In vitro release of histamine from murine mast cells by block co-polymers composed of polyoxyethylene and polyoxypropylene

A series of block co-polymers composed of polyoxyethylene and polyoxypropylene were investigated for their ability to induce in vitro activation of mouse mast cells. We found that six of these co-polymers could cause histamine release from mouse mast cells in vitro. At low concentrations, the most efficacious co-polymer, T130R2, caused rapid and extensive concentration-dependent release of histamine from mouse mast cells. The release process was not cytotoxic; it required metabolic energy and was not accompanied by release of lactate dehydrogenase. Optimal release of histamine was dependent on both calcium and sodium ions in the extracellular medium. The degree of in vitro histamine release correlated with in vivo inflammation and in vitro ionophore activity. We believe that this represents the first report of the activation of mediator-containing cells by an ionophore selective for monovalent cations. These copolymers may therefore represent new reagents for investigations of cellular excitation.     

Histamine release from cultured human basophils: lack of histamine resynthesis after antigenic release.

Human peripheral basophils can be maintained in cultured for up to 72 hr. These cells retain their functional integrity as judged by total histamine content and by their ability to release histamine by an IgE-mediated reaction in response to a specific antigen challenge. Cells cultured after suboptimal antigenic challenge could be activated to release histamine upon the addition of antigen. In contrast, cells culture after supra-optimal antigenic challenge did not fully recover their ability to release histamine even after 24 hr. With a variety of culture conditions, it was impossible to demonstrate any net synthesis of histamine in cells. Cells cultured after depletion of their stores by reaction with antigen did not show any net histamine formation. The experiments suggest that the basophil in peripheral blood is functionally an end-stage cell and can participate in the histamine release reaction only once.     

Induced loss of Syk in human basophils by non-IgE-dependent stimuli

In the general population, Syk expression in human basophils is highly variable and correlates well with the IgE-mediated responsiveness of these cells. Previous studies established that IgE-mediated stimulation results in loss of Syk expression. The current studies investigated whether stimulation through other receptors results in loss of Syk. Two classes of stimulation were examined, those that operate through the kinase Syk and those that operate through a GTP-binding protein. These studies demonstrated that aggregation of leukocyte Ig-like receptor LILRA-2 resulted in phosphorylation of Syk and c-Cbl, was inhibited by a third generation Syk inhibitor with an expected IC(50), and induced histamine release in strict proportion to release induced by anti-IgE Ab. Stimulation of LILRA-2 for 18 h resulted in modest loss of Syk that correlated with the more profound loss of Syk induced by anti-IgE Ab. Human recombinant histamine-releasing factor has also recently been shown to induce Syk phosphorylation and in the current studies has also been shown to induce loss of Syk in 18-h cultures. fMLP stimulation for 18 h was also found to induce modest loss of Syk. fMLP induced phosphorylation of c-Cbl that was sustained for at least 45 min. Phosphorylation of c-Cbl was inhibited by a Syk kinase inhibitor but with an IC(50) that was not consistent with Syk activity, suggesting another kinase was responsible for Cbl phosphorylation following fMLP. These studies demonstrate that it is possible to induce the loss of Syk expression in human basophils by a non-IgE-dependent mechanism and even by a mechanism that does directly involve Syk in the reaction complex.     

Qualitative characteristics of histamine release from human basophils by covalently cross-linked IgE

We have studied the effects of permanent oligomers of human IgE produced using the cross-linking reagent, dimethyl suberimidate, on histamine release from human basophils. IgE dimers were found to be sufficient stimuli for both release and desensitization of these cells; monomeric IgE had no effect. Histamine release was augmented by deuterium oxide (D2O) in the medium, but D2O was not an absolute requirement to observe release. Desensitization by the dimeric IgE was specific in that the response to anti-IgE was not affected by preincubation of the leukocytes with the IgE dimer under suboptimal releasing conditions. IgE trimers and higher oligomers of IgE also caused both release and desensitization. IgE trimers were 3- to 4-fold more effective than IgE dimers with regard to the amount required for 50% histamine release. Dilution studies with monomeric IgE suggested that the difference was due to the presence of more "active" dimers in the trimeric IgE fractions. We conclude that dimeric IgE, by juxtaposing 2 receptors on the basophil membrane, is the "unit signal" for both release and desensitization of these cells.     

Cyclosporin A rapidly inhibits mediator release from human basophils presumably by interacting with cyclophilin.

We have examined the effects of cyclosporin A (CsA) and a series of CsA analogs that bind with decreasing affinity to cyclophilin, to evaluate the involvement of this protein in the release of preformed (histamine) and de novo synthesized (peptide leukotriene C4; LTC4) mediators of inflammatory reactions from human basophils. CsA (8 to 800 nM) concentration-dependently inhibited (5 to 60%) histamine release from peripheral blood basophils challenged with anti-IgE. CsA was more potent (92.6 +/- 1.8 vs 59.1 +/- 4.5%; p less than 0.001) and, at low concentrations, more effective when the channel-operated influx of Ca2+ was bypassed by the ionophore A23187 (IC40 = 24.1 +/- 3.9 vs 105.5 +/- 22.2 nM; p less than 0.05). CsA had no effect on the release of histamine caused by phorbol myristate and bryostatin 1 that activate different isoforms of protein kinase C. Inhibition of histamine release from basophils challenged with anti-IgE was not abolished by washing (three times) the cells before anti-IgE challenge. CsA also inhibited the de novo synthesis of LTC4 from basophils challenged with anti-IgE. The inhibitory effect of CsA was very rapid, and the drug, added from 1 to 10 min during the reaction, inhibited the ongoing release of histamine caused by anti-IgE and by A23187. The experiments with CsA analogs (CsG, CsC, CsD, and CsH) showed that CsH, which has an extremely low affinity for cyclophilin, has no effect on basophil mediator release. In addition, there is a significant correlation between the concentrations of CsA, G, C, and D that inhibited by 30% the histamine release induced by anti-IgE (r = 0.99; p less than 0.001) and by A23187 (r = 0.87; p less than 0.001) and their affinity for cyclophilin.     

The mechanism of mediator release from human basophils induced by platelet-activating factor.

Platelet-activating factor (PAF) is a lipid mediator able to induce a variety of inflammatory processes in human peripheral blood cells. We have investigated the effect of PAF on the release of chemical mediators from human basophils of allergic and normal donors. PAF (10 nM to 1 microM) caused a concentration-dependent, noncytotoxic histamine release (greater than or equal to 10% of total) in 27 of 44 subjects tested (24 atopic and 20 nonatopic donors). The release process was either very rapid (t1/2 approximately equal to 10 s) or quite slow (t 1/2 approximately equal to 10 min), temperature- and Ca2(+)-dependent (optimal at 37 degrees C and 5 mM Ca2+). Coincubation of PAF with cytochalasin B (5 micrograms/ml) enhanced the release of histamine induced by PAF and activated the release process in most donors (42 of 44). Atopics did not release significantly more histamine than normal subjects, and the percentage of PAF responders (greater than or equal to 10% of total) was nearly the same in the two groups. Histamine release was accompanied by the synthesis and release of leukotriene C4, although this lagged 1 to 2 min behind histamine secretion. Lyso-PAF (100 nM to 10 microM), alone or together with cytochalasin B, did not release significant amounts of histamine. The release of histamine activated by PAF was inhibited by the specific PAF receptor antagonist, L-652,731, with an IC50 of 0.4 microM. There was a partial desensitization to PAF when the cells were preincubated with PAF (100 nM to 1 microM) for 2 min in the absence of Ca2+, whereas the cells remained responsive to anti-IgE (0.1 micrograms/ml). If neutrophils were removed from the basophil preparation by a Percoll gradient or a countercurrent elutriation technique, there was a significant decrease in PAF-induced histamine release. PAF (1 microM) was able to induce a very rapid, transient rise (peak less than 10 s) in [Ca2+]i in purified basophils analyzed by digital video microscopy. Finally, among human histamine-containing cells, the basophils are unique in degranulating following a PAF challenge. Mast cells from human lung, skin, or uterus failed to respond to PAF (10 nM to 1 microM) regardless of the presence or absence of cytochalasin B (5 micrograms/ml). Our results demonstrate that PAF is able to induce the release of inflammatory mediators from human basophils, and that neutrophils can influence this response. It is suggested that PAF-induced basophil activation can play a role in the pathogenesis of allergic disorders.     

The effect of pertussis toxin on mediator release from human basophils

We have found that basophils (n = 9) treated with pertussis toxin (1.0 microgram/ml) fail to respond to a subsequent challenge with either 1.0 microM f-Met peptide (p less than 0.0005) or 0.24 microgram/ml of C5a (p less than 0.0005) although their responses to anti-IgE (0.1 microgram/ml) and A23187 (1.0 microgram/ml) were unaltered. These results were confirmed in purified (average purity = 89 +/- 3%) basophils (n = 4). Leukotriene C4 release was also reduced to 15 +/- 5% of control (p less than 0.005) when pertussis toxin-treated basophils were exposed to 1.0 microM f-Met peptide, although no inhibition was noted when anti-IgE or A23187 were used as the stimuli. The effect of pertussis toxin on basophils appears to be independent of the presence of contaminating mononuclear cells. We found that pertussis toxin inhibited f-Met peptide-induced histamine release regardless of the magnitude of the stimulus (0.01 microM to 1.0 microM f-Met peptide), although anti-IgE-induced release was unaffected over a dose-response curve. The effect of pertussis toxin was found to be both time- and concentration-dependent. The maximum effects were obtained after a 3-hr incubation with 1 microgram/ml of toxin. Lower (0.01 to 0.05 microgram/ml) concentrations of toxin or shorter (30 to 60 min) incubation periods did not significantly (p greater than 0.05) inhibit mediator release.     

Generation and release of platelet-activating factor (PAF) from enriched preparations of rabbit basophils; failure of human basophils to release PAF

Rabbit mononuclear cells containing up to 20% basophils and uncontaminated by neutrophils release PAF when stimulated with goat antiserum to rabbit IgE. The amount of PAF detected was a function of basophil concentration but decreased on a per cell basis at high basophil or high total cell concentrations. Calcium ionophore A23187, but not protein A, C5a, or FMLP, initiated rabbit basophil degranulation and PAF release. By contrast, extensive studies using a variety of human leukocyte preparations failed to demonstrate the release of significant levels of PAF from human basophils by IgE-dependent or -independent mechanisms. These results suggest that cells other than the peripheral blood basophil (e.g., the neutrophil) may act as the primary site of PAF production in man.     

Effect of synthetic cyclic nucleotides on immunologic histamine release from calf granulocytes.

Sensitized bovine granulocytes release histamine when exposed to specific antigens. A unique modulation of histamine release by adrenergic agents has been shown in the bovine; beta-adrenergic agonists enhance and alpha-adrenergic agonists inhibit histamine release. This is an opposite response to that reported in other species. The present study was undertaken to determine the possible relationship between cyclic nucleotides and adrenergic agents in this species. Dibutyryl cAMP enhanced antigen-induced histamine release over the complete concentration range tested (10(-6)--10(-3)M); it also overcame, in a dose-dependent manner, the inhibition of antigen-induced histamine release produced by 10(-4) M phenylephrine. The 8-bromo cGMP AND 0-MONOBUTYRYL CGMP had no significant effect on antigen-induced histamine release nor did 8-bromo-cGMP have any significant effect on the enhancement of histamine release produced by 10(-4) M dibutyryl cAMP. These findings suggest that only cAMP has a role in the modulation of antigen-induced histamine release from bovine granulocytes.     

Complement-induced histamine release from human basophils. II. Mechanism of the histamine release reaction.

The activation of human serum complement by incubation with zymosan generates C5a which releases histamine from autologous basophils. The characteristics of the C5a-induced histamine release were investigated. It is similar to IgE-mediated reactions in requiring Ca++ and in being inhibited by EDTA. However, it has marked differences from IgE-mediated reactions. C5a, at all concentrations, released histamine completely in less than 2 min. The C5a reaction has a narrow pH optimum that antigen-induced release and occurs well at 17 degrees to 37 degreesC but not at 0 degreesC. The optimal reaction temperature is 25 degrees to 30 degrees C. Unlike the antigen-induced release, no two-stage activation with C5a for the release of histamine could be demonstrated. There was additive release between C5a- IgE-mediated reactions. Leukocytes could be desensitized to the C5a-mediated reaction by 1) incubating the cells at 37 degrees C for 45 min, 2) pretreating the leukocytes with activated serum in the presence of EDTA, and 3) adding the activated serum to the leukocytes at 0 degrees C before transferring to the optimal reaction temperatures. Cells desensitized to the complement-induced release have normal reactions to IgE-mediated histamine release. In parallel experiments, cells from allergic donors desensitized for IgE-mediated reactions by incubation with antigen under sub-optimal conditions release histamine normally upon the addition of C5a. The results indicate that histamine release by C5a involves a mechanism of basophil activation that is different from the pathway involved in the IgE-induced reaction.     

Interferon-induced enhancement of IgE-mediated histamine release from human basophils requires RNA synthesis.

Incubation of human leukocytes with certain viruses results in the enhancement of IgE-mediated release of histamine. This enhancement is produced by interferon. The present experiments show that an induction period of 6 to 9 hr and new RNA synthesis are required for interferon to enhance histamine release. This points to the possibility that interferon may exert its antiviral and histamine-release enhancing activities by acting through a common pathway.     

Recombinant human IL-1 alpha and -1 beta potentiate IgE-mediated histamine release from human basophils

In this study, we have explored the relationship between interleukins and human basophil activation. Previous studies by ourselves and others have found that recombinant human (rh) IL-3 causes histamine release. The ability to release histamine has also been claimed for IL-1 but we cannot confirm this. In experiments with the basophils of 29 donors (excluding one D2O responder), histamine release with 100 ng/ml rhIL-1 alpha was 1.3 +/- 1% (SEM), whereas with rhIL-1 beta, it was 0.8 +/- 1%. Both IL-1 alpha and -1 beta were also used at concentrations of 0.01 to 1000 ng/ml without causing release. Neither increasing the Ca2+ concentration nor adding D2O or cytochalasin B caused IL-1 alpha and -1 beta to become secretagogues. rhIL-1, however, did augment IgE-dependent histamine release. The enhancement was similar with both rhIL-1 alpha and -1 beta, i.e. they were dose-dependent between 0.1 and 3 ng/ml and reached a plateau from 3 to 100 ng/ml. At submaximal histamine release (less than 10%), there was enhancement of three IgE-dependent secretagogues: 125% with goat anti-human IgE (n = 7), 215% with Ag E (n = 10), and 260% with a histamine releasing factor (n = 7). Non-IgE-dependent stimuli (formyl-methionine-leucine-phenylalanine and the ionophore A23187, n = 10) were enhanced less than 5%. rhIL-1-enhancement persisted after cell washing (n = 10). rhIL-1 was active in preparations of 50 to 75% pure basophils in which mononuclear cells were reduced by greater than 95% (n = 4), and mAbH34 to IL-1 beta blocked the enhancement caused by that molecule. We postulate that basophils have an IL-1 receptor which, when occupied, upregulates the response to IgE-related signals. Thus, this work characterizes a second interaction between interleukins and the cells central to the allergic response.     

Complement-induced histamine release from human basophils. I. Generation of activity in human serum.

The incubation of zymosan, endotoxin, or immune aggregates with normal human serum activates a factor which induces release of histamine from autologous basophils. The reaction can be divided into two steps: in the first, complement must be activated and in the second, the histamine-releasing factor interacts with basophils. The generation of histamine-releasing activity in serum occurs at 17 to 37 degrees C but not at 0 degrees C, is inhibited by heating the serum at 56 degrees C for 30 min, or by the addition of EDTA to the serum. Once generated, the histamine-liberating activity is stable to heating at 56 degrees C for 30 min. Gel filtration of the activated serum demonstrated that this factor eluted in the same region as a factor with chemotactic activity. Both factors have a molecular weight of about 16,000 daltons and their activities were inhibited by antibody to human C5. This is therefore a pathway for histamine release by C5a where the activation of the basophil is unrelated to the membrane bound IgE.     

Complement-induced histamine release from human basophils. III. Effect of pharmacologic agents.

Human serum activated with zymosan generates a factor (C5a) that releases histamine from autologous basophils. Previously we have presented evidence that this mechanism for C5a-induced release differs from IgE-mediated reactions. The effect of several pharmacologic agents known to alter IgE-mediated release was studied to determine whether they have a similar action on serum-induced release. Deuterium oxide (D2O), which enhances allergic release, inhibited in a concentration-dependent fashion the serum-induced reaction at incubation temperatures of 25 and 32 degrees C. The colchicine-induced inhibition was not reversed by D2O. Cytochalasin B, which gives a variable enhancement of IgE-mediated release, had a marked enhancing effect on the serum-induced reaction in all subjects tested. The following agents known to inhibit the IgE-mediated reaction also inhibited serum-induced release at 25 degrees C: colchicine, dibutyryl cyclic AMP, aminophylline, isoproterenol, cholera toxin, chlorphenesin, diethylcarbamazine, and 2-deoxy-D-glucose. These results suggest that the serum-induced release is modulated by intracellular cyclic AMP, requires energy, and is enhanced by the disruption of microfilaments. The lack of an effect by D2O would suggest that microtubular stabilization is not required. The data can be interpreted to indicate that IgE- and C5a-mediated reactions diverge at a late stage in the histamine release pathway.     

Morphologic and immunologic characterization of human basophils developed in cultures of cord blood mononuclear cells

Selective growth of human basophilic granulocytes was obtained in suspension cultures of mononuclear cells from umbilical cord blood. Approximately 50 to 80% of nonadherent cells recovered from 2- to 3-wk-old cultures contained metachromatic granules, and these cells were identified as human basophilic granulocytes by electron microscopy. Histamine content of cultured human basophils was comparable to that in peripheral blood basophils. Cultured basophils bear 2.7 to 3.7 X 10(5) IgE receptors per cell that bind both human IgE and rodent IgE with comparable affinity. Average equilibrium constants of the receptors for human IgE and mouse IgE were 2.56 +/- 0.88 X 10(9) M-1 and 1.85 +/- 0.86 X 10(9) M-1, respectively. The cell-surface component of the IgE receptors on cultured basophils has a m.w. of 64,000. Cultured basophils could be passively sensitized with human IgE and mouse IgE monoclonal antibody, and sensitized basophils released characteristic cytoplasmic granules and both histamine and arachidonate upon challenge with either anti-human IgE or antigen. Incubation of cultured basophils with ionophore A23187 or F-Met-Leu-Phe resulted in histamine release. However, compound 48/80 failed to induce histamine release from the cells.     

Characteristics of the effect of ketotifen on the selective and non-selective histamine release from human basophils

Administration of ketotifen at concentrations from 0.125 to 1.0 mM did not lead to the release of histamine from basophils of normal persons but induced a dose-dependent inhibition of histamine release provoked by IgE antibodies or Con A. The inhibitory action of ketotifen developed shortly after addition to the cells, being retained in the presence of the drug throughout the whole time of experiment (up to 40 min). Washing of the cells from ketotifen was followed by rapid recovery (at any rate within the first 5 min) of cell sensitivity to the histamine liberator. Ketotifen did not produce any influence on histamine release provoked by the cytotoxic secretion activators, chlorpromazine and mellitin.