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.

Cytokine induction of heat shock protein expression in human oligodendrocytes: an interleukin-1-mediated mechanism.

In this study, we examined the role of cytokines, known to be in elevated levels in multiple sclerosis (MS) plaques, in regulating oligodendrocyte (ODC) expression of heat shock protein (hsp) in human brain-derived glial cell cultures. Using dual-stain immunohistochemistry, we initially compared the ability of a mixture of cytokines (IL-1 alpha, IL-1 beta, IL-2, IL-6, IL-8, TNF-alpha, TNF-beta, IFN-beta and IFN-gamma) with that of physical stimuli such as heat shock and peroxide, to increase cellular expression of the mainly inducible hsp72 species in mixed glial cell cultures (containing ODC, astrocytes and microglia). Similar to heat shock and peroxide, the cytokine mixture induced hsp72 expression only in ODC (70 +/- 5% vs. a baseline of 3 +/- 1% positive cells). When used individually, however, only IL-1 alpha (79 +/- 3%), IFN-gamma (70 +/- 2%) and TNF-alpha (65 +/- 5%) induced ODC hsp72 expression in mixed glial cell cultures. In purified ODC preparations, only IL-1 alpha induced hsp72 expression (84 +/- 4%). An IL-1 receptor antagonist (IL-1ra), abrogated hsp72 induction by IL-1 alpha (16 +/- 3%) as well as that due to IFN-gamma (14 +/- 1%) and TNF-alpha (13 +/- 2%) in mixed glial cell cultures. Furthermore, ODC express IL-1 receptors, detected by confocal laser scanning microscopy. Our data indicate that cytokines mediate hsp induction in ODC possibly via a final common pathway involving IL-1 binding to its receptor on ODC. Such interaction could enhance any putative ODC-immune interactions which are dependent on hsp molecule recognition.

Fas expression on human fetal astrocytes without susceptibility to fas-mediated cytotoxicity.

Fas (APO-1/CD95) is a cell surface receptor, initially identified in lymphoid cells, but more recently detected in the central nervous system under pathologic conditions. Ligation of the fas receptor by fas ligand or by agonist antibodies induces apoptotic cell death in most fas-expressing cells. In the current study, using dissociated cultures of human fetal central nervous system-derived cells, we detected fas expression on astrocytes but not on neurons. Such expression differs from our previous results using cultures of human adult central nervous system-derived cells, which demonstrated fas expression on oligodendrocytes but not on astrocytes; the oligodendrocytes were susceptible to cell death via this pathway. Using multiple assays of cell death, including nuclear propidium iodide and TUNEL staining to detect nuclear-directed injury, cytofluorometric propidium iodide inclusion, and lactate dehydrogenase release to detect membrane-directed injury, we found that fas ligation, however, did not induce cell death in the cultured fetal astrocytes. Cytokines that augmented (gamma-interferon) or inhibited (interleukin-4) fetal astrocyte proliferation did not alter fas expression or resistance to fas ligation. Cells obtained immediately ex vivo from human fetal but not from adult central nervous system tissue expressed fas; such expression was restricted to astrocytes as assessed by dual-stain immunohistochemistry. The fetal central nervous system cells did not express fas ligand. Our findings indicate that fas expression on central nervous system cells may reflect their state of maturity; expression may not, however, always be coupled to susceptibility to cell death via this pathway.

Ciliary neurotrophic factor selectively protects human oligodendrocytes from tumor necrosis factor-mediated injury.

Oligodendrocytes (OLs) and their myelin membranes are the apparent injury targets in the putative human autoimmune disease multiple sclerosis. The basis for this selective injury remains to be defined. OLs in vitro have been shown to be susceptible to both tumor necrosis factor (TNF) and non-TNF-dependent immune effector mechanisms. The former involves initial nuclear injury (apoptosis); the latter, when mediated by activated T cells, involves initial cell membrane injury (lysis). In the current study, we determined whether human adult CNS-derived OLs could be protected from the above immune effector mechanisms by selected neurotrophic factors (CNTF, BDNF, NGF, NT-3, and NT-4/5) or cytokines demonstrated to protect from human or experimental autoimmune demyelinating diseases (beta-interferon [IFN], IL-10, and TGF-beta). Nuclear injury was assessed in terms of DNA fragmentation using a DNA nick-end-labelling technique; cell membrane injury was assessed by lactate dehydrogenase or chromium 51 release. MTT and cell counting assays were used to assess cell viability and cell loss, respectively. Amongst the neurotrophic factors and cytokines tested, only CNTF significantly protected the OLs from TNF-mediated injury. CNTF also protected the OLs from serum deprivation-induced apoptosis. CNTF, however, did not protect the OLs from injury induced by activated CD4+ T cells. CNTF also did not protect human fetal cortical neurons from serum deprivation or TNF-induced DNA fragmentation, nor did it protect the U251 human glioma cell line from DNA fragmentation induced by a combination of TNF and reduced serum concentration in the culture media. Our results indicate that potential protective effects of neurotrophic factors or cytokines on neural cell populations can be selective both for cell type involved and mechanism of immune-mediated injury. CNTF is the protective factor selective for nuclear-directed injury of OLs.