Selective Proinflammatory Activation of Astrocytes by High-Mobility Group Box 1 Protein Signaling 1 Marco Pedrazzi, 2 * Mauro Patrone, † Mario Passalacqua,* Elia Ranzato, † Diego Colamassaro,* Bianca Sparatore,* Sandro Pontremoli,* and Edon Melloni* Extracellular high-mobility group box 1 protein (HMGB1) triggers inflammatory events in the brain. We demonstrate that astrocytes, the main glial cells in the brain, acquire a specific reactive phenotype when exposed to HMGB1. This cell activation, which involves the receptor for advanced glycation end-products and the MAPK/ERK1/2 cascade, results in the transcriptional/ translational induction of a restricted number of inflammatory mediators, including cyclooxygenase-2, matrix metalloproteinase-9, and several chemokines of the CC and CXC families. The mixture of factors released by HMGB1-reactive astrocytes displays a potent chemotactic activity on human monocytic cells. This study is the first to suggest that HMGB1/astrocyte interaction plays a specific functional role in the progression of inflammatory processes in the CNS by facilitating local leukocyte infiltration. The Journal of Immunology, 2007, 179: 8525– 8532. H igh-mobility group box 1 protein (HMGB1) 3 is a widely distributed DNA-binding protein in mammalian tissues (1). Maximal expression of HMGB1 is detectable during embryonic life and in several proliferating normal or transformed phenotypes (2– 4). Once exported, HMGB1 behaves as a potent cytokine at a local or systemic level (5). Although extracellular HMGB1 can contribute to tissue development and regeneration, it is also able to sustain pathological conditions by enhancing in- flammatory responses, tumor cell invasiveness and lethal endotox- emia (6). Recently, high levels of HMGB1 have been detected in specific areas of the adult brain, including the extracellular matrix (7). The local extracellular accumulation of this cytokine in the brain, as a result of its intracerebroventricular administration or its release from dying neurons during cerebral ischemia, elicits local inflammatory events in mice (8 –10). Moreover, HMGB1 also pro- motes neuroinflammation in postischemic rat brain (9) and in the neurodegenerative processes associated to Alzheimer’s disease (11, 12). Hence, new information on the role of single phenotypes in the brain’s responses to extracellular HMGB1 is of crucial value with regard to possible therapeutic applications. The present report focuses on the proinflammatory effect exerted by HMGB1 on primary astrocytes. These cells are the most abun- dant glial phenotype selectively localized in the CNS (13). The ability of astrocytes to play alternative roles in several physiolog- ical and pathological contexts has been related to their uncommon ability to display specific reactive profiles, depending on the type and concentration of inflammatory cytokines present in their ex- tracellular milieu (14, 15). Although various chemical stimuli pro- mote the active release of HMGB1 from rat astrocytes (16), the sensitivity of these cells to HMGB1 has not yet been explored. Previous evidence has indicated that HMGB1 activates the release of excitatory amino acids from subcellular re-sealed particles of astrocyte origin (17). Thus, it is conceivable that astrocytes may be a target of extracellular HMGB1. The local concentration of extracellular HMGB1 can rapidly increase in the CNS through active exportation of this leaderless cytokine or as a consequence of passive cell leakage. It has pre- viously been shown that several cells of the CNS can actively export HMGB1 when exposed to appropriate stimuli (9, 16, 18) and a massive accumulation of HMGB1 outside necrotic cells oc- curs immediately after cerebral ischemic injury (9). Once released, HMGB1 can interact with several components of the extracellular matrix such as the proteoglycans neurocan and phosphacan, which have been identified as high-affinity ligands for HMGB1 in the CNS (19, 20). The receptor for advanced glycation end products (RAGE), TLR2 and TLR4 are further transmembrane receptors, which can be engaged and activated by HMGB1 in brain cells (21, 22). Hence, HMGB1 can trigger neuronal and glial cell responses through RAGE-dependent and -independent mechanisms (16, 23–25). To determine whether HMGB1 promotes a specific proinflam- matory program on primary astrocytes, we analyzed the effect of this cytokine on the expression of inflammatory gene markers re- lated to cell stress and death, immunity, signaling, adhesion, me- tabolism, and proteolysis. Our findings indicate that astrocytes ex- posed to HMGB1 acquire a reactive profile that is characterized by the release of potent mediators involved in the local progression of physiopathological processes with an inflammatory component. Materials and Methods Animals Rats of the Milan strain (26) were housed at constant temperature (22  1°C) and relative humidity (50%) under a regular light-dark schedule *Department of Experimental Medicine-Biochemistry Section and Centre of Excel- lence for Biomedical Research, University of Genoa, Viale Benedetto XV, Genoa, Italy; and † Department of Environmental and Life Sciences, University of Eastern Piedmont “Amedeo Avogadro”, Via Bellini 25G, Alessandria, Italy Received for publication June 14, 2007. Accepted for publication October 3, 2007. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by the Italian Ministero dell’Istruzione, dell’Universita `e della Ricerca (FIRB-Post Genoma Project) and by the University of Genoa, Italy. 2 Address correspondence and reprint requests to Dr. Marco Pedrazzi, Department of Experimental Medicine-Biochemistry Section, University of Genoa, Viale Benedetto XV, 1-16132 Genoa, Italy. E-mail address: marcopedraz@libero.it 3 Abbreviations used in this paper: HMGB1, high mobility group box 1; RAGE, receptor for advanced glycation end-products; CCM, complete cytokine mixture; MMP, matrix metalloproteinase; CASP, caspase; IFIT, interferon-induced protein with tetratricopeptide repeat; IRF, interferon regulatory factor; TIMP, tissue inhibitor of the matrix metalloproteinase; COX, cyclooxygenase. Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 The Journal of Immunology www.jimmunol.org