Cytokine secretion in decidual mononuclear cells from term human pregnancy with or without labour: ELISPOT detection of IFN-γ, IL-4, IL-10, TGF-β and TNF-α

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Abstract

Cytokines are believed to be important in maintaining pregnancy and in the process of labour induction in humans. The aim of this study was to investigate the secretion of the cytokines interferon-γ (IFN-γ), interleukin-4 (IL-4), IL-10, transforming growth factor-β (TGF-β) and tumour necrosis factor-α (TNF-α) in decidual tissue with or without labour.

Decidual tissue was collected from 32 healthy women undergoing elective caesarean sections before the onset of labour (n = 17) or after normal vaginal delivery (n = 15). Mononuclear cells were analysed for cytokine secretion with ELISPOT. To validate the widely used method of tissue collected at caesarean sections and after vaginal deliveries as a representative of before and after labour, respectively, placenta biopsies were collected from 12 healthy women to study the expression of the prostaglandin pathway enzymes cyclooxygenase-2 (COX-2) and microsomal prostaglandin E2 synthase (mPGES).

Decidual mononuclear cells from term human pregnancy spontaneously secrete IFN-γ, IL-4, IL-10, TGF-β and TNF-α. No difference was seen in cytokine secretion with or without labour, indicating that decidual leukocytes are not the main cell population responsible for plausible cytokine regulation in the process of termination of pregnancy. Placental tissues obtained after vaginal delivery showed a higher mRNA expression of the prostaglandin regulating molecules COX-2 and mPGES than tissues from caesarean sections before the onset of labour, validating that the model can be used as a representative of the state before and after labour.

Introduction

The immune system is tightly regulated during pregnancy in order to avoid rejection of the semi-allogenic foetus. In addition, the immune system also seems to contribute to the development of a normal pregnancy including induction of parturition. It is widely accepted that prostaglandins (PGs) play a crucial role in the onset of labour. Prostaglandins and their receptors are regulated by, for example, hormonal changes, mechanical pressure of the uterine wall and the secretion of cytokines (Hertelendy and Zakar, 2004, Keelan et al., 2003, Yellon et al., 2003). The secretion and effect of different cytokines and prostaglandins seem to vary between different tissues and fluids in the uterine environment (Alfaidy et al., 2003, Marvin et al., 2002, Mitchell et al., 2004, Osman et al., 2003, Young et al., 2002). To elucidate the role of different sets of molecules in the induction of labour in humans, samples taken from caesarean section were used to represent a state before labour, whereas samples from vaginal deliveries are used to represent the state after labour. Regulation of cytokines is important in both maintaining pregnancy and the process of labour induction. Pro-inflammatory cytokines, such as interleukin-1β (IL-1β), IL-6, IL-8 and tumour necrosis factor-α (TNF-α) have been shown to increase in cervical tissues along with labour (Sennström et al., 2000, Winkler et al., 1998), although opposing results on TNF-α have been reported (Osman et al., 2003, Young et al., 2002). Interferon-γ (IFN-γ) represents cell-mediated type I immunity and has been shown to be detrimental to pregnancy in mice (reviewed in Raghupathy, 1997). Labour, in contrast, has been proposed to be a type I phenomenon (Wegmann et al., 1993). As for TNF-α, IFN-γ expression in labouring/non-labouring tissues show different patterns with regard to the methods and tissues used in different studies (Hanna et al., 2004, Veith and Rice, 1999). IL-10 is a potent cytokine that counteracts the effects of inflammatory and type I actions. We have previously demonstrated high spontaneous secretion of IL-10 in leukocytes as well as in enriched macrophages from decidua in early normal pregnancy, indicating a role in maintaining pregnancy in humans (Ekerfelt et al., 2002b, Lidström et al., 2003). IL-10 has also been shown to prevent LPS-induced preterm birth in a mouse model (Dudley et al., 1996b). A withdrawal of IL-10 would thus be a potential mechanism in the induction of labour, and a decrease in IL-10 production in placental and decidual tissues along with gestation and with labour has indeed been shown (Hanna et al., 2000, Simpson et al., 1998), although functional studies show opposite effects of IL-10 in different tissues (Mitchell et al., 2004). Other down-regulatory agents include the anti-inflammatory cytokine transforming growth factor-β (TGF-β) and the major type 2 immunity cytokine IL-4, which have both been detected in decidua, at mRNA and protein levels for TGF-β and at mRNA levels for IL-4 (Marvin et al., 2002, Wilczynski et al., 2002). However, their role at term pregnancy seems uncertain since no differences were found in tissues from caesarean sections compared with tissues from vaginal deliveries.

Although often used, the model of caesarean section as a representative of before labour and vaginal delivery as a representative of after labour can be questioned. A differential expression in these samples of key molecules in the PG pathway would be in favour of the model. The complex and multifactorial regulation of prostaglandin synthesis is dependent on the enzyme cyclooxygenase-2 (COX-2) and both mRNA levels and protein concentrations of this enzyme have been shown to increase in foetal membranes with gestational age and in association with labour (Slater et al., 1999). Prostaglandin E2 synthase (PGES) is the terminal enzyme of the COX-2 dependent synthesis of PGE2. Microsomal PGES is the inducible form of PGES, whose expression is markedly increased following pro-inflammatory stimuli (Murakami et al., 2000). The importance of PGES in regulating prostaglandin production at term labour is uncertain, but a recent study showed increased levels of mPGES protein expression in chorion after labour (Alfaidy et al., 2003).

With the aim of clarifying a part of the complex cytokine network in the uterus in term pregnancy and at parturition, we investigated the secretion of the anti-inflammatory cytokines IL-10 and TGF-β, the type 1/type 2 immunity cytokines IFN-γ and IL-4, and the inflammatory cytokine TNF-α in decidual leukocytes. With the use of the sensitive ELISPOT method, we were able to detect spontaneously secreted proteins even at very low levels, which was particularly useful for IL-4, a cytokine many other methods fail to detect. The use of tissues from caesarean sections and vaginal deliveries as being representative of the state before and after labour, respectively was validated by studying differential mRNA expression of the strategic PG pathway enzymes COX-2 and PGES.

Section snippets

Participants

Decidual tissue was obtained from 32 healthy women with normal pregnancies, undergoing elective caesarean section before the onset of labour (n = 17) or after normal vaginal delivery (n = 15) at the Department of Gynecology and Obstetrics, Linköping University Hospital, Sweden. The patients’ characteristics are summarized in Table 1. The purity of decidual mononuclear cells was studied using flow cytometry in six of the women, three from the vaginal and three from the caesarean section groups.

Cytokine secreting mononuclear cells in decidua

All cytokines analysed, IFN-γ, IL-4, IL-10, TGF-β and TNF-α, were spontaneously secreted in mononuclear cells from decidual tissues both from women undergoing elective caesarean section prior to labour and from women after vaginal delivery (Fig. 1). There were no significant differences in the number of secreting cells for any of the cytokines when comparing decidual cells from caesarean section with those obtained from vaginal delivery.

There were significant correlations between the secretion

Discussion

We have previously shown spontaneous secretion of IFN-γ, IL-4 and IL-10 in decidual mononuclear cells as well as in decidual macrophages and natural killer cells (NK cells) from early normal pregnancies (Ekerfelt et al., 2002b, Lidström et al., 2003). We report here the secretion of these cytokines and in addition, that of TNF-α and TGF-β in decidual mononuclear cells from normal pregnancies at term. These cytokines, among others, were previously detected at the mRNA level from term

Acknowledgements

This study was supported by grants from the Swedish Research Council (project 73X-15335-01A), The Health Research Council in the South-East of Sweden (FORSS), the County Counsil of Östergötland (ÖLL), Linköping University and Linköping University Hospital. We thank Jeannette Svartz and Karin Backteman for help with the flow cytometry. We also thank Jenny Mjösberg for help with control experiments and David Engblom for help with the PCR-analyses.

References (43)

  • T.G. Wegmann et al.

    Bidirectional cytokine interactions in the maternal–fetal relationship: is successful pregnancy a TH2 phenomenon?

    Immunol. Today

    (1993)
  • J. Yui et al.

    Functional, long-term cultures of human term trophoblasts purified by column-elimination of CD9 expressing cells

    Placenta

    (1994)
  • G. Aboagye-Mathiesen et al.

    Isolation and characterization of human placental trophoblast subpopulations from first-trimester chorionic villi

    Clin. Diagn. Lab. Immunol.

    (1996)
  • N. Alfaidy et al.

    Expression of membrane prostaglandin E synthase in human placenta and fetal membranes and effect of labor

    Endocrine

    (2003)
  • D.M. Bullens et al.

    Effects of anti-IL-4 receptor monoclonal antibody on in vitro T cell cytokine levels: IL-4 production by T cells from non-atopic donors

    Clin. Exp. Immunol.

    (1998)
  • D.J. Dudley et al.

    Inflammatory cytokine mRNA in human gestational tissues: implications for term and preterm labor

    J. Soc. Gynecol. Investig.

    (1996)
  • D.J. Dudley et al.

    Interleukin-10 (IL-10) prevents preterm birth in a mouse model of infection-mediated preterm labor

    J. Soc. Gynecol. Investig.

    (1996)
  • C. Ekerfelt et al.

    Th1-like responses to peptides from peripheral nerve myelin proteins in patients with polyneuropathy associated with monoclonal gammopathy

    Clin. Exp. Immunol.

    (1997)
  • C. Ekerfelt et al.

    Spontaneous secretion of interleukin-4, interleukin-10 and interferon-gamma by first trimester decidual mononuclear cells

    Am. J. Reprod. Immunol.

    (2002)
  • M. Eriksson et al.

    Unique phenotype of human uterine NK cells and their regulation by endogenous TGF-beta

    J. Leukoc. Biol.

    (2004)
  • N. Hanna et al.

    Gestational age-dependent expression of IL-10 and its receptor in human placental tissues and isolated cytotrophoblasts

    J. Immunol.

    (2000)
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