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Anesth Analg 2004;99:1508-1515
© 2004 International Anesthesia Research Society
doi: 10.1213/01.ANE.0000132976.19021.1B

CRITICAL CARE AND TRAUMA

Dobutamine Inhibits Phorbol-Myristate-Acetate-Induced Activation of Nuclear Factor-{kappa}B in Human T Lymphocytes In Vitro

Torsten Loop, MD, Tobias Bross, Matjaz Humar, PhD, Alexander Hoetzel, MD, Rene Schmidt, MD, Heike L. Pahl, PhD, Klaus K. Geiger, MD, and Benedikt H. J. Pannen, MD

Department of Anesthesiology and Critical Care Medicine, University Hospital, Freiburg, Germany

Address correspondence and reprint requests to Benedikt H. J. Pannen, MD, Anaesthesiologische Universitätsklinik Hugstetterstrasse 55, D-79106 Freiburg, Germany. Address e-mail to pannen{at}nz11.ukl.uni-freiburg.de


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Adrenergic drugs are often used for hemodynamic support of cardiac output and vasomotor tone in critically ill patients. Recent evidence shows that the administration of these vasoactive drugs may affect cytokine release and could influence the inflammatory response. However, the mechanism of this immunomodulatory effect remains unknown. The nuclear transcription factor-{kappa}B (NF-{kappa}B) regulates the expression of many cytokines and plays a central role in the immune response. Therefore, we examined the effects of various adrenergic drugs (dobutamine, xamoterol, clenbuterol, epinephrine, norepinephrine, and phenylephrine) on the activation of NF-{kappa}B, on the NF-{kappa}B-driven reporter gene activity, and on the expression of the NF-{kappa}B target gene interleukin (IL)-8. In addition, we quantified the amount of the NF-{kappa}B inhibitors I{kappa}B{alpha} and IL-10. Here we report that dobutamine inhibited the activation of NF-{kappa}B in primary human CD3+ T lymphocytes. Suppression of NF-{kappa}B involved the stabilization of its inhibitor, I{kappa}B{alpha}. The effect appears to be ß2-receptor specific, because ß1-adrenergic and {alpha}-adrenergic substances (i.e., xamoterol, epinephrine, norepinephrine, and phenylephrine) did not affect NF-{kappa}B activation and because dobutamine-mediated inhibition of NF-{kappa}B could be prevented by a specific ß2-antagonist. Our results demonstrate that dobutamine is a potent and specific inhibitor of NF-{kappa}B, and they thus provide a possible molecular mechanism for the immunomodulation associated with ß-adrenergic therapy.

IMPLICATIONS: Dobutamine is a specific inhibitor of nuclear factor-{kappa}B, which may provide a molecular mechanism for the immunomodulation associated with ß-adrenergic therapy.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Adrenergic drugs are often used in critically ill patients to exert inotropic, vasotonic, and bronchodilating effects for hemodynamic support. In addition to these well documented effects, adrenergic agonists have also been shown to modulate various immunological functions. For example, in vivo epinephrine infusion increases the number of circulating lymphocytes and augments natural killer (NK) cell activity (1,2). In contrast, in vitro epinephrine and norepinephrine have been demonstrated to decrease stimulated interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF) production in whole blood from patients with sepsis (3–7). Likewise, in endotoxin-induced acute lung injury, adrenergic agonists can significantly decrease proinflammatory cytokine expression (8). Depending on the chemical structure of the sympathomimetic drug, catecholamines exert their pharmacologic effects by directly or indirectly activating either the {alpha}- or ß-adrenergic receptors. Different lymphocytes express different adrenergic receptors. For example, ß2-adrenergic receptors are expressed on T-helper Type 1 cells, but not on T-helper Type 2 cells (9). Epinephrine and norepinephrine inhibit TNF-{alpha} release and increase TNF-receptor expression by a ß2-receptor-dependent pathway (4). Furthermore, catecholamines increase lipopolysaccharide (LPS)-induced production of IL-6 in endothelial cells, a process mediated by both ß1- and ß2-receptor stimulation (10). It is speculated that antiinflammatory effects are mediated by adenylate cyclase stimulation and the resulting increase in production of cyclic adenosine monophosphate (cAMP). Because other drugs that increase cAMP have been shown to reduce TNF mRNA transcription and since TNF gene transcription is mainly regulated by nuclear transcription factor-{kappa}B (NF-{kappa}B), it is possible that adrenergic drugs may influence the activation of NF-{kappa}B (11).

The transcription factor NF-{kappa}B plays a central role in the expression of a wide range of immunomodulatory genes, including proinflammatory cytokines (such as IL-1, IL-2, IL-6, IL-8, and TNF-{alpha}), genes encoding immunoreceptors, cell adhesion molecules, hematopoietic growth factors, growth factor receptors, and acute phase proteins (12). In most unstimulated cell types, NF-{kappa}B proteins are sequestered in the cytosol as an inactive complex bound to I{kappa}B, the inhibitory subunit (13). A large variety of stimuli induce NF-{kappa}B DNA-binding activity via activation of an I{kappa}B kinase complex (14). Activation of the I{kappa}B kinase signalsome is followed by I{kappa}B phosphorylation, ubiquitination, and rapid proteolytic degradation of the inhibitor (15,16). This allows translocation of free, active NF-{kappa}B into the nucleus, where it binds to its cognate DNA elements and activates gene transcription.

Regulation of the activation of NF-{kappa}B is implicated in the antiinflammatory or immunosuppressive effects of various drugs, such as glucocorticoids (17), aspirin (18), and thiopental (19). We therefore studied whether the NF-{kappa}B pathway is involved in the antiinflammatory effects of the ß-adrenergic agonist dobutamine in primary human CD3+ T lymphocytes.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
The following adrenergic drugs were obtained from Tocris (Bristol, UK): clenbuterol, dobutamine, ICI 118.551, propranolol, and xamoterol. All other reagents were purchased from Sigma (Deisenhofen, Germany) unless otherwise specified.

Peripheral blood mononuclear cells were isolated from buffy coats obtained from healthy donors by using density centrifugation on Ficoll-Hypaque® (Amersham Pharmacia, Freiburg, Germany) according to the manufacturer’s recommendations. The cells were microscopically analyzed and counted in a Neubauer chamber. For the isolation of CD3+ T lymphocytes, peripheral blood mononuclear cells (3–4 x 108) were incubated for 15 min on ice with anti-CD3 antibodies conjugated to magnetic beads (Miltenyi Biotech, Bergisch-Gladbach, Germany). Separation of CD3+ cells was performed with an L/S column (Miltenyi Biotech) and confirmed by fluorescence-activated cell sorting (>90% CD3+ cells; <1% CD14+ cells). For electrophoretic mobility shift assays (EMSAs), >2 x 106 T lymphocytes were analyzed per sample.

Jurkat T cells (ACC 282; DSMZ, Braunschweig, Germany) and primary human T lymphocytes, which had been isolated as described above, were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum, 1% glutamine, and 50 mg/mL penicillin and streptomycin (all from Gibco-BRL, Karlsruhe, Germany) and were grown in a humidified atmosphere containing 5% CO2 at 37°C.

Total cell extracts were prepared by using a high-salt detergent buffer (Totex; 20 mM HEPES [pH 7.9], 350 mM NaCl, 20% [vol/vol] glycerol, 1% [wt/vol] Nonidet P-40, 1 mM MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol [DTT], 0.1% phenylmethylsulfonyl fluoride [PMSF], and 1% aprotinin). Cells were harvested by centrifugation, washed once in ice-cold phosphate-buffered saline, and resuspended in 4 cell volumes of the detergent buffer. The cell lysate was incubated for 30 min on ice and was then centrifuged for 5 min at 13,000g at 4°C. EMSAs were performed with a 32P-labeled NF-{kappa}B oligonucleotide. The kinase reaction consisted of 37 µL of purified water, 1 µL of NF-{kappa}B oligonucleotides (25 ng/µL; Promega, Madison, WI), 5 µL of kinase buffer, 5 µL of {gamma}-32P-deoxyadenosine triphosphate (Amersham International, Braunschweig, Germany), and 1.5 µL of T4 kinase (polynucleotide kinase buffer and polynucleotide T4 kinase; New England Biolabs, Schwalbach, Germany) and was incubated for 30 min at 37°C. The protein content of the cell lysates was determined with a Bradford-Assay system (Bio-Rad Laboratories, München, Germany), and equal amounts of protein (30 µg) were added to a 20-µL EMSA reaction mixture containing 20 µg of bovine serum albumin, 2 µg of poly(dI-dC) (Roche, Mannheim, Germany), 2 µL of buffer D+ (20 mM HEPES [pH 7.9], 20% glycerol, 100 mM KCl, 0.5 mM EDTA, 0.25% Nonidet P-40, 2 mM DTT, and 0.1% PMSF), 4 µL of 5x Ficoll buffer (20% Ficoll 400, 100 mM HEPES, 300 mM KCl, 10 mM DTT, and 0.1% PMSF), 4 µL of double-distilled (dd) H2O, and 1 µL of NF-{kappa}B 32P-labeled oligonucleotide. These samples were incubated at room temperature for 30 min and then loaded on an acrylamide gel containing 60 mL of ddH2O, 10 mL of 30% acrylamide, 3.8 mL of 10x Tris-borate-EDTA buffer (900 mM Tris-HCl, 900 mM boric acid, and 20 mM EDTA [pH 8.0]), 400 µL of ammonium persulfate, and 40 µL of tetramethylethylenediamine. After the gel was run in 0.5x Tris-borate-EDTA running buffer, gels were vacuum-dried (Gel Dryer 543; Bio-Rad, Hercules, CA, USA) for 30 min on a 3 MM chromatography filter (Whatman, Maidstone, England) and exposed to radiograph film (Kodak, Stuttgart, Germany).

To evaluate the effects of ß1- and ß2-agonistic sympathomimetics on NF-{kappa}B activation, cells were treated with the ß1-agonist, xamoterol, the ß2-agonist, clenbuterol, or the combined ß1/ß2-agonist dobutamine. Thirty minutes before harvesting, the cells were stimulated with phorbol-myristate-acetate (PMA; 50 ng/mL), after which total cell protein extracts were prepared and analyzed by EMSA for the DNA-binding activity of NF-{kappa}B.

To determine whether the inhibitory effect on NF-{kappa}B activation is mediated mainly through an {alpha}- or ß-receptor-dependent mechanism, we performed EMSAs with total cell extracts from CD3+ T cells after a 2-h incubation with the {alpha}-agonist phenylephrine or the combined {alpha}- and ß-agonists norepinephrine and epinephrine.

To test whether the inhibitory effect on NF-{kappa}B activation is mediated by a ß-receptor-dependent mechanism, we performed EMSAs by using total cell extracts from CD3+ T cells after preincubation with the nonselective ß-adrenergic receptor-blocking drug propranolol or the ß2-adrenoreceptor antagonist ICI 118.551 for 1 h, followed by a 2-h incubation with dobutamine and subsequent stimulation with PMA (50 ng/mL for 30 min).

The activation and translocation of NF-{kappa}B to the nucleus is preceded by the phosphorylation and proteolytic degradation of the inhibitory I{kappa}B{alpha} proteins. This process is readily detectable in Western blots. To determine whether dobutamine, clenbuterol, or xamoterol may interfere with the degradation of I{kappa}B{alpha}, CD3+ T lymphocytes were pretreated with different doses of these substances (100 or 500 µM) for 105 min and subsequently stimulated with PMA (50 ng/mL) for 15 min. Total cell extracts of CD3+ T lymphocytes were boiled in Laemmli sample buffer and subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Before transfer, gels were equilibrated for 15 min in cathode buffer (25 mM Tris, 40 mM glycine, and 10% methanol). Proteins were transferred at 0.8 mA/cm2 for 1 h onto Immobilon P membranes (Millipore Corp., Eschborn, Germany) preequilibrated in methanol (15 s), ddH2O (2 min on each side), and anode buffer II (25 mM Tris/10% methanol) by using a semidry blotting apparatus (Bio-Rad Laboratories). Equal loading and transfer were monitored by Ponceau S staining of the membranes. Nonspecific binding sites were blocked by immersing the membrane in blocking solution (TBST [10 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.1% Tween-20] containing 2% bovine serum albumin) overnight at 4°C. Membranes were washed in TBST and incubated in a 1:1000 dilution of anti-I{kappa}B{alpha} antibody (Catalog No. 9241; Cell Signaling Technology Inc., Beverly, MA) in blocking solution for 1 h at room temperature, followed by extensive washing with TBST. Bound antibody was decorated with goat anti-rabbit/horseradish peroxidase conjugate (Amersham Pharmacia) diluted 1:5000 in blocking solution for 30 min at room temperature. After washing four times (5 min each), the immunocomplexes were detected with enhanced chemiluminescence Western blotting reagents (Amersham Pharmacia) according to the manufacturer’s instructions. Exposure to Kodak XAR-5 films was performed for 15 s to 1 min.

Jurkat cells were plated 12–16 h before transfection at a density of 3 x 105 cells per well in a 6-well plate. Cells were transiently transfected with a luciferase reporter gene driven either by the minimal thymidine kinase (tk) promotor alone or by the tk promotor preceded by six NF-{kappa}B binding sites. The vectors were generous gifts of Dr. Markus Mayer (EMBL, Heidelberg, Germany) and have been previously described (19). Transfections were performed by using the reagent SuperFect (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. To exclude the possibility of differences in transfection efficiency, all cells were pooled at 6 h after transfection, gently mixed, and then equally distributed into individual wells for all further determinations. Moreover, the experiment was repeated six times. Cells were pretreated with dobutamine, clenbuterol, or xamoterol for 1 h before stimulation with PMA (50 ng/mL) and were harvested in situ after 17 h with a commercial lysis buffer and luciferase assay system (Promega Corp., Heidelberg, Germany). Luciferase activity was determined with a microplate luminometer (Eg & G-Berthold, Bad Wilbach, Germany) by measuring light emission over 30 s, and the results are expressed as a percentage of the arbitrary light units of the respective positive control.

Cell culture supernatants were analyzed for IL-8 and IL-10 24 h after stimulation with PMA (50 ng/mL). Cells were pretreated with different concentrations of dobutamine, clenbuterol, or xamoterol for 1 h before stimulation. Measurements were performed with enzyme-linked immunosorbent assay kits purchased from R&D Systems (Minneapolis, MN) according to the manufacturer’s instructions.

Differences in measured variables between the experimental conditions were assessed by using a one-way analysis of variance on ranks followed by a nonparametric Student-Newman-Keuls test for multiple comparisons. Results were considered statistically significant if P < 0.05. The tests were performed with the SigmaStat software package (Jandel Scientific, San Rafael, CA).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
The results obtained with xamoterol, clenbuterol, and dobutamine alone did not differ from control conditions (Fig. 1; compare Lanes 6–8 with Lane 1). Treatment of CD3+ T cells with PMA induced NF-{kappa}B DNA-binding activity (Fig. 1; compare Lanes 1 and 2). Incubation of the cells with xamoterol (100, 500, and 1000 µM) had no effect on NF-{kappa}B DNA-binding activity (Fig. 1A; Lanes 3–5). In contrast, incubation of CD3+ T cells with increasing concentrations (100, 500, and 1000 µM) of clenbuterol (Fig. 1B; Lanes 3–5) or dobutamine (Fig. 1C; Lanes 3–5) inhibited the activation of NF-{kappa}B. Whereas activation of NF-{kappa}B DNA binding was not affected by pretreatment with 100 µM clenbuterol (Fig. 1B; Lane 3) or dobutamine (Fig. 1C; Lane 3), 500 and 1000 µM completely abolished the PMA-stimulated NF-{kappa}B activation (Fig. 1, B and C; Lanes 4 and 5).



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  		Figure 1. The effect of xamoterol, clenbuterol, and dobutamine on nuclear factor (NF)-{kappa}B DNA binding after phorbol-myristate-acetate (PMA) stimulation. CD3+ T lymphocytes were treated for 1.5 h with xamoterol, clenbuterol, or dobutamine at the concentrations indicated (Lanes 3–8) and subsequently stimulated with PMA 50 ng/mL for 30 min (Lanes 2–5) or the respective volumes of ddH2O (Lanes 6–8) as vehicle control. Equal amounts of protein from cell extracts were analyzed for NF-{kappa}B activity by electrophoretic mobility shift assay. A section of a fluorogram is shown. ({blacktriangleleft}) Position of NF-{kappa}B DNA complexes. ({circ}) Nonspecific activity binding to the probe. The data shown are representative of six independent experiments.

 
To evaluate whether the suppressing action of clenbuterol and dobutamine on NF-{kappa}B activation is mediated exclusively through ß-receptors or also affects {alpha}-receptors, we repeated the experiments described above by using phenylephrine, norepinephrine, or epinephrine (Fig. 2). However, in contrast to the results described above, neither phenylephrine (Fig. 2A; Lanes 3–5), nor norepinephrine (Fig. 2B; Lanes 3–5), nor epinephrine (Fig. 2C; Lanes 3–5) inhibited NF-{kappa}B activation in CD3+ T cells.



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  		Figure 2. The effect of phenylephrine, norepinephrine, and epinephrine on nuclear factor (NF)-{kappa}B DNA binding after phorbol-myristate-acetate (PMA) stimulation. CD3+ T lymphocytes were treated for 1.5 h with phenylephrine, norepinephrine, and epinephrine at the concentrations indicated (Lanes 3–8) and subsequently stimulated with PMA 50 ng/mL for 30 min (Lanes 2–5) or the respective volumes of ppH2O (Lanes 6–8) as vehicle control. Equal amounts of protein from cell extracts were analyzed for NF-{kappa}B activity by electrophoretic mobility shift assay. A section of a fluorogram is shown. ({blacktriangleleft}) Position of NF-{kappa}B DNA complexes. ({circ}) Nonspecific activity binding to the probe. The data shown are representative of six independent experiments.

 
To determine whether the inhibition of NF-{kappa}B activation by catecholamines is mediated through ß-receptors, we performed the same experiments as described above after pretreatment with either the nonselective ß-receptor antagonist propranolol or the ß2-receptor-specific antagonist ICI 118.551. Treatment of CD3+ T cells with PMA induced NF-{kappa}B DNA-binding activity (Fig. 3, A and B; Lanes 1 and 2), which was inhibited by pretreatment of cells with 500 and 1000 µM dobutamine (compare with Fig. 1). Incubation of CD3+ T cells with the ß-receptor antagonist propranolol (20 and 100 µM) alone had an inhibitory effect on the activation of NF-{kappa}B by PMA (Fig. 3A; Lanes 6 and 7). The pretreatment of CD3+ T cells with propranolol did not prevent the inhibition of NF-{kappa}B by dobutamine (Fig. 3A; Lanes 8 and 9). Incubation of cells with the specific ß2-receptor-antagonist ICI 118.551 alone (1 and 10 µM) did not alter the extent of NF-{kappa}B activation in response to PMA (Fig. 3B; Lanes 6 and 7). However, pretreatment of the cells with ICI 118.551 completely antagonized the inhibitory effect of dobutamine 500 µM, but not 1000 µM, on NF-{kappa}B activation (Fig. 3B; Lane 8).



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  		Figure 3. The effect of dobutamine, of the ß-receptor-antagonist propranolol (A), and of the ß2-receptor antagonist ICI 118.551 (B) on nuclear factor (NF)-{kappa}B DNA binding after phorbol-myristate-acetate (PMA) stimulation. CD3+ T lymphocytes were treated for 1.5 h with dobutamine (Lanes 3–5) or propranolol (Lanes 6 and 7) at the concentrations indicated and subsequently stimulated with PMA 50 ng/mL for 1 h (Lanes 2–7). Alternatively, CD3+ T cells were pretreated for 1 h with either propranolol (A) or ICI 118.551 (B) at the concentrations indicated, subsequently incubated with dobutamine (500 or 1000 µM), and stimulated with PMA as described above. Equal amounts of protein from cell extracts were analyzed by electrophoretic mobility shift assay for NF-{kappa}B activity. A section of a fluorogram is shown. ({blacktriangleleft}) Position of NF-{kappa}B DNA complexes. ({circ}) Nonspecific activity binding to the probe. The results are representative of six independent experiments.

 
To determine whether ß-agonists may interfere with the degradation of I{kappa}B{alpha}, CD3+ T cells were pretreated with different doses of dobutamine, clenbuterol, and xamoterol (100 and 500 µM) and subsequently stimulated with PMA (50 ng/mL). Incubation of the cells with PMA in the absence of dobutamine, clenbuterol, or xamoterol led to a reduction in I{kappa}B{alpha} steady-state protein levels (Fig. 4; Lane 2). Xamoterol at all concentrations tested, and small concentrations of dobutamine or clenbuterol (100 µM; Fig. 4; Lanes 3, 5, 7, and 8), had no effect on I{kappa}B{alpha} levels compared with PMA-stimulated cells alone. In contrast, pretreatment of the cells with 500 µM dobutamine and clenbuterol partially prevented the PMA-induced degradation of I{kappa}B{alpha} (Fig. 4; Lanes 4 and 6).



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  		Figure 4. The effect of dobutamine, clenbuterol, and xamoterol on I{kappa}B{alpha} degradation. CD3+ T lymphocytes were incubated with different concentrations of dobutamine, clenbuterol, or xamoterol (100 or 500 µM; Lanes 3–8) for 105 min before stimulation with phorbol-myristate-acetate (PMA) (50 ng/mL) for 15 min (Lanes 2–8). Cell extracts were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting with an anti-I{kappa}B{alpha} polyclonal antibody. The results are representative of three independent experiments.

 
The observed inhibition of NF-{kappa}B DNA binding (compare with Fig. 1) raises the question of whether dobutamine or clenbuterol also decreases NF-{kappa}B-dependent gene expression. PMA stimulation resulted in a profound increase in reporter gene activity in six-copy NF-x{kappa}B construct (6x{kappa}B-tk-luciferase) transfected cells (Fig. 5). Whereas xamoterol had no effect on the activation of the {kappa}B-driven promotor, incubation of the cells with dobutamine and clenbuterol (500 µM) attenuated the PMA-induced increase in reporter gene activity by 50%–80% under these experimental conditions (Fig. 5).



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  		Figure 5. The effect of dobutamine, clenbuterol, and xamoterol on nuclear factor (NF)-{kappa}B promoter-driven reporter gene activity. Jurkat T cells were transiently transfected with a six-copy NF-{kappa}B reporter construct (6x-{kappa}B-tk-luciferase). At 6 h after transfection, all cells were pooled, equally distributed into individual wells, and pretreated with dobutamine, clenbuterol, or xamoterol (100 or 500 µM; Lanes 3–8) for 1 h before stimulation with phorbol-myristate-acetate (PMA) (50 ng/mL). After 17 h, the cells were harvested, and luciferase activity was determined. The results are given in relative light units (RLU). These data are representative of six independent experiments.

 
To determine whether inhibition of NF-{kappa}B transcriptional activity by ß-agonists is associated with altered cytokine release by CD3+ T lymphocytes, the effect of dobutamine, clenbuterol, or xamoterol on stimulated IL-8 and IL-10 production was studied. In CD3+ T cells, the release of both IL-8 and IL-10 increased upon PMA stimulation (Fig. 6, top). Dobutamine and clenbuterol attenuated the increase in the production of IL-8, a typical target gene of NF-{kappa}B, whereas xamoterol had no effect (Fig. 6, top). IL-10 release was completely abrogated in the presence of dobutamine and clenbuterol (Fig. 6, bottom), whereas again xamoterol had no effect on the stimulated release of IL-10 (Fig. 6, bottom).



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  		Figure 6. The effect of dobutamine, clenbuterol, and xamoterol on phorbol-myristate-acetate (PMA)-induced interleukin (IL)-8 and IL-10 production in primary CD3+ T lymphocytes. CD3+ T lymphocytes were isolated from the blood of healthy donors and pretreated with different concentrations of dobutamine, clenbuterol, or xamoterol (100 or 500 µM) for 1 h before stimulation by PMA (50 ng/mL) for 24 h. The cell culture supernatant was collected and analyzed for the concentration of IL-8 and IL-10 by enzyme-linked immunosorbent assay. The data shown are representative of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Our findings support by different criteria that the ß-adrenergic agonist dobutamine is an inhibitor of the transcription factor NF-{kappa}B, a central regulator of the immune response (20–22). The effect of dobutamine had the following characteristics: 1) It appeared to be a special pharmacological characteristic of ß2-receptor agonists (dobutamine and clenbuterol), because it occurred in a dose-dependent manner, and the predominant {alpha}/ß-adrenergic agonists tested did not exert any inhibitory effect on NF-{kappa}B activation. Additionally, pretreatment with a specific ß2-receptor antagonist prevented the NF-{kappa}B inhibition by dobutamine. 2) It could be important for the function of immune cells, because it was associated with an inhibition of NF-{kappa}B-driven reporter gene activity and of the expression of NF-{kappa}B target genes such as IL-8. 3) IL-10 is not involved in the inhibition of NF-{kappa}B, because dobutamine inhibited the production of this NF-{kappa}B inhibitor. 4) Dobutamine-mediated NF-{kappa}B inhibition appeared to involve the stabilization of I{kappa}B{alpha} degradation, because the disappearance of immunoreactive I{kappa}B{alpha} that could otherwise be observed upon stimulation was prevented by dobutamine. Finally, 5) it may be of clinical relevance because dobutamine-mediated inhibition of NF-{kappa}B activation appeared in human primary CD3+ T lymphocytes from healthy donors and occurred at in vitro concentrations comparable to those attained in the plasma of patients under certain clinical conditions. We thus propose that dobutamine may exert some of its antiinflammatory effects through inhibition of the activation of NF-{kappa}B.

Several previously reported effects of adrenergic agonists on the function of immune cells could be explained by their inhibitory effect on NF-{kappa}B. NF-{kappa}B plays a crucial role in controlling the activation, proliferation, and differentiation of neutrophils, macrophages, and T and B lymphocytes (21). Therefore, increases in the total number and variations in the subsets of circulating lymphocytes and in the activity of NK cells during catecholamine infusion would be consistent with an inhibition of NF-{kappa}B (1,2,23,24). Moreover, it has been shown that nadolol, a ß-receptor antagonist, blocks stress- and ß-adrenergic-mediated immune alterations, as well as surgery-induced reduction in B- and T-cell proliferation in rats in vivo (25,26). The sum of these results suggests a crucial role of ß-adrenergic receptor activation in modulating NF-{kappa}B-dependent immune functions.

Our observation raises the question of which underlying mechanism is responsible for dobutamine’s possible inhibition of NF-{kappa}B activation. Adrenergic drugs exert their pharmacologic action via potent agonism of {alpha}- and ß-receptors. Dobutamine, with ß1- and ß2-agonistic properties, is used clinically. In this regard, it is of particular interest that Sanders et al. (9) have recently provided substantial evidence for the presence of functional ß2-adrenergic receptors on T cells. In this study, radioligand binding showed a detectable number of ß2-adrenoreceptor binding sites on T-helper Type 1 cells, but not on T-helper Type 2 cells. These results showed that the expression of adrenergic receptors establishes a physiologic mechanism by which endogenous catecholamines or administered vasopressors affect their antiinflammatory actions. Thus, it would be tempting to speculate that the inhibitory effect of dobutamine on NF-{kappa}B described in this study could involve ß2-adrenergic receptor stimulation. In fact, our results revealed that suppression of NF-{kappa}B DNA binding was attenuated by preincubation of the cells with ICI 118.551, a selective ß2-receptor antagonist, and not by pretreatment of T cells with propranolol, a nonselective ß-antagonist. In contrast, propranolol showed inhibitory properties on NF-{kappa}B activation, and this would be consistent with an intrinsic activity in primary CD3+ T lymphocytes at this large concentration. Furthermore, clenbuterol, a selective ß2-agonist, exerted an inhibitory effect on NF-{kappa}B activation comparable to dobutamine treatment. These results are consistent with previous observations that other ß-adrenergic agonists (i.e., isoproterenol) inhibited LPS-induced TNF-{alpha} and IL-8 production in a human monocytic cell line (27). The sum of these findings strongly argues for a key role of the ß2-adrenergic receptor complex in mediating the inhibition of NF-{kappa}B by dobutamine.

Another hint about which signal transduction pathway is involved can be derived from the fact that other drugs that increase intracellular cAMP, e.g., forskolin (28), prostaglandin E2 (29), or theophylline (11), have antiinflammatory properties similar to those of ß-adrenergic agonists. This is in agreement with the results of a previous study that showed a suppressive effect of theophylline on NF-{kappa}B activation and I{kappa}B{alpha} degradation in human pulmonary epithelial cells (30). In addition, dibutyryl cAMP, which increases intracellular cAMP by catecholamine-independent mechanisms, inhibited NF-{kappa}B, TNF-{alpha}, and tissue factor expression at the level of transcription (31). The induction of NF-{kappa}B-dependent gene expression in transiently transfected human monocytic cell line and human umbilical vein endothelial cells was inhibited by increased cAMP and by overexpression of the catalytic subunit of protein kinase A (31). Protein kinase A mediates the phosphorylation of cAMP response element binding protein, which competes with p65 for limiting amounts of cAMP response element binding protein, resulting in reduced NF-{kappa}B activation (32,33). Previously published data revealed that epinephrine increased LPS-induced IL-10 production in human mononuclear cells, an antiinflammatory cytokine that has recently been shown to inhibit NF-{kappa}B by blocking {kappa}B kinase activity (7,34). In contrast, our results demonstrated suppressed IL-10 release in dobutamine- and clenbuterol-treated PMA-stimulated T lymphocytes. This is consistent with previously reported data showing that dobutamine decreased pulmonary IL-10 expression after endotoxin administration in mice (8). However, the fact that other adrenergic drugs tested—such as xamoterol, phenylephrine, epinephrine, and norepinephrine—are also cAMP-increasing substances but did not exert an inhibitory effect on NF-{kappa}B activation in our experimental setting argues against a major role of increased cAMP in NF-{kappa}B inhibition under these conditions.

Because Western blot analyses revealed that dobutamine and clenbuterol prevented the disappearance of immunoreactive I{kappa}B{alpha} after PMA stimulation, these findings would be consistent with previously described mechanisms of other antiinflammatory drugs, in which suppression of NF-{kappa}B activation was due to stabilization and accumulation of I{kappa}B, the natural inhibitor of NF-{kappa}B (17,35). However, it has been shown that the I{kappa}B{alpha} gene contains a {kappa}B-binding site. Therefore, signals such as PMA that induce NF-{kappa}B activation may also stimulate I{kappa}B{alpha} gene expression (36,37). These results, combined with the increased half-life of I{kappa}B{alpha} protein in ß-adrenergic agonist-treated monocytic cells, support the hypothesis that stabilization of newly synthesized I{kappa}B{alpha} by dobutamine could also be responsible for NF-{kappa}B inhibition in T cells (27).

The following lines of evidence suggest that the dobutamine-mediated inhibition of NF-{kappa}B described here may be of clinical relevance. First, the suppressing effect of dobutamine could be observed in human primary CD3+ T lymphocytes obtained from healthy donors. Second, the inhibitory action occurred at in vitro concentrations comparable to those attained in the plasma of patients during the administration of dobutamine (38–41). Finally, incubation of cells with 100 µM dobutamine caused a profound attenuation of NF-{kappa}B-driven reporter gene activity and of cytokines such as IL-8 that are expressed in a NF-{kappa}B-dependent fashion.

In conclusion, our data suggest that dobutamine, a drug that is frequently used for inotropic therapy, is a potent and specific inhibitor of NF-{kappa}B, its trans-acting potency, and its downstream effects on immune cell function. Therefore, our data may describe a molecular mechanism for the antiinflammatory effects associated with ß-adrenergic treatment.


   Acknowledgments
 
This work was supported by departmental funding and a grant from the Deutsche Forschungsgemeinschaft (Bonn, Germany; Heisenberg Stipend DFG PA 533/3-2; BHJP).


   References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Kappel M, Poulsen TD, Galbo H, Pedersen BK. Effects of elevated plasma noradrenaline concentration on the immune system in humans. Eur J Appl Physiol Occup Physiol 1998; 79: 93–8.[Medline]
  2. Kappel M, Tvede N, Galbo H, et al. Evidence that the effect of physical exercise on NK cell activity is mediated by epinephrine. J Appl Physiol 1991; 70: 2530–4.[Abstract/Free Full Text]
  3. Bergmann M, Gornikiewicz A, Sautner T, et al. Attenuation of catecholamine-induced immunosuppression in whole blood from patients with sepsis. Shock 1999; 12: 421–7.[ISI][Medline]
  4. Guirao X, Kumar A, Katz J, et al. Catecholamines increase monocyte TNF receptors and inhibit TNF through ß2-adrenoreceptor activation. Am J Physiol 1997; 273: E1203–8.
  5. Severn A, Rapson NT, Hunter CA, Liew FY. Regulation of tumor necrosis factor production by adrenaline and ß-adrenergic agonists. J Immunol 1992; 148: 3441–5.[Abstract]
  6. van der Poll T, Jansen J, Endert E, et al. Noradrenaline inhibits lipopolysaccharide-induced tumor necrosis factor and interleukin-6 production in human whole blood. Infect Immun 1994; 62: 2046–50.[Abstract/Free Full Text]
  7. van der Poll T, Coyle SM, Barbosa K, et al. Epinephrine inhibits tumor necrosis factor-{alpha} and potentiates interleukin-10 production during human endotoxemia. J Clin Invest 1996; 97: 713–9.[ISI][Medline]
  8. Dhingra VK, Uusaro A, Holmes CL, Walley KR. Attenuation of lung inflammation by adrenergic agonists in murine acute lung injury. Anesthesiology 2001; 95: 947–53.[ISI][Medline]
  9. Sanders VM, Baker RA, Ramer-Quinn DS, et al. Differential expression of the ß2-adrenergic receptor by Th1 and Th2 clones: implications for cytokine production and B cell help. J Immunol 1997; 158: 4200–10.[Abstract]
  10. Gornikiewicz A, Sautner T, Brostjan C, et al. Catecholamines up-regulate lipopolysaccharide-induced IL-6 production in human microvascular endothelial cells. FASEB J 2000; 14: 1093–100.[Abstract/Free Full Text]
  11. Talmadge J, Scott R, Castelli P, et al. Molecular pharmacology of the ß-adrenergic receptor on THP-1 cells. Int J Immunopharmacol 1993; 15: 219–28.[ISI][Medline]
  12. Pahl HL. Activators and target genes of Rel/NF-{kappa}B transcription factors. Oncogene 1999; 18: 6853–66.[ISI][Medline]
  13. Baeuerle PA, Baltimore D. Activation of DNA-binding activity in an apparently cytoplasmic precursor of the NF-{kappa}B transcription factor. Cell 1988; 53: 211–7.[ISI][Medline]
  14. DiDonato JA, Hayakawa M, Rothwarf DM, et al. A cytokine-responsive I{kappa}B kinase that activates the transcription factor NF-{kappa}B. Nature 1997; 388: 548–54.[Medline]
  15. Beg AA, Finco TS, Nantermet PV, Baldwin AS Jr. Tumor necrosis factor and interleukin-1 lead to phosphorylation and loss of I{kappa}B{alpha}: a mechanism for NF-{kappa}B activation. Mol Cell Biol 1993; 13: 3301–10.[Abstract/Free Full Text]
  16. Henkel T, Machleidt T, Alkalay I, et al. Rapid proteolysis of I{kappa}B{alpha} is necessary for activation of transcription factor NF-{kappa}B. Nature 1993; 365: 182–5.[Medline]
  17. Auphan N, DiDonato JA, Rosette C, et al. Immunosuppression by glucocorticoids: inhibition of NF-{kappa}B activity through induction of I{kappa}B synthesis. Science 1995; 270: 286–90.[Abstract/Free Full Text]
  18. Kopp E, Ghosh S. Inhibition of NF-{kappa}B by sodium salicylate and aspirin. Science 1994; 265: 956–9.[Abstract/Free Full Text]
  19. Loop T, Liu Z, Humar M, et al. Thiopental inhibits the activation of nuclear factor {kappa}B. Anesthesiology 2002; 96: 1202–13.[ISI][Medline]
  20. Baldwin AS. The NF-{kappa}B and I{kappa}B proteins: new discoveries and insights. Annu Rev Immunol 1996; 14: 649–83.[ISI][Medline]
  21. Ghosh S, May MJ, Kopp EB. NF-{kappa}B and Rel proteins: evolutionarily conserved mediators of immune responses. Annu Rev Immunol 1998; 16: 225–60.[ISI][Medline]
  22. Tak PP, Firestein GS. NF-{kappa}B: a key role in inflammatory diseases. J Clin Invest 2001; 107: 7–11.[ISI][Medline]
  23. Nomoto Y, Jhonokosi H, Karasawa S. Natural killer cell activity and lymphocyte subpopulations during dobutamine infusion in man. Br J Anaesth 1993; 71: 218–21.[Abstract/Free Full Text]
  24. Burns AM, Keogan M, Donaldson M, et al. Effects of inotropes on human leucocyte numbers, neutrophil function and lymphocyte subtypes. Br J Anaesth 1997; 78: 530–5.[Abstract/Free Full Text]
  25. Nelson CJ, Lysle DT. Severity, time, and ß-adrenergic receptor involvement in surgery-induced immune alterations. J Surg Res 1998; 80: 115–22.[ISI][Medline]
  26. Shakhar G, Ben-Eliyahu S. In vivo ß-adrenergic stimulation suppresses natural killer activity and compromises resistance to tumor metastasis in rats. J Immunol 1998; 160: 3251–8.[Abstract/Free Full Text]
  27. Farmer P, Pugin J. ß-Adrenergic agonists exert their antiinflammatory effects in monocytic cells through the I{kappa}B/NF-{kappa}B pathway. Am J Physiol Lung Cell Mol Physiol 2000; 279: L675–82.[Abstract/Free Full Text]
  28. Taffet SM, Singhel KJ, Overholtzer JF, Shurtleff SA. Regulation of tumor necrosis factor expression in a macrophage-like cell line by lipopolysaccharide and cyclic AMP. Cell Immunol 1989; 120: 291–300.[ISI][Medline]
  29. Spengler RN, Spengler ML, Lincoln P, et al. Dynamics of dibutyryl cyclic AMP- and prostaglandin E2-mediated suppression of lipopolysaccharide-induced tumor necrosis factor-{alpha} gene expression. Infect Immun 1989; 57: 2837–41.[Abstract/Free Full Text]
  30. Ichiyama T, Hasegawa S, Matsubara T, et al. Theophylline inhibits NF-{kappa}B activation and I{kappa}B{alpha} degradation in human pulmonary epithelial cells. Naunyn Schmiedebergs Arch Pharmacol 2001; 364: 558–61.[ISI][Medline]
  31. Ollivier V, Parry GC, Cobb RR, et al. Elevated cyclic AMP inhibits NF-{kappa}B-mediated transcription in human monocytic cells and endothelial cells. J Biol Chem 1996; 271: 20828–35.[Abstract/Free Full Text]
  32. Gerritsen ME, Williams AJ, Neish AS, et al. CREB-binding protein/p300 are transcriptional coactivators of p65. Proc Natl Acad Sci U S A 1997; 94: 2927–32.[Abstract/Free Full Text]
  33. Parry GC, Mackman N. Role of cyclic AMP response element-binding protein in cyclic AMP inhibition of NF-{kappa}B-mediated transcription. J Immunol 1997; 159: 5450–6.[Abstract]
  34. Schottelius AJ, Mayo MW, Sartor RB, Baldwin ASJ. Interleukin-10 signaling blocks inhibitor of {kappa}B kinase activity and nuclear factor {kappa}B DNA binding. J Biol Chem 1999; 274: 31868–74.[Abstract/Free Full Text]
  35. Scheinman RI, Cogswell PC, Lofquist AK, Baldwin ASJ. Role of transcriptional activation of I{kappa}B{alpha} in mediation of immunosuppression by glucocorticoids. Science 1995; 270: 283–6.[Abstract/Free Full Text]
  36. Brown K, Park S, Kanno T, et al. Mutual regulation of the transcriptional activator NF-{kappa}B and its inhibitor, I{kappa}B{alpha}. Proc Natl Acad Sci U S A 1993; 90: 2532–6.[Abstract/Free Full Text]
  37. Sun SC, Ganchi PA, Ballard DW, Greene WC. NF-{kappa}B controls expression of inhibitor I{kappa}B{alpha}: evidence for an inducible autoregulatory pathway. Science 1993; 259: 1912–5.[Abstract/Free Full Text]
  38. Daly AL, Linares OA, Smith MJ, et al. Dobutamine pharmacokinetics during dobutamine stress echocardiography. Am J Cardiol 1997; 79: 1381–6.[ISI][Medline]
  39. Klem C, Dasta JF, Reilley TE, Flancbaum LJ. Variability in dobutamine pharmacokinetics in unstable critically ill surgical patients. Crit Care Med 1994; 22: 1926–32.[ISI][Medline]
  40. Klem C, Dasta JF, Reilley TE, Flancbaum LJ. Pulmonary extraction of dobutamine in critically ill surgical patients. Anesth Analg 1995; 81: 287–91.[Abstract]
  41. Knoll R, Brandl M. Determination of dobutamine levels in human plasma: methodological and clinical aspects. Anasth Intensivther Notfallmed 1986; 21: 34–7.[Medline]
Accepted for publication May 5, 2004.




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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press