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Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine
Published on: May 21, 2012
Differential subcellular localization of CD86 in human PBMC-derived macrophages and DCs, and ultrastructural
Christine M Smyth1, Grant Logan, Ross Boadle
1Gene Therapy Research Unit, Children's Medical Research Institute and The Children's Hospital at Westmead, Locked Bag 23, Wentworthville, NSW, 2145.
Abstract:
We have previously reported the presence of a discrete reservoir of the costimulatory molecule CD86 in the cytoplasm of human monocytes freshly isolated from peripheral blood mononuclear cells (PBMC). In the current study, we have extended analysis of the subcellular localization of this molecule to in vitro PBMC-derived dendritic cells (DCs) and macrophages. In a sub-population of DCs, we observed by confocal microscopy an intracellular focal concentration of CD86 that bore striking similarities to that previously reported in monocytes. Further analyses revealed that this intracellular CD86 was not localized to the Golgi apparatus, MHC II compartments or endocytic structures, and required intact microtubules to retain structural integrity. A similar concentration of CD86 was not present in PBMC-derived macrophages. Electron microscopy revealed two distinct DC phenotypes containing either sparse or abundant cytoplasmic vesicles, and CD86 was found to be concentrated within the vesicular compartment of this latter phenotype. Collectively, these data not only identify and characterize a novel CD86-containing cytoplasmic compartment in human PBMC-derived DCs, but also define micro-structurally distinct DC subsets that differentially concentrate CD86 within cytoplasmic vesicles. Although the functional significance of these observations remains to be established, available evidence supports the conclusion that the focal concentration of CD86 is a storage reservoir that facilitates rapid deployment of this molecule to the DC surface when increased costimulatory capacity is required.
Insights
Human monocytes store costimulatory molecule CD86 in cytoplasmic reservoirs. This study identifies similar CD86 storage in dendritic cells (DCs), suggesting rapid surface deployment for enhanced immune responses.
Area of Science:
- Immunology
- Cell Biology
Background:
- The costimulatory molecule CD86 is crucial for T cell activation.
- Previous work identified cytoplasmic CD86 reservoirs in human monocytes.
Purpose of the Study:
- To investigate the subcellular localization of CD86 in dendritic cells (DCs) and macrophages.
- To characterize the nature and potential function of intracellular CD86 compartments.
Main Methods:
- Confocal microscopy to visualize CD86 localization in vitro PBMC-derived DCs and macrophages.
- Analysis of CD86 association with cellular organelles and requirement for microtubules.
- Electron microscopy to identify distinct DC phenotypes and vesicular compartments.
Main Results:
- A sub-population of DCs displayed intracellular focal concentrations of CD86, similar to monocytes.
- This intracellular CD86 was not found in Golgi, MHC II compartments, or endosomes, and required intact microtubules.
- Two distinct DC phenotypes were identified: one with sparse vesicles and another with abundant vesicles containing CD86.
- PBMC-derived macrophages did not exhibit similar CD86 concentrations.
Conclusions:
- Human PBMC-derived DCs possess a novel intracellular CD86-containing cytoplasmic compartment.
- Distinct DC subsets differentially concentrate CD86 within cytoplasmic vesicles.
- This focal CD86 concentration likely serves as a storage reservoir for rapid surface expression, enhancing costimulatory capacity.
