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Progesterone and the immunology of pregnancy
Journal of Steroid Biochemistry & Molecular Biology 97 (2005) 389–396
Progesterone and the immunology of pregnancy夽 Ren´e Druckmann ∗ , Marc-Alexandre Druckmann A.N.E.M.O.-Centre de m´enopause, 10-12 Rue de France, F 06000 Nice, France
Abstract The foetal–placental unit is a semi-allograft and the immunological recognition of pregnancy, together with the subsequent response of the maternal immune system, is necessary for a successful pregnancy. This recognition of pregnancy results in an upregulation of progesterone receptors on activated lymphocytes amongst placental cells and decidual CD56+ cells. In the presence of sufficient progesterone, these cells synthesise progesterone induced blocking factor (PIBF), a mediator that exerts substantial anti-abortive activities. PIBF affects B cells and induces an increased production of asymmetric, non-cytotoxic antibodies. It also alters the profile of cytokine secretion by activated lymphocytes resulting in an increase in the production of non-inflammatory, non-cytotoxic interleukins (IL) (e.g. IL-3, IL-4 and IL-10) and a reduction in the production of inflammatory, cytotoxic cytokines (e.g. interferon (IFN)-␦, tumour necrosis factor (TNF)-␣ and IL-2). PIBF also inhibits the cytotoxity of natural killer (NK) cells by blocking their degranulation and perforin release, as well as inhibiting IFN-␦, TNF-␣ and IL-2-mediated transformation of NK cells into detrimental lymphokine activated killer (LAK) cells. © 2005 Elsevier Ltd. All rights reserved. Keywords: Pregnancy; Progesterone; Spontaneous abortion; Immune system; Progesterone-induced blocking factor; T-helper cells; Cytokines
1. Introduction For more than 50 years it has been suspected that a distinct interaction between female sex hormones and the overall immune system might exist. “How does the pregnant mother contrive to nourish a foetus within herself for many months that is an antigenically foreign body?” was the question raised by P.B. Medawar in 1953 [1]. Spontaneous miscarriage (abortion) occurs in 15% of all clinically recognised pregnancies and it is the most common complication of pregnancy. Approximately 50–60% of spontaneous pregnancy losses can be explained by chromosomal anomalies of the foetus, infectious aetiologies, maternal endocrinology or anatomical comorbidity, but still 40–50% remain “unexplained”. A substantial portion of these “unexplained” cases could be attributable to an abnormal, abortogenic immune response of the mother towards potential antigens on the foetus. Recently, a growing body of evidence has been accumulating to suggest that proges夽 Presented at the European Progestin Club Scientific Meeting Amsterdam, The Netherlands, 05 October 2004. ∗ Corresponding author. Tel.: +33 493820608; fax: +33 493162743. E-mail address: [email protected] (R. Druckmann).
0960-0760/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2005.08.010
terone might play a significant role in establishing an adequate immune response during the early stages of successful pregnancy [2]. The implantation of the human embryo is a double paradox in that it is immunological and biological. The immunological paradox is that it consists of a heterologous graft in which the uterine immune system (via the cytokines) and the antigenicity of the embryo (HLA-G) collaborate to make both implantation and maintenance of the pregnancy possible [3]. The biological paradox arises because several different mechanisms must be successively implemented for these two epithelia to fuse and then for one to allow invasion by the other (i.e. for the endometrium to be decidualised by the trophoblast): these mechanisms include preparation of the endometrium throughout the menstrual cycle under the influence of oestrogens and then progesterone, the involvement of growth factors (epidermal growth factor, transforming growth factor and insulin-like growth factors), neoangiogenisis (oestradiol, fibroblast growth factors and vascular endothelial growth factor), recognition by the trophoblastic cells of the various components of the decidua and the extracellular matrix (integrins and cadherin) and the progressive invasion of the decidua to the depth of the spinal arteries (by the trophoblastic secretion of metalloproteases). A defective
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or excessive trophoblastic invasion can result in complications of pregnancy [4]. 2. The classical immune system In order to comprehend the range of immuno-modulatory effects of oestrogen and progesterone, it is important to remember the essential functions of the classical immune system. The immune system eliminates antigenically foreign material in two ways: (1) A general response is caused by natural immunity preventing access of pathogens to the human body which does not need a ‘knowledge library’ of similar pathogens from previous attacks. Macrophages (which, it is important to point out, are oestrogen sensitive) and granulocytes attack these invading micro-organisms as soon as they enter the human body. (2) Additionally, a more complex and ‘professional’ response to pathogens is caused by adaptive immunity processes. This highly specific immune response is based on antigen representations on the ‘invader’s’ surface and is also sensitive to sex hormones. Adaptive immunity can be either of a cellular nature (using specialised T cells) or of a humoral nature (producing antibodies). In both ways, it is characterized by an anamnestic response that results in ‘remembering’ the foreign antigenic enemy. This enables the human immune system to react faster to subsequent exposures to the same antigen. Immune cells act by producing cytokines and then releasing them into their environment, thus creating particular microenvironments. The T-helper (Th) cell is an important
precursor cell on that pathway. According to the type of cytokines the immune cells produce, they differentiate into Th-1 or Th-2 lymphocytes, which secrete different types of interleukin and interferon to establish the microenvironment. Female sex hormones can reinforce this differentiation (Fig. 1) [5]. Cytokines have a similarly important role in reproduction. Some (e.g. transforming growth factor (TGF), Fig. 1) counteract the action of progesterone. Oestrogen receptors are found on T lymphocytes [6] and representations of intracellular oestrogen receptors show that T cells are target cells for oestrogens [6,7]. Oestrogen shows its immunomodulatory effect by binding to these oestrogen receptors, the expression of which has been characterised in human peripheral monocytes and in tissue macrophages. It was known that sex hormones could regulate the immune response and invoke the thymus even before the discovery of the importance of the thymus itself [7]. 3. The role of progesterone in early pregnancy 3.1. The foetal–placental unit as a semi-graft Initially, the foetal–placental unit is a semi-allograft due to the paternal genetic contributions. Subsequently, there is a maternal immune reaction to the allogeneic pregnancy. The constituents of the maternal immune reaction to the allogeneic stimulus are not different from any other immune reaction and the allogeneic conceptus (trophoblast) is like all other allogeneic tissue grafts. The special characteristic, however, that distinguishs the trophoblast from other tissues is its ability to eliminate abortogenic maternal B cell and T cell responses. The trophoblast induces an immunmodulation
Fig. 1. The pathways of Th-1 and Th-2 lymphocytes. Adapted from Aschkenazzi et al. [5]. ER: oestrogen receptor; IL: interleukin; IFN: interferon; LT: lymphotoxin; MS: multiple sclerosis; RA: rheumatoid arthritis; TGF: transforming growth factor; PDGF: platelet derived growth factor; LIF: leukaemia inhibitory factor; SLE: systemic lupus erythematosus.
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and thus actively defends itself from the maternal immune attack [8]. The presence of progesterone, and its interaction with progesterone receptors at the decidual level, appears to play a major role in this defence strategy. Most pregnancies are characterised by the predominance of humoral immunity and an increased total antibody production. In essence, fertilisation provides a signal for the immune system and decidual ␥/␦ T cells appear to play a major role [3,9–11]. The trophoblast is glycosylated whereby, due to changes in lectin binding, the glycosylation undergoes continuous gestational age-specific changes. It has been shown that there is a difference of glycosylation between normal trophoblasts and trophoblasts derived from aborted placentae [9]. Inadequate glycosylation might result in an inadequate recognition of foetal antigens and subsequently in a failed pregnancy. Activation of the immune system is necessary for a normal pregnancy; for example, it has been shown that human leukocyte antigen (HLA) matching between the parents is associated with spontaneous abortion) [12,13]. The increase in the number of ␥/␦ T cell receptor (TCR) positive cells in the decidua facilitates the recognition of the foetal antigens. Specifically, trophoblast recognition appears to be medicated by the V␥l subset of ␥/␦ T cells that recognise a conserved mammalian sequence on the trophoblast [9]. 3.2. Up-regulation of progesterone receptors and release of PIBF The outcome of the immunological recognition of pregnancy is an upregulation of progesterone receptors on natural killer (NK) cells in the decidua or on lymphocytes amongst placental cells [14]. In the presence of progesterone, activated lymphocytes and decidual CD56+ cells synthesise progesterone-induced blocking factor (PIBF), which exerts a substantial anti-abortive effect in vivo. Its pregnancy protective activity is mediated by its effects on the humoral (B cell) and cellular (T cell) immune system and by the reduction of NK cell activity [9]. 3.3. Effector mechanisms in the maternal immunoresponse 3.3.1. The humoral B cell system When the antigen-specific B cells bind to the antigen, a proliferating process is put into action. Subsequently, B cells secrete immunoglobulins, the antigen-specif¨ıc antibodies responsible for eliminating the target. Antibodies consist of two identical heavy chains and two identical light chains that are held together by disulfide bonds. The N terminal of each chain possesses a variable domain that binds the antigen. The C terminal domains of the heavy and light chains form the constant region that is responsible for the activation of effector functions such as complement fixation, phagocytosis and cytotoxicity [15].
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3.3.2. The cellular T cell system Still within the context of immunomodulatory effects in early pregnancy, the most important components of the cellular immunosystem are the Th cells. These cells can be differentiated into two different polarised forms that mobilise different types of effector responses. The information processing within the immune system depends largely on the production and release of cytokines. As regards their impact on the immune response, cytokines can be divided into two categories: Th-1 cytokines (i.e. tumour necrosis factor (TNF)␣, interferon (IFN)-␥, interleukin (IL)-2, IL-12, IL-18) which induce several cell-mediated cytotoxic and inflammatory reactions, and Th-2 cytokines (i.e. IL-4, IL-5, IL-6, IL-10, and IL-13) which are associated with B cell antibody production. Th-2 cytokines are necessary for the trophoblast to secrete human placental lactogen (hPL) and human chorionic gonadotropin (hCG). In addition, Th-2 cytokines downregulate Th-1-type reactivity [16–18]. In successful pregnancy, the normal profile is a Th-2 type immunity. A shift towards Th-1-dominance is thought to be associated with unexplained habitual abortion [19]. 3.3.3. Natural killer cells The NK cell is part of the innate response of the immune system. It first recognises, and then kills, abnormal cells and its activation relies upon killer-activating and killer-inhibitory receptors. If the killer-activating receptors are engaged, a “kill” instruction is issued to the NK cell. This signal can be overridden by an inhibitory signal sent by the killer-inhibitory receptor. The “killing” of the target cell is carried out by inserting the pore-forming molecule perforin into the membrane of the target cell and then injecting it with cytotoxic granzymes [15].
4. Immunomodulation in successful versus abortogenic pregnancy 4.1. Immunmodulatory mechanisms in successful pregnancy 4.1.1. Asymmetric antibodies An immunological deviation in the maintenance of normal pregnancy is the induction of blocking, or asymmetric, antibodies. Asymmetric and symmetric antibodies are synthesized by the same cellular clone [20]. The asymmetric structure is achieved by a mannose-rich oligosaccharide group that is present on only one of the Fab regions. Owing to their asymmetric glycosylation, they are unable to activate effector functions such as complement fixation, phagocytosis and cytotoxicity. However, as they can combine with the antigen, they compete with precipitating antibodies of the same specificity and thus act as “blocking” antibodies [21]. The switch from the effector-type, symmetric immunoglobulin (Ig)G to the non-precipitating, blocking-type, asymmetric IgG constitutes a substantial part of the control of the equi-
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librium of maternal anti-foetal immunresponses. It prevents immunological eradication of the foetus by blocking subsequent effector events such as the formation of the membrane attack complex (C5b-9) via the “classical” means of complement activation. In a prevalence study, approximately 9% of the total IgG in nulliparous and multiparous women was found to be asymmetric, compared with 29% in healthy pregnant women (95% confidence interval (C.I.): 24–35%) and 3% in women who were habitual aborters (95% C.I.: 2–5%) (p < 0.001 versus controls) [21]. 4.1.2. Th-2/Th-1 bias Several studies investigating the ratio of Th-2 to Th-1 in terms of circulatory levels of serum cytokines reveal a Th-2 bias in normal pregnancy and a Th-1 bias in cases of recurrent miscarriages. IL-13, a Th-2-type cytokine, has been shown to be produced by human trophoblast cells [17]. In women with a successful pregnancy, there are markedly higher concentrations of IL-4 and IL-10 -producing T-cell clones, in both peripheral blood mononuclear cells (PBMC) and at the maternal–foetal interface, than are found in recurrent aborters [22]. To summarise, there is dominance of a Th-2 biased situation in the first trimester of a successful human pregnancy, whereas a Th-1 type bias may lead to pregnancy failure. In a more detailed analysis, it was shown that it is not the absolute concentrations of Th-1 or Th-2 -type cytokines that indicate a bias, but the ratios of Th-1 to Th-2 cytokines. In a study in 24 women with a history of successful pregnancies and 23 women with a history of unexplained habitual abortion, a comparison at the same gestational age showed that for every permutation of Th-l toTh-2 cytokines, the ratios were higher in the habitual abortion group [19]. In successful pregnancies, there was a clear Th-2 bias during the first trimester. In animal experiments, administration of IL-10, a Th-2type anti-inflammatory cytokine, prevents foetal wastage in mice prone to this effect (model for abortion) [23]. It was proposed that the important immunregulatory role for IL-10 is not primarily a direct one (like the role of colony-stimulating factor [CSF]-1, IL-3 or granulocyte-macrophage [GM]-CSF on the development of the placenta [24]), but that it may aid the survival of the conceptus by down-regulating Th-1 type reactivity, e.g., by inhibiting IFN-␥ production [25]. 4.1.3. Reduced NK cell activity NK cells are observed in the uterus during early pregnancy in several species [26–28]. Their role in normal pregnancy is, however, still controversial. CD16-negative, perforinpositive, but non-cytotoxic NK cells have been reported in first trimester human decidua [29]. Thus, the relative proportion of decidual NK cells is increased in normal pregnancy compared with the non-pregnant uterus. However, decidual NK activity is lower in normal compared with anembryonic pregnancies and in recurrent spontaneous abortions [30].
Fig. 2. Protective immunomodulatory mechanisms associated with a successful pregnancy. AB: antibody; IL: interleukin; IFN: interferon; TNF: tumour necrosis factor; NK: natural killer; Th: T-helper.
The protective immunomodulatory mechanisms associated with a successful pregnancy are summarised in Fig. 2. 4.2. Immunmodulatory mechanisms in abortogenic pregnancy 4.2.1. Symmetric antibodies Approximately 80% of the IgG specifically reactive to endometrial antigens are symmetric antibodies (which can be cytotoxic) in women with habitual abortion; in healthy pregnant women, the percentage is much lower at approximately 25%. Some investigators consider that, whether or not the antibodies are symmetric (i.e. effector-type with the potential to activate the complement system, or non-precipitating asymmetric-type that allows immunological tolerance) they are the major determining factor for successful pregnancy [21,31]. 4.2.2. Th-1/Th-2 bias The particularity of Th-1 cells is that they induce several cell-mediated cytotoxic and inflammatory reactions mainly via IL-2, IFN-␥ and TNF, whilst Th-2 cells support B cell antibody production via the cytokines IL-4, IL-5, IL-6 and IL-10. Th-2-type immunity is the normal profile in successful pregnancy, with a shift towards Th-1 dominance occurring in unexplained habitual abortion. Concentrations of Th-1 type cytokines, both at the maternal–foetal interface and in PBMC, are higher in women with unexplained habitual abortion. Th-1 and Th-2 cytokines are mutually inhibitory and a shift towards a Th-1 bias tends to further down-regulate Th-2 reactivity. A comparison of the ratios of various Th-1 and Th-2 cytokines in a total of 47 women (23 with a history of unexplained habitual abortion and 24 with a history of successful pregnancies) showed a higher Th-1:Th-2 ratio for all permutations (e.g. IL-2:IL-10 was 80-fold higher in the aborter group, IFN-
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␥:IL-10 was 30 times higher and TNF-:IL-10 was 40 times higher) [19]. Under experimental conditions, the outcome of pregnancy can be influenced by modulating the cytokine balance. Administration of TNF-␣, IFN-␥ or IL-2 to normal pregnant mice caused abortion [32,33], whilst anti-TNF-␣ reduced the resorption rate in a murine model of spontaneous immunologically-mediated abortion [34]. Note that although TNF-␣ is produced by both Th-1 and Th-2 cells, it is secreted at higher levels by Th-1 responses than by Th2 responses. Furthermore it elicits the production of IFN-␥, a Th-1 cytokine. Thus, TNF-␣ is often referred to as Th-1like [35]. IFN-␥ and TNF-␣ destroy trophoblast cells [36] and inhibit the implantation of mouse embryos [37] and the proliferation of human trophoblasts in vitro [38]. IL-12 has been shown to be present in significantly elevated levels in the serum of pregnant women with habitual abortion [39]. It significantly augments the cytolytic activity of lymphocytes against trophoblast cells [40] and the administration of IL-12, along with IL-18, to pregnant mice has also been shown to result in pregnancy loss [41]. The final step by which abortion is mediated is a Th-1 cytokine-triggered thrombotic and inflammatory process [42]. 4.3. Increased NK cell activity and lymphokine activated killer cell transformation Spontaneous pregnancy termination is linked to increased NK activity [3,43]; uterine resorption sites in a murine model of recurrent abortion were shown to be infiltrated by NK cells. However, it is not the presence of NK cells that is detrimental to the trophoblast, as it is able to resist NK-mediated lysis in vitro [3,9,44]. In fact, NK cells are able to induce trophoblast lysis and cause foetal loss if they are converted to lymphokine activated killer (LAK) cells by IFN-␥, TNF-␣ and IL-2 [44–46]. NK cells are not only the target of cytokines, they are also producers of proinflammatory cytokines such as IFN-␥ [47]. Hence, there is a potential for the initiation of a vicious circle. The final mechanism by which activated NK cells kill their target cells is exocytosis of perforin and serine esterase -containing granules in the contact area between effector (LAK) and target (trophoblast) cells [29,48]. The immune reactions associated with spontaneous pregnancy loss are summarised in Fig. 3.
5. The role of progesterone Following recognition of the antigens presented by the trophoblast, peripheral blood lymphocytes and decidual CD56+ and ␥␦+ cells develop specific progesterone receptors [3,9,49] In vitro, progesterone has been shown to exert a significant and dose-dependant inhibitory effect on lymphocyte cytotoxicity at serum concentrations comparable to those present during pregnancy. The effects of progesterone are achieved via a mediator molecule.
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Fig. 3. Immune reactions associated with spontaneous pregnancy loss. AB: antibody; IL: interleukin; IFN: interferon; TNF: tumour necrosis factor; NK: natural killer; Th: T-helper; LAK: lymphokine activated killer.
6. Progesterone-induced blocking factor In the presence of progesterone, progesterone receptorpositive lymphocytes and decidual CD56+ cells synthesize a 34 kDa immunomodulatory protein known as progesterone induced blocking factor (PIBF) [50]. PIBF has several antiabortive effects in vivo and appears to be the pivotal mediator in progesterone-dependent immunomodulation [9]. In recurrent spontaneous aborters and women showing clinical symptoms of threatened preterm pregnancy termination, PIBF expression and the percentage of PIBF+ lymphocytes was found to be significantly lower than in healthy pregnant women [9,51,52].
7. The pregnancy protecting role of PIBF 7.1. PIBF enhances asymmetric antibody production PIBF affects B cells, both directly and via cytokines [52,53], and induces increased production of asymmetric antibodies. This has a regulatory effect on anti-foetal immune responses during pregnancy. In animal experiments, blockade of the progesterone receptor resulted in reduced PIBF production together with a reduced amount of nonprecipitating (asymmetric) antibodies and elevated foetal resorption rates. As a corollary, the blocking of progesterone receptors by RU486 was shown to reduce the production of asymmetric antibodies in pregnant mice. In humans, RU486 has a proven efficacy in medically-induced, elective abortion. 7.2. PIBF causes a Th-2/Th-1 shift In essence, PIBF alters the profile of cytokine secretion by activated lymphocytes. It increases the production of Th-2 cytokines IL-3, IL-4 and IL-10 and decreases the
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Fig. 4. The pivotal role of progesterone receptor-mediated immune modulation in successful pregnancy. PBMC: peripheral blood mononuclear cells; AB: antibody; NK: natural killer; Th: T-helper; LAK: lymphokine activated killer.
production of the Th-l cytokine IL-12 [54]. In pregnant mice, neutralisation of endogenous PIBF activity resulted in a decrease in IL-10 and an increase in IFN-␥ production, leading to a high resorption rate and increased NK activity [55]. 7.3. PIBF inhibits NK activity NK activity in pregnant women is inversely related to the rate of PIBF-positive lymphocytes [51]. PIBF keeps NK activity at a low level both by controlling IL-12 production and also by inhibiting perforin liberation [9,52,56]. Decidual NK cells express a high level of perforin and are therefore potentially cytotoxic [29,52]; nevertheless, they have low cytotoxic activity. In vitro studies in decidual cells obtained from elective pregnancy termination suggest that it is PIBF that inhibits the cytotoxicity of NK cells via blockade of degranulation, and thus contributes to the low decidual NK activity [54]. Neutralisation of endogenous PIBF in pregnant mice results in abortion due to increased NK activity; these NK-mediated resorptions can be counteracted by simultaneous PIBF treatment [9]. The pivotal role of progesterone receptor-mediated immune modulation in successful pregnancy is summarised in Fig. 4.
8. Sequence of events To summarise, the immunogenic process at the foetal– maternal interphase can be divided into four stages [3,9]. (1) Recognition of foetal antigens. Foetally-derived antigens on the trophoblast are recognized by V␥l cells, a subset of the ␥/␦ T cells, in the decidua. (2) Upregulation of progesterone receptors. Following recognition of the antigens presented by the trophoblast, V␥l (CD56+) cells become activated and develop progesterone receptors. (3) Production and release of PIBF. In the presence of progesterone, decidual CD56+ and 76+ cells, as well as progesterone receptor-positive decidual NK cells, synthesise PIBF, a 34 kDa immunomodulatory protein. (4) Protection of pregnancy. Sufficient release of PIBF contributes to the success of early pregnancy by exerting its anti-abortogenic effects on three pathways. PIBF induces - an increase in asymmetric, “blocking” antibodies; - a Th-2 dominated, cytoprotective immune response (Th-2:Th-l bias); - a reduction in NK cell activity. In doing so, PIBF prevents inflammatory and thrombotic secondary reactions towards the trophoblast.
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9. Conclusions
References
There is an immunological relationship between the mother and the foetus which is a bi-directional communication determined on the one hand by foetal antigen presentation and on the other hand by recognition of, and reaction to, these antigens by the maternal immune system. We now have evidence that immunological recognition of pregnancy is important for the maintenance of gestation, and that an inadequate recognition of foetal antigens might result in failed pregnancy. Whereas HLA-A and HLA-B-I genes are downregulated in human trophoblast cells, nonpolymorphic class I molecules, e.g. HLA-G Class Ib, are expressed in extravillous cytotrophoblast and endothelial cells of foetal vessels in the chorionic villi, as well as in amnion cells and amniotic fluid. The trophoblast does not induce transplantation immunity and resists NK-and cytotoxic T-lymphocyte-mediated lysis in vitro. To date, we know that HLA-G presents antigens for ␥/␦ T cells and, at the same time, defends the trophoblast from cytotoxic effector mechanisms. Unfortunately, since polymorphic major histocompatability complex (MHC) is absent from the trophoblast, presentation of foetally-derived antigens is MHC-restricted. The ␥/␦ T cells recognise a distinct group of ligands with a smaller receptor repertoire than ␣/ T cells [57]. Most ␥/␦ T cells recognise unprocessed foreign antigens without MHC. The ␥/␦ TCR-positive cells in the decidua significantly increase in number and the majority of decidual ␥/␦ T cells are activated due to recognition of conserved mammalian molecules on the trophoblast. Following recognition of foetally derived antigens, the immune system reacts with a wide range of protective mechanisms. Many observations suggest that pregnancy is associated with an altered Th-1:Th-2 balance. The maternal immune response is biased towards humoral immunity and away from cellmediated immunity that could be harmful to the foetus. Cytokines of maternal origin influence placental development, whilst the maternal cytokine pattern is determined by antigen expression on the placenta. Normal human pregnancy is characterised by low peripheral NK activity, and increased NK activity appears to play a role in spontaneous abortions of unknown aetiology. In early human pregnancy, the majority of uterine lymphocytes are CD56 granulated NK cells, which do not express CD16 or CD3. In rodents and humans, uterine NK cells are under hormonal control and in early pregnancy they are enriched at sites where the foetal trophoblast infiltrates the deciduas. The dynamics of the appearance of uterine NK cells suggest that one of the functions of these cells is to control placentation. Another protective mechanism operating in favour of pregnancy is progesterone-dependent immunomodulation. Upon stimulation by foetally-derived antigens, pregnancy lymphocytes develop progesterone receptors and, in the presence of progesterone, produce a mediator (PIBF) that, through altering the cytokine balance, inhibits NK activity and produces an anti-abortive effect.
[1] R. Druckmann, Review: female sex hormones, autoimmune diseases and immune response, Gynecol. Endocrinol. 5 (Suppl. 6) (2001) 69–76. [2] A.E. Schindler, Immunology and progestins in pregnancy, Gynecol. Endocrinol. 13 (Suppl. 4) (1999) 47–50. [3] J. Szekeres-Bartho, Immunological relationship between the mother and the foetus, Int. Rev. Immunol. 21 (2002) 471–495. [4] P. Merviel, D. Evain-Brion, J.C. Challlier, J. Salat-Baroux, S. Uzan, The molecular basis of embryo implantation in human, Zentralbl. Gynakol. 123 (2001) 328–339. [5] S. Aschkenazzi, F. Naftolin, G. Mor, Menopause, sex hormones, and the immune system, Menopause Manag. 9 (2000) 6–13. [6] R.G. Lahita, Sex hormones and the immune system – part 1. Human data, Baillieres Clin. Rheumatol. 4 (1990) 1–11. [7] A.S. Ansar, N. Talal, Sex hormones and the immune system – part 2. Animal data, Baillieres Clin. Rheumatol. 4 (1990) 13–29. [8] D.H. Munn, M. Zhou, J.T. Attwood, I. Bondarev, S.J. Conway, B. Marshall, C. Brown, A.L. Mellor, Prevention of allogeneic foetal rejection by tryptophan catabolism, Science 281 (1998) 1191–1193. [9] J. Szekeres-Bartho, A. Barakonyi, B. Polgar, G. Par, Z. Faust, T. Palkovics, L. Szereday, The Role of g/d T cells in progesteronemediated immunomodulation during pregnancy: a review, Am. J. Reprod. Immunol. 42 (1999) 44–48. [10] L. Szereday, A. Barakonyi, E. Miko, P. Varga, J. Szekeres-Bartho, Gamma/delta T cell subsets, NKJ 2A expression and apoptosis of Vdelta+ T cells in pregnant women with or without risk of premature pregnancy termination, Am. J. Reprod. Immunol. 50 (2003) 490– 496. [11] A. Barakonyi, K.T. Kovacs, E. Miko, L. Szereday, P. Varga, J. Szekeres-Bartho, Recognition of nonclassical HLA class 1 antigens by ␥/␦T cells during pregnancy, J. Immunol. 168 (2002) 2683– 2688. [12] L. Komlos, R. Zamir, H. Joshua, I. Halbracht, Common HLA antigens in couples with repeated abortions, Clin. Immunol. Immunopathol. 7 (1977) 330–335. [13] C. Ober, T. Hyslop, S. Elias, L.R. Weikamp, W.W. Hauck, Human leucocyte antigen matching and foetal loss: result of a 10 year prospective study, Hum. Reprod. 13 (1998) 33–38. [14] R.G. Roussev, N.G. Higgins, J.A. McIntyre, Phenotypic characterization of normal human placental mononuclear cells, J. Reprod. Immunol. 25 (1993) 15–29. [15] P.J. Delves, I.M. Roitt, The immune system. Advances in Immunology, N. Engl. J. Med. 343 (2000) 37–49. [16] G. Par, J. Geli, N. Kozma, P. Varga, J. Szekeres-Bartho, Progesterone regulates IL-12 expression in pregnancy lymphocytes by inhibiting phospholipase A2, Am. J. Reprod. Immunol. 49 (2003) 1–5. [17] G.B. Dealtry, D.E. Clark, A.M. Sharkey, S. Charnock-Jones, S.K. Smith, Expression and localization of the Th2-type cytokine interleukin-13 and its receptor in the placenta during human pregnancy, Am. J. Reprod. Immunol. 40 (1998) 283–290. [18] T.R. Mosmann, S. Sad, The expanding universe of T-cell subsets, Immunol. Today 17 (1996) 138–146. [19] R. Raghuphaty, M. Makhseed, F. Azizieh, A. Omu, M. Gupta, R. Farhat, Cytokine production by maternal lymphocytes during normal human pregnancy and in unexplained recurrent abortion, Hum. Reprod. 15 (2000) 713–718. [20] M.O. Labeta, R.A. Margni, J. Leoni, R.A. Binaghi, Structure of asymmetric non-precipitating antibody: presence of a carbohydrate residue in only one Fab region of the molecule, Immunology 57 (1986) 311–317. [21] A.C. Eblen, C. Gercel-Taylor, L.B.E. Shields, J.S. Sanfilippo, S.T. Nakajima, D.D. Taylor, Alterations in humoral immune responses associated with recurrent pregnancy loss, Fertil. Steril. 73 (2000) 305–313.
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[22] M.-P. Piccinni, L. Beloni, C. Livi, E. Maggi, G. Scarselli, S. Romagnani, Defective production of both leukemia inhibitory factor and type 2 T-helper cytokines by decidual T cells in unexplained recurrent abortions, Nat. Med. 4 (1998) 1020–1024. [23] G. Chaouat, A.A. Meliani, J. Martal, R. Raghupathy, J.J. Elliot, T.R. Mosmann, T.G. Wegmann, IL-10 prevents naturally occurring fetal loss in the CBA x DBA/2 mating combination, and local defect in IL-10 production in this abortion-prone combination is corrected by in vivo injection of IFN-tau, J. Immunol. 154 (1995) 4261–4268. [24] S.A. Robertson, M. Br¨annstr¨om, R.F. Seamark, Cytokines in rodent reproduction and the cytokine–endocrine interaction, Curr. Opin. Immunol. 4 (1992) 585–590. [25] D.F. Fiorentino, M.W. Bond, T.R. Mosmann, Two types of mouse T helper cell. IV. Th-2 clones secrete a factor that inhibits cytokine production by Th-l clones, J. Exp. Med. 170 (1989) 2081–2095. [26] P.M. Starkey, L.L. Sargent, C.W.G. Redman, Cell populations in human early pregnancy decidua: Characterization and isolation of large granular lymphocytes by flow cytometry, Immunology 65 (1988) 129–134. [27] B.A. Croy, P. Gambel, J. Rossant, T.G. Wegmann, Characterization of murine decidual natural killer (NK) cells and their relevance to the success of pregnancy, Cell. Immunol. 93 (1985) 315–326. [28] B.A. Croy, A. Waterfˆıeld, W. Wood, G.J. Kind, Normal murine and porcine embryos recruit NK cells to the uterus, Cell. Immunol. 115 (1988) 471–480. [29] D. Rukavina, G. Rubesa, L. Gudelj, H. Haller, E.R. Podack, Characteristics of perforin expressing lymphocytes within the first trimester decidua of human pregnancy, Am. J. Reprod. Immunol. 33 (1995) 394–404. [30] K.-H. Chao, Y.-S. Yang, H.-N. Ho, S.-U. Chen, H.-F. Chen, H.J. Dai, S.-C. Huang, T.J. Gill, Decidual natural killer cytotoxicity decreased in normal pregnancy but not in anembryonic pregnancy and recurrent spontaneous abortion, Am. J. Reprod. Immunol. 34 (1995) 274–280. [31] I. Malan Borel, S. Miranda, S. Menezes Freire, A. Canellada, M. Eslave Bueno, R. Binaghi, Modulation of the humoral immune response by placental secretory factors, Am. J. Reprod. Immunol. 35 (1996) 529–533. [32] M. Parant, The role of TNF in non-specific stimulation of the mouse resistance to infection, Immunobiology 175 (1987) 26. [33] B.V. Tezabwala, A.P. Johnstone, Effect of administration of interleukin 2 in pregnancy, J. Reprod. Immunol. 1 (Suppl.) (1986) 147. [34] G. Chaouat, E. Menu, T.G. Wegmann, Role of lymphokines of the CSF family and of TNF, gamma-interferon and IL-2 on placental growth and foetal survival studied in 2 murine models of spontaneous resorptions, in: G. Chaouat, G. Mowbry (Eds.), Cellular and Molecular Biology of the Materno-Fetal Relationship, Vol. 212, Colloque INSERM John Libbey Eurotext Ltd., Paris, London, 1991, pp. 91–100. [35] R. Raghupathy, M. Makhseed, F. Azizieh, N. Hassan, M. Al-Azemi, E. Al-Shamali, Maternal Th-l- and Th-2-type reactivity to placental antigens in normal human pregnancy and unexplained recurrent spontaneous abortions, Cell. Immunol. 196 (1999) 122–130. [36] J. Yui, M. Garcia-Lloret, T.G. Wegmann, L.J. Guilbert, Cytotoxicity of tumour necrosis factor-alpha and gamma-interferon against primary human placental trophoblasts, Placenta 15 (1994) 819– 835. [37] F. Haimovici, J.A. Hill, D.J. Andersen, The effects of soluble products of activated lymphocytes and macrophages on blastocyst implantation events in vitro, Biol. Reprod. 44 (1991) 69–75. [38] J.A. Hill, The rationale for leukocyte immunization in women with recurrent spontaneous abortion: Fact or fiction? in: G. Chaouat, G. Mowbry (Eds.), Cellular and Molecular Biology of the Materno-Fetal Relationship, Vol. 212, Colloque INSERM John Libbey Eurotext Ltd., Paris, London, 1991, pp. 263–269.
[39]] R. Wilson, I. McInnes, B. Leung, J.H. McKillop, J.J. Walker, Altered interleukin 12 and nitric oxide levels in recurrent miscarriage Eur. J. Obstet. Gynecol. Reprod. Biol. 75 (1997) 211–214. [40] S. Hayakawa, N. Nagai, T. Kanaeda, M. Karasaki-Suzuki, M. Ishi, F. Chishima, F. Satoh, Interleukin-12 augments cytolytic activity of peripheral and decidual lymphocytes against choriocarcinoma cell lines and primary culture human placental trophoblasts, Am. J. Reprod. Immunol. 41 (1999) 320–329. [41] J. Muranaka, Y. Tsuji, A. Hasegawa, A. Ida, Y. Nakata, R. Kanazawa, K. Koyama, Interleukin-18 as an important new cytokine in pregnancy, Am. J. Reprod. Immunol. 40 (1998) 278. [42] D.A. Clark, G. Chaouat, P.C. Arck, H.W. Mittruecker, G.A. Levy, Cytokine-dependent abortion in CBA x DBA/2 mice is mediated by the procoagulant fgl2 prothrombinase [correction of prothombinase], J. Immunol. 160 (1998) 545–549. [43] J. Szekeres-Bartho, P. Varga, A.S. Pasca, Immunological factors contributing to the initiation of labour-lymphocyte reactivity in term labour and threatened pre-term delivery, Am. J. Obstet. Gynecol. 155 (1986) 108–113. [44] B.L. Drake, J.R. Head, Murine trophoblast cells can be killed by lymphokine-activated killer cells, J. Immunol. 143 (1989) 9–14. [45] D.A. Clark, Are there immune abortions? Res. Immunol. 141 (1990) 104–109. [46] B.L. Ferry, P.M. Starkey, I.L. Sargent, G.M. Watt, M. Jackson, C.W. Redman, Cell populations in the human early pregnancy decidua: natural killer activity and response to interleukin-2 of CD56positive large granular lymphocytes, Immunology 70 (1990) 446– 452. [47] D.A. Clark, G. Chaouat, Spontaneous abortion in DBA/2 mated mice is due to cytokines and not trophoblast killing by asialo GM1+ NK cells or NO-producing macrophages, Am. J. Reprod. Immunol. 35 (1996) 454. [48] C.-C. Liu, C.M. Walsh, J.D. Young, Perforin: structure and function, Immunol. Today 16 (1995) 194–201. [49] J. Szekeres-Bartho, G. Szekeres, P. Debre, B. Autran, G. Chaouat, Reactivity of lymphocytes to a progesterone receptor-specific monoclonal antibody, Cell. Immunol. 125 (1990) 273–283. [50] J. Szekeres-Bartho, F. Kilar, G. Falkay, V. Csernus, A. Torok, A.S. Pacsa, Progesterone-treated lymphocytes of healthy pregnant women release a factor inhibiting cytotoxicity and prostaglandin synthesis, Am. J. Reprod. Immunol. Microbiol. 9 (1985) 15–19. [51] J. Szekeres-Bartho, Z. Faust, P. Varga, The expression of a progesterone-induced immunomodulatory protein in pregnancy lymphocytes, Am. J. Reprod. Immunol. 34 (1995) 342–348. [52] J. Szekeres-Bartho, A. Barakonyi, G. Par, B. Polgar, T. Palkovics, L. Szereday, Progesterone as an immunomodulary molecule, Int. Immunopharmacol. 1 (2001) 1037–1048. [53] K. Kelemen, I. Bognar, M. Paal, J. Szekeres-Bartho, A progesteroneinduced protein increases the synthesis of asymmetric antibodies, Cell. Immunol. 167 (1996) 129–134. [54] Z. Faust, G. Laskarin, D. Rukavina, J. Szekeres-Bartho, Progesterone-induced blocking factor inhibits degranulation of natural killer cells, Am. J. Reprod. Immunol. 42 (1999) 71–75. [55] L. Thompson-Snipes, V. Dhar, M.W. Bond, T.R. Mosmann, K.W. Moore, D.M. Rennick, Interleukin-10: a novel stimulatory factor for mast cells and their progenitors, J. Exp. Med. 173 (1991) 507– 510. [56] G. Laskarin, N. Strbo, V. Sotosek, D. Rukavina, Z. Faust, J. Szekeres-Bartho, E.R. Podack, Progesterone directly and indirectly affects perforin expression in cytolytic cells, Am. J. Reprod. Immunol. 45 (1999) 312–320. [57] J. Szekeres-Bartho, A. Barakonyi, E. Miko, B. Polgar, T. Palkovics, The role of gamma/delta T cells in the feto-maternal relationship, Semin. Immunol. 13 (2001) 229–233.
Progesterone and the immunology of pregnancy夽 Ren´e Druckmann ∗ , Marc-Alexandre Druckmann A.N.E.M.O.-Centre de m´enopause, 10-12 Rue de France, F 06000 Nice, France
Abstract The foetal–placental unit is a semi-allograft and the immunological recognition of pregnancy, together with the subsequent response of the maternal immune system, is necessary for a successful pregnancy. This recognition of pregnancy results in an upregulation of progesterone receptors on activated lymphocytes amongst placental cells and decidual CD56+ cells. In the presence of sufficient progesterone, these cells synthesise progesterone induced blocking factor (PIBF), a mediator that exerts substantial anti-abortive activities. PIBF affects B cells and induces an increased production of asymmetric, non-cytotoxic antibodies. It also alters the profile of cytokine secretion by activated lymphocytes resulting in an increase in the production of non-inflammatory, non-cytotoxic interleukins (IL) (e.g. IL-3, IL-4 and IL-10) and a reduction in the production of inflammatory, cytotoxic cytokines (e.g. interferon (IFN)-␦, tumour necrosis factor (TNF)-␣ and IL-2). PIBF also inhibits the cytotoxity of natural killer (NK) cells by blocking their degranulation and perforin release, as well as inhibiting IFN-␦, TNF-␣ and IL-2-mediated transformation of NK cells into detrimental lymphokine activated killer (LAK) cells. © 2005 Elsevier Ltd. All rights reserved. Keywords: Pregnancy; Progesterone; Spontaneous abortion; Immune system; Progesterone-induced blocking factor; T-helper cells; Cytokines
1. Introduction For more than 50 years it has been suspected that a distinct interaction between female sex hormones and the overall immune system might exist. “How does the pregnant mother contrive to nourish a foetus within herself for many months that is an antigenically foreign body?” was the question raised by P.B. Medawar in 1953 [1]. Spontaneous miscarriage (abortion) occurs in 15% of all clinically recognised pregnancies and it is the most common complication of pregnancy. Approximately 50–60% of spontaneous pregnancy losses can be explained by chromosomal anomalies of the foetus, infectious aetiologies, maternal endocrinology or anatomical comorbidity, but still 40–50% remain “unexplained”. A substantial portion of these “unexplained” cases could be attributable to an abnormal, abortogenic immune response of the mother towards potential antigens on the foetus. Recently, a growing body of evidence has been accumulating to suggest that proges夽 Presented at the European Progestin Club Scientific Meeting Amsterdam, The Netherlands, 05 October 2004. ∗ Corresponding author. Tel.: +33 493820608; fax: +33 493162743. E-mail address: [email protected] (R. Druckmann).
0960-0760/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2005.08.010
terone might play a significant role in establishing an adequate immune response during the early stages of successful pregnancy [2]. The implantation of the human embryo is a double paradox in that it is immunological and biological. The immunological paradox is that it consists of a heterologous graft in which the uterine immune system (via the cytokines) and the antigenicity of the embryo (HLA-G) collaborate to make both implantation and maintenance of the pregnancy possible [3]. The biological paradox arises because several different mechanisms must be successively implemented for these two epithelia to fuse and then for one to allow invasion by the other (i.e. for the endometrium to be decidualised by the trophoblast): these mechanisms include preparation of the endometrium throughout the menstrual cycle under the influence of oestrogens and then progesterone, the involvement of growth factors (epidermal growth factor, transforming growth factor and insulin-like growth factors), neoangiogenisis (oestradiol, fibroblast growth factors and vascular endothelial growth factor), recognition by the trophoblastic cells of the various components of the decidua and the extracellular matrix (integrins and cadherin) and the progressive invasion of the decidua to the depth of the spinal arteries (by the trophoblastic secretion of metalloproteases). A defective
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or excessive trophoblastic invasion can result in complications of pregnancy [4]. 2. The classical immune system In order to comprehend the range of immuno-modulatory effects of oestrogen and progesterone, it is important to remember the essential functions of the classical immune system. The immune system eliminates antigenically foreign material in two ways: (1) A general response is caused by natural immunity preventing access of pathogens to the human body which does not need a ‘knowledge library’ of similar pathogens from previous attacks. Macrophages (which, it is important to point out, are oestrogen sensitive) and granulocytes attack these invading micro-organisms as soon as they enter the human body. (2) Additionally, a more complex and ‘professional’ response to pathogens is caused by adaptive immunity processes. This highly specific immune response is based on antigen representations on the ‘invader’s’ surface and is also sensitive to sex hormones. Adaptive immunity can be either of a cellular nature (using specialised T cells) or of a humoral nature (producing antibodies). In both ways, it is characterized by an anamnestic response that results in ‘remembering’ the foreign antigenic enemy. This enables the human immune system to react faster to subsequent exposures to the same antigen. Immune cells act by producing cytokines and then releasing them into their environment, thus creating particular microenvironments. The T-helper (Th) cell is an important
precursor cell on that pathway. According to the type of cytokines the immune cells produce, they differentiate into Th-1 or Th-2 lymphocytes, which secrete different types of interleukin and interferon to establish the microenvironment. Female sex hormones can reinforce this differentiation (Fig. 1) [5]. Cytokines have a similarly important role in reproduction. Some (e.g. transforming growth factor (TGF), Fig. 1) counteract the action of progesterone. Oestrogen receptors are found on T lymphocytes [6] and representations of intracellular oestrogen receptors show that T cells are target cells for oestrogens [6,7]. Oestrogen shows its immunomodulatory effect by binding to these oestrogen receptors, the expression of which has been characterised in human peripheral monocytes and in tissue macrophages. It was known that sex hormones could regulate the immune response and invoke the thymus even before the discovery of the importance of the thymus itself [7]. 3. The role of progesterone in early pregnancy 3.1. The foetal–placental unit as a semi-graft Initially, the foetal–placental unit is a semi-allograft due to the paternal genetic contributions. Subsequently, there is a maternal immune reaction to the allogeneic pregnancy. The constituents of the maternal immune reaction to the allogeneic stimulus are not different from any other immune reaction and the allogeneic conceptus (trophoblast) is like all other allogeneic tissue grafts. The special characteristic, however, that distinguishs the trophoblast from other tissues is its ability to eliminate abortogenic maternal B cell and T cell responses. The trophoblast induces an immunmodulation
Fig. 1. The pathways of Th-1 and Th-2 lymphocytes. Adapted from Aschkenazzi et al. [5]. ER: oestrogen receptor; IL: interleukin; IFN: interferon; LT: lymphotoxin; MS: multiple sclerosis; RA: rheumatoid arthritis; TGF: transforming growth factor; PDGF: platelet derived growth factor; LIF: leukaemia inhibitory factor; SLE: systemic lupus erythematosus.
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and thus actively defends itself from the maternal immune attack [8]. The presence of progesterone, and its interaction with progesterone receptors at the decidual level, appears to play a major role in this defence strategy. Most pregnancies are characterised by the predominance of humoral immunity and an increased total antibody production. In essence, fertilisation provides a signal for the immune system and decidual ␥/␦ T cells appear to play a major role [3,9–11]. The trophoblast is glycosylated whereby, due to changes in lectin binding, the glycosylation undergoes continuous gestational age-specific changes. It has been shown that there is a difference of glycosylation between normal trophoblasts and trophoblasts derived from aborted placentae [9]. Inadequate glycosylation might result in an inadequate recognition of foetal antigens and subsequently in a failed pregnancy. Activation of the immune system is necessary for a normal pregnancy; for example, it has been shown that human leukocyte antigen (HLA) matching between the parents is associated with spontaneous abortion) [12,13]. The increase in the number of ␥/␦ T cell receptor (TCR) positive cells in the decidua facilitates the recognition of the foetal antigens. Specifically, trophoblast recognition appears to be medicated by the V␥l subset of ␥/␦ T cells that recognise a conserved mammalian sequence on the trophoblast [9]. 3.2. Up-regulation of progesterone receptors and release of PIBF The outcome of the immunological recognition of pregnancy is an upregulation of progesterone receptors on natural killer (NK) cells in the decidua or on lymphocytes amongst placental cells [14]. In the presence of progesterone, activated lymphocytes and decidual CD56+ cells synthesise progesterone-induced blocking factor (PIBF), which exerts a substantial anti-abortive effect in vivo. Its pregnancy protective activity is mediated by its effects on the humoral (B cell) and cellular (T cell) immune system and by the reduction of NK cell activity [9]. 3.3. Effector mechanisms in the maternal immunoresponse 3.3.1. The humoral B cell system When the antigen-specific B cells bind to the antigen, a proliferating process is put into action. Subsequently, B cells secrete immunoglobulins, the antigen-specif¨ıc antibodies responsible for eliminating the target. Antibodies consist of two identical heavy chains and two identical light chains that are held together by disulfide bonds. The N terminal of each chain possesses a variable domain that binds the antigen. The C terminal domains of the heavy and light chains form the constant region that is responsible for the activation of effector functions such as complement fixation, phagocytosis and cytotoxicity [15].
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3.3.2. The cellular T cell system Still within the context of immunomodulatory effects in early pregnancy, the most important components of the cellular immunosystem are the Th cells. These cells can be differentiated into two different polarised forms that mobilise different types of effector responses. The information processing within the immune system depends largely on the production and release of cytokines. As regards their impact on the immune response, cytokines can be divided into two categories: Th-1 cytokines (i.e. tumour necrosis factor (TNF)␣, interferon (IFN)-␥, interleukin (IL)-2, IL-12, IL-18) which induce several cell-mediated cytotoxic and inflammatory reactions, and Th-2 cytokines (i.e. IL-4, IL-5, IL-6, IL-10, and IL-13) which are associated with B cell antibody production. Th-2 cytokines are necessary for the trophoblast to secrete human placental lactogen (hPL) and human chorionic gonadotropin (hCG). In addition, Th-2 cytokines downregulate Th-1-type reactivity [16–18]. In successful pregnancy, the normal profile is a Th-2 type immunity. A shift towards Th-1-dominance is thought to be associated with unexplained habitual abortion [19]. 3.3.3. Natural killer cells The NK cell is part of the innate response of the immune system. It first recognises, and then kills, abnormal cells and its activation relies upon killer-activating and killer-inhibitory receptors. If the killer-activating receptors are engaged, a “kill” instruction is issued to the NK cell. This signal can be overridden by an inhibitory signal sent by the killer-inhibitory receptor. The “killing” of the target cell is carried out by inserting the pore-forming molecule perforin into the membrane of the target cell and then injecting it with cytotoxic granzymes [15].
4. Immunomodulation in successful versus abortogenic pregnancy 4.1. Immunmodulatory mechanisms in successful pregnancy 4.1.1. Asymmetric antibodies An immunological deviation in the maintenance of normal pregnancy is the induction of blocking, or asymmetric, antibodies. Asymmetric and symmetric antibodies are synthesized by the same cellular clone [20]. The asymmetric structure is achieved by a mannose-rich oligosaccharide group that is present on only one of the Fab regions. Owing to their asymmetric glycosylation, they are unable to activate effector functions such as complement fixation, phagocytosis and cytotoxicity. However, as they can combine with the antigen, they compete with precipitating antibodies of the same specificity and thus act as “blocking” antibodies [21]. The switch from the effector-type, symmetric immunoglobulin (Ig)G to the non-precipitating, blocking-type, asymmetric IgG constitutes a substantial part of the control of the equi-
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librium of maternal anti-foetal immunresponses. It prevents immunological eradication of the foetus by blocking subsequent effector events such as the formation of the membrane attack complex (C5b-9) via the “classical” means of complement activation. In a prevalence study, approximately 9% of the total IgG in nulliparous and multiparous women was found to be asymmetric, compared with 29% in healthy pregnant women (95% confidence interval (C.I.): 24–35%) and 3% in women who were habitual aborters (95% C.I.: 2–5%) (p < 0.001 versus controls) [21]. 4.1.2. Th-2/Th-1 bias Several studies investigating the ratio of Th-2 to Th-1 in terms of circulatory levels of serum cytokines reveal a Th-2 bias in normal pregnancy and a Th-1 bias in cases of recurrent miscarriages. IL-13, a Th-2-type cytokine, has been shown to be produced by human trophoblast cells [17]. In women with a successful pregnancy, there are markedly higher concentrations of IL-4 and IL-10 -producing T-cell clones, in both peripheral blood mononuclear cells (PBMC) and at the maternal–foetal interface, than are found in recurrent aborters [22]. To summarise, there is dominance of a Th-2 biased situation in the first trimester of a successful human pregnancy, whereas a Th-1 type bias may lead to pregnancy failure. In a more detailed analysis, it was shown that it is not the absolute concentrations of Th-1 or Th-2 -type cytokines that indicate a bias, but the ratios of Th-1 to Th-2 cytokines. In a study in 24 women with a history of successful pregnancies and 23 women with a history of unexplained habitual abortion, a comparison at the same gestational age showed that for every permutation of Th-l toTh-2 cytokines, the ratios were higher in the habitual abortion group [19]. In successful pregnancies, there was a clear Th-2 bias during the first trimester. In animal experiments, administration of IL-10, a Th-2type anti-inflammatory cytokine, prevents foetal wastage in mice prone to this effect (model for abortion) [23]. It was proposed that the important immunregulatory role for IL-10 is not primarily a direct one (like the role of colony-stimulating factor [CSF]-1, IL-3 or granulocyte-macrophage [GM]-CSF on the development of the placenta [24]), but that it may aid the survival of the conceptus by down-regulating Th-1 type reactivity, e.g., by inhibiting IFN-␥ production [25]. 4.1.3. Reduced NK cell activity NK cells are observed in the uterus during early pregnancy in several species [26–28]. Their role in normal pregnancy is, however, still controversial. CD16-negative, perforinpositive, but non-cytotoxic NK cells have been reported in first trimester human decidua [29]. Thus, the relative proportion of decidual NK cells is increased in normal pregnancy compared with the non-pregnant uterus. However, decidual NK activity is lower in normal compared with anembryonic pregnancies and in recurrent spontaneous abortions [30].
Fig. 2. Protective immunomodulatory mechanisms associated with a successful pregnancy. AB: antibody; IL: interleukin; IFN: interferon; TNF: tumour necrosis factor; NK: natural killer; Th: T-helper.
The protective immunomodulatory mechanisms associated with a successful pregnancy are summarised in Fig. 2. 4.2. Immunmodulatory mechanisms in abortogenic pregnancy 4.2.1. Symmetric antibodies Approximately 80% of the IgG specifically reactive to endometrial antigens are symmetric antibodies (which can be cytotoxic) in women with habitual abortion; in healthy pregnant women, the percentage is much lower at approximately 25%. Some investigators consider that, whether or not the antibodies are symmetric (i.e. effector-type with the potential to activate the complement system, or non-precipitating asymmetric-type that allows immunological tolerance) they are the major determining factor for successful pregnancy [21,31]. 4.2.2. Th-1/Th-2 bias The particularity of Th-1 cells is that they induce several cell-mediated cytotoxic and inflammatory reactions mainly via IL-2, IFN-␥ and TNF, whilst Th-2 cells support B cell antibody production via the cytokines IL-4, IL-5, IL-6 and IL-10. Th-2-type immunity is the normal profile in successful pregnancy, with a shift towards Th-1 dominance occurring in unexplained habitual abortion. Concentrations of Th-1 type cytokines, both at the maternal–foetal interface and in PBMC, are higher in women with unexplained habitual abortion. Th-1 and Th-2 cytokines are mutually inhibitory and a shift towards a Th-1 bias tends to further down-regulate Th-2 reactivity. A comparison of the ratios of various Th-1 and Th-2 cytokines in a total of 47 women (23 with a history of unexplained habitual abortion and 24 with a history of successful pregnancies) showed a higher Th-1:Th-2 ratio for all permutations (e.g. IL-2:IL-10 was 80-fold higher in the aborter group, IFN-
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␥:IL-10 was 30 times higher and TNF-:IL-10 was 40 times higher) [19]. Under experimental conditions, the outcome of pregnancy can be influenced by modulating the cytokine balance. Administration of TNF-␣, IFN-␥ or IL-2 to normal pregnant mice caused abortion [32,33], whilst anti-TNF-␣ reduced the resorption rate in a murine model of spontaneous immunologically-mediated abortion [34]. Note that although TNF-␣ is produced by both Th-1 and Th-2 cells, it is secreted at higher levels by Th-1 responses than by Th2 responses. Furthermore it elicits the production of IFN-␥, a Th-1 cytokine. Thus, TNF-␣ is often referred to as Th-1like [35]. IFN-␥ and TNF-␣ destroy trophoblast cells [36] and inhibit the implantation of mouse embryos [37] and the proliferation of human trophoblasts in vitro [38]. IL-12 has been shown to be present in significantly elevated levels in the serum of pregnant women with habitual abortion [39]. It significantly augments the cytolytic activity of lymphocytes against trophoblast cells [40] and the administration of IL-12, along with IL-18, to pregnant mice has also been shown to result in pregnancy loss [41]. The final step by which abortion is mediated is a Th-1 cytokine-triggered thrombotic and inflammatory process [42]. 4.3. Increased NK cell activity and lymphokine activated killer cell transformation Spontaneous pregnancy termination is linked to increased NK activity [3,43]; uterine resorption sites in a murine model of recurrent abortion were shown to be infiltrated by NK cells. However, it is not the presence of NK cells that is detrimental to the trophoblast, as it is able to resist NK-mediated lysis in vitro [3,9,44]. In fact, NK cells are able to induce trophoblast lysis and cause foetal loss if they are converted to lymphokine activated killer (LAK) cells by IFN-␥, TNF-␣ and IL-2 [44–46]. NK cells are not only the target of cytokines, they are also producers of proinflammatory cytokines such as IFN-␥ [47]. Hence, there is a potential for the initiation of a vicious circle. The final mechanism by which activated NK cells kill their target cells is exocytosis of perforin and serine esterase -containing granules in the contact area between effector (LAK) and target (trophoblast) cells [29,48]. The immune reactions associated with spontaneous pregnancy loss are summarised in Fig. 3.
5. The role of progesterone Following recognition of the antigens presented by the trophoblast, peripheral blood lymphocytes and decidual CD56+ and ␥␦+ cells develop specific progesterone receptors [3,9,49] In vitro, progesterone has been shown to exert a significant and dose-dependant inhibitory effect on lymphocyte cytotoxicity at serum concentrations comparable to those present during pregnancy. The effects of progesterone are achieved via a mediator molecule.
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Fig. 3. Immune reactions associated with spontaneous pregnancy loss. AB: antibody; IL: interleukin; IFN: interferon; TNF: tumour necrosis factor; NK: natural killer; Th: T-helper; LAK: lymphokine activated killer.
6. Progesterone-induced blocking factor In the presence of progesterone, progesterone receptorpositive lymphocytes and decidual CD56+ cells synthesize a 34 kDa immunomodulatory protein known as progesterone induced blocking factor (PIBF) [50]. PIBF has several antiabortive effects in vivo and appears to be the pivotal mediator in progesterone-dependent immunomodulation [9]. In recurrent spontaneous aborters and women showing clinical symptoms of threatened preterm pregnancy termination, PIBF expression and the percentage of PIBF+ lymphocytes was found to be significantly lower than in healthy pregnant women [9,51,52].
7. The pregnancy protecting role of PIBF 7.1. PIBF enhances asymmetric antibody production PIBF affects B cells, both directly and via cytokines [52,53], and induces increased production of asymmetric antibodies. This has a regulatory effect on anti-foetal immune responses during pregnancy. In animal experiments, blockade of the progesterone receptor resulted in reduced PIBF production together with a reduced amount of nonprecipitating (asymmetric) antibodies and elevated foetal resorption rates. As a corollary, the blocking of progesterone receptors by RU486 was shown to reduce the production of asymmetric antibodies in pregnant mice. In humans, RU486 has a proven efficacy in medically-induced, elective abortion. 7.2. PIBF causes a Th-2/Th-1 shift In essence, PIBF alters the profile of cytokine secretion by activated lymphocytes. It increases the production of Th-2 cytokines IL-3, IL-4 and IL-10 and decreases the
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Fig. 4. The pivotal role of progesterone receptor-mediated immune modulation in successful pregnancy. PBMC: peripheral blood mononuclear cells; AB: antibody; NK: natural killer; Th: T-helper; LAK: lymphokine activated killer.
production of the Th-l cytokine IL-12 [54]. In pregnant mice, neutralisation of endogenous PIBF activity resulted in a decrease in IL-10 and an increase in IFN-␥ production, leading to a high resorption rate and increased NK activity [55]. 7.3. PIBF inhibits NK activity NK activity in pregnant women is inversely related to the rate of PIBF-positive lymphocytes [51]. PIBF keeps NK activity at a low level both by controlling IL-12 production and also by inhibiting perforin liberation [9,52,56]. Decidual NK cells express a high level of perforin and are therefore potentially cytotoxic [29,52]; nevertheless, they have low cytotoxic activity. In vitro studies in decidual cells obtained from elective pregnancy termination suggest that it is PIBF that inhibits the cytotoxicity of NK cells via blockade of degranulation, and thus contributes to the low decidual NK activity [54]. Neutralisation of endogenous PIBF in pregnant mice results in abortion due to increased NK activity; these NK-mediated resorptions can be counteracted by simultaneous PIBF treatment [9]. The pivotal role of progesterone receptor-mediated immune modulation in successful pregnancy is summarised in Fig. 4.
8. Sequence of events To summarise, the immunogenic process at the foetal– maternal interphase can be divided into four stages [3,9]. (1) Recognition of foetal antigens. Foetally-derived antigens on the trophoblast are recognized by V␥l cells, a subset of the ␥/␦ T cells, in the decidua. (2) Upregulation of progesterone receptors. Following recognition of the antigens presented by the trophoblast, V␥l (CD56+) cells become activated and develop progesterone receptors. (3) Production and release of PIBF. In the presence of progesterone, decidual CD56+ and 76+ cells, as well as progesterone receptor-positive decidual NK cells, synthesise PIBF, a 34 kDa immunomodulatory protein. (4) Protection of pregnancy. Sufficient release of PIBF contributes to the success of early pregnancy by exerting its anti-abortogenic effects on three pathways. PIBF induces - an increase in asymmetric, “blocking” antibodies; - a Th-2 dominated, cytoprotective immune response (Th-2:Th-l bias); - a reduction in NK cell activity. In doing so, PIBF prevents inflammatory and thrombotic secondary reactions towards the trophoblast.
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9. Conclusions
References
There is an immunological relationship between the mother and the foetus which is a bi-directional communication determined on the one hand by foetal antigen presentation and on the other hand by recognition of, and reaction to, these antigens by the maternal immune system. We now have evidence that immunological recognition of pregnancy is important for the maintenance of gestation, and that an inadequate recognition of foetal antigens might result in failed pregnancy. Whereas HLA-A and HLA-B-I genes are downregulated in human trophoblast cells, nonpolymorphic class I molecules, e.g. HLA-G Class Ib, are expressed in extravillous cytotrophoblast and endothelial cells of foetal vessels in the chorionic villi, as well as in amnion cells and amniotic fluid. The trophoblast does not induce transplantation immunity and resists NK-and cytotoxic T-lymphocyte-mediated lysis in vitro. To date, we know that HLA-G presents antigens for ␥/␦ T cells and, at the same time, defends the trophoblast from cytotoxic effector mechanisms. Unfortunately, since polymorphic major histocompatability complex (MHC) is absent from the trophoblast, presentation of foetally-derived antigens is MHC-restricted. The ␥/␦ T cells recognise a distinct group of ligands with a smaller receptor repertoire than ␣/ T cells [57]. Most ␥/␦ T cells recognise unprocessed foreign antigens without MHC. The ␥/␦ TCR-positive cells in the decidua significantly increase in number and the majority of decidual ␥/␦ T cells are activated due to recognition of conserved mammalian molecules on the trophoblast. Following recognition of foetally derived antigens, the immune system reacts with a wide range of protective mechanisms. Many observations suggest that pregnancy is associated with an altered Th-1:Th-2 balance. The maternal immune response is biased towards humoral immunity and away from cellmediated immunity that could be harmful to the foetus. Cytokines of maternal origin influence placental development, whilst the maternal cytokine pattern is determined by antigen expression on the placenta. Normal human pregnancy is characterised by low peripheral NK activity, and increased NK activity appears to play a role in spontaneous abortions of unknown aetiology. In early human pregnancy, the majority of uterine lymphocytes are CD56 granulated NK cells, which do not express CD16 or CD3. In rodents and humans, uterine NK cells are under hormonal control and in early pregnancy they are enriched at sites where the foetal trophoblast infiltrates the deciduas. The dynamics of the appearance of uterine NK cells suggest that one of the functions of these cells is to control placentation. Another protective mechanism operating in favour of pregnancy is progesterone-dependent immunomodulation. Upon stimulation by foetally-derived antigens, pregnancy lymphocytes develop progesterone receptors and, in the presence of progesterone, produce a mediator (PIBF) that, through altering the cytokine balance, inhibits NK activity and produces an anti-abortive effect.
[1] R. Druckmann, Review: female sex hormones, autoimmune diseases and immune response, Gynecol. Endocrinol. 5 (Suppl. 6) (2001) 69–76. [2] A.E. Schindler, Immunology and progestins in pregnancy, Gynecol. Endocrinol. 13 (Suppl. 4) (1999) 47–50. [3] J. Szekeres-Bartho, Immunological relationship between the mother and the foetus, Int. Rev. Immunol. 21 (2002) 471–495. [4] P. Merviel, D. Evain-Brion, J.C. Challlier, J. Salat-Baroux, S. Uzan, The molecular basis of embryo implantation in human, Zentralbl. Gynakol. 123 (2001) 328–339. [5] S. Aschkenazzi, F. Naftolin, G. Mor, Menopause, sex hormones, and the immune system, Menopause Manag. 9 (2000) 6–13. [6] R.G. Lahita, Sex hormones and the immune system – part 1. Human data, Baillieres Clin. Rheumatol. 4 (1990) 1–11. [7] A.S. Ansar, N. Talal, Sex hormones and the immune system – part 2. Animal data, Baillieres Clin. Rheumatol. 4 (1990) 13–29. [8] D.H. Munn, M. Zhou, J.T. Attwood, I. Bondarev, S.J. Conway, B. Marshall, C. Brown, A.L. Mellor, Prevention of allogeneic foetal rejection by tryptophan catabolism, Science 281 (1998) 1191–1193. [9] J. Szekeres-Bartho, A. Barakonyi, B. Polgar, G. Par, Z. Faust, T. Palkovics, L. Szereday, The Role of g/d T cells in progesteronemediated immunomodulation during pregnancy: a review, Am. J. Reprod. Immunol. 42 (1999) 44–48. [10] L. Szereday, A. Barakonyi, E. Miko, P. Varga, J. Szekeres-Bartho, Gamma/delta T cell subsets, NKJ 2A expression and apoptosis of Vdelta+ T cells in pregnant women with or without risk of premature pregnancy termination, Am. J. Reprod. Immunol. 50 (2003) 490– 496. [11] A. Barakonyi, K.T. Kovacs, E. Miko, L. Szereday, P. Varga, J. Szekeres-Bartho, Recognition of nonclassical HLA class 1 antigens by ␥/␦T cells during pregnancy, J. Immunol. 168 (2002) 2683– 2688. [12] L. Komlos, R. Zamir, H. Joshua, I. Halbracht, Common HLA antigens in couples with repeated abortions, Clin. Immunol. Immunopathol. 7 (1977) 330–335. [13] C. Ober, T. Hyslop, S. Elias, L.R. Weikamp, W.W. Hauck, Human leucocyte antigen matching and foetal loss: result of a 10 year prospective study, Hum. Reprod. 13 (1998) 33–38. [14] R.G. Roussev, N.G. Higgins, J.A. McIntyre, Phenotypic characterization of normal human placental mononuclear cells, J. Reprod. Immunol. 25 (1993) 15–29. [15] P.J. Delves, I.M. Roitt, The immune system. Advances in Immunology, N. Engl. J. Med. 343 (2000) 37–49. [16] G. Par, J. Geli, N. Kozma, P. Varga, J. Szekeres-Bartho, Progesterone regulates IL-12 expression in pregnancy lymphocytes by inhibiting phospholipase A2, Am. J. Reprod. Immunol. 49 (2003) 1–5. [17] G.B. Dealtry, D.E. Clark, A.M. Sharkey, S. Charnock-Jones, S.K. Smith, Expression and localization of the Th2-type cytokine interleukin-13 and its receptor in the placenta during human pregnancy, Am. J. Reprod. Immunol. 40 (1998) 283–290. [18] T.R. Mosmann, S. Sad, The expanding universe of T-cell subsets, Immunol. Today 17 (1996) 138–146. [19] R. Raghuphaty, M. Makhseed, F. Azizieh, A. Omu, M. Gupta, R. Farhat, Cytokine production by maternal lymphocytes during normal human pregnancy and in unexplained recurrent abortion, Hum. Reprod. 15 (2000) 713–718. [20] M.O. Labeta, R.A. Margni, J. Leoni, R.A. Binaghi, Structure of asymmetric non-precipitating antibody: presence of a carbohydrate residue in only one Fab region of the molecule, Immunology 57 (1986) 311–317. [21] A.C. Eblen, C. Gercel-Taylor, L.B.E. Shields, J.S. Sanfilippo, S.T. Nakajima, D.D. Taylor, Alterations in humoral immune responses associated with recurrent pregnancy loss, Fertil. Steril. 73 (2000) 305–313.
396
R. Druckmann, M.-A. Druckmann / Journal of Steroid Biochemistry & Molecular Biology 97 (2005) 389–396
[22] M.-P. Piccinni, L. Beloni, C. Livi, E. Maggi, G. Scarselli, S. Romagnani, Defective production of both leukemia inhibitory factor and type 2 T-helper cytokines by decidual T cells in unexplained recurrent abortions, Nat. Med. 4 (1998) 1020–1024. [23] G. Chaouat, A.A. Meliani, J. Martal, R. Raghupathy, J.J. Elliot, T.R. Mosmann, T.G. Wegmann, IL-10 prevents naturally occurring fetal loss in the CBA x DBA/2 mating combination, and local defect in IL-10 production in this abortion-prone combination is corrected by in vivo injection of IFN-tau, J. Immunol. 154 (1995) 4261–4268. [24] S.A. Robertson, M. Br¨annstr¨om, R.F. Seamark, Cytokines in rodent reproduction and the cytokine–endocrine interaction, Curr. Opin. Immunol. 4 (1992) 585–590. [25] D.F. Fiorentino, M.W. Bond, T.R. Mosmann, Two types of mouse T helper cell. IV. Th-2 clones secrete a factor that inhibits cytokine production by Th-l clones, J. Exp. Med. 170 (1989) 2081–2095. [26] P.M. Starkey, L.L. Sargent, C.W.G. Redman, Cell populations in human early pregnancy decidua: Characterization and isolation of large granular lymphocytes by flow cytometry, Immunology 65 (1988) 129–134. [27] B.A. Croy, P. Gambel, J. Rossant, T.G. Wegmann, Characterization of murine decidual natural killer (NK) cells and their relevance to the success of pregnancy, Cell. Immunol. 93 (1985) 315–326. [28] B.A. Croy, A. Waterfˆıeld, W. Wood, G.J. Kind, Normal murine and porcine embryos recruit NK cells to the uterus, Cell. Immunol. 115 (1988) 471–480. [29] D. Rukavina, G. Rubesa, L. Gudelj, H. Haller, E.R. Podack, Characteristics of perforin expressing lymphocytes within the first trimester decidua of human pregnancy, Am. J. Reprod. Immunol. 33 (1995) 394–404. [30] K.-H. Chao, Y.-S. Yang, H.-N. Ho, S.-U. Chen, H.-F. Chen, H.J. Dai, S.-C. Huang, T.J. Gill, Decidual natural killer cytotoxicity decreased in normal pregnancy but not in anembryonic pregnancy and recurrent spontaneous abortion, Am. J. Reprod. Immunol. 34 (1995) 274–280. [31] I. Malan Borel, S. Miranda, S. Menezes Freire, A. Canellada, M. Eslave Bueno, R. Binaghi, Modulation of the humoral immune response by placental secretory factors, Am. J. Reprod. Immunol. 35 (1996) 529–533. [32] M. Parant, The role of TNF in non-specific stimulation of the mouse resistance to infection, Immunobiology 175 (1987) 26. [33] B.V. Tezabwala, A.P. Johnstone, Effect of administration of interleukin 2 in pregnancy, J. Reprod. Immunol. 1 (Suppl.) (1986) 147. [34] G. Chaouat, E. Menu, T.G. Wegmann, Role of lymphokines of the CSF family and of TNF, gamma-interferon and IL-2 on placental growth and foetal survival studied in 2 murine models of spontaneous resorptions, in: G. Chaouat, G. Mowbry (Eds.), Cellular and Molecular Biology of the Materno-Fetal Relationship, Vol. 212, Colloque INSERM John Libbey Eurotext Ltd., Paris, London, 1991, pp. 91–100. [35] R. Raghupathy, M. Makhseed, F. Azizieh, N. Hassan, M. Al-Azemi, E. Al-Shamali, Maternal Th-l- and Th-2-type reactivity to placental antigens in normal human pregnancy and unexplained recurrent spontaneous abortions, Cell. Immunol. 196 (1999) 122–130. [36] J. Yui, M. Garcia-Lloret, T.G. Wegmann, L.J. Guilbert, Cytotoxicity of tumour necrosis factor-alpha and gamma-interferon against primary human placental trophoblasts, Placenta 15 (1994) 819– 835. [37] F. Haimovici, J.A. Hill, D.J. Andersen, The effects of soluble products of activated lymphocytes and macrophages on blastocyst implantation events in vitro, Biol. Reprod. 44 (1991) 69–75. [38] J.A. Hill, The rationale for leukocyte immunization in women with recurrent spontaneous abortion: Fact or fiction? in: G. Chaouat, G. Mowbry (Eds.), Cellular and Molecular Biology of the Materno-Fetal Relationship, Vol. 212, Colloque INSERM John Libbey Eurotext Ltd., Paris, London, 1991, pp. 263–269.
[39]] R. Wilson, I. McInnes, B. Leung, J.H. McKillop, J.J. Walker, Altered interleukin 12 and nitric oxide levels in recurrent miscarriage Eur. J. Obstet. Gynecol. Reprod. Biol. 75 (1997) 211–214. [40] S. Hayakawa, N. Nagai, T. Kanaeda, M. Karasaki-Suzuki, M. Ishi, F. Chishima, F. Satoh, Interleukin-12 augments cytolytic activity of peripheral and decidual lymphocytes against choriocarcinoma cell lines and primary culture human placental trophoblasts, Am. J. Reprod. Immunol. 41 (1999) 320–329. [41] J. Muranaka, Y. Tsuji, A. Hasegawa, A. Ida, Y. Nakata, R. Kanazawa, K. Koyama, Interleukin-18 as an important new cytokine in pregnancy, Am. J. Reprod. Immunol. 40 (1998) 278. [42] D.A. Clark, G. Chaouat, P.C. Arck, H.W. Mittruecker, G.A. Levy, Cytokine-dependent abortion in CBA x DBA/2 mice is mediated by the procoagulant fgl2 prothrombinase [correction of prothombinase], J. Immunol. 160 (1998) 545–549. [43] J. Szekeres-Bartho, P. Varga, A.S. Pasca, Immunological factors contributing to the initiation of labour-lymphocyte reactivity in term labour and threatened pre-term delivery, Am. J. Obstet. Gynecol. 155 (1986) 108–113. [44] B.L. Drake, J.R. Head, Murine trophoblast cells can be killed by lymphokine-activated killer cells, J. Immunol. 143 (1989) 9–14. [45] D.A. Clark, Are there immune abortions? Res. Immunol. 141 (1990) 104–109. [46] B.L. Ferry, P.M. Starkey, I.L. Sargent, G.M. Watt, M. Jackson, C.W. Redman, Cell populations in the human early pregnancy decidua: natural killer activity and response to interleukin-2 of CD56positive large granular lymphocytes, Immunology 70 (1990) 446– 452. [47] D.A. Clark, G. Chaouat, Spontaneous abortion in DBA/2 mated mice is due to cytokines and not trophoblast killing by asialo GM1+ NK cells or NO-producing macrophages, Am. J. Reprod. Immunol. 35 (1996) 454. [48] C.-C. Liu, C.M. Walsh, J.D. Young, Perforin: structure and function, Immunol. Today 16 (1995) 194–201. [49] J. Szekeres-Bartho, G. Szekeres, P. Debre, B. Autran, G. Chaouat, Reactivity of lymphocytes to a progesterone receptor-specific monoclonal antibody, Cell. Immunol. 125 (1990) 273–283. [50] J. Szekeres-Bartho, F. Kilar, G. Falkay, V. Csernus, A. Torok, A.S. Pacsa, Progesterone-treated lymphocytes of healthy pregnant women release a factor inhibiting cytotoxicity and prostaglandin synthesis, Am. J. Reprod. Immunol. Microbiol. 9 (1985) 15–19. [51] J. Szekeres-Bartho, Z. Faust, P. Varga, The expression of a progesterone-induced immunomodulatory protein in pregnancy lymphocytes, Am. J. Reprod. Immunol. 34 (1995) 342–348. [52] J. Szekeres-Bartho, A. Barakonyi, G. Par, B. Polgar, T. Palkovics, L. Szereday, Progesterone as an immunomodulary molecule, Int. Immunopharmacol. 1 (2001) 1037–1048. [53] K. Kelemen, I. Bognar, M. Paal, J. Szekeres-Bartho, A progesteroneinduced protein increases the synthesis of asymmetric antibodies, Cell. Immunol. 167 (1996) 129–134. [54] Z. Faust, G. Laskarin, D. Rukavina, J. Szekeres-Bartho, Progesterone-induced blocking factor inhibits degranulation of natural killer cells, Am. J. Reprod. Immunol. 42 (1999) 71–75. [55] L. Thompson-Snipes, V. Dhar, M.W. Bond, T.R. Mosmann, K.W. Moore, D.M. Rennick, Interleukin-10: a novel stimulatory factor for mast cells and their progenitors, J. Exp. Med. 173 (1991) 507– 510. [56] G. Laskarin, N. Strbo, V. Sotosek, D. Rukavina, Z. Faust, J. Szekeres-Bartho, E.R. Podack, Progesterone directly and indirectly affects perforin expression in cytolytic cells, Am. J. Reprod. Immunol. 45 (1999) 312–320. [57] J. Szekeres-Bartho, A. Barakonyi, E. Miko, B. Polgar, T. Palkovics, The role of gamma/delta T cells in the feto-maternal relationship, Semin. Immunol. 13 (2001) 229–233.