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. 2009 Apr 7;106(14):5767-72.
doi: 10.1073/pnas.0901173106. Epub 2009 Mar 20.

HLA-G Homodimer-Induced Cytokine Secretion Through HLA-G Receptors on Human Decidual Macrophages and Natural Killer Cells

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Free PMC article

HLA-G Homodimer-Induced Cytokine Secretion Through HLA-G Receptors on Human Decidual Macrophages and Natural Killer Cells

Changlin Li et al. Proc Natl Acad Sci U S A. .
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Abstract

Human decidual CD14(+) macrophages and CD56(+) NK cells were isolated from material obtained after first-trimester pregnancy terminations. Each cell type expressed a specific surface receptor for histocompatibility leukocyte antigen (HLA)-G (an MHC class Ib protein that is expressed on extravillous trophoblasts), LILRB1 on CD14(+) macrophages and KIR2DL4 on CD56(+) NK cells. Cross-linking with anti-LILRB1 or anti-KIR2DL4 resulted in up-regulation of a small subset of mRNAs including those for IL-6, IL-8, and TNFalpha detected using a microarray representing 114 cytokines. Incubation with transfectants expressing the HLA-G homodimer (but not with transfectants expressing the HLA-G monomer) resulted in secretion of the same cytokine proteins from both leukocyte sets. Moreover, cytokine secretion from both leukocyte sets was blocked by both the appropriate anti-receptor mAb and by anti-HLA-G. The amount of these cytokines secreted by decidual macrophages was substantially greater than that secreted by decidual NK cells. VEGF was constitutively secreted by both cell types. LILRB1, which contains an immunoreceptor tyrosine-based switch motif, functions here as an activating receptor, although it has been known as an inhibitory receptor. KIR2DL4 also functions as an activating receptor, although it also has the potential to function as an inhibitory receptor. Secretion of proinflammatory and proangiogenic proteins supports a role for these leukocytes in important processes that are essential for successful pregnancy, but they may represent only a portion of the proteins that are secreted.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
LILRB1 and KIR2DL4 surface expression on peripheral and decidual CD14+ and CD56+ cells assessed by flow cytometry. (A) LILRB1 expression on peripheral and decidual CD14+ cells. (B) LILRB1 expression on peripheral and decidual CD56+ cells. (C) KIR2DL4 expression on peripheral and decidual CD14+ cells. (D) KIR2DL4 expression on peripheral and decidual CD56+ cells. Staining relative to an isotype control antibody (shaded) is shown.
Fig. 2.
Fig. 2.
HLA-G homodimer induced cytokine protein secretion by human peripheral and decidual CD14+ macrophages and CD56+ NK cells. Peripheral CD56+ (A), decidual CD56+ cells (B), peripheral CD14+ cells (C), or decidual CD14+ cells (D) were cocultured in a 1:1 ratio with HLA class I-negative 721.221 cells (white bars), 721.221 cells transfected to express HLA-G monomer (gray bars), or 721.221cells transfected to express HLA-G homodimer (black bars). Supernatants were collected after 24 h for CD14+ macrophages or after 48 h for CD56+ NK cells. The concentration of cytokines (IL-6, IL-8, IL-10, TNFα, or VEGF) in each supernatant was measured by a multiplex cytokine assay. The SD was calculated from the mean of 4 experiments, each with an individual donor. Statistical significance: *, P < 0.05; **, P < 0.01; and ***, P < 0.001. Note that the scale of C is compressed ≈4-fold relative to D because the secretion of IL-8 by peripheral CD14+ cells is so much larger than that of decidual CD14+ cells.
Fig. 3.
Fig. 3.
Cytokine secretion induced by the HLA-G homodimer was blocked in the presence of specific antibodies against HLA-G or its receptors. Decidual CD14+ cells (Left) or decidual CD56+ cells (Right) were cocultured in a 1:1 ratio with 721.221 cells transfected with HLA-G homodimer in the presence of various mAb as shown. Supernatants were collected after 24 h for CD14+ macrophages or after 48 h for CD56+ NK cells. The concentration of cytokines (IL-6, IL-8, IL-10, IFNγ, TNFα, or VEGF) in each supernatant was measured by a multiplex cytokine assay. The SD was calculated from the mean of 3 experiments for decidual CD56+ cells and 4 experiments for decidual CD14+ cells, each with an individual donor. Statistical significance: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

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