Mechanisms underlying TNF-alpha 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

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have previously shown that tumor necrosis factor (TNF)-alpha , 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-alpha modulates ASM cell responsiveness to such agents. In human ASM cells loaded with fura 2, TNF-alpha and interleukin (IL)-1beta significantly enhanced thrombin- and bradykinin-evoked elevations of intracellular Ca2+. In TNF-alpha -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-1beta -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-alpha 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-alpha on Ca2+ signals. We also found that TNF-alpha 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-alpha -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-alpha . The percentage of change in PI accumulation over control increased from 280 ± 35% in control cells to 437 ± 92% in TNF-alpha -treated cells. Taken together, these results show that TNF-alpha 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-alpha

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

TUMOR NECROSIS FACTOR (TNF)-alpha may be an important mediator in airway inflammatory diseases such as asthma and bronchiolitis (20). Increased levels of TNF-alpha have been found in the bronchoalveolar fluid of symptomatic asthmatic patients (6), and administration of TNF-alpha to healthy volunteers (40) and animals (21, 42) induces a bronchial hyperreactivity, a characteristic feature of asthma. The role of TNF-alpha 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-alpha in asthma. The mechanism(s), however, by which TNF-alpha induces bronchial hyperreactivity remains unknown.

In isolated airways, TNF-alpha or interleukin (IL)-1beta alters the contractile response of tracheal smooth muscle to cholinergic agonists (30) as well as of tracheal smooth muscle relaxation to beta -adrenergic stimulation (13, 44, 45). In cultured human airway smooth muscle (ASM) cells, we have shown that TNF-alpha 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-alpha elicited its cellular effects by activation of the p55 TNFR subtype (TNFRp55) (4). The TNFRp55 has been shown to mediate most effects of TNF-alpha , such as apoptosis (19, 27), proliferation of fibroblast, and adhesion molecule expression (reviewed in Ref. 39). TNF-alpha 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-kappa B (18). In ASM cells, investigators have recently shown that TNF-alpha 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-alpha induced by bronchoconstrictor agents. The current study demonstrates that TNF-alpha 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-alpha on Ca2+ responsiveness may be due to a direct modulatory effect on G protein-mediated signal transduction. We also show that IL-1beta , 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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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(beta -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-alpha -treated cells in which TNF-alpha 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). alpha -Thrombin, IL-1, and TNF-alpha were purchased from Boehringer Mannheim (Indianapolis, IN) and Calbiochem (La Jolla, CA).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

TNF-alpha and IL-1 potentiate the increase in cytosolic free Ca2+ concentration induced by bradykinin in human ASM cells. The effects of TNF-alpha and IL-1beta (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-alpha 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-alpha -treated cells, the net [Ca2+]i increase to bradykinin was 573 ± 93 vs. 247 ± 48 nM in nontreated cells (P < 0.0001). In IL-1beta -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)-alpha on bradykinin-evoked cytosolic free Ca2+ concentration ([Ca2+]i). TNF-alpha (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-alpha . C and D: values for the peak (C) and sustained (D) Ca2+ phase from cells incubated in the absence or presence of TNF-alpha . 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)-1beta on bradykinin-induced [Ca2+]i. IL-1beta (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-1beta . C and D: values for the peak (C) and sustained (D) Ca2+ phase from cells incubated in the absence or presence of IL-1beta . 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.

To define the time course by which TNF-alpha modulates bradykinin-induced cytosolic peak Ca2+ responses, cells were treated for 4, 6, 8, and 12 h with TNF-alpha , 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-alpha 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-alpha -mediated potentiation of Ca2+ responsiveness. Time dependence for TNF-alpha -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.)

TNF-alpha 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-alpha 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-alpha -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-alpha 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-alpha on thrombin-induced [Ca2+]i. TNF-alpha (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-alpha . 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-alpha . 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.

Effect of TNF-alpha on bradykinin- and NaF-stimulated PI formation. To further dissect the effect of TNF-alpha on Ca2+ responses, we postulated that TNF-alpha augmented bradykinin-induced PI formation. ASM cells prelabeled with [3H]inositol for 48 h were then incubated with or without 10 ng/ml TNF-alpha 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-alpha 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-alpha -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-alpha 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-alpha , 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-alpha -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-alpha -treated and untreated cells (data not shown). We next investigated whether TNF-alpha 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-alpha 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-alpha 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-alpha enhances bradykinin-induced phosphoinositide (PI) turnover. Time-dependent inositol monophosphate generation in response to bradykinin in TNF-alpha -treated (bullet ) and TNF-alpha -untreated (open circle ) 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-alpha -treated (bullet ) and TNF-alpha -untreated (open circle ) 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-alpha . Cells prelabeled with [3H]inositol were preincubated with TNF-alpha 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).

Effect of cycloheximide on TNF-alpha -induced potentiation of Ca2+ signals. To determine whether the TNF-alpha 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-alpha . In a dose-dependent manner, 1 and 10 µM cycloheximide significantly inhibited TNF-alpha -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-alpha ; Fig. 8). Interestingly, cycloheximide had no effect on the bradykinin-induced Ca2+ transients in untreated cells. These results suggest that TNF-alpha -mediated potentiation of agonist-evoked Ca2+ transients required de novo protein synthesis. These findings are consistent with our studies that showed TNF-alpha effects on Ca2+ mobilization require a minimum of 6 h of cytokine stimulation.


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Fig. 8.   Cycloheximide (CHX) inhibits TNF-alpha -mediated potentiation of agonist-induced Ca2+ responses. Cells preincubated with cycloheximide (1 and 10 µM) for 15 min were treated with TNF-alpha 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-alpha alone, statistical significance using Student's t-test).

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

In this study, we extended our previous work to examine the mechanisms by which TNF-alpha enhances Ca2+ responsiveness to bronchoconstrictor agents such as bradykinin and carbachol (3, 4). We show that TNF-alpha 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-alpha 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-alpha effects. The ability of TNF-alpha to enhance Ca2+ responses by a variety of agonists suggests that the TNF-alpha 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-alpha modulates PI turnover induced by bradykinin. Bradykinin-induced PI formation was significantly enhanced in ASM cells that were pretreated with TNF-alpha . Augmented Ca2+ responses induced by TNF-alpha 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-alpha 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-alpha may enhance coupling of agonist receptors to downstream signaling events and that PLC activity may be altered by TNF-alpha . 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-alpha 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-alpha on G protein-mediated signal transduction. In rat cardiomyocytes and in human leukocytes, TNF-alpha 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-alpha enhanced transmembrane signaling remains unknown. The NaF data suggest that treatment of cells with TNF-alpha increases receptor coupling at a level downstream from the receptor located at the level of either the G protein or PLC. Because TNF-alpha has been shown to directly activate G proteins in osteoblast cells (46), it is also possible that TNF-alpha regulates the activity of G protein by enhancing, for example, the formation of the alpha beta gamma -complex upon stimulation by an agonist. In this manner, the transduction through the G proteins could be increased. Alternatively, TNF-alpha 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 Gialpha in some cells (14, 22, 32, 35). Interestingly, this increase in Gialpha protein induced by TNF-alpha 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-alpha 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-alpha may be located at the level of PLC activation. TNF-alpha 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-alpha effects in ASM cells (4), also stimulates neutral sphingomyelinase-induced PLA2 activity (5). The arachidonate pathway may represent another pathway that mediates TNF-alpha effects on cell activation. To support this notion, others reported that treatment of rat ASM cells with either TNF-alpha or IL-1beta 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-alpha 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-alpha 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-alpha 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-alpha (2, 4). Based on these data, we conclude that, in addition to the receptor coupling, TNF-alpha 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-alpha and IL-1beta , 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-alpha 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-1beta had enhanced agonist-evoked Ca2+ responses to agonists compared with those treated with diluent alone. The ability of IL-1beta to mimic the effect of TNF-alpha on Ca2+ responses may have important implications for understanding the mechanisms that regulate ASM hyperreactivity in asthma. High levels of IL-1beta and TNF-alpha 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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
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].

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