Test of a theory relating to the cross-linking of IgE antibody on the surface of human basophils

Recent mathematical models of bivalent hapten-induced histamine release from basophils predict that under appropriate conditions histamine release is maximum when cross-link formation is maximum, at a hapten concentration equal to 1/(2Ka), where Ka is the average affinity constant of the hapten for a single IgE binding site. To test this prediction we sensitized human basophils with a monoclonal anti-dinitrophenol IgE and generated histamine release dose-response curves with a bivalent hapten, alpha, epsilon-DNP-lysine. The monoclonal IgE has a published affinity constant of 7.1 X 10(7) M-1 for epsilon-DNP-lysine as determined by equilibrium dialysis. From the position of the maximum of the histamine dose-response curves, both in the presence and in the absence of monovalent DNP hapten, we determine that the sensitizing IgE has an intrinsic affinity constant of 6.9 +/- 0.5 X 10(7) M-1 for epsilon-DNP-lysine and 1.2 +/- 0.6 X 10(6) M-1 for alpha-DNP-lysine. The agreement between the two estimates of the epsilon-DNP-lysine affinity constant, one from histamine release experiments involving surface bound IgE and one from binding experiments involving IgE free in solution, 1) is consistent with a central prediction of the theory of cross-linking and 2) indicates that the hapten-binding properties of the IgE are unaffected by its being bound to Fc epsilon receptors on the basophil surface.     

Anti-human IgG causes basophil histamine release by acting on IgG-IgE complexes bound to IgE receptors.

We have reexamined the ability of anti-human IgG antibodies to induce histamine release from human basophils. A panel of purified murine mAbs with International Union of Immunological Societies-documented specificity for each of the four subclasses of human IgG was used. Of the 24 allergic subjects studied, the basophils of 75% (18/24) released greater than 10% histamine to one or more anti-IgG1-4 mAb, whereas none of the 13 nonatopic donor's basophils released histamine after stimulation with optimal amounts of anti-IgG mAb. The basophils of 85% (11/13) of the nonatopic donors did respond to anti-IgE challenge, as did 92% (22/24) of the atopic donor cells. Histamine release was induced most frequently by anti-IgG3, and 10/18 anti-IgG responder cells released histamine with mAb specific for two or more different subclass specificities. The rank order for induction of histamine release was anti-IgG3 greater than anti-IgG2 greater than IgG1 greater than anti-IgG4. As in our previous study using polyclonal anti-IgG, 100- to 300-micrograms/ml quantities of the anti-IgG mAb were required for maximal histamine release, about 1000-fold higher than those for comparable release with anti-human IgE. Specificity studies using both immunoassays and inhibition studies with IgE myeloma protein indicated that anti-IgG induced histamine release was not caused by cross-reactivity with IgE. Ig receptors were opened by lactic acid treatment so that the cells could be passively sensitized. Neither IgE myeloma nor IgG myeloma (up to 15 mg/ml) proteins could restore the response to anti-IgG mAb. However, sera from individuals with leukocytes that released histamine upon challenge with anti-IgG mAb could passively sensitize acid-treated leukocytes from both anti-IgG responder and nonresponder donors for an anti-IgG response. The only anti-IgG mAb that induced release from these passively sensitized cells were those to which the serum donor was responsive. Sera from non-IgG responders could not restore an anti-IgG response. These data led to the hypothesis that the IgG specific mAb were binding to IgG-IgE complexes that were attached to the basophil through IgE bound to the IgE receptor. This was shown to be correct because passive sensitization to anti-IgG could be blocked by previous exposure of the basophils to IgE. We conclude that anti-IgG-induced release occurs as a result of binding to IgG anti-IgE antibodies and cross-linking of the IgE receptors on basophils.     

Demonstration of Fcgamma receptors on human basophil granulocytes.

Fcgamma receptors were detected on human basophil granulocytes. The mononuclear cell fraction of human peripheral blood was incubated with heat-aggregated human IgG (HGG) followed by 125I-anti-HGG. Autoradiography of the cells showed that the majority of basophil granulocytes gave a significant number of grains. Basophils were not labeled by preincubation of the same cells with monomeric HGG followed by 125I-anti-HGG. However, the binding of aggregated HGG to basophils was inhibited by the presence of a high concentration of monomeric HGG or its Fc fragment but not by the Fab fragment. Evidence was obtained that Fcgamma receptors are distinct from IgE receptors on the same cells: i) Saturation of basophils with IgE did not affect the binding of aggregated HGG to the cells. ii) Preincubation with and the presence of aggregated HGG failed to affect the binding of 125I-IgE to basophils, or to block passive sensitization of the cells with IgE antibodies. iii) The Fcgamma receptors did not co-cap with IgE receptors. Aggregated HGG failed to induce histamine release from basophils even in the presence of D2O. It was also found that the presence of aggregated HGG on basophils did not modulate IgE-mediated histamine release from the cells.     

Effects of ADP-ribosylation of GTP-binding protein by pertussis toxin on immunoglobulin E-dependent and -independent histamine release from mast cells and basophils

Pretreatment of rat peritoneal mast cells, human basophils, bone marrow-derived mouse mast cells (BMMC) and mouse mast cell line PT-18 cells with 1 microgram/ml pertussis toxin (PT) failed to inhibit immunoglobulin E (IgE)-dependent histamine release from the cells. In BMMC and PT-18 cells, even 20-hr incubation of the cells with 1 microgram/ml PT, which ADP-ribosylates more than 97% of 41 kDa, alpha-subunit of Ni in the cells, failed to affect the IgE-dependent release of histamine or arachidonate. The results indicate that GTP-binding protein, Ni, is not involved in the transduction of triggering signals induced by cross-linking of IgE receptors. In contrast, pretreatment of rat mast cells with 1 ng/ml to 0.1 microgram/ml PT for 2 hr inhibited histamine release induced by compound 48/80 in a dose-dependent manner. A similar pretreatment with PT inhibited thrombin-induced histamine release from BMMC and N-formyl-L-methionyl-L-leucyl-L-phenylalanine-induced histamine release from human basophils in a similar dose-dependent fashion. However, even 20 hr of incubation of sensitized BMMC with 1 microgram/ml PT failed to inhibit either thrombin-induced or antigen-induced breakdown of phosphatidylinositides (PI), i.e., the formation of inositol triphosphate and diacylglycerol, Quin-2 signal, and the release of arachidonic acid. The results indicate that the inhibition of thrombin-induced histamine release by PT-treatment is not due to the inhibition of PI-turnover, and that Ni is not involved in thrombin-induced or antigen-induced (IgE-dependent) hydrolysis of phosphatidylinositides in mast cells.     

Comparison of the Fc receptors for IgE on human lymphocytes and monocytes

Fc receptors for IgE (Fc epsilon R) on human peripheral blood lymphocytes and monocytes and cultured lymphoblastoid and macrophage-like cell lines were compared with respect to: 1) binding affinity for radiolabeled IgE, 2) inhibition of IgE-specific rosette formation and inhibition of binding of radiolabeled IgE by an antiserum raised against Fc epsilon R isolated from a lymphoblastoid cell line, and 3) m.w. of radiolabeled cell surface proteins precipitated with the anti-Fc epsilon R serum. Scatchard analysis of 125I-IgE binding to lymphocytes, monocytes, and their corresponding cell lines showed biphasic binding curves with all cell types, from which 2 binding affinities were calculated to be KA = 6.2 +/- 1.1 and 2.0 +/- 0.5 x 10(7) M-1. The anti-Fc epsilon R serum inhibited both IgE rosette formation and binding of radiolabeled IgE by lymphocytes and monocytes but did not inhibit IgE rosettes formed by basophils. The inhibitory activity of the anti-Fc epsilon R serum could be absorbed with Fc epsilon R(+) but not with Fc epsilon R(-) cell lines. The anti-Fc epsilon R serum precipitated 2 peptides having m.w. of approximately 47,000 and 23,000 daltons from lysates of both cell surface-labeled lymphocyte and macrophage cell lines. These data indicate that Fc epsilon R on normal lymphocytes and monocytes, as well as on cultured lymphoblastoid and macrophage-like cells, are related structurally, since they share antigenic determinants, bind IgE with a similar affinity, and have similar m.w. However, they differ in all 3 parameters from Fc epsilon R on basophilic granulocytes.     

Neutrophils and mast cells. Comparison of neutrophil-derived histamine-releasing activity with other histamine-releasing factors

Human neutrophil-derived histamine-releasing activity (HRA-N) was partially purified and found to contain a heat-stable 1400 to 2300-Da fraction which caused human basophils and rat basophil leukemia cells (RBL) to degranulate. The capacity of HRA-N to activate basophils was not related to the gender or atopic status of the basophil donor, but was related to anti-IgE responsiveness. Several lines of evidence suggest that HRA-N and anti-IgE induce histamine release through distinctly different mechanisms: 1) the time course of HRA-N- and anti-IgE-induced RBL histamine release are different; 2) HRA-N causes histamine release from RBL with and without surface-bound IgE; 3) lactic acid stripping of IgE from human basophils reduces anti-IgE-induced histamine release, but has no consistent effect on HRA-N-induced histamine release; and 4) passive sensitization of lactic acid-stripped basophils with IgE restores anti-IgE-induced histamine release but not HRA-N-induced histamine release. Several histamine-releasing factors (HRF) were compared with HRA-N. Human nasal HRF (HRF-NW, crude and partially purified fractions of 15 to 30, 3.5 to 9, and less than 3.5 kDa), like HRA-N, caused equal histamine release from both native and IgE-sensitized RBL. However, only the 15- to 30-kDa fraction caused histamine release from human basophils in the doses tested. Mononuclear cell HRF (HRF-M, crude and a partially purified 25 kDa Mr fraction) and platelet HRF (HRF-P, crude preparation) failed to cause histamine release from either native or IgE-sensitized RBL but caused 30 +/- 5.5% and 20 +/- 10% net histamine release from human basophils, respectively. HRA-N and HRF-NW were both stable to boiling. These data, taken together, suggest that the capacity of HRA-N to induce RBL and human basophil histamine release and of HRF-NW to stimulate RBL histamine release is independent of IgE. The data further suggest that HRA-N and HRF-NW can be distinguished by size, and that they both differ from mononuclear cell HRF and platelet HRF. Thus, it appears that inflammatory cells generate a family of distinct HRF.     

Development of mast cells in vitro. II. Biologic function of cultured mast cells.

Mast cells were obtained by long term culture of rat thymus cells on rat embryonic fibroblast monolayers. Pure mast cell preparations obtained culture were incubated with 125I-labeled rat E myeloma protein to study receptors for IgE on their surface. When the cells were obtained after 35 to 45 days culture, the average number of receptors per mast cell was 100,000 to 400,000. An equilibrium constant of the binding reaction between their receptor and rat IgE was in the order of 108 M-1. The histamine content of the cultured mast cells was 0.2 to 5 mug/106 cells. The measurement of histamine content in mast cells recovered after different periods of culture suggested that the histamine content increased with maturation. Even after 45 to 50 days culture, the histamine content of cultured mast cells was significantly lower than that in rat peritoneal mast cells. The cultured mast cells were passively sensitized in vitro with rat IgE antibody against Nippostrongylus brasiliensis. The sensitized cells released histamine upon incubation with the antigen. It was also found that cultured mast cells released histamine upon exposure to compound 48/80. These results indicated that cultured mast cells have physiologic functions similar to those of normal rat mast cells, but they have not reached full maturation.     

Mast cells and pulmonary fibrosis. Identification of a histamine releasing factor in bronchoalveolar lavage fluid.

As elevated bronchoalveolar lavage (BAL) fluid histamine levels are noted in patients with pulmonary fibrosis (PF), we assayed BAL fluid from 16 patients with PF for the presence of a histamine releasing factor (HRF). HRF activity was assayed by measuring release of the preformed mast cell-derived mediators, histamine, or beta-hexosaminidase (beta-hex) from a purified population of IL-3 dependent mouse bone marrow derived mast cells (MBMMC) or human blood basophils. Mean BAL cell free histamine levels in the patients with PF was 1226 +/- 1349 pg/ml, whereas BAL histamine levels in a comparison group of six non-PF patients was 118 +/- 60 pg/ml. HRF was significantly elevated in BAL fluid of patients with PF (mean beta-hex release 24.5 +/- 12.9%; range 6.8 to 52.4%) compared to the non-PF group of patients (mean beta-hex release 7.9 +/- 7.7%; range 1.8 to 20.7%). The PF HRF not only degranulated MBMMC, but also induced the generation of the arachidonic acid metabolite leukotriene C4 from MBMMC (24.6 +/- 4.2 ng leukotriene C4/10(6) MBMMC). The PF HRF did not appear to be a cytokine previously identified in BAL fluid of patients with PF (i.e., platelet derived growth factor or insulin growth factor-1) or a human cytokine able to degranulate human basophils (i.e., IL-1, or granulocyte-macrophage-CSF) as these recombinant human cytokines did not induce MBMMC beta-hex release. Physicochemical characterization of the HRF revealed that it was relatively heat stable, pronase sensitive and on Sephadex G-75 and G-200 column chromatography had an apparent molecular mass of 30 to 50 kDa. The ability of PF BAL to induce beta-hex release from MBMMC was not dependent on IgE as unsensitized or lactic acid treated MBMMC release similar amounts of beta-hex compared to MBMMC sensitized with IgE. Thus, BAL fluid of patients with PF contains an HRF that induces beta-hex release from MBMMC via an IgE-independent mechanism. The presence of the HRF could explain elevated BAL histamine levels in patients with PF.     

Biochemical analysis of glucocorticoid-induced inhibition of IgE-mediated histamine release from mouse mast cells

Pretreatment of mouse mast cells with 10(-7) to 10(-6) M dexamethasone (DM) during overnight sensitization with mouse IgE antibody resulted in inhibition of antigen-induced histamine release and degranulation. The inhibition of both degranulation and histamine release increased linearly with the duration of the treatment; maximal inhibition was obtained after approximately 16 hr with DM. The addition of DM to sensitized mast cells immediately before antigen challenge did not affect the antigen-induced histamine release. DM interacted directly with mast cells by binding to DM-specific cytoplasmic receptors. The treatment of mast cells with DM did not affect the binding of IgE to mast cells or intracellular cAMP levels. Bridging of cell-bound IgE anti-DNP antibody on mouse mast cells either by multivalent DNP-HSA or by anti-IgE induced phospholipid methylation at the plasma membrane and Ca++ influx into the cells. Pretreatment of mast cells with DM inhibited the antigen-induced phospholipid methylation and Ca++ uptake but failed to affect histamine release by Ca++ ionophore A23187. The results suggest that DM treatment inhibits histamine release by the inhibition of the early stage of biochemical processes leading to opening Ca++ channels but does not affect the process distal to Ca++ influx or the binding of IgE molecules to IgE receptors.     

Human monocytes generate basophil histamine-releasing activities

Human peripheral blood monocytes generated activities during 24-h culture that were capable of triggering histamine release from 17 of 18 human basophil donors. Monocytes and their in vitro transformed macrophages continued to elaborate these basophil histamine-releasing activities for at least 3 wk in culture. In the 18 basophil donors tested, maximum histamine release induced by monocyte supernatants was 33.8 +/- 5.9% (mean +/- SEM) of total basophil histamine content; optimum anti-IgE-induced release was 38.8 +/- 6.2%. Basophil histamine release in response to monocyte activities was optimal at 37 degrees C and at calcium concentrations of 2 to 5 mM. Release was greater than 90% complete 1 min after challenge and was inhibited by anti-allergic drugs. The mechanism of release appeared to be independent of IgE binding. Gel filtration of supernatants derived from both day 1 (monocyte stage) and day 14 (macrophage stage) cultures demonstrated activity peaks with approximate m.w. of 12,000 and 30,000. In contrast to the marked responsiveness of basophils, only 2 of 10 human lung mast cell preparations responded; release in those preparations was low: 3% and 13% histamine release, respectively. Thus, monocytes produce potent histamine-releasing activities with differential actions on basophils and mast cells.     

Identification of antigen-capturing cells as basophils

Binding of intact Ag is a hallmark of Ag-specific B cells. Apart from B cells, a small number of non-B cells can bind Ag with comparable efficacy as B cells and are found in the peripheral blood, spleen, and bone marrow of mice. This population has been observed for a long time and recently named "Ag-capturing cells." Their identity remained enigmatic. In this study, we show that these cells are basophilic granulocytes. Their ability to capture Ags is dependent on surface IgE receptors and on Ag-specific plasma IgE molecules appearing after immunization. Several surface markers including surface bound IgE, IL-3R, CD45, CD16/32, and the chemokine receptor CCR2 were used to clearly identify these cells. Cross-linkage of surface Igs results in the release of large amounts of IL-4 and IL-6. The data identify basophils as Ag-capturing cells and support the concept of basophils as important regulators of humoral immune responses.     

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.     

Some quantitative aspects of the passive sensitization of human basophils.

We review the theory for the binding of IgE to Fc receptors on basophil surfaces. We then use the theory to analyze binding experiments of Malveaux et al. and T. Ishizaka on the passive sensitization of basophils from a patient with chronic myelocytic leukemia and 75% basophilia. From their data we determine that the forward rate constant for the binding of human IgE to receptors on these human basophils is (3.0 +/- 1.0) x 10(4) M-1 sec-1.     

Presence and functional role of an epitope of the second receptor for IgE (FcE RII) in mast cells and basophils in the rat]

Mast cells and basophils express the high affinity IgE receptor (FcERI) whereas the low affinity receptor for monomeric IgE (FcE RII) is present on macrophages, lymphocytes, eosinophils, platelets and Langerhans cells. Recent studies confirmed that the two receptors were totally distinct. The present work shows that a monoclonal antibody (BB10), able to bind to FcE RII on different cell populations, interacts with FcE RI expressing cells: rat peritoneal mast cells and a rat basophilic leukemia cell line (RBL 2 H 3). The structure recognized by BB10 is distinct from FcE RI and modulates the IgE-dependent histamine release. In conclusion, it appears that a common epitope with FcE RII is present on mast cells and basophils and that a functional relation might exist between this structure and FcE RI.     

Dissociation of IgE from receptors on human basophils. I. Enhanced passive sensitization for histamine release

Leukocytes of only one of 11 nonatopic donors could be passively sensitized for histamine release elicited by ragweed extract. A short incubation in an unbuffered isotonic saline at pH 3.9 or in an 0.01 M lactic acid/lactate-buffered isotonic saline at pH 3.9 dissociated from 4 X 10(5) to less than 3 X 10(4) IgE molecules per basophil from washed leukocytes of several in a series of six atopic and 11 nonatopic donors. After such treatment, leukocytes of only one of the 11 nonatopic donors could not be sensitized for histamine release. Basophils of the four ragweed-sensitive donors lost their sensitivity to ragweed after the treatment, but all could be passively resensitized; for three of these donors the level of release approximated their original reactivity. Leukocytes of the two mold-sensitive donors could be passively sensitized to ragweed allergens after but not before treatment. Four plasma samples from histamine release-positive volunteers were used for sensitization of treated leukocytes of each cell donor; three were consistently effective and one was consistently ineffective. The positive plasmas had concentrations of antigen E-specific IgE of over 100 ng/ml, which accounted for 17 to 23% of the total IgE; the inactive one had less than 5 ng/ml of specific IgE. For each cell donor, all three samples of active plasma mediated quite similar histamine release, but there was a spectrum of donor cell reactivity ranging from 23 to 70% release. These results suggest that basophils from each donor, atopic or nonatopic, had a maximal potential for in vitro sensitization, which was only attained if the plasma contained appropriate, but yet to be fully defined, concentrations of specific and total IgE. Several unexpected results were obtained. Treated leukocytes from some individuals were sensitized for mediator release to a greater extent by sixfold diluted than undiluted plasma. In addition, a 4-hr incubation with plasma at 37 degrees C, but not at 25 degrees C or 0 degrees C, was less effective than were shorter incubation periods. Treated leukocytes should be useful in studying kinetic and equilibrium parameters of IgE binding to specific receptors on human basophils. Analogous treatments should also be useful in sensitization and measurement of IgE-receptor interactions of mast cell populations.     

Biochemical and phenotypic characterization of human basophilic cells derived from dispersed fetal liver with murine T cell factors

Metachromatically granulated cells were generated from human fetal liver stem cells cultured in heterologous mouse conditioned medium rich in interleukin 3. After 2 to 3 wk of culture with biweekly changes of medium and selection of nonadherent cells, all cells present in five cultures had cytoplasmic granules, and 60 to 95% of the cells stained metachromatically with toluidine blue or with alcian blue but not with the safranin counterstain. Ultrastructurally, many granules contained fibrillar material or electron-dense cores with fibrils and vesicular fragments. In addition, the granules of many cells were filled with electron-dense material, which in some cases had a fine structure consisting of concentric whorls or a reticular pattern. Analysis of high-affinity IgE receptors on the cultured cells by flow cytometry demonstrated a unimodal fluorescence pattern, suggesting that most cells were in the basophil or mast cell lineage. The cultured cells lacked the lymphoid cell surface determinants B1, B4, T3, and T11, the myeloid determinants Mo2 and MY9, the natural killer cell determinant 901, and Ia histocompatibility antigens, but expressed the myeloid determinant MY7. The cells contained 52 ng/10(6) cells of histamine and incorporated [35S]sulfate at an average rate of 31,300 cpm/10(6) cells/4 hr into 175,000 m.w. chondroitin sulfate A proteoglycans. Upon activation with 1 microM calcium ionophore A23187, the cultured cells released 53% of their cell-associated histamine and metabolized arachidonic acid to 15.0 ng/10(6) cells of immunoreactive leukotriene C4 equivalents, 0.5 ng/10(6) cells of leukotriene B4, and 3.1 ng/10(6) cells of prostaglandin D2 (means, n = 3). Thus, stem cells present in human fetal liver give rise, as do stem cells in mouse fetal liver, to metachromatically granulated cells when cultured in the presence of mouse interleukin 3. In both species, the cultured cells bear IgE receptors, lack characteristic lymphoid and most myeloid cell surface determinants, and contain histamine and chondroitin sulfate proteoglycans. The human fetal liver-derived cells are similar in morphology and T cell factor dependence to basophil-like cells derived from umbilical cord blood, but are novel in their capacity to generate leukotrienes and prostaglandin D2.     

Triggering of cultured neoplastic mast cells by antibodies to the receptor for IgE.

Cell surface receptors for IgE were isolated from detergent lysates of iodinated, IgE-saturated, rat basophilic leukemia cells by precipitation with anti-IgE antibodies followed by chromatography at acid pH. The isolated material showed a single 125I-band (m.w. approximately 58,000) on gel electrophoresis in sodium dodecyl sulfate and was used to immunize a rabbit. The resulting anti-serum was reacted with lysates of surface iodinated mouse or rat tumor mast cells. Analysis of the precipitates on (10%) gel electrophoresis revealed one major peak comprising greater than 80% of the detectable counts and having an estimated m.w. of approximately 58,000. The antiserum reacted with detergent-solubilized and cell-bound receptors in the presence or absence of excess IgE; it also inhibited the binding of 125I-IgE. Cultured mouse mastocytoma cells never exposed to IgE released 3H-serotonin when incubated with F(ab')2, but not Fab' fragments of the antiserum, which had been rigorously freed of IgE and anti-IgE. The release was inhibited in the presence of excess IgE, was Ca++ dependent, and equaled 80% of the maximum obtained with IgE and anti-IgE. We conclude that aggregation of the receptors for IgE provides the critical signals for cell activation.     

Regulation of human basophil and lung mast cell function by cyclic adenosine monophosphate

Immunologic activation of purified human lung mast cells (HLMC) and basophils with anti-IgE induced histamine release but failed to elicit any changes in cAMP levels. In contrast, histamine release and monophasic rises in cAMP were observed in both rat peritoneal mast cells (RPMC) challenged with concanavalin A (73% enhancement over basal cAMP 20 sec after activation) and a cultured mouse bone marrow-derived mast cell (PT18 cell line) passively sensitized with dinitrophenol-specific IgE and stimulated with antigen (39% increase above basal at 15 sec). The adenylate cyclase activators isoprenaline, prostaglandin E2 (PGE2), and forskolin and the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) all induced elevations in cAMP levels in both basophils and HLMC. In basophils, PGE2 and isoprenaline produced approximately twofold increases in cAMP that were maximal at 1 min and decayed thereafter. Forskolin and IBMX produced threefold increases in cAMP that peaked 10 min after activation and persisted for up to 20 min. In HLMC, isoprenaline provoked a rapid monophasic fourfold increase in cAMP that was maximal at 1 min after addition. Levels of cAMP subsequently declined but remained significantly elevated over resting levels for up to 30 min. PGE2, forskolin, and IBMX all produced approximately threefold rises in HLMC cAMP that peaked around 5 min and persisted for 30 min. In both the basophil and HLMC, agonist-induced elevations in cAMP correlated well with the inhibition of mediator release. In basophils, the order IBMX greater than forskolin greater than PGE2 greater than isoprenaline held for both the inhibition of histamine and leukotriene C4 release and the augmentation of cAMP levels. In HLMC, individual agonists elevated cAMP levels to similar degrees and inhibited the release of histamine, leukotriene C4, and PGD2 to comparable extents, although the release of the arachidonate metabolites was generally more sensitive to the inhibitory actions of these agonists. These results suggest that elevations in cAMP, in both the basophil and HLMC, are associated with the inhibition of mediator release but not the initiation of the secretory process.     

Inhibition of IgE-mediated histamine release by myosin light chain kinase inhibitors.

Wortmannin, a specific inhibitor of myosin light chain kinase (MLCK) blocked IgE mediated histamine release from rat basophilic leukemia cell (RBL-2H3) and human basophils dose-dependently. Its IC50 was 20 nM for RBL-2H3 cells and 30 nM for human basophils. There was complete inhibition at the concentration of 1 microM. Wortmannin inhibited partially the A23187 induced histamine release from RBL-2H3 cells (40% inhibition at 1 microM). This inhibition was not accompanied by any significant effect on cytosolic free calcium concentration [( Ca2+]i). KT5926, another MLCK inhibitor, inhibited histamine release comparably with wortmannin and blocked to some degree the increase of [Ca2+]i in RBL-2H3 cells. Thus, the phosphorylation of myosin seems to be involved in signal transduction through Fc epsilon RI.