title: Proinflammatory responses induced by CD40 in retinal endothelial and Müller cells are inhibited by blocking CD40-Traf2,3 or CD40-Traf6 signaling.
abstract: OBJECTIVE
  The cell surface receptor CD40 is required for the development of retinopathies induced by diabetes and ischemia/reperfusion. The purpose of this study was to identify signaling pathways by which CD40 triggers proinflammatory responses in retinal cells, since this may lead to pharmacologic targeting of these pathways as novel therapy against retinopathies.
  
  
  METHODS
  Retinal endothelial and Müller cells were transduced with vectors that encode wild-type CD40 or CD40 with mutations in sites that recruit TNF receptor associated factors (TRAF): TRAF2,3 (ΔT2,3), TRAF6 (ΔT6), or TRAF2,3 plus TRAF6 (ΔT2,3,6). Cells also were incubated with CD40-TRAF2,3 or CD40-TRAF6 blocking peptides. We assessed intercellular adhesion molecule-1 (ICAM-1), CD40, monocyte chemoattractant protein-1 (MCP-1), VEGF, and prostaglandin E₂ (PGE₂) by fluorescence-activated cell sorting (FACS), ELISA, or mass spectrometry. Mice (B6 and CD40(-/-)) were made diabetic using streptozotocin. The MCP-1 mRNA was assessed by real-time PCR.
  
  
  RESULTS
  The CD40-mediated ICAM-1 upregulation in endothelial and Müller cells was markedly inhibited by expression of CD40 ΔT2,3 or CD40 ΔT6. The CD40 was required for MCP-1 mRNA upregulation in the retina of diabetic mice. The CD40 stimulation of endothelial and Müller cells enhanced MCP-1 production that was markedly diminished by CD40 ΔT2,3 or CD40 ΔT6. Similar results were obtained in cells incubated with CD40-TRAF2,3 or CD40-TRAF6 blocking peptides. The CD40 ligation upregulated PGE₂ and VEGF production by Müller cells, that was inhibited by CD40 ΔT2,3 or CD40 ΔT6. All cellular responses tested were obliterated by expression of CD40 ΔT2,3,6.
  
  
  CONCLUSIONS
  Blockade of a single CD40-TRAF pathway was sufficient to impair ICAM-1, MCP-1, PGE₂, and VEGF upregulation in retinal endothelial and/or Müller cells. Blockade of CD40-TRAF signaling may control retinopathies.
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: JA
authors.lastname: Portillo
authors.affiliation: Division of Infectious Diseases and HIV Medicine, Department of Medicine, Case Western Reserve University, Cleveland, Ohio, United States.
authors.forename: Isaac
authors.initials: I
authors.lastname: Schwartz
authors.affiliation: Department of Pharmacology, University of Colorado Denver, Aurora, Colorado, United States.
authors.forename: Simona
authors.initials: S
authors.lastname: Zarini
authors.affiliation: Department of Pediatrics, Case Western Reserve University/Rainbow Babies and Children's Hospital, Cleveland, Ohio, United States.
authors.forename: Reena
authors.initials: R
authors.lastname: Bapputty
authors.affiliation: Division of Clinical and Molecular Endocrinology, Department of Medicine, Case Western Reserve University, Cleveland, Ohio, United States.
authors.forename: Timothy S
authors.initials: TS
authors.lastname: Kern
authors.affiliation: Department of Pediatrics, Case Western Reserve University/Rainbow Babies and Children's Hospital, Cleveland, Ohio, United States.
authors.forename: Rose A
authors.initials: RA
authors.lastname: Gubitosi-Klug
authors.affiliation: Department of Pharmacology, University of Colorado Denver, Aurora, Colorado, United States.
authors.forename: Robert C
authors.initials: RC
authors.lastname: Murphy
authors.affiliation: Veterans Administration Medical Center, Research Service 151, Cleveland, Ohio, United States Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, Case Western Reserve University, Cleveland, Ohio, United States.
authors.forename: M Cecilia
authors.initials: MC
authors.lastname: Subauste
authors.affiliation: Division of Infectious Diseases and HIV Medicine, Department of Medicine, Case Western Reserve University, Cleveland, Ohio, United States Department of Ophthalmology and Visual Sciences, Case Western Reserve University, Cleveland, Ohio, United States Department of Pathology, Case Western Reserve University, Cleveland, Ohio, United States.
authors.forename: Carlos S
authors.initials: CS
authors.lastname: Subauste
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D019013:CD40 Antigens / Q000037:antagonists & inhibitors / Q000276:immunology*; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000378:metabolism; D003921:Diabetes Mellitus, Experimental / Q000276:immunology* / Q000378:metabolism; D015232:Dinoprostone / Q000378:metabolism; D042783:Endothelial Cells / Q000276:immunology* / Q000378:metabolism; D063928:Ependymoglial Cells / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D051379:Mice; D051381:Rats; D012160:Retina / Q000276:immunology* / Q000378:metabolism; D015398:Signal Transduction / Q000276:immunology / Q000502:physiology; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins / Q000037:antagonists & inhibitors / Q000276:immunology*; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
chemical_list: D015415:Biomarkers; D019013:CD40 Antigens; D018932:Chemokine CCL2; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins; D042461:Vascular Endothelial Growth Factor A; D018799:Intercellular Adhesion Molecule-1; D015232:Dinoprostone
keywords: CD40; chemokine; diabetes; intercellular adhesion molecules