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Published ahead of print on September 27, 2007, doi:10.1164/rccm.200609-1288OC
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American Journal of Respiratory and Critical Care Medicine Vol 176. pp. 1251-1260, (2007)
© 2007 American Thoracic Society
doi: 10.1164/rccm.200609-1288OC

Original Article

Inhibition of Nuclear Factor-{kappa}B in T Cells Suppresses Lung Fibrosis

Hajime Fujimoto1, Corina N. D'Alessandro-Gabazza1, Moorthy S. S. Palanki5, Paul E. Erdman5, Takehiro Takagi1, Esteban C. Gabazza1,2, Nelson E. Bruno3, Yutaka Yano3, Tatsuya Hayashi4, Shigenori Tamaki6, Yasuhiro Sumida3, Yukihiko Adachi1, Koji Suzuki4 and Osamu Taguchi1

Departments of 1 Pulmonary and Critical Care Medicine, 2 Immunology and Allergy, 3 Diabetes and Endocrinology, and 4 Molecular Pathobiology, Mie University Graduate School of Medicine, Tsu City, Japan; 5 Celgene Signal Research Division, San Diego, California; and 6 Mie Central Hospital, Tsu City, Japan

Correspondence and requests for reprints should be addressed to Dr. Corina N. D'Alessandro-Gabazza, Department of Pulmonary and Critical Care Medicine, Mie University Graduate School of Medicine, Edobashi 2-174, Tsu City, Mie, Japan 514-8507. E-mail: gabazza{at}clin.medic.mie-u.ac.jp


   ABSTRACT
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 ABSTRACT
AT A GLANCE COMMENTARY
 METHODS
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 DISCUSSION
 REFERENCES
 
Rationale: Cytokines secreted by T cells play a pivotal role in the pathogenesis of lung injury and fibrosis, and the transcription factors nuclear factor (NF)-{kappa}B and activator protein (AP)-1 are involved in the expression of cytokines from T cells during lung injury.

Objectives: We assessed the potential therapeutic effect of SP100030, a specific inhibitor of T-cell NF-{kappa}B and AP-1 in lung fibrosis.

Methods: The effect of SP100030 was evaluated using a mouse model of chronic lung fibrosis.

Measurements and Main Results: Mice treated with SP100030, as compared with untreated mice, had significantly less cachexia and less lung injury and had decreased levels of inflammatory cytokines and growth factors, decreased activation of coagulation activation, and decreased collagen deposition in the lung. The inhibitory activity of SP100030 was dose dependent and was effective in acute and chronic phases of lung fibrosis. SP100030 inhibited the activation of the protein kinase C{theta}-isoform in T-cell lines and suppressed NF-{kappa}B–driven cytokine expression in CD4+ and CD8+ T cells.

Conclusions: These results suggest that the specific inhibition of NF-{kappa}B could be useful for the treatment of lung fibrosis.

Key Words: fibrosis • transcription factor • T cells



   AT A GLANCE COMMENTARY
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 ABSTRACT
 AT A GLANCE COMMENTARY
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Scientific Knowledge on the Subject
The role of T cells in pulmonary fibrosis is uncertain. Prior studies suggested that inhibiting nuclear factor-{kappa}B in T cells might help some immune diseases, but the effect on lung fibrosis is unknown.

What This Study Adds to the Field
This study suggests that T cells are important in pulmonary fibrosis because specific inhibition of the transcription factors NF-{kappa}B and AP-1 in T cells blocked the development of bleomycin-induced pulmonary fibrosis in mice.

 
Lung fibrosis is the most advanced stage of a group of inflammatory disorders of the lower respiratory tract. It results from injury to the lung parenchyma, which causes increased proliferation and migration of fibroblasts and the excessive accumulation of connective-tissue matrix proteins in the lung (1). Lung damage results in the recruitment of inflammatory mediators and the immune cells targeted to the lung play a crucial role in lung damage and in subsequent fibrosis. T lymphocytes are particularly important for the initiation and maintenance of lung fibrosis (2). Pulmonary fibrosis cannot be induced in T-cell–deficient mice, and the disease can be successfully induced by the passive transfer of T cells (3, 4). Different T-cell subpopulations are believed to contribute to the pathogenesis of lung fibrosis by their ability to secrete cytokines, which promote inflammation and attract other inflammatory cells at sites of lung injury during the early phases of the disease. Cytokines may also sustain fibrogenesis in the chronic stages of lung fibrosis by stimulating the proliferation of mesenchymal cells and the secretion of inflammatory cytokines and growth factors from surrounding cells (5). The expression of cytokines, chemokines, and growth factors is regulated by several transcription factors, including nuclear factor (NF)-{kappa}B and activator protein (AP)-1 (6). NF-{kappa}B regulates the expression of IL-1β, IL-2, IL-6, IL-8, IL-12, tumor necrosis factor-{alpha}, monocyte chemoattractant protein (MCP)-1, and granulocyte-macrophage colony-stimulating factor (7). NF-{kappa}B is also involved in the transcription of the fibrogenic growth factors platelet-derived growth factor (PDGF) and transforming growth factor (TGF)-β1 (8, 9).

SP100030 is a recently identified T-cell–specific inhibitor of the NF-{kappa}B and AP-1 transcription factors (10). Its chemical formula is 2-chloro-4-(trifluoromethyl)pyrimidine-5-N-(3',5'-bis[trifluoromethyl]phenyl)-carboxamide. SP100030 has been demonstrated to have therapeutic efficacy in experimental animal models of rheumatic arthritis, inflammatory bowel disease, allograft rejection, and delayed-type hypersensitivity, but its effect on the development of lung fibrosis has not been assessed (10–13). In the present study, we evaluated the therapeutic efficacy of SP100030 on bleomycin-induced lung fibrosis in a mouse model, focusing on NF-{kappa}B and its role in lung fibrosis. Some of the results of this study have been reported in the form of abstracts (14, 15).


   METHODS
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 METHODS
RESULTS
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Mouse Model of Lung Fibrosis
Mice used for evaluating the effect of SP100030 on lung fibrosis were 8- to 10-week-old, female C57BL/6 mice, weighing 18 to 22 g, and were purchased from Nihon SLC (Hamamatsu, Japan) and maintained in the animal house of Mie University. The Mie University's Committee on Animal Investigation approved the experimental protocol. Lung damage was induced by bleomycin (100 mg/kg) dissolved in sterile saline (Nihon Kayaku, Tokyo, Japan) by constant (7 d) subcutaneous infusion through osmotic minipumps (model 2001; Alza Corp., Palo Alto, CA) (16). In experiments with knockout mice, animals were categorized into six groups distinguished by genetic background and treatment with saline or bleomycin (BLM). Saline (SAL)-treated mice included wild type (WT/SAL), NF-{kappa}B knockout (NF{kappa}BKO/SAL), CD4KO (CD4KO/SAL), and CD8KO (CD8KO/SAL). BLM-treated mice included wild type (WT/BLM), NF{kappa}BKO (NF{kappa}BKO/BLM), CD4KO (CD4KO/BLM), and CD8KO (CD8KO/BLM).

Effect of SP100030 on Lung Fibrosis: Study Design
There were four treatment groups of animals: (1) mice treated with subcutaneous sterile saline (SAL) plus intraperitoneal polyethylene glycol 200 (PEG200) (SAL/PEG), (2) mice treated with subcutaneous BLM plus intraperitoneal PEG200 (BLM/PEG), (3) mice treated with subcutaneous saline plus intraperitoneal SP100030 (SAL/SP), and (4) mice treated with subcutaneous BLM plus intraperitoneal SP00030 (BLM/SP). For assessment in the acute phase of lung injury, the animals received subcutaneous administration of BLM or saline on Day 0 and were treated daily for 12 days with intraperitoneal injections of SP100030 (10 mg/kg) dissolved in PEG200 in a total volume of 50 µl or 50 µl of PEG200 alone. For assessment in the chronic phase of lung injury, the animals were treated daily with S100030 (10 mg/kg) or PEG200 alone in a similar manner as described previously but were injected starting on Day 12 until Day 20 after BLM or saline administration. To evaluate the dose dependency of S100030-mediated effects, mice were treated with 10, 5, or 0 mg S100030/kg body weight, starting on Day 0 until Day 12 after BLM or saline administration (16).

Biochemical Analysis
The concentration of total protein was measured using a dye-binding assay (Bio-Rad Laboratories, Hercules, CA) and lactate dehydrogenase was measured using a commercial kit (LDH ICII kit; Wako Pure Chemical Industry, Osaka, Japan). Cytokines, growth factors, and coagulation markers were measured by enzyme immunoassays. The concentrations of hydroxyproline, soluble collagen, elastase, and myeloperoxidase were measured by colorimetric assays as described (see online supplement) (16).

Histology and Immunochemistry
Mice were killed on Day 21 after BLM or saline subcutaneous infusion. The left lung was perfused with 10% neutral buffered formalin and fixed in formalin for 24 hours. The tissue sections were embedded in paraffin and then prepared for hematoxylin–eosin staining. Immunocytochemistry of bronchoalveolar lavage fluid (BALF) cells was performed using anti–NF-{kappa}B (p65) antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and FITC-labeled goat anti-rabbit antibody (Santa Cruz Biotechnology) (see online supplement).

Statistical Analysis
All data are expressed as the mean ± SEM. The statistical difference between three or more variables was calculated by analysis of variance with post hoc analysis using Fisher's predicted least significant difference test.


   RESULTS
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 RESULTS
DISCUSSION
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Lung Injury and Fibrosis in NF-{kappa}B p50 KO Mice
In mammals, the NF-{kappa}B family of transcription factors comprises five polypeptides, including RelA (p65), RelB, c-Rel, NF-{kappa}B1 p50, and NF-{kappa}B2 p52. Each polypeptide contains a Rel homology domain, which mediates DNA binding, nuclear localization, and subunit dimerization (17). The NF-{kappa}B members dimerize to form homo- or heterodimers, which have different effects on gene transcription. Unlike RelA, RelB, and c-Rel, NF-{kappa}B1 p50 and NF-{kappa}B2 p52 lack transcriptional activation domains, and thus their homodimers (p50/p50) act as repressors of gene expression (18, 19). Therefore, if NF-{kappa}B plays an important role in the development of lung fibrosis, then mice deficient in the NF-{kappa}B p50 subunit would be expected to demonstrate more rapid onset or more severe forms of the disease. To examine disease progression in this situation, lung fibrosis was induced in mice deficient for NF-{kappa}B p50 (NF-{kappa}Bp50 KO/BLM mice) by administering BLM, and the resultant disease state was compared with that of BLM-treated wild-type mice. We examined the number of inflammatory cells and the total protein levels as markers of tissue inflammation in (BALF) and found that in NF-{kappa}Bp50 KO/BLM mice, these indices were significantly elevated as compared with the wild-type BLM-treated mice (see Figure E1 in the online supplement). Lung fibrotic changes and the Ashcroft score (which measures the progression of lung fibrosis) were markedly enhanced in NF-{kappa}Bp50 KO/BLM mice as compared with wild-type BLM-treated mice (Figure E1).

Specificity of SP100030
To examine the T-cell specificity of SP100030, Jurkat T cells and A549 alveolar epithelial cells were stimulated after serum starvation with 100 nM phorbol 12-myristate 13-acetate plus 50 µM phytohemaglutinin in the presence or absence of SP100030, and NF-{kappa}B activation was assessed by gel shift assays. SP100030 inhibited NF-{kappa}B activation in Jurkat T cells (Figure 1a) but not in A549 epithelial cells (Figure 1b). To examine the ability of SP100030 to function in vivo, lung fibrosis was induced by the injection of BLM into wild-type mice previously treated with the delivery vehicle polyethylene glycol 200 (PEG200) (BLM/PEG) or with SP100030 (BLM/SP). The spleen cells were removed to compare the extent of NF-{kappa}B activation in lymphocytes and fibroblasts. As controls, spleen cells from mice treated with saline and vehicle (SAL/PEG) or saline and SP100030 (SAL/SP) were prepared. Gel-shift assays show that in the BLM/SP mice, there was notable suppression of NF-{kappa}B activation in lymphocytes as compared with lymphocytes from the BLM/PEG mice. However, there was no difference in NF-{kappa}B activation in spleen fibroblasts between the BLM/PEG and BLM/SP groups (Figure 1c). The binding of homologous complexes of p65 was significantly inhibited in whole lung tissue in the BLM/SP group as compared with the BLM/PEG group (Figure 1c). Inhibition of NF-{kappa}B was assessed by immunostaining of BALF cells with anti–NF-{kappa}B p65 antibody. We observed strong cytoplasmic and nuclear immunoreactivity in BALF cells from the BLM/PEG group, indicating that NF-{kappa}B was activated and translocated into the nucleus. However, there was no nuclear staining for NF-{kappa}B in BALF cells from the BLM/SP group, indicating that NF-{kappa}B had been inhibited. NF-{kappa}B staining was not detected in the nucleus of BALF cells from the SAL/PEG and SAL/SP groups; this was expected because these mice were not activated for NF-{kappa}B (Figure 1d). We also evaluated the NF-{kappa}B–driven secretion of the cytokine IL-8 from Jurkat T cells, A549 alveolar epithelial cells, and BEAS-2B lung epithelial cells and found that SP100030 inhibited IL-8 secretion in Jurkat cells but not in epithelial cell lines (Figure 1e). The secretion of MCP-1 from spleen lymphocytes taken from SAL/SP, BLM/PEG, and BLM/SP and cultured ex vivo was also evaluated; SP100030 inhibited the secretion of MCP-1 in lymphocytes from BLM/SP (7.8 ± 1.9 pg/ml) compared with spleen cells from the BLM/PEG group (25.1 ± 6.7 pg/ml). However, SP100030 failed to inhibit the secretion of MCP-1, a NF-{kappa}B-driven chemokine, in mouse alveolar macrophages (Figure 1e).


Figure 1
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  		Figure 1. Specific inhibition of nuclear factor (NF)-{kappa}B and activator protein (AP)-1 activation in lymphocytic cells. (a, b) NF-{kappa}B was activated with phorbol 12-myristate 13-acetate (PMA, 100 nM) and phytohemaglutinin (PH, 1 µg/ml) in Jurkat T cells (a) and A549 epithelial cells (b) in the presence or absence of SP100030 (SP, 2 µM), nuclear proteins were purified, and gel-shift assays were performed. A representative image of three experiments is shown. (c) Nuclear proteins were prepared from lymphocytes and fibroblasts from spleen and from lung tissue of mice that were treated with saline plus vehicle (SAL/PEG), bleomycin plus SP100030 (BLM/SP), or saline plus SP100030 (SAL/SP). Nuclear protein was electrophoresed in a gel-shift assay. Representative autoradiographs of two experiments are shown. (d) NF-{kappa}B antibody staining of bronchoalveolar lavage fluid cells from each group of mice described in (c). NF-{kappa}B antibodies were directed against the p65 subunit. (e) Analysis of cytokine IL-8 levels in culture supernatants after NF-{kappa}B activation with thrombin (20 U/ml) and lipopolysaccharide (10 µg/ml) in Jurkat T cells, A549 cells, and BEAS-2B lung epithelial cells in the presence or absence of SP100030 (2 µM) as indicated. The effect on MCP-1 secretion from mouse alveolar macrophages was also assessed. Data represent the mean ± SEM of two experiments carried out in triplicate. Statistical analysis was performed using analysis of variance with Fisher's predicted least significant difference test. Cold wild-type AP-1 or NF-{kappa}B; nonlabeled double-stranded competitor oligonucleotide (100-fold molar excess) for AP-1 or NF-{kappa}B; cold mutant AP-1 or NF-{kappa}B, nonlabeled double-stranded mutant (noncompetitor) oligonucleotide (100-fold molar excess) for AP-1 or NF-{kappa}B; nuclear protein (–), loading without nuclear protein. *P < 0.05 compared with thrombin + LPS.

 
Phosphoprotein Analysis of Jurkat Cells Treated with SP100030
To examine which signaling molecules were involved in the ability of SP100030 to inhibit NF-{kappa}B activation, a cocktail of stimulants was used to activate NF-{kappa}B in Jurkat cells that had been pretreated with SP100030 for 30 minutes. The phosphorylation status of 30 kinases in whole cell lysates was then evaluated with Kinexus phosphorylated protein screening assays (Kinexus Bioinformatics Corp., Vancouver, BC, Canada), which revealed that the phosphorylation of several kinases was markedly affected in cells pretreated with SP100030 (protein kinase C{theta}, 49% inhibited; p85 S6 kinase, 44% inhibited; Rafl, 32 to 34% inhibited; see Figure E2). It has been reported that protein kinase C{theta} translocates to membrane rafts during cell activation (20). To evaluate the degree of protein kinase C{theta} phosphorylation in membrane rafts, in a separate experiment, Jurkat T cells that had been stimulated in the presence or absence of SP100030 were processed using the Kinetworks KCSS screening service (Kinexus Bioinformatics Corp.). Results showed that SP100030 markedly reduced the level of phosphorylated protein kinase C{theta} in the membrane fraction (Figure E2).

Effect of SP100030 on the Acute Phase of Pulmonary Fibrosis
Mice were injected with BLM or saline and treated daily with either vehicle alone (BLM/PEG, SAL/PEG) or SP100030 (BLM/SP, SAL/SP) for 12 successive days. There was a gradual increase in body weight in the SAL/PEG and SAL/SP groups. Body weight decreased markedly in the BLM/PEG mice from Day 9 compared with the SAL/PEG and SAL/SP mice, although weight remained relatively stable in BLM/SP mice. There was a substantial difference in body weight between the BLM/PEG and BLM/SP groups from Day 11 after BLM injection (Figure 2a). The ratio of wet-to-dry lung weight, which is a marker of pulmonary edema, was greater in BLM/PEG mice compared with BLM/SP and SAL/PEG mice; no difference was observed between the SAL/PEG and SAL/SP mice (Figure 2b).


Figure 2
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  		Figure 2. SP100030 inhibits mouse cachexia and inflammation during the acute phase of lung injury. Mice were treated with saline plus vehicle (SAL/PEG), saline plus SP100030 (SAL/SP), bleomycin plus vehicle (BLM/PEG), or bleomycin plus SP100030 (BLM/SP) (n = 6–15 mice per group). SP100030 was administered intraperitoneally once a day for 12 days after bleomycin administration. Body weight was measured daily for 21 days, as shown in (a), and samples of plasma and bronchoalveolar lavage fluid (BALF) were collected on Day 21. *P < 0.05 compared with BLM/PEG group. (b) Wet/dry lung weight ratio, which is an indicator of pulmonary edema. *P < 0.05 compared with SAL/PEG and SAL/SP groups. {ddagger}P < 0.05 compared with BLM/PEG group. (c) Extent of lung injury as assessed in BALF by levels of total protein (open bars), myeloperoxidase (open bars), elastase (solid bars), and IL-1β (open bars) and in plasma by levels of lactate dehydrogenase (solid bars) and IL-1β (solid bars). *P < 0.05 compared with SAL/PEG and SAL/SP groups. {ddagger}P < 0.05 compared with BLM/PEG group. Statistical analysis was performed using analysis of variance with Fisher's predicted least significant difference test. Values represent the mean ± SEM. LDH, lactate dehydrogenase.

 
Mice were killed on Day 21 and lungs were examined for inflammation markers, cytokines, growth factors and collagen deposition, and the concentrations of total protein and lactate dehydrogenase, which serve as markers of lung injury, were measured. The BALF concentration of total protein and the plasma concentration of lactate dehydrogenase were markedly higher in the BLM/PEG group than in the SAL/PEG and BLM/SP groups (Figure 2c). Because neutrophil-derived proteases are important mediators of lung injury, we also measured the BALF concentrations of myeloperoxidase and elastase and found that they were significantly increased in BLM/PEG mice as compared with SAL/PEG and BLM/SP mice (Figure 2c). There was no difference in the BALF levels of total protein, myeloperoxidase, and elastase between mice in the SAL/PEG and SAL/SP groups. We also examined levels of specialized cells known to be involved in inflammation. Table 1 shows that in BLM/PEG mice, the numbers of polymorphonuclear cells and lymphocytes were significantly increased and the number of macrophages was markedly decreased as compared with SAL/PEG mice. In BLM/SP mice, the number of polymorphonuclear cells and lymphocytes was significantly decreased as compared with BLM/PEG mice, whereas the number of alveolar macrophages was markedly increased in BLM/SP mice.


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  		TABLE 1. CELL COUNT IN BRONCHOALVEOLAR LAVAGE FLUID

 
Cytokine Expression
The proinflammatory cytokine IL-1β acts as an accessory growth factor for T cells and may directly stimulate fibrosis by inducing fibroblast proliferation and increased collagen deposition (1). The plasma and BALF concentrations of IL-1β were significantly increased in BLM/PEG mice as compared with SAL/PEG mice. However, they were significantly lower in BLM/SP mice than in BLM/PEG mice (Figure 2c). We also analyzed levels of MCP-1 and the Th2 cytokine IL-13, which may also promote lung fibrosis (21). The plasma and BALF concentrations of MCP-1 and the BALF concentration of IL-13 were significantly increased in BLM/PEG mice as compared with the SAL/PEG and BLM/SP mice (Figure 3a). There was no statistically significant difference in the BALF level of IFN-{gamma} between the different groups of animals (data not shown). There was impairment of the Th1/Th2 cytokine balance, as evidenced by an increased IL-13/IFN-{gamma} ratio in the BLM/PEG group compared with the SAL/PEG group; this imbalance was restored in mice treated with SP100030 (BLM/SP) (Figure 3a). There was no difference in cytokine expression between the SAL/PEG and SAL/SP groups.


Figure 3
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  		Figure 3. SP100030 inhibits inflammatory mediators and fibrosis during the acute phase of lung injury. Mice were treated with saline plus vehicle (SAL/PEG), saline plus SP100030 (SAL/SP), bleomycin plus vehicle (BLM/PEG), or bleomycin plus SP100030 (BLM/SP) (n = 6–15 mice per group). SP100030 was administered intraperitoneally once a day for 12 days after bleomycin administration. Samples of plasma and bronchoalveolar lavage fluid (BALF) were drawn on Day 21. (a) BALF and plasma concentrations of monocyte chemoattractant protein (MCP)-1 (open bars, BALF; solid bars, plasma) and thrombin–antithrombin complex (TAT) (open bars, BALF; solid bars, plasma) and the level of IL-13 (open bars) and the IL-13:IFN-{gamma} (Th2/Th1 balance) ratio (solid bars) in BALF. (b) The urokinase:TAT ratios (open bars) and the level of plasminogen activator inhibitor (PAI)-1 (solid bars) in BALF, as well as the BALF concentrations of TGF-β1 (open bars), platelet-derived growth factor (PDGF) (solid bars), total soluble collagen (open bars), and the lung content of hydroxyproline (HDP; solid bars). *P < 0.05 compared with SAL/PEG and SAL/SP groups. {ddagger}P < 0.05 compared with BLM/PEG groups. Statistical analysis was performed using analysis of variance with Fisher's predicted least significant difference test. Values represent the mean ± SEM. (c) Representative photomicrographs of fibrotic lesions and inflammation in the lung from each treatment group are shown with hematoxylin–eosin staining (original magnification, x20). TGFβ1, transforming growth factor-β1.

 
Activation of the Coagulation System and Development of Lung Fibrosis
The activation of the coagulation system and the occurrence of hypofibrinolysis (decreased ability to break down fibrin) in the intraalveolar space play fundamental roles in the establishment of lung fibrosis (22). We measured the levels of the thrombin–antithrombin complex (TAT) and soluble tissue factor as markers of coagulation system activation and the ratio of urokinase activity to TAT as a marker of fibrinolysis activity. Levels of TAT in plasma and in BALF were significantly increased in BLM/PEG mice as compared with SAL/PEG and BLM/SP mice (Figure 3a). The level of tissue factor in BALF tended to be high in BLM/PEG mice (26.6 ± 2.6 pg/ml) as compared with SAL/PEG (18.8 ± 5.8 pg/ml) and BLM/SP (21.2 ± 4.5 pg/ml) mice. BLM/PEG mice exhibited a significantly lower urokinase/TAT ratio than SAL/PEG mice, and BLM/SP mice exhibited a significantly higher urokinase/TAT ratio than BLM/PEG mice (Figure 3b). We also analyzed levels of the plasminogen activator inhibitor (PAI)-1 because elevated PAI-1 levels have been assigned as the primary cause of the low fibrinolytic activity observed in lung fibrosis (23). The BALF level of PAI-1 was significantly elevated in mice from the BLM/PEG group as compared with the SAL/PEG and BLM/SP groups (Figure 3b); there was no difference between the SAL/PEG and SAL/SP groups. TGF-β1 plays a role in the pathogenesis of lung fibrosis by stimulating the secretion of extracellular matrix proteins such as collagen, fibronectin, and glycosaminoglycans, and PDGF promotes the proliferation and chemotaxis of fibroblasts (6). The concentrations of TGF-β1 and PDGF in BALF were significantly increased in BLM/PEG mice as compared with the SAL/PEG and BLM/SP mice. There was no difference in PDGF or TGF-β1 levels between the SAL/PEG and SAL/SP groups (Figure 3b).

Lung Collagen Levels and Lung Pathology
Collagen metabolism in the lung was assessed by measuring levels of hydroxyproline (a component of collagen) in lung tissue and by measuring the BALF levels of total soluble collagen in each group of mice. Both were significantly increased in BLM/PEG mice as compared with SAL/PEG mice, and both were markedly higher in mice treated with polyethylene glycol alone (BLM/PEG) than in mice treated with SP100030 (BLM/SP). There were no significant differences in hydroxyproline content or soluble collagen between the SAL/PEG and SAL/SP groups (Figure 3b).

When mice were analyzed on Day 21 after BLM administration, the BLM/PEG mice showed severe fibrotic changes in the central regions of the lungs and in the perivascular and peribronchiolar areas and showed areas of consolidation in the subpleural regions as compared with the SAL/PEG mice. By contrast, lungs from BLM/SP mice showed fewer fibrotic lesions in the subpleural areas, and central areas of lung parenchyma seemed to be almost normal. No changes were observed in the SAL/SP mice (Figure 3c).

SP100030-mediated Effects Are Dose Dependent
To study whether SP100030-mediated effects were dose dependent, mice were treated daily with SP100030 at 5 or 10 mg/kg body weight for 12 days after the administration of BLM. After Day 14, there was a significant reversal of weight loss in the BLM/SP mice treated with 10 mg/kg of SP100030 but not in mice treated with 5 mg/kg of SP100030, as compared with BLM/PEG mice (Figure 4a). In BLM/SP mice treated with SP100030 at 10 mg/kg body weight (but not in those treated at 5 mg/kg), the BALF concentrations of total protein, MCP-1, and hydroxyproline were significantly reduced as compared with the BLM/PEG group, whereas the BALF level of soluble collagen was significantly decreased in mice treated with 10 or 5 mg/kg of SP100030 as compared with the BLM/SP group (Figure 4b).


Figure 4
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  		Figure 4. Dose-dependent SP100030-mediated inhibition of tissue injury and fibrosis in the lung. Mice were treated with saline plus SP100030 (SAL/SP), bleomycin plus vehicle (BLM/PEG), or bleomycin plus SP100030 at 5 mg/kg body weight (BLM/SP [5 mg/kg]) or at 10 mg/kg body weight (BLM/SP [10 mg/kg]) (n = 4–8 mice per group). SP100030 was administered intraperitoneally once daily for 12 days after bleomycin administration. Mice were killed on Day 21, and samples were collected. (a) Cachexia (severe wasting/weight loss) in the different treatment groups. Solid diamonds, SAL/SP; open circles, BLM/SP (10 mg/kg); solid circles, BLM/PEG; open diamonds, BLM/SP (5 mg/kg). (b) Differences in lung edema (as measured by bronchoalveolar lavage fluid [BALF] total protein) the BAL concentration of monocyte chemoattractant protein (MCP)-1 and the lung content of hydroxyproline in the different treatment groups. *P < 0.05 compared with the SAL/PEG and SAL/SP groups. {ddagger}P < 0.05 compared with the BLM/PEG group. Statistical analysis was performed using analysis of variance with Fisher's predicted least significant difference test. Values represent the mean ± SEM. HDP, hydroxyproline.

 
Effect of SP100030 on the Chronic Phase of Lung Fibrosis
To evaluate the effect of SP100030 on chronic BLM-induced lung fibrosis, mice were treated daily with 10 mg/kg of SP100030 starting on Day 12 after BLM administration and continuing to Day 20. The SAL/PEG and SAL/SP groups gradually gained body weight, whereas the BLM/PEG and BLM/SP groups experienced significant weight loss. However, significant weight recovery was observed in BLM/SP mice from Day 17 as compared with BLM/PEG mice (Figure 5a). In BLM/SP mice, there was a significant decrease in the BALF levels of total protein, TAT, IL-13, PDGF, soluble collagen, and hydroxyproline as compared with BLM/PEG mice. There was also a significant decrease in the BALF IL-13/IFN-{gamma} ratio and a markedly increased BALF urokinase/TAT ratio in BLM/SP mice as compared with BLM/PEG mice (Figure 5a). A histological assessment revealed a low grade of fibrosis in the lungs of BLM/SP mice as compared with those of BLM/PEG mice. There was no significant difference between SAL/PEG mice and SAL/SP mice in any of the above categories (Figure 5b).


Figure 5
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  		Figure 5. SP100030 inhibits inflammation and fibrosis during the chronic phase of lung injury. Mice were treated with saline plus vehicle (SAL/PEG), saline plus SP100030 (SAL/SP), bleomycin plus vehicle (BLM/PEG), or bleomycin plus SP100030 (BLM/SP) (n = 3–4 mice per group). SP100030 was administered intraperitoneally once a day for 9 days, starting at Day 12 after bleomycin administration. Samples of plasma and bronchoalveolar lavage fluid (BALF) were drawn on Day 21. (a) Differences in body weight; lung edema (as indicated by the concentrations of total protein); levels of TAT, IL-13, PDGF, and TGF-β1; fibrinolysis/coagulation balance (as indicated by the urokinase/thrombin–antithrombin complex [TAT] ratio); Th2/Th2 balance (as indicated by the IL-13/IFN-{gamma} ratio); and the total lung content of hydroxyproline (HDP) in the different treatment groups. *P < 0.05 compared with SAL/PEG and SAL/SP groups. {ddagger}P < 0.05 compared with BLM/PEG groups. Statistical analysis was performed using analysis of variance with Fisher's predicted least significant difference test. Values represent the mean ± SEM. (b) Representative photomicrographs of fibrotic lesions and inflammation in lungs from each group of mice are shown with hematoxylin–eosin staining (original magnification, x20). PDGF, platelet-derived growth factor.

 
Effect of SP100030 on T-Cell Subpopulations in Lung Fibrosis
Two subpopulations of T cells, CD4+ and CD8+, have been implicated in the pathogenesis of lung fibrosis (21). To examine the role of these cells in our model, we induced lung fibrosis in CD4+ (CD4KO) and CD8+ (CD8KO) knockout mice. In mice with BLM-induced lung fibrosis (WT/BLM, CD4KO/BLM, and CD8KO/BLM), we found that weight loss, the number of inflammatory cells in the lung, the BALF concentration of total protein, and the total lung tissue content of collagen were significantly increased as compared with animals treated with saline instead of BLM (WT/SAL, CD4KO/SAL, and CD8KO/SAL). We also noted that the pathologic findings of lung fibrosis were markedly pronounced in mice treated with BLM (Figure 6a and 6b). There were no significant differences in these categories between WT/BLM, CD4KO/BLM, and CD8KO/BLM mice, indicating that BLM induces lung fibrosis equally in all three mouse types. However, the lung distribution of fibrotic changes was predominantly peribronchial and perivascular in CD4KO/BLM mice and subpleural in CD8KO/BLM mice (Figure 6b).


Figure 6
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  		Figure 6. Bleomycin induces lung fibrosis in mice deficient in CD4 or CD8 T cells. Wild-type (WT/BLM), CD4KO (CD4KO/BLM), and CD8KO (CD8KO/BLM) mice were treated with bleomycin over 7 days. Wild-type (WT/SAL), CD4KO (CD4KO/SAL), and CD8KO (CD8KO/SAL) mice treated with saline were used as controls (n = 4 mice per group). All mice were killed on Day 21. (a) Weight loss, the total number of inflammatory cells, and the concentration of total protein in saline- and bleomycin-treated mice. *P < 0.05 compared with WT/SAL CD4KO/SAL and CD8KO/SAL groups. Statistical analysis was performed using analysis of variance with Fisher's predicted least significant difference test. Values represent the mean ± SEM. (b) Representative photomicrographs of fibrotic lesions and inflammation in lungs from each group of mice are shown with hematoxylin–eosin staining (original magnification, x20). (c) Concentration of macrophage inhibitory protein (MIP)-1{alpha} in spleen CD4+ and CD8+ T cells isolated from wild-type mice with bleomycin-induced lung fibrosis. CD4+ and CD8+ T cells were stimulated with thrombin plus LPS with or without SP100030 before measurement of MIP-1{alpha}. *P < 0.05 compared with cells stimulated with thrombin plus LPS. Statistical analysis was performed using analysis of variance with Fisher's predicted least significant difference test. Data represent the mean of two independent experiments, with each experiment internally carried out in triplicate. BALF, bronchoalveolar lavage fluid; BLM, bleomycin; SAL, saline; SP, SP100030.

 
To examine whether SP100030 inhibits NF-{kappa}B–driven cytokine expression from CD4+ and CD8+ T cells, these cells were isolated from wild-type (CD4+ and CD8+) mice with BLM-induced lung fibrosis and, in the presence of SP100030, were stimulated with thrombin and lipopolysaccharide to activate NF-{kappa}B. Macrophage inhibitory protein (MIP)-1{alpha}, which is a NF-{kappa}B–driven proinflammatory cytokine, was measured in the cell supernatant. SP100030 significantly suppressed MIP-1{alpha} secretion from mouse CD4+ and CD8+ T cells, suggesting that the inhibitory activity of SP100030 is effective in both types of T cells (Figure 6c).

Effect of SP100030 on Lung Fibrosis Induced by Intratracheal Instillation of BLM
The effect of SP100030 on lung fibrosis induced by intratracheal instillation of BLM was evaluated. The concentration of MCP-1 in BALF and the lung hydroxyproline content were significantly decreased in the BLM/SP group compared with the BLM/PEG group. Tissue consolidation and fibrosis of the lung were more conspicuous in the lung from BLM/PEG mice than in that from BLM/SP mice (Figure E3).


   DISCUSSION
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We found that the T-cell–specific NF-{kappa}B inhibitor SP100030 suppressed inflammation, the development of fibrosis, and the expression of cytokines, chemokines, and growth factors in a mouse model of lung injury. We also demonstrated that SP100030 decreased the activation of protein kinase C{theta} and decreased the secretion of cytokines from CD4+ and CD8+ T cells.

Activation of NF-{kappa}B is linked to the aberrant expression of numerous genes involved in the initiation and progression of several diseases (2, 24). Previous studies in animal models have shown that enhanced activation of the NF-{kappa}B family of transcription factors occurs in lung fibrosis (24). To evaluate the role of NF-{kappa}B in our model of lung fibrosis, we compared the development of BLM-induced lung fibrosis in wild-type and in mice that were deficient for the NF-{kappa}B1 p50 subunit. Among the five members of the NF-{kappa}B family proteins (RelA[p65], RelB, c-Rel, p50, and p52), only the p50 and p52 subunits lack transactivation domains, so when p50 homodimers are present in the nucleus, they act as repressors of NF-{kappa}B–mediated gene transcription (16). NF-{kappa}B1 p50-deficient mice exhibited enhanced inflammation, collagen deposition, and lung fibrosis as compared with their wild-type counterparts, supporting a pivotal role for NF-{kappa}B activation in the development of lung injury and fibrosis.

A previous study has shown that SP100030 inhibits NF-{kappa}B in various murine and human T cell lines but not in non–T-cell lines such as monocytes, fibroblasts, endothelial cells, and epithelial cells (11). Most of the T-cell lines tested were transformed lines, so no information regarding the specificity of SP100030 for lymphocytes in vivo was provided. We found that in mice with lung fibrosis, treatment with SP100030 decreased NF-{kappa}B activation in lymphocytes, but not in fibroblasts, as compared with cells isolated from control, untreated mice with lung fibrosis, suggesting that the SP100030 was specific for lymphocytes in vivo. We also found that SP100030 inhibits NF-{kappa}B–driven cytokine in spleen lymphocytes isolated from mice treated with the compound but not in mouse alveolar macrophages cultured and stimulated in vitro. However, we used only one dose of the compound in these experiments; whether SP100030 exerts inhibitory activity on nonlymphocytic cells when used at higher doses in vivo remains to be clarified.

Activation of NF-{kappa}B occurs in response to ligation of several receptors, and numerous intermediate pathways and kinases are involved in the transduction route from receptor-mediated signaling to NF-{kappa}B activation (25, 26). To define the means by which SP100030 exerts its inhibitory effect, we examined changes in the phosphorylation state of a panel of kinases and found that SP100030 treatment was associated with the strong inhibition of protein kinase C{theta}, p85 S6 kinase, and Raf1, all of which are known to be involved in NF-{kappa}B activation (27). The expression pattern of protein kinase C{theta}, unlike that of other kinases and other protein kinase C isoforms, is restricted to T cells and to skeletal muscle, which suggests that the inhibition of protein kinase C{theta} may be the basis for the observed lymphocyte specificity of SP100030 (28, 29).

Inflammation plays a pivotal role in the initiation of the fibrotic process in the lung, and the development of lung fibrosis is believed to result from repeated episodes of lung injury (30). To create a model that resembles the likely developmental course of the disease in humans, we induced lung fibrosis in mice by the continuous subcutaneous infusion of BLM over 7 days, and to figure out whether control of inflammation results in reduced lung fibrosis, SP100030 was administered to mice during the acute phase of BLM-induced inflammation. In mice treated with SP100030, there was a substantial decrease in the systemic and lung-specific levels of the proinflammatory cytokine IL-1β and the chemokine MCP-1, with a concomitant reduction in cachexia, cell infiltration, pulmonary edema, and lung injury. The expression of the growth factors TGF-β1 and PDGF is also partially regulated by NF-{kappa}B activation, and the lung-specific levels of these growth factors were also reduced by SP100030 treatment (8, 9). Previous work has shown that this reduction of growth factors is associated with less fibrotic changes in the lung and inhibition of collagen deposition (8). Overall, these observations suggest that inhibition of acute inflammation by SP100030 reduces collagen deposition in the lung. The role of inflammation in idiopathic pulmonary fibrosis is controversial, but antiinflammatory agents (e.g., immunosuppressant, corticosteroids) have been shown to be beneficial in pulmonary fibrosis secondary to other diseases, such as collagen vascular disorders and hypersensitivity pneumonitis (31). NF-{kappa}B inhibitors would be more beneficial in pulmonary fibrosis associated with diseases in which inflammation is the predominant underlying mechanism.

In humans, lung fibrosis such as idiopathic pulmonary fibrosis is progressive and unrelenting; the general assumption is that the fibrosis self-perpetuates once it starts (32, 33). The mechanism by which the self-perpetuation occurs remains obscure, but activation of the coagulation system and fibrogenic cytokines may be involved (32). To evaluate the effect of SP100030 on established lung fibrosis, we started treatment with this compound on Day 12 (Figure 5). In our mouse model of chronic lung fibrosis, the lung concentrations of IL-13, PDGF, and coagulation activation activity were markedly increased, but this increase was significantly reduced by SP100030 treatment. In SP100030-treated mice, cachexia, lung protein leakage, collagen deposition, and histological evidence of lung fibrosis were reduced. A shift in the hemostatic balance toward a predominance of coagulation over fibrinolysis also promotes lung fibrosis (23, 32). Compared with untreated mice, reversal of the coagulation/fibrinolysis imbalance was observed in SP100030-treated mice. T cells may indirectly activate the coagulation system by inducing the expression of tissue factor, the initiator of coagulation activation, from other cells (34). Once activated, the coagulation system releases thrombin, which may stimulate resident cells to secrete growth factors and cytokines (23). The high level of thrombin formation, as demonstrated by the high BALF level of TAT, may explain the increased level of PDGF and IL-13 in the lung from our lung fibrosis model. Overall, these findings suggest that activated T cells may promote the generation of thrombin, which stimulates the secretion of growth factors such as IL-13 and PDGF, leading to enhanced fibrogenic responses. SP100030 could have a palliative effect on the development of lung fibrosis by inhibiting T-cell activation and suppressing this cycle. Unlike our findings with SP100030, Inayama and colleagues recently reported that an I{kappa}B kinase-β inhibitor could not block BLM-induced lung fibrosis in the chronic phase (35). One explanation for this discrepancy may be the higher dose of BLM (125 or 150 mg/kg) they used, which resulted in more conspicuous fibrosis in the late phase. Another explanation may be the differential effects of NF-{kappa}B inhibitors on the coagulation system. Inayama and colleagues did not measure markers of coagulation system before and after treatment. A recent study showing that anticoagulant therapy improves survival in patients with idiopathic pulmonary fibrosis supports the importance of the coagulation activation in the pathogenesis of lung fibrosis (36).

Different subpopulations of T lymphocytes, including CD4+ and CD8+, have been implicated in the pathogenesis of lung fibrosis by virtue of their ability to secrete fibrogenic cytokines (21, 37). In lung fibrosis, type 2 cytokines (IL-4, IL-5, and IL-13) predominate over type 1 cytokines (IFN-{gamma} and IL-2), and CD4+ and CD8+ T cells can secrete type 2 cytokines (29, 37). Because the T-cell target of SP100030 is unknown, as a first step toward clarification we wished to define the role of each T-cell subset in our chronic model of lung fibrosis. We found that CD4+ and CD8+ knockout mice developed lung fibrosis, but the distribution of the disease in the lung varied, suggesting that CD4+ and CD8+ cells may have a role in our model of lung fibrosis but may function in different areas of the lung. SP100030 inhibited cytokine expression in isolated CD4+ and CD8+ T cells that were collected from wild-type mice, suggesting that the therapeutic effect of this compound on lung fibrosis may result from its ability to inhibit both types of T cells.


   FOOTNOTES
 
Supported by grants-in-aid (nos. 17590788, 18590846) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by grants-in-aid from the Mie Medical Research Foundation, the Suzuken Memorial Foundation, and the Mie University COE Project Fund.

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

Originally Published in Press as DOI: 10.1164/rccm.200609-1288OC on September 27, 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 September 11, 2006; accepted in final form September 21, 2007


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