Effects Of MM-CSF-expressing Hematopoietic Malignant Cells In The Functional Polarization Of Macrophages

Macrophages have long been considered to be important immune effector cells, with great plasticity in their phenotype and function. Under different microenvironment, macrophages can be polarized to assume different phenotypes and show different functions. As everything has the pros and cons, in tumor tissues, macrophages can act both pro-and anti-tumor functions. Macrophage colony-stimulating factor (M-CSF) is a multifunctional cytokine, whose expression and function is closely associated with the development of many diseases. The membrane form of macrophage colony-stimulating factor (mM-CSF) is an alternative splicing variant of this cytokine. Our previous research showed that mM-CSF could play adhesion molecule-like roles and partly replaced M-CSF functions by juxtacrine mechanism. High expression of mM-CSF was detected in leukemia and lymphoma, which suggested mM-CSF might play important roles in hematopoietic malignancies. Basing on our previous studies on mM-CSF, we are trying to explore the roles of mM-CSF on the functional polarization of macrophages.In this study, high-expressing mM-CSF hematopoietic malignant cells were subcutaneous injected into nude mouse. Immunofluorescent assay were used to detect the distribution and phenotype of macrophages in the tumor tissues. Results showed that tumors formed by mM-CSF-expressing malignant cells had more macrophages infiltrated and those infiltrating macrophages demonstrated the high expression of CD206, surface marker of alternatively activated macrophages, compared to the control group. In order to further investigate the immunotype and function of macrophages in tumor tissues, FACS were employed. Although we can obtain some macrophages, but the number was far from that can be used for further study. So we began to use in vitro cocultured system to investigate the anti-inflammatory/immune modulatory effects of high-expressing membrane bound M-CSF-hematopoietic malignant cells on macrophages. After coculturing murine macrophage cell line, RAW264.7, with Namalwa-M, a cell line stably expressing mM-CSF, and Namalwa-V, a cell line stably transfected with the empty vector as the control, we used Flow Cytometry to detect the expression of the marker of alternatively activated macrophages, CD206, and intracellular cytokine expression IL10, IL-12, IL-6 and TNF a to study the immunophenotype of RAW264.7; phagocytic assays to investigate their functional activity in vitro.Results demonstrated that RAW264.7 cocultured with Namalwa-M consistently showed high-level expression of CD206. Furthermore, these RAW264.7 expressed high levels of IL-10, TNF a and low levels of IL-12, IL-6, as determined by intracellular staining. At gene level, RAW264.7 cocultured with Namalwa-M up regulated the expression of ARG1, VEGFA, CXCL4, MMP9, UPa and down regulated the expression of IL-18. Functionally, RAW264.7 cocultured with Namalwa-M showed a higher level of phagocytic activity.In conclusion, We describe a novel type of macrophage generated in vitro after coculture with mM-CSF-expressing lymphoma cell line that assumes an immunophenotype defined as IL-10-high, IL-12-low, IL-6-low and TNFα-high secreting cells. These mM-CSF-educated macrophages may be a unique and novel type of functional polarized macrophages with a significant role in tumor development.