Abstract
Caspases are a family of proteins important for the elimination of infected cells through the induction of apoptosis as well as the initiation of inflammatory cytokines including IL-1β and IL-18. Morphine exposure to animals and/or cells has been associated with the induction of apoptosis. The most common practices of apoptosis detection have involved removing tissues from animal or humans and the analysis of apoptosis on cells or tissues. These methods can potentially induce spontaneous apoptosis that is unrelated to the actual treatment. The objective of this study was to develop an in vivo detection method for assessing caspase activity induced both by morphine directly and by morphine combined with lipopolysaccharide (LPS)–immune activation. Mice were administered saline, morphine, LPS, or a combination of morphine and LPS. Prior to sacrifice, mice were injected with a poly-caspase-specific apoptosis detection probe to detect internal caspase activity in vivo. Results revealed that morphine alone did not directly induce caspase activity. However, morphine significantly enhanced the LPS-induced caspase activity in spleen, thymus, and bone marrow-derived immune cells. The use of a poly-caspase detection probe methodology to label caspase activity in vivo provides a powerful quantitative tool for the in vivo analysis of caspase activity.
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Aldeguer X, Debonera F, Shaked A, Krasinkas AM, Gelman AE, Que X, Zamir GA, Hiroyasu S, Kovalovich KK, Taub R, Olthoff KM (2002) Interleukin-6 from intrahepatic cells of bone marrow origin is required for normal murine liver regeneration. Hepatology 35:40–48
Amstad P, Yu G, Johnson G, Lee B, Dhawan S, Phelps D (2001) Detection of caspase activation in situ by fluorochrome-labeled caspase inhibitors. Biotechniques Vol 31:608–614
Bedner E, Smolewski P, Amstad P (2000) Activation of caspases measured in-situ by binding of fluorochrome-labeled inhibitors of caspases (FLICA): correlation with DNA fragmentation. Exp Cell Res 259:308–313
Boronat MA, Garcia-Fuster MJ, Garcia-Sevilla JA (2001) Chronic morphine induces up-regulation of the pro-apoptotic Fas receptor and down-regulation of the anti-apoptotic Bcl-2 oncoprotein in rat brain. Br J Pharmacol 134:1263–1270
Cursio R, Colosetti P, Auberger P, Gugenheim J (2008) Liver apoptosis following normothermic ischemia–reperfusion: in vivo evaluation of caspase activity by FLIVO assay in rats. Transplant Proc 40:2038–2041
Cursio R, Miele C, Filippa N, Colosetti P, Auberger P, Van Obberghen E, Gugenheim J (2009) Tyrosine phosphorylation of insulin receptor substrates during ischemia/reperfusion-induced apoptosis in rat liver. Langenbecks Arch Surg 394:123–131
Di Francesco DB, Tavazzi R, Gaziano G, Lazzarino IA, Casalinuovo D, Pierro D, Garaci E (1998) Differential effects of acute morphine administrations on polymorphonuclear cell metabolism in various mouse strains. Life Sci 63:2167–2174
Duan RW, Garner DS, Williams SD (2003) Comparison of immunohistochemistry for activated caspase-3 and cleaved cytokeratin 18 with TUNEL method for quantification of apoptosis in histological sections of PC-3 subcutaneous xenografts. J Pathol 199:221–228
Emeterio EP, Tramullas M, Hurle MA (2006) Modulation of apoptosis in the mouse brain after morphine treatments and morphine withdrawal. J Neurosci Res 83:1352–1361
Ghayur T, Banerfee S, Hugunin M, Butler D, Herzog L, Carter A, Quintal L, Sekut L, Talanian R, Paskind M (1997) Caspase-1 processes IFN-γ-inducing factor and regulated LPS-induced IFN-γ production. Nature 366:619–623
Goudas LCA, Langlade AS, Matson W, Milbury P, Thurel C, Sandouk P, Carr DB (1999) Acute decreases in cerebrospinal fluid glutathione levels after intracerebroventricular morphine for cancer pain. Anesth Analg 89:1209–1215
Griffin RJ, Williams BW, Bischof JB, Olin MR, Johnson GL, Lee BW (2007) Use of a fluorescently labeled poly-caspase inhibitor for in vivo detection of apoptosis related to vascular-targeting agent arsenic trioxide for cancer therapy. Technol Cancer Res Treat 6:651–654
Hu S, Sheng WS, Lokensgard JR, Peterson PK (2002) Morphine induces apoptosis of human microglia and neurons. Neuropharmacology 42:829–836
Lee BW, Johnson GL, Hed SA, Darzynkiewicz Z, Talhouk J, Mehrotra S (2003) DEVDase detection in apoptotic cells using the cell permeant fluorogenic substrate, (z-DEVD)2-cresyl violet. BioTechniques 35:1080–1085
Li X, Du L, Darzynkiewicz Z (2000) During apoptosis of HL-60 and U-937 cells caspases are activated independently of dissipation of mitochondrial electrochemical potential. Exp Cell Res 257:290–297
Lin J, Lai Y, Liu C, Wu A (2007) Effects of lotus plumule supplementation before and following systemic administration of lipopolysaccharide on the splenocyte responses of BALB/c mice. Food Chem Toxicol 45:486–493
Liu B, Wang K, Gao HM, Mandavilli B, Wang JY, Hong JS (2001) Molecular cconsequences of activated microglia in the brain: overactivation induces apoptosis. J Neurochem 77:182–189
Liu HC, Anday JK, House SD, Chang SL (2004) Dual effects of morphine on permeability and apoptosis of vascular endothelial cells: morphine potentiates lipopolysaccharide-induced permeability and apoptosis of vascular endothelial cells. J Neuroimmunol 146:13–21
Macchia AT, Palamara C, Bue P, Savini M, Ciriolo R, Di Francesco P (1999) Increased replication of Sendai virus in morphine-treated epithelial cells: evidence for the involvement of the intracellular levels of glutathione. Int J Immunopharmacol 21:185–193
Malhotra R, Lin Z, Vincenz C, Brosius FC (2001) Hypoxia induces apoptosis via two independent pathways in Jurkat cells: differential regulation by glucose. Am J Physiol Cell Physiol 281:C1596–C1603
Mao J, Sung B, Ji RR, Lim G (2002) Neuronal apoptosis associated with morphine tolerance: evidence for an opioid-induced neurotoxic mechanism. J Neurosci 22:7650–7661
Mizukami S, Kikuchi K, Higuchi T, Urano Y, Mashima T, Tsuruo T, Nagano T (1999) Imaging of caspase-3 activation in HeLa cells stimulated with etoposide using a novel fluorescent probe. FEBS Lett 453:356–360
Nelson CJ, Schneider GM, Lysle DT (2000) Involvement of central mu—but not delta- or kappa-opioid receptors in immunomodulation. Brain Behav Immun 14:170–184
Ocasio FM, Jiang Y, House SD, Chang SL (2004) Chronic morphine accelerates the progression of lipopolysaccharide-induced sepsis to septic shock. J Neuroimmunol 149:90–100
Patel J, Manjappa N, Bhat R, Mehrotra P, Bhaskaran M, Singhal PC (2003) Role of oxidative stress and heme oxygenase activity in morphine-induced glomerular epithelial cell growth. Am J Physiol Renal Physiol 285:F861–F869
Payabvash S, Beheshtian A, Salmasi AH, Kiumehr S, Ghahremani MH, Tavangar SM, Sabzevari O, Dehpour AR (2006) Chronic morphine treatment induces oxidant and apoptotic damage in the mice liver. Life Sci 79:972–980
Poot M, Zhang YZ, Kramer, Wells KS, Jones LJ, Hanzel DK, Lugade AG, Singer VL, Haugland RP (1996) Analysis of mitochondrial morphology and function with novel fixable fluorescent stains. J Histochem Cytochem 44:1363–1372
Portenoy RK, Lesage P (1999) Management of cancer pain. Lancet 353:1695–1700
Radbranch L, Grond S, Zech DJ, Bischoff A (1996) High-dose oral morphine in cancer pain management: a report of twelve cases. J Histochem Cytochem 8:144–150
Riol-Blanco L, Delgado-Martín C, Sánchez-Sánchez N, Alonso-C LM, Gutiérrez-López MD, Del Hoyo GM, Navarro J, Sánchez-Madrid F, Cabañas C, Sánchez-Mateos C, Rodríguez-Fernández JL (2009) Immunological synapse formation inhibits, via NF-kappaB and FOXO1, the apoptosis of dendritic cells. Nat Immunol 7:753–760
Roy S, Charboneau RG, Barke RA (1999) Morphine synergizes with lipopolysaccharide in a chronic endotoxemia model. J Neuroimmunol 95:107–114
Salvioli S, Ardizzoni A, Franceschi C, Cossarizza A (1997) JC-1 but not DiOC6(3) or rhodamine 123, is a reliable fluorescent probe to assess delta psi changes in intact cells: implications for studies on mitochondrial functionality during apoptosis. FEBS Lett 411:77–82
Singhal PC, Sharma P, Kapasi AA, Reddy K, Franki N, Gibbons N (1998) Morphine enhances macrophage apoptosis. J Immunol 160:1889–1893
Smolewski P, Grabarek J, Lee BW, Johnson GL, Darzynkiewick Z (2002) Kinetics of HL-60 cell entry to apoptosis during treatment with TNF-alpha or camptothecin assayed by the stathmo-apoptosis method. Cytometry 47:143–149
Stahelin BJ, Marti U, Solioz M, Zimmermann H, Reichen J (1998) False positive staining in the TUNEL assay to detect apoptosis in liver and intestine is caused by endogenous nucleases and inhibited by diethyl pyrocarbonate. Mol Pathol 51:204–208
Wang J, Charboneau R, Balasubramanian S, Barke RA, Loh HH, Roy S (2001) Morphine modulates lymph node-derived T lymphocyte function: role of caspase-3, -8, and nitric oxide. J Leuk Biol 70:527–536
Yin D, Mufson RA, Wang R, Shi Y (1999) Fas-mediated cell death promoted by opioids. Nature 397:218–224
Acknowledgments
Special thanks are due to Rick Charboneau and Brian Lee for their assistance in the laboratory and manuscript preparation as well as to the Department of Immunochemistry for the donation of the caspase probes. The study was supported by NIH/NIDA funding R01 DA12104, DA015349, DA022935, and DA011806. Michael Olin was supported by the National Institute of Health, National Research Service Award T32 DA07097 from the National Institute on Drug Abuse.
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Drs. Molitor and Roy contributed equally to this manuscript.
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Olin, M.R., Roy, S. & Molitor, T. In Vivo Morphine Treatment Synergistically Increases LPS-Induced Caspase Activity in Immune Organs. J Neuroimmune Pharmacol 5, 546–552 (2010). https://doi.org/10.1007/s11481-010-9209-8
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DOI: https://doi.org/10.1007/s11481-010-9209-8