Immature myeloid cells (IMCs) are bone marrow–derived cells that normally differentiate into granulocytes, macrophages, and dendritic cells (DCs) but expand in pathological conditions such as malignancy. DCs are antigen-presenting cells that regulate the immune response. Both IMCs and DCs were shown to take part in angiogenesis; however, little is known of their function in the placenta. We sought to determine whether alterations in DC and IMC populations in the placenta precede the onset of delivery.
Experiments were performed on 6-8 week old C57Bl/6 female mice. Placentas from pregnant mice that were killed on designated days, immunostained using fluorescently labeled anti-CD11b, Gr-1, CD11c, major histocompatibility II (MHCII), and CD45, and analyzed by flow cytometry and immunofluorscent microscopy.
Throughout the latter part of pregnancy toward labor and delivery, the CD45 + CD11b + Gr1 + -IMC population decreased 29 ± 9.1% (day 12) and 30 ± 9.9% (day 15), vs 21 ± 8.1% (day18, n = 21, 15, and 27; P = .006 and P = .004, respectively), whereas the CD45 + CD11c + MHCII + -DC population increased 0.87 ± 0.3% (day 12) and 0.70 ± 0.3% (day 15) vs 1.81 ± 1.3% (day 18, n = 21, 15, and 27, P = .002 and P = .001, respectively). Both myeloid cell populations were localized adjacent to CD31 + endothelial cells in sites of placental angiogenesis.
Labor and delivery are preceded by proangiogenic-myeloid cell alterations, reflected by a decrease in IMCs and an increase in DCs populating the mouse placenta. The intriguing possibility that delivery is preceded by the maturation of IMCs in part into DCs warrants further studies.
Tumor progression depends on the recruitment of blood vessels and the successful induction of a complete process of blood vessel formation. This complex process of tumor angiogenesis is orchestrated by a variety of stromal cells and mainly by various types of bone marrow derived cells. Immature myeloid cells (IMCs), also known as myeloid derived suppressor cells (MDSCs), are a heterogeneous family of myeloid cells that differentiate in the steady state into mature granulocytes, macrophages, or dendritic cells (DCs). In other conditions, such as cancer or pregnancy, a partial block in the differentiation of IMCs/MDSCs into mature myeloid cells results in the expansion of this population. Although IMCs were initially described to suppress the immune system, thereby allowing tumor expansion and growth, a growing body of evidence suggests that they also play a significant role in tumor angiogenesis.
DCs are terminally differentiated myeloid cells that specialize in antigen processing and presentation. In malignant states, tumor-derived factors and the presence of other myeloid cells lead to restricted DC maturation and function. Tumors are thus populated by an abundance of immature DCs with an impaired capacity to stimulate adaptive antitumor immunity. Accordingly, multiple clinical studies have indicated that the presence of mature DCs is decreased and their function is impaired in patients with various tumors. In addition to their classic role as antigen-presenting cells, DCs have been found to promote angiogenesis in tumors, endometriosis, uterine deciduas, and choroidal neovascularization.
Many of the proliferative, invasive, and immune tolerance mechanisms that are used to support normal pregnancies have been shown to be similarly exploited by tumors to establish a nutrient supply and to evade immune surveillance. We have previously demonstrated in both mice and humans that IMCs infiltrate the placentas in the proximity of blood vessels. When isolated from either tumors or placentas, IMCs directly promoted angiogenesis in both Matrigel plug and endothelial tube formation assays. DCs were shown to take part in angiogenesis in other models; however, little is known of their function in the placenta. In the present study, we sought to determine whether alterations in DC and IMC populations in the placenta precede the onset of delivery.
Materials and Methods
Animal studies were carried out using 6-8 week old female mice. C57Bl/6J mice were purchased from Harlan Laboratories (Jerusalem, Israel). Timed pregnancies were dated according to vaginal plug appearance at morning inspection. Mice were killed at the indicated stage, and the uterine horns were exposed by midline laparotomy. Placentas were carefully dissected from the decidual tissue and used for further analysis. Specifically, 6 separate experiments were performed, with a total of 10 pregnant mice. Sixty-three placentas were analyzed: 21 from day 12, 15 from day 15, and 27 from day 18. All animal procedures were performed in compliance with Weizmann Institute of Science guidelines and protocols approved by the Institutional Animal Care and Use Committee.
Placentas were digested with an enzyme mixture including 25 μg/mL hyaluronidase (MP Biomedical, Solon, OH), 25 μg/mL DNase (Sigma-Aldrich, St. Louis, MO), and 3 U/mL Liberase (Roche, Nutley, NJ) dissolved in phosphate-buffered saline (PBS) at 37°C for 30 minutes. Digested tissue was then filtered through a 40 μM cell strainer and resuspended in fluorescence-activated cell sorting (FACS) buffer (PBS, 5 mM EDTA, 1% bovine serum albumin, and 0.05% sodium azide). Immunostaining was performed in the presence of rat antimouse Fc receptor III/II (FcgammaRIII/II) (CD16/32; Pharmingen, San Diego, CA) by incubating the cells with monoclonal antibodies for 30 minutes on ice. Staining reagents included fluorochrome (allophycocyanin, phycoerythrin, or fluorescein isothiocyanate)-labeled anti-CD11b, CD45, CD31, Gr1, CD11c, and IA/IE (mouse major histocompatibility complex class II) (all purchased from BD Biosciences, San Diego, CA). Flow cytometry was performed with a FACS Calibur (Becton Dickinson, Mountain View, CA).
Frozen sections (12 μm) of placental tissue were fixed with acetone (–20°C), washed in PBS, and blocked with 20% horse serum in PBS. After immunostaining with primary antibodies anti-CD11b, anti-Gr1 (BD Biosciences) for IMCs and anti-CD11c for DCs, and antiplatelet endothelial cell adhesion molecule-1 (PECAM1; CD31) for endothelial cells (eBioscience, San Diego, CA) followed by secondary antibodies, mounting was performed on glass slides with Vectashield HardSet mounting media with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA) for nuclear staining. Optical sections were scanned using a Zeiss LSM 510 Meta Duoscan confocal system (Carl Zeiss, Jena, Germany).
Continuous data are presented as mean ± SD. A nonparametric Kruskall-Wallis test was performed to compare the distributions of samples. P < .05 was considered statistically significant.
We first analyzed mouse placentas for the population of IMCs in the latter part of pregnancy. Placental single cell suspensions were stained with fluorescent-conjugated antibodies and analyzed by flow cytometry. The concentrations of CD45 + CD11b + Gr1 + IMCs ( Figure 1 ) as a percentage of total placental bone marrow–derived cells (CD45 + ) showed a statistically significant decline toward labor and delivery: 29 ± 9.1% (day 12) and 30 ± 9.9% (day 15) vs 21 ± 8.1% (day18, n = 21, 15, and 27; analysis of variance [ANOVA], P = .002).
We next stained frozen placental sections with fluorescent-conjugated antibodies and examined them by confocal microscopy. IMCs expressing both CD11b and Gr1 were localized adjacent to endothelial cells (identified by the expression of PECAM1), consistent with the involvement of these cells in angiogenesis ( Figure 2 ).
Because a possible explanation for the observed decrease in IMCs toward labor and delivery could be their terminal differentiation into more mature cells, we next determined the concentrations of DCs in the placenta. Indeed, the concentrations of CD45 + CD11c + IA/IE + DCs ( Figure 3 ) as a percentage of total placental bone marrow–derived cells (CD45 + ) increased toward labor and delivery: 0.87 ± 0.3% (day 12) and 0.70 ± 0.3% (day 15) vs 1.81 ± 1.3% (day 18, n = 21, 15, and 27, ANOVA, P < .001).
When examining frozen placental sections by confocal microscopy ( Figure 4 ), as observed with IMCs, CD11c-expressing DCs were also found to be localized adjacent to endothelial cells (identified by the expression of PECAM1); nevertheless, their abundance at these sites was rare.