Anthony J. Frew MD FRCP
Professor of Allergy & Respiratory Medicine
Inflammation, Infection & Repair Division (AIR), Mailpoint 810, Southampton General Hospital, Southampton SO16 6YD UK e-mail: A.J.Frew@soton.ac.uk
Asthma is an increasing problem throughout the developed world. The rate of increase suggests that environmental factors have played a critical, and several plausible arguments have been put forward, including the possibility that air pollution may have influenced the development and expression of asthma and other allergic diseases. Clean air legislation has dramatically reduced the black smoke and sulphur dioxide that used to pollute our cities, but we now experience new forms of air pollution, particularly “summer smogs” in which fine particulates and ozone accumulate over our cities during periods of high pressure in the summer. In cities such as Los Angeles or Mexico City, the ozone concentrations commonly reach levels that are associated with adverse affects on normal and asthmatic subjects.
The functional responses of asthmatic patients to ozone, NO2, and SO2, have been extensively assessed in controlled exposure studies. Ozone has direct effects on the airways, causing reduced inspiratory capacity. This effect is more marked in patients with asthma and is biologically important in that epidemiological studies have shown direct and linear associations between ozone concentrations and admission rates for asthma and related respiratory diseases [1]. In human exposure studies, ozone induces an acute neutrophilic inflammatory response [2] together with release of chemokines (IL-8 and GRO-) [3]. NO2 has less direct effect on human airways, but increases patients’ sensitivity to inhalation allergen challenge [4].
Concerns have also been raised about the effects of particulate pollution, especially diesel exhaust particles. It has been shown that individuals living close to traffic routes in Japan are more likely to be sensitised to mountain cedar than those who live in areas with lower concentrations of DEP [5]. In vitro, DEP act as an adjuvant for the production of IgE [6] and stimulate cultured epithelial cells to release IL-8, GM-CSF and sICAM-1 [7].
Human exposure studies have found an acute inflammatory response to DE (PM10 300g/m3) in the airways of normal healthy subjects with increase numbers of neutrophils, mast cells and T cells, as well as upregulation of the adhesion molecule ICAM-1 [8]. However, in asthmatic subjects, exposure to DE increased bronchial reactivity, but did not induce an acute neutrophil influx or any worsening of the pre-existent eosinophilic inflammation. Further work is needed to enable us to explain the epidemiological findings of increased clinical sensitivity to ambient PM pollution among asthmatics. Possible explanations include damage to cilia, epithelial cell activation, induction of epithelial apoptosis, and/or mucus gland hyperplasia.
Health effects of Particulate Matter
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 [9]. Over the last ten 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 [10]. 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 m) 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, that have been reported to produce alterations in blood coagulability and increased rates of cardiovascular disorders as well as increased carcinogenicity [11], and potentiation of autoimmune disorders [12],
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 g/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 [13].
The role of DEP in the increased prevalence of asthma
Diesel exhaust is a major contributor to particulate pollution. Diesel exhaust particles (DEP) may promote inflammation and the allergic state through three broad mechanisms. (1) DEP may act as a carrier for the transport of allergens into the airways by adsorbing allergenic proteins onto their surface. (2) DEP may act as an adjuvant in promoting the switching of B cells to produce allergen-specific IgE. (3) DEP may affect a number of downstream immunological mechanisms, leading to increased prevalence or increased severity of allergic and asthmatic disorders.
DEP as a carrier
Some allergens, such as the major rye grass pollen allergen Lol p1, bind specifically to DEP [14]. In an in vitro study of allergen adsorption to indoor-suspended particulate matter (ISPM) and DEP, 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 DEP [15]. By acting as a carrier of allergens, DEP might deliver allergenic material to the airways in increased amounts during pollution episodes, and trigger asthma attacks in those who are sensitised [14].
DEP as an adjuvant for IgE
Co-presentation to the immune system of allergens and DEP may lead to an altered immune response to the allergen. Allergenic proteins can be identified bound to DEP 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 [16]. When these DEP and pollen grains are ingested by antigen-presenting cells, the allergenic proteins will undergo processing in an environment which is simultaneously affected by one or more components of the DEP. Moreover, the binding of pollen or other inhaled allergens to DEP may modulate the allergenic epitopes and thereby increase their allergenicity [17].
DEP 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 DEP rather than their carbon core, since PAH extracted from DEP enhance IgE production from purified human B-cells stimulated by IL-4 [18]. Importantly, PAH-DEP 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 DEP, had a similar effect on IgE production by a human B cell line [19]. In vivo, DEP enhanced total IgE and cedar pollen-specific IgE production in mice given ovalbumin (OA) mixed with DEP either intraperitoneally or intranasally [20,21]. 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 immunised four times with either OA, OA with DEP or OA with carbon black (CB). Specific IgE for OA was then analysed. Both DEP and CB showed an adjuvant activity for specific IgE production after intra-nasal instillation, indicating that they were both responsible for the effect [22]. This effect has been confirmed in other studies in mice [23].
The effect of DEP on antibody responses is dose- and time-dependent [24,27]. Even at low doses that have little demonstrable effect on IgE, exposure to DEP enhances the effect of allergen challenge on the airways, with increased eosinophilic inflammation and goblet cell hyperplasia [24,25].
In humans, diesel particles have been shown to potentiate nasal IgE production four days after challenge with DEP[28]. 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 DEP and allergen has been shown to induce higher ragweed-specific IgE and IgG4 responses compared with DEP or ragweed alone but with similar total IgE levels [29]. There was also a change in the cytokine pattern favouring allergic sensitisation. This implies that synergism between DEP and natural allergens may be a key feature in increasing the prevalence of allergen-induced respiratory disease.
DEP and allergic inflammation/downstream mediator effects
In addition to these allergen-specific effects, exposure to DEP 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 DEP damages the bronchial epithelium and cilia, thereby impairing their ability to act as a biological barrier against inhaled substances [30]. 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 DEP exposure may include up-regulation of histamine receptor gene expression [31] and increased penetration of allergen across the respiratory mucosa [32].
The cytotoxicity of DEP, their phagocytosis, and the resulting immune response have been studied in human bronchial and nasal epithelial cell cultures. DEP exposure 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) DEP 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, [33] even though it was phagocytosed to a similar degree.
Exposure to DEP 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 DEP exposure 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 [34].
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 DEP for 24 hours. 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 to10 g/ml of DEP exposure 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 g/ml led to a decrease in the release of IL-8 and RANTES. In contrast these higher concentrations of DEP led to a significant increase in the release of IL-8 and GM-CSF in the cells cultured from non-asthmatics [35]. In another study the cytokine response to DEP was shown to be synergistic with the response to TNF [36].
These effects may be mediated through the transcription factor NF-kB. Using electrophoretic mobility shift assay, DEP has been shown to increase binding to the specific motif of NF-kB but not of transcription factor AP-1 [37]. Moreover, DEP can modulate chemokine pathways at the transcriptional level [38].
In vitro, DEP 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 [39].
DEP may also act through the complement system [40]. Pre-treatment of human serum with DEP extracts (500 - 2.500 g/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 [41,42] and in particular acid aerosols [43]. 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 by more than 100% [44]. 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.
Conclusion:
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 characterised by enhanced production in the airways of Th-2 cytokines encoded in a cluster on chromosome 5q31-33. While 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. 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 due to altered signalling between the bronchial epithelium and underlying mesenchymal cells [Davies]. These studies have demonstrated that the asthmatic bronchial epithelium is more susceptible to oxidant-induced injury and that epithelial repair in asthma is attenuated [Bucchieri] The injured epithelium becomes a potent source of growth factors such as PDGF, bFGF, TGF that cause increased proliferation and biosynthetic activity of the underlying myofibroblasts which are responsible for increased matrix deposition and airway remodelling.
While 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. Air pollution could contribute to this process: in rats, DEP exposure has been found to cause increased collagen deposition in the airways during lung development. 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 DEP, ozone, or NO2 may activate or reactivate morphogenetic mechanisms that cause structural changes in the airways [. 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.
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FACULTY
Luísa Geraldes, Ana Todo Bom, Isabel Carrapatoso, Fernando Rodrigues,
Rosário Cunha, Celso Chieira.
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