CSF1 up-regulates Tie2 receptor on CD14+ human monocytes.

<p>(<b>A</b>) CD14+ monocytes were isolated from whole blood using CD14+ microbeads. Cells were fixed and immunostained using anti-human Tie2 receptor antibody or isotype control antibody immediately following isolation (<i>Freshly isolated</i>) or after treated without (-<i>CSF1</i>) or with rhCSF1 (100 ng/ml) (+<i>CSF1</i>) for 24 hours. N = 10 per group and results represent the mean ± SEM of Tie2-positivity. (<b>B</b>) CD14+ monocytes treated with rhANG1 (100 ng/ml), rhANG2 (100 ng/ml) or a dose-response of rhCSF1 (0, 0.1, 1, 10, 100 ng/ml). ANG2 up-regulated Tie2 expression compared to ANG1 and CSF1 induces a dose-escalation of Tie2 on CD14+ monocytes. N = 10 per group and results represent the mean ± SEM of Tie2-positivity. (<b>C</b>) CD14+ monocytes were left untreated (<i>Utx</i>) or treated with rhANG2 (100 ng/ml) (<i>ANG2</i>), rhCSF1 (100 ng/ml) (<i>CSF1</i>), CSF1R neutralizing antibody alone, or pre-treated with the CSF1R Nab for 30 minutes prior to stimulation with rhCSF1 (100 ng/ml) (<i>CSF1R NAb+CSF1</i>) for 24 hours. ANG2- and CSF1-treatment significantly increased Tie2 expression while the CSF1R NAb abrogated this effect. N = 8 per group and results represent the mean ± SEM of Tie2-positivity by flow cytometry. (<b>D</b>) CD14+ monocytes were left untreated (<i>Untreated</i>), pre-treated with CSF1R NAb (40 µg or 80 µg) for 30 minutes then treated with rhCSF1 (100 ng/ml) (<i>CSF1R NAb+CSF1</i>), or with rhCSF1 (100 ng/ml) alone (<i>CSF1</i>) for 10 minutes. Western blot analysis indicates that the CSF1R NAb was effective at reducing Akt1 phosphorylation.</p

CSF1 and HIF pathways independently and synergistically regulate Tie2 receptor expression on monocytes.

<p>(<b>A</b>) Bone marrow-derived macrophages were differentiated from age-matched wild type LysMcre and HIF-1α<i>^fl/fl</i>/LysMcre mice over five days in non-adherent tubes. After, the cells were serum-starved in endotoxin-free RMPI for 24 hours. The cells were treated with PBS or CSF1 (100 ng/ml) for 24 hours followed by immunostaining with antibodies specific for F4/80 and Tie2 receptor. CSF1 induced an increase in F4/80+/Tie2+ cells over PBS-treated cells from the macrophages derived from the bone marrow of wild type LysMcre mice. The macrophages derived from the HIF-1α<i>^fl/fl</i>/LysMcre bone marrow and treated with CSF1 had a significantly smaller percentage of F4/80+ Tie2+ cells than those from the wild type mice. N = 5 per group and results represent the mean ± SEM of percent F4/80+/Tie2+ cells of total F4/80+ cells. (<b>B</b>) Macrophages were derived from the bone marrow of wild type female mice over five days in non-adherent tubes. The cells were serum-starved in endotoxin-free RMPI for 24 hours. The cells were pre-treated with DMSO (<i>vehicle</i>), the PI3 kinase/Akt inhibitor LY294002 (50 µM) (<i>LY294002</i>), the MEK inhibitor U0126 (10 µM) (<i>U0126</i>), or the NF-Kb inhibitor PDTC (100 µM) (<i>PDTC</i>) for 30 minutes. After, the cells were treated with CSF1 (100 ng/ml) (<i>Vehicle+CSF1</i>), LY294002+CSF1 (100 ng/ml) (<i>LY294002+CSF1</i>), U0126+CSF1 (100 ng/ml) (<i>U0126+CSF1</i>), or PDTC+CSF1 (100 ng/ml) (<i>PDTC+CSF1</i>) or left untreated for 24 hours followed by immunstaining with antibodies specific for F4/80 and Tie2. CSF1 induced an increase in the percent of F4/80+/Tie2+ cells compared to vehicle alone. The inhibitors LY294002, U0126, and PDTC alone had no effect on TEM levels. LY294002 pre-treatment significantly reduced the percent of TEMs regulated by CSF1 while U0126 and PDTC had no effect on CSF1 expansion of TEMs. Results represent the mean ± SEM of percent F4/80+/Tie2+ cells of total F4/80+ cells. (<b>C</b>) Bone marrow-derived macrophages were differentiated from age-matched wild type LysMcre and HIF-1α<i>^fl/fl</i>/LysMcre mice over five days in non-adherent tubes. After, the cells were serum-starved in endotoxin-free RMPI for 24 hours. The cells were then pre-treated with DMSO or LY294002 (50 µM) for 30 minutes followed by CSF1 (100 ng/ml) (<i>LY294002+CSF1</i>) or not (<i>vehicle</i>) for 24 hours and then immunostained with antibodies specific for F4/80 and Tie2. The macrophages derived from HIF-1α<i>^fl/fl</i>/LysMcre mice in combination with the PI3 kinase inhibitor LY294002 significantly reduced the percent TEMs to that similar to untreated levels. N = 5 per group and results represent the mean ± SEM of percent F4/80+/Tie2+ cells of total F4/80+ cells.</p

CSF1 pre-treatment augments the migratory response to ANG2 by CD14+ monocytes.

<p>(<b>A</b>) CD14+ monocytes were isolated and cultured in Boyden chemotaxis chambers in minimal media alone (<i>Media</i>), in media containing 0.1, 1, 10, 100 or 300 ng/ml rhANG2, or with the same ANG2 doses but first pre-treated for 24 hours with media containing 10 ng/ml rhCSF1 and analyzed for their migratory ability. A significant synergistic effect of CSF1 pre-treatment was first observed at 1 ng/ml rhANG2 and peaked at 100 ng/ml rhANG2. N = 8 and results represent the mean ± SEM of CD14+ monocyte migration through the Boyden chamber. (<b>B</b>) CD14+ monocytes were treated with rhANG2 (10 ng/ml) <i>(ANG2</i>) or rhCSF1 (10 ng/ml) (<i>CSF1</i>) alone, or pre-treated with rhCSF1 (10 ng/ml) for 24 hours, washed 3x, then treated with rhANG2 (10 ng/ml) for another 24 hours and transfected with a scrambled siRNA or an siRNA targeting the human Tie2 receptor. While ANG2 and CSF1 did not induce significant migration, the CSF1-pre-treated cells transfected with the scrambled siRNA migrated significantly more than those cells transfected with siRNA targeting Tie2. N = 8 and results represent the mean ± SEM of CD14+ monocyte migration through the Boyden chamber.</p

CSF1 has no effect on tumor growth but increases percent tumor TEMs and augments angiogenesis.

<p>(<b>A</b>) After two weeks of treatment, tumors were removed, homogenized and immunostained with antibodies specific for F4/80 and Tie2 to identify total F4/80+ cells and F4/80+/Tie2+ cells (Tie2-expressing macrophages, TEMs). While there was a marked increase in total F4/80+ macrophages with CSF1 treatment, the percent of F4/80+/Tie2+ TEMs was significantly increased in response to CSF1 suggesting a regulatory role for CSF1 in expanding the TEM population. N = 5 mice per group and results represent the mean ± SEM of total F4/80+ and F4/80+/Tie2+ TEMs within the tumors. (<b>B</b>, <i>top and bottom left</i>) PyMT tumors without CSF1 treatment and (<i>top and bottom right</i>) with CSF1 treatment immunostained with CD31 for blood vessels, F4/80 for macrophages, Tie2 for F4/80+/Tie2+ TEMS, and DAPI. Confocal images (using 60× objective (<i>top</i>) and with 3× zoom (<i>bottom</i>) suggest an increase in both F4/80 macrophages and F4/80+/Tie2+ TEMS in the CSF1-treated tumors. <i>Multiply overlap</i> indicates those areas where F4/80 and Tie2 positivity overlap. Individual stains are in <i>Supplementary </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098623#pone-0098623-g003" target="_blank"><i>Figure 3</i></a>. (<b>C</b>, <i>top</i>) Orthotopically implanted PyMT mammary tumors in wild type C57Bl/6 female mice were allowed to become palpable then intraperitoneally treated with PBS (<i>PBS</i>), CSF1 (100 ng in 100 µls) (<i>CSF1</i>), a neutralizing antibody for the CSF1R (50 mg/kg) 4 hours prior to CSF1 treatment (100 ng in 100 µls) (<i>CSF1R NAb+CSF1</i>), the CSF1R antibody alone (<i>CSF1R NAb</i>), an isotype antibody (50 mg/kg) 4 hours prior to CSF1 (100 ng in 100 µls) treatment (<i>CSF1+IgG</i>), or the isotype antibody alone (<i>IgG</i>) three times per week for two additional weeks. The tumors were immunostained with a CD31-Alexa Flour 546 antibody to recognize endothelial cells that comprise blood vessels. Qualitatively, CSF1 treatment increased the percent of CD31-postitive pixels per high powered field compared to PBS treated tumors, while the neutralizing antibody to CSF1R suppressed the CSF1 effect on angiogenesis. (<b>B</b>, <i>bottom</i>) Quantitatively, the percent of CD31+ pixels per high powered field were quantified as blood vessels (angiogenesis) using Adobe Photoshop histogram analysis. CSF1 treatment significantly increased CD31-positive pixels (angiogenesis) compared to PBS. The neutralizing antibody for CSF1R significantly reduced the ability of CSF1 to up-regulate angiogenesis. N = 5 mice per group and results represent the mean ± SEM of percent CD31-positive pixels per high powered field (HPF).</p