Figure 1.
NE regulates macrophage proliferation and maturation from BM.
Unfractionated BM cells were plated in a 24-well plate at 2 x 106 cells/well and cultured for 7 days in hormone-deficient medium with murine M-CSF alone, or in varying concentrations of NE (1 x 10-8 M or 1 x 10-6 M) added at day 0, or in the presence of 1 x 10-6 M of NE added at different time points (day 0, 3 and 6). BMM proliferation was examined using the CyQUANT Cell Proliferation Assay Kit. Briefly, 2 x 105 BM cells were seeded into each well of a 96-well plate and treated with various doses of NE or without NE. At day 7, cells were collected and total cell numbers were counted based on the standard curve (A). To phenotype BMM, cells stained with Abs for CD11b, MHC II and F4/80. Representative dot plot data of the percentage of MHC II+/F4/80+ Mφ are shown in (B). The graphic format of the percentage of MHC II+/F4/80+ Mφ is shown in (C). The time course of NE effects is shown in (D). Data show mean ± SD of 4 independent experiments. Significant difference is indicated as * p<0.05 or **p<0.01, compared to untreated control or different time points.
Figure 2.
High dose of NE decreases CSF-1R expression.
Unfractionated BM cells were cultured with murine M-CSF alone, or in the presence of varying concentrations of epinephrine. At day 7, cells were collected and stained with Abs for CD11b, F4/80 and CSF-1R. Based on the gating of CD11b+/F4/80+ BMM cells, representative histogram data of CSF-1R expression on Mφ is shown in (A). The corresponding graphic format data of CSF-1R expression on Mφ is shown in (B). Their corresponding MFI of CSF-1R expression on Mφ is shown in (C). Data show mean ± SD of 4 independent experiments. Significant difference is indicated as * p<0.05, compared to untreated control.
Figure 3.
NE inhibits macrophage migration by decreasing CCR2 expression.
BM cells were cultured in hormone-deficient medium with murine M-CSF alone, or in the presence of varying concentrations of NE. At day 7, cells were collected and stained with Abs for CD11b, F4/80 and CCR2. Based on the gating of CD11b+/F4/80+ cells, representative dot plot for percentage of CCR2+/F4/80+ Mφ is shown in (A). The corresponding graphic format data of percentage of CCR2+/F4/80+ Mφ is shown in (B). The comparison of cells that passed through insert filter after 4 hours incubation with MCP-1 (final concentration, 50 ng/mL) in the lower chamber are shown in Figure 2 (C). Data show mean ± SD of 4 independent experiments. Significant difference is indicated as * p<0.05, compared to untreated control.
Figure 4.
In the last 24 hours of 7 day culture of BM cells with the treatment of NE, LPS (50 ng/mL) was added to the culture. At the end of culture, cells were collected and stained with Abs for CD11b, MHC II and CCR2 antibodies. Representative dot plot data and their corresponding graphic format data of percentage of MHC II+/F4/80+ Mφ are shown in (A) and (B) respectively. Representative dot plot data and their correspondent graphic format data of percentage of CCR2+/F4/80+ Mφ shown in (C) and (D) respectively. Data show mean ± SD of 4 independent experiments. Significant difference is indicated as * p<0.05 and ** p<0.01, compared to untreated control.
Figure 5.
NE regulates antigen uptake by BMM.
The whole bone marrow cells from C57BL/C mice were cultured with M-CSF in the presence of NE (1 x 10-8 M and 1 x 10-6 M) for 7 days. At day 7, FITC-Dextran (200µg/mL) was added to the culture for further 30 min culture at 37°C. As a negative control, FITC-Dextran-added wells were incubated at 4°C for 30 min. At end of the experiment, cells were collected and stained with antibodies for CD11b and F4/80. The percentage of FITC-Dextran-positive CD11b+/F4/80+ BMM was obtained by gating on CD11b+/F4/80+ BMM. Representative flow cytometric data are shown in (A) and corresponding graphic data are shown in (B). Bars represent FITC-Dextran-positive DCs as the mean of 4 independent experiments ± SD. *p<0.05; ** p<0.01, compared to untreated control (0 M).
Figure 6.
NE enhances TNF-α production from BMM.
Bone marrow cells were cultured with M-CSF in the presence of NE (1 x 10-8 M and 1 x 10-6 M). At day 7, LPS (50 ng/mL) and the protein transport inhibitor GolgiPlug (final concentration, 1 µg/mL) were added to the culture for further 4 hour culture and then cells were collected and sequentially stained with membrane antibodies for CD11b and F4/80, and intracellular antibody of TNF-α. Representative flow cytometric data was shown in (A) and corresponding graphic data in (B). Graph bars represent TNF-α+ BMM as the means ± SD of 2 independent experiments in triplicate each time. ** p<0.01, compared to untreated control (0 M).
Figure 7.
NE regulates MafB expression of BMM.
At the end of the 7 day BMM culture, cells were harvested and stained with antibodies for phenotypic markers CD11b and F4/80 followed by sequential intracellular staining with primary MafB antibody and FITC-conjugated secondary antibodies. Representative dot plot data of percentage of MafB+ BMM were shown in (A) and their corresponding graphic format in (B). The MFI of MafB+ BMM were shown in (C). Data represent samples in triplicate for each time of two independent determinations. * p<0.05, ** p<0.01, compared to non-hormone treated control (0 M).