Megumi Kikuchi

Accumulation of CRTH2-positive leukocytes in human allergic nasal mucosa.

BACKGROUND Prostaglandin D2 (PGD2) has been thought to be a potent mediator involved in allergic rhinitis because PGD2 has been recovered from the nasal lavage fluid of patients with allergic rhinitis after allergen provocation and because PGD2 receptor antagonists relieved nasal allergic symptoms in an animal model of allergic rhinitis. The inflammatory effects of PGD2 are exerted through high-affinity interactions with 2 G protein-coupled receptors: D-prostanoid receptor 1 and chemoattractant-homologous receptor expressed on TH2 cells (CRTH2). CRTH2 may mediate the recruitment of leukocytes during a nasal allergic response. OBJECTIVE To evaluate the number of CRTH2-expressing cells in allergic and nonallergic human nasal mucosa by means of immunohistochemical analysis. METHODS Human turbinates were obtained after turbinectomy from 14 patients with nasal obstruction refractory to medical therapy. To identify cells expressing the CRTH2 protein, double immunostaining was performed using anti-CRTH2 antibody and monoclonal anti-leukocyte antibodies. RESULTS The immunohistochemical study revealed that anti-CRTH2 antibody labeled eosinophils, macrophages, mast cells, T lymphocytes, epithelial cells, and submucosal glands in the nasal mucosa. CRTH2 expressions of these leukocytes in allergic nasal mucosa are significantly up-regulated compared with those in nonallergic nasal mucosa. CONCLUSION These results suggest that CRTH2 may play an important role in the recruitment of leukocytes into allergic nasal mucosa.

Localization and Up-Regulation of Cysteinyl Leukotriene-2 Receptor in Human Allergic Nasal Mucosa

BACKGROUND Cysteinyl leukotrienes (CysLTs) are lipid mediators that have been implicated in the pathogenesis of allergic rhinitis. Pharmacological studies using CysLTs indicate that 2 classes of receptors exist, namely, CysLT1 and CysLT2 receptors. The former class of receptors is sensitive to the CysLT1 antagonists currently used to treat asthma and allergic rhinitis, and its localization has been previously examined by our group using immunohistochemistry and in situ hybridization techniques. We investigated the expression and localization of the CysLT2 receptor in human nasal mucosa by western blot and immunohistochemical analyses. METHODS Human turbinates were obtained after turbinectomy from 16 patients with nasal obstruction refractory to medical therapy. To identify the cells expressing the CysLT2 receptor, double immunostaining was performed by using anti-CysLT2 receptor antibody and anti-CD31 (endothelial cell) antibody or anti-smooth muscle actin antibody. RESULTS A 39 kDa band was detected on the western blots of human turbinates samples by using the anti-CysLT2 receptor antibody. The expression level of the CysLT2 receptor in patients with nasal allergy was higher than that in patients with non-allergic rhinitis. The immunohistochemical study also showed an intense immunoreactivity for CysLT2 receptor in both vascular endothelial cells and vascular smooth muscles. CONCLUSIONS The results indicated that the CysLT2 receptor plays a primary role in the vascular responses in the upper respiratory tract.

Agonist-and TH2 cytokine-induced up-regulation of cysteinyl leukotriene receptor messenger RNA in human monocytes

Background The cysteinyl leukotrienes (CysLTs) are lipid mediators that have been implicated in the pathogenesis of allergic diseases, and their actions are mediated via specific receptors named CysLT1 receptor (CysLT1R) and CysLT2 receptor (CysLT2R). Little information is known about the role of T h 2 cytokines in the regulation of both CysLT1R and CysLT2R expression. Objective To investigate the possible modulation of both CysLT1R and CysLT2R messenger RNA (mRNA) expression, we have developed a real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay based on the TaqMan fluorescence method to quantify CysLT1R and CysLT2R mRNA in human monocytes. Methods Human monocytes were stimulated with leukotriene D 4 or interleukin (IL) 4 or IL-13, and the levels of CysLT1R and CysLT2R mRNA were measured by the quantitative RT-PCR. Results CysLT1R and CysLT2R mRNA was increased after stimulation with leukotriene D 4 . CysLT1R mRNA was augmented 150-fold after treatment with IL-4; however, no significant increase was observed in CysLT2R mRNA level. IL-13 could induce a biphasic augmentation of CysLT1R mRNA level. In contrast to IL-4, IL-13 enhanced CysLT2R mRNA level, with a maximal effect at 2 hours of incubation. Conclusions CysLT1R and CysLT2R expression can be regulated by CysLT itself and T H 2 cytokines at the transcriptional level.

Expression and localization of platelet-activating factor receptor in human nasal mucosa.

BACKGROUND Platelet-activating factor (PAF) has been thought to be a potent mediator of allergic rhinitis because PAF was recovered from the nasal lavage fluid of patients with allergic rhinitis after allergen provocation. Furthermore, PAF receptor antagonist attenuates the antigen-induced increase in nasal airway resistance and nasal vascular permeability in sensitized guinea pigs. OBJECTIVE To clarify the expression of PAF receptor in human nasal mucosa by investigating PAF receptor messenger RNA (mRNA) expression and its protein localization using polymerase chain reaction (PCR) and immunohistochemical analyses, respectively. METHODS Human turbinates were obtained after turbinectomy from 6 patients with nasal obstruction refractory to medical therapy. Total RNA was isolated from human nasal mucosa, and PAF receptor mRNA was detected in these tissues by using reverse transcriptase-PCR analysis. To identify the cells expressing PAF receptor protein, double immunostaining was performed using anti-PAF receptor antibody and monoclonal antileukocyte antibodies. RESULTS Reverse transcriptase-PCR analysis of total nasal RNA demonstrated the expression of PAF receptor mRNA. The immunohistochemical studies revealed that anti-PAF receptor antibody-labeled eosinophils, macrophages, neutrophils, mast cells, lymphocytes, vascular endothelial cells, epithelial cells, and submucosal glands in nasal mucosa. CONCLUSIONS These results may have important clinical implications for understanding the role of PAF receptor on upper airway diseases such as allergic and nonallergic rhinitis.

Distribution of specific binding sites for cysteinyl leukotriene 1 receptor antagonist in human nasal mucosa

Conclusion. The high density of [3H]-pranlukast binding sites on the local leukocytes in human nasal mucosa suggests that CysLT1 receptor antagonists may directly modulate cellular function of the local leukocytes through binding to CysLT1 receptor on allergic nasal mucosa. Objectives. The cysteinyl leukotrienes (CysLTs) are lipid mediators that have been implicated in the pathogenesis of allergic diseases. Pharmacological studies using CysLTs indicate that two classes of receptors named CysLT1 and CysLT2 receptor exist. The former is sensitive to the CysLT1 receptor antagonist currently used to treat asthma and allergic rhinitis. To confirm the binding sites of CysLT1 receptor antagonist in human nasal mucosa, the autoradiographic distribution of CysLT1 receptor was studied in human nasal inferior turbinates. Materials and methods. Cryostat sections were incubated with [3H]-pranlukast for autoradiography. Nonspecific binding was determined by adding unlabelled pranlukast. Results. Autoradiograms indicated [3H]-pranlukast densely labeled on the interstitial cells. Blood vessels were sparsely labeled. There was no specific labeling in the submucosal glands or epithelium. These results support our previous report from in situ hybridization and immunohistochemistry of CysLT1 receptor.

Expression and Localization of Purinergic P2Y12 Receptor in Human Nasal Mucosa

BACKGROUND Extracellular nucleotides such as ATP and UTP are released from essentially all cells, and they interact with cell surface P2 receptors to produce a broad range of physiological responses. P2Y12 receptor is the major platelet receptor that mediates ADP-induced aggregation, P2Y12 receptor inhibitors such as clopidogrel and prasugrel inhibit platelet aggregation, and thus, they are used in the treatment and prevention of coronary artery disease. Recently, studies have focused on the P2Y12 receptor as a receptor for leukotriene E4 (LTE4), because this receptor is required for LTE4-mediated pulmonary inflammation. To establish the presence of P2Y12 receptor in human nasal mucosa, we investigated the expression and the localization of the P2Y12 receptor in human nasal mucosa. METHODS Human turbinates were obtained by turbinectomy from 12 patients with nasal obstruction refractory to medical therapy. The expression of P2Y12 receptor was evaluated by RT-PCR, western blotting, and immunohistochemical analysis. RESULTS RT-PCR analysis of total RNA extracted from human nasal turbinate, primary cultured human nasal epithelial cells and nasal vascular endothelial cells demonstrated the expression of P2Y12 receptor mRNA. A band of approximately 55 kDa was detected in human turbinates by western blot analysis using anti-P2Y12 receptor antibody. We could not find any differences between P2Y12 receptor levels in allergic and non-allergic nasal mucosa. An immunohistochemical study revealed that epithelial cells, submucosal glands and vascular endothelial cells showed intense immunoreactivity for the P2Y12 receptor. CONCLUSIONS The results may have important clinical implications for understanding the role of P2Y12 receptor in upper airway diseases such as allergic rhinitis and non-allergic rhinitis.

Platelet-activating factor (PAF) increases NO production in human endothelial cells—real-time monitoring by DAR-4M AM

BACKGROUND Platelet-activating factor (PAF) is a potent inflammatory lipid mediator that increases vascular permeability and vasodilation. Several studies have addressed the effect of PAF on nitric oxide (NO) production from microvessels in vivo. OBJECTIVE The aim of present study was to evaluate the effect of PAF on NO production in primary cultured human vascular endothelial cells. METHODS Human umbilical vein endothelial cells (HUVECs) were loaded with diaminorhodamine-4M acetoxymethyl ester (DAR-4MAM), and the cells were stimulated with PAF. Intracellular NO production was monitored as increase in fluorescence intensity. Also, NO production was visualized at cellular levels using DAR-4M AM and fluorescence imaging. RESULTS Significant increases in NO production in HUVECs were soon after the PAF stimulation, reaching a plateau after 10 min of the stimulation. The increase of NO production at 10 min after the stimulation was statistically significant (p<0.05) for 0.01-10 microM PAF. PAF-induced NO production was abolished by pretreatment of HUVECs with a NOS inhibitor N(G)-monomethyl-L-arginine (L-NMMA) or PAF receptor antagonist BN 52021. LysoPAF, the inactive metabolite of PAF, did not exert a significant effect on intracellular NO levels. CONCLUSIONS These results provide direct evidence that PAF cause intracellular NO production via activation of PAF receptors in human vascular endothelial cells.

Clinical benefit of component-resolved diagnosis in Japanese birch-allergic patients with a convincing history of apple or peach allergy

OBJECTIVE In northern Japan, birch pollen is the major allergen in pollinosis, while oral allergy syndrome (OAS) is caused primarily by apple and peach, and is almost exclusively related to birch pollinosis. To clarify the clinical benefit of allergen-based component-resolved diagnosis (CRD) in Japanese birch-allergic patients with OAS, we present an analysis of IgE profiles in response to crude extracts and recombinant component-resolved allergen to birch pollen and Rosaceae fruits allergens. METHODS The sera of 30 patients with birch pollen-related OAS to apple or peach were analyzed for specific IgE reactivity to pathogenesis-related class 10 (PR-10) family (birch: rBet v 1, apple: rMal d 1, and peach: rPru p 1), profilin (birch: rBet v 2 and peach: rPru p 4), and lipid transfer protein (LTP) (apple: rMal d 3 and peach: rPru p 3) allergens, as well as to conventional crude, unfractionated extracts (birch: T3, apple: f49, and peach: f95) using the ImmunoCAP System™. Allergen-specific IgE values <0.35kUA/L were considered negative. RESULTS Of the 30 sera CAP-positive for natural birch pollen extract, 28 (93.3%) exhibited specific IgE against Bet v1, and two (6.7%) contained specific IgE against Bet v2. Of the 26 sera of OAS to apple patients, only 17 were positive for specific IgE against f49 extract (65.4%); however, 24 were positive for specific IgE against rMal d 1 (92.3%). Similarly, only 17 of the 23 sera of OAS to peach patients contained specific IgE against the f95 extract (73.9%); however, 22 were positive for specific IgE against rPru p 1 (95.7%). CONCLUSION Our data suggest that CRD constitutes a reliable tool for the diagnosis of birch pollen-related OAS.

Nasal Mucosal Expression of the Receptors for Inflammatory Chemical Mediators

The nasal allergic response is a complex process involving interactions between many chemical mediators such as histamine, bradykinin, cysteinyl leukotrienes, platelet-activating factor, prostaglandin D2 and thromboxane A2. The actions of these chemical mediators are facilitated by specific cell surface receptors that are coupled to G-proteins. Current therapeutic strategies against nasal allergic responses are mainly based on drugs that target these chemical mediators. To understand the role of these chemical mediators in allergic rhinitis, determining the identity and distribution of their receptors is of considerable interest. We have examined the expression and localization of the receptors for these chemical mediators in human nasal mucosa. Here, we review our data on the expression and localization of these receptors in allergic rhinitis, and we discuss the roles of chemical mediators in allergic rhinitis.