beta -Adrenergic agonists exert their "anti-inflammatory" effects in monocytic cells through the Ikappa B/NF-kappa B pathway

Pierre Farmer and Jérôme Pugin

Division of Medical Intensive Care, Department of Internal Medicine, University Hospital of Geneva, 1211 Geneva 14, Switzerland


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In addition to their well-studied bronchodilatory and cardiotonic effects, beta -adrenergic agonists carry anti-inflammatory properties by inhibiting cytokine production by human mononuclear cells. In a model of human promonocytic THP-1 cells stimulated with lipopolysaccharide (LPS), we showed that beta -agonists inhibited tumor necrosis factor-alpha and interleukin-8 production predominantly via the beta 2-adrenergic receptor through the generation of cAMP and activation of protein kinase A. This effect was reproduced by other cAMP-elevating agents such as prostaglandins and cAMP analogs. Activation and nuclear translocation of the transcription factor nuclear factor-kappa B induced by LPS were inhibited with treatment with beta -agonists, an effect that was prominent at late time points (>1 h). Although the initial Ikappa B-alpha degradation induced by LPS was minimally affected by beta -agonists, the latter induced a marked rebound of the cytosolic Ikappa B-alpha levels at later time points (>1 h), accompanied by an increased Ikappa B-alpha cytoplasmic half-life. This potentially accounts for the observed nuclear factor-kappa B sequestration in the cytoplasmic compartment. We postulate that the anti-inflammatory effects of beta -agonists reside in their capacity to increase cytoplasmic concentrations of Ikappa B-alpha , possibly by decreasing its degradation.

lipopolysaccharide; nuclear factor-kappa B; Ikappa B; adenosine 3',5'-cyclic monophosphate


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

beta -ADRENERGIC AGONISTS are utilized in a variety of clinical situations, mostly for their bronchodilating and cardiotonic effects. It has been recognized that these pharmacological agents modulate the production of inflammatory mediators. It was, for example, shown that catecholamines could inhibit tumor necrosis factor (TNF)-alpha , interleukin (IL)-1, and IL-6 production by human mononuclear cells (16, 35, 42). It has been postulated that the generation of cAMP by these agents was necessary and that the regulation of this effect was at the level of inflammatory gene transcription (37-39).

Nuclear factor (NF)-kappa B is a transcription factor that has been implicated in control of the expression of numerous inflammatory genes including TNF-alpha and IL-8 (7). NF-kappa B is sequestered in an inactive form in the cytoplasm by its natural inhibitor, Ikappa B-alpha . Ikappa B-alpha phosphorylation and degradation induces the nuclear translocation of NF-kappa B and the binding of the protein to specific responding elements in the promoter regions of inflammatory genes (6). Several studies (1, 3, 33, 47) indicated that the regulation of the activation of this transcription factor is implicated in the anti-inflammatory or immunosuppressive effects of agents such as glucocorticoids, transforming growth factor-beta 1, and aspirin. We have therefore investigated whether the NF-kappa B pathway is implicated in the anti-inflammatory effects induced by beta -agonists. In a model of human monocytic cells (THP-1 cells) stimulated with endotoxin, we found that the beta 2-adrenergic receptor was primarily implicated in the inhibition of IL-8 production observed with beta -agonists. This inhibitory effect was cAMP- and protein kinase (PK) A-dependent and resided in the capacity of beta -agonists to block the NF-kappa B pathway. We show here evidence that beta -agonists modulate lipopolysaccharide (LPS) responses by inducing a marked increase of cytoplasmic Ikappa B-alpha concentration in the presence of LPS, an effect that was observed only at late time points (>1 h).


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture and stimulation. Human promonocytic THP-1 cells (American Type Culture Collection) were maintained in RPMI 1640 medium containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, and antibiotics (all from Life Technologies, Paisley, UK). THP-1 cells were differentiated for 3 days with 10-7 M 1,25-dihydroxyvitamin D3 (Hoffmann La Roche, Basel, Switzerland) (43). The cells were washed and distributed into sterile microtiter plates (Costar, Corning, NY) at a concentration of 100,000 cells/well in RPMI 1640 medium containing 2% FBS. In some experiments, 1,25-dihydroxyvitamin D3-differentiated THP-1 cells were substituted with undifferentiated THP-1 cells transfected with CD14 (31). The cells were stimulated with nanomolar concentrations of Escherichia coli 0111:B4 LPS (List, Campbell, CA) for 8 h (unless indicated otherwise) at 37°C in the presence and absence of beta -adrenergic agonists and antagonists. The following pharmacological agents were tested: isoproterenol (IMS, South El Monte, CA), albuterol (Glaxo Wellcome, Stevenage, UK), epinephrine (Sintetica, Mendrisio, Switzerland), norepinephrine (Aventis, Frankfurt, Germany), propranolol (Zeneca, Blackley, UK), metoprolol (Novartis, Basel, Switzerland), esmolol (Gensia, Bracknel, UK), phentolamine (Novartis), and iloprost (Ilomedin, Schering, Berlin, Germany). In most experiments, isoproterenol, a beta 1- and beta 2-adrenergic agonist, was used as a prototypic beta -adrenergic agonist to inhibit IL-8 production in THP-1 cells. IL-8 and TNF-alpha concentrations were measured in conditioned supernatants with a sandwich ELISA with paired monoclonal antibodies available commercially (Endogen, Cambridge, MA) as described elsewhere (29).

In some experiments, the effect of beta -agonists on both intracellular and secreted IL-8 production was assessed. To measure intracellular concentrations of IL-8, 5 × 106 cells were pelleted after 15 h of incubation and washed once with PBS, pH 2.0, to remove membrane-associated IL-8. The cells were then lysed in 20 mM Tris buffer, pH 7.5, containing 1% Triton X-100, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml of leupeptin, and 200 U/ml of aprotinin (Sigma, St. Louis, MO). Measurement of IL-8 concentrations was determined in parallel in conditioned supernatants and cell lysates.

Preparation of nuclear extracts and electrophoretic mobility shift assay. 1,25-Dihydroxyvitamin D3-differentiated THP-1 cells (7.5 × 106) were stimulated with 10 ng/ml of LPS in RPMI 1640 medium with 2% FBS for various times. After stimulation, the cells were rapidly chilled on ice, washed twice with ice-cold PBS, pH 7.4. Nuclear extracts were prepared as described elsewhere (28). Nuclear proteins were used for electrophoretic mobility shift assay. Twenty to fifty femtomoles of 32P-labeled NF-kappa B double-stranded oligonucleotide probe (30,000-50,000 counts/min; 5'-AGT TGA GGG GAC TTT CCC AGG-3'; Promega, Madison, WI) were added to the nuclear proteins (5-8 µg) in a binding buffer containing 5 mM HEPES, pH 8.5, 5 mM MgCl2, 50 mM dithiothreitol, 0.4 mg/ml of poly(dI-dC) (Amersham Pharmacia Biotech, Uppsala, Sweden), 0.1 mg/ml of sonicated double-stranded salmon sperm DNA (Sigma), and 10% glycerol and incubated for 10 min at room temperature. Samples were migrated on a nondenaturing 5% acrylamide gel made in Tris-glycine-EDTA buffer. Gels were transferred onto Whatman paper, dried, and subjected to autoradiography.

Detection of Ikappa B-alpha protein by Western blot. After LPS stimulation in the presence and absence of beta -agonists, THP-1 cells were lysed in 10 mM HEPES, pH 7.9, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride. In some experiments, the cells were pretreated for 30 min with the PKA inhibitor H-89. In one experiment, isoproterenol was substituted for PGE2, another cAMP-increasing agent. Ten micrograms of cytoplasmic protein extracts were separated by SDS-PAGE (10% acrylamide-bis-acrylamide gel) and electrotransferred onto a nitrocellulose membrane (Bio-Rad, Hercules, CA). Ikappa B-alpha was detected with a rabbit polyclonal anti-human Ikappa B-alpha antibody (Santa Cruz Biotechnology, Santa Cruz, CA), a mouse anti-rabbit horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA), and enhanced chemiluminescence (Amersham Pharmacia). In one experiment, the cells were treated with 10 µg/ml of cycloheximide (Sigma) 3 h after LPS treatment (32). Nuclear extracts were then prepared after various incubation times as described in Preparation of nuclear extracts and electrophoretic mobility shift assay. Quantification of Ikappa B-alpha levels was done with densitometry of the Ikappa B-alpha bands with a Molecular Dynamics densitometer and ImageQuant software (Sunnyvale, CA).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We used 1,25-dihydroxyvitamin D3-differentiated THP-1 cells stimulated by bacterial endotoxin as a model for monocyte/macrophage activation (4, 30, 31). The concentration-dependent effects of beta -agonists on LPS-induced IL-8 production by differentiated THP-1 cells are shown in Fig. 1A. Results are expressed as a percentage of inhibition relative to the activation induced by LPS alone. All beta -agonists inhibited production of IL-8 at concentrations as low as 10-9 M and showed similar affinities. Fenoterol, albuterol, isoproterenol, and epinephrine had EC50 values of 3, 9, 13, and 15 nM, respectively. Norepinephrine was slightly less potent than the other agonists, with an EC50 value of 38 nM. Fenoterol, epinephrine, isoproterenol, and norepinephrine showed similar efficacies, with maximal inhibition levels of 65, 68, 71, and 73%, respectively. The efficacy of albuterol was found to be consistently less than that of other beta -agonists in inhibiting LPS-induced IL-8 production, with a maximal inhibition level of ~ 25%. This may reflect a partial agonistic activity of this compound as previously reported by others (9, 14). Similar results were obtained with beta -agonists in undifferentiated THP-1 cells transfected with CD14 (IL-8 production). The pharmacological activity of isoproterenol was independent of the LPS concentrations used to activate THP-1 cells (tested from 1 ng/ml to 10 µg/ml). This effect was not restricted to the production of IL-8. We found that TNF-alpha production by THP-1 cells was inhibited to a similar extent (Fig. 1B). CD14 surface expression, determined by fluorescence-activated cell sorter analysis, was not influenced by treatment with LPS and beta -agonists (data not shown). In all experiments described in MATERIALS AND METHODS, the cells were found fully viable by the cell viability MTT assay (12).


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Fig. 1.   A: inhibitory effect of various beta -agonists on interleukin (IL)-8 production by 1,25-dihydroxyvitamin D3-differentiated THP-1 cells stimulated for 8 h with lipopolysaccharide (LPS). Isoproterenol is a nonselective beta 1- and beta 2-agonist; albuterol and fenoterol are beta 2-selective agonists. Values are means in percent of inhibition relative to LPS alone from 3-5 independent experiments done with each beta -agonist in triplicate. SEs were omitted for clarity; they ranged between 0.4 and 12%. B: inhibitory effect of 1 µM isoproterenol (I) on tumor necrosis factor (TNF)-alpha production by THP-1 cells stimulated for 8 h with 1 µg/ml of LPS. Control, cells treated with medium only. Values are means ± SD of 3 separate experiments.

We next pharmacologically characterized the beta -adrenergic receptor involved in these effects by measuring the potencies of several beta -adrenergic antagonists (Fig. 2). The results are expressed as a percentage of the LPS response. Isoproterenol decreased the response of LPS by 69%. The addition of <= 10-7 M metoprolol, a selective beta 1-antagonist, practically unaltered the effect of isoproterenol. When added at concentrations >=  10-6 M, the LPS response was restored. Similar results were obtained with another specific beta 1-antagonist, esmolol (data not shown). ICI-118551, a selective beta 2-antagonist, and propranolol, a nonselective beta 1- and beta 2-antagonist, showed the highest potency to interfere with the agonistic (anti-inflammatory) activity of isoproterenol. ICI-118551 and propranolol at concentrations as low as 10-8 M partially inhibited and at concentrations >=  10-6 M completely inhibited the effects of isoproterenol. Both were markedly more potent than the beta 1-antagonist metoprolol. An alpha -antagonist, phentolamine, did not modify the beta -agonistic effect of isoproterenol (data not shown).


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Fig. 2.   A: effect of beta -agonists and antagonists on IL-8 production by THP-1 cells stimulated for 8 h with 10 ng/ml of LPS. ICI-118551, a selective beta 2-antagonist, and propranolol, a beta 1- and beta 2-antagonist both had a greater potency than metoprolol, a selective beta 1-antagonist, to antagonize the effects of isoproterenol. All conditions were in the presence of LPS (L) and isoproterenol (Iso) except for effect of the vehicle only [medium (M)]. Values are means ± SD of 6 independent experiments. B: effect of cAMP-elevating agents (PGE2 and iloprost), a cAMP analog [dibutyryl cAMP (DbcAMP)], and a protein kinase (PK) A inhibitor (H-89) on IL-8 production by THP-1 cells stimulated with LPS. PGE2, iloprost (a prostacyclin analog), and DbcAMP all decreased IL-8 secretion in response to LPS. The PKA inhibitor H-89 blocked the inhibitory effect of 10-6 M isoproterenol [ISO (6) +H89]. C, control [corresponds to LPS plus isoproterenol (H-89 left out)]. PGE2, iloprost, DbcAMP, and H-89 alone had no effect on IL-8 secretion (data not shown). Values are means ± SD of 6 independent experiments.

To determine whether the inhibitory effect of isoproterenol was cAMP dependent, we studied the effect of other cAMP-elevating agents, a cAMP analog, and a PKA inhibitor. PGE2 and iloprost (a prostacyclin analog) are two cAMP-elevating agents in monocytes. Both inhibited IL-8 production induced by 10 ng/ml of LPS, with potencies of 0.3 and 0.9 nM for PGE2 and iloprost, respectively. However, these mediators showed lower efficacies than the beta -agonists (PGE2, 43%; iloprost, 51%). The plasma membrane-permeable cAMP analog dibutyryl cAMP decreased LPS-induced IL-8 release to the levels observed with isoproterenol. A concentration-dependent effect of dibutyryl cAMP was observed, with a maximal inhibitory response of 60% obtained with a 0.1 mM concentration. The PKA inhibitor H-89 abrogated the pharmacological activity of isoproterenol at a 10 µM concentration. Together, these results indicate that isoproterenol acted as an "anti-inflammatory" agent principally through its interaction with a beta 2-adrenergic receptor at the surface of THP-1 cells, leading to the generation of cAMP and activation of PKA.

Because inhibitory effects of beta -agonists could be at the level of protein secretion per se (44), we measured the intra- and extracellular concentrations of IL-8 in THP-1 cells stimulated with LPS in the presence and absence of beta -agonists. IL-8 production was increased in both intra- and extracellular compartments in response to LPS. Isoproterenol blocked both intra- and extracellular IL-8 to similar levels, an effect that could be reversed by propranolol (Table 1). These experiments suggest that the inhibitory effect of isoproterenol takes place before the secretion process.

                              
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Table 1.   Effect of isoproterenol on LPS-induced IL-8 production by differentiated THP-1 cells

We next addressed whether isoproterenol influenced the activation and nuclear translocation of NF-kappa B because this transcription factor has been shown to be of importance for the activation of proinflammatory mediators such as IL-8 and TNF-alpha (20, 36). NF-kappa B was found to be activated in nuclear extracts from THP-1 cells treated with LPS but not in those cells treated with medium only (Fig. 3). The NF-kappa B-oligonucleotide bands from electrophoretic mobility shift assay gels were quantified by densitometry in three separate experiments. The addition of isoproterenol did not modify NF-kappa B activation by LPS at 20 and 60 min but significantly decreased the NF-kappa B signal at 120 and 180 min (results for 20 and 180 min shown in Fig. 3). The loading of the nuclear protein-oligoprobe mixture was similar in all lanes (data not shown).


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Fig. 3.   Isoproterenol inhibits nuclear factor (NF)-kappa B activation in THP-1 cells stimulated with 10 ng/ml of LPS in the presence and absence of 10-6 M isoproterenol. Representative electrophoretic mobility shift assay (of 3 similar experiments) of THP-1 nuclear extracts mixed with a 32P-labeled NF-kappa B probe is shown.

We next postulated that the inhibition of NF-kappa B activation and nuclear translocation induced by beta -agonists was at the level of the regulation by its natural inhibitor Ikappa B-alpha . Figure 4A shows, with a Western blot technique on cytosolic THP-1 cell extracts, that treatment with LPS resulted in the early disappearance (5-30 min) of Ikappa B-alpha due to its degradation. Repeated experiments (n = 3) indicated that isoproterenol induced a significantly more rapid degradation of Ikappa B-alpha (results for 1 representative experiment shown in Fig. 4A). This was followed by the reappearance of Ikappa B-alpha after ~1 h due to its resynthesis. Isoproterenol dramatically and significantly increased the cytoplasmic concentration of Ikappa B-alpha at late time points (>1 h; P < 0.01; 1 representative experiment shown in Fig. 4B and pooled results from 5 independent experiments shown in Fig. 4C). This effect was particularly marked at 3 h. This effect was not observed in the absence of LPS treatment; i.e., isoproterenol did not increase the cytoplasmic Ikappa B-alpha levels by itself (1 representative experiment shown in Fig. 4D). This was confirmed in three separate experiments (data not shown). Because modulation of the cAMP-PKA pathway modified IL-8 secretion in THP-1 cells treated with LPS, we next addressed whether another cAMP-increasing agent, PGE2, could also influence Ikappa B-alpha cytosolic concentrations. As shown in Fig. 5A, PGE2 had a similar inhibitory effect compared with isoproterenol. This was a consistent finding observed in four separate experiments (Fig. 5B). Increased Ikappa B-alpha cytoplasmic levels induced by isoproterenol at 150 min could be blocked by the addition of the PKA inhibitor H-89 (Fig. 5B). These results strongly suggested that this effect was not specific for beta -agonists but was observed with other cAMP-increasing agents such as PGE2 and that a cAMP-PKA-dependent pathway was directly involved in the modulation of Ikappa B-alpha in response to these pharmacological agents.


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Fig. 4.   Effect of the beta -adrenergic agonist isoproterenol (10-6 M) on cytosolic levels of Ikappa B-alpha in differentiated THP-1 cells treated with 10 ng/ml of LPS. A: early Ikappa B-alpha degradation after LPS treatment in THP-1 cells as shown in Western blot. Isoproterenol does not inhibit LPS- induced degradation of Ikappa B-alpha at early time points (5-30 min). B: representative Western blot showing the effect of isoproterenol on cytoplasmic Ikappa B-alpha levels after LPS treatment at late time points (2-3 h; top) and at 3 and 8 h (bottom). C: density of the Ikappa B-alpha bands as measured by gel densitometry. AU, arbitrary density units. Values are means ± SD from 5 different Western blots. P = 0.06 between groups at 2 h. P < 0.01 between groups at 3 h. D: isoproterenol alone does not induce an increase in cytoplasmic Ikappa B-alpha levels at a late time point (150 min) but potentiates the effect of LPS as shown in a representative experiment.



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Fig. 5.   Cytoplasmic Ikappa B-alpha levels in differentiated THP-1 cells treated with 10 ng/ml of LPS for 180 min. Two different cAMP-increasing agents (isoproterenol and PGE2) augmented LPS-induced cytoplasmic Ikappa B-alpha levels. The increasing effect of isoproterenol was blocked by the PKA inhibitor H-89. A: a representative Western blot. B: density of the Ikappa B-alpha bands as measured by gel densitometry. Values are means ± SD from 3 different Western blots. P < 0.05 between LPS+I and LPS+I+H-89.

The marked increase in cytoplasmic levels of Ikappa B-alpha protein induced by isoproterenol may result from an increase in Ikappa B-alpha synthesis or a decrease in its degradation. To address this, we performed an experiment in which we tested the stability of the Ikappa B-alpha protein in the cytoplasm after stimulation with LPS in the presence and absence of isoproterenol. The cells were stimulated for 3 h with LPS in the presence and absence of isoproterenol. The protein synthesis inhibitor cycloheximide (10 µg/ml) was then added, and cytoplasmic Ikappa B-alpha levels were measured with Western blots at different times over the next 2 h. Figure 6 shows that after LPS stimulation, Ikappa B-alpha levels remained high, with greater stability in the presence of the beta -agonist than that measured in its absence (half life ~60 and 20 min, respectively).


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Fig. 6.   Kinetics of Ikappa B-alpha degradation in differentiated THP-1 cells treated with 10 ng/ml of LPS for 180 min in the presence and absence of isoproterenol. Cycloheximide (CHX; 10 µg/ml) was then added, and Ikappa B-alpha protein cytoplasmic levels were measured at indicated times during the next 120 min. A: representative Western blot showing the effect of isoproterenol on cytosolic Ikappa B-alpha protein levels after LPS treatment of differentiated THP-1 cells. -CHX, Ikappa B-alpha levels 120 min after the end of LPS treatment in the absence of CHX. B: density of the Ikappa B-alpha bands. Values are means ± SD of 2 independent experiments measured by gel densitometry. To compare kinetics, densities are ratios of the density at a given time point divided by the density of the band at 180 min.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Here we show that beta -adrenergic agonists inhibit the production of IL-8 by human promonocytic THP-1 cells in response to LPS. The inhibitory effect of beta -adrenergic agonists on LPS-induced IL-8 production was predominantly mediated by their interaction with the beta 2-adrenergic receptor. This conclusion was based on experiments with beta -agonists of different selectivities for the beta 1- and beta 2-adrenergic receptors and the use of selective and nonselective beta -blockers. We found that albuterol and fenoterol, selective beta 2-agonists, had similar potencies compared with epinephrine and isoproterenol in inhibiting LPS-dependent mononuclear cell activation. In contrast, norepinephrine, known for its greater avidity for the beta 1-adrenergic receptor, was three times less potent than the other agonists tested. In addition, the compound ICI-118551, a selective beta 2-blocker, and propranolol, a nonselective beta 1- and beta 2-antagonist, were much more efficient in reverting the beta -agonistic effect than the selective beta 1-agonist metoprolol. This is in accordance with the work of Sekut et al. (34). These authors reported the inhibitory effect of the beta 2-selective agonists albuterol and salmeterol on LPS-induced TNF-alpha production by these cells, an effect that was blocked by the specific beta 2-antagonist oxprenolol (34). In addition, the beta 2-adrenoceptor was previously found to be the adrenoceptor prominently expressed and functional in monocyte/macrophage-like cells (8, 17, 18, 21, 25). In contrast, Talmadge et al. (39) found that in undifferentiated promonocytic THP-1 cells, the beta 1-adrenoceptor mediated the inhibition of LPS-induced TNF synthesis (39). This is probably due to a different beta 1- to beta 2-adrenoceptor ratio of surface expression in undifferentiated THP-1 cells as well as a different readout (mRNA vs. protein). The cell type utilized in our study (macrophages) as well as the agonist and antagonist potency orders indicate that participation of the beta 3-adrenoceptor is unlikely.

The observed decreased IL-8 production by beta -agonists was dependent on the generation of cAMP and on the activation of PKA. Indeed, the cAMP analog dibutyryl cAMP and other cAMP-elevating agents such as PGE2 and iloprost reproduced the effects observed with beta -agonists. H-89, a PKA inhibitor, completely reversed the anti-inflammatory effects of isoproterenol. These findings are in agreement with several studies (2, 10, 37, 38) that tested the effects of cAMP-elevating agents. Whether downstream effectors of this pathway [e.g., cAMP response element binding protein (CREB) transcription factor] are implicated remains to be determined.

Because NF-kappa B has been implicated in the transcriptional regulation of many inflammatory genes including TNF-alpha (46) and IL-8 (13, 20), we addressed whether beta -agonists would interfere with this pathway. We found that this effect was likely due to decreased activation and nuclear translocation of NF-kappa B, and this was observed only after >1 h of LPS stimulation. At earlier time points, the drug did not influence the level of NF-kappa B activation. We hypothesized that this was due to a secondary increase of cytoplasmic concentrations of its natural inhibitor Ikappa B-alpha , a mode of action already described for other anti-inflammatory agents (3, 33). Cytoplasmic levels of Ikappa B-alpha as measured by Western blot indicated that the treatment of cells with isoproterenol did not prevent the initial Ikappa B-alpha degradation on LPS stimulation but rather induced a subsequent marked increase in cytosolic Ikappa B-alpha levels. Even a small increase in cytosolic concentrations of Ikappa B-alpha was previously shown to negatively affect NF-kappa B nuclear translocation (19). Importantly, and in contrast with the mode of action described for glucocorticoids, an increased production of Ikappa B-alpha was not observed in cells treated only with beta -agonists. The addition of a proinflammatory stimulus such as LPS was necessary to observe this effect.

Ollivier et al. (24) previously showed that cAMP induced by forskolin inhibited LPS-induced NF-kappa B activation in THP-1 cells. In their study, the rate of NF-kappa B nuclear translocation was not affected, but it was the transcription efficiency of NF-kappa B at early times (1 h) that was reduced with increased cAMP concentrations. However, these authors did not investigate the activation of NF-kappa B at later time points. Our results unravel another possible mechanism, which may explain the inhibitory effect observed with beta -agonists. After the initial NF-kappa B activation by LPS, beta -agonists may induce the activation of transcription factors such as CREB, which might cooperate with NF-kappa B at the level of the Ikappa B-alpha promoter to increase its transcription. Such a cooperative mechanism has been described for NF-kappa B and other transcription factors (20). This could be the reason for the need of the presence of both LPS and beta -agonists to observe the increased Ikappa B-alpha cytoplasmic levels. Interestingly, recently published data (11) indicated that a transcriptionally active glucocorticoid receptor was translocated into the nucleus on treatment with beta 2-agonists, which may also cooperate with NF-kappa B to increase Ikappa B-alpha transcription.

Another possibility is that the regulation of Ikappa B-alpha by cAMP is at the level of protein degradation as suggested by the experiment shown in Fig. 5. Such an effect was proposed by Neumann et al. (22) in a study where forskolin increased the cytoplasmic levels of Ikappa B-alpha . It is also conceivable that beta -agonists decrease Ikappa B-alpha phosphorylation and degradation through the activation of second messengers, which will, in turn, inhibit upstream kinases such as IL-1 receptor-associated kinase, TNF receptor-associated kinase-6, or members of the mitogen-activated protein kinase kinase kinase (MEKK)-1-NF-kappa B-inducing kinase-Ikappa B kinase complex involved in LPS signaling (15, 23). The delayed appearance of Ikappa B-alpha protein in the cytoplasm could also suggest that beta -agonists may induce or activate a cytosolic inhibitor of kinases upstream of NF-kappa B. Candidate inhibitors are those of the family of antiapoptotic factors. Indeed, it was recently demonstrated that increased expression of Bcl-2 and Bcl-XL, which are under the control of both NF-kappa B and CREB (40, 45), prevented Ikappa B-alpha degradation (5). In another study, it was shown that the LPS-induced A20 protein could directly inhibit MEKK-1 (Kravchenko VV, personal communication). It is also possible that an anti-inflammatory cytokine such as IL-10 is produced on beta -agonist treatment, which may turn down IL-8 production induced by LPS in an autocrine fashion via an Ikappa B-dependent mechanism (27, 41). Finally, Parry and Mackman (26) have proposed that NF-kappa B inhibition by cAMP occurred at the level of the differential binding of CREB and NF-kappa B to the CREB-binding protein, a protein necessary for efficient gene transcription (26).

In conclusion, we hereby provide clues as to the mechanisms by which cAMP-increasing agents such as beta -agonists exert their anti-inflammatory effects. These pharmacological agents block NF-kappa B activation and nuclear translocation and, secondarily, inflammatory gene transcription by increasing Ikappa B-alpha cytoplasmic concentration. Our results also make an important link between the cAMP and NF-kappa B pathways.


    ACKNOWLEDGEMENTS

We thank Pierre Weber (Hoffmann La Roche, Basel, Switzerland) and Ursula Lang and Alessandro Capponi (University of Geneva, Geneva, Switzerland) for the gift of precious reagents, and A. Nials, M. Skingle and T. N. C. Wells for stimulating discussions and constant support.


    FOOTNOTES

This work was supported by Swiss National Foundation for Scientific Research Grant SNF 32-50764 (to J. Pugin) and grants from the 3R and Carlos and Elise de Reuter Foundations and Glaxo Wellcome.

P. Farmer received a scholarship from the Canadian Heart and Stroke Foundation and the Fonds pour la Formation de Chercheur et l'Aide à la Recherche. J. Pugin is the recipient of a fellowship from the Prof. Dr. Max Cloëtta Foundation.

Address for reprint requests and other correspondence: J. Pugin, Division of Medical Intensive Care, Dept. of Internal Medicine, University Hospital of Geneva, 1211 Geneva 14, Switzerland (E-mail: pugin{at}cmu.unige.ch).

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

Received 28 January 2000; accepted in final form 26 April 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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