Introduction of the interleukin-10 gene into mice inhibited bleomycin-induced lung injury in vivo

Toru Arai1, Kin'Ya Abe2, Hiroto Matsuoka1, Mitsuhiro Yoshida3, Masahide Mori1, Sho Goya1, Hiroshi Kida1, Kazumi Nishino1, Tadashi Osaki1, Isao Tachibana1, Yasufumi Kaneda4, and Seiji Hayashi1

1 Department of Molecular Medicine and 4 Division of Gene Therapy Science, Osaka University Medical School, Suita, Osaka 565-0871; 2 Department of Pulmonary Disease, Higashiosaka City General Hospital, Higashiosaka 578-8588, Japan; and 3 Department of Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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Interleukin (IL)-10 has been shown to reduce many inflammatory reactions. We investigated the in vivo effects of IL-10 on a bleomycin-induced lung injury model. Hemagglutinating virus of Japan (HVJ)-liposomes containing a human IL-10 expression vector (hIL10-HVJ) or a balanced salt solution as a control (Cont-HVJ) was intraperitoneally injected into mice on day -3. This was followed by intratracheal instillation of bleomycin (0.8 mg/kg) on day 0. Myeloperoxidase activity of bronchoalveolar lavage fluid and tumor necrosis factor-alpha mRNA expression in bronchoalveolar lavage fluid cells on day 7 and hydroxyproline content of the whole lung on day 21 were inhibited significantly by hIL10-HVJ treatment. However, Cont-HVJ treatment could not suppress any of these parameters. We also examined the in vitro effects of IL-10 on the human lung fibroblast cell line WI-38. IL-10 significantly reduced constitutive and transforming growth factor-beta -stimulated type I collagen mRNA expression. However, IL-10 did not affect the proliferation of WI-38 cells induced by platelet-derived growth factor. These data suggested that exogenous IL-10 may be useful in the treatment of pulmonary fibrosis.

transforming growth factor-beta ; pulmonary fibrosis; collagen


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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IDIOPATHIC PULMONARY FIBROSIS (IPF) is a disease characterized by progressive pulmonary insufficiency due to a fibrotic process of unknown etiology. The pathophysiology of pulmonary fibrosis has not been fully elucidated. However, a number of investigations have indicated sustained and augmented expression of some cytokines in the cells from lung fibrosis, and abnormal expression was suggested to be related to the progression of the disease. Tumor necrosis factor (TNF)-alpha (48), interleukin (IL)-1beta (29), IL-8 (25), monocyte chemoattractant protein-1 (20), platelet-derived growth factor (PDGF) (30), and transforming growth factor (TGF)-beta (6) are supposed to play important roles in pulmonary inflammation and fibrosis of IPF. A previous clinical investigation suggested that corticosteroid sometimes ameliorates lung inflammation (3), but in most cases of IPF, it hardly prevents the progression of fibrosis (2) and the prognosis is poor, with a mean survival of 5-6 yr (8, 21). Therefore, new treatment strategies are urgently required.

Intratracheal instillation of bleomycin (Bleo) in mice has been shown to cause lung inflammation and fibrosis (24). This lung injury model in mice caused by Bleo has been used to study the mechanism of lung fibrosis and the antifibrotic effects of many drugs. Anti-TNF-alpha antibody (33), soluble TNF-alpha receptor (34), IL-1-receptor antagonist (35), and anti-TGF-beta antibody (17) were demonstrated to be useful in suppressing Bleo-induced lung injury. These investigations suggested that inhibiting the function of cytokines related to lung inflammation and fibrosis may be an effective means of treating IPF.

IL-10, which is produced by Th2 cells, B cells, monocytes, macrophages, and keratinocytes, is known to suppress many inflammatory reactions (28). IL-10 reduces the synthesis of proinflammatory cytokines such as IL-1, IL-6, IL-8, and TNF-alpha by monocytes/macrophages (10, 13) and polymorphonuclear leukocytes (44), and it also reduces free radical release (15) and nitric oxide synthesis (5) by macrophages and synthesis of interferon-gamma by T cells (14). IL-10 also exerts anti-inflammatory effects by inducing apoptosis of activated neutrophils (9). Anti-inflammatory effects of IL-10 have been identified in vivo in a collagen-induced arthritis model (43). Therefore, it is of interest to determine whether IL-10 can ameliorate pulmonary inflammation induced by Bleo. In this study, we investigated the inhibitory effects of IL-10 on lung injury induced by Bleo in mice. We also examined whether IL-10 can suppress collagen production and proliferation by human lung fibroblasts.


    MATERIALS AND METHODS
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Animals. Seven-week-old male C57BL/6 mice were purchased from SLC Japan (Hamamatsu, Japan) and were kept under specific pathogen-free conditions in our animal facility.

Plasmid vector. Human IL-10 cDNA (American Type Culture Collection) (42) was cloned into the expression vector pCAGGS (31) that contains the chicken beta -actin promoter. Plasmids were grown in Escherichia coli JM109 and prepared with a Qiagen (Chatsworth, CA) Endofree Plasmid Mega Kit. To confirm the capacity of human IL-10 expression, pCA-hIL10 was transfected into the human lung adenocarcinoma cell line A549 with FuGENETM6 transfection reagent (Boehringer Mannheim, Mannheim, Germany). The concentration of human IL-10 in the culture supernatant was measured by enzyme-linked immunosorbent assay (ELISA) with a human IL-10 ELISA kit (Endogen, Cambridge, MA) with a sensitivity of 3 pg/ml. Although mock-transfected cells did not produce significant amounts of human IL-10, 55.2 ng/ml of human IL-10 were detected in the culture supernatant of human IL-10-transfected cells

Hemagglutinating virus of Japan-liposome preparation. Hemagglutinating virus of Japan (HVJ)-liposomes were prepared as previously described (23). Briefly, lipids (phosphatidylcholine, phosphatidylserine, and cholesterol) were mixed in a ratio of 24:5:10 (wt/wt/wt). The lipid mixture (10 mg) in tetrahydrofuran was placed in a rotary evaporator. Plasmids and high mobility group-1 (Wako, Osaka, Japan) were incorporated into liposomes by shaking and sonication. The liposomes and HVJ, inactivated by ultraviolet irradiation (198 mJ/cm2), were incubated at 4°C for 10 min and then at 37°C for 60 min with gentle shaking. This solution was centrifuged on a sucrose gradient. The top layer was collected and diluted with balanced salt solution (BSS). HVJ-liposomes containing pCA-hIL10 (hIL10-HVJ) or BSS only [control (Cont)-HVJ] were intraperitoneally injected into mice in a total volume of 0.3 ml.

Bleo treatment and sample acquisition. C57BL/6 mice aged 7 wk received a single dose of intratracheal Bleo (0.8 mg/kg) in a volume of 0.05 ml. The mice were killed by inhalation of sevoflurane (Maruishi Pharmaceutical, Osaka, Japan), the lungs were excised, and blood was obtained from the right ventricle. Bronchoalveolar lavage (BAL) was performed as follows: 1 ml of isotonic saline was instilled five times and withdrawn from the lungs via an intratracheal cannula.

Myeloperoxidase activity of BAL fluid. Myeloperoxidase (MPO) activity of BAL fluid (BALF) was determined as previously described (11). Briefly, BALF was centrifuged at 400 g for 5 min. The cell pellet was resuspended in 0.1 M K2HPO4 buffer and sonicated for 90 s. After centrifugation at 12,000 g for 10 min, the supernatant (0.35 ml) was mixed with 0.3 ml of Hanks' BSS containing 0.25% bovine serum albumin, 0.25 ml of 0.1 M K2HPO4 (pH 7.0), 0.05 ml of 1.25 mg/ml of o-dianisidine (Sigma, St. Louis, MO), and 0.05 ml of 0.05% H2O2 and incubated for 10 min at 25°C. The reaction was terminated by adding 0.05 ml of 1% NaN3, and absorbance at 460 nm was measured.

Hydroxyproline content. For total lung collagen estimation, tissue hydroxyproline content was measured as previously described (47). Briefly, the lungs were excised, minced, and hydrolyzed with 6 N HCl for 18 h at 105°C. Hydroxyproline in the hydrolysate was assessed colorimetrically at 562 nm with p-dimethylaminobenzaldehyde. Data are expressed as micrograms of hydroxyproline per pair of lungs.

Titration of cytokines in the serum and lavage fluid. Human IL-10 in the serum and abdominal lavage fluid was measured with an UltraSensitive human IL-10 immunoassay kit (Biosource International, Camarillo, CA) with a sensitivity of 208 fg/ml. Mouse TNF-alpha in the serum was measured with a mouse TNF-alpha ELISA kit (R&D Systems, Minneapolis, MN) with a detection limit of 5.1 pg/ml. Mouse IL-10 in the serum was measured with a mouse IL-10 ELISA kit (Endogen, Cambridge, MA) with a detection limit of 12 pg/ml.

cDNA synthesis. Total RNA was isolated with Isogen (Nipponegene, Tokyo, Japan) from BALF cells or human lung fibroblasts. RNA (800 ng) was reverse transcribed in 20 µl of a solution containing 10 mM Tris · HCl (pH 8.3), 50 mM KCl, 5 mM MgCl2, 1 mM each deoxynucleotide triphosphate, 20 U of RNase inhibitor, 2.5 µM random primer, and 5 U of reverse transcriptase with an RNA PCR kit version 2.1 (Takara, Kyoto, Japan) for 10 min at 30°C, for 30 min at 42°C, for 5 min at 99°C, and then for 5 min at 5°C.

PCR. Reaction mixtures contained 1× PCR buffer (Takara), 0.2 mM each deoxynucleotide triphosphate, 400 nM sense and antisense primers, 1.25 U of Taq polymerase (Takara), and cDNA in 50 µl. Amplification was performed in a thermal cycler MP (Takara) for the appropriate number of cycles of denaturation at 94°C for 30 s, annealing for 30 s, and extension at 72°C for 1 min. The optimal number of PCR cycles and annealing temperature for each primer set were as follows: 32 cycles and 57°C for mouse TNF-alpha , 27 cycles and 57°C for mouse glyceraldehyde-3-phospate dehydrogenase (GAPDH), 30 cycles and 56°C for human type I collagen (alpha 1-chain), and 30 cycles and 60°C for human GAPDH. PCR products were electrophoresed on 2% agarose gels and stained with ethidium bromide. The density of the product was calculated with FMBIO II multiview system (Takara).

PCR primer sequences were designed as follows: mouse TNF-alpha sense primer, 5'-GCAGGTCTACTTTAGAGTCATTGC-3'; mouse TNF-alpha antisense primer, 5'-TCCCTTTGCAGAACTCAGGAATGG-3'; mouse GAPDH sense primer, 5'-GGTGA- AGGTCGGTGTGAACGGATT-3'; mouse GAPDH antisense primer, 5'-ATGCCAAAGTTGTCATGGATGACC-3'; human type I collagen (alpha 1-chain) sense primer, 5'-CTGGTCCCAAG- GGTAACAG-3'; human type I collagen (alpha 1-chain) antisense primer, 5'-GCCAGGAGAACCACGTTC-3'; human GAPDH sense primer, 5'-TGCCTCCTGCACCACCAACTGC-3'; and human GAPDH antisense primer, 5'-AATGCCAGCCCCAGCGTCAAAG-3'.

Semiquantitative analysis of mouse TNF-alpha mRNA in BALF cells by RT-PCR. cDNA of mouse BALF cells was synthesized, and half of the cDNA was diluted 10-fold with water. PCR was performed with specific sets of primers in 50-µl mixtures including 4 µl of cDNA for mouse TNF-alpha or 2 µl of diluted cDNA for mouse GAPDH. PCR products were electrophoresed on 2% agarose gels and stained with ethidium bromide. The density of the product was quantified with the FMBIO II multiview system (Takara). The amount of TNF-alpha mRNA was assessed by calculating the ratio of the density of TNF-alpha to that of GAPDH.

Cell culture. The human lung fibroblast cell line WI-38 was obtained from Riken Gene Bank (Saitama, Japan). The cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml of penicillin, and 10 µg/ml of streptomycin. Cells were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO2 and were used between passages 5 and 15. Cells were trypsinized and seeded in DMEM containing 10% FBS at a density of 2.25 × 104 cells/well of a 96-well microtiter plate or at a density of 1.5 × 105 cells/well of a 12-well plate. At confluence, the medium of the 12-well plates was replaced with DMEM containing 0.4% FBS and 50 µg/ml of ascorbate. After 24 h, the cells were cultured in the presence of recombinant TGF-beta (R&D Systems) and/or recombinant IL-10 (PharMingen) for 24 h and used for quantification of type I collagen mRNA. Another 12-well plate was cultured under the same conditions. The cells were recovered by trypsinization, and cell viability was determined with trypan blue staining. Ninety-six-well microtiter plates were used for cell proliferation assay. Cells were seeded at a density of 2.25 × 104 cells/well. After 24 h, before confluence was reached, the medium was replaced with DMEM containing 0.4% FBS (sparse condition). The cells were cultured in the presence of IL-10 and/or recombinant PDGF (R&D Systems) for 24 h.

Quantification of human type I collagen mRNA in human lung fibroblasts by competitive PCR. Competitor DNA, which could be amplified by specific sets of primers for human type I collagen (alpha 1-chain) or GAPDH were constructed with a competitive DNA construction kit (Takara). For competitive PCR, 1 µl of cDNA from WI-38 cells and the competitor DNA (109 to 105 copies) were added to the reaction mixture, and PCR was performed. The PCR products were electrophoresed on 2% agarose gels and stained with ethidium bromide. Densities of the target DNA (type I collagen or GAPDH) and the respective competitor DNA were quantified with the FMBIO II multiview system, and the ratio of target DNA to competitor DNA was calculated. The ratio was plotted against the copy number of competitor DNA added to the reaction mixture, and a regression curve was fitted. The copy number of target DNA in sample cDNA was calculated from the curve. The amount of type I collagen mRNA was assessed by calculating the ratio of the copy number of type I collagen to that of GAPDH.

Cell proliferation assay. Cell proliferation was estimated by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (7). Cells in 96-well microtiter plates were stimulated with IL-10 and/or PDGF. After 24 h, 50 µg of MTT (Sigma) were added to each well. After 6 h of incubation, the culture medium was replaced with 150 µl of DMSO to solubilize formazan crystals. Proliferation is expressed as absorbance at 490 nm recorded with a microtiter plate reader.

Statistical analysis. All data are expressed as means ± SE. The significance of differences between groups was assessed with analysis of variance in conjunction with Fisher's least squares difference test. Only the difference in serum TNF-alpha levels induced by Bleo treatment with and without IL-10 was analyzed by nonparametric Mann-Whitney U-test. Probability values of <0.05 were considered significant.


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Time course of lung inflammation and fibrosis in Bleo-induced lung injury. To investigate the time course of lung injury induced by intratracheal instillation of Bleo, MPO activity of BALF and hydroxyproline content of pairs of lungs were quantified. MPO activity, which was taken as an indicator of the extent of inflammation, increased significantly on day 7 (0.123 ± 0.017) compared with the baseline level on day 0 (0.007 ± 0.001; P < 0.01) and decreased significantly on day 21 (0.029 ± 0.017; P < 0.05; Fig. 1A). In addition, serum TNF-alpha level, which is another parameter of inflammation, was significantly elevated on day 7 (P < 0.05), and its peak concentration was 15.20 ± 5.38 pg/ml (Fig. 1B). TNF-alpha concentration then decreased gradually. After the peak concentration of TNF-alpha , serum IL-10 began to increase and reached a plateau 7 days after the peak concentration of TNF-alpha . Serum IL-10 was elevated significantly on day 21 (67.29 ± 25.79 pg/ml; P < 0.05; Fig. 1B). Hydroxyproline content, which was used to monitor the extent of fibrosis, was increased significantly on days 7, 14, and 21 (P < 0.05), with a maximum of 417.0 ± 76.0 µg/pair of lungs on day 21 (Fig. 1C).


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Fig. 1.   Changes in parameters of pulmonary inflammation and fibrosis after intratracheal (it) bleomycin (Bleo) injection (0.8 mg/kg). Values are means ± SE; n = 4 animals/group. A: myeloperoxidase (MPO) activity of bronchoalveolar lavage (BAL) fluid (BALF) was increased significantly on day 7 compared with baseline level (day 0; * P < 0.01) and decreased significantly on day 21 compared with maximum level (day 7). B: serum tumor necrosis factor-alpha (TNF-alpha ) level was increased significantly on days 7 and 14 (** P < 0.05), but serum interleukin (IL)-10 level was elevated significantly on day 21 after elevation in TNF-alpha (** P < 0.05). C: hydroxyproline content of lungs, a parameter of fibrosis, was increased significantly on days 7, 14 (* P < 0.01), and 21 (** P < 0.05) and showed maximum level on day 21.

In the following experiments, the effects of IL-10 on Bleo-induced lung injury were evaluated by determining MPO activity on day 7 and hydroxyproline on day 21 after Bleo treatment.

Human IL-10 expression in the hIL10-HVJ-treated mice. To confirm successful IL-10 gene transfer and expression, hIL10-HVJ or Cont-HVJ was intraperitoneally injected into mice, and serum levels of human IL-10 were determined by ELISA. Elevated serum levels of human IL-10 (2.23 ± 1.33 pg/ml) were observed 1 day after hIL10-HVJ treatment (Fig. 2A). However, IL-10 expression returned to the background level 2 days after HVJ-liposome injection, and significant elevation of IL-10 was not detected on day 3 or 7. We also tested human IL-10 expression in the peritoneal cavity. Three days after HVJ-liposome treatment, the peritoneal cavity was washed with 5 ml of phosphate-buffered saline, and human IL-10 concentration was measured. In hIL10-HVJ-treated mice, 2.47 ± 0.42 pg/ml of IL-10 were detected. However, significant elevation of IL-10 was not detected in Cont-HVJ-treated mice (Fig. 2B).


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Fig. 2.   Human IL-10 (hIL-10) expression after intraperitoneal (ip) injection of hemagglutinating virus of Japan (HVJ)-liposomes containing hIL-10 expression vector (hIL10-HVJ) or balanced salt solution [control (Cont)-HVJ] into 7-wk-old male C57BL/6 mice. Values are means ± SE. A: serum hIL-10 level of hIL10-HVJ-treated mice increased significantly on day 1 (2.23 ± 1.33 pg/ml) compared with that in Cont-HVJ-treated mice (n = 6/group) and baseline level (* P < 0.05). B: hIL-10 level in abdominal lavage fluid of hIL10-HVJ-treated mice (n = 3/group) was increased significantly on day 3 (2.47 ± 0.42 pg/ml).

Inhibitory effects of IL-10 on Bleo-induced lung injury. To determine whether IL-10 can suppress pulmonary inflammation induced by Bleo treatment, MPO activity (Fig. 3) and TNF-alpha mRNA in the lungs (Fig. 4) were quantified. Mice received an intraperitoneal injection of HVJ-liposomes on day -3, and Bleo was instilled intratracheally on day 0. Then the mice were killed on day 7, and BAL was performed. MPO activity was increased significantly in the with Bleo (Bleo+), without HVJ (HVJ-) group (0.202 ± 0.036) compared with the without Bleo (Bleo-), HVJ- group (0.006 ± 0.002; P < 0.01). However, the increase in MPO activity was significantly reduced by pretreatment with hIL10-HVJ (0.110 ± 0.013; P < 0.05). The HVJ-liposomes without the IL-10 expression vector (Cont-HVJ) showed no suppression (0.232 ± 0.040; Fig. 3). Treatment with Cont-HVJ or hIL10-HVJ without Bleo instillation did not affect the MPO activity level in BALF (0.005 ± 0.001 and 0.006 ± 0.001, respectively).


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Fig. 3.   MPO activity in BALF on day 7 after Bleo treatment. Values are means ± SE. MPO activity was increased significantly by intratracheal Bleo compared with baseline level [without HVJ (HVJ-), without Bleo (Bleo-); n = 4 mice; * P < 0.01]. Increase in MPO activity induced by Bleo [HVJ-, with Bleo (Bleo+); n = 6 mice] was significantly reduced by IL-10 gene introduction (hIL10-HVJ, Bleo+; n = 9 mice; P < 0.05) but not by balanced salt solution (BSS) introduction (Cont-HVJ, Bleo+; n = 7 mice). Basal level of MPO activity was not affected by IL-10 gene introduction (hIL10-HVJ, Bleo-; n = 4 mice) or by BSS introduction (Cont-HVJ, Bleo-; n = 4 mice). NS, not significant.



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Fig. 4.   Effects of hIL10-HVJ treatment on TNF-alpha expression induced by Bleo. On day 7 after Bleo injection, BAL and collection of blood were performed. A: TNF-alpha and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression by BALF cells were assessed by RT-PCR. Densities of PCR products were calculated with FMBIO II multiview system. TNF-alpha mRNA level is presented as ratio of TNF-alpha to GAPDH PCR product densities. Values are means ± SE; n, no. of mice/group. TNF-alpha mRNA level of mice treated with hIL10-HVJ and Bleo+ was decreased significantly compared with mice treated with Cont-HVJ and Bleo+. B: serum TNF-alpha level induced by Bleo was slightly decreased by hIL10-HVJ treatment (by Mann-Whitney U-test). , Means ± SE.

According to the same schedule, the TNF-alpha mRNA level in BALF cells was quantified by RT-PCR (Fig. 4A). hIL10-HVJ treatment resulted in a significant decrease in TNF-alpha mRNA in BALF cells (0.120 ± 0.023) compared with Cont-HVJ treatment (0.229 ± 0.034; P < 0.05). Serum levels of TNF-alpha were lower in hIL10-HVJ-treated mice (1.78 ± 1.95 pg/ml) than in Cont-HVJ-treated mice (28.54 ± 17.62 pg/ml), but this difference was not significant (P < 0.10; Fig. 4B).

Next, the effects of IL-10 on pulmonary fibrosis 21 days after Bleo instillation were assessed. Hydroxyproline content in the Bleo+, HVJ- group (396.6 ± 36.8 µg/pair of lungs) was significantly increased compared with that in the Bleo-, HVJ- group (221.4 ± 3.2 µg/pair of lungs; P < 0.01). However, the increase in hydroxyproline content was significantly reduced by pretreatment with hIL10-HVJ (307.8 ± 8.8 µg/pair of lungs). Cont-HVJ treatment showed no suppression (405.5 ± 24.5 µg/pair of lungs; Fig. 5). Treatment with Cont-HVJ or hIL10-HVJ without Bleo injection did not affect the hydroxyproline content (231.1 ± 7.5 and 226.9 ± 2.4 µg/pair of lungs, respectively).


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Fig. 5.   Hydroxyproline content of whole lungs on day 21 after Bleo treatment. Values are means ± SE. Hydroxyproline content was increased significantly by intratracheal Bleo compared with baseline level (HVJ-, Bleo-; n = 3 mice; * P < 0.01). Increase in hydroxyproline content induced by Bleo (HVJ-, Bleo+; n = 8 mice) was significantly reduced by IL-10 gene introduction (hIL10-HVJ, Bleo+; n = 7 mice; P < 0.05) but not by BSS introduction (Cont-HVJ, Bleo+; n = 8 mice). Basal hydroxyproline content was not affected by IL-10 gene introduction (hIL10-HVJ, Bleo-; n = 3 mice) or BSS introduction (Cont-HVJ, Bleo-; n = 3 mice).

Human type I collagen mRNA expression. To determine the direct effects of IL-10 on collagen production by human lung fibroblasts in a static state or under TGF-beta stimulation, the following in vitro studies were conducted. A human lung fibroblast cell line, WI-38, was cultured in the presence of IL-10 (0.2, 2.0, and 20 ng/ml) and/or TGF-beta (0.1, 1.0, and 10 ng/ml), and expression of type I collagen mRNA was quantified by competitive PCR. Constitutive type I collagen mRNA expression in WI-38 cells was suppressed by IL-10 in a dose-dependent manner (Fig. 6A). Twenty nanograms per milliliter of IL-10 reduced type I collagen mRNA expression to 41%. In contrast, TGF-beta augmented type I collagen mRNA expression dose dependently (Fig. 6B). To assess the interaction of these factors, WI-38 cells were cultured with 10 ng/ml of TGF-beta and 20 ng/ml of IL-10. Only the constitutive level of type I collagen gene expression was detected in cells cultured with TGF-beta and IL-10 (Fig. 6C). Cell viability as determined by trypan blue staining was not affected by stimulation of IL-10 and/or TGF-beta (data not shown).


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Fig. 6.   Effects of IL-10 on type I collagen gene expression. Human lung fibroblast cell line WI-38 was cultured in presence of IL-10 and/or TGF-beta , and mRNAs of type I collagen and GAPDH were quantified by competitive PCR as described in MATERIALS AND METHODS. Gene expression is denoted as ratio of copy number of type I collagen to that of GAPDH. Type I collagen mRNA expression was suppressed by IL-10 (A) and augmented by transforming growth factor (TGF)-beta (B) in a dose-dependent manner. C: competitive PCR of type I collagen. s, Sample cDNA; c, competitor DNA; +, with; -, without. Values are means ± SE. Experiment was performed in triplicate. Induction of type I collagen mRNA expression by TGF-beta (10 ng/ml) was significantly suppressed by IL-10 (20 ng/ml; * P < 0.05).

Human fibroblast proliferation. To verify the direct effect of IL-10 on fibroblast proliferation, cells were cultured under sparse conditions in the presence of IL-10 and/or PDGF. PDGF induced fibroblast proliferation at concentrations of 5 and 20 ng/ml (Fig. 7B), but IL-10 did not influence fibroblast proliferation (Fig. 7A). IL-10 also did not affect fibroblast proliferation induced by PDGF (20 ng/ml; Fig. 7C).


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Fig. 7.   Effects of IL-10 on lung fibroblast proliferation. Human lung fibroblast cell line WI-38 was cultured in presence of IL-10 and/or platelet-derived growth factor (PDGF), and proliferation of cells was assessed by MTT assay. OD 490, optical density at 490 nm. Values are means ± SE. Experiment was performed in quadruplicate. Proliferation was not affected by IL-10 at indicated concentrations (A). PDGF significantly augmented cell proliferation at indicated concentrations (* P < 0.01; B) compared with baseline level. PDGF (20 ng/ml) without IL-10 significantly augmented cell proliferation (* P < 0.01; C). However, IL-10 at indicated concentrations had no effect on proliferation induced by PDGF (C).


    DISCUSSION
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ABSTRACT
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A number of investigations (5, 9, 10, 13-15, 28, 44) have shown that IL-10 is involved in regulation of various aspects of inflammation. Production of TNF-alpha , which is one of the most important factors in the pathogenesis of interstitial pneumonia, is inhibited by IL-10. TNF-alpha induces the expression of adhesion molecules by vascular endothelial cells (22) and intensifies the recruitment of inflammatory cells into the lungs (38). Thus suppressing the effects of TNF-alpha is supposed to weaken pulmonary inflammation. For example, it has been reported that the intratracheal instillation of recombinant IL-10 can suppress TNF-alpha content in BALF and the expression of intercellular adhesion molecule-1 on pulmonary endothelial cells in immune complex-induced lung injury (40). It has also been shown that IL-10 treatment attenuates silica-induced pulmonary inflammation by reducing the production of chemokines, superoxide anions, and nitric oxide by BALF cells (12). However, the effects of IL-10 on pulmonary inflammation induced by Bleo have not been reported previously.

In our experiments, the endogenous TNF-alpha level in the serum of mice treated with intratracheal instillation of Bleo was elevated transiently on day 7 compared with the baseline level on day 0 and decreased gradually thereafter. However, serum IL-10 began to increase after the peak concentration of TNF-alpha , and the maximum concentration of IL-10 was detected from days 14 to 21. The endogenous IL-10 expression may be interpreted as a negative feedback reaction in response to inflammation. Hence it is reasonable to suppose that endogenous IL-10 before the elevation in TNF-alpha can attenuate lung inflammation induced by Bleo. To test the anti-inflammatory effect, we introduced the human IL-10 gene into the peritoneum of mice before Bleo treatment. As anticipated, MPO activity in BALF and TNF-alpha mRNA expression in BALF cells on day 7 after Bleo instillation were significantly inhibited by in vivo human IL-10 gene transfer with HVJ-liposomes. In addition, hydroxyproline content, which is an indicator of the extent of pulmonary fibrosis, was also reduced in our experiments on day 21 after Bleo treatment. The results of the present investigation suggested that IL-10 inhibited not only pulmonary inflammation but also the pulmonary fibrosis induced by Bleo.

Previous in vitro experiments indicated that TNF-alpha is relevant to the induction of fibrosis by augmenting synthesis of fibronectin (4), prostaglandin (18), and TGF-beta (32). Fibrogenic effects of TNF-alpha have also been demonstrated by in vivo experiments. Sime et al. (41) reported that the transfer of TNF-alpha into rat lungs by an adenovirus vector induced severe pulmonary inflammation and patchy interstitial fibrogenesis with the induction of TGF-beta . Moreover, it has been shown that lung-specific persistent expression of TNF-alpha in transgenic mice results in the development of chronic inflammation and severe fibrosis of the lungs (27). These observations are compatible with our findings, and it is reasonable to assume that blocking the effects of TNF-alpha would lead to inhibition of pulmonary fibrosis. Thus our observations have shown that IL-10 exerts its antifibrotic activity partially through reducing TNF-alpha expression.

It is also important to determine whether IL-10 modulates the functions of lung fibroblasts because IL-4, which is a Th2 cytokine and has anti-inflammatory effects similar to IL-10, is known to directly upregulate collagen synthesis by human fibroblasts (36). In addition, it may be possible that endogenous IL-10 elevation in the serum induced by Bleo treatment, which we have shown, intensifies collagen synthesis. The direct effects of IL-10 on collagen production and proliferation of lung fibroblasts have not been extensively investigated. In skin fibroblasts (37, 46), it has been reported that IL-10 can suppress collagen production in the static state. Hepatic stellate cells, the cells that produce collagen in the liver, produce more collagen when cultured with anti-IL-10 antibody (45), and IL-10 is supposed to modulate collagen synthesis by activated hepatic stellate cells in the fibrotic liver. In addition, proliferation of smooth muscle cells is inhibited by IL-10 (39). Thus IL-10 may directly influence the functions of lung fibroblasts. We have shown in this investigation that IL-10 inhibited collagen production by WI-38 cells, a human lung fibroblast cell line, in the steady state and also in the presence of TGF-beta . Proliferation of WI-38 cells was not influenced by IL-10, and proliferation induced by PDGF was not suppressed. The precise inhibitory mechanisms of IL-10 on collagen production have not yet been fully clarified. In addition to prevention of TNF-alpha expression, the present results strongly suggested that IL-10 may exert its in vivo antifibrotic effects through the direct inhibition of collagen gene expression. Hence it may be possible that the elevation in endogenous IL-10 in the serum was too late to prevent pulmonary fibrosis induced by Bleo treatment.

In this investigation, we clarified the biological effects of IL-10 by in vivo gene transfer. The in vivo effects of IL-10 on lung disease have also been studied in IL-10-deficient mice. Huaux et al. (19) reported that in a silica-induced lung fibrosis model, the amount of total protein, lactate dehydrogenase activity, and number of total cells in BALF were increased in IL-10 knockout mice compared with those in normal littermates. However, the hydroxyproline content of lungs on day 30 after silica exposure was decreased in IL-10 knockout mice. They concluded that IL-10 suppresses pulmonary inflammation but may promote the process of pulmonary fibrosis. The apparent contradiction between our results and their observations may be explained by the differences between the Bleo and silica models. In the silica exposure model, intensity of pulmonary fibrosis is inversely correlated with strength of initial inflammation. Adamson et al. (1) explained this by suggesting that intratracheal instillation of leukocyte chemotactic factor can enhance clearance of silica from the lung interstitium by intensifying pulmonary inflammation and thus reduce pulmonary fibrosis. Therefore, the results of this experiment with IL-10 knockout mice do not necessarily indicate that IL-10 exerts fibrogenic effects, and it remains to be determined whether Bleo causes more severe fibrosis in IL-10 knockout mice than in normal mice.

Martinez et al. (26) studied the clinical significance of IL-10 expression in IPF. They reported that macrophages from patients with IPF expressed increased levels of IL-10 mRNA compared with those from healthy control subjects, but less IL-10 was detected in the BALF from IPF patients compared with that from healthy control subjects. The clinical significance of this discrepancy between IL-10 mRNA and protein levels is not clear, but it is possible that the low concentration of IL-10 in the lungs enhances pulmonary inflammation and fibrosis in IPF. The inflammatory and fibrotic foci are present in the same area and at the same time in the lungs of IPF patients. Histopathological observations strongly suggested that IL-10, which exerts both anti-inflammatory and antifibrotic effects, may be useful in the treatment of IPF.

We used in vivo gene transfer utilizing HVJ-liposomes for the administration of IL-10. Because the half-life of IL-10 in mouse serum is no more than 20 min (16), we supposed that in vivo persistent expression of human IL-10 by gene transfer would be an effective strategy to achieve high serum levels of this cytokine. Although the actual duration of IL-10 expression was <2 days, the gene expression was sufficient to suppress lung injury induced by Bleo. There are some problems to be resolved in administering IL-10 by in vivo gene transfer with the use of HVJ-liposomes. The major problem is that the efficiency and duration of IL-10 expression may not be sufficient to ameliorate the lung disease completely. By improving these drawbacks, gene transfer may become a feasible method for the treatment of lung fibrosis.


    ACKNOWLEDGEMENTS

We thank investigators in the Division of Gene Therapy Science (Osaka University Medical School, Osaka, Japan) for supplying hemagglutinating virus of Japan, Dr. Jun'ichi Miyazaki for supplying the plasmid vector pCAGGS, Dr. Junko Mochiduki for technical assistance and instruction, and Yoko Habe for secretarial assistance.


    FOOTNOTES

This work was supported by grants from the Japanese Ministry of Science, Culture and Education; the Japanese Ministry of Health and Welfare; and the Osaka Foundation for Promotion of Clinical Immunology.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: T. Arai, Dept. of Molecular Medicine, Osaka University Medical School, 2-2, Yamada-oka, Suita, Osaka 565-0871, Japan (E-mail: arai{at}imed3.med.osaka-u.ac.jp).

Received 15 September 1999; accepted in final form 13 December 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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