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Division of Cell Biology (D.C., Y.I.), Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan; Department of Bioregulation (T.I.), Institute of Development and Aging Sciences, Nippon Medical School, Kanagawa 211-8533, Japan; and Third Department of Internal Medicine (T.S.), Hirosaki University School of Medicine, Hirosaki 036-8562, Japan
Address all correspondence and requests for reprints to: Yoichiro Iwakura, D. Sc., Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. E-mail: iwakura{at}ims.u-tokyo.ac.jp.
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Abstract |
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/ß and IL-6 are endogenous modulator of hypothalamo-pituitary-adrenal axis (HPAA) and are thought to play key roles in immune-neuroendocrine interactions during inflammation. Here, we show IL-1 induced a normal HPAA activation in IL-1/ß knockout (KO) and IL-6 KO mice at 1 h; however, at 6 h HPAA activation was reduced relative to wild-type mice, indicating a role for endogenous IL-1/ß and IL-6 in prolonged HPAA activation. We found that the induction of proopiomelanocortin (POMC) transcript in the anterior pituitary (AP) at 6 h in response to IL-1 was reduced in IL-1/ß KO and IL-6 KO mice, as well as in CRH KO mice, suggesting IL-1/ß, IL-6, and CRH are all required for POMC induction. The induction of CRH transcript in the paraventricular nucleus at 6 h and plasma IL-6 levels, in response to IL-1, were reduced in IL-1/ß KO mice. Because IL-1-induced activation of signal transducer and activator of transcription 3 in the AP was also suppressed in IL-6 KO mice, we suggest that plasma IL-6 is first induced by IL-1, and IL-6 activates signal transducer and activator of transcription 3 in the AP, leading to the induction of POMC in concert with CRH. Our results suggest a role for IL-1/ß in the induction of POMC in the AP through the induction of two independent pathways, CRH and IL-6. |
Introduction |
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Proinflammatory cytokines, released during systemic and localized inflammation, elicit a number of responses in the host, including fever and anorexia. The landmark studies by Besedovsky et al. and Blalock and colleagues (5, 6) indicated that IL-1/ß and IL-6 could be the extrahypothalamic CRH released by injured tissue. Several reports have shown that IL-1/ß stimulates HPAA mainly through the hypothalamus, and its action depends on CRH release (7). We have previously demonstrated that IL-1/ß not only induce CRH release, but also induces expression of CRH in the PVN and POMC in the AP, which is a precursor of ACTH (8). Moreover, we showed the importance of IL-1/ß in in vivo HPAA activation induced by turpentine; the corticosterone response in IL-1/ß knockout (KO) mice was completely abolished 8 h after injection of turpentine, whereas it was normal 2 h after injection of turpentine (9). Although IL-1/ß is known to be important for the activation of HPAA, the molecular mechanism by which these cytokines induce HPAA activation is poorly understood. IL-6 is another proinflammatory cytokine whose effects on the HPAA have been investigated extensively (10, 11, 12). Its levels in the circulation are increased during physical, psychological, and inflammatory stresses (2). Peripheral IL-6 administration in rodents induces ACTH and glucocorticoid secretion. Because peripheral injection of IL-1 or IL-1ß induces plasma IL-6 (13), some of the actions of IL-1/ß on the neuroendocrine network is thought to be mediated by the action of IL-6 (14, 15). Although a synergism between gp130 family cytokines and CRH on HPAA activation and POMC gene expression in vitro was reported by several studies (10, 16, 17), in vivo significance of each pathway remains to be elucidated.
In this report, we have examined the HPAA response in CRH KO mice, IL-1/ß KO mice, and IL-6 KO mice to elucidate the signaling cascade induced by periphery-administered IL-1. We demonstrate that IL-1 induces POMC in the AP, and both IL-1/ß and IL-6 are involved in the prolonged activation of HPAA and the induction of POMC. It is suggested that IL-1 activates HPAA through the induction of CRH in the PVN and IL-6 in the plasma.
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Materials and Methods |
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(rmIL-1) was obtained from Pepro Tech EC LTD (London, UK). The lyophilized protein was dissolved in 0.9% NaCl (saline) containing 0.1% BSA (A9306; Sigma, St. Louis, MO).
Animals
IL-1/ß double-KO mice were produced as described (9) and IL-6 KO mice were kindly provided by Dr. Manfred Kopf (18). These mice were back-crossed to C57BL/6J mice for eight generations, and C57BL/6J (SLC Inc., Shizuoka, Japan) mice were used as controls. CRH KO mice generated by targeted mutation in embryonic stem cells were used in this study (19). Mice were housed individually after weaning at 4 wk of age, and age-matched (8–12 wk of age) male mice were used for each experiment. Mice were kept under specific pathogen-free conditions in an environmentally controlled clean room at the Center for Experimental Medicine, the Institute of Medical Science, the University of Tokyo. Mice were housed at an ambient temperature of 24 C and a daily cycle of 12 h light (0800–2000 h) and 12 h darkness. All experiments were carried out according to the institutional ethical guidelines for animal experiments and according to the safety guidelines for gene manipulation experiments.
Hormone assays
IL-1/ß double-KO, IL-6 KO, CRH KO, and wild-type (WT) mice were injected with IL-1 (20 µg per kg of body weight, ip) or saline at 1000 h and killed 1, 3, or 6 h after administration. Mice were rapidly anesthetized with diethyl-ether, and blood samples were collected from the heart. Plasma corticosterone levels were determined by RIA (detection limit: 0.6 ng/ml; Amersham Biosciences, Buckinghamshire, UK). The intra- and interassay assay coefficients of variation were 5.0% and 5.9%, respectively. Plasma ACTH concentration was determined by immunoradiometric assay (detection limit: 5 pg/ml; Mitsubishi, Tokyo, Japan). The intra- and interassay assay coefficients of variation were 3.5% and 5.0%, respectively. Plasma IL-6 levels were measured by ELISA (detection limit: 10 ng/ml; PharMingen, San Diego, CA) according to the manufacturer’s instructions.
In situ hybridization
Mice were deeply anesthetized and perfused transcardially with 4% neutralized paraformaldehyde. Frozen sections (30 µm) were cut on a sliding microtome, mounted onto silane-coated slides (Matsunami, Tokyo, Japan), and air-dried. The hybridization protocol was similar to that previously described (20). Before hybridization, sections were dried overnight under vacuum, digested with proteinase K (10 µg/ml, 37 C, 15–20 min), acetylated, and dehydrated. After vacuum drying, 100 µl of the hybridization mixture (106 cpm/ml, with 10 mM dithiothreitol) was spotted onto each slide, sealed under a coverslip, and incubated at 65 C overnight. The coverslips were then removed and the slides were rinsed in 4x SSC [1x SSC = 15 mM trisodium citrate buffer (pH 7.0)/0.15 M NaCl] at room temperature. The sections were digested with ribonuclease A (20 µg/ml, 37 C, 30 min) and washed in 0.1x SSC for 30 min at 65 C. These sections were then exposed to double-sided x-ray film (XAR-5; Eastman Kodak, Rochester, NY) at 4 C for periods of 7–14 d (depending upon the nature of the probes used), dipped in NTB2 nuclear emulsion (1:1 with water) (Kodak), exposed for 14–30 d, and developed. The slides were counterstained with thionin. An adjoining series of sections were stained with thionin to provide better cytoarchitectonic definition for analysis. All samples from a single experiment were assayed simultaneously.
Probe labeling
A pGEM-4 plasmid containing rat CRH cDNA (1.2 kb, a gift from Dr. K. Mayo, Northwestern University, Chicago, IL) was linearized with HindIII. Mouse POMC cDNA (923 bp, a gift from Dr. Douglass, Vollum Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland, OR), subcloned into Psp65O, was linearized with HindIII. The EcoRI fragment of rat c-fos cDNA (2.0 kb, Dr. I. Verma, Salk Institute, San Diego, CA) was subcloned into p-Bluescript SK-I and linearized with BamHI. Radioactive antisense cRNA copies were synthesized by incubating 0.1 µg linearized plasmid with SP6 (Roche Molecular, Indianapolis, IN) for CRH and POMC probes or T7 (Roche Molecular) for c-fos probe, in a reaction mixture containing 6 mM MgCl2, 2 mM spermidine, 8 mM dithiothreitol, 25 mM ATP/GTP/CTP, 5 mM unlabeled uridine triphosphate, (-35S)-uridine triphosphate (370 MBr/ml, Amersham Biosciences), 1 U RNAsin (Promega, Madison, WI), 36 mM Tris (pH 7.5), for 60 min at 37 C. All probes were purified on resin columns (Nensorb 20; NEN Life Science Products, Wilmington, MA). The specific activity of each probe was approximately 1.0 x 108 cpm/µl.
The densities of CRH and c-fos mRNA in the PVN or POMC mRNA in the AP were semiquantified from the film autoradiograms using an MCID image analysis system (Imaging Research, St. Catherines, Canada) (21). The levels obtained were converted to relative ODs (RODs) using the formula: ROD = log10 (256/levels). Using the mouse brain atlas of Paxinos and Watson (22) as an anatomical guide, we enclosed the area of the medial parvocellular PVN by a rectangle (300 x 520 µm), forming a fixed window. The ROD within the window was measured and the background was assessed by measuring the ROD when the window was placed over another area of the brain where no specific hybridization for CRH was detected. The OD of the PVN was measured bilaterally for each subject.
Western blot analysis
IL-6 KO and WT mice were injected with IL-1 (20 µg per kg of body weight, ip) at 1000 h and decapitated at 1, 3, or 6 h after administration. Whole cell lysate from the pituitary was prepared and separated on a 7.5% SDS-PAGE, transferred to a polyvinylidene fluoride membrane (Millipore, Bedford, MA), probed with monoclonal antiphospho-STAT3 (signal transducer and activator of transcription 3) (Tyr705) antibodies (no. 9131; Cell Signaling, Beverly, MA), and visualized by ECL (RPN 2131; Amersham Biosciences). Blots were stripped and reprobed with the anti-STAT3 antibody (no. 9132; Cell Signaling).
Statistical analysis
All values were calculated as means ± SEM. Comparisons of two groups was analyzed by the Student’s t test; for the comparisons of more than two groups, one- or two-way ANOVA was performed followed by Fisher’s protected least significant difference, Dunnett’s or Tukey’s tests were used to analyze statistical differences in each group. In all analyses, a two-tailed probability of less than 5% (i.e. P < 0.05) was considered statistically significant, and significance was confirmed in at least two independent experiments.
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Results |
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/ß, or IL-6 in the activation of HPAA in response to exogenous IL-1, we injected recombinant murine IL-1 into CRH KO, IL-1/ß KO, and IL-6 KO mice, respectively, and measured the plasma corticosterone, ACTH, and IL-6 levels. Firstly, we analyzed the time course of the HPAA activation after IL-1 stimulation. When IL-1 was administered to WT mice (20 µg/kg of body weight, ip), corticosterone levels (Fig. 1A
) and ACTH levels (Fig. 1B) were significantly elevated at 1, 3, and 6 h after injection.
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, we measured plasma corticosterone and plasma ACTH levels at 1 h after peripheral injection of IL-1 in CRH KO mice. CRH KO mice failed to induce corticosterone (Fig. 2A) and ACTH in the plasma (Fig. 2B), indicating that endogenous CRH is required for the activation of HPAA at 1 h.
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-induced endogenous IL-1 and IL-1ß in the periphery as well as in the brain (9, 23, 24, 25), we next analyzed the role of these endogenously induced IL-1/ß components in IL-1/ß KO mice. We measured plasma corticosterone (Fig. 3, A and C) and plasma ACTH levels (Fig. 3, B and D) in IL-1/ß KO mice at 1 and 6 h after ip injection of IL-1. IL-1/ß KO mice showed similar corticosterone and ACTH levels to these of WT mice at 1 h after injection (Fig. 3, A and B), indicating that endogenous IL-1/ß expression is not required for HPAA activation at 1 h. In contrast, IL-1/ß KO mice showed reduced plasma corticosterone and ACTH levels at 6 h after injection (Fig. 3, C and D) relative to WT mice, suggesting that endogenous IL-1/ß is required for a prolonged HPAA response at 6 h.
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/ß induces IL-6. Furthermore, it was suggested that IL-6 plays an important role in turpentine- or lipopolysaccharide (LPS)-induced HPAA activation (10, 11). Then we examined the effect of IL-6 deficiency on HPAA activation. Significant reduction of corticosterone and ACTH levels was observed in IL-6 KO mice at 6 h after injection (Fig. 3
, C and D), whereas the levels of these hormones were not reduced at 1 h after injection consistently with a previous report (26) (Fig. 3, A and B). Consistently with previous observations, plasma IL-6 levels were significantly elevated upon IL-1 stimulation in WT mice (Fig. 4). However, plasma IL-6 levels were reduced in IL-1/ß KO mice at 3 h compared with WT mice (Fig. 4), indicating that endogenous IL-1/ß is also necessary for the induction of IL-6. These results indicate that endogenous IL-6 is required for the prolonged HPAA activation at 6 h but is not required for the HPAA response at 1 h.
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in IL-1/ß KO mice/ß and IL-6 in prolonged activation of the HPAA in response to IL-1, we analyzed the expression of POMC, the precursor of ACTH, in the AP. After ip administration of IL-1, POMC expression was significantly induced in the AP after 6 h, but not at 1 h, in WT mice, as examined by in situ hybridization (Fig. 5A). In contrast, POMC induction was not observed after IL-1 injection after 6 h in IL-1/ß KO mice (Fig. 5B) and IL-6 KO mice (Fig. 5C). The suppression of POMC induction in IL-1/ß KO mice was confirmed in another experiment using 6 mice (see supplemental Fig. 1, which is published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). These results suggest a possibility that blunted induction of POMC is responsible for the reduced activation of the HPAA in IL-1/ß KO and IL-6 KO mice. We found that IL-1 also failed to induce POMC expression at 6 h after injection in CRH KO mice (Fig. 5D).
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in IL-1/ß KO and IL-6 KO mice/ß KO, and IL-6 KO mice. CRH transcript was clearly induced in the PVN of both WT and IL-6 KO mice at 6 h after IL-1 injection (Fig. 5, E and G). However, the induction of CRH was not observed in IL-1/ß KO mice (Fig. 5F). These results indicate that IL-1/ß, but not IL-6, is necessary for the induction of CRH in the PVN.
Activation of STAT3 in the pituitary in response to IL-1 is dependent on IL-6
Because IL-6 activates STAT3, and STAT3 is suggested to play an important role in the expression of POMC in the pituitary (27, 28), we next analyzed the activation of STAT3 in the pituitary. Activation of STAT3 was observed 1–3 h after injection of IL-1 in WT mice (Fig. 6A), whereas it was blunted at 1 h after injection of IL-1 in IL-6 KO mice, clearly correlated with the levels of IL-6 in the plasma (Fig. 6B). Low-level activations of STAT3 were observed at later time points (data not shown). These results indicate that activation of STAT3 in response to IL-1 depends on plasma IL-6.
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Discussion |
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and demonstrated that the HPAA activation mechanism at 6 h is different from that at 1 h after stimulation; the activation depends only on CRH, but not IL-/ß or IL-6, at 1 h, whereas it depends on both IL-1/ß and IL-6 expression at 6 h. We found that POMC was induced at 6 h in the AP in a IL-1/ß-, IL-6-, and CRH-dependent manner, although it was not induced at 1 h. In agreement with our observations, Melmed and colleagues (27, 28) suggested that two independent pathways, CRH and gp130-STAT3, are important for the regulation of POMC gene expression in the AP.
We showed recently that, upon induction of fever by peripheral IL-1 injection, endogenous IL-1/ß expression is not necessary and IL-1-induced PGE2 and IL-6 in the brain play important roles (25). However, it is not known whether endogenous IL-1/ß expression is required for the HPAA activation or CRH neuron activation in the PVN (29). In this report, we showed that the endogenous expression of IL-1/ß is not necessary for the activation of HPAA at 1 h after injection with IL-1. In contrast to the febrile response, however, an important role for endogenous IL-1/ß was suggested in the prolonged activation of HPAA at 6 h. The difference between the febrile response may be explained by the fact that HPAA, in contrast to fever, is regulated at the level of the pituitary, which is considered to be the peripheral part of the neuroendocrine system.
Several lines of studies, including ours, have demonstrated that a variety of stresses that induces IL-1/ß in the brain (9, 30), such as hypertonic saline injection (31), insulin-induced hypoglycemia (32), foot shock stress (33), and restraint stress (34), as well as peripheral injection of IL-1 or ß, induces CRH transcripts in the PVN (8). Furthermore, it was demonstrated that intracerebroventricular infusion of IL-1Ra attenuates the corticosterone response 24 h after tail shock stress (35) and that continuous intracerebroventricular infusion of IL-1Ra completely prevents the rise of CRH mRNA in PVN observed 8 h after administration of LPS (36). These results suggest that IL-1/ß in the brain plays an important role in the activation of HPAA by inducing CRH mRNA in the PVN. Consistently with this notion, induction of CRH by peripherally injected IL-1 was abolished in IL-1/ß KO mice (Fig. 5F), indicating that endogenous brain IL-1/ß, which is induced by IL-1 is important for the induction of CRH. On the other hand, IL-1 induced CRH in IL-6 KO mice, consistently with our previous observation that IL-1/ß is normally induced in the brain by the peripheral injection of IL-1 in IL-6 KO mice (25). These results indicate that endogenous expression of IL-1/ß, but not IL-6, is required for the induction of CRH in response to IL-1. It should be noted here that IL-1/ß KO mice have normal responsiveness to exogenously administered IL-1 because the induction of c-fos in the PVN in response to IL-1 in IL-1/ß KO mice was similar to that in WT mice (see supplemental Fig. 2).
We found that STAT3 activation in the pituitary after administration of IL-1 correlated with the induction of POMC. It is still controversial whether or not IL-1/ß directly acts on the pituitary (37, 38). On the other hand, it was suggested that IL-6 can directly stimulate the pituitary because IL-6 receptor is expressed on the pituitary, plasma corticosterone levels after bacterial LPS injection in IL-6 KO mice are significantly lower than in WT mice, and administration of IL-6 induces ACTH release (11). Consistently with this idea, we found that peripheral injections of IL-1 induced STAT3 activation in the pituitary, which was abolished in IL-6 KO mice (Fig. 6). Furthermore, we found that POMC expression in the AP was reduced in IL-6 KO mice (Fig. 5G). Thus, it was suggested that IL-1 -induced IL-6 directly induces POMC in the AP through activation of STAT3. Although it was reported that LIF expression in the AP is important for the induction of POMC in response to IL-1ß (39), we could not detect significant change of LIF expression under our experimental conditions (data not shown).
Consistently with our notion, Venihaki et al. (12) demonstrated that, upon turpentine injection, immunoneutralization of ACTH abolished corticosterone rise in CRH KO mice despite the concomitant very high circulating IL-6 levels, suggesting that ACTH, which is induced in the AP by circulating IL-6, is the major mediator for HPAA activation. However, because Bethin et al. (11) demonstrated that IL-6 receptor is expressed on the adrenal glands, it is possible that IL-6 directly activates adrenal glands to secrete corticosterone. Actually, we detected STAT3 activation in the adrenal glands of WT mice in response to IL-1 (Chida, D., Y. Iwakura, unpublished results). The direct effect of IL-6 on the adrenal glands may be examined in the absence of CRH, in which ACTH and POMC are not induced (12) (Fig. 5D). However, as the zona fasciculate of the adrenal gland of CRH KO mice is atrophic due to chronic CRH deficiency (19), the lack of corticosterone response to IL-1 in CRH KO mice does not necessarily mean that IL-1-induced IL-6 cannot induce corticosterone response in the adrenal gland. Direct effect of IL-6 on adrenal gland might be observed if CRH were acutely deficient or adrenal size of CRH KO mice was restored by previous CRH or ACTH administration (40). Analysis of ACTH receptor (melanocortin receptor type II) KO mice should be useful to discriminate whether the effect of IL-6 on the HPAA depends on ACTH activity or not (i.e. direct effects of IL-6 on the adrenal glands).
Taken together, we demonstrated that endogenous IL-1/ß induction is important for prolonged activation of HPAA in response to IL-1, and that IL-1 induces CRH in the PVN and also induces IL-6, both of which are independently important for the POMC induction in the AP.
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Acknowledgments |
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Footnotes |
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First Published Online September 1, 2005
Abbreviations: AP, Anterior pituitary; HPAA, hypothalamic-pituitary-adrenal axis; KO, knockout; LPS, lipopolysaccharide; POMC, proopiomelanocortin; PVN, paraventricular nucleus; rm, recombinant murine; ROD, relative OD; STAT3, signal transducer and activator of transcription 3; WT, wild type.
Received April 8, 2005.
Accepted for publication August 15, 2005.
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References |
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, IL-1ß, IL-1/ß, and IL-1 receptor antagonist shows that IL-1ß is crucial in turpentine-induced fever development and glucocorticoid secretion. J Exp Med 187:1463–1475. Endocrinology 145:5044–5048This article has been cited by other articles:
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