Soluble CD83 Inhibits Human Monocyte Differentiation Into Dendritic Cells In Vitro

Human CD83 mainly expressed on mature dendritic cells(DCs), It is Type I transmembrane glycoprotein, so it was first described as a molecular marker for mature DC. However, An increasing number of studies have shown that CD83 is not just a marker for DC maturation, but also a immunomodulatory molecule. On the protein level, two different isoforms of CD83 exist: a membrane-bound form and a extracellular domain-released form, called soluble form. Intriguingly, the m CD83 possesses stimulatory effects on immune response, on the contrary, the s CD83 has inhibitory effects. In the process of monocyte differentiation into DCs,(m CD83) has the immune stimulation ability which enhance the body’s immune ability.But whether the s CD83 has the inhibitory effects on human monocyte differentiation into DCs is unknown.To this end, the buffy coat containing peripheral blood leukocytes(PBL) separated from the anti-coagulated blood sample from healthy donors were obtained from a local blood donation centre. The buffy coats centrifuge through a Ficoll-Paque gradient. PBLs were collected from the gradient interface.The washed cells were re-suspended cultured in T75 flask for 2 h. DCs were generated from monocytes for 6 or 7-day culture in the presence of GM-CSF. On the other way,we prepare recombinant s CD83 fused with His-tag, the PCR fragment containing human CD83 extracellular domain was amplified by RT-PCR,in which Kpn I and Age I sites were antisense primers, respectively. The recombinant s CD83 expression was in the HEK293 T cells,which cultured in antibiotic-free DMEM medium were transfected with pc DNA-s CD83/H plasmids or pc DNA3.1A as a negative control using the calcium phosphate transfection method. Some of the HEK 293 T cells were treated with tunicamycin for inhibiting s CD83 glycosylation. The transfected cells were cultured for 48-72 h in the serum-free conditioned medium for s CD83/His expression. The recombinant s CD83/His proteins were isolated and purified with Ni-NTA agarose beads from the culture medium collected after transfection,and identified for s CD83 expression by Western-blot. The mononuclear cells are treated by recombinant CD83 to differentiated to dendritic cells in different conditions,respectively. The cells were immunostained and analyzed on a FACScalibur flow cytometer.The results showed that the s CD83, especially glycosylated s CD83 could bind the monocytes and significantly inhibited the depression of CD14 expressions(P<0.01) and reduced CD1 a, CD80, CD86 and MHC II expressions(P<0.01 or P<0.05) during the differentiation, indicating that the s CD83 can inhibit monocyte differentiation into DCs, and suggesting that a negative feedback regulation may exist in monocyte differentiation into DCs based on s CD83 released from the mature DCs.