TNF and its receptors in the CNS: The essential, the desirable and the deleterious effects.

Tumor necrosis factor (TNF) is the prototypic pro-inflammatory cytokine. It is central to host defense and inflammatory responses but under certain circumstances also triggers cell death and tissue degeneration. Its pleiotropic effects often lead to opposing outcomes during the development of immune-mediated diseases, particularly those affecting the central nervous system (CNS). The reported contradictions may result from lack of precision in discussing TNF. TNF signaling comprises at minimum a two-ligand (soluble and transmembrane TNF) and two-receptor (TNFR1 and TNFR2) system, with ligands and receptors both differentially expressed and regulated on different cell types. The "functional multiplicity" this engenders is the focus of much research, but there is still no general consensus on functional outcomes of TNF signaling in general, let alone in the CNS. In this review, evidence showing the effects of TNF in the CNS under physiological and pathophysiological conditions is placed in the context of major advances in understanding of the cellular and molecular mechanisms that govern TNF function in general. Thus the roles of TNF signaling in the CNS shift from the conventional dichotomy of beneficial and deleterious, that mainly explain effects under pathological conditions, to incorporate a growing number of "essential" and "desirable" roles for TNF and its main cellular source in the CNS, microglia, under physiological conditions including regulation of neuronal activity and maintenance of myelin. An improved holistic view of TNF function in the CNS might better reconcile the expansive experimental data with stark clinical evidence that reduced functioning of TNF and its dominant pro-inflammatory receptor, TNFR1, are risk factors for the development of multiple sclerosis. It will also facilitate the safe translation of basic research findings from animal models to humans and propel the development of more selective anti-TNF therapies aimed at selectively inhibiting deleterious effects of this cytokine while maintaining its essential and desirable ones, in the periphery and the CNS.

Mesenchymal stem cells protect CNS neurons against glutamate excitotoxicity by inhibiting glutamate receptor expression and function.

Mesenchymal stem cells (MSC) promote functional recovery in experimental models of central nervous system (CNS) pathology and are currently being tested in clinical trials for stroke, multiple sclerosis and CNS injury. Their beneficial effects are attributed to the activation of endogenous CNS protection and repair processes as well as immune regulation but their mechanisms of action are poorly understood. Here we investigated the neuroprotective effects of mouse MSC in rodent MSC-neuron co-cultures and mice using models of glutamate excitotoxicity. A 24h pre-culture of mouse primary cortical neurons with MSC protected them against glutamate (NMDA) receptor-induced death and conditioned medium from MSC (MSC CM) was sufficient for this effect. Protection by MSC CM was associated with reduced mRNA levels of genes encoding NMDA receptor subunits, and increased levels for genes associated with non-neuronal and stem cell types, as shown by RT-PCR and cDNA microarray analyses. Changes in gene expression were not associated with alterations in cell lineage representation within the cultures. Further, MSC CM-mediated neuroprotection in rat retinal ganglion cells was associated with reduced glutamate-induced calcium influx. The adoptive transfer of EGFP(+)MSC in a mouse kainic acid epilepsy model also provided neuroprotection against glutamate excitotoxicity in vivo, as shown by reduced neuron damage and glial cell activation in the hippocampus. These results show that MSC mediate direct neuroprotection by reducing neuronal sensitivity to glutamate receptor ligands and altering gene expression, and suggest a link between the therapeutic effects of MSC and the activation of cell plasticity in the damaged CNS.

Positive and negative implications of tumor necrosis factor neutralization for the pathogenesis of multiple sclerosis.

Multiple sclerosis (MS) is a progressive, presumably autoimmune, degenerative disease of the central nervous system (CNS). The mechanisms which trigger the disease are unknown, but the pathology of MS is caused by the host's own immune system, which invades the CNS and attacks the myelin sheath that protects and insulates the axons of the nerve cells. Although this inflammatory assault selectively destroys myelin, it is believed that the neurological deficits of MS are rather the consequence of damage to axons, which occurs secondary to inflammation. The inflammatory mediators are generally secreted by myelin-specific, CD4+ T cells, CD8+ T cells, macrophages and activated glial cells and include a large number of cytokines, chemokines and other proinflammatory proteins.

Glial expression of tumor necrosis factor in transgenic animals: how do these models reflect the "normal situation"?

Recent progress in the field of experimental genetics, which enables the selective and conditional ablation or dysregulation in the expression of specific genes in mice, and its application to the study of experimentally inducible models for human disease, have contributed enormously to our understanding of the molecules and mechanisms that underlie autoimmunity and inflammation in the CNS. This article describes the lessons learned from the application of such technology to the study of the tumor necrosis factor-alpha (TNF) ligand/receptor system in the CNS. Important roles for TNF and its two membrane-bound receptors in the initiation and support of CNS inflammation, the development of CNS autoimmunity, and possibly in the resolution of T-cell-mediated disease, as well as their implications for our understanding of the "normal" cellular and molecular mechanisms that underlie CNS pathology, are discussed.

Treatment of experimental allergic encephalomyelitis (EAE) induced by guinea pig myelin basic protein epitope 72-85 with a human MBP(87-99) analogue and effects of cyclic peptides.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory and demyelinating disease of the central nervous system and is an animal model of multiple sclerosis (MS). In the present report, a linear analogue and a series of cyclic semi-mimetic peptides were designed and synthesized based on the human myelin basic protein (MBP(87-99)) epitope (Val87-His-Phe-Phe-Lys-Asn-Ile-Val-Thr-Pro-Arg-Thr-Pro90) and on Copolymer I (a mixture of random polymers of Ala, Gln, Lys and Tyr used to treat MS). These analogues were designed looking for suppressors of EAE induced by guinea pig MBP(72-85) epitope (Gln-Lys-Ser-Gln-Arg-Ser-Gln-Asp-Glu-Asn-Pro-Val) in Lewis rats. The linear analogue [Arg91,Ala96]MBP(87-99), in which Arg substitutes Lys91 and Ala substitutes Pro96, was found to be a strong inhibitor which when administered to Lewis rats together with the encephalitogenic agonist MBP(72-85) completely prevented the induction of EAE. In contrast, three N- and C-termini amide-linked cyclic semi-mimetic peptides, [cyclo-Phe-Arg-Asn-Ile-Val-Thr-Ala-Acp (1), cyclo-Phe-Ala-Arg-Gln-Acp (2), cyclo-Tyr-Ala-Lys-Gln-Acp (3)] as well as a Lys side chain and C-terminous cyclic semi mimetic peptide cyclo(Lys, Acp)-Phe-Lys-Asn-Ile-Val-Thr-Ala-Acp (4) which contain segments of MBP(87-99) or are constituted from immunophoric residues of copolymer 1, were ineffective in inducing or inhibiting EAE in Lewis rats. However co-injection of cyclic analogues with MBP(72-85) delayed the onset of EAE indicating a modulatory effect on the EAE activity of MBP(72-85). These findings suggest that molecule length, size of cyclic moiety and backbone conformation are important elements for immunogenic activity. Moreover blockade of MBP(72-85) induced EAE by the unrelated peptide [Arg91,Ala56]MBP(87-99) could indicate that the mechanism of inhibition is not due to binding competition but rather due to the delivery of a negative signal by the antagonist which overcomes the agonist response possibly through the activation of antigen specific regulatory T cells.

TNFR1 signalling is critical for the development of demyelination and the limitation of T-cell responses during immune-mediated CNS disease.

In this review we summarize the essential findings about the function of tumour necrosis factor (TNF) and its cognate receptors TNFR1 and TNFR2, and lymphotoxin alpha (LT-alpha) ligands in immune-mediated CNS inflammation and demyelination. The advent of homologous recombination technology in rodents provides a new method which has been used during the last 5 years and has led to insights into the pathophysiology of experimental autoimmune encephalomyelitis (EAE) in an unprecedented way. Studies with knockout mice in which genes of the TNF ligand/receptor superfamily are not expressed and studies with transgenic mice overexpressing TNF and TNFR reveal the critical role of the TNFR1 signalling pathway in the control of CNS demyelination and inflammation. These studies provide novel findings and at the same time shed light on the complex pathophysiology of EAE. Together, these findings may contribute to better understanding of EAE and open new avenues in experimental therapies for multiple sclerosis.

Learning performances, brain NGF distribution and NPY levels in transgenic mice expressing TNF-alpha.

Tumor necrosis factor-alpha (TNF-alpha) is a cytokine involved in a variety of neurobiological activities including changing behavior and regulation of both neurotrophin and neuropeptide levels. In this study we used two lines of transgenic mice overexpressing brain TNF-alpha characterized by neurological deficits (line Tg6074) or phenotypically normal (line TgK3). We analyzed whether or not impairments in learning and memory processes due to TNF-alpha overexpression were associated with changes in endogenous brain NGF, NPY and beta-amyloid. The results indicate that full TNF-alpha transgene expression disrupted the learning capabilities of transgenic mice (both Tg6074 and TgK3). NGF decreased in the hippocampus of both transgenic mice whereas hippocampal NPY slightly potentiated in Tg6074. The decrease in NGF is correlated with deficits in spatial learning and memory whereas inflammation in the brain of Tg6074 could be responsible of the hippocampal increase in NPY. As a whole, these results show that transgenic mice overexpressing TNF-alpha in the brain represent a useful model for studying neuronal degeneration and brain inflammatory processes.

Treatment of experimental allergic encephalomyelitis (EAE) by a rationally designed cyclic analogue of myelin basic protein (MBP) epitope 72-85.

In this report the rational design, synthesis and pharmacological properties of an amide-linked cyclic antagonist analogue of the guinea pig myelin basic protein epitope MBP(72-85) are described. Design of the potent cyclic analogue was based on 2D NOESY nuclear magnetic resonance and molecular dynamics studies carried out in the linear antagonist Ala81MBP(72-85). The cyclic antagonist completely prevented the induction of experimental allergic/autoimmune encephalomyelitis when coinjected with linear and cyclic agonist analogues MBP(72-85) and cyclo(2-9)MBP(72-85).

Learning abilities, NGF and BDNF brain levels in two lines of TNF-alpha transgenic mice, one characterized by neurological disorders, the other phenotypically normal.

In this study we used two lines of transgenic mice overexpressing tumor necrosis factor alpha (TNF-alpha) in the central nervous system (CNS), one characterized by reactive gliosis, inflammatory demyelination and neurological deficits (Tg6074) the other showing no neurological or phenotypical alterations (TgK3) to investigate the effect of TNF-alpha on brain nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) levels and learning abilities. The results showed that the amount of NGF in the brain of Tg6074 and TgK3 transgenic mice is low in the hippocampus and in the spinal cord, increases in the hypothalamus of Tg6074 and showed no significant changes in the cortex. BDNF levels were low in the hippocampus and spinal cord of TgK3. BDNF increased in the hypothalamus of TgK3 and Tg6074 while in the cortex, BDNF increased only in Tg6074 mice. Transgenic mice also had memory impairments as revealed by the Morris Water Maze test. These findings indicate that TNF-alpha significantly influences BDNF and NGF synthesis, most probably in a dose-dependent manner. Learning abilities were also differently affected by overexpression of TNF-alpha, but were not associated with inflammatory activity. The possible functional implications of our findings are discussed.

Effects of hTNFalpha expression in T cells on haematopoiesis in transgenic mice.

A transgenic line of mice carrying one copy of the hTNFalpha gene under the control of its own promoter and the CD2 locus control region has been analysed for the effects of TNFalpha on haematopoiesis. A low level constitutive expression of hTNFalpha in lymphoid tissue was observed. Human TNFalpha binds to and activates the murine p55 receptor, but not the p75 receptor. This implies that the observed effects of hTNFalpha in mice were mediated only through the p55 receptor. Various lymphoid tissues were depleted of lymphocytes, especially thymus, spleen and peripheral blood. Effects on thymus development were detected already at 3 wk of age, more general effects on haematopoiesis were evident by 5 wk: a drop in total blood leukocytes, mainly due to a 67% decline in lymphocytes. At 16 wk the mice had developed anaemia, whereas platelets, neutrophils and monocytes had increased. The fall in lymphocytes was due to lowered levels of T cells as well as B cells. The cause of the shortened lifespan of the transgenic mice was probably not the haematological effects of hTNFalpha directly. Absence of trophic factors supplied by the normal T cell population remains possible.

Protein glycosylation and advanced glycosylated endproducts (AGEs) accumulation: an avian solution?

The objectives of this study were to determine the effect of diet restriction (DR) and the crosslinking inhibitor, aminoguanidine (AG), on PMA-induced respiratory burst, concentrations of uric acid, and the rate of pentosidine accumulation in the skin (Ps) of naturally hyperglycemic broiler breeder hens. Female chicks (n = 450) were randomly assigned to four groups from 8 to 92 weeks after hatch: ad libitum (AL), diet restricted (DR), AL and DR groups supplemented with 400 ppm AG each (AL + AG and DR + AG). No consistent effects of treatments were observed on plasma concentrations of glucose. The accumulation of Ps in AL birds increased linearly with age (p < .001) and was significantly retarded in all treatment groups (p < .001). Ps in the AL + AG group was comparable to that in the DR or DR + AG groups. PMA-induced respiratory bursts in blood leukocytes were significantly retarded in DR or AG-supplemented (p < .0001) groups. Although there was a marginal increase in overall mean concentrations of plasma uric acid for the DR group, no consistent differences were observed on individual time points. It is concluded that the glycosylation process may not be the primary cause of glucose-derived crosslinks and that the accumulation of Ps can be retarded by DR and AG in broiler breeder hens.

Design and synthesis of a potent cyclic analogue of the myelin basic protein epitope MBP72-85: importance of the Ala81 carboxyl group and of a cyclic conformation for induction of experimental allergic encephalomyelitis.

Experimental allergic encephalomyelitis (EAE) is induced in susceptible animals by immunodominant determinants of myelin basic protein (MBP), such as guinea pig sequence MBP72-85. Two linear and one cyclic analogues based on MBP72-85 have been synthesized and evaluated for EAE induction in Lewis rats. The linear peptide Gln1-Lys2-Ser3-Gln4-Arg5-Ser6-Gln7-+ ++Asp8-Glu9-Asn10-Pro11-Val12 (1) was found to induce EAE, while substitution of the Asp residue at position 8 with Ala resulted in an analogue (2) which suppressed the induction of EAE by its parent peptide. Nuclear magnetic resonance studies of analogue 1 in dimethyl sulfoxide (DMSO) using TOCSY/ROESY techniques revealed a head-to-tail intramolecular interaction (ROE connectivity between betaVal12-gammaGln1), indicating a pseudocyclic conformation for the immunogenic peptide 1. A conformational model was developed using NMR constraints and molecular dynamics. Based on this model, a novel amide-linked cyclic analogue has been designed and synthesized by connecting the side-chain amino and carboxyl groups of Lys and Glu at positions 2 and 9, respectively, of linear analogue 1. The cyclic analogue (3) had similar activity to the linear peptide 1, and the EAE effects induced by cyclic analogue 3 were completely suppressed by co-injection with the Ala81-substituted analogue 2 in Lewis rats. The similar potencies of analogues 1 and 3 support the proposed cyclic comformation suggested for analogue 1 from NMR studies and computer modeling and provides the basis for designing more potent molecules with improved properties such as increased resistance to degradation.15 The present findings suggest that a cyclic conformation for the MBP72-85 epitope positions the carboxyl group of Asp81 correctly and presumably other side groups of the peptide such as Arg78 in a manner which enables functional binding of the trimolecular complex MHC-peptide-T cell receptor resulting in EAE.

TNF accelerates the onset but does not alter the incidence and severity of myelin basic protein-induced experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) induction in TNF gene-targeted mice has resulted in conflicting reports in part due to the strong association of TNF with the MHC locus. To define the participation of TNF in EAE development, we back-crossed TNF-deficient mice (H-2b) into the SJL/J strain and directly compared them to H-2b congenic SJL or inbred SJL/J mice. Induction of EAE with myelin basic protein (MBP) revealed that H-2b congenic SJL mice are fully susceptible, indicating that the H-2b haplotype does not affect disease susceptibility. Using H-2b congenic SJL mice we show here that TNF deficiency modifies the normal course of EAE by considerably delaying the onset for approximately 5 days, suggesting that TNF is required for the normal initiation of MBP-induced EAE. However, TNF-deficient mice eventually developed severe EAE with perivascular inflammation and primary demyelination similar to wild-type controls, indicating that TNF is not essential during these processes. Taken together, these results indicate that although TNF is not required for the progression of MBP-induced EAE, it contributes positively by advancing the onset of disease.

A tumor necrosis factor-induced model of human primary demyelinating diseases develops in immunodeficient mice.

We have reported previously that in the central nervous system (CNS) local expression of tumor necrosis factor (TNF) transgenes can trigger the development of oligodendrocyte apoptosis, primary inflammatory demyelination and neurological dysfunction, accompanied by lymphocyte and macrophage infiltration into the CNS. To distinguish between the local effects of transgene-encoded TNF and the potential encephalitogenic effects of immune infiltrates upon CNS disease pathogenesis, we have backcrossed Tg6074 TNF-transgenic mice to mice deficient in CD4, beta2-microglobulin (beta2m), immunoglobulin mu chain (Igmu) or recombination activation gene-1 (Rag-1). TNF was capable of triggering undiminished primary demyelination in all of the immunodeficient mice, in the presence of activated cells of the macrophage/microglial lineage. We conclude that TNF is sufficient to induce primary inflammatory demyelination and neurological deficits even in the absence of adaptive immunity.

Overexpression of tumour necrosis factor alpha in the brain of transgenic mice differentially alters nerve growth factor levels and choline acetyltransferase activity.

Tumour necrosis factor alpha (TNF-alpha) is a pleiotrophic cytokine synthesized primarily by macrophages and monocytes, which exerts a variety of biological activities during inflammatory responses, immune reactions, and wound healing. Within the central nervous system (CNS), the basal levels of TNF-alpha are almost undetectable, but increase after neurological insults. Using transgenic mice expressing high levels of TNF-alpha in the CNS, we investigated the effect of this cytokine on the levels of brain nerve growth factor (NGF), a neurotrophin playing a crucial role in the development, maintenance and regeneration of basal forebrain cholinergic neurons. The immunoenzymatic assay and in situ hybridization revealed that the constitutive expression of NGF decreased in the hippocampus, increased in the hypothalamus, while remained unchanged in the cortex. Moreover, septal cholinergic neurons which receive trophic support from NGF produced in the hippocampus display loss of choline acetyltransferase immunoreactivity, suggesting that the reduced availability of NGF may influence negatively the synthesis of brain cholinergic neurons. These observations indicate that the basal level of brain NGF can be influenced negatively or positively by local expression of TNF-alpha and that this cytokine, through dose-dependent regulation of NGF synthesis and release, may be involved in neurodegenerative events associated with aging.

Oligodendrocyte apoptosis and primary demyelination induced by local TNF/p55TNF receptor signaling in the central nervous system of transgenic mice: models for multiple sclerosis with primary oligodendrogliopathy.

The scientific dogma that multiple sclerosis (MS) is a disease caused by a single pathogenic mechanism has been challenged recently by the heterogeneity observed in MS lesions and the realization that not all patterns of demyelination can be modeled by autoimmune-triggered mechanisms. To evaluate the contribution of local tumor necrosis factor (TNF) ligand/receptor signaling pathways to MS immunopathogenesis we have analyzed disease pathology in central nervous system-expressing TNF transgenic mice, with or without p55 or p75TNF receptors, using combined in situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling and cell identification techniques. We demonstrate that local production of TNF by central nervous system glia potently and selectively induces oligodendrocyte apoptosis and myelin vacuolation in the context of an intact blood-brain barrier and absence of immune cell infiltration into the central nervous system parenchyma. Interestingly, primary demyelination then develops in a classical manner in the presence of large numbers of recruited phagocytic macrophages, possibly the result of concomitant pro-inflammatory effects of TNF in the central nervous system, and lesions progress into acute or chronic MS-type plaques with axonal damage, focal blood-brain barrier disruption, and considerable oligodendrocyte loss. Both the cytotoxic and inflammatory effects of TNF were abrogated in mice genetically deficient for the p55TNF receptor demonstrating a dominant role for p55TNF receptor-signaling pathways in TNF-mediated pathology. These results demonstrate that aberrant local TNF/p55TNF receptor signaling in the central nervous system can have a potentially major role in the aetiopathogenesis of MS demyelination, particularly in MS subtypes in which oligodendrocyte death is a primary pathological feature, and provide new models for studying the basic mechanisms underlying oligodendrocyte and myelin loss.

Exploratory and displacement behavior in transgenic mice expressing high levels of brain TNF-alpha.

Studies reported recently have shown that tumor necrosis factor-alpha (TNF-alpha) a cytokine released by macrophages and monocytes plays a key role in inflammatory processes and immune and neuro-endocrine regulation. TNF-alpha is also produced in the central nervous system (CNS). However, the role of this cytokine in the CNS is largely unknown, although evidence indicates that it is involved in various neurobehavioral manifestations. Using transgenic mice expressing high amounts of murine TNF-alpha transgene in the neurons of the CNS, we investigated the stereotyped, exploratory, and displacement activities in the hole-board and black/white box. Transgenic mice and their normal control littermates were hybrids of the CBA x C57BL/6 genetic backgrounds and were obtained by backcrossing the CBA x C57BL/6 founder female and her progeny with F1 hybrid mates. Transgenic mice did not show changes in the stereotyped behavior on the hole-board, but they displayed several alterations in the exploratory activities both in the hole-board and black/white box. Transgenic mice also exhibited an increase in grooming when exposed to a highly unfamiliar environmental stimuli in the black/white box. The study suggests that supranormal endogenous TNF-alpha in the brain affects the behavioral responses to stressful conditions.

Interleukin 6 is required for the development of collagen-induced arthritis.

Interleukin-6 (IL-6) is overproduced in the joints of patients with rheumatoid arthritis (RA) and, based on its multiple stimulatory effects on cells of the immune system and on vascular endothelia, osteoclasts, and synovial fibroblasts, is believed to participate in the development and clinical manifestations of this disease. In this study we have analysed the effect of ablating cytokine production in two mouse models of arthritis: collagen-induced arthritis (CIA) in DBA/1J mice and the inflammatory polyarthritis of tumor necrosis factor alpha (TNF-alpha) transgenic mice. IL-6 was ablated by intercrossing an IL-6 null mutation into both arthritis-susceptible genetic backgrounds and disease development was monitored by measuring clinical, histological, and biochemical parameters. Two opposite responses were observed; while arthritis in TNF-alpha transgenic mice was not affected by inactivation of the IL-6 gene, DBA/1J, IL-6(-/-) mice were completely protected from CIA, accompanied by a reduced antibody response to type II collagen and the absence of inflammatory cells and tissue damage in knee joints. These results are discussed in the light of the present knowledge of cytokine networks in chronic inflammatory disorders and suggest that IL-6 receptor antagonists might be beneficial for the treatment of RA.

Design and synthesis of small semi-mimetic peptides with immunomodulatory activity based on myelin basic protein (MBP).

Experimental allergic encephalomyelitis (EAE) is induced in susceptible animals by immunodominant determinants of myelin basic protein (MBP). Analogs of these disease-associated peptides have been identified with disease progression upon coimmunization. Usage of peptides, with disease-specific immunomodulatory capacity in vivo is limited, however, due to their sensitivity to proteolytic enzymes. Alternative approaches include the development of mimetic molecules which maintain the biological function of an original peptide, yet are stable and able to elicit their response in pharmacological quantities. A novel technique was employed to design a series of semi-mimetic peptides, based on the guinea pig MBP72-85 peptide used to induce EAE in Lewis rats. We used isonipecotic (iNip) and aminocaproic (Acp) acids as templates. Acp-MBP72-85 peptide derived analogues were effective in inducing EAE compared to iNip-peptide analogues which were ineffective at 350 micrograms. These findings suggest that the design and synthesis of semi-mimetic peptide molecules with immunomodulatory potential is possible and that eventually these molecules may form the basis for the development of novel and more effective disease-specific therapeutic agents.