Our recent studies indicate that lipocortin 1 (LC1), a putative second messenger protein for the anti-inflammatory steroids in peripheral tissues, may also contribute to the regulatory actions of the glucocorticoids on the hypothalamo-pituitary-adrenal axis. In the present study we have used in vitro and in vivo models to compare the effects of adrenalectomy, LC1 and dexamethasone on the cytokine-induced secretion of the 41-amino acid corticotrophin releasing factor (CRF-41) and arginine vasopressin (AVP) by the rat hypothalamus. In addition, western blot analysis was used to examine the influence of dexamethasone on the expression of LC1 in the hypothalamus. In vitro, interleukins- (IL-) 1α (100 and 200 pg/ml), 1β (0.5 and 1.0 ng/ml) and 8 (0.25-1.0 ng/ml) readily initiated the release of CRF-41 and AVP from hypothalami removed from intact rats. IL-6 (10 and 20 ng/ml) was also an effective CRF-41 secretagogue but, unlike the other interleukins tested, it was ineffective with regard to AVP. Adrenalectomy 7-14 days prior to autopsy increased significantly (p < 0.01) the magnitude of the CRF-41 responses to IL-1α, IL-1β and IL-6 but not to IL-8. In contrast however, while hypothalamic tissue from adrenalectomized rats, unlike that from intact animals, responded to IL-6 (5-20 ng/ml) with a pronounced hypersecre-tion of AVP, the AVP responses to IL-1α and IL-1β were largely unaffected by adrenalectomy as too were those to IL-8. The marked increases in CRF-41 and AVP release from hypothalami from adrenalectomized rats initiated in vitro by IL1α, IL-1β, IL-6 and IL-8 were readily overcome by preincubation of the tissue with dexamethasone (10-⁷M). In addition, the steroid caused ‘externalization’ of two species of immunoreactive (ir-) LC1 (37 and 58 kDa) by the hypothalamic cells but failed to influence the total LC1 content of the tissue. The inhibitory effects of dexamethasone on the cytokine-induced release of CRF-41 in vitro were mimicked by LC 1(10 ng/ml) which alone had no effect on the basal release of the peptide. However, unlike dexamethasone, LC1 failed to influence the concomitant release of AVP from the hypothalamic tissue elicited by IL1α, IL-1β or IL-8 and potentiated that evoked by IL-6. In vivo central administration of IL-1β (5-10 ng in 3 µl) or IL-6 (7.5-30 ng) in 3 µl) via an indwelling intracerebroventricular (i.c.v.) cannula initiated significant (p < 0.01) dose-dependent increases in the serum corticosterone concentration; these responses were inhibited by prior injection into the third ventricle of LC1 (0.3-1.2 µg/3 µl, i.c.v.) which alone, in the highest dose tested, precipitated a small increase in corticosterone release. The results support the concept that cytokines play a key role in initiating the release of CRF-41 from the hypothalamus and provide novel evidence to suggest that the acute inhibitory effects of the corticosteroids on these responses may be mediated in part by LC1. In addition they demonstrate the ability of certain cytokines to stimulate the release of AVP from iso...

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Our recent studies suggest that lipocortin 1 (LC1), a potential mediator of the anti-inflammatory, antiproliferative and anti-fever actions of glucocorticoids in peripheral tissues, may also contribute to the powerful negative feedback actions of the steroids on the hypothalamo-pituitary-adrenal (HPA) axis. In the present study we have used (1) an in vitro model to examine the influence of a specific neutralizing monoclonal anti-LC1 antibody (anti-LC1 mAb) on the capacity of dexamethasone to suppress the cytokine-induced release of the 41-amino acid corticotropin-releasing factor (CRF-41) and arginine vasopressin (AVP) from the rat hypothalamus and (2) a passive immunization protocol to assess the contribution of LCI to the inhibitory actions of dexamethasone on the HPA responses to immunological (i.p. injection of interleukin 1β, IL-1β) and surgical (laparotomy under ether anaesthesia) stress. In vitro, Il-1α (0.2 ng/ml), IL-1β (0.5 ng/ml), IL-6 (10ng/ml) and IL-8 (1 ng/ml) each caused significant increases in the release of immunoreactive (ir)-CRF-41 and ir-AVP from hypo-thalami removed from rats adrenalectomized 10-12 days before autopsy; these responses were readily inhibited by preincubation of the tissue with dexamethasone (10-⁷M). The inhibitory actions of the steroid were attenuated and, in many instances, abolished by inclusion in the medium of a monoclonal anti-LCI antibody (LCI mAb, diluted 1:15,000); an isotype-matched control antibody (antispectrin α+β, diluted 1:15,000) was ineffective in this regard. IL-1α (0.2 ng/ml), IL-1β (0.5 ng/ml) and IL-6 (10 ng/ml) also initiated similar increases in the release of CRF-41 and AVP from hypothalami from intact rats which were effectively blocked by dexamethasone (10^–7M). However, although the inhibitory actions of the steroid on the pharmacologically evoked release of CRF-41 were specifically overcome by anti-LC1 mAb (diluted 1:15,000), the steroid blockade of AVP release was not. In vivo, rats pretreated with either a polyclonal anti-LC 1 antibody (anti-LCI pAb, 1 ml/day s.c. for 2 days) or a corresponding volume of a nonimmune sheep serum (NSS) responded to immunological (IL-1β, 3 µg/kg i.p.) or surgical (laparotomy under ether anaesthesia) trauma with significant increases in the serum ACTH and corticosterone concentrations. In the NSS-treated groups, dexamethasone (100 µg/kg), which had no effect on the prestress concentrations of ACTH and corticosterone in the blood, completely prevented the HPA responses to both IL-1β and laparotomy. The steroid treatment also abolished the HPA responses to laparotomy in rats pretreated with anti-LC1 pAb. By contrast, passive immunization against LC1 largely overcame the ability of dexamethasone to inhibit the hypersecretions of ACTH and corticosterone provoked by IL-1β (3 µg/kg i.p.). The results suggest that LC1 plays an important role in mediating the inhibitory actions of dexamethasone on the HPA responses to cytokines in vitro and in vivo.

Lipocortin 1 (LC1: also called annexin 1) was first described as a putative second messenger protein for the anti-inflammatory steroids in peripheral tissues. In the present study, in vitro and in vivo methods were used to examine its potential role within the hypothalamus as a mediator of the regulatory actions of the glucocorticoids on the hypothalamo-pituitary-adrenocortical axis of the rat. In the in vitro studies, the effects of human recombinant LC1 (hu-r-LC1) on the concomitant release of the two major corticotrophin-releasing factors (CRF-41 and arginine vasopressin, AVP) from isolated hypothalami removed from chronically adrenalectomized rats were compared with those of dexamethasone in the presence and absence of appropriate secretagogues, namely phospholipase A2 (PLA2), interleukin-6 (IL-6) and a non-specific depolarizing agent, K+ (56 mM). The spontaneous release of CRF-41 in vitro was unaffected by either hu-r-LC1 (5 to 100 ng/ml) or dexamethasone (1 microM). Both compounds however reduced the release of the neuropeptide evoked by IL-6 (5 ng/ml) but failed to modify the secretory responses to PLA2 (25 U/ml) or K+ (56 mM). Dexamethasone (1 microM) had no effect on the basal release of AVP but effectively blocked the secretion of the peptide induced by either IL-6 (10 ng/ml) or PLA2 (25 U/ml). In complete contrast, hu-r-LC1 (5 to 100 ng/ml) stimulated the release of AVP and potentiated the secretory responses to IL-6 (10 ng/ml) and PLA2 (25 U/ml) but not to K+ (56 mM). The hypothalamic responses to PLA2 stimulation (25 U/ml) were associated with significant (P < 0.01) increases in prostaglandin E2 release which, in some instances, were potentiated by hu-r-LC1 (5 to 20 ng/ml). In vivo, administration of histamine (0.6 mg/100 g body wt, ip) produced significant (P < 0.01) increases in the serum corticosterone concentration and in the hypothalamic LC1 content. Neither hu-r-LC1 (0.6 to 1.2 micrograms) nor a polyclonal anti-LC1 antibody (3 microliters, diluted 1:200), injected intracerebroventricularly (icv), influenced either the resting serum corticosterone concentration or the hypersecretion of the steroid evoked by histamine stress. A lower dose of the recombinant protein (0.3 micrograms icv) also failed to alter basal corticosterone release but, in contrast to the higher doses, potentiated the pituitary-adrenocortical responses to histamine. The results suggest that LC1 may contribute to some aspects of peptide release in the hypothalamus but that its actions are not necessarily related to those of the glucocorticoids.

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