Dr. Ivan bubanovic

reproductive immunology and tumor immunology

 

Medica Centre, Novosadska 1/c, Nis, Serbia

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IMMUNOSTIMULATORY EFFECTS OF INDOMETHACINE ON ALLOGENEIC AND SYNGENEIC PREGNANCY

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SUMMARY

Factors of making and keeping immunotolerance in pregnancy mainly affect immunocompetent cells by immunosuppressive factors or moving of Th1/Th2 balance in favour of Th2 response.  Errors made in making and keeping immunotolerance in pregnancy can lead to clinic entities such as resorption of fetoplacental unit, miscarriage and reduced placental and fetal weight. Besides, production of sex steroids in the yellow body of gravidity and later in the fetoplacental unit depends on the factors such as prostaglandins and cytokines. In our experiment the significance of paternal and maternal MHC (in)compatibility of rats on the number of embryos, percentage of embryos in resorption, placental and fetal weight and the level of serum steroids were tested. A specimen for this investigation syngeneic Sprague Doley (SD) and Wistar (W) were taken. The rats of SD and W lineage were treated by indomethacine of 2.1-2.8 mg/kg daily by giving them drinking water in the period of 6-18 day of pregnancy. Stimulation of Th1 response by indomethacine in allogeneic pregnancy led to resorption of 56% embryos while the rate of resorption in the control group of W rats was 12%. Syngeneic SD animals treated by indomethacine did not show a significantly larger rate of embryos in resorption (8.14%) than those ones in the control group (5.13%) Acceleration of Th1 response by indomethacine led to activation of decidual CTL and NK cells as well as to intensified expression of MHC antigens on the cells of trophoblast. In allogeneic pregnancy this resulted in the increased resorption of embryos, while in syngeneic pregnancy acceleration of Th1 response did not result in increased resorption of embryos. Besides, indomethacine leads to the significant fall of the level of serum progesterone and estradiol no matter the MHC compatibility of pregnancy However, the level of serum progesterone at W rats treated by indomethacine is significantly lower than at SD rats also treated by indomethacine as well. This result shows the possibility of greater damage of trophoblast at MHC incompatible pregnancies under the influence of indomethacine or greater activities of cytokine of Th1 group, which inhibits synthesis of sex steroids.

Key words: Pregnancy, indomethacine, progesterone, estradiol, prostaglandine, immunomodulation, Th1 and Th2 response.

INTRODUCTION

From its early days the embryo shows a good expression of MHC antigens class II and I and from the point of view of MHC incompatibility, it can be defined as allogeneic transplant [1,2]. Placenta has embryonic origin and it is directly connected with maternal immunocompetent cells, but the character of expression of MHC antigens on the placental tissues is selective, restrictive and conditional. Varied limitations at the expression of MHC antigens impede the definition of placenta as allogeneic transplant [1,2]. Fetoplacental unit under certain conditions can become a target of aggression by maternal immune system. As a counterpoise to the potential immunodestructive mechanisms, the whole series of immunoprotective mechanisms of pregnancy immunotolerance have been developed [3]. One of the main factors of making and keeping immunotolerance in pregnancy is prostaglandins activity of decidua and trophoblast. Placenta mainly synthesizes immunomodulatory PGE2 which major role is suppression of immunocompetent cells over the inhibition of secretion IL-2 and the inhibition of expression of receptors for IL-2 [2,3]. The whole series of research by Lala et al. shows in the best way the role of prostaglandins in pregnancy. These authors got the high percentage of resorption fetoplacental unit and miscarriages, which raised even to 89% in experiment based on chronically treatment of allogravid female rats by indomethacine or IL-2. The authors explained this phenomenon by immune mechanisms, which were mediated by rejection of fetoplacental unit as allogeneic transplant [3]. In the researches that appeared later the same group of authors confirmed that for the fetoplacental unit resorption stimulated by indomethacine or IL-2 are mainly responsible decidual NK cells, which are otherwise suppressed by decidual prostaglandins and Th2 cytokines. Decidual NK cells show a very low rate of activity to YAC-1 lymphoma cells. Decidual NK cells from allogeneic pregnant animals treated by indomethacine or IL-2 show a high degree of anti-YAC-1 activity [3,4]. In reference to the importance IL-10 compared by the process of marring and keeping immunotolerance in pregnancy, it is significant to mention that PGE2 fairly increases concentration IL-10 and IL-8 in the maternal serum and decidual compartment [3,4]. One the other hand, pregnancy is the condition where a great concentration of serum sex steroids occurs. This is the consequence of increased synthesis of these hormones, first in the yellow body of pregnancy and later in placenta or more precisely in the fetoplacental unit [5]. Hypotalamic-hypophysis hormones and chorionic gonadotropins, there are other mediators that have influence on the intensity of biosynthesis of sex steroids, first of all those which have influence on the level of cAMP, such as prostaglandins and histamine [1,2,6]. Indomethacine efficiently suppresses luteolytic characteristics of prostaglandins so that it is proved that the yellow body persists, even at non-pregnant animals, until there is a significant suppression of synthesis of prostaglandins by indomethacine [7,8]. As for ovarian steroidogenesis it is more significant lipooxigenic way of metabolism of arahydonic acid than cyclooxygenic way. Inhibition of lipooxygenic way results in decrease of intensity of ovarian steroidogenesis, while the inhibition of cyclooxygenase (COX) by indomethacine except anti-luteolytic effects has no any significant effect on ovarian steroidogenesis [1,9]. The other mechanisms of including prostaglandins in regulation biosynthesis of sex steroids in more significant for pregnancy and is based on stimulation of adneil-cyclase and the increase of concentration of cAMP in trophoblast cells, that results in intensifying of the process of basic steroidogenesis and synthesis of all steroid hormones. Placenta is place of very intensive synthesis of prostaglandins that by all means take part in the stimulation of the process of biosynthesis of placental sex steroids. Sendrani et al. affirm that PGE2 stimulates synthesis of all steroid hormones [2,7,9]. Treatment of female rabbits by indomethacine in the early pregnancy makes a significant slow down of steroidogenesis and decrease of the level of serum progesterone and estradiol. Parallel treatment of female rabbits by indomethacine and PGE2 return the level of all products of steroidogenesis to the physiological level [7,9]. Cytokines are very often mentioned as factors that can have influence on the level of sex steroids, and as well as on ovarian and placental production of these hormones. Ohno et al. in their investigations in vitro got the suppression of production of progesterone from granular cells cultivated with IL-2, while in their research IL-1 did not show such effects. They came to conclusion that the inhibition of production of progesterone depends on the dosage related to concentrations IL-2 in medium [2,10,11]. In contrast to granular cells, trophoblast cells intensify steroidogenesis when IL-1 stimulates them. This cytokine stimulates aromatase of steroidogenetic enzymatic system of trophoblast cells and in that way intensified secretion of progesterone and estrogenic hormones. This is explained by intensified production of prostaglandins in cultured trophoblast cells stimulated by IL-1. By adding anti-IL-1 antibody, the level of progesterone and estrogenic hormones to the level of control is given back. The effects of IL-2 on trophoblast cells and their steroidogentic potential are negative. It is proved that IL-2 inhibits product on of sex steroids [2,10]. One of the aims of this research was to establish the significance of MHC incompatibility on the number of embryos, the percentage of embryos in resorption, placental and fetal weight. As the aim of research is also set the observation of these parameters in the conditions of acceleration Th1 response by indomethacine in allogeneic and syngeneic pregnancy and establishing of effects of indomethacine on synthesis of sex steroids in pregnancy. As the aim of research is also set a comparison of effects of indomethacine as an accelerator of Th1 type immune response on synthesis of sex steroids in allogeneic and syngeneic pregnancy.

MATERIALS AND METHODS

The animal have been divided into 4 groups, two of them control ones for W and SD rats and two experimental groups of W and SD rats, which have been treated by indomethacine. Animals for experiment have been female, 12-16 week old and weighed 190±10 g. Female rats from all four groups have been mated in harem. Indomethacine has been included in the drinking water to the experimental groups of W and SD rats from 6th day of pregnancy. Regarding the fact that rat of 200 g weight drinks 30-40 ml water daily, concentration of indomethacine is adapted to 14 mg/ml, so that a daily dosage of indomethacine, which animals consumed, was 2.1-2.8 mg/kg. The animals were sacrificed on the 18th day of pregnancy in Nesdonal anaesthesia by heart puncture. The blood got by heart was kept at room temperature for 2 hours in order to form a blood cake and to separate serum. After that it was centrifuged at 3000 rpm in the period of 5 minutes. This serum was taken by a automatic pipette and put aside at -20^o C to the moment of further treatment. The level of serum progesterone and estradiol has been determined by RIA methods on the RIA System-Junior F-520, by means of original kits of monoclonal antibodies (INEP-Zemun).

 RESULTS

The results of this research show that the number of embryos in allogeneic pregnancy is significantly larger (p<0.05) than in syngeneic pregnancy, while the treatment by indomethacine did not significantly have any influence on the total number of embryos of SD and W rats (table 1). The percentage of embryos in resorption of allogravid female rats treated by indomethacine is significantly larger (p<0.05) than the percentage and the number of embryos in resorption of untreated allogravid animals and syngeneic pregnant rats treated by indomethacine (table 2, graph 1).

Table 1. Average values of the number of embryos found in the uterus of gravid rats.

	Sprague Doley rats		Wistar rats
	Control (n = 8)	Treated by indomethacine (n = 9)	Control (n = 8)	Treated by indomethacine (n = 9
Altogether	78	86	104	109
Average	9.75	9.56	13.00	12.11
St.Dev.	0.85	1.24	2.56	1.96

 

 

 

 

 

 

 

 

 

Table 2. Average values of the number of embryos in resorption found in the uterus of gravid rats.

	Sprague Doley rats		Wistar rats
	Control (n = 8)	Treated by indomethacine (n = 9)	Control (n = 8)	Treated by indomethacine (n = 9)
Altogether	4	7	13	61
Average	0.5	0.78	1.63	6.78
St.Dev.	0.53	0.67	1.19	2.11

 

 

 

 

 

 

 

 

The values of serum progesterone in allogeneic pregnancy do not significantly differ (p<0.05) from the values of serum progesterone of syngeneic pregnancy. Treatment by indomethacine had a significant influence (p<0.05) on the little value of serum progesterone in allogeneic and syngeneic pregnancy. Recorded fall of concentration of serum progesterone under the influence of indomethacine in allogeneic pregnancy is more significant (p<0.05) than the one in syngeneic pregnancy (graph 2). The values of serum estradiol in allogeneic pregnancy do not significantly differ (p<0.05) from the values of serum estradiol of syngeneic pregnancy. Treatment by indomethacine had a significant influence (p<0.05) on the fall of value of serum estradiol in allogeneic and syngeneic pregnancy (graph 2).

% Of vital embryos

% Of embryos in resorption

 

 

 

Graph 1. Percentage of the vital embryos and the embryos in resorption at all groups of rats. 

Level of serum progesterone

Level of serum estradiol

 

 

 

Graph 2. The level of serum progesterone and estradiol at al four groups of rats.

DISCUSSION

Result related to comparison of number of embryos in allogeneic and syngeneic pregnancy are in accordance with some citations from literature that alloimmunization can be factor that will contribute to a larger number of descendants [12]. There is opinion that activation of Th2 response in allogeneic pregnancy contributes to a more successful implantation and keeping a large number of embryos in the conditions of multifetal pregnancy [13]. Mechanisms that induced a significantly rate of embryos in resorption at allogravid animals treated by indomethacine are probably related to pushing out of synthesis PGE2. Inhibition of placental production of prostaglandin induces activation Th1 response [2,3]. As a result of the intensified Th1 response, an intensified secretion of cytokine such as IL-2, TNF, and IFN-g is induced. These cytokines contribute to the intensified resorption of fetoplacental unit activating decidual NK cells and Tc cells. Above mentioned cytokines stimulate the expression of MHC antigens on the trophoblast cells and in that way is emphasized the immune reaction focused against fetoplacental unit [1,13]. Lala et al. got in parenteral treatment by indomethacine of allogravid mice the percentage of embryos in resorption of 89%, and they got the same percentage of embryos in resorption in the groups of mice treated by IL-2. This group of authors affirms that NK cells are the main effector cells in the process of fetoplacental resorption mediated by immune mechanisms [3,4]. For the phenomenon of fetoplacental unit resorption mediated by immune mechanisms two prerequisites are necessary and they are: (i) the presence of paternal MHC alloantigens and (ii) acceleration of Th1 responses. One fact shows us that there is not statistically significant difference in the percentage of embryos in resorption of control group of SD rats and in the percentage of embryos in resorption of the group of SD rats treated by indomethacine. Namely, besides the treatment by indomethacine of gravid syngeneic SD rats and factor of acceleration of Th1 response it did not happen to come to the significant increase in the percentage of resorption of embryos, and that is so because of compatibility of paternal and maternal MHC phenotypes of the trophoblast cells (table 1,2 and graph 1). The fact that there is no statistically significant difference in the resorption between control groups W and SD rats, shown that in the conditions of normal pregnancy when there is balance of Th1 and Th2 response, MHC incompatibility of allogeneic pregnancy of W rats does not have any significance for developing the phenomenon of fetoplacental unit resorption. Judging by the percentage of fetal resorption, it can be said that allogeneic and syngeneic pregnant animals in the condition of balance Th1 and Th2 response behave in the same way. This proves that trophoblast show a very low rate of expression of MHC antigens, but only to the moment of imbalance between Th1 and Th2 responses in favour of Th1 response [1,2]. The results of our research show that MHC incompatibility in allogene pregnancy has no any significance for the synthesis of progesterone and its serum level. We come to this conclusion by observation that at syngeneic pregnant animals treated by indomethacine, the level of serum progesterone is significantly lower than at non-treated syngeneic animals and this decrease of the level of progesterone is proportionate to decrease of the level of progesterone at treated out bred animals. Prostaglandins have the role to regulate the biosynthesis of sex steroids, which is included in the stimulation of enzyme of adenilcylase, so that by the increase of concentration of cAMP, they active proteinkinase and the process of phosphorylation of ester-cholesterols, and as consequence of that intensifying of the process of basic steroidogenesis and synthesis of all steroid hormones [1,2,15]. The inhibition of synthesis of prostaglandins by indomethacine could be responsible for the decrease of steroidogenic potential of trophoblast and for the decrease of value of concentration of serum progesterone, as well as in allogeneic and syngeneic pregnancy. The level of serum progesterone of allogravid animals treated by indomethacine is significantly lower than the level of serum progesterone of syngeneic animals treated by indomethacine as well, and that is so because of greater damage of placental tissue in relation to treated syngeneic animals, and that is why we come to conclusion that the significance of MHC incompatibility in terms of the level of serum progesterone can be emphasized in the conditions of immunostimulation of maternal immune system and probably intensified expression of MHC antigen on the trophoblast. This might show that MHC incompatibility itself is not of essential significance for some pregnancy parameters, while in the condition of immunostimulation and likely intensified expression of MHC antigens on the placental cells result in disordered homeostasis in pregnancy. It is well known that cytokines can have some influence on the level of sex steroids, and on the ovarian as well as on the placental production of sex steroids hormones [1,2,10]. The results related to the serum level of estradiol in allogeneic and syngeneic pregnancy, show the little importance of MHC incompatibility for the intensity of synthesis and level of this hormone in pregnancy. The mechanisms of the inhibitory effect of indomethacine on the synthesis of estrogens hormones in pregnancy are the same as if we talk about the relation of indomethacine and progesterone. It might be said that indomethacine generally inhibits all steroidogenetic process by the inhibition of prostaglandins synthesis and by the significant changes in network of cytokines, above all trophoblast-decidual union.

CONCLUSION

Based on the given data from literature and analyzing the given results, we could wake the following conclusions:
MHC incompatibility in allogene pregnancy is the factor that has a considerable influence on the total number of embryos.
The treatment by indomethacine can considerably increase the percentage of embryos in the resorption, but only in case of presence incompatibility between maternal and embryonic MHC phenotype, while in syngeneic pregnancy there is not a significant influence in the percentage of embryos in the resorption.
MHC incompatibility of allogeneic pregnancy does not have a considerable influence on the average values of serum progesterone.
Indomethacine considerably decreases the average values of serum progesterone regardless the fact pregnancy is allogeneic or syngeneic.
Indomethacine efficiently inhibits the synthesis of progesterone in allogene pregnancy that is probably related to MHC incompatibility and acceleration of Th1 response.
MHC incompatibility of allogene pregnancy does not have a considerable influence on average values of serum estradiol.
Indomethacine considerably decreases average values of serum estradiol regardless the fact pregnancy is allogeneic or syngeneic.
 

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