Effects Of Fumonisin B1 On HLA Class I Antigen, LMP2 And TAP1 In GES-1 Cells In Vitro

Objective: The classical HLA class I is composed of both units of HLA-A,B,C andβ2m, which refer to present antigens derived from intracellular proteins. The intracellular proteins are degraded by the proteasome, of which LMP2 and LMP7 were both units. Subsequently,the generation of peptide fragments of 8-11 residues in length is transported into the endoplasmic reticulum (ER) by the transporter associated with antigen presentation (TAP) complex, which is a heterodimer of TAP1 and TAP2. Here, nascent class I heavy chain,β2m and peptide fragments are assembled into a heterotrimeric complex and transported through the Golgi apparatus to the cell surface. By binding to a specific peptide/HLA class I complex, the CD8+ T cells with complementary T-cell receptors (TCRs) are stimulated to proliferate and destroy the abnormal target cell.Fumonisins are a group of fungal toxins produced by Fusarium moniliforme that are commonly found on corn and other cereals grains in animal feed and human foods. Fumonisin B1 (FB1) is the major fumonisin produced in culture and as well as naturally occurring in maize and maize-based feeds and foods.FB1 is reported to be carcinogenic in rodents. Moreover, epidemiological investigation was reported that the high rates of esophageal cancer have been correlated with FB1. The current research of FB1 mainly focus on the detection of animal toxicology and cell line experiments without immunal function. Hence, the present research was carried out to determine the immunological effects of FB1 on HLA class I antigen processing and presentation in human gastric epithelial GES-1 cells cultured in vitro to furtherly investigate the mechanism of FB1 on HLA class I antigen presention pathway. This may produce some evidences for antigen presenting defects induced by FB1.Methods:1 cellular culture and treatmentGES-1 cells were cultured in DMEM medium supplemented with 10% new-born calf serum, streptomycin (100μg/ml), penicillin (100U/ml) at 37℃, 5% CO2 . FB1 was added into culture flasks with GES-1 cells in the exponential growth phase to obtain the concentration of FB1 as designed. After durations of the incubations, cells were scraped and collected for extraction of RNA and protein.2 RNA extraction, cDNA synthesis and PCRThe impact of FB1 on the expression of HLA-A,-B,-C,β2m, LMP2 and TAP1 on mRNA levels in GES-1 cells were determined with RT–PCR method.3 Protein extraction and Western blotThe expressed status of HLA-A,B,C,β2m, LMP2, and TAP1 at protein levels was analysized using Western blot, respectively.4 Immunocytochemistry analysisThe immunocytochemistry pattern was carried out to show the status of HLA-A,B,C,β2m, LMP2, and TAP1 at protein levels in the cytoplasm and/or membrane.5 Statistical analysisAll results were expressed as mean±S.D. from each group and the statistical analysis was performed with one-way analysis of variance (ANOVA). The dose-effect relationship was analysised with Pearson analysis. All statistical analysis were calculated by SPSS 13.0 statistical software. The P-values less than 0.05 were considered to be significant.Results:1 The effect of FB1 on HLA-A, B and C1.1 The dose-effect of FB1 on HLA-A, B and C The expression of HLA-A mRNA was decreased significantly (P<0.05) in different concentration of FB1 treated groups compared with control group(r=-0.767,P<0.01). FB1 could also downregulate the expression of HLA-B mRNA only in 10μM and 20μM treatment groups (P<0.05). However, no significant differences could be detected about the dose-effect of FB1 on HLA-C mRNA (P>0.05).The results of Western blot showed that the expression of HLA-A,B,C was decreased significantly in a dose-dependent manner(r=-0.848, P<0.01).The result of ICC was the same as that of Western blot(P<0.05).1.2 The time-effect of FB1 on HLA-A, B and C The results of expression of HLA-A mRNA showed a tendency of decrease in a time-dependent manner(r=-0.792, P<0.01)from 2 to 24h. And then, a tendency of increase could be detected range in 24 to 72h with no significant relationship with the time of exposure(r=0.182, P>0.05).The expression of HLA-B mRNA was decreased gradually from 2 to 12h(r=-0.683, P<0.01)and then increased from 12 to 72h without in a time-dependent manner.No significant difference could be detected about the expression of HLA-C mRNA in 72h compared with control groups, respectively(P>0.05).The results of HLA-A,B,C at protein level showed a tendency of decrease in 24 hours(r=-0.770, P<0.01)and then increase until 72h(r=0.976, P<0.01). However, the relative expression of HLA-A,B,C is lower than that in control group, respectively.2 The effect of FB1 onβ2mBoth dose- and time- effects of FB1 revealed that there was no significant differences between control and FB1 treatment groups on the expression ofβ2m mRNA. It suggested that there is no effect of FB1 on the expression ofβ2m mRNA.3 The effect of FB1 on LMP23.1 The dose-effect of FB1 on LMP2The expression of LMP2 mRNA revealed significantly decreased at 10 and 20μM FB1 treatment groups (P<0.05). However, FB1 did not cause any significant changes at the concentration of 5μM(P> 0.05).The results of Western blot showed a tendency of decrease in a dose-dependent manner ( r=-0.812, P<0.01 ) . The predominant differences were also observed in each FB1 treatment group in the results of ICC compared with control group(P<0.01).3.2 The time-effect of FB1 on LMP2The RT-PCR results showed a significant decrease before 12h(r=-0.452, P>0.05). And then, the trend of increase with the time was detected from 12 to 72h(r=0.803, n=3, P <0.01).The expression LMP2 at protein level in time-effect reseach showed a decreased tendency in 24 hours(r=-0.972, P<0.01), and then increased compared with control groups, respectively(r=0.061, P>0.05).4 The effect of FB1 on TAP14.1 The dose-effect of FB1 on TAP1The result of RT-PCR showed the significant differences between 20μM FB1 treatment group and control group(P<0.01), while no significant difference was found in 5μM and 10μM FB1 treatment group(P>0.05).The results of Western blot showed the higher conentration of FB1(10 and 20μM) caused significantly decrease on the expression of TAP1 at protein level compared to control group with no significant change in 5μM FB1 treatment group. The results of ICC the expression of TAP1 both in 10 and 20μM FB1 treatmnet groups were predominantly decreased (P<0.01), while no significant change could be observed in 5μM group.4.2 The time-effect of FB1 on TAP1For 20μM FB1 treatment groups, the expression of TAP1 mRNA showed a significant decrease ranging in 2 to 12 hours(r=-0.783, P<0.05)and then increased gently from 12 to 72h (r=0.679, P<0.01).The time-effect of FB1 on the expression of TAP1 at protein level showed a tendency of decreased in 12 hours(r=-0.860, P<0.01) and then increased with no significance association between the expression of TAP1 and the time of exposure(r=0.560, P>0.05).Conclusion:1 FB1 could inhibit the expression of HLA-A, HLA-B, LMP2 and TAP1 at mRNA level and decrease the expression of HLA-A,B,C, LMP2 and TAP1 at protein level in GES-1 cells in vitro.2 The expression of HLA-A,B,C, LMP2 and TAP1 in GES-1 cells treated by 20μM FB1 showed a tendency of decrease and then increase both at mRNA level and protein level from 2 to 72h.3 No signifficant difference was found about the effect of FB1 on HLA-C at mRNA level and onβ2m both at mRNA level and protein level.4 The results abovementioned suggesteds that FB1 could decrease HLA class I molecular by downregulation the expression of classical HLA-A,B,C and/or LMP2, TAP1.