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B. Liu, Z. Li, S. P. Mahesh, S. Pantanelli, F. S. Hwang, W. O. Siu, R. B. Nussenblatt; GITR Negatively Regulates Activation of Primary Human NK Cells by Blocking Proliferative Signals and Increasing NK Cell Apoptosis. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5144.
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Our previous studies have shown glucocorticoid-induced TNF-related receptor (GITR) ligand was constitutively expressed in human eye and its expression abrogated immunosuppressive function of ocular tissue. This study is to investigate GITR signaling mechanisms and the potential role of GITR in regulating NK cell activation.
Human PBMCs from normal donors were isolated from their buffy coat using Ficoll gradient centrifugation. Highly purified NK cells were obtained from isolated PBMCs by magnetic sorting technique using a negative NK isolation kit. IL15 (10 ng/ml) and poly(I:C) (25 µg/ml) were used to stimulate NK cells. Total RNA was purified using a Qiagen RNAeasy isolation kit. For cDNA microarray analysis, an array of genes selectively involved in NF-KB pathway was used for analysis. Apoptotic cells were detected by staining of cells with the combination of annexin-V-FITC and Via-probeTM (7-Amino-actinomycin D, 7-AAD) followed by flow cytometry. SDS-PAGE and western blot analysis were used to detect apoptosis related signaling molecules.
GITR was constitutively expressed on human primary NK cells at low levels. It was up-regulated upon stimulation by either TLR ligand or NK cell growth factor, IL-15. cDNA microarray analysis showed that engagement of GITR primarily suppressed the activation of NF-KB pathway of NK cells and up-regulated anti-inflammatory genes heme oxygenase-1 and IL-10. Further analysis revealed that GITR activation suppressed NK cell proliferation in response to IL-15. GITR activation also suppressed pro-inflammatory cytokines secretion and increased NK cell apoptosis. GITR activation resulted in blocked phosphorylation of Stat5 and Akt which may have contributed to the observed anti-proliferative effect of GITR on NK cells. Increased apoptosis was independent of the Fas-FasL pathway but Bcl-XL and phospho-Bad protein expressions were diminished, suggesting involvement of mitochondrial apoptosis pathway.
The results suggest that although GITR is an activation marker for NK cells similar to that for T cells, GITR serves as a negative regulator for NK cell activation. Our studies demonstrate a novel physiological role of GITR on NK cells.
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