Effects of controlled diesel exhaust exposure on apoptosis and proliferation markers in bronchial epithelium – an in vivo bronchoscopy study on asthmatics, rhinitics and healthy subjects
BMC Pulmonary Medicine volume 15, Article number: 99 (2015)
Epidemiological evidence demonstrates that exposure to traffic-derived pollution worsens respiratory symptoms in asthmatics, but controlled human exposure studies have failed to provide a mechanism for this effect. Here we investigated whether diesel exhaust (DE) would induce apoptosis or proliferation in the bronchial epithelium in vivo and thus contribute to respiratory symptoms.
Moderate (n = 16) and mild (n = 16) asthmatics, atopic non-asthmatic controls (rhinitics) (n = 13) and healthy controls (n = 21) were exposed to filtered air or DE (100 μg/m3) for 2 h, on two separate occasions. Bronchial biopsies were taken 18 h post-exposure and immunohistochemically analysed for pro-apoptotic and anti-apoptotic proteins (Bad, Bak, p85 PARP, Fas, Bcl-2) and a marker of proliferation (Ki67). Positive staining was assessed within the epithelium using computerized image analysis.
No evidence of epithelial apoptosis or proliferation was observed in healthy, allergic or asthmatic airways following DE challenge.
In the present study, we investigated whether DE exposure would affect markers of proliferation and apoptosis in the bronchial epithelium of asthmatics, rhinitics and healthy controls, providing a mechanistic basis for the reported increased airway sensitivity in asthmatics to air pollutants. In this first in vivo exposure investigation, we found no evidence of diesel exhaust-induced effects on these processes in the subject groups investigated.
Outdoor air pollution is of growing concern worldwide, and is linked to an increase in both cardiovascular and respiratory disease . Children, the elderly and subjects with pre-existing respiratory or cardiovascular diseases have been demonstrated to be susceptible groups [2–4]. There is evidence that exacerbations of pre-existing asthma are attributed to exposure to pollutants related to road traffic . Diesel exhaust exposures have also been shown to induce oxidative injury to the airways, leading to inflammation and increased risk of sensitisation .
In an attempt to understand the mechanisms responsible for these adverse health effects in humans, controlled exposure studies employing diesel exhaust (DE) have been performed both in healthy individuals and in subjects with established allergic diseases (asthma and rhinitis). These studies have shown that healthy individuals respond acutely to DE challenge with a marked neutrophilic airway inflammation [7–11]. In asthmatics, increased bronchial hyperresponsiveness to methacholine is evident following exposure to high ambient DE concentrations . However, this occurs with no evidence of augmented airway inflammation in terms of diesel exhaust-induced changes in submucosal inflammatory cell number or adhesion molecule expression . The only responses reported in the asthmatic airway following DE have been increased epithelial expression of IL-10  and enhanced sputum IL-6 levels . In another study, asthmatics were exposed to DE in combination with cat allergen, again without any signs of an enhanced airway inflammation .
The bronchial epithelium forms a barrier between the external environment and the underlying lung mucosa. Epithelial integrity is usually maintained due to a balance between apoptotic and proliferative processes , with the relative activation of the positive and negative regulatory pathways of apoptosis ultimately determining cell fate . The Bcl-2 family are key regulators of the mitochondrial apoptosis pathway and include the pro-apoptosis members Bad and Bak and the anti-apoptosis member Bcl-2 [15–17]. Another pathway of importance is the TNFα receptor pathway, which includes the receptor Fas. Binding to these receptors leads to activation of initiator caspases. These two pathways are also termed the intrinsic and extrinsic pathways and they converge at the effector caspases, including caspase 3, and lead to cleavage of poly (ADP-ribose) polymerase (PARP) into a C terminal 85 kDa peptide fragment .
There have been some studies in human subjects, or samples derived from humans, investigating apoptosis in asthma and following exposure to DE. Evidence supports increased apoptosis in asthma, in that the damaged bronchial epithelium found in asthmatics has been related to the degree of bronchial hyperresponsiveness [19, 20]. In a study by Bucchieri employing bronchial biopsies from asthmatics, increased epithelial apoptosis was demonstrated in terms of a greater expression of the caspase cleavage product p85 PARP, compared to healthy controls . Furthermore, using primary epithelial cell cultures, the asthmatic epithelium was shown to be more susceptible to oxidant-induced apoptosis compared with normal controls . In vitro studies using epithelial cell lines (A549) have demonstrated that exposure to particulate matter (PM) leads to increased apoptosis [22–24]. Apoptosis is further enhanced in cystic fibrosis epithelial cell lines compared to controls . The effects of PM on the apoptosis pathway in vivo and on subjects with pre-existing lung disease are unknown. The lack of an increased airway inflammation following exposure to DE has led us to hypothesize that other mechanisms, apart from airway inflammatory responses, such as apoptosis, may account for the clinical outcomes observed in asthmatics following exposure to traffic-derived pollution.
In the present study, we hypothesised that increased apoptosis, or impaired proliferative responses could potentially enhance the penetration of bronchoconstrictive stimuli to the airway wall, potentially explaining the increased sensitivity of asthmatics to symptoms during periods of high pollution. The specific aim in this study was to explore whether DE-exposure would affect pro-apoptotic and anti-apoptotic proteins and markers of proliferative responses in the bronchial airways. This study included both mild and moderate asthmatics along with healthy controls, as previously described . In addition, due to the observed differential responses to DE in asthmatics and healthy control subjects, atopic non-asthmatics (subjects with allergic rhinitis) were also included. We and others have shown evidence of lower airway inflammation of atopic non-asthmatics that is intermediary between that observed in the bronchial mucosa of asthmatics versus healthy subjects [26–28]. Furthermore, in a controlled DE exposure study of allergic rhinitics, we found that the DE-induced airway inflammatory response was similar to that observed in asthmatic airways .
This current study was performed as a follow-up investigation of a larger study addressing DE-induced responses in the airways of healthy, rhinitic and asthmatic subjects [7, 29], using archived biopsies. Subjects included healthy controls (non-asthmatic, non-atopic) (n = 21), allergic rhinitics without asthma (atopic controls) (n = 13), asthmatics on short acting β-2 agonists on demand (n = 16) and asthmatics on inhaled corticosteroids (200–1200 μg daily) (n = 16). Baseline demographics of each group are summarized in Table 1. All participants gave their informed consent and the local ethics committee of Umeå University approved the study, which was performed in accordance with the declaration of Helsinki.
Subjects were exposed to filtered air or diesel exhaust (100 μg/m3) for 2 h, on two separate occasions, at least 3 weeks apart, in a randomised order, as previously described [7, 29]. During the exposures, subjects alternated exercise on a bicycle ergometer (minute ventilation = 20 L/min/m2 body surface area) with rest at 15-min intervals. This exposure setup was used to model a moderate level of outdoor activity in a high-polluted environment. The diesel exhaust was generated from an idling 1991 Volvo diesel engine (Volvo TD45, 4.5 l, four cylinders, 680 rpm). The air within the exposure chamber was monitored continuously and the steady state concentration of PM10, gases and semi-volatiles during the diesel exposures were 100 ± 12 μg/m3 for PM10, 9.1 ± 2.6 parts per million (ppm) for carbon monoxide, 1.2 ± 0.1 ppm for nitrogen monoxide, 0.39 ± 0.05 ppm for nitrogen dioxide, 1.7 ± 0.4 ppm for oxides of nitrogen and 1.1 ± 0.8 ppm for total gaseous hydrocarbons (C3H8-equivalents), expressed as mean ± SD. The PM mass was dominated by fine and ultrafine particles (<1 μm), and the mass median particle diameter was 0.18 μm. Further details of the methods used are described in detail in previous papers [7, 29].
Bronchoscopy with collection of bronchial biopsies was performed at the Department of Medicine, Division of Respiratory Medicine and Allergy, University Hospital, Umeå, Sweden, 18 h after exposure. The biopsies were processed into glycol methacrylate (GMA) resin for immunohistochemistry using standardised protocols . Immunohistochemical staining was performed using the streptavidin biotin-peroxidase technique and monoclonal antibodies directed against the pro-apoptotic markers, Bad (Serotec, Kidlington, UK) and Bak (Serotec); the anti-apoptotic marker Bcl-2 (Dako, Ely, UK); the death receptor Fas (CD95) (TCS Biosciences ltd, Botolph Claydon, UK); the caspase cleavage product p85 PARP (Promega, Southampton, UK); and the cell proliferation marker Ki67 (Dako). Staining was visualised with diaminobenzidine (DAB) and sections counterstained with haematoxylin.
Positive staining was analysed in lengths of intact well orientated epithelium with the assistance of computerised image analysis (KS400 software with a Zeiss Axioskop 2 microscope and Axiocam, Zeiss, Bicester, UK). For Ki67 the percentage positive nuclear area was determined and for all other markers the percentage positive of the total epithelium was assessed, based on the red/green/blue (RGB) colour composition of the DAB staining . In brief, following standardisation of the computerised image analysis system for light level and white balance digitised images of the epithelium were captured. Threshold RGB settings for the DAB staining were applied and adjusted to select all the positive staining within the section. For epithelial markers, the area of the epithelium was then delineated interactively and the percentage of positive staining within the epithelium calculated. For Ki67, DAB positive thresholding was measured against RGB thresholding for blue nuclear counterstaining.
Data were not normally distributed and are therefore presented as medians with interquartile ranges (IQR). All paired air versus diesel exhaust comparisons were performed using the Wilcoxon signed rank test. The Kruskall Wallis ANOVA test was initially used to test for differences in the response (change) to diesel exhaust versus air between the four subject groups and differences at baseline. If a significant difference was observed between the four groups, the Mann Whitney U test was used for further analyses. All statistical analyses were performed using SPSS, version 21.0 (SPSS, Cary, North Carolina, USA). A p-value of <0.05 was considered statistically significant.
Representative images of the immunohistochemical staining are shown in Fig. 1. There were no differences in baseline (post air exposure) in the levels of any of the markers of apoptosis or proliferation examined between the four subject groups (Table 2). In the asthmatic and rhinitic subjects, there was also no significant change in expression (response) following exposure to DE compared to air in any of these markers. However, in the healthy control subjects the expression of Bcl-2 was significantly lowered (p = 0.02) after DE compared to filtered air: 4.5 % (IQR 0–11.44) after air versus 0.06 % (IQR 0.01–0.62) following DE exposure. However, comparison of the response (change) to diesel versus air exposure across the groups was not significant for Bcl-2 or any of the other markers investigated (Fig. 2).
In this study, we investigated the hypothesis that DE would lead to an increase in apoptosis accompanied by impaired repair reflected by decreased proliferation in the bronchial epithelium of asthmatics, providing a mechanistic basis for their reported increased sensitivity to air pollutants. Contrary to our hypothesis, in this first in vivo exposure investigation, we found no evidence of the induction of these processes in either mild-moderate asthmatics or allergic rhinitics following DE challenge. Whilst we observed a decrease in Bcl-2 in the healthy subjects following diesel exposure compared to air exposure, no significant changes were found when comparing the Bcl-2 responses across the groups.
Previous DE exposure studies have used different exposure setups. We have reported a marked neutrophilic inflammatory response in the airways of healthy volunteers following exposure to 300 μg/m3 DE for one hour, 6 h post-exposure . In a previous study in which healthy and asthmatics were exposed to DE at PM10 108 μg/m3 for 2 h, an increase in airway resistance of similar magnitude was observed in both healthy and asthmatics. Healthy subjects also developed airway inflammation 6 h after DE exposure, a response that was absent in asthmatic subjects. However, epithelial staining for the cytokine IL-10 was increased after DE in the asthmatic group, a cytokine with potential anti-inflammatory effects . In the present study, we hypothesised that the inflammatory response would be delayed in the allergic/asthmatics subjects. We therefore exposed them to this lower concentration (100 μg/m3) for 2 h and performed the bronchoscopies 18 h post exposure. The dose was chosen to mimic real world exposure more closely. This is also a dose observed at kerbside locations for example in London (http://www.londonair.org.uk/). Again we found a neutrophilic airway inflammation in the healthy subjects but not in subjects with allergic rhinitis or asthma. We therefore hypothesised that other mechanisms such as apoptosis would be affected by the DE exposure.
The failure of this study to confirm the previous in vitro studies demonstrating increased apoptosis in A549 cells in response to PM could suggest that these responses do not extrapolate to the in vivo setting or may reflect differences in methodologies between in vitro and in vivo systems [22–25]. The A549 cell line is a transformed adenocarcinoma alveolar type II cell line. These cells are not normal and have a squamous phenotype; therefore they are likely to respond differently to PM than the in vivo stratified columnar bronchial epithelium. Also, these investigators used different methodologies to assess apoptosis, measuring different end-points and features of apoptosis, which again may account for the diverse outcomes observed. Their techniques included: TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) to assess DNA fragmentation [22, 23]; measurement of DNA nucleosomal fragmentation by ELISA; [23, 25] colourimetric assays for caspase 3 and 9 activation;  and Western blot to measure active caspase 3 and fragmentation of cytokeratins. In the immortalised BEAS-2B bronchial epithelial cell line, which resembles the in vivo bronchial epithelium more closely, no effect on apoptosis following exposure to DE was observed , which concurs with our findings. In their study, Cao et al. measured antibody immunoreactivity to detect Bcl2 and p85 protein by Western blotting, which is similar to the immunohistochemical approach we employed.
There was variability in the expression of the markers explored both at baseline (Table 1) and following DE exposure (Table 2 and Fig. 2). It is well known that asthma has a heterogeneous immunopathology that could to some extent impact on this variability. However, this did not result in any outliers in clinical responses following DE exposure. The participants in the present study were all stable in their disease. If the study had included subjects with uncontrolled asthma, the results may have been different.
Whilst none of the examined markers were altered in the asthmatics or allergic rhinitics after exposure to DE, this study is the first to report an in vivo effect of DE exposure on Bcl-2 expression in healthy subjects. However, as this change in expression was not significant in comparison with the other subject groups, this finding should be considered with caution. Bcl-2 is known to be a key apoptosis regulatory protein of the mitochondrial death pathway, and has an anti-apoptotic function that is closely associated with its level of expression . Oxidative stress has been shown to downregulate Bcl-2 expression and thus promote apoptosis . Exposure of A549 cells to parabenzoquinone, a component of DE, leads to decreased expression of Bcl-2 . Also, over-expression of Bcl-2 has been shown to delay apoptosis of macrophages induced by chemical extracts from DE  and to promote survival of cultured retinal pigment epithelial cells exposed to oxidative damage .
The observed decrease in the anti-apoptotic marker Bcl-2 would suggest an increase in apoptosis following acute exposure to oxidative air pollution, which may be a protective mechanism to remove damaged epithelium. It has also been suggested that decreased apoptosis, if accompanied by increased proliferation, may be a repair response to protect the epithelium . However, in the present study, the decrease in Bcl-2 was not paralleled by an increase in p85 PARP, and so we could therefore not confirm that exposure to DE leads to increased apoptosis, or an increase in Ki67 expression that would suggest a proliferative response.
Whilst the lack of a measurable response to DE within the bronchial epithelium in the asthmatic subjects may reflect a difference between in vitro and in vivo exposure (as discussed earlier), it is possible this may be due to epithelial shedding of damaged cells. An impaired epithelial barrier due to epithelial shedding [19, 20, 39] is a characteristic feature of asthma, as is increased sensitivity to oxidative damage . In the present study the number of epithelial cells in bronchial lavages was very low, and there were no signs of epithelial shedding in any of the groups. Neither was there any sign of shredding in the biopsy material. Another possible scenario could have been that the exposure of an already fragile sensitive epithelium to DE could already have led to apoptosis and shedding into the bronchial lumen by 18 h. It may therefore be more appropriate to look at an earlier time point after DE exposure. In this study we did not observe a response assessed by changes in markers of apoptosis or proliferation in the bronchial epithelium of the allergic rhinitic subjects when exposed to DE, suggesting their responses are similar to those seen in asthmatics.
Whilst previous studies have reported increased markers of apoptosis and proliferation in asthmatics [40–42], we did not observe any differences in the expression of the pro-apoptotic markers, Bad and Bak; the anti-apoptotic marker Bcl-2; the death receptor Fas; the caspase cleavage product p85 PARP; or the cell proliferation marker Ki67 between the subject groups when exposed to air. Druilhe reports increased expression of Fas in the bronchial epithelium of asthmatics compared to controls, but no difference in Fas L, Bcl-2 or PCNA . p85 PARP was found by Western blot in asthmatic epithelial cells, accompanied by an increase in immunohistochemical staining for Ki67. Such responses were not seen in healthy controls . In another study, Bcl-2 expression was increased in the bronchial epithelium of asthmatic subjects compared to controls, implying an anti-apoptotic effect, but in the absence of PCNA positivity . Following an asthma exacerbation, an increase in Bcl-2 expression in BAL lymphocytes has been shown, but contrary to our study, these asthmatics had a FEV1 that ranged between 62 and 83 % predicted, compared with 98 % in our current study. The increase in Bcl-2 could therefore be related to asthma severity . There are also other previous studies employing less severe asthmatics that also failed to demonstrate differences in Bcl-2, Fas or Ki67 compared to healthy controls .
These initial data do not support our original contention that the heightened sensitivity of asthmatics to traffic derived particulate matter might reflect an underlying imbalance between cell clearance and proliferation in response to DE; effectively enhancing the penetration of bronchoconstrictive stimuli to the airway wall. We acknowledge that this interpretation is limited by the single time point examined, and further studies will be required before we can fully exclude the involvement of these pathways in the induction of airway hyper-responsiveness after exposure to particulate matter air pollution.
The volume exhaled during the first second of a forced expiratory maneuver
Poly ADP-ribose polymerase
Histamine concentration causing a 20 % drop in FEV1
Proliferating cell nuclear antigen
Huang YC. Outdoor air pollution: a global perspective. J Occup Environ Med. 2014;56 Suppl 10:S3–7.
Janssen NA, Brunekreef B, van Vliet P, Aarts F, Meliefste K, Harssema H, et al. The relationship between air pollution from heavy traffic and allergic sensitization, bronchial hyperresponsiveness, and respiratory symptoms in Dutch schoolchildren. Environ Health Perspect. 2003;111(12):1512–8.
Clark NA, Demers PA, Karr CJ, Koehoorn M, Lencar C, Tamburic L, et al. Effect of early life exposure to air pollution on development of childhood asthma. Environ Health Perspect. 2010;118(2):284–90.
Kunzli N, Bridevaux PO, Liu LJ, Garcia-Esteban R, Schindler C, Gerbase MW, et al. Traffic-related air pollution correlates with adult-onset asthma among never-smokers. Thorax. 2009;64(8):664–70.
Perez L, Declercq C, Iniguez C, Aguilera I, Badaloni C, Ballester F, et al. Chronic burden of near-roadway traffic pollution in 10 European cities (APHEKOM network). Eur Respir J. 2013;42(3):594–605.
Gowers AM, Cullinan P, Ayres JG, Anderson HR, Strachan DP, Holgate ST, et al. Does outdoor air pollution induce new cases of asthma? Biological plausibility and evidence; a review. Respirology. 2012;17(6):887–98.
Behndig AF, Larsson N, Brown JL, Stenfors N, Helleday R, Duggan ST, et al. Proinflammatory doses of diesel exhaust in healthy subjects fail to elicit equivalent or augmented airway inflammation in subjects with asthma. Thorax. 2011;66(1):12–9.
Nightingale JA, Maggs R, Cullinan P, Donnelly LE, Rogers DF, Kinnersley R, et al. Airway inflammation after controlled exposure to diesel exhaust particulates. Am J Respir Crit Care Med. 2000;162(1):161–6.
Nordenhall C, Pourazar J, Blomberg A, Levin JO, Sandstrom T, Adelroth E. Airway inflammation following exposure to diesel exhaust: a study of time kinetics using induced sputum. Eur Respir J. 2000;15(6):1046–51.
Salvi SS, Nordenhall C, Blomberg A, Rudell B, Pourazar J, Kelly FJ, et al. Acute exposure to diesel exhaust increases IL-8 and GRO-alpha production in healthy human airways. Am J Respir Crit Care Med. 2000;161(2 Pt 1):550–7.
Salvi S, Blomberg A, Rudell B, Kelly F, Sandstrom T, Holgate ST, et al. Acute inflammatory responses in the airways and peripheral blood after short-term exposure to diesel exhaust in healthy human volunteers. Am J Respir Crit Care Med. 1999;159(3):702–9.
Nordenhall C, Pourazar J, Ledin MC, Levin JO, Sandstrom T, Adelroth E. Diesel exhaust enhances airway responsiveness in asthmatic subjects. Eur Respir J. 2001;17(5):909–15.
Stenfors N, Nordenhall C, Salvi SS, Mudway I, Soderberg M, Blomberg A, et al. Different airway inflammatory responses in asthmatic and healthy humans exposed to diesel. Eur Respir J. 2004;23(1):82–6.
Riedl MA, Diaz-Sanchez D, Linn WS, Gong Jr H, Clark KW, Effros RM, et al. Allergic inflammation in the human lower respiratory tract affected by exposure to diesel exhaust. Res Rep. 2012;165:5–43. discussion 45–64.
Bortner CD, Cidlowski JA. Cellular mechanisms for the repression of apoptosis. Annu Rev Pharmacol Toxicol. 2002;42:259–81.
Cory S, Huang DC, Adams JM. The Bcl-2 family: roles in cell survival and oncogenesis. Oncogene. 2003;22(53):8590–607.
Huang DC, Strasser A. BH3-Only proteins-essential initiators of apoptotic cell death. Cell. 2000;103(6):839–42.
Kaufmann SH, Desnoyers S, Ottaviano Y, Davidson NE, Poirier GG. Specific proteolytic cleavage of poly(ADP-ribose) polymerase: an early marker of chemotherapy-induced apoptosis. Cancer Res. 1993;53(17):3976–85.
Laitinen LA, Heino M, Laitinen A, Kava T, Haahtela T. Damage of the airway epithelium and bronchial reactivity in patients with asthma. Am Rev Respir Dis. 1985;131(4):599–606.
Jeffery PK, Wardlaw AJ, Nelson FC, Collins JV, Kay AB. Bronchial biopsies in asthma. An ultrastructural, quantitative study and correlation with hyperreactivity. Am Rev Respir Dis. 1989;140(6):1745–53.
Bucchieri F, Puddicombe SM, Lordan JL, Richter A, Buchanan D, Wilson SJ, et al. Asthmatic bronchial epithelium is more susceptible to oxidant-induced apoptosis. Am J Respir Cell Mol Biol. 2002;27(2):179–85.
Alfaro-Moreno E, Martinez L, Garcia-Cuellar C, Bonner JC, Murray JC, Rosas I, et al. Biologic effects induced in vitro by PM10 from three different zones of Mexico City. Environ Health Perspect. 2002;110(7):715–20.
Upadhyay D, Panduri V, Ghio A, Kamp DW. Particulate matter induces alveolar epithelial cell DNA damage and apoptosis: role of free radicals and the mitochondria. Am J Respir Cell Mol Biol. 2003;29(2):180–7.
Ackland ML, Zou L, Freestone D, van de Waasenburg S, Michalczyk AA. Diesel exhaust particulate matter induces multinucleate cells and zinc transporter-dependent apoptosis in human airway cells. Immunol Cell Biol. 2007;85(8):617–22.
Kamdar O, Le W, Zhang J, Ghio AJ, Rosen GD, Upadhyay D. Air pollution induces enhanced mitochondrial oxidative stress in cystic fibrosis airway epithelium. FEBS Lett. 2008;582(25–26):3601–6.
Djukanovic R, Lai CK, Wilson JW, Britten KM, Wilson SJ, Roche WR, et al. Bronchial mucosal manifestations of atopy: a comparison of markers of inflammation between atopic asthmatics, atopic nonasthmatics and healthy controls. Eur Respir J. 1992;5(5):538–44.
Brown JL, Behndig AF, Sekerel BE, Pourazar J, Blomberg A, Kelly FJ, et al. Lower airways inflammation in allergic rhinitics: a comparison with asthmatics and normal controls. Clin Exp Allergy. 2007;37(5):688–95.
Foresi A, Leone C, Pelucchi A, Mastropasqua B, Chetta A, D’Ippolito R, et al. Eosinophils, mast cells, and basophils in induced sputum from patients with seasonal allergic rhinitis and perennial asthma: relationship to methacholine responsiveness. J Allergy Clin Immunol. 1997;100(1):58–64.
Larsson N, Brown J, Stenfors N, Wilson S, Mudway IS, Pourazar J, et al. Airway inflammatory responses to diesel exhaust in allergic rhinitics. Inhal Toxicol. 2013;25(3):160–7.
Britten KM, Howarth PH, Roche WR. Immunohistochemistry on resin sections: a comparison of resin embedding techniques for small mucosal biopsies. Biological Stain Commission. 1993;68(5):271–80.
Puddicombe SM, Polosa R, Richter A, Krishna MT, Howarth PH, Holgate ST, et al. Involvement of the epidermal growth factor receptor in epithelial repair in asthma. FASEB J. 2000;14(10):1362–74.
Cao D, Bromberg PA, Samet JM. Diesel particle-induced transcriptional expression of p21 involves activation of EGFR, Src, and Stat3. Am J Respir Cell Mol Biol. 2010;42(1):88–95.
Azad N, Iyer A, Vallyathan V, Wang L, Castranova V, Stehlik C, et al. Role of oxidative/nitrosative stress-mediated Bcl-2 regulation in apoptosis and malignant transformation. Ann N Y Acad Sci. 2010;1203:1–6.
Azad N, Iyer AK, Manosroi A, Wang L, Rojanasakul Y. Superoxide-mediated proteasomal degradation of Bcl-2 determines cell susceptibility to Cr(VI)-induced apoptosis. Carcinogenesis. 2008;29(8):1538–45.
Das A, Chakrabarty S, Choudhury D, Chakrabarti G. 1,4-Benzoquinone (PBQ) induced toxicity in lung epithelial cells is mediated by the disruption of the microtubule network and activation of caspase-3. Chem Res Toxicol. 2010;23(6):1054–66.
Hiura TS, Li N, Kaplan R, Horwitz M, Seagrave JC, Nel AE. The role of a mitochondrial pathway in the induction of apoptosis by chemicals extracted from diesel exhaust particles. J Immunol. 2000;165(5):2703–11.
Godley BF, Jin GF, Guo YS, Hurst JS. Bcl-2 overexpression increases survival in human retinal pigment epithelial cells exposed to H(2)O(2). Exp Eye Res. 2002;74(6):663–9.
Bayram H, Ito K, Issa R, Ito M, Sukkar M, Chung KF. Regulation of human lung epithelial cell numbers by diesel exhaust particles. Eur Respir J. 2006;27(4):705–13.
Montefort S, Roche WR, Holgate ST. Bronchial epithelial shedding in asthmatics and non-asthmatics. Respir Med. 1993;87(Suppl B):9–11.
Druilhe A, Wallaert B, Tsicopoulos A, Lapa e Silva JR, Tillie-Leblond I, Tonnel AB, et al. Apoptosis, proliferation, and expression of Bcl-2, Fas, and Fas ligand in bronchial biopsies from asthmatics. Am J Respir Cell Mol Biol. 1998;19(5):747–57.
Comhair SA, Xu W, Ghosh S, Thunnissen FB, Almasan A, Calhoun WJ, et al. Superoxide dismutase inactivation in pathophysiology of asthmatic airway remodeling and reactivity. Am J Pathol. 2005;166(3):663–74.
Vignola AM, Chiappara G, Siena L, Bruno A, Gagliardo R, Merendino AM, et al. Proliferation and activation of bronchial epithelial cells in corticosteroid-dependent asthma. J Allergy Clin Immunol. 2001;108(5):738–46.
Abdulamir AS, Hafidh RR, Abubakar F. Different inflammatory mechanisms in lungs of severe and mild asthma: crosstalk of NF-kappa-B, TGFbeta1, Bax, Bcl-2, IL-4 and IgE. Scand J Clin Lab Invest. 2009;69(4):487–95.
Cohen L, E X, Tarsi J, Ramkumar T, Horiuchi TK, Cochran R, et al. Epithelial cell proliferation contributes to airway remodeling in severe asthma. Am J Respir Crit Care Med. 2007;176(2):138–45.
The authors would like to thank Annika Johansson, Helena Tjällgren, Helen Bertilsson, Jamshid Pourazar, Ann-Britt Lundström and Maj-Cari Ledin and the Histochemistry Research Unit for technical contribution to the project. We also would like to express our gratitude to the volunteers who participated in this investigation. This study was funded by Swedish Heart and Lung Foundation, European Commission HEPMEAP project (QLRT 1999–01582) and Umeå University, Sweden.
The authors declare that they have no competing interests.
AFB participated in the design of the study, recruited and screened study subjects, performed the bronchoscopies, drafted and finalised the manuscript. JB supervised the diesel exhaust exposures. KS and LW carried out the immunohistochemistry. AJF, FJK and TS participated in the design of the study. NS participated in the design of the study and performed the bronchoscopies. ISM participated in the design of the study and drafting of the manuscript. SJW conceived of the study, participated in its design and coordination, supervised the IHC work and helped to draft the manuscript. All authors read and approved the final manuscript.
About this article
Cite this article
Behndig, A.F., Shanmuganathan, K., Whitmarsh, L. et al. Effects of controlled diesel exhaust exposure on apoptosis and proliferation markers in bronchial epithelium – an in vivo bronchoscopy study on asthmatics, rhinitics and healthy subjects. BMC Pulm Med 15, 99 (2015). https://doi.org/10.1186/s12890-015-0096-x
- Allergic Rhinitics
- Diesel Exhaust
- Bronchial Epithelium
- Asthmatic Subject
- Asthmatic Airway