Inhibitory effects of geranium essential oil and its major component, citronellol, on degranulation and cytokine production by mast cells.

We investigated the effects of geranium essential oil (GEO) on anaphylaxis. GEO can exert antioxidant and anti-inflammatory effects, but its roles in allergic reactions are incompletely understood. Here, we used mouse cells to show that GEO inhibited the degranulation of cultured mast cells (CMCs). Citronellol is the major component of GEO and inhibited CMC degranulation. The l-enantiomer of citronellol more effectively suppressed CMC degranulation than did d-citronellol. We also examined whether citronellol could inhibit the immunoglobulin (Ig) E-induced production of tumor necrosis factor (TNF)-α. Treatment with various concentrations of citronellol before CMC activation with IgE significantly inhibited the induction of TNF-α in a dose-dependent manner. Mechanistically, citronellol suppressed the phosphorylation of mitogen-activated protein kinase (ERK), which is critical for ERK activation and the production of inflammatory cytokines in mast cells. These findings suggest that citronellol may represent a candidate compound for the effective treatment of allergic diseases.

Suppression of allergic and inflammatory responses by essential oils derived from herbal plants and citrus fruits.

The aim of the present study was to investigate the biological activity of 20 essential oils (EOs) derived from herbal plants and citrus fruits. The in vitro anti-allergic and anti-inflammatory activities of these oils were investigated, and the EO which was found to have the strongest activity of the 20 EOs examined, was investigated further to identify its components and bioactive compounds. The in vitro anti-allergic activity was determined by measuring the release of ß-hexosaminidase from rat basophilic leukemia (RBL-2H3) cells treated with the calcium ionophore, A23187. The in vitro anti-inflammatory activity was determined by measuring the production of tumor necrosis factor-α (TNF-α) in RAW264.7 murine macrophages treated with lipopolysaccharide. Among the EOs examined, lemongrass [Cymbopogon citratus (DC.) Stapf] elicited the strongest anti-allergic and anti-inflammatory effects. A principal component of this EO is citral (3,7-dimethyl-2,6-octadien-1-al) (74.5%), a mixture of the stereoisomers, geranial (trans-citral, 40.16%) and neral (cis-citral, 34.24%), as determined by chromatography-mass spectrometry analysis. The activities of citral and geranial are similar to those of lemongrass EO. These compounds elicited significant in vivo anti-allergic and anti-inflammatory effects, suppressing an immunoglobulin E (IgE)-induced passive cutaneous anaphylactic reaction in mice and a 12-O-tetradecanoylphorbol-13-acetate-induced inflammatory mouse ear edema, respectively. Our data demonstrate that lemongrass EO and its constituents, citral and geranial, may be a therapeutic candidate for allergic and inflammatory diseases.

Effects of essential oils from herbal plants and citrus fruits on DNA polymerase inhibitory, cancer cell growth inhibitory, antiallergic, and antioxidant activities.

In this study, the biological activity of 20 essential oils (EOs) from herbal plants and citrus fruits were investigated in terms of mammalian DNA polymerase (pol) inhibitory activity, cancer cell (human colon carcinoma, HCT116) growth inhibitory activity, antiallergic activity, as anti-ß-hexosaminidase release activity in rat basophilic leukemia RBL-2H3 cells treated with calcium ionophore A23187, and antioxidant activity by a lipophilic-oxygen radical absorbance capacity method. These EOs showed patterns of inhibition of pol α, a DNA replicative pol, similar to their cancer cell growth inhibitory activity, and their inhibitory activity on pol λ, a DNA repair/recombination pol, by the EOs showed correlation with anti-ß-hexosaminidase release activity. Among these EOs, chamomile (Matricaria chamomilla L.) was the strongest inhibitor of pols α and λ and showed significant effects on both cancer cell growth and mast cell degranulation. On the basis of these results, chamomile EO can be recommended as a potentially useful, bioactive candidate for therapeutic applications.

Inhibitory effects of water-soluble low-molecular-weight ß-(1,3-1,6) D-glucan isolated from Aureobasidium pullulans 1A1 strain black yeast on mast cell degranulation and passive cutaneous anaphylaxis.

We investigated the effects of water-soluble low-molecular-weight ß-(1,3-1,6) D-glucan isolated from Aureobasidium pullulans 1A1 strain black yeast (LMW-ß-glucan) on mast cell-mediated anaphylactic reactions. Although it is known that LMW-ß-glucan has anti-tumor, anti-metastatic and anti-stress effects, the roles of LMW-ß-glucan in immediate-type allergic reactions have not been fully investigated. We examined whether LMW-ß-glucan could inhibit mast cell degranulation and passive cutaneous anaphylaxis (PCA). LMW-ß-glucan dose-dependently inhibited the degranulation of both rat basophilic leukemia (RBL-2H3) and cultured mast cells (CMCs) activated by calcium ionophore A23187 or IgE. However, LMW-ß-glucan had no cytotoxicity towards RBL-2H3 cells and CMCs. Furthermore, orally administered LMW-ß-glucan inhibited the IgE-induced PCA reaction in mice. These results show LMW-ß-glucan to be a possible compound for the effective therapeutic treatment of allergic diseases.

Inhibitory effects of guarana seed extract on passive cutaneous anaphylaxis and mast cell degranulation.

This study investigated the effects of guarana seed extract (GSE) on an anti-allergic mechanism. GSE orally administered inhibited the anti-dinitrophenol IgE-induced passive cutaneous anaphylaxis reaction in mice. Furthermore, it inhibited the degranulation of rat basophilic leukemia RBL-2H3 cells. It had no cytotoxicity on RBL-2H3 cells. These results show that GSE is a candidate for effective therapeutic material for allergic diseases.

Number of mast cells in the peritoneal cavity of mice: influence of microphthalmia transcription factor through transcription of newly found mast cell adhesion molecule, spermatogenic immunoglobulin superfamily.

The mi (microphthalmia) locus of mice encodes a transcription factor, MITF. B6-tg/tg mice that do not express any MITF have white coats and small eyes. Moreover, the number of mast cells decreased to one-third that of normal control (+/+) mice in the skin of B6-tg/tg mice. No mast cells were detectable in the stomach, mesentery, and peritoneal cavity of B6-tg/tg mice. Cultured mast cells derived from B6-tg/tg mice do not express a mast cell adhesion molecule, spermatogenic immunoglobulin superfamily (SgIGSF). To obtain in vivo evidence for the correlation of nonexpression of SgIGSF with decrease in mast cell number, we used another MITF mutant, B6-mi(vit)/mi(vit) mice that have a mild phenotype, ie, black coat with white patches and eyes of normal size. B6-mi(vit)/mi(vit) mice had a normal number of mast cells in the skin, stomach, and mesentery, but the number of peritoneal mast cells decreased to one-sixth that of +/+ mice. Cultured mast cells and peritoneal mast cells of B6-mi(vit)/mi(vit) mice showed a reduced but apparently detectable level of SgIGSF expression, demonstrating the parallelism between mast cell number and expression level of SgIGSF. The number of peritoneal mast cells appeared to be influenced by MITF through transcription of SgIGSF.

Roles of MITF for development of mast cells in mice: effects on both precursors and tissue environments.

The mutant tg/tg mice, which do not express mi transcription factor (MITF), lack mast cells in most tissues. Since MITF is expressed in both mast cells and tissues where mast cells develop, there is a possibility that the tg/tg mice may show abnormalities in both mast cell precursors and tissue environments. We examined this possibility by bone marrow and skin transplantation. When bone marrow cells of tg/tg mice were transplanted to W/W(v) mice that possess normal tissue environment, mast cells did not develop in all tissues examined. The number of developing mast cells in the skin of W/W(v) mice was much lower when grafted to tg/tg recipients than when grafted to normal (+/+) recipients. These results indicated that mast cell precursors of tg/tg mice were defective. When bone marrow cells of +/+ mice were transplanted, the number of developing mast cells was significantly lower in examined tissues of tg/tg recipients than in those of W/W(v) recipients, suggesting that the tissue environment for mast cell development was defective in tg/tg mice. MITF appeared essential for the function of both mast cell precursors and tissue environments for their development.

Contribution of the SgIGSF adhesion molecule to survival of cultured mast cells in vivo.

Spermatogenic immunoglobulin superfamily (SgIGSF) is a recently identified adhesion molecule, and the microphthalmia transcription factor (MITF) was essential for its expression in mast cells. Since the tg mutant allele is practically a null mutation of the MITF gene, cultured mast cells (CMCs) derived from (WBxC57BL/6)F(1) (F(1))-tg/tg mice did not express SgIGSF whereas CMCs from F(1)-wild-type (+/+) mice expressed it abundantly. When cocultured with NIH/3T3 fibroblasts, F(1)-tg/tg CMCs showed poor adhesion to NIH/3T3 fibroblasts. When injected intraperitoneally, F(1)-tg/tg CMCs showed poor survival in the peritoneal cavity of mast cell-deficient F(1)-W/Wv mice. SgIGSF was expressed in tg/tg CMCs ectopically through retroviral transfection and through expression of a transgene. The resulting tg/tg CMCs showed not only a better adhesion to NIH/3T3 fibroblasts but also a better survival in the peritoneal cavity than control F(1)-tg/tg CMCs. SgIGSF-mediated adhesion seemed to play a role in the survival of CMCs in the peritoneal cavity.

Effect of anatomical distribution of mast cells on their defense function against bacterial infections: demonstration using partially mast cell-deficient tg/tg mice.

Mast cells were depleted in the peritoneal cavity of WBB6F1-tg/tg mice that did not express a transcription factor, MITF. When acute bacterial peritonitis was induced in WBB6F1-+/+, WBB6F1-W/Wv, and WBB6F1-tg/tg mice, the proportion of surviving WBB6F1-+/+ mice was significantly higher than that of surviving WBB6F1-W/Wv or WBB6F1-tg/tg mice. The poor survival of WBB6F1-W/Wv and WBB6F1-tg/tg mice was attributed to the deficient influx of neutrophils into the peritoneal cavity. The injection of cultured mast cells (CMCs) derived from WBB6F1-+/+ mice normalized the neutrophil influx and reduced survival rate in WBB6F1-W/Wv mice, but not in WBB6F1-tg/tg mice. This was not attributable to a defect of neutrophils because injection of TNF-alpha increased the neutrophil influx and survival rate in both WBB6F1-W/Wv and WBB6F1-tg/tg mice. Although WBB6F1-+/+ CMCs injection normalized the number of mast cells in both the peritoneal cavity and mesentery of WBB6F1-W/Wv mice, it normalized the number of mast cells only in the peritoneal cavity of WBB6F1-tg/tg mice. Mast cells within the mesentery or mast cells in the vicinity of blood vessels appeared to play an important role against the acute bacterial peritonitis. WBB6F1-tg/tg mice may be useful for studying the effect of anatomical distribution of mast cells on their antiseptic function.

SgIGSF: a new mast-cell adhesion molecule used for attachment to fibroblasts and transcriptionally regulated by MITF.

Microphthalmia transcription factor (MITF) is a basic-helix-loop-helix-leucine zipper-type transcription factor. The mutant mi and Mi(wh) alleles encode MITFs with deletion and alteration of a single amino acid, respectively, whereas the tg is a null mutation. In coculture with NIH/3T3 fibroblasts, the numbers of cultured mast cells (CMCs) derived from C57BL/6 (B6)(mi/mi), B6(Miwh/Miwh), and B6(tg/tg) mice that adhered to NIH/3T3 fibroblasts were one third as large as the number of B6(+/+) CMCs that adhered to NIH/3T3 fibroblasts. From a cDNA library of B6(+/+) CMCs, we subtracted messenger RNAs expressed by B6(mi/mi) CMCs and found a clone encoding SgIGSF, a recently identified member of the immunoglobulin superfamily. Northern and Western blot analyses revealed that SgIGSF was expressed in B6(+/+) CMCs but not in CMCs derived from MITF mutants. Immunocytochemical analysis showed that SgIGSF localized to the cell-to-cell contact areas between B6(+/+) CMCs and NIH/3T3 fibroblasts. Transfection of B6(mi/mi) and B6(tg/tg) CMCs with SgIGSF cDNA normalized their adhesion to NIH/3T3 fibroblasts. NIH/3T3 fibroblasts did not express SgIGSF, indicating that SgIGSF acts as a heterophilic adhesion molecule. Transfection of B6(tg/tg) CMCs with normal MITF cDNA elevated their SgIGSF expression to normal levels. These results indicated that SgIGSF mediated the adhesion of CMCs to fibroblasts and that the transcription of SgIGSF was critically regulated by MITF.

Additive effect of mouse genetic background and mutation of MITF gene on decrease of skin mast cells.

The mi transcription factor (MITF) is a basic-helix-loop-helix leucine zipper transcription factor and is encoded by mi locus. The mi/mi mutant mice showed a significant decrease of skin mast cells in C57BL/6 (B6) genetic background but not in WB genetic background. Kit ligand (KitL) is the most important growth factor for development of mast cells, and the decrease of skin mast cells in B6-mi/mi mice was attributable to the reduced expression of c-kit receptor tyrosine kinase (KIT) that is a receptor for KitL. However, the expression level of KIT in WB-mi/mi mast cells was comparable with that of B6-mi/mi mast cells, suggesting that a factor compensating the reduced expression of KIT was present in WB-mi/mi mice. By linkage analysis, such a factor was mapped on chromosome 10. The mapped position was closely located to the KitL locus. Two alternative spliced forms are known in KitL mRNA: KL-1 and KL-2. Soluble KitL, which is important for development of skin mast cells, is produced more efficiently from KL-1 mRNA than from KL-2 mRNA. The KL-1/KL-2 ratio was higher in WB-mi/mi than in B6-mi/mi mice, suggesting that the larger amount of soluble KitL may compensate for the reduced expression of KIT in WB-mi/mi mice.

Effect of MITF on mast cell differentiation.

The mi transcription factor (MITF) is a basic helix-loop-helix leucine zipper (bHLH-Zip) transcription factor and encoded by the mi locus of mice. Double gene dose of mutant allele at the mi locus results the decrease of mast cells and phenotypic abnormalities of mast cells. Various mutations have been reported at the mi locus. We divided them to null and inhibitory mutations. The tg is a typical null mutation due to the insertion of a transgene into the promoter region of MITF gene. Adult mice of tg/tg genotype can easily obtained and are a potentially useful tool for studying development and function of mast cells.

Dual abnormal effects of mutant MITF encoded by Mi(wh) allele on mouse mast cells: decreased but recognizable transactivation and inhibition of transactivation.

MITF is a basic helix-loop-helix leucine zipper-type transcription factor and is important for development of mast cells. MITF encoded by Mi(wh) allele (Mi(wh)-MITF) was mutated at a single amino acid of basic domain, and possessed a deficient but apparent DNA-binding ability. Here, we characterized the unique effects of Mi(wh)-MITF on the expression of mast cell-related genes. The expression level of mouse mast cell protease (mMCP)-4, -5, and -6 genes in Mi(wh)/Mi(wh) cultured mast cells (CMCs) was intermediate between levels of normal (+/+) CMCs and tg/tg CMCs, which did not express any MITFs. Mi(wh)-MITF appeared to show the positive transactivation effect through the remaining DNA-binding ability. On the other hand, the expression level of tryptophan hydroxylase gene was lower in Mi(wh)/Mi(wh) CMCs than in tg/tg CMCs, suggesting the inhibitory effect of Mi(wh)-MITF on the transactivation. Mi(wh)-MITF possessed dual abnormal effects on transactivation of mast cell-related genes.

Regulation of mast cell phenotype by MITF.

The development of mast cells is controlled through the cooperative effects of growth factors and nuclear transcription factors. The signals generated by the binding of stem cell factor (SCF) to c-kit receptor tyrosine kinase (KIT) are essential for their development and survival. A double gene dose of mutant alleles at either the SCF or KIT locus results in a decrease of mast cells. A double gene dose of mutant alleles at the mi transcription factor (MITF) locus also results in mast cell deficiency. Although the phenotype of the few mast cells remaining in SCF and KIT mutant mice appeared to be normal, the phenotype of mast cells was abnormal in MITF mutant mice. We describe here the abnormalities of mast cells observed in MITF mutant mice.

Isoforms of mi transcription factor preferentially expressed in cultured mast cells of mice.

MITF is a basic helix-loop-helix leucine zipper transcription factor, which is important for normal phenotypic expression of mast cells. Three isoforms of MITF have been known in mice, MITF-A, -H, and -M. Since cultured mast cells (CMCs) are useful for studying the function of MITF, we examined isoforms of MITF expressed in CMCs using 5'-RACE, and found a new isoform of MITF, MITF-E. We assessed the relative mRNA amount of various MITF isoforms with reverse transcription-PCR. When the mRNA amount of MITF-E was used as a standard, that of MITF-M was approximately 10%, that of MITF-H was approximately 1%, and that of MITF-A was approximately 0.1%. Although MITF-E was the preferential isoform in CMCs, peritoneal mast cells expressed only MITF-M. The expression profile of MITF isoforms appeared to be influenced by the developing process of mast cells.