George Kassiotis

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.

On the role of tumor necrosis factor and receptors in models of multiorgan failure, rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease.

Summary: The specific role of the tumor necrosis factor (TNF)/TNF receptor (TNFR) system in disease pathogenesis still remains an unresolved puzzle. Recent studies in transgenic and knockout animals, where the pathogenic influence of genetically perturbed TNF expression has been evaluated, indicate that several pathways of TNF/TNFR action may contribute independently or in concert to initiate, promote or downregulate disease pathogenesis. Evidently, organ‐specific inflammatory or autoimmune pathology may ensue due co sustained activation by TNF of innate immune cells and inflammatory responses, which may consequently lead to tissue damage and co organ‐specific chronic pathology. However, more cryptic functions of this molecule may be considered Co play a significant pare in che development of TNF‐mediated pathologies. Direct interference of TNF with the differentiation, proliferation or death of specific pathogenic cell targets may be an alternative mechanism for disease initiation or progression. In addition Co these activities, there is now considerable evidence to suggest that TNF may also directly promote or downregulate the adaptive immune response. It is therefore evident that no general scenario may adequately describe the role of TNF in disease pathogenesis. In this article, we aim to place these diverse functions of TNF/TNFRs into context with the development of specific pathology in murine models of multiorgan failure, rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease.

TNF-α transgenic and knockout models of CNS inflammation and degeneration

Tumour necrosis factor-alpha (TNF-alpha) plays a central role in inflammatory events including those taking place in the central nervous system (CNS), and has been implicated as a key pathogenic mediator in several human inflammatory, infectious and autoimmune CNS disorders. Using transgenic and gene knockout mice we have investigated the role of deregulated TNF-alpha production in the CNS. We show that the overexpression of wild-type murine or human TNF-alpha transgenes by resident CNS astrocytes or neurons in sufficient to trigger a neurological disorder characterised by ataxia, seizures and paresis, with histopathological features of chronic CNS inflammation and white matter degeneration. Furthermore, we show that transmembrane human TNF-alpha is sufficient to trigger CNS inflammation and degeneration when overexpressed by astrocytes but not by neurons, indicating that target cells mediating the neuroinflammatory activities of TNF-alpha localise in the vicinity of astrocytes rather than neurons. Our results establish that both soluble and transmembrane molecular forms of TNF-alpha can play critical roles in vivo in the pathogenesis of CNS inflammation and demyelination, and validate TNF-alpha transgenic and mutant mice as important models for the further study of related human CNS diseases.

Learning abilities, NGF and BDNF brain levels in two lines of TNF-α 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.

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.

Dissection of the pathologies induced by transmembrane and wild-type tumor necrosis factor in transgenic mice.

With increasing awareness that seemingly diverse immune‐mediated diseases involve similar pathogenetic mechanisms, and the identification of a growing number of key effector molecules, it is becoming possible to design and generate effective transgenic models for such diseases. Tumor necrosis factor (TNF) plays a prominent role in immune and host defense responses and there is strong evidence that abnormal TNF production contributes to disease initiation and progression in rheumatoid arthritis, systemic inflammatory response syndrome, diabetes, multiple sclerosis, and many other immune‐mediated disorders. The generation of TNF transgenic mice, in which TNF production is deregulated, has provided us with direct evidence that, in vivo, this cytokine can indeed trigger the development of such complex disease phenotypes. Transgenic mice that have been engineered to overexpress human or murine TNF molecules in peripheral joints, T cells, or neurons of the central nervous system represent important animal models for human rheumatoid arthritis, systemic inflammation, and multiple sclerosis, respectively. In addition to establishing a central role for TNF in such diseases, these animal models have already proved valuable for identifying additional important disease‐effector molecules, and for gaining an insight into the complex in vivo mechanisms that are involved in disease pathogenesis. For example, in the case of arthritis, TNF has been found to transmit its pathogenic effects entirely through interleukin‐1, which may therefore represent an additional important target for therapeutic intervention in the human disease. In summary, TNF transgenic models of human disease are expected to serve as important in vivo tools for defining details of disease pathogenesis, potential targets for therapeutic intervention, and for evaluating the possible involvement of additional genetic and environmental factors on the disease state.

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, β2‐microglobulin (β2m), immunoglobulin μ  chain (Igμ ) 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.

Transgenic Models of Tnf Induced Demyelination

The nervous system and the immune system are intimately connected within an extensive network of anatomical and molecular communications, in which molecules of one system can directly stimulate the other. Immune organs such as the spleen are innervated by sympathetic nerves and immunological and inflammatory responses can be modulated by neuropeptides (Payan, 1986; Sternberg, 1997). In the opposite direction cytokines and cytokine receptors, classically associated with the peripheral immune system, are now known to play important roles in CNS functioning (Hopkins and Rothwell, 1995) and cytokine disbalances are associated with CNS disease (Merrill and Benveniste, 1996). TNF is a pleiotropic cytokine which is believed to play a central role in the pathogenesis of human immune-mediated diseases such as multiple sclerosis (MS) (Raine, 1995), Alzheimers Disease (Meda et al., 1995), bacterial meningitis (Leist et al., 1988), cerebral malaria (Grau et al, 1989), and AIDS dementia complex (Tyor et al., 1992). The cell types that contribute to the local production of TNF within the CNS during the pathogenesis of neuronal disease are resident CNS cells, mainly microglia and astrocytes, and infiltrating cells of the immune system particularly macrophages (Hofman et al., 1989; Selmaj et al., 1991; Canella and Raine, 1995).

Role for TNF in CNS Inflammation, Demyelination and Neurodegeneration Studied in Transgenic Mice

Tumor necrosis factor (TNF) is a pluripotent cytokine which is be-lieved to play a central role in the pathogenesis of human im-mune-mediated diseases. In neuroimmunologic diseases TNF is produced locally in the central nervous system (CNS) and in vitro experiments have shown that it is capable of exerting proliferative and/or cytotoxic effects upon isolated CNS cells depending upon the identity of the target cell. Through the recent application of transgenic and gene knockout technology to the study of TNF neurobiology, it has become possible to directly assess the contribution of TNF action to CNS pathology. Our aim in this chapter is to discuss the cellular interactions and molecular pathways through which TNF can induce neuropathology in vivo. Special emphasis is given to the TNF/p55 TNFR-dependent inflammation, demyelination and neurodegeneration that develop when TNF is chronically expressed by resident CNS cells of transgenic mice. TNF transgenic mice, which model the pathology observed in neuroimmunologic diseases, provide important information concerning the etiopathogenesis of such diseases in humans and support the use of therapeutic approaches designed to target TNF/p55 TNFR signaling pathways in their treatment.