title: Putative role of protein kinase C in neurotoxic inflammation mediated by extracellular heat shock protein 70 after ischemia-reperfusion.
abstract: BACKGROUND
  Sterile inflammation occurs in the absence of live pathogens and is an unavoidable consequence of ischemia-reperfusion (IR) injury in the central nervous system (CNS). It is known that toll-like receptor 4 (Tlr4) contributes to damage and sterile inflammation in the CNS mediated by IR. However, the mechanism of Tlr4 activation under sterile conditions in ischemic tissue is poorly understood. We performed this study to clarify the mechanism. To this end, we focused on the extracellular heat shock protein 70 (Hsp70), the prototypic Tlr4 ligand.
  
  
  METHODS
  Tlr4-, Myd88- and Trif-knockout animals, as well as C57BL/6 mice, were used for the wild type control. For the in vivo study, we used a mouse model of retinal IR injury. To test the role of protein kinase C (PKC) in IR injury, IR retinas were treated with the PKC inhibitors (polymyxin B and Gö6976) and retinal damage was evaluated by directly counting neurons in the ganglion cell layer of flat-mounted retinas seven days after IR. Primary retinal neurons (retinal ganglion cells) and glial cells were used for in vitro experiments. Quantitative RT-PCR, ELISA and western blot analysis were used to study the production of pro-inflammatory factors in IR retinas and in primary cell cultures.
  
  
  RESULTS
  We found significant accumulation of extracellular Hsp70 in a model of retinal IR injury. We noted that PKC was involved in Tlr4 signaling, and found that PKC inhibitors promoted neuroprotection by reducing pro-inflammatory activity in ischemic tissue. To put all of the pieces in the signaling cascade together, we performed an in vitro study. We found that PKC was critical to mediate the Hsp70-dependent pro-inflammatory response. At the same time, the contamination of Hsp70 preparations with low-dose endotoxin was not critical to mediate the production of pro-inflammatory factors. We found that extracellular Hsp70 can promote neuronal death at least, by mediating production of cytotoxic levels of tumor necrosis factor alpha, predominantly due to the Tlr4/Myd88 signaling cascade.
  
  
  CONCLUSIONS
  Our findings suggest that PKC acts as a switch to amplify the pro-inflammatory activity of Hsp70/Tlr4 signaling, which is sufficient to mediate neuronal death.
authors.forename: Galina
authors.initials: G
authors.lastname: Dvoriantchikova
authors.forename: Andrea Rachelle C
authors.initials: AR
authors.lastname: Santos
authors.forename: Ali M
authors.initials: AM
authors.lastname: Saeed
authors.forename: Xenia
authors.initials: X
authors.lastname: Dvoriantchikova
authors.affiliation: Bascom Palmer Eye Institute, Department of Ophthalmology, University of Miami Miller School of Medicine, 1638 NW 10th Ave, Miami, FL 33136, USA. divanov@med.miami.edu.
authors.forename: Dmitry
authors.initials: D
authors.lastname: Ivanov
mesh_terms: D000818:Animals; D002452:Cell Count; D016923:Cell Death / Q000187:drug effects; D002478:Cells, Cultured; D004195:Disease Models, Animal; D004731:Endotoxins / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology / Q000627:therapeutic use; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D007249:Inflammation / Q000209:etiology* / Q000378:metabolism; D007511:Ischemia / Q000150:complications / Q000188:drug therapy; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009457:Neuroglia / Q000187:drug effects / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D018696:Neuroprotective Agents / Q000494:pharmacology / Q000627:therapeutic use; D011493:Protein Kinase C / Q000378:metabolism*; D015424:Reperfusion; D012160:Retina / Q000166:cytology / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology*
chemical_list: D004731:Endotoxins; D004791:Enzyme Inhibitors; D018840:HSP70 Heat-Shock Proteins; D018696:Neuroprotective Agents; D011493:Protein Kinase C
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