Transient receptor potential vanilloid 1 - a polymodal nociceptive receptor - plays a crucial role in formaldehyde-induced skin inflammation in mice.

Formaldehyde (FA) is irritating to the skin and is the main cause of sick building syndrome. However, the cutaneous reaction induced by long-term FA exposure has not been fully investigated. In our previous study, we demonstrated that repeated painting of 2% - 10% FA on mouse ears caused marked ear swelling and increased mRNA expression of transient receptor potential vanilloid 1 (TRPV1) and neurotrophins in the ear. TRPV1 is reported to be involved in neurogenic inflammation; therefore, in the present study, we investigated the role of TRPV1 in FA-induced skin inflammation using TRPV1 gene-knockout mice. Mice were painted with 5% FA once a week for 5 weeks, and ear swelling and mRNA expression were investigated. Ear swelling and increased expression of neurotrophins mRNA by FA provocation in wild-type mice were attenuated by disruption of the TRPV1 gene. Furthermore, painting with a threshold dose of capsaicin, which does not induce ear swelling in intact mice, caused marked ear swelling after painting the ear 5 times with FA, indicating that inflamed tissues after FA application are hypersensitive to various ligands of TRPV1 in mice. These results demonstrated that neurogenic inflammation via TRPV1 and neurotrophins could be involved in FA-induced dermatitis.

Endotoxin tolerance attenuates airway allergic inflammation in model mice by suppression of the T-cell stimulatory effect of dendritic cells.

Prior exposure of dendritic cells (DCs) and monocytes/macrophages to LPS causes unresponsiveness to subsequent LPS stimulation, a phenomenon called endotoxin tolerance (ET). ET impairs antigen presentation of these cells to T cells by down-regulating expression of MHC class II and co-stimulatory molecules such as CD86 and CD40. Some epidemiological studies have shown that endotoxin acts as a protective factor for allergic diseases. Accordingly, LPS has beneficial effects on the onset of airway allergic inflammation in model animals by T(h)1 skewing or induction of regulatory T cells. However, results derived from asthma model animals are controversial, probably due to the difficulty of handling LPS. We previously generated a monoclonal agonistic antibody against Toll-like receptor (TLR) 4, named UT12, which mimics the biological activities of LPS, exhibiting more potent and sustained ET than does LPS. In this study, we took advantage of UT12 to generate prolonged ET to explore the possibility that ET is involved in the inhibitory effects of the TLR4 signals on asthma model mice. Induction of ET by UT12 inhibited the capacity of DCs to expand ovalbumin (OVA)-specific T(h)2 and T(h)17 cells, without inducing T(h)1 cell or regulatory T-cell populations or producing inhibitory cytokines. Accordingly, administration of UT12 before the OVA sensitization significantly suppressed airway allergic inflammation by OVA inhalation. Taken together, these results demonstrate that ET induced by activating TLR4 signals attenuates airway allergic inflammation through direct suppression of the T-cell stimulatory effect of DCs in asthma model mice.

Characterization of skin inflammation induced by repeated exposure of toluene, xylene, and formaldehyde in mice.

Volatile organic compounds (VOCs) are considered the main cause of sick building syndrome and they are likely to irritate the skin, eyes, and mucous membrane; however, the toxic threshold and the mechanisms of cutaneous reaction induced by long-time VOC exposure have not been clarified. In the present study, we investigated the effect of repeated painting of VOCs onto mouse skin. Various concentrations of toluene, xylene, and formaldehyde (FA) were applied once a week for 5 weeks. While FA solution (2-10%) induced remarkable ear swelling and caused evident infiltration of inflammatory cells, high concentrations of toluene and xylene (50 or 100%) evoked mild ear swelling and marginal inflammatory cell invasion. In addition, FA exposure markedly increased the expression of interleukin-4 (IL-4), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and transient receptor potential vanilloid-1 (TRPV-1) mRNAs in the ears and IL-4 and NT-3 mRNAs in the cervical lymph nodes. Furthermore, capsazepine, a TRPV-1 antagonist, significantly suppressed ear swelling caused by repeated painting of 5% FA. These findings demonstrate that FA has more potent irritancy against skin than toluene or xylene and suggest that the Th2 response, neurotrophins and TRPV-1 play important roles in FA-induced skin inflammation.

Effect of diesel exhaust particles on house dust mite-induced airway eosinophilic inflammation and remodeling in mice.

Recent research has focused on the effects of ambient particulate pollution and much evidence has indicated that particulate pollution is associated with the onset of asthma and allergy; however, the effect of diesel exhaust particles (DEP) on the development of allergen-induced airway remodeling has not been fully investigated in vivo. In the present study, we examined the effects of DEP on Dermatophagoides farinae allergens (Der f)-induced asthma-like phenotypes in mice. Mice were administered i.t. 8 times with Der f. DEP were injected i.t. with Der f 4 times throughout the experiment or twice at the sensitization period. In both cases, DEP aggravated Der f-induced increases in airway responsiveness to acetylcholine, the number of eosinophils and neutrophils in the bronchoalveolar lavage fluid (BALF), serum Der f-specific IgG1 levels, Th2 cytokines and transforming growth factor-beta1 levels in BALF, and amount of hydroxyproline in the right lungs. Furthermore, goblet cell hyperplasia and subepithelial fibrosis were also markedly aggravated. These findings indicate that DEP can potentiate airway remodeling induced by repeated allergen challenge as well as Th2-drived airway hyperresponsiveness, eosinophilic inflammation, and IgG1 production and that DEP can exhibit adjuvant activity for airway remodeling, probably due to the enhancement of allergen sensitization and/or of Th2 polarizing pathways.

A novel CC-chemokine receptor 3 antagonist, Ki19003, inhibits airway eosinophilia and subepithelial/peribronchial fibrosis induced by repeated antigen challenge in mice.

CC-chemokine receptor 3 (CCR3) is a chemokine receptor for which major ligands, CC-chemokine ligand (CCL) 11, CCL24, and CCL26, are known to be involved in chemotaxis for eosinophils. In the present study, we evaluated the effect of a low molecular weight CCR3-receptor antagonist, Ki19003 (4-[[5-(2,4-dichlorobenzylureido)pentyl][1-(4-chlorophenyl)ethyl]amino]butanoic acid), on airway remodeling in a mouse model of allergic asthma. BALB/c mice were sensitized twice by intraperitoneal injection of ovalbumin (OA) and exposed daily to 1% OA for 3 weeks. Twenty-four hours after the final antigen challenge, bronchoalveolar lavage and histological examinations were carried out. Ki19003 clearly inhibited antigen-induced increase in the number of eosinophils in bronchoalveolar lavage fluid (BALF), but did not affect the number of other cell types examined in this study. Ki19003 also inhibited the increased production of transforming growth factor-beta1 in BALF and the amount of hydroxyproline in the lungs in a dose-dependent manner. Furthermore, Ki19003 significantly attenuated allergen-induced subepithelial and peribronchial fibrosis. These findings indicate that CCR3 antagonism prevents not only the infiltration of eosinophils into the airways but also the development of allergen-induced subepithelial and peribronchial fibrosis. Therefore, a CCR3 antagonist may be useful in the treatment of airway remodeling, especially subepithelial and peribronchial fibrosis, in allergic asthma.

The effects of inhaled KP-496, a novel dual antagonist for cysteinyl leukotriene receptor and thromboxane A(2) receptor, on allergic asthmatic responses in guinea pigs.

AIMS: The aim of this study was to evaluate the effects of inhaled KP-496, a novel dual antagonist for cysteinyl leukotriene receptor 1 and thromboxane A(2) receptor, on the allergic asthmatic responses in guinea pigs. METHODS: Actively sensitized animals were repeatedly exposed to antigen, and KP-496 (0.01 and 0.1%) was inhaled for 5 min before every antigen exposure. After evaluating the effects of KP-496 on asthmatic responses, such as immediate and late asthmatic response (IAR and LAR) and airway hyperresponsiveness (AHR), histopathological analyses of the lungs of asthmatic animals were made. RESULTS: KP-496 significantly inhibited both antigen-induced LAR and AHR to acetylcholine, and slightly inhibited antigen-induced IAR. Furthermore, histopathological analyses of the lungs of the asthmatic animals demonstrated the following: (1) KP-496 suppressed infiltration of eosinophils around airway smooth muscle, (2) KP-496 suppressed airway epithelial hypertrophy, and (3) KP-496 suppressed increased mucus production in the airway. CONCLUSION: In addition to suppression of LAR and AHR, our findings demonstrated that KP-496 inhibits features of airway inflammation. Since these broad ameliorative effects of KP-496 on asthmatic pathology are thought to result from the inhibition of multiple chemical mediators, KP-496 will be a potent agent in the treatment of bronchial asthma.

A small amount of tetrachloroethylene ingestion from drinking water accelerates antigen-stimulated allergic responses.

Previously, we observed that tetrachloroethylene (perchloroethylene, PCE) increased histamine release and inflammatory mediator production from antigen-stimulated mast cells. In this study, we examined the enhancing effect of low concentrations of PCE in drinking water on antigen-stimulated allergic responses. After exposure of Wistar rats to PCE in drinking water for 2 or 4 weeks, we performed a passive cutaneous anaphylaxis (PCA) reaction. PCE exposure for 4 weeks enhanced PCA reaction in a dose-dependent manner. In pathological studies, PCE exposure for 2 weeks exacerbated inflammation characterized by infiltration of lymphocytes and accumulation of mast cells around the vessel. Non-purified mast cells (NPMCs) from rats treated with 1mg/L PCE in drinking water for 2 weeks increased antigen-stimulated histamine release. Furthermore, the leukocytes of rats treated with PCE in drinking water for 4 weeks showed increased interleukin (IL)-4 expression. The mechanism of enhancing the PCA reaction is assumed to be that PCE increases IL-4 production and PCE causes T helper (Th) 1/Th2-type helper T-cell imbalance and increases histamine release from excessively accumulated mast cells. The results suggest that the intake of PCE in drinking water, even at a low concentration, leads to the initiation and acceleration of allergic diseases.

Immunomodulatory effects of CpG oligodeoxynucleotides on house dust mite-induced airway inflammation in mice.

BACKGROUND: CpG oligodeoxynucleotides (CpG ODNs) are reported to protect against airway eosinophilia and hyperresponsiveness in animal models of asthma. However, little is known about the effects of CpG ODNs on house dust mites, one of the most common environmental allergens, causing allergic asthma. In the present study, we evaluated the immunomodulatory effects of CpG ODNs on the development of house dust mite-induced airway inflammation and remodeling in mice. METHODS: Mice were instilled with Dermatophagoides farinae (Der f) into the trachea 8 times without any additional adjuvants. 48 h after the final allergen instillation, the airway responsiveness to acetylcholine (Ach) was measured, and bronchoalveolar lavage (BAL) and histopathological examination were carried out. CpG ODNs were instilled into the trachea mixed with Der f at the first allergen instillation. RESULTS: Repeated instillation of Der f induced increases in airway responsiveness to Ach, the numbers of inflammatory cells, the levels of T-helper type 2 cytokines and transforming growth factor-beta(1) in the BAL fluid. Furthermore, goblet cell hyperplasia, the thickness of the epithelium and subepithelial fibrosis were observed. The simultaneous instillation of CpG ODNs with Der f at the first allergen instillation showed significant inhibition of these parameters dose dependently. CONCLUSIONS: Our results demonstrate that CpG ODNs have inhibitory effects on Der f-induced airway hyperresponsiveness and eosinophilia, as well as airway remodeling, and that CpG ODNs can be a therapeutic approach for the treatment of house dust mite-induced asthma.

Repeated instillations of Dermatophagoides farinae into the airways can induce Th2-dependent airway hyperresponsiveness, eosinophilia and remodeling in mice: effect of intratracheal treatment of fluticasone propionate.

Dermatophagoides farinae are known to be a common environmental allergen causing allergic asthma; however, little is known about their pathophysiological effect via the allergenicities in vivo. Therefore, we first established a mouse model of asthma induced by repeated instillations of D. farinae. Second, to investigate whether the asthmatic responses are Th2-dependent, we examined the effect of the deficiency of interleukin-4 (IL-4) receptor alpha chain gene. Finally, we examined the effect of fluticasone propionate on this model. Mice were instilled with D. farinae without additional adjuvants into the trachea 8 times. After the final allergen instillation, the airway responsiveness to acetylcholine was measured, and bronchoalveolar lavage and histological examination were carried out. The instillation of the allergen-induced airway hyperresponsiveness, the accumulation of inflammatory cells and increases in the levels of Th2 cytokines and transforming growth factor-beta(1) production in the bronchoalveolar lavage fluid dose dependently. The number of goblet cells in the epithelium and the extent of the fibrotic area beneath the basement membrane were also increased in the morphometric study. In contrast, the defect of IL-4/IL-13 signaling through IL-4 receptor alpha chain completely abrogated all these responses. Furthermore, the simultaneous instillation of fluticasone propionate with the allergen showed significant inhibition or an inhibitory tendency of these changes. These findings demonstrate that the repetitive intratracheal instillations of D. farinae can induce airway remodeling through Th2-type inflammation, and that fluticasone propionate inhibits D. farinae-induced airway remodeling in mice, and this model would be useful for studying mechanisms involved in the development of allergic asthma.

Inhibitory effects of Piper betle on production of allergic mediators by bone marrow-derived mast cells and lung epithelial cells.

The leaves of the Piper betle Linn. (Piperaceae) are used in traditional medicine and possess anti-oxidant, anti-bacterial, anti-fungal, anti-diabetic and radioprotective activities. However, little is known about their anti-allergic activity. Therefore, the effects of P. betle ethanolic extract (PE) on the production of histamine and granulocyte macrophage-colony-stimulating factor (GM-CSF) by murine bone marrow mast cells (BMMCs) and on the secretion of eotaxin and IL-8 by the human lung epithelial cell line, BEAS-2B, were investigated in vitro. PE significantly decreased histamine and GM-CSF produced by an IgE-mediated hypersensitive reaction, and inhibited eotaxin and IL-8 secretion in a TNF-alpha and IL-4-induced allergic reaction. The results suggest that P. betle may offer a new therapeutic approach for the control of allergic diseases through inhibition of production of allergic mediators.

Enhancing effect of chlorinated organic solvents on histamine release and inflammatory mediator production.

We investigated the effect of several chlorinated organic solvents on antigen-induced histamine release and inflammatory mediator production. Non-purified rat peritoneal mast cells (NPMC) and rat basophilic leukemia (RBL-2H3) cells were sensitized with anti-dinitrophenol (DNP) monoclonal IgE antibody, and then stimulated with DNP-conjugated bovine serum albumin (DNP-BSA) and several chlorinated organic solvents. Trichloroethylene (TCE) and tetrachloroethylene (PCE) enhanced histamine release from antigen-stimulated NPMC and RBL-2H3 in a dose-dependent manner. In addition, TCE and PCE increased IL-4 and TNF-alpha production from antigen-stimulated RBL-2H3. In an in vivo study, we investigated the effect of TCE and PCE on passive cutaneous anaphylaxis (PCA) reaction. TCE and PCE enhanced PCA reaction markedly. These results suggest that TCE and PCE increase histamine release and inflammatory mediator production from antigen-stimulated mast cells via the modulation of immune responses. In addition, exposure to TCE and PCE may lead to the augmentation of allergic diseases.

Augmentation of antigen-stimulated allergic responses by a small amount of trichloroethylene ingestion from drinking water.

In previous report, we have shown that trichloroethylene (TCE) increases histamine release and inflammatory cytokine production from antigen-stimulated mast cells. In this study, we examined the enhancing effect of a small amount of TCE ingestion from drinking water on antigen-stimulated allergic responses. After exposure of Wistar rats to TCE ingestion for 2 or 4 weeks, we performed a passive cutaneous anaphylaxis (PCA) reaction. TCE ingestion for 2 and 4 weeks enhanced PCA reaction in a dose-dependent manner. On histological examination, TCE ingestion for 2 weeks exacerbated inflammation characterized by infiltration of lymphocytes and accumulation of mast cells around the vessel in the skin. After TCE ingestion for 4 weeks, the mesenteric lymph nodes (MLNs) showed increase of the size and wet weight, and germinal centers changed distinctly. The interleukin-4 (IL-4) mRNA levels on spleen, MLNs and leukocytes were increased. Moreover, serum total IgE levels of TCE ingestion increased in a time-dependent manner. Our results suggest that TCE ingestion induces pro-inflammatory responses and causes Th1/Th2-type helper T-cell imbalance. And more, a small amount of TCE ingestion may lead to the initiation and acceleration of type I allergic reaction.

Inhibition of scratching behavior associated with allergic dermatitis in mice by tacrolimus, but not by dexamethasone.

Itching is the most important problem in many allergic and inflammatory skin diseases especially in atopic dermatitis. However, animal models for allergic dermatitis useful for the study of itching have rarely been established. We established a mouse allergic dermatitis model involving frequent scratching behavior by repeated painting with 2,4-dinitrofluorobenzene (DNFB) acetone solution onto the mouse skin, and comparatively examined the effects of tacrolimus and dexamethasone on the dermatitis and associated scratching behavior. Repeated DNFB painting caused typical dermatitis accompanied by elevated serum immunoglobulin E (IgE) and frequent scratching behavior. An apparent thickening of the epidermis and dermis, and the significant accumulation of inflammatory cells were observed. Increased interferon (IFN)-gamma mRNA expression and the induction of interleukin (IL)-4 and IL-5 mRNA expression were also observed in the skin lesion. The scratching behavior was inhibited by dibucaine and naloxone. Although tacrolimus reduced the increased expression of IFN-gamma and IL-4 mRNA, dexamethasone potently depressed that of IFN-gamma, IL-4 and IL-5 mRNA. Dexamethasone inhibited the accumulation of lymphocytes and eosinophils, although tacrolimus did not. Both drugs failed to inhibit the elevation of serum IgE levels. Tacrolimus significantly inhibited the scratching behavior that was associated with the inhibition of nerve fiber extension into the epidermis, whereas dexamethasone failed to have any effect. The mouse dermatitis model seems to be beneficial for the study of itching associated with allergic dermatitis, such as atopic dermatitis, and tacrolimus seems to exhibit an anti-itch effect through the inhibition of nerve fiber extension at least in part.

Minocycline inhibits oxidative stress and decreases in vitro and in vivo ischemic neuronal damage.

The neuroprotective effects of minocycline-which is broadly protective in neurologic-disease models featuring cell death and is being evaluated in clinical trials-were investigated both in vitro and in vivo. For the in vivo study, focal cerebral ischemia was induced by permanent middle cerebral artery occlusion in mice. Minocycline at 90 mg/kg intraperitoneally administered 60 min before or 30 min after (but not 4 h after) the occlusion reduced infarction, brain swelling, and neurologic deficits at 24 h after the occlusion. For the in vitro studies, we used cortical-neuron cultures from rat fetuses in which neurotoxicity was induced by 24-h exposure to 500 microM glutamate. Furthermore, the effects of minocycline on oxidative stress [such as lipid peroxidation in mouse forebrain homogenates and free radical-scavenging activity against diphenyl-p-picrylhydrazyl (DPPH)] were evaluated to clarify the underlying mechanism. Minocycline significantly inhibited glutamate-induced cell death at 2 microM and lipid peroxidation and free radical scavenging at 0.2 and 2 microM, respectively. These findings indicate that minocycline has neuroprotective effects in vivo against permanent focal cerebral ischemia and in vitro against glutamate-induced cell death and that an inhibition of oxidative stress by minocycline may be partly responsible for these effects.

Role of Th2 responses in the development of allergen-induced airway remodelling in a murine model of allergic asthma.

(1) To clarify the involvement of Th2 responses in the development of allergen-induced airway remodelling, we investigated the effect of anti-CD4 monoclonal antibody (mAb) and anti-CD8 mAb, and the responses of IL-4 gene-knockout (KO) mice in a murine model of allergic asthma. (2) Mice were immunized twice by intraperitoneal injections of ovalbumin (OA), and exposed to aeroallergen (OA, 1% w v(-1)) for 3 weeks. Twenty-four hours after the final challenge, airway responsiveness to acetylcholine was measured, and bronchoalveolar lavage (BAL) and histological examinations were carried out. (3) Anti-CD4 mAb (1 mg kg(-1)) clearly inhibited allergen-induced increases in airway responsiveness to acetylcholine, the number of eosinophils in BAL fluid, serum OA-specific IgE levels, IL-13 and transforming growth factor-beta1 levels in BAL fluid, and amount of hydroxyproline in the lung by 100, 99, 100, 100, 84, and 60%, respectively. Furthermore, the antibody (1 mg kg(-1)) also attenuated allergen-induced goblet cell hyperplasia in the epithelium and subepithelial fibrosis by 72 and 83%, respectively. In contrast, anti-CD8 mAb (1 mg kg(-1)) showed no effect on each parameter. Furthermore, all these parameters were attenuated in IL-4KO mice by 57, 93, 100, 45, 84 and 60%, and also 72 and 83%, respectively. (4) These findings suggest that Th2 responses play a critical role for the development of allergen-induced airway remodelling, and that the inhibition of Th2 responses, e.g. using anti-CD4 mAb, is a therapeutic approach for the treatment of airway remodelling in asthma.

Augmentation of allergic inflammation in prostanoid IP receptor deficient mice.

1 To evaluate the role of prostaglandin I(2) (PGI(2)) in allergic inflammation, allergic responses in the airway, skin and T cells were studied in mice lacking the receptor for PGI(2) (the prostanoid IP receptor) through gene disruption. 2 Three inhalations of antigen caused an increase in plasma extravasation, leukocyte accumulation and cytokine (interleukin (IL)-4 and IL-5) production in the airway of sensitized mice. These airway inflammatory responses were significantly greater in IP receptor deficient mice than in wild-type mice. 3 The vascular leakage caused by passive cutaneous anaphylaxis, substance P and 5-hydroxytryptamine was markedly increased in the skin of IP receptor deficient mice, compared with comparably treated wild-type mice. 4 The inhalation of antigen in sensitized mice resulted in increased serum antigen specific IgE, total IgE and IgG levels. The magnitude of the elevations of each immunoglobulin level in IP receptor deficient mice is notably higher than that in wild-type mice. To elucidate the mechanism of an enhancement of immunoglobulin production, the activity of T cells in sensitized and non-sensitized mice was studied by means of the production of cytokines. The antigen-induced IL-4 production by spleen cells from sensitized IP receptor deficient mice was almost three times greater than that in wild-type mice. On the contrary, the anti-CD3 antibody-induced interferon-gamma production by CD4(+) T cells from non-sensitized IP receptor deficient mice was significantly lower than that in wild-type mice. 5 The present data indicate that IP receptor deficiency reinforced an allergic airway and skin inflammation by augmentation of vascular permeability increase and the T helper 2 cell function. These findings suggest a regulatory role of PGI(2) in allergic inflammation.

Induction of PYPAF1 during in vitro maturation of mouse mast cells.

Coculture of mouse bone marrow-derived immature mast cells (BMMC) with Swiss 3T3 fibroblasts in the presence of stem cell factor (SCF) promotes morphological and functional maturation toward a connective tissue mast cell (CTMC)-like phenotype, which is accompanied by increased expression of several unique genes. Here we report the molecular identification of one of them, mast cell maturation-associated inducible gene (MMIG)-1. The MMIG-1 cDNA encodes a 117-kDa cytosolic protein that comprises an N-terminal PYRIN domain, a central nucleotide-binding domain, and nine C-terminal leucine-rich repeats. MMIG-1 shows >85% sequence similarity to human cryopyrin/PYPAF1, a causal gene for familial cold urticaria and Muckle-Wells syndrome. MMIG-1 was distributed in the cytosol of CTMC-like differentiated BMMC. MMIG-1 underwent alternative splicing in the leucine-rich repeats and each variant was induced differently in BMMC during coculture. Moreover, its expression was increased in the ears of mice with experimental atopic dermatitis. Thus, MMIG-1, a likely mouse PYPAF1 ortholog, may play a role in mast cell-directed inflammatory diseases.

Involvement of unique mechanisms in the induction of scratching behavior in BALB/c mice by compound 48/80.

Compound 48/80 induced scratching behavior in BALB/c mice, and the role of mast cell mediators in this behavior was examined. Mouse scratching behavior was detected and evaluated using a new apparatus, MicroAct. Compound 48/80 increased the incidence of scratching behavior and scratching time in a dose-dependent manner, accompanied by a potent activation of mast cells and a potent increase in vascular permeability. Dibucaine and mu-opioid receptor antagonists inhibited the scratching behavior. Although histamine H(1) receptor antagonists potently inhibited the vascular permeability increase, they did not affect the scratching behavior. Methysergide inhibited the scratching behavior slightly without affecting the vascular permeability increase, whereas cyproheptadine inhibited both. A cyclooxygenase inhibitor, a 5-lipoxygenase-activating protein inhibitor and a PAF receptor antagonist did not affect the scratching behavior. High doses of serotonin induced scratching behavior less frequently than did compound 48/80. Furthermore, mast cell-deficient WBB6F1-W/W(v) mice exhibited frequent scratching behavior after injection of compound 48/80. These results clearly indicate that compound 48/80 can induce scratching behavior in mice independent of mast cell mediators.

Effect of MX-68 on airway inflammation and hyperresponsiveness in mice and guinea-pigs.

MX-68 is a newly synthesized antifolate, which is a derivative of methotrexate (MTX). In this paper, the effect of MX-68 on allergic airway responses in mice and guinea-pigs was studied. In the first experiment, antigen-induced airway inflammation and airway hyperresponsiveness (AHR) to acetylcholine in mice were examined and compared with the effects of classical antifolate methotrexate and prednisolone. Mice were sensitized with ovalbumin as an antigen and challenged with ovalbumin inhalation three times. After the last inhalation, AHR and airway inflammation were observed. An increase in Th2 cytokines (IL-4 and IL-5) and a decrease in a Th1 cytokine (IFN-gamma) in the bronchoalveolar lavage fluid (BALF), as well as an elevation of the immunoglobulin level in serum, were observed in sensitized mice. Oral administration of MX-68 had no effect on changes of body weight, but prednisolone reduced body weight during the experiment. The antigen-induced AHR and increases in the number of eosinophils and lymphocytes in BALF were significantly inhibited by MX-68. MX-68 interfered with the elevation of IL-4 and IL-5 levels in BALF, but had no effect on the decrease in IFN-gamma. Moreover, MX-68 significantly inhibited the elevation of serum IgE and IgG levels. In the guinea-pig model for bronchial asthma, biphasic increases in airway resistance (immediate asthmatic response, IAR, and late asthmatic response, LAR), as well as accumulated inflammatory cells in BALF, were observed after repeated antigen challenge. These asthmatic responses and inflammatory signs were significantly decreased by administration of MX-68. These results suggest that MX-68 obviously has an anti-inflammatory effect in an animal model of asthma and would be useful clinically for the treatment of bronchial asthma.