Selective involvement of interleukin-6 in the transcriptional activation of the suppressor of cytokine signaling-3 in the brain during systemic immune challenges.

Cytokine-inducible proteins named as suppressors of cytokine signaling (SOCS) are rapidly induced by interleukin-6 (IL-6) and other members sharing the gp130 receptor subunit after activation of the Janus kinases (JAK) and the signal transducers and activators of transcription (STAT). These inhibitory proteins generally prevent tyrosine phosphorylation of IL-6 receptor signaling subunit gp130, specific JAK and STAT or in acting at steps distal to JAK activation. Expression of these inhibitory proteins is therefore a useful tool to investigate the signaling events occurring in the brain during immunogenic stimuli that involve cytokines of the IL-6 family. This study investigated the effect of ip lipopolysaccharide (LPS) administration on the expression of one key member of the SOCS family, SOCS-3, in both rats and mice. In rats, the endotoxin caused a profound transcriptional activation of the inhibitory factor in the circumventricular organs subfornical organ, organum vasculosum of the lamina terminalis, arcuate nucleus/median eminence, area postrema, choroid plexus, leptomeninges, ependymal lining cells, and along the endothelium of the brain blood vessels. The hybridization signal for SOCS-3 messenger RNA was low at 1 h, but robust at 3 and 6 h and declined to return to basal levels 12 h after the single ip LPS injection. The pattern of SOCS-3 expression was similar in the brain of wild-type mice, although induction of the inhibitory factor was no longer observed in the ependymal lining cells of the cerebral ventricles and the blood microvessels of IL-6-deficient animals at all the times evaluated, i.e. from 1-8 h post-LPS injection. The endothelium of the brain capillaries also exhibited up-regulation of both IL-6 receptor and gp130 subunits during systemic inflammation, which allowed SOCS-3 expression in response to circulating IL-6. The present data indicate that the JAK/STAT transduction pathways that lead to SOCS-3 transcription are activated within cells accessible from the blood circulation, but not within deep parenchymal elements of the brain during endotoxemia. Induction of SOCS-3 followed the cascade of events that take place during the acute phase response and the contribution of IL-6 in activating the inhibitory factor is site specific and not generalized throughout the central nervous system.

How the blood talks to the brain parenchyma and the paraventricular nucleus of the hypothalamus during systemic inflammatory and infectious stimuli.

There are exciting new developments regarding the molecular mechanisms involved in the influence of circulating proinflammatory molecules within cells of the blood-brain barrier (BBB) during systemic immune challenges. These molecules, when present in the circulation, have the ability to trigger a series of events in cascade, leading to either the mitogen-activated protein (MAP) kinases/nuclear factor kappa B (NF-kappaB) or the janus kinase (JAK)/signal transducer and activator of transcription (STAT) transduction pathways in vascular-associated cells of the central nervous system (CNS). The brain blood vessels exhibit both constitutive and induced expression of receptors for different proinflammatory ligands that have the ability to stimulate these signaling molecules. Depending on the challenges and the cytokines involved, the transduction signal(s) solicited in cells of the BBB may orient the neuronal activity in a very specific manner in activating the transcription and production of soluble factors, such as prostaglandins (PGs). It is interesting to note that cytokines as well as systemic localized inflammation stimulate the cells of the BBB in a nonselective manner (i.e., within both large blood vessels and small capillaries across the brain). This nonselectivity raises several questions with regard to the localized neuronal activation induced by different experimental models of inflammation and cytokines. It is possible that the selectivity of the neuronal response is a consequence of the fine interaction between nonparenchymal synthesis of soluble mediators and expression of specific receptors for these ligands within parenchymal elements of different brain nuclei. This review will present the recent developments on this concept and the mechanisms that take place in cells of the BBB, which lead to the neuronal circuits involved in restoring the body's homeostasis during systemic immunogenic challenges. The induction of fever, the hypothalamic-pituitary adrenal (HPA) axis, and other autonomic functions are among the physiological outcomes necessary for the protection of the mammalian organism in the presence of foreign material.

Interleukin-6 is a needed proinflammatory cytokine in the prolonged neural activity and transcriptional activation of corticotropin-releasing factor during endotoxemia.

Interleukin-6 (IL-6) is a proinflammatory cytokine that plays multiple roles in the central nervous system during infections and injuries. Although this molecule is capable of stimulating the release of ACTH and glucocorticoids, it has been demonstrated that a single injection of IL-6 fails to activate the paraventricular nucleus (PVN) neurons that control the hypothalamic-pituitary-adrenal axis. The observation that IL-6 receptor (IL-6R) is up-regulated in the brain during endotoxemia led us to hypothesize that prior induction of IL-6R synthesis could amplify the effect of circulating IL-6 on the neuroendocrine response. Rats received a first iv injection of either bacterial lipopolysaccharide (LPS; 5 microg) or vehicle solution. After a 6-h waiting period, they received a second iv injection of either recombinant rat IL-6 or vehicle solution and were killed 1 h thereafter. Using in situ hybridization, we observed that IL-6R was barely expressed in the PVN under basal conditions, but was rapidly produced in response to LPS. IL-6 itself was also able to induce the synthesis of its own receptor along cerebral blood vessels, and this effect extended to several parenchymal structures, including the PVN, when the cytokine was administrated after LPS. In agreement with our hypothesis, we found that IL-6 injected in LPS-pretreated rats stimulated PVN neurons, as revealed by the expression of CRF primary transcript and c-fos messenger RNA, an immediate early gene used as a marker of cellular activation. A significant increase in plasma corticosterone levels was also found in animals that received iv IL-6 injection after being pretreated 6 h before with the very low dose of LPS. The fact that IL-6 alone or injected after LPS treatment was unable to induce cyclooxygenase-2 synthesis is an argument in favor of a PG-independent mechanism. The relative contribution of IL-6 in stimulating CRF expression in the PVN and neural activity throughout the brain during endotoxemia was also investigated in IL-6-deficient mice after an ip injection of LPS. The endotoxin induced similar c-fos and CRF expression patterns in knockout and wild-type mice, but the expression levels were generally higher and/or lasted longer in wild-type animals. Taken together, physiological changes that may include the induction of IL-6R synthesis seem to be necessary for IL-6 to activate PVN neurons. Moreover, although IL-6 does not appear essential during the early phases of endotoxemia, this cytokine is required during the later phases to prolong the activation of neural cells throughout the brain and to maintain CRF expression in the PVN neurons that control the hypothalamic-pituitary-adrenal axis.

Regulation of the genes encoding interleukin-6, its receptor, and gp130 in the rat brain in response to the immune activator lipopolysaccharide and the proinflammatory cytokine interleukin-1beta.

Interleukin-6 (IL-6) is a pleiotropic cytokine believed to play key roles in the neuroimmune interactions. This molecule may act on the nervous system by interacting with its specific receptor subunit (IL-6R) and the signal transducer gp130. The purposes of the present study were to describe the central distribution of IL-6, IL-6R, and gp130 mRNAs under basal conditions and to verify the influence of the immune activator lipopolysaccharide (LPS) and the proinflammatory cytokine interleukin-1beta (IL-1beta) on the expression of IL-6 and its related genes throughout the rat brain. Rats were killed at multiple times after intraperitoneal injection of the bacterial endotoxin and intravenous administration of the recombinant rat IL-1beta (rrIL-1beta), and their brains were cut into 30-microm coronal sections from the olfactory bulb to the end of the medulla. Each transcript was localized by in situ hybridization histochemistry using 35S-labeled rat riboprobes. The results show that IL-6 mRNA was undetectable in the brain under basal conditions and following the injection of rrIL-1beta. Injection of LPS rapidly stimulated transcription of this gene in the choroid plexus and the sensorial circumventricular organs (CVOs), including the organum vasculosum laminae terminalis (OVLT), subfornical organ, median eminence, and area postrema. Conversely, IL-6R and gp130 mRNAs were heterogeneously distributed throughout the brain under basal conditions. The injection of LPS stimulated the biosynthesis of IL-6R in the CVOs, medial preoptic area, bed nucleus stria terminalis, central nucleus of the amygdala, hippocampus, hypothalamic paraventricular nucleus, cerebral cortex, and blood vessels. Increased levels of IL-6R mRNA were also observed in the microvasculature following rrIL-1beta injection. Finally, gp130 mRNA expression was increased in the OVLT and throughout the endothelium of brain capillaries of LPS-treated rats but remained unchanged after administration of rrIL-1beta. These results demonstrate that expression of the genes encoding IL-6, IL-6R, and gp130 can be up-regulated in selective regions of the brain in response to the bacterial endotoxin LPS and the proinflammatory cytokine IL-1beta (only for IL-6R expression). This fine genetic regulation might be of great importance in the neuroimmune interplay and provides the evidence that sensorial CVOs and microvasculature are in a privileged position to mediate the action of IL-6 of central and/or systemic origin in the brain of immune-challenged animals.

Influence of interleukin-6 on neural activity and transcription of the gene encoding corticotrophin-releasing factor in the rat brain: an effect depending upon the route of administration.

Interleukin-6 (IL-6) is a pleiotropic cytokine produced by various lymphoid and neural cells. In addition to its classic role during immune and inflammatory responses, IL-6 acts on the central nervous system to elicit changes, such as activation of the hypothalamic-pituitary-adrenal (HPA) axis. This study investigated the effects of systemic and central injection of IL-6 on neural activity and transcription of the corticotrophin-releasing factor (CRF) gene in the brain of conscious rats. The animals were killed 1 and 3 h after a single infusion of IL-6 into the right jugular vein (0.83 or 3.0 microg) or the right lateral ventricle (0.2 microg) and their brains cut from the olfactory bulb to the end of the medulla in 30-microm coronal sections. Messenger RNA encoding the protein Fos, a marker of neural activity, and the neuropeptide CRF were localized by in situ hybridization histochemistry using 35S-labelled exonic and intronic riboprobes. The results show that systemic injection of IL-6 induced specific transcription of c-fos gene in most of the sensorial circumventricular organs, including the organum vasculosum lamina terminalis, subfornical organ, median eminence, and area postrema, as well as in the central nucleus of the amygdala and bed nucleus of the stria terminalis. On the other hand, central injection of IL-6 increased cellular level of c-fos mRNA in the ependymal layer and the walls of the ventricles, meninges, nucleus of the solitary tract, and circumventricular organs. These effects were rapid and transient, since the signals for c-fos mRNA were detected 1 h after both treatments and vanished 3 h afterwards. Moreover, the CRF gene was not activated by either systemic or central administration of IL-6 in the paraventricular nucleus of the hypothalamus. Taken together, these results suggest that circumventricular organs hold a privileged position in mediating the central effects of systemic IL-6 and that centrally injected IL-6 can strongly activate cells of the ventricular system and surrounding structures. Although this differential circuitry may explain distinct origin-dependent functions of IL-6, this cytokine seems insufficient, in itself, to induce transcription of the gene encoding neuroendocrine CRF, the neuropeptide responsible for control of the HPA axis.

C-fos mRNA pattern and corticotropin-releasing factor neuronal activity throughout the brain of rats injected centrally with a prostaglandin of E2 type.

The present study investigated the effect of central administration of the prostaglandin of E2 type (PGE2) on the distribution of the immediate early gene (IEG) c-fos mRNA and the transcriptional activity of corticotropin-releasing factor (CRF) and its type 1 receptor in the brain of conscious rats. Adult male rats were sacrificed 30 min and 2 h after a single infusion of PGE2 into the right lateral ventricle (2 micrograms/10 microliters) and their brains cut from the olfactory bulb to the end of the medulla in 30 micrometer coronal sections. mRNAs encoding the IEG c-fos and CRF1 receptor were assayed by in situ hybridization histochemistry using 35S-labeled exonic riboprobes whereas the primary transcript (heteronuclear (hn)RNA) for CRF was detected using intronic probe technology as an index of CRF transcriptional activity. Colocalization of c-fos mRNA within CRF, vasopressin (AVP), and oxytocin (OT) neurons was determined by means of a combination of immunocytochemistry and in situ hybridization techniques on the same brain sections. Thirty min after PGE2 injection, a moderate to strong positive signal for c-fos mRNA was detected in multiple structures of the brain such as the medial preoptic area/organum vasculosum of the lamina terminalis, supraoptic nucleus (SON), parvocellular and magnocellular divisions of the paraventricular nucleus (PVN) of the hypothalamus, central nucleus of the amygdala, nucleus of the solitary tract, dorsal motor nucleus of the vagus, area postrema, dorsal division of the ambiguus nucleus, and throughout the choroid plexus and leptomeninges. A smaller but significant c-fos expression was observed in various structures including the subfornical organ, bed nucleus of the stria terminalis, arcuate nucleus, and periventricular nucleus of the hypothalamus. Two h after treatment with the PG, the signal for c-fos mRNA in most of these brain nuclei vanished. In the parvocellular nucleus of the PVN, c-fos was expressed in CRF-immunoreactive (ir) and OT-ir neurons, whereas in the magnocellular part of that nucleus and in the SON, this transcript was essentially colocalized in OT-ir neurons. Activation of CRF neuroendocrine cells was also associated with an increase in CRF transcription as revealed by the selective presence of CRF primary transcript (hnRNA), which was stimulated only in the PVN but not in any other nuclei in the brains of PGE2-treated rats. Central administration of PGE2 also induced expression of the CRF type 1 receptor in the parvocellular PVN. Taken together, these results provide clear anatomical evidence that central PGE2 injection causes specific and selective expression of c-fos in several brain structures recognized to be activated in the brains of endotoxin-challenged rats. It is therefore possible that PG of E2 type plays a crucial role within the CNS in the interface between the immune and nervous systems to modulate neuroendocrine responses, such as the hypothalamic-pituitary-adrenal axis.

The rat growth hormone proximal silencer contains a novel DNA-binding site for multiple nuclear proteins that represses basal promoter activity.

Cell-type-specific expression of the rat growth hormone (rGH) gene is determined by the interaction of both positive as well as negative regulatory proteins with cis-acting elements located upstream of the rGH mRNA start site. We have recently shown that the rat liver transcription factor NF1-L binds to the proximal rGH silencer (called silencer-1) to repress its transcriptional activity. However, this single factor proved to be insufficient by itself to confer cell-specific gene repression. We therefore attempted to identify other regulatory proteins interacting with silencer 1, which might be needed to achieve full cell-specific repression of that gene. A common recognition site for three yet uncharacterized nuclear proteins (designated as SBP1, SBP2 and SBP3) which bind a DNA sequence adjacent to the NF1-L-binding site in the rGH silencer-1 element were identified. UV crosslinking of DNA/protein complexes and nuclear protein fractionation/renaturation from SDS/polyacrylamide gels further indicated that the molecular masses for SBP1-3 are 41, 26 and 17 kDa respectively, the major species being the 26-kDa protein (SBP2) which account for 83% of the shifted SBP double-stranded oligonucleotide in gel mobility-shift assays. For this reason, most of this study focussed on the characterization of SBP2. We demonstrated that binding of NF1-L and SBP2 to their respective recognition sequence is a mutually exclusive event. Although an SBP-binding activity has been found in every non-pituitary tissue or cell line tested, no such activity could be detected in either rat pituitaries or rat pituitary GH4C1 cells. Insertion of the SBP element upstream of the basal promoter of the mouse p12 heterologous gene resulted in a consistent decrease in chloramphenicol acetyl transferase reporter gene expression following transient transfections in non-pituitary cells only, suggesting that the related SBP1-3 proteins might be involved in generally repressing gene transcription in a cell-specific manner.