Analysis of the IFN-γ-induced signal transduction pathway in fetal rejection

The placenta, one of the most important fetal tissues during gestation, ensures nutrition, development and protection of the fetus. Although placenta lacks expression of class II MHC antigens, they can be induced either by interferon-gamma (IFN-γ) on the spongiotrophoblast zone, or by 5-azacytidine (5-azaC) on the labyrinthine trophoblast zone, two agents actively participating in a plethora of immunological and inflammatory reactions. This induction is correlated with fetal abortion and fetal developmental abnormalities. In this work the in vitro and in vivo signal transduction pathways followed by IFN-γ or 5-azaC to induce class H antigen expression on placental cells by using specific pathway inhibitors has been studied. It is shown that at least three intracellular pathways are implicated in the Ia induction, p21ras is the first protein activated by the two agents while further signalling requires Ca2+ mobilization and PKC activations. When the in vitro results are transferred to live animals using the same inducing agents and pathway inhibitors, it is found that theophylline (Ca2+/CaM inhibitor) and anti-p21ras are the most potent suppressors of the IFN-γ- and 5-azaC-induced side effects during pregnancy. The data presented here point to novel directions not only as to the intracellular signalling, but also to the use of pathway inhibitors in vivo to treat aberrant antigen expression associated with fetal loss.

THE placenta, one of the most important fetal tissues during gestation, ensures nutrition, development and protection of the fetus. Although placenta lacks expression of class II MHC antigens, they can be induced either by interferon-gamma (IFN-y) on the spongiotrophoblast zone, or by 5-azacytidine (5-azaC) on the labyrinthine trophoblast zone, two agents actively participating in a plethora of imInunological and inflammatory reactions. This induction is correlated with fetal abortion and fetal developmental abnormalities. In this work the in vitro and in vivo signal transduction pathways followed by IFN-y or 5-azaC to induce class H antigen expression on placental cells by using specific pathway inhibitors has been studied. It is shown that at least three intracellular pathways are implicated in the Ia induction, p21 is the first protein activated by the two agents while further signalling requires Ca 2+ mobilization and PKC activations. When the in vitro results are transferred to live animals using the same inducing agents and pathway inhibitors, it is found that theophylline (Ca2+/CaM inhibitor) and anti-p21 are the most potent suppressors of the IFN-y-and 5-azaCinduced side effects during pregnancy. The data presented here point to novel directions not only as to the intracellular signalling, but also to the use of pathway inhibitors in vivo to treat aberrant antigen expression associated with fetal loss.

Introduction
Cellular crosstalk is the fundamental basis of development, differentiation and survival of any pluricellular organism. Protein binding to a cell membrane receptor initiates intracellular signalling reactions leading to the regulation of any physiological process. The principal architecture of the signal transduction pathways known up to now, includes specific receptors, GTP-binding proteins, second messenger generating protein kinases, target functional proteins and regulatory proteins.
This study is an analysis of the signal transduction pathway initiated by IFN-/leading to class II major histocompatibility complex (MHC) antigen expression on the adherent placental cells, which previous studies have identified as spongiotrophoblasts. IFN-,/ is known to induce class II antigens in different human and murine cells and tissues, including leukocytes, monocytes, glioblastomas and astrocytes.'--However, the intracellular pathway followed each time depends on (C) 1995 Rapid Communications of Oxford Ltd the species and the cell tp+e studied. In humans, IFNq, activates the Ca /Calmodulin (Ca2+/ CaM) pathway in most cases. 5's The second messenger calcium ions bind to calmodulin, triggering conformational changes in this protein, and allowing the activation of many enzymes, including protein kinases. This Ca2+/CaM pathway can be blocked by inhibitors like theophylline and W7 (see the Methods section and Reference 5). In the murine system, the induction of class II antigens by IFNq, also involves the protein kinase 6 8 C (PKC) pathway.-Stimulation of receptors or opening of Ca 2+ channels initiates hydrolysis of phosphatidyl inositol (Ptdlns) by phospholipase C where the subsequently produced diacylglycerol (DAG) activates PKC, thus providing a linkage between extracellular and intracellular responses. Although DAG is the most important stimulator of PKC, the latter can also be regulated by other phospholipid derived messengers. Upon activation of PKC, multiple positive and negative regulatory signals may affect a pie-L Athanassakis thora of target functional proteins. A way of abortion and in most cases (depending on the studying the events occurring downstream of inhibitor used) lead the feto-placental develop-PKC activation is by applying specific inhibitors, ment to physiological levels. such as sphingosine and staurosporine. <8 Another important system shown in some Materials and Methods cases to be involved in class II antigen induction by IFN-7 in humans as well as in mice, is the G-Mice: BALB/cJ and C3H/HeJ mice were mainprotein system. 9'x2 It consists of a complicated tained in our Animal Breeding Unit at the Unicascade of GTP-binding proteins located in the versity of Crete (Greece). Each BALB/cJ or C3H/ inner cell membrane, able to cross-link many cell HeJ female was individually caged with a BALB/cJ surface receptors to effector proteins and initiate or C3H/HeJ male overnight and the following many intracellular events, x2 This pathway was morning the females were examined for the preapproached by applying mevalonic acid lactone sence, of a vaginal plug. The morning where the (MEV) to the cells which, although it does not plug was observed was considered as day zero of have well defined properties, x'4 is employed as pregnancy. a G-protein (ras) suppressor.
Study of the signal transduction mechanisms Pathway inhibitors: Theophylline (TPH) and W7 leading to class II antigen expression on murine (N-(6-aminohexyl)-5-chloro-l-napthalenesulfonatrophoblasts are very important because these mide HCl) were used to block the Ca2+/CaM cells directly regulate the feto-placental unit pathway. 5 TPH blocks the interaction between development and control whether the outcome CaM and enzymes (inactive adenyl cyclase). This of pregnancy is successful or not. Placental tro-drug is able to block the class II MHC antigen phoblasts have been found not to express class induction by IFN-7 on the human cell lines HL-II MHC antigens '5'6 and this negative state is 60, HeLa, U937, a Burkitt's lymphoma, and Daudi one of the protective mechanisms evoked by the cells. 5 From the literature, dose-response experifetus to escape maternal immune attack and ments place the best working concentration at avoid autoimmune reactions. It has been shown 0.15 mg/ml. For this work and cellular system previously in mice that the two trophoblast layers (the trophoblast portion of adherent placental of the placenta differ in their inducibility to cells), doses of 0.2 and 0.1mg/ml (Sigma, St express class II antigens. Spongiotrophoblast Louis, MO) were applied. The higher dose derived trophoblasts are found to be under regu-(0.2 mg/ml) gave the best results as to the reduclatory control and are susceptible to induction of tion of class II MHC antigen expression by IFN-7 class II antigens by IFN-7 in vitro and in and 5-azaC. W7 is a CaM antagonist that binds ViVO. 8'17'18 Trophoblasts in the inner placental CaM and inhibits calcium ion-CaM regulated layer (labyrinthine trophoblast zone)can only be activities. In the cellular systems used by Ina et induced to express these antigens upon stimula-aL5, dose-response experiments (10, 20, 30btM) tion with the demethylating agent 5-azaC in vitro showed that the best working conditions are as well as in vivo. 8'9 In both systems studied, obtained with 10 and 20 l.tM of W7. For the class II antigen induction is preceded by the acti-present system, W7 (Sigma) was used in vitro at vation of p21ras. 2 the concentration of 15 l.tM, which gave the best In the present communication, a possible responses. signal transduction pathway is proposed, trig-Sphingosine (SPH) and staurosporine (St) are gered by IFN-7 and 5-azaC leading to class II potent inhibitors of the PKC pathway, which are antigen and p21 ras expression on placental known to inhibit the activity of PKC. SPH has adherent cells. It is shown that class II antigen been used in several cell lines to block the class induction by both agents requires activation of II antigen induction by IFN-7, including human Cai+/CaM, PKC and p21r Intracellular signal-THP-1, murine WEHI-3, bone marrow macroling begins with p21 ras activation since only MEV phages, where dose-response experiments (1, inhibits induction of p21 ras and class II antigen 10, 50tM) have shown that 501.tM offers 68 expression. This step is followed by Ca2+/CaM maximal inhibition.' In the present work 50btM and PKC activation, whereas the pathway of tyro-and 25 btM of SPH (Sigma) were applied to the sine kinase (TK) is not involved in this process, adherent placental cells and it was found that the The importance of these results lies in in vivo best reduction of class II antigen expression experiments where an attempt was made to induced by IFN-7 and 5-azaC is obtained at the reverse the IFN-7-and 5-azaC-induced harmful concentration of 501M (151g/ml). St inhibits the effects by injecting the treated pregnant mice phospholipid calcium ion dependent protein with specific pathway inhibitors. The results kinase C. This product was purchased from UBI show that appropriate treatments can rescue fetal (Lake Placid, NY) and showed the best inhibitory effect on the induction of class II antigen expression by IFN-7 and 5-azaC at the concentration of 50nM, which is in agreement with the dose-res_.ponse experiments performed by Gumina et a12 Mevalonic acid (MEV) is an intermediate product in cholesterol synthesis and it is claimed to interfere with the G protein pathway through HMG-CoA and farnesylation of cysteins. 12 MEV was purchased from Sigma and tested at the concentrations of 5 mM (0.65 mg/ml) and 50 mM (6.5 mg/ml). Since both concentrations effectively inhibited class II and p21 ras expression induced by IFN-7 and 5-azaC with no apparent toxic effects, the lowest concentration was used. Genistein (Gen, 4',5,7-trihydroxyisoflavone), a potent inhibitor of tyrosine-specific protein kinase (TK)9, was purchased from UBI and tested at concentrations of 10, 30 and 50 l.tg/ml. None of these concentrations was able to block the class II or p21 ras antigen expression induced by IFN-7 and 5-azaC. However, this drug when applied to a tyrosine kinase dependent system (T98G human glioblastoma) shows its inhibitory activity even at the dose of 10 l.tg/ml. Antibodies: Rat anti-p21 ra neutralizing monoclonal antibody (Ab-1, Oncogene Sc. Inc. Mand k d hasset, NY), mouse anti-IA or IA or K or K k monoclonal antibodies (Becton Dickinson, Meshelen, Belgium), mouse monoclonal anticytokeratin antibody (PKK1, Labsystems, Helsinki, Finland), mouse monoclonal anti-vimentin antibody (PK-V, Labsystems) and rat anti-mouse Mac-1 antibody (Caltag Lab, San Francisco, CA) were used at a concentration of 1 l.tg/ml for in vitro detection of the equivalent proteins. Anti-mouse or anti-rat IgG coupled to FITC antibodies (Sigma) were used at a dilution of 1/100 and 1/150, respectively. Mouse IgG or rat IgG were used as negative controls to the above antibodies.  (Fig. I(A)).
Although both inhibitors block the Cai+/CaM pathway, their action is localized at different levels (see Methods), which may explain the difference in the degree of inhibition. These results indicate that class II antigen induction by IFN-/ requires mobilization of Ca 2+ (see Discussion).
In order to assess intracellular cross-talk with other pathways, placental cell cultures were treated with SPH and staurosporine, two potent inhibitors of PKC activation. These agents caused 82 and 71% inhibition (p< 0.001) of class II expression, respectively, as compared with IFN-7induced cells (Fig. I(B)). Thus, PKC is also involved in the IFN-7 signal transduction pathway leading to class II antigen expression.
It has been previously reported that the expression of p21 ras is stimulated in the IFN-2treated trophoblast cells. 2 In order to further test whether intracellular cross-talk with the Gprotein pathway is necessary to IFN-7-induced class II antigen expression, MEV was applied to the adherent placental cells. The application of this agent completely suppressed class II antigen induction, giving only background staining of the cells (Fig. 1(C)). MEV was unable to suppress IFN-7-induced expression of class II antigens on human HL-60 and HeLa cells as well as murine WEHI-3 and 70Z cell lines (data not shown). Finally, the involvement of TK in ,his system was excluded, since application of genistein to the cultures did not have any effect on class II antigen expression (Fig. I(D)).
Following the same reasoning and experimental protocols, the signal transduction pathway PK( Pathway TK Pathway riced and the weight of placenta, ferns and maternal spleen as well as the percentage of fetal abortion was recorded: TPH and SPH, when administered during the IFN-( treatment, reduce fetal abortion to normal levels, whereas IFN-g treatment alone increases the percentage of fetal loss 3-fold, as compared with untreated controls (Table 1). However, only TPH significantly, but not completely, corrects the effect of IFN-/ in fetal and spleen weight (Table 1). Thus, TPH causes an 88% increase in fetal weight (p< 0.001), and a 46% reduction in spleen weight (p< 0.005), as compared with IFN-g treatment alone. SPH gives a 38% reduction (p < 0.005) in spleen weight but it does not significantl increase fetal size (Table 1). Placental weight is not affected in either case. While testing the toxicity of TPH and SPH during pregnancy, control experiments show that the doses used do not affect fetal viability or maternal spleen weight, but reduce fetal weight non-significantly.
It was then attempted to confirm, in an additional way, the above described results by inject- (p< 0.005), whereas the percentage of fetal leading to p21s antigen expression after IFNq, abortion returns to physiological levels ( Table 1). administration was studied. The results show that The combination IFNq, + anti-Ia reduced fetal only mevalonic acid lactone completely ablated abortion to normal levels and increased placental p21 ras expression, whereas all the other inhibi-and fetal weight by 43% and 100% respectively, tors did not significantly affect this expression as compared with IFNq,-treated pregnant mice. (Fig. 2). These results indicate that p21 ras is an However, this treatment did not significantly intermediate product to the class II antigen reduce the maternal spleen weight ( In vitro and in vivo study of signal transduction In vivo study of signal transduction pathways pathways leading to class II and p21 ras expresleading to class II and p2I ras expression by IFNsion by 5-azaC: It is known that the demethylat-2: It has been shown previously by this laboraing agent 5-azaC induces class II and p21 ras tory that administration of IFNq, to pregnant antigen expression on trophoblasts derived from mice from day 6 to 11 of gestation increases fetal the labyrinthine trophoblast zone of the plaabortion, reduces fetal size and, among other centa. 1'2 It is shown that class II antigen expresdysfunctions obseeeed at the maternal level, it sion is affected by the same inhibitors as for the increases the size of the spleen approximately'3-IFNq,-induced expression of these antigens. fold. These IFNq,-induced events were asso-Specifically, TPH and W7 reduce the 5-azaCciated with the expression of class II MHC anti-included class II antigen expression by 54% and gens as well as the p21 a protein on the 38% respectively (p< 0.005), as compared with spongiotrophoblast layer of the placenta. 5-azaC treated cells (Fig. 3(A)). Furthermore, In order to test whether the in vitro results SPH and St inhibit the expression of these antidescribed above can also be applied in vivo, gens by 75% and 97% (p< 0.001) respectively pregnant mice were injected with IFN-, and inhi- (Fig. 3(B)), whereas the application of MEV gives bitors (doses and timing given in Methods). On 100% inhibition of Ia antigen expression the I2th day of pregnancy, the mice were sacri- (Fig. 3(C)). The TK pathway does not seem to *Pregnant mice were treated as described in Methods. Briefly, IFN-7 (5 000 U/injection) was administered i.p. to the animals from day 6 to 11 of gestation. TPH (0.15 mg/injection) was given i.p. either alone or with IFN-7 on days 6 and 10 of pregnancy. SPH (15 Ig/ injection) was given i.p., alone or with IFN-y on days 6, 8 and 10 of pregnancy. Anti-p21 (51g/injection) was administered i.p., either alone or with IFN-y on days 6, 7, 9 and 10 of gestation. Finally, anti-class II monoclonal antibody (anti-lAd, 50 Ig/injection) was administered i.v., either alone or with IFN-y on days 8 and 10 of gestation, p values are given in the text.
**Resorbed sites were not included in the means. be involved in this system, since genistein does not affect the 5-azaC-induced expression of these antigens on placental cells (Fig. 3(D)). All these inhibitors were used at the same doses as those described for IFN-(. Application of these inhibitors in order to examine changes at the p21 ras expression levels, showed that only MEV could block the appear-ance of the p21 ra= proteins (Fig. 4). Administration of 101.tg of 5-azaC to pregnant mice i.p. from days 6 to 11 of gestation, results either in complete fetal abortion or in very high percentages of fetal loss, where the surviving embryos 9 ed (10 15%) have abnormal development. Bas on the in vitro results described above, the reversal of 5-azaC-induced events by TPH or *Pregnant mice were treated as described in Methods. Briefly, 5-azaC (10 lg/injection) was administered i.p. to the animals from day 6 to 11 of gestation. TPH (0.15 mg/injection) was given i.p., either alone or with 5-azaC on days 6 and 10 of pregnancy. SPH (15 Ig/ injection) was given i.p., either alone or with 5-azaC on days 6, 8 and 10 of pregnancy. Anti-p21 (5 Ig/injection) was administered i.p., either alone or with 5-azaC on days 6, 7, 9 and 10 of gestation. Finally, anti-class II monoclonal antibody (anti-lAd, 50 lg/injection) was administered i.v., either alone or with 5-azaC on days 8 and 10 of gestation, p values are given in the text. **Resorbed sites were not included in the means.
SPH, or anti-p21 ras or anti-class II monoclonal antibodies, was attempted. All treatments rescued fetal abortion induced by 5-azaC alone and increased placental size to normal ( Table 2).
Fetal size was not significantly different from that in the untreated animals and spleen weight was not affected in any case (data not shown).

Discussion
When a physiological or chemical agent comes in contact with the surface of a cell, second messenger signals are transmitted not only to downstream functional proteins, but also to upstream compounds and members of other signal transduction pathways in order to give this cell specific instructions about its function(s) and phenotype. The major event studied here is the induction of class II MHC antigens by IFN-, on the placenta. Although the mechanisms of induction of these antigens by IFN-, have been examined in many systems, including different cell types of different species, my interest in trophoblasts comes from the biological significance of this mechanism and the role it may play in escaping fetal abortion and ameliorating conditions of fetal survival. The results show that at least three pathways are involved in the signal transduction mechanism of class II antigen expression on 2+ trophoblasts, including Ca mobilization, PKC and p21 ras activation. In cases such as pregnancy, graft implantation and autoimmune disorders, the expression of class II antigens has harmful effects on the organism and such activation has to be depressed. This is in contrast with systems studied by other investigators, where MHC class II induction benefits the organism by stimulating the immune system. Thus, after using adherent placental cells, the in vitro findings concerning inhibition of class II antigens via the three described pathways were transferred to in vivo experiments. It was shown that the inhibitors which block the induction of these antigens protect the fetus from abortion and ameliorate the conditions of fetal development.
In this study specific inhibitors of four different pathways were employed: TPH and W7 to suppress the Cai+/CaM system; SPH and staurosporine to inhibit PKC activation; mevalonic acid to block G-proteins (ras); and genistein to inhibit the TK system. All but genistein were able to block class II antigen expression induced by IFN-T, on adherent placental cells.
It has been shown previously that the induction of class II antigens on trophoblasts is closely linked to the cellular ras oncogene, which was also found to be stimulated by the IFN-, treatment on the same cells, i Using mevalonic acid lactone as an inhibitor of p21 ras, and consequently of G-proteins, it was possible to block the class II antigen induction by IFN-T. Results indicated that p21 ras activation occurs upstream to the stimulation of class II antigens.
In order to place p21 rs activation within the signal transduction pathway of IFN-T and/or examine possible degrees of intracellular crosstalk, the inhibitors to Cai+/CaM, PKC and TK were tested for their ability to block the expression of p21 rs. With the exception of mevalonate, none of the inhibitors was able to inhibit such induction, indicating that p21 ras activation is the first step in the signal transduction pathway leading to class II antigen expression.
It has been reported from this and other laboratories that administration of IFN-, to pregnant mice affects feto-placental development as well as maternal physiology. 18'2'22 In view of the present in vitro results, I attempted to block the in vivo IFNq,-induced side effects by specific pathway inhibitors (TPH and SPH), anti-class II or anti-p21 ras antibodies. All these treatments rescued the IFN-,-induced abortion, TPH and anti-p21 ras being the most efficient.
In the last section of this work, I concentrated on the mechanisms inducing class II antigen expression on the labyrinthine trophoblast after 5-azaC treatment. It is demonstrated that the same inhibitors blocking the IFN-'f-induced class II and p21 as expression, also inhibit the 5-azaCinduced stimulation, both in vitro and in vivo.
It is thus proposed that the sequence of events leading to class II expression and ultimately fetal rejection after IFN-7 or 5-azaC administration begins with an initial p21 ras activation followed by either Ca 2+ mobilization and PKC activation or vice versa. This does not exclude the possibility that the two agents may follow or share other means of propagating their signals leading, however, to the same endpoint. In vivo application of pathway inhibitors is able to reverse the side effects not only at the feto-placental but also the maternal level. It is premature to speculate that the use of TPH, anti-p21 ras SPH and to a lesser degree anti-class II antibodies, can be used as therapeutic agents. However, further experimentation may prove that administration of these or analogue reagents may have a potential role in the future. It is clear that aberrant expression of class II antigens on the murine placenta lead to fetal rejection. '8'2 These findings can be generalized to the role of IFNq,-mediated class II upregulation in the induction of immune responses, graft rejection or autoimmune diseases. Although in the present study the class II inducers were given exogenously, multiple mechanisms including viral or microbial infection, cytokine overproduction, and hormonal disequilibrium, may lead to the production of IFN-/ and/or other inflammatory cytokines with synergistic potential like TNFz, IL-6, IL-1 and IL-12. All possibilities are valid until definitive proof can be provided, a task that is being undertaken at this laboratory.