 2 Production in Cultured Human Bronchial Epithelial
Cells Is Attenuated by Interferon-
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
Abstract |
|---|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Cytokines derived from lymphocytes are believed to play key
roles in a variety of diseases, including airway diseases such as
asthma. The current study was designed to evaluate the hypothesis that cytokines derived from Th2 cells, interleukin
(IL)-4 and IL-13, might contribute to tissue remodeling by
modulating the production of transforming growth factor
(TGF)-
. In addition, the ability of interferon (IFN)-
, a cytokine derived from Th1 cells that can antagonize many effects
of IL-4 and IL-13, was also assessed for its effects on TGF-
production. IL-4 and IL-13 both stimulated production of TGF-2
release from human bronchial epithelial cells in a time- and
concentration-dependent manner. Both with and without
acidification, TGF-2 were detected. Neither TGF-1 nor TGF-3
was released. In contrast to the stimulatory effect on human
bronchial epithelial cells, neither IL-4 nor IL-13 stimulated release of any TGF- isoform from human lung fibroblasts. IFN-
reduced both basal, IL-4-, and IL-13-stimulated release of
TGF-2 in human bronchial epithelial cells. The stimulatory effects of IL-4 and IL-13 and the inhibitory effect of IFN- on TGF-2 release were paralleled by mRNA levels, as assessed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). In summary, the Th2-derived cytokines, IL-4 and IL-13,
can stimulate production of TGF- from airway epithelial cells
but not from lung fibroblasts. IFN-, in contrast, can inhibit
TGF-2 release both under basal conditions and following IL-4
or IL-13 stimulation. The ability of these cytokines to modulate
TGF- release may contribute to both normal airway repair
and to the development of subepithelial fibrosis in asthma.
| |
Introduction |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
The transforming growth factor-s (TGF-s) are a family
of dimeric polypeptide growth factors consisting of three
isoforms: TGF-1, TGF-2, and TGF-3, which are expressed in many cell types including epithelial and mesenchymal cells (1). TGF- regulates cellular proliferation
and differentiation in several settings, including embryonic
development, wound healing, and angiogenesis (2). An
increase or a decrease in the production of TGF- has been linked to several disease states, including atherosclerosis and fibrotic disease of the kidney, liver, and lung (6,
7). In asthma and chronic obstructive pulmonary diseases
(COPD), chronic inflammation and injury of both the airways and the alveolar structures of the lung are observed
and both peribronchiolar fibrosis and subepithelial fibrosis
are also often seen (8). In addition, TGF- immunoreactivity is increased in epithelial cells and submucosa of
those with asthma and bronchitis (11). TGF- has been
suggested to function in the airway both as a mediator of
normal repair processes and a contributor to the development of peribronchiolar fibrosis.
In asthma, cytokines produced by activated Th2 lymphocytes are believed to play critical roles in regulating
the inflammatory process. Interleukin (IL)-4 and IL-13 in
particular have been suggested to be key factors contributing to the chronic inflammatory state characterizing asthma
(12, 13). These cytokines may also be involved in the connective tissue alterations that characterize airway remodeling in asthma. Both cytokines have been demonstrated to stimulate fibroblasts (14). Conversely, interferon (IFN)- is a cytokine derived from a variety of cell types, including Th1 lymphocytes (15). This cytokine is believed to be deficient in asthma and is believed to antagonize some of the
effects of IL-4 and IL-13 (16, 17).
The current study was designed to determine if the Th2
lymphocyte-derived cytokines, IL-4 and IL-13, might modulate airway remodeling by altering production of TGF-.
In addition, the ability of IFN- to interact with IL-4 and
IL-13 was assessed. These studies demonstrate that IL-4
and IL-13 may have a profibrotic effect mediated by the
stimulation of TGF- production by airway epithelial cells, an effect which is not observed with lung fibroblasts. IFN-, in contrast, appears to downregulate TGF- production and to antagonize the IL-4 and IL-13 effect.
| |
Materials and Methods |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Recombinant human IL-4, IL-13, and IFN-, recombinant human TGF-1 and -2, mouse anti-human TGF-1 (clone: 9016-2)
and -2 (clone: 8,607.211), monoclonal antibodies (used for capture in enzyme-linked immunosorbent assay [ELISA]), and biotinylated anti-human TGF-1 and -2 antibodies (for detection) were
purchased from R&D Systems Inc. (Minneapolis, MN). Tetramethylbenzidine was purchased from Sigma Chemical Co. (St.
Louis, MO).
Cell Culture
Human bronchial epithelial cells. Adult human bronchial epithelial cells (HBECs) were obtained by the explant method (18). Briefly, bronchi obtained at autopsy were cut into 4- to 5-mm squares with a sterile scalpel. Individual explants were placed onto 35-mm culture dishes coated with Vitrogen 100 (Collagen, Palo Alto, CA) in serum-free medium comprised of a 1:1 mixture of Laboratory of Human Carcinogenesis (LHC)-9 and RPMI 1,640 (GIBCO Life Technologies, Grand Island, NY). LHC-9 containing LHC basal medium (Biofluids, Rockville, MD) was supplemented as previously described (19). Culture dishes containing explants were then incubated at 37°C in a humidified atmosphere of 5% CO2. After cells grew to confluence, cells were trypsinized and passaged onto Vitrogen-coated dishes in LHC-9/ RPMI. Third-passage cultures were stored in liquid N2. Cells of passages 4-8 were used for experiments. Cells were routinely checked with anti-human cytokeratin antibody (MAK-6; Triton, Alameda, CA) and anti-vimentin (DAKO, Santa Barbara, CA) for purity. All cells were found to be keratin positive and, in general, vimentin negative. Under some conditions, slight vimentin positivity could be observed. This was in marked contrast to the strong vimentin positivity and clear keratin negativity routinely observed with fibroblasts. Cell preparations, therefore, were free of fibroblast contamination to the limit of the immunohistochemical techniques.
Human fetal lung fibroblasts.Human fetal lung fibroblasts (HFL-1) were purchased from the American Type Culture Collection (Rockville, MD). The cells were cultured in 100-mm tissue culture dishes (FALCON) (Becton Dickinson Labware, Lincoln Park, NJ) in Dulbecco's modified Eagle's medium (DMEM) (GIBCO) supplemented with 10% fetal calf serum (FCS) (Biofluid), 50 U/ml penicillin G sodium, 50 µg/ml streptomycin sulfate (penicillin-streptomycin, GIBCO), and 1 µg/ml amphotericin B (Parma-Tek, Huntington, NY). The fibroblasts were passaged weekly by trypsinization (Trypsin-ethylenediaminetetraacetic acid; 0.05% trypsin, 0.53 mM EDTA-4Na). Passages 14-19 were used for experiments.
Experimental Protocol
To evaluate the effect of cytokines on the production of TGF-2
by HBECs, 1 × 105 HBECs were seeded in LHC-9/RPMI in 12-well plates (FALCON) and grown for 3-4 d to reach confluence.
After washing, fresh unsupplemented media, LHC-D/RPMI,
containing various concentrations of IL-4, IL-13, or IFN- were
added and incubated for 24 h. To examine the effect of incubation time with cytokines on TGF-2 production by HBECs, the
cells were incubated with media containing 10 ng/ml of IL-4 or
IL-13, 200 U/ml of IFN-, or the combinations of IL-4 or IL-13
and IFN- for up to 48 h. To examine the effect of IFN- on the
IL-4- or IL-13-induced production of TGF-2 by HBECs, cells were incubated with 10 ng/ml of IL-4 or IL-13, together with various concentrations of IFN- for 24 h.
For HFL-1 fibroblasts, cells were cultured to confluence in
10% FCS/DMEM in humidified 5% CO2/95% air at 37°C in 35 mm
tissue culture dishes and growth-arrested in serum-deprived media for 24 h before experiments. Immediately before each experiment, fresh serum-free media containing IL-4, IL-13, IFN-, or
combinations of these cytokines were added and incubated for 48 h.
At the indicated times, all the culture media were harvested
and stored at 
80°C until ELISA for TGF-s was performed.
Measurement of TGF-s by ELISA
TGF-1 and -2 concentrations were determined by ELISA. Briefly,
ELISA plates were coated overnight at 4°C with 100 µl of mouse
anti-human TGF--coating antibodies that had been diluted in
1× Voller's buffer (pH 9.6). For the assay of total TGF-s, samples to be tested were first activated by 1N HCl for 10 min and
then neutralized by 1.2 N NaOH/0.5 M N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid at room temperature. In addition,
samples were directly assayed without acid activation. After rinsing, 100 µl of sequential dilutions of samples or standards containing known amounts of human TGF-s were added and incubated
at room temperature for 2 h. Following this, 100 µl of biotinylated
TGF-s antibodies diluted in phosphate-buffered saline (pH 7.4)
containing 0.05% Tween 20 was added for 1 h. Next, 100 µl of
streptavidin-horseradish peroxide (HRP) conjugate that had been
diluted to 1:20,000 in dilution buffer was added for 1 h. Finally,
200 µl of the substrate buffer containing the HRP substrate tetramethylbenzidine and hydrogen peroxide in 0.05 M phosphate-citrate buffer (pH 5.0) was added for 30-60 min and color-developed in relation to the amount of TGF-s present. The reaction
was stopped by adding 50 µl of stop solution (1 M sulfuric acid)
and the degree of color that had been generated was determined
by measuring the optical density at 450 nm in a Benchmark microplate reader (Bio-Rad, Hercules, CA). The standard curve was
linearized and subjected to regression analysis. The concentration
of TGF-s in unknown samples was estimated using this standard
curve with commercially available software (MPM III-Vs 1.57, Bio Rad). The results are expressed as picograms per milliliter of
culture medium and then corrected by the cell number. These assays are isoform-specific and detect an epitope expressed as the
active TGF- forms, but are not a measure of bioactivity.
RNA Preparation
To determine whether changes in TGF- protein levels were related
to mRNA levels, real-time reverse transcriptase/polymerase chain reaction (RT-PCR) was done. Cells were cultured until confluence and
exposed to 10 ng/ml of IL-4 or IL-13 in the absence or presence of 200 U/ml of IFN- for 24 h, as described previously. Total RNA was isolated by a single-step guanidinium-thiocyanate-phenol-chloroform extraction procedure described by Chomczynski (20) and treated with RNase free DNaseI. After denaturation of the freshly prepared RNA at 65°C for 10 min and 95°C for 5 min, a single-stranded cDNA was produced by reverse transcription described below.
Taqman Real-Time RT-PCR Assay
The PCR primers and Taqman probes for human TGF-1, TGF-2,
and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a constitutively expressed gene, were a gift from Dr. Paula Belloni from
Roche. The sequences used were: TGF-1 primers, 5'-CGA GCC
TGA GGC CGA CTA C-3' (forward) and 5'-AGA TTT CGT
TGT GGG TTT CCA-3' (reverse); TGF-2 primers, 5'-CCA
TTA AGT GGA GCT GTA CGT-3' (forward) and 5'-GTG CCT
ATT GCA TAG CAA TAC AGA A-3' (reverse); GAPDH primers, 5'-CCA GGA AAT GAG CTT GAG AAA GT-3'(forward)
and 5'-CCC ACT CCT CCA CCT TTG AC-3'(reverse). Taqman
probes were labeled with a reporter fluorescent dye, FAM (6-carboxyfluorescein), at the 5' end and a fluorescent dye quencher,
TAMRA (6-carboxy-tetramethyll-rhodamine), at the 3' end. The
probe sequence for TGF-1 was FAM-CCA AGG AGG TCA
CCC GCG TGC-TAMRA; for TGF-2 was FAM-CCG TTC
CTA TCC CGC GCC TCA CT-TAMRA; for GAPDH was FAM-CGT TGA GGG CAA TGC CAG CCC-TAMRA.
RT and PCR were performed using Taqman Reverse Transcription Reagents and a TaqMan Gold RT-PCR kit (Perkin-Elmer, Norwalk, CT) according to the manufacturer's specifications. A two-step RT-PCR was performed. The RT reaction was performed with 500 ng total RNA in a total volume of 40 µl containing 1× Taqman PCR buffer, 5.5 mM MgCl2, 500 µM of each deoxynucleotide triphosphate, 2.5 µM oligo d(T)16 primers, 0.4 U/µl RNase Inhibitor, and 1.25 U/µl MultiScribe Reverse Transcriptase. The RT reaction was performed at 25°C for 10 min, 48°C for 30 min, and 95°C for 10 min.
A thermal stable AmpliTaq Gold DNA polymerase was used
for the second strand cDNA synthesis and DNA amplification.
Real-time PCR was performed with 4 µl of RT products, 1×
Taqman probe buffer A, 5.5 mM MgCl2, 200 µM dATP/dCTP/
dGTP, 400 µM dUTP, 300 nM primers (forward and reverse),
200 nM Taqman probe, 0.01 U/µl AmpErase, and 0.025 U/ml
AmpliTaq Gold DNA Polymerase in a total volume of 50 µl.
PCR was performed at 50°C for 2 min, 95°C for 10 min, and then
run for 40 cycles at 95°C for 15 sec, 60°C for 1 min on the ABI
PRISM 7,700 Detection System. Each sample was run in duplicate, and the 
Rn (the ratio for the amount of reporter dye emission to the quenching dye emission) and threshold cycle (Ct) values were averaged from each reaction. Data were analyzed using a Sequence Detector V1.6 program (Perkin-Elmer).
Statistical Analysis
Each condition in every experiment included three replicate dishes and the data presented are the mean ± standard error of the mean of these triplicates. Each experiment was repeated on multiple occasions and each figure represents composite data from all experiments as indicated in the figure legends. Data were evaluated by one-way ANOVA in the experiments examining dose-dependent effect of cytokines unless indicated. If the overall F statistic was significant at the 0.05 level, subsequent inter-group significance testing was assessed post hoc by the Scheffe's F-test. In the experiments examining the effect of incubation time, two-way analysis of variance (ANOVA) was performed. A two-tailed unpaired Student's t test with the Bonferoni correction was also performed to compare paired samples.
| |
Results |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Time Course of IL-4, IL-13, and IFN- on TGF-2
Release by HBECs
Figure 1 shows TGF-2 production by HBECs after incubation with 10 ng/ml of IL-4, IL-13, or 200 U/ml of IFN- alone
and with the combination of either IL-4 or IL-13 and IFN-
for various times. TGF-2 release from IL-4- or IL-13-
treated cells was significantly increased compared with control cells (P < 0.01). The stimulatory effect of IL-4 and IL-13
was observed at 12 h after the start of the incubation and
continued for the 48 h-incubation period. The TGF-2 release from IFN--treated HBECs was significantly suppressed compared with control cells (P < 0.01). The suppressive effect of IFN- was observed at 24 h after the start of the
incubation and continued for the 48 h-incubation period.
|
When HBECs were incubated in media containing either IL-4 or IL-13 together with IFN-, the stimulatory effect of IL-4 and IL-13 on TGF-2 production was significantly reduced (P < 0.01). A significant decrease in TGF-2
production was observed at 24 h, at which time it was close
to the control levels; after 48 h of incubation, it was lower
than that of control.
Effect of IL-4, IL-13, and IFN- on TGF-2 Release by
HBECs: Concentration Dependence
Figure 2 shows the release of TGF-2 by HBECs treated
with various concentrations of IL-4, IL-13, or IFN- for 24 h.
The concentration of total TGF-2 released from control
HBECs cultures was ~ 2,460 ± 210 pg/106 cells. IL-4 and
IL-13 significantly increased the production of TGF-2 by
HBECs in a concentration-dependent manner (P < 0.01). IL-4 and IL-13 caused a 140%, 180%, and 250% increase of
the control in the release of TGF-2 from HBECs at 0.1, 1, and 10 ng/ml, respectively. IFN- moderately but significantly suppressed the production of TGF-2 by HBECs in a
concentration-dependent manner (P < 0.01). IFN- caused
a 10%, 30%, and 40% decrease in the release of TGF-2 from HBECs at 2, 20, and 200 U/ml, respectively.
|
Effect of IL-4, IL-13, and IFN- on TGF-1 Release from
HFL-1 Fibroblasts
In contrast to HBECs, which released only TGF-2, HFL-1
fibroblasts cultured for 48 h in serum-free DMEM released detectable amounts of both latent TGF-1 and
TGF-2. The addition of IL-4, IL-13 or IFN- alone or the
combination of either IL-4 or IL-13 with IFN- had little
effect on the release of TGF-1 compared with control.
Similarly, the release of TGF-2 by HFL-1 fibroblasts was
not affected by the addition of IL-4, IL-13, or IFN- alone, or the combination of these cytokines (Figure 3).
|
Effect of IFN- on TGF-2 Release by HBECs Treated
with IL-4 or IL-13: Concentration Dependence
IFN- also inhibited the augmented TGF-2 production in
HBECs that were treated with either 10 ng/ml of IL-4 (Figure 4A), or IL-13 (Figure 4B) for 24 h. Addition of IFN-
to the medium containing IL-4 or IL-13 significantly suppressed the production of TGF-2 from HBECs in a concentration-dependent manner in all cases (P < 0.01). As
seen in Figure 4A, 200 U/ml of IFN- reduced the concentration of TGF-2 45% from 5,820 ± 370 to 3,210 ± 100 pg/
106 cells in HBECs treated with IL-4. Similarly, as seen in
Figure 4B, the concentration of TGF-2 was reduced 57%
from 6,080 ± 210 to 2,610 ± 440 in cells treated with IL-13.
|
Effect of IL-4, IL-13, and IFN- on TGF-2
mRNA Expression
To determine whether IL-4, IL-13, or IFN- regulated
TGF-2 production by altering mRNA expression, real
time RT-PCR analysis was done (Figure 5). IL-4 or IL-13
treatment resulted in 4- to 5-fold increases in TGF-2
mRNA expression in HBECs after 24-h-incubation (P < 0.01). IFN- treatment significantly inhibited not only the spontaneous TGF-2 mRNA expression but also the IL-4-
or IL-13-stimulated TGF-2 mRNA expression in HBECs
(P < 0.01). The cytokines tested did not significantly change
mRNA expression for TGF-1 in HFL-1 fibroblasts; however, a slight, but significant increase in mRNA expression
for TGF-2 was noted in HFL-1 fibroblasts treated with IL-13. These results demonstrate that TGF-2 production
by HBECs regulated by IL-4, IL-13, and IFN- is, at least
in part, due to changes in mRNA levels.
| |
Discussion |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
The present study demonstrates that the Th2 cytokines
IL-4 and IL-13 enhance the release of TGF-2 by human
bronchial epithelial cells, whereas the Th1 cytokine IFN-
suppresses this release. In combination, IFN- suppresses
the enhanced production of TGF-2 induced by IL-4 or
IL-13. The decrease in the production of TGF-2 by
HBECs treated with IFN- together with IL-4 or IL-13
was more marked than that seen with IFN- alone. Parallel changes were observed in TGF-2 mRNA in HBECs.
None of the cytokines affected HFL-1's release of TGF-1
or TGF-2. These results, therefore, suggest that IL-4,
IL-13, and IFN- can modulate the production of TGF-2
by human airway epithelial cells.
TGF- is believed to play an important role in repair
and remodeling processes in general (21, 22). In asthma,
the airway epithelium is frequently subject to damage, and
current concepts suggest that TGF- could play an important role in modulating repair of the airways in asthma (23,
24). Consistent with this, airway epithelial cells have been
noted to increase TGF- by histochemical methods, and
the airway wall in asthma has increased TGF- as well (11,
25). TGF- may contribute to epithelial repair by altering epithelial cell adhesion, modulating epithelial cell proliferation and differentiation, and by modulating epithelial cell production of other mediators (29, 30).
TGF- may also play a role in regulating mesenchymal
cell repair in the airways (22, 31, 32). In this context, TGF- is a potent stimulus for fibroblast and myofibroblast production of the connective tissue matrix (33, 34). It is possible, therefore, that TGF- may modulate both restoration
of a damaged epithelium in asthma and also contribute to
the altered connective tissue that characterizes the asthmatic
airway. In this context, epithelial cells can produce a number of mediators that can modulate mesenchymal cell recruitment, accumulation, and proliferation. Repair and remodeling processes in the airway, therefore, likely depend
on a complex balance of a network of mediators in which TGF- may play a crucial role.
Among the cytokines that are believed to play a prominent role in the pathogenesis of asthma are IL-13 and IL-4
(12, 13). These cytokines are produced by several cell types
including, prominently, Th2 lymphocytes (34). IL-13 and
IL-4 are believed to contribute to the characteristic inflammatory response that is present in the asthmatic airway.
These cytokines also can modulate the behavior of mesenchymal cells, including fibroblasts (14). In this regard, IL-4
has been demonstrated to stimulate fibroblast chemotaxis
and proliferation (35, 36), and IL-13 and IL-4 have both
been demonstrated to stimulate fibroblast-mediated contraction of extracellular matrix, a model of tissue remodeling characteristic of fibrotic lesions (37). The current study
suggests that IL-4 and IL-13 may also contribute to mesenchymal cell participation in airway remodeling in asthma
indirectly; that is, by stimulating TGF- production. Both
IL-4 and IL-13 interact with specific receptors which are
capable of activating several signal transduction pathways leading to altered gene expression (38). The current study
demonstrates that IL-4- and IL-13-stimulated TGF- production is paralleled by an increase in mRNA expression,
although the exact signal transduction pathways involved
remain to be defined. The specific ELISA for TGF- used
in the current study recognizes an epitope expressed only
when TGF- is dissociated from the latency associated
peptide. This is not a direct assay of TGF- activity. Some
TGF-2 was detected without acidification, and IL-4 and
IL-13 increased this by ~2-fold (data not shown). This immunoassay suggests that some of the TGF-2 produced by
the airway epithelial cells may have been released in its active form, but bioassay would be needed to establish this.
Many cells are capable of releasing TGF-. Most of the
TGF- released by cells, however, is inactive due to the
presence of the latency-associated peptide (39). A number
of mechanisms exist for activating TGF-, including proteolytic cleavage and nonproteolytic conformational changes
(40). Moreover, TGF- is capable of stimulating its own
production in a positive feedback loop (41). By virtue of
the ability of IL-4 and IL-13 to stimulate the production of
TGF-, IL-4 and IL-13 could initiate a TGF--driven positive feedback cascade, thereby contributing to mesenchymal cell participation in airway remodeling. Several studies
have reported that airway epithelial cells make TGF-1
(11, 27, 42). Earlier work from this laboratory, however,
demonstrated only TGF-2 production by bovine bronchial epithelial cells (43). Using the mink lung cell bioassay, some of this TGF- was demonstrated to be released
in its active form. A very recent study by Richter and colleagues has also reported that IL-4 and IL-13 increased TGF-2 release from bronchial epithelial cells (44). The
current study, therefore, is consistent both with earlier
work from our laboratory and with the study of Richter.
In contrast to IL-13 and IL-4, IFN- is a cytokine believed to be decreased in asthma (16, 17). It is produced by
a number of cells, including Th1 lymphocytes (15), and it is
thought that the balance between Th1 and Th2 cells in the
airway actually depends on the balance between IFN- and
cytokines such as IL-13 and IL-4 (45). In the current study,
IFN- was found to antagonize the IL-4 and IL-13 effect
of stimulating TGF-2 release.
The current study is entirely an in vitro study. The demonstration that Th2 cytokines can "network" with TGF-
raises the possibility that they can directly participate in
remodeling events. Further evaluation of this hypothesis
will require appropriate evaluation using in vivo models
and clinical materials.
IFN- is also known to have a number of other "antifibrotic" activities. It can inhibit fibroblast chemotaxis, proliferation, and production of extracellular matrix macromolecules (46). IFN- can also directly antagonize TGF-
effects. In this context, by stimulating STAT-1, IFN- can
lead to upregulation of the expression of SMAD-7 (49).
SMAD-7 is a so-called inhibitory SMAD and is capable of
binding to the TGF- receptor preventing activation of
SMAD-2 and -3 which, when phosphorylated by the TGF-
receptor, mediate TGF- effects (50). The current study
extends the antagonistic effects of IFN- on TGF- signaling by demonstrating that IFN- can also suppress TGF-
production, particularly in response to IL-4 and IL-13.
The full significance of altered airway structure in
asthma remains to be determined. Some individuals with
asthma appear to experience progressive loss of lung function, and altered structure may be a contributing factor. It
is also reasonable that the altered airway structure creates
a milieu suitable for the persistence of asthmatic inflammation. The mechanisms by which cytokines regulate tissue remodeling, therefore, may determine whether asthma
is persistent or progressive. The current study provides
evidence that the cytokines believed to regulate the inflammatory response in asthma can, by regulating TGF-
production by airway epithelial cells, contribute to tissue
remodeling in asthma as well.
|
| |
Footnotes |
|---|
Address correspondence to: Stephen I. Rennard, M.D., Pulmonary and Critical Care Medicine Section, Department of Internal Medicine, University of Nebraska Medical Center, 985125 Nebraska Medical Center, Omaha, Nebraska 68198-5125. E-mail: srennard{at}unmc.edu
(Received in original form November 21, 2001 and in revised form January 8, 2002).
Abbreviations: chronic obstructive pulmonary disease, COPD; Dulbecco's modified Eagle's medium, DMEM; fetal calf serum, FCS; human bronchial epithelial cell, HBEC; human fetal lung fibroblast, HFL-1; interferon , IFN-; interleukin, IL; reverse transcriptase-polymerase chain
reaction, RT-PCR; transforming growth factor-, TGF-.
Acknowledgments: This work was supported by grant #HL64088-03 from National Heart Lung and Blood Institute. The authors greatly appreciate and acknowledge the assistance in manuscript preparation by Ms. Lillian Richards and Ms. Mary Tourek.
| |
References |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
1. Roberts, A. B., and M. B. Sporn. 1990. The transforming growth factor-b. In Handbook of Experimental Pharmacology: Peptide Growth Factors and Their Receptors, vol. 95. M. B. Sporn, and A. B. Roberts, editors. Springer-Verlag, New York. 419-472.
2.
Blobe, G. C.,
W. P. Schiemann, and
H. F. Lodish.
2000.
Role of transforming growth factor beta in human disease.
N. Engl. J. Med.
342:
1350-1358
3.
Roberts, A. B..
1996.
Transforming growth factor : activity and efficacy in
animal models of wound healing.
Wound Repair Regen.
3:
408-418
.
4.
Singer, A. J., and
R. A. F. Clark.
1999.
Cutaneous wound healing.
N. Engl.
J. Med.
341:
738-746
5.
Sankar, S.,
N. Mahooti-Brooks,
L. Bensen,
T. L. McCarthy,
M. Centrella, and
J. A. Madri.
1996.
Modulation of transforming growth factor receptor levels on microvascular endothelial cells during in vitro angiogenesis.
J.
Clin. Invest.
97:
1436-1446
[Medline].
6. Grainger, D. J., P. R. Kemp, A. C. Liu, R. M. Lawn, and J. C. Metcalfe. 1994. Activation of transforming growth factor-beta is inhibited in transgenic apolipoprotein(a) mice. Nature 370: 460-462 [Medline].
7.
Border, W. A., and
N. A. Noble.
1994.
Transforming growth factor beta in
tissue fibrosis.
N. Engl. J. Med.
331:
1286-1292
8. Roche, W. R., R. Beasley, J. H. Williams, and S. T. Holgate. 1989. Subepithelial fibrosis in the bronchi of asthmatics. Lancet i: 520-524 .
9. Jeffery, P. K., A. J. Wardlaw, F. C. Nelson, J. V. Colins, and A. B. Kay. 1989. Bronchial biopsies in asthma. Am. Rev. Respir. Dis. 140: 1745-1753 [Medline].
10. Rennard, S., and A. Floreani. 1997. Pathophysiology of chronic obstructive pulmonary disease. In Asthma. P. J. Barnes, M. M. Grunstein, A. R. Leff, and A. J. Woolcock, editors. Lippincott-Raven Publishers, Philadelphia. 1375-1388.
11.
Vignola, A. M.,
P. Chanez,
G. Chiappara,
A. M. Nerendino,
E. Pace,
A. Rizzo,
A. M. la Rocca,
V. Bellia,
G. Bonsignore, and
J. Bousquet.
1997.
Transforming growth factor-beta expression in mucosal biopsies in asthma
and chronic bronchitis.
Am. J. Respir. Crit. Care Med.
156:
591-599
12.
Wills-Karp, M.,
J. Luyimbazi,
X. Xu,
B. Schofield,
T. Y. Neben,
C. L. Karp, and
D. D. Donaldson.
1998.
Interleukin-13: central mediator of allergic
asthma.
Science
282:
2258-2261
13. Zhu, Z., R. J. Homer, Z. Wang, Q. Chen, G. P. Geba, J. Wang, Y. Zhang, and J. A. Elias. 1999. Pulmonary expression of interleukin-13 causes inflammation, mucus hypersecretion, subepithelial fibrosis, physiologic abnormalities, and eotaxin production. J. Clin. Invest. 103: 779-788 [Medline].
14. Doucet, C., D. Brouty-Boye, C. Pottin-Clemenceau, G. W. Canonica, C. Jasmin, and B. Azzarone. 1998. Interleukin (IL)4 and IL-13 act on human lung fibroblasts. J. Clin. Invest. 101: 2129-2139 [Medline].
15. Mosmann, T. R., and S. Sad. 1996. The expanding universe of T-cell subsets: Th1, Th2 and more. Immunol. Today 17: 138-146 [Medline].
16.
Renzi, P. M.,
J. P. Turgeon,
J. E. Marcotte,
S. P. Drblik,
D. Berube,
M. F. Gagnon, and
S. Spier.
1999.
Reduced interferon-gamma production in infants with bronchiolitis and asthma.
Am. J. Respir. Crit. Care Med.
159:
1417-1422
17.
Cohn, L.,
C. Herrick,
N. Niu,
R. Homer, and
K. Bottomly.
2000.
IL-4 promotes airway eosinophilia by suppressing IFN-gamma production: defining a novel role for IFN-gamma in the regulation of allergic airway inflammation.
J. Immunol.
166:
2760-2767
18. Gruenert, D. C., C. B. Basbaum, and J. H. Widdicombe. 1990. Long-term culture of normal and cystic fibrosis epithelial cells grown under serum-free conditions. In Vitro Cell. Dev. Biol. 26: 411-418 [Medline].
19. Beckmann, J. D., H. Takizawa, D. Romberger, M. Illig, L. Claassen, K. Rickard, and S. I. Rennard. 1992. Serum-free culture of fractionated bovine bronchial epithelial cells. In Vitro Cell. Dev. Biol. 28A:39-46.
20. Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidine thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159 [Medline].
21. Tipton, D. A., and M. K. Dabbous. 1998. Autocrine transforming growth factor beta stimulation of extracellular matrix production by fibroblasts from fibrotic human gingiva. J. Periodontol. 69: 609-619 [Medline].
22. Pittet, J. F., M. J. Griffiths, T. Geiser, N. Kaminski, S. L. Dalton, X. Huang, L. A. Brown, P. J. Gotwals, V. E. Koteliansky, M. A. Matthay, and D. Sheppard. 2001. TGF-beta is a critical mediator of acute lung injury. J. Clin. Invest. 107: 1537-1544 [Medline].
23. Oddera, S., M. Silvestri, A. Balbo, B. O. Jovovich, R. Penna, E. Crimi, and G. A. Rossi. 1996. Airway eosinophilic inflammation, epithelial damage, and bronchial hyperresponsiveness in patients with mild-moderate, stable asthma. Allergy 51: 100-107 [Medline].
24. Holgate, S.. 2000. Epithelial damage and response. Clin. Exp. Allergy 30(Suppl.): 137-141 .
25. Redington, A. E., W. R. Roche, S. T. Holgate, and P. H. Howarth. 1998. Co-localization of immunoreactive transforming growth factor-beta 1 and decorin in bronchial biopsies from asthmatic and normal subjects. J. Pathol. 186: 410-415 [Medline].
26. Hoshino, M., Y. Nakamura, and J. J. Sim. 1998. Expression of growth factors and remodeling of the airway wall in bronchial asthma. Thorax 53: 21-27 [Abstract].
27. Aubert, J.-D., B. I. Dalal, T. R. Bai, C. R. Roberts, S. Hayashi, and J. C. Hogg. 1994. Transforming growth factor beta-1 gene expression in human airways. Thorax 49: 225-232 [Abstract].
28.
Redington, A.,
J. Madden,
A. J. Frew,
R. Djukanovic,
W. R. Roche,
S. T. Holgate, and
P. H. Howarth.
1997.
Transforming Growth Factor-B1 in
Asthma.
Am. J. Respir. Crit. Care Med.
156:
642-647
29. Jetten, A. M., J. E. Shirley, and G. Stoner. 1986. Regulation of proliferation and differentiation of respiratory tract epithelial cells by TGF beta. Exp. Cell Res. 167: 539-549 [Medline].
30. Boland, S., E. Boisvieux-Ulrich, O. Houcine, A. Baeza-Squiban, M. Pouchelet, D. Schoevaert, and F. Marano. 1996. TGF beta 1 promotes actin cytoskeleton reorganization and migratory phenotype in epithelial tracheal cells in primary culture. J. Cell Sci. 109: 2207-2219 [Abstract].
31. Knight, D.. 2001. Epithelium-fibroblast interactions in response to airway inflammation. Immunol. Cell Biol. 79: 160-164 [Medline].
32. Holgate, S. T., D. E. Davies, P. M. Lackie, S. J. Wilson, S. M. Puddicombe, and J. L. Lordan. 2000. Epithelial-mesenchymal interactions in the pathogenesis of asthma. J. Allergy Clin. Immunol. 105: 193-204 [Medline].
33.
Reed, M. J.,
R. B. Vernon,
I. B. Abrass, and
E. H. Sage.
1994.
TGF-1 induces the expression of type I collagen and SPARC, and enhances contraction of collagen gels, by fibroblasts from young and aged donors.
J.
Cell. Physiol.
158:
169-179
[Medline].
34. Abbas, A. K., K. M. Murphy, and A. Sher. 1996. Functional diversity of helper T lymphocytes. Nature 383: 787-793 [Medline].
35. Postlethwaite, A. E., and J. M. Seyer. 1991. Fibroblast chemotaxis induction by human recombinant interleukin-4. J. Clin. Invest. 87: 2147-2152 .
36. Kraft, M., C. Lewis, D. Pham, and H. W. Chu. 2001. IL-4, IL-13, and dexamethasone augment fibroblast proliferation in asthma. J. Allergy Clin. Immunol. 107: 602-606 [Medline].
37. Liu, X. D., Y. Zhu, K. H. Wang, T. Kohyama, F. Q. Wen, D. J. Romberger, and S. I. Rennard. 2000. Cytokine regulation of Type I collagen gel contraction mediated by human airway cells. Am. J. Respir. Crit. Care Med. 161: A440 .
38. Nelms, K., A. D. Keegan, J. Zamorano, J. J. Ryan, and W. E. Paul. 1999. The IL-4 receptor: signaling mechanisms and biologic functions. Annu. Rev. Immunol. 17: 701-738 [Medline].
39. Lawrence, D. A.. 2001. Latent-TGF-beta: an overview. Mol. Cell. Biochem. 219: 163-170 [Medline].
40. Massague, J.. 1998. TGF-beta signal transduction. Annu. Rev. Biochem. 67: 753-791 [Medline].
41.
Wen, F. Q.,
T. Kohyama,
C. M. Skold,
Y. K. Zhu,
R. Pineda,
D. J. Romberger, and
S. I. Rennard.
2001.
Glucocorticoids modulate TGF- production by human fetal lung fibroblasts.
Am. J. Respir. Crit. Care Med.
163:
A911
.
42.
Minshall, E. M.,
D. Y. M. Leung,
R. J. Martin,
Y. L. Song,
L. Cameron,
P. Ernst, and
Q. Hamid.
1997.
Eosinophil-associated TGF-1 mRNA expression and airways fibrosis in bronchial asthma.
Am. J. Respir. Cell Mol. Biol.
17:
326-333
43. Sacco, O., D. Romberger, A. Rizzino, J. Beckmann, S. I. Rennard, and J. R. Spurzem. 1992. Spontaneous production of transforming growth factor beta 2 by primary cultures of bronchial epithelial cells: effects on cell behavior in vitro. J. Clin. Invest. 90: 1379-1385 .
44.
Richter, A.,
S. M. Puddicombe,
J. L. Lordan,
F. Bucchieri,
S. J. Wilson,
R. Djukanovic,
G. Dent,
S. T. Holgate, and
D. E. Davies.
2001.
The contribution of interleukin (IL)-4 and IL-13 to the epithelial-mesenchymal trophic
unit in asthma.
Am. J. Respir. Cell Mol. Biol.
25:
385-391
45. Ray, A., and L. Cohn. 2000. Altering the Th1/Th2 balance as a therapeutic strategy in asthmatic diseases. Curr. Opin. Investig. Drugs 1: 442-448 . [Medline]
46. Adelmann-Grill, B. C., R. Hein, F. Wach, and T. Krieg. 1987. Inhibition of fibroblast chemotaxis by recombinant human interferon gamma and interferon alpha. J. Cell. Physiol. 130: 270-275 [Medline].
47. Elias, J. A., S. A. Jimenez, and B. Freundlich. 1987. Recombinant gamma, alpha, and beta interferon regulation of human lung fibroblast proliferation. Am. Rev. Respir. Dis. 135: 62-65 [Medline].
48.
Ghosh, A. K.,
W. Yuan,
Y. Mori,
S. Chen, and
J. Varga.
2001.
Antagonistic
regulation of type I collagen gene expression by interferon-gamma and
transforming growth factor-beta: integration at the level of p300/CBP transcriptional coactivators.
J. Biol. Chem.
276:
11041-11048
49. Ulloa, L., J. Doody, and J. Massague. 1999. Inhibition of transforming growth factor-beta/SMAD signalling by the interferon-gamma/STAT pathway. Nature 397: 710-713 [Medline].
50.
Nakao, A.,
S. Miike,
M. Hatano,
K. Okumura,
T. Tokuhisa,
C. Ra, and
I. Iwamoto.
2000.
Blockade of transforming growth factor beta/Smad signaling in T cells by overexpression of Smad7 enhances antigen-induced airway inflammation and airway reactivity.
J. Exp. Med.
192:
151-158
This article has been cited by other articles:
 |
|
 |
|
  H. P. Gaide Chevronnay, P. B. Cornet, D. Delvaux, P. Lemoine, P. J. Courtoy, P. Henriet, and E. Marbaix Opposite Regulation of Transforming Growth Factors-{beta}2 and -{beta}3 Expression in the Human Endometrium Endocrinology, March 1, 2008; 149(3): 1015 - 1025. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.-Y. Nam, Y.-H. Kim, J.-S. Do, Y.-H. Choi, H.-J. Seo, H.-K. Yi, P.-H. Hwang, C.-H. Song, H.-K. Lee, J.-S. Kim, et al. CD30 supports lung inflammation Int. Immunol., February 1, 2008; 20(2): 177 - 184. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. W. J. Young, O. W. Williams, D. Chandra, L. K. Bellinghausen, G. Perez, A. Suarez, M. J. Tuvim, M. G. Roy, S. N. Alexander, S. J. Moghaddam, et al. Central Role of Muc5ac Expression in Mucous Metaplasia and Its Regulation by Conserved 5' Elements Am. J. Respir. Cell Mol. Biol., September 1, 2007; 37(3): 273 - 290. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Fang, X. Liu, M. Al-Mugotir, T. Kobayashi, S. Abe, T. Kohyama, and S. I. Rennard Thrombin and TNF-{alpha}/IL-1beta Synergistically Induce Fibroblast-Mediated Collagen Gel Degradation Am. J. Respir. Cell Mol. Biol., December 1, 2006; 35(6): 714 - 721. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. G. R. Thompson, J. D. Mih, T. B. Krasieva, B. J. Tromberg, and S. C. George Epithelial-derived TGF-beta2 modulates basal and wound-healing subepithelial matrix homeostasis Am J Physiol Lung Cell Mol Physiol, December 1, 2006; 291(6): L1277 - L1285. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Kiwamoto, Y. Ishii, Y. Morishima, K. Yoh, A. Maeda, K. Ishizaki, T. Iizuka, A. E. Hegab, Y. Matsuno, S. Homma, et al. Transcription Factors T-bet and GATA-3 Regulate Development of Airway Remodeling Am. J. Respir. Crit. Care Med., July 15, 2006; 174(2): 142 - 151. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Bergeron and L.-P. Boulet Structural changes in airway diseases: characteristics, mechanisms, consequences, and pharmacologic modulation. Chest, April 1, 2006; 129(4): 1068 - 1087. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. W. Chu, S. Balzar, G. J. Seedorf, J. Y. Westcott, J. B. Trudeau, P. Silkoff, and S. E. Wenzel Transforming Growth Factor-{beta}2 Induces Bronchial Epithelial Mucin Expression in Asthma Am. J. Pathol., October 1, 2004; 165(4): 1097 - 1106. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Kikuchi, J. D. Shively, J. S. Foley, J. M. Drazen, and D. J. Tschumperlin Differentiation-dependent responsiveness of bronchial epithelial cells to IL-4/13 stimulation Am J Physiol Lung Cell Mol Physiol, July 1, 2004; 287(1): L119 - L126. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F.-Q. Wen, X. Liu, T. Kobayashi, S. Abe, Q. Fang, T. Kohyama, R. Ertl, Y. Terasaki, L. Manouilova, and S. I. Rennard Interferon-{gamma} Inhibits Transforming Growth Factor-{beta} Production in Human Airway Epithelial Cells by Targeting Smads Am. J. Respir. Cell Mol. Biol., June 1, 2004; 30(6): 816 - 822. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. I. Rennard and S. G. Farmer Exacerbations and Progression of Disease in Asthma and Chronic Obstructive Pulmonary Disease Proceedings of the ATS, April 1, 2004; 1(2): 88 - 92. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. E. Evans, T. V. Colby, J. H. Ryu, and A. H. Limper Transforming Growth Factor-{beta}1 and Extracellular Matrix-Associated Fibronectin Expression in Pulmonary Lymphangioleiomyomatosis Chest, March 1, 2004; 125(3): 1063 - 1070. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. W. Wynes and D. W. H. Riches Induction of Macrophage Insulin-Like Growth Factor-I Expression by the Th2 Cytokines IL-4 and IL-13 J. Immunol., October 1, 2003; 171(7): 3550 - 3559. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||