Beta 2-adrenergic receptor agonists are novel regulators of macrophage activation in diabetic renal and cardiovascular complications

Abstract

Macrophage activation is increased in diabetes and correlated with the onset and progression of vascular complications. To identify drugs that could inhibit macrophage activation, we developed a cell-based assay and screened a 1,040 compound library for anti-inflammatory effects. Beta2-adrenergic receptor (β2AR) agonists were identified as the most potent inhibitors of phorbol myristate acetate-induced tumor necrosis factor-α production in rat bone marrow macrophages. In peripheral blood mononuclear cells isolated from streptozotocin-induced diabetic rats, β2AR agonists inhibited diabetes-induced tumor necrosis factor-α production, which was prevented by co-treatment with a selective β2AR blocker. To clarify the underlying mechanisms, THP-1 cells and bone marrow macrophages were exposed to high glucose. High glucose reduced β-arrestin2, a negative regulator of NF-κB activation, and its interaction with IκBα. This subsequently enhanced phosphorylation of IκBα and activation of NF-κB. The β2AR agonists enhanced β-arrestin2 and its interaction with IκBα, leading to downregulation of NF-κB. A siRNA specific for β-arrestin2 reversed β2AR agonist-mediated inhibition of NF-κB activation and inflammatory cytokine production. Treatment of Zucker diabetic fatty rats with a β2AR agonist for 12 weeks attenuated monocyte activation as well as pro-inflammatory and pro-fibrotic responses in the kidneys and heart. Thus, β2AR agonists might have protective effects against diabetic renal and cardiovascular complications.

Keywords: diabetes; fibrosis; inflammation; macrophages.

Figure 1

High glucose (HG) and diabetes increase TNF-α production and PKC activity in BM-derived macrophages (A–C). A: Rat BM-derived macrophages were cultured under control glucose (CG, 5.6 mM) or HG (25 mM) for 72 h. TNF-α production was measured in conditioned medium (n=17). B: PKC activity in rat BM-derived macrophages stimulated with HG for 72 h or PMA (100 nM) for 30 min (n=3). C: PKC activity in BM-derived macrophages isolated from control and diabetic mice at 12 weeks of diabetes (n=4, also indicates the number of mice studied individually). Diabetes was induced in 6-week-old male C57Bl6/J mice fasted for 12 h, with intraperitoneal injections of STZ in citrate buffer (90 mg/kg) for 2 consecutive days. D: β2AR agonists decreased LPS-induced TNF-α production in rat BM-derived macrophages. Cells were preincubated with metaproterenol or terbutaline hemisulfate for 1 h and then stimulated with LPS (50 ng/mL) for 6 or 48 h (n=3). E: β2AR agonists decreased diabetes-induced TNF-α production in rat PBMCs isolated from control and diabetic rats at 4 weeks of diabetes. Cells were incubated in RPMI medium with 10% FBS for 1 h, and then treated with 500 nM metaproterenol or terbutaline hemisulfate for an additional 16 h with or without 1 h pretreatment with ICI118551 (100 nM), a selective β2AR blocker (n=9~11, also indicates the number of rats studied individually). ^* p < 0.001, ^†p < 0.01, and ^‡p < 0.05 vs. control, ^§p < 0.01, ^||p < 0.001, and ^¶p < 0.005 vs. LPS or DM, ^#p < 0.05, and ^** p < 0.001 vs. corresponding control without ICI118551.

Figure 2

β2AR agonists inhibit high glucose (HG)-induced proinflammatory responses via β-arrestin2-NF-κB pathway in THP-1 cells. A: Cells were treated with control glucose (CG, 5.6 mM), HG (25 mM), or mannitol (25 mM) and real-time RT-PCR was performed to measure the level of gene expression (n=3). B–H: Cells were preincubated with 10 μM metaproterenol or salbutamol for 2 h and then stimulated with HG for 72 h (n=3–7). Real time RT-PCR and flow cytometry were performed to measure the level of mRNA expressions of MCP-1, TNF-α and IL-1β (B) and CD36 positive cells (C). Representative western blots show the protein levels of β-arrestin2 (D) and phospho-IκBα (F). Equal amounts of protein were subjected to immunoprecipitation with anti-β-arrestin2 antibody followed by immunoblotting with antibody against β-arrestin2 or IκBα (E). NF-κB activity was measured in nuclear protein extract (G). Cells were transfected with luciferase reporter vector for NF-κB and luciferase activity was measured (H). ^* p < 0.001, ^‡p < 0.05, ^††p < 0.01, and ^‡‡p < 0.005 vs. CG, ^†p < 0.001, and ^§p < 0.05 vs. mannitol, ^||p < 0.01, ^¶p < 0.005, ^#p < 0.05, and ^** p < 0.001 vs. HG.

Figure 3

β2AR agonists inhibit high glucose (HG)-induced proinflammatory responses via β-arrestin2-NF-κB pathway in rat BM-derived macrophages. Cells were preincubated with 10 μM metaproterenol or salbutamol for 2 h and then stimulated with HG for 72 h (n=3–6). Real time RT-PCR was performed to measure the level of mRNA expressions of MCP-1, TNF-α and IL-1β (A). Representative western blots show the protein levels of β-arrestin2 (B) and phospho-IκBα (D). Equal amounts of protein were subjected to immunoprecipitation with anti-β-arrestin2 antibody followed by immunoblotting with antibody against β-arrestin2 or IκBα (C). NF-κB activity was measured in nuclear protein extract (E). Cells were transfected with luciferase reporter vector for NF-κB and luciferase activity was measured (F). ^* p < 0.005, ^§ p < 0.01, and ^¶p < 0.05 vs. CG, ^†p < 0.001, ^‡p < 0.005, ^||p < 0.05, and ^#p < 0.01 vs. HG.

Figure 4

Suppression of β-arrestin2 or β2AR using siRNA significantly blocks effects of β2AR agonists on NF-κB activation and proinflammatory cytokine production. THP-1 cells were treated with high glucose (HG) for 72 h in the presence or absence of 10 μM metaproterenol or salbutamol pretreatment for 2 h (n=4–8). A: Representative western blot shows that protein level of β-arrestin2 was decreased by siRNA transfection. B and E: NF-κB activity was measured in nuclear protein extract. C and F: Real time RT-PCR was performed to measure the level of mRNA expressions of MCP-1, TNF-α and IL-1β. D: Quantitative RT-PCR shows that mRNA level of β2AR was decreased by siRNA transfection. ^* p < 0.001, ^†p < 0.01, and ^§ p < 0.05 vs. control siRNA.

Figure 5

β2AR agonist inhibits monocyte activation in ZDF rats (A–C). A: Monocyte adhesion to human microvascular endothelial cells was assessed in PBMCs isolated from ZF lean and ZDF rats with or without salbutamol treatment. B: CD11b/c and CCR2-positive cells were measured using flow cytometry in PBMCs and BM mononuclear cells. C: CD36 positive cells were measured in BM mononuclear cells after 24 h stimulation with LPS (1 μg/mL). β2AR agonist inhibits NF-κB pathway in BM-derived macrophages in ZDF rats (D–H). Rat BM-derived macrophages were differentiated for 7 days in RPMI medium containing L929 cells conditioned medium and then stimulated with LPS (1 μg/mL) for an additional 24 h. Representative western blots show the protein levels of β-arrestin2 (D) and phospho-IκBα (F). Equal amounts of protein were subjected to immunoprecipitation with anti-β-arrestin2 antibody followed by immunoblotting with antibody against β-arrestin2 or IκBα (E). NF-κB activity was measured in nuclear protein extract (G). Cells were transfected with luciferase reporter vector for NF-κB and luciferase activity was measured (H). ^* p < 0.001, ^‡p < 0.005, ^§p < 0.05, and ^#p < 0.01 vs. vehicle-treated ZF lean, ^†p < 0.05, ^||p < 0.001, and ^¶p < 0.005 vs. vehicle-treated ZDF, n=6–9.

Figure 6

β2AR agonist reduces renal changes in ZDF rats. Representative kidney sections stained with PAS (A), Masson’s trichrome (E) and immunohistochemical staining for CD68 positive macrophages (F), original magnification ×100 and ×400, scale bar: 200 and 50 μm. B–D, I, K: Protein expression of collagen I and IV, fibronectin, phosphorylated p65, and β-arrestin2 was analyzed using immunoblot analysis. G, H, J: Real-time RT-PCR was performed to measure the mRNA expression of NOS2, MCP-1, TNF-α, IL-1β, and arginase-1. ^* p < 0.05, ^†p < 0.005, and ^||p < 0.001 vs. vehicle-treated ZF lean, ^‡p < 0.005, ^§p < 0.001, ^¶p < 0.05, and ^#p < 0.01 vs. vehicle-treated ZDF, n=6–9.

Figure 7

β2AR agonist reduces cardiac changes in ZDF rats. A–C, G: Protein expression of collagen I and IV, fibronectin, and phosphorylated p65 was analyzed using immunoblot analysis. Representative heart sections stained for CD68 positive macrophages by immunohistochemistry (D, original magnification ×100 and ×400). Scale bar: 200 and 50 μm. E, F, H: Real-time RT-PCR was performed to measure mRNA expression of NOS2, MCP-1, TNF-α, IL-1β, and arginase-1. I: Representative M-mode echocardiograms and quantification of left ventricular ejection fraction and fractional shortening. ^* p < 0.001, ^§p < 0.05, ^||p < 0.005, and ^#p < 0.01 vs. vehicle-treated ZF lean, ^†p < 0.001, ^‡p < 0.05, and ^¶p < 0.005 vs. vehicle-treated ZDF, n=6–9.