Multiple G-protein-coupling specificity of beta-adrenoceptor in macrophages

Abstract

Adrenergic signalling of the immune system is one of the important modulator pathways of the inflammatory immune response realized via G protein-mediated pathways. The resulted signal depends on the type of the receptor-coupled G-protein (GPCR) that, according to the classical paradigm in the case of beta-adrenergic receptor (beta-AR), is Gs-type. Recently, alternate and/or multiple G protein coupling specificity of GPCRs have been demonstrated including a switch from Gs to Gi binding. The possibility of a Gs/Gi switch and its role in the immune response of macrophages has not been investigated yet. In this study, we demonstrate that beta-adrenergic stimulation itself is able to induce a transient mitogen-activated protein kinase phosphorylation in murine peritoneal macrophages in a pertussis toxin-sensitive manner, suggesting that the Gs/Gi switch also occurs in the immune system. Although this process is very rapid, it can influence different signalling pathways and can reprogramme effector functions suggesting that sympathetic modulation of the defence mechanism of the innate immune system has an additional, Gs/Gi switch-dependent component.

Figure 1

Isoproterenol-induced ERK (a) and p38 (b) phosphorylation in murine peritoneal macrophages. In each experiment, 5 × 10⁶ peritoneal macrophages were treated and 50 µg of whole cell protein extracts were analysed by Western blot using specific antibodies recognizing phospho-ERK1/2, phospho-p38, or total ERK1/2 and p38 proteins, as described in Materials and methods. Experiments were repeated three times from separate pooled macrophage preparations and each experiment was analysed by Western blot two times. Macrophages were treated with 10 µm or 100 µm isoproterenol for 10 min (upper and middle parts) or for 5, 10, 30, 60, 90 and 210 min with 100 µm isoproterenol (lower parts). Upper parts: One typical immunoblot out of n = 6. Middle panels show the quantification of these experiments and lower panels show the quantification of ERK and p38 phosphorylation in time. In the middle and lower panels, data are given as the optical densities of the bands obtained by Western blot and determined by BioScan v1.0. Protein loading was standardized to the total ERK1/2 protein levels of the cellular extracts and values were normalized to the control, nontreated cells. Columns represent the mean values and error bars represent the ±SE where n = 6. The asterisks represent the significances as they are given in the Materials and methods section. ***Indicates significance at level P < 0·001.

Figure 2

PTX sensitivity of isoproterenol-induced ERK and p38 phosphorylation in peritoneal macrophages. Peritoneal macrophages (5 × 10⁶) were pretreated with 100 ng/ml PTX overnight and that was followed by the administration of 100 µm isoproterenol for 10 min. Western blots were performed as described in the legend to Fig. 1. Immunoblot (upper panels) and quantification (lower panels) of ERK (a) and p38 (b) phosphorylation in macrophages are presented. Immunoblots show the results of one typical experiment from the three independent experiments, each with duplicate determination. Evaluation and standardization were carried out as described in the legend to Fig. 1. Columns represent the mean values and error bars represent the ±SE from the mean (n = 6). ***Indicates significance at level P < 0·001.

Figure 3

Effect of PTX and isoproterenol pretreatments on LPS induced TNF-α production and ERK phosphorylation in peritoneal macrophages. Peritoneal macrophages (2–3 × 10⁵) were pretreated with or without 100 ng/ml PTX overnight and then with or without 100 µm isoproterenol for 10 min, followed by the stimulation with 10 µg/ml LPS(a) Supernatants were collected after 24 hr for determination of TNF-α levels. The quantitative results were obtained from five independent experiments each in triplicate. Error bars represent ±SE from the mean. **Indicates significance at level P < 0·01 and *** at level P < 0·001. (b and c) Peritoneal macrophages (5 × 10⁶) were pretreated with or without 100 ng/ml PTX overnight and that was followed by the administration of 100 µm isoproterenol for 10 min, followed by the stimulation with 10 µg/ml LPS for 5 min 50 µg whole cell protein extracts were analysed by Western blot, immuno-stained with phospho-specific ERK1/2 antibodies and reprobed with antibodies raised against non-phosphorylated ERK1/2 to reveal total ERK1/2 protein levels. Immunoblot (b) and quantification (c) of three independent experiments, each with duplicate determination. Quantification and standardization of the data were carried out as described in the legend to Fig. 1. Error bars represent ±SE from the mean (n = 6). The asterisks represent the significances as they are given in the Materials and methods section.

Figure 4

Modulation of PMA-induced TNF-α production by isoproterenol. Effect of PTX or KT-5620 pretreatments. Peritoneal macrophages (2–3 × 10⁵) were pretreated with or without 100 ng/ml PTX overnight and then with or without 100 µm isoproterenol for 10 min, followed by the stimulation of 100 nm PMA for 24 hr (a) Macrophages without PTX treatment were preincubated with 200 nm KT-5720 for 30 min prior to isoproterenol and PMA addition (b) Supernatants were collected after 24 hr for the determination of TNF-α. The quantitative results were obtained from three independent experiments, each tested in triplicate. Error bars represent ±SE from the mean (n = 9). The asterisks represent the significances as they are given in the Materials and methods section.

Figure 5

Modulation of PMA-induced ERK phosphorylation by isoproterenol in macrophages. The effect of PTX. Peritoneal macrophages (5 × 10⁶) were pretreated with or without 100 ng/ml PTX overnight that was followed by the administration of 100 µm isoproterenol for 10 min and stimulated with 100 nm PMA for 5 min 50 µg whole cell protein extracts were analysed by Western blot, immuno-stained with phospho-specific ERK1/2 antibodies and reprobed with antibodies raised against non-phosphorylated ERK1/2 to reveal total ERK1/2 protein levels. Immunoblot (a) and quantification (b) of three independent experiments, each with duplicate determination. Quantification and standardization of the data were carried out as described in the legend to Fig. 1. Error bars represent ±SE from the mean (n = 6). The asterisks represent the significances as they are given in the Materials and methods section.

Figure 6

Effect of PKC inhibitors on the mitogen-stimulated TNF-α production in murine peritoneal macrophages. Cells were prepared and plated as described in the Methods section. 50 nm Bim or 50 nm Gö6976 were added to the cells 30 min before LPS (10 µg/ml) or PMA (100 nm) induction. Cells were incubated for 24 hr and TNF-α levels where determined from the supernatants by the ELISA technique. Columns represent the TNF-α level expressed as the percentage of the total stimulation observed in the presence of LPS or PMA alone. Results were obtained from three independent experiments, each tested in triplicate. Error bars represent ±SE from the mean (n = 9) and ** indicates significance at level P < 0·01 and *** at level P < 0·001.