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. 2007 Aug 28;104(36):14436–14441. doi: 10.1073/pnas.0702811104

Fig. 1.

Fig. 1.

Rat alveolar macrophages and mouse bone marrow-derived macrophages express CD200 upon multinucleation. (A) Freshly isolated rat alveolar macrophages were plated at confluency over 50% of the surface of each well to promote fusion and multinucleation. After 5 days, they were subjected to immunohistochemical analysis. Note that mononucleated macrophages were positive for MFR/SIRPα and CD44 but not for CD200. (Scale bar, 1 mm.) Also note that multinucleate rat alveolar macrophages contained hundreds of nuclei that were stained with DAPI (blue). (B) Freshly isolated rat alveolar macrophages were plated as in A and subjected to Western blot analysis at the indicated times. Note that CD200 was not detected in macrophages for the first 24 h. (C) Mouse bone marrow-derived macrophages were cultured in the presence of M-CSF (30 ng/ml) and RANKL (50 ng/ml) for the indicated times to induce the differentiation of multinucleate osteoclasts. Cells were analyzed by RT-PCR. Note that mouse bone marrow-derived macrophages expressed transcripts for CD200 receptor (CD200R) but not for CD200. The abundance of CD200 mRNA relative to that of GAPDH, in response to M-CSF (30 ng/ml) and increasing doses of RANKL, was determined. (Scale bars represent standard deviations; n = 3.) (D) Mouse bone marrow-derived macrophages were cultured in the presence of M-CSF (30 ng/ml) and RANKL (50 ng/ml) for the indicated times to induce the differentiation of multinucleate osteoclasts. Cells were subjected to Western blot analysis by using antibodies directed against the indicated antigens. (E) Flow-cytometric analysis (in a FACS) of the expression of CD200. Mouse bone marrow-derived macrophages were isolated from CD200+/+ and CD200−/− mice, cultured in the presence of M-CSF (30 ng/ml) and RANKL (50 ng/ml) and subjected to flow-cytometric analysis at the indicated times with an antibody directed against CD200 and a control isotype antibody. Bone marrow-derived macrophages expressed increasing amounts of CD200 with time in the presence of M-CSF and RANKL, which promote fusion, multinucleation and osteoclastogenesis.

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