title: Ligation of CD40 in Human Müller Cells Induces P2X7 Receptor-Dependent Death of Retinal Endothelial Cells.
abstract: Cluster of differentiation 40 (CD40) is required for retinal capillary degeneration in diabetic mice, a process mediated by the retinal endothelial cells (REC) death. However, CD40 activates prosurvival signals in endothelial cells. The purpose of this study was to identify a mechanism by which CD40 triggers programmed cell death (PCD) of RECs and address this paradox.
  
  
  
  Human RECs and Müller cells were incubated with CD154 and L-N6-(1-Iminoethyl)lysine (L-Nil, nitric oxide synthase 2 inhibitor), α-lipoic acid (inhibitor of oxidative stress), anti-Fas ligand antibody, or A-438079 (P2X7 adenosine triphosphate [ATP] receptor inhibitor). Programmed cell death was analyzed by fluorescence-activated cell sorting (FACS) or Hoechst/propidium iodide staining. Release of ATP was measured using a luciferase-based assay. Mice were made diabetic with streptozotocin. Expression of P2X7 was assessed by FACS, quantitative PCR, or immunohistochemistry.
  
  
  
  Ligation of CD40 in primary RECs did not induce PCD. In contrast, in the presence of primary CD40+ Müller cells, CD40 stimulation caused PCD of RECs that was not impaired by L-Nil, α-lipoic acid, or anti-Fas ligand antibody. We found CD40 did not trigger TNF-α or IL-1β secretion. Primary Müller cells released extracellular ATP in response to CD40 ligation. Inhibition of P2X7 (A-438079) impaired PCD of RECs; CD40 upregulated P2X7 in RECs, making them susceptible to ATP/P2X7-mediated PCD. Diabetic mice upregulated P2X7 in the retina and RECs in a CD40-dependent manner.
  
  
  
  Cluster of differentiation 40 induces PCD of RECs through a dual mechanism: ATP release by Müller cells and P2X7 upregulation in RECs. These findings are likely of in vivo relevance since CD40 upregulates P2X7 in RECs in diabetic mice and CD40 is known to be required for retinal capillary degeneration.
authors.affiliation: Division of Infectious Diseases and HIV Medicine, Department of Medicine, Case Western Reserve University, Cleveland, Ohio, United States.
authors.forename: Jose-Andres C
authors.initials: JC
authors.lastname: Portillo
authors.affiliation: Division of Infectious Diseases and HIV Medicine, Department of Medicine, Case Western Reserve University, Cleveland, Ohio, United States 2Department of Pathology, Case Western Reserve University, Cleveland, Ohio, United States.
authors.forename: Yalitza
authors.initials: Y
authors.lastname: Lopez Corcino
authors.affiliation: Department of Biophysics, Case Western Reserve University, Cleveland, Ohio, United States.
authors.forename: George R
authors.initials: GR
authors.lastname: Dubyak
authors.affiliation: Division of Molecular Endocrinology, Department of Medicine, Case Western Reserve University, Cleveland, Ohio, United States 5Department of Ophthalmology and Visual Sciences, Case Western Reserve University, Cleveland, Ohio, United States 6Veterans Administration Medical Center, Research Service 151, Cleveland, Ohio, United States.
authors.forename: Timothy S
authors.initials: TS
authors.lastname: Kern
authors.affiliation: Department of Medicine, Case Western Reserve University, Cleveland, Ohio, United States 8Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, Ohio, United States.
authors.forename: Shigemi
authors.initials: S
authors.lastname: Matsuyama
authors.affiliation: Division of Infectious Diseases and HIV Medicine, Department of Medicine, Case Western Reserve University, Cleveland, Ohio, United States 2Department of Pathology, Case Western Reserve University, Cleveland, Ohio, United States 5Department of Ophthalmology and Visual Sciences, Case Western Reserve University, Cleveland, Ohio, United States.
authors.forename: Carlos S
authors.initials: CS
authors.lastname: Subauste
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D023201:CD40 Ligand / Q000378:metabolism*; D002454:Cell Differentiation; D002478:Cells, Cultured; D003921:Diabetes Mellitus, Experimental; D003930:Diabetic Retinopathy / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D042783:Endothelial Cells / Q000378:metabolism* / Q000473:pathology; D004797:Enzyme-Linked Immunosorbent Assay; D063928:Ependymoglial Cells / Q000378:metabolism* / Q000473:pathology; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D058920:Purinergic P2X Receptor Antagonists / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D060888:Real-Time Polymerase Chain Reaction; D058486:Receptors, Purinergic P2X7 / Q000235:genetics* / Q000378:metabolism; D012160:Retina / Q000378:metabolism / Q000473:pathology; D013777:Tetrazoles / Q000494:pharmacology
chemical_list: C523668:3-(5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl)methylpyridine; D058920:Purinergic P2X Receptor Antagonists; D011725:Pyridines; D058486:Receptors, Purinergic P2X7; D013777:Tetrazoles; D023201:CD40 Ligand; D000255:Adenosine Triphosphate