In the following experiments, characteristics of 4 types of cells were compared: monocytes, unfractionated decidual cells containing macrophages, primary cultures of purified decidual macrophages, and in vitro–generated monocyte-derived macrophages. Potential conditions associated with driving monocytes into the decidual macrophage phenotype that were tested included activation, differentiation, and environmental conditioning.
shows the locations of CD14pos macrophages in term extraplacental membranes. Fetal macrophages (open arrowheads) are located in the connective tissue between the amnion epithelium and the chorion membrane. These fetal cells are eliminated during dissection of the tissue. Maternal macrophages are scattered randomly thoughout the decidua and are clustered near the chorion membrane. As shown in , the isotype-specific control antibody failed to yield positive signals.
Figure 1 Immunohistochemical localization of macrophages in term extraplacental membranes. (A) Anti-CD14. (B) Isotype matched control. Open arrows mark fetal macrophages between the amnion and chorion membranes. Closed arrows mark decidual macrophages close to (more ...)
Decidual Macrophages Maximally Display Markers for Activation
In the first set of experiments, cell ELISA assays were used to assess the ability of a macrophage activation factor, IFN-γ, and a macrophage differentiation factor, M-CSF, to drive monocytes into a phenotype similar to that of the decidual macrophage. Levels of 2 IFN-γ–responsive markers involved in antigen presentation, HLA-DR and CD86 (B7-2), were compared using a cell ELISA. A growth factor for NK cells11
that has been implicated in decidualization30,31
and is displayed on monocyte cell surfaces,32
IL-15, was investigated by the same method.
As shown in , monocytes, purified decidual macrophages, and monocyte-derived macrophages displayed the 3 surface markers. IFN-γ is known to enhance mononuclear phagocyte display of Fc receptors, which could cause misinterpretation of data in these assays. Therefore, binding of isotype control antibodies to cells treated with or without IFN-γ was calculated. For both IgG1 (control for anti-CD86, -IL-15, -LILRB1, -LILRB2) and IgG2a (control for anti-HLA-DR), the absorbance values (0.07 for untreated, 0.08 for IFN-γ treated) were not significantly different when comparing IFN-γ–treated and -untreated groups.
Figure 2 Analysis of activation markers on monocytes, purified decidual macrophages, and monocyte-derived macrophages using cell enzyme-linked immunosorbent assay (ELISA). Antibodies used to obtain the results were (A) anti-HLA-DR, (B) anti-CD86, and (C) anti-IL-15. (more ...) Activation
show that monocyte responsiveness to IFN-γ treatment was significantly greater than purified decidual macrophage responsiveness. Following exposure to IFN-γ, levels of markers in monocytes were increased by 4.4-, 2.1-, and 1.6-fold for HLA-DR, CD86, and IL-15, respectively. Brackets and P values indicate statistical significance between fold changes in response to IFN-γ treatment. When monocyte induction was compared with decidual macrophage induction for HLA-DR, the P value was .0016. For induction of CD86 between the same 2 populations, the P value was .006. Although the trend persisted, statistical significance was not reached for differences between monocytes and decidual macrophages in induction of IL-15.
These data imply that activation by IFN-γ differentially increased similarities between monocytes and purified decidual macrophages, enhancing molecules involved in antigen presentation (HLA-DR, CD86) but not expression of a surface cytokine involved in lymphocyte activation (IL-15).
Activation and differentiation
As shown in , in the resting stage, levels of HLA-DR on monocytes that had been differentiated (ie, monocyte-derived macrophages) were similar to levels in purified decidual macrophages, and levels of CD86 and IL-15 appeared slightly lower, although the differences were not statistically significant. Monocyte-derived macrophages were responsive to IFN-γ activation, showing 1.4-, 1.3-, and 1.1-fold induction of HLA-DR, CD86, and IL-15, respectively. Although consistent among the 3 preparations of cells tested, none of the comparisons between monocyte-derived macrophages and decidual macrophages were statistically significant with regard to levels of markers expressed or responsiveness to IFN-γ.
This lack of statistical significance indicates that M-CSF treatment increased the resemblances between monocytes and primary decidual macrophages.
Decidual Macrophages and Monocyte-Derived Macrophages Display Similar Cellular Morphologies
In the next set of experiments, we compared the morphologies of activated decidual macrophages and activated monocyte-derived macrophages. shows the morphological characteristics of decidual macrophages following the IFN-γ activation protocol. Activated decidual macrophages were plastic adherent and demonstrated heterogeneous morphologies that include elongated cells with fine extensions, small rounded cells, large flattened cells, and cells containing cytoplasmic vesicles.
Figure 3 Morphologic appearances of (A) decidual macrophages cultured for 48 hours in medium containing interferon-γ (IFN-γ) and (B) monocyte-derived macrophages, which had been cultured for 5 days in medium containing 150 IU/mL macrophage colony-stimulating (more ...)
Differentiation of monocytes with M-CSF induced an increase in size, adherence to plastic, and ruffling of the membrane, but the cells remained rounded (not shown). As illustrated in , when the activation factor, IFN-γ, was added, the monocyte-derived macrophages exhibited heterogenous morphologies with elongated, round, and flattened cells in the cultures that were similar to those of decidual macrophages. However, neither differentiation nor activation induced the smaller cell size that characterized the decidual macrophage.
Thus, these experiments indicate that as with expression of the surface markers tested above, both differentiation and activation induce decidual macrophage morphology. However, reduction in cell size was uniquely associated with residency in tissue.
Comparison of Inhibitory Receptors for HLA-G
HLA-G is composed of a group of related proteins produced by cytotrophoblast cells in human placentas.33
Primary decidual macrophages in early and late gestation decidua contain mRNAs encoding 2 receptors for HLA-G, LILRB1 (ILT2), and LILRB2 (ILT4).34
Binding of HLA-G to these receptors would be expected to block activation signals and influence their production of proinflammatory and anti-inflammatory cytokines. In this group of experiments, we tested the expression of the receptor proteins by cell ELISA to predict whether activation and/or differentiation of monocytes would induce the decidual macrophage phenotype.
shows that the expression of LILRB1 and LILRB2 was similar in the 3 cell populations, monocytes, purified decidual macrophages, and monocyte-derived macrophages, prior to stimulation with IFN-γ. Activation effectively increased levels of both LILRB1 and LILRB2 on monocytes but not decidual macrophages. Brackets and P values indicate statistical significance between fold changes in response to IFN-γ treatment, with statistically significant differences of P = .044 and P = .024, respectively, between the responses of the 2 populations. Differentiation obviated this induction, with no statistically significant changes in monocyte-derived macrophages.
Figure 4 Analysis of HLA-G receptors on monocytes, purified decidual macrophages, and monocyte-derived macrophages by cell surface enzyme-linked immunosorbent assay. (A) Leukocyte immunoglobulin-like receptor (LILR)B1 and (B) LILRB2. Blood monocytes (Mo), purified (more ...)
Thus, unlike expression of HLA-DR, CD86, and IL-15 as well as decidual cell morphology, neither activation by IFN-γ nor differentiation by M-CSF appeared to be involved in regulating levels of expression of the receptors for HLA-G displayed by the purified decidual macrophages taken from term extraplacental membranes.
Differentiation and Activation Do Not Recapitulate Decidual Macrophage Production of IL-10
The previous experiments suggest that activation and differentiation program monocytes into a decidual macrophage phenotype that is mirrored in many but not all respects by exposing monocytes to differentiation by M-CSF and activation by IFN-γ in vitro. In the next set of experiments, we compared the production of 2 cytokines, TGF-β1 and IL-10, in the 3 cell populations. Both cytokines are known to be products of mononuclear phagocytes, and both could support an immunosuppressive environment in the decidua. Cell culture supernatants were collected from 3 sets each of monocytes, freshly isolated decidual macrophages, and monocyte-derived macrophages for IL-10 and TGF-β1 assays.
When evaluated by using an ELISA assay, TGF-β1 was undetectable in supernatant culture media from monocytes, purified decidual macrophages, and monocyte-derived macrophages regardless of time of incubation (data not shown).
By contrast, IL-10 was absent from the culture supernatants of monocytes and monocyte-derived macrophages (data not shown) but was readily detected in culture supernatants from purified decidual macrophages (see ). This cytokine was identified after overnight culture, and levels increased after 48 hours and 72 hours of culture. Absolute values differed in the 3 preparations, although temporal profiles were similar, with peak production reached by culturing the cells for 48 hours.
Interleukin-10 Production by Purified Decidual Macrophages
These data indicate that as with reduction in cell size, programming of monocytes (TGF-β1neg, IL-10neg) into the cytokine secretory profile of decidual macrophages (TGF-β1neg, IL-10pos) is not achieved by activation or differentiation, suggesting that the uteroplacental environment is engaged in this programming step.
Comparisons of Purified Decidual Macrophages and Unfractionated Decidual Cells
In the final group of experiments, the goal was to learn whether the expression patterns described above for purified decidual macrophages would be the same or different in cultures of unfractionated decidual cells.
As shown in , activation marker (HLA-DR, CD86) levels were significantly higher in unfractionated decidual cells (n = 3) when compared with purified decidual macrophages (P = .018). By contrast, neither IL-15 levels (n = 3; ) nor LILRB1 and LILRB2 levels (n = 2, data not shown) were different. Brackets and P values indicate statistical significance between absorbance values regardless of IFN-γ treatment. In 1 experiment, the unfractionated and purified preparations were matching (ie, from the same placenta), whereas in 2 experiments, they were not. The outcomes were the same for all preparations.
Figure 5 Analysis of (A) HLA-DR, (B) CD86, and (C) IL-15 on resting (−IFN-γ) and activated (+IFN-γ) purified decidual macrophages and unfractionated decidual cells using a cell enzyme-linked immunosorbent assay. OD indicates optical density. (more ...)
IL-10 levels in supernatant culture media from purified decidual macrophages (n = 3) and mixed decidual cell cultures (n = 4) were similar in the 2 populations (data not shown), whereas TGF-(β1 was present only in the supernatant culture media from the unfractionated decidual cells (n = 6; ). After overnight incubation, TGF-β1 was detectable in 1 of the 6 preparations (85 pg/mL). After 48 hours of culture, the cytokine was detectable in 3 of the 6 preparations (range, 34-1131 pg/mL), and after 72 hours of culture, TGF-β1 was present in 5 of the 6 cultures (range, 27-1251 pg/mL).
Transforming Growth Factor–β1 Production by Unfractionated Decidual Cells
These results indicate that unfractionated decidual cells exhibit a higher density of antigen presentation molecules (HLA-DR, CD86) and increased secretion of a multipurpose cytokine with immunosuppressive properties, TGF-β1, than purified decidual macrophages. By contrast, the display of IL-15, expression of HLA-G receptors, and secretion of IL-10 were not different when values for purified decidual macrophages and unfractionated decidual cells were compared.
Summary of Results
illustrates the markers that characterize monocytes during in vitro activation and differentiation. The phenotype of the final product of in vitro conditioning, the activated monocyte-derived macrophage, is compared with that of the decidual macrophage.
Figure 6 Schematic illustration showing marker expression of decidual macrophages (left panel) in comparison with in vitro–activated monocytes, differentiated monocytes, and monocytes that were both differentiated and activated (right panel). IFN, interferon; (more ...)