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HIGHLIGHTED TOPICS
Airway Hyperresponsiveness: From Molecules to Bedside

Selected Contribution: TNF- modulates murine tracheal rings responsiveness to G-protein-coupled receptor agonists and KCl

Pulmonary, Allergy and Critical Care Division, Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Submitted 10 February 2003 ; accepted in final form 16 April 2003

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES ACKNOWLEDGMENTS REFERENCES

 
Although the mechanisms that underlie airway hyperresponsiveness in asthma are complex and involve a variety of factors, evidence now suggests that intrinsic abnormalities in airway smooth muscle (ASM) may play an important role. We previously reported that TNF-, a cytokine involved in asthma, augments G-protein-coupled receptor (GPCR) agonist-evoked calcium responses in cultured ASM cells. Here we have extended our previous studies by investigating whether TNF- also modulates the contractile and relaxant responses to GPCR activation using cultured murine tracheal rings. We found that in tracheal rings treated with 50 ng/ml TNF-, carbachol-induced isometric force was significantly increased by 30% compared with those treated with diluent alone (P < 0.05). TNF- also augmented KCl-induced force generation by 70% compared with rings treated with diluent alone (P < 0.01). The enhancing effect of TNF- on carbachol-induced isometric force generation was completely abrogated in the tracheal rings obtained from TNF- receptor (TNFR)1-deficient mice and in control rings treated with a TNF- mutant that solely activates TNFR2. TNF- also attenuated relaxation responsiveness to isoproterenol but not to PGE₂ or forskolin. TNF- modulatory effects on GPCR-induced ASM responsiveness were completely abrogated by pertussis toxin, an inhibitor of G_i proteins. Taken together, these data suggest that TNF- may participate in the development of airway hyperresponsiveness in asthma via the modulation of ASM responsiveness to both contractile and -adrenoceptor GPCR agonists.

asthma; airway smooth muscle; tumor necrosis factor-

TNF-, a potent proinflammatory cytokine, plays an important role in the pathogenesis of asthma (16, 48). Increased levels of TNF- have been reported in the sputum (56) as well as in the bronchoalveolar fluid of patients with symptomatic asthma (13). Other studies in healthy human subjects and in animals showed that administration of TNF- induced AHR to a variety of G-protein-coupled receptor (GPCR) agonists such as histamine, methacholine, and serotonin (35, 57, 58). Ro-45-2081, a potent TNF- receptor antagonist, also significantly reduced allergen-induced AHR in ovalbumin-sensitized guinea pigs (49). More recently, the role of TNF- receptors (TNFR) in AHR has also been confirmed in various studies using TNFR-deficient mice (15, 34, 54). The mechanism(s) by which TNF- regulates AHR remains unknown, but evidence from our laboratory showed a direct effect of TNF- on ASM function. We reported that TNF- or IL-1 can "prime" cultured human ASM cells to become nonspecifically hyperresponsive to a variety of GPCR agonists because TNF- augmented calcium responses to bradykinin, thrombin, and acetylcholine (2, 3, 5, 9, 18). Together these studies support the central hypothesis that cytokine-induced alterations in GPCR function in ASM may represent a key mechanism involved in AHR.

In the present study, we examine whether TNF- modulates ASM responsiveness to both contractile and relaxant GPCR agonists by using a murine tracheal ring organ culture model that expresses both TNF- receptors. We show that TNF- significantly enhanced isometric force generation induced by carbachol, an effect that was abrogated in TNFR1-deficient mice or with pretreatment with pertussis toxin (PTX). In addition, TNF- attenuated tracheal ring responsiveness to -adrenergic stimulation by also involving a PTX-sensitive pathway. Taken together, these data expand our previous studies concerning the effects of TNF- on ASM by demonstrating that TNF- also modulates smooth muscle responsiveness to both contractile and relaxant GPCR agonists, thus showing a possible mechanism by which cytokines may promote AHR in chronic airway inflammatory diseases such as asthma and chronic obstructive pulmonary disease.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES ACKNOWLEDGMENTS REFERENCES

 
Tissue preparation and organ culture. In our studies, we used 7- to 20-wk-old female Balb/c mice (Harlan Sprague Dawley, Indianapolis, IN) or C3H/HeJ mice (NCI, Bethesda, MD). Briefly, mice were killed by CO₂ inhalation, and the tracheae were rapidly excised, dissected free of adherent connective tissue, and sectioned into 3–4 mm in length. The tracheal rings were then cultured in an equal mixture of Ham's F-12 and DMEM (vol/vol) medium for 24 h. The organ culture medium also contained 100 mM HEPES, 1.0 M NaOH, 10% fetal bovine serum (Hy-Clone, Logan, UT), 0.2 M glutamine, 1.0 M CaCl₂, 2.5 µg/ml fungizone, 5 µg/ml insulin, 100 U/ml penicillin, and 100 µg/ml streptomycin. This medium optimally maintained agonist-induced contractile responses for 18 h compared with those obtained from tracheae harvested immediately from mice (data not shown). The use of a tracheal ring model offers several advantages over tracheal strips because the constrictor responses are directly related to in vivo airway narrowing (29). The Institutional Review Boards of the Wistar Institute and the University of Pennsylvania approved use of all animals.

Measurement of isometric force generation and relaxation. Tracheae were supported longitudinally by a Plexiglas rod with a stainless steel pin into the base of a double-jacketed, glass organ bath filled with 10 ml of Krebs-Henseleit (K-H) solution at 37° C. The K-H solution contained (in mM): 118 NaCl, 4.7 KCl, 1.2 KH₂PO_4, 11.1 dextrose, 1.2 MgSO₄, 2.8 CaCl₂, and 25 NaHCO₃ and was continuously aerated with a 5% CO₂-95% O₂ mixture; a pH of 7.40–7.45 was established for the duration of the experiments. The upper support was attached by a loop of silk thread to an FT03 isometric transducer (Astro-Med, West Warwick, RI), and changes in tension of the rings were measured. The tracheal murine rings were oriented perpendicularly to the silk mounting thread connected to the transducer. Concentration-response curves were synchronously recorded with an MP 100WS system (BIOPAC Systems, Santa Barbara, CA) and displayed on a Macintosh computer. All initial tensions of tracheal rings were set at 0.5 g and maintained for 1 h until agonists were given after a steady state of tension level had been reached.

RT-PCR analysis. Total RNA was extracted from total murine tracheal rings by using the SV total RNA isolation system (Promega, Madison, WI) according to the manufacturer's instructions. RT-PCR reactions were performed with the use of TNFR1, TNFR2, and -actin primers for semiquantitative analysis. The following sense and antisense primer sequences were used: TNFR1, 5'-CCGGGCCACCTGGTCCG-3', 5'-CAAGTAGGTTCCTTTGTG-3'; TNFR2, 5'-GTCGCGCTGGTCTTCGAACTG-3', 5'-GGTATACATGCTTGCCTCACAGTC-3'; -actin, 5'-TGGAATCCTGTGGCATCCATGAAAC-3', 5'-TAAAACGCAGCTCAGTAACAGTCCG-3' (47). All the reactions were carried out at a primer concentration of 0.12 µM in 25 µl containing 10 x Taq DNA polymerase buffer, 1.5 mM MgCl₂, 1 mM of each 2-deoxynucleotide 5'-triphosphate, and 2.5 U Taq DNA polymerase. After initial denaturation (94°C/3 min), the cycling for each gene was conducted as follows (indicated in the order of denaturation, annealing, and extension): TNFR1: 94°C/1.5 min, 55°C/2 min and 72°C/2 min; TNFR2: 94°C/1 min, 62°C/1 min and 72°C/2 min; and -actin: 94°C/30 s, 59°C/20 s and 72°C/20 s. In addition, a final 10-min extension at 72°C was included at the end of 35 cycles for all the genes. The PCR products (TNFR1, 307 bp, TNFR2 234 bp, -actin 385 bp) were resolved on 1.8% agarose gel electrophoresis, stained with ethidium bromide, and photographed.

Experimental protocols. Murine tracheae were harvested and prepared for organ culture as described above. The organ cultures were treated with 10 or 50 ng/ml TNF- or diluent alone for 18 h and then washed with K-H solution at 10-min intervals x 5 times. The concentrations of TNF- used in these studies are comparable to those used in previous studies (10, 26, 55). Contractile and relaxant concentration-response curves were constructed by using a range of concentrations from 10^-8 to 10^-5 M, respectively. In some experiments, tracheal rings pretreated with either TNF- and diluent were also incubated with either PTX (0.5 µg/ml) or cholera toxin (CTX) (2 µg/ml) for 1 h before the contractile concentration-response curves were performed as previously described (60). Similar experiments were also performed using tracheal rings harvested from mice that were deficient in either TNFR1 or TNFR2 (33, 42). For the relaxation experiments, tracheae were precontracted with 1 µM carbachol, a concentration that induces 75% of the maximum contraction before relaxation concentration-response curves to agonists PGE₂, isoproterenol, and forskolin (10^-8 to 10^-5 M) were performed. Papaverine (a phosphodiesterase inhibitor, 200 µM) was added at the end of each concentration-response curve to obtain maximal relaxation capacity of the smooth muscle in a receptor-independent manner. At the end of all experiments, tracheae were blotted on a gauze pad and weighed.

Data analysis. Tension was calculated as milligram tensions per milligram tracheal smooth muscle weight (mg/mg) and expressed as an individual percentage (%) of 10^-5 M carbacholor 100 mM KCl-evoked force of the cultured tracheal rings in the absence of TNF- for contraction studies. The concentrations of agonists required to produce half-maximal contraction (EC₅₀) were determined, and the EC₅₀ of carbachol were then converted to log values. In relaxation studies, results were expressed as an individual percentage (%) of a papaverine-induced response in control rings. Concentration of agonists required to produce a half-maximal relaxation (pD₂) was determined with -log values of the EC₅₀. All values were expressed as means ± SE. Comparisons among groups with or without TNF- were performed by a one-way ANOVA. Student's unpaired t-test was used to compare the effect of drug treatment. A P value of <0.05 was considered significant.

Materials and reagents. Recombinant human TNF- (specific activity of 10⁸ U/mg) was purchased from Roche (Indianapolis, IN). Carbachol, PTX, CTX, PGE₂, isoproterenol, forskolin, and papaverine were purchased from Sigma Chemical (St. Louis, MO).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES ACKNOWLEDGMENTS REFERENCES

 
TNF- augments carbachol- and KCl-induced isometric force generation. Our laboratory has previously shown that TNF- renders cultured human ASM cells nonspecifically hyperreactive to various GPCR agonists (reviewed in Ref. 8). In the present study, we used an organ culture model, i.e., isolated murine tracheal rings, to determine whether cytokines also modulate ASM contractile responses to GPCR activation. As shown in Fig. 1A, carbachol in a concentration-dependent manner evoked isometric tension in murine tracheal rings, showing that this ex vivo "isolated tracheal rings" model retains contractile properties to agonists. To address whether cytokines modulate agonist-induced force generation, tracheal rings were cultured in the presence or the absence of 10 or 50 ng/ml TNF- for 18 h, because our previous studies suggest that cytokine effects on GPCR responsiveness in ASM cells required at least 8 h and were dependent on protein synthesis (3). TNF- at 10 ng/ml had no significant effect on carbachol-induced force generation (Fig. 1A). In rings treated with 50 ng/ml TNF-, however, there was a significant enhancement of the carbachol-evoked contractile response, at concentrations ranging from 10^-7 to 10^-5 M, (P < 0.05) compared with diluent-treated rings. In the TNF--treated rings, maximal tensions were increased from 251 ± 20 to 329 ± 21 mg (P < 0.05), which represents approximately a 30% increase in tension compared with the diluent-treated controls. Interestingly, the effect of TNF- on carbachol-evoked maximal force generation was not associated with an alteration of the pD₂ values (-logEC₅₀), which were 6.67 ± 0.07 and 6.72 ± 0.03 in control and TNF--treated rings, respectively.

View larger version (19K): [in this window] [in a new window]   Fig. 1. TNF- enhances carbachol and KCl-induced contraction in cultured trachea. A: cumulative concentration-response curves to carbachol were completed in cultured controls (, n = 18) in the presence of TNF- at 10 ng/ml (, n = 12) and 50 ng/ml (, n = 12). B: cumulative concentration-response curves to KCl were completed in the absence (, n = 7) and presence of TNF- at 10 ng/ml (, n = 5) and 50 ng /ml (, n = 6). All tension measurements from groups are expressed as means ± SE. *P < 0.05 compared with rings treated with diluent.

Next, we examined whether TNF- also modulates isometric force generated by KCl, which evokes smooth muscle contraction by a receptor-independent depolarization of the cell membrane (30, 31). As shown in Fig. 1B, treatment of tracheal rings with KCl induced a concentration-dependent increase in isometric force (maximal force 215 ± 14 mg and pD₂ = 1.38 ± 0.19). Maximal force was evoked at 80 mM KCl and represented 85% of the maximal response induced by carbachol. Although 10 ng/ml TNF- had little effect on KCl-evoked force generation, 50 ng/ml TNF- significantly augmented KCl-induced force generation compared with that obtained from diluent-treated controls. Maximal tension generated in TNF--treated rings was 169 ± 18% above that observed in diluent-treated KCl controls. TNF- treatment, however, did not alter the EC₅₀ for KCl-evoked force generation compared with rings treated with diluent, with pD₂ values of 1.4 ± 0.037 and 1.37 ± 0.026 in diluent and TNF- treated rings, respectively. Taken together, these data suggest that TNF- modulates maximal force generation induced by carbachol and KCl in murine tracheal rings without affecting the receptor affinity.

TNFR1 mediates TNF- effects on agonist-induced force generation. Using RT-PCR analysis, we next examined whether murine tracheal tissues express both TNF- receptors. As shown in Fig. 2A, murine tracheal rings express steady-state levels of mRNA of both TNFR1 and TNFR2. To determine the TNF- receptor subtype mediating TNF- effect on carbachol-induced isometric force, we compared the effect of TNF- on carbachol-induced isometric tension in tracheal rings harvested from wild-type and from TNFR1-deficient mice. As shown in Fig. 2B, treatment of rings with 50 ng/ml TNF- augmented carbachol-induced isometric force in wild-type mice; however, this effect was completely abrogated in TNFR1-deficient mice. Importantly, there were no differences found in the EC₅₀ values for carbachol-induced isometric force among rings harvested from wild-type and TNFR1-deficient mice with pD₂ values of 6.62 ± 0.032 and 6.66 ± 0.033, respectively. These data suggest that TNF--enhancing effects on agonist-evoked isometric force are mediated by the TNFR1. To further support this hypothesis, we used a recombinant TNF- generated by site-directed mutagenesis with amino-acid mutations Asp143 Asn and Ala145 Arg (D143N-A145R-TNF-) that allow the mutant to activate TNFR2 but not TNFR1 (9, 37). As shown in Fig. 2C, treatment of murine rings from wild-type animals with D143N-A145R-TNF- had no effect on carbachol-induced contractile responses, supporting the notion that activation of TNFR1 plays a major role in the augmented contractile responses to carbachol.

View larger version (29K): [in this window] [in a new window]   Fig. 2. Role of TNF- receptor (TNFR)1 in mediating the TNF- effect on agonist-evoked contraction. A: steady-state mRNA expression of TNF- receptors in murine tracheal rings. One microgram of total RNA was subjected to RT-PCR with specific primers for -actin, TNFR1, and TNFR2, as described in MATERIALS AND METHODS. PCR products were separated on 1.8% agarose gel and stained with ethidium bromide. Data are representative of three different tracheal rings. B: cumulative concentration-response curves to carbachol were performed in wild-type mice (WT) treated with diluent (, n = 4) or with 50 ng/ml of TNF- (, n = 5). TNFR1-deficient mice (, n = 8) were also treated with 50 ng/ml TNF-. *P < 0.05 compared with rings treated with diluent. C: cumulative concentration-response curves to carbachol were performed in wild-type mice treated with diluent (, n = 4) or with 50 ng/ml D143N-A145R-TNF-, a TNF- mutant that only activates TNFR2 (, n = 5). These tension measurements from groups are expressed as means ± SE.

Effect of PTX and CTX on the potentiation of carbachol and KCl-evoked contraction induced by TNF-. Using PTX, we next examined whether G_i protein-dependent pathways mediate TNF- regulatory effects on agonist-induced contractile responses. As shown in Fig. 3A, pretreatment of murine tracheal rings with PTX had no effect on carbachol-induced maximal contraction (108 ± 13% of carbachol response in diluent-treated rings) or carbachol receptor affinity (pD₂ = 6.78 ± 0.084) but completely abrogated the potentiation of agonist-evoked contraction induced by TNF- (maximal contraction of 111 ± 10%). In contrast, CTX, an activator of G_s, did not prevent the enhancing effect of TNF- on carbachol-induced ASM contraction with maximal tensions of 131 ± 10 to 190 ± 14% (P < 0.05) in murine rings treated with CTX alone or in the presence of TNF- (Fig. 4A).

View larger version (16K): [in this window] [in a new window]   Fig. 3. Pertussis toxin (PTX) completely abrogates the enhanced contractile responses to carbachol induced by TNF-. Cumulative concentration-response curves to carbachol (A) or KCl (B) were performed on PTX (0.5 µg/ml), 1-h-treated rings preincubated overnight with diluent (, n = 6) or 50 ng /ml of TNF- (, n = 6). All tension measurements from different groups are expressed as means ± SE.

View larger version (18K): [in this window] [in a new window]   Fig. 4. Cholera toxin (CTX) has no effect on the enhanced contractile responses to carbachol induced by TNF-. Cumulative concentration-response curves to carbachol (A) or KCl (B) were performed on CTX (2 µg/ml), 1-h-treated rings preincubated overnight with diluent (, n = 8) or 50 ng /ml of TNF- (, n = 8). *P < 0.05 compared with rings treated with diluent. All tension measurements from different groups are expressed as means ± SE.

We also investigated whether PTX modulates the effect of TNF- on KCl-induced force generation. As shown in Fig. 3B, pretreatment of murine tracheal ring with PTX had no effect on either KCl-induced maximal contraction (93 ± 18% of KCl response in rings treated with diluent) or the EC₅₀ (41 ± 1.35 and 42.75 ± 1.17 mM in control and PTX-treated rings). Interestingly, PTX completely abrogated TNF--induced potentiation of KCl-evoked contraction (Fig. 3B). In contrast, CTX, which significantly decreased carbachol receptor affinity (pD₂ = 6.5 ± 0.039) compared with untreated rings, did not prevent the enhancing effect of TNF- on KCl-induced ASM contraction (Fig. 4B).

Effect of TNF- on agonist-induced relaxation of murine isolated tracheal rings. We studied the effect of TNF- on relaxant responses induced by PGE₂, isoproterenol, and forskolin, which directly activates adenylate cyclase. As shown in Fig. 5A, isoproterenol (10^-8 to 10^-5 M) caused a concentration-dependent relaxation of murine tracheal rings with a pD₂ of 7.28 ± 0.11. Pretreating the rings with 50 ng/ml TNF- significantly reduced isoproterenol-induced maximal relaxation responses (P < 0.05). The initial and maximal relaxant forces (%) were shown as 6.57 ± 2.87 and 59.64 ± 8.42 (n = 8) in the control rings and 7.49 ± 2.05 and 28.70 ± 2.19 (n = 7, P < 0.05) in TNF--treated rings. Interestingly, the inhibitory effect of TNF- on isoproterenol-induced maximal relaxation was abrogated in PTX-pretreated rings (Fig. 5B) with maximal relaxation responses of 65 ± 12% (n = 7) in the control rings and 52 ± 5% (n = 9) in TNF--treated rings. In separate experiments, we also investigated the effect of TNF- on forskolin and PGE₂. Figure 6 shows that the concentration-response relaxant responses to forskolin or PGE₂ were unaffected by TNF- with maximal responses (%) of 100 ± 11 (n = 9) and 62 ± 16 (n = 9) in the control rings and 98.3 ± 8.7 (n = 8) and 50 ± 11 (n = 7) in TNF--treated rings, respectively.

View larger version (17K): [in this window] [in a new window]   Fig. 5. PTX abrogates the attenuated relaxant responses to isoproterenol induced by TNF-. Cumulative concentration-response curves to isoproterenol were performed on treated rings overnight with diluent (, n = 8) or 50 ng /ml of TNF- (, n = 8) in the presence (B) or the absence (A) of PTX (0.5 µg/ml, 1 h). All tension measurements from different groups are expressed as means ± SE. *P < 0.05 and **P < 0.01 compared with rings treated with diluent.

View larger version (15K): [in this window] [in a new window]   Fig. 6. TNF- does not modulate the relaxant responses to PGE2 and to forskolin. Cumulative concentration-response curves to PGE2 (A) or forskolin (B) were performed in rings preincubated overnight with diluent (, n = 6) or 50 ng /ml of TNF- (, n = 6). All tension measurements from different groups are expressed as means ± SE.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES ACKNOWLEDGMENTS REFERENCES

 
The major finding of this study shows that TNF-, a potent cytokine involved in the pathogenesis of asthma, alters airway smooth muscle responsiveness to a variety of G-protein-coupled receptor agonists, i.e., TNF- enhanced the contractile responses to carbachol and selectively inhibited the relaxant responses to isoproterenol, a -adrenergic agonist. TNF- also increased the contractile responses to KCl, a nonreceptor-dependent stimulus. Interestingly, PTX, an inhibitory G_i protein, completely abrogated the modulatory effect of TNF- on murine tracheal rings.

The mechanisms underlying AHR, a characteristic feature of asthma, are complex and likely involve a variety of factors. The use of isolated airways preparation provided many investigators with a model 1) to describe the nature of the AHR (hyperreactivity characterized by an upward shift of the dose-response curve vs. hyperexcitability, which is a leftward shift of the curve) and 2) to determine the factors that modulate ASM responsiveness (reviewed in Refs. 8 and 32). Previous studies showed that human ASM passively sensitized with asthmatic serum exhibits a nonspecific increase in smooth muscle contractility to GPCR agonists, such as histamine (11, 39), leukotriene C4 (52), and acetylcholine (22). Asthmatic sensitized tissues also exhibit attenuation in the maximal relaxant capacity to -adrenergic stimulation (22, 24, 53). The nature of the mediators present in the serum of asthmatic patients that modulate smooth muscle responsiveness remains to date unknown. The present study demonstrates that cytokines may play a critical role in altering ASM responsiveness to GPCR agonists. We demonstrate that murine tracheal rings exposed to TNF- have impaired relaxant responses to isoproterenol (60% reduction), an effect that was not associated with a decreased -adrenoceptor receptor affinity (no change in EC₅₀) as previously reported in guinea pig tracheae treated with TNF- (60). Cultured murine ASM cells may represent an interesting approach to investigate the mechanisms by which TNF- modulated -adrenergic receptor function, but unfortunately, such an in vitro model is not available. Previous studies using cultured human ASM cells provide important observations such as the ability of TNF- to decrease isoproterenol-induced adenylyl cyclase activity (19) and cAMP-dependent gene expression (36). Here we found that TNF- did not attenuate the relaxant response induced by PGE₂ or forskolin, a nonreceptor-dependent relaxant agent that directly activates adenylate cyclase, suggesting that TNF- likely acts at the level of -adrenergic receptor activation. This hypothesis is supported by the fact that IL-1 can also decrease isoproterenol relaxant responses by possibly uncoupling the -adrenergic receptor to G_s (53). TNF- may also decrease -adrenergic receptor expression on ASM cells because TNF- significantly reduces the expression of muscarinic receptors on cultured ASM cells while increasing calcium responses (reviewed in Ref. 8). Whether these mechanisms are involved in the cytokine effect on -adrenergic responsiveness remains to be investigated.

In addition to its suppressive effect on -adrenergic-mediated relaxation, TNF- enhanced carbachol-induced maximal contractile responses without altering the muscarinic receptor affinity (no change in pD₂ value by TNF-). It is interesting to note that similar findings were reported in both guinea pig and human airways, where TNF- increased the contractile responses to cholinergic agents, without changing the receptor sensitivity (43, 44, 55). These data demonstrate that TNF- may use similar pathways in both human and mice to "prime" ASM to become hyperreactive to cholinergic stimulation. The effect of TNF- on ASM responsiveness seems to be both stimuli and species dependent because in other animal species, such as bovine tracheal rings, TNF- increases both the maximal contractile responses (i.e., hyperreactivity) and receptor sensitivity (50). The ability of TNF- to increase the contractile responses induced by several GPCR agonists and in a variety of animal species suggests that TNF- may modulate GPCR responsiveness by using similar mechanisms, i.e., receptor-G protein-effector complex, unlike the selective effect of TNF- observed on -adrenergic responsiveness (Fig. 5). In previous studies, we and others have shown that, in cultured human and canine ASM cells, TNF- or IL-1 significantly enhanced GPCR agonist-associated calcium homeostasis (2, 3, 5, 9, 28, 46, 51, 61). Interestingly, the enhancing effect of TNF- on agonist-evoked calcium signals was also not associated with changes in GPCR receptor affinity (3, 5). Because increases in the cytosolic calcium concentration and phosphorylation of the regulatory light chains of myosin II by the myosin light chain kinase represent key events that modulate ASM shortening and contraction (7, 30, 32), our data suggest that modulation of GPCR-associated calcium signaling may play a potential role in the development of bronchial hyperresponsiveness induced by cytokines. We also found that TNF- was more effective in enhancing the contractile response mediated by KCl, an agent that promotes contraction via calcium influx through the opening of voltage-operated channels (30). The ability of TNF- to enhance nonreceptor but calcium-dependent contractile responses supports the hypothesis that TNF- may also regulate ASM responsiveness by modulating events downstream to the GPCR receptor. In that regard, TNF- may act at the level of the contractile apparatus by increasing calcium sensitivity, as previously reported in guinea pig tracheal muscle (41, 43). Another mechanism explaining the effect of TNF- on the KCl-induced contractile response may involve a possible increase in ASM mass. This hypothesis seems unlikely given TNF-'s weak mitogenic activity on ASM cells, an effect that also would require longer incubation time (96 h) than that used in the present study (24 h) (9). Together these data suggest that the effect of TNF- on ASM responsiveness in murine tracheal rings may be complex and may involve mechanisms that include a modulation of the GPCR-associated signal transduction as well as an increased calcium sensitivity of the contractile elements.

Using RT-PCR analysis, we showed that both TNFR1 and TNFR2 are expressed in murine tracheal preparations, although the contribution of each receptor in TNF--induced ASM hyperresponsiveness remains unknown. Using TNFR-deficient mice (33, 42), we showed that TNFR1 is the predominant receptor mediating the enhancing effect of TNF- on agonist-evoked contractile responses. This finding was also suggested by the fact that D143N-A145R-TNF-, a mutant of TNF- that specifically activates TNFR2 (9, 37, 38), did not increase carbachol-induced ASM contractile responses. This is an important finding because recent reports using TNFR-deficient mice showed the involvement of both TNFR1 and TNFR2 in various aspects of the pathogenesis of allergic asthma such as AHR (34, 54) and airway inflammation (14, 45). Previously, we have shown that TNFR1 is expressed on ASM cells (1, 4, 9) and mediates the potentiating effect of TNF- on GPCR-coupled calcium signaling in human cultured ASM cells (8, 40). Interestingly, we also show that PTX, a G_i inhibitor (but not CTX, an activator of G_s) prevented the modulatory effects of TNF- on both cholinergic and -adrenergic responsiveness in agreement with previous observations showing the involvement of a PTX-sensitive pathway in the modulation of ASM responsiveness induced either by cytokines (23, 60) or by asthmatic serum (21). The mechanisms by which PTX abrogates cytokine effects on GPCR responsiveness remain unknown. Muscarinic M2 and M3 receptors that are coupled to both G_i and G_q are expressed on human ASM cells (20, 25, 59). Recent evidence now shows that the M2 receptor may act in concert with the M3 receptor to enhance ASM contractile responses to muscarinic agonists by inducing "calcium sensitization," a phenomenon that increases the sensitivity of the contractile apparatus to calcium (17). Such a calcium sensitization process induced by acetylcholine appears to involve a PTX-sensitive pathway. It is therefore possible that the modulation of G_i expression and/or activity in ASM by cytokines may augment ASM contraction to cholinergic stimulation by enhancing calcium sensitization. Along this line of investigation, it is interesting that levels of both G_i and G_q are significantly increased in human ASM cells after TNF- stimulation (27). Additional experiments are required to better define the mechanisms by which cytokines including IFN- (6) modulate GPCR responsiveness in ASM.

In conclusion, we now show that murine tracheal smooth muscle treated with TNF- has an enhanced contraction to muscarinic agonists and decreased relaxation to -adrenergic agonists, an effect that involves TNFR1 activation and PTX-sensitive pathways. The modulatory effects of TNF- on ASM muscle responsiveness to GPCR agonists may play an important role in AHR in chronic lung diseases such as asthma.

	DISCLOSURES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES ACKNOWLEDGMENTS REFERENCES

 
This work was supported by National Heart, Lung, and Blood Institute Grants 2R01-HL55301 and 1P50 [PDB] -HL67663 (both to R. A. Panettieri) and by an American Lung Association grant RG-062-N (to Y. Amrani). Yassine Amrani is a Parker B. Francis Fellow in Pulmonary Research.

	ACKNOWLEDGMENTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES ACKNOWLEDGMENTS REFERENCES

 
We thank Drs. W. Lesslauer and H. Loetscher for providing TNF- mutants and Dr. Phillip Scott (Veterinary School of the University of Pennsylvania) for generously donating TNFR-deficient mice. We thank Mary McNichol for assistance in the preparation of the manuscript.

	FOOTNOTES

 

Address for reprint requests and other correspondence: Y. Amrani, Univ. of Pennsylvania Medical Center, Pulmonary, Allergy and Critical Care Division, 848 Biomedical Research Bldg. II/III, 421 Curie Blvd., Philadelphia, PA (E-mail: amrani{at}mail.med.upenn.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES ACKNOWLEDGMENTS REFERENCES

 

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