Anti-anti-IgE idiotypic antibodies mimic IgE in their binding to the Fc epsilon receptor

The binding site of some anti-idiotypic antibodies (anti-Id) can appear as a structural image of the antigen and as such may mimic its biologic activity. We raised anti-anti-IgE antibodies in an attempt to obtain anti-Id capable of interacting with the Fc epsilon receptor (Fc epsilon R). Guinea pigs were immunized with purified murine monoclonal antibodies (mAb) that had been found to react with epitopes closely related to the site on the IgE molecule which is recognized by the Fc epsilon R. After only two injections, we could detect in the immune sera anti-Id that inhibited the binding of IgE to the anti-IgE mAb used as immunogens. However, only after 10 immunizations over a period of about 6 months could we detect antibodies that competed efficiently with the binding of IgE to rat basophilic leukemia (RBL) cells. The "IgE-like" anti-Id could be affinity purified from immunosorbents made of the anti-IgE mAb. F(ab')2 and Fab' fragments were as effective inhibitors of IgE binding as the intact anti-anti-Id antibodies. Some of the anti-Id caused RBL degranulation and all of them, like IgE, inhibited the binding of specific anti-Fc epsilon R mAb to RBL cells. In summary, by hyperimmunization with anti-IgE mAb we could obtain anti-Id whose antigen-binding site is recognized by the mast cell receptor specific to the Fc portion of IgE.     

Anti-IgE monoclonal antibodies directed at the Fc epsilon receptor binding site

In a search for the region in the IgE molecule, which is recognized by the Fc epsilon receptor (Fc epsilon R) on mast cells and basophils, we have generated and characterized anti-IgE monoclonal antibodies (MAbs). The novel rat anti-mouse IgE MAb described herein (denoted 84-1c) interacts with an antigenic determinant which is associated with the Fc epsilon R recognition site on the IgE molecule. The MAb can bind to the Fc epsilon of IgE and block its binding to rat basophil leukemia (RBL) cells. The epitope recognized by 84-1c MAb was completely masked by the Fc epsilon R either in its cellular or soluble form. This epitope was dependent on the native conformation of the IgE molecule and differed from the ones that were recognized by the anti-IgE MAbs we described before.     

Monoclonal antibodies defining epitopes on human IgE.

Twelve monoclonal antibodies (mAb) were isolated that bound to six clusters of epitopes on the constant region of the epsilon chain of human IgE. Four of the mAb bound to the C epsilon 1 or early C epsilon 2 regions; three of these bound to the IgE myeloma protein PS and to serum IgE but not to the IgE myeloma protein ND. These mAb probably recognize an allotypic marker. Another mAb reacted with heat-denatured, but not native IgE. Four of the mAb failed to release histamine; the epitopes recognized by these mAb are in the C epsilon 1, C epsilon 2 and C epsilon 3-4 regions of IgE. Three of these non-histamine releasing mAb did not bind to IgE on the basophil surface. These mAb recognize epitopes in C epsilon 2 and C epsilon 3-4 that are not accessible when IgE is bound to its receptor. Four mAb inhibited IgE binding to basophils; two of these did not release histamine, and two others that bind to epitopes in the C epsilon 2-4 domain, released histamine and therefore blocked IgE binding by steric hindrance. Inhibition of IgE binding by different mAb suggest that the Fc epsilon RI and Fc epsilon RII bind to partly overlapping regions of the IgE molecule although the sites do not appear to be identical. A number of sites on C epsilon 1 and C epsilon 3-4 were accessible when IgE is bound to its basophil receptor. The data support the concept that only part of the Fc portion of IgE is hidden in the receptor and that portions of C epsilon 1-4 are accessible on the cell surface. These mAb should be useful in determining the domains of IgE that are critical for its biological activity.     

Characterization of new rat anti-mouse IgE monoclonals and their use along with chimeric IgE to further define the site that interacts with Fc epsilon RII and Fc epsilon RI.

Three rat monoclonal antibodies specific for mouse IgE (C12B9, 23G3, and B1E3) were established by using monoclonal anti-DNP mouse IgE (mIgE) as immunogen. These antibodies, as well as a fourth, (R1E4) were characterized. It was found that one antibody (C12B9) recognizes an allotypic determinant (Igh-7a) found on the C epsilon chain of mIgE. Antibody cross-blocking studies and epitope mapping studies using recombinant mIgE indicated that 3 antibodies (C12B9, R1E4 and 23G3) were directed against the C epsilon 3 domain while one (B1E3) was directed against the C epsilon 4 domain. A highly specific sandwich RIA for mIgE was developed using these antibodies. Use of these monoclonal anti-mIgE antibodies in conjunction with recombinant chimeric mIgE-human IgG1 molecules, demonstrated that the C epsilon 3 domain is important in the binding of mIgE to the murine B cell Fc epsilon RII as well as to the murine mast cell F epsilon RI. The presence of the C epsilon 4 domain influenced the binding of the recombinant IgE to the Fc epsilon RII; in contrast to the C epsilon 4 domain had no effect on binding to the Fc epsilon RI.     

Binding properties and histamine release in variants of rat basophilic leukemia cells with changes in the IgE receptor

Variants of the rat basophilic leukemia cell line were selected that had a decreased number of high affinity IgE receptors (Fc epsilon R). Cloned lines with fewer than 10,000 Fc epsilon R on their surface could release histamine following IgE-mediated stimulation. These variant lines were further characterized by binding studies with 125I-labeled IgE or a series of monoclonal antibodies (mAb) that inhibit IgE binding. Three of these mAb (mAb BC4, mAb CA5 and mAb CD3) bind competitively with IgE. A fourth mAb (AA4) inhibits IgE binding but its binding is not inhibited by IgE. In all the variant cloned lines, binding by the 3 mAb was highly correlated to IgE binding. Therefore, these epitopes are closely related. In contrast, there was poor correlation between mAb AA4 and IgE binding. However, even in these lines, mAb AA4 inhibited labeled IgE binding. Therefore, there is independent variation of the mAb AA4 epitope compared to the sites to which the other mAb and IgE bind.     

Effect of anti-IgE antibodies on IgE binding to CD23

Human sera contain anti-IgE autoantibodies. Using a human B lymphoblastoid cell line (Wil-2WT cells) and monoclonal murine anti-IgE antibodies (BSW17 and Le27) we investigated a possible role of such anti-IgE antibodies. A 100-fold excess of monoclonal anti-IgE antibodies inhibited binding of 125I labeled IgE to Fc epsilon RII on Wil-2WT cells. Further, both monoclonal anti-IgE antibodies dissociated surface bound IgE from Fc epsilon RII on Wil-2WT cells. However, BSW17 which does not trigger histamine release from human leucocytes, was much more effective in dissociating Fc epsilon RII bound IgE than Le27 which triggers histamine release. These results may suggest that naturally occurring IgG anti-IgE antibodies are able to inhibit binding of IgE to its receptor.     

Inhibition of IgE binding to mast cells and basophils by monoclonal antibodies to murine IgE

In an attempt to identify the site on IgE which binds with high affinity to the Fc? receptor (Fc?R) on mast cells, we established monoclonal anti-IgE antibodies (mAb) by fusion of myeloma cells with rat splenocytes immunized with purified murine IgE mAb. Six individual mAb were found to react with various IgE mAb of different specificities and not with immunoglobulins of other classes. Three different clusters of epitopes on the Fc? portion could be detected by antibody competition studies. These antigenic determinants were expressed on the Fc? portion and required the two heavy chains in their native conformation. Two groups of mAb and their Fab′ fragments completely inhibited the binding of ¹²⁵I-labeled IgE to rat basophilic leukemia cells (RBL), and one mAb inhibited the specific IgE binding only partially (55–65%). Likewise, the Fab′ fragments of the purified mAb inhibited the antigen-mediated, IgE-dependent, serotonin release of RBL cells. These in vitro findings were confirmed by in vivo experiments, which demonstrated that the anti-IgE mAb could specifically block passive cutaneous anaphylaxis reaction when injected i.d., before challenging with the antigen. The differences in blocking reactivity of the various anti-IgE mAb are discussed in view of heterogeneity in the IgE-Fc?R interaction.     

Identification of peptide motifs recognized by a human IgG autoanti-IgE antibody using a phage display library

BACKGROUND: The potential of murine monoclonal anti-IgE antibodies as long-term therapy for atopic diseases will have to rely, for the time being, on passive antibody administration. There is therefore considerable interest in developing a peptide-based vaccine for active immunization to elicit long-term protective anti-IgE antibodies in the patient. It has been shown that some human IgG autoanti-IgE antibodies have the ability to partially block the binding of IgE to Fc receptors such as Fc epsilonRI. Therefore, the epitopes recognized by such antibodies could have vaccine potential. OBJECTIVE: To determine the epitope specificity of one such human IgG anti-IgE antibody. METHODS: A 15-mer phage-peptide library was used to establish the epitope specificity of an IgG anti-IgE antibody isolated from the serum of an asthma patient. RESULTS: The SRPSP sequence, or part of it (i.e. RPS, RPSP, SPS or PSP), was present in all 18 phage-peptides that have been sequenced. This common motif was found to be within the human epsilon chain sequence Ser341-Thr355 near the N-terminus of the C epsilon3 domain. According to the human Fc epsilon model, the most accessible residues in this sequence are Arg342, Ile350, Arg351, Lys352 and Ser353. CONCLUSIONS: The present data should provide the molecular basis for the rational design of a suitable peptide immunogen (vaccine) for boosting the production of protective autoanti-IgE antibodies.     

Generation of canine-human Fc IgE chimeric antibodies for the determination of the canine IgE domain of interaction with Fc epsilon RI alpha

Identification of the domain(s) of canine IgE that interact with Fc epsilon RI alpha may lead to novel therapeutic intervention strategies that inhibit the ability of canine IgE to engage Fc epsilon RI alpha. A panel of canine-human Fc IgE chimeric antibodies was constructed to investigate this interaction by replacing canine IgE-Fc domains with the corresponding human IgE-Fc domains since human IgE-Fc does not recognize canine Fc epsilon RI alpha. beta-Hexosaminidase release assays were performed to assess the ability of the chimeric antibodies to bind to and sensitize a novel RBL cell line transfected with canine Fc epsilon RI alpha for antigen induced mediator release. Replacing canine C epsilon2 with human C epsilon2 resulted in similar levels of release as those elicited by canine Fc IgE from RBL-2H3 cells transfected with either canine Fc epsilon RI alpha or human Fc epsilon RI alpha. Substitution of canine C epsilon4 with human C epsilon4 resulted in approximately 10% lower levels of release compared to cells sensitized with canine Fc IgE. Receptor binding by flow cytometry and cell activation could not be detected when transfected RBL cells were incubated with chimeric constructs where canine C epsilon2 and C epsilon4 were substituted with human C epsilon2 and C epsilon4. However, when this construct was incubated with cognate antigen prior to cell challenge mediator release was observed, albeit at a 20% lower level, indicating that while canine C epsilon3 is the only domain essential for binding to canine or human Fc epsilon RI alpha, species specific residues in canine Cepsilon2 and C epsilon4 inhibit dissociation of the ligand from the receptor.     

Potential role of anti-IgE antibodies in vivo

Murine monoclonal antibodies which recognize similar epitopes as the naturally occurring human IgG anti-IgE antibodies were used to study their role in interfering with the effector functions of IgE. Two types of antibodies were found which were either anaphylactogenic or did not release histamine from human basophils. However, both types of antibodies were capable of inhibiting binding of IgE to Fc epsilon RII. Furthermore, the nonanaphylactic antibody was capable of removing IgE from Fc epsilon RII+ cells, but no antibodies were found which removed IgE from Fc epsilon RI+ cells. Thus, anti-IgE antibodies may interfere with the pathophysiological role of IgE.     

Inhibition of human cord blood-derived mast cell responses by anti-Fc epsilon RI mAb 15/1 versus anti-IgE Omalizumab

Aggregation of the alpha-chain of the high affinity IgE receptor (Fc epsilon RI alpha) on mast cells or basophils after cross-linking of receptor-bound IgE by its antigen or an anti-IgE antibody results in cell activation and release of inflammatory mediators. Omalizumab (Xolair), Novartis Pharmaceuticals; Genentech Inc.) is a recombinant humanized anti-IgE mAb developed for the treatment of severe allergic asthma. It complexes with free serum IgE, which prevents its binding to Fc epsilon RI and thereby interrupts the allergic cascade. Administration of an inhibitory anti-Fc epsilon RI alpha mAb may represent an alternative strategy to neutralize IgE-mediated receptor activation. In the present report, for the first time, we have performed direct side of side comparison between the inhibitory anti-Fc epsilon RI alpha mAb designated 15/1 and Omalizumab for their effects on human cord blood-derived mast cells. We provide the first evidence that both 15/1 mAb and Omalizumab efficiently inhibit Fc epsilon RI-mediated human mast cell responses in vitro (degranulation, activation, release of IL-8 and IL-13, phosphorylation of Akt) and that mAb 15/1 is a non-anaphylactogenic antibody, which compared to Omalizumab, displays markedly higher inhibitory potency in the presence of high IgE levels.     

Inhibition of IgE-dependent histamine release from human peripheral blood basophils by humanized Fab fragments that recognize the membrane proximal domain of the human Fc epsilon RI alpha-chain

BACKGROUND: Inhibition of the interaction between IgE and the alpha-chain of Fc epsilon RI (Fc epsilon RI alpha) is a straightforward strategy to develop therapeutic reagents for IgE-mediated allergic diseases. OBJECTIVE: The purpose of this study is the humanization of CRA2 and/or CRA4, mouse anti-human Fc epsilon RI alpha monoclonal antibodies (mAbs) which recognize the IgE-binding membrane proximal immunoglobulin-like domain of Fc epsilon RI alpha. METHODS: The two mAbs were humanized by CDR grafting onto human V region frameworks encoded by human germline V and J genes. The activities of the recombinant antibodies to bind Fc epsilon RI alpha and inhibit IgE binding to Fc epsilon RI alpha were analyzed by flow cytometry and ELISA. Human peripheral blood basophils were pretreated with the Fab fragments of the humanized CRA2 and stimulated with IgE and an anti-IgE polyclonal antibody. The released histamine was measured. RESULTS: The humanized CRA2 had almost the same activities of binding and inhibition of IgE binding to Fc epsilon RI alpha as the original mouse CRA2. Although the Fc epsilon RI-binding activity was maintained following humanization of the CRA4 light chain V region, it was lost by the humanization of the CRA4 heavy chain V region. Pretreatment of human peripheral blood basophils with the Fab fragments of the humanized CRA2 inhibited their subsequent degranulation activated by cross-linking of the Fc epsilon RI. CONCLUSION: In the humanized CRA2, all amino acid residues except CDR are replaced with the residues encoded by human germline genes. The humanization of CRA2 might be an important step in the development of immunotherapy to manipulate the IgE network in which mast cells, basophils, and various types of Fc epsilon RI alpha expressing cells are involved.     

Production and characterization of rabbit anti-idiotypic antibodies specific to mouse monoclonal anti-human IgE and reacting with IgE-binding factors and lymphocyte receptors for IgE

A mouse monoclonal antibody specific to human IgE (mAb 75) was employed to immunize a rabbit to obtain anti-idiotypes (aId) bearing the internal image of human IgE determinants and reacting with IgE-binding factors (IgE-BF) and/or lymphocyte receptors for IgE (Fc epsilon R). mAb 75 was selected on the basis of inhibition assays where the binding of mAb 75 to radiolabeled IgE was blocked by IgE-BF. The latter were produced by a lymphoblastoid cell line (RPMI 8866) expressing Fc epsilon R. Sequential samples of rabbit serum, collected during the immunization period, were extensively absorbed on mouse and human Ig-Sepharose 4B. The IgG fractions of the rabbit serum displayed the following activities: (a) they reacted with 125I-labeled mAb 75 but not with other labeled mouse Ig including mAb-aIgE, (b) this binding was inhibited in a dose-dependent fashion by human IgE but not by other human Ig classes nor by heat-inactivated IgE, (c) they reacted with a polyclonal rabbit anti-human IgE and (d) they blocked the binding of 125I-labeled IgE to mAb 75. It was concluded that the rabbit IgG contained aId (RaId) bearing the internal image of heat labile determinants of human IgE. The rosetting of IgE-coated bovine erythrocytes with Fc epsilon R-bearing cells was inhibited by preincubating the receptor-bearing cells with IgG RaId or its F(ab')2 but not with normal rabbit IgG. The ability of RaId to react with IgE-BF as well as with Fc epsilon R was also shown in inhibition experiments where IgE-BF and solubilized Fc epsilon R blocked the binding of mAb 75 to RaId. Finally, Western blot analysis of human colostrum, known to contain IgE-BF, indicated that radioiodinated RaId and IgE identified the same 12-16-kDa molecules corresponding to IgE-BF. It is concluded that RaId expresses the internal image of a heat-labile determinant of IgE which is involved in the binding of IgE to IgE-BF and Fc epsilon R. An alternative interpretation is that RaId reacts with an idiotypic determinant of mAb 75 which is shared by IgE-BF and Fc epsilon R.     

Quantitative and qualitative analysis of the Fc receptor for IgE (Fc epsilon RII) on human eosinophils

In order to characterize the Fc receptor for IgE (Fc epsilon RII) on human eosinophils, we have compared the binding of human IgE myeloma protein to that of a monoclonal antibody (mAb BB10) directed against a common antigenic determinant of the Fc epsilon RII present on eosinophils, platelets and macrophages. Scatchard analysis of the binding to human eosinophils of the BB10 mAb revealed a linear monophasic binding curve, with a binding affinity of 1.17 x 10(7) M-1 and a number of 10(5) binding sites per cell. Biochemical analysis of the human eosinophil Fc epsilon R, performed by immunosorbent chromatography with either BB10 mAb or IgE, showed under nonreducing conditions a major component of 200 kDa. Under reducing conditions, 3 peptide fragments were obtained, with molecular masses of 45-50, 23 and 15 kDa. Finally, comparative analysis suggested that the Fc epsilon RII of human eosinophils and of a human macrophage cell line (U937) are structurally related and differ from the high-affinity Fc epsilon RI present on basophilic granulocytes.     

Thermoinactivation of human IgE: antigenic and functional modifications.

The thermoinactivation kinetics of IgE were studied in experimental models revealing the antigenic properties and the basophil-sensitizing capacity of these immunoglobulins. A pool of human sera containing anti-Dactylis glomerata (Dg) IgE was heated from 5 min up to 4 hr at 56 degrees. The IgE antigenicity was tested by two polyclonal 125I-labelled anti-IgE antibodies; one anti-IgE was specific of the whole Fc epsilon region, while the other had a specificity restricted to the D epsilon 2 domain. Radioimmunoassays showed that the D epsilon 2 epitopes were more rapidly altered than the D epsilon 1 epitopes. The capacity of IgE to bind to basophil Fc epsilon receptors was assayed by passive sensitization experiments. Basophil sensitivity towards the Dg pollen extract was tested by histamine release experiments in the presence of this allergen. A progressive decrease in cell sensitivity was observed when IgE samples used for cell sensitization were heated for longer than 5 min. Thermoinactivation kinetics of IgE revealed an unexpected increase in the apparent quantity and biological activity of IgE heated for 5 min at 56 degrees. This fact could be due to auto-anti-IgE antibodies linked to the unheated IgE and which interfere with the biological activities of IgE and their quantification.     

Elucidation of the epitope locations of human autoanti-IgE: recognition of two epitopes located within the C epsilon 2 and the C epsilon 4 domains.

IgG autoanti-IgE is detectable in a large proportion of individuals with allergic asthma, where it is suggested to be potentially involved in modulating IgE-mediated hypersensitivity. Using a series of overlapping recombinant human epsilon-chain peptides, we have shown that circulating IgG anti-IgE antibodies recognise at least 2 epitopes located within the C epsilon 2 and the C epsilon 4 domains, respectively. The C epsilon 2 recognition site is located within the C-terminal portion of the C epsilon 2 domain (i.e. aa301-339) which is thought to contribute residues to the Fc epsilon RI-binding site on IgE. The recognition by autoanti-IgE of an effector function site of such pivotal importance in IgE-mediated hypersensitivity suggests that it plays a possible modulatory role during mast cell and basophil activation.     

Fc epsilon receptors on rat B-lymphocytes: specificity and binding kinetics

The binding kinetics of radiolabelled rat IgE to Fc epsilon receptors (R) of rat B-cells have already been studied in IgE-stimulated animals. The receptors expressed after Nippostrongylus brasiliensis infection or 2 injections of 5 mg IgE/100 g body wt were class-specific: IgE binding was not hindered by rat IgM, IgD, IgA, IgG1, IgG2a, IgG2b and IgG2c in immunocompetitive-binding assays. The rat B-cell Fc epsilon R were not species-specific, since mouse IgE competes with rat IgE for binding to these receptors. The apparent number of Fc epsilon R on rat mesenteric lymph node B-cells varied with temp and was 1.1-2.4 X 10(5) at 4 degrees C and 5.9-7.7 X 10(5) at 37 degrees C. The experimental Ka was not influenced by temp and had an average value of 1.38 X 10(8) M-1. At 4 degrees C the IgE binding to B-cell Fc epsilon R had an association rate of 4.9 X 10(3) M-1 sec-1 and a dissociation rate of 4.65 X 10(-5) sec-1. After a very strong stimulation produced by injecting 5 mg IgE/100 g body wt every 24 hr for 5 days, the equilibrium binding curve became diphasic, indicating a probable heterogeneity of the B-cell Fc epsilon R.     

IgE receptors on human lymphocytes. II. Detection of cells bearing IgE receptors in unstimulated mononuclear cells by means of a monoclonal antibody

A monoclonal antibody (mAb) specific for lymphocyte IgE receptors (ER) was employed in a rosette assay for the detection of cells bearing IgE receptors (Fc epsilon R). The specificity of the assay was documented by inhibition studies with soluble immunoglobulins (Ig) and anti-Ig antibodies. Moreover, similar results were obtained by employing the F(ab')2 fragment of mAbER instead of intact molecule. Circulating mononuclear cells isolated from normal or allergic adults and from umbilical cord blood contained approximately 8% of Fc epsilon R-bearing cells with values ranging from 0.3 to 17%. Tonsillar lymphocytes contained about 30% of Fc epsilon R+ cells. After the removal of adherent cells, there was a small but significant reduction of the proportion of Fc epsilon R+ cells. When mononuclear cells were separated into T and B cell fractions by two-cycle rosetting with 2-aminoethylisothiouronium bromide hydrobromide-treated sheep red blood cells, most of the Fc epsilon R+ cells were in the B cell fraction; however, a small proportion of Fc epsilon R+ was also found in the enriched T cells and double-labeling experiments confirmed that these cells were indeed T lymphocytes. Fc epsilon R+ cells were purified by rosetting with mAbER-coated erythrocytes and their phenotype was compared to that of Fc epsilon R- cells; Fc epsilon R+ cells contained about 90% of B cells (B1+) together with a small proportion of OKT3+, Leu 7+ and Mo2+ cells. The bulk of T cells, macrophages and natural killer (NK) cells was found in the Fc epsilon R- cells which contained fewer B cells than the fraction of Fc epsilon R+ cells. These data thus indicated that the great majority of Fc epsilon R-bearing cells are B cells but that a small proportion of NK cells, macrophages and T lymphocytes also express Fc epsilon R. Upon incubation at 37 degrees C, B cells lost their Fc epsilon R and this phenomenon was selectively inhibited by IgE; however, purified T cells seemed to express more Fc epsilon R after overnight incubation at 37 degrees C and this was not influenced by IgE. It is finally shown that the expression of Fc epsilon R is cyclic and that Fc epsilon R-bearing B cells do not represent a functionally distinct subpopulation of B lymphocytes.     

The human mast cell receptor binding site maps to the third constant domain of immunoglobulin E.

The characterization of the site on the IgE molecule which accommodates the high affinity receptor for IgE (Fc epsilon RI) should allow the design of IgE analogues which can be utilized to block allergic responses. Using chimeric human IgE molecules in which different constant region domains were exchanged with their murine homologues, we demonstrate here that the C epsilon 3 in its native configuration is essential for the binding to the alpha subunit of the human Fc epsilon RI. Deletion of the human C epsilon 2 from such chimeric molecules did not impair their ability to interact with the Fc epsilon RI, indicating that C epsilon 2 is not directly involved in the human Fc epsilon RI binding site and that C epsilon 3 alone is necessary and sufficient to account for most of the human Fc epsilon RI-binding capacity.     

Mast cell stimulation by monoclonal antibodies specific for the Fc epsilon receptor yields distinct responses of arachidonic acid and leukotriene C4 secretion

The release of arachidonic acid ([3H]AA) and leukotriene C4 (LTC4) from the rat mucosal mast cells of the RBL-2H3 line stimulated by Fc epsilon receptor-specific monoclonal antibodies (mAb), by IgE and multivalent antigen, or by Ca2+ ionophores, was investigated. In parallel, secretion of the granular enzyme beta-hexosaminidase was also assayed. The release of [3H]AA and LTC4 in response to stimulation by three Fc epsilon RI-specific mAb shows similar quantitative differences to those observed for the secretion of granule-stored mediators. The mAb F4 induced a substantial release of both [3H]AA and LTC4, which is as high as that induced by IgE and multivalent antigen. mAb J17 and H10 were found to induce an insignificant release of [3H]AA, but while J17 did induce release of LTC4, H10 failed to induce it, even though both J17 and H10 caused substantial release of beta-hexosaminidase. Ca2+ ionophores were found to be relatively more effective in inducing release of [3H]AA and LTC4 than in causing the secretion of granular mediators, as compared to cell stimulation by Fc epsilon RI aggregation. These results illustrate that the cell responses of degranulation and de novo synthesis and release of mediators have different sensitivities to stimulation by (a) configurationally distinct Fc epsilon RI dimers, (b) Fc epsilon RI aggregates induced by IgE and multivalent antigen, and (c) Ca2+ ionophores.