Immunological characteristics of dendritic cells marker CD83 in flounder (Paralichthys olivaceus)

Abstract

Dendritic cells (DCs) are the professional antigen presenting cells, which play important roles in regulating the immune response and tolerance. In mammals, CD83 is considered as a maturation marker for DCs, which is a type I membrane glycoprotein. In this research, the gene sequence of the CD83 in Paralichthys olivaceus was obtained on NCBI, the sequence characteristics were studied and CD83 was detected in the head kidney, spleen, gut, gill, liver and muscle, with lower expression in the liver and muscle and higher expression in the head kidney and spleen. And then the CD83 recombinant protein was expressed and the specific antibodies (Abs) were prepared, transiently expressed CD83 eukaryotic proteins could be identified by western blot and indirect immunofluorescence. Then single cell suspensions of head kidney were obtained and DCs were collected by density gradient centrifugation after 7 days of cultivation. Giemsa staining revealed that the nuclei were kidney and dumbbell shaped, and the cell surfaces had dendritic or pseudopod-like protrusions. In addition, the DCs could phagocytose fluorescent microspheres, and the phagocytosis rate was 79.27±1.01%. After incubation with allogeneic lymphocytes, it was found that the lymphocytes clustered and proliferated significantly with the ratio ranging from 39.0% to 59.8% in a dose-dependent manner, which proved that the DCs could elicit mixed lymphocyte responses. After LPS immunization in vivo, the relative expression of CD83 in the head kidney and spleen showed a trend of increasing and then decreasing, peaking at 6 h. Meanwhile, the expression of CD83, MHC II, CD80/86 and CD40 were significantly up-regulated in DCs after stimulated with LPS for 24 h in vitro. And then IIF revealed that CD83⁺ cells could be detected in the LPS-stimulated group using CD83 Abs, while no positive signal was detected in the control group, which suggested that CD83 protein may be considered as a specific marker for the maturation of DCs in teleost.