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Effects of interleukin-1β on the steroid-induced luteinizing hormone surge: Role of norepinephrine in the medial preoptic area
Brain Research Bulletin, Vol. 58, No. 4, pp. 405–409, 2002 Copyright © 2002 Elsevier Science Inc. All rights reserved. 0361-9230/02/$–see front matter
PII: S0361-9230(02)00809-2
Effects of interleukin-1β on the steroid-induced luteinizing hormone surge: Role of norepinephrine in the medial preoptic area Sheba M. J. MohanKumar∗ and P. S. MohanKumar Neuroendocrine Research Laboratory, Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, East Lansing, MI, USA [Received 8 March 2002; Revised 3 May 2002; Accepted 6 May 2002] ovariectomized animals [6,7]. While the mechanisms involved are still unclear, evidence points to the hypothalamus as being the main site of action [5–7]. In the rat, luteinizing hormone releasing hormone (LHRH) neurons that regulate the LH surge are localized in specific areas of the hypothalamus such as the suprachiasmatic nucleus, medial preoptic area (MPA), and the arcuate nucleus. Of these, the MPA has the largest number of LHRH perikarya [8]. These neurons are influenced by a wide variety of neurochemicals [9,10]. Of these, the catecholamines, norepinephrine (NE), and dopamine (DA) have been the most widely studied [8,11]. The activity of catecholamine synthesizing enzymes in the MPA is known to change paralleling LH levels during different stages of the estrous cycle [12,13]. Moreover, NE concentration, turnover, and release increase significantly in the MPA during the afternoon of proestrus at the time of preovulatory LH surge [14–18]. Thus, it is possible that IL-1’s effects on LH secretion may involve central noradrenergic systems, especially NE levels in the MPA. The present study was, therefore, done to examine the role of both NE and DA levels in the MPA in the IL-induced inhibition of the LH surge. For this purpose, push–pull perfusion in combination with high performance liquid chromatography (HPLC) was used to obtain catecholamine release profiles in the MPA before, during, and after the LH surge in ovariectomized steroid-primed rats. Concurrent sampling of blood from a jugular catheter was used to measure simultaneous changes in LH levels in the periphery.
ABSTRACT: Interleukin-1β (IL-1β), a cytokine, is known to inhibit the preovulatory surge of luteinizing hormone (LH); however, the mechanism by which it does so is unclear. This study was done to see if this effect is mediated through hypothalamic catecholamines. Adult female Sprague–Dawley rats were ovariectomized and implanted with a push–pull cannula in the medial preoptic area (MPA) of the hypothalamus. They were injected subcutaneously with 30 µg of Estradiol on the day 8 after surgery and with 2 mg of Progesterone on day 10 at 1000 h. On the day of perfusion (day 10), the rats were injected with IL-1β or its vehicle at 1300 h. Perfusate samples from the MPA and blood samples from a jugular catheter were collected from 1300 to 1800 h. Catecholamine concentrations in the perfusate were measured using high performance liquid chromatography (HPLC)-EC and LH levels in the serum using RIA. Norepinephrine release in the MPA of control rats increased significantly at 1530, 1600, and 1630 h paralelling an increase in LH at 1600 h. In contrast, IL-1β treatment blocked the LH surge and the rise in norepinephrine release in the MPA. No changes were observed in dopamine release, both in control and IL-treated animals. These results demonstrate for the first time that IL-induced suppression of the LH surge is probably mediated through inhibition of norepinephrine release in the MPA. © 2002 Elsevier Science Inc. All rights reserved. KEY WORDS: Hypothalamus, Neurotransmitters, Cytokines, Neuroendocrine regulation, HPLC-EC.
INTRODUCTION
MATERIALS AND METHODS
Infection and disease processes are known to produce changes in the central nervous system and the neuroendocrine system [1] and there is a bidirectional connection between these two systems and the immune system [2]. One of the pronounced effects of infection on the neuroendocrine system is the suppression of the hypothalamo–pituitary–gonadal (HPG) axis [1,3]. Interleukin-1β (IL-1β), a cytokine, is known to be an important mediator of this effect. IL-1 has been shown to suppress LH levels in castrated male rats [4]. It also inhibits the preovulatory luteinizing hormone (LH) surge and ovulation during the afternoon of proestrus in intact female rats [5] and blocks the steroid-induced LH surge in
Animals Three- to four-month-old female Sprague–Dawley rats were used in the experiments. They were housed in light-controlled (lights on from 0500 to 1900 h) and air-conditioned (23 ± 2◦ C) animal rooms and were given rat chow and water ad lib. Push–Pull Cannula Implantation and Ovariectomy The animals were weighed and randomly divided into two groups. All animals were bilaterally ovariectomized and were implanted with a push–pull cannula in the MPA as described before
∗ Address for correspondence: Dr. Sheba M.J. MohanKumar, Neuroendocrine Research Laboratory, Department of Pathobiology and Diagnostic Investigation, A 522 E. Fee Hall, College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824, USA. Tel.: +1-517-432-4680; Fax: +1-517-432-7480; E-mail: [email protected]
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406 [14]. Briefly, the animals were given Atropine sulphate (2.2 mg/kg, intraperitoneal (i.p.)) to reduce salivary secretions and to keep the airways open and were anesthetized using sodium pentobarbital (50 mg/kg, i.p.). The ovaries were removed through a midventral incision after ligation of the uterine horns and the surrounding vasculature. The abdominal muscles were sutured using sterile cotton sutures and the skin incision was closed using sterile autoclips. Immediately after ovariectomy, the rats were implanted with a push–pull cannula in the MPA using a stereotaxic apparatus (Kopf, Tujunga, CA). Construction of the push–pull cannula has been described previously [19]. It consisted of an 8.5 mm-long outer cannula made from a 22-ga hypodermic needle. The coordinates used for implantation of the cannula were 8.5 mm ventral, 0.3 mm posterior, and 0.3 mm lateral to the bregma [20]. The cannula was secured in place with screws and dental cement. After implantation, a 29 ga stainless steel stylet was introduced so that it extended 0.5 mm beyond the tip of the outer cannula. Rats were housed individually in flat-bottomed cages after recovery. They were periodically monitored for signs of infection or discomfort and were used in the experiments 8 days after surgery. Rats that showed any sign of infection were excluded from the study. All procedures involving animals were approved by the Institutional animal care and use committee.
MOHANKUMAR AND MOHANKUMAR milliliter. Blood samples were stored at 4◦ C for 4 h before they were centrifuged at 2000 rpm for 20 min to separate the serum. Serum samples were stored at −20◦ C until they were used for LH-RIA. Histology After completion of perfusion, the animals were sacrificed, and the brains were immediately removed and frozen. The perfusion site was determined by examination of serial brain sections (40 µm) which were obtained using a cryostat (Slee, London, UK) maintained at −10◦ C and stained with cresyl violet. HPLC-EC
We followed the steroid treatment protocol used by Kalra et al. [6]. On the day 8 after surgery, rats in both groups were treated with a subcutaneous (s.c.) injection of estrogen (30 µg in 0.1 ml corn oil) at 1000 h. On the day 9, they were implanted with indwelling catheters in the jugular vein as described previously [14]. On the day 10, the rats received an s.c. injection of 2 mg of progesterone in 0.1 ml of corn oil at 1000 h and were subjected to push–pull perfusion.
The HPLC-EC system has been described before [13–15,19,21, 23]. Briefly, it consisted of an LC-4B amperometric detector (Bioanalytical Systems, West Lafayette, IN, USA), a glassy carbon working electrode, a phase II, 5 µm ODS reverse phase, 75 mm × 3.2 mm, C-18 column, and a C-R6A Chromatopac integrator (Shimadzu, Columbia, MD, USA). The mobile phase consisted of monochloroacetic acid (14.5 g/l), sodium hydroxide (4.675 g/l), octanesulfonic acid disodium salt (0.3 g/l), ethylenediaminetetraacetic acid (0.25 g/l), and acetonitrile (35 ml/l) in pyrogen-free, degassed water, filtered through a Milli-Q purification system (Millipore Co., Bedford, MA, USA). The pH of the mobile phase was adjusted to 3.1 using NaOH. The flow rate of the pump (Shimadzu LC-6A) was 1.1 ml/min. The sensitivity of the detector was 1.0 nA full scale, and the potential of the working electrode was 0.65 V. The column and the working electrode were kept in a Shimadzu CTO-6A oven at a temperature of 37◦ C. At the time of HPLC-EC analysis, the samples were thawed at 60◦ C for 1 min. A mixture of 75 µl of the sample and 25 µl of the internal standard (0.05 M isoproterenol) was injected into the HPLC system. Neurotransmitter release was expressed as picogram per minute.
Push–Pull Perfusion Procedure
LH-RIA
Push–pull perfusion was performed as described earlier [14,19,21,22]. On the day of push–pull perfusion, the stylet was replaced with an inner cannula assembly, which consisted of two 29-ga stainless steel tubes of unequal lengths. The longer tube (3.5 cm), which protruded 0.5 mm beyond the outer cannula, was used to introduce (push) the perfusion medium at the implantation site. The shorter tube (2.0 cm) was used to collect (pull) perfusate from the implantation site. The two tubes were kept together in a 2 mm-long piece of Silastic tubing which was mounted with epoxy resin in the lower part of a tuberculin syringe cut at the 0.05 ml mark. The push and pull tubes were connected to two identically calibrated peristaltic pumps (Pharmacia, Uppsala, Sweden). Before starting the perfusion, care was taken to make sure that the pumps were perfectly balanced. Artificial cerebrospinal fluid (ACSF) was used as the perfusion medium. It consisted of CaCl2 (0.087 g/l), NaCl (7.188 g/l), KCl (0.358 g/l), MgSO4 (0.296 g/l), and Na2 HPO4 (1.703 g/l) and had a pH of 7.3. Pump speeds were adjusted to achieve a flow rate of 10 µl/min. The rats were introduced into the perfusion cages at 1000 h and perfusion was started at 1200 h. Animals in the control group (n = 7) were injected intraperitoneally with 250 µl of PBS-0.1% BSA at 1300 h while animals in the experimental group (n = 8) were treated with 5 µg of human recombinant IL-1β in 250 µl of PBS-0.1% BSA. Push–pull perfusates were collected in both groups from 1300 to 1800 h at 30-min intervals at the rate of 10 µl/min. Perfusates were mixed with 0.5 M HClO4 at the rate of 25:1 v/v and stored at −70◦ C until HPLC analysis. Venous blood was collected through the jugular catheter at 1-h intervals from 1300 to 1800 h. Blood was replaced by an equal volume of heparinized saline containing 10 units of heparin per
A double antibody RIA was used to determine LH levels in the serum samples as described before [14]. LH label was obtained from Hazelton Washington (Vienna, VA) and the LH standards and antibody were obtained from NIDDK. The reference preparation for LH was NIDDK rLH-RP-3. The first antibody used was anti rLH-S11. One hundred microliters of the serum samples were assayed in duplicate. The assay had a sensitivity of less than 10 pg and an interassay variability of 5.26 ± 1.06% and an intraassay variability of 6.89 ± 1.7%.
Treatment
Statistical Analysis The differences in the profiles of NE and DA release and LH levels in the serum during the entire observation period were analyzed using repeated measures ANOVA followed by post hoc Fisher’s LSD test. RESULTS Location of the Push–Pull Cannulae Fig. 1 depicts the locations of the push–pull cannulae in the two groups of animals. Histological examination revealed that the tips of the push–pull cannulae in all the animals were in the MPA. The area of perfusion around each cannula tip was about 0.5 mm. Effects of IL-1β on NE Release NE release in control animals that were treated with PBS-BSA and experimental animals that were treated with 5 µg of hrIL-1β are shown in Fig. 2A. NE levels (pg/min, mean ± SE) in the control animals were 8.1 ± 2.0 at 1300 h and increased gradually to
EFFECTS OF INTERLEUKIN-1β ON THE STEROID-INDUCED LUTEINIZING HORMONE SURGE
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Fig. 1. Schematic representation of the sagittal section of a rat brain indicating the locations of the push–pull cannulae in (ovx + EP + vehicle)-treated group (䊊; n = 7) and in (ovx + EP + IL)-treated group (䊉; n = 8). The numbers A1–P3 represent coronal plates extending 1 mm anterior (A1) to 3 mm posterior (P3) from the bregma (AP0). MPA = medial preoptic area, SCh = suprachiasmatic nucleus, AH = anterior hypothalamus, LA = lateroanterior hypothalamic nucleus, AVPO = anteroventral preoptic nucleus, MPO = median preoptic nucleus, StHy = striohypothalamic nucleus, VMH = ventromedial hypothalamus, OX = optic chiasm, and SOX = supraoptic decussation. The location of the push–pull cannula in individual animals was determined by examining stained serial brain sections under a light microscope.
11.7 ± 2.7 at 1530 h and reached a peak at 1630 h (13.2 ± 3.4, p < 0.05) before declining to basal levels. In contrast, treatment with 5 µg of IL-1β completely blocked the rise in NE release. NE levels in these animals were 5.0 ± 2.1 at 1300 h and remained at about the same level during the rest of the period of observation. There were differences in the basal levels, the timing of the peaks, and the amplitude of the peaks but the consistent feature observed in all the control animals was the persistent and progressive increase in NE release at the time of the LH surge. In contrast to the control animals, NE release and LH levels in the IL-treated animals remained low and were free of any significant change during the entire period of observation. Effects of IL-1β on LH Serum LH levels (ng/ml, mean ± SE; Fig. 2B) in the control animals were 0.3 ± 0.2 at 1300 h and increased progressively to reach a peak at 1600 h (15.9 ± 7.3, p < 0.05) before declining to basal levels at 1800 h (0.6 ± 0.3). A distinct LH peak was observed in all the control animals. In contrast, treatment with IL-1β blocked the increase in LH observed in the control animals. In the IL-treated animals, LH was 0.27 ± 0.1 at 1300 h and remained at that level during the rest of the period of observation. LH levels were free of any change in five out of eight animals. Two animals showed a modest peak in LH at 1600 h. Effects of IL-1β on DA In contrast to NE, DA release profiles in individual animals, in both the control and IL-treated groups, remained unchanged during the entire observation period. There was no significant difference in DA release (pg/min, mean ± SE) between control and experimental animals (Fig. 2C). DA levels at 1300 h were 4.5 ± 1.8 in the control group and 3.4 ± 1.4 in the IL-1-treated group. It remained at about that level in both the groups throughout the entire period of observation.
Fig. 2. The effects of IL-1β on NE and DA release in the medial preoptic area and LH levels in the serum. Average (mean±SE) NE release (pg/min) profile in the medial preoptic area measured at 30-min intervals in ovariectomized estrogen and progesterone-treated animals that were either treated with 5 µg of IL-1β (n = 8) or the vehicle (n = 7) for IL-1β, ∗ p < 0.05 (A). Average (mean ± SE) LH (ng/ml) profile measured at hourly intervals from 1300 to 1800 h in ovariectomized estrogen and progesterone-treated animals that were either treated with 5 µg of IL-1β or the vehicle for IL-1β, ∗ p < 0.05 (B). Average (mean ± SE) DA release (pg/min) profile in the medial preoptic area measured at 30-min intervals in ovariectomized estrogen and progesterone-treated animals that were either treated with 5 µg of IL-1β or the vehicle for IL-1β (C).
DISCUSSION Results from this study provide evidence for the first time that IL-1-induced suppression of LH may be mediated through changes in central noradrenergic activity. NE release in the MPA increased significantly during the afternoon, paralleling an in-
408 crease in LH in control ovariectomized steroid-primed rats. Intraperitoneal injection of 5 µg of IL-1β blocked the rise in NE levels in the MPA and completely suppressed the LH surge in five out of eight animals. This indicates that IL-1β probably blocks the LH surge in ovariectomized steroid-primed rats by decreasing NE release in the MPA. In contrast to NE, DA probably plays little or no role in the LH surge observed in ovariectomized steroid-primed rats since DA release remained essentially free of change during the entire period of observation in control animals. Systemic administration of IL-1 did not affect DA release either. These results also indicate that the effects of IL-1β are highly specific affecting one catecholamine in a specific brain area while leaving another untouched. This implies that IL-1 is capable of influencing different catecholaminergic neurons in different ways. This is supported by another study in which IL-1β was shown to affect catecholamines and indoleamines differentially depending on the area of the hypothalamus that was involved [23]. In the present study, we focussed on the MPA because it is rich in LHRH cell bodies and is part of the preopticosuprachiasmatic tuberoinfundibular system of LHRH neurons [8]. It is believed to be a critical area where NE acts to regulate LH release. Increases in NE content, concentration, turnover, and release during the afternoon of proestrus have been reported in the MPA [11,14,15,17,18]. Also, the activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of catecholamines, has been reported to increase in the MPA during the afternoon of proestrus [13]. The MPA receives its noradrenergic innervation from the brain stem [8,10]. Recent studies involving the use of noradrenergic depletors and lesioning of the noradrenergic brain stem nuclei have produced decreases in NE content and LHRH mRNA levels in the MPA and this caused the suppression of pulsatile LH secretion [24,25]. All these support findings from the present study in which we observe increases in NE release in the MPA at the time of LH surge in ovariectomized, steroid-primed rats (control). The interesting finding in this study was that IL-1β blocked the rise in NE release in the MPA and simultaneously suppressed the LH surge. These results demonstrate for the first time that this could probably be one of the mechanisms by which IL-1 suppresses LH secretion. The mechanism by which IL-1 suppresses the steroid-induced release of NE in the MPA in ovariectomized animals is not clear. It is possible that IL-1 could act at the level of noradrenergic terminals in the MPA to inhibit NE release. This would require a local increase in the level of IL-1 in the MPA. In fact, a recent study has shown that IL-1β mRNA levels increased in the MPA, when animals were subjected to chronic stress. A more interesting observation in this study was that this was accompanied by a reduction in LHRH mRNA in this area [26]. Another possible site of action could be the A1 and A2 brain stem noradrenergic regions, which provide noradrenergic innervation to the MPA [10]. It is possible that IL-1 could act at the level of these nuclei to inhibit NE release in the MPA. These regions are also activated when animals are injected intraperitoneally with IL-1 or lipopolysaccharide [27–29]. The exact mechanism by which this could transduce to a decrease in NE release in the MPA needs to be examined. The decrease in LH levels associated with IL-1 administration may involve other neurochemicals as well. Previous studies have shown that the inhibitory effect of IL-1 on basal gonadotropin secretion can be reversed by the administration of corticotropin-releasing hormone (CRH) antibodies suggesting the involvement of CRH in this sequence of events [30]. Naloxone, a potent opioid antagonist has been shown to overturn the IL-1-induced suppression of the LH surge in ovariectomized steroid-primed rats indicating a role for endogenous opioids in this
MOHANKUMAR AND MOHANKUMAR phenomenon [7]. Other neurochemicals, such as prostaglandins, nitric oxide, excitatory amino acids, neuropeptide Y, and gamma amino butyric acid, may also be involved in the IL-1-induced suppression of LH release [3,28,31–35]. The long list of substances that may contribute to this phenomenon is not surprising because the regulation of LH secretion is complex in nature involving a host of neurochemicals [8,10,11]. Unlike NE, the role of DA in LH regulation is not understood clearly [8,10,11]. Recent studies measuring DA release in the MPA and the medial basal hypothalamus have discounted the possibility that DA might have a stimulatory role in LH surge [14,22]. In these studies, DA release either did not change or decreased marginally during the afternoon of proestrus indicating that it either has an inhibitory role or is probably not involved in the regulation of the LH surge. The results from the present study support this conclusion. These results are more convincing, since IL-1 was capable of blocking the rise in NE release but left DA release unaffected. This differential action of IL-1 is believed to contribute to its specific effects on the neuroendocrine system [23]. The route of IL-1 administration has also been reported to play a role in the suppression of LH secretion. When given intracerebroventricularly, both IL-1α and -1β were able to inhibit the LH surge in young cycling female rats. However, when given peripherally, these cytokines were incapable of affecting LH secretion. Central administration of IL-1 also interfered with ovulation while systemic IL-1 did not [4–6]. However, in the present study, i.p. administration of IL-1β blocked the LH surge and this effect was accompanied by inhibition of NE release in the MPA. This could be attributed to the dose of IL-1β used in the present study (5 µg) compared to doses ranging from 250 ng to 1 µg that were used in the other studies. However, the dose of 5 µg, we feel, is not excessive since it did not completely suppress the LH surge in three out of eight animals. The routes by which IL-1 affect the brain to produce its central effects are being studied. There is evidence to show that IL-1 can cross the blood-brain barrier [36]. It may also bind to IL-1 receptors located on abdominal paraganglia of the vagus to activate brain stem nuclei [29]. It could even pass through regions where the blood–brain barrier is weak and bind to its receptors that have been identified in different parts of the brain [27,28]. In this process, it could affect a variety of neurotransmitters that regulate LH secretion. Results from the present study demonstrate for the first time that NE could be a possible mediator of the IL-induced inhibition of the LH surge. ACKNOWLEDGEMENTS
This work was partially supported by the Grant NIH AG05980. The authors thank Drs. Michael Widmer and Steven Gillis, Immunex Corporation, for the kind gift of IL-1β. The authors also thank Mr. Shawn A. Taylor for his technical assistance.
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PII: S0361-9230(02)00809-2
Effects of interleukin-1β on the steroid-induced luteinizing hormone surge: Role of norepinephrine in the medial preoptic area Sheba M. J. MohanKumar∗ and P. S. MohanKumar Neuroendocrine Research Laboratory, Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, East Lansing, MI, USA [Received 8 March 2002; Revised 3 May 2002; Accepted 6 May 2002] ovariectomized animals [6,7]. While the mechanisms involved are still unclear, evidence points to the hypothalamus as being the main site of action [5–7]. In the rat, luteinizing hormone releasing hormone (LHRH) neurons that regulate the LH surge are localized in specific areas of the hypothalamus such as the suprachiasmatic nucleus, medial preoptic area (MPA), and the arcuate nucleus. Of these, the MPA has the largest number of LHRH perikarya [8]. These neurons are influenced by a wide variety of neurochemicals [9,10]. Of these, the catecholamines, norepinephrine (NE), and dopamine (DA) have been the most widely studied [8,11]. The activity of catecholamine synthesizing enzymes in the MPA is known to change paralleling LH levels during different stages of the estrous cycle [12,13]. Moreover, NE concentration, turnover, and release increase significantly in the MPA during the afternoon of proestrus at the time of preovulatory LH surge [14–18]. Thus, it is possible that IL-1’s effects on LH secretion may involve central noradrenergic systems, especially NE levels in the MPA. The present study was, therefore, done to examine the role of both NE and DA levels in the MPA in the IL-induced inhibition of the LH surge. For this purpose, push–pull perfusion in combination with high performance liquid chromatography (HPLC) was used to obtain catecholamine release profiles in the MPA before, during, and after the LH surge in ovariectomized steroid-primed rats. Concurrent sampling of blood from a jugular catheter was used to measure simultaneous changes in LH levels in the periphery.
ABSTRACT: Interleukin-1β (IL-1β), a cytokine, is known to inhibit the preovulatory surge of luteinizing hormone (LH); however, the mechanism by which it does so is unclear. This study was done to see if this effect is mediated through hypothalamic catecholamines. Adult female Sprague–Dawley rats were ovariectomized and implanted with a push–pull cannula in the medial preoptic area (MPA) of the hypothalamus. They were injected subcutaneously with 30 µg of Estradiol on the day 8 after surgery and with 2 mg of Progesterone on day 10 at 1000 h. On the day of perfusion (day 10), the rats were injected with IL-1β or its vehicle at 1300 h. Perfusate samples from the MPA and blood samples from a jugular catheter were collected from 1300 to 1800 h. Catecholamine concentrations in the perfusate were measured using high performance liquid chromatography (HPLC)-EC and LH levels in the serum using RIA. Norepinephrine release in the MPA of control rats increased significantly at 1530, 1600, and 1630 h paralelling an increase in LH at 1600 h. In contrast, IL-1β treatment blocked the LH surge and the rise in norepinephrine release in the MPA. No changes were observed in dopamine release, both in control and IL-treated animals. These results demonstrate for the first time that IL-induced suppression of the LH surge is probably mediated through inhibition of norepinephrine release in the MPA. © 2002 Elsevier Science Inc. All rights reserved. KEY WORDS: Hypothalamus, Neurotransmitters, Cytokines, Neuroendocrine regulation, HPLC-EC.
INTRODUCTION
MATERIALS AND METHODS
Infection and disease processes are known to produce changes in the central nervous system and the neuroendocrine system [1] and there is a bidirectional connection between these two systems and the immune system [2]. One of the pronounced effects of infection on the neuroendocrine system is the suppression of the hypothalamo–pituitary–gonadal (HPG) axis [1,3]. Interleukin-1β (IL-1β), a cytokine, is known to be an important mediator of this effect. IL-1 has been shown to suppress LH levels in castrated male rats [4]. It also inhibits the preovulatory luteinizing hormone (LH) surge and ovulation during the afternoon of proestrus in intact female rats [5] and blocks the steroid-induced LH surge in
Animals Three- to four-month-old female Sprague–Dawley rats were used in the experiments. They were housed in light-controlled (lights on from 0500 to 1900 h) and air-conditioned (23 ± 2◦ C) animal rooms and were given rat chow and water ad lib. Push–Pull Cannula Implantation and Ovariectomy The animals were weighed and randomly divided into two groups. All animals were bilaterally ovariectomized and were implanted with a push–pull cannula in the MPA as described before
∗ Address for correspondence: Dr. Sheba M.J. MohanKumar, Neuroendocrine Research Laboratory, Department of Pathobiology and Diagnostic Investigation, A 522 E. Fee Hall, College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824, USA. Tel.: +1-517-432-4680; Fax: +1-517-432-7480; E-mail: [email protected]
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406 [14]. Briefly, the animals were given Atropine sulphate (2.2 mg/kg, intraperitoneal (i.p.)) to reduce salivary secretions and to keep the airways open and were anesthetized using sodium pentobarbital (50 mg/kg, i.p.). The ovaries were removed through a midventral incision after ligation of the uterine horns and the surrounding vasculature. The abdominal muscles were sutured using sterile cotton sutures and the skin incision was closed using sterile autoclips. Immediately after ovariectomy, the rats were implanted with a push–pull cannula in the MPA using a stereotaxic apparatus (Kopf, Tujunga, CA). Construction of the push–pull cannula has been described previously [19]. It consisted of an 8.5 mm-long outer cannula made from a 22-ga hypodermic needle. The coordinates used for implantation of the cannula were 8.5 mm ventral, 0.3 mm posterior, and 0.3 mm lateral to the bregma [20]. The cannula was secured in place with screws and dental cement. After implantation, a 29 ga stainless steel stylet was introduced so that it extended 0.5 mm beyond the tip of the outer cannula. Rats were housed individually in flat-bottomed cages after recovery. They were periodically monitored for signs of infection or discomfort and were used in the experiments 8 days after surgery. Rats that showed any sign of infection were excluded from the study. All procedures involving animals were approved by the Institutional animal care and use committee.
MOHANKUMAR AND MOHANKUMAR milliliter. Blood samples were stored at 4◦ C for 4 h before they were centrifuged at 2000 rpm for 20 min to separate the serum. Serum samples were stored at −20◦ C until they were used for LH-RIA. Histology After completion of perfusion, the animals were sacrificed, and the brains were immediately removed and frozen. The perfusion site was determined by examination of serial brain sections (40 µm) which were obtained using a cryostat (Slee, London, UK) maintained at −10◦ C and stained with cresyl violet. HPLC-EC
We followed the steroid treatment protocol used by Kalra et al. [6]. On the day 8 after surgery, rats in both groups were treated with a subcutaneous (s.c.) injection of estrogen (30 µg in 0.1 ml corn oil) at 1000 h. On the day 9, they were implanted with indwelling catheters in the jugular vein as described previously [14]. On the day 10, the rats received an s.c. injection of 2 mg of progesterone in 0.1 ml of corn oil at 1000 h and were subjected to push–pull perfusion.
The HPLC-EC system has been described before [13–15,19,21, 23]. Briefly, it consisted of an LC-4B amperometric detector (Bioanalytical Systems, West Lafayette, IN, USA), a glassy carbon working electrode, a phase II, 5 µm ODS reverse phase, 75 mm × 3.2 mm, C-18 column, and a C-R6A Chromatopac integrator (Shimadzu, Columbia, MD, USA). The mobile phase consisted of monochloroacetic acid (14.5 g/l), sodium hydroxide (4.675 g/l), octanesulfonic acid disodium salt (0.3 g/l), ethylenediaminetetraacetic acid (0.25 g/l), and acetonitrile (35 ml/l) in pyrogen-free, degassed water, filtered through a Milli-Q purification system (Millipore Co., Bedford, MA, USA). The pH of the mobile phase was adjusted to 3.1 using NaOH. The flow rate of the pump (Shimadzu LC-6A) was 1.1 ml/min. The sensitivity of the detector was 1.0 nA full scale, and the potential of the working electrode was 0.65 V. The column and the working electrode were kept in a Shimadzu CTO-6A oven at a temperature of 37◦ C. At the time of HPLC-EC analysis, the samples were thawed at 60◦ C for 1 min. A mixture of 75 µl of the sample and 25 µl of the internal standard (0.05 M isoproterenol) was injected into the HPLC system. Neurotransmitter release was expressed as picogram per minute.
Push–Pull Perfusion Procedure
LH-RIA
Push–pull perfusion was performed as described earlier [14,19,21,22]. On the day of push–pull perfusion, the stylet was replaced with an inner cannula assembly, which consisted of two 29-ga stainless steel tubes of unequal lengths. The longer tube (3.5 cm), which protruded 0.5 mm beyond the outer cannula, was used to introduce (push) the perfusion medium at the implantation site. The shorter tube (2.0 cm) was used to collect (pull) perfusate from the implantation site. The two tubes were kept together in a 2 mm-long piece of Silastic tubing which was mounted with epoxy resin in the lower part of a tuberculin syringe cut at the 0.05 ml mark. The push and pull tubes were connected to two identically calibrated peristaltic pumps (Pharmacia, Uppsala, Sweden). Before starting the perfusion, care was taken to make sure that the pumps were perfectly balanced. Artificial cerebrospinal fluid (ACSF) was used as the perfusion medium. It consisted of CaCl2 (0.087 g/l), NaCl (7.188 g/l), KCl (0.358 g/l), MgSO4 (0.296 g/l), and Na2 HPO4 (1.703 g/l) and had a pH of 7.3. Pump speeds were adjusted to achieve a flow rate of 10 µl/min. The rats were introduced into the perfusion cages at 1000 h and perfusion was started at 1200 h. Animals in the control group (n = 7) were injected intraperitoneally with 250 µl of PBS-0.1% BSA at 1300 h while animals in the experimental group (n = 8) were treated with 5 µg of human recombinant IL-1β in 250 µl of PBS-0.1% BSA. Push–pull perfusates were collected in both groups from 1300 to 1800 h at 30-min intervals at the rate of 10 µl/min. Perfusates were mixed with 0.5 M HClO4 at the rate of 25:1 v/v and stored at −70◦ C until HPLC analysis. Venous blood was collected through the jugular catheter at 1-h intervals from 1300 to 1800 h. Blood was replaced by an equal volume of heparinized saline containing 10 units of heparin per
A double antibody RIA was used to determine LH levels in the serum samples as described before [14]. LH label was obtained from Hazelton Washington (Vienna, VA) and the LH standards and antibody were obtained from NIDDK. The reference preparation for LH was NIDDK rLH-RP-3. The first antibody used was anti rLH-S11. One hundred microliters of the serum samples were assayed in duplicate. The assay had a sensitivity of less than 10 pg and an interassay variability of 5.26 ± 1.06% and an intraassay variability of 6.89 ± 1.7%.
Treatment
Statistical Analysis The differences in the profiles of NE and DA release and LH levels in the serum during the entire observation period were analyzed using repeated measures ANOVA followed by post hoc Fisher’s LSD test. RESULTS Location of the Push–Pull Cannulae Fig. 1 depicts the locations of the push–pull cannulae in the two groups of animals. Histological examination revealed that the tips of the push–pull cannulae in all the animals were in the MPA. The area of perfusion around each cannula tip was about 0.5 mm. Effects of IL-1β on NE Release NE release in control animals that were treated with PBS-BSA and experimental animals that were treated with 5 µg of hrIL-1β are shown in Fig. 2A. NE levels (pg/min, mean ± SE) in the control animals were 8.1 ± 2.0 at 1300 h and increased gradually to
EFFECTS OF INTERLEUKIN-1β ON THE STEROID-INDUCED LUTEINIZING HORMONE SURGE
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Fig. 1. Schematic representation of the sagittal section of a rat brain indicating the locations of the push–pull cannulae in (ovx + EP + vehicle)-treated group (䊊; n = 7) and in (ovx + EP + IL)-treated group (䊉; n = 8). The numbers A1–P3 represent coronal plates extending 1 mm anterior (A1) to 3 mm posterior (P3) from the bregma (AP0). MPA = medial preoptic area, SCh = suprachiasmatic nucleus, AH = anterior hypothalamus, LA = lateroanterior hypothalamic nucleus, AVPO = anteroventral preoptic nucleus, MPO = median preoptic nucleus, StHy = striohypothalamic nucleus, VMH = ventromedial hypothalamus, OX = optic chiasm, and SOX = supraoptic decussation. The location of the push–pull cannula in individual animals was determined by examining stained serial brain sections under a light microscope.
11.7 ± 2.7 at 1530 h and reached a peak at 1630 h (13.2 ± 3.4, p < 0.05) before declining to basal levels. In contrast, treatment with 5 µg of IL-1β completely blocked the rise in NE release. NE levels in these animals were 5.0 ± 2.1 at 1300 h and remained at about the same level during the rest of the period of observation. There were differences in the basal levels, the timing of the peaks, and the amplitude of the peaks but the consistent feature observed in all the control animals was the persistent and progressive increase in NE release at the time of the LH surge. In contrast to the control animals, NE release and LH levels in the IL-treated animals remained low and were free of any significant change during the entire period of observation. Effects of IL-1β on LH Serum LH levels (ng/ml, mean ± SE; Fig. 2B) in the control animals were 0.3 ± 0.2 at 1300 h and increased progressively to reach a peak at 1600 h (15.9 ± 7.3, p < 0.05) before declining to basal levels at 1800 h (0.6 ± 0.3). A distinct LH peak was observed in all the control animals. In contrast, treatment with IL-1β blocked the increase in LH observed in the control animals. In the IL-treated animals, LH was 0.27 ± 0.1 at 1300 h and remained at that level during the rest of the period of observation. LH levels were free of any change in five out of eight animals. Two animals showed a modest peak in LH at 1600 h. Effects of IL-1β on DA In contrast to NE, DA release profiles in individual animals, in both the control and IL-treated groups, remained unchanged during the entire observation period. There was no significant difference in DA release (pg/min, mean ± SE) between control and experimental animals (Fig. 2C). DA levels at 1300 h were 4.5 ± 1.8 in the control group and 3.4 ± 1.4 in the IL-1-treated group. It remained at about that level in both the groups throughout the entire period of observation.
Fig. 2. The effects of IL-1β on NE and DA release in the medial preoptic area and LH levels in the serum. Average (mean±SE) NE release (pg/min) profile in the medial preoptic area measured at 30-min intervals in ovariectomized estrogen and progesterone-treated animals that were either treated with 5 µg of IL-1β (n = 8) or the vehicle (n = 7) for IL-1β, ∗ p < 0.05 (A). Average (mean ± SE) LH (ng/ml) profile measured at hourly intervals from 1300 to 1800 h in ovariectomized estrogen and progesterone-treated animals that were either treated with 5 µg of IL-1β or the vehicle for IL-1β, ∗ p < 0.05 (B). Average (mean ± SE) DA release (pg/min) profile in the medial preoptic area measured at 30-min intervals in ovariectomized estrogen and progesterone-treated animals that were either treated with 5 µg of IL-1β or the vehicle for IL-1β (C).
DISCUSSION Results from this study provide evidence for the first time that IL-1-induced suppression of LH may be mediated through changes in central noradrenergic activity. NE release in the MPA increased significantly during the afternoon, paralleling an in-
408 crease in LH in control ovariectomized steroid-primed rats. Intraperitoneal injection of 5 µg of IL-1β blocked the rise in NE levels in the MPA and completely suppressed the LH surge in five out of eight animals. This indicates that IL-1β probably blocks the LH surge in ovariectomized steroid-primed rats by decreasing NE release in the MPA. In contrast to NE, DA probably plays little or no role in the LH surge observed in ovariectomized steroid-primed rats since DA release remained essentially free of change during the entire period of observation in control animals. Systemic administration of IL-1 did not affect DA release either. These results also indicate that the effects of IL-1β are highly specific affecting one catecholamine in a specific brain area while leaving another untouched. This implies that IL-1 is capable of influencing different catecholaminergic neurons in different ways. This is supported by another study in which IL-1β was shown to affect catecholamines and indoleamines differentially depending on the area of the hypothalamus that was involved [23]. In the present study, we focussed on the MPA because it is rich in LHRH cell bodies and is part of the preopticosuprachiasmatic tuberoinfundibular system of LHRH neurons [8]. It is believed to be a critical area where NE acts to regulate LH release. Increases in NE content, concentration, turnover, and release during the afternoon of proestrus have been reported in the MPA [11,14,15,17,18]. Also, the activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of catecholamines, has been reported to increase in the MPA during the afternoon of proestrus [13]. The MPA receives its noradrenergic innervation from the brain stem [8,10]. Recent studies involving the use of noradrenergic depletors and lesioning of the noradrenergic brain stem nuclei have produced decreases in NE content and LHRH mRNA levels in the MPA and this caused the suppression of pulsatile LH secretion [24,25]. All these support findings from the present study in which we observe increases in NE release in the MPA at the time of LH surge in ovariectomized, steroid-primed rats (control). The interesting finding in this study was that IL-1β blocked the rise in NE release in the MPA and simultaneously suppressed the LH surge. These results demonstrate for the first time that this could probably be one of the mechanisms by which IL-1 suppresses LH secretion. The mechanism by which IL-1 suppresses the steroid-induced release of NE in the MPA in ovariectomized animals is not clear. It is possible that IL-1 could act at the level of noradrenergic terminals in the MPA to inhibit NE release. This would require a local increase in the level of IL-1 in the MPA. In fact, a recent study has shown that IL-1β mRNA levels increased in the MPA, when animals were subjected to chronic stress. A more interesting observation in this study was that this was accompanied by a reduction in LHRH mRNA in this area [26]. Another possible site of action could be the A1 and A2 brain stem noradrenergic regions, which provide noradrenergic innervation to the MPA [10]. It is possible that IL-1 could act at the level of these nuclei to inhibit NE release in the MPA. These regions are also activated when animals are injected intraperitoneally with IL-1 or lipopolysaccharide [27–29]. The exact mechanism by which this could transduce to a decrease in NE release in the MPA needs to be examined. The decrease in LH levels associated with IL-1 administration may involve other neurochemicals as well. Previous studies have shown that the inhibitory effect of IL-1 on basal gonadotropin secretion can be reversed by the administration of corticotropin-releasing hormone (CRH) antibodies suggesting the involvement of CRH in this sequence of events [30]. Naloxone, a potent opioid antagonist has been shown to overturn the IL-1-induced suppression of the LH surge in ovariectomized steroid-primed rats indicating a role for endogenous opioids in this
MOHANKUMAR AND MOHANKUMAR phenomenon [7]. Other neurochemicals, such as prostaglandins, nitric oxide, excitatory amino acids, neuropeptide Y, and gamma amino butyric acid, may also be involved in the IL-1-induced suppression of LH release [3,28,31–35]. The long list of substances that may contribute to this phenomenon is not surprising because the regulation of LH secretion is complex in nature involving a host of neurochemicals [8,10,11]. Unlike NE, the role of DA in LH regulation is not understood clearly [8,10,11]. Recent studies measuring DA release in the MPA and the medial basal hypothalamus have discounted the possibility that DA might have a stimulatory role in LH surge [14,22]. In these studies, DA release either did not change or decreased marginally during the afternoon of proestrus indicating that it either has an inhibitory role or is probably not involved in the regulation of the LH surge. The results from the present study support this conclusion. These results are more convincing, since IL-1 was capable of blocking the rise in NE release but left DA release unaffected. This differential action of IL-1 is believed to contribute to its specific effects on the neuroendocrine system [23]. The route of IL-1 administration has also been reported to play a role in the suppression of LH secretion. When given intracerebroventricularly, both IL-1α and -1β were able to inhibit the LH surge in young cycling female rats. However, when given peripherally, these cytokines were incapable of affecting LH secretion. Central administration of IL-1 also interfered with ovulation while systemic IL-1 did not [4–6]. However, in the present study, i.p. administration of IL-1β blocked the LH surge and this effect was accompanied by inhibition of NE release in the MPA. This could be attributed to the dose of IL-1β used in the present study (5 µg) compared to doses ranging from 250 ng to 1 µg that were used in the other studies. However, the dose of 5 µg, we feel, is not excessive since it did not completely suppress the LH surge in three out of eight animals. The routes by which IL-1 affect the brain to produce its central effects are being studied. There is evidence to show that IL-1 can cross the blood-brain barrier [36]. It may also bind to IL-1 receptors located on abdominal paraganglia of the vagus to activate brain stem nuclei [29]. It could even pass through regions where the blood–brain barrier is weak and bind to its receptors that have been identified in different parts of the brain [27,28]. In this process, it could affect a variety of neurotransmitters that regulate LH secretion. Results from the present study demonstrate for the first time that NE could be a possible mediator of the IL-induced inhibition of the LH surge. ACKNOWLEDGEMENTS
This work was partially supported by the Grant NIH AG05980. The authors thank Drs. Michael Widmer and Steven Gillis, Immunex Corporation, for the kind gift of IL-1β. The authors also thank Mr. Shawn A. Taylor for his technical assistance.
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