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.     

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.     

Recombinant IL-3 induces histamine release from human basophils

Human rIL-3 induces histamine release from some human basophils, with cells from atopics responding to a greater extent than non-atopic donors. The dose response curves were highly variable. IL-3 was active on purified basophils and the release process was slower and required more calcium than anti-IgE. Removal of surface IgE from basophils rendered them unresponsive to IL-3. The response could be restored by passive sensitization of basophils with IgE+, IgE known to bind histamine-releasing factors, and not IgE-, IgE unreactive with histamine-releasing factors. Thus, IL-3 uncovers IgE heterogeneity. IL-3 does not, however, directly interact with IgE+. Rather, passive sensitization with IgE+ or stimulation of basophils with low concentrations of several secretagogues renders the cells sensitive to IL-3. IL-3 may well play a pro-inflammatory role by potentiating the effects of IgE+ or various secretagogues.     

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.     

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.     

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.     

Measurement of IgE on human basophils: relation to serum IgE and anti-IgE-induced histamine release.

The number of IgE molecules bound to human basophils was calculated from direct measurements of the IgE dissociated after exposing leukocytes to pH 3.7 acetate buffer in the cold. In 18 donors studied, cell-bound IgE ranged from 4000 to 500,000 molecules/basophil and correlated with the serum IgE concentration (r = 0.89, p less than 0.001) which ranged from 5 to 3,000 ng/ml. Sensitivity of these cells to anti-IgE was tested to explore the relationship between cell-bound IgE and the concentration of anti-IgE required for histamine release. Cells from some nonatopic donors (4000 to 100,000 IgE molecules/basophil) were as sensitive as cells from allergic donors (100,00 to 500,000 IgE molecules/basophil). Moreover, cells from donors having approximately the same cell-bound IgE concentration varied widely in their sensitivity to anti-IgE. We conclude that an intrinsic property of human basophils ("releasability") is an important parameter in determing mediator release.     

H2 antihistamines augment antigen-induced histamine release from human basophils in vitro

H2 antihistamines, including cimetidine, burimamide, metiamide, and tiotidine, consistently augmented antigen-induced histamine release from human basophils in vitro when control histamine release was less than 20% of total. This effect was specific to the H2-receptor blocking activity of these drugs: equivalent degrees of receptor blockade by four different H2 antihistamines resulted in equipotent enhancement; H1-receptor antagonists did not alter histamine release; and aminoguanidine and amodiaquine, agents that inhibit histamine metabolism but do not block H2 receptors, did not enhance histamine release. Cimetidine did not enhance release when present a) when basophils were "activated" but did not release histamine ("first stage"), or b) when basophils were no longer susceptible to histamine inhibition ("second stage"). Thus, H2 antagonists enhanced histamine release by blocking the capacity of released histamine to act on H2 receptors to inhibit release. Because it is likely that only small percentages of histamine are released in vivo, it is possible that H2 antihistamines amplify the inflammatory process by blocking the inhibitory effects of the released histamine.     

Adenosine receptor on human basophils: modulation of histamine release.

Adenosine, at physiologic concentrations, inhibits in vitro IgE-mediated human basophil histamine release in a dose-dependent fashion. The inhibition dose-response curve is paralleled by an adenosine-induced increase in cAMP levels of human leukocyte preparations. Further evidence that the adenosine effect is related to changes in cAMP levels is that the nucleoside inhibits only in the first stage of antigen-induced histamine release and fails to inhibit the release caused by ionophore A23187. A poorly metabolized derivative of adenosine, 2-chloroadenosine inhibits as effectively as adenosine; dipyridamole, which blocks adenosine uptake, does not impair the inhibition caused by adenosine. Finally, theophylline, which is a competitive antagonist of adenosine in human lymphocytes also blocks the inhibition of release caused by adenosine. These data suggest that adenosine acts via a specific cell-surface receptor linked to adenylate cyclase. It appears that the human basophil has a specific receptor for adenosine and that this nucleoside may modulate the in vivo release of the mediators of immediate hypersensitivity reactions.     

Complement-mediated release of histamine from human basophils. II. Biochemical characterization of the reaction.

Release of histamine from human basophils was induced by activation of complement using zymosan. The histamine-releasing factor resembled C5a on the basis of m.w. (15,000) as well as previous studies showing inactivation by anti-C5. Complement-induced release of histamine was compared with allergic release of histamine which is mediated through appropriate allergens and reaginic IgE. Previously we demonstrated that the former reaction occurred more quickly. Both reactions were inhibited by drugs which increase intracellular concentrations of cAMP3 (theophylline, prostaglandin E1, and histamine) or which mimic the action of cAMP (its dibutyrly derivative). Calcium was required for complement-mediated release of histamine and an increasing response was observed up to physiologic concentrations (2 mM). Magnesium (0 to 1 mM) did not affect the amount of histamine released. Also, glycolysis was probably required for optimal release by complement, since both 2-deoxyglucose and iodoacetamide were inhibitory. When basophils were partly enriched by depletion of neutrophils and eosinophils, the percentage of histamine released by complement was unchanged. Finally, it was shown that activated complement desensitized basophils from responding to a second challenge by the same stimulus. Cross-desensitization was not observed between complement and pollen allergens.     

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.     

Interleukin 8-inhibitor and inducer of histamine and leukotriene release in human basophils.

We have shown previously that interleukin 8 (IL-8) induces histamine and leukotriene release in human basophils exposed to interleukin 3 (IL-3). We now found that pretreatment with low concentrations of IL-8 selectively inhibits this response. Inhibition was significant at 0.01 nM IL-8 and virtually complete at 1 nM, which is about 100-fold lower than the concentration required for induction of mediator release. IL-8 dependent responses were also inhibited, albeit to a lesser extent, by preincubation with neutrophil-activating peptide 2 (NAP-2), but not with connective tissue-activating peptide III (CTAP-III) or platelet factor 4 (PF4). Release induced by C5a, fMet-Leu-Phe, or anti-IgE antibody, by contrast, was not affected.     

Low molecular weight thymic factor inhibits histamine release from basophils

Low molecular weight thymic factor stimulates the suppressor function of T-lymphocytes, increases cAMP content (but not cGMP) in lymphocytes and inhibits histamine release from sensitized basophils. The mechanisms of LTF action are discussed.     

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.     

Very low density lipoproteins (VLDL) trigger the release of histamine from human basophils

Circulating basophils are well established sources of the granule-associated mediator, histamine. The physiological control, however, of histamine release from human basophils is poorly understood. Because histamine may play a role in the transendothelial transport of various compounds, including very low density lipoprotein (VLDL) and its hydrolysis products, we investigated the possibility that VLDL regulates mediator release from basophils. The incubation of VLDL (at physiological concentrations) with basophils (isolated as mixed leukocyte preparations) resulted in a significant release of histamine. Histamine release was dependent on VLDL concentration (half-maximal stimulation occurring at VLDL-protein concentration of 15-20 micrograms/ml), length of incubation (half-maximal release at 5-12 min), temperature (37 degrees C optimum) and required calcium (concentration 0.5-2.0 mM). Furthermore, VLDL-induced histamine release was inhibited by three different mediator-release inhibitors: dimaprit, dibutyryl cAMP and nordihydroguaiaretic acid. Incubation of basophils with LDL or HDL under the same experimental conditions did not result in significant histamine release from basophils. The histamine-secretory response of basophils obtained from different donors varied considerably. Basophils isolated from 28 donors and challenged with 100 micrograms/ml VLDL released 23 +/- 5% of their cellular histamine (mean +/- S.E.; with a range of 0-94%). Desensitization of VLDL-induced histamine release could be accomplished by preincubation of basophils with either VLDL or anti-IgE but not with N-formyl-L-methionyl-L-leucyl-L-phenylalanine. Through the secretion of histamine, a potent vasoactive mediator (and also possibly through granule-associated glycosaminoglycans, stimulants of the enzyme lipoprotein lipase), this novel effect of VLDL may be part of a physiological loop for the regulation of VLDL hydrolysis and lipid transport. This effect of VLDL may also have deleterious consequences, because of the atherogenic properties of histamine.     

Usefulness of testing histamine release from isolated human basophils in anti-allergic and anti-asthmatic drug assessment]

Histamine release from isolated human basophils test was used to evaluate an activity of: histamine receptors H1 and H2 blockers, agonists of beta-receptors, calcium channel blocking agents, hydrocortisone, and disodium cromoglycate (Intal). The study involved 84 patients hospitalized for the bronchial asthma. Basophils were isolated with Day's technique modified by Shov and Norn. Histamine was measured with Shov's spectrofluorimetric technique. It was found that histamine release from isolated human basophils may be used in both evaluation of the mechanism of action and efficiency of drugs used in allergic diseases therapy.