Corticotropin-releasing hormone links pituitary adrenocorticotropin gene expression and release during adrenal insufficiency

Corticotropin-releasing hormone (CRH)-deficient (KO) mice provide a unique system to define the role of CRH in regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Despite several manifestations of chronic glucocorticoid insufficiency, basal pituitary proopiomelanocortin (POMC) mRNA, adrenocorticotrophic hormone (ACTH) peptide content within the pituitary, and plasma ACTH concentrations are not elevated in CRH KO mice. The normal POMC mRNA content in KO mice is dependent upon residual glucocorticoid secretion, as it increases in both KO and WT mice after adrenalectomy; this increase is reversed by glucocorticoid, but not aldosterone, replacement. However, the normal plasma levels of ACTH in CRH KO mice are not dependent upon residual glucocorticoid secretion, because, after adrenalectomy, these levels do not undergo the normal increase seen in KO mice despite the increase in POMC mRNA content. Administration of CRH restores ACTH secretion to its expected high level in adrenalectomized CRH KO mice. Thus, in adrenal insufficiency, loss of glucocorticoid feedback by itself can increase POMC gene expression in the pituitary; but CRH action is essential for this to result in increased secretion of ACTH. This may explain why, after withdrawal of chronic glucocorticoid treatment, reactivation of CRH secretion is a necessary prerequisite for recovery from suppression of the HPA axis.     

IL-25 regulates Th17 function in autoimmune inflammation

Interleukin (IL)-25 is a member of the IL-17 family of cytokines. However, unlike the other members of this family, IL-25 promotes T helper (Th) 2 responses. We now show that IL-25 also regulates the development of autoimmune inflammation mediated by IL-17-producing T cells. We have generated IL-25-deficient (il25-/-) mice and found that they are highly susceptible to experimental autoimmune encephalomyelitis (EAE). The accelerated disease in the il25-/- mice is associated with an increase of IL-23 in the periphery and a subsequent increase in the number of inflammatory IL-17-, IFNgamma-, and TNF-producing T cells that invade the central nervous system. Neutralization of IL-17 but not IFNgamma in il25-/- mice prevented EAE, suggesting that IL-17 is a major disease-promoting factor. IL-25 treatment at several time points during a relapse-remitting model or chronic model of EAE completely suppressed disease. IL-25 treatment induced elevated production of IL-13, which is required for suppression of Th17 responses by direct inhibition of IL-23, IL-1beta, and IL-6 expression in activated dendritic cells. Thus, IL-25 and IL-17, being members of the same cytokine family, play opposing roles in the pathogenesis of organ-specific autoimmunity.     

IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron regulatory hormone hepcidin

Hypoferremia is a common response to systemic infections or generalized inflammatory disorders. In mouse models, the development of hypoferremia during inflammation requires hepcidin, an iron regulatory peptide hormone produced in the liver, but the inflammatory signals that regulate hepcidin are largely unknown. Our studies in human liver cell cultures, mice, and human volunteers indicate that IL-6 is the necessary and sufficient cytokine for the induction of hepcidin during inflammation and that the IL-6-hepcidin axis is responsible for the hypoferremia of inflammation.     

Upregulation of IL-6, IL-8 and CCL2 gene expression after acute inflammation: Correlation to clinical pain

Tissue injury initiates a cascade of inflammatory mediators and hyperalgesic substances including prostaglandins, cytokines and chemokines. Using microarray and qRT-PCR gene expression analyses, the present study evaluated changes in gene expression of a cascade of cytokines following acute inflammation and the correlation between the changes in the gene expression level and pain intensity in the oral surgery model of tissue injury and acute pain. Tissue injury resulted in a significant upregulation in the gene expression of interleukin-6 (IL-6; 63.3-fold), IL-8 (8.1-fold), chemokine (C-C motif) ligand 2 (CCL2; 8.9-fold), chemokine (C-X-C motif) ligand 1 (CXCL1; 30.5-fold), chemokine (C-X-C motif) ligand 2 (CXCL2; 26-fold) and annexin A1 (ANXA1; 12-fold). The upregulation of IL-6 gene expression was significantly correlated to the upregulation of IL-8, CCL2, CXCL1 and CXCL2 gene expression. Interestingly, the tissue injury-induced upregulation of IL-6, IL-8 and CCL2 gene expression, was positively correlated to pain intensity at 3 h post-surgery, the onset of acute inflammatory pain. However, ketorolac treatment did not have a significant effect on the gene expression of IL-6, IL-8, CCL2, CXCL2 and ANXA1 at the same time point of acute inflammation. These results demonstrate that the upregulation of IL-6, IL-8 and CCL2 gene expression contributes to the development of acute inflammation and inflammatory pain. The lack of effect of ketorolac on the expression of these gene products may be related to the ceiling analgesic effects of non-steroidal anti-inflammatory drugs.     

Corticotropin-releasing hormone binding to the syncytiotrophoblast membranes

The human placenta secretes large amounts of corticotropin-releasing hormone (CRH) which was thought to exert a paracrine action in the placenta. We have recently characterized high-affinity binding sites for CRH in the human placenta. However, our studies utilized whole placental membranes, which did not identify the site of binding of CRH in the plasma membrane. In this study we investigated the characteristics of CRH binding to purified mother-facing, brush border membranes (BBM) and fetus-facing, basal plasma membranes (BPM) of the syncytiotrophoblast. The two membranes were separated by a series of differential and density-gradient centrifugations. The purity of the membranes was determined by measuring alkaline phosphatase, as a marker of BBM and Na+/K+ATPase as a marker of BPM. Each membrane showed specific and high-affinity binding. Scatchard analysis revealed a high-affinity binding site for CRH with Kd of 1.0 +/- 0.15 and 1.3 +/- 0.176 for BBM and BPM, respectively. The maximal number of binding sites was significantly different (P < 0.01) in the two plasma membranes: Bmax of 79 +/- 6.4 fmol/mg protein for BBM and 23 +/- 3.9 fmol/mg protein for BPM. Both the mother-facing and fetus-facing membranes of the syncytiotrophoblast contain binding proteins for CRH, with significantly more binding sites on the mother-facing membranes. The functional consequences of CRH binding could be different for the two polar membranes due to differential localization of second messenger systems between the two membrane types. It is proposed that partial purification of BBM and BPM provides a better system to study CRH action in the placenta, than whole placental membrane preparations.     

Interplay between IFN-gamma and IL-6 signaling governs neutrophil trafficking and apoptosis during acute inflammation

Regulated recruitment and clearance of neutrophils (PMN) is the hallmark of competent host defense and resolution of inflammation. We now report that IFN-gamma controls PMN infiltration and modulates IL-6 signaling through its soluble receptor (sIL-6R) to promote their apoptosis and clearance. Induction of peritoneal inflammation in IFN-gamma-deficient (IFN-gamma-/-) mice emphasized that the initial rate of PMN recruitment was impaired. This defect in PMN recruitment was also associated with the suppressed intraperitoneal expression of IL-1beta and IL-6. Reconstitution of IFN-gamma signaling restored the rate of PMN infiltration and IL-6 levels and was accompanied by normalization of PMN-activating CXC chemokine expression. To test whether local IL-6 signaling modulated PMN recruitment, inflammation was induced in IFN-gamma-/- and IL-6-/- mice and cytokine signaling adapted by intraperitoneal sIL-6R-IL-6 fusion protein (HYPER-IL-6) or IFN-gamma. Although HYPER-IL-6 attenuated PMN influx in IFN-gamma-/- mice, IFN-gamma had no effect on PMN infiltration in IL-6-/- mice. Examination of the leukocyte infiltrate from IFN-gamma-/-, IL-6-/-, and wild-type mice showed that apoptosis was aberrant in the absence of IFN-gamma and IL-6 as a result of impaired sIL-6R signaling. These data emphasize a pivotal role for IFN-gamma in regulating innate immunity through control of both the recruitment and clearance phases of PMN trafficking.     

IL-23 is essential for T cell-mediated colitis and promotes inflammation via IL-17 and IL-6

Uncontrolled mucosal immunity in the gastrointestinal tract of humans results in chronic inflammatory bowel disease (IBD), such as Crohn disease and ulcerative colitis. In early clinical trials as well as in animal models, IL-12 has been implicated as a major mediator of these diseases based on the ability of anti-p40 mAb treatment to reverse intestinal inflammation. The cytokine IL-23 shares the same p40 subunit with IL-12, and the anti-p40 mAbs used in human and mouse IBD studies neutralized the activities of both IL-12 and IL-23. IL-10-deficient mice spontaneously develop enterocolitis. To determine how IL-23 contributes to intestinal inflammation, we studied the disease susceptibility in the absence of either IL-23 or IL-12 in this model, as well as the ability of recombinant IL-23 to exacerbate IBD induced by T cell transfer. Our study shows that in these models, IL-23 is essential for manifestation of chronic intestinal inflammation, whereas IL-12 is not. A critical target of IL-23 is a unique subset of tissue-homing memory T cells, which are specifically activated by IL-23 to produce the proinflammatory mediators IL-17 and IL-6. This pathway may be responsible for chronic intestinal inflammation as well as other chronic autoimmune inflammatory diseases.     

Neutrophil AKT2 regulates heterotypic cell-cell interactions during vascular inflammation

Interactions between platelets, leukocytes, and activated endothelial cells are important during microvascular occlusion; however, the regulatory mechanisms of these heterotypic cell-cell interactions remain unclear. Here, using intravital microscopy to evaluate mice lacking specific isoforms of the serine/threonine kinase AKT and bone marrow chimeras, we found that hematopoietic cell–associated AKT2 is important for neutrophil adhesion and crawling and neutrophil-platelet interactions on activated endothelial cells during TNF-α–induced venular inflammation. Studies with an AKT2-specific inhibitor and cells isolated from WT and Akt KO mice revealed that platelet- and neutrophil-associated AKT2 regulates heterotypic neutrophil-platelet aggregation under shear conditions. In particular, neutrophil AKT2 was critical for membrane translocation of αMβ2 integrin, β2-talin1 interaction, and intracellular Ca²⁺ mobilization. We found that the basal phosphorylation levels of AKT isoforms were markedly increased in neutrophils and platelets isolated from patients with sickle cell disease (SCD), an inherited hematological disorder associated with vascular inflammation and occlusion. AKT2 inhibition reduced heterotypic aggregation of neutrophils and platelets isolated from SCD patients and diminished neutrophil adhesion and neutrophil-platelet aggregation in SCD mice, thereby improving blood flow rates. Our results provide evidence that neutrophil AKT2 regulates αMβ2 integrin function and suggest that AKT2 is important for neutrophil recruitment and neutrophil-platelet interactions under thromboinflammatory conditions such as SCD.     

Corticotropin-releasing hormone, proopiomelanocortin, and glucocorticoid receptor gene expression in adrenocorticotropin-producing tumors in vitro.

To differentiate between ectopic ACTH syndrome and Cushing's disease, gene expression of corticotropin-releasing hormone (CRH), proopiomelanocortin (POMC), and glucocorticoid receptor was examined in 10 pituitary adenomas (Cushing's disease) and in 10 ectopic ACTH-producing tumors. CRH increased plasma ACTH levels in all patients with Cushing's disease and in five patients with ectopic ACTH syndrome whose tumors contained CRH and CRH mRNA. In five CRH nonresponders, CRH was not detected in tumors that contained no CRH mRNA or that contained only long-size CRH mRNA. Dexamethasone (Dex) decreased plasma ACTH levels in all patients with Cushing's disease and in three patients with ectopic ACTH-producing bronchial carcinoid. These tumors contained glucocorticoid receptor mRNA. CRH increased and Dex decreased ACTH release and POMC mRNA levels in pituitary adenoma and bronchial carcinoid cells. PMA increased POMC mRNA levels only in carcinoid cells. These results reveal characteristics of ectopic ACTH-producing tumors: long-size CRH mRNA and PMA-induced POMC gene expression. In addition, there are two ectopic ACTH syndrome subtypes: tumors containing ACTH with CRH (CRH responder) and tumors without CRH. Dex decreases ACTH release and POMC mRNA levels in some bronchial carcinoids. Therefore, CRH and Dex tests have limited usefulness in differentiating between Cushing's disease and ectopic ACTH syndrome.     

Commensal-dependent expression of IL-25 regulates the IL-23-IL-17 axis in the intestine

Alterations in the composition of intestinal commensal bacteria are associated with enhanced susceptibility to multiple inflammatory diseases, including those conditions associated with interleukin (IL)-17-producing CD4(+) T helper (Th17) cells. However, the relationship between commensal bacteria and the expression of proinflammatory cytokines remains unclear. Using germ-free mice, we show that the frequency of Th17 cells in the large intestine is significantly elevated in the absence of commensal bacteria. Commensal-dependent expression of the IL-17 family member IL-25 (IL-17E) by intestinal epithelial cells limits the expansion of Th17 cells in the intestine by inhibiting expression of macrophage-derived IL-23. We propose that acquisition of, or alterations in, commensal bacteria influences intestinal immune homeostasis via direct regulation of the IL-25-IL-23-IL-17 axis.     

Corticotropin-releasing hormone and opioid peptides in reproduction and stress.

Increased knowledge on the mechanisms whereby corticotropin releasing hormone (CRH) and opioid peptides mediate the effects of stress has helped us to understand the relationship between stress and disturbed reproductive function. Increases of CRH and beta-endorphin in the hypothalamus in stressful situations inhibits the secretion of gonadotropins, oxytocin and vasopressin. This may lead to amenorrhea, which often is a consequence of intensive training or psychological stress, or it may disrupt parturition and lactation. There is a relationship between ovarian function and opioid peptides in the hypothalamus. Opioid peptides increase during puberty and fall at the menopause. Oestradiol and progesterone increase beta-endorphin concentrations in the luteal phase of the menstrual cycle, and this is followed by a rapid fall at menstruation. These changes may mediate symptoms typical of the premenstrual syndrome. Rather intensive exercise is required to increase plasma concentrations of beta-endorphin and corticotropin. During labour the amounts of beta-endorphin and corticotropin reach the values found in athletes during maximal exercise. The placenta produces increasing amounts of CRH towards the end of pregnancy which may help the mother and fetus to withstand the increased demands of labour. The placenta may thus be involved in the adaptation of the stress mechanism during pregnancy. CRH has also a paracrine function in different biological processes of the placenta and fetal membranes. It is possible to counteract the deleterious effects of stress on reproductive function by the administration of opiate antagonists. Induction of ovulation with naltrexone has been shown in patients with hypothalamic amenorrhea but the effect on fertility is not known.     

Methyltransferase G9A regulates T cell differentiation during murine intestinal inflammation

Inflammatory bowel disease (IBD) pathogenesis is associated with dysregulated CD4⁺ Th cell responses, with intestinal homeostasis depending on the balance between IL-17–producing Th17 and Foxp3⁺ Tregs. Differentiation of naive T cells into Th17 and Treg subsets is associated with specific gene expression profiles; however, the contribution of epigenetic mechanisms to controlling Th17 and Treg differentiation remains unclear. Using a murine T cell transfer model of colitis, we found that T cell–intrinsic expression of the histone lysine methyltransferase G9A was required for development of pathogenic T cells and intestinal inflammation. G9A-mediated dimethylation of histone H3 lysine 9 (H3K9me2) restricted Th17 and Treg differentiation in vitro and in vivo. H3K9me2 was found at high levels in naive Th cells and was lost following Th cell activation. Loss of G9A in naive T cells was associated with increased chromatin accessibility and heightened sensitivity to TGF-β1. Pharmacological inhibition of G9A methyltransferase activity in WT T cells promoted Th17 and Treg differentiation. Our data indicate that G9A-dependent H3K9me2 is a homeostatic epigenetic checkpoint that regulates Th17 and Treg responses by limiting chromatin accessibility and TGF-β1 responsiveness, suggesting G9A as a therapeutic target for treating intestinal inflammation.     

Corticotropin-releasing factor receptor 1 and central heart rate regulation in mice during expression of conditioned fear

The present study was performed to 1) determine heart rate (HR) effects mediated through central corticotropin-releasing factor receptor subtypes 1 (CRF(1)) investigate and 2 (CRF(2)) and 2) to the contribution of endogenous CRF to baseline HR and its fear-induced adjustment in freely moving mice. CRF ligands were injected into both lateral ventricles (i.c.v.) 15 min before the presentation of a conditioned auditory fear stimulus (CS). Initial behavioral results suggest an ovine CRF (oCRF)-mediated enhanced baseline fear and mildly enhanced conditioned auditory fear. In contrast, i.c.v. injection of oCRF (35-210 ng/mouse) dose-dependently decreased baseline HR, increased HR variability, and attenuated the CS-induced tachycardia. This effect is suggested to depend on a combined activation of sympathetic and parasympathetic activity referred to as enhanced sympathovagal antagonism. An extreme bradycardia was elicited by oCRF injection into the lower brainstem. All HR effects were probably mediated by CRF(1) because injection of the CRF(2)-selective agonist mouse urocortin II was ineffective, and the baseline bradycardia by i.c.v. CRF was preserved in CRF(2)-deficient mice. Injection of various CRF receptor antagonists including the CRF(2)-selective antisauvagine-30 did not affect the conditioned HR response. This finding suggests that endogenous CRF does not contribute to the fear-mediated tachycardia. Thus, the hypothesis of an involvement of CRF in HR responses of mice to acute aversive stimulation is rejected. Pharmacological evidence points at the involvement of CRF(1) in enhanced sympathovagal antagonism, a pathological state contributing to elevated cardiac risk, whereas the physiological role of the brain CRF system in cardiovascular regulation remains to be determined.     

The IL-23/IL-17 axis in inflammation

IL-23 induces the differentiation of naive CD4(+) T cells into highly pathogenic helper T cells (Th17/Th(IL-17)) that produce IL-17, IL-17F, IL-6, and TNF-alpha, but not IFN-gamma and IL-4. Two studies in this issue of the JCI demonstrate that blocking IL-23 or its downstream factors IL-17 and IL-6, but not the IL-12/IFN-gamma pathways, can significantly suppress disease development in animal models of inflammatory bowel disease and MS (see the related articles beginning on pages 1310 and 1317). These studies suggest that the IL-23/IL-17 pathway may be a novel therapeutic target for the treatment of chronic inflammatory diseases.     

Glucocorticoid hormone regulates the circadian coordination of micro-opioid receptor expression in mouse brainstem

The 24-h variation in glucocorticoid secretion from the adrenal cortex is observed not only in nocturnally active rodents but also in diurnally active humans. Although the cyclic change in circulating glucocorticoid levels is thought to influence the efficacy and/or toxicity of many drugs, the mechanism underlying the influence remains poorly understood. In this study, we demonstrate that the 24-h variation in circulating glucocorticoid levels modulates the analgesic effect of morphine by regulating the expression of the mu-opioid receptor. Significant time-dependent variations in the mRNA levels of the mu-opioid receptor and its binding capacity were observed in mouse brainstem. The analgesic effect of morphine was enhanced by administering the drug when mu-opioid receptor levels were increased. However, corticotrophin-releasing hormone (CRH)-deficient mice, disrupting the 24-h rhythm of glucocorticoid secretion, showed no significant time-dependent variation in the expression of the mu-opioid receptor. As a consequence, there was no significant dosing time-dependent difference in the analgesic effect of morphine in CRH-deficient mice. A single administration of corticosterone significantly induced the expression of the mu-opioid receptor in the CRH-deficient mouse brainstem and also enhanced the analgesic effect of morphine. These findings suggest a mechanism underlying the time-dependent variation in mu-opioid receptor function and provide clues to select the most appropriate time of day for administration of morphine.     

Corticotropin-releasing hormone (CRH) in psychiatry: from stress to psychopathology

Corticotropin-releasing hormone (CRH) plays a central role in the adaptation of the organism to stress. It serves as the main regulating hormone of the hypothalamic pituitary adrenal (HPA) axis, which is activated within seconds after exposure to acute stress. Furthermore, it acts as a neurotransmitter in numerous other brain regions. Globally, CRH leads to a number of metabolic, neuroendocrine and autonomic adaptations, which are vitally important for an adequate reaction to acute stress, but can lead to pathological somatic and psychological effects in chronic stress situations. The adequate functioning of CRH is a delicate equilibrium, which can be permanently disturbed by early experiences of physical or sexual abuse, leading to psychopathology in adulthood. This review discusses the physiological functions of CRH as the stress response hormone. Subsequently, the emerging data on the disruptive effects of early trauma on the CRH system are summarized. The third part is devoted to CRH and HPA axis abnormalities in major depression and other psychiatric disorders. This rapidly accumulating evidence will change our understanding of psychopathology, and might challenge the established classification of psychiatric disorders.     

Blocking IL-1 in systemic inflammation

A growing number of systemic inflammatory diseases characterized in part by recurrent fevers, leukocytosis, anemia, and elevated acute phase proteins are linked to interleukin (IL)-1 activity since rapid and sustained resolution is observed upon specific blockade of IL-1 receptors. Rapid resolution of systemic and local inflammation is now also reported in systemic onset juvenile idiopathic arthritis (SoJIA). Loss of control of the secretion of IL-1beta might be a common mechanism explaining the aberrant activity of IL-1 in these diseases.