Phagocytosis of myelin by microglia in vitro

Previous experiments from this laboratory have shown that peritoneal macrophages in culture will phagocytize myelin. Myelin preopsonized with myelin antibodies is phagocytized to a much greater extent than untreated myelin, indicating that macrophages ingest myelin by an Fc receptor. The present work was undertaken to determine the characteristics of myelin phagocytosis by microglia, the resident macrophages of the central nervous system. Microglia isolated from 4–5 day primary cultures of newborn rat brains were shown to bind and phagocytize myelin labeled in the lipids by ¹⁴C-acetate. Both binding and phagocytosis as shown by the appearance of ¹⁴C-cholesterol ester were greatly increased if labeled myelin was preopsonized with antiserum to myelin basic protein or galactocerebroside. Both preopsonized and untreated myelin were phagocytized more actively by microglia than by peritoneal macrophages under the same culture conditions. Microglia cultured in the presence of GM-CSF showed slightly increased cholesterol ester production from opsonized myelin, but the effect of GM-CSF was significantly greater than myelin pretreated with control serum (34% increase) or untreated myelin (154% increase). There was no significant effect of GM-CSF on myelin phagocytosis by peritoneal macrophages. Cerebrospinal fluid containing immunoglobulin drawn from rabbits with acute EAE also opsonized myelin to increase phago cytosis by microglia, as has been previously shown with peritoneal macrophages. These results indicate that microglia may actively participate in myelin destruction in demyelinating diseases where myelin antibodies or a source of GM-CSF may be present. © 1993 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

2′3′cyclic nucleotide 3′phosphodiesterase immunohistochemistry shows an impairment on myelin compaction in hypothyroid rats

The effects of hypothyroidism on oligodendroglial differentiation and myelination are for the first time studied by immunohistochemical localization of an early oligodendroglial marker, the 2′3′cyclic nucleotide 3′phosphodiesterase (E.C. 3.1.4.37-CNPase), in developing rats. Two groups received methimazol; one during gestation (H) and another postnatally (PN). One H sub-group received thyroxine after birth (T). We observed a delay in CNPase expression followed by a decrease in the number of CNPase immunoreactive fibers in both H and PN groups. The T sub-group was not different from controls. Furthermore, the immunoreactive fibers, in mature hypothyroid animals, showed a continuous pattern of staining in contrast with a discontinuous one in controls.

Myelinogenesis is a highly regulated timed event. CNPase links myelin related proteins to the cytoskeleton also interacting with membrane lipids during extension and wrapping of the oligodendroglial process around the axon (ensheathment phase). In mature myelinated fiber the CNPase is absent from compact myelin sheath, being located only in the inner and outer loops and in paranodal loops. Thus, our data suggest a disorder in myelin compaction and point once more to the post-natal period as critical for the mechanisms that are thyroid hormone regulated in myelinogenesis.     

Transferrin binding protein is expressed by oligodendrocytes in the avian retina

It has been documented that some axons of ganglion cells in the nerve fiber layer of avian retina are wrapped in a myelin sheath. However, the identity of myelin-forming cells has not been established. In this study we demonstrated immunohistochemical evidence for the existence of a large population of oligodendrocytes in avian retina, using an antiserum against transferrin binding protein (TfBP), the avian homologue of the mammalian GRP 94 family of stress-regulated proteins. TfBP⁺ cells were mostly confined to the ganglion cell and optic nerve fiber layers of the retina, in which they were closely associated with the soma and axons of ganglion cells. The double-labeling experiments clearly show that TfBP is specific to oligodendrocytes. The morphology, distribution, and antigenic properties indicated by our findings suggest that TfBP⁺ cells are retinal oligodendrocytes that may be responsible for the myelination of ganglion cell axons in avian retina. A putative tropic role of TfBP⁺ oligodendrocytes to the ganglion cells is also discussed in conjunction with the physical properties of TfBP and avascular retinae of birds.     

Multifocal pattern of postnatal development of the macroglial framework of the rat fimbria

The development of the rat fimbria over the first postnatal month is associated with an approximate doubling of the tract diameter, a large increase in the number of glial cells, and the transformation of the prenatal radial glial skeleton into the adult interfascicular glial rows of solitary astrocytes and contiguous myelinating oligodendrocytes. The ventricular zone is reduced from a heterogeneous germinal layer of three or more cells thick at birth to the mature adult unicellular ependyma of homogeneous pale, mitotically inactive cells by the end of the second postnatal week. Mitoses are present throughout the body of the tract at all times, and persist, at reduced levels, in the adult. At birth the interior of the fimbria has only few scattered glial cell nuclei, largely solitary, or at most in longitudinal pairs. Over the first two postnatal weeks, the numbers and density of the interfascicular glia increase continuously. The scattered cells and cell clusters become progressively transformed into longer unicellular rows, which are aligned along the longitudinal axis of the tract, and which finally coalesce to form the continuous regular astrocyte/oligodendrocyte units that make up the interfascicular glial rows of the adult fimbrial glial skeleton. The increased cell packing density of the developing fimbrial glia is associated with a substantial decrease in nuclear and cytoplasmic size. From the end of the second postnatal week, the characteristic, large pale solitary astrocytes, and the smaller, more numerous, densely stained, closely packed oligodendrocytes are recognisable. Immunostaining for glial fibrillary acidic protein shows that immediately after birth the characteristic embryonic pattern of regular parallel radial glial processes starts to be modified by the progressive accumulation of longitudinal astrocytic processes, so the prenatal radial glial framework is rapidly transformed into the adult type of rectilinear array of radial and longitudinal processes. The development of the oligodendrocytes is shown clearly by immunostaining for myelin basic protein in enlarged, cytoplasm-rich, symmetrically placed cell pairs first seen at around P7. At P8-P10, there is a characteristic pattern of simultaneous multifocal maturation in which a single oligodendrocyte in each cluster develops a full complement of parallel, rather varicose myelinating processes. By P14 myelination is becoming confluent, oligodendrocytes are smaller, darker, with little cytoplasm, and individual myelinating processes cannot be discerned. Even at the end of the first postnatal month there are still many immature glia of indeterminate morphology. Myelination tends at first to be concentrated in the region adjacent to the hippocampus, and only reaches strengthen the hope that it may in future become possible to devise some form of self-reconstruction of the damaged adult glial tract structure in traumatic lesions completion by the end of the second month. During the first postnatal week, the entire array of fimbrial axons is traversed by the entire population of dentate neuroblasts. The highly vascular connective tissue on the pial surface of the fimbria is occupied by a prominent but transient population of mast cells which disappear in the adult.     

The effects of hypoxic preconditioning on white matter damage following hypoxic-ischaemic injury in the neonatal rat brain

Myelination is an essential process in human development that is carried out by oligodendrocytes in the central nervous system. Hypoxic-ischaemic (HI) brain injury can disrupt myelination by causing oxidative stress, inflammation and excitotoxicity, resulting in the loss of myelin as well as cells of the oligodendrocyte lineage. We have previously shown that hypoxic preconditioning (HP) can protect against HI injury, however, to date there have been no reports of its effects on white matter injury. Sprague-Dawley rat pups (postnatal day (P) 6) were placed into control and HP groups. On P7, pups were further separated into HI and sham surgery groups. HI pups underwent a unilateral common carotid artery occlusion and then exposed to 8% oxygen for 3 h. Sham pups underwent the same procedure without occlusion and were maintained in room air. Brains were removed 5 days post-surgery for analysis. In HI-only pups there was a significant reduction in brain volume observed. Consequently, when HP was performed prior to HI, the loss of brain tissue was prevented. The number of early and late oligodendrocyte progenitors (preOLs) in the corpus callosum was unaffected by HI, however, HI reduced the amount of myelin basic protein, indicating that HI may inhibit the maturation of preOLs. Whilst HP did not affect preOL density, it was found to prevent the loss of myelin caused by HI. This indicates that HP may either protect myelin directly or possibly promote the maturation of preOLs to regenerate the lost or damaged myelin.     

A sulfatide-reactive human monoclonal antibody obtained from a multiple sclerosis patient selectively binds to the surface of oligodendrocytes

In a previous paper, we described the production of a sulfatide-reactive IgM antibody-secreting B cell line that was obtained by Epstein-Barr virus transformation of peripheral B cells from a patient with multiple sclerosis (MS) (Uhlig and Dernick, 1989). In the present study, we demonstrate that this human monoclonal antibody (humAb) DS1F8 selectively binds to the surface of living oligodendrocytes in mixed brain cell cultures of newborn rats. Since a mouse mAb reactive with sulfatide was shown to inhibit oligodendrocyte progenitor differentiation, autoantibodies with binding specificities similar to DS1F8 could play a role in the demyelinating process in the CNS.     

Studies on hexachlorophene-induced myelin lesions in the trigeminal root transitional region in developing and adult mice

Summary Hexachlorophene (HCP)-induced intramyelinic vacuolation was studied in the transitional region of the trigeminal root of suckling and adult mice. HCP produced an extensive vacuolation in the central compartment of the region in both suckling and adult mice, while in the peripheral compartment myelin lesions were only seen in mice less than 16 days of age. Gas chromatographic measurements showed that in suckling mice the blood concentration of HCP decreased with age, apparently reflecting a faster elimination of HCP from the blood. By substantially increasing the HCP dose, higher blood concentrations were obtained in adult than less the 16-day-old mice; in spite of this, PNS myelin changes occurred only in the latter. Thus, by observing the HCP effect on the transitional region, where CNS and PNS directly meet, it is concluded that CNS of both suckling and adult mice is more severely effected by HCP than PNS, and that the reaction of the PNS myelin is age-dependend during the period of myelinogenesis; it is particularly vulnerable to a cytotoxic edema inducing substance.     

Release of myelin basic protein-degrading proteolytic activity from microglia and macrophages after infection with Theiler's murine encephalomyelitis virus: comparison between susceptible and resistant mice

Theiler's murine encephalomyelitis virus (TMEV) produces a chronic inflammatory demyelinating disease in its natural host, the mouse. A delayed-type hypersensitivity (DTH) response to viral antigens generally correlates with susceptibility to the disease and is thought to play an important role in the pathogenesis of demyelination in this model of human multiple sclerosis (MS). The hallmark of DTH responses is the recruitment by activated Th-1 cells of lymphoid cells and especially macrophages in infected areas. It is believed that soluble factors released by these cells would produce tissue damage, particularly myelin breakdown. In the present study, we compared TMEV-infected macrophages and microglia, isolated from both susceptible SJL/J and resistant C57BL/6 mice, for their ability to secrete proteolytic enzymes capable of degrading myelin basic protein. In addition, we studied whether supernatants from infected microglia/macrophages were also capable of killing oligodendrocytes in the same in vitro system. As detected by SDS-PAGE, MBP-degrading proteolytic activity was found only in supernatants from infected SJL/J microglia and macrophages, but not in supernatants collected from infected C57BL/6 microglia and macrophages, or in supernatants from mock-infected SJL/J and C57BL/6 cells. Similarly, incubation of E20.1 cells, an immortalized line of oligodendrocytes, with infected SJL/J, but not C57BL/6 supernatants, resulted in cytotoxic activity. When cells from resistant C57BL/6 mice were treated with LPS, they became susceptible to infection and also secreted proteolytic enzymes. The proteolytic activity released from infected microglia and macrophages was found to be dose-dependent, was inactivated by heat, and was inhibited by phenylmethylsulphonyl fluoride (PMSF). These results indicate that a serine protease is released from infected microglia and macrophages and suggest a role for proteases in TMEV-induced myelin injury.     

Polarity Development in Oligodendrocytes: Sorting and Trafficking of Myelin Components

In vertebrates, myelination is required for the saltatory signal conductance along the axon. At the onset of myelination, the myelinating cells, i.e., oligodendrocytes in the central nervous system and Schwann cells in the peripheral nervous system, are heavily engaged in the biogenesis of membranes that are wrapped around the axon to form the myelin sheath. Although the membrane of the myelin sheath is continuous with the plasma membrane surrounding the cell body, the composition of both membrane domains is clearly distinct implying that myelinating cells are polarized cells. The coordinated manner of myelin sheath formation requires the existence of sorting and trafficking pathways to establish and maintain this highly polarized phenotype. Although in vitro data show that the formation of myelin-like membranes is an intrinsic property of oligodendrocytes, exogenous factors modulate myelination and are required for the subcompartmentation and compaction of the myelin sheath in vivo. In this paper, we discuss the sorting and trafficking of myelin proteins and lipids in oligodendrocytes in relation to polarity development and maintenance, including the role of exogenous factors, and give examples how the perturbation of trafficking pathways may contribute to the development of demyelinating diseases of the central nervous system.     

Distribution of myelin basic protein messages relative to the cytoskeleton

Abstract

In situ hybridization is performed on oligodendrocytes using oligonucleotide probes to determine the subcellular distribution of myelin basic protein (MBP) encodulg. messages which, are associated with the cytoskeleton, relative to sub-cellular distribution of all MBP-encodlng messages In the cells. [Neural Res 1997; 19: 451-453]     

Carbonic anhydrase II in microglia in forebrains of neonatal rats

In the brains of adult rodents carbonic anhydrase II (CA) immunoreactivity has been observed in the choroid plexus and in oligodendrocytes, astrocytes, and myelin. Localization and functions of CA in the neonatal brain, however, have been controversial. One issue is whether the CAII-immunopositive round and ameboid cells in the corpus callosum and cingulum in the rat CNS during the first postnatal week are oligodendrocytes or microglia. Colocalization of C AH with the microglial antigen, EDI, and the microglia-specific isolectin, BSI-B4, suggested that most (approx. 60%) of the CAII-positive round and ameboid cells in rat brain during the first postnatal week were, indeed, macrophages and microglia. During that initial week, some CAII-positive protoplasmic astrocytes (approx. 40%) were observed as well. At the end of the first postnatal week smooth-surfaced CAII-positive cells began to appear in the corpus callosum. Those cells also bound MAbO4, a marker for the oligodendrocyte cell line. We conclude that during the first postnatal week most of the CAII-positive cells are macrophages and microglia, and that some are protoplasmic astrocytes. During the second postnatal week CAII-positive cells in the oligodendrocyte lineage become apparent, and by the end of that week there are few CAII-positive microglia. Confocal microscopy suggests that in brains of three-day-old rats the ameboid microglia are associated with nerve fibers, where they may perform phagocytosis of axons, directional guidance of axons, or disinhibition of axonal growth.     

Neonatal hypothyroidism and early undernutrition affect myelin and myelin precursor membranes in a different way

The lipid and protein composition as well as the activity of 2′3′ cyclic nucleotide 3′ phosphohydrolase (CNPH) and the distribution of individual proteins separated by SDS-PAGE were studied in myelin and in a fraction closely related to myelin or assumed to be a precursor membrane of mature myelin (fraction SN₄) isolated from 20-day-old rats made hypothyroid at birth or submitted to early malnutrition.In both experimental conditions lipid and protein components were found to be reduced in myelin when data were expressed as mg/g fresh tissue, but the results were close to those obtained in normal controls when data were expressed as mg/mg total protein of each fraction. CNPH activity was normal in myelin but markedly reduced in fraction SN₄. Although the results appear to suggest that both experimental conditions produce a reduction in the amount of myelin but no qualitative changes, the data obtained with SDS-PAGE show that the distribution of the various types of proteins present in this fraction and fraction SN₄ was abnormal. Myelin and fraction SN₄ isolated from malnourished animals displayed a protein profile which was quite similar to that found in fraction SN₄ isolated from normal rats, indicating a delay in the process of myelin maturation. The changes in protein composition of myelin and fraction SN₄ produced by neonatal hypothyroidism on the other hand differed clearly from those produced by early malnutrition; the ratio small basic protein: large basic protein (SBP:LBP) was found to be reduced in both membrane fractions in the former condition and the protein patterns of myelin and that of fraction SN₄ were different, at variance with what was found in the case of malnourished animals.Our findings appear to suggest that the effects of early malnutrition and neonatal hypothyroidism upon myelin and myelin-related membranes are different, and that myelination is more affected in the latter condition.     

Distribution of myelin lipid antigens in adult and developing rat spinal cord

We examined the distribution of myelin antigens recognized by monoclonal antibodies (mAbs) 01 and 04 in the developing ventral white matter of the cervical spinal cord of the rat using immunogold-labeled ultrathin cryosections. From the beginning of myelination after birth to multilamellar myelin in adult animals, we observed colocalization of 04 and 01 label in myelin. In the oligodendrocyte soma, immunolabel was found primarily over Golgi cisternae. In the oligodendrocyte processes, immunolabeling was also found in the cytoplasm and along the plasmalemma. More cytoplasmic 04 and 01 label was found in the external loop of myelin than in the internal loop. The amount of 01 and 04 label increased over compact myelin in proportion to the number of lamellae, but the label density per unit length of membrane remained approximately the same in compact myelin as in oligodendrocyte plasmalemma. We did not see a concentration gradient for either 04 or 01 label across, or along multilamellar myelin sheaths.     

Heterosis for myelin content is limited to the central nervous system

We have recently described a murine model for studying aspects of myelination (Seyfried and Yu, 1980; Ebato et al., 1983; Miskimins et al., 1986). This mouse shows hypermyelination during the period of most active synthesis of myelin, 9 to 21 days post-natal. The myelin parameters showing an increase were all measured in the central nervous system. We investigated here whether this effect extends into the peripheral nervous system. Our results indicate that the hypermyelination is limited to the central nervous system.     

CIDP患者血清和脑脊液中硫脂抗体、P2蛋白抗体的临床意义

目的　通过测定慢性炎性脱髓鞘性多发性神经病 (CIDP)患者血清和脑脊液 (CSF)中抗硫脂抗体及P2蛋白抗体水平 ,探讨其临床意义和可能的致病机制。方法　应用ELISA法检测 2 4例CIDP患者血清和脑脊液中抗硫脂抗体和P2蛋白抗体水平。结果　 (1 )CIDP组血清中高滴度抗硫脂抗体与其它周围神经病 (OPN)组和其它神经系统疾病 (OND)组比较无显著的差异 (P >0 .0 5) ;脑脊液中IgM 抗硫脂抗体与各对照组比较差异有显著性 (P <0 .0 5)。 (2 )CIDP组血清中高滴度抗P2抗体与OPN、OND组比较无显著的差异 (P >0 .0 5) ;脑脊液中抗P2抗体与各对照组比较有显著性差异 (P <0 .0 5 ,P <0 .0 1 )。 (3) 1 3例抗硫脂抗体阳性CIDP患者中 9例 (69.2 % )为感觉轴索性损害 ,1 1例阴性患者中 3例 (2 7.4 % )为感觉轴索性损害 ,差异有显著性 (P <0 .0 5)。 (4) 1 8例抗P2抗体阳性CIDP患者中 ,1 3例 (72 .2 % )为轴索性损害 ,6例抗P2抗体阴性患者中 3例 (50 % )为轴索性损害 ,差异无显著性 (P >0 .0 5)。结论　CIDP患者脑脊液中IgM 抗硫脂抗体可以作为感觉轴索型周围神经病的临床诊断参考指标 ;血清和脑脊液中P2抗体的检测对CIDP诊断参考价值不大 ,可能与疾病的修复有关。     

Plasma membrane of cultured oligodendrocytes: III. Relatedness to myelin

We have compared highly purified fractions of oligodendrocyte plasma membrane to myelin by one- and two dimensional gel electrophoresis and found them to be distinct. The major myelin proteins--proteolipid protein (PLP), DM-20, and myelin basic protein (MBP), which dominate the sodium dodecyl sulfate polyacrylamide gel electrophoresis pattern of myelin--were minor components of the plasmalemma. However, 2',3', cyclic nucleotide phosphodiesterase (CNPase) and myelin-associated glycoprotein (MAG) were represented equally in both membranes. Labeling the cells with various precursors followed by isolation of plasmalemma revealed that newly synthesized PLP, DM-20, CNPase, and MAG were incorporated into the plasma membrane of "floating" oligodendrocytes (i.e., nonattached to substratum). This was not so with MBP. Nevertheless, scattered patches of MBP were localized on the plasma membrane of intact cells using the immunogold method at the electron microscopic level. The data are consistent with the notion that MBP is not a constituent of the plasma membrane of mature oligodendrocytes (the MBP patches on intact cells are likely remnants from past association with myelin) but is rapidly associated with the plasmalemma of myelinating oligodendrocytes (i.e., attached cells). It is suggested that phosphorylation of MBP provides the triggering signal for plasma membrane association. In order to analyze the minor proteins in myelin and compare them to the plasma membrane by two-dimensional gel electrophoresis, myelin was extracted with chloroform:methanol to remove PLP, DM-20, and MBP. Even in the absence of PLP, DM-20, and MBP the pattern of extracted myelin still differed from that of plasmalemma indicating that their minor protein compositions were not the same. Myelin was characterized by a group of proteins that clustered at pI 5.5-6.5 and Mr 40,000-60,000 of which alpha-tubulins, beta-tubulins, and actin are part: the plasmalemma had tubulins and actin but in different proportions. Our findings indicate that in addition to PLP, DM-20, and MPB, myelin is also enriched relative to the plasmalemma in another group of proteins.     

Stimulation of myelin gene expression in vitro and of sciatic nerve remyelination by interleukin-6 receptor-interleukin-6 chimera

Induction of myelin gene expression denotes the last stage of differentiation of myelinating glial cells. Following peripheral nerve transection, Schwann cells (SC) lose myelin gene expression and proliferate, resembling premyelinating embryonic SC (eSC). We show that a fusion protein of the soluble interleukin-6 receptor to interleukin-6 (IL6RIL6), a potent activator of the gp130 signaling receptor, is an inducer of MBP and Po gene products in rat E18 embryonic dorsal root ganglia (DRG) 3 day cultures. Cells whose growth is dependent on the IL6RIL6 chimera were isolated from DRG. These cells (designated CH cells) express Krox-20, as do promyelinating and myelinating SC (mSC). IL6RIL6 induces Po and MBP in CH cells and their cocultures with neurons. In addition, IL6RIL6 leads to a disappearance of Pax-3, a marker of eSC and nonmyelinating Schwann cells (nmSC). Glial fibrillary acidic protein, present in nmSC, is not significantly induced by IL6RIL6. The CH cells acquire glial morphology when exposed to IL6RIL6 and cover axons in cocultures. In a sciatic nerve-derived SC line, IL6RIL6 also induces Po and triggers a rapid attachment along axons. In vivo administration of IL6RIL6 intraperitoneally to rats after sciatic nerve transection and resuture increases 4-fold the number of myelinated nerve fibers (MF) measured on day 12, 2.5-5 mm distal to the suture. The stimulation by IL6RIL6 treatment is highest (7.1-fold) at the more distant 5 mm site, and the thickness of myelin sheaths is increased. Compared to known SC growth factors, the gp130 activator IL6RIL6 appears to combine both in vitro mitogenic effects and promotion of myelin gene expression.     

Juvenile metachromatic leucodystrophy

Summary Clinical history, histopathological, ultrastructural and biochemical observations are presented in a juvenile MLD with onset at 8 years and death at 20 years. The findings are compared with related ones in different age groups, and current views on sulfatide metabolism and enzymatic pathogenesis in MLD are briefly entertained. It is postulated that the relatively well established morphological and chemical stability of the nervous tissue at the time of appearance of the metabolic derangement was an important factor in the pathomechanism of the disease process in this particular variant.     

Myelin-associated glycoprotein is produced before myelin basic protein in cultured oligodendrocytes

Mixed glial cell cultures from neonatal dog cerebellum were harvested daily between 3 and 21 days after seeding and studied with immunocytochemical techniques for the demonstration of myelin-associated glycoprotein (MAG) and myelin basic protein (MBP). Both MAG and MBP were detected in the cultures and by means of double labelling techniques shown to be produced by the same cells. MAG+ cells occurred earlier and were always more numerous than MBP+ cells. These results suggest that the oligodendrocyte in vitro expresses MAG before MBP. The findings are discussed in respect to oligodendroglial differentiation and myelination in vivo.