53.1. Serum myelin degrading Eactors
In vitro myelinotoxic sera effects are not just confined to MS as activity has been reported with sera from healthy individuals and other neurological diseases (see Table 5.1). Of the OND patients* sera tested, myelin degradation was only seen with serum from one of the three GBS patients and with one of two sera from patients with peripheral neuropathy. It has previously been reported that sera from patients with GBS cause complement dependent in vitro demyelination of both PNS and CNS organotypic cultures (Cook et a/., 1971; Dubois- Dalq et a/., 1971; Bradbury et a/., 1985). Sanders and colleagues (1986) reported the finding of terminal complement C5b-9 complexes in the CSF of 90% of GBS patients tested. In comparison, 75% of MS patients’ CSF contained these complexes but at a lower concentration and only 27% of CSF from non-infiammatoiy OND patients contained the C5b-9 complexes. They suggested that as it has been shown that complement-fixing peripheral nerve myelin antibodies are detected in 100% of patients with acute monophasic GBS which correlates with the clinical course of the disease, then this finding of C5b-9 complexes in the CSF may indicate that terminal complement components are participating in the peripheral myelin damage which occurs in GBS thereby implicating a possible role of serum complement in demyelination. Reduced levels of CSF C9 have been documented in MS patients as compared to OND and this was thought to reflect the possibility that local consumption of the complement terminal component may indicate the involvement of MAC in tissue damage (Compston et aL, 1986b).
Demyelinating activity has also been reported with sera from MND patients (Hughes and Field, 1967; Ulrich and Lardi, 1978) although none of the six serum samples tested on isolated myelin caused a significant loss in CNPase activity. Demyelinating activity of OND sera has not only been demonstrated with GBS and MND sera as Ulrich and Lardi (1978) showed that 49% of sera from a wide range of different OND’s produced in vitro demyelination including sera from patients with Parkinson’s disease, cerebral tumours, epilepsy, viral meningitis, amyotrophic lateral sclerosis, headaches and other diagnosis. In comparison only 14% of MS sera had demyelinating properties in the same assay system. Whether serum induced in vitro demyelination is an indicator of m vivo demyelination, as was previously thought, is probably not so and is more likely to be a reflection of chronic CNS
disease. In diseases where there is CNS damage such as in stroke patients who have suffered focal CNS damage anti-CNS antibodies may be produced along with toxic products of cellular breakdown which could cause in vitro demyelination (Bradbury et cd., 1985).
As a further control RA sera was tested as this is, like MS, an inflammatory condition thought to have an autoimmune basis. A higher percentage of RA sera with degradative activity was seen (15%, 2/13) than with any of the other patient groups as none o f the MS sera (n=40) and 8% of the OND sera (2/25) tested caused myelin degradation. Indicating that serum induced myelinotoxicity is not just confined to diseases of the CNS but can also be caused by chronic inflammatory conditions such as RA. Finally in conflict with the reports that healthy individuals sera is capable of in vitro demyelination (Hughes and Field, 1967; Silberberg et a l, 1984; Bradbury et a l, 1985), none of the sera from ten healthy individuals that were tested on whole myelin preparations caused any significant loss in CNPase activity.
As can be seen with the results presented here and those of others, serum induced in vitro demyelination is not a specific property of MS sera but has been previously shown to be produced by MS and OND and even healthy individuals' sera. Using isolated myelin, in vitro myelin degradation was rarely seen, with a low percentage of OND and RA sera causing a significant loss of myelin CNPase activity and no myelinotoxic effects being produced by MS and healthy individuals sera (see Table 5.1).
The general lack of serum induced myelin degradation in this assay system suggests that the previously reported sera induced demyelinative effects may have been due to its action on the oligodendrocyte rather than directly on myelin. Only direct damage to myelin can be quantified in the assay system described here whereas problems of incompatibility could occur with the heterogeneic systems used by others, as human serum was added to rodent myelinated cultures. Nevertheless, the fact that RA and OND sera can induce significant losses in CNPase activity of myelin in vitro suggests that serum induced myelin degradation may be a non-specific indicator of chronic disease with a variety of different mechanisms being involved (antibody, complement, cytokines or degradative enzymes secreted by circulating leukocytes). This however, does not explain why healthy control sera in some reports has been able to induce demyelination. It has been suggested that serum induced demyelination (all disease states) could be due to activation of the alternate complement
pathway (Silberberg et al., 1984) as myelin can activate complement directly without the involvement of immunoglobulin (Vanguri et al., 1982).
S 3 2 , Cellular myelin degrading factors
It has been demonstrated that PBMC from 42% of MS and 58% of RA patients can cause a significant release of CNPase from myelin. In contrast, only 10% of OND cells caused a significant loss in CNPase from the myelin, and healthy individuals' PBMC samples possessed no myelin degrading ability. Thus the action of cells or their products is not restricted to MS but is probably a non-specific cellular response shown by chronically stimulated immune cells occurring in inflammatory diseases. In the OND control group, of the two GBS samples tested one caused in vitro myelin degradation. It has previously been demonstrated that PBMCs and a factor ^ th e siz e d in vitro by these cells had a myelin-destructive effect on cultures of spinal ganglia (Cook et al., 1971). The role of lymphocytes in the pathogenesis of GBS has been implied as lymphocytic infiltrates have been seen associated with myelin destruction in spinal nerve roots and circulating lymphocytes are sensitised to peripheral nerve myelin (Wisniewsl^ et al., 1969). More recently it has been shown that, as in MS, there are circulating activated T cells in the blood of GBS patients as assessed by augmented expression of HLA-DR antigen, IL-2 receptor and also increased levels of serum ID 2 and the soluble IL-2 receptor have been seen (Hartung and Tokya, 1990). Circulating monocytes have also been shown to be activated in GBS patients which may contribute to the pathogenesis of the disease (Hartung et al., 1991). Therefore as in MS, the demonstration of myelinotoxic activity by circulating mononuclear cells may imply a role for these cells in myelin destruction in vivo.
There are many similarities between the immunological abnormalities seen in MS and RA. Rheumatoid arthritis is a chronic inflammatory disease with autoimmune features which are primarily localised to the synovial joints. The joints are characterised by infiltration of leucocytes many of which show signs of activation (HLA class II expression). There is intra joint IgG synthesis with production of oligoclonal bands. Oligoclonal anti-measles antibody
represent a minor fraction of the oligoclonal IgG however, the production is not compartmentalised to the joint as the antibody bands have identical migration patterns in synovial fluid and serum (Cruz et al, 1991). As well as elevated class II expression on infiltrating macrophages and T cells many resident cells such as endothelial cells and
fibroblasts are activated which is of critical importance for antigen presentation and the perpetuation of an immune response. Both the infiltrating and resident cells are capable of producing large amounts of inflammatory mediators such as prostaglandin Ej (P G E j, degradative enzymes, reactive oxygen species and cytokines such as I l^ la and 6, TNF-a, H r 6, n ^ 8 and GM-CSF (Brennan et al., 1990). These cytokines may be responsible for many manifestations of the disease process, including bone and cartilage destruction (Bertolini et al., 1986) as well as the persistence of the inflammatory state by attracting other inflammatory cells to the joint and the augmentation of HLA class II expression (Haworth et a l, 1991).
The demonstration of in vitro myelin degradation by both RA and MS PBMC may therefore indicate that activated cells, both lymphocytes and monocytes, which are producing inflammatory mediators that could lead to tissue damage whether it be in the CNS or the joint have entered the circulation and are equally able to cause non-specific myelin damage in vitro.
In the next chapter an examination of the MS and RA PBMC cell type / factor causing in vitro myelin degradation will be performed as this may provide an insight into the pathogenesis of MS and RA.
CHAPTER SIX