Effects Of Human Mesenchymal Stem Cells On Dendritic Cells Via CD200-CD200R Pathway

Objectives:Mesenchymal stem cells are bone marrow (BM) derived cells with the capacity of self-renewal,immunosuppression and to differentiate in vitro into several tissues. MSCs were shown to exert a strong inhibitory effect on cells belonging to either innate or adaptive immunity, including T-cells natural killer (NK) cells,dendritic cells (DCs),B cells.A pioneering clinical study reveal that infusion of MSCs could successfully treat severe acute GVHD.Futher study showed the effect of MSC on GVHD probably reflects the inhibition of DC maturation and function.Dendritic cells (DCs) play a key role in the induction of immunity and tolerance.MSCs alter differentiation,maturation and function of DCs.The mechanisms that MSCs inhibit DC cells involve soluble factors and cell-cell contact.Regarding the MSC-mediated inhibition of DC maturation and function,a major inhibitory role was ascribed,in previous studies,to soluble factors.But some studies showed cell-cell contact probably play a more important role.CD200 is a potent immunoregulatory molecule and elicits suppressive effects on myelo-id cells including microglia by interacting with the CD200 receptor (CD200R).Recently, CD200 was found to be expressed in in normal mesenchymal stem cells.CD200 receptor is almost exclusively expressed by myeloid cells, including macrophages and dendritic cells. Binding of CD200 to CD200R imparts a unidirectional negative signal to CD200R bearing cells by cell-cell contact. So,we consider CD200-CD200R pathway may be a mechanism that MSCs inhibit DC cells.Part one The expression of CD200 in mesenchymal stem cellsMethods 1. BM-MSC were isolated from bone marrow cells and cultured using a previously reported method. BM-MSC were recovered from passages 2-6 for experiment. The proportion of CD200+cells was assessed by flow cytometry and RT-PCR for each passage, respectively.2. BM-MSCs were cocultured with PBMCs in the presence or in the absence of mitogen PHA for 72 hours. Then CD200 was assessed by flow cytometry and PBMC proliferation was measured by means of ELIS A.4. All results were expressed as mean±SD. The statistical comparision were performed using the One-Way ANOVA with the use of SPSS 11.5 software. A P value less than or equal to 0.05 was considered significantly different.Results 1. The expression of CD200 on MSCs from P2 to P6 was assessed by flow cytometry:P2 (17.35%±10.38%,n=11),P3 (26.35%±19.79%,n=11),P4 (34.20%±21.29%,n=11),P5(40.44%±26.28%,n=11). P6(50.19%±26.29%,n=11). The expression of CD200 on MSCs from P2 to P6 was assessed by RT-PCR:P2 (0.33%±0.26%,n=5,P3 (0.48%±0.52%,n=7),P4 (0.88%±0.68%,n=8),P5 (1.34%±0.96%,n=7),P6 (1.99%±1.74%,n=7).2. The presence of PHA in the cultures increased the expression of CD200 on MSC ([44.842±12.958] vs [33.742±7.938], P=0.017). The suppression was stronger when PHA was added into the MSC-PBMC coculture (OD:[0.62±0.03] vs [0.44±0.01], P=0.000)Part Two The effects of human mesenchymal stem cells on dendritic cells via CD200-CD200R pathwayMethods 1 PBMCs were cultured with or without MSCs and the anti-CD200 antibody was added to monocyte-MSC cocultures. Every 2 days 0.3mL was removed and 0.5 mL of media containing plasma and cytokines was added. We got imDC at day 5 and cytofluorimetric analysis was performed to evaluate imDC.To analyze imDCphenotype by FACS.2 ImDCs were cultured with or without MSCs and anti-CD200 antibody was added to imDC-MSC cocultures. ImDCs differentiated under standard conditions,were induced to mature at day 5 by LPS stimulation for 2 additional days.To analyze mDC phenotype by FACS;The PBMC proliferation was measured by means of ELISA;The supernatant were then collected for detection of IL-12 secretion by means of ELISA.3 The MSCs of passage 2 have higher expression of CD200 and the MSCs of passage 5 have lower expression of CD200. MSCs were cultured with or without imDCs under standard conditions and imDC were induced to mature at day 5 by LPS stimulation for 2 additional days. To analyze mDC phenotype by flow cytometry;The PBMC proliferation was measured by means of ELISA; The supernatant were then collected for detection of IL-12 secretion by means of ELISA.Results 1 The imDC phenotype differ between cells cultured under standard conditions(ie,with GM-CSF and IL-4) and cells cultured in the presence of MSCs at day 0 (CD14:[35.13%±14.75%] vs [5.05%±3.66%], P=0.005; CD1a:[11.15%±5.22%] vs [79.86%±4.59%], P=0.000). The imDC phenotype did not differ between cells cultured in the presence of MSCs and the cells added a blocking anti-CD200 antibody (CD14: [48.25%±12.57%] vs [35.13%±14.75%], P=0.138; CDla:[10.52%±12.91%] vs [11.15%±5.22%], P=0.919.2 The mDC phenotype differ between cells cultured under standard conditions(ie,with GM-CSF and IL-4) and cells cultured in the presence of MSCs at day 5(CD83:[46.34%±6.77%] vs [78.31%±6.51%],P=0.002;CD86:[81.42%±8.61%] vs [94.04%±2.62%], P=0.026). The mDC phenotype differ between cells cultured in the presence of MSCs and the cells added a blocking anti-CD200 antibody at day 5 (CD83: [65.95%±13.28%] vs [46.34%±6.77%], P=0.039; CD86:[92.96%±3.42%] vs [81.42%±8.61%], P=0.04).ImDC cultured in the presence of MSCs displayed a severely impaired capability of stimulating PBMC responses compared with control cultures in the absence of MSCs (OD vaule:[0.767±0.036] vs [0.944±0.017], P=0.000).But imDC cultured in the presence of MSCs added anti-CD200 antibody have a stronger suppression index compared with control coltures in the presence of MSCs (OD vaule:[0.859±0.024] vs [0.767±0.036], P=0.001).The IL-12 production was impaired on DCs induced to mature in the presence of MSCs added at day 5 of culture compared with control coltures in the absence of MSCs ([216pg/ml±12pg/ml] vs [346pg/ml±7pg/ml], P=0.000).But imDC cultured in the presence of MSCs added anti-CD200 antibody have more IL-12 production compared with control coltures in the presence of MSCs ([272pg/ml±13pg/ml] vs [216pg/ml±12pg/ml], P=0.000)3 The mDC phenotype differ between cells cultured in the presence of MSCs which have a higher expression of CD200 and cells cultured in the presence of MSCs which have a lower expression of CD200 (CD83:[55.58%±0.46%] vs [68.81%±8.49%], P=0.017; CD86:[74.34%±1.28%] vs [81.86%±3.27%], P=0.005). ImDC cultured in the presence of MSCs which have a higher expression of CD200 displayed a severely impaired capability of stimulating PBMC responses compared with cultured in the presence of MSCs which have a lower expression of CD200 (OD vaule:[0.773±0.011] vs [0.870±0.004], P=0.000). The IL-12 production was impaired on DCs induced to mature in the presence of MSCs which have a higher expression of CD200 (IL-12:[226±6.8]ng/L vs [272±8.0]ng/L, P= 0.001).Conclusions 1 The effects of MSC may be related to CD200 in local immune response environment.2 We show that CD200-CD200R pathway plays a important role in MSC-mediated inhibition of DC.