The role of complement in myelin phagocytosis during PNS wallerian degeneration

Myelin removal in nerves undergoing wallerian degeneration mainly depends on invading, non-resident macrophages. The present study clarifies the role of serum complement components in this process in vitro and in vivo. Macrophages cocultured with degenerating nerves in vitro were unable to invade these nerves in the presence of C3-deficient serum. Application of C3-deficient serum subsequent to cellular invasion abolished the myelin phagocytic capacity of the invaded macrophages. This indicates that opsonization of myelin by complement components is necessary in myelin ingestion via macrophage receptors. In vivo, a monoclonal antibody to the macrophage complement receptor type 3 (CR3) significantly reduced myelin phagocytosis. Immunohistochemistry with anti-C3 antibodies showed a marked reaction in degenerating nerves. Immunoelectron microscopy localized C3 particles at the degenerating myelin sheaths. Haematogenous cells, invading the degenerating nerves, also showed a strong reaction for C3 in their cytoplasm. These results indicate that complement components play a critical role both in macrophage invasion of degenerating nerves and in the ingestion of myelin by these cells.     

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.     

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.     

Involvement of intercellular adhesion molecule-1 in myelin recognition by macrophages

Macrophages play a crucial role in myelin removal during nerve degeneration and demyelination. The exact mechanisms of myelin recognition and uptake are not yet defined. The present experiments aimed at defining the role of intercellular adhesion molecule-1 (ICAM-1) in this process. Myelin phagocytosis was studied in an established in vitro model of cultured macrophages and sciatic nerves. Cocultures of wild-type C57BL macrophages with sciatic nerves resulted in a massive invasion of the nerves by macrophages with subsequent removal of myelin. In contrast, when macrophages of ICAM-1-deficient animals were cocultured with wild-type nerves, myelin phagocytosis was significantly retarded, whereas cell invasion was completely undisturbed. These data indicate that the ICAM-1 molecule acts as a costimulatory signal in myelin recognition and uptake by macrophages. Received: 3 January 2000 / Revised, accepted: 7 February 2000     

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.     

The Role of the Mouse Macrophage Scavenger Receptor in Myelin Phagocytosis

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

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.     

Modulation (inhibition and augmentation) of complement receptor-3-mediated myelin phagocytosis.

The removal of damaged myelin is central to repair after injury to axons and in autoimmune demyelinating diseases. Complement receptor 3 (CR3/MAC-1) plays a major role in mediating the phagocytosis of damaged myelin by macrophages and microglia. We studied the modulation (inhibition and augmentation) of CR3/MAC-1 mediated myelin phagocytosis by mAbs that bind to distinct epitopes of subunits alphaM and beta2 of CR3/MAC-1. mAb M1/70 anti-alpha(M) and mAb 5C6 anti-alpha(M) inhibited, whereas mAb M18/2 anti-beta2 augmented myelin phagocytosis. This mAb-induced modulation of myelin phagocytosis occurred in the presence and absence of active complement. Inhibition induced by M1/70 or 5C6 did not add when the two were combined. Combining M1/70 or 5C6 with M18/2 reduced the augmentation induced by M18/2 alone. CR3/MAC-1-mediated myelin phagocytosis may thus be subjected to modulation between efficient and inefficient functional/activation states. These observations and conclusions may offer an explanation for the observed discrepancy between efficient myelin phagocytosis in experimental allergic encephalomyelitis and inefficient myelin phagocytosis after injury to CNS axons, although in both instances macrophages/microglia express CR3/MAC-1.     

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.     

Millipore diffusion chambers allow dissociation of myelin phagocytosis by non-resident cells and of allogenic nerve graft rejection

Allogenic graft rejection leads to rapid tissue destruction of nerves transplanted directly into a muscle lodge. If the nerves are enclosed in 5.0 micron pore chambers and transplanted into the peritoneal cavity, there is no allogenic graft rejection. The phagocytosis of myelin by invading cells is, however, not disturbed, showing that these cells can distinguish the degenerating myelin from the Schwann cell without being responsive to the Schwann cell's allotype. If the allografts are allowed to predegenerate for 4 wk in 0.22 micron pore chambers which do not admit any cells, there is a striking mitigation of the allogenic graft rejection if the nerves are subsequently released from the chamber. Myelin phagocytosis in such nerves is also reduced. These observations indicate the existence of a hierarchy of cellular recognition mechanisms involved in nerve tissue degradation. Phagocytosis of the myelin sheath by macrophages involves recognition mechanisms which differ from those of the allogenic rejection of the Schwann cell, presumably mediated by T lymphocytes.     

Myelin phagocytosis in Wallerian degeneration

Summary Myelin phagocytosis in Wallerian degeneration was studied using a model of murine sciatic nerve degeneration in millipore diffusion chambers in the peritoneal cavity of host mice. Immunocytological investigations showed the dependence of myelin digestion on the invasion of Fc-positive, Mac-1-positive and partly Ia-positive monocytes. Lymphocytes did not play a prominent role. Compared to Wallerian degeneration in situ, phagocytosis was decreased in nerves enclosed by millipore membranes on both sides of the chamber. The membrane acted as a trap for invading monocytes/macrophages. Neither tissue integrity nor genetic strain influenced the degree of phagocytosis. A modification of the experimental technique is introduced which permits myelin phagocytosis in the peritoneal cavity in a degree comparable to that in Wallerian degeneration in situ.Supported by a grant from the Deutsche Forschungsgemeinschaft (609)     

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.     

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.     

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.     

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.     

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.

     

Changes of myelin proteins during Wallerian degeneration in situ and in millipore diffusion chambers preventing active phagocytosis

Changes of myelin proteins in mouse sciatic nerves were studied comparing nerves degenerating in situ with nerves enclosed in millipore diffusion chambers which eliminate invasion of non-resident cells. Nerves kept in chambers showed nearly complete preservation of myelin sheaths with a very slow degradation of myelin proteins. Nerves degenerating in situ showed rapid myelin phagocytosis by macrophages with almost complete disappearance of myelin proteins after 28 days. These data elucidate the role of macrophages for removal of myelin proteins.     

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.     

Complement depletion affects demyelination and inflammation in experimental allergic neuritis

The effect of systemic complement depletion by cobra venom factor (CVF) on experimental allergic neuritis (BAN) was studied in rats immunized with variable amounts of bovine peripheral nerve myelin. Low-dose myelin EAN rats treated with CVF i.p. (n = 10) had lower clinical scores (0.3 ± 0.7 vs. 1.1 ± 1.1), less demyelination (0.4 ± 0.8 vs. 1.9 ± 1.1) and inflammation (0.6 ± 1.2 vs. 2 ± 1) than EAN animals treated with i.p. saline (n = 10). Endoneurial infiltrates had fewer EDI-positive (phagocytic) macrophages (if0.4 ± 0.5 vs.> 1.6 ± 1.1) and CD11bc-positive (expressing iC3b receptor or CR3) cells (1 ± 0.8 vs. 2.5 ± 0.8) (mean ± S.D.) detected by immunocytochemistry. This effect was partially abrogated by immunizing animals with a higher dose of myelin. Our studies suggest that complement may play a role in the recruitment of macrophages into the endoneurium and in opsonizing myelin for phagocytosis.