title: Identification of Therapeutic Targets of Inflammatory Monocyte Recruitment to Modulate the Allogeneic Injury to Donor Cornea.
abstract: OBJECTIVE
  We sought to test the hypothesis that monocytes contribute to the immunopathogenesis of corneal allograft rejection and identify therapeutic targets to inhibit monocyte recruitment.
  
  
  METHODS
  Monocytes and proinflammatory mediators within anterior chamber samples during corneal graft rejection were quantified by flow cytometry and multiplex protein assays. Lipopolysaccharide or IFN-γ stimulation of monocyte-derived macrophages (MDMs) was used to generate inflammatory conditioned media (CoM). Corneal endothelial viability was tested by nuclear counting, connexin 43, and propidium iodide staining. Chemokine and chemokine receptor expression in monocytes and MDMs was assessed in microarray transcriptomic data. The role of chemokine pathways in monocyte migration across microvascular endothelium was tested in vitro by chemokine depletion or chemokine receptor inhibitors.
  
  
  RESULTS
  Inflammatory monocytes were significantly enriched in anterior chamber samples within 1 week of the onset of symptoms of corneal graft rejection. The MDM inflammatory CoM was cytopathic to transformed human corneal endothelia. This effect was also evident in endothelium of excised human cornea and increased in the presence of monocytes. Gene expression microarrays identified monocyte chemokine receptors and cognate chemokines in MDM inflammatory responses, which were also enriched in anterior chamber samples. Depletion of selected chemokines in MDM inflammatory CoM had no effect on monocyte transmigration across an endothelial blood-eye barrier, but selective chemokine receptor inhibition reduced monocyte recruitment significantly.
  
  
  CONCLUSIONS
  We propose a role for inflammatory monocytes in endothelial cytotoxicity in corneal graft rejection. Therefore, targeting monocyte recruitment offers a putative novel strategy to reduce donor endothelial cell injury in survival of human corneal allografts.
authors.affiliation: Division of Infection and Immunity University College London, London, United Kingdom 2Eye Center, Albert-Ludwigs-University of Freiburg, Freiburg, Germany.
authors.forename: Thabo
authors.initials: T
authors.lastname: Lapp
authors.affiliation: Division of Infection and Immunity University College London, London, United Kingdom 3NIHR Moorfields Biomedical Centre, Moorfields Eye Hospital, London, United Kingdom.
authors.forename: Sarah S
authors.initials: SS
authors.lastname: Zaher
authors.affiliation: Division of Infection and Immunity University College London, London, United Kingdom.
authors.forename: Carolin T
authors.initials: CT
authors.lastname: Haas
authors.affiliation: Division of Biosciences, University College London, London, United Kingdom.
authors.forename: David L
authors.initials: DL
authors.lastname: Becker
authors.affiliation: Division of Biosciences, University College London, London, United Kingdom.
authors.forename: Chris
authors.initials: C
authors.lastname: Thrasivoulou
authors.affiliation: Division of Infection and Immunity University College London, London, United Kingdom.
authors.forename: Benjamin M
authors.initials: BM
authors.lastname: Chain
authors.affiliation: NIHR Moorfields Biomedical Centre, Moorfields Eye Hospital, London, United Kingdom.
authors.forename: Daniel F P
authors.initials: DF
authors.lastname: Larkin
authors.affiliation: Division of Infection and Immunity University College London, London, United Kingdom.
authors.forename: Mahdad
authors.initials: M
authors.lastname: Noursadeghi
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001082:Aqueous Humor / Q000378:metabolism; D002465:Cell Movement; D002648:Child; D002675:Child, Preschool; D016039:Corneal Transplantation; D016207:Cytokines / Q000378:metabolism; D004728:Endothelium, Corneal / Q000276:immunology / Q000378:metabolism / Q000473:pathology*; D005260:Female; D006084:Graft Rejection / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D006801:Humans; D008297:Male; D008875:Middle Aged; D009000:Monocytes / Q000166:cytology*; D014184:Transplantation, Homologous; D055815:Young Adult
chemical_list: D016207:Cytokines