Dr. Cedric S. Raine's
Multiple Sclerosis Investigations

The molecules that comprise the Tumor Necrosis Factor Ligand and Receptor (TNF-L and TNF-R) families play important roles in tissue homeostasis and in Multiple Sclerosis (MS).

For example, levels of the TNF Ligand (TNF-; Cachectin) correlate with disease progression and LymphoToxin (LT, TNF-ß) has been localized in MS lesions.

Members of the TNF-R family are typical signal sensors which upon binding with Ligand aggregate and recruit signal transducers.

To date, no TNF-R molecules have been reported in MS although TNF-RI and RII have been localized to Oligodendrocytes in culture.

In the present study, the expression of TNF, LT- (the soluble form of LT), LT-ß (the ß chain of LT- ß, the membrane-bound form of LT), TNF-RI, TNF-RII, LT-ß-R, FasL, and Fas receptor in MS lesions has been examined by ImmunoHistoChemistry for protein and by RT-PCR for mRNA.

In addition, the TUNEL technique for DNA fragmentation was applied to detect Apoptosis. The results have shown that contrarily to predictions, Oligodendrocytes around active MS lesions frequently expressed TNF-R molecules belonging to the Apoptotic cascade.

However, these cells did not undergo Apoptosis, as judged by TUNEL. On the other hand, Lymphocytes (and a few Microglia Cells) in the same tissue displayed Apoptosis.

Microglial Cells were the major Effector Cells in the CNS and expressed TNF, LT- and FasL. LT-ß expression was seen on Astrocytes and Oligodendrocytes, and LT-ß-R on Astrocytes.

We conclude that TNF-L and TNF-R molecules are extensively expressed in MS, that their expression occurs at high levels but is not specific for MS, and that Oligodendrocytes are depleted by a CytoLytic mechanism, not by Apoptosis.

Glial Growth Factor 2 (GGF2) is a Neuronal signal that promotes the proliferation and survival of the Oligodendrocyte, the Myelinating cell of the Central Nervous System (CNS).

The present study examined whether recombinant human GGF2 (rhGGF2) could effect clinical recovery and repair to damaged Myelin in Chronic Relapsing Experimental AutoImmune EncephaloMyelitis (EAE) in the mouse, a major animal model for the human DeMyelinating Disease, Multiple Sclerosis.

Mice with EAE were treated with rhGGF2 during both the acute and Relapsing phases. Clinically, GGF2 treatment delayed signs, decreased severity, and resulted in statistically significant reductions in relapse rate.

rhGGF2-treated groups displayed CNS lesions with more ReMyelination than in controls.

This correlated with increased mRNA expression of Myelin Basic Protein Exon 2, a marker for ReMyelination, and with an increase in the CNS of the regulatory Cytokine, InterLeukin 10, at both the RNA and protein levels.

Thus, a beneficial effect of a NeuroTrophic Growth Factor has been demonstrated on the clinical, pathologic, and molecular manifestations of AutoImmune DeMyelination, an effect that was associated with increased expression of a T-Helper 2 Cytokine.

rhGGF2 treatment may represent a novel approach to the treatment of Multiple Sclerosis.

The ProInflammatory Th1 Cytokine, Tumor Necrosis Factor-alpha (TNF ), the cell death signaling molecule FasL, and several ExtraCellular Matrix degrading MetalloProteinases have been implicated in the PathoGenesis of Multiple Sclerosis (MS).

The latter enzymes, as well as TNF -converting enzyme and FasL-converting enzyme, can be blocked by Matrix MetalloProteinase Inhibitors (MMPIs).

In this study, we show that a potent MMPI was clinically effective in an animal model for MS, Experimental AutoImmune EncephaloMyelitis (EAE) in the SJL/J mouse.

Efficacy was remarkable, as indicated by blocking and reversal of acute disease and reduced number of relapses and diminished mean cumulative disease score in Chronic Relapsing animals.

Also, DeMyelination and Glial scarring were significantly decreased in MMPI-treated mice with Chronic Relapsing EAE, as was Central Nervous System Gene expression for TNF- and fasL.

It is interesting that expression of the beneficial Cytokine InterLeukin-4 (IL-4) was increased, and IL-4 was expressed on Glial Cells.

The relevance of these compounds for MS was underscored by their ability to specifically inhibit TNF- shedding and CytoToxicity of Myelin-AutoReactive human Cytotoxic CD4⁺ T-Cell clones.

This is the first report to show a positive effect by MMPIs on Chronic Relapsing EAE, its Central Nervous System Cytokine profile, and on TNF- shedding by human Myelin-Autoreactive T-Cells.

The mechanisms involved in the elimination of Oligodendrocytes and Myelin from the DeMyelinated plaque of Multiple Sclerosis (MS) are inextricably intertwined and yet most investigations tend to consider them separately.

This short review revisits the problem of Oligodendrocyte pathology in MS and attempts to put the topic into perspective by examining the numerous Immunologically-active molecules associated with the Oligodendrocyte, some, but not all, cross-reactive with Myelin.

The consensus of opinion is that Myelin is the primary target in MS but that Oligodendrocytes are eventually lost from the lesion.

Reappraisal of recent and past works brings into focus a possible key role for soluble mediators, in particular AntiBody and the ProInflammatory Cytokine, TNF-, in Oligodendrocyte loss and Myelin in MS.

Despite extensive NeuroPathologic investigation by a number of laboratories, no evidence has yet been found to support the concept that Apoptosis might account for Oligodendrocyte depletion in MS, even though molecules belonging to the Apoptotic Cascade can be expressed by Oligodendrocytes in and around lesions.

Indeed, abundant evidence has been presented to show that Oligodendrocytes initially respond to the DeMyelinating insult in MS by proliferating and elaborating new Myelin but, no doubt due to the relentless progression of inflammatory events, the cells are eventually lost, probably via a CytoLytic Pathway.

Strategies to block the progression of CNS inflammation in EAE and MS appear to promote the survival of Oligodendrocytes and to enhance ReMyelination. Such strategies appear to hold much promise for the MS patient.

To investigate the structural role of Glial Fibrillary Acidic Protein (GFAP) in vivo, mice carrying a null mutation in GFAP were generated.

In 7/14 mutant animals older than 18 months of age, Hydrocephalus associated with White Matter loss was detected.

Mutant mice displayed abnormal Myelination including the presence of actively Myelinating Oligodendrocytes in adults, NonMyelinated Axons in Optic Nerve, and reduced Myelin thickness in Spinal Cord.

White Matter was poorly vascularized and the Blood-Brain Barrier was structurally and functionally impaired.

Astrocytic structure and function were abnormal, consisting of shortened Astrocytic Cell processes, decreased septation of White Matter, and increased CNS ExtraCellular Space.

Thus, GFAP expression is essential for normal White Matter architecture and Blood-Brain Barrier integrity, and its absence leads to late-onset CNS DysMyelination.

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 (Cytokines).

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 (Microglia).

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.

The stress proteins belonging to the Heat Shock Protein 60 (hsp6O) family of molecular chaperones with known ImmunoGenic properties are expressed at increased levels in a number of AutoImmune conditions.

Because previous studies from this laboratory suggested that hsp6O may be involved in the PathoGenesis of the chronic Multiple Sclerosis (MS) plaque, we have examined autopsied Central Nervous System tissue from 10 cases of MS, ranging in clinical history from acute to chronic inactive.

MS Lesions ranged from acute, actively DeMyelinating and edematous, to fibrous Astrogliotic and chronically DeMyelinated. As controls, Central Nervous System tissue from other Neurologic Diseases and NonNeurologic conditions was used.

Frozen, paraffin, and epoxy-embedded sections were studied ImmunoCytoChemically with the ML30 mAb to hsp6O.

Acute MS lesions displayed the greatest reactivity, with particularly prominent staining of hypertrophic Astrocytes, reactive Macrophages, and hyperplastic Oligodendrocytes.

In all these cells, elevated expression occurred in the constitutive site for hsp6O (Mitochondria) and within the Cytosol, which is suggestive of a shift in expression.

The hsp6O-reactive Oligodendrocytes were structurally intact. Chronic active MS lesions also revealed the highest levels of hsp6O in HyperTrophic Astrocytes and Oligodendrocytes.

Chronic silent MS lesions displayed elevated hsp6O in HyperTrophic Astrocytes only while constitutive expression occurred elsewhere in the Central Nervous System at levels slightly higher than normal.

Other Neurologic Disease tissue displayed expression elevated above that found in NonNeurologic cases, but this was considerably less than that seen in acute MS.

Of the other Neurologic Diseases, AIDS Encephalitis revealed the greatest activity for hsp6O, with both Mitochondrial and cytosolic staining of Astrocytes.

It is proposed that the high levels of hsp6O in hyperplastic, structurally intact Oligodendrocytes in acute MS Lesions may bespeak a protective mechanism, whereas hsp6O in chronic active lesions may serve a Pathogenic role in the later depletion of these cells.

The fundamental message emerging from Immunologic and ImmunoPathologic analyzes of the Brain and Spinal Cord in Multiple Sclerosis (MS) is that during inflammation, the Central Nervous System (CNS) is capable of interactions with the Lymphoid System.

Mainly through induced (as opposed to constitutive) expression of Immune System-specific molecules on CNS elements.

CNS Endothelium, Astrocytes and Microglia Cells are the main participants, with Oligodendrocytes and Neurons remaining essentially inert.

There appears to be nothing unique about the manner in which the CNS responds to inflammation or in the molecules expressed.

The ensuing Adhesion Molecules, pro-inflammatory and regulatory Cytokines, HistoCompatibility Molecules, and T and B-Cell markers, are difficult to distinguish from those occurring in Peripheral Lymphoid tissue.

However, differences certainly exist in the outcome of an inflammatory insult in the CNS versus other, peripheral tissues, whereby there is generally a poor reparatory response.

Reasons for the latter appear to lie in the anatomical complexity of the CNS, its vulnerability to damage by soluble mediators, and in the White Matter (the battlefield for the inflammatory attack in MS), the exquisite sensitivity of the Oligodendrocyte and its Myelin to exogenous factors.

With the aid of examples drawn from Experimental Allergic EncephaloMyelitis, the prime animal model for MS, a number of approaches to prevent or downregulate CNS Inflammation during Immune-mediated DeMyelination are presented as possible therapeutic avenues for MS, some of which are already under investigation.