Multiple sclerosis : Distribution of T cells, T cell subsets and Ia-positive macrophages in lesions of different ages

Using monoclonal antibodies in combination with the PAP technique, total (T11+) T cells, helper-inducer (T4+) T cells, suppressor-cytotoxic (T8+) T cells and Ia+ cells (macrophages and B cells) were localized in frozen sections of multiple sclerosis (MS) lesions with varied disease activity. In acute MS, T11+, T4+, T8+ cells and Ia+ macrophages were found in large numbers throughout the lesion but were virtually absent from normal white matter. In active chronic MS lesions, the numbers of T11+, T4+ and T8+ cells increased from the center towards the edge of the lesion. T11+ and T4+ cells penetrated deeply into the normal-appearing white matter adjacent to the lesion, while T8+ cells were more confined to the lesion edge. Ia+ macrophages displayed a reverse distribution pattern to that of T cells. They showed the highest density in the lesion center and their numbers decreased slightly towards the lesion edge. Small numbers of T11+, T4+, T8+ and Ia+ cells were always present in normal white matter. In silent chronic MS lesions, the numbers of both T cells and Ia+ cells were significantly lower than in active chronic MS. While T11+ and T4+ cells were found throughout the central nervous system (CNS), T8+ cells were virtually absent from the lesion center. Ia+ macrophages were also present in small numbers throughout the CNS and, sometimes, showed some accumulation at the lesion edge. Thus, T cells and T cell subsets have been demonstrated to be involved in lesion pathogenesis in MS in that lesion progression was associated with T4+ cells while ongoing demyelination depended upon the presence of Ia+ macrophages.     

Multiple sclerosis. Evidence for antigen presentation in situ by endothelial cells and astrocytes

To investigate the possibility of local antigen presentation within the central nervous system (CNS) in multiple sclerosis (MS), frozen sections of active chronic MS lesions were stained by the peroxidase-antiperoxidase technique for the demonstration of Ia antigen using a monoclonal antibody. Ia antigen could be localized on some endothelial cells and astrocytes as well as on macrophages. While Ia+ endothelial cells were randomly distributed throughout the CNS, Ia+ astrocytes were mainly found at the edge of active chronic MS lesions. These findings in MS suggest that antigen might be presented locally within the CNS on endothelial cells, where it might lead to new lesion formation by the initiation of a local immune response; on astrocytes, where it might be involved in lesion growth at the periphery.     

Multiple sclerosis: relevance of class I and class II MHC-expressing cells to lesion development

Expression of Class I (HLA-ABC) and Class II (HLA-Dr; Ia) major histocompatibility (MHC) antigens on endothelial cells and astrocytes was investigated in multiple sclerosis (MS) lesions of variable disease activity and in normal central nervous system (CNS) using immunocytochemical techniques. Findings were correlated to lesion pathology and to the presence and distribution of T cells, T cell subsets, and interleukin-2 (IL-2) receptor-bearing cells. HLA-ABC was present on virtually all endothelial cells in normal and pathologic tissue samples. Ia was absent from controls and was detectable on about 10% of CNS endothelial cells in MS. In normal CNS, astrocytes were Ia-negative and rarely expressed HLA-ABC. In MS, Class I and II MHC-positive astrocytes were found, and both displayed a high frequency in active lesions. Class I-reactive glia were primarily associated with T cell infiltrates and were less common in older lesions in which macrophages predominated. In contrast, Class II-positive astrocytes were found in all active MS lesions independent of the composition of inflammatory cells. Expression of HLA-ABC and Ia molecules on astrocytes in MS lesions could indicate their involvement in local presentation of antigen to cytotoxic (T8+) and helper/inducer (T4+) T cells, respectively. The observed distinct distribution patterns of HLA-ABC and Ia-positive astrocytes might suggest that cytotoxic T8+ cells are operative early during lesion development in MS. This could be followed by a more extensive Class II MHC-restricted helper T cell-mediated immune response which leads to selective destruction of myelin via activated macrophages.     

Multiple sclerosis: involvement of interferons in lesion pathogenesis

To investigate a possible role of interferons (IFNs) in lesion pathogenesis, central nervous system tissue from multiple sclerosis patients and control subjects was stained by immunocytochemical techniques in combination with monoclonal antibodies or polyclonal antisera for the demonstration of IFN-alpha, IFN-beta, and IFN-gamma. The results were correlated to lesion activity, to the presence of class I and class II major histocompatibility antigen-positive astrocytes, and to the composition of cellular infiltrates. IFNs were detectable in active but not in inactive chronic multiple sclerosis lesions. In acute multiple sclerosis plaques, all three types of IFN were widely distributed on glial elements and infiltrating cells. In active chronic multiple sclerosis, labeling of cells for IFN-alpha, IFN-beta, and IFN-gamma was most pronounced at the lesion edge and displayed distinct distribution patterns. Overall, IFN-gamma was more common than IFN-alpha and IFN-beta and was predominantly found on astrocytes. IFN-alpha was detectable mainly on macrophages. Distribution of IFN-beta partially overlapped with that of IFN-gamma and IFN-alpha, in that it was present on some astrocytes and on some macrophages. IFNs were rare in normal white matter remote from lesions and in the gliotic lesion center. Ia antigen and human leukocyte antigen ABC on astrocytes were distributed similarly to IFN-gamma and IFN-beta, respectively. These findings indicate that IFN-gamma may play a role in active lesion growth in multiple sclerosis, whereas IFN-alpha and IFN-beta may exert some local immunosuppressive effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Localization of the CD44 glycoprotein to fibrous astrocytes in normal white matter and to reactive astrocytes in active lesions in multiple sclerosis.

The CD44 antigen is a proteoglycan recently implicated in several adhesion events including that of lymphocytes to endothelium. The CD44 antigen, reactive with monoclonal antibody (MAb) 44D10, has been shown previously to be expressed in normal human white matter homogenates and to be found at higher concentrations in brain homogenates of victims of multiple sclerosis (MS). The cellular localization of CD44 in human brain of normal individuals and in those afflicted with MS has now been determined. Monoclonal antibody 44D10 reacted with astrocyte-like cells in 40 microns thick paraformaldehyde-fixed sections but not in thin (6 microns) fixed sections. A double labeling experiment performed on a frozen brain section with MAb 44D10 and rabbit anti-glial fibrillary acidic protein (GFAP), a cytoplasmic marker of astrocytes, confirmed the co-localization of these two antigens. The reactivity with brain tissue sections of a rabbit antiserum produced against lymphocyte-CD44 could be absorbed by a preparation of the CD44 glycoprotein, purified 2,100-fold from a white matter homogenate. The antiserum was shown by Western blot analysis to be specific for p80 glycoprotein in brain extracts derived from a normal and MS patients. This antibody reacted with fibrous astrocytes predominantly in white matter; staining was also noted in subependymal and subpial regions. Inhibition studies using a cellular radioimmunoassay indicated that the highest concentrations of CD44 in three MS victims were found in plaques, followed by periplaques and non-involved areas of white matter which were higher than normal white matter. Reactive astrocytes, identified in active lesions, expressed high levels of CD44 on their surfaces. Thus, CD44 is associated with astrocytes in human brain and the increased expression observed in MS brain may reflect activation and/or proliferation of astrocytes implicated in the pathogenesis of this disease.     

A role for CXCL12 (SDF-1alpha) in the pathogenesis of multiple sclerosis: regulation of CXCL12 expression in astrocytes by soluble myelin basic protein

The pathogenic mechanisms that contribute to multiple sclerosis (MS) include leukocyte chemotaxis into the central nervous system (CNS) and the production of inflammatory mediators, resulting in oligodendrocyte damage, demyelination, and neuronal injury. Thus, factors that regulate leukocyte entry may contribute to early events in MS, as well as to later stages of lesion pathogenesis. CXCL12 (SDF-1alpha), a chemokine essential in CNS development and a chemoattractant for resting and activated T cells, as well as monocytes, is constitutively expressed at low levels in the CNS and has been implicated in T cell and monocyte baseline trafficking. To determine whether CXCL12 is increased in MS, immunohistochemical analyses of lesions of chronic active and chronic silent MS were performed. CXCL12 protein was detected on endothelial cells (EC) in blood vessels within normal human brain sections and on a small number of astrocytes within the brain parenchyma. In active MS lesions, CXCL12 levels were high on astrocytes throughout lesion areas and on some monocytes/macrophages within vessels and perivascular cuffs, with lesser staining on EC. In silent MS lesions, CXCL12 staining was less than that observed in active MS lesions, and also was detected on EC and astrocytes, particularly hypertrophic astrocytes near the lesion edge. Experiments in vitro demonstrated that IL-1beta and myelin basic protein (MBP) induced CXCL12 in astrocytes by signaling pathways involving ERK and PI3-K. Human umbilical vein EC did not produce CXCL12 after treatment with MBP or IL-1beta. However, these EC cultures expressed CXCR4, the receptor for CXCL12, suggesting that this chemokine may activate EC to produce other mediators involved in MS. In agreement, EC treatment with CXCL12 was found to upregulate CCL2 (MCP-1) and CXCL8 (IL-8) by PI3-K and p38-dependent mechanisms. Our findings suggest that increased CXCL12 may initiate and augment the inflammatory response during MS.     

Astrocyte characterization in the multiple sclerosis glial scar

Introduction:  Dense astrocytic scarring in chronic multiple sclerosis (MS) plaques inhibits tissue repair. However, at the rim of a lesion the glial scar fails to form despite the presence of reactive astrocytes. Animal studies have shown that astrocyte antigen expression varies depending on astrocyte type and location. Characterization of human astrocytes in MS tissue may identify markers relevant to the glial scar.
Materials and methods:  Astrocyte antigenic phenotype was investigated in subventricular white matter by immunocytochemistry and Western blotting. Snap-frozen tissue from normal controls ( n  = 4), MS normal appearing white matter ( n  = 5) and lesions [acute ( n  = 7), subacute ( n  = 7) and chronic ( n  = 13)] was studied.
Results:  As expected, glial fibrillary acidic protein and vimentin expression was elevated in scar astrocytes. In addition there was increased expression of nestin, embryonic neural cell adhesion molecule, epidermal growth factor receptor, nerve growth factor and its receptor p75, and in a subpopulation of scar astrocytes, basic fibroblast growth factor.
Conclusion:  Changes in expression of proteins associated with development, growth factors and growth factor receptors are characteristic of the scar astrocyte phenotype in chronic MS lesions. These proteins may be of relevance to glial scar formation and tissue repair.     

Immunocytochemical characterisation of the immune reaction in the central nervous system in multiple sclerosis. Possible role for microglia in lesion growth

As there is evidence that in multiple sclerosis T-cell activation occurs in the central nervous system rather than outside, the inflammatory lesion may be extended through antigen presentation by cells at the edge of the plaque. In this study we present an immunocytochemical report on CNS tissue from an active case of MS, with an analysis of the distribution of CD4 and CD8 binding T cells and the expression of class I and II MHC determinants in plaques and white matter. Perivascular cuffs of early lesions, as judged by hypercellularity and minimal demyelination, contained activated T (Tac+) cells, which reacted with an anti-IL-2 monoclonal antibody. Thus sufficient T-cell growth factor would appear to be present to fuel the immune reaction in a growing lesion. The preponderance of T cells of the cytotoxic/suppressor (CD8) phenotype in the CNS parenchyma was found in conjunction with widespread staining of class I MHC antigen, a prerequisite for activity of cytotoxic T cells. Potential antigen presenting cells were demonstrated in MS plaques with a monoclonal antibody against the cytoplasmic, invariant chain of class II MHC. Macrophages and astrocytes, contributed to the staining in the hypercellular plaque border while the distribution of class II+ microglia in white matter suggest they may also be of importance in local antigen presentation.     

Immunohistochemical staining of human brain with monoclonal antibodies that identify lymphocytes, monocytes, and the Ia antigen

Using immunoperoxidase histochemistry, human brain sections obtained at biopsy were labeled with monoclonal antibodies which identify human lymphocytes subsets, monocytes, and the Ia antigen. Staining of a population of cells in white matter was present with the anti-Ia and the anti-M1 (monocyte-associated) antibodies but not with any of the 8 monoclonal antibodies which react with human T-cell subsets (anti-T1, 3, 4, 5, 6, 8, 10 and 12). The Ia antigen was present on 1–2% of cells in white matter, and approximately 5% of cells in white matter were M1-positive. Ia-positive cells demonstrated a pattern of diffuse surface membrane staining whereas the M1 antigen appeared to cluster at proximal cell processes. Definitive identification of these cells as microglial cells, astrocytes or oligodendrocytes was not possible.These findings demonstrate that: (1) cells which beam the Ia and M1 determinants can be found in histologically normal human white matter, and (2) human oligodendrocytes do not react with monoclonal antibodies (anti-T5 and anti-T8) that identify human suppressor/cytotoxic cells.     

The blood-brain barrier in cortical multiple sclerosis lesions

The blood-brain barrier (BBB) is composed mainly of specialized endothelial cells characterized by the presence of intercellular tight junctions. Additionally, perivascular cells, astrocytes, and surrounding basement membranes determine BBB integrity. BBB disruption is an early phenomenon in the formation of new white matter multiple sclerosis (MS) lesions; however, knowledge of the extent of BBB changes in gray matter MS lesions is lacking. Here, we studied several markers for BBB integrity in well-characterized brain tissue of patients with MS. Plasma protein leakage was enhanced in white matter lesions compared with that in normal-appearing white matter, whereas plasma protein leakage was absent in gray matter lesions. White matter lesions showed irregular basement membranes and parenchymal depositions of collagen type IV, whereas purely gray matter lesions lacked basement membrane alterations. Similarly, we observed no evidence for astrogliosis and tight junction changes in cortical MS lesions. Although BBB dysfunction is a common feature of white matter MS lesions, cortical MS lesions lack markers for BBB disruption or astrogliosis. Our data may indicate that BBB breakdown is not a critical event in the formation of gray matter MS lesions.     

Proliferating oligodendrocytes are present in both active and chronic inactive multiple sclerosis plaques

The proliferation marker Ki-67 labels cell nuclei in the G(1), S, M, and G(2) phases of the cell cycle. We used Ki-67 immunohistochemistry to quantify proliferating glial cells in brain tissue sections from twenty-four patients, comprised of multiple sclerosis, normal brains, and other neurological disease controls. Glial proliferation was greatly increased in MS lesions when compared with control brain white matter. Both actively demyelinating/early remyelinating plaques and chronic inactive plaques of long standing often displayed large numbers of glial cells in the proliferative cycle. The bulk of these proliferating cells were of oligodendroglial lineage in the MS plaques. Ki-67 positive macrophage/microglial lineage cells were largely restricted to acute lesions. The finding of increased numbers of proliferating oligodendroglia in most MS plaques, regardless of disease duration or activity state, indicates that the MS brain is capable of recruiting unexpectedly large numbers of new oligodendrocytes over long periods of time. The factors within the MS plaque microenvironment that provoke new oligodendrocyte generation and their subsequent loss still need to be identified.     

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.     

Fos RNA accumulation in multiple sclerosis white matter tissue.

In order to better characterize the molecular events that accompany lesion development in multiple sclerosis (MS), we studied the accumulation of RNA specific to the nuclear proto-oncogenes c-fos and c-myb in post mortem white matter brain tissue. RNA was prepared from plaque and periplaque regions of 6 different MS brains, from "normal" white matter regions of 3 MS brains and from 6 normal control samples. Quantitation of specific RNA corresponding to each proto-oncogene was performed by Northern blot hybridization and by scanning densitometry. Results indicate a 2-fold increase in c-fos RNA in MS white matter, compared to control tissue. No c-myb signal was identified in any sample. In situ hybridization studies confirmed the selective upregulation of c-fos RNA levels in MS tissue, and suggested that glial cells and not inflammatory cells were responsible for the enhanced c-fos signal. These results suggest that persistent glial cell activation is present within chronic MS lesions irrespective of whether the lesions are active (e.g., inflammatory) or inactive.     

Astrocytes in chronic active multiple sclerosis plaques express MHC class II molecules

To initiate the inflammatory cascade leading to demyelination in multiple sclerosis (MS) T cells have to recognize their specific myelin antigen, which needs to be presented in the context of major histocompatibility (MHC) class II molecules expressed on antigen presenting cells. Whether astrocytes can express MHC class II molecules in vivo is a controversial issue. We performed double labeling immunohistochemistry in postmortem samples from nine patients with MS, three patients with a cerebral infarction and six controls. Astrocytes in controls, in normal appearing white matter in MS, and at the boundary of infarctions were MHC class II negative. In contrast, a subset of astrocytes in active chronic plaques immunostained for MHC class II, indicating potential antigen presenting interactions of astrocytes in MS.     

Distribution of transferrin and ferritin binding in normal and multiple sclerotic human brains.

Delivery of iron to the brain traditionally has been considered the responsibility of transferrin. However, transferrin receptors in brain are located primarily within gray matter areas rather than in the iron rich white matter tracts. In this report we present the first demonstration of ferritin binding sites in human brain and provide evidence that these binding sites are primarily in white matter tracts. This distribution of ferritin binding is opposite of that seen for the distribution of the transferrin receptor in normal adult human brain. Ferritin binds to human brain tissue in a competitive and saturable manner with a dissociation constant of 0.35 nM and a binding site density of 116.7 fmol/mg protein. In brain tissue from multiple sclerotic (MS) patients the normal pattern of transferrin and ferritin binding distributions is disrupted. Ferritin binding is absent in the lesion itself and in the immediate periplaque region within the white matter but returns to normal as the distance from the lesion becomes greater. In direct contrast to ferritin binding, transferrin binding in the MS tissue is present in the white matter tracts, but only in the periplaque region. The periplaque region also contains transferrin receptor positive cells (as determined by immunocytochemistry) morphologically consistent with oligodendrocytes. Gray matter binding of transferrin in MS patients appears normal. These data provide the initial evidence of ferritin binding in human brain, address the enigma of the apparent absence of an iron delivery system to the iron-rich white matter, and suggest loss of ferritin binding is involved in or is a consequence of demyelination associated with MS.     

Tenascin-R and C in multiple sclerosis lesions: relevance to extracellular matrix remodelling

In the present study the distribution of the inhibitory extracellular molecules tenascin-R (TN-R) and tenascin- C (TN-C) was examined by immunocytochemistry during evolution of the multiple sclerosis (MS) lesion, in which astrogliosis is a prominent feature. Sections were cut from five control cases and from 22 blocks containing lesions representing different pathological stages in 18 cases of secondary progressive MS. Widespread expression of TN-R was found in the normal human central nervous system (CNS), while that of TN-C was in general restricted to white matter. In acute MS plaques however, there was a similar striking loss of both TN-R and TN-C up to the edge of the lesion, where the macrophage density is greatest, extending into the apparently normal white matter. In subacute lesions a TN-C and/or TN-R-immunopositive reactive astrocyte subpopulation was prominent, reflecting synthesis of extracellular matrix molecules. Both tenascins were expressed throughout chronic MS plaques at levels similar to those seen in adjacent white matter. The loss of TN-R and TN-C in acute plaques is indicative of enzyme-mediated breakdown of the matrix which may be a marker of blood-brain barrier breakdown and leucocyte extravasation. Subsequent production of tenascins by reactive astrocytes may result in glial scar formation impeding remyelination and axonal repair in MS lesions.     

Multiple sclerosis: a role for astroglia in active demyelination suggested by class II MHC expression and ultrastructural study

Central nervous system (CNS) tissue was studied by immunocytochemistry and electron microscopy from three cases of multiple sclerosis (MS) in which evidence of ongoing myelin breakdown could be documented. The study focussed upon the role of glial cells in the pathogenesis of demyelination. In acute MS, demyelination involved the vesicular dissolution of myelin from intact axons and a paucity of fibrillary astrogliosis. Foamy macrophages, many of them probably derived from transformed and recently proliferated microglia, contained recognizable myelin debris and lipid droplets and were abundant throughout the lesions. These cells formed the major phagocytic population and stained positively for class II major histocompatibility complex antigens (HLA-DR; Ia). In acute MS lesions, rounded astrocytes were encountered which possessed membrane-bound compartments enclosing phagocytosed fragments of myelin basic protein-positive debris. Despite the superficial resemblance of these cells to foamy macrophages, the presence of intermediate filaments, glycogen granules and diffuse glial fibrillary acidic protein positivity supported an astroglial identity. Astrocyte processes were involved in myelin removal and invested recently demyelinated axons. Hypertrophic fibrous astrocytes were common in chronic active lesions, were capable of myelin degradation and on occasion, contained myelin debris attached to clathrin-coated pits. These astrocytes were sometimes Ia+. Oligodendrocytes were depleted from the center of active lesions but were numerous at the lesion margin, suggesting survival and proliferation. They stained positively for myelin-associated glycoprotein, a marker for immature oligodendrocytes. However, they were invariably Ia-. The findings confirm and further support a role for the astrocyte as both an antigen presenting cell and a phagocyte in the CNS during MS.     

Insulin-like growth factor-I receptors in normal appearing white matter and chronic plaques in multiple sclerosis

Preclinical studies suggest that insulin-like growth factor-I (IGF-I) plays an important role in oligodendrocyte survival and myelination. We used human recombinant [¹²⁵I]IGF-I to study IGF-I receptors in post-mortem brain tissue from patients with multiple sclerosis (MS). In normal appearing white matter, we found that IGF-I receptor densities and binding characteristics were not different between MS patients and controls. In chronic plaques, histologically characterized by astrogliosis, we found densities of IGF-I receptors which were in the same range as those measured in the normal appearing white matter. In vitro studies have shown that IGF-I also acts as a mitogenic factor for astrocytes. Since MS lesions are rapidly invaded by reactive astrocytes, IGF-I may not only protect oligodendrocytes and stimulate remyelination but also enhance the astrogliosis that limits repair.