Celia F. Brosnan

Identification of lymphotoxin and tumor necrosis factor in multiple sclerosis lesions.

Multiple sclerosis (MS) brain tissue, spleen, and PBMC were studied using immunocytochemistry and FACS for immunoreactivity for lymphotoxin (LT) and TNF. Both cytokines were identified in acute and chronic active MS lesions but were absent from chronic silent lesions. LT was associated with CD3+ lymphocytes and Leu-M5+ microglia cells at the lesion edge and to a lesser extent, in adjacent white matter. TNF was associated with astrocytes in all areas of the lesion, and with foamy macrophages in the center of the active lesion. In acute lesions, immunoreactivity for TNF in endothelial cells was noted at the lesion edge. No LT or TNF reactivity was detected in Alzheimers or Parkinsons disease brain tissues but was present at lower levels in central nervous system (CNS) tissue from other inflammatory conditions, except for adrenoleucodystrophy which displayed high levels of LT in microglia. No increase in LT and TNF reactivity was detected in spleen and PBMC of MS patients suggesting specific reactivity within the CNS. These results indicate that LT and TNF may be involved in the immunopathogenesis of MS, and can be detected in both inflammatory cells and cells endogenous to the CNS.

Quantitative aspects of reactive gliosis a review

Recent studies of gliosis in a variety of animal models are reviewed. The models include brain injury, neurotoxic damage, genetic diseases and inflammatory demyelination. These studies show that reactive gliosis is not a stereotypic response, but varies widely in duration, degree of hyperplasia, and time course of expression of GFAP immunostaining, content and mRNA. We conclude that there are different biological mechanisms for induction and maintenance of reactive gliosis, which, depending on the kind of tissue damage, result in different expressions of the gliotic response.

Multiple sclerosis: Re-expression of a developmental pathway that restricts oligodendrocyte maturation

During mammalian central nervous system (CNS) development, contact-mediated activation of Notch1 receptors on oligodendrocyte precursors by the ligand Jagged1 induces Hes5, which inhibits maturation of these cells. Here we tested whether the Notch pathway is re-expressed in the adult CNS in multiple sclerosis (MS), an inflammatory demyelinating disease in which remyelination is typically limited. We found that transforming growth factor-β1 (TGF-β1), a cytokine upregulated in MS, specifically re-induced Jagged1 in primary cultures of human astrocytes. Within and around active MS plaques lacking remyelination, Jagged1 was expressed at high levels by hypertrophic astrocytes, whereas Notch1 and Hes5 localized to cells with an immature oligodendrocyte phenotype, and TGF-β1 was associated with perivascular extracellular matrix in the same areas. In contrast, there was negligible Jagged1 expression in remyelinated lesions. Experiments in vitro showed that Jagged1 signaling inhibited process outgrowth from primary human oligodendrocytes. These data are the first to implicate the Notch pathway in the limited remyelination in MS. Thus, Notch may represent a potential target for therapeutic intervention in this disease.

P2X7 Receptors Mediate ATP Release and Amplification of Astrocytic Intercellular Ca2+ Signaling

Modulation of synaptic transmission and brain microcirculation are new roles ascribed to astrocytes in CNS function. A mechanism by which astrocytes modify neuronal activity in the healthy brain depends on fluctuations of cytosolic Ca2+ levels, which regulate the release of “gliotransmitters” via an exocytic pathway. Under pathological conditions, however, the participation of other pathways, including connexin hemichannels and the pore-forming P2X7R, have been proposed but remain controversial. Through the use of genetically modified 1321N1 human astrocytoma cells and of spinal cord astrocytes derived from neonatal Cx43- and P2X7R-null mice, we provide strong evidence that P2X7Rs, but not Cx43 hemichannels, are sites of ATP release that promote the amplification of Ca2+ signal transmission within the astrocytic network after exposure to low divalent cation solution. Moreover, our results showing that gap junction channel blockers (heptanol, octanol, carbenoxolone, flufenamic acid, and mefloquine) are antagonists of the P2X7R indicate the inadequacy of using these compounds as evidence for the participation of connexin hemichannels as sites of gliotransmitter release.

MCP-1, MCP-2 and MCP-3 expression in multiple sclerosis lesions: an immunohistochemical and in situ hybridization study

Chemokines are low molecular weight chemotactic cytokines that have been shown to play a central role in the perivascular transmigration and accumulation of specific subsets of leukocytes at sites of tissue damage. Two major families have been defined depending on the positioning of four conserved cysteines. The CXC chemokines predominantly attract neutrophils, whereas the CC chemokines predominantly attract monocytes and other leukocyte cell types. Members of the monocyte chemotactic protein (MCP)-1 family form a major component of the CC family of chemokines and are considered the principal chemokines involved in the recruitment of monocytes/macrophages and activated lymphocytes. In this study we addressed the expression and distribution of MCP-1, -2 and -3 in multiple sclerosis (MS) lesions of differing ages and levels of inflammatory activity using immunohistochemistry and in situ hybridization. In acute and chronic-active MS lesions immunoreactivity for MCP-1, -2 and -3 was prominent throughout the lesion center with reactivity diminishing abruptly at the lesion edge. Hypertrophic astrocytes were strongly reactive and inflammatory cells showed variable reactivity. Outside of the lesion only hypertrophic astrocytes were reactive. The results obtained by in situ hybridization for MCP-1 were in agreement with those obtained by immunostaining. In more chronic lesions immunoreactivity for MCP-1, -2 and -3 was considerably diminished, and in chronic-silent lesions immunoreactivity was restricted to a few scattered reactive astrocytes. Normal control brains showed no immunoreactivity for MCP-1, -2 and -3. Although the cellular distribution of all three members of this family was similar, antibodies to MCP-3 gave prominent staining of the extracellular matrix that was not noted for MCP-1 and -2. These results support the conclusion that members of the MCP family of chemokines are involved in the development of MS lesions in the central nervous system.

Induction of nitric oxide synthase activity in human astrocytes by interleukin-1β and interferon-γ

Nitric oxide (NO) is a short-lived, diffusible molecule that has a variety of biological activities including vasorelaxation, neurotransmission, and cytotoxicity. In the central nervous system, a constitutive form of nitric oxide synthase (NOS) has been localized in a subset of neurons and in endothelial cells. In addition, both constitutive and LPS-inducible NOS has been demonstrated in rat astrocytes and microglia in vitro. In this report, we present evidence for the production of NO, as measured by the production of nitrite, in highly enriched human fetal astrocyte cultures stimulated with IL-1β. The production of nitrite paralleled the induction of NADPH diaphorase enzyme activity in the perikarya of the majority of stimulated astrocytes. The IL-1β-induced nitrite production by astrocytes was markedly enhanced when cells were co-stimulated with IFN-γ or TNF-α (IFN-γ > TNF-α); LPS had no effect used as a single agent or in combination with other cytokines. NGMMA and NG-nitro-arginine, competitive inhibitors of NOS, diminished the accumulation of nitrite, but calmodulin antagonists (trifluoperazine, W-5 and W-7) had little or no inhibitory effect. Human fetal microglia, in contrast to astrocytes, failed to secrete significant amounts of nitrite in response to various stimuli. The results demonstrate the presence of an inducible form of NOS in human fetal astrocytes; human microglia, in turn, may control astrocyte NO production by providing IL-1β as an activating signal.

Developmental plasticity of CNS microglia

Microglia arise from CD45+ bone marrow precursors that colonize the fetal brain and play a key role in central nervous system inflammatory conditions. We report that parenchymal microglia are uncommitted myeloid progenitors of immature dendritic cells and macrophages by several criteria, including surface expression of “empty” class II MHC protein and their cysteine protease (cathepsin) profile. Microglia express receptors for stem cell factor and can be skewed toward more dendritic cell or macrophage-like profiles in response to the lineage growth factors granulocyte/macrophage colony-stimulating factor or macrophage colony-stimulating factor. Thus, in contrast to other organs, where terminally differentiated populations of resident dendritic cells and/or macrophages outnumber colonizing precursors, the majority of microglia within the brain remain in an undifferentiated state.

Peroxisome proliferator-activated receptor-γ agonists prevent experimental autoimmune encephalomyelitis

The development of clinical symptoms in multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE) involves T‐cell activation and migration into the central nervous system, production of glial‐derived inflammatory molecules, and demyelination and axonal damage. Ligands of the peroxisome proliferator‐activated receptor (PPAR) exert anti‐inflammatory effects on glial cells, reduce proliferation and activation of T cells, and induce myelin gene expression. We demonstrate in two models of EAE that orally administered PPARγ ligand pioglitazone reduced the incidence and severity of monophasic, chronic disease in C57BL/6 mice immunized with myelin oligodendrocyte glycoprotein peptide and of relapsing disease in B10.Pl mice immunized with myelin basic protein. Pioglitazone also reduced clinical signs when it was provided after disease onset. Clinical symptoms were reduced by two other PPARγ agonists, suggesting a role for PPARγ activation in protective effects. The suppression of clinical signs was paralleled by decreased lymphocyte infiltration, lessened demyelination, reduced chemokine and cytokine expression, and increased inhibitor of kappa B (IkB) expression in the brain. Pioglitazone also reduced the antigen‐dependent interferon‐γ production from EAE‐derived T cells. These results suggest that orally administered PPARγ agonists could provide therapeutic benefit in demyelinating disease.

Expression of Inducible Nitric Oxide Synthase and Nitrotyrosine in Multiple Sclerosis Lesions

Nitric oxide generated by the inducible form of nitric oxide synthase (iNOS) may contribute to the pathogenesis of multiple sclerosis (MS). In this report, we studied postmortem tissues of MS patients for the expression of iNOS by in situ hybridization and immunocytochemistry. Immunocytochemistry for nitrotyrosine, a putative footprint for peroxynitrite formation was also performed. In acute MS lesions, intense reactivity for iNOS mRNA and protein was detected in reactive astrocytes throughout the lesion and in adjacent normal appearing white matter. Staining of macrophages, inflammatory cell infiltrates, and endothelial cells was variable from case to case, but generally detected only in acute lesions. In chronic MS lesions reactive astrocytes at the lesion edge were positive for iNOS whereas the lesion center was nonreactive. Normal appearing white matter demonstrated little reactivity, as did tissues from noninflamed control brains. Staining for nitrotyrosine was also detected in acute but not chronic MS lesions, and displayed a diffuse parenchymal, membranous, and perivascular pattern of immunoreactivity. These results support the conclusion that iNOS is induced in multiple cell types in MS lesions and that astrocyte-derived nitric oxide could be important in orchestrating inflammatory responses in MS, particularly at the blood-brain barrier.

MECHANISMS OF IMMUNE INJURY IN MULTIPLE SCLEROSIS

In this review, we address current concepts regarding the mechanisms of tissue damage that lead to demyelination and oligodendrocyte loss in multiple sclerosis. Particular emphasis has been placed on examining the MS lesion for evidence for pathogenetic processes that have been implicated from various in vivo and in vitro model systems. Central in this analysis has been the evaluation of the various effector cell types and their products. The results strongly support the conclusion that proinflammatory cytokines are major mediators of tissue damage, through the activation of inflammatory cells and resident glial cells. A role for antibody is also discussed, particularly as part of an antibody‐dependent cell‐mediated demyelinating process. Minor populations of lymphocytes may also participate by defining the nature of the immunological microenvironment.