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Previous work from this laboratory has shown that both macrophages and microglia phagocytize relatively little myelin in vitro under basal conditions. In an effort to better simulate the conditions within the central nervous system (CNS), we have co-cultured these cells with astrocytes, the most numerous of the neural cells in the CNS, and have compared myelin phagocytosis in the co-cultures with that in cells cultured alone. Both macrophages and microglia in company with astrocytes phagocytized about three times as much myelin as controls, as measured by the formation of cholesterol ester, while astrocytes alone showed little evidence of myelin phagocytosis. Astrocyte-conditioned medium increased phagocytic activity in macrophages by 2.3-fold, and by 3.5-fold in microglia. A number of adhesion molecules and extracellular matrices were tested for their effects on myelin phagocytosis. Matrigel was most effective in activating the macrophages, and in the presence of conditioned medium, stimulated these cells to phagocytize as much myelin as when co-cultured with astrocytes. On the other hand, Matrigel inhibited myelin phagocytosis in microglia. These results indicate that activation of macrophages by astrocytes may be due to an adhesion component, as well as to soluble factors secreted by the astrocytes. While microglia were also stimulated by conditioned medium, adhesion to astrocytes or Matrigel induced a downregulation in phagocytic activity.

In the cell-mediated demyelinating diseases such as experimental allergic encephalomyelitis and multiple sclerosis, as well as their peripheral nerve counterparts, the phagocytic cells are the agent of myelin destruction. Both resident microglia and peripheral macrophages invading the nervous system have been shown to phagocytize myelin, although microglia appear to be more active, especially at early stages of disease. Several different receptors on these cells have been implicated as myelin receptors, with the Fc- and complement receptors receiving the most attention. Other receptors, especially the macrophage scavenger receptor with its broad specificity deserves further exploration, especially in view of its affinity for phosphatidylserine, which becomes externalized with membrane disruption. Evidence is shown for cytokine regulation of phagocytic activity in both macrophages and microglia. Further investigation of the pathways of cytokine action on myelin phagocytosis through signal transduction molecules will be important for a further understanding of the events leading to myelin destruction in demyelinating diseases.

The mechanisms for phagocytosis of myelin in cell-mediated demyelinating diseases have not been clarified. We have previously shown with cultured phagocytic cells that myelin opsonized with antiserum to myelin constituents is phagocytized in much higher amounts than untreated myelin, indicating that Fc receptors may be involved in the demyelinating process. Using various treatments of antisera, such as heating to destroy complement, and purification of IgG, we show here that complement is a necessary factor for maximal myelin phagocytosis by cultured macrophages. If myelin is sonicated to decrease its particle size, however, complement is not an active factor. Cultured microglia, on the other hand, required complement for maximal phagocytosis of both unsonicated and sonicated myelin. Addition of serum complement greatly increased phagocytosis of untreated CNS and PNS myelin, both unsonicated and sonicated, by macrophages and microglia. From these results it appears that the most important effect of complement is to fragment the myelin, making it more easily phagocytized. Prefragmentation of myelin by sonication can substitute for complement. Complement receptors may, in addition, be important for maximal myelin phagocytosis by microglia.

Previous studies from this laboratory have shown that CNS myelin is phagocytized and metabolized by cultured rat macrophages to a much larger extent when myelin is pretreated with serum containing antibodies to myelin constituents than when it is left untreated or pretreated with non-specific serum. In this study the effect of cerebrospinal fluid (CSF) from rabbits with experimental allergic encephalomyelitis (EAE) in promoting myelin phagocytosis was examined. Fourteen rabbits were immunized with purified myelin in Freund's complete adjuvant, seven of which developed clinical EAE symptoms. Serum and CSF were collected from EAE and control rabbits, and the CSF was centrifuged to remove cells. Sera and CSF from these rabbits and from Freund's adjuvant-immunized controls and untreated controls were measured for IgG content by radial diffusion assay, their myelin antibody characteristics were analyzed by immunoblots, and the ability of these serum and CSF samples to promote myelin phagocytosis when used for myelin opsonization was examined. The ability of a CSF sample to enhance radioactive myelin uptake and phagocytosis by cultured macrophages as measured by the appearance of radioactive cholesterol ester was linearly proportional to its total IgG titer, and correlated approximately both with clinical symptoms of the animal and the presence of antibody against the myelin constituents myelin basic protein, proteolipid protein, and galactocerebroside. The cholesterol esterification activities of EAE sera correlated to a lesser extent with IgG levels and clinical symptoms.(ABSTRACT TRUNCATED AT 250 WORDS)

Experimental allergic encephalomyelitis is characterized by invasion of lymphocytes and macrophages into the central nervous system resulting in inflammation, edema, and demyelination. Sera from Lewis rats from 7-95 days after immunization with purified guinea pig CNS myelin were examined with respect to their ability to opsonize myelin. This was correlated with the appearance of antibody components and the relative amounts of antibody to myelin basic protein (MBP) and proteolipid protein (PLP). Sera from rats 10-95 days after immunization preincubated with purified myelin induced phagocytosis of myelin by cultured macrophages with the resulting production of cholesterol ester. This opsonization activity as measured by the percentage of cholesterol esterified reached a peak at 26-27 days after immunization but remained significantly elevated up to 95 days post-immunization compared to the activity of serum from the Freund's adjuvant-injected controls. Immunoblots of the sera revealed a gradual increase in antibody activity against myelin components. ELISA assays for MBP and PLP antibody showed a similar pattern. Antibody to galactocerebroside (GC) was not detected by immunostains nor by the ELISA assay. Areas of demyelination were observed histologically by luxol-fast blue stained spinal cords up to 60 days post-immunization. These results indicate that antibodies to myelin protein when given access to myelin through or within the blood brain barrier could initiate or enhance the phagocytic response by peripheral or resident macrophages.

Four inhibitors of oligosaccharide processing were used to investigate their effects on the transport of PNS myelin glycoproteins through the secretory pathway, as well as to gain further insight into the structure of the oligosaccharide chains of the P0 and 19-kDa glycoproteins. Several different inhibitors of oligosaccharide processing were incubated with chopped peripheral nerves from young rats (21-24 days of age) and the uptake of 14C-amino acid and [3H]fucose or [3H]mannose was measured in P0 and the 19-kDa glycoprotein after separation of homogenate and myelin proteins on polyacrylamide gels. [3H]Mannose was not found as suitable as [3H]fucose as an oligosaccharide precursor because glucose used as an energy source profoundly inhibited the uptake of [3H]mannose. The substitution of pyruvate as an energy source, however, resulted in incomplete glycosylation, poor amino acid uptake, and truncated oligosaccharide chains. Endoglycosidase H cleaved approximately 50% of the P0 labeled with [3H]fucose and 14C-amino acid. The lower molecular weight protein resulting from endoglycosidase H cleavage contained approximately one-half the [3H]fucose label on the protein, whereas one-half remained on the oligosaccharide chain of the undegraded P0, indicating that at least one-half the P0 has a hybrid structure. Deoxynojirimycin, deoxymannojirimycin, and castanospermine inhibited incorporation of [3H]fucose into the oligosaccharide chains of P0 and the 19-kDa glycoprotein as predicted from their action in blocking various stages of trimming of high mannose structures before the addition of fucose. P0 synthesized in the presence of these inhibitors was cleaved to a greater extent by endoglycosidase H than the normal protein, indicating increased vulnerability to this enzyme with arrest of normal processing. Similar results were obtained for the 19-kDa glycoprotein. Both the incompletely processed P0 and the 19-kDa glycoprotein formed in the presence of these inhibitors appeared to be transported normally into myelin.

Activated macrophages harvested from rat peritoneum were shown to contain phospholipase A1, A2 and lysophospholipase activities which were defined on a series of radiolabelled phospholipid substrates. During in vitro culture of these elicited macrophage populations, phospholipase enzymes were secreted into the culture medium. Radiolabelled myelin, prepared from young rats after intracerebral injection of 14C acetate, was used as a substrate to analyze the susceptibility of central nervous system (CNS) myelin to attack by cell-associated and secreted macrophage enzymes. Homogenates of peritoneal macrophages degraded the myelin lipids at acid pH; phosphatidyl choline (PC) and ethanolamine phosphatide (EP) were both degraded with liberation of free fatty acid and small amounts of lysolipids. The ethanolamine lipids were most vulnerable; up to 20% of this fraction was degraded in six hours. Selected batches of macrophage culture supernatant similarly degraded the myelin EP at acid pH. These results suggest that phospholipase enzymes, released from activated macrophages in close proximity to the myelin sheath, may participate in primary demyelination in inflammatory CNS lesions.

Results from this laboratory have demonstrated that 14C-labeled myelin opsonized with antibodies raised to purified CNS myelin in rabbit is phagocytized by cultured macrophages in larger amounts than untreated myelin or myelin opsonized with preimmune serum. The cultured macrophages produced high amounts of radioactive cholesterol ester and triglyceride from the antibody-treated myelin while much less was formed from preimmune serum-treated or untreated myelin. Antiserum to galactocerebroside also greatly enhanced the formation of radioactive cholesterol ester, while that to myelin basic protein as well as to other myelin constituents had little or no effect. Serum from Lewis rats with acute EAE 13-14 days after immunization with whole CNS myelin also stimulated radioactive cholesterol ester formation compared to serum from Freund's adjuvant-injected controls (FAC). Serum from EAE rats as a result of myelin basic protein injection was as active as that from rats with whole myelin injection. No galactocerebroside antibody could be demonstrated in the EAE sera, although a strong immunostaining to myelin basic protein and proteolipid protein was demonstrated. IgG prepared from EAE serum also showed stimulatory effects compared to IgG from FAC serum, but much of the activity was lost, and the possibility that other factors may be involved is discussed. These experiments provide evidence that myelin phagocytosis and digestion by macrophages is enhanced by the presence of antibody to myelin. In EAE this antibody may leak into CNS with the breakdown of the blood-brain barrier.(ABSTRACT TRUNCATED AT 250 WORDS)

Sensitization of Lewis rats with whole central nervous system tissue or with purified myelin induced both experimental allergic encephalomyelitis (EAE) and a serum factor which inhibited myelin formation in vitro. Sensitization with the encephalitogenic factor, myelin basic protein, induced EAE, but not the myelination inhibition factor. Sensitization with cerebroside induced neither EAE nor myelination inhibition factor. The serums from control animals without EAE as well as from animals sensitized with all of the above antigens blocked evoked electrical responses in vitro.

The effect of an inhibitor of N-glycosylation of glycoproteins, tunicamycin, on synthesis of PNS myelin proteins was investigated in vitro by using chopped sciatic nerves or spinal roots of 21-day-old Wistar rats. Tunicamycin when incubated with these nerves in the presence of 3H-labeled fucose, mannose, or glucosamine inhibited the uptake of radioactivity into myelin proteins including some high-molecular-weight proteins, P0, 23K protein, and 19K protein by amounts ranging from 42 to 79%. Uptake of 14C amino acid mixture was inhibited much less by tunicamycin, but a new radioactive protein peak appeared when the protein mixtures had been separated by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. This protein ran directly in front of the P0 peak, did not correspond to any bands stained by Fast green, and was not labeled by fucose. This peak appeared in increasing larger proportions with progressive time of incubation of nerves with 3H amino acids in the presence of tunicamycin. The new protein, which cross-reacts with P0 antiserum, was tentatively identified as a nonglycosylated P0 protein that appears to be almost as well incorporated as P0 into the subcellular fraction containing myelin. At this time it is not possible to determine whether the unglycosylated P0 is actually assembled into a site and configuration like that of P0.

Border disease (BD) was induced in lambs by inoculation of their dams at 50 days gestation with Border disease virus (BDV) isolate #31. At birth, the clinically affected lambs had diffuse spinal cord hypomyelination, confirmed by immunocytochemical staining for myelin-associated glycoprotein and myelin basic protein. In the BD lambs, large numbers of thyroid follicular epithelial cells and scattered pituitary cells contained BDV antigen by immunofluorescence staining. Double labeling techniques demonstrated the BDV-infected pituitary cells to contain growth hormone, adrenocorticotrophic hormone, prolactin, or luteinizing hormone. Cells containing thyroid stimulating hormone were rare and were not positive for BDV antigen. Infection of the pituitaries and thyroid glands caused no detectable morphologic changes as compared to controls. The BD lambs had statistically significantly (p less than 0.05) lower mean serum concentrations of thyroxine and L-3,3',5-triiodothyronine as compared to age-matched uninfected controls. Similar significant differences in the mean plasma levels of growth hormone and thyroid stimulating hormone were not found. In addition, the BD lambs had a statistically significant (p less than 0.05) lower mean activity of the myelin-associated, thyroid hormone dependent enzyme, 2',3'-cyclic nucleotide-3'-phosphodiesterase in spinal cord tissue. Although not conclusive, these results indicate that the hypomyelination in BD may be due to depressed levels of circulating thyroid gland hormones secondary to a noninflammatory and noncytolytic infection of the thyroid gland by BDV. This is one of the first reports indicating that a virus-induced hormonal alteration may cause a congenital lesion in animals.

Measurements of several different protein determinants correlated with the time and rate of myelination in five areas of the central nervous system are presented. The deposition of protein in the subcellular fraction corresponding to the density of adult myelin, the appearance of basic protein characteristic myelin, the change in proportions of the individual myelin proteins, the appearance and distribution of the myelin marker 2':3'-cyclic nucleotide3'-phosphohydrolase, and the results of morphological studies of purified myelin are compared. According to these various criteria, and in agreement with the morphological observations of others, myelin appears earliest in the spinal cord, then in the brain stem, and latest in the cerebral hemispheres. Multilamellar myelin was observed in the rat brain stem and spinal cord as early as 5 days of age. The relative proportion of the individual myelin proteins changed with myelin maturation in all areas, with the larger basic protein decreasing reciprocally with increase of the smaller basic protein. The proportion of Wolfgram protein also decreased with maturation. Larger proportions of the enzyme 2':3'-cyclic nucleotide 3'-phosphohydrolase were located in the microsomal fraction at early ages. During development the enzyme activity gradually became associated more with a fraction of a density corresponding to adult myelin, suggesting the presence of precursor membrane fragments in microsomal fractions in the early stages of myelination before compact myelin formation. A significant proportion of the total nucleotide phosphohydrolase activity of the homogenate could not be recovered in subcellular fraction at early ages, but the recovers of the enzyme increased with maturation and the activity was found more in the myelin fraction.

The enzyme UDP-N-acetylglucosamine: dolichyl phosphate, N-acetylglucosamine-1-phosphate transferase initiates the synthesis of the oligosaccharide chain of complex-type glycoproteins. In view of the high content of glycoprotein in peripheral nerve myelin, the properties of this enzyme, its changes with age, and the effect of the specific inhibitor tunicamycin were investigated. The enzyme activity in rat peripheral nerve homogenate was completely dependent on the presence of exogenous dolichyl phosphate as well as Mg2+ and a detergent (Triton X-100) and was also greatly stimulated by a high salt concentration (0.4 M KCl) and AMP. The highest specific activity was present in the postmitochondrial membranes. The specific activity in postmitochondrial membranes in the presence of exogenous dolichyl phosphate reached a maximum at 17 days and remained relatively high throughout development, up to 2 years of age, but the activity was much lower when dolichyl phosphate was not added. This indicates that the enzyme level does not decrease with age, but that the content of the lipid cofactor may limit glycoprotein synthesis in vivo. Tunicamycin (5 micrograms) was injected intraneurally into 24-day-old rat sciatic nerve, and the enzyme was assayed from 1 to 24 days after injection. The specific activity of the transferase remained at low levels (5-40% of the level in control nerve) in most injected nerves assayed throughout this postinjection period. A protein previously identified as the unglycosylated P0 protein was synthesized in vitro by the tunicamycin-injected nerve and could be demonstrated to be incorporated into myelin in large amounts at 2 days and in small amounts at 6 days after injection.(ABSTRACT TRUNCATED AT 250 WORDS)

In most demyelinating diseases, macrophages are believed to be active agents of myelin destruction. In experimental encephalomyelitis, these cells appear to strip off and ingest the myelin lamellae, and myelin debris has been observed within the cell body. We show here in vitro conditions in which rat peritoneal macrophages phagocytose and metabolize CNS myelin lipids. Purified rat myelin, prelabeled in vivo with [14C]acetate, was incubated with preimmune serum or rabbit antiserum to rat CNS myelin and added to macrophage monolayers. Myelin opsonized with antimyelin antibodies was more readily phagocytosed and metabolized by cultured macrophages than untreated myelin or that preincubated with preimmune serum. In the presence of macrophages, levels of myelin polar lipids and cholesterol decreased, whereas radioactive cholesterol ester and triglyceride accumulated. Up to five times as much radioactive cholesterol ester and about twice as much triglyceride accumulated in macrophage cultures containing antibody-treated myelin as in cultures fed preimmune serum-treated myelin or in those incubated with untreated myelin. Both the fatty acid and the cholesterol from cholesterol ester contained radioactive label; therefore, both were derived at least partly from the radioactive myelin lipid. Antiserum to myelin purified from peripheral nerve was almost as effective as that to CNS myelin in stimulating cholesterol metabolism, whereas antiserum to galactocerebroside was about 70% as active. Antiserum to basic protein had less effect, whereas antiserum to the myelin-associated glycoprotein and proteolipid protein was inactive. Of the polar lipids, ethanolamine phosphatide was most degraded in both the antiserum- and preimmune serum-treated myelin, with the diacyl form and plasmalogen form degraded about equally. These experiments indicate that myelin-specific antibodies in inflammatory CNS lesions may participate in and stimulate macrophage-mediated demyelination.

Lysophosphatidylcholine (LPC) was injected intraneurally into the right sciatic nerve of a series of rats, leaving the left nerve as a control. At various time points up to 30 days after LPC treatment, the injected and control nerves were removed and incubated with [3H]amino acids, then purified myelin was prepared from the nerves. At all time points investigated the uptake of labeled amino acids was much higher in myelin proteins from the LPC-treated nerve than in those from the intact control nerve. [3H]Fucose uptake was also slightly increased. In the first week after LPC injection the increased amino acid incorporation was much greater in the myelin proteins of molecular weight higher than P0. From 10 days on, the smaller myelin proteins including P0, 19K, P1 and P2 showed the largest increase. From comparison of the morphology and biochemistry at 3 and 7 days after LPC injection, we propose that in the first 7 days, while myelin is degenerating, those proteins associated with Schwann cell or myelin reaction, interaction, and recognition functions are most stimulated metabolically, while after ten days the structural myelin proteins are actively resynthesized and the axon is remyelinated.

A survey of differences in composition and metabolism of myelin from five areas of the central nervous system was made in brain and spinal cord slices of the rat from 20 days to 20 months postnatal age. Purified myelin from the forebrain areas showed a composition characteristic of immaturity longer than did myelin from the hindbrain and spinal cord. The trend of chemical maturity is in agreement with the anatomical observations that myelination begins in the hindbrain and proceeds rostrally. Myelin recovery per 100-mg slice increased continually from 20 days to 20 months of age, while the uptake of [1-(14)C]acetate into myelin lipid and of [1-(14)C]leucine into myelin protein decreased precipitously with age. Taking into account the continuous increase in myelin during maturation, a calculation was made of the total amount of incorporation of labeled material into lipids or proteins per 100-mg slice for each region at each age. The metabolic characteristics of myelin from the cerebral cortex (including the corpus callosum), the thalamic area, and the cerebellum were very similar, while myelin from brainstem and spinal cord was metabolically more active, especially at the early ages. Synthesis of lipid in the myelin sheath represents about 50% of the lipid synthesis of the whole brain and about 75% of that of the spinal cord. The proportion of myelin-related protein synthesis is much less, probably less than 10% of the protein synthesis occurring in whole brain and about 15% of that in the spinal cord except at early ages.