Effect of soluble factors produced by CCL18-stimulated monocyte/macrophages. After 3 days in culture, monocytes/macrophages (5 × 106 cells/condition) were incubated in serum-free RPMI 1640 for 20 hours in the presence of buffer control or 40 nM CCL18. (A) Effect of monocyte-conditioned medium on colony formation. CFUs were determined as described in “CFU assay.” The following additions were made: medium (RPMI 1640) only (bar 1); 1:10 dilution of conditioned medium obtained from monocytes prepared, as described, in the absence of stimulus (control CM; bar 2); 1:10 dilution of conditioned medium from monocytes treated with 40 nM CCL18 (CCL18 CM; bar 3). Mean ± SD of 3 experiments. (B) Image of colony cultured in the presence of CCL18-stimulated conditioned media or control-conditioned media (Leica 10×/0.22 NA objective). (C) Lack of direct effect of CCL18 on CFUs: medium only (bar 1); 40 nM CCL18 added to CFU assay (bar 2); 1:10 dilution of monocyte-conditioned medium (bar 3); 1:10 dilution of conditioned medium of CCL18-stimulated monocytes (bar 4). Mean ± SD from 1 experiment. (D) Antibody arrays were incubated with supernatants from unstimulated or CCL18-stimulated cultures (n = 6 from 6 different donors) using 2 different arrays with partially overlapping specificity. Results of 1 representative experiment are shown. Duplicate spots are represented in the following sequence, as indicated in the figure: positive control, angiogenin, EGF, ENA78/CXCL5, basic fibroblast growth factor, gro-α/CXCL1, interferon-γ, insulin-like growth factor 1, IL-6, IL-8, leptin, MCP-1/CCL2, platelet-derived growth factor, placental growth factor, RANTES/CCL5, transforming growth factor-β, tissue inhibitor of metalloproteinases-1, tissue inhibitor of metalloproteinases-2, thrombopoietin, VEGF, VEGF-B. CCL18 consistently inducedan increase in IL-6, IL-8, MCP-1, MIP-1α (present in an alternate blot), and gro-α, though the baseline of IL-6 production ranged from barely detectable to the highest signal in the unstimulated blot. Similar results were seen with monocytes purified from bone marrow and cultured for 3 days (n = 2; results not shown). (E) ELISA for CCL3/MIP-1α and IL-6 (n = 6) using supernatants prepared in the same way. MIP-1α concentrations were significantly different (Student t test; P < .01), and IL-6 concentrations were always increased, but no significant difference was reached because of the large variability in different donors. (F) Effect of antibody inhibition of SCF on colony formation by CM from CCL18-stimulated cultured monocytes: control CM (bar 1); CCL18 (40 nM) stimulated–CM (bar 2); control CM + control IgG (10 μg/mL; bar 3); control CM + anti-SCF (10 μg/mL; bar 4); CCL18-stimulated CM + control IgG (bar 5); CCL18-stimulated CM + anti-SCF (bar 6). Mean ± SD of 3 experiments performed in triplicate. A statistical difference was observed between CCL18-stimulated CM + control IgG and CCL-stimulated CM + anti-SCF (unpaired Student t test; n = 9; P < .05).
