Modulation of inflammatory response via alpha2-adrenoceptor blockade in acute murine colitis

Abstract

Inflammatory bowel disease (IBD) is characterized by heavy production of proinflammatory cytokines such as tumour necrosis factor (TNF)-alpha and interleukin (IL)-1beta. Interactions of the autonomic nervous system with local immune cells play an important role in the development of IBD, and the balance of autonomic nerve function is broken in IBD patients with sympathetic overactivity. However, the function of catecholamines in the progress of colitis is unclear. In this study, we examined the role of catecholamines via alpha2-adrenoreceptor in acute murine colitis. The expression of tyrosine hydroxylase (TH) and dopamine b-hydroxylase (DBH), two rate-limiting enzymes in catecholamine synthesis, was detected by immunohistochemistry in murine colitis. Murine colitis was induced by dextran sodium sulphate or trinitrobenzene sulphonic acid (TNBS), and the mice were administered RX821002 or UK14304, alpha2-adrenoceptor antagonists or agonists. Colitis was evaluated by clinical symptoms, myeloperoxidase assay, TNF-alpha and IL-1beta production and histology. Lamina propria mononuclear cells (LPMCs) from mice with TNBS colitis were cultured in the absence or presence of RX821002 or UK14304, and stimulated further by lipopolysaccharide. TH and DBH are induced in LPMCs of inflamed colon, the evidence of catecholamine synthesis during the process of colitis. RX821002 down-regulates the production of proinflammatory cytokines from LPMCs, while UK14304 leads to exacerbation of colitis. Together, our data show a critical role of catecholamines via alpha2-adrenoreceptors in the progress of acute colitis, and suggest that use of the alpha2-adrenoceptor antagonist represents a novel therapeutic approach for the management of colitis.

Fig. 1

Tyrosine hydroxylase (TH) and dopamine b-hydroxylase (DBH) expressions were induced in colonic mucosa during the process of trinitrobenzene sulphonic acid (TNBS)- (day 4) and dextran sulphate sodium (DSS)-induced colitis (day 10). TH and DBH expressions were assessed by immunochemical staining in colonic mucosa of mice with experimental colitis and normal mice (n = 5 in each group). All images are shown at the same magnification (×200).

Fig. 2

Blocking α2-adrenoceptor treatment after initiation of colitis inhibits the progress of trinitrobenzene sulphonic acid (TNBS)-induced disease. Colitis was induced by intracolonic administration of TNBS. RX821002 (10 mg/kg) or UK14304 (2 mg/kg) were administered intraperitoneally 2 h after TNBS instillation and repeated daily. Mice treated with ethanol alone were used as control. (a) Body weight changes in the mice in each group. *P < 0·05 between RX821002-treated mice and the UK14304 group or TNBS mice. (b) Colonic inflammation was scored by histological analysis at the end of the experiment. *P < 0·05; RX821002 mice versus the UK14304 group or TNBS mice. (c) Haematoxylin and eosin staining of colonic tissues of four groups of mice. All images are shown at the same magnification (×40).

Fig. 3

Blocking α2-adrenoceptor treatment inhibits the inflammatory response of trinitrobenzene sulphonic acid (TNBS)-induced colitis. Myeloperoxidase (MPO) activities in colonic tissues and concentrations of tumour necrosis factor (TNF)-α and interleukin (IL)-1β in colonic homogenates of each group of mice were determined by MPO activity assay and specific sandwich enzyme-linked immunosorbent assay (ELISA) kit, respectively, n = 8 in each group. (a) MPO activities in colonic tissues of each group of mice. *P < 0·05; RX821002-treated mice versus treatment-free mice with TNBS colitis. (b) Concentration of TNF-α in colonic homogenates of each group of mice was determined by ELISA. *P < 0·05 compared with the RX821002 group; **P < 0·01, with RX821002. (c) Concentration of IL-1β in colonic homogenates of each group of mice. *P < 0·05 compared with the RX821002 group; **P < 0·01; RX821002.

Fig. 4

Blocking α2-adrenoceptor treatment down-regulates immune response in trinitrobenzene sulphonic acid (TNBS)-induced colitis. Lamina propria mononuclear cells (LPMCs) were isolated from colonic tissues of mice with TNBS-induced colitis at the peak of the disease (day 4) and cultured with medium in the absence or presence of RX821002 (2 nM, 10 nM) or UK14304 (1 nM, 5 nM). After 15 min, the cells were stimulated further in the presence or absence of lipopolysaccharide (LPS) (50 ng/ml) for 8 h, then culture supernatants were collected and determined by enzyme-linked immunosorbent assay (ELISA), n = 5 in each group. (a) Concentration of tumour necrosis factor (TNF)-α in culture supernatants was measured by ELISA. *P < 0·01 compared with LPS + UK14304 (1 nM) or LPS + UK14304 (5nM); #P < 0·05 compared with LPS. (b) Concentration of interleukin (IL)-1β was measured by ELISA. *P < 0·01 compared with LPS, LPS + UK14304 (1nM) or LPS + UK14304 (5nM); #P < 0·05 compared with UK14304 (5 nM). (c) Concentration of IL-10 in culture supernatants was measured; no significant difference was found among those groups with or without stimulation of LPS.

Fig. 5

Blocking α2-adrenoceptor management decreases systemic and colonic inflammatory responses in the dextran sulphate sodium (DSS) model of colitis. Colitis was induced in mice by administration of 5% DSS dissolved in drinking water. RX821002 (10 mg/kg) or UK14304 (2 mg/kg) was administered intraperitoneally 24 h after induction of colitis, and repeated daily until the mice were killed on day 10. Control mice drank only distilled water. (a) Body weight changes in the mice in each group, nine mice in each group. *P < 0·05 compared with DSS group mice or UK14304 group mice. (b) Disease activity index (DAI) was scored on day 10 using the following parameters: stool consistency, presence or absence of faecal blood and weight loss. *P < 0·05 compared with DSS group mice or UK14304 group mice. (c) Colonic inflammation was scored by histological analysis at the end of the experiment. *P < 0·05 compared with DSS mice; #P < 0·01 compared with UK14304 mice. (d) Haematoxylin and eosin staining of colonic tissues of four groups of mice. All images are at shown the same magnification (×40).

Fig. 6

Blocking α2-adrenoceptor treatment inhibits the inflammatory response of DSS-induced colitis. Myeloperoxidase (MPO) activities in colonic tissues and concentrations of tumour necrosis factor (TNF)-α and by interleukin (IL)-1β in colonic homogenates of each group of mice were determined by MPO activity assay and specific sandwich enzyme-linked immunosorbent assay (ELISA) kit respectively. *P < 0·05 compared with the RX821002 group; **P < 0·01, with RX821002. (a) MPO activities in colonic tissues of each group of mice. (b) Concentration of TNF-α in colonic homogenates of each group of mice was determined by ELISA. (e) Concentration of IL-1β in colonic homogenates of each group of mice.