Trigger Histamine Release Research Articles

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.

Monoclonal antibodies specific to the alpha-subunit of the mast cell's Fc epsilon R block IgE binding and trigger histamine release.

In an attempt to block the interactions between IgE and its receptor on mast cells (Fc epsilon R), we have established anti-Fc epsilon R monoclonal antibodies (mAb) by fusion of myeloma cells with mouse splenocytes immunized with irradiated rat basophilic leukemia (RBL) cells. Two anti-Fc epsilon R mAb were obtained (denoted 4.7 and 5.14) that could specifically bind to RBL and mast cells. This binding could be inhibited by IgE. The mAb and their F(ab')2 fragments inhibited 125I-IgE binding to RBL cell and triggered cell degranulation. The Fab' fragments, on the other hand, could only inhibit IgE binding but did not stimulate cell degranulation. Furthermore, these monovalent fragments inhibited RBL and mast cell degranulation induced by IgE-antigen complexes both in vitro and in vivo in the passive cutaneous anaphylaxis reaction. The number of mAb 4.7 and 5.14 molecules bound per RBL cells was similar to that of IgE; nevertheless, mAb 4.7 and 5.14 recognized different epitopes on the IgE receptor. Immunoprecipitation and immunoblotting analysis demonstrated that the mAb reacted with the alpha-subunit of the Fc epsilon R. Our findings establish the anti-Fc epsilon R mAb as a useful reagent for the isolation and characterization of the Fc epsilon R's alpha-subunit and the monomeric (Fab') for blocking the IgE-Fc epsilon R interactions.

Anaphylaxis with Schistosoma mansoni extracts in normal and infected mice

Methods generally utilized for studies on anaphylaxis to protein antigens such as determination of histamine release to the blood, hemoconcentration, histamine release from peritoneal mast cells and passive cutaneous anaphylaxis (PCA) were used to investigate some aspects of the anaphylaxis to parasite antigens in Schistosoma mansoni infected mice. The release of histamine to the blood and significant rates of hemoconcentration were induced by intravenous injection of schistosomula or cercarial extracts into 10-13 weeks infected mice. Cercarial, schistosomula, worm tegument and soluble egg antigens were able to trigger histamine release from peritoneal mast cells from chronically infected mice. In spite of the PCA reaction beeing detected within 2 hours of sensitization (IgG1antibodies) in 6 of 8 tested sera from chronically infected mice, no detectable reactions were obtained after 48 hours sensitization (IgE antibodies). Although IgE was not detected in the circulation, by the PCA technique, the results indicate that the infected mice contained IgE antibodies bound to their mast cells.

Biochemical analysis of desensitization of mouse mast cells.

Biochemical mechanisms of desensitization were explored by using peritoneal mouse mast cells saturated with monoclonal mouse IgE anti-DNP antibody. It was found that a 1-min incubation of the sensitized cells with 0.01 micrograms/ml DNP-HSA in the absence of Ca2+ was sufficient to desensitize the cells completely. The treated cells failed to release a detectable amount of histamine upon incubation with an optimal concentration (0.1 to 1.0 micrograms/ml) of DNP-HSA and Ca2+. Determination of the number of antigen molecules bound to mast cells revealed that only a small (less than 10%) fraction of cell-bound IgE antibody molecules reacted with desensitizing antigen, and that desensitized cells and untreated (sensitized) cells could bind comparable amounts of antigen upon incubation with rechallenging antigen. However, the binding of antigen molecules to desensitized cells failed to induce any of the early biochemical events, i.e., phospholipid methylation, cAMP rise, and 45Ca uptake, as well as histamine release. It was also found that intracellular cAMP levels in desensitized cells were comparable to those in sensitized cells. Desensitization by a suboptimal concentration of DNP-HSA was prevented by inhibitors of methyltransferases, such as 3-deaza adenosine plus L-homocysteine thiolactone. Sensitized cells pretreated with 0.01 micrograms/ml DNP-HSA in the absence of Ca2+ and in the presence of the methyltransferase inhibitors responded to an optimal concentration of antigen for histamine release when they were rechallenged in the presence of Ca2+. Inhibition of desensitization by methyltransferase inhibitors was reversed by the addition of S-adenosyl-L-methionine to the system. The results indicated that the activation of methyltransferases, induced by receptor bridging, is involved in the process of desensitization. Desensitization was inhibited by reversible inhibitors of serine proteases, such as p-aminobenzamidine, indole, and synthesized substrates of rat mast cell proteases. It was also found that diisopropylfluorophosphate (DFP), an irreversible inhibitor of serine proteases, completely blocked desensitization at the concentration of 10 to 40 nM. This concentration of DFP did not affect the antigen-induced histamine release, whereas 100- to 1000-fold higher concentrations of DFP did inhibit histamine release. The results suggest that serine proteases are involved in both the induction of histamine release and desensitization, and that the protease involved in desensitization is distinct from that involved in triggering histamine release.

Production of a monoclonal antibody against a major allergen of Dactylis glomerata pollen (Dg1)

A mouse monoclonal antibody was produced against the most frequent allergen Ag Dg1ief, purified from the Dactylis glomerata grass pollen by isoelectric focusing. Using the immunoprint technique, this monoclonal antibody detected Ag Dg1ief in D. glomerata pollen and some cross-reacting molecules in four other grass pollens. This hybridoma was grown in ascitic fluid. An immunosorbent was made from it and, by affinity chromatography, was used to purify monoclonal Ag Dg1m from the pollen extract. Ag Dg1m (6,000 to 8,000 daltons) appeared to be 4 to 5 times smaller than Ag Dg1ief. It appeared as a weak allergen in passive cutaneous anaphylaxis in rats, but was more potent in triggering histamine release on human basophils from grass-pollen sensitive patients. These results suggest that Ag Dg1m could be a part of the Ag Dg1ief used to induce production of this monoclonal antibody.

Purification and Characterization of a Major Allergen from <i>Dactylis glomerat</i><i>a</i> Pollen: The Ag Dg1

We have isolated an allergen (Ag Dg1) from Dactylis glomerata pollen which is recognized by the serum of 95% of human patients sensitive to D. glomerata pollen, as has been shown by the nitrocellulose immunoprint technique. After two successive purifications by preparative isoelectric focusing (IEF), Dg1 was characterized as a single band in analytical agarose IEF with a pI of 5.9 and was found to display 3 bands by sodium dodecyl sulfate polyacrylamide gel with the respective molecular weights: 21,000, 31,000 and 33,000 daltons. The high recognition frequency by IgE antibodies of Dg1 in the sera of allergic patients and its ability to trigger histamine release from sensitized human basophils allow to consider that Ag Dg1 is the main major allergen extracted from D. glomerata pollen.

Failure of covalently cross-linked human IgG myeloma subclass protein to release histamine from human leukocytes.

We have examined the ability of IgG subclass antibodies to release histamine from human leukocytes using covalently cross-linked oligomers of human myeloma proteins. Purified IgG1, G2, G3, G4, (or IgE) was incubated with dimethyl suberimidate to induce cross-linking. The resulting dimers, trimers, and higher molecular weight oligomers were isolated using gel filtration columns (Sephadex G200 and Ultrogel AcA 22) connected in tandem. None of the oligomers of IgG1, G2, G3, or G4 released histamine from leukocytes of donors whose basophils released histamine when challenged with IgE dimer. Furthermore, preincubation with subclass specific oligomers did not desensitize cells to challenge with IgE dimer or to anti-IgE. We conclude that, under our experimental conditions, oligomers of human IgG myeloma subclass antibodies do not trigger histamine release nor modulate IgE-mediated reactions.

Analysis of triggering events in mast cells for immunoglobulin E-mediated histamine release

It has been established that mast cells and basophil granulocytes bear specific receptors for IgElP4 and that the reaction of cell-bound IgE molecules, either by multivalent antigen or with divalent anti-IgE antibody, induces release of a variety of chemical mediators from the cells.j* 6 With respect to the mechanisms of mediator release, it has been suggested that polymerization of cell-bound IgE molecules may activate membrane-associated enzymes that in turn initiate the process of histamine release.iq 8 In order to analyze initial triggering events on the mast cells for IgE-mediated histamine release, I prepared antibodies against IgE receptors on rat mast cells (anti-RBL).” Analysis of the antibody preparation revealed that the major antibodies in the preparation are directed toward the binding sites for IgE in the receptor molecules.‘O Using this antireceptor antibody (anti-RBL), it was demonstrated that bridging of receptor molecules by divalent antireceptor antibody or its F(ab’), fragments induced histamine release without participation of IgE, while the binding of Fab’ monomer fragments with the receptor was not sufficient for triggering histamine release. However, when receptor-bound Fab’ monomer was bridged with divalent antirabbit y-globulin (anti-RGG), mast cells were triggered. This situation is similar to anti-IgEinduced histamine release in which receptor-bound IgE molecules are bridged by anti-IgE (Fig. 1). From

Theory of equilibrium binding of a bivalent ligand to cell surface antibody: the effect of antibody heterogeneity on cross-linking.

We investigate the equilibrium binding of symmetric bivalent ligands to a heterogeneous population of symmetric bivalent cell surface receptors. The receptors are heterogeneous in their binding affinities (equilibrium binding constants) for the ligand. For any distribution of receptor binding affinities we show how to calculate the total concentration of receptors that are cross-linked by the ligand, i.e., the concentration of cell surface aggregates composed of two or more receptors, as well as the concentration of any given aggregate. We show that certain qualitative properties of cross-linking which hold for homogeneous antibody populations fail to hold in the heterogeneous case. We use our results to interpret certain in vitro experiments in which synthetic bivalent haptens are used to trigger histamine release from basophils which have on their surface antibody specific for the hapten.

HISTAMINE METABOLISM IN CELLS OF THE ALLERGIC RESPONSE

We have developed sensitive and rapid assays to investigate histamine synthesis (histidine decarboxylase activity) (HD) and degradation (histaminase activity) during specific allergen triggered histamine release. HD activity was determined in isolated mast cells and mixed human leukocytes using 2-14C histidine (50uM, 0.5uc) as substrate. Synthesized histamine was separated by thin layer chromatography on cellulose sheets and quantitated in cut sections of chromatograms by liquid scintillation spectrometry. In isolated mast cells (>98%pure) HD activity was linear for at least 6 hr. with an activity of 20ng histamine synthesized /106 mast cells/hr. Histaminase activity was determined by a similar thin layer chromatographic technique. The degradative products formed from the interaction of soluble cell sonicates and 2-14C histamine (8uM, 0.1uc) were quantitated by scintillation spectrometry. Histaminase activity was undetectable in “mononuclear cell fractions” (>98% lymphocytes, monocytes, basophils) but was present in a granulocyte fractions (>98% neutrophils and eosinophils) of human leukocytes. In granulocytes, histaminase activity was associated principally with eosinophils. In eosinophil-rich fractions (95% pure), histaminase activity was linear for at least 5 hr. with an activity of 50 pmol/5×106 eosinophils/hr. These data suggest that the human eosinophil may modulate the allergic response by neutralizing histamine during allergen stimulated histamine release.