Prevention of allergic rhinitis by aldose reductase inhibition in a murine model.

BACKGROUND: Allergic rhinitis, one of the most common atopic diseases, is known to be elicited by Th2 cytokine-mediated inflammatory response. We have shown earlier that a polyol pathway enzyme aldose reductase (AR) regulates airway inflammation; however its role in allergic rhinitis is not known. We have investigated the role of AR in mediating pathological symptoms associated with allergic rhinitis in mice. METHODS: The wild-type (WT) mice treated without or with AR inhibitor and AR knock out (AR(-/-)) mice were sensitized by two intraperitoneal injections of ragweed pollen extract (RWE) with adjuvant alum on days 0 and 4 followed by challenge on day 11 and/or 18 and 25. The allergic rhinitis symptoms were assessed by monitoring the nasal scratch, mast cell degranulation and release of tryptase in nasal lavage, infiltration of inflammatory cells, production of inflammatory cytokines and nasal epithelium remodeling. RESULTS: Sensitization and challenge of mice with RWE produced robust and reproducible pathological symptoms of allergic rhinitis as compared to control mice. AR inhibitor, fidarestat administered mice showed markedly reduced early phase response to allergen exposure such as nasal scratches, mast cells degranulation and release of tryptase in the nasal passage as well as late phase response such as inflammatory cell infiltration and release of Th2 type cytokines and nasal epithelial remodeling. Further, prevention of these events in AR(-/-)) mice suggests the role of AR in the mediation of allergic rhinitis. CONCLUSION: These results indicate an important role of AR in the mediation of RWE-induced allergic rhinitis in mice and prevention by AR inhibitor, fidarestat offers a novel therapeutic approach to ameliorate allergic rhinitis.

Association of the MMP9 gene with childhood cedar pollen sensitization and pollinosis.

Matrix metalloproteinase 9 (MMP9) gene has been shown to be involved in the pathogenesis of allergic rhinitis (AR) and asthma. Previous studies suggested that single-nucleotide polymorphisms (SNPs) of the MMP9 gene conferred a risk for childhood asthma. However, whether the SNPs confer a risk for AR has not been previously investigated. The objective of this study was to investigate whether SNPs of the MMP9 gene are associated with risk of seasonal AR (pollinosis), perennial AR and allergen sensitization. A total of 670 school children were recruited in Japan and genotyped for functional polymorphism in the promoter (-1590C/T: rs3918242) and three amino-acid substitutions (R297Q: rs17576; P574R: rs2250889; R668Q: rs17577). Serum levels of total and specific IgE were determined. Disease status and other clinical characteristics of the subjects were investigated using a questionnaire. Associations between the MMP9 SNPs and both AR and serum IgE levels were evaluated. -1590C/T showed significant association with cedar pollinosis (corrected P (Pcor)=0.039). R668Q was in strong linkage disequilibrium (LD) with -1590C/T and showed significant association with cedar pollinosis (Pcor=0.023) and serum cedar pollen-specific IgE level (Pcor=0.022). A haplotype associated with -1590T and 668Q showed a significant association with cedar pollinosis, orchard grass pollinosis and cedar pollen-specific IgE (Pcor=0.0012, Pcor=0.0059 and Pcor=0.0041, respectively). R297Q and P574R were in weak LD with the rest of the SNPs and did not show significant association with disease. Compared with wild-type MMP9 protein (279R-574P-668R), a variant enzyme (279R-574P-668Q) that showed association with pollinosis had lower activity. However, lower enzyme activity was not associated with disease risk because another variant (279Q-574R-668R) showed lower enzyme activity but was not associated with pollinosis. The -1590T allele and its corresponding haplotype was associated with higher promoter activity and with pollen-specific IgE levels and pollinosis, suggesting that -1590C/T may have more impact on sensitization and disease development than R668Q. Our results suggest that the MMP9 gene confers susceptibility to cedar pollinosis in Japanese children. The MMP9 gene may be associated with pollinosis through sensitization processes.

Aldose reductase deficiency in mice protects from ragweed pollen extract (RWE)-induced allergic asthma.

BACKGROUND: Childhood hospitalization related to asthma remains at historically high levels, and its incidence is on the rise world-wide. Previously, we have demonstrated that aldose reductase (AR), a regulatory enzyme of polyol pathway, is a major mediator of allergen-induced asthma pathogenesis in mouse models. Here, using AR null (AR-/-) mice we have investigated the effect of AR deficiency on the pathogenesis of ragweed pollen extract (RWE)-induced allergic asthma in mice and also examined the efficacy of enteral administration of highly specific AR inhibitor, fidarestat. METHODS: The wild type (WT) and AR-/- mice were sensitized and challenged with RWE to induce allergic asthma. AR inhibitor, fidarestat was administered orally. Airway hyper-responsiveness was measured in unrestrained animals using whole body plethysmography. Mucin levels and Th2 cytokine in broncho-alveolar lavage (BAL) were determined using mouse anti-Muc5A/C ELISA kit and multiplex cytokine array, respectively. Eosinophils infiltration and goblet cells were assessed by H&E and periodic acid Schiff (PAS)-staining of formalin-fixed, paraffin-embedded lung sections. T regulatory cells were assessed in spleen derived CD4+CD25+ T cells population. RESULTS: Deficiency of AR in mice led to significantly decreased PENH, a marker of airway hyper-responsiveness, metaplasia of airway epithelial cells and mucus hyper-secretion following RWE-challenge. This was accompanied by a dramatic decrease in infiltration of eosinophils into sub-epithelium of lung as well as in BAL and release of Th2 cytokines in response to RWE-challenge of AR-/- mice. Further, enteral administration of fidarestat significantly prevented eosinophils infiltration, airway hyper-responsiveness and also markedly increased population of T regulatory (CD4+CD25+FoxP3+) cells as compared to RWE-sensitized and challenged mice not treated with fidarestat. CONCLUSION: Our results using AR-/- mice strongly suggest the role of AR in allergic asthma pathogenesis and effectiveness of oral administration of AR inhibitor in RWE-induced asthma in mice supports the use of AR inhibitors in the treatment of allergic asthma.

Simultaneous flow cytometric detection of basophil activation marker CD63 and intracellular phosphorylated p38 mitogen-activated protein kinase in birch pollen allergy.

BACKGROUND: Phosphorylation of p38 MAPK is a crucial step in IgE-receptor signaling in basophils. The relation of p38 MAPK to the well-validated diagnostic cell surface marker CD63 has not been evaluated in a clinical allergy model. METHODS: Expression of CD63 and phosphorylation of p38 MAPK were analyzed flow cytometrically in anti-IgE-gated basophils from 18 birch pollen allergic patients, five grass pollen allergic patients, and five healthy individuals, after 3 and 20 min of stimulation with recombinant major birch pollen allergen (rBet v 1). Additional time points and the influence of p38 MAPK inhibitor SB203580 were studied in birch pollen allergic patients. RESULTS: Phospho-p38 MAPK and CD63 were expressed dose-dependently in birch pollen allergic patient basophils within 1 minute of rBet v 1 stimulation. P38 MAPK phosphorylation was fastest and subsided gradually while CD63 expression remained elevated for at least 20 min. Inhibition of p38 MAPK significantly inhibited CD63 upregulation. With optimal stimulation of the cells (1 µg/mL), sensitivity and specificity for the discrimination between patients and a group of control individuals (grass pollen allergic patients and healthy controls) were 94% and 100% for CD63 at 3 and 20 min and for phospho-p38 MAPK at 3 min. CONCLUSION: Antigen-induced p38 MAPK phosphorylation in human basophils essentially contributes to CD63 upregulation. It is a sensitive and specific intracellular marker for allergy diagnosis and offers new insight into the mechanisms of basophil activation.

Protease activity of allergenic pollen of cedar, cypress, juniper, birch and ragweed.

BACKGROUND: Pollen is an important trigger of allergic rhinitis, conjunctivitis, and/or asthma, and an exacerbating factor in atopic dermatitis. Although it is proposed that protease activity from allergen sources, such as mites, enhances allergenicity, little information is available on that from relevant allergenic pollens such as Japanese cedar and Japanese cypress pollens, which are the major cause of pollinosis in Japan. METHODS: We analyzed the protease activities derived from allergenic pollen of Japanese cedar, Japanese cypress, and Rocky mountain juniper, which belong to the Cupressaceae/Taxodiaceae family, and white birch and short ragweed, using synthetic substrates and class-specific inhibitors. RESULTS: We found that the pollen of the three members of the Cupressaceae/Taxodiaceae family contained serine protease activity, that the pollen of white birch and short ragweed contained not only serine protease activity but also cysteine protease activity, that all five types of pollen tested contained at least one other type of serine protease, whose sensitivity to a serine protease-specific inhibitor was relatively low, and that the content and releasability of the pollen-derived proteases differed according to the plant families. CONCLUSIONS: Clinically relevant allergenic pollens tested in the present study can release serine and/or cysteine endopeptidases. Information on the spectrum of the endopeptidase activities from these allergenic pollen grains will be useful for investigating their contribution to the pathogenesis of allergies.

Increased expression of lipoxygenase enzymes during pollen season in nasal biopsies of pollen-allergic patients.

BACKGROUND: Exposure of patients sensitized to pollen triggers development of seasonal allergic rhinitis symptoms (SAR). Eicosanoids are a group of arachidonic acid metabolites contributing to the symptoms of SAR. The aim of this study was to investigate seasonal changes in the expression of enzymes of the eicosanoid pathway in the nasal mucosa of patients with SAR. METHODS: Twenty SAR patients allergic to birch or grass and eight healthy subjects were included in the study. Patients registered rhinoconjunctivitis symptoms and use of rescue medication before and during the pollen season. Nasal biopsies were obtained before and around the peak of the season, sectioned and stained using markers for eosinophils, mast cells, T cells and neutrophils. Antibodies against the following enzymes were also used: cyclo-oxygenase (COX-1, COX-2), 5-lipoxygenase (5-LO), 5-lipoxygenase-activating factor (FLAP), LTA4 hydrolase (LTA4h) and LTC4 synthase (LTC4s). RESULTS: During the pollen season symptoms of rhinoconjunctivitis and medication score increased significantly (P=0.001; P=0.001 respectively). During the pollen season numbers of eosinophils (P=0.02) and cell positive 5-LO (P=0.02), LTC4s (P=0.04) and LTA4h (P=0.02) increased significantly. During season number of mast cells and cells expressing 5-LO and LTA4h were higher in SAR than in healthy controls group (P=0.02; P=0.01; P=0.03 respectively). CONCLUSION: In sensitized patients exposure to pollen allergen results in increased expression of enzymes of the eicosanoid pathway.

Spring brings breezes, wheezes, and pollen oxidases.

While the release of pollen into the air is essential for the reproduction of plants, the accidental yet inevitable uptake of pollen into human airways can cause symptoms of seasonal allergies and asthma. The symptomatic response to pollen is caused by granulocytes that produce inflammation, which is due in part to oxidative stress through the action of NADPH oxidases. The recruitment of these inflammatory granulocytes was previously thought to depend entirely on the activation of an adaptive immune response. In this issue of the JCI, Boldogh et al. demonstrate that pollens contain endogenous NADPH oxidase activity, which functions to generate local "danger signals" in nearby airway epithelium. These signals in turn trigger the early recruitment of granulocytes, even in the absence of the adaptive immune response. These findings suggest that inhibition of the pollen oxidase may provide a way to antagonize allergic inflammation at a very early step.

Fixation with Carnoy's fluid reduces the number of chymase-positive mast cells: not all chymase-positive mast cells are also positive for tryptase.

Mast cells in the nasal mucosa can be studied by means of monoclonal antibodies (mAb) against tryptase (T+MC) and chymase (C+MC). Fixation with acetone gives more positive cells than does fixation with Carnoy's fluid. In frozen biopsy specimens of allergic nasal mucosa fixed with acetone, the number of T+MC equals that of C+MC. When fixed with Carnoy's fluid, however, the number of T+MC is larger than the number of C+MC. The decrease in both T+MC and C+MC resulting from fixation with Carnoy's fluid is time-related and depends on the type of mAb used. Carnoy fixation time gives a decrease in the number of C+MC within 1 min, whereas the number of T+MC decreases only after 10 min. Within 1 min, the number of C+MC decreases to a level where continued fixation no longer gives further decreases in the number of cells. Two populations of mast cells can be distinguished here: one sensitive and the other insensitive to Carnoy's fluid. When double-staining is used, fixation with acetone gives three populations of mast cells: one positive for tryptase (T+C-MC), another positive for tryptase and chymase (T+C+MC), and a third one positive for chymase (T-C+MC). These three populations were found in lymph node, spleen, thymus, dermis, lung parenchyma, small intestinal submucosa, and nasal mucosa.

Metachromatic, IgE-bearing and tryptase-containing cells on the nasal mucosal surface in provoked allergic rhinitis.

We studied the relationship between mast cells or basophils and symptoms in provoked allergic rhinitis. Nasal brush and lavage samples were obtained before nasal allergen challenge and every 2 h for 12 h after the challenge in 10 allergics and 3 controls. The cells were identified by their metachromatic staining properties (brush and lavage samples) or with immunohistochemical methods using specific antibodies to IgE and tryptase, a selective mast-cell marker (brush samples). Histamine was determined in the brush samples using liquid chromatography. After an initial decrease, the numbers of metachromatic, IgE-bearing and tryptase-containing cells, as well as the histamine content of the cells in the brush samples, increased during the subsequent observation hours. The prechallenge cell and histamine values correlated with the symptom score 15 min after the challenge. The prechallenge lavage samples lacked metachromatic cells, but these cells were found in increasing numbers after the provocation. Three of the patients differed from the remaining seven in that their prechallenge brush samples contained many positively stained cells. All patients showed a positive cellular response to the allergen challenge, but these three individuals showed the most vivid response. The morphology of the metachromatic cells in the prechallenge brush samples agreed with that of mast cells, but the morphology of metachromatic cells which outnumbered tryptase-containing cells in samples at 8 to 12 h rather agreed with their being basophils.

Changes of serum histaminase activity in pollinosis.

In order to study changes in serum histaminolytic enzyme activity in allergic individuals following allergen challenge, serum histaminase activity was measured using the 0-dianisidine peroxidase method in patients with Japanese cedar pollinosis. Significantly higher serum histaminase activity was detected in pollen season than out of season and a significant relationship was recognized between serum histaminase activity and the severity of nasal symptoms.