Biology of microglia

a. Microglia in Normal and Injured CNSs

Microglia constitute a distinct glial population in the CNS [1-4]. Unlike neurons and macroglia that are of neuroepithelial origin, microglia are mesodermal (i.e., bone marrow) in origin and seed the brain early in embryogenesis: during development, mono-cytes migrate to the brain through the vessels located in specific regions of the brain

(called "glial fountains" in humans). These areas are concentrated around the subven-tricular zones where active neurogenesis occurs. These ameboid tissue macrophages then migrate throughout the entire brain parenchyma and differentiate into resident microglial cells. In the mature CNS, microglia are ubiquitously present as highly ramified cells ("resting" microglia) [5,6]. They respond to changes in the CNS microenvironment in a variety of disorders with or without the participation of the systemic monocytes. Although in degenerative disorders such as AD and Parkinson's disease there is little evidence to support recruitment of monocytes from the periphery, in infectious and autoimmune diseases such as HIVE and multiple sclerosis (MS) and in stroke, there is frank infiltration of monocyte-derived macrophages as well as other inflammatory cells. Even in these diseases in which monocytes are known to contribute significantly to the disease process, studies using sensitive markers of microglia invariably demonstrate that parenchymal microglia are one of the earliest reacting cell types in the brain [7,8]. Unlike perivascular macrophages, the highly ramified cell processes of parenchymal and juxtavascular microglia (see below) make them ideal candidates for intercellular interactions with other glia and with neurons. Consequently, they are more likely to be implicated in neurodegenerative disease processes in which there is no overt disruption of the blood-brain barrier (BBB). Their close contact with neurons and other glia makes them a leading suspect as the source of inflammatory soluble mediators that contribute to neurotoxicity in neurodegenerative conditions.

Determining the relative contribution of monocyte-derived macrophages and intrinsic microglia to the disease process, although conceptually important, is hampered by the fact that no single surface marker reliably differentiates intrinsic microglia from blood-borne monocytes [2] (see also below). There are several animal models that have contributed significantly to an understanding of micro-glial cell biology. The bone marrow chimera studies by Hickey and colleagues provided the first evidence that perivascular microglia (macrophages) in normal brain are a distinct population of CNS macrophages, different from parenchymal ramified microglia. Perivascular macrophages turn over more rapidly, and they are capable of presenting antigen in immune response reactions although intrinsic microglia represent a stable population with limited antigen presenting potential [9,10].

Contrary to these views, somewhat different results are presented in more recent studies using stem cell transplantation. When bone marrow stem cells (expressing the green fluorescent protein, GFP) are transplanted into the systemic circulation of irradiated mice, the GFP+ cells are shown to migrate into the adult CNS across the BBB and differentiate into the ramified parenchymal microglial cells [11]. These results are similar to those obtained earlier, which showed that up to a quarter of the regional microglial population was donor-derived by 4 months after transplantation [12]. However, a very similar study reported previously by Vallieres and Sawchenko demonstrated that the vast majority of the transplanted cells become perivascular macrophages in the CNS [13]. The latter study agrees with an earlier one on human subjects who received sex-mismatched bone marrow transplantation, which showed that Y chromosome marker-bearing CD45+ cells (donor-derived mononuclear leukocytes) entered the normal-appearing brains of female recipients and transformed into "perivascular cells" [10]. Therefore, the potential for blood-borne macrophages to migrate into the brain parenchyma and differentiate into microglia in mature brain is still an issue open for debate.

The third type of resident brain macrophages that has received relatively little attention is the juxtavascular microglia. Juxtavascular microglia are characterized by the parenchymal location of the cell body, ramified cell processes, and direct contact with the basal lamina of blood vessels by the cell processes. According to Dailey and colleagues, who studied them in rat hippocampal slice cultures, approximately 10-30 % of total brain microglia belong to this population of juxtavascular microglia [14]. The authors identified them as a mobile subpopulation of parenchy-mal microglia that activate rapidly and that are preferentially recruited to the surfaces of blood vessels following brain tissue injury. As such, this particular subpopulation of microglia may represent cells specialized to facilitate signaling between the injured brain parenchyma and components of the bloodnbrain barrier in vivo [14].

Regardless of their ontogeny and relationship to blood-borne monocytes, it is now well established that microglial cells are a distinct brain macrophage population capable of mounting various reactive and reparative responses. Using the rat facial nerve axotomy model, Kreutzberg and colleagues have elegantly illustrated the various cellular and molecular changes that occur in microglial cells within the degenerating facial nucleus [15,16]. They demonstrated that microglial activation is a key factor in the defense of the neural parenchyma against various insults and have shown that microglia function as scavenger cells as well as in tissue repair and neural regeneration, ultimately facilitating the return to tissue homeostasis.

b. Microglia and Innate Immunity

The early phase of an effective immune response to invading pathogens is essential for the survival of organisms and is known as the innate immune response. It is a type of immunity that is not dependent upon memory T- or B-cells but is dependent on secretory factors and macrophages [17]. The inflammation that is characteristic of neurodegenerative disorders (referred to as neuroinflammation) has been compared to innate immunity. The innate immune response can be driven through specific recognition systems, the best examples being the interactions between microbial components and the Toll-like receptors (TLR). In the CNS, the cells that bear the appropriate receptors to interact with these microbial components are monocyte-derived macrophages and resident brain microglia. Innate immunity is characterized by the de novo production of mediators that directly contribute to antimicrobial activity or set off secondary inflammatory cascades that could ultimately result in inflammation and host injury. Some of the best characterized gene products that are induced as a result of innate immune response are cytokines.

c. Activation of Microglia through Surface Immune Receptors

By far the most potent activator of microglia and macrophages is lipopolysaccharide (LPS) from the Gram (-) bacterial cell wall. LPS complexed with LPS-binding protein (LBP) has been shown to bind cells through the specific receptor CD14 [18], but because CD14 lacks a functional intracellular domain, it has been unclear how the receptor signal is transduced within cells. Recently, members of the microbial pattern recognition receptor (PRR) family called Toll-like receptors have been found to interact with specific microbial components [19-21]; TLR4 is now shown to be the signaling partner for CD14, whereas similar cell signaling pathways have been found to be activated following binding of the double stranded (ds) viral RNA to TLR3. Peptidoglycan, a Gram (+) bacterial cell wall component, triggers signaling through TLR2, whereas the bacterial nucleotide CpG sequence specifically binds to and triggers signaling through TLR9. The primary function of microbial PRRs is in innate immunity and studies implicate these receptors in defense against bacteria, yeast, and viruses and possibly factors released by dead and dying cells.

1. TLR4 Signal Transduction

LPS induces responses in leukocytes by interacting with a soluble binding protein present in serum, LBP (Figure 1.1). The LPS/LBP complex then initiates its biological activities through a heteromeric receptor complex containing CD 14, TLR4, and at least one other protein, myeloid differentiation protein-2 (MD-2) [22]. The intracellular signaling domain (termed TIR [Toll-IL-1 receptor homologous region]) of TLR4 is homologous to that of the IL-1 receptor, and TIR involves downstream activators such as the receptor-associated adapter protein, myeloid differentiation factor 88 (MyD88), IL-1 receptor-associated protein kinase (IRAK), and TNF receptor activated factor 6 (TRAF6), leading to activation of nuclear factor k-B (NFk-B) and mitogen-activated protein (MAP) kinases [19]. This signaling cascade leads to the robust production of cytokines such as IL-1, TNFa, or IL-6 or chemokines such as IL-8/CXCL8. Although the activation of NFk-B is a conserved response following activation of most TLRs, activation of interferon regulatory factor-3 (IRF-3) is a response unique to TLR3 and TLR4 pathways. Specifically, activation of IRF3 through TLR3/TLR4, together with NFk-B, results in the induction of IFNP (and several other primary response genes); IFNP then activates a group of secondary response genes (Figure 1.1 and Figure 1.2). Activation of the IRF3 pathway through TLR3/TLR4 is shown to be MyD88-independent but dependent on the MyD88-like adaptor known as TIR-domain containing adaptor inducing IFNP (TRIF) [23]. IRF3 had been known as a constitutively expressed transcription factor that can be activated by phosphorylation by certain RNA viruses [24]. In Sendai virus-infected cells, both IFNP and the chemokine RANTES have been found to be induced in an IRF3-dependent manner. The identities of the kinases that are responsible for IRF3 phosphorylation have only recently been identified. They are now known to be the Ik-B kinase (IKK)-related kinases, IKK and TANK-binding kinase-1 (TBK1), kinases previously implicated in NFk-B activation [25,26]. Thus, IKK and TBK1 have a pivotal role in coordinating the activation of IRF3 and NFk-B in the innate immune response.

2. Activation of Microglia through CD14/TLR4 In Vivo

Although microglial activation by bacterial products like LPS is well documented in vitro, whether intrinsic microglia in the brain can respond to similar signals has

The MyD88-dependent Pathway

FIGURE 1.1 TLR and IL-1 receptor signaling: the MyD88-dependent pathway. The MyD88-dependent pathway of NFk-B activation occurs through the IL-1R or TLRs [99,100]. The IL-1 receptor complex is composed of the type 1 receptor (IL-1RI) and the receptor accessory protein (IL-1RAcp). LPS/LBP binds to a heteromeric receptor complex containing CD14, TLR4, and MD2 [22]. IL-1/TLR signaling pathways arise from intracytoplasmic TIR domains and TIR domain-containing adaptors such as MyD88, Toll-interacting protein (Tollip), or TIR domain-containing adaptor protein (TIRAP). Tollip is specifically involved in IL-1R signaling [101], whereas TIRAP is specifically involved in MyD88-dependent TLR4 (and TLR2) signaling [102,103]. Upon ligand binding, the cytosolic adaptor proteins are recruited to the receptor complex. IRAK is recruited and phosphorylated by IRAK4, and then TRAF6 is subsequently recruited to the complex. IRAK brings TRAF6 to TGF^-activated kinase

FIGURE 1.1 TLR and IL-1 receptor signaling: the MyD88-dependent pathway. The MyD88-dependent pathway of NFk-B activation occurs through the IL-1R or TLRs [99,100]. The IL-1 receptor complex is composed of the type 1 receptor (IL-1RI) and the receptor accessory protein (IL-1RAcp). LPS/LBP binds to a heteromeric receptor complex containing CD14, TLR4, and MD2 [22]. IL-1/TLR signaling pathways arise from intracytoplasmic TIR domains and TIR domain-containing adaptors such as MyD88, Toll-interacting protein (Tollip), or TIR domain-containing adaptor protein (TIRAP). Tollip is specifically involved in IL-1R signaling [101], whereas TIRAP is specifically involved in MyD88-dependent TLR4 (and TLR2) signaling [102,103]. Upon ligand binding, the cytosolic adaptor proteins are recruited to the receptor complex. IRAK is recruited and phosphorylated by IRAK4, and then TRAF6 is subsequently recruited to the complex. IRAK brings TRAF6 to TGF^-activated kinase been questioned due to the lack of appropriate receptor expression in the normal brain. In the periphery, CD14 is expressed by monocytes and neutrophils. Monocytes isolated from the blood show time-dependent loss of CD14 in culture, whereas normal microglia isolated from the brain acquire CD14 expression in culture [27,28]. These results suggest that downregulation of surface CD14 is a feature that accompanies monocyte differentiation into tissue macrophages. Microglia are derived from CD14+ monocytes during embryogenesis with subsequent loss of CD14 and other myeloid-lineage markers during brain maturation. Upon injury, however, many of the macrophage surface proteins reappear in reactive microglial cells; reexpression of CD14 in human CNS has been documented following stroke and HIVE [8,29]. The ability of parenchymal ramified microglia to express CD14 de novo has been elegantly illustrated by Rivest and colleagues [30]: adult rats that received LPS peripherally (thus leaving the BBB intact) show diffuse parenchymal induction of CD 14 mRNA following the waves of TNFa induction in the circum ventricular organs, areas of the brain that lack a BBB.

Although the principal ligand for CD14 is the LPS/LBP complex, other less well-known ligands might also trigger CD14/TLR4 cell activation pathways. For instance, Fassbender et al. [31] found that CD14 interacts with fibrils of Alzheimer amyloid peptide (AP): anti-CD14 antibodies as well as mice with a genetic deficiency for CD14 showed reduced AP peptide-induced microglial activation and neuronal death in a mouse model of AD. CD14 mRNA was strongly induced in activated microglia in this model. These results suggest that CD 14 may contribute to the neuroinflammatory response to amyloid peptide and highlighted the possibility that the progress being made in the field of innate immunity could be extended to the research of neurodegenerative disorders.

Gene profiling studies are useful in determining the cell response programs activated by specific ligand/receptors. To gain insight into the transcriptional machinery activated by TLR4 in CNS glial cells, we performed microarray analyses of LPS-activated human microglial cells and compared their response with that of IL-1-activated human astrocytes in culture. These studies revealed that the mRNAs induced in the two cell populations are very similar: the genes that are activated include those involved in TLR4 signal transduction such as IFN-P, IRF-3, and NFk-B-related genes, as well as those that are known end products of TLR4 signal transduction, that is, cytokines, chemokines, and other inflammatory products (Table 1.1) [32]. These results suggest a synergism between microglia and astrocytes during

(TAK1), a member of the MAPK kinase kinase (MAPKKK) family, and TAB1 and TAB2, two proteins that bind to TAK1, which are preassociated on the membrane. This leads to phosphorylation of TAK1 and TAB2 followed by dissociation of TRAF6-TAK1-TAB1-TAB2 from IRAK and subsequent translocation to the cytosol. IRAK stays in the membrane and is degraded. Through ubiquitin E2 ligases and other unknown mechanisms, TRAF6 activates TAK1 [100]. Activated TAK1 then activates the IKK complex consisting of IKKa, IKKP, and IKKy, which induces phosphorylation and degradation of IKBa releasing NFK-B. NFK-B translocates to the nucleus and activates transcription of NFK-B-dependent genes.

The MyD88-independent Pathway

FIGURE 1.2 TLR and IL-1 receptor signaling: the MyD88-independent pathway. The MyD88-independent pathway is activated by ligand binding to TLR3 or TLR4 involving TRIF as the TIR domain-containing adaptor protein [23,104]. LPS stimulation of MyD88-null macrophages leads to delayed activation of NF-kB, as well as activation of the transcription factor IRF-3, thereby inducing IFN^ (primary response gene). IFN^, in turn, activates the induction of several IFN-inducible genes such as RANTES/CCL5, IP-10/CXCL10, and ISG-54 (secondary response genes). Virus and viral-derived dsRNA are potent activators of IRF-3,

FIGURE 1.2 TLR and IL-1 receptor signaling: the MyD88-independent pathway. The MyD88-independent pathway is activated by ligand binding to TLR3 or TLR4 involving TRIF as the TIR domain-containing adaptor protein [23,104]. LPS stimulation of MyD88-null macrophages leads to delayed activation of NF-kB, as well as activation of the transcription factor IRF-3, thereby inducing IFN^ (primary response gene). IFN^, in turn, activates the induction of several IFN-inducible genes such as RANTES/CCL5, IP-10/CXCL10, and ISG-54 (secondary response genes). Virus and viral-derived dsRNA are potent activators of IRF-3, innate immune response. Furthermore, IL-1 produced by microglia in virtually all human CNS pathologies (see below) could activate TLR-like signaling cascades in astrocytes contributing to neuroinflammation that is indistinguishable from an innate as immune response (see Section IVB).

3. Activation of Microglia through Scavenger Receptors

Scavenger receptors (SR) are a class of heterogeneous macrophage receptors that bind acetylated or other modified lipoproteins with high affinity and are involved in lipid metabolism, phagocytosis, and cell signaling (for review, see [33]). The SR family includes class A SR (SR-A), class B type 1 SR (SR-B1), CD36, receptor for advanced glycation product (RAGE), macrophage receptor containing collagenous domain (MARCO), and CD68. Most SRs are highly expressed in macrophages and microglia during development and injury, but their expression is downregulated in normal microglia in vivo. Amyloid plaque-associated microglia demonstrate enhanced expression of surface antigens, including class B SRs [34,35]. Additionally, astrocytes and endot-helial cells have been shown to express SR-B1 [33]. In human macrophages, CD36 is involved in reactive oxygen radical formation and cytokine production following binding of acetylated low-density lipoprotein (LDL). Ap has been shown to activate microglia through the receptor complexes that include CD36 and to trigger intracellular signaling cascades involving tyrosine kinase activation and expression of IL-ip [36]. Another SR that is relevant to microglial function is MARCO. MARCO is a type of SR-A that is involved in cytoskeletal rearrangement and the downregulation of antigen uptake function during dendritic cell and microglial cell maturation [37]. MARCO appears to be important in innate immunity, and its expression has been shown to be upregulated by LPS and GM-CSF [38]. CD68 (the human homologue of mouse macrosialin) is closely related to the family of lysosomal-associated mucin-like membrane proteins (lamps). The biological function of CD68 is not fully understood, but it is thought to play a role in cholesterol metabolism and foam cell (lipid-laden macrophage) formation by binding to oxidized LDL [39]. In human CNS, CD68 is widely used as a marker of resting and activated microglia (Figure 1.3).

4. Activation of Microglia through the Fc Receptors

The receptors for the immunoglobulin (Ig) constant region (Fc) are expressed primarily by myeloid-lineage cells. In human brain, resting microglia express FcR with further upregulation during diseases such as AD, multiple sclerosis (MS) [40,41], which leads to the initial phase of IFNP induction. The noncanonical IKKs, TBK-1, and IKK £ (also called inducible IKK or IKKi) are shown to be IRF-3 kinases [25,26]. It has been shown that LPS can activate the similar mechanism of IRF-3 activation in macrophages [105,106], but whether this pathway is activated following IL-1 stimulation is yet to be determined. Studies with TBK1 knockout cells demonstrate that the expression of TBK1 and IKK £ is cell type-dependent and that TBK1 plays a more general role [105,106].

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