Abstract
Purpose: :
Amplified generation of reactive oxygen species (ROS), which is also evident during glaucomatous neurodegeneration, has been associated with immune system activation. Based on the evidence of immune system activation in glaucoma, this study aimed to determine the effects of ROS on the phenotype and antigen presenting function of the retina and optic nerve head glia, in vitro.
Methods: :
Primary cultures of rat retinal macroglial and microglial cells, and optic nerve head astrocytes were treated with different compounds generating superoxide anion, nitric oxide, or hydroxyl radical for 24 and 48 hours. The glial cells pre–treated with ROS generating compounds were then co–cultured with syngeneic CD4+ T cells in the presence of antigen. These co–cultures were also incubated in the absence and presence of a non–specific ROS scavenger, trolox. ROS generation and cell viability were assessed using dihydroethidium and calcein assays, respectively. Flow cytometry and immunocytochemistry were performed to determine major histocompatibility complex (MHC) class II molecules on glial cells. In addition, functional experiments were performed to determine proliferation and cytokine secretion of T cells using [3H]–thymidine incorporation and TNF–α enzyme–linked immunosorbent assay, respectively.
Results: :
The MHC class II expression was up–regulated on glial cells, predominantly microglial cells, exposed to ROS. Compared with the control glia, glial cells in ROS generating systems were found to be more potent inducers of T cell activation in a cell density– and time–dependent manner as assessed by increased proliferation (approximately 3–fold) and increased TNF–α secretion (approximately 6–fold) of T cells. Despite mitomycin C treatment of glial cells before mixing them with T cells, TNF–α levels were higher in the co–culture medium (p<0.05). This excludes the possibility that TNF–α secretion in co–cultures was significantly contributed by glial cells. When the ROS scavenging treatment was applied, ROS–induced MHC class II up–regulation on glial cells was persisted; however, antigen–mediated T cell activation was significantly decreased (p<0.01). This indicates an additional co–stimulatory function of ROS during antigen presentation.
Conclusions: :
These in vitro findings suggest that by stimulating the antigen presenting ability of glial cells as well as functioning as co–stimulatory factor for antigen presentation, ROS regulate the immune response which likely contributes to the neurodegenerative process of glaucoma.
Keywords: oxidation/oxidative or free radical damage • retinal glia • astrocytes: optic nerve head