| Objective: AGEs and RAGE interaction have been widely studied for their involvement in diabetic complication. These complication are largely vascular and accompanied by inflammation. Because T lymphocyte initiate and modulate inflammatory response, we hypothesized that AGEs might exert immunomodulatory effects in T lymphocyte. To test this hypothesis, we investigated effects of AGEs on the expression of RAGE, cytokines and associated signaling pathway in Jurkat cells.Methods:①Jurkat cells were incubated in presence or absence of AGEs or BSA for 24h, the cells viability was detected by MTT.②Jurkat cells which prestimulated by PHA (0.25μg/ml) for 24h were incubated in different concentrations of AGEs or BSA for another 24h, then the expression of RAGE was determined by western blot; The production of TNF-αand IFN-γwere investigated by ELSIA; To further explore the role of RAGE in expression of cytokines, neutralizing antibody for RAGE was used before AGEs treated.③Prestimulated Jurkat cells were treated with identical concentration of AGEs or BSA for 0, 15, 30 and 60 min respectively, the nuclear translocation of CaMKIV was observed by fluorescence microscopy and confocal microscopy.④Treat prestimulated Jurkat cells with anti-RAGE(1μg/ml)for 24h, we employed the fluorescence microscopy and confocal microscopy for the translocation of CaMKIV.Results:①No changes in cell viability was observed when Jurkat cells were treated with 50-200μg/ml AGEs or BSA for 24h (P>0.05).②Exposure of PHA-actived Jurkat cells to AGEs enhanced the expression of RAGE than did exposure to BSA .③From ELISA, we found a significant increase in TNF-αand IFN-γexpression in Jurkat cells exposed to AGEs compared with BSA. Furthermore, the AGEs-stimulated TNF-αand IFN-γwere significantly suppressed by a neutralizing Abs for RAGE.④After exposure to the AGEs for 30min, the translocation of CaMKIV protein was accelerated and detected by fluorescence microscopy and confocal microscope.⑤In addition, the nuclear translocation of CaMKIV induced by AGEs were blocked by a neutralizing RAGE antibody.Conclusion: The findings from this study suggested that AGEs can up-regulate the expression of RAGE in Jurkat cells, and promote the secretion of inflammatory cytokines TNF-αand IFN-γ. AGEs induced the nuclear translocation of CaMKIV through RAGE. |
