Quantitative analysis of peripheral blood Th0, Th1, Th2 and the Th1:Th2 cell ratio during normal human pregnancy and preeclampsia.

We calculated the percentage of Th1, Th2, Th0 cells and the Th1:Th2 cell ratio of peripheral blood from normal pregnant subjects and preeclampsia patients using flow cytometry which can analyse both the surface marker, CD4, and intracellular cytokines, interleukin (IL)-4 and interferon (IFN)-gamma. In normal pregnancy, the percentage of Th1 cells was significantly lower in the third trimester, and the ratios of Th1:Th2 were significantly lower in the second and third trimester than in nonpregnant subjects. In contrast, the percentage of Th1 cells and the ratios of Th1:Th2 in preeclampsia were significantly higher than in normal third trimester pregnant subjects. The percentage of Th2 cells in preeclampsia was significantly lower than in third trimester of normal pregnancy. Additionally, peripheral blood mononuclear cells from these subjects and patients were cultured with phytohemagglutinin stimulation, and IL-4 and IFN-gamma concentrations were determined in the supernatant by enzymed linked immunosorbent assays. The percentage of Th1 and Th2, and the ratios of Th1:Th2 were correlated with cytokine (IFN-gamma and IL-4) secretion level. These results demonstrated that Th2 cells were predominant in the second and third trimesters of normal pregnancy, but Th1 cells predominated in preeclamptic patients.     

A novel surface molecule of Th2- and Tc2-type cells, CRTH2 expression on human peripheral and decidual CD4+ and CD8+ T cells during the early stage of pregnancy

It has been demonstrated that pregnancy induces the immunomodulation of cytokine responses away from the Th1 paradigm and towards the Th2 paradigm. In this study, we examined the expression of CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2) on decidual CD4+ and CD8+ T cells during the early stages of pregnancy. Examination of the cytokine profile revealed that CRTH2 was expressed on CD4+-type-2 T helper (Th2-type) and CD8+-type 2 T cytotoxic (Tc2-type) cells. The percentages of CRTH2+ cells in CD3+/CD4+ T cells and CD3+/CD8+ T cells were significantly higher in the decidua than in the peripheral blood. These results indicate the significance of Th2- and Tc2-type cells of the decidua in the maternal immune system, presumably through their production of cytokines which may contribute to the maintenance of pregnancy.     

Proliferative activity in endometrial stromal granulocytes throughout menstrual cycle and early pregnancy.

The proliferative activity in endometrial stromal granulocytes was studied using two approaches. Firstly, mitotic activity was studied in paraffin wax embedded sections of normal non-pregnant endometrium and early pregnancy decidua stained with phloxine-tartrazine. Secondly, the monoclonal antibody Ki67 was applied to cryostat sections of similar tissues. Endometrial stromal granulocytes were identified by their labelling with NKH1, Dako T11, UCHL1 or Dako-LC. The percentage of cases in which endometrial stromal granulocytes showed mitosis was 25%, 75%, 86%, and 93%, respectively in proliferative, early secretory, mid secretory, and late secretory phases, and 14% in early decidua. There were at most one or two endometrial stromal granulocytes in mitosis per 10 high power fields. Double labelled cells were present in small numbers in proliferative endometrium and in moderate numbers in secretory endometrium. Only a few cells in early decidua double labelled with Ki67/T11; moderate numbers of cells double labelled with Ki67/Dako-LC. It is concluded that proliferative activity does occur in endometrial stromal granulocytes and is particularly prominent in the late secretory phase.     

Regulation of fetal allograft survival by hormone-controlled Th1- and Th2-type cytokines

There is clear evidence to suggest that the maternal immune system during pregnancy can enhance or inhibit the development of the fetoplacental unit. Recent data support the view that some cytokines produced by both T cells and non-T cells (IL-3, GM-CSF, TGF-β, IL-4, IL-10), favor fetal survival and growth. In contrast, other cytokines, such as IFN-γ, TNF-β and TNF-α, can rather compromise pregnancy. Accordingly, we show here that T-cell clones generated from the decidua of women with unexplained recurrent abortion produced significantly lower concentrations of IL-4 than clones derived from the decidua of voluntary abortions or the endometrium of nonpregnant women. Thus, despite the complexity of the cytokine network, it appears that cytokines favoring the maintenance of fetal survival mainly belong to the Th2 pathway, whereas the failure of pregnancy rather associates with the predominance of Th1-type cytokines and/or the absence of Th2-type cytokines. Interestingly, we also found that, at least in vitro, progesterone promotes the preferential development of Th2-like cells and induces transient IL-4 production by established Th1 cells, whereas relaxin, another corpus luteum-derived hormone, mainly promotes the development of Th1-like cells. These data provide an excellent basis for investigating the relationship between the endocrine and the immune system in the regulation of the maternal-fetal interaction.     

Predominance of Th2-promoting dendritic cells in early human pregnancy decidua

Dendritic cells (DCs) are specialized antigen-presenting cells required for the priming and activation of T cells and promote the differentiation of na?ve CD4+ T cells toward the T helper cell type 1 (Th1) or Th2 phenotype. Here, we describe the characterization of CD45+CD3-CD14-CD16-CD19-CD20-CD56-HLA-DRbright DCs from early human pregnancy decidua by flow cytometry. The percentage of DCs to mononuclear cells (leukocytes) in the decidua was significantly higher than that in the peripheral blood. Moreover, decidual DCs expressed costimulatory molecules such as CD80 and CD86 and a mature marker such as CD83 on their surface. The percentage of CD11c+CD123- myeloid DCs in the decidua was significantly higher than that in the peripheral blood. Conversely, the ratio of CD11c-CD123+ lymphoid DCs in the decidua was significantly lower than that in the peripheral blood. The number of interleukin (IL)-12-producing cells in the total DC population and the myeloid DCs in the decidua was significantly lower than that in the peripheral blood. IL-12 secretion by activated decidual myeloid DCs was significantly lower than that by peripheral DCs. Na?ve CD4+ T cells primed with decidual myeloid DCs led to a higher percentage of Th2 cells in comparison with that with peripheral myeloid DCs. This finding was abolished by exogenous IL-12 administration with decidual myeloid DCs. Thus, the DCs in the decidua could regulate the Th1/Th2 balance to maintain a Th2-dominant state, leading to maintenance of pregnancy.     

Questioning the Th1/Th2 paradigm in reproduction: peripheral levels of IL-12 are down-regulated in miscarriage patients

PROBLEM: It has been postulated that a T helper (Th)1 response is associated with pregnancy failure, whereas a Th2 response contributes to pregnancy maintenance. However, this Thl/Th2 dichotomy has recently been hypothesized to be an oversimplification. To prove this novel hypothesis, we investigated the levels of the Th1-inducer cytokine interleukin (IL)-12 in immunocompetent cells of patients with normal pregnancies (NP) and spontaneous abortion (SA). METHODS: Presence of intracellular IL-12 was evaluated in CD8+ and CD56-blood and decidual lymphocytes as well as in monocytes and granulocytes by flow cytometry from NP and SA individuals. IL-12 serum levels were measured by enzyme-linked immunosorbent assay (ELISA). We further investigated the effect of recombinant human (rh) IL-12 on the production of interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha in peripheral leukocytes ex vivo. RESULTS: In patients suffering from SA we observed lower percentages of IL-12 in lymphocytes, monocytes and granulocytes derived from peripheral blood and decidua, compared with women with normally progressing pregnancies. No differences could be observed when evaluating the levels of IL-12 in the granulocyte population. The IL-12 serum levels were below the ELISA sensitivity limit. Ex vivo stimulation of the peripheral blood cells with increasing doses of IL-12 resulted in a significant decrease of IFN-gamma+, whereas levels of TNF-alpha+ in lymphocytes were unaffected. CONCLUSIONS: The classical Th1/Th2 paradigm appears to be insufficient to exclusively explain the causes of pregnancy loss. Our current results render us to requestion the role of Th1 cytokines during pregnancy and suggest some protective function of the Th1-inducer cytokine IL-12.     

Th1 and Th2 cytokine profiles in recurrent aborters with successful pregnancy and with subsequent abortions

BACKGROUND: This study compared Th1-Th2 cytokine profiles in a subgroup of recurrent aborters who had an abortion with those in a subgroup of recurrent aborters who had a successful pregnancy. METHODS: Fifty-four women with a history of at least three normal pregnancies, 24 women with a history of recurrent spontaneous abortion (RSA) followed by abortion (RSA-->A) and 39 women with a history of RSA followed by normal pregnancy (RSA-->N) were studied. Blood samples and placentas were obtained at the time of delivery or abortion; peripheral blood mononuclear cells were stimulated separately with phytohaemagglutinin and with autologous placental cells, and the secreted cytokines estimated. RESULTS: Peripheral blood mononuclear cells from the RSA-->N subgroup secreted higher concentrations of Th1-type cytokines as compared with normal pregnant women, indicating a higher Th1 bias in these women. However, women in the RSA-->N subgroup had significantly higher concentrations of Th2 cytokines as compared with women in the RSA-->A subgroup. A comparison of Th1:Th2 cytokine ratios indicated a higher Th2 bias in RSA-->N women as compared with RSA-->A women. CONCLUSIONS: We conclude that abortion-prone women who proceed to have successful pregnancy are more Th2-biased than abortion-prone women who abort, and that recurrent aborters who undergo spontaneous abortion have a stronger Th1 bias than aborters who have normal pregnancy.     

Intravenous immunoglobulin treatment in women with recurrent abortions: increased cytokine levels and reduced Th1/Th2 lymphocyte ratio in peripheral blood

PROBLEM: The aim of this study was to investigate changes in peripheral blood Th1/Th2 cytokine levels and lymphocyte ratios after massive intravenous immunoglobulin (MIVIg) treatment for women with recurrent spontaneous abortion (RSA) of unexplained etiology. METHOD OF STUDY: Serum Th1 (IFN-gamma, TNF-alpha) and Th2 cytokine (IL-4, IL-10) levels were assessed by ELISA methods (n = 9) and peripheral blood Th1/Th2 lymphocyte ratios (n = 4) by flow cytometry before and after MIVIg treatments in women with four or more consecutive RSA. RESULTS: Pre-treatment serum IFN-gamma (0.06 +/- 0.09 pg/mL, mean +/- SD), TNF-alpha (0.21 +/- 0.45 pg/mL), IL-4 (0.70 +/- 1.16 pg/mL), and IL-10 (1.12 +/- 1.67 pg/mL) increased to 0.17 +/- 0.16 pg/mL, 0.77 +/- 0.28 pg/mL, 1.82 +/- 0.89 pg/mL, and 3.44 +/- 0.48 pg/mL, respectively, after MIVIg treatments (P < 0.05). CD4-positive IFN-gamma/IL-4 lymphocyte ratios (17.3 +/- 9.1) were reduced to 11.5 +/- 7.1 after treatment (P < 0.05). CONCLUSIONS: Massive intravenous immunoglobulin treatments increased peripheral blood cytokine levels and decreased Th1/Th2 lymphocyte ratios; thus, MIVIg treatments modify the peripheral Th1/Th2 balance.     

Immunohistochemical localization of the growth hormone in human endometrium and decidua

PROBLEM: Recent evidence of growth hormone (GH) receptor expression in rat endometrium and human myometrium have focused our attention on the role of the GH in endometrial development. We tested the expression of GH in the human endometrium throughout the menstrual cycle and during pregnancy. METHOD OF STUDY: Immunohistochemical study was performed on endometrial specimens of fertile women in different periods of the menstrual cycle and in decidua of pregnant women. RESULTS: Glandular cells of the human endometrium were positive for GH in the mid and late luteal phase. Furthermore, the glandular cells of decidua showed intense staining for GH, while the stromal cells were negative. No immunostaining was expressed in the proliferative or early luteal phase. The intensity levels of staining for GH in decidual specimens were significantly higher than in glandular cells of secretory endometrium specimens (P < 0.01). CONCLUSIONS: The glandular cells of the human endometrium express GH from the late luteal phase throughout pregnancy in the decidual tissue. We suppose that GH plays an important role in blastocyst implantation.     

Reduced uterine indoleamine 2,3-dioxygenase versus increased Th1/Th2 cytokine ratios as a basis for occult and clinical pregnancy failure in mice and humans

PROBLEM: Indoleamine 2,3-dioxygenase (IDO) expression in fetal trophoblast and decidual antigen-presenting cells has been proposed to inactivate maternal T cells and thereby prevent rejection of the "fetal allograft" in early pregnancy. Psychic stress has been proposed to cause miscarriages as well as infertility, at the same time in pregnancy when blockade of IDO causes loss, but the suggested mechanism of stress-triggered loss has been an increased ratio of pro-rejection Th1-type cytokines to anti-rejection Th2/3 cytokines. Could stress act by reducing IDO expression? METHODS: Using DBA/2-mated A/J mice where stress causes early pregnancy failure, we examined the role of stress in reducing IDO versus increasing Th1/Th2 ratio in deciduas. IDO loss was also examined in human decidua associated with pregnancy failure. RESULTS: A post-implantation sonic stress increased the pregnancy failure rate, increased the Th1/Th2 ratio, but did not reduce IDO. IDO was reduced, and Th1/Th2 ratios increased in A/J mice pre-immunized against paternal DBA/2 antigens, and concomitant stress increased these effects. The rate of pregnancy failure was not further increased consistent with recent discoveries of factors that limit the impact of Th1 cytokines at the feto-maternal interface. In deciduas from spontaneous miscarriage patients, IDO(+) cell frequencies were low in only 30% of patients. CD3(+) T-cell numbers and percentage terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end-labelling (TUNEL)(+) apoptotic T cells were increased, but the level of IDO did not correlate with likelihood of apoptosis. CONCLUSIONS: Loss of an allogeneic embryo in early pregnancy is more likely to be due to a high Th1/Th2 ratio than loss of putative protection by IDO.     

Characterization of NKT Cells in Human Peripheral Blood and Decidual Lymphocytes

PROBLEM: To examine whether natural killer (NKT) cells are present in human pregnancy decidua. METHOD OF STUDY: We calculated the percentage of CD3+CD161+Valpha 24+-NKT cells in peripheral blood and early pregnancy decidua, and analyzed intracellular cytokines, interleukin (IL)-4 and interferon (IFN)gamma in NKT cells using flow cytometry. RESULTS: A distinct subset of CD3+ CD161+ lymphocytes expressing an invariant antigen receptor encoded by the Valpha24 and Vbeta11 segment was accumulated in the decidua. In pregnant subjects the percentages of NKT cells were significantly increased in the decidua compared with peripheral blood. Both NKT cells in the decidua and the peripheral blood had an ability to rapidly produce cytokine associated with Th1 (IFNgamma) and Th2 (IL-4). Interestingly, the percentages of IL-4 and IFNgamma producing NKT cells were significantly higher in the decidua compared with the peripheral blood. CONCLUSIONS: These findings suggest that NKT cells might control the Th1/Th2 balance by producing IL-4 and IFNgamma at the feto-maternal interface.     

Characteristic changes of large granular lymphocytes that strongly express CD56 in endometrium during the menstrual cycle and early pregnancy

Large granular lymphocytes that strongly express CD56 (CD56++ LGL) constitute a major population of leukocytes in the secretory endometrium and pregnancy decidua and are considered to be involved in reproductive immunity and in maintaining the pregnancy. The present study aimed to reveal the relationship between the characteristic changes of CD56++ LGL and altered hormonal environment and/or trophoblast invasion in the endometrium. Cell surface markers of CD56++ LGL obtained from the endometrium during the menstrual cycle and early pregnancy were analysed using flow cytometry. The percentages of both CD56++ LGL that express activation antigens (CD69, HLA-DR) and those that express lymphocyte function associated antigen-1 (LFA-1) (CD11a/CD18) were highest in the proliferative phase and decreased gradually throughout the menstrual cycle. Expression of these antigens was further suppressed in the late secretory phase, as well as in the early stage of pregnancy. However, the percentage of CD56++ LGL that express these antigens was significantly higher in spontaneous abortions than in normal pregnancies. On the other hand, the percentage of CD56++ LGL that express CD45RA was lower during normal pregnancy than during the menstrual cycle. The present results suggest that characteristics alterations of CD56++ LGL are regulated by altered hormonal environment and by trophoblast invasion.      

SHORT COMMUNICATION: Circulating and Decidual Th17 Cell Levels in Healthy Pregnancy

Citation Nakashima A, Ito M, Yoneda S, Shiozaki A, Hidaka T, Saito S. Circulating and decidual Th17 cell levels in healthy pregnancy. Am J Reprod Immunol 2010; 63: 104–109 Problem The Th1/Th2 paradigm has recently been reconstituted to include a third population, Th17 cells. It has been reported that Th2 type immunity is predominantly present in normal pregnancy. However, the level of Th17 cells during pregnancy is still unclear. We investigated the level of peripheral Th17 cells in healthy pregnancy subjects. Method of study To evaluate the levels of Th17 cells, we investigated the proportion of peripheral blood mononuclear cells that produced IL‐17 in the first, second, and third trimester pregnancy subjects using flow cytometry. We further studied the proportion of decidual lymphocytes that produced IL‐17 in early pregnant subjects. Results Most of the IL‐17‐producing cells were CD4⁺ T cells. The number of circulating Th17 cells did not change during pregnancy. In a paired t‐test of early normal pregnant subjects, the proportion of IL‐17⁺ decidual lymphocytes was significantly higher than that of peripheral blood lymphocytes. Conclusion Th17 levels in peripheral blood lymphocytes do not change during normal pregnancy.     

Progesterone receptor subtype B is differentially regulated in human endometrial stroma

Two anti-progesterone receptor (PgR) antibodies, a new one specific to PgRB, the other to PgR subtypes A + B, have been used to examine the cellular location of PgR subtypes A and B in normal endometrium throughout the menstrual cycle and in early human decidua by standard immunohistochemical techniques. PgR(A+B) is the receptor detected by the antibody recognizing both isoforms of the receptor. PgRB is the receptor detected by the new antibody specific to the B isoform. Since it is not possible to raise antibody specific to PgR subtype A, all immunohistochemical analysis of the PgRA subtype is by subtractive inference. Thus we refer to PgRA as the subtype responsible for positive immunoreactivity when the PgRB subtype cannot be specifically detected. Endometrial biopsies were collected from 40 women with regular menses (n = 5 each stage of cycle: menstrual; early, mid and late proliferative; ovulatory; early, mid, and late secretory). Decidual tissue was obtained from 10 women undergoing first trimester surgical termination of pregnancy. As previously reported, the PgR(A+B) antibody stained glandular and stromal nuclei during the proliferative phase but only stromal nuclei during the secretory phase and early pregnancy. The new PgRB antibody also stained both cell types intensely during the proliferative phase, but failed to stain either stromal or glandular nuclei strongly during the secretory phase and early pregnancy. We concluded that, while both PgR subtypes were present in glands and stroma in the proliferative phase, and both subtypes were dramatically reduced in the glands during the secretory phase, PgRA remained as the predominant type in the stroma during the secretory phase and early pregnancy. The profound effects of progesterone on endometrium during the secretory phase and early pregnancy appear to be mediated primarily by PgRA in the stroma.      

Polarity of helper T cell subsets represents disease nature and clinical course of experimental autoimmune myocarditis in rats

The mechanisms of progression, remission and relapse of myocarditis remain unclear. To clarify these mechanisms, we focused on T helper-1 (Th1)/T helper-2 (Th2) subsets balance of peripheral lymphocytes and serum cytokine levels during disease progression in rats with experimental autoimmune myocarditis (EAM). Lewis rats were immunized with cardiac myosin on day 0. Blood samples were collected on days 0, 7, 15, 18, 21, 28, 35, 42, 49 and 56 following immunization. We examined percentages of interferon (IFN)-gamma and/or interleukin (IL)-4 producing cells in stimulated peripheral CD4-positive lymphocytes using flow cytometry analysis. Serum IFN-gamma, IL-2, IL-6 and IL-10 levels were measured by enzyme-linked immunosorbent assay (ELISA). The percentage of Th1/Th2 subsets in EAM on days 0, 15, 28 and 56 were 2.5 +/- 0.5/0.5 +/- 0.1%, 19.4 +/- 3.2/1.6 +/- 0.3%, 2.0 +/- 0.5/22.1 +/- 5.7% and 3.0 +/- 0.4/1.7 +/- 0.3%, respectively. Serum levels of Th1 cytokines, IFN-gamma and IL-2 significantly increased in the acute phase (from day 15-18) and immediately decreased in the early recovery phase. On the other hand, serum levels of Th2 cytokine, IL-10 significantly increased in the early recovery phase (from day 24-30). These results suggest that induction of acute myocarditis might be associated with systemic Th1 dominance, while recovery is related to systemic Th2 polarity. Thus, analysis of Th1/Th2 balance in peripheral T cells may be useful in disease monitoring in patients with myocarditis and postmyocarditic dilated cardiomyopathy.     

自身免疫性溶血性贫血患者外周血Th1／Th2、Th3／Tr1细胞的检测及意义

目的探讨自身免疫性溶血性贫血（AIHA）患者外周血Th1／Th2、Th3／Tr1细胞状态,分析它们在AIHA发病机制中的作用。方法收集AIHA患者及健康者外周抗凝静脉血,分离纯化淋巴细胞。运用FITC-CD3单抗,Cy5-CD4单抗,PE—CRTH2单抗,以CD3／CD4设门作三色流式细胞术检测Th1／Th2细胞,ELISA法检测血清中Th3细胞相关的细胞因子TGF-β1的含量和Tr1细胞的相关因子IL-10的含量。结果与正常对照相比．AIHA患者外周血CD3^＋CD4^＋CRTH2-T细胞（Th1）百分率、CD3^＋CD4^＋CRTH2^＋T细胞（Th2）百分率均下降（P〈0．05）,而CD4^＋CRTH2^-T／CD4^＋CRTH2^＋T比例（Th1／Th2）均明显升高（P〈0．01）,Th3／Tr1细胞分泌的相关细胞因子TGF-β1和IL-10的含量均降低（P〈0．05）。结论AIHA患者外周血存在细胞免疫功能失调,T细胞亚群极化状态发生改变,呈Th1型细胞优势,Th3／Tr1细胞因子含量下降,可能与AIHA的免疫学发病机制有关。