title: Activated monocytes induce human retinal pigment epithelial cell apoptosis through caspase-3 activation.
abstract: Dysfunction and loss of human retinal pigment epithelial (HRPE) cells is a significant component of many ocular diseases, in which mononuclear phagocyte infiltration at the HRPE-related interface is also observed. In this study, we investigated whether HRPE cell apoptosis may be induced by overlay of IFN-gamma-activated monocytes. Human monocytes primed with IFN-gamma overlaid directly onto HRPE cells elicited significant increases in terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive HRPE cells (p < 0.0001) and decreases of proliferating cell nuclear antigen-positive (p < 0.0001) HRPE cells. The activated monocytes also induced HRPE cell caspase-3 activation, which was inhibited by the caspase-3 inhibitor, Z-DEVD-fmk. However, co-incubations in which activated monocytes were prevented from direct contact with HRPE cells or in which the monocytes were separated from the HRPE cells after 30 minutes of direct contact, did not induce significant HRPE cell apoptosis. Function-blocking anti-CD18 and anti-intercellular adhesion molecule-1 (ICAM-1) antibodies significantly reduced activated monocyte-induced TUNEL-positive HRPE cells by 48% (p = 0.0051) and 38% (p = 0.046), respectively. Anti-CD18 and anti-ICAM-1 antibodies significantly inhibited caspase-3 activity by 56% (p < 0.0001) and 45% (p < 0.0001), respectively. However, antibodies to vascular cell adhesion molecule-1, TNF-alpha, IL-1beta, or TNF-related apoptosis-inducing ligand did not inhibit apoptosis or caspase-3 activation. Direct overlay of monocytes also induced reactive oxygen metabolites (ROM) within HRPE cells. The intracellular HRPE cell ROM production was inhibited by the anti-CD18 and anti-ICAM-1 antibodies, but not by superoxide dismutase, presumably due to its failure to penetrate into HRPE cells. Accordingly, neither superoxide dismutase nor N(G)-monomethyl-L-arginine had significant effects on HRPE cell apoptosis or caspase-3 activation. Our results suggest that activated monocytes may induce ROM in HRPE cells through cell-to-cell contact, in part via CD18 and ICAM-1, and promote HRPE cell apoptosis. These mechanisms may compromise HRPE cell function and survival in a variety of retinal diseases.
authors.affiliation: Department of Ophthalmology, University of Michigan, Ann Arbor, Michigan 48105, USA.
authors.forename: Ayako
authors.initials: A
authors.lastname: Yoshida
authors.forename: Susan G
authors.initials: SG
authors.lastname: Elner
authors.forename: Zong-Mei
authors.initials: ZM
authors.lastname: Bian
authors.forename: Andrei L
authors.initials: AL
authors.lastname: Kindezelskii
authors.forename: Howard R
authors.initials: HR
authors.lastname: Petty
authors.forename: Victor M
authors.initials: VM
authors.lastname: Elner
mesh_terms: D017209:Apoptosis; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002450:Cell Communication; D002452:Cell Count; D002478:Cells, Cultured; D018920:Coculture Techniques; D004789:Enzyme Activation; D006801:Humans; D020287:In Situ Nick-End Labeling; D007371:Interferon-gamma / Q000494:pharmacology; D009000:Monocytes / Q000187:drug effects / Q000502:physiology*; D009842:Oligopeptides / Q000494:pharmacology; D010857:Pigment Epithelium of Eye / Q000473:pathology*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism
chemical_list: D061945:Caspase Inhibitors; D009842:Oligopeptides; D018809:Proliferating Cell Nuclear Antigen; C110772:benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone; D007371:Interferon-gamma; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases