Multiple Sclerosis

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Multiple sclerosis (MS) is a chronic, often progressive, inflammatory demyeli-nating disease of the CNS. Large numbers of immunocompetent cells are present in brain and spinal fluid and concentrations of immunoglobulins also are increased in these compartments. The target of the inflammatory response is presumed to be the oligodendrocyte-myelin complex. As a result, there is extensive, multifocal demyelination, little in the way of remyelination, and frequent astrocytic proliferation.

The evidence that MS is an autoimmune disease, or for that matter, an immunologically mediated disease, is circumstantial. Numerous laboratories tried to find immune responses to myelin and other antigens that were unique to patients with MS, but none succeeded. Persons with MS do have increased immune responses to a variety of myelin antigens, including myelin basic protein, proteolipid protein, and myelin/oligodendrocyte glycoprotein (18-24), and several of these are potent autoantigens capable of inducing EAE (25,26). However, it is not clear whether the heightened immune responses to these antigens are primary or secondary to the disease process.

A well-documented clinical phenomenon in MS is the association between disease activity and infections (27; personal observations). The study by Sibley and associates showed a clear association between antecedent infections, usually viral, and attacks or worsening of disease. Although antecedent symptoms of MS will worsen during the acute phase of an infection, especially if there is an associated fever, true disease exacerbations occur 3-14 days after the acute infection during recovery.

There are several mechanisms whereby infections could cause exacerbations of an inflammatory, possibly autoimmune, disease:

1. Infections result in increased concentrations of circulating cytokines. Some of these, such as tumor necrosis factor or interferon gamma, in addition to having direct effects on oligodendrocytes, could alter the blood brain-barrier, allowing easier entry of myelin-specific immunocompetent cells and/or antibodies. Cytokines could also nonspecifically activate antimyelin T cells resident within the CNS of patients with MS.

2. Infections result in immune responses to the infectious organism's stress proteins. These immune responses could cross react with phylogeneti-cally conserved stress proteins expressed by oligodendrocytes in areas of inflammation.

3. Immune responses to an infectious agent's stress proteins could cross react with myelin antigens, resulting in myelin destruction.

4. A combination of above effects may be important.

Different approaches were used to determine whether stress proteins, or immune responses to stress proteins, are involved in the pathogenesis of MS. These included studies on

1. Expression of stress proteins in the brains of persons with MS

2. Distribution of y8 T lymphocytes in MS brains

3. Immune responses to stress proteins in patients with MS

4. Studies on cross reactivity between stress proteins and CNS myelin

1. Expression of Stress Proteins in MS CNS

Several laboratories studied expression of stress proteins in areas of MS demy-elination. Selmaj and his associates (28,29) demonstrated expression of hsp 65 in immature oligodendrocytes at the edges of MS plaques. There also was constitutive expression of hsp 72 in MS and non-MS brain, especially in astrocytes. Wucherpfenning et al. (30) demonstrated expression of hsp 60 in foamy mac-

rophages at the edges of acute plaques and expression of hsp 90 in reactive astrocytes. As noted above, we utilized a panel of 20 monoclonal and polyvalent antibodies to human and mycobacterial stress proteins to immunocytochemi-cally stain fixed, frozen sections of normal and MS brains (1). Patterns of staining varied among antibodies. Some antibodies stained cell bodies of neurons and astrocytes, others stained neurofilaments, and still others stained normal myelin. No increased staining was noted at the edges of either acute or chronic plaques and demyelinated areas did not stain at all.

Using a different approach, a number of laboratories studied the expression of stress proteins by glial cells in vitro. Again, results varied with the techniques used and the antibodies used for the assays. Selmaj et al. (29), Freedman et al. (31), and Satoh et al. (32) identified oligodendrocytes as cells expressing stress proteins. Selmaj et al. detected constitutive hsp 65 expression in oligodendrocytes but not astrocytes, yet noted some hsp 70/72 expression in astrocytes. Satoh et al. detected constitutive expression of hsp 60 in cultured murine oligodendrocytes and "marginally detectable" levels in most astrocytes. Expression of hsp 60 was increased in oligodendrocytes following heat stress. Freedman et al. detected constitutive expression of hsp 60 and hsp 70/72 expression in oligodendrocytes with increased expression of hsp 70/72 following heat stress. No astrocytes expressed hsp 70/72, and only a small percentage of astrocytes expressed hsp 60 proteins. In contrast, Marini and coworkers (11), using tissue cultures of rat astrocytes and neurons, demonstrated heat-inducible expression of hsp 70 stress proteins predominantly in astrocytes, but oligodendrocytes may have been absent from these cultures. Using purified cultures of rat astrocytes, Dwyer et al. (33) demonstrated that such cells synthesized stress proteins of 30-34, 68, 70, 89, and 97 kDa and that exposure to heat readily induced expression of hsp 65.

Since MS is an inflammatory disease, concentrations of cytokines are increased in MS brain (34,35). De Souza et al. (36) studied the effects of cytokines on the expression of stress proteins in mixed cultures of human glial cells (33, 34). A mixture of cytokines induced expression of hsp 72, predominantly in oligodendrocytes. The specific cytokines involved in this induction were inter-leukin-1 (IL-1), interferon gamma (INF-y), and tumor necrosis factor alpha (TNF-a). Birnbaum et al. (manuscript in preparation) prepared cultures of purified murine astrocytes and exposed them to heat shock with or without exposure to a mixture of cytokines. Cytokines alone induced small amounts of hsp 70/72. However, the combination of heat shock and cytokine exposure augmented expression of hsp 70/72 2- to 11-fold. The particular cytokines involved in this were INF-y and TNF-a.

It is apparent from the above data, depending on the specificities of the antibodies used and the particular methods, results can vary widely. Nevertheless, there is good evidence that some antibodies to stress proteins bind to nor mal myelin, whereas others bind to oligodendrocytes or astrocytes. In addition, the inflammatory milieu within MS brains increases the expression of stress proteins within glia, increasing the possibility that immune responses to infectious agents' stress proteins could cross react with their human homologues.

2. The Distribution of y/8 T Cells in MS Brains

Although the vast majority of T cells responding to stress proteins express antigen-specific receptors composed of a and p chains, their numbers comprise only a small fraction of the total a/p-expressing T-cell pool. In contrast is the population of T cells expressing antigen-specific receptors composed of y and 8 chains. These cells comprise only about 1-5% of the total T-cell pool, yet a majority of this cells respond to stress proteins (37-41). Investigators, therefore, studied the association between areas of MS demyelination, expression of stress proteins, and the distribution of y/8 T cells. Their assumption was that the presence of y/8 T cells could indicate the presence of an immune response to stress proteins in that region.

Selmaj et al. (28) and Wucherpfenning et al. (30) found accumulations of y/ 8 T cells in areas of MS demyelination. Selmaj et al. found them in regions of chronic demyelination in proximity to immature oligodendrocytes that expressed hsp 65. Wucherpfenning et al. found y/8 T cells in regions of acute demyelination. The 60- and 90-kDa stress proteins were found in regions of normal myelin but were "overexpressed" in acute MS plaques. Cells expressing these stress proteins were foamy macrophages and reactive astrocytes, respectively. On the basis of sequence analyses, Wucherpfenning postulated there were oligo-clonal expansions of y/8 cells in MS brains, but since the repertoire of T-cell receptors in this population is already restricted, these data do not necessarily indicate reactivity to a single antigen. Hvas and coworkers (42) looked for the presence of y/8 cells in MS brains utilizing the technique of polymerase chain reaction amplification of T-cell receptor mRNA. They found y/8 messages in all of their 12 MS brains, in one of their 10 nonneurological disease controls, and in 2 of their three neurological controls. Sequence analyses of the MS brain-derived cDNA failed to show evidence of clonal expansions of particular populations of y/8 cells. Two groups studied y/8 T cells in MS and control spinal fluids. Shimonkevitz et al. (43) found increased numbers of y/8 cells in the spinal fluid of patients with MS with recent onset of disease but not in patients with chronic MS or in disease controls. Sequence analyses of the junctional regions of these cells' antigen receptors suggested an oligoclonal expansion of this population, perhaps in response to a particular antigen. Perella et al. (44) found equivalent numbers of y/8 T cells in MS and other neurological disease (OND) spinal fluids, and did not think that y/8 cells had a unique role to play in the pathogenesis of MS.

Since y/8 T cells are present in brains of MS and other neurological disease brains and spinal fluids, their presence per se is not disease specific. However, their presence does suggest an in situ immune response to stress proteins. There may be disease specificity in these responses, since it is possible that the antigens stimulating these cells may be different in persons with MS than in persons with OND.

3. Immune Responses to Stress Proteins in Patients with MS and Controls

Work in this area can be divided into studies involving cellular immune responses and those involving humoral responses to stress proteins.

Two groups that described cellular immune responses to heat shock proteins in persons with MS and OND are Salvetti et al. (45) and our laboratory (46). Salvetti et al. studied peripheral blood T-cell proliferative responses to recombinant hsp 65 and hsp 70 from M. bovis in 31 persons with MS, 19 individuals with OND, and 19 normal controls. Proliferative responses to hsp 70 were significantly more frequent in persons with MS compared with OND and healthy controls. Responses to hsp 65 were equivalent in the three groups. Lines of T cells were established from 10 patients with MS and 12 healthy controls using PPD as the antigen. Again, hsp 70-reactive lines were significantly more common in patients with MS than in healthy controls. Interestingly, cytofluorometric analyses of PPD-responsive lines revealed that only a minority of responding cells expressed y/8 T-cell receptors. Our laboratory studied T-cell proliferative responses to mycobacterial stress proteins, tetanus toxoid, and recombinant hsp 65 from M. leprae. The T cells were concurrently collected from the peripheral bloods and spinal fluids of 20 persons with MS and 9 persons with inflammatory neurological diseases other than MS. Cells were cultured in vitro and stimulated with the above antigens. Significantly increased spinal fluid lymphocyte proliferative responses to mycobacterial sonicate, relative to responses from paired peripheral blood lymphocytes, were present in 14 of the 20 specimens from patients with MS compared with 2 of 9 specimens from patients with other neurological diseases (p < 0.025). Spinal fluid lymphocytes also responded to tetanus toxoid, but differences between blood and spinal fluid were not statistically significant. Lymphocytes from one patient with MS responded only to recombinant hsp 65. When patients with MS were classified according to duration of disease, 9 of 10 with duration less than 2 years had spinal fluid T cells responding to M. tuberculosis compared with 5 of 10 with disease longer than 2 years (p < 0.012). These data supplement the observations of Shimonkevitz et al. (43), who described increased numbers of activated y/8 cells predominantly in spinal fluids from persons with recent-onset MS.

Additional data in support of the hypothesis that immune responses to stress proteins play a role in autoimmune demyelination are the observations of Mor et al. (47). These investigators prepared T cells from the spinal cords, blood, spleen, and lymph nodes of rats during the acute phase of EAE or during recovery from EAE. Using limiting dilution analyses, they determined the frequency of T-cell responses to the myelin protein, myelin basic protein (MBP), and recombinant hsp 65 and hsp 70. As expected, responses to MBP were enriched in the spinal cords of rats during and after acute EAE. However, there was also enrichment of T cells responsive to hsp 65. T-cell lines established from spinal cord lymphocytes responded to MBP, hsp 65, and hsp 70. When EAE was induced from an anti-MBP-responsive T-cell line, similar patterns of enrichment for MBP and hsp 65-reactive T cells were noted, indicating that responses to stress proteins occurred in the absence of exposure to adjuvant mycobacteria.

Studies of humoral immune responses to stress proteins in MS are mainly the work of in our laboratory (48,49) and Freedman et al. (50). We used immu-noblots to detect antibodies to native and recombinant mycobacterial stress proteins and to bacterial stress proteins in spinal fluids and paired sera from persons with MS and OND. Antibodies to many stress proteins, including those of the 60-kDa and 70-kDa families, were present in CSF and sera from all patient groups. Patterns of antibodies varied between CSF and sera and between patients, but no disease-distinctive pattern was seen. When anti-stress protein antibodies were analyzed for isotypes, patients with MS had higher concentrations of anti-stress protein immunoglobulin A (IgA) antibodies than did patients with OND. This suggested an in situ synthesis of such antibodies within the CNS in persons with MS. Freedman et al. studied antibody concentrations to recombinant hsp 60 using an ELISA. Titers of antibodies in MS spinal fluids were significantly higher than those seen in persons with OND, and the higher titers correlated with the presence of oligoclonal bands in MS CSF but not OND CSF.

4. Studies on Cross Reactivity Between Stress Proteins and CNS Myelin

As noted above, certain anti-stress antibodies stain normal myelin. We studied this phenomenon using immunoblots (Birnbaum, et al., submitted for publication). A panel of 20 anti-stress protein antibodies was assayed for the ability to bind to myelin proteins separated on SDS-PAGE. Three monoclonal antibodies specific for either mycobacterial or human stress proteins stained bands of normal myelin proteins. One murine monoclonal to M. leprae hsp 65, IIH9, stained a 44- to 46-kDa doublet. This doublet was the same size as the myelin protein 2', 3'-cyclic 3'-nucleotide phosphodiesterase (CNP). To study this observation further, purified CNP was used in immunoblots. IIH9 bound to this protein in a pattern identical to that seen with whole myelin. A nonapeptide region of sequence homology was identified between the epitope of hsp 65 rec ognized by IIH9 and CNP. This peptide was synthesized and used in immunoblots. IIH9 strongly bound to this peptide, proving that this region of sequence homology was responsible for the observed cross-reactivity.

In a more circumspect manner, data from several other laboratories suggest that cross reactivity occurs between stress proteins and myelin. In 1975, Wis-niewski and Bloom (51) noted primary demyelination in the brains of guinea sensitized to tuberculin, who were then subsequently challenged with PPD in-tracranially. The investigators interpreted these data to indicate that demyelination was the result of a passive bystander effect induced by the localized delayed hypersensitivity response. In retrospect, an alternative explanation is that demyelination occurred because of a specific cross reactivity between myelin and PPD. Additional observations that support this alternative conclusion come from several different laboratories (52-55), all of them demonstrating that exposure of EAE susceptible animals to either Bordetella pertussis or M. tuberculosis rendered them highly resistant to subsequent development of EAE.

It thus appears that at both the humoral and cellular level there is cross reactivity between infectious agents' stress proteins and normal myelin components.

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