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Published on: June 14, 2021
MCP-1-activated monocytes induce apoptosis in human retinal pigment epithelium
Dongli Yang1, Susan G Elner, Xun Chen
1Department of Ophthalmology and Visual Sciences, University of Michigan, Ann Arbor, Michigan 48105-0714, USA.
Purpose:
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.
Insights
Monocyte chemoattractant protein-1 (MCP-1) activated monocytes induce retinal pigment epithelium (RPE) apoptosis via cluster of differentiation antigen 14 (CD14), calcium (Ca2+), and reactive oxygen species (ROS). These findings offer insights into age-related macular degeneration (AMD) pathogenesis.
Area of Science:
- Ophthalmology
- Immunology
- Cell Biology
Background:
- Age-related macular degeneration (AMD) involves inflammation and retinal pigment epithelium (RPE) cell loss.
- Mononuclear leukocyte infiltration across the RPE barrier is a key feature of AMD.
Purpose of the Study:
- To investigate if monocyte chemoattractant protein-1 (MCP-1) activated monocytes trigger RPE apoptosis.
- To determine the roles of intracellular calcium (Ca2+) and reactive oxygen species (ROS) in this process.
Main Methods:
- RPE cells were exposed to MCP-1 activated human monocytes.
- Intracellular Ca2+ and ROS levels were measured using fluorometric assays.
- Apoptosis was assessed via caspase-3 activation, Hoechst staining, and ELISA.
Main Results:
- Activated monocytes increased RPE cell Ca2+, ROS, and apoptosis.
- Inhibition by 8-bromo-cyclic adenosine diphosphoribosyl ribose (8-Br-cADPR) suggests cyclic adenosine diphosphoribosyl ribose (cADPR) involvement.
- ROS scavengers reduced ROS and apoptosis, but not Ca2+ levels.
- An antibody against cluster of differentiation antigen 14 (CD14) inhibited Ca2+ and apoptosis.
Conclusions:
- CD14, Ca2+, and ROS mediate monocyte-induced RPE apoptosis.
- cADPR plays a role in these inflammatory changes.
- Understanding these pathways may lead to new treatments for AMD and other retinal diseases.
