American Society of Hematology

Figure 1

$Figure 1. Maturation and polarization of human macrophages and expression of characteristic M1 and M2 markers. (A) Isolation of distinct cell populations from peripheral blood. Granulocytes, monocytes, and lymphocytes were fractionated from peripheral blood of healthy donors. Smears of whole blood and isolated cells were stained to analyze the purity of distinct cell populations. The representative images of stained cells demonstrate high purity fractions of neutrophils (97-99% of granulocytic fraction), lymphocytes (96-99%), and monocytes (92-98%). Images were acquired using an Olympus BX60 microscope (Olympus America, Melville, NY), equipped with a digital DVC video camera and high-performance ImageJ plugin acquisition software (DVC Company, Austin, TX), and processed using Adobe Photoshop. Objective lens, ×40. (B) Maturation and polarization of macrophages. Purified monocytes were incubated for 7 days with M-CSF to differentiate into mature macrophages (M0 phenotype). The M0 macrophages were then polarized for additional 2 days into M1 macrophages by substituting M-CSF for a mixture of LPS and IFNγ and into M2 macrophages by stimulation with IL-4. Note the changes in morphology of rounded, loosely adherent monocytes to elongated, firmly adherent M0 macrophages, which retained their M0 morphology being polarized into M1 macrophages and become more rounded and less adhesive after M2 polarization. Phase contrast images were acquired using an Olympus CKX-41 microscope equipped with Olympus U-LS30-3 video camera (Olympus America, Center Valley, PA) and Infinity Capture software (Lumenera, Ottawa, Canada), and processed using Adobe Photoshop. Objective lens, 20×. (C) Analysis of MMR expression. Immunocytochemical staining was performed for MMR (CD206) on permeabilized cells to visualize both cell surface and intracellular pools of the protein. The staining indicates that MMR (green) is not detectable in freshly isolated neutrophils and monocytes. MMR expression is moderately induced during differentiation of monocytes into M0 macrophages but is inhibited during polarization into M1 macrophages. In contrast, M2 polarization is accompanied by substantial increase in the levels of MMR expression. Cell nuclei were stained with 4,6 diamidino-2-phenylindole (blue). Images were acquired using a Carl Zeiss AxioImager M1m microscope equipped with AxioVision Re.4.6 software (Carl Zeiss Microscopy, Thornwood, NY) and processed using Adobe Photoshop. Objective lens, ×40. Inserted numerical data represent the levels of cell surface expression of MMR as determined by flow cytometry. Indicated is mean fluorescence intensity determined after subtraction of nonspecific values for mouse IgG. (D) Analysis of α-arginase-1 expression. (Upper) Western blot analysis for α-arginase-1 was conducted on cell lysates separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions (40 µg protein per lane). The 38-kDa α-arginase-1 is detectable in M0 and M2 macrophages. (Lower) The blot was reprobed for β-actin (42 kDa) to provide a loading control. Position of molecular weight markers in kilodaltons is indicated on the left. Bar graph, analysis of ARG1 gene expression. Quantitative analysis of ARG1 was performed on mRNA preparations isolated from monocytes and macrophages in comparison with the corresponding levels of housekeeping genes GAPDH and ACTB. Gene expression levels were determined relative to monocytes (1.0). Both protein and gene expression analyses indicate that α-arginase-1 expression is induced during maturation of macrophages from arginase-negative monocytes and is dramatically inhibited during M0 macrophage polarization into the M1, but not M2, phenotype. (E) Analysis of iNOS expression. (Upper) Cell lysates from mature and polarized macrophages (20 µg per lane) were analyzed for iNOS by western blotting under reducing conditions, confirming the induction of 130-kDa iNOS in M2 macrophages. (Lower) The blot was reprobed for α-tubulin (Biolegend) to provide equal loading control. Position of molecular weight markers in kilodaltons is indicated on the left. Bar graph, analysis of NOS2 gene expression. Quantitative analysis of NOS2 was performed on mRNA preparations isolated from macrophages in comparison with the corresponding levels of β-actin. Gene expression levels were determined relative to M0 macrophages (1.0). Both protein and gene expression analyses indicate that iNOS expression is induced during polarization of M0 macrophages toward the M1, but not M2, phenotype.$

Maturation and polarization of human macrophages and expression of characteristic M1 and M2 markers. (A) Isolation of distinct cell populations from peripheral blood. Granulocytes, monocytes, and lymphocytes were fractionated from peripheral blood of healthy donors. Smears of whole blood and isolated cells were stained to analyze the purity of distinct cell populations. The representative images of stained cells demonstrate high purity fractions of neutrophils (97-99% of granulocytic fraction), lymphocytes (96-99%), and monocytes (92-98%). Images were acquired using an Olympus BX60 microscope (Olympus America, Melville, NY), equipped with a digital DVC video camera and high-performance ImageJ plugin acquisition software (DVC Company, Austin, TX), and processed using Adobe Photoshop. Objective lens, ×40. (B) Maturation and polarization of macrophages. Purified monocytes were incubated for 7 days with M-CSF to differentiate into mature macrophages (M0 phenotype). The M0 macrophages were then polarized for additional 2 days into M1 macrophages by substituting M-CSF for a mixture of LPS and IFNγ and into M2 macrophages by stimulation with IL-4. Note the changes in morphology of rounded, loosely adherent monocytes to elongated, firmly adherent M0 macrophages, which retained their M0 morphology being polarized into M1 macrophages and become more rounded and less adhesive after M2 polarization. Phase contrast images were acquired using an Olympus CKX-41 microscope equipped with Olympus U-LS30-3 video camera (Olympus America, Center Valley, PA) and Infinity Capture software (Lumenera, Ottawa, Canada), and processed using Adobe Photoshop. Objective lens, 20×. (C) Analysis of MMR expression. Immunocytochemical staining was performed for MMR (CD206) on permeabilized cells to visualize both cell surface and intracellular pools of the protein. The staining indicates that MMR (green) is not detectable in freshly isolated neutrophils and monocytes. MMR expression is moderately induced during differentiation of monocytes into M0 macrophages but is inhibited during polarization into M1 macrophages. In contrast, M2 polarization is accompanied by substantial increase in the levels of MMR expression. Cell nuclei were stained with 4,6 diamidino-2-phenylindole (blue). Images were acquired using a Carl Zeiss AxioImager M1m microscope equipped with AxioVision Re.4.6 software (Carl Zeiss Microscopy, Thornwood, NY) and processed using Adobe Photoshop. Objective lens, ×40. Inserted numerical data represent the levels of cell surface expression of MMR as determined by flow cytometry. Indicated is mean fluorescence intensity determined after subtraction of nonspecific values for mouse IgG. (D) Analysis of α-arginase-1 expression. (Upper) Western blot analysis for α-arginase-1 was conducted on cell lysates separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions (40 µg protein per lane). The 38-kDa α-arginase-1 is detectable in M0 and M2 macrophages. (Lower) The blot was reprobed for β-actin (42 kDa) to provide a loading control. Position of molecular weight markers in kilodaltons is indicated on the left. Bar graph, analysis of ARG1 gene expression. Quantitative analysis of ARG1 was performed on mRNA preparations isolated from monocytes and macrophages in comparison with the corresponding levels of housekeeping genes GAPDH and ACTB. Gene expression levels were determined relative to monocytes (1.0). Both protein and gene expression analyses indicate that α-arginase-1 expression is induced during maturation of macrophages from arginase-negative monocytes and is dramatically inhibited during M0 macrophage polarization into the M1, but not M2, phenotype. (E) Analysis of iNOS expression. (Upper) Cell lysates from mature and polarized macrophages (20 µg per lane) were analyzed for iNOS by western blotting under reducing conditions, confirming the induction of 130-kDa iNOS in M2 macrophages. (Lower) The blot was reprobed for α-tubulin (Biolegend) to provide equal loading control. Position of molecular weight markers in kilodaltons is indicated on the left. Bar graph, analysis of NOS2 gene expression. Quantitative analysis of NOS2 was performed on mRNA preparations isolated from macrophages in comparison with the corresponding levels of β-actin. Gene expression levels were determined relative to M0 macrophages (1.0). Both protein and gene expression analyses indicate that iNOS expression is induced during polarization of M0 macrophages toward the M1, but not M2, phenotype.

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