The membrane attack complex of complement mediates peripheral nervous system demyelination in vitro

The present study used cocultures of rat dorsal root ganglia (DRG) and peritoneal macrophages to define the role of activated complement components during demyelination. The complement cascade was activated in vitro by treatment of the cultures with natural rat serum and lipopolysaccharides. Complement activation was examined by detection of the membrane attack complex of complement (MAC) with an antibody directed against rat C5–9. Detection of MAC in vitro by immunoelectron microscopy was associated with morphological changes of the myelin sheath. The sheath's regular structure was disrupted. Myelin lamellae were split and showed signs of decompaction. These changes were followed by a selective macrophage attack on myelin sheaths resulting in demyelination. Schwann cell viability was not affected by complement activation. Axons and sensory ganglion cells also survived this attack. The specificity of the complement effect was tested in experiments using treatment regimens with natural rat serum or lipopolysaccharides alone. In these experiments, no morphological changes of the myelin sheath were observed as well as no macrophage attack on myelin.     

Activation of macrophages by recombinant interferon-gamma has no effect on myelin phagocytosis but hinders invasion of nerves in organ culture

Myelin phagocytosis in nerves undergoing Wallerian degeneration was shown to depend on their invasion by non-resident, hematogenous macrophages. This process can be studied in vitro using organ cultures of peripheral nerves exposed to cultured peritoneal macrophages. The present report concerns the effect of recombinant interferon-gamma (rIFN-gamma) on luminol-dependent chemiluminescence, macrophage migration and myelin phagocytosis in organ cultures. Chemiluminescence was activated by rIFN-gamma compared to untreated cells. The macrophage population was capable of activation at any phase of exposure to organ cultures. The engagement of macrophages in myelin phagocytosis, however, varied with the timing of the application of rIFN-gamma. Application from the start of the experiment led to activation of chemiluminescence and also to a complete inhibition of macrophage invasion of the organ culture, thus preventing myelin removal. Application of rIFN-gamma at a later phase of the experiment had no effect on cell invasion and also no detectable effect on the efficiency of myelin phagocytosis. There was no indication that myelin phagocytosis by itself activated chemiluminescence in untreated cultures. Phagocytosis of myelin appears to be a function of macrophages independent of activation causing production of oxygen radicals.     

Increased vulnerability to demyelination in streptozotocin diabetic rats

Demyelination is a prominent feature in nerve biopsies of patients with diabetic neuropathy. The mechanism is unknown because diabetic rodents, unlike humans, do not consistently develop segmental demyelination. We examined how diabetes influences toxicant-induced demyelination, remyelination, Schwann cell nerve growth factor receptor (p75) expression, and endoneurial macrophage apolipoprotein E (apo E) synthesis in diabetic rats. Postnatal day 17 (P17) rats were given 110 mg/kg streptozotocin intraperitoneally and then fed a diet containing metallic tellurium (Te) from P20 to P27 to induce demyelination. Transverse electron micrographs and immunostained longitudinal cryosections were prepared from sciatic nerve during demyelination and remyelination. Diabetic rats had a mean serum glucose concentration of 490 mg/dl and consumed equivalent doses of peroral Te. The number of demyelinated fibers in electron micrographs was increased significantly by 17% (P < .0011). Endoneurial density of p75-stained Schwann cells was increased in diabetic rats in proportion to the increased number of injured internodes. Density of apo E- and ED1-positive macrophages also was significantly increased in diabetes. There was no delay in macrophage myelin clearance, and remyelination was not compromised. Increased Schwann cell vulnerability to stress, by increasing the turnover rate of myelinated units, may explain why myelin defects accumulate after long-standing diabetes. © 1996 Wiley-Liss, Inc.     

Macrophage recruitment in different models of nerve injury: Lysozyme as a marker for active phagocytosis

Macrophages play critical roles in both degenerative and regenerative processes following peripheral nerve injury. These include phagocytosis of debris, stimulation of Schwann cell dedifferentiation and proliferation, and salvage of myelin lipids for reutilization during regeneration. To better define the role of macrophages, we studied models of primary demyelination (tellurium intoxication) and secondary demyelination (nerve crush and cut). Sections of paraformaldehyde-fixed rat sciatic nerves at various stages of demyelination were stained with monoclonal antibody ED1, a standard macrophage marker, and a polyclonal antiserum specific for lysozyme (LYS). Near the peak of demyelination in all three models, LYS immunoreactivity colocalized with ED1 staining. Macrophages present in nerve after the period of maximal phagocytosis of myelin were much less immunoreactive for LYS. These results suggest LYS is a good marker for macrophages which are active in phagocytosis. Tellurium intoxication, which causes synchronous demyelination and subsequent remyelination of only about 25% of myelin internodes, recruited more macrophages (and induced more lysozyme expression) than either nerve crush or cut, which cause demyelination of all internodes distal to the injury site. This suggests that Schwann cells may recruit macrophages soon after metabolic insult and prior to actual demyelination. The final signal for macrophage recruitment is not directly related to the amount of damaged myelin. In the models listed above, steady state mRNA levels for apolipoprotein E (ApoE; possible mediator of cholesterol salvage), LYS, and Po (major structural protein of PNS myelin), were analyzed by Northern blot analysis. LYS mRNA levels peaked sharply in all models, with a temporal pattern consistent with the expected presence of activated, phagocytic macrophages. The temporal pattern for ApoE mRNA levels differed in the 3 models, but ApoE expression was consistent with its proposed role in salvage of cholesterol during remyelination. © 1995 Wiley-Liss, Inc.     

L-fucosidase treatment blocks myelin phagocytosis by macrophages in vitro

Myelin phagocytosis in nerves undergoing Wallerian degeneration has been shown to depend on their invasion by non-resident, hematogenous macrophages. This process can be studied in vitro using organ cultures of peripheral nerves exposed to cultured peritoneal macrophages. The present report concerns the role of cell surface carbohydrates in the invasion of degenerating nerves and in the recognition and ingestion of myelin by the phagocytic cells. Additional experiments explored the effect of pH, calcium and cytochalasin D on myelin phagocytosis. Organ cultures with peritoneal macrophages were treated with 14 simple or complex sugars or with eight sugar-splitting enzymes. Macrophage invasion was diminished by many simple or complex sugars, but exposure to sugars had no effect on the recognition or ingestion of myelin by the invading macrophages. Macrophage invasion was abolished upon treatment with beta-mannosidase. Exposure to L-fucosidase abolished the myelin phagocytic capacity of invading macrophages completely without affecting their capacity to ingest carbon or latex particles. The results indicate that the phagocytosis of myelin by macrophages is an L-fucosidase-sensitive process, probably by interaction with their complement receptor type C3.     

L-fucosidase treatment blocks myelin phagocytosis by macrophages in vitro

Myelin phagocytosis in nerves undergoing Wallerian degeneration has been shown to depend on their invasion by non-resident, hematogenous macrophages. This process can be studied in vitro using organ cultures of peripheral nerves exposed to cultured peritoneal macrophages. The present report concerns the role of cell surface carbohydrates in the invasion of degenerating nerves and in the recognition and ingestion of myelin by the phagocytic cells. Additional experiments explored the effect of pH, calcium and cytochalasin D on myelin phagocytosis. Organ cultures with peritoneal macrophages were treated with 14 simple or complex sugars or with eight sugar-splitting enzymes. Macrophage invasion was diminished by many simple or complex sugars, but exposure to sugars had no effect on the recognition or ingestion of myelin by the invading macrophages. Macrophage invasion was abolished upon treatment with β-mannosidase. Exposure to L-fucosidase abolished the myelin phagocytic capacity of invading macrophages completely without affecting their capacity to ingest carbon or latex particles. The results indicate that the phagocytosis of myelin by macrophages is an L-fucosidase-sensitive process, probably by interaction with their complement receptor type C3.     

Hyperglycemia and downregulation of caveolin-1 enhance neuregulin-induced demyelination

Neuregulins (NRGs) are growth factors which bind to Erb receptor tyrosine kinases that localize to Schwann cells (SCs). Although NRGs can promote cell survival, mitogenesis, and myelination in undifferentiated SCs, they also induce demyelination of myelinated co-cultures of SCs and dorsal root ganglion (DRG) neurons. We have shown previously that Erb B2 activity increased in premyelinating SCs in response to hyperglycemia, and that this correlated with the downregulation of the protein caveolin-1 (Cav-1). As myelinated SCs undergo substantial degeneration in diabetic neuropathy, we used myelinated SC/DRG neuron co-cultures to determine if hyperglycemia and changes in Cav-1 expression could enhance NRG-induced demyelination. In basal glucose, NRG1 caused a 2.4-fold increase in the number of damaged myelin segments. This damage reached 3.8-fold under hyperglycemic conditions, and was also associated with a robust decrease in the expression of Cav-1 and compact myelin proteins. The loss of Cav-1 and compact myelin proteins following hyperglycemia and NRG treatment was not due to neuronal loss, since the axons remained intact and there was no loss of PGP 9.5, an axonal marker protein. To examine if changes in Cav-1 were sufficient to alter the extent of NRG-induced demyelination, SC/DRG neurons co-cultures were infected with antisense or dominant-negative Cav-1(P132L) adenoviruses. Either antisense-mediated downregulation or mis-localization of endogenous Cav-1 by Cav-1(P132L) resulted in a 1.5- to 2.4-fold increase in NRG-induced degeneration compared to that present in control cultures. These data support that hyperglycemia and changes in Cav-1 are sufficient to sensitize myelinated SC/DRG co-cultures to NRG-induced demyelination.     

Myelin phagocytosis by peritoneal macrophages in organ cultures of mouse peripheral nerve. A new model for studying myelin phagocytosis in vitro

Organ cultures of degenerating nerve fascicles were exposed to cultured macrophages obtained by peritoneal lavage. Invasion of the nerve fascicle by phagocytes was shown by prelabeling with carbon and with electron microscopy. There was massive active phagocytosis of degenerating myelin sheaths. The invading phagocytic cell population was identified as Fc receptor-positive, Mac-1-positive macrophages by immunocytochemistry. The Schwann cell population persisted without significant myelin phagocytosis. The vitality of the Schwann cell population was shown by subsequent reimplantation of the organ cultures into host animals. The reimplants had retained their ability to remyelinate regenerating axon sprouts. These observations were made in cultures exposed to cytostatic agents. If cytostatic agents were omitted, there was proliferation of endogenous phagocytes in the nerve fascicles without added peritoneal cells. These endogenous phagocytes were identified as proliferating resident monocytes and were positive for the Fc receptor and Mac-1 markers. This model allows studies on how monocytes recognize and digest degenerating myelin apart from surviving Schwann cells.     

Studies with antisera against peripheral nervous system myelin and myelin basic proteins. II. Immunohistochemical studies in cultures of rat dorsal root ganglion neurons and Schwann cells

Antiserum against rat peripheral nervous system (PNS) myelin contained immunoglobulins which bound preferentially to the extracellular surfaces of myelin-related Schwann cells in intact cultures of dorsal root ganglion (DRG) neurons and Schwann cells, while antiserum against basic protein (BP) from central nervous system myelin or the PNS basic protein P₂ did not. We demonstrate the presence of PNS myelin proteins P₁ (identical to BP) and P₂ by immunoperoxidase techniques in DRG cultures that had been treated to disrupt cellular membranes. These observations suggest that P₁ and P₂ are not exposed on the extracellular surfaces of myelin-related Schwann cells in culture. The results also supported the hypothesis concerning the possible mechanisms by which anti-PNS myelin serum demyelinates DRG cultures, while anti-BP serum and anti-P₂ serum do not.     

Ross River virus--induced demyelination: II. Ultrastructural studies

Focal central nervous system demyelination is a prominent feature of Ross River virus encephalitis in mice. The present ultrastructural study shows that oligodendrocytes are a primary site of viral replication. The earliest myelin disruption occurs in association with an inflammatory infiltrate composed primarily of polymorphonuclear leukocytes, which are later replaced by macrophages. Viral particles are found in oligodendrocytes, selected neuronal populations, macrophages, and polymorphonuclear leukocytes through the end of the first week of infection as macrophages remove myelin from normal-appearing axons. Between the second and third weeks of infection, axons within foci of demyelination partially remyelinate with central myelin. Schwann cells are not found within regions of central remyelination. Cyclophosphamide treatment does not prevent or delay demyelination or remyelination. Results of this and previous studies strongly suggest that Ross River virus--induced demyelination is not immune mediated but rather the direct result of viral infection of oligodendrocytes.     

DELAYED REMYELINATION IN RAT SPINAL CORD FOLLOWING ETHIDIUM BROMIDE INJECTION

Areas of demyelination were produced by injecting ethidium bromide into the white matter of the lumbar spinal cord of rats. There was variation in the nature of the process of demyelination and a difference in the speed with which Schwann cells remyelinated the demyelinated axons. In some lesions, or areas within lesions, myelin debris was rapidly processed by macrophages and axons were rapidly remyelinated by Schwann cells, while in other lesions of similar duration, or in areas within the same lesion, the myelin was transformed into lattices of membranous profiles which persisted around axons for long periods of time. In the lesions containing such myelin derived membranes, there were few macrophages and remyelination by Schwann cells was delayed compared to that seen in the more rapidly resolving lesions. It was concluded that the slow resolution of some lesions resulted from the delay between intoxication and cell disintegration (7-10 days) which meant that the cell responses to demyelination took place in a glial free area which could not support cell movement needed for removal of myelin debris and remyelination. This study indicates that the tempo and results of demyelination can be altered by the cellular events which accompany degeneration of oligodendrocytes.     

Oxygen radical-induced neurotoxicity in spinal cord neuron cultures

The neurotoxic effects of oxygen radical on spinal cord neuron cultures derived from fetal mouse have been studies. Oxygen radicals, superoxide radical and hydrogen peroxide, were generated by adding xanthine oxidase and hypoxanthine in the culture medium. Exposure of neurons to this oxygen radical generating system resulted in a significant cell death and decrease of choline acetyltransferase (ChAT) activity in a time-dependent manner in spinal cord neuron cultures. The decrease in cell viability and ChAT enzyme activity induced by the oxygen radicals was blocked by scavengers such as superoxide dismutase (SOD), catalase, and tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a metal chelator. Antagonista of the N-methyl-D -aspartate (NMDA) receptor, including MK801 (a noncompetitive NMDA antagonist), D -2-amino-5-phosphonovaleric acid (APV) (a competitive NMDA antagonist), and 7-chlorokynurenic acid (an antagonist at the glycine site associated with the NMDA receptor), similarly blocked oxygen radical-induced decrease in cell viability and ChAT activity in spinal cord neuron cultures. These results indicate that both oxygen radicals and excitotoxic amino acids were involved in the oxidant-ititiated neurotoxicity of spinal cord neurons. © 1994 Wiley-Liss, Inc.     

Peripheral nerve demyelination induced by intraneural injection of experimental allergic encephalomyelitis serum.

Intraneural injection of sera from rabbits with experimental allergic encephalomyelitis, induced by sensitization with bovine brain white matter in complete Freund's adjuvant, produced focal primary demyelinative lesions in rat sciatic nerves. Demyelinating activity was removed by prior incubation of antisera with central (CNS) and peripheral nervous system (PNS) myelin but not with liver or kidney, and was heat-labile and complement-dependent. Recipient animals developed a sensorimotor disturbance of their toes and ankles on the side injected with antiserum. Twenty minutes after antiserum injection, Schwann cells showed focal cytoplasmic outpouching and their external mesaxons opened. Between 1 and 8 hours after injection vacuolation, splitting and vesiculation of myelin became increasingly prominent at Schmidt-Lanterman clefts and paranodal regions, with concomitant degenerative changes in Schwann cell cytoplasm. Polymorphonuclear cell infiltration and endoneurial edema were apparent at this time. Substantial demyelination occurred before the appearance of phagocytic cells. Between 8 hours and 3 days many nerve fibers were surrounded and attacked by invading macrophages. Axons became demyelinated progressively over several internodes by macrophage phagocytosis. Early signs of remyelination were observed by 5 days. These findings suggest that antibodies directed against antigens common to both CNS and PNS myelin can produce in vivo peripheral nerve demyelination.     

The Role of the Mouse Macrophage Scavenger Receptor in Myelin Phagocytosis

Myelin phagocytosis during Wallerian degeneration and immune-mediated demyelination depends on the action of mononuclear cells of the monocyte/macrophage system. The present study investigated the role of the macrophage scavenger receptor, a trimeric membrane glycoprotein, in myelin uptake by macrophages. Two in vitro models of myelin phagocytosis were used: an organ culture model of mouse peripheral nerves exposed to cocultured macrophages and a quantitative flow cytometric assay. Different concentrations of the monoclonal rat anti-mouse scavenger receptor antibody (2F8) were applied to these systems to selectively block the macrophage scavenger receptor. Concentration-dependent effects on macrophage migration and myelin uptake were seen when the macrophage scavenger receptor was blocked by the antibody 2F8. Low concentrations reduced myelin phagocytosis by the invading macrophages; higher concentrations completely abolished macrophage invasion of the nerves. Using a quantitative flow cytometric assay it was also shown that the 2F8 antibody inhibits phagocytosis of myelin in a dose-dependent manner. These data indicate that the macrophage scavenger receptor is involved in myelin phagocytosis by macrophages.     

Monocyte chemoattractant protein-1 is a pathogenic component in a model for a hereditary peripheral neuropathy

Macrophages are critically involved in the pathogenesis of genetically caused demyelination, as it occurs in models for inherited demyelinating neuropathies. It is presently unknown which factors link the Schwann cell-based myelin mutation to the activation of endoneurial macrophages. Here we identified the chemokine monocyte chemoattractant protein-1 (MCP-1) as a first and crucial factor upregulated in Schwann cells of mice heterozygously deficient for the myelin protein zero. The chemokine could be identified as an important mediator of macrophage immigration into peripheral nerves. Furthermore, a 50% reduction of chemokine expression by crossbreeding with MCP-1-deficient mice reduced the increase in macrophage numbers in the mutant nerves and lead to a robust amelioration of pathology. Surprisingly, the complete absence of MCP-1 aggravated the disease. Our findings show that reducing but not eliminating chemokine expression can rescue genetically caused demyelination that may be an interesting target in treating demyelinating diseases of the peripheral nervous system.     

CHRONOLOGICAL STUDY OF ULTRASTRUCTURAL CHANGES IN THE PERIPHERAL NERVES IN MAREK'S DISEASE

Chronological study of ultrastructural changes in the peripheral nerves in Marek's disease
A chronological study was made of the ultrastructural changes in peripheral nerves following inoculation of 1-day-old chicks with a neurogenic strain of Marek Disease virus. No virus particles were found in nerves. Cellular infiltration of nerves was detected as early as 5 days after inoculation and by 3 weeks some nerves contained proliferative lesions which possessed many of the ultrastructural features characteristic of normal, reactive lymphoid tissue. About 4 weeks after inoculation, coinciding with the onset of neurological signs, areas of widespread demyelination appeared within these lesions; lymphocytes and macrophages penetrated and destroyed the myelin sheath, but spared Schwann cells and most axons. Later oedematous, sparsely infiltrated B type lesions were observed, some of which contained demyelin-ated nerve fibres undergoing repair; these were therefore a stage in the regression of the proliferative lesions. Our observations do not favour the hypothesis that cellular infiltration of nerves in Marek's disease is the direct result of auto-sensiti-zation to normal myelin. They are consistent with the hypothesis that demyelination is a secondary feature and that the primary lesions are preferential sites for immune demyelination.     

Inflammatory mediators in demyelinating disorders of the CNS and PNS

Work in both experimental models and human disorders of the central and peripheral nervous system has delineated multiple effector mechanisms that operate to produce inflammatory demyelination. The role of various soluble inflammatory mediators generated and released by both blood-borne and resident cells in this process will be reviewed. Cytokines such as interleukin (IL)-1, interferon (IFN)-γ, and tumor necrosis factor (TNF)- are pivotal in orchestrating immune and inflammatory cell-cell interactions and represent potentially noxious molecules to the myelin sheath, Schwann cells, and/or oligodendrocytes. Arachidonic acid metabolites, synthesized by and liberated from astrocytes, microglial cells and macrophages, are intimately involved in the inflammatory process by enhancing vascular permeability, providing chemotactic signals and modulating inflammatory cell activities. Reactive oxygen species can damage myelin by lipid peroxidation and may be cytotoxic to myelin-producing cells. They are released from macrophages and microglial cells in response to inflammatory cytokines. Activation of complement yields a number of inflammatory mediators and results in the assembly of the membrane attack complex that inserts into the meylin sheath-creating pores. Activated complement may contribute both to functional disturbance of neural impulse propagation, and to full-blown demyelination. Proteases, abundantly present at inflammatory foci, can degrade myelin. Vasoactive amines may play an important role in breaching of the blood-brain/blood-nerve barrier. The importance of nitric oxide metabolites in inflammatory demyelination merits investigation. A better understanding of the multiple effector mechanisms operating in inflammatory demyelination may help to devise more efficacious antigen non-specific therapy.     

Failure of remyelination in areas of demyelination produced in the spinal cord of old rats

The extent of remyelination was determined one month after injecting 1μl of 1.0% lysolecithin into the dorsal columns of adult rats of three age groups—juvenile, young and old. In the juvenile group (2 months) all axons were remyelinated by either oligodendrocytes or Schwann cells. In the young rats (5 months) nearly all axons were remyelinated. However, in the oldest age group (> 12 months) many axons remained demyelinated and there was a decrease in both oligodendrocyte and Schwann cell remyelination. Also, myelin sheaths formed by oligodendrocytes in the old rats were thinner than those found in the young rats; evidence of impaired Schwann cell remyelination was also seen. The appearance of the lesions in the old animals was variable and many contained myelin debris despite the presence of macrophages within the demyelination area. Although some astrocytes were present in the lesion, many of the demyelinated axons were not separated by astrocyte processes. It is suggested that the failure of remyelination in the old rats following lysolecithin-induced demyelination may be related to sluggish responses of astrocytes and/or macrophages to demyelination. However, a lack of recruitable myelin-forming cells in old animals cannot be excluded.