Introduction

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IL-6 is a multifunctional cytokine produced by a variety of cells, including immune cells, fibroblasts, endothelial cells and astrocytes, in response to infection, trauma and shock. It possesses a broad range of activities on immune responses, hematopoiesis and acute phase reactions (for review, see Hirano, 1994). It has been observed that immobilization stress induces an increase in plasma IL-6 levels in rats (Zhou et al., 1993; Takaki et al., 1994) and mice (Song et al., 1996). Recently, we showed that i.c.v. injection of MK-801, an NMDA receptor blocker, increased the basal and stress-induced levels of plasma IL-6, which suggests that there is a tonic inhibitory control mechanism via the NMDA receptors for the regulation of the plasma IL-6 level (Song et al., 1996). One mechanism by which the NMDA receptor system might tonically suppress the plasma IL-6 levels is that NMDA receptors might exert a tonic facilitatory effect on inhibitory neurotransmitters that subsequently inhibit plasma IL-6 levels.

GABA is a major inhibitory neurotransmitter in the mammalian brain, which acts through receptors generally divided into GABA_A and GABA_B subtypes (Bowery, 1993; Macdonald and Olsen, 1994). However, the potential roles of central GABA_A and GABA_B receptors in the regulation of plasma IL-6 levels have not been characterized. Furthermore, immunomodulatory actions of benzodiazepines, agents that act through a GABA_A receptor complex, have been reported (Fride et al., 1990; Galdiero et al., 1995; Benschop et al., 1996).

In the present study, we examined the effects of the i.c.v. injection of GABA receptor agonists (muscimol and baclofen, GABA_A and GABA_B receptor agonists, respectively) and antagonists (SR-95,531 and 2-hydroxysaclofen, GABA_A and GABA_B receptor antagonists, respectively) on basal and restraint stress-induced plasma IL-6 levels in mice.

	Materials and Methods

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Animals. Male ICR mice weighing 25 to 30 g, supplied from Myung-Jin, Inc. (Seoul, Korea), were used in all the experiments. The animals were housed five per cage in a room maintained at 22°C ± 1°C with an alternating 12-h light-dark cycle. Food and water were available ad libitum.

Compounds. Muscimol hydrobromide, R(+)-baclofen hydrochloride, SR-95,531, 2-hydroxysaclofen and MK-801 were purchased from Research Biochemicals International (Natick, MA). 2-Hydroxysaclofen was dissolved in 0.1 N NaOH and diluted to the appropriate volume with 0.2 N HCl. Muscimol, baclofen, SR-95,531 and MK-801 were dissolved in normal saline solution (0.9% NaCl). The doses of all drugs represent the salt.

Intracerebroventricular injection. The i.c.v. administration was performed in accordance with the procedure established by Laursen and Belknap (1986), which was modified from the method of Haley and McCormick (1957). Briefly, each conscious mouse was grasped firmly by the loose skin behind the head, and its snout was gently pushed into the mouth of a 1.5-ml Eppendorf tube, which was horizontally fixed on the edge of a table. The cone of the Eppendorf tube was cut off to secure ventilation. The animal was injected 1 to 2 mm lateral to bregma with a 50-µl Hamilton microsyringe fitted with a 26-gauge needle the tip of which was adjusted to be inserted 2.4 mm deep. The i.c.v. injection volume was 5 µl, and injection sites were verified by injecting the same volume of 1% methylene blue into the site and then observing the distribution of the injected dye in the ventricular space. The dye injected i.c.v. was found to be distributed in the ventricular spaces and ventral surface of the brain and in the upper cervical portion of the spinal cord.

Restraint stress and plasma IL-6 assay. Mice were pretreated i.c.v. with saline (5 µl), various doses of muscimol (20-200 ng), baclofen (5-20 ng), SR-95,531 (1-10 ng) and 2-hydroxysaclofen (1-10 µg) for 10 min before the start of the restraint stress. The stress procedure consisted of restraining each animal for 1 h in a 50-ml Corning conical tube, with the nose of the mouse at the tip of the tube. Adequate ventilation was provided by means of a hole at the tip of the tube. Immediately after completion of the restraint stress, blood was collected by puncturing the retro-orbital venous plexus of the conscious animals. For the nonstressed control mice, blood was collected 70 min after the i.c.v. injection of vehicle or drugs. Each mouse was bled once and then sacrificed. The time consumed was 30 to 40 s for each animal. Plasma was separated by centrifugation of the freshly drawn blood and stored at 80°C until assayed. The plasma IL-6 level was determined with an enzyme-linked immunosorbent assay (ELISA) kit (Genzyme, Cambridge, MA). The detection limit of the assay was 5 pg/ml. The plasma IL-6 level of 14% of nonstressed animals given i.c.v. saline was below the detection limit at 1 h after injection. Assays were performed exactly as described by the manufacturers. The plasma IL-1 and TNF- levels were determined 90 min after i.c.v. injection of saline, SR-95,531 (10 ng) or 2-hydroxysaclofen (10 µg) with an ELISA kit (Genzyme, Cambridge, MA).

Pretreatment with 6-OHDA. For the 6-OHDA study, 6-OHDA hydrobromide (Sigma), dissolved in sterile 1% ascorbic acid, was injected either i.p. or i.c.v. 3 days before GABA antagonist injection. An i.p. (100 mg/kg) and an i.c.v. (50 µg) injection of 6-OHDA decreased norepinephrine splenic and hypothalamic content equally, to 22% of control values (14.9 ± 1.0 vs. 3.2 ± 0.5 ng/100 mg wet weight for spleen; 16.3 ± 0.5 vs. 3.5 ± 0.2 ng/10 mg wet weight for hypothalamus), 3 days after the injection.

Statistical analysis. Statistical analysis was carried out by one-way (fig. 6) or two-way (figs. 1-5, 7, 8) analysis of variance (ANOVA). A Bonferroni test was used for post-hoc comparisons. P values less than .05 were considered to indicate statistical significance.

	Results

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Effects of i.c.v. injection of muscimol or baclofen on basal and restraint stress-induced plasma IL-6 levels. Plasma IL-6 levels in the nonstressed control mice injected i.c.v. with saline (5 µl) were 6 ± 1 and 14 ± 3 pg/ml (n = 15-20, mean ± S.E.) at 0 and 1 h after injection, respectively. One hour of restraint stress induced a marked elevation of plasma IL-6 levels to 46 ± 4 pg/ml in mice injected i.c.v. with saline (fig. 1). To determine whether stimulation of GABA_A or GABA_B receptors by muscimol and by baclofen, respectively, affects the restraint stress-induced rise of plasma IL-6, we injected various doses of muscimol (20-200 ng) or baclofen (5-20 ng) i.c.v. 10 min before the start of the 1-h restraint procedure. As shown in figure 1A, muscimol injected i.c.v. did not affect the basal plasma IL-6 levels in nonstressed control animals, but it dose-dependently inhibited the stress-induced rise in plasma IL-6 levels; at the doses of 100 and 200 ng, the stress-induced rise in plasma IL-6 levels was completely inhibited (P < .001). As shown in figure 1B, baclofen (5-20 ng) injected i.c.v. did not affect the basal plasma IL-6 levels in the nonstressed control animals, but it significantly inhibited the stress-induced rise in plasma IL-6 levels from the dose of 15 ng i.c.v. (P < .05). Muscimol (200 ng) and baclofen (20 ng) injected 10 min before the start of the 30-min restraint procedure also significantly inhibited the stress-induced rise in plasma IL-6 levels (fig. 2A). Post-treatment of animals with an i.c.v. injection of muscimol (200 ng) or baclofen (20 ng) 15 or 30 min after the start of the 1-h restraint also significantly inhibited the stress-induced rise in plasma IL-6 levels (fig. 2B).

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Effects of muscimol (panel A) and of baclofen (panel B) injected i.c.v. on plasma IL-6 levels in non-stressed control and restraint-stressed mice. Either saline or various doses of muscimol (20-200 ng) or baclofen (5-20 ng) were given as pretreatment i.c.v. 10 min before the start of the restraint-stress procedure. Mice were restrained for 1 h, and blood samples were obtained immediately after completion of the procedure. The data were means ± S.E. (n = 10). + P < .05, ++ P < .01, significantly different from saline-treated stressed animals.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   A) Effects of pretreatment with muscimol or baclofen injected i.c.v. on plasma IL-6 levels in 30-min restraint-stressed mice. Saline, muscimol (200 ng) or baclofen (20 ng) was given as pretreatment i.c.v. 10 min before the start of the 30-min restraint-stress procedure. Blood samples were obtained immediately after completion of the 30-min restraint-stress procedure. B) Effects of post-treatment with muscimol or baclofen injected i.c.v. on plasma IL-6 levels in 1-h restraint-stressed mice. Saline, muscimol (20 ng) or baclofen (20 ng) was given as post-treatment i.c.v. 15 or 30 min after the start of the 1-h restraint-stress-procedure. Blood samples were obtained immediately after completion of the 1-h restraint-stress procedure. The data were means ± S.E. (n = 10). ** P < .01, *** P < .001, significantly different from nonstressed control animals; + P < .05, ++ P < .01, +++ P < .001, significantly different from saline-treated stressed animals.

Effects of i.c.v. injection of muscimol or baclofen on the i.c.v. MK-801-induced increases in basal plasma IL-6 levels. Next, we examined whether the MK-801 (an NMDA receptor blocker)-induced increase in the basal plasma IL-6 level could also be suppressed by GABA receptor agonists. When GABA receptor agonists were co-administered with MK-801, either muscimol (100 ng i.c.v.), or baclofen (20 ng i.c.v.) completely blocked the MK-801 (1 µg i.c.v.)-induced increase in basal plasma IL-6 (fig. 3).

View larger version (32K): [in this window] [in a new window]   Fig. 3.   Effects of muscimol or baclofen on the MK-801-induced increase in plasma IL-6 levels. Saline, muscimol (100 ng) or baclofen (20 ng) was co-injected i.c.v. with either saline or MK-801 (1 µg). Blood samples were obtained 1 h after the injection. The data were means ± S.E. (n = 8). ** P < .001, significantly different from saline-saline-treated animals.

Effects of i.c.v. injection of SR-95,531 or 2-hydroxysaclofen on basal and stress-induced plasma IL-6 levels. To examine whether blockade of GABA_A or GABA_B receptors by SR-95,531 or 2-hydroxysaclofen, respectively, affects the basal and restraint stress-induced rise in plasma IL-6, we injected i.c.v. various doses of SR-95-531 (0.3-10 ng) or 2-hydroxysaclofen (1-10 µg) 10 min before the start of the 1-h restraint. As shown in figure 4A, SR-95,531 (0.3-10 ng) injected i.c.v. increased the basal plasma IL-6 from the dose of 1 ng (P < .01) when measured 70 min after the injection. In the stressed animals, SR-95,531 caused a significant enhancement of the 1-h restraint stress-induced rise in plasma IL-6 (P < .01 and P < .05, at doses of 3 and 10 ng i.c.v., respectively). As shown in figure 4B, 2-hydroxysaclofen (1-10 µg) injected i.c.v. dose-dependently increased the basal plasma IL-6 (P < .01 and P < .001, at doses of 3 and 10 µg i.c.v., respectively) when measured 70 min after the injection. In the stressed animals, 2-hydroxysaclofen caused an enhancement of the stress-induced plasma IL-6 at the dose of 10 µg i.c.v. (P < .01). SR-95,531 (10 ng i.c.v.) and 2-hydroxysaclofen (10 µg i.c.v.) also increased the basal plasma IL-6 (P < .01) when measured 30 min after the injection (31 ± 3, 35 ± 2 and 9 ± 2 pg/ml for SR-95,531-, 2-hydroxysaclofen- and saline-treated animals, respectively). To determine the specificity of GABA receptor antagonists at the dose used in this study, we co-administered GABA receptor agonist with GABA receptor antagonist. Muscimol (100 ng i.c.v.), but not baclofen (20 ng i.c.v.), completely blocked the SR-95,531 (3 ng i.c.v.)-induced increase in the basal plasma IL-6 (fig. 5A). On the other hand, baclofen (20 ng i.c.v.), but not muscimol (100 ng i.c.v.), completely blocked the 2-hydroxysaclofen (10 µg i.c.v.)-induced increase in the basal plasma IL-6 (fig. 5B). IL-6, IL-1 and TNF- are major proinflammatory cytokines. Thus it was of interest to study the effects of GABA receptor antagonists on the plasma levels of IL-1 and TNF-. As shown in figure 6A, SR-95,531 (10 ng i.c.v.), but not 2-hydroxysaclofen (10 µg i.c.v.), also significantly increased plasma IL-1 levels. However, neither SR-95,531 nor 2-hydroxysaclofen affected plasma TNF- levels (fig. 6B).

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Effects of SR-95,531 (panel A) or 2-hydroxysaclofen (panel B) injected i.c.v. on plasma IL-6 levels in nonstressed control and restraint-stressed mice. Either saline or various doses of SR-95541 (0.3-10 ng) or 2-hydroxysaclofen (1-10 µg) were given as pretreatment i.c.v. 10 min before the start of the restraint stress. Mice were restrained for 1 h, and blood samples were obtained immediately after completion of the procedure. The data were means ± S.E. (n = 10-14). * P < .05, ** P < .01, significantly different from saline-treated nonstressed animals; + P < .05, ++ P < .01, significantly different from saline-treated stressed animals.

View larger version (38K): [in this window] [in a new window]   Fig. 5.   Effects of muscimol or baclofen on SR-95,541 (panel A)- or 2-hydroxysaclofen (panel B)-induced increase in basal plasma IL-6 levels. Saline, muscimol (100 ng) or baclofen (20 ng) was co-injected with either saline or GABA antagonists (SR-95,541, 3 ng or 2-hydroxysaclofen, 10 µg). Blood samples were obtained 1 h after the injection. The data were means ± S.E. (n = 10). * P < .01, ** P < .001, significantly different from saline-saline-treated animals.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Effects of SR-95,531 or 2-hydroxysaclofen injected i.c.v. on plasma IL-1 (panel A) and TNF- (panel B) levels in nonstressed mice. Plasma levels of cytokines were measured 90 min after i.c.v. injection of saline, SR-95,541 (10 ng) or 2-hydroxysaclofen (10 µg). The data were means ± S.E. (n = 10). ** P < .01, significantly different from saline-treated animals.

Effects of i.c.v. or i.p. pretreatment with 6-OHDA on SR-95,531- or 2-hydroxysaclofen-induced plasma IL-6 levels. Central as well as peripheral catecholaminergic systems are involved in the restraint stress-induced increase in plasma IL-6 levels (Takaki et al., 1994). To study the involvement of central and peripheral catecholaminergic systems in the GABA antagonists-induced increase in plasma IL-6 levels, we pretreated animals either i.c.v. or i.p. with 6-OHDA, which depletes catecholamines. Pretreatment of animals with 6-OHDA (50 µg i.c.v.) for 3 days affected neither the SR-95,531 (3 ng i.c.v.)- nor the 2-hydroxysaclofen (10 µg i.c.v.)-induced increase in basal plasma IL-6 levels (figs. 7A and 8A). On the other hand, pretreatment of animals with 6-OHDA (100 mg/kg i.p.) for 3 days significantly inhibited the SR-95,531 (3 ng i.c.v.)- but not the 2-hydroxysaclofen (10 µg i.c.v.)-induced increase in basal plasma IL-6 levels (figs. 7B and 8B).

View larger version (37K): [in this window] [in a new window]   Fig. 7.   Effects of pretreatment with i.p. or i.c.v. 6-OHDA on the SR-95,531 (3 ng, i.c.v.)-induced increase in the plasma IL-6 levels. Mice were injected with either vehicle (i.p. or i.c.v.) or 6-OHDA (100 mg/kg i.p. or 50 µg i.c.v.). Three days later, we evaluated the effect of SR-95,531 (3 ng i.c.v.) on the plasma IL-6 levels. The data are means ± S.E. of 12 animals. * P < .05, ** P < .01, significantly different from the respective saline-treated controls. ++ P < .01.

View larger version (38K): [in this window] [in a new window]   Fig. 8.   Effects of pretreatment with i.p. or i.c.v. 6-OHDA on the 2-hydroxysaclofen (10 µg i.c.v.)-induced increase in the plasma IL-6 levels. Mice were injected with either vehicle (i.p. or i.c.v.) or 6-OHDA (100 mg/kg i.p. or 50 µg i.c.v.). Three days later, we evaluated the effect of 2-hydroxysaclofen (10 µg i.c.v.) on the plasma IL-6 levels. The data are means ± S.E. of 14 to 18 animals. ** P < .01, significantly different from the respective saline-treated controls.

	Discussion

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The major findings of the present study are that i.c.v. injection of either muscimol (a GABA_A receptor agonist) or baclofen (a GABA_B receptor agonist) inhibited both restraint stress-induced and MK-801-induced increases in plasma IL-6 levels, whereas i.c.v. injection of either SR-95,531 (a GABA_A receptor antagonist) or 2-hydroxysaclofen (a GABA_B receptor antagonist) increased basal and restraint stress-induced plasma IL-6 levels. And i.p. pretreatment with 6-OHDA inhibited SR-95,531-induced but not 2-hydroxysaclofen-induced plasma IL-6 levels.

Previously, the immobilization stress-induced rise in plasma IL-6 levels was shown to be significantly reduced by i.v. pretreatment of the animals with 6-OHDA, a catecholamine neuronal toxin that does not cross the blood-brain barrier; this result implied the involvement of peripheral sympathetic nerves in the response (Takaki et al., 1994). The inhibition by muscimol or baclofen of the stress-induced plasma IL-6 increase in the present study suggests that these GABA receptor agonists inhibit central neural circuits that may be positively involved in the stress-induced, peripheral sympathetic nervous system-mediated increase in plasma IL-6. In line with these results, central administration of muscimol (Unger et al., 1984; Wible et al., 1988; Takenaka et al., 1995) and baclofen (Takenaka et al., 1995, 1996) has been reported to suppress peripheral sympathetic outflow in rats. Central injection of muscimol also inhibits stress-induced peripheral manifestations, such as gastric lesions (Bhargava et al., 1985) and cardiovascular (Lisa et al., 1989; Stotz-Potter et al., 1996) and neuroendocrine changes (Stotz-Potter et al., 1996). GABA_A receptor-mediated inhibition of the stress-induced rise in plasma IL-6 levels in the present study may involve the previously reported modulatory effects of benzodiazepines on immune responses to stress (Freire-Garabal et al., 1993; Benschop et al., 1996).

In addition to the suppressive effect of muscimol and baclofen on stress-induced plasma IL-6 levels, they also inhibited MK-801-induced plasma IL-6 responses. We previously showed that i.c.v. injection of MK-801, an NMDA receptor blocker, increased basal and stress-induced levels of plasma IL-6, which suggests the presence of a tonic inhibitory control mechanism via NMDA receptors the regulation of the plasma IL-6 level (Song et al., 1996). The NMDA receptor system may suppress the plasma IL-6 levels through a tonic facilitatory effect on inhibitory neurotransmitters, such as GABA. Tonic activation of GABAergic neurons via a glutamate receptors system was reported in various areas of brain (Wellis and Kauer, 1993; Salin and Prince, 1996). Thus it is tempting to speculate that tonic activation of NMDA receptors may induce GABA release, which subsequently inhibits plasma IL-6 levels through both GABA_A and GABA_B receptors. The complete blockade of the MK-801-induced increase in the plasma IL-6 levels by either muscimol or baclofen may support that interpretation, although other possibilities cannot be excluded.

The increase in the basal plasma IL-6 levels by both SR-95,531 and 2-hydroxysaclofen indicates that endogenous GABA tonically inhibits plasma IL-6 levels via both GABA_A and GABA_B receptors. For GABA_A receptors, this tonic inhibition of plasma cytokine levels extended to plasma IL-1. However, plasma levels of TNF-, another potent proinflammatory cytokine, were not tonically regulated by central GABA receptors.

Intracerebroventricular pretreatment with 6-OHDA, which markedly reduced central norepinephrine levels, affected neither the SR-95,531- nor the 2-hydroxysaclofen-induced increase in plasma IL-6 levels. This suggests that central noradrenergic systems may not be involved in the GABA receptor antagonists-induced plasma IL-6 increase. This is in contrast to restraint stress-induced plasma IL-6 levels, which have been shown to be inhibited markedly by i.c.v. pretreatment with 6-OHDA (Takaki et al., 1994). On the other hand, i.p. pretreatment with 6-OHDA, which markedly reduced peripheral norepinephrine levels, significantly inhibited the SR-95,531- but not the 2-hydroxysaclofen-induced increase in plasma IL-6 levels, which suggests that the peripheral sympathetic nervous system is involved, at least in part, in the SR-95,531- but not the 2-hydroxysaclofen-induced increase in plasma IL-6 levels. Central administration of bicuculline methiodide, a GABA_A antagonist, was reported to elevate plasma catecholamines, which indicates tonic inhibition of the peripheral sympathetic outflow via GABA_A receptors (Martin et al., 1991). Interestingly, nicotine, another drug that increases peripheral sympathetic activity (Yokotani et al., 1987; Van Loon et al., 1989), induced a pattern of plasma IL-6 changes similar to that induced by SR-95,531 injection: it increased basal plasma IL-6 levels, and the increase was inhibited by pretreatment with i.p. 6-OHDA but not by i.c.v. 6-OHDA (unpublished observation). Additionally, i.p. pretreatment with 6-OHDA was also shown to inhibit plasma IL-6 responses induced by restraint stress, a stimulus that increases peripheral sympathetic activity (Takaki et al., 1994).

It is not known whether the source of plasma IL-6 induced by either SR-95,531 or 2-hydroxysaclofen is identical to the source of plasma IL-6 induced by restraint stress, which has been reported to be liver (Takaki et al., 1995; Kitamura et al., 1997). Experiments on the localization of the source of plasma IL-6 induced by either SR-95,531 or 2-hydroxysaclofen are currently being executed in this laboratory. In the present study, wherein we used the i.c.v. injection method in mice, the anatomical sites of action of GABA agonists and antagonists in modulating plasma IL-6 levels remain obscure. Further localization studies in rats, wherein intracerebral microinjection is used, will help to delineate the exact sites (anatomical and synaptic) and neuronal pathways involved in the GABA receptor modulation of plasma IL-6 levels.

In conclusion, the results of this study indicate that basal plasma IL-6 levels are tonically inhibited by endogenous GABA acting at GABA_A and GABA_B receptors and that the restraint stress-induced as well as the MK-801-induced increases in plasma IL-6 levels can be inhibited by stimulation of both GABA_A and GABA_B receptors. These results suggest that central GABA_A and GABA_B receptors may play important roles in the suppressive modulation of plasma IL-6 levels in mice.