Figure 1.
CD163 is differentially expressed on monocyte subsets at the protein and gene level.
(A) Monocytes from whole blood were identified by forward and side scatter properties and expression of CD14 vs. CD16 with subsets defined as CD14++CD16− (P2, lower right), CD14++CD16+ (P3, upper right) and CD14+CD16++ (P4, upper left). (B) Expression of CD163 protein (dark grey) in each monocyte subset is shown compared to matched isotype control (light grey) in the histograms. Data illustrate results from a representative experiment. (C) CD163 mRNA was determined by Q-PCR in isolated monocyte subsets from 8 HIV-1 negative donors. Bars indicate median values. P values were calculated using the non-parametric, Mann-Whitney U test adjusted for multiple comparisons.
Figure 2.
Surface CD163 expression is reduced on CD14++CD16− monocytes after density gradient separation and LPS stimulation.
(A) Comparison of CD163 protein expression on the classical CD14++CD16− subset was carried out on 13 samples from HIV-1 negative donors and on all monocyte subsets from a further eight donors before and after Ficoll density gradient purification of PBMC from whole blood. Bars represent median values. Differences were analysed using the Wilcoxon matched pairs test. WB: whole blood, PBMC: peripheral blood mononuclear cells. (B) Whole blood from HIV-uninfected donors was incubated with 1 µg/ml LPS for 2 h and CD163 surface expression measured by flow cytometry. CD163 expression on each subset (mean fluorescence intensity) is plotted as the ratio of expression in treated compared to untreated monocytes. Data represent mean ± SEM for 4 independent donors. ∧ data from 1 donor was excluded from the CD14++CD16+ data set due to insufficient cell number.
Figure 3.
Intracellular and surface CD163 expression increases during maturation of monocytes to macrophages.
Monocytes purified by countercurrent elutriation from healthy HIV-1 uninfected donors were cultured in vitro for the times indicated, then stained for CD163 with (solid line) and without (dashed line) permeabilisation to measure total (surface and intracellular) and surface only CD163, respectively. At the same time, cells were stained for expression of mannose receptor to confirm maturation of monocytes into macrophages. Percent of monocytes/macrophages positive for CD163 is presented in upper graph and mean fluorescence intensity of expression in middle graph. Percent of monocytes/macrophage positive for mannose receptor is presented in lower graph. Data represent mean ± SEM using 5 independent donors.
Table 1.
Mean percent of cells expressing CD163 after culture of the monocytes in M-CSF or GM-CSF.
Figure 4.
Membrane bound and soluble CD163 in monocyte subsets from HIV-1 infected donors.
(A.i) CD163 expression on each monocyte subset was determined using a whole blood flow cytometric assay for HIV-1 uninfected donors (closed circles, n = 27) and infected donors (open squares, n = 38). Bars represent median values. (A.ii) The proportion of total monocytes in each of the three monocyte subsets was determined for each donor analysed in (A.i). The event count for that subset was divided by the sum of events in each subset. Bars represent median values. (B) Soluble CD163 in serum from 12 HIV uninfected donors, 38 HIV-1infected donors receiving antiretroviral therapy was determined by sandwich ELISA. Bars represent median values. P values were calculated using the non-parametric Mann Whitney U test.
Figure 5.
Membrane CD163 expression is related to CD4 T cell count.
Frequency of CD163 positive cells in CD14++CD16− monocytes (top row) and CD14++CD16+ monocytes (bottom row) was correlated with CD4 T cell count for donors below 500 cells/µl (left column) and above 500 cells/µl (right column). Donors not currently receiving antiretroviral therapy are represented by open circles and donors receiving protease inhibitors are represented by dotted squares. rs and P values after analysis excluding patients receiving PI are presented in parentheses after pooled data. rs value was determined by Spearman correlation coefficient.