Primary human airway epithelial cell-dependent inhibition of human lung mast cell degranulation

Introduction

Chronic mast cell activation is a characteristic feature of asthma. BEAS-2B human airway epithelial cells (AEC) profoundly inhibit both constitutive and IgE-dependent human lung mast cell (HLMC) histamine release. The aim of this study was to examine the regulation of HLMC degranulation by primary AEC from healthy and asthmatic subjects, and investigate further the inhibitory mechanism.

Methods

HLMC were co-cultured with both BEAS-2B and primary AEC grown as monolayers or air-liquid interface (ALI) cultures.

Results

Both constitutive and IgE-dependent HLMC histamine release were attenuated by BEAS-2B, primary AEC monolayers and ALI cultures. This occurred in the absence of HLMC-AEC contact indicating the presence of a soluble factor. Unlike healthy ALI AEC, asthmatic ALI-AEC did not significantly reduce constitutive histamine release. AEC inhibitory activity was transferable in primary AEC monolayer supernatant, but less active than with Transwell co-culture, suggesting that the inhibitory factor was labile. The AEC inhibitory effects were attenuated by both AEC wounding and pertussis toxin, indicating the involvement of a G₀/G_i receptor coupled mechanism. Solid phase extraction of lipids (<10 kDa) removed the AEC inhibitory activity. The lipid derivatives resolvin D1 and D2 and lipoxin A₄ attenuated HLMC histamine release in a dose-dependent fashion but were not detectable in co-culture supernatants.

Conclusions

Primary AEC suppress HLMC constitutive and IgE-dependent histamine secretion through the release of a soluble, labile lipid mediator(s) that signals through the G₀/G_i receptor coupled mechanism. Manipulation of this interaction may have a significant therapeutic role in asthma.     

The Effects of Overexpression of Histamine Releasing Factor (HRF) in a Transgenic Mouse Model

Background

Asthma is a disease that affects all ages, races and ethnic groups. Its incidence is increasing both in Westernized countries and underdeveloped countries. It involves inflammation, genetics and environment and therefore, proteins that exacerbate the asthmatic, allergic phenotype are important. Our laboratory purified and cloned a histamine releasing factor (HRF) that was a complete stimulus for histamine and IL-4 secretion from a subpopulation of allergic donors'' basophils. Throughout the course of studying HRF, it was uncovered that HRF enhances or primes histamine release and IL-13 production from all anti-IgE antibody stimulated basophils. In order to further delineate the biology of HRF, we generated a mouse model.

Methodology/Principal Findings

We constructed an inducible transgenic mouse model with HRF targeted to lung epithelial cells, via the Clara cells. In antigen naïve mice, overproduction of HRF yielded increases in BAL macrophages and statistical increases in mRNA levels for MCP-1 in the HRF transgenic mice compared to littermate controls. In addition to demonstrating intracellular HRF in the lung epithelial cells, we have also been able to document HRF''s presence extracellularly in the BAL fluid of these transgenic mice. Furthermore, in the OVA challenged model, we show that HRF exacerbates the allergic, asthmatic responses. We found statistically significant increases in serum and BAL IgE, IL-4 protein and eosinophils in transgenic mice compared to controls.

Conclusions/Significance

This mouse model demonstrates that HRF expression enhances allergic, asthmatic inflammation and can now be used as a tool to further dissect the biology of HRF.     

Proton Pump Inhibitors Exert Anti-Allergic Effects by Reducing TCTP Secretion

Background

Extracellular translationally controlled tumor protein (TCTP) is known to play a role in human allergic responses. TCTP has been identified outside of macrophages, in activated mononuclear cells, and in biological fluids from allergic patients. Even TCTP devoid of signal sequences, is secreted to extracellular environment by an yet undefined mechanism. This study is aimed at understanding the mechanism of TCTP release and its regulation. A secondary goal is to see if inhibitors of TCTP release can serve as potential anti-allergic asthmatic drugs.

Methodology/Principal Findings

Using Western blotting assay in HEK293 and U937 cells, we found that TCTP secretion is reduced by omeprazole and pantoprazole, both of which are proton pump inhibitors. We then transfected HEK293 cells with proton pump expression vectors to search for the effects of exogeneously overexpressed H⁺/K⁺-ATPase on the TCTP secretion. Based on these in vitro data we checked the in vivo effects of pantoprazole in a murine model of ovalbumin-induced allergy. Omeprazole and pantoprazole reduced TCTP secretion from HEK293 and U937 cells in a concentration-dependent fashion and the secretion of TCTP from HEK293 cells increased when they over-expressed H⁺/K⁺-ATPase. In a murine model of ovalbumin-induced allergy, pretreatment with pantoprazole reduced infiltration of inflammatory cells, increased goblet cells, and increased TCTP secretion induced by OVA challenge.

Conclusion

Since Omeprazole and pantoprazole decrease the secretion of TCTP which is associated with the development of allergic reaction, they may have the potential to serve as anti-allergic (asthmatic) drugs.     

Astragalin inhibits autophagy-associated airway epithelial fibrosis

Background

Fibrotic remodeling of airway and lung parenchymal compartments is attributed to pulmonary dysfunction with an involvement of reactive oxygen species (ROS) in chronic lung diseases such as idiopathic pulmonary fibrosis and asthma.

Methods

The in vitro study elucidated inhibitory effects of astragalin, kaempferol-3-O-glucoside from leaves of persimmon and green tea seeds, on oxidative stress-induced airway fibrosis. The in vivo study explored the demoting effects of astragalin on epithelial to mesenchymal transition in BALB/c mice sensitized with ovalbumin (OVA).

Results

The exposure of 20 μM H₂O₂ for 72 h accelerated E-cadherin loss and vimentin induction in airway epithelial BEAS-2B cells, which was reversed by non-toxic astragalin at 1–20 μM. Astragalin allayed the airway tissue levels of ROS and vimentin enhanced by OVA challenge. Collagen type 1 production increased in H₂O₂–exposed epithelial cells and collagen fiber deposition was observed in OVA-challenged mouse airways. This study further investigated that the oxidative stress-triggered autophagic regulation was responsible for inducing airway fibrosis. H₂O₂ highly enhanced the expression induction of the autophagy-related beclin-1 and light chains 3A/B (LC3A/B) within 4 h and astragalin blocked such induction by H₂O₂. This compound deterred the ROS-promoted autophagosome formation in BEAS-2B cells. Consistently, in OVA-sensitized mice the expression of beclin-1 and LC3A/B was highly induced, and oral administration of astragalin suppressed the autophagosome formation with inhibiting the induction of these proteins in OVA-challenged airway subepithelium. Induction of autophagy by spermidine influenced the epithelial induction of E-cadherin and vimentin that was blocked by treating astragalin.

Conclusion

These results demonstrate that astragalin can be effective in allaying ROS-promoted bronchial fibrosis through inhibiting autophagosome formation in airways.     

Site of allergic airway narrowing and the influence of exogenous surfactant in the Brown Norway rat

Background

The parameters R_N (Newtonian resistance), G (tissue damping), and H (tissue elastance) of the constant phase model of respiratory mechanics provide information concerning the site of altered mechanical properties of the lung. The aims of this study were to compare the site of allergic airway narrowing implied from respiratory mechanics to a direct assessment by morphometry and to evaluate the effects of exogenous surfactant administration on the site and magnitude of airway narrowing.

Methods

We induced airway narrowing by ovalbumin sensitization and challenge and we tested the effects of a natural surfactant lacking surfactant proteins A and D (Infasurf®) on airway responses. Sensitized, mechanically ventilated Brown Norway rats underwent an aerosol challenge with 5% ovalbumin or vehicle. Other animals received nebulized surfactant prior to challenge. Three or 20 minutes after ovalbumin challenge, airway luminal areas were assessed on snap-frozen lungs by morphometry.

Results

At 3 minutes, R_N and G detected large airway narrowing whereas at 20 minutes G and H detected small airway narrowing. Surfactant inhibited R_N at the peak of the early allergic response and ovalbumin-induced increase in bronchoalveolar lavage fluid cysteinyl leukotrienes and amphiregulin but not IgE-induced mast cell activation in vitro.

Conclusion

Allergen challenge triggers the rapid onset of large airway narrowing, detected by R_N and G, and subsequent peripheral airway narrowing detected by G and H. Surfactant inhibits airway narrowing and reduces mast cell-derived mediators.     

Invasive versus noninvasive measurement of allergic and cholinergic airway responsiveness in mice

Background

This study seeks to compare the ability of repeatable invasive and noninvasive lung function methods to assess allergen-specific and cholinergic airway responsiveness (AR) in intact, spontaneously breathing BALB/c mice.

Methods

Using noninvasive head-out body plethysmography and the decrease in tidal midexpiratory flow (EF₅₀), we determined early AR (EAR) to inhaled Aspergillus fumigatus antigens in conscious mice. These measurements were paralleled by invasive determination of pulmonary conductance (GL), dynamic compliance (Cdyn) and EF₅₀ in another group of anesthetized, orotracheally intubated mice.

Results

With both methods, allergic mice, sensitized and boosted with A. fumigatus, elicited allergen-specific EAR to A. fumigatus (p < 0.05 versus controls). Dose-response studies to aerosolized methacholine (MCh) were performed in the same animals 48 h later, showing that allergic mice relative to controls were distinctly more responsive (p < 0.05) and revealed acute airway inflammation as evidenced from increased eosinophils and lymphocytes in bronchoalveolar lavage.

Conclusion

We conclude that invasive and noninvasive pulmonary function tests are capable of detecting both allergen-specific and cholinergic AR in intact, allergic mice. The invasive determination of GL and Cdyn is superior in sensitivity, whereas the noninvasive EF₅₀ method is particularly appropriate for quick and repeatable screening of respiratory function in large numbers of conscious mice.     

Synthesis of tenascin and laminin beta2 chain in human bronchial epithelial cells is enhanced by cysteinyl leukotrienes via CysLT1 receptor

Background

Cysteinyl leukotrienes (CysLTs) are key mediators of asthma, but their role in the genesis of airway remodeling is insufficiently understood. Recent evidence suggests that increased expression of tenascin (Tn) and laminin (Ln) β2 chain is indicative of the remodeling activity in asthma, but represents also an example of deposition of extracellular matrix, which affects the airway wall compliance. We tested the hypothesis that CysLTs affect production of Tn and Ln β2 chain by human bronchial epithelial cells and elucidated, which of the CysLT receptors, CysLT₁ or CysLT₂, mediate this effect.

Methods

Cultured BEAS-2B human bronchial epithelial cells were stimulated with leukotriene D₄ (LTD₄) and E₄ (LTE₄) and evaluated by immunocytochemistry, Western blotting, flow cytometry, and RT-PCR. CysLT receptors were differentially blocked with use of montelukast or BAY u9773.

Results

LTD₄ and LTE₄ significantly augmented the expression of Tn, whereas LTD₄, distinctly from LTE₄, was able to increase also the Ln β2 chain. Although the expression of CysLT₂ prevailed over that of CysLT₁, the up-regulation of Tn and Ln β2 chain by CysLTs was completely blocked by the CysLT₁-selective antagonist montelukast with no difference between montelukast and the dual antagonist BAY u9773 for the inhibitory capacity.

Conclusion

These findings suggest that the CysLT-induced up-regulation of Tn and Ln β2 chain, an important epithelium-linked aspect of airway remodeling, is mediated predominantly by the CysLT₁ receptor. The results provide a novel aspect to support the use of CysLT₁ receptor antagonists in the anti-remodeling treatment of asthma.     

Evidence for a novel Kit adhesion domain mediating human mast cell adhesion to structural airway cells

Background

Human lung mast cells (HLMCs) infiltrate the airway epithelium and airway smooth muscle (ASM) in asthmatic airways. The mechanism of HLMC adhesion to both cell types is only partly defined, and adhesion is not inhibited by function-blocking anti-Kit and anti-stem cell factor (SCF) antibodies. Our aim was to identify adhesion molecules expressed by human mast cells that mediate adhesion to human ASM cells (HASMCs) and human airway epithelial cells.

Methods

We used phage-display to isolate single chain Fv (scFv) antibodies with adhesion-blocking properties from rabbits immunised with HLMC and HMC-1 membrane proteins.

Results

Post-immune rabbit serum labelled HLMCs in flow cytometry and inhibited their adhesion to human BEAS-2B epithelial cells. Mast cell-specific scFvs were identified which labelled mast cells but not Jurkat cells by flow cytometry. Of these, one scFv (A1) consistently inhibited mast cell adhesion to HASMCs and BEAS-2B epithelial cells by about 30 %. A1 immunoprecipitated Kit (CD117) from HMC-1 lysates and bound to a human Kit-expressing mouse mast cell line, but did not interfere with SCF-dependent Kit signalling.

Conclusion

Kit contributes to human mast cell adhesion to human airway epithelial cells and HASMCs, but may utilise a previously unidentified adhesion domain that lies outside the SCF binding site. Targeting this adhesion pathway might offer a novel approach for the inhibition of mast cell interactions with structural airway cells, without detrimental effects on Kit signalling in other tissues.     

Comparison of Airway Responses in Sheep of Different Age in Precision-Cut Lung Slices (PCLS)

Background

Animal models should display important characteristics of the human disease. Sheep have been considered particularly useful to study allergic airway responses to common natural antigens causing human asthma. A rationale of this study was to establish a model of ovine precision-cut lung slices (PCLS) for the in vitro measurement of airway responses in newborn and adult animals. We hypothesized that differences in airway reactivity in sheep are present at different ages.

Methods

Lambs were delivered spontaneously at term (147d) and adult sheep lived till 18 months. Viability of PCLS was confirmed by the MTT-test. To study airway provocations cumulative concentration-response curves were performed with different allergic response mediators and biogenic amines. In addition, electric field stimulation, passive sensitization with house dust mite (HDM) and mast cells staining were evaluated.

Results

PCLS from sheep were viable for at least three days. PCLS of newborn and adult sheep responded equally strong to methacholine and endothelin-1. The responses to serotonin, leukotriene D₄ and U46619 differed with age. No airway contraction was evoked by histamine, except after cimetidine pretreatment. In response to EFS, airways in PCLS from adult and newborn sheep strongly contracted and these contractions were atropine sensitive. Passive sensitization with HDM evoked a weak early allergic response in PCLS from adult and newborn sheep, which notably was prolonged in airways from adult sheep. Only few mast cells were found in the lungs of non-sensitized sheep at both ages.

Conclusion

PCLS from sheep lungs represent a useful tool to study pharmacological airway responses for at least three days. Sheep seem well suited to study mechanisms of cholinergic airway contraction. The notable differences between newborn and adult sheep demonstrate the importance of age in such studies.     

Mast cells contribute to double-stranded RNA-induced augmentation of airway eosinophilia in a murine model of asthma

Background

Clinical studies showed the contribution of viral infection to the development of asthma. Although mast cells have multiple roles in the pathogenesis of allergic asthma, their role of in the virus-associated pathogenesis of asthma remains unknown. Most respiratory viruses generate double-stranded (ds) RNA during their replication. dsRNA provokes innate immune responses. We recently showed that an administration of polyinocinic polycytidilic acid (poly IC), a mimetic of viral dsRNA, during allergen sensitization augments airway eosinophilia and hyperresponsiveness in mice via enhanced production of IL-13.

Methods

The effect of poly IC on allergen-induced airway eosinophilia was investigated for mast cell-conserved Kit^+/+ mice and -deficient Kit^W/Kit^W-v mice. The outcome of mast cell reconstitution was further investigated.

Results

Airway eosinophilia and IL-13 production were augmented by poly IC in Kit^+/+ mice but not in Kit^W/Kit^W-v mice. When Kit^W/Kit^W-v mice were reconstituted with bone marrow-derived mast cells (BMMCs), the augmentation was restored. The augmentation was not induced in the mice systemically deficient for TIR domain-containing adaptor-inducing IFN-β (TRIF) or interferon regulatory factor (IRF)-3, both mediate dsRNA-triggered innate immune responses. The augmentation was, however, restored in Kit^W/Kit^W-v mice reconstituted with TRIF-deficient or IRF-3-deficient BMMCs. Although leukotriene B₄ and prostaglandin D₂ are major lipid mediators released from activated mast cells, no their contribution was shown to the dsRNA-induced augmentation of airway eosinophilia.

Conclusions

We conclude that mast cells contribute to dsRNA-induced augmentation of allergic airway inflammation without requiring direct activation of mast cells with dsRNA or involvement of leukotriene B₄ or prostaglandin D₂.     

Diesel Exhaust Particles Induce Cysteine Oxidation and S-Glutathionylation in House Dust Mite Induced Murine Asthma

Background

Diesel exhaust particle (DEP) exposure enhances allergic inflammation and has been linked to the incidence of asthma. Oxidative stress on the thiol molecules cysteine (Cys) and glutathione (GSH) can promote inflammatory host responses. The effect of DEP on the thiol oxidation/reduction (redox) state in the asthmatic lung is unknown.

Objective

To determine if DEP exposure alters the Cys or GSH redox state in the asthmatic airway.

Methods

Bronchoalveolar lavage fluid was obtained from a house dust mite (HDM) induced murine asthma model exposed to DEP. GSH, glutathione disulfide (GSSG), Cys, cystine (CySS), and s-glutathionylated cysteine (CySSG) were determined by high pressure liquid chromatography.

Results

DEP co-administered with HDM, but not DEP or HDM alone, decreased total Cys, increased CySS, and increased CySSG without significantly altering GSH or GSSG.

Conclusions

DEP exposure promotes oxidation and S-glutathionylation of cysteine amino acids in the asthmatic airway, suggesting a novel mechanism by which DEP may enhance allergic inflammatory responses.     

The Impact of Allergic Rhinitis and Asthma on Human Nasal and Bronchial Epithelial Gene Expression

Background

The link between upper and lower airways in patients with both asthma and allergic rhinitis is still poorly understood. As the biological complexity of these disorders can be captured by gene expression profiling we hypothesized that the clinical expression of rhinitis and/or asthma is related to differential gene expression between upper and lower airways epithelium.

Objective

Defining gene expression profiles of primary nasal and bronchial epithelial cells from the same individuals and examining the impact of allergic rhinitis with and without concomitant allergic asthma on expression profiles.

Methods

This cross-sectional study included 18 subjects (6 allergic asthma and allergic rhinitis; 6 allergic rhinitis; 6 healthy controls). The estimated false discovery rate comparing 6 subjects per group was approximately 5%. RNA was extracted from isolated and cultured epithelial cells from bronchial brushings and nasal biopsies, and analyzed by microarray (Affymetrix U133+ PM Genechip Array). Data were analysed using R and Bioconductor Limma package. For gene ontology GeneSpring GX12 was used.

Results

The study was successfully completed by 17 subjects (6 allergic asthma and allergic rhinitis; 5 allergic rhinitis; 6 healthy controls). Using correction for multiple testing, 1988 genes were differentially expressed between healthy lower and upper airway epithelium, whereas in allergic rhinitis with or without asthma this was only 40 and 301 genes, respectively. Genes influenced by allergic rhinitis with or without asthma were linked to lung development, remodeling, regulation of peptidases and normal epithelial barrier functions.

Conclusions

Differences in epithelial gene expression between the upper and lower airway epithelium, as observed in healthy subjects, largely disappear in patients with allergic rhinitis with or without asthma, whilst new differences emerge. The present data identify several pathways and genes that might be potential targets for future drug development.     

Neutralisation of Interleukin-13 in Mice Prevents Airway Pathology Caused by Chronic Exposure to House Dust Mite

Background

Repeated exposure to inhaled allergen can cause airway inflammation, remodeling and dysfunction that manifests as the symptoms of allergic asthma. We have investigated the role of the cytokine interleukin-13 (IL-13) in the generation and persistence of airway cellular inflammation, bronchial remodeling and deterioration in airway function in a model of allergic asthma caused by chronic exposure to the aeroallergen House Dust Mite (HDM).

Methodology/Principal Findings

Mice were exposed to HDM via the intranasal route for 4 consecutive days per week for up to 8 consecutive weeks. Mice were treated either prophylactically or therapeutically with a potent neutralising anti-IL-13 monoclonal antibody (mAb) administered subcutaneously (s.c.). Airway cellular inflammation was assessed by flow cytometry, peribronchial collagen deposition by histocytochemistry and airway hyperreactivity (AHR) by invasive measurement of lung resistance (R_L) and dynamic compliance (C_dyn). Both prophylactic and therapeutic treatment with an anti-IL-13 mAb significantly inhibited (P<0.05) the generation and maintenance of chronic HDM-induced airway cellular inflammation, peribronchial collagen deposition, epithelial goblet cell upregulation. AHR to inhaled methacholine was reversed by prophylactic but not therapeutic treatment with anti-IL-13 mAb. Both prophylactic and therapeutic treatment with anti-IL-13 mAb significantly reversed (P<0.05) the increase in baseline R_L and the decrease in baseline C_dyn caused by chronic exposure to inhaled HDM.

Conclusions/Significance

These data demonstrate that in a model of allergic lung disease driven by chronic exposure to a clinically relevant aeroallergen, IL-13 plays a significant role in the generation and persistence of airway inflammation, remodeling and dysfunction.     

Prolonged cigarette smoke exposure alters mitochondrial structure and function in airway epithelial cells

Background

Cigarette smoking is the major risk factor for COPD, leading to chronic airway inflammation. We hypothesized that cigarette smoke induces structural and functional changes of airway epithelial mitochondria, with important implications for lung inflammation and COPD pathogenesis.

Methods

We studied changes in mitochondrial morphology and in expression of markers for mitochondrial capacity, damage/biogenesis and fission/fusion in the human bronchial epithelial cell line BEAS-2B upon 6-months from ex-smoking COPD GOLD stage IV patients to age-matched smoking and never-smoking controls.

Results

We observed that long-term CSE exposure induces robust changes in mitochondrial structure, including fragmentation, branching and quantity of cristae. The majority of these changes were persistent upon CSE depletion. Furthermore, long-term CSE exposure significantly increased the expression of specific fission/fusion markers (Fis1, Mfn1, Mfn2, Drp1 and Opa1), oxidative phosphorylation (OXPHOS) proteins (Complex II, III and V), and oxidative stress (Mn-SOD) markers. These changes were accompanied by increased levels of the pro-inflammatory mediators IL-6, IL-8, and IL-1β. Importantly, COPD primary bronchial epithelial cells (PBECs) displayed similar changes in mitochondrial morphology as observed in long-term CSE-exposure BEAS-2B cells. Moreover, expression of specific OXPHOS proteins was higher in PBECs from COPD patients than control smokers, as was the expression of mitochondrial stress marker PINK1.

Conclusion

The observed mitochondrial changes in COPD epithelium are potentially the consequence of long-term exposure to cigarette smoke, leading to impaired mitochondrial function and may play a role in the pathogenesis of COPD.     

Volatile organic compounds enhance allergic airway inflammation in an experimental mouse model

Background

Epidemiological studies suggest an association between exposure to volatile organic compounds (VOCs) and adverse allergic and respiratory symptoms. However, whether VOCs exhibit a causal role as adjuvants in asthma development remains unclear.

Methods

To investigate the effect of VOC exposure on the development of allergic airway inflammation Balb/c mice were exposed to VOCs emitted by new polyvinylchloride (PVC) flooring, sensitized with ovalbumin (OVA) and characterized in acute and chronic murine asthma models. Furthermore, prevalent evaporated VOCs were analyzed and mice were exposed to selected single VOCs.

Results

Exposure of mice to PVC flooring increased eosinophilic lung inflammation and OVA-specific IgE serum levels compared to un-exposed control mice. The increased inflammation was associated with elevated levels of Th2-cytokines. Long-term exposure to PVC flooring exacerbated chronic airway inflammation. VOCs with the highest concentrations emitted by new PVC flooring were N-methyl-2-pyrrolidone (NMP) and 2,2,4-trimethyl-1,3-pentanediol diisobutyrate (TXIB). Exposure to NMP or TXIB also increased the allergic immune response in OVA-sensitized mice. In vitro or in vivo exposure to NMP or TXIB reduced IL-12 production in maturing dendritic cells (DCs) and enhanced airway inflammation after adoptive DC transfer into Balb/c mice. At higher concentrations both VOCs induced oxidative stress demonstrated by increased isoprostane and glutathione-S-transferase-pi1 protein levels in the lung of non-sensitized mice. Treatment of PVC flooring-exposed mice with N-acetylcysteine prevented the VOC-induced increase of airway inflammation.

Conclusions

Our results demonstrate that exposure to VOCs may increase the allergic immune response by interfering with DC function and by inducing oxidative stress and has therefore to be considerate as risk factor for the development of allergic diseases.     

Early phase resolution of mucosal eosinophilic inflammation in allergic rhinitis

Background

It is widely assumed that apoptosis of eosinophils is a central component of resolution of allergic airway disease. However, this has not been demonstrated in human allergic airways in vivo. Based on animal in vivo observations we hypothesised that steroid-induced resolution of human airway eosinophilic inflammation involves inhibition of CCL5 (RANTES), a CC-chemokine regulating eosinophil and lymphocyte traffic, and elimination of eosinophils without evident occurrence of apoptotic eosinophils in the diseased tissue.

Objective

To determine mucosal eosinophilia, apoptotic eosinophils, general cell apoptosis and cell proliferation, and expression of CCL5 and CCL11 (eotaxin) in human allergic airway tissues in vivo at resolution of established symptomatic eosinophilic inflammation.

Methods

Twenty-one patients with intermittent (birch and/or grass) allergic rhinitis received daily nasal allergen challenges for two seven days'' periods separated by more than two weeks washout. Five days into these "artificial pollen seasons", nasal treatment with budesonide was instituted and continued for six days in a double blinded, randomized, placebo-controlled, and crossover design. This report is a parallel group comparison of nasal biopsy histochemistry data obtained on the final day of the second treatment period.

Results

Treatments were instituted when clinical rhinitis symptoms had been established. Compared to placebo, budesonide reduced tissue eosinophilia, and subepithelial more than epithelial eosinophilia. Steroid treatment also attenuated tissue expression of CCL5, but CCL11 was not reduced. General tissue cell apoptosis and epithelial cell proliferation were reduced by budesonide. However, apoptotic eosinophils were not detected in any biopsies, irrespective of treatment.

Conclusions

Inhibition of CCL5-dependent recruitment of cells to diseased airway tissue, and reduced cell proliferation, reduced general cell apoptosis, but not increased eosinophil apoptosis, are involved in early phase steroid-induced resolution of human allergic rhinitis.     

Effects of a dual CCR3 and H1-antagonist on symptoms and eosinophilic inflammation in allergic rhinitis

Background

The CC-chemokine receptor-3 (CCR3) has emerged as a target molecule for pharmacological intervention in allergic inflammation.

Objective

To examine whether a dual CCR3 and H₁-receptor antagonist (AZD3778) affects allergic inflammation and symptoms in allergic rhinitis.

Methods

Patients with seasonal allergic rhinitis were subjected to three seven days'' allergen challenge series. Treatment with AZD3778 was given in a placebo and antihistamine-controlled design. Symptoms and nasal peak inspiratory flow (PIF) were monitored in the morning, ten minutes post challenge, and in the evening. Nasal lavages were carried out at the end of each challenge series and α₂-macroglobulin, ECP, and tryptase were monitored as indices of allergic inflammation.

Results

Plasma levels of AZD3778 were stable throughout the treatment series. AZD3778 and the antihistamine (loratadine) reduced rhinitis symptoms recorded ten minutes post challenge during this period. AZD3778, but not the anti-histamine, also improved nasal PIF ten minutes post challenge. Furthermore, scores for morning and evening nasal symptoms from the last five days of the allergen challenge series showed statistically significant reductions for AZD3778, but not for loratadine. ECP was reduced by AZD3778, but not by loratadine.

Conclusions

AZD3778 exerts anti-eosinophil and symptom-reducing effects in allergic rhinitis and part of this effect can likely be attributed to CCR3-antagonism. The present data are of interest with regard to the potential use of AZD3778 in allergic rhinitis and to the relative importance of eosinophil actions to the symptomatology of allergic rhinitis.

Trial registration

EudraCT No: 2005-002805-21.     

Prostaglandin E2 Prevents Hyperosmolar-Induced Human Mast Cell Activation through Prostanoid Receptors EP2 and EP4

Background

Mast cells play a critical role in allergic and inflammatory diseases, including exercise-induced bronchoconstriction (EIB) in asthma. The mechanism underlying EIB is probably related to increased airway fluid osmolarity that activates mast cells to the release inflammatory mediators. These mediators then act on bronchial smooth muscle to cause bronchoconstriction. In parallel, protective substances such as prostaglandin E₂ (PGE₂) are probably also released and could explain the refractory period observed in patients with EIB.

Objective

This study aimed to evaluate the protective effect of PGE₂ on osmotically activated mast cells, as a model of exercise-induced bronchoconstriction.

Methods

We used LAD2, HMC-1, CD34-positive, and human lung mast cell lines. Cells underwent a mannitol challenge, and the effects of PGE₂ and prostanoid receptor (EP) antagonists for EP_1–4 were assayed on the activated mast cells. Beta-hexosaminidase release, protein phosphorylation, and calcium mobilization were assessed.

Results

Mannitol both induced mast cell degranulation and activated phosphatidyl inositide 3-kinase and mitogen-activated protein kinase (MAPK) pathways, thereby causing de novo eicosanoid and cytokine synthesis. The addition of PGE₂ significantly reduced mannitol-induced degranulation through EP₂ and EP₄ receptors, as measured by beta-hexosaminidase release, and consequently calcium influx. Extracellular-signal-regulated kinase 1/2, c-Jun N-terminal kinase, and p38 phosphorylation were diminished when compared with mannitol activation alone.

Conclusions

Our data show a protective role for the PGE₂ receptors EP₂ and EP₄ following osmotic changes, through the reduction of human mast cell activity caused by calcium influx impairment and MAP kinase inhibition.     

Pulmonary cryptococcosis induces chitinase in the rat

Background

We previously demonstrated that chronic pulmonary infection with Cryptococcus neoformans results in enhanced allergic inflammation and airway hyperreactivity in a rat model. Because the cell wall of C. neoformans consists of chitin, and since acidic mammalian chitinase (AMCase) has recently been implicated as a novel mediator of asthma, we sought to determine whether such infection induces chitinase activity and expression of AMCase in the rat.

Methods

We utilized a previously-established model of chronic C. neoformans pulmonary infection in the rat to analyze the activity, expression and localization of AMCase.

Results

Our studies indicate that intratracheal inoculation of C. neoformans induces chitinase activity within the lung and bronchoalveolar lavage fluid of infected rats. Chitinase activity is also elicited by pulmonary infection with other fungi (e.g. C. albicans), but not by the inoculation of dead organisms. Enhanced chitinase activity reflects increased AMCase expression by airway epithelial cells and alveolar macrophages. Systemic cryptococcosis is not associated with increased pulmonary chitinase activity or AMCase expression.

Conclusion

Our findings indicate a possible link between respiratory fungal infections, including C. neoformans, and asthma through the induction of AMCase.