In vitro binding ability of transglutaminase 3 to dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin / 中华皮肤科杂志

ABSTRACT

Objective To evaluate the recognition and uptake of transglutaminase 3 (TG3) by dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) receptors on the membrane surface of DC-SIGN-transfected human embryonic kidney (HEK) 293T cells and monocytederived dendritic cells (MDDCs).Methods The eukaryotic expression vector pGCMV-enhanced green fluorescent protein (EGFP) containing DC-SIGN gene fragments was transfected into HEK293T cells to prepare DC-SIGN-EGFP-HEK293T cells by using liposome transfection method.CD14+ monocytes were isolated from peripheral blood samples by magnetic bead-based negative selection,and then were induced by granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) to prepare MDDCs.Laser confocal microscopy and flow cytometry were performed to evaluate the recognition and uptake of TG3 protein by DC-SIGN receptors on the surface of HEK293T cells and MDDCs.MDDCs treated without Alexa Fluor 647 dye-tagged TG3 served as blank control group,and those treated with Alexa Fluor 647 dye alone served as negative control group.Results After co-culture with TG3 for 3 hours,laser confocal microscopy and flow cytometry both showed that TG3 could be recognized by and uptaken through DC-SIGN receptors into HEK293T cells and MDDCs.Flow cytometry also revealed that the binding of TG3 to MDDCs could be partially blocked by DC-SIGN blocking antibodies.Neither the negative control group nor the blank control group showed the recognition and binding of TG3 to HEK293T cells and MDDCs.Conclusion TG3 can serve as a kind of autoantigen to be recognized and bound by DC-SIGN receptors,followed by uptake by dendritic cells.