Chronic Active Multiple Sclerosis Lesions Research Articles

Enhanced expression of NGF receptors in multiple sclerosis lesions.

The receptor for nerve growth factor (NGF) comprises a 75-kDa (NGFRp75) and a tyrosine kinase A (TrkA) subunit. In view of conflicting opinions on the identity of glial targets of NGF in human central nervous system (CNS), we examined the cellular distribution of both NGF receptor subunits in normal CNS and in chronic multiple sclerosis (MS) lesions. For this, we compared the pattern of recognition of 2 monoclonal antibodies (mAbs) and a polyclonal antiserum to NGFRp75. Only the 2 mAbs specifically recognized NGFRp75, while the polyclonal antiserum showed widespread reactivity. In normal CNS and silent MS lesions, immunohistochemistry with anti-NGFRp75 mAbs and for TrkA revealed perivascular cell reactivity. At the edge of chronic active MS lesions, selective NGFRp75 staining was prominent on reactive astrocytes, while throughout the lesion, NGFRp75 was expressed on microglia/macrophages. The vast majority of mature or precursor oligodendrocytes did not express NGFRp75. Both NGF receptors were co-expressed on a subset of inflammatory cells. Immunoreactivity for NGFRp75 on glial and immune cells did not correlate with the distribution of apoptotic figures, as detected by TUNEL. Thus, expression of NGF receptors in active MS lesions suggests a role for NGF in regulating the autoimmune response at both immune and glial cell levels.

Neuregulin: An Oligodendrocyte Growth Factor Absent in Active Multiple Sclerosis Lesions

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) which results in demyelination and axonal injury. Conventional therapy for MS is immune suppression in the absence of agents that promote neural and glial survival or remyelination. Neuregulins are a family of ligands that exert trophic effects on both neurons and glia. Using mice bearing a null mutation in the neuregulin gene, here we demonstrate that neuregulins are necessary for the normal development of oligodendrocytes. In addition, neuregulins are produced in the normal human CNS by astrocytes as well as neurons. Astrocyte-derived neuregulin is functionally active in bioassays and exists in secreted and membrane-associated β-isoforms. In active and chronic active MS lesions, however, the expression of astrocyte neuregulin is dramatically reduced. The absence of neuregulin in active MS lesions may contribute to the paucity of remyelination in MS.

Expression of the β-chemokine receptors CCR2, CCR3 and CCR5 in multiple sclerosis central nervous system tissue

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) characterised by perivascular inflammatory cell infiltrates and plaques of demyelination. Chemokines have been shown to play an important role in the activation and directional migration of cells to sites of CNS inflammation. The action of chemokines requires the expression of their complementary chemokine receptors by their target cells. We have examined the expression of the β-chemokine receptors CCR2, CCR3 and CCR5 in post-mortem MS CNS tissue using single- and double-labelling immunocytochemistry techniques. Low levels of CCR2, CCR3 and CCR5 were expressed by microglial cells throughout control CNS tissue. In chronic active MS lesions CCR2, CCR3 and CCR5 were associated with foamy macrophages and activated microglia. CCR2 and CCR5 were also present on large numbers of infiltrating lymphocytes. A smaller number of CCR3-positive lymphocytes were present, but we also noted CCR3 and CCR5 on astrocytes in five of the 14 cases of MS investigated, particularly associated with processes around vessels and at the glia limitans. Ligands for CCR2 and CCR3 include MCP-1 and MCP-3 which were co-localised around vessels with the infiltrating leukocytes, but were also present in unaffected areas of cortex. The elevated expression of CCR2, CCR3 and CCR5 in the CNS in MS suggests these β-chemokine receptors and their ligands play a role in the pathogenesis of MS.

Activation of NF-κB and c- jun Transcription Factors in Multiple Sclerosis Lesions : Implications for Oligodendrocyte Pathology

Oligodendrocytes are a major target of the purported autoimmune response in multiple sclerosis (MS) lesions, but little is known about the mechanisms underlying their demise. Despite the expression of proapoptotic receptors, these cells are rarely seen to undergo apoptosis in situ. On the other hand, cytotoxic mediators present in MS lesions, such as tumor necrosis factor-alpha, are known to generate survival signals through the activation of the transcription factors NF-kappaB and c-jun. The aim of this study was to investigate in chronic active and silent MS lesions and control white matter the expression of c-jun, its activating molecule, JNK, as well as NF-kappaB complex and its inhibitor, IkappaB. By immunohistochemistry we found negligible reactivity for these molecules in control white matter and silent MS plaques. In active MS lesions, double-label immunohistochemistry with oligodendrocyte markers showed up-regulation of the nuclear staining for both NF-kappaB and JNK on a large proportion of oligodendrocytes located at the edge of active lesions and on microglia/macrophages throughout plaques. Oligodendrocytes showed no reactivity for IkappaB, which was predominantly confined to the cytoplasm of microglia/macrophages. We hypothesize that activation of these transcriptional pathways may be one mechanism accounting for the paucity of oligodendrocyte apoptosis reported in MS.

Expression of transforming growth factor (TGF)-beta1, -beta2, and -beta3 isoforms and TGF-beta type I and type II receptors in multiple sclerosis lesions and human adult astrocyte cultures.

It is known that the pleiotropic cytokine transforming growth factor beta (TGF-beta) has a regulatory role in the process of tissue repair and remodelling following injury. As reports on these molecules in multiple sclerosis (MS) lesion with different lesional activity are rare, we studied the cellular localization of TGF-beta1, -beta2, and -beta3 isoforms, and TGF-beta receptor type I (TGF-betaR-I) and TGF-betaR-II expression by immunohistochemistry on postmortem brain tissue from MS and normal control cases. To validate the TGF-beta staining results we demonstrated that cultured human adult astrocytes that produce biological active TGF-beta2, and to a lesser extent TGF-beta1, were immunoreactive for all 3 TGF-beta isoforms. Moreover, at mRNA level TGF-beta1 was detected in MS and normal control brain tissue. In normal control brain tissue, TGF-beta isoforms were expressed in ramified microglia and TGF-beta2, and -beta3 on neuronal cells in the gray matter TGF-betaR-I and TGF-betaR-II expression was found on endothelial cells, astrocytes, microglia, and neurons. In active demyelinating MS lesions a strong to intense immunoreactivity was detected for all 3 TGF-beta isoforms in perivascular and parenchymal (foamy) macrophages and in hypertrophic astrocytes. Strong immunoreactivity for TGF-betaR-I and TGF-betaR-II was found on macrophages in both parenchymal and perivascular areas and on hypertrophic astrocytes and endothelial cells in active demyelinating MS lesions. In chronic active and inactive MS lesions, all 3 TGF-beta isoforms and their receptors were strongly expressed in hypertrophic astrocytes. Our findings strongly suggest that the expression of the various TGF-beta isoforms and their receptor types found in MS lesions with different cellular activity participate in reactive processes leading to the formation of chronic MS lesions.

Expression of MCP-1 by Reactive Astrocytes in Demyelinating Multiple Sclerosis Lesions

The pathology of multiple sclerosis (MS) is characterized by breakdown of the blood-brain barrier (BBB), accompanied by infiltration of macrophages and T lymphocytes into the central nervous system (CNS). The migration of these cells into the CNS parenchyma may be partly regulated by chemokines. The aim of this study was therefore to investigate the cellular localization of the potent monocyte- and T-cell-attracting chemokine monocyte chemoattractant protein (MCP)-1 by immunohistochemistry on postmortem brain tissue from MS and normal control cases. Brain tissue samples of six MS patients and four patients without a history of brain disease were neuropathologically classified according to characteristic (immuno)histochemical staining patterns. Frozen tissue sections of active demyelinating MS lesions, chronic active demyelinating MS lesions, and normal control brain were immunohistochemically stained with a monoclonal antibody directed against MCP-1. In active demyelinating MS lesions as well as in chronic active MS lesions, reactive hypertrophic astrocytes were strongly immunoreactive for MCP-1, whereas perivascular and parenchymal foamy macrophages did not express MCP-1 protein. These results suggest a significant role for the beta-chemokine MCP-1, synthesized in vivo by reactive hypertrophic astrocytes, in the recruitment and activation of myelin-degrading macrophages and thereby contributing to the evolution of MS lesions.

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.

Immunocytochemical characterization of the expression of inducible and constitutive isoforms of nitric oxide synthase in demyelinating multiple sclerosis lesions.

The cellular localization and distribution of inducible and constitutive nitric oxide synthase (iNOS/cNOS) was determined in tissue sections from multiple sclerosis (MS) and control brain and spinal cord. Immunocytochemical techniques were applied using specific iNOS- and cNOS-directed antibodies. In addition, NADPH-diaphorase histochemistry was performed. To establish the identity of iNOS-, cNOS- and NADPH-diaphorase-positive cells single and double staining was performed on tissue sections with the macrophage marker KP1 (CD68) and with the astrocyte marker glial fibrillary acidic protein (GFAP). Areas of myelin breakdown and demyelination were determined using a staining for neutral lipids, Oil Red O (ORO). Furthermore, macrophages isolated from active demyelinating MS lesions were stained for iNOS, cNOS, KP1 and ORO. In active MS lesions strong iNOS immunoreactivity was found exclusively in perivascular and parenchymal macrophages distributed within regions of active demyelination. In these active MS lesions immunoreactivity for cNOS was also found in macrophages. Macrophages isolated from active MS lesions also showed immunoreactivity for iNOS and cNOS. Moreover, these isolated macrophages produced nitric oxide (NO; >30 microM) in vitro. NADPH-diaphorase activity was detected in KP1-positive perivascular and parenchymal macrophages and in GFAP-positive reactive astrocytes in active MS lesions and in reactive astrocytes located in the hypercellular rims of chronic active MS lesions. cNOS-positive reactive astrocytes were detected in both active and chronic active MS lesions. Inside chronic active lesions some residual macrophages were weakly iNOS-positive. In control brain and spinal cord no iNOS immunoreactivity could be detected. These results suggests an important role for human macrophages capable of producing the free radical nitric oxide (NO), which may contribute to the cytotoxicity of oligodendrocytes and destruction of myelin in MS brain and spinal cord.

Enhanced expression of chemotactic receptors in multiple sclerosis lesions

We have previously shown that astrocytes and microglia express the receptors for C5a, interleukin-8 (IL-8) and N-formyl-Met-Leu-Phe (FMLP) in vitro. The expression and function of chemotactic receptors in the central nervous system (CNS) is, however, largely unexplored. In this study, we examined tissue sections from normal human brain and active, chronic active and chronic silent multiple sclerosis (MS) lesions for the expression of the receptors for C5a, IL-8 and FMLP by immunohistochemistry. In normal brain tissue, the expression of all three receptors was seen at low levels on astrocytes and microglia. In contrast, expression for all three receptors was markedly elevated on foamy macrophages in the acute and chronic active MS lesions. In addition, fibrous astrocytes stained intensely for the C5a receptor in the chronic active disease. Receptor expression in the chronic silent lesion was low and similar to that seen in normal brain, with staining confined to a few hypertrophic astrocytes and foamy macrophages. These are the first studies to demonstrate expression of these receptors in the CNS and elevated receptor expression in inflammatory MS lesions. The data suggest that these chemotactic receptors may play a role in inflammatory responses in MS and possibly in other CNS diseases.

Multiple sclerosis: Fas signaling in oligodendrocyte cell death.

Fas is a cell surface receptor that transduces cell death signals when cross-linked by agonist antibodies or by fas ligand. In this study, we examined the potential of fas to contribute to oligodendrocyte (OL) injury and demyelination as they occur in the human demyelinating disease multiple sclerosis (MS). Immunohistochemical study of central nervous system (CNS) tissue from MS subjects demonstrated elevated fas expression on OLs in chronic active and chronic silent MS lesions compared with OLs in control tissue from subjects with or without other neurologic diseases. In such lesions, microglia and infiltrating lymphocytes displayed intense immunoreactivity to fas ligand. In dissociated glial cell cultures prepared from human adult CNS tissue, fas expression was restricted to OLs. Fas ligation with the anti-fas monoclonal antibody M3 or with the fas-ligand induced rapid OL cell membrane lysis, assessed by LDH release and trypan blue uptake and subsequent cell death. In contrast to the activity of fas in other cellular systems, dying OLs did not exhibit evidence of apoptosis, assessed morphologically and by terminal transferase-mediated d-uridine triphosphate-biotin nick-end-labeling staining for DNA fragmentation. Other stimuli such as C2-ceramide were capable of inducing rapid apoptosis in OLs. Antibodies directed at other surface molecules expressed on OLs or the M33 non-activating anti-fas monoclonal antibody did not induce cytolysis of OLs. Our results suggest that fas-mediated signaling might contribute in a novel cytolytic manner to immune-mediated OL injury in MS.

CDlb is expressed in multiple sclerosis lesions

Recent observations have shown that CD1 molecules act as restriction elements in the presentation of antigens to specialized subsets of T cells. To examine the expression of CD1 molecules in multiple sclerosis (MS) lesions, frozen sections of central nervous system (CNS) tissues from nine MS and three other neurological disease (OND) patients, one patient with Wilson's disease, and one non-neurological control were stained by immunocytochemistry. In chronic-active MS lesions, CDlb immunoreactivity was prominent on perivascular inflammatory cells whereas macrophages within the lesion showed little reactivity. At the lesion edge, intense immunoreactivity for CDlb was found on hypertrophie astrocytes. High level expression of CDlb in MS lesions was found to colocalize with the presence of GM-CSF in astrocytes. In chronic-silent lesions, CDlb expression was found on only a few perivascular astrocytic foot processes and the occasional perivascular macrophage. CDlb was not found in the tissues studied for control purposes. In contrast, MHC class II expression was detected on microglia in all tissues examined. The relatively low level expression of CDlb in normal-appearing tissues, chronic-silent lesions and in the OND controls supports the conclusion that the expression of CDlb in active MS lesions is significantly upregulated and could contribute to lesion development.

The Dale E. McFarlin memorial lecture: The immunology of the multiple sclerosis lesion

This review surveys the structural terrain of the multiple sclerosis (MS) lesion from the standpoint of its immunologic responsiveness. Similarities in lymphocyte trafficking patterns are noted between MS and its laboratory model, experimental allergic encephalomyelitis (EAE), and in both conditions, the inflammatory response is selective for the central nervous system (CNS). While adhesion molecules abound during the genesis of the MS lesion, none has yet been found that is unusual to this condition and, indeed, many occur in other neurodegenerative states in which inflammation is not a component of the lesion. Cytokines are effective regulators of lymphocyte traffic and adhesion events and most can be located in MS lesions. Of these, tumor necrosis factor-alpha (TNF-alpha) occurs in abundance. Together with its known affinity to effect myelin and oligodendrocyte destruction and to up-regulate adhesion molecule expression, the presence of TNF-alpha renders it an important player in lesion pathogenesis. Demyelination is described as a rapid lytic event, perhaps involving cytokines and immunoglobulin, and structural similarities are common in the patterns seen in MS and EAE. Oligodendrocytes survive the initial stages of lesion formation. Moreover, they are now known to proliferate and elaborate new myelin at the same time as myelin is being degraded. This paradoxical reparatory scenario is apparently a transient event although rims of remyelination persist about the margins of chronic lesions. The speculation is reiterated that the demise of the oligodendrocyte in MS may occur late in lesion formation and may be in part related to the expression of heat shock proteins (specifically, members of the hsp 60 family), potent stimulators of T-cell receptor-gamma delta T cells that have been claimed to have cytolytic activity and that have been located in chronic active MS lesions. In sum, while no single immune system molecule can be assigned as unusual to the CNS in MS, and while there appears to be nothing unique about the manner in which the CNS responds to the inflammation, the true uniqueness of the situation in MS is probably related to the many, normally sequestered, specific antigens within the myelin sheath and the biology of the myelinating cell, the oligodendrocyte.

Astrocyte Expression of Major Histocompatibility Complex Gene Products in Multiple Sclerosis Brain Tissue Obtained by Stereotactic Biopsy

Major histocompatibility complex (MHC) class I and class II antigens were characterized by immunocytochemistry in two chronic-active multiple sclerosis lesions in tissue obtained from two patients by stereotactic biopsy. We examined in particular astrocytic MHC-positive cells in relation to lesion architecture. The MHC class I (HLA-A,B,C)-positive astrocytic cells were widely dispersed, being present at the lesion edge, in the gliotic lesion center, and in normal-appearing white matter as well. Morphologically astrocytic MHC class II (HLA-DR)-positive cells were confined exclusively to the lesion edge. By staining serial sections with antisera to glial-fibrillary acidic protein and HLA-DR, we confirmed the lineage of several MHC class II-positive astrocytes. The demonstration of MHC antigen-positive astrocytes in multiple sclerosis tissue obtained by stereotactic biopsy is novel; the differential distribution of MHC class I- and class II-positive astrocytes in the multiple sclerosis lesion may provide suggestive clues about the regulation of MHC expression on these cells in vivo.

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 Alzheimer's or Parkinson's 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.