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BACKGROUND: Exposure to particulate matter (PM) air pollution especially derived from traffic is associated with increases in cardiorespiratory morbidity and mortality. In this study, we evaluated the ability of novel vehicle cabin air inlet filters to reduce diesel exhaust (DE)-induced symptoms and markers of inflammation in human subjects.

METHODS: Thirty healthy subjects participated in a randomized double-blind controlled crossover study where they were exposed to filtered air, unfiltered DE and DE filtered through two selected particle filters, one with and one without active charcoal. Exposures lasted for one hour. Symptoms were assessed before and during exposures and lung function was measured before and after each exposure, with inflammation assessed in peripheral blood five hours after exposures. In parallel, PM were collected from unfiltered and filtered DE and assessed for their capacity to drive damaging oxidation reactions in a cell-free model, or promote inflammation in A549 cells.

RESULTS: The standard particle filter employed in this study reduced PM10 mass concentrations within the exposure chamber by 46%, further reduced to 74% by the inclusion of an active charcoal component. In addition use of the active charcoal filter was associated by a 75% and 50% reduction in NO2 and hydrocarbon concentrations, respectively. As expected, subjects reported more subjective symptoms after exposure to unfiltered DE compared to filtered air, which was significantly reduced by the filter with an active charcoal component. There were no significant changes in lung function after exposures. Similarly diesel exhaust did not elicit significant increases in any of the inflammatory markers examined in the peripheral blood samples 5 hour post-exposure. Whilst the filters reduced chamber particle concentrations, the oxidative activity of the particles themselves, did not change following filtration with either filter. In contrast, diesel exhaust PM passed through the active charcoal combination filter appeared less inflammatory to A549 cells.

CONCLUSIONS: A cabin air inlet particle filter including an active charcoal component was highly effective in reducing both DE particulate and gaseous components, with reduced exhaust-induced symptoms in healthy volunteers. These data demonstrate the effectiveness of cabin filters to protect subjects travelling in vehicles from diesel exhaust emissions.

In this study aerosol samples collected in an Asian mega-city (Kabul, Afghanistan) were compared to PM samples collected in a European location with traffic (Umea, Sweden) and a reference urban dust material (SRM 1649b). The toxicity of each sample towards normal human bronchial epithelial (NHBE) cells and a human bronchial epithelial cell line (BEAS-2B) was tested along with their ability to induce reactive oxygen species (ROS) formation and inflammatory responses. The extracts' morphology and elemental composition was studied by SEM-EDXRF, and filter samples were analyzed for metals and organic compounds. The PM from Kabul contained a larger fraction of fine particles, 19 times more polyaromatic hydrocarbons (PAH) and 37 times more oxygenated PAH (oxy-PAH) compared to samples from timed. The PM-samples from Kabul and the reference material (SRM 1649b) induced significantly stronger oxidative stress responses than the samples from Umea. Furthermore, samples collected in Kabul induced significantly higher secretion of the cytokines IL-6, IL-8 and GM-CSF while SRM1649b induced a cytokine pattern more similar to samples collected in Umea. Several properties of the particles could potentially explain these differences, including differences in their size distribution and contents of PAH and oxy-PAH, possibly in combination with their relative transition metal contents. 

Throughout many parts of the world house dust mites (HDM) are considered as a major source of indoor aeroallergens and they are powerful inducers of allergic diseases. Proteolytic HDM allergens are recognised as being able to directly activate respiratory epithelial cells and thereby actively participate in innate immune responses. Although several major HDM allergens lack proteolytic activity, their possible ability to similarly interact with epithelial cells is not known.

The overall aim of this thesis was therefore to elucidate if and how major non-proteolytic group 2 allergens from different mite species interact with respiratory epithelial cells. The effects of the structurally related Der p 2, Der f 2 and Eur m 2 from different HDM species as well as the storage mite allergen Lep d 2 were studied in vitro using human respiratory epithelial cells. Also the non-proteolytic, but structurally dissimilar, Fel d 1 from cat, Can f 2 from dog, Bet v 1 from birch and Phl p 5a from timothy were studied.

In this thesis evidence that major group 2 mite allergens activate bronchial epithelial cells is presented. Following allergen exposure the secreted amount of the inflammatory mediators G-CSF, GM-CSF, IL-6, IL-8, MCP-1, MIP-3α and sICAM-1 was increased. Surface expression of ICAM-1 was also increased following allergen exposure. Moreover, Fel d 1 and Can f 2 induced secretion of the same mediators from bronchial epithelial cells, representing two additional protein structures being able to directly induce cell activation. In experiments using specific inhibitors and siRNA transfection, it was shown that the mite allergens engage TLR4 and activation through MyD88, MAPK and NF-κB signal transduction pathways.

In conclusion, the novel findings in this thesis provide knowledge on how major aeroallergens, in addition to their ability to provoke specific adaptive immune responses, may aggravate a respiratory airway disease by adjuvant-like activation of inflammatory responses in bronchial epithelial cells. This differs from previously reported allergen-induction of epithelial cells by the clear independency of proteolytic activation.

Background: Exposure to seasonal or indoor allergens may cause sensitisation and development of allergic airway diseases. We have previously demonstrated that the non-proteolytic major house dust mite (HDM) allergen Der p 2 stimulates pro-inflammatory responses in bronchial epithelial cells. We aimed to determine if other clinically relevant non-proteolytic aeroallergens originating from HDMs, storage mites, cat, dog, birch and timothy also activate respiratory epithelial cells.

Methods: Cultures of human bronchial epithelial cell line BEAS-2B, normal human bronchial epithelial cells and alveolar epithelial cell line A549 were exposed to recombinant (r)Der p 2, natural (n)Der f 2, rEur m 2, rLep d 2, rFel d 1, nFel d 1, rCan f 2, rBet v 1 or rPhl p 5a. A panel of secreted mediators and expression of cell adhesion receptors involved in recruitment, survival and adhesion of inflammatory cells in asthmatic airways was assessed.

Results: The mite allergens rDer p 2, nDer f 2, rEur m 2 and rLep d 2 as well as the cat and dog allergens rFel d 1, nFel d 1 and rCan f 2 induced granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, interleukin (IL)-6, IL-8, monocyte-chemotactic protein-1 and macrophage inflammatory protein-3α secretion from bronchial epithelial cells as well as surface expression of intracellular adhesion molecule-1. The pollen allergens rBet v 1 and rPhl p 5a from birch and timothy did not activate the cells. None of the studied allergens affected the alveolar epithelial cells.

Conclusion: These results show that both mite and structurally unrelated cat and dog allergens can activate respiratory epithelial cells by adjuvant-like protease-independent mechanisms.

BACKGROUND: House dust mites (HDM) are well-known as a source of indoor aeroallergens and for causing allergic airway diseases. Some proteolytic HDM allergens are known to activate respiratory epithelial cells to produce pro-inflammatory mediators, while there is limited knowledge regarding such activity among non-proteolytic HDM allergens.

OBJECTIVE: To investigate whether Der p 2, a major non-proteolytic allergen of Dermatophagoides pteronyssinus, activates respiratory epithelial cells to produce mediators involved in asthma pathogenesis and to elucidate the mechanism of such activation.

METHODS: The human bronchial epithelial cell line BEAS-2B, normal human bronchial epithelial (NHBE) cells and the alveolar epithelial cell line A549 were exposed to recombinant Der p 2. Following exposure, we analysed a panel of soluble mediators and cell adhesion receptors involved in asthma pathogenesis by promoting recruitment, survival and binding of inflammatory cells. The involvement of nuclear factor (NF)-kappaB and mitogen-activated protein kinases (MAPKs) was studied using specific inhibitors.

RESULTS: Der p 2 activated bronchial BEAS-2B and NHBE cells, but not alveolar A549 cells. In BEAS-2B cells Der p 2 induced dose-dependent up-regulation in both mRNA level and protein secretion of granulocyte-macrophage colony-stimulating factor, IL-6, IL-8, monocyte-chemotactic protein-1 and macrophage inflammatory protein-3alpha. Secretion as well as surface expression of intercellular adhesion molecule (ICAM)-1 was also up-regulated, which was associated with increased adhesion of monocytes to the epithelial cells. The release of cytokines and chemokines was regulated by NF-kappaB and MAPK activation in different ways, while expression of ICAM-1 was solely dependent on NF-kappaB activation.

CONCLUSION: These results show that Der p 2 activates respiratory epithelial cells, indicating that this non-proteolytic allergen, in addition to its immunogenic properties, can aggravate respiratory airway disease by adjuvant-like activation of the lung epithelium.