Figure 1.
Purity of leukocytes and presence of fetal cells after enrichment from placenta and uterus (16.5 dpc).
A. Enriched leukocyte preparations from non-pregnant (NP) uterus, pregnant (16.5 dpc) uterus (P) and placenta were analyzed by flow cytometry. Viable cells excluding propidium iodide were gated on the basis of forward (FSC) and side scatter (SSC) criteria. The average leukocyte purity assessed by the presence of the CD45.2 marker was 89.3% for NP uterus, 97% for pregnant uterus, and 95% for placenta, compared to 98% for the spleen (positive control for the assessment of purity) from the same mice. Analyses were performed on at least 3 animals per group B. CD45.2+ B6 females were crossed with CD45.1+ B6 males. CD45+ leukocyte populations from the uterus and placenta were analyzed by flow cytometry on day 16.5 pc. The detection of CD45.1+2+ cells revealed the presence of a small percentage (<5%) of fetal leukocytes. The experiment has been repeated 3 times.
Figure 2.
Drastic changes in the scatter distribution of leukocytes from NP, or pregnant uterus, and placenta at various stages of a syngeneic pregnancy.
The same protocol as Figure 1 was followed. Viable cells excluding propidium iodide were gated on the basis of forward (FSC) and side scatter (SSC) criteria, from uterus (A) or placenta (D). Mean percentages (B, E) and total cell numbers (C, F) in « granular » (R1 gate, white bars) or « lymphoïd/monocytoïd » (R2 gate, black bars) are presented. NP uteri were pooled from 4 to 15 mice in all phases of œstrus cycle and the experiment was repeated 6 times. At each stage of pregnancy or post-partum, the data were collected from 4 to 14 mice assayed in 2 to 9 separate experiments. (*) p≤0.05, (**) p≤0.005, (***) p≤0.001.
Table 1.
Antibody and dilutions used for flow cytometry staining.
Figure 3.
Analyses of immune cell populations present in the uterus of NP or pregnant mice, and in placenta (16.5 dpc).
Only viable cells excluding propidium iodide were analysed. Enriched leukocytes from NP (A,B) or pregnant (16.5 dpc) uterus (C,D) and placenta (E,F) were analysed by flow cytometry. R2-gated cells (left, cf. Figure 2) were analysed on the basis of the following cell surface markers: TCRβ+/CD4+ (CD4 T cells); TCRβ+/CD8+ (CD8 T cells); NK1.1+/TCRβ− (NK cells); TCRβ+/NK1.1+ (NKT cells); CD19+/B220+ (B cells); Gr1+/CD11b+ (myeloid Gr1+ cells including monocytes); Gr1−/CD11c+/CD11bHi/low (myeloid CD11c+ cells including DCs). R1-gated cells (right, cf. Figure 2) were analysed on the basis of the following cell surface markers: Gr1+/CD11b+ (Granulocytes); CD11bHi/Gr1+/Gr1+/−/CD11c+/− (Highly granulosity cells or HGC). The results are representative from a typical experiment of a pool of 7 mice (A, B) or from a single mouse (C, D, E, F). The experiment was repeated at least twice with 3–6 animals per assay.
Figure 4.
High granulosity cells in non pregnant uterus are primarily eosinophils.
Viable R1- or R2- gated CD45.2+ cells from NP uterus were analysed by FACS stained for APC and granulocytes markers (CD11b, CD24, CD11b, F4/80, Ly6C, Ly6G, MHC class II and CCR2) (A, B and Table 1) and for NK and T cell markers (CD11b, NK1.1, CD3ε, CD8α and CD4) (B and Table 1). The experiment was repeated twice with 6 females each time (A, B). Viable CD45.2+, CD11b+, F4/80+ R1 (C) or R2 (D) cells were sorted by flow cytometry and spun onto Super + glass microscope slides using a Cytospin cytofuge. Cells were fixed with methanol and stained with Wright-Giemsa. Pictures were taken on a Nikon H600L microscope equipped with a DS-Fi2-Nikon camera (C, D). The experiment was performed twice with at least 5 females each time.
Figure 5.
Similarities and differences in leukocyte subpopulation frequencies and cytokine/chemokine production between NP, pregnant uterus and placenta.
A, B, C, D: comparison of leukocyte subpopulation frequencies E, F, G, H: comparison of cytokine/chemokine productions (a) syngeneic pregnancy (day 16.5 pc) (b) cytokines/chemokines from leukocyte-enriched preparations (c) cytokines/chemokines from whole uterus preparations Similarities referred to comparable cell percentages or cytokine/chemokine quantities, varying at most by a factor of 2. Differences corresponded to large statistically significant differences by factors ranging from 4 to 700.
Figure 6.
Comparison of syngeneic versus allogeneic pregnancies from the mean percentages and cell numbers of uterine and placental leukocyte populations (day 16.5 pc).
Mean cell percentages among all viable leukocytes (A and C) and corresponding mean cell numbers, per uterus (B) or per placenta (D), were measured by flow cytometry in leukocyte subpopulations from NP-uterus (striped bars, A–B), compared to the uterus from syngeneic (grey bars, A–B) or allogeneic pregnancies (black bars, A–B), and in placenta from syngeneic (grey bars) or allogeneic pregnancies (black bars, C–D), on day 16.5 pc. NP-uteri were pooled from 4 to 15 mice in all phases of estrus cycle, in at least 3 different experiments. For each experiment, 3 to 9 placentas or implantation sites were collected. The experiment was performed 4 times. (*) p≤0.05, (**) p≤0.005, (***) p≤0.001.
Figure 7.
Quantification of uterine and placental cytokines and chemokines in syngeneic pregnancy (day 16.5 pc).
Uterine and placental tissues were prepared as described in Materials and Methods : (A) Uteri from NP mice, (B) Uteri from syngeneic pregnancy (day 16.5 dpc), (C) Placenta from syngeneic pregnancy (same animals). Samples were analyzed simultaneously for the following 22 cytokines: IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12(p70), IL-13, IL-15, IL-17, CSF2 (GM-CSF), CSF3 (G-CSF), IFNγ, CXCL10 (IP-10), CXCL1 (KC), CCL2 (MCP-1), CCL3 (MIP-1α), CCL5 (RANTES), TNF-α. Only IL-9, IL-10, GM-CSF (CSF2), G-CSF (CSF3), CXCL10 (IP-10), IL-1α, CCL3 (MIP-1α), CXCL1 (KC), CCL2 (MCP-1) yielded reproducibly significant measurements and have been presented. Striped bars: whole organ, black bars: enriched leukocytes from the same organ. Data are from 4 to 5 different samples. Statistically significant differences: (*) p≤0.05, (***) p≤0.001.
Figure 8.
Cytokine and chemokine expression levels in uterus from NP mice versus syngeneic or allogeneic pregnancies (day 16.5 pc).
The same protocol as in Figure 7 was followed. Proteins were measured from whole uterus (A), or from enriched uterine leukocytes (B) from NP uterus, or syngeneic or allogeneic pregnancies (note the important scale variations). Striped bars: NP uterus, grey bars: syngeneic pregnancy at 16.5 dpc, black bars: allogeneic pregnancy at 16.5 dpc. Data are from 4 to 5 different samples. (*) p≤0.05, (**) p≤0.005, (***) p≤0.001.
Figure 9.
Cytokine and chemokine expression levels in placentae from syngeneic or allogeneic pregnancies (day 16.5 pc).
Analyses were performed on the placentas from the same pregnant mice as in Figure 8. Proteins were measured from whole placenta (A), or from enriched placental leukocytes (B) from syngeneic or allogeneic pregnancies (note the important scale variations). Grey bars: syngeneic pregnancy, black bars: allogeneic pregnancy. Data are from 4 to 5 different samples. (*) p≤0.05, (**) p≤0.005, (***) p≤0.001.