Cholinergic stimulation blocks endothelial cell activation and leukocyte recruitment during inflammation

Abstract

Endothelial cell activation plays a critical role in regulating leukocyte recruitment during inflammation and infection. Based on recent studies showing that acetylcholine and other cholinergic mediators suppress the production of proinflammatory cytokines via the alpha7 nicotinic acetylcholine receptor (alpha7 nAChR) expressed by macrophages and our observations that human microvascular endothelial cells express the alpha7 nAChR, we examined the effect of cholinergic stimulation on endothelial cell activation in vitro and in vivo. Using the Shwartzman reaction, we observed that nicotine (2 mg/kg) and the novel cholinergic agent CAP55 (12 mg/kg) inhibit endothelial cell adhesion molecule expression. Using endothelial cell cultures, we observed the direct inhibitory effects of acetylcholine and cholinergic agents on tumor necrosis factor (TNF)-induced endothelial cell activation. Mecamylamine, an nAChR antagonist, reversed the inhibition of endothelial cell activation by both cholinergic agonists, confirming the antiinflammatory role of the nAChR cholinergic pathway. In vitro mechanistic studies revealed that nicotine blocked TNF-induced nuclear factor-kappaB nuclear entry in an inhibitor kappaB (IkappaB)alpha- and IkappaBepsilon-dependent manner. Finally, with the carrageenan air pouch model, both vagus nerve stimulation and cholinergic agonists significantly blocked leukocyte migration in vivo. These findings identify the endothelium, a key regulator of leukocyte trafficking during inflammation, as a target of anti-inflammatory cholinergic mediators.

Figure 1.

Cholinergic agonists block endothelial cell activation in vivo. Mice previously injected with a preparatory dose of LPS in the ear received vehicle (saline), nicotine (2 mg/kg), or CAP55 (12 mg/kg) 15 min before systemic LPS challenge (Shwartzman reaction model). Five h after LPS challenge, mouse ears were analyzed for (A) VCAM-1 and E-selectin mRNA expression by quantitative RT-PCR. Data are shown as the average relative expression of VCAM-1 and E-selectin mRNA copy number (normalized to GAPDH) in samples obtained from saline- versus nicotine-treated mice. *, P < 0.05 comparing nicotine treated vs. saline treated using the Student's t test. (B) VCAM-1 and (C) E-selectin expression within the ears by immunostaining methods (arrows indicate vessels).

Figure 2.

Structure and antiinflammatory activity of CAP55 in vitro. (A) Chemical structure of nicotine and CAP55. (B) CAP55 and nicotine inhibit TNF production by LPS-stimulated macrophages in vitro. Experiments were performed as described in Materials and methods. *, P < 0.05; **, P < 0.001 comparing CAP55 + LPS– or nicotine + LPS–treated cells versus LPS-treated cells alone, as determined using the Student's t test.

Figure 3.

ACh and cholinergic agonists block adhesion molecule expression by TNF-treated endothelial cells in vitro. Confluent monolayers of HuMVECs were untreated, treated with TNF (1 ng/ml) alone or were treated with (A) ACh, (B) nicotine, or (C) CAP55 before TNF stimulation (1 ng/ml). Cell surface expression of ICAM-1 (▪), E-selectin (♦), or VCAM-1 (▴) was determined using a cell-based ELISA method. 100% represents 0.55 (0.04), 1.12 (0.05), and 0.864 (0.03) OD (±SD) at 450 for E-selectin, ICAM-1, and VCAM-1, respectively. *, P < 0.05, **, P < 0.01 comparing treatment + TNF versus TNF alone using the Student's t test. Data are shown as the percent control (±SD), with TNF alone as control. (D) ICAM-1 expression was assessed using flow cytometry methods (mean fluorescence intensity, MFI) using ICAM-1–specific antibodies: (1) untreated cells (isotype control), MFI = 29.6; (2) untreated cells, MFI = 164; (3) nicotine (10⁻⁶ M) + TNF (1 ng/ml), MFI = 380; and (4) TNF (1 ng/ml) alone, MFI = 677.

Figure 4.

HuMVECs express the α7 nAChR. The expression of α7 nAChR by HuMVECs (EC1, EC2) was assessed by (A) RT-PCR and (B) Western blotting methods. (C) HuMVECs bind α-BGT-FITC (0–2.5 μg), a specific α7 nAChR antagonist, as determined by flow cytometry.

Figure 5.

Nicotine and CAP55 block TNF-induced adhesion molecule expression in vitro via the nAChR pathway. HuMVECs were untreated, treated with TNF (1 ng/ml) alone, or treated with mecamylamine (Mec, 2 μM) before (A) nicotine (10⁻⁶–10⁻⁷ M) or (B) CAP55 (CAP55, 10⁻⁶–10⁻⁷ M) plus TNF (1 ng/ml) for 18 h. ICAM-1 expression (black bars) was determined using a cell-based ELISA technique. Data are shown as the percent control (±SD), with TNF alone as control. 100% represents 0.574 (0.04) and 0.497 (0.04) OD 450 for the nicotine and CAP55 sets, respectively. *, P < 0.05; **, P < 0.01, comparing treatment plus TNF versus treatment plus mecamylamine plus TNF using the Student's t test.

Figure 6.

ACh and cholinergic agonists block chemokine production by TNF-treated HuMVECs. HuMVECs were untreated, treated with TNF (1 ng/ml) alone, or treated with (A) ACh, (B) nicotine, or (C) CAP55 before TNF stimulation (1 ng/ml) for 18 h. The production of IL-8 (♦), MCP-1 (▪), or RANTES (▴) was determined by ELISA. Data are shown as the percent control (±SD), with TNF alone as control. 100% represents 18,848 (430), 63,436 (9,194), 3,122 (64) pg/ml (±SD) for IL-8, MCP-1, and RANTES, respectively. *, P < 0.05; **, P < 0.01 comparing treatment plus TNF versus TNF alone using the Student's t test.

Figure 7.

Nicotine blocks monocyte and neutrophil adhesion to HuMVECs. HuMVECs were untreated, treated with TNF alone, or treated with nicotine (Nic, 10⁻⁷ M) before TNF stimulation (1 ng/ml) or no TNF stimulation. HuMVECs were washed and incubated with either (A) human monocytes or (B) neutrophils (previously labeled with Calcein AM) for 0.5 h. After washing, bound leukocytes were quantified using a cytofluorescence assay method. The data are shown as the number of cells bound to the HuMVEC monolayers expressed as percent control (±SD), with the control being TNF-treated HuMVECs alone. 100% represents ∼16% (or 3.2 × 10⁴) and 27% (or 5.4 × 10⁴) of input monocytes and neutrophils, respectively (based on a standard curve of labeled cells). *, P < 0.05 nicotine plus TNF versus TNF alone using the Student's t test.

Figure 8.

Nicotine blocks NF-κB nuclear translocation in an IκBα- and IκBɛ-dependent manner. HuMVECs were either untreated or treated with nicotine (10⁻⁶–10⁻⁷ M) before TNF stimulation or no stimulation. Cytoplasmic and nuclear fractions were isolated 15 min after TNF addition. Samples were electrophoresed, transferred, and Western blotted using (A) NF-κB, (B) IκBα, or (C) IκBɛ antibodies. Lamin A/C and β-actin were used as controls for loading the nuclear and cytoplasmic fractions, respectively. Data are also shown as the ratio of NF-κB, IκBα, or IκBɛ to control (nuclear or cytoplasmic) protein.

Figure 9.

Cholinergic stimulation blocks leukocyte recruitment in vivo. (A) The effect of vehicle (saline) or nicotine (0.2 and 2 mg/kg, i.p.) on leukocyte recruitment was examined using the carrageenan air pouch model. (B) The effect of saline or CAP55 (1, 4, or 12 mg/kg, i.p.) on leukocyte recruitment. (A, inset) Mice were pretreated with mecamylamine (Mec, 200 μg/mouse) before treatment with nicotine (2 mg/kg) or (B inset) CAP55 (12 mg/kg). (C) VNS blocks leukocyte recruitment (compared with sham surgery treatment) using the carrageenan air pouch model. The data are shown as the average number of inflammatory cells per pouch (± SEM), presented as percent control vehicle-treated animals as the control. 100% represents 2.7 × 10⁶, 1.4 × 10⁶, and 1.8 × 10⁶ cells per pouch in the groups of animals treated with nicotine, CAP55, and VNS, respectively. *, P < 0.05; **, P <0.01; ***, P < 0.001, using the Student's t test comparing treatment versus vehicle or treatment versus treatment plus mecamylamine (insets).