Change in excitability and phenotype of substance P and its receptor in cat Aβ sensory neurons following peripheral inflammation

The effect of peripheral inflammation on spontaneous firing and level of substance P (SP) and its receptor in electrophysiologically identified cat Aβ neurons of dorsal root ganglion (DRG) was studied in vivo using a combination of intracellular recording, dye injection and immunohistochemical techniques. Following injection of carrageenan (Carg) into cat hindpaw, the number of Aβ neurons with spontaneous firing was enhanced significantly (42.9%, n=182) in comparison with control (16.8%, n=149, P<0.01). DRG Aβ neurons became less depolarized 2–4 h following Carg injection. After identifying the cell properties, Lucifer Yellow was injected and SP-like immunoreactivity (SP-LI) was then detected. A total of 17% of Aβ sensory neurons exhibited SP-LI in inflammatory cat. We also found in rat DRGs that the number of SP-LI positive large cells (>35 μm) was also significantly increased in Carg-treated DRG (11.8±1.2, n=8) compared with untreated DRG (1.8±0.8, n=8, P<0.01). In control cat, the topical use of SP in DRG did not induce any response of Aβ neurons. However, in Carg-treated cat, SP depolarized the membrane potential in most Aβ neurons (68.2%, n=22). L668,169, an antagonist of SP receptor, completely blocked the SP-induced responses. Furthermore, repeated application of SP did not induce obvious desensitization of Aβ neurons. These data suggest that peripheral inflammation increased the excitability, SP level and sensitivity of SP receptor of Aβ neurons. Therefore, we concluded that Aβ sensory neurons appear to contribute to inflammatory allodynia.     

The antihyperalgesic activity of a selective P2X7 receptor antagonist, A-839977, is lost in IL-1αβ knockout mice

The pro-inflammatory cytokine interleukin-1β (IL-1β) has been implicated in both inflammatory processes and nociceptive neurotransmission. Activation of P2X7 receptors is the mechanism by which ATP stimulates the rapid maturation and release of IL-1β from macrophages and microglial cells. Recently, selective P2X7 receptor antagonists have been shown to reduce inflammatory and neuropathic pain in animal models. However, the mechanisms underlying these analgesic effects are unknown. The present studies characterize the pharmacology and antinociceptive effects of a structurally novel P2X7 antagonist. A-839977 potently (IC₅₀ = 20–150 nM) blocked BzATP-evoked calcium influx at recombinant human, rat and mouse P2X7 receptors. A-839977 also potently blocked agonist-evoked YO-PRO uptake and IL-1β release from differentiated human THP-1 cells. Systemic administration of A-839977 dose-dependently reduced thermal hyperalgesia produced by intraplantar administration of complete Freund's adjuvant (CFA) (ED₅₀ = 100 μmol/kg, i.p.) in rats. A-839977 also produced robust antihyperalgesia in the CFA model of inflammatory pain in wild-type mice (ED₅₀ = 40 μmol/kg, i.p.), but the antihyperalgesic effects of A-839977 were completely absent in IL-1αβ knockout mice. These data demonstrate that selective blockade of P2X7 receptors in vivo produces significant antinociception in animal models of inflammatory pain and suggest that the antihyperalgesic effects of P2X7 receptor blockade in an inflammatory pain model in mice are mediated by blocking the release of IL-1β.     

Mechanisms of tumor necrosis factor-α (TNF-α) hyperalgesia

Activation of immune cells by pathogens induces the release of a variety of proinflammatory cytokines, including IL-1β and TNF-α. Previous studies using IL-1β have demonstrated that this cytokine can alter brain function, resulting in a variety of ‘illness responses’ including increased sleep, decreased food intake, fever, etc. We have recently demonstrated that i.p. IL-1β also produces hyperalgesia and that this hyperalgesia (as well as most illness responses) is mediated via activation of subdiaphragmatic vagal afferents. The present series of studies were designed to provide an initial examination of the generality of proinflammatory cytokine-induced hyperalgesia by examining the effects of i.p. TNF-α on pain responsivity. These studies demonstrate that: (a) i.p. TNF-α produces dose-dependent hyperalgesia as measured by the tailflick test, (b) this hyperalgesia is mediated via the induced release of IL-1β, (c) hyperalgesia is mediated via activation of subdiaphragmatic vagal afferents, and (d) the effects of subdiaphragmatic vagotomy cannot be explained by a generalized depression of neural excitability.     

Effect of tumor necrosis factor α and β on human oligodendrocytes and neurons in culture

Cytokines produced by infiltrating hematogenous cells or by glial cells activated during the course of central nervous system disease or trauma are implicated as mediators of tissue injury. In this study, we have assessed the extent and mechanism of injury of human-derived CNS oligodendrocytes and neurons in vitro mediated by the cytokines tumor necrosis factor α and β and compared these with the tumor necrosis factor independent effects mediated by activated CD4⁺ T-cells. We found that activated CD4⁺ T-cells, but not tumor necrosis factor α or β, could induce significant release of lactate dehydrogenase, a measure of cell membrane lysis, from oligodendrocytes within 24 hr. Neither induced DNA fragmentation as measured using a fluorescence nick-end labelling technique. After a more prolonged time period (96 hr), tumor necrosis factor α did induce nuclear fragmentation changes in a significant proportion of oligodendrocytes without increased lactate dehydrogenase release. The extent of DNA fragmentation was comparable to that induced by serum deprivation. Tumor necrosis factor β effects were even more pronounced. In contrast to oligodendrocytes, the extent of DNA fragmentation, assessed by propidium iodide staining, induced in neurons by tumor necrosis factor α was less than that induced by serum deprivation. In-situ hybridization studies of human adult glial cells in culture indicated that astrocytes, as well as microglia, can express tumor necrosis factor α mRNA.     

Lack of effect of interleukins 1α and 1β, during in vitro perifusion,on anterior pituitary release of adrenocorticotropic hormone and β endorphin in the male rat

It has been demonstrated that interleukin 1 (IL1) injection provokes a great variety of biological effects, notably an activation of the corticotropic axis, increasing plasma adrenocorticotropic hormone (ACTH) and corticosterone. However, the primary site of action of IL1 is still controversial. In the present study, we first verified the in vivo capability of human interleukins 1α (hIL1α) and 1β (hIL1β) to release ACTH and β endorphin (β EP) in the normal male rat, before investigating, through an anterior pituitary (AP) perifusion system, the hIL1α and hIL1β effects on basal and corticotropin-releasing factor (CRF)-induced ACTH and β EP secretions. This system enabled the examination of a dynamic profile of hormones secretion, avoiding the possibility of feedback mechanisms, as is the case with the use of regular but very often longtime incubations. The results showed that in a perifusion system, with a short duration treatment (below 2 hr) compatible with the kinetics of action observed in vivo, basal and CRF-induced ACTH and β EP release were not modified in the presence of a broad range of concentrations (from 10^?12 to 10^?9 M) of hIL1α or hIL1β. Taken together, these results clearly show that in an in vitro situation close to physiological conditions, the primary site of action of hIL1α and hIL1β on ACTH and β EP release is not located at the AP level in the male rat. © 1993 Wiley-Liss, Inc.     

Tumor necrosis factor α and transforming growth factor β upregulate astrocyte expression of monocyte chemoattractant protein-1

Astrocytes participate in the pathophysiology of central nervous system (CNS) inflammatory disease. Astrocyte expression of adhesion molecules, cytokines, and major histocompatibility complex antigens may contribute to these inflammatory processes. In addition, recent data suggested that astrocytes may be a source of monocyte chemoattractant protein-1 (MCP-1). MCP-1 is a member of the chemokine family of small cytokines and functions both as a chemoattractant as well as a stimulator of monocytes. To further characterize the role of astrocytes in CNS inflammation, we examined the effect of inflammatory cytokines on MCP-1 expression by astrocytes. Results of these studies demonstrate that the pro-inflammatory cytokine tumor necrosis factor alpha (TNFa) upregulates MCP-1 message and protein expression. The pleiotropic cytokine transforming growth factor beta (TGFβ) also stimulated MCP-1 expression. When astrocytes were exposed to both cytokines simultaneously, an additive effect on MCP-1 message, but not MCP-1 protein expression, was observed. These data suggest that TNFa and TGFβ, each present during CNS inflammatory disease, may upregulate the expression of MCP-1 which, in turn, may function to both recruit monocytes to the site of inflammation as well as to activate those monocytes already present in an inflammatory lesion.     

α2 and α1-adrenoceptors mediate opposing actions on parasympathetic neurons

We used intracellular recording methods to analyze the membrane responses to norepinephrine in cat vesical parasympathetic ganglia. In parasympathetic neurons, norepinephrine (NE) produces a membrane hyperpolarization, a membrane depolarization often accompanied by cell firing and a biphasic potential, a hyperpolarization followed by a depolarization. We found that the NE hyperpolarization is mediated through alpha 2-adrenoceptors while the NE depolarization is mediated through alpha 1-adrenoceptors. This situation is different than in sympathetic neurons where beta-adrenoceptors mediate a NE depolarization.     

Brain interleukin-1β and tumor necrosis factor-α are involved in lipopolysaccharide-induced delayed rectal allodynia in awake rats

Recently, we have developped a model of delayed (12 h) increase in sensitivity (allodynia) to rectal distension (RD) induced by intraperitoneal lipopolysaccharide (LPS) in awake rats. Thus, we examined whether central interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) are involved in LPS response. Abdominal contractions (criterion of visceral pain) were recorded in rats equipped with intramuscular electrodes. RDs were performed at various times after pharmacological treatments. RD induced abdominal contractions from a threshold volume of distension of 0.8 ml. At lowest volume (0.4 ml), this number was significantly increased 12 h after LPS. Intracerebroventricular (i.c.v.) injection of IL-1 receptor antagonist, IL-1β converting enzyme inhibitor or recombinant human TNF-α soluble receptor reduced LPS-induced increase of abdominal contractions at 0.4 ml volume of distension. When injected i.c.v., recombinant human IL-1β and recombinant bovine TNF-α reproduced LPS response at 9 and 12 h and at 6 and 9 h, respectively. These data suggest that IL-1β and TNF-α act centrally to induce delayed rectal hypersensitivity and that central release of these cytokines is responsible of LPS-induced delayed (12 h) rectal allodynia.     

Attenuation of β‐amyloid‐induced tauopathy via activation of CK2α/SIRT1: Targeting for cilostazol

β‐Amyloid (Aβ) deposits and hyperphosphorylated tau aggregates are the chief hallmarks in the Alzheimer's disease (AD) brains, but the strategies for controlling these pathological events remain elusive. We hypothesized that CK2‐coupled SIRT1 activation stimulated by cilostazol suppresses tau acetylation (Ac‐tau) and tau phosphorylation (P‐tau) by inhibiting activation of P300 and GSK3β. Aβ was endogenously overproduced in N2a cells expressing human APP Swedish mutation (N2aSwe) by exposure to medium containing 1% fetal bovine serum for 24 hr. Increased Aβ accumulation was accompanied by increased Ac‐tau and P‐tau levels. Concomitantly, these cells showed increased P300 and GSK3β P‐Tyr216 expression; their expressions were significantly reduced by treatment with cilostazol (3–30 μM) and resveratrol (20 μM). Moreover, decreased expression of SIRT1 and its activity by Aβ were significantly reversed by cilostazol as by resveratrol. In addition, cilostazol strongly stimulated CK2α phosphorylation and its activity, and then stimulated SIRT1 phosphorylation. These effects were confirmed by using the pharmacological inhibitors KT5720 (1 μM, PKA inhibitor), TBCA (20 μM, inhibitor of CK2), and sirtinol (20 μM, SIRT1 inhibitor) as well as by SIRT1 gene silencing and overexpression techniques. In conclusion, increased cAMP‐dependent protein kinase‐linked CK2/SIRT1 expression by cilostazol can be a therapeutic strategy to suppress the tau‐related neurodegeneration in the AD brain. © 2013 Wiley Periodicals, Inc.     

Interleukin-1β-induced brain injury in the neonatal rat can be ameliorated by α-phenyl-n-tert-butyl-nitrone

To examine the possible role of inflammatory cytokines in mediating perinatal brain injury, we investigated effects of intracerebral injection of interleukin-1beta (IL-1β) on brain injury in the neonatal rat and the mechanisms involved. Intracerebral administration of IL-1β (1 μg/kg) resulted in acute brain injury, as indicated by enlargement of ventricles bilaterally, apoptotic death of oligodendrocytes (OLs) and loss of OL immunoreactivity in the neonatal rat brain. IL-1β also induced axonal and neuronal injury in the cerebral cortex as indicated by elevated expression of β-amyloid precursor protein, short beaded axons and dendrites, and loss of tyrosine hydroxylase-positive neurons in the substantia nigra and the ventral tegmental areas. Administration of α-phenyl-n-tert-butyl-nitrone (PBN, 100 mg/kg i.p.) immediately after the IL-1β injection protected the brain from IL-1β-induced injury. Protection of PBN was linked with the attenuated oxidative stress induced by IL-1β, as indicated by decreased elevation of 8-isoprostane content and by the reduced number of 4-hydroxynonenal or malondialdehyde or nitrotyrosine-positive cells following IL-1β exposure. PBN also attenuated IL-1β-stimulated inflammatory responses as indicated by the reduced activation of microglia. The finding that IL-1β induced perinatal brain injury was very similar to that induced by lipopolysaccharide (LPS), as we previously reported and that PBN was capable to attenuate the injury induced by either LPS or IL-1β suggests that IL-1β may play a critical role in mediating brain injury associated with perinatal infection/inflammation.     

The effects of α- and β-neurotoxins from the venoms of various snakes on transmission in autonomic ganglia

We have previously shown that certain commercially available lots of α-bungarotoxin block transmission in ciliary and choroid neurons of both pigeon and chicken ciliary ganglia at a concentration of 10 μg/ml (1.2 μM). The blockade is antagonized by pre-incubation with 100 μM tubocurarine.Further evidence that this blockade is produced by a postsynaptic action, as one would expect of an α-neurotoxin, are our findings that: (a) exposure to the toxin prevents the depolarization of ganglion cells normally seen in response to the cholinergic agonist, carbachol; and (b) the blocking activity of the toxin is removed by treatment with membranes purified from Torpedo electric organ containing an excess of α-neurotoxin binding sites.A high affinity binding site for [¹²⁵I]α-bungarotoxin was characterized in the chicken ciliary ganglion. However, since it is labelled equally well by lots of α-bungarotoxin which block transmission and those that do not, this site does not appear to be involved in the blockade of transmission.α-Cobratoxin (fromNaja naja siamensis), the α-neurotoxin L.s. III (fromLaticauda semifasciata) and certain lots of α-bungarotoxin produce a partial blockade of transmission in ciliary neurons of the pigeon ciliary ganglion at a concentration of 10 μg/ml (1.2 μM), but have no effect on transmission in choroid neurons. Two other α-neurotoxins fromLaticauda semifasciata, erabutoxin a and erabutoxin b, have no effect on transmission in either cell population at this concentration. None of the α-neurotoxins tested had any effect on transmission in either the rat superior cervical ganglion or the rat pelvic ganglion at concentrations up to 100 μg/ml (12 μM). Collagenase treatment of these ganglia, in an attempt to increase access of the toxins to ganglion cells, did not alter these negative results.β-Bungarotoxin (0.5 μg/ml, 0.02 μM) produces a complex blockade of transmission in both avian ciliary ganglia and rat superior cervical ganglia. Unlike the action of α-bungarotoxin, the blockade of ciliary ganglion transmission by β-bungarotoxin is irreversible and is not prevented by pretreatment with tubocurarine.     

α-Melanotropin and β-endorphin-like immunoreactivities are contained within neurons and nerve fibers of the rat duodenum

Both α-melanotropin and β-endorphin were revealed by immunofluorescence microscopy studies within neurons and nerve fibers of the rat duodenum. An immunohistochemical staining for α-melanotropin was seen within neuronal cell bodies and nerve fibers bundles of the myenteric and submucous plexus. A β-endorphin immunofluorescence was visualized within perikarya and nerve fibers of both the myenteric and submucous plexus. α-Melanotropin as well as β-endorphin immunoreactivities were strictly localized to structures of the enteric nervous system. In crypts and epithelial cells only a non-specific staining was observed.     

Hypoxia‐independent mechanisms of HIF‐1α expression in astrocytes after ischemic preconditioning

We recently demonstrated that ischemic tolerance was dependent on astrocytes, for which HIF‐1α had an essential role. The mild ischemia (preconditioning; PC) increased HIF‐1α in a biphasic pattern, that is, a quick and transient increase in neurons, followed by a slow and sustained increase in astrocytes. However, mechanisms underlying such temporal difference in HIF‐1α increase remain totally unknown. Here, we show that unlike a hypoxia‐dependent mechanism in neurons, astrocytes increase HIF‐1α via a novel hypoxia‐independent but P2X7‐dependent mechanism. Using a middle cerebral artery occlusion (MCAO) model of mice, we found that the PC (a 15‐min MCAO period)‐evoked increase in HIF‐1α in neurons was quick and transient (from 1 to 3 days after PC), but that in astrocytes was slow‐onset and long‐lasting (from 3 days to at least 2 weeks after PC). The neuronal HIF‐1α increase was dependent on inhibition of PHD2, an oxygen‐dependent HIF‐1α degrading enzyme, whereas astrocytic one was independent of PHD2. Astrocytes even do not possess this enzyme. Instead, they produced a sustained increase in P2X7 receptors, activation of which resulted in HIF‐1α increase. The hypoxia‐independent but P2X7‐receptor‐dependent mechanism could allow astrocytes to cause long‐lasting HIF‐1α expression, thereby leading to induction of ischemic tolerance efficiently. GLIA 2017;65:523–530     

Cytokines in relapsing experimental autoimmune encephalomyelitis in DA rats: persistent mRNA expression of proinflammatory cytokines and absent expression of interleukin-10 and transforming growth factor-β

Experimental autoimmune encephalomyelitis (EAE) in rats is typically a brief and monophasic disease with sparse demyelination. However, inbred DA rats develop a demyelinating, prolonged and relapsing encephalomyelitis after immunization with rat spinal cord in incomplete Freund's adjuvant. This model enables studies of mechanisms related to chronicity and demyelination, two hallmarks of multiple sclerosis (MS). Here we have investigated, in situ, the dynamics of cytokine mRNA expression in the central nervous system (CNS) and peripheral lymphoid organs (lymph node cells and splenocytes) of diseased DA rats. We demonstrate that peripheral lymphoid cells stimulated in vitro with encephalitogenic peptides 69–87 and 87–101 of myelin basic protein responded with high mRNA expression for proinflammatory cytokines; Interferon-7, interleukin-12 (IL-12), tumour necrosis factors α and β, IL-1β and cytolysin. A high expression of mRNA for these proinflammatory cytokines was also observed in the CNS where it was accompanied by classical signs of inflammation such as expression of major histocompatibility complex class I and II, CD4, CDS and IL-2 receptor. The expression of mRNA for proinflammatory cytokines was remarkably long-lasting in DA rats as compared to LEW rats which display a brief and monophasic EAE. Furthermore, mRNAs for putative immunodownmodulatory cytokines i.e. transforming growth factor-β (TGF-/3), IL-10 and IL-4 were almost absent in DA rats, in both the CNS and in vitro stimulated peripheral lymphoid cells, while their levels were elevated in the CNS of LEW rats during the recovery phase. We conclude that the MS-like prolonged and relapsing EAE in DA rats is associated with a prolonged production of proinflammatory cytokines and/or low or absent production of immunodownmodulatory cytokines.     

Identification and topography of neuronal cell populations expressing; TNFα and IL-1α in response to hippocampal lesion

In previous studies, we have shown that a traumatic lesion to the hippocampus of adult mice induces the transitory expression of TNFα and IL-1α by neurons of different brain areas and also by glial cells at the site of injury. The aim of the present study was to establish whether the expression of TNFα and IL-1α is restricted to defined subpopulations, or else is common to most of the central neuronal populations. Using polyclonal anti-GAD 67, anti-TH and monoclonal anti-ChAT, and anti-5-HT antibodies in a double-labeling immunohistochemical procedure in combination with murine anti-TNFα and anti-IL-1α polyclonal antibodies, we show that most GABAergic, catecholaminergic, and serotoninergic neurons, and a subgroup of the cholinergic neurons, express these cytokines. Although not immunohistochemically characterized, neurons in some glutamatergic structures such as the hippocampus and the prefrontal cortex also express these cytokines. Thus, we conclude that the capacity of central neurons to express cytokines like TNFα and IL-1α in reaction to a brain Injury is not restricted to peculiar neuronal subtypes, but could include most of the neuronal populations of the brain. © 1996 Wiley-Liss, Inc.     

α‐Synuclein activates innate immunity but suppresses interferon‐γ expression in murine astrocytes

Glial activation and neuroinflammation contribute to pathogenesis of neurodegenerative diseases, linked to neuron loss and dysfunction. α‐Synuclein (α‐syn), as a metabolite of neuron, can induce microglia activation to trigger innate immune response. However, whether α‐syn, as well as its mutants (A53T, A30P, and E46K), induces astrocyte activation and inflammatory response is not fully elucidated. In this study, we used A53T mutant and wild‐type α‐syns to stimulate primary astrocytes in dose‐ and time‐dependent manners (0.5, 2, 8, and 20 μg/ml for 24 hr or 3, 12, 24, and 48 hr at 2 μg/ml), and evaluated activation of several canonical inflammatory pathway components. The results showed that A53T mutant or wild‐type α‐syn significantly upregulated mRNA expression of toll‐like receptor (TLR)2, TLR3, nuclear factor‐κB and interleukin (IL)‐1β, displaying a pattern of positive dose–effect correlation or negative time–effect correlation. Such upregulation was confirmed at protein levels of TLR2 (at 20 μg/ml), TLR3 (at most doses), and IL‐1β (at 3 hr) by western blotting. Blockage of TLR2 other than TLR4 inhibited TLR3 and IL‐1β mRNA expressions. By contrast, interferon (IFN)‐γ was significantly downregulated at mRNA, protein, and protein release levels, especially at high concentrations of α‐syns or early time‐points. These findings indicate that α‐syn was a TLRs‐mediated immunogenic agent (A53T mutant stronger than wild‐type α‐syn). The stimulation patterns suggest that persistent release and accumulation of α‐syn is required for the maintenance of innate immunity activation, and IFN‐γ expression inhibition by α‐syn suggests a novel immune molecule interaction mechanism underlying pathogenesis of neurodegenerative diseases.     

Intercellular adhesion molecule-1 expression induced by interleukin (IL)-1β or an IL-1β fragment is blocked by an IL-1 receptor antagonist and a soluble IL-1 receptor

The effects of a recombinant human interleukin-1 (IL-1) receptor antagonist (IL-1ra) and a recombinant human soluble IL-1 receptor (sIL-1R) on cytokine-induced intercellular adhesion molecule-1 (ICAM-1) expression in a human glioblastoma cell line and a neuroblastoma cell line were determined. Cells were incubated with IL-1β, tumor necrosis factor (TNF)α and interferon (IFN)γ. Cells were also tested under identical conditions with an IL-1β synthetic peptide fragment (IL-1β_208–240) previously shown to possess biological activity. IL-1β, TNFα and IFNγ potentiated ICAM-1 expression in both cell lines in a dose-related manner. The IL-1β_208–240 fragments, corresponding to the rabbit, rat and human sequences, enhanced ICAM-1 expression in glioblastoma cells at high doses. ICAM-1 expression induced by IL-1β, rabbit IL-1β_208–240 and human IL-1β_208–240 was blocked by the IL-1ra, while TNFα- and IFNγ-induced ICAM-1 expression were not. ICAM-1 expression induced by IL-1β and human IL-1β_208–240 was also blocked by the sIL-1R. Our findings suggest that IL1β_208–240 acts as an IL-1β agonist in enhancing ICAM-1 expression in vitro and that this effect is receptor-mediated.