D A Carter1, A D Dick. 1. Division of Ophthalmology, University of Bristol, Bristol Eye Hospital, Lower Maudlin Street, Bristol BS1 2LX, UK.
Abstract
BACKGROUND: /aims: The retina possesses a rich network of CD45(+) positive myeloid derived cells that both surround inner retinal vessels and lie within the retina (microglia). Microglia migrate and accumulate in response to neurodegeneration and inflammation. Although microglia express MHC class II, their role remains undefined. The aims of this study are to investigate changes in human microglia phenotype, migration, and activation status in response to pro-inflammatory and anti-inflammatory stimulation. METHODS: Donor eyes were obtained from the Bristol Eye Bank with consent and whole retina was removed. 5 mm retinal trephines were cultured in glucose enhanced RPMI on cell culture insert membranes for up to 72 hours. The effects of lipopolysaccharide/interferon-gamma (LPS/IFNgamma) and transforming growth factor beta inhibits (TGFbeta) stimulation, alone or in combination, on migration, phenotype, and activation status (iNOS expression) of microglia were studied using immunofluorescence and cytokine analysis by ELISA. RESULTS: CD45(+) MHC class II(+) retinal microglia were observed within retinal explants, and in culture microglia readily migrated, adhered to culture membrane, downregulated MHC class II expression, and produced interleukin 12 (IL-12) and tumour necrosis factor alpha (TNFalpha). Following LPS/IFNgamma stimulation microglia remained MHC class II(-) iNOS(-), and secreted IL-10. Migration was suppressed and this could be reversed by neutralising IL-10 activity. TGFbeta did not affect ability of microglia to migrate and was unable to reverse LPS/IFNgamma induced suppression. CONCLUSIONS: Microglia readily migrate from retinal explants and are subsequently MHC class II(-), iNOS(-), and generate IL-12. In response to LPS/IFNgamma microglia produce IL-10, which inhibits both their migration and activation. TGFbeta was unable to counter LPS/IFNgamma effects. The data infer that microglia respond coordinately, dependent upon initial cytokine stimulation, but paradoxically respond to classic myeloid activation signals.
BACKGROUND: /aims: The retina possesses a rich network of CD45(+) positive myeloid derived cells that both surround inner retinal vessels and lie within the retina (microglia). Microglia migrate and accumulate in response to neurodegeneration and inflammation. Although microglia express MHC class II, their role remains undefined. The aims of this study are to investigate changes in human microglia phenotype, migration, and activation status in response to pro-inflammatory and anti-inflammatory stimulation. METHODS:Donor eyes were obtained from the Bristol Eye Bank with consent and whole retina was removed. 5 mm retinal trephines were cultured in glucose enhanced RPMI on cell culture insert membranes for up to 72 hours. The effects of lipopolysaccharide/interferon-gamma (LPS/IFNgamma) and transforming growth factor beta inhibits (TGFbeta) stimulation, alone or in combination, on migration, phenotype, and activation status (iNOS expression) of microglia were studied using immunofluorescence and cytokine analysis by ELISA. RESULTS:CD45(+) MHC class II(+) retinal microglia were observed within retinal explants, and in culture microglia readily migrated, adhered to culture membrane, downregulated MHC class II expression, and produced interleukin 12 (IL-12) and tumour necrosis factor alpha (TNFalpha). Following LPS/IFNgamma stimulation microglia remained MHC class II(-) iNOS(-), and secreted IL-10. Migration was suppressed and this could be reversed by neutralising IL-10 activity. TGFbeta did not affect ability of microglia to migrate and was unable to reverse LPS/IFNgamma induced suppression. CONCLUSIONS: Microglia readily migrate from retinal explants and are subsequently MHC class II(-), iNOS(-), and generate IL-12. In response to LPS/IFNgamma microglia produce IL-10, which inhibits both their migration and activation. TGFbeta was unable to counter LPS/IFNgamma effects. The data infer that microglia respond coordinately, dependent upon initial cytokine stimulation, but paradoxically respond to classic myeloid activation signals.
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