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.     

Macrophage and microglial responses to cytokines in vitro: Phagocytic activity,proteolytic enzyme release,and free radical production

Certain cytokines are believed to play a key role in the development of autoimmune demyelinating diseases. Little is known, however, about the effects of these cytokines in the regulation of the key event in myelin destruction, the phagocytosis of myelin by phagocytic cells. We investigated the effects of certain cytokines and growth factors on cultured peritoneal macrophages and microglia in respect to their various functions, phagocytosis, secreted proteolytic activity, and oxidative activity. Interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and lipopolysaccharide (LPS), all proinflammatory factors, actually decreased (IFN-γ and LPS), or had no effect (TNF-α) on myelin phagocytosis by macrophages, but substantially increased phagocytic activity by microglia. Surprisingly, interleukins 4 and 10 (IL-4 and IL-10), considered to be downregulating cytokines, increased phagocytic activity by macrophages, while with microglia, IL-4 had no effect, but IL-10 almost doubled myelin phagocytosis. Transforming growth factor-β (TGF-β) had no significant effect on either cell. These cytokines did not affect proteolytic secretion in microglia, while IFN-γ and LPS induced a doubling of the secreted proteases. This proteolytic activity was almost completely suppressed by calpain inhibitors, although some gelatinase appeared to be present. Microglia exerted much more oxidative activity on the membranes than macrophages, and granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 1β (IL-1β) significantly increased microglial oxidative activity. The pattern of responses of macrophages and microglia to the cytokine types indicate that in cytokine-driven autoimmune demyelinating disease, microglia may be the more aggressive cell in causing tissue injury by phagocytosis and oxidative injury, while infiltrating macrophages may produce most of the proteolytic activity thought to contribute to myelin destruction. J. Neurosci. Res. 54:68–78, 1998. © 1998 Wiley-Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.     

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.     

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)     

Microglia and macrophage activation and the regulation of complement-receptor-3 (CR3/MAC-1)-mediated myelin phagocytosis in injury and disease

Microglia and macrophages play critical roles in the response of the central and peripheral nervous systems (CNS and PNS, respectively) to injury and disease, one of which is the removal of degenerated myelin by phagocytosis. Myelin removal is efficient during Wallerian degeneration, which follows injury to PNS axons, and in CNS autoimmune demyelinating diseases (e.g., multiple sclerosis) but is inefficient after injury to CNS axons. We suggest that inefficient myelin removal results from deficient microglia activation, reflected by the failure to up-regulate Galectin-3/MAC-2 expression, which marks a state of activation correlated with efficient myelin phagocytosis. Surprisingly, whether or not executing myelin phagocytosis, CNS microglia express the alphaM/beta2 integrin complement receptor-3 (CR3/MAC-1), which has the potential of mediating efficient myelin phagocytosis. We hypothesize that CR3/MAC-1 might be present in distinct inactive and active states that determine, respectively, efficient and inefficient CR3/MAC-1-mediated myelin phagocytosis. We present evidence that CR3/MAC-1-mediated myelin phagocytosis is regulated in microglia and macrophages. First, CR3/MAC-1- mediated myelin phagocytosis has complement-dependent and -independent components. Second, an active complement system augments CR3/MAC-1-mediated myelin phagocytosis. Third, anti-alphaM monoclonal antibodies (MAbs) inhibit and anti-beta2 MAbs augment CR3/MAC-1-mediated myelin phagocytosis in the presence and absence of an active complement system. Fourth, an active complement system modulates MAb-induced regulation of CR3/MAC-1-mediated myelin phagocytosis. Overall, MAb-induced phagocytosis regulation might range three- to sevenfold from inefficient to efficient. We suggest that one of the mechanisms underlying MAb-induced phagocytosis regulation is the induction/stabilization of inactive and active conformational changes. Monoclonal antibody-induced phagocytosis regulation must reveal a mechanism by which native extracellular molecules bind to and regulate CR3/MAC-1-mediated myelin phagocytosis in microglia and macrophages.     

Complement-receptor-3 and scavenger-receptor-AI/II mediated myelin phagocytosis in microglia and macrophages

Microglia and macrophages express the alpha(M)/beta(2) integrin complement-receptor-3 (CR3/MAC-1; CD11b/CD18) and scavenger-receptor-AI/II (SRAI/II). Both can mediate myelin phagocytosis. We document that CR3/MAC-1 mediated myelin phagocytosis in microglia is modulated by complement and anti-CR3/MAC-1 mAbs. Complement augmented phagocytosis twofold. Anti-alpha(M) mAbs M1/70 and 5C6 inhibited and anti-beta(2) mAb M18/2 augmented myelin phagocytosis in the presence and absence of active complement. Active complement modulated phagocytosis inhibition by M1/70 and 5C6 and phagocytosis augmentation by M18/2. CR3/MAC-1 mediated myelin phagocytosis may thus be, at least partially, independent of but modulated by complement. Anti-beta(2) mAb Game-46 did not affect phagocytosis. However, combining M18/2 with Game-46 resulted in phagocytosis augmentation that was larger in magnitude than that induced by M18/2 alone. Thus, phagocytosis augmentation induced by one anti-beta(2) mAb was potentiated by another anti-beta(2) mAb. Combining M1/70 or 5C6 with M18/2 inhibited M18/2-induced augmentation. Overall, mAbs-induced phagocytosis modulation ranged three- to sevenfold from inhibition to augmentation. Anti-CR3/MAC-1 mAbs may reveal a mechanism by which native extracellular molecules bind to and modulate CR3/MAC-1 mediated myelin phagocytosis in microglia and macrophages. We further document SRAI/II mediated myelin phagocytosis in microglia and CR3/MAC-1 contributing to myelin phagocytosis two- to threefold more than SRAI/II when the two receptors function together.     

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.     

The role of macrophages in demyelination in experimental allergic neuritis

The role of macrophages and serum factors in demyelination in experimental allergic neuritis (EAN) was examined by a simple in vitro method.Cultivated rabbit peritoneal macrophages, preincubated with serum obtained from rabbit EAN produced by sensitization with bovine spinal nerve roots, could agglutinate and phagocytize purified bovine or rabbit peripheral nerve myelin. Sera from normal animals or from controls given adjuvant alone could not. Adhesion and phagocytosis were inhibited if EAN sera were absorbed with peripheral nerve myelin. Rabbit red blood cells were not phagocytized by macrophages exposed to EAN serum.Concomitant to these observations, three lyosomal acid hydrolases: acid proteinase, acid phosphatase and β-glucuronidase, were assayed with respect to their topographical and chronological distribution. In the group examined at clinical onset, increases in the specific activities were 1.5–3.0-fold in the spinal roots and 1.0–1.5-fold in the sciatic nerves compared with control. The degree of increase in total activities per whole root or sciatic nerve was much higher than for specific activities. The topographical distribution of the increase closely corresponded to the histological distribution of EAN lesions. These observations suggested that the increased lysosomal activity originated from lysosomal-rich infiltrating cells.These observations strongly indicated the significant role of macrophages activated by EAN serum in the demyelination of EAN.     

Phagocytosis of peripheral nerve myelin in vitro: effect of antibody.

We have previously shown that antisera to whole CNS myelin, whole PNS myelin, galactocerebroside (GC), and myelin basic protein (MBP) promote the uptake of CNS myelin by cultured macrophages, and stimulate the conversion of myelin lipids to cholesterol ester and triglycerides. Here we report the results of similar studies using PNS myelin purified from the rat sciatic nerve. Antisera to whole CNS myelin, whole PNS myelin, GC, and MBP preincubated with 14C-labeled PNS myelin increased the production of radioactive cholesterol ester by macrophages in culture to a level about twice that with preimmune serum, and five to six times that of untreated myelin. The amounts of [14C]triglyceride were similarly increased with these antisera, whole P0 and P2 antisera had little or no effect. IgG prepared from the antisera stimulated lipid metabolism to almost the same extent, while heating the antisera did not decrease the stimulatory effect, indicating that myelin was opsonized by IgG, but not likely by complement. With a few exceptions, the four active sera and their IgGs promoted the macrophage metabolism of CNS and PNS myelin almost equally. The cultured macrophages converted about 3% of untreated CNS myelin and about 6% PNS myelin cholesterol to cholesterol ester. Under phase contrast microscopy it was noted that vesicles of CNS myelin appeared to bind individually to macrophages, whereas PNS myelin vesicles tended to self-associate to form large clumps which were found to macrophages. Binding studies showed PNS myelin to be bound more firmly to macrophages than CNS myelin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phagocytic microglia and macrophages in brain injury and repair

AimsPhagocytosis is the cellular digestion of extracellular particles, such as pathogens and dying cells, and is a key element in the evolution of central nervous system (CNS) disorders. Microglia and macrophages are the professional phagocytes of the CNS. By clearing toxic cellular debris and reshaping the extracellular matrix, microglia/macrophages help pilot the brain repair and functional recovery process. However, CNS resident and invading immune cells can also magnify tissue damage by igniting runaway inflammation and phagocytosing stressed—but viable—neurons.DiscussionMicroglia/macrophages help mediate intercellular communication and react quickly to the “find‐me” signals expressed by dead/dying neurons. The activated microglia/macrophages then migrate to the injury site to initiate the phagocytic process upon encountering “eat‐me” signals on the surfaces of endangered cells. Thus, healthy cells attempt to avoid inappropriate engulfment by expressing “do not‐eat‐me” signals. Microglia/macrophages also have the capacity to phagocytose immune cells that invade the injured brain (e.g., neutrophils) and to regulate their pro‐inflammatory properties. During brain recovery, microglia/macrophages engulf myelin debris, initiate synaptogenesis and neurogenesis, and sculpt a favorable extracellular matrix to support network rewiring, among other favorable roles. Here, we review the multilayered nature of phagocytotic microglia/macrophages, including the molecular and cellular mechanisms that govern microglia/macrophage‐induced phagocytosis in acute brain injury, and discuss strategies that tap into the therapeutic potential of this engulfment process.ConclusionIdentification of biological targets that can temper neuroinflammation after brain injury without hindering the essential phagocytic functions of microglia/macrophages will expedite better medical management of the stroke recovery stage.     

Phagocytosis of apoptotic inflammatory cells by microglia and its therapeutic implications: termination of CNS autoimmune inflammation and modulation by interferon-beta

Apoptosis of autoaggressive T-cells in the CNS is an effective, noninflammatory mechanism for the resolution of T-cell infiltrates, contributing to clinical recovery in T-cell-mediated neuroinflammatory diseases. The clearance of apoptotic leukocytes by tissue-specific phagocytes is critical in the resolution of the inflammatory infiltrate and leads to a profound downregulation of phagocyte immune functions. Adult human microglia from surgically removed normal brain tissue was used in a standardized, light-microscopic in vitro phagocytosis assay of apoptotic autologous peripheral blood-derived mononuclear cells (MNCs). Microglia from five different patients had a high capacity for the uptake of apoptotic MNCs in contrast to nonapoptotic target cells with the phagocytosis rate for nonapoptotic MNCs amounting to only 61.6% of the apoptotic MNCs. A newly described phosphatidylserine receptor, critical in the phagocytosis of apoptotic cells by macrophages, is also expressed at similar levels on human microglia. The effects of the therapeutically used immunomodulatory agent interferon-beta (IFNbeta) were investigated using Lewis rat microglia and apoptotic, encephalitogenic, myelin basic protein-specific autologous T-cells. Also, rat microglia had a high capacity to phagocytose apoptotic T-cells specifically. IFNbeta increased the phagocytosis of apoptotic T-cells to 36.8% above the untreated controls. The enhanced phagocytic activity was selective for apoptotic T-cells and was not mediated by increased IL-10 secretion. Apoptotic inflammatory cells may be efficiently and rapidly removed by microglial cells in the autoimmune-inflamed human CNS. The in vitro increase of phagocytosis by IFNbeta merits further investigations whether this mechanism could also be therapeutically exploited.     

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.     

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.     

Galectin-3/MAC-2 in experimental allergic encephalomyelitis

The removal of degenerating myelin by phagocytosis is central to pathogenesis and repair in traumatized and diseased nervous system. Galectin-3/MAC-2 is a differentiation and activation marker of murine and human monocytes/macrophages/microglia. Galectin-3/MAC-2, along with MAC-1 that mediates myelin phagocytosis, marks an in vivo activation state in macrophages, which are involved in myelin degeneration and phagocytosis in injured mouse peripheral nerves. In contrast, high levels of MAC-1 but extremely low levels of Galectin-3/MAC-2 are expressed in vivo in injured CNS where myelin degeneration and phagocytosis progress extremely slowly. The present study was aimed at testing whether an activation state marked by Galectin-3/MAC-2 is present in vivo in the CNS of EAE mice concomitant with autoimmune induced myelin degeneration and phagocytosis. EAE was inflicted by mouse spinal cord homogenate. Demyelination was assessed by light microscopy and Galectin-3/MAC-2, MAC-1, and F4/80 expression by immunocytochemistry. We presently document that Galectin-3/MAC-2 expression is up regulated, along with MAC-1 and F4/80, in spinal cords and optic nerves of EAE mice in areas of demyelination and myelin degeneration, in myelin phagocytosing microglia and macrophages. Copolymer 1 (Glatiramer acetate) suppresses EAE, demyelination, and Galectin-3/MAC-2 expression. EAE pathogenesis thus involves a state of activation in microglia and macrophages characterized by the expression Galectin-3/MAC-2 along with MAC-1. Furthermore, the in vivo responses to injury and autoimmune challenge in the CNS differ in the activation pattern of microglia and macrophages with regard to Galectin-3/MAC-2 expression and the corresponding occurrence of myelin degeneration and phagocytosis.     

Phagocytosis of apoptotic inflammatory cells by microglia and modulation by different cytokines: mechanism for removal of apoptotic cells in the inflamed nervous system

Apoptosis of autoaggressive T cells in the central nervous system (CNS) is an effective, nonphlogistic mechanism for the termination of autoimmune inflammation in experimental autoimmune encephalomyelitis (EAE). The clearance of apoptotic leukocytes by tissue-specific phagocytes is a critical event in the resolution of the inflammatory attack. To investigate the role of microglia in the removal of apoptotic cells and potential regulatory mechanisms of microglial phagocytosis, an in vitro phagocytosis assay was established, using Lewis rat microglia. Microglia exhibited a high capacity for the uptake of apoptotic autologous thymocytes, as well as apoptotic encephalitogenic myelin basic protein (MBP)-specific T cells, in contrast to nonapoptotic target cells. Pretreatment of microglia with interferon-gamma (IFN-gamma) raised the proportion of microglia capable of phagocytosing apoptotic cells to 75% above the untreated controls. The increased phagocytic activity was selective for apoptotic target cells and was not dependent on phosphatidylserine-mediated recognition mechanisms. In contrast, preincubation of microglia with interleukin-4 (IL-4) inhibited the uptake of apoptotic cells, whereas tumor-necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta) did not alter phagocytosis. Phagocytic clearance of apoptotic inflammatory cells by microglia may be an important mechanism for the termination of autoimmune inflammation in the CNS. Augmentation of microglial phagocytosis by the Th-1-type cytokine IFN-gamma suggests a feedback mechanism for the accelerated clearance of the inflammatory infiltrate in the CNS.     

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.     

Chemotaxis by a CNS macrophage, the microglia

Microglia demonstrate many characteristics similar to those seen in monocytes and tissue-specific macrophages, including phagocytosis, production of oxygen radicals, and growth factors and expression of MHC antigens. We have examined the ability of microglia, cultured from the cerebral cortices of neonatal rats, to demonstrate another important functional characteristic of monocytic-derived cells, that is, chemotaxis. Our results show that cultured rat microglia demonstrate chemotaxis to complement dependent chemoattractants such as recombinant C5a, zymosan activated serum, and to rat serum as well as to transforming growth factor-beta, a chemoattractant produced by platelets. Microglia fail to migrate to bacterial dependent chemoattractants such as the N-formyl peptides. The failure to respond is not dependent on maturational state of the microglia. Treatment with DMSO or casein, agents known to induce morphological and functional changes in cultured microglia reminescent of a "resting" and an "activated" macrophage, respectively, do not alter the response to fMet-Leu-Phe. In addition, the chemotactic response to serum in DMSO or casein-treated cells is the same as the response seen in untreated day 10 cultured microglia or untreated age-matched controls. The ability of microglia to migrate in response to inflammatory mediators suggests that these cells can move to sites of injury, thereby enabling them to participate in an inflammatory response.     

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.     

Phagocytosis of polystyrene latex beads by rat brain microglia cell cultures is increased by treatment with gangliosides

Cultures of microglial cells were prepared from primary rat brain cultures of astrocytes. Microglia appeared under two main forms: ameboid and ramified. Only the former were capable of ingesting opsonized latex particles of 1 and 6 micron; while the latter, which appeared later in culture, were not capable of phagocytosis. When gangliosides (10(-6) M), which are known for their neuronotrophic capacity in vitro, were added to the growth medium (for 20 min) they stimulated the phagocytosis of ameboid microglia 2-fold.