Introduction

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₂ agonists are the mainstay bronchodilator agents used in the treatment of asthma, their effects being moderated by activation of adenylyl cyclase (AC) and subsequent elevation in cell cyclic adenosine monophosphate (cAMP) content (1). This pathway has been reported to be hyporesponsive under a variety of conditions, including chronic exposure to the inflammatory mediators interleukin (IL)-1 and tumor necrosis factor (TNF)- (2). IL-1 is a potent proinflammatory mediator released by a wide variety of cells and is able to modulate many cell types by activation of a number of important pathways, including cyclooxygenase II (COX-2), nuclear factor-B, and mitogen-activated protein kinase (3). In the airways, IL-1 may both initiate and perpetuate inflammatory responses; levels of this cytokine are elevated in the bronchoalveolar lavage fluid of patients with symptomatic asthma (4).

In addition to these properties, IL-1 has been reported to be instrumental in the production of a proinflammatory cellular phenotype observed after viral infection of the airways (5). Respiratory viral infections constitute the most frequent trigger of asthma exacerbations and are the major cause of wheezing in people with asthma. During asthma exacerbations, the viral pathogen detected in around 65% of patients is rhinovirus (RV) (6). Previous studies have shown that viral infection has a number of deleterious effects in the airways, including diminished ₂ adrenergic receptor (₂AR)-induced relaxation and enhanced contractile responses of airway smooth muscle (9). Chronic RV 16 exposure has been reported to directly induce secretion of IL-1 by human airway smooth-muscle (HASM) cells in parallel to the proasthmatic phenotypic changes observed in airway smooth-muscle tissue (5). In contrast to the typically procontractile effects of acute exposure to inflammatory mediators in the airways, we have recently reported that chronic stimulation with some agents typically considered as proinflammatory or procontractile (e.g., muscarinic receptor agonists) has differential effects on cAMP formation depending on whether activation is acute or chronic. In particular, we were able to show that chronic muscarinic M2 receptor activation leads to Gi-dependent sensitization of AC in HASM cultures (10).

In the present study, we have defined the effects on AC activation of chronic exposure to IL-1 and RV. The data presented here demonstrate that chronic exposure of HASM cells to either IL-1 or RV induces sensitization of AC-mediated cAMP formation, implying that a homeostatic feedback mechanism exists in these cells to counterbalance the effects of these agents on ₂AR coupling.

	Materials and Methods

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Materials

2,8-[³H]adenine (26 µCi/mmol), 8-[¹⁴C]cAMP (42.4 µCi/mmol), and [¹²⁵I]adenosine 3', 5' -cyclicphosphoric acid (2,200 Ci/mmol) were purchased from New England Nuclear (Stevenage, UK; and Boston, MA). IL-1 and IL-1 receptor antagonist (IL-1ra) were purchased from Peprotech (London, UK), and forskolin (FSK) was purchased from R&D Systems (Abingdon, UK) Biotrak human IL-1 enzyme-linked immunosorbent assay (ELISA) kit was obtained from Amersham Pharmacia Biotech (Little Chalfont, UK). cAMP antibody was a gift from Mario Ascoli (University of Iowa). All other chemicals were obtained from Sigma Chemical Co. (Poole, UK). Plasticware was obtained from Costar (UK) Ltd. (High Wycombe, UK).

Culture of HASM Cells

Primary cultures of HASM cells were prepared from explants of trachealis muscle obtained from individuals without respiratory disease within 12 h of death, as described previously (11). All primary cell cultures from each donor were examined using anti- smooth-muscle  actin antibody (1:100 dilution) (Sigma) to confirm the presence of smooth muscle-type cells using standard immunocytochemical techniques. Primary cell cultures used for the experiments described in this paper showed > 95% of cells staining for smooth-muscle  actin.

Determination of cAMP Response

Accumulation of [³H]cAMP was measured by a modification of a previously described method (12). In brief, HASM cells were allowed to reach confluency in 24-well plates. Because in preliminary experiments we found that IL-1ra was ineffective in the presence of fetal calf serum (FCS) (possibly because of binding to FCS components), in experiments with IL-1ra the cells were serum-starved for 24 h before the addition of the IL-1ra, and serum-free media were used throughout the assay. Where appropriate, cells were inoculated for 1 h by replacing 1 ml full growth media with RV (5 × 10^3.2 infectious dose required to infect 50% of cell cultures inoculated with virus (ID₅₀  )/ 200 µl). This dose was selected because cytopathic effects were observed in HASM cells inoculated with RV at concentrations > 10 × 10^4.2 ID₅₀/200 µl. After inoculation, cells were washed twice with 1 ml media, then full growth media were added and the cells left for 22 h in an incubator constantly gassed with air/CO₂ (5%). Where appropriate, at this stage supernatants were removed and stored at 80°C for subsequent confirmation of RV infection (via inoculation of MRC-5 fibroblasts before titration assays) and/or the presence of IL-1 (via ELISA). HASM cells were then washed twice with Dulbecco's modified Eagle's medium (DMEM) before the media were replaced with 1 ml DMEM containing [³H] adenine (2 µCi/well). Where required, IL-1 were added to full growth media and cells were left to incubate for 16 h before the cells being washed. IL-1 was re-added immediately after the loading of the cells for 2 h at 37°C. At the end of this period, cells were washed three times with 1 ml of Hanks'/N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid buffer and allowed to rewarm to 37°C for 10 min. Agonists were added 10 min before termination of the reactions by the addition of 50 µl concentrated HCl. Cells were then stored at 20°C. After lysate thawing, [³H]cAMP was determined by column chromatography as described previously (12). Aliquots of [¹⁴C]cAMP were added to each sample and the counts obtained from this recovery marker were used to correct for variations in recovery from each column. In addition, a 100-µl aliquot, taken from each well of the plate after reactions were stopped, was counted for tritium to correct for variations in the number of cells per well.

In separate experiments examining the accumulation of endogenous, unlabeled cAMP, HASM cultures were grown in serum-free media as described previously (13) and initially pretreated with either vehicle, bisindolymaleimide I (Bis I) (10 µM) or cycloheximide (50 µM), for 30 min or with pertussis toxin (PTX) (100 ng/ml) for 8 h. Cultures were then treated for 18 h with IL-1, then washed twice with phosphate-buffered saline (PBS) before stimulation with vehicle, 1 µM isoproterenol, or 100 µM FSK for 10 min at 37°C. Additional washes with PBS did not appreciably affect experimental responses. cAMP was isolated and quantitated by radioimmunoassay as described previously (13).

Preparation of RV

Wild-type RV isolated from a throat swab from a patient with upper respiratory tract symptoms was cultured using human embryonic lung fibroblasts (MRC-5). The cultures were grown in modified Eagle's minimum essential medium supplemented with Earle's balance salt solution, 1% FCS, 2 mM L-glutamine, 40 U/ml gentamycin, 200 U/ml penicillin, and 1 µg/ml amphoterecin B. When obvious cytopathic effects were observed, cell supernatants were harvested, clarified by low-speed centrifugation, and titrated in triplicate to determine the ID₅₀ titer before being frozen in aliquots at 70°C.

Determination of IL-1 in HASM Cell Supernatant

HASM cell supernatants were assayed for the presence of human IL-1 using the Biotrak human IL-1 ELISA system (catalogue no. RPN 2751; Amersham Pharmacia Biotech) as outlined in the manufacturer's instructions. The lower limit of detection for this assay is 10 pg/ml.

Data Analysis

The concentration that produces half-maximum effect (EC₅₀) for IL-1 was defined in each individual experiment and used to calculate mean values. Each data point in individual experiments was calculated from the mean of triplicate determinations. Statistical analysis of data was performed by using GraphPad Prism to perform paired t tests or one-way analysis of variance with Bonferroni post-test being used as appropriate (14). All values in the text represent means ± standard error of the mean (SEM) of n separate experiments.

	Results

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Chronic IL-1 Treatment Induces AC Sensitization

[³H]cAMP formation was assayed in HASM cells treated acutely with drugs activating the ₂AR-AC-cAMP signaling cascade at different levels. The ₂AR agonist isoproterenol (10 min, 1 µM) and the direct activator of AC, FSK (10 min, 10 µM), were both observed to increase cAMP formation significantly (3.0 ± 0.2-fold and 5.8 ± 1.0-fold compared with basal, respectively; both P < 0.0001, n = 4- 7), as previously reported (12). Interestingly, the observed cAMP responses to FSK and isoproterenol after preincubation for 18 h with the cytokine IL-1 (400 pg/ml) before acute exposure with effector was differentially altered. Chronic treatment with IL-1 resulted in inhibition of the cAMP response to isoproterenol (50.4 ± 3.7% inhibition versus 10 min isoproterenol alone; P < 0.05, n = 4) (Figure 1A), a heterologous desensitization previously reported by others (9, 15, 16), presumably mediated by an induction of COX-2 and accompanying prostaglandin (PG) E₂ synthesis by IL-1.

View larger version (9K): [in this window] [in a new window]   Figure 1.   cAMP formation in cultured HASM cells after stimulation with isoproterenol (10 min, 1 µM) (ISO) or FSK (10 min, 10 µM). Filled bars indicate 18-h pretreatment with IL-1 (400 pg/ml) (IL+) followed by 10 min stimulation with Isoproterenol (ISO + IL) or FSK (IL+FSK). Experiments were performed in the presence (A) and absence (B) of serum, using previously described methods for each (A, Ref. 12; B, Ref. 13). Each bar represents the mean ± SEM fold increase compared with basal of four to seven (A) or seven (B) experiments. In all experiments, each response was determined in triplicate. *P < 0.05; **P < 0.001.

In stark contrast to the effects on isoproterenol-stimulated cAMP production, chronic IL-1 treatment of HASM caused a significant augmentation of the response to FSK (3.4 ± 0.4-fold compared with FSK alone; P < 0.001, n = 7) (Figure 1A), suggesting that sensitization of AC occurred after chronic IL-1 exposure. The robustness of this effect on AC responsiveness was demonstrated in separate experiments performed under different conditions. In these experiments, HASM cultures were grown in serum-free media and accumulation of endogenous cAMP in response to isoproterenol or FSK was assessed. Again, chronic IL-1 treatment significantly increased FSK-stimulated cAMP formation (1.9 ± 0.1-fold compared with FSK alone; P < 0.05, n = 7) (Figure 1B). However, under these conditions the absolute cAMP production elicited by isoproterenol was slightly increased in IL-1-treated cells (25.5 + 3.4-fold basal versus 21.3 + 3.1-fold in vehicle-treated [CON] cells), this disparity with results in Figure 1A likely reflecting the relative inability of IL-1 to induce COX-2 and PGE₂ production in serum-starved cells (17).

IL-1 Sensitizes AC in a Concentration- and Time-Dependent Manner

The ability of 18 h IL-1 pretreatment to augment FSK-stimulated cAMP formation was observed to be highly potent with an EC₅₀ of 67.8 ± 0.3 pg/ml (n = 4). Maximal augmentation was obtained with 400 pg/ml (Figure 2). The augmentation of FSK-induced cAMP formation was observed after varying lengths of pretreatment with IL-1 (Figure 3). Significant effects were observed after 6 h pretreatment and the response was maximal at 24 h (2.3 ± 0.4-fold and 6.9 ± 1.0-fold compared with FSK alone, respectively; P < 0.05, n = 4).

View larger version (12K): [in this window] [in a new window]   Figure 2.   cAMP formation in cultured HASM cells after 18 h preincubation with a range of concentrations of IL-1 followed by acute stimulation with FSK (EC50 67.8 ± 0.3 pg/ml). The response to FSK alone (10 min, 10 µM) is seen in the filled bar. Each point represents the mean ± SEM fold increase compared with basal of three experiments. In all experiments, each response was determined in triplicate. *P < 0.05; **P < 0.001.

View larger version (13K): [in this window] [in a new window]   Figure 3.   cAMP formation in cultured HASM cells after varying preincubation times with IL-1 (400 pg/ml) followed by stimulation with FSK (10 min, 10 µM). The response to FSK alone is seen in the bar marked FSK. Each point represents the mean ± SEM fold increase compared with basal of six experiments. In all experiments, each response was determined in triplicate. *P < 0.05.

IL-1-Induced AC Sensitization Is Mediated via the IL-1 Receptor 1

To confirm that the apparent sensitization of AC by IL-1 was mediated via specific activation of the IL-1 receptor 1 (IL-1R1), cells were pretreated with IL-1ra (100 ng/ml, 1 h) before addition of IL-1 (400 pg/ml, 18 h). FSK (10 min, 10 µM) was then added and [³H]cAMP formation was determined. IL-1ra was observed to completely abrogate the IL-1-induced augmentation of FSK-stimulated cAMP formation (P < 0.001, n = 4; Figure 4). IL-1ra did not affect the cAMP responses in naive cells or the response in cells exposed to FSK without prior exposure to IL-1 (data not shown).

View larger version (10K): [in this window] [in a new window]   Figure 4.   cAMP formation in cultured HASM cells after stimulation with vehicle (CON) or FSK (10 min, 10 µM) with and without 1 h preincubation with IL-1ra (ra) (100 ng/ml). Filled bars indicate 18 h pretreatment with IL-1 (IL) (400 pg/ml). Each bar represents the mean ± SEM fold increase compared with basal of four experiments. In all experiments, each response was determined in triplicate. **P < 0.001.

IL-1-Induced AC Sensitization Is Not Protein Kinase C-/Gi-Dependent

To elucidate the possible mechanism involved in IL-1-induced AC sensitization, HASM cells were preincubated with inhibitors of a range of pathways that could potentially induce AC sensitization based upon the observed properties of the AC isoforms known to be expressed in HASM (10, 18). The pathways and agents studied were: protein kinase (PK) C (inhibited by the staurosporine analogue Bis I) and Gi (inhibited by PTX). Preincubation with the relevant agent was followed by chronic exposure to IL-1, then acute stimulation with FSK. Bis I had no significant effects upon the sensitization observed in response to chronic IL-1 exposure, regardless of the culture conditions, with serum (Figure 5A) or serum-free (Figure 5B), or method of cAMP analysis. PTX produced no significant effects in experiments performed in the presence of serum (Figure 5A); however, under serum-free conditions, PTX caused a small (19 ± 5%; P < 0.05, n = 5), but statistically significant decrease in the magnitude of FSK-stimulated cAMP generation in IL-1-treated cells, suggesting a possible contribution of a Gi-dependent mechanism. However, PTX also caused a small decrease in the response in vehicle-treated (CON) cells, and thus the comparative effect of IL-1 in PTX-treated cells was similar (53 ± 13% increase, n = 5) to that which occurred between groups not receiving PTX treatment (70 ± 9%, n = 5).

View larger version (9K): [in this window] [in a new window]   Figure 5.   (A) The effects of preincubating with two selective inhibitors on cAMP formation in cultured HASM cells determined as described previously (12), followed by stimulation of all cells with FSK (10 min, 10 µM): PTX (50 ng/ml, 30 min) (+PTX); Bis I (1 µM, 10 min) (+Bis). Filled bars indicate additional 18-h pretreatment with IL-1 (+IL) (400 pg/ml). The response to FSK alone has been designated 100% and is shown in the unlabeled bar. (B) cAMP formation determined as described previously (13) after preincubation with PTX (100 ng/ml, 8 h)(+PTX) or Bis I (10 µM, 30 min) (+BIS) before IL-1 exposure (filled bars, +IL) (400 pg/ml, 18 h) and finally acute addition of FSK. Each bar represents the mean ± SEM fold increase compared with basal of four experiments. In all experiments, each response was determined in triplicate. *P < 0.05; **P < 0.001.

IL-1-Induced AC Sensitization Requires De Novo Protein Synthesis

To determine whether de novo protein synthesis is required for IL-1-induced AC sensitization, HASM cells were preincubated with the protein synthesis inhibitor cycloheximide (50 µM) for 45 min before the addition of IL-1 (18 h, 400 pg/ml). The presence of cycloheximide resulted in a complete abrogation of the observed sensitization of AC while having no effect on the cAMP responses seen in naive HASM cells or HASM cells exposed solely to FSK regardless of the presence (Figure 6A) or absence (Figure 6B) of serum.

View larger version (9K): [in this window] [in a new window]   Figure 6.   cAMP formation in cultured HASM cells using previously described methods (A, Ref. 12; B, Ref. 13) after stimulation with FSK (10 min, 10 µM) with and without 45 min (A) or 30 min (B) preincubation with cycloheximide (CHX) (50 µM). Filled bars indicate 18 h pretreatment with IL-1 (+IL) (400 pg/ml). The response to FSK alone is seen in the bar marked FSK. Each bar represents the mean ± SEM fold increase compared with basal of four (A) or three (B) experiments. In all experiments, each response was determined in triplicate. *P < 0.05; **P < 0.001.

RV Infection Induces AC Sensitization

Following previous observations suggesting that IL-1 may be a major mediator of the cellular effects of RV in HASM (5), we directly studied the effects of RV inoculation on AC sensitization and ₂AR desensitization to determine whether RV mirrors the effects observed with IL-1 (Figure 7). Wild-type RV was isolated from an individual with viral exacerbation of airway disease and subcultured in MRC-5 fibroblasts as detailed. As previously reported (9), after RV infection of HASM cells (24 h, 5 × 10^3.2 ID₅₀/ 200 µl), the cAMP response to isoproterenol (10 min, 100 µM) showed a significant reduction (39 ± 18% inhibition compared with isoproterenol alone, P < 0.05, n = 5) (Figure 7A). In contrast, and in accordance with our data on IL-1, RV infection followed by acute FSK addition showed a significant augmentation of the response to 10 µM FSK (1.6 ± 0.13-fold compared with FSK alone; P < 0.05, n = 10) (Figure 7B), although the degree of sensitization observed was less than that seen with treatment with IL-1.

View larger version (11K): [in this window] [in a new window]   Figure 7.   cAMP formation in cultured HASM cells after stimulation with 100 µM isoproterenol (ISO) (10 min) (A) or a range of concentrations of FSK (10 min) (B). Filled bars indicate 1 h inoculation with RV (RV) (5 × 103.2 ID50/200 µl) followed by washes, media replacement, and 22 h incubation. Each bar represents the mean ± SEM fold increase compared with basal of five (A) or three to 10 (B) experiments. In all experiments, each response was determined in triplicate. *P < 0.05.

RV-Induced AC Sensitization Does Not Appear to be Mediated via IL-1 Secretion

To determine whether RV-induced AC sensitization in HASM cells is mediated by secreted IL-1, we preincubated cells with IL-1ra (100 ng/ml, 1 h) before infection with RV (1 h infection, then 22 h incubation) and ultimately, acute addition of FSK. Interestingly, RV-induced sensitization of AC was not abrogated by IL-1ra (Figure 8). To establish whether the observed RV-induced AC sensitization could, contrary to our initial hypothesis, be unrelated to that induced by IL-1, we examined the levels of IL-1 in HASM cell supernatants collected after RV infection. IL-1 was not detected at levels above the assay sensitivity concentration (10 pg/ml) in any of the supernatants studied (n = 23).

View larger version (11K): [in this window] [in a new window]   Figure 8.   cAMP formation in naive cultured HASM cells (CON) or in HASM cells after stimulation with FSK (10 min, 10 µM) with and without 1 h preincubation with IL-1ra (ra) (100 ng/ml). Filled bars indicate 22 h inoculation with RV (RV) (5 × 103.2 ID50/200 µl). The response to FSK alone is seen in the bar marked FSK. Each bar represents the mean ± SEM fold increase compared with basal of three experiments. In all experiments, each response was determined in triplicate. *P < 0.05.

RV-Induced AC Sensitization Is PKC-Dependent

Having determined that RV-induced AC sensitization is not mediated via induction of IL-1 secretion, we attempted to elucidate the mechanisms involved by using selective inhibitors to PKC and Gi. The adenosine diphosphate (ADP) ribosylator PTX (50 ng/ml, 30 min preincubation) was observed to be ineffective (data not shown); however, 10 min preincubation with the PKC inhibitor Bis I (1 µM) significantly inhibited RV-induced AC sensitization (68 ± 17% inhibition compared with response to RV and FSK; P < 0.05, n = 4) (Figure 9), suggesting that PKC may be an important component of this response.

View larger version (14K): [in this window] [in a new window]   Figure 9.   cAMP formation in naive cultured HASM cells (CON) or in HASM cells after stimulation with FSK (10 min, 10 µM) with and without 10 min preincubation with Bis I (Bis) (1 µM). Filled bars indicate 22 h inoculation with RV (RV) (5 × 103.2 ID50/200 µl). The response to FSK alone is seen in the bar marked FSK. Each bar represents the mean ± SEM fold increase compared with basal of four experiments. In all experiments, each response was determined in triplicate. *P < 0.05.

	Discussion

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In this paper we report the effects of chronic exposure to IL-1 and RV on the ₂AR-AC-cAMP pathway in cultured HASM cells. The elevation of cAMP formation seen after acute treatment with isoproterenol or FSK was observed to be diversely altered after chronic IL-1 stimulation, depending on the exact point of activation of the ₂AR-AC cascade. Chronic IL-1 treatment followed by acute activation of the ₂AR by isoproterenol resulted in a significant decrease in cAMP formation when compared with isoproterenol alone, suggesting a desensitization of the ₂AR. This effect was dependent on cell culture conditions, being observed in cultures maintained in serum-containing, but not serum-free, media. The ability of chronic IL-1 treatment to induce ₂AR hyporesponsiveness has been reported in a number of airway systems, including guinea-pig and rabbit trachea (2, 19), human cultured airway epithelial cells (15), and rabbit and human cultured airway smooth-muscle cells (20). Specifically in airway smooth muscle, the observed ₂AR hyporesponsiveness has been reported to occur in combination with a number of cellular changes, including increased PG formation via COX-2 induction (16) and, in rabbit airway smooth muscle tissue, increased expression of specific Gi isoforms (2).

Interestingly, in stark contrast to its effects on isoproterenol-stimulated cAMP, chronic IL-1 treatment significantly increased cAMP production in response to FSK, in both a time- and concentration-dependent manner. This effect has not previously been described and suggests the existence of a novel homeostatic cellular mechanism. These data are in contrast to the lack of effect of chronic IL-1 exposure to FSK-stimulated cAMP formation previously reported in the literature by Shore and colleagues (20). This is probably due to differing experimental conditions; for example, in the study by Shore and associates (20), an important difference was that HASM cells were seeded 3 to 6 h before the addition of IL-1, which is likely to activate a number of pathways (e.g., p42/p44/focal adhesion kinases) which themselves could effect new protein synthesis and AC sensitization.

Subsequent experiments focused on identifying the mechanisms underlying IL-1-induced AC sensitization. Inclusion of IL-1ra during the period of IL-1 pretreatment reversed this AC sensitization, suggesting that the IL-1 effect is mediated via the IL-1R1. One potential explanation for our data is that AC sensitization is an artifactual phenomenon resulting from an inability to deal with residual PGE₂ that results from COX-2 induction.We are very confident that this is not the case, for a number of reasons: (1) AC sensitization was observed in cells grown under both serum-containing and serum-free conditions, the latter having previously been shown to exhibit little if any induction of COX-2 and PGE₂ by IL-1 (17). (2) On the basis of preliminary data we know that TNF- induces an equivalent sensitization, yet has no effect on COX and PGE₂ (21). (3) We can demonstrate that AC sensitization occurs regardless of the extent of washing before agonist challenge. (4) AC sensitization by IL-1 is slow to reverse, for if cells are treated with IL-1, washed extensively, re-fed media and allowed to recover for 2 h, then washed again and challenged with FSK, the sensitization is only slightly decreased (data not shown). These findings are consistent with our observation that AC sensitization requires new protein synthesis and suggest that it is dissociated from PGE₂ induction and the mechanism which mediates ₂AR desensitization. We have previously demonstrated that treatment of HASM cells with PGE₂ induces ₂AR hyporesponsiveness via a PKA-dependent mechanism (12, 13), and have recently determined that chronic exposure of HASM cultures to exogenous PGE₂ causes a slight loss of FSK-stimulated cAMP production (21). Thus, PGE₂ is an unlikely effector of IL-1-induced AC sensitization.

Because we have previously determined that Gi-coupled receptor agonists can mediate AC sensitization in HASM, we considered the possibility that a Gi-activating autocrine factor mediates the AC sensitization induced by IL-1. ADP ribosylation of Gi by PTX failed to abrogate the IL-1-induced AC sensitization compared with its effects on FSK alone, suggesting that this phenomenon is not Gi- mediated. Previous reports of elevated levels of specific Gi isoforms in rabbit trachea after chronic IL-1 exposure do not reconcile easily with our findings and may, as suggested by Hakonarson and colleagues, be species-specific (2).

Given the known sensitivity of some AC isoforms to PKC modulation we also studied the effects of Bis I, an effective inhibitor of PKC-mediated events in airway smooth-muscle cells (22). However, Bis I also failed to alter the sensitization of AC seen in response to IL-1, suggesting no role for PKC in IL-1-induced AC sensitization.

The only clear inhibitor of IL-1-induced augmentation of FSK-stimulated cAMP formation was cycloheximide; this finding suggests that de novo protein synthesis is required for AC sensitization to occur. Given that the observed sensitization of AC by IL-1 does not appear to be mediated at the receptor level, we therefore hypothesize that the critical point of interaction after stimulation with IL-1 is at the level of Gs or AC itself. Overexpression studies in HASM with ₂AR/Gs/AC suggest that AC is the rate-limiting factor in this pathway (10); hence, AC appears to be the most likely mechanistic candidate. We have made a considerable effort to characterize AC 5/6 isoform protein levels in both control and IL-1-treated airway smooth muscle using a commercially available antibody. Despite using numerous methods of cell lysate preparation and immunoblotting conditions we were unable to obtain unequivocal results. For some blots we did observe faint bands of the proper molecular weight but no large differences between control and IL-1-treated samples were apparent. On the basis of these results we believe any changes in AC6 protein are likely to be small and cannot be quantified with any degree of sensitivity. Hence, it is difficult to determine whether an increase in AC expression or altered sensitivity of AC (perhaps secondary to induced synthesis of an autocrine factor) is the major mechanism underlying this response to IL-1, although the latter possibility seems more likely.

RV infections have been shown to be one of the major causes of asthma exacerbation. Although a number of effects of RV on airway function probably contribute to this response, one possibility is that RV directly modulates signal transduction pathways in the major effector cells that control airway tone. Given that in airway smooth muscle RV has previously been reported to modulate signaling through the ₂AR via an IL-1-dependent pathway (5), we investigated the effects of RV infection on direct activation of AC. As was observed with IL-1, significant augmentation of FSK-driven cAMP formation occurred, although this effect was less marked than with IL-1. In addition to this novel observation, we were also able to demonstrate a significant decrease in the absolute amount of isoproterenol-stimulated cAMP formation after RV exposure, suggesting the occurrence of RV-induced ₂AR desensitization, as previously described (9). RV 16 has previously been reported to stimulate cultured HASM cells to secrete IL-1 to concentrations of 250 pg/ml 24 h after infection (5), a concentration where we observed near-maximal AC sensitization. Hence, we considered it probable that RV-induced AC sensitization was being mediated via autocrine IL-1 production. However, the absence of IL-1 in RV-infected HASM cell supernatant, coupled with the lack of effect of the IL-1ra on RV-induced AC sensitization, strongly suggested an alternative pathway to be involved in this response. Subsequent experiments revealed RV-induced AC sensitization to occur through a Gi-independent pathway. However, RV-induced AC sensitization was reversed in part by an inhibitor of PKC, suggesting a role for PKC in this process. It thus seems clear that the AC sensitization that occurs after IL-1 and RV exposure is achieved through different mechanisms.

In summary, we report here the ability of chronic exposure to IL-1 or RV to significantly augment FSK-induced cAMP formation. The mechanisms involved in these processes, however, appear to be different, occurring through PKC-independent and -dependent pathways, respectively. In addition, we confirmed previous reports of IL-1 and RV inducing ₂AR hyporesponsiveness in HASM cells. We hypothesize, therefore, that the ability of IL-1 and RV to induce AC sensitization is a protective homeostatic mechanism favoring a prorelaxant phenotype by mitigating the ₂AR desensitization induced by the inflammatory response in the context of inflammation and/or viral exacerbations of asthma.