INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Several lines of evidence point to the involvement of the proinflammatory cytokines interleukin (IL)-1 and tumor necrosis factor (TNF)- in mediating the airway inflammatory processes and the altered airway responsiveness, which are characteristic features of asthma (1). Apart from being significantly increased in bronchoalveolar lavage fluid from symptomatic patients with asthma (4), both IL-1 and TNF- have been demonstrated to induce a pronounced airway eosinophilia in guinea pigs (5). Moreover, IL-1 and TNF- may also contribute to the alterations in airway responsiveness, as it was shown that inhalation of TNF- or administration of IL-1 caused an increase in airway responsiveness (6, 7). Furthermore, an IL-1 receptor antagonist suppressed bronchial hyperreactivity and inflammatory cell infiltration following allergen challenge in sensitized guinea pigs, confirming an important role for IL-1 and TNF- in allergic inflammation and in the development of late asthmatic responses (2). Recently, IL-1 and TNF- have been demonstrated to act directly on airway smooth muscle cells, resulting in -adrenergic hyporesponsiveness (8) and potentiation of Ca²⁺ responses by bradykinin (9), providing mechanisms underlying bronchial hyperreactivity in patients with asthma.

To further address this hypothesis, we have examined whether IL-1 and TNF- might also induce alterations in histamine H₁-receptor (H₁R) signaling in human airway smooth muscle. Histamine is an important inflammatory mediator in asthma and bronchial hyperreactivity to histamine is one of the diagnostic features of asthma. Histamine-induced airway smooth muscle contractions are mediated via H₁R, coupled via a regulatory G_q protein to phospholipase C. Stimulation of H₁R leads to the formation of two secondary messengers, inositol 1,4,5-triphosphate (IP₃) and diacylglycerol (DAG) (10). IP₃ increases intracellular Ca²⁺ from the endoplasmic reticulum, via interacting with a specific receptor, resulting in an initial transient contractile response, whereas DAG activates protein kinase C (PKC), which is believed to underlie the sustained phase of the smooth muscle contraction (11).

In this study we have investigated whether IL-1 and TNF- affected histamine-induced inositol phosphate (IP) accumulation, which reflects the level of IP₃ formation, in human airway smooth muscle cells and the contractile responses to histamine in human bronchial rings.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Culture of Human Airway Smooth Muscle Cells

Human airway smooth muscle cells (HASMC) were grown from explants of human bronchial smooth muscle, as previously described (12). Briefly, airway tissue was obtained from resections of patients undergoing surgery for lung carcinoma. None of the patients had characteristics of asthma. Bronchial smooth muscle tissue was carefully dissected free of surrounding tissue. Small explants (2 × 2 mm) of the bronchial muscle were prepared and placed in a petri dish. After allowing the explants to adhere, Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 µg/ml), and amphotericin B (1.25 µg/ml) was added, just to cover the explants. The medium was changed every day until the cells started to grow, whereafter the cultures were supplemented with fresh DMEM containing FCS, glutamine, antibiotics, and amphotericin B every 3 d. When the cells were reaching confluency in some parts of the petri dish, the explants were removed and 24 h later the cells were harvested with trypsin/EDTA and plated into a 75-cm² flask where they were grown to confluency. After subculturing the cells twice, the cultures were characterized immunohistochemically, using an anti-human smooth muscle actin antibody. Primary cell cultures used for the experiments showed > 95% of cells staining for smooth muscle actin. All experiments were carried out between passage 3 and 9.

Accumulation of Total [³H]Inositol Phosphates

Accumulation of total [³H]inositol phosphates ([³H]IP) in primary cultures of HASMC, which reflects the level of IP₃ formation, was performed as described by Daykin and coworkers (13). In brief, HASMC were seeded in 24-well plates and grown to confluency. The medium was aspirated and replaced by 300 µl inositol-free DMEM, supplemented with 2 µCi ml¹ [³H]myoinositol for 24 h. Where IL-1 and/or antagonists were present for 24 h, they were added with the [³H]myoinositol. After 24 h the medium was removed and the cells were washed twice with 1 ml Hanks/HEPES buffer. The cells were incubated for 15 min with 300 µl of Hanks/HEPES buffer, containing 20 mM LiCl. Finally, agonists were added in a volume of 10 µl. After 30-min incubation at 37° C the reactions were stopped by removing the medium of each well and adding 1 ml of methanol/HCl (1:1, vol/vol) which was previously kept 20° C. Samples were then stored at 20° C for at least 2 h. An 800 µl aliqout of each sample was then neutralized with a mixture of 25 mM Tris/0.5 M NaOH/H₂O (0.238/0.025/0.737 vol/ vol/vol). Total [³H]IP was finally separated from free [³H]myoinositol by anion-exchange chromatography on Dowex-Cl columns (13).

Measurement of Airway Smooth Muscle Responsiveness

Contraction measurements of human airway smooth muscle was performed as described by Verleden and coworkers (14). Briefly, macroscopically normal bronchial tissue was obtained from thoracotomy specimens of patients undergoing surgery, mostly for resection of bronchial carcinoma. None of the patients had characteristics of asthma. Immediately after surgical resection, a macroscopically normal part of the lung tissue was immersed in cooled (4° C) and aerated (5% CO₂ in 95%O₂) Krebs-Henseleit solution (KHS) of the following composition: NaCl 118, KCl 5.9, MgSO₄ 1.2, CaCl₂ 2.5, NaH₂PO₄ 1.2, NaHCO₃ 25.5, and dextrose 5.5 mM (pH 7.4). The airways were carefully stripped from surrounding lung tissue and cut into strips (main bronchi) or ring segments (segmental and subsegmental bronchi). The tissues were incubated in the absence or presence of IL-1 (10 ng/ml) for 24 h in oxygenated KHS at room temperature, after which the tissue preparations were washed and mounted in 10-ml organ baths containing oxygenated KH at 37° C. Thin silk threads were tied to both ends of the strips and through the bronchial rings. One thread was connected to a steel hook at the bottom of the organ bath and the other was connected to the arm of a Grass FT 03 force displacement transducer (Stag Instruments Ltd., Chalgrove, Oxon, UK). The preparations contracted against a load of 2 g, which has previously been shown to produce optimal repeatable responses in similar preparations (14). The tissues were allowed to equilibrate for 30 min during which they were washed with fresh KH every 10 min, after which a stable baseline tension was achieved. Contractility to histamine was subsequently assessed by cumulative administration of histamine in final bath concentrations ranging from 1 µM to 10 mM. The results were expressed as a percentage of the contraction to KCl (100 mM), which induces receptor-independent contraction and which was determined at the beginning of the experiment.

Immunoblot Analysis of Thr/Tyr-phosphorylated SAPK/JNK and p38 MAP Kinase

Extraction of cytosolic proteins. Following treatment, the cells were rinsed with cold phosphate-buffered saline (PBS) and scraped into lysis buffer (25 mM HEPES, 0.3 M NaCl, 1.5 mM MgCl₂, 20 mM -glycerolphosphate, 2 mM EDTA, 2 mM EGTA, 1 mM dithiothreitol [DTT], 1% Triton X, 10% glycerol, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 200 nM microcystin, and 1 mM sodium orthovanadate), vortex mixed, and incubated on ice for 30 min, followed by centrifugation (20 min, 12,000 × g). The cytosolic proteins were stored at 20° C until used for Western blot analysis.

Western blot analysis. The threonine and tyrosine phoshorylation of SAPK/JNK and p38 MAP kinase was analyzed by Western blot analysis, using antiactive SAPK/JNK polyclonal antibodies (Promega, Leiden, The Netherlands) and Phospho-p38 MAP kinase polyclonal antibodies (New England Biolabs Inc., Westburg, Leusden, The Netherlands). Protein samples (100 µg, in sample buffer: 62.5 mM Tris, 10% glycerol, 2% sodium dodecyl sulfate [SDS], and 10 mM 2-mercaptoethanol) were separated by SDS-polyacrylamide gel electrophoresis on 10% acrylamide gels and then transferred to a nitrocellulose membrane. To block nonspecific protein binding, the membrane was incubated at room temperature for 1 h in blocking buffer (0.1% Tween 20 in Tris-buffered saline containing 5% wt/vol skim powdered milk). The membrane was then incubated overnight at 4° C with antiactive SAPK/JNK polyclonal antibodies (1:5,000) and Phospho-p38 MAP kinase polyclonal antibodies (1:1,000; Promega) in blocking buffer without Tween 20. The membrane was washed with blocking buffer, without skim milk, six times for 5 min, incubated with a 1:10 000 dilution of alkaline phosphatase-conjugated anti-rabbit secondary antibody in blocking buffer without Tween 20. The membrane was washed as before, and protein detection was carried out using enhanced chemiluminescence reagent and exposed against Hyperfilm (Amersham Pharmacia Biotech, Roosendaal, The Netherlands). To reprobe the membrane, antibodies were stripped using 2% SDS, 100 mM 2-mercaptoethanol, and 62.5 mM Tris, pH 6.7, at 50° C for 30 min.

Data Analysis

All data are expressed as mean ± SE mean. The effect of IL-1, NaF, anisomycin, and PGE₂ on the histamine-induced IP accumulation and the effect of the inhibitors (PDTC, SB203580, PD98059, indomethacin, and H-89) on the IL-1-induced inhibitory effect were carried out by analysis of variance (ANOVA). Probability values of < 0.05 were considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The Effect of IL-1 and TNF- on Histamine-Induced Inositol Phosphate Accumulation in HASMC

Histamine induced a concentration-dependent accumulation of [³H]IP in cultured HASMC (Figure 1A). The histamine-induced inositol phosphate formation was completely antagonized, using mepyramine (1 µM), a selective H₁R antagonist (results not shown), providing evidence that this response was mediated through the H₁R subtype. A 24-h incubation period with IL-1 (10 ng/ml) resulted in a significant decrease in histamine-induced IP accumulation (n = 7, p < 0.05) (Figure 1A). There was, however, no difference between control and TNF- (10 ng/ml) pretreated HASMC with regard to the histamine-induced IP accumulation (Figure 1A). This is in contrast to IP accumulation following bradykinin stimulation, which was clearly enhanced by pretreatment (24 h) with both IL-1 and TNF- (n = 3, p < 0.05) (Figure 1A). Figure 1B demonstrates the dose-dependent inhibition of histamine-induced IP accumulation following IL-1 (0.01-10 ng/ml, n = 3) stimulation, as well as the time-dependent effect of IL-1 (10 ng/ml, n = 3) on the histamine-induced IP accumulation. To determine whether the IL-1-induced changes occurred at the level of the H₁R or downstream in the signaling pathway, IP accumulation to sodium fluoride (NaF), a G-protein activator, was performed. We observed no difference in NaF-induced IP formation between control and IL-1-treated HASMC, indicating that the observed IL-1-induced desensitization of the H₁R is mediated at the level of the receptor or at the coupling with its G-protein (Figure 1C).

View larger version (39K): [in this window] [in a new window]   Figure 1.   Functional desensitization of H1R by IL-1 and TNF-. HASMC were treated with IL-1 (10 ng/ml) or TNF- (10 ng/ml) for 24 h, after which the cells were stimulated with increasing concentrations of histamine (0.1 nM-1 mM, A), bradykinin (0.1 pM-10 µM, A), or NaF (1-30 mM, C ). B shows the dose (at 24 h) and time (at 10 ng/ml) effect of IL-1 on histamine (10 µM)-induced IP accumulation. The accumulation of [3H]inositol phosphates was then determined as described (see METHODS). Data are the mean ± SE of three to six independent experiments.

IL-1-induced Alterations in ASM Responsiveness to Histamine

To examine whether the observed attenuation in histamine-induced IP accumulation by IL-1 could be associated with an altered airway smooth muscle responsiveness to histamine, we examined airway constrictor responses in isolated human bronchial smooth muscle preparations. As shown in Figure 2, IL-1 (10 ng/ml) pretreatment induced a significant loss of subsequent histamine-induced contractions (n = 3, p < 0.001), demonstrating a correlation between IL-1-induced reduction in inositol phospolipid hydrolysis in HASMC and the loss of contractile response to histamine in bronchial smooth muscle preparations.

View larger version (14K): [in this window] [in a new window]   Figure 2.   Effect of IL-1 on histamine-induced contractile responses in human bronchial tissue. Comparison of histamine-induced cumulative concentration-response curves in control (open circles) and IL-1-treated (10 ng/ ml for 24 h) (closed circles) human bronchial tissue. Data are the mean ± SE of 3 independent experiments.

Effect of IL-1 and Anisomycin on the Thr/Tyr Phosphorylation of SAPK/JNK and p38 MAP Kinase

In Figure 3 it is demonstrated that IL-1 (10 ng/ml) phosphorylates and activates SAPK/JNK and p38 MAP kinase in HASMC. Furthermore it is demonstrated that anisomycin, at a concentration below that required for inhibiting translation (0.1 µM), induces Thr/Tyr phosphorylation of SAPK/JNK and p38 MAP kinase (n = 3).

View larger version (50K): [in this window] [in a new window]   Figure 3.   Activation of SAPK/JNK and p38 MAP kinase by IL-1 and anisomycin. HASMC were stimulated with IL-1 (10 ng/ml) or with anisomycin (A, 0.1 µM) for 15 min. The HASMC lysates were then separated in an SDS-polyacrylamide gel, transferred to nitrocellulose membranes, and probed with a specific antibody directed against the phosphorylated threonine and tyrosine of p38 MAP kinase (upper panel ). The blots were stripped and reprobed using a specific antibody directed against the phosphorylated threonine and tyrosine of SAPK/ JNK (lower panel ). U, untreated cells.

Effect of MAP Kinase Inhibitors on the IL-1-induced H₁R Desensitization

In separate experiments we attempted to characterize the intracellular pathways leading to the IL-1-induced H₁R desensitization. Treatment of HASMC with IL-1 in the presence of a p38 MAPK inhibitor, SB 203580 (10 µM), could completely block IL-1-induced H₁R desensitization (n = 6, p < 0.01), suggesting that activation of the p38 MAP kinase pathway might be involved in the observed effect (Figure 4A). Moreover, anisomycin (0.1 µM), a strong activator of SAPK/ JNK and p38 MAP kinase, could mimic the IL-1-induced reduction in histamine-induced IP accumulation (n = 4, p < 0.001). PD 98059 (10 µM), a selective and potent inhibitor of p44/42 MAP kinase cascade, was without effect on the IL-1-induced desensitization of the H₁R (Figure 4C). Further, PDTC (10 µM), an inhibitor of NF-B activation, partly inhibited H₁R desensitization by IL-1, suggesting a role for NF-B in the mechanism leading to uncoupling of the H₁R (Figure 5). None of the antagonists used had any effect on their own on the histamine-induced IP accumulation (Figures 4 and 5).

View larger version (19K): [in this window] [in a new window]   Figure 4.   Effect of MAP kinase inhibitors or activators on IL-1-induced H1R desensitization. HASMC were treated with anisomycin (0.1 µM for 24 h, B) or with IL-1 (10 ng/ml) in the absence or presence of SB203580 (10 µM, A) or PD 98059 (10 µM, C ) for 24 h, after which the cells were stimulated with increasing concentrations of histamine (0.1 nM-1 mM). The accumulation of [3H]inositol phosphates was then determined as described (see METHODS). Data are the mean ± SE of four to six independent experiments.

View larger version (21K): [in this window] [in a new window]   Figure 5.   Effect of the inhibition of NF-B on IL-1-induced H1R desensitization. HASMC were treated with IL-1 (10 ng/ml) in the absence or presence of PDTC (10 µM for 24 h), after which the cells were stimulated with increasing concentrations of histamine (0.1 nM-1 mM). The accumulation of [3H]- inositol phosphates was then determined as described (see METHODS). Data are the mean ± SE of six independent experiments.

Effect of Indomethacin and PGE₂ on the IL-1-induced H₁R Desensitization

To examine whether IL-1-induced prostaglandin synthesis might play a role in H₁R desensitization, we looked at the effect of indomethacin (10 µM), a nonselective cyclooxygenase inhibitor. Indomethacin blocked the IL-1-induced effect (n = 6, p < 0.05), suggesting a role for prostaglandin synthesis in H₁R desensitization (Figure 6A). It is known that HASMC release prostanoids, with PGE₂ as a principal product, upon stimulation with IL-1. Because indomethacin blocked H₁R desensitization by IL-1, we suggested an involvement of PGE₂. Therefore, we investigated the effect of PGE₂ on histamine-induced IP accumulation, and as shown in Figure 6B, PGE₂ (1 µM) preincubation of HASMC resulted in a desensitization of the H₁R (n = 6, p < 0.05). Taken together, we hypothesized that phosphorylation of the H₁R by PKA, activated by PGE₂-induced cAMP formation, could be the mechanism of action. To support the above hypothesis we examined the effect of H-89 (3 µM), a selective inhibitor of PKA activation, on IL-1-induced H₁R desensitization. H-89 completely antagonized the IL-1-induced loss of IP formation in response to histamine (n = 4, p < 0.01), indicating a role for PKA in the above observed desensitization (Figure 6C). None of the antagonists used had any effect on histamine-induced IP accumulation.

View larger version (20K): [in this window] [in a new window]   Figure 6.   Effect of PGE2 on H1R-mediated accumulation and the effect of indomethacin and H-89 on H1R desensitization by IL-1. HASMC were treated with PGE2 (1 µM for 1 h, B) or with IL-1 (10 ng/ml) in the absence or presence of indomethacin (indo, 10 µM, A) or H-89 (3 µM, C ) for 24 h, after which the cells were stimulated with increasing concentrations of histamine (0.1 nM-1 mM). The accumulation of [3H]inositol phosphates was then determined as described (see METHODS). Data are the mean ± SE of four or five independent experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Recent reports have provided some insight into the potential mechanisms involved in the impaired airway relaxation to ₂-adrenoceptor stimulation (15) and in the exaggerated bronchoconstriction to contractile agonists (16), which are characteristic features of bronchial asthma. It is suggested that IL-1 and TNF-, which have been implicated in orchestrating and perpetuating asthmatic airway inflammation (3), can alter airway responsiveness by direct effects on airway smooth muscle, such as enhanced Ca²⁺ response to bradykinin (9) and uncoupling of ₂-adrenoceptors (8), and therefore represent a mechanism underlying bronchial hyperreactivity in patients with asthma. The unexpected finding in the present study, however, is that IL-1 attenuates histamine-induced IP formation in HASMC and decreases contraction of bronchial tissue to histamine, which suggests a protective effect of IL-1 in histamine-induced airway contractile responses, which has been suggested before in dog airway smooth muscle (17). Furthermore, our findings indicate that IL-1-induced changes in H₁R signaling do not correlate with the characteristic bronchial hyperreactivity to histamine, which is one of the diagnostic features of asthma.

Surprisingly, the effect at the level of the H₁R cannot be repeated with TNF-, a proinflammatory cytokine that may also contribute, although to a lesser extent, to the altered airway responsiveness in asthma (6). There also seems to be no clear synergistic effect on histamine-induced IP accumulation, following stimulation of HASMC with the combination of IL-1 and TNF-. This is in contrast to bradykinin-mediated IP accumulation, which was significantly enhanced by both IL-1 and TNF-, indicating important differences in the regulation of these two receptors, which are both, via a G-protein, coupled to phospholipase C to induce IP₃ formation, Ca²⁺ release, and subsequent contraction of airway smooth muscle (10, 18). The above findings, together with the lack of effect of IL-1 on IP accumulation to NaF, which bypasses the receptor-mediated process of the signal transduction pathway (19), suggest that the IL-1-induced effect is likely mediated at the receptor itself or at the coupling with its G-protein rather than mediated "downstream" of the G-protein.

In this study we have also attempted to unravel the mechanism leading to IL-1-induced H₁R desensitization. Uncoupling of the receptor as a result of phosphorylation by protein kinases is often involved in desensitization of G-protein-coupled receptors (20), which, with regard to the H₁R, has so far been demonstrated following activation of protein kinase C, protein kinase A, calcium/calmodulin protein kinase II, and a putative H₁R-specific kinase (21). In the present study, there is evidence to suggest that activation of PKA is likely to be involved in H₁R desensitization, as an inhibitor of PKA, H-89, completely blocked the IL-1-induced reduction in IP formation by histamine. Moreover, cloning of H₁Rs has indeed revealed a potential PKA phosphorylation site in the primary sequence of the receptor protein, besides protein kinase C phosphorylation sites and many threonine and serine residues, that could be phosphorylated by other kinases (25). Furthermore, a potential role for PKA in the regulation of H₁R-mediated responses has already been suggested as agents that increase the accumulation or mimic the action of cyclic AMP, such as salbutamol, forskolin, VIP, and 8-bromo-cyclic AMP, resulted in a subsequent decrease in histamine-induced inositol phospholipid hydrolysis (28).

If a PKA-mediated mechanism is involved in IL-1-induced H₁R desensitization, this implies an increase in intracellular cAMP upon IL-1 stimulation, and the latter has indeed recently been shown in HASMC (15). It was suggested that prostanoids, released as a result of IL-1-induced increase in cox-2 expression, contribute to the increased cAMP formation. IL-1 has been demonstrated to induce a marked cox-2 expression in HASMC, resulting in the synthesis and release of prostanoids of which PGE₂, and to a lesser extent 6-keto-PGE₁, are the predominant arachidonic metabolites (29). In airway smooth muscle, PGE₂ presumably activates EP2 or EP4 receptors, which are positively coupled, via a G_S-protein, to adenylyl cyclase resulting in cAMP formation, which finally leads to the activation of PKA (30).

In support of the above hypothesis we have demonstrated that indomethacin, a nonselective cox inhibitor, was able to prevent the IL-1-induced attenuation of IP formation by histamine, implicating the involvement of newly synthesized prostaglandins. Furthermore, pretreatment of the HASMC cells with PGE₂ mimicked the effects of IL-1, supporting the involvement of PGE₂ in IL-1-induced H₁R desensitization. Interestingly, in smooth muscle cells, TNF- failed to cause a significant increase in PGE₂ release and cox-2 induction (29), providing a potential explanation for the lack of effect of TNF- on the H₁R functional response in our study, and further supporting a role for PGE₂ in IL-1-induced effects at the H₁R. PGE₂ has been described as having a wide range of effects in the airways, such as the inhibition of cholinergic neurotransmission, of smooth muscle proliferation, of mast-cell mediator release, and of eosinophil chemotaxis and survival (31). Moreover, PGE₂ is the most important bronchoprotective metabolite yet identified in the airways (32); PGE₂ seems to be involved in hyporesponsiveness to histamine and ₂-adrenoceptor agonists (8) and it plays a role in hyperresponsiveness to bradykinin (33). Collectively, the above findings suggest that PGE₂ may be significantly involved in mediating both the proinflammatory processes in the airways and the changes in airway responsiveness that characterize subjects with asthma. Furthermore, the evidence pointing to the airway smooth muscle as the major source of PGE₂ production in the airway (34, 35) and the fact that HASMC can release multiple pro-inflammatory chemokines (36) suggest an important role for HASMC in participating and coordinating the inflammatory response in the airways, in addition to its contractile function.

To further characterize the intracellular signaling pathway leading to IL-1-stimulated cox-2 induction, PGE₂ release, and subsequent H₁R desensitization, we have investigated which IL-1 receptor downstream signaling events are involved. Nuclear factor-B (NF-B) is a prominent transcription factor, which has been shown to be activated by IL-1 in HASMC (39), and it may be relevant here as analysis of the human cox-2 gene promotor region revealed two putative NF-B binding sites (40). We therefore speculated that activation of NF-B was involved in H₁R desensitization. To block the activation of NF-B in our study, we have used an antioxidant, pyrrolidine dithiocarbamate (PDTC), which has been shown to specifically inhibit the activation of NF-B (41) and the transcription of genes, which are regulated by NF-B (42). Pretreatment of the HASMC with PDTC partly blocked IL-1-induced desensitization of the H₁R, suggesting the involvement of NF-B. Another signaling cascade known to be triggered by IL-1 is activation of the mitogen-activated protein kinase (MAPK) pathways. At least three distinct MAP kinase signal transduction pathways have been characterized in mammalian cells, leading to the activation of extracellular signal-regulated kinase (p44/42 MAP kinase), stress-activated protein kinase or c-Jun N-terminal kinase (SAPK/JNK), or p38 MAP kinase (43). Proinflammatory cytokines, such as IL-1, preferentially activate the SAPK/ JNK and p38 MAP kinase pathway, with little activation of the p44/42 MAP kinase pathway, which is primarily activated by mitogenic stimuli (44, 45). It has, however, been demonstrated that all three MAP kinases are activated by IL-1 in human airway smooth muscle cells (46). In our study, PD 98059, a selective inhibitor of the p44/42 MAP kinase signaling cascade, failed to attenuate IL-1-induced H₁R desensitization, indeed suggesting no involvement of this MAP kinase cascade.

Using Western blot analysis, we have demonstrated that IL-1 phosphorylates p38 MAP kinase and SAPK/JNK. Because SB203580, a very selective inhibitor of p38 MAP kinase, completely inhibited the IL-1-induced effect, we suggest the involvement of p38 MAP kinase in H₁R desensitization by IL-1. The role of SAPK/JNK, however, remains speculative, as so far no specific antagonists for this kinase have been developed. Furthermore, anisomycin, a translational inhibitor, which strongly activates the stress-activated MAP kinase cascades SAPK/JNK and p38 MAP kinase (as demonstrated by Western blot analysis), at a concencentration below that required for inhibiting translation (47), mimicked IL-1-induced H₁R desensitization. Taken together, all the above findings suggest that the IL-1-induced H₁R desensitization is mediated by the activation of the p38 MAP kinase pathway and hence cox-2 induction and PGE₂ release, without any clear evidence at the time for the involvement of the SAPK/JNK and the p44/42 MAP kinase pathway. The events upstream of p38 MAP kinase activation following IL-1 stimulation in HASMC remain to be elucidated, although there is some evidence, at least in rat renal mesangial cells, that IL-1 activates MKK4/ SEK1, MKK3, and MKK6, resulting in cox-2 expression and PGE₂ synthesis (48). To conclude, we suggest that the mechanism of IL-1-induced H₁R desensitization probably involves activation of the p38 MAP kinase and, at least in part, activation of NF-B, as schematically outlined in Figure 7. Activation of p38 MAP kinase and NF-B will subsequently induce cox-2 expression with an increase in PGE₂ production and release. PGE₂ increases intracellular cAMP formation, presumably via EP2 or EP4 receptors, resulting in the activation of PKA, which will finally lead to phosphorylation and uncoupling of the H₁R.

View larger version (40K): [in this window] [in a new window]   Figure 7.   Proposed mechanism underlying IL-1-induced H1R desensitization. IL-1 activates p38MAP kinase and NF-B, which produces PGE2 synthesis by inducing cox-2 expression. Activation of EP2 or EP4 receptors, by the released PGE2, increases intracellular cAMP. cAMP-dependent activation of PKA will finally result in phosphorylation and functional uncoupling of the H1R. ILR, IL-1 receptor; MAPK, mitogen-activated protein kinase; MAPKK, MAPK kinase; MAPKKK, MAPKK kinase; AC, adenylyl cyclase; PKA, protein kinase A; PLC, phospholipase C.

In summary, we have demonstrated that IL-1 protects airway smooth muscle to histamine-induced inositol phospholipid hydrolysis and subsequent airway constriction, by desensitizing the H₁R. The intracellular mechanism leading to H₁R desensitization involves cAMP-induced activation of PKA, probably as a result of IL-1-induced cox-2 expression and PGE₂ release. Therefore, our observations also suggest a potential mechanism for IL-1-induced transcriptional activation of the cox-2 gene and PGE₂ production, which involves activation of p38 MAP kinase and NF-B. Moreover, our results indicate that bronchial hyperreactivity to histamine, which characterizes the patient with asthma, is not associated with proinflammatory cytokine-induced enhancement of H₁R signaling.