Differences of Gpnmb expression in M1 and M2 bone marrow-derived macrophages in mouse

Abstract

Objective To investigate the differences of glycoprotein non-metastatic melanoma b （Gpnmb） expression between M1 and M2 bone marrow-derived macrophages （BMMφs） in mouse. Meth- ods Primary BMMφs were cultured and then identified by immunofluorescenee staining for F4/80 and flow cytometry testing of CDllb. Interferon-γ and lipopolysaccharide were used to induce differentiation of BMMφ towards M1 macrophages and interleukin-4 was adopted to induce differentiation of M2 macropha- ges. Realtime PCR was performed to analyze mRNA expressions of tumor necrosis factor （TNF-α）, induc- ible NO synthase （ iNOS）, macrophage mannose receptor （MMR）, arginase-1 （ Arg-1 ） and Gpnmb. Pro- teins of Gpnmb and MMR were detected by double immunofluorescence staining, Western blot and flow cy- tometry. Results （1） Immunofluorescence staining showed high expression of F4/80 in BMMφs and flow cytometry results showed that CDllb was expressed in 92.7% ±6.1% of BMMφs, suggesting that primary BMMφs were successfully cultured. （2） Compared with M0 BMMφs, mRNAs of TNF-α and iNOS were highly up-regulated in M1 BMMφs （ both P〈0. 01 ）, and mRNAs of MMR and Arg-1 were highly up-regula- ted in M2 BMMφs （ both P〈0.01 ）, indicating that differentiation of BMMφs towards M1 and M2 BMMφs were successfully induced. （3） Expressions of Gpnmb mRNA and Gpnmb protein were predominantly up- regulated in M2 BMMφs in comparison with those in M0 and M1 BMMφs （ both P〈0. 01 ）. Gpnmb and MMR were co-expressed in M2 BMMφs and 83.2% ±9.7% of MMR positive BMMφs expressed Gpnmb. Conclusion Gpnmb expression is significantly increased in M2 macrophages than that in M1 macrophages in vitro, indicating that Gpnmb which takes part in the differentiation of macrophages might be used as a marker for identification of M1 and M2 macrophages. Key words: Gpnmb;  Bone marrow-derived macrophages;  M1 macrophages;  M2 macrophages;  MMR