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Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting.
A L Ford1, A L Goodsall, W F Hickey
1Centenary Institute of Cancer Medicine and Cell Biology, Sydney, Australia.
Abstract:
Ramified microglia in the adult central nervous system (CNS) are the principal glial element up-regulating MHC class I and II expression in response to inflammatory events or neuronal damage. A proportion of these cells also express MHC class II constitutively in the normal CNS. The role of microglia as APCs for CD4+ T cells extravasating into the CNS remains undefined. In this study, using irradiation bone marrow chimeras in CD45-congenic rats, the phenotype CD45lowCD11b/c+ is shown to identify microglial cells specifically within the CNS. Highly purified populations of microglia and nonmicroglial but CNS-associated macrophages (CD45highCD11b/c+) have been obtained directly from the adult CNS, by using flow cytometric sorting. Morphologically, freshly isolated microglia vs other CNS macrophages are quite distinct. Of the two populations recovered from the normal CNS, it is the minority CD45highCD11b/c+ transitional macrophage population, and not microglia, that is the effective APC for experimental autoimmune encephalomyelitis-inducing CD4+ myelin basic protein (MBP)-reactive T cells. CD45highCD11b/c+ CNS macrophages also stimulate MBP-reactive T cells without addition of MBP to culture, suggesting presentation of endogenous Ag. This is the first study in which microglia vs other CNS macrophages have been analyzed for APC ability directly from the CNS, with substantial cross-contamination between the two populations eliminated. The heterogeneity of these populations in terms of APC function is clearly demonstrated. Evidence is still lacking that adult CNS microglia have the capacity to interact with and stimulate CD4+ T cells to proliferate or secrete IL-2.
Insights
In the central nervous system (CNS), transitional macrophages, not microglia, effectively present antigens to CD4+ T cells. This study clarifies the distinct antigen-presenting cell (APC) roles of CNS macrophages and microglia.
Area of Science:
- Neuroimmunology
- Cellular immunology
Background:
- Microglia are ramified glial cells in the CNS that up-regulate MHC class I and II expression during inflammation or neuronal damage.
- Some microglia constitutively express MHC class II in the normal CNS.
- The function of microglia as antigen-presenting cells (APCs) for CD4+ T cells in the CNS is not well-defined.
Purpose of the Study:
- To investigate and differentiate the antigen-presenting capabilities of microglia versus other CNS-associated macrophages.
- To determine which CNS cell population is responsible for presenting antigens to CD4+ T cells involved in experimental autoimmune encephalomyelitis.
Main Methods:
- Utilized irradiation bone marrow chimeras in CD45-congenic rats.
- Employed flow cytometric sorting to isolate highly purified populations of microglia (CD45lowCD11b/c+) and CNS-associated macrophages (CD45highCD11b/c+).
- Analyzed the APC function of isolated populations using myelin basic protein (MBP)-reactive T cells.
Main Results:
- Microglia (CD45lowCD11b/c+) and CNS-associated macrophages (CD45highCD11b/c+) were morphologically and phenotypically distinct.
- The CD45highCD11b/c+ transitional macrophage population, not microglia, effectively acted as APCs for experimental autoimmune encephalomyelitis-inducing CD4+ MBP-reactive T cells.
- CNS macrophages (CD45highCD11b/c+) stimulated MBP-reactive T cells without exogenous antigen, suggesting presentation of endogenous antigens.
Conclusions:
- Demonstrated significant heterogeneity in APC function between microglia and other CNS macrophages.
- Highlighted the critical role of transitional CNS macrophages as APCs in the context of neuroinflammation.
- Provided evidence that adult CNS microglia may not effectively stimulate CD4+ T cells for proliferation or IL-2 secretion.