Activation of peroxisome proliferator-activated receptor gamma suppresses mast cell maturation involved in allergic diseases.

BACKGROUND: Mast cells play a central role in allergic and inflammatory diseases. Several reports indicated role of peroxisome proliferator-activated receptor gamma (PPARgamma) on mast cell function. However, there is no report about the role of PPARgamma on differentiation of mast cells from the progenitors. In this study, we investigated the role of PPARgamma in regulating bone marrow-derived mast cell maturation and the therapeutic implications for mast cell-related diseases such as atopic or contact dermatitis. METHODS: We used in vitro cell culture system for mast cell differentiation from bone marrow-progenitors using specific ligands and lentiviral-mediated short hairpin RNA of PPARgamma, and in vivo murine dermatitis models. RESULTS: Activation of PPARgamma inhibited the maturation of bone marrow progenitors into connective tissue-type mast cells (CTMCs) through up-regulation of GATA-4 and GATA-6 resulting in a decrease in expression of histidine decarboxylase and mast cell histamine content. In comparison, the differentiation of bone marrow progenitors into CTMCs was significantly accelerated by the knockdown of PPARgamma expression by lentiviral-mediated short hairpin RNA. Peroxisome proliferator-activated receptor gamma ligand administration to mice inhibited the maturation of mast cells resulting in attenuation of atopic and contact dermatitis via diminishment of the number of mature mast cells. CONCLUSION: Our results indicate that PPARgamma is one of master regulators on mast cell maturation and potentially useful for the therapy in various disorders involving mast cell activation.

Increase of histidine decarboxylase activity in murine myelomonocytic leukemia cells (WEHI-3B) in parallel to their differentiation into macrophages.

When cells of mouse myelomonocytic leukemia cell line, WEHI-3B, were cultured in the presence of actinomycin D plus the serum which was obtained from mice injected with bacterial endotoxin, i.e., lipopolysaccharide, their histidine decarboxylase (L-histidine carboxy-lyase, EC 4.1.1.22) (HDC) activity increased about 100-fold with a peak at 48 h. According to the increase in HDC activity, the expression of surface antigens associated with macrophages, such as Mac II, Mac III and Iad, increased markedly on WEHI-3B cells as well as their morphological changes to macrophages. Histamine levels in the culture medium increased concomitantly with the increase in the HDC activity in WEHI-3B cells, whereas the histamine contents inside the cells did not increase remarkably. Furthermore, the addition of lipopolysaccharide to the culture medium caused an additional 2-fold increase in the HDC activity of WEHI-3B cells. These results indicate that the increase in HDC activity in WEHI-3B cells may represent an event in the process of the differentiation to macrophages.

Pharmacology of alpha-fluoromethylhistidine, a specific inhibitor of histidine decarboxylase.

alpha-Fluoromethyl-[S]-histidine (FMH) is a specific and potent inhibitor of histidine decarboxylase, which forms histamine from histidine. It acts selectively and irreversibly by formation of a covalent linkage, possibly with the serine residue in the active site of the enzyme. A single administration of FMH decreases the histamine content only of non-mast cells in the brain and stomach of rodents, but repeated administration gradually decreases the histamine content of mast cells in all tissues. Thus, FMH can be used to deplete histamine in pharmacological studies. As no marked side-effects have been observed during administration of FMH, it may be useful in pathological conditions, such as some allergic diseases, peptic ulcers and mastocytosis, in which excess production of histamine is involved.

Determination of imidazole acetic acid and its conjugate(s) levels in urine, serum and tissues of rats: studies on changes in their levels under various conditions.

A convenient and reproducible method for assay of imidazole acetic acid (ImAA) was developed as a modification of that described previously (Watanabe et al., 1983). ImAA conjugate(s) (ImAA-C), mainly consisting of imidazole acetic acid riboside, could be measured by this method after its hydrolysis to ImAA. The ImAA and ImAA-C levels in various tissues of rats were measured and the effects of various agents on these levels were studied. The renal clearance values of ImAA-C in rats and man were similar to the creatinine clearance values, but those of ImAA were 1/40 of those of ImAA-C, suggesting that the latter is readily excreted in the urine. Consistent with this idea, the urinary excretion of ImAA-C was found to increase much more than that of other histamine metabolites during late pregnancy, when the foetus produces much histamine.

Involvement of apamin-sensitive K+ channels in antigen-induced spasm of guinea-pig isolated trachea.

1. In order to examine whether K+ channels play a role in antigen-induced airway responses, the effect of K+ channel blockers on antigen-induced airway smooth muscle contraction and mediator release was examined in vitro in guinea-pigs actively sensitized with ovalbumin (OA). 2. Tracheal strips from sensitized animals were suspended in organ baths under a resting tension of 1 g and isometric tension was continuously measured. Cumulative concentration-response curves to OA (0.1-1000 ng ml-1) or histamine (10 nM-1 mM) were obtained in the presence and absence of K+ channel blockers. 3. OA (10, 100 or 1000 ng ml-1) was incubated with minced lung tissues from the same animals for 15 min in the presence and absence of K+ channel blockers, and released histamine and leukotriene C4 (LTC4) in the incubating medium were measured. 4. Apamin, a small conductance Ca(2+)-activated K+ channel (PK,Ca) blocker, (0.1, 0.3 and 1 microM) significantly inhibited OA-induced smooth muscle contraction, while charybdotoxin (ChTX, 10 nM), an intermediate and large conductance PK,Ca blocker, and iberiotoxin (IbTX, 3 nM), a large conductance PK,Ca blocker, were without effect. Apamin (0.3 microM) had no effect on exogenously administered histamine-induced airway smooth muscle contraction, suggesting that the inhibition of OA-induced contraction by apamin did not occur at the smooth muscle level. 5. The inhibition of OA-induced contraction by apamin (0.3 microM) was not significantly affected by pretreatment with a leukotriene antagonist, ONO-1078 (10 microM), but was abolished by pretreatment with a histamine H1-receptor blocker, pyrilamine (1 microM). 6. Apamin by itself (up to 0.1 MicroM) had no effect on spontaneous histamine release from minced lung tissues. Histamine release induced by low and intermediate concentrations of OA (10 and 100 ng ml-1)was significantly suppressed by apamin pretreatment (P<0.05 and P<0.001), whereas LTC4 release was not affected. ChTX (0.1 MicroM) and IbTX (10 nM) had no significant effect on either spontaneous or OA (100 ng ml-1)-induced histamine release.7. These results suggest that apamin partially but substantially inhibits antigen-induced smooth muscle contraction, presumably by inhibiting antigen-induced histamine release from airway mast cells through small conductance PKca closure.

Isolation of the third component of complement and its derivative with anaphylatoxin-like activity from the plasma of the newt Cynops pyrrhogaster.

The third component of complement (C3) of a newt, Cynops pyrrhogaster, was purified using a fast protein liquid chromatography technique. The purified newt C3 consists of two polypeptide chains (the molecular masses of the alpha and beta-chains of C3 were 120,000 and 70,000, respectively) linked by disulfide bonds. The alpha-chain retained an internal thiolester bond that was cleaved with methylamine, and the N-terminal amino acid sequence of the alpha-chain was XVQLIDAKAGKAAKF. Digestion of newt C3 with trypsin yielded fragments that induced significant histamine release from newt peritoneal cells. These results indicate that newt C3 retains structural and functional properties shared with mammalian C3.

Tissue distribution of histamine in a mutant mouse deficient in mast cells: clear evidence for the presence of non-mast-cell histamine.

The contents of histamine in various tissues of mutant mice deficient in mast cells (W/Wv) and in congenic normal mice (+/+) were determined by high-performance liquid chromatography and were compared. In spite of the absence of mast cells in W/Wv mice, the histamine content of their whole bodies was about 5-10% of that of +/+ mice. The skin, heart and lungs of W/Wv mice contained negligible amounts of histamine (about 2% of that in +/+ mice), but the liver, kidneys and spleen contained appreciable histamine (8-15% of that in +/+ mice), and the brain and stomach contained much histamine (45 and 34%, respectively, of that in +/+ mice). These results indicate the presence of non-mast-cell histamine, especially in the brain and stomach, where it may play important physiological roles.

Histidine decarboxylase in human basophilic leukemia (KU-812-F) cells. Characterization and induction by phorbol myristate acetate.

The human leukemic cell line KU-812-F is known to differentiate into mature basophil-like cells under serum-free culture conditions. In the present study, the activity of histidine decarboxylase (HDC), a histamine-forming enzyme, in KU-812-F cells was found to be high, ranging from 10 to 57 pmol/min/mg protein. The great variation in HDC activity appeared to be due to different percentages and degrees of maturity of basophil-like cells during differentiation of this cell line. The enzyme was inhibited by alpha-fluoromethylhistidine but not by carbidopa, was unable to form dopamine from L-3,4-dihydroxyphenylalanine, and had a Km value for histidine of 0.27 mM, indicating that it was HDC and not aromatic amino acid decarboxylase. The HDC activity increased 1.8-fold when the cells were stimulated by phorbol myristate acetate, which is known to activate protein kinase C, and this increase was blocked by staurosporine, a potent inhibitor of protein kinase C.

Further studies on endo-beta-N-acetylglucosaminidase D1.

The purification procedure for endo-beta-N-acetylglucosaminidase D was improved to yield an enzyme preparation which was homogeneous upon gel electrophoresis. The molecular weight of the enzyme as estimated by Sephadex G-200 column chromatography was 280,000, while SDS-gel electrophoresis after reduction with 2-mercaptoethanol gave a value of 150,000. The purified enzyme did not show any chitinase, hyaluronidase or lysozyme activity. In the presence of exoglycosidases removing peripheral sugars, the endoglycosidase acted on serum glycoproteins such as transferrin and fetuin. The enzyme also hydrolyzed an oligosaccharide, (Man)5(GlcNAc)2, indicating that the peptide portion of substrates does not have much effect on susceptibility to the enzyme.

Effect of food intake on urinary excretions of histamine, N tau-methylhistamine, imidazole acetic acid and its conjugate(s) in humans and mice.

The urinary excretions by young healthy men of histamine and its metabolites, N tau-methylhistamine, imidazole acetic acid, and imidazole acetic acid conjugate(s), increased 1-3 h after food intake. The increase was seen even after the intake of konnyaku (mannan) as a protein-deficient food, suggesting that physical stimulation of the gastric mucosa by food is the main cause of histamine release. This suggestion was confirmed by the following findings in patients and mice. In patients with stomach diseases, gastrectomy resulted in decreases in the excretion of histamine and its metabolites in the urine, and patients subjected to intravenous hyperalimentation excreted less histamine and its metabolites in the urine than normal subjects. In mice, a correlation of histamine excretion with food intake was demonstrated experimentally. Namely, mice fed only during the night (21:00-0:00) showed increased excretions of histamine and its metabolites at 23:00-3:00, whereas those fed in the morning (9:00-12:00) showed increased excretions of those compounds at 11:00-15:00. All these results are consistent with the idea that urinary histamine and its metabolites mainly originate from the stomach.

A novel reaction for annulation onto alpha,beta-unsaturated ketones: W(CO)(5).L promoted exo- and endo-selective cyclizations of omega-acetylenic silyl enol ethers prepared by 1,4-addition of propargyl malonate to enones.

A highly useful method for five- and six-membered ring annulation onto alpha,beta-unsaturated ketones is described. 1,4-Addition of propargylmalonate to alpha,beta-unsaturated ketones in the presence of silyl triflate gives 7-siloxy-6-en-1-yne derivatives in good yield. W(CO)(5).L-catalyzed cyclization of these substrates can be induced to give preferentially either exo- or endo-cyclized products in good yield simply by changing the reaction solvent. [reaction: see text]

Ozone increases susceptibility to antigen inhalation in allergic dogs.

To determine whether O3 exposure increased airway responsiveness to antigen inhalation, we studied airway responsiveness to acetylcholine (ACh) and Ascaris suum antigen (AA) before and after O3 in dogs both sensitive and insensitive to AA. Airway responsiveness was assessed by determining the provocative concentration of ACh and AA aerosols that increased respiratory resistance (Rrs) to twice the base-line value. O3 (3 parts per million) increased airway responsiveness to ACh in dogs both sensitive and insensitive to AA, and it significantly decreased the ACh provocation concentration from 0.541 +/- 0.095 to 0.102 +/- 0.047 (SE) mg/ml (P less than 0.01; n = 10). AA aerosols, even at the highest concentration in combination with O3, did not increase Rrs in dogs insensitive to AA. However, O3 increased airway responsiveness to AA in AA-sensitive dogs and significantly decreased log AA provocation concentration from 2.34 +/- 0.22 to 0.50 +/- 0.17 (SE) log protein nitrogen units/ml (P less than 0.01; n = 7). O3-induced hyperresponsiveness to ACh returned to the base-line level within 2 wk, but hyperresponsiveness to AA continued for greater than 2 wk. The plasma histamine concentration after AA challenge was significantly higher after than before O3 (P less than 0.01). Intravenous infusion of OKY-046 (100 micrograms.kg-1.min-1), an inhibitor of thromboxane synthesis, inhibited the O3-induced increase in responsiveness to ACh, but it had no effects on the O3-induced increase in responsiveness to AA and the increase in the plasma histamine concentration. These results suggest that O3 increases susceptibility to the antigen in sensitized dogs via a different mechanism from that of O3-induced muscarinic hyperresponsiveness.

Brain penetration of the histamine H3 receptor antagonists thioperamide and clobenpropit in rat and mouse, determined with ex vivo [125I]iodophenpropit binding.

We investigated the brain penetration of the histamine H3 receptor antagonists thioperamide and clobenpropit using ex vivo [125I]iodophenpropit binding. Homogenates of the rat cortex, striatum and mouse whole brain were prepared 1 h after subcutaneous injection of the H3 antagonists and incubated with [125I]iodophenpropit, a radiolabeled H3 receptor antagonist, to determine the H3 receptor occupancy. Specific [125I]iodophenpropit binding to the rat cortex and striatum was inhibited by thioperamide with IC30 values of 1.0 and 1.5 mg/kg, respectively. Clobenpropit also inhibited [125I]iodophenpropit binding, but was less potent (IC30: 18 and 19 mg/kg in the rat cortex and striatum, respectively) than thioperamide. Similar results were obtained in experiments with mouse whole brain (3.5 and 13 mg/kg for thioperamide and clobenpropit), indicating that there is no important species differences in the brain penetration of these drugs between rats and mice. These findings suggest that after peripheral injection both in rat and mouse thioperamide penetrates the blood-brain barrier more efficiently compared to clobenpropit.

The lack of compound 48/80-induced contraction in isolated trachea of mast cell-deficient Ws/Ws rats in vitro: the role of connective tissue mast cells.

In the rat trachea, two types of mast cells have been identified, connective tissue mast cells and mucosal mast cells. Their different characteristics may account for their different biological functions. The role of connective tissue mast cells in tracheal contraction as one feature of the immediate reaction of asthma was studied in vitro in isolated trachea, using tissue derived from mast cell-deficient (Ws/Ws) rats, heterozygous (Ws/+) rats and control (+/+) rats, and compound 48/80 as a potent inducer of mast cell degranulation. The contractile response of tracheas from the three types of rats was also studied upon exposure to the following spasmogens: histamine, 5-hydroxytryptamine (5-HT), and carbachol. Histamine content in tissues reflected the differing mast cell numbers in strips from the three rat types. It was found that carbachol and 5-HT elicited tracheal contraction in a similar manner in strips from the three types of rats. Histamine had no contractile effect. Compound 48/80, at a dose of 25 microg/ml, elicited contraction in tracheas from both control (+/+) and heterozygous (Ws/+), but not in trachea from Ws/Ws rats. Compound 48/80-induced contractions in tracheas from +/+ rats were inhibited by 0.1 microM ketanserin and 0.1 microM nedocromil, but not by 0.1 microM mepyramine. Enzyme histochemistry confirmed that the degranulation occurred in connective tissue mast cells, but not in mucosal mast cells. We concluded that connective tissue mast cells play an important role in rat tracheal contraction via 5-HT release induced by compound 48/80. In addition, the specific mast cell-deficient (Ws/Ws) rats provide a good tool for studying the roles of mast cells in airway system.

Role of mast cell histamine in the formation of rat paw edema: a microdialysis study.

We determined the endogenous histamine concentration in the subplantar space of rat hind paws using an in vivo microdialysis technique. A microdialysis probe was implanted into the rat hind paw and the histamine content in dialysates was measured by high performance liquid chromatography-fluorometry. In wild type (+/+) rats, the histamine output (basal level 25.7 +/- 0.9 pmol/ml) increased 115-, 199- and 426-fold rapidly after subplantar injection of compound 48/80 at doses of 0.5, 5 and 50 microg/paw, respectively. In genetically mast cell-deficient (Ws/Ws) rats, the basal level of histamine was one third of that obtained from +/+ rats, and was not increased by compound 48/80 injection. With this treatment, marked, dose dependent, but relatively gradual development of the paw edema was found in +/+ rats. However, no edema formation was observed in Ws/Ws rats. Histological observations showed neither mast cells nor edema to be present in the paw skin of Ws/Ws rats. These findings indicate the critical role of histamine as a trigger for the development of edema in vivo. In addition, Ws/Ws rats will provide important information as to the roles of mast cells in the inflammatory response.

Biphasic elevation of plasma histamine induced by water immersion stress, and their sources in rats.

The effect of water immersion stress on the plasma concentration of histamine, in Wistar and mast cell-deficient (Ws/Ws) rats, was investigated. The histamine content of the plasma, skin and gastric mucosa, as well as the level of activity of histidine decarboxylase in the gastric mucosa, were determined by high performance liquid chromatography (HPLC)-fluorometry. In Wistar rats exposed to water immersion stress for a total of 6 h, an initial, acute, four-fold, transient increase in the plasma histamine level, followed by a sustained, though lower, elevation of the plasma histamine level, was observed. The initial acute increase in plasma histamine level was also seen in gastrectomized Wistar rats exposed to water immersion stress, but not in Ws/Ws rats exposed to stress. The sustained elevation of the plasma histamine level was observed in the Ws/Ws rats. However, in both the gastrectomized Wistar rats and gastrectomized Ws/Ws rats, the sustained elevation in plasma histamine level was not observed. The histamine content of the skin of Wistar rats after 15 min or more exposure to water immersion stress, was 20% lower than that of control rats. The mucosal histamine content of both Wistar rats and Ws/Ws rats, was 20% lower, whereas histidine decarboxylase activity in the gastric mucosa was enhanced by two-fold, during exposure to stress for 4 h. These findings indicate that water immersion stress causes a biphasic increase in plasma histamine concentration in Wistar rats; the initial acute increase in plasma histamine level originates from mast cells, and the second, sustained increase is attributed to enterochromaffin-like cells.

Effect of rabeprazole on histamine synthesis in enterochromaffin-like cells of mast cell-deficient (Ws/Ws) rats.

The effect of rabeprazole, the latest proton pump inhibitor, on the serum gastrin concentration, histidine decarboxylase activity and histamine content of the oxyntic mucosa in Wistar rats, mast cell-deficient (Ws/Ws) rats, and their normal type, +/+, rats was investigated. In Wistar rats, 2 weeks of treatment with rabeprazole (30 mg/kg/day, s.c.) induced a 1.8-fold increase in serum gastrin concentration and a 3.9-fold increase in histidine decarboxylase activity of the oxyntic mucosa over the control levels, whereas neither 2- nor 4-week treatment affected the histamine content of the oxyntic mucosa. In Ws/Ws and +/+ rats, the serum gastrin concentration, histidine decarboxylase activity and even histamine content of the oxyntic mucosa were increased significantly as compared with control levels after the 4-week treatment with rabeprazole. Immunohistochemistry using a histamine antibody confirmed the increase in the histamine content of the oxyntic mucosa after the 4-week treatment with rabeprazole. The finding that there were no differences in serum gastrin concentration and histidine decarboxylase activity between Ws/Ws and +/+ rats, both with and without the 4-week treatment, indicates that mast cells do not respond to endogenous hypergastrinemia elicited by acid-inhibitory treatment. Moreover, the present study clarified for the first time that enterochromaffin-like (ECL) cells in Ws/Ws rats synthesize and store histamine in response to gastrin.

Clobenpropit (VUF-9153), a new histamine H3 receptor antagonist, inhibits electrically induced convulsions in mice.

The effect of clobenpropit (VUF-9153), a new histamine H3 receptor antagonist, on electrically induced convulsions was studied in mice. Clobenpropit significantly and dose dependently decreased the duration of each convulsive phase. Its anticonvulsant effects were prevented by pretreatment with (R)-alpha-methylhistamine and imetit (VUF-8325), histamine H3 receptor agonists. These findings suggest that the effect of clobenpropit on electrically induced convulsions is due to an increase in endogenous histamine release in the brain, which is consistent with biochemical results that clobenpropit increased brain histidine decarboxylase activity dose dependently. The anticonvulsive effect of clobenpropit was antagonized by mepyramine, a histamine H1 receptor antagonist, but not by zolantidine, a histamine H2 receptor antagonist, indicating that histamine released by the anticonvulsant effect of clobenpropit interacts with histamine H1 receptors of postsynaptic neurons. The present findings of the effect of clobenpropit on electrically induced convulsions are fully consistent with those of thioperamide as described previously (Yokoyama et al., 1993, Eur. J. Pharmacol. 234, 129), supporting the hypothesis that the central histaminergic neuron system is involved in the inhibition of seizures.

Activation of sensory nerves participates in stress-induced histamine release from mast cells in rats.

To elucidate the mechanism by which stress induces rapid histamine release from mast cells, Wistar rats, pretreated as neonates with capsaicin, were subjected to immobilization stress for 2 h, and histamine release was measured in paws of anesthetized rats by using in vivo microdialysis after activation of sensory nerves by electrical or chemical stimulation. Immobilization stress studies indicated that in control rats stress induced a 2.7-fold increase in the level of plasma histamine compared to that in freely moving rats. Whereas pretreatment with capsaicin significantly decreased stress-induced elevation of plasma histamine. Microdialysis studies showed that electrical stimulation of the sciatic nerve resulted in a 4-fold increase of histamine release in rat paws. However, this increase was significantly inhibited in rats pretreated with capsaicin. Furthermore, injection of capsaicin into rat paw significantly increased histamine release in a dose-dependent manner. These results suggest that activation of sensory nerves participates in stress-induced histamine release from mast cells.

Changes in the electrical activity of locus coeruleus neurons in pregnant rats.

Under urethane anesthesia, unit activity of locus coeruleus (LC) neurons was recorded extracellularly in pregnant and non-pregnant rats. The spontaneous firing of LC neurons was found to be reduced in pregnant rats. In addition, biochemical studies indicated that noradrenaline contents in the cerebral cortex, one of the major projection sites of LC neurons, were significantly higher in pregnant than non-pregnant rats. These results suggest that the electrical activity of noradrenergic neurons in the LC is reduced during gestation.