IL-6 production by stromal cells. (A-B) Purified CLL cells were treated with stromal cell conditioned media (final concentration 30%) in the presence or absence of actemra for 4 hours. Levels of the indicated phosphorylated and native signaling molecules, along with p65 and p100/52 were then measured in whole cell lysates by immunoblotting with β-actin as a loading control. (C) Conditioned media from stromal cells and primary CLL cells were incubated with the custom antibody microarray described in “Methods.” Stromal cells made IL-6, granulocyte macrophage colony-stimulating factor, vascular endothelial growth factor, Axl, growth hormone, IL-9, and IL-11 (marked 1, 2, 3, 4, 5, 6, and 7, respectively) with IL-6 produced in highest amounts. The upper left four spots and lower right two spots (marked ‘0”) are positive controls. (D) IL-6 mRNA was measured by real-time PCR in the stromal line, a BM stromal cell line from another CLL patient (BS), primary CLL cells, and MCF-7 and MDA-231 breast cancer cells. Splenic stromal cells expressed high levels of IL-6 compared with these cells.