Ingresos

Brain macrophages consist of several distinct populations of specialised cells. Macrophages located in the ventricular margins are known as supraependymal cells; epiplexus cells are found in the choroid plexus; subarachnoid macrophages are located in the meninges; and microglia are found in the parenchyma. Brain macrophages therefore cover every area of the brain, where they act as immune cells and survey the CNS for infiltrating pathogens or damaged neurons. They can phagocytose or repair damaged neurons, and also interact with the adaptive immune system to elicit an immune response, thereby enabling the entry of systemic immune cells into the parenchyma. Microglia are the focus of this study and will be discussed in greater detail.

31 1.2 Microglia in heath and disease

Microglia, the immune-competent cells of the CNS (Färber & Kettenmann 2005), represent 12% of the total cell number in the brain and 20% of glial cells (Block et al. 2007) and can instigate innate immunity within the brain parenchyma following immune challenge (Walter

& Neumann 2009). Microglia were first charachterised by Rio Hortega (1932), who recognised focal areas of invasion following brain lesions, termed “fountains of microglia” in the corpus callosum and other white matter areas (reviewed in Färber & Kettenmann 2005), which has lead to further investigations into the protective and immune roles of microglia.

Microglia are derived from myeloid precursors, and appear in the neuroepithelium during early embryonic development (Walter & Neumann 2009) before invasion into the CNS during late embryonic development where they transform into microglia (Ling & Wong 1993). The monocytic origin of microglia was determined in the 1980‟s in studies showing that ramified microglia express complement and Fc receptors as well as macrophage specific membrane glycoprotein (Perry et al. 1985). Furthermore, (Streit & Kreutzberg 1987) demonstrated that ramified and amoeboid microglia could be visualised by lectin binding, a histochemical marker typically used to label blood monocytes.

Microglia exist in a range of activation states, enabling them to respond appropriately to their environment (Stence et al. 2001) (Fig. 1.5). In the normal brain, microglia have a ramified morphology, and are termed “resting microglia”, however in this state, microglia are highly dynamic, enabling them to constantly survey their microenvironment through extension and retraction of their processes (Nimmerjahn et al. 2005; Davalos et al. 2005). In this way, microglia monitor a defined area of the CNS and respond rapidly to any changes in their microenvironment without making physical contact with other cells (Färber & Kettenmann 2005). Resting microglia do not express the full repertoire of immune receptors seen on activated microglia, however, they do express enzymes implicated in the removal of

32 neurotransmitters from the CSF, and are involved in maintaining neuronal function and structure (Booth & Thomas 1991; Rimaniol et al. 2000).

There has been much debate over the role of microglia in the normal brain under physiological conditions; however, their highly dynamic resting state suggests that their main role is in monitoring the CNS for damage. This suggestion has been supported by in vivo imaging of GFP - expressing microglia, which has shown that in situ, microglia continuously re-organise their processes, enabling them to probe their environment for activating signals radiating from damaged neurons (Davalos et al. 2005; Fetler & Amigorena 2005). The signals responsible for this continuous remodelling of microglial processes are not well understood (Garden & Möller 2006), although microglia are retained in this ramified state by a range of neuronal interactions.

The interaction between the neuronal membrane glycoprotein CD200 (OX2) and the microglial CD200 receptor (CD200R) regulates microglial activity (Hoek et al. 2000).

Microglia are less ramified, aggregated, and express higher levels of immune receptors typically found on activated microglia in CD2000 knockout mice, demonstrating the importance of this interaction in microglial quiescence (Hoek et al. 2000). Microglia are also actively repressed by electrically active neurons, and the removal of this tonic inhibition following neuronal damage leads to microglial activation (Neumann 2001). Two photon microscopy has revealed that microglia are present at, and make contacts with neuronal synapses approximately once an hour, with each contact lasting around 5 min, and microglia make fewer contacts with less active synapses, which promotes microglial activation (Wake et al. 2009). Furthermore, the chemokine fractalkine expressed on neurons also regulates microglial activation, and cleavage of fractalkine from neurons is a microglial activating stimulus which promotes chemotaxis (Chapman et al. 2000). Neurons therefore play an important role in microglial activation state.

33 Microglia are activated by a variety of stimuli released from damaged or dying neurons, and other cells of the CNS. In response to immunological stimulation, activated microglia exhibit an amoeboid morphology enabling increased mobility to sites of neuronal damage (Fig. 1.5).

Microglial activation is a plastic process, with cells changing from a ramified morphology, to a hyper-ramified morphology before finally becoming amoeboid, which facilitates migration to sites of neuronal injury (Raivich 2005). Microglia respond to signals released from damaged neurons, such as adenosine tri-phosphate (ATP) (Booth & Thomas 1991). Microglia fail to respond to CNS injury following blockade of ATP signalling, whilst application of ATP to microglia induces activation to levels seen during CNS injury (Davalos et al. 2005).

Furthermore, physical damage to CNS tissue through the formation of lesions or damage to blood vessels or the BBB which results in an influx of immune cells from the periphery, also promotes microglial activation, and the damage of blood vessels specifically may induce this activation through exposure of microglia to blood-derived factors such as thrombin (Möller et al. 2000), albumin or leukocytes (Nimmerjahn et al. 2005).

Activated microglia are characterised by a number of phenotypic and morphological changes.

A hallmark of microglial activation is the retraction of processes and enlargement of the cell body and nucleus, characteristic of the amoeboid morphology (Fig. 1.5). Activated microglia also express a number of immune receptors and immunomodulatory proteins such as the major histocompatibilty complex II (MHC II), complement factors, and growth factors (Rimaniol et al. 2000). In addition, activation also promotes the release of mediator substances such as cytotoxic proteases, reactive oxygen species (ROS), reactive nitrogen species (RNS) and glutamate (reviewed in Block et al. 2007). Microglial activation also correlates with proliferation and recruitment of immune cells to the site of neuronal injury, and activated microglia also exhibit a phagocytic phenotype (Ling & Wong 1993).

34 Figure 1.5 Microglial activation states. Microglia are found in a resting, highly ramified state where they extend and retract their processes in response to changes in the microenvironment. Activation induces a retraction of processes and the expression of immune receptors and enhances the antigen presenting properties of microglia, and also induces the release of mediator substances. Activated microglia can become migratory and move to the sites of neuronal injury, or phagocytic, enabling them to engulf pathogens and toxic proteins.

35 Microglial activation can be either protective or toxic (Streit 2002). Activated microglia can maintain and support neuronal survival by releasing trophic and anti-inflammatory molecules, such as transforming growth factor-β (TGF-β) and interleukin-6 (IL-6) (Boche et al. 2006); removing invading pathogens or toxic products (Liu et al. 2002), and facilitating the guidance of stem cells to lesion sites to promote neurogenesis (Walton et al. 2006).

However, microglial activation can also release cytotoxic substances such as NO or peroxynitrite (Colton & Gilbert 1987), superoxide (Chéret et al. 2008), and pro-inflammatory cytokines such as interleukin-1β (IL-1β) and tumour necrosis factor-α (TNF-α) (Bruce et al.

1996). Furthermore, microglial activation can be perpetuated by autocrine pathways, in which cytokines released from microglia may enhance microglial activation (Walter & Neumann 2009).