CCL21-induced calcium transients and proliferation in primary mouse astrocytes: CXCR3-dependent and independent responses Hilmar R.J. van Weering, Arthur P.H. de Jong 1 , Alexander H. de Haas 2 , Knut P.H. Biber * , Hendrikus W.G.M. Boddeke Department of Neuroscience, Section Medical Physiology, University Medical Center Groningen (UMCG), Rijksuniversiteit Groningen (RUG), University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands article info Article history: Received 9 February 2009 Received in revised form 19 April 2009 Accepted 20 April 2009 Available online 3 May 2009 Keywords: Astrocyte Chemokine CCL21 CXCL10 CXCR3 Calcium Proliferation abstract CCL21 is a homeostatic chemokine that is expressed constitutively in secondary lymph nodes and attracts immune cells via chemokine receptor CCR7. In the brain however, CCL21 is inducibly expressed in dam- aged neurons both in vitro and in vivo and has been shown to activate microglia in vitro, albeit not through CCR7 but through chemokine receptor CXCR3. Therefore, a role for CCL21 in CXCR3-mediated neuron-microglia signaling has been proposed. It is well established that human and mouse astrocytes, like microglia, express CXCR3. However, effects of CCL21 on astrocytes have not been investigated yet. In this study, we have examined the effects of CCL21 on calcium transients and proliferation in primary mouse astrocytes. We show that similar to CXCR3-ligand CXCL10, CCL21 (10 9 M and 10 8 M) induced calcium transients in astrocytes, which were mediated through CXCR3. However, in response to high con- centrations of CCL21 (10 7 M) calcium transients persisted in CXCR3-deficient astrocytes, whereas CXCL10 did not have any effect in these cells. Furthermore, prolonged exposure to CXCL10 or CCL21 pro- moted proliferation of wild type astrocytes. Although CXCL10-induced proliferation was absent in CXCR3-deficient astrocytes, CCL21-induced proliferation of these cells did not significantly differ from wild type conditions. It is therefore suggested that primary mouse astrocytes express an additional (che- mokine-) receptor, which is activated at high CCL21 concentrations. Ó 2009 Elsevier Inc. All rights reserved. 1. Introduction Chemokines are small chemotactic cytokines of approximately 8–14 kDa and are classified into four distinct groups (CXC, CC, XC and CX3C) based on the position of their conserved cysteine resi- dues in the N-terminus (Bacon et al., 2002; Murphy et al., 2000). Chemokines are produced locally and exert their actions on seven-transmembrane, G-protein coupled chemokine receptors (Allen et al., 2007; Murphy et al., 2000). In general, activation of chemokine receptor-induced signaling pathways results in an increase in intracellular calcium and is in most cases pertussis toxin sensitive, suggesting that most of these receptors are G i -protein linked (Murphy, 1996). In recent years approximately twenty chemokine receptors and over forty chemokines have been identified (Bacon et al., 2002; Murphy et al., 2000; Murphy, 2002). CXC-, CC-, XC- and CX3C-chemokines predominantly induce their actions through their respective CXC-, CC-, XC- and CX3C receptors. One exception is chemokine C-C motif ligand 21 (CCL21, formerly known as TCA-4, Secondary Lymphoid-organ Chemokine (SLC), Exodus-2, 6CKine), which not only binds its pri- mary receptor CCR7, but also functionally binds to CXCR3, a mem- ber of the CXC-chemokine receptor family (Soto et al., 1998). In the periphery, CCL21 is constitutively expressed in secondary lym- phoid organs and controls the homing of mature dendritic cells and of naive T- and B-cells (Dieu et al., 1998; Sallusto et al., 1998; Stein et al., 2000; Yanagihara et al., 1998). In the CNS how- ever, CCL21 is rapidly expressed by endangered neurons during an ischemic insult or spinal cord injury (Biber et al., 2001; Zhao et al., 2007). CCL21 expression was detected in damaged neurons but not in other brain-resident cells (Biber et al., 2001). In addition, CCL21 0889-1591/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbi.2009.04.007 Abbreviations: CCL21, chemokine (C-C motif) ligand 21; CXCL10, chemokine (C- X-C motif) ligand 10; CXCR3, chemokine (C-X-C motif) receptor 3; CXCR3 / , CXCR3-deficient; DMEM, Dulbecco’s modified Eagle’s medium; FCS, fetal calf serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HBSS, Hank’s bal- anced salt solution; PBS, phosphate-buffered saline; RT-PCR, reverse transcriptase- polymerase chain reaction; SLC, secondary lymphoid-organ chemokine. * Corresponding author. Fax: +31 50 3632751. E-mail address: k.p.h.biber@med.umcg.nl (K.P.H. Biber). 1 Present address: Department of Functional Genomics, Center for Neurogenomics and Cognitive Research (CNCR), VU University Amsterdam, Amsterdam, The Netherlands. 2 Present address: Department of Pathology and Laboratory Medicine, University Medical Center Groningen (UMCG), Rijksuniversiteit Groningen (RUG), Groningen, The Netherlands. Brain, Behavior, and Immunity 24 (2010) 768–775 Contents lists available at ScienceDirect Brain, Behavior, and Immunity journal homepage: www.elsevier.com/locate/ybrbi