Con A-induced Histamine Release Research Articles

Intestinal Mucosa-Associated Bacteria Modulate Rat Mast Cell Reactivity

It is well known that in some conditions bacteria of physiological flora of gastrointestinal tract may become pathogenic. Each complaint which causes injury of gastrointestinal wall integrity permits bacteria to penetrate the tissues and affect the tissue cells. Since mast cells represent one of very important and numerous cellular elements of the gastrointestinal tract walls, bacteria can exert the effects on them. Therefore, the aim of our study was to examine the influence of four strains of intestinal mucosa-associated bacteria--Bacteroides thetaiotaomicron, Bacteroides fragilis, Bifidobacterium adolescentis and Escherichia coli on the mast cell reactivity. Our experiments were performed in vitro on isolated rat peritoneal mast cells and the reactivity of these cells was estimated on the basis of histamine release. We used the suspensions of whole bacteria, killed by heating at 65 degrees C. We have noticed that the magnitude of bacteria-induced histamine release from mast cells was very low (up to 6.0%) when compared with histamine release induced with Con A, compound 48/80 and TNF-alpha. However, all studied bacteria changed the reactivity of mast cells in anaphylactic (with ConA) and anaphylactoid (with compound 48/80) reactions. After 40 min preincubation with B. thetaioataomicron, B. fragilis, B. adolescentis or E. coli ConA-induced histamine release was diminished up to 25%, 71%, 58% and and 68% of maximal histamine release, respectively. Preincubation of rat mast cells with B. thetaioataomicron, B. fragilis, B. adolescentis or E. coli also changed their reactivity in anaphylactoid reaction with compound 48/80 (histamine release was diminished up to 70%, 63%, 63% and 60%, respectively).

Concanavalin A-induced activation of hamster mast cells: morphological changes and histamine secretion.

Peritoneal mast cells of Syrian hamsters release histamine to the action of concanavalin A (Con A) in dose-dependent fashion. The rate of release was very rapid in the first seconds of cell activation and completed in 60 s after the challenge. Morphological changes concomitant to the lectin treatment, followed by electron microscopy, show that early signs of exocytosis are seen after 10 s. The process starts in peripherally located granules which swell, have a decreased density and form pores by fusion of the cellular membrane and the perigranular membranes. Then it spreads toward the cell interior by fusion of granules and forming intracytoplasmic cavities. Some extruded granules are also observed. Preincubation of lectin with rat IgE or with rat serum induced an inhibition of its histamine releasing action. Immunization increased the Con A-induced histamine release in young but not in older hamsters. An IgE-mediated mechanism is suggested for the parallel ultrastructural changes and histamine release effects induced by Con A on the hamster mast cell.

The influence of H1-, H2- and H3-receptors on the spontaneous and ConA induced histamine release from human adenoidal mast cells.

The effects of the H3-agonist R-alpha-methylhistamine (R-alpha-MeHA) and the H3-antagonist thioperamide on the spontaneous and concanavalin A (ConA) induced histamine release from human mast cells were tested and compared with the effect of some H1- and H2-receptor active substances. R-alpha-MeHA (10(-9)-10(-7) M) exerted no effect on histamine release whereas thioperamide increased the spontaneous release at 10(-6)-10(-4) M but inhibited the ConA induced release in a narrow concentration range (10(-6)-10(-5) M). This enhancement might be taken as an indication of the existence of H3-receptor dependent autoregulation although presently other mechanism cannot be excluded.

Cutaneous and basophilic sensitivity to substance P and gastrin in non-atopic versus atopic subjects

We compared the cutaneous reaction to intradermal injection of substance P, gastrin and histamine in asymptomatic atopic subjects with a history of hay fever and/or asthma versus non-atopic healthy volunteers. We also studied in these two groups the basophilic histamine release induced by substance P and gastrin with that obtained with anti-human IgE and Con A. Intradermal injection of substance P (3-300 pM) and gastrin (3-30 pM) caused a wheal and flare reaction which was comparable in both groups of subjects. Substance P 10(-4)M caused a mean basophilic histamine release of about 15% in atopic and non-atopic subjects. Gastrin was not effective in this model. Anti-IgE and Con A-induced histamine release was significantly higher in atopic than in non-atopic volunteers.

Effects of calmodulin antagonists on human adenoidal mast cells

N-(6-aminohexyl)-1-naphthalenesulfonamide (W5), N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) and Triflupromazine (TFPr) are substances with calmodulin antagonistic properties. All of these compounds inhibited the Con A-induced histamine release from human adenoidal mast cells. Maximal inhibition for W5 was observed at 200 microM, for W7 at 50 microM and for TFPr at 20 microM. Higher concentrations of each substance induced a marked histamine release. The kinetics of the mediator release were found to be different for Con A and W7 only after 5 mins incubation. The kinetics of TFPr were found to be biphasic: a slow onset was followed by a fast release reaction.

The effects of metoclopramide and cloxacepride on human mast cells from adenoidal tissues

Cloxacepride is an amide of the dopamine antagonist metoclopramide and has been reported to possess oral antiallergic properties in the rat PCA model. Both substances have now been tested in isolated mast cell preparations from human adenoidal tissues to determine whether any therapeutic antiallergic potential in man could be expected. Metoclopramide at concentrations 10(-5)-10(-3) M had no inhibitory effect but instead enhanced Con A-induced histamine release at concentrations greater than 10(-4) M. Cloxacepride at concentrations 10(-5)-10(-4) M significantly inhibited Con A-induced histamine release. This inhibitory effect was not diminished by increasing the preincubation time for up to 30 min. In contrast, cloxacepride concentrations greater than 4 x 10(-5) M caused a substantial histamine release. This effect could not be alleviated by an increase in the number of mast cells per sample. These results then suggest a very narrow range of therapeutic potential for cloxacepride.

Modulation of histamine release from canine fundic mucosal mast cells

To study the control of histamine release, we developed techniques for culturing fundic mucosal mast cells. After enzyme dispersion, enrichment by elutriation, and overnight suspension culture, mast cells accounted for 30% of the cells present. Histamine release into the medium, measured by radioenzymatic assay, was stimulated by the lectin concanavalin A (Con A). Ragweed antigen released histamine in antisera-sensitized cultures. Con A-induced histamine release was enhanced by adenosine, but adenosine alone was inactive. The relative potency of adenosine analogues was consistent with interaction at an adenosine A1-receptor site. The calcium ionophore A23187 (0.1-1 microM) also induced histamine release. Phorbol esters that activate protein kinase C, such as phorbol 12-myristate 13-acetate, did not release histamine but enhanced release when added to low concentrations of A23187. In contrast, inactive phorbols, such as 4 alpha-phorbol 12,13-didecanoate, failed to enhance A23187-induced release. Parallel studies with canine hepatic mast cells yielded comparable results. We conclude that canine fundic mast cells possess receptors for immunoglobulin E and adenosine. Our data are consistent with increases in cytosolic calcium and protein kinase C activation working synergistically to stimulate fundic mast cells.

The role of a Ca2+/calmodulin dependent plasma membrane Ca2+ channel during Concanavalin A activation of MC9 mast cells

The effects of Con A on free cytoplasmic calcium concentrations in the cloned murine mast cell, MC9, have been measured using the fluorescent calcium indicator quin 2. Con A causes a rapid, small yet sustained rise in free cytosolic calcium (up to 245 nM) followed closely by increased 45calcium uptake and more slowly by histamine release. The increases in 45calcium uptake and histamine release require extracellular calcium. However, the Ca2+ influx blockers, nifedipine and verapamil inhibit these responses only at concentrations significantly higher than those used in smooth muscle to oppose potential-dependent events, and diltiazem is inactive. These observations suggest that, in these mast cells, other types of channels control Ca2+ entry. In contrast, the intracellular Ca2+ blocker, TMB-8, inhibits both the Con A-induced histamine release and the Ca2+ changes. The calmodulin antagonists calmidazolium, trifluoperazine and W-7 are also highly effective inhibitors of both the Ca2+ changes and histamine release in direct proportion to their potency against calmodulin-dependent phosphodiesterase, implicating calmodulin in the regulation of stimulus-secretion in MC9 cells. These data imply that histamine release follows increases in intracellular Ca2+ concentration. Free intracellular Ca2+ results from rapid release from internal stores and is followed by a slower but more sustained influx of extracellular Ca2+.

Study of the inhibition of histamine release by inosine pranobex

The effects of Inosine pranobex and its parent compounds inosine and dimethylamino-2-propanol-p-acetamidobenzoic acid (DipPacBa) on the Concanavalin A (ConA)-induced histamine release from human adenoidal mast cells were investigated. Inosine pranobex inhibited the ConA-induced histamine release at concentrations greater than 10(-3) M. Inosine itself (10(-5)-10(-2) M) enhanced the ConA-induced histamine release whereas DipPacBa inhibited the release significantly at concentrations between 10(-6) and 10(-2) M. These results are consistent with the assumption that the effect of Inosine pranobex is due to the DipPacBa moiety.

The effects of prostaglandins of the E and I type on histamine release from human adenoidal mast cells

(PgE1 (10(-8)-10(-6) M) inhibited dose dependently the Con A-induced histamine release from human adenoidal mast cells. Out of two stable PgI analogues tested, EL 784 had a slight inhibitory effect at 10(-10) M, and SE 63 at 10(-4) M. PgI2 itself was ineffective.

The effects of adenosine and of some adenosine analogues on the concanavalin A- or acetylcholine-induced histamine release from human adenoidal mast cells

Histamine release induced by concanavalin A (Con A) or acetylcholine is enhanced by adenosine or the adenosine analogues PIA and NECA. The enhancement is not affected by preincubation with adenosine deaminase. The degree of the Con A-induced histamine release decreases with increasing incubation time. Under these conditions, the enhancing effect of adenosine on histamine release is either abolished or even reversed to inhibition.

Anti-IgE-and ConA-induced histamine release from human basophilic leukocytes: the existence of differences in relative responses within individuals.

Histamine release was induced from human blood leukocytes with Concanavalin A (ConA) and two different preparations of anti-IgE. A dose-response curve was constructed for each cell population and secretagogue. No qualitative difference in response pattern was observed with the two anti-IgEs; however, the relative release-inducing efficacy of ConA and anti-IgE seemed to vary with the individual providing the cells. These findings indicate that release induced by ConA is not completely equivalent to that induced by anti-IgE.

Recognition of the 'calcium paradox' and the effects of verapamil and gallopamil in human adenoidal mast cells.

Human mast cells from adenoids show when resuspended in medium containing 10(-3) M CaCl2 after their temporary exposure to Ca2+-free saline for about 20 min an irreversible reduction of responsiveness to a variety of stimuli: The histamine release induced by concanavalin A or ionophore A 23187 is only 30-50% of the one obtained in cells which were kept in 10(-3) M Ca2+ throughout the experiment. This phenomenon called 'calcium paradox' can be almost entirely avoided if the cells are temporarily exposed to 10(-4) M Ca2+ instead of Ca2+-free saline. Number yields, average histamine contents of mast cells and the rate of the spontaneous histamine release are not affected by the transitory lack of Ca2+, nor is the histamine release enhancing effect of adenosine. At 10(-3) M Ca2+ concentration the calcium antagonists verapamil or gallopamil cause a significant inhibition of the Con A-induced histamine release only at concentrations much higher (10(-4) M than those effective in smooth muscle preparations. The actions of both calcium antagonists were not affected by the presence of added extracellular adenosine.

Inhibition by azelastine of nonallergic histamine release from rat peritoneal mast cells

The ability of azelastine and selected antiallergic drugs to inhibit compound 48 80-induced and PS-potentiated, Con A-induced histamine release from RPMC was investigated. Azelastine, ketotifen, theophylline, and DSCG added simultaneously with the secretagogues or preincubated with the RPMC for 10 min before the addition of secretagogues produced concentration-dependent inhibition of histamine release. In general, the relative order of potency at calculated IC 50 level was as follows: azelastine > ketotifen > theophylline > DSCG. The preincubation of RPMC with azelastine for 10 min exerted 3.5 times greater inhibition of Con A plus PS-stimulated histamine release but did not influence the inhibitory activity on compound 48 80-induced release. The duration of preincubation did not influence the inhibitory effects of ketotifen with either secretagogue. Theophylline and DSCG exerted significantly greater inhibition when they were added simultaneously with Con A plus PS. The inhibitory activity of DSCG was also significantly improved upon simultaneous addition with compound 48 80 . These data demonstrated that azelastine is the most potent inhibitor of nonallergic histamine release from RPMC among the four antiallergic drugs examined.

In vitro inhibition of histamine release from mouse mast cells and human basophils by an anthranilic acid derivatives

Optimal conditions for in vitro histamine release mediated either by Con A (3μg/ml) or by Ionophore A23187 (1μg/ml), were established for mouse peritoneal mast cells and human normal basophils. In both systems N-5′ exerts potent inhibiting effects on histamine release after short term (1 – 5 minutes) in vitro preincubation. At concentrations of 1mM for mouse mast cells and 3mM for normal human basophils, N-5′ inhibits up to 95% Con A-induced histamine release and more than 50% ionophore-induced histamine release. Such in vitro effects are more potent than DSCG-mediated inhibition, under similar experimental conditions, and are resistant to challenge with exceeding doses of the two releasing agents, particularly in the Con A system. Interestingly, basophils with apparent normal morphology, from a CML patient, were resistant to both challenges.

Anti-IgE and Con A-induced histamine release from mast cells of four rat strains: correlation with total serum IgE.

Anti-IgE- and Con A-induced histamine release from serosal mast cells were compared to each other and to total serum levels of IgE in non-immunized, alum-injected, and Silica gel-injected rats of the BN, Fischer, PVG, and SD strains. The results indicate that the degree of anti-IgE- and Con A-induced release is strain-dependent and varies with immunization conditions. Furthermore, there is a gross but not complete correlation between the degree of serosal mast cell histamine release induced by the two secretagogues. However, Con A- or anti-IgE-induced release could significantly be correlated to serum levels of total IgE only in the Fischer strain but not in the BN or the PVG strains. In the SD strain, Con A-induced release correlated to serum IgE levels in Silica gel-injected but not in alum-injected animals.

Concanavalin A-Induced Histamine Release from Normal Rat Mast Cells

Concanavalin A- (con A) induced release of histamine from normal rat mast cells was studied. In the presence of phosphatidylserine (PS) con A induced a concentration and temperature-dependent, noncytotoxic histamine release at con A concentrations ranging from 0.1 to 100 mug/ml. The optimal con A concentration, 100 mug/ml, caused a 27.3% (+/- 2.7 S.E.M.) net histamine release. Release began approximately 30 sec after addition of con A and was complete within 45 min. In the absence of PS, no net con A-induced release occurred. The effect of PS was concentration dependent from 1 to 100 mgg/ml. PS alone, however, did not cause histamine release. Binding studies indicated that mast cells bound up to 16 X 10(6) con A molecules per cell without histamine release. Upon removal of unbound con A and the addition of PS, normal histamine release occurred. Alpha-Methyl-D-mannose (50 mM) prevented both con A binding and histamine release and if added after Con A, caused a rapid cessation of histamine release and a reversal of con A binding. This study indicates several important advantages of the con A-induced histamine release system. Binding of con A to mast cells can be dissociated from histamine release by omitting PS from the medium. Release can then be induced by the addition of PS. Alpha-Methyl-D-mannose can be used to terminate rapidly the ongoing release reaction at any phase of the interaction. This system is potentially very useful for investigation of metabolic events during histamine release.

Mechanism of Action of Concanvalin A on Human Basophils

Abstract Concanavalin A (Con A) activates human basophils to release histamine. This reaction has great similarities to that of antigen-or anti-IgE-induced release. It is characterized by a two stage reaction: the initial stage is the binding of Con A to the cell and the second stage is a secretory process of the cell. Cells desensitized by incubation with excess amounts of Con A or by prolonged incubation in the absence of cations cannot be activated to release with antigen or anti-IgE. When the native (tetravalent) Con A molecule is modified by succinic anhydride to become divalent it is much less active in releasing histamine. There is wide variation in the response of cells from different individuals to Con A. However, a close correlation exists between the histamine release induced by Con A and anti-IgE. Therefore, in general, cells from allergic donors release more histamine with Con A than those from non-allergic individuals. Evidence is presented to demonstrate that Con A acts by binding to carbohydrate groups on the IgE molecule located on the basophil membrane. On a molar basis, IgE is 4000-fold more active than α-methyl-D-mannoside in inhibiting the Con A-induced histamine release. Succinyl Con A blocks the ability of Con A and anti-IgE equally well and to a lesser extent the ability of antigen to activate cells to release histamine.

Involvement of IgE in con A-induced histamine release from human basophils in vitro

CONCANAVALIN A (con A) induces the release of histamine from rat1 and hamster mast cells2 and human basophils. Time course, cationic requirements, enzymatic sequences and dose-response seem to be the same for con A-induced release as for antigen-induced release, which is mediated by reaginic antibody (reagin), believed to be bound by its Fc portion to a receptor in the membrane of the histamine-releasing cell. Human reagin is an antibody of the IgE class3. Antibodies4 specific for IgE can induce histamine release whereas the papain digest5 of those antibodies (which produces monomer anti-IgE) fails to elicit a response and can inhibit the intact anti-IgE-induced release6. The requirement for bridging to initiate histamine release has been established7–13. The types of sugar moieties to which con A binds have been studied14–17, and the physicochemical properties of con A15,18–22 indicate that at physiological pH it can bridge two or more of those sugar moieties. The question is raised as to whether con A is bridging sugar moieties at the receptor for reagin, or whether it is bridging sugar moieties which are directly attached to the reagin. The nature of the receptor remains to be established and the observation that con A can induce histamine release raised expectations that this interaction could elucidate some of the properties of this receptor.