Abstract
Hyperresponsiveness to bronchoconstrictor stimuli is a major pathophysiologic feature of asthma, but the molecular mechanisms behind this are not fully understood. The release of TNF-α and IL-1β during the inflammatory process is believed to play an important role in airway hyperresponsiveness. We have previously demonstrated, using a murine in vitro model of chronic airway inflammation, that TNF-α up-regulated bradykinin B1 and B2 receptors in the airway smooth muscle. By using the same model, the present study was designed to investigate the effects of IL-1β and its interaction with TNF-α on the expression of bradykinin B1 and B2 receptors in mouse tracheal smooth muscle. IL-1β up-regulated bradykinin B1 and B2 receptor expression and increased contractile response to bradykinin B1 and B2 receptor agonists (des-Arg9-bradykinin and bradykinin, respectively) in the tracheal smooth muscle. Transcriptional inhibitor actinomycin D, c-Jun N-terminal kinase (JNK) inhibitors SP600125 and TAT-TI-JIP153–163, but not extracellular signal–regulated kinase 1 and 2 (ERK 1/2) inhibitor PD98059, significantly attenuated this up-regulation, indicating that a transcriptional mechanism and intracellular JNK signal transduction pathway were involved. In addition, IL-1β did not affect bradykinin B1 and B2 receptor mRNA stability. Remicade, an anti–TNF-α antibody, markedly suppressed IL-1β–induced up-regulation of bradykinin B1 and B2 receptors, suggesting that TNF-α was involved in the up-regulation, which is further supported by the fact that IL-1β enhanced TNF-α mRNA expression in the tracheae. Intracellular JNK pathway and TNF-α might provide key links between inflammatory mediators like IL-1β and airway hyperresponsiveness to bradykinin.
This article demonstrates how IL-1β that appears in bronchoalveolar lavage from individuals with asthma, induces airway hyperresponsiveness via up-regulation of bradykinin B1 and B2 receptors. This process involves TNF-α and JNK, which might be of importance for the development of asthma.
Bradykinin is an important airway constrictor, formed from the kininogen precursor after proteolytic cleavage by kallikrein, which is further converted to des-Arg9-bradykinin by carboxypeptides N (7). The effects of different kinins are mediated by two G protein–coupled receptors termed bradykinin B1 and B2. Bradykinin B2 receptors show a high affinity for bradykinin and kallidin, whereas bradykinin B1 receptors show a high affinity for their metabolites, des-Arg9-bradykinin and des-Arg10-kallidin (8). Bradykinin is a weak constrictor of isolated human bronchi, but a potent bronchoconstrictor in vivo, in patients with asthma (9). The bradykinin B2 receptor is constitutively expressed, whereas the bradykinin B1 receptor is inducible (10, 11).
IL-1β and TNF-α have close relationships during inflammation. Both of them are agonists for intracellular mitogen-activated protein kinase (MAPK) pathways that consist of three major intracellular signal pathways: c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1 and 2 (ERK 1/2), and p38 (12, 13). We have recently demonstrated, using a murine in vitro model of chronic airway inflammation, that TNF-α could up-regulate bradykinin B1 and B2 receptors in the tracheal smooth muscle via activation of the intracellular JNK and ERK 1/2 pathways (14). By using the same model, the present study was designed to investigate effects of IL-1β on bradykinin B1 and B2 receptor expression in the airway smooth muscle, to study the underlying intracellular MAPK signal transduction mechanisms and to evaluate roles of TNF-α in this process.
Tracheae from 9- to 10-wk-old male BALB/c J mice (MB A/S, Ry, Denmark) was dissected and cut into three to four segments and placed individually into wells of a 96-well plate (Ultra-low attachment; Sigma, St Louis, MO) with 300 μl serum-free Dulbecco's Modified Eagle's Medium (DMEM) supplemented with penicillin (100 U/ml) and streptomycin (100 μg/ml) (15). The segments were incubated for 1, 2, 4, or 8 d at 37°C in humidified 5% CO2 in air in the absence and presence of murine IL-1β (IL-1β), murine TNF-α (mTNF-α), or human TNF-α (hTNF-α), with and without inhibitors (Remicade, actinomycin D, SP600125, TAT-TI-JIP153–163, or PD98059). The segments were moved into new wells containing fresh medium with the supplements of the cytokines and inhibitors every day.
The cultured segments were immersed in temperature-controlled (37°C) myographs containing 5 ml Krebs-Henseleit buffer-solution continuously equilibrated with 5% CO2 in O2 to result in a stable pH of 7.4 for recording of isometric tension. A pre-tension of 0.8 mN was applied to each segment, which has been demonstrated to be optimal (15). The segments were incubated with 3 μM indomethacin 30 min before administration of des-Arg9-bradykinin or bradykinin.
All data were expressed as mean values ± SEM. Each agonist concentration–effect curve was fitted to the Hill equation using an iterative, least square method (GraphPad Prism, San Diego, CA), to provide estimates of maximal contraction (Emax) and pEC50 values (negative logarithm of the agonist concentration that produces 50% of the maximal effect). Unpaired Student's t test or two-way ANOVA with Bonferroni post-test were used when two sets of data were compared, and one-way ANOVA with Dunnett's post-test was used for comparisons of more than two sets of data. P ⩽ 0.05 was considered to be statistically significant (n = number of experiments performed).
Recombinant murine IL-1β, normal control IgG, murine and human TNF-α (R&D Systems, Abingdon, UK), bradykinin and des-Arg9-bradykinin (Neosystem S.A., Strasbourg, France), PD98059 (2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one), N-monomethyl-l-arginine (l-NMMA), actinomycin D, indomethacin, DMEM and Krebs-Henseleit buffer (Sigma), Remicade (Centocor B.V., Leiden, The Netherlands), SP600125 (anthrax(1,9-cd)pyrazol-6(2H)-one), and TAT-TI-JIP153–163 (Calbiochem, Bad Sodem, Germany) were used. The vehicle for SP600125, TAT-TI-JIP153–163, and PD98059 was dimethyl sulfoxide (DMSO).
Tracheal smooth muscle strips or whole tracheae were homogenized and total RNA was extracted using RNeasy Mini-kit (QIAGEN GmbH, Hilden, Germany). Reverse transcription of total RNA (absorption ratio 1.6–2.0) to cDNA was performed using Omniscript reverse-transcriptase-kit (QIAGEN) in 20 μl volume reaction at 37°C for 1 h.
Real-time polymerase chain reaction (real-time PCR) was performed with QuantiTect SYBR Green PCR-kit (QIAGEN) in 25-μl reaction volumes and performed with heating 95°C for 15 min followed by touch down PCR—that is, denaturing at 94°C for 30 s and annealing at 66°C for the first PCR cycle; thereafter, a decrease of 2°C for the annealing temperature in every cycle until down to 56°C. Finally, 40 thermal cycles with 94°C for 30 s and 55°C for 1 min were performed (Smart Cycler II system; Cepheid, Sunnyvale, CA). The data were analyzed with the threshold cycle (Ct) method, and the specificity of the PCR products were checked by the dissociation curves.
Specific primers were synthesized by DNA Technology A/S (Aarhus, Denmark). The sequences are as follows. Bradykinin B1 receptor: forward, 5′-CCA TAG CAG AAA TCT ACC TGG CTA AC-3′; reverse, 5′-GCC AGT TGA AAC GGT TCC-3′. Bradykinin B2 receptor: forward, 5′-ATG TTC AAC GTC ACC ACA CAA GTC-3′; reverse, 5′-TGG ATG GCA TTG AGC CAA C-3′. TNF-α: forward, 5′-GAC TCA AAT GGG CTT TCC GA-3′; reverse, 5′-TCC AGC CTC ATT CTG AGA CAG AG-3′. β-actin: forward, 5′-TGG GTC AGA AGG ACT CCT ATG TG-3′; reverse, 5′-CGT CCC AGT TGG TAA CAA TGC-3′.
The relative amount of mRNA was obtained by the Ct values of mRNA for bradykinin B1 or B2 receptor or TNF-α in relation to the Ct values of mRNA for the housekeeping gene β-actin in the same sample. Data are presented as mean ± SEM. Statistical analyses were performed with unpaired Student's t test, and P ⩽ 0.05 was considered to be significant.
After the organ culture, the tracheae were homogenized in SDS sample buffer (0.5 M Tris pH 6.8, glycerol, Coomassie blue, 20% SDS, 0.05 M DTT, 0.1 M PSMF). After the homogenization, the samples were heated at 95–100°C for 5 min and centrifuged at 10,000 rpm for 10 min. The total protein was measured by Advanced Protein Assay Kit (Sigma-Aldrich Fluka, Buchs, Switzerland). Equal amounts of the protein were loaded onto NuPAGE Bis-Tris 4–12% gel (Invitrogen, Carlsbad, CA) and separated by electrophoresis (Mini vertical gel system; Thermo EC, Waltham, MA). The blot was transferred to Immobilon-P PVDF membranes (Millipore, Billerica, MA) and blocked in Tris-buffered saline (10 mM Tris-HCl pH 7.4, 0.9% NaCl) containing 5% milk for 1 h at room temperature, then incubated with primary antibody against mouse bradykinin B2 receptor (1: 2,000; Alexis Biochemicals, Lausen, Switzerland) overnight at 4°C, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (20 ng/ml) for 2 h at room temperature in Tris-buffered saline containing 5% milk. The immunoreactive signals were visualized by using SuperSignal West Pico chemiluminescent substrate (Pierce Biotechnology, Rockford, IL) and detected by using MAN-X X-ray system (Fujifilm Science Imaging Systems, Tokyo, Japan).
The tracheal segments were cultured for 4 d with different concentrations of IL-1β (0.1, 1, 10, or 100 ng/ml). An increase of the contractile responses to des-Arg9-bradykinin and bradykinin was observed. This increase was concentration-dependent on IL-1β (Table 1). The maximal contractile response for both agonists was reached with 10 ng/ml IL-1β. The potency for the agonist-induced contraction also increased with IL-1β concentrations, and reached statistical significance at 10 ng/ml for both agonists.
IL-1β (ng/ml) | des-Arg9-Bradykinin | Bradykinin | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| n | Emax (mN) | pEC50 | n | Emax (mN) | pEC50 | |||||
| 0 (control) | 18 | 0.89 ± 0.16 | 6.67 ± 0.17 | 13 | 1.93 ± 0.35 | 5.78 ± 0.19 | ||||
| 0.1 | 7 | 0.79 ± 0.19 | 7.14 ± 0.32 | 6 | 2.78 ± 0.93 | 6.12 ± 0.30 | ||||
| 1 | 9 | 2.01 ± 0.59* | 6.81 ± 0.29 | 6 | 2.88 ± 0.54 | 6.38 ± 0.32 | ||||
| 10 | 8 | 2.04 ± 0.22* | 7.38 ± 0.11 | 12 | 4.37 ± 0.41* | 7.43 ± 0.26* | ||||
| 100 | 5 | 1.60 ± 0.22 | 7.09 ± 0.35 | 6 | 4.92 ± 0.79* | 7.38 ± 0.31 | ||||
To assess time-course of IL-1β, tracheal segments were cultured for 1, 2, 4, or 8 d in the absence and presence of IL-1β (10 ng/ml). At Day 1, a significant increase of the response induced by des-Arg9-bradykinin and bradykinin in presence of IL-1β was seen. The maximal contractions for des-Arg9-bradykinin were increased at the similar level as after 2, 4, and 8 d of culture (Figures 1A, 1C, 1E, and G), whereas bradykinin-induced contraction reached the maximum at 4 d of culture and then, after 8 d, returned toward the same level as Day 1 (Figures 1B, 1D, 1F, and 1H). A general increase in potency was also seen during the culture periods, reaching statistical significance at 1 and 2 d for des-Arg9-bradykinin (Figures 1A, 1C, 1E, and 1G) and at 2 and 4 d for bradykinin (Figures 1B, 1D, 1F, and 1H).
Figure 1. Time-course of IL-1β on the contractile response to des-Arg9-bradykinin (A, C, E, G) and bradykinin (B, D, F, H). The tracheal segments were cultured for 1 d (A, B), 2 d (C, D), 4 d (E, F) and 8 d (G, H) in the absence (control) and presence of 10 ng/ml IL-1β. Each data point is derived from 8–18 experiments and presented as mean ± SEM. Statistical analysis was performed by two-way ANOVA with Bonferroni post-test. Asterisks indicate control versus IL-1β: *P < 0.05, **P < 0.01, ***P < 0.001.
[More] [Minimize]The relative amount of mRNA for bradykinin B1 and B2 receptors was quantified after 4 d organ culture by real-time PCR. Compared with the control (organ culture without IL-1β), mRNA levels of bradykinin B1 and B2 receptors were increased in the smooth muscle isolated from the tracheae treated by IL-1β (Figures 2A and 2B, P < 0.05).
Figure 2. Bradykinin B1 (A) and B2 receptor (B) mRNA expression in the tracheal smooth muscle. The tracheae were cultured for 4 d in the absence (organ culture) and presence of 10 ng/ml IL-1β, with and without 1 mg/ml Remicade or 10 μM SP600125 or vehicle (DMSO). Each data point is derived from three to four experiments and presented as mean ± SEM. Unpaired Student's t test was used. Organ culture versus Remicade or IL-1β; IL-1β versus IL-1β + Remicade; IL-1β + vehicle versus IL-1β + SP600125, N.S. = not significant; *P < 0.05 and ***P < 0.001. (C) Western blot demonstrated an increase in bradykinin B2 receptor protein expression. The tracheae were cultured for 4 d in the absence (control) and presence of IL-1β (10 ng/ml). Three identical experiments were performed. Each sample was derived from two mice. Lanes 1 and 3, control (Ctrl); lanes 2 and 4, IL-1β (10 ng/ml).
[More] [Minimize]Protein expression for bradykinin B2 receptors was examined by Western blot. There was a marked increase of bradykinin B2 receptor protein expression in the trachea cultured for 4 d in the presence of IL-1β, compared with the control (without IL-1β treatment) (Figure 2C).
IL-1β (10 ng/ml) did not affect carbachol-induced the maximal contraction in the segments cultured for 1 d (control Emax = 7.9 ± 0.7 versus IL-1β Emax = 7.1 ± 1.0 mN, n = 6, P > 0.05), 2 d (control Emax = 6.5 ± 0.7 versus IL-1β Emax = 6.6 ± 0.7 mN, n = 7, P > 0.05), 4 d (control Emax = 6.1 ± 0.4 versus IL-1β Emax = 6.3 ± 1.0 mN, n = 6, P > 0.05), and 8 d (control Emax = 3.6 ± 0.5 versus IL-1β Emax = 3.5 ± 0.5 mN, n = 12, P > 0.05).
To investigate transcriptional mechanisms, actinomycin D (a general transcriptional inhibitor) was used. The results showed that actinomycin D almost completely abolished IL-1β–enhanced contractile responses to des-Arg9-bradykinin and bradykinin (Figures 3A and 3B, P < 0.001). Accordingly, the mRNA levels were decreased after treatment with actinomycin D, but the receptor mRNA expression for either bradykinin B1 or B2 receptors mRNA was not altered by IL-1β (Figures 3C and 3D). Thus, the up-regulation of bradykinin B1 and B2 receptor mRNA expression by IL-1β was not mediated through an increase of receptor mRNA stability.
Figure 3. Effects of actinomycin D on des-Arg9-bradykinin (A)- and bradykinin (B)-induced contraction, and of IL-1β on bradykinin B1 (C) and B2 (D) receptor mRNA stability. The tracheae were cultured in the absence and presence of 10 ng/ml IL-1β with 5 μg/ml actinomycin D (ACD) for 24 h. Actinomycin D was added to the culture 30 min before IL-1β administration. Each data point is derived from 5–11 (for contraction) and 3–4 (for mRNA stability) experiments and presented as mean ± SEM.
[More] [Minimize]Inhibitors for intracellular JNK (SP600125) and ERK 1/2 (PD98059) were used to investigate if intracellular JNK and ERK 1/2 signal transduction pathways were required for the up-regulation of bradykinin B1 and B2 receptors by IL-1β. Tracheal segments were cultured for 4 d with IL-1β (10 ng/ml) together with either SP600125 (1, 10 μM), PD98059 (100 μM), or vehicle (DMSO). SP600125 at 10 μM almost completely abolished IL-1β–enhanced contraction induced by des-Arg9-bradykinin and bradykinin (Figures 4A and 4B, P < 0.001), while PD98059 had no effects (Figures 4A and 4B, P > 0.05). In addition, IL-1β–enhanced mRNA expression for bradykinin B1 and B2 receptors in the tracheal smooth muscle was significantly attenuated by SP600125 (Figures 2A and 2B, P < 0.05).
Figure 4. Effects of SP600125, TAT-TI-JIP 153–163 and PD98059 on IL-1β–enhanced contractile responses to des-Arg9-bradykinin (A) and bradykinin (B). The tracheal segments were cultured for 4 d in the presence of 10 ng/ml IL-1β with and without 10 μM SP600125 (SP), 1 μM TAT-TI-JIP153–163 (TAT-JIP), 100 μM PD98059 (PD), or vehicle (DMSO). Each data point is derived from 6–14 experiments and presented as mean ± SEM.
[More] [Minimize]Since SP600125 is relatively specific to JNK pathway (16), verification of the results was assessed by using another highly specific JNK inhibitor TAT-TI-JIP153–163, a small peptide and structurally unrelated to SP600125 (17). TAT-TI-JIP153–163 significantly inhibited IL-1β–enhanced contractile response to des-Arg9-bradykinin and bradykinin (Figures 4A and 4B, P < 0.05), which agrees well with SP600125 and that the JNK pathway at least in part was involved.
To ascertain if TNF-α was involved in IL-1β–enhanced the expression of bradykinin B1 and B2 receptors, the tracheal segments were cultured for 4 d with IL-1β (10 ng/ml) in the absence and presence of Remicade (0.1 and 1 mg/ml), a chimeric monoclonal anti–TNF-α antibody. Remicade inhibited IL-1β–enhanced bradykinin B1 and B2 receptor-mediated contraction (Figures 5A and 5B). The inhibition was more marked in des-Arg9-bradykinin-induced contraction (∼ 90% reduction) than in bradykinin-induced contraction (∼ 60% reduction). In the control experiment, normal control IgG did not affect the contractile responses to des-Arg9-bradykinin and bradykinin in the tracheal segments cultured for 4 d in the presence of IL-1β (Figures 5A and 5B).
Figure 5. Effects of Remicade on IL-1β (A, B)–, mTNF-α (C, D)–, and hTNF-α (E, F)–enhanced contractile response to des-Arg9-bradykinin (A, C, E) and bradykinin (B, D, F). The tracheal segments were cultured for 4 d in the presence of 10 ng/ml IL-1β, 100 ng/ml mTNF-α, or 10 ng/ml hTNF-α, with and without Remicade (0.1, 1 mg/ml) (Rem) or normal control IgG (0.1 mg/ml) (IgG). Each data point is derived from 7–18 experiments and presented as mean ± SEM.
[More] [Minimize]Furthermore, Remicade (1 mg/ml) abolished IL-1β–enhanced bradykinin B1 receptor mRNA expression by 100% and B2 receptor mRNA expression by 70% in the tracheal smooth muscle (Figures 2A and 2B, P < 0.05).
However, in the tracheal segments without IL-1β treatment, Remicade (1 mg/ml) had no inhibitory effects on des-Arg9-bradykinin–induced contraction (control Emax = 1.03 ± 0.23 versus Remicade Emax = 1.83 ± 0.36 mN, n = 5, P > 0.05) and bradykinin-induced contraction (control Emax= 1.93 ± 0.35 versus Remicade Emax = 2.28 ± 0.49 mN, n = 5, P > 0.05). In the same way, Remicade did not inhibit bradykinin B1 and B2 receptor mRNA expression (Figures 2A and 2B).
Remicade (0.1, 1 mg/ml) concentration-dependently decreased bradykinin B1 and B2 receptor-mediated contraction in the segments cultured for 4 d in the presence of mTNF-α (100 ng/ml) (Figure 5C-D). To confirm the results from mTNF-α, effects of Remicade on hTNF-α were tested. Remicade (1 mg/ml) almost totally revoked the hTNF-α-enhanced bradykinin B1 and B2 receptor-mediated contraction (Figure 5E-F). The inhibitory effect of Remicade was stronger in the hTNF-α than in the mTNF-α group, indicating that Remicade may have stronger affinity for hTNF-α than for mTNF-α.
In addition, Remicade 1 mg/ml significantly suppressed both mTNF-α- and hTNF-α-enhanced bradykinin B1 and B2 receptor mRNA expression in the tracheal smooth muscle (Figures 6A and 6B).
Figure 6. Bradykinin B1 (A) and B2 receptor (B) mRNA expression in the tracheal smooth muscle. The tracheae were cultured for 4 d in the presence of 100 ng/ml mTNF-α or 10 ng/ml hTNF-α, with and without 1 mg/ml Remicade (Rem). Each data point is derived from three to four experiments and presented as mean ± SEM. Unpaired Student's t test was used. TNF-α versus TNF-α + Rem; *P < 0.05.
[More] [Minimize]To further investigate interactions between IL-1β and TNF-α, TNF-α mRNA expression was quantified by real-time PCR. The results showed that IL-1β (10 ng/ml) increased TNF-α mRNA expression in the whole tracheae with epithelium (Figure 7). Similarly, IL-1β (10 ng/ml) increased TNF-α mRNA expression in the tracheal smooth muscle; however, the level of TNF-α mRNA expression in the tracheal smooth muscle was much lower than in the whole tracheae with epithelium (Figure 7).
Figure 7. TNF-α mRNA expression in the tracheal smooth muscle (4 d of culture) and the whole trachea with epithelium (2 d of culture). The tracheae were cultured in the absence (control) and presence of 10 ng/ml IL-1β for 2 or 4 d. Each data point is derived from three to five experiments and presented as mean ± SEM. Unpaired Student's t test was used. **P < 0.01, ***P < 0.001.
[More] [Minimize]To test if nitric oxide (NO) production was involved in our system, the NOS inhibitor l-NMMA (100 μM) was added to the tissue bath 30 min before the administration of des-Arg9-bradykinin and bradykinin. There were no significant difference between l-NMMA and control groups in the segments cultured for 4 d with IL-1β (10 ng/ml), for des-Arg9-bradykinin (control group Emax 2.05 ± 0.55 and pEC50 7.83 ± 0.19 versus l-NMMA group Emax 3.01 ± 0.33 and pEC50 7.73 ± 0.25, n = 5, P > 0.05) and for bradykinin (control group Emax 4.59 ± 0.39 and pEC50 7.45 ± 0.19 versus L-NMMA group Emax 4.97 ± 0.44 and pEC50 7.51 ± 0.09, n = 5, P > 0.05).
The inflammatory mediator TNF-α, IL-1β, and bradykinin play critical roles in asthmatic inflammation, but the interactions between these mediators are far from known (1). We have developed an in vitro model of airway inflammation, and demonstrated that TNF-α up-regulated the expression of bradykinin B1 and B2 receptors in the airway smooth muscle and that intracellular JNK and ERK 1/2 pathways were involved in this process (14). The present study revealed a similar phenomenon that IL-1β–up-regulated bradykinin B1 and B2 receptor–mediated contraction as well as the receptor expression in mouse tracheal smooth muscle. JNK inhibitor SP600125 and TAT-TI-JIP153–163, but not ERK 1/2 inhibitor PD98059 inhibited this up-regulation, suggesting that intracellular JNK signal transduction pathway was involved. A general transcriptional inhibitor, actinomycin D, completely abolished the IL-1β–enhanced contraction-induced by des-Arg9-bradykinin and bradykinin, indicating a transcriptional mechanism that was responsible and further supported by IL-1β did not affect bradykinin B1 and B2 receptor mRNA stability. Remicade, a chimeric monoclonal anti–TNF-α antibody, markedly inhibited IL-1β–up-regulated bradykinin B1 and B2 receptor–mediated contraction and the receptor mRNA expression in the airway smooth muscle. This reveals that the effect of IL-1β was at least partly mediated by TNF-α production. In addition, IL-1β increased the expression of TNF-α mRNA in the airway.
It has been reported that IL-1β acted on the Rho/Rho-kinase pathway and up-regulated contractile protein expression in coronary artery smooth muscle in vivo in the porcine model (18, 19). However, we have previously demonstrated that IL-1β induced a down-regulation of ETB, while it did not affect ETA receptor–mediated contraction in the tracheal smooth muscle in an in vitro model (20). By using the same methods, the present study has revealed that IL-1β up-regulated bradykinin B1 and B2 receptors; however, it did not alter carbachol-induced contraction. Taking all the results from our in vitro model together, the data suggest that IL-1β induces a receptor alteration, rather than the general hyperresponsiveness (i.e., increase in contractile protein expression). This discrepancy is most likely due to the differences between the airway and the artery smooth muscle cells, animals, experimental assays, in vitro and in vivo models.
In mice, bradykinin and des-Arg9-bradykinin are commonly used to activate bradykinin B2 and B1 receptors, respectively. The potency for bradykinin B2 receptor is Lys-bradykinin = bradykinin > >>Lys-des-Arg9-bradykinin = des-Arg9-bradykinin; for the B1 receptor it is Lys-des-Arg9-bradykinin > des-Arg9-bradykinin ⩾ Lys-bradykinin > bradykinin (21). We have revealed that simultaneous use of bradykinin B1 and B2 receptor antagonists, [Des-Arg10]HOE140 (100 nM) and HOE140 (100 nM), caused a further rightward shift and depressed maximal contraction of bradykinin concentration–effect curves, suggesting that bradykinin at high concentrations might also activate bradykinin B1 receptors (14). Bradykinin B1 and B2 receptors display low sequence homology and they exhibit different expression patterns. Bradykinin B1 receptor expression is inducible (11). The expression level of contractile bradykinin B1 receptors is generally low during normal circumstances, but is up-regulated or synthesized de novo during inflammatory conditions (22). Our studies revealed that in fresh tracheal segments from mouse, there is only negligible response to des-Arg9-bradykinin (14), while the segments after culture in presence of IL-1β have strong contractile response to des-Arg9-bradykinin, and this is in parallel with an increase in bradykinin B1 receptor mRNA expression, suggesting that bradykinin B1 receptors are induced.
Remicade is a mouse Fv-human IgG1-k antibody with high affinity for human TNF-α (23). It binds with specificity to both soluble and membrane-bound forms of TNF-α, but does not interact with TNF-β (24). Remicade is successfully used in the treatment of rheumatoid arthritis and Crohn's disease (25, 26). In the present study, we demonstrated that both mTNF-α and hTNF-α enhanced des-Arg9-bradykinin– and bradykinin-induced airway contraction and that Remicade could abolish this enhancement with different degrees. Remicade at 1 mg/ml almost completely abolished hTNF-α–increased bradykinin B1 and B2 receptor–mediated contraction, while it inhibited only 50–60% of the corresponding response in the mTNF-α–treated group. This difference in effects of Remicade is probably due to a higher affinity to hTNF-α than to mTNF-α.
Proinflammatory cytokines are mainly produced de novo in response to inflammatory stimuli. They generally act over a short distance (intercellular space) and exists therefore at low circulating levels (27). In the present study, the concentrations of the cytokines used in culture medium and the agonists in tissue bath seem relatively high, but presumably, the concentrations gained in the intercellular space should reflect paracrine or autocrine secretion. Since the levels of enhanced bradykinin responses by IL-1β and TNF-α were very similar and given the ability of Remicade to inhibit the effect of IL-1β, the TNF-α concentration given in the well might reflect the IL-1β–induced secretion of TNF-α in the intercellular space. Our finding of IL-1β–enhanced bradykinin B1 and B2 receptor expression is well in line with in vivo data demonstrating that bradykinin B1 and B2 receptors were involved in allergen-induced airway hyperresponsiveness in rat (28), and bradykinin-induced airway contraction was enhanced in human subjects with asthma (8). Thus, our in vitro finding is relevant to whole animal and human pathophysiology.
The present study showed that IL-1β increased TNF-α mRNA expression in the whole tracheae with epithelium. Similarly, IL-1β increased TNF-α mRNA expression in the tracheal smooth muscle; however, the level of TNF-α mRNA expression in the tracheal smooth muscle was much lower than in the whole tracheae with epithelium. The up-regulation of TNF-α mRNA expression by IL-1β in the tracheae suggests a local increase in TNF-α production under IL-1β stimulation. It has been reported that IL-1β induces an increase of TNF-α secretion in an alveolar macrophage cell line (MH-S murine) (29) and an increase in the production of TNF-α in antigen-challenged guinea pig airways (30). However, we were not able to measure any TNF-α in the cultured medium by enzyme-linked immunosorbant assay (ELISA) after organ culture of the tracheal segments in the presence and absence of IL-1β. This might be due to the fact that the ELISA is rather insensitive, or the release of TNF-α is similar to the release of endothelin-1, which is mainly directed toward the smooth muscle cells (31).
Both IL-1β and TNF-α can activate MAPK. The intracellular MAPK signal pathways consist of JNK, ERK1/2, and p38 and their downstream transcription factors (12, 32). The effectors of these pathways include proliferation, contraction, migration, apoptosis, receptor expressions, and peptides synthesis in airway (33–35). We have recently demonstrated that the intracellular JNK and ERK 1/2 pathways were involved TNF-α–induced up-regulation of bradykinin B2 receptors (14). In the present study, SP600125 has powerfully inhibitory effects on IL-1β-enhanced upregulation of bradykinin B1 and B2 receptors, while the intracellular ERK 1/2 inhibitor PD98059 had no effects. Interestingly, the lack of inhibition by PD98059 on the IL-1β–induced up-regulation of B2 receptors can partly be due to a direct effect of IL-1β on the smooth muscle and partly due to a secretion of TNF-α, since it was not blocked by more than 60% by Remicade. Conversely, Yang and coworkers has reported that the intracellular ERK 1/2 pathway was involved in IL-1β–induced up-regulation of bradykinin B2 receptors in canine cultured tracheal smooth muscle cells (36). The long-term culture procedure along with the use of different species and different models might be one explanation for this dissimilarity.
SP600125 was developed as an inhibitor of JNK, but it may inhibit other kinases as well (16). Thus, a more selective and specific JNK inhibitor, TAT-TI-JIP153–163 (a small peptide, cell membrane permeable and structurally unrelated to SP600125), was used to confirm the involvement of JNK pathway. TAT-TI-JIP153–163 had a significant inhibitory effect on the IL-1β–induced up-regulation of bradykinin B1 and B2 receptor expression, suggesting that the JNK pathway was involved. Since the signaling transduction inhibitors had been washed away before the tracheal segments were mounted in tissue bath, and then the tracheal segments have incubated more than 2 h before the administration of agonists, it abolishes a direct effect of the inhibitors on contractility of the segments (data not shown). The contractility of the tracheal segments was tested on carbachol-induced contraction, with no differences in the presence or absence of the inhibitors (SP600125 and PD98059) (14). In addition, we have previously revealed that PD98059 and SP600125 could inhibit organ culture (the control)-induced up-regulation of bradykinin receptor expression (14). The present study showed that IL-1β further enhanced this up-regulation. SP600125, but not PD98059, could inhibit IL-1β-induced up-regulation of bradykinin B1 and B2 receptor expression in the tracheal smooth muscle.
The intracellular JNK inhibitor SP600125 exhibited a much stronger inhibition on bradykinin B1 receptor–mediated contraction than on bradykinin B1 receptor mRNA expression. This difference is most likely due to a “time-window” between transcription of the receptor mRNA and translation of the receptor protein for the function of the receptors. However, there was no evidence that angiotensin-converting enzyme (ACE), endothelial function and cholinergic activity were involved in our model (14). In the present study, higher levels of bradykinin B1 receptor mRNA than bradykinin B2 receptor mRNA were in all groups, while the level of bradykinin B1 and B2 receptors-mediated contractions were more or less identical. Particularly, in the fresh tracheal segments, there was a considerable amount of bradykinin B1 receptor mRNA (data not shown), but bradykinin B1 receptor only elicited a negligible contraction (14). This indicates that the levels of receptor mRNA is not necessarily in concert with the level of the achieved receptor-mediated contraction, although we in most cases found that changes of the receptor mRNA paralleled with the altered levels of the receptor-mediated contraction (37).
In conclusion, IL-1β induced a transcriptional up-regulation of bradykinin B1 and B2 receptor expression in mouse tracheal smooth muscle. The intracellular JNK signal transduction pathway was involved in this up-regulation. In addition, TNF-α was at least partly participated in this process. Finding a link between airway hyperresponsiveness and inflammatory mediators during airway inflammation may provide a new way for the treatment of asthmatic inflammation.
The authors are grateful to Ann Reutherborg and Ingegerd Larsson for the RNA isolation.
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