The Role Of Human Proximal Tubule Epithelium In The Recruitment Of Th17by CCL20

BackgroundMore recently, Th17cells have been identified as a distinct lineage of CD4+T cells. Th17cells play critical roles in the clearance of extracellular pathogens. However, under certain conditions, these cells can also be associated with the pathogenesis of various autoimmune and inflammatory diseases. IL-17is the main effector molecules of Th17cells. Th17cells express high level of CCR6. CCR6and its only highly specific ligand CCL20form a chemokine-chemokine receptor pair inducing the recruitment of Th17cells. The recent report showed that CCL20expression localized to tubulointerstitial infiltrates and in dilated tubular cells. And IL-17could enhance CCL20expression in airway epitheliums. Based on the above, our study was designed to address a major issue:Can IL-17induce CCL20expression by tubule epithelium, which may lead to further recruitment of other Th17cells in renal inflammation? To figure out this issue is critically important for acquiring a better understanding of the immunopathogenesis of inflammatory autoimmune renal disease.ObjectiveOur study was trying to figure out whether IL-17can induce CCL20expression by tubule epithelium. Methods1. Cell cultureThe human proximal tubule epithelial cell line HK-2was grown in1:1DMEM/F12containing10%FBS,1%streptomycin-penicillin mixture in an atmosphere of5%CO2-95%air at37℃in a humidified incubator.2. Immunocytochemical stainingBriefly, HK-2cells cultured on coverslips were prepared before being incubated with the specific primary antibodies (E-cad, CK-18). To visualize the primary antibodies, cells were stained with horseradish-coupled secondary antibody and signals were detected by DAB.3. Treatment of IL-17For dose-dependent effects of IL-17on CCL20expression HK-2was treated with different concentrations (0,1,10,100ng/ml) recombinant human IL-17. Cultures were harvested24h after the treatment and analyzed by RT-PCR or48h after the treatment and analyzed by ELISA. For time-dependent effects of IL-17on CCL20expression HK-2was treated with or without10ng/ml IL-17. Cultures were harvested at various times (6h,12h,24h,48h) after the treatment for RT-PCR or ELISA analysis.4. RT-PCR expression analysisTotal RNA isolated with TRIzol was reverse transcribed with All-in-One First-Strand cDNA Synthesis Kit (Gene Copoeia). cDNA was analyzed using All-in-One qPCR Mix(Gene Copoeia). The relative mRNA amount in each sample was calculated based on its threshold cycle in comparison to the threshold cycle of GAPDH. The results were presented as2-ΔΔC in arbitrary units. This procedure was performed in at least three independent experiments.5. Measurement of CCL20secretion by ELISACCL20secretion was analyzed by CCL20ELISA Kit (CUSABIO). The CCL20concentration in each sample was calculated based on its O.D. and the standard curve. This procedure was performed in at least three independent experiments.6. Statistical analysisData are expressed as mean±SE. Group differences were calculated by one-way ANOVA. Values of p<0.05were considered significant.Results1. Compared with the controls, E-cad and CK18were positive staining in HK-2cells.2. Compared with the controls, IL-17significantly stimulated CCL20gene expression and protein secretion (P<0.05). The effect of IL-17on CCL20was dose-and time-dependent.ConclusionIL-17could induce CCL20expression by tubule epithelium, which may lead to further recruitment of other Thl7cells in renal inflammation...