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     

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.

     

Differential effects of central and peripheral nerves on macrophages and microglia

The poor ability of injured central nervous system (CNS) axons to regenerate has been correlated, at least partially, with a limited and suppressed postinjury inflammatory response. A key cell type in the inflammatory process is the macrophage, which can respond in various ways, depending on the conditions of stimulation. The aim of this study is to compare the activities of macrophages or microglia when encountering CNS and peripheral nervous systems (PNS), on the assumption that nerve-related differences in the inflammatory response may have implications for tissue repair and thus for nerve regeneration. Phagocytic activity of macrophages or of isolated brain-derived microglia was enhanced upon their exposure to sciatic (PNS) nerve segments, but inhibited by exposure to optic (CNS) nerve segments. Similarly, nitric oxide production by macrophages or microglia was induced by sciatic nerve segments but not by optic nerve segments. The previously demonstrated presence of a resident inhibitory activity in CNS nerve, could account, at least in part, for the inhibited phagocytic activity of blood-borne macrophages in CNS nerve as well as of microglia resident in the brain. It seems that the CNS microglia are reversibly immunosuppressed by the CNS environment, at least with respect to the activities examined here. It also appears from this study that the weak induction of early healing-related activities of macrophages/microglia in the environment of CNS might explain the subsequent failure of this environment to acquire growth-supportive properties in temporal and spatial synchrony with the needs of regrowing axons. GLIA 23:181–190, 1998. © 1998 Wiley-Liss, Inc.     

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)     

Normal polyclonal immunoglobulins ('IVIg') inhibit microglial phagocytosis in vitro

Phagocytosis removes pathogens and tissue debris during inflammatory reactions, but also plays an important role in autoimmune reactions. The main phagocytes in the central nervous system (CNS) are microglial cells that are activated during CNS inflammation. In the treatment of inflammatory demyelinating diseases like multiple sclerosis (MS), administration of intravenous immunoglobulins (IVIg) has become a promising immunomodulatory therapy. Although a large number of potential mechanisms for the effects of IVIg has been suggested, the precise mode of action in CNS inflammation is unknown. We assessed the influence of IVIg on phagocytosis and endocytosis in microglia in vitro. IVIg had little effect on non-specific phagocytosis of latex particles in untreated microglia, while there was a dose-dependent inhibition in microglia activated with LPS and IFNgamma. Endocytosis of soluble myelin basic protein (MBP) was downregulated by IVIg in both untreated and activated microglia. The effect was mediated by an F(ab')(2) preparation of immunoglobulins, suggesting that Fc receptor-mediated phagocytosis is not involved. Intact IVIg, but not F(ab')(2) fragments also suppressed Fc receptor-mediated phagocytosis of opsonised erythrocytes in both untreated and activated microglia. These results show that IVIg can inhibit the phagocytic activity of microglia via different mechanisms. Such an effect could contribute to the immunomodulatory capacity of IVIg in inflammatory CNS diseases.     

Peripheral nerve demyelination induced by intraneural injection of experimental allergic encephalomyelitis serum.

Intraneural injection of sera from rabbits with experimental allergic encephalomyelitis, induced by sensitization with bovine brain white matter in complete Freund's adjuvant, produced focal primary demyelinative lesions in rat sciatic nerves. Demyelinating activity was removed by prior incubation of antisera with central (CNS) and peripheral nervous system (PNS) myelin but not with liver or kidney, and was heat-labile and complement-dependent. Recipient animals developed a sensorimotor disturbance of their toes and ankles on the side injected with antiserum. Twenty minutes after antiserum injection, Schwann cells showed focal cytoplasmic outpouching and their external mesaxons opened. Between 1 and 8 hours after injection vacuolation, splitting and vesiculation of myelin became increasingly prominent at Schmidt-Lanterman clefts and paranodal regions, with concomitant degenerative changes in Schwann cell cytoplasm. Polymorphonuclear cell infiltration and endoneurial edema were apparent at this time. Substantial demyelination occurred before the appearance of phagocytic cells. Between 8 hours and 3 days many nerve fibers were surrounded and attacked by invading macrophages. Axons became demyelinated progressively over several internodes by macrophage phagocytosis. Early signs of remyelination were observed by 5 days. These findings suggest that antibodies directed against antigens common to both CNS and PNS myelin can produce in vivo peripheral nerve demyelination.     

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 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.     

A survey of neurological mutant mice. II. Lipid composition of myelinated tissue in possible myelin mutants

The lipids of white matter and peripheral nerve from neurological mutant mice with possible myelin abnormalities were analyzed by thin-layer chromatography and quantitated by densitometry. Eight mutants had major abnormalities in the central nervous system (CNS) and/or peripheral nervous system (PNS) tissues examined (optic nerve, and trigeminal and sciatic nerves). In the optic nerve of axJ/axJ, there were increases of 20-30% in the levels of the major phospholipids; peripheral nerve was normal. In bc3J/bc3J CNS, the major phospholipids and cholesterol were increased by 25-40%; the PNS was normal. In myd/myd CNS, there were increases of about 20% in the levels of both forms of cerebrosides and in the major phospholipids; in the PNS the lipids were normal. ot/ot CNS had 20-40% reductions of all the glycolipids and minor alterations in some of the phospholipids and cholesterol; the PNS had 20% losses of both forms of cerebrosides. In the PNS of ji/ji, there were decreases of 10-40% among the glycolipids and of 15-25% in three of the major phospholipids; the CNS was virtually normal. In the PNS of dtJ/dtJ, vb/vb and wr/wr, almost all lipids were significantly decreased. The CNS of dtJ/dtJ and vb/vb were normal; wr/wr had minor reductions of certain glycolipids and phospholipids. Six mutants had relatively minor lipid abnormalities in their myelinated tissues. In cr/cr PNS, there were elevated levels of the cerebrosides and major phospholipids; the CNS was virtually normal. In db/db CNS and PNS, there were reduced levels of the nonhydroxy forms of cerebroside and sulfatide. The major change in htr/htr was the elevation of all the glycolipids in the CNS. In the CNS of Lc/+, nonhydroxy cerebroside was reduced. In shm/shm PNS, nonhydroxy sulfatide was elevated and there were small decreases in some of the phospholipids. wl/wl CNS showed decreases among most of the glycolipids. Mutants homozygous for du, mto, spa and tg had virtually normal lipid levels in both the optic and peripheral nerves. Cholesterol ester, lysophospholipids and other unusual lipid species were not detected in any of the mutants. The plasmalogen forms of ethanolamine and choline phosphatides were at normal levels in all mutants that otherwise had significant alterations among their lipids. Although many alterations in lipid composition were found in these mutants, the changes were moderate compared to the classical myelin mutants and indicate that none of the mutants are severely myelin-deficient.(ABSTRACT TRUNCATED AT 400 WORDS)     

Peripheral nerve-stimulated macrophages simulate a peripheral nerve-like regenerative response in rat transected optic nerve

We have previously demonstrated that the failure of the mammalian central nervous system (CNS) to regenerate following axonal injury is related to its immunosuppressive nature, which restricts the ability of both recruited blood-borne monocytes and CNS-resident microglia to support a process of repair. In this study we show that transected optic nerve transplanted with macrophages stimulated by spontaneously regenerating nerve tissue, e.g., segments of peripheral nerve (sciatic nerve), exhibit axonal regrowth at least as far as the optic chiasma. Axonal regrowth was confirmed by double retrograde labeling of the injured optic axons, visualized in their cell bodies. Transplanted macrophages exposed to segments of CNS (optic) nerve were significantly less effective in inducing regrowth. Immunocytochemical analysis showed that the induced regrowth was correlated with a wide distribution of macrophages within the transplanted-transected nerves. It was also correlated with an enhanced clearance of myelin, known to be inhibitory for regrowth and poorly eliminated after injury in the CNS. These results suggest that healing of the injured mammalian CNS, like healing of any other injured tissue, requires the partnership of the immune system, which is normally restricted, but that the restriction can be circumvented by transplantation of peripheral nerve-stimulated macrophages. GLIA 24:329–337, 1998. © 1998 Wiley-Liss, Inc.     

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.     

Phospholipase A2 plays an important role in myelin breakdown and phagocytosis during Wallerian degeneration

Phospholipase A(2) (PLA(2)) hydrolyzes phosphatidylcholine to lysophosphatidylcholine and arachidonic acid. The former can induce myelin breakdown and the latter, via eicosanoids, can stimulate inflammatory responses. Immunohistochemical analysis of secreted (sPLA(2)) and cytosolic (cPLA(2)) forms of the enzyme was assessed in the injured adult rat sciatic and optic nerves. sPLA(2) and cPLA(2) are expressed in the first 2 weeks in the injured sciatic nerve, which correlates with rapid Wallerian degeneration in peripheral nerves. In contrast, both forms of PLA(2) were not expressed in the optic nerve for the first 3 weeks after crush injury, which correlates with slow Wallerian degeneration in the central nervous system (CNS). In addition, PLA(2) is not expressed in the lesioned sciatic nerve of C57BL/Wld(s) mutant mice in which Wallerian degeneration is severely retarded. Blocking cPLA(2) in the transected sciatic nerve of C57BL/6 mice, which have a naturally occurring null mutation for the major from of sPLA(2), resulted in a marked slowing of myelin and axonal degradation and phagocytosis in the distal nerve segment. These results provide direct evidence of an important role for cPLA(2) in Wallerian degeneration.     

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.     

Neuronal age influences the response to neurite outgrowth inhibitory activity in the central and peripheral nervous systems.

Axonal regeneration is abortive in the central nervous system (CNS) of adult mammals, but readily occurs in the injured peripheral nervous system (PNS). Recent experiments indicate an important role for both intrinsic neuronal features and extrinsic substrate properties in determining the propensity for axonal regrowth. In particular, certain components of adult mammalian CNS myelin have been shown to exert a strong inhibitory influence on neurite outgrowth. To determine whether the potent neurite outgrowth inhibitory activity found in CNS myelin may also be present in PNS myelin and to study the influence of neuronal age on neurite outgrowth, we used a cryoculture assay in which dissociated rat dorsal root ganglion (DRG) neurons of different ages were challenged to extend neurites on fractionated myelin and cryostat sections from the PNS (sciatic nerve and myelin-free degenerated sciatic nerve) and CNS (optic nerve) of adult rats. The CNS environment of the optic nerve did not support E17 to P8 DRG neurite adhesion or outgrowth. E17 DRG neurons, unlike their older counterparts, however, were able to attach and extend neurites onto normal sciatic nerve and onto purified PNS myelin. In contrast, a vigorous neurite outgrowth response from all the ages tested was observed on the myelin-free degenerated sciatic nerve. These results indicate that PNS myelin is a potent inhibitor of neurite outgrowth and that DRG neuronal age plays an important role in determining the propensity for neurite outgrowth and regenerative response on inhibitory PNS and CNS substrata.     

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.     

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.     

Polyclonal immunoglobulins (IVIg) modulate nitric oxide production and microglial functions in vitro via Fc receptors

Controlled trials in multiple sclerosis (MS) and case reports in acute demyelinating encephalomyelitis (ADEM) have shown that intravenous immunoglobulins (IVIg) are of therapeutic benefit in central nervous system (CNS) inflammatory diseases. Studies in experimental autoimmune encephalomyelitis (EAE) have suggested these effects are mediated by modulation of the cytokine network and T cell responses. However, there are no data on the influence of IVIg on the local immune reaction in the CNS, the site of inflammation in EAE. We have therefore studied the effect of IVIg on cultured rat microglia, the main immune cell in the CNS. IVIg increased nitric oxide (NO) production in a dose-dependent manner in microglia stimulated with IFNgamma. The increase was only marginal in LPS-treated cells, and no effect was seen in untreated microglia or after stimulation with TNFalpha or PMA. This enhancement of NO production depended on the Fc portion of IVIg and could be abrogated by the pharmacological inhibition of Syk and phosphatidylinositol 3-kinase, two enzymes involved in the signalling cascade of Fc receptors. TNFalpha secretion was dose-dependently stimulated by IVIg in both untreated microglia and after stimulation with LPS or IFNgamma. Again, this effect was mediated through the Fc portion. Finally, we showed that Fc receptor-mediated phagocytosis was inhibited by IVIg, presumably by blockade of the Fc receptor. These different effects may protect oligodendrocytes from antibody mediated phagocytosis and on the other hand could terminate the immune reaction by induction of apoptosis in infiltrating T cells via NO and TNFalpha. We propose that IVIg, in addition to known effects on the peripheral immune system, may also modulate the local immune reaction in CNS inflammatory disease.     

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.     

ApoD,a glia‐derived apolipoprotein,is required for peripheral nerve functional integrity and a timely response to injury

Glial cells are a key element to the process of axonal regeneration, either promoting or inhibiting axonal growth. The study of glial derived factors induced by injury is important to understand the processes that allow or preclude regeneration, and can explain why the PNS has a remarkable ability to regenerate, while the CNS does not. In this work we focus on Apolipoprotein D (ApoD), a Lipocalin expressed by glial cells in the PNS and CNS. ApoD expression is strongly induced upon PNS injury, but its role has not been elucidated. Here we show that ApoD is required for: (1) the maintenance of peripheral nerve function and tissue homeostasis with age, and (2) an adequate and timely response to injury. We study crushed sciatic nerves at two ages using ApoD knock‐out and transgenic mice over‐expressing human ApoD. The lack of ApoD decreases motor nerve conduction velocity and the thickness of myelin sheath in intact nerves. Following injury, we analyze the functional recovery, the cellular processes, and the protein and mRNA expression profiles of a group of injury‐induced genes. ApoD helps to recover locomotor function after injury, promoting myelin clearance, and regulating the extent of angiogenesis and the number of macrophages recruited to the injury site. Axon regeneration and remyelination are delayed without ApoD and stimulated by excess ApoD. The mRNA and protein expression profiles reveal that ApoD is functionally connected in an age‐dependent manner to specific molecular programs triggered by injury. © 2010 Wiley‐Liss, Inc.     

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