Mechanisms of pollution-induced allergy and asthma

REVUE FRAN~AISE D'ALLERGOLOGIE

ET D'IMMUNOLOGIE CLINIQLIE

Mechanisms of pollution-induced allergy and asthma C. RUSZNAK, S. JENKINS, ER. MILLS, R.J. SAPSFORD, J.L. DEVALIA, R.J. DAVIES

SUMMARY

RESUM~

Evidence from a n u m b e r of epidemiological and exposure chamber studies suggests that an increase in air pollutants, such as ozone (03), oxides of nitrogen (NOx), respirable particulates (PMlo) and volatile organic chemicals (VOCs), resulting from increased use of liquid petroleum gas or kerosene, may be linked to an increase in the prevalence of allergic disease, particularly in the developed countries. Several studies have indicated that there is an association between hospital emergency room visits due to asthma and increased air pollution. Studies from Japan and Germany have demonstrated that there is a significant association between increased vehicle exhaust pollution and increased incidence of rhino-conjunctivitis and hay-fever. Exposure chamber studies have shown that acute inhalation of air pollutants such as 03, nitrogen dioxide (NO2) and sulphur dioxide (SO2), either individually or in combination, may increase the airway response to inhaled allergen in atopic asthma. Studies investigating the mechanisms underlying pollution-induced pathogenesis of allergic airways disease have demonstrated that there is an association between air pollutants and an increase in mean total/specific serum IgE level and increased positive skin reactions. Bronchial and nasal lavage studies have shown that O 3 and NO 2 can induce significant increase in epithelial damage and permeability, an influx of inflammatory cells and release of pro-inflammatory cytokines into the respiratory tract. We have shown that exposure of human bronchial epithelial cells to NO 2, 03, diesel exhaust particles (DEP) and cigarette smoke (CS), in vitro, leads to significant epithelial cell dysfunction a n d significant release of proinflammatory cytokines such as interleukin-8 (IL-8),

M6canismes de l'induction d'aUergie et d'asthme par la p o l l u t i o n . - U n certain nombre d'6tudes 6pid6miologiques ou en chambres d'exposition, sugg6rent l'existence de liens entre l'angmentation des polluants a6riens comme l'ozone (OB), les oxydes d'azote (NOx), les particules inhalables (PM10) et les produits organiques volatiles (POV) r6sultant de l'usage croissant de gaz naturel et de k6ros~ne et la pr6valence des maladies allergiques, particuli6rement dans les pays industrialis6s. Plusieurs 6tudes ont conclu une relation entre les consultations hospitali6res d'urgence due A l'asthme et les pics de pollution. Des 6tudes, au Japon et en Allemagne ont montr6 qu'il y avait des relations significatives entre l'augmentation de la pollution par les gaz d'4chappement des v6hicules et l'incidence accrue des rhino-conjonctiviteset rhumes des foins. Les 6tudes en chambres d'exposition ont mesur4 q u ' u n e forte inhalation de polluants a6riens comme l'O B, le dioxyde d'azote (NO2) et le dioxyde de soufre (SO2), soit s6parfment, soit associ6s, pouvait augmenter la r6ponse des voies respiratoires aux allerg~nes inhal6s darts l'asthme atopique. Des 6tudes sur les m&anismes conditionnant la pathog6nie des affections respiratoires allergiques induites par la pollution ont d6montr4 qu'il existe un lien entre les polluants a6riens et une augmentation du taux s6rique moyen d'IgE totales ou sp&ifiques ainsi q u ' u n e majoration des tests cutan6s. Des 6tudes par lavage bronchique et nasal ont montr6 que 03 et NO 2 peuvent induire une augmentation significative de l'alt6ration et de la perm6abilit6 6pith6liales, u n affiux de cellules inflammatoires et la lib6ration de cytokines pro-inflammatoires dans le tractus respiratoire. Nous avons montr6 que chez l'homme l'exposition des

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Academic Department of Respiratory Medicine, St. Bartholomew's and the Royal London School of Medicine and Dentistry, The London Chest Hospital, LONDON E29JX, (United Kingdom). Correspondence: RJ. Davies, MA MD FRCP. Professor of Respiratory Medicine, Academic Department of Respiratory Medicine, St. Bartholomew's and the Royal London, School of Medicine and Dentistry,the London Chest Hospital, Bonner Road, LONDON E29JX, (United Kingdom). Interasma Marrakech' 98.

RUSZNAK C., JENKINS S. MILLS RR., SAPSFORD R.J., DEVALIA J.L., DAVIES R.J. - Mechanisms of pollution-induced allergy and asthma. Rev. ft. Allergol., 1998, 38 (7S), $80-$90.

© Expansion Scientifique Publications, 1998

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tumour necrosis factor-c~ (TNF-cQ, granulocytemacrophage colony stimulating factor (GM-CSF) and regulated on activation, normal T-cell expressed and secreted (RANTES). We have also shown that epithelial cells of atopic individuals release significantly greater amounts of these cytokines. The epidemiological and direct in vivo and in vitro exposure studies presented in this paper provide evidence that exposure to pollutants precipitate attacks of asthma and may also be one of several causes of the increase in the prevalence of this disorder. The mechanisms by which pollutants exert their effect are likely to be direct and dose-dependent actions on epithelial cells leading to changes in permeability and generation of pro-inflammatory mediators including cytokines.

cellules 6pith~liales des bronches au NO 2,/t l'Os, aux particules de l'6chappement des moteurs diesels et gtla fum6e de cigarettes, in vitro, conduit/t une dysfonction significafive de la cellule 6pith61iale et/t une lib6ration significative de cytokines pro-inflammatoires telles que l'interleukine 8 (IL8), le TNF-c~, le GM-CSF et le RANTES. Nous avons aussi montr(? que les cellules ~pith61iales des individus atopiques lib6raient des quantit6s significativement plus grandes de ces cytokines. Les 6tudes 6pid6miologiques et les &udes d'exposition directe in vivo et in vitro ici pr~sent6es montrent que l'exposition aux polluants pr6cipite les attaques d'asthme et peut aussi 6tre une cause, parmi diverses autres, de l'augmentation de la pr&alence de cette affection. Le m6canisme par lequel les polluants exercent leur influence semble bien ~tre une action directe et dose-d~pendante sur les cellules dpith61iales entra~nant des changements de la perm~abilit6 et la formation de m6diateurs pro-inflammatoires, y compris les cytokines.

KEY-WORDS: Allergic diseases - air pollution - allergenpollutant interaction - airway epithelium.

MOTS-CLI~S: Maladies allergiques - Pollution adrienne Interaction allerg6ne-polluant - Epith61ium bronchique.

INTRODUCTION

Several studies have shown that allergic diseases such as asthma, rhinitis, a n d e c z e m a have b e c o m e m o r e c o m m o n over the last 50 years. B u r r a n d colleagues [1] have c o n d u c t e d two surveys 15 years apart in 12 year-old school c h i l d r e n in South Wales (UK), a n d d e m o n s t r a t e d that the n u m b e r o f c h i l d r e n suffering f r o m asthma, e c z e m a a n d hay fever increased significantly f r o m 6% to 12%, 5% to 16% a n d 9% to 15%, respectively, d u r i n g this p e r i o d . Similarly, t h r e e s e p a r a t e surveys c o n d u c t e d by Russell a n d colleagues [2,3], in school c h i l d r e n in A b e r d e e n (UK) over a thirty year p e r i o d b e t w e e n 1964-1994, indicated that the p r e v a l e n c e o f r e s p i r a t o r y symptoms a n d atopy was increased, as d e m o n s t r a t e d by increased diagnosis o f asthma ( f r o m 4.1% in 1964 to 10.2% in 1989 to 19.5% in 1994), hay fever (from 3.2% in 1964 to 11.9% in 1989 to 12.9% in 1994) a n d e c z e m a (from 5.3% in 1964 to 12% in 1989 to 17.7% in 1994). Most r e c e n t data indicate that this increase has b e e n sustained since 1994. Analysis o f the p r e v a l e n c e o f allergic airway disease in 1996 has d e m o n s t r a t e d t h a t whilst t h e p r e v a l e n c e o f asthma has risen to 21% a m o n g s t 12-13 years old school children, the p r e v a l e n c e o f allergic rhinitis has i n c r e a s e d even f u r t h e r to 30% [4].

ASSOCIATION BETWEEN AIR POLLUTION A N D A I J , E R G I C AIRWAY DISEASE

E p i d e m i o l o g i c a l studies have d e m o n s t r a t e d that levels o f air pollutants, including 03, NO2 a n d Rev. fi: Allergol., 1998, 38, 7S

PM10 resulting f r o m i n c r e a s e d use o f liquid p e t r o l e u m a n d gas in t h e t r a n s p o r t a n d m a n u f a c t u r i n g industries a n d domestic settings, have progressively increased in the last d e c a d e a n d have occasionally e x c e e d e d the E u r o p e a n a n d W H O safety guidelines in several m a j o r cities in the U K [5,8]. S o m e studies have d e m o n s t r a t e d that the increase in N O 2 a n d 0 3 are associated with s y m p t o m s o f r h i n i t i s , c h r o n i c c o u g h , phlegm, decreased morning p e a k flow, emergency room visits and increased hospitalisation d u e to a s t h m a in asthmatic adults a n d c h i l d r e n , a n d t h a t t h e effects o f t h e s e pollutants may p r e d o m i n a t e after 1-2 days [5, 8]. A m o r e r e c e n t study by Studnicka a n d colleagues has c o n f i r m e d s o m e o f t h e s e f i n d i n g s a n d d e m o n s t r a t e d that the p r e v a l e n c e o f a s t h m a a n d symptoms such as -wheeze,, a n d - c o u g h a p a r t f r o m colds,,, in seven-year old Austrian children, was significantly associated with traffic related levels o f a t m o s p h e r i c N O 2 [9]. Similarly, s t u d i e s o f r e s p i r a b l e p a r t i c u l a t e m a t t e r have d e m o n s t r a t e d that increased levels o f PM10 are also associated with worsening p e a k flow, i n c r e a s e d i n h a l e r u s a g e , r e s p i r a t o r y symptoms, emergency room visits, a n d c a r d i o p u l m o n a r y a n d l u n g c a n c e r mortality [10]. More recently it has b e e n r e p o r t e d that e x p o s u r e o f asthmatic adults to b o t h ultrafine particulates (smaller t h a n 0.1 ~tm in d i a m e t e r ) a n d fine particulates (0.1-2.5 btm in d i a m e t e r ) in the a t m o s p h e r e was associated with a decrease in peak e x p i r a t o r y flow (PEF) a n d an increase in c o u g h a n d feeling o f b e i n g ill d u r i n g the day [11]. H e a l t h effects o f the five-day m e a n o f the n u m b e r o f ultrafine particulates were larger t h a n those for

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the five day mean of the mass of fine particulates. Moreover, the effect of the ultrafine particulates on PEF were greater than the effects of PM10 on the PEF of the asthmatic individuals. Although epidemiological studies, particularly from the developed countries, suggest that the increase in the prevalence of allergic disease such as asthma may be associated with air pollution resulting from increased use of liquid petroleum fuel [12-14], the findings from these studies have been difficult to interpret due to confounding effects of cigarette smoke, exposure to allergens, meteorological conditions and socioeconomic factors [15]. Additionally, these studies have investigated the effects of only the m a j o r p o l l u t a n t s individually, w i t h o u t taking into account the potential additive a n d / o r synergistic effects of combinations of pollutants which are more relevant. However, studies from Japan have demonstrated that the incidence of rhino-conjunctivitis in residents living alongside old cedar tree-lined main roads with heavy traffic all day long was much higher than that in residents living in the cedar forest but with less traffic, despite the cedar pollen counts being similar in both areas [16]. These studies suggest that the disparity in the incidence of rhino-conjunctivitis in the different areas may be a result of vehicle exhaust pollution, which was the predominating factor in areas with high incidence. Early studies from Germany, particularly by von Mutius a n d colleagues, demonstrated that allergic conditions such as hay fever were more c o m m o n in West German cities than in East G e r m a n cities, w h e r e chronic bronchitis was more prevalent, and suggested that this was likely to be a consequence of the different types of air pollutants predominating in West and East Germany [17]. In a more recent study, these authors have found that the prevalence of hay fever amongst schoolchildren (aged 9-11 years) in Leipzig (East Germany) has risen from 2.3% (1991-92) to 5.1% (1995-96) whilst the prevalence of atopic sensitisation has increased from 19.3% (1991-92) to 26.7% (1995-96) [18]. Other studies from Germany have demonstrated that whilst the prevalence of atopic diseases has not altered significantly in H a m b u r g (West Germany), there has been a significant increase in the prevalence of these diseases in Erfurt (East Germany), suggesting that there is a converging tendency of self reported asthma and asthma symptoms [19]. Overall, the d i f f e r e n c e s in the r e s p i r a t o r y conditions have largely disappeared following unification of East and West Germany, and it has

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been suggested that this is likely to be a result of changes in environment and living habits in the former East Germany [20]. The indoor environment is likely to be an important factor in the increased incidence of allergic disease, since as much as 90 % of the time is spent indoors [21]. Von Mutius and colleagues have investigated the association between skin test reactivity and methods of heating and cooking in the homes of over 5,000 schoolchildren in West Germany and demonstrated that the prevalence of atopy and hay fever was significantly higher in children living in homes with gas ovens, oilfurnaces and central heating, c o m p a r e d to children living in homes where coal or wood was used for heating or cooking, indicating the possible deleterious effects of gas and oil-derived air pollutants [22]. Luczynska and colleagues have investigated skin sensitivity to a panel of indoor and outdoor allergens in 10-11 years old school children living in Tower Hamlets and Eltham, two areas of London segregated on the basis of their socio-economic status and traffic pollution and demonstrated that the prevalence of skin sensitivity to house dust mite, grass pollen and cockroach allergen was higher in Tower Hamlets, the more deprived and polluted of the two areas [23].

LABORATORY STUDIES OF EXPOSURE T O AIR POLLUTANTS O N L U N G F U N C T I O N

Laboratory based studies have shown inconsistent effects of NO 2 exposure on lung function and nonspecific bronchial responsiveness. In one study, NO 2 at low concentration (400ppb) did not affect lung function in normals b u t caused bronchoconstriction and increased bronchial responsiveness in asthmatics [24]. In contrast, n u m e r o u s studies have a g r e e d in f i n d i n g significant effects of 03, in both asthmatics and non-asthmatics, who appear to be equally sensitive to the effects of this pollutant gas [5]. Studies of O3 inhalation have also demonstrated that this agent produces concentration- and exposure timerelated changes in symptoms and lung function, including an increase in airway resistance, a reduction in lung volumes and an increase in airway responsiveness to b r o n c h o c o n s t r i c t o r agents [5]. Seal and colleagues [25] investigated the lowest 0 3 concentrations and the minimum exposure time required to cause significant c h a n g e s in either p u l m o n a r y f u n c t i o n or Rev.fr. Allergol., 1998, 38, 7S

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respiratory symptoms in healthy male volunteers exposed to 0-240ppb 0 3 for 0-6.8 hours. Although cough was induced at a minimum concentration of 180ppb 03, significant adverse changes in forced expiratory volume in 1 second (FEV1) were induced at a minimum concentration of 240ppb 03. Furthermore, a minimum exposure time of 6.8 hours to 0 3 was required to see these changes. Other studies have demonstrated that there is rapid adaptation to continuing exposure to ozone [26]. Studies involving i n h a l a t i o n of SO 2 have d e m o n s t r a t e d that this gas also leads to bronchoconstriction in both normal healthy and asthmatic subjects and that deep breathing and intermittent exercise may potentiate this effect [6]. Although the response to SO 2 inhalation is variable in individuals, concentrations of SO 2 that have little or no effect on normal healthy subjects can p r o d u c e m a r k e d s y m p t o m a t i c bronchoconstriction in patients with asthma. Some studies have demonstrated that the effects of SO 2 on lung function are potentiated by prior exposure to either NO 2 [27] or 0 3 [28], and therefore suggest that exposure to combinations of pollutants is likely to be more harmful than exposure to individual pollutants.

INFLUENCE OF AIR P O L L U T I O N O N AIRWAY RESPONSIVENESS T O INHALED AIJJERGEN

T h e r e is now evidence that e x p o s u r e to pollutants such as 03, NO2 and a combination of NO 2 and SO 2 may increase the airway responsiveness of asthmatics to inhaled allergen. J6rres and colleagues [29] exposed groups of intermittently exercising allergic asthmatics, allergic rhinitics without asthma and healthy subjects for 3 hours to 250ppb O3 or filtered air, in randomised manner, followed by challenge with doubling concentrations of m e t h a c h o l i n e or allergen, until the FEV 1 dropped by 20% from baseline. Exposure to 0 3 itself significantly decreased the lung function in all groups of individuals, as indicated by decreased baseline FEV 1, compared with exposure to filtered air. Preexposure for 3 hours to 250ppb 0 3 also increased the bronchial responsiveness to allergen in both the asthmatic and rhinitic subjects, as indicated by significant decreases in the amounts of allergen required to reduce the mean FEV 1 by 20% from baseline, compared with pre-exposure for 3 hours Rev. fr. Allergol., 1998, 38, 7S

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to air. F u r t h e r m o r e , p r e - e x p o s u r e to 0 3 significantly increased the airway responsiveness to methacholine in the asthmatic patients, but not the patients with rhinitis nor the healthy subjects [29]. Tunnicliffe and colleagues [30] have investigated the effect of randomised exposure at rest for 1 hour to either air, 100ppb NO 2 or 400ppb NO 2, followed by challenge with a predetermined dose of house dust mite (HDM) allergen required to produce a 15% fall in FEV1, in mild asthmatics. M t h o u g h exposure to 400ppb NO 2 did not significantly alter the baseline FEV1 in these individuals, this significantly increased the airway response to inhaled allergen during b o t h the i m m e d i a t e a n d late phase, w h e n compared with exposure to air. We have investigated the effect of randomised exposure for 6 hours to either air, 400ppb NO 2, 2 0 0 p p b SO 2 or a c o m b i n a t i o n of the two pollutants, followed by inhalation of increasing concentrations of Dermatophagoides pteronyssinus (Der p) allergen, on lung function and airway responsiveness in non-exercising mild asthmatic p a t i e n t v o l u n t e e r s to [31]. C o m p a r e d with exposure to air, exposure to neither NO2, SO2, n o r the c o m b i n a t i o n of the two pollutants significantly altered FEV 1 or forced vital capacity (FVC). Although exposure to NO 2 and SO 2 d e c r e a s e d the m e a n allergen c o n c e n t r a t i o n required to cause a 20% fall in FEV1 (PD20FEV1) by 41.2% and 32.2%, respectively, these were not significantly different compared with exposure to air. In contrast, exposure to the combination of the two pollutants significantly decreased the mean allergen PD20FEV 1 by 60.5%, compared with exposure to air. In view of the epidemiological evidence that the effects of air pollutants may be lagged by 1-2 days, we investigated the possibility that such a lag effect may be r e p r o d u c i b l e u n d e r strictly controlled laboratory conditions and studied the time course over which the airway response enhancing effects of pollutants may persist [32]. Non-exercising mild asthmatic patient volunteers were exposed in randomised manner for 6 hours to either air or 400ppb NO 2 + 200ppb SO 2. Following e x p o s u r e to air the individuals u n d e r w e n t challenge immediately with Der p allergen as above. Following exposure to 400ppb NO 2 + 200ppb SO 2 the individuals underwent randomised allergen challenge either immediately, 24 hours or 48 hours later. Exposure to the combination of NO 2 + SO 2 significantly decreased the mean allergen PD20FEV 1 by 37.0%, 63.0% and 49.0%, immediately, 24 hours and 48

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hours after exposure, respectively, compared with exposure to air. Additionally, the mean allergen PD20FEV 1 at 24 h o u r s after e x p o s u r e was significantly lower, w h e n c o m p a r e d to that immediately after exposure to the pollutant mixture, suggesting that the enhanced airway r e s p o n s e to i n h a l e d allergen in asthmatic individuals, resulting from exposure to pollutants, was likely to be lagged over a period of 24-48 hours and was maximal 24 hours after exposure. More recently we have tested that hypothesis that prior exposure for a longer period to low concentrations of pollutants has a similar effect to e x p o s u r e for a s h o r t e r p e r i o d to high concentrations of these pollutants, delivering the same total dose, by comparing the effect of prior exposure for 6 hours to 100ppb 03, 200ppb NO2, or their combination, with prior exposure for 3 hours to 200ppb O3, 400ppb NO2, or their combination, on the airway response to inhaled allergen in asthmatics [33]. These studies have demonstrated that whilst exposure for 6 hours to either 100ppb O~, 200ppb NO 2 or 100ppb O~+200ppb NO 2 did not have any significant effect, exposure for half the time to double the c o n c e n t r a t i o n s o f these pollutants, b o t h individually and in combination, significantly i n c r e a s e d the airway r e s p o n s e to i n h a l e d individuals in the same individuals. These studies suggest that the effect of p o l l u t a n t s are d e p e n d e n t on a threshold concentration, rather than total inhaled dose.

MECHANISMS UNDERLYING INCREASED AIRWAY R E S P O N S I V E N E S S T O INHAI,F~D A L L E R G E N F O L L O W I N G EXPOSURE TO POLLUTANTS

Although there is only circumstantial evidence to suggest a link between an increase in the prevalence of allergic airway disease and an increase in air pollution, there is little doubt that exposure to certain air pollutants enhances the airway r e s p o n s e to i n h a l e d allergens in susceptible individuals. However, the mechanisms underlying these effects are not fully understood. Some studies have suggested, that air pollutants may p r o m o t e sensitization and s u b s e q u e n t development of allergic disease, by modulating the allergenicity of airborne allergens. Behrendt and colleagues have demonstrated that pollen collected from roadsides with heavy traffic and

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other areas with high levels of air pollution are c o v e r e d with large n u m b e r s of a i r b o r n e particulates (-5gin in size), and that incubation of pollen for 2-5 hours in aqueous solutions prepared from these particulates led to morphological alterations.in pollen and extravasation of allergens with altered antigenicity [34]. Similarly, Thomas and colleagues have studied the effect of exposure for 4 hours to 50-200ppb NO 2 on viability, germination and protein release from freshly collected birch, rye, alder and hazel pollens and demonstrated that exposure to 100ppb NO 2 adversely affected that ability of all species of pollen to germinate [35]. The detrimental effect on germination was most pronounced for alder pollen (reduction of 55%). The viability of alder and hazel pollens was also reduced and the release of protein from the various pollens was enhanced maximally by exposure to 100ppb NO 2. Knox and colleagues have investigated the interaction b e t w e e n DEP and purified rye-grass pollen allergens Lol pl and Lol p5 and demonstrated that Lol pl binds strongly to the DEP [36]. Collectively, these studies suggest that air pollutants may modulate allergenicity of the pollen allergens and also act as environmental triggers for exacerbation of allergic airway disease, particularly during episodes of increased air pollution in the pollen season. H u m a n and animal studies have suggested that pollutant exposure increase synthesis of IgE [37, 40]. A study of 363 healthy non-atopic children, under the age of 12 years, demonstrated that the degree of air pollution in the areas where these children lived was significantly correlated with an increase in their mean total serum IgE levels [41]. Diaz-Sanchez and colleagues have investigated the effects of nasal challenge with 0.30 mg DEP on the local immune response of healthy volunteers and shown that this led to a four to five-fold increase in the amounts of IgE, but not IgG, IgA or IgM, measured in nasal lavage of these subjects, four days after challenge [42]. Additionally, these authors demonstrated that the n u m b e r of IgE secreting cells and the levels of epsilon mRNA coding for specific IgE proteins were increased approximately 20-25 fold following challenge with DEE Recently, these authors investigated the effect of DEP on the synthesis of cytokines known to influence the production of IgE and lead to allergic reactions in the nasal mucosa of subjects allergic to ragweed allergen [43]. These studies have demonstrated that the levels of mRNA for IL2, 4, 5, 6, 10 and 13 and interferon gamma (IFNy) were increased and readily detectable in the nasal mucosal cells of these individuals, 18 hours Re~f~Al~rgoL, 1998,38, 7S

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after intranasal challenge with DEP [43]. Interestingly, the enhancement in cytokine mRNA following challenge with DEP was considerably g r e a t e r than that n o t e d following allergen challenge alone and the levels of mRNAs for IL-4, 5, 6, 10 and 13 were increased even further when allergen and DEP challenges were a p p l i e d simultaneously. In contrast the levels of IL-2 were unchanged, whilst the levels of IFN-y mRNA were decreased suggesting that DEP upregulated the activity of TH2-1ike lymphocytes. More recently, these authors have investigated the ability of DEP to act as an adjuvant to antigen in ragweed sensitive individuals undergoing nasal provocation challenges with either DEE the ragweed allergen A m b a / o r a combination of DEP and A m b a I [44]. Nasal washes were performed 18 hours, as well as four and eight days after challenge and analyzed for total and ragweed specific IgE. Challenge with ragweed allergen led to an increase in both total and ragweed-specific IgE in the nasal lavage fluid. Although challenge with DEP also led to an increase in total, but not ragweed specific IgE, a c o m b i n e d challenge with DEP and ragweed allergen increased the levels of ragweed specific IgE 16-fold, compared to levels seen after ragweed challenge alone. In vitro studies by the same group of authors have demonstrated that the effects of DEP may at least partly be as a result of the polyaromatic hydrocarbons (PAHs) present on the DEP [45, 46]. Takenaka and colleagues have investigated the effect of PAils extracted from DEP on IgE production in h u m a n peripheral blood mononuclear cells or purified tonsillar B cells. These authors demonstrated that although PAIl extract itself did not induce de novo IgE synthesis by either cell type pre-treated with either anti-IL-4 or anti-CD40 m o n o c l o n a l antibody, the e x t r a c t enhanced the IgE production from these cells by 20-360%, w h e n the cells were treated simultaneously with IL-4 and CD40, suggesting that PAHs were possibly acting by modifying on-going transcriptional programs related to IgE production, rather than inducing such programs de novo [46]. Similarly, animal studies have shown that specific IgE to ovalbumin and platinum salts is increased when the animals are exposed to 0 3 [38,39] or DEP [40] compared to air. Sato and colleagues have investigated the effects of airborne particulates collected from urban and industrialised areas of Sao Paulo, on mutagenicity in salmonella [47]. These authors demonstrated that particulates collected from urban areas of Sao Paulo, where vehicle emissions are the major Rev. fr. Allergol., 1998, 38, 7S

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pollution source, were much more mutagenic to the salmonella than the particulates collected from the industrialised areas of Sao Paulo. Similarly, Osornio-Vargas and colleagues have investigated the effect of PM10 samples collected from different parts of Mexico City on viability 0f human lung fibroblast cultures in vitro [48]. Preliminary data from these studies have demonstrated that the PM10 collected from the more industrialised northern part of Mexico City contained more Cu+V+Zn+Pb and were more cytotoxic, than PM10 collected from the less industrialised but more traffic congested areas of central and southern Mexico City. These authors suggest that the increased cytotoxicity of PM10 from northern Mexico City was likely to be a result of the increased transition metal load of these PM10. Several studies have suggested that pollutioninduced airway epithelial damage and impaired mucociliary clearance may allow easier penetration and access of inhaled allergens to cells of the i m m u n e system. Studies investigating the pathophysiological effects resulting from inhalation of 0 3 have demonstrated that this agent leads to epithelial damage and an increased inflammatory response in the upper and lower airways, as indicated by leakage of lactate dehydrogenase, albumin and total protein, and increase in neutrophils, eosinophils, mononuclear cells, fibronectin, (z-l-antitrypsin, IL-6 and IL-8, GM-CSF and prostaglandin E2, in nasal lavage (NAL), proximal airway lavage (PAL) a n d bronchoalveolar lavage (BAL) [49-51]. Basha and colleagues have demonstrated that although there were no significant differences in spirometric values and symptom scores between asthmatics and healthy volunteers, after exposure for 6 hours to 200ppb 0 3, there were significant increases in IL-6, IL-8 and PMN numbers in BAL fluid obtained 18 hours post-exposure in asthmatics only [52]. These studies suggest that O~ may preferentially increase the production of cytokines and inflammatory cells in asthmatics, possibly leading to an acute exacerbation at a later stage. Sandstrgm and colleagues have investigated the effects of NO 2 inhalation in healthy non-smoking and lightly exercising individuals and demonstrated that this agent increases the numbers of lymphocytes, lysozyme-positive alveolar macrophages and mast cells in BAL [53]. More recently, this group demonstrated that exposure for 20 minutes to 1.53.5ppm NO 2 significantly reduced the mucociliary activity in healthy non-smoking volunteers [54]. Peden and colleagues have investigated the effect of prior exposure for two hours to either air or

$86 400ppb 0 3 on s u b s e q u e n t allergen-induced changes in the nasal mucosa of perennially allergic asthmatic patients and d e m o n s t r a t e d that exposure to 0 3 significantly increased the allergeninduced release of eosinophil cationic protein (ECP) in nasal lavage of these indMduals, without significantly affecting the numbers of eosinophils, 4 hours after allergen challenge [55]. These results suggest that exposure to O3 may ~prime,, the eosinophils to subsequent activation by inhaled allergen. Similarly, we have exposed seasonal allergic rhinitics for 6 hours to either 400ppb NO 2 or air _+ allergen challenge, following 30 minutes after exposure, and have evaluated the changes in nasal airway resistance (NAR) and presence of inflammatory mediators in nasal lavage [56]. Although exposure to NO 2 alone did not significantly alter the NAR or increase the concentration of ECR tryptase or myeloperoxidase in nasal lavage, exposure to NO 2 prior to allergen challenge significantly increased the concentration of ECP, but not tryptase or myeloperoxidase, compared with exposure to air. Our findings are in accordance with those of Peden and colleagues and suggest that acute exposure to NO 2 may also ~prime,, the eosinophils for subsequent activation by allergen in seasonal allergic rhinitics. In order to investigate whether treatment with steroids can alter the inflammatory response in the nasal airways under these conditions, we have investigated seasonal allergic rhinitics randomised to receive either topical fluticasone propionate aqueous nasal spray (FP) 200~tg once daily or matched placebo for 4 weeks in a double blind, crossover design [57]. Analysis of ECP in lavage samples of these individuals, demonstrated that this was significantly increased following exposure to NO2+allergen, when the individuals were treated with placebo. In contrast, there was a much smaller effect of exposure to NO2+allergen challenge on ECP levels when these individuals were treated with FE The difference in changes of ECP levels between placebo and FP treatments was significant suggesting that FP may down-grade NO2+allergen-induced eosinophil activation in allergic rhinitics. More recently, we have investigated the effect of prior exposure to 0 3 on allergen-induced inflammatory cell changes in the lower airways of mild asthmatics randomized to receive either inhaled FP 500~tg bd or matched placebo for 4 weeks [58]. Following treatment, indMduals in both groups underwent randomised exposure for 1 hour to either air or 120ppb 03, followed by bronchoscopy and saline challenge in the right upper lobe anterior segmental orifice and allergen

• C. R U S Z N A K E T A L . /

challenge in the right middle lobe medial segmental orifice. Bronchoscopy was repeated after 24 hours and bronchial biopsies were obtained from the challenge sites. After a 4 weeks' washout period, treatment with FP or placebo was repeated and biopsy samples were obtained after prior exposure for 1 hour to the alternate atmosphere of air or 120ppb 03. Immunohistochemical analysis of the biopsy tissue demonstrated that prior exposure for 1 hour to 0 3 did not significantly increase the numbers of neutrophils, EG1-, or EG2-staining cells, when c o m p a r e d with exposure to air. However, allergen challenge following exposure to either air or ozone significantly increased the numbers of EG2-staining cells in the placebotreated group, an effect that was markedly reduced in patients receiving FP. Although there are no equivalent inhalation studies investigating the effects of respirable particulates in humans, preliminary studies in rats and mice have demonstrated that exposure by inhalation for 30 minutes to concentrations of 105 to 5x105 ultrafine particles/cm ~ can lead to i) acute p u l m o n a r y i n f l a m m a t i o n , ii) severe haemorrhagic pulmonary oedema with increased n u m b e r s of PMNs in lung lavage samples collected within 4 hours of exposure and iii) significantly increased concentrations of IL-lc~, IL-6, TNF-c~, inducible nitric oxide synthase and manganese superoxide dismutase, in the lung lavage samples [59]. Additionally, these studies demonstrated that the ultrafine particles could be t r a n s l o c a t e d to epithelial, interstitial and endothelial sites showing marked cell membrane injuries and cell necrosis. It has been proposed that the ultrafine particles are inhaled as single particles and are deposited in the alveolar region of the lung, w h e r e r a t h e r than b e i n g p h a g o c y t i z e d by alveolar m a c r o p h a g e s they penetrate into and interact with the alveolar epithelial, interstitial and endothelial cells. This leads to the release of p r o - i n f l a m m a t o r y mediators and subsequent infiltration by large numbers of activated PMNs which contribute further to oxidative lung injury.

R O L E O F AIRWAY E P I T H E L I A L CELLS IN THE DEVELOPMENT O F P O L L U T I O N - I N D U C E D AIRWAY DISEASE

T h e r e is increasing e v i d e n c e that airway epithelial cells play a pivotal role in the pathogenesis of allergic airways disease, since they Rev.fr Allergol.,

1998,38, 7S

/ MECHANISMS OF POLLUFION-INDUCED ALLERGY AND ASTHMA °

can express and synthesise a variety o f inflammatory cytokines and adhesion molecules including IL-16, IL-6, IL-8, IL-16, GM-CSF, TNFot, RANTES and intercellular adhesion molecule-1 (ICAM-1), which i n f l u e n c e the activity of eosinophils a n d lymphocytes, which play important roles in the allergic reaction [60-64]. RANTES, IL-8 and GM-CSF in combination are chemoattractant for eosinophils and GM-CSF plays an important part in delaying @optosis of eosinophils. Studies from our laboratory have shown that e x p o s u r e of h u m a n b r o n c h i a l epithelial cells to 400-800ppb NO 2, in vitro, leads to increased epithelial permeability and damage of these cells, decreased ciliary activity, and release of pro-inflammatory mediators, including LTC4, GM-CSF, TNF-a and IL-8 [65, 66]. We have also demonstrated that exposure of these cells for six hours to ambient concentrations of 10-50ppb 03, well below the WHO safety guidelines, i n d u c e d significant release of IL-8, GM-CSF, TNF-c~ and soluble (s)ICAM-1, of which release o f GM-CSF, TNF-c~ and slCAM-1 could be blocked by treatment of the cells with 10-SM nedocromil s o d i u m [67]. L u c h t e l a n d colleagues have investigated the effect of exposure for 3 hours to 300ppb O 3 on the synthesis of extracellular matrix molecules in monkey nasal epithelial cell cultures, and demonstrated that the mRNA levels for tenascin and decorin were increased 1.4 fold and 1.6 fold, respectively, 24 hours after exposure to 03, compared with exposure to air [68]. These studies suggest that air pollutants may influence epithelial integrity also by m o d u l a t i n g the expression and synthesis of cell structural proteins. More recently we have hypothesised that air pollutants, such as DEP and CS facilitate the d e v e l o p m e n t of atopy by compromising the barrier function of the epithelium. This would lead to i) decreased clearance of allergens as a result of impaired ciliary activity and ii) increased access of the allergen to the intra- and subepithelially resident i m m u n o c o m p e t e n t cells, such as L a n g e r h a n s cells a n d T a n d Blymphocytes, due to increased permeability of the epithelium. It is likely that increased interaction between the allergen and the cells of the i m m u n e system leads to synthesis o f specific proinflammatory mediators and allergen specific IgE, which subsequently leads to sensitisation of the airways. To test this hypothesis, we have investigated the effect of incubation with 10100~g/ml DEP on cultured h u m a n bronchial epithelial cells (HBEC) and demonstrated that this pollutant significantly attenuated the ciliary Rev. fr. AllergoL, 1998, 38, 7S

S87

beat frequency of these cells and increased the release of IL8, GMCSF and soluble intercellular adhesion molecule-1 (slCAM1) from these cells [69]. More recently, we have investigated the effect of exposure to DEP on the release of inflammatory mediators from human bronchial epithelial cells cultured from bronchial biopsy specimens of three matched groups; (i) 6 nonatopic non-smokers with n o r m a l p u l m o n a r y function, (ii) 6 non-atopic smokers with normal p u l m o n a r y f u n c t i o n a n d (iii) 6 non-atopic patients with COPD [70]. Preliminary studies have demonstrated that although constitutive release of IL-8 from the cells of smokers was not different from that of non-smokers, the release of IL-8 f r o m the cells of COPD patients was significantly lower compared with the non-smoker group. Furthermore, whilst incubation with 1050~tg/ml DEP significantly increased the release of IL-8 release from the cells of the non-smokers, the release of IL-8 from the cells of neither smokers with normal lung function nor COPD patients was affected by exposure to DEE These results suggest that IL-8 p r o d u c t i o n by the b r o n c h i a l epithelial cells in COPD a n d smokers with n o r m a l p u l m o n a r y function is downregulated. Reed and colleagues [71] have reported that residual oil fly ash (ROFA), a particulate containing soluble compounds of V, Ni, and Fe and produced by burning oil for electricity., may act synergistically with IFN-y to enhance the production of RANTES by airway epithelial cells. These authors have suggested that this may possibly contribute to the increased disease activity in asthma, o b s e r v e d after increased ambient particulate exposure. We have also investigated the effect of exposure for 20 minutes to CS, in the absence or presence of 3 0 0 n g / m l Der p, on the permeability of c o n f l u e n t h u m a n b r o n c h i a l epithelial cell cultures and passage of Derp across these epithelial culture [71]. These studies demonstrated that a l t h o u g h e x p o s u r e to CS alone did n o t significantly alter the p e r m e a b i l i t y o f the epithelial cell cultures, when compared with exposure to air, this significantly enhanced the Der p-induced permeability of the epithelial cell cultures. Additionally, the t r a n s e p i t h e l i a l migration of the Der p itself was significantly increased by prior exposure to CS, suggesting that although short term exposure to CS may not increase non-specific epithelial permeability, it may r e n d e r the epithelium more susceptible to adverse effects of allergens such as Derp.

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Recent studies have demonstrated that there are differences in the ability of epithelial cells of atopic and non-atopic individuals, to synthesise different amounts a n d / o r profiles of pro-inflammatory cytokines, and suggest that genetic pre-disposition and manifestation of the symptoms of allergic airway disease in the atopic individuals may, at least in part, be a consequence of these differences. Studies of epithelial cells cultured from nasal tissue of non-atopic non-rhinitic subjects, patients with allergic rhinitis and patients with nasal polyps have d e m o n s t r a t e d that the epithelial cells from rhinitics a n d individuals with nasal polyps synthesise significantly greater quantities of GMCSF and IL-8, than cells of healthy non-atopic nonrhinitic individuals [72,73]. We have also shown that epithelial cells cultured from nasal biopsies of atopic non-rhinitic and atopic rhinitic patients, release significantly greater amounts of IL-8, GMCSF and TNF-(x, than the epithelial cells from the non-atopic non-rhinitic normal volunteers and that this release of cytokines, particularly RANTES, is significantly enhanced during the pollen season [74]. Additionally, our studies have demonstrated that the cells of atopic rhinitics are also significantly more susceptible to the effects of 0 3 and NO 2 during the pollen season, compared with cells of atopic rhinitics outside the pollen season. Similarly, studies of bronchial epithelial cells have demonstrated that the cells of asthmatic patients synthesize greater quantities of IL-I[3, ILl6, GM-CSF and MCP-1, than the cells of nonasthmatic subjects [75-78], in vivo, and that the expression of GM-CSF and IL-16 correlates well with the numbers of activated eosinophils [79] and CD4+ cells [78], respectively. Studies of epithelial cells cultured from bronchial biopsies of well characterised groups of asthmatic and nonasthmatic subjects in our laboratory have also demonstrated that bronchial epithelial cells of atopic asthmatics release significantly greater amounts of constitutive IL-8, GM-CSF, RANTES and sICAM-1, than the cells of non-atopic nonasthmatics [80]. Similar to our findings for nasal epithelial cells of atopic rhinitic and non-atopic non-rhinitic individuals, the cells of atopic asthmatics also release significantly more RANTES, and are also more susceptible to the effects of exposure to DEP, 0 3 and NO 2, compared with the cells of non-atopic non-asthmatics.

• C. R U S Z N A K E T A L . /

Collectively, these studies suggest that genetic pre-disposition a n d m a n i f e s t a t i o n of the symptoms of allergic airway disease in atopic individuals may, at least in part, be a consequence of increased expression, synthesis and release of specific pro-inflammatory mediators from their airway epithelial cells, both constitutively and following exposure to external factors such as allergens and pollutants.

CONCLUSIONS

Although epidemiological evidence suggests that there may be a link between increased air pollution resulting from excessive use of liquid petroleum and an increase in exacerbations of allergic airway disease, there is little direct evidence to substantiate this. Laboratory based studies have d e m o n s t r a t e d that exposure to pollutants such as O 3 and NO 2 + SO 2 can enhance the airway response, of particularly susceptible individuals such as asthmatics and rhinitics, to inhaled allergen. Although such studies cannot simulate exactly the meteorological/atmospheric conditions u n d e r which natural exposure to pollutants may occur and which possibly lead to different responses in the airways of asthmatics and rhinitics, they do, however, provide an invaluable means for elucidating the putative mechanisms underlying these changes. Studies investigating the effects of pollutants at the cellular and sub-cellular levels have suggested that a variety of cells are likely to be involved and will interact with one another to determine the overall inflammation and airway hyperresponsiveness observed in allergic airways disease. At the molecular level, the cell-cell and irritant/allergencell interactions lead to the synthesis of proinflammatory mediators, including cytokines and cell adhesion molecules, which modulate the activity and function of the inflammatory cells (eosinophils, mast cells and neutrophils) and i m m u n o c o m p e t e n t cells (such as Langerhans cells, T-lymphocytes and B-lymphocytes), which t o g e t h e r lead to an overall increase in sensitisation and incidence of allergic disease.

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