Roles of complement receptor 3 on murine macrophages in recognition of Penicillium marneffei / 中华皮肤科杂志

ABSTRACT

Objective To evaluate the role of complement receptor 3 (CR3) on murine macrophages in the recognition of Penicillium marneffei.Methods RAW264.7 murine macrophage cells were cultured in vitro,and divided into four groups to be cocultured with inactivated and live Penicillium mameffei yeast cells as well as inactivated and live Penicillium marneffei conidia respectively at 37 ℃ in 5％ CO2 for one hour.The RAW264.7 cells incubated with phosphate-buffered saline (PBS) served as the blank control group.Then,reverse transcription-PCR was conducted to detect CR3 mRNA expression,Western blot to measure CR3 protein expression,flow cytometry to determine phagocytosis rate,enzyme-linked immunosorbent assay (ELISA) to quantify cytokine levels in culture supernatant.Some RAW264.7 macrophages were transfected with a specific siRNA targeting CR3 gene and cocultured with inactivated Penicillium marneffei conidia,subsequently,phagocytosis rate and supematant cytokine levels were determined.Data were processed by the SPSS 16.0 software,and one-way analysis of variance (ANOVA) was conducted for inter-group comparisons of these parameters.Results No significant differences were observed in the mRNA or protein expressions of CR3 among the four groups of RAW264.7 cells cocuhured with different forms of Penicillium marneffei (both P ＞ 0.05).The phagocytosis rate was 95.14％,89.56％,91.03％ and 90.78％ in RAW264.7 cells cocultured with inactivated conidia and yeast cells,as well as live conidia and yeast cells of Penicillium marneffei,respectively (P ＞ 0.05).The levels of interleukin (IL)-2,interferon (IFN)-γ,IL-4 and IL-10 in culture supernatant were increased at different degrees after one-hour coculture in the four coculture groups compared with the blank control group,but no statistical difference was noted among the four coculture groups in the supernatant levels of these cytokines (all P ＞ 0.05).After coculture with inactivated Penicillium marneffei conidia,the siRNA-transfected RAW264.7 cells showed a statistical decrease in phagocytosis rate (10.89％ vs.92.78％,P ＜ 0.05) and supernatant levels of IL-2,IFN-γ IL-4 and IL-10 compared with untransfected RAW264.7 cells.Conclusions In early stage of innate immunity,CR3 on macrophages may be one of the pattern recognition receptors participating in the recognition and mediation of phagocytosis of Penicillium marneffei.It's possible that both Thl-and Th2-type cytokines,such as IL-2,IFN-γ,IL-4 and IL-10,are involved in the immune response of macrophages against Penicillium marneffei.