Original Paper Neuroendocrinology 1992;55:708-713
Neuropeptide Division, Department of Physiology, The University of Texas Southwestern Medical Center, Dallas, Tex., USA
Involvement of Arachidonic Acid Cascade Pathways in lnterleukin-6-Stimulated Corticotropin-Releasing Factor Release in vitro
Key Words Dexamethasone Indomethacin 5,8,11-Eicosatriynoic acid Clotrimazole Medial basal hypothalami
Abstract We have demonstrated that centrally administered interleukin-6 (IL-6) stimu lates adrenocorticotropin (ACTH) secretion by a direct effect on cortico tropin-releasing factor (CRF) release from the hypothalamus. Since metabo lites of the arachidonic acid cascade (AAC) have been implicated in mediating actions of cytokines in different tissues and some AAC inhibitors were able to block pyrogenic effects of cytokines and suppress IL-1-induced ACTH secre tion, we decided to examine the mechanism of IL-6 action on CRF release in vitro. After a 60-min preincubation in Krebs-Ringer bicarbonate buffer, me dial basal hypothalami (MBH) were preincubated for 30 min with dexametha sone (DEX), a phospholipase A 2 (PLA2) inhibitor, to block arachidonic acid (AA) formation, or with inhibitors of AA metabolism: a cyclooxygenase in hibitor - indomethacin (IND); a lipoxygenase inhibitor - 5,8,11-eicosatriynoicacid (ETI), and an epoxygenase inhibitor-clotrimazole (CLO). Then, the medium was discarded and MBH were incubated with medium or the above compounds and/or IL-6 for 30 min, and CRF release into the incuba tion medium was measured by radioimmunoassay. As reported previously, 10"I3A/ IL-6 increased CRF release, which was significantly suppressed by DEX in a dose-dependent manner. The suppression was already highly signif icant at a concentration of 10"" M DEX and became maximal at 10'7A/, at which concentration CRF release was no longer stimulated by IL-6. The re sponse to IL-6 was completely blocked at the highest DEX concentration evaluated (10-5M). CLO also suppressed IL-6-induced CRF release with a minimal effective dose of 10"9A/. Suppression was complete at 10~7 and 10-5 M. IL-6-stimulated CRF release was suppressed significantly only by higher than micromolar concentrations of IND and ETI. The results suggest that IL-6 stimulates CRF release by the activation of the AAC system and show that PLA2 and epoxygenase inhibitors are far more effective than cy clooxygenase and lipoxygenase inhibitors to block IL-6-stimulated CRF re lease in vitro. The results suggest an important role for the PLA2 pathway and the epoxygenase pathway in IL-6-induced CRF and ACTH release.
This work was supported by NIH grants DKI0073 and DK40994. A preliminary report of a pan of this work was presented at the 73rd Annual Meeting of The Endocrine Society, Washington, D.C., June 19-22. 1991.
Received: July 26. 1991 Accepted after revision : October 17. 1991
S.M. McCann Department o f Physiology The University o f Texas Southwestern Medical Center 5323 Harry Hines Blvd.. Dallas, TX 75235-9040 (USA)
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/16/2019 2:20:17 AM
Krzysztof Lyson Samuel M. McCann
Materials and Methods Adult male, Sprague-Dawley-derived rats (Holtzman, Ma dison, Wise., USA), weighing 220-260 g, were decapitated be tween 12.00 and 13.00 h. The mediobasal hypothalamus (MBH) was rapidly excised. The boundaries were the rostral extent of the optic chiasm anteriorly, the caudal extent of mammillary bodies posteriorly, the hypothalamic sulci laterally and 2 mm from the base of the hypothalamus dorsally. The whole MBHs (15-19 mg) were incubated in vitro according to a previously described method [II] with some minor modifications. Briefly, the explants were preincubated for 60 min in polyethylene tubes (12 x 75 mm, Sarstedt, Inc., Newton, N.C., USA) containing 0.2 m l/ explant of Krebs-Ringer bicarbonate buffer (KRB), pH 7.4, sup plemented with 20 \iM bacitracin (Sigma, St. Louis, Mo., USA) to inhibit peptidases, at 37 °C under an atmosphere of 95% Ch/5% COj in a Dubnoff metabolic shaker (50 cycles/min). After remo val of preincubation medium, the hypothalami were resuspended in 0.2 ml/explant of medium alone or medium containing DEX (9a-fluoro-16a-methylprednisolone, Sigma), IND (Sigma), 5,8,11-eicosatriynoic acid (ETI; Calbiochem, La Jolla, Calif., USA) and clotrimazole [CLO; 1-(o-chloro-a,a-diphenylbenzyl) imidazole, Sigma] for an additional 30-min preincubation time, then medium was discarded and the hypothalamic explants were incubated with medium or the above compounds and/or IL-6 (recombinant human IL-6, Boehringer-Mannheim Biochemicals, Indianapolis, Ind., USA) for 30 min. After the incubation, me dium was removed and used directly for CRF assay. During each experiment 4 separate incubation vials per group were used. The experiments were replicated at least 3 times. The AAC inhibitors (in substance) were dissolved in absolute ethanol and the drugs were further diluted in the KRB to the desired concentration. The final concentration of ethanol in the incubation medium was below 0.1% and did not affect CRF release. Radioimmunoassay CRF concentrations in the incubation media were assayed di rectly. A specific rat CRF antiserum (Peninsula Lab., Belmont, Calif, USA) and rat [,:5I-Tyr°-]CRF (NEN, DuPont, Wilmington. Del., USA), 3,000 cpm/assay tube, were used for the CRF radioim munoassay. Rat CRF-41 standard (Peninsula Lab.) added to incu bation media or media samples were incubated at 4 °C with CRF antibody according to a previously described method [II], The sen sitivity of the assay (ED90) was 20 pg/assay tube. The intraassay and interassay coefficients of variation were 6 and 10%, respectively. Statistic! Results were analyzed by one-way analysis of variance followed by the Student-Newman-Keuls test. A p value ofless than 0.05 was considered significant.
Results None of the AAC inhibitors significantly changed basal CRF release from the MBH (fig. 1-4). IL-6 (10~13 M) significantly increased CRF levels in the incu bation medium (fig. 1-4), as reported previously [11],
709
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Increasing evidence supports the important role of cytokines in the response of the hypothalamic-pituitaiyadrenal axis to infection. Among them interleukin-1 (IL1) has been extensively studied and stimulates the release of corticotropin-releasing factor (CRF) followed by adrenocorticotropin (ACTH) and glucocorticoid release [for review, see ref. 1 and 2]. However, IL-1 activities in some tissues can be explained either by synergism with IL-6 or by induction of IL-6 production [3-7], which is often coproduced with IL-1 and resembles IL-1 in its ac tions at the hypothalamic and pituitary level [4], IL-1 in jected into the lateral brain ventricle of rats increases cir culating IL-6 levels in hypophysectomized and adrenalectomized rats, even after pretreatment with indomethacin (IND), which blocks the fever induced by IL-1 [8], These results indicate that IL-1 acts centrally on a path way dissociated from its pyrogenic activity, to induce re lease of IL-6 from the brain. Centrally administered IL-6 rapidly exerts a stimulatory effect on ACTH release in conscious male rats, which is also at least partially inde pendent of temperature-regulating centers [9]. IL-6 also increases CRF release from the hypothala mus in vitro [10, 11], which can be diminished by preincu bation with micromolar concentrations of cyclooxygenase but not lipoxygenase inhibitors [10], indicating involve ment of prostaglandins rather than lipoxygenase metabo lites in the mediation of IL-6 action at the hypothalamic level. Phospholipase Aj (PLA:) is the enzyme which cleaves arachidonic acid (AA) from phospholipids, whereas cyclooxygenase, lipoxygenase and epoxygenase are the three key enzymes initiating further metabolism of AA [12]. Since AA cascade (AAC) metabolites are in volved in the control of CRF release [13] and some cyto kines (IL-1, tumor necrosis factor) have been shown to stimulate the activity of PLA? and cyclooxygenase [14, 15], as well, a role of prostaglandins in the central action of IL-1 to stimulate ACTH release in vivo has been sug gested [16, 17]. However, the importance of PLA2 and epoxygenase pathways in cytokine-induced CRF release remained unknown. Because the anti-inflammatory prop erties of glucocorticoids are best explained so far by the inhibition of PLA2 activity [18-20], we decided to examine the effects of a synthetic glucocorticoid, dexamethasone (DEX), on IL-6-stimulated CRF release from the hypothalamus in vitro and compare it to the effective ness of inhibitors of the pathways of AA metabolism via lipoxygenase [21, 22], cyclooxygenase [23, 24] and epox ygenase [25].
p
Neuropeptide Division, Department of Physiology, The University of Texas Southwestern Medical Center, Dallas, Tex., USA
Involvement of Arachidonic Acid Cascade Pathways in lnterleukin-6-Stimulated Corticotropin-Releasing Factor Release in vitro
Key Words Dexamethasone Indomethacin 5,8,11-Eicosatriynoic acid Clotrimazole Medial basal hypothalami
Abstract We have demonstrated that centrally administered interleukin-6 (IL-6) stimu lates adrenocorticotropin (ACTH) secretion by a direct effect on cortico tropin-releasing factor (CRF) release from the hypothalamus. Since metabo lites of the arachidonic acid cascade (AAC) have been implicated in mediating actions of cytokines in different tissues and some AAC inhibitors were able to block pyrogenic effects of cytokines and suppress IL-1-induced ACTH secre tion, we decided to examine the mechanism of IL-6 action on CRF release in vitro. After a 60-min preincubation in Krebs-Ringer bicarbonate buffer, me dial basal hypothalami (MBH) were preincubated for 30 min with dexametha sone (DEX), a phospholipase A 2 (PLA2) inhibitor, to block arachidonic acid (AA) formation, or with inhibitors of AA metabolism: a cyclooxygenase in hibitor - indomethacin (IND); a lipoxygenase inhibitor - 5,8,11-eicosatriynoicacid (ETI), and an epoxygenase inhibitor-clotrimazole (CLO). Then, the medium was discarded and MBH were incubated with medium or the above compounds and/or IL-6 for 30 min, and CRF release into the incuba tion medium was measured by radioimmunoassay. As reported previously, 10"I3A/ IL-6 increased CRF release, which was significantly suppressed by DEX in a dose-dependent manner. The suppression was already highly signif icant at a concentration of 10"" M DEX and became maximal at 10'7A/, at which concentration CRF release was no longer stimulated by IL-6. The re sponse to IL-6 was completely blocked at the highest DEX concentration evaluated (10-5M). CLO also suppressed IL-6-induced CRF release with a minimal effective dose of 10"9A/. Suppression was complete at 10~7 and 10-5 M. IL-6-stimulated CRF release was suppressed significantly only by higher than micromolar concentrations of IND and ETI. The results suggest that IL-6 stimulates CRF release by the activation of the AAC system and show that PLA2 and epoxygenase inhibitors are far more effective than cy clooxygenase and lipoxygenase inhibitors to block IL-6-stimulated CRF re lease in vitro. The results suggest an important role for the PLA2 pathway and the epoxygenase pathway in IL-6-induced CRF and ACTH release.
This work was supported by NIH grants DKI0073 and DK40994. A preliminary report of a pan of this work was presented at the 73rd Annual Meeting of The Endocrine Society, Washington, D.C., June 19-22. 1991.
Received: July 26. 1991 Accepted after revision : October 17. 1991
S.M. McCann Department o f Physiology The University o f Texas Southwestern Medical Center 5323 Harry Hines Blvd.. Dallas, TX 75235-9040 (USA)
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/16/2019 2:20:17 AM
Krzysztof Lyson Samuel M. McCann
Materials and Methods Adult male, Sprague-Dawley-derived rats (Holtzman, Ma dison, Wise., USA), weighing 220-260 g, were decapitated be tween 12.00 and 13.00 h. The mediobasal hypothalamus (MBH) was rapidly excised. The boundaries were the rostral extent of the optic chiasm anteriorly, the caudal extent of mammillary bodies posteriorly, the hypothalamic sulci laterally and 2 mm from the base of the hypothalamus dorsally. The whole MBHs (15-19 mg) were incubated in vitro according to a previously described method [II] with some minor modifications. Briefly, the explants were preincubated for 60 min in polyethylene tubes (12 x 75 mm, Sarstedt, Inc., Newton, N.C., USA) containing 0.2 m l/ explant of Krebs-Ringer bicarbonate buffer (KRB), pH 7.4, sup plemented with 20 \iM bacitracin (Sigma, St. Louis, Mo., USA) to inhibit peptidases, at 37 °C under an atmosphere of 95% Ch/5% COj in a Dubnoff metabolic shaker (50 cycles/min). After remo val of preincubation medium, the hypothalami were resuspended in 0.2 ml/explant of medium alone or medium containing DEX (9a-fluoro-16a-methylprednisolone, Sigma), IND (Sigma), 5,8,11-eicosatriynoic acid (ETI; Calbiochem, La Jolla, Calif., USA) and clotrimazole [CLO; 1-(o-chloro-a,a-diphenylbenzyl) imidazole, Sigma] for an additional 30-min preincubation time, then medium was discarded and the hypothalamic explants were incubated with medium or the above compounds and/or IL-6 (recombinant human IL-6, Boehringer-Mannheim Biochemicals, Indianapolis, Ind., USA) for 30 min. After the incubation, me dium was removed and used directly for CRF assay. During each experiment 4 separate incubation vials per group were used. The experiments were replicated at least 3 times. The AAC inhibitors (in substance) were dissolved in absolute ethanol and the drugs were further diluted in the KRB to the desired concentration. The final concentration of ethanol in the incubation medium was below 0.1% and did not affect CRF release. Radioimmunoassay CRF concentrations in the incubation media were assayed di rectly. A specific rat CRF antiserum (Peninsula Lab., Belmont, Calif, USA) and rat [,:5I-Tyr°-]CRF (NEN, DuPont, Wilmington. Del., USA), 3,000 cpm/assay tube, were used for the CRF radioim munoassay. Rat CRF-41 standard (Peninsula Lab.) added to incu bation media or media samples were incubated at 4 °C with CRF antibody according to a previously described method [II], The sen sitivity of the assay (ED90) was 20 pg/assay tube. The intraassay and interassay coefficients of variation were 6 and 10%, respectively. Statistic! Results were analyzed by one-way analysis of variance followed by the Student-Newman-Keuls test. A p value ofless than 0.05 was considered significant.
Results None of the AAC inhibitors significantly changed basal CRF release from the MBH (fig. 1-4). IL-6 (10~13 M) significantly increased CRF levels in the incu bation medium (fig. 1-4), as reported previously [11],
709
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/16/2019 2:20:17 AM
Increasing evidence supports the important role of cytokines in the response of the hypothalamic-pituitaiyadrenal axis to infection. Among them interleukin-1 (IL1) has been extensively studied and stimulates the release of corticotropin-releasing factor (CRF) followed by adrenocorticotropin (ACTH) and glucocorticoid release [for review, see ref. 1 and 2]. However, IL-1 activities in some tissues can be explained either by synergism with IL-6 or by induction of IL-6 production [3-7], which is often coproduced with IL-1 and resembles IL-1 in its ac tions at the hypothalamic and pituitary level [4], IL-1 in jected into the lateral brain ventricle of rats increases cir culating IL-6 levels in hypophysectomized and adrenalectomized rats, even after pretreatment with indomethacin (IND), which blocks the fever induced by IL-1 [8], These results indicate that IL-1 acts centrally on a path way dissociated from its pyrogenic activity, to induce re lease of IL-6 from the brain. Centrally administered IL-6 rapidly exerts a stimulatory effect on ACTH release in conscious male rats, which is also at least partially inde pendent of temperature-regulating centers [9]. IL-6 also increases CRF release from the hypothala mus in vitro [10, 11], which can be diminished by preincu bation with micromolar concentrations of cyclooxygenase but not lipoxygenase inhibitors [10], indicating involve ment of prostaglandins rather than lipoxygenase metabo lites in the mediation of IL-6 action at the hypothalamic level. Phospholipase Aj (PLA:) is the enzyme which cleaves arachidonic acid (AA) from phospholipids, whereas cyclooxygenase, lipoxygenase and epoxygenase are the three key enzymes initiating further metabolism of AA [12]. Since AA cascade (AAC) metabolites are in volved in the control of CRF release [13] and some cyto kines (IL-1, tumor necrosis factor) have been shown to stimulate the activity of PLA? and cyclooxygenase [14, 15], as well, a role of prostaglandins in the central action of IL-1 to stimulate ACTH release in vivo has been sug gested [16, 17]. However, the importance of PLA2 and epoxygenase pathways in cytokine-induced CRF release remained unknown. Because the anti-inflammatory prop erties of glucocorticoids are best explained so far by the inhibition of PLA2 activity [18-20], we decided to examine the effects of a synthetic glucocorticoid, dexamethasone (DEX), on IL-6-stimulated CRF release from the hypothalamus in vitro and compare it to the effective ness of inhibitors of the pathways of AA metabolism via lipoxygenase [21, 22], cyclooxygenase [23, 24] and epox ygenase [25].
p
