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Cited by 107Neuroinflammation and Neurodegeneration MechanismsNeurogenesis and neuroplasticity mechanismsNeuroscience and Neuropharmacology Research

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M-CSF increases proliferation and phagocytosis while modulating receptor and transcription factor expression in adult human microglia

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Smith, Amy M, Gibbons, Hannah M, Oldfield, Robyn L, Bergin, Peter M, Mee, Edward W, Curtis, Maurice A, Faull, Richard L M, Dragunow, Mike·2013·Journal of Neuroinflammation
DOI 10.1186/1742-2094-10-85PMC PMC3729740
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Abstract

BACKGROUND: Microglia are the primary immune cells of the brain whose phenotype largely depends on their surrounding micro-environment. Microglia respond to a multitude of soluble molecules produced by a variety of brain cells. Macrophage colony-stimulating factor (M-CSF) is a cytokine found in the brain whose receptor is expressed by microglia. Previous studies suggest a critical role for M-CSF in brain development and normal functioning as well as in several disease processes involving neuroinflammation. METHODS: Using biopsy tissue from patients with intractable temporal epilepsy and autopsy tissue, we cultured primary adult human microglia to investigate their response to M-CSF. Mixed glial cultures were treated with 25 ng/ml M-CSF for 96 hours. Proliferation and phagocytosis assays, and high through-put immunocytochemistry, microscopy and image analysis were performed to investigate microglial phenotype and function. RESULTS: We found that the phenotype of primary adult human microglia was markedly changed following exposure to M-CSF. A greater number of microglia were present in the M-CSF- treated cultures as the percentage of proliferating (BrdU and Ki67-positive) microglia was greatly increased. A number of changes in protein expression occurred following M-CSF treatment, including increased transcription factors PU.1 and C/EBPβ, increased DAP12 adaptor protein, increased M-CSF receptor (CSF-1R) and IGF-1 receptor, and reduced HLA-DP, DQ, DR antigen presentation protein. Furthermore, a distinct morphological change was observed with elongation of microglial processes. These changes in phenotype were accompanied by a functional increase in phagocytosis of Aβ1-42 peptide. CONCLUSIONS: We show here that the cytokine M-CSF dramatically influences the phenotype of adult human microglia. These results pave the way for future investigation of M-CSF-related targets for human therapeutic benefit.

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Figure 1 The macrophage colony-stimulating factor (M-CSF) receptor (CSF-1R) is expressed by adult human microglia in vitro. (A) Adult human microglia in culture labeled for CD45 cell surface protein (green). (B) Same field of view showing microglial expression of PU.1 transcription factor (red). (C) Brightfield image showing 3,3′-diaminobenzidine tetrahydrochloride (DAB)-labeling of CSF-1R in the same field of view. (D) Overlay of images A, B and C demonstrating co-localization of CSF-1R with microglial markers CD45 and PU.1. (E) CSF-1R (brown) is not co-expressed by glial fibrillary acid protein (GFAP)-positive astrocytes (green) in mixed glial cultures (all nuclei labeled with Hoechst, blue). (F) CSF-1R (brown) is not expressed by prolyl-4-hydroxylase-positive fibroblast-like leptomeningeal cells (green) in mixed glial cultures (all nuclei labeled with Hoechst, blue). Scale bar = 50 μm.
Figure 1 The macrophage colony-stimulating factor (M-CSF) receptor (CSF-1R) is expressed by adult human microglia in vitro. (A) Adult human microglia in culture labeled for CD45 cell surface protein (green). (B) Same field of view showing microglial expression of PU.1 transcription factor (red). (C) Brightfield image showing 3,3′-diaminobenzidine tetrahydrochloride (DAB)-labeling of CSF-1R in the same field of view. (D) Overlay of images A, B and C demonstrating co-localization of CSF-1R with microglial markers CD45 and PU.1. (E) CSF-1R (brown) is not co-expressed by glial fibrillary acid protein (GFAP)-positive astrocytes (green) in mixed glial cultures (all nuclei labeled with Hoechst, blue). (F) CSF-1R (brown) is not expressed by prolyl-4-hydroxylase-positive fibroblast-like leptomeningeal cells (green) in mixed glial cultures (all nuclei labeled with Hoechst, blue). Scale bar = 50 μm.CC BY · PubMed Central
Figure 2 Macrophage colony-stimulating factor (M-CSF) treatment increases the number of microglia and expression of PU.1 in primary adult human mixed glial cultures. (A) PU.1-positive microglia (red nuclei) are present in glial cultures from human brain tissue. (B) There are more PU.1-positive cells with M-CSF treatment compared to vehicle treatment. The increase in microglia number is also seen by immunostaining for microglial surface receptor CD45 (C and D). (E) Treatment of human glial cultures with 25 ng/ml M-CSF for 96 hours significantly increases the number of microglia present. P < 0.0001, n = 6. (F) M-CSF significantly increases the intensity of PU.1 expression (amount of PU.1 protein) in adult human microglia. P < 0.05, n = 6. Scale bar = 100 μm.
Figure 2 Macrophage colony-stimulating factor (M-CSF) treatment increases the number of microglia and expression of PU.1 in primary adult human mixed glial cultures. (A) PU.1-positive microglia (red nuclei) are present in glial cultures from human brain tissue. (B) There are more PU.1-positive cells with M-CSF treatment compared to vehicle treatment. The increase in microglia number is also seen by immunostaining for microglial surface receptor CD45 (C and D). (E) Treatment of human glial cultures with 25 ng/ml M-CSF for 96 hours significantly increases the number of microglia present. P < 0.0001, n = 6. (F) M-CSF significantly increases the intensity of PU.1 expression (amount of PU.1 protein) in adult human microglia. P < 0.05, n = 6. Scale bar = 100 μm.CC BY · PubMed Central
Figure 3 Macrophage colony-stimulating factor (M-CSF) increases the division of adult human microglia. (A and B) Immunocytochemical images of CD45 microglial cell surface marker (red) and Ki67 cell division marker (green). Adult human microglia undergo limited proliferation in culture, as demonstrated by a lack of co-expression of Ki67 by CD45-immunoreactive microglia in (A). However, M-CSF treatment increases the number of dividing microglia, as shown by microglial nuclear expression of Ki67 (B). Arrows indicate examples of Ki67-immunopositive microglia. (C) Quantification of the percentage of microglia that incorporate BrdU under control conditions and with M-CSF treatment showing a significant increase in microglial division with M-CSF. P < 0.01, n = 6. Scale bar = 50 μm.
Figure 3 Macrophage colony-stimulating factor (M-CSF) increases the division of adult human microglia. (A and B) Immunocytochemical images of CD45 microglial cell surface marker (red) and Ki67 cell division marker (green). Adult human microglia undergo limited proliferation in culture, as demonstrated by a lack of co-expression of Ki67 by CD45-immunoreactive microglia in (A). However, M-CSF treatment increases the number of dividing microglia, as shown by microglial nuclear expression of Ki67 (B). Arrows indicate examples of Ki67-immunopositive microglia. (C) Quantification of the percentage of microglia that incorporate BrdU under control conditions and with M-CSF treatment showing a significant increase in microglial division with M-CSF. P < 0.01, n = 6. Scale bar = 50 μm.CC BY · PubMed Central
Figure 4 Macrophage colony-stimulating factor (M-CSF) increases microglial phagocytosis of amyloid-β1-42. (A) Microglial nuclei labeled with PU.1 (red) are surrounded by fluorescently labeled amyloid-β1-42 peptide (green) that has been phagocytosed by the microglia. (B) M-CSF treatment for 96 hours increases the amount of amyloid-β1-42 that has been phagocytosed by microglia. Another demonstration of this is shown in (C) and (D) where all nuclei are labeled with Hoechst and microglia are labeled with cell surface marker CD45 (red). Arrows indicate highly phagocytic microglia. (E) M-CSF significantly increases the percentage of microglia that undergo phagocytosis. P < 0.0001, n = 6. Scale bar = 50 μm.
Figure 4 Macrophage colony-stimulating factor (M-CSF) increases microglial phagocytosis of amyloid-β1-42. (A) Microglial nuclei labeled with PU.1 (red) are surrounded by fluorescently labeled amyloid-β1-42 peptide (green) that has been phagocytosed by the microglia. (B) M-CSF treatment for 96 hours increases the amount of amyloid-β1-42 that has been phagocytosed by microglia. Another demonstration of this is shown in (C) and (D) where all nuclei are labeled with Hoechst and microglia are labeled with cell surface marker CD45 (red). Arrows indicate highly phagocytic microglia. (E) M-CSF significantly increases the percentage of microglia that undergo phagocytosis. P < 0.0001, n = 6. Scale bar = 50 μm.CC BY · PubMed Central
Figure 5 Macrophage colony-stimulating factor (M-CSF)-treated microglia assume a rod-like shape. (A and B) Immunocytochemical labeling of CD45 microglial cell surface protein shows the morphology of adult human microglia in vitro. (A) Basal adult human microglial morphology is heterogeneous - some microglia are more rounded and others are more ramified. (B) A shift in microglial shape to a rod-like morphology is evident after 48 hours M-CSF treatment. This significant elongation of microglia with M-CSF can be quantified using Metamorph Elliptical Form Factor (elongation; P < 0.001, n = 16) (C) and Shape Factor (roundness; P < 0.01, n = 16) (D) image analysis tools. Scale bar = 100 μm.
Figure 5 Macrophage colony-stimulating factor (M-CSF)-treated microglia assume a rod-like shape. (A and B) Immunocytochemical labeling of CD45 microglial cell surface protein shows the morphology of adult human microglia in vitro. (A) Basal adult human microglial morphology is heterogeneous - some microglia are more rounded and others are more ramified. (B) A shift in microglial shape to a rod-like morphology is evident after 48 hours M-CSF treatment. This significant elongation of microglia with M-CSF can be quantified using Metamorph Elliptical Form Factor (elongation; P < 0.001, n = 16) (C) and Shape Factor (roundness; P < 0.01, n = 16) (D) image analysis tools. Scale bar = 100 μm.CC BY · PubMed Central
Figure 6 Microglial expression of HLA-DP, DQ, DR is reduced by macrophage colony-stimulating factor (M-CSF). (A) Representative image of basal HLA expression (green) in cultured adult human microglia. HLA expression is highly variable between cases and is expressed by some, but not necessarily all, microglia (PU.1-positive nuclei, red). (B) Following 96 hours exposure to M-CSF, the level of microglial HLA expression was found to be decreased compared to basal levels. (C) Quantification of the effect of M-CSF on HLA expression shows a significant reduction in the number of microglia expressing HLA. P < 0.0001, n = 18. Scale bar = 100 μm.
Figure 6 Microglial expression of HLA-DP, DQ, DR is reduced by macrophage colony-stimulating factor (M-CSF). (A) Representative image of basal HLA expression (green) in cultured adult human microglia. HLA expression is highly variable between cases and is expressed by some, but not necessarily all, microglia (PU.1-positive nuclei, red). (B) Following 96 hours exposure to M-CSF, the level of microglial HLA expression was found to be decreased compared to basal levels. (C) Quantification of the effect of M-CSF on HLA expression shows a significant reduction in the number of microglia expressing HLA. P < 0.0001, n = 18. Scale bar = 100 μm.CC BY · PubMed Central
Figure 7 Macrophage colony-stimulating factor (M-CSF) increases adult human microglial expression of C/EBPβ. (A-C) C/EBPβ is expressed by PU.1-immunopositive adult human microglia in mixed glial cultures. (D-F) M-CSF increases the intensity of C/EBPβ labeling in adult human microglia and the number of microglia which express C/EBPβ. (G) Quantification of microglial C/EBPβ expression showing that M-CSF increases the percentage of microglia which express C/EBPβ. P < 0.001, n = 12. Scale bar = 100 μm.
Figure 7 Macrophage colony-stimulating factor (M-CSF) increases adult human microglial expression of C/EBPβ. (A-C) C/EBPβ is expressed by PU.1-immunopositive adult human microglia in mixed glial cultures. (D-F) M-CSF increases the intensity of C/EBPβ labeling in adult human microglia and the number of microglia which express C/EBPβ. (G) Quantification of microglial C/EBPβ expression showing that M-CSF increases the percentage of microglia which express C/EBPβ. P < 0.001, n = 12. Scale bar = 100 μm.CC BY · PubMed Central
Figure 8 Macrophage colony-stimulating factor (M-CSF) increases adult human microglial expression of DAP12. (A) DAP12 monocyte/microglial adaptor protein is expressed by human microglia isolated from biopsy tissue. (B) Treatment of microglia with M-CSF increases their expression of DAP12 as seen by increased intensity of immunochemical staining. (C) Quantification of DAP12 staining intensity shows a significant increase in DAP12 expression with M-CSF treatment. P < 0.0001, n = 8. Scale bar = 100 μm.
Figure 8 Macrophage colony-stimulating factor (M-CSF) increases adult human microglial expression of DAP12. (A) DAP12 monocyte/microglial adaptor protein is expressed by human microglia isolated from biopsy tissue. (B) Treatment of microglia with M-CSF increases their expression of DAP12 as seen by increased intensity of immunochemical staining. (C) Quantification of DAP12 staining intensity shows a significant increase in DAP12 expression with M-CSF treatment. P < 0.0001, n = 8. Scale bar = 100 μm.CC BY · PubMed Central
Figure 9 Macrophage colony-stimulating factor (M-CSF) increases adult human microglial expression of CSF-1R and IGF-1R. M-CSF treatment does not significantly alter the total number of Hoechst-labeled nuclei in human adult mixed glial cultures (A and E), but it does increase the number of microglia (B and F), as shown by labeling for the microglial transcription factor PU.1. Together with an increase in microglia number, M-CSF produces an increase in microglial expression of its receptor CSF-1R (C and G) and the receptor for IGF-1 (D and H). A significant increase in intensity of receptor labeling is found for both CSF-1R (I; P < 0.0001, n = 8) and IGF-1R (J; P < 0.01, n = 8) following M-CSF treatment. Scale bar = 50 μm.
Figure 9 Macrophage colony-stimulating factor (M-CSF) increases adult human microglial expression of CSF-1R and IGF-1R. M-CSF treatment does not significantly alter the total number of Hoechst-labeled nuclei in human adult mixed glial cultures (A and E), but it does increase the number of microglia (B and F), as shown by labeling for the microglial transcription factor PU.1. Together with an increase in microglia number, M-CSF produces an increase in microglial expression of its receptor CSF-1R (C and G) and the receptor for IGF-1 (D and H). A significant increase in intensity of receptor labeling is found for both CSF-1R (I; P < 0.0001, n = 8) and IGF-1R (J; P < 0.01, n = 8) following M-CSF treatment. Scale bar = 50 μm.CC BY · PubMed Central

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Metadata

Authors
Smith, A. M., Gibbons, H. M., Oldfield, R. L., Bergin, P. M., Mee, E. W., Curtis, M. A., Faull, R. L. M., & Dragunow, M.
Affiliations
University of Auckland (NZ)
Corresponding author
Dragunow, Mike
ORCID
Venue
Journal of Neuroinflammation
Publisher
Springer Science and Business Media LLC
Published
2013-07-17
Volume
10
Issue
1
Pages
85-85
ISSN
1742-2094 (ISSN-L)
DOI
10.1186/1742-2094-10-85
PMC
PMC3729740
OpenAlex
W1973529716
Cited by
107
References
72
Citations by year
2026: 2 · 2025: 14 · 2024: 10 · 2023: 7 · 2022: 7 · 2021: 10 · 2020: 7 · 2019: 8 · 2018: 10 · 2017: 6 · 2016: 7 · 2015: 7 · 2014: 9 · 2013: 3
FWCI
4.86
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Neuroinflammation (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
  • Tertiary Education Commission
  • Hugh Green Foundation
  • Neurological Foundation of New Zealand
  • Health Research Council of New Zealand
Topics
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Life Sciences › Neuroscience › Developmental Neuroscience › Neurogenesis and neuroplasticity mechanisms
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
Keywords
macrophage colony-stimulating factor, microglia, microglial proliferation, phagocytosis, CSF-1 receptor, PU.1, C/EBPβ, DAP12, IGF-1 receptor, HLA antigen expression, microglial morphology, amyloid-beta, temporal lobe epilepsy, neuroinflammation, microglial phenotype
MeSH terms
Proto-Oncogene Protein Spi-1, Antimetabolites, Autopsy, Biopsy, Bromodeoxyuridine, Cells, Cultured, HLA Antigens, Humans, Image Processing, Computer-Assisted, Immunohistochemistry, Macrophage Activation, Membrane Proteins, Phagocytosis, Proto-Oncogene Proteins, Transcription Factors, Trans-Activators, Macrophage Colony-Stimulating Factor, Receptor, Macrophage Colony-Stimulating Factor, Microglia, Ki-67 Antigen, CCAAT-Enhancer-Binding Protein-beta, Adaptor Proteins, Signal Transducing, Cell Proliferation

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