For double immunofluorescence labeling, tissue sections were incubated with a mixture of primary antibodies at 4 oC overnight. Slides were next reacted with a mixture of donkey anti rabbit IgG R, DyLight 488 conjugated don key anti mouse IgG, and 40, 6 diamidine 20 phenylindole dihydrochloride for 1 h. Images were acquired and processed digitally under a fluorescence microscope. Cell lines and treatment MH7A was a generous gift from Dr. David Yu, which was isolated from intra articular soft tis sues of the knee joints of RA patients and established by transfection with the SV40 T antigen. Cells were cultured in DMEM supplemented with 10% FBS, peni cillin and streptomycin in a humidified atmosphere of 5% CO2 at 37oC. Cells were exposed to recombinant human IL 29 at various concentrations or TNF a.
At 24 h and 48 h following incubation, cells were collected for the detection of IL 6, IL 8, IL 10, IL 17 and matrix metalloproteinase 3 by real time PCR. Immunofluorescence staining of IL 28Ra in cells The biological activity of IL 29 was determined in part by the expression level of its specific receptor chain IL 28Ra. MH7A cells were washed in PBS twice for 1 min and then fixed with 4% paraformaldehyde for 15 min. The cells were incubated with rabbit anti human IL 28Ra antibody for 1 h in 37oC. After incubation, the cells were washed twice and further incubated with goat anti rabbit IgG/ TRITC for 1 h in room temperature. Finally, the cells were washed and incubated with DAPI staining solution for 2 min, and analyzed by fluorescence microscopy.
IL 28Ra was stained red and nuclei were stained in blue. Statistical analysis Statistical Dacomitinib analyses were performed with SPSS version 18. 0 software. Data were expressed as mean SD. Differences between two groups were per formed with Students t test for parametric data and Mann Whitney U test for nonparametric data. The Pear son correlation test was used to evaluate the correlation between serum IL 29 levels and laboratory values and clin ical features. For all experiments, P 0. 05 was considered as significant. Results Upregulated expression of IL 29 and its receptor transcripts in PBMCs from RA patients To explore whether IL 29 was involved in the pathogen esis of RA, we first examined the expression of IL 29 mRNA and its receptor IL 28Ra in PBMC by real time PCR.
It was found that expression of IL 29 and IL 28Ra mRNA was significantly higher in RA PBMCs when compared to HC. Increased serum and SF levels of IL 29 in RA patients To examine the protein level of IL 29 in serum and SF, we measured the concentrations of IL 29 in the serum of RA patients and HC and in SF from patients with RA and OA. Our results showed that serum levels of circu lating IL 29 were significantly higher in RA than those in HC. Similar to serum samples, the mean level of IL 29 in SF was increased in RA compared to OA .