Up-regulation of cytokines and activation of oncogenic intracellular signaling molecules in myeloid cells of c-fms-rtTA/(TetO)₇-CMV-MMP12 bitransgenic mice. (A) Plasma samples were collected from 3-month doxycycline-treated wild-type (WT) mice, doxycycline-treated (+DOX), or untreated (−DOX) bitransgenic mice. The concentrations of IL-1 β, IL-6, MIP-2, and TNF-α were measured by ELISA. Results are the mean ± SD, n = 4. (B) Intracellular staining of phosphor-Stat3 in BM CMP and GMP progenitor cells of 3-month wild-type (WT, blue line), doxycycline-treated (+DOX, red line), or untreated (−DOX, green line) bitransgenic mice by flow cytometric analysis. The shaded areas were isotype controls. (C) Intracellular staining of phosphor-Stat3 in CD11b⁺/Gr-1⁺ cells from the blood, spleen, and lung of 3-month doxycycline-treated (+DOX, red line) or untreated (−DOX, green line) bitransgenic mice by flow cytometric analysis. The shaded areas were isotype controls. (D) Lin⁻ progenitor cells were isolated from the BM of wild-type mice and cultured in vitro in the absence and presence of inactivated (inact) or activated (act) MMP12. After 12 hours, cultured cells were stained with CD11b and Gr-1 Abs for flow cytometric analysis. (E) The concentrations of secreted IL-6 and IL-10 in the above cultured medium were measured by ELISA. Results are the mean ± SD, n = 4. (F) In the above study, intracellular staining of phospho-Stat3, NFκBp65, and C/EBPα in CD11b⁺/Gr-1⁺ cells was analyzed by flow cytometry. The shaded areas were isotype controls. (G) Lin⁻ progenitor cells were isolated from the BM of wild-type and bitransgenic mice, and cultured in vitro. Cells were treated with doxycycline for 4 days followed by flow cytometric analysis with CD11b and Gr-1 Ab staining. (H) The concentrations of secreted IL-6 and IL-10 in the above cultured medium were measured by ELISA. Results are the mean ± SD, n = 4. (I) In the above study, intracellular staining of phospho-Stat3 in CD11b⁺/Gr-1⁺ cells was analyzed by flow cytometry. The shaded area was isotype control. (J) Wild-type CD4⁺ T cells from the spleen were cultured and stimulated with anti-CD3 mAb plus anti-CD28 mAb with or without doxycycline treatment. After 72 hours, T cells were stained with anti-CD69 and CD4 Abs. A representative flow cytometric analysis is demonstrated.