The Role of Nuclear Factor-κ B in Pulmonary Diseases

impact of basic research on tomorrow’s medicine The Role of Nuclear Factor-␬ B in Pulmonary Diseases* John W. Christman, MD, FCCP; Ruxana T. Sadikot, MBBS; and Timothy S. Blackwell, MD

Nuclear factor-␬ B (NF-␬B) is a family of DNA-binding protein factors that are required for transcription of most proinflammatory molecules, including adhesion molecules, enzymes, cytokines, and chemokines. NF-␬B activation seems to be a key early event in a variety of cell and animal model systems developed to elucidate the pathobiology of lung diseases. The purpose of this short review is to describe what is known about the molecular biology of NF-␬B and to review information that implicates NF-␬B in the pathogenesis of lung disease, including ARDS, systemic inflammatory response syndrome, asthma, respiratory viral infections, occupational and environmental lung disease, and cystic fibrosis. (CHEST 2000; 117:1482–1487) Key words: air pollution; ARDS; asbestosis; asthma; ozone; respiratory syncytial virus; rhinovirus; sepsis; silicosis Abbreviations: ALI ⫽ acute lung injury; CF ⫽ cystic fibrosis; ICAM-1 ⫽ intercellular adhesion molecule 1; I␬B ⫽ inhibitory ␬ B; IKK ⫽ I␬B kinase; IL ⫽ interleukin; LPS ⫽ lipopolysaccharide; NF-␬B ⫽ nuclear factor-␬ B; NIK ⫽ NF-␬B-inducing kinase; NLS ⫽ nuclear localization peptide sequence signals; SIRS ⫽ systemic inflammatory response syndrome; TNF ⫽ tumor necrosis factor

factor-␬ B (NF-␬B) is a protein transcripN uclear tion factor that is required for maximal transcription of many proinflammatory molecules that are thought to be important in the generation of inflammation, including certain adhesion molecules (intercellular adhesion molecule 1 [ICAM-1]), critical enzymes (inducible nitric oxide synthase, cyclooxygenase-2), most cytokines (interleukin [IL]-1␤, tumor necrosis factor [TNF]-␣, IL-6), and chemokines (IL-8).1–3 Because these molecules are regulated at the level of transcription and are involved in the inflammatory cascade, by inference NF-␬B is a critical intracellular mediator of the inflammatory *From the Division of Allergy, Pulmonary, and Critical Care Medicine, Department of Medicine, Vanderbilt University School of Medicine, and the Department of Veterans Affairs, Nashville, TN. Supported by The Cystic Fibrosis Foundation, the US Department of Veterans Affairs, and grants HL 61419 and HL 07123 from the National Heart, Lung, and Blood Institute, National Institutes of Health. Manuscript received September 30, 1999; accepted October 1, 1999. Correspondence to: John W. Christman, MD, FCCP, Division of Allergy, Pulmonary, and Critical Care Medicine, Vanderbilt University School of Medicine, T-1217 MCN, Nashville, TN 27232-2650; e-mail: [email protected] 1482

cascade. Most studies that have been reported have used in vitro cell culture systems. There is a rapidly expanding body of literature investigating the role of NF-␬B in animal models of inflammatory disease; however, data proving the involvement of NF-␬B in human lung diseases are just now emerging. The purpose of this short review is to describe what is known about the activation pathway and molecular biology of NF-␬B and to review information that implicates NF-␬B in the pathogenesis of lung disease, including ARDS, the systemic inflammatory response syndrome (SIRS), asthma, respiratory viral infections, occupational and environmental lung disease, and the inherited lung disease cystic fibrosis (CF; Table 1). Molecular Biology of NF-␬B As with other transcription factors, NF-␬B binds to DNA in the promoter regions of target genes as a dimer usually composed of two Rel family proteins, p50 (also called NF-␬B1) and RelA (also called p65). In the NF-␬B heterodimer, both subunits contact DNA, but only RelA contains a transactivation domain that activates transcription by direct interaction Impact of Basic Research on Tomorrow’s Medicine

Table 1—Accumulation of Data Implicating Activation of NF-␬B in the Pathogenesis of Lung Disease*

Condition ALI/ARDS SIRS/sepsis Asthma Ozone exposure Rhinoviral infections RSV infection Asbestosis/silicosis Particulate air pollutants CF/P aeruginosa infections

Evidence Using in Vitro Cell Culture Systems

Evidence Using Animal Models of Disease

⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹ ⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹

⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹ ⫹

Supportive Human Studies ⫹ ⫹ ⫹⫹

I␬B-␣ and I␬B-␤ have a role in activation and the generation of acute inflammation, I␬B-␣ has a prominent role in down-regulation of NF-␬B, and I␬B-␤ is capable of resulting in sustained activation of NF-␬B. NF-␬B Activation Pathway Although a wide variety of stimuli can activate NF-␬B, among the most potent inducers are Gram-

⫹⫹

⫹⫹

*RSV ⫽ respiratory syncytial virus; ⫹⫹⫹⫹ indicates many reports; ⫹⫹⫹ indicates some reported studies; ⫹⫹ indicates few supportive publications; ⫹ indicates as single report or abstract.

with the basal transcription apparatus.4 In quiescent cells, NF-␬B is sequestered in the cytoplasm by its interaction with a member of the inhibitory kappa B (I␬B) family, which includes I␬B-␣ and I␬B-␤. After cell stimulation, I␬B-␣ and I␬B-␤ are phosphorylated, polyubiquitinated, and degraded by the 26S proteasome. I␬B degradation unmasks nuclear localization peptide sequence signals (NLS) that allow NF-␬B to be transported to the cell nucleus, where these dimers are free to bind DNA containing the sequence that activates gene transcription (5⬘-GGGPuNNPyPyCC-3⬘), where Pu ⫽ purine, N ⫽ any base; and Py ⫽ pyrimidine. New synthesis of I␬B␣ is involved in limiting the intracellular inflammatory cascade. Active NF-␬B causes an up-regulation of I␬B-␣ messenger RNA levels by binding to NF-␬B sites in the I␬B-␣ promoter.5,6 The newly synthesized I␬B-␣ helps terminate the NF-␬B response by resequestering NF-␬B in the cytoplasm. In contrast, I␬B-␤ plays a role in persistent activation of NF-␬B. I␬B-␤ exists as a basal phosphorylated form that masks the NLS on NF-␬B. On cell stimulation, I␬B-␤ is polyubiquitinated and degraded by the proteasome complex and is resynthesized as an unphosphorylated (or hypophosphorylated) form.7 Unlike I␬B-␣ and the basally phosphorylated form of I␬B-␤, hypophosphorylated I␬B-␤ is unable to mask the NLS and the DNA-binding domain of NF-␬B.7 Therefore, NF-␬B bound to hypophosphorylated I␬B-␤ is protected from inactivation by I␬B-␣ and can enter or remain in the nucleus and mediate persistent transcription activation of proinflammatory genes. Thus, both

Figure 1. TNF-␣ binds to the type 1 TNF receptor (TNFR1), which results in an association with TNFR1-associated death domain protein (TRADD), the receptor-interacting protein (RIP), and the TNF receptor-associated factor-2 (TRAF-2). These cytoplasmic proteins form an active signaling complex that interacts with NF-␬B-inducing kinase (NIK). Activation of NIK results in phosphorylation of I␬B kinases (IKK), which cause phosphorylation of I␬B. Phosphorylated I␬B is targeted for destruction by the ubiquitinization/proteasome degradation pathway, allowing the translocation of NF-␬B to the nucleus. IL-1␤ binds to the type 1 IL-1 receptor (IL-1R1) and the IL-1 receptor accessory protein (IL-1RAcP), which facilitates an interaction between IL-1 receptor-associated kinase (IRAK) and TNF receptor-associated factor-6 (TRAF-6). The interaction between IRAK and TRAF-6 can also be triggered by endotoxin (LPS). LPS binds with high affinity to CD14, and to toll-like receptor 2 (TLR2). These proteins form an active signaling complex that also results in activation of NIK and IKK, leading to the sequence of events that results in activation of NF-␬B. Activation of NF-␬B results in expression of messenger RNA for a variety of proinflammatory mediators that are involved in the pathogenesis of lung inflammation. I␬B is also induced by NF-␬B activation and contributes to the down-regulation of this intracellular signaling cascade. CHEST / 117 / 5 / MAY, 2000

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negative endotoxin or lipopolysaccharide (LPS), TNF-␣, and IL-1␤. A simplified version of the key signaling events that link LPS, TNF-␣, and IL-1␤ through their cell surface receptors to NF-␬B activation is shown in Figure 1. Mercurio and Manning8 have recently written a concise and scholarly review that deciphers much of the most current information about activation of NF-␬B. As indicated above, NF-␬B activation results from signaled phosphorylation and proteolytic degradation of I␬B by the proteasome. The I␬B kinase or IKK signalsome consists of IKK␣ and IKK␤, which catalyze phosphorylation of serine residues on both I␬B␣ and I␬B␤. Activation of the IKK signalsome also requires a phosphorylation event, which is mediated by a member of the mitogen-activated protein kinase kinase kinase family.8 LPS, TNF-␣, and IL-1␤ all result in activation of a specific mitogen-activated protein kinase kinase kinase, which is referred to as NF-␬B-inducing kinase, or NIK. The fundamental role of NIK, the IKK signalsome, and members of the I␬B family in the activation state of NF-␬B make them attractive targets for molecular inventions in the intracellular inflammatory cascades that lead to production of proinflammatory mediators and the generation of inflammation and injury.

Role of NF-␬B in the Pathogenesis of Acute Lung Injury and SIRS Although many animal and cell studies have implicated the NF-␬B pathway in the pathogenesis of acute lung injury (ALI) and SIRS, only a few clinical studies have been published. The hypothesis is that in ALI and SIRS, the triad of endotoxin (LPS), TNF␣, and IL-1␤ results in activation of NF-␬B in the lung and other organs, which leads to cytokine and chemokine gene expression and neutrophilassociated organ dysfunction, clinically recognized as multiple organ dysfunction syndrome. In this regard, there is a recent report that activation of NF-␬B occurs in alveolar macrophages obtained by BAL from patients with ARDS.9 In contrast, basal activation of NF-␬B in alveolar macrophages from normal volunteers appears to be minimal.10 Small animal models of ARDS-like lung inflammation have demonstrated that macrophage depletion with liposomeencapsulated dichloromethylene diphosphoneate has largely abrogated both activation of NF-␬B in whole lung tissue and the development of neutrophilic alveolitis.11 This seems to indicate that the alveolar macrophage has a critical sentinel role in mediating NF-␬B activation in the lung and in generating neutrophilic inflammation. Therapeutic regulation of NF-␬B activation in alveolar macro1484

phages is a desirable goal, which should lead to the ability to modulate exuberant lung inflammation. We measured NF-␬B activation in mixed BAL cells (50 to 70% neutrophils) in 22 patients receiving mechanical ventilation who met generally accepted criteria for ALI or ARDS.12 Substantial NF-␬Bbinding activity was present in nuclear protein extracts of nearly three fourths of the BAL samples, and there was good correlation between a sample taken in the first 24 h of meeting study criteria and 72 h later. Although this correlation seems to validate our technique, we did not find a clear correlation between activation of NF-␬B, the percentage of neutrophils or IL-8 concentrations in BAL, pulmonary gas exchange variables, or mortality. Although activation of NF-␬B in the mixed cellular constituents of the alveolar space has a potential role as a mediator of lung inflammation, we found that it is not a predictive marker and certainly is not the sole determinant of pathophysiology or outcome. A recent review has highlighted the many cell and animal studies that support a potential role of NF-␬B in SIRS,2 but clinical data are few. In a recent clinical study, NF-␬B activation was detected in peripheral blood monocytes of 15 septic patients. NF-␬B activation was more intense in 5 patients who ultimately died compared with 10 survivors and correlated with the acute physiology and chronic health evaluation II score.13 This seems to indicate that intensity of NF-␬B activation within the vascular space has prognostic significance in SIRS, possibly as a marker of generalized inflammation. We have developed a novel animal model to investigate generalized multiple organ inflammation using a transgenic line of mice in which an NF-␬B-responsive promoter activates a luciferase reporter gene. In these mice, a single intraperitoneal injection of endotoxin results in a coordinated sequence of reporter gene expression in multiple organs, including lung, liver, kidney, bone marrow, and spleen.14 In addition, lung expression of several important NF-␬B-dependent cytokine genes and neutrophilic alveolitis correlated well with reporter gene expression in the lungs. Taken together, these data emphasize the systemic nature of the sepsis reaction and suggest that organ interactions and dysfunction are mediated by NF-␬Bdependent cytokine and chemokine gene expression.

Role of NF-␬B in the Pathogenesis of Asthma Asthma is an inflammatory disease of the airways that is associated with reversible bronchial hyperreactivity. The pathogenesis of asthma seems to involve expression of a broad array of inflammatory proteins, Impact of Basic Research on Tomorrow’s Medicine

including cytokines, enzymes, and adhesion molecules, that are regulated by NF-␬B. Three lines of evidence suggest a central role of NF-␬B in the pathogenesis of asthma: (1) activated NF-␬B has been identified in key locations in the airways of asthmatic patients; (2) agents such as allergens, ozone, and viral infections, which are associated with exacerbation of asthma, stimulate activation of NF␬B; and (3) the major effective treatment for asthma, corticosteroids, is a potent blocker of NF-␬B activation. Certain NF-␬B-dependent chemokines, like RANTES (regulated upon activation normal T-cell expressed and secreted) and eotaxin, function to recruit eosinophils in the airway, which is a typical feature of asthma.15 Increased NF-␬B binding activity has been identified in airway samples from asthmatics, in alveolar macrophages recovered from sputum, and in airway epithelial cells from bronchial mucosal biopsies.16 The stimuli for NF-␬B activation in asthmatic airways have not been defined, but agents that are associated with exacerbations of asthma, in general, activate NF-␬B. For example, allergens have been shown in vitro to activate NF-␬B in asthmatic bronchial epithelial cells,17 and exposure to aerosolized ovalbumin results in profound activation of NF-␬B and expression of inducible nitric oxide synthase in the airways of sensitized Brown Norway rats.18 Also, respiratory tract irritants like ozone may worsen asthma symptoms and activate inflammation through NF-␬B. Exposure of A549 cells to ozone results in activation of NF-␬B and gene expression of IL-8.19,20 Exposure of rats to ozone results in time- and dosedependent activation of NF-␬B and an orchestrated expression of CXC and CC chemokines that are related to influxes of neutrophils and monocytes, respectively, into the lavageable airspace.21,22 Viral infections of the upper respiratory tract may exacerbate asthma through activation of NF-␬B. Rhinoviruses activate NF-␬B and increase ICAM-1 gene expression in bronchial epithelial cells. ICAM-1 is a cellular receptor for rhinovirus as well as having a central role in recruitment of inflammatory cells.23 Rhinovirus induces oxidative stress in cultured bronchial epithelial cells as well as NF-␬B activation and IL-8 gene expression, which, in turn, could play a role in recruiting neutrophils into the upper airway.24 Respiratory syncytial virus (RSV) has been shown to activate NF-␬B and result in IL-8 and IL-11 gene expression in human type II–like alveolar epithelial cells (A549 cells).25–28 Replication of RSV seems to be an essential condition for activation of NF-␬B by RSV and inducing RANTES gene expression in a bronchial epithelial cell line.29 Although pleiotropic in action, a major anti-inflammatory mechanism of corticosteroids is to in-

hibit NF-␬B activation. In this regard, treatment of asthmatic patients with inhaled budesonide dramatically decreases NF-␬B-binding activity in bronchial mucosa biopsy samples.30 Despite several lines of evidence linking NF-␬B activation to the pathogenesis of bronchial hyperreactivity, proof of a critical role of NF-␬B activation in asthma awaits the availability of safe, specific inhibitors.

Role of NF-␬B in the Pathogenesis of Mineral Dust Disease Two groups have intensely investigated the role of NF-␬B in asbestosis using in vitro cell lines and animal models.31–36 The hypothesis is that iron present in asbestos fibers induces cellular redox changes by facilitating production of intracellular reactive oxygen species through Fenton-like chemistry, resulting in activation of NF-␬B. Reactive oxygen species are potent stimuli, which activate at least two transcription factors, NF-␬B and activator protein-1, through a mechanism that is not yet understood.37 In vitro exposure to asbestos results in increased NF-␬B-binding activity to sites in the IL-8 and IL-6 gene promoters in bronchial epithelial cells31 and in alveolar macrophages.34 Nuclear translocation and increased DNA-binding activity of RelA has been shown to occur by a variety of techniques in rat airway epithelial and pleural mesothelial cells after inhalation of crocidolite and chrysotile asbestos and in tracheal epithelial cells after in vitro exposure to asbestos.35,36 Activation of NF-␬B also occurs in non–ironcontaining minerals, because inhalation of silicon dioxide in rats activates NF-␬B in lungs and correlates with the intensity of neutrophilic alveolitis.38 This may accompany phagocytic activation inasmuch as other particulates are capable of activation of NF-␬B. Many in vitro studies have shown that a wide array of particulate air pollutants result in activation NF-␬B, including residual oil fly ash,39 copper ion-containing particulate from the atmosphere of Provo, UT,40 and diesel exhaust particles.41 Although human data are lacking, these cell and animal data strongly suggest that NF-␬B-related events contribute to the pathogenesis of asbestosis and silicosis and possibly other occupational lung diseases.

CF CF is a chronic inflammatory airway disease that is caused by mutations of the CFTR gene. Lung disease in CF presumably results from chronic airway inCHEST / 117 / 5 / MAY, 2000

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flammation related to increased viscosity of respiratory secretions and colonization with Pseudomonas and other bacterial species. Pseudomonas aeruginosa apparently leads to activation of NF-␬B and could have a role in overproduction of mucin through activation of MUC2 mucin transcription.42 Although little in vivo data exist, increased activation of NF-␬B and overproduction of IL-8 can be demonstrated in bronchial epithelial cells that exhibit CFTR mutations (IB3 cells) compared with normal bronchial epithelial cells or a corrected CF cell line (C38 cells).43,44 Inhibition of NF-␬B activation has been suggested as a method of decreasing sputum viscosity in CF45 as well as an effective treatment for reducing airway inflammation and attenuating detrimental changes in lung function. These data seem to indicate that a link between CFTR mutations and airway inflammation involves altered intracellular signals that modify the NF-␬B activation pathway.

Summary NF-␬B has been shown to regulate production of acute inflammatory mediators in a variety of cell and animal models developed to elucidate the pathobiology of lung diseases. In addition, there are some emerging clinical data relating NF-␬B activation to the pathogenesis of ARDS, SIRS, and asthma. In ARDS and SIRS, NF-␬B activation in alveolar macrophages and other lung cell types very likely modulates neutrophilic alveolitis and lung injury. In asthma, NF-␬B activation in airway epithelial cells and other cell types may affect initiation or maintenance of the inflammatory phenotype that characterizes the disease. NF-␬B activation may also be a pivotal factor in other lung diseases in which cytokine-mediated inflammation is important, such as occupational lung disease and CF. NF-␬B could be involved in many other lung diseases, including idiopathic pulmonary fibrosis and primary pulmonary hypertension, but data are lacking. There is an intense interest in defining nuances that govern subtle differences in the regulation of NF-␬B, which could account for a varied role of the same transcription factor in remarkably different types of lung disease. Regardless of the details, NF-␬B and other transcription factors seem to be the critical link between genotype, environmental stresses, and phenotypic cellular responses that result in generation of a wide range of inflammatory diseases. Direct proof of the importance of NF-␬B activation for directing the inflammatory processes that result in lung disease will require large-scale clinical studies using specific inhibitors of the NF-␬B activation pathway. 1486

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