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and IL-13 Synergistically Increase Eotaxin-1 Production in Human Airway Fibroblasts1
National Jewish Medical and Research Center and University of Colorado Health Sciences Center, Denver, CO 80206
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1 (innate response factor associated with wound repair) in
combination with IL-13 (Th2 factor) might augment inflammatory
processes associated with asthma. Airway fibroblasts were cultured from
asthmatic subjects and normal controls. These fibroblasts were exposed
to TGF-1 and IL-13 alone or in combination, and eotaxin-1 expression
and production were evaluated. At 48 h, eotaxin-1 production was
markedly increased with the combination of TGF-1 and IL-13
(p < 0.0001) compared with either stimulus alone.
mRNA increased slightly at 1 h with IL-13 or TGF-1 plus IL13,
peaked, and became significantly increased over IL-13 alone at 24
h. Protein was measurable from 6 h with IL-13 and TGF-1 plus
IL-13, but greater levels were measured over time with the combination.
Actinomycin ablated the increase in mRNA and protein seen with IL-13
alone and with TGF-1 plus IL-13. Cycloheximide blocked the increase
in mRNA at 6 h in both conditions, but also blocked the increase
at 24 h with TGF-1 plus IL-13. STAT-6 was rapidly activated
with both IL-13 and the combination, without difference. Finally,
eotaxin-1-positive fibroblasts were identified in severe asthma
biopsies in greater numbers than in normals. These results support the
concept that interactions of innate and adaptive immune systems may be
important in promoting the tissue eosinophilia of asthma, particularly
in those with more severe disease. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-1 or
TGF-2 in asthma and the eosinophilia associated with it (3, 4). Whether these two systems interact in any way in asthma, or
in other diseases, is not clear.
Although tissue eosinophilia is a hallmark of asthmatic inflammation,
the precise mechanisms behind the eosinophilia remain unclear. Th2
cytokines such as IL-4 and/or IL-13 have been associated with
eosinophilia in murine and human models (2, 5). However,
because these cytokines do not directly attract eosinophils, indirect
stimulation of a second eosinophil chemoattractant may be required
(6). In a manner similar to Th2 cytokines, the innate
factors TGF-1 and TGF-2 have been associated with airway
eosinophils, especially in subjects with more severe disease (3, 4, 7). Like IL-4 and IL-13, TGF- is not known to have direct
chemotactic effects on eosinophils, and the mechanism behind the
associated eosinophil increases remains unclear.
Eotaxin-1, a C-C chemokine with potent direct chemoattractant effects
on eosinophils, is known to be regulated by Th2 factors such as IL-4
and IL-13, likely involving a STAT-6 pathway (8).
Eotaxin-1 can be produced by resident cells including fibroblasts and
smooth muscle cells, and this resident cell production has been
suggested to contribute directly to tissue eosinophilia (9, 10). Interestingly, eotaxin-1 production from fibroblasts,
smooth muscle cells, and epithelial cells has been shown to
synergistically increase after stimulation with IL-4/-13 and other
innate response factors, namely TNF-
and IL-1
(11, 12, 13).
Given the previously reported increases in TGF-1/2 and IL-13 in
asthma and the association of each with eosinophils, we hypothesized
that these innate and adaptive immune factors could synergistically
activate resident cells such as fibroblasts. To explore this
relationship, primary fibroblasts were obtained from asthmatic and
normal subjects and exposed to TGF-1 in combination with IL-13.
Eotaxin-1 protein and mRNA were measured and activation of STAT-6 was
determined. For comparison, IL-8 protein was also measured. Finally,
human airway tissue was evaluated for the presence of
eotaxin-1+ fibroblasts.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Endobronchial biopsies were obtained from asthmatics ranging in severity from mild to severe, as well as from normal control subjects as previously described (4). Briefly, severe asthmatics were all on high-dose inhaled or oral glucocorticoids and had continual symptoms and airway obstruction. Mild-moderate asthmatics were stable asthmatics on no or low-moderate doses of inhaled glucocorticoids, but with chronic airflow limitation. Normal controls had no evidence of any respiratory disease. All subjects were nonsmokers and none had a history of smoking >5 pack years (4). The study was approved by the National Jewish Institutional Review Board and all subjects gave informed consent. Biopsies were obtained from third to fifth order subcarinae, placed in chilled medium (DMEM (Life Technologies, Rockville, MD) with 10% FBS (Gemini, Woodland, CA) and penicillin/streptomycin/gentamicin) and minced into small pieces using a scalpel. The pieces were placed into plastic 60-mm dishes (BD Biosciences, Franklin Lakes, NJ) and incubated at 37°C/5% CO2 for 5–7 days until fibroblasts advanced out from the tissue. The tissue was then removed. Fibroblasts were passed during the proliferative state and were all studied at the third passage. In disease group comparison and dose response experiments, where the primary outcome was eotaxin-1 protein, the fibroblasts were plated in 24-well dishes and serum starved. After 24 h, FBS (0.5%) and the stimuli were then added in the described concentrations. Time course studies required adequate numbers of fibroblasts at the early time periods and were done on third- to fourth-passage fibroblasts grown to near confluency (80–90%) in 60-mm dishes (with 10% FBS) before being serum starved for 24 h. Medium was removed and replaced with DMEM/0.5% FBS and the appropriate stimuli. Supernatants and cells were harvested at the described time points and stored for analysis of eotaxin-1 protein and mRNA, STAT-6, and phosphorylated STAT (pSTAT)3-6 protein.
Enzyme immunoassay for eotaxin-1 and IL-8
Sandwich ELISAs were used to quantify these chemokines. Eotaxin-1 Abs and standard were purchased from R&D Systems (Minneapolis, MN), with Ab MAB320 used to coat plates and biotinylated BAF320 used as a detection Ab. A matched Ab pair (MAB208 and BAF208) was used to measure IL-8 (R&D Systems). Avidin-HRP was purchased from BD PharMingen (San Diego, CA) and tetramethylbenzidene substrate was from KPL Laboratories (Gaithersburg, MD). Assay sensitivity ranged from 10 to 20 pg/ml for all assays and cell culture supernatants were assayed without any purification or concentration.
Northern blot for eotaxin-1
Total RNA was extracted from fibroblasts by TRIzol reagent (Life Technologies). RNA samples (18 µg) were separated on a 1% agarose/formaldehyde gel and transferred to a Hybond-N membrane (Amersham Pharmacia Biotech, Piscataway, NJ) in 10x SSC overnight at room temperature. The membrane was fixed using an UV autocrosslinker and was then prehybridized at 42°C for 6–8 h. The blot was hybridized with a 32P-labeled cDNA probe for human eotaxin-1 (corresponding to bases 274–773; GenBank accession no. D49372) at 42°C overnight, followed by washing with 0.5% SDS in 2x SSC twice (10 min each wash) at room temperature, and then 0.1% SDS in 0.1x SSC at 65°C for 30 min. The membrane was then exposed to Hyperfilm MP (Amersham Pharmacia Biotech) for 24 h. After removing the eotaxin-1 probe, the membrane was rehybridized with a 32P-labeled ubiquitin cDNA probe (Clontech Laboratories, Palo Alto, CA).
Real-time PCR for measurement of eotaxin-1 gene expression
Eotaxin-1 mRNA expression in fibroblasts was determined by
reverse transcription (RT), followed by real-time quantitative PCR.
Total RNA of fibroblasts was extracted using TRIzol reagent (Life
Technologies). RT was performed using 1 µg of total RNA and random
hexamers in a 50-µl reaction according to the manufacturer’s
protocol (PE Applied Biosystems, Foster City, CA). The eotaxin-1
primers and the probe, labeled with 5'-reporter dye FAM and TAMRA, were
designed using Primer Express software (PE Applied Biosystems). The
following are sequences for eotaxin-1 primers and probe: forward
primer, 5'-AGGAGAATCACCAGTGGCAAAT-3'; reverse primer,
5'-GCACAGATATCCTTGGCCAGTT-3'; probe,
5'-TCCCCAGAAAGCTGTGATCTTCAAGACC-3'. Real-time PCR was performed on the
ABI Prism 7700 sequence detection system (PE Applied Biosystems). The
50-µl PCR contained 30 ng cDNA, 100 nM fluogenic probe, and 50 nM
primers and other components from the TaqMan RT-PCR kit. GAPDH was
evaluated using the same PCR protocol as eotaxin-1. The specificity of
PCR for both eotaxin-1 and GAPDH was verified by no signal in
no-template controls or RT (-) fibroblast samples. The threshold cycle
(CT) was recorded for each sample to reflect the mRNA expression
levels. A validation experiment proved the linear dependence of the CT
value for both eotaxin-1 and GAPDH on the template RNA concentrations
and the consistency of 
CT (eotaxin-1 average CT - GAPDH
average CT) in a given sample at different RNA concentrations.
Therefore, CT was used to reflect the relative eotaxin-1 mRNA
expression levels. To determine the effects of different stimuli on
eotaxin-1 gene expression as compared with nonstimulated cells,
CT was calculated (CT = CT stimulus - CT
nonstimulated cells). Eotaxin-1 mRNA was indexed to the GAPDH using the
formula 1/(2DCT) x 100%.
2DDCT was calculated to demonstrate the fold
change of eotaxin-1 gene expression in stimulated cells as compared
with the nonstimulated cells.
Western blot STAT-6 and pSTAT-6
Fibroblasts (1 x 106) were lysed in buffer (0.1% SDS, 1.0% Nonidet P-40 in 50 mM Tris-HCl and 150 mM NaCl, and 10 mM EDTA with 1% protease inhibitor mixture (Sigma-Aldrich, St. Louis, MO)). Samples were bath-sonicated on ice and boiled, and the whole cell lysate protein was electrophoresed on a 7.5% acrylamide/bis-SDS gel and transferred to a Hybond ECL nitrocellulose membrane (Amersham, Arlington Heights, IL). The blots were blocked with 5% nonfat milk/Tween (1/10,000), followed by overnight 4°C exposure in pSTAT-6 polyclonal Ab (1/1000 in 5% BSA in TBS/Tween (1/1000)) (Tyr641; Cell Signaling Technology, St. Louis, MO). After extensive washing, the HRP-conjugated secondary Ab (F(ab')2) was added (1/10,000), and the blot was washed again, developed using the ECL Western blot detection reagents, and immediately exposed to Hyperfilm (Amersham). The membrane was then stripped, checked for complete Ab removal, then reblocked and reprobed with STAT-6 polyclonal Ab (1/750; Santa Cruz Biotechnology, Santa Cruz, CA) followed by the same washings and development. Specific bands were scanned and semiquantitated by densitometry (Scion Image Analysis software; National Institutes of Health, Bethesda, MD). Quantification was performed by evaluating the ratio of the density of the STAT-6 bands to that of the pSTAT-6 bands for all conditions.
Tissue double immunofluorescent staining for eotaxin-1/fibroblasts
To confirm the eotaxin-1 expression by fibroblasts, double immunofluorescent staining was performed in endobronchial biopsy tissue sections from seven severe asthmatics and six normal controls. The acetone-fixed and glycol methacrylate-embedded 2-µm tissue sections were incubated with a rabbit polyclonal Ab against human eotaxin-1 (BioSource International, Camarillo, CA) and a mouse mAb against human fibroblast Ag (Ab-1; Calbiochem, San Diego, CA). These sections were then rinsed and incubated with FITC-conjugated anti-rabbit and rhodamine-conjugated anti-mouse Abs (DAKO, Carpinteria, CA). The nuclei were counterstained with DAPI. The slides were mounted and observed with an Olympus BX51 fluorescence microscope (Olympus, Melville, NY). The tissue triple-colored fluorescent images were processed and overlaid using the Adobe Photoshop 6.0 software (Adobe Systems, Mountain View, CA. Negative control slides were similarly processed but with the primary Abs omitted or replaced with nonimmune serum. No staining was seen in the negative control slides.
Statistics
Data were checked for normalcy of distribution. Data that were
normally distributed were represented as means ± SEM.
Log-transformation was done on variables that were heavily right
skewed, producing variables that were roughly normally distributed.
Graphic representation of log-transformed data was done by reconverting
the mean - SEM, the mean, and the mean + SEM back to the original
scale. This produces SEs that are not equal, with the larger SE being
that described in the positive direction. Differences between
conditions and subject groups were analyzed by ANOVA on both normal and
log-transformed data, because the log-transformation produced data that
were roughly normally distributed. When there were significant
differences between the conditions, specific intercondition differences
were assessed by Tukey-Kramer testing. A p value of 
0.05
was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eotaxin-1 protein was measured in the supernatants of primary lung
fibroblasts cultured for 48 h in the presence of TGF-1 (0.5
ng/ml) and IL-13 (30 ng/ml), alone and in combination. Thirty separate
primary cultures were performed, all on third-passage fibroblasts. The
30 primary cultures were obtained from 12 normal subjects, 6
mild-moderate asthmatics, and 12 severe asthmatics. The combination of
TGF-1 and IL-13 produced a synergistic and significant increase in
eotaxin-1 protein from cultures in all groups as compared with
unstimulated TGF-1 alone or IL-13 alone (overall p
< 0.0001; combination significantly greater than any other condition)
(Fig. 1
). When the three subject groups
were analyzed separately, there was no difference in the eotaxin
protein response to TGF-1 plus IL-13 among the groups (normal =
2253 ± 559 pg/ml, mild-moderate = 4457 ± 1262 pg/ml,
severe = 3411 ± 987 pg/ml, p = 0.30).
Although the response pattern was consistent among the groups, there
was considerable variability from subject to subject in the absolute
amount of eotaxin-1 produced. IL-4 (10 ng/ml) was used in combination
with TGF-1 (0.5 ng/ml) in three cultures with similar results (data
not shown) (12). Likewise, TGF-2 (at similar doses to
TGF-1) could also be substituted for TGF-1 with nearly identical
results (data not shown). Because the results were similar and the
initial studies had been done with IL-13 and TGF-1, the remainder of
the studies were done only with this combination.
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To evaluate the specificity of the synergistic effect of TGF-1
and IL-13 on eotaxin-1, IL-8 was measured for comparison from cultures
treated similarly to those described above. IL-8 levels (10 normal
controls, 5 mild-moderate asthmatics, and 10 severe asthmatics) were
low after 48 h in culture, increased significantly
(p < 0.05) after TGF-1, did not change with
IL-13, and were significantly (but not synergistically) increased after
the combination of TGF-1 and IL-13 (p <
0.05), although at a lower numerical amount than for TGF-1 alone
(Fig. 2).
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Northern analysis was performed on four fibroblast cultures (two
severe asthmatics and two normal controls). In each case, mRNA for
eotaxin-1 was only marginally detected at 48 h in unstimulated
wells, or with TGF-1 (0.5 ng/ml) or IL-13 (30 ng/ml) alone. In a
similar manner as to what was seen for protein, there was a large
increase in eotaxin-1 mRNA after the addition of both, without
difference between normal controls and asthmatics (Fig. 3). Real-time PCR was then used to
quantify eotaxin-1 mRNA from 10 normal controls, 5 mild-moderate, and
10 severe asthmatic primary fibroblast cultures at 48 h. Eotaxin-1
mRNA was present at low levels in most fibroblasts at baseline. There
was little effect of TGF-1 (0.5 ng/ml). IL-13 (30 ng/ml) generated a
small increase in eotaxin-1 mRNA levels, while the combination
significantly and synergistically increased the levels of fibroblast
eotaxin-1 mRNA (p < 0.0001) (Fig. 4). Similar to protein levels, there were
no differences in response among the asthmatic or normal subject
groups.
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1 and IL-13
Dose-response curves evaluating eotaxin-1 protein were constructed
in six separate primary third-passage cultures. The doses used ranged
from 0.05 to 50 ng/ml TGF-1 and from 0.3 to 30 ng/ml IL-13. The
dose-response range chosen was larger for TGF-1 because of the
possibility of a bimodal effect for TGF-1. As shown in Fig. 5, the minimum TGF-1 dose required was
0.5 ng/ml, while the minimum IL-13 dose required was 3 ng/ml. Between
0.05 and 0.5 ng/ml TGF-1 and between 0.3 and 3 ng/ml IL-13 there was
a large increase in the total response. The effect appeared to plateau
at 5 ng/ml TGF-1 and 30 ng/ml IL-13. TGF-1 at 50 ng/ml with IL-13
at 30 ng/ml produced less eotaxin-1 than the 5 ng/ml dose in two of the
three subjects studied at that dose (data not shown). The maximum
response appears to occur with the combination of 5 ng/ml TGF-1 and
30 ng/ml IL-13. However, due to the small, nonsignificant differences
between 0.5 and 5 ng/ml TGF-1 and the number of studies done with
the lower dose, 0.5 ng/ml TGF-1 and 30 ng/ml IL-13 were chosen for
the time course studies and for analysis of the STAT-6 pathway.
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Responses to medium alone, TGF-1 (0.5 ng/ml), IL-13 (30 ng/ml),
or the combination were evaluated at 0 min, 15 min, 1 h, 6 h,
24 h, and 48 h. Four separate fibroblasts cultures (one
normal and three asthmatic) were used. Eotaxin-1 protein was not
present consistently in the medium above the detection limit of the
assay until 6 h. At 6 h, a low level of eotaxin-1 could be
measured in the cells stimulated with both IL-13 alone and TGF-1
plus IL-13 in similar amounts (119 ± 61 and 78 ± 53 pg/ml,
respectively). However, by 24 h, differences between IL-13 alone
and TGF-1 plus IL-13 appeared (750 ± 176 and 2303 ± 221
pg/ml, respectively) which were maintained at 48 h (Fig. 6A).
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1
there was a slight but consistent decline in eotaxin-1 mRNA through
6 h. At 24 and 48 h there were small but significant
increases in mRNA over that of medium alone (Fig. 6
B). With
either IL-13 or TGF-1 plus IL13, increases in eotaxin-1 mRNA were
seen as early as 1 h after stimulation (Fig. 6C; note
the marked increase in range of the y-axis over that in Fig. 6B). At 6, 24, and 48 h, the increase in eotaxin mRNA
was significantly greater than that seen with medium or TGF-1 alone.
The increase in mRNA with IL-13 slowly increased out to 48 h. In
contrast, the increase in mRNA with TGF-1 plus IL-13 increased
significantly over that of IL-13 alone at 24 h. By 48 h, the
mRNA level with the combination declined by 
80%, no longer
different from that of IL-13 alone. Effects of actinomycin and cycloheximide (CHX) on protein and mRNA
Actinomycin (100 nM) inhibited (>90%) the increase in mRNA and
protein at all time points for IL-13 alone and for the combination. It
also significantly blocked the small increase in mRNA with TGF-1 at
24 and 48 h. CHX blocked the protein production from all
conditions by >90%. Additionally, CHX (100 nM) completely inhibited
the increase in mRNA at 6 h under both IL-13 and TGF-1 plus
IL-13 conditions. CHX had no effect on the IL-13-stimulated mRNA at
later time points (Fig. 6C). In contrast, CHX significantly
blunted the peak increase in mRNA after TGF-1 plus IL-13 at 24
h, while having no effect on the mRNA at 48 h. Of interest, the
small increase in mRNA at 24 and 48 h with TGF-1 alone was
blocked by CHX (Fig. 6C). These data suggest that the
increase in eotaxin-1 is at least partially transcriptionally regulated
and that one or more proteins produced between 6 and 24 h after
stimulation with TGF-1 is responsible for the synergistic increase
in eotaxin-1 mRNA and protein that occurs with TGF-1 plus IL-13.
Activation of STAT-6
Western blots of pSTAT-6 (Fig. 7)
evaluated the time course of activation at 15 min, 1 h, 2 h,
6 h, and 24 h post-stimulation with medium, TGF-1 (0.5
ng/ml), IL-13 (30 ng/ml), or the combination. Neither TGF-1 alone
nor the unstimulated control demonstrated phosphorylation of STAT-6.
Both IL-13 and TGF-1 plus IL-13 induced phosphorylation of STAT-6
within 15 min of stimulation. The increase in pSTAT-6 was partially
maintained through 2 h and decreased substantially (but was still
present) at 6 h. There were no differences in the amount of
pSTAT-6 between those cells treated with IL-13 alone or in combination
with TGF-, although there was a tendency for the phosphorylation to
be greater with IL-13 alone. Nonphosphorylated STAT-6 was present at
baseline and there were no differences at any time point or under any
type of stimulation.
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Endobronchial tissue from seven severe asthmatics and six normal
controls was analyzed for the presence of eotaxin-1-positive
fibroblasts (Fig. 8). Four of the seven
severe asthmatic subject tissue sections contained cells that expressed
both eotaxin-1 and Ab-1 (fibroblast marker) (median = 4.7 cells
(interquartile range 0–11.8)/10 mm sub-basement membrane vs 0 cells
(0–0 interquartile range)/10 mm sub-basement membrane). The difference
in expression was statistically significant between groups
(p < 0.05). There were no correlations between
the amount of eotaxin-1 produced in vitro and the number of
eotaxin-1+ fibroblasts.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 or TGF-2) and a Th2 cytokine (IL-13 or
IL-4) induces a response in primary human airway fibroblasts that could
markedly enhance tissue eosinophilia. This synergistic up-regulation of
eotaxin-1 occurred in human fibroblasts from asthmatic and normal
subjects, suggesting that the increase in eotaxin-1 can occur in airway
tissue fibroblasts at any time, given the right convergence of stimuli
in the environment of the cells. Because both Th2 cytokines and
TGF-1/2 have been identified in asthma biopsies, especially those
with more severe disease, it is conceivable this combination of factors
contributed to the significant increase in
eotaxin-1+ fibroblasts observed in the severe
asthmatic tissue studied here.
In earlier studies, TGF- (pan isoforms) from multiple cell sources
was found to be significantly elevated in a subgroup of severe
steroid-dependent asthmatics with persistent eosinophilia (3, 4, 14). A parallel study found that levels of the eicosanoid
15-hydroxyeicosatetraenoic acid were elevated in bronchoalveolar lavage
fluid in the same group of severe eosinophilic asthmatics
(15). Th2 cytokines such as IL-4/-13 up-regulate the
enzymatic source for 15-hydroxyeicosatetraenoic (15-lipoxygenase-1),
thus indirectly suggesting the presence of TGF- and Th2 cytokines in
the same subjects. Other groups have directly reported increases in
IL-13 and IL-4 (1, 16). Interestingly, a recent report
suggested that IL-13 can up-regulate tissue TGF-1 in a murine model
(17). Combined, these data suggest that both TGF-1 and
IL-13 are present in the submucosa of asthma patients with access to
airway fibroblasts. Dose-response studies demonstrate that the effect
of the combination to increase eotaxin-1 follows a dose-response
paradigm, although the large increase from 0.05 to 0.5 ng/ml TGF-1
and from 0.3 to 3 ng/ml IL-13 suggests that some element of a
"threshold" response exists. These responses occurred without
difference in asthmatics (of all groups) and normal controls.
Additionally, the doses required for the effect were low and within
physiologic ranges.
The increase in human airway fibroblast eotaxin-1 production to TGF-
and IL-13 occurred at both the protein and mRNA levels. The
up-regulation of the mRNA occurred quickly (within 1 h of
stimulation) in both IL-13 alone and TGF-1 plus IL-13-stimulated
cells. However, by 24 h the magnitude of the increase in mRNA was
significantly greater in the cells treated with both TGF-1 and
IL-13. Actinomycin suppressed both eotaxin-1 mRNA and the protein
response to 90% of the untreated response, supporting a
transcriptional level regulation of these increases. Interestingly, CHX
also inhibited the increase in mRNA at 6 h (for both IL-13 and
TGF-1 plus IL-13 stimulation). In cells treated with IL-13 alone,
this effect was lost by 24 h. In contrast, the peak increase in
mRNA at 24 h with TGF-1 and IL-13 was also significantly
blocked by CHX. Interestingly, the smaller but significant increase in
eotaxin-1 mRNA at 24 and 48 h in fibroblasts treated with TGF-1
alone was also blocked by CHX. These results suggest that synthesis of
specific proteins between 6 and 24 h may be responsible for the
synergistic increase in mRNA, either by increasing transcription at
later time points or by stabilizing the mRNA. A similar effect of
TGF-1 to stabilize mRNA (for elastin) has been previously described
in fibroblasts (18).
Because IL-13 (and IL-4) can signal through an IL-4/IL-13R
complex/STAT-6 pathway and eotaxin-1 has been described to have a
STAT-6 response element in its promoter, the activation of the STAT-6
pathway was evaluated (19, 20). Not surprisingly, IL-13
alone produced marked phosphorylation of STAT-6. The addition of
TGF-1 to the system did not appear to enhance this activation and,
if anything, may have decreased it slightly. These findings suggest
that, although STAT-6 may be involved, the mechanism behind the
synergistic effect of TGF-1 on eotaxin-1 production is not due to
augmented phosphorylation of STAT-6. Further studies of the promoter
region of eotaxin-1 for involvement of other transcription factors,
including those newly synthesized by the combination, should help
elucidate the mechanisms of activation and production.
The combination of IL-13 with TNF- or IL-1 has also been reported to
synergistically increase eotaxin-1 production in fibroblasts,
epithelial cells, and smooth muscle cells (11, 12, 13, 20).
Studies in our laboratory with IL-13 and TNF- demonstrated that the
production of eotaxin-1 was similar to that seen with TGF-1 and
IL-13, although variability existed from cell culture to cell culture
(data not shown). Because the signaling mechanisms for these factors
(TGF- compared with TNF- or IL-1) are quite disparate, the
similar eotaxin-1 responses in the presence of IL-4 or IL-13 are
surprising. TNF- and IL-1 signal primarily through protein-protein
interactions and serine/threonine kinases leading to activation of
NF-
B. In contrast, TGF- signals primarily through Smads
and AP-1 transcription factors. Synergy with neither TNF-/IL-1 nor
TGF-1 appears to be completely dependent on enhanced activation of
STAT-6 (12, 20). It is also not clear whether there are
further additive or synergistic effects when all three stimuli are
used. Studies to evaluate the commonalities in the signaling and
protein responses to TNF-/IL-1 vs TGF- should prove helpful.
The increased eotaxin-1 levels seen with combinations of IL-1 or
TNF- with IL-13 were associated with high levels of IL-8 in the
supernatants (12, 13). In contrast, the combination of
TGF- and IL-13 did not lead to high levels of IL-8, but rather to
increased procollagen I (21). It is possible that an early
inflammatory reaction, commonly involving IL-1 and TNF-, in the
presence of IL-4/-13 could induce a fibroblast phenotype augmenting
both eosinophilia and neutrophilia. In contrast, as the situation
becomes more chronic (in the presence of TGF-), eosinophils could
become dominant with associated increases in profibrotic factors.
In an IL-13-driven murine system, eotaxin-1 appears to contribute to the maintenance of airway tissue eosinophilia (10, 22). The cellular source for eotaxin-1 was not determined, but the tissue location of the eosinophils and the findings described here strongly implicate the fibroblast. These findings may also explain why an Ab to IL-5, a cytokine believed to be important in eosinophilia and asthma-like reactions in mice, failed to improve asthmatic responses in humans, even though it profoundly decreased sputum (luminal) eosinophils (23). A recently reported follow-up study suggested that the IL-5 Ab did not completely reverse tissue (in contrast to luminal and blood) eosinophilia, supporting additional tissue factors (24). It is conceivable that eotaxin-1-positive airway fibroblasts, confirmed in the current study in severe asthmatic subjects, could have contributed to the persistent tissue eosinophilia seen in those patients. However, studies in humans specifically designed to evaluate that question, including the use of CCR3 antagonists currently in development, will be required before the relationship of eotaxin to eosinophilia can be confirmed.
Finally, this study supports a specific and substantial interaction of
the innate immune system and the Th2 immune system. This study suggests
that the addition of an atopic background to the repair of a wound,
specifically a chronically healing one as seen in asthma, may
substantially alter the repair process from that seen in the absence of
a Th2 background. It is likely that up-regulation of eotaxin-1 is only
one example. If the interaction proves to involve some modification of
the STAT-6 response, then multiple other STAT-6 responsive genes, such
as VCAM-1, could also be involved. The relevance of these findings to
human disease is suggested by the confirmation of
eotaxin-1+ fibroblasts in a group of subjects
with known increases in TGF-1 and probable increases in
IL-4/-13. Uncovering the mechanisms driving the interactions
should prove beneficial to understanding the relationships between the
innate and the Th2 immune systems, which may eventually lead to
improvement in the understanding of diseases such as asthma.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Sally E. Wenzel, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. E-mail address: wenzels{at}njc.org
3 Abbreviations used in this paper: pSTAT, phosphorylated STAT; CHX, cycloheximide; RT, reverse transcription; CT, threshold cycle.
Received for publication May 28, 2002. Accepted for publication August 9, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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