Central injection of nitric oxide synthase inhibitors increases peripheral interleukin-6 and serum amyloid A: involvement of adrenaline from adrenal medulla

Abstract

1. Accumulating evidence suggests that plasma levels of interleukin-6 (IL-6), a major cytokine stimulating the synthesis of acute phase proteins, are intimately regulated by the central nervous system (CNS). 2. In the present study, effects of intracerebroventricular (i.c. v) injection of N(G)-nitro-L-arginine methyl ester (L-NAME) or 7-nitroindazole, nitric oxide synthase (NOS) inhibitors, on plasma IL-6 levels and peripheral IL-6 mRNA expression were examined in mice. 3. L-NAME (0.1 - 2 microg per mouse i.c.v.) and 7-nitroindazole (0.2 - 2 microg per mouse i.c.v.) induced a dose-dependent increase in plasma IL-6 levels and a subsequent increase in circulating serum amyloid A, a liver acute-phase protein. In contrast, an intraperitoneal (i.p.) injection of L-NAME up to the dose of 25 microg per mouse had no effect. 4. Pretreatment with yohimbine (alpha(2)-adrenergic antagonist; 1 mg kg(-1) i.p.), or ICI-118,551 (beta(2)-adrenergic antagonist; 2 mg kg(-1) i.p.), but not with prazosin (alpha(1)-adrenergic antagonist; 1 mg kg(-1) i.p.), nor betaxolol (beta(1)-adrenergic antagonist; 2 mg kg(-1) i.p.), significantly inhibited the central L-NAME-induced plasma IL-6 levels. 5. I.c.v. (50 microg per mouse) or i.p. (100 mg kg(-1)) pretreatment with 6-hydroxydopamine had no effect on central L-NAME-induced plasma IL-6 levels. However, intrathecal (i.t.) pretreatment with 6-hydroxydopamine (20 microg per mouse) markedly inhibited central L-NAME-induced plasma IL-6 levels. Both yohimbine (1.5 microg per mouse i.t.) and ICI-118,551 (1.5 microg per mouse i. t.) were effective in inhibition of central L-NAME-induced plasma IL-6 levels. 6. There was an elevation of base-line plasma IL-6 levels in adrenalectomized animals. The adrenalectomy-enhanced levels were not further increased by central L-NAME. 7. L-NAME (2 microg per mouse i.c.v.) induced an increase in IL-6 mRNA expression in liver, spleen, and lymph node. 8. These results suggest that NOS activity in the brain tonically down-regulates peripheral IL-6 by inhibiting adrenaline release from the adrenal medulla.

Figure 1

(A) Effects of L-NAME injected i.c.v. on the plasma IL-6 levels. Either saline (5 μl per mouse i.c.v.) or various doses of L-NAME (0.1–2 μg per mouse) were administered i.c.v. and blood was collected 1.5 h after the injection. For restraint group, the stress was applied for 1.5 h immediately after the L-NAME injection. (B) Time course of the effect of L-NAME injected i.c.v. on plasma IL-6 levels. Blood samples were obtained from one group of animals immediately after L-NAME (2 μg per mouse i.c.v.) or saline injection (value at time point 0), whereas other groups of animals were allowed to rest for the indicated intervals before blood samples were obtained. (C) Dose-dependent increase in plasma IL-6 levels by an i.c.v. injection of 7-nitroindazole, a selective inhibitor of neuronal NOS. (D) Effects of L-NAME and 7-nitroindazole injected i.c.v. on plasma IL-1β and TNF-α levels. The data are means±s.e.mean (n=8–16). ^*P<0.05; ^**P<0.01; ^***P<0.001, significantly different from saline-treated control.

Figure 2

Effects of an i.c.v. injection of L-NAME and 7-nitroindazole on the plasma SAA levels. Either saline (5 μl per mouse), L-NAME (2 μg per mouse), or 7-nitroindazole (2 μg per mouse) was administered i.c.v. and blood was collected 24 h after the injection. The data are means±s.e.mean (n=8–16). ^*P<0.05, significantly different from saline-treated control.

Figure 3

Effects of adrenoceptor antagonists, on the i.c.v. L-NAME-induced plasma IL-6 levels. Animals were intraperitoneally pretreated with phentolamine mesylate (2 mg kg⁻¹), propranolol HCl (10 mg kg⁻¹), prazosin HCl (0.5 mg kg⁻¹), yohimbine HCl (1 mg kg⁻¹), betaxolol HCl (2 mg kg⁻¹), or ICI-118,551 HCl (2 mg kg⁻¹) 15 min prior to the central L-NAME (2 μg per mouse i.c.v.) injection, and blood was collected 2 h after the L-NAME injection. The data are means±s.e.mean of 8–14 animals. ^*P<0.05, ^**P<0.01, ^***P<0.001, significantly different from the respective saline-treated controls. ††P<0.01.

Figure 4

Effects of pretreatment of mice with (A) i.c.v. or (B) i.p. 6-hydroxydopamine (6-OHDA) on the L-NAME (2 μg per mouse)-induced increase in the plasma IL-6 levels. Animals were pretreated with 6-OHDA (A) i.c.v. (50 μg per mouse), (B) i.p. (100 mg kg⁻¹) 3 days prior to the central L-NAME (2 μg per mouse i.c.v.) injection, and blood was collected 2 h after the L-NAME injection. The data are means±s.e.mean of 8–14 animals. ^**P<0.01, significantly different from the respective saline-treated controls.

Figure 5

Effects of intrathecal (i.t.) pretreatment with (A) 6-hydroxydopamine (6-OHDA), (B) yohimbine or ICI-118,551 on the i.c.v. L-NAME-induced plasma IL-6 levels. Animals were pretreated with i.t. (A) 6-OHDA (20 μg per mouse) and (B) yohimbine HCl (1.5 μg per mouse) or ICI-118,551 HCl (1.5 μg per mouse), 3 days and 15 min, respectively, prior to the central L-NAME (2 μg per mouse i.c.v.) injection, and blood was collected 2 h after the L-NAME injection. The data are means±s.e.mean of 8–14 animals. ^*P<0.05, ^**P<0.01, ^***P<0.001, significantly different from the respective saline-treated controls. †P<0.05, ††P<0.01.

Figure 6

Effects of L-NAME and MK-801 on plasma IL-6 levels in sham operated or adrenalectomized (ADX) mice. Sham operation or adrenalectomy was performed 2 weeks prior to the central injection of L-NAME (2 μg per mouse) or MK-801 (1 μg per mouse), and blood was collected 2 h after the injection. The data are means±s.e.mean of 8–10 animals. ^*P<0.05, significantly different from the respective saline-treated controls.

Figure 7

Effect of L-NAME on IL-6 mRNA expression in various peripheral organs. Either saline or L-NAME (2 μg per mouse) was administered i.c.v. and IL-6 mRNA expression was evaluated at 1 h after the injection. The gel shown is a representative one from three repeated experiments. The number of animals used for each experiment was seven for the pituitary, five for the adrenals, and three for the rest of the organs.