identified as non-T-cell activation linker (NTAL), an adaptor molecule similar to linker for activated T cells (LAT). Unlike the Fc epsilon RI response, SCF induced NTAL phosphorylation in the absence of detectable LAT phosphorylation. When SCF and antigen were added concurrently, there was a marked synergistic effect on NTAL phosphorylation, however, SCF did not enhance the phosphorylation of LAT induced by Fc epsilon RI aggregation. Fc epsilon RI- and SCF-mediated NTAL phosphorylation appear to be differentially regulated by Src kinases and/or Kit kinase, respectively. Diminution of NTAL expression by silencing RNA oligonucleotides in HuMCs resulted in a reduction of both Kit- and Fc epsilon RI-mediated degranulation. NTAL, thus, appears to be an important link between the signaling pathways that are initiated by these receptors, culminating in mast cell degranulation.     

Fc epsilon RI-mediated tyrosine phosphorylation and activation of the 72-kDa protein-tyrosine kinase, PTK72, in RBL-2H3 rat tumor mast cells.

In RBL-2H3 rat tumor mast cells, cross-linking the high-affinity IgE receptor Fc epsilon RI causes tyrosine phosphorylation of multiple proteins. These phosphoproteins include phospholipase C gamma 1, the beta and gamma subunits of the Fc epsilon RI, the Src family protein-tyrosine kinase Lyn, and a 72-kDa protein that coimmunoprecipitates from lysates of antigen-stimulated cells with antibody to the receptor beta subunit. We now present evidence that the 72-kDa Fc epsilon RI-associated protein is the protein-tyrosine kinase PTK72 that forms part of the antigen receptor complex in B lymphocytes. The identification is based on immunoreactivity with anti-PTK72 antiserum, chromatographic profiles on the affinity resin heparin/agarose, and one-dimensional phosphopeptide mapping studies. Enzymatic activity of the kinase is increased in anti-PTK72 immune complexes prepared from lysates of antigen-activated RBL-2H3 cells. The 72-kDa protein-tyrosine kinase is the principal substrate for in vitro tyrosine phosphorylation in anti-phosphotyrosine immunoprecipitates of RBL-2H3 cells. The discovery that RBL-2H3 mast cells share a receptor-activated protein-tyrosine kinase, PTK72, with B lymphocytes provides additional support for the existence of common signaling pathways initiated by multichain immune recognition receptors.     

Impaired FcεRI stability, signaling, and effector functions in murine mast cells lacking glycosylphosphatidylinositol-anchored proteins

A key event and potential therapeutic target in allergic and asthmatic diseases is signaling by the IgE receptor FcεRI, which depends on its interactions with Src family kinases (SFK). Here we tested the hypothesis that glycosylphosphatidylinositiol-anchored proteins (GPI-AP) are involved in FcεRI signaling, based on previous observations that GPI-AP colocalize with and mediate activation of SFK. We generated mice with a hematopoietic cell-specific GPI-AP deficiency by targeted disruption of the GPI biosynthesis gene PigA. In these mice, IgE-mediated passive cutaneous anaphylaxis was largely abolished. PigA-deficient mast cells cultured from these mice showed impaired degranulation in response to stimulation with IgE and antigen in vitro, despite normal IgE binding and antigen-induced FcεRI aggregation. On stimulation of these cells with IgE and antigen, coprecipitation of the FcεRI α-chain with the γ-chain and β-chain was markedly reduced. As a result, IgE/antigen-induced FcεRI-Lyn association and γ-chain tyrosine phosphorylation were both impaired in PigA-deficient cells. These data provide genetic evidence for an unanticipated key role of GPI-AP in FcεRI interchain interactions and early FcεRI signaling events, necessary for antigen-induced mast cell degranulation.     

Development of human mast cells from umbilical cord blood cells by recombinant human and murine c-kit ligand.

Both human and mouse c-kit ligand induced differentiation of human mast cells in a long-term culture of the mononuclear cells of umbilical cord blood. Growth factor activity for human mast cells present in conditioned medium of BALB/3T3 fibroblasts was due to mouse c-kit ligand. Recombinant c-kit ligand induced differentiation and proliferation of mast cell progenitors in early stages of culture. However, apparent selective growth of mast cells by c-kit ligand in cord blood cell cultures is mainly due to the effect of the cytokine to selectively maintain survival of immature mast cells. Electron microscopic analysis indicated that human mast cells developed by c-kit ligand were similar to human mast cells in the lung and gut mucosa, while those developed in coculture of cord blood cells with Swiss albino/3T3 fibroblasts were similar to skin mast cells. This conclusion was supported by the fact that the majority of mast cells developed by c-kit ligand contained only tryptase in their granules, whereas those developed in the cocultures contained both tryptase and chymase. It was also found that mast cells developed by c-kit ligand were immature even after culture for 14 weeks. Nevertheless, these cells express Fc epsilon RI, and could be sensitized with human IgE for anti-IgE-induced release of histamine, prostaglandin D2, and leukotriene C4.     

Aggregated IgE mimic interleukin-3-induced histamine synthesis by murine hematopoietic progenitors

Similar to interleukin-3 (IL-3), IgE acts on murine bone marrow cells by inducing histamine production. This effect does not result from degranulation of histamine-containing cells, but from histamine synthesis, as assessed by the following findings. (1) The histamine content of freshly isolated bone marrow cells is too low to account for the increase in extracellular histamine levels. (2) Neither IL-3 nor IgE induced histamine production in the presence of the specific inhibitor of histidine decarboxylase (HDC), the histamine-forming enzyme. (3) Both the enzymatic activity and the mRNA expression of HDC were enhanced in response to IL-3 or IgE. Artificial aggregation or formation of IgE immune complexes augmented ther effect on histamine synthesis, indicating that the aggregated form is responsible for this biologic activity. Yet, it is apparently not mediated by Fc epsilon RI because their cross-linkage by dinitrophenyl bovine serum albumin after presensitization with IgE did not induce histamine production by hematopoietic progenitors. Among other aggregated isotypes tested, only IgG2a and, to a lesser extent, IgG1 had a consistent but lower effect, whereas IgM and IgA were completely inactive. The target cells of IL-3 and IgE in terms of histamine synthesis do not belong to mature bone marrow populations, especially mast cells. They copurify with hematopoietic progenitors in the low-density layers of a discontinuous Ficoll gradient where they represent around 5% of the cells, as determined by in situ hybridization. This percentage remained the same, regardless of whether the cells were stimulated by IgE or IL-3 alone or by a combination of both, suggesting a common responder cell. In accordance with this notion, histamine-producing cells could not be distinguished from each other on the basis of density, size and internal structure, or rhodamine (Rh) retention. Finally, the effect of IgE is not caused by the induction of IL-3 because anti-IL-3 antibodies did not abrogate the effect of IgE.