Mechanisms underlying TNF-
effects on agonist-mediated
calcium homeostasis in human airway smooth muscle cells
Yassine
Amrani,
Vera
Krymskaya,
Christopher
Maki, and
Reynold A.
Panettieri Jr.
Pulmonary and Critical Care Division, Department of Medicine,
University of Pennsylvania Medical Center, Philadelphia, Pennsylvania
19104
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ABSTRACT |
We have previously shown that tumor necrosis
factor (TNF)-
, a cytokine involved in asthma, enhances
Ca2+ responsiveness to
bronchoconstrictor agents in cultured human airway smooth muscle (ASM)
cells. In the present study, we investigated the potential mechanism(s)
by which TNF-
modulates ASM cell responsiveness to such agents. In
human ASM cells loaded with fura 2, TNF-
and interleukin (IL)-1
significantly enhanced thrombin- and bradykinin-evoked elevations of
intracellular Ca2+. In
TNF-
-treated cells, Ca2+
responses to thrombin and bradykinin were 350 ± 14 and 573 ± 93 nM vs. 130 ± 17 and 247 ± 48 nM in nontreated cells,
respectively (P < 0.0001).
In IL-1
-treated cells, the Ca2+
response to bradykinin was 350 ± 21 vs. 127 ± 12 nM in
nontreated cells (P < 0.0001). The
time course for TNF-
potentiation of agonist-induced
Ca2+ responses requires a minimum
of 6 h and was maximum after 12 h of incubation. In addition,
cycloheximide, a protein synthesis inhibitor, completely blocked the
potentiating effect of TNF-
on
Ca2+ signals. We also found that
TNF-
significantly enhanced increases in phosphoinositide (PI)
accumulation induced by bradykinin. The percentage of change in PI
accumulation over control was 115 ± 8 to 210 ± 15% in control
cells vs. 128 ± 10 to 437 ± 92% in TNF-
-treated cells for 3 × 10
9 to 3 × 10
6 M bradykinin. The PI
turnover to 10 mM NaF, a direct activator of G proteins, was also found
to be enhanced by TNF-
. The percentage of change in PI accumulation
over control increased from 280 ± 35% in control cells to 437 ± 92% in TNF-
-treated cells. Taken together, these results show
that TNF-
can potently regulate G protein-mediated signal
transduction in ASM cells by activating pathways dependent on protein
synthesis. Our study demonstrates one potential mechanism underlying
the enhanced Ca2+ response to
bronchoconstrictor agents induced by cytokines in human ASM cells.
asthma; bronchial hyperreactivity; cytokines; inflammation; hyperresponsiveness; tumor necrosis factor-
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INTRODUCTION |
TUMOR NECROSIS FACTOR (TNF)-
may be an important
mediator in airway inflammatory diseases such as asthma and
bronchiolitis (20). Increased levels of TNF-
have been found in the
bronchoalveolar fluid of symptomatic asthmatic patients (6), and
administration of TNF-
to healthy volunteers (40) and animals (21,
42) induces a bronchial hyperreactivity, a characteristic feature of
asthma. The role of TNF-
in modulating airway hyperresponsiveness in
vivo has recently been shown using Ro-45-2081, a potent TNF receptor
(TNFR) antagonist (33). This receptor antagonist prevented allergen-induced bronchial hyperreactivity. These data provide evidence
for an implication for TNF-
in asthma. The mechanism(s), however, by
which TNF-
induces bronchial hyperreactivity remains unknown.
In isolated airways, TNF-
or interleukin (IL)-1
alters the
contractile response of tracheal smooth muscle to cholinergic agonists
(30) as well as of tracheal smooth muscle relaxation to
-adrenergic
stimulation (13, 44, 45). In cultured human airway smooth muscle (ASM)
cells, we have shown that TNF-
significantly enhanced the
Ca2+ responsiveness to
bronchoconstrictor agents, i.e., bradykinin and carbachol, and induced
cell adhesion molecule expression (1, 3, 4, 23). In addition, we showed
that TNF-
elicited its cellular effects by activation of the p55
TNFR subtype (TNFRp55) (4). The TNFRp55 has been shown to
mediate most effects of TNF-
, such as apoptosis (19, 27),
proliferation of fibroblast, and adhesion molecule expression (reviewed
in Ref. 39). TNF-
effects mediated by activation of the p75 TNFR
subtype (TNFRp75) appear to be more restricted; TNFRp75 is involved in
T-cell development, activation (11), and apoptosis (17). Recent studies
have revealed several downstream signaling events induced by TNFRp55
activation, namely activation of GTP-binding proteins (46),
phosphatidylcholine-specific phospholipase (PL) C (24), and nuclear
factor-
B (18). In ASM cells, investigators have recently shown that
TNF-
also activates the c-Jun
NH2-terminal kinase in rabbit ASM
cells (36). However, the downstream signaling events coupling the
TNFRp55 to Ca2+
responses remain unknown.
The aim of the present study was to dissect the mechanisms underlying
the enhancement of Ca2+
responsiveness by TNF-
induced by bronchoconstrictor agents. The
current study demonstrates that TNF-
markedly enhanced
phosphoinositide (PI) accumulation induced by bradykinin and NaF, an
agonist that bypasses membrane receptors and directly activates G
proteins. Therefore, it is conceivable that the effects of TNF-
on
Ca2+ responsiveness may be due to
a direct modulatory effect on G protein-mediated signal transduction.
We also show that IL-1
, a cytokine that also has been described to
alter ASM responsiveness in vitro (37, 45), enhances agonist-induced
Ca2+ responses. Taken together,
these data suggest that cytokine-induced alteration of
Ca2+ homeostasis in ASM cells may
represent, in part, a mechanism underlying bronchial hyperreactivity in
asthmatic patients.
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MATERIALS AND METHODS |
Cell culture.
The methods of cell culture for human ASM are identical to that
previously described (29). Briefly, human trachea was obtained from
lung transplant donors, in accordance with procedures approved by the
University of Pennsylvania Committee on Studies Involving Human Beings.
A segment of trachea just proximal to the carina was removed under
sterile conditions, and the trachealis muscles were isolated. Through
the use of this technique, ~0.5 mg of wet tissue was obtained,
minced, centrifuged, and resuspended in 10 ml of buffer containing 0.2 mM CaCl2, 640 U/ml collagenase, 1 mg/ml soybean trypsin inhibitor, and 10 U/ml elastase. Enzymatic dissociation of the tissue was performed for 90 min in a shaking water
bath at 37°C. The cell suspension was filtered through 105-mm Nytex
mesh, and the filtrate was washed with equal volumes of cold Ham's
F-12 medium supplemented with 10% fetal bovine serum, 100 U/ml
penicillin, 0.1 mg/ml streptomycin, and 2.5 mg/ml amphotericin B and
was replaced every 72 h. Cell counts were obtained in triplicate wells
with 0.5% trypsin in 1 mM EDTA solution.
Measurement of cytosolic free
Ca2+
concentration.
ASM cells were plated at low density onto 15-mm coverslips
3-5 days before experiments. All experiments were done with the use of subconfluent cells between the third and fifth passages. Cells
were loaded with 2.5 µM fura 2-acetoxymethyl ester [AM; in
medium 199 supplemented with 1 mg/ml bovine serum albumin (BSA)] for 20 min at 37°C. After fura 2 was loaded, cells were washed with
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES)-buffered saline solutions as previously described (26) and
were placed in a thermostatically controlled cell chamber on a Nikon
inverted microscope (Diaphot). Cells were imaged using a ×40
(oil) fluorescence objective lens. Excitation energy was switched
between 340 and 380 nm wavelength using a 75-watt xenon lamp (Nikon)
source and a fura 2 dichroic mirror (Chroma Technology, Brattleboro,
VT). The emitted fluorescence (510 nm) was diverted to an
image-intensified charge-coupled device camera (Hamamatsu) attached to
the video analog-to-port digital conversion board (Maatrox). Image
analysis of individual cursor-defined regions corresponding to
individual cells was accomplished using the Image-1 AT/Fluor program
(Universal Imaging, West Chester, PA). The ratio of fluorescence at 340 nm to fluorescence at 380 nm was converted to an estimate of cytosolic free Ca2+ using previously
described calibration methods (12, 26). Calibration
measurements were made with 10 mM ionomycin and added Ca2+ (total extracellular
Ca2+ 12 mM) to measure maximum
fluorescence ratio (Rmax) or
addition of a stoichiometric excess of ethylene
glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic
acid to achieve the minimum fluorescence ratio
(Rmin). Values used for
calibration equation were Rmin = 0.3 and Rmax = 5, dissociation
constant = 224, and ratio of minimum to maximum fluorescence at 380 nm = 5.
Accumulation of total
[3H]PI.
[3H]PI formation in
cultures of human tracheal smooth muscle cell was quantified by an
assay as described by Daykin et al. (9). The medium was aspirated from
confluent monolayers of cells in 24 wells and was replaced with
inositol-free medium containing 0.1% BSA and 5 µCi/ml
myo-[2-3H]inositol
(NEN, Boston, MA). The cells were then further incubated for 48 h
except for TNF-
-treated cells in which TNF-
at a concentration of
1,000 IU/ml was added for the last 24 h. This medium was
removed, and cells were washed three times with HEPES buffer and then
were incubated with the same buffer containing 15 mM LiCl and 0.1% BSA
for 30 min at 37°C. Agonists were added, and the reaction was then
stopped by adding 5% trichloroacetic acid. The 24 flasks were
incubated at 4°C for 10-15 min, and the total
[3H]PI was finally
separated from free
myo-[3H]inositol
by anion-exchange chromatography on Dowex-Cl columns (9, 16).
Drugs.
Bradykinin and fura 2-AM were purchased from Sigma Chemical (St. Louis,
MO). Dulbecco's modified Eagle's medium-F-12, fetal calf serum,
trypsin, and antibiotics (penicillin and streptomycin) were obtained
from GIBCO-BRL (Grand Island, NY).
-Thrombin, IL-1, and TNF-
were
purchased from Boehringer Mannheim (Indianapolis, IN) and Calbiochem
(La Jolla, CA).
 |
RESULTS |
TNF-
and IL-1 potentiate the increase in cytosolic
free Ca2+
concentration induced by bradykinin in human ASM cells.
The effects of TNF-
and IL-1
(10 ng/ml for 24 h) on the increase
in cytosolic free Ca2+
concentration
([Ca2+]i)
induced by 1 µM bradykinin are shown in Figs.
1 and 2. As shown in Figs. 1A and
2A, bradykinin induced a typical
biphasic increase in
[Ca2+]i,
characterized by a rapid transient peak followed by a sustained elevation of
[Ca2+]i.
Addition of 40 mM KCl during the sustained phase rapidly lowered the
sustained elevation of Ca2+ to
baseline. In cells treated with TNF-
or IL-1 for 24 h (Figs. 1B and
2B), both phases of the
[Ca2+]i
increase were potentiated compared with those treated with diluent.
These data are summarized as the net values of the peak and sustained
increase in
[Ca2+]i,
respectively, obtained in treated versus untreated cells in Figs. 1,
C and
D, and 2,
C and
D. In TNF-
-treated cells, the net
[Ca2+]i
increase to bradykinin was 573 ± 93 vs. 247 ± 48 nM in
nontreated cells (P < 0.0001). In
IL-1
-treated cells, the Ca2+
response to bradykinin was 350 ± 21 vs. 127 ± 12 nM in
nontreated cells (P < 0.0001, unpaired Student's t-test). For the
sustained phase of increase in
[Ca2+]i
(Figs. 1D and
2D), there was also a significant
difference in cells treated with both cytokines
(P < 0.01, unpaired Student's t-test).

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Fig. 1.
Effect of tumor necrosis factor (TNF)- on bradykinin-evoked
cytosolic free Ca2+ concentration
([Ca2+]i).
TNF- (10 ng/ml) was preincubated for 24 h before cells were exposed
to bradykinin. A and
B: typical
Ca2+ traces from cells incubated
in the absence (A) or presence
(B) of TNF- .
C and
D: values for the peak
(C) and sustained
(D)
Ca2+ phase from cells incubated in
the absence or presence of TNF- . Results are expressed as the net
increase in
[Ca2+]i
over basal (unstimulated) levels. Values are means ± SE of 4 separate experiments and are significantly different from control
(untreated cells; P < 0.01).
Statistical significance was determined using unpaired Student's
t-test.
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Fig. 2.
Effect of interleukin (IL)-1 on bradykinin-induced
[Ca2+]i.
IL-1 (10 ng/ml) was preincubated for 24 h before cells were exposed
to bradykinin. A and
B: typical
Ca2+ traces from cells incubated
in the absence (A) or presence
(B) of IL-1 .
C and
D: values for the peak
(C) and sustained
(D)
Ca2+ phase from cells incubated in
the absence or presence of IL-1 . Results are expressed as the net
increase in
[Ca2+]i
over basal (unstimulated) levels. Values are expressed as means ± SE of 4 separate experiments and are significantly different from
control (untreated cells; P < 0.01).
Statistical significance was determined using unpaired Student's
t-test.
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To define the time course by which TNF-
modulates bradykinin-induced
cytosolic peak Ca2+ responses,
cells were treated for 4, 6, 8, and 12 h with TNF-
, and then the
degree of stimulation (percent change in
[Ca2+]i)
was compared with those that were treated with diluent alone. As shown
in Fig. 3, the potentiating effects of
TNF-
on bradykinin-induced cytosolic
Ca2+ peak reached a maximal level
after 12 h of pretreatment and was sustained over the subsequent time
points (n = 60-100 cells/time point, mean ± SE). In nontreated cells, no significant change was
observed in the bradykinin-induced
Ca2+ transient (Fig. 3). These
data suggest that cytokines directly modulate
Ca2+ homeostasis in a
time-dependent manner in human ASM cells.

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Fig. 3.
Time course of TNF- -mediated potentiation of
Ca2+ responsiveness. Time
dependence for TNF- -mediated potentiation of the
Ca2+ transient induced by
bradykinin. Values are expressed as means ± SE from 60-100
cells/time point. (* P < 0.01 by unpaired Student's
t-test.)
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TNF-
and IL-1 also augment the increase in
[Ca2+]i
induced by thrombin.
We have previously shown that thrombin increased
[Ca2+]i
in ASM cells (29). We next examined whether TNF-
also alters
Ca2+ responses induced by
thrombin. As shown in Fig. 4,
A and
B, the biphasic increase in
[Ca2+]i
induced by 1 IU/ml thrombin was greater in TNF-
-treated cells compared with control cells. The net
[Ca2+]i
increases to thrombin were 350 ± 14 vs. 130 ± 17 nM in
nontreated cells for the peak and 110 ± 7 vs. 150 ± 9 nM for
the sustained phase in nontreated cells
(P < 0.0001, statistical
significance using unpaired Student's
t-test). Similar results were also
obtained when cells were pretreated with IL-1 (data not shown). Because cytokines modulated both bradykinin- and thrombin-evoked
Ca2+ responses and because our
previous studies showed that TNF-
has no effect on cell surface
expression of agonist receptors (3), these data suggest that cytokines
modulate Ca2+ responses downstream
from the contractile agonist receptor.

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Fig. 4.
Effect of TNF- on thrombin-induced
[Ca2+]i.
TNF- (10 ng/ml) was preincubated for 24 h before the cells were
exposed to thrombin. A and
B: typical
Ca2+ traces from cells incubated
in the absence (A) or presence
(B) of TNF- .
C and
D: respective values for the peak
(C) and sustained
(D)
Ca2+ phase from cells incubated in
the absence or the presence of TNF- . Results are expressed as the
net increase in
[Ca2+]i
over basal (unstimulated) levels. Values are means ± SE of 4 separate experiments and are significantly different from control
(untreated cells; P < 0.01).
Statistical significance was determined using unpaired Student's
t-test.
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Effect of TNF-
on bradykinin- and NaF-stimulated PI
formation.
To further dissect the effect of TNF-
on
Ca2+ responses, we postulated that
TNF-
augmented bradykinin-induced PI formation. ASM cells prelabeled
with [3H]inositol for
48 h were then incubated with or without 10 ng/ml TNF-
for 24 h and
were stimulated with bradykinin. Figure 5
shows the time course of the inositol monophosphate
(IP1) generation to bradykinin.
In our study,
[3H]IP1
was used as a measure of PI turnover by this method, but similar
results were also obtained when measuring
[3H]inositol
1,4,5-trisphosphate
([3H]IP3)
accumulation (data not shown). Addition of
10
6 M bradykinin to
cultured ASM cells stimulated a rapid accumulation of
[3H]IP1.
This rise was observed as early as 15 s and increased until 30 min.
Treatment of cells with TNF-
for 24 h markedly increased bradykinin-induced IP1 formation.
The percentage of change over basal level [basal counts/min (cpm)
were 159 ± 18] after 5, 10, 20, and 30 min of stimulation
with bradykinin was 590 ± 86, 1,324 ± 130, 3,134 ± 268, and
4,361 ± 488%, respectively, in TNF-
-treated cells compared with
273 ± 22, 736 ± 138, 1,406 ± 47, and 2,305 ± 341%,
respectively, in nontreated cells
[P < 0.05, analysis of
variance (ANOVA), n = 5 experiments
performed in quadruplicate for each point]. The effect of TNF-
on the concentration-response curve for
[3H]IP1
accumulation is shown in Fig. 6. Bradykinin
(10
9 to
10
6 M) induced a
dose-dependent increase in total PI turnover. The percentage of change
in PI accumulation over control (basal cpm were 86 ± 10) was 115 ± 8 to 210 ± 15% for 3 × 10
9 to 3 × 10
6 M bradykinin,
respectively. When cells were pretreated with TNF-
, bradykinin-stimulated PI accumulation was significantly enhanced, and
the percentage of change in PI accumulation over control was 128 ± 10, 217 ± 24, 388 ± 75, and 437 ± 92% for 3 × 10
9, 3 × 10
8, 3 × 10
7, and 3 × 10
6 M bradykinin,
respectively (P < 0.01, statistical
significance using ANOVA for n = 6 experiments performed in quadruplicate). Taken together, these data
suggest that TNF-
-induced enhancement of bradykinin
Ca2+ responsiveness may be, in
part, mediated by an increase in PI accumulation. To determine whether
PI turnover in response to bradykinin was due to PLC activation, we
performed separate experiments using U-73122, a PLC inhibitor.
Preincubation of human ASM cells with 5 µM U-73122 (for 15 min)
completely inhibited bradykinin-induced IP generation in
TNF-
-treated and untreated cells (data not shown). We next
investigated whether TNF-
treatment also alters upstream signaling
events from PLC activation. Cells were treated with NaF, which directly
activates all G proteins, and PI turnover was measured. As shown in
Fig. 7, 10 mM NaF induced a 280 ± 35-fold increase in PI formation. Pretreatment of ASM cells with
TNF-
significantly enhanced NaF-induced PI accumulation to 415 ± 30% compared with cells treated with diluent alone
(P < 0.01). These results suggest
that the potentiating effect of TNF-
on the bradykinin-induced PI
response may be due to an effect at the level of the G protein that is
coupled to PLC activation.

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Fig. 5.
TNF- enhances bradykinin-induced phosphoinositide (PI) turnover.
Time-dependent inositol monophosphate generation in response to
bradykinin in TNF- -treated ( ) and TNF- -untreated ( ) airway
smooth muscle (ASM) cells. Values are means ± SE from 5 separate
experiments performed in quadruplicate
[* P < 0.01, statistical
significance using analysis of variance (ANOVA)].
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Fig. 6.
Concentration-dependent inositol monophosphate generation induced by
bradykinin. Concentration-dependent inositol monophosphate generation
in response to bradykinin in TNF- -treated ( ) and
TNF- -untreated ( ) ASM cells. Values are expressed as means ± SE from 6 separate experiments performed in quadruplicate
(* P < 0.01, statistical
significance using ANOVA).
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Fig. 7.
NaF, a direct activator of G proteins, evoked inositol monophosphate
generation that was enhanced by TNF- . Cells prelabeled with
[3H]inositol were
preincubated with TNF- for 24 h and then were stimulated with 10 mM
NaF for 15 min. Values are expressed as means ± SE from 6 separate
experiments performed in quadruplicate
(* P < 0.01, statistical
significance using ANOVA).
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Effect of cycloheximide on TNF-
-induced potentiation
of Ca2+ signals.
To determine whether the TNF-
effects on
Ca2+ homeostasis were due to
protein synthesis, ASM cells were pretreated with cycloheximide, a
protein synthesis inhibitor, and then were stimulated with TNF-
. In
a dose-dependent manner, 1 and 10 µM cycloheximide significantly inhibited TNF-
-induced increases in
Ca2+ responses to bradykinin by 24 and 99% inhibition, respectively (P < 0.001; statistical significance using unpaired Student's t-test compared with cells treated
with TNF-
; Fig. 8). Interestingly, cycloheximide had no effect on the bradykinin-induced
Ca2+ transients in untreated
cells. These results suggest that TNF-
-mediated potentiation of
agonist-evoked Ca2+ transients
required de novo protein synthesis. These findings are consistent with
our studies that showed TNF-
effects on
Ca2+ mobilization require a
minimum of 6 h of cytokine stimulation.

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Fig. 8.
Cycloheximide (CHX) inhibits TNF- -mediated potentiation of
agonist-induced Ca2+ responses.
Cells preincubated with cycloheximide (1 and 10 µM) for 15 min were
treated with TNF- for 24 h.
Ca2+ responses to 1 µM
bradykinin were then studied as described in MATERIALS
AND METHODS. Values are expressed as means ± SE
from 5 separate experiments (* P < 0.01 and
** P < 0.001 compared with
cells exposed to TNF- alone, statistical significance using
Student's t-test).
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DISCUSSION |
In this study, we extended our previous work to examine the mechanisms
by which TNF-
enhances Ca2+
responsiveness to bronchoconstrictor agents such as bradykinin and
carbachol (3, 4). We show that TNF-
pretreatment enhances cytosolic
Ca2+ mobilization induced by
thrombin, which increases
[Ca2+]i
by activating a non-Gi coupled
receptor (29). In addition, the effects of TNF-
on the
Ca2+ mobilization were seen only
after 6 h of stimulation and were inhibited by cycloheximide,
suggesting that protein expression may be involved in mediating TNF-
effects. The ability of TNF-
to enhance
Ca2+ responses by a variety of
agonists suggests that the TNF-
may modulate signaling events
downstream from the receptor at either the level of G protein coupling
or a more distal site in the signal transduction pathway.
Because agonist-stimulated tracheal smooth muscle contraction is
dependent on PLC activation and because generation of
IP3 evokes increases in
[Ca2+]i
(8), we next examined whether TNF-
modulates PI turnover induced by
bradykinin. Bradykinin-induced PI formation was significantly enhanced
in ASM cells that were pretreated with TNF-
. Augmented Ca2+ responses induced by TNF-
may be directly due to enhanced PI metabolism or potentially to
inhibition of specific phosphatases. Similar results have been reported
using human epidermoid carcinoma cells. In these cells, TNF-
treatment enhanced PI turnover generated by bradykinin (34).
Interestingly, the authors also found a decrease in cell surface
expression of bradykinin receptors. This suggests that TNF-
may
enhance coupling of agonist receptors to downstream signaling events
and that PLC activity may be altered by TNF-
. Similarly, the
increased PI turnover induced by NaF, which has been described to
stimulate inositol formation in ASM cells by directly stimulating G
proteins (15, 16, 25), supports the notion that TNF-
effects on
Ca2+ mobilization also occur
downstream from the receptor and possibly at the level of the G
protein. Our data are consistent with two previous studies that
showed a direct modulatory effect of TNF-
on G
protein-mediated signal transduction. In rat cardiomyocytes and in
human leukocytes, TNF-
significantly enhanced isoproterenol-mediated G protein activation of adenylyl cyclase (32) and
N-formyl-methionyl-leucyl-phenylalanine-stimulated G protein
activation (22), respectively. The mechanism by which TNF-
enhanced
transmembrane signaling remains unknown. The NaF data suggest that
treatment of cells with TNF-
increases receptor coupling at a level
downstream from the receptor located at the level of either the G
protein or PLC. Because TNF-
has been shown to directly activate G
proteins in osteoblast cells (46), it is also possible that TNF-
regulates the activity of G protein by enhancing, for
example, the formation of the 

-complex upon stimulation by an
agonist. In this manner, the transduction through the G proteins could
be increased. Alternatively, TNF-
may increase the quantity of G
protein involved in the receptor coupling. It has already been shown
that this cytokine stimulates de novo synthesis of
Gi
in some cells (14, 22, 32,
35). Interestingly, this increase in
Gi
protein induced by TNF-
was associated with an alteration in cell responsiveness to subsequent
receptor stimulation coupled to the G protein (22, 32, 35). Recently,
cytokines have also been reported to increase
Gi expression in rabbit ASM cells
(14). It is unlikely, however, that this mechanism can account for the
observed effects of TNF-
on thrombin-induced Ca2+ mobilization because the
thrombin receptor that evokes Ca2+
transients in human ASM is coupled to a non-pertussis toxin-sensitive G protein (28, 29). The effect of TNF-
may be located at the level of PLC activation. TNF-
may directly stimulate or
"prime" ASM cells for increased PLC through the activation of
PLA2. In some cell types,
activation of the TNFRp55 receptor, which mediates TNF-
effects in
ASM cells (4), also stimulates neutral sphingomyelinase-induced PLA2 activity (5). The
arachidonate pathway may represent another pathway that mediates
TNF-
effects on cell activation. To support this notion, others
reported that treatment of rat ASM cells with either TNF-
or IL-1
increased the expression of inducible cyclooxygenase and
PLA2 (41). Effects of both
cytokines were also inhibited by treating ASM cells with either
dexamethasone or cycloheximide. In human bronchial ASM cells, the
mitogenic effect of TNF-
was also completely abolished by
dexamethasone and aspirin (38). Other investigators who used Swiss 3T3
fibroblasts (7) and osteoblast-like cell lines (46) have reported that
TNF-
effects on signal transduction were inhibited by aspirin.
Clearly, more studies are needed to dissect whether cytokines can
directly modulate G protein expression or
PLA2 activation. These two
potential mechanisms may not be mutually exclusive because cytokines
can alter multiple signaling pathways.
Another possibility by which TNF-
potentiated
Ca2+ responses is by affecting the
Ca2+ pools activated by the
bronchoconstrictor agents. We have shown that the response to
thapsigargin, which directly releases
Ca2+ from the internal stores, was
potentiated by pretreating cells with TNF-
(2, 4). Based on these
data, we conclude that, in addition to the receptor coupling, TNF-
could also affect the intracellular stores of
Ca2+. Because the sustained phase
of elevation of Ca2+ induced by
bradykinin and thrombin was also potentiated by TNF-
and IL-1
, it
could be possible that both cytokines enhanced
Ca2+ influx in our cells. This may
result from a direct effect on Ca2+ channels as reported for both
cytokines in rat vascular smooth muscle cells (43). In a previous
report, we have also shown that, in human ASM cells, the
Ca2+ influx pathway was modulated
by the filling state of the internal store of
Ca2+ (2). These data are
consistent with the capacitive model described by Putney (31). The
increased Ca2+ influx observed
after TNF-
treatment may, therefore, simply be a consequence of the
increased magnitude of Ca2+
depletion from the IP3-sensitive
intracellular Ca2+ stores because
both phases of elevation of Ca2+,
namely the transient and sustained phases, were shown to be linked (2).
In the current study, we also show that ASM cells treated with IL-1
had enhanced agonist-evoked Ca2+
responses to agonists compared with those treated with diluent alone.
The ability of IL-1
to mimic the effect of TNF-
on
Ca2+ responses may have important
implications for understanding the mechanisms that regulate ASM
hyperreactivity in asthma. High levels of IL-1
and TNF-
have been
detected in the airways of patients with symptomatic asthma (6).
Moreover, these cytokines induce bronchial hyperreactivity to agonists
either in vivo (40) or in vitro in tracheal and bronchial ASM
preparations (30, 37). Because
Ca2+ is known to play an important
role in the regulation of contractile responses to agonists (reviewed
in Ref. 8), our data suggest that cytokine-mediated alteration in
Ca2+ homeostasis may represent one
mechanism underlying bronchial hyperreactivity in asthma. Because both
cytokines were also found to stimulate proliferation of ASM cells (4,
10), the subsequent ASM hyperplasia and hypertrophy may, in part,
contribute to bronchial hyperreactivity via an enhanced contractile
response or, alternatively, via a decrease in luminal caliber (reviewed
in Ref. 28). Additional studies are needed to clarify the precise
mechanism(s) by which cytokines alter ASM cell function before new
therapeutic interventions can be developed to abrogate these effects.
 |
ACKNOWLEDGEMENTS |
We thank Mary McNichol for assistance in preparation of the
manuscript.
 |
FOOTNOTES |
Y. Amrani was supported by a postdoctoral fellowship of the Association
Francaise pour la Recherche Therapeutique (Paris, France). This study
was also supported by National Heart, Lung, and Blood Institute Grant
R01-HL-55301 (to R. A. Panettieri, Jr.), National Aeronautics and Space
Administration Grant NRA-94-OLMSA-02 (to R. A. Panettieri, Jr.), and a
Career Investigator Award, American Lung Association (to R. A. Panettieri, Jr.).
Address for reprint requests: Y. Amrani, Pulmonary and Critical Care
Division, Univ. of Pennsylvania Medical Center, Rm. 808 East Gates
Bldg., 3400 Spruce St., Philadelphia, PA 19104-4283.
Received 18 April 1997; accepted in final form 12 August 1997.
 |
REFERENCES |
1.
Amrani, Y.,
and
C. Bronner.
Tumor necrosis factor alpha potentiates the increase in cytosolic free calcium induced by bradykinin in guinea-pig tracheal smooth muscle cells.
C. R. Acad. Sci. Paris
316:
1489-1494,
1993[Medline].
2.
Amrani, Y.,
C. Magnier,
F. Wuytack,
J. Enouf,
and
C. Bronner.
Ca2+ increase and Ca2+ influx in human tracheal smooth muscle cells: role of Ca2+ pools controlled by sarco-endoplasmic reticulum Ca2+-ATPase 2 isoform.
Br. J. Pharmacol.
115:
1204-1210,
1995[Abstract].
3.
Amrani, Y.,
N. Martinet,
and
C. Bronner.
Potentiation by tumour necrosis factor-
of calcium signals induced by bradykinin and carbachol in human tracheal smooth muscle cells.
Br. J. Pharmacol.
114:
4-5,
1995[Abstract].
4.
Amrani, Y.,
R. A. Panettieri, Jr.,
N. Frossard,
and
C. Bronner.
Activation of the TNF
-p55 receptor induces myocyte proliferation and modulates agonist-evoked calcium transients in cultured human tracheal smooth muscle cells.
Am. J. Respir. Cell Mol. Biol.
15:
55-63,
1996[Abstract].
5.
Belka, K.,
K. Wiegmann,
D. Adam,
R. Holland,
M. Neuloh,
F. Herrmann,
M. Kronke,
and
M. Brach.
Tumor necrosis factor (TNF)-
activates c-raf-1 kinase via the p55 TNF receptor engaging sphingomyelinase.
EMBO J.
14:
1156-1165,
1995[Abstract].
6.
Broide, D. H.,
M. Lotz,
A. J. Cuomo,
D. A. Coburn,
E. C. Federburn,
and
S. I. Wasserman.
Cytokines in symptomatic asthma airways.
J. Allergy Clin. Immunol.
89:
958-967,
1992[Medline].
7.
Burch, R. M.,
and
C. W. Tiffany.
Tumor necrosis factor causes amplification of arachidonic acid metabolism in responses to interleukin-1, bradykinin, and other agonists.
J. Cell. Physiol.
141:
85-89,
1989[Medline].
8.
Coburn, R. F.,
and
C. B. Baron.
Coupling mechanisms in airway smooth muscle.
Am. J. Physiol.
258 (Lung Cell. Mol. Physiol. 2):
L119-L133,
1990[Abstract/Free Full Text].
9.
Daykin, K.,
S. Widdop,
and
I. P. Hall.
Control of histamine-induced inositol phospholipid hydrolysis in cultured human smooth muscle cells.
Eur. J. Pharmacol.
264:
135-140,
1993.
10.
De, S.,
E. T. Zelazny,
J. F. Souhrada,
and
M. Souhrada.
Interleukin-1
stimulates the proliferation of cultured airway smooth muscle cells via platelet-derived growth factor.
Am. J. Respir. Cell Mol. Biol.
9:
645-651,
1993[Medline].
11.
Erickson, S. L.,
F. J. de Sauvage,
K. Kikly,
K. Carver-Moore,
S. Pitts-Meek,
N. Gillett,
K. C. F. Sheehan,
R. D. Schrelber,
D. V. Goeddel,
and
M. W. Moore.
Decreased sensitivity to tumor-necrosis factor but normal T-cell development in TNF receptor-2-deficient mice.
Nature
372:
560-563,
1994[Medline].
12.
Grynkiewicz, G.,
M. Poenie,
and
Y. R. Tsien.
A new generation of Ca2+ indicators with greatly improved fluorescence properties.
J. Biol. Chem.
260:
3340-3450,
1985.
13.
Hakonarson, H.,
D. J. Herrick,
P. Gonzalez Serrano,
and
M. M. Grunstein.
Autocrine role of interleukin 1
in altered responsiveness to atopic asthmatic sensitized airway smooth muscle.
J. Clin. Invest.
99:
117-124,
1997[Abstract/Free Full Text].
14.
Hakonarson, H.,
D. J. Herrick,
and
M. M. Grunstein.
Mechanism of impaired
-adrenoceptor responsiveness in atopic sensitized airway smooth muscle.
Am. J. Physiol.
269 (Lung Cell. Mol. Physiol. 13):
L645-L652,
1995[Abstract/Free Full Text].
15.
Hall, I. P.,
J. Donaldson,
and
S. J. Hill.
Modulation of fluoroaluminate-induced inositol phosphate formation by increases in tissue cyclic AMP content in bovine tracheal smooth muscle.
Br. J. Pharmacol.
100:
646-650,
1990[Abstract].
16.
Hardy, E.,
M. Farahni,
and
I. P. Hall.
Regulation of histamine receptor coupling by dexamethasone in human cultured airway smooth muscle.
Br. J. Pharmacol.
118:
1079-1084,
1996[Abstract].
17.
Heller, R. A.,
K. Song,
N. Fan,
and
D. J. Chang.
The p70 tumor necrosis factor receptor mediates cytotoxicity.
Cell
70:
47-52,
1992[Medline].
18.
Hsu, H.,
J. Xiong,
and
D. V. Goeddel.
The TNF receptor 1-associated protein TRADD signals cell death and NF-
B activation.
Cell
81:
495-504,
1995[Medline].
19.
Jarvis, W. D.,
A. J. Turner,
L. F. Povirk,
R. S. Traylor,
and
S. Grant.
Induction of apoptotic DNA fragmentation and cell death in HL-60 human promyelocytic leukemia cells by pharmacological inhibitors of protein kinase C.
Cancer Res.
54:
1707-1714,
1994[Abstract].
20.
Kips, J. C.,
J. H. Tavernier,
G. F. Joos,
R. A. Peleman,
and
R. A. Pauwels.
The potential role of tumor necrosis factor in asthma.
Clin. Exp. Allergy
23:
247-250,
1993[Medline].
21.
Kips, J. C.,
J. H. Tavernier,
and
R. A. Pauwels.
Tumor necrosis factor (TNF) causes bronchial hyperresponsiveness in rats.
Am. Rev. Respir. Dis.
145:
332-336,
1992[Medline].
22.
Klein, J. B.,
J. A. Scherzer,
G. Harding,
A. A. Jacobs,
and
K. R. McLeish.
TNF-
stimulates increased plasma membrane guanine nucleotide binding protein activity in polymorphonuclear leukocytes.
J. Leukoc. Biol.
57:
500-506,
1995[Abstract].
23.
Lazaar, A. L.,
S. M. Albelda,
J. M. Pilewski,
B. Brennan,
E. Puré,
and
R. A. Panettieri.
T lymphocytes adhere to airway smooth muscle cell integrins and CD44 and induce muscle cell DNA synthesis.
J. Exp. Med.
180:
807-816,
1994[Abstract].
24.
Machleidt, T.,
B. Kramer,
D. Adam,
B. Neumann,
S. Schutze,
K. Wiegmann,
and
M. Kronke.
Function of the p55 tumor necrosis factor receptor "death domain" mediated by phosphatidylcholine-specific phospholipase C.
J. Exp. Med.
184:
725-733,
1996[Abstract].
25.
Marsh, K. A.,
and
S. J. Hill.
Bradykinin B2 receptor-mediated phosphoinositide hydrolysis in bovine cultured tracheal smooth muscle cells.
Br. J. Pharmacol.
107:
443-447,
1992[Abstract].
26.
Murray, R. K.,
B. K. Fleischmann,
and
M. I. Kotlikoff.
Receptor-activated Ca2+ influx in human airway smooth muscle: use of Ca2+ imaging and perforated patch-clamp techniques.
Am. J. Physiol.
264 (Cell Physiol. 33):
C485-C490,
1993[Abstract/Free Full Text].
27.
Obeid, L. M.,
C. M. Linardic,
L. A. Karolok,
and
Y. A. Hannun.
Programmed cell death induced by ceramide.
Science
259:
1769-1771,
1993[Medline].
28.
Panettieri, R. A., Jr.
Regulation of growth of airway smooth muscle by second messenger systems.
In: Airway Wall Remodelling in Asthma, edited by A. G. Stewart. Boca Raton, FL: CRC, 1996, p. 269-293.
29.
Panettieri, R. A., Jr.,
I. P. Hall,
C. S. Maki,
and
R. K. Murray.
-Thrombin increases cytosolic calcium and induces human airway smooth muscle cell proliferation.
Am. J. Respir. Cell Mol. Biol.
13:
205-216,
1995[Abstract].
30.
Pennings, H. J.,
K. Kramer,
A. Bast,
W. Buurman,
and
E. Wouters.
Tumour necrosis factor causes hyperresponsiveness in tracheal smooth muscle of the guinea-pig model in vitro (Abstract).
Eur. Respir. J.
70:
325s,
1993.
31.
Putney, J. W.
Excitement about calcium signaling in inexcitable cells.
Science
262:
676-678,
1993[Medline].
32.
Reithmann, C.,
P. Gierschik,
K. Werdan,
and
K. H. Jakobs.
Tumor necrosis factor-
up-regulates Gi
and G
proteins and adenylyl cyclase responsive in rat cardiomyocytes.
Eur. J. Pharmacol.
206:
53-60,
1991[Medline].
33.
Renzetti, L. M.,
P. M. Paciorek,
S. A. Tannu,
N. C. Rinaldi,
J. E. Tocker,
M. A. Wasserman,
and
P. R. Gater.
Pharmacological evidence for tumor necrosis factor as a mediator of allergic inflammation in the airways.
J. Pharmacol. Exp. Ther.
278:
847-853,
1996[Abstract].
34.
Sawutz, D. G.,
S. S. Singh,
L. Tiberio,
E. Koszewski,
C. G. Johnson,
and
C. L. Johnson.
The effect of TNF-
on bradykinin receptor binding, phosphotidylinositol turnover and cell growth in human A431 epidermoid carcinoma cells.
Immunopharmacology
24:
1-10,
1992[Medline].
35.
Scherzer, J. A.,
Y. Lin,
K. R. McLeich,
and
J. B. Klein.
TNF translationally modulates the expression of G protein
i2 subunits in human polymorphonuclear leukocytes.
J. Immunol.
158:
913-918,
1997[Abstract].
36.
Shapiro, P. S,
J. N. Evans,
R. J. Davis,
and
J. A. Posada.
The seven transmembrane-spanning receptors for endothelin and thrombin cause proliferation of airway smooth muscle cells and activation of the extracellular regulated kinase and c-Jun NH2-terminal kinase groups of mitogen-activated protein kinases.
J. Biol. Chem.
271:
5750-5754,
1996[Abstract/Free Full Text].
37.
Souhrada, M.,
and
J. F. Souhrada.
Potentiation of electrical and contractile response of sensitized airway smooth muscle to a specific antigen by interleukin-1
(Abstract).
Am. J. Respir. Cell Mol. Biol.
147:
A52,
1993.
38.
Stewart, A. G.,
P. R. Tomlinson,
D. J. Fernandes,
J. W. Wilson,
and
T. Harris.
Tumor necrosis factor
modulates mitogenic responses of human cultured airway smooth muscle.
Am. J. Respir. Cell Mol. Biol.
12:
110-119,
1995[Abstract].
39.
Tartaglia, L. A.,
and
D. V. Goeddel.
Two TNF receptors.
Immunol. Today
13:
151-153,
1992[Medline].
40.
Thomas, P. S.,
D. H. Yates,
and
J. P. Barnes.
Tumor necrosis factor-alpha increases airway responsiveness and sputum neutrophilia in normal human subjects.
Am. J. Respir. Crit. Care Med.
152:
76-80,
1995[Abstract].
41.
Vadas, P.,
E. Stefanski,
M. Wloch,
B. Grouix,
H. Van Den Bosch,
and
B. Kennedy.
Secretory non-pancreatic phospholipase A2 and cyclooxygenase-2 expression by tracheobronchial smooth muscle cells.
Eur. J. Biochem.
235:
557-563,
1996[Abstract].
42.
Wheeler, A. P.,
W. D. Hardie,
and
G. R. Bernard.
The role of cyclooxygenase products in lung injury induced by tumor necrosis factor in sheep.
Am. Rev. Respir. Dis.
145:
632-639,
1992[Medline].
43.
Wilkinson, M. F.,
M. L. Earle,
C. R. Triggle,
and
S. Barnes.
Interleukin-1
, tumor necrosis factor-1
and LPS enhance calcium channel current in isolated vascular smooth muscle cells of rat tail artery.
FASEB J.
10:
785-796,
1996[Abstract/Free Full Text].
44.
Wills-Karp, M.,
J. Jinot,
J. Y. Lee,
and
F. Hirata.
Interleukin-1 alters guinea-pig tracheal smooth muscle responsiveness to
-adrenergic stimulation.
Eur. J. Pharmacol.
83:
1185-1190,
1990.
45.
Wills-Karp, M.,
Y. Uchida,
J. Y. Lee,
J. Jinot,
A. Hirata,
and
F. Hirata.
Organ bath proinflammatory cytokines reproduce impairment of the
-adrenoceptor-mediated relaxation of tracheas in a guinea-pig model.
Am. J. Respir. Cell Mol. Biol.
8:
153-159,
1993[Medline].
46.
Yanaga, F.,
M. Abe,
T. Koga,
and
M. Hirata.
Signal transduction by tumor necrosis factor
is mediated through a guanine nucleotide-binding protein in osteoclast-like cell line, MC3T3-E1.
J. Biol. Chem.
267:
5114-5121,
1992[Abstract/Free Full Text].
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