Characterization of defective phorbol ester responses in a low secreting rat basophilic leukemia (RBL-2H3) cell variant

The role of protein kinase C (PKC) in the signaling mechanism that stimulates the release of mediators from rat mast cells, for which the RBL-2H3 cell line is a model, is at present unresolved. Current evidence suggests that PKC activation alone is an insufficient stimulus, although it can modulate mast cell exocytosis induced by other agents. In this article we characterize a variant of the RBL-2H3 cell line that has a reduced capacity for mediator secretion in response to an IgE-mediated antigen-induced stimulation. The outcome of our study suggests that at least two PKC isotypes are active in RBL-2H3 cells, and affect the positive and negative modulation of the secretory response.     

Inhibitory effects of apple polyphenol on induced histamine release from RBL-2H3 cells and rat mast cells

The anti-allergic activities of polyphenol fractions extracted from immature fruits of apple (Rosaceae, Malus sp.) were evaluated by in vitro assays. A crude apple polyphenol (CAP) fraction, which had been obtained from the juice of immature apples by reverse-phase column chromatography, was further purified by LH-20 column chromatography to obtain an apple condensed tannin (ACT) fraction consisting of linear oligomeric epicatechins from the dimer to pentadecamer. ACT strongly inhibited the release of histamine from rat basophilic leukemia (RBL-2H3) cells stimulated by the antigen-stimulation and from rat peritoneal mast cells stimulated by compound 48/80. The IC50 values for histamine release were 30 micrograms/ml and 25 micrograms/ml, respectively. ACT also inhibited hyaluronidase activity and the increase in intracellular free calcium concentration in RBL-2H3 cells stimulated with the antigen. These results suggest that ACT affected early signal transduction including the calcium influx.     

CGP 41251, a novel protein kinase inhibitor with in vitro selectivity for protein kinase C, strongly inhibits immunological activation of human skin mast cells and human basophils

The process of high-affinity IgE receptor (Fc epsilon RI)-mediated signal transduction in human basophils and mast cells is accompanied by activation of protein kinase C (PKC). The present study investigated the effects of a novel protein kinase inhibitor with in vitro selectivity for PKC (CGP 41251) in comparison with the potent but non-selective PKC inhibitor staurosporine on the activation of human peripheral basophilic leukocytes and enzymatically isolated human skin mast cells. CGP 41251 exerted strong concentration-dependent inhibitory effects on Fc epsilon RI-mediated histamine release from both cell populations. In addition, the IgE-mediated generation of arachidonic acid metabolites (leukotriene C4/D4 and prostaglandin E2) from human basophils was also significantly inhibited by this compound. Its action was not significantly different from the action of staurosporine. Direct activation of cellular PKC by the phorbol ester 12-o-tetradecanoyl-phorbol-13-acetate and subsequent histamine release from basophils was also inhibited by both compounds. CGP 41251 did not suppress N-formyl-met-leu-phe- or A23187-induced activation of basophils, whereas A23187-induced mediator release from human skin mast cells was inhibited in a concentration-dependent fashion. We conclude that an increase of in vitro selectivity for PKC does not significantly enhance inhibitory effects on immunological activation of histamine-containing cells. Moreover, nonimmunological pathways of signal transduction in basophils and mast cells appear to be mediated by distinct biochemical events.     

Heterogeneous effects of protamine on human mast cells and basophils

To investigate the mechanisms of anaphylactoid reactions to protamine, we have examined the in vitro effects of increasing concentrations of protamine (10(-6)-3 x 10(-4) mol litre-1) on the release of preformed (histamine and tryptase) and de novo synthesized (peptide leukotriene C4 (LTC4) or prostaglandin D2 (PGD2)) mediators from human basophils and mast cells isolated from lung parenchyma, heart, skin and synovial tissues. Protamine 10(-6)-3 x 10(-4) mol litre-1 induced release of histamine, but not de novo synthesis of LTC4 from basophils. At concentrations from 10(-5) to 3 x 10(-4) mol litre-1 it induced histamine release from human heart (mean 6.5 (SEM 1.5)%), skin (17.7 (4.1)%) and to a lesser extent from synovial mast cells, but not from lung mast cells. Protamine also caused the release of tryptase from heart mast cells (12.8 (3.2) micrograms/10(7) cells), but did not induce de novo synthesis of LTC4 and PGD2 from lung and skin mast cells. In these experiments cross-linking of IgE by anti-IgE caused release of LTC4 or PGD2 from human basophils or mast cells. These results demonstrate that protamine acted as an incomplete secretagogue, causing the release of preformed mediators from human basophils and mast cells.     

Effects of dental amalgam and its components of histamine release from human basophils and tissue mast cells

Recent studies have shown that metal ions can be released from dental amalgam or other dental materials, and can cause toxic effects on various cells. In this study, the effects of amalgam-conditioned culture medium (ACCM), components of amalgam (Ag+, Cu2+, Sn2+, Hg2+) and dental composite-conditioned culture medium (CCCM) on histamine release from human blood basophils (healthy subjects, n = 3) and tissue mast cells (n = 3) were analyzed. ACCM and CCCM were prepared using either fresh or 6-weeks-aged specimens. Of the metal ions tested, Ag+, and Hg2+ were found to induce histamine release from basophils (Ag+, 0.33 mM: 83 +/- 11% vs Hg2+, 0.33 mM: 100% vs control medium: 5 +/- 5%) and mast cells (Ag+, 0.33 mM: 91 +/- 16% vs Hg2+, 0.33 mM: 99 +/- 1% vs control: 2 +/- 1%), whereas no effects were seen with Cu2+ and Sn2+. Neither ACCM from freshly prepared amalgam nor ACCM from 6-weeks aged amalgam, produced histamine release in basophils or mast cells. Inductively coupled plasma atomic emission spectrometry (ICP) revealed that the Ag(+)- and Hg(2+)-concentrations in ACCM were below the range in which histamine release occurred. Similar to ACCM, no effects on basophils or mast cells were observed with CCCM. In summary, our data show that distinct metal ions present in dental amalgam, can induce (toxic) histamine liberation from basophils and mast cells. However, the amounts of metal ions released from amalgam apparently were too low, to cause histamine release.     

Effect of an active metabolite of the antiallergic agent tazanolast on histamine release from rat mast cells

WP-871 (3'-(1H-tetrazol-5-yl)oxanilic acid monohydrate, CAS 114607-46-4) is a monohydrate of a main active metabolite of tazanolast (butyl 3'-(1H-tetrazol-5-yl) oxanilate, CAS 82989-25-1), an orally active antiallergic drug. WP-871 inhibited dose-dependently compound 48/80-induced histamine release from rat peritoneal mast cells. In a similar dose range, WP-871 was effective in inhibiting compound 48/80-induced 45Ca uptake into mast cells from extracellular medium and compound 48/80-induced translocation of protein kinase C from the cytosol to the membrane fraction of mast cells. WP-871 also inhibited inositol trisphosphate production but did not exhibit a direct inhibitory effect on phospholipase C in mast cells. WP-871 caused no increase in cAMP content in mast cells. These results suggest that WP-871 may inhibit histamine release mainly by preventing the increase in intracellular Ca2+ concentration, which is a critical event in signal transduction leading to histamine release in mast cells.     

Endogenous superallergen protein Fv induces IL-4 secretion from human Fc epsilon RI+ cells through interaction with the VH3 region of IgE

We investigated the mechanism whereby protein Fv (pFv), a human sialoprotein found in normal liver and largely released in the intestinal tract in patients with viral hepatitis, induces mediator release from basophils and mast cells and evaluated whether it also induces IL-4 synthesis and secretion in basophils. pFv is a potent stimulus for histamine and IL-4 release from purified basophils. Histamine and IL-4 secretion from basophils activated by pFv was significantly correlated (rs = 0.70; p < 0.001). There was also a correlation (rs = 0.58; p < 0.01) between the maximum pFv- and anti-IgE-induced IL-4 release from basophils. The average t1/2 for pFv-induced histamine release was lower (3.5+/-1.5 min) than for IL-4 release (79.5+/-8.5 min; p < 0.01). IL-4 mRNA, constitutively present in basophils, was increased after stimulation by pFv and was inhibited by cyclosporin A and tacrolimus. Basophils from which IgE had been dissociated by brief exposure to lactic acid no longer released IL-4 in response to pFv and anti-IgE. The response to an mAb cross-linking the alpha-chain of Fc epsilon RI was unaffected by this treatment. Three human VH3+ monoclonal IgM concentration-dependently inhibited pFv-induced secretion of IL-4 and histamine from basophils and of histamine from human lung mast cells. In contrast, VH6+ monoclonal IgM did not inhibit the release of IL-4 and histamine induced by pFv. These results indicate that pFv, which acts as an endogenous superallergen, interacts with the VH3 domain of IgE to induce the synthesis and release of IL-4 from human Fc epsilon RI+ cells.     

Substitution of an aspartic acid results in constitutive activation of c-kit receptor tyrosine kinase in a rat tumor mast cell line RBL-2H3

The c-kit protooncogene encodes a receptor tyrosine kinase that mediates signals required for differentiation, proliferation and survival of mast cells. We have already shown the constitutive activation of c-kit receptor tyrosine kinase (KIT) in a human mast cell leukemia line (HMC-1) and a murine mastocytoma cell line (P-815). We here examined whether such constitutive activation of KIT occurred in the rat tumor mast cell line RBL-2H3 as well, which is frequently used as a tool for studying functions of mast cells. In RBL-2H3 cells, KIT was constitutively phosphorylated on tyrosine and activated in the absence of autocrine production of its ligand, stem cell factor (SCF). Sequencing analysis revealed that one of c-kit genes of RBL-2H3 cells had a point mutation, resulting in amino acid substitution of Tyr for Asp in codon 817. When rat wild-type c-kit cDNA and mutant-type c-kit cDNA encoding KITTyr817 were transfected into cells of a human embryonic kidney cell line (293T), only mutant form KITTyr817 was constitutively phosphorylated on tyrosine and activated in the absence of SCF. Since mutations at the same Asp codon constitutively activated KIT in all the human HMC-1, murine P-815, and rat RBL-2H3 cell lines, and since the incorporation of antisense oligonucleotides of c-kit messenger RNA significantly suppressed the proliferation of RBL-2H3 cells, the activating mutations in the Asp codon of the c-kit gene appeared to be involved in neoplastic growth of mast cells.     

Effect of contignasterol on histamine release induced by anti-immunoglobulin E from rat peritoneal mast cells

In rat peritoneal mass cells induced by anti-immunoglobulin E (anti-IgE), contignasterol (1) inhibited histamine release in a dose-dependent manner. On the other hand, a reduction product of contignasterol (2) did not inhibit histamine release from mast cells induced by anti-IgE.     

Regulation of human basophil function by phosphatase inhibitors

1. Okadaic acid, a cell permeant inhibitor of protein serine/threonine phosphatases (PPs), attenuated the IgE-mediated release of the pre-formed mediator, histamine from human basophils in a time- and dose-dependent manner. Optimal inhibition (77 +/- 4%, P < 0.0001) of histamine release was observed following a 2 h incubation with 1 microM okadaic acid. 2. Okadaic acid and two analogues of okadaic acid were also studied and were found to inhibit the IgE-dependent release of histamine. Concentrations required to inhibit release by 50% (IC50) were 0.6 microM for okadaic acid and 7.5 microM for okadaol, whereas okadaone was inactive. 3. The structurally-unrelated PP inhibitor, calyculin A, also inhibited IgE-dependent histamine release from basophils dose-dependently and was approximately six fold more potent than okadaic acid. 4. The IgE-mediated generation of sulphopeptidoleukotrienes (sLT) from basophils was inhibited by okadaic acid and related analogues with the following rank order of potency; okadaic acid (approx. IC50 0.3 microM) > okadaol (3 microM) > okadaone (inactive). 5. Okadaic acid, okadaol and okadaone (all at 3 microM) inhibited the IgE-mediated generation of the cytokine interleukin 4 (IL4) from human basophils by 67 +/- 9% (P < 0.002), 48 +/- 14% (P < 0.05) and 8 +/- 7% (P = 0.31), respectively. 6. Extracts of purified human basophils liberated 32P from radiolabelled glycogen phosphorylase and this PP activity was inhibited by 17 +/- 3% (P < 0.0005) by a low (2 nM) concentration of okadaic acid and was inhibited by 96 +/- 1% (P < 0.0001) by a higher (5 microM) concentration of okadaic acid. Because a low (2 nM) concentration of okadaic acid inhibits PP2A selectively whereas a higher (5 microM) concentration inhibits both PP1 and PP2A, these findings suggest that both PP1 and PP2A are present in basophils. 7. In total these data suggest that PPs are resident in human basophils and that PPs may be important in the regulation of basophil function.     

The identification and characterization of umbilical cord blood-derived human basophils

Cross-linking allergen-specific immunoglobin E on human peripheral blood basophils results in the release of histamine and other inflammatory mediators that initiate allergy and asthma. The signaling pathways leading from IgE binding to mediator release have not been well established, mainly due to the difficulty in obtaining adequate numbers of highly purified basophils. It was the goal of this study to easily obtain Fc epsilonRI-positive human basophils in high yield and purity for studies of signal transduction pathways. We describe an in vitro culture system in which pulsing normal human cord blood leukocytes with interleukin-3 (IL-3) for 3-4 h followed by incubation in medium with fetal bovine serum generates a cell population that is predominately Fc epsilonRI positive between 14 and 28 days of culture. These cells resemble peripheral blood basophils when examined by light and electron microscopy. Like normal blood basophils, they express the integrins, CD11b, CD18, CD29, and CD49d. A majority of the IL-3-pulsed cells also express a marker recognized by the basophil-specific antibody, 2D7. Fc epsilonRI cross-linking results in a time and dose-dependent release of histamine. Fc epsilonRI cross-linking also stimulates protein-tyrosine phosphorylation, thought to be the first event leading to the IgE-mediated activation of peripheral blood basophils. These studies establish cord blood as an accessible source from which basophil-like cells can be developed to examine Fc epsilonRI-mediated signal transduction.     

Cholera toxin increases IL-6 synthesis and decreases TNF-alpha production by rat peritoneal mast cells

Mast cells have been traditionally associated with an acute allergic response. However, their role in regulating chronic inflammatory processes must also be considered in view of evidence that mast cells synthesize and release a number of cytokines. In this study, we have examined the effect of cholera toxin (CT) on peritoneal mast cell IL-6 and TNF-alpha production. Highly purified, freshly isolated, rat peritoneal mast cells from Brown Norway rats were cultured in the presence of CT or its B subunit (CTB) alone or in combination with anti-IgE or bacterial LPS. Histamine release was measured after 10 min; IL-16 and TNF-alpha production was assessed in supernatants after 18 h. We found that CT or CTB alone did not affect histamine release; however, mast cell IL-6 production was significantly enhanced by CT but not by CTB. In contrast, constitutive production of TNF-alpha was inhibited by CT. The effects of CT were similar to our previous observations of the actions of prostaglandin E2 on mast cells. We also examined the effects of CT in combination with other mast cell activating agents. CT had no significant effect on anti-IgE-induced histamine release. An additive effect on IL-6 production was observed in the context of LPS. Forskolin, an agent known to increase intracellular cAMP levels, also induced a significant increase in IL-6 production, whereas TNF-alpha production was decreased. These data have important implications for our understanding of the regulation of mast cell cytokine production and the effects of CT on local cytokine production.     

Inhibition of rat peritoneal mast cell exocytosis by frusemide: a comparison with disodium cromoglycate

Among the loop diuretics, frusemide possesses unique airway protective activities which may be due to the inhibition of airway inflammatory cells such as the mast cell. We previously reported that frusemide and disodium cromoglycate (DSCG) demonstrated a similar profile of inhibitory activities against histamine release from rat peritoneal mast cells activated by various stimuli which increased intracellular calcium via different routes. Furthermore, the inhibitory activities of both compounds demonstrated marked tachyphylaxis and we hence postulated that frusemide and DSCG might share the same mechanism of action which involves the prevention of extracellular calcium influx into the mast cell cytoplasm. The present study confirmed the postulation by (a) demonstrating that cross-tachyphylaxis exists between the two compounds and (b) extending the observations on histamine release to the influx of extracellular calcium (45Ca) into rat peritoneal mast cells.     

The Na+/K(+)-pump in rat peritoneal mast cells: some aspects of regulation of activity and cellular function

The mast cell contains potent mediators of inflammation which are released after IgE-directed and non-IgE-directed stimulation of the cell. This highly specialized cell is therefore ascribed a role in the pathogenesis of disease states in which the inflammatory response plays a role for the development of the clinical symptoms. Thus, besides being of interest in basic research, studies of the cellular processes leading to release of inflammatory mediators from the mast cell also have important clinical implications. The aim of the present work has been to document the existence of the Na+/K(+)-pump in rat peritoneal mast cells, to investigate the regulation of the pump activity and to explore whether modulation of the pump activity interferes with the cellular stimulus/secretion coupling mechanism. The Na+/K(+)-pump activity following stimulation of the mast cell was also investigated. The pump activity was assessed as the ouabain-sensitive cellular potassium uptake with 86Rb+ as a tracer for potassium. The histamine release from the mast cell following IgE-directed and non-IgE directed stimulation of the cell was used as a parameter for cellular degranulation. Histamine was measured by spectrofluorometry. The finding of an ouabain-sensitive uptake mechanism in the mast cell documents the presence of a functional Na+/K(+)-pump in this cell. The pump activity is inhibited by lanthanides and by the divalent cations calcium, magnesium, barium and strontium. The pump has a large reserve capacity which probably is caused by a low intracellular concentration of sodium. This enables the pump to respond to changes in the intracellular sodium concentration. The inhibitory effect of di- and trivalent ions on the pump activity is probably a result of the inhibitory effect of these ions on the cellular sodium uptake. The digitalis glycosides, ouabain and digoxin, but not the more lipophilic drug digitoxigenin, increase both IgE-directed and non-IgE-directed histamine release from the mast cell in a calcium-free medium, while there is no effect of digitalis glycosides in a medium containing physiologically relevant concentrations of calcium. The effect of digitalis glycosides on the histamine release is dependent on the drug concentrations used and the time of preincubation. An increase in the intracellular concentration of sodium secondary to inhibition of the Na+/K(+)-pump is the effector mechanism likely to explain the effect of digitalis glycosides on the mast cell histamine release. Increases in intracellular sodium might affect the intracellular concentration of calcium via changes in Na+/Ca(2+)-exchange. IgE-directed and non-IgE-directed stimulation of the mast cell activates the Na+/K(+)-pump. In case of compound 48/80-induced histamine release, the pump is stimulated for at least 2 hr. It is proposed, that the poststimulatory activation of the Na+/K(+)-pump is due to increased cellular sodium uptake associated with the release process. This sodium uptake may occur via Na+/Ca(2+)-exchange, Na+/H(+)-exchange, Na+/K+/2Cl(-)-cotransport or a non-selective ion channel. Besides describing aspects of the function and regulation of the Na+/K(+)-pump in the rat peritoneal mast cells, this thesis points to the potential role of sodium transport mechanisms in mast cell physiology. Pharmacological manipulations of such transport mechanisms might in the future add to the treatment of allergic diseases.     

Complement peptide C3a inhibits IgE-mediated triggering of rat mucosal mast cells

The relationship between mast cells' secretory response to stimulation via their type 1 Fc epsilon receptors (Fc epsilon RI) and that provided by the C3a fragment of the complement system was investigated in the rat mucosal-type mast cell line RBL-2H3. These cells are known to be unresponsive to the so-called 'peptidergic' stimulus provided by cationic agents, such as anaphylatoxins, neuropeptides or polyamines. We now observed that C3a effectively inhibits the Fc epsilon RI clustering induced secretion of RBL-2H3 cells. This inhibition is dose-dependent and takes place at a C3a concentration range of 0.4-12.5 nM, i.e. at least three orders of magnitude lower than those where this anaphylatoxin exerts its secretory stimulus to 'serosal' mast cells. In order to identify where C3a interferes in the Fc epsilon RI coupling cascade, we have studied its effect on the cells' protein phosphorylation pattern, hydrolysis of phosphatidyl inositides, transient rise in free cytosolic Ca2+ ion concentration and Ca2+ uptake. All these processes were found to be inhibited by a similar C3a concentration range.     

Histamine increases anti-CD3 induced IL-5 production of TH2-type T cells via histamine H2-receptors

Besides its proinflammatory functions histamine released from basophils and mast cells during immediate-type hypersensitivity reactions is known to inhibit several lymphocyte functions like IL-2 and gamma-IFN production. Recently, it has been shown that T helper cells of type 2 phenotype (TH2) represent the T cell fraction which may play a pivotal role in the promotion of the allergic inflammatory eosinophilic late-phase reaction by secretion of cytokines, especially IL-4 and IL-5. We have investigated the effect of histamine on anti-CD3 induced IL-4 and IL-5 production by TH2 cells. Histamine in concentrations between 10(-7) and 10(-5) mol/l concentration-dependently increased anti-CD3 induced IL-5 production up to 120%, whereas IL-4 production was not affected. The activity of histamine in increasing IL-5 production was mimicked by the H2-receptor agonist dimaprit. Histamine induced increase in IL-5 production was inhibited by histamine H2-receptor antagonists, but remained unaffected by H1- or H3-receptor antagonists. Administration of forskolin which directly stimulates the production of cAMP, the second messenger of the H2-receptor, also resulted in an increase in anti-CD3 induced IL-5 production. These results indicate that the histamine-mediated increase in anti-CD3 induced IL-5 production is mediated via H2-receptors. Consequently, histamine released from mast cells and basophils during the early-phase allergic reaction may act as an important stimulatory signal for the initiation of the allergic inflammatory late-phase reaction by increasing local IL-5 production of allergen triggered TH2 cells.     

Studies on cytosolic 5SrRNA-L5 protein particles (5SRNP) of rat liver

Effects of antiallergic drugs on bradykinin-induced histamine release and intracellular Ca2+ release from peritoneal mast cells were studied in rats. Bradykinin caused a concentration-dependent histamine release as well as Ca2+ release from the intracellular Ca store of peritoneal mast cells. Antiallergic drugs used in this study showed an inhibition of not only histamine release but also Ca2+ release. The Ca2+ release from the intracellular Ca store induced by bradykinin was more sensitive to antiallergic drugs than histamine release from mast cells. Mequitazine and terfenadine caused potent inhibitory effects on both responses, whereas effects of ketotifen and cromolyn sodium were relatively weak. In conclusion, histamine release from mast cells and intracellular C2+ release induced by bradykinin were inhibited by antiallergic drugs similar to those induced by substance P and compound 48/80.     

Immunocytochemical localization of chymase to cytoplasmic vesicles after rat peritoneal mast cell stimulation by compound 48/80

The subcellular events responsible for release of mediators by mast cells may help to clarify roles for mast cells in health and disease. In this study we show that the granule-associated protease chymase is also within cytoplasmic vesicles in appropriately stimulated rat peritoneal mast cells. Rat peritoneal mast cells were recovered before or 1-10 sec after exposure to the secretogogue compound 48/80 (10 micrograms/ml) and then were examined by radioimmunoassay to quantify histamine release or were processed, using routine methods for postembedding immunoelectron microscopy, to identify the subcellular localization of chymase. In comparison to unstimulated cells, compound 48/80 stimulated cells in two independent experiments showed an increase (15%, 28%) in the surface area of the cell and a decrease (12%, 6%) in the surface area of the total granule compartment before degranulation channel formation. These global cellular changes occurred in a background of transient but significant (p < 0.01) increases in the area and number of chymase-immunoreactive vesicles per microns2 cytoplasm. These changes were detectable at 5 or 7 sec after stimulation with compound 48/80 but returned to near prestimulation levels by 9 or 10 sec after addition of compound 48/80 (total cumulative histamine release was 28% by 8 sec and 47% by 14 sec). These observations suggest that vesicles participate in the early stages of regulated secretion of chymase from rat peritoneal mast cells.