Functional and biochemical characterization of rat bone marrow derived mast cells

The functional and biochemical characterization of rat bone marrow derived mast cells (RBMMC) confirms both species-related differences between rat and mouse bone marrow-derived mast cells (MBMMC) as well as mast cell heterogeneity in a single species. Such RBMMC have the staining characteristics of mucosal mast cells and contain the mucosal mast cell protease. The RBMMC release the preformed granule mediator beta-hexosaminidase both in response to immunologic stimulation with 200 ng Ag (net release 15.8 +/- 3.8%) and in response to 1 microM calcium ionophore A23187 (net release 21.8 +/- 6.8%). However, compound 48/80, substance P, and somatostatin did not induce mast cell degranulation. In experiments with optimal beta-hexosaminidase release, the RBMMC generated similar quantities of the newly formed arachidonic acid metabolites leukotriene C4 and PGD2 when stimulated with either Ag or calcium ionophore A23187. The RBMMC incorporate [35S]sulfate into proteoglycans consisting of 90% chondroitin sulfates and 10% heparin. The chondroitin sulfates were comprised of chondroitin 4 sulfate and chondroitin sulfate diB sulfated disaccharides in a ratio of 4/1. Although we show that RBMMC and MBMMC share a low histamine content, functional IgE receptors and unresponsiveness to cromolyn and selective secretagogues (compound 48/80, substance P, and somatostatin), we also provide evidence that RBMMC differ from MBMMC in their profile of newly generated mediators, preformed granule proteoglycan, and lack of proliferative response to mouse IL-3.     

Ionophore A-23187 induced histamine release from rat mast cells and rat basophil leukemia (RBL-1) cells.

Ionophore A-23187 releases histamine from normal mast cells apparently by promoting Ca++ influx (Foreman et al, Nature 245: 249, 1973). In our hands at concentrations of greater than 0.2 mug/ml release occurs in 1 to 2 min, is blocked by metabolic inhibitors, and is unaccompanied by cytotoxicity (trypan-blue uptake, lactic dehydrogenase (LDH) release). At higher doses (0.5 mug/ml) histamine release is followed by significant cytotoxicity, but again Ca++ is required. In parallel studies, we examined cultured rat basophilic leukemia (RBL-1) cells. These cells, which apparently have normal surface receptors for IgE, contained approximately 700 ng histamine/10(6) cells but did not release histamine when IgE-mediated release was looked for. They do not respond to doses of ionophore which would be expected to give non-cytotoxic histamine release. At higher doses histamine release is preceded by progressive LDH release: LDH release is 75% complete at 5 min whereas 10 min are required for 75% maximal histamine release. This reaction requires Ca++ and is temperature dependent but is not inhibited by metabolic poisons (2-deoxyglucose, dinitrophenol, CN-). These studies suggest that either Ca++ does not enter into these cells normally or that one or more mechanisms which are ordinarily triggered by the changes in Ca++ flow are unresponsive in the RBL-1 cells. These studies also underline the importance of ruling out cytotoxicity in ionophore-induced phenomena.     

Transforming growth factor-beta 1 selectively inhibits IL-3-dependent mast cell proliferation without affecting mast cell function or differentiation

Transforming growth factor-beta 1 (TGF-beta 1) is an important regulator of cell growth, differentiation, and function. We show that TGF-beta 1 selectively inhibits IL-3-dependent mouse bone marrow derived mast cell (MBMMC) proliferation without affecting MBMMC function or differentiation. TGF-beta 1 significantly decreased [3H]thymidine uptake by IL-3-dependent MBMMC in a dose-dependent manner with 50% inhibition of proliferation occurring with a TGF-beta 1 concentration of 0.1 ng/ml. A brief (i.e., 30 min) incubation of MBMMC with TGF-beta 1 is sufficient to inhibit IL-3-induced proliferation of MBMMC (cultured in the absence of TGF-beta 1) for 24 to 48 h. The inhibitory effect of TGF-beta 1 on the IL-3-dependent proliferation of MBMMC is not cytotoxic as evident from the absence of MBMMC trypan blue staining, the retained functional characteristics of the MBMMC cultured in TGF-beta 1, and the reversibility of the TGF-beta 1 induced inhibition of IL-3 dependent MBMMC proliferation. MBMMC grown in TGF-beta 1 acutely (24 to 48 h) or chronically (7 to 14 days) do not exhibit functional differences in performed or newly generated mediator secretion (Ag/IgE or calcium ionophore A23187 induced MBMMC beta-hexosaminidase or leukotriene C4 release) from MBMMC grown in the absence of TGF-beta 1. In addition, MBMMC cultured for 2 wk in TGF-beta 1 do not show evidence of differentiation as assessed by cellular histamine content or Alcian blue/safranin staining. Thus, TGF-beta 1 is an important negative regulator of IL-3-dependent mast cell proliferation in vitro, selectively inhibiting IL-3-dependent MBMMC proliferation without affecting MBMMC function or differentiation.     

Biochemical analysis of glucocorticoid-induced inhibition of IgE-mediated histamine release from mouse mast cells

Pretreatment of mouse mast cells with 10(-7) to 10(-6) M dexamethasone (DM) during overnight sensitization with mouse IgE antibody resulted in inhibition of antigen-induced histamine release and degranulation. The inhibition of both degranulation and histamine release increased linearly with the duration of the treatment; maximal inhibition was obtained after approximately 16 hr with DM. The addition of DM to sensitized mast cells immediately before antigen challenge did not affect the antigen-induced histamine release. DM interacted directly with mast cells by binding to DM-specific cytoplasmic receptors. The treatment of mast cells with DM did not affect the binding of IgE to mast cells or intracellular cAMP levels. Bridging of cell-bound IgE anti-DNP antibody on mouse mast cells either by multivalent DNP-HSA or by anti-IgE induced phospholipid methylation at the plasma membrane and Ca++ influx into the cells. Pretreatment of mast cells with DM inhibited the antigen-induced phospholipid methylation and Ca++ uptake but failed to affect histamine release by Ca++ ionophore A23187. The results suggest that DM treatment inhibits histamine release by the inhibition of the early stage of biochemical processes leading to opening Ca++ channels but does not affect the process distal to Ca++ influx or the binding of IgE molecules to IgE receptors.     

Strain differences in histamine release from mouse peritoneal mast cells induced by compound 48/80 or A23187

Strain differences among BALB/c, BDF1, CDF1, C3 H/He, C57 BL/6, DBA/2, ddy and ICR mice were investigated with respect to the ratios of histamine release from mouse peritoneal mast cells induced by compound 48/80, a Ca2+ dependent histamine releaser, and the Ca2+ ionophore A23187. The ratios of histamine release from mouse peritoneal mast cells induced by compound 48/80 were found to be high in BALB/c, ddY and ICR mice, but low in BDF1, CDF1, C3 H/He, C57 BL/6 and DBA/2 mice. Those induced by Ca2+ ionophore A23187 were high in BALB/c, BDF1, CDF1, C3 H/He, DBA2, ddy and ICR mice but low in C57 BL/6 mice. These results indicate that differences in histamine release from mouse peritoneal mast cells are strain dependent.     

Modulatory effect of HCO3- on rat mast cell exocytosis: cross-talks between bicarbonate and calcium.

HCO-3 modulation of histamine release and its relationship with the Ca2+ signal were studied in serosal rat mast cells. Histamine release was induced by Ca2+ mobilizing stimuli, namely compound 48/80, thapsigargin, Ca2+ chelators, ionophore A23187, and PMA and ionophore A23187 in a HCO-3-buffered medium or a HCO-3-free medium. The presence of HCO-3 reduced histamine release by 48/80, Ca2+ chelators, A23187, and PMA/A23187, but increased histamine release induced by thapsigargin. Histamine release by PMA was significantly higher in a HCO-3-free medium than in a HCO-3-free medium, as it was the PMA potentiation of histamine release by A23187. [Ca2+]i changes induced by these drugs were measured in fura-2-loaded mast cells. In thapsigargin and EGTA or BAPTA preincubated mast cells [Ca2+]i increase was higher in a HCO-3-buffered medium than in a HCO-3-free medium in the presence of Ca2+. On the contrary, in compound 48/80 and PMA/A23187 activated mast cells the [Ca2+]i increase is the same both in the presence and in the absence of HCO-3. The effect of HCO-3 on histamine release in serosal rat mast cells depends on the stimulus, but it is not related to the presence of Cl-. In thapsigargin-stimulated mast cells the effect of HCO-3 on histamine release may be related to the Ca2+ signal, but in compound 48/80, EGTA, and PMA/A23187-activated mast cells there is no relationship between intracellular Ca2+ and the inhibitory effect of HCO-3 on histamine release. Additionally, the PKC pathway is implicated in the inhibitory effect of HCO-3 on histamine release, the higher the chelation of calcium rendering the higher the enhancement of the response after adding calcium in the absence of HCO-3.     

Regulation of adhesion of mouse bone marrow-derived mast cells to laminin

We have reported that mast cells adhere to laminin after activation with PMA. In this study, we demonstrate that the cross-linking of cell surface high-affinity IgE-R on mast cells derived from mouse bone marrow cultured for 3 wk in the presence of WEHI-3-conditioned media acts as a highly sensitive physiologic stimulus for this attachment and that receptor activation is also induced by calcium ionophore A23187. Adherence occurred at threefold log concentrations less of A23187 and Ag than required for histamine release in a selective subpopulation comprising 20 to 30% of the total cells. At higher concentrations of agonist that permitted histamine release, the time course for degranulation was shown to be more rapid than that of adherence. Adherence was inhibited by antibodies to laminin and laminin receptor and was calcium ion and temperature dependent. Treatment of cells with dibutyryl cAMP, which activates protein kinase A, inhibited both adherence and histamine release induced by Ag or calcium ionophore. Treatment of cells with staurosporin, which inhibits protein kinase C, also inhibited adherence and histamine release induced by calcium ionophore, but was not significantly active against either adherence or histamine release induced by Ag. It thus appears that agents which modulate intracellular signaling mechanisms are equally effective toward histamine release and adherence, suggesting these two events are intimately linked in stimulus secretion coupling. Specific cytokines stimulating mast cell adhesion to laminin could not be found; however, culture of mast cells with TGF-beta 1 was determined to enhance IgE-mediated adherence to laminin. Hence, the high-affinity IgE-R on the mast cell functions not only in exocytosis but also facilitates the process of mast cell adherence to laminin.     

Calmodulin antagonism: a pharmacological approach for the inhibition of mediator release from mast cells

Several Ca2+ antagonists with either Ca2+-entry blocking or calmodulin (CaM) antagonistic properties and antiallergic drugs were investigated for their effects on mediator release from mast cells induced by different secretagogues (compound 48/80, concanavalin A, antigen-IgE and Ca2+ ionophore A23187) and for their ability to inhibit the function of CaM or phospholipid/Ca2+-dependent protein kinase (C-kinase). The effects of the different agents--with the only exception of cromolyn sodium--on histamine release elicited by compound 48/80 correlated well with their actions on two CaM-dependent enzymes whereas the activity of C-kinase was far less altered, or not altered at all. CaM antagonism of cloxacepride, picumast, oxatomide, fendiline and bepridil correlated not only with the inhibition of exocytosis evoked by compound 48/80 but also with that induced by A23187, concanavalin A and antigen-IgE. This indicates an action of these substances distal to the generation of the Ca2+ signal since the various secretagogues elevate the intracellular Ca2+ concentration by different mechanisms. However, prenylamine and thioridazine inhibited concanavalin A- and antigen-IgE-induced mediator release more potently and more effectively than that elicited by compound 48/80 or A23187. Therefore inhibition of allergic histamine release by these drugs may in part be dependent on an impairment of the Ca2+ signal. Since for each of two agents inhibition of histamine release (evoked by different releasers) parallels that of serotonin release it may be concluded that these mediators are secreted via the same mechanism. The results obtained with agents exhibiting different pharmacological properties but which share one common property, namely antagonism of CaM, strengthen the view that CaM is involved in exocytosis of mediators from mast cells.     

The role of mast cells in atrial natriuretic peptide-induced cutaneous inflammation

Atrial natriuretic peptide (ANP) is widely distributed throughout the heart, skin, gastrointestinal and genital tracts, and nervous and immune systems. ANP acts to mediate vasodilation and induces mast cell activation in both human and rats in vitro. However, the mechanisms of ANP-induced mast cell activation, the extent to which ANP can induce tissue swelling, mast cell degranulation, and granulocyte infiltration in mouse skin are not fully understood. This issue was investigated by treatment with ANP in rat peritoneal mast cells (RPMCs) and mouse peritoneal mast cells (MPMCs) in vitro and by injection of ANP into the skin of congenic normal WBB6F1/J-Kit+/Kit+ +/+, genetically mast cell-deficient WBB6F1/J-Kit(W)/Kit(W-v) (W/W(v)) and mast cell-engrafted W/W(v) (BMCMC→W/W(v)) mice in vivo. ANP induced the release of histamine and TNF-α from RPMCs and enhanced serotonin release from MPMCs, in a dose-dependent fashion, as well as reduced cAMP level of RPMCs in vitro. In +/+ mice, ANP induced significant tissue swelling, mast cell degranulation, and granulocyte infiltration in a dose-dependent manner, whereas not in genetically mast cell-deficient W/W(v) mice. However, ANP-induced cutaneous inflammation has been restored in BMCMC→W/W(v) mice. These data indicate that mast cells play a key role in the ANP-induced cutaneous inflammation.     

Human skin mast cells: their dispersion, purification, and secretory characterization

Digestion of human foreskin with collagenase and hyaluronidase disperses approximately 3.4 X 10(7) nucleated cells per gram of tissue, of which mast cells constitute 4.7%. These may be purified to 80% by use of density gradient centrifugation. The majority of mast cells (79%) measured between 9 and 13 micron in diameter, and the mean histamine content was 4.6 pg/cell. Viability was demonstrated by trypan blue exclusion by 93% of the cells and the low spontaneous histamine secretion of less than 7% in functional studies. Anti-IgE released up to 17.5% of cell-associated histamine within 5 to 7 min. Calcium ionophore-induced release was optimal with 0.3 microM A23187 when 28.6% histamine was released. Unlike human lung mast cells, skin mast cells released histamine in response to compound 48/80 and poly-L-lysine. This release, which was complete within 20 sec, was totally dependent on intact glycolysis and oxidative phosphorylation and partially dependent on extracellular calcium. The same characteristics were observed with secretion induced by substance P and morphine. The weak activity of eledoisin and physalaemin suggests that the substance P receptor, like that of the rat mast cell, is not of the classical types described for smooth muscle. Morphine-induced secretion was partially blocked by naloxone in a manner not compatible with competitive antagonism at a classical opioid receptor. The sensitivity of skin mast cells to nonimmunologic stimulation clearly distinguishes them from mast cells of the lung and lymphoid tissues and provides evidence of functional heterogeneity within human mast cells.     

Ca2+ and protein kinase C signaling for histamine and sulfidoleukotrienes released from human cultured mast cells

Human cultured mast cells (HCMC) release histamine and sulfidoleukotrienes (LTs) upon IgE-FcepsilonRI-mediated mast cell activation. We analyzed the Ca2+ and PKC signaling in HCMC and compared it to that in rodent mast cells. In HCMC, after IgE-mediated stimulation, an elevation of [Ca2+]i and PKC translocation to the membrane fraction was observed. As concerns Ca2+ signaling, 1) IgE-mediated histamine and LTs release was abolished after Ca2+ depletion, and the reconstitution of Ca2+ recovered the release of histamine and LTs. As regards PKC signaling, 1) staurosporine inhibited IgE-mediated mediator release. 2) PKC-downregulated mast cells did not release histamine and LTs. A23187 and PMA synergistically potentiated the activation of extracellular-regulated kinase and synergistically induced histamine and LTs release. These results demonstrated that HCMC might be useful for analysis of the signal transduction pathway for mediator release, such as histamine and LTs.     

Differential calcium effects on prostaglandin D2 generation and histamine release from isolated rat peritoneal mast cells

We examined the role of Ca2+ mobilization in prostaglandin (PG) D2 generation and histamine release induced by A23187 from rat peritoneal mast cells. Both PGD2 generation and histamine release accompanied with 45Ca uptake were observed above 0.1 microM A23187. Although an increase of PGD2 generation was not exactly correlated with that of Ca2+ uptake, histamine release occurred in proportion to Ca2+ uptake. In contrast to PGD2 generation, below 0.1 microM A23187, about 20% of the total histamine was released without Ca2+ uptake and this response was inhibited by 10 microM 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), which is an intracellular Ca2+ antagonist. However, TMB-8 had no effect on PGD2 generation. These results suggest that Ca2+ dependency of histamine release is clearly different from that of PGD2 generation, and that histamine release is induced by not only Ca2+ uptake but also intracellular Ca2+ mobilization.     

Mast cells and pulmonary fibrosis. Identification of a histamine releasing factor in bronchoalveolar lavage fluid.

As elevated bronchoalveolar lavage (BAL) fluid histamine levels are noted in patients with pulmonary fibrosis (PF), we assayed BAL fluid from 16 patients with PF for the presence of a histamine releasing factor (HRF). HRF activity was assayed by measuring release of the preformed mast cell-derived mediators, histamine, or beta-hexosaminidase (beta-hex) from a purified population of IL-3 dependent mouse bone marrow derived mast cells (MBMMC) or human blood basophils. Mean BAL cell free histamine levels in the patients with PF was 1226 +/- 1349 pg/ml, whereas BAL histamine levels in a comparison group of six non-PF patients was 118 +/- 60 pg/ml. HRF was significantly elevated in BAL fluid of patients with PF (mean beta-hex release 24.5 +/- 12.9%; range 6.8 to 52.4%) compared to the non-PF group of patients (mean beta-hex release 7.9 +/- 7.7%; range 1.8 to 20.7%). The PF HRF not only degranulated MBMMC, but also induced the generation of the arachidonic acid metabolite leukotriene C4 from MBMMC (24.6 +/- 4.2 ng leukotriene C4/10(6) MBMMC). The PF HRF did not appear to be a cytokine previously identified in BAL fluid of patients with PF (i.e., platelet derived growth factor or insulin growth factor-1) or a human cytokine able to degranulate human basophils (i.e., IL-1, or granulocyte-macrophage-CSF) as these recombinant human cytokines did not induce MBMMC beta-hex release. Physicochemical characterization of the HRF revealed that it was relatively heat stable, pronase sensitive and on Sephadex G-75 and G-200 column chromatography had an apparent molecular mass of 30 to 50 kDa. The ability of PF BAL to induce beta-hex release from MBMMC was not dependent on IgE as unsensitized or lactic acid treated MBMMC release similar amounts of beta-hex compared to MBMMC sensitized with IgE. Thus, BAL fluid of patients with PF contains an HRF that induces beta-hex release from MBMMC via an IgE-independent mechanism. The presence of the HRF could explain elevated BAL histamine levels in patients with PF.     

Release of arylsulfatase A but not B from rat mast cells by noncytolytic secretory stimuli.

The net percentage of release of arylsulfatase activity from purified rat mast cells induced by rabbit anti-rat F(ab')2 was consistently only about 1/3 that of histamine. Isoelectric focusing of the released and residual arylsulfatase activities demonstrated specific release of the A type without B and a net percentage of immunologic release of arylsulfatase A equivalent to that of histamine. When the net percentage of histamine and arylsulfatase A release were nearly maximal (88 and 76%) in response to the calcium ionophore A23187, specific release of arylsulfatase B did not occur. Thus, arylsulfatase A and not B was associated with the secretory granule released from the rat mast cell by reversed anaphylaxis or the calcium ionophore. In contrast, subcellular fractionation of water-lysed mast cells yielded arylsulfatase B with the heparin- and chymase-containing granule fraction and arylsulfatase A in the aqueous fraction comprised of cell sap and granule water eluate. It may be that arylsulfatase B resides in a minor second granule, whereas arylsulfatase A is loosely associated with the predominant secretory granule of the rat mast cell.     

Characterization of the anti-inflammatory effect of FK-506 on human mast cells.

We have examined the effects of FK-506 and of the struturally related macrolide rapamycin, which bind with high affinity to a specific binding protein (FKBP), to evaluate the involvement of this protein in the release of preformed (histamine) and de novo synthesized inflammatory mediators (sulfidopeptide leukotriene C4 and prostaglandin D2) from mast cells isolated from human lung parenchyma. FK-506 (0.1 to 300 nM) concentration dependently inhibited histamine release from lung parenchymal mast cells activated by anti-IgE. FK-506 was more potent in lung mast cells than in basophils (IC50 = 1.13 +/- 0.46 nM vs 5.28 +/- 0.88 nM; p less than 0.001), whereas the maximal inhibitory effect was higher in basophils than in lung mast cells (88.4 +/- 2.5% vs 76.4 +/- 3.8%; p less than 0.01). FK-506 had little or no inhibitory effect on histamine release from lung mast cells challenged with compound A23187, whereas it completely suppressed A23187-induced histamine release from basophils. FK-506 also inhibited the de novo synthesis of 5-lipoxygenase (sulfidopeptide leukotriene C4) and cyclo-oxygenase (prostaglandin D2) metabolites of arachidonic acid from mast cells challenged with anti-IgE. Unlike in basophils, Il-3 (3 to 30 ng/ml) did not modify anti-IgE- or A23187-induced histamine release from lung mast cells nor did it reverse the inhibitory effect of FK-506. Rapamycin (3 to 300 nM) had little or no effect on the release of histamine from lung mast cells, but it was a competitive antagonist of the inhibitory effect of FK-506 on anti-IgE-induced histamine release from human mast cells with a dissociation constant of about 12 nM. These data indicate that FK-506 is a potent anti-inflammatory agent that acts on human lung mast cells presumably by binding to a receptor site (i.e., FKBP).     

Evidence for de novo synthesis of phosphatidylinositol coupled with histamine release in activated rat mast cells

Phospholipid metabolisms in rat mast cells activated by ionophore A23187 and compound 48/80 were examined with reference to 'phosphatidylinositol (PI) cycle'. The addition of A23187 to [3H]glycerol-prelabeled mast cells induced a marked accumulation of the radioactivity in 1,2-diacylglycerol(DG) and phosphatidic acid(PA) within 10 to 30 sec. A great enhancement of [3H]glycerol incorporation into PA and PI was also detected during histamine release. On the other hand, 48/80 was far less effective than A23187 both in producing 1,2- DG and PA and in accerelating [3H]glycerol incorporation into PA and PI, despite the comparable ability of histamine release. The activity of Ca2+ uptake into mast cells, as measured by pulse-labeling with 45Ca2+, was increased when exposed to both of two agents. These data provide circumstantial evidence that phospholipid metabolisms, mainly de novo PI synthesis, may be a part of the triggering events for Ca2+ mobilization and secretory process. The PI metabolism induced by two different stimulants appears to behave in a different manner.     

Evidence of an adriamycin binding site in the secretory granules of the mast cell

Adriamycin induced significant non-cytotoxic histamine release from rat peritoneal mast cells to which the drug showed a very high affinity. The relationship between adriamycin-induced exocytosis and its uptake by purified rat peritoneal mast cells was studied. Adriamycin induced histamine release and was highly concentrated in mast cells at 37 degrees C but not at 0 degrees C. However, if exocytosis was provoked by other secretagogues like compound 48/80, protamine, concanavalin A, and ionophore A23187, and cells were then treated with adriamycin at 0 degrees C, the concentrations of the antineoplastic drug significantly increased. Adriamycin binding to purified granular material was similar to that of intact cells treated at 37 degrees C, but was not modified by metabolic inhibitors, extremes of temperature (0 or 45 degrees C) or by the carboxylic ionophore monensin. On the contrary, sodium cromoglycate limited adriamycin binding to granular materials as well. In addition, sodium cromoglycate, but not monensin, displaced the antineoplastic drug from mast cells, even when added after adriamycin. We conclude that the high affinity of adriamycin for mast cells is ascribable to the externalization of a granular binding site, as a consequence of the exocytotic process. The experiments with sodium cromoglycate suggest that this binding site could be in common with the antiallergic drug.     

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.     

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.     

Biochemical analysis of desensitization of mouse mast cells

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