Dave Gayle

Pro-inflammatory and anti-inflammatory cytokine mRNA induction in the periphery and brain following intraperitoneal administration of bacterial lipopolysaccharide

Gram-negative bacteria-derived lipopolysaccharide (LPS or endotoxin) is known to play an important role in immune and neurological manifestations during bacterial infections. LPS exerts its effects through cytokines, and peripheral or brain administration of LPS activates cytokine production in the brain. In this study, we investigated cytokine and neuropeptide mRNA profiles in specific brain regions and peripheral organs, as well as serum tumor necrosis factor (TNF)-alpha protein levels, in response to the intraperitoneal administration of LPS. For the first time, the simultaneous analysis of interleukin (IL)-1beta system components (ligand, signaling receptor, receptor accessory proteins, receptor antagonist), TNF-alpha, transforming growth factor (TGF)-beta1, glycoprotein 130 (IL-6 receptor signal transducer), OB protein (leptin) receptor, neuropeptide Y, and pro-opiomelanocortin (opioid peptide precursor) mRNAs was done in samples from specific brain regions in response to peripherally administered LPS. The same brain region/organ sample was assayed for all cytokine mRNA components. Peripherally administered LPS up-regulated pro-inflammatory cytokine (IL-1beta and/or TNF-alpha) mRNAs within the cerebral cortex, cerebellum, hippocampus, spleen, liver, and adipose tissue. LPS also increased plasma levels of TNF-alpha protein. LPS did not up-regulate inhibitory (anti-inflammatory) cytokine (IL-1 receptor antagonist and TGF-beta1) mRNAs in most brain regions (except for IL-1 receptor antagonist in the cerebral cortex and for TGF-beta1 in the hippocampus), while they were increased in the liver, and IL-1 receptor antagonist was up-regulated in the spleen and adipose tissue. Overall, peripherally administered LPS modulated the levels of IL-1beta system components within the brain and periphery, but did not affect the neuropeptide-related components studied. The data suggest specificity of transcriptional changes induced by LPS and that cytokine component up-regulation in specific brain regions is relevant to the neurological and neuropsychiatric manifestations associated with peripheral LPS challenge.

Kindling modulates the IL-1β system, TNF-α, TGF-β1, and neuropeptide mRNAs in specific brain regions

Cytokines and neuropeptides may be involved in seizure-associated processes. Following amygdala kindling in rats, we determined alterations of IL-1β, IL-1 receptor antagonist (IL-1Ra), IL-1 receptor type I (IL-1RI), IL-1 receptor accessory proteins (IL-1R AcPs) I and II, TNF-α, TGF-β1, neuropeptide Y (NPY), glycoprotein 130 (gp 130) and pro-opiomelanocortin (POMC) mRNA levels in the parietal, prefrontal and piriform cortices, amygdala, hippocampus and hypothalamus. Messenger RNAs expression in all brain regions was determined 2 h or 3 weeks following the last generalized convulsive seizure triggered from the ipsilateral kindled amygdala. The same brain region sample was used to assay for changes of all mRNA components. The results show that the 2 h-kindled group exhibited a significant up-regulation of IL-1β, IL-1RI, TNF-α and TGF-β1 mRNAs in all three cortical brain regions, amygdala and hippocampus. The largest up-regulation occurred in the prefrontal cortex (about 30-fold induction for IL-1β and TNF-α mRNAs). IL-1R AcP I and II mRNA levels were also up-regulated in the cortical regions. No changes in IL-1β, IL-1RI or TNF-α mRNA levels occurred in the 3 week-kindled group. NPY mRNA levels increased in the hippocampus, prefrontal and piriform cortices in the 2 h-kindled group, while IL-1Ra, gp 130, or POMC mRNA levels did not change in any group. The overall profile of mRNA changes shows specificity of transcriptional modulation induced by amygdala kindling. The data support a role of cytokines and NPY in the adaptive mechanisms associated with generalized seizure activity, with implications for neuroprotection, neuronal dysfunction and vulnerability associated with epileptic activity.

Central nervous system IL-1β system and neuropeptide Y mrnas during IL-1β-induced anorexia in rats

Abstract Interleukin-1β (IL-1β) induces anorexia and neuropeptide Y (NPY) increases feeding by direct action in the central nervous system (CNS). IL-1β, depending on the dose, attenuates or blocks NPY-induced feeding. This suggests that IL-1β-NPY interactions may be involved in IL-1β-induced anorexia. Here, RNase protection assays were used to investigate the effects of the chronic intracerebroventricular (ICV) administration of IL-1β (at a dose that yields estimated pathophysiological concentrations in the cerebrospinal fluid) on mRNA levels of IL-1β system components and NPY in the cerebellum, parietofrontal cortex, hippocampus, hypothalamus, and midbrain. The results show that the chronic ICV administration of IL-1β (8.0 ng/24 h for 72 h) differentially induced IL-1β system components across brain regions in anorectic rats. IL-1β mRNA and IL-1 receptor antagonist (IL-1Ra) mRNA were induced similarly, exhibiting highest and lowest expression levels in the hypothalamus and hippocampus, respectively. IL-1 receptor type I (IL-1RI) mRNA and the soluble form of IL-1 receptor accessory protein (IL-1R AcP II) mRNA were also induced in the hypothalamus and cerebellum. NPY mRNA expression showed a small, but significant decrease in the hypothalamus. Heat-inactivated IL-1β (8.0 ng/24 h for 72 h) had no effect on the behavioral or molecular profiles. The results suggest that endogenous upregulation of IL-1β contributes to IL-1β-induced anorexia, and that modification of NPY mechanisms also may be involved.

Neither acute nor chronic exposure to a naturalistic (predator) stressor influences the interleukin-1β system, tumor necrosis factor-α, transforming growth factor-β1, and neuropeptide mRNAs in specific brain regions

Physical (neurogenic) stressors may influence immune functioning and interleukin-1β (IL-1β) mRNA levels within several brain regions. The present study assessed the effects of an acute or repeated naturalistic, psychogenic stressor (predator exposure) on brain cytokine and neuropeptide mRNAs. Acute predator (ferret) exposure induced stress-like behavioral effects, including elicitation of a startle response and reduced exploratory behaviors; these responses diminished after 30 sessions. Moreover, acute and repeated predator exposure, like acute restraint stress, increased plasma corticosterone levels measured 5 min later, but not 2 h after stressor exposure. In contrast, none of the stressors used influenced IL-1β, IL-1 receptor antagonist, IL-1 receptor type I, IL-1 receptor accessory proteins I and II, or tumor necrosis factor-α mRNA levels in the prefrontal cortex, amygdala, hippocampus, or hypothalamus. Likewise, there were no stressor effects on transforming growth factor-β1, neuropeptide Y, glycoprotein 130, or leptin receptor mRNAs in brain regions. Thus, the naturalistic/psychogenic stressor used does not affect any of the brain cytokine component mRNAs studied. It is suggested that this type of stressor activates homeostatic mechanisms (e.g., glucocorticoid release), which act to preclude brain cytokine alterations that would otherwise favor neuroinflammatory/neuroimmunological responses and the consequent increase of brain sensitivity to neurotoxic and neurodegenerative processes.

Interleukin-1β System (Ligand, Receptor Type I, Receptor Accessory Protein and Receptor Antagonist), TNF-α, TGF-β1 and Neuropeptide Y mRNAs in Specific Brain Regions During Bacterial LPS-Induced Anorexia

Abstract Bacterial lipopolysaccharide (LPS) or endotoxin induces neurological manifestations including anorexia. It is proposed that LPS-induced cytokine production is involved in the generation of neurological manifestations and in neuroinflammatory/immunological responses during Gram-negative infections. For example, LPS-induced effects can be blocked or ameliorated by the interleukin-1 receptor antagonist (IL-1Ra). Here, sensitive and specific RNase protection assays were used to investigate the effects of the intracerebroventricular (i.c.v.) administration of LPS on mRNA levels of interleukin-1β (IL-1β) system components, tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β1, and neuropeptide Y (NPY) in the cerebellum, hippocampus, and hypothalamus. The same brain region sample was analyzed with all of the antisense probes. The data show simultaneous local induction of multiple cytokine components messenger ribonucleic acids (mRNAs) within specific brain regions in anorectic rats responding to i.c.v. administered LPS (500 ng/rat). Interleukin-1β and IL-1Ra had a similar mRNA induction profile (hypothalamus > cerebellum > hippocampus). Interleukin-1 receptor type I (IL-1RI) mRNA also increased in all three brain regions examined, and the soluble form of IL-1 receptor accessory protein (IL-1R AcP II) mRNA was induced in the hypothalamus. Tumor necrosis factor-α mRNA levels increased in the hypothalamus > hippocampus > cerebellum. Levels of membrane bound IL-1R AcP, TGF-β1, and NPY mRNAs did not change significantly in any brain region. The results suggest that: (1) endogenous up-regulation of IL-1β and TNF-α in the hypothalamus contribute to LPS-induced anorexia; and (2) the ratio IL-1Ra/IL-1β, and IL-1β ↔ TNF-α interactions may have implications for Gram-negative infections associated with high levels of LPS in the brain-cerebrospinal fluid.

Lipopolysaccharide (LPS)- and muramyl dipeptide (MDP)-induced anorexia during refeeding following acute fasting: characterization of brain cytokine and neuropeptide systems mRNAs

We investigated the effectiveness of lipopolysaccharide (LPS) and muramyl dipeptide (MDP) administered into the brain to induce anorexia in acutely fasted Wistar rats allowed to refeed. We also assayed for changes in mRNA levels of IL-1 system components, TNF-alpha, TGF-beta1, glycoprotein 130 (gp 130), leptin receptor (OB-R), pro-opiomelanocortin (POMC), neuropeptide Y (NPY), glucocorticoid receptor (GR), and CRF receptor (CRF-R) in selected brain regions. The data show that LPS and MDP induced anorexia differentially during refeeding. LPS-induced anorexia was of a stronger magnitude and duration than that of MDP. RNase protection assays showed that LPS and MDP significantly increased the expression of IL-1beta, IL-1 receptor type I, and TNF-alpha mRNAs in the cerebellum, hippocampus, and hypothalamus; LPS was more potent in all cases. MDP treatment, on the other hand, induced a stronger increase in hypothalamic levels of IL-1 receptor antagonist (IL-1Ra) and TGF-beta1 mRNAs relative to LPS. In addition, competitive RT-PCR analysis showed that LPS induced an eleven-fold increase in IL-1alpha mRNA in the hypothalamus relative to vehicle. These findings suggest that LPS and MDP mediate anorexia through different cytokine mechanisms. A stronger up-regulation of anti-inflammatory cytokines (IL-1Ra and TGF-beta1) mRNA expression by MDP may be involved in the weaker MDP-induced anorexia relative to LPS. No significant changes were observed in the peptide components examined except for an up-regulation in cerebellar gp 130 mRNA and down-regulation of hypothalamic GR mRNA expression in response to LPS or MDP. This study shows that LPS and MDP induce anorexia in fasted rats allowed to refeed, and suggests an important role for endogenous cytokine-cytokine interactions.

Persistent Borna disease virus infection of neonatal rats causes brain regional changes of mRNAs for cytokines, cytokine receptor components and neuropeptides.

Borna disease virus (BDV) replicates in brain cells. The neonatally infected rat with BDV exhibits developmental-neuromorphological abnormalities, neuronal cytolysis, and multiple behavioral and physiological alterations. Here, we report on the levels of interleukin-1beta (IL-1beta), IL-1 receptor antagonist (IL-1Ra), tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-beta1 (TGF-beta1), IL-1 receptor type I (IL-1RI), IL-1 receptor accessory protein (IL-1R AcP) I and II, glycoprotein 130, and various neuropeptide mRNAs in the cerebellum, parieto-frontal cortex, hippocampus and hypothalamus of BDV-infected rats at 7 and 28 days postintracerebral BDV inoculation. The data show that cytokine and neuropeptide mRNA components are abnormal and differentially modulated in brain regions. IL-1beta, TNF-alpha and TGF-beta1 mRNA levels were up-regulated in all brain regions following BDV inoculation. The same cerebellar samples from BDV-infected animals exhibited the highest levels of IL-1beta, IL-1Ra, TNF-alpha, IL-1RI, and IL-1R AcP II mRNA expression. The profiles of IL-1beta, IL-1Ra, TNF-alpha, and TGF-beta1 mRNA induction in the cerebellar samples were highly intercorrelated, indicating an association among cytokine ligand mRNAs. Cytokine mRNA induction was differentially up-regulated among brain regions, except for TGF-beta1. Specificity of transcriptional changes in response to BDV infection is also suggested by the up-regulation of cytokine and neuropeptide Y mRNAs associated with down-regulation of pro-opiomelanocortin, and with no change of IL-1R AcPI, dynorphin and leptin receptor mRNAs in the same brain region samples. Other data also show a differential mRNA component modulation in distinct brain regions obtained from the same rats depending on the stage of BDV infection. The conclusion of these studies is that cytokines may play a role in the neuropathophysiology of neonatally BDV-infected rats.

Feeding status and bacterial LPS-induced cytokine and neuropeptide gene expression in hypothalamus.

This study determined the effects of feeding status on basal and lipopolysaccharide (LPS)-stimulated cytokine and neuropeptide gene expression in the hypothalamus. With the use of RNase protection assays, we measured mRNA levels of interleukin-1beta (IL-1beta), IL-1 receptor antagonist (IL-1RA), IL-1 receptor type I (IL-1RI), IL-1R accessory proteins (AcP I and II), tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-beta1 (TGF-beta1), glycoprotein 130 (Gp 130), leptin receptor (OB-R), neuropeptide Y (NPY), preprodynorphin, and proopiomelanocortin (POMC). Analyses were done in ad libitum-fed, fasted, and fasted and refed rats treated with the intracerebroventricular administration of physiological saline or LPS. The data show that food deprivation increases the basal mRNA expression of IL-1beta, IL-1RA, TNF-alpha, IL-1RI, and IL-1R AcP I, whereas mRNA levels of POMC showed a decrease. Five hours of refeeding returned cytokine levels to those observed in the ad libitum-fed group. LPS administration induced a robust upregulation of IL-1beta, TNF-alpha, and IL-1RI during all three feeding conditions. Acute food deprivation did not modulate LPS-induced changes in hypothalamic cytokine mRNA profiles. These findings show that 1) cytokine modulation occurs as an adaptive response to the stress of acute fasting and 2) acute fasting does not affect LPS-induced cytokine mRNA modulation in the hypothalamus. The data have implications to gram-negative infections associated with acute anorexia.This study determined the effects of feeding status on basal and lipopolysaccharide (LPS)-stimulated cytokine and neuropeptide gene expression in the hypothalamus. With the use of RNase protection assays, we measured mRNA levels of interleukin-1β (IL-1β), IL-1 receptor antagonist (IL-1RA), IL-1 receptor type I (IL-1RI), IL-1R accessory proteins (AcP I and II), tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1), glycoprotein 130 (Gp 130), leptin receptor (OB-R), neuropeptide Y (NPY), preprodynorphin, and proopiomelanocortin (POMC). Analyses were done in ad libitum-fed, fasted, and fasted and refed rats treated with the intracerebroventricular administration of physiological saline or LPS. The data show that food deprivation increases the basal mRNA expression of IL-1β, IL-1RA, TNF-α, IL-1RI, and IL-1R AcP I, whereas mRNA levels of POMC showed a decrease. Five hours of refeeding returned cytokine levels to those observed in the ad libitum-fed group. LPS administration induced a robust upregulation of IL-1β, TNF-α, and IL-1RI during all three feeding conditions. Acute food deprivation did not modulate LPS-induced changes in hypothalamic cytokine mRNA profiles. These findings show that 1) cytokine modulation occurs as an adaptive response to the stress of acute fasting and 2) acute fasting does not affect LPS-induced cytokine mRNA modulation in the hypothalamus. The data have implications to gram-negative infections associated with acute anorexia.

Basal and IL-1β-stimulated cytokine and neuropeptide mRNA expression in brain regions of young and old Long–Evans rats

Young and old Long-Evans rats respond with fevers of equal magnitude and duration to the brain administration of interleukin-1beta (IL-1beta). Here, we characterized brain regional mRNA expression of cytokine and neuropeptide components in response to the brain administration of IL-1beta. We used specific and highly sensitive RNase protection assays to determine mRNA changes for IL-1beta, IL-1 receptor type I (IL-1RI), IL-1R accessory proteins I and II (IL-1R AcP I and II), IL-1 receptor antagonist (IL-1Ra), transforming growth factor-beta1 (TGF-beta1), glycoprotein 130 (gp 130), leptin receptor (OB-R), neuropeptide Y (NPY) and pro-opiomelanocortin (POMC) in the cerebellum, parieto-frontal cortex, hippocampus, hypothalamus, and midbrain of male young (3-5 months) and old (24-26 months) Long-Evans rats. In both young and old rats, IL-1beta induced a significant up-regulation of cerebellar IL-1Ra, IL-1RI, and TGF-beta1 mRNAs; hippocampal TGF-beta1 mRNA; hypothalamic IL-1beta, IL-1Ra, TGF-beta1, and gp 130 mRNAs; and midbrain IL-1beta and TGF-beta1 mRNAs. There were no age-related differences in any cytokine mRNA levels under basal or IL-1beta-stimulated conditions. Levels of hypothalamic POMC mRNA were different between age groups under basal and stimulated conditions. IL-1R AcP I and leptin receptor did not change in any brain region from either young or old rats, suggesting specificity of transcriptional changes. The data show that old Long-Evans rats are not defective in their capacity to develop an appropriate cytokine response to the brain administration of IL-1beta. The implications of these findings for neuroimmunological-neuroinflammatory and neurotoxic/neurodegenerative processes are discussed.

Autoregulation of the interleukin-1 system and cytokine-cytokine interactions in primary human astrocytoma cells.

Cytokines are proposed to play important roles in brain tumor biology. Previous studies reported on interleukin-1beta (IL-1beta) production and IL-1 receptor type I (IL-1RI, signaling receptor) expression in human astrocytomas, and on IL-1beta action in astrocytoma cell lines. However, all studies that have tested the direct action of cytokines have used exclusively astrocytoma cell lines, which do not recapitulate the in situ astrocytoma. Here, we demonstrate that astrocytoma cells obtained shortly after tumor neurosurgical resection respond to the direct application of human IL-1beta with a significant upregulation of IL-1alpha, IL-1beta, IL-1RI, and tumor necrosis factor-alpha (TNF-alpha) mRNAs. IL-1 receptor antagonist (IL-1Ra, an endogenous inhibitor that blocks IL-1alpha and IL-1beta actions) mRNA was not upregulated. Application of heat-inactivated IL-1beta had no effect on any cytokine component examined, demonstrating specificity of action. On the other hand, IL-1beta application did not modulate any cytokine component in acutely resected and dissociated primitive neuroectodermal tumor cells. The data have implications for a positive autoregulatory IL-1beta feedback system and synergistic IL-1beta <=> TNF-alpha interactions, which can be involved in the growth of pilocytic astrocytomas. The results together with our previous studies also support the notion that IL-1Ra or a compound with similar cytokine inhibitory activity could be useful for brain immunotherapy of astrocytomas.