Mouse resident microglia: isolation and characterization of immunoregulatory properties with naïve CD4+ and CD8+ T-cells.
We describe a non-enzymatic procedure designed to isolate in high purity resident microglia from the brains of normal mice. This procedure allowed for the characterization of the cells without concern that their surface features had been enzymatically altered during tissue processing. A cell population was obtained and judged to consist primarily of microglia because essentially all the cells were Mac-1+, Mac-3+, F4/80+, CD44+, CD54+, and CD86+, and they expressed CD45 with a mean fluorescence intensity value of about one-half that of tissue macrophages. The cells also expressed marginal levels of MHC class I, CD14, CD40, and CD80, but lacked detectable MHC class II, CD4, CD8, CD45R, and CD102 molecules. Molecular phenotyping revealed that the purified microglial population contained mRNA transcripts encoding the receptor for colony stimulating factor-1 (CSF-1), the macrophage growth factor, and contained few, if any, transcripts for glial fibrillary acidic protein, an astrocyte-specific marker. Ex vivo, the microglia constitutively stimulated, in the mixed leukocyte reaction, the proliferation of naive allogeneic CD8+, but not CD4+ T-cells. However, they failed to present a protein antigen to naive antigen-specific CD4+ T-cells unless pretreated with interferon-gamma, a response that was inhibited by antibodies to CD86. Agar-cloning experiments confirmed that normal mouse brain contains a CSF-1-responsive cell that gave rise to cells with identical immunophenotypic characteristics as freshly isolated resident microglia. Moreover, the microglial progenitor cell located in a density fraction that was different from that containing the mature resident microglia.
Havenith
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CE
Department of Immunology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
Askew
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D
Walker
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WS
D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D002469:Cell Separation; D002478:Cells, Cultured; D003470:Culture Media; D005260:Female; D005434:Flow Cytometry; D006651:Histocytochemistry; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D017628:Microglia / Q000276:immunology*; D010587:Phagocytosis; D016133:Polymerase Chain Reaction; D012756:Sheep / Q000276:immunology
D003470:Culture Media