Leukotriene D4 induces chemotaxis in human eosinophilc cell line, EoL-1 cells via CysLT1 receptor activation.

Numerous reports have shown that cysteinyl leukotrienes (CysLTs) contribute to tissue accumulation of eosinophils in allergic airway inflammation. To date, only a few studies have reported that CysLTs promote chemotactic activity of human eosinophils in vitro. The purpose of this study was to investigate whether CysLTs promote chemotaxis in the human eosinophilic cell line, EoL-1. EoL-1 cells were induced to differentiate into mature eosinophil-like cells via incubation with butyric acid and cytokines (IL-3, IL-5 and GM-CSF). The chemotactic activity of the differentiated EoL-1 cells was assessed using the commercial cell migration assay kit. LTD4 elicited dose-related chemotactic activity in the differntiated EoL-1 cells in the range of 1-100 nM. A typical bell-shaped dose-response curve was observed with optimal activity at 10 nM. The chemotactic activity elicited by LTD4 (10 nM) was significantly inhibited by montelukast (control, 345 ± 19.2 × 103 RFU; LTD4 10 nM alone, 511 ± 39.2 × 103 RFU; LTD4 10 nM plus montelukast 100 nM, 387 ± 28.2 × 103 RFU). LTD4 induces migration in eosinophilic cells via activation of CysLT1 receptor. The present in vitro model may be useful for elucidation of the mechanism underlying CysLT-induced tissue eosinophilia.

Expression and localization of GPR99 in human nasal mucosa.

OBJECTIVE: The cysteinyl leukotrienes (CysLTs) are lipid mediators that have been implicated in the pathogenesis of allergic rhinitis. Pharmacological studies of CysLTs indicate that two classes of receptors, CysLT1R and CysLT2R exist. CysLT1R is a high affinity LTD4 receptor with lower affinity for LTC4, and a CysLT1R antagonist is currently used to treat asthma and allergic rhinitis. CysLT2R binds to LTC4 and LTD4 with equal affinity. GPR99 (also called GPR80), previously described as an oxoglutarate receptor (OXGR1), has recently emerged as a potential novel receptor with LTE4. The purpose of this study was to determine the expression and localization of GPR99 protein in the human nasal mucosa. METHODS: Human turbinates were obtained after turbinectomy from 12 patients with nasal obstruction refractory to medical therapy. GPR99 protein expression was evaluated by western blotting, and the specific cells expressing GPR99 protein identified by immunostaining using a commercial anti-GPR99 (OXGR1) monoclonal antibody. RESULTS: A 38-kDa band was detected in the western blots of human nasal samples by using the anti-GPR99 monoclonal antibody. We did not find any differences in GPR99 protein levels between allergic and non-allergic nasal mucosa. The immunohistochemical studies revealed that the anti-GPR99 monoclonal antibody mainly labeled vascular smooth muscle cells in the nasal mucosa. CONCLUSION: These immunohistochemical results suggest that GPR99 may play some roles in the vascular response. Further functional studies will be necessary to clarify the biological significance of the GPR99 receptor in nasal vasculature.

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.

Immunohistochemical localization of alpha and beta adrenergic receptors in the human nasal turbinate.

OBJECTIVE: Adrenergic receptors (ARs) include four general types (α1, α2, ß1 and ß2), which are found in different target tissues. α-AR agonists are commonly used for decongestant therapy of upper airway diseases. In order to clarify the roles of AR subtypes in the upper airways, we investigated the localization of these receptors by immunohistochemistry. METHODS: Human turbinates were obtained after turbinectomy from 12 patients with nasal obstruction refractory to medical therapy. The specific cells expressing α- and ß-AR proteins were identified by immunostaining using an anti-human AR subtype-specific antibodies (α1A-, α1D-, α2C- and ß2-ARs) antibody. RESULTS: Immunohistochemical analysis revealed that immunoreactivities for α1D- and ß2-ARs were densely distributed in submucosal glands. In contrast, immunoreactivities for α1A- and 2C-ARs were densely distributed in vascular smooth muscle. CONCLUSION: Our results suggested that adrenergic receptor (AR) subtypes had different roles in upper airway diseases, such as allergic rhinitis and nonallergic rhinitis.

Leukotriene E4 induces MUC5AC release from human airway epithelial NCI-H292 cells.

BACKGROUND: Hypersecretion of mucin in the airway epithelium is an important feature of allergic airway diseases. Of the 3 cysteinyl leukotrienes (CysLTs; LTC4 LTD4 and LTE4), only LTE4 is sufficiently stable to be detectable in extracellular fluids. However, LTE4 has received little attention because it binds poorly to the CysLT1 and CysLT2 receptors; therefore, little is known about the effects of LTE4 on mucous secretion. Recently, studies have focused on the P2Y12 receptor as a potential receptor for LTE4, because this receptor is required for LTE4-mediated pulmonary inflammation. In our previous study, we confirmed the expression of P2Y12 receptor in human airway epithelial cells. To clarify the roles of LTE4 in airway epithelial cells, we investigated mucus secretion by LTE4 in vitro. METHODS: Confluent NCI-H292 cells were stimulated with LTE4 (0.01-1 µM) for 24 h. The release and production of MUC5AC protein, a gel-forming mucin, were evaluated with an enzyme-linked immunosorbent assay. RESULTS: Western blot analysis revealed that NCI-H292 cells expressed P2Y12 receptor protein. LTE4 significantly induced the release of MUC5AC mucin in a dose-dependent manner. Th2 cytokines such as IL-4 (10 ng/mL) and IL-13 (10 ng/mL) accelerated the LTE4-induced release of MUC5AC protein. MRS2935, a P2Y12 receptor antagonist, partially inhibited the LTE4-induced release of MUC5AC protein in the airway. In contrast, MK571, a CysLT1 receptor antagonist, did not affect the release of MUC5AC protein elicited by LTE4. CONCLUSIONS: These results suggest that LTE4 may play some important roles in allergic mucus secretion partially via activation of P2Y12 receptor.

Expression and localization of purinergic P2Y(12) 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.

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.

Localization and upregulation of the nasal histamine H1 receptor in perennial allergic rhinitis.

In the present study, we have investigated the expression of histamine H1 receptor in human turbinates by RT-PCR, western blotting, and immunohistochemistry. Human turbinates were obtained by turbinectomy from 12 patients with nasal obstruction refractory to medical therapy. 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 histamine H1 receptor mRNA. About 56 kDa band was detected in human turbinates by western blot analysis using anti-H1 receptor antibody. The expression level of H1 receptor protein was marked in patients with nasal allergy than in patients with nonallergic rhinitis. The immunohistochemical study revealed that epithelial cells and vascular endothelial cells showed intense immunoreactivity for histamine H1 receptor. In addition, the blood vessels in superficial area expressed higher level of H1 receptor immunoreactivity than that in deeper area in the nasal mucosa. These results may have an important clinical implication for understanding the role of histamine H1 receptor on upper airway diseases such as allergic rhinitis and nonallergic rhinitis.

Correlation of Local FOXP3-Expressing T Cells and Th1-Th2 Balance in Perennial Allergic Nasal Mucosa.

Regulatory T cells (Treg) play some important roles in allergic rhinitis. The most specific marker for Treg is FOXP3, a recently identified transcription factor that is essential for Treg development. In order to clarify the levels of Treg in allergic nasal mucosa, we studied the relationship between FOXP3-expressing cells and Th1-Th2 balance in nasal mucosa by means of immunohistochemistry. Human turbinates were obtained after turbinectomy from 26 patients (14 patients with perennial allergic rhinitis and 12 patients with nonallergic rhinitis). To identify the cells expressing the FOXP3 protein, double immunostaining was performed by using anti-FOXP3 antibody and anti-CD3 antibody. There was no significant difference in the percentage of FOXP3+CD3+ cells among CD3+ cells in the nasal mucosa of two groups. The proportion of FOXP3+CD3+ cells tend to be correlated positively with GATA3+CD3+ cells/T-bet+CD3+ cells ratio (R = 0.56, P = 0.04). A positive correlation with GATA3+CD3+/T-bet+CD3+ ratio and FOXP3+CD3+/CD3+ ratio suggests the role of local regulatory T cells as a minimal control of the chronic allergen exposure in nasal mucosa.

Immunohistochemical localization of the bradykinin B1 and B2 receptors in human nasal mucosa.

Bradykinin (BK) has been tobe thought a potent mediator involved in allergic rhinitis because BK was recovered from the nasal lavage fluid of allergic rhinitis patients after allergen provocation and BK receptor antagonists relief nasal allergic symptoms. Two mammalian BK receptor subtypes, B1 and B2, have been defined based on their pharmacological properties. We investigated the localization of these receptors by immunohistochemistry. Human turbinates were obtained after turbinectomy from 12 patients with nasal obstruction refractory to medical therapy. The immunohistochemical study revealed that epithelial cells, submucosal glands, fibroblast, vascular smooth muscle, vascular endothelial cells, and macrophages showed immunoreactivity for both B1 and B2 receptors. The B2 receptor expression was found in peripheral nerve fibers, whereas the B1 expression was not observed in nerves. The results may have an important clinical implication for understanding the differential roles of BK receptor subtypes on upper airway diseases such as allergic rhinitis and nonallergic rhinitis.

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.

Agonist- and T(H)2 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 D4 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 D4. 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 the thromboxane A2 receptor in human nasal mucosa.

Thromboxane A2 (TXA2) has been thought a potent mediator involved in allergic rhinitis, because TXA2 was recovered from the nasal lavage fluid of allergic rhinitis patients after allergen provocation and TXA2 receptor antagonists relief nasal allergic symptoms. In order to clarify the expression of TXA2 receptor in human nasal mucosa, we investigated TXA2 receptor mRNA expression and its protein localization by polymerase chain reaction (PCR) and immunohistochemistry, respectively. Human turbinates were obtained after turbinectomy from 10 patients with nasal obstruction refractory to medical therapy. RT-PCR analysis of total RNA from nasal mucosa demonstrated the expression of TXA2 receptor alpha mRNA. The immunohistochemical studies revealed that anti-TXA2 receptor alpha antibody labeled vascular smooth muscle cells, vascular endothelial cells, epithelial cells and submucosal glands in the nasal mucosa. The results may have an important clinical implication for understanding the role of TXA2 receptor on upper airway diseases such as allergic rhinitis and non-allergic 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 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.

Expression and localization of steroid receptors in human nasal mucosa.

OBJECTIVE: To investigate the expression of glucocorticoid receptor (GR), oestrogen receptor (ER), progesterone receptor (PR) and androgen receptor (AR) in nasal mucosa. MATERIAL AND METHODS: Human turbinates were obtained after turbinectomy from seven patients. The expression and localization of steroid receptors were examined using reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. RESULTS: Using RT-PCR, GR and ER alpha mRNA were detected in all cases. In contrast, ER beta, PR and AR mRNA were found in five, four and six cases, respectively. Using immunohistochemistry, antibodies to GR showed the presence of GR within all cells of nasal mucosa, with the highest quantities of GR being localized in epithelial cells, submucosal glands and inflammatory leukocytes. Immunohistochemical analysis of sex steroid receptor revealed that anti-ER alpha antibody labelled mainly mast cells and anti-ER beta antibody labelled submucosal glands, and that no PR or AR expression was detected in any of the samples tested. CONCLUSIONS: The role of ER in mast cells and submucosal glands has not been well clarified. However, precise knowledge of the identity and distribution of sex steroid receptor should be of considerable interest in understanding the role of sex hormones in upper airway diseases such as allergic and non-allergic rhinitis.

Expression of neurokinin a receptor mRNA in human nasal mucosa.

In airway tissues, it has been suggested that tachykinins act as the transmitter for afferent sensory nerves which respond to various irritants and may be involved in airway allergic reactions. Three classes of tachykinin receptor have been recognized, denoted NK1, NK2 and NK3, which exhibit preferential affinity for substance P, neurokinin A and neurokinin B, respectively. We used molecular probes to study the gene expression and distribution of NK2 receptor in human nasal mucosa. Total RNA was isolated from human nasal mucosa and NK2 receptor mRNA was detected in these tissues using reverse transcriptase polymerase chain reaction (RT-PCR). For an in situ hybridization study of human nasal mucosa, we utilized the PCR directly to incorporate a T7 RNA polymerase promoter sequence onto the NK2 receptor cDNA, and these PCR products were used as the DNA template for producing digoxigenin-labeled antisense and sense RNA probes. These studies revealed that NK2 receptor mRNA was expressed in blood vessels. The results suggest a primary role for neurokinin A in the form of vascular responses in the upper respiratory tract.

Effects of glucocorticoids on infiltrating cells and epithelial cells of nasal polyps.

Glucocorticoids are known to be effective in the treatment of nasal polyps (NPs). To examine the mechanisms of their effect, we evaluated 1) the ability of glucocorticoids to induce the apoptosis of eosinophils and T lymphocytes in NPs, and 2) the ability of dexamethasone to down-regulate epithelial cell functions that relate to eosinophilic inflammation. In vitro and in vivo, glucocorticoids increased the apoptosis of both eosinophils and T lymphocytes in NPs. Dexamethasone inhibited the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) from both NP epithelial cells that were unstimulated and NP epithelial cells that were stimulated with interleukin-4 or tumor necrosis factor alpha. These results suggest that the clinical efficacy of glucocorticoids on NPs may be due to 1) induction of apoptosis in both eosinophils and T lymphocytes that infiltrate NPs, and 2) down-regulation of epithelial GM-CSF production, which prolongs eosinophil survival.