Effect of ozone and nitrogen dioxide on the permeability of bronchial epithelial cell cultures of non-asthmatic and asthmatic subjects.

BACKGROUND: Although epidemiological as well as in vivo exposure studies suggest that ozone (O3) and nitrogen dioxide (NO2) may play a role in airway diseases such as asthma, the underlying mechanisms are not clear. OBJECTIVE: Our aim was to investigate the effect of O3 and NO2 on the permeability of human bronchial epithelial cell (HBEC) cultures obtained from non-atopic non-asthmatic (non-asthmatics) and atopic mild asthmatic (asthmatics) individuals. METHODS: We cultured HBECs from bronchial biopsies of non-asthmatics and asthmatics, and exposed these for 6 h to air, 10 to 100 parts per billion (p.p.b.) O3, or to 100 to 400 p.p.b. NO2, and assessed changes in electrical resistance (ER) and movement of 14C-BSA across the cell cultures. RESULTS: Although exposure to either O3 or NO2 did not alter the permeability of HBEC cultures of non-asthmatics, 10 to 100 p.p.b. O3 and 400 p.p.b. NO2 significantly decreased the ER of HBEC cultures of asthmatics, when compared with exposure to air. Additionally, 10, 50 and 100 p.p.b. O3 led to a significant increase in the movement of 14C-BSA across asthmatic HBEC cultures, after 6 h of exposure (medians = 1.73%; P < 0.01, 1.50%; P < 0.05 and 1.53%, P < 0.05, respectively), compared with air exposed cultures (median = 0.89%). Similarly, exposure for 6 h to both 200 and 400 p.p.b. NO2 significantly increased the movement of 14C-BSA across asthmatic HBEC cultures, when compared with air exposure. A comparison of data obtained from the two study groups demonstrated that 10 to 100 p.p.b. O3- and 200 to 400 p.p.b. NO2-induced epithelial permeability was greater in cultures of asthmatics compared with non-asthmatics. CONCLUSION: These results suggest that HBECs of asthmatics may be more susceptible to the deleterious effects of these pollutants. Whether in patients with asthma the greater susceptibility of bronchial epithelial cells to O3 and NO2 contributes to the development of the disease, or is a secondary characteristic of this condition, remains to be determined.

Cigarette smoke decreases the expression of secretory component in human bronchial epithelial cells, in vitro.

Epithelial secretory component (SC) is thought to be essential for immunologic protection of the respiratory tract from viral and bacterial infection, since it transports polymeric IgA from the basolateral to the luminal surface of epithelial cells. We have hypothesized that recurrent infection in airways of cigarette smokers is at least partly a consequence of cigarette smoke-induced downregulation of the expression and/or release of SC from airway epithelial cells, subsequently resulting in decreased transcytosis of secretory IgA to the airway lumen. To test this hypothesis, we have cultured human bronchial epithelial cells (HBEC) from surgical tissues and exposed these for 20 minutes to either air or cigarette smoke. Following exposure to cigarette smoke the HBEC cultures were incubated for a further period of up to 24 h, during which time separate cultures were processed by immunocytochemistry for the presence of SC, in a time-dependent manner. The stained HBEC cultures were evaluated by colour image analysis for the percentage of total cells staining for SC. Exposure to cigarette smoke significantly decreased the percentage of total HBEC staining for secretory component from a baseline value (median and interquartile[IQ]1, IQ3) of 35.9% (26.5, 41.6) to 15.7% (8.2, 25.4; p < 0.05) 1 h after exposure, compared with exposure to air. The percentage of cells staining for secretory component were further reduced to 5.3% (3.3, 6.4; p < 0.01), 6 h after exposure, compared to exposure to air. After incubation for 24 h following exposure to cigarette smoke, there was gross cell damage and the cells were not suitable for immunocytochemical analysis. These results suggest that short-term exposure to cigarette smoke may compromise the immune barrier function of the airway mucosa by decreasing the expression and/or release of epithelial SC, thereby decreasing the transcytosis of IgA necessary for inactivating the microbial pathogens in the airway lumen.

Interaction of cigarette smoke and house dust mite allergens on inflammatory mediator release from primary cultures of human bronchial epithelial cells.

Several studies have shown that exposure to cigarette smoke and/or house dust mite (HDM) can lead to increased airway inflammation in susceptible individuals. The underlying mechanisms, however, are not defined. To investigate the interaction between cigarette smoke and HDM allergen on mediator release from primary cultures of human bronchial epithelial cells. Confluent human bronchial epithelial cell cultures were exposed to cigarette smoke in the absence or presence of HDM allergen and investigated for the release of IL-8, IL-1beta, and sICAM-1. Damage to the epithelial cells themselves was assessed by release of 51Cr. On separate occasions, we investigated the effect of PTL11028, a highly potent and selective Der p1 inhibitor, on HDM allergen-induced release of IL-8, following activation of HDM allergen by incubation with cysteine. The effect of cigarette smoke exposure on the stability of these released mediators in prepared solutions in the absence/presence of reduced glutathione was also studied. Both HDM allergens and short-term (20 min) cigarette smoke exposure led to a significantly increased release of IL-8, IL-1beta and sICAM-1 from the epithelial cell cultures. Longer exposure (1-6 h) to cigarette smoke led to a dramatic decrease in the amount of these mediators detected in the culture medium. Whilst incubation of epithelial cultures with HDM allergen did not cause any significant change in the release of 51Cr from pre-loaded cells, cigarette smoke on its own led to a marked, exposure and incubation-time dependent increase in the release of 51Cr. Incubation with HDM allergen led to a significant, dose and time-dependent increase in the release of IL-8, which was further enhanced when the allergen extract was pre-activated with cysteine. This effect was completely abrogated by PTL11028, a novel Der p1 inhibitor. Prepared solutions of various concentrations of IL-8, IL-1beta and sICAM-1 exposed to cigarette smoke demonstrated a dramatic exposure time-dependent decrease in the detectable amount of these mediators, an effect which was abrogated by GSH. HDM-induced airway inflammation may include Der p-mediated release of inflammatory mediators from epithelial cells. Additionally, short-term cigarette smoke exposure may induce airway inflammation by release of inflammatory mediators from these cells, an effect which may be potentiated by Der p allergens. Longer term cigarette smoke exposure may cause damage to epithelial cells and changes in the structure of inflammatory mediators.

Effect of cigarette smoke on the permeability and IL-1beta and sICAM-1 release from cultured human bronchial epithelial cells of never-smokers, smokers, and patients with chronic obstructive pulmonary disease.

Although cigarette smoking is of paramount importance in the development of chronic obstructive pulmonary disease (COPD), only a small proportion of smokers develop the disease. We tested the hypothesis that the response of the bronchial epithelium to cigarette smoke (CS) differs in patients with COPD. Such a difference might explain in part why only some cigarette smokers develop the disease. We established primary explant cultures of human bronchial epithelial cells (HBEC) from biopsy material obtained from never-smokers who had normal pulmonary function, smokers with normal pulmonary function, and smokers with COPD, and exposed these for 20 min to CS or air. Measurements were subsequently made over a period of 24 h of transepithelial permeability and release of interleukin (IL)-1beta and soluble intercellular adhesion molecule-1 (sICAM-1). In addition, intracellular reduced glutathione (GSH) levels were measured after 24 h incubation. Exposure to CS increased the permeability of these cultures in all study groups, but the most marked effect was observed in cultures from patients with COPD (mean increase, 85.5%). The smallest CS-induced increase in the permeability was observed in HBEC cultured from smokers with normal pulmonary function (mean, 25.0%), and this was significantly lower than that of HBEC from never-smokers (mean, 53.4%) (P<0.001). Compared with exposure to air, exposure to CS led to a significantly increased release of these mediators from cultures of the never-smoker group (mean 250.0% increase in IL-1beta and mean 175.3% increase in sICAM-1 24 h after exposure) and COPD group (mean 383.3% increase in IL-1beta and mean 97.4% increase in sICAM-1 24 h after exposure). In contrast, CS exposure did not influence significantly the release of either mediator from the cells of smokers with normal pulmonary function. Levels of intracellular GSH were significantly higher in cultures of HBEC derived from smokers, both those with normal pulmonary function and those with COPD, compared with cultures from healthy never-smokers. Exposure to CS significantly decreased the concentration of intracellular GSH in all cultures. However, the fall in intracellular GSH was significantly greater in cells from patients with COPD (mean 72.9% decrease) than in cells from never-smokers (mean 61.4% decrease; P = 0.048) or smokers with normal pulmonary function (mean 43.9% decrease; P = 0.02). These results suggest that whereas smokers with or without COPD demonstrate increased levels of GSH within bronchial epithelial cell cultures, those with COPD have a greater susceptibility to the effects of CS in reducing GSH levels and causing increased permeability and release of proinflammatory mediators such as IL-1beta and sICAM-1.

Functional effects of the inhibition of the cysteine protease activity of the major house dust mite allergen Der p 1 by a novel peptide-based inhibitor.

BACKGROUND: The house dust mite (HDM) Dermatophagoides pteronyssinus is an important source of allergens, which can cause allergic conditions. The cysteine protease activity of Der p 1 may enhance the potency of this major mite allergen through cleavage of CD23 and CD25 from the surface of immune cells, IgE independent mast cell activation, increases in epithelial cell permeability and inactivation of an endogenous serine protease inhibitor. Inhibition of the enzymatic activity of Der p 1 may therefore be of therapeutic benefit. OBJECTIVE: To examine the activity of PTL11028, a newly developed Der p 1 inhibitor, in a range of assays that directly or indirectly measure Der p 1 protease activity and to compare its activity to endogenous cysteine protease inhibitors. METHODS: The proteolytic activities of purified Der p 1 or HDM extract and inhibitory properties of PTL11028 were examined through cleavage of an artificial peptidyl substrate, cleavage of CD23 from human B cells and permeability studies on primary human bronchial epithelial cells. RESULTS: PTL11028 is a highly potent and specific Der p 1 inhibitor, being effective against both purified protease and Der p 1 within HDM extract. PTL11028 can completely inhibit Der p 1-mediated CD23 cleavage from human B cells and also reduces HDM-induced human bronchial epithelial cell permeability by 50%. Der p 1 is potently inhibited by cystatin A and to a lesser extent by cystatins C and E/M. CONCLUSION: PTL11028 is a highly potent and selective irreversible inhibitor of the cysteine protease activity of Der p 1, an activity that may be modulated in vivo by some human cystatins. PTL11028 prevents the Der p 1-mediated cleavage of CD23 from human B cells and significantly reduces HDM-induced permeabilization of the epithelial barrier. PTL11028 is an important tool to examine the biological effects of Der p 1 in a range of in vitro and in vivo model systems.

The effect of exposure to ozone and nitrogen dioxide on the airway response of atopic asthmatics to inhaled allergen: dose- and time-dependent effects.

Eleven mild atopic asthmatic patients were exposed for 6 h, in randomized order, to air, 100 ppb O3, 200 ppb NO2, and 100 ppb O3 + 200 ppb NO2, followed immediately by bronchial allergen challenge. Subsequently 10 of these patients were exposed for 3 h to air, 200 ppb O3, 400 ppb NO2, and 200 ppb O3 + 400 ppb NO2, followed immediately by bronchial allergen challenge. All exposures were carried out in an environmental chamber, with intermittent moderate exercise, and a minimal interval of 2 wk. Exposure for 6 h to 100 ppb O3, 200 ppb NO2, and 100 ppb O3 + 200 ppb NO2 did not lead to any significant increase in the airway response of these individuals to inhaled allergen, when compared with exposure for 6 h to air. In contrast, exposure for 3 h to 200 ppb O3, 400 ppb NO2, and 200 ppb O3 + 400 ppb NO2 significantly decreased the dose of allergen (in log cumulative breath units [CBU]) required to decrease FEV1 by 20% (allergen PD20FEV1), compared with exposure to air (geometric mean CBU: 3.0 for air versus 2.66 for O3 [p = 0.002]; 2.78 for NO2 [p = 0. 018]; 2.65 for O3 + NO2 [p = 0.002]). These results suggest that the pollutant-induced changes in airway response of mild atopic asthmatics to allergen may be dependent on a threshold concentration rather than the total amount of pollutant inhaled over a period of time.

Cigarette smoke potentiates house dust mite allergen-induced increase in the permeability of human bronchial epithelial cells in vitro.

Although studies have suggested that exposure to cigarette smoke (CS) may be associated with the development of atopy, the mechanisms underlying this are not clearly understood. It has been suggested that CS impairs the barrier function of the airway epithelium, leading to increased access of allergens such as those of the house dust mite (HDM) Dermatophagoides pteronyssinus (Der p) to antigen-presenting cells, with subsequent allergic sensitization. In order to test this hypothesis, we established primary explant cultures of human bronchial epithelial cells (HBEC) in cell culture inserts, and exposed these for 20 min, 1 h, 3 h, and 6 h to CS or air in the absence or presence of 300 ng/ml Der p, and then further incubated the cultures over a period of 24 h. The HBEC cultures were assessed for changes in permeability as measured by changes in: (1) electrical resistance (ER); and (2) passage of 14C-labeled bovine serum albumin (14C-BSA) and Der p allergens across the HBEC cultures. We also assessed the effects of protease inhibitors and the antioxidant glutathione (GSH) in this experimental system. Damage to HBEC cultures was assessed by the release of [51Cr]sodium chromate from prelabeled cells, and by release of lactate dehydrogenase (LDH). Twenty minutes of exposure to CS as compared with exposure to air did not significantly alter either the ER or passage of 14C-BSA across the HBEC cultures. In contrast, incubation with Der p led to a significant increase in the permeability of HBEC cultures, an effect that was enhanced by exposure to CS but was abrogated by the specific protease inhibitors and GSH. Passage of Der p was also increased by exposure to CS. Exposure of HBEC cultures to CS led to a significant release of 51Cr and LDH from these cells as compared with cells exposed to air. This effect was augmented further when HBEC cultures were incubated with Der p. Exposure of HBEC cultures for 1 h, 3 h, and 6 h to CS led to a markedly significant dose- and time-dependent increase in the permeability of these cells. These results suggest that exposure to CS significantly enhances Der p-induced decreases in electrical resistance and the increased passage across HBEC cultures of 14C-BSA and of the Der p allergen itself.

Effect of fluticasone propionate aqueous nasal spray on allergen-induced inflammatory changes in the nasal airways of allergic rhinitics following exposure to nitrogen dioxide.

BACKGROUND: The authors have recently demonstrated that prior exposure for 6 h to 400 p.p.b. nitrogen dioxide significantly enhances the early phase response of eosinophils in the nasal airways of allergic rhinitics to subsequent allergen provocation. OBJECTIVE: To investigate whether treatment with fluticasone propionate aqueous nasal spray (FP) can alter the inflammatory response in the nasal airways under these conditions. METHODS: Sixteen allergic, rhinitic patients were recruited for this double-blind, randomized, cross-over study and received either topical FP 200 microg once daily or matched placebo for 4 weeks. At the end of treatment, all underwent nasal lavage followed by a 6 h exposure to 400 p.p.b. NO2. Following exposure to NO2, nasal allergen challenge was performed and nasal lavage repeated. After a 4 week washout period, patients were given alternate treatment and tested as above. RESULTS: Analysis of eosinophil cationic protein (ECP) in lavage samples from patients treated with placebo, demonstrated that this was significantly increased from a median value of 2.3 ng/mL (range: 1.0-7.1) to 15.1 ng/mL (range: 1.5-40.0; P = 0.001) following exposure to NO2 and allergen challenge. However, in patients treated with FP, ECP concentrations only increased from 3.3 ng/mL (range: 0.2-9.2) to 5.1 ng/mL (range: 0.3-20.0; P = 0.034) following exposure to NO2 and allergen challenge. The difference of the changes in ECP concentration between the placebo and the FP-treated group was significant (P = 0.003). Similarly, there was a significant increase in the number of eosinophils in nasal lavage after exposure to NO2 and allergen challenge in the placebo group, and this increase was inhibited in FP group (P = 0.002). CONCLUSION: These results suggest that FP influences NO2- and allergen-induced changes in eosinophil function, as well as eosinophil number in the nasal airway of allergic rhinitics.

Why is allergy increasing?--environmental factors.

Data from epidemiological studies have shown that allergic conditions have increased over the last 30-40 years, particularly in developed countries, despite a decrease in the severity of grass pollen seasons. Other epidemiological studies suggest an interaction between allergic diseases and traffic pollution, and laboratory findings indicate that diesel exhaust particles enhance sensitivity to allergens. In an in vitro study, we found evidence to suggest that cigarette smoke may render the airway epithelium more susceptible to adverse effects of allergens. Evidence from other studies indicates that O3 and NO2, with or without SO2, can enhance the airway allergic response in susceptible individuals such as those with asthma and rhinitis. Studies investigating cellular and subcellular mechanisms suggest that pollutants are likely to influence the actions and interactions of a variety of cells, and lead to the synthesis of proinflammatory mediators that modulate the activity and functions of inflammatory cells.

The effect of nedocromil sodium on human airway epithelial cell-induced eosinophil chemotaxis and adherence to human endothelial cell in vitro.

Although some studies have shown that long-term treatment of asthmatics with nedocromil sodium can reduce airway hyperresponsiveness and improve symptoms and lung function, the mechanisms underlying its effects are not well understood. We have investigated the effect of nedocromil sodium on eosinophil chemotaxis, eosinophil adherence to human endothelial cells and release of soluble intercellular adhesion molecule-1 (sICAM-1) from endothelial cells, induced by conditioned medium collected from cultured human bronchial epithelial cells. Conditioned medium significantly increased eosinophil chemotaxis from a baseline median value of 2.1 (range 1.9-4.5) cells-high power field(-1) (HPF) to 10.5 (range 7.8-12.3) cells-HPF(-1) (p<0.05). Similarly, conditioned medium significantly increased eosinophil adherence to endothelial cells from a baseline value of 9 (range 8-12)% to 23 (range 21-30)% (p<0.05). Nedocromil sodium, at 10(-5) M concentration, significantly attenuated the eosinophil chemotaxis and adherence induced by conditioned medium. Conditioned medium also significantly increased the release of sICAM-1 from endothelial cells, from a baseline value of 11.5 (range 8.1-15.4) pg x microg(-1) protein to 67.6 (range 55.6-73.5) pg x microg(-1) protein (p<0.05). This was significantly attenuated by anti-tumour necrosis factor-alpha (TNF-alpha), anti-interleukin-1beta (IL-1beta) and 10(-5) M nedocromil sodium. These findings suggest that human bronchial epithelial cell-derived mediators may potentiate eosinophil activity, and that this can be modulated by nedocromil sodium, suggesting a possible mechanism underlying its anti-inflammatory effect.

New insights into the understanding of asthma.

The prevalence of asthma is increasing, despite better understanding of its pathogenesis and improved treatments. During the past 10 years, the perception of asthma has shifted from a disease primarily characterized by altered smooth muscle function to one mainly characterized by chronic inflammation. This article reviews the evidence supporting the relationship of inflammation in both the upper and lower airways, focusing on intermittent seasonal disease as well as on the more chronic and severe forms of asthma, including that associated with aspirin intolerance. It also presents evidence to support a pivotal role for the epithelial cell, together with the mast cell and the eosinophil, in initiating and maintaining inflammation in the upper and lower airways.

Mechanisms of pollution-induced airway disease: in vitro studies in the upper and lower airways.

Evidence from both epidemiological and laboratory-based studies suggests that increased exposure to liquid petroleum and gas-derived air pollutants [nitrogen dioxide (NO2), ozone, and respirable particulate matter] may play a role in the clinical manifestation of both allergic and non-allergic airway disease. The mechanisms and cell types involved in pollutant-mediated effects in the airways, however, are not clear. In vitro studies have suggested that human fibroblasts, B-lymphocytes, alveolar macrophages, and epithelial cells/cell lines may be involved. Studies of fibroblasts and macrophages have demonstrated that exposure to ozone results in decreased cell viability and increased release of pro-inflammatory mediators from macrophages. Similarly, studies of B-lymphocytes have demonstrated that exposure to diesel exhaust particles (DEP) enhances the synthesis of immunoglobulin E by these cells. The airway epithelial cells have received the greatest attention in mechanistic studies of air pollution-induced airway disease and suggest that these cells are likely to play a pivotal role in the pathogenesis of airways disease. Various studies have demonstrated that exposure of nasal or bronchial epithelial cells to NO2, ozone, and DEP results in significant synthesis and release of pro-inflammatory mediators, including eicosanoids, cytokines, and adhesion molecules. Additionally, evidence suggests that epithelial cells of atopic individuals release significantly greater amounts of cytokines such as granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-6 (IL-6), IL-8, and regulated on activation, normal T-cell expressed and secreted (RANTES), on exposure to NO2 and ozone. Studies investigating the biological relevance of epithelial cell-derived pro-inflammatory mediators have shown that these enhance eosinophil chemotaxis and eosinophil adherence to endothelial cells, suggesting that pollution-induced inflammation of the airways is likely to be influenced by modulation of epithelial synthesis and release of these mediators.

Allergen-irritant interaction and the role of corticosteroids.

Studies of exposure to air pollutants, such as ozone and nitrogen dioxide (NO2) +/- sulphur dioxide (SO2), have demonstrated that these agents, either individually or in combination, increase the airway response of both asthmatics and allergic rhinitics to inhaled allergen. Other studies have demonstrated that exposure to these pollutants significantly increased the levels of eosinophil cationic protein (ECP) in the nasal secretions of both asthmatics and allergic rhinitics, suggesting that pollutants may prime eosinophils for subsequent activation by allergen. More recently, our studies have demonstrated that treatment with inhaled corticosteroids, such as fluticasone propionate, significantly attenuated pollution+ allergen-induced release of ECP in allergic rhinitics. Although the mechanisms underlying the potentiating effects of pollutants on allergen-induced changes in the airways of allergic individuals are not fully understood, in vitro studies have suggested that airway epithelial cells may play an important role, since they can synthesize a variety of cytokines and adhesion molecules which influence the activity of eosinophils and other inflammatory cells. Studies of nasal epithelial cells cultured from biopsies of atopic rhinitic and atopic non-rhinitic individuals have shown that they constitutively release significantly greater quantities of pro-inflammatory cytokines than nasal epithelial cells of non-atopic individuals, and that the release of these cytokines is greater from cells of atopic rhinitics during the pollen season. Furthermore, exposure of the cells of rhinitics to ozone led to an even greater release of these cytokines, and this effect was attenuated by treatment with fluticasone propionate and beclomethasone dipropionate.

Nedocromil sodium and airway inflammation in vivo and in vitro.

We conducted a series of studies investigating the antiinflammatory effects of nedocromil sodium, with particular reference to its effects on human bronchial epithelial cells and eosinophils in vitro and on eosinophils in vivo. Nedocromil sodium produced a dose-related inhibition of ozone-induced IL-8 release from human bronchial epithelial cells and also attenuated the release of granulocyte macrophage colony-stimulating factor, tumor necrosis factor-alpha, and soluble intercellular adhesion molecule 1. The culture medium from human bronchial epithelial cell cultures, containing the proinflammatory cytokines IL-8, granulocyte macrophage colony-stimulating factor, "regulated on activation, normal T expressed and secreted," IL-1 beta, and tumor necrosis factor-alpha, increased eosinophil chemotaxis and eosinophil adhesion to cultured human endothelial cells. The chemotaxis and increased adhesion were blocked in the presence of nedocromil sodium. The drug also abrogated the epithelial cell dysfunction (assessed as ciliary beat frequency) induced by the presence of activated eosinophils and blocked the release of eosinophil cationic protein from the eosinophils. We also conducted a double-blind placebo-controlled study of the effects of regular albuterol 200 micrograms or nedocromil sodium 4 mg, both given four times daily for 16 weeks, on inflammatory cell numbers in bronchial biopsy and bronchoalveolar lavage samples. Assessed in terms of total and activated eosinophils in biopsy samples, inflammation decreased with nedocromil sodium and was significantly different from a deterioration with albuterol, although neither of these changes was significantly different from that with placebo treatment. Levels of eosinophil cationic protein in bronchoalveolar lavage samples showed a similar trend.