Figure 1
Figure 1. IRF5 protein is highly expressed in MDDCs and control late-phase TNF secretion. (A) Monocyte-derived dendritic cells (MDDCs) and monocyte-derived macrophages (MDMs) were stimulated with lipopolysaccharide (LPS; 10 ng/mL) for 4 hours and 24 hours, and secreted tumor necrosis factor (TNF) was measured by ELISA. Data show means ± standard error of the mean (SEM) of 5 independent experiments each using monocytes derived from a different donor. **P < .01 (Student t test). (B) MDDCs were stimulated with LPS for 2 hours and then were cultured with T lymphocytes. Anti-TNFR1 or anti-IgG control antibodies were added 6 hours or 24 hours after coculture start. IFN-γ secretion was determined by ELISA after 72 hours of coculture. Data show means ± SEM of 3 independent experiments. *P < .05. (C) Cells were collected at day 0 (monocytes); days 1, 3, 5, and 7 (MDDCs) after differentiation with granulocyte-macrophage colony-stimulating factor (GM-CSF; 50 ng/mL) and IL-4 (10 ng/mL); and days 1, 3, and 5 after differentiation with M-CSF (50 ng/mL; MDMs); total protein extracts were then subjected to Western blot analysis. p38 MAPK was used as loading control. Representative blots of 5 independent experiments each using monocytes derived from a different donor. (D) MDMs were left untreated (cells) or infected with adenoviral vectors encoding IRF5 or empty vector (pBent), stimulated with LPS for 2 hours, and cultured with T lymphocytes. IFN-γ secretion was determined by ELISA after 72 hours of coculture. Data show means ± SD and are representative of 3 independent experiments each using MDMs derived from a different donor. (E) MDMs were left untreated (cells) or infected with adenoviral vectors encoding IRF5, IRF3, or empty vector (pBent) and stimulated with LPS for the indicated time. The amount of secreted TNF protein was determined by ELISA. Data show means ± SD and are representative of 3 independent experiments each using MDMs derived from a different donor. (F) MDDCs were transfected with siRNAs targeting IRF5 (siIRF5) and stimulated with LPS (10 ng/mL) for the indicated time. TNF secretion was compared with control cells transfected with nontargeting siRNA (siC). Data shown are the means ± SD and are representative of 2 independent experiments each using MDDCs derived from a different donor.

IRF5 protein is highly expressed in MDDCs and control late-phase TNF secretion. (A) Monocyte-derived dendritic cells (MDDCs) and monocyte-derived macrophages (MDMs) were stimulated with lipopolysaccharide (LPS; 10 ng/mL) for 4 hours and 24 hours, and secreted tumor necrosis factor (TNF) was measured by ELISA. Data show means ± standard error of the mean (SEM) of 5 independent experiments each using monocytes derived from a different donor. **P < .01 (Student t test). (B) MDDCs were stimulated with LPS for 2 hours and then were cultured with T lymphocytes. Anti-TNFR1 or anti-IgG control antibodies were added 6 hours or 24 hours after coculture start. IFN-γ secretion was determined by ELISA after 72 hours of coculture. Data show means ± SEM of 3 independent experiments. *P < .05. (C) Cells were collected at day 0 (monocytes); days 1, 3, 5, and 7 (MDDCs) after differentiation with granulocyte-macrophage colony-stimulating factor (GM-CSF; 50 ng/mL) and IL-4 (10 ng/mL); and days 1, 3, and 5 after differentiation with M-CSF (50 ng/mL; MDMs); total protein extracts were then subjected to Western blot analysis. p38 MAPK was used as loading control. Representative blots of 5 independent experiments each using monocytes derived from a different donor. (D) MDMs were left untreated (cells) or infected with adenoviral vectors encoding IRF5 or empty vector (pBent), stimulated with LPS for 2 hours, and cultured with T lymphocytes. IFN-γ secretion was determined by ELISA after 72 hours of coculture. Data show means ± SD and are representative of 3 independent experiments each using MDMs derived from a different donor. (E) MDMs were left untreated (cells) or infected with adenoviral vectors encoding IRF5, IRF3, or empty vector (pBent) and stimulated with LPS for the indicated time. The amount of secreted TNF protein was determined by ELISA. Data show means ± SD and are representative of 3 independent experiments each using MDMs derived from a different donor. (F) MDDCs were transfected with siRNAs targeting IRF5 (siIRF5) and stimulated with LPS (10 ng/mL) for the indicated time. TNF secretion was compared with control cells transfected with nontargeting siRNA (siC). Data shown are the means ± SD and are representative of 2 independent experiments each using MDDCs derived from a different donor.

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