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Plays a Critical Role in the Pathogenesis of Cigarette Smoke-Induced Pulmonary Emphysema and Inflammation1
,
* Section of Pulmonary and Critical Care Medicine,
Department of Pathology, and
Department of Surgery, Yale University School of Medicine, New Haven, CT 06520;
Department of Pathology and Laboratory Medicine Service, Veterans Administration-Connecticut Health Care System, West Haven, CT 06516; and
¶ Division of Pulmonary Medicine, Department of Internal Medicine, Hanyang University Hospital, College of Medicine, Seoul, Korea
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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(IL-18R–/–). CS was a potent stimulator and activator of IL-18 and caspases 1 and 11. In addition, although CS caused inflammation and emphysema in wild-type mice, both of these responses were significantly decreased in IL-18R–/– animals. CS also induced epithelial apoptosis, activated effector caspases and stimulated proteases and chemokines via IL-18R-dependent pathways. Importantly, the levels of IL-18 and its targets, cathepsins S and B, were increased in pulmonary macrophages from smokers and patients with chronic obstructive lung disease. Elevated levels of circulating IL-18 were also seen in patients with chronic obstructive lung disease. These studies demonstrate that IL-18 and the IL-18 pathway are activated in CS-exposed mice and man. They also demonstrate, in a murine modeling system, that IL-18R signaling plays a critical role in the pathogenesis of CS-induced inflammation and emphysema. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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plays an important role in the pathogenesis of emphysema in murine modeling systems (12, 13, 14). Interestingly, chronic obstructive pulmonary disease (COPD)3 (the disease spectrum that includes pulmonary emphysema) may not be mediated solely by Th1 responses because tissue eosinophilia and Th2 cytokines are well documented in tissues and sputum from these patients and individuals with features of COPD and asthma (a Th2-dominated disease) are frequently encountered (15, 16, 17, 18, 19, 20). Surprisingly, the contributions of Th1 cytokines other than IFN- and the mechanisms by which type I and type II immune responses can both be induced in COPD have not been addressed.
IL-18 was described in 1995 as IFN--inducing factor and shown to be a member of the IL-1 cytokine superfamily (21, 22, 23). It is produced as a 23- to 24-kDa inactive propeptide, is activated by caspase-1 to an 18-kDa active moiety, and mediates its effects by binding to a heterodimeric receptor made up of a ligand binding subunit and a signaling 
subunit (reviewed in Refs. 21, 22, 23). It is now appreciated to be an important regulator of innate and adaptive immunity. It has impressive effects on type I immune responses where it induces Th1/Tc1 lineage differentiation and T and NK cell maturation, stimulates IFN- production, and regulates macrophage and neutrophil accumulation and function and cellular apoptosis (21, 22, 23). Interestingly, in the presence of IL-4 or IL-2, it can also contribute to Th2 responses by stimulating IgE production and Th2 cell differentiation (21, 22, 23). However, despite its important contributions to type I and II immune responses, the role of IL-18 in the pathogenesis of pulmonary emphysema has not been addressed.
We hypothesized that IL-18 is induced during and plays a key role in the pathogenesis of pulmonary emphysema. To test this hypothesis, we characterized the effects of cigarette smoke (CS) on IL-18 and the caspases that activate it and characterized the inflammatory and destructive effects of CS in wild-type (WT) mice and mice with null mutations of IL-18R. These studies demonstrate that IL-18, caspase-1 and caspase 11 are induced by CS, that signaling via IL-18R is a critical event in the pathogenesis of CS-induced inflammation and emphysema and that CS induces epithelial apoptosis, stimulates proteases and chemokines and activates a variety of caspases via IL-18R-dependent mechanisms. We also demonstrate that IL-18 is present in exaggerated quantities in the lungs from smokers and patients with COPD and that elevated levels of IL-18 can be detected in the serum from patients with COPD.
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Materials and Methods |
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C57BL/6 WT mice and IL-18R–/– mice were purchased from The Jackson Laboratory. Starting at 10 wk of age, they were exposed twice a day, 5 days a week, to room air (RA) or the smoke from two nonfiltered standard research cigarettes (2R4, University of Kentucky) (CS) using the smoking apparatus described by Hautamaki et al. (24). At the 0.5- to 3-mo time points, bronchoalveolar lavage fluid (BAL) and TUNEL evaluations were undertaken as described below. After 6 mo, the mice were anesthetized and sacrificed, and the trachea was cannulated. After ligation of the right main bronchus, the left lung was inflated with 0.5% low temperature-melting agarose in 10% PBS-buffered formalin at a constant pressure of 25 cm. This allowed for homogenous expansion of lung parenchyma as described by Halbower et al. (25). The lungs were then fixed in 10% PBS-buffered formalin for 24 h, sectioned, and evaluated using histologic, immunohistologic, and morphologic methods as described below.
Immunologic interventions
Eight- to 10-wk-old C57BL/6 transgene (–) mice were randomized to receive a neutralizing Ab raised against murine anti-IL-18R (20 mg/mouse i.p. every other day) (catalog no. MAB12161; R&D Systems) or an appropriate control. One day later, they were randomized to RA or CS as described above. BAL and tissue analyses were undertaken 1 mo later.
BAL and quantification of IL-18
Mice were euthanized, the trachea was isolated by blunt dissection, and tubing was secured in the airway. Three volumes of 0.6 ml of PBS were then instilled and gently aspirated and pooled. Each BAL fluid sample was centrifuged, and the supernatants were stored in –70°C until used. Lung lysates were also prepared. The levels of IL-18 were determined using a commercial ELISA kit (R&D Systems) as per the manufacturer’s instructions.
Histologic analysis
Animals were anesthetized, a median sternotomy was performed and right heart perfusion was accomplished with calcium- and magnesium-free PBS to clear the pulmonary intravascular space. The lungs were then fixed to pressure (25 cm) with neutral-buffered 10% formalin, fixed overnight in 10% formalin, embedded in paraffin, sectioned, and stained. H&E stains were performed in the Research Histology Laboratory of the Department of Pathology at Yale University School of Medicine.
mRNA analysis
mRNA levels were assessed using real-time RT-PCR as previously described by our laboratory (12, 13, 26). In these assays, gene-specific primers were used to amplify selected regions of each target moiety. The primers for targeted genes have been described in our publications or are detailed below: caspase-1, forward, 5'-acc ctc aag ttt tgc cct tt-3', and reverse, 5'-tct ttc aag ctt ggg cac tt-3'; caspase-11, forward, 5'-gac aag cgt tgg gtt ttt gt-3', and reverse, 5'-tct ggt tcc tcc att tcc ag-3'; and IL-18, forward, 5'-aca act ttg gcc gac ttc ac-3', and reverse, 5'-ggg ttc act ggc act ttg at-3'.
Immunohistochemistry (IHC)
IHC was undertaken as described previously (12, 13, 26). Primary Abs against murine IL-18 (Santa Cruz Biotechnology), cathepsin-S (Santa Cruz Biotechnology), or activated caspase-3 (Promega) were used.
Chemokine measurements
The levels of selected chemokines in BAL were evaluated by ELISA using commercial assays (R&D Systems) as described by the manufacturer.
Lung volume, morphometric, and compliance assessment
Lung volume, alveolar size, and lung compliance were assessed via volume displacement and morphometric chord length assessments as described previously (12, 13, 26).
TUNEL evaluations
End labeling of exposed 3'-OH ends of DNA fragments in paraffin-embedded tissue was undertaken with the TUNEL in situ cell death detection kit AP (Roche Diagnostics) using the instructions provided by the manufacturer. Staining specificity was assessed by comparing the signal that was seen when terminal transferase was included and excluded from the reaction. After staining, a minimum of 20 fields of alveoli were randomly chosen, and 500 nuclei were counted per lung. The labeled cells were expressed as a percentage of total nuclei.
Murine caspase and immunoblot evaluations
Whole lung lysates were prepared, and Western blot evaluations were undertaken as previously described by our laboratory (12, 13, 26). Abs that selectively reacted to IL-18, caspase-3, caspase-8, cathepsin-S, cathepsin-B, and -tubulin were purchased from Santa Cruz Biotechnology. Commercial sources were also used for the Abs against caspase-1 (Upstate Biotechnology), caspase-11 (Abcam), and caspase-12 (Cell Signaling Technology).
Immunohistochemical evaluation of human lung tissues
We identified 44 patients who had undergone lung resection at Yale between 1995 and 1999 who had adequate clinical data and tissue blocks available for evaluation. Clinical data were extracted using a standardized chart abstraction form by an investigator blinded to the results of the tissue staining. Pulmonary function tests performed within the 1 year before surgery were obtained from the chart. Patients were divided into groups based on smoking status and the presence or absence of COPD. The smoking status categories included current smokers (any use within 1 year before surgery), former smokers (last use >1 year before surgery), and never-smokers. The never-smokers were generally undergoing resection for a metastatic neoplasm. The current smokers and former smokers were generally undergoing resection for non-small cell lung cancer. Patients were defined as having COPD if they had 1) documentation of COPD according to physician notes in the medical record, 2) an active smoking status with a forced expiratory volume in 1 s/forced vital capacity ratio < 70% without another explanation, and 3) an active smoking status without any other lung disease with a carbon monoxide diffusing capacity (DLCO) < 80% predicted and computerized tomography scan evidence of emphysema. Classification using guidelines from the Global Initiative for Chronic Obstructive Lung Disease (GOLD) was also undertaken. These patients are described in Table I. This study was approved by the Yale Human Investigation Committee.
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Quantification of serum IL-18
Informed consent was obtained on all study participants. A total of 29 patients with COPD and 78 age-matched controls seen in the pulmonary and primary care clinics of the West Haven Veterans Administration Medical Center between January 2005 and April 2006 was enrolled. The diagnosis of COPD, the demographics of the patients, and their comorbid diseases were obtained from a standardized chart review. Smoking histories were obtained from the chart reviews and standardized interviews performed by one investigator (A. Gallo) who was blinded to the results of the patient’s IL-18 evaluations. Subjects with a history of asthma, other lung diseases, lung resection, HIV, or hepatitis C were excluded. Subjects that were immunocompromised or taking oral corticosteroids were also excluded. Once the chart review and interview were completed, the patients were placed into two groups: patients without COPD and patients with COPD (a physician diagnosis and a forced expiratory volume in 1 s/forced vital capacity ratio < 70%) (see Table III). In all cases, peripheral blood samples were collected in noncoated tubes, the serum was separated by centrifugation, and the specimens were stored at –80°C until used. The levels of IL-18 were determined by ELISA using commercial kits according to the manufacturer’s instructions (R&D Systems). This research was approved by the Yale University and West Haven Veterans Administration Institutional Review Boards. There was no overlap between these patients and the patients whose lung resection specimens were evaluated immunohistochemically.
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Normally distributed data are expressed as mean ± SEM and were assessed for significance by Student’s t test or ANOVA as appropriate. Data that were not normally distributed are expressed as media with interquartile ranges and were assessed for significance using the Mann-Whitney U test or the Kruskal-Wallis test with Dunn’s posttest for multiple comparisons as appropriate. Statistical analysis was performed using Stata version 9.0 (StataCorp), Deltagraph (RockWare), and Prism version 4 (GraphPad). Statistical significance was defined at a level of p 
0.05.
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Results |
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To address the role of IL-18 in the pathogenesis of CS-induced pulmonary responses, studies were undertaken to determine whether CS regulated the expression and/or production of this cytokine in the murine lung. IL-18 mRNA and protein were readily appreciated in lungs from mice breathing RA (Fig. 1, A and B). Interestingly, the levels of IL-18 mRNA and protein were increased significantly after CS exposure (Fig. 1, A and B). The increases in mRNA were readily apparent after as little as 2 wk of CS exposure, were readily appreciated after 1–3 mo of CS exposure, and persisted throughout the study interval (Fig. 1A and data not shown). In all cases, they were associated with comparable increases in BAL and lysate IL-18 protein when assessed by ELISA (Fig. 1, B and C, and data not shown), immunoblot (Fig. 1D), or IHC evaluations (Fig. 1E). The immunoblots also demonstrated that this stimulation was associated with modest increases in the levels of the IL-18 propeptide and more impressive increases in the levels of the mature IL-18 cytokine (Fig. 1D). The IHC demonstrated that the major site of IL-18 protein localization was in alveolar macrophages (Fig. 1E).
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Studies were next undertaken to determine whether CS regulated the production and/or activation of the IL-18 activator, caspase-1. Caspase-1 mRNA and protein were readily detected in lungs from mice breathing RA (Fig. 2, A and B). At the protein level, the majority of this pool was made up of inactive caspase-1 precursor moieties (Fig. 2B). In contrast to mice breathing RA, increased levels of caspase-1 mRNA and activated caspase-1 protein were noted in lungs from mice exposed to CS (Fig. 2, A and B). This increase in activated caspase-1 could be detected after as little as 2 wk and persisted with longer periods (2 mo) of CS exposure (data not shown). It was also associated with significantly increased levels of activated caspase-11 and the mRNA encoding this known caspase-1 activator (Fig. 2, B and C). Thus, CS increases the levels of active caspase-1 and stimulates caspase-11 while simultaneously increasing the production and activation of IL-18 in the murine lung.
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To determine whether IL-18 signaling contributes to the pathogenesis of CS-induced emphysema, WT and IL-18R-null (IL-18R–/–) mice were exposed to CS for 6 mo and the alveoli in lungs from these animals were compared. In accord with studies from our laboratory and others (12, 24), 6 mo of cigarette exposure caused modest alveolar destruction, which could be appreciated in histologic sections (Fig. 3A) and morphometric evaluations (Fig. 3B). This alveolar simplification was significantly ameliorated in IL-18R–/– animals (Fig. 3). Overall, a null mutation of IL-18R caused a 51.5% decrease in the CS-induced increase in alveolar chord length (p < 0.01). Thus, IL-18R plays a critical role in the pathogenesis of CS-induced pulmonary emphysema.
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in CS-induced inflammation
The current concept of emphysema disease pathogenesis suggests that inflammation contributes, in a causal way, to the genesis of pulmonary emphysema. Thus, studies were undertaken to determine whether genetic alterations in the IL-18 receptor altered CS-induced inflammation in a manner that is analogous to their effects on alveolar remodeling. When compared with mice breathing RA, BAL from mice exposed for 1–3 mo to CS manifest significant increases in total cell recovery and increases in macrophage, and to a lesser extent, neutrophil recovery (Fig. 4, A–C, and data not shown). These BAL alterations were associated with patchy increases in tissue cellularity where increased numbers of macrophages were readily appreciated (data not shown). In all cases, these responses were IL-18R dependent because the BAL and tissue cellular responses were both significantly decreased in IL-18R–/– animals and WT mice treated with neutralizing Abs against IL-18R (Fig. 4). Thus, CS induces pulmonary inflammation via an IL-18R-dependent mechanism.
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Studies were next undertaken to determine whether CS induced tissue apoptosis and IL-18 contributed to the pathogenesis of this cell death response. This was done by comparing the levels of DNA injury and cell death (using TUNEL evaluations) and the levels of activated caspases 3 and 8 in lungs from WT and IL-18R–/– mice exposed to RA or CS. Cells that were TUNEL positive were not readily appreciated and only low levels of activated caspases –3 and –8 were appreciated in lungs from WT and IL-18R-null mice breathing RA (Fig. 5). In contrast, apoptosis of alveolar type II epithelial cells and occasional airway epithelial cells was readily appreciated and increased levels of activated caspase-3 and caspase-8 were detected in lungs from WT mice exposed to CS for 1–3 mo (Fig. 5 and data not shown). Similarly, the caspase-12 proenzyme was readily appreciated in lungs from mice breathing RA, while increased levels of this proenzyme and activated caspase-12 were seen after CS exposure (Fig. 5D). Importantly, each of these responses was significantly ameliorated in mice with null mutations of IL-18R (Fig. 5). Thus, IL-18R signaling contributes, in a significant fashion, to the pathogenesis of CS-induced cell injury and death and caspase activation in the murine lung.
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Studies were next undertaken to determine whether IL-18R contributed to CS-induced alterations in proteases in the murine lung. This was done by comparing the expression of matrix metalloproteinase (MMP)-12 and cathepsins -S and -B in lungs from RA and CS-exposed IL-18R+/+ and IL-18R–/– mice. Comparable levels of mRNA encoding these proteases were appreciated in lungs from mice with WT and null IL-18R loci breathing RA (Fig. 6, A–C). In addition, the levels of mRNA encoding these moieties increased after 1–3 mo of CS exposure in WT mice (Fig. 6, A–C). In contrast, comparable increases in the levels of mRNA encoding these moieties were not appreciated in lungs from IL-18R-null animals exposed to CS (Fig. 6, A–C). In all cases, alterations in mRNA were associated with comparable alterations in protease protein (Fig. 6D and data not shown). IHC also demonstrated that the cathepsin alterations were largely localized to alveolar macrophages (Fig. 6E and data not shown). Thus, IL-18R signaling plays an important role in the pathogenesis of CS-induced protease alterations in macrophages in the murine lung.
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Previous studies from our laboratory and others have demonstrated that CS induces chemokine responses that play an important role in the pathogenesis of pulmonary emphysema (12, 30). Thus, the roles of IL-18 signaling in the generation of these responses was evaluated. In accord with the prior studies (12, 30), CS induced MIP-1/CCL-3, MCP-1/CCL-2, and MCP-3/CCL-7 mRNA and or protein in lungs from WT mice (Fig. 7). Importantly, the levels of expression of MIP-1/CCL-3, MCP-1/CCL-2, and MCP-3/CCL-7 in lungs from CS-exposed IL-18R–/– animals were significantly diminished when compared with WT controls (Fig. 7). Thus, CS stimulates MIP-1/CCL-3, MCP-1/CCL-2, and MCP-3/CCL-7 via IL-18R-dependent mechanisms.
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To evaluate the applicability of our murine finding to human disease, studies were undertaken to determine whether IL-18, or the gene products it regulates, are expressed in an exaggerated fashion in CS-exposed humans. This was done by comparing the levels of immunoreactive IL-18 and cathepsins in lung tissues from current smokers, former smokers, and never-smokers (Fig. 8). The clinical characteristics of these patients are shown in Table I. Modest levels of immunoreactive IL-18 were seen in airway epithelial cells but not in macrophages in lungs from never-smokers (Fig. 8A and data not shown). This pattern of staining in normal lung has been reported previously (29, 31). The airway epithelial staining was not significantly altered in tissues from current smokers (Fig. 8A). In contrast, increased levels of immunoreactive IL-18 were readily appreciated in the alveolar macrophages from current smoker tissues (Fig. 8A). Using a semiquantitative scoring system, there was a correlation of smoking status and the levels of IL-18 staining (p < 0.0001, Kruskal-Wallis test) (Fig. 8A). Pairwise comparisons using Dunn’s test showed significantly higher levels of IL-18 in tissues from current smokers when compared with never-smokers (p < 0.01) and current smokers vs former smokers (p < 0.001) (Fig. 8A). Similar results were seen with evaluations of cathepsin-S and cathepsin-B. Cathepsin-S and cathepsin-B were both only minimally expressed in lungs from never smokers but were significantly up-regulated in alveolar macrophages from current smokers (Fig. 8, B and C). Cathepsin-S was also increased in smooth muscle and some epithelial cells from current smokers (data not shown). Quantitation showed a strong overall correlation with smoking status (cathepsin-S p < 0.0001, cathepsin-B p < 0.0023, Kruskal-Wallis test) with higher levels of cathepsin-S and cathepsin-B in current vs never-smokers (cathepsin-S, p < 0.001, cathepsin-B p < 0.05 using Dunn’s test) and in current vs former smokers (cathepsin-S p < 0.01, cathepsin-B p < 0.01 using Dunn’s test) (Fig. 8, B and C).
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Evaluations were also undertaken to determine whether the levels of IL-18 and the cathepsins correlated with the presence or absence of COPD. Once again, statistically significantly elevated levels of IL-18, cathepsin-S, and cathepsin-B were seen in the tissues from the patients with COPD compared with the patients without COPD (Table II). When viewed in combination with the above studies, these studies demonstrate that CS exposure increases the levels of macrophage IL-18, cathepsin-S, and cathepsin-B in the human lung and that the levels of IL-18 and the cathepsins are increased in patients with COPD.
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To further understand the regulation of IL-18 in CS-exposed humans, we also compared the levels of immunoreactive IL-18 in the serum of age-matched patients with and without COPD. The demographics of these populations are illustrated in Table III. The two populations were similar with respect to other comorbidities, yet more patients in the COPD group had smoking histories with a greater number of overall pack-years. As can be seen in Fig. 8D, IL-18 was readily apparent in the circulation of our age-matched control patients. It was, however, significantly higher in the serum from patients with COPD (Fig. 8D) (p = 0.024) (Mann-Whitney U test). When comparing each population to the combined median, 223 pg/ml, 72% of the COPD patients had IL-18 levels above 223 pg/ml, in contrast to 42% of the control patients (p = 0.006). Of the 42% of control patients with IL-18 levels over 223 pg/ml, 73% had smoking histories raising the possibility that they will develop COPD in the future. There was no significant difference in the IL-18 levels when we stratified for the other comorbidities listed in Table III (data not shown). Thus, elevated levels of IL-18 are detectable in the circulation of patients with COPD.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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and that CS-induction of apoptosis and stimulation of caspases (3, 8, and 12), proteases (MMP-12, cathepsin-S, and cathepsin-B) and chemokines (MCP-1, MCP-3, and MIP-1) are mediated by IL-18R-dependent pathways. Lastly, human disease relevance is also provided by demonstrating that the levels of immunoreactive IL-18 and its downstream targets, cathepsin-S and cathepsin-B, are increased in macrophages in lungs from smokers and patients with COPD and that the levels of circulating IL-18 are increased in the serum of patients with COPD. When viewed in combination, these studies highlight the impressive activation of the IL-18-IL-18R pathway in response to CS in mice and man and demonstrate that IL-18R signaling plays a critical role in the pathogenesis of CS-induced inflammation and remodeling.
IL-18 is an inactive propeptide that is cleaved by caspase-1 to an active 18-kDa ligand that binds a heterodimeric receptor that contains a ligand binding subunit and a signal transducing subunit (21, 22). Our studies demonstrate that the IL-18 propeptide and mature peptide is induced by CS. They also demonstrate that these inductive events are associated with increased levels of caspase-1 and the caspase-1 stimulator, caspase-11 (32). Interestingly, CS induction of caspase-11 was mediated via an IL-18R-dependent pathway, whereas the stimulation of caspase-1 was not (M.-J. Kang and J. A. Elias, unpublished observation). These observations suggest that caspase-1 and caspase-11 play important roles in the activation of IL-18 in the CS-exposed lung. They also highlight a potential positive feedback loop in which IL-18 induces caspase-11, which, in turn, drives caspase-1 to further increase the levels of active IL-18. However, the ultimate importance of this pathway will need validation because, in addition to caspase-1, proteinase 3 and cathepsin-B can contribute to IL-18 activation (21, 33).
Our current concepts of the emphysema pathogenesis have focused on the destructive effects of protease excess in the lung (34, 35). In accord with this conceptualization, our studies demonstrate that IL-18R-dependent signaling plays an important role in regulating the levels of pulmonary proteases with CS induction of MMP-12, cathepsin-S, and cathepsin-B being mediated via IL-18R-dependent mechanisms. These findings are in accord with the established importance of many of these proteases in murine models of emphysema (14, 24), the known ability of IL-18 to contribute to the tissue destruction in murine models of arthritis (21, 36), diabetes (37, 38). and colitis (39) and the established ability of IL-18 to regulate MMP-2 and other MMP (40, 41, 42, 43). They also extend our knowledge of IL-18-induced protease effector responses to MMP-12 and cathepsins S and B and demonstrate that IL-18 is an important mediator of the regulatory effects of CS on these important proteolytic moieties.
Recent studies demonstrating increased levels of structural cell apoptosis in tissues from patients with emphysema and models of this disorder (44, 45, 46, 47, 48) have led to the speculation that this cell death response plays an important role in the pathogenesis of emphysematous tissue destruction. Our studies demonstrate that CS induces apoptosis and activates caspases 3 and 8 in the murine lung and that signaling via IL-18R is required for the optimization of these responses. These observations suggest that both the extrinsic (death receptor) and intrinsic (mitochondrial) cell death pathways are being activated by IL-18. This observation is in keeping with the results of studies using a variety of extrapulmonary cells and tissues, which have highlighted the ability of IL-18 to induce apoptosis and tissue injury via intrinsic and extrinsic pathways (49, 50, 51, 52). In addition to the cell death and mitochondrial apoptosis pathways, endoplasmic reticulum stress regulates cell viability via a pathway involving caspase-12 (53, 54). Our studies demonstrate that caspase-12 is also activated by CS via an IL-18R-dependent mechanism. In so doing, these observations extend our knowledge of CS-induced and IL-18-induced apoptosis and cell death responses to include the unique endoplasmic reticulum stress, cell death pathway.
The British Hypothesis, which dominates present day thinking in airways disease, suggests that asthma and COPD are clinically, mechanistically, and pathologically distinct entities (15, 19). In accord with this traditional concept, asthma is now viewed as a chronic Th2 inflammatory disorder of the airway characterized by reversible airflow obstruction that does not develop significant lung destruction (55, 56). In contrast, the emphysema in COPD is characterized by Th1 inflammation, macrophage, and neutrophil tissue infiltration, partially reversible or irreversible airflow obstruction and alveolar destruction (7, 12, 13, 34, 35). However, in real life, these distinctions may not be as clear as initially perceived because patients are commonly encountered that manifest clinical features of asthma and COPD (15, 19), Th2 cytokines have been shown to be induced during and generate asthma-like, bronchitic and emphysematous tissue responses (16, 17, 26), and polymorphisms in Th2 cytokines have been noted to associate with COPD (57). In keeping with this complexity, the Dutch Hypothesis was proposed in 1961 (15, 19). This hypothesis suggests that, in some patients, there is clinical and mechanistic overlap between asthma and COPD. To date, however, a common abnormality or mechanism upon which these Th2- and Th1-driven responses could be based has not been defined. Our studies highlight impressive alterations in the production and activation of the IL-18/IL-18R pathway in CS-exposed mice and man. IL-18 can promote Th1 or Th2 lineage maturation, depending on underlying genetic influences and the ambient cytokine milieu (21, 23, 58). Thus, when viewed in combination, it is tempting to speculate that CS-induced alterations in the IL-18/IL-18R pathway are proximal events in the pathogenesis of pulmonary emphysema and that classic British Hypothesis manifestations and mixed asthma/COPD (Dutch Hypothesis) manifestations can occur depending on the nature of subsequent insults and underlying genetic predispositions. If true, this would provide the first mechanistic link between patients with these two related, but different, disease syndromes. It would also suggest that interventions that control the IL-18/IL-18R pathway could be effective therapies for patients with both of these patterns of disease presentation.
Previous studies from our laboratory demonstrated that IFN- plays an important role(s) in the pathogenesis of the pulmonary emphysema in a variety of experimental modeling systems (12, 13, 14). When viewed in combination with the well-known ability of IL-18 to regulate Th1 responses (21, 23, 55), it is tempting to speculate that IFN- plays an important role in the pathogenesis of the effects of IL-18 in CS-exposed mice. This hypothesis must, however, be viewed with caution because IL-18 can have IFN--dependent and -independent effects and, as noted above, can contribute to Th2 as well as Th1 responses (21, 23, 55). The present studies do not formally address the relationship(s) between IFN- and IL-18 in CS-induced emphysema. This relationship will need to be addressed in subsequent experimentation.
To evaluate the applicability of our murine findings to human COPD, we characterized the expression of IL-18 in lung tissues from never-smokers, current smokers, and former smokers. These studies revealed elevated levels of immunoreactive IL-18 in macrophages from CS-exposed individuals. They also demonstrated that the levels of the IL-18 targets, cathepsin-S and cathepsin-B, were increased in the tissues from these patients and that these IL-18 and cathepsin findings were independent of the presence of pulmonary malignancies. Furthermore, they demonstrated that, when COPD was defined based on historical, physiologic, and chest computerized tomography scan criterion, significantly elevated levels of IL-18, cathepsin-S, and cathepsin-B were seen in tissues from patients with COPD. Interestingly, the levels of IL-18 and the cathepsins did not correlate with disease severity as assessed by GOLD staging (M.-J. Kang and J. A. Elias, unpublished observation). This conclusion, however, must be viewed with caution in light of the single point in time study design that was used and the limited number of patients in each GOLD category. Overall, these studies highlight remarkably similar responses to CS in mice and man. The demonstration that CS increases the levels of cathepsins in COPD also allows for the intriguing speculation that the novel cathepsin-S-mediated epithelial apoptosis response that has been seen in murine models of emphysema (14) is also operative in the human disorder. Additional investigations will be required to define the relationships between the CS-induced IL-18 pathway abnormalities and disease natural history and the mechanisms that underlie these relationships.
In an attempt to get a glimpse at the status of the IL-18 system in vivo, we measured the levels of IL-18 in the serum of patients with COPD. These studies demonstrated that the levels of IL-18 are higher in patients with COPD than in the patients without COPD. Importantly, although diseases other than COPD have been associated with elevated levels of IL-18 (6, 21, 29, 59, 60, 61, 62, 63, 64), our findings could not be attributed to these potential confounders because these diseases, when present, were similarly distributed in our patient populations. These findings raise the intriguing possibility that the levels of circulating IL-18 represent a readily accessible biomarker that reflects that activation of the IL-18 pathway in tissues. As an extension of this possibility, it would be very exciting if the IL-18 levels predicted the onset, rate of progression, or response to an IL-18 pathway-based therapeutic intervention in smokers or patients with established COPD. A biomarker of this sort would be particularly useful in COPD because only a minority of cigarette smokers actually get the disease, and the severity of the disease that they acquire varies on an individual-to-individual basis (34, 35). In accord with this possibility, the levels of circulating IL-18 are elevated in an early preclinical stage of diabetes where they foreshadow the development of the full blown disease (62). Lastly, if an IL-18 biomarker is appropriately validated it could facilitate the subsequent evaluation of IL-18-based therapeutic interventions. Specifically, the levels of circulating IL-18 could represent a rational basis on which target populations could be stratified thereby allowing therapeutic effectiveness to be evaluated in more mechanistically homogeneous patient populations.
In summary, our studies demonstrate that CS induces and activates IL-18 in mice and man and demonstrate, in a murine modeling system, that IL-18R plays a critical role in the pathogenesis of CS-induced inflammation and alveolar destruction. These studies suggest that interventions that block IL-18R may be therapeutically useful in patients with CS-induced pulmonary pathologies. Additional investigations of the roles of IL-18 and the IL-18R in the pathogenesis of COPD and other pulmonary disorders and the utility of IL-18R-based therapeutics in their treatment are warranted.
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1 These studies were funded by National Institutes of Health Grants HL-56389, HL-064242, and HL-078744 (to J.A.E.). 
2 Address correspondence and reprint requests to Dr. Jack A. Elias, Section of Pulmonary and Critical Care Medicine, Yale University School of Medicine, P.O. Box 208057, 300 Cedar Street (S441 TAC), New Haven, CT 06520-8040. E-mail address: jack.elias{at}yale.edu
3 Abbreviations used in this paper: COPD, chronic obstructive pulmonary disease; BAL, bronchoalveolar lavage fluid; CS, cigarette smoke; GOLD, Global Initiative for Chronic Obstructive Lung Disease; IHC, immunohistochemistry; MMP, matrix metalloproteinase; RA, room air; WT, wild type.
Received for publication June 12, 2006. Accepted for publication November 10, 2006.
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T cell receptor-positive cells and interferon , but not inducible nitric oxide synthase, are critical for granuloma necrosis in a mouse model of mycobacteria-induced pulmonary immunopathology. J. Exp. Med. 194: 1847-1859. cells. Am. J. Ther. 12: 580-591. [Medline]-induced and cigarette smoke-induced emphysema. J. Clin. Invest. 115: 3460-3472. [Medline] induction of pulmonary emphysema in the adult murine lung. J. Exp. Med. 192: 1587-1600. -induced alveolar remodeling and pulmonary emphysema. J. Immunol. 174: 8106-8115. , IFN-, IL-6, IL-8, IL-12, IL-17, and IL-18 in patients with active psoriasis and correlation with disease severity. Mediators Inflamm. 2005: 273-279. [Medline]-inducing cytokine interleukin-18 are increased in individuals at high risk of developing type I diabetes. Diabetologia 44: 309-311. [Medline]This article has been cited by other articles:
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  C. A. Nold-Petry, M. F. Nold, J. W. Nielsen, A. Bustamante, J. A. Zepp, K. A. Storm, J.-W. Hong, S.-H. Kim, and C. A. Dinarello Increased Cytokine Production in Interleukin-18 Receptor {alpha}-deficient Cells Is Associated with Dysregulation of Suppressors of Cytokine Signaling J. Biol. Chem., September 18, 2009; 284(38): 25900 - 25911. [Abstract] [Full Text] [PDF]
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 A. Le, R. Zielinski, C. He, M. T. Crow, S. Biswal, R. M. Tuder, and P. M. Becker Pulmonary Epithelial Neuropilin-1 Deletion Enhances Development of Cigarette Smoke-induced Emphysema Am. J. Respir. Crit. Care Med., September 1, 2009; 180(5): 396 - 406. [Abstract] [Full Text] [PDF]
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 D. A. Ngan, S. V. Vickerman, D. J. Granville, S. F. P. Man, and D. D. Sin The possible role of granzyme B in the pathogenesis of chronic obstructive pulmonary disease Therapeutic Advances in Respiratory Disease, June 1, 2009; 3(3): 113 - 129. [Abstract] [PDF]
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 A. Churg, S. Zhou, X. Wang, R. Wang, and J. L. Wright The Role of Interleukin-1{beta} in Murine Cigarette Smoke-Induced Emphysema and Small Airway Remodeling Am. J. Respir. Cell Mol. Biol., April 1, 2009; 40(4): 482 - 490. [Abstract] [Full Text] [PDF]
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A. Churg, S. Zhou, O. Preobrazhenska, H. Tai, R. Wang, and J. L. Wright Expression of Profibrotic Mediators in Small Airways versus Parenchyma after Cigarette Smoke Exposure Am. J. Respir. Cell Mol. Biol., March 1, 2009; 40(3): 268 - 276. [Abstract] [Full Text] [PDF]
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 M. A. Williams, T. Rangasamy, S. M. Bauer, S. Killedar, M. Karp, T. W. Kensler, M. Yamamoto, P. Breysse, S. Biswal, and S. N. Georas Disruption of the Transcription Factor Nrf2 Promotes Pro-Oxidative Dendritic Cells That Stimulate Th2-Like Immunoresponsiveness upon Activation by Ambient Particulate Matter J. Immunol., October 1, 2008; 181(7): 4545 - 4559. [Abstract] [Full Text] [PDF]
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 M. T. Borchers, S. C. Wesselkamper, B. L. Eppert, G. T. Motz, M. A. Sartor, C. R. Tomlinson, M. Medvedovic, and J. W. Tichelaar Nonredundant Functions of {alpha}{beta} and {gamma}{delta} T Cells in Acrolein-Induced Pulmonary Pathology Toxicol. Sci., September 1, 2008; 105(1): 188 - 199. [Abstract] [Full Text] [PDF]
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 K. F. Chung and I. M. Adcock Multifaceted mechanisms in COPD: inflammation, immunity, and tissue repair and destruction Eur. Respir. J., June 1, 2008; 31(6): 1334 - 1356. [Abstract] [Full Text] [PDF]
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 A. Churg, M. Cosio, and J. L. Wright Mechanisms of cigarette smoke-induced COPD: insights from animal models Am J Physiol Lung Cell Mol Physiol, April 1, 2008; 294(4): L612 - L631. [Abstract] [Full Text] [PDF]
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H. Imaoka, T. Hoshino, S. Takei, T. Kinoshita, M. Okamoto, T. Kawayama, S. Kato, H. Iwasaki, K. Watanabe, and H. Aizawa Interleukin-18 production and pulmonary function in COPD Eur. Respir. J., February 1, 2008; 31(2): 287 - 297. [Abstract] [Full Text] [PDF]
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T. Hoshino, H. A. Young, and H. Aizawa Cytokines in Pulmonary Emphysema: Can Results in Mice Be Translated to Humans? Am. J. Respir. Crit. Care Med., January 15, 2008; 177(2): 238 - 239. [Full Text] [PDF]
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