Cytokines

Cytokines are signalling molecules (secreted, cell-membrane associated or stored in the extracellular matrix) that transmit signals from the extracellular environment to the nucleus through specific receptors and intracellular signal transduction or second messenger molecular pathways. They are key regulators of innate and adaptive immune responses.

Fig. 2 Schematic representation of the blood-brain barrier. The cerebral capillaries lack fenestrations and have a dense basement membrane; endothelial cells form tight junctions between each other; several footplates of astrocytes are tightly apposed to the endothelium (adapted from Francis et al. 2003).

In the brain a number of cytokines are induced in response to cerebral ischemia, injury, infectious and autoimmune diseases; they include the proinflammatory 1α, 1β, 2, IL-6, IL-8 and TNF-α, as well as some anti-inflammatory cytokines such as TGF-β, IL-10 (Aloisi 2001). The sources of cytokines in the CNS are mainly microglia and astrocytes, but also tissue infiltrating immune cells and CNS-associated macrophages (Raivich and Banati 2004).

The receptors for most cytokines have been described in the CNS (Szelenyi 2001), some of them at very low level, although rapid up-regulation can occur after injury. Cytokines have a multitude of actions that are important in the process of neurodegeneration. The effects of cytokines may depend on which cell type they act upon and whether it is a direct or indirect effect (Allan and Rothwell 2001).

1.2.1.1 Pro-inflammatory cytokines

The two main pro-inflammatory cytokines with pleiotropic and largely overlapping functions are IL-1 and TNF-α. Intracerebrally produced TNF-α can be involved both in initiating CNS tissue destruction and inflammation (Akassoglou et al. 1998) as well as maintaining autoimmune inflammation (Taupin et al. 1997).

Two different TNF-α receptors have been characterised: TNF-RI (or p55), mediating most actions of soluble TNF-α and known to induce apoptosis, and TNF-RII (p75), more sensitive to transmembranous TNF-α and shown to have anti-apoptotic effects (Pan et al. 1997a; Pan et al. 1997b). Moreover, members of the TNF-receptor family activate several transcription factors, including NF-κB, which themselves induce the transcription of mediators of inflammation (O'Neill and Kaltschmidt 1997; Koedel et al. 2000).

TNF-α is reported to have neurotoxic effects in vivo, as mice overexpressing TNF-α developed neuropathological symptoms (Probert et al. 1995). In vitro, TNF-α has been shown to stimulate secretion of glutamate from glial cells (Piani and Fontana 1994) and to potentiate glutamate neurotoxicity in human foetal brain cells (Chao and Hu 1994).

IL-1β induces inflammatory effects similar to TNF-α. In many CNS diseases IL-1β concentrations have been shown to correlate significantly with TNF-α concentrations and with neurological complications (McCracken et al. 1989; van Deuren 1994; Jain et al. 2000).

Convincing evidence suggests a prominent role of IL-1β in acute neuronal injury. Increased expression of IL-1β is seen in the CNS after a variety of brain insults and administration of exogenous IL-1β to animals undergoing ischemic or excitotoxic challenges leads to a dramatic increase in cell death. On the other hand, the administration of a selective IL-1 receptor antagonist (IL-1ra) markedly reduces the extent of cell death induced by ischemic or

excitotoxic injury in rats (Mulcahy et al. 2003; Hailer et al. 2005). Similarly, inhibition of caspase-1 (which is required for the release of active IL-1β) and the administration of anti-IL-1β antibodies, have been also shown to decrease neuronal injury (Rothwell and Luheshi 2000;

Touzani et al. 2002). Recent studies have employed transgenic animals with specific modifications of selected genes to investigate the contribution of inflammation to neurodegeneration. Mice deficient in pro-inflammatory cytokines (Boutin et al. 2001; Ohtaki et al. 2003) show a reduction in cell death in response to different insults when compared to their wild-type counterparts, whereas in mice deficient in anti-inflammatory cytokines neuronal injury is increased (Grilli et al. 2000).

TNF-α and IL-1β are known to cause blood-brain barrier breakdown, mainly through their ability to induce in cerebrovascular endothelial cells and astrocytes the expression of adhesion molecules and of chemokines, which facilitate leukocyte extravasation and recruitment into the CNS (Lee and Benveniste 1999; Sedgwick et al. 2000). Intrathecal injection of TNF-α or IL-1β leads to blood-brain barrier injury, influx of leukocytes and serum proteins across the BBB into the CSF, brain oedema, increase in intracranial pressure and CSF lactate levels, reduction in cerebral oxygen uptake and in cerebral blood flow, and neuronal apoptosis (Allan 2000; Allan and Rothwell 2001). They also up-regulate endothelial derived adhesion molecules (P-selectin, E-selecin) in cerebral vasculature, which promotes the recruitment of granulocytes to the site of inflammation (Tang et al. 1996). TNF-α and IL-1β have been shown to induce the expression of iNOS and trigger the release of NO from glial cells, as well as vascular endothelium (Bonmann et al. 1997). TNF-α and IL-1β are also known to induce glial reactions and to promote astrogliosis and microgliosis (Selmaj et al. 1990; Balasingam et al. 1994; Probert et al. 1995; Herx and Yong 2001; Basu et al. 2002).

Another cytokine produced in the CNS by microglia and astrocytes is interleukin-6 (IL-6), a multifunctional mediator, which plays an important role in cell-cell signalling under normal and pathophysiological conditions (Chao et al. 1995; Gadient and Otten 1995; Gruol and Nelson 1997). IL-6 can exert both pro-and anti-inflammatory effects (Gadient and Otten 1997). The IL-6 levels, which are low in the normal brain, are strongly up-regulated following brain injury or inflammation in vivo (Clark et al. 1999), and in response to LPS or TNF-α treatment in vitro (Gruol and Nelson 1997). The main role of IL-6 in the brain is its neurotrophic and neuroprotective effect (Chao et al. 1995). Both in vitro and in vivo studies demonstrated that IL-6 promotes the survival of neurons against several neurotoxic agents (glutamate, MPP+, NMDA), presumably through inhibition of apoptosis (Hama et al. 1991;

Yamada and Hatanaka 1994; Hirota et al. 1996; Umegaki et al. 1996). Apart from its

protective effects on neurons, 6 can also mediate glial activation. Mice overexpressing IL-6 have showed severe astrogliosis and increased microglial reactions. IL-IL-6 had also mitogenic effects in astrocytes cultured in vitro (Selmaj et al. 1990).

Glial cells are also considered the major CNS sources of cytokines that stimulate the humoral and cell-mediated immune responses such as IL-12 (Becher et al. 1996; Aloisi et al.

1997), and IL-18 (Prinz and Hanisch 1999). All these cytokines have been shown to be produced during infections and autoimmune diseases and to be critically involved in the development of Experimental Allergic Encephalomyelitis (EAE, an animal model of multiple sclerosis) (Segal et al. 1998; Shevach et al. 1999).

1.2.1.2 Anti-inflammatory cytokines

Although most studies concentrate on glia-derived pro-inflammatory cytokines, recently more attention has been devoted to the role of CNS cells in the anti-inflammatory processes that down-modulate inflammation and immunity. Evidence has provided that microglia produce anti-inflammatory cytokines, such as TGF-β, IL-10, and IL-1 receptor antagonist (IL-1ra), whereas astrocytes secrete TGF-β and IL-10 (Jander et al. 1998; Kiefer et al. 1998; Liu et al.

1998; De Groot et al. 1999; Aloisi et al. 1999). IL-1ra has a major role in counteracting the biological effects of IL-1, thanks to its ability to bind to IL-1 receptor I (IL-1RI) without initiating signal transduction, and thus blocking the IL-1 receptor. IL-10 is found in large amounts in the CSF during bacterial meningitis (van Furth at al. 1995). It inhibits the production of IL-1β, IL-6, IL-8 and TNF-α by monocytes and the release of reactive oxygen species (ROS) by macrophages (Fiorentino et al. 1991; Bogdan et al. 1991; Cunha et al.

1992). TGF-β is also an anti-inflammatory cytokine that can deactivate microglia by suppressing the hydrogen peroxide release (Tsunawaki et al. 1988), as well as release of nitric oxide (Matsuno et al. 2001). It also inhibits endothelial granulocyte adhesion and the production of several cytokines including IL-1, TNF-α, IL-6 and IFN-γ. In addition, TGF-β protects neurons from N-methyl-D-aspartate (NMDA) induced calcium overload and thus from excitotoxic death (Hailer et al. 2001) as well as from β-amyloid induced neurotoxicity (Chao et al. 1994). In microglial cells TGF-β, IL-4 and IL-10 are known to inhibit microglia activation, down-regulate the expression of molecules associated with antigen presentation on these cells, and inhibit the production of pro-inflammatory cytokines, chemokines, nitrogen and oxygen radicals (Aloisi et al. 1999; O'Keefe et al. 1999).