Mechanisms of glucocorticoid induced tumor necrosis factor ligand in regulating the inflammatory reaction

Objective　To investigate the role of glucocorticoid　induced tumor necrosis factor ligand (GITRL) in regulating the inflammatory reaction of kupffer cells. Methods　The kupffer cells and T cells of mice were isolated and divided into 6 groups after being co－cultured: control group, kupffer cells and T cells were cultured in DMEM only; lipopolysaccharide (LPS) group, kupffer cells and T cells were cultured in media　with　LPS (1 mg/L); LPS+GITRL siRNA group, kupffer cells and T cells were cultured in media as the LPS group after transfected with GITRL siRNA; LPS+control siRNA group, kupffer cells and T cells were cultured in media as the LPS group after transfected with control siRNA; LPS+pEGFPN1 GITRL group, kupffer cells and T cells were cultured in media as the LPS group after transfected with pEGFPN1 GITRL plasmid; LPS+pEGFPN1 control group, kupffer cells and T cells were cultured in media as the LPS group after transfected with control plasmid. After 24 hours of treatment, the expressions of GITRL and PDL1 of kupffer cells were detected by immunofluorescence and western blot, respectively. The proliferation and apoptosis of T cells were measured by MTT assay and Annexin Ⅴ/PI flow cytometry, respectively. The expression of tumor necrosis factor (TNFα) in the supernatant fluid was measured by ELISA. All data were analyzed using the independent　t　test and one－way analysis of variance. Results　The transfection efficiencies of GITRL siRNA and pEGFPN1 GITRL were 90% and 85%, respectively. Compared with normal kupffer cells, the protein expression of GITRL of kupffer cells transfected with GITRL siRNA was significantly decreased, while the protein expression of GITRL of kupffer cells transfected with pEGFPN1 GITRL was significantly increased (t=41.72, 13.10, P<0.05). There was no significant difference in the protein expressions of GITRL between normal kupffer cells and those in the control groups (F=2.27, P>0.05). The fluorescence intensity of GITRL in the LPS group was significantly higher than that in the control group t=49.29, P<0.05). Compared with LPS group, the activation of GITRL expression by the LPS was significantly suppressed in the LPS + GITRL siRNA group (t=9.84, P<0.05), while the expression　of GITRL in the LPS + pEGFPN1 GITRL group was significantly increased (t=5.78, P<0.05). There was no significant difference in the GITRL expression among the LPS+control siRNA group, LPS+pEGFPN1 control group and LPS group (F=0.86, P>0.05). The expression of PDL1 in the LPS group was significantly lower than that in the control group (t=18.83, P<0.05). Compared with LPS group, the expression of PDL1 in the LPS+pEGFPN1 GITRL group was significantly suppressed (t=11.79, P<0.05), while the expression of PDL1 in the LPS+GITRL siRNA group was significantly stronger (t=19.08, P<0.05). There was no significantly difference in the expression of PDL1 in the LPS+control siRNA group, LPS+pEGFPN1 control group and LPS group (F=2.22, P>0.05). The proliferation of T cells was increased and the number of apoptotic T cell was decreased in the LPS group when compared with control group (t=49.43, 40.11, P<0.05). Compared with LPS group, the proliferation and apoptosis of T cells in the LPS+pEGFP　N1 GITRL group had the same trend(t=5.77, 12.64, P<0.05); while the proliferation of T cells was decreased and the apoptosis of T cells was increased in the LPS+GITRL siRNA group (t=17.00, 49.90, P<0.05). There was no significant difference in the proliferation and apoptosis of T cells among the LPS+control siRNA group, LPS+pEGFP　N1 control group and LPS group (F=1.87, 1.35, P>0.05). The expression of TNFα was significantly higher in the LPS group than that in the control group (t=125.68, P<0.05). Compared with the LPS group, the expression of TNF　α was significantly decreased in the LPS+GITRL siRNA group t=119.65, P<0.05), while the expression of TNFα in the LPS+pEGFPN1 GITRL group was significantly increased (t=147.70, P<0.05). There was no significant difference in the TNFα expression among the LPS + control siRNA group, LPS+pEGFPN1 control group and LPS group (F=0.14, P>0.05). Conclusion　Kupffer cells suppress the expression of PDL1 by upregulating GITRL, and thus activate the proliferation of T cells and promote the inflammatory reaction. The immunologic balance may be recovered　after　 the interference of GITRL to restrain the inflammatory reaction.