Maternal decidual macrophages inhibit NK cell killing of invasive cytotrophoblasts during human pregnancy

Abstract

Human pregnancy is an immunological paradox. Semiallogeneic (fetal) placental cells (extravillous cytotrophoblasts [CTBs]) invade the uterine lining (decidua), which contains a unique decidual natural killer (dNK) cell population, identified by the cell surface phenotype CD56(bright) CD16(-) CD3(-) and CD14(+) CD206(+) macrophages (dMac). Previous reports suggested that human dNK cells are not a threat to the fetoplacental unit because they are anergic. In contrast, here we showed that purified and exogenously stimulated dNK cells are capable killers of cellular targets, including semiallogeneic CTBs. However, dMacs in the decidual leukocyte (DL) population restrained dNK killing through a transforming growth factor beta1 (TGF-beta1)-dependent mechanism. Our findings support a new model whereby dNK cells, capable of killing CTBs, are prevented from doing so by neighboring macrophages, thus protecting the fetal cells from NK cell attack. We speculate that this mechanism would inhibit dNK cell-mediated killing, even under conditions where high levels of cytokines may stimulate dNK cells, which could pose a threat to the developing placenta.

Keywords: decidua; macrophage; natural killer cells; placenta; pregnancy; trophoblast.

FIG. 1

Decidual NK cells lysed K562 targets and CTBs. A) In ⁵¹Cr-release assays, purified CD56⁺ dNK cells were potent killers of K562 targets in the presence of IL-2 (n = 3). B) dNK cells killed CTBs isolated from the same pregnancy (semiallogeneic) or an unrelated pregnancy (heterologous). There were no significant differences between the groups (n = 3). C) NK92 cells lysed K562 and primary CTB targets equally well (n = 6; no significant differences).

FIG. 2

Total decidual leukocytes and decidual CD14⁺ cells were potent inhibitors of cytotoxicity. A) With CTB targets, purified IL-2-treated dNK cells had higher cytotoxic activity than unfractionated DLs, which contained other leukocytes types (*P ≤ 0.05; n = 5). B) NK92 killing of K562 targets was inhibited by the addition of DLs (n = 9). C) NK92 cells lysed K562 targets in the absence or in the presence of DLs depleted of CD14⁺ cells. Unfractionated DLs or CD14-depleted DLs reconstituted with CD14⁺ cells inhibited NK92 cell killing. Purified CD14⁺ cells inhibited NK92-mediated killing to the greatest extent (*P ≤ 0.05; n = 4). D) Unlike DLs, adult peripheral blood (pb) CD14⁺ cells failed to inhibit CTB lysis (n = 5; no significant differences).

FIG. 3

Decidual macrophages, which expressed CD14 and CD206, were abundant at the maternal–fetal interface and inhibited cytotoxicity. Viable DLs were gated based on PI exclusion (A) and expression of CD45 and CD14 (B). dMacs also expressed CD206 (C), but few stained with anti-CD163 (D). Data are representative of 5 experiments. CD206⁺ macrophages were localized in tissue sections of human decidua; DAPI staining identified nuclei. E) At lower magnification, CD14⁺ (green) and CD206⁺ (red) cells were abundant. F–H) At higher magnification, many of these macrophages coexpressed CD14 (green) and CD206 (red). Bars = 100 μm. I) NK92 cells killed K562 targets in the absence or presence of DLs depleted of CD206⁺ macrophages. Unfractionated DLs or CD206-depleted DLs reconstituted with CD206⁺ cells inhibited NK92 lysis of K562 targets. The addition of purified CD206⁺ macrophages inhibited cytotoxicity to the greatest degree (*P ≤ 0.05; n = 5).

FIG. 4

Confocal immunolocalization demonstrated close interactions among dNK cells, dMacs, and CTBs within the human uterine wall and at the maternal–fetal interface. A) At low magnification, CD206⁺ macrophages (red) were detected throughout the interstitial compartment of the pregnant human uterus at 10 wks of gestation. CTBs, identified by CK7 staining (green), occupied the interstitium (box) and lined maternal blood vessels (*). B) Close interactions between CTBs and CD206⁺ macrophages were evident. C) At higher magnification of the box shown in (B), an overlay of the phase contrast and fluorescent images showed contact between the plasma membranes of the two cell types. D) In 8-wk decidua, CD206⁺ dMacs formed close cell-cell contacts with CD56⁺ dNK cells. A representative image from one of four samples tested is shown. E) Immunolocalization of CK7⁺ CTBs (green) and CD206⁺ dMacs (red) at 16 wks of gestation. An anchoring villus (Av) is shown attached to the decidua (Dec). CTBs that invaded the uterine wall were found in close proximity to numerous dMacs. A representative image from one of four samples is shown. Bars = 100 μm.

FIG. 5

TGF-β₁ was a potent inhibitor of dNK cell-mediated cytotoxicity. A) In ⁵¹Cr-release assays, NK92 cells lysed K562 targets in the presence of anti-TGF-β₁ or control IgG antibodies. The addition of DLs and control antibody inhibited killing and this suppression was partially relieved by anti-TGF-β₁ (n = 6; *P ≤ 0.05). B) NK92 cells killed CTBs in the presence of control antibodies and DL-mediated inhibition of cytotoxicity was relieved by the addition of neutralizing anti-TGF-β₁ (n = 5; *P ≤ 0.05). C) The cytotoxic activity of purified DLs was increased by the addition of anti-TGF-β₁ antibody at the E:T ratio of 10:1 (n = 2; error bars reflect SEM of three technical replicates). D) In Transwell assays, NK92 cell lysis of K562 targets was inhibited by DLs whether or not they were cultured in the same or separate wells (n = 4). E) Identical results were obtained when CTBs were used as targets (n = 4). F) Exogenous TGF-β₁ decreased purified dNK cell-mediated lysis of K562 cells at the E:T ratio of 5:1 (n = 2; error bars reflect SEM of 3 technical replicates).

FIG. 6

Decidual macrophages produced TGF-β. TMLC reporter assays showed that conditioned medium (CM) from decidual CD14⁺ cells contained larger amounts of active TGF-β than CTB-conditioned medium (n = 2; error bars reflect SEM of three technical replicates).