 1 mRNA Expression by
Taurine and Niacin in the Bleomycin Hamster Model of Lung Fibrosis
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Abstract
Introduction
Materials & Methods
Results
Discussion
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We have reported that taurine (T) and niacin (N) inhibit the expression of procollagen type I and type III
genes at the level of gene transcription in the bleomycin (BL) hamster model of lung fibrosis. In the
present study, we have investigated the effects of TN in diet on the temporal expression of transforming
growth factor-
1 (TGF-1) mRNA and TGF-1 protein production in the same model of lung fibrosis to
determine whether the decreased transcription of procollagen genes is associated with downregulation of
TGF-1 mRNA. Our results demonstrate that expression of TGF-1 mRNA in lungs is increased in BL-treated hamsters in the BL + control diet (CD) group, compared to saline controls in the saline-instilled (SA) + CD group, by 3.5-, 2.5-, 4-, and 2-fold at 3, 7, 14, and 21 d, respectively, and TN treatment caused
significant decreases in TGF-1 mRNA expression in BL-treated animals in the BL + TN group from Day
3 through Day 21. In addition, TN treatment also reduced TGF-1 protein in bronchoalveolar lavage fluid
(BALF) from BL-treated animals in the BL + TN group. These decreases in TGF-1 mRNA and TGF-1
protein correlated with decreased lung collagen content in hamsters in the BL + TN group as demonstrated in our earlier study. To confirm that the TGF-1 activity observed in BALF is reflected at the transcriptional level, total RNA was isolated from lavaged cells. Reverse transcriptase-polymerase chain reaction analysis demonstrated maximal expression of TGF-1 mRNA transcripts in BL-treated lavaged cells
from animals in the BL + CD group and only low levels were detected in both saline control groups, and
in BL + TN-treated lavaged cells. Nuclear runoff analysis indicated that TN-mediated reduction of TGF-1 mRNA steady-state levels was a result of decreased gene transcription, suggesting a transcriptional downregulation mechanism. Our results indicate that the combined treatment with TN ameliorates BL-induced lung fibrosis, at least in part, via inhibition of TGF-1 mRNA expression.
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Introduction |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Interstitial lung fibrosis (ILF), a common final pathway for
many forms of lung injury, is characterized by alteration in the amount and organization of the extracellular matrix of
the lung (1). Bleomycin (BL)-induced lung fibrosis is a complex process that involves the participation of many cell
types. Increased synthesis and decreased degradation of
collagen result in an increased lung collagen content and
distortion of the pulmonary structure and architecture (2).
Alterations in the expression of a variety of cytokines and
growth factors are believed to contribute to the fibroproliferative process that leads to the development of fibrosis
(3). Evidence from animal models and human studies suggests that the cytokine transforming growth fractor-1
(TGF-1) plays a central role in a variety of fibroproliferative disorders, including pulmonary fibrosis. Various lines
of in vivo and in vitro studies have demonstrated that a
number of cellular constituents of the lung, including alveolar macrophages, bronchial epithelial cells, fibroblasts,
and endothelial cells, have the capacity to contribute to a
large pool of TGF-1 (4).
TGF-1 plays a critical role in the pathogenesis of lung
fibrosis through stimulation of collagen and fibronectin
production in fibroblasts (5), as well as through inhibition of biosynthesis of proteases that degrade the extracellular matrix (9). TGF-1 promotes wound healing (10) and
its presence has been demonstrated in lung macrophages
of BL-induced lung fibrosis in animal models (11). TGF-1
has been shown to be increased in BL-induced lung fibrosis (12, 13). TGF-1 mRNA is elevated before increases in
type I and type III procollagen mRNAs are found during
the course of development of pulmonary fibrosis (12, 13).
It has been demonstrated that the secretion of biologically
active TGF-1 by alveolar macrophages is transiently elevated in BL-induced pulmonary inflammation, whereas
latent (L)-TGF-1 secretion remains elevated for a prolonged length of time and it is likely that plasmin determines the quantity of active TGF-1, which in turn determines the extent of inflammation in this model (14).
Therapeutic advances in the management of ILF have been less than satisfactory despite the evaluation of a diversified group of compounds to prevent collagen accumulation in rodent models of lung fibrosis (15). Taurine (T) has been demonstrated to protect against tissue damage in various in vivo and in vitro models of inflammation and exposure to oxidants (16, 17). Similarly, niacin (N) has also been shown to prevent injury and tissue damage (18).
Previous studies from our laboratory have shown that
collagen accumulation is decreased in vivo by TN in a
multidose BL-hamster model of lung fibrosis (21, 22). This
TN-mediated reduction in collagen accumulation is manifested by decreases in both types I and III procollagen
mRNAs, as demonstrated in our earlier study (22). In view
of the reports that the levels of TGF-1 rise before increases in type I and type III procollagen mRNAs in BL-induced lung fibrosis (12, 13, 23), the present study was
designed to evaluate whether TN treatment could block
BL-induced increased accumulation of TGF-1 mRNAs at
the level of gene transcription. We have also determined the
steady state TGF-1 mRNA levels in lungs and lavaged
cells as well as TGF-1 protein levels in bronchoalveolar lavage fluid (BALF).
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Materials and Methods |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Treatment of Animals
Male golden Syrian hamsters weighing 90-110 g were purchased from Simonsens, Inc. (Gilroy, CA). Hamsters were housed in groups of four, in facilities with filtered air and constant temperature and humidity. All care was in accordance with the guidelines of the National Institutes of Health (NIH) for animal welfare. The hamsters were allowed to acclimate in facilities for 1 wk before all treatments. A 12 h/12 h light/dark cycle was maintained and hamsters had access to water and either pulverized Rodent Laboratory Chow 5001 (Purina Mills, St. Louis, MO) or the same pulverized chow containing 2.5% T and 2.5% N (wt/wt). The animals were fed these diets starting 3 d before the first intratracheal (IT) instillation and continuing throughout the course of the experiment. Under pentobarbital anesthesia hamsters were IT instilled with three consecutive doses of saline or BL (2.5, 2.0, or 1.5 U/5 ml/kg) at weekly intervals as described in our earlier article (22). Animals were randomly divided into four experimental groups: Saline-instilled (SA) with a control diet (CD) (SA + CD); saline-instilled with the TN diet (SA + TN); BL-instilled with the control diet (BL + CD); and BL-instilled with the TN diet (BL + TN).
Animals were killed by decapitation at 0, 3, 7, 14, and
21 d after the last BL or saline instillation and their lungs
(n = 4) were removed and quickly frozen in liquid N2,
then stored at 
80°C until used for mRNA analysis. Five
animals from each group at the same time interval were
killed by intraperitoneal injection of sodium pentobarbital
(90-120 mg/kg), followed by exsanguination. Immediately
thereafter, lungs were lavaged in situ according to the
method of Giri and coworkers (24). After the lavage, the lung tissue was frozen and stored at 80°C until use.
BALF was centrifuged at 4°C for 10 min at 1,500 rpm. The
supernatant was aspirated for TGF-1 assay and the sedimented cells were assayed for TGF-1 mRNA. Both were
stored at 80°C until use.
Molecular Probes
Plasmid pBas containing human TGF-1 cDNA 1.05-kb
EcoRI fragment was obtained from R. Derynk (Genentech, San Francisco, CA). Clone HHCPF19 containing the
cDNA insert of glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1-kb EcoRI fragment) and 18S ribosomal
RNA clone PN29III with a 0.752-kb BamHI and SphI cDNA
insert were obtained from the American Type Culture Collection (Rockville, MD). The plasmids pBR322 and pUC18
were purchased from Pharmacia (Piscataway, NJ). Using
standard procedures, plasmids were isolated and inserts
were purified with a Qiagen gel extraction kit (Qiagen, Chatsworth, CA) after complete restriction endonuclease
digestion. 18S rRNA has been used as an alternative standard with which to normalize the amount of RNA applied
to Northern blots.
Total RNA Isolation and Hybridization Analyses
Total RNA isolation from hamster whole-lung samples
was done according to Gurujeyalakshmi and coworkers (22).
Northern blot experiments were performed as described
previously (23) to determine the effect of dietary intake of
TN on the level of TGF-1 mRNA in BL-treated hamster
lungs. Briefly, total RNA (10 µg/lane) was electrophoresed through 1% agarose-2.2 M formaldehyde gels and transferred to a nylon membrane. The samples were prehybridized at 42°C for 2 h in a solution containing 50%
formamide, 5× SSPE (1× SSPE is 0.15 M NaCl, 10 mM
NaH2PO4, and 1 mM EDTA [pH 7.4]), 0.3% sodium dodecyl sulfate (SDS), and 200 µg/ml sheared salmon sperm
DNA. The membranes were hybridized either with TGF-
cDNA or 18S rRNA cDNA probe, 2 × 106 cpm of probe
per milliliter of hybridization solution at 42°C for 16 h. RNA
hybridization and washings were done as described elsewhere (22). We normalized the TGF-1 mRNA steady state
levels relative to 18S rRNA. Relative intensities of hybridized probe in each band were determined using a dual-wavelength flying spot scanning densitometer (model CS-9301PC; Shimadzu, Columbia, MD). The signal intensity
for TGF-1 mRNA bands was divided by the signal intensity of 18S rRNA bands in the same lanes to control for
variations in the quantity of RNA loaded in each lane.
Nuclear Run-off Transcription Analysis
Isolation of nuclei from lungs and in vitro transcriptional
reactions were performed by the method of Gurujeyalakshmi and coworkers (22). Plasmids containing cDNA inserts
(namely TGF-1, GAPDH, and 18S rRNA) were linearized
with the appropriate restriction enzymes and processed
before RNA extraction. Ten micrograms of plasmid with
cDNA inserts were slot-blotted onto nylon membrane and
UV cross-linked. Insert-free pBR322 and pUC18 were included as a control for nonspecific binding. The membrane
strips were prehybridized and then hybridized with buffer
containing 3.5 to 4.3 × 106 cpm/ml of 32P-labeled transcripts. RNA binding was evaluated by autoradiography and
densitometry.
RNA Extraction and Reverse Transcribed-Polymerase Chain Reaction
BAL cells obtained from lung lavages (n = 5) were washed
in ice-cold sterile isotonic saline and total cellular RNA
was extracted by the RNeasy total RNA extraction protocol (Qiagen, Chatsworth, CA) according to the manufacturer instructions. For the synthesis of cDNA, 1 µg of RNA,
measured by spectrophotometer from each sample, was
mixed with 1 µl of oligo(dT)18 primer and heated at 70°C
for 2 min. After quenching the samples on ice, the following components were added to make a 20-µl final volume
of reaction buffer: 50 mM Tris-HCl (pH 8.3), 75 mM KCl,
3 mM MgCl2, a 0.5 mM concentration of each dNTP, RNase
inhibitor (1 U/µl), and moloney-murine leukemia virus
(MMLV) reverse transcriptase (
200 units/µg RNA). The reaction was incubated at 42°C for 1 h, and stopped by
denaturing the enzyme at 94°C for 5 min. The mixture was
then diluted with RNase-free water to a volume of 100 µl.
Polymerase chain reaction (PCR) amplification was
performed using commercially available PCR primers (Clontech Laboratories, Palo Alto, CA) for human TGF-1 and
GAPDH. The latter was used as an indicator of constitutive expression. The sequences of the primers used were as
follows:
TGF-1: 5' primer: 5'-GCCCTGGACACCAACTATTGCT-3'
3' primer: 5'-AGGCTCCAAATGTAGGGGCAGG-3'
GAPDH: Sense: 5'-TGAAGGTCGGTGTCAACGGATTTGGC-3'
Antisense: 5'-CATGTAGGCCATGAGGTC-CACCAC-3'
Five-microliter aliquots of the synthesized cDNA were added to 45 µl of PCR mix containing 4 µl of 10× PCR buffer, 1 µl of deoxynucleotides (1 mM each), 1 µl of 3' and 5' RNA-specific sense and antisense primers (0.15 µM), and 0.25 µl of DNA polymerase (GeneAmp PCR kit; Perkin-Elmer, Cetus, Norwalk, CT). The reaction mixture was covered with 50 µl of mineral oil (Perkin-Elmer Cetus). Amplification was initiated by 5 min of denaturation at 94°C for 1 cycle followed by 30 cycles at 94°C for 45 s, 60°C for 45 s, and 72°C for 2 min using a GeneAmp PCR 480 DNA thermal cycler (Perkin-Elmer Cetus). After the last cycle of amplification, the samples were incubated for 7 min at 72°C for extension. The PCR products were visualized by UV illumination after electrophoresis through 2.0% agarose (UltraPure; BRL, Gaithersburg, MD) in 1× TAE buffer and stained with ethidium bromide (0.5 µg/ml).
Quantitative analysis of TGF-1 message was conducted by means of competitive PCR using stepwise dilutions
with the synthetic competitor (PCR MIMIC; Clontech
Laboratories) as a template. The competitors are sequences complementary to the TGF-1 primers but with
different-size PCR products. Five-microliter aliquots of
cDNA were amplified for 30 cycles with the TGF-1 primers in the presence of TGF-1 MIMIC (competitor), ranging from 10
2 to 101 amol of competitor. The amplified
PCR product from TGF-1 mRNA was 161 bp, whereas
the size for TGF-1 MIMIC was 270 bp. The relative amount
of mRNA transcripts was determined visually and by laser
densitometry. The ratio of the target densitometry value to the corresponding competitor densitometry value was
plotted against the reciprocal of the molar concentration
of the competitor.
TGF-1 Quantitation in BALF
BALF was obtained from five hamsters in each group.
BALF recovery in saline-treated hamsters in SA + CD
and SA + TN groups was 84-90% and it was 85-88% in
BL-treated hamsters in BL + CD and BL + TN groups.
There were no statistically significant differences in the
volume of BALF recovered among the four groups. TGF-1 in the BALF was assayed using a commercially available Predicta TGF-1 enzyme-linked immunsorbent assay (ELISA)
kit (Genzyme Diagnostics, Cambridge, MA). This kit contains a 96-well microtiter plate with immobilized mouse
monoclonal antibody to TGF-1. The reported sensitivity
for this ELISA is 0.05 ng/ml and the assays were performed according to manufacturer recommendations. We generated standard curves using TGF-1 provided with the kit
and reported the results as the mean of five samples (in pg/
ml) of BALF.
Statistical Analysis
Data were expressed as the mean ± SEM. The data were
compared within the four groups at the corresponding
times using a one-way analysis of variance and the Fisher's
least significant difference test (SAS Institute Inc. [25]). A
value of P 
0.05 was considered to be the minimum level
of statistical significance.
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Results |
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Abstract
Introduction
Materials & Methods
Results
Discussion
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Regulation of TGF-1 mRNA Accumulation by T and N
Northern blot analysis was used to determine the kinetics
of TGF-1 mRNA expression from four groups of hamster
lungs (SA + CD, SA + TN, BL + CD, and BL + TN)
treated with either saline or BL, with and without TN
treatment. The normalized TGF-1 mRNA expressed for
each experimental group is shown in Figure 1. IT instillation of saline in the SA + CD or SA + TN group did not induce a significant signal for TGF-1 mRNA. In contrast,
a 2.5-kb TGF-1 mRNA transcript was readily detected in
BL-treated hamsters in the BL + CD group. The results
show that TGF-1 mRNA steady state levels in the latter
group were upregulated significantly (P < 0.05) at all times
except at 0 and 7 d and maximized at 14 d following the IT
instillation of the last dose of BL relative to controls. The
steady state levels of TGF-1 mRNA for the BL + CD
group were 3.5-, 2.5-, 4.0-, and 2.0-fold higher than the saline control in the SA + CD group at 3, 7, 14, and 21 d, respectively. T and N treatment dramatically inhibited BL-induced overexpression of TGF-1 mRNA in the lungs of
hamsters in the BL + TN group at all time points. TGF-1
mRNA reductions in the BL + TN group were significant
(P < 0.05) when compared with the corresponding BL + CD group at 3, 7, 14, and 21 d after the IT instillation of the last dose of BL. The downregulation at these time points
ranged from 55 to 76% in the BL + TN group relative to
the BL + CD group. Figure 2 represents the Northern blot
showing the effect of T and N on TGF-1 mRNA accumulation in BL-instilled hamster lungs.
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Regulation of TGF-1 Protein Production by T and N
We hypothesized that if TGF-1 mRNA expression in lungs
was less in the BL + TN group than the BL + CD group,
we may find a corresponding lower level of TGF-1 protein in the BALF of this group. To investigate whether or
not dietary intake of TN decreases TGF-1 protein levels
in the BALF of BL-treated hamsters, an ELISA was used
to quantitate TGF-1 with the assay sensitivity of about 0.05 ng/ml. TGF-1 protein was detected in the BALF of
all control animals and it ranged from 61 to 90 pg/ml. The
TGF-1 protein content of the BALF from BL-treated
hamsters in the BL + CD group increased to 264, 304, 313 and 217% of the corresponding SA + CD group values at
0, 3, 7, and 14 d after the last dose of BL instillation, respectively. The combined treatment with T and N reduced
the BL-induced increased secretion of TGF-1 protein in
BALF samples from the BL + TN group. Statistically significant reductions in TGF-1 protein in BALF were seen
from Days 3 through 14 in the BL + TN group (Figure 3).
The decreases were 26% at Day 0, 49% at Day 3, 68% at
Day 7, 42% at Day 14, and 22% at Day 21 relative to the
corresponding BL + CD groups.
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TGF-1 mRNA Expression in BAL Cells
Total cell counts of BAL cells showed that there were no
significant differences between SA + CD and SA + TN
groups. In contrast, the total and differential cell counts
were significantly higher in the BL + CD and BL + TN
groups compared with the control groups (data not shown).
However, we did not find any noticeable differences in
differential cell counts of BAL cells (macrophages, neutrophils, and lymphocytes) between BL + CD and BL + TN groups. Differential cell count analysis of BAL cells
revealed that macrophages were the predominant cell type
that was increased both in BL + CD- and BL + TN-treated groups in this multidose BL-hamster model. Therefore, we further investigated whether the observed increase in TGF-1 synthesis during lung injury by BL was
due to TGF-1 released mainly by alveolar macrophages
(or other inflammatory cells) accumulated in BALF. A
competitive reverse transcribed (RT)-PCR technique was
employed to analyze the TGF-1 gene expression in BAL
cells from saline- or BL-treated groups with and without the dietary intake of TN to determine whether the TGF-1
activity observed in BALF of all four groups is reflected at
the transcriptional level. Because the proportion of macrophages among BAL cells was always above 95-98% in
both BL-treated BL + CD and BL + TN groups, we did
not purify them from total BAL cell samples in order to avoid the effects of various ingredients present in the culture media on the activity of macrophages.
RNA was isolated from lavaged cells of all four groups
14 and 21 d after the last IT instillation. The purified RNA
from these cells was reverse transcribed and the resulting
cDNA was amplified using specific primers. The cDNA
samples were also used to detect GAPDH transcripts as a
check for successful PCR amplification (Figure 4A). The
intensity of GAPDH transcript staining was similar in all
samples, whereas the intensity of TGF-1 transcript staining was variable. We preferred TGF-1 MIMIC to measure TGF-1 mRNA transcripts in BAL cells because, in
our preliminary experiments, we found that mRNA levels
of the so-called housekeeping genes, -actin and GAPDH,
increased twofold 7 d after BL administration. The induction of these genes after lung injury has been previously reported by other investigators as well (3, 13). We therefore decided to quantitate TGF-1 mRNA expression using competitive RT-PCR (Figure 4B). Maximal TGF-1
expression was observed in BAL cells from BL-treated
hamsters in the BL + CD group and only low levels were
detected in other groups as revealed by densitometric analysis (Figures 4C and 4D). The constitutively expressed gene GAPDH was used to normalize cDNA concentrations.
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X174 HaeIII digest;
BRL); lanes 2-5, SA + CD; lanes 6-9, SA + TN; lanes 10-13, BL + CD; lanes 14-17, BL + TN; lane 18, TGF-Downregulation of TGF-1 Gene Expression by
T and N at the Transcriptional Level
To elucidate the nature of this apparent inhibition of gene
expression by TN treatment in the BL + TN group, a nuclear runoff transcriptional assay was performed to determine whether altered TGF-1 mRNA and subsequently
TGF-1 protein levels occur at the transcriptional level.
The assay was performed as described previously by Gurujeyalakshmi and coworkers (22). Newly synthesized RNA transcripts from lung nuclei of hamsters in SA + CD, SA + TN, BL + CD, and BL + TN groups were isolated 14 d after IT instillation of saline or BL as described in MATERIALS AND METHODS. As shown in Figure 5 and Table 1, the
labeled transcripts that hybridized to the cDNA of TGF-1
were decreased by almost 50% in the BL + TN group.
Transcription of TGF-1 was readily detected in nuclei prepared from lungs of hamsters in the BL + CD group.
In contrast, transcription of TGF-1 was barely detectable
in nuclei prepared from the lungs of hamsters in the BL + TN group. The hamsters in the SA + CD and SA + TN
groups also exhibited levels of gene transcription similar
to that found in the BL + TN group (data not shown).
There was no alteration in the rate of transcription of the
gene encoding 18S rRNA in lung nuclei of hamsters either in the BL + CD or BL + TN group. Interestingly, TN
treatment caused an approximately 50-60% decrease in
BL-induced TGF-1 gene transcription, mRNA accumulation, and subsequently TGF-1 protein production. These
results suggest that TN may have transcriptional effects on
TGF-1 gene expression although the exact nature of the
transcriptional control remains to be elucidated.
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Discussion |
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Abstract
Introduction
Materials & Methods
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Discussion
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We have previously suggested a possible role of TGF-1 in
the downregulation of BL-induced procollagen gene expression by TN in the BL-hamster model of lung fibrosis
(22). In the present study, we have investigated the temporal expression of TGF-1 and protein and their attenuations by TN treatment in the same model of lung fibrosis.
It is interesting that an increase in the levels of TGF-
mRNA correlates well with an increase in the levels of
TGF-1 followed by increases in type I and type III procollagen mRNAs as reported during the course of development of pulmonary fibrosis (12, 13).
Mechanisms for the initiation and progression of lung
fibrosis may involve growth factors that act alone or in concert with other cytokines to result in the accumulation of
collagen in the lung. TGF-1 is a growth factor with multiple effects on the homeostasis of the extracellular matrix
and its mechanisms may include (1) increased synthesis
and secretion of matrix proteins; (2) increased transcription, translation, and processing of receptors for the extracellular matrix-cell adhesion proteins; (3) decreased synthesis of matrix-degrading proteinases; and (4) increased
synthesis of specific inhibitors of these proteinases (26).
We observed a marked upregulation in the production
of TGF-1 mRNA in the present multidose BL-hamster
model of lung fibrosis in the BL + CD group. Steady state
mRNA analysis by Northern blot demonstrated elevated
levels of TGF-1 mRNA, suggesting the abundance of
TGF-1 mRNA in this group is probably due to transcriptional activation or prolongation of the half-life of the
mRNA. The results of the present study demonstrate that
TGF-1 mRNA synthesis, TGF-1 mRNA steady state
levels, and TGF-1 protein are elevated in BL-treated hamster lungs in the BL + CD group and that their elevations
are significantly suppressed by TN treatment in the BL + TN group. Increases or decreases in mRNA may result
from a variety of mechanisms, including changes in gene
copy number (DNA), message stability, and alterations in
gene transcription rate (27). Our results also suggest
that the inhibition by TN treatment occurs, at least in part,
at the transcriptional level. This conclusion is based on the
analyses of TGF-1 message level by Northern hybridization, competitive RT-PCR, and nuclear run-off assays using lung and BAL cells.
The biochemical and molecular mechanisms of increased collagen synthesis in pulmonary fibrosis involves
the interaction among the various factors essential to the
genesis of fibrosis. The role of TGF-1 has been elegantly
demonstrated in the increased synthesis of collagen and fibronectin in fibroblasts (5). TGF-1 also appears to
downregulate various proteases responsible for degradation of extracellular matrix (30). The present study identified a constitutive level of expression of TGF-1 mRNA
transcripts in lavaged cells of control groups. This TGF-1
mRNA expression was increased in cells collected from
BALF of hamsters treated with BL intratracheally in the
BL + CD group. However, treatment with TN reduced
the BL-induced increases in TGF-1 transcripts in BAL
cells from hamsters in the BL + TN group. These data
suggest that if TGF-1 mRNA abundance is important in
BL-induced lung fibrosis, TN are likely to be beneficial in
diminishing the fibrosis.
TGF-1 has been localized in macrophages in the lungs
of animals undergoing BL-induced lung fibrosis (11). Elevated levels of TGF-1 mRNA expression in alveolar macrophages in lungs from patients with idiopathic pulmonary
fibrosis have also been reported (31). Macrophages are
known to have an important role in chronic inflammation
and wound repair (32). Activated macrophages secrete a
number of proinflmmatory and fibrogenic cytokines such
as platelet-derived growth factor (PDGF), interleukin-1
(IL-1), tumor necrosis factor-
(TNF-), basic fibroblast
growth factor (bFGF), and TGF-. These cytokines are
chemotactic to inflammatory cells and promote the fibroblast proliferation, which in turn, increases connective tissue synthesis. It is also known that TGF- secreted by
macrophages regulates inflammation and fibrosis (33, 34).
The cell types in lung responsible for this increase in TGF-1
mRNA are fibroblasts (35), macrophages (11), bronchial
epithelial cells (36), and endothelial cells (37). At lower
concentrations, TGF-1 is a potent chemoattractant for
macrophages and monocytes (38), whereas at higher concentrations it activates these cells to secrete IL-1, TNF-,
PDGF, and bFGF and autostimulates its own production (38), thereby providing various growth factors known to
be involved in the fibroproliferative diseases including
lung fibrosis.
The present study by RT-PCR analysis tested the hypothesis that TN treatment could modulate the TGF-1
mRNA abundance in cells from the BALF of BL-treated
hamsters. Although BL-treated hamsters in both BL + CD and BL + TN groups had a significantly higher number of cells in BALF than did hamsters in the SA + CD and SA + TN groups, there were no significant differences
in the differential cell counts between the two BL-treated
groups (data not shown). Analysis of differential cell
counts revealed that the macrophages were the only main
type of BAL cell that were increased in both BL + CD
and BL + TN groups, suggesting that macrophages in the
former group play a central role in the pathogenesis of
lung fibrosis. The finding that TN treatment downregulates the BL-induced overexpression of TGF-1 mRNA in the
BAL cells (mostly macrophages) suggests that treatment
with TN has compromised the ability of these cells to produce TGF-1; one of the mechanisms by which this could
be achieved is by a suppression of TGF-1 gene activation.
It is believed that the activation of growth factor genes in
lung macrophages is important in the pathogenesis of lung
fibrosis. Previous studies have suggested that macrophages play a central role by releasing mediators that may be involved in inflammation, fibroblast proliferation, and increasing the amount of extracellular matrix in pulmonary
fibrosis (39). Therefore, it is highly likely that there is a
direct relationship between the potency of potential antifibrotic agents and their ability to suppress the activation of
growth factor genes, particularly TGF-1. Furthermore, it
has been shown that BL treatment stimulates TGF- production by fibroblasts (35), and a positive feedback loop
could also increase procollagen mRNA transcription and
collagen synthesis. The secreted TGF-1 after activation
could interact with TGF- receptors on the fibroblasts. Rat
lung fibroblasts have type I, II, and III TGF- receptors,
subsets of which appear to have a higher affinity for TGF-1
than TGF-2 (42). However, the effects of TN treatment
on TGF-1 receptors are not known at this time.
The role of TGF-1 in the development of BL-induced
lung fibrosis is further supported by the finding that treatment with anti-TGF-1 antibody (43) caused a marked reduction in BL-induced lung fibrosis. In addition, long-term
dietary supplementation with vitamin E in animals undergoing CCl4-induced liver fibrosis produces a net inhibition
of both hepatic TGF-1 and procollagen 2(I) mRNA levels (44). Although the initial stimulus for TGF- synthesis
may be different in different types of pulmonary fibrosis, interferon- (IFN-) has also been reported to inhibit
TGF-, which in turn arrests the ongoing fibrotic reactions
(45). Castilla and coworkers (46) were able to decrease
hepatitis C-induced liver fibrosis in 75% of patients treated
with IFN-. All these patients had normal levels of TGF-
mRNA in the liver at the end of the therapy. Matyne and
coworkers (47) also showed decreased TGF- levels in
platelets of patients with myelofibrosis with myeloid metaplasia after treatment with IFN-
. Similarly, we also found
that in vivo treatment with IFN- reduced the BL-induced lung fibrosis in mice and the TGF- mRNA levels followed by decreased procollagen mRNA levels and lung collagen content (23).
It is also known that T offers protection against oxidant-induced lung injury by inhibiting production of nitric oxide
and the release of TNF-, which are known to be directly
linked to tissue injury (48, 49). Taurine acts as a trap for
toxic hypochlorous acid and forms the less reactive metabolite, N-chlorotaurine. It is tempting to speculate that the
anti-inflammatory effect of T in suppressing BL-induced
lung inflammation and increased influx of inflammatory
cells might be responsible for decreased levels of TGF-1
mRNA and protein in the BL + TN group. However, this
is highly unlikely, because there was no difference in the differential cell counts between the BL + CD and BL + TN groups in this multidose BL-hamster model of lung fibrosis.
The results of this study suggest the involvement of a
transcriptional mechanism, at least in part, for decreased
levels of TGF-1 mRNA and protein in the BL + TN group.
Transcriptional activation normally requires the interaction of an active transcriptional factor(s) with a specific cis
element located in the 5' flanking region of a gene. TGF-1
has previously been shown to stimulate the type I collagen
gene through binding elements in the 5' flanking region
(50, 51). The activity of the procollagen 2(I) promoter is
increased by TGF- through the nuclear factor 1 site (50), whereas procollagen 1(I) promoter activity is increased at
a site more distant from the initiation site of transcription
(51). In addition to TN-mediated reduction of collagen
content in the BL-hamster model of lung fibrosis, as reported previously (22), we now report a decrease in TGF-1
mRNA and TGF-1 protein levels in the present study.
These data indicate that TN may regulate collagen synthesis, at least in part, through the mediation of TGF-1 in
BL-induced lung fibrosis. Whether T and N administered alone will produce a similar effect is not known at this
time. However, it should be noted that these two compounds were demonstrated to produce an antifibrotic effect independently in the single-dose BL-hamster model
of lung fibrosis (19, 52).
On the basis of the results obtained from this study,
we conclude that the overexpression of TGF-1 is closely
linked with the development of BL-induced lung fibrosis,
and that treatment with T and N can effectively abrogate
this BL-induced lung fibrosis by downregulating the expression of this gene at the mRNA level.
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Footnotes |
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Abbreviations: bronchoalveolar lavage fluid, BALF; basic fibroblast
growth factor, bFGF; bleomycin, BL; control diet, CD; glyceraldehyde-3-phosphate dehydrogenase, GAPDH; interferon-, IFN-; interleukin-1,
IL-1; interstitial lung fibrosis, ILF; intratracheal, IT; latent, L; niacin, N;
polymerase chain reaction, PCR; platelet-derived growth factor, PDGF;
reverse transcribed, RT; saline-instilled, SA; taurine, T; transforming
growth factor-1, TGF-1; tumor necrosis factor-, TNF-.
(Received in original form December 4, 1996 and in revised form July 28, 1997).
Acknowledgments: The authors thank S. N. Iyer for assistance in feeding the animals. This work was supported by a grant (R01 HL 56262-02) from the National Heart, Lung, and Blood Institute of the National Institutes of Health and the Biomedical Research Program of the School of Medicine at U.C. Davis (M.A.H.).
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References |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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