The role of pollutants in allergic sensitization and the development of asthma
Authors: S. Parnia, J. L. Brown, A. J. Frew
Journal: Allergy (2002)

There is no longer any doubt that the past 30–40 years have witnessed an increase in the prevalence and incidence of asthma and other allergic diseases (1, 74). Epidemiological data indicate that this rise has been most marked in affluent westernised countries, although there is also data suggestive of a parallel increase in less developed parts of the world including parts of Africa where the nature and degree of affluence is different. Although atopy and asthma both have a significant genetic component, there is no reason to think there has been a shift in the gene pool over a single generation. This means that we must seek out one or more aspects of the western lifestyle which may have interacted with a susceptible subgroup who now show asthma and allergies, whereas they would not have been affected if they had been born 50 years ago. In thinking about this we must always remember that there is also a subgroup who have asthma now and would still have had it even if they had been born 50 years ago. Clinical data show that patients with asthma are more vulnerable to the effects of air pollution than normal healthy individuals. This can be seen with direct effects on lung function, increased rates of symptomatic episodes and increased rates of hospital admissions for asthma during episodes of ozone or particulate pollution. However, it is less clear whether air pollutants affect the process of allergic sensitization or the initial development of asthma, as opposed to exacerbating the condition once it has arisen for other reasons. In broad terms, the affluent west has seen a dramatic reduction in traditional forms of pollution, such as black smoke and SO2. Air quality control legislation has helped but the temporal changes also reflect the decline in heavy industry and the replacement of coal by gas for domestic power and heating. However, three pollutants have increased in the affluent west and thus deserve consideration as possible causes of the rise in allergic diseases and asthma: ozone, NO2 and particulate matter. Ozone is a secondary pollutant formed in the troposphere through a series of sunlight-driven reactions of atmospheric oxygen with volatile organic compounds and nitrogen oxides, which are produced through combustion (2, 3). Levels of ozone tend to be higher during hot summer days and in cities with large (3) amounts of traffic and with temperature inversions (Los Angeles, Mexico City), but may also be very high in workplaces such as welding plants and paper mills. Ozone is virtually insoluble in water and therefore may be deposited anywhere along the airway, but as it is highly reactive it is thought that very little of the inspired ozone reaches the alveolar epithelium (4, 5). The majority of the effects of ozone are mediated by a cascade of secondary products derived from free radicals that cause cellular damage when antioxidant defences have been overwhelmed (6, 7). Experimental exposure of humans to high concentrations of ozone causes irritation and cough, with decrements in forced vital capacity and forced expiratory volume at 1 s (FEV1). In healthy subjects ozone induces neutrophilic inflammation of the airway mucosa accompanied by increased levels of inflammatory mediators and proteins in bronchoalveolar lavage (BAL). Albumin, IgG and α1-antitrypsin are increased in the epithelial lining fluid, indicating increased vascular permeability (8). Exposure to ozone also induces release of the neutrophil chemoattractant IL-8 as well as GM-CSF, and increases expression of the vascular endothelial adhesion molecule ICAM-1 (9) suggesting a likely mechanism for neutrophil recruitment. Some studies have reported recruitment of eosinophils and mast cells after ozone exposure. In vitro, cultured human epithelial cells exposed to ozone release increased amounts of lipid mediators PGE2, LTC4, LTD4 and LTE4 (10). Fibronectin and the proinflammatory cytokines IL-6 and IL-8 are also released by epithelial cells in response to ozone exposure (11, 12). Other in vitro studies carried out using human epithelial cells have shown that both NFkB and AP-1, which regulate the IL-8 gene, increase in response to ozone exposure in respiratory epithelial cells (13). Exposure to ozone may affect asthma and allergy through a number of mechanisms. Firstly, the increased permeability of the airways to protein may allow allergen particles and proteins to penetrate deeper into the airways mucosa, and either increase or alter the immune response to the allergen. Data from chronic exposure studies in humans are limited, but animal studies have shown that exposure to ozone does increase and enhance the allergic response to allergens. In one study in mice, the effects of 4 weeks ozone exposure on the allergic response to ovalbumin was investigated both in "high responder" (BALB/c) and "low responder" (C57BL/6) strains. In both mice strains, ozone exposure led to an increase in immediate cutaneous hypersensitivity and anti-ovalbumin IgG1, with a parallel reduction in anti-ovalbumin IgG2. In the "high IgE responder" BALB/c mice there was also a dose-dependent increase in specific IgE, IL-5, eosinophils and lymphocytes (14). In guinea pigs, exposure to ozone increased nasal allergic responses to ovalbumin, as shown by increased nasal responsiveness and eosinophil infiltration, paralleled by an increase in allergen-specific IgG (15). However, in asthmatic humans, the effect of ozone on airway allergen responsiveness remains unclear, with some acute exposure studies showing an increase in responsiveness (16–18) whereas others found no effect (19, 20). This may be caused by technical differences or could reflect individual genetic susceptibility (21). Yet further studies suggest that the combination of ozone with other pollutants, such as nitrogen dioxide, may further increase the airway allergen responsiveness of people with mild IgE-mediated asthma (22). Nitrogen dioxide (NO2) is a primary pollutant produced by the combustion of fossil fuels. It is a major component of photochemical smog and a common indoor air pollutant as well as an occupational hazard, especially in shipyards, mines, garages and tunnels. NO2 is a poorly water-soluble gas and may therefore deposit in the peripheral airspaces. It is highly reactive, and the most toxic of the various nitrogen compounds produced by combustion. The main chemical effects of NO2 in the airways are thought to involve lipid peroxidation of cell membranes and various actions of free radicals. In vitro, as a result of its oxidizing properties, NO2 causes damage and inflammation of epithelial cells, with increases in supernatant concentrations of GM-CSF and TNF-α (23). Human exposure studies at concentrations below 2 p.p.m. (the recommended maximum exposure levels in an 8-h work shift) have shown an increase in neutrophils in the proximal airways. At higher concentrations, single exposures to NO2 induce dose-dependent increases in BAL mast cells, neutrophils and lymphocytes, which resolve within 72 h (24). After repeated exposures, the airway physiological responses become accommodated, but the neutrophil response persists. Markers of inflammation such as albumin, β2-microglobulin, leukocyte elastase, lactate dehydrogenase, total protein, LTB4, PGE2, thromboxane A2 and TNFα do not seem to be affected by NO2 exposure. However, in vitro studies of human bronchial epithelial cells have shown an increase in the synthesis of pro-inflammatory cytokines such as GM-CSF, IL-8 and TNFα. NO2 appears to increase the morbidity of asthma, but not its incidence (25). It is possible that NO2 may act to decrease the threshold of sensitization to allergen exposure, but this remains uncertain. An increase in bronchoconstrictive response to house dust mite has been seen in mild asthmatics after exposure to NO2 (26), although an earlier study showed little response (27). However, the combination of nitrogen dioxide and another pollutant, such as ozone and sulphur dioxide, has been shown to increase the airway responsiveness of mild IgE-mediated asthmatics to allergen (22, 28, 29). NO2 may also influence asthma by increasing the likelihood of respiratory virus infections taking hold in the airway, and by increasing the likelihood that virus infection may cause lower airway symptoms (30, 31). Over 60 epidemiological studies conducted in different parts of the world have demonstrated a consistent association between ambient levels of air particles and various health outcomes, including mortality, increased exacerbation of asthma, chronic bronchitis, respiratory tract infections, ischaemic heart disease and stroke (32). Over the last 10 years major concerns have been raised about the health effects of fine particulates (less than 10 microns in aerodynamic diameter – PM10). In 1993, a seminal paper described an association of PM10 levels with cardiovascular and respiratory mortality and morbidity in six US cities (33). Initially this observation met with some scepticism but the association has subsequently been confirmed in a wide number of settings and in different countries. Interestingly, the association seems to also apply across settings even where the composition of PM is quite different. The precise mechanisms underlying the cardiovascular effects of PM remain uncertain. Current attention is particularly focused on ultrafine particles (diameter < 0.05–0.10 µm) which are highly reactive and are present in large numbers in the urban environment. Ultrafine particles can penetrate through the epithelium and vascular walls and enter the blood stream, and in animal models, have been reported to produce alterations in blood coagulability and increased rates of cardiovascular disorders as well as increased carcinogenicity (34), and potentiation of autoimmune disorders (35). Epidemiological studies have reported that patients with asthma are adversely affected by PM pollution. As with the cardiovascular effects, there seems to be no threshold below which PM effects disappear. Across the full range of PM concentrations, there is a clear and detectable gradient for asthma symptoms, lung function and hospital admissions. For every 10 µg/m3 increase in PM concentrations, there is a 3% increase in asthma attacks, a 3.4% increase in emergency room visits for asthma, a 2.9% increase in bronchodilator use, and a 0.15% decrease in FEV1. This problem is accentuated by the fact that about 63% of US patients with asthma live in areas that fail to meet the US EPA standards for PM pollution. Reports from Japan suggest that children living close to roads with heavy traffic are more likely to develop allergies. A higher prevalence of allergy to cedar pollen has been observed in people living close to motorways, as compared with people living in a more rural environment close to a cedar forest and being exposed to the same number of cedar pollens (36). Diesel exhaust is a major contributor to particulate pollution. Diesel exhaust particles (DEPs) may promote inflammation and the allergic state through three broad mechanisms. (1) DEPs may act as a carrier for the transport of allergens into the airways by adsorbing allergenic proteins onto their surface. (2) DEPs may act as an adjuvant in promoting the switching of B cells to produce allergen-specific IgE. (3) DEPs may affect a number of downstream immunological mechanisms, leading to increased prevalence or increased severity of allergic and asthmatic disorders. Some allergens, such as the major grass pollen Lolium perenne− 1 (Lol p1) allergen, bind specifically to DEPs (37). In an in vitro study of allergen adsorption to indoor-suspended particulate matter (ISPM) and DEPs, cat, dog and birch pollen major allergens (Fel d1, Can f1 and Bet v1) were all found on the surface of ISPM, whereas house dust mite allergen (Der p1) was not. However, all four allergens were found to be absorbed to DEPs (38). By acting as a carrier of allergens, DEPs might deliver allergenic material to the airways in increased amounts during pollution episodes, and trigger asthma attacks in those who are sensitized (37). Co-presentation to the immune system of allergens and DEPs may lead to an altered immune response to the allergen. Allergenic proteins can be identified bound to DEPs and SPM, while in urban areas pollen grains can become coated with fuel residues and combustion products. Birch pollen grains collected during the pollen season in the north of Stockholm were found to be coated with a complex mixture, about 80% of which consisted of n-alkanes and n-alkenes, but methylketones, ethers, alcohols and amino alcohol were also identified (39). When these DEPs and pollen grains are ingested by antigen-presenting cells, the allergenic proteins will undergo processing in an environment that is simultaneously affected by one or more components of the DEPs. Moreover, the binding of pollen or other inhaled allergens to DEPs may modulate the allergenic epitopes and thereby increase their allergenicity (40). DEPs have been shown to have a direct enhancing effect on IgE production. This effect seems to be driven by polyaromatic hydrocarbons (PAH) on the surface of the DEPs rather than their carbon core, since PAH extracted from DEPs enhance IgE production from purified human B-cells stimulated by IL-4 (41). Importantly, PAH–DEPs did not induce IgE production in unstimulated B-cells, indicating that the PAH only enhance ongoing IgE production. Phenanthrene, a major polyaromatic hydrocarbon and an important component of DEPs, had a similar effect on IgE production by a human B cell line (42). In vivo, DEPs enhanced total IgE and cedar pollen-specific IgE production in mice given ovalbumin (OA) mixed with DEPs either intra-peritoneally or intra-nasally (43, 44). In order to elucidate which part of the DEP particle (the carbon core or the adsorbed organic chemicals) is responsible for the adjuvant effect, mice were immunized four times with either OA, OA with DEPs or OA with carbon black (CB). Specific IgE for OA was then analysed. Both DEPs and CB showed an adjuvant activity for specific IgE production after intra-nasal instillation, indicating that they were both responsible for the effect, although DEPs had a more pronounced effect (45). This effect has been confirmed in other studies in mice (46). The effect of DEPs on antibody responses is dose- and time-dependent (47, 50). Even at low doses that have little demonstrable effect on IgE, exposure to DEPs enhances the effect of allergen challenge on the airways, with increased eosinophilic inflammation and goblet cell hyperplasia (47, 48). In humans, diesel particles have been shown to potentiate nasal IgE production 4 days after challenge with DEPs (51). There was also an increase in the number of IgE-secreting cells in lavage but no increase in IgA-secreting cells. Similarly, nasal challenge with a combination of DEPs and allergen has been shown to induce higher ragweed-specific IgE and IgG4 responses compared with DEPs or ragweed alone but with similar total IgE levels (52). There was also a change in the cytokine pattern favouring allergic sensitization. This implies that synergism between DEPs and natural allergens may be a key feature in increasing the prevalence of allergen-induced respiratory disease. In addition to these allergen-specific effects, exposure to DEPs alone causes an inflammatory response in the airways, which may then prime the airways and increase the magnitude of the response to allergen challenge. Inhalation of DEPs damages the bronchial epithelium and cilia, thereby impairing their ability to act as a biological barrier against inhaled substances (53). Allergens may therefore remain on the epithelial surface for longer, perhaps allowing them to diffuse into the epithelial layer and increasing the probability of them coming into contact with cells of the immune system. Other mechanisms which might affect asthma development after exposure to DEPs may include up-regulation of histamine receptor gene expression (54) and increased penetration of allergen across the respiratory mucosa (55). The cytotoxicity of DEPs, their phagocytosis, and the resulting immune response have been studied in human bronchial and nasal epithelial cell cultures. Exposure to DEPs led to time- and dose-dependent membrane damage. Transmission electron microscopy showed that DEPs underwent endocytosis by epithelial cells and were then translocated through the epithelial cell sheet. Flow cytometric measurements confirmed the time and dose dependency of this phagocytosis and its non-specificity (DEPs, carbon black, and latex showed similar responses). DEPs led to a time-dependent increase in IL-8, GM-CSF, and IL-1beta release. This inflammatory response occurred later than phagocytosis and appeared to depend on the extent of adsorbed compounds, as in this study carbon black had no effect on cytokine release, (56) even though it was phagocytosed to a similar degree. Exposure to DEPs attenuated the ciliary beat frequency of human bronchial epithelial cells and increased the release of IL-8, GM-CSF and soluble ICAM-1. The observations support the concept that exposure to DEPs may lead to functional changes and the release of pro-inflammatory mediators, which may influence the development of airway disease, especially in those disorders in which neutrophils are implicated (57). The epithelial cells of asthmatic subjects seem to be more sensitive than those of non-atopic non-asthmatics. When cultured human bronchial cells were exposed to DEPs for 24 h, the ciliary beat frequency was similarly attenuated in both groups, but the asthmatic cell cultures constitutively released significantly greater amounts of IL8, GM-CSF, and sICAM-1 and RANTES compared to cells from non-asthmatics. In response to 10 µg/ml of exposure to DEPs there was a significant increase in the release of IL-8, GM-CSF, and sICAM-1 in cells from asthmatics. However, exposure to doses of 50 and 100 µg/ml led to a decrease in the release of IL-8 and RANTES. In contrast these higher concentrations of DEPs led to a significant increase in the release of IL-8 and GM-CSF in the cells cultured from non-asthmatics (58). In another study the cytokine response to DEPs was shown to be synergistic with the response to TNFα (59). These effects may be mediated through the transcription factor NF-kB. Using electrophoretic mobility shift assay, DEPs have been shown to increase binding to the specific motif of NF-kB but not to transcription factor AP-1 (60). Moreover, DEPs can modulate chemokine pathways at the transcriptional level (61). In vitro , DEPs induce eosinophil degranulation and adhesiveness to epithelial cells without changing the eosinophil survival rate. These results indicate that DEPs may play a significant role in the promotion of airway hypersensitivity induced by enhanced eosinophil infiltration and degranulation ( 62 ). DEPs may also act through the complement system (63). Pre-treatment of human serum with DEP extracts (500–2500 µg/ml) demonstrated activation of the alternative complement pathway, resulting in a dose-dependent reduction in haemolytic activity (of up to 20%). Finally, it is important to remember that PM pollution episodes do not exist in isolation, and in most PM episodes, there will also be an increase in gaseous pollutants. Patients with asthma have been found to be more adversely affected than the normal population to inhalation of gaseous components of air pollution (64, 65) and in particular acid aerosols (66). Various studies have shown that overall lung deposition is increased in patients with obstructed airways and abnormal geometry. A 30% reduction in airway cross sectional area results in a deposition increase in the bifurcating airways of more than 100% (67). This will contribute to the deposition of PM and enhance both the immunological changes and non-specific inflammatory effects brought about in the airways by particulate pollution. Asthma is a chronic inflammatory disorder involving recruitment and activation of mast cells, eosinophils and T cells, associated with the development of bronchial hyperresponsiveness and variable airflow obstruction. All forms of the disease are characterized by enhanced production in the airways of Th-2 cytokines encoded in a cluster on chromosome 5q31−33 (1). Although the human exposure studies discussed above implicate air pollutants in the acute inflammatory response, questions remain about the chronic effects of air pollutants on the lung. Patients with chronic, severe asthma have thickened airways with thickening and increased density of the sub-basement membrane (SBM) collagen layer, as well as increases in smooth muscle, and in microvascular and neural networks (68). The deposition of interstitial (repair) collagens in the lamina reticularis and an associated increase in the number of myofibroblasts is a unique feature of asthma, irrespective of aetiology, and is thought to be caused by altered signalling between the bronchial epithelium and underlying mesenchymal cells (69). These studies have demonstrated that the asthmatic bronchial epithelium is more susceptible to oxidant-induced injury and that epithelial repair in asthma is attenuated (70). The injured epithelium becomes a potent source of growth factors such as PDGF, bFGF and TGFβ, that cause increased proliferation and biosynthetic activity of the underlying myofibroblasts, which are responsible for increased matrix deposition and airway remodelling. Whereas traditionally it has been thought that airway inflammation precedes, and is responsible for, airways remodelling, a recent study in children found that children who subsequently developed asthma had airway eosinophilia and increased epithelial SBM collagen thickening present up to 4 years before asthma was clinically expressed (71). Air pollution could contribute to this process: in rats, exposure to DEPs has been found to cause increased collagen deposition in the airways during lung development (72). Thus, remodelling of the airways in asthma may occur in parallel with inflammation, while epithelial stress and injury, such as that caused by the air pollutants DEPs, ozone, or NO2 may activate or reactivate morphogenetic mechanisms that cause structural changes in the airways similar to that occurring during bone formation (73). Further studies in humans will no doubt help to elucidate the relevant mechanisms, but for the moment, some caution is needed in defining appropriate air quality standards. This work is supported by project grants from the British Lung Foundation and the European Union (Framework Programme 5).