Diesel engine exhaust has been demonstrated to induce inflammation in the bronchial epithelium, which apart from its classical barrier function, increasingly has been demonstrated to carry important immune regulatory properties. The EGFR has been shown to be of importance in these principal functions, as highlighted in respiratory diseases such as asthma, the most common condition recognised to be affected by particulate air pollution [19
]. In this first in vivo
study examining the involvement of EGFR in the human airways responses to DE, analyses of bronchial mucosal biopsies demonstrated a significantly increased expression of EGFR in the bronchial epithelium six hours after challenge. This was associated with a significantly increased phosphorylation of the Tyr 1173 auto-phosphorylation site on the EGFR C-terminal. Src was not found to be involved in the EGFR activation as indicated by unchanged phosphorylation of Src Tyr 416 and EGFR Tyr 845. At this time post DE exposure, the EGFR downstream MEK/ERK signalling pathway was also unaffected.
It is recognized that certain transcription factors, which regulate cytokine production and inflammatory responses may be activated by MAPKs and their upstream signalling pathways. This has been confirmed to take place in the human airway epithelium after DE exposure, by analyses of archived human bronchial biopsies [15
]. The nuclear expression of phosphorylated p38 MAPK was significantly upregulated, together with increased nuclear translocation of p-JNK MAPK and c-jun of the AP-1 complex. Additionally, the nuclear expression of NFκB was increased. These transcription factors are known to control the production of a wide range of cytokines. DE exposure has been shown to enhance the production of IL-8 and Gro-α in the bronchial epithelium, accompanied by a pronounced neutrophil influx in the bronchial wall. This neutrophilia was mediated by the upregulation of the vascular adhesion molecule expression of P-selectin and ICAM-1 [10
]. Furthermore, a DE mediated increase in the bronchial epithelial expression of IL-13 has been reported [9
]. When considering upstream signalling and regulation of these signal transduction pathways, it is clear that EGFR could have the potential to regulate, or assist in regulating, all the above mentioned events. Therefore a critical question to address was whether activation of EGFR would occur after inhalation of diesel exhaust, which is a complex mixture of gases and particulates.
Most likely, nitrogen dioxide (NO2
), a major gaseous component in DE does not play any major role in the bronchial mucosal effects of DE in humans [23
]. In contrast to DE, exposure to NO2
at levels similar or higher than employed here, has failed to elicit any bronchial mucosal inflammatory responses [23
]. Indeed, there are several indications in the literature that metal and hydrocarbon induced oxidative stress in the airways is involved in the airway response to DE. Even though the metal content in DEP is usually not as extensive as in residual oil fly ash (ROFA), it is clear that the content of transition metals may contribute to oxidative stress [8
Oxidative stress related to hydrocarbons has de facto
been suggested to be a common denominator for many DEP-induced cellular effects. Reactive oxygen species (ROS) can be produced following metabolism and bioactivation of hydrocarbons by cytochrome P-450 1A1 (CYP1A1). The polar quinones have the capacity to generate oxidative stress by redox cycling. Additionally, the metabolism of semiquinones by NADPH-cytrochrome P-450 reductase followed by autooxidation also results in ROS production [24
]. Development of oxidative stress in the airways following DE challenge has been implied by analyses of human airway lavage samples [8
]. Furthermore, genetic polymorphisms in Glutathione-S-transferases (GSTs) have been indicated to be critical for the defence against ROS and detoxification of DEP. The GSTM1 and GSTP1 genotypes have been confirmed to modify the allergen response by DEP in the nose in human subjects [27
]. There are several studies indicating that EGFR is activated by metals, organic components and oxidative stress, supporting the notion that this receptor tyrosine kinase may play a major regulatory role in the inflammatory response to DE exposure [16
]. While only providing indirect evidence, two recent studies have more specifically investigated the role of EGFR after exposure to DEP in-vitro
]. In these studies, the authors were able to demonstrate that DEP triggered the secretion of amphiregulin, a ligand of EGFR, from bronchial epithelial cells, which could be blocked by ERK and EGFR tyrosine kinase inhibition as well as antioxidant supplementation. Furthermore, DEP quinone compounds have been shown to induce contraction of smooth muscle cells, mediated by activated phospholipase A2. This signalling pathway could be blocked by PTK and EGFR inhibitors. Taken together, these studies demonstrate the ability of DEP to both activate and transactivate EGFR.
EGFR has an extracellular ligand-binding domain, a membrane-spanning domain and a cytoplasmatic protein tyrosine kinase domain with a carboxyl terminal that contains tyrosine residues that undergo autophosphorylation during receptor activation [31
]. Three major tyrosine sites, Y1068, Y1173 and Y1148 and two minor tyrosine sites, Y992 and Y1086, serve as sites of autophosphorylation following ligand binding or transphosphorylation by other stimuli (figure ). These autophosporylation sites function as binding sites for Src homology 2 (SH2) and protein tyrosine binding (PTB) domains of a variety of signalling proteins with enzyme activity such as phospholipase C-γ (PLC-γ), signal transducers such as PI3-K and adaptor proteins such as Growth factor receptor-binding protein 2 (Grb2) and Src-homology and collagen protein (Shc). These create binding sites for SH2 or protein tyrosine binding (PTB) domains of proteins or adaptor molecules that link EGFR activation to the downstream signalling pathway.
Figure 2 Description of possible involved signalling pathways. Schematically description of possible involved signalling pathways in the activation and phosphorylation of EGFR, protein tyrosine kinases and downstream signalling following DE exposure. Extended (more ...)
Following ligand binding, epidermal growth factor (EGF), transforming growth factor-α (TGF-α) and amphiregulin may cause downstream activation of the Ras/MAPK pathway. Importantly, EGFR transduces not only its own ligands, but also various stimuli, such as cytokines via cytokine receptors and/or G-protein-coupled receptor activation, as well as oxidative stress all of which result in transactivation. The increased EGFR expression demonstrated after DE in this study is expected to be related to a ligand – activated receptor and inhibition of endocytosis and degradation of receptor which leads to increased receptor expression.
The observation of enhanced phosphorylation of Tyr 1173 is in accordance with the previously demonstrated DE-induced increase in epithelial expression of NFkB, JNK, c-jun and p38 MAPK together with cytokines under their regulation such as IL-8 and GRO-α. Increased phosphorylated Tyr 1173 can function as a docking site for signalling systems such as PLC-γ or PI3-K (figure , yellow box) and exert oxidative responses following DE exposure, linking to NFkB as well as Ras activation [35
]. Tyr 1173 is suggested to be the primary autophosphorylated site that is involved in the PLC-γ association with EGFR [37
], and that signal transduction across membrane coupled to PI3-Kinase may be involved in the activation of PLC-γ [38
]. The autophosphorylated Tyr1173 also acts as a docking site for Shc (Src-homology and collagen protein) which in turn can bind to Grb2 (Growth factor receptor-binding protein 2) and build Shc-Grb2-Sos complex which leads to downstream activation [39
The adaptor protein Shc binding to Grb2, complex building and subsequence MEK kinase-1 (MEKK1) activation may specifically regulate the demonstrated JNK and p38 MAPK activation (figure blue box). It has been suggested that EGFR mediated JNK signalling is regulated by Shc and a transient interaction of Grb2 and MEKK1 [39
]. In contrast, ERK pathway induction is depending on binding of the Grb2-Sos complex to Tyr 1068 and mediated by MEK1 phosphorylation, which was not increased in the present material [42
]. The ERK pathway was expected to be upregulated in the present diesel challenge scenario, as were the JNK and p38 MAPK pathways. ERK activation transduces proliferative and differentiation responses which could be in demand after diesel challenge. The absence of ERK pathway activation contrasts with that seen in a recent study by Blanchet et al
] who demonstrated PM2.5 and archived DEP to cause specific ERK activation, with amphiregulin secretion, by use of different blocking agents for the MAPK pathways. Amphiregulin is an EGFR ligand known to contribute to GM-CSF release, which can be important for sustaining a proinflammatory response. Analyses of the present biopsy material not only failed to show any DE-induced increase in ERK activation but, as previously reported, GM-CSF expression was unaltered [11
]. The difference between this study and that of Blanchet et al, may be due to a dose threshold effect. Another possibility is the time course of events, since in the current study bronchial mucosal biopsies were sampled at 6 hours after in-vivo DE exposure and Blanchet et al
determined the in-vitro response in the 16-HBE cells after 18 hours and in absence of cooperation with other signalling involved in an in-vivo
system. The question whether the ERK pathway is activated at a later time point after in-vivo diesel exposure in humans in-vivo
, will shortly be addressed in archived biopsies sampled 18 hours post exposure [7
]. Of even more importance, EGFR pathway activation after DE exposure, can also be addressed in archived biopsies from asthmatic subjects [12
], in which EGFR pathways are of major importance in terms of epithelial barrier integrity, airway remodelling and signal transduction [21
To examine the potential role of metals in DE induced EGFR activation, cellular PTK or non-receptor protein tyrosine kinase such as Src were considered. Src can act as co-transducer of EGFR signals and has been demonstrated to be involved in the responses of Zn2+
-induced Ras activation via the EGFR. Src-dependent EGFR signalling has been reported to be mediated by phosphorylation of Tyr 845 and Tyr 1101 [45
]. In this study we did not find any changes either in Tyr 845 (EGFR tyrosine) or Tyr 416, Src related tyrosine (which is an autophosphorylation site on c-Src). Even though a role for Src in transphosphorylating EGFR tyrosine in the time course after a DE exposure in-vivo
cannot completely be ruled out, the present study has not given any support for its involvement at the 6 hour post exposure sampling time.
IL-13 is a Th2
cytokine that has been implicated in allergy and asthma airway inflammation, airway remodelling and bronchial hyperresponsiveness. Increased bronchial epithelial expression of IL-13 has previously been shown after DE exposure in healthy non-atopic subjects [9
]. This response could potentially have been mediated by EGFR downstream, such as via AP-1. Interestingly, IL-13 and EGFR may interact in epithelial and goblet cell regulation. IL-13 has been shown to activate neutrophils, and may by interaction with EGFR, lead to increased goblet cell mucin production and metaplasia [47
]. The present findings could therefore be of particular importance in asthmatic and COPD subjects, who may experience exacerbations after exposure to particulate matter air pollution [1