Interplay between T helper type 1 and type 2 cytokines and soluble major histocompatibility complex molecules: a paradigm in pregnancy

The balance between T helpers type 1 (Th1) and type 2 (Th2) is being postulated to regulate almost any kind of immune response. Thus, we refer to the Th1/Th2 balance in autoimmunity, antibody production, antiviral and antimicrobial responses, graft rejection, allergies, etc. Every type of immune response to various stimuli is favoured either by Th1- or Th2-type cytokines, which also depend on the genetic background of the individual, the stage of infection, and the dose and nature of stimulant. Pregnancy has long been considered to be a Th2 phenomenon.1 This conclusion, drawn essentially from murine experimental models, is based on the beneficial role of Th2 cytokines, such as interleukin-10 (IL-10), granulocyte–macrophage colony-stimulating factor (GM-CSF) and IL-3, in feto–placental growth2–4 and the harmful effect of Th1 cytokines, such as interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), during the same period.5,6 However, accumulating data show that Th1 cytokines, although harmful during mid-gestation, are necessary during the early stages of pregnancy and during labour.7 Thus, TNF-α release is stimulated by gonadotrophins and plays an important role in the pathway of ovulation by inducing prostaglandin and nitrogen oxide production for follicle rupture.8 Similarly, ovulatory follicles produce IFN-γ, which also promotes nitric oxide production.8 At higher doses, however, it inhibits follicle-stimulating hormone-stimulated oestradiol production by human luteinized granulosa cells, as well as human chorionic gonadotropin-stimulated progesterone secretion, which results, respectively, in ovulation and implantation effects.9,10 Both IFN-γ and TNF-α are produced by human preimplantation embryos in small quantities,8,11 whereas higher concentrations become embryotoxic and inhibit development of two-cell stage mouse embryos to blastocysts.12 Kinetic experiments in mice show peaks of IFN-γ and TNF-α production on day 7/9 and day 12 of gestation in the maternal serum, respectively, that decline thereafter.13,14 The period of IFN-γ overproduction correlates with ectoplacental cone trophoblast differentiation into the spongio- and labyrinthine-trophoblast populations of the mature placenta.13,15 At this stage of pregnancy, IFN-γ also induces expression of CCR3 chemokine receptors on murine as well as human trophoblasts,16,17 which may play an important role in T-cell chemotaxis at the maternal–fetal interface. However, overproduction of IFN-γ in the maternal serum has been correlated with fetal rejection during the first trimester of pregnancy in humans.18 In murine experimental models, exogenous administration of IFN-γ has been shown to increase the levels of fetal resorption.3,5,6 IL-10, which has been shown to exert a beneficial action during mid-gestation and decrease the fetal abortion rate in the CBA X DBA/2 abortion model,3 also appears in the pregnant uterus after implantation, is expressed in the trophectoderm and inner cell mass and is found at higher levels in the maternal serum as compared to non-pregnant controls.4,19 IL-3 is undetectable in the murine uterus during the menstrual cycle but appears during early pregnancy on days 2 and 3 in the uterus and the trophectoderm of blastocysts to facilitate implantation. It decreases, however, thereafter until mid-gestation14,19 and starts promoting placental growth.2,14 GM-CSF is found to be involved in ovulation and implantation and its production in the uterus declines until day 5 of pregnancy in mice whereas it reappears in the serum on day 8.14 Thus, it is obvious that the Th1/Th2 balance during pregnancy is essential to fetal survival, a balance that leans towards Th1 or Th2 responses according to the stage of pregnancy. Due to the high numbers of cytokines involved in pregnancy and their specific regulation during the different stages of embryo development, it becomes mandatory to search for new parameters to characterize these phenomena and focus on events prior to cytokine production. T cells possess a central role not only in pregnancy but in the whole immune system. Elimination of T cells during pregnancy in murine experimental models results in increased levels of fetal abortion and significant reduction of placental growth.2,20 More precisely, depletion of the CD8+ cell population from the maternal organism considerably reduces placental cell proliferation and phagocytosis, whereas elimination of CD4+ T cells results only in reduction of placental cell proliferation.21 Although various cell types, including epithelial cells, trophoblasts, macrophages and natural killer (NK) cells, can produce cytokines, T cells are considered to be the major source for most of these factors. The general term ‘T cells’ includes all Th populations from Th0 to Th1 and Th2, cytotoxic T-cell (Tc) populations from pre-Tc to Tc1, Tc2, etc., T regulatory, T suppressor and contra-suppressor populations, without excluding the low Thy-1-expressing NK cells. T-cell populations express receptors that recognize antigen in association with class I or class II major histocompatibility complex (MHC) antigens. Binding of the antigen–MHC complex to the T-cell receptor (TCR) initiates a plethora of cascade reactions, each one leading to the activation of a specific pathway and gene activation. For example, one pathway that could be followed after binding of TCR with an antigen–MHC complex is the activation of tyrosine kinases (ZAP70, Lck, Fyn) which involves transducing the signal through activation of Grb2-SOS, Ras, c-Raf, stimulating JNK, c-jun, NF-κB, resulting in activation of the IL-2 promoter.22 According to similar mechanisms, the various cytokine genes become activated, producing the corresponding factors either locally at the feto–placental unit or systemically. The local production, however, is also reflected at the periphery because these cytokines are detected in the maternal serum14,18 indicating that the stimulatory signals are not solely anchored to the feto–placental unit. Circulating fetal cells in the maternal blood cannot obviously interfere with the early events necessary for implantation and early embryo development because they are detectable at the periphery as early as after the 4th week of gestation and at very low numbers.23,24 There should, however, exist other circulating molecules that trigger TCR on T lymphocytes, or similar structures on NK cells [immunoglobulin-like transcript-2 (ILT-2), protien 29 (p29), killer cell immunoglobulin-like receptors (KIR), netural killer receptor for HLA-G (NKG) receptors25] and stimulate cytokine gene expression and production. Such candidate circulating molecules are serum-soluble MHC antigens, whose presence has been reported by many laboratories but their role remains unclear. Soluble class I MHC antigens (sMHC-I) are being detected in the serum of healthy individuals but have also been correlated with various pathological disorders.26 Soluble class I molecules maintain the structure of surface class I proteins and their ability to bind anti-MHC antibodies. They form complexes with surface CD8 molecules27,28 and have the ability to bind endogenous and exogenous peptides,29 indicating that they can specifically modulate the immune response. In various pathological disorders the levels of sMHC-I have been shown to differ from those found in healthy individuals. Thus, sMHC-I levels increase in human immunodeficiency virus-infected patients,30 in ankylosing spondylitis, systemic lupus erythematosus patients, sensory neural deafness, and head and brain injury patients.26 Treatment of hepatitis C virus-positive patients with IFN-γ was shown to increase sMHC-I and soluble CD8 (sCD8) levels in the serum31 whereas immunosuppressive drugs like steroids and cyclosporin A in patients with renal transplants inhibit gene expression and reduce transcription.32 Increased levels of sMHC-I are also detected in the serum of patients undergoing an acute graft rejection process.33 The levels of sMHC-I in the serum vary within individuals depending on the human leucocyte antigen (HLA) phenotype (some phenotypes give higher concentrations of sMHC-I26), as well as age (sMHC-I increase with age34). Different mechanisms for class I antigen release from the cells have been proposed:35–39 alternative splicing alters the α3-exon of the class I gene and excludes the transmembrane/cytoplasmic exons, thus leading to truncated molecules, post-translational removal of the transmembrane and cytoplasmic tail and release from the cell through secretory particles, proteolytic cleavage of membrane-bound class I molecules, and release of full-length empty class I molecules. During pregnancy, sMHC-I levels vary depending on the gestational stage of the mother. Some laboratories report reduced sMHC-I levels during the third trimester as compared to the first and second trimesters,40,41 whereas others show reduction of sMHC-I levels during the first trimester and an increase thereafter.42–44 From our experience, this discrepancy depends on the exact time of serum collection during each trimester in the different laboratories, because significant differences exist even within weekly examinations that may go undetected if all trimester samples are pooled. Indeed, the physiological levels of sMHC-I during the first trimester of pregnancy were shown to be time-dependent and vary within a specific range of values. In cases of recurrent abortion, sMHC-I levels are found outside this specific range of values.42,43 Although sMHC-I are consistently detected in the serum, with the exception of HLA-G, only limited information is available. Soluble HLA-G (sHLA-G) exists in three isoforms HLA-G5, -G6 and -G7 deriving from HLA-G by alternative splicing. HLA-G5 and -G6 consist of three domains (α1, α2, α3) missing the transmembrane/cytoplasmic tail and are associated or not with β2-microglobulin, respectively, whereas, in HLA-G7 the presence of a stop codon in intron 2 gives rise to a soluble isoform with a molecular weight of 17 000, corresponding to the α1 domain. HLA-G5 and HLA-G6 have been found to inhibit NK cytolysis and induce apoptosis of activated T cells.45,46 Although HLA-G and especially the HLA-G2 isoform are found to circulate in the maternal blood,47 soluble classical HLA-A, -B, -C follow specific patterns of production during the different gestational stages,41,48 a model not applicable to β2-migroglobulin because β2-microglobulin levels do not change significantly during pregnancy and do not correlate with sHLA-I levels.41 Soluble class I antigens have been shown to reduce the cytotoxic activity of CD8+ anti-HLA-B7-specific cytotoxic T lymphocytes in an MHC-restricted manner.49 Other studies, however, show that sHLA-B7 blocked the generation of CD8+ cytotoxic T lymphocytes in a non-MHC-restricted manner.50 Although it seems that MHC restriction offers a higher affinity of binding, interaction with other surface molecules, i.e. CD8, can sufficiently block the cytotoxic T lymphocyte response. Recent studies have shown that sMHC-I also induces apoptosis by stimulating expression of CD95-L, thus regulating the Fas/FasL system. However, ligation of CD28 was found to rescue T cells from death, indicating that apoptosis takes place through selective stimulation of TCR.51 The cell origin of sMHC-I has not yet been determined. In cases of lung transplants, significantly higher levels of donor sMHC-I as compared to kidney or heart allografts were detected.52 Zhang et al. describe the acquisition of soluble donor MHC class I alloantigen from regulatory T cells53 whereas He and Le describe the release of soluble HLA class I heavy-chain proteins from fibroblasts after IFN-γ or TNF treatment through alternative splicing of HLA class I transcripts.54 During pregnancy, trophoblast cells seem to account for the production of at least some portion of soluble MHC class I antigens because such molecules can be detected in the culture supernatants of short-term IFN-γ-treated (4–6 hr) murine and human trophoblasts (unpublished data). Information of soluble class II MHC antigens (sMHC-II) is limited and although their presence in the circulation was described 25 years ago,55,56 sMHC-II are found increased in the serum during viral infections, acute graft rejection and graft versus host episodes.29 Thus, soluble HLA-DR (sHLA-DR) significantly increases in patients with acute anterior uveitis, a B27-associated inflammatory disease, as compared to healthy controls.57 Furthermore, sHLA-DR levels are significantly higher in patients with chronic hepatitis C, but decline upon treatment with IFN-α in IFN-α-responder patients and not in non-responders.58 During pregnancy, specific levels of sMHC-II characterize each stage of fetal development and correlate with maternal immunostimulation.42–44 Women who have recurrent abortions show increased levels of sMHC-II,42,43 which decline to normal after intravenous immunoglobulin treatment.43,44 Similar to sMHC-I, subunits of sHLA-DR molecules have been detected in the serum of healthy individuals.29 Thus, soluble CD74 (invariant chain), HLA-DR heterodimer or isolated entities have been reported.59 The sMHC-II molecules have been detected in the culture supernatants of primary human or murine trophoblasts or the murine trophoblasts cell line TROPHO-160 upon a 6-hr stimulation with IFN-γ, indicating that although these cells do not express surface class II molecules,61 they contain intracellular pools that can be released from the cells and account for the increased levels of sMHC-II observed during pregnancy.60 Indeed, cell fractionation experiments and confocal microscopy analysis localize class II pools in the endosomal compartments of the cells, which upon treatment with IFN-γ are released from the cells.60,62 Recent observations from our laboratory using the reverse transcription–polymerase chain reaction demonstrated that the regulation of classical H-2A class II antigens in the TROPHO-1 trophoblast cell line implicates constitutive expression of H-2O and H-2A and is inducible by IFN-γ expression of H-2M (submitted manuscript, to be presented in the 8th IFPA Meeting). Secretion of the intracellular pools of class II antigens can be inhibited by the protease inhibitor pepstatin A and by the protein synthesis inhibitor cyclohexamide, a finding demonstrating that IFN-γ is involved in novel H-2M class II antigen synthesis and activation of the secretory pathway of the cells during the 6 hr of stimulation.62 Similar experiments with macrophages and epithelial cells, however, could not reproduce the phenomenon of class II antigen release. Thus, besides the gestational period, the cell origin of sMHC-II antigens is still undetermined. From the knowledge acquired thus far it seems that both soluble class I and II antigens are involved in immune regulation but apparently account for different regulatory pathways. Thus, a significant increase of sHLA-DR was observed in the serum of cases of acute anterior uveitis57 as compared to healthy controls, whereas sMHC-I in these patients was only slightly increased. The use of various class I and II specific monoclonal antibodies has allowed the physiological levels of serum HLA in human pregnancy to be defined.42 It has been shown that a specific pattern of production is followed during the different stages of gestation and that the levels of class I and class II serum antigens correlate and reflect maternal immunosuppression and immunostimulation levels, respectively, findings that permit diagnosis and treatment for women suffering from recurrent abortions.42,43 In support of the above, Pfeiffer et al. show that patients experiencing an early abortion after in vitro fertilization have lower soluble HLA class I and class II levels as compared to intact pregnancies.63 As proposed by Vassiliadis et al. however, maternal immunostimulation and immunosuppression vary within a specific range of soluble values and early abortion occurs when serum HLA are found outside this range, either with higher or lower values depending on the individual.43 Deviation from the physiological serum HLA class I and class I levels is also observed in endometriosis where, upon treatment with danazol, women may recover to physiological serum antigen levels.64 Identical results are also obtained from renal transplant patients undergoing acute rejection who, after receiving an immunosuppressive treatment, exhibited restored levels of HLA class I antigens.32 The fact that sMHC-I antigens interact with CD8+ T cells indicates that sMHC antigens are indeed capable of regulating the immune response. Similar interactions should be expected for sMHC-II antigens and CD4+ T cells. In vitro experiments show that sHLA-G stimulated production of TNF-α, IFN-γ and IL-10 and at the same time, reduced the release of IL-3 from peripheral blood mononuclear cells.65 In contrast to sHLA-G, surface HLA-G-expressing cells in contact with decidual mononuclear cells were shown to decrease the release of TNF-α and IFN-γ,66 pointing to a differential cytokine regulation at the periphery and the maternal–fetal interface. During pregnancy in humans, as well as in murine experimental models, a Th1-inducing immune response is accompanied by increased serum class I antigen levels, whereas a Th2-inducing immune response is accompanied by increased serum class II levels.18,42 Administration of protein A in pregnant mice results in a significant increase of serum IL-10 correlating with an increase of serum class II levels as compared to untreated pregnant controls. Similarly, muramic acid administration to pregnant mice decreases the levels of IFN-γ and TNF-α in the serum, an observation that correlates with a decrease of soluble class I antigens. Lipopolysaccharide treatment of pregnant mice, which decreases both Th1- and Th2-type cytokine production,67 results in a suppression of class I and class II antigen levels in the serum. Interestingly, direct administration of IFN-γ or TNF-α to pregnant mice results in an increase of sMHC-II in the serum, indicating that other mechanisms also take place in the regulation of sMHC secretion.42 These observations favour the findings concerning the role of IFN-γ to induce the release of intracellular pools of class II antigens.60 The description of regulatory T cells has complicated the profile of Th1/Th2 because the identification of mouse and human CD4+ T cells, distinct in surface and/or cytokine production from Th1 and Th2 cells, were found to suppress Th1 and Th2 effector cell functions.68–71 This suppressive activity was shown to function through cytotoxic T-lymphocyte antigen-4 co-stimulation to produce IL-10 and transforming growth factor-β, which were then able to suppress autoimmunity.72–74 Furthermore, co-stimulation of CD4+ T cells through inducible costimulator (ICOS) promotes Th2 CD4+ cell responses, while it does not regulate Th1-mediated inflammation.75 Except from T cells, soluble class I antigens could bind to CD8+ macrophages or mast cells to increase nitric oxide or TNF-α levels76 during the development of inflammatory reactions.67,77 Therefore, from the known literature, it is possible that there is a degree of correlation between Th/MHC suggesting that the Th1/Th2 profile may be regulated by the soluble class I/class II balance, a link with many important applications concerning the study of tolerance, autoimmunity, graft rejection, inflammation and response to bacterial and viral infections. Th1/Th2 reflects a very general term that does not characterize the type of cytokine produced. There is increasing evidence that cytokine production depends on the co-stimulatory signals on T cells. Thus, the elevated levels of sMHC-II observed during recurrent abortion could activate CD4+ T cells bearing ICOS co-stimulator and become the stimulus for IFN-γ and TNF-α production (Fig. 1). It turns out that the affinity of acquired soluble moiety of class I or II HLA molecules for the TCR/CD3 and co-stimulators (which is closely linked to the genetic background of the individual) can be the basis of immune regulation, where cytokine production follows as a During pregnancy is and Although the same effect could be obtained by in Th and sHLA-I on these should not have the same at the same For example, could be on Th and sHLA-I on leading to a specific balance during immune activation. of a model concerning immune regulation by soluble major histocompatibility complex (MHC) Soluble class I and class II human leucocyte antigens can trigger T cytotoxic (Tc) and T cells through specific of T-cell receptor and T-cell receptor respectively, to exert their regulatory The sHLA-I may also bind CD8 on macrophages or cells. on the co-stimulatory signals or associated with the different regulatory T-cell populations, can be In pregnancy one of the for sMHC molecules upon IFN-γ stimulation are However, the cell origin of these molecules in other normal or pathological has not yet been determined. IFN-γ, TNF-α, necrosis