The emerging role for chemokines in epilepsy

Journal of Neuroimmunology 224 (2010) 22–27

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Journal of Neuroimmunology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n e u r o i m

The emerging role for chemokines in epilepsy Paolo F. Fabene a,⁎, Placido Bramanti b, Gabriela Constantin c a b c

Department of Morphological and Biomedical Sciences, Section of Anatomy and Histology, University of Verona, Verona, Italy IRCCS Centro Neurolesi “Bonino-Pulejo”, Messina, Italy Department of General Pathology, Section of Pathology, University of Verona, Verona, Italy

a r t i c l e

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Article history: Received 26 April 2010 Accepted 4 May 2010 Keywords: Seizures Chemokines Pilocarpine Epileptogenesis Leukocyte trafficking

a b s t r a c t Epilepsy has been considered mainly a neuronal disease, without much attention to non-neuronal cells. In recent years growing evidence suggest that astrocytes, microglia, blood leukocytes and blood–brain barrier breakdown are involved in the pathogenesis of epilepsy. In particular, leukocyte–endothelium interactions and eventually subsequent leukocyte recruitment in the brain parenchyma seem to represent key players in the epileptogenic cascade. Chemokines are chemotactic factors controlling leukocyte migration under physiological and pathological conditions. In the light of recent advances in our understanding of the role of inflammation mechanisms in the pathogenesis of epilepsy, pro-inflammatory chemokines may play a critical role in epileptogenesis. © 2010 Elsevier B.V. All rights reserved.

1. Epilepsy Epilepsy is a neurological condition characterized by a paroxysmal event due to abnormal and hypersynchronous discharges from an aggregate of neurons in the central nervous system (CNS). Epilepsy affects 1% of the general world population, resulting in a condition in which a person has recurrent seizures due to a chronic, underlying pathologic process. Epilepsy affects around 50 million people worldwide, and nearly 90% of them are found in developing areas (WHO Fact sheet N°999; http://www.who.int/mediacentre/factsheets/ fs999/en/index.html). Temporal lobe epilepsy (TLE) is the most common form of focal epilepsy in adults, and often represents a treatment-refractory disorder (Hauser et al., 1996; Engel, 2001; Wieser, 2004). Given the fact that, by definition, epilepsy is a neuronal malfunctioning, many of the studies have been historically focused almost exclusively on the consequences on neuronal alterations (see Fig. 1), and, in particular, on the unbalance between excitability and inhibition (Holmes, 2005). TLE is often associated with a characteristic pattern of selective and extensive hippocampal atrophy, referred as hippocampal sclerosis (HS; see, e.g., Meldrum and Bruton, 1992). The sclerotic hippocampus is considered to be the source of the electrical events that cause spontaneous epileptic seizures (Spencer, 1998). The indirect evidence that surgical removal of HS produces clinical improvement (Falconer and Taylor, 1968) strengthened the concept that HS itself is an

⁎ Corresponding author. Department of Morphological and Biomedical Sciences, Section of Anatomy and Histology, Faculty of Medicine, Strada Le Grazie 8, 37134 Verona, Italy. Tel.: + 39 045 8027 267; fax: + 39 045 8027163. E-mail address: [email protected] (P.F. Fabene). 0165-5728/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2010.05.016

epileptogenic area (Falconer, 1974). However, whether hippocampal sclerosis is the consequence of repeated seizures, or whether it plays a role in the development of the epileptic focus is still debated (Jefferys, 1999). Both clinical and preclinical data suggest that HS can be associated but not necessary for long-lasting epileptic condition. In particular, we recently demonstrated the occurrence of spontaneous recurrent seizures (SRSs) in rats with preserved hippocampal (and extrahippocampal) morphology and even in absence of status epilepticus (SE) (Navarro Mora et al., 2009). Furthermore, we have provided evidences that modulating leukocyte–endothelium interaction we can reduce the SRSs frequency up to 60%, even in presence of a severe HS (Fabene et al., 2008). These considerations indicate that we should carefully interpret the experimental data obtained in animal models of epilepsy and that neuroinflammation has a more important role in the etiopathogenesis of epilepsy than previously considered.

1.1. Pilocarpine model of TLE Systemic administration of single dose of pilocarpine, a muscarinic cholinergic agonist, leads to SE and, after a seizure-free period, to a chronic condition determined by SRSs (see, for review, Turski et al., 1989). Pilocarpine, which, together with kainic acid (KA), is probably the most commonly studied chemical-inductive model for TLE, has been recently also proposed as a model of pharmacoresistance in TLE (Chakir et al., 2006) and for a non-SE SRSs model (Navarro Mora et al., 2009). Pilocarpine-induced SE is usually characterized by lesions similar to those of patients with TLE, like HS, loss of GABAergic interneurons in the dentate hilus and pyramidal cell death within CA3 and CA1 strata of hippocampus (Sarkisian, 2001). Furthermore, an enhancement of

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Fig. 1. Pie diagram based on a literature analysis, indicating that only a limited minority (b 3%) of the manuscripts on experimental epilepsy are focused on chemokines/ cytokines or other non-neural/inflammatory players. In particular, less than 1% of the manuscripts are based on cytokines and chemokines (0.57%; string used for the search: (epilepsy OR seizure* OR convulsion* OR status epilepticus) AND (cytokine* OR chemokine*)), or inflammatory cells (0.59%; (epilepsy OR seizure* OR convulsion* OR status epilepticus) AND (WC OR WBC OR leukocyte* Or granulocyte* OR lymphocyte*)). A limited number of studies focused instead on vascular alterations or BBB disruption (1.75%; (epilepsy OR seizure* OR convulsion* OR status epilepticus) AND (BBB OR vascular OR blood vessel* OR endothelial cell*)). The large majority (N97%) do not focus primarily to non-neuronal players ((epilepsy OR seizure* OR convulsion* OR status epilepticus)). The literature search has been performed on Pubmed website (http:// www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed), using as limit “preclinical studies” and is updated to October 15th, 2009.

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cells to blood albumin or potassium ions, respectively (Seiffert et al., 2004; Ivens et al., 2007; van Vliet et al., 2007; Marchi et al., 2007). We have recently demonstrated that leukocyte trafficking mechanisms induce BBB damage leading to seizure generation in animal models of epilepsy (Fabene et al., 2008). The role of immune cells in epilepsy was further supported by the study of Kim and colleagues demonstrating that leukocyte migration through the brain endothelium breaks down BBB and causes severe seizures in an animal model of meningitis (Kim et al., 2009). Our recent results have shown that spontaneous recurrent seizures lead to chronic expression of VCAM-1, the ligand for VLA-4 integrin, potentially contributing to BBB permeability, neuroinflammation and brain damage that could explain, at least in part, the evolution of chronic disease with spontaneous seizure generation (Fabene et al., 2008). In support of our work, a recent study showed that epileptiform activity is able to rapidly induce expression of adhesion molecules on brain endothelium (Librizzi et al., 2007) suggesting that each seizure may induce pro-inflammatory mediators able to activate brain endothelium, which in turn may favor the generation of other seizures. In support to the results obtained from animal models of seizures and epilepsy, it has been shown that BBB disruption by intraarterial injection of mannitol in human patients suffering from cerebral lymphoma induces focal motor seizures (Marchi et al., 2007). Vascular alterations and lymphocyte accumulation into the brain parenchima were documented in patients with refractory epilepsy (Hildebrandt et al., 2008). Increased number of leukocytes was observed in brain parenchyma of patients with epilepsy (mostly patients with refractory epilepsy) independently on the disease etiology (Fabene et al., 2008). All together, data obtained in experimental models of epilepsy and results obtained from humans strongly suggest a key role for leukocytes and vascular inflammation mechanisms in the induction of seizures. 3. Chemokines — brief description

GABAA-current run-down in the hippocampus, occurring at the first SRS after the latent phase have been reported in the pilocarpine model (Mazzuferi et al., 2010), mimicking data reported in human brain specimen (Palma et al., 2007). This model has been reported to be highly isomorphic with the human disease (Curia et al., 2008), but the much more widespread structural alterations should be taken into account in the data interpretation (Fabene et al., 2003, 2007). Pilocarpine-induced non-neural alterations leading to epileptogenesis have been recently more clearly indicated: seizures can induce leukocyte–endothelial interactions (Fabene et al., 2008; Kleen and Holmes, 2008; Ransohoff, 2009), blood–brain barrier (BBB) leakage (Janigro, 2007) and angiogenesis characterized by a poor barrier function (Rigau et al., 2007). The role of other non-neuronal cells, such as astrocytes, as critical signaling elements that contribute in the induction of neuronal death following pilocarpine-induced SE has been also clearly demonstrated (Ding et al., 2007).

2. Vascular inflammation mechanisms in epilepsy Recently, it has been shown that inflammation mechanisms, such as pro-inflammatory cytokines, play a role in the pathogenesis of epilepsy (see, for review, Vezzani and Granata, 2005). CNS inflammation is associated with BBB breakdown, and BBB leakage has been implicated both in the induction of seizures and in the progression to epilepsy with chronic seizure generation (Seiffert et al., 2004; Ivens et al., 2007; Marchi et al., 2007; van Vliet et al., 2007). In addition, BBB opening leads by itself to neuronal hypersynchronization and epileptiform activity mediated by exposure of astrocytes and neuronal

The chemokines are homologous 8-to-10-kd proteins that are subdivided into families on the basis of the relative position of the cysteine residues in the mature protein. The two major structural subfamilies are distinguished by the arrangement of the two NH2terminal Cys residues, which are either separated by a single amino acid (CXC) or are in adjacent (CC) positions. C and CX3C chemokines are minor structural subfamilies and include two single Cys-residue chemokines (XCL1, XCL2) and one with three amino acids separating the two NH2-terminal Cys residues (CX3CL1) (Moser et al., 2004). It is widely known that chemokines control leukocyte trafficking by rapid integrin triggering in circulating leukocytes during physiological and pathological conditions (Ley et al., 2007). Chemokines and chemokine receptors generate a regulatory network characterized by specificity and robustness and involved in regulating the diversity of leukocyte recruitment (Oppermann and Forster, 2005). Although all are characterized by chemotactic activity on different leukocyte subtypes, and thus involved in microenvironmental positioning, some chemokines are expressed by the unactivated or inflamed endothelium to be able to fully activate leukocyte integrins under flow (Campbell et al., 1998). Leukocytes generally operate in a high shear stress environment represented by the blood flow, and, remarkably, pro-adhesive chemokines are able to trigger full integrin activation and dependent lymphocyte arrest under flow within few milliseconds representing the most powerful physiological integrin activators (Campbell et al., 1998). Endothelial chemokines bind to G proteincoupled receptors (GPCRs) exposed by lymphocytes and trigger intravascular lymphocyte arrest to endothelial cells. The following chemokines are able to induce activation of β1, β2 and β7 integrindependent adhesion: CCL2, CCL3, CCL4, CXCL10, CXCL9, CCL5, CXCL12, CCL19, CCL21, CCL20, CCL17 and CCL22 (Laudanna et al., 2002).

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4. Chemokine-induced neuromodulation Increasing evidence indicates that cytokines, which have been traditionally identified as systemic mediators of both innate and acquired immune responses, are actually key factors in the modulation of intercellular signaling within the CNS (Pickering and O'Connor, 2007; Viviani et al., 2002). Chemoattractant cytokines, referred as chemokines, secreted by immune cells after pathogen detection, have been proposed as a new class of neuromodulators (Rostene et al., 2007; Cardona et al., 2008; Mélik-Parsadaniantz and Rostène, 2008; Lauro et al., 2008). In particular, CXCL12 has been demonstrated to induce a slow inward current followed by a spontaneous synaptic activity via ionotropic glutamatergic receptors (Ragozzino et al., 2002), as well as changes in dopaminergic neuronal activity via presynaptic GABA and glutamate release (Guyon et al., 2006). Moreover, it has been recently shown that CX3CL-1 may modulate glutamatergic AMPA-currents (Lauro et al., 2008). Overall, chemokines can modulate neuronal activity under physiological and in pathological conditions by: a) modulation of voltage-dependent channels (sodium, potassium, and calcium); b) activation of the G-protein-activated inward rectifier potassium current; and c) increasing neurotransmitter release (gamma-amino butyric acid (GABA), glutamate, and dopamine), often through Ca-dependent mechanisms (Lauro et al., 2008; Guyon and Nahon, 2007). Considering that seizures are characterized by an altered neuronal firing frequency and susceptibility, it becomes thus important to uncover the potential neuromodulatory role of chemokines in experimental and human epilepsy. 5. Chemokines in the control of acute and chronic neuroinflammation Chemokines were studied in CNS diseases mainly in acute brain inflammation in ischemic stroke and during chronic inflammation in multiple sclerosis. Acute inflammation during experimental stroke associates with an increased expression of CCL2 and CCL3 chemokines in the ischemic area, but only CCL2 was found on brain endothelium suggesting a role for this chemokine in the recruitment of blood leukocytes during early phases of brain ischemia (Kim et al., 1995). In addition, blockade with monoclonal antibody or genetic deficiency of CCL2 or of its receptor CCR2 were protective against brain ischemia in rats and mice (Hughes et al., 2002; Kumai et al., 2004; Dimitrijevic et al., 2007). CX3CL1/fractalkine is a chemokine synthesized in endothelial cells in the presence of pro-inflammatory cytokines, and CX3CR1, the specific receptor for fractalkine, is expressed in monocytes, lymphocytes and microglia (Soriano et al., 2002; Imaizumi et al., 2004). The expression of fractalkine seems to play an important role in the interaction between leukocytes and endothelial cells and mice deficient in fractalkine are less susceptible to cerebral ischemia-reperfusion injury (Soriano et al., 2002). Overall our current knowledge suggests a role for CCL2 and CX3CL1 in ischemic stroke but further studies are needed to complete a broader picture. In humans, the majority of T cells present in the cerebrospinal fluid (CSF) have a central memory phenotype expressing CCR7 suggesting that activated T cells access CNS by using, among other chemokine receptors, CCR7 (Kivisäkk et al., 2004). In support of this idea, lymphoid chemokines CCL19 and CCL21, previously shown to mediate arrest of naïve lymphocytes in high endothelial venules (HEVs) of secondary lymphoid organs, are indeed expressed in inflamed brain venules, whereas CCR7 expressing cells accumulate in inflammatory lesions during experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis, which represents the prototype of chronic inflammatory CNS disease (Alt et al., 2002; Columba-Cabezas et al., 2003). These data suggest a dual role for CCL19 and CCL21 both in the regulation of naïve lymphocyte homing

and T cell arrest in brain vessels and migration into chronically inflamed CNS. Interestingly, CCL19, but not CCL21, is transcribed in normal human brain and detectable as a protein in tissue lysates and in cerebrospinal fluid, whereas in both active and inactive multiple sclerosis (MS) lesions CCL19 transcripts were elevated, suggesting that CCL19 plays a role in both the physiological immunosurveillance of the healthy CNS and the pathological maintenance of immune cells in the CNS of MS patients (Krumbholz et al., 2007). As shown for CCL19, CXCL12 is also constitutively expressed in human CNS parenchyma on blood vessel walls, but is elevated in MS lesions, suggesting a role of this chemokine in leukocyte extravasation in human brain (Krumbholz et al., 2005). A clear demonstration of the role of endothelial CCL2 and CCL5 chemokines in leukocyte arrest in brain venules was elegantly performed by Carvalho-Tavares and colleagues in intravital microscopy studies in inflamed brain microcirculation at EAE onset (dos Santos et al., 2005). CCL2 and CCL5 expression is also abundant within MS lesions suggesting a role for these chemokines in lymphocyte migration into the inflamed human brain (Sørensen et al., 1999, 2004; Mahad et al., 2006). CXCL9, CXCL10, and CXCL11 bind to chemokine receptor CXCR3, which has been shown to be able to trigger rapid integrin-dependent adhesion. Although with some controversies, CXCR3 deficient mice show increased EAE severity, which seems to be explained by a reduction of Foxp3 regulatory T cell recruitment into the brain (Fife et al., 2001; Liu et al., 2005; Narumi et al., 2002; Klein et al., 2004; Müller et al., 2007). However, CXCR3+ T cells are also found in CSF of healthy subjects and patients with MS and active MS lesions have high frequency of T cells expressing CXCR3, suggesting that CXCR3 might represent a key inflammatory chemokine receptor involved in intrathecal accumulation of T cells in MS (Sørensen et al., 1999; Kivisäkk et al., 2002). Recent studies highlighted an important role for CCR6 chemokine receptor in the induction and/or regulation of EAE. CCR6 has been first shown to be important in the migration of both Th17 and regulatory T cells in sites of inflammation (Yamazaki et al., 2008). However, contrasting results have been obtained in CCR6-/- mice. One study showed that CCR6-/- mice were resistant to EAE induction, whereas another work demonstrated an increase disease severity in the presence of CCR6 deficiency due to reduced migration capacity of regulatory T cells (Reboldi et al., 2009; Villares et al., 2009). These controversies may be due to different immunization protocols that may emphasize or reduce the generation and importance of regulatory T cells (Cassan et al., 2006; Chen et al., 2006). 6. Chemokines in experimental and human epilepsy Considering that, as reported above, chemokines are key players in the modulation of the neuronal excitability, as well as in the leukocyte recruitment and inflammatory CNS diseases, it becomes thus interesting to assess whether these molecules are involved also in the pathogenesis of experimental epilepsy (Fig. 2). Few, but convincing studies are nowadays present in the literature for testing this hypothesis. 6.1. Chemokines in human epilepsy Recent DNA microarray analysis performed by Lee and colleagues has shown the presence of genes for chemokines CCL2, CCL3, and CCL4 in hippocampi surgically removed from TLE patients (Lee et al., 2007). However, the precise cellular source of the correspondent proteins and the presence of their receptors have not been characterized in these studies. CCL2 and CCL3 proteins can attract polymorphonuclear leukocytes, monocytes, memory T cells and dendritic cells in the hippocampus of TLE patients. In support of these results, neutrophils and activated T cells with Th1 phenotype have been shown to interact

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Fig. 2. A schematic drawing illustrating the possible contribution of chemokines and chemokines receptors in leukocyte vascular adhesion to seizure pathogenesis. CNS vascular activation with upregulation of adhesion molecules (AMs) and involvement of chemokines and chemokines receptors can be provoked by the chemoconvulsant agents or by inflammatory pathologies. Activation of brain endothelium leads to leukocyte adhesion, which compromises the endothelial barrier function resulting in leakage of plasma constituents into the brain parenchyma. Exposure of neuronal cells to plasma proteins and K+ accumulation contributes to neuronal electrical hyperactivity. BBB leakage, and neuronal hyperactivity may thus synergize with each other, amplifying the vascular and neuronal changes that ultimately lead to seizures (modified from Fabene et al., 2008). (A) Selectins, chemokine receptors and integrins, with the relative counterligands, mediating the firm adhesion (B); (C) plasma protein (albumin) extravasation to brain parenchyma.

with the endothelium of brain vessels and migrate perivascularly or inside brain parechima after pilocarpine-induced status epilepticus in mice (Fabene et al., 2008; Fabene and Constantin, unpublished observations). Moreover DNA microarray analysis of human hippocampus of TLE patients showed also expression of VCAM-1 suggesting that vascular adhesion molecules and chemokines may direct together leukocyte trafficking during epilepsy. Astrocytes, perivascular microglia, and infiltrating leukocytes have been identified as major cellular sites of CCL3 and CCL2 production in the brain (Karpus and Ransohoff, 1998) and their receptors CCR1 and CCR2 are expressed on the abluminal surface of human brain endothelial cells (Andjelkovic and Pachter, 2000) potentially directing leukocyte trafficking inside brain parenchyma during inflammatory responses (Fabene et al., 2008; Hildebrandt et al., 2008). Increased CXCR4 expression has been reported in the hippocampus of TLE patients mainly on microglia and a small population of astrocytes (Lee et al., 2007). Increased expression of CXCR4 could allow for increased CXCL12 binding, thereby inducing microglia to release TNFα, which potentiates prostaglandin-dependent Ca2+ activation and glutamate release (Lee et al., 2007). After the initial study by Lee and colleagues, CCL2 mRNA upregulation have been successively confirmed in human epileptic patients (Wu et al., 2008), as well as CCL3 and CCL4 (van Gassen et al., 2008), which have been reported to be highly upregulated (N10-fold) in human TLE.

induced in reactive glial cells, as well as in the blood vessel, at late time-points after pilocarpine-induced SE in mice potentially related to changes in permeability of the blood–brain barrier and leukocyte recruitment during epileptogenesis (Xu et al., 2009). Moreover, CCL2 upregulation has been shown also following kainate-induced seizure in the rat hippocampus (Manley et al., 2007). In this later study, CCL2 expression has been correlated to the temporal profile of BBB permeability and immune cell recruitment at the injury site. The same authors also reported that BBB permeability increased prior to upregulation of CCL2, whereas CCL2 upregulation and immune cell recruitment occurred concurrently, a few hours after BBB leakage (Manley et al., 2007). These results are in agreement with studies performed in other experimental models of brain inflammatory diseases such as EAE in which it has been shown increased BBB permeability preceding consistent leukocyte recruitment in the brain (Floris et al., 2004). Other chemokines receptors, such as CCR7, CCR8, CCR9 and CCR10 have been studied also in the normal Swiss mouse hippocampus. In particular, during pilocarpine-induced SE, downregulation of CCR7, CCR9 and CCR10 in hippocampal neurons has been reported, but the significance of these results is not yet understood (Liu et al., 2007). Overall, and in agreement to human data, the majority of the results obtained in experimental animal models of epilepsy point to a key role for CCL2 in the control of neuroinflammatory reactions during epileptogenesis.

6.2. Chemokines in experimental epilepsy 7. Conclusions In agreement with human data, CCR2 and CCL2 have been shown to increase in the hippocampus following pilocarpine-induced seizures in rats (Foresti et al., 2009). After SE hypertrophied astrocytes exhibited CCR2-labeling, especially in CA1 and dentate gyrus, whereas microglial cells were more closely apposed to the CCR2-labeled cells in SE rats (Foresti et al., 2009), supporting the involvement of nonneuronal cells in the pathology of epilepsy. In addition, CCL2 was

Endothelial chemokines induce complex signal transduction pathways leading to integrin activation and controlling leukocyte recruitment into the brain during physiological and pathological conditions. Despite growing evidence suggesting a role for leukocyte trafficking and BBB vascular alterations in the induction of seizure, little is known about the role of chemokines in the pathogenesis of

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epilepsy and further studies are required to expand our knowledge of the mechanisms leading to seizure generation. As antagonists for chemokine receptors are now being tested in preclinical and clinical settings, more comprehensive studies in experimental models of seizures and on samples derived from human subjects with epilepsy may identify novel targets for potential therapeutic interventions in brain inflammatory diseases. Acknowledgments This work was also supported by grants from the Fondazione Cariverona (P.F.F and G.C.); the European Community [EU Research Grants LSH-CT-2006-037315] (EPICURE), thematic priority LIFESCIHEALTH (P.F.F); Italian Ministry of Education and Research (MIUR) (G.C and P.F.F); Fondazione Italiana Sclerosi Multipla (FISM) (G.C.); and Fondazione San Paolo (P.F.F.). References Alt, C., Laschinger, M., Engelhardt, B., 2002. 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