Figure 6.
Figure 6. The CCL3–CCR1 axis is required for accumulation of MAMs and after extravasation of cancer cells. (A) Levels of chemokine receptors were assessed by flow cytometry in circulating inflammatory monocytes (IMs) and lung MAMs, as indicated. Cells were isolated from WT mice 24 h after E0771-LG tumor cell injection (n = 3). Dotted lines and shaded areas show control isotype matched IgG for each cell type. Representative histograms from three independent experiments are shown. (B) Levels of Ccr1 and Ccr5 mRNA were assessed in IMs, MAMs, and E0771-LG cancer cells. Cells were isolated from WT mice 24 h after E0771-LG tumor cell injection (n = 4). Data are means ± SEM. *, P < 0.05 versus IM. (C) Levels of chemokine receptors were assessed in CD45−CD31+ endothelial cells from the tumor cell–challenged lung. Cells were isolated from WT mice 24 h after E0771-LG tumor cell injection (n = 3). Dotted lines and shaded area show control isotype matched IgG for each receptor. A representative histogram from two independent experiments is shown. (D) Levels of chemokine receptors on cultured E0771-LG tumor cells were assessed by flow cytometry. Dotted lines and shaded areas show control isotype matched IgG for each receptor. A representative histogram from two independent experiments is shown (n = 2). (E) Relative numbers of lung CD11b+ macrophages (top) and circulating CD11b+CD115+ monocytes (bottom) were assessed by flow cytometry in C57BL/6 (WT; n = 14), Ccl3−/− (n = 4), Ccr1−/− (n = 7), and Ccr5−/− (n = 7) mice challenged with E0771-LG cells 24 h before measurement (at least two independent experiments/genotype). Data are means ± SEM. *, P < 0.05 versus WT. (F) Number and size of lung foci were assessed in mice transplanted with BM cells from C57BL/6 (WT, n = 14), Ccl3−/− (n = 11), or Ccr1−/− (n = 9) mice. Mice were injected with E0771-LG cells (two independent experiments). Data are means ± SEM. *, P < 0.01. (G) Number of transmigrated E0771-LG cells was measured in the transendothelial migration assay in the presence of BMDMs from C57BL/6 or Ccr1−/− mice (n = 6, three independent experiments). Mean cell number in each insert was determined by the counts from five fields. Data are means ± SEM. *, P < 0.01. (H) CCR1 protein expression was assessed by flow cytometry in BMDMs, 3B-11 endothlial cells, and Met-1 tumor cells. A representative histogram from two independent experiments is shown (n = 2).

The CCL3–CCR1 axis is required for accumulation of MAMs and after extravasation of cancer cells. (A) Levels of chemokine receptors were assessed by flow cytometry in circulating inflammatory monocytes (IMs) and lung MAMs, as indicated. Cells were isolated from WT mice 24 h after E0771-LG tumor cell injection (n = 3). Dotted lines and shaded areas show control isotype matched IgG for each cell type. Representative histograms from three independent experiments are shown. (B) Levels of Ccr1 and Ccr5 mRNA were assessed in IMs, MAMs, and E0771-LG cancer cells. Cells were isolated from WT mice 24 h after E0771-LG tumor cell injection (n = 4). Data are means ± SEM. *, P < 0.05 versus IM. (C) Levels of chemokine receptors were assessed in CD45−CD31+ endothelial cells from the tumor cell–challenged lung. Cells were isolated from WT mice 24 h after E0771-LG tumor cell injection (n = 3). Dotted lines and shaded area show control isotype matched IgG for each receptor. A representative histogram from two independent experiments is shown. (D) Levels of chemokine receptors on cultured E0771-LG tumor cells were assessed by flow cytometry. Dotted lines and shaded areas show control isotype matched IgG for each receptor. A representative histogram from two independent experiments is shown (n = 2). (E) Relative numbers of lung CD11b+ macrophages (top) and circulating CD11b+CD115+ monocytes (bottom) were assessed by flow cytometry in C57BL/6 (WT; n = 14), Ccl3−/− (n = 4), Ccr1−/− (n = 7), and Ccr5−/− (n = 7) mice challenged with E0771-LG cells 24 h before measurement (at least two independent experiments/genotype). Data are means ± SEM. *, P < 0.05 versus WT. (F) Number and size of lung foci were assessed in mice transplanted with BM cells from C57BL/6 (WT, n = 14), Ccl3−/− (n = 11), or Ccr1−/− (n = 9) mice. Mice were injected with E0771-LG cells (two independent experiments). Data are means ± SEM. *, P < 0.01. (G) Number of transmigrated E0771-LG cells was measured in the transendothelial migration assay in the presence of BMDMs from C57BL/6 or Ccr1−/− mice (n = 6, three independent experiments). Mean cell number in each insert was determined by the counts from five fields. Data are means ± SEM. *, P < 0.01. (H) CCR1 protein expression was assessed by flow cytometry in BMDMs, 3B-11 endothlial cells, and Met-1 tumor cells. A representative histogram from two independent experiments is shown (n = 2).

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