NK cytotoxicity to endothelial cell mediated by its surface major histocompatibility complex class I-related chain A molecule

Abstract

Objective To observe natural killer cell(NK) cytotoxicity to endothelial cell mediated by major histocompatibility complex class Ⅰ-related chain A (MICA) molecule on the surface of endothelial cell.Methods HUVECs, an endothelial line, of experimental group were cultured for 48 h induced by 10 ng/ml recombinant MICA antigen. Another part of HUVECs was added with equal of volume of phosphote buffer salution(PBS) and worked as control group. MICA molecule on endothelial cell surface was detected by flow cytometery (FCM). NK cells were collected by using immumomagnetic beads ( CD56 positive isolation kit) according to the manufacturer's recommended conditions. NK cells and HUVECs induced by MICA antigen were cocultured for 10 h. All the cells were stained by fluoresceins acridine orange(AO) and ernidium bromide(EB).The dead and alive HUVECs were measured under fluorescence microscope, respectively. Killing effect of NK cells was calculated by counting of dead cells among total cells. Levels of IFN-γ and perforin in culfure supernate were detected by ELISA. Results Expression of MICA molecule on endothelial cell surface of experimental group was (32 ± 5.6) % and control group was (6.0 ± 2.4) %. The killing effect of NK cells in experimental group was(35.5 ± 6.4)%, which was significant higher than that in control group, ( 12.6 ± 3.2)%( P = 0.0087 ). Levels of IFN-γ and perforin in experimental group were significant higher than that of control group (P =0.0025,0.0078). Conclusion Expression of MICA molecule on the surface of HUVECs can enhance NK cytotoxicity to HUVECs. IFN-γ and perforin might be involved in the killing effect. Key words: Major histocompatibility complex class Ⅰ-related chain A; Natural killer cell; NKG2D receptor; Cytotoxicity