Gene transfer of interleukin-1 receptor antagonist into HIG-82 cells

Objective: We had confirmed the increased of interleukin-1β (IL-1β) level and the decreased interleukin-1 receptor antagonist (IL-1ra) level in the synovial fluid from the patients with temporomandibular joint arthritis. Acceleration of suppressive mechanism of inflammation would be mandatory in management of arthritic conditions. In the present study, we tried to establish the sonoporation method as a new gene therapy.

Materials and Methods: HIG-82 cells, a synovial cell line derived from a rabbit knee, were used in this experiment. pVIVO1-GFP/LacZ and pCI-neo-IL-1ra were transfected into HIG-82 cells by sonoporation. To enhance the transfection efficiency, we used SonoVueTM as a micro bubble. The transfection efficiency of pVIVO-1-GFP/LacZ was examined by X-gal staining. The appearance of IL1-ra mRNA was confirmed by RT-PCR, and the expression of IL-1ra was examined by immunocytochemical analysis. IL-1ra-transfected HIG-82 cells were stimulated with lipopolysaccharide (LPS), and the amounts of IL-1β, IL-1ra and prostaglandin E2 (PGE2) were examined by ELISA.

Result: Human IL-1ra mRNA was remarkably expressed in IL-1ra-transfected cells. In immunocytochemical analysis, the expression of hIL-1ra was detected in HIG-82 cells. The treatment of LPS enhanced the production of IL-1β in HIG-82 cells and IL-1ra-transfected HIG-82 cells. IL-1ra-transfected HIG-82 cells spontaneously release IL-1ra in culture supernatant, and its level increased when the cells were cultured with LPS for 48 h. Interestingly, when IL-1ra-transfected HIG-82 cells were cultured with LPS for 60 h, a significant decrease of PGE2 release was observed.

Conclusion: We showed that in vitro sonoporation-mediated transfer of IL-1ra plasmid had a remarkable effect on the regulation of inflammatory mediators. These findings suggest the possible application of sonoporation gene therapy using IL-1ra for the treatment of the inflammation in arthritis.