Figure 6

Differentiation into CCR9+Mφs in BM derived monocytes was mediated by interaction with activated HSCs.

Total BM, PB, or liver mononuclear cells were cultured for 6 hours with extracts of whole liver or spleen from WT mice treated with PBS or Con A (6 hr or 12 hr). All extracts were prepared at same protein concentration (7 mg/mL). (a) Left: Representative CCR9 and CD11b staining on total BM cells cultured with whole liver extracts (upper) or spleen extracts (lower). Middle: Representative CCR9 expression on CD11b⁺ gated BM cells. Right: Percentage of CCR9⁺ cells in CD11b⁺ cells cultured with whole liver extracts or spleen extracts. Data show mean ± SEM of triplicate samples. (b) Upper: Representative histogram of CCR9 expression on gated BM, PB, or liver CD11b⁺ cells cultured with PBS or Con A injected liver extracts. Lower: Percentage of CCR9⁺ cells in BM, PB, or liver CD11b⁺ cells cultured with whole liver extracts. Data show mean ± SEM of triplicate samples. (c) Fold induction of CCR9, CD80, and MHC class II expression on CD11b⁺ gates BM cells cultured with extracts of LSECs, HSCs or hepatocytes from Con A-treated mice compared to those from PBS-treated mice. Data show mean ± SEM of triplicate samples. (d) Upper: Histogram of CCR9 expression on CD11b⁺ BM cells co-cultured with HSCs isolated from Con A or PBS treated mice for 4 days. Lower: Percentage of CCR9⁺ cells in BM CD11b⁺ cells co-cultured with HSCs. Data show mean ± SEM of triplicated samples. (e) Fluorescence immunohistochemistry of the liver from Con A treated mice. CCR9 (green), F4/80 (red) and GFAP (blue) were shown in a single immunofluorescence for each expression as well as merged co-immunofluorescence (yellow). (f) Percentage of CCR9⁺ cells in CD11b⁺ BM cells cultured with retinoic acid in vitro for 4 days. Data show mean ± SEM of triplicated samples. *p < 0.05, **p < 0.01, ^††p < 0.01, n.s.: not significant.