1 Physiology Program, Harvard School of Public Health, Boston, Massachusetts 02115; 2 Pulmonary and Critical Care Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19002-2209; and 3 GSF National Research Center for Environment and Health Institute of Inhalation Biology, Oberschleissheim, Germany
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ABSTRACT |
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We have previously reported that pretreatment of
cultured human airway smooth muscle (HASM) cells with interleukin-1
(IL-1
) results in decreased
-adrenergic responsiveness. The
purpose of this study was to determine whether prostanoids released as a result of cyclooxygenase-2 (COX-2) induction by IL-1
contribute to
this effect of the cytokine. Confluent serum-deprived HASM cells were
studied in passages 4-7. IL-1
(20 ng/ml for 22 h) reduced the ability of the
-agonist isoproterenol (Iso) to decrease stiffness of HASM cells as measured by magnetic twisting cytometry. The
effect of IL-1
on Iso-induced changes in cell stiffness was abolished by nonselective [indomethacin (Indo),
10
6 M] and selective
(NS-398, 10
5 M) COX-2
inhibitors. Indo and NS-398 also inhibited both the increased basal
cAMP and the decreases in Iso-stimulated cAMP production induced by
IL-1
. IL-1
(20 ng/ml for 22 h) caused an increase in both basal
(15-fold) and arachidonic acid (AA)-stimulated (10-fold)
PGE2 release. Indo blocked basal
and AA-stimulated PGE2 release in
both control and IL-1
-treated cells. NS-398 also markedly reduced
basal and AA-stimulated PGE2
release in IL-1
-treated cells but had no significant effect on
AA-stimulated PGE2 release in
control cells. Western blot analysis confirmed the induction of COX-2
by IL-1
. Exogenously administered
PGE2
(10
7 M, 22 h) caused a
significant reduction in the ability of Iso to decrease cell stiffness,
mimicking the effects of IL-1
. Cycloheximide (10 µg/ml for 24 h),
an inhibitor of protein synthesis, also abolished the effects of
IL-1
on Iso-induced cell stiffness changes and cAMP formation. In
summary, our results indicate that IL-1
significantly increases
prostanoid release by HASM cells as a result of increased COX-2
expression. The prostanoids appear to contribute to
-adrenergic hyporesponsiveness, perhaps by heterologous desensitization of the
2 receptor.
prostaglandin E2; cytoskeletal
mechanics; indomethacin; NS-398; adenosine 3',5'-cyclic
monophosphate; interleukin-1
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INTRODUCTION |
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-adrenergic hyporesponsiveness is a characteristic feature
of asthma. Decreased bronchodilator responses to
-agonists have been
observed in asthmatic subjects and also in animal models of asthma (2,
3, 14, 33). It is possible that cytokines are involved in mediating
this phenomenon. Increased levels of interleukin-1
(IL-1
) have
been observed in bronchoalveolar fluid of patients with
symptomatic asthma (32, 50), and IL-1
has been shown to decrease
-adrenergic responsiveness in a number of cells and tissues (11, 17,
25, 53), including cultured human airway smooth muscle (HASM) cells
(45).
The cyclooxygenase (COX) enzyme converts arachidonic acid (AA) to
PGH2 (19), which is then
metabolized to various PGs, such as
PGE2,
PGD2, and
PGI2, and thromboxane
A2
(TxA2). It is now recognized
that two COX enzymes exist. COX-1 was initially cloned from ovine
seminal vesicles (13). The second isoenzyme, COX-2, was initially
identified as a member of a group of genes in which expression is
elevated in chicken fibroblasts after transformation by Rous sarcoma
virus (46). The mouse homologue of COX-2 gene was later identified as
one of several inducible proteins from 3T3 fibroblasts (26). The major
difference between these isoenzymes appears to be their regulation.
COX-1 is expressed constitutively in most cells and is probably
responsible for the production of prostaglandins under physiological
conditions (47). In contrast, COX-2 is induced in cells by
proinflammatory stimuli such as mitogens (23), cytokines (13, 5, 47),
and bacteria lipopolysaccharide (27, 34). One of the cytokines that
induces COX-2 is IL-1. IL-1
has been shown to induce the
expression of COX-2 in a variety of cell types, including airway smooth
muscle cells (15, 16, 31, 35, 47, 48).
PGE2 is the primary prostanoid
produced by airway smooth muscle, and IL-1
increases its synthesis
(39, 49).
In some systems, prostanoids generated as a result of COX-2 induction
appear to mediate the effects of IL-1. For example, IL-1
activates endothelial cells of the brain vasculature to induce COX-2
expression, which is possibly responsible for the elevated level of
prostaglandins important during fever (10). The enhancement of the
production of PGE2 by human dermal
fibroblasts and sinovial fibroblast cells by IL-1
may play an
important role in the process of the inflammatory response and the
persistence of the inflammation in the chronic inflammatory diseases
(9).
We have previously reported that IL-1 decreases the ability of
cultured HASM cells to respond to
-agonists (45). Responses to
isoproterenol (Iso) were assessed by measuring cAMP formation and by
measuring changes in cytoskeletal stiffness by magnetic twisting
cytometry (22, 45, 51, 52). Using this technique, we have previously
shown that HASM cells decrease their stiffness in response to any of a
panel of bronchodilator agonists known to cause relaxation of airway
smooth muscle, whereas stiffness increases in response to contractile
agonists known to increase cytosolic calcium concentrations in these
cells (22, 45). To determine whether COX products are involved in this
effect of IL-1
, we first examined the effect of nonselective
[indomethacin (Indo)] and selective (NS-398) COX-2 inhibitors on
IL-1
-induced changes in HASM cell responses to the
-agonist Iso.
To confirm previous reports of COX-2 induction by IL-1
in our system
and to verify the efficacy of the inhibitors used, we also examined the
effect of IL-1
on PGE2 release
from HASM cells and measured IL-1
-induced COX-2 expression by
Western blot analysis. Because our results indicated that COX products
are implicated in IL-1
-induced changes in the response to
-agonists, we examined the effect of prolonged pretreatment with
PGE2 on HASM cell stiffness in response to Iso. Because our results also indicated that
induction of COX-2 by IL-1
appears to be required for the cytokine
to exert its effect on
-adrenergic responsiveness, we examined the
effect of the protein synthesis inhibitor cycloheximide on
IL-1
-induced changes in HASM cell stiffness and cAMP formation
responses to Iso.
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METHODS |
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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 trachea as previously described (38). Briefly, a segment of trachea just proximal to the carina was dissected under sterile conditions, and the trachealis muscle was isolated. Approximately 1 g of wet tissue was obtained from each donor. The tissue was minced, centrifuged, and resuspended in 10 ml of buffer containing 0.2 mM CaCl2, 640 units of collagenase, 10 mg of soybean trypsin inhibitor, and 10 U/ml elastase. Tissue was incubated with enzymes for 90 min in a shaking water bath at 37°C. The cell suspension was then filtered through 127-µm Nytex mesh, and the filtrate was washed with an equal volume of cold Ham's F-12 medium supplemented with 10% fetal bovine serum (FBS). Cells were plated in plastic flasks at 1.0 × 104 cells/cm2 in Ham's F-12 medium supplemented with 10% FBS, 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). Medium was replaced every 3-4 days. 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 insulin and 5 µg/ml tranferrin 24 h before use. Cells from 13 different donors studied in passages 4-7 were used in the studies.
Experimental protocol. For experiments
in which we examined the effect of the COX inhibitor Indo on
IL-1-induced changes in cell stiffness responses to Iso or
dibutyryl-cAMP (DBcAMP), four flasks of HASM cells from the same
passage of the same donor cells were serum deprived and hormone
supplemented. Approximately 10 h later, two flasks were treated with
Indo (10
6 M). Two hours
later, IL-1
was added to one of the flasks treated with Indo and
also to an untreated flask. Eighteen hours later, cells were harvested
by brief exposure to trypsin and EDTA, resuspended in serum-free medium
with or without IL-1
and Indo, 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. Cumulative concentration-response curves to Iso or DBcAMP 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 agonist
Iso or DBcAMP 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 obtained again. This procedure was repeated with
increasing concentration of the agent. The concentration ranges used
were as follows: Iso, 10
8
to 10
5 M; DBcAMP,
10
4 to 3 × 10
3 M. Only one agonist was
studied per well. The effects of the selective COX-2 inhibitor NS-398
(10
5 M) and the protein
synthesis inhibitor cycloheximide (10 µg/ml) were examined in an
identical fashion. Details of the methodology for magnetic twisting
cytometry are found in Magnetic twisting cytometry.
We also examined the effect of exogenous
PGE2 on cell stiffness responses
to Iso. For these experiments, two flasks of confluent serum-deprived,
hormone-supplemented HASM cells were used. One flask was treated with
PGE2
(107 M), and one served as
a control. Twenty hours later, the cells were harvested and used to
obtain cell stiffness dose responses to Iso as described above.
PGE2 was washed from the cells
~20 min before the initiation of cell stiffness measurements. The dose of PGE2
(10
7 M) was chosen for the
following reason. In some experiments, we harvested
supernatant of the IL-1
-treated cells just before harvesting of the
cells for plating in Removawells. In those supernatants, which we
believed were representative of what the cells were continuously exposed to during their IL-1
treatment period, the
PGE2 concentration was
10
7 M or higher, depending
on the donor line used.
For experiments in which we examined the effects of Indo, NS-398, and
cycloheximide on IL-1-induced changes in cAMP formation, cells were
treated identically as in the cell stiffness experiments, except that,
once harvested, the cells were plated at
105/well in 24-well
plates. cAMP generation in response to Iso was measured 4 h after plating, at which time the medium was replaced with 0.5 ml of
phosphate-buffered saline (PBS) containing 0.1 mM IBMX (to prevent
degradation of cAMP by phosphodiesterases), ascorbic acid (300 µM; to
prevent oxidation of Iso), Indo
(10
6 M), NS-398
(10
5 M), and cycloheximide
(10 µg/ml). Thirty minutes later, Iso
(10
7 to
10
5 M) was added to the
cells. Only one concentration of agonist was used per well. Some cell
wells were used to measure basal cAMP release and did not receive Iso.
Cells were incubated for an additional 10 min and then were removed
from the incubator and placed on ice. Ice-cold ethanol (1 ml) was added
to lyse the cells. The lysate was centrifuged at 2,000 g for 15 min at 4°C, and the
supernatant was removed, evaporated to dryness, and stored at
70°C. On the day of the assay, the cell lysate was
resuspended in 200-500 µl of assay buffer. Some samples were
diluted to ensure that they fell within the limits of the standard
curve. cAMP was assayed using a Rainen cAMP
125I radioimmunoassay kit (New
England Nuclear).
We examined the effect of IL-1 on
PGE2 release from HASM cells in
the presence or absence of Indo or NS-398. For these experiments, the
cells were treated with IL-1
and COX inhibitors and harvested as
described in Experimental protocol. The cells
were plated at 105 cells/well in
24-well plates. COX inhibitors and IL-1
were readded to the cells,
and the cells were incubated for 4 h. The medium was then replaced with
0.5 ml of fresh medium. Indo and NS-398 were readded to each treated
well. The effect of IL-1
on COX activity was examined by adding AA
(10
5 M) to some of the cell
wells at this time. Fifteen minutes later, the supernatants were
harvested and stored at
20°C until subsequent assay with a
PGE2 enzyme immunoassay kit
(Cayman Chemical, Ann Arbor, MI). The antibody to
PGE2 had <1% cross-reactivity
to 6-keto-PGF1
and <0.01% to
TxB2 and other prostaglandins
according to the manufacturer's specifications.
In other experiments, we assessed the effect IL-1 on the expression
of COX-2 by Western blotting. For these experiments, two flasks of HASM
cells from the same passage of the same donor cells were grown to
confluence and serum deprived and 10 h later were treated with IL-1
(20 ng/ml) or control medium. Approximately 24 h later, cell medium was
removed, and cells were washed with PBS. HASM cells were harvested by
brief exposure to 0.25% trypsin and 1 mM EDTA and then were washed
with Ham's F-12 medium. Cells were incubated with 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% sodium
dodecyl sulfate (SDS), 1% Triton X-100, 0.2 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 1 µg/ml pepstatin, and
10
2 U/ml aprotinin]
and then were passed through a 25.625-gauge needle. Cell lysates
were clarified by centrifugation at 4,000 g for 10 min to remove debris. Protein
concentration of the supernatant was determined with Bio-Rad dye
reagent (Bio-Rad, Richmond, CA).
Western blotting. Supernatant of cell
lysates from control and IL-1-treated cells were mixed with equal
volumes of loading buffer [0.062 M Tris · HCl
(pH 6.8), 10% glycerol, 2% SDS, 5%
-mercaptoethanol, and 0.01%
(wt/vol) bromphenol blue] and then were boiled for
5 min. Solubilized proteins (100 µg/lane) were separated by
SDS-polyacrylamide gel electrophoresis (125 V, 2 h) on 12%
Tris-glycine gel (Novex, San Diego, CA) under nonreducing conditions
and transferred electrophorically (16 V, 1 h) to a nitrocellulose
membrane in transfer buffer (Pierce, Rockford, IL). Recombinant COX-2
(Oxford Biomedical Research, Oxford, MI) was also loaded on the gel as
a positive control. After transfer to nitrocellulose, the blot was
incubated in blocking solution (10 mM Tris saline buffer containing 150 mM NaCl, 0.1% Tween 20, and 4% bovine serum albumin) and then primed
with rabbit polyclonal antibody raised to COX-2 protein (dilution of
1:1,000; Oxford Biomedical Research) for 2 h. The blot was then
incubated with a goat anti-rabbit IgG linked to peroxidase (dilution
1:20,000; Calbiochem, La Jolla, CA) for 1 h and then visualized by
light emission on film with enhanced chemiluminescent substrate
(Pierce). The band visualized at ~70 kDa was quantified using a laser
densitometer. Band density values were expressed in arbitrary optical
density units.
Magnetic twisting cytometry. Details of the magnetic twisting cytometry technique have been previously described (51, 52). The principle of magnetic twisting cytometry is as follows: ferromagnetic beads are first coated with a prescribed ligand [Arg-Gly-Asp (RGD)-containing peptides in this case] and then bound to the surface of the cells through the corresponding receptor system (integrins in this case). Individual wells containing adherent cells bound to ligand-coated ferromagnetic beads are placed into the magnetic twisting chamber, are maintained in defined medium, and are held at 37°C using a circulating water bath that is built into the system. The cell well is oriented horizontally, rotated around the vertical axis (10 Hz), and shielded with four external superalloy cylinders to minimize the disturbances from external magnetic field and hence the signal-to-noise ratio. The attached beads are magnetized with a brief 1,000-G pulse so that their magnetic moments are aligned in one direction, parallel to the surface on which the cells are plated. The magnetic field vector generated by the beads in the horizontal direction is measured by the in-line magnetometer. The sensitivity of the magnetometer is 0.1 nT (1,000 G = 0.1 mT). Subsequently, a much smaller magnetic field is applied in the vertical direction to the first applied torque (or twisting stress) using coils 1.9 cm in diameter. This twisting stress causes the beads to rotate as would a compass needle, but bead rotation is opposed by reaction forces developed within the cytoskeleton to which the beads are bound through the integrin molecules. Magnetic twisting cytometry measures the applied twisting stress and the resulting angular rotation of the magnetic bead and expresses the ratio as cell stiffness. Bead rotation increases with the strength of the applied twisting field and is inversely proportional to the cell's resistance to shape distortion.
Ferromagnetic beads (4.5 µm in diameter,
Fe3O4)
were coated with synthetic RGD-containing peptide (Peptite 2000, Telios) by incubation overnight at 4°C with 50 mg · ml
peptide1 · mg
beads
1 in carbonate buffer
(pH 9.0). Beads were then washed two times in serum-free medium
containing 1% BSA. Approximately 5 × 104 beads were added to wells
containing 20,000 HASM cells and were allowed to incubate 20 min at
37°C to permit binding of beads to cells through integrin receptors
that recognize the RGD sequence. Nonadherent beads were then removed by
washing the cells with serum-free medium, and the wells containing the
cells were placed within the magnetometer and maintained at 37°C.
Magnetic twisting cytometry was used to characterize HASM cell
mechanics on the basis of three variables: angular strain, applied
shear stress (), and apparent stiffness (51, 52). The
,
which is directly proportional to the strength of the magnetic twisting
field, was defined as torque per bead volume and is expressed as dynes
per square centimeter. In this study, we set the strength of the
twisting field so that, initially, a
of 80 dyn/cm2 was applied to the cells.
Because the angle between the twisting field and the bead's magnetic
moment vector decreases as the beads rotate toward the twisting field,
decreases with angular strain (and time). The
was calibrated by
placing beads in known viscosity standard, and thus a relationship
between the twisting field and applied stress was obtained (52).
Average angular strain was defined as the fractional rotation of beads
during a 1-min twist and was expressed as the angle (
) between the
bead's original magnetic moment and the magnetic moment 1 min after
the twisting field was applied. Therefore,
= cos
1
(M80/M20)
where M80 is the magnetic field
measured at the end of the 1-min twist (i.e., 80 s after the original
pulse) and M20 is the magnetic
field 20 s after the original pulse (i.e., just before the application
of the twisting field). Apparent stiffness is defined as the ratio
between
and shear strain, both measured 60 s after the twisting
field is applied. Thus stiffness =
ocos
/
, where
o is the magnitude of the
stress at the instant when it is applied. Stiffness is analogous to the
shear modulus of the cell and is also expressed in dynes per square
centimeter. The magnetic field obtained after pulsing, but without the
second imposed twisting field, was used to control for the normal decay of the magnetic field induced by random movement of the beads. This
decay is usually very small when compared with the bead's rotation due
to twisting and is usually <3% of the original magnetic field over
the 2.5-min period during which measurements are made.
Reagents. Tissue culture reagents and
drugs used in this study were obtained from Sigma (St. Louis, MO), with
the exception of amphotericin B and trypsin-ETDA solution which were
purchased from GIBCO (Grand Island, NY), IL-1 which was obtained
from Genzyme (Cambridge, MA), and
PGE2
(10
2 M in DMSO) which was
obtained from BioMol (Philadelphia, PA). DBcAMP was dissolved at
10
1 M in distilled water,
frozen in aliquots, and diluted appropriately in medium on the day of
use. Iso (10
1 M in
distilled water) was made fresh each day. Because Iso is rapidly
oxidized, dilution of Iso in medium was made immediately before
addition to the cells. Indo and cycloheximide were dissolved in ethanol
and diluted in medium on the day of use. NS-398 was dissolved in DMSO
at 10
2 M and diluted in
medium.
Statistics. The effect of the
inhibitors (cycloheximide, Indo, and NS-398) on IL-1-induced changes
in cell stiffness responses to Iso was examined by repeated-measures
ANOVA, using treatment (control, IL-1
, inhibitor, and IL-1
+ inhibitor) and experimental day as main effects. Follow-up
t-tests were used to determine where
the treatment effect lay. Repeated-measures ANOVA was also used to
determine the effect of pretreatment with
PGE2 on Iso-induced changes in
cell stiffness. Changes in cAMP formation induced by Iso were expressed
as percent of basal cAMP and log transformed, and ANOVA was performed
using experimental day and drug treatment (control, IL-1
, inhibitor,
and IL-1
+ inhibitor) as main effects. Changes in basal and
AA-stimulated PGE2 release induced
by IL-1
were examined by repeated-measures ANOVA, using treatment
and experimental day as main effects. A
P value <0.05 was considered significant.
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RESULTS |
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We have previously reported that IL-1 decreases the ability of
cultured HASM cells to respond to
-agonists. To determine whether
COX products are involved in this effect of IL-1
, we first examined
the effect of nonselective (Indo) and selective (NS-398) COX-2
inhibitors on IL-1
-induced changes in HASM cell responses to Iso.
Results are shown in Fig. 1. Neither Indo,
IL-1
, nor their combination had any effect on baseline cell
stiffness. Baseline stiffness averaged 108.03 ± 7.85 dyn/cm2 in control, 122.59 ± 12.01 dyn/cm2 in IL-1
-treated
cells, 116.02 ± 9.2 dyn/cm2 in
Indo-treated cells, and 125.84 ± 7.21 dyn/cm2 in IL-1
+ Indo-treated
cells. Acute treatment with Indo (5-30 min) also did not change
the baseline cell stiffness. In control cells, Iso caused a
dose-related decrease in cell stiffness (Fig. 1). Repeated-measures
ANOVA indicated a significant effect of drug treatment
(P < 0.001) on Iso-induced changes
in cell stiffness. Follow-up analysis indicated that the treatment
effect lay in the response to IL-1
, which reduced the capacity of
Iso to decrease cell stiffness, as previously described. Compared with
control treatment, Indo caused a more pronounced decrease in cell
stiffness at 10
8 and
10
5 M Iso. Importantly,
Indo abolished the effect of IL-1
on Iso-induced changes in cell
stiffness.
|
We have previously reported that, whereas IL-1 significantly
inhibits the ability of HASM cells to decrease their stiffness in
response to Iso, it has no effect on cell stiffness responses to DBcAMP
(45), suggesting that the effect of IL-1
lies upstream of the
effects of activation of protein kinase A (PKA) by cAMP. To ensure that
the observed effects of Indo (Fig. 1) were not related to any
downstream effects of the drug, we also examined the effects of Indo
and IL-1
on cell stiffness responses to DBcAMP. DBcAMP induced a
concentration-related decrease in cell stiffness in all four groups of
cells, and an effect of neither IL-1
nor Indo was observed (Fig.
2).
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To determine whether it was COX-2-induced prostanoid formation that was
important in IL-1-induced changes in
-adrenergic responses of
HASM cells, we examined the effect of the more specific COX-2 inhibitor
NS-398 (10
5 M, 24 h) on
IL-1
-induced changes in cell stiffness responses to Iso (Fig.
3). Neither NS-398, IL-1
, nor their
combination had any effect on baseline cell stiffness. Baseline
stiffness averaged 87.6 ± 2.9 dyn/cm2 in control cells, 85.5 ± 1.1 dyn/cm2 in
NS-398-treated cells, 85.4 ± 5.9 dyn/cm2 in IL-1
-treated cells,
and 97.4 ± 5.3 dyn/cm2 in
NS-398 + IL-1
-treated cells [not significant (NS)].
Repeated-measures ANOVA indicated a significant effect of drug
treatment on cell stiffness responses to Iso
(P < 0.001; Fig. 3). Follow-up
analysis indicated that, as previously observed, the treatment effect
lay in the response to IL-1
(20 ng/ml), which reduced the capacity of Iso to decrease cell stiffness (P < 0.001). Compared with control cells, NS-398 alone had no effect on
cell stiffness responses to any concentration of Iso. However, as with
Indo treatment, NS-398 abolished the effects of IL-1
on cell
stiffness responses to Iso.
|
We have previously reported that IL-1 causes a slight (~30%)
increase in basal cAMP formation but substantially decreases the
ability of Iso to stimulate cAMP in HASM cells in culture (45). To
determine whether prostanoids might be involved in these effects and,
if so, whether the prostanoids were derived from COX-2, we examined the
effect of Indo and NS-398 on changes in basal and Iso-stimulated cAMP
formation induced by IL-1
. Figure 4
shows the effect of Indo and NS-398 on basal cAMP formation in control
and IL-1
(2 ng/ml for 22 h)-treated cells. In both experiments,
ANOVA indicated a significant treatment effect
(P < 0.05 for NS-398 experiment and
P < 0.001 for Indo experiment). IL-1
increased basal cAMP formation
(P < 0.05). Indo reduced basal cAMP in both control (P < 0.05) and IL-1
-treated cells (P < 0.001) such that there was no
effect of IL-1
on basal cAMP in Indo-treated cells. NS-398 had no
significant effect on basal cAMP in control cells but reduced basal
cAMP formation in IL-1
-treated cells
(P < 0.02).
|
The effects of Indo and NS-398 on IL-1-induced changes in
Iso-stimulated cAMP formation are shown in Fig.
5. As shown, Iso (10
7 and
10
6 M) caused approximately
twofold and threefold increases in cAMP formation over baseline in
control cells, respectively. In both experiments, ANOVA indicated a
significant drug treatment effect (P < 0.001 for Indo experiment and
P < 0.03 for NS-398
experiment). Follow-up analysis indicated that, in both cases, the
treatment effect lay in the IL-1
-treated cells, in which cAMP
formation was reduced relative to the controls
(P < 0.01). Neither Indo nor NS-398
alone had any effect on Iso-stimulated cAMP formation. However, Indo
blocked the effect of IL-1
such that Iso
(10
6 and
10
7 M)-stimulated cAMP
formation in cells treated with IL-1
+ Indo was not different from
that in cells treated with Indo alone. NS-398 also blocked the effect
of IL-1
such that Iso
(10
6 M)-stimulated cAMP
formation was significantly different in cells treated with IL-1
+ NS-398 compared with cells treated with IL-1
, and Iso
(10
7 M)-stimulated cAMP
formation was not different in cells treated with IL-1
+ NS-398
compared with cells treated with NS-398 alone.
|
To verify the efficacy of the inhibitors used and to confirm, in our
system, previous reports of increased
PGE2 release by IL-1, we also
examined the effect of IL-1
(20 ng/ml for 22 h) on basal and AA (10 µM)-stimulated release of PGE2
by HASM cells in the presence or absence of the COX inhibitors Indo and
NS-398 (Fig. 6). Compared with control
cells, IL-1
caused a significant increase in both basal
PGE2 release (15-fold increase)
and in AA-stimulated PGE2 release
(10-fold increase; P < 0.0001 in
each case). Indo markedly reduced basal and AA-stimulated
PGE2 release in both control and
IL-1
-treated cells (P < 0.0001 in
each case). NS-398 caused a marked and significant reduction in basal
and AA-stimulated PGE2 release in
IL-1
-treated cells (P < 0.0001 in
each case), but NS-398 had no significant effect on AA-stimulated PGE2 release in control cells.
Western blot analysis confirmed the induction of COX-2 by IL-1
(20 ng/ml for 24 h; Fig. 7). In four different
IL-1
-stimulated HASM cell extracts, the COX-2 antibody revealed one
band corresponding to molecular mass of 70 kDa, as described by others
(15, 39, 50). In contrast, in control cells taken from the same
passages of the same donor cells, no band corresponding to molecular
mass of 70 kDa was observed.
|
|
Because our results suggested that prostanoid formation was involved in
IL-1-induced changes in
-adrenergic responses of HASM cells, we
examined the effect of exogenous
PGE2
(10
7 M for 22 h) on changes
in cell stiffness responses to Iso.
PGE2 was washed from the cells
before the initiation of cell stiffness measurements. Baseline
stiffness averaged 126.52 ± 16.4 dyn/cm2 in control and 125.6 ± 7.9 dyn/cm2 in
PGE2-treated cells (NS).
PGE2 caused a significant
reduction in the cell stiffness response to Iso
(P < 0.01 by repeated-measures ANOVA; Fig. 8). Follow-up
t-tests indicated that the effect of PGE2 was observed at all
concentrations of Iso examined.
|
Because our results suggested that IL-1-induced COX-2 synthesis was
important in IL-1
-induced changes in
-adrenergic responses of
HASM cells, we examined the effect of the protein synthesis inhibitor
(cycloheximide: 10 µg/ml for 24 h) on IL-1
-induced changes in HASM
cell stiffness responses to Iso (Fig. 9).
In contrast to experiments described above in which cell treatment did
not alter baseline cell stiffness, baseline stiffness was significantly decreased by treatment with cycloheximide
(P < 0.02 by repeated-measures ANOVA). Baseline stiffness averaged 123.4 ± 11.6, 132.31 ± 8.4, 90.6 ± 5.9, and 97.5 ± 4.7 dyn/cm2 in control cells, cells
treated with IL-1
alone, cells treated with cycloheximide alone, and
cells treated with cycloheximide + IL-1
, respectively. A possible
explanation is that cycloheximide might influence the formation of
molecules important for cell adhesion to the extracellular matrix and
thereby influence basal cell stiffness (22). As previously observed,
IL-1
(20 ng/ml) reduced the capacity of Iso to decrease cell
stiffness (P < 0.001). Cycloheximide
blocked this effect of IL-1
such that there was no significant
difference in cell stiffness responses to Iso between control and
IL-1
-treated cells that had been pretreated with the protein
synthesis inhibitor.
|
The effects of cycloheximide on IL-1-induced changes in Iso
(10
6 M)-stimulated cAMP
formation are shown in Fig. 10. ANOVA
indicated a significant drug treatment effect
(P < 0.0001). Follow-up analysis indicated that the treatment effect lay in the IL-1
-treated cells, in which cAMP formation was reduced relative to the controls
(P < 0.003). Cycloheximide alone had
no effect on Iso-stimulated cAMP formation. However, cycloheximide
blocked the effect of IL-1
such that Iso-stimulated cAMP formation
in cells treated with IL-1
+ cycloheximide was not different from
that in cells treated with cycloheximide alone.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our results indicate that Indo and NS-398 inhibited the effects of
IL-1 on cell stiffness and cAMP responses to
-agonists (Figs. 1
and 3-5), whereas pretreatment of HASM cells with
PGE2 mimicked the effects of
IL-1
(Fig. 8). IL-1
significantly increased PGE2 release, and both
nonselective (Indo) and selective (NS-398) COX-2 inhibitors (Fig. 6)
prevented this augmented release, suggesting that it was the result of
the increased COX-2 expression induced by IL-1
(Fig. 7). A protein
synthesis inhibitor prevented the IL-1
-induced changes in
-adrenergic responses of HASM cells (Figs. 9 and 10). Taken
together, the results support the hypothesis that prostanoids released
as a result of increased COX-2 expression contribute to the
-adrenergic hyporesponsiveness induced in HASM cells by IL-1
.
We have previously reported that IL-1 decreased the ability of HASM
cells to both reduce their stiffness and to increase cAMP formation in
response to Iso (45). We have also shown that IL-1
decreases cell
stiffness and cAMP generation responses to PGE2 (45). In contrast, HASM cell
responses to the cell-permeant cAMP analog DBcAMP and to forskolin,
which directly activates adenylyl cyclase, were not altered. The latter
results suggest that neither changes in PKA activation and responses
nor changes in adenylyl cyclase expression contributed to the effects
of IL-1
. Pretreatment with IL-1
had no significant effect on
2-adrenoceptor number, nor was
there any significant effect of IL-1
on
Gs expression. Furthermore, the
effect of IL-1
was not altered by pretreatment of cells with
pertussis toxin, indicating that the effect of IL-1
was not mediated
by changes in Gi. We suggested
that the
-adrenergic hyporesponsiveness induced by IL-1
was
mediated by uncoupling of
-receptors from
Gs-induced activation of adenylyl
cyclase. In this report, we have shown that COX inhibitors block the
ability of IL-1
to reduce cell stiffness responses of HASM cells to
Iso, suggesting that prostanoids contribute to the decreased
responsiveness to
-agonists that is induced by IL-1
. A unifying
explanation for these observations is outlined schematically in Fig.
11. IL-1
induces COX-2 expression and
markedly increases the production of
PGE2 by HASM cells. The release of
PGE2 is sufficient to increase cAMP formation by the cells activating PKA. PKA causes phosphorylation of the
-receptor, inhibiting its ability to bind and activate Gs and resulting in decreased
ability of Iso to induce responses.
|
In support of this hypothesis, our results indicate that IL-1 causes
a marked increase in PGE2
formation in HASM cells. Basal and AA-stimulated
PGE2 release increased ~15- and
10-fold, respectively, in response to IL-1
. The observation that the
COX-2 inhibitor NS-398 blocked the increase in basal and AA-stimulated
PGE2 release (Fig. 6) in
conjunction with the observation that IL-1
induced expression of
COX-2 (Fig. 7) supports the hypothesis that the increase in
PGE2 release results from
increased COX-2 activation. NS-398 also blocked the basal release of
PGE2 but had no effect on
AA-stimulated release in control cells. These results may suggest that
NS-398 is a weak inhibitor of COX-1 inhibitor, at least at very low
concentrations of the substrate. Alternatively, it is possible that
NS-398 interferes with the assay of low concentrations of
PGE2. Our results are consistent
with those of Pang and Knox (39), who demonstrated that 1 and 10 ng/ml
IL-1
increased PGE2 release by
~10-fold and induced COX-2 expression in HASM cells after 24 h of
treatment. Vigano et al. (49) have shown that IL-1
(1-50 U/ml)
induced COX-2 expression and increased
PGE2 formation in HASM cells
sevenfold after 6 h of treatment. Other investigators
have reported that COX-2 expression is also induced by phorbol esters
and by tumor necrosis factor-
in a human bronchial smooth muscle
cell line and in rat tracheobronchial smooth muscle cells (1, 39, 47).
The observation that cycloheximide prevented IL-1-induced changes in
both cell stiffness and cAMP responses to
-agonists indicates that
this effect of IL-1
is dependent on de novo protein synthesis. These
experiments do not allow us to pinpoint the precise protein whose
synthesis is important to the effect of IL-1
. However, we believe
that inhibition of COX-2 synthesis is the basis for the efficacy of
cycloheximide. We have also observed that
anti-inflammatory steroid treatment, such as dexamethasone, which
inhibits IL-1
-induced COX-2 expression and
PGE2 release (5, 35, 49), also
reverses the effect of IL-1
on
-adrenergic responses of HASM
cells (37).
In support of the hypothesis outlined in Fig. 11, our results reported
both here (Fig. 4) and previously (45) indicate that IL-1 causes a
small but significant increase in basal cAMP formation in HASM cells.
In airway smooth muscle cells,
PGE2, like Iso, results in cAMP
formation (18, 45), and it is likely that the small increase in basal
cAMP induced by IL-1
(Fig. 4) is the result of increased
PGE2 formation by these cells;
both Indo and NS-398 prevented IL-1
from augmenting basal cAMP (Fig.
4). IL-1
has also been shown to induce cAMP formation in other types (25, 36, 44). In addition, IL-1
-induced cAMP formation was also
associated with a marked increase in prostacyclin production in human
vascular smooth muscle cells and 3T3 fibroblasts (4, 8).
PGE2 has the capacity to induce
-adrenergic hyporesponsiveness in HASM cells. We observed reduced
cell stiffness responses to
-agonists after prolonged exposure to
PGE2 (Fig. 6). This occurred
without any effect of PGE2 on
baseline cell stiffness, suggesting that the
PGE2 had been effectively washed
from the cells at the time of Iso application. Similarly, Hall et al.
(18) have reported that the same concentration of
PGE2 (10 µM) produced a 44%
attenuation of subsequent cAMP responses to Iso
(10
8 to
10
5 M).
PGE2 causes cAMP formation, and it
has been shown that activation of PKA by cAMP can phosphorylate the
2-adrenoceptor and induce desensitization of the receptor (6). The
2-adrenoceptor possesses sites
for PKA phosphorylation in the COOH-terminal portion of its third
intracellular loop and in its cytoplasmic tail. These regions are
important in the receptor's interaction with
Gs and appear to be involved in
the heterologous densitization of the receptor by agonists that induce
cAMP formation (12, 21, 29, 28, 41). In contrast, phosphorylation of
the receptor by
-adrenergic receptor kinase, which permits binding
to
-arrestin, requires receptor occupation with ligand and appears
to be important in homologous desensitization (30, 43).
HASM cells produce other prostanoids such as
PGI2,
TxA2, and
PGF2 in response to IL-1
.
Although the amounts released are smaller than for
PGE2 (39),
PGI2
, like
PGE2 and Iso, can act on cell
surface receptors that couple to
Gs protein and may also contribute
to the increased basal cAMP induced by IL-1
. Another potential
mechanism whereby prostanoids might uncouple
-receptors from
Gs-induced activation of adenylyl
cyclase is phosphorylation of the
-receptor or
Gs by protein kinase C (7, 24, 40,
42). Both TxA2 and
PGF2 act on cell surface receptors that couple to Gq and activate
phospholipase C. Phospholipase C can cause diacylglycerol formation and
activation of protein kinase C (20).
Cytoskeletal stiffness, as defined here, is a measure of the ability of
cells to resist distortions of shape in response to shear stresses
applied through magnetic beads linked to the cytoskeleton via integrin
receptors. Actin and myosin form part of the cytoskeleton, and
cross-bridge formation appears to increase cytoskeletal stiffness, since application of a variety of contractile agonists to HASM cells
results in increased stiffness, whereas bronchodilating agonists reduce
stiffness (22, 45). Changes in cell adhesion to the
extracellular matrix can also influence cytoskeletal stiffness (51),
and we have previously reported that HASM cells plated on high-density
collagen are more spread out and develop more pronounced decreases in
cell stiffness in response to Iso than cells plated on low-density
collagen matrix (22). Although it is theoretically possible that COX
inhibitors might influence cell adhesion and thereby influence cell
stiffness responses to Iso, we believe that such an explanation is very
unlikely. First, changes in cell adhesion influence basal cell
stiffness (22, 51), but neither IL-1, COX inhibitors, nor their
combination altered baseline cell stiffness in these experiments.
Second, such changes would have been expected to alter cell stiffness responses to any dilating agonist, but responses to DBcAMP were unaffected either by IL-1
or by Indo. Finally, the observation that
COX inhibitors also reversed the effects of IL-1
on Iso-induced cAMP
formation suggests that the effect of the prostanoids lies upstream of
the mechanical responses to the PKA activation.
In summary, our results indicate that IL-1 significantly increases
PGE2 release by HASM cells as a
result of increased COX-2 expression. The increased
PGE2 formation appears to
contribute to
-adrenergic hyporesponsiveness perhaps by heterologous
desensitization of the
-receptor.
-Adrenergic hyporesponsiveness
is a characteristic feature of human asthma. Although we do not know
that IL-1
contributes to this phenomenon in asthma, the observations
that IL-1
is produced in greater amounts in airways of asthmatic
than of normal subjects and that IL-1
can decrease responses of HASM
cells to
-agonists suggest that it may be important. Understanding
the role of prostanoids in the mechanism of action of cytokines in
leading to
-adrenergic receptor dysfunction may provide new avenues
for improvements in the safety and efficacy of these agents.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dale Youngkin, Joseph Abraham, and Hadi Danaee for technical assistance.
![]() |
FOOTNOTES |
---|
This study was supported by National Heart, Lung, and Blood Institute Grants HL-55301 and HL-33009. J. Laporte was supported by a fellowship from the Medical Research Council of Canada and the Canadian Lung Association.
Address for reprint requests: J. Laporte, Physiol. Prgm., Harvard School of Public Health, 665 Huntington Ave., Boston, MA 02115.
Received 3 November 1997; accepted in final form 19 May 1998.
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