Multiple sclerosis: Fas signaling in oligodendrocyte cell death

Abstract

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.

Figure 1

Expression of fas and related molecules in normal and MS CNS tissue. (a) Normal human white matter reacted with M3 anti-fas mAb. Note the faintly positive cells (brown) that have the morphology of oligodendrocytes. Frozen section: DAB-reacted and counterstained with hematoxylin. (b) Normal human white matter immunoreactive for fasL. Note the faint positivity on the fine processes of the ramified microglial cells. Frozen section: DAB and hematoxylin. (c) White matter adjacent to a chronic active MS lesion shows numerous fas-positive interfascicular oligodendrocytes (brown). DAB and hematoxylin. (d ) Detail of fas-positive oligodendrocytes from the edge of a chronic silent MS lesion. Note the typical bipolar outline of the cells. DAB and hematoxylin. (e) Interfascicular oligodendrocytes in white matter adjacent to a chronic silent MS lesion show positive immunoreactivity for Leu-7. Similar, but less intense staining was obtained with anti-CNPase antiserum. DAB and hematoxylin. (  f  ) Double-staining of interfascicular oligodendrocytes from white matter adjacent to a chronic silent lesion with M3 and Leu-7 (Brown and blue, respectively). DAB and NBT/BCIP. No counterstain. (  g) The edge of a chronic active MS lesion shows fas-positive infiltrating cells around a blood vessel. DAB-immunoreacted, no counterstain. (h) Microglial cells at the periphery of a chronic silent MS lesion display intense immunoreactivity for fasL. DAB and hematoxylin. Original magnifications: (A, D, F, G) ×750; (B, C, E, H) ×300.

Figure 2

Fas expression on human adult CNS-derived oligodendrocytes as assessed by confocal laser scanning microscopy. Human oligodendrocytes were maintained in 5% serum-supplemented culture medium for 3 wk and assessed for (A) fas immunoreactivity (green) and (B) CNPase immunoreactivity (red). (C) Double-staining is depicted by superimposing the fas (green) and CNPase (red) images, in which yellow indicates colocalization of the fas and CNPase signal. (D) An irrelevant IgG₁ mAb isotype control for oligodendrocyte immunostaining.

Figure 3

Fas expression on human adult CNS-derived astrocytes, microglial cells, and known fas-expressing (U251 glioma cells, Jurkat T cells, and U937 myeloid leukemia cells) and fas-negative cells (L929 fibroblast cells). Panels A and B represent adult astrocytes: (A) lack of fas immunoreactivity on astrocytes and (B) GFAP immunoreactivity of the same field as in A to identify astrocytes. Panels C and D represent adult microglial cells. (C) lack of fas immunoreactivity on microglia and (D) Leu M5 immunoreactivity of the same field as in C to identify microglia. The remainder of the panels represent fas immunoreactivity on (E ) U251 glioma cells, (F) Jurkat T cells, (G) U937 myeloid leukemia cells, and (H ) L929 fibroblast cells. Original magnifications: (A, B, E, H) ×400; (C, D, F) ×250.

Figure 4

Mean LDH release in units per milliliter (unshaded bars) and mean percentage of fragmented nuclei as assessed by PI staining (shaded bars) for the indicated cell targets treated with anti-fas IgG₁ (M3) (25 μg/ml; 24 h) mAb followed by cross-linking with anti-IgG₁ mAb (1:100 dilution; 24 h). Mean LDH release for control target cells or target cells treated with anti-fas (M3) mAb alone or nonactivating fas (M33) mAb followed by cross-linking with anti-IgG₁ or anti-IgG₁ alone was <200 U/ml. n represents the number of LDH experiments performed; for PI staining studies, n = 3. For LDH and PI studies, each test condition was assessed in triplicate per experiment.

Figure 5

Induction of DNA fragmentation in human adult CNS-derived oligodendrocytes, as assessed by the TUNEL technique (A) after exposure to C-2 ceramide (50 μM) for 18 h. B represents oligodendrocytes under control culture conditions. Original magnification ×250.