In vivo relationship of tumor necrosis factor-α to blood-brain barrier damage in patients with active multiple sclerosis

Tumor necrosis factor-alpha (TNF-alpha) has well recognized effects on cerebral endothelial cells and, therefore, may mediate disruption of blood-brain barrier in patients with multiple sclerosis (MS). To evaluate the in vivo relationship of TNF-alpha to blood-brain barrier impairment in MS, levels of this cytokine in cerebrospinal fluid (CSF) and serum samples from 38 patients with active MS and 48 controls were correlated with CSF to serum albumin ratios. TNF-alpha was detected in the serum of 74% and the CSF of 66% of patients with active MS. CSF levels of TNF-alpha were significantly higher in active MS compared to stable MS or other controls, and were significantly higher than corresponding serum levels. In patients with active MS, only those with detectable TNF-alpha showed signs of blood-brain barrier damage. Moreover, intrathecal levels of TNF-alpha in active MS correlated with albumin ratios and with the degree of barrier damage. Our findings are important in understanding some of the pathological changes in active multiple sclerosis.     

Detection of tumor necrosis factor-α-positive cells in cerebrospinal fluid of patients with HTLV-I-associated myelopathy

Tumor necrosis factor (TNF)-α-positive cells constituted 1.6–18% and 8.2–23.5% of the total number of cerebrospinal fluid cells from six of 12 patients with HTLV-I-associated myelopathy and in all samples obtained from inflammatory cases, respectively. However, in non-inflammatory cases no TNF-α-positive cells were detected. These results suggest that some of the infiltrating CSF cells produce TNF-α, which plays a role in host immune defenses against causative agents including HTLV-I and in lesion formation within the central nervous system in inflammatory diseases.     

Tumor necrosis factor modulates transcription of myelin basic protein gene through nuclear factor kappa B in a human oligodendroglioma cell line

Tumor necrosis factor-alpha (TNF-alpha) is a major mediator of inflammation and it is involved in many neurological disorders such as multiple sclerosis. Levels of TNF-alpha and lymphotoxin-alpha have been found elevated in plaques, bloods, and cerebral spinal fluids from multiple sclerosis patients. The expression of myelin basic protein (MBP), a major protein of the myelin sheath, is affected by cytokines secreted by activated immune cells. To determine the signal transduction pathway involving tumor necrosis factor's action in myelination and demyelination, we have cloned and analyzed cis-elements on promoters of the human and mouse MBP genes. There are two putative nuclear factors kappa-B (NF-kappaB) cis-elements on the human and one on the mouse gene promoter. In an electrophoretic mobility shift assay, all three NF-kappaB cis-elements showed binding to a protein, which was recognized by an antibody against NF-kappaB P65 component. The specificity of the binding was demonstrated in a competitive assay using NF-kappaB consensus oligonucleotides. A two base pair site-directed mutation on the mouse NF-kappaB cis-element abolished its binding activity. We created a DNA construct by linking the mouse MBP gene promoter containing the NF-kappaB cis-element to luciferase gene. Transfection of this construct into a human oligodendroglioma cell line showed TNF-alpha increased the transgene expression. Furthermore the mutation of NF-kappaB site abolished TNF-alpha -induction of the transgene. The data demonstrate that NF-kappaB is the mediator between tumor necrosis factor's action and MBP gene expression. Elucidating the molecular mechanisms underlying TNF-alpha regulation of MBP gene expression provides new scientific bases for the development of therapy against oligodendrocyte-specific and myelin-related disorders such as multiple sclerosis.     

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.     

Cytokine modulation of murine microglial cell superoxide production

Activated microglia may contribute to two opposite effects during inflammation within the central nervous system: host defense against microorganisms and neuronal injury. Each of these processes may be mediated by the generation of reactive oxygen intermediates by activated microglia. We investigated the effects of two proinflammatory cytokines, interferon (IFN)-β and tumor necrosis factor (TNF)-α, and of the anti-inflammatory cytokine, transforming growth factor (TGF)-β, on murine microglial cell superoxide (O₂) production upon stimulation with phorbol myristate acetate (PMA). Priming of microglia with IFN-β or TNF-α resulted in a dose-dependent enhancement of O₂ release in response to PMA. The priming effects of these two cytokines were additive, suggesting that they acted by independent mechanisms. We also found that IFN-β and TNF-β stimulated the release of bioactive TGF-β and that treatment of microglial cell cultures with TGF-β antagonized the priming effects of IFN-β and TNF-α on O₂ production. The results of this study have implications for understanding the mechanisms by which cytokines and microglia may contribute to host defense as well as to injury of the brain. © 1995 Wiley-Liss, Inc.     

High innate production of interleukin-10 and tumor necrosis factor-alpha contributes to susceptibility for non-paraneoplastic Lambert-Eaton myasthenic syndrome

Non-paraneoplastic Lambert-Eaton myasthenic syndrome (LEMS) is an antibody-mediated autoimmune disorder, in which genetically determined interleukin-10 (Il-10) and tumor necrosis factor-alpha (TNF-alpha) could play a role in the susceptibility for the disease. Therefore, we analyzed the production of Il-10 and TNF-alpha after whole-blood stimulation in first-degree family members of patients with LEMS without malignancy, as a measure of innate production in the patients. Thirty-six first-degree family members of 10 patients and 80 healthy controls were studied. Both Il-10 (p=0.037) and TNF-alpha (p=0.0016) production were increased in the family members, but had no relation with the severity of LEMS or HLA-B8DR3 carriership. Our findings suggest that high innate production of Il-10 and TNF-alpha is a susceptibility factor for non-paraneoplastic LEMS.     

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.     

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.     

Cooperative regulation of nerve growth factor synthesis and secretion in fibroblasts and astrocytes by fibroblast growth factor and other cytokines

Acidic fibroblast growth factor (aFGF) enhances nerve growth factor (NGF) synthesis by astrocytes obtained from various brain regions. NGF secretion by fibrous-shaped astrocytes transformed by dibutyryl-cAMP (db-cAMP) pretreatment was less than that by untreated astrocytes. However, aFGF also enhanced NGF secretion by fibrous-shaped astrocytes. The effects of various kinds of intracellular signaling modulators on NGF synthesis were examined. None of the following second messenger effectors had an effect on NGF synthesis: protein kinase C (PKC) agonist (phorbol myristate acetate (PMA)) or antagonist (sphingosine (SP)). LiCl, and ionomycin (Iono). Further, increases of intracellular cAMP by forskolin (FK) or db-cAMP have no significant effect on NGF synthesis in astrocytes under a standard culture condition. However, NGF synthesis by astrocytes in the presence of aFGF was significantly enhanced by db-cAMP, but not by FK or sodium butyrate. These results indicate that an excessive amount of cAMP enhances the effect of aFGF on NGF synthesis in astrocytes. NGF synthesis in astrocytes was not affected by treatment with anti-aFGF or anti-bFGF neutralizing antibodies, indicating that FGFs are not involved in the autocrine regulation of NGF synthesis in astrocytes. Transforming growth factor-beta 1 (TGF-beta 1), which inhibits some effects of FGFs, increased NGF synthesis in concert with aFGF. Furthermore, the highest NGF synthesis was observed when astrocytes were stimulated by all of the following cytokines: aFGF, interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha) and TGF-beta 1. The mechanism regulating NGF synthesis in fibroblasts obtained from prenatal rat skin was also investigated. Acidic FGF, basic FGF (bFGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factor-alpha (TGF-alpha), TGF-beta 1, IL-1 beta, and TNF-alpha were found to be regulators of NGF synthesis in skin fibroblasts. Among these cytokines, aFGF is the most potent regulator of NGF synthesis in fibroblasts. NGF synthesis by skin fibroblasts, either in the presence or absence of aFGF, was not modified by any of the following: FK, PMA, SP, LiCl, and Iono. However, db-cAMP significantly enhanced NGF synthesis in both conditions. Sodium butyrate enhanced NGF synthesis in the presence of aFGF, but not in the absence of aFGF. These results suggest that an excessive amount of cAMP and butyrate moiety regulate NGF synthesis in skin fibroblasts in different ways.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tumor necrosis factor-α in immune-mediated demyelination and Wallerian degeneration of the rat peripheral nervous system

This study reports on the immunocytochemical localization of tumor necrosis factor-alpha (TNFα) in immune-mediated demyelination and Wallerian degeneration of the rat peripheral nervous system (PNS) using teased nerve fiber preparations. In experimental autoimmune neuritis induced by active immunization (EAN) or by adoptive transfer of autoreactive T cells (AT-EAN), macrophages passing blood vessels as well as macrophages adherent to nerve fibers were TNFα-positive. Large post-phagocytic macrophages at later stages of demyelination were TNFα-negative. Intraperitoneal application of an anti-TNFα antibody to EAN rats significantly reduced the degree of inflammatory demyelination, suggesting a pathogenic role for TNFα. After nerve transection only macrophages located within degenerating nerve fibers were TNFα-positive, while those entering and leaving nerves were negative. TNFα produced by macrophages seems to bevolved in immune-mediated demyelination and non-immune myelin degradation after axotomy. While interferon-gamma (IFNγ) is present in EAN nerves and may act as a local stimulus for TNF expression, the nature of this signal in Wallerian degeneration in the absence of IFNγ is unknown.     

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.     

Myelin-induced experimental allergic encephalomyelitis in Lewis rats: tumor necrosis factor α levels in serum and cerebrospinal fluid Immunohistochemical expression in glial cells and macrophages of optic nerve and spinal cord

Tumor necrosis factor a (TNFa) activity was measured in serum and cerebrospinal fluid (CSF) of Lewis rats after experimental allergic encephalomyelitis (EAE) induction and during the clinical course of acute disease. TNFa bioactivity expression preceded the clinical symptoms and paralleled the severity of disease. We further investigated the identity of the central nervous system (CNS) cells involved in TNFa expression and their regional localization during EAE. Tissue sections of brain, cerebellum, dorsal spinal cord and optic nerve were studied by indirect double labelling immunofluorescence. Spinal cord white matter and optic nerve showed a widespread TNFa immunoreactivity at critical stages of EAE in macrophages/microglia and astrocytes. We have shown changes in CSF/serum albumin ratio and immunoglobulin G index during EAE. Our results confirm the very important role of TNFa in EAE.     

Tumor necrosis factor-α and X-linked adrenoleukodystrophy

The two most common forms of X-linked adrenoleukodystrophy (X-ALD), the childhood cerebral form (CCER) and the adult form, adrenomyeloneuropathy (AMN), arise from the same mutations in the X-ALD gene at Xq28. These two forms are distinguished by the degree of cerebral inflammation. Segregation analysis suggests that an autosomal modifying gene may be a major determinant of phenotype in X-ALD. Thus, a modifying gene could be involved in initiating or promoting the inflammatory response. In this study we detected a difference in tumor necrosis factor-α (TNF-α) bioactivity, but not TNF-α protein levels, in serum from some advanced CCER patients. Early-stage CCER patients and AMN patients were in the normal range. Allelic differences in TNF-α or levels of soluble TNF receptor did not account for bioactivity differences or phenotypic heterogeneity in X-ALD.     

Microglial cell upregulation of HIV-1 expression in the chronically infected promonocytic cell line U1: the role of tumor necrosis factor-α

Culture supernatants from lipopolysaccharide (LPS)-treated murine microglial cells were found to markedly induce the expression of human immunodeficiency virus (HIV)-1 in the chronically infected human promonocytic cell line U1 as detected by measurements of HIV-1 p24 antigen release into U1 culture supernatants. Antibody to tumor necrosis factor (TNF)-α had an inhibitory effect on the induction of virus by microglial cell supernatants. Also, treatment of microglia with pentoxifylline, an inhibitor of TNF-α production, resulted in suppressed amounts of TNF in the supernatants of LPS-treated microglia and in a reduced stimulatory capacity of these supernatants on HIV-1 expression in U1 cells. These findings support the concept that TNF-α production by glial cells plays a pathogenetic role in HIV-1-associated brain disease by promoting the expression of the virus in infected cells.     

Tumor necrosis factor and interleukin-1β: suppression of food intake by direct action in the central nervous system

Intracerebroventricular microinfusion of recombinant human tumor necrosis factor (rhTNF) and recombinant human interleukin-1β (rhIL-1β) suppressed food intake in rats. Central infusion of heat-inactivated rhTNF and rhIL-1β, bovine serum albumin, heparin or transforming growth factor-β had no such effect. Central infusion of rhIL-1β did not affect the dipsogenic response to central administration of angiotensin II. Peripheral administration of rhTNF and rhIL-1β in doses equivalent to or higher than those administered centrally had no effect. Electrophoretically applied rhTNF and rhIL-1β specifically suppressed the activity of glucose-sensitive neurons in the lateral hypothalamic area. Glucose-insensitive neurons were little affected. The results suggest that TNF and IL-1β act directly in the central nervous system to suppress feeding, and this effect may be operative during acute and chronic disease.     

gb-adrenergic receptor regulation of macrophage-derived tumor necrosis factor-α production from rats with experimental arthritis

Prostaglandin E₂ (PGE₂) and β-adrenergic agonists can suppress lipopolysaccharide-induced tumor necrosis factor-α (TNF) production from elicited macrophages. We assessed the responsiveness of rat peritoneal macrophages to PGE₂ and the β-adrenergic agonist isoproterenol during immunologically-mediated arthritis. We assessed macrophage sensitivity to these mediators from resident macrophages and macrophages elicited with either streptococcal cell wall or complete Freund's adjuvant. Peritoneal macrophages were obtained from female Lewis rats that were (1) injected with complete Freund's adjuvant and non-arthritic (CFA); (2) injected with streptococcal cell wall and arthritic (ART); (3) injected with streptococcal cell wall and non-reactive (NON) and (4) non-elicited resident macrophages (RES). When challenged with graded concentrations of lipopolysaccharide (0.1 to 10,000 ng/ml), macrophages obtained from each group of rats released TNF in a concentration-dependent manner, with macrophages from arthritic rats (ART) producing the greatest amount of TNF (p < 0.001). While PGE₂ suppressed lipopolysaccharide (100 ng/ml) stimulated TNF production in a concentration-dependent manner in all groups, the greatest sensitivity to PGE₂ was observed with macrophages obtained from rats which received streptococcal cell wall when compared to both complete Freund's adjuvant-elicited and resident macrophages (p < 0.05). The β-adrenergic agonist isoproterenol also inhibited lipopolysaccharide-stimulated TNF production from macrophages in all groups. In addition, the specific β₂-adrenergic antagonist, ICI 118.551, shifted isoproterenol concentration-effect curves to the right (p < 0.01). Minimal responsiveness to isoproterenol was observed with resident peritoneal macrophages. Maximum isoproterenol-induced inhibition of TNF production was observed with complete Freund's adjuvant-elicited macrophages, and significantly less in macrophages of streptococcal cell wall-injected rats. Of particular interest, macrophages obtained from streptococcal cell wall-injected rats, which became arthritic, were significantly less sensitive to isoproterenol than those which did not develop arthritis (p < 0.02). In addition, these changes in sensitivity were not reflected by changes in the sensitivity of both CFA and ART groups to dibutyryl cAMP. The present study demonstrates a shift in the balance between inhibitory mediator responses in rats inoculated with one of two different adjuvants. These investigations support the role of PGE₂ and a neurotransmitter as immunomodulating compounds which may effectively maintain an inflammatory lesion such as arthritis.     

Brain-IL-1β induces local inflammation but systemic anti-inflammatory response through stimulation of both hypothalamic–pituitary–adrenal axis and sympathetic nervous system

It is well established that systemic inflammation induces a counter-regulatory anti-inflammatory response particularly resulting in deactivation of monocytes/macrophages. However, recently we demonstrated a systemic anti-inflammatory response without preceding signs of systemic inflammation in patients with brain injury/surgery and release of cytokines into the cerebrospinal fluid (CSF). In order to analyze the mechanisms and pathways of systemic immunodepression resulting from sterile cerebral inflammation we established an animal model using continuous intra-cerebroventricular (i.c.v.) or intra-hypothalamic (i.h.) infusion of rat recombinant (rr) tumor necrosis factor (TNF)-α and interleukin (IL)-1β for 48 h. Controls received intra-venous (i.v.) cytokine administration. Interestingly, i.c.v. and i.h. infusion of IL-1β but not TNF-α produced distinct signs of central nervous system (CNS) inflammation. Correspondingly, i.c.v. infusion of IL-1β particularly diminished the TNF-α but increased the IL-10 concentration in whole blood cultures after endotoxin stimulation. All parameters normalized within 48 h after termination of the infusion. Blocking the hypothalamic–pituitary–adrenal (HPA) axis by hypophysectomy (HPX) led to complete recovery of the diminished TNF-α concentration and temporarily inhibited the IL-10 increase. Blocking the sympathetic nervous system (SNS) transmission by application of the β₂-adrenoreceptor antagonist propranolol not only inhibited the increase but further downregulated the endotoxin induced IL-10 concentration in the media of whole blood cell cultures, whereas the TNF-α decrease was only partially prevented. Interestingly, HPX and propranolol also diminished the cell invasion into the CSF. In summary, activation of both the HPA axis and the SNS plays an important role in systemic anti-inflammatory response resulting from cytokines in brain and cerebral inflammation.     

Functional characterization of substance P receptors on cultured human spinal cord astrocytes: Synergism of substance P with cytokines in inducing interleukin-6 and prostaglandin E2 production

Following brain injury, astrocytes express receptors for cytokines and neuropeptides and secrete several regulatory mediators that have a well established role in inflammation, immunity, and tissue development or repair. To elucidate the role of substance P (SP), a neurotransmitter peptide of the tachykinin family, in inducing astrocyte secretory activities, we have examined the expression of SP receptors and the functional consequences of their activation in cultured astrocytes from the human embryonic brain or spinal cord. Radioligand binding studies revealed that only one type of SP receptors, the high affinity NK-1 receptor, was present on human astrocytes and that spinal cord astrocytes expressed about 6 times as many SP binding sites as brain astrocytes. Following SP treatment, a substantial inositol phosphate formation was observed in spinal cord astrocytes only. Stimulation of spinal cord astrocytes with SP alone did not induce secretion of cytokines [interleukin-6 (IL-6), granulocyte-macrophage-CSF, macrophage chemoattractant protein-1 or leukemia inhibitory factor] or prostaglandin E₂ (PGE₂). Interestingly, however, SP selectively potentiated the inducing effect of IL-1β on IL-6 and PGE₂ secretion by spinal cord astrocytes without affecting the IL-1-β-evoked secretion of other cytokines. SP also enhanced the small inducing effect of tumor necrosis factor-α (TNF-α) on IL-6 and PGE₂ secretion and that of transforming growth factor-β on PGE₂ secretion. These results suggest that SP can enhance immunoregulatory and neurotrophic astroglial functions mediated by IL-6 and PGE₂ by acting in concert with a set of cytokines whose cerebral expression has been reported during development and in a variety of diseases. GLIA 21:183–193, 1997. © 1997 Wiley-Liss, Inc.     

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.     

An anti-tumor necrosis factor antibody suppresses sleep in rats and rabbits

It is hypothesized that tumor necrosis factor (TNF) is an endogenous sleep-promoting substance. In the present experiments we studied the effects of a monoclonal anti-TNF antibody in rats and rabbits. Seven rats and 14 rabbits were implanted with electroencephalographic electrodes, a brain thermistor and an intracerebroventricular guide cannula. The animals were injected with saline, control IgG, and monoclonal hamster anti-murine-TNF antibodies (TNFab) on 3 separate days. Ten μg TNFab suppressed non-rapid-eye-movement sleep (NREMS) in rats. In rabbits, 2.5 μg TNFab did not affect sleep but decreased brain temperature; in contrast, 25 μg TNFab suppressed NREMS without affecting brain temperature. These results are consistent with the hypothesis that endogenous TNF plays an important role in sleep regulation.