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- Materials and methods
Thrombin (THR) plays a key role in the brain under physiological and pathological conditions. Several of the biological activities of thrombin have been shown to be mainly driven through activation of protease-activated receptor-1 (PAR-1)-type thrombin receptor. Here we have studied the effect of THR and PAR-1-activating peptide (PAR1-AP), SFLLRN, on cytokine-induced expression of inducible nitric oxide (iNOS), a prominent marker of astroglial activation using the rat C6 glioma cells. In this cell line, THR (1–10 U/mL) and PAR1-AP (1–100 µm) induced a significant concentration-dependent increase both of IFN-γ- (250 U/mL) or TNF-α- (500 U/mL) induced NO release. The observed increase of NO production was related to an enhancement of iNOS expression as measured in cell lysates prepared from different treatments by using SDS–PAGE followed by western blot analysis. The effect of THR, but not that of PAR1-AP, was significantly inhibited by hirulogTM (60 µg/mL), a specific and stochiometric THR inhibitor or by cathepsin-G (40 mU/mL), an inhibitor of PAR-1. In conclusion our data suggest a role for THR through activation of PAR-1 in the induction of astroglial iNOS, and further support the hypothesis that THR may function as an important pathophysiological modulator of the inflammatory response.
Following trauma, injury, or diseases, breakdown of blood–brain barrier (BBB) occurs, causing activation of several different repairing mechanisms. In the complex pattern of events involved the protease thrombin (THR), generated after vascular injury as part of the coagulation cascade, plays an important role. Indeed, in addition to its central role in coagulation, THR has numerous biological functions, that are related to inflammation, tissue remodelling, and healing (for a review see Cirino et al. 2000).
Recent observations suggest an equally important role for THR in the brain under normal conditions and following injury (Nishino et al. 1993; Turgeon and Houenou 1997). Many of its actions are mediated by the activation of protease-activated receptor-1 (PAR-1)-type thrombin receptor, a member of the G-protein coupled receptor family. There are four known members of this receptor family of PARs, namely PAR-1, PAR-3 and PAR-4, which are activated by THR, while PAR-2 is a receptor for trypsinlike-enzymes (Coughlin 2000). The peculiar mechanism whereby THR activates its receptor is a proteolytic unmasking of a new –NH2 terminus, which acts as a new self-activating ligand. In particular activation of PAR-1, localised to neurones and glial cells, leads to a widespread effects such as extensive retraction of processes on neurones and astrocytes, inhibition of neurite outgrowth (Gurwitz and Cunningham 1988), stellation reverse in type-1 astrocytes (Cavanaugh et al. 1990; Grabham and Cunningham 1995), and cell proliferation (Grabham and Cunningham 1995). All these effects contribute to gliosis commonly seen at sites of injury in the brain, and limiting tissue regeneration. In addition, astroglia has been proposed to have a role in the cerebral injury as it can synthesise and secrete inflammatory cytokines (Benveniste 1992; Norris et al. 1994; Campbell et al. 1997), and express adhesion molecules (Aloisi et al. 1992), responsible for interaction with T-lymphocytes and extracellular matrix protein. The potential importance of THR effects in the brain is also strengthened by studies where THR inhibitors have been shown to produce beneficial effects. In particular glia-derived nexin, a specific THR inhibitor, secreted mainly by astrocytes, blocks astrocyte stellation reversal by THR (Cavanaugh et al. 1990) and argatroban or hirudin, reduces secondary brain damage following inflammation (Motohashi et al. 1997; Kubo et al. 2000).
Nitric oxide (NO) has been shown to be involved in many physiological and pathological processes in the brain. Small amount of NO synthesised in the brain during neural activity mediates physiological functions, such as neural morphogenesis, development and plasticity, while excess NO production contributes to neuronal injury during cerebral ischemia and may lead to neurodegeneration in various pathological conditions (Koprowski et al. 1993). NO is synthesised by three different types of NO-synthase (NOS) including the constitutive neuronal (nNOS) and endothelial (eNOS) isoforms and the inducible isoform (iNOS) (Knowles and Moncada 1994). The contribution of iNOS to delayed ischemic injury was demonstrated by using iNOS knockout mice (Iadecola et al. 1997) and a selective iNOS inhibitor (Parmentier et al. 1999).
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- Materials and methods
The inflammatory nature of ischemic damage has led to investigate on the involvement of several mediators and enzymes that are characteristic of this phenomenon. It goes without any doubt that activation of the inflammatory pathways leads invariably to the activation of the coagulation cascade. Here by using the rat C6 glioma cell line, where it has been demonstrated the existence of PAR-1 receptor (Ubl et al. 1998), we have evaluated the effect of THR and PAR-1AP in basal and inflammatory conditions. C6 glioma cell line exhibits many properties of astrocytes, as expression of astrocyte-specific markers, including glial fibrillary acidic protein (GFAP) and S-100 (Pfeiffer et al. 1970; Bissel et al. 1974). In addition, compared with primary culture of astrocytes, which can contain small amounts of contaminating microglia, C6 provide a pure source of astroglia-derived cells. Astrocytes have been suggested to play a role in cerebral injury by synthesising and secreting cytokines and inflammatory mediators, such as NO (del Zoppo et al. 2000).
Astrocyte-derived NO could contribute to neuronal cell death in a variety of pathological conditions. The cytotoxic activity of NO is due not only to a direct proinflammatory effect on brain tissue, but also to an increase of cerebral vessel permeability with an ischemia- and inflammation-induced disruption of the BBB that allows the recruitment of other proinflammatory cells, such as neutrophils and lymphocytes (Boje and Lakhman 2000; Mayhan 2000).
In our experimental conditions both THR and PAR-1AP at the concentrations used did not cause a significant increase in nitrite release in C6 cell supernatants. Conversely, they both potentiated IFN-γ-induced nitrite production and this effect was paralleled by an increased iNOS expression. In pathological situations, such as viral or allergic encephalitis, multiple sclerosis and brain diseases associated with inflammation, there are infiltrates of activated T-lymphocytes, which could be the main source of different inflammatory cytokines such as IFN-γ, TNF-α and IL-6. Moreover, it is well known that stimulation of cytokine receptors located on the astroglial cell membrane is followed by important biochemical changes including the induction of iNOS and consequent formation of NO (Feinstein et al. 1994; Sakai et al. 1995). Thus, THR released following an injury could induce, in co-operation with IFN-γ, iNOS expression and in turn NO release contributing to sustain the ongoing inflammatory response.
THR effect was inhibited by the specific THR inhibitor hirulogTM, while PAR-1 AP effect was unchanged. This latter result outlines the importance of THR proteolytic activity to exert the effect observed. It is well known that hirulogTM, a 20 amino acid peptide, binds the fibrinogen site of THR and inhibits THR proteolytic effect by a catalytic site inhibition domain (Maraganore et al. 1990). HirulogTM did not inhibit the response mediated by PAR-1AP, as would be expected based on the direct activation of the peptide agonist compared with the proteolitically response of THR.
The observation that PAR-1AP reproduces THR effect suggests that THR receptor PAR-1 is involved. This evidence is strengthened by the evidence that cathepsin-G, an inhibitor of PAR-1, abolishes THR but not PAR-1AP effect. Cathepsin-G is known to cleave PAR-1 downstream of the thrombin cleavage site, generating a ‘disarmed’ receptor, unresponsive to subsequent proteolytic activation of TRH, but still responsive to non-proteolytic activity of PAR-1AP (Vergnolle 2000).
In order to investigate whether the observed effects were solely linked to IFN-γ or even to another pro-inflammatory cytokine, we have also evaluated the capability of THR and PAR-1AP to potentiate TNF-α induction of iNOS. Indeed, TNF-α plays a major role in inflammation and it is also a potent inducer of iNOS. In our experiments, TNF-α-induced nitrite production was strongly increased in a concentration-dependent fashion by THR and PAR-1AP. Also with this different cytokine the increased nitrite production well correlated with enhanced and concentration-dependent up-regulation of iNOS protein expression. Our observation that cytokines synergised with THR and PAR-1AP to release NO from C6 cells suggests that activation of both cytokine and THR receptors may be a key interaction associated with inflammatory responses. Several possibilities can be considered to account for this synergy. One possibility is that cytokines increase the expression of functional THR receptors necessary for stimulation of NO synthesis. This mechanism is unlikely, as in our experimental conditions IFN-γ did not affect THR-induced cell proliferation. The exact mechanism leading to the this synergistic up-regulation of iNOS is presently unclear. However, it is likely that differential pathways of cellular activation are involved; in fact, it is well known that iNOS induction by cytokines occurs through pathways involving JAK/STAT proteins, while PARs, that are G protein coupled receptors, can activate alternative signalling transduction mechanisms (Kitamura et al. 1996).
In the system studied PAR-1AP is less efficacious than THR. This difference in potency could be due to different causes such as (1) an inefficient presentation of the soluble peptide to the binding domain of PAR-1, when compared with the tethered peptide, (2) the rapid inactivation of peptide by proteolysis and (3) the involvement of other receptors activated by THR but not by PAR-1AP.
The involvement of other PARs and non-PARs related activation can not be excluded. Till now there is no evidence for PAR-3 and PAR-4 expression on rat C6 cells, while Kaufman et al. (2000) reported a role for PAR-1 and PAR-4 in THR-induced calcium signalling in an astrocytoma cell line of human origin.
As injury of the CNS causes an astrogliosis, characterised by cell swelling and proliferation, similar to the effects of the serine protease THR on astrocytes (Pindon et al. 1998), we also evaluated the proliferative effect of THR and PAR-1AP on C6 cells. This effect was significant at the highest concentration of THR and uneffected by IFN-γ, while PAR-1AP was ineffective. We hypothesise that THR released concomitant to inflammatory cytokines such as TNF-α and IFN-γ present at the site of injury might amplify and/or sustain CNS inflammation.
In conclusion, the results of the present study show that THR has the potential to increase the production of proinflammatory mediators, such as NO, by activating glial cells in the CNS, supporting a role for THR as an important pathophysiological modulator of the inflammatory response.