1 Physiology Program, Harvard School of Public Health, Boston, Massachusetts 02115; and 2 Pulmonary and Critical Care Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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Interleukin (IL)-1 induces
cyclooxygenase (COX)-2 expression and prostanoid formation in cultured
human airway smooth muscle (HASM) cells. In other cell types, IL-6
family cytokines induce COX-2 or augment IL-1
-induced COX-2
expression. The purpose of this study was to determine whether IL-6
family cytokines were involved in COX-2 expression in HASM cells.
RT-PCR was used to demonstrate that the necessary receptor components
for IL-6-type cytokine binding are expressed in HASM cells. IL-6 and
oncostatin M (OSM) each caused a dose-dependent phosphorylation of
signal transducer and activator of transcription-3, whereas IL-11 did not. IL-6, IL-11, and OSM alone had no effect on COX-2 expression. However, OSM caused dose-dependent augmentation of COX-2 expression and
prostaglandin (PG) E2 release induced by IL-1
. In
contrast, IL-6 and IL-11 did not alter IL-1
-induced COX-2
expression. IL-6 did increase IL-1
-induced PGE2
formation in unstimulated cells but not in cells stimulated with
arachidonic acid (AA; 10
5 M), suggesting that IL-6
effects were mediated at the level of AA release. Our results indicate
that IL-6 and OSM are capable of inducing signaling in HASM cells. In
addition, OSM and IL-1
synergistically cause COX-2 expression and
PGE2 release.
oncostatin; cyclooxygenase; prostaglandin; signal transducer and activator of transcription
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INTRODUCTION |
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CYCLOOXYGENASE (COX)
is an enzyme that catalyzes the conversion of arachidonic acid (AA) to
prostaglandins (PG) and thromboxane. There are two isoforms of the
enzyme. COX-1 is constitutively expressed in most mammalian cells,
whereas COX-2 is induced by certain cytokines, lipopolysaccharide, and
mitogens depending on the cell type (8, 18, 19, 34).
Interleukin (IL)-1 and tumor necrosis factor-
are among the
cytokines with the most potent effects on COX-2 expression. In human
airway smooth muscle (HASM) cells, IL-1
at concentrations as low as
0.2 ng/ml induces COX-2 expression and PGE2 release. In
these cells, the induction of COX-2 by IL-1
has important functional
consequences, including a decrease in
2-adrenergic
receptor responsiveness (27).
In some cell types, members of the IL-6 family of cytokines, which
includes IL-6, IL-11, and oncostatin M (OSM), increase COX-2
expression. For example, IL-6 alone and in conjunction with IL-1 has
been demonstrated to augment COX-2 expression and PGE2 production in mouse osteoblasts (40). IL-6 also has been
shown to promote PGE2 release in a canine basilar artery
model (32). OSM induces COX-2 expression both alone and
synergistically with IL-1
in human vascular smooth muscle
(9). Because IL-1 has been shown to induce large
quantities of IL-6 family cytokines in HASM cells (11) and
other human cell types (35) and because members of the
IL-6 family are capable of inducing COX-2 in other cell types, we
hypothesized that these cytokines might in part mediate the effect of
IL-1
on COX-2 expression in HASM.
Members of the IL-6 cytokine family share a common receptor subunit,
glycoprotein (gp)-130. IL-6 signaling occurs through binding of the
cytokine to an IL-6 receptor (IL-6R) coupled to two gp130 subunits.
Signaling through the IL-11 receptor (IL-11R) occurs in a similar
manner, with IL-11 binding to the IL-11R coupled to two gp130 subunits.
OSM acts through an OSM receptor coupled to a single gp130 subunit.
Janus kinases associated with the gp130 component of each receptor type
then become phosphorylated and in turn phosphorylate the gp130 subunit.
The phosphorylated gp130 receptor is able to bind signal transducer and
activator of transcription (STAT), particularly STAT3, through SH2
domains on STAT3. Once bound to the receptor, STAT3 itself is
phosphorylated. The phosphorylated STAT3 is then released from the
receptor, dimerizes with other phosphorylated STAT3 molecules, and
translocates to the nucleus, where it activates target genes such as
2-macroglobulin in the rat and
1-antichymotrypsin in humans (7, 16, 22, 29, 38). IL-6 also induces extracellular signal-regulated kinase (ERK) and p38 mitogen-associated protein kinase (MAPK) activation in
some cell types (39). There is also evidence that ERK may phosphorylate STAT3 near its COOH terminus in some cell types, enhancing its activity (23).
To examine the hypothesis that IL-1-induced formation of IL-6 family
cytokines might, in part, mediate the effects of IL-1
on COX-2
formation and PGE2 production, we first determined whether IL-6, IL-11, and OSM were capable of acting on HASM cells by examining the expression of IL-6 family receptors. We also measured the phosphorylation of STAT3 in response to IL-6, IL-11, and OSM to ensure
that these cytokines did indeed signal in these cells. Finally, we
measured COX-2 expression and PGE2 production in HASM cells
in response to IL-6, IL-11, and OSM in the presence and absence of
IL-1
. Our results indicate that receptors for IL-6 family cytokines
are present on HASM cells and that ligation of these receptors induces
STAT3 activation. In addition OSM, but not IL-6 or IL-11, synergizes
with IL-1
to augment COX-2 expression.
We have previously reported that activation of ERK by IL-1 is
required for COX-2 formation and PGE2 release (25,
26). Because our results indicated that OSM also induced ERK, we
hypothesized that ERK might mediate the ability of OSM to augment
IL-1
-induced COX-2 expression. To address this hypothesis, we
measured ERK phosphorylation by OSM alone and in combination with
IL-1
. We also examined the effect of U-0126, an inhibitor of
MAPK/ERK (MEK) (12), the enzyme upstream of ERK, on the
ability of OSM to augment IL-1
-induced COX-2 expression.
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METHODS |
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Cell culture. HASM cells were obtained from lung transplant donor tracheae in accordance with procedures approved by the University of Pennsylvania (Philadelphia) Committee on Studies Involving Human Beings. A segment of trachea just proximal to the carina was dissected under sterile conditions. The trachealis muscle was isolated, and the tissue was prepared as previously described (33). For culture, the cells were plated in plastic flasks or six-well plates at 104 cells/cm2 in Ham's F-12 medium supplemented with 10% fetal bovine serum, 100 U/ml of penicillin, 0.1 mg/ml of streptomycin, 2.5 mg/ml of amphotericin B, 12 mM NaOH, 1.6 µM CaCl2, 2 mM L-glutamine, and 25 mM HEPES. The medium was replaced every 3-4 days. The cells were passaged with 0.25% trypsin and 1 mM EDTA every 10-14 days. Confluent cells were serum deprived and supplemented with 5.7 µg/ml of insulin and 5 µg/ml of transferrin 24 h before use. Cells in passages 4-7 from 12 different donors were used in the studies described below.
Western blotting for measurement of phosphorylated STAT3,
phosphorylated ERK, phosphorylated p38 levels, and COX-2 expression.
HASM cells were grown to confluence in six-well plates and serum
deprived for 24 h as described above. For measurement of phosphorylated STAT3, ERK, and p38, cells were treated with IL-6, IL-11, or OSM (20 ng/ml) for 5, 10, 15, 30, and 60 min. We also examined the effect of IL-6 and OSM at varying doses from 0.2 to 50 ng/ml for 15 min. The effect of IL-6 and OSM on IL-1-induced ERK
activation was measured using 20 ng/ml of IL-6 or OSM in the presence
and absence of IL-1
(2 ng/ml). The medium was then removed, and the
cells were washed with PBS and lysed with 100 µl of extraction buffer
[10 mM Tris · HCl buffer with 50 mM NaCl, 10 mM
D-serine, 1 mM EDTA, I mM EGTA, 1% sodium dodecyl sulfate
(SDS), 1% Triton X-100, 0.2 mM phenylmethylsulfonyl fluoride, 5 µg/ml of leupeptin, 1 µg/ml of pepstatin, and 10
2
U/ml of aprotinin]. The cells were scraped from the plates, passed through a 25
on COX-2 expression, cells were treated with
IL-6 (20 ng/ml), IL-11 (20 ng/ml), or OSM (at 0.5, 2, and 20 ng/ml) in
the presence and absence of IL-1
at 0.2 or 2 ng/ml. The effect of
OSM (20 ng/ml) on IL-1
(2 ng/ml)-induced COX-2 expression was also
examined after 2 h of pretreatment with either the MEK inhibitor
U-0126 (10 µM; Promega, Madison, WI) or vehicle (DMSO 0.01%). We
have previously reported that U-0126 causes a marked inhibition of
IL-1
-induced ERK phosphorylation (24), indicating its
efficacy. Cytokines were added simultaneously and protein was extracted
24 h later.
PGE2 release.
To examine the ability of IL-6 family cytokines alone or in combination
with IL-1 to induce PGE2 formation, cells were grown to
confluence in 24-well plates and serum deprived for 24 h. Wells were either left untreated or were treated with IL-6, IL-11, or OSM
(all at 20 ng/ml) alone, IL-1
alone (0.2 and 2.0 ng/ml), or the
combination of either IL-6, IL-11, OSM, and IL-1
(both concentrations). After incubation at 37°C for 20 h, the medium was replaced with 0.5 ml of fresh serum-free medium and incubated at
37°C for 15 min in the presence and absence of AA (10
5
M) and the supernatant was then harvested. Supernatants were stored at
20°C until assayed with a PGE2 enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI).
IL-6 and OSM release.
To determine whether IL-1-induced prostanoids contributed to
IL-1
-induced IL-6 and OSM release, HASM cells were grown to confluence in 24-well plates and serum deprived for 24 h. Wells were either left untreated or treated with IL-1
(2 ng/ml) for 20 h. One-half of the IL-1
-treated cells were pretreated with indomethacin (10
6 M) 2 h before addition of IL-1
.
Some cells not treated with cytokines were also treated with
indomethacin. After incubation for 20 h, the supernatants were
harvested and stored at
20°C until assayed with a human IL-6 enzyme
immunoassay kit (Cayman Chemical). The assay had 100% specificity for
IL-6 and <0.01% specificity for IL-1
and IL-1
per
manufacturer's specifications. A human OSM enzyme immunoassay kit (R&D
Systems, Minneapolis, MN) was used to assay OSM. The minimum detectable
concentration of OSM was less than 6 pg/ml per manufacturer's specifications.
PCR for IL-6R, IL-11R, OSM receptor, and gp130. HASM cells from two different donors were serum deprived and hormone supplemented for 24 h. Total RNA was isolated using RNeasy spin columns (QIAGEN, Valencia, CA) according to the manufacturer's specifications. For each sample, ~0.5 µg of total RNA was reverse transcribed using Advantage RT-for-PCR (Clontech, Palo Alto, CA) according to the manufacturer's specifications. PCR was then performed to assess the expression by HASM cells of IL-6R and gp130. gp130 was amplified using two sets of primers. The first set, 5'-TGACGTTGCAGACTTGGGTA-3' (forward primer) and 5'-TTCTGTTCAAGCTGTCCGAA-3' (reverse primer), yielded a 337-bp product. The second set, 5'-TGGAGTGAAGAAGCAAGTGG-3' (forward primer) and 5'-AACAGCTGCATCTGATTTGC-3' (reverse primer), yielded a 303-bp product. Each PCR contained 200 ng of cDNA, 0.5 µl of Taq polymerase (Promega, Madison, WI), 200 µM dNTPs (Clontech), and 20 pmol of each primer in a total volume of 50 µl of PCR buffer (Promega). Conditions for PCR were 94°C for 3 min, followed by 35 cycles for 45 s at 94°C, 45 s at 60°C, and 2 min at 72°C, with a final extension time of 7 min at 72°C. Amplication of the human IL-6R was accomplished using the amplimer set from Clontech. The PCR was performed with the same conditions as described previously for gp130.
PCR was also performed to assess the expression of IL-11R and OSM receptor (OSM-R) by HASM. IL-11R was amplified using the following primers: 5'-CCAAACCTGTAGAGGACCCA-3' (forward primer) and 5'-CGTTCCTTGAGCAGAACTCC-3' (reverse primer), yielding a 224-bp product. OSM-R was amplified using 5'-TCACGTGCTGGTGGATACAT-3' (forward primer) and 5'-TGAATCAGCATCGAGGAGTG-3' (reverse primer), yielding a 342-bp product. The PCR conditions for both the IL-11R and OSM-R were as follows: 94°C for 3 min, followed by 35 cycles for 45 s at 94°C, 45 s at 58°C, and 1 min at 72°C, with a final extension time of 7 min at 72°C.Reagents.
Drugs and reagents for tissue culture used in this study were obtained
from Sigma (St. Louis, MO), with the exception of the following.
Amphotericin B and trypsin-ETDA solution were purchased from GIBCO BRL
(Life Technologies, Grand Island, NY). Recombinant human IL-1, IL-6,
IL-11, and OSM were purchased from R&D Systems.
Data analysis and statistics.
ANOVA was used to examine the statistical significance among the
changes in PGE2 release induced by IL-1, IL-6, IL-11,
and OSM, using treatment and experimental days as main effects.
P < 0.05 was considered significant.
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RESULTS |
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HASM cells express IL-6R, gp130, IL-11R, and OSM-R.
To determine whether IL-6 family cytokines were able to act on HASM, we
used RT-PCR to examine the expression of IL-6 family receptor
components. As shown in Fig. 1, both
components of the IL-6 receptor, gp130 and IL-6R (gp80), are expressed
in HASM cells from two different donors. The gp130 products are visible
in lanes 2-5 (Fig. 1A). Two different sets
of primers were used to examine the expression of gp130. Lanes
2 and 3 show a band consistent with the 337-bp product
expected using the first set of primers, whereas lanes 4 and
5 show a slightly smaller molecular mass band consistent
with the 303-bp product expected using the second set of primers.
Figure 1B shows bands in lanes 2 and 3 consistent with the 251-bp product representing the IL-6R. The presence
of the IL-11R and OSM-R is displayed in Fig. 1C. The 224-bp
product in lanes 2 and 3 represents the IL-11R,
whereas the higher molecular mass bands in lanes 4 and
5 are consistent with the 334-bp product predicted for the
OSM-R. In all these instances, PCR performed without cDNA or without
the RT step yielded no discernible bands.
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IL-6 and OSM activate STAT3.
To determine whether ligation of the IL-6 receptor is capable of
inducing signaling in HASM cells, we measured STAT3 phosphorylation after the addition of IL-6 (20 ng/ml). Maximal phosphorylation of STAT3
by IL-6 occurred at 15 min (Fig.
2A). By 1 h, the effect of IL-6 had markedly diminished. Treatment with IL-11 (20 ng/ml) did
not produce a significant increase in phosphorylated STAT3 (Fig.
2B), whereas OSM at 20 ng/ml induced a large signal that peaked at 10 min and remained elevated even 1 h later (Fig.
2C). Similar results were obtained in cells from three
different donors (Fig. 3) and confirmed
that OSM induced a more substantial and prolonged activation of STAT3
than IL-6. Fifteen-minute treatment with IL-6 caused a dose-dependent
phosphorylation of STAT3 beginning at 2 ng/ml and peaking at 20 ng/ml
(Fig. 4A). A similar dose
response was observed for OSM-induced phospho-STAT3 production (Fig.
4B). IL-6 did not induce phosphorylation of ERK in HASM.
However, OSM (20 ng/ml) caused an increase in ERK phosphorylation,
which was maximal at 15 min and markedly waned by 1 h. (Fig.
5). Whereas both OSM (20 ng/ml) and
IL-1 (0.2 or 2 ng/ml) induced ERK phosphorylation at 15 min, there
were no additive or synergistic effects observed when both were given
in combination (data not shown). None of the IL-6 family cytokines
tested induced phosphorylation of p38 (data not shown).
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Effect of IL-6, IL-11, and OSM on IL-1-induced COX-2 expression
and PGE2 release.
To determine whether IL-1
-induced production of IL-6 family
cytokines contributes to the induction of COX-2 by IL-1
, we examined
the effect of IL-6, IL-11, and OSM alone on COX-2 expression by Western
blotting (Fig. 6). IL-6 (20 ng/ml for
24 h) treatment alone did not result in COX-2 expression, whereas
treatment of HASM cells with IL-1
at concentrations of 0.2 and 2 ng/ml resulted in significant COX-2 expression, as previously described
(Fig. 6A) (16). No further increase in COX-2
expression was observed when cells were treated with both IL-1
and
IL-6. Similar results were obtained in cells from four donors. IL-11
also failed to induce COX-2 expression or to augment IL-1
-induced
COX-2 expression (Fig. 6B). OSM alone did not induce COX-2
expression. However, there was marked synergy between IL-1
(2 ng/ml)
and OSM (Fig. 6B). These results were reproduced in cells
from three donors (Fig. 7). The effect of
OSM was dose dependent and observed at concentrations as low as 0.5 ng/ml (Fig. 6C).
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Indomethacin does not alter IL-1-induced IL-6 production.
In human cancer cells (14), human osteoblasts
(41), and mouse macrophages (29),
PGE2 has been found to increase IL-6 production. Because
IL-1
induces both marked increases in PGE2 release (see
above) as well as increases in IL-6 production (11), we
hypothesized that IL-1
-induced IL-6 formation might be mediated by
IL-1
-induced PGE2. To test this hypothesis, we treated
HASM cells with IL-1
and measured IL-6 release in the presence and absence of the COX inhibitor indomethacin. IL-1
(2 ng/ml) caused a
marked increase in IL-6 release into the supernatant in HASM, consistent with previous reports (11). This increase in
IL-6 was not affected by pretreatment with the cyclooxygenase inhibitor indomethacin at 10
6 M (Fig.
10). In contrast, indomethacin
virtually abolished IL-1
-induced PGE2 formation,
indicating that the lack of effect on IL-6 production was not due to a
lack of efficacy. Production of PGE2 averaged 16 ± 5 pg/ml (n = 3), 649 ± 96 pg/ml (n = 6), and 59 ± 22 pg/ml (n = 6) in control cells,
cells treated with IL-1
(2 ng/ml for 24 h), and cells treated
with both IL-1
and indomethacin, respectively. OSM was below the
limit of detection both in unstimulated HASM cells and in cells treated
with IL-1
(2 ng/ml).
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DISCUSSION |
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Our data indicate that IL-6 family cytokines are capable of
activating HASM cells and altering their function; the components necessary for IL-6, IL-11, and OSM to bind to the cell membrane, namely
gp130 and IL-6R, IL-11R, and OSM-R, are expressed, and both IL-6 and
OSM cause a dose- and time-dependent phosphorylation of STAT3.
Furthermore, both IL-6 and OSM synergize with IL-1 to increase
PGE2 release, although their mechanisms of action differ.
Activation of STAT3 by IL-6 family cytokines has been described in other cell types. The phosphorylation of STAT3 by IL-6 has been well documented in mouse hepatocytes (28), rat Sertoli cells (20), and rat cerebral cortex (10). Significant activation of STAT3 in response to IL-6 also has been described in human breast carcinoma (42) and hepatoma (39) cells. It is therefore not surprising that we found increased STAT3 phosphorylation in response to IL-6. Although IL-11 also has been shown to result in phosphorylation of STAT3 in other cell types, such as human umbilical vein endothelium (30), there was no significant STAT3 effect seen in our cells. Whereas the presence of the IL-11R was established by PCR, the actual receptor number was not quantified. It is possible that the level of receptor expression was too low to induce detectable signaling. We found that OSM induced a more potent phosphorylated STAT3 signal compared with either IL-6 or IL-11 in HASM cells (Fig. 3). Similar results were found in human brain tumor cells, with OSM producing an earlier, more potent activation of STAT3 compared with the other IL-6 family cytokines (37).
IL-6 does have a role in the expression of COX-2 in some cell types. In
mouse osteoblasts, IL-6 both alone and in conjunction with IL-1 was
found to stimulate PGE2 production and COX-2 gene transcription (40). Similar effects were seen in a canine
basilar artery model, although the degree to which IL-6-induced
PGE2 was less than with IL-1
(32). Ferreira
et al. (13) have also demonstrated that IL-6
synergistically acts with IL-1
to induce COX-2 expression in a rat
hyperalgesia model. The expression of COX-2 induced by serum from
preeclamptic subjects was inhibited by anti-IL-6 antibody in uterine
endothelial cells (1). However, IL-6 alone did not induce
COX-2 expression in HASM cells, and IL-6 did not synergize with IL-1
to augment COX-2. These findings also have been observed in rat uterus,
as well as in rat microglial cells and hepatocytes (5, 6,
31).
PGE2 is produced when membrane phospholipids are converted
by phospholipase A2 to AA. AA becomes a substrate for COX,
resulting in the release of PGE2. Even though IL-6 did not
augment COX-2 expression caused by IL-1, it did increase
IL-1
-induced PGE2 release. The increase in
PGE2 by IL-6 was seen only in unstimulated but not in
AA-stimulated cells. Therefore, the increased production of
PGE2 by IL-6 in unstimulated cells may result from effects of the cytokine on the expression or activation of phospholipase A2. If the effect of IL-6 occurred at the level of COX-2
expression, an increase in the amount of PGE2 release would
have been expected after the administration of AA.
We observed a marked synergy between OSM and IL-1 in the induction
of COX-2 (Figs. 6B and 7). Synergy between OSM and IL-1
has also been observed in human aortic smooth muscle cells
(9). However, in the aortic cells, COX-2 was induced by
OSM alone, whereas in HASM cells, it was not. Differences in signaling
may account for these differences in COX-2 expression. In contrast to
our results, STAT1, not STAT3, was the major STAT involved in the OSM
signaling pathway in aortic smooth muscle.
It is possible that differences in STAT3 phosphorylation by OSM and
IL-6 (Fig. 3) may be responsible for the differences in their effects
on COX-2. STAT3 has been shown to activate the junB promoter in a
hepatoma cell line (22) and 2-macroglobulin
in human breast carcinoma cells (42) as well as c-Fos
(16, 24). The transcription factor AP-1 is composed of
dimers of Fos and Jun and there are AP-1-like elements in the promoter
of the COX-2 gene (4).
It is also possible that the differences in effects of IL-6 and OSM on
IL-1-induced COX-2 expression are due to differences in their
effects on ERK phosphorylation. Whereas both OSM and IL-6, to a lesser
degree, have been shown to phosphorylate ERK in HepG2 cells and or
hepatoma cells (21, 39), OSM, but not IL-6, caused ERK
phosphorylation in HASM cells. ERK phosphorylation is known to be
important in IL-1
-induced COX-2 expression in HASM cells
(25). However, there was no synergism between IL-1
and
OSM in their ability to activate ERK. The MEK inhibitor U-0126 did
appear to decrease COX-2 expression in cells treated with IL-1
and
OSM, but this effect is likely due to the effects of U-0126 on
IL-1
-induced effects. In fact, the ability of OSM to enhance
IL-1
-induced COX-2 expression was not altered in cells treated with
U-0126, suggesting that ERK is not involved in the mechanism of action
of OSM.
Because PGE2 has been shown to be capable of inducing IL-6
in some cell types and because IL-1 evokes the release of large amounts of PGE2 and IL-6, we sought to determine whether
IL-1
-induced prostanoids might contribute to the release of IL-6 by
IL-1
. The ability of IL-1
to induce production of IL-6 was not
affected by the nonsteroidal anti-inflammatory drug indomethacin (Fig. 10). This suggests that in HASM cells PGE2 is not
primarily responsible for IL-6 production. In contrast, studies on
human osteoblasts have revealed a suppression of IL-1
-induced IL-6
production by treatment with the specific COX-2 inhibitor NS-398
(41). Similar results were seen in human macrophages,
suggesting regulation of IL-6 by COX-2 (44). Animal models
of inflammation also demonstrated that COX-2 was involved in the
regulation of IL-6 production (3, 29).
No production of OSM was observed in either control or IL-1-treated
HASM cells, whereas both IL-6 and IL-11 were produced (11). OSM is produced by macrophages and neutrophils
(15, 36). Hence, it is possible that OSM might contribute
to COX-2 expression and PGE2 release with illnesses
characterized by airway inflammation, such as asthma and other chronic
obstructive pulmonary diseases. In these disease states, the
above-implicated cells, macrophages and neutrophils, are known to have
a major role.
In summary, we found that members of the IL-6 cytokine family activate
STAT3 and play a role in COX-2 expression and/or PGE2 release in HASM cells. OSM, synergistically with IL-1, induced a
significant increase in both COX-2 expression and PGE2
release. Although OSM also phosphorylated ERK, ERK did not appear to
have a role in the ability of OSM to enhance IL-1
-induced COX-2
expression. Although IL-6 was found to increase IL-1
-induced
PGE2 release, this effect did not appear to occur at the
level of COX-2 expression.
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ACKNOWLEDGEMENTS |
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We acknowledge Igor Schwartzman and Trudi Church for the technical assistance they provided in performing these experiments.
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FOOTNOTES |
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This work was supported by the National Heart, Lung, and Blood Institute Grant HL-56383. J. L. Laporte and P. E. Moore were supported by an American Lung Association (ALA) Fellowship and an ALA grant.
Address for reprint requests and other correspondence: S. Shore, Physiology Program, Harvard School of Public Health, 665 Huntington Ave, Boston, MA 02115.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 27 September 2000; accepted in final form 8 January 2001.
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