Quantification of CD4, CCR5, and CXCR4 levels on lymphocyte subsets, dendritic cells, and differentially conditioned monocyte-derived macrophages.
CCR5 and CXCR4 are the major HIV-1 coreceptors for R5 and X4 HIV-1 strains, respectively, and a threshold number of CD4 and chemokine receptor molecules is required to support virus infection. Therefore, we used a quantitative fluorescence-activated cell sorting assay to determine the number of CD4, CCR5, and CXCR4 antibody-binding sites (ABS) on various T cell lines, T cell subsets, peripheral blood dendritic cells (PBDC), and monocyte-derived macrophages by using four-color fluorescence-activated cell sorting analysis on fresh whole blood. Receptor levels varied dramatically among the various subsets examined and typically varied from 2- to 5-fold between individuals. CCR5 was expressed at much higher levels in CD4+/CD45RO+/CD62L-true memory cells compared with CD4+/CD45RO+/CD62L+ cells. Fresh PBDC had the highest number of CCR5 ABS among the leukocyte subsets examined but had few CXCR4 ABS, affording a strategy for sort-purifying PBDC. In vitro maturation of PBDC resulted in median 3- and 41-fold increases in CCR5 and CXCR4 ABS, respectively. We found that macrophage colony-stimulating factor caused the greatest up-regulation of both CCR5 and CXCR4 on macrophage maturation (from approximately 5,000 to approximately 50, 000 ABS) whereas granulocyte-macrophage colony-stimulating factor caused a marked decrease of CXCR4 (from approximately 5,000 ABS to <500) while up-regulating CCR5 expression (from approximately 5,000 to approximately 20,000 ABS). Absolute ABS for CD4 and the major HIV-1 coreceptors serve as a more quantitative measure of cell surface expression, and we propose that this be used for future studies looking at the modulation of CD4 or chemokine receptor expression by cytokines, HIV-1 infection, or receptor polymorphisms.
Lee
B
B
Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Sharron
M
M
Montaner
L J
LJ
Weissman
D
D
Doms
R W
RW
D015704:CD4 Antigens / Q000032:analysis*; D002454:Cell Differentiation; D002460:Cell Line; D003713:Dendritic Cells / Q000276:immunology / Q000378:metabolism*; D015658:HIV Infections / Q000276:immunology; D015497:HIV-1 / Q000502:physiology; D006801:Humans; D008264:Macrophages / Q000276:immunology / Q000378:metabolism*; D009000:Monocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D019713:Receptors, CCR5 / Q000032:analysis*; D019718:Receptors, CXCR4 / Q000032:analysis*; D016176:T-Lymphocyte Subsets / Q000276:immunology / Q000378:metabolism*; D014779:Virus Replication
D015704:CD4 Antigens; D019713:Receptors, CCR5; D019718:Receptors, CXCR4