Dorota Darmochwal-Kolarz

T helper 1- and T helper 2-type cytokine imbalance in pregnant women with pre-eclampsia.

OBJECTIVES The purpose of our study was to investigate T helper 1/T helper 2 balance in pregnant women with pre-eclampsia. STUDY DESIGN 18 patients with pre-eclampsia and 20 healthy pregnant women were included in the study. Peripheral blood mononuclear cells (PBMC) were stimulated with phytohaemagglutinin (PHA) for 48 h. Cytokine: interleukin-2 (11-2), interferon-gamma (IFN-gamma) and interleukin-10 (I1-10) concentrations in culture supernatants were determined using the ELISA method. Statistical analysis was performed using a standard non-parametric Mann-Whitney U-test. RESULTS We found that in pre-eclamptic patients PHA-stimulated 11-2 and IFN-y production was significantly higher (P<0.001) and I1-10 production significantly lower (P<0.005) in comparison with the control group. CONCLUSION These results could suggest that there is Th1/Th2 imbalance in pre-eclamptic patients with predominant Th1-type immunity.

The predominance of Th17 lymphocytes and decreased number and function of Treg cells in preeclampsia.

The aim of this study was to estimate the prevalence of CD3(+)CD4(+) T lymphocytes producing IL-17, IL-2, IFN-γ, and IL-4, plus CD4(+)CD25(+)FoxP3(+) T regulatory (Treg) cells, in peripheral blood of patients with preeclampsia and healthy women in the third trimester of normal pregnancy. Another purpose was to assess the immunosuppressive activity of Treg cells from patients with preeclampsia compared with controls. Thirty-four preeclampsia patients and 27 healthy pregnant women were included. The percentages of CD4(+)CD25(+)FoxP3(+) Treg cells and CD3(+)CD4(+) T lymphocytes with intracellular expressions of cytokines were estimated using monoclonal antibodies and flow cytometry. In vitro functional assays were performed using a Treg Cell Isolation Kit and (3)H-thymidine incorporation assays. The percentage of CD3(+)CD4(+) T lymphocytes producing IL-17A was significantly higher in preeclampsia than in healthy, normotensive pregnant women in the third trimester (p<0.001). The population of CD4(+)CD25(+)FoxP3(+) Treg cells was significantly lower in the study group compared with controls (p<0.05). There was no change in the stimulation index of CD3(+)CD4(+)CD25(-) T lymphocytes from preeclampsia patients without Treg cells and after addition of autologous Treg cells. In normal pregnancy, the stimulation index of CD3(+)CD4(+)CD25(-) T lymphocytes was significantly higher without Treg cells compared with the response after addition of autologous Treg cells (p<0.05). The results suggest up-regulation of the Th17 immune response in preeclampsia. The decreased number and function of Treg cells may be responsible for activating the inflammatory response characteristic of this disorder. In preeclampsia, the predominance of Th17 immunity could act through modulating the Th1/Th2 immune balance.

The expressions of intracellular cytokines in the lymphocytes of preeclamptic patients

Darmochwal‐Kolarz D, Rolinski J, Leszczynska‐Gorzelak B, Oleszczuk J. The expressions of intracellular cytokines in the lymphocytes of preeclamptic patients. AJRI 2002; 48:381–386

Activated T lymphocytes in pre-eclampsia.

The aim of our study was to investigate the activation markets of T CD3+, T helper CD4+ and T cytotoxic CD8+ cells, as well as, the populations of T naïve CD4+ CD45RA+, T memory CD4+ CD45RO+ and T regulatory lymphocytes in PE and healthy pregnant women.

Blood myeloid and lymphoid dendritic cells are stable during the menstrual cycle but deficient during mid-gestation

The aim of this study was to estimate the populations of peripheral blood myeloid and lymphoid dendritic cells (CD1c(+), BDCA-2(+), BDCA-4(+)) and the CD1c(+):BDCA-2(+) ratio in phases of the ovarian cycle and in normal pregnant patients. 18 non-pregnant women and 17 normal pregnant women were included. Dendritic cells were isolated from peripheral blood, stained with monoclonal antibodies (mAbs) against blood dendritic cell antigens (anti-BDCA-1, BDCA-2, BDCA-4) and estimated using flow cytometry. CD1c(+), BDCA-2(+) and BDCA-4(+) dendritic cells were present in the follicular and luteal phases of the ovarian cycle and in all trimesters of normal pregnancy. The percentages of CD1c(+) dendritic cells did not differ between the follicular and luteal phases of the ovarian cycle. The percentage of BDCA-2(+) dendritic cells was lower in the luteal phase of the ovarian cycle compared with the follicular phase, but the differences were not statistically significant. The CD1c(+):BDCA-2(+) cell ratio was significantly lower in the luteal phase compared with the follicular phase of the ovarian cycle. The numbers of dendritic cells were significantly lower in the second trimester when compared with the first and third trimesters of normal pregnancy. Furthermore, in the second trimester, the CD1c(+):BDCA-2(+) ratio was higher than in the other trimesters of normal pregnancy. All populations of dendritic cells and the CD1c(+):BDCA-2(+) ratio did not differ in the first and third trimesters of physiological pregnancy. Our results suggest that myeloid and lymphoid dendritic cells are not affected by steroid hormones during the menstrual cycle. The deficiency of peripheral blood dendritic cells observed during the second trimester of normal pregnancy can be associated with their migration to the uterus during the second physiological invasion by cytotrophoblast.

The expression and concentrations of Fas/APO-1 (CD95) antigen in patients with severe pre-eclampsia.

PROBLEM Apoptosis has been proposed as a mechanism for maintaining homeostasis in the immune system. Activated lymphocytes are removed by a Fas/FasL-mediated programmed cell death process called activation induced cell death (AICD). The aim of the study was to investigate surface Fas antigen (APO-1, CD95) expression on T lymphocytes and NK cells and also soluble Fas antigen concentrations in pre-eclamptic patients and healthy pregnant women. MATERIALS AND METHODS Sixteen pre-eclamptic patients and 18 healthy pregnant women were studied. Peripheral blood lymphocytes were isolated, labeled by direct staining with anti-CD95 monoclonal antibodies and analyzed using the flow cytometric method. Furthermore, the concentrations of soluble CD95 molecule in serum of patients with severe pre-eclampsia and women with uncomplicated pregnancy were measured using ELISA method. RESULTS We found that Fas antigen expression and fluorescence intensity on T CD8+ lymphocytes were higher in patients with severe pre-eclampsia in comparison with healthy pregnant women (P<0.05). Furthermore, the concentrations of soluble CD95 molecule were higher in the group of pre-eclamptic patients when compared to controls (P<0.001). CONCLUSIONS These findings suggest that T CD8+ lymphocytes in patients with severe pre-eclampsia can be activated. Moreover, higher concentrations of soluble CD95 antigen can suggest altered possibilities to undergo Fas/FasL-mediated activation induced cell death process of lymphocytes in severe pre-eclampsia.

Fas Antigen Expression on the Decidual Lymphocytes of Pre‐Eclamptic Patients

PROBLEM: Apoptosis has been proposed as a mechanism for maintaining the homeostasis in the immune system. Activated lymphocytes are removed by a programmed cell death process Fas/FasL‐mediated called activation induced cell death. The aim of the study was to investigate Fas antigen expression on decidual cells (T CD4+ lymphocytes, T CD8+ lymphocytes and Natural Killer (NK) cells) of pre‐eclamptic patients and healthy pregnant women.
 METHOD OF STUDY: 12 pre‐eclamptic patients and 10 healthy pregnant women were studied. Lymphocytes were isolated from decidual tissues mechanically, labeled by direct staining with monoclonal antibodies, and analyzed using the flow cytometric method.
 RESULTS: We found Fas antigen expression on decidual NK cells and T lymphocytes. CD 95 molecule expression and fluorescence intensity on NK cells of pre‐eclamptic patients were lower when compared with controls (P<0.05).
 CONCLUSIONS: These findings suggest that decidual NK cells and T lymphocytes are able to undergo Fas/FasL‐mediated apoptosis. It seems that NK cells’ ability to undergo Fas/FasL‐mediated apoptosis in pre‐eclamptic patients can be altered because of lower CD95 molecule expression.

The concentrations of soluble HLA-G protein are elevated during mid-gestation and decreased in pre-eclampsia

The aim of our study was to investigate the dynamics of the alterations of soluble human leukocyte antigen-G (sHLA-G) concentrations in sera of healthy non-pregnant women, as well as healthy pregnant women and patients with pre-eclampsia. Thirty five patients with pre-eclampsia, 52 healthy pregnant women, and 24 healthy non-pregnant women were included in the study. Sera concentrations of sHLA-G protein were determined using the immunoenzymatic ELISA method. Statistical analysis was performed using ANOVA and Mann-Whitney U tests. The concentrations of sHLA-G protein in sera of pregnant women in the first, as well as the second and third, trimesters of normal pregnancy were significantly higher in comparison with healthy nonpregnant women. The sera concentrations of sHLA-G in pregnant women in the second trimester of pregnancy were significantly higher compared to the first and third trimesters. The concentrations of sHLA-G in sera of patients with pre-eclampsia were significantly lower than in pregnant women in the third trimester of physiological pregnancy. The results of our study suggest that normal physiological pregnancy is associated with elevated sera concentrations of sHLA-G molecule. The increased concentrations of sHLA-G molecule in mid-gestation could suggest a role for the protein in the second phase of a physiological invasion of extravillous cytotrophoblast to spiral arteries. Furthermore, the results could suggest a role for the decreased sera concentrations of sHLA-G in the pathogenesis of pre-eclampsia.

The immunophenotype of patients with recurrent pregnancy loss

OBJECTIVE The aim of the study was to estimate the alterations in the phenotype of lymphocytes of women with unexplained pregnancy failures in comparison with healthy women. MATERIALS AND METHODS Fourteen women with unexplained habitual miscarriages and 18 healthy, fertile women with the history of successful pregnancies were included in the study. The lymphocytes were isolated from peripheral blood and stained with monoclonal antibodies. The expression of selected surface molecules was estimated using the flow cytometric method. RESULTS We found that the percentage of T CD4(+) lymphocytes, CD3(-)16/56(+) cells, and T CD8(+)11b(-) cells was significantly higher in patients with recurrent pregnancy loss in comparison with healthy women. The percentage of B-1 CD19(+)5(+) lymphocytes was also significantly higher in women with unexplained habitual miscarriages in comparison with healthy women. Furthermore, we found higher expression of CD25 molecule on T CD3(+) and T CD4(+) lymphocytes in the study group, when compared to controls. Moreover, the percentages of B CD19(+) and T suppressor CD8(+)11b(+) lymphocytes were lower in women with pregnancy failures in comparison with the control group. The percentage of T CD3(+) lymphocytes and T CD8(+) cells did not differ in both studied groups. Similarly, the expression of CD25 antigen and HLA-DR molecule on T CD8(+) did not differ in the study group, when compared to controls. CONCLUSION Our results can suggest that the immunological alterations may be involved in the etiopathogenesis of unexplained recurrent pregnancy loss.

Apoptosis Signaling Is Altered in CD4+CD25+FoxP3+ T Regulatory Lymphocytes in Pre-Eclampsia

The aim of our study was to estimate the surface expressions of CD95 (APO-1/Fas) antigen and the intracellular expressions of anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax in CD4+CD25+FoxP3+ T regulatory lymphocytes (Tregs) as well as the percentage of CD8+CD28+ T cytotoxic cells in peripheral blood of patients with pre-eclampsia in comparison with healthy pregnant women in the third trimester of physiological pregnancy. Twenty-four women with pre-eclampsia and 20 normal third trimester pregnant women were included in the study. The lymphocytes were isolated from peripheral blood samples and labeled with monoclonal antibodies. The expressions of surface antigens and intracellular proteins were estimated using flow cytometry. The population of CD4+CD25+FoxP3+ Treg cells was significantly lower in peripheral blood of patients with pre-eclampsia when compared to normal third trimester pregnant women. The percentages of CD4+CD25+FoxP3+ Treg cells that express Bcl-2 protein were significantly lower in peripheral blood of patients with pre-eclampsia when compared to healthy pregnant women, whereas the percentages of CD4+CD25+FoxP3+ Treg cells with the expressions of Bax protein did not differ in both groups. Moreover, the mean fluorescence intensity (MFI) of Bcl-2 protein in CD4+CD25+FoxP3+ Treg cells was significantly lower and MFI of Bax protein significantly higher in pre-eclampsia when compared to the control group. The percentage of CD8+CD28+ T cells did not differ in both studied groups but MFI of CD28 antigen on T CD8+ cells was significantly higher in pre-eclampsia when compared to the control group. The obtained results suggest that the deficit of CD4+CD25+FoxP3+ Treg lymphocytes which is observed in pre-eclampsia may be associated with altered apoptosis signaling in Tregs.