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Published ahead of print on August 12, 2004, doi:10.1165/rcmb.2003-0306OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 31, pp. 619-625, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0306OC

Enhanced Expression of Interleukin-18 and its Receptor in Idiopathic Pulmonary Fibrosis

Yasuhiko Kitasato, Tomoaki Hoshino, Masaki Okamoto, Seiya Kato, Yoshiro Koda, Nobuhiko Nagata, Masaharu Kinoshita, Hideyuki Koga, Do-Young Yoon, Hironobu Asao, Hiroshi Ohmoto, Takeharu Koga, Toru Rikimaru and Hisamichi Aizawa

Departments of Internal Medicine 1, Pathology, and Forensic Medicine, Kurume University School of Medicine, Kurume; Department of Respiratory Medicine, the National Kyushu Medical Center, Fukuoka; Department of Respiratory Medicine, the National Omuta Hospital, Omuta; Nagata Hospital, Yanagawa; Department of Microbiology and Immunology, School of Medicine, Tohoku University, Sendai; Carna Biosciences, Inc., Kobe, Japan; and Laboratory of Cellular Biology, Korea Research Institute of Bioscience and Biotechnology, Taejon, Korea

Address correspondence to: Dr. Tomoaki Hoshino, 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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Idiopathic pulmonary fibrosis (IPF)/usual interstitial pneumonia (UIP) is a major interstitial lung disease (ILD). Recently, we established a new mouse model for ILD in which daily administration of interleukin (IL)-18 with IL-2 induces lethal lung injury, suggesting that IL-18 is involved in the pathogenesis of ILD. Here, utilizing immunohistochemistry, we have analyzed IL-18 and IL-18 receptor (IL-18R) {alpha} expression in the lungs of 18 patients with IPF/UIP and 13 control subjects by using monoclonal anti–IL-18 antibodies and a new monoclonal antibody for IL-18R{alpha} (H44). IL-18 was expressed in bronchoalveolar epithelium, alveolar macrophages, and the endothelium of small vessels in control subjects, and was abundantly expressed in the majority of pulmonary cells in patients with IPF. IL-18R{alpha} was expressed in bronchoalveolar epithelium and alveolar macrophages in control subjects, and was strongly expressed in interstitial cells in patients with IPF, especially in the fibroblastic foci (FF). Interestingly, IL-18R{alpha} expression was only weakly observed in areas showing established fibrosis. Semiquantitative analysis revealed that the histologic FF score was significantly correlated with the IL-18R{alpha} expression level in FF lesions. Moreover, IL-18 levels in the serum and bronchoalveolar lavage fluid of patients with IPF were significantly higher than those in control subjects. Our findings suggest IL-18 and IL-18R are involved in the pathogenesis of IPF/UIP.

Abbreviations: antibody, Ab • alveolar macrophage, AM • bronchoalveolar lavage fluid, BALF • lung diffusing capacity for carbon monoxide, DLCO • established fibrosis, EF • fibroblastic foci, FF • high-resolution computed tomography, HRCT • interferon, IFN • immunoglobulin, Ig • interleukin, IL • IL-18 receptor {alpha}, IL-18R{alpha} • interstitial lung disease, ILD • interstitial mononuclear cell, IMNC • idiopathic pulmonary fibrosis, IPF • monoclonal Ab, mAb • natural killer, NK • observation field, OF • transforming growth factor, TGF • T helper cell type, Th • tumor necrosis factor, TNF • usual interstitial pneumonia, UIP


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Acute and chronic lung disorders with variable degrees of interstitial or intraalveolar inflammation and fibrosis are collectively referred to as interstitial lung diseases (ILD) (for reviews, see Refs. 1–4). Idiopathic pulmonary fibrosis (IPF) is a major human ILD of unknown etiology that has a grave prognosis. IPF is currently classified as one type of the idiopathic interstitial pneumonias. IPF is histopathologically characterized as usual interstitial pneumonia (UIP), and shows progressive interstitial fibrosis accompanied by random and nonuniform foci of inflammation. A rodent model of bleomycin-induced lung fibrosis has been widely used for the investigation of human IPF. Piguet and colleagues previously reported that tumor necrosis factor (TNF)-{alpha} played a critical role in bleomycin-induced lung fibrosis in mice (5). Other cytokines and chemokines (e.g., transforming growth factor [TGF]-ß, interleukin [IL]-1, IL-2, interferon [IFN]-{gamma}, and macrophage inflammatory protein [MIP]-2) are also reported to be involved in the pathogenesis of human IPF. Furthermore, it has become clear that multiple mediators may be involved in the establishment of IPF, including oxygen radicals, eicosanoids, prostaglandin, and apoptosis-related genes (2–4). However, the precise mechanisms involved in the development of IPF are not well understood.

IL-18, discovered as IFN-{gamma}–inducing factor (6), plays an important role in T helper cell type (Th) 1 polarization and various diseases, such as tuberculosis, Leishmaniasis, tuberculoid leprosy, Sjögren's syndrome, rheumatoid arthritis, and Crohn's disease (see review in Ref. 7). Furthermore, we and other groups have reported that, in the absence of IL-12, IL-18 can induce Th2 or Th2-like polarization (7–11). We have established a line of IL-18–transgenic mice that produce increased amounts of both Th1 and Th2 cytokines in vivo (12). IL-18 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-1ß converting enzyme. It has been reported that pro–IL-18 is expressed in a wide range of cells, including Kupffer cells, macrophages, T cells, B cells, osteoblasts, keratinocytes, dendritic cells, astrocytes, and microglia. In contrast, mature IL-18 is barely detectable in these tissues (7). Moreover, a previous report suggested that IL-18 mRNA and protein could be found in the airway epithelium of normal individuals and rodents (13). The IL-18 receptor (IL-18R) complex is composed of at least two chains, IL-18R{alpha} and IL-18Rß, and has homology with the IL-1R/Toll receptor family. IL-18R{alpha} is the extracellular signaling domain of the IL-18R complex (14, 15). It is expressed in a number of tissues, including keratinocytes, vascular endothelial cells, smooth muscle cells, and macrophages (16, 17). Recently, we reported a new murine model of human ILD in which daily administration of IL-18 plus IL-2 induces lethal lung injury. Histologic analysis revealed that the treated mice showed massive infiltration of polymorphonuclear and mononuclear cells in the pulmonary interstitium, followed by thickening of alveolar walls, which is characteristic of human interstitial pneumonia (18). Our findings suggest that the IL-18/IL-18R complex is involved in the pathogenesis of human ILD, including IPF/UIP.

Here, we establish a monoclonal antibody (mAb) against human IL-18R{alpha} (H44), and used immunohistochemical staining to analyze the expression of IL-18 and IL-18R{alpha} in the lungs of 18 patients with IPF/UIP and 13 control subjects. We also measured the levels of IL-18 in the serum of 26 patients with IPF and 20 control subjects, and in the bronchoalveolar lavage fluid (BALF) of 37 patients with IPF and 16 control subjects. In the patients with IPF, the majority of pulmonary cells, especially interstitial fibroblastic foci (FF), (intra)alveolar macrophages (AM), and interstitial mononuclear cells (IMNC), strongly expressed both IL-18 and IL-18R{alpha}. It has been reported that high FF is characteristic of ongoing fibrosis in the lung of patients with IPF (19). Our semiquantitative analysis revealed a strong correlation between histologic FF scores and the IL-18R{alpha} expression levels in FF lesions, suggesting that enhanced IL-18R{alpha} expression in FF manifests severe disease activity. Furthermore, levels of IL-18 were markedly elevated in the serum and BALF of patients with IPF. Finally, we discuss the possible role of IL-18/IL-18R in the pathogenesis of ILD/IPF.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Subjects
Seventy-two patients (45 males and 27 females, aged 41–86 [66.5 ± 8.8] yr) diagnosed between 1982 and 2002 as having IPF were monitored at Kurume University Hospital (Kurume, Japan), the National Kyushu Medical Center (Fukuoka, Japan), the National Omuta Hospital (Omuta, Japan), Yame General Hospital (Yame, Japan), and the Japan Social Insurance Tagawa Hospital (Tagawa, Japan). Forty-nine subjects (29 males and 20 females, aged 39 to 78 [60.4 ± 12.0] years) were used as controls in this study. Among the 72 patients, 3 were sampled for both lung tissue and serum, 1 was sampled for both lung tissue and BALF, and 5 were sampled for both serum and BALF. No patients were sampled for lung tissues, serum, and BALF. All patients with IPF were diagnosed on the basis of clinical history, physical examination, chest X-ray, chest computed tomography, lung function tests, and/or high-resolution computed tomography (HRCT) according to the clinical criteria for diagnosis of IPF by the American Thoracic Society/European Respiratory Society (1, 3, 4). A total of 18 of the 72 patients with IPF underwent an independent histopathologic review by two pathologists, and showed a pattern of UIP: 4 had undergone open-lung biopsies, 9 had video-assisted thoracoscopic surgery, and 5 were examined at autopsy (Table 1). In five autopsies, one died in an accident, two died from brain damage (stroke), and two patients, who had been treated with prednisolone alone (50 mg daily) and methylprednisolone alone (125 mg daily), respectively, died from respiratory failure. None of the patients, except the two patients who died from respiratory failure, had been treated with medication at the time of sampling. Patients with congestive heart failure, infectious diseases, collagen diseases, and other ILD, such as pneumoconiosis, pulmonary sarcoidosis, hypersensitivity pneumonitis, eosinophilic pneumonitis, nonspecific interstitial pneumonia, or cryptogenic organizing pneumonia were excluded from this study. We used 13 lung sections obtained from control subjects (9 males and 4 females) for histopathologic analysis: 6 from patients who had died in accidents (obtained from the Department of Forensic Medicine, Kurume University) and 7 noncancerous lung sections of patients who had received surgery for lung cancer (Table 1). We obtained sera from 26 patients with IPF and 20 control subjects chosen from age- and sex-matched healthy volunteers without lung disease. We analyzed the BALF of 37 patients with IPF. As controls, we used 16 control subjects that visited Kurume University Hospital and underwent BALF analysis. We carefully excluded lung diseases (e.g., lung cancer, sarcoidosis, infectious diseases) from the 16 control subjects. The cell number and cell population (CD4+, CD8+ T cells) were normal, and no malignant cells were found in the control BALF.


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  		TABLE 1. Histopathologic and immunoreactivity scores of patients with idiopathic pulmonary fibrosis/usual interstitial pneumonia

 
Establishment of Anti–Human IL-18R{alpha} mAb (H44)
Human IL-18R{alpha} cDNA (14) was inserted into the EcoRV site of the pcdef3 vector (12). The human IL-18R{alpha}/pcdef3 plasmid was then transfected into P815 cells by using a Gene Pulser II (Bio-Rad, Hercules, CA). Several cell line clones established from the P815–IL-18R{alpha} transfectants were used for hybridoma screening. An anti–human IL-18R{alpha} mAb (H44, mouse immunoglobulin [Ig] G1, {kappa}) was established by fusion of the mouse myeloma cell line NS1 with spleen cells isolated from a BALB/c mouse immunized with the human natural killer (NK) cell line NK0 (20). Currently, this H44 mAb is commercially available from PharMingen (San Diego, CA), eBioscience (San Diego, CA), and Serotec (Oxford, UK).

Histology, Immunohistochemical Assay, and Immunoreactivity Score
Samples of lung tissue obtained by open-lung biopsies, video-assisted thoracoscopic surgery, or autopsy were fixed with 10% formalin and embedded in paraffin wax for conventional analysis with hematoxylin and eosin (H&E) staining. For the analysis of UIP, the histologic features of the lung were scored using a modified method reported by Nicholson (19). A semiquantitative assessment of histologic score was defined for each individual H&E section by using a scale of 0–6 for 4 individual histologic features: (i) extent of FF; (ii) extent of AM; (iii) extent of IMNC infiltrate; and (iv) extent of established fibrosis (EF). Each H&E section was viewed at low-power magnification (40x). One square field (2.5 mm x 3.5 mm; 8.75 mm2), designated as observation field (OF), where disease activity appeared to be most marked, was selected from each H&E section at 40x magnification. The number of FF was counted in a square field (1 mm x 1.4 mm; 1.4 mm2) at 100x magnification within OF (Figure 2A, i, v, and ix). FF score of "x" demonstrates "x" foci per mm2. For example, if 7 FF are found in a square field (1 mm x 1.4 mm) at 100x magnification, then the FF score is 5. For FF, the absence of fibroblastic proliferation was scored as 0, as seen in control subjects (Figure 2A, i), and a level equivalent to that seen in Figure 2A, v, was scored as 5. For AM, a score of 0 or 1 was considered within normal limits (Figure 2A, i), and a score of 6 represented the degree of accumulation seen in desquamative interstitial pneumonia. For IMNC, a score of 0 represented no infiltrating mononuclear cells (Figure 2A, i), and a score of 6 represented the degree of infiltration that would be expected in lymphoid interstitial pneumonia. For EF, a scale of 0 represented no fibrosis, as seen in control subjects (Figure 2A, i), and a score of 5 (Figure 2A, ix) or 6 represented end-stage lung fibrosis or honeycombing. When lung sections were obtained from more than two different sites in a patient with UIP, an average score was taken. Anti–human IL-18 (clone 8 [IgG2a; ascites dilution 1:400–1:1000] and clone 81 [IgM; ascites dilution 1:400 to 1:1,000]) (20) and anti–human IL-18R{alpha} (H44 [IgG1, 0.1 to 2 µg/ml]) mAbs were used at 4°C for 18 h to detect human IL-18 and IL-18R{alpha}, respectively. Mouse purified IgG2a, IgM, and IgG1 Abs (Caltag Laboratories, Burlingame, CA) were used as negative controls. Positive reactivity was identified by 3–3'-diaminobenzidine- 4HCl using an LSAB2 kit (Dako, Kyoto, Japan). Alternatively, we used a New Fuchsin avidin–biotin–alkaline phosphatase detection system (Dako), as previously reported (21). Recombinant human IL-18 (10 µg/ml) (Pepro Tech Inc., Nutley, NJ) and human IL-18R{alpha}/Fc chimera protein (10 µg/ml) (R&D Systems, Minneapolis, MN) were used at room temperature for 60 min for blocking assays to enhance the specific reactivity of anti–human IL-18 and -IL-18R{alpha} mAb, respectively. Using serial sections, we further selected specific areas in the OF in which four individual histologic features (FF, AM, IMNC, and EF) appeared to be the most markedly observed. IL-18 and IL-18R{alpha} immunoreactivity scores for each histologic feature were defined by counting the numbers of positively stained cells in these areas at 400x magnification. Levels of IL-18 or IL-18R{alpha} expression were defined as follows: score 0, < 5% of cells stained with anti–IL-18 or anti–IL-18R{alpha} mAb; score 1, 5–24% of cells stained; score 2, 25–49%; score 3, 50–75%; and score 4, > 75%. Two independent pathologists examined these sections without prior knowledge of the patients' clinical status. Sample collection and all procedures were approved by the institutional ethics committees.

Statistical Analysis
Results are expressed as the mean ± SD. The Mann-Whitney test was used to compare differences between groups, and correlations were analyzed by simple regression. Correlations involving histologic scores were evaluated by Spearman's rank correlation coefficient. P < 0.05 or R2 > 0.5 was considered to represent statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
A New mAb (H44) Recognizes Human IL-18R{alpha}
The H44 mAb reacted with the P815–human IL-18R{alpha} transfectant, but not with the P815–pcdef3 vector transfectant or with parental P815 cells. Flow cytometric analysis revealed that H44 did not react with mouse IL-18R{alpha} (data not shown). Moreover, preincubation of H44 mAb (0.5 µg) with excess recombinant IL-18R{alpha} protein (10 µg) almost completely eliminated the binding activity (Figure 1A). These results confirm the specificity of H44 mAb against human IL-18R{alpha}. H44 had neutralizing activity against recombinant human IL-18 (rhIL-18) as IFN-{gamma} production by NK0 cells in response to rhIL-2 (25 IU/ml) and rhIL-18 (50 ng/ml) was neutralized by H44 mAb in a dose-dependent manner (0.1 to 20 µg/ml). Almost 100% inhibition of IFN-{gamma} production by NK0 was observed with H44 mAb at 10 µg/ml (Figure 1B). Immunoprecipitation analysis revealed that H44 mAb recognized an 80-kD IL-18R{alpha} chain on the 125I-labeled NK0 cells (data not shown) and the P815–human IL-18R{alpha} transfectant, but not the parental P815 cells (Figure 1C).





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  		Figure 1. Establishment of anti–human IL-18R{alpha} mAb (H44). (A) Anti–human IL-18R{alpha} mAb (H44) recognizes human IL-18R{alpha}. P815–pcdef vector transfectant (left panel) and P815–human IL-18R{alpha} transfectant (middle panel) were stained with biotin-labeled H44 mAb (solid) or control isotype-matched biotin mouse IgG1 (gray line). Preincubation of biotin-labeled H44 mAb (0.5 µg) with excess recombinant IL-18R{alpha} protein (10 µg) at room temperature for 20 min almost completely eliminated the activity against P815–human IL-18R{alpha} transfectant (right panel). Streptavidin-phycoerythrin was used for second-step staining, and fluorescence-activated cell sorter analysis was performed as previously reported (30). (B) Anti–human IL-18R{alpha} mAb (H44) has a neutralizing effect on recombinant human IL-18. NK0 cells were pretreated with anti–human IL-18R{alpha} (H44, 0.003–20 µg/ml) for 30 min. Then, NK0 (1 x 106 cells/ml) cells were treated with recombinant human (rh) IL-2 (25 IU/ml) plus rhIL-18 (50 ng/ml) for 18 h, and IFN-{gamma} production from NK0 cells was analyzed with an enzyme-linked immunosorbent assay kit (R&D Systems). (C) Immunoprecipitation analysis revealed that anti–human IL-18R{alpha} mAb (H44) recognized the 80 kD IL-18R{alpha}. We radiolabeled 1 x 107 cells with Na125I and solubilized them in 500 µl of lysis buffer (140 mM NaCl, 25 mM Tris·HCl [pH 7.5], 10 mM ethylenediaminetetraacetic acid, 10 mM Na4P2O7, 1% aprotinin, and 1 mM phenylmethylsulfonyl fluoride) supplemented with 1% Nonidet P-40 or 1% Brij-35 (Pierce, Rockford, IL). The lysates were incubated for 4–8 h at 4°C with anti–human IL-18R{alpha} (H44) or control isotype-matched mouse IgG1 mAbs and protein A-sepharose pretreated with anti-mouse IgG. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis was used to analyze the immunoprecipitation, as described previously (31).

 
Immunohistochemical Analysis and Semiquantitative Assessment of Lung Tissues
We analyzed H&E-stained lung sections of 18 patients with IPF and 13 control subjects (Table 1). ILD or specific inflammatory responses were not observed in the lung tissues of control subjects. In contrast, progressive interstitial fibrosis accompanied by random and nonuniform foci of inflammation, exudation of alveolar macrophages, mononuclear cell infiltration, degeneration of bronchoalveolar epithelium, and thickening of vascular walls was widely detected in the lungs of all tested patients with IPF. Semiquantitative analysis (19) revealed that the mean histologic scores of FF, AM, IMNC, and EF were 3.3, 2.4, 2.7, and 3.1, respectively (Table 1). Our analysis revealed that the bronchoalveolar epithelium, AMs, and endothelium of small vessels constitutively expressed IL-18 in control subjects (Figure 2A, iii). Bronchoalveolar epithelium and AMs also constitutively expressed IL-18R{alpha} in control lung tissues (Figure 2A, iv). In patients with IPF/UIP, the expression of both IL-18 (presumably both the pro- and mature forms) and IL-18R{alpha} was enhanced in native pulmonary cells, as well as in inflammatory infiltrating cells, especially in FF (Figures 2A, vii and viii, amd 2B). In contrast, areas of established lung fibrosis only weakly expressed IL-18R{alpha} in the patients with IPF, irrespective of maintained IL-18 expression (Figure 2A, xi and xii). Both the streptavidin–biotin–peroxidase and avidin–biotin–alkaline phosphatase detection systems showed similar patterns of IL-18 and IL-18R{alpha} expression in the lungs (data not shown). Excess recombinant human IL-18 (10 µg/ml) and IL-18R{alpha}/Fc chimera proteins (10 µg/ml) completely blocked all positive reactivity with anti–human IL-18 mAb and IL-18R{alpha} mAb, respectively (Figure 2B). Moreover, no staining was observed in the lung tissues when utilizing control mouse Ig, nonimmune serum, or omitting the second Ab (data not shown). These results clearly showed that these mAbs specifically recognized IL-18 and IL-18R{alpha} expression in the lungs of patients with IPF.




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  		Figure 2. IL-18 and IL-18R{alpha} expression in the lungs of patients with idiopathic pulmonary fibrosis (IPF). (A) Lung tissues were obtained from a patient with IPF with a high FF score (Patient 1, v–viii: FF=5), a patient with IPF with a high EF score (Patient 2, ix–xii: EF = 5), and a 66-yr-old male control subject (i–iv). Representative histologic results (i, v, ix: H&E staining, original magnification at observation, 100x; ii, vi, x: H&E staining, original magnification at observation, 400x; iii, vii, xi: immunostaining by anti–IL-18 mAb [clone 81], original magnification at observation, 400x; iv, viii, xii: immunostaining by anti–IL-18R{alpha} mAb [H44], original magnification at observation, 400x) are shown. IL-18 and IL-18R{alpha} immunoreactivity scores of FF in Patient 1 are 3 and 3, respectively (vii, viii). IL-18 and IL-18R{alpha} immunoreactivity scores of EF in Patient 2 are 3 and 0, respectively (xi, xii). (B) Excess recombinant human IL-18 or IL-18R{alpha}/Fc chimera proteins blocked positive reactivity with anti–human IL-18 mAb (clone 81) or IL-18R{alpha} mAb (H44), respectively (original magnification, 400x). Moreover, control mouse Ig, nonimmune serum, or omitting second Ab did not result in stain in the lung tissues (data not shown).

 
Next, we attempted to semiquantitatively analyze the relationship between the levels of IL-18 or IL-18R{alpha} immunoreactivity and each of the four different histologic features (FF, AM, IMNC, and EF). The individual histologic and immunoreactivity scores in the patients with IPF/UIP are shown in Table 1. Both IL-18 and IL-18R{alpha} immunoreactivity scores were significantly elevated in FF, IMNC, and EF lesions, whereas expression of IL-18 and IL-18R{alpha} in AM was constant in both patients with IFF and controls. There was a strong correlation between the histologic FF score and the immunoreactivity score for IL-18R{alpha}, but not for IL-18, in FF lesions of patients with IPF (Figure 3). In contrast, no significant correlation was observed between the AM, IMNC, or EF scores and their IL-18 and IL-18R{alpha} immunoreactivity scores in these patients.



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  		Figure 3. Correlation between FF scores and IL-18R{alpha} immunoreactivity in FF lesions of patients with IPF. FF score and IL-18R{alpha} immunoreactivity of FF in the lungs of patients with IPF (n = 18) were analyzed.

 
Increase in Serum or BALF Levels of Mature IL-18, but not of IL-2, IL-4, IL-12, IL-13, TNF-{alpha}, or IFN-{gamma} in Patients with IPF
Serum levels of mature IL-18 in 26 patients with IPF (536.5 ± 273.6 pg/ml) were significantly higher than those in 20 control subjects (82 ± 48 pg/ml) (Figure 4A; P = 9.55 x 10–9). Levels of mature IL-18 in the BALF of 37 patients with IPF (58.2 ± 52.7 pg/ml) were significantly higher than those in 16 control subjects (9.65 ± 17.34 pg/ml) (Figure 4B; P = 4.9 x 10–5). Mean levels of serum IL-2, IL-4, IL-12, IL-13, TNF-{alpha}, and IFN-{gamma} in 26 patients with IPF were < 7 pg/ml, < 10 pg/ml, < 5 pg/ml, < 32 pg/ml, 4.72 pg/ml, and 9.53 pg/ml, and mean levels of the same cytokines in the BALF of patients with IPF were < 7 pg/ml, < 10 pg/ml, < 5 pg/ml, < 32 pg/ml, < 4.4 pg/ml, and < 8 pg/ml, respectively. None of the levels of these other cytokines was significantly increased in the serum or BALF of the patients with IPF as compared with the healthy control subjects. We further analyzed the correlation between the serum and BALF levels of mature IL-18 and various clinical parameters in the patients with IPF. Levels of mature IL-18 in patient serum and BALF were not significantly correlated with sex, age, percent vital capacity, percent lung diffusing capacity for carbon monoxide (DLCO), arterial oxygen pressure, peripheral white blood cell numbers, lactate dehydrogenase levels, or erythrocyte sedimentation rate (ESR) levels (data not shown). KL-6 is a high-molecular-weight mucin–like glycoprotein expressed by type II epithelial cells, and its serum level has been reported to be well correlated with disease activity in patients with IPF (22). Serum KL-6 levels in 26 patients with IPF (1,244.2 ± 810.1 U/ml) were significantly (P = 2.03 x 10–7) higher than those in control subjects (206.6 ± 50.3 U/ml). However, levels of mature IL-18 in the serum and BALF were not significantly correlated with KL-6 levels in the patients with IPF or the healthy control subjects. Moreover, the IL-18R{alpha} immunoreactivity scores of FF were not significantly correlated with KL-6 levels in the patients with IPF tested (data not shown).




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  		Figure 4. Increased levels of IL-18 in serum and BALF of patients with IPF. (A) Serum levels of mature IL-18 in patients with IPF (n = 20) and age- and sex-matched healthy control subjects (n = 26) were measured with an enzyme-linked immunosorbent assay kit (MBL, Nagoya, Japan). (B) BALF levels of mature IL-18 in patients with IPF (n = 37) and control subjects (n = 9) were measured with an enzyme-linked immunosorbent assay kit.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We found that bronchoalveolar epithelium, AMs, and the endothelium of small vessels constitutively expressed IL-18 in control subjects, and that the IL-18R{alpha} receptor was also expressed in the bronchoalveolar epithelium and alveolar macrophages of control subjects (Figure 2A). Small amounts of IL-18 were detectable in the BALF of control subjects (Figure 4B), suggesting that basal expression of IL-18 and endogenous IL-18 signaling may exist in normal lung tissue. A previous study reported that IL-18 mRNA and protein was observed in the bronchoalveolar epithelium of normal individuals and BALB/c mice. The study also revealed that there was altered IL-18 expression during lung inflammation (13). The present study also showed that IL-18 and IL-18R{alpha} expression in normal pulmonary tissue, and expression of both proteins, was enhanced in patients with IPF. In particular, interstitial fibroblasts, regenerative bronchoalveolar epithelium, and AMs were recognized as major sources of IL-18 and IL-18R{alpha} in the lungs of patients with IPF. However, IL-18R{alpha} was expressed poorly in areas of established fibrosis in patients with IPF (Figure 2A, Table 1). There was a strong correlation between histologic FF scores and the IL-18R{alpha} (but not IL-18 ligand) immunoreactivity scores of FF in patients with IPF (Figure 3). FF are thought to be hyperplastic features of IPF (18, 24), and they strongly express both IL-18R{alpha} and its ligand. Our data suggest that IL-18 may contribute not only to the inflammatory process, but also to processes of pulmonary tissue remodeling, such as interstitial fibrosis, thickening of the vascular wall, and epithelial regeneration. King and coworkers showed that an increase in the number of FF was correlated with increased mortality rates in patients with IPF (23). Our data suggest that increased IL-18R{alpha} expression level in FF is associated with a greater extent of FF in the lungs of patients with IPF (i.e., higher disease activity). Recently, Nicholson and colleagues reported that increasing FF scores were associated with increased mortality and decline of both forced vital capacity and DLCO at 6 and 12 mo after lung biopsy (19). Therefore, we are analyzing whether increased IL-18R{alpha} immunoreactivity of FF was associated with increased mortality and the decline of both forced vital capacity and DLCO after lung biopsy in patients with IPF/UIP. There was no significant correlation between FF score and the IL-18 immunoreactivity of FF, but the correlation between FF score and IL-18R{alpha} immunoreactivity was significant. As our anti–human IL-18 mAbs (clones 81 and 8) recognized both the pro-IL-18 and mature forms of IL-18 (20), we could not determine which form of IL-18 was expressed in patients with IPF, and it is possible that the FF score is correlated with either pro-IL-18 or mature IL-18 expression in FF in patients with IPF. Further analysis is needed to clarify this issue.

Our data demonstrate that IL-18 levels in serum and BALF were increased in patients with IPF but not in healthy subjects. Recently, it has been reported that IL-18 levels are increased in the serum or lungs of patients with advanced tuberculosis (24) or sarcoidosis (25). These findings and our results suggest that increased expression of IL-18 and its receptor in the lung might be involved in the pathogenesis of several lung diseases, including IPF, tuberculosis, and sarcoidosis. The routine laboratory evaluation of a patient suspected of having IPF is often not helpful, although some laboratory markers (e.g., ESR, lactate dehydrogenase, KL-6) are increased in some patients with IPF (3, 19). Analysis of IL-18 and its receptor in the lungs, or of serum/BALF IL-18 levels, may be helpful in diagnosis, analysis of disease activity, and in the prognosis of IPF.

Previous studies have demonstrated that expression of the proinflammatory cytokine TNF-{alpha} is induced in bleomycin- and silica-induced lung fibrosis (5, 26). Therefore, TNF-{alpha} is thought to be an important mediator in the establishment of lung fibrosis. IFN-{gamma} also mediates bleomycin-induced pulmonary inflammation and fibrosis in mice (27). Moreover, previous studies have shown that various Th1-type cytokines, including IL-2, granulocyte macrophage colony–stimulating factor, and IFN-{gamma}, are released from CD4+ T cells in the lungs of patients with IPF, including those with sarcoidosis (28). A recent study has shown that lung-specific expression of the Th2 cytokine IL-13 in transgenic mice, in which IL-13 is overexpressed under the control of the CC10 promoter, causes pulmonary fibrosis through activation of TGF-ß (29). More recently, we reported that daily administration of IL-18 with IL-2 in mice results in death from interstitial pneumonia (18). In the present study, we found enhanced pulmonary cell expression of IL-18 and increased serum/BALF levels of mature IL-18 in patients with IPF. These results suggest that a number of cytokines, including IL-13, IL-18, IFN-{gamma}, TNF-{alpha}, and TGF-ß, may play important roles in the establishment of human IPF.

In conclusion, the majority of pulmonary cells, and typically those components of FF, strongly expressed both IL-18 and IL-18R{alpha} in patients with IPF/UIP. There was a close correlation between FF score and IL-18R{alpha} immunoreactivity score of FF in patients with IPF. Serum and BALF levels of IL-18 in patients with IPF were significantly higher than those in normal control subjects. Our results suggest that the proinflammatory cytokine IL-18 plays an important role in the lung pathology of human IPF/UIP. Currently, anti-inflammatory drugs, such as corticosteroids, azathioprine, and cyclophosphamide, are used alone or in combination for the treatment of IPF, but such regimens have demonstrated only marginal efficacy and cannot halt the progression of the disease (2, 3). Our results raise the possibility that blocking of IL-18 expression in vivo (e.g., with anti–IL-18 or anti–IL-18R mAbs or with IL-18 binding protein) may be of clinical benefit in the treatment of IPF.


   Acknowledgments
 
The authors thank Dr. Howard A. Young (NCI-Frederick, Frederick, MD) for helpful discussions, and Drs Akihiro Hayashi, Shinzo Takamori (Department of Surgery, Kurume University), Mikiko Emori (the National Omuta Hospital, Fukuoka, Japan), Yukari Ikedou, Kazuhiko Yamada (General Yame General Hospital, Fukuoka, Japan), and Mamoru Nishiyama (the Japan Social Insurance Tagawa Hospital, Fukuoka, Japan) for offering the lung sections. This work was supported by grants from the Long-Range Research Initiative by the Japan Chemical Industry Association (JCIA, Tokyo, Japan), Uehara Memorial (Tokyo, Japan), Kanae (Osaka, Japan), Nagao Memorial (Tokyo, Japan), Ishibashi (Tokyo, Japan), Japan Allergy (Tokyo, Japan), and Mitsui Medical Science Promotion (Tokyo, Japan) Foundations, and by a Grant-in-Aid for Scientific Research on Priority Areas (C) "Medical Genome Science" from the Ministry of Education, Science, Sports, and Culture of Japan to T.H.

Received in original form August 15, 2003

Received in final form July 8, 2004


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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