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Keywords:

  • microglia;
  • immune effectors;
  • phagocytosis;
  • cytokines;
  • neurogenesis;
  • disease;
  • gene therapy

Abstract

  1. Top of page
  2. Abstract
  3. HISTORICAL VIEW OF MICROGLIA
  4. MICROGLIA AS PHAGOCYTIC CELLS OF THE CNS
  5. SURFACE ANTIGENS SPECIFIC FOR MICROGLIA
  6. MICROGLIAL PRODUCTION OF CYTOKINES AND CHEMOKINES
  7. REGULATION OF CYTOKINE PRODUCTION IN MICROGLIA
  8. HUMAN MICROGLIA ARE NOT THE NITRIC OXIDE PRODUCER
  9. MICROGLIA AS PRODUCERS OF NEUROTROPHIC MOLECULES
  10. MICROGLIA AND NEUROGENESIS
  11. MICROGLIAL PROLIFERATION INDUCED BY CYTOKINES
  12. PERMANENT STABLE CELL LINES OF MICROGLIA
  13. MICROGLIA AND NEUROLOGIC DISEASES
  14. MICROGLIA AS A VEHICLE FOR TRANGENE IN GENE THERAPY
  15. REFERENCES

Microglia, one of three glial cell types in the central nervous system (CNS), play an important role as resident immunocompetent and phagocytic cells in the CNS in the event of injury and disease. It was del Rio Hortega in 1927 who determined that microglia belong a distinct glial cell type apart from astrocytes and oligodendrocytes, and since 1970s there has been wide recognition that microglia are immune effectors in the CNS that respond to pathological conditions and participate in initiation and progression of neurological disorders including Alzheimer's disease, Parkinson's disease, multiple sclerosis, and acquired immune deficiency syndrome dementia complex by releasing potentially cytotoxic molecules such as proinflammatory cytokines, reactive oxygen intermediates, proteinases and complement proteins. There is also evidence to suggest that microglia are capable of secreting neurotrophic or neuron survival factors upon activation via inflammation or injury. It is thus timely to review current status of knowledge on biology and immunology of microglia, and consider new directions of investigation on microglia in health and disease. © 2005 Wiley-Liss, Inc.

Microglia cells are a major glial cell element of the central nervous system (CNS), play a critical role as resident immunocompetent and phagocytic cells in the CNS (Perry and Gordon, 1988; Kreutzberg, 1996), and serve as scavenger cells in the event of infection, inflammation, trauma, ischemia, and neurodegeneration in the CNS (Thomas, 1992; McGeer and McGeer, 1995; Gonzalez-Scarano and Baltuch, 1999).

Microglia have at various times been described as mesodermal, hematomonocytic, or ectodermal origin (Kitamura et al., 1984; Hickey and Kimura, 1988; Ashwell, 1990), but the prevailing view is that monocytes in the bloodstream enter brain during embryonic development and differentiate into brain resident microglia displaying many of the cell surface antigens found for macrophages. Activation of microglia is associated with cell transformation to phagocytes, capable of releasing potentially cytotoxic substances such as oxygen radicals, proteases, and proinflammatory cytokines (Colton and Gilbert, 1987; Banati et al., 1993). Recent studies have indicated that activation of microglia precedes or is concomitant with neuronal and glial cell degeneration in neurologic disorders including Alzheimer's disease (AD; McGeer and McGeer, 1995), Parkinson's disease (PD; McGeer et al., 1993), multiple sclerosis (MS; Bo et al., 1994), and acquired immune deficiency syndrome dementia complex (AIDS-DC; Dickson et al., 1991; Gelman, 1993).

HISTORICAL VIEW OF MICROGLIA

  1. Top of page
  2. Abstract
  3. HISTORICAL VIEW OF MICROGLIA
  4. MICROGLIA AS PHAGOCYTIC CELLS OF THE CNS
  5. SURFACE ANTIGENS SPECIFIC FOR MICROGLIA
  6. MICROGLIAL PRODUCTION OF CYTOKINES AND CHEMOKINES
  7. REGULATION OF CYTOKINE PRODUCTION IN MICROGLIA
  8. HUMAN MICROGLIA ARE NOT THE NITRIC OXIDE PRODUCER
  9. MICROGLIA AS PRODUCERS OF NEUROTROPHIC MOLECULES
  10. MICROGLIA AND NEUROGENESIS
  11. MICROGLIAL PROLIFERATION INDUCED BY CYTOKINES
  12. PERMANENT STABLE CELL LINES OF MICROGLIA
  13. MICROGLIA AND NEUROLOGIC DISEASES
  14. MICROGLIA AS A VEHICLE FOR TRANGENE IN GENE THERAPY
  15. REFERENCES

CNS is composed of two major types of cells, nerve cells and glial cells, and glial cells are consisted of astrocytes, oligodendrocytes, and microglia. The microglia as a distinct cell type was first recognized by Nissl who named them Staebchenzellen (rod cells) for their rod-shaped nuclei and considered them as reactive neuroglia (Nissl, 1899). It was del Rio Hortega, however, who advanced microglia as a distinct cell type apart from astrocytes and oligodendrocytes after his studies of brains from young animals using his silver carbonate staining method (del Rio Hortega, 1927). He believed that microglia originate from mononuclear cells of the circulating blood and have the ability to transform from resting ramified form into ameboid macrophages (del Rio Hortega, 1932). Currently, it is a general view that the microglia are derived from circulating monocytes or precursor cells in the monocyte–macrophage lineage that originates in bone marrow. These precursor cells invade the developing brain during the embryonic, fetal, or perinatal stages, and they transform from actively phagocytic globoid–ameboid form into resting ramified form of microglia of the normal mature CNS (Ling et al., 1980; Barron, 1982; Perry et al., 1985; Hickey and Kimura, 1988). There have been others who have proposed microglia of non-monocyte–macrophage lineage origin (Kitamura et al., 1984; Schelper and Adrian, 1980).

Microglia were defined originally as a distinct cell type in the CNS based solely on in situ morphology. According to the classic morphologic studies based on silver carbonate staining technique, microglia cells were divided into three types: ameboid, ramified, and intermediate forms (del Rio Hortega, 1932; Kershman, 1939; Fig. 1 and 2). Recent studies have also adopted similar classification of microglia into three classes to describe their activation status: resting microglia, activated microglia, and ameboid phagocytic microglia (Streit et al., 1989; Sasaki et al., 1993). In the CNS, ramified resting microglia constitute 5–20% of the neuroglial population, are less numerous in white matter than in gray matter, and they modify their morphology and expression of cell surface antigens according to their microenvironment (Lawson et al., 1990, 1992).

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Figure 1. Human microglial cells demonstrated by del Rio Hortega silver carbonate staining method. Temporal cortex of 68-year-old male.

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Figure 2. Microglia represented by earlier investigators. Brain sections were prepared after the silver carbonate method of del Rio Hortega. A: del Rio Hortega (1932). B: Glees (1955). C: Penfield (1932). D: Kershman (1939) noted that the amoeboid form of microglia originates from cerebral capillary wall, penetrates into various layers of the brain tissue, where the cell body begins to send out branches until the final resting state is reached. His study of the origin of microglia was undertaken in 22 human embryo brains of 8–27 weeks.

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At the ultrastructural level, the microglial cytoplasm is electron-dense, cisterns of Golgi complexes are narrow and closely apposed, and there are few microtubules and intermediate filaments. Dense bodies are numerous and complex in structure, and these intracytoplasmic organelles are the cell type-specific marker of microglia at the ultrastructure level. The nuclear chromatin is clumped and located perinuclearly. The most characteristic structure of microglia is numerous filopodia and pseudopodia surrounding the cell surface, and well-developed dense bodies and vacuoles suggest their active phagocytotic activity (Peters et al., 1991; Fig. 3).

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Figure 3. Electron micrograph of human microglia in culture. Note numerous filopodia and pseudopodia surrounding the cell surface, and well-developed dense bodies and vacuoles in the cytoplasm. Cultures were prepared from human brain from a 62-year-old male.

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MICROGLIA AS PHAGOCYTIC CELLS OF THE CNS

  1. Top of page
  2. Abstract
  3. HISTORICAL VIEW OF MICROGLIA
  4. MICROGLIA AS PHAGOCYTIC CELLS OF THE CNS
  5. SURFACE ANTIGENS SPECIFIC FOR MICROGLIA
  6. MICROGLIAL PRODUCTION OF CYTOKINES AND CHEMOKINES
  7. REGULATION OF CYTOKINE PRODUCTION IN MICROGLIA
  8. HUMAN MICROGLIA ARE NOT THE NITRIC OXIDE PRODUCER
  9. MICROGLIA AS PRODUCERS OF NEUROTROPHIC MOLECULES
  10. MICROGLIA AND NEUROGENESIS
  11. MICROGLIAL PROLIFERATION INDUCED BY CYTOKINES
  12. PERMANENT STABLE CELL LINES OF MICROGLIA
  13. MICROGLIA AND NEUROLOGIC DISEASES
  14. MICROGLIA AS A VEHICLE FOR TRANGENE IN GENE THERAPY
  15. REFERENCES

Recently, cell culture techniques have provided new avenues of research in investigation of microglia. It is now possible to isolate microglia in mass from the brains of experimental animals and humans in the absence of any other contaminating cell types so that properties of microglia can be investigated in detail. Microglia cultures used most commonly were derived from newborn rat brain, and produced very high cytotoxic molecules including proinflammatory cytokines, reactive oxygen intermediates, proteinases, and complement proteins (Giulian and Baker, 1985; Rieske et al., 1989; Rogers et al., 1992; McGeer et al., 1993; McGeer and McGeer 1995; Gonzalez-Scarano and Baltuch, 1999). Microglia-enriched populations could be prepared from primary cultures derived from human fetal telencephalon by collecting microglial cells that float freely in the medium in culture flasks. The purity of microglia in these cultures is more than 99% as determined by surface staining by Ricinus communis agglutinin 1 lectin (RCA) or CD11b (Satoh and Kim, 1994, 1995; Lee et al., 1996, 2000, 2002; Nagai et al., 2001).

After incubation for 2 hr in medium containing latex beads (1 μm size), active phagocytotic activity of human microglia is apparent by showing cells laden with numerous bright beads (Fig. 4). In vivo, microglia cells phagocytize cellular debris during the prenatal and early postnatal stages of brain development (Ashwell, 1990; Pearson et al., 1993). Axonal degeneration and neuronal cell death occur as a genetically programmed event during the CNS development to eliminate overproduced neurons and glial cells (Cunningham, 1982; Oppenheim et al., 1990). The dead and dying neurons and their degenerating processes provide the stimulus for the monocyte–macrophage lineage cell invasion of the CNS. Heightened phagocytic activity of microglia in vivo has also been investigated in experimental models of facial nerve transaction and deafferented dentate gyrus in which microglia/macrophages remove cell debris by phagocytosis (Rieske et al., 1989; Streit et al., 1989).

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Figure 4. Human microglia in culture prepared from a 14-week-gestation fetal brain. A: Phase contrast microscopy of live human microglia. B: Human microglia actively incorporate latex beads by phagocytotic process. Scale bars = 20 μm.

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SURFACE ANTIGENS SPECIFIC FOR MICROGLIA

  1. Top of page
  2. Abstract
  3. HISTORICAL VIEW OF MICROGLIA
  4. MICROGLIA AS PHAGOCYTIC CELLS OF THE CNS
  5. SURFACE ANTIGENS SPECIFIC FOR MICROGLIA
  6. MICROGLIAL PRODUCTION OF CYTOKINES AND CHEMOKINES
  7. REGULATION OF CYTOKINE PRODUCTION IN MICROGLIA
  8. HUMAN MICROGLIA ARE NOT THE NITRIC OXIDE PRODUCER
  9. MICROGLIA AS PRODUCERS OF NEUROTROPHIC MOLECULES
  10. MICROGLIA AND NEUROGENESIS
  11. MICROGLIAL PROLIFERATION INDUCED BY CYTOKINES
  12. PERMANENT STABLE CELL LINES OF MICROGLIA
  13. MICROGLIA AND NEUROLOGIC DISEASES
  14. MICROGLIA AS A VEHICLE FOR TRANGENE IN GENE THERAPY
  15. REFERENCES

Microglia are immune effectors in the CNS. As such, they respond to any pathologic conditions involving immune system activation. They share phenotypic markers of monocyte–macrophage lineage and many of them are surface antigens/receptors with significant functional properties (Williams et al., 1992; McGeer and McGeer, 1995). They are β2-integrins (CD11a, CD11b, CD11c, and CD18), leukocyte common antigen (LCA; CD45), immunoglobulin Fcγ receptors, major histocompatibility complex (MHC) class I glycoproteins (HLA-ABC; β-2 microglobulin), and MHC class II glycoproteins (HLA-DR, HLA-DP, and HLA-DQ; Fig. 5). Microglia constitutively express β2-integrins (CD11a, CD11b, and CD11c); the ligand for CD11a is ICAM-1, whereas the ligands for CD11b and CD11c are complement proteins. LCA is a family of tyrosine protein phosphatase receptors that have a signal transduction function and are expressed exclusively nucleated cells of hematopoietic origin. Receptors for Fc chain of immunoglobulins are highly expressed in phagocytic cells including microglia. Because microglia are immunocompetent cells of the CNS, they express MHC class II antigens (Matsumoto and Fujiwara, 1986; Hickey and Kimura, 1988; Akiyama and McGeer, 1990; Woodroofe et al., 1991; McGeer et al., 1993). Microglia also express MHC class I antigens particularly in reactive microglia (Woodroofe et al., 1991; Tooyama et al., 1992), and some microglia express both class I and class II MHC antigens, and interact with T-helper/inducer (T4) and T-cytotoxic/suppressor (T8) classes of lymphocytes simultaneously.

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Figure 5. Immunohistochemical demonstration of cell type-specific markers for human microglia. Human microglia cultures were prepared from a 14-week-gestation fetal brain, grown for 2–4 weeks, and then processed for immunocytochemistry. A: CD11b (Mac1). B: CD11C (LeuM5). C: CD64 (FcγR immunoglobulin receptor). D: CD45 (leukocyte common antigen, LCA). E: HLA-ABC (MHC class I antigen). F: HLA-DR (MHC class II antigen).

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Phenotypic markers of human microglia grown in culture are shown in Figure 5 and represent cell type-specific markers of monocyte–macrophage lineage. They are β2-integrins CD11b (Mac1) and CD11c (Leu M5), immunoglobulin Fcγ receptors (CD64), leukocyte common antigen (LCA, CD45), MHC class I glycoproteins (HLA-ABC, β-2 microglobulin) and MHC class II glycoproteins (HLA-DR) (Fig. 5).

Microglia are known as antigen presenting cells (APCs) in the CNS by virtue of their expression of MHC class II antigens and pronounced phagocytic ability (Frei et al., 1987; McGeer and McGeer, 1995). Although a previous study has claimed that murine astrocytes primed with interferon-γ (IFN-γ) are capable of effectively presenting antigens to T cells (Fontana et al., 1984), in our experiments human astrocytes are poor candidate for APCs due to lack of costimulating signals, B7-1 or B7-2 (Satoh et al., 1995). B7-1 and B7-2 are expressed in a cytokine-regulated manner in human microglia but not in human astrocytes. B7 is a family of cell surface glycoproteins that are comprised of two major members of B7-1 (CD80) and B7-2 (CD86). The antigens are expressed only by fully immunocompetent APCs of the lymphoid system such as activated monocytes/macrophages, B cells, dendritic cells, epidermal Langerhans cells, and a subpopulation of T cells (June et al., 1994). These APCs play a major role in the initiation of T cell-mediated immune responses at the sites of inflammatory lesions in human. The expression of B7 on APCs is upregulated by various cytokines such as interleukin (IL)-2, IL-4, IFN-γ, and granulocyte macrophage-colony stimulating factor (GM-CSF) and is downregulated by IL-10 (Azuma et al., 1993; Ding et al., 1993). Previous studies have also reported that adult human microglia in culture also express B7 antigens and act as immunocompetent APCs (Williams et al., 1992; De Simone et al., 1995).

MICROGLIAL PRODUCTION OF CYTOKINES AND CHEMOKINES

  1. Top of page
  2. Abstract
  3. HISTORICAL VIEW OF MICROGLIA
  4. MICROGLIA AS PHAGOCYTIC CELLS OF THE CNS
  5. SURFACE ANTIGENS SPECIFIC FOR MICROGLIA
  6. MICROGLIAL PRODUCTION OF CYTOKINES AND CHEMOKINES
  7. REGULATION OF CYTOKINE PRODUCTION IN MICROGLIA
  8. HUMAN MICROGLIA ARE NOT THE NITRIC OXIDE PRODUCER
  9. MICROGLIA AS PRODUCERS OF NEUROTROPHIC MOLECULES
  10. MICROGLIA AND NEUROGENESIS
  11. MICROGLIAL PROLIFERATION INDUCED BY CYTOKINES
  12. PERMANENT STABLE CELL LINES OF MICROGLIA
  13. MICROGLIA AND NEUROLOGIC DISEASES
  14. MICROGLIA AS A VEHICLE FOR TRANGENE IN GENE THERAPY
  15. REFERENCES

Microglia, in common with other cells of monocyte–macrophage lineage, elaborate and secrete cytokines (Lee et al., 1993, 2002; Hanisch, 2002). Cytokines are low molecular-weight proteins and include ILs, IFNs, tissue necrosis factors (TNFs), colony stimulating factors (CSFs), and transforming growth factors (TGFs) among others. Cytokines participate in a wide range of biological responses, including growth, development, and modulation of inflammation and immune responses and regulation of homeostasis. Chemokines constitute a superfamily of small (8–10 kDa), inducible, secreted proinflammatory cytokines that are involved in a variety of immune and inflammatory responses, acting primarily as chemoattractants and activators of specific types of leukocytes. Three classes of chemokines have been defined by the arrangement of conserved cysteine (C) residues of the mature proteins: the C-X-C or α chemokines, the C-C or β chemokines, and the C or γ chemokines (Locati and Murphy, 1999; Murphy, 2001).

Previous studies reporting on the expression of cytokines/chemokines in microglial cells have dealt mostly with murine microglia and focused only on several selected cytokines (McDonald et al., 1998; Pyo et al., 1998; Sawada et al., 1999; Combs et al., 2001; Szczepanik et al., 2001). To understand the autocrine and paracrine regulation of cytokine/chemokine expression in the CNS, it is also important to identify in microglia what types of receptors that respond to each specific cytokine/chemokine in microglial cells. We have prepared enriched populations of human microglial cells from primary cultures of fetal human telencephalon tissues (Fig. 4 and 6A), and gene expression and protein production of cytokines, chemokines and receptors specific for cytokines/chemokines were investigated (Lee et al., 1996, 2000, 2002; Miyamoto and Kim, 1999; Nagai et al., 2001). Human microglia under nonstimulated conditions constitutively express transcripts for mRNAs of IL-1β, IL-6, IL-8, IL-10, IL-12, IL-15, TNF-α, macrophage inflammatory protein (MIP)-1α, MIP-1β, and MCP-1 (Fig. 6B). After stimulation with lipopolysaccharide (LPS), expression of all the cytokines/chemokines except IL-15 is markedly upregulated.

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Figure 6. RT-PCR studies of gene expression in human microglia cultures under normal nonstimulated conditions. Cultures were prepared from a 14-week-gestation fetal brain. A: Human microglia preparations express mRNA transcripts for CD68 and B7-2, both cell type-specific markers for microglia, but do not express neurofilament protein (NF-L; cell-type marker for neurons), glial fibrillary acidic protein (GFAP; cell-type marker for astrocytes), or myelin basic protein (MBP; cell-type marker for oligodendrocytes).The results indicate that the microglia preparations are consisted of pure populations of human microglia. B: Gene expression of chemokine receptor CXCR4 and chemokines macrophage inflammatory protein (MIP)-1α, MIP-1β, and MCP-1 in human microglia. Gene expression of cytokines (C) and cytokine receptors (D) in human microglia.

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Human microglial cells express mRNA transcripts for cytokine receptors IL-1RI, IL-1RII, IL-5R, IL-6R, IL-8R, IL-9R, IL-10R, IL-12R, IL-13R, IL-15R, TNFRI, and TNFRII, whereas expression of IL-2R, IL-3R, IL-4R, IL-7R or IL-11R was not detected (Fig. 6C). In addition, microglia express gp130, a common receptor component for IL-6, leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), and oncostatin M, as well as chemokine receptor CXCR4, which has drawn considerable attention as a coreceptor for human immunodeficiency virus (HIV) entry into human microglia (Doms and Peiper, 1997). It is noteworthy that the human microglia express receptors for cytokines IL-1, IL-6, IL-8, and TNF-α, indicating that human microglia could respond to and be activated by these proinflammatory cytokines via receptors and further propagate inflammatory reaction in the CNS. In addition, it is important that human microglia express receptors for immunomodulatory cytokines, IL-5R, IL-12R and IL-15R, and receptors for antiinflammatory cytokines IL-10R and IL-13R. Although an earlier study in mouse microglia reported the expression of transcripts for IL-2R, IL-3R, IL-4R, and IL-7R (Sawada et al., 1993), none of these receptors was found in human microglia. These opposing results could be attributed to the difference in species (human vs. mouse) or methods of microglial isolation. The pattern of cytokine expression in human microglia is in good agreement with general view that human microglia express not only inflammatory cytokines IL-1β, IL-6, IL-8, and TNF-α, but also express immunomodulatory cytokines IL-12 and IL-15, and antiinflammatory cytokine IL-10 at the same time. It is noteworthy that the pretreatment of human microglia with IL-10 is effective in blocking the LPS-mediated production of TNF-α. These results indicate that the activated microglia is a major cellular source of proinflammatory cytokines that cause inflammatory response in the CNS, but microglia also have an opposite function by producing IL-10 that inhibits inflammatory response of microglia via an autocrine feedback loop.

There is growing evidence that the overproduction of proinflammatory cytokines by CNS cells contributes to pathophysiologic changes seen in various neurologic diseases and brain injury and that the major source of these cytokines are microglia. Activated microglia are considered to play an important role in initiation and progression of neurodegenerative diseases (Dickson et al., 1991; Thomas, 1992; McGeer et al., 1993; Kreutzberg, 1996; Gonzalez-Scarano and Baltuch, 1999).

REGULATION OF CYTOKINE PRODUCTION IN MICROGLIA

  1. Top of page
  2. Abstract
  3. HISTORICAL VIEW OF MICROGLIA
  4. MICROGLIA AS PHAGOCYTIC CELLS OF THE CNS
  5. SURFACE ANTIGENS SPECIFIC FOR MICROGLIA
  6. MICROGLIAL PRODUCTION OF CYTOKINES AND CHEMOKINES
  7. REGULATION OF CYTOKINE PRODUCTION IN MICROGLIA
  8. HUMAN MICROGLIA ARE NOT THE NITRIC OXIDE PRODUCER
  9. MICROGLIA AS PRODUCERS OF NEUROTROPHIC MOLECULES
  10. MICROGLIA AND NEUROGENESIS
  11. MICROGLIAL PROLIFERATION INDUCED BY CYTOKINES
  12. PERMANENT STABLE CELL LINES OF MICROGLIA
  13. MICROGLIA AND NEUROLOGIC DISEASES
  14. MICROGLIA AS A VEHICLE FOR TRANGENE IN GENE THERAPY
  15. REFERENCES

The three mitogen-activated protein kinases (MAP kinases), extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), and p38 MAP kinase, differ in their responses to extracellular stimuli. ERKs are most responsive to growth factors and phorbol esters (Cobb and Goldsmith, 1995), whereas JNK and p38 MAP kinase are activated in response to stress signals, including UV irradiation, heat, osmotic stress, LPS, and proinflammatory cytokines such as IL-1β, IL-6, and TNF-α (Kyriakis et al., 1994; Raingeaud et al., 1995). A previous study has demonstrated that human microglia and astrocytes express TNF-α, but after treatment with LPS in microglia and IL-1β in astrocytes, high levels of TNF-α mRNA and secreted protein were demonstrated in these cells (Lee et al., 2000). These authors demonstrated that in the presence of a specific inhibitor of p38 MAP kinase expression of TNF-α was reduced markedly, indicating that p38 MAP kinase plays a key role in upregulation of TNF-α production in activated microglia. It is noteworthy that the amount of TNF-α secreted from LPS-stimulated microglia is tenfold that found in cytokine-activated astrocytes; thus, microglial cells are major players in immune response and major source of TNF-α in the CNS.

HUMAN MICROGLIA ARE NOT THE NITRIC OXIDE PRODUCER

  1. Top of page
  2. Abstract
  3. HISTORICAL VIEW OF MICROGLIA
  4. MICROGLIA AS PHAGOCYTIC CELLS OF THE CNS
  5. SURFACE ANTIGENS SPECIFIC FOR MICROGLIA
  6. MICROGLIAL PRODUCTION OF CYTOKINES AND CHEMOKINES
  7. REGULATION OF CYTOKINE PRODUCTION IN MICROGLIA
  8. HUMAN MICROGLIA ARE NOT THE NITRIC OXIDE PRODUCER
  9. MICROGLIA AS PRODUCERS OF NEUROTROPHIC MOLECULES
  10. MICROGLIA AND NEUROGENESIS
  11. MICROGLIAL PROLIFERATION INDUCED BY CYTOKINES
  12. PERMANENT STABLE CELL LINES OF MICROGLIA
  13. MICROGLIA AND NEUROLOGIC DISEASES
  14. MICROGLIA AS A VEHICLE FOR TRANGENE IN GENE THERAPY
  15. REFERENCES

Recent studies have indicated that nitric oxide (NO) generated intracerebrally in the brain contributes significantly to the physiology and pathophysiology of the nervous system (Moncada and Higgs, 1991; Murphy et al., 1993). NO is a signal molecule that diffuse freely across cell boundaries to activate target cells. There are two constitutively expressed nitric oxide synthase (NOS): neural NOS (nNOS) and endothelial NOS (eNOS). A third NOS isotype, inducible NOS (iNOS), which is absent in resting cells, can be induced in various cell types by stimulation with cytokines and LPS. Previous studies have reported that NO generated by iNOS in the brain causes injury and cell death of neurons and oligodendrocytes (Boje and Arora, 1992; Chao et al., 1992; Merrill et al., 1993), and that NO is implicated in pathogenesis of AD, PD, AIDS-DS, MS, and stroke (Hyman et al., 1992; Brosnan et al., 1994). Previous studies have reported that the major source of NO in the CNS is microglia, and microglia as such are responsible for injury and cell death of neurons and oligodendrocytes in AD, PD, and MS (Boje and Arora, 1992; Merrill et al., 1993). Interestingly, all these investigators employed in their studies microglial cells derived from adult or neonatal rats or mice. Primary microglial cells isolated from rat or mouse brain carry mRNA transcript for iNOS, the enzyme responsible for NO production, and are good producers of NO as determined by Griess reaction (Ding et al., 1988). We have investigated expression of iNOS mRNA transcript and NO production in purified populations of human microglia and astrocytes derived from human fetal telencephalon tissues. The results indicate that iNOS expression is absent in human microglia but well represented in human astrocytes. Similarly high levels of NO production are clearly found in conditioned media of human astrocytes but not in media taken from human microglia cultures (Kim, unpublished data). Microglia as the principal immune cells in the CNS are known as a source of cells producing very highly cytotoxic substances including proinflammatory cytokines, reactive oxygen intermediates, proteinases, and complement proteins (Rieske et al., 1989; Rogers et al., 1992; McGeer et al., 1993; Giulian et al., 1995). Unlike in microglia of murine origin, however, human microglial cells are not the NO producer at all.

MICROGLIA AS PRODUCERS OF NEUROTROPHIC MOLECULES

  1. Top of page
  2. Abstract
  3. HISTORICAL VIEW OF MICROGLIA
  4. MICROGLIA AS PHAGOCYTIC CELLS OF THE CNS
  5. SURFACE ANTIGENS SPECIFIC FOR MICROGLIA
  6. MICROGLIAL PRODUCTION OF CYTOKINES AND CHEMOKINES
  7. REGULATION OF CYTOKINE PRODUCTION IN MICROGLIA
  8. HUMAN MICROGLIA ARE NOT THE NITRIC OXIDE PRODUCER
  9. MICROGLIA AS PRODUCERS OF NEUROTROPHIC MOLECULES
  10. MICROGLIA AND NEUROGENESIS
  11. MICROGLIAL PROLIFERATION INDUCED BY CYTOKINES
  12. PERMANENT STABLE CELL LINES OF MICROGLIA
  13. MICROGLIA AND NEUROLOGIC DISEASES
  14. MICROGLIA AS A VEHICLE FOR TRANGENE IN GENE THERAPY
  15. REFERENCES

Microglia are now generally accepted as resident immunocompetent and phagocytic cells in the CNS (Perry and Gordon, 1988; McGeer and McGeer, 1995; Kreutzberg, 1996). There is growing evidence that the overproduction of proinflammatory cytokines by microglia contributes to pathophysiologic changes seen in various neurologic diseases and brain injury (Dickson et al., 1991; Thomas, 1992; McGeer et al., 1993; Gonzalez-Scarano and Baltuch, 1999). Previous studies have indicated that microglial cells are capable of secreting neurotrophic or neuron survival factors upon activation via inflammation or injury in the CNS (Nakajima and Kohsaka, 1993). A recent study has reported that primary microglia cultures derived from fetal mouse brain release soluble factors that direct migration of neural stem/precursor cells and promote differentiation of neural stem cells to neuronal fate, although the exact nature of the soluble factors is not known (Aarun et al., 2003). Neurotrophic molecules identified in microglia and microglia conditioned media include nerve growth factor (NGF) and neurotrophin (NT)3 (Elkabes et al., 1996), NGF and brain-derived neurotrophic factor (BDNF; Miwa et al., 1997; Elkabes et al., 1998), BDNF (Nakajima et al., 2001), basic fibroblast growth factor (bFGF; Shimojo et al., 1991), hepatocyte growth factor (HGF; Hamanoue et al., 1996), and plasminogen (Nakajima et al., 1993). In rat microglia preparations, members of the neurotrophin family of trophic factors are expressed in a region-specific manner so that NT-3 was identified in the cerebral cortex, striatum, and medulla but not in ependyma, external capsule, or choroids plexus (Elkabes et al., 1996). Because only subpopulations of rat microglia express NGF and NT-3 genes but not BDNF, as studied by in situ hybridization, these authors concluded that microglial subtypes serve a trophic role in normal brain. As it is important to identify signal transduction pathways involved in microglia production of trophic molecules, a recent study has suggested that the protein kinase C signaling cascade is associated closely with BDNF secretion in rat microglia culture (Nakajima et al., 2001). It is not known, however, why activated microglia should produce cytotoxic proinflammatory cytokines that are involved in progression of injury and disease in the brain and at the same time are capable of producing neurotrophic factors that support the survival of neurons.

MICROGLIA AND NEUROGENESIS

  1. Top of page
  2. Abstract
  3. HISTORICAL VIEW OF MICROGLIA
  4. MICROGLIA AS PHAGOCYTIC CELLS OF THE CNS
  5. SURFACE ANTIGENS SPECIFIC FOR MICROGLIA
  6. MICROGLIAL PRODUCTION OF CYTOKINES AND CHEMOKINES
  7. REGULATION OF CYTOKINE PRODUCTION IN MICROGLIA
  8. HUMAN MICROGLIA ARE NOT THE NITRIC OXIDE PRODUCER
  9. MICROGLIA AS PRODUCERS OF NEUROTROPHIC MOLECULES
  10. MICROGLIA AND NEUROGENESIS
  11. MICROGLIAL PROLIFERATION INDUCED BY CYTOKINES
  12. PERMANENT STABLE CELL LINES OF MICROGLIA
  13. MICROGLIA AND NEUROLOGIC DISEASES
  14. MICROGLIA AS A VEHICLE FOR TRANGENE IN GENE THERAPY
  15. REFERENCES

In the adult brain, hippocampus is the one of exceptional places to produce new neurons, throughout adult life as it contains neural progenitor cells (NPS). A question of whether the cognitive decline in patients with brain tumor who received irradiation treatment could be due to a reduction in hippocampal neurogenesis was investigated, and it was found that the irradiation blocks differentiation of neural stem cells in vivo but not in vitro (Monje et el., 2003). In a follow-up study, they demonstrated that the irradiation-induced damage in hippocampus was due to inflammatory response caused by activated microglia. Blocking inflammation elicited by either irradiation or injection of bacterial LPS in the rat brain with a nonsteroid antiinflammatory drug indomethacin restored hippocampal neurogenesis (Monje et al., 2003). In another study, hippocampal neurogenesis in adult rat was impaired by LPS-induced inflammation, but this inhibition of neurogenesis was reversed by minocycline, a drug that blocks microglial activation (Ekdahl et al., 2003). These studies indicate that the inflammatory mediators/cytokines released by microglia during an immune response to injury or disease strongly influence neurogenesis and their ability to function (Kempermann and Neumann, 2003). The next challenge for these investigators is to identify the exact molecule or molecules that cause this impairment of hippocampal neurogenesis. Neurogenesis was inhibited when neural stem cells were exposed to activated microglia in culture. Proinflammatory cytokine IL-6 was the key regulator of this inhibition, because an IL-6 antibody selectively blocked the IL-6 effect and restored hippocampal neurogenesis (Monje et al., 2003). During the brain inflammation in response to infection or physical damage such as irradiation, secretion of proinflammatory cytokines such as IL-1β, IL-6, and TNFα increases in activated microglia. IL-6, one of the proinflammatory cytokines, evidently interferes with neurogenesis of new neurons by perturbing the microenvironment of neural stem cells.

MICROGLIAL PROLIFERATION INDUCED BY CYTOKINES

  1. Top of page
  2. Abstract
  3. HISTORICAL VIEW OF MICROGLIA
  4. MICROGLIA AS PHAGOCYTIC CELLS OF THE CNS
  5. SURFACE ANTIGENS SPECIFIC FOR MICROGLIA
  6. MICROGLIAL PRODUCTION OF CYTOKINES AND CHEMOKINES
  7. REGULATION OF CYTOKINE PRODUCTION IN MICROGLIA
  8. HUMAN MICROGLIA ARE NOT THE NITRIC OXIDE PRODUCER
  9. MICROGLIA AS PRODUCERS OF NEUROTROPHIC MOLECULES
  10. MICROGLIA AND NEUROGENESIS
  11. MICROGLIAL PROLIFERATION INDUCED BY CYTOKINES
  12. PERMANENT STABLE CELL LINES OF MICROGLIA
  13. MICROGLIA AND NEUROLOGIC DISEASES
  14. MICROGLIA AS A VEHICLE FOR TRANGENE IN GENE THERAPY
  15. REFERENCES

Microglial proliferation and activation in the CNS after injury and infection are well documented, but little is known about how these processes are regulated. Cytokines secreted by microglia themselves and other CNS cell elements are involved in these processes. Cytokines such as IL-1β, IL-4, and IFN-γ have some effect on microglial proliferation in vitro, but the most effective mitogens for microglia are CSFs (Suzumura et al., 1990; Lee et al., 1994). Primary cultures of human fetal and adult microglia were investigated for their proliferation capacity and the results indicate that both macrophage-colony stimulating factor (M-CSF) and GM-CSF induced microglial proliferation both in fetal and adult microglia cultures, but GM-CSF was a much more effective mitogen (Lee et al., 1994). These authors also reported that LPS abolished proliferation in adult microglia but induced a weak increase in proliferation in fetal microglia. It is noteworthy that microglia are responsive to the members of NGF neurotrophin family, BDNF, and NT-3, and incorporate larger amount of tritiated-thymidine (Elkabes et al., 1996).

PERMANENT STABLE CELL LINES OF MICROGLIA

  1. Top of page
  2. Abstract
  3. HISTORICAL VIEW OF MICROGLIA
  4. MICROGLIA AS PHAGOCYTIC CELLS OF THE CNS
  5. SURFACE ANTIGENS SPECIFIC FOR MICROGLIA
  6. MICROGLIAL PRODUCTION OF CYTOKINES AND CHEMOKINES
  7. REGULATION OF CYTOKINE PRODUCTION IN MICROGLIA
  8. HUMAN MICROGLIA ARE NOT THE NITRIC OXIDE PRODUCER
  9. MICROGLIA AS PRODUCERS OF NEUROTROPHIC MOLECULES
  10. MICROGLIA AND NEUROGENESIS
  11. MICROGLIAL PROLIFERATION INDUCED BY CYTOKINES
  12. PERMANENT STABLE CELL LINES OF MICROGLIA
  13. MICROGLIA AND NEUROLOGIC DISEASES
  14. MICROGLIA AS A VEHICLE FOR TRANGENE IN GENE THERAPY
  15. REFERENCES

To investigate complex function of microglia, it is necessary to obtain sufficient numbers of homogenous populations of the cell type. Primary cultures of microglia obtained from mouse, rat, or human grow slowly and contaminated by other cell types such as fibroblasts or astrocytes. An ideal solution to circumvent this difficulty in securing pure populations of microglia is the generation of immortalized microglia cell lines that retain properties of their original counterparts.

Previously, many stably immortalized cell lines of CNS cell type have been produced using replication-defective retrovirus vectors encoding oncogenes and several mouse or rat microglia cell lines were generated (Righi et al., 1989; Blasi et al., 1990; Hosaka et al., 1992; McLaurin et al., 1995). These microglia cell lines and clones were positive for microglia/macrophage phenotypic markers including MHC class I and II, complement 3 receptor, and CD4 homologue antigen in short-term culture, but in long-term culture they showed a gradual downregulation of microglia cell-type markers (McLaurin et al., 1995). More recently, Nagai et al. (2001) have reported generation of an immortalized human microglia cell line using a retroviral vector encoding v-myc, and the cell line HMO6 shows cell type-specific antigens for microglia/macrophage lineage cells including CD11b (Mac-1), CD68, CD86 (B7-2), HLA I and II, and RCA-1, and the cells actively phagocytosed latex beads. This cell line, as in its normal human microglia counterpart, expresses cytokine gene of IL-1β, IL-6, IL-8, IL-10, IL-12, IL-15, and TNF-α. Treatment of this cell line with β amyloid (Aβ) peptides or bacterial LPS resulted in upregulation of gene expression and protein production of proinflammatory cytokines and chemokine in the cell line. The details of the immortalized human microglial cell line are found elsewhere in this journal issue (Nagai et al., 2005).

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Figure 7. Cytotoxic effect of amyloid peptide in human microglia. Cultures were prepared from a 14-week-gestation fetal brain. A: Control normal human microglia. B: Human microglia exposed to amyloid-β (Aβ)1–42 peptide for 12 hr. Extensive vacuolization indicates that microglia are undergoing degeneration.

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MICROGLIA AND NEUROLOGIC DISEASES

  1. Top of page
  2. Abstract
  3. HISTORICAL VIEW OF MICROGLIA
  4. MICROGLIA AS PHAGOCYTIC CELLS OF THE CNS
  5. SURFACE ANTIGENS SPECIFIC FOR MICROGLIA
  6. MICROGLIAL PRODUCTION OF CYTOKINES AND CHEMOKINES
  7. REGULATION OF CYTOKINE PRODUCTION IN MICROGLIA
  8. HUMAN MICROGLIA ARE NOT THE NITRIC OXIDE PRODUCER
  9. MICROGLIA AS PRODUCERS OF NEUROTROPHIC MOLECULES
  10. MICROGLIA AND NEUROGENESIS
  11. MICROGLIAL PROLIFERATION INDUCED BY CYTOKINES
  12. PERMANENT STABLE CELL LINES OF MICROGLIA
  13. MICROGLIA AND NEUROLOGIC DISEASES
  14. MICROGLIA AS A VEHICLE FOR TRANGENE IN GENE THERAPY
  15. REFERENCES

Microglia are the main immune effector cells of the CNS (McGeer et al., 1988; Perry and Gordon, 1988; Streit et al., 1988). They are involved in variety of human neurologic diseases including AD, PD, MS, AIDS-DC, and stroke (McGeer et al., 1988, 1993; Dickson et al., 1991; Carpenter et al., 1993; Lees, 1993; Gelman, 1993; Gonzales-Scarano and Baltuch, 1999). Microglia under these pathologic conditions are characterized by increases in proliferation, expression of surface antigens, migration, and finally phagocytic activity.

AD is characterized pathologically by the presence of senile plaques containing Aβ core and intraneuronal neurofibrillary tangles (NFTs) that chiefly consist of hyperphosphorylated tau protein in the AD brain. The presence of large numbers of activated microglia in the AD brain tissues indicates that microglia play a significant role in the progression of the disease. The senile plaques found in AD brains are surrounded by clusters of reactive microglia (McGeer et al., 1988; Permutter et al., 1992; Carpenter et al., 1993). Reactive microglia found closely associated with plaques in AD brain are strongly immunopositive for CD11a, CD11b, CD11c (members of the β2 integrin family), LCA (CD45), immunoglobulin Fcγ receptors (CD64), and MHC class II glycoprotein (HLA-DR; McGeer et al., 1988; Masliah et al., 1991; Peress et al., 1993; Akiyama et al., 1994; Mrak et al., 1995). In addition, expression of complement proteins C1–C4 in human microglia derived from AD brains has been reported previously (Walker et al., 1995). The presence of complement proteins and immunoglobulin receptors on microglia is associated with their role in phagocytosis of complement and immunoglobulin-opsonized tissue debris. The elevated secretion of proinflammatory cytokines from the reactive microglia in the vicinity of senile plaques or NFTs induces an increase in immunoglobulin Fcγ receptor expression in microglia in an autocrine manner (Dickson et al., 1991). Histopathologic observations as described above implicate reactive microglia in AD brain as plaque-attacking scavenger cells and same time a source of cells producing very highly cytotoxic substances including proinflammatory cytokines, reactive oxygen intermediates, proteinases, and complement proteins (Giulian and Baker, 1985; Rieske et al., 1989; Rogers et al., 1992; McGeer et al., 1993).

When primary human microglia were treated with Aβ1–42 peptide for 6–24 hr, microglial cells were laden with vacuoles and became fully vacuolated cells (Fig. 7). Expression pattern of proinflammatory cytokines in Aβ-activated microglia was investigated by exposing human microglia cultures to Aβ1–42 or Aβ25–35 for 6 hr (Nagai et al., 2001). The results with RT-PCR showed that there were increased mRNA levels of IL-1β, IL-8, IL-10, IL-12, TNF-α, MIP-1β, and MCP-1 (Fig. 8). Production of IL-1β, IL-8, TNF-α, and MIP-1α increased significantly in primary human microglia after treatment with Aβ25–35 for 6 hr as determined by specific enzyme-linked immunosorbent assay (ELISA). The results demonstrate that treatment of human primary microglia with Aβ peptides upregulates gene expression and protein production of proinflammatory cytokines and chemokines in these cells. Taken together, these results in Aβ-treated human microglia cultures strongly support that reactive microglia in the vicinity of plaques and NFTs play a significant role in the initiation and propagation of immune responses, as resident CNS APCs and inflammatory mediators during the continuing process of AD pathogenesis.

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Figure 8. RT-PCR study of gene expression of cytokines and chemokines in human microglia after 6-hr treatment with amyloid-β peptides (Aβ25–45 and Aβ1–42). Aβ treatment induced increased expression of mRNA transcripts for selected cytokines. Cultures were prepared from a 14-week-gestation fetal brain.

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MICROGLIA AS A VEHICLE FOR TRANGENE IN GENE THERAPY

  1. Top of page
  2. Abstract
  3. HISTORICAL VIEW OF MICROGLIA
  4. MICROGLIA AS PHAGOCYTIC CELLS OF THE CNS
  5. SURFACE ANTIGENS SPECIFIC FOR MICROGLIA
  6. MICROGLIAL PRODUCTION OF CYTOKINES AND CHEMOKINES
  7. REGULATION OF CYTOKINE PRODUCTION IN MICROGLIA
  8. HUMAN MICROGLIA ARE NOT THE NITRIC OXIDE PRODUCER
  9. MICROGLIA AS PRODUCERS OF NEUROTROPHIC MOLECULES
  10. MICROGLIA AND NEUROGENESIS
  11. MICROGLIAL PROLIFERATION INDUCED BY CYTOKINES
  12. PERMANENT STABLE CELL LINES OF MICROGLIA
  13. MICROGLIA AND NEUROLOGIC DISEASES
  14. MICROGLIA AS A VEHICLE FOR TRANGENE IN GENE THERAPY
  15. REFERENCES

Gene therapy by gene transfer to the diseased or injured CNS provides the basis for the development of potentially powerful new therapeutic strategies for a broad spectrum of human neurologic disorders. Previously, several viral systems encoding genes of interest have been injected into brains with variable success. The retroviral vectors with high integration efficiency and potential for permanent gene expression have been accepted most widely; however, they have major deficiencies including low and unstable viral titer and the requirement for target cell division (Mulligan, 1993; Dranoff and Mulligan, 1995). Adenovirus could provide efficient gene transfer and viral stability, but its strong antigenicity could result in serious consequences (Kozarsky and Wilson, 1993). An earlier clinical trial with an adenoviral system in 1999 led to the death of a patient (Thomas et al., 2003). Viral vectors such as adeno-associated viruses, herpes simplex virus, and HIV-based vectors have been available for gene transfer, but they are mostly in exploratory phases and are not accepted widely. In addition, all these gene transfer methods require surgical procedures to introduce viral vectors into the desired brain region.

A previous study has demonstrated that it is feasible to use microglia as a vehicle to deliver a gene of interest to the brain selectively by bypassing other tissues or organs. Intraarterial injection of immortalized rat microglia cells transfected with β-galactosidase into the rat subclavian artery resulted in migration of microglia into the brain 2 days later, and enzyme activity was detected for up to 23 days postinjection. The enzyme activity was detected only in the brain and very little, if any, enzyme activity was found in liver, lung, or spleen of the experimental animals (Sawada et al., 1999). This brain-targeting gene delivery system utilizing microglial cells should facilitate gene therapy of neurologic disorders.

Gene therapy via microglia could be applied in brain tumor therapy. An approach for cancer therapy is based on delivery of a gene encoding an enzyme that transforms an inert prodrug into a toxic product (Aghi et al., 2000). Cells expressing “the suicide gene” are thus killed after prodrug administration. The cytosine deaminase (CD) gene is such a gene and this bacterial enzyme catalyzes the deamination of nontoxic 5-fluorocytosine (5-FC) into the highly toxic 5-fluorouracil (5-FU). In this CD/5-FC system, 5-FU released by CD-expressing cells is able to diffuse and kill adjacent cancer cells. Neural stem cells have extensive migratory and tumor-tropic capacities, and thus could be utilized to deliver a CD/5-FC system to treat malignant brain tumors. When neural stem cells encoded with the CD gene were implanted directly into or distant sites from the brain tumor, or injected intravenously, they distributed efficiently throughout the tumor mass and tracked down advancing tumor cells. After application of prodrug 5-FC, an 80% reduction in brain tumor size was demonstrated (Aboody et al., 2000; Brown et al., 2003). As in the neural stem cells, microglial cells are capable of migrating into the brain after intravenous or intraarterial injection. We have demonstrated recently that human microglia transduced with β-galactosidase also show remarkable migratory and tumor-tropic properties when they were injected directly into the adult rat tail vein. We have also generated an immortalized cell line of human microglia carrying CD gene, and intraarterial injection of these cells in brain tumor-carrying rats resulted in reduction in tumor size (Kim, unpublished data).

REFERENCES

  1. Top of page
  2. Abstract
  3. HISTORICAL VIEW OF MICROGLIA
  4. MICROGLIA AS PHAGOCYTIC CELLS OF THE CNS
  5. SURFACE ANTIGENS SPECIFIC FOR MICROGLIA
  6. MICROGLIAL PRODUCTION OF CYTOKINES AND CHEMOKINES
  7. REGULATION OF CYTOKINE PRODUCTION IN MICROGLIA
  8. HUMAN MICROGLIA ARE NOT THE NITRIC OXIDE PRODUCER
  9. MICROGLIA AS PRODUCERS OF NEUROTROPHIC MOLECULES
  10. MICROGLIA AND NEUROGENESIS
  11. MICROGLIAL PROLIFERATION INDUCED BY CYTOKINES
  12. PERMANENT STABLE CELL LINES OF MICROGLIA
  13. MICROGLIA AND NEUROLOGIC DISEASES
  14. MICROGLIA AS A VEHICLE FOR TRANGENE IN GENE THERAPY
  15. REFERENCES