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
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ABSTRACT |
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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-
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-
-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-; pulmonary fibrosis; collagen
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INTRODUCTION |
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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)- (48), interleukin (IL)-1
(29), IL-8 (25), monocyte
chemoattractant protein-1 (20), platelet-derived growth factor (PDGF)
(30), and transforming growth factor (TGF)-
(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-
antibody (33), soluble TNF-
receptor (34), IL-1-receptor antagonist
(35), and anti-TGF-
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- 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-
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.
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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 -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- in the
serum was measured with a mouse TNF-
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-, 27 cycles and
57°C for mouse glyceraldehyde-3-phospate dehydrogenase (GAPDH), 30 cycles and 56°C for human type I collagen (
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- sense
primer, 5'-GCAGGTCTACTTTAGAGTCATTGC-3'; mouse TNF-
antisense primer, 5'-TCCCTTTGCAGAACTCAGGAATGG-3'; mouse
GAPDH sense primer, 5'-GGTGA- AGGTCGGTGTGAACGGATT-3'; mouse
GAPDH antisense primer, 5'-ATGCCAAAGTTGTCATGGATGACC-3';
human type I collagen (
1-chain) sense primer,
5'-CTGGTCCCAAG- GGTAACAG-3'; human type I collagen (
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- 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-
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-
mRNA was assessed by calculating
the ratio of the density of TNF-
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- (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 (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- 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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RESULTS |
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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-
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-
concentration then decreased gradually. After the peak concentration of
TNF-
, serum IL-10 began to increase and reached a plateau 7 days
after the peak concentration of TNF-
. 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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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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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- 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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According to the same schedule, the TNF- mRNA level in BALF cells
was quantified by RT-PCR (Fig. 4A). hIL10-HVJ treatment resulted in a significant decrease in TNF-
mRNA in BALF cells (0.120 ± 0.023) compared with Cont-HVJ treatment (0.229 ± 0.034; P < 0.05). Serum levels of TNF-
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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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- 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-
(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-
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-
and 20 ng/ml of IL-10. Only the constitutive level of type I
collagen gene expression was detected in cells cultured with TGF-
and IL-10 (Fig. 6C). Cell viability as determined by trypan
blue staining was not affected by stimulation of IL-10 and/or TGF-
(data not shown).
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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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DISCUSSION |
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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-, which is one of the most important
factors in the pathogenesis of interstitial pneumonia, is inhibited by
IL-10. TNF-
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-
is
supposed to weaken pulmonary inflammation. For example, it has been
reported that the intratracheal instillation of recombinant IL-10 can
suppress TNF-
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- 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-
, 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-
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-
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- is relevant to the
induction of fibrosis by augmenting synthesis of fibronectin (4),
prostaglandin (18), and TGF-
(32). Fibrogenic effects of TNF-
have also been demonstrated by in vivo experiments. Sime et al. (41)
reported that the transfer of TNF-
into rat lungs by an adenovirus
vector induced severe pulmonary inflammation and patchy interstitial
fibrogenesis with the induction of TGF-
. Moreover, it has been shown
that lung-specific persistent expression of TNF-
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-
would lead to inhibition of pulmonary fibrosis. Thus our observations
have shown that IL-10 exerts its antifibrotic activity partially
through reducing TNF-
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-. 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-
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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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