Investigation of the possible functions of PACAP in human trophoblast cells.

Pituitary adenylate cyclase activating polypeptide (PACAP) is an endogenous neuropeptide having a widespread distribution both in the nervous system and peripheral organs including the female reproductive system. Both the peptide and its receptors have been shown in the placenta but its role in placental growth, especially its human aspects, remains unknown. The aim of the present study was to investigate the effects of PACAP on invasion, proliferation, cell survival, and angiogenesis of trophoblast cells. Furthermore, cytokine production was investigated in human decidual and peripheral blood mononuclear cells. For in vitro studies, human invasive proliferative extravillous cytotrophoblast (HIPEC) cells and HTR-8/SVneo human trophoblast cells were used. Both cell types were used for testing the effects of PACAP on invasion and cell survival in order to investigate whether the effects of PACAP in trophoblasts depend on the examined cell type. Invasion was studied by standardized invasion assay. PACAP increased proliferation in HIPEC cells, but not in HTR-8 cells. Cell viability was examined using MTT test, WST-1 assay, and annexin V/propidium iodide flow cytometry assay. Survival of HTR-8/SVneo cells was studied under oxidative stress conditions induced by hydrogen peroxide. PACAP as pretreatment, but not as co-treatment, significantly increased the number of surviving HTR-8 cells. Viability of HIPEC cells was investigated using methotrexate (MTX) toxicity, but PACAP1-38 could not counteract its toxic effect. Angiogenic molecules were determined both in the supernatant and the cell lysate by angiogenesis array. In the supernatant, we found that PACAP decreased the secretion of various angiogenic markers, such as angiopoietin, angiogenin, activin, endoglin, ADAMTS-1, and VEGF. For the cytokine assay, human decidual and peripheral blood lymphocytes were separated and treated with PACAP1-38. Th1 and Th2 cytokines were analyzed with CBA assay and the results showed that there were no significant differences in control and PACAP-treated cells. In summary, PACAP seems to play various roles in human trophoblast cells, depending on the cell type and microenvironmental influences.

Changes in the expression of pituitary adenylate cyclase-activating polypeptide in the human placenta during pregnancy and its effects on the survival of JAR choriocarcinoma cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide with survival-promoting actions, has been observed in endocrine organs and is thought to play a role in reproductive functions, including pregnancy. PACAP occurs in two forms, 27 and 38 amino acid residues, with PACAP38 being the predominant form in human tissues. In the present study, we determined the concentrations of PACAP38 and PACAP27 in first-trimester and full-term human placentas using radioimmunoassay. We found high levels of PACAP38 and lower levels of PACAP27 in different parts of the full-term human placenta. PACAP38 content increased in the placenta during pregnancy, both on the maternal side and on the fetal side. The effects of PACAP on the survival of JAR human choriocarcinoma cells were investigated using flow cytometry and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) cell viability assay in cells exposed to the widely used chemotherapeutic agent methotrexate (MTX). It was found that PACAP neither influenced the survival of JAR cytotrophoblast cells nor affected cellular response to the death-inducing effect of the chemotherapeutic agent MTX. The present observations further support the significance of PACAP in the human placenta. The observation that PACAP did not influence the effects of MTX may have future clinical importance, showing that PACAP does not decrease the effects of certain chemotherapeutic agents.

Progesterone-dependent immunomodulation.

The biological effects of progesterone are mediated by a 34-kDa protein named the progesterone-induced blocking factor (PIBF). PIBF, synthesized by lymphocytes of healthy pregnant women in the presence of progesterone, inhibits arachidonic acid release as well as NK activity, and modifies the cytokine balance. Within the cell the full-length PIBF is associated with the centrosome, while secretion of shorter forms is induced by activation of the cell. PIBF induces nuclear translocation of STAT6 as well as PKC phosphorylation and exerts a negative effect on STAT4 phosphorylation. The concentration of PIBF in pregnancy urine is related to the positive or negative outcome of pregnancy; furthermore, premature pregnancy termination is predictable by lower than normal pregnancy PIBF values. In vivo data suggest the biological importance of the above findings. Treatment of pregnant Balb/c mice with the antiprogesterone RU 486 results in an increased resorption rate, which is associated with the inability of spleen cells to produce PIBF. High resorption rates induced by progesterone receptor block as well as those due to high NK activity are corrected by simultaneous PIBF treatment.

The role of gamma/delta T cells in the feto-maternal relationship.

Polymorphic MHC is absent from the trophoblast, therefore, it resists NK as well as CTL-mediated lysis in vitro. Activated gamma / delta TCR positive cells are significantly enriched in the decidua as well as in peripheral blood of healthy pregnant women. Human peripheral gamma / delta lymphocytes preferentially express the V gamma 9/V delta 2 TCR, whereas those of the decidua use the V delta 1 chain. These subpopulations are functionally polarized, the former being Th1, the latter Th2. Potentially cytotoxic V delta 2+ lymphocytes recognize HLA-E on the trophoblast via the CD94/NKG2A receptor, which induces an inhibitory signal, thus potentially inhibiting Th1 type cytokine production.

Progesterone as an immunomodulatory molecule.

Increased progesterone sensitivity of pregnancy lymphocytes is due to activation-induced appearance of progesterone binding sites in the lymphocytes. Following recognition of fetally derived antigens gamma/delta TCR+ cells develop progesterone receptors. Progesterone binding results in the synthesis of a mediator protein named the progesterone-induced blocking factor (PIBF). PIBF by acting on the phospholipase A2 enzyme interferes with arachidonic acid metabolism, induces a Th2 biased immune response, and by controlling NK activity exerts an anti-abortive effect.

The role of gamma/delta T-cell receptor-positive cells in pregnancy: part II.

PROBLEM: We have previously demonstrated a significantly increased ratio of gamma/delta T-cell receptor (TCR)-positive progesterone receptor(PR)-positive cells in the peripheral blood of healthy pregnant women compared to that of recurrent aborters or non-pregnant individuals. Treatment of pregnancy lymphocytes with a pan anti-gamma/delta TCR antibody inhibits progesterone-induced blocking factor (PIBF) production, increases natural killer (NK) activity, and alters the cytokine profile. The present study was aimed at investigating the role of the different gamma/delta subpopulations in these phenomena. METHOD OF STUDY: Peripheral blood lymphocytes from healthy pregnant women were incubated with either anti-gamma1.4 and delta1, or anti-gamma9 and delta2 antibodies. The effect of these treatments on PR induction and interleukin (IL)-10 and IL-12 expression were tested by immunocytochemistry. NK activity of anti-gamma/delta treated lymphocytes was also determined. RESULTS: In peripheral blood of healthy pregnant women, the most frequently occurring chain combination was gamma1.4/delta1, whereas in recurrent aborters, the gamma9/delta2 combination was predominant. Treatment of normal pregnancy lymphocytes with a mixture of gamma1.4 and delta1 antibodies resulted in a significantly reduced NK activity and increased PR and IL-10 expression, whereas treatment with a mixture of gamma9 and delta2 antibodies significantly reduced IL-10 production and slightly increased IL-12 production and NK activity. These data suggest the presence of two functionally distinct subpopulations in the peripheral blood of pregnant women.

The role of gamma/delta T cells in progesterone-mediated immunomodulation during pregnancy: a review.

PROBLEM: To determine if pregnancy is recognized by the immune system and if inadequate recognition of fetal antigens might result in failed pregnancy. METHOD OF STUDY: Review of literature and current data. RESULTS: In the decidua gamma/delta TCR positive cells significantly increase in number. A subset of gamma/delta T cells reacts with nonpolymorphic Class I or Class I like molecules. Trophoblast recognition is mediated by the V gamma 1 subset which recognize a conserved mammalian sequence on the trophoblast. Almost all gamma/delta T cells in the decidua are activated and use the V delta 1 chain, whereas the majority of human peripheral gamma/delta lymphocytes expresses V gamma 9/V delta 2 TCR. Peripheral gamma/delta T cells of healthy pregnant women preferentially use V gamma V delta 1 chains, on the other hand, those of recurrent aborters use the V gamma 9V delta 2 combination. Signaling via the V gamma 1.4V delta 1 receptor induces a Th2 type response, whereas activation of the lymphocytes via the V gamma 9V delta 2 receptor results in increased IL-12 production and natural killer (NK) activity. In the presence of progesterone, activated lymphocytes synthesize the progesterone induced blocking factor (PIBF), which inhibits NK activity and exerts an anti abortive effect in vivo. Decidual CD56+ and gamma delta+ cells are to a high extent the same population. CONCLUSION: All decidual CD56+ cells express PIBF, thus it cannot be excluded that local production of this substance contributes to low decidual NK activity and thus to the success of the pregnancy.

The role of gamma/delta T cell receptor positive cells in pregnancy.

PROBLEM: Due to the lack of classical HLA antigens on the trophoblast, fetal antigens are possibly presented in a non major histocompatibility complex (MHC) restricted way. Decidual gammadelta T cells, which significantly increase in number during pregnancy, might play a role in recognition of fetal antigens and also in determining the quality of the response to these antigens. Our study was aimed at investigating the role of this cell population in progesterone-dependent immunomodulation. METHOD OF STUDY: Peripheral lymphocytes from healthy pregnant women and from habitual aborters were tested by immunocytochemistry for the presence of gamma/delta T cell receptor (TCR) and progesterone receptor. To investigate the effect of treatment with a pan anti gamma/delta antibody, lymphocytes were incubated for 3 hr with the antibody, and then interleukin (IL)-10, IL-12 and progesterone-induced blocking factor (PIBF) expression (by immuno-cytochemistry) as well as natural killer (NK) cell activity were determined. RESULTS: In peripheral blood of healthy pregnant women the percentage of gamma/delta TCR+ cells was significantly higher (P < 0.001) than in that of recurrent aborters or of non-pregnant individuals. Ninety-seven percent of gamma/delta TCR+ pregnancy lymphocytes expressed progesterone receptor. Binding of a specific antibody to the gamma/delta TCR inhibited PIBF- as well as IL-10 production, whereas it increased NK activity and IL-12 expression. CONCLUSIONS: These data suggest the role of gamma/delta TCR-bearing lymphocytes in progesterone-dependent immunomodulation.