Abstract
Particles generated from numerous anthropogenic sources have the potential to cause or exacerbate lung diseases, including asthma, bronchitis, and COPD. Fibrotic reactions are a component of all of these pulmonary diseases, and involve the progressive deposition of collagen by pulmonary fibroblasts. The reactivity, toxicity, and fibrogenic potential of particles in the lung depends on a variety of factors including particle size, surface area, and composition. Smaller particles, particularly in the nanosized range, have more toxic and fibrogenic capacity due to a higher surface-to-mass ratio and greater oxidant-generating potential. Composition is also an important determinant in the fibrotic response to particles. Transition metals, bacterial lipopolysaccaride, and polycyclic aromatic hydrocarbons are some of the toxic components of particles that activate intracellular signaling pathways that culminate in the production of profibrotic cytokines and growth factors.
Introduction
Not only can the exposure of the lungs to inhaled particles have numerous pathologic effects on the respiratory system, it can also affect other organ systems. For example, air pollution particle exposure is associated with increased morbidity and mortality due to impact on the respiratory and cardiovascular systems (Schwartz, 1993). Metal particles such as manganese are deposited within the nasal mucosa and rapidly transported to the brain where they exert neurotoxicity (Erikson et al., 2005). In the lung, particles mediate their toxic effects by exacerbating preexisting respiratory diseases, including asthma, bronchitis, and COPD (Pope et al., 1995; Anderson et al., 1998). All of these diseases involve considerable lung remodeling, including fibrotic reactions that are defined by increased numbers of fibroblasts and deposition of collagen by these cells. In general, particle size and composition are critical determinants of reactivity, toxicity, and fibrogenic potential of inhaled particles in the lung.
Particle Size
Particle size is a major factor in determing lung toxicity. Inhaled particles in the 1 to 10 micron size range have the capacity to reach the distal lung where they deposit on the airway epithelium. Much smaller particles in the nanosized range also reach the distal lung after exposure and have more potential to cause tissue injury due to a higher surface to mass ratio and greater capacity to generate reactive oxygen species (ROS). Thus, there is a direct relationship between the surface area, ROS-generating capability, and proinflammatory effects of nanoparticles in the lung. For example, studies with ultrafine and fine titanium dioxide (TiO2) showed that nanosized ultrafine particles (20 nm), when instilled intratracheally, induced a much greater neutrophilic inflammatory response than fine particles (250 nm) at the same mass dose (Oberdorster et al., 2005). Additionally, inhaled nanosized (ultrafine) vanadium pentoxide (V2O5) particles cause more neutrophilic infiltration and inflammation than inhaled fine V2O5 particles (Hahn et al., 2005). On an equal mass basis, nanosized particles may also have the capacity to physically hinder macrophage clearance and function as compared to fine particles (Moss and Wong, 2006).
Particle Composition
A mixture of organic and inorganic agents contribute to the composition of particles, including transition metals released during the burning of petrochemicals (Dreher et al., 1997; Kodavanti et al., 1998), polycyclic aromatic hydrocarbons derived from diesel exhaust (Diaz-Sanchez et al., 2000; Nightingale et al., 2000), and endotoxins from bacterial sources (Becker et al., 1996; Bonner et al., 1998a). Some of these particle constituents and the cellular targets they influence are illustrated in Figure 1. Because it is difficult to study mechanisms of lung fibrosis caused by a complex particle mixture, many studies have focused on a specific particle constituent. For example, the transition metals vanadium and copper were found to mediate cytokine gene expression induced by residual oil fly ash (ROFA) emission source particles and Utah Valley urban air particles (UAP), respectively (Kennedy et al., 1998; Dye et al., 1999). LPS appears to be a major constituent of UAP from several major cities, including Mexico City (Becker et al., 1996; Osornio-Vargas et al., 2003). Polycyclic aromatic hydrocarbons (PAH) in diesel exhaust particles (DEP) are major constituents of these particles that cause inflammatory responses (Boland et al., 1999; Terada et al., 1999). Therefore, the toxicity of a given particle depends on source and composition of organic and inorganic constituents.
Particle-Induced Oxidative Stress
Oxidative injury is thought to play a major role in lung fibrogenesis. A variety of organic and inorganic constituents of particles have the potential to cause ROS generation, either directly via redox chemistry or through stimulating pulmonary cells to increase ROS production. Inhaled particles that deposit within the distal lung have the potential to generate ROS, depending on the surface area of the particle and the reactive composition of the particle. Many transition metals present on particles serve as catalysts for ROS production. ROS are produced by Fenton-like reactions, or by stimulating an oxidative burst in cells that have taken up metal-containing particles through phagocytosis. For example, residual oil fly ash particles rich in vanadium induce oxidant generation in alveolar macrophages and epithelial cells (Kadiiska et al., 1997; Goldsmith et al., 1998; Hiura et al., 1999). Diesel exhaust particles (DEP) and lipopolysaccharide (LPS) from gram negative bacteria have also been shown to stimulate generation of ROS in alveolar macrophages. Phagocytosis of DEP by macrophages induces rapid oxidant generation leading to activation of apoptotic pathways (Hiura et al., 1999). Additionally, semi-quinone radicals derived from combustion of DEP mediate redox cycling and sustained production of ROS (Squadrito et al., 2001). Extraction of PAHs and quinones from DEP reduces ROS generation and apoptosis, indicating that organic constituents of DEP mediate these effects.
ROS generated by particle exposure serve as signaling intermediates to activate intracellular signaling targets, including receptor tyrosine kinases, mitogen-activated protein (MAP) kinases, and transcription factors. These signaling intermediates drive transcriptional activation and the expression of genes involved in inflammation and fibrosis. Some of the signaling molecules targeted by particle-derived ROS are illustrated in Figure 1. Increased generation of reactive oxygen species, such as superoxide anion (O.2 −) and hydrogen peroxide (H2O2), has been associated with inflammation following tissue injury (Rosen et al., 1995). In addition to ROS, reactive nitrogen species (RNS) are also generated by particle exposure in the lung. Nitric oxide (NO.) released by inflammatory cells reacts with O.2 − to form peroxynitrite (ONOO−) (Pryor et al., 1995). ONOO− causes nitration of tyrosine residues on proteins and thereby modifies protein function. Nitrated proteins have been identified in lung tissue following exposure to particles and fibers (Rosen et al., 1995; Zhu et al., 1998). Therefore, ONOO− generation and subsequent tyrosine nitration leading to protein dysfunction appear to contribute to disease progression following particle-induced lung injury.
Oxidants serve as signaling intermediates required for receptor tyrosine kinase function and downstream activation of mitogen-activated protein (MAP) kinases. Low levels of oxidants (<10 μM) are essential mediators of normal cell physiologic function, including proliferation, migration and differentiation (Sundaresan et al., 1995; Bae et al., 1997). Quiescent lung fibroblasts in cell culture systems generate micromolar levels of H2O2 that likely maintain their proliferative potential when stimulated with growth factors (Wang et al., 2003). This endogenous pool of H2O2 could also react with constituents of particles to affect cytotoxic responses of cells. Particle-associated metals such as vanadium react with cell-derived H2O2 to form the protein tyrosine phosphatase (PTP) inhibitor, peroxovanadate. PTP inhibition by peroxovanadate is irreversible and results in prolonged MAP kinase activation and cellular stress (Ingram et al., 2003). Therefore, vanadium causes prolonged phosphorylation of intracellular signaling molecules, resulting in cellular stress, altered gene expression patterns, and apoptosis.
Particle-Induced Intracellular Signaling
Particles stimulate pulmonary cells to release inflammatory mediators by activating intracellular signaling pathways that lead to an increase or decrease in gene expression. The reactivity of particles is due in large part to the capacity to generate ROS, which activate receptor tyrosine kinases, MAP kinases and transcription factors such as NF-κB and STAT-1 (Figure 1). The epidermal growth factor receptor (EGFR) is a major target of particle-induced cellular activation (Bonner et al., 2002). ROS reversibly inhibit protein tyrosine phosphatases (PTPs) associated with the intracellular domain of the EGFR. Inactivation of PTPs leads to EGFR phosphorylation and downstream activation of MAP kinase pathways. Vanadium is capable of generating H2O2 and forms peroxovanadate, which is structurally similar to the phosphate molecule and acts as a potent irreversible PTP inhibitor (Samet et al., 1997). Through PTP inhibiton, vanadium is capable of activating the EGFR via a ligand-independent mechanism. Metals also cause the release of cell-associated EGFR ligands to bind and activate the EGFR through ligand-dependent (i.e., ligand-receptor binding) mechanisms. Zinc and vanadium have been shown to activate the EGFR in human bronchial epithelial cells at least in part by cleavage of the membrane-tethered EGFR ligand, HB-EGF (Zhang et al., 2001; Wu et al., 2004). Despite the fact that zinc and vanadium operate through some similar mechanisms, gene profiling experiments have shown that these two metals do not induce identical patterns of gene expression (Li et al., 2005). EGFR activation by particles results in the activation of downstream signaling cascades, including MAP kinases pathways. MAP kinases are pivotal intracellular signaling proteins that function in cell growth, development, and differentiation (Davis, 1995). Three major classes of MAP kinases; extracellular signal-regulated kinases (ERKs), c-Jun-N-terminal kinases (JNKs), and p38 MAP kinases, are all activated by particle-induced cellular stress. Co-activation of MAP kinases may be required for particle-induced increases in growth factor production by lung cells. For example, the co-activation of ERK and p38 MAP kinases were found to be required for vanadium-induced stimulation of HB-EGF by human bronchial epithelial cells and human lung fibroblasts (Zhang et al., 2001; Ingram et al., 2003).
Transcription factors are activated by particles through ROS, metals, or LPS. Vanadium is a strong activator of NF-κB and STAT-1, which mediate the production of proinflammatory cytokines and apoptosis, respectively (Wang et al., 2003). Vanadium has been shown to activate NF-κB in airway epithelial cells through peroxide-induced activation of EGFR and p38 MAP kinase (Jaspers et al., 2000; Wu et al., 2002). Vanadium-induced STAT-1 activation in lung fibroblasts also requires p38 MAP kinase and EGFR (Wang et al., 2003). LPS present on some particles, such as UAP, activates the toll-like receptor termed TLR4 to activate NF-κB (Bowie and O’Neill, 2000; Soukup and Becker, 2001).
Elucidating the intracellular signaling pathways that mediate the various pathologic outcomes associated with particle exposure is complex due to the diversity of constituents associated with particles. Moreover, little is known about the interactive effects of these constituents in stimulating biologic responses. Further research is needed to better understand how mixtures of metals and/or organic consitituents (LPS, PAH) interact to stimulate cell signaling and the production of inflammatory mediators.
Particle-Induced Expression of Pro-Fibrotic Cytokines and Growth Factors
A variety of growth factors and cytokines have been implicated in the pathogenesis of fibrosis. A variety of air pollution or emission source particles stimulate the release of IL-1β and TNF-α. While these cytokines do not directly promote fibroblast growth or the deposition of extracellular matrix proteins, they increase the expression of pro-fibrotic growth factors and their receptors. For example, TNF-α stimulates the production of TGF-β1, a major stimulator of collagen deposition (Sime et al., 1998). TGF-β1 also causes increased production of connective tissue growth factor (CTGF), which further increases the fibrotic response. IL-1β increases the expression of PDGF-AA and its receptor, PDGF receptor-α (PDGFRα), on lung fibroblasts (Raines et al., 1989; Lindroos et al., 1997). The increased expression of PDGFRα by IL-1β has been implicated in the progression of particle and metal-induced airway fibrosis. PDGFRα is up-regulated in vivo following the intratracheal instillation of ROFA or vanadium pentoxide (V2O5) in rats (Lindroos et al., 1997; Bonner et al, 1998b). Vanadium-induced oxidative stress first causes IL-1β release by alveolar macrophages, which then stimulates increased PDGF-Rα expression. Air pollution particles from Mexico City increase PDGF-Rα on lung fibroblasts primarily through LPS adsorbed to the particles (Bonner et al., 1998a). Purified E. Coli LPS is a potent inducer of the PDGF-Rα (Coin et al., 1996). The secretion of PDGF isoforms are increased by a variety of different particles and fibers. Macrophages stimulated with particles or fibers release PDGF-B chain isoforms (PDGF-AB and PDGF-BB), while fibroblasts produce PDGF-AA (Bonner et al., 1991; Lasky et al., 1995). Therefore, the proliferation of lung fibroblasts during particle-induced fibrosis likely involves the coordinated secretion of PDGF isoforms and increased levels of PDGF-Rα. In general, particle exposure activates numerous cytokine/growth factor cascades that lead to fibrotic reactions in chronically exposed individuals.
Particle-Induced Exacerbation of Asthma and Airway Fibrosis
Exposure to certain types of particles increases symptom severity in allergic asthmatics (Salvi and Holgate, 1999). Asthma pathogenesis involves an immune response that features the recruitment of eosinophils and production of Th2 type cytokines, including IL-13 and IL-4 (Wills-Karp et al., 1998). For the most part, particle studies in mouse models have utilized a single exposure strategy that does not result in increased Th2 cytokines. However, under the appropriate experimental conditions wherein mice are pre-exposed to a sensitizing agent, particles have the ability to increase Th2 cytokines. For example, residual oil fly ash particles increase Th2 cytokine production, eosinophil recruitment, and airway hyperresponsiveness in mice sensitized to ovalbumin (Gavett et al., 1999). DEP enhances the production of Th2 cytokines, including IL-13, in individuals exposed to ragweed allergen (Diaz-Sanchez et al., 1997). In turn, IL-13 stimulates the expression of profibrotic growth factors, including TGF-β1, a potent stimulator of collagen deposition (Lee et al., 2001), and PDGF-AA, a strong inducer of fibroblast growth (Ingram et al., 2004). Therefore, IL-13 appears to be a key mediator of particle induced fibrosis that contributes to fibroblast replication and collagen deposition. Overall, these studies suggest that particles exacerbate allergic asthma at least in part by enhancing Th2 cytokine production.
Epithelial-Mesenchymal Cell Interactions in Particle-Induced Bronchitis
Airway fibrosis is a common feature of asthma, chronic bronchitis, and COPD. It is likely that epithelial-fibroblast interactions are important in mediating the pathogenesis of airway fibrosis. Communication between the airway epithelium and underlying fibroblasts and smooth muscle cells via paracrine signaling is referred to as the “epithelial-mesenchymal cell trophic unit.” Epithelial-mesenchymal cell interactions are thought to play an important role in the pathogenesis of asthma (Holgate et al., 2000). A variety of cytokines, growth factors, and lipid mediators produced by the epithelium activate mesenchymal cells to proliferate, differentiate or produce their own soluble mediators. Air pollution particles also activate the epithelium to produce inflammatory mediators that likely activate the underlying mesenchymal cells. However, the majority of studies with particles have focused on cultured epithelial cells and have not directly addressed the issue of epithelial-mesenchymal cell communication.
The epithelial-mesenchymal cell trophic unit does appear to be important in V2O5-induced lung injury and the subsequent development of airway fibrosis. Exposure of rats to V2O5 causes airway remodeling similar to that seen in humans with chronic bronchitis arising from occupational exposure to V2O5-rich particles (Bonner et al., 2000; Woodin et al., 2000). The pathology of V2O5-induced lesions in rats includes airway fibrosis, smooth muscle thickening, and mucous cell metaplasia (Figure 2). Experiments with human bronchial epithelial cells exposed to V2O5-induced oxidative stress in vitro show that these cells release heparin-binding epidermal growth factor (HB-EGF), a potent mitogen for human lung fibroblasts (Zhang et al., 2001). HB-EGF binds to the EGFR on fibroblasts and epithelial cells, thereby promoting proliferation of fibroblasts and differentiation of epithelial cells, respectively. Moreover, pharmacologic inhibition of the EGFR reduces V2O5-induced fibrotic responses in rats (Rice et al., 1999). Therefore, HB-EGF appears to be important to airway fibrogenesis and remodeling after exposure to particles rich vanadium. In general, the combined use of in vivo rodent models of V2O5-induced airway remodeling and in vitro cell culture systems with epithelial cells and fibroblast have proven useful in dissecting the cellular and molecular mechanisms that mediate V2O5-induced occupational bronchitis.
Summary
The potential of particles to cause fibrotic reactions within the lung depend on the both particle size and composition. Particles containing certain transition metals, such as vanadium and zinc, have a greater capacity to generate ROS and cause tissue injury. Decreasing particle size is also a critical determinant of toxicity due to the greater surface area of an equal mass of smaller particles. ROS generated by particle exposure activates intracellular signaling proteins, including MAP kinases and redox-sensitive transcription factors. These activated molecules play key roles in cell survival and the production of growth factors that drive fibrotic reactions within the lung following exposure to toxic particles.