anti-macrophage cr3 antibody blocks myelin phagocytosis by macrophages in vitro

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Acta Neuropathol (1990) 80:4t5-418 NActa . europatholog:ca Springer-Verlag1990 Anti-macrophage CR3 antibody blocks myelin phagocytosis by macrophages in vitro* W. Briick and R. L. Friede Abteilung ffir Neuropathologie, Universitfit G6ttingen~ Robert-Koch-Strasse 40, D-3400 G6ttingen, Federal Republic of Germany Received December 28, 1989/Revised March 5, 1990/Accepted March 14, 1990 Summary. Myelin phagocytosis in Wallerian degener- ation of peripheral nerves depends on invasion of nerves by non-resident macrophages. The present study was done to clarify the role of the macrophage complement receptor type 3 (CR3) in myelin removal. Myelin phago- cytic capacity of invading macrophages was abolished by treatment of cultured nerves and macrophages with anti- CR3 antibody or by serum complement depletion with cobra venom factor. This indicates that myelin phago- cytosis is mediated by the macrophage CR3. Key words: Myelin phagocytosis - Macrophages - Complement receptor type 3 - Organ culture series, we tested the effect of 14 simple or complex sugars and 8 sugar-splitting enzymes on organ cultures of nerves cocultured with macrophages; only treatment with L-fucosidase was effective in blocking myelin phagocy- tosis [6]. The striking inhibitory effect of L-fucosidase sug- gested a potential involvement of the complement recep- tor type 3 (CR3) in myelin phagocytosis: this receptor is activated by a lymphokine binding to fucose [10]. The major function of the macrophage CR3 is the binding and ingestion of opsonized targets [15]; its molecular structure is identical to the Mac-1 antigen found in mice. In the following we report experiments showing a critical role of the macrophage CR3 in myelin phagocytosis. Non-resident, hematogenous ceils of the macrophage sys- tem are massively engaged in myelin phagocytosis during Wallerian degeneration [3]. Nerve segments degenerating within millipore diffusion chambers were actively invaded by Fc-positive and Mac-l-positive monocytes [19, 20]. Similar results were obtained in vitro by exposing organ cultures of mouse peripheral nerves to cultured macro- phages [12]. The latter invaded the nerves and ingested the degenerating myelin sheaths. The invading phago- cytes were also Fc- and Mac-l-positive. Schwann cells did not show significant myelin phagocytosis under these experimental conditions. Further studies aimed at defining the way in which phagocytes recognize degenerating myelin, particularly the involvement of their numerous plasma membrane receptors [9]. Non-specific damage of monocytes with silica retarded myelin degradation in vivo [4], while phagocytosis mediated by Fc-receptors did not seem to play a significant role in myelin removal [11]. In another * Supported by grant 609/2-1 from the Deutsche Forschungsge- meinschaft Offprint requests to: R. L. Friede Materials and methods Preparation of macrophages and peripheral nerves Adult mice of either sex of the C57B1J6 strain (Zentralinstitut ffir Tierzucht, Hannover, FRG) were used. Peritoneal macrophages were harvested 4 days after intraperitoneal thioglycollate injection (2.9%, NIH, thioglycollate broth, Difco Nr. 0257 01) by peritoneal lavage with cold phosphate-buffered saline, pH 7.4. Macrophages were cultured in Dulbecco's minimal essential medium, supplement- ed with 10% fetal calf serum (FCS), 100 U/ml penicillin, 100 gg/ml streptomycin and 58 mg% L-glutamine containing 10 gM cytosine arabinoside. Experiments were done both in the presence or the absence of fresh mouse serum. A constant cell number of 2 x 106/ ml was added to the culture vessels (Petriperm, N. C., Hanau, FRG). Sciatic nerves were dissected after decapitation of the animals in deep anesthesia. The nerve sheaths were removed with fine forceps and the desheathed nerves were cut into 3- to 4-mm segments. The segments with added macrophages were maintained in tissue culture medium at 37~ in a humidified atmosphere containing 5% COz. A culture period of 10 days was generally used. Treatment of cell cultures Rat anti-mouse macrophage CR3 (Mac-l) monoclonal antibody (clone M 1/70) [2] from Serotec was added at 10 gg/ml for the entire culture period. Other monoclonal antibodies used were directed

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Page 1: Anti-macrophage CR3 antibody blocks myelin phagocytosis by macrophages in vitro

Acta Neuropathol (1990) 80:4t5-418

NActa . europatholog:ca

�9 Springer-Verlag 1990

Anti-macrophage CR3 antibody blocks myelin phagocytosis by macrophages in vitro* W. Briick and R. L. Friede

Abteilung ffir Neuropathologie, Universitfit G6ttingen~ Robert-Koch-Strasse 40, D-3400 G6ttingen, Federal Republic of Germany

Received December 28, 1989/Revised March 5, 1990/Accepted March 14, 1990

Summary. Myelin phagocytosis in Wallerian degener- ation of peripheral nerves depends on invasion of nerves by non-resident macrophages. The present study was done to clarify the role of the macrophage complement receptor type 3 (CR3) in myelin removal. Myelin phago- cytic capacity of invading macrophages was abolished by treatment of cultured nerves and macrophages with anti- CR3 antibody or by serum complement depletion with cobra venom factor. This indicates that myelin phago- cytosis is mediated by the macrophage CR3.

Key words: Myelin phagocytosis - Macrophages - Complement receptor type 3 - Organ culture

series, we tested the effect of 14 simple or complex sugars and 8 sugar-splitting enzymes on organ cultures of nerves cocultured with macrophages; only treatment with L-fucosidase was effective in blocking myelin phagocy- tosis [6].

The striking inhibitory effect of L-fucosidase sug- gested a potential involvement of the complement recep- tor type 3 (CR3) in myelin phagocytosis: this receptor is activated by a lymphokine binding to fucose [10]. The major function of the macrophage CR3 is the binding and ingestion of opsonized targets [15]; its molecular structure is identical to the Mac-1 antigen found in mice. In the following we report experiments showing a critical role of the macrophage CR3 in myelin phagocytosis.

Non-resident, hematogenous ceils of the macrophage sys- tem are massively engaged in myelin phagocytosis during Wallerian degeneration [3]. Nerve segments degenerating within millipore diffusion chambers were actively invaded by Fc-positive and Mac-l-positive monocytes [19, 20]. Similar results were obtained in vitro by exposing organ cultures of mouse peripheral nerves to cultured macro- phages [12]. The latter invaded the nerves and ingested the degenerating myelin sheaths. The invading phago- cytes were also Fc- and Mac-l-positive. Schwann cells did not show significant myelin phagocytosis under these experimental conditions.

Further studies aimed at defining the way in which phagocytes recognize degenerating myelin, particularly the involvement of their numerous plasma membrane receptors [9]. Non-specific damage of monocytes with silica retarded myelin degradation in vivo [4], while phagocytosis mediated by Fc-receptors did not seem to play a significant role in myelin removal [11]. In another

* Supported by grant 609/2-1 from the Deutsche Forschungsge- meinschaft

Offprint requests to: R. L. Friede

Materials and methods

Preparation of macrophages and peripheral nerves

Adult mice of either sex of the C57B1J6 strain (Zentralinstitut ffir Tierzucht, Hannover, FRG) were used. Peritoneal macrophages were harvested 4 days after intraperitoneal thioglycollate injection (2.9%, NIH, thioglycollate broth, Difco Nr. 0257 01) by peritoneal lavage with cold phosphate-buffered saline, pH 7.4. Macrophages were cultured in Dulbecco's minimal essential medium, supplement- ed with 10% fetal calf serum (FCS), 100 U/ml penicillin, 100 gg/ml streptomycin and 58 mg% L-glutamine containing 10 gM cytosine arabinoside. Experiments were done both in the presence or the absence of fresh mouse serum. A constant cell number of 2 x 106/ ml was added to the culture vessels (Petriperm, N. C., Hanau, FRG). Sciatic nerves were dissected after decapitation of the animals in deep anesthesia. The nerve sheaths were removed with fine forceps and the desheathed nerves were cut into 3- to 4-mm segments. The segments with added macrophages were maintained in tissue culture medium at 37~ in a humidified atmosphere containing 5% COz. A culture period of 10 days was generally used.

Treatment of cell cultures

Rat anti-mouse macrophage CR3 (Mac-l) monoclonal antibody (clone M 1/70) [2] from Serotec was added at 10 gg/ml for the entire culture period. Other monoclonal antibodies used were directed

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Fig. 1. a A control nerve degenerated in organ culture for 10 days is massively invaded by cocultured macrophages, x 500. b In the presence of 10 gg/ml anti-Mac-l, numerous small cells are dispersed in the nerve (arrows), but there is no indication of active myelin ingestion, x 500. c In controls, invaded macrophages preloaded

with latex beads contain myelin debris, lipid droplets (+) and latex particles (arrows) in their cytoplasm, x 8900. d With anti-Mac-1 treatment, the invading cells contain lipid droplets (+) and latex beads (arrows), but they do not ingest myelin, x 11,000

against class I and II MHC antigens: monoclonal anti-mouse H-2D and monoclonal anti-mouse I-A (Camon, Wiesbaden, FRG). These antibodies were also added for the entire culture period. Cobra venom factor (CVF) was obtained from Sigma and added at 10 gg/ml.

Preparation of myelin particles and phagocytosis assay

Mouse sciatic nerves were desheathed and shredded with a razor. The nerve segments were then homogenized at 4~ using the homogenizer Potter S (type 853 202; Braun, Melsungen, FRG). Macrophages were prepared as described above and incubated with the anti-mouse macrophage CR3 antibody for 4 h before the myelin particles were added for 2 h. Conventional electron microscopic methods were used for specimen preparation.

Results

Control cultures of nerves with cocultured macrophages which were not treated with anti-CR3 antibody showed

massive invasion of nerves by macrophages which pro- fusely ingested myelin fragments (Fig. l a). Cultures treated with 10 gg/ml anti-Mac-1 were invaded by numer- ous small cells containing abundant lipid droplets in their cytoplasm, but there were no enlarged macrophages con- taining myelin debris (Fig. I b). Most invading cells were freely dispersed between the profiles of nerve fibers. The electron microscope showed a few of them also lodging within a basal laminar envelope. Such cells had many finger-like pseudopodia without specific contacts to the fibers. Attachment of cells to myelin sheaths or myelin figures was rare. The cytoplasm contained many lipid droplets but hardly any myelin fragments. Results were similar in the presence or the absence of fresh mouse serum in the FCS-supplemented culture medium.

Additional experiments aimed at verifying the origin of the invading cell population using macrophages pre- loaded with latex beads before their addition to the organ cultures. Latex beads were ingested by macrophages upon

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Fig. 2. Mouse sciatic nerve degenerated for 10 days in organ culture with added macrophages in the presence of cobra venom factor. Numerous cells with abundant cytoplasmic vacuoles (arrows) invade the nerve, but there is no indication of active myelin phagocytosis. x 270

Fig. 3. Treatment with anti-I-A antibody (major histocompatibility complex class II) has no effect on nerve invasion or myelin ingestion by the cocultured macrophages compared with untreated controls. Experimental conditions same as in Figs. 1 and 2. x 270

2-h exposure in vitro. The preloaded macrophages were then added to the organ cultures of nerves, with or with- out anti-Mac-I treatment. Invasion of the nerves oc- curred in both experiments but there were striking differ- ences in macrophage structure. Without anti-Mac-1 treatment, the macrophages contained an admixture of latex beads and myelin debris (Fig. 1 c). In the presence of anti-Mac-l, ceils containing only latex beads and lipid droplets accumulated between the degenerating nerve fibers without ingesting myelin (Fig. 1 d). The partly degraded latex beads often accumulated in large cyto- plasmic vacuoles. Phagocytosis of latex particles by macrophages was not affected by anti-Mac-1 treatment.

Similar results were obtained when complement was depleted in vitro with CVF. In these experiments there was also no indication of active myelin phagocytosis. Degenerating nerves were invaded by many cells contain- ing abundant lipid droplets in their cytoplasm (Fig. 2). Rarely, myelin profiles were seen in these cells. Electron microscopy revealed no morphological differences to the anti-Mac-l-treated macrophages.

Quite different results were obtained when cultured macrophages were incubated with homogenized myelin particles. Control macrophages ingested myelin particles upon 2-h exposure. Electron microscopy revealed numer- ous myelin profiles in their cytoplasm. With anti-Mac-1 treatment, similar results were obtained. The cultured macrophages also contained myelin debris in their cyto- plasm, indicating that anti-Mac-i treatment had no effect on the ingestion of homogenized myelin particles.

Addition of monoclonal antibodies to major histo- compatibility complex (MHC) class I or to class II anti- gens affected neither macrophage invasion of degenera- ting nerves nor the amount of myelin debris ingested by the invaded cells compared with untreated controls (Fig. 3). Like in controls, these cells contained lipid drop- lets, myelin debris and vacuoles in their cytoplasm. Elec-

tron microscopy revealed no morphological differences between treated and untreated macrophages.

Discussion

The experiments presented here show the involvement in myelin uptake of the macrophage CR3. Myelin phago- cytosis was blocked in vitro with anti-CR3 antibody. The major function of this cell surface receptor is the phagocytosis of opsonized particles by specific binding to the iC3b fragment of the complement component C3. Anti-CR3 antibodies inhibit this interaction [1]. Opsonization of myelin is necessary for its phagocytosis via the macrophage CR3. Myelin was shown to be opsonized by normal serum and anti-myelin antibodies [8]. In the in vitro model of Wallerian degeneration used here, degenerating myelin must be opsonized either by the added fresh mouse serum or, if fresh mouse serum is absent, the calf serum must act as a complement source. This hypothesis was confirmed by depletion of serum complement with CVF. In these experiments, myelin in- gestion by macrophages was also blocked.

The massive blockage of myelin phagocytosis by anti-CR3 antibody or by complement depletion stands out when compared with the preceding series in which we found no evidence for a competitive blocking of lectinophagocytosis upon testing 14 simple or complex sugars and 8 sugar-splitting enzymes, except for the effect of L-fucosidase [6]. The present data supplement the screening of surface receptors with antibodies directed against MHC class I or II antigens, which also had no effect. Previously we found no evidence for an involve- ment of Fc-receptor, using a different model [11]. The blockage with anti-CR3 antibody did not affect the viability or the general phagocytic capacities of the macrophage. The effect, accordingly, was more specific than that obtained upon non-specific silica damage [4],

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with inappropriate concentrations of sugars [6] or with cytochalasin D [6].

Blocking phagocytosis with anti-CR3 ant ibody was specific not only when compared with blocking of other macrophage surface receptors; it was also substrate specific: latex beads were ingested in the presence of anti- CR3 antibody. Interestingly, there was a similar differ- ence between myelin in organ cultures and homogenized myelin. One must conclude that mechanical disinte- gration and physicochemical properties of the myelin itself are additional, still undetermined factors affecting the accessibility of myelin to phagocytosis. These obser- vations also show that exposing macrophages in vitro to homogenized myelin is not an appropriate model to explore the behavior of macrophages in a diseased tissue.

The function of the macrophage CR3 requires the presence of divalent cations. In our model, we found calcium to be necessary for an unimpaired myelin re- moval by macrophages [6]. Release of toxic oxygen metabolites is not coupled to phagocytosis mediated by CR3 [22] in contrast to Fc receptor-mediated activity. We could also show that myelin phagocytosis occurred independently of an oxidative burst [5]. These obser- vations, combined with data presented here, corroborate the critical role of the macrophage CR3 in myelin re- moval.

Our studies used Wallerian degeneration as the most elementary system of myelin phagocytosis. However, the biological and immunological means of myelin recog- nition by macrophages may be the same in many situ- ations. There is evidence that the complement system also plays an important role in the pathogenesis of demyelinating lesions [17]. Serum complement depletion was shown to suppress the clinical expression of acute experimental allergic encephalomyelitis, an effect quite similar to that in our organ cultures. Also, the com- plement component C3 was detected in the brain of patients with multiple sclerosis [18]. The terminal com- plement complex C9 is deposited in multiple sclerosis [7] and in experimental allergic encephalomyelitis [16]. The terminal components of the complement system are also found in patients with demyelinating peripheral nervous system diseases, especially with the Guillain-Barr6 syn- drome. A possible role of the membrane attack complex in myelin destruction was suggested in these diseases [14]. Isolated myelin was also shown to activate the classic [21] as well as the alternative [13] pathway of complement.

In conclusion, myelin ingestion by macrophages via their CR3 appears to be a key factor both in Wallerian degeneration and in demyelination by way of com- plement activation causing opsonization of myelin.

Acknowledgement. The authors wish to thank Mrs. Brigitte Maruschak for her excellent technical assistance.

References

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3. Beuche W, Friede RL (1984) The role of nonrresident cells in Wallerian degeneration. J Neurocytol 13:767-796

4. Beuche W, Friede RL (1986) Myelin phagocytosis in Wallerian degeneration of peripheral nerves depends on silica-sensitive, bg/bg-negative and Fc-positive monocytes. Brain Res 378: 97-106

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