Reactive gliosis is a characteristic response of astrocytes to inflammation and trauma of the central nervous system. To investigate whether soluble factors (cytokines) from inflammatory mononuclear cells that accumulate at lesion sites can provide the cellular signals to initiate gliosis and to identify such cytokines, we have tested and found that supernatants derived from subsets of activated human T lymphocytes (CD8' or CD41) are potent mitogens for cultured human adult astrocytes. This effect is blocked by a neutralizing antibody to -interferon (IFN). Recombinant A consideration in relating relevance of in vitro proliferation results to reactive gliosis in vivo is that gliotic scars seldom develop after insults to the embryonic brain (22-24) but are common features in adult brain injuries. Thus, it becomes important to assess proliferation of astrocytes derived from adult animals, rather than from neonatal ones. We have adopted such a strategy by using adult human astrocytes isolated from surgical brain biopsies and have addressed the following questions. Do activated human T lymphocytes produce sufficient cytokines to induce proliferation of cultured adult human astrocytes; if so, which cytokine (s) (see Results), were treated with trysin and seeded on poly(L-lysine)-coated (10 jag/ml) 9-mm Aclar fluorocarbon coverslips at 10,000 cells per coverslip. These mixed cells were used for the present study; methods for eliminating microglia from rodent glia cultures (leucine methyl ester and silica ingestion) (28-30) were not effective for human preparations. Culture medium was Eagle's minimum essential medium supplemented with 5% (vol/vol) fetal calf serum, Gentamicin (20 gg/ml), and dextrose (1 mg/ml) (all from GIBCO).

T cells are postulated to contribute to the injury of the oligodendrocyte-myelin complex underlying the demyelinating disease multiple sclerosis (MS). The apparent lack of class I or II major histocompatibility complex (MHC) expression in situ on human oligodendrocytes and the consistent failure to identify a universal myelin antigen in MS suggest that the immune damage might be mediated by effector T cells that are capable of reacting in an antigen-nonspecific and possibly MHC-unrestricted manner, such as T cells expressing the gamma-delta T-cell receptor. Since gamma-delta T cells are reported to be present in MS plaques and an increased number are found in the cerebrospinal fluid of patients with MS, we directly examined whether gamma-delta T cells are capable of inducing injury to human oligodendrocytes. We found, using a 6-hour 51Cr release assay, that oligodendrocytes cultured from surgically resected human brain specimens were effectively lysed in a dose-dependent manner by human gamma-delta T cells (28 +/- 5% mean specific lysis, n = 6, at an effector-target ratio of 20:1). Although heat shock protein HSP72, a putative gamma-delta T-cell recognition molecule, could be induced in vitro in our oligodendrocytes, an antibody to HSP72 did not inhibit gamma-delta T cell-mediated lysis of oligodendrocytes. These results suggest that gamma-delta T cells gaining entry into the central nervous system may be deleterious to oligodendrocytes and thus may contribute to the pathogenesis of MS.

Using immunohistochemical techniques, we examined major histocompatibility complex (MHC) antigen expression on astrocytes, oligodendrocytes, and macrophages-microglia derived from surgically resected tissue from young adults and maintained in dissociated cell cultures supplemented with either fetal calf or human AB serum. The majority of these cells in culture expressed class I MHC antigens. MHC class II expression was observed on only a restricted proportion of astrocytes either under basal or induction conditions (gamma-interferon, activated lymphocyte supernatants), on the majority of macrophages-microglia under inducing conditions, and not on oligodendrocytes. MHC class II expression on astrocytes in culture did not correlate with the extent of in situ gliosis or with in vitro cell morphology. MHC antigen expression was not detected in situ immunohistochemically. These data extend observations on the dissociation of in vivo and in vitro expression of MHC antigens on glial cells. The apparent greater expression of MHC class II antigens on macrophages-microglia compared to astrocytes raises the issue of the relative roles of each of these cell types in promoting immune reactivity under pathologic conditions.

The majority of human oligodendrocytes in enriched glial cell cultures expresses class I major histocompatibility complex (MHC) antigens. We used a 51Cr release assay to study the susceptibility of oligodendrocyte-enriched glial cells to MHC-restricted and non-restricted immune-mediated cytolysis. Mitogen-activated mononuclear cells induced significant lysis in a lectin-dependent cytotoxicity assay. Mononuclear cells allo-activated in a one-way mixed lymphocyte culture with E- cells from the glial cell donor induced a significantly higher degree of oligodendrocyte cytolysis than mononuclear cells activated with E- cells bearing MHC-class I antigens discordant with the glia. Cytolysis by alloactivated unfractionated lymphocytes and by purified CD8+ lymphocytes was reduced by an anti-class I antibody (W6/32). Our findings suggest that human oligodendrocytes can be susceptible targets for MHC class I-restricted lysis.

Expression of Class II major histocompatibility complex (MHC) antigens on astrocytes has been implicated as contributing to the immune responses characteristic of chronic autoimmune diseases of the central nervous system. We examined the properties and regulation of HLA-DR on cultured human adult astrocytes. We found that a proportion of human astrocytes from each of fifteen individual donors expressed HLA-DR under basal culture condition; while this proportion differed among the human subjects (range 3-65%), the results for each individual remained relatively constant when analyzed at several time points (up to 125 days in vitro). Attempts to modulate HLA-DR expression by a variety of cytokines likely to be present in inflammatory infiltrates in the brain showed that only gamma-interferon could increase the proportion of human astrocytes that expressed HLA-DR. Whether the variability of HLA-DR expression on astrocytes between different individuals reflects a genetic trait which can influence susceptibility to autoimmune central nervous system diseases remains to be determined.