title: MCP-1-activated monocytes induce apoptosis in human retinal pigment epithelium.
abstract: OBJECTIVE
  The inflammatory response in age-related macular degeneration (AMD) is characterized by mononuclear leukocyte infiltration of the outer blood-retina barrier formed by the retinal pigment epithelium (RPE). A key mechanistic element in AMD progression is RPE dysfunction and apoptotic cell loss. The purpose of this study was to evaluate whether monocyte chemoattractant protein (MCP)-1-activated monocytes induce human RPE apoptosis and whether Ca(2+) and reactive oxygen species (ROS) are involved in this process.
  
  
  METHODS
  A cell-based fluorometric assay was used to measure intracellular Ca(2+) concentrations ([Ca(2+)](i)) in RPE cells loaded with fluorescent Ca(2+) indicator. Intracellular RPE ROS levels were measured by using the 5- and 6-chloromethyl-2',7'-dichlorodihydrofluorescence diacetate acetyl ester (CM-H(2)DCFDA) assay. RPE apoptosis was evaluated by activated caspase-3, Hoechst staining, and apoptosis ELISA.
  
  
  RESULTS
  MCP-1-activated human monocytes increased [Ca(2+)](i), ROS levels, and apoptosis in RPE cells, all of which were inhibited by 8-bromo-cyclic adenosine diphosphoribosyl ribose (8-Br-cADPR), an antagonist of cADPR. Although the ROS scavengers pyrrolidinedithiocarbamate (PDTC) and N-acetylcysteine (NAC) significantly inhibited ROS production and apoptosis induced by activated monocytes, they did not affect induced Ca(2+) levels. The induced Ca(2+) levels and apoptosis in RPE cells were inhibited by an antibody against cluster of differentiation antigen 14 (CD14), an adhesion molecule expressed by these cells.
  
  
  CONCLUSIONS
  These results indicate that CD14, Ca(2+), and ROS are involved in activated monocyte-induced RPE apoptosis and that cADPR contributes to these changes. Understanding the complex interactions among CD14, cADPR, Ca(2+), and ROS may provide new insights and treatments of retinal diseases, including AMD.
authors: Department of Ophthalmology and Visual Sciences, University of Michigan, Ann Arbor, Michigan 48105-0714, USA.
authors: Dongli
authors: D
authors: Yang
authors: Susan G
authors: SG
authors: Elner
authors: Xun
authors: X
authors: Chen
authors: Matthew G
authors: MG
authors: Field
authors: Howard R
authors: HR
authors: Petty
authors: Victor M
authors: VM
authors: Elner
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D000906:Antibodies / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000276:immunology*; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000276:immunology* / Q000378:metabolism; D036563:Cyclic ADP-Ribose / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors / Q000494:pharmacology; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000276:immunology; D008268:Macular Degeneration / Q000276:immunology* / Q000473:pathology; D008875:Middle Aged; D009000:Monocytes / Q000276:immunology* / Q000473:pathology; D017382:Reactive Oxygen Species / Q000037:antagonists & inhibitors / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000276:immunology* / Q000473:pathology
chemical_list: C493642:8-bromo-cyclic-ADP-ribose; D000906:Antibodies; C111835:CCL2 protein, human; D018932:Chemokine CCL2; D018950:Lipopolysaccharide Receptors; D017382:Reactive Oxygen Species; D036563:Cyclic ADP-Ribose; D002118:Calcium