Extensive characterization of the immunophenotype and pattern of cytokine production by distinct subpopulations of normal human peripheral blood MHC II+/lineage- cells

Abstract

Dendritic cells (DC) represent the most powerful professional antigen-presenting cells (APC) in the immune system. The aim of the present study was to analyse, on a single-cell basis by multiparametric flow cytometry with simultaneous four-colour staining and a two-step acquisition procedure, the immunophenotypic profile and cytokine production of DC from 67 normal whole peripheral blood (PB) samples. Two clearly different subsets of HLA-II+/lineage- were identified on the basis of their distinct phenotypic characteristics: one DC subset was CD33strong+ and CD123dim+ (0.16 +/- 0.06% of the PB nucleated cells and 55.9 +/- 11. 9% of all PB DC) and the other, CD33dim+ and CD123strong+ (0.12 +/- 0.04% of PB nucleated cells and 44.53 +/- 11.5% of all PB DC). Moreover, the former DC subpopulation clearly showed higher expression of the CD13 myeloid-associated antigen, the CD29 and CD58 adhesion molecules, the CD2, CD5 and CD86 costimulatory molecules, the CD32 IgG receptor and the CD11c complement receptor. In addition, these cells showed stronger HLA-DR and HLA-DQ expression and a higher reactivity for the IL-6 receptor alpha-chain (CD126) and for CD38. In contrast, the CD123strong+/CD33dim+ DC showed a stronger reactivity for the CD4 and CD45RA molecules, whereas they did not express the CD58, CD5, CD11c and CD13 antigens. Regarding cytokine production, our results show that while the CD33strong+/CD123dim+ DC are able to produce significant amounts of inflammatory cytokines, such as IL-1beta (97 +/- 5% of positive cells), IL-6 (96 +/- 1.1% of positive cells), IL-12 (81.5 +/- 15.5% of positive cells) and tumour necrosis factor-alpha (TNF-alpha) (84 +/- 22.1% of positive cells) as well as chemokines such as IL-8 (99 +/- 1% of positive cells), the functional ability of the CD123strong+/CD33dim+ DC subset to produce cytokines under the same conditions was almost null. Our results therefore clearly show the presence of two distinct subsets of DC in normal human PB, which differ not only in their immunophenotype but also in their functionality, as regards cytokine production.

Fig. 1

Morphological characteristics of human peripheral blood MHC II⁺/lineage⁻ cells after immunomagnetic isolation (May–Grünwald–Giemsa, × 1000). Dendritic processes can only be clearly seen in some of the isolated cells, while others, even lacking cytoplasmic veils, show irregular nuclei shapes.

Fig. 2

Light scatter characteristics of normal human peripheral blood (PB) MHC II⁺/lineage⁻ cells (black). (a) All nucleated cells present in PB are shown. (b) Only the (CD3⁻, CD14⁻, CD19⁻, CD56⁻)/HLA-DR⁺ gated cells (MHC II⁺/lineage⁻ cells) from the same PB sample are displayed.

Fig. 3

Phenotypic identification of distinct subsets of MHC II⁺/lineage⁻ cells present in normal human peripheral blood. One MHC II⁺/lineage⁻ cell population is identified by its strong reactivity for CD123 (grey dots in (a)) and the dim CD33 expression (grey dots in (b)), while the other MHC II⁺/lineage⁻ subset was CD123^dim+ (black dots in (a)) and CD33^strong+ (black dots in (b)).

Fig. 4

Illustrative dot plots of the phenotypic differences between the two peripheral blood (PB) MHC II⁺/lineage⁻ cell subpopulations. CD33^strong+/CD123^dim+ MHC II⁺/lineage⁻ cells are shown in black and CD123^strong+/CD33^dim+ MHC II⁺/lineage⁻ cells are displayed in grey.

Fig. 5

Comparative analysis of cytokine production by the normal human peripheral blood (PB) CD33^strong+/CD123^dim+ and CD123^strong+/CD33^dim+ subsets of MHC II⁺/lineage⁻ cells. The right panels show representative dot plots of IL-1β, IL-6, IL-12, tumour necrosis factor-alpha (TNF-α) and IL-8 production by the CD33^strong+/CD123^dim+ (black points) and the CD123^strong+/CD33^dim+ (grey dots) PB MHC II⁺/lineage⁻ cell subpopulations after stimulation for 6 h in the presence of brefeldin A with lipopolysaccharide (LPS) plus IFN-γ. Left panels show the production of the same cytokines by the two MHC II⁺/lineage⁻ cell subsets from the same PB sample in the absence of LPS plus IFN-γ stimuli (negative control).