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International limflunopharmacolagy ELSEVIER Internal!onat immunopharmacology 2 (2002] 191 -200 www.elsevier.cora/locatc/intirap Review Diseases caused by asbestos: mechanisms of injury and disease development Christopher B. Manning3, Val Vallyathanb, Brooke T. Mossmana'* `Department of Pathology, College ofMedicine, University of Vermont, Soule Alum. Bldg. A-I45, Burlington. VT 05405. USA bPathology and Physiology Research Branch. Health Effects Laboratory Division. National Institutefor Occupational Safety and Health, Morgantown, WV 26505, USA Abstract Asbestos is a ubiquitous, naturally occurring fiber that has been linked to the development ofmalignant and fibrotic diseases of the lung and pleura. These diseases may be initiated by injury to epithelial cells and mesothelial cells by asbestos fibers through the formation of reactive oxygen intermediates. Elaboration of oxidants are also a consequence of inflammation, a hallmark of exposure to asbestos after inhalation or injection of asbestos fibers into animals. The type, size, and durability of asbestos fibers may be important in toxicity and pathogenicity of asbestos types. This review discusses the pathways of oxidant generation by asbestos fibers, cell-cell interaction that may initiate and peipetuate inflammation, cytokine release and proliferative responses to asbestos, and cell signaling pathways implicated in these events. 2002 Published by Elsevier Science B.V, . Keywords: Asbestos; Cancer; NF-k.8 1. Introduction Epidemiologic and animal studies indicate that in halation of asbestos can result in pulmonary fibrosis, lung cancer, and mesothelioma. Occupational expo sure to asbestos has been associated with the develop ment of pulmonary fibrosis (asbestosis), lung cancer, malignant mesothelioma, and other pleuropulmonary disorders [l-3a,b]. The National Institutes of Health in 1978 estimated that approximately 11 million individuals had been exposed to asbestos in the * Corresponding author. Tel.: +1-802-656-0382; fax; +1-802 656-8892. E-mail address: bmossman@zoo.uvin.edu (B.T. Mossman). United States since 1940 [4]. The Work-Related Lung Disease Surveillance Report, published by the Na tional Institute for Occupational Safety and Health (NIOSH) in 1999, determined that between 1987 and 1996, the total number of deaths associated with as bestosis was 96 J 4, of which 5207 were from malig nant neoplasms of the pleura [5], Due to these health concerns, the use of asbestos has been limited or prohibited in the United States and in several other countries, but in developing countries, the use of asbestos continues to increase [6]. The mechanisms of injury and disease develop ment caused by asbestos fibers are presumed to be related to the greater fibrogenic and carcinogenic properties in comparison to other minerals. The exact mechanisms of injury by asbestos fibers to cells of 1567-5769/02/$ - see front matter 2002 Published by Elsevier Science B.V. Pit: 51567-5769(01)00172-2 192 C.B. Manning et al 1International Immunopharmacotogy 2 (2002} 191-200 the lung and pleura arc unclear, but generation of oxidants by fibers due to their surface redox proper ties or upon interaction and uptake of fibers by cells has been shown to be an important factor in many cellular responses to asbestos [7-10]. Inflammation observed both in animal models and in the lungs of patients with asbestos-related lung disease may be an other source of reactive oxygen and nitrogen species (ROS/RNS). In this article, we first describe briefly the diseases caused by asbestos, their chemical and phys ical properties, pathways of oxidant generation by fibers in vivo and in vitro, and how these may relate to the development of asbestos-associated pathologies. We then discuss the role ofasbestos and oxidants in the stimulation of cytokines and pro-inflammatory medi ators, describing how cell-cell interactions may be im portant to the initiation of diseases and/or repair of lung injury. Lastly, we provide an overview ofcell sig naling pathways, activation of transcription factors, and gene expression implicated in the development of inflammation, fibroproliferahve diseases, and cancers induced by asbestos. 2. Diseases caused by asbestos 2.1. Asbestosis Development of bilateral diffuse interstitial pulmo nary fibrosis is caused by the inhalation of asbestos fibers [1,1-1], The most common clinical symptoms of asbestosis are dyspnea on exertion with progression over time leading to restrictive impairment and de creased diffusing capacity. Radiographically, the dis ease is usually confined to the lower zones of the lungs as rcticulonodular infiltrates with the presence of calcified pleural plaques suggestive of exposure of asbestos. High resolution computer tomography (CT) scans arc sometimes helpful in the diagnosis of subtle parenchymal changes, although the results are often debatable. Gross pathology reveals bilateral interstitial fibro sis involving the lower zones of the lungs with severe disease closer to the pleura. In advanced asbestosis, honeycombing is common [1], Moderate or initial subtle stages of asbestosis can only be identified by critical light microscopic evaluation of the lung by several representative sections of the lung. For the pathologic diagnosis of asbestosis, it is mandatory to demonstrate discrete foci of fibrosis in the walls of respiratory bronchioles associated with the presence of asbestos bodies or fibers which are coated with iron and protein. A history of exposure to asbestos is sup portive evidence to document disease with at) etiologic association of a specific fiber type in the lung tissue and in an air sample from the work site. 2.2. Pleural plagues ' Pleural plaques appear as white or yellow smooth surfaced lesions on parietal, visceral and diaphrag matic pleura. They are often considered hallmarks of exposure to asbestos or other fibrous materials [12]. They can become extensive with calcification and en case the lungs. Pleural thickening and or plaques can impair lung function [13]. Microscopically, pleural plaques are acellular, nonvascular dense strands of hyalinized collagen showing a "basket-weave" pat tern of mesh appearance. On the pleural surface, it is covered by a single layer of mesothelial cells. Asbes tos bodies are very uncommon, and occasional fibers can be found by critical evaluation. 2.3. Bronchogenic carcinoma An increased risk for developing lung cancer in workers with heavy occupational exposure to asbestos and a dose-response relationship between asbestos exposure and cancer incidence has been well docu mented [14,15].. However, the presence of asbestosis and the severity and extent ofexposure required for an excess lung cancer risk are disputed especially when cancer risk is not observed for 10 or more years from initial exposure, whereas with longer exposures, tocreased risk is evident On the other hand, it was shown that radiographic asbestosis is not a prerequisite for asbestos-associated increased lung cancer risk [16,17]. In a consensus report by the Helsinki Criteria for Diagnosis and Attribution on Asbestos, Asbestosis, and Cancer, it wag stated that it is not necessary to demonstrate asbestosis on the chest X-ray or to biopsied tissue to order to attribute a causal role to asbestos to cases of lung cancer [18]. Cigarette smoking and asbestos exposure have additive or synergistic interactions to inducing cancer ofthe lung [2,19]. Compared to cigarette smokers with C.B. Manning ei al. / International Immunopkarmacology 2 (2002) 191-200 m , no asbestos exposure, there is a substantial increase in f mortality rate in cigarette-smoking asbestos workers ! [19,20]. : Histological cell types associated with lung cancer in asbestos workers are similar in cellular features to other primary cancers of the lung. In a number of studies, a predominant incidence of adenocarcinomas has been reported with an increased frequency of oc currence in the peripheral and lower lung zones [21 24]. However, there is no consistent reliable evidence on the association of asbestos exposure and genesis of a specific cell type and site or lobe of tumor origin. Recent studies have shown no difference between can cers associated with asbestos exposure and those as sociated with cigarette smoking [25], types of cellular features observed in mesotheliomas such as epithelial, sarcomatous, and mixed types show ing different cellular patterns. Definitive diagnosis of mesothelioma is made on the basis of, gross appear ance, histological examination, and with the aid of special immunohistochemical stains and/or electron microscopy. There are a number of other disorders presumed to be associated with or implicated to asbestos exposure. These include benign pleural effusions, generalized pleural thickening, carcinoma of the larynx, carcino mas of the gastrointestinal tract, small airway disease; Caplan's syndrome, honeycombing, and bronchiecta sis. These topics are outside the scope and purpose of this review. 2.4. Mesothelioma Diffuse malignant mesotheliomas of the pleura and peritoneum are well associated with asbestos exposure. However, in addition to asbestos, there are idiopathic and other accepted causes of mesothelioma including fibrous minerals such as erionite, radiation, pleural scarring, and SV 40 virus. Overall, there is an increased prevalence of pleural mesothelioma and lower prevalence of peritoneal mesothelioma in asbes tos-exposed workers [1], Peritoneal mesotheliomas are predominantly found in heavily exposed asbestos workers. Asbestos-induced mesothelioma has a long latency period, usually 30 or more years, and the la tency increases with lower levels of exposure [26], Unlike carcinoma associated with asbestos exposure, mesothelioma is not associated with cigarette smoking [3b, 14], The disease potential to induce malignant meso thelioma varies significantly between amphiboles and chrysotile. Commercial amphiboles like amosite, cro- cidolite, or tremolite have the greatest potential and chrysotile the least potential to induce mesothelioma. In millers and miners with heavy chrysotile exposure, pleural mesotheliomas were reported [3b,24]. How ever, the potential to induce mesothelioma by chrys otile is debated because there is lung fiber analysis evidence suggesting that tremolite, a component of the ore, is the actual etiologic agent [27], Malignant mesothelioma rapidly spreads over the surfaces of the lung, thoracic, and abdominal cavities. Metastasis to other organs is very rare, There are three 2.4.1. Physical and chemical characteristics of asbestos Asbestos is a group of naturally occurring crystal line fibrous silicate minerals used extensively in the past due to their valuable insulating and electrical properties, and in other uses requiring high tensile strength and/or chemical and heat resistance. Its ex tensive use in over 3000 commercial products, includ ing roofing materials, floor tiles, cement pipes, and textiles, has resulted in wide distribution of asbestos in public places and. the environment. There are six commercially used types of asbestos derived from two groups: serpentines (curly fibers) and amphiboles (straight fibers). Chrysotile, a serpentine asbestos, is the only serpentine asbestos commercially used and has many industrial applications worldwide. It is very flexible, curly and heat resistant, but is damaged in acid environments. Chrysotile fibers are made of fib rils with a layered silicate structure formed of cylin drical tubes. It was estimated that more than 95% of commercially used asbestos in North America in the past was chrysotile, and most of it was derived from Canadian and Russian mines [1,28]. Amphiboles (amosite, crocidolite, tremolite, actinolite, and anthophyllite) on the other hand are straight, needle-shaped, not pliable, relatively acid-resistant fibers. These types of asbestos were used extensively as insulation against seawater corrosion and as a fire retardant in ships and other applications. The basic subunit of amphibole asbestos is a silicon dioxide tetrahedron arranged in parallel chains and linked late rally by cations. Among the five amphiboles, amosite 194 C.B. Manning et al. /International Immunopharmacology 2 (2002) 191-200 and crocidolite were used extensively in North Amer ica and the United Kingdom and were imported from South Africa, In North America, amosite was used extensively in shipyards, while in the United Kingdom, crocidolite was preferentially used in several commer cial applications. Other amphiboles (actinolite, anthophyllite, and tremolite) are not extensively used com mercially due to the lack of huge deposits, except for antbophyllite in Finland. . The most commonly used two amphiboles, amo site and crocidolite, have chemical and physical com positions, shapes, sizes, durability, and pulmonary penetration abilities disparate from that of chrysotile. The higher potency of these two types of asbestos to increase health risk is correlated with the fiber gra dient or ampbibole hypothesis for disease develop ment [8], 2.4.2, Surface properties and oxidant generation by asbestos Toxicity, fibrogenicity, and carcinogenicity of as bestos fibers are dependent on many chemical and physical properties of the fibers. Biopersistence as well as chemical and physical characteristics play important roles in toxicity, pathogenicity, and carci nogenicity. Durability of asbestos fibers has a direct correlation to toxicity and pathogenicity. Clearance and dissolution kinetics of amphibole fibers in ani mal studies and occupationally exposed populations are slower than chrysotile fibers [29]. Elements, such as magnesium and iron are known to leach from inhaled fibers mediating toxicity through redox reac tions. Leaching of elements from chrysotile asbestos changes its surface charge from positive to negative and reduces potential toxicity [30]. Because of the leaching of elements from chrysotile fibers, the re lative toxicity and pathogenicity of these fibers has been suggested to be relatively low compared to that for amphiboles. Several studies have suggested the presence of transition metals on the fiber surface is associated with the ability of fibers to generate ROS and induce injury [31-34]. The generation of ROS by different types of asbestos and cellular interactions have been well documented [33,35-38]. The results show that generation of ROS* from the interaction of phagocytes with target cells, correlates with toxicity and patho genicity of asbestos types. In general, asbestos fibers. which contain more iron and longer fibers, have been shown to generate more ROS. Iron-dependent ROS generation from fibers results in the generation of hydroxyl radicals through file Fenton reaction and die Haber-Weiss cycle. In addition, fiber length and biopersistence are important in ROS generation and the resulting toxicity and pathogenicity. Experimental studies suggest that "frustrated phagocytosis" appears to have a dramatic influence on the sustained gener ation of ROS [33,38], Repeated "frustrated phagocy tosis" would be expected to attract more phagocytes, resulting in an enhanced generation of ROS. 3. Oxidant release and pro-inflammatory mediators . The pathogenesis of asbestos-associated diseases is associated with a persistent inflammatory response initiated directly or indirectly by ROS, cytokines, chemokines, growth factors, and pro-inflammatory factors. These secretions trigger activation of tran scription factors and mitogen-activated protein kinases (MAPK), which are linked to early response genes. Pro-inflammatory gene responses appear to be regu lated at the transcriptional level by DNA binding proteins that are under the influence of ROS. Modu lation of several transcription factor-mediated, proinflammatory responses has been shown in cell sys tems and animal models. Time and dose-responsive increases in nuclear factor-kappa B (NF-kB) activation following exposure of cells to different types of as bestos have been demonstrated (see below). The in creases in NF-kB activation can be accompanied by increases in tumor necrosis factor-alpha (TNF-a) pro duction by the exposed cells. TNF is reported to be an important mediator of pulmonary fibrogenesis [29]. Asbestos fibers induce release of TNF-a from macro phages and cultured cells, which is mediated by ROS and dependent on fiber length [39,40]. Increased re lease of TNF has also been shown in animals exposed to crocidolite asbestos [41,42] and in humans exposed in the workplace [43], The ROS production initiated by inhalation of as bestos contributes to the inflammatory response that is thought to play a key role in the development of fi brosis. TNF, a cytokine agonist for chemotactic chemokine production, is upregulated in alveolar ma- **** I ....C.B. Manning el al. I International Immunopharmacology 2 (2002) 191-200 ' crophages exposed in vitro to crocidolite or chrysotile 4. Activation of cell signaling pathways in 195 ? asbestos [44]. The mechanism of TNF induction in inflammation and fibroprollferative responses macrophages following asbestos exposure involves the activation of NF-k,B by ROS '[39]. While TNF is not In vitro experiments have shown that the mitogen- by itself chemotactic for neutrophils or macrophages, activated protein kinase (MAPK) cascade is involved at least three TNF-inducible chemotactic chemokines in both apoptotic and proliferative responses to as are thought to play a role in the recruitment of in bestos. The extracellular signal-regulated kinases flammatory cells to the site of asbestos exposure: (ERKs), c-Jun N-terminal kinases (JNKs), and p38 macrophage inflammatory protein-2 (MIP-2), interleu kinases are all important components of the MAPK kin-8 (IL-8), and cytokine-induced neutrophil chemo cascade, which plays a role in cellular responses to a attractant (OINC) [45]. Additionally, asbestos ex variety of stimuli [55]. Activation of MAPK pathways posure induces production ofIL-1 and 1L-6, in alveolar can lead to a number of outcomes including cell pro macrophages (AMs) [46]. Though both of these cyto liferation, cell survival, or apoptosis. Western blots kines are involved in the recruitment of inflammatory performed on rat pleural mesothelial cells (RPMs) cells, IL-6 also encourages fibroblast proliferation have shown that treatment with crocidolite asbestos [45], While TNF encourages the production of IL-6, stimulates the phosphorylation of the ERK isofoims there is also evidence that the cellular redox state plays designated as ERKl and ERK2, enzymes that play a a direct role in the induction-of IL-6 [47]. The de- role in cellular proliferation under some circumstances : monstration, using antibodies directed at TNF and em [56,57]. Kinase assays performed on crocidolite-trea- ploying IL-1 receptor antagonists, that the neutral ted cells confirmed that the phosphorylation of ERKl ization of these mediators abrogates the development and ERK2 corresponded with an increase in ERK of asbestos-induced fibrosis in mice provides convinc activity. Further work showed that the compound ing evidence for a critical role for these mediators in tyrphosrin AG 1478, a specific inhibitor of the tyrosine initiating the inflammatory response involved in asbes kinase activity of EGFR, blocked phosphorylation and tos-induced pathology [48,49], Other evidence activation of these enzymes by both EGF and croci strongly implicating TNF in the pathogenesis of as- dolite. Treatment with AG1478 has also been shown bestosis comes from in vivo models utilizing trans to prevent the induction of the early response proto genic mice. Mice overexpressing TNF in type II al oncogene ofos, thought to be important in asbestos veolar epithelial cells spontaneously develop fibrosis toxicity, thus providing further evidence that EGFR similar to that seen in asbestosis [50]. Furthermore, is involved in responses to asbestos [58]. In vitro expe knockout mice lacking the TNF receptor produce riments, using alveolar type II epithelial cells (CIO), increased levels of TNF when exposed to chrysotile have established that EGFR and ERK also play a role asbestos, but do not develop the fibrotic lesions seen in in initiating cell cycle alterations in lung epithelial wild-type mice [51). cells [55). In vivo studies have also suggested a role for The exact nature ofthe interaction between asbestos transforming growth factor (TGF) in the development and EGFR is unknown, but a variety of methods have of asbestosis. Both TGF-(3 and TGF-ot are produced in provided some clues. Using fluorescence microscopy, bronchoalveolar duct regions of developing asbestotic it has been shown that crocidolite blocks the interac lesions [52,53]. The spontaneous development of tion of EGF and its receptor [58], While both crocido fibrotic lesions resembling those found in asbestosis lite treatment and EGF cause increased levels ofEGFR in transgenic mice overexpressing TGF-a in the lungs protein and mRNA, EGF causes an increase in degra further implicates TGF in the pathogenesis of asbes dation of EGFR whereas crocidolite does not. It has tosis [54]. There are a number of other cytokines pro also been found that the increase in EGFR protein posed as putative mediators in the inflammatory expression correlates with the carcinogenicity of var response to asbestos; however, more research is need ious mineral fibers [59], Experiments using confocal ed to characterize what part, if any, these mediators scanning laser microscopy demonstrated that immu- play in the pathogenesis of asbestos-related disease [29], noreactivity to EGFR in cells exposed to crocidolite asbestos is concentrated around the contact area m C.B. Manning et al. / International Immunopharmacolagy 2 (2002) J91-200 between the fiber and the cell [60]. The same technique has also demonstrated that crocidolite fibers longer than 60 pm are associated with immunoreactivity to EGFR much more often than smaller fibers. In vivo studies have also suggested a role for ERK in asbestos-induced damage. Immunoperoxidase stain ing oflung sections from animals exposed to chrysotile asbestos in inhalation experiments have demonstrated that immunoreactivity to phospho-ERK is concen trated within pulmonary epithelial cells at sites of developing fibrotic lesions after both 14 and 30 days ofasbestos exposure [57]. These studies are the first to demonstrate activation of the MARK cascade in key cell types of fibrosis and carcinogenesis. 5. Activation of transcription factors and early response genes The cell signaling pathways activated by asbestos are of importance as they initiate the transactivation of genes that may be critical to the development of inflammation and/or fibroproliferative diseases of the lung and pleura, including .fibrosis, mesothelioma, and lung cancer. Thus, knowledge of these signaling pathways and their relationship to the activation or inactivation of transcription factors and gene expres sion critical to changes in cell phenotype and function are critical to the design of strategies for prevention and intervention of asbestos-related diseases. Thus far, two classical transcription factors, activator protein-1 (AP-1) and NF-kB, have been studied in our labo ratories in both cells and rodent models after expo sures to asbestos. 5.1. Activator protein-1 (AP-1) AP-1 is a family of transcription factors comprised of homo- and heterodimers of the Jwn and Fos family of proteins. As summarized above, these proteins are phosphorylated and activated by MAPK signaling cascades [61,62], We first showed in both rodent tracheal epithelial and mesothelial cells that asbestos and erionite, a type of fiber that morphologically re sembles crocidolite asbestos and causes mesothelioma in both rodents and humans, induced increased mRNA levels ofthe early response proto-oncogenes, c-fos and c-jun, in contrast to a number of nonpathogenic fibers and particles (glass, polystyrene beads, riebeckite, and antigorite) [63--65]. Since complexes of Jun and Fos family members interact with regulatory DNA sequen ces known as TPA (12-O-tetradecanoylphorbol 12myristate 13-acetate) response elements (TREs), we explored, using electrophoretic mobility shift analyses (EMSAs), the formation of AP-1 complexes in these cells by asbestos, TPA, and H202 [63,64], Studies revealed that asbestos fibers, particularly crocidolite, in contrast to other stresses, caused delayed and protracted expression of early response genes and AP-1 binding to DNA. In mesothelial cells, inhibition of protein kinase C or its down modulation by phorbol ester dibutyrate inhibited crocidolite asbestos-induced elevations in c-fos and c-jun mRNA levels, whereas tyrosine kinase inhibitors were effective in decreasing c-fos, but not c-jun, levels in these cells [66], We also demonstrated AP-1-dependent gene transactivation by H202 and crocidolite asbestos in tracheal epithe lial cells as well as a functional role for c-jun in cell proliferation and transformation using transient trans fection techniques [67]. Induction of c-jun and c-fos by asbestos is oxidant-related and ameliorated when stores of intracellular glutathione are increased [64], A murine AP-1 luciferase stable type II epithelial cell line developed in this laboratory has recently allowed confirmation of elevated AP-1-dependent gene expression by silica and asbestos, and revealed other members of the Fos/Jun family, i.e. Fra-1, which are oxidant-inducible and important in AP-1 complex formation governing cell cycle alterations [68], A critical question is whether in vitro increases in Fos/Jun expression and AP-1 transactivation occur in rodent lungs after in vivo exposures to asbestos. In comparative studies using rats exposed to crocidolite or chrysotile asbestos by inhalation, increased c-jun in lung homogenates was observed by Northern blot analyses in crocidolite-exposed rats which subse quently developed pulmonary fibrosis [69,70], We subsequently showed, in a murine inhalation model using AP-1 luciferase reporter transgenic mice [71] and anti-luciferase antibody, that exposures to croci dolite caused increases in AP-1-dependent gene ex pression in bronchiolar and alveolar type II epithelial cells [62], Further studies at the National Institute for Occu pational Safety and Health, on a well characterized C.B. Manning et at. / International Immunopharmacology 2 (2002) 191-200 197 mouse epidermal JB6+ cell line system for studying tumor promotion and neoplastic transformation responses, and in mice canying the TRE-luciferase transgene mice with AIM luciferase reporter, showed that crocidolite asbestos activates AP-1 through the generation of ROS [72,73], Asbestos exposure caused a time- and dose-dependent activation of AP-1 in cultured JB6+ cells and transgenic mice, which was inhibited by OH radical scavengers. These studies suggest that the carcinogenic effect of asbestos may be mediated through the generation of OH leading to the induction of AP-1 activation. Studies using trans genic animals further support this hypothesis because when the AP-1 activation increased 10-fold in lung tissue, the corresponding increase in the bronchial tissue was significantly higher (22-fold), suggesting that AP-1 activation may play a pivotal role in cancer development. The induction of AP-1 activation appears to be mediated through MAPK family mem bers such as ERK 1 and ERK 2 [72,73]. Thus, increases in AP-1-dependent gene expression by asbestos in epithelial cells of the lung are likely tq be important in the epithelial cell proliferation ob served after inhalation of asbestos [74]. A key area of future investigation is to determine, the causal relation ship of AP-1-dependent gene expression to asbestosinduced cell proliferation, fibrosis, arid cancers. In addition, identification of AP-1 regulated genes that are critical to these processes is vital to understanding the pathogenesis of cell injury, proliferation, and repair after exposures to asbestos, 6. NF-kB The NF-kB transcription factor family is activated in response to physical and oxidative stress, mitogens, microbial products such as endotoxin, and inflamma tory cytokines [75,76], Like AP-1, NF-kB is com prised of protein dimers, including the transcription activating heterodimer consisting of p50 and p65 (RelA) subunits. NF-kB activity is controlled by members of the IttB family which bind directly to NF-kB dimers in the cytoplasm, preventing its nuclear localization which is required for DNA binding. Upon stimulation by oxidants, IkB family members are phosphorylated at specific serine residues, causing the dissociation of KB, which is subsequently ubiq- uitinated and degraded. Thus, the NF-kB nuclear localization sequence enters the nucleus and interacts at specific sites, i.e. kB motifs. DNA binding then leads to recruitment of essential components allowing transcription and enhanced expression of genes prox imal to the kB motif. These include a number of inflammatoiy chemokines and cytokines that are cau sally related to inflammation and asbestosis [29], as well as cell adhesion molecules, growth factors, etc. that are associated with fibrogenesis and carcinogen esis. In addition, NF-kB activation causes increased cell survival and anti-apoptosis in a number of cell types [76]. . We first showed that crocidolite asbestos caused protracted and dose-related increases in proteins bind ing to nuclear NF-kB binding DNA elements in tracheal epithelial cells. NF-kB binding to DNA was decreased by elevation of intracellular glutathione levels [77]. Transient transfection assays with a con struct containing NF-KB-binding sequences linked to a luciferase reporter gene showed that asbestos in duced transcriptional activation of NF-kB regulated genes. Some of these have subsequently been identi fied as c-myc [77] and iNOS [78], After inhalation of crocidolite asbestos, increases in immunolocalization of the p65 transcriptionally active subunit were ini tially observed in the epithelial cells of rats in both the distal bronchioles aDd at sites of developing inflam matory and fibrotic lesions [79]. With increasing time of exposure to asbestos, immunostaining increased in fibrotic lesions. This indicated a gradual pattern of increased NF-kB activation occurring first in prolif erating epithelial cells, and later identified as key to the initiation of inflammatory responses after inhala tion of asbestos or silica [29]. 7. Summary and relationship to the development of asbestos-associated fibroproliferation and cancers An overall hypothetical schema relating the cellu lar signaling events and outcomes of asbestos-induced exposures to inflammation and die development of asbestos-related diseases is provided in Fig. 1. Studies in our laboratories have shown that multiple signaling pathways and transcription factors are activated by asbestos fibers through oxidant-dependent pathways involving the elaboration of oxidants from redox 198 C.B. Manning el al. /InternationalImmunophamacofagy 2 (2002) 191-200 ASBESTOS l BOS kcBBK&innases AW activation kB p53 Inactivation Acnvakon . oe Activation Early response genes Inflammatory aeries Z&ssfEsT f-------- Cytokines TNF-O.IL-1J5 IL-6.IL-1I 1L-4, TL-5 Pro-lnfiaiwnatpry Mediators T, Chcmoldnes MIP-lo M1P-3 IL-8 * Enzymes iNOS. PLA-2 5-LO, COX-2 i Ad-Md ICAM-1 v CAM-2 E-Seleciin Fig. 1. Activation oftranscription factors and related gene expression by asbestos, ROS have been shown to be important mediators of asbestos-induced cellular events in a number of cell types. reactions on the fiber surface driven primarily by iron. In addition, production of oxidants from cells after interaction with and phagocytosis of fibers during the respiratory burst is a likely source of oxidant produc tion, particularly after "frustrated phagocytosis" of long (> 5 tun) fibers that may cause chronic oxidant elaboration from both epithelial cells and macro phages. Oxidants are known to interact with macro molecules, such as proteins and DNA, and the precise alterations in signaling molecules by asbestos and other oxidant stresses are fields ripe for investigation. From the complex interactions illustrated in Fig. 1, it is likely that a number of signaling pathways coo perate in cellular responses to asbestos. Moreover, these pathways may have different roles in various cell types in different phases of the cell cycle. The ability to study these cascades in transgenic mouse models after inhalation of asbestos and other partic ulates will allow dissection of the key components of these pathways as well as genes that contribute to inflammation and the pathogenesis of asbestos-asso ciated diseases. Acknowledgements Work in Dr. Mossman's laboratory is supported by NIH grants ES/HL09213 and P01HL67004. References [1] Craighead JE, Abraham JL, Churg A, Green FHY, KJciner- man J, Pratt PC, et al. Asbestos-associated diseases. Arch Pathol Lab Med 1982;106:541-96, [2] Mossman BT, Gee JBL. Medical progress. Asbeslos-related diseases. N Engl J Med 1989;320:1721-338. [3] (a) Churg A. Nonneoplastic disease caused by asbestos. In: Churg A, Green FHY, editors. Pathology of occupational lung disease. Baltimore, MD: Williams and Wilkins; 1999. p. 277 338; . . (b) Churg A. Neoplastic asbestos-induced disease. In: Churg FHY, Green FHY, editors. Pathology of occupational lung disease. 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