Differential subcellular localization of CD86 in human PBMC-derived macrophages and DCs, and ultrastructural characterization by immuno-electron microscopy.
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.
Smyth
Christine M
CM
Gene Therapy Research Unit, Children's Medical Research Institute and The Children's Hospital at Westmead, Locked Bag 23, Wentworthville, NSW, 2145.
Logan
Grant
G
Boadle
Ross
R
Rowe
Peter B
PB
Smythe
Jason A
JA
Alexander
Ian E
IE
D015703:Antigens, CD / Q000032:analysis*; D051940:B7-2 Antigen; D003513:Cycloheximide / Q000494:pharmacology; D022162:Cytoplasmic Vesicles / Q000737:chemistry / Q000648:ultrastructure; D003713:Dendritic Cells / Q000737:chemistry* / Q000187:drug effects / Q000648:ultrastructure*; D006056:Golgi Apparatus / Q000737:chemistry / Q000648:ultrastructure; D000949:Histocompatibility Antigens Class II / Q000032:analysis; D006801:Humans; D008264:Macrophages / Q000737:chemistry* / Q000648:ultrastructure*; D008562:Membrane Glycoproteins / Q000032:analysis*; D016253:Microscopy, Immunoelectron; D009000:Monocytes / Q000502:physiology; D014176:Protein Biosynthesis; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D015703:Antigens, CD; D051940:B7-2 Antigen; C497964:CD86 protein, human; D000949:Histocompatibility Antigens Class II; D008562:Membrane Glycoproteins; D014409:Tumor Necrosis Factor-alpha; D003513:Cycloheximide