Ad-NK4 Reverses The Inhibition Of Hepatocyte Growth Factor On Dendritic Cell Immunity

Objectives:To verify the inhibition of hepatocyte growth factor on dendritic cell immunity.Observed the reversion of HGF on DCs via constructing adenovirus carrier recombination with NK4(Ad-NK4)and transfecting it into DCs.Thereby it could provide new ideas and strategies for the treatment of MM with DCs vaccine.Method: Peripheral blood mononuclear cells(PBMNCs)were obtained from peripheral blood of healthy volunteers by density gradient centrifugation and separated and purified by CD14 magnetic beads.The purified CD14+ PBMNCs were induced to differentiate into DCs in the medium containing 100 ng/ml(final concentration)rh GM-CSF and 50 ng/ml(final concentration)rh IL-4.These DCs were transfected by Ad-NK4 and Ad-GFP.The appropriate multiplicity of infection was determined and the transfection efficiency of Ad-NK4 towards DC was verified,using flow cytometry,Western Blot and PCR.There were 4 groups of cells with DCs without adenovirus transinfection and HGF treatment as the blank control group and 3experimental groups with the addition of 50 ng/ml(final concentration)HGF in the culture medium,including DCs without adenovirus transinfection(HGF group),DCs with Ad-NK4(HGF+Ad-NK4 group)and DCs with Ad-GFP(HGF+Ad-GFP group).Cells in culture were collected and the following assays were performed.(1)The cells were collected at d 5 of culture and regarded as immature DCs(im DCs),resuspended into 2×105/ml,added with FITC-dextran(1mg/ml)and blended and cultivated in an incubator at 37 ℃ for another 2 h.Then the cells were washed by PBS and detected in flow cytometry.The dextran positive cells was calculated as the indicator of phagocytic ability.(2)The im DCs were collected for detection of surface molecules of CD209,CD206 and CD16.(3)The im DCs were collected for detection of surface molecules of CD40,CD80 and CD86 in flow cytometry.(4)On the fifth day,TNF-αwas added into the culture medium and cells were collected at d 7 and regarded as mature DCs(m DCs).Flow cytometry was used to detect the surface molecules of CD40,CD80 and CD86.(5)The migration ability of im DCs was tested by Transwell cell migration assay.(6)The activities of matrix metalloproteinase MMP2 and MMP9 were detected by gelatin zymography.(7)The expressions of protein MMP2 and MMP9 were detected by Western Blot.Results(1)With differentiation induction of PBMNCs by GM-CSF and IL-4,DCs with the characteristic immunophenotype could be obtained.After the stimulation of TNF-α,obvious dendrite like protuberances were found under microscope and the cells appeared to be of typical DCs morphology.(2)Ad-NK4 had a high infection rate towards DCs and the appropriate MOI was 100.(3)HGF can inhibit im DCs’ capacity engulfing antigens.The rates of dextran positive cells in HGF group and control group were 26.50 ± 4.78 and 72.53 ± 3.42(P<0.01),respectively.Ad-NK4 reversed the inhibition exerted by HGF on im DCs and the rate of dextran positive cells in HGF-Ad-NK4 group was 70.56 ± 3.46(P<0.05).(4)HGF down-regulated the expressions of antigen phagocytosis related molecules CD206(expression rates of HGF group vs blank control group: 36.43 ± 6.45 vs 54.40± 8.72,P<0.05)and CD16(expression rates of HGF group vs blank control group: 24.25 ± 7.56 vs 38.03 ± 4.96,P<0.05);Ad-NK4 reversed the action of HGF and the expressions rates of CD206 and CD16 were 56.23 ± 4.48(P<0.05)and 31.43±3.79(P<0.05),respectively.(5)HGF down-regulated the expressions of im DCs antigen presentation related molecules CD80(expression rates of HGF group vs blank control group: 40.28 ± 6.52 vs 80.48 ± 11.2,P<0.05)and CD86(expression rates of HGF group vs blank control group: 34.36 ± 7.56 vs76.01 ± 10.23,P<0.05).Ad-NK4 reversed the action of HGF.The expression rates of CD80 and CD86 were 78.3 ± 4.48(P<0.05)and 69.43±8.7(P<0.05),respectively.(6)The migrated cell number of HGF group and blank controlgroup were 20.34 ± 2.13 and 18.49 ± 1.97,respectively.(P<0.05).However,the difference is trivial and make no sense practically.The migrated cell number in HGF-Ad-NK4 group was 19.11 ± 1.87.(7)The gelatin zymography and Western Blot results indicated that HGF increased the activity of MMP9 in DCs;Ad-NK4 reversed this effect of HGF.Conclusions(1)Ad-NK4 might effectively infect DCs.(2)HGF might decrease im DCs’ antigen phagocytic capacity and lower the expressions of antigen phagocytosis related molecules CD206 and CD16,and Ad-NK4 might reverse the inhibitory effect of HGF.(3)HGF might decrease the expression of im DCs antigen presentation related molecules,and Ad-NK4 might reverse this effect of HGF.(4)The promotion effect of HGF on the migration of im DCs is less obvious.(5)HGF might increase the activity of MMP9 in im DCs and Ad-NK4 reverse the activation of HGF.