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.     

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.     

Epitope spreading initiates in the CNS in two mouse models of multiple sclerosis

Chronic progression of two T cell-mediated central nervous system (CNS) demyelinating models of multiple sclerosis, relapsing EAE (R-EAE) and Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) is dependent on the activation of T cells to endogenous myelin epitopes (epitope spreading). Using transfer of carboxyfluorescein succinyl ester (CFSE)-labeled T-cell receptor (TCR)-transgenic T cells and mixed bone marrow chimeras, we show that activation of naive proteolipid protein (PLP)139-151-specific T cells in SJL mice undergoing PLP178-191-induced R-EAE or TMEV-IDD occurs directly in the CNS and not in the cervical lymph nodes or other peripheral lymphoid organs. Examination of the antigen-presentation capacity of antigen-presenting cell (APC) populations purified from the CNS of mice with PLP178-191-induced R-EAE shows that only F4/80-CD11c+CD45hi dendritic cells (DCs) efficiently present endogenous antigen to activate naive PLP139-151-specific T cells in vitro. In contrast, DCs as well as F4/80+CD45hi macrophages and F4/80+CD45lo microglia activate a PLP139-151-specific helper T cell line. The data suggest that naive T cells enter the inflamed CNS and are activated by local APCs, possibly DCs, to initiate epitope spreading.     

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.     

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     

A Role for Complement in Phagocytosis of Myelin

The mechanisms for phagocytosis of myelin in cell-mediated demyelinating diseases have not been clarified. We have previously shown with cultured phagocytic cells that myelin opsonized with antiserum to myelin constituents is phagocytized in much higher amounts than untreated myelin, indicating that Fc receptors may be involved in the demyelinating process. Using various treatments of antisera, such as heating to destroy complement, and purification of IgG, we show here that complement is a necessary factor for maximal myelin phagocytosis by cultured macrophages. If myelin is sonicated to decrease its particle size, however, complement is not an active factor. Cultured microglia, on the other hand, required complement for maximal phagocytosis of both unsonicated and sonicated myelin. Addition of serum complement greatly increased phagocytosis of untreated CNS and PNS myelin, both unsonicated and sonicated, by macrophages and microglia. From these results it appears that the most important effect of complement is to fragment the myelin, making it more easily phagocytized. Prefragmentation of myelin by sonication can substitute for complement. Complement receptors may, in addition, be important for maximal myelin phagocytosis by microglia.This work was done at the VA Medical Center in fulfillment of the research requirement at the University of Amsterdam     

Upregulation of Myelin Gene Expression by a Physically-Modified Saline via Phosphatidylinositol 3-Kinase-Mediated Activation of CREB: Implications for Multiple Sclerosis

An increase in central nervous system (CNS) remyelination and a decrease in CNS inflammation are important steps to halt the progression of multiple sclerosis (MS). RNS60 is a bioactive aqueous solution generated by subjecting normal saline to Taylor–Couette–Poiseuille flow under elevated oxygen pressure. Recently we have demonstrated that RNS60 exhibits anti-inflammatory properties. Here, we describe promyelinating property of RNS60. RNS60, but not normal saline (NS), RNS10.3 (TCP-modified saline without excess oxygen) or PNS60 (saline containing excess oxygen without TCP modification), stimulated the expression of myelin-specific genes and proteins (myelin basic protein, MBP; myelin oligodendrocyte glycoprotein, MOG and proteolipid protein, PLP) in primary mouse oligodendroglia and mixed glial cells. While investigating the mechanisms, we found that RNS60 treatment induced the activation of cAMP response element binding protein (CREB) in oligodendrocytes, ultimately leading to the recruitment of CREB to the promoters of myelin-specific genes. Furthermore, activation of type 1A p110β/α, but not type 1B p110γ, phosphatidylinositol-3 (PI-3) kinase by RNS60 together with abrogation of RNS60-mediated activation of CREB and upregulation of myelin genes by LY294002 (a specific inhibitor of PI-3 kinase) suggest that RNS60 upregulates the activation of CREB and the expression of myelin-specific molecules in oligodendrocytes via activation of PI3 kinase. These results highlight a novel promyelinating property of RNS60, which may be of benefit for MS and other demyelinating disorders.     

The role of antigen presenting cells in multiple sclerosis

Multiple sclerosis (MS) is a debilitating T cell mediated autoimmune disease of the central nervous system (CNS). Animal models of MS, such as experimental autoimmune encephalomyelitis (EAE) and Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) have given light to cellular mechanisms involved in the initiation and progression of this organ-specific autoimmune disease. Within the CNS, antigen presenting cells (APC) such as microglia and astrocytes participate as first line defenders against infections or inflammation. However, during chronic inflammation they can participate in perpetuating the self-destructive environment by secretion of inflammatory factors and/or presentation of myelin epitopes to autoreactive T cells. Dendritic cells (DC) are also participants in the presentation of antigen to T cells, even within the CNS. While the APCs alone are not solely responsible for mediating the destruction to the myelin sheath, they are critical players in perpetuating the inflammatory milieu. This review will highlight relevant studies which have provided insight to the roles played by microglia, DCs and astrocytes in the context of CNS autoimmunity.     

Phagocytosis of Microglia in the Central Nervous System Diseases

Microglia, the resident macrophages of the central nervous system, rapidly activate in nearly all kinds of neurological diseases. These activated microglia become highly motile, secreting inflammatory cytokines, migrating to the lesion area, and phagocytosing cell debris or damaged neurons. During the past decades, the secretory property and chemotaxis of microglia have been well-studied, while relatively less attention has been paid to microglial phagocytosis. So far there is no obvious concordance with whether it is beneficial or detrimental in tissue repair. This review focuses on phagocytic phenotype of microglia in neurological diseases such as Alzheimer’s disease, multiple sclerosis, Parkinson’s disease, traumatic brain injury, ischemic and other brain diseases. Microglial morphological characteristics, involved receptors and signaling pathways, distribution variation along with time and space changes, and environmental factors that affecting phagocytic function in each disease are reviewed. Moreover, a comparison of contributions between macrophages from peripheral circulation and the resident microglia to these pathogenic processes will also be discussed.     

Myelin-associated oligodendrocytic basic protein: identification of an encephalitogenic epitope and association with multiple sclerosis

Myelin-associated oligodendrocytic basic protein (MOBP) is an abundant myelin constituent expressed exclusively by oligodendrocytes, the myelin-forming cells of the CNS. We report that MOBP causes experimental allergic encephalomyelitis and is associated with multiple sclerosis. First, we note that purified recombinant MOBP inoculated into SJL/J mice produces CNS disease. Tests of overlapping peptides spanning the murine MOBP molecule map the encephalitogenic site to amino acids 37-60. MOBP-induced experimental allergic encephalomyelitis shows a severe clinical course and is characterized by a prominent CD4+ T lymphocyte infiltration and a lesser presence of CD8+ T cells and microglia/macrophages around vessels and in the white matter of the CNS. Second, PBL obtained from patients with relapsing/remitting multiple sclerosis mount a proliferative response to human MOBP, especially at amino acids 21-39. This response equals or exceeds the response to myelin basic protein and an influenza virus hemagglutinin peptide, both serving as internal controls. Thus, a novel myelin Ag, MOBP aa 37-60, plays a role in rodent autoimmune CNS disease, and its human MOBP counterpart is associated with the human demyelinating disease multiple sclerosis.     

Comparison of a Chemically Mediated and an Immunologically Mediated Demyelinating Lesion Model

The production of two animal models for the central nervous system degenerative condition multiple sclerosis is described in detail. The first is a chemically mediated noninflammatory demyelinating lesion of the brain stem induced by the injection of a trypanocidal DNA binding dye, ethidium bromide, into the cerebellomedullary cistern. The injection does not involve any physical damage to the blood–brain barrier or the CSF–brain barrier and is simple to perform. The second lesion model is an immunologically mediated demyelinating condition involving the injection of a T-cell line specific for myelin basic protein, followed by injection of a monoclonal antibody against the myelin surface protein, myelin/oligodendrocyte glycoprotein. We describe the production of the antigen-specific T-cell line in detail. This model is characterized by widespread inflammatory infiltrates accompanied by areas of demyelination. Both of these models are produced in the Lewis rat, allowing the direct comparison of mechanisms involved in demyelination and repair in the presence or absence of invading inflammatory cells. Despite the very different etiologies of the two lesion models, they are both acute and result in efficient remyelination.     

Targeted expression of baculovirus p35 caspase inhibitor in oligodendrocytes protects mice against autoimmune-mediated demyelination

The mechanisms underlying oligodendrocyte (OLG) loss and the precise roles played by OLG death in human demyelinating diseases such as multiple sclerosis (MS), and in the rodent model of MS, experimental autoimmune encephalomyelitis (EAE), remain to be elucidated. To clarify the involvement of OLG death in EAE, we have generated transgenic mice that express the baculovirus anti-apoptotic protein p35 in OLGs through the Cre-loxP system. OLGs from cre/p35 transgenic mice were resistant to tumor necrosis factor-alpha-, anti-Fas antibody- and interferon-gamma-induced cell death. cre/p35 transgenic mice were resistant to EAE induction by immunization with the myelin oligodendrocyte glycoprotein. The numbers of infiltrating T cells and macrophages/microglia in the EAE lesions were significantly reduced, as were the numbers of apoptotic OLGs expressing the activated form of caspase-3. Thus, inhibition of apoptosis in OLGs by p35 expression alleviated the severity of the neurological manifestations observed in autoimmune demyelinating diseases.     

Myelin-induced microglial neurotoxicity can be controlled by microglial metabotropic glutamate receptors

Microglia are present in an activated state in multiple sclerosis lesions. Incubation of primary cultured rat microglia with rat-brain derived myelin (0.1–1 μg/mL) for 24 h induced microglial activation; cells displayed enhanced ED1 staining, expression of inducible nitric oxide synthase, production and release of the cytokine tumour necrosis factor-α and glutamate release. Exposure of microglia to myelin induced the expression of neuronal caspases and ultimately neuronal death in cultured cerebellar granule cell neurons; neurotoxicity was directly because of microglial-derived soluble toxins. Co-incubation of microglia with agonists or antagonists of different metabotropic glutamate receptor (mGluR) subtypes ameliorated microglial neurotoxicity by inhibiting soluble neurotoxin production. Activation of microglial mGluR2 exacerbated myelin-evoked neurotoxicity whilst activation of mGluR3 was protective as was activation of group III mGluRs. These data show that myelin-induced microglial neurotoxicity can be prevented by regulation of mGluRs and suggest these receptors on microglia may be promising targets for therapeutic intervention in multiple sclerosis.     

Iron and copper in progressive demyelination – New lessons from Skogholt's disease

The pathophysiological mechanisms of progressive demyelinating disorders including multiple sclerosis are incompletely understood. Increasing evidence indicates a role for trace metals in the progression of several neurodegenerative disorders. The study of Skogholt disease, a recently discovered demyelinating disease affecting both the central and peripheral nervous system, might shed some light on the mechanisms underlying demyelination. Cerebrospinal fluid iron and copper concentrations are about four times higher in Skogholt patients than in controls. The transit into cerebrospinal fluid of these elements from blood probably occurs in protein bound form. We hypothesize that exchangeable fractions of iron and copper are further transferred from cerebrospinal fluid into myelin, thereby contributing to the pathogenesis of demyelination. Free or weakly bound iron and copper ions may exert their toxic action on myelin by catalyzing production of oxygen radicals. Similarities to demyelinating processes in multiple sclerosis and other myelinopathies are discussed.     

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)¹. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)². Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers³. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation⁴ and pathologies such as inflammation⁵. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages⁶ and microglia⁷. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer''s disease and brain tumors.     

IL-17A Promotes Granulocyte Infiltration,Myelin Loss,Microglia Activation,and Behavioral Deficits During Cuprizone-Induced Demyelination

Recent evidence suggests a pivotal role of the proinflammatory cytokine interleukin - 17A (IL-17) in demyelinating autoimmune diseases of the central nervous system (CNS) such as multiple sclerosis (MS). Nevertheless, it remains unclear if this cytokine exerts direct effects on CNS resident cells during MS or modulates the function of infiltrating immune cells towards a more detrimental phenotype. Here, we investigated the effects of locally produced IL-17 during experimental demyelination of the CNS using the cuprizone (CPZ) model in mice with (GF/IL17) or without transgenic production of IL-17 by astrocytes in the CNS. During early demyelination, GF/IL17 mice demonstrated enhanced activity and decreased anxiety-related behavior in the elevated plus maze suggesting a more severe disease course. Furthermore, in GF/IL17 mice, toxic demyelination was accelerated and synthesis of myelin proteins was reduced. Early demyelination was accompanied by an increased ratio of infiltrating granulocytes in GF/ILl17 mice. The presence of IL-17 during CPZ treatment increased the accumulation of activated microglia and sustained microglial proliferation during myelin loss. Taken together, our results argue for a detrimental role of IL-17 during demyelinating diseases.     

Experimental Demyelination and Remyelination of Murine Spinal Cord by Focal Injection of Lysolecithin

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system characterized by plaque formation containing lost oligodendrocytes, myelin, axons, and neurons. Remyelination is an endogenous repair mechanism whereby new myelin is produced subsequent to proliferation, recruitment, and differentiation of oligodendrocyte precursor cells into myelin-forming oligodendrocytes, and is necessary to protect axons from further damage. Currently, all therapeutics for the treatment of multiple sclerosis target the aberrant immune component of the disease, which reduce inflammatory relapses but do not prevent progression to irreversible neurological decline. It is therefore imperative that remyelination-promoting strategies be developed which may delay disease progression and perhaps reverse neurological symptoms. Several animal models of demyelination exist, including experimental autoimmune encephalomyelitis and curprizone; however, there are limitations in their use for studying remyelination. A more robust approach is the focal injection of toxins into the central nervous system, including the detergent lysolecithin into the spinal cord white matter of rodents. In this protocol, we demonstrate that the surgical procedure involved in injecting lysolecithin into the ventral white matter of mice is fast, cost-effective, and requires no additional materials than those commercially available. This procedure is important not only for studying the normal events involved in the remyelination process, but also as a pre-clinical tool for screening candidate remyelination-promoting therapeutics.     

CD36 null mice reveal a key role for CD36 in β-amyloid induced microglial activation

Microglia and macrophages are important antigen-presenting cells (APCs) in the central nervous system (CNS). By virtue of their ability to express class II MHC antigens and costimulatory molecules such as CD40 and B7, microglia/macrophages promote Th1 cell activation and subsequent immune/inflammatory responses within the CNS. We have previously demonstrated that IFN-γ is the most potent inducer of CD40 expression on microglia. Our more recent studies have focused on the molecular basis of IFN-γ induced CD40 expression, and mechanisms by which this gene can be inhibited. The suppressive effects of IL-4 on CD40 expression will be discussed, as will the involvement of SH2 containing proteins called SOCS (for Suppressors Of Cytokine Signalling). Expression of CD40 by activated microglia/macrophages may contribute to the complex neuroimmunologic cascades that result in inflammation, demyelination and neuronal dysfunction. As such, understanding the mechanisms of inhibition of this molecule will be beneficial in diseases such as multiple sclerosis, HIV-1 associated dementia and Alzheimer's disease.