Phagocytosis of Myelin in Demyelinative Disease: A Review

In the cell-mediated demyelinating diseases such as experimental allergic encephalomyelitis and multiple sclerosis, as well as their peripheral nerve counterparts, the phagocytic cells are the agent of myelin destruction. Both resident microglia and peripheral macrophages invading the nervous system have been shown to phagocytize myelin, although microglia appear to be more active, especially at early stages of disease. Several different receptors on these cells have been implicated as myelin receptors, with the Fc- and complement receptors receiving the most attention. Other receptors, especially the macrophage scavenger receptor with its broad specificity deserves further exploration, especially in view of its affinity for phosphatidylserine, which becomes externalized with membrane disruption. Evidence is shown for cytokine regulation of phagocytic activity in both macrophages and microglia. Further investigation of the pathways of cytokine action on myelin phagocytosis through signal transduction molecules will be important for a further understanding of the events leading to myelin destruction in demyelinating diseases.     

Receptor-mediated phagocytosis of myelin by macrophages and microglia: Effect of opsonization and receptor blocking agents

Myelin is phagocytosed by microglia (MG) and to a somewhat lesser extent by peritoneal macrophages (Mϕ) in a dose- and time-dependent manner. In serum-free medium opsonization of rat myelin significantly enhances binding and ingestion, more by rat macrophages than by microglia. Furthermore the requirement for opsonization is not restricted to anti-myelin antibodies as the difference in the rate of myelin uptake by macrophages is largely eliminated when they are cultured in 10% fetal calf serum. Binding and ingestion of both myelin and opsonized myelin are inhibited to the same dose-dependent extent by zymosan, oxidized LDL, peroxidase-antiperoxidase (PAP), opsonized erythrocytes and the anti-CR3 antibody OX42 implicating lectin, scavenger, Fc and complement receptors in the phagocytosis of myelin. Thus while the differential uptake of myelin and opsonized myelin by macrophages would indicate a central role for the Fc receptor, binding inhibition studies implicate a range of membrane receptors which would obviate the need for antigen-antibody complexing to stimulate phagocytosis. Uptake of both myelin preparations by macrophages or microglia is stimulated by interferon-γ and inhibited by TGF-β, and the process of ingestion results in increased nitric oxide release and decreased superoxide production, the effect being more pronounced when myelin is opsonized. Special issue dedicated to Dr. Marion E. Smith.     

Myelin-Derived Lipids Modulate Macrophage Activity by Liver X Receptor Activation

Multiple sclerosis is a chronic, inflammatory, demyelinating disease of the central nervous system in which macrophages and microglia play a central role. Foamy macrophages and microglia, containing degenerated myelin, are abundantly found in active multiple sclerosis lesions. Recent studies have described an altered macrophage phenotype after myelin internalization. However, it is unclear by which mechanisms myelin affects the phenotype of macrophages and how this phenotype can influence lesion progression. Here we demonstrate, by using genome wide gene expression analysis, that myelin-phagocytosing macrophages have an enhanced expression of genes involved in migration, phagocytosis and inflammation. Interestingly, myelin internalization also induced the expression of genes involved in liver-X-receptor signaling and cholesterol efflux. In vitro validation shows that myelin-phagocytosing macrophages indeed have an increased capacity to dispose intracellular cholesterol. In addition, myelin suppresses the secretion of the pro-inflammatory mediator IL-6 by macrophages, which was mediated by activation of liver-X-receptor β. Our data show that myelin modulates the phenotype of macrophages by nuclear receptor activation, which may subsequently affect lesion progression in demyelinating diseases such as multiple sclerosis.     

Antibody to myelin constituents: A possible factor in induction of cell-mediated demyelination

Results from this laboratory have demonstrated that¹⁴C-labeled myelin opsonized with antibodies raised to purified CNS myelin in rabbit is phagocytized by cultured macrophages in larger amounts than untreated myelin or myelin opsonized with preimmune serum. The cultured macrophages produced high amounts of radioactive cholesterol ester and triglyceride from the antibody-treated myelin while much less was formed from preimmune serum-treated or untreated myelin. Antiserum to galactocerebroside also greatly enhanced the formation of radioactive cholesterol ester, while that to myelin basic protein as well as to other myelin constituents had little or no effect. Serum from Lewis rats with acute EAE 13–14 days after immunization with whole CNS myelin also stimulated radioactive cholesterol ester formation compared to serum from Freund's adjuvant-injected controls (FAC). Serum from EAE rats as a result of myelin basic protein injection was as active as that from rats with whole myelin injection. No galactocerebroside antibody could be demonstrated in the EAE sera, although a strong immunostaining to myelin basic protein and proteolipid protein was demonstrated. IgG prepared from EAE serum also showed stimulatory effects compared to IgG from FAC serum, but much of the activity was lost, and the possibility that other factors may be involved is discussed. These experiments provide evidence that myelin phagocytosis and digestion by macrophages is enhanced by the presence of antibody to myelin. In EAE this antibody may leak into CNS with the breakdown of the blood-brain barrier. A humoral involvement in demyelination in EAE is implicated, and these findings may be extended eventually to the demyelinative mechanism in multiple sclerosis where IgG is found in large amounts in the CNS.Special Issue Dedicated to Dr. Abel Lajtha.     

Effects of Phorbol Myristate Acetate (PMA) on Functions of Macrophages and Microglia In Vitro

Peripheral macrophages infiltrating the central nervous system and resident microglia phagocytize myelin in cell-mediated demyelinating diseases, including experimental autoimmune encephalomyelitis and multiple sclerosis. A cascade of cytokines is believed to modulate the immunological sequence of events occurring in these conditions, and several of these mediate their effects through the protein kinase C pathway. Therefore, we compared the effects of phorbol myristate acetate (PMA), an activator of protein kinase C, on various functions of cultured macrophages and microglia. PMA at moderate concentrations induced apoptosis in macrophages, and this process appeared to be increased in the presence of myelin. In contrast, microglia were activated by PMA, and greatly increased their phagocytosis of myelin. Control macrophages released a considerable amount of proteolytic activity into the medium, as measured by the breakdown of myelin basic protein, and in the process of undergoing apoptosis from PMA-treatment, even higher amounts were released. The enzyme activity in control macrophage medium was inhibited mainly by PMSF and calpain inhibitors, while that from PMA-treated macrophages was inhibited by calpain inhibitors only. An ICE inhibitor was ineffective in inhibiting activity in medium from PMA-treated cells undergoing apoptosis. Medium from microglia contained very little proteolytic activity, and this was not increased by PMA. Cultured macrophages showed little evidence of oxygen free radical release as measured by the TBARS procedure, and PMA had no effect. Microglia, on the other hand, produced higher levels of reactive oxygen species, with a further increase of 18% by PMA. Thus major functions of these phagocytic cells appear to be modulated by the protein kinase C pathway, although the two cell types show very different responses to an activator of this signal.Medical Student at the     

Attenuation of experimental autoimmune demyelination in complement-deficient mice

The exact mechanisms leading to CNS inflammation and myelin destruction in multiple sclerosis and in its animal model, experimental allergic encephalomyelitis (EAE) remain equivocal. In both multiple sclerosis and EAE, complement activation is thought to play a pivotal role by recruiting inflammatory cells, increasing myelin phagocytosis by macrophages, and exerting direct cytotoxic effects through the deposition of the membrane attack complex on oligodendrocytes. Despite this assumption, attempts to evaluate complement's contribution to autoimmune demyelination in vivo have been limited by the lack of nontoxic and/or nonimmunogenic complement inhibitors. In this report, we used mice deficient in either C3 or factor B to clarify the role of the complement system in an Ab-independent model of EAE. Both types of complement-deficient mice presented with a markedly reduced disease severity. Although induction of EAE led to inflammatory changes in the meninges and perivascular spaces of both wild-type and complement-deficient animals, in both C3(-/-) and factor B(-/-) mice there was little infiltration of the parenchyma by macrophages and T cells. In addition, compared with their wild-type littermates, the CNS of both C3(-/-) and factor B(-/-) mice induced for EAE are protected from demyelination. These results suggest that complement might be a target for the therapeutic treatment of inflammatory demyelinating diseases of the CNS.     

Quantifying complement-mediated phagocytosis by human monocyte-derived macrophages

The present study demonstrates that SRBC can be opsonized with untreated human serum such that lysis by active complement components is minimal but sufficient opsonization occurs to permit high rates of complement-mediated phagocytosis. Phagocytosis of SRBC opsonized with 2% whole human serum by human monocyte-derived macrophages was quantified in a colourimetric assay. Ingestion of SRBC was shown to occur solely via complement receptors because no phagocytosis was observed when SRBC were coated with heat- inactivated human serum, phagocytosis was augmented by the phorbol ester, PMA, and phagocytosis was inhibited by a protein kinase C (PKC)-specific inhibitor RO 31-8220. This method was used to demonstrate directly that HIV-1 infection of human monocyte-derived macrophages inhibits complement-mediated phagocytosis and will provide a useful tool for pharmacological investigations on complement-mediated phagocytosis by adherent macrophages.     

Phagocytosis of polymorphonuclear leukocytes by guinea pig peritoneal macrophages: effects of serum and temperature in vitro

Abstract The regulation of phagocytosis of neutrophils by peritoneal macrophages was studied in vitro. Peritoneal exudate cells (PECs) of guinea pigs were lavaged 15 h after the i.p. injection of thioglycollate medium and were cultured in chamberslides. When PECs were cultured in RPMI 1640 medium in the absence of serum, approximately 20% of the macrophages phagocytized autologous neutrophils during 48–72 h of culture. Addition of guinea pig serum to the culture (2.5–20% v/v) suppressed the extent of the phagocytosis. The suppression was induced by globulin-rich ammonium sulfate fractions of the serum. Sera from rat, mouse, hamster, horse or calf also suppressed the phagocytosis, but fetal bovine serum (FBS) supported the phagocytosis, which was inhibited by globulin-rich Cohn fractions of bovine serum. The rate of neutrophil-phagocytosing macrophages was proportional to the rate of the pyknotic change of neutrophils. At a high temperature (42°C), the autophagocytosis took place at 12 h of culture when fresh, but not heat-inactivated, autologous serum was added, implying that complement components may play a role in the hyperthermia-induced phagocytosis of neutrophils by macrophages. At 42°C, ingested neutrophils did not show the pyknotic changes, indicating that intact neutrophils were ingested by macrophages.     

Iron Is a Sensitive Biomarker for Inflammation in Multiple Sclerosis Lesions

MRI phase imaging in multiple sclerosis (MS) patients and in autopsy tissue have demonstrated the presence of iron depositions in white matter lesions.The accumulation of iron in some but not all lesions suggests a specific, potentially disease-relevant process, however; its pathophysiological significance remains unknown.Here, we explore the role of lesional iron in multiple sclerosis using multiple approaches: immunohistochemical examination of autoptic MS tissue, an in vitro model of iron-uptake in human cultured macrophages and ultra-highfield phase imaging of highly active and of secondary progressive MS patients.Using Perls'' stain and immunohistochemistry, iron was detected in MS tissue sections predominantly in non-phagocytosing macrophages/microglia at the edge of established, demyelinated lesions. Moreover, iron-containing macrophages but not myelin-laden macrophages expressed markers of proinflammatory (M1) polarization.Similarly, in human macrophage cultures, iron was preferentially taken up by non-phagocytosing, M1-polarized macrophages and induced M1 (super) polarization. Iron uptake was minimal in myelin-laden macrophages and active myelin phagocytosis led to depletion of intracellular iron.Finally, we demonstrated in MS patients using GRE phase imaging with ultra-highfield MRI that phase hypointense lesions were significantly more prevalent in patients with active relapsing than with secondary progressive MS.Taken together, our data provide a basis to interpret iron-sensitive GRE phase imaging in MS patients: iron is present in non-phagocytosing, M1-polarized microglia/macrophages at the rim of chronic active white matter demyelinating lesions. Phase imaging may therefore visualize specific, chronic proinflammatory activity in established MS lesions and thus provide important clinical information on disease status and treatment efficacy in MS patients.     

Macrophages in CNS Remyelination: Friend or Foe?

Hematogenous macrophages and resident brain microglia are agents of demyelination in multiple sclerosis (MS) and paradoxically may also participate in remyelination. In vitro studies have shown that macrophage enrichment of aggregate brain cultures promotes myelination per se and enhances the capacity to remyelinate following a demyelinating episode. It has been hypothesized that remyelination in MS is implemented by surviving dedifferentiated oligodendrocytes or by newly recruited progenitors that migrate, proliferate and synthesize myelin in response to signalling molecules in the local environment. We postulate that macrophage-derived cytokines or growth factors may directly or indirectly promote oligodendroglial proliferation and differentiation, contributing to myelin repair in inflammatory demyelinating disease.     

Depletion of Blood-Borne Macrophages Does Not Reduce Demyelination in Mice Infected with a Neurotropic Coronavirus

Mice infected with the neurotropic coronavirus mouse hepatitis virus strain JHM (MHV-JHM) develop a chronic demyelinating disease with symptoms of hindlimb paralysis. Histological examination of the brains and spinal cords of these animals reveals the presence of large numbers of activated macrophages/microglia. In two other experimental models of demyelination, experimental allergic encephalomyelitis and Theiler's murine encephalomyelitis virus-induced demyelination, depletion of hematogenous macrophages abrogates the demyelinating process. In both of these diseases, early events in the demyelinating process are inhibited by macrophage depletion. From these studies, it was not possible to determine whether infiltrating macrophages were required for late steps in the process, such as myelin removal. In this study, we show that when macrophages are depleted with either unmodified or mannosylated liposomes encapsulating dichloromethylene diphosphate, the amount of demyelination detected in MHV-infected mice is not affected. At a time when these cells were completely depleted from the liver, approximately equivalent numbers of macrophages were present in the spinal cords of control and drug-treated animals. These results suggest that blood-borne macrophages are not required for MHV-induced demyelination and also suggest that other cells, such as perivascular macrophages or microglia, perform the function of these cells in the presence of drug.     

Opsonization with Antimyelin Antibody Increases the Uptake and Intracellular Metabolism of Myelin in Inflammatory Macrophages

In most demyelinating diseases, macrophages are believed to be active agents of myelin destruction. In experimental encephalomyelitis, these cells appear to strip off and ingest the myelin lamellae, and myelin debris has been observed within the cell body. We show here in vitro conditions in which rat peritoneal macrophages phagocytose and metabolize CNS myelin lipids. Purified rat myelin, prelabeled in vivo with [14C]acetate, was incubated with preimmune serum or rabbit antiserum to rat CNS myelin and added to macrophage monolayers. Myelin opsonized with antimyelin antibodies was more readily phagocytosed and metabolized by cultured macrophages than untreated myelin or that preincubated with preimmune serum. In the presence of macrophages, levels of myelin polar lipids and cholesterol decreased, whereas radioactive cholesterol ester and triglyceride accumulated. Up to five times as much radioactive cholesterol ester and about twice as much triglyceride accumulated in macrophage cultures containing antibody-treated myelin as in cultures fed preimmune serum-treated myelin or in those incubated with untreated myelin. Both the fatty acid and the cholesterol from cholesterol ester contained radioactive label; therefore, both were derived at least partly from the radioactive myelin lipid. Antiserum to myelin purified from peripheral nerve was almost as effective as that to CNS myelin in stimulating cholesterol metabolism, whereas antiserum to galactocerebroside was about 70% as active. Antiserum to basic protein had less effect, whereas antiserum to the myelin-associated glycoprotein and proteolipid protein was inactive. Of the polar lipids, ethanolamine phosphatide was most degraded in both the antiserum- and preimmune serum-treated myelin, with the diacyl form and plasmalogen form degraded about equally. These experiments indicate that myelin-specific antibodies in inflammatory CNS lesions may participate in and stimulate macrophage-mediated demyelination.     

Phagocytic functions of microglial cells in the central nervous system and their importance in two neurodegenerative diseases: multiple sclerosis and Alzheimer’s disease

Microglial cells are the resident phagocytic cells of the central nervous system (CNS). They possess a wide range of receptors allowing them to identify and internalize numerous pathogens. We will discuss here the role of the most important receptors of microglia involved in non-opsonin-dependent phagocytosis (mannose receptor, β-glucan receptor, scavenger receptor) and that of receptors involved in the opsonin-dependent phagocytosis, namely the complement 3 (CR3) and the Fcγ receptors (FcγR). First, the molecular and cellular mechanisms induced when these receptors are conducting a phagocytic event are presented. In the second part, we will discuss the role these receptors may play in multiple sclerosis and Alzheimer’s disease, in the elimination by phagocytosis of myelin and beta amyloid peptide respectively. The first two authors contributed equally to this work.     

Differential response of macrophage subpopulations to myelin degradation in the injured rat sciatic nerve

Molecular mechanisms of myelin removal by macrophages were explored by examining the immunophenotypes of macrophages following injury of rat sciatic nerve, using a combined method of immunohistochemistry and confocal laser microscopy. In the crush injury model, the involvement in myelin clearance of a cytoplasmic antigen specific for monocytes/macrophages, ED1, was evident. The obvious recruitment of ED1-immunoreactive (-ir) cells was detected first at the crush injury site and then in the distal stump within which Wallerian degeneration had occurred. Double labelling revealed that the ED1-ir cells, except for monocyte-like round cells, always phagocytosed myelin basic protein-ir myelin debris. On the other hand, the expression of ED2, a surface antigen specific for resident macrophages, was significantly different; ED2-ir cells also increased while myelin removal was progressing from day 3 to day 7, but only some of the cells were engaged in myelin phagocytosis. The poor capacity of myelin phagocytosis by ED2-ir cells was supported by the transection model, in which the proximal stump was ligated to suppress regeneration. ED2 may be involved in events other than myelin removal, providing a local environment conducive to axonal regeneration. Our findings thus seem to suggest that ED1 is one of the most reliable markers for cells carrying out myelin phagocytosis, whereas ED2 may participate in entirely different functions. The expression of complement receptor type 3, OX42, was similar to that of ED1 in terms of the swift recruitment of immunopositive cells, their distribution with close association to myelin debris and their high phagocytotic capacity. This supports previously reported in vitro evidence that myelin phagocytosis by macrophages may be complement-mediated.     

Serum opsonic activity in rodent malaria: functional and immunochemical characteristics in vitro.

The functional and immunochemical characteristics of serum opsonic activity in rodent malaria were examined in the present study. Schizont- and late trophozoite-enriched populations of Plasmodium berghei-infected red blood cells (IRBC) were isolated on a Ficoll density-gradient and used in an in vitro phagocytosis system composed of serum and monolayer cultures of rat peritoneal macrophages. Hyperimmune serum augmented the phagocytosis of IRBC to a greater degree than did nonimmune serum. When either IRBC or macrophages were pre-incubated with serum, the phagocytosis-promoting factors acted on the IRBC rather than on the macrophages in a manner characteristic of serum opsonins. The opsonic activity was specific for IRBC since noninfected red blood cells were rarely phagocytized and were unable to absorb opsonic activity from serum. The opsonic activity of both hyperimmune and nonimmune sera was heat stable, and unaffected by agents known to inactivate or inhibit complement (cobra venom factor and ethylenediaminetetraacetic acid). Finally, the opsonic activity was identified in preparations of purified IgG isolated from both hyperimmune and nonimmune sera.     

NPP1 is responsible for potent extracellular ATP hydrolysis as NTPDase1 in primary cultured murine microglia

The movement of microglia is regulated mainly by P1 and P2 purinergic receptors, which are activated by various nucleotides and their metabolites. Recently, such purinergic signalling has been spotlighted because of potential roles in the pathophysiologies of neurodegenerative and neuropsychiatric disorders. To understand the characteristics of microglia in relation of P1 and P2 signalling, we investigated the ectoenzymes expressed in microglia. At first, we profiled the expression of all known ectoenzymes in cultured microglia. We found that, like NTPDase1 (ectonucleoside triphosphate diphosphohydrolase 1, CD39), NPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1, PC-1) is also highly expressed in primary cultured murine microglia. Knockdown of NPP1 significantly reduced ATP hydrolysis and P_i production in cultured microglia. In addition, the knockdown of NPP1 enhanced basal nucleotide-stimulating responses of cultured microglia, such as phagocytosis and cell migration, and these results were very similar to NTPDase1 knockdown results. Moreover, inhibition of the adenosine receptors by caffeine treatment reduced phagocytosis of NPP1 knock downed-cultured microglia. In conclusion, we suggest that these potent ectoenzymes of primary cultured murine microglia, NPP1 together with CD73 (ecto-5′-nucleotidase) maintain the adenosine levels for triggering nucleotide-stimulating responses.     

Direct activation of innate and antigen-presenting functions of microglia following infection with Theiler's virus

Microglia are resident central nervous system (CNS) macrophages. Theiler's murine encephalomyelitis virus (TMEV) infection of SJL/J mice causes persistent infection of CNS microglia, leading to the development of a chronic-progressive CD4(+) T-cell-mediated autoimmune demyelinating disease. We asked if TMEV infection of microglia activates their innate immune functions and/or activates their ability to serve as antigen-presenting cells for activation of T-cell responses to virus and endogenous myelin epitopes. The results indicate that microglia lines can be persistently infected with TMEV and that infection significantly upregulates the expression of cytokines involved in innate immunity (tumor necrosis factor alpha, interleukin-6 [IL-6], IL-18, and, most importantly, type I interferons) along with upregulation of major histocompatibility complex class II, IL-12, and various costimulatory molecules (B7-1, B7-2, CD40, and ICAM-1). Most significantly, TMEV-infected microglia were able to efficiently process and present both endogenous virus epitopes and exogenous myelin epitopes to inflammatory CD4(+) Th1 cells. Thus, TMEV infection of microglia activates these cells to initiate an innate immune response which may lead to the activation of naive and memory virus- and myelin-specific adaptive immune responses within the CNS.     

Accelerated phagocytosis of amyloid-beta by mouse and human microglia overexpressing the macrophage colony-stimulating factor receptor

Microglia surrounding A beta plaques in Alzheimer's disease and in the APPV717F transgenic mouse model of Alzheimer's disease have enhanced immunoreactivity for the macrophage colony-stimulating factor receptor (M-CSFR), encoded by the proto-oncogene c-fms. Increased expression of M-CSFR on cultured microglia results in proliferation and release of pro-inflammatory cytokines and expression of inducible nitric-oxide synthase. We transfected mouse BV-2 and human SV-A3 microglia to overexpress M-CSFR and examined microglial phagocytosis of fluorescein-conjugated A beta. Flow cytometry and laser confocal microscopy showed accelerated phagocytosis of A beta in mouse and human microglia because of M-CSFR overexpression that was time- and concentration-dependent. In contrast, microglial uptake of 1-microm diameter polystyrene microspheres was not enhanced by M-CSFR overexpression. Microglial uptake of A beta was blocked by cytochalasin D, which inhibits phagocytosis. M-CSFR overexpression increased the mRNA for macrophage scavenger receptor A, and fucoidan blocking of macrophage scavenger receptors inhibited uptake of A beta. M-CSFR antibody blocking experiments demonstrated that increased A beta uptake depended on the interaction of the M-CSFR with its ligand. These results suggest that overexpression of M-CSFR in APPV717F mice may prime microglia for phagocytosis of A beta after immunization.     

Macrophage hydrogen peroxide production and phagocytic function are decreased following phagocytosis mediated by Fc receptors but not complement receptors

Previous in vivo and in vitro studies have shown that the phagocytosis of IgG-coated erythrocytes results in a depression of macrophage function. The present study compared the effect of phagocytosis mediated by Fc receptors with that mediated by complement receptors. The phagocytosis of IgG-coated erythrocytes by elicited peritoneal macrophages depressed their capacity to produce hydrogen peroxide as well as phagocytic function. Phagocytosis of erythrocytes coated with IgM and complement had neither of these effects. These results implicate the intracellular signaling that results from Fc receptor mediated phagocytosis in the depression of macrophage function that is caused by phagocytosis.     

Effect of antilysosomal sera on the phagocytosis of corpuscular antigens in a macrophage culture]

A study was made of the effect of the antilysosomal sera on various phases of phagocytosis Macrophages fluorochromized with acridine orange served as a model for the assessment of the effect of the antilysosomal sera on the phagocytosis: the fluorescent-microscopic method permitted to evaluate quantitatively the activity of the antilysosomal sera and to study the intracellular changes in the phagocytized antigen associated with its interaction with the lysosomes. The data obtained showed the antisera to the enzymes and the lysosome membranes to inhibit the phagocytosis both in the presence of a complement and without it. It was also demonstrated that the antilysosomal sera influenced the activity of the acid phosphatase and its distribution in the macrophages.