Magainin-2 releases histamine from rat mast cells.

The magainins are basic 23 amino acid peptides with a broad spectrum of antimicrobial activity. Their bactericidal effect has been attributed to their capacity to interact with lipid bilayer membranes. We observed histamine release by magainin-2 amide from rat peritoneal mast cells (ED50 = 13 micrograms/ml) but not from human basophils. This histamine-releasing reaction from peritoneal mast cells was due to a secretory rather than cytolytic effect, i.e., release occurred without concomitant liberation of lactic dehydrogenase. Furthermore, the pretreatment of mast cells with magainin-2 amide did not desensitize cells against subsequent challenge with other secretagogues. Maximum histamine release occurred in less than a minute at 25 and 37 degrees C. The addition of Ca2+ was not required for histamine release, although release was enhanced by the addition of 0.3-1 mM Ca2+. The addition of 3 mM Ca2+ or Mg2+ was markedly inhibitory. The presence of Na+ or Cl- ions in the medium was not required for release. Therefore, histamine release is not due to the formation of anion-selective channels in the membrane of mast cells. The results indicated that the characteristics of histamine secretion induced by magainin-2 amide were unlike IgE-mediated release but were similar to the mechanism of release attributed to some other basic peptides and to compound 48/80.     

Effect of colchicine on rat mast cells

In the mast cell, a well-developed array of microtubules is centered around the centrioles. Complete loss of microtubules is observed when mast cells are treated with 10(-5) M colchicine for 4 h at 37 degrees C. The loss of ultrastructurally evident microtubules is associated with a marked change in the shape of mast cells from spheroids to highly irregular, frequently elongated forms with eccentric nuclei. In colchicine-treated cells the association of nucleus, Golgi apparatus, and centrioles is also lost. Mast cells exposed to 10(-5) M colchicine for 4 h at 37 degrees C retain 80% of their capacity to release histamine when stimulated by polymyxin B. Exocytosis is evident in stimulated cells pretreated with colchicine and lacking identifiable microtubules. When the conditions of exposure of mast cells to colchicine are varied with respect to the concentration of colchicine, the length of exposure, and the temperature of exposure, dissociation between deformation of cell shape and inhibition of histamine secretion is observed. These observations indicate that microtubules are not essential for mast cell histamine release and bring into question the assumption that the inhibitory effect of colchicine on mast cell secretion depends on interference with microtubule integrity.     

Ouabain-induced enhancement of rat mast cells response. Modulation by protein phosphorylation and intracellular pH

The digitalic glicoside ouabain induces potentiation of rat mast cell histamine release in response to several stimuli, which is mediated by Na+/Ca2+ exchanger. In this work, we studied the effect of ouabain on cytosolic calcium, intracellular pH and histamine release with Ca2+ ionophore A23187 in conditions designed to maximize ouabain-induced potentiation of rat mast cells response. The effect of protein kinase C (PKC), cAMP and phosphatase inhibition was also tested. Ouabain induced an enhancement in histamine release, cytosolic calcium and intracellular pH. The adenylate cyclase activator forskolin reduced the effect of ouabain on histamine release and intracellular pH, but enhanced the effect on cytosolic calcium. PKC activator PMA enhanced the effect of ouabain on histamine release and cytosolic calcium, without affecting intracellular pH. A PKC inhibitor, GF-109203X, reduced ouabain-induced enhancement of histamine release and intracellular pH, but increased the enhancement on cytosolic calcium. Finally, inhibition of protein phosphatases 1 and 2A with okadaic acid, increased the effect of ouabain on histamine release and intracellular pH, but reduced cytosolic calcium in presence of ouabain. This result suggest that ouabain-induced potentiation of rat mast cell histamine release with A23187 is modulated by kinases, and this modulation may be carried out by changes in intracellular alkalinization. However, the mechanism underlying cellular alkalinization remains to be elucidated.     

IgE-mediated release of histamine from human cutaneous mast cells

We investigated the ability of antigen-IgE interactions to stimulate histamine release from human infant cutaneous mast cells. Skin obtained at circumcision contained numerous perivascular mast cells, as assessed by light and electron microscopy. The histamine content of this tissue averaged 17.7 ng (+/- 1.5 SEM)/mg wet weight. Challenge of 200-microns thick sections of unsensitized skin with varying concentrations of monoclonal murine antibodies to human IgE caused no net release of histamine. After skin sections were incubated in the presence of 5 micrograms/ml of human myeloma IgE (S) for 120 min at 37 degrees C, monoclonal anti-IgE challenge resulted in 40.1% (+/- 6.0 SEM) histamine release. Similar passive sensitization with 1/20 dilutions of serum from humans expressing IgE to purified Juniperus sabinoides (JS) antigen rendered the tissue responsive to specific antigen challenge. Dose-related histamine release occurred over 30 min with optimal release of 12.6% (+/- 2.4 SEM) after stimulation with 100 ng/ml of JS antigen. This reaction required sensitization with serum containing IgE to JS and was antigen-specific. Optimal reactions to antigen occurred at 3 mM added Ca++, 34 degrees C to 37 degrees C, pH 7.2. Antigen-induced release was markedly influenced by the added Ca++ concentration; no release occurred in the absence of Ca++, 54% of the optimal response was observed at 2 mM Ca++, and 28% of the optimal response occurred at 4 mM Ca++. The addition of Mg++ did not influence antigen-induced release. The results of this study provide functional evidence that 1) human infant cutaneous mast cells express Fc-epsilon receptors; 2) these receptors are largely unoccupied in vivo; and 3) stimulation of passively sensitized infant mast cells with anti-IgE or specific antigen leads to immediate histamine release. This new system should permit detailed in vitro studies of immediate hypersensitivity reactions in human skin.     

Mechanisms of mouse mast cell activation and inactivation for IgE-mediated histamine release.

The IgE-mediated histamine release from mouse mast cells requires Ca++, is optimal at 37 degrees C, and is enhanced by phosphatidylserine. The rate of release is relatively slow. The mast cells can be activated to release histamine by either anti-IgE or anti-Fab antibodies and, in the case of cells from sensitized mice, by the immunizing antigen. The incubation of mast cells with antigen in the absence of Ca++ or phosphatidylserine fails to release histamine. Such cells are desensitized to the further addition under optimal conditions of the same antigen. Desensitization is antigen specific, requires optimal levels of antigen, and occurs at both 30 degrees and 37 degrees C. In contrast, anti-IgE desensitizes all IgE-mediated histamine release reactions.     

Protein L. A bacterial Ig-binding protein that activates human basophils and mast cells.

Peptostreptococcus magnus strain 312 (10(6) to 10(8)/ml), which synthesizes a protein capable of binding to kappa L chains of human Ig (protein L), stimulated the release of histamine from human basophils in vitro. P. magnus strain 644, which does not synthesize protein L, did not induce histamine secretion. Soluble protein L (3 x 10(-2) to 3 micrograms/ml) induced histamine release from human basophils. The characteristics of the release reaction were similar to those of rabbit IgG anti-Fc fragment of human IgE (anti-IgE): it was Ca2(+)- and temperature-dependent, optimal release occurring at 37 degrees C in the presence of 1.0 mM extracellular Ca2+. There was an excellent correlation (r = 0.82; p less than 0.001) between the maximal percent histamine release induced by protein L and that induced by anti-IgE, as well as between protein L and protein A from Staphylococcus aureus (r = 0.52; p less than 0.01). Preincubation of basophils with either protein L or anti-IgE resulted in complete cross-desensitization to a subsequent challenge with the heterologous stimulus. IgE purified from myeloma patients PS and PP (lambda-chains) blocked anti-IgE-induced histamine release but failed to block the histamine releasing activity of protein L. In contrast, IgE purified from myeloma patient ADZ (kappa-chains) blocked both anti-IgE- and protein L-induced releases, whereas human polyclonal IgG selectively blocked protein L-induced secretion. Protein L acted as a complete secretagogue, i.e., it activated basophils to release sulfidopeptide leukotriene C4 as well as histamine. Protein L (10(-1) to 3 micrograms/ml) also induced the release of preformed (histamine) and de novo synthesized mediators (leukotriene C4 and/or PGD2) from mast cells isolated from lung parenchyma and skin tissues. Intradermal injections of protein L (0.01 to 10 micrograms/ml) in nonallergic subjects caused a dose-dependent wheal-and-flare reaction. Protein L activates human basophils and mast cells in vitro and in vivo presumably by interacting with kappa L chains of the IgE isotype.     

Calcium requirement for the inhibition by theophylline of histamine release from mast cells

When added to Ca2+-free Hanks' solution, Ca2+ (0.1-2.5 mM) had no significant effect on antigen-induced histamine release from rat mast cells, but Sr2+ (1.0-3.0 mM) dose-dependently increased the release. Ba2+ (1.0 and 2.0 mM) also enhanced the release. Ca2+ and Ba2+ inhibited compound 40/80-induced histamine release, in a dose-dependent manner. In ordinary Hanks' medium, theophylline and 3-isobutyl-1-methylxanthine (IBMX) dose-dependently inhibited the antigen-induced histamine release but these drugs were ineffective in Ca2+-free medium. Theophylline (1.0 mM) also inhibited compound 48/80-induced histamine release in the presence but not absence of Ca2+. There was an optimal Ca2+ concentration for the theophylline effect. Sr2+ but not Ba2+ could substitute for Ca2+ in supporting the theophylline effect. Theophylline (1.0 mM) and IBMX (1.0 mM) increased mast cell cyclic AMP levels both in the presence and absence of Ca2+. These results suggest that Ca2+ is required in the interaction of theophylline and specific sites on mast cells or in the mast cell response to theophylline which probably does not involve the cyclic AMP increase and is linked to the inhibition of histamine release.     

Mode of action of protamine sulfate on histamine secretion in the rat mast cells

Protamine sulfate, known for a long time as a histamine releaser, was labeled with a fluorescent dye (FITC). This conjugate was shown to stain selectively the mast cell fraction of rat peritoneal cells. Within a few seconds, the protamine was found inside the cells. Although the cells had lost their histamine completely, no granules were found outside the cells. In the electron microscope, the protamine treated mast cells showed a loss of the electron density of their granules, a vacuolization, and other signs of histamine release. Evidence for a direct connection between the vacuoles and the extracellular fluid was gained by incubating mast cells in FITC-labeled human serum albumin followed by the addition of unlabeled protamine. After washing, the fluorescence was found to be located inside the cells, demonstrating an influx of the FITC-HSA under the influence of protamine. The protamine-induced release reaction is increased after addition of Ca2+, reduced by lowering the temperature, addition of 2-deoxyglucose, or cytochalasin B. Disodium cromoglycate also diminished the histamine release in a dose dependent manner. Protamine did not induce a loss of lactate dehydrogenase from the mast cells. The release reaction is mediated by the cell membrane, as shown by the releasing activity of insolubilized protamine. We conclude that the protamine-induced release is a non-cytotoxic reaction, fulfilling some criteria of the anaphylactic histamine release.     

Enhancement of IGE-induced mediator release from rat peritoneal mast cells by serum-treated zymosan

Phagocytosis of zymosan (Z) treated with rat serum (ZX) by rat peritoneal mast cells caused only a small amount of [3H]serotonin release, and prior release of mediators from mast cells did not affect phagocytosis of sheep erythrocytes bearing IgG and C3b, indicating the independence of these two phenomena. When, however, mast cells were exposed to ZX, subsequent IgE-mediated release of histamine, [3H]serotonin, and beta-hexosaminidase was greatly enhanced. Prevention of complement activation by the presence of EDTA during the treatment of Z with the serum or prior heating of the serum at 56 degrees C for 30 min only slightly impaired the ability of ZX to augment mediator release, whereas prior absorption of the serum with zymosan at 0 degree C greatly diminished the enhancement. Exposure of fresh Z to variable amounts of either the acid or the high-salt eluate of ZX also generated ZX capable of enhancing [3H]serotonin release in a dose-dependent fashion. IgG, IgM, C3, and albumin were detected in the eluates by immunodiffusion. When IgG was depleted from the high-salt eluate by treating with Sepharose-anti-IgG, the enhancement was significantly reduced, indicating that IgG but not C3 or other immunoglobulins was required for the enhancement.     

Mechanism of histamine release by formyl methionine-containing peptides.

Small, acylated, methionine-containing peptides release histamine from human basophils. The characteristics of this reaction were compared to that of C5a- and IgE-induced release. fMet peptide-induced release requires Ca++ and is inhibited by EDTA in a manner similar to IgE- and C5a-mediated reactions. The fMet-Phe-Met-initiated reaction is complete within 2 min at temperatures of 25, 30, and 37 degrees C; but does not occur at 0 degrees C. There was a large variation in the capacity of leukocytes from different donors to release histamine with fMet peptides. However, there was no correlation in the capacity of leukocytes to release histamine with fMet-Phe-Met and their release with C5a or anti-IgE. The release by fMet-Phe-Met (but not by C5a or anti-IgE) was reversibly inhibited by a nonreleasing tripeptide. Leukocytes could be desensitized to the action of active fMet-peptide by preincubation with the peptide in the absence of cations. After washing, these cells released normally with C5a or anti-IgE. Conversely, cells desensitized to the action of C5a- or IgE-mediated reactions released normally with fMet peptides. There was cross-desensitization between different active peptides, and inactive peptides could not desensitize the leukocytes. Pharmacologic agents had similar effects on C5a and fMet peptide-induced release (e.g., lack of enhancement with deuterium oxide; enhancement with cytochalasin B; and inhibition with aminophylline and dibutyryl cyclic AMP). Therefore, histamine release with fMet peptides is initiated by their binding to and activation of a specific receptor on the basophil; the reaction beyond that point is similar to the C5a-mediated reaction.     

The involvement of protein kinase C in exocytosis in mast cells.

Diacylglycerol generated from inositolphospholipid hydrolysis and tumor-promoting phorbol esters stimulate protein kinase C. The synthetic diacylglycerol 1-oleoyl-2-acetyl-rac-glycerol and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) have been used in pure rat peritoneal mast cells. Both caused histamine release associated with exocytosis. The release by the stimulation of protein kinase C alone in the absence of secretagogues was slow although up to 50% of the histamine content was released by TPA in 120 min. Remarkable potentiation of histamine release was observed when the mast cells were preincubated with TPA before exposure to the calcium ionophore A23187. The potentiation of histamine release corresponded with an intensification of exocytosis. The potentiation is consistent with a participation of protein kinase C in the secretory process. An inhibitory effect due to protein kinase C activity was also demonstrated using TPA and mast cells from sensitized rats. When sensitized mast cells preincubated with 50 nM TPA for 5 min were exposed to the antigen, the histamine release was substantially reduced compared to the sum of the release by the antigen and TPA or by the antigen alone. There was a corresponding decrease in exocytosis. The inhibition of exocytosis and histamine release seems to reflect a regulatory function of protein kinase C for the termination of the response, as demonstrated in other types of cells apparently acting through an inhibition of inositolphospholipid hydrolysis.     

Adherence of rat basophilic leukemia (RBL-2H3) cells to fibronectin-coated surfaces enhances secretion.

Rat basophilic leukemia (RBL-2H3) cells are a useful in vitro model for studies of mast cells and basophils. We examined the adherence of RBL-2H3 cells to different extracellular matrix proteins and the effect of such attachment on secretion. The cells bound to fibronectin-coated surfaces with maximum binding by 1 h at 37 degrees C. There was less attachment to laminin, collagen type I, and collagen type IV. There was no adherence to uncoated surfaces or in the absence of Ca2+. Binding to fibronectin was blocked by a synthetic peptide containing the sequence Arg-Gly-Asp. Therefore, the binding of RBL-2H3 cells to fibronectin may be mediated by surface molecules that belong to the integrin family. Adherence to fibronectin-coated surfaces resulted in cell spreading, a reorganization of the cytoskeletal elements, and a redistribution of the secretory granules. Attachment to fibronectin also dramatically enhanced high affinity IgE receptor-mediated histamine release. This enhancement was maximum by 1 h of adherence and lasted for at least 6 h. There was also enhanced secretion by the Ca2+ ionophore A23187. Thus, adherence to fibronectin can enhance both receptor and non-receptor-mediated release. Addition of soluble fibronectin to RBL-2H3 cells in suspension had no effect on secretion. Therefore, enhanced histamine release required cell attachment to immobilized fibronectin. These results suggest that secretion from mast cells/basophils may be modulated by their interaction with the extracellular matrix.     

High rate of IgE-mediated histamine release from rat mesenteric mast cells.

IgE-dependent histamine release from rat mesenteric mast cells was investigated. Excised mesenterium was cut into pieces and incubated with IgE overnight at 4 degrees C for sensitization. Over 10 pieces of mesenterium specimen could be prepared from a rat. Antigen-induced histamine release from mesenterium specimen was initiated rapidly and reached a plateau in 5 min. In an optimal condition, over 50% of total histamine was released. In contrast, unpurified and purified peritoneal mast cells released only 22.5% and 5.3% of total histamine, respectively, upon IgE stimulation. Tranilast, a mast cell stabilizer, inhibited the histamine release from mesenteric mast cells significantly. The mesenterium might be useful material for studying tissue-associated mast cell activation.     

Mouse mast cell activation and desensitization for immune aggregate-induced histamine release

Immune aggregate-induced histamine release and desensitization were studied in mouse mast cells. Maximal histamine release was rapid, occurred at 37 degrees C, and required the addition of alpha-L-phosphatidyl-L-serine and Ca2+. The amount of histamine released varied with the composition of the immune aggregates and was dependent on the antibody concentration. Saturation of mast cell Fc epsilon receptors with rat or mouse IgE had no effect on subsequent immune aggregate-induced release. The incubation of mouse mast cells with immune aggregates in the absence of cations of alpha-L-phosphatidyl-L-serine did not stimulate the release of histamine but resulted in desensitization of the cells for release with the addition of the same or unrelated immune aggregates. Such cells are capable, however, of IgE-mediated histamine release. Mast cells desensitized for IgE-mediated histamine release by incubation with anti-IgE were capable of immune aggregate-induced release. These data suggest that IgE-mediated and immune aggregate-induced triggering of mouse mast cells occurs through separate receptors.     

Effect of nonhydrolyzable guanosine phosphate on IgE-mediated activation of phospholipase C and histamine release from rodent mast cells

Rat mast cells and bone marrow-derived mouse mast cells (BMMC) were sensitized with mouse IgE mAb, and permeabilized by ATP to introduce guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) and/or guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) into the cells. After ATP-induced lesions were resealed with Mg2+, the cells were challenged by Ag to determine the effect of the nonhydrolyzable guanosine phosphate on Ag-induced hydrolysis of phosphoinositides and histamine release. Introduction of GTP gamma S into permeabilized rat mast cells or BMMC, followed by exposure of the cells to extracellular Ca2+, resulted in histamine release, but failed to induce hydrolysis of phosphoinositides. It was also found that introduction of GTP gamma S into the cells did not synergistically enhance Ag-induced histamine release. Introduction of GDP beta S into sensitized BMMC inhibited the GTP gamma S-dependent, Ca2+-induced histamine release but failed to inhibit Ag-induced histamine release. The results suggest that GTP gamma S-dependent, Ca2+-induced histamine release and Ag-induced histamine release go through independent biochemical pathways. It was also found that introduction of GTP gamma S or GDP beta S into sensitized BMMC neither enhanced nor inhibited Ag-induced formation of inositol phosphates. These results together with previous findings that pretreatment of BMMC with either pertussis toxin or cholera toxin does not affect Ag-induced hydrolysis of phosphoinositides, indicate that a G protein is not involved in the transduction of IgE-mediated triggering signals to phospholipase C in rodent mast cells.     

Temperature dependence of benzyl alcohol- and 8-anilinonaphthalene-1-sulfonate-induced aggregation of recombinant human interleukin-1 receptor antagonist

The critical role played by temperature in ligand-induced protein aggregation was investigated. Recombinant human interleukin-1 receptor antagonist (rhIL-1ra) and the ligands benzyl alcohol and 8-anilinonaphthalene-1-sulfonate (ANS) were used. We investigated aggregation kinetics and the conformation and cysteine reactivity of rhIL-1ra in buffer alone or in the presence of 0.9% (w/v) benzyl alcohol or 4.2 or 21 mM ANS at 25 and 37 degrees C. In buffer, protein aggregation was not detected at 25 degrees C but occurred at 37 degrees C. At 25 degrees C, neither benzyl alcohol nor 4.2 mM ANS enhanced aggregation. However, at 37 degrees C, both compounds greatly accelerated protein aggregation. With 21 mM ANS, rhIL-1ra aggregation was accelerated at both temperatures, but the effect was more pronounced at 37 degrees C than at 25 degrees C. Increasing the temperature from 25 to 37 degrees C caused a minor perturbation in the tertiary structure of rhIL-1ra in buffer but no detectable alteration in secondary structure. Benzyl alcohol enhanced the tertiary structural perturbation at 37 degrees C, but the secondary structure was not affected by the ligand. The reactivity of buried free cysteines of rhIL-1ra was enhanced by benzyl alcohol at 37 degrees C but not at 25 degrees C, consistent with the structural results. Isothermal titration calorimetry documented that the interaction of benzyl alcohol with rhIL-1ra was hydrophobic and that the degree of hydrophobic interactions increased with temperature. At 25 degrees C, the interaction of ANS with rhIL-1ra was electrostatic, but at 37 degrees C, both electrostatic and hydrophobic interactions were important. Taken together, our results support the conclusion that benzyl alcohol and ANS interact hydrophobically with partially unfolded aggregation-prone protein molecules, resulting in temperature-dependent increases in their levels and acceleration of protein aggregation.     

Inhibitory effect of diethylstilbestrol on histamine release by rat mast cells and its relation to the cellular ATP content

The effect of diethylstilbestrol, a synthetic estrogen, on mast cell secretion was investigated. The results showed that 50 microM diethylstilbestrol inhibited histamine release from rat peritoneal mast cells in the presence and absence of glucose, but did not affect 45Ca uptake stimulated by concanavalin A. Diethylstilbestrol also inhibited histamine release induced by compound 48/80, exogenous ATP, or ionophore A23187. Since estradiol benzoate, hexestrol and daidzein were not inhibitory, the inhibitory action of diethylstilbestrol must be independent of its estrogenic activity. The ATP content of mast cells decreased to less than 0.1 nmol/10(6) cells on treatment with 50 microM diethylstilbestrol at 37 degrees C for 15 min. This effect of diethylstilbestrol in decreasing the ATP content of mast cells correlated well with its inhibitory effect on histamine release. Diethylstilbestrol at 50 microM depleted the cells of ATP at 37 degrees C, but not at 0 degrees C, whereas [3H]diethylstilbestrol ( [monoethyl-3H]diethylstilbestrol) binding to rat mast cells was the same at 0 and 37 degrees C. It is concluded that diethylstilbestrol reduced the ATP content of rat mast cells by inhibiting metabolism of the cells, and consequently inhibited degranulation.     

Perturbation of protein tertiary structure in frozen solutions revealed by 1-anilino-8-naphthalene sulfonate fluorescence

Although freeze-induced perturbations of the protein native fold are common, the underlying mechanism is poorly understood owing to the difficulty of monitoring their structure in ice. In this report we propose that binding of the fluorescence probe 1-anilino-8-naphthalene sulfonate (ANS) to proteins in ice can provide a useful monitor of ice-induced strains on the native fold. Experiments conducted with copper-free azurin from Pseudomonas aeruginosa, as a model protein system, demonstrate that in frozen solutions the fluorescence of ANS is enhanced several fold and becomes blue shifted relative free ANS. From the enhancement factor it is estimated that, at -13 degrees C, on average at least 1.6 ANS molecules become immobilized within hydrophobic sites of apo-azurin, sites that are destroyed when the structure is largely unfolded by guanidinium hydrochloride. The extent of ANS binding is influenced by temperature of ice as well as by conditions that affect the stability of the globular structure. Lowering the temperature from -4 degrees C to -18 degrees C leads to an apparent increase in the number of binding sites, an indication that low temperature and /or a reduced amount of liquid water augment the strain on the protein tertiary structure. It is significant that ANS binding is practically abolished when the native fold is stabilized upon formation of the Cd(2+) complex or on addition of glycerol to the solution but is further enhanced in the presence of NaSCN, a known destabilizing agent. The results of the present study suggest that the ANS binding method may find practical utility in testing the effectiveness of various additives employed in protein formulations as well as to devise safer freeze-drying protocols of pharmaceutical proteins.     

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.     

Concerted stimulation of PI-turnover, Ca2+-influx and histamine release in antigen-activated rat mast cells

Phospholipid metabolism in rat mast cells activated by antigen was examined with reference to phosphatidylinositol (PI) turnover. Upon antigen stimulation, histamine release from passively sensitized mast cells with IgE was potentiated by adding phosphatidylserine (PS). The addition of antigen to [3H]glycerol-prelabeled and sensitized mast cells induced a marked loss of radioactivity of PI and a concurrent accumulation of 1,2-diacylglycerol (DG) and phosphatidic acid (PA) within 5 to 60 sec. Furthermore, this antigen-induced PI breakdown was enhanced in the presence of Mg2+. Histamine release occurred in parallel with PI breakdown. On the other hand, the transient Ca2+ influx into mast cells, as measured by uptake of 45Ca2+, was found to occur quickly after cells were activated by antigen, which was concerted with PI breakdown. These results suggest that enhanced PI turnover may be an important step in the biochemical sequence of events leading to release of histamine, and that not only Ca2+ but also Mg2+ appears to take a part in stimulus-response coupling in rat mast cells.