Intracellular expression and release of Fc epsilon RI alpha by human eosinophils.

Although Fc epsilon R have been detected on human eosinophils, levels varied from moderate to extremely low or undetectable depending on the donor and methods used. We have attempted to resolve the conflicting data by measuring levels of IgE, Fc epsilon RI, and Fc epsilon RII in or on human eosinophils from a variety of donors (n = 26) and late-phase bronchoalveolar lavage fluids (n = 5). Our results demonstrated little or no cell surface IgE or IgE receptors as analyzed by immunofluorescence and flow cytometry. Culture of eosinophils for up to 11 days in the presence or absence of IgE and/or IL-4 (conditions that enhance Fc epsilon R on other cells) failed to induce any detectable surface Fc epsilon R. However, immunoprecipitation and Western blot analysis of eosinophil lysates using mAb specific for Fc epsilon RI alpha showed a distinct band of approximately 50 kDa, similar to that found in basophils. Western blotting also showed the presence of FcR gamma-chain, but no Fc epsilon RI beta. Surface biotinylation followed by immunoprecipitation again failed to detect surface Fc epsilon RI alpha, although surface FcR gamma was easily detected. Since we were able to detect intracellular Fc epsilon RI alpha, we examined its release from eosinophils. Immunoprecipitation and Western blotting demonstrated the release of Fc epsilon RI alpha into the supernatant of cultured eosinophils, peaking at approximately 48 h. We conclude that eosinophils possess a sizable intracellular pool of Fc epsilon RI alpha that is available for release, with undetectable surface levels in a variety of subjects, including those with eosinophilia and elevated serum IgE. The biological relevance of this soluble form of Fc epsilon RI alpha remains to be determined.     

Characterization of the IgE Fc receptors on monocytes and macrophages

Subpopulations of human monocytes (15%) and alveolar macrophages (AM phi, 8%) and rat and mouse AM phi (89%) and peritoneal M phi (57%) bear Fc receptors for IgE (Fc epsilon R) as shown by IgE-specific rosette formation. Cells from M phi-like cell lines of human, rat, and mouse origins also express Fc epsilon R. Monomeric IgE binds to Fc epsilon R on M phi with an equilibrium association constant Ka congruent to 10(7) M-1. The Fc epsilon R on human monocytes and M phi are antigenically similar to Fc epsilon R on lymphocytes but differ from Fc epsilon R on basophilic granulocytes. The Fc epsilon R on human and mouse M phi promote phagocytosis and lysis of IgE-coated erythrocytes. Patients with active IgE-mediated allergic diseases have elevated percentages of Fc epsilon R(+) monocytes (56%) that show allergic increased lytic activity against IgE-coated erythrocytes as compared to monocytes from normal humans. M phi from rats infested with Nippostrongylus brasiliensis parasites express more Fc epsilon R than normal M phi. The data indicate that Fc epsilon R expressed on M phi differ from those on mast cells and basophils, increase in number during IgE immune responses, and are likely to play an important role in the host's defense against parasites and in the pathogenesis of allergic diseases.     

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.     

Regulation of mast-cell and basophil function and survival by IgE

Mast cells and basophils are important effector cells in T helper 2 (T(H)2)-cell-dependent, immunoglobulin-E-associated allergic disorders and immune responses to parasites. The crosslinking of IgE that is bound to the high-affinity receptor Fc epsilon RI with multivalent antigen results in the aggregation of Fc epsilon RI and the secretion of products that can have effector, immunoregulatory or autocrine effects. This response can be enhanced markedly in cells that have been exposed to high levels of IgE, which results in the increased surface expression of Fc epsilon RI. Moreover, recent work indicates that monomeric IgE (in the absence of crosslinking) can render mast cells resistant to apoptosis induced by growth-factor deprivation in vitro and, under certain circumstances, can induce the release of cytokines. So, the binding of IgE to Fc epsilon RI might influence mast-cell and basophil survival directly or indirectly, and can also regulate cellular function.     

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.     

Animal models for IgE-meditated cancer immunotherapy

Although most monoclonal antibodies developed for cancer therapy are of the IgG class, antibodies of the IgE class have certain properties that make them attractive as cancer therapeutics. These properties include the superior affinity for the Fc epsilon receptors (FcεRs), the low serum level of IgE that minimizes competition of endogenous IgE for FcεR occupancy, and the ability to induce a broad and vigorous immune response through the interaction with multiple cells including mast cells, basophils, monocytes, macrophages, dendritic cells, and eosinophils. Tumor-targeted IgE antibodies are expected to harness the allergic response against tumors and activate a secondary, T-cell-mediated immune response. Importantly, the IgE antibody can be used for passive immunotherapy and as an adjuvant of cancer vaccines. However, there are important limitations in the use of animal models including the fact that human IgE does not interact with rodent FcεRs and that there is a different cellular distribution of FcεRs in humans and rodents. Despite these limitations, different murine models have been used with success to evaluate the in vivo anti-cancer activity of several IgE antibodies. These models include wild-type immunocompetent animals bearing syngeneic tumors, xenograft models using immunocompromised mice bearing human tumors and reconstituted with human effector cells, and human FcεRIα transgenic mice bearing syngeneic tumors. In addition, non-human primates such as cynomolgus monkeys can be potentially used for toxicological and pharmacokinetic studies. This article describes the advantages and disadvantages of these models and their use in evaluating the in vivo properties of IgE antibodies for cancer therapy.     

IgE enhances Fc epsilon receptor I expression and IgE-dependent release of histamine and lipid mediators from human umbilical cord blood-derived mast cells: synergistic effect of IL-4 and IgE on human mast cell Fc epsilon receptor I expression and mediator release

We investigated the effects of IgE versus IL-4 on Fc epsilon RI surface expression in differentiated human mast cells derived in vitro from umbilical cord blood mononuclear cells. We found that IgE (at 5 micrograms/ml) much more strikingly enhanced surface expression of Fc epsilon RI than did IL-4 (at 0.1-100 ng/ml); similar results were also obtained with differentiated mouse mast cells. However, IL-4 acted synergistically with IgE to enhance Fc epsilon RI expression in these umbilical cord blood-derived human mast cells, as well as in mouse peritoneal mast cells derived from IL-4-/- or IL-4+/+ mice. We also found that: 1) IgE-dependent enhancement of Fc epsilon RI expression was associated with a significantly enhanced ability of these human mast cells to secrete histamine, PGD2, and leukotriene C4 upon subsequent passive sensitization with IgE and challenge with anti-IgE; 2) preincubation with IL-4 enhanced IgE-dependent mediator secretion in these cells even in the absence of significant effects on Fc epsilon RI surface expression; 3) when used together with IgE, IL-4 enhanced IgE-dependent mediator secretion in human mast cells to levels greater than those observed in cells that had been preincubated with IgE alone; and 4) batches of human mast cells generated in vitro from umbilical cord blood cells derived from different donors exhibited differences in the magnitude and pattern of histamine and lipid mediator release in response to anti-IgE challenge, both under baseline conditions and after preincubation with IgE and/or IL-4.     

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.     

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.     

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.     

Regulation of highly cytokinergic IgE-induced mast cell adhesion by Src, Syk, Tec, and protein kinase C family kinases

Mast cells play a critical role in IgE-dependent immediate hypersensitivity. Recent studies have shown that, contrary to the traditional view, binding of monomeric IgE to Fc epsilon RI results in a number of biological outcomes in mast cells, including survival. However, IgE molecules display heterogeneity in inducing cytokine production; highly cytokinergic (HC) IgEs cause extensive Fc epsilon RI aggregation, which leads to potent enhancement of survival and other activation events, whereas poorly cytokinergic (PC) IgEs can do so inefficiently. The present study demonstrates that HC, but not PC, IgEs can efficiently induce adhesion and spreading of mouse mast cells on fibronectin-coated plates in slow and sustained kinetics. HC IgE-induced adhesion through beta1 and beta7 integrins promotes survival, IL-6 production, and DNA synthesis. Importantly, we have identified Lyn and Syk as requisite tyrosine kinases and Hck, Btk, and protein kinase C theta as contributory kinases in HC IgE-induced adhesion and spreading, whereas protein kinase C epsilon plays a negative role. Consistent with these results, Lyn, Syk, and Btk are activated in HC IgE-stimulated cells in a slower but more sustained manner, compared with cells stimulated with IgE and Ag. Thus, binding of HC IgEs to Fc epsilon RI induces adhesion of mast cells to fibronectin by modulating cellular activation signals in a unique fashion.     

Investigation of early events in Fc epsilon RI-mediated signaling using a detailed mathematical model

Aggregation of Fc epsilon RI on mast cells and basophils leads to autophosphorylation and increased activity of the cytosolic protein tyrosine kinase Syk. We investigated the roles of the Src kinase Lyn, the immunoreceptor tyrosine-based activation motifs (ITAMs) on the beta and gamma subunits of Fc epsilon RI, and Syk itself in the activation of Syk. Our approach was to build a detailed mathematical model of reactions involving Fc epsilon RI, Lyn, Syk, and a bivalent ligand that aggregates Fc(epsilon)RI. We applied the model to experiments in which covalently cross-linked IgE dimers stimulate rat basophilic leukemia cells. The model makes it possible to test the consistency of mechanistic assumptions with data that alone provide limited mechanistic insight. For example, the model helps sort out mechanisms that jointly control dephosphorylation of receptor subunits. In addition, interpreted in the context of the model, experimentally observed differences between the beta- and gamma-chains with respect to levels of phosphorylation and rates of dephosphorylation indicate that most cellular Syk, but only a small fraction of Lyn, is available to interact with receptors. We also show that although the beta ITAM acts to amplify signaling in experimental systems where its role has been investigated, there are conditions under which the beta ITAM will act as an inhibitor.     

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 and IgG are synergistic in antigen-mediated release of thromboxane from human lung macrophages.

The mechanism of IgE-mediated release of thromboxane A2 from human lung macrophages has been studied using a monoclonal chimeric human/mouse IgE antibody and its specific antigen. The cells could be sensitized at 37 degrees C but not at 4 degrees C by incubation with IgE, and released a significant amount of thromboxane A2 (TXA2), measured as the stable hydrolysis product TXB2, in response to an anti-chimeric IgE antibody. In contrast, stimulation of IgE-sensitized macrophages with the specific antigen produced less than 10% of this response. A similar time course for the release of TXB2 and the formation of inositol monophosphate in the presence of LiCl was observed. Cleavage of the Fc domain of the anti-chimeric IgE antibody substantially eliminated its capacity to stimulate IgE-sensitized cells. However, the weak or undetectable response to chimeric IgE plus specific antigen was substantially potentiated by an antigen-specific chimeric IgG antibody. IgG-sensitized macrophages did not respond to antigen challenge by the release of TXB2. Preincubation of the cells with a monoclonal antibody against the low affinity receptor for IgE (Fc epsilon RII/CD23) did not prevent IgE sensitization. We conclude that cell-bound IgE antibody cannot induce the release of TXB2 but has fixed antigen which then must interact with specific IgG antibody and IgG receptors to induce mediator release.     

Conservation of signal transduction mechanisms via the human Fc epsilon RI alpha after transfection into a rat mast cell line, RBL 2H3.

The high affinity receptor for IgE (Fc epsilon RI) is present on mast cells and basophils, and the aggregation of IgE-occupied receptors by Ag is responsible for the release of allergic mediators. The Fc epsilon RI is composed of at least three different subunits, alpha, beta, and gamma, with the alpha subunit binding IgE. The series of biochemical events linking receptor aggregation to the release of mediators has not been fully delineated. As a step towards understanding these processes, and for the development of functional cell lines, we have transfected the human Fc epsilon RI alpha subunit into the rat mast cell line RBL 2H3. These human Fc epsilon RI alpha-transfected cell lines have been characterized with respect to the association of the human alpha subunit with endogenous rat beta and gamma subunits and the ability of aggregated Fc epsilon RI alpha subunits to mediate a variety of biochemical events. The signal transduction events monitored include phosphoinositide hydrolysis, Ca2+ mobilization, tyrosine phosphorylation, histamine release, and arachidonic acid metabolism. In all cases, the events mediated by aggregating human Fc epsilon RI alpha subunits were indistinguishable from those produced via the rat Fc epsilon RI alpha. These results demonstrate that the human Fc epsilon RI alpha subunit can functionally substitute for the rat Fc epsilon RI alpha subunit during signal transduction. The availability of this cell line will provide a means of evaluating potential Fc epsilon RI antagonists.     

The human IgA-Fc alpha receptor interaction and its blockade by streptococcal IgA-binding proteins

IgA plays a key role in immune defence of the mucosal surfaces. IgA can trigger elimination mechanisms against pathogens through the interaction of its Fc region with Fc alpha Rs (receptors specific for the Fc region of IgA) present on neutrophils, macrophages, monocytes and eosinophils. The human Fc alpha R (CD89) shares homology with receptors specific for the Fc region of IgG (Fc gamma Rs) and IgE (Fc epsilon RIs), but is a more distantly related member of the receptor family. CD89 interacts with residues lying at the interface of the two domains of IgA Fc, a site quite distinct from the homologous regions at the top of IgG and IgE Fc recognized by Fc gamma R and Fc epsilon RI respectively. Certain pathogenic bacteria express surface proteins that bind to human IgA Fc. Experiments with domain-swap antibodies and mutant IgAs indicate that binding of three such proteins (Sir22 and Arp4 of Streptococcus pyogenes and beta protein of group B streptococci) depend on sites in the Fc interdomain region of IgA, the binding region also used by CD89. Further, we have found that the streptococcal proteins can inhibit interaction of IgA with CD89, and have thereby identified a mechanism by which a bacterial IgA-binding protein may modulate IgA effector function.     

Inhibition of IgE binding to RBL-2H3 cells by a monoclonal antibody (BD6) to a surface protein other than the high affinity IgE receptor.

A mAb was isolated (mAb BD6) that recognized a surface glycoprotein on rat basophilic leukemia cells (RBL-2H3). The antibody bound to 2 x 10(6) molecules/cell and specifically blocked IgE binding (50% inhibition with 3.48 +/- 0.51 micrograms/ml; mean +/- SEM), although neither IgE nor anti-high affinity IgE receptor (anti-Fc epsilon RI) mAb blocked mAb BD6 binding to the cells. mAb BD6 did not affect the rate of dissociation of cell-bound IgE, nor did it induce or inhibit the internalization of IgE. mAb BD6 did not release histamine. However, it did cause rapid spreading of the cells. By 1 h the cells had retracted to a spherical shape with their surface covered with membranous spikes, and they could easily be detached from the tissue culture plate. These changes differed from those observed after Fc epsilon RI activation. mAb BD6 immunoprecipitated a complex of two proteins, 38 to 50 kDa and 135 kDa from 125I-surface labeled rat basophilic leukemia cells that are not subunits of Fc epsilon RI. Chemical cross-linking studies showed that these molecules are associated on the cell surface. By immunoblotting, mAb BD6 reacted with a 40-kDa protein. Therefore, mAb BD6 binds to a surface protein that is close to the Fc epsilon RI and sterically inhibits 125I-IgE binding.     

Analysis of Fc(epsilon)RI-mediated mast cell stimulation by surface-carried antigens

Clustering of the type I receptor for IgE (Fc[epsilon]RI) on mast cells initiates a cascade of biochemical processes that result in secretion of inflammatory mediators. To determine the Fc(epsilon)RI proximity, cluster size, and mobility requirements for initiating the Fc(epsilon)RI cascade, a novel experimental protocol has been developed in which mast cells are reacted with glass surfaces carrying different densities of both antigen and bound IgE, and the cell's secretory response to these stimuli is measured. The results have been analyzed in terms of a model based on the following assumptions: 1) the glass surface antigen distribution and consequently that of the bound IgE are random; 2) Fc(epsilon)RI binding to these surface-bound IgEs immobilizes the former and saturates the latter; 3) the cell surface is formally divided into small elements, which function as a secretory stimulus unit when occupied by two or more immobilized IgE-Fc(epsilon)RI complexes; 4) alternatively, similar stimulatory units can be formed by binding of surface-carried IgE dimers to two Fc(epsilon)RI. This model yielded a satisfactory and self-consistent fitting of all of the different experimental data sets. Hence the present results establish the essential role of Fc(epsilon)RI immobilization for initiating its signaling cascade. Moreover, it provides independent support for the notion that as few as two Fc(epsilon)RIs immobilized at van der Waals contact constitute an "elementary stimulatory unit" leading to mast cell (RBL-2H3 line) secretory response.