Experimental allergic encephalomyelitis

Journal of the Neurological Sciences, 1981, 50:63-79 © Elsevier/North-Holland Biomedical Press

63

EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS Characterization of Serum Factors Causing Demyelination and Swelling of Myelin

INGE G R U N D K E - I Q B A L 12, CEDRIC S. RAINE 34, A N N E B. JOHNSON 34, CELIA F. BROSNAN 3 and M U R R A Y B. BORNSTEiN 4'5 1New York State Institute .[or Basic Research in Mental Retardation, Staten Island, N Y 10314; 2 Department of Neurology, S U N Y Downstate Medical Center, Brooklyn, N Y 11203; and Departments of 3 Pathology, 4 Neuroscience and 5 Neurology, Albert Einstein College of Medicine of Yeshiva UniversiO', Bronx, N Y 10461 (U.S.A.)

(Received 18 August, 1980) (Accepted 18 September, 1980)

SUMMARY

Serum factors in rabbits with white matter-induced experimental allergic encephalomyelitis (WM-EAE) were studied with respect to their role in demyelination in vitro in organotypic central nervous system (CNS) tissue cultures and in vivo in the myelinated retina of the rabbit eye. By absorption with staphylococcal protein A, lgG was quantitatively separated from the other serum proteins. No IgG was demonstrable in the absorbed IgG-depleted sera by Ouchterlony double diffusion, immunoelectrophoresis and SDS-polyacrylamide gel electrophoresis. Both the IgG-depleted WM-EAE sera and the IgG fractions had complement-dependent demyelinating activity on CNS cultures, and both contained immunoglobulin binding to myelin and oligodendroglia of the cultures, as demonstrated by an immunoperoxidase technique. However, only the purified IgG fractions in the absence of complement induced swelling of myelin and proliferation of oligodendroglial processes with redundant myelin in tissue cultures. The IgG-depleted complement-

This study was supported by United States Public Health Service Grants NS-11920 and NS-08952, and National Multiple Sclerosis Society, Grant 1335. Presented in part at the 53rd Annual Meeting of the American Association of Neuropathologists, Chicago, June 1977. Reprint requests and all correspondence to: Dr. Inge Grundke-Iqbal, Department of Neuropathology, New York State Institute for Basic Research in Mental Retardation, 1050 Forest Hill Road, Staten Island, NY 10314, U.S.A. Abbreviations: CFA, complete Freund's adjuvant ; CNS, central nervous system ; EA E, experimental allergic encephalomyelitis, MS, multiple sclerosis; WM-EAE, white matter-induced EAE.

64 inactivated WM-EAE sera produced no morphological changes, in the rabbit cyc model, antibody-dependent cell-mediated demyelination was observed only ~ith the IgG fractions but not with the lgG-depleted EAE sera. No oligodendrogtial proliferation occurred. These studies demonstrate for the first time that in (!NS cultures, non-IgG immunoglobulins as well as lgG mediate complement-dependent demyelination and that these bind to myelin and oligodendrocytes, whereas only lgG causes myelin swelling and oligodendrocyte proliferation.

INTRODUCTION

Experimental allergic encephalomyelitis (EAE), the most applied animal model for multiple sclerosis (MS) research, is an autoimmune demyelinating disease of the central nervous system (CNS) which is readily induced in a variety of animals by injection of whole white matter or myelin basic protein in complete Freund's adjuvant (CFA). While it is well established that the T-cell system is essential in the induction of EAE, it is not clear whether humoral factors also participate in the disease process. Immunoglobulins are present within the CNS lesions of acute (Oldstone and Dixon 1968; Prineas and Raine 1976: Paterson 1976) and chronic relapsing EAE (Grundke-Iqbal et al. 1980). Complement is also present in acute EAE lesions (Oldstone and Dixon 1968; Grundke-Iqbat et al. 1980) and a possible role for proteases in demyelination has been suggested (Cammer et al. 1978; Sibley et al. 1978). Concerning a role for antibody, serum and also isolated IgG from rabbits or guinea pigs with white matter-induced EAE (WM-EAE) readily produce primary demyelination in organotypic CNS tissue cultures. This reaction is complement dependent (Appel and Bornstein 1964; Lebar et al. 1976) and associated with the binding of immunoglobulin to myelin (Johnson and Bornstein 1978). WM-EAE serum that has been heated to inactivate complement does not demyetinate cultures, but it produces swelling of myelin lamellae with a doubling of the intraperiod line (Bornstein and Raine 1976) and excessive proliferation of oligodendroglial processes with aberrant myelin formation (Raine et al. 1978). With heated WM-EAE serum, immunoglobulin binds to oligodendroglial plasmalemmae and to the intraperiod line of swollen myelin (Johnson et al. 1979). Demyelination can also be induced in vivo in the myelinated retina of normal rabbits by injection of WM-EAE serum together with supernates from nonspecifically stimulated lymphocytes, an event probably representing antibody-dependent cellmediated demyelination (Brosnan et al. 1977). Recent studies have also demonstrated demyelination of peripheral nerve after intraneural injection of WM-EAE serum (Saida et al. 1979), a reaction which seems to be linked primarily to the action of antibodies and complement as in the tissue culture system. These studies on EAE serum imply a role of antibodies in demyelination. Unlike the situation in EAE, in MS, using serum, in vitro demyelination is linked to non-immunoglobulin serum factors (Grundke-lqbal and Bornstein 1980).

65 Whether the same factors also occur in EAE serum is not known. The present study was undertaken to elucidate further in EAE the role of immunoglobulin as well as non-immunoglobulin serum factors in demyelination in vitro in organotypic tissue cultures and in vivo in the rabbit eye model. MATERIALS A N D M E T H O D S

Sera WM-EAE sera were obtained from New Zealand white rabbits inoculated for acute EAE with bovine brain white matter in CFA, and were of proven strong demyelinating activity. Sera from rabbits injected with CFA served as a control. Aliquots of sera were stored at -90 °C.

Analysis of immunoglobulins IgG, IgA and IgM were examined in rabbit serum and serum fractions by Ouchterlony double diffusion test using 1~ agarose plates in phosphate-buffered saline (PBS). Immunoelectrophoresis was performed with a modification of the micro-method of Scheidegger (1955), using 1~o agarose in a barbital buffer, pH 8.8 (Gelman Instruments, Ann Arbor, MI). Polyvalent antiserum against rabbit immunogtobulins and monospecific antisera against rabbit IgG, IgA and IgM were purchased from Cappel Laboratories, Cochranville, PA. Antiserum against rabbit serum was purchased from Biorad Laboratories, Richmond, CA. The concentration of IgG in protein A-treated and control-treated EAE sera was determined by single radial immunodiffusion using the "rabbit IgG Kit" from Miles Research Products, Elkhart, IN. Protein determination and gel patterns Protein was determined by the method of Lowry et al. (1951). The protein patterns of whole sera and serum fractions were analyzed by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis (Maizel 1971) using Tris-glycine discontinuous system (Laemmli et al. 1970) and a linear acrylamide gradient of 7.5-30~o. Fractionation of sera by DEAE cellulose chromatography Distribution of the demyelinating serum factors was studied by DEAE cellulose chromatography. Whole WM-EAE sera were equilibrated by dialysis against 0.01 M sodium phosphate buffer, pH 8.0. The samples (30 mg) were layered on columns containing 20 ml DE 52 cellulose (Whatman, Maidstone, Kent, U.K.) equilibrated with the phosphate buffer. Proteins were eluted stepwise with the same buffer containing increasing amounts of sodium chloride, dialyzed against PBS and concentrated to the original sample volume. Fractionation of sera by absorption with protein A IgG was removed from WM-EAE and control serum samples (0.3 ml) by passing them over a column containing 2 mg protein A coupled to 1 ml Sepharose

66 C1-4B (Pharmacia, Piscataway, N J) as previously described (Grundke-lqbal and Bornstein 1979). The IgG was recovered from the protein A column by elution with 0.1 M glycine-HC1 buffer pH 2.8. In some cases, the recovered lgGdepleted fraction was further fractionated by DEAE cellulose chromatography.

Role of complement (1) Removal of complement activity. To test for a role for complement in demyelination of the tissue cultures, complement from whole and IgG-depleted WM-EAE sera was inactivated by heating at 56°C for 30 min. In addition, in the case of IgG-depleted sera, complement was also inactivated by treating 1 ml samples for 1 h at 37 °C with one of the following preparations : (i) 20 mg Zymosan (Sigma, St. Louis, MO); (ii) 1 mg lipopolysaccharide B from S. typhosa (Difco Laboratories, Detroit, MI) and (iii)15 mg of pelleted, well-washed immune complexes of goat anti-rabbit IgG (Calbiochem, Rutherford, N J) and rabbit IgG (Cappel, Cochranville, PA) which had been reacted at antigen excess for 1 h at 37 °C and 2 h on ice. As a control, an aliquot of IgG-depleted WM-EAE serum alone was incubated at 37°C for 1 h. The samples were then centrifuged at 10,000 × g for 30 rain and the clear supernatants tested for demyelinating activity. For each treatment, the effectiveness of complement depletion was measured by the lysis of antibody-sensitized sheep red cells (Cordis, Miami, FL) with the micromethod of Wasserman and Levine (1961). In order to eliminate the alternative pathway of complement activation, some of the IgG-depleted sera were heated at 50 °C for 30 min. (2) Effect of enzyme inhibitors. Enzyme inhibitors were employed to test for a possible role in serum demyelinating activity of plasmin or trypsin-type proteinases which, among other effects, can activate complement. Immediately before application to the tissue cultures, aliquots of IgG-depleted WM-EAE sera were incubated at room temperature for 1 h with soybean trypsin inhibitor, 10 mg/ml (Sigma, St. Louis, MO) or Trasylol, 500 kallikrein units/ml (Mobay Chemical Corp., New York, NY). Both treatments completely inhibited the plasmin activity of these sera, as monitored with aliquots of the sera in which plasmin previously had been activated under optimal conditions with the plasminogen activator urokinase (Biorad Laboratories, Richmond, CA). Plasmin was measured semiquantitatively using agarose plates containing 1~ casein (Biorad Laboratories).

Biological activity (1) Studies with cultures. The WM-EAE and control sera and serum fractions, reconstituted to their approximate concentration in whole serum, were diluted to 25~ in nutrient medium and tested on organotypic cultures of fetal mouse spinal cord maintained in Maximow slide assemblies (Bornstein 1973). For demyelination studies, well myelinated cultures at 18 days in vitro were used, and the nutrient medium was additionally supplemented with 0.2 mg/ml gentamycin and 10~ normal non-demyelinating rabbit serum as a source of complement. Demyelination was scored as previously described (Grundke-Iqbal and Bornstein 1979). For

67 morphological studies, cultures were exposed to the sera or serum fractions (heated and unheated) for 6, 24 and 48 h, processed for embedment in plastic, and examined by light and electron microscopy as previously described (Bornstein and Raine 1976). For immunocytochemical studies, CNS cultures 3~1 weeks in vitro were incubated 1 h at room temperature with 25~o heated sera, or serum fractions. Then they were fixed, incubated with horseradish peroxidase-labeled antibody against rabbit IgG (Pasteur Institute, France) and reacted with diamino benzidine, as previously described (Johnson and Bornstein 1978). Bound immunoglobulin was thus localized light-microscopically. Immunoelectrophoresis of the antibody conjugate followed by diamino benzidine staining showed that it reacted with rabbit IgG, IgM and IgA. (2) Studies in the rabbit eye. The demyelinating activity of some IgG fractions and IgG-depleted sera was also tested in vivo in the rabbit eye. Test samples (0.1 ml) were admixed with an equal volume of supernates from Concanavalin A-stimulated rabbit peripheral lymphocytes and injected into the vitreous of normal rabbits. The rabbits were killed 5 days after injection by whole body perfusion with 4~o glutaraldehyde and the retina examined by light and/or electron microscopy (Brosnan et al. 1977). RESULTS

Biological studies on C N S cultures

Initial attempts to study a possible role for serum components other than IgG in demyelination by WM-EAE serum were made by removing IgG with DEAE cellulose chromatography. This produced a fraction (fraction I) which contained only about 80~ of the total IgG, as determined by quantitative radial immunodiffusion, and the remaining 20-30~ IgG was recovered in another 3 fractions (fraction II, III and IV). The isolated IgG (fraction I), had strong demyelinating activity in CNS cultures. However, fraction IV (see Table 1), which had been eluted with high ionic strength buffer and contained only small amounts of IgG, also demyelinated the cultures equally well. Demyelination with fractions I and IV occurred only if fresh, normal, non-demyelinating rabbit serum was added as a source of complement. IgG-depleted sera. Next, an attempt was made to study serum components other than IgG in demyelination, by removing IgG quantitatively from the sera by absorption with staphylococcal protein A. In most of the protein A-treated sera, no IgG was detected by immunoelectrophoresis, Ouchterlony tests, or SDS-polyacrylamide gel electrophoresis (Figs. 1 and 2). Analysis by radial immunodiffusion revealed that more than 99~o of the total IgG had been removed by absorption with protein A. Eleven IgG-depleted WM-EAE sera were tested on CNS cultures and all had considerable demyelinating activity (Table 2). No demyelination occurred if the fractions had been heat-inactivated at 56 °C. To elucidate further whether IgGdepleted WM-EAE sera required complement to induce demyelination in vitro,

68 TABLE 1 WM-EAE SERUM -

D E M Y E L I N A T I N G ACTIVITY OF DEAE Cf~LLULOSI~ FRACTIONS

The amount of IgG was assayed by quantitative radial immunodiffusion. The presence of lgA and IgM was determined by Ouchterlony double diffusion test

Fraction No.

Molarity of NaCI in the elution buffer a

lgG % of total serum IgG

I II III IV

0 0.05 0.10 0.50

70 80 1~15 9 4

lgM

+ +

lgA

+ +

Demyeliuating activity at 48 h b

4.3 2.0 • 3.8

~ 0.01 M sodium phosphate buffer, pH 8.0. b Each sample was tested on 6 different explants, and the degree ofdemyelination determined 24 and 48 h after exposure. The values listed in the table represent the arithmetic means of the individual scores: 0 = no change, 1 = some swelling and ballooning of myelin without demyelination, 2 = loss of up to 25% of myelin, 3 = demyelination up to 50%, 4 = demyelination up to 75%, 5 = demyelination up to 100% (Grundke-Iqbal and Bornstein 1979). c Cytotoxic.

! .......~-?.:

ID

3

Fig. 1 lmmunoelectrophoresis patterns of WM-EAE serum depleted of lgG by absorption with protein A (1), undepleted WM-EAE serum (2) and the protein fraction eluted with pH 2.8 buffer from protein A (3). The trough in panel a contains antiserum against rabbit gamma-globulins and in panel b antiserum against normal rabbit serum.

69

Fn

Fig. 2. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of WM-EAE serum (A); protein A-treated, lgG depleted WM-EAE serum (B) ; IgG fraction eluted with pH 2.8 buffer from protein A (C); same fraction as in panel C, 4-fbld amount (D); IgG isolated from WM-EAE serum by chromatography on DEAE cellulose (E); same as in panel E, 4-fold amount (F).

other complement inactivation procedures were studied. Unlike heating at 56 °C, treatment at 50°C did not reduce the demyelinating activity. However, the demyelinating activity was abolished by pretreatment with Zymosan, antigenantibody precipitates or bacterial lipopolysaccharide (Table 3). Hemolysis tests for the presence of complement showed that these treatments had reduced the hemolytic activity of the IgG-depleted serum by about 9 0 ~ . Addition of fresh, non-demyelinating, normal rabbit serum restored the demyelinating activity in all cases except in the lipopolysaccharide-treated sample. No decrease in demyelinating activity was observed if IgG-depleted serum was treated with soybean trypsin inhibitor or Trasylol (not shown in Table 3), under conditions which completely inhibited the urokinase-activated caseinolytic activity of W M - E A E serum. To characterize further the n o n - I g G demyelinating activity, IgG-depleted EAE sera were fractionated on D E A E cellulose (Table 4). The demyelinating activity was about 6-fold enriched in fraction IV, which contained the proteins with the most acidic charge, including part of the IgA and IgM. No demyelination occurred if this fraction was heated at 56°C prior to application to the tissue cultures, but the demyelinating activity could be fully restored with fresh serum

70 -FABLE 2 E F F E C T OF I g G - D E P L E T I O N BY P R O T E I N A ON TH E D E M Y E L I N A T I N G A C TIV ITY OF W M - E A E A N D C O N T R O L SERA

EAE rabbit No.

1 2 3 4 5 6 7 8 9 10 ll Control rabbit d 12

Demyelinating activity ~ lgG-depleted serum b .... 24 h 48 h

undepleted, whole serum

IgG fractions c eluted from Prot. A 24 h

24 h

48 h

2.1 1.8 2.8 1.6 2.1 1.8 2.1 3.5 l.l 1.6 3.8

3.6 3.7 4.8 3.6 4.0 3.0 3.8 4.7 2.5 3.8 4.6

5 3.8 4.5 3.6 5 5 5 5 2.0 5 4.0

5 4.8 4.8 5 5 5 5 5 3.0 5 5

5 ND 5 2.3 5 5 2.3 4.3 3.3 4.8 4.8

0.5

0.8

0.3

0.9

1.0

a Determined as in Table 1. b IgG below detectable limits. c With addition of normal rabbit serum as a source of complement. d CFA-injected. ND = not determined.

TABLE 3 I g G - D E P L E T E D W M - E A E S E R U M - - E F F E C T O F C O M P L E M E N T INHIB1TORS O N T H E DEMYELINATING ACTIVITY

Treatment

None 50°C, 30 min 56 °C, 30 min b Zymosan (20 mg/ml) b I m m u n e precipitate (15 mg/ml) b LPS (1 mg/ml)

Demyelinating activity at 48 h a

no addition

addition of NRS c

3.0 3.3 0.5 1.0 0.3 1.5

3.5 3.5 3.2 3.8 2.5 1.0

a Determined as in Table 1. b IgG-depleted EAE serum and complement inhibitors were incubated at 37°C for t h. Any insoluble material was then removed by centrifugation prior to in vitro tests. c Fresh normal rabbit serum.

71 TABLE 4 IgG-DEPLETED WM-EAE SERUM FRACTIONS Fraction No.

I II Ill IV

DEMYELINAT1NG ACTIVITY OF DEAE CELLULOSE

Molarity of NaCI in the elution buffer b

Protein IgG (mg/ml)

0 0.05 0.10 0.50

0.05 1.96 17.0 3.9

trace -

IgM

+ +

IgA

+ +

Demyelinating activity ~ at 48 h Heated c

Complement d added

ND ND ~' 1.0

1.5 1.3 e 3.8

a Determined as in Table 1. b 0.01 M sodium phosphate buffer, pH 8.0. c 56 °C for 30 rain. d 5~, fresh normal rabbit serum. e Cytotoxic. ND = not determined from a n o r m a l rabbit. Fraction III, which contained the less acidic IgA, I g M and other serum proteins, f r o m 3 out o f 4 sera did not substantially demyelinate the cultures, but was highly cytotoxic and p r o d u c e d swollen cell bodies with granular cytoplasm and finally cell death. This activity was not labile to heating at 56 °C, nor could it be enhanced by the addition o f n o r m a l rabbit serum (Table 4). IgG fractions. The I g G a b s o r b e d by protein A from W M - E A E sera was quantitatively recovered by elution with acidic buffer. I m m u n o l o g i c and biochemical analysis showed that this fraction was comprised o f I g G o f high purity c o m p a r a b l e to the I g G fraction isolated by DEAE-cellulose c h r o m a t o g r a p h y o f W M - E A E serum. By immunoelectrophoresis, only I g G and a trace o f I g M could be detected using antiserum against total rabbit serum (Fig. 1). Only when SDS-polyacrylamide gels were overloaded did a few trace c o n t a m i n a n t s become visible (Fig. 2). N o IgA was detected in a n y o f the I g G preparations either by O u c h t e r l o n y agar gel diffusion test or by immunoelectrophoresis. W h e n applied to C N S cultures, the I g G fractions induced strong demyelination (Table 2). This reaction required n o r m a l rabbit serum, p r e s u m a b l y as a source o f complement. N o demyelination occurred if n o r m a l rabbit serum was absent or if this serum was substituted by n o r m a l guinea pig serum. Similarly prepared I g G f r o m serum o f a CFA-injected control rabbit had no demyelinating activity.

Morphological and immunocytochemical studies on CNS cultures IgG-depleted sera. Examination o f 1-/~m sections f r o m cultures treated with IgG-depleted E A E serum revealed the same light microscope a n d ultrastructural changes as observed in sister cultures treated with undepleted W M - E A E serum, i.e. demyelination and oligodendroglial cell death. With IgG-depleted W M - E A E

72

Fig. 3. lmmunoglobulin bound to myelin of CNS culture incubated with heated, lgG-depteted WM-EAE serum, lmmunoperoxidase method, ×320.

serum which had been previously heated at 56 °C, no demyelination or abnormal morphology of any of the structural elements of the cultures was noted at both the light and electron microscope levels. This was in marked contrast to the effect of heated, undepleted whole WM-EAE serum, which caused swelling and ballooning of myelin sheaths as in previous studies. Immunoperoxidase studies with 4 WM-EAE sera in which IgG had been removed to below detectable levels with protein A gave the same immunostaining of myelin seen with undepleted whole serum, although less myelin appeared stained with the IgG-depleted serum (Fig. 3). In addition, 3 of the 4 IgG-depleted sera also immunostained the cell membranes of cells with elaborate branching processes which are believed to be oligodendrocytes on the basis of previous studies. These cells were more prominent with the IgGdepleted than with the whole serum. No demyelination, morphological change or immunostaining of myelin or cells occurred with IgG-depleted fractions from control serum. IgG.fractions. Examination of cultures treated with IgG fractions without added complement revealed morphological changes similar to those observed in

73

Fig. 4. CNS culture exposed for 48 h to an IgG fraction isolated from WM-EAE serum by absorption with protein A. Note how the myelinating oligodendrocyte (nucleus below) has proliferated copious amounts of abnormal myelin and of cell processes around itself, x27,500.

Fig. 5. Detail from Fig. 4. Note the packing of the oligodendroglial cell processes into layers of abnormal myelin with a 22-nm periodicity and 4 linearities (arrows) in place of the normally bilamellar intraperiod line. × 150,000.

74

Fig. 6. Sameculture as Fig. 4. Note the swollen myelinsheath around tile normal ~xon (center) x 75.()1)(L cultures treated with heated undepleted WM-EAE serum. Myelin sheaths surrounding axons in the living cultures appeared strongly birefringent. Electron microscope examination revealed that oligodendrocytes had proliferated copious amounts of swollen myelin with a doubling of the periodicity of myelin lametlae from approximately 11 nm to 22 nm (Figs. 4 and 5). An additional pair of electron-dense linearities replaced the intraperiod line, which now consisted of" 4 instead of 2 leaflets. Myelin sheaths around axons displayed a similar swelling (Fig. 6). Immuno-peroxidase studies revealed that the isolated IgG fractions immuno-stained both myelin and oligodendrocytes and the appearance closely resembled that with the undepleted, whole serum samples. The amount of reactive myelin was greater than with the IgG-depleted serum, and the oligodendrocytes were less prominent. Studies on the rabbit eye

Strong primary demyelination of the retinal fibers was observed in eyes injected with IgG fractions from W M - E A E sera, together with supernatants from non-specifically stimulated lymphocytes. No demyelination or proliferation of oligodendrocytes was observed after injection with control IgG and lymphocyte supernatants, with IgG fractions from W M - E A E serum alone, or with IgG-depleted WMoEAE serum plus lymphocyte supernatants. DISCUSSION The present study demonstrates unequivocally for the first time that in addition to IgG, complement-dependent non-igG factors, most probably immunoglobulins, are responsible for a considerable part of the in vitro demyelinating activity of sera

75 from rabbits with WM-EAE. Previous attempts to study non-IgG factors gave conflicting results (Appel and Bornstein 1964; Berg and Bergstrand 1968). In previous studies, IgG was depleted from the sera by chromatography on DEAE Sephadex or cellulose. With this method, however, only about 80~o of the IgG can be removed from the sera; the residual 20~ are retained by the column because of their more acidic net charge and will elute together with the other serum proteins (Fahey 1967). Thus, in order to investigate whether demyelinating serum factors other than IgG are present in EAE sera~ it has been found necessary to separate IgG from other serum proteins more effectively than can be achieved by conventional ion-exchange chromatography. The present studies have shown that absorption with staphylococcal protein A removes IgG from the sera beyond detectable limits, thus facilitating the unequivocal detection of acidic, non-IgG factors which demonstrate demyelinating activity on cultures. IgG-depleted MS sera have also been reported previously to demyelinate CNS cultures (Grundke-Iqbal and Bornstein 1979). However, with MS sera, most if not all of the activity was found to be due to non-immunoglobulins (Grundke-Iqbal and Bornstein 1980), whereas the present data on the IgG-depleted WM-EAE sera strongly indicate that most, if not all of the activity on cultures is antibodyrelated. Myelin-binding immunoglobulin has been demonstrated by immunocytochemistry, both in IgG fractions and in IgG-depleted WM-EAE sera, whereas with MS sera, no binding of immunoglobulin to CNS tissue cultures was observed (Johnson and Bornstein 1978). The present study has also indicated that the demyelinating activity of IgG-depleted WM-EAE serum on cultures is complementdependent, as it is with IgG fractions. This activity was destroyed by heating the IgG-depleted EAE sera at 56 °C, a treatment destroying or diminishing certain complement components and other heat-labile enzymes. |t was also abolished by the more specific treatments with Zymosan or bacterial lipopolysaccharide which inactivate mainly complement components C3-C9, and with immune complexes, which inactivate mainly C1-C4 (Gewurz et al. 1968). The addition of normal, non-demyelinating rabbit serum fully restored the demyelinating activity in all but the lipopolysaccharide-treated IgG-depleted serum samples. Lipopolysaccharide is partly water soluble, and most of it could not be removed from the serum. Residual lipopolysaccharide in the serum, therefore, probably interfered with the added complement. Complement can be activated not only by immunoglobulin complexes but also by non-immunoglobulin compounds. With such compounds, however, activation does not occur via the classical pathway, which involves complement components C1 through C9. Instead, C1, C2 and C4 are by-passed and C3 is directly activated by the enzymes of the properdin system, or by other serum proteases. Since no decrease in demyelinating activity was observed by heating the IgG-depleted EAE sera at 50 °C, a treatment which destroys factor B of the properdin system (Theofilopoulos and Perrin 1977), complement activation via the alternate pathway was not likely. There was also no indication that complement activation was initiated by the serine proteases in serum, like trypsin or plasmin,

76 which convert C3 to C3a and C3b. No decrease in in vitro demyelmating actiwt\ was observed when lgG-depleted serum was treated with soybean trypsin inhibit~r or Trasylol, which are potent inhibitors for these enzymes. Therefore, complement activation via the classical pathway by immunoglobulin complexes, formed by non-IgG class antibodies reacting with CNS tissue, is in this case the most likely explanation. IgG fractions of high purity were prepared from WM-EAE rabbit sera either by DEAE cellulose chromatography or by absorption with protein A. These fractions demyelinated CNS tissue cultures when non-demyelinating serum from a normal rabbit was added, presumably as a source of complement. Complement-dependent demyelinating activity of lgG fractions isolated t¥om serum by chromatography on DEAE cellulose has also been reported in rabbits and guinea pigs with WM-EAE (Appel and Bornstein 1964, Lebar et al. 1976). It is interesting that no demyelination was observed in the present study if normal rabbit serum was replaced by guinea pig serum as a source of complement. Similarly, demyelination of cultures by IgG isolated from sera of certain MS patients did not take place if the human complement was replaced by guinea pig complement (Grundke-Iqbal and Bornstein 1979). This effect may be due to species differences between certain components of the complement system (Tamura 1970) required for IgG-mediated demyelination of cultures. When applied to CNS tissue cultures, heat-inactivated whole WM-EAE sera cause excessive proliferation of oligodendroglial processes and swelling ot" myelin (Bornstein and Raine 1976; Raine et al. 1978). The present studies demonstrate that it is IgG by itself which induces these characteristic changes without the participation of other serum factors. IgG-depleted EAE sera from which complement had been removed, did not induce myelin swelling or oligodendroglial proliferation. This is remarkable since the same IgG-depleted EAE sera with their complement system intact demyelinated the tissue cultures and since immunocytochemical studies with the IgG-depleted sera clearly demonstrated the presence of immunoglobulin binding to myelin and oligodendroglia. Two principal possibilities may explain the different effect on myelin of lgG and non-lgG fractions. The most direct explanation would be that both IgG and non-IgG antibodies are directed against the same antigen on oligodendroglia and myelin and that binding of antibodies in the absence of complement induced the oligodendroglia to produce the prolific array of processes forming redundant myelin. Complement, when present~ would be activated by these immunoglobulin complexes and induce demyelination. That proliferating myelin was only observed in the presence of IgG would then suggest that the concentration of antibodies in the non-IgG population was too low to stimulate myelin to swell or the oligodendroglia to proliferate myelin, although sufficient to cause activation of complement to induce demyelination. Alternatively, it is also possible that demyelination and proliferation of myelin are caused by antibodies binding to different antigenic sites: i.e. that antibody binding to antigen A would result in demyelination, whereas antibody-binding to antigen B would give the signal to

77 the oligodendroglial cell to produce aberrant myelin. That such myelin was only detected with IgG and not with non-IgG fractions would then suggest insufficient amount of antibody specific for antigen B in the non-IgG fractions. An additional possibility is that steric effects prevent the non-IgG immunoglobulins (probably lgM, which is much larger than IgG) from causing the morphological alteration of the myelin intraperiod line. Masses of redundant and abnormal myelin also have been found in the spinal cords of animals with acute and chronic WM-EAE (Lampert 1965; Prineas et al. 1969; Madrid and Wisniewski 1979). The cause of the myelin proliferation in this in vivo model is not as yet known, but a role of antibodies in the pathogenesis of this lesion is suggested, especially in light of the present tissue culture studies. Furthermore, IgG is bound to the parenchyma in the periphery of demyelinative lesions of similarly immunized guinea pigs (Grundke-Iqbal et al. 1980) which also points to a possible role for immunoglobulins in in vivo demyelination. In MS, the presence of immunoglobulin at the periphery of plaques has been reported frequently (Tourtellotte 1972; Prineas and Raine 1976; Woyciechowska and Brzoski 1977) but as yet no swollen myelin with doubling of the intraperiod line or abnormally proliferating oligodendroglia have been observed. The rabbit eye model contrasts with the tissue culture system in that demyelination but no proliferation of oligodendroglial processes or swelling of myelin were observed with the IgG fractions from WM-EAE sera. Perhaps in the rabbit optic nerve, antigenic sites responsible for proliferation of oligodendroglia are not present or not available to the immunoglobulins, or the IgG concentration achieved was too low. The rabbit eye model also differs from the culture system in that no demyelination was found with the IgG-depleted WM-EAE serum. This supports the previous suggestion (Brosnan et al. 1977) that only IgG can participate in antibody-dependent, cell-mediated demyelination in this system. The present study on CNS cultures exposed to WM-EAE serum fractions has confirmed that the IgG binds to myelin and oligodendrocytes and causes complement-dependent demyelination. It has also shown that it is the IgG which produces myelin swelling and oligodendroglial proliferation with aberrant myelinogenesis in the absence of complement. For the first time, IgG-depleted WM-EAE serum has been demonstrated to contain immunoglobulin binding to myelin and oligodendrocytes and to mediate complement-dependent demyelination. Surprisingly, the non-IgG immunoglobulin in the absence of complement produced no discernible morphological effect on cultures. ACK NOW LEDG EM ENTS

The authors gratefully acknowledge the skilled technical assistance of YunnChyn Tung, Michael Diaz, Miriam Pakingan, Howard Finch, Everett Swanson, Norma Blum, the secretarial help of Judy Jones and the photographic work of Richard Weed.

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