Activation of microglia

Microglia are the immunocompetent cells specifically equipped to monitor the CNS environment. They represent the endogenous brain defence and immune system, which is responsible for CNS protection against all types of pathogenic factors. Microglial cells are not neural cells by their origin (see Chapter 3.5), nonetheless after invading and setting down in the CNS, they acquired a very specific phenotype, which clearly distinguish them from their ancestors, the blood-derived macrophages. The most important parts of this microglial phenotype are the very specialized appearance of resting microglial cells, and the peculiar complement of receptor molecules expressed by microglia. Microglial cells are endowed with

Resting Activated microglia microglia

Resting Activated microglia microglia

Resting Microglia

Figure 9.4 Resting and activated microglia as drawn by Pio del Rio-Hortega (1933). The left panel represents normal resting microglia, which were described by Rio-Hortega as 'the brain nerve cells have bodyguards which extend their tentacles in every direction, and hold back whatever might be noxious'. Right panel shows an encephalitic brain where these cells 'resemble voracious monsters and are valuable assistants in cleaning the tissue of whatever has damaged the nerve cells' (cited from Somjen GG (1988) Nervenkitt: notes on the history of the concept of neuroglia. Glia 1, 2-9)

Figure 9.4 Resting and activated microglia as drawn by Pio del Rio-Hortega (1933). The left panel represents normal resting microglia, which were described by Rio-Hortega as 'the brain nerve cells have bodyguards which extend their tentacles in every direction, and hold back whatever might be noxious'. Right panel shows an encephalitic brain where these cells 'resemble voracious monsters and are valuable assistants in cleaning the tissue of whatever has damaged the nerve cells' (cited from Somjen GG (1988) Nervenkitt: notes on the history of the concept of neuroglia. Glia 1, 2-9)

Phagocytic or 'amoeboid' microglia k

Figure 9.5 Schematic representation of microglial activation stages. The resting, or 'ramified' microglia has a small soma and several thin and long processes. Insult to the brain results in release of vascular, neuronal or astroglial factors (e.g. ATP, thrombin or cytokines), which trigger activation of microglia. The activated microglia is characterized by shorter and thicker processes and larger soma. The final stage of activation is represented by a phagocytic or 'amoeboid' microglia, which acts as a tissue microphage both classical immunological receptors (e.g. complement receptors, cytokine and chemokine receptors, immunoglobulin receptors of the Fc family, thrombin and scavenger receptors) and classical neuro-ligand receptors (represented by glutamate, GABA, and P2X purinoreceptors).

Under physiological conditions microglia in the CNS exist in the 'resting' state (Figures 9.4, 9.5). The resting microglial cell is characterized by a small cell body and much elaborated thin processes, which send multiple branches and extend in all directions. Similar to astrocytes, every microglial cell has its own territory, about 30-15 ^m wide; there is very little overlap between neighbouring cells. In essence the term 'resting' microglia can be somewhat misleading, as microglia in unperturbed brain are far from being quiescent. The processes of resting microglial cells are constantly moving through its territory; this is a relatively rapid movement with a speed ~ 1.2-1.5 ^m/min. At the same time microglial processes also constantly send out and retract small protrusions, which can grow and shrink by 2-3 ^m/min. The movement of microglial processes does not have any manifest pattern; the microglia seem to be randomly scanning through their domains (Figure 9.6). Considering the velocity of this movement, the brain parenchyma can be completely scanned by microglial processes every several hours. The motility of the processes is not affected by neuronal firing, but it is sensitive to activators (ATP and its analogues) and inhibitors of purinoreceptors. Focal neuronal damage induces a rapid (~1.5 ^m) and concerted movement of many microglial processes towards the site of lesion, and very soon the latter is completely surrounded by these processes. This injury-induced motility is also governed, at least in part, by activation of purinoreceptors; it is also sensitive to the inhibition of gap junctions,

Microglial Activation

Phagocytic or 'amoeboid' microglia

Figure 9.5 Schematic representation of microglial activation stages. The resting, or 'ramified' microglia has a small soma and several thin and long processes. Insult to the brain results in release of vascular, neuronal or astroglial factors (e.g. ATP, thrombin or cytokines), which trigger activation of microglia. The activated microglia is characterized by shorter and thicker processes and larger soma. The final stage of activation is represented by a phagocytic or 'amoeboid' microglia, which acts as a tissue microphage

Normal conditions

Normal conditions

Resting Microglia

Microglia

Figure 9.6 Microglial cells constantly scan their territories and send their processes towards the site of injury. In the resting condition every microglial cell occupies a distinct territory. Processes of resting microglial cells are constantly moving, scanning this territory for possible damage signals. In the case of local insult microglial processes rapidly move to the source of the damage signal; the latter is most likely represented by ATP that stimulates microglial process movement through activation of metabotropic P2Y purinoreceptors

Microglia

Figure 9.6 Microglial cells constantly scan their territories and send their processes towards the site of injury. In the resting condition every microglial cell occupies a distinct territory. Processes of resting microglial cells are constantly moving, scanning this territory for possible damage signals. In the case of local insult microglial processes rapidly move to the source of the damage signal; the latter is most likely represented by ATP that stimulates microglial process movement through activation of metabotropic P2Y purinoreceptors which are present in astrocytes, but not in microglia; inhibition of gap junctions also affects physiological motility of astroglial processes. Therefore, it appears that astrocytes signal to the microglia by releasing ATP (and possibly some other molecules) through connexin hemichannels. All in all, microglial processes act as a very sophisticated and fast scanning system. This system can, by virtue of receptors residing in the microglial cell plasmalemma, immediately detect injury and initiate the process of active response, which eventually triggers the full blown microglial activation.

When a brain insult is detected by microglial cells, they launch a specific programme that results in the gradual transformation of resting microglia into a phagocyte; this process is generally referred to as 'microglial activation' and proceeds through several steps (Figures 9.4 and 9.5). The first stage of microglial activation produces 'normally' activated or reactive microglia. During this transition, resting microglia retract their processes, which become fewer and much thicker, increase the size of their cell bodies, change the expression of various enzymes and receptors, and begin to produce immune response molecules. At this activated stage, some microglial cells return into a proliferative mode, and microglial numbers around the lesion site start to multiply. Finally, microglial cells become motile, and using amoeboid-like movements they gather around sites of insult. If the damage persists and CNS cells begin to die, microglial cells undergo further transformation and become phagocytes. This is, naturally, a rather sketchy account of complex and highly coordinated changes which occur in microglial cells; the process of activation is gradual and most likely many sub-states exist on the way from resting to phagocytic microglia. Furthermore, activated microglial cells may display quite heterogeneous properties in different types of pathology and in different parts of the brain.

The precise nature of the initial signal that triggers the process of microglial activation is not fully understood; it may be associated either with withdrawal of some molecules (the 'off-signal') released during normal CNS activity, or by the appearance of abnormal molecules or abnormal concentrations of otherwise physiologically present molecules (the 'on-signal'). Both types of signalling can provide microglia with relevant information about the status of brain parenchyma within their territorial domain.

The 'off-signals' that may indicate deterioration in neural networks are not yet fully characterized. A good example of this type of communication is represented by neuronal firing: depression of the latter affects neighbouring microglia, turning them if not into an activated, then into an 'alerted' state - in response to blockade of neuronal activity microglial cells start to up-regulate several immunocompetent molecules. In fact these 'off-signals' can be defensively important as they allow microglia to sense some disturbances even if the nature of the damaging factor cannot be identified.

The 'on-signalling' is conveyed by a wide array of molecules, either associated with cell damage or with foreign matter invading the brain. In particular, damaged neurones can release high amounts of ATP, nucleotides, neuropeptides, growth factors, neurotransmitters, etc. Practically all of these factors can be sensed by microglia (which are endowed with a remarkable array of receptors, see Table 5.1 and Chapter 5) and trigger activation. It might well be that different molecules can activate various subprogrammes of this routine, regulating therefore the speed and degree and peculiar features of microglial activation. Some of these molecules can carry both 'off' and 'on' signals: for example low concentrations of ATP may be indicative of normal on-going synaptic activity, whereas high concentrations signal cell damage. Microglia are also capable of sensing disturbances in brain metabolism: for example, accumulation of ammonia, which follows grave metabolic failures (e.g. during hepatic encephalopathy) can activate microglial cells either directly or via intermediates such as NO or ATP.

Astroglial cells are also capable of releasing a variety of molecules that can activate microglia; this is especially characteristic of activated astroglia, which can up-regulate the synthesis and begin to release biologically active molecules. Microglial cells can also be activated by signals released from their sister cells, which have already undergone activation. An important activator signal is conveyed by molecules arriving with infectious agents, e.g. by lypopolysaccha-rides, forming the bacterial cellular wall, by prions, or by viral components, such as g120 protein from HIV. Finally, microglia can be activated by a number of molecules which can infiltrate the brain following damage to the blood-brain barrier, e.g. by coagulation factors, immunoglobulins, albumin, thrombin etc.

Intracellularly, most of the 'on' signals produce elevations in [Ca2+]j, the amplitude and shape of which vary significantly depending on the type and concentration of molecule/receptor complex involved. Most likely, these variabilities of the Ca2+ signals are instrumental for information encoding, although the precise nature of this type of informational processing remains unknown.

Activation that follows the recognition of damage signals can be a very rapid process indeed. The initial changes in cellular biochemistry occur within minutes after the presenting signal, and the full activation of microglial cells can follow within several hours. Activated microglial cells change their physiological and biochemical properties considerably. First, activated microglia start to up-regulate potassium channels, initially Kir and then delayed K+ channels. Second, activated microglia can change the repertoire and levels of expression of numerous receptors, e.g. by down-regulating 'neuronal' receptors and up-regulating the 'immunocompetent' ones. Furthermore, activated microglial cells alter their biochemistry, by stimulating the synthesis of numerous enzymes. Finally, microglial cells change their motility as they gather at the site of damage, first by sending their processes and then by appearing there in soma.

All these changes allow the execution of the primary function of microglia -defence. This function requires the ability to rapidly attack and kill the invader; subsequently, the remnants of the aggressor, its victims and the collateral damage must be effectively removed. Thus, activated microglial cells are fully equipped with cytotoxic tools, like reactive oxygen species, or NO (most notably the NO system is absent in human microglia, although it is fully operative in rodents), or indeed the cytokines and chemokines. Not only, however, do activated microglial cells aim to destroy the foreign cells, but also to assist neurones in overcoming the damage. In line with this, microglial cells express and release various growth factors (NGF, BDNF, NT-3, NT-4 etc.). Microglial activation often leads to high expression of glutamate transporter (GLT-1), which assists in clearing the excess of glutamate. It has been suggested that microglial cells after arriving at the location of damage, can selectively remove excitatory glutamatergic synapses (so-called 'synaptic stripping'), which further limits glutamate release into compromised brain regions. Finally, microglial cells produce an incredible array of immunocompetent molecules, which include numerous interleukins (IL-1a/p; IL-3, IL-6, IL-8, IL-10, IL-12, IL-15, IL-18), tumour necrosis factor a, interferon inducing factor (IGIF), inflammatory proteins, transforming growth factor TGFp, etc. All these molecules regulate the inflammatory processes and control the immune response of the brain.

Many of these, such as TGFp, are also important regulators of astrogliosis and are important in the orchestration of glial scar formation.

Microglial activation is a reversible process (except the final, phagocytic, stage), which occurs when the pathological factor is defeated. The nature of the signals regulating the deactivation of microglia is unknown. Most likely, the waning of pathological stimulation may be sufficient, although some active 'terminating' message is not excluded. Some in vitro experiments have shown the existence of certain astrocyte-derived factors that may initiate deactivation of microglia.

The final transmutation from activated microglia into a phagocytic one is also initiated by factors released from dying or already dead neurones or astrocytes. The nature of these 'death signals' is not very clear; it seems likely that vanishing cells can release certain chemoattractants (e.g. phosphatidylserine and lysophos-phatidylcholine), which can initiate the ultimate transformation of microglial cells into phagocytes; the latter engulf and devour the remnants of dying cells. Importantly, the actual killing of neurones by microglia is confined to the damaged area, and, normally, it does not extend to undamaged areas. When this constraint fails, however, activated microglia may assume the role of brain destroyer, which does happen in certain pathological conditions.

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