Antibody-dependent cellular cytotoxicity in antimyelin antibody-induced oligodendrocyte damage in vitro

Journal of Neuroimmunology, 33 (1991) 145-155

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© 1991 Elsevier Science Publishers B.V. 0165-5728/91/$03.50 JNI 02026

Antibody-dependent cellular cytotoxicity in antimyelin antibody-induced oligodendrocyte damage in vitro M. G r i o t - W e n k t, C. G r i o t

1 H.

Pfister 2 a n d M. V a n d e v e l d e 1

Institutes of 1Animal Neurology and 2 Veterinary Virology, University of Berne, CH-3001 Berne, Switzerland

(Received 16 July (1990) (Revised, received 2 October 1990,2 November 1990,4 February 1991,28 February 1991) (Accepted 1 March 1991)

Key words: Antibody-dependent cellular cytotoxicity;Antimyelin antibody; Demyelination; Macrophage; Oligodendrocyte

Summary Treatment of dissociated murine brain cell cultures with an antibody recognizing galactocerebroside (GalC) led to degeneration of oligodendrocytes with loss of their cell processes. F(ab') 2 fragments prepared from this antibody showed no effect. The anti-GalC antibody - - but not its F(ab') 2 fragments b2 was able to stimulate macrophages in these cultures as seen in a chemiluminescence assay. Therefore, antibodies bound to oligodendrocytes stimulated nearby macrophages by interacting with their Fc receptors. The oligodendroglial damage coincided with the release of toxic compounds by the stimulated macrophages, since treatment of the cultures with the anti-GalC antibody and a variety of other macrophage stimulating agents led to secretion of reactive oxygen species and - - in some experiments - - tumor necrosis factor, both known to be toxic for oligodendrocytes. These in vitro experiments show evidence that antibody-dependent cellular cytotoxicity may be an important mechanism of tissue destruction in inflammatory demyelinating diseases.

Introduction In inflammatory demyelinating diseases, such as multiple sclerosis (MS), experimental allergic encephalitis (EAE) and canine distemper encephalitis (CDE), macrophages are an important feature within the lesions and are believed to be

Address for correspondence: M. Vandevelde, lnstitut fiir Tierneurologie, Universit~it Bern, P.O. Box 2735, CH-3001 Bern, Switzerland.

active mediators of myelin destruction (Wisniewski et al., 1972; Raine, 1983; Traugott et al., 1983; Prineas et al., 1984; Esiri and Reading, 1987). Recently, we have shown (Biirge et al., 1989; Griot et al., 1989) that anti-canine distemper virus (CDV) antibodies are capable of stimulating brain macrophages in the presence of persistently CDV-infected glial cells in vitro leading to the release of reactive oxygen species (ROS). Some ROS can cause considerable tissue damage through reaction with DNA, protein and membrane lipids (Halliwell and Gutteridge, 1984). Es-

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pecially brain tissue appears to be vulnerable because of its high content of oxidizable substrates (Halliwell and Gutteridge, 1985; Konat and Wiggins, 1985). We have shown that the release of ROS depends on the presence of viral antigens on the surface of infected glial cells and is mediated by the interaction of antigen-bound antibody with Fc receptors on macrophages in their immediate vicinity (Biirge et al., 1989; Griot et al., 1989). These observations showed how antiviral antibodies may worsen the lesions in CDE by stimulating macrophages. Antimyelin antibodies have been shown to cause considerable lesion progression in EAE and it was postulated that an antibody-dependent cellular cytotoxicity (ADCC) mechanism may be involved in this process (Linington and Lassmann, 1987; Lassmann et al., 1988). This view is supported by our finding that anti-galactocerebroside (GalC) antibodies binding to oligodendrocytes in mixed glial cell cultures are also able to stimulate macrophages (Griot et al., 1989). In the present study we incubated primary mouse brain cell cultures with a monoclonal anti-GalC antibody. This treatment resulted in the stimulation of macrophages and led to degenerative changes in the oligodendrocytes. The results of our experiments strongly suggest that an ADCC mechanism is responsible for the observed oligodendroglial damage.

Materials and methods

Mouse brain cell cultures Cultures of mouse brain cells (MBCC) were prepared as described elsewhere (Wiesmann et al., 1975). Briefly, brains of newborn mice were aseptically removed immediately after decapitation and dissociated in tissue culture medium by chopping and repeated aspiration through 10 ml glass pipettes. The suspension (1.5 × 106 cells/ml) was seeded in Petri dishes (3003, Falcon) containing Dulbecco's modified Eagle's medium (07402100, Gibco BRL) supplemented with 10% heat-inactivated fetal calf serum, penicillin (250 U / m l ) and streptomycin (100 p~g/ml). Eight glass coverslips (1000, 18 × 18 mm, Assistent, F.R.G.) were mounted onto the Petri dishes with silicon

grease. The cultures were kept at 37°C in a water-saturated atmosphere of 5% CO 2 and 95% air. Medium was changed every other day.

Treatment of cultures After 21 days in culture, MBCC were exposed to the following reagents: Phorbol myristate acetate (PMA). PMA (P8139, Sigma) was dissolved in dimethyl sulfoxide (DMSO) and used at a final concentration of 70 nM (Sonderer et al., 1987). The final concentration of DMSO in the working solution was less than 0.1%, a concentration that had no effect on viability of cultured brain cells (Sonderer et al., 1987). Lipopolysaccharide (LPS). LPS (L-3012, Sigma) was dissolved in 0.9% NaC1 and added at a concentration of 0.1 ixg/ml. Opsonized zymosan. Z y m o s a n (Z-4250, Sigma) was opsonized with fresh bovine serum and resuspended in 0.9% NaC1 (Biirge et al., 1989). The final concentration was 100 txg/ml. Anti-galactocerebroside (GalC) antibody. Supernatant of a murine hybridoma cell line producing the monoclonal antibody (MAB) I 6G1, an antibody binding to GalC (Zurbriggen et al., 1987) and of the IgG 3 subclass was purified by ammonium sulfate precipitation and concentrated by subsequent membrane ultrafiltration (PM 10, Amicon, Switzerland). The IgG 3 concentration of the obtained fraction was 130 /xg/ml as measured by a sandwich enzyme-linked immunosorbent assay (ELISA). MAB I 6G1 was added to brain cell cultures at a concentration of 0.13 /xg/ml. For some experiments, IgG fractions were pepsinized in order to prepare F(ab') 2 fragments (Fey et al., 1976), which were used in the same concentration as MAB I 6G1. IgG-coated erythrocytes. Bovine erythrocytes were opsonized with rabbit IgG to the erythrocyte stroma (8879, Nordic Immunological Laboratories, The Netherlands) as described for sheep erythrocytes (Jungi et al., 1988). Erythrocytes coated with this antibody were added to the cultures (106 erythrocytes/ml). It was shown that they are capable of stimulating brain macrophages by binding to Fc receptors (Sonderer et al., 1987). MAB D 110. MAB D 110 (directed to the nucleocapsid protein of CDV and of IgG 1 sub-

147 class; Bollo et al., 1986) was used as a mockstimulating antibody. All reagents (except the IgG-coated erythrocytes) were kept frozen in small aliquots at - 20°C and were thawed immediately before use. IgGcoated erythrocytes were kept at 4°C. Twenty-four hours after the last medium change, the reagents were added to the cultures in the concentrations described above. Coverslips from treated and corresponding control cultures were harvested 24, 48 and 72 h afterwards and oligodendrocytes were monitored by immunocytochemistry and ELISA. These experiments were repeated 15-20 times. In selected experiments, cultures were maintained for additional periods of time and harvested 6, 7 and 8 days after treatment.

Immunocytochemistry Glial cells were specifically labelled using indirect immunofluorescence or peroxidase-antiperoxidase (PAP) techniques as described previously (Zurbriggen et al., 1987). Oligodendrocytes were immunolabelled with MAB I 6G1, which binds specifically to GalC (Dumas et al., 1985; Zurbriggen et al., 1987), a well-established marker for oligodendrocytes (Raft et al., 1978) or with MAB V 7E5 which binds specifically to oligodendrocytes (Dumas et al., 1985; Zurbriggen et al., 1987). For demonstration of astrocytes, a commercially available anti-glial fibrillary acidic protein antibody (GFAP, Z 334, Dakopatts, Denmark) was used. Macrophages were labelled by a rat MAB, which recognizes the membrane antigen Mac-1 (MCA 74S, Serotec, U.K.) or by the erythrocyte-rosetting assay as described (Taffet et al., 1981). In selected experiments, the number of macrophages per coverslip was counted 24 h after treatment. ELISA for oligodendroglial antigen An ELISA for quantitative evaluation of oligodendrocytes in MBCC was used. To this end, MAB V 7E5 was applied as primary antibody. This MAB recognizes a fixation-resistant oligodendrocyte specific glycolipid (Dumas et al., 1985; Zurbriggen et al., 1987) and d o e s not compete with MAB I 6G1. Six coverslips from each experi-

ment (harvested at the different time points described above) plus six corresponding untreated control coverslips were assayed simultaneously. Coverslips, fixed with 3.7% phosphate-buffered formalin for 20 rain at room temperature (RT), were blocked by normal goat serum, incubated with MAB V 7E5 (2 h, 37°C) followed by goat anti-mouse IgG (30 rain, RT) and finally by PAP (P-850, Dakopatts, Denmark) (30 rain, RT). The coverslips were then reacted with 200 p~l each of o-phenylenediamine dihydrochloride (P 8287, Sigma) dissolved in citric acid phosphate buffer. After 5 rain at RT, 150 ~i of this solution from each coverslip was transferred to a 96-well microtiter plate (2797, Costar, The Netherlands). The enzyme-substrate reaction was stopped by adding 50/~1 of H2SO 4 (2 N) and optical densities (OD) were measured at 492 nm with a Titertek Multiscan MC (Flow Laboratories, Switzerland). OD values of six coverslips for each reagent were averaged and expressed as percentage of the values in the control cultures. The values for 0D492 nm obtained were reproducible within one given experiment, but varied, however, between different batches of MBCC due to various factors (Gard et al., 1988).

Nitroblue tetrazolium assay Cells were covered with a solution of nitroblue tetrazolium (NBT; 2 mg/ml in phosphatebuffered saline (PBS)) (24823, Merck, F.R.G.) containing PMA (10 -v M) and sodium azide (10 -3 M). After 10 min, the solution was removed and the cultures were fixed with 95% ethanol-5% acetic acid for 5 rain at -20°C. Cells capable of producing ROS are able to reduce NBT (Volkman et al., 1984; Sonderer et al., 1987). This reduction can be recognized by the formation of blue-purple granules of formazan monitored by light microscope. In some experiments, the NBT assay was combined with immunofluorescence staining for macrophages with anti-Mac1 antibody. Localization of opsonized zymosan and IgG-coated erythrocytes in brain cell cultures In order to determine to which cells opsonized zymosan and IgG-coated erythrocytes bind,

148 MBCC treated with zymosan were harvested, fixed with 95% ethanol-5% acetic acid and subjected to double-immunofluorescent labelling using goat anti-bovine IgG-fluorescein isothiocyanate (FITC) (65-164-2, Miles Scientific, U.S.A.) (labelling zymosan coated with bovine IgG) and rat anti-Mac-1 IgG. The latter antibody was visualized in a second step using goat anti-rat IgG-tetramethyl rhodamine isothiocyanate (TRITC) (03-16-06, KPL, U.S.A.). A similar procedure was applied to the IgG-coated erythrocyte-treated cultures whereby erythrocytes were directly visible by their size and autofluorescence in ultraviolet light.

Chemiluminescence In order to detect macrophage stimulation by the agents used in these experiments, luminol-dependent chemiluminescence (CL) experiments were carried out. On stimulation with a variety of particulate and soluble agents, phagocytic cells reduce molecular oxygen to O 2, from which by enzymatic and non-enzymatic mechanisms an array of other oxygen species, collectively referred to as ROS, are derived (Allen, 1986; Peterhans, 1987). In the presence of suitable indicators such as luminol (5-amino-2,3-dihydro-l,4-phthalazinedione; A 8511, Sigma), some of these species result in CL (for review see Allen, 1986; Peterhans, 1987). CL measurement in glial cell cultures has been described in detail previously (Sonderer et al., 1987; Biirge et al., 1989; Griot et al., 1989). Briefly, CL assays were carried out with 20-dayold cultures in a modified liquid scintillation spectrometer. Glass coverslips with cultured glial cells were removed from the Petri dishes, washed and transferred into glass tubes containing 1.5 ml Hanks' buffered salt solution with 5 mM glucose and 5 p~M luminol. After dark adaptation and twice measuring background light emission, zymosan (300/zg/ml), PMA (70 nM), MAB I 6G1 (13 /xg/ml), IgG-coated erythrocytes (10 6 erythrocytes/vial), uncoated erythrocytes, rabbit anti-bovine IgG (15/zg/vial) and lipopolysaccharide (LPS) (0.1 /zg/ml) were added to the vials. Light emission was measured up to 30 min and curves were plotted relative to curves of mockstimulated controls (for details on the equipment see Peterhans, 1987).

Measurement of tumor necrosis factor (TNF) activity TNF activity in supernatant of MBCC was determined in a biological assay on actinomycin D-treated mouse L-929 fibroblasts grown in microtiter plates (163320, Nunclon, Denmark) (for a review see Meager et al., 1989). Supernatants were taken from 30 min to 72 h after treatment of MBCC with PMA, LPS, MAB I 6G1, opsonized zymosan, IgG-coated erythrocytes and F(ab') z fragments of MAB I 6G1. Upon near confluency, medium was removed and replaced by 50 /zl serial dilutions of human recombinant TNF (rhTNF; 87650, W.H.O. International Laboratory for Biological Standards, U.K.) as a positive control. Supernatant of MBCC was pipetted into the medium of the L-929 cells. After incubation overnight (37°C, 5% CO2), 50 /zl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (3 mg/ml, M-2128, Sigma), a yellow tetrazolium salt which is reduced to a purple formazan by the mitochondrial dehydrogenase of live cells, was added. After 3 h incubation at 37°C, medium was replaced with 100/zl isopropanol containing 0.04 N HC1 and 20 /.d 3% sodium diphenyl sulfate. After 1 h, absorbance (591 nm) was read using an automatic spectrometer. The concentration of TNF was quantified by comparing the dilution of test supernatant with a known amount of rhTNF (added as control in each experiment). Propidium iodide and trypan blue staining Propidium iodide (which when intercalated with DNA becomes strongly fluorescent) and trypan blue are two substances which enter damaged cells. MBCC were incubated with propidium iodide (1 /zg/ml, 10 min) (PI, 33671, Serva, F.R.G.) (Parks et al., 1986) or with trypan blue (0.05%, 1 min) (TP, 47285, Serva, F.R.G.). After rinsing with Tris-buffer, cultures stained with TP were fixed with 95% ethanol-5% acetic acid and evaluated with the light microscope. PI-stained cultures were examined without fixation using a fluorescent microscope. Following this treatment selected coverslips were immunostained with MAB I 6G1 (anti-GalC) in order to determine the percentage of PI-positive oligodendrocytes. Cell damage was calculated as percentage of PIor TP-positive cells compared to untreated con-

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Fig. 1. a: Oligodendrocytes m normal untreated mouse brain cell cultures (MBCC). Characteristic morphology of oligodendr6cytes with extensively branching processes covering large areas of the culture. Mouse anti-galactocerebroside (GalC) IgG-goat anti-mouse IgG-fluorescein isothiocyanate (FITC). X400. b-d: Clear reduction of GalC-positive processes of oligodendrocytes 72 h after treatment with the murine monoclonal antibody (MAB) I 6G1 (b), opsonized zymosan (c) and IgG-coated erythrocytes (d). Mouse anti-GalC IgG-goat anti-mouse IgG-FITC. x 400.

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Fig. 2. a: Untreated control MBCC. Erythrocyte-rosette forming macrophages which are in close contact to an oligodendrocyte immunostained with M A B V 7E5-peroxidase-antiperoxidase. x400. b: M B C C 72 h after treatment with IgG-coated erythrocytes. Macrophages increased in n u m b e r and enlarged. Oligodendrocyte has lost its processes. Immunocytochemical staining as in a. x400. c: M B C C 72 h after treatment with opsonized zymosan. Two enlarged macrophages are in close contact with an oligodendroglial process and have incorporated most erythrocytes used for rosetting assay (increased phagocytosis). Immunocytochemical staining as in a. x 1000. d: M B C C treated with opsonized zymosan. Double-immunofluorescent labelling for Mac-1 antigen of macrophages (rat anti-Mac-1 IgG-goat anti-rat IgG-tetramethyl rhodamine isothiocyanate) and zymosan particles opsonized with bovine IgG (goat anti-bovine IgG-FITC). Zymosan particles are attached to the surface of Mac-l-positive cells. x 1000.

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trois using a fluorescence microscope (PI) or a light microscope (TP).

Results

Morphological studies of mouse brain cell cultures After 10 to 14 days in culture, MBCC became confluent. These cultures predominantly contained astrocytes, flbroblasts, oligodendrocytes and brain macrophages. The latter two cell types were mainly distributed in clusters superimposed on the other cells in the culture. GalC-positive oligodendrocytes had a striking morphology characterized by an irregularly shaped perikaryon and few large cell processes that were extensively branched. Most ceils had also processes which flattened into GalC-positive sheaths (Fig. la). A few GalC-positive cells were small and round with dense cytoplasm and few, unbranched pro-

cesses. Macrophages were mostly of the amoeboid type as described by Frei et al. (1987) (Fig. 2a). Few were elongated displaying at their ends long filopods. Ramified microglia, with a varying number of relatively long cytoplasmic processes, were found occasionally. Oligodendrocytes were often in close contact with macrophages (Fig. 2a). Astrocytes were predominantly of the fibrous type with thin, unbranched, strongly GFAP-positive cell processes. The morphology of oligodendrocytes, astrocytes and brain macrophages in treated cultures was evaluated 72 h after exposure. Treatment with MAB I 6G1 (Fig. lb), IgG-coated erythrocytes (Fig. lc) and opsonized zymosan (Fig. ld) led to varying degrees of oligodendroglial changes, which were mainly seen at the level of cell processes with loss of GalC-positive sheaths and finer branches. LPS induced also a marked reduction in the number of cell processes. PMA

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Fig. 3. Result of enzyme-linked immunosorbent assay (ELISA) for oligodendroglial antigen after treatment of MBCC with a variety of agents in one selected experiment. OD492 n m values obtained of six separate coverslips after the treatments with each reagent have been averaged (standard deviations are indicated) and expressed as a percentage of the OD492n m values obtained in corresponding untreated control cultures. Marked decrease in amount of oligodendroglial antigens is found 24-72 h after treatment with all agents except F(ab') 2 fragments of MAB I 6GI and the mock-stimulating MAB D 110 (control: column 1; F(ab') 2 of MAB I 6Gl: column 2; MAB D 110: column 3; PMA: column 4; MAB I 6Gl: column 5; zymosan: column 6; LPS: column 7; IgG-coated erythrocytes: column 8).

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treatment resulted in nearly complete loss of cell processes. F ( a b ' ) 2 fragments of M A B I 6G1 and MAB D 110 had no visible effect. In cultures which were kept for prolonged periods of time after treatment, oligodendroglial morphology returned to normal within 8 days. T h e r e was a 2- to 10-fold increase in the number of macrophages per coverslip already 24 h after treatment with all reagents (Fig. 2b), except with F ( a b ' ) 2 fragments of MAB I 6G1 and mock-stimulating MAB D 110. Macrophages clearly increased in size after treatment with opsonized zymosan and IgG-coated erythrocytes (Fig. 2b). Opsonized zymosan also induced ruffling of their surface (Fig. 2c). Opsonized zymosan and IgG-coated erythrocytes produced elongation of cell processes and resulted in increased phagocytosis of opsonized erythrocytes used for labelling macrophages (Fig. 2c). Astrocytes in the treated cultures did not differ from those in the control cultures. There was no significant increase in PI- and TP-positive cells after treatment.

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ELISA for oligodendroglial antigen There was a marked and progressive decrease in the amount of oligodendroglial antigen recognized by M A B V 7E5 in the cultures treated with PMA, LPS, opsonized zymosan, M A B I 6G1 and IgG-coated erythrocytes, but not after treatment with F ( a b ' ) 2 fragments of M A B I 6G1 and MAB D 110 (Fig. 3). The decrease varied considerably from one experiment to the other (all experiments were repeated 15-20 times) but was highly reproducible as such. The amount of V 7E5-positive antigen returned to normal levels in cultures that were maintained up to 8 days after treatment; in some experiments the values in cultures kept up to 8 days markedly exceeded those of the corresponding control cultures.

bound to macrophage surfaces and not to other cell types. Formazan granules in the NBT test were clearly present in Mac-l-positive cells but also - - to a lesser extent - - in other cell types.

Measurement of ROS in mouse brain cell cultures After treatment with LPS, PMA, opsonized zymosan and MAB I 6G1 the oxidative burst measured with a luminol-dependent CL assay reached its maximum within 5 - 1 0 min (Fig. 3) and 30-40 min after addition of IgG-coated erythrocytes. Addition of Hanks' buffered salt solution, F ( a b ' ) 2 fragments of MAB I 6G1 (Fig. 4) and MAB D 110 failed to induce CL.

Localization of reagents Immunostaining for macrophages with MAB anti-Mac-1 after treatment with opsonized zymosan and IgG-coated erythrocytes revealed that zymosan particles (labelled with goat anti-bovine I g G - F I T C ) w e r e p r e d o m i n a n t l y b o u n d to macrophages and phagocytized by them (Fig. 2d). Likewise, IgG-coated erythrocytes were only

Measurement of TNF activity Significant concentrations of T N F activity could only be measured in MBCC supernatant after treatment with LPS. Four hours after initiation of the experiments, 30 I U of T N F were detected. After 72 h the amount decreased to 28 IU.

153 Discussion

It has been shown that antibodies against myelin surface antigens can considerably modulate lesion formation in EAE (Linington and Lassmann, 1987; Lassmann et al., 1988) and alter the morphology of oligodendrocytes in vitro (Diaz et al., 1978; Lehrer et al., 1979). Our results demonstrate that a murine monoclonal antibody (MAB) against galactocerebroside (GalC) damages oligodendrocytes in mixed mouse brain cell cultures. The damage was not the result of the mere presence of immunoglobulin in the system since addition of another MAB not directed against brain antigens did not cause any damage. Treatment with the anti-GalC antibody also led to stimulation of the macrophages as seen in the chemoluminescence assay, macrophages being the only cell type in MBCC capable of producing ROS (Biirge et al., 1989). Since F(ab') 2 fragments of the anti-GalC antibody failed to produce a response, stimulation took place by way of the macrophage Fc receptors, which are not present on other cell types in these cultures (Raft et al., 1979; Giulian and Baker, 1980; Raedler and Raedler, 1984; DuBois et al., 1985; Ditrich, 1986; Jordan and Thomas, 1987; Zucker-Franklin et al., 1987). The use of the F(ab') 2 fragments alone also failed to induce lesions indicating that the binding of the antibody to the cell surface was by itself not sufficient to cause cell damage. The binding of the anti-GalC antibody to the oligodendrocytes was essential for macrophage stimulation to occur since treatment of isolated purified macrophages with the antibody failed to provoke a response (Griot et al., 1989). Therefore, we conclude that the observed oligodendroglial damage after anti-GalC antibody treatment is probably mediated by an ADCC mechanism whereby the antibodies bound to the oligodendrocytes stimulate nearby macrophages by interacting with their Fc receptors. Although it is clear from our results that macrophages were stimulated and that interaction between oligodendrocytes, anti-myelin antibody and macrophages took place in our experiments, we cannot exclude that other cell types in the culture were also involved by some unknown mechanism. Therefore, it would be desirable to repeat these

experiments in pure oligodendrocyte cultures. However, while it is quite easy to produce enriched oligodendrocyte cultures, 100% purity which would be required is extremely difficult to obtain and certainly not in quantities needed for this type of experiments. We believe that the damage inflicted upon the oligodendrocytes was mediated by soluble factors secreted by stimulated macrophages; both cell types are on the surface of the cultures superimposed on the other cells and very frequently in close apposition to each other. This view was supported by our experiments using other reagents to stimulate macrophages. These included opsonized zymosan and opsonized erythrocytes which bind to macrophages only, as shown in our double-labelling studies, as well as PMA and LPS. It is known that the latter two agents can act on other cell types (Robbins et al., 1987; Sonderer et al., 1987) and it cannot be completely excluded that the two immune complexes used could have had - - in some mysterious way - - some direct effect on oligodendrocytes. We could show, however, that all these reagents induced ROS secretion and similar oligodendroglial changes. It is known that ROS are highly toxic for many different biological tissues including central nervous system cells and we have recently shown that ROS provided by a chemically defined system are far more injurious for oligodendrocytes than for other glial ceils in vitro (Griot et al., 1991). Therefore, it is possible that oligodendroglial changes resulted from ROS activity. Our search for TNF, another mediator of oligodendroglial damage (Selmaj and Raine, 1988) - - possibly by way of ROS activity (Zimmerman et al., 1989) - was only successful in PMA-treated cultures. The failure to find enhanced activity in supernatants after treatment with the other reagents could be due to kinetic reasons since TNF may act locally in a membrane-associated form killing in close cell apposition (Decker et al., 1987) and does not exclude its possible involvement. Pathological changes were only obvious in oligodendrocytes and mostly restricted to the cell processes. Trypan blue and propidium iodide staining did not indicate widespread cell killing. Such a selective effect supports our view that

154 m a c r o p h a g e p r o d u c t s are involved. T h e selective v u l n e r a b i l i t y of o l i g o d e n d r o c y t e s to R O S may be related to the p r e s e n c e of iron c o m p o u n d s in these cells. It has b e e n shown in vivo a n d in vitro that o l i g o d e n d r o c y t e s selectively a c c u m u l a t e t r a n s f e r r i n (iron m o b i l i z a t i o n protein), ferritin (iron storage p r o t e i n ) a n d u n b o u n d iron (Bloch et al., 1985; C o n n o r a n d F i n e , 1987; G r i o t a n d V a n develde, 1988; G e r b e r a n d C o n n o r , 1989; C o n n o r a n d Menzies, 1990). I r o n ions are necessary for the c o n v e r s i o n of the superoxide a n i o n radical (secreted by m a c r o p h a g e s ) to the highly destructive hydroxyl radical causing lipid p e r o x i d a t i o n (Halliwell a n d G u t t e r i d g e , 1989). It r e m a i n s to be d e t e r m i n e d how iron ions are mobilized from their storage sites in the o l i g o d e n d r o c y t e s in order to b e c o m e available for this reaction. I n conclusion, we could show that a n t i - m y e l i n a n t i b o d y - i n d u c e d o l i g o d e n d r o c y t e pathology in mixed glial cell cultures is due to an A D C C m e c h a n i s m . T h e i n t e r a c t i o n of a n t i b o d i e s with m a c r o p h a g e s leads to secretion of R O S a n d cytokines, k n o w n to be toxic for oligodendrocytes. W e believe that the observed m e c h a n i s m in vitro may be r e l e v a n t to tissue d e s t r u c t i o n in inflamm a t o r y d e m y e l i n a t i n g diseases such as m u l t i p l e sclerosis, e x p e r i m e n t a l allergic e n c e p h a l i t i s a n d c a n i n e d i s t e m p e r encephalitis.

Acknowledgements This work was s u p p o r t e d by the Swiss N a t i o n a l Science F o u n d a t i o n G r a n t No. 3.956.87, the Swiss M u l t i p l e Sclerosis Society a n d the W a n d e r F o u n dation. T h e a u t h o r s wish to t h a n k Prof. Dr. E. P e t e r h a n s for critically r e a d i n g the m a n u s c r i p t a n d B. G l a u s a n d A. R i c h a r d for excellent technical assistance.

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