Journal:

Article Title: Toll-Like Receptors on Hematopoietic Progenitor Cells Stimulate Innate Immune System Replenishment

doi: 10.1016/j.immuni.2006.04.008

Figure Lengend Snippet: Lin- c-Kit+ progenitors in bone marrow express TLRs and their co-receptors.(A) TLR2, TLR4, TLR4-MD-2, or CD14 were analyzed by flow cytometry on mature peripheral cells or Lin- c-Kit+ bone marrow cells. Peritoneal lavage cells from C57BL/6 mice were stained with mAbs to F4/80 together with TLR2, TLR4, TLR4-MD-2, or CD14. Whole spleen cells from C57BL/6 mice were stained with mAbs to B220, Mac-1, Gr-1, and CD11c together with TLR2, TLR4, TLR4-MD-2, or CD14. Whole bone marrow cells from C57BL/6 mice were stained with mAbs to lineage markers as described in Methods and c-Kit together with TLR2, TLR4, TLR4-MD-2, or CD14. Open histograms depict staining with the isotype matched Abs. The results shown are representative of three independent experiments. (B) Whole bone marrow cells from C57BL/6 mice were stained with mAbs to lineage markers, Sca-1, c-Kit, and Flk-2 together with TLR2, TLR4-MD-2, or CD14. Open histograms depict staining with the isotype matched Abs. The results shown are representative of two independent experiments.

Article Snippet: Reagents Recombinant mouse IL-7, stem cell factor (SCF), Flt-3 ligand (FL), M-CSF, GM-CSF, recombinant mouse CD14/Fc chimera protein, and recombinant human CD14 were purchased from R&D Systems (Minneapolis, MN).

Techniques: Flow Cytometry, Staining

Journal:

Article Title: Toll-Like Receptors on Hematopoietic Progenitor Cells Stimulate Innate Immune System Replenishment

doi: 10.1016/j.immuni.2006.04.008

Figure Lengend Snippet: TLRs and related molecules are expressed by hematopoietic stem cells and progenitor cells.(A) Lineage marker negative cells were enriched from bone marrow suspensions before staining with antibodies to hematopoietic subsets and TLR2, TLR4, TLR4-MD-2, or CD14. Open histograms depict staining with the appropriate isotype matched Abs. The results shown are representative of three independent experiments. (B) Total RNA was extracted from each progenitor subset and semi-quantitative RT-PCR was conducted to detect mRNA encoding TLR4, MD-2, TLR2, and CD14. The results are shown as values normalized to peak expression for each of the transcripts and representative of two independent experiments.

Article Snippet: Reagents Recombinant mouse IL-7, stem cell factor (SCF), Flt-3 ligand (FL), M-CSF, GM-CSF, recombinant mouse CD14/Fc chimera protein, and recombinant human CD14 were purchased from R&D Systems (Minneapolis, MN).

Techniques: Marker, Staining, Quantitative RT-PCR, Expressing

Journal:

Article Title: Toll-Like Receptors on Hematopoietic Progenitor Cells Stimulate Innate Immune System Replenishment

doi: 10.1016/j.immuni.2006.04.008

Figure Lengend Snippet: TLR stimulation causes a progression of Lin+ cells from LKS+ cells and soluble CD14 augments the acquisition of Mac-1 and F4/80.(A) Sorted LKS+ cells (10,000 cells/well) from C57BL/6 mice were cultured in the presence of FL and SCF with medium alone, LPS (10 μg/ml) or Pam3CSK4 (1 μg/ml). After 24 or 48 hr in culture, cells were analyzed by flow cytometry for expression of lineage markers and percentages of Lin+ or Lin- cells are indicated. (B) Sorted LKS+ cells (10,000 cells/well) from C57BL/6 mice were cultured in the presence of FL and SCF with medium alone, LPS (10 μg/ml) or Pam3CSK4 (1 μg/ml). After 24 hr in culture, cells were analyzed by flow cytometry for expression of lineage markers and FcγR. (C) Sorted LKS+ cells from C57BL/6 mice were cultured in the presence of FL and SCF with a range of concentrations of LPS (Left, open circles), Pam3CSK4 (Right, open circles) or a combination of mouse CD14-Fc protein (1 μg/ml) plus LPS (Left, filled circles) or Pam3CSK4 (Right, filled circles). After 72 hr in culture, cells were analyzed by flow cytometry for expression of lineage markers. Data represent mean values with standard deviations from triplicate cultures (*P<0.001). The results are representative of three independent experiments. (D) Left, sorted LKS+ cells from C57BL/6 mice were cultured in the presence of FL and SCF with 1 μg/ml LPS (Left, open circles) or a combination of recombinant mouse CD14-Fc protein (1 μg/ml) plus LPS (Left, filled circles). After 24, 48, or 72 hr in culture, cells were analyzed by flow cytometry for expression of lineage markers. The graph depicts cell yields and the results are representative of two independent experiments. Right, sorted LKS+ cells from C57BL/6 mice were cultured in the presence of FL and SCF with a range of concentrations of LPS (Right, open circles) or a combination of recombinant mouse CD14-Fc protein (1 μg/ml) plus LPS (Right, filled circles). After 72 hr in culture, cells were analyzed by flow cytometry for expression of Mac-1 and F4/80. The graph depicts cell yields and the data are representative of two independent experiments. (E) Sorted Flk-2- or Flk-2+ LKS+ cells (10,000 cells/well) were cultured with medium alone, LPS (10 μg/ml) or Pam3CSK (1 μg/ml). Flk-2- LKS+ cells were cultured with SCF. Flk-2 LKS+ cells were cultured with SCF and FL. After 72 h in culture, cells were analyzed by flow cytometry for expression of lineage markers (left panels). Percentages given in quadrants indicate the frequencies of Lin+ or Lin- cells and the bar graphs on the right depict cell yields. The data represent mean values with standard deviations from triplicate cultures and the results are representative of three independent experiments. (*P<0.03: Flk-2- LKS+, *P<0.002: Flk-2+ LKS+)

Article Snippet: Reagents Recombinant mouse IL-7, stem cell factor (SCF), Flt-3 ligand (FL), M-CSF, GM-CSF, recombinant mouse CD14/Fc chimera protein, and recombinant human CD14 were purchased from R&D Systems (Minneapolis, MN).

Techniques: Cell Culture, Flow Cytometry, Expressing, Recombinant

Journal:

Article Title: Toll-Like Receptors on Hematopoietic Progenitor Cells Stimulate Innate Immune System Replenishment

doi: 10.1016/j.immuni.2006.04.008

Figure Lengend Snippet: TLR stimulation allows lymphoid biased progenitors to produce dendritic cells at the expense of B lymphopoiesis.(A) Sorted CLPs (5,000/well) from C57BL/6 or Myd88-/- mice were stimulated in X-VIVO15 medium alone, LPS (10 μg/ml) or Pam3CSK4 (100 ng/ml) plus SCF, FL and IL-7. After 7 days in culture, cells were analyzed by flow cytometry for expression of CD19 and Mac-1 (Left). The bar graphs depict cell yields for CD19+ cells or Mac-1+ cells (Right) and the data represent mean values with standard deviations from triplicate cultures (*P<0.02). The results are representative of those obtained in five independent experiments. (B) Subsets of the recovered cells described in panel (A) were sorted and used to prepare Giemsa-May-Grünwal stained slides. (C) Cultured cells from C57BL/6 mice were also analyzed by flow cytometry for expression of Gr-1 and CD11c. (D) Sorted CLPs from C57BL/6 mice were cultured in the presence of SCF, FL and IL-7 with a range of concentrations of LPS or combinations of recombinant mouse CD14-Fc protein (1 μg/ml) plus LPS. After 7 days in culture, cells were analyzed by flow cytometry for expression of CD19 and Mac-1 (Left). The bar graph depicts cell yields for CD19+ cells or Mac-1+ cells (Right). Data represent mean values with standard deviations from triplicate cultures (*P<0.003). The results are representative of three independent experiments.

Article Snippet: Reagents Recombinant mouse IL-7, stem cell factor (SCF), Flt-3 ligand (FL), M-CSF, GM-CSF, recombinant mouse CD14/Fc chimera protein, and recombinant human CD14 were purchased from R&D Systems (Minneapolis, MN).

Techniques: Flow Cytometry, Expressing, Staining, Cell Culture, Recombinant