Lipopolysaccharide suppresses activation of the tuberomammillary histaminergic system concomitant with behavior: a novel target of immune-sensory pathways.

Infection and inflammation strongly inhibit a variety of behaviors, including exploration, social interaction, and food intake. The mechanisms that underlie sickness behavior remain elusive, but appear to involve fatigue and a state of hypo-arousal. Because histaminergic neurons in the ventral tuberomammillary nucleus of the hypothalamus (VTM) play a crucial role in the mediation of alertness and behavioral arousal, we investigated whether the histaminergic system represents a target for immune activation and, if so, whether modulation by ascending medullary immune-sensitive projections represents a possible mechanism. Rats were injected intraperitoneally with either the pro-inflammatory stimulus lipopolysaccharide (LPS) or saline, and exposed to one of various behavioral tests that would induce motivated behavior (exploration, play behavior, social interaction, sweetened milk consumption). Upon kill, brains were processed for c-Fos and histidine decarboxylase immunoreactivity. LPS treatment reduced behavioral activity and blocked behavioral test-associated c-Fos induction in histaminergic neurons of the VTM. These effects of LPS were prevented by prior inactivation of the caudal medullary dorsal vagal complex (DVC) with a local anesthetic. To determine whether LPS-responsive brainstem projection neurons might provide a link from the DVC to the VTM, the tracer Fluorogold was iontophoresed into the VTM a week prior to experiment. Retrogradely labeled neurons that expressed c-Fos in response to LPS treatment included catecholaminergic neurons within the nucleus of the solitary tract and ventrolateral medulla. These findings support the hypothesis that the histaminergic system represents an important component in the neurocircuitry relevant for sickness behavior that is linked to ascending pathways originating in the lower brainstem.

Neural-immune interface in the rat area postrema.

The area postrema functions as one interface between the immune system and the brain. Immune cells within the area postrema express immunoreactivity for the pro-inflammatory cytokine, interleukin-1beta following challenge with immune stimulants, including lipopolysaccharide (from bacterial cell walls). As a circumventricular organ, the area postrema accesses circulating immune-derived mediators, but also receives direct primary viscerosensory signals via the vagus nerve. Neurons in the area postrema contribute to central autonomic network neurocircuitry implicated in brain-mediated host defense responses. These experiments were directed toward clarifying relationships between immune cells and neurons in the area postrema, with a view toward potential mechanisms by which they may communicate. We used antisera directed toward markers indicating microglia (CR3/CD11b; OX-42), resident macrophages (CD163; ED-2), or dendritic cell-like phenotypes (major histocompability complex class II; OX-6), in area postrema sections from lipopolysaccharide-treated rats processed for light, laser scanning confocal, and electron microscopy. Lipopolysaccharide treatment induced interleukin-1beta-like immunoreactivity in immune cells that either associated with the vasculature (perivascular cells, a subtype of macrophage) or associated with neuronal elements (dendritic-like, and unknown phenotype). Electron microscopic analysis revealed that some immune cells, including interleukin-1beta-positive cells, evinced membrane apposition with neuronal elements, including dendrites and terminals, that could derive from inputs to the area postrema such as vagal sensory fibers, or intrinsic area postrema neurons. This arrangement provides an anatomical substrate by which immune cells could directly and specifically influence individual neurons in the area postrema, that may support the induction and/or maintenance of brain responses to inflammation.

Staphylococcal enterotoxin B induces fever, brain c-Fos expression, and serum corticosterone in rats.

The paraventricular nucleus of the hypothalamus (PVH) occupies a pivotal point within the network of brain nuclei coordinating critical host-defense responses. In mice, T cell-dependent immune stimuli, including the bacterial superantigen staphylococcal enterotoxin B (SEB), can activate the PVH. To determine whether T cell-dependent immune stimuli activate the PVH in rats, we assessed plasma corticosterone (Cort) levels, fever responses, and c-Fos expression in the PVH in animals treated with intraperitoneal injections of SEB. In animals with previously implanted abdominal thermisters, intraperitoneal injection of 1 mg/kg SEB resulted in a significant rise in body temperature, with a latency of 3.5-4 h. In separate animals, intraperitoneal injection of 1 mg/kg SEB resulted in a significant elevation of plasma Cort and induced c-Fos expression in parvocellular neurons within the PVH. These results support the idea that T cell-dependent immune stimuli activate brain pathways mediating host-defense responses such as fever and neuroendocrine changes.

The contribution of the vagus nerve in interleukin-1beta-induced fever is dependent on dose.

It has been suggested that proinflammatory cytokines communicate to the brain via a neural pathway involving activation of vagal afferents by interleukin-1beta (IL-1beta), in addition to blood-borne routes. In support, subdiaphragmatic vagotomy blocks IL-1beta-induced, brain-mediated responses such as fever. However, vagotomy has also been reported to be ineffective. Neural signaling would be expected to be especially important at low doses of cytokine, when local actions could occur, but only very small quantities of cytokine would become systemic. Here, we examined core body temperature after intraperitoneal injections of three doses of recombinat human IL-1beta (rh-IL-1beta). Subdiaphragmatic vagotomy completely blocked the fever produced by 0.1 microg/kg, only partially blocked the fever produced by 0.5 microg/kg, and had no effect at all on the fever that followed 1.0 microg/kg rh-IL-1beta. Blood levels of rh-IL-1beta did not become greater than normal basal levels of endogenous rat IL-beta until the 0.5-microg/kg dose nor was IL-1beta induced in the pituitary until this dose. These results suggest that low doses of intraperitoneal IL-1beta induce fever via a vagal route and that dose may account for some of the discrepancies in the literature.

Vagotomy does not inhibit high dose lipopolysaccharide-induced interleukin-1beta immunoreactivity in rat brain and pituitary gland.

In the present study, we examined whether the vagus nerve is involved in mediating lipopolysaccharide (LPS)-induced appearance of IL-1beta immunoreactive cells in the brain and pituitary gland. Rats were either sham-operated or subjected to subdiaphragmatic vagotomy. Four weeks later, pyrogen free saline or 400 microg/kg LPS was administered to the rats intraperitoneally. Four and 8 h later, the animals were intracardially perfused with 4% paraformaldehyde and tissues were prepared for IL-1beta immunocytochemistry. IL-1beta positive cells were observed at both time-intervals after LPS administration in the choroid plexus, meninges, circumventricular organs and pituitary gland of both sham-operated and vagotomized rats. We conclude that under the conditions studied, the vagus nerve does not mediate LPS-induced appearance of IL-1beta in the rat brain and pituitary gland.

Effects of vagotomy on serum endotoxin, cytokines, and corticosterone after intraperitoneal lipopolysaccharide.

The vagus nerve appears to play a role in communicating cytokine signals to the central nervous system, but the exact extent of its involvement in cytokine-to-brain communication remains controversial. Recently, subdiaphragmatic vagotomy was shown to increase bacterial translocation across the gut barrier and thus may cause endotoxin tolerance. The current experiment tested whether or not vagotomized animals have similar systemic responses to endotoxin challenge as do sham-operated animals. Subdiaphragmatically vagotomized and sham-operated animals were injected intraperitoneally with one of three doses (10, 50, 100 microg/kg) of lipopolysaccharide (LPS) or vehicle, and blood samples were taken at 15, 30, 60, 90, and 120 min after the injection. The intraperitoneal injection of LPS increased circulating LPS levels at all time points examined. In addition, all three doses of LPS significantly increased serum interleukin (IL)-1beta, IL-6, and corticosterone in both control and vagotomized rats. In conclusion, vagotomy itself has no marked effect on circulating endotoxin levels or the production of IL-1beta, IL-6, or corticosterone in blood after an intraperitoneal injection of LPS.

Effects of vagotomy on lipopolysaccharide-induced brain interleukin-1beta protein in rats.

The production of interleukin-1beta (IL-1beta) in brain is thought to be a critical step in the induction of central manifestations of the acute phase response, and the vagus nerve has been implicated in immune-to-brain communication. Thus, this study examined the effects of intraperitoneal (i.p.) injections of lipopolysaccharide (LPS) on brain IL-1beta protein levels in control and subdiaphragmatically vagotomized rats. In the first experiment, vagotomized and sham-operated male Sprague-Dawley rats were injected i.p. with one of three doses (10, 50, 100 microg/kg) of LPS or vehicle (sterile, pyrogen-free saline) and sacrificed 2 h after the injection. In the second experiment, vagotomized and sham-operated rats were injected i.p. with 100 microg/kg LPS or vehicle and sacrificed 1 h after the injection. The i.p. injection of LPS dose-dependently increased IL-1beta protein levels in the hypothalamus, hippocampus, dorsal vagal complex, cerebellum, posterior cortex, and pituitary 2 h after the injection. Brain and pituitary IL-1beta levels were also significantly increased 1 h after the injection of 100 microg/kg LPS. There were no significant differences in brain IL-1beta levels between sham-operated and vagotomized rats at either the 2 h or 1 h time points. The current data are consistent with previous studies showing increases in brain IL-1beta after peripheral injections of LPS, and support the notion that brain IL-1beta is a mediator in the illness-induction pathway. Furthermore, these data indicate that, at the doses and times tested, subdiaphragmatic vagal afferents are not crucial for LPS-induced brain IL-1beta protein.

Vagal immune-to-brain communication: a visceral chemosensory pathway.

The immune system operates as a diffuse sensory system, detecting the presence of specific chemical constituents associated with dangerous micro-organisms, and then signalling the brain. In this way, immunosensation constitutes a chemosensory system. Several submodalities of this sensory system function as pathways conveying immune-related information, and can be classified as either primarily brain barrier associated or neural. The vagus nerve provides the major neural pathway identified to date. The initial chemosensory transduction events occur in immune cells, which respond to specific chemical components expressed by dangerous micro-organisms. These immune chemosensory cells release mediators, such as cytokines, to activate neural elements, including primary afferent neurons of the vagal sensory ganglia. Primary afferent activation initiates local reflexes (e.g. cardiovascular and gastrointestinal) that support host defense. In addition, at least three parallel pathways of ascending immune-related information activate specific components of the illness response. In this way, immunosensory systems represent highly organized and coherent pathways for activating host defense against infection.

Subdiaphragmatic vagotomy blocks interleukin-1beta-induced fever but does not reduce IL-1beta levels in the circulation.

Peripheral interleukin-1beta has been implicated in the initiation of fever responses, yet the pathways by which it influences brain function are still unclear. Sectioning the abdominal vagus has been reported to inhibit fever after intraperitoneal administration of interleukin-1beta, suggesting that vagal afferents participate in signaling the brain to mount a fever response to interleukin-1beta. However, the inhibitory effect of subdiaphragmatic vagotomy could be due to alterations in pharmacokinetics such that the intraperitoneally injected cytokine does not reach the general circulation in sufficient quantities to activate the brain via blood-borne signaling. We measured both fever and plasma levels of interleukin-1beta in vagotomized and sham-operated rats after intraperitoneal administration of 1 microg/kg human recombinant interleukin-1beta to determine whether vagotomy reduces fever and levels of circulating interleukin-1beta after intraperitoneal injection. Plasma levels of human recombinant and endogenous rat interleukin-1beta were measured in separate enzyme-linked immunosorbent assays. While intraperitoneal administration of human recombinant interleukin-1beta elevated plasma levels of this cytokine similarly in vagotomized and sham-operated animals, only sham-operated rats responded with fever. Plasma levels of endogenous rat interleukin-1beta were unchanged by any treatment. These results demonstrate that the blockade of intraperitoneal interleukin-1beta-induced fever after subdiaphragmatic vagotomy cannot be accounted for by alterations of interleukin-1beta levels in the general circulation.

Subdiaphragmatic vagotomy does not block intraperitoneal lipopolysaccharide-induced fever.

Several recent findings, including the inability of subdiaphragmatic vagotomy to block lipopolysaccharide (LPS)-induced interleukin-1beta (IL-1beta) protein in brain, have made it necessary to reexamine the role of the subdiaphragmatic vagal afferents in immune-to-brain communication. In this study, we examined the effects of intraperitoneal (i.p.) injections of LPS on core body temperature in control and subdiaphragmatically vagotomized rats. Vagotomized and sham-operated male Sprague-Dawley rats were injected i.p. with vehicle (pyrogen-free saline) on the control day and LPS (1, 10 or 50 microg/kg) on the experimental day, and core body temperature was monitored by telemetry for 6 h after the injection. At this time, rats were sacrificed, and serum, liver, and pituitary samples were collected. The i.p. injection of LPS increased core body temperature in both sham-operated and vagotomized rats compared to the saline injection. In addition, LPS significantly increased IL-1beta levels in serum, liver, and pituitary compared to saline-injected controls. There were no significant differences in the magnitude of the fever or in the levels of IL-1beta in serum, liver, or pituitary between sham-operated and vagotomized rats. Thus, the current data indicate that, at the doses tested, subdiaphragmatic vagal afferents are not crucial for i.p. LPS-induced fever. Because several effects of vagotomy have been shown to be dependent on dose, we are currently investigating whether vagal afferents are involved in lower-dose i.p. LPS-induced fever.

Interleukin-1beta in immune cells of the abdominal vagus nerve: a link between the immune and nervous systems?

Intraperitoneal administration of the cytokine interleukin-1beta (IL-1beta) induces brain-mediated sickness symptoms that can be blocked by subdiaphragmatic vagotomy. Intraperitoneal IL-1beta also induces expression of the activation marker c-fos in vagal primary afferent neurons, suggesting that IL-1beta is a key component of vagally mediated immune-to-brain communication. The cellular sources of IL-1beta activating the vagus are unknown, but may reside in either blood or in the vagus nerve itself. We assayed IL-1beta protein after intraperitoneal endotoxin [lipopolysaccharide (LPS)] injection in abdominal vagus nerve, using both an ELISA and immunohistochemistry, and in blood plasma using ELISA. IL-1beta levels in abdominal vagus nerve increased by 45 min after LPS administration and were robust by 60 min. Plasma IL-1beta levels increased by 60 min, whereas little IL-1beta was detected in cervical vagus or sciatic nerve. IL-1beta-immunoreactivity (IR) was expressed in dendritic cells and macrophages within connective tissues associated with the abdominal vagus by 45 min after intraperitoneal LPS injection. By 60 min, some immune cells located within the nerve and vagal paraganglia also expressed IL-1beta-IR. Thus, intraperitoneal LPS induced IL-1beta protein within the vagus in a time-frame consistent with signaling of immune activation. These results suggest a novel mechanism by which IL-1beta may serve as a molecular link between the immune system and vagus nerve, and thus the CNS.

Interleukin-1 induces c-Fos immunoreactivity in primary afferent neurons of the vagus nerve.

Peripheral administration of bacterial endotoxin, an immune stimulant, induces evidence of activation in vagal primary afferent neurons. To determine whether interleukin-1beta (IL-1beta) is part of the molecular pathway leading to this activation, we assessed the expression of the neuronal activation marker c-Fos in vagal primary afferent neurons after intraperitoneal injections of IL-1beta (2 microg/kg). IL-1beta, but not vehicle, induced c-Fos expression, demonstrating that IL-1beta is likely an important signal from the immune system to the vagus nerve, and thus the brain.

Bacterial endotoxin induces fos immunoreactivity in primary afferent neurons of the vagus nerve.

Subdiaphragmatic vagotomy inhibits brain-mediated illness responses to peripherally administered bacterial endotoxin, including fever, hyperalgesia, sickness behavior, and activation of the hypothalamic-pituitary-adrenal axis. However, direct evidence implicating vagal afferents specifically in conveying information about peripheral immune activation to the brain is still lacking. This study assessed whether (1) endotoxin induces the expression of the functional activation marker Fos in the vagal sensory ganglia, and (2) vagotomy abrogates endotoxin-induced Fos expression in these ganglia. Male rats, which had previously received vagotomy or sham surgery, were injected intraperitoneally or intravenously with either endotoxin or saline. Fos immunolabeling was absent in saline-treated rats. In contrast, scattered cells within the vagal sensory ganglia showed Fos immunoreactivity after both intraperitoneal and intravenous endotoxin administration in sham-operated rats. Vagotomy abolished Fos expression after intraperitoneal endotoxin administration, whereas after intravenous administration Fos expression was strongly attenuated, but not eliminated. These findings implicate vagal afferents as a potential signaling pathway to brain regions that generate illness responses to pro-inflammatory mediators.

Thermogenic and corticosterone responses to intravenous cytokines (IL-1beta and TNF-alpha) are attenuated by subdiaphragmatic vagotomy.

The brain orchestrates changes in behavior and physiology as a consequence of peripheral immune activation and infection. These changes require that the brain receives signals from the periphery that an immunological challenge has occurred. Previous research has established that cytokines play a role in signalling the brain. What remains unclear, however, is how peripheral cytokines signal the central nervous system. A recent proposal is that cytokines signal the brain by stimulating peripheral nerves. The hypothesis states that following infection and the release of cytokines such as IL-1beta into local tissue or microvasculature, IL-1beta stimulates IL-1 receptors on vagal afferent terminals, or more likely on cells of vagal paraganglia. Vagal afferents, in turn, signal the brain. Previous work has demonstrated that transection of the vagus below the level of the diaphragm blocks or attenuates many illness consequences of intraperitoneally (i.p.) administered lipopolysaccharide (LPS) or IL-1beta. The present studies extend these findings by examining the effect of subdiaphragmatic vagotomy on illness consequences following intravenously (i.v.) administered IL-1beta and TNF-alpha. Subdiaphragmatic vagotomy attenuated both the fever response and corticosterone response produced by i.v. administered cytokines. This effect was dose dependent. The results add support to the hypothesis that vagal afferents are involved in peripheral cytokine-to-brain communication.

The role of the vagus nerve in cytokine-to-brain communication.

Peripheral interleukin-1 beta (IL-beta) and inflammatory stimuli that induce the synthesis and release of IL-1 beta produce a variety of central nervous system responses. Most proposals designed to explain how peripheral IL-1 beta influences the CNS have focused on blood-borne routes of communication. We will review data that indicate that at least some of the CNS response to peripheral IL-1 beta are instead mediated by a neural route of communication between the periphery and the CNS. IL-1 beta activates afferent vagal fibers that terminate in the nucleus tractus solitarius, and communication via the vagus is responsible for much of the hyperalgesia, fever, anorexia, taste aversions, increased levels of plasma corticosteroid, and brain norepinephrine changes produced by intraperitoneal injections of IL-1 beta and LPS. Data extending this analysis to TNF-alpha and intravenous routes will be described.

Subdiaphragmatic vagotomy does not prevent fever following intracerebroventricular prostaglandin E2: further evidence for the importance of vagal afferents in immune-to-brain communication.

Brain-mediated sickness responses can be blocked by subdiaphragmatic vagotomy, suggesting that vagal afferents signal peripheral inflammation or infection. This study tested whether subdiaphragmatic vagotomy disrupts sickness responses by interrupting effector pathways. If this explanation is correct, intracerebroventricular prostaglandin E2-induced fever should be blocked by this procedure. Fever was unaffected by subdiaphragmatic vagotomy, thus these data provide support for the conclusion that vagal afferents signal the brain during immune activation.

Vagal paraganglia bind biotinylated interleukin-1 receptor antagonist: a possible mechanism for immune-to-brain communication.

Interleukin-1 beta is a proinflammatory cytokine released by activated immune cells. In addition to orchestrating immune responses to infection, interleukin-1 beta is a key mediator of immune-to-brain communication. Interleukin-1 beta and endotoxin (which releases IL1 beta from immune cells) cause centrally mediated illness responses such as fever, aphagia, etc. These effects are blocked by intraperitoneal IL1 receptor antagonist (IL1ra), suggesting critical involvement of peripheral IL1 receptors. Centrally mediated illness responses are also blocked by vagotomy, suggesting that IL1 beta directly or indirectly activates vagal afferents. To test for IL1 beta binding whole vagus (abdominal, laryngeal, and thoracic) and sections of hepatic vagus and liver hilus were incubated with biotinylated IL1ra and processed for avidin-biotin complex (ABC) or avidin-FITC histochemistry. Glomus cells of vagal paraganglia were labeled in all regions of the vagus. Biotinylated IL1ra also labeled smooth muscle and endothelial cells of blood vessels and lymphoid tissues. No label was present in omission or competition controls. These data suggest that centrally mediated illness responses result from IL1 activation of vagal paraganglia.

TNF-alpha-induced corticosterone elevation but not serum protein or corticosteroid binding globulin reduction is vagally mediated.

Immune activation leads to production of mediators such as cytokines, which act to induce both brain-mediated and peripheral defense processes. We used intraperitoneal administration of the cytokine tumor necrosis factor-alpha (TNF-alpha) to investigate whether defense processes induced by this cytokine are mediated by vagal afferents and/or interleukin-1 (IL-1) receptors. Because some effects of TNF-alpha are mediated, at least in part, by the brain [plasma corticosterone (CORT) elevation] and some are mediated by peripheral organs [reduction of serum protein and corticosteroid binding globulin (CBG)], we also investigated whether the effects of vagotomy are specific to those defense processes mediated by the brain. Both vagotomy and IL-1 receptor antagonist attenuated serum CORT elevation, but had no effects on serum protein or CBG reduction. These results support the idea that vagal afferents provide a true immune-to-brain pathway that may include IL-1 receptors.

Visceral afferent and efferent columns in the spinal cord of the teleost, Ictalurus punctatus.

In tetrapod vertebrates, neural circuitries subserving visceral and somatic reflexes are each represented in distinct columns of cells within the gray area of the spinal cord. To determine the location of visceral elements of the spinal cord of a teleost fish, crystals of the carbocyanine dye 1,1'dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI), were placed on either the abdominal sympathetic (mesenteric) nerves, the coeliac ganglia, or on the rostral three somatic spinal nerves, in fixed specimens of the channel catfish, Ictalurus punctatus. In fish in which DiI had been placed on the mesenteric nerves, labeled fibers coursed along the lateral margin of the dorsal horn within the first and second spinal segments, and appeared to terminate in a region at the base of the dorsal horn. In contrast, when DiI crystals were placed on the somatic spinal nerves, labeled primary afferents terminated in the dorsalmost two thirds of the dorsal horn, as well as in ventral and ventromedial areas of the medial funicular nuclear complex. Labeled somata (motor neurons) were situated in the ventral horn. When DiI crystals were placed bilaterally on the coeliac ganglia, labeled piriform and fusiform preganglionic neurons occurred in intermediate positions adjacent to the central canal, corresponding to the paracentral nucleus of Herrick, and in the lateral funiculus. These results demonstrate that somatic and visceral afferent and efferent functional columns are distinct in a teleost fish as they are in amniote vertebrates.

Calcitonin gene-related peptide innervation of the rat hepatobiliary system.

The digestive system is densely innervated by calcitonin gene-related peptide (CGRP)-immunoreactive neurons. The present study investigated a) the distribution and origin of CGRP-immunoreactive fibers in the rat hepatobiliary tract, and b) their relation with substance P/tachykinin (SP/TK) immunoreactivity using immunohistochemical and radioimmunoassay techniques. CGRP-containing fibers form dense networks in the fibromuscular layer of the biliary tree and surrounding the portal vein. Thin, varicose fibers are present at the base of the mucosa of the ducts. In the liver, labeled fibers are restricted to the portal areas and the stromal compartment. Neonatal treatment with capsaicin, a neurotoxin for primary afferent neurons, or celiac/superior mesenteric ganglionectomy depletes CGRP-containing fibers in the biliary tract, and reduces those associated with the portal vein. In contrast, subdiaphragmatic vagotomy does not appreciably modify the density of these fibers. Radioimmunoassay studies show a reduction of CGRP-immunoreactive contents in the biliary tract and portal vein by 84% and 65%, respectively, following capsaicin treatment, and by 80% and 66%, respectively, following ganglionectomy. By contrast, CGRP concentrations in vagotomized animals are comparable to those of controls. Most CGRP-positive fibers appear to contain SP/TK immunoreactivity, as indicated by double-label studies. These results demonstrate that the rat hepatobiliary tract is prominently innervated by CGRP- and CGRP/SP/TK-immunoreactive fibers, which are likely to originate from spinal afferent neurons. The abundance of these fibers and their association with a variety of targets are in line with the involvement of these peptidergic visceral afferents in regulating hepatobiliary activities, including hemodynamic functions of the hepatic vasculature.