Multiple sclerosis: Oligodendrocytes in active lesions do not express class II major histocompatibility complex molecules

Journal of Neuroimmunology, 25 (1989) 261-266

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Elsevier JNI 00911

Short Communication

Multiple sclerosis: Oligodendrocytes in active lesions do not express class II major histocompatibility complex molecules Sunhee C. Lee * and Cedric S. Raine Departments of Pathology (Neuropathology) and Neuroscience, The Rose F Kennedy Center for Research in Mental Retardation and Human Development, Albert Einstein College of Medicine, The Bronx, N Y 10461, U.S.A.

(Received 8 August 1989) (Revised, received 13 September 1989) (Accepted 19 September 1989)

Key words: Multiple sclerosis; Oligodendrocyte; Plaque; Demyelination; Major histocompatibility complex class II; Myelin-associ-

ated glycoprotein; Remyelination

Summary The expression of major histocompatibility complex (MHC) molecules by oligodendrocytes has been proposed as evidence for their involvement in the multiple sclerosis (MS) lesion although the literature on the subject is controversial and based largely upon observations in vitro. With a modified immunocytochemical procedure on 1 ffm epoxy sections, the present study has examined the expression of class II M H C molecules (Ia) on cells within actively demyelinating lesions in a central nervous system biopsy from a case of acute MS. White Ia was readily demonstrable on microglial cells and astrocytes, it was never detected on adjacent surviving oligodendrocytes. Unexpectedly, in parallel sections, the oligodendrocytes stained positively for myelin-associated glycoprotein, a marker for immature oligodendrocytes. The unequivocal lack of Ia expression by oligodendrocytes in MS makes it unlikely that they serve as immunomodulators in lesion pathogenesis.

Introduction Selective depletion of oligodendrocytes is a common feature of the demyelinated plaque of

Address for correspondence: Dr. Cedric S. Raine, Department of Pathology, K433, Albert Einstein College of Medicine, 1300 Morris Park Avenue, The Bronx, NY 10461, U.S.A. * Present address: Department of Pathology (Neuropathology), University of Maryland School of Medicine, 22 S. Greene Street, Baltimore, MD 21201, U.S.A.

multiple sclerosis (MS), the paradigm of the inflammatory demyelinating diseases of the human central nervous system (CNS) (Raine, 1985). Since the oligodendrocyte is responsible for the production and maintenance of CNS myelin, its primary involvement in lesion pathogenesis in MS has been the recurrent theme of a number of hypotheses which have variably attributed the cell's demise to enzyme-, antibody- or cytokine-mediated mechanisms (Lumsden, 1955; Abramsky et al., 1977; Robbins et al., 1987), cross-antigenicity with T cell epitopes (Oger et al., 1982), or expression of

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major histocompatibility complex (MHC) molecules (Suzumura et al., 1986). In this regard, class II M H C (HLA-DR; Ia) has been demonstrated on CNS cells in MS (Traugott et al., 1983, 1985a; Hauser et al., 1986) and this has brought into focus the possibility that CNS elements might under certain circumstances serve as antigen presenting ceils (APCs), presenting antigen in the context of Ia to specifically sensitized, CD4 + (helper) T cells (Allen, 1987). While Ia has been demonstrated in MS lesions on astrocytes, microglial cells (resident macrophages in the CNS) and endothelial cells, whether it occurs on oligodendrocytes remains unknown and immunocytochemical studies to date have only employed frozen sections in which cellular identification can be difficult. Studies on Ia expression by oligodendrocytes in vitro have produced conflicting results (Wong et al., 1984; Kim, 1985; Suzumura et al., 1986). The presence of Ia molecules on oligodendrocytes in MS might have important pathogenetic ramifications in that it could confer upon these cells the role of an APC or render them vulnerable to immunologic attack by their bearing an inappropriate surface antigen.

Materials and methods

For the present study, a CNS biopsy from an 18-year-old woman with radiologically-documented white matter disease of about 12 weeks duration was found to contain actively demyelinating lesions consistent with acute MS (Lee et al., in press). For immunocytochemistry, 1 btm epoxy sections of glutaraldehyde-fixed, postosmicated lesion material were etched with sodium ethoxide and reacted with mouse monoclonal antibodies to human H L A - D R (Dako, Carpinteria, CA, U.S.A.; 1 : 50) and human CNS myelin-associated glycoprotein (MAG (GEN-S3); 1 : 100; provided by Dr. N. Latov, Columbia University, New York City, U.S.A.), and rabbit anti-human glial fibrillary acidic protein antiserum (GFAP; 1 : 100; provided by Dr. J.E. Goldman, Columbia University, New York City, U.S.A.) using a conventional three-step peroxidase-antiperoxidase technique. The anti-MAG antibody has been fully char-

acterized elsewhere (Nobile-Orazio et al., 1984). The anti-HLA-DR monoclonal is marketed by Dako as a marker for Ia in formalin-fixed, paraffin-embedded tissue. Negative controls consisted of incubation with normal serum or phosphatebuffered saline (PBS) and positive controls for Ia were spleen and lymph node sections prepared identically. In addition, antibodies to HLA-ABC, myelin basic protein, galactocerebroside, human natural killer cells, 2',3'-cyclic n u c l e o t i d e - 3 ' - p h o s phohydrolase and interleukin-2 (IL-2) receptor, were tested, l /xm epoxy sections stained with toluidine blue as well as paraffin sections of formaldehyde-fixed material were used for neuropathologic analysis.

Results

In sections reacted for Ia, positive reaction product was seen predominantly on the surface of rounded, lipid-laden, foamy macrophages (microglia) which occurred throughout the lesion material (Fig. 1). In the same preparations, oligodendrocytes failed to show staining, for Ia (Fig. 1). Hypertrophic astrocytes were common, were G F A P + and showed occasional Ia positivity. Adjacent sections of the same tissue reacted with anti-human M A G antibody to confirm the identity of the oligodendrocytes, showed them to be M A G ÷ and all other cells, particularly lipid-laden macrophages, to be M A G (Fig. 2). There was some M A G positivity in the background parenchymal matrix, probably related to myelin breakdown products. The number of oligodendrocytes at the lesion edge was increased and highly suggestive of cell proliferation. Ultrastructural examination of the tissue confirmed the identification of these cells as oligodendroglia (Raine et al., 1981). Toluidine blue-stained preparations documented the presence of myelin destruction, oligodendrocyte survival and microglial cells containing myelin debris and neutral lipid (Fig. 3), all occurring against a background of inflammation by small lymphocytes. The results of tests with the other antibodies on this material were inconsistent and inconclusive.

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Fig. 1. The edge of an acute MS lesion obtained at biopsy and embedded in epoxy resin is reacted for HLA-DR (Ia). While reactive microglia (foamy macrophages) display strong surface reactivity for Ia, oligodendroglia (arrows) are Ia negative and show background level staining only. PAP staining; no counterstain; 1/~ m epoxy section, x 750.

Fig. 2. In an adjacent 1 ~m epoxy section reacted with anti-MAG, oligodendroglia (arrows) are positively stained. Note the sharp contrast with the rounded microglial cells ( * ) in the same section, which show no staining for MAG. The diffuse positive staining in the background matrix probably represents extracellular myelin debris. PAP staining; no counterstain, x 750.

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Fig. 3. A toluidine blue-stained 1 #m epoxy section shows the perimeter of an active MS plaque. At least five oligodendroglia are present (arrows). Ongoing demyelination is evident by the occurrence of extracellular myelin debris (small black ringlets) and foamy microglia containing myelin debris and neutral lipid. × 750.

Discussion The present study supports the view that oligodendrocytes play no direct role in the immunopathogenesis of lesion formation in MS and, as depicted in Fig. 1, may actually occur in increased numbers suggestive of proliferation. In general, immune system molecules have rarely been shown on oligodendrocytes in situ. In one instance, Ting et al. (1981) claimed to show Ia positivity on interfascicular oligodendrocytes in normal mouse brain but more recent immunocytochemical studies on mouse CNS have failed to confirm this observation (Traugott et al., 1985b, 1986). A recent immuno-electron microscope study by Rodriguez et al. (1987) described oligodendroglial cytoplasmic reactivity for Ia in mice infected with Theiler's murine encephalomyelitis virus. This observation awaits confirmation. Likewise, studies of Ia expression on oligodendrocytes in vitro have produced conflicting results. K i m (1985) reported that a number of oligodendrocytes in dissociated human brain cell cultures expressed Ia. However, Wong et al. (1984) showed that treatment of murine CNS cultures with interferon-7, a potent

up-regulator of class II M H C , induced Ia expression on astrocytes but not on oligodendrocytes. Similarly, Suzumura et al. (1986) reported the inducibility of class I M H C (H-2), but not class I1, on murine oligodendrocytes. The latter authors speculated that the ability to bear surface H-2 might render oligodendrocytes vulnerable to MHC-restricted, cytotoxic T cell attack. To date, however, there has been no convincing demonstration of M H C molecules (class I or II) on human oligodendrocytes in situ to support this notion (Traugott, 1987; Sobel and Ames, 1988) and the present demonstration of an absence of Ia reactivity in oligodendrocytes adjacent to Ia + microglial cells represents the first of its kind on human MS tissue. The positive demonstration by K i m (1985) of Ia on normal h u m a n oligodendrocytes in culture may be related to technical differences (e.g. in vitro versus in vivo material; species and monoclonal antibody epitope variation) or to differences in level of antigen concentration (e.g. whole cells versus tissue sections). Nevertheless, the ability to demonstrate Ia ÷ microglia in the same section as Ia oligodendroglia might argue against differences in antigen concentration and

265 speak in favor of the specificity of the reaction. Our observation of Ia negativity by oligodendrocytes might also be supported by the recent studies of Calder et al. (1988) and Mauerhoff et al. (1988) which showed that oligodendrocytes appear to be refractory to a variety of conditions used to induce class II M H C expression. In this regard, it was demonstrated that O-2A progenitor cells (oligodendrocyte precursors) were capable of expressing Ia but further differentiation to galactocerebroside-positive oligodendrocytes resulted in a loss of Ia inducibility. While we attributed our inconsistency in galactocerebroside staining of oligodendrocytes in the present MS tissue to technical vagaries, the possibility that the general lack of galactocerebroside positivity was related to variation in expression during differentiation has not been excluded. The present documentation of oligodendrocyte survival and proliferation in MS is in keeping with the reported observation of remyelination at the periphery of MS plaques occurring simultaneously with active, ongoing demyelination (Raine et al., 1981; Prineas et al., 1984). In accord with the observed proliferation, oligodendrocytes in these locations expressed the adhesion molecule, MAG, a myelin antigen usually associated with early development (Sternberger et al., 1979). Whether specific factors underlie this cellular proliferation or whether glial progenitor cells (Raft et al., 1983) are involved, is open to speculation. It has recently been demonstrated that the cytokine, IL-2, a secretory product of activated T cells, induces proliferation and maturation of oligodendrocytes in vitro (Benveniste and Merrill, 1986). Furthermore, the observation of IL-2 and IL-2 receptorbearing cells within MS lesions (Hofman et al., 1986) might suggest that this cytokine serves a mitogenic or growth promoting role for oligodendrocytes in these areas. IL-2 receptor was documented on cultured oligodendrocytes in a study which concluded that IL-2 was inhibitory for these cells (Saneto et al., 1986). Taken in concert, we have shown with a modified high resolution immunocytochemical procedure, that oligodendrocytes at the edge of active MS lesions do not express class II M H C and stain positively for the adhesion molecule MAG. Thus, anti-MAG antibody may serve as a marker for

oligodendrocytes in areas of disease activity and remyelination. Since active MS lesions have previously been associated with abortive remyelination (Raine et al., 1981; Prineas et al., 1984) and are heavily infiltrated by small lymphocytes (Prineas and Raine, 1976) of the appropriate (CD4) phenotype (Traugott et al., 1983) for class II MHC interactions, these are the most likely areas where one might anticipate expression of M A G and Ia, respectively. In the event that endogenous CNS elements might participate in immune responses in MS at the level of accessory cells, one should focus perhaps on microglial cells, endothelial cells and astrocytes, upon which Ia expression has already been unequivocally documented (Traugott et al., 1985b).

Acknowledgements The authors thank Drs. N. Latov, D. Colman and J.E. Goldman for antibodies; Drs. G.R.W. Moore, G. Golenwsky, D. Dickson, B. Cannella and A.H. Cross for helpful discussion; E. Swanson, M. Pakingan and H. Finch for skilled technical assistance; and M. Briggs for careful preparation of the manuscript. Supported by research grants from the National Multiple Sclerosis Society (RG 1001-G-7) and the National Institutes of Health (NS 08952; NS 11920; and NS 07098).

References Abrarnsky, O., Lisak, R.P., Silberberg,D.H. and Pleasure, D.E. (1977) Antibodies to oligodendrogliain patients with multiple sclerosis. New Engl, J. Med. 297, 1207-1281. Allen, P.M. (1987) Antigen processing at the molecular level. Irnmunol. Today 8, 270-273. Benveniste, E.N. and Merrill, J.E. (1986) Stimulation of oligodendroglial proliferation and maturation by interleukin-2. Nature 321,610-613. Calder, V.L., Wolswijk, G. and Noble, M. (1988) The differentiation of O-2A progenitor cells into oligodendrocytesis associated with a loss of inducibilityof la antigens. Eur. J. Immunol. 18, 1195-1201. Hauser, S.L., Chan, A.K., Gilles, F., Kemp, M., Kerr, C. and Weiner, H. (1986) Immunocytochemical analysis of the cellular infiltrates in multiple sclerosis lesions. Ann. Neurol. 19, 578-587. Hofman, F.M., von Hanwehr, R.I., Dinarello, C.A., Mizel, S.B., Hinton, D. and Merrill, J.E. (1986) Immunoregulatory

266 molecules and IL-2 receptors identified in multiple sclerosis brain. J. Immunol. 136, 3239-3245. Kim, S.U. (1985) Antigen expression by glial cells grown in culture. J. Neuroimmunol. 8, 255-282. Lee, S.-H., Moore, G.R.W., Golenwsky, G. and Raine, C.S. (in press) Multiple sclerosis: a role for astroglia in active demyelination suggested by Class I1 MHC expression and ultrastructural study. J. Neuropathol. Exp. Neurol. Lumsden, C.E. (1955) In: D. McAlpine, N.D. Compston and C.E. Lumsden (Eds.), Multiple Sclerosis, Livingston, Edinburgh, p. 231. Mauerhoff, T., Pujol-Borrell, R., Mirakian, R. and Bottazzo, G.F. (1988) Differential expression and regulation of major histocompatibility complex (MHC) products in neural and glial cells of the human fetal brain. J. Neuroimmunol. 18, 271 289. Nobile-Orazio, E., Hays, A.P., Latov. N., Perman, G., Golier, J., Shy, M.E. and Freddo, L. (1984) Specificity of mouse and human monoclonal antibodies to myelin-associated glycoprotein. Neurology 34, 1336-1342. Oger, J., Szuchet, S., Antel, J. and Arnason, B.G.W. (1982) A monoclonal antibody against human T suppressor lymphocytes binds specifically to the surface of cultured oligodendrocytes. Nature 295, 66-68. Prineas, J.W. and Raine, C.S. (1976) Electron microscopy and immunoperoxidase studies of early multiple sclerosis lesions. Neurology 26, 29-32. Prineas, J.W., Kwon, E.E., Cho, E.S. and Sharer, L.R. (1984) Continual breakdown and regeneration of myelin in progressive multiple sclerosis plaques. In: L.C. Scheinberg and C.S. Raine (Eds.), Multiple Sclerosis: Experimental and Clinical Aspects. Ann. N.Y. Acad. Sci. 436, 11-32. Raft, M.C., Miller, R.H. and Noble, M. (1983) A glial progenitor cell that develops in vitro into an astrocyte or an oligodendrocyte depending on culture medium. Nature 303, 390-396. Raine, C.S. (1985) Demyelinating diseases. In: R.L. Davis and D.M. Robertson (Eds.), Textbook of Neuropathology, Williams and Wilkins, Baltimore, MD, pp. 468-547. Raine, C.S., Scheinberg, L.C. and Waltz, J.M. (1981) Multiple sclerosis: oligodendrocyte survival and proliferation in an active, established lesion. Lab. Invest. 45, 534-546. Robbins, D.S., Shirazi, Y., Drysdale, B.E., Lieberman, A., Shin, H.S. and Shin, M.L. (1987) Production of cytotoxic factor for oligodendrocytes by stimulated astrocytes. J. lmmunol. 139, 2593 2597. Rodriguez, M., Pierce, M.L. and Howie, E.A. (1987) Immune

response gene product (la antigens) on glial and endothelial cells in virus-induced demyeUnation. J. lmmunol. 138, 3438 3442. Saneto, R.P., Airman, A., Knobler, R.L., Johnson, H.M. and DeVellis, J. (1986) Interleukin-2 mediates the inhibition of oligodendrocyte progenitor cell proliferation in vitro. Proc. Natl. Acad. Sci. U.S.A. 83, 9221-9225. Sobel, R.A. and Ames, M.B. (1988) Major histocompatihility complex molecule expression in the human central nervous system: immunohistochemical analysis of 40 patients. J. Neuropathol. Exp. Neurol. 47, 19-28, Sternberger, N.H., Quarles, R.H., hoyama, Y. and Webster, H.deF. (1979) Myelin-associated glycoprotein demonstrated immunocytochemically in myelin and myelin-forming cells of developing rat. Proc. Natl. Acad. Sci. U.S.A. 76. 1510-1514. Suzumura, A., Silberberg, D.H. and Lisak, R.P. (1986) The expression of MHC antigens on oligodendrocytes: induction of polymorphic H-2 expression by lymphokines. J. Neuroimmunol. 11, 179-190. Ting, J.P.Y., Shigekawa, B.L., Linthicum, D.S., Weiner, L.P. "and Frelinger, J.A. (1981) Expression and synthesis of murine immune response-associated (Ia) antigens by brain cells. Proc. Natl. Acad. Sci. U.S.A. 78, 3170-3174. Traugott, U. (1987) Multiple sclerosis: relevance of class I and class II MHC-expressing cells to lesion development. J. Neuroimmunol. 16, 283-302. Traugott, U., Reinherz, E.L. and Raine, C.S. (1983) Multiple sclerosis: distribution of T cells, T cell subsets and la-positive macrophages in lesions of different ages. J. Neuroimmunol. 4, 201-221. Traugott, U., Raine, C.S. and McFarlin, D.E. (1985a) Acute experimental allergic encephalomyelitis in the mouse: immunopathology of the developing lesion. Cell. Immunol. 91,240-254. Traugott, U., Scheinberg, L.C. and Raine, C.S. (1985b) On the presence of Ia-positive endothelial cells and astrocytes in multiple sclerosis lesions and its relevance to antigen presentation. J. Neuroimmunol. 8, 1-14. Traugott, U., McFarlin, D.E. and Raine, C.S. (1986) lmmunopathology of the lesion in chronic relapsing experimental autoimmune encephalomyelitis in the mouse. Cell. Immunol. 99, 395-410. Wong, G.H.W., Bartlett, P.F., Clark-Lewis, I., Battye, F. and Schrader, J.W. (1984) Inducible expression of H-2 and la antigens on brain cells. Nature 310, 688-691.