Proc. Natl. Acad. Sci. USA Vol. 88, pp. 2763-2767, April 1991 Physiology/Pharmacology
Interleukin la inhibits prostaglandin E2 release to suppress pulsatile release of luteinizing hormone but not folliclestimulating hormone (third-ventricular injection/in vitro hypothalamic incubation/plasma gonadotropins/Iuliberin)
VALERIA RETTORI*, MARTHA F. GIMENOt, ARMANDO KARARAf, M. CARMEN GONZALEZ§, AND SAMUEL M. MCCANN*¶ *Neuropeptide Division, Department of Physiology, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-9040; tCEFYBO, Serrano 665, Buenos Aires 1414, Argentina; tDepartment of Nephrology, Vanderbilt University, Nashville, TN 37232; and §Departamento de Fisiologia, Universidad de La Laguna, Tenerife, E-38320 Spain
Contributed by Samuel M. McCann, December 31, 1990
Interleukin la (IL-la), a powerful endogeABSTRACT nous pyrogen released from monocytes and macrophages by bacterial endotoxin, stimulates corticotropin, prolactin, and somatotropin release and inhibits thyrotropin release by hypothalamic action. We injected recombinant human IL-la into the third cerebral ventricle, to study its effect on the pulsatile release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in conscious, freely moving, ovariectomized rats. Intraventricular injection of 0.25 pmol of IL-la caused an almost immediate reduction of plasma LH concentration; this decrease was statistically significant 20 min after i jection and occurred through a highly significant reduction in the number of LH pulses, with no effect on pulse amplitude. In contrast, there was no change in pulse frequency but a small significant elevation in amplitude of FSH pulses. Intraventricular iujection of the diluent had no effect on gonadotropin release. The results provide further evidence for separate hypothalamic control mechanisms for FSH and LH release. To determine the mechanism of the suppression of LH release, mediobasal hypothalamic fragments were incubated in vitro with IL-1a (10 pM) and the release of LH-releasing hormone (LHRH) and prostaglandin E2 into the medium was measured by RIA in the presence or absence of norepinephrine (50 jpM). IL-la reduced basal LHRH release and blocked LHRH release induced by norepinephrine. It had no effect on the basal release of prostaglandin E2; however, it completely inhibited the release of PGE2 evoked by norepinephrine. To evaluate the possibility that IL-la might also interfere with the epoxygenase pathway of arachidonic acid metabolism, epoxyeicosatrienoic acids were also measured. IL-la had no effect on the content of epoxyeicosatrienoic acids in the hypothalamic fragments as measured by gas chromatography and mass spectrometry. In conclusion, IL-la suppresses LH but not FSH release by an almost complete cessation of pulsatile release of LH in the castrated rat. The mechanism of this effect appears to be by inhibition of prostaglandin E2-mediated release of LHRH.
accompanied by an increase in corticotropin secretion as evidenced by a rise in plasma cortisol concentration (3). Endogenous pyrogen was also shown to evoke corticotropin release (4). Finally, one of these active substances, interleukin 1 (IL-1), was isolated, its structure was determined, and it was synthesized (5). It is released from macrophages and other cells of the immune system following interaction with bacterial pyrogen, a lipopolysaccharide that reacts with specific receptors on the cells to induce the release of IL-1, which is an important endogenous pyrogen (5). Research with IL-1 showed that it not only induces fever but also induces the release of corticotropin by a hypothalamic action that involves release of corticotropin-releasing factor (6, 7) and possibly by a direct pituitary action (8). It also alters release of other anterior pituitary hormones, including prolactin and growth hormone, and inhibits thyrotropin release (9). The pattern of pituitary hormone release induced by IL-1 is quite similar to that evoked by stressful stimuli in humans: release of corticotropin, prolactin, and growth hormone and inhibition of thyrotropin release (10). Because stress also inhibits gonadotropin secretion (11), it is reasonable to evaluate the effects of this monokine on gonadotropin secretion. IL-la has been shown to suppress the release of luteinizing hormone (LH) in castrated animals (12) and to block the preovulatory release of LH (13). Since LH release is pulsatile in castrated rats (14), the present experiments were performed to determine the mechanism of suppression of LH release, to determine whether there was any effect on release of follicle-stimulating hormone (FSH), and to examine the subcellular mechanism by which the release of LH was suppressed.
MATERIALS AND METHODS
(2).
Rats. Male (230-270 g) and female (200-230 g) rats (Holtzmann, Madison, WI) were used for in vitro and in vivo experiments, respectively. They were housed in group cages (10 rats per cage) under controlled conditions of light (14 hr of light, 10 hr of darkness) and temperature (24 iP°C); rat chow and water were freely available. In Vivo Experiments. The female rats were ovariectomized 3 weeks prior to the cannulation. A 23-gauge stainless steel cannula was implanted into the third cerebral ventricle (3V) as described (15). After cannulation the rats were kept in individual cages. Five to 8 days after implantation of the cannula into the 3V, an indwelling catheter was placed into
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviations: IL-1, interleukin 1; LH, luteinizing hormone; LHRH, LH-releasing hormone; FSH, follicle-stimulating hormone; PGE2, prostaglandin E2; EET, epoxyeicosatrienoic acid; MBH, medial basal hypothalamus; 3V, third cerebral ventricle. ITo whom reprint requests should be addressed.
Bacterial infection induces fever and alterations of pituitary hormone secretion (1). Because bacterial pyrogens induce fever, it was first believed that these hormonal changes might be mediated directly by the bacterial pyrogens. However, early work revealed a delay between the injection of such pyrogens and the onset of fever, which suggested that an endogenous pyrogen was produced by the bacterial pyrogens
Indeed, a purified preparation of bacterial pyrogen injected intravenously into dogs induced fever, after a delay, that was
2763
2764
Proc. Natl. Acad. Sci. USA 88 (1991)
Physiology/Pharmacology: Rettori et al.
the right atrium of the heart through the right jugular vein (16). The evening before an experiment the animals were brought to the laboratory. Between 0900 and 1000, after the connection of extension tubing to the jugular catheter, the rats were allowed to rest for at least 1 hr. One heparinized blood sample (0.3 ml) was withdrawn from the jugular catheter before 3V injection of IL-la (2.5 A.l, 0.25 pmol) or vehicle (2.5 1.d of 76 mM Na2HPO4/10 mM NaH2PO4/73 mM sucrose/0.5% human plasma albumin, pH 7.4). Blood samples (0.3 ml) were obtained at 10-min intervals for 2 hr after the injection. After removal of each blood sample, an equal volume of 0.9% NaCl (saline) was injected to maintain blood volume. Care was taken not to disturb the animals during the experiment. Experiments were begun between 1000 and 1100. After centrifugation, plasma samples were stored at -20bC until assayed for hormones. The IL-1 was recombinant human IL-la produced in Escherichia coli and obtained from Collaborative Research (catalogue no. 40041, lot 881385). One half-maximal unit was equivalent to 1 ng of IL-1 with a molecular weight of 17,000. In Vitro Experiments. Rats were decapitated, their brains were removed, and medial basal hypothalami (MBHs) were dissected as described (15). The MBH fragments were incubated in 1 ml of Krebs-Ringer bicarbonate/glucose buffer (pH 7.4) in an atmosphere of 95% 02/5% CO2 with constant shaking at 60 cycles per min at 370C. In all cases, the tissues were preincubated for 30 min, after which the medium was replaced by fresh medium containing IL-1 (10 pM) and/or norepinephrine (50 ,uM; Sigma). At the end of the incubations, the media were transferred to centrifuge tubes and centrifuged for 2 min in a microcentrifuge; Aliquots were assayed immediately for prostaglandin E2 (PGE2) and the rest was stored at -200C until assayed for LH-releasing hormone (LHRH). RIA of PGE2, LHRH, and Gonadotropins. The amount of PGE2 released into the incubation medium was determined by RIA (125I RIA kit, NEN). The sensitivity of the assay was 0.25 pg per tube and the curve was linear up to 25 pg per tube. The crossreactivity was 3% with PGEI and negligible with arachidonic acid and other prostaglandins. LHRH was assayed (17) using antiserum R11373 kindly provided by V. D. Ramirez (Univ. of Illinois, Champaign-Urbana, IL). The sensitivity of the assay was 0.6 pg per tube and the curve was linear up to 100 pg of LHRH. Plasma levels of LH and FSH were assayed according to the directions in the National Institute of Diabetes and Digestive and Kidney Diseases kits and results were expressed in terms of the RP-2 reference preparations for LH and FSH, respectively. Pulsatile release of FSH and LH was analyzed as described (18, 19). Measurement of Endogenous Epoxyeicosatrienoic Acids (EETs). MBH fragments were incubated in Krebs-Ringer bicarbonate/glucose solution at 370C as described above for PGE2 and LHRH measurements. After preincubation for 30 min, the medium was replaced by fresh medium containing IL-1 (10 pM) and/or norepinephrine (50 ttM) and incubated for 1 hr. Four MBHs were pooled from each experimental group, and after addition of 0.2 ,uCi of [3H8]EET mixture (200 Ci/mmol; 1 Ci - 37 GBq) as recovery standards, the samples were homogenized in 2 ml of CH30H/H20 (2:3, vol/vol) containing triphenylphosphine (0.1 mM) and were extracted into acidified CHC13/CH30H. The combined organic phases were evaporated under N2 and hydrolyzed, and the free EETs were purified by reverse-phase HPLC (20). The endogenous EETs were derivatized into pentafluorobenzyl esters (EET-PFB) and purified by reverse-phase HPLC. After the biological samples were mixed with known amounts of [2H8]EET-PFB mixtures as internal standards, they were analyzed by gas chromatography/mass spectrometry (20).
Quantifications were done by gas chromatography/negativeion chemical ionization (NICI) mass spectral analysis of the PFB esters by selected ion monitoring at m/z 319 (loss of PFB from endogenous EET-PFB) and 327 (loss of PFB from [2H8]EET-PFB standards). Based on these ion ratios and the final percent recoveries, the total endogenous EET content was calculated and expressed as nanograms of EETs per MBH. Statistics. Data were analyzed by one-way analysis of variance and the Student-Newman-Keuls multiple comparison test for unequal replicates. Differences with P < 0.05 were considered significant.
RESULTS Effect of Intracerebroventricular Injection of IL-la on Pulsatile Release of FSH and LH in Ovariectomized Rats. To determine the action of the monokine on pulsatile release of LH and FSH, either the diluent or IL-la (2.5 A.l, 0.25 pmol) was microinjected into the 3V after removal of an initial blood sample from the conscious, freely moving animals. Samples were then collected every 10 min for 2 hr. In diluent-injected controls, pulsatile release of LH was unaltered (Figs. 1-3); there were approximately three pulses during the 2 hr of observation and plasma LH concentration was unchanged. In marked contrast, the release of plasma LH declined in all of the animals microinjected with IL-la, although there were differences in the delay before a decline occurred. This delay
10 -
-7.5
'5f
5I3r E
-J
OL 2.5
I
I
i
--I
I
I
0 10 20 30 40 50 60 70 80 90 100 110120
Time (min) 200
0150 I..
(U- 1 00 E
I1
FL 50 I3v
0 10 20 30 40 50 60 70 80 90 100110120
Time (min) FIG. 1. Mean (+ SEM) plasma LH (Upper) and FSH (Lower) concentrations after microinjection of 0.25 pmol of IL-la (E, n = 10 rats) or 2.5 /Al of diluent (e, n = 7) into the 3V. Asterisks indicate P < 0.05 (*) or P < 0.01 (**) versus diluent-injected controls.
Physiology/Pharmacology: Rettori et al. CONTROLS 20
3v 10 .
E
so
z
CONTROL ILla
1mu
3
&D2 p
Interleukin la inhibits prostaglandin E2 release to suppress pulsatile release of luteinizing hormone but not folliclestimulating hormone (third-ventricular injection/in vitro hypothalamic incubation/plasma gonadotropins/Iuliberin)
VALERIA RETTORI*, MARTHA F. GIMENOt, ARMANDO KARARAf, M. CARMEN GONZALEZ§, AND SAMUEL M. MCCANN*¶ *Neuropeptide Division, Department of Physiology, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-9040; tCEFYBO, Serrano 665, Buenos Aires 1414, Argentina; tDepartment of Nephrology, Vanderbilt University, Nashville, TN 37232; and §Departamento de Fisiologia, Universidad de La Laguna, Tenerife, E-38320 Spain
Contributed by Samuel M. McCann, December 31, 1990
Interleukin la (IL-la), a powerful endogeABSTRACT nous pyrogen released from monocytes and macrophages by bacterial endotoxin, stimulates corticotropin, prolactin, and somatotropin release and inhibits thyrotropin release by hypothalamic action. We injected recombinant human IL-la into the third cerebral ventricle, to study its effect on the pulsatile release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in conscious, freely moving, ovariectomized rats. Intraventricular injection of 0.25 pmol of IL-la caused an almost immediate reduction of plasma LH concentration; this decrease was statistically significant 20 min after i jection and occurred through a highly significant reduction in the number of LH pulses, with no effect on pulse amplitude. In contrast, there was no change in pulse frequency but a small significant elevation in amplitude of FSH pulses. Intraventricular iujection of the diluent had no effect on gonadotropin release. The results provide further evidence for separate hypothalamic control mechanisms for FSH and LH release. To determine the mechanism of the suppression of LH release, mediobasal hypothalamic fragments were incubated in vitro with IL-1a (10 pM) and the release of LH-releasing hormone (LHRH) and prostaglandin E2 into the medium was measured by RIA in the presence or absence of norepinephrine (50 jpM). IL-la reduced basal LHRH release and blocked LHRH release induced by norepinephrine. It had no effect on the basal release of prostaglandin E2; however, it completely inhibited the release of PGE2 evoked by norepinephrine. To evaluate the possibility that IL-la might also interfere with the epoxygenase pathway of arachidonic acid metabolism, epoxyeicosatrienoic acids were also measured. IL-la had no effect on the content of epoxyeicosatrienoic acids in the hypothalamic fragments as measured by gas chromatography and mass spectrometry. In conclusion, IL-la suppresses LH but not FSH release by an almost complete cessation of pulsatile release of LH in the castrated rat. The mechanism of this effect appears to be by inhibition of prostaglandin E2-mediated release of LHRH.
accompanied by an increase in corticotropin secretion as evidenced by a rise in plasma cortisol concentration (3). Endogenous pyrogen was also shown to evoke corticotropin release (4). Finally, one of these active substances, interleukin 1 (IL-1), was isolated, its structure was determined, and it was synthesized (5). It is released from macrophages and other cells of the immune system following interaction with bacterial pyrogen, a lipopolysaccharide that reacts with specific receptors on the cells to induce the release of IL-1, which is an important endogenous pyrogen (5). Research with IL-1 showed that it not only induces fever but also induces the release of corticotropin by a hypothalamic action that involves release of corticotropin-releasing factor (6, 7) and possibly by a direct pituitary action (8). It also alters release of other anterior pituitary hormones, including prolactin and growth hormone, and inhibits thyrotropin release (9). The pattern of pituitary hormone release induced by IL-1 is quite similar to that evoked by stressful stimuli in humans: release of corticotropin, prolactin, and growth hormone and inhibition of thyrotropin release (10). Because stress also inhibits gonadotropin secretion (11), it is reasonable to evaluate the effects of this monokine on gonadotropin secretion. IL-la has been shown to suppress the release of luteinizing hormone (LH) in castrated animals (12) and to block the preovulatory release of LH (13). Since LH release is pulsatile in castrated rats (14), the present experiments were performed to determine the mechanism of suppression of LH release, to determine whether there was any effect on release of follicle-stimulating hormone (FSH), and to examine the subcellular mechanism by which the release of LH was suppressed.
MATERIALS AND METHODS
(2).
Rats. Male (230-270 g) and female (200-230 g) rats (Holtzmann, Madison, WI) were used for in vitro and in vivo experiments, respectively. They were housed in group cages (10 rats per cage) under controlled conditions of light (14 hr of light, 10 hr of darkness) and temperature (24 iP°C); rat chow and water were freely available. In Vivo Experiments. The female rats were ovariectomized 3 weeks prior to the cannulation. A 23-gauge stainless steel cannula was implanted into the third cerebral ventricle (3V) as described (15). After cannulation the rats were kept in individual cages. Five to 8 days after implantation of the cannula into the 3V, an indwelling catheter was placed into
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviations: IL-1, interleukin 1; LH, luteinizing hormone; LHRH, LH-releasing hormone; FSH, follicle-stimulating hormone; PGE2, prostaglandin E2; EET, epoxyeicosatrienoic acid; MBH, medial basal hypothalamus; 3V, third cerebral ventricle. ITo whom reprint requests should be addressed.
Bacterial infection induces fever and alterations of pituitary hormone secretion (1). Because bacterial pyrogens induce fever, it was first believed that these hormonal changes might be mediated directly by the bacterial pyrogens. However, early work revealed a delay between the injection of such pyrogens and the onset of fever, which suggested that an endogenous pyrogen was produced by the bacterial pyrogens
Indeed, a purified preparation of bacterial pyrogen injected intravenously into dogs induced fever, after a delay, that was
2763
2764
Proc. Natl. Acad. Sci. USA 88 (1991)
Physiology/Pharmacology: Rettori et al.
the right atrium of the heart through the right jugular vein (16). The evening before an experiment the animals were brought to the laboratory. Between 0900 and 1000, after the connection of extension tubing to the jugular catheter, the rats were allowed to rest for at least 1 hr. One heparinized blood sample (0.3 ml) was withdrawn from the jugular catheter before 3V injection of IL-la (2.5 A.l, 0.25 pmol) or vehicle (2.5 1.d of 76 mM Na2HPO4/10 mM NaH2PO4/73 mM sucrose/0.5% human plasma albumin, pH 7.4). Blood samples (0.3 ml) were obtained at 10-min intervals for 2 hr after the injection. After removal of each blood sample, an equal volume of 0.9% NaCl (saline) was injected to maintain blood volume. Care was taken not to disturb the animals during the experiment. Experiments were begun between 1000 and 1100. After centrifugation, plasma samples were stored at -20bC until assayed for hormones. The IL-1 was recombinant human IL-la produced in Escherichia coli and obtained from Collaborative Research (catalogue no. 40041, lot 881385). One half-maximal unit was equivalent to 1 ng of IL-1 with a molecular weight of 17,000. In Vitro Experiments. Rats were decapitated, their brains were removed, and medial basal hypothalami (MBHs) were dissected as described (15). The MBH fragments were incubated in 1 ml of Krebs-Ringer bicarbonate/glucose buffer (pH 7.4) in an atmosphere of 95% 02/5% CO2 with constant shaking at 60 cycles per min at 370C. In all cases, the tissues were preincubated for 30 min, after which the medium was replaced by fresh medium containing IL-1 (10 pM) and/or norepinephrine (50 ,uM; Sigma). At the end of the incubations, the media were transferred to centrifuge tubes and centrifuged for 2 min in a microcentrifuge; Aliquots were assayed immediately for prostaglandin E2 (PGE2) and the rest was stored at -200C until assayed for LH-releasing hormone (LHRH). RIA of PGE2, LHRH, and Gonadotropins. The amount of PGE2 released into the incubation medium was determined by RIA (125I RIA kit, NEN). The sensitivity of the assay was 0.25 pg per tube and the curve was linear up to 25 pg per tube. The crossreactivity was 3% with PGEI and negligible with arachidonic acid and other prostaglandins. LHRH was assayed (17) using antiserum R11373 kindly provided by V. D. Ramirez (Univ. of Illinois, Champaign-Urbana, IL). The sensitivity of the assay was 0.6 pg per tube and the curve was linear up to 100 pg of LHRH. Plasma levels of LH and FSH were assayed according to the directions in the National Institute of Diabetes and Digestive and Kidney Diseases kits and results were expressed in terms of the RP-2 reference preparations for LH and FSH, respectively. Pulsatile release of FSH and LH was analyzed as described (18, 19). Measurement of Endogenous Epoxyeicosatrienoic Acids (EETs). MBH fragments were incubated in Krebs-Ringer bicarbonate/glucose solution at 370C as described above for PGE2 and LHRH measurements. After preincubation for 30 min, the medium was replaced by fresh medium containing IL-1 (10 pM) and/or norepinephrine (50 ttM) and incubated for 1 hr. Four MBHs were pooled from each experimental group, and after addition of 0.2 ,uCi of [3H8]EET mixture (200 Ci/mmol; 1 Ci - 37 GBq) as recovery standards, the samples were homogenized in 2 ml of CH30H/H20 (2:3, vol/vol) containing triphenylphosphine (0.1 mM) and were extracted into acidified CHC13/CH30H. The combined organic phases were evaporated under N2 and hydrolyzed, and the free EETs were purified by reverse-phase HPLC (20). The endogenous EETs were derivatized into pentafluorobenzyl esters (EET-PFB) and purified by reverse-phase HPLC. After the biological samples were mixed with known amounts of [2H8]EET-PFB mixtures as internal standards, they were analyzed by gas chromatography/mass spectrometry (20).
Quantifications were done by gas chromatography/negativeion chemical ionization (NICI) mass spectral analysis of the PFB esters by selected ion monitoring at m/z 319 (loss of PFB from endogenous EET-PFB) and 327 (loss of PFB from [2H8]EET-PFB standards). Based on these ion ratios and the final percent recoveries, the total endogenous EET content was calculated and expressed as nanograms of EETs per MBH. Statistics. Data were analyzed by one-way analysis of variance and the Student-Newman-Keuls multiple comparison test for unequal replicates. Differences with P < 0.05 were considered significant.
RESULTS Effect of Intracerebroventricular Injection of IL-la on Pulsatile Release of FSH and LH in Ovariectomized Rats. To determine the action of the monokine on pulsatile release of LH and FSH, either the diluent or IL-la (2.5 A.l, 0.25 pmol) was microinjected into the 3V after removal of an initial blood sample from the conscious, freely moving animals. Samples were then collected every 10 min for 2 hr. In diluent-injected controls, pulsatile release of LH was unaltered (Figs. 1-3); there were approximately three pulses during the 2 hr of observation and plasma LH concentration was unchanged. In marked contrast, the release of plasma LH declined in all of the animals microinjected with IL-la, although there were differences in the delay before a decline occurred. This delay
10 -
-7.5
'5f
5I3r E
-J
OL 2.5
I
I
i
--I
I
I
0 10 20 30 40 50 60 70 80 90 100 110120
Time (min) 200
0150 I..
(U- 1 00 E
I1
FL 50 I3v
0 10 20 30 40 50 60 70 80 90 100110120
Time (min) FIG. 1. Mean (+ SEM) plasma LH (Upper) and FSH (Lower) concentrations after microinjection of 0.25 pmol of IL-la (E, n = 10 rats) or 2.5 /Al of diluent (e, n = 7) into the 3V. Asterisks indicate P < 0.05 (*) or P < 0.01 (**) versus diluent-injected controls.
Physiology/Pharmacology: Rettori et al. CONTROLS 20
3v 10 .
E
so
z
CONTROL ILla
1mu
3
&D2 p
