Figure 1. Lyst is selectively involved in TLR3- and TLR4-mediated cytokine production. (A and B) Quantitative real-time PCR analysis of Lyst mRNA expression in different tissues (A) and different immune cell types (purified B and T lymphocytes, BMDMs, and BMDCs; B). (C) Lyst mRNA expression in BMDMs stimulated with 500 ng/ml LPS for the indicated times. The expression level of Lyst in unstimulated BMDMs was set as 1. (A–C) Error bars represent mean ± SD from duplicate samples. rel., relative. (D) Lyst mRNA expression levels in WT and Bg-J BMDMs. Data (mean ± SD) are pooled from three independent experiments. (E and F) BMDMs and BMDCs were differentiated by culturing bone marrow cells from WT and Bg-J mice in the presence of either M-CSF (BMDMs; E) or GM-CSF (BMDCs; F). The progress of cell differentiation was monitored at the indicated days of culture by flow cytometric analysis. (E) For BMDM differentiation cultures, CD11b and Gr-1 cell-surface expression is shown. (F) For BMDC differentiation cultures, CD11c and Ly6C cell-surface expression is shown. (G) Mean cell number in BMDM and BMDC cultures obtained from 106 bone marrow cells from WT and Bg-J mice. BMDMs: day 7; BMDCs: day 9. WT, n = 3; Bg-J, n = 4. Data are mean ± SD. (H–M) BMDCs (H–J) or BMDMs (K-M) from WT and Bg-J mice were stimulated for 6 h with the indicated TLR ligands. Release of TNF (H and K), IFN-β (I and L), and IL-12 (J and M) into culture supernatants was measured by ELISA. Graphs show means ± SD from two cultures derived from different mice. UT, untreated. (N) BMDMs from WT and Bg-J mice were analyzed by flow cytometry for expression of TLR3 (intracellular staining) and TLR4/MD2 (cell-surface staining). Shaded histograms show staining with specific antibodies. Dashed lines depict matching isotype controls. GeoMFI, geometric mean fluorescence intensity. (O) Quantitative real-time PCR analysis of Tlr3 and Tlr4 mRNA expression levels in WT and Bg-J BMDMs. Data are mean ± SD from duplicate samples. (P) BMDMs form WT and Bg-J mice were infected with S. Typhimurium at a multiplicity of infection of 5:1. Supernatants were collected at the indicated times after infection, and TNF levels were determined by ELISA. Data are mean ± SD from two cultures derived from different mice. All data are representative of at least three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Student’s t test).
Figure 1.

Lyst is selectively involved in TLR3- and TLR4-mediated cytokine production. (A and B) Quantitative real-time PCR analysis of Lyst mRNA expression in different tissues (A) and different immune cell types (purified B and T lymphocytes, BMDMs, and BMDCs; B). (C) Lyst mRNA expression in BMDMs stimulated with 500 ng/ml LPS for the indicated times. The expression level of Lyst in unstimulated BMDMs was set as 1. (A–C) Error bars represent mean ± SD from duplicate samples. rel., relative. (D) Lyst mRNA expression levels in WT and Bg-J BMDMs. Data (mean ± SD) are pooled from three independent experiments. (E and F) BMDMs and BMDCs were differentiated by culturing bone marrow cells from WT and Bg-J mice in the presence of either M-CSF (BMDMs; E) or GM-CSF (BMDCs; F). The progress of cell differentiation was monitored at the indicated days of culture by flow cytometric analysis. (E) For BMDM differentiation cultures, CD11b and Gr-1 cell-surface expression is shown. (F) For BMDC differentiation cultures, CD11c and Ly6C cell-surface expression is shown. (G) Mean cell number in BMDM and BMDC cultures obtained from 106 bone marrow cells from WT and Bg-J mice. BMDMs: day 7; BMDCs: day 9. WT, n = 3; Bg-J, n = 4. Data are mean ± SD. (H–M) BMDCs (H–J) or BMDMs (K-M) from WT and Bg-J mice were stimulated for 6 h with the indicated TLR ligands. Release of TNF (H and K), IFN-β (I and L), and IL-12 (J and M) into culture supernatants was measured by ELISA. Graphs show means ± SD from two cultures derived from different mice. UT, untreated. (N) BMDMs from WT and Bg-J mice were analyzed by flow cytometry for expression of TLR3 (intracellular staining) and TLR4/MD2 (cell-surface staining). Shaded histograms show staining with specific antibodies. Dashed lines depict matching isotype controls. GeoMFI, geometric mean fluorescence intensity. (O) Quantitative real-time PCR analysis of Tlr3 and Tlr4 mRNA expression levels in WT and Bg-J BMDMs. Data are mean ± SD from duplicate samples. (P) BMDMs form WT and Bg-J mice were infected with S. Typhimurium at a multiplicity of infection of 5:1. Supernatants were collected at the indicated times after infection, and TNF levels were determined by ELISA. Data are mean ± SD from two cultures derived from different mice. All data are representative of at least three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Student’s t test).

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