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and IL-1
in airway smooth
muscle cells: implications for -adrenergic responsiveness
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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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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In human cultured airway smooth muscle cells, interleukin
(IL)-1
increases cyclooxygenase (COX)-2 expression and
PGE2 release, ultimately resulting in decreased
-adrenergic responsiveness. In this study, we aimed to determine
whether tumor necrosis factor-
(TNF-) synergizes with IL-1 in
the induction of these events. TNF- alone, at concentrations up to
10 ng/ml, had no effect on COX-2 protein expression; at concentrations
as low as 0.1 ng/ml, it significantly enhanced the ability of IL-1
(0.2 ng/ml) to induce COX-2 and to increase PGE2 release.
IL-1 and TNF- in combination also significantly enhanced COX-2
promoter activity, indicating that synergism between the cytokines is
mediated at the level of gene transcription. Although IL-1 and
TNF- each increased nuclear factor-
B activation and induced
extracellular regulated kinase and p38 phosphorylation, combined
administration of the cytokines did not enhance either nuclear
factor-B or mitogen-activated protein kinase activation. Combined
administration of IL-1 (0.2 ng/ml) and TNF- (0.1 or 1.0 ng/ml)
reduced the ability of isoproterenol to decrease human airway smooth
muscle cell stiffness, as measured by magnetic twisting cytometry, even
though individually these cytokines, at these concentrations, had no
effect on isoproterenol responses. Treatment with the selective COX-2
inhibitor NS-398 abolished the synergistic effects of TNF- and
IL-1 on -adrenergic responsiveness. Our results indicate that low
concentrations of IL-1 and TNF- synergize to promote
-adrenergic hyporesponsiveness and that effects on COX-2 expression
and PGE2 are responsible for these events. The data suggest
that the simultaneous release in the airway, of even very small amounts
of cytokines, can have important functional consequences.
isoproterenol; cyclooxygenase-2; mitogen-activated protein
kinase; nuclear factor-B; magnetic twisting cytometry; interleukin-1; tumor necrosis factor-
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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THE CYCLOOXYGENASE (COX)
enzyme catalyzes the conversion of arachidonic acid to prostaglandins
and thromboxane. Two isoforms of the enzyme have been described. COX-1
is constitutively expressed in most mammalian cells, whereas COX-2 is
induced under conditions of inflammation, for example, by certain
cytokines, lipopolysaccharide, and mitogens (6, 15, 21,
28). Interleukin (IL)-1 is among the cytokines with the most
potent effects on COX-2 expression. In human airway smooth muscle
(HASM) cells, IL-1 induces COX-2 expression and PGE2
release. In these cells, the induction of COX-2 by IL-1 has
important functional consequences, including 2-adrenergic hyporesponsiveness (18, 27), a
characteristic feature of asthma (4, 5, 12). The precise
role of COX-2 in these events has not been firmly established but is
thought to involve the release of PGE2, consequent increase
in cAMP formation, protein kinase A activation, and phosphorylation of
the 2-adrenergic receptor by protein kinase A,
uncoupling it from Gs (18).
Although tumor necrosis factor- (TNF-) has also been shown to
induce COX-2 expression in some cell types (13, 16, 34), HASM cells do not increase their expression of COX-2 even after treatment with very high (100 ng/ml) concentrations of TNF-
(28). TNF- alone also has no effect on -adrenergic
responsiveness in HASM cells (27). However, when TNF-
is administered in conjunction with IL-1, it increases the ability
of IL-1 to induce COX-2 (6, 28). The ability of TNF-
to enhance the effects of IL-1 may be particularly important, since
increased levels of both of these cytokines have been observed in
bronchoalveolar lavage (BAL) fluid of patients with symptomatic asthma
(7, 8, 33). Whether TNF- also enhances the ability of
IL-1 to induce -adrenergic responsiveness has not been established.
To date, synergism between TNF- and IL-1 in HASM cells has been
demonstrated only at fairly high concentrations of these cytokines (10 ng/ml each) and the signaling cascade leading to this interaction is
not known. The first purpose of this study was to determine whether
there is also synergism between TNF- and IL-1 at lower
concentrations (in the 0.1-1.0 ng/ml range) and to examine the
mechanistic basis for this synergism. Such effects may be particularly
important to establish because concentrations of TNF- and IL-1
measured in BAL fluid of asthmatic subjects fall within this range
(7, 8). To determine whether the interaction between the
cytokines occurs at the transcriptional level, we used a COX-2
promoter/luciferase reporter construct. Because the extracellular
regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinases
and the transcription factor nuclear factor (NF)-B have been
implicated in the induction of COX-2 by IL-1 and/or TNF- either
in HASM cells or in other cell types (16, 17, 19, 24, 34),
we also determined whether there was synergism between IL-1 and
TNF- at the level of MAP kinase or NF-B activation.
The second purpose of the study was to determine whether TNF- and
IL-1 can also synergize in the induction of -adrenergic hyporesponsiveness. -Adrenergic responsiveness was determined by
measuring isoproterenol-induced changes in cell stiffness using magnetic twisting cytometry. We and others have established that changes in stiffness can be used as a surrogate for force generation in
these cells (9, 14, 20, 31). Because our results indicated synergism at the level of COX-2 and prostanoid synthesis as well as
-adrenergic responsiveness and because we have previously reported
that COX-2 expression is required for the -adrenergic hyporesponsiveness induced by IL-1 alone (18),
we also assessed the effects of the selective COX-2 inhibitor NS-398 on
the decreased responsiveness to -agonists that was observed in cells
treated with low concentrations of TNF- and IL-1.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Cell culture. Human tracheas were obtained from lung transplant donors, in accordance with procedures approved by the University of Pennsylvania Committee on Studies Involving Human Beings. Tracheal smooth muscle cells were harvested from the tracheas as previously described (18, 26, 29). Cells were plated in plastic flasks at 104 cells/cm2 in Ham's F-12 medium supplemented with 10% fetal bovine serum, penicillin (103 U/ml), streptomycin (1 mg/ml), amphotericin-B (2 mg/ml), NaOH (12 mM), CaCl2 (1.7 µM), L-glutamine (2 mM), and HEPES (25 mM). Culture medium was replaced every 3-4 days. Cells were passaged with 0.25% trypsin and 1 mM EDTA every 10-14 days. Confluent serum-deprived HASM cells in passages 5-7 were used in the studies described below. When cells were serum deprived, they were supplemented with 5.7 µg/ml insulin and 5.0 µg/ml transferrin 24-48 h before use.
Western blotting for measurement of COX-2 expression and ERK and
p38 phosphorylation.
Confluent HASM cells were serum deprived and treated with IL-1 (0.2 ng/ml) alone or in combination with TNF- (0.1 and/or 1.0 ng/ml).
Cytokine treatment was for 15 min in the case of ERK and p38 activation
and for 20 h in the case of COX-2 expression. Medium was removed,
and cells were washed with PBS and then lysed in 400 µl of extraction
buffer (10 mM Tris · HCl buffer with 50 mM NaCl, 50 mM NaF, 10 mM D-serine, 1 mM EDTA, 1 mM EGTA, 1% SDS, 1% Triton
X-100, 0.2 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 1 µg/ml pepstatin, 10
2 U/ml aprotinin). Cells were
scraped off flasks, passed through a 25 and 5/8-gauge needle, and
solubilized by sonication. Western blot analysis using antibodies to
phosphorylated p38, phosphorylated p42/p44 ERK (New England Biolabs,
Beverly, MA), or COX-2 (Oxford Biomedical Research, Oxford, MI) was
performed as previously described (17-19). The
standards for phosphorylated p42 ERK and phosphorylated p38 and for COX
were also obtained from New England Biolabs and Oxford Biomedical
Research. The bands visualized at 38 kDa (p38), 42 and 44 kDa (ERK), or
70 kDa (COX-2) were quantified by laser densitometry.
PGE2 release.
Cells were plated in 24-well plates, grown to confluence, and serum
deprived for 24. Cells were then either left untreated or treated with
IL-1 (0.02-2.0 ng/ml), TNF- (0.01-1.0 ng/ml), or
combinations of these cytokines. Approximately 22 h later, cell
medium was removed, cells were washed with PBS, and 0.5 ml of fresh
medium was added to each well. After a 15-min incubation at 37°C, the
supernatants were harvested and stored at 
20°C until assayed with a
PGE2 enzyme immunoassay kit (Caymen Chemical, Ann Arbor,
MI). The antibody to PGE2 had <1% cross-reactivity to
6-keto-PGF1
and <0.01% to thromboxane B2
and other prostaglandins, according to the manufacturer's specifications.
Magnetic twisting cytometry.
Confluent cells were serum deprived for 24 h and treated with
TNF- (0.1 or 1.0 ng/ml) alone or in combination with IL-1 (0.2 ng/ml). Eighteen hours later, cells were harvested by brief exposure to
trypsin and EDTA, resuspended in serum-free hormone supplemented medium
with or without cytokines, and plated at 20,000 cells/well on collagen
I (500 ng/cm2)-coated bacteriological plastic dishes (6.4 mm, 96-well Removawells, Immunlon II). Two to six hours later,
measurements of cell stiffness were made using magnetic twisting
cytometry as previously described (17-19, 22, 23,
29). Cumulative concentration-response curves to isoproterenol
were performed as follows. First, three to five measurements of cell
stiffness were made under baseline conditions. After these
measurements, 2 µl of a solution containing the isoproterenol were
added to the well that contained 200 µl of medium. After a 1-min
incubation with agent, two to four measurements of cell stiffness were
again obtained. This procedure was repeated with concentrations of
isoproterenol increased from 108 to 105 M.
- and TNF--induced changes
in cell stiffness responses to isoproterenol, four flasks of cells from
the same passage of the same donor were used. Two flasks were treated
with NS-398 (105 M). Two hours later, IL-1 (0.2 ng/ml)
and TNF- (0.2 ng/ml) were added to one of the flasks treated with
NS-398 and was also added to an untreated flask. Eighteen hours later,
the cells were harvested and used for cell stiffness measurements as
described above.
HASM cell transfection.
After passage, HASM cells were grown for 72 h (60-80%
confluence) in complete medium in six-well tissue culture plates.
Before transfection with the COX-2 promoter/luciferase reporter
construct (COX2-S), the medium was changed to contain only serum-free
media, to avoid growth factor induction of COX-2. HASM cells were
cotransfected with 0.5 µg of COX2-S and 0.5 µg of a
-galactosidase control vector (Promega, Madison, WI), using Fugene 6 (Roche, Indianapolis, IN) according the manufacturer's protocol. The
-galactosidase control vector was used to normalize for differences
in transfection efficiency. The ~500-bp fragment of the promoter
region of human COX-2 was constructed by creating SacI
(471 bp) and NheI (4 bp upstream of ATG) sites from a
larger 2.0-kb fragment of the COX-2 promoter (gift of Stephen M. Prescott), and this fragment was cloned into the respective sites of
the pGL3-basic vector (Promega). Cells were then incubated for 15 h with either IL-1 (0.2 ng/ml) or TNF- (1.0 ng/ml) alone or with
the two cytokines in combination. Cells were lysed with reporter lysis
buffer (Promega), harvested, and assayed for luciferase activity by
scintillation counting after addition of luciferin and for
-galactosidase activity by spectrophometry using the
-galactosidase enzyme assay system (Promega). The results of the
experiments are reported as mean luciferase activity normalized for
-galactosidase activity. With the use of this system, transfection
efficiency typically ranges between 10 and 20%, as assessed by flow
cytometry of cells transfected with a green fluorescence
protein-expressing vector.
and TNF- to synergize in the
activation of NF-B, HASM cells were cotransfected with 0.5 µg of
pNF-B-Luc, designed for monitoring the NF-B signal transduction pathway (Clontech, Palo Alto, CA) and with 0.5 µg of a
-galactosidase control vector as described above, except that, for
these experiments, the medium was changed from 10% to 1% fetal bovine
serum just before transfection. Cells were treated with cytokines and
luciferase and -galactosidase activities were measured as described above.
Reagents.
Tissue culture reagents and drugs used in this study were obtained from
Sigma Chemical (St. Louis, MO), with the exception of amphotericin and
trypsin-EDTA, which were obtained from GIBCO (Grand Island, NY), and
TNF- and IL-1, which were obtained from R&D Systems (Minneapolis,
MN). Isoproterenol (101 M in distilled water) was made
fresh each day. Because isoproterenol is rapidly oxidized, dilutions of
isoproterenol in medium were made immediately before addition to the
cells. NS-398 was dissolved in DMSO at 102 M and diluted
in culture medium.
Statistics.
The effect of TNF- and IL-1 on changes in stiffness induced by
isoproterenol was examined by repeated-measures ANOVA using drug and
experimental day as main effects. Follow-up tests were performed to
determine at which concentration of isoproterenol the drug treatment
effect was observed. The Bonferroni rule was used to correct for
multiple comparisons. The effect of TNF- and IL-1 on
PGE2 release was assessed by ANOVA using experimental day,
TNF- dose, and IL-1 dose as main effects. The effect of TNF-
and IL-1 on COX-2 expression, COX-2 promoter activity, and NF-B
luciferase activity was determined by ANOVA using experimental day and
drug treatment as main effects. Follow-up paired t-tests were performed to compare among drug treatment groups. A P
value <0.05 was considered significant.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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TNF- at concentrations up to 10 ng/ml had no effect on COX-2
expression in HASM cells from four different donors (data not shown).
However, TNF- did synergize with IL-1 in the induction of COX-2
expression. For these experiments, cells were treated with a
concentration of IL-1 (0.2 ng/ml) that induced a small but less than
maximal increase in COX-2 expression (18) and with
concentrations of TNF- in the same range (0.1-1.0 ng/ml). A
representative COX-2 Western blot is shown in Fig.
1A. As previously described,
COX-2 was not expressed in control, untreated cells. IL-1 induced
COX-2 expression, whereas TNF- did not. However, when the two
cytokines were administered simultaneously, there was a marked increase
in COX-2 expression compared with cells treated with IL-1 alone.
Similar results were obtained in cells from six HASM cell donors (Fig.
1B). There was also synergism between IL-1 and TNF- at
the level of PGE2 release. IL-1 caused a
concentration-related increase in PGE2 release
(P < 0.001), as previously described
(18), whereas TNF- failed to increase PGE2
release. PGE2 release into HASM cell supernatants averaged 0.40 ± 0.15 ng/ml in control cells and 0.36 ± 0.10, 0.37 ± 0.15, 0.62 ± 0.25, and 0.47 ± 0.16 ng/ml in
cells treated with 0.01, 0.1, 1.0, or 10.0 ng/ml TNF- for 20 h
(P > 0.05). Although TNF- alone had no effect on
PGE2 release, it significantly augmented the effects of
IL-1 (P < 0.05 by ANOVA) (Fig.
2). This was particularly apparent at 1.0 ng/ml TNF- but was also observed at 0.1 ng/ml TNF-.
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To determine whether the effects of IL-1 and TNF- we observed are
mediated at the level of COX-2 transcription, HASM cells were
transiently transfected with a COX-2 promoter/luciferase construct and
treated with IL-1 (0.2 ng/ml) and TNF- (1.0 ng/ml) alone or in
combination. As shown in Fig. 3, neither
cytokine on its own increased COX-2 promoter activity; however, in
combination, there was a significant increase (P < 0.05) in COX-2 promoter activity that was only slightly smaller in
magnitude than that induced by IL-1 at a 100-fold higher
concentration (20 ng/ml). We reasoned that this effect on COX-2
promoter activity might be the result of IL-1 and TNF-
synergizing in their ability to activate NF-B, since consensus
binding sites for NF-B are present in the COX-2 gene in the region
of the promoter that we used in our construct (2). To
address this possibility, HASM cells were transiently transfected with
an NF-B/luciferase reporter construct (Fig.
4). Although IL-1 (0.2 ng/ml) and
TNF- (1.0 ng/ml) each increased NF-B promoter activity, the
combinations of these cytokines did not significantly augment the
increase observed with IL-1 on its own.
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Because we have previously demonstrated that both the ERK and p38 MAP
kinases are required for induction of COX-2 by higher concentrations of
IL-1 (2.0 ng/ml) (17, 19), we reasoned that the
synergism between IL-1 and TNF- might be mediated at the level of
MAP kinase activation. Hence, we treated HASM cells with IL-1 (0.2 ng/ml) and TNF- (0.1 ng/ml) alone or in combination and examined
their effects on ERK and p38 phosphorylation. As shown in Fig.
5, IL-1 and TNF- at these
concentrations each caused phosphorylation of ERK and p38. However,
when the two cytokines were combined, there was no further augmentation
of the effects of IL-1 by TNF-. Similar results were obtained in
cells from three donors (data not shown).
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We have previously reported that COX-2 expression and PGE2
release are involved in the decreased -adrenergic responsiveness induced by IL-1 in HASM cells (18). Hence, we sought to
determine whether the ability of TNF- to synergize with IL-1 in
the induction of COX-2 expression and PGE2 release (Figs. 1
and 2) might lead to increased -adrenergic desensitization.
Isoproterenol-induced changes in HASM cell stiffness, as measured by
magnetic twisting cytometry, were used as the index of -adrenergic
responsiveness. Because the greatest synergism in terms of
PGE2 release was observed when IL-1 at 0.2 ng/ml was
administered with TNF- at 0.1 or 1.0 ng/ml (Fig. 2), we examined the
effects of these combinations of cytokines on responses to
isoproterenol. We have previously reported that IL-1 (0.2 ng/ml)
alone has no effect on cell stiffness responses to isoproterenol
(29), although higher concentrations do influence these
responses. Similarly, TNF- alone at 0.1 ng/ml did not alter
responses to isoproterenol: isoproterenol caused a
concentration-dependent decrease in cell stiffness in control cells,
and the magnitude of this decrease in stiffness was not significantly
different in cells treated with TNF- (Fig.
6). TNF- at 1.0 ng/ml did have a
statistically significant effect on cell stiffness responses to
isoproterenol, but the magnitude of the effect was small and was
apparent only at 107 and 106 M
isoproterenol. Note that baseline stiffness was not affected by
TNF-, averaging 117 ± 7.0, 137 ± 11.6, and 125 ± 8.4 dyn/cm2 in control cells and cells treated with 0.1 or
1.0 ng/ml TNF-, respectively (P > 0.05). Whereas no
effect of IL-1 and either no or a very small effect of TNF-
depending on the concentration was observed when the cytokines were
administered separately, the cytokines had profound effects on HASM
cell responses to isoproterenol when they were administered together,
virtually abolishing the response to isoproterenol (Fig.
7). None of the cytokine combinations had
any significant effect on baseline cell stiffness.
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To determine whether the effects of combined IL-1 and TNF-
treatment on -adrenergic responsiveness were the result of COX-2 expression and PGE2 release, we examined the effect of the
selective COX-2 inhibitor NS-398 (105 M) on IL-1 and
TNF--induced changes in the ability of isoproterenol to evoke cell
stiffness changes (Fig. 8). Neither
NS-398 nor the combination of IL-1 and TNF- alone or with NS-398
had any significant effect on baseline stiffness. Repeated-measures
ANOVA indicated a significant effect of drug treatment on cell
stiffness responses to isoproterenol (P < 0.01).
Follow-up analysis indicated that the treatment effect lay in the
combined IL-1 and TNF- treatment group in which ISO responses
were significantly different from control at all isoproterenol
concentrations (P < 0.05). In contrast, neither cells
treated with NS-398 alone nor cells treated with NS-398 in combination
with IL-1 and TNF- were significantly different from control
cells. Note that the data in Fig. 8 were derived from donors different
from those in Fig. 7, which likely accounts for the difference in the
efficacy of isoproterenol in the control cells.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Our results indicate that TNF- alone has no effect on COX-2
expression and PGE2 release. However, even very small
concentrations of TNF- can augment the ability of IL-1 to induce
these effects in HASM cells (Figs. 1 and 2). Furthermore, synergism
between IL-1 and TNF- appears to occur at the level of COX-2 gene
transcription (Fig. 3), but this transcriptional effect does not appear
to involve increased activation of NF-B (Fig. 4); NF-B activation
in cells treated with IL-1 and TNF- was similar to that induced
by IL-1 alone. Similarly, both IL-1 and TNF- alone induced p38
and ERK phosphorylation, but we did not observe any synergism between IL-1 and TNF- in their effects on MAP kinase activation (Fig. 5).
The combination of TNF- and IL-1 also caused marked
-adrenergic hyporesponsiveness in HASM cells (Fig. 7), and this
effect was abolished by the selective COX-2 inhibitor, NS-398 (Fig. 8).
TNF- alone did not increase COX-2 expression or PGE2
release. These results confirm those of Pang and Knox (28)
in HASM cells. TNF- also has no effect on COX-2 expression in the
human macrophage U-937 cell line (3), although it does
induce COX-2 expression and/or PGE2 release in human
monocytes, human HT-29 epithelial cells, and the murine MC3T3-E1
osteogenic cell line (13, 16, 34). It is not clear why
there are cell type-related differences in the expression of COX-2 by
TNF-.
Synergism between IL-1 and TNF- in their effects on COX-2
expression and PGE2 in HASM cells has been reported by
other investigators (6, 28). However, these investigators
used 10- to 100-fold higher concentrations of the cytokines in their
studies and did not examine effects of cytokine combinations on
-adrenergic responsiveness or the mechanism of interaction between
IL-1 and TNF-. To our knowledge, we are the first to report
synergistic effects of these cytokines at these lower concentrations on
HASM cells. These findings may be of particular importance, since
TNF- and IL-1 are often released together, and the concentrations
of TNF- and IL-1 measured in BAL fluid of symptomatic asthmatics
fall within this range (7, 8). We do not know whether the
concentrations of IL-1 and TNF- in the vicinity of the airway
smooth muscle are greater or less than the concentrations in BAL fluid,
but our results suggest that even very mild airway inflammation, if it
results in the release of both IL-1 and TNF-, may lead to COX-2
expression and -adrenergic hyporesponsiveness.
Synergism between IL-1 and TNF- in their effects on COX-2 protein
expression and/or PGE2 release has also been reported in
other cell types (10, 32, 35). Because the cytokines also
exert synergistic effects on COX-2 mRNA expression (10, 35), their effects are likely to be mediated at the level of COX-2 transcription or message stabilization. Indeed, both IL-1 and
TNF- have been shown to increase COX-2 transcription in some cell
types (10, 24, 34). Our results (Fig. 3), using a COX-2 promoter/luciferase reporter construct, suggest that the synergistic effects of IL-1 and TNF- are due to transcriptional effects, at
least in part. IL-1 and TNF- have also been demonstrated to
stabilize COX-2 mRNA in a murine osteogenic cell line
(13). There is an AU-rich region in the 3'-untranslated
region of the COX-2 gene, and protein binding to this region has been
postulated to increase COX-2 mRNA stability (30). We
cannot rule out the possibility that such effects also contribute to
the synergism between IL-1 and TNF-.
The promoter region of the COX-2 gene contains consensus sequences for
binding of the transcription factor NF-B (2). In addition, activation of NF-B occurs in response to both IL-1 and
TNF- in HASM cells (1, 19, 23) and has been reported to
play a role in the transcriptional regulation of COX-2 by IL-1 and
TNF- in some cell types (16, 24, 34). Although NF-B does not appear to contribute to IL-1-induced COX-2 expression in
HASM cells (19), we reasoned that it might still be
involved in the synergistic effects exerted by IL-1 and TNF-.
However, our results suggest that this is unlikely. Although both
IL-1 and to a lesser extent TNF- caused activation of NF-B
even at fairly low concentrations, the combination of the two cytokines did not increase the magnitude of NF-B activation that was effected by IL-1 alone (Fig. 4).
We have previously established that both the ERK and p38 MAP kinases
are required for the COX-2 expression and -adrenergic hyporesponsiveness induced by higher concentrations of IL-1 (2 ng/ml) in HASM cells (17, 19). ERK and p38 are activated
by IL-1, and relatively selective inhibitors of each of these
pathways cause marked decreases in IL-1-induced COX-2 expression and
PGE2 release and abolish the effects of IL-1 on
-adrenergic responsiveness in HASM cells. Because TNF- has also
been demonstrated to induce ERK and p38 activation in HASM cells
(25), we reasoned that one explanation for the synergistic
effects of TNF- and IL-1 in HASM cells might be effects at the
level of ERK and/or p38 activation. Our results confirm that both
IL-1 and TNF- cause p38 and ERK phosphorylation even at fairly
low concentrations. However, when administered together, the cytokines
did not augment phosphorylation of these MAP kinases above the levels
induced by either cytokine alone (Fig. 5). Our results also indicate
that, although activation of p38 and ERK are required for induction of
COX-2 expression (17, 19), their activation is not
sufficient for COX-2 expression because TNF- alone induced
phosphorylation of both proteins but did not induce COX-2.
We have previously reported that IL-1 at concentrations 2.0 ng/ml
and greater causes -adrenergic hyporesponsiveness in HASM cells and
that the mechanistic basis for this effect is likely to involve
uncoupling of the -receptor from Gs (29).
The observations that COX-2 is induced by IL-1, that
PGE2 mimics the effects of IL-1, and that COX-2
inhibitors ablate the effects of IL-1 on -adrenergic responses
suggest that COX-2-generated prostanoids are involved in the signal
transduction pathway leading from IL-1 to effects on -adrenergic
responsiveness (18) (27). One hypothesis is
that COX-2-generated PGE2 leads to increased cAMP
formation, activation of protein kinase A, and consequent
phosphorylation of the -receptor (18). We now report
that TNF- at concentrations that on their own have either no effect
or very minimal effects on responses to -agonists (Fig. 6) has
marked effects on -adrenergic responsiveness (Fig. 7) if
administered simultaneously with IL-1 at a concentration that on its
own has no effect (29). The same concentrations of IL-1
and TNF- synergize in the induction of COX-2 expression and
PGE2 release (Figs. 1 and 2). Taken together with the
observation that the relatively selectively COX-2 inhibitor, NS-398,
abolishes the effects of combined treatment with low concentrations of
IL-1 and TNF- (Fig. 8), the results suggest that COX-2-generated prostanoids mediate the effects of the combination of IL-1 and TNF- on -adrenergic responsiveness.
Decreased -adrenergic responsiveness is a characteristic feature of
human asthma. Decreased bronchodilator responses to -agonists have
been observed in asthmatic airways both in vivo (5) and in
vitro (4, 12), as well as in animal models of asthma
(11). -Agonists are currently one of the most important
forms of therapy for asthma, and understanding the mechanistic basis
for the -adrenergic receptor dysfunction in asthma may prove to be
an important step in improving the efficacy of these agents. Our
results suggest that the release of IL-1 and TNF- in the
asthmatic airway may contribute to the -adrenergic
hyporesponsiveness of asthma. Furthermore, the data suggest that the
release of even very small amounts of these cytokines can have
important functional consequences if they are released simultaneously.
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ACKNOWLEDGEMENTS |
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We thank Joseph Abraham and Igor Schwartzman for technical assistance.
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FOOTNOTES |
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This study was supported by National Heart, Lung, and Blood Institute Grants HL-56383, HL-33009, and HL-04395, the American Lung Association, and the Charles H. Hood Foundation.
Address for reprint requests and other correspondence: S. A. Shore, Physiology Program, Harvard School of Public Health, 665 Huntington Ave., Boston, MA 02115 (E-mail: sshore{at}hsph.harvard.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 3 April 2001; accepted in final form 14 June 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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1.
Amrani, Y,
Lazaar AL,
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