Published ahead of print on March 30, 2007, doi:10.1164/rccm.200603-316OC
© 2007 American Thoracic Society doi: 10.1164/rccm.200603-316OC
Pulmonary Inflammation and EmphysemaRole of the Cytokines IL-18 and IL-131 Department of Internal Medicine 1, and 2 Pathology, Kurume University School of Medicine, Kurume, Japan; 3 Laboratory of Experimental Immunology, National Cancer Institute, Center for Cancer Research, Frederick, Maryland; 4 Internal Medicine 3, and Cardiovascular Research Institute, and 5 Endocrinology and Metabolism, Kurume University School of Medicine, Kurume, Japan; 6 Division of Pathology and Cell Biology, Graduate School and Faculty of Medicine, University of the Ryukyus, Okinawa, Japan Correspondence and requests for reprints should be addressed to Tomoaki Hoshino, M.D., Ph.D., Department of Internal Medicine 1, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830–0011, Japan. E-mail: hoshino{at}med.kurume-u.ac.jp
Rationale: Chronic obstructive pulmonary disease (COPD) is believed to be an inflammatory cytokine–driven disease, but a causal basis that can be associated with a specific cytokine has not been directly demonstrated. We have previously reported that proinflammatory cytokine IL-18 expression is important in the pathogenesis of pulmonary inflammation and lung injury in mice. Our results demonstrate that IL-18 overproduction in the lungs can induce lung diseases, such as pulmonary inflammation, lung fibrosis, and COPD. Objectives: We analyzed the role of IL-18 in the pathogenesis of COPD. Methods: Using the human surfactant protein C promoter to drive expression of mature mouse IL-18 cDNA, we developed two different lines of transgenic (Tg) mice that overproduced mouse mature IL-18 in the lungs either constitutively or in response to doxycycline.
Measurements and Main Results: Constitutive overproduction of IL-18 in the lungs resulted in the increased production of IFN- Conclusions: Our results indicate that IL-18 and IL-13 may have an important role in the pathogenesis of COPD.
Key Words: emphysema • IFN-
Chronic obstructive pulmonary disease (COPD) includes several clinical respiratory syndromes, including emphysema and chronic bronchitis, and is referred to as a pulmonary inflammatory disease. Cigarette smoke and air pollution can induce pulmonary inflammation and directly damage the lungs (1–3). In patients with COPD, inflammation is observed in the bronchial tree and the parenchyma of lungs, and is closely correlated with a history of cigarette smoking (4). Tissue remodeling accompanied by inflammation is also observed in the small airways of lungs from patients with severe COPD (5). Increased numbers of CD8+ T cells, alveolar macrophages, and neutrophils are characteristic pathologic features of the lung in COPD, and lymphocyte infiltration with enhanced accumulation of CD8+ T cells is a prominent finding (6, 7). Eosinophil numbers are increased during exacerbations and possibly also during a stable phase of the disease in a subset of patients (8). These activated inflammatory cells can release various mediators, including leukotriene B4 and cytokines, such as IL-8, IFN-  , and tumor necrosis factor (TNF)- (see review in Reference 3). Previous studies in transgenic (Tg) mice have reported that overproduction of cytokines, including IFN- (9), TNF- (10), IL-1 (11), and IL-13 (12), in the lungs induces emphysema, suggesting that cytokines may be involved in the pathogenesis of COPD. The chronic pulmonary inflammation of COPD is believed to result in progressive respiratory disorders.
IL-18, a proinflammatory cytokine, is produced intracellularly from a biologically inactivated precursor, pro–IL-18, and the mature IL-18 is secreted after cleavage of pro–IL-18 by caspase-1, originally identified as IL-1 We have previously demonstrated that IL-18 expression is important in the pathogenesis of pulmonary inflammation and lung injury in mice (19) and human idiopathic pulmonary fibrosis (IPF) (15). Our results suggest that IL-18 overproduction in the lungs can induce lung diseases, such as pulmonary inflammation, lung fibrosis, and COPD. Therefore, to evaluate the role of IL-18 in the pathogenesis of COPD, we developed two different lines of lung-specific IL-18 Tg mice that overproduced mouse mature IL-18 in the lungs, either constitutively or in response to doxycycline (DOX). In this report, we demonstrate a role for IL-18 in the pathogenesis of COPD using these Tg mice. Some of the results of this study have been previously reported in the form of an abstract (20).
Generation of Tg Mice Constitutively Overproducing Mouse Mature IL-18 in the Lung Mature IL-18 cDNA fused with the signal peptide from the V-J2-C region of the mouse Igµ-chain (IL-18SP) was generated by polymerase chain reaction (PCR) using mouse pro–IL-18 cDNA, as previously reported (18). IL-18SP was subcloned into the BamHI site of a 3.7SPC/SV40 vector containing the surfactant protein (SP) C promoter, the SV40 small T intron, and a polyA signal (21) (kindly provided by Dr. Jeffrey A. Whitsett, Cincinnati Children's Hospital Medical Center, Cincinnati, OH), and was designated as SPC-IL-18SP. The NdeI and NotI-digested linear DNA fragment (see Figure 1A) was injected into fertilized eggs of BDF1 mice. Hemizygous Tg mice were generated by mating founder mice with C57BL/6N (B6) mice, and the offspring were screened by PCR using genomic DNA prepared from their tails. B6 background Tg mice were generated by mating more than five times with wild-type (WT) B6 mice. We established B6 IFN- –deficient (–/–) SPC-IL-18 Tg mice and B6 IL-13–/– SPC-IL-18 Tg mice by backcrossing line A of SPC-IL-18 Tg mice with IFN-–/– and IL-13–/– mice, as previously reported (22). Littermates were used as control animals in our experiments.
ELISA Assays and Western Blot See the online supplement for details.
Histologic Examinations
Morphometric Analysis to Measure Alveolar Size
The Destructive Index
Assessment of Lung Compliance
Echocardiography and Hemodynamic Analysis
Establishment of Inducible Lung-specific IL-18 Tg Mice
Statistical Analysis
Generation of Tg Mice Constitutively Overproducing Mature IL-18 in the Lung Previous studies reported that overexpression of transgenes in the lungs was obtained through the use of either the Clara cell 10-kD (CC10) (26) or surfactant protein (SP) C (21) promoters. The SP-C promoter directs gene expression exclusively to the developing and mature epithelium of the primordial lung buds, with maintenance of expression in the alveolar and bronchiolar epithelial cells after birth (27, 28). In contrast, the CC10 promoter can direct the expression of transgenes in the alveolar and bronchiolar epithelial cells (26). Therefore, we used the SP-C promoter to drive the expression of mature mouse IL-18 cDNA in alveolar and bronchiolar epithelial cells. As described previously here, mature IL-18 is secreted after the cleavage of a biologically inactivated precursor, pro–IL-18, by caspase-1 (14). To avoid the need for such cleavage, we generated a mature mouse IL-18 cDNA fused to the signal peptide (designated IL-18SP [18], Figure 1A) for use in a Tg model. Four founders (A, B, C, and D) that demonstrated germ line transmission of the transgene were obtained. Offspring were generated at a male-to-female ratio of approximately 1:1. SPC-IL-18 Tg mice were healthy at birth, and grew normally. We first analyzed IL-18 levels in the sera and lungs of the Tg mice at 10 weeks of age. In the first founder line that we analyzed, the mean lung IL-18 level was 1,675 ± 282 pg/ml, and in the control transgene-negative WT mice it was 96 ± 11 pg/ml (n = 4 per group). Mean serum IL-18 levels were 1,053 ± 48 and 150 ± 27 pg/ml (n = 4 per group), respectively. Three founder lines (A, B, and C) of SPC-IL-18 Tg mice showed significantly higher IL-18 levels in their sera and lungs than in those of control WT mice (Figure 1B). Pulmonary inflammation and emphysematous changes were also observed in all three lines of the Tg mice. Thus, we used the offspring from these three founder lines (line A, B, and C) for this study. We have not analyzed line D.
IL-18 Proteins Are Strongly Expressed in the Lungs but Not Other Tissues of SPC-IL-18 Tg Mice
Th1- and Th2-type Cytokine Production in SPC-IL-18 Tg Mice
Histologic Effects of Constitutive IL-18 Overproduction in the Lung, Heart, and Other Organs
Based on the development of increased lung volume with age, we next performed histologic analysis of the lungs during growth. Lung formation was normal, as described previously here, but mild interstitial inflammation in the alveolar wall was observed in Tg mice at approximately 4 weeks after birth. Little mucus metaplasia or alveolar damage was observed in Tg mice of this age (Figure 3A). Around 8 weeks after birth, alveolar enlargement accompanied by infiltration of inflammatory cells was observed in Tg but not WT mice. The inflammatory cells contained increased numbers of mononuclear cells, neutrophils, and eosinophils in Tg mice of this age. In particular, enlarged macrophages and exudates were observed in some of the pulmonary alveoli (Figure 3B). Moreover, thickening of alveolar walls and interstitium accompanied with severe pulmonary inflammation was observed in a part of the lung in aged Tg mice (24 weeks). However, Azan and Elastica van Gieson staining revealed that severe lung fibrosis was not apparent in the lungs (Figure 3C).
Microscopic examination revealed that the hearts of the Tg mice were enlarged when compared with those of WT mice, and the right ventricle (RV) in some Tg mice showed hypertrophy at 8 weeks of age (Figure 3D). Next, we analyzed other organs of SPC-IL-18 Tg mice at necropsy. Enteritis, glomerulonephritis, or hepatitis was not observed in the Tg mice. Moderate splenomegaly was observed, and lymphoid follicle was enlarged in spleens of the Tg mice (Figure 3E). No brain damage was found in the Tg mice (data not shown). Thus, striking pathologic changes in the Tg mice were limited to the lungs and heart.
Alveolar Size and Static Lung Compliance in SPC-IL-18 Tg Mice
Increased lung compliance is also a characteristic feature of human COPD (2). Consistent with our model, static lung compliance in Tg mice (20.98 ± 1.89 µl/cm H2O; n = 4) was significantly higher (p < 0.0048) than in the control WT mice (13.83 ± 1.24 µl/cm H2O; n = 4) at 11 weeks of age. Next, we measured static lung compliance in aged Tg mice when severe interstitial inflammation was seen in the lungs. Static lung compliance in 24-week-old Tg mice (8.22 ± 1.89 µl/cm H2O; n = 6) was significantly lower (p < 0.01) than in the control WT mice (11.42 ± 1.16 µl/cm H2O; n = 6) (Figure 4D). These results demonstrate that decreased lung compliance is observed in aged Tg mice.
Body Weight of SPC-IL-18 Tg Mice
Alveolar Macrophages, CD8+ T Cells, CD4+ T Cells, CD3+ T Cells, Neutrophils, and Eosinophils Are Increased in the Bronchoalveolar Lavage Fluid of SPC-IL-18 Tg Mice
SPC-IL-18 Tg Mice Display Signs of Right-Sided Heart Failure with Aging Echocardiography showed significant (p < 0.03) dilatation of the RV dimension (RVD) in aged (15- to 17-week-old) Tg mice when compared with that of WT mice (Table 2). The aortic dimension, interventricular septal thickness, left atrial dimension, left ventricular end-systolic dimension, and left ventricular end-diastolic dimension did not differ between Tg and WT mice. In contrast, RVD was normal in juvenile SPC-IL-18 Tg mice (data not shown). The results suggest that the aged Tg mice suffered from right-sided heart failure.
Pulmonary Hypertension in SPC-IL-18 Tg Mice with Aging Pulmonary hypertension is often observed in patients with severe COPD (2). Therefore, we analyzed RV pressure by right-sided cardiac catheterization to determine the presence of pulmonary hypertension in 20-week-old SPC-IL-18 Tg mice (Figure 6). The right ventricular systolic pressure in the Tg mice (36.0 ± 3.2 mm Hg; n = 6) was significantly (p < 0.002) higher than in WT mice (16.9 ± 4.0 mm Hg; n = 6). The right ventricular end-diastolic pressure was also significantly higher (p < 0.002) in Tg mice (n = 6; 4.0 ± 1.0 mm Hg) than in WT mice (2.0 ± 0.6 mm Hg; n = 6). In contrast, RV end-diastolic pressure was normal in juvenile (< 9-week-old) Tg mice (16.1 ± 2.1 mm Hg; n = 5). These results revealed the presence of mild pulmonary hypertension in the aged Tg mice.
IFN- –Independent Pulmonary Emphysema and Inflammation in SPC-IL-18 Tg MiceWe found that IFN- was produced at high levels in the lungs of SPC-IL-18 Tg mice (see Figures 1D and 1E). The results suggest that overproduction of IL-18 in the lungs of Tg mice induced emphysema and pulmonary inflammation through up-regulation of IFN-. Therefore, we established IFN-–/– (GKO) SPC-IL-18 Tg mice by backcrossing our initial founder line A with IFN-–/– mice on a B6 background. Morphometric and histopathologic analyses were then performed on mice that were confirmed to be IFN-–/– but Tg-positive (+). The mean alveolar chord lengths of 14-week-old IFN-+/+ Tg– (WT), IFN-+/+ Tg+ (SPC-IL-18), and IFN-–/– Tg+ (SPC-GKO) littermates (n = 4 per group) were 19.7 (± 3.8), 28.0 (± 4.6), and 32.0 (± 3.2) µm, respectively. Surprisingly, the mean alveolar chord length of the IFN-–/– Tg+ mice was significantly greater (p < 0.001) than that of the WT and IFN-+/+ Tg+ mice (Figure 7A). Lung volume (1.07 ± 0.29 ml; n = 4) in the IFN-–/– Tg+ mice was also significantly higher (p < 0.05) than in the IFN-+/+ Tg+ (0.61 ± 0.18 ml; n = 4) or WT mice (0.45 ± 0.03 ml; n = 4) (Figure 7B). Histopathologic analysis revealed that more severe emphysematous change and pulmonary inflammation with swelled alveolar macrophages were observed in the IFN-–/– Tg+ mice than in the IFN-+/+ Tg+ mice or WT littermates (Figure 7C). Thus, the changes we have observed were independent of IFN- gene expression.
IL-18 Levels Were Increased in IFN- –Deficient MiceThe above results suggest that the lack of an effect of IFN- expression may influence IL-18 production in vivo. Thus, we analyzed mature IL-18 levels in BALFs, lungs, and sera of B6 background IFN-–/– mice and control B6 WT mice (n = 11 per group) by ELISA (Figure 7D). IL-18 levels in BALF of 14-week-old IFN-–/– mice and WT mice were 21.2 (± 9.5) and 13.1 (± 3.7) pg/ml, respectively. IL-18 levels in BALF were not significantly increased in IFN-–/– mice. IL-18 levels in the lungs were 130.5 (± 24.8) and 105.6 (± 20.2) pg/ml, respectively. IL-18 levels in sera were 252.8 (± 92.7) and 55.3 (± 16.5) pg/ml, respectively. In contrast to BALF, IL-18 levels in lungs and sera were significantly increased in IFN-–/– mice when compared with control WT B6 mice. These results suggest that endogenous IFN- expression may suppress IL-18 expression in vivo.
IL-13 Involves Pulmonary Emphysema and Inflammation in SPC-IL-18 Tg Mice
Inducible IL-18 Overproduction in the Lungs Leads to Pulmonary Inflammation but Not Emphysema in Adult Mice In order to transiently induce IL-18 gene in the lung tissues, we used a DOX-dependent SP-C promoter to generate inducible lung-specific SP-C-rtTA/tetO-Cre mice (21). Using a CAG-loxp-CAT-loxp-IL-18SP construct (Figure 9A), we established CAG-loxp-CAT-loxp-IL-18SP mice. Triple transgene–positive mice were established by mating with (CAT)-loxp-IL-18SP Tg and SP-C-rtTA/(tetO)-Cre mice. DOX-inducible and lung-specific IL-18 Tg mice are shown in Figure 9B. We first determined if this construct could generate soluble IL-18 in the presence of Cre recombinase (25) in vitro in transient transfection experiments. No mouse IL-18 protein was found in the supernatants of pPGK-expression vector Cre gene alone- or CAG-loxp-CAT-loxp-IL-18SP alone-transfected 293T cells. In contrast, large amounts of IL-18, but not IFN- , were found in the supernatants of pPGK-Cre and CAG-loxp-CAT-loxp-IL-18SP double-transfected 293T cells (Figure 9C). On the basis of this result, we established three lines of CAG-loxp-CAT-loxp-IL-18 Tg mice. Triple transgene–positive mice were established by mating with CAG-loxp-CAT-loxp-IL-18 Tg and SP-C-rtTA/(tetO)-Cre mice. Five-week-old triple transgene–positive mice and control littermates were kept on their usual diet plus water with 2 mg/ml DOX for 4 weeks (n = 4 per group). The mice were then killed and the sera and lung tissues analyzed. DOX administration caused a significant increase (p < 0.05) in IL-18 (180 ± 58 pg/ml) and IFN- (19 ± 5 pg/ml) production in the lungs of the transgene-positive mice, but not their transgene-negative littermates. No significant increases in IL-18 or IFN- production were found in the sera of the transgene-positive mice (Figure 9D). Furthermore, Th2-type cytokines, including IL-5 and IL-13, were not detectable in the transgene-positive mice. In the absence of DOX administration, no significant induction of IL-18 or IFN- production was observed in the sera or lungs of the transgene-positive mice (data not shown).
The mean alveolar chord length was estimated in the triple transgene–positive mice and control transgene-negative WT littermates, and was 17.7 ± 2.6 and 19.8 ± 3.0 µm, respectively. The mean alveolar chord length of the triple transgene–positive mice was significantly lower (p < 0.001) than that of the WT littermates (Figure 9E). Histopathologic analysis revealed that DOX administration caused pulmonary inflammation, with infiltration of mononuclear cells in the alveolar wall and general interstitium, which is characteristic of interstitial pneumonia in humans. We did not observe severe emphysema, swelled alveolar macrophages, hyaline membranes, or proteinaceous debris in the triple transgene–positive mice after DOX administration (Figure 9F). Moreover, no evidence of enhanced lung volumes or cardiomegaly was observed in the triple transgene–positive mice without or with DOX administration (data not shown), suggesting that the triple transgene–positive mice showed normal lung and heart development.
Using the human surfactant promoter, SP-C, we developed new IL-18 Tg mice in which murine IL-18 protein was overproduced in lung tissue. We found that constitutive overproduction of IL-18 induced the subsequent production of both Th1- and Th2-type cytokines, including IL-13 and IFN- , in the lungs. Pulmonary inflammatory cells (including CD8+ T cells, macrophages, and eosinophils), progressive airspace enlargement, enhanced static lung compliance, and right-sided heart failure with mild pulmonary hypertension were observed in the Tg mice. Previously, by using mouse Ig VH and human keratinocyte K5 promoters, we established two different lines of IL-18 Tg mice, Ig-IL-18 Tg (18) and K5-IL-18 Tg (34), in which IL-18 was overproduced in lymphocytes and keratinocytes. However, we did not observe pulmonary inflammation or lung conditions, such as injury, fibrosis, and emphysema, in either of these two lines, suggesting that lung-specific production of IL-18 is essential for the induction of pulmonary inflammation and emphysema. Mice may develop enlarged airspaces due to abnormal lung development or destruction of normal adult lung. It is very important in the mouse models of COPD to clarify whether mice develop enlargement of airspaces due to destruction of normal adult lung or due to failed septation, impairing alveogenesis during lung development (35). Thus, IL-18, expressed as a transgene, may induce abnormal lung development or destruction of normal lung (pulmonary emphysema) in mice. We found that lung maturation with changes in alveolar size was normal during the first 2 weeks in the Tg mice (data not shown). Little interstitial inflammation in the alveolar wall without airspace enlargement was observed until 5 weeks after birth in constitutive IL-18–expressing Tg mice. Airspace enlargement, accompanied by severe pulmonary inflammation, was observed at 6–8 weeks after birth in the Tg mice (Figure 3A). Furthermore, although the mean alveolar chord length was not significantly increased at 5 weeks of age, it was greatly increased at 10 weeks of age in the Tg mice (Figure 4A). The destructive index is known to indicate the severity of alveolar wall destruction (24, 32), and the destructive index in 10-week-old Tg mice was greater than in control WT mice (Figure 4C). Moreover, the body weights of Tg mice and control WT mice were not significantly different until 10 weeks of age. These results suggest that the IL-18 transgene may induce destruction of normal lungs (i.e., pulmonary emphysema) in mice, but may not interfere with the alveogenesis beyond 5–6 weeks of age. In addition, the alterations of airspace enlargement in SPC-IL-18 Tg mice are not simply the result of differences in animal size. However, it is possible that there is abnormal lung development with a progressive destructive component in the lungs of Tg mice, although pulmonary inflammation developed when IL-18 production was induced in the lungs of 5-week-old inducible IL-18 Tg mice for 4 weeks (Figure 9E). These results suggest that 4 weeks may not be enough time to develop emphysematous changes in the lungs of the inducible IL-18 Tg mice. Long (e.g., > 8 weeks) DOX treatment may be necessary to cause emphysema development in these mice, but we could not complete experiments to test this hypothesis, as only a limited number of triple transgene–positive mice can be obtained. Cigarette smoke is the major cause of COPD. It can induce pulmonary inflammation and directly damage the lungs, and the chronic pulmonary inflammation induced by long-term smoking is thought to result in the progressive respiratory disorders seen in COPD (2, 3). Based on the mouse model presented here, we propose that long-term cigarette smoking induces chronic IL-18 overproduction in the lung tissues and, thus, chronic pulmonary inflammation in COPD. Inflammatory cells recruited in the lungs, such as CD8+ T cells, macrophages, and neutrophils, produce large amounts of several mediators, and these substances may result in alveolar destruction in patients with COPD. In support of our hypothesis, we have found that serum IL-18 levels in patients with very severe COPD (Global Initiative for Chronic Obstructive Lung Disease stage IV) were significantly higher than those in smokers or nonsmokers (data not shown). However, further analysis is necessary to more fully substantiate our model.
It has been reported that some patients with COPD have had pulmonary hypertension with right-sided heart failure, whereas others have not. Recent studies have indicated that the pathogenesis of this disease includes pulmonary vascular remodeling, endothelial dysfunction, and pulmonary inflammation (36). However, the precise mechanisms involved in the development and establishment of pulmonary hypertension in COPD are still unknown. Previous reports, using Tg mice, including TNF- Inflammatory cells contained increased numbers of enlarged macrophages, exudates, mononuclear cells, neutrophils, and eosinophils in the lungs of SPC-IL-18 Tg mice. In particular, pulmonary vessels were severely destroyed in some of the pulmonary alveoli in Tg mice (Figure 3B). These results suggest that lower numbers of pulmonary vessels and severe pulmonary inflammation resulted in two different colors in the lungs of aged Tg mice (Figure 2A, right). In aged Tg mice, emphysema and thickening of alveolar walls and interstitium accompanied by severe pulmonary inflammation was observed in part of the lungs. However, server fibrosis was not apparent in the lungs of aged Tg mice (Figure 3C). Interestingly, in aged Tg mice with severe lung interstitial inflammation, decreased static lung compliance is observed, whereas static lung compliance is enhanced in young Tg mice (Figure 4D). These results suggest that aged Tg mice have both emphysema and interstitial inflammation in the lungs. A recent article reported that lung-specific rtTA expression causes emphysema in Clara cell secretory protein (CCSP)-rtTA single-Tg mice and SPC-rtTA single-Tg mice (39). These CCSP-rtTA single-Tg mice and SPC-rtTA single-Tg mice are generated from an offspring of C57BL/6 x SJL background Tg mice. As described previously here, our SPC-rtTA Tg mice are on a C57BL/6N background. It is possible that this strain of mouse is resistant to the induction of emphysema in mice containing an rtTA construct.
Pulmonary lymphocytes that produce IFN-
The Th2-type cytokine IL-13 is believed to play an important role in allergic diseases, including asthma, atopic dermatitis, and contact hypersensitivity (14, 41). In addition, IL-13 also plays a central role in the pathogenesis of several fibrotic disorders, including airway remodeling, progressive systemic sclerosis, IPF, and hepatic fibrosis (42, 43). Here, we have demonstrated that production of the Th2-type cytokines, IL-5 and IL-13, was induced in the lungs of SPC-IL-18 Tg mice. Around 8 weeks after birth, we observed alveolar destruction and infiltration of pulmonary inflammatory cells accompanied by alveolar macrophages, CD8+ T cells, neutrophils, and eosinophils. It has been shown that constitutive IL-13 overproduction in the lungs of CC10-IL-13 Tg mice under the control of the CC10 promoter induces pulmonary fibrosis accompanied by pulmonary inflammation (mainly eosinophils and alveolar macrophages), goblet cell hyperplasia, and the deposition of Charcot-Leyden–like crystals (42). Both lines of Tg mice have phenotypic similarities (e.g., increased pulmonary inflammation with eosinophil infiltration and enlarged alveolar macrophages). However, unlike CC10-IL-13 Tg mice, we did not observe any marked pulmonary fibrosis, goblet cell hyperplasia, or deposition of Charcot-Leyden–like crystals in our SPC-IL-18 Tg mice. The original CC10-IL-13 Tg mice were generated on a CBA x C57BL/6 genetic background. In contrast, our IL-18 Tg mice are on a C57BL/6N background. It is possible that the differences in the mouse genetic background may explain this phenotype discrepancy. Moreover, we established C57BL/6N background IL-13–/– IL-18 Tg+ mice by backcrossing with C57BL/6N IL-13–/– mice. Histologic analysis revealed that pulmonary inflammation and emphysematous changes were prevented in IL-13–/– SPC-IL-18 Tg mice (Figures 8A and 8B). Our results suggest that IL-13, but not IFN- Although some patients with IPF have COPD, including emphysema and chronic bronchitis, COPD is not referred to as an interstitial lung disease (ILD) at this time (2). In an attempt to develop relevant mouse models for these diseases, we have reported that daily administration of IL-18 plus IL-2 induces lethal lung injury in normal mice (19). The treated mice show massive infiltration of polymorphonuclear and mononuclear cells in the pulmonary interstitium, followed by thickening of the alveolar walls, which is characteristic of human interstitial pneumonia. Recently, in another study, we observed increased IL-18 levels in the sera and lungs in patients with IPF (15). These results suggest that IL-18 may play an important role in the pathogenesis of ILD. We then set out to determine whether IL-18 could induce different lung diseases (i.e., ILD or emphysema). We therefore established a line of conditional lung-specific IL-18 Tg (triple-transgene–positive) mice by using lung-specific Cre mice under the control of a DOX-dependent SP-C promoter (21). Dox administration caused a significant increase in IL-18 levels (180 ± 58 pg/ml) in the lungs of the triple-transgene–positive mice (see Figure 9D). In contrast, approximately 10 times the IL-18 levels (1675 ± 282 pg/ml) were found in the lungs of SPC-IL-18 Tg mice (see Figure 1B). The conditional lung-specific IL-18 Tg mice showed interstitial pneumonia accompanied by inflammatory cell infiltration of the pulmonary interstitium, but less emphysema (alveolar destruction) in the lung tissues. These results showed that the histologic features of the lungs of DOX-treated triple-transgene–positive mice were dissimilar to those of SPC-IL-18 Tg mice. These differences might be attributable to the fact that the inducible mice expressed significantly lower levels of IL-18 than the SPC-IL-18 Tg mice, in which IL-18 expression was constitutive. Elias and colleagues reported a similar observation in IL-13 Tg mice (i.e., constitutive IL-13 overproduction in the lungs induced pulmonary fibrosis, which presumably can be referred to as interstitial pneumonia) (42). In contrast, conditional lung-specific IL-13 overproduction in the lungs induces emphysema in adult mice (43). Therefore, we speculate that constitutive and/or strong expression of IL-18 can induce progressive and severe pulmonary inflammation and alveolar destruction, and results in emphysema in mice and, possibly, in humans. In contrast, transient and/or weak expression of IL-18 can induce pulmonary inflammation, and results in interstitial pneumonia. Further analysis is needed to confirm this hypothesis.
The treatment strategy for COPD includes the use of bronchodilators, such as
The authors thank Ms. Keiko Nomiyama and Emiko Kuma (Kurume University, Kurume, Japan) for their technical assistance. They express their appreciation to Dr. Tomoko Betsuyaku (Hokkaido University, Sapporo, Japan) and Dr. Kazutetsu Aoshiba (Tokyo Women's Medical University, Tokyo, Japan) for helpful discussions. The 7SPC/SV40 vector and SPC-reverse tetracycline transactivator/polymeric tetracycline operator–Cre mice were kindly provided by Dr. Jeffrey A. Whitsett (Cincinnati Children's Hospital Medical Center, Cincinnati, OH). IL-13–/– mice were kindly provided by Dr. Andrew N. McKenzie (Medical Research Council, UK). CAG-loxp-CAT-loxp-HES-polyA vector was kindly provided by Dr. Kenichi Yamamura (Kumamoto University, Kumamoto, Japan). The authors also express their appreciation to Mr. Michael Sanford, Ms. Della Reynolds, and Mr. John Wine (National Cancer Institute at Frederick, MD) for performing the RNAse protection assay analysis and animal experiments.
Supported by Grant-in-Aid for Scientific Research (B) 17390244 and Grant-in-Aid for Exploratory Research 18659244 from the Ministry of Education, Science, Sports, and Culture of Japan, a grant from the Kakihara Foundation (Fukuoka, Japan), a grant from the Takeda Science Foundation (Osaka, Japan) (T.H.), and a Grant-in-Aid for Scientific Research (B) 18390244 (H.A.). This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org Originally Published in Press as DOI: 10.1164/rccm.200603-316OC on March 30, 2007 Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form March 3, 2006; accepted in final form March 27, 2007
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ABSTRACT
AT A GLANCE COMMENTARY
chain was used for the Tg mouse generation, the molecular weight of IL-18 in the lungs of the Tg mice was higher than that of recombinant mouse IL-18 protein (18 kD), as previously reported (
