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Published ahead of print on June 19, 2003, doi:10.1164/rccm.200209-982OC
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American Journal of Respiratory and Critical Care Medicine Vol 168. pp. 1075-1083, (2003)
© 2003 American Thoracic Society

Redox-active Protein Thioredoxin Prevents Proinflammatory Cytokine- or Bleomycin-induced Lung Injury

Tomoaki Hoshino, Hajime Nakamura, Masaki Okamoto, Seiya Kato, Shinichi Araya, Keiko Nomiyama, Kotaro Oizumi, Howard A. Young, Hisamichi Aizawa and Junji Yodoi

Departments of Internal Medicine 1 and Pathology, Kurume University, Kurume, Fukuoka; Department of Biological Responses, Institute for Virus Research, Kyoto University, Kyoto, Japan; and Laboratory of Experimental Immunology, DBS, NCI-Frederick, Frederick, Maryland

Correspondence and requests for reprints should be addressed to 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
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Thioredoxin (TRX) is a multifunctional redox (reduction/oxidation)-active protein that scavenges reactive oxygen species by itself or together with TRX-dependent peroxiredoxin. TRX also has chemotaxis-modulating functions and suppresses leukocyte infiltration into sites of inflammation. Leukocyte infiltration and oxidative stress may be involved in the pathogenesis of several diseases, including interstitial lung diseases (ILD). We examined the effects of TRX in two mouse models of human ILD. Recently, we established a new mouse model for human ILD in which daily administration of proinflammatory cytokine interleukin (IL)-18 with IL-2 induces lethal lung injury accompanied by acute interstitial inflammatory responses. Administration of recombinant TRX suppressed IL-18/IL-2–induced interstitial infiltration of cells and prevented death and lung tissue damage. TRX-transgenic mice also showed resistance to lethal lung injury caused by IL-18/IL-2. Administration of bleomycin induces the infiltration of polymorphonuclear and mononuclear leukocytes in the pulmonary interstitium, followed by progressive fibrosis. Wild-type mice given recombinant TRX treatment and TRX-transgenic mice demonstrated a decrease in bleomycin-induced cellular infiltrates and fibrotic changes in the lung tissue. These results suggest that TRX modulates pulmonary inflammatory responses and acts to prevent lung injury. TRX may have clinical benefits in human ILD, including lung fibrosis, for which no effective therapeutic strategy currently exists.

Key Words: thioredoxin • redox • interstitial lung diseases • cytokine • bleomycin

Acute and chronic lung disorders with variable degrees of pulmonary inflammation and fibrosis are collectively referred to as interstitial lung diseases (ILD) (for a review, see References 1–3). Acute interstitial pneumonia is characterized clinically by a rapid onset of respiratory failure and has a grave prognosis with over 70% mortality in 3 months, despite mechanical ventilation. Acute interstitial pneumonia resembles acute respiratory distress syndrome (ARDS), which is induced by diverse causes such as highly concentrated oxygen, poisonous gases, severe infections, and shock status. The histologic basis of acute interstitial pneumonia is the infiltration of leukocytes into the pulmonary interstitial space and diffuse alveolar destruction. The majority of chronic ILD are referred to as idiopathic pulmonary fibrosis. Recently it has become clear that multiple mediators, including reactive oxygen species (ROS), cytokines, chemokines, eicosanoids, prostaglandin, and apoptosis-related genes may be involved in the establishment of ILD. However, the pathogenesis of human ILD is still not well understood (1–5).

Interleukin (IL)-18 is a proinflammatory cytokine and was discovered as an IFN-{gamma} inducing factor in a Propionibacterium acnes–induced mouse model of toxic shock (6). IL-18 is considered to be a strong cofactor in Th1 cell development (reviewed in References 7 and 8). However, we and other investigators have reported that IL-18 induces production of not only the Th1 cytokine IFN-{gamma} but also Th2 cytokines, including IL-4, IL-5, IL-10, and IL-13, in murine model systems (8–11). It has been reported that IL-18 also plays a critical causative role in the pathogenesis of various diseases, including asthma, organ failure, and lethal endotoxemia (7, 8). Recently we reported a new mouse model for human ILD: in this model, daily administration of proinflammatory cytokine IL-18 with IL-2 induces rapid lung injury but no other tissue damage and results in death from interstitial pneumonia (12).

Bleomycin, a member of the glycopeptide group of antibiotics, is a chemotherapeutic drug used clinically for a variety of human malignancies. It has been reported that administration of a high dose of bleomycin often leads to lung injury and pulmonary fibrosis in bleomycin-treated patients. Bleomycin-induced lung fibrosis is a widely used animal model for human idiopathic pulmonary fibrosis (4, 5). Several studies have indicated that ROS (e.g., oxygen radicals) are involved in bleomycin-induced lung injury because the antioxidants superoxide dismutase (13) and N-acetyl-L-cysteine (14) can partly inhibit bleomycin-induced lung injury. Therefore, bleomycin-induced lung fibrosis is mediated, at least in part, by the generation of ROS.

Thioredoxin (TRX) is a 12-kD ubiquitous protein with a redox (reduction/oxidation)-active dithiol/disulfide at the highly conserved active site (–Cys–Gly–Pro–Cys–) found in both prokaryotic and eukaryotic genomes. This protein has been shown to catalyze a protein disulfide reduction in combination with TRX reductase and nicotinamide-adenine dinucleotide phosphate. TRX was originally identified as an electron donor for ribonucleotide reductase in Escherichia coli (15, 16). We cloned human TRX as adult T cell leukemia–derived factor produced by human lymphotropic virus type-I–transformed T cells (17). Recent studies have shown that TRX is induced by a variety of stress conditions including viral infection, ischemic insult, exposure to UV light, X-ray irradiation, and hydrogen peroxide exposure (18). It has been reported that TRX is a scavenger of ROS. In vitro and in vivo studies have demonstrated that TRX has a protective effect against ROS-induced cellular damage (19, 20). TRX-transgenic (TRX-Tg) mice, in which TRX expression is driven by the human ß-actin promoter, are more resistant to focal cerebral ischemia than are control wild-type (WT) C57BL/6 mice (21). Furthermore, our recent study showed that intravenous administration or overexpression of TRX suppresses neutrophil extravasation into inflammatory sites in the mouse chemotaxis air pouch model (22, 23).

The aim of our present study was to evaluate the possible therapeutic effect of TRX in preventing lung injury. We investigated the effects of exogenous recombinant TRX (rTRX) administration and TRX overexpression in transgenic mice using bleomycin- and IL-18/IL-2–induced mouse models of human ILD.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice and Reagents
We used juvenile (< 10-week-old) female C57BL/6 (B6) mice. B6 background TRX-Tg mice (21) and recombinant human TRX were kindly supplied by Ajinomoto, Co. Inc. (Kawasaki, Japan).

Analysis of Pharmacologic Kinetics of rTRX
Mice were intraperitoneally injected with 40 µg of rTRX. They were then bled and killed at 0 (untreated control group), 1, 3, 6, 18, 24, or 48 hours after the injection. The lung tissues were homogenized in 1 ml of lysis buffer. The concentrations of TRX were measured by sandwich ELISA.

In Vivo Treatment of Mice with rTRX
Mice were treated daily or every second day with an intraperitoneal injection of 40 µg rTRX suspended in 100 µl sterile phosphate-buffered saline (PBS), and 40 µg ovalbumin (OVA, Sigma, St. Louis, MO) was used as a control.

IL-18/IL-2–induced Lung Injury Model
Mice were treated daily with an intraperitoneal injection of 200 µl of recombinant human IL-2 and/or recombinant mouse IL-18, as previously reported (12).

Bleomycin-induced Lung Fibrosis Model and Hydroxyproline Assay
Mice were treated with an intraperitoneal injection of bleomycin (100 mg/kg; Nippon Kayaku, Tokyo, Japan), dissolved in 200 µl sterile PBS. The hydroxyproline assay was performed by capillary electrophoresis (24).

Histologic Examinations
For the histologic analysis, the lungs were fixed as previously reported (25). For the semiquantitative analysis of IL-18/IL-2–induced lung injury, the cell numbers in the pulmonary interstitium were measured. Total numbers of pulmonary cells in the alveolar wall and general interstitium were hand-counted in 10 random high-power fields (hpfs) (observation at x400) of the lung hematoxylin and eosin section of each mouse. For the analysis of bleomycin-induced lung fibrosis, the fibrotic responses of the lung were scored as previously reported (26).

Cytokine Assays of the Lung and Serum
Total RNA was isolated from the lungs with an RNeasy Midi Kit (Qiagen Inc., Valencia, CA). Cytokine and chemokine messenger RNA (mRNA) expression was analyzed by ribonuclease protection assay using a RiboQuant kit (PharMingen, San Diego, CA), as previously described (27). For ELISA measurement of cytokines (28), the lung tissue was suspended and homogenized 1:4 (w:v) in sterile ice-cold PBS containing 0.1% Tween 20, and then centrifuged at 20,000 x g for 15 minutes. The supernatants were collected and stored at -80°C until assay.

Statistical Analysis
Unpaired Student's t tests were used to compare differences between groups. Survival curves were analyzed by the Kaplan–Meyer log-rank test. A p value of less than 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Pharmacologic Kinetics of rTRX
B6 mice were given 40 µg of rTRX by intraperitoneal injection, and the concentrations of TRX in the lung tissues and sera were measured 0, 1, 3, 6, 18, 24, or 48 hours after the injection. The TRX levels in the lung tissues were less than 0.1, 22.2, 10.1, 6.9, 4.9, 4.6, and 4.6 ng/ml, respectively. The TRX levels in the sera were less than 0.1, 858.1, 217.5, 77.2, 7.9, 4.4, and 0.7 ng/ml, respectively. The half-life (t1/2) of rTRX in the lung and serum was 51.3 and 8.5 hours, respectively. In the following studies we therefore gave 40 µg rTRX intraperitoneally every second day.

Treatment with rTRX Suppresses Lethal Lung Injury Caused by IL-18 plus IL-2
We previously reported the IL-18/IL-2–induced lung injury model (12). Daily administration (once/day) of IL-18 (0.04 to 2 µg/day) or IL-2 alone (1,000–1 million IU/day) did not induce lethality in normal B6 mice. However, daily administration of IL-18 plus IL-2 was lethal in B6 mice in an IL-18– and IL-2–dose-dependent manner. We found that both male and older than 10-week-old female mice succumbed to IL-18/IL-2 treatment but were more resistant than juvenile female mice, suggesting that body weight and sex could influence the lethality induced by IL-18/IL-2 treatment. Therefore, we used younger than 10-week-old female TRX-Tg and age-matched WT mice in all of the following studies. Juvenile female B6 mice were treated with an intraperitoneal injection of rTRX (40 µg) or control OVA (40 µg) every second day from Day -1. Then the mice were injected daily with 0.2 µg IL-18 and 50,000 IU IL-2 from Day 0. Two repeated experiments found that 18 days after the beginning of treatment 8 of 10 (80%) rTRX-treated mice had survived, compared with only 1 of 10 (10%) OVA-treated control mice. Survival analysis revealed that the rTRX-treated group survived significantly longer than the OVA-treated group (p = 0.0027) (Figure 1A) . Histologic analysis revealed that administration of rTRX, but not of OVA, dramatically inhibited the inflammatory infiltration in the interstitium and collapse of the alveolar spaces induced by IL-18/IL-2 treatment (Figure 1B). We counted cell numbers in the inflamed pulmonary interstitium to semiquantitatively evaluate the effect of rTRX in IL-18/IL-2–induced lung injury model. In the sections obtained at Day 8, cell numbers in the pulmonary interstitium were 734 ± 108 cells/hpf in untreated B6 mice, 1,466 ± 76 cells/hpf in OVA-treated mice, and 932 ± 53 cells/hpf in rTRX-treated mice (p = 0.0005 vs. OVA-treated mice, Figure 1C).





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  		Figure 1. Recombinant thioredoxin (rTRX) treatment prevented lethal lung injury caused by interleukin (IL)-18 plus IL-2. Juvenile (< 10-week-old) female C57BL/6 (B6) mice were treated with an intraperitoneal injection of rTRX (40 µg) or control ovalbumin (OVA) (40 µg) every second day from Day -1. The mice were then treated daily with an intraperitoneal injection of IL-18 (0.2 µg) plus IL-2 (50,000 IU) suspended in 200 µl phosphate-buffered saline (PBS) (control mice were treated with 200 µl PBS) from Day 0, and the surviving mice were killed at Day 18. None of the mice treated with control PBS, rTRX, or OVA alone died (n = 10, each group). p = 0.0027: OVA-treated group versus rTRX-treated group (A). The lung tissue was stained with hematoxylin and eosin (H&E) and was microscopically observed at x200. OVA/IL-18 + IL-2– (B, left panel) and TRX/IL-18 + IL-2–treated mice were killed at Day 8 (B, right panel). (C) For the semiquantitative analysis of IL-18/IL-2–induced lung injury, the cell numbers in the pulmonary interstitium were measured at Day 8. Total numbers of pulmonary cells in the alveolar wall and general interstitium were hand-counted in 10 random high-power fields (hpfs) (observation at x400) of the lung H&E section of each mouse. Results are expressed as the mean ± SD in 10 random hpfs for 5 mice per group.

 
TRX-Tg Mice Are Resistant to the Lethal Effects and Lymphocyte Infiltration Induced by IL-18 plus IL-2
We used TRX-Tg mice to test the hypothesis that TRX could suppress lung injury caused by IL-18 plus IL-2. Eight-week-old female B6 background TRX-Tg (n = 8) and age-matched female WT B6 mice (n = 10) were treated daily with 0.2 µg IL-18 plus 50,000 IU IL-2, as described previously. TRX-Tg mice were more resistant to IL-18/IL-2 administration than were control B6 mice (p = 0.00108) (Figure 2A) . Histologically, at Day 8, lymphocytic infiltration was minimally observed in the pulmonary interstitium of the IL-18/IL-2–treated TRX-Tg mice, whereas severe lung injury was observed in the lungs of control B6 mice (Figure 2B). Semiquantitative analysis performed at this time point revealed that cell numbers in the pulmonary interstitium were 1,356 ± 61 cells/hpf in control B6 mice versus 882 ± 72 cells/hpf in TRX-Tg mice (p = 0.005, Figure 2C).





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  		Figure 2. Thioredoxin-transgenic (TRX-Tg) mice were more resistant to lethal lung injury caused by interleukin (IL)-18 plus IL-2. Eight-week-old female B6 background TRX-Tg (n = 8) and control wild-type (WT) B6 (n = 10) mice were treated daily with IL-18 (0.2 µg) plus IL-2 (50,000 IU), and killed at Day 26. p = 0.00108: WT versus TRX-Tg mice (A). The lung tissue was microscopically observed with hematoxylin and eosin (H&E) staining. Original magnification at x200. IL-18/IL-2–treated control WT mice (B, left panel) and TRX-Tg mice were killed on Day 8 (B, right panel). (C) The numbers of cells in the pulmonary interstitium were measured on Day 8. Total numbers of pulmonary cells in the alveolar wall and general interstitium were hand-counted in 10 random high-power fields (hpfs) (observation at x400) of the lung H&E section of each mouse. Results are expressed as the mean ± SD for 5 mice per group.

 
TRX Can Modulate Cytokine and Chemokine Expression as well as Cell Infiltration in the Lung
Cytokine and chemokine gene expression is strongly induced in the lungs of IL-18/IL-2–treated mice (12). We analyzed whether TRX can modulate cytokine and chemokine expression induced by IL-18/IL-2. Lymphotactin, macrophage inflammatory protein-1ß, macrophage inflammatory protein-1{alpha}, IFN-{gamma}–inducible protein-10, tumor necrosis factor (TNF)–-ß, TNF-{alpha}, and IFN-{gamma} mRNA levels induced by IL-18/IL-2 (lane 2 of Figure 3A) were 3.4-, 176.7-, 510.0-, 782.4-, 59.0-, 2.5-, and 22.0-fold higher, respectively, than with control PBS treatment (lane 1). The respective mRNA levels induced by IL-18/IL-2/rTRX (lane 3) were 0.1-, 0.1-, 0.1-, 104.4-, 0.1-, 1.1-, and 1.1-fold higher than with control PBS treatment (lane 1). Therefore, the mRNA levels induced by IL-18/IL-2 (lane 2) were, respectively, 34.0-, 1,767.0-, 5,100.0-, 7.5-, 590.0-, 2.3-, and 20.0-fold higher than with IL-18/IL-2/rTRX (lane 3), when normalized with control PBS treatment (lane 1). TRX was able to suppress lymphotactin, macrophage inflammatory protein-1{alpha}/ß, inducible protein-10, TNF-{alpha}ß, and IFN-{gamma} mRNA induction in the lungs of the IL-18/IL-2–treated mice at this time point. On the basis of these results, we evaluated the protein levels of macrophage inflammatory protein-1{alpha}, IFN-{gamma}, TNF-{alpha}, and IL-1ß by using ELISA kits. We found that rTRX treatment significantly suppressed macrophage inflammatory protein-1{alpha} and IFN-{gamma} protein levels in the lungs of IL-18/IL-2–treated mice (n = 10) (Figure 3B). However, rTRX treatment did not significantly suppress TNF-{alpha} and IL-1ß protein expression in the lung. We also analyzed cell infiltration in the lungs of control PBS, IL-18/IL-2/OVA, and IL-18/IL-2/rTRX–treated mice. The total numbers of harvested cells were, respectively, 2.05 ± 0.99 x 107, 3.92 ± 1.60 x 107, and 2.24 ± 1.01 x 107 cells (n = 6 in each group). rTRX treatment significantly suppressed cell infiltration in the lungs of IL-18/IL-2–treated mice (p = 0.04 vs. IL-18/IL-2/OVA–treated mice). The same observation was obtained in the lung hematoxylin and eosin sections (Figure 1C). Flow cytometric analysis revealed that ~ 2 to 5, 40, and 40% of cells, respectively, were CD3- NK1.1+ natural killer (NK) cells. The percentage and absolute number of CD3- NK1.1+ NK cells was increased in the lungs of IL-18/IL-2/OVA–treated mice. However, there were no dramatic differences in the percentages of CD3- NK1.1+ NK cells, CD4+ T cells, CD8+ T cells, and Gr1+ CD11b (Mac1{alpha})+ F4/80+ macrophages or granulocytes in the lungs of IL-18/IL-2/OVA– and IL-18/IL-2/rTRX–treated mice (data not shown).




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  		Figure 3. Recombinant thioredoxin (rTRX) treatment prevented cytokine and chemokine induction in the lungs of interleukin (IL)-18/IL-2–treated mice. (A) Juvenile female B6 mice were treated with an intraperitoneal injection of rTRX (40 µg) every day from Day -1. The mice were then treated daily with IL-18 (1 µg) plus IL-2 (50,000 IU) suspended in 200 µl phosphate-buffered saline (PBS) or control PBS from Day 0 for 3 days. Two hours after the third injection, the mice were killed. The lung tissue was immediately harvested, and total RNA (1 µg) was used for messenger RNA (mRNA) analysis using a multiprobe ribonuclease protection assay. Lane 1, control PBS; lane 2, IL-18 plus IL-2; and lane 3, IL-18 plus IL-2 with rTRX treatment. (B) With or without rTRX treatment, B6 mice (n = 10 each) were treated with control PBS, IL-2 (50,000 IU) and/or IL-18 (1 µg) once a day, as described previously. Six hours after the third injection, the mice were killed. The lung tissue supernatants were assayed by sandwich ELISA as described in METHODS. Macrophage inflammatory protein (MIP)-1{alpha} and IFN-{gamma} levels in the lung of IL-18/IL-2– and TRX/IL-18/IL-2–treated B6 mice were significantly (p < 0.05) higher than those in control PBS-, IL-2–, and IL-18–treated B6 mice.

 
rTRX Treatment Prevents Bleomycin-induced Lung Fibrosis
Juvenile female B6 mice were given bleomycin (100 mg/kg) at Days 0, 7, 14, and 21 (total four times) plus rTRX (40 µg every other day), and were killed at Day 28. In this model, rTRX treatment did not suppress this bleomycin (4x)-induced lung fibrosis (data not shown). However, rTRX treatment significantly suppressed lung fibrosis in B6 mice given bleomycin (3x) at Days 0, 7, and 14. Treatment with rTRX strongly suppressed lung fibrosis in B6 mice given bleomycin (2x) at Days 0 and 7 (Table 1 , Figure 4A) . Both hydroxyproline content and wet lung weight in the lungs of B6 mice treated with bleomycin (2x) plus rTRX was significantly lower than that in surviving B6 mice treated with bleomycin plus OVA (n = 5 in each group, Figure 4B). However, rTRX treatment could not completely prevent bleomycin-induced lung injury. It is worth noting that none of the B6 mice treated with bleomycin (2x) plus rTRX died, but 5 of the 10 B6 mice treated with bleomycin (2x) plus OVA died.


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  		TABLE 1. Effect of recombinant thioredoxin on bleomycin-induced lung fibrosis: fibrotic scoring

 



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  		Figure 4. Recombinant thioredoxin (rTRX)-treatment prevented bleomycin-induced lung fibrosis. (A) Juvenile female B6 mice were treated with an intraperitoneal injection of rTRX (40 µg) or control OVA (40 µg) every second day from Day -1. The mice were intraperitoneally treated with bleomycin (100 mg/kg), on Days 0 and 7. Mice were killed on Days 3, 7, or 28. The lung tissue was microscopically observed with hematoxylin and eosin (H&E) (i–iii, vi–viii), Azan (iv, ix), or Elastica van Gieson (EVG) (v, x) staining. Original magnification at observation x200. (B) Hydroxyproline content of the lungs and wet lung weights were measured at Day 28 (n = 5 in each group).

 
Bleomycin-induced Lung Fibrosis Is Prevented in TRX-Tg Mice
Juvenile female TRX-Tg and control WT B6 mice (n = 5 in each group) were treated with bleomycin (100 mg/kg) at Days 0, 7, 14, and 21, and histologic analysis was performed at Days 3, 7, or 28. The histologic changes induced by bleomycin were reduced in TRX-Tg mice at all days examined, when compared with control WT B6 mice (Figure 5A) . The fibrotic score (26) counted at Day 28 in bleomycin-treated TRX-Tg mice was also significantly lower than that in control WT mice (Table 2) . Hydroxyproline content in the lungs of bleomycin-treated TRX-Tg mice was significantly lower than that in control WT mice. Moreover, the mean wet lung weight in bleomycin-treated WT mice was significantly higher than that in bleomycin-treated TRX-Tg mice (Figure 5B).




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  		Figure 5. Bleomycin-induced lung fibrosis was prevented in thioredoxin-transgenic (TRX-Tg) mice. (A) Juvenile female B6 TRX-Tg and control wild-type (WT) B6 mice were intraperitoneally treated with bleomycin (100 mg/kg) at Days 0, 7, 14, and 21. Mice were killed at Days 3, 7, or 28. The lung tissue was microscopically observed with hematoxylin and eosin (H&E) (i–iii, vi–viii), Azan (iv, ix), or EVG (v, x) staining. Original magnification at observation x200. (B) TRX-Tg and control WT B6 mice were treated with bleomycin (100 mg/kg) at Days 0, 7, and 14. The mice were killed at Day 28, and the hydroxyproline content of the lungs and wet lung weights were measured. Untreated TRX-Tg and WT B6 (n = 5 in each group) mice were used as control mice.

 

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  		TABLE 2. Prevention of bleomycin-induced lung fibrosis in thioredoxin-transgenic mice: fibrotic scoring

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Initially, we evaluated the pharmacologic kinetics of TRX. After intraperitoneal injection of 40 µg of rTRX in mice, the TRX levels in the lung tissues and in the sera were detectable for 48 hours. The half-life of rTRX in the lung and serum was 51.3 and 8.5 hours, respectively. We previously reported that serum TRX levels in TRX-Tg mice were ~ 100 to 200 ng/ml (22). Here we found that B6 TRX-Tg mice were more resistant to bleomycin-induced lung fibrosis than were WT B6 mice treated intraperitoneally with 40 µg of rTRX every second day. These results suggest that the high TRX levels in TRX-Tg mice may given them more resistance to bleomycin-induced lung injury than rTRX-treated WT mice.

We have demonstrated that TRX can suppress the cytokine-induced (IL-18/IL-2) infiltration of leukocytes into the pulmonary interstitial space and can attenuate the subsequent lethal lung injury. There were more neutrophils and mononuclear cells after bleomycin injection in WT mice than in TRX-Tg mice. Treatment with rTRX also prevented bleomycin-induced cell infiltration into the lung tissues. These results suggest that infiltration of leukocytes, including neutrophils, might be associated with the establishment of bleomycin-induced lung fibrosis. Previous reports have shown that TRX works as a strong scavenger of ROS and shows antioxidant effects, which may be involved in the suppression of cytokine and chemokine production in the lung, resulting in the attenuation of leukocyte infiltration. More recently, we have shown that TRX has a variety of antiinflammatory and antichemotaxis effects that may not be directly explained by its antioxidant effect. Intravenous injection of rTRX suppresses LPS-induced leukocyte (mainly neutrophil) infiltration in the mouse air pouch model (22), suggesting that circulating TRX can prevent extravasation of leukocytes into inflammatory sites. Thus, TRX prevents cytokine- or bleomycin-induced lung injury directly and/or indirectly through an antioxidant effect. Modified TRX, which is changed at the redox-active site (–Cys–Gly–Pro–Cys–), does not exhibit antioxidant effect and antichemotaxis effect (22). Thus, it may not prevent cytokine- or bleomycin-induced lung injury in vivo. Further analysis is needed to clarify this issue.

We used an IL-18/IL-2–induced lethal lung injury model. In this model, infiltration of CD3- NK1.1+ NK cells into the lung tissues was observed. This lung injury is NK-cell–dependent, as anti-NK1.1 monoclonal antibody or anti-asialoGM1 antibody treatment completely prevents this lethality (12). We speculate that IL-18 plus IL-2 can synergistically activate NK cells to induce lung injury. The inhibitory effect of TRX in the present model may be explained by the direct suppression of NK cell infiltration or alternatively by the indirect suppression of the initial endothelial damage that leads to the infiltration of NK cells. Moreover, TRX may provide resistance against NK cell–mediated cytotoxicity because previous reports show that TRX inhibits TNF- and Fas-dependent cytotoxicity (29) and ROS-induced cytotoxicity (19).

In our present study, rTRX treatment inhibited cytokine and chemokine induction in the lungs of IL-18/IL-2–treated mice. However, these total lung analyses could reflect either changes in the cellular expression of cytokines and cytokines or changes in the cell populations in the lung, or both. Therefore, we examined whether exogenous TRX could modulate cytokine expression in murine NK or T cells in vitro. We isolated murine NK cells, and treated the cells with TRX and/or IL-18 plus IL-2. We found that exogenous TRX did not suppress IFN-{gamma} expression induced by IL-18 plus IL-2 in murine NK cells at both the mRNA and protein levels. The same result was found in the murine T cell line 7(3)H (data not shown). In addition, we found that TRX suppressed cell infiltration (of mainly NK and polymorphonuclear cells) into the lungs of IL-18/IL-2–treated mice. These results suggest that TRX may alter the cell populations in the lung but may not directly affect expression of cytokines or chemokines in cells.

In this study, TRX-Tg mice showed a significant decrease in bleomycin (4x)-induced lung fibrosis. However, treatment with rTRX (40 µg every second day) did not suppress bleomycin (4x)-induced lung fibrosis. Treatment with rTRX weakly suppressed lung fibrosis in mice given bleomycin (3x). Treatment with rTRX strongly suppressed lung fibrosis in mice given bleomycin (2x). Moreover, none of the B6 mice treated with bleomycin (2x) plus rTRX died, but some B6 mice treated with bleomycin (2x) plus OVA died. These results suggest that (1) treatment with rTRX can suppress bleomycin-induced lung fibrosis in B6 mice but not as well as it does in TRX-Tg mice; (2) rTRX treatment suppresses bleomycin-induced lung fibrosis in a dose-dependent manner; (3) four times administration of bleomycin overcame the effects of rTRX treatment; and (4) rTRX treatment prevented bleomycin-induced lethality.

There are several possible mechanisms by which TRX plays a protective role in bleomycin-induced lung fibrosis. Bleomycin induces the infiltration of neutrophils and mononuclear cells in the interstitial space of the lung, and this process can be directly suppressed by TRX. Several studies have indicated that ROS (e.g., oxygen radicals) are involved in bleomycin-induced lung injury because the antioxidants superoxide dismutase (13) and N-acetyl-L-cysteine (14) can partly inhibit bleomycin-induced lung injury. Thus, overexpression of TRX can act as a powerful scavenger of ROS, resulting in the prevention of bleomycin-induced lung fibrosis. We have found that TRX is associated with procollagen and modulates collagen synthesis in vivo in the liver (manuscripts in preparation), suggesting that TRX may suppress bleomycin-induced collagen synthesis in the lung. We have also found that bleomycin increases the expression of mature IL-18 (manuscript in preparation). TRX might modulate IL-18 signaling in the bleomycin-induced lung fibrosis model; we are currently investigating this hypothesis. Our recent study demonstrated that bleomycin upregulates TRX expression in both in vivo and in vitro systems (30). Our present study showed that overexpression of exogenous TRX attenuates bleomycin-induced lung injury. These results raise the possibility that loss of TRX function increases sensitivity to bleomycin, although TRX deficiency is lethal (31). Further analysis is needed to confirm this hypothesis.

ILD, including idiopathic pulmonary fibrosis and acute interstitial pneumonia/ARDS, is a serious lung disease with a poor clinical prognosis. Current therapeutic strategies for interstitial pneumonia are far from satisfactory, although glucocorticoids (steroids), antibiotics, and antiviral agents are often used. Here we have demonstrated that TRX can prevent both bleomycin-induced lung fibrosis and acute interstitial pneumonia caused by IL-18/IL-2. In addition to its antioxidant effect, TRX also has a variety of antiinflammatory and antichemotaxis effects. Thus, as a therapy, TRX may differ from any other drugs (e.g., N-acetyl-L-cysteine) by augmenting antioxidant activity. Recently, we found that geranylgeranylacetone, which is widely used as an antiulcer drug, can induce endogenous TRX, and pretreatment with geranylgeranylacetone protects cells against oxidative stress (32). Collectively, administration of rTRX and/or TRX-inducing agents may have clinical benefits in human ILD.


   FOOTNOTES
 
Supported by grants from Long-range Research Initiative (LRI) by 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 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., and a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science to H.N. and Technology, Japan, and a grant from Research and Development Program for New Bio-industry Initiatives to J.Y.

This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Conflict of Interest Statement: T.H. has no declared conflict of interest; H.N. has no declared conflict of interest; M.O. has no declared conflict of Interest; S.K. has no declared conflict of interest; S.A. has no declared conflict of interest; K.N. has no declared conflict of interest; K.O. has no declared conflict of interest; H.A.Y. has no declared conflict of interest; H.A. has no declared conflict of interest; J.Y. has no declared conflict of interest.

Received in original form September 3, 2002; accepted in final form June 16, 2003


   REFERENCES
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 RESULTS
 DISCUSSION
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