Galectin-3/MAC-2, Ras and PI3K activate complement receptor-3 and scavenger receptor-AI/II mediated myelin phagocytosis in microglia

The removal of degenerated myelin is essential for repair in Wallerian degeneration that follows traumatic injury to axons and in autoimmune demyelinating diseases (e.g., multiple sclerosis). Microglia can remove degenerated myelin through phosphatidylinositol-3-kinase (PI3K)-dependent phagocytosis mediated by complement receptor-3 (CR3/MAC-1) and scavenger receptor-AI/II (SRAI/II). Paradoxically, these receptors are expressed in microglia after injury but myelin is not phagocytosed. Additionally, Galectin-3/MAC-2 is expressed in microglia that phagocytose but not in microglia that do not phagocytose, suggesting that Galectin-3/MAC-2 is instrumental in activating phagocytosis. S-trans, trans-farnesylthiosalicylic (FTS), which inhibits Galectin-3/MAC-2 dependent activation of PI3K through Ras, inhibited phagocytosis. K-Ras-GTP levels and PI3K activity increased during normal phagocytosis and decreased during FTS-inhibited phagocytosis. Galectin-3/MAC-2, which binds and stabilizes active Ras, coimmunoprecipitated with Ras and levels of the coimmunoprecipitate increased during normal phagocytosis. A role for Galectin-3/MAC-2 dependent activation of PI3K through Ras, mostly K-Ras, is thus suggested. An explanation may thus be offered for deficient phagocytosis by microglia that express CR3/MAC-1 and SRAI/II without Galectin-3/MAC-2 and efficient phagocytosis when CR3/MAC-1 and SRAI/II are co-expressed with Galectin-3/MAC-2.     

cAMP cascade (PKA, Epac, adenylyl cyclase, Gi, and phosphodiesterases) regulates myelin phagocytosis mediated by complement receptor-3 and scavenger receptor-AI/II in microglia and macrophages

The removal by phagocytosis of degenerated myelin is central for repair in Wallerian degeneration that follows traumatic injury to axons and in autoimmune demyelinating diseases (e.g., multiple sclerosis). We tested for roles played by the cAMP cascade in the regulation of myelin phagocytosis mediated by complement receptor-3 (CR3/MAC-1) and scavenger receptor-AI/II (SRAI/II) separately and combined in mouse microglia and macrophages. Components of the cAMP cascade tested are cAMP, adenylyl cyclase (AC), Gi, protein kinase A (PKA), exchange protein directly activated by cAMP (Epac), and phosphodiesterases (PDE). PKA inhibitors H-89 and PKI(14-22) amide inhibited phagocytosis at normal operating cAMP levels (i.e., those occurring in the absence of reagents that alter cAMP levels), suggesting activation of phagocytosis through PKA at normal cAMP levels. Phagocytosis was inhibited by reagents that elevate endogenous cAMP levels to above normal: Gi-inhibitor Pertussis toxin (PTX), AC activator Forskolin, and PDE inhibitors IBMX and Rolipram. Phagocytosis was inhibited also by cAMP analogues whose addition mimics abnormal elevations in endogenous cAMP levels: nonselective 8-bromo-cAMP, PKA-specific 6-Benz-cAMP, and Epac-specific 8-CPT-2'-O-Me-cAMP, suggesting that abnormal high cAMP levels inhibit phagocytosis through PKA and Epac. Altogether, observations suggest a dual role for cAMP and PKA in phagocytosis: activation at normal cAMP levels and inhibition at higher. Furthermore, a balance between Gi-controlled cAMP production by AC and cAMP degradation by PDE maintains normal operating cAMP levels that enable efficient phagocytosis.     

Phosphatidylinositol 3-kinase, phosphoinositide-specific phospholipase-Cgamma and protein kinase-C signal myelin phagocytosis mediated by complement receptor-3 alone and combined with scavenger receptor-AI/II in macrophages

Complement-receptor-3 (CR3/MAC-1), scavenger-receptor-AI/II (SRAI/II) and Fcgamma-receptor (FcgammaR) can mediate phagocytosis of degenerated myelin in macrophages and microglia. However, CR3/MAC-1 and SRAI/II, but not FcgammaR, mediate phagocytosis after axonal injury. We tested for phosphatidylinositol 3-kinase (PI3K), phosphoinositide-specific phospholipase-Cgamma (PLCgamma) and protein kinase-C (PKC) signaling in myelin phagocytosis mediated by CR3/MAC-1 alone and by CR3/MAC-1 combined with SRAI/II. Phagocytosis was inhibited by PI3K inhibitors wortmannin and LY-294002, PLCgamma inhibitor U-73122, classical PKC (cPKC) inhibitor Go-6976, general PKC inhibitors Ro-318220 and calphostin-C, and BAPTA/AM which chelates intracellular Ca(2+) required for cPKC activation. PKC activator PMA augmented phagocytosis and further alleviated inhibitions induced by PI3K and PLCgamma inhibitors. Overall, altering PKC activity modulated phagocytosis 4- to 6-fold between inhibition and augmentation. PLCgamma activation did not require tyrosine phosphorylation. Thus, signaling of myelin phagocytosis mediated by CR3/MAC-1 alone and by CR3/MAC-1 combined with SRAI/II involves PI3K, PLCgamma and cPKC, the cascade PI3K-->PLCgamma-->cPKC, and wide-range modulation by PKC. This pathway may thus be targeted for in vivo modulation, which may explain differences in the efficiency of CR3/MAC-1-mediated myelin phagocytosis in different pathological conditions.     

Differential regulation of myelin phagocytosis by macrophages/microglia, involvement of target myelin, Fc receptors and activation by intravenous immunoglobulins.

Macrophages/microglia are the key effector cells in myelin removal. Differences exist in the amount and time course of myelin uptake in the central (CNS) and peripheral nervous system (PNS), the basis of this difference, however, is not yet clarified. In the present experiments we studied the phagocytosis rate of CNS or PNS myelin by macrophages and microglia in vitro. Additionally, the effects of intravenous immunoglobulins (IVIg) on this process were investigated. In the PNS experiments, sciatic nerves were cocultured with peritoneal macrophages. Optic nerve fragments were used to characterize the myelin-removing properties of microglia. Cocultures with peritoneal macrophages aimed at investigating the differences in phagocytosis between resident microglia and added macrophages. The myelin phagocytosis in sciatic nerve fragments was higher than in optic nerves, indicating differences in the myelin uptake rate between peripheral macrophages and microglia. IVIg increased the phagocytosis of PNS myelin by macrophages, but not by microglia in optic nerves. The addition of peritoneal macrophages to optic nerve fragments did not lead to an increase in the phagocytosis of CNS myelin either. The IVIg induced phagocytosis of PNS myelin by peripheral macrophages was associated with an increased expression of macrophage Fc receptors measured by FACS. Blocking of Fc receptors by anti-Fc receptor antibody reduced the IVIg induced PNS myelin phagocytosis to basic levels, indicating that the induced but not the basic myelin uptake by macrophages is Fc receptor dependent. In contrast to peripheral macrophages, IVIg did not increase Fc receptor density on microglia. These data indicate that phagocytosis of PNS and CNS myelin by macrophages or microglia is differentially regulated. Local factors within the CNS or PNS may affect this process by modulating the surface receptor profile and activation state of the phagocytic cell or the structure of the myelin sheath.     

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

Summary Myelin phagocytosis in Wallerian degeneration 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 phagocytic 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 phagocytosis is mediated by the macrophage CR3.Supported by grant 609/2-1 from the Deutsche Forschungsgemeinschaft     

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.     

Distinct inflammatory stimuli induce different patterns of myelin phagocytosis and degradation in recruited macrophages

Injury and demyelinating diseases result in the disruption of the myelin sheath that surrounds axons in the nervous system. The removal of degenerating myelin by macrophages and microglia is central to repair mechanisms that follow. The efficiency of myelin removal depends on magnitudes and rates of myelin phagocytosis and degradation. In the present study we test whether environmental conditions within a tissue can control patterns of myelin removal. We document that macrophages that are recruited to the same tissue but by distinct inflammatory stimuli differ in their ability to phagocytose and degrade myelin. These observations may apply to the nervous system where different pathological conditions that involve distinct inflammatory stimuli may induce different functional states in microglia and macrophages.     

Leukemia inhibitory factor modulates production of inflammatory mediators and myelin phagocytosis by macrophages

Leukemia inhibitory factor (LIF) promotes survival of glial cells and neurons during autoimmune and injury responses in the central nervous system (CNS). While various studies indicate that LIF also modulates ongoing inflammatory responses, data on underlying events are lacking. In this study we demonstrate that LIF modulates macrophage function. LIF inhibits the production of oxygen radicals and TNFalpha and stimulates myelin uptake by macrophages. These effects of LIF are accompanied by activation of the JAK/STAT3 signalling pathway. Our findings demonstrate that LIF has anti-inflammatory properties and enhances myelin clearance, implicating that LIF may be an important factor in CNS inflammatory disease.     

EAE cerebrospinal fluid augments in vitro phagocytosis and metabolism of CNS myelin by macrophages.

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)     

Dissimilar and similar functional properties of complement receptor‐3 in microglia and macrophages in combating yeast pathogens by phagocytosis

Central nervous system (CNS) microglia (MG) and peripheral tissue macrophages (MO) remove pathogens by phagocytosis. Zymosan, a model yeast pathogen, is a β‐glucan rich particle that readily activates the complement system and then becomes C3bi‐opsonized (op). Complement receptor‐3 (CR3) has initially been implicated in mediating the phagocytosis of both C3bi‐op and non‐opsonized (nop) zymosan by MO through C3bi and β‐glucan binding sites, respectively. Later, the role of CR3 as a phagocytic β‐glucan receptor has been questioned and the supremacy of β‐glucan receptor Dectin‐1 advocated. We compare here between primary mouse CNS MG and peripheral tissue MO with respect to CR3 and Dectin‐1 mediated phagocytosis of C3bi‐op and nop zymosan. We report that MG and MO display similar as well as dissimilar functional properties in this respect. Although CR3 and Dectin‐1 function both as β‐glucan/non‐opsonic receptors in MG during nop zymosan phagocytosis, Dectin‐1, but not CR3, does so in MO. CR3 functions also as a C3bi/opsonic receptor in MG and MO during C3bi‐op zymosan phagocytosis, leading to phagocytosis which is more efficient than that of nop zymosan. Dectin‐1 contributes, albeit less than CR3, to phagocytosis of C3bi‐op zymosan in MG and further less in MO, suggesting that C3bi‐opsonization does not block all β‐glucan sites on zymosan from binding Dectin‐1 on phagocytes. Thus, altogether CR3 and Dectin‐1 contribute both to phagocytosis of nop and C3bi‐op zymosan in MG, whereas MO switch from CR3‐independent/Dectin‐1‐dependent phagocytosis of nop zymosan to phagocytosis of C3bi‐op zymosan where CR3 dominates over Dectin‐1. © 2010 Wiley‐Liss, Inc.     

Macrophage phagocytosis of myelin in vitro determined by flow cytometry: phagocytosis is mediated by CR3 and induces production of tumor necrosis factor-α and nitric oxide

Demyelination of axons in the central nervous system (CNS) during multiple sclerosis (MS) and its animal model experimental allergic encephalomyelitis (EAE) is a result of phagocytosis and digestion by macrophages (M?) and the local release of inflammatory mediators like tumor necrosis factor-α (TNF-α) and nitric oxide (NO). We have investigated the process of myelin phagocytosis by M? in vitro using flow cytometric analysis. The binding and uptake of CNS-derived myelin was dose dependent, was abolished in the presence of EDTA and was enhanced after opsonization with complement. The phagocytosis of opsonized myelin could be inhibited by antibodies directed against complement receptor type 3 (CR3). Furthermore, CR3 also contributes to phagocytosis of non-opsonized myelin, e.g. under serum-free conditions. The phagocytosis of CNS-derived myelin induced the production of substantial amounts of TNF-α and NO by the M?. Our results indicate an important role for CR3 in myelin phagocytosis. The induction of TNF-α and NO which accompanies this phagocytosis may further contribute to the overall process of demyelination during MS or EAE.     

The role of anti-myelin (auto)-antibodies in the phagocytosis of myelin by macrophages

Plasma cells secreting antibodies directed to myelin components are present in CNS of MS patients and although the pathogenic role of such antibodies has yet to be established it is apparent from animal studies that anti-myelin antibodies are involved in myelin damage. In this study, we have investigated the effect of disease-promoting anti-myelin mAb on the phagocytosis of myelin by macrophages. Monoclonal antibodies directed to myelin basic protein (MBP)--clones 1, 12, 17, 22, 26, proteolipid protein (PLP), galactocerebroside (GalC) and myelin oligodendrocyte glycoprotein (MOG)--clones Y1, Y4, Y6, Y7, Y9, Y10, Y11 and Z12 were incubated with purified murine myelin labeled with DiI. The degree of phagocytosis of antibody-treated myelin by murine macrophages in vitro was determined using a quantitative flow cytometric assay. In comparison to untreated myelin pretreatment with myelin-specific mAb modified the degree of phagocytosis. The degree of opsonization of myelin was dependent on the isotype of antibody and the epitope recognized in addition to the ability of the mAb to fix complement. The greatest degree of opsonization of myelin was observed with the monoclonal antibody MOG Z12 that has previously been shown to enhance EAE and augment demyelination. These findings suggest a major role for anti-myelin antibodies, in particular antibodies directed to MOG, for the phagocytosis of myelin by macrophages in vitro. This may have relevance to the pathogenesis of myelin damage in vivo and provide a helpful tool for the classification of heterogeneous diseases such as MS.     

Non-PKC DAG/phorbol-ester receptor(s) inhibit complement receptor-3 and nPKC inhibit scavenger receptor-AI/II-mediated myelin phagocytosis but cPKC, PI3k, and PLCgamma activate myelin phagocytosis by both

Complement-receptor-3 (CR3/MAC-1), scavenger-receptor-AI/II (SRAI/II), and Fcgamma-receptor (FcgammaR) can mediate myelin phagocytosis in macrophages and microglia. Paradoxically, after injury to CNS axons these receptors are expressed but myelin is not phagocytosed, suggesting that phagocytosis is subject to regulation between efficient and inefficient states. In the present work, we focus on CR3/MAC-1 and SRAI/II-mediated myelin phagocytosis. Phagocytosis by CR3/MAC-1 and SRAI/II was inhibited by cPKC inhibitor Go-6976, general-PKC inhibitors Ro-318220 and calphostin-C, and BAPTA/AM, which chelates intracellular Ca2+ required for cPKC activation. Signaling/activation by cPKC are thus suggested. PMA, which mimics diacylglycerol (DAG) as an activator of cPKC, novel-PKC (nPKC), and non-PKC DAG-driven molecule(s), produced a dose-dependent dual effect on phagocytosis by CR3/MAC-1 and SRAI/II, i.e., augmentation at low concentrations and inhibition at high concentrations. Inhibition of phagocytosis by CR3/MAC-1 was enhanced by combining inhibiting concentrations of PMA with PKC inhibitors Go-6976 or Ro-318220, suggesting inhibition by PMA/DAG-driven non-PKC molecule(s). In contrast, inhibition of phagocytosis by SRAI/II was enhanced by combining inhibiting concentrations of PMA with cPKC inhibitor Go-6976 but not with general-PKC inhibitor Ro-318220, suggesting inhibition by nPKC. Phagocytosis by CR3/MAC-1 and SRAI/II was further inhibited by PI3K inhibitors wortmannin and LY-294002 and PLCgamma inhibitor U-73122. Altogether, our observations suggest that CR3/MAC-1 and SRAI/II-mediated myelin phagocytosis share activation by PI3K, PLCgamma and cPKC. The two differ, however, in that non-PKC DAG-driven molecule(s) inhibit CR3/MAC-1-mediated phagocytosis, whereas nPKC inhibit SRAI/II-mediated phagocytosis. Each of these signaling steps may be targeted for regulating CR3/MAC-1 and/or SRAI/II-mediated phagocytosis between efficient and inefficient states.     

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.     

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.

     

Distribution and characterization of microglia/macrophages in human brain tumors

The role of inflammatory reactions in brain tumors is still unclear. In particular, there is little information about the participation of the microglia/macrophage cell system. We therefore investigated 72 surgical biopsy samples of brain tumors (astrocytoma, glioblastoma, oligodendroglioma, ependymoma, medulloblastoma, cerebral lymphoma, gangliocytoma, neurocytoma and germinoma) and the brains of eight cases with malignant gliomas that came to autopsy, using immunohistochemical markers for the monocyte/macrophage lineage (Ki-M1P, HLA-DR, KP1, My4, My7, Ki-M1, Ki-M6, EBM 11). These markers allowed us to characterize four subtypes of the microglia/macrophage cell system: ramified microglia, ameboid microglia, perivascular microglia and brain macrophages. Among the different tumors, glioblastomas and anaplastic gliomas showed the largest number of mixed cell populations, which consisted of macrophages and ramified and ameboid microglia. In glial tumors of low malignancy fewer, predominantly ameboid, microglia were found. Neuronal tumors showed only a mild increase of microglia. Cerebral lymphomas contained macrophages diffusely distributed within the tumor center, while activated microglia were prominent at the border zone and in the adjacent brain tissue. The autopsy cases were used to study the morphometric distribution of microglia/macrophages. There was a significant increase of microglia/macrophages within the tumor, but no differences were seen between central and peripheral tumor areas. The non-neoplastic gray and white matter contained more microglial cells than controls. We conclude that the distribution pattern of ameboid and ramified microglial cells and macrophages is distinct in most of the investigated tumor types, underlining the complex immunological function of the microglia/macrophage cell system. Received: 24 July 1995 / Revised: 11 December 1995 / Accepted: 9 February 1996     

Stimulation of in vitro myelin synthesis by microglia

Central nervous system myelin is elaborated by oligodendrocytes, which have been studied extensively in cell culture. Dissociated brain cultures allow in vitro analysis of events in myelinogenesis, including cell-cell interactions. Microglia, the primary phagocytic cell of the central nervous system, appear in developing fiber tracts prior to the onset of myelination in vivo. To gain insight into potential oligodendrocytemicroglial interactions during development, these cells were co-cultured and various parameters of myelin synthesis were measured. In co-culture, microglia stimulated the synthesis of sulfatide, a myelin-specific galactolipid, in oligodendrocytes, as well as the expression of the myelin-specific proteins myelin basic protein and proteolipid protein. Activity of the oligodendrocyte cytoplasm-specific enzyme 2′,3′-cyclic nucleotide 3′-phosphohydrolase was not elevated, suggesting that the effects of microglia were not due to stimulation of oligodendrocyte proliferation. This was confirmed by the inability of microglia to induce significant DNA synthesis. Conditioned medium from cultured microglia provided a similar stimulatory activity, suggesting that the increase in myelin synthesis does not require contact between oligodendrocytes and microglia. These findings suggest a stimulatory role for microglia during myelinogenesis. © 1994 Wiley-Liss, Inc.     

Astrocytes modulate macrophage phagocytosis of myelin in vitro

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.     

TNF-alpha suppresses CR3-mediated myelin removal by macrophages.

Mononuclear cells of the monocyte/macrophage system play an important role in myelin ingestion during Wallerian degeneration. The present in vitro study clarifies the role in this process of two macrophage-secreted cytokines, TNF-alpha and interleukin-1. Treatment with TNF-alpha massively reduced the amount of myelin ingested by macrophages via their complement receptor type 3 (CR3). Anti-TNF-alpha antibodies reversed the effect. Immunofluorescence of macrophages indicated that TNF-alpha caused a reduced expression of the CR3 by phagocytic cells. Further experiments revealed an interaction of TNF-alpha with its receptor on the macrophage cell membrane. Interleukin-1 had no effect on myelin ingestion in the in vitro system used in these experiments.