Role of the cysteine protease cathepsin S in neuropathic hyperalgesia Jane Barclay a , Anna K. Clark c , Pam Ganju a,1 , Clive Gentry a,c , Sadhana Patel a , Glen Wotherspoon a , Frank Buxton b , Chuanzheng Song b , Jakir Ullah a,2 , Janet Winter a , Alyson Fox a,3 , Stuart Bevan a,c , Marzia Malcangio a,c, * a Novartis Institutes for Biomedical Research, 5 Gower Place, London WC1E 6BS, UK b Department of Functional Genomics, Novartis Institutes for Biomedical Research, 100 Technology Square, Cambridge, MA 02139, USA c Wolfson CARD, King’s College London, Guy’s Campus, London SE1 1UL, UK Received 22 June 2006; received in revised form 11 October 2006; accepted 29 November 2006 Abstract Using a gene expression analysis approach we found that the mRNA encoding the lysosomal cysteine protease cathepsin S (CatS) was up-regulated in rat dorsal root ganglia (DRG) following peripheral nerve injury. CatS protein was expressed in infiltrating mac- rophages in DRG and near the site of injury. At both sites CatS expression progressively increased from day 3 to day 14 after injury. In naı ¨ve rats, intraplantar injection of activated rat recombinant (rr) CatS (0.3, 1 lg/rat) induced a mechanical hyperalgesia that developed within half-an-hour, diminished by 3 h and was absent after 24 h. Activated rrCathepsin B (CatB) and non-activated rrCatS injected intraplantarly at the same or higher doses than activated rrCatS had no effect on rat nociceptive thresholds. In nerve-injured rats, mechanical hyperalgesia, but not allodynia, was significantly reversed for up to 3 h by systemic administration of a non-brain penetrant, irreversible CatS inhibitor (LHVS, 3–30 mg/kg s.c.). Depletion of peripheral macrophages by intravenous injection of liposome encapsulate clodronate (1 ml, 5 mg/ml) partially reduced established mechanical hyperalgesia but not allodynia, and abolished the anti-hyperalgesic effect of LHVS. Our results demonstrate a pro-nociceptive effect of CatS and indicate that endogenous CatS released by peripheral macrophages contributes to the maintenance of neuropathic hyperalgesia following nerve injury. Ó 2006 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. Keywords: Macrophages; Hyperalgesia; Sensory neurons; Cysteine proteases; Allodynia 1. Introduction Chronic neuropathic pain can occur as a result of trauma or injury to a peripheral nerve. At present, neu- ropathic pain is treated by opiates and other treatments (tricyclic anti-depressants, anti-convulsants including gabapentin) that have limited efficacy and evoke signifi- cant side effects. The lack of suitable therapies has stim- ulated investigations in animal models to discover the molecular mechanisms underlying neuropathic pain and to identify novel targets for analgesic therapies. In animals, damage to the peripheral axons of senso- ry neurons causes a long lasting thermal and mechanical allodynia and mechanical hyperalgesia that are due to multiple mechanisms operating both peripherally and centrally. These mechanisms include an increase in peripheral nerve excitability that can lead to spontane- ous activity and changes in the expression of some pep- tides, receptors and ion channels in the sensory nerves. These changes may be evident at the site of nerve injury, 0304-3959/$32.00 Ó 2006 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.pain.2006.11.017 * Corresponding author. Tel.: +44 20 7848 8141. E-mail address: marzia.malcangio@kcl.ac.uk (M. Malcangio). 1 Present address: Bristol-Myers Squibb Pharmaceuticals Ltd., 141–149 Staines Road, Middlesex TW3 3JA, UK. 2 Present address: Novartis Institutes for Biomedical Research, CH-4002 Basel, Switzerland. 3 Present address: Novartis Institutes for Biomedical Research, Wimblehurst Road, Horsham RH12 5AB, UK. www.elsevier.com/locate/pain Pain 130 (2007) 225–234