The novel use of the human nasal epithelial cell line RPMI 2650 as an in vitro model to study the influence of allergens and cytokines on transforming growth factor-beta gene expression and protein release.

BACKGROUND: The epithelial accumulation of mast cells is a feature of allergic rhinitis and this has been linked to the expression of the known mast cell chemoattractant transforming growth factor-beta (TGF-beta) at this site. Little is known concerning the regulation of TGF-beta gene expression or protein release by nasal epithelial cells. To address this we have utilized the RPMI 2650 human nasal epithelial cell line, which has some features that closely resemble normal nasal epithelium and has been reported to secrete a TGF-beta-like molecule. OBJECTIVES: To investigate the regulation of TGF-beta gene expression and protein secretion in RPMI 2650 nasal epithelial cells following exposure to allergens (house dust mite (HDM) and grass pollen) and mast cell associated T-helper type 2 (Th2) cytokines (IL-4, IL-13, and TNF-alpha). Methods Light and scanning electron microscopy was used to evaluate the morphology of RPMI 2650 cells in culture, enzyme-linked immunosorbent assay was used to investigate their TGF-beta secretory capacity and the identification of the TGF-beta isotype(s) involved, flow cytometry was used to demonstrate the presence of TGF-beta receptors on the RPMI 2650 cells, and the quantitative real-time TaqMan PCR was used to measure TGF-beta gene expression. RESULTS: TGF-beta(2) was identified as the main isotype secreted by the RPMI 2650 cells. HDM allergens and TNF-alpha increased both TGF-beta gene expression and protein release from these cells, whereas grass pollen, IL-4, and IL-13 were without effect. CONCLUSIONS: The RPMI 2650 nasal epithelial cell line represents a valid in vitro model to evaluate the regulation of TGF-beta biology. In this system HDM allergens have stimulatory activity that is fundamentally different from that of grass pollen allergens, and the Th2 cytokines IL-4 and IL-13 are without effect. The ability of TNF-alpha to up-regulate both TGF-beta gene expression and protein release indicates that mast cell-epithelial interactions concerning TGF-beta are bi-directional and this may be fundamental to epithelial immunoregulation. The availability of a model system, such as the RPMI 2650 cells, will enable the early evaluation of future novel and targeted interventions directed toward the aberrant responses of upper airway structural cells.

Assessment of antihistamine efficacy and potency.

H1 antihistamines are widely used in the treatment of conditions such as seasonal and perennial allergic rhinitis, urticaria and angioedema, as well as being an adjunct therapy for anaphylaxis. The development of new compounds within this class of pharmacological agents is based on preclinical and clinical assessments prior to registration. Preclinical assessment of novel compounds has been advanced both by the definition of the molecular structure of the H1 receptor and by high throughput screening, which allows the definition of potent and selective candidate compounds. The elimination, in subsequent evaluations, of those compounds with potential for central nervous system and cardiotoxic effects and those with inappropriate pharmacokinetics restricts the subsequent toxicological and clinical evaluation of novel H1 antihistamines. Clinical evaluation is based initially on human volunteer studies to define pharmacodynamic, pharmacokinetic and safety parameters. Local challenge models such as in the skin or nose for oral therapy or nose and conjunctiva for topical drug development allow definition of potential dose for administration, speed of onset of activity and duration of effect, both in comparison to placebo and to reference compounds. While a range of laboratory allergen challenge models, pollen chamber challenge studies and acute 'day in the park' study designs have been used in the early clinical evaluation of H1 antihistaminic activity, ultimately it is the efficacy of novel compounds in naturally occurring disease that is of importance. The assessment of the magnitude and profile of clinical disease improvement in specific disease areas is thus the crucial evaluation. The appropriate selection and characterization of patients, the optimization of the trial design, dependent upon the nature of the disease, and the appropriate selection of endpoints for analysis are thus critical considerations, as well as evaluation of appropriate safety criteria.

Effect of systemic beta-agonist therapy on IgE production in allergic subjects in vivo.

BACKGROUND: Although beta2-adrenoceptor agonists are widely used in the treatment of asthma, a number of studies have suggested that their long-term use may exacerbate the condition. One possible mechanism for this stems from the in vitro observation that beta2-agonists increase IgE synthesis by human blood mononuclear cells. OBJECTIVE: We sought to examine the effect of regular beta2-agonist therapy on IgE production in vivo in human volunteers. METHODS: Placebo or salbutamol (8 mg BD) tablets were given in a double-blind, randomized fashion to 25 volunteers allergic to grass pollen throughout a period encompassing the UK grass pollen season (April through September). Levels of serum IgE were measured monthly, and nasal IgE was measured at the height and end of the season. Efficacy was assessed through monthly recordings of symptoms of blocked nose (vascular) and other symptoms of rhinitis (nonvascular). RESULTS: For the whole group the geometric mean of serum IgE levels rose from a baseline of 58.7 IU/mL (range, 0 to 1027 IU/mL) to 140 IU/mL (range, 12 to 878 IU/mL) at the height of the pollen season (P =.0001). There was no significant difference between the magnitude of the rise in IgE between the groups with a ratio of increase for salbutamol/placebo of 1.17 (confidence interval = 0.78 to 1.75). There was no change in nasal IgE levels. Total and nonvascular symptom scores were reduced by salbutamol, reaching statistical significance at the height of the pollen season (P <.05). CONCLUSION: An oral dose of the beta2-agonist salbutamol, sufficient to maintain therapeutic levels and provide clinical benefit, does not accentuate the seasonal increase of IgE in human atopic volunteers.

Effect of thromboxane A2-receptor antagonist on bradykinin-induced bronchoconstriction in asthma.

The role of the thromboxane A2 (TxA2) receptor in bradykinin-induced bronchial responses was investigated in this study by using a selective and potent TxA2-receptor antagonist BAY u 3405. Eleven asthmatic subjects were randomized to receive 50 mg of BAY u 3405 or matched placebo in a crossover and double-blind fashion. Ninety minutes after dosing, serum was taken for drug assay, and subjects underwent provocation with bradykinin or prostaglandin D2 (PGD2) to determine bronchial responsiveness [provocative concentration of agonist required to produce a 20% fall in forced expiratory volume in 1 s from the postdiluent baseline (PC20)]. Pretreatment with BAY u 3405 caused a twofold doubling-dilution reduction in bronchial reactivity to PGD2; the geometric mean PC20 values were 0.132 (0.015-0.871) and 0.034 (0.008-0.095) mg/ml, respectively, for active and placebo days (P = 0.001). There was, however, no significant difference in PC20 values for bradykinin between active and placebo treatment days. We have demonstrated that BAY u 3405 caused a significant inhibition of bronchconstriction induced by inhaled PGD2 but had no influence on bronchial responsiveness to inhaled bradykinin. This study suggests therefore that TxA2 receptors do not play a role in bradykinin-induced bronchoconstriction in asthma.

Effect of natural allergen exposure during the grass pollen season on airways inflammatory cells and asthma symptoms.

BACKGROUND: Bronchial challenge with allergen causes a specific form of airways inflammation consisting of an influx of neutrophils, eosinophils, and T cells. Because the relevance of the challenge model to clinical asthma is uncertain, the cellular changes that occur in the lungs of asthmatic subjects during natural seasonal allergen exposure were investigated. METHODS: Seventeen grass pollen sensitive asthmatic subjects with previously reported seasonal exacerbations of asthma kept records of symptoms and underwent fibreoptic bronchoscopy with bronchoalveolar lavage (BAL) and endobronchial biopsy before and during the peak of the grass pollen season. The BAL cells were analysed for differential cell counts and by flow cytometry for T cell subsets and surface activation markers. The biopsy samples were processed into glycol methacrylate resin and immunohistochemical analysis was performed for mast cells, activated eosinophils, T cells and interleukin 4 (IL-4), a cytokine with a pivotal role in allergen-induced inflammation. RESULTS: In the pollen season there was an increase in T lymphocyte activation in the BAL fluid as identified by increased expression of interleukin 2 receptor (IL-2R). In the submucosa these changes were paralleled by an increase in CD4+ T cells. By contrast, the numbers of metachromatic cells in BAL fluid staining with toluidine blue were reduced, possibly because of degranulation following allergen stimulation. In keeping with mast cell activation, the number of mucosal mast cells staining for secreted IL-4 increased during the season. In comparison with the period shortly before the onset of the season, all but two subjects experienced an asthma exacerbation which followed the rise in pollen counts but, compared with the period preceding the first bronchoscopic examination, asthma symptoms were not increased during the pollen season. CONCLUSIONS: The data suggest that natural allergen exposure, leading to a clinical exacerbation of asthma, may induce an inflammatory response involving T cells, mast cells and eosinophils. The relationship between allergen exposure, cellular infiltration and activation, and clinical symptoms appears to be complex, with factors other than allergen also contributing to asthmatic activity.

The effect of local hyperthermia on allergen-induced nasal congestion and mediator release.

BACKGROUND: Local hyperthermia reduces mast cell degranulation, the severity of acute lung injury, and exercise-induced asthma and decreases symptoms of rhinitis. We have investigated the effect of local hyperthermia on mast cell degranulation and symptom generation in allergic rhinitis to assess its effect and mechanism of action within the nose. METHODS: In a randomized, double-blind, placebo-controlled, crossover study, 10 subjects with rhinitis were treated for 30 minutes with local hyperthermia or placebo, which was followed 30 minutes later by nasal allergen challenge. During the first two visits nasal lavages were performed to assess vascular leakage and mediator release. During the last two visits nasal airway resistance, the number of sneezes, and mucus secretion were monitored. RESULTS: Local hyperthermia significantly reduced both nasal airway resistance (p < 0.05) and vascular leakage (p < 0.02) but had no significant effect on the number of sneezes, on mucus secretion, or on tryptase release. CONCLUSION: Local hyperthermia reduces allergen-provoked nasal blockage and vascular leakage but has no effect on sneezing, rhinorrhea, or tryptase release. Nasal blockage occurs predominantly via newly formed lipid mediators and kinins, whereas sneezing and rhinorrhea occur predominantly via preformed mediators. We propose that local hyperthermia inhibits newly formed mediator production or release or reduces the sensitivity of the vasculature to inflammatory mediators in general. Further investigation into the mechanisms and potential uses of local hyperthermia is warranted.

Astemizole, a potent histamine H1-receptor antagonist: effect in allergic rhinoconjunctivitis, on antigen and histamine induced skin weal responses and relationship to serum levels.

The efficacy of astemizole, a new, long acting, oral histamine H1-receptor antagonist was compared to placebo for the treatment of allergic rhinitis and conjunctivitis during the grass pollen season of 1982. Sixty-three patients with a positive skin prick test to grass pollen and current symptoms participated in an 8 week, double-blind, randomized study. Astemizole, 10 mg, was significantly better than placebo in alleviating both nose (P less than 0.05) and eye (P less than 0.01) symptoms despite significantly greater use of the reserve medication, clemastine, by the placebo group (P less than 0.003). There was a lag period of 5 days after initiation of therapy before treatment benefit became manifest. Subdivision of nasal symptoms indicated significant improvement compared to placebo over the 8 weeks for sneezing (P less than 0.05) and runny nose (P less than 0.05) but not blocked nose. The absence of effect on nasal blockage was confirmed by parallel measurement of nasal calibre by body plethysmography. The antihistaminic potency of astemizole was indicated by an 80% inhibition of the histamine induced skin weal response after 8 weeks therapy. A positive correlation was found between serum drug levels and % inhibition of histamine skin weal (r = 0.64, P less than 0.001). Astemizole was free from adverse sedative or anticholinergic effects but did cause a mean increase in weight of 1.3 kg (P less than 0.01) after 8 weeks therapy, not found with placebo.