Cross-linking Of IgE Research Articles

5-Iodonaphthyl-1-azide labeling of plasma membrane proteins adjacent to specific sites via energy transfer

We have examined conditions optimal for 5-iodonaphthyl-1-azide (INA 4) labeling of membrane proteins proximal to known membrane sites. Membrane-bound INA can be indirectly activated by energy transfer from visible chromophores. We demonstrate that the efficiency of this sensitized activation is enhanced by use of triplet-forming chromophores such as eosin and by deoxygenation. Variation of sensitized activation efficiency with INA concentration indicates that the critical distance for eosin-INA energy transfer in solution is 8–14 Å. We suggest that photosensitization occurs through triplet exchange and present an improved labeling protocol based on these findings. This protocol was used to examine whether different accessory proteins are associated with isolated and crosslinked Type I Fc ϵ receptors on 2H3 rat basophilic leukemia cells. 2H3 cells were incubated with eosin-conjugated IgE and irradiated at 514 nm yielding [ 125I]INA derivatized peptides at 53, 38, 34, and 29 kDa. Crosslinking IgE with mouse anti-rat IgE prior to irradiation labeled three additional proteins at 60, 54, and 43 kDa. These results demonstrate the utility of sensitized INA labeling in characterizing protein–protein interactions in membranes of intact cells and indicate the importance of considering photophysical factors when selecting sensitizers and reaction conditions. We discuss estimation of the size of the membrane region surrounding a sensitizing chromophore within which INA labeling of membrane proteins occurs.

Biologic allergen assay for in vivo test allergens with an in vitro model of the murine type I reaction

Background: The determination of the biologic activity of allergenic extracts in human beings is limited for ethical and practical reasons. The establishment of a simplified in vitro model, which mimics a main event of the type I reaction, should provide true benefit for manufacturers, researchers, and clinicians. Objective: This study was designed to develop and evaluate a mediator release assay based on rat basophil leukemia cells for the purpose of investigating allergenic extracts. Methods: Rat basophil leukemia cells were passively sensitized with murine IgE raised against allergens and stimulated by serial dilutions of allergenic extracts in a dose-related manner. The allergen-specific degranulation was monitored by measuring the release of β-hexosaminidase. Results: The investigation of standardized commercial allergen products for in vivo diagnostics (birch pollen, cat dander, and bee venom) allowed a quantitative description of differences in biologic activity in accordance with the declared activity units. The Fel d 1 content was determined in 17 cat dander extracts and correlated well with the results of a two-site binding ELISA ( r= 0.93, log/log). Extremely low allergen amounts in the range of 10 to 100 pg/ml could be easily detected. Moreover, the cross-reactivity pattern of patients allergic to birch pollen could be reproduced with extracts of hazel, alder, apple, and celery. Conclusion: The assay is suitable for supplementing quality control of allergenic extracts and for the determination of biologic activity of final allergen products. As a research tool, it allows the study of IgE cross-linking properties of modified and recombinant allergens at an early stage before they are tested in human beings. (J Allergy Clin Immunol 1997;99:227-32.)

Cyclic AMP-Elevating Agents Inhibit Mite-Antigen-Induced IL-4 and IL-13 Release from Basophil-Enriched Leukocyte Preparation

Human peripheral blood basophils are known to secrete interleukin (IL)-4 and IL-13 after cross-linking of cell surface IgE. However, little is known about the pharmacological regulation of allergic cytokine release from basophils. In the present study, we investigated the effects of cyclic 3',5'-adenosine monophosphate (cAMP)-elevating agents on antigen-induced IL-4 and IL-13 release from basophil-enriched leukocyte preparations. We obtained venous blood from 27 atopic asthmatic patients (mean age was 45.8+/-3.6 years, all patients were sensitive to mite antigen) and prepared basophil-enriched leukocyte preparations by double-Percoll gradients (basophil purity was 13.4+/-1.6%). The cell preparations were treated with phosphodiesterase (PDE) inhibitors, dexamethasone, forskolin or dibutyryl cAMP for 10 min and were challenged with mite antigen for 6 h. The released IL-4 and IL-13 in the supernatants were measured by enzyme-linked immunosorbent assay systems. No IL-4 or IL-13 was detected in the supernatant of the basophil-depleted preparation after the challenge with mite antigen, suggesting that basophils specifically produce these cytokines. A nonselective PDE inhibitor, theophylline, and a PDE IV-selective inhibitor, rolipram, significantly suppressed the release of IL-4 and IL-13 from the basophil-enriched preparation. Although several concentrations of cilostazol, a PDE III-selective inhibitor, had no effect on the release of both cytokines, cilostazol suppressed the release of IL-4 additively when applied with rolipram. Forskolin and dibutyril cAMP also significantly suppressed the release of both cytokines, suggesting that the suppressive effects by PDE inhibitors were accompanied by the elevations in cAMP levels. We conclude that basophil-enriched leukocyte preparations produce IL-4 and IL-13 in response to antigen and that the release of these cytokines could be regulated by cAMP-modulating agents.

Dexamethasone or cyclosporin A inhibits stem cell factor-dependent secretory responses of rat peritoneal mast cells in vitro

Stem cell factor (SCF) is a potent activator of degranulation of rat peritoneal mast cells in vitro and may promote mast cell activation under certain circumstances in vivo. In this study we report that the anti-inflammatory glucocorticoid dexamethasone (DEX) and the immunosuppressive cyclosporin A (CsA) are both effective inhibitors of SCF-induced degranulation of rat peritoneal mast cells in vitro, measured as release of serotonin (5-HT). Of the two drugs, DEX was the more potent with near maximal inhibition reached at 10-8 M, whereas a graded inhibition was seen with CsA in the range 10−8-10−6 M. DEX was equally effective in inhibiting the release of 5-HT induced by either SCF or anti-IgE, but was less effective in inhibiting release induced by compound 48/80 or calcium ionophore A23187. CsA produced a similar degree of inhibition of degranulation induced by SCF, anti-IgE or ionophore, but was without effect on the response to compound 48/80. Neither DEX nor CsA had any significant effect on mast cell surface expression of the SCF receptor or IgE antibody. We conclude that both DEX and CsA inhibit components of the secretion-coupling pathways that are triggered following either SCF receptor engagement or cross-linking of IgE, but that these drugs differentially influence mast cell secretion induced by compound 48/80 or the calcium ionophore A23187.

Human synovial mast cells. I. Ultrastructural in situ and in vitro immunologic characterization.

To examine the ultrastructure of human synovial mast cells in situ, to identify immunologic and nonimmunologic stimuli that activate these cells in vitro, and to quantify a number of preformed and de novo-synthesized mediators. We conducted an ultrastructural study of synovial mast cells in situ and performed immunoelectron microscopy localization of tryptase and chymase. Isolated synovial mast cells were analyzed biochemically, immunologically, and functionally in vitro and compared with cells from human lung, heart, and skin. Ultrastructural study of synovial tissue revealed mast cells with homogeneously dense, scrolled, crystal, and mixed granules, and lipid bodies in the cytoplasm. A small percentage of mast cells showed evidence of degranulation. Immunoelectron microscopy demonstrated the subcellular localization of tryptase and chymase over granules of > 90% of the mast cells, which were of the MCTC subtype. Isolated synovial mast cells released histamine in response to immunologic (anti-IgE and anti-Fc epsilon receptor I [anti-Fc epsilon RI]) and nonimmunologic (substance P, recombinant human stem cell factor, and 48/80) stimuli, but did not respond to recombinant human C5a in vitro. Synovial mast cells differed from those isolated from other human tissues, in a variety of immunologic and biochemical features. There was a linear correlation between the percentage of histamine secretion and tryptase release (r = 0.79, P < 0.001) induced by cross-linking of Fc epsilon RI. Cross-linking of IgE with anti-IgE on synovial mast cells induced de novo synthesis of prostaglandin D2 (mean +/- SEM 87.5 +/- 4.9 ng/10(6) cells) and of leukotriene C4 (57.6 +/- 17.8 ng/10(6) cells). Mast cells ultrastructurally characterized in situ in synovial tissue were seen to differ from mast cells previously isolated from other human tissues. This raises the possibility that the local microenviroment influences their phenotype. Isolation of mast cells from human synovia can be useful for studying their role and their mediators in patients with arthritis.

Nonreleasing basophils convert to releasing basophils by culturing with IL-3

The extent of basophil histamine release initiated by IgE cross-linking stimuli has been known to vary greatly among donors. Studies on anti-IgE nonreleasing basophils are useful in understanding the IgE-specific control mechanism of mediator release. We attempted to determine (1) whether a mutation of FcϵRI is present in nonreleasing basophils and (2) whether treatment with IL-3 converts anti-IgE nonreleasing basophils to releasing basophils. Basophils were purified from normal human blood and donors were divided into releasers (maximal histamine release >5%) and nonreleasers (<5%). The mutation of FcϵRIα, β, and γ was evaluated by reverse transcriptase–polymerase chain reaction, and the DNA sequence was determined from amplified polymerase chain reaction products. Although antibodies against FcϵRI failed to cause histamine release in anti-IgE nonreleasing basophils, no primary structural change of FcϵRI was observed in nonreleaser basophils. After culturing with IL-3 for 7 days, nonreleasing basophils released histamine in response to anti-IgE, and dose-response curves of anti-IgE were equal in both releasers and nonreleasers. The conversion of nonreleasing basophils to releasing basophils was evident after 3 days of culture with IL-3. These findings indicate that nonreleasing basophils have recoverable defect(s) in the signal transduction pathway after IgE cross-linking. (J ALLERGY CLIN IMMUNOL 1996;97:1279-87.)

Multiple loss of effector cell functions in FcR gamma-deficient mice.

Immunoglobulin Fc receptor (FcR) gamma subunit is a component of low affinity receptor for IgG, Fc gamma RIII, as well as high affinity receptor for IgE, Fc epsilon RI. This subunit is required for efficient surface expression of these FcRs on various cells in immune system. The FcR gamma-deficient mice, generated by gene targeting in embryonic stem cells, exhibit multiple defects in FcR-mediated effector cell responses, including absence of phagocytic activity against opsonized red blood cells by activated macrophages, loss of antibody-dependent cell-mediated cytotoxicity manifested by IL-2-induced splenic NK cells, and unresponsiveness of mast cells to crosslinking of IgE on these cells. These results demonstrate an indispensable role of FcR gamma for functional expression of FcRs, and clearly indicate the importance of Fc gamma RIII as well as Fc epsilon RI for these effector functions. Since FcR gamma-deficient mice is unable to mount the type II and type III hypersensitivity reactions, it is suggested that FcRs play pivotal roles in initiating these reaction cascades. The mutant mice should prove to be useful in evaluating FcRs in various humoral and cellular immune responses, and in developing new strategies for treatment of immunodeficiency as well as autoimmune disorders.

Proximity relationships between the type I receptor for Fcεe (FcεeRI) and the mast cell function‐associated antigen (MAFA) studied by donor photobleaching fluorescence resonance energy transfer microscopy

Clustering of the mast cell function-associated antigen (MAFA) on the surface of rat mucosal type mast cells line 2H3 (RBL-2H3) leads to suppression of the secretory response induced by the type I Fc epsilon receptor (Fc epsilon RI). In order to establish a possible association between MAFA and Fc epsilon RI we measured fluorescence resonance energy transfer (FRET) between the MAFA-specific monoclonal antibody (mAb) G63 and Fc epsilon RI-bound ligands as well as between Fc epsilon RI-bound ligands themselves using the donor photobleaching FRET (pbFRET) technique. Average FRET efficiencies between 6 and 9% were determined after low-temperature incubation with fluorescent dye conjugated mAb G63 bound to MAFA (donor) and IgE bound to Fc epsilon RI (acceptor) on RBL-2H3 cells. Subsequent cross-linking of IgE by a polyvalent antigen caused no change in FRET efficiencies. These results suggest that the MAFA is located in the vicinity of the Fc epsilon RI on resting cells, and that clustering of the Fc epsilon RI leads to no significant change in the proximity of the two molecular species. In view of the sequence motif identified in the cytosolic tail of the MAFA and the observed changes in its phosphorylation upon antigen stimulation (Guthmann et al., Proc. Natl. Acad. Sci. USA 1995, 92: 9397-9401), the present study suggests that the secretory response inhibition by MAFA interferes with the signal transduction cascade initiated via the Fc epsilon RI. An additional finding was that clustering of the Fc epsilon RI by antigen showed a clear increase in the efficiency of FRET between Fc epsilon RI-bound IgE molecules conjugated with fluorescent donor and acceptor.

Multiple defects in the immune system of Lyn-deficient mice, culminating in autoimmune disease

Mice homozygous for a disruption at the Lyn locus display abnormalities associated with the B lymphocyte lineage and in mast cell function. Despite reduced numbers of recirculating B lymphocytes, Lyn -/- mice are immunoglobulin M (IgM) hyperglobulinemic. Immune responses to T-independent and T-dependent antigens are affected. Lyn -/- mice fail to mediate an allergic response to IgE cross-linking, indicating that activation of LYN plays an indispensable role in FcεRI signaling. Lyn -/- mice have circulating autoreactive antibodies, and many show severe glomerulonephritis caused by the deposition of IgG immune complexes in the kidney, a pathology reminiscent of systemic lupus erythematosus. Collectively, these results implicate LYN as having an indispensable role in immunoglobulin-mediated signaling, particularly in establishing B cell tolerance.

The central rôle of T-cells in allergic sensitization and IgE regulation.

The atopic phenotype develops on the basis of a genetic predisposition. Several candidate genes and chromosomal regions have been recently identified that may play a rôle in the development of allergic sensitization and total IgE production, including genes encoding MHC and T-cell receptor (TCR) molecules, cytokines and others. Genetic predisposition triggers and immunological dysregulation which is controlled by CD4+ T-cells. (Specialized) antigen presenting cells process and present allergenic peptides (T-cell epitopes) on MHC class II molecules to T-cells that recognize MHC plus peptide using the TCR. Cognate and non-cognate interaction results in T-cell activation. Selective stimulation of the allergen specific T-cells is the result of allergen-specific sensitization. These T-cells are characterized by (simultaneous) production of IL-3, IL-4, IL-5 (and may be IL-13). These cytokines control the production of IgE by B cells and play a critical rôle in the activation and differentiation of effector cells of the allergic response (such as eosinophils and mast cells). In addition to MHC-TCR interaction and cytokine production, ligation of CD40 and CD40L represents an additional requirement for the production of functional IgE molecules. Immediate hypersensitivity responses are characterized by an early phase response (triggered by many mediators released from effector cells following allergen exposure, IgE cross-linking and activation of signal transduction pathways) and a late phase response that is mediated to a large extend by the influx of T-cells and effector cells into the site of allergic inflammation. Deliniation of the immunological mechanisms that result in allergic sensitization will contribute to the development of specific immunomodulatory strategies aimed to prevent the development of allergies.

LOCAL ANESTHETICS AND NEUROMUSCULAR BLOCKING AGENTS

Although a number of neuromuscular blockers may cause nonspecific histamine release, serious systemic reactions are anaphylactic in type and have been shown to be due to crosslinking of IgE by these bivalent molecules. Properly performed skin testing appears to be the most reliable method of detecting sensitivity to these agents, which can occur in patients who have never been exposed to them. A negative skin test appears to be reliable in predicting that an agent can be given safely; conversely, the considerable cross-reactivity demonstrated by in vitro tests is often not clinically significant. Routine skin testing to one or more agents before surgery should be investigated for cost-effectiveness in preventing systemic reactions. Systemic reactions require prompt recognition and administration of epinephrine to prevent an adverse outcome.

Compounds that induce autoimmunity in the brown Norway rat sensitize mast cells for mediator release and interleukin-4 expression.

Brown Norway (BN) rats given mercuric chloride (HgCl2), gold (Au) salts or D-penicillamine develop a T helper 2 (Th2) cell-mediated autoimmune syndrome. The recent observation of tissue injury within 24 h of HgCl2 treatment suggested the involvement of a non-T cell. We therefore examined the effect of these compounds on rat mast cells in vitro. Incubation of BN rat peritoneal mast cells with HgCl2 enhanced the release of serotonin in response to IgE cross-linking agents. Mast cells from Lewis rats, a strain not susceptible to the autoimmune syndrome in vivo, were affected to a lesser extent. The effect was observed with purified BN mast cells, suggesting a direct action. Similar effects were seen with D-penicillamine in the presence of copper ions, a combination that produces hydrogen peroxide, and Au. HgCl2 caused significant induction of interleukin (IL)-4 mRNA in mast cells from BN, but not Lewis rats. The data demonstrate a novel enhancing effect of a number of compounds on mast cell mediator release, and an inducing effect of HgCl2 on mast cell IL-4, expression. These findings are consistent with our hypotheses that mast cells may contribute to early tissue injury, and also, via production of IL-4, may initiate and/or augment, the Th2 response in the BN rat model of chemical-induced autoimmunity.

Tyrosine phosphorylation of p95Vav in myeloid cells is regulated by GM-CSF, IL-3 and steel factor and is constitutively increased by p210BCR/ABL.

Vav is a recently described proto-oncogene expressed only in hematopoietic cells which contains an SH2 and two SH3 domains and shares homology with the Dbl GDP-GTP exchange factor and BCR. p95Vav is phosphorylated on tyrosine residues in response to stimulation of the T cell antigen receptor, cross-linking of IgE or IgM receptors and stimulation of immature hematopoietic cells by Steel factor. Monoclonal antibodies to human Vav were generated and used to examine the events which regulate tyrosine phosphorylation of p95Vav in myeloid cells. In the factor-dependent MO7e cell line, p95Vav was rapidly phosphorylated on tyrosine residues in a dose- and time-dependent manner by GM-CSF, IL-3 and Steel factor. Introduction of the BCR/ABL oncogene into this cell line resulted in factor-independent proliferation and constitutive phosphorylation of p95Vav. Tyrosine phosphorylation of p95Vav was also substantially increased by treatment of cytokine-deprived cells with the tyrosine phosphatase inhibitor sodium vanadate. Since many of the cytokines known to induce tyrosine phosphorylation of p95Vav are also known to activate JAK family tyrosine kinases, we looked for an interaction of p95Vav with JAK kinases. p95Vav co-precipitated with JAK2 in MO7e cells stimulated with GM-CSF, but not in unstimulated cells. Also, JAK2 was found to be constitutively associated with p95Vav in vivo when expressed at high levels in insect cells using baculovirus vectors. A fusion protein consisting of glutathione-S-transferase and the SH2 domain of p95Vav (GST-Vav-SH2) precipitated JAK2, suggesting that this interaction is mediated by the SH2 domain of p95Vav.(ABSTRACT TRUNCATED AT 250 WORDS)

Paracrine Ca2+ signaling in vitro: serotonin-mediated cell-cell communication in mast cell/smooth muscle cocultures.

Mast cells are tissue-resident immune cells that are capable of signaling many different cell types in vascularized tissue including epithelia and smooth muscle. We have developed an in vitro coculture system in which secretion of serotonin by a mucosal mast cell line (RBL-2H3) can be studied at a single cell level by measuring Ca2+ transients in fura-2 loaded mast cells and serotonin-sensitive A7r5 smooth muscle cells using fluorescence video microscopy and digital image processing. A7r5 cells elevate intracellular Ca2+ via 5HT2 receptors in response to bath-applied serotonin with an ED50 for serotonin of 550nM. Crosslinking IgE receptors with antigen caused Ca2+ transients in the mucosal mast cells. Ca2+ responses in the smooth muscle were detected approximately 30-240 sec after the initiation of the mast cell Ca2+ responses. Smooth muscle Ca2+ responses were dependent on preloading mast cells with serotonin and were blocked by the 5HT2 antagonist ketanserin. The timing and magnitude of the smooth muscle responses indicated that secretion from mast cells can lead to local concentrations of serotonin in the range of 300 nM within 1 min of antigen stimulation. This coculture technique has allowed the first direct demonstration of serotonin-mediated signaling between immune cells and vascular elements.

Stimulation of mitogen-activated protein kinase activity by different secretory stimuli in rat basophilic leukemia cells.

Ag-induced cross-linking of IgE bound to its high affinity receptor (Fc epsilon RI) at the surface of basophils or mast cells triggers a number of biochemical events culminating in the release of several inflammatory mediators. In rat basophilic leukemia (RBL-2H3) cells expressing the G protein-coupled m1 muscarinic receptor, Ag/IgE-induced cross-linking of Fc epsilon RI, calcium ionophore A23187, and carbachol through M1 receptors stimulated tyrosine phosphorylation of several proteins, including two of 42 and 44 kDa. Proteins of identical molecular masses were recognized by anti-MAP-kinase antibodies, and these immunoreactive proteins exhibited in part a slightly increased molecular mass on SDS polyacrylamide gels after incubation of cells with secretory stimuli. All stimuli led to the activation of MAP kinase, which co-purified on Mono Q chromatography with 42- and 44-kDa proteins, which were tyrosine phosphorylated in response to secretory stimuli and reacted with anti-(MAP kinase) antibodies. Finally, 42- and 44-kDa proteins immunoprecipitated by anti-MAP-kinase antibodies and anti-phosphotyrosine antibodies were recognized by anti-phosphotyrosine and anti-MAP-kinase antibodies, respectively. Primarily threonine and tyrosine residues were found to be phosphorylated in 42- and 44-kDa proteins immunoprecipitated from [32P]phosphate-labeled cells that had been treated with secretory stimuli. The dose dependence of secretagogue-induced MAP kinase activation correlated with that of increases in serotonin release from activated cells, and the maximum of MAP kinase activation coincided with the maximum rate of secretion. Down-regulation or inhibition of protein kinase C as well as incubation of cells with the tyrosine kinase inhibitor genistein markedly inhibited MAP kinase activation in parallel with serotonin release. Taken together, these findings demonstrate that 42- and 44-kDa MAP kinases are activated in response to secretory stimuli and provide some evidence for a functional link between MAP kinase activation and signaling events leading to mediator release in RBL cells.

Peptidergic pathway in human skin and rat peritoneal mast cell activation

The common pathway of heterogenous mast cell activation as mediated by antigens is through the cross-linking of IgE bound to FcϵRI receptors. The peptidergic pathway of mast cell activation, achieved by cationic secretagogues, is restricted to “serosal” mast cells, the experimental models being rat peritoneal and human skin mast cells. Cationic secretagogues include positively charged peptides but also various amines such as compound 48/80 and natural polyamines. An early intracellular event of this pathway is the activation of pertussis toxin-sensitive G proteins. The correlation observed between the ability of basic compounds to trigger mast cell exocytosis and their potency to activate purified G proteins strongly suggests that cationic compounds activate mast cell G proteins via a receptor-independent but membrane-assisted process. In this paper, alternative mechanisms are discussed. The consequence of G protein stimulation is the activation of phospholipase C with an increase in inositol triphosphates. Natural polyamines are relatively poor triggers of mast cells (10−4 to 10−2 M). Neuropeptides such as substance P, neuropeptide Y or vasoactive intestinal peptide, peptidic hormones such as kinins, and venoms such as mastoparan and mast cell degranulating peptide, are all active in a concentration range from 10−7 to 10−4 M. The cationic anaphylatoxin C3a also stimulates mast cells at concentrations below precursor complement C3 blood levels. The component C3 of the complement system is one of only a few plasma proteins having activation fragments (i.e. C3a) that can be generated at micromolar levels. The effects of basic secretagogues defines a peptidergic pathway of mast cell activation, which represents a potentially toxic process considering the tissue effects caused by exogenous basic compounds such as venom peptides and certain amine containing drugs. Peptidergic activation of mast cells may also be a pathophysiological process having an important role in neurogenic inflammation and in diseases involving extensive activation of the blood complement cascade.

3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibition in a rat mast cell line. Impairment of tyrosine kinase-dependent signal transduction and the subsequent degranulation response

IgE molecules bind mast cells via a heterotetrameric receptor termed Fc epsilon RI. Cross-linking of bound IgE by specific allergen (Ag) initiates a signal transduction cascade resulting in a degranulation response. Inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in isoprenoid and sterol biosynthesis, by the cholesterol-lowering drug, lovastatin, blocks Fc epsilon RI-dependent [3H] serotonin ([3H]5HT) release from the mast cell line, RBL-2H3. We studied the mode and locus of action of lovastatin in these cells. Lovastatin inhibited Ag-stimulated degranulation, as well as that evoked by ionomycin or by phorbol 12-myristate 13-acetate and ionomycin, stimuli which bypass early receptor events. Inhibition was concentration-dependent, occurred at levels which reduce lipid synthesis, and was reversible by addition of mevalonic acid, the product of the reductase reaction. The effects of lovastatin were not mimicked by treatment with the sterol demethylase inhibitor, econazole, suggesting that nonsterol isoprenoid synthesis is required for the degranulation response. Conversely, tyrosine kinase inhibitors from three disparate chemical classes reduced stimulus-evoked [3H]5HT release in a manner similar to lovastatin, suggesting that these agents share similar loci of action. Accordingly, lovastatin altered the phosphorylation pattern in unstimulated RBL-2H3, and reduced the phosphorylation response to IgE cross-linking. By analogy to 5HT release, this effect was concentration-dependent and mevalonic acid-reversible. The tyrosine kinase inhibitor, geldanamycin, also reduced the phosphorylation response to Ag. Lyn, a Src-related tyrosine kinase activated upon IgE cross-linking, was little influenced by either lovastatin or geldanamycin. Thus, lipid synthesis inhibition by lovastatin results in impaired tyrosine phosphorylation in RBL-2H3. This impairment is reflected in the subsequent exocytotic response. While lovastatin may inhibit tyrosine phosphorylation via an indirect mechanism, our results with tyrosine kinase inhibitors support the concept that multiple tyrosine kinases participate in the Fc epsilon RI-dependent signal transduction process.

Biphasic Ca2+ responses in human basophils. Evidence that the initial transient elevation associated with the mobilization of intracellular calcium is an insufficient signal for degranulation.

Abstract Human basophils exhibited biphasic cytosolic Ca2+ responses after stimulation with both the bacterial peptide, FMLP and IgE cross-linking agents such as goat polyclonal anti-IgE PS myeloma (anti-IgE) antibody and Ag. The biphasic response to anti-IgE antibody was not apparent unless supraoptimal concentrations of anti-IgE antibody were used to stimulate the cells. Biphasic response characteristics were also observed in single cells. As in many cell types, the first phase of the Ca2+ response appeared independent of extracellular Ca2+. Challenging basophils with anti-IgE in the presence of EGTA, added simultaneously or 3 to 5 min before the stimulus, had little effect on the first transient Ca2+ elevation while completely eliminating the second phase of the response. We noted that while simultaneous addition of EGTA had little effect on the histamine release after challenge with FMLP, there was no histamine release after the simultaneous addition of anti-IgE and EGTA. An examination of the kinetics of histamine release after anti-IgE also demonstrated that no histamine release occurs during the initial transient response. Since the initial Ca2+ transient was intact after both stimuli in the presence of EGTA, we concluded that the initial transient was not sufficient to induce histamine release. The hypothesis that distinct sources of calcium were responsible for the initial Ca2+ transient that followed challenge with FMLP peptide or anti-IgE antibody was also tested. We found that prior treatment of basophils with thapsigargin inhibited the initial Ca2+ response that followed both FMLP and anti-IgE antibody. Furthermore, stimulation with FMLP, in the presence of EGTA, depleted the calcium source responsible for the anti-IgE response. The experiment conducted in reverse order showed that anti-IgE antibody could discharge the calcium source responsible for the initial FMLP Ca2+ response. These experiments indicated that FMLP and anti-IgE antibody use the same internal source of calcium to produce the initial Ca2+ response. Taken together with previous studies on FMLP, anaphylatoxin split product of the C component, C5; and platelet activating factor-induced release in human basophils, these results suggest that the initial transient elevation, which is relatively independent of extracellular Ca2+, is not sufficient to induce degranulation.

Regulation of mouse peritoneal mast cell secretory function by stem cell factor, IL-3 or IL-4.

Abstract We examined whether three cytokines that promote mouse mast cell development, the c-kit ligand stem cell factor (SCF), IL-3, or IL-4, also can directly stimulate or modulate mouse peritoneal mast cell (PMC) mediator release. Challenge of purified PMC with rat rSCF164 at 20 to 100 ng/ml for 30 min induced a modest release of serotonin (5-HT), whereas IL-3 or IL-4 did not directly stimulate 5-HT release. Experiments in which PMC were exposed to each cytokine for 15 min, and then to DNP-HSA Ag or anti-IgE antibody for a further 15 min, showed that SCF, but not IL-3 or IL-4, had an additive effect on the 5-HT release induced by either of the IgE cross-linking agents. In longer term experiments, SCF (0.16 to 500 ng/ml), IL-3 (2.5 to 100 ng/ml), or IL-4 (0.06 to 2.5 ng/ml) was added to peritoneal cell cultures for 48 h, during which the cells were passively sensitized with IgE anti-DNP antibody. Incubation of either unfractionated or highly purified PMC preparations with each of the three cytokines resulted in a concentration-related increase in 5-HT release upon subsequent challenge of the cells with DNP-HSA Ag. However, after pretreatment of peritoneal cells for 48 h with each cytokine, only IL-4 (10 ng/ml) enhanced release of 5-HT induced by calcium ionophore A23187 (0.25 microM); IL-3 (100 ng/ml) had no effect, whereas SCF (100 ng/ml) significantly inhibited ionophore-induced release. Although IL-3 or SCF up-regulate responsiveness to IgE-dependent stimuli, we detected no effect of these cytokines on the binding of [125I]IgE to PMC. This suggests that the enhancing effects of SCF or IL-3 on IgE-dependent 5-HT release did not simply reflect changes in the amount of IgE bound to the cells. In conclusion, we found that SCF, IL-3, or IL-4 each exerted a different spectrum of stimulatory, costimulatory, or regulatory effects on the secretory function of mouse PMC.

Restricted Lymphokine Synthesis in CD4+ Helper T Cells: New Insights into the Regulation of IgE Synthesis and the Pathogenesis of Allergic Disease

Over the past 5 years, a greater appreciation has developed regarding the role that IgE plays in the pathogenesis of allergic disease and asthma. Clinical investigators have shown that elevated IgE levels strongly correlate with the presence of asthma. In addition, other investigators have demonstrated that crosslinking of IgE on the surface of mast cells not only results in the release of histamine and other mediators but also produces a cascade of events leading to late phase reactions, bronchial hyperreactivity, and an allergic inflammatory response. Recent studies of the effects of a number of factors produced by CD4+ helper T lymphocytes—in particular, interleukin (IL) 4, IL-5 and IL-3—indicate that a distinct subtype of CD4+ T lymphocytes producing a specific profile of lymphokines greatly increases the secretion of IgE from B cells and enhances the accumulation of eosinophils and mast cells in the allergic inflammatory response. Thus, a complex network of cell types, including B cells producing IgE, mast cells, eosinophils, and subsets of CD4+ T lymphocytes secreting specific profiles of lymphokines, is involved in the development and regulation of allergic inflammation. This new understanding of allergic inflammatory disease and its dependence on IgE and on lymphokine synthesis in subsets of CD4+ T cells will lead to improved strategies in the treatment and control of allergic diseases.