Table 1.
Patient characteristics of nonpregnant, healthy pregnant and preeclamptic women.
Figure 1.
Flow cytometric gating strategy for monocyte subsets in human peripheral blood.
Dead cells were excluded by selecting all live cells from the forward/side scatter plot (A), after which monocytes were selected (B). Subsequently, cells were plotted for their CD14 and CD16 expression, after which three distinct subpopulations of monocytes (classical, nonclassical and intermediate) could be selected and analyzed (C).
Figure 2.
Flow cytometric gating strategy for monocyte subsets in rat peripheral blood.
Dead cells and red blood cells were excluded by selecting all live cells from the forward/side scatter plot (A), after which a wide gate around the monocytes and granulocytes was selected (B) and copied to a CD172a/CD43 plot (C). The total monocyte population (used for calculating the total monocyte numbers) was selected from the CD172a/CD43 plot (C) and subsequently plotted in a new graph to calculate the percentages of the two classical (CD43-lo) and nonclassical (CD43-hi) subsets in the total monocyte population (D).
Table 2.
Leukocyte and monocyte counts in the human peripheral blood samples of nonpregnant, healthy pregnant and preeclamptic women.
Figure 3.
Human monocyte subsets in peripheral blood.
Lower percentages of classical monocytes were observed in healthy pregnant women (Pr, squares) as compared to nonpregnant women (NP, circles) and even less in preeclamptic women (PE, triangles) compared to healthy pregnant women (A). Higher percentages of combined nonclassical/intermediate monocytes were found in pregnant women compared to nonpregnant women and even higher percentages in preeclamptic patients compared to healthy pregnant women (B). When subdividing into nonclassical and intermediate monocytes, no significant changes were observed in nonclassical monocytes (C), though a trend towards lower nonclassical monocytes was observed in preeclamptic women compared to healthy pregnant women. In contrast, higher percentages of intermediate monocytes were found in healthy pregnant women compared to nonpregnant women and even higher percentages in preeclamptic patients compared to healthy pregnant women (D). Medians are shown, #p<0.1, *p<0.05; **p<0.01; ***p<0.001; Mann Whitney U test.
Figure 4.
Rat monocyte subsets in peripheral blood on day 20 of pregnancy.
At the end of pregnancy (day 20) in rats (Pr, squares), the percentage of classical monocytes decreases (A) and the percentage of nonclassical monocytes rises (B) as compared to nonpregnant rats (NP, circles). Medians are shown, **p<0.01; Mann Whitney U test.
Table 3.
Leukocyte and monocyte counts in peripheral blood samples of nonpregnant and pregnant rats.
Figure 5.
Rat monocyte subsets after ATP or saline infusion.
No changes in classical monocytes (as percentage of live cells) (A) were found after ATP (solid line, solid symbols) or saline infusion (dashed line, open symbols) between pregnant (Pr, squares) and nonpregnant (NP, circles) rats infused on day 14 of pregnancy, while the percentage of nonclassical monocytes (B) increased after ATP-infusion in the pregnant rats only. Medians with the interquartile range are shown, *p<0.05 in a Friedman repeated measures test followed by Dunn's posttest compared to day 13 (pre-infusion). a = p<0.05 and b = p<0.1 in pregnant saline vs nonpregnant saline as tested with Mann Whitney U for the same day. NP + saline n = 5; NP + ATP n = 6; Pr + saline n = 7; Pr + ATP n = 9.
Figure 6.
Activation of nonclassical monocytes after ATP infusion in pregnant rats.
Activation of nonclassical monocytes was assessed by lower mean fluorescent intensity (MFI) of two activation markers, CD62L (A, early activation marker) and CD4 (B, late activation marker) [21], [40]. Decreased expression of CD62L was found in ATP-infused rats only on nonclassical monocytes on day 15 (trend) of pregnancy compared to pre-infusion day 13, while CD4 expression on nonclassical monocytes was decreased on day 20 of pregnancy compared to pre-infusion day 13 in the same experimental group. Percentage deviations from pre-infusion day 13 are shown. Medians with the interquartile range are shown, #p<0.1 (trend), *p<0.05 in a Friedman repeated measures test followed by Dunn's posttest compared to day 13 (pre-infusion). NP + saline n = 5; NP + ATP n = 6; Pr + saline n = 7; Pr + ATP n = 9.
Figure 7.
Total and CD206-positive macrophages in placentas of saline and ATP-infused pregnant rats.
No changes in total, CD68-positive macrophages (as percentage positively stained tissue) were found after ATP-infusion (squares) in the decidua (A) and mesometrial triangle (B) as compared to saline-infusion (circles). Staining for CD206-positive macrophages was found to be lower in mesometrial tissue (D) of rats infused with ATP compared to saline. No differences were found for CD206 in decidual tissue between ATP and saline-infused rats (C). Medians are shown, #p<0.1; Mann Whitney U test.