2-Hydroxy-3-methoxybenzoic acid attenuates mast cell-mediated allergic reaction in mice via modulation of the FcεRI signaling pathway.

Mast cells are important effector cells in immunoglobulin (Ig) E-mediated allergic reactions such as asthma, atopic dermatitis and rhinitis. Vanillic acid, a natural product, has shown anti-oxidant and anti-inflammatory activities. In the present study, we investigated the anti-allergic inflammatory effects of ortho-vanillic acid (2-hydroxy-3-methoxybenzoic acid, o-VA) that was a derivative of vanillic acid isolated from Amomum xanthioides. In mouse anaphylaxis models, oral administration of o-VA (2, 10, 50 mg/kg) dose-dependently attenuated ovalbumin-induced active systemic anaphylaxis and IgE-mediated cutaneous allergic reactions such as hypothermia, histamine release, IgE production and vasodilation; administration of o-VA also suppressed the mast cell degranulator compound 48/80-induced anaphylaxis. In cultured mast cell line RBL-2H3 and isolated rat peritoneal mast cells in vitro, pretreatment with o-VA (1-100 µmol/L) dose-dependently inhibited DNP-HSA-induced degranulation of mast cells by decreasing the intracellular free calcium level, and suppressed the expression of pro-inflammatory cytokines TNF-α and IL-4. Pretreatment of RBL-2H3 cells with o-VA suppressed DNP-HSA-induced phosphorylation of Lyn, Syk, Akt, and the nuclear translocation of nuclear factor-κB. In conclusion, o-VA suppresses the mast cell-mediated allergic inflammatory response by blocking the signaling pathways downstream of high affinity IgE receptor (FcεRI) on the surface of mast cells.

Ultramicronized palmitoylethanolamide counteracts the effects of compound 48/80 in a canine skin organ culture model.

BACKGROUND: Ultramicronized palmitoylethanolamide (PEA-um) has been reported to reduce pruritus and skin lesions in dogs with moderate atopic dermatitis and pruritus. HYPOTHESIS/OBJECTIVES: A canine ex vivo skin model was used to investigate the ability of PEA-um to counteract changes induced by compound 48/80, a well-known secretagogue that causes mast cell degranulation. ANIMALS: Normal skin was obtained from three donor dogs subjected to surgery for reasons unrelated to the study. METHODS: Cultured skin biopsy samples in triplicate were treated with 10 and 100 µg/mL compound 48/80, without or with 30 µM PEA-um. Mast cell (MC) degranulation, histamine release into the culture medium, local microvascular dilatation, epidermal thickness, keratinocyte proliferation and epidermal differentiation markers were evaluated. RESULTS: Exposure of the skin organ culture to PEA-um 24 h before and 72 h concomitantly to compound 48/80 resulted in a significant decrease of degranulating MCs. PEA-um also reduced the histamine content in the culture medium by half, although the effect did not reach statistical significance. PEA-um significantly counteracted vasodilation induced by 100 µg/mL compound 48/80. Finally, PEA-um alone did not induce changes in epidermal thickness, differentiation markers, keratinocyte proliferation, MC density and/or degranulation. CONCLUSIONS AND CLINICAL IMPORTANCE: Collectively, these results support the protective action PEA-um on the skin of dogs undergoing allergic changes.

Pretreatment with Evans blue, a stimulator of BK(Ca) channels, inhibits compound 48/80-induced shock, systemic inflammation, and mast cell degranulation in the rat.

The present study demonstrated that intravenous injection of a high dose of compound 48/80 to the rat induced 50% drop, within a few min, in the mean arterial pressure and pulse pressure as well as systemic inflammatory plasma leakage that might lead to circulatory and respiratory failure. We also investigated whether pretreatment with Evans blue, a stimulator of BK(Ca) channels, could exert inhibitory effect against compound C48/80-induced allergic circulatory shock and systemic inflammation. Different groups of Sprague-Dawley rats received an intravenous injection of a dose of Evans blue (0, 5, 10, or 50 mg/kg) just 20 s prior to injection of compound 48/80 (200 µg/kg, over 2 min). The present study found that pretreatment with Evans blue in a dose of 10 or 50 mg/kg exerted acute inhibitory effect on compound 48/80-induced sudden drop in mean arterial and pulse pressures. We also showed that pretreatment with Evans blue in a dose of 5, 10, or 50 mg/kg significantly inhibited compound 48/80-induced extensive plasma extravasation, mast cell degranulation, and edema formation in various organs including the airways, esophagus, and skin. Pretreatment with Evans blue 50 mg/kg 1 h earlier exhibited longer-term inhibitory effect on compound 48/80-induced arterial hypotension and systemic inflammation. We concluded that Evans blue pretreatment prevented rats from compound 48/80-triggered allergic shock and systemic inflammation, possibly mainly through inhibition of mast cell degranulation. Evans blue might be potentially useful in elucidating the mechanism and acting as a therapeutic agent of allergic shock and systemic inflammation.

Agonist-promoted kappa opioid receptor (KOR) phosphorylation has behavioral endpoint-dependent and sex-specific effects.

We reported previously that the selective agonist U50,488H promoted phosphorylation of the mouse kappa opioid receptor (mKOR) in vitro at four residues in the C-terminal domain. In this study, we generated a mutant mouse line in which all the four residues were mutated to Ala (K4A) to examine the in vivo functional significance of agonist-induced KOR phosphorylation. U50,488H promoted KOR phosphorylation in brains of the wildtype (WT), but not K4A, male and female mice. Autoradiography of [3H] 69,593 binding to KOR in brain sections showed that WT and K4A mice had similar KOR distribution and expression levels in brain regions without sex differences. In K4A mice, U50,488H inhibited compound 48/80-induced scratching and attenuated novelty-induced hyperlocomotion to similar extents as in WT mice without sex differences. Interestingly, repeated pretreatment with U50,488H (80 mg/kg, s.c.) resulted in profound tolerance to the anti-scratch effects of U50,488H (5 mg/kg, s.c.) in WT mice of both sexes and female K4A mice, while in male K4A mice tolerance was attenuated. Moreover, U50,488H (2 mg/kg) induced conditioned place aversion (CPA) in WT mice of both sexes and male K4A mice, but not in female K4A mice. In contrast, U50,488H (5 mg/kg) caused CPA in male, but not female, mice, regardless of genotype. Thus, agonist-promoted KOR phosphorylation plays important roles in U50,488H-induced tolerance and CPA in a sex-dependent manner, without affecting acute U50,488H-induced anti-pruritic and hypo-locomotor effects. These results are the first to demonstrate sex differences in the effects of GPCR phosphorylation on the GPCR-mediated behaviors.

Effects of aegeline, a main alkaloid of Aegle Marmelos Correa leaves, on the histamine release from mast cells.

Aegeline or N-[2-hydroxy-2(4-methoxyphenyl) ethyl]-3-phenyl-2-propenamide is a main alkaloid isolated from Aegle marmelos Correa collected in Yogyakarta Indonesia. In our study, we investigated the effects of aegeline on the histamine release from mast cell. The study was performed by using (1) rat basophilic leukemia (RBL-2H3) cell line, and (2) rat peritoneal mast cells (RPMCs). DNP(24)-BSA, thapsigargin, ionomycin, compound 48/80 and PMA were used as inducers for histamine release from mast cell. In our study, aegeline inhibited the histamine release from RBL-2H3 cells induced by DNP(24)-BSA. Indeed, aegeline showed strong inhibition when RBL-2H3 cells induced by Ca(2+) stimulants such as thapsigargin and ionomycin. Aegeline is suggested to influence the intracellular Ca(2+) pool only since could not inhibit the (45)Ca(2+) influx into RBL-2H3 cells. Aegeline showed weak inhibitory effects on the histamine release from RPMCs, even though still succeed to inhibit when the histamine release induced by thapsigargin. These findings indicate that aegeline altered the signaling pathway related to the intracellular Ca(2+) pool in which thapsigargin acts. Based on the results, the inhibitory effects of aegeline on the histamine release from mast cells depended on the type of mast cell and also involved some mechanisms related to intracellular Ca(2+) signaling events via the same target of the action of thapsigargin or downstream process of intracellular Ca(2+) signaling in mast cells.

Antiallergic drug cromolyn may inhibit histamine secretion by regulating phosphorylation of a mast cell protein.

Cromolyn inhibited histamine release from mast cells that was induced by a classic secretagogue and correspondingly increased incorporation of radioactive phosphate into a 78,000-dalton protein. These effects on histamine secretion and on protein phosphorylation were rapid in onset and both showed tachyphylaxis. Cromolyn may therefore act by altering the phosphorylation of a protein involved in the regulation of secretion.

Inhibition of basic secretagogue-induced signaling in mast cells by cell permeable G alpha i-derived peptides.

BACKGROUND: Basic secretagogues of connective tissue mast cells act as receptor mimetic agents that trigger mast cells by activating G proteins. This leads to simultaneous propagation of two signaling pathways: one that culminates in exocytosis, while the other involves protein tyrosine phosphorylation and leads to release of arachidonic acid metabolites. We have previously shown that introduction of a peptide that comprises the C-terminal end of G alpha i3 into permeabilized mast cells inhibits basic secretagogue-induced exocytosis [Aridor et al., Science 1993;262:1569-1572]. We investigated whether cell-permeable peptides, composed of the C-terminus of G alpha i3 fused with importation sequences, affect mast cell function. METHODS: Following preincubation with the fused peptides, rat peritoneal mast cells were activated by compound 48/80 and analyzed for histamine and prostaglandin D2 release and protein tyrosine phosphorylations. RESULTS: We demonstrate that out of three importation sequences tested only G alpha i3 peptide fused with the Kaposi fibroblast growth factor importation sequence (ALL1) inhibited release of histamine. ALL1 as well as a cell-permeable peptide that corresponds to G alpha i2 also blocked compound 48/80-stimulated protein tyrosine phosphorylation, though the latter did not block histamine release. ALL1 effect was G protein-specific, as it was incapable of blocking protein tyrosine phosphorylation stimulated by pervanadate. CONCLUSION: ALL1, a transducible G alpha i3-corresponding peptide, blocks the two signaling pathways in mast cells: histamine release and protein tyrosine phosphorylation. Cell permeable peptides that block these two signaling cascades may constitute a novel approach for preventing the onset of the allergic reaction.

Tea catechins have dual effect on mast cell degranulation induced by compound 48/80.

Green tea catechins are emerging as one of the most efficient and safest ingredient in health promoting food. We investigated catechin's effects on intracellular ROS generation in mast cell activation and degranulation. Compound 48/80, receptor mimetic basic secretagogues for mast cell, induced ROS generation dose-dependently with bell-shaped degranulation pattern in canine cutaneous mastocytoma cells (CM-MC). When intracellular ROS level was relatively low, catechins decreased both ROS and the degranulation. However, when intracellular ROS level was remarkably high, catechins decreased ROS level but increased the degranulation paradoxically. Gallocatechins showed the stronger effects than non-gallated catechins. Exogenous H(2)O(2) also shows dual effect on degranulation dose-dependently. EGCG shows the dual effect on the tyrosine and threonine phosphorylation depending on the concentration of compound 48/80. Particularly, 60 kDa protein tyrosine-phosphorylated by EGCG with 3 microg/ml of compound 48/80 might be a negative regulator for the degranulation. Taken together, there is an optimal level of ROS for the degranulation, and the catechins have a dual function by controlling ROS level.

Inhibitory effects of epigallocatechin gallate on compound 48/80-induced mast cell activation and passive cutaneous anaphylaxis.

Epigallocatechin gallate (EGCG) is a principle phenolic antioxidant found in a variety of plants, including green and black tea. The anti-allergic effect of EGCG is unknown. The purpose of this study is to investigate the effects of EGCG on compound 48/80-induced mast cell activation and passive cutaneous anaphylaxis. For this, the influences of EGCG on the compound 48/80-induced cutaneous reaction were measured in vivo and the effects of EGCG on the compound 48/80-induced mast cell activations were examined in vitro. Results are below: as 1) EGCG significantly inhibited compound 48/80-induced passive cutaneous anaphylaxis, 2) the compound 48/80-induced degranulation, calcium influx and histamine release of rat peritoneal mast cells (RPMCs) were significantly inhibited by the pretreatment with EGCG, and 3) the compound 48/80-mediated inhibition of cAMP level in RPMCs was significantly increased by the pretreatment with EGCG. These results suggested that EGCG, the most abundant polyphenol in green tea, inhibits the compound 48/80-induced mast cell activation and the increase of vascular permeability, and potentially serve as effective therapeutic tools for allergic diseases.

Inhibitory effects of Okbyungpoong-Gamhmi on anaphylactic responses.

We investigated the effect of a herbal formulation Okbyungpoong-Gamhmi (OG) on mast cell-dependent anaphylactic reactions by intra-rectal administration. OG concentration dependently inhibited compound 48/80-induced anaphylaxis-like response and ear swelling response with doses of 0.01-1g/kg. OG also inhibited the passive cutaneous anaphylaxis at the same concentrations. The histamine release induced by compound 48/80 or IgE from the rat peritoneal mast cells was reduced by 64.2 and 63.6%, respectively, at 1g/l. These results provide evidence that intra-rectal therapy of OG may be beneficial in the treatment of anaphylactic response.

Cold urticaria: inhibition of cold-induced histamine release by doxantrazole.

Thirteen patients with cold urticaria were studied to assess the effect of the systemic drug doxantrazole, which has actions resembling disodium cromoglycate, on cold evoked histamine release. The patients, all of whom developed an immediate local whealing response after cooling of the forearm, demonstrated release of histamine into venous blood draining that forearm. Following doxantrazole treatment, significant suppression of histamine release occurred. In some but not all patients this was accompanied by diminution of urtication in response to cooling. A double-blind study was carried out in 3 subjects, all of whom showed diminished cold-stimulated histamine release after doxantrazole. Two of these showed clinical improvement. Doxantrazole had no effect on erythema due to intradermal histamine, but did suppress the erythematous reaction to intradermal injection of compound 48/80. Our results suggest that doxantrazole or related anti-allergic agents might be useful in the treatment of cold urticaria.

Effect of 8-methoxypsoralen plus long-wave ultraviolet (PUVA) radiation on mast cells. II. In vitro PUVA inhibits degranulation of rat peritoneal mast cells induced by compound 48/80.

Rat peritoneal mast cells incubated with a histamine liberator, compound 48/80, showed a significantly reduced capacity for releasing histamine following in vitro treatment with 0.1 micrograms/ml of 8-methoxypsoralen (8-MOP) plus 1-5 J/cm2 of long-wave ultraviolet (UVA) irradiation (PUVA). No remarkable inhibition in histamine release was observed in the cells treated with 8-MOP only. Irradiation with 5 J/cm2 of UVA alone exerted an inhibitory effect on histamine release, to a lesser extent than PUVA. PUVA irradiation did not bring any decrease in cell viability or any spontaneous release of histamine from irradiated cells as shown by phase-contrast microscopy and by histamine assay, respectively. These results suggest that PUVA treatment may cause a noncytotoxic disturbance at mast cell membranes or on surface receptors, leading to a decreased capacity for secreting chemical mediators.

Changes in afferent impulse activity of small intestine mesenteric nerves in response to antigen challenge.

Sprague-Dawley rats (weight 130-150 g) were sensitized by an intraperitoneal injection of 1 mg chicken egg albumin with 0.25 ml Freund's adjuvant to stimulate immunoglobulin E antibody production. Leukocyte migration inhibitory factor was used as an indicator of animal sensitization. In acute electrophysiological experiments on sensitized animals, an intra-arterial or intraluminal chicken egg albumin (100 microg) challenge evoked a 10% enhancement of the activity of mesenteric nerves of the small intestine, regardless of the injection site chosen. Afferent nerve activity in control animals was not changed during the chicken egg albumin challenge. Morphometry at the light microscope level showed activation of mast cell degranulation after the antigen challenge to presensitized rats. Intraluminal injections of a stimulator of mast cell degranulation, compound 48/80 (20-30 mg), were found to increase afferent discharges in intact rats. An antagonist of H1 histamine receptors, clemastine, reduced the effect of compound 48/80. The results obtained provide direct evidence for the stimulation of sensory nerve endings by mast cell mediators released during mast cell degranulation.

The influence of the sesquiterpene lactones from Geigeria on mast cell degranulation.

The sesquiterpene lactones isolated from Geigeria were found to be incapable of inducing rat peritoneal mast cell degranulation at levels of 0.3-1.6 mM. The sulphydryl reagent, N-ethylmaleimide, too was unable to trigger mast cell secretion. Instead, it was observed that these compounds inhibited the release of histamine induced by Compound 48/80. Pretreatment of the lactones and N-ethylmaleimide with the amino acid, L-cysteine, reduced their inhibition ability of histamine release to a considerable extent, but not completely. Geigerin(V), which lacks an alpha-methylene group and the chemically prepared cysteine-adduct of dihydrogriesenin(I), were also capable of inhibiting mast cell secretion by Compound 48/80, but to a lesser extent.

Toad (Bufo paracnemius) mast cell degranulation by compound 48/80 and by chlorpromazine: a non-lytic process.

Toad mast cells are degranulated by compound 48/80 and by chlorpromazine. The former was effective at rather high (100 micrograms/ml) and the latter at low (0.1 mM) concentrations. Toad mast cell degranulation induced by these drugs was characterized by cytoplasmic granule dissolution without cell lysis. The metabolic inhibitor, dinitrophenol, blocked the degranulation induced by both agents. Glucose reversed the inhibition of compound 48/80-induced degranulation. The results indicate that toad mast cell degranulation by compound 48/80 and by chlorpromazine is a non-lytic process.

Relationship between phosphatidylserine and cromolyn in histamine release.

The opposing actions of phosphatidylserine (PS) and cromolyn on histamine release were studied in rat peritoneal mast cells in vitro. Histamine-releasing drugs could be separated into 2 groups on the basis of whether they were potentiated by PS and inhibited by muM cromolyn (dextran, antigen (BSA), concanavalin A) or neither potentiated by PS nor inhibited by muM cromolyn (48/80, polymyxin, phospholipase C and ATP). Compound 48/80 and polymyxin chemically combined with PS, but this could be circumvented by preincubating the mast cells in PS for one hour, and then washing them free of PS in solution prior to addition of the drugs. These data define at least 2 pathways for induction of histamine release: (1) a PS-potentiated pathway inhibited by muM cromolyn, and (2) a PS-independent pathway unaffected by muM cromolyn. Dose-response curves of the muM cromolyn inhibition of PS-potentiated release revealed a parallel shift, suggesting that cromolyn may compete with PS.

Glucocorticoid-induced vasoconstriction in human skin. An inhibitory role on phospholipase A2 activity.

The ability of topically applied betamethasone valerate to reduce erythema produced by a variety of vasodilators was assessed. The steroid significantly reduced the erythema induced by topical arachidonic acid, intradermal histamine, and compound 48/80. We postulated that the steroid reduced endogenous phospholipase activity either before or after application of the vasodilators, or both. This action may explain the mechanism of glucocorticoid-induced vasoconstriction in the skin and part of the steroids' action in acute inflammatory skin disease.

Inhibition of compound 48/80--induced vascular protein leakage by pretreatment with capsaicin and a substance P antagonist.

Intravenous injection of compound 48/80 (1 mg X kg-1) induced an acute increase in vascular permeability to plasma proteins in various organs of rats. The compound 48/80 response was partly inhibited by histamine H1 and H2 receptor blockade in the urinary bladder and in the duodenum, but not in the trachea, the oesophagus, the ureter and the paw skin. Blockade of 5-hydroxytryptamine receptors with methysergide led to a reduction of the permeability response in the oesophagus and in the urinary bladder, leaving responses in other organs unchanged. Pretreatment of neonatal rats with capsaicin almost abolished the 48/80 response in all organs except in the duodenum. Pretreatment of rats with [D-Arg1, D-Trp7,9, Leu11]-substance P, a substance P antagonist, also caused a partial inhibition of the permeability response to compound 48/80 in several organs. Topical administration of compound 48/80 (1 mg X ml-1) onto the tracheal mucosa induced local Evans blue extravasation. This response was resistant to pretreatment with histamine receptor antagonists, but was largely inhibited after neonatal capsaicin pretreatment. Topical administration of compound 48/80 (1 mg X ml-1 or 10 mg X ml-1) into the eye did not cause visible Evans blue extravasation in the conjunctiva, nor any signs of pain reaction as indicated by the absence of the wiping response, usually seen upon noxious chemical stimuli in the eye. In guinea-pigs, 10 mg X kg-1 compound 48/80 i.v. were required to induce vascular protein leakage in different organs. This response was blocked by pretreatment with H1 and H2 receptor antagonists, but only slightly reduced after systemic capsaicin pretreatment of guinea-pigs.(ABSTRACT TRUNCATED AT 250 WORDS)

Novel findings in inhibition of mast cell-dependent immediate-type cutaneous reactions by Gahmi-Shini-San.

This report describes an inhibitory effect of Gahmi-Shini-San (GSS) on mast cell-mediated immediate-type allergic reactions. GSS is an Oriental herbal medication, which has been successfully used in Korea for the treatment of allergic disorders, mainly skin anaphylactic diseases. GSS inhibited the ear swelling response induced by intradermal injection of compound 48/80 in a mouse model on a concentration-dependent basis. The mast cells in mouse ear tissue were stained by alcian blue/nuclear fast red. GSS significantly inhibited the compound 48/80-induced degranulation from mast cells in ear tissue. GSS dose-dependently inhibited the histamine release from the rat peritoneal mast cells by compound 48/80. We also studied the effect of GSS on mast cell-dependent passive cutaneous anaphylaxis activated by dinitrophenyl IgE antibody. GSS showed inhibition of passive cutaneous anaphylaxis following oral administration. These results indicated that GSS has inhibitory effect on mast cell-dependent immediate type cutaneous reactions.

Effect of the new theophylline derivatives on degranulation of mast cells and phosphodiesterase activity in smooth muscles.

Effect of the new theophylline derivative (beta-hydroxy-benzylopiperazinopropyl-theophylline), designed as R6 on degranulation of the mast cells from rat mesenteries, was tested in vitro and in vivo. In vitro it was found that R6 significantly inhibited mast cell degranulation induced both by the histamine liberator C 48/80 and by rabbit globulin directed against rat serum proteins. The effect was comparable to that revealed by mepyramine, but much stronger than the effects of DSCG and aminophylline. The influence of R6 on mast cells degranulation was less pronounced in the in vivo systems. It was demonstrated by histochemical methods that R6 did not change activity of lactic and succinic dehydrogenases, neither activity of lysosomal hydrolases. In separate system including small intestine from rat it was shown that R6 inhibited activity of the phosphodeisterase in smooth muscles. It was suggested that the effect of R6 on mast cells degranulation is mediated by an increase in cAMP as well as by indirect influence on Ca++ influx.