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84. The impact of systemic Tumour Necrosis Factor Alpha on CNS function and inflammation
PNIRS meeting abstracts / Brain, Behavior, and Immunity 25 (2011) S179–S242
84. The impact of systemic tumour necrosis factor alpha on CNS function and inflammation E. Hennessy, C. Cunningham Trinity College Institute of Neuroscience, School of Biochemistry & Immunology, Trinity College Dublin, Dublin, USA There is evidence that systemic inflammation has deleterious effects on existing chronic neurodegenerative disease. We have shown that microglia are primed by neurodegeneration to react more robustly to subsequent systemic challenges. Based on these observations we have demonstrated that systemic TNF-alpha is associated with accelerated cognitive decline in Alzheimer’s Disease patients. There is limited information on the CNS effects of elevated peripheral TNF-alpha. In this study we have examined the CNS effects of systemic TNF-alpha in normal mice and in those with chronic neurodegeneration (ME7 prion disease). Female C57/BL6 mice were challenged with TNF-alpha (50 lg/kg i.p) and sacrificed at 1, 2, 4, 8 or 24 h. There were robust time-dependent effects on TNF-alpha, IL-6, CXCL-1, MCP-1, uPAR and Fas mRNA in the hippocampus. Minimal changes occurred in IL-1beta, iNOS and microglial markers CD11b and CD68 in the hippocampus. There was marked induction of IL-1beta in the hypothalamus. We examined whether TNF-alpha induced a differential response in the degenerating brain using ME7 mice at 16 weeks post-inoculation. Systemic TNF-alpha induced IL-1beta and TNF-alpha mRNA in the hippocampus of normal and prion animals. Levels were higher in ME7 + TNF-alpha than NBH + TNF-alpha for both cytokines however these levels were not more than additive. These studies provide insights into the CNS consequences of systemic TNF-alpha and continuing investigations will elucidate the contribution of TNF-alpha to progression of chronic neurodegenerative disease. doi:10.1016/j.bbi.2011.07.087
85. GRK2 in sensory neurons regulates epinephrine-induced signalling and duration of mechanical hyperalgesia H. Wang a,b, C.J. Heijnen a, N. Eijkelkamp a,c, K.W. Kelley d, R. Dantzer d, A. Kavelaars a,d a University Medical Center Utrecht, Laboratory for Neuroimmunology and Developmental Origins of Disease (NIDOD), Lundlaan 6, Utrecht, 3584 EA, USA b Department of Pharmacology, Shanghai Medical College, Fudan University, China c Molecular Nociception Group, University College London, London WC1E 6BT, United Kingdom d Integrative Immunology and Behavior Program, College of Medicine and College of ACES, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
Epinephrine (EPI) contributes to hyperalgesia in inflammatory and stress conditions. EPI signals via adrenoceptors, which are regulated by G protein-coupled receptor kinase 2 (GRK2). We already reported that GRK2 is decreased in nociceptors during chronic inflammation. Here, we investigated whether GRK2 modulates EPIinduced mechanical and thermal hyperalgesia by using GRK2+/ mice which express 50% of the GRK2 protein. We demonstrate for the first time that EPI-induced mechanical as well as thermal hyperalgesia is prolonged to 21 days in GRK2+/ mice, whereas it lasts only 3–4 days in WT mice. Using cell- specific GRK2-deficient mice we further show that low GRK2 in primary sensory neurons is critical for this prolongation of EPI-induced hyperalgesia. Low GRK2 in
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microglia had only a small effect on EPI-induced hyperalgesia. Low GRK2 in astrocytes did not alter EPI-induced hyperalgesia. EPIinduced hyperalgesia was prolonged similarly in mice with tamoxifen-induced homozygous or heterozygous deletion of GRK2. In terms of EPI signalling pathways, the PKA inhibitor H-89 inhibited EPIinduced mechanical hyperalgesia in WT mice, whereas H-89 had no effect in mice with low GRK2 in sensory neurons (SNS-GRK2+/ mice). Conversely, intraplantar injection of the PKCe inhibitor TATPKCev1 2 inhibited hyperalgesia in SNS-GRK2+/ mice and not in WT mice. These results indicate that low GRK2 in primary sensory neurons switches EPI-induced signalling from a PKA-dependent towards a PKCe-dependent pathway that ultimately mediates prolonged EPI-induced hyperalgesia. doi:10.1016/j.bbi.2011.07.088
86. Low GRK2 in microglia/macrophages prevents silencing of spinal cord microglia and promotes transition to chronic pain H.L. Willemen a, C.J. Heijnen a, N. Eijkelkamp a,b, R. Dantzer c, K.W. Kelley c, A. Kavelaars a,c a
University Medical Center Utrecht, Laboratory for Neuroimmunology and Developmental Origins of Disease (NIDOD), Lundlaan 6, Utrecht, 3584 EA, Netherlands b Molecular Nociception Group, University College London, London WC1E 6BT, United Kingdom c Integrative Immunology and Behavior Program, College of Medicine and College of ACES, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA Chronic pain associated with inflammation is a major clinical problem, but the underlying mechanisms are incompletely understood. Recently, we showed that spinal microglia/macrophage GRK2 is reduced during chronic inflammation-induced hyperalgesia. In addition, we reported that LysM-GRK2+/ mice with a 50% reduction of GRK2 in microglia/macrophages, develop markedly prolonged hyperalgesia following a single intraplantar injection of interleukin-1b (IL-1b). Two days after intraplantar IL-1b increased microglial/macrophage activity occurs in the lumbar but not thoracic spinal cord of GRK2-deficient mice. Intrathecal pre-treatment with minocycline, an inhibitor of microglia/macrophage activation, accelerates resolution of hyperalgesia in both WT and GRK2-deficient mice. Moreover, minocycline treatment prevents transition to chronic hyperalgesia in GRK2+/ mice. These findings indicate that normal levels of GRK2 in microglia/macrophages are required to silence the activity of these cells in the spinal cord and prevent transition to chronic pain. Further investigation of the underlying mechanism revealed that GRK2-deficient microglia produce more pro-inflammatory cytokines (IL-1b and TNFa) and less anti-inflammatory cytokines (IL-10). These differences in cytokine production are important because inhibition of TNF signaling by intrathecal administration of Etanercept or of IL-1 signaling by IL1-RA, prevented transition from acute to chronic pain in GRK2deficient mice. Conversely, intrathecal administration of anti-IL-10 antibody markedly prolonged IL-1b-induced hyperalgesia in WT mice. We propose that chronic inflammation decreases spinal microglial/macrophage GRK2, which prevents silencing of microglia/macrophage activity and thereby contributes to chronic pain. doi:10.1016/j.bbi.2011.07.089
84. The impact of systemic tumour necrosis factor alpha on CNS function and inflammation E. Hennessy, C. Cunningham Trinity College Institute of Neuroscience, School of Biochemistry & Immunology, Trinity College Dublin, Dublin, USA There is evidence that systemic inflammation has deleterious effects on existing chronic neurodegenerative disease. We have shown that microglia are primed by neurodegeneration to react more robustly to subsequent systemic challenges. Based on these observations we have demonstrated that systemic TNF-alpha is associated with accelerated cognitive decline in Alzheimer’s Disease patients. There is limited information on the CNS effects of elevated peripheral TNF-alpha. In this study we have examined the CNS effects of systemic TNF-alpha in normal mice and in those with chronic neurodegeneration (ME7 prion disease). Female C57/BL6 mice were challenged with TNF-alpha (50 lg/kg i.p) and sacrificed at 1, 2, 4, 8 or 24 h. There were robust time-dependent effects on TNF-alpha, IL-6, CXCL-1, MCP-1, uPAR and Fas mRNA in the hippocampus. Minimal changes occurred in IL-1beta, iNOS and microglial markers CD11b and CD68 in the hippocampus. There was marked induction of IL-1beta in the hypothalamus. We examined whether TNF-alpha induced a differential response in the degenerating brain using ME7 mice at 16 weeks post-inoculation. Systemic TNF-alpha induced IL-1beta and TNF-alpha mRNA in the hippocampus of normal and prion animals. Levels were higher in ME7 + TNF-alpha than NBH + TNF-alpha for both cytokines however these levels were not more than additive. These studies provide insights into the CNS consequences of systemic TNF-alpha and continuing investigations will elucidate the contribution of TNF-alpha to progression of chronic neurodegenerative disease. doi:10.1016/j.bbi.2011.07.087
85. GRK2 in sensory neurons regulates epinephrine-induced signalling and duration of mechanical hyperalgesia H. Wang a,b, C.J. Heijnen a, N. Eijkelkamp a,c, K.W. Kelley d, R. Dantzer d, A. Kavelaars a,d a University Medical Center Utrecht, Laboratory for Neuroimmunology and Developmental Origins of Disease (NIDOD), Lundlaan 6, Utrecht, 3584 EA, USA b Department of Pharmacology, Shanghai Medical College, Fudan University, China c Molecular Nociception Group, University College London, London WC1E 6BT, United Kingdom d Integrative Immunology and Behavior Program, College of Medicine and College of ACES, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
Epinephrine (EPI) contributes to hyperalgesia in inflammatory and stress conditions. EPI signals via adrenoceptors, which are regulated by G protein-coupled receptor kinase 2 (GRK2). We already reported that GRK2 is decreased in nociceptors during chronic inflammation. Here, we investigated whether GRK2 modulates EPIinduced mechanical and thermal hyperalgesia by using GRK2+/ mice which express 50% of the GRK2 protein. We demonstrate for the first time that EPI-induced mechanical as well as thermal hyperalgesia is prolonged to 21 days in GRK2+/ mice, whereas it lasts only 3–4 days in WT mice. Using cell- specific GRK2-deficient mice we further show that low GRK2 in primary sensory neurons is critical for this prolongation of EPI-induced hyperalgesia. Low GRK2 in
S203
microglia had only a small effect on EPI-induced hyperalgesia. Low GRK2 in astrocytes did not alter EPI-induced hyperalgesia. EPIinduced hyperalgesia was prolonged similarly in mice with tamoxifen-induced homozygous or heterozygous deletion of GRK2. In terms of EPI signalling pathways, the PKA inhibitor H-89 inhibited EPIinduced mechanical hyperalgesia in WT mice, whereas H-89 had no effect in mice with low GRK2 in sensory neurons (SNS-GRK2+/ mice). Conversely, intraplantar injection of the PKCe inhibitor TATPKCev1 2 inhibited hyperalgesia in SNS-GRK2+/ mice and not in WT mice. These results indicate that low GRK2 in primary sensory neurons switches EPI-induced signalling from a PKA-dependent towards a PKCe-dependent pathway that ultimately mediates prolonged EPI-induced hyperalgesia. doi:10.1016/j.bbi.2011.07.088
86. Low GRK2 in microglia/macrophages prevents silencing of spinal cord microglia and promotes transition to chronic pain H.L. Willemen a, C.J. Heijnen a, N. Eijkelkamp a,b, R. Dantzer c, K.W. Kelley c, A. Kavelaars a,c a
University Medical Center Utrecht, Laboratory for Neuroimmunology and Developmental Origins of Disease (NIDOD), Lundlaan 6, Utrecht, 3584 EA, Netherlands b Molecular Nociception Group, University College London, London WC1E 6BT, United Kingdom c Integrative Immunology and Behavior Program, College of Medicine and College of ACES, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA Chronic pain associated with inflammation is a major clinical problem, but the underlying mechanisms are incompletely understood. Recently, we showed that spinal microglia/macrophage GRK2 is reduced during chronic inflammation-induced hyperalgesia. In addition, we reported that LysM-GRK2+/ mice with a 50% reduction of GRK2 in microglia/macrophages, develop markedly prolonged hyperalgesia following a single intraplantar injection of interleukin-1b (IL-1b). Two days after intraplantar IL-1b increased microglial/macrophage activity occurs in the lumbar but not thoracic spinal cord of GRK2-deficient mice. Intrathecal pre-treatment with minocycline, an inhibitor of microglia/macrophage activation, accelerates resolution of hyperalgesia in both WT and GRK2-deficient mice. Moreover, minocycline treatment prevents transition to chronic hyperalgesia in GRK2+/ mice. These findings indicate that normal levels of GRK2 in microglia/macrophages are required to silence the activity of these cells in the spinal cord and prevent transition to chronic pain. Further investigation of the underlying mechanism revealed that GRK2-deficient microglia produce more pro-inflammatory cytokines (IL-1b and TNFa) and less anti-inflammatory cytokines (IL-10). These differences in cytokine production are important because inhibition of TNF signaling by intrathecal administration of Etanercept or of IL-1 signaling by IL1-RA, prevented transition from acute to chronic pain in GRK2deficient mice. Conversely, intrathecal administration of anti-IL-10 antibody markedly prolonged IL-1b-induced hyperalgesia in WT mice. We propose that chronic inflammation decreases spinal microglial/macrophage GRK2, which prevents silencing of microglia/macrophage activity and thereby contributes to chronic pain. doi:10.1016/j.bbi.2011.07.089