Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of California, Los Angeles School of Medicine, Los Angeles, California 90095-1922
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ABSTRACT |
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Interleukin (IL)-12 is a potent inducer of interferon (IFN)-.
We postulated that IL-12 would attenuate bleomycin-induced pulmonary
fibrosis. To test this hypothesis, we administered IL-12 or murine
serum albumin to bleomycin-treated mice by daily intraperitoneal injection until day 12. Mice treated with IL-12 demonstrated
decreased hydroxyproline levels compared with control treated mice.
Furthermore, administration of IL-12 led to a time-dependent increase
in both lung and bronchoalveolar lavage fluid IFN-
. The antifibrotic effect of IL-12 could be attenuated with simultaneous administration of
neutralizing anti-IFN-
antibodies. These findings support the notion
that IL-12 attenuates bleomycin-induced pulmonary fibrosis via
modulation of IFN-
production.
lung; chemokines; inflammation; in vivo animal models
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INTRODUCTION |
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IDIOPATHIC
PULMONARY FIBROSIS (IPF) is a chronic and often fatal pulmonary
disorder with prevalence rates of 27-29 cases per 100,000 (9). The incidence of IPF appears to be on the rise in
certain parts of the world (10). Conventional treatment
with immunosuppressive therapy has been disappointing, with objective response rates of <30% and a median survival of 5 yr. A recent study
suggests that interferon (IFN)- may have a beneficial role in the
treatment of IPF (43). One of the major inducers of
IFN-
production is interleukin (IL)-12, which is produced by
activated macrophages and antigen-presenting cells and acts on NK and T cells (24). IL-12 has been described to have a variety of
in vivo activities, including antitumor and antiangiogenic activities (8, 11, 37). These effects appear to be mediated through IFN-
(8, 22, 37, 41).
Bleomycin sulfate has been used in rodents to initiate fibrotic lung
lesions, which have many of the histological components of IPF
(1, 4). Bleomycin administration results in a route-, dose-, and strain-dependent pulmonary inflammatory response
characterized by increases in leukocyte accumulation, fibroblast
proliferation, and collagen content. Although these pathological
changes occur in a more rapid fashion than human IPF, the rodent
pulmonary inflammatory response to intratracheal bleomycin challenge
constitutes a representative model of human IPF. The hallmark of
fibrosis is collagen deposition. Because collagen contains significant
amounts of hydroxyproline, measurement of hydroxyproline is a good
index of fibrosis (18, 19, 25, 26). In this study, we show
that IL-12 attenuates bleomycin-induced pulmonary fibrosis.
Furthermore, administration of IL-12 leads to a time-dependent increase
in intrapulmonary IFN-. Moreover, the beneficial effects of IL-12
can be inhibited by simultaneous administration of anti-IFN-
antibodies. These findings provide further support for IFN-
as an
inhibitor of fibrosis.
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MATERIALS AND METHODS |
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Reagents.
Murine IL-12 and IFN- were purchased from R&D Systems (Minneapolis,
MN). Polyclonal anti-murine IFN-
antibodies were produced by the
immunization of rabbits with murine recombinant IFN-
(R&D Systems)
in multiple intradermal sites with complete Freund's adjuvant. The
specificity of the antibody was assessed by Western blot analysis and
ELISA against a panel of other recombinant cytokines. The antibody was
specific in our sandwich ELISA without cross-reactivity to a panel of
cytokines, including IL-1 receptor antagonist protein, IL-1, IL-2,
IL-4, IL-6, tumor necrosis factor-
, interferon-inducible protein-10,
monokine induced by IFN-
, and members of the C-X-C and C-C chemokine
families (2, 3). The IFN-
antibody is a
neutralizing antibody as determined by its ability to block IFN-
activity in an antiviral assay using L-929 cells infected with
encephalomyocarditis virus (38). The
"anti-protease" buffer for tissue homogenization consisted of 1×
PBS with one Complete tablet (Boehringer Mannheim, Indianapolis, IN)
per 50 ml.
Animal model of pulmonary fibrosis.
Female CBA/J mice (6-8 wk) were purchased from Jackson Laboratory
(Bar Harbor, ME). We used CBA/J mice because they are a well-characterized inbred strain of mice which are susceptible to
bleomycin-induced pulmonary fibrosis. Mice were maintained in specific
pathogen-free conditions and provided with food and water ad libitum.
To induce pulmonary fibrosis, mice were treated with intratracheal
bleomycin (Blenoxane, a gift from Bristol Myers, Evansville, IN; 1 U/kg) on day 0 as previously described (18, 19). Control animals received only sterile saline as previously described (18, 19). Briefly, mice were anesthetized with
250 µl of 12.5 µg/ml ketamine injected intraperitoneally followed by intratracheal instillation of 0.025 U of bleomycin in 25 µl of
sterile isotonic saline. Bleomycin-treated mice were given daily
intraperitoneal injections of recombinant murine IL-12 [1 µg in 100 µl of 0.25% mouse serum albumin (MSA)] or MSA (100 µl of 0.25%
MSA) until day 16. Mice were killed on days
2, 4, 8, 12, and
16 for ELISA and on day 12 for hydroxyproline
assay. In separate experiments, mice were again given daily
intraperitoneal injections of IL-12 as described previously; on
days 2, 4, 6, and 8, these
IL-12-treated mice were given either anti-IFN- antibodies or normal
rabbit serum by intraperitoneal injection. These mice were killed on
day 12 for hydroxyproline measurement.
Lung tissue preparation.
Bleomycin- or saline-treated lungs were homogenized and sonicated in
anti-protease buffer as previously described (3, 18, 19,
31). Specimens were centrifuged at 900 g for 15 min,
filtered through 1.2-µm Sterile Acrodiscs (Gelman Sciences, Ann
Arbor, MI), and frozen at 70°C until thawed for assay by specific
IFN-
ELISA or Western blot analysis.
IFN- ELISA.
Antigenic murine IFN-
was quantitated using a modification of an
ELISA as previously described (2, 3). The sensitivity of
our ELISA is
50 pg/ml. Briefly, flat-bottomed 96-well microtiter plates (Nunc) were coated with 50 µl/well of the polyclonal
anti-murine IFN-
antibody (1 ng/µl in 0.6 M NaCl, 0.26 M
H3BO4, and 0.08 N NaOH, pH 9.6) for 24 h
at 4°C and then washed with PBS and 0.05% Tween 20 (wash buffer).
Nonspecific binding sites were blocked with 2% BSA. Plates were rinsed
and samples were added (50 µl/well), followed by incubation for
1 h at 37°C. Plates were then washed and 50 µl/well of the
appropriate biotinylated polyclonal antibody (3.5 ng/µl in wash
buffer and 2% FCS) were added for 45 min at 37°C. Plates were washed
three times, streptavidin-peroxidase conjugate (Bio-Rad Laboratories,
Richmond, CA) was added, and the plates were incubated for 30 min at
37°C. Chromogen substrate (DAKO, Carpinteria, CA) was then added, and
the plates were incubated at room temperature to the desired
extinction. Plates were read at 490 nm in an automated microplate
reader (Bio-Tek Instruments, Winooski, VT). Standards were 1/2 log
dilutions of recombinant IFN-
from 100 ng to 1 pg/ml (50 µl/well).
Western blot analysis of IFN-.
Western blot analysis was performed as described previously
(19). Total protein extracts were made by homogenizing
lungs in TNE lysis buffer (20 mM Tris · HCl, pH 8, 150 mM NaCl,
1% Nonidet P-40, and 2.5 mM EDTA) supplemented with 2 ng/ml aprotinin
and 35 ng/ml phenylmethylsulfonyl fluoride. Cell extracts were
incubated on ice for 30 min, followed by centrifugation at 4°C for 30 min. Supernatants were then removed and assayed for total protein
content using bicinchoninic acid protein assay reagents (Pierce,
Rockford IL) and comparison to known amounts of bovine serum
albumin. Total protein (1 µg) was loaded in each well of a
12% polyacrylamide gel, and extracts were subjected to SDS-PAGE. The
separated proteins were transferred to polyvinylidene difluoride
membrane (Pierce) by electrophoretic transfer overnight in Tris-glycine
buffer [20 mM Tris and 150 mM glycine, pH 8.0, methanol added to a
final concentration of 20% (vol/vol)]. Blots were blocked in 5% skim milk in TBS-T buffer (10 mM Tris · HCl, pH 8.0, 150 mM NaCl,
and 0.05% Tween 20) for 2 h at room temperature, followed by
incubation in rabbit primary antibody serum against IFN-
diluted
1:1,000 in blocking solution for 2 h at room temperature. Blots
were washed for three 10-min washes in TBS-T and were incubated for
1 h at room temperature in goat anti-rabbit horseradish
peroxidase-conjugated secondary antibody (Bio-Rad, Hercules CA) at a
1:10,000 dilution. Blots were again washed for four 10-min washes in
TBS-T, and proteins were visualized after incubation of the blots in
SuperSignal chemiluminescent substrate solution according to the
manufacturer's protocol (Pierce) and exposure to XAR-5 film (Kodak,
Rochester NY).
Hydroxyproline assay. Total lung collagen was determined by analysis of hydroxyproline as previously described (18, 19). Briefly, lungs were harvested on day 12 or 16 post-bleomycin administration and homogenized in 1 ml of PBS, pH 7.4, with a Tissue Tearor. One-half milliliter of each sample (both lungs) was then digested in 1 ml of 6 N HCl for 8 h at 120°C. Five microliters of citrate-acetate buffer (5% citric acid, 7.24% sodium acetate, 3.4% sodium hydroxide, and 1.2% glacial acetic acid, pH 6.0) and 100 µl of chloramine T solution (282 mg of chloramine T, 2 ml of n-propanol, 2 ml of H2O, and 16 ml of citrate-acetate buffer) were added to 5 µl of sample, and the samples were left at room temperature for 20 min. Next, 100 µl of Ehrlich's solution [2.5 g of 4-(dimethylamino)-benzaldehyde (4-DMAB, Aldrich, Milwaukee, WI)], 9.3 ml of n-propanol, and 3.9 ml of 70% perchloric acid (Eastman Kodak) were added to each sample and the samples were incubated for 15 min at 65°C. Samples were cooled for 10 min and read at 550 nm on a Beckman DU 640 spectrophotometer (Fullerton, CA). Hydroxyproline (Sigma Immunochemicals, St. Louis, MO) concentrations from 0 to 400 µg/ml were utilized to construct a standard curve.
Statistical analysis.
Data were analyzed on a Power Macintosh 7500 computer using the
Statview 4.5 statistical package (Abacus Concepts, Berkeley, CA).
Two-group comparisons were made using the unpaired t-test. Multiple groups were compared using ANOVA followed by Bonferroni post
hoc test where appropriate. Data were considered statistically significant at P 0.05. All values are means ± SE.
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RESULTS |
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IL-12 attenuates bleomycin-induced pulmonary fibrosis.
We assessed whether administration of exogenous IL-12 by repeated
intraperitoneal injection during bleomycin-induced pulmonary fibrosis
would attenuate the fibrotic response. We used lung hydroxyproline levels as an index of total lung collagen and fibrosis. Administration of IL-12 on days 1-12 led to reduced total lung
hydroxyproline on day 12 compared with control treated mice
(Fig. 1).
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IL-12 administration leads to augmented intrapulmonary and
bronchoalveolar lavage levels of IFN-.
Having shown that administration of IL-12 led to a reduction in
bleomycin-induced pulmonary fibrosis, we were next interested to see
whether IL-12 augmented intrapulmonary IFN-
levels. We first
measured levels of IFN-
by specific ELISA in lung tissue from
bleomycin-treated mice compared with controls. We found that there was
a deficiency of IFN-
in lung tissue from bleomycin-treated mice
compared with saline-treated controls (Fig.
2). We next looked at IFN-
levels from
lung tissue of bleomycin-treated mice who received IL-12. Lungs were
harvested on days 2, 4, 6,
8, 12, and 16 and assayed for IFN-
by specific ELISA. Before lung harvest, bronchoalveolar lavage (BAL)
was performed. Administration of IL-12 on days 1-16 led
to a time-dependent increase in both intrapulmonary and BAL fluid
IFN-
levels (Fig. 3). This is
consistent with the known effects of IL-12 as a potent
inducer of IFN-
from NK and T cells (24).
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Antifibrotic effects of IL-12 are reversed by antibodies to
IFN-.
Having shown that IL-12 leads to both a reduction in pulmonary fibrosis
and an increase in intrapulmonary IFN-
, we were next interested in
establishing that the antifibrotic effect of IL-12 was mediated through
IFN-
. IL-12-treated mice were given either anti-IFN-
antibodies
or normal rabbit serum (control) on days 2, 4,
6, and 8. We chose these time points based on the
temporal increase in IFN-
that we saw after administration of IL-12.
IL-12 failed to attenuate bleomycin-induced pulmonary fibrosis when simultaneous neutralizing anti-IFN-
antibodies were administered (Fig. 4A). This suggests that
the antifibrotic actions of IL-12 are mediated through IFN-
. This is
consistent with previous studies that have demonstrated that IL-12
induces tumor regression and this effect is mediated through IFN-
(8, 22, 37, 41). Furthermore, administration of
anti-IFN-
antibodies attenuates the IL-12-induced increase in
IFN-
levels, as assessed by Western blot analysis (Fig.
4B). We used Western blot analysis as opposed to an ELISA
because the polyclonal anti-IFN-
antibody used to treat the mice is
the same antibody that we use in our ELISA.
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DISCUSSION |
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Traditional approaches to the treatment of pulmonary fibrosis have
involved immunosuppressive agents, with corticosteroids being the most
commonly used (33). Despite objective response rates of no
more than 30%, the standard of care remains corticosteroids either
with or without a second immunosuppressive agent (33). Whereas experimental models have traditionally focused on attenuating the inflammatory response (32, 35, 42), more recent
approaches have involved attenuation of epithelial injury
(21) or the targeting of the transforming growth
factor- pathway and the associated fibrotic response
(23).
In the present study, we assessed whether administration of exogenous
IL-12 during bleomycin-induced pulmonary fibrosis would attenuate the
fibrotic response. Administration of IL-12 on days 1-12
led to reduced total lung hydroxyproline on day 12 compared with control treated mice. Furthermore, we found that there was a
relative deficiency of IFN- levels in bleomycin-treated lung tissue
compared with controls and that administration of IL-12 led to a
time-dependent increase in both intrapulmonary and BAL fluid IFN-
levels from bleomycin-treated mice. This is consistent with the known
effects of IL-12 as a potent inducer of IFN-
from NK and T cells
(24). Although we did not assess the predominant cellular
source of IFN-
in our study, we would speculate that IL-12 is
inducing its expression from both NK and T cells.
IL-12 failed to attenuate bleomycin-induced pulmonary fibrosis when
simultaneous neutralizing anti-IFN- antibodies were administered. This suggests that the antifibrotic actions of IL-12 are mediated through IFN-
. This is consistent with previous studies that have demonstrated that IL-12 induces tumor regression and that this effect
is mediated through IFN-
(8, 22, 37, 41). Furthermore, the antitumor effects of IL-12 appear to be mediated through the induction of IFN-inducible chemokines, which result in tumor necrosis (17, 30).
IFN- is a known inhibitor of wound repair (34) and has
been shown to attenuate fibrosis in bleomycin-induced pulmonary fibrosis (14, 15). IFN-
can inhibit both fibroblast and
chondrocyte collagen production in vitro, as well as decrease the
expression of steady-state type I and III procollagen mRNA
(5-7, 12, 13, 16). A recent study demonstrated a
beneficial role for IFN-
-1b in the treatment of IPF and suggested
that this effect was mediated through inhibition of transforming growth
factor-
1 and connective tissue growth factor (43). This
would suggest that IFN-
has the potential to inhibit several
pathways involved in the fibrotic response. It has been shown that
there is a deficiency of IFN-
production in fibrosing interstitial
lung diseases (27, 43). This suggests an underlying
deficiency in the natural antifibrotic mechanisms in the lung in IPF.
Moreover, in bleomycin-induced pulmonary fibrosis, there is a
deficiency of IFN-
and of IFN-inducible chemokines
(18). In this study, we have shown that this deficiency of
IFN-
can be corrected by the administration of IL-12, with subsequent attenuation of fibrosis.
Furthermore, IL-12 has the potential to switch the response from a fibrotic T helper type 2 (Th2) phenotype to a more favorable type 1 (Th1) phenotype (36, 40). The realization that Th1 and Th2 cytokines are expressed by a variety of cells and that the function of these cytokines is different suggests that an imbalance in the expression of Th1 and Th2 cytokines may be important in dictating different immunopathological responses (28, 29). Th2 cytokines are predominantly involved in mediating allergic inflammation and chronic fibroproliferative disorders such as asthma, atopic dermatitis, IPF, and systemic sclerosis (28, 29). Thus it may be appropriate to define certain diseases in terms of the predominant cytokine profile.
Although animal models of pulmonary fibrosis have provided insight into
a role for Th2 cytokines in the mediation of pulmonary fibrosis, recent
studies have confirmed this profile in IPF. Lung tissue of patients
with IPF have been examined for the presence of a Th1 vs. Th2 pattern
of cytokine expression by in situ hybridization and immunolocalization
of cytokine protein (39). Although there is evidence for
the existence of both Th1 (characterized by the expression of IFN-)
and Th2 (characterized by the expression of IL-4 and IL-5) cytokines in
IPF lung tissue, the presence of Th2 cytokines predominated over the
expression of IFN-
(39). This pattern of cytokine
expression may be related to the potential role for the humoral
response in the pathogenesis of IPF or related to the inability of
IL-12 and IFN-
to tilt the balance that may favor an
IL-4/IL-13-dependent profibrotic environment. In further support of an
imbalance in the presence of Th2 cytokines compared with IL-12 and
IFN-
is the finding that IFN-
levels are inversely related to the
levels of type III procollagen in the BAL fluid of IPF patients
(20). The levels of IFN-
were especially
correlated with patients that demonstrate progression of their
pulmonary fibrosis by evidence of further deterioration of their
pulmonary function (20). These findings suggest that the
persistent imbalance in the expression of Th1 and Th2 cytokines in the
lung may be a mechanism for the progression of pulmonary fibrosis.
IL-12 has the ability to correct this abnormality by directly promoting a Th1 response and indirectly through the induction of IFN-
, which
itself promotes a Th1 response.
In summary, we have shown that the administration of IL-12 leads to
increased IFN- production and a reduction in pulmonary fibrosis,
suggesting that the serial pathway of IL-12 to IFN-
represents an
important pathway in attenuating the fibrotic response. Furthermore, it
suggests that a Th1 cytokine profile is beneficial in attenuating the
fibrotic response. Future studies will address the possibility that the
antifibrotic effects of IL-12 may be mediated in part through the
induction of interferon-inducible chemokines and the inhibition of angiogenesis.
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ACKNOWLEDGEMENTS |
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This work was supported in part by National Institutes of Health Grants P50-HL-67665 (M. P. Keane and R. M. Strieter), HL-03906 (M. P. Keane), and CA-87879 and HL-60289 (R. M. Strieter).
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. P. Keane, Dept. of Medicine, Div. of Pulmonary and Critical Care Medicine, 900 Veteran Ave, 14-154 Warren Hall, Los Angeles, CA 90095-1922 (E-mail: mpkeane{at}mednet.ucla.edu).
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. Section 1734 solely to indicate this fact.
Received 28 June 2000; accepted in final form 14 February 2001.
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