Figure 8.

TNF is highly expressed in the Sh3bp2 gain-of-function mutant DCs and macrophages. (a,b) Bone marrow cells were isolated from 14- to 15-week-old WT, Sh3bp2^KI/+, Fas^lpr/lpr, and Sh3bp2^KI/+Fas^lpr/lpr mice and pre-cultured with GM-CSF (20 ng/mL) and IL-4 (5 ng/mL) for 8 days; resulting BMDCs were used for the experiments. (a) Tnf mRNA levels relative to that of Hprt were determined by qPCR. (b) TNF protein levels in culture supernatants. Culture supernatants were collected at the end of BMDC culture, and TNF levels were determined by ELISA. (c) Tnf mRNA expression in BMMs. Bone marrow cells were isolated from 10- to 12-week-old WT, Sh3bp2^KI/+, Fas^lpr/lpr, and Sh3bp2^KI/+Fas^lpr/lpr mice and stimulated with M-CSF (25 ng/mL) for 2 days, after which the yielded BMMs were treated with LPS (1 ng/mL) in the presence of M-CSF (25 ng/mL). Tnf mRNA levels relative to that of Hprt were determined by qPCR. (d) Phagocytic capacity of BMMs. Apoptotic Jurkat cells were labeled with pH-sensitive fluorescent dye and co-cultured with BMMs, followed by the measurement of fluorescence intensity derived from the engulfed apoptotic cells. Values are presented as the mean ± SD. Note: * P < 0.05. SH3BP2, SH3 domain-binding protein 2; WT, wild-type; KI, knock-in; BMDC, bone marrow-derived dendritic cell; BMM, bone marrow-derived macrophage; GM-CSF, granulocyte-macrophage colony stimulating factor; M-CSF, macrophage colony stimulating factor; IL-4, interleukin-4; LPS, lipopolysaccharide; Hprt, hypoxanthine phosphoribosyltransferase.