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. 2001 May 22;98(11):6295-300.
doi: 10.1073/pnas.111152498.

Developmental plasticity of CNS microglia

L Santambrogio  1 , S L Belyanskaya, F R Fischer, B Cipriani, C F Brosnan, P Ricciardi-Castagnoli, L J Stern, J L Strominger, R Riese
Affiliations

Developmental plasticity of CNS microglia

L Santambrogio et al. Proc Natl Acad Sci U S A. .

Abstract

Microglia arise from CD45(+) bone marrow precursors that colonize the fetal brain and play a key role in central nervous system inflammatory conditions. We report that parenchymal microglia are uncommitted myeloid progenitors of immature dendritic cells and macrophages by several criteria, including surface expression of "empty" class II MHC protein and their cysteine protease (cathepsin) profile. Microglia express receptors for stem cell factor and can be skewed toward more dendritic cell or macrophage-like profiles in response to the lineage growth factors granulocyte/macrophage colony-stimulating factor or macrophage colony-stimulating factor. Thus, in contrast to other organs, where terminally differentiated populations of resident dendritic cells and/or macrophages outnumber colonizing precursors, the majority of microglia within the brain remain in an undifferentiated state.

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Figures

Figure 1
Figure 1
Neonatal microglia can be skewed toward a DC-like or macrophage-like phenotype by GM-CSF or M-CSF. (A) Neonatal microglia express c-kit, but not flt3, as analyzed by flow cytometry (c-kit, clone 2B8; flt3, A2F10.1). (B) Phenotypic markers are differentially expressed on neonatal microglia after administration of myeloid growth factors. Neonatal microglia were cultured with or without 5 ng/ml GM-CSF or 1 ng/ml M-CSF from the beginning of the mixed glial culture. (Upper) Phase-contrast confocal microscopy of microglia skewed as described above (magnification: microglia, ×69; GMCSF and MSCF, ×100). (Lower) Cell-surface expression of CD11b (clone M1/70), CD11c (clone HL3), mannose receptor (clone Dec205), B7–1 (clone 16–10A1), and B7–2 (clone GL1) was analyzed by flow cytometry.
Figure 2
Figure 2
Microglial cells express surface-empty class II MHC molecules. (A) Resting neonatal microglia express empty, but not peptide-loaded class II MHC molecules, as detected by flow cytometry by using mAb KL304 (empty) and Y3P (peptide-loaded). Primary cultures of neonatal microglial cells and two microglial cell lines (N9, N1P)21 were stained with CD45, a marker for bone marrow-derived cells, to distinguish microglia from other CNS components before class II analysis. (B) Empty/peptide-receptive class II MHC molecules are expressed throughout the brain parenchyma, but are particularly evident in the submeningeal spaces. Brain sections were immunostained with anti I-A (Y3P, KL-304) or isotype control (mouse IgG2b) to detect MHC class II conformation. (C) Empty class II molecules do not colocalize with PECAM-1, a marker for endothelial cells. Brain sections were double-stained with CD31 (PECAM-1) and KL-304. (D) Empty MHC class II do colocalize with CD45 in isolated brain capillaries, implying expression of empty/peptide-receptive class II MHC on perivascular microglia.
Figure 3
Figure 3
Expression and conformation of class II MHC molecules are regulated by proinflammatory cytokines. (A) IFN-γ induces expression of SDS-stable MHC class II on N9 cells. Cells were incubated with or without 100 units/ml IFN-γ for 48 h before the MHC class II proteins were immunoprecipitated (αβ-SDS stable MHC class II complexes; α−α chain of class II, and β-β chain of class II; nb, nonboiled; b, boiled). (B) IFN-γ up-regulates peptide-loaded class II MHC and down-regulates empty class II surface expression on N9 cells. Confocal microscopy (Upper) and flow cytometry (Lower) of similarly treated cells, using anti I-A mAb KL304 (green) and Y3P (red). (C) Microglia cells, differentially skewed by myeloid growth factors, are able to translocate class II MHC protein to their surfaces in response to proinflammatory cytokines. Neonatal microglial cells incubated with M-CSF and GM-CSF as described in Fig. 1 were washed and recultured with 10 ng/ml IFN-γ, 100 ng/ml LPS, or 100 units/ml TNFα for 48 h. Cells were collected and analyzed for surface MHC class II expression by staining with the Y3P mAb. Normalized fluorescence represents the ratio between the geometric mean of the Y3P staining divided for the geometric mean of the isotype control.
Figure 4
Figure 4
Microglia express cathepsins S and L but not F. (A and B) Cell lysates from cell lines (LS109, Jaws II, N9, and N1P), primary cultures of neonatal microglia (μGlia), bone marrow-derived DC (BM DC), splenocytes (Spl), and peritoneal macrophages (MΦ) were labeled with 125I-JPM and analyzed by 13% SDS/PAGE. All APC express active cathepsins S and B. Up-regulation of cathepsin S with IFN-γ is seen in many, but not all, cell lines. (C) Cathepsin L Western blot of cell lysates showing the active form of cathepsin L in N9 cells, microglia, and macrophages but not splenocytes. (D) Cathepsin F Western analysis of the same blot as in C demonstrating expression of active cathepsin F in macrophages and, to a lesser extent, in N9 cells. Microglia, but not other cells, were stimulated with IFN-γ (100 units/ml) for 72 h before analysis to maximize expression of cathepsin F. (E) RT-PCR of cathepsin F from mouse tissues and cell suspensions. Cathepsin F mRNA is more highly expressed in N9 cells and lung (Lu) compared with neonatal microglia, thymocytes (Thy), and splenocytes.
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