Research Paper Mediators of Inflammation, 7, 69–72 (1998)

Corresponding Author Tel: (+30) 081 394 355 Fax: (+30) 081 243 656 Email: simon@biology.uch.gr DISTURBANCE of th e cytokine equilibrium has been accused for m any pathological disorders . Microbial in fections, autoim m une disease s , graft r e jection have been correlated to over or under-production of specific cytokine s wh ich are produced as re sponder m olecules to the various im m une stim uli. Th e sole naturally occurring im m une reaction in th e organ is m is deve loped durin g the ge stational per iod where, de spite the pre sence of a sem i-allogeneic graft, m ater nal im m unoreactiv ity is driven to support fe tal grow th . Th e successful em bryo developm ent has been attributed to th e im portant in tervention of cytokines where som e have been characte rized as in dispensable and others de leterious to fe tal grow th. How ever, th e physiological levels of m any factors during th e gestational process have not be en determ in ed. Th us, in th e present s tudy we have m easured and established the values of IL-1 a , IL-2, IL-3, IL-4, IL6, IL-10, IL-12, GM-CSF, TNF-a and IFN-g dur in g all phases of hum an pregnancy (firs t, second and th ird trim ester of pregnancy, labour, abortions of the firs t trim ester ) as w ell as in the non-pregn an t con trol state. This is an attem pt to assess se rum protein concentrations and present th e physio logical leve ls of th ese cytokines at ce rtain tim e intervals providing thus a diagn ostic advantage in pregnancy cases w here the m other cannot im m unologically support the fe tus . Ex ploitation of th is knowledge and further research m ay be use ful for the rapeutic in te rven tions in the future.


Introduction
Despite the unique ability of the immune system to highly specific antigen recognition, its susceptibility to cytokines allows these molecules to dominate all kinds of immune reactions. These proteins, produced in an autocrine or paracrine fashion, bind to specific receptors initiating thus a cascade of reactions on different targets having beneficial or harmful effects since their redundancy and/or pleiotropic nature may account for all possible reactions. With T helper (Th) lymphocytes being the major producers of cytokines, it is believed that the equilibrated balance of Th1 versus Th2 cytokines defines the welfare of the organism. 1,2 The immune system, in order to ensure protection from microbial infections, autoimmune reactions, graft rejection, allergies, etc., leans the balance towards one or the other family of cytokines.
Pregnancy is a natural example of an immune reaction occurring for a determined time period in the organism w hich opposes the rules of graft rejection. The semi-or allogeneic fetal components growing in the privileged site of uterus, not only escape maternal immune attack but are supported by the maternal immune system. An important role in the maintenance of pregnancy is played by cytokines, where successful fetal outcome is correlated to the production of Th2 cytokines and fetal rejection to Th1 cytokines. Although the protective role of the Th2 cytokine IL-10 during mid-gestation, 3 as well as the harmful effects of Th1 cytokines IL-2, 4 TNF-a 5-7 and IFN-g 5,8 have been demonstrated in mice, other studies show that each cytokine follows a specific pattern of expression each day of pregnancy in mice. 9 These observations indicate that despite the beneficial or harmful effect of a cytokine during a specific time course of the gestational cycle, a determined upor downregulation of these factors must be followed, pattern that will decide for the successful or not outcome of pregnancy. It becomes thus mandatory to define the physiological levels of cytokines during human pregnancy, w hich may finally have a prognostic character for the outcome of pregnancy.
In the present report, we examined the serum levels of 10 cytokines during the first, second and third trimesters of pregnancy, before pregnancy and labour and compared the values of physiological first trimester of pregnancy with cases of fetal rejection.

Patients
After informed consent of all patients, we analysed the serum of 15 non-pregnant-state women, the serum of 19, 17 and 18 first, second and third trimester women respectively, the serum of 15 women in labour and 19 women who underwent spontaneous abortion. All sera, isolated from 5 ml peripheral blood, were provided by the Division of Gynaecology of the University Hospital (Heraklion, Crete).

Reagents and cytokine determination
Cytokine detection in the serum was achieved by commercial kits purchased from Endogen (Cambridge, MA, USA; IL-1a , IL-2, IL-4, IL-6, IL-10, GM-CSF, TNF-a , IFN-g ) and R&D systems (Oxfordshine, UK; IL-3, IL-12). Serum cytokine measurement was performed using an ELISA method as specified by the suppliers at test and reference wavelengths of 450 and 550 nm respectively. The results are expressed in ng/ml and were compared with standard curves obtained from titrations of the corresponding recombinant factors provided by the suppliers. The Endogen ELISA kits for IL-1a , IL-4, GM-CSF and IFN-g detect <2 pg/ml of the equivalent protein, w hereas the IL-2 kit detects <6 pg/ ml, IL-6 <1 pg/ml, IL-10 <3 pg/ml and TNF-a <5 pg/ml. The R&D Systems ELISA kits for IL-3 and IL-12 detect 7.4 and 5 pg/ml of protein respectively. In all cases the above values given for the lower limit of detection is the smallest dose of protein that is not zero with 95% confidence. Both manufacturers guarantee the specificity of each individual kit.

Statistical analysis
Since the abortions studied occurred during the first trimester of pregnancy, the Student's t-test was employed in order to compare the significance levels (P) betw een the abortion values of the different cytokines and those of the actual first trimester of gestation. In this particular group of analysis all P values equal or lower than 0.025 are considered to be non-significant. Degrees of freedom: 18 (n-1).

Results and Discussion
Cytokine levels in the serum seem to reflect the pathologic state of the organism and may, in many cases, have a prognostic character for therapy subscription. In this study, concentrating our interest to human pregnancy w here the rates of fetal rejection dangerously increase, we define the physiological levels of 10 different cytokines before, during and after pregnancy.

Assessment of cytokine levels during pregnancy and in the non-pregnant state
The goal of this work was to determine the actual (physiological) levels of diverse cytokines during pregnancy. For this, serum from non-pregnant women as well as during the first, second and third trimester of pregnancy and labour was collected and analysed by ELISA as to the cytokine content. In addition, serum from women w ho underwent abortion during the first trimester of their pregnancy was also examined and the same cytokines were evaluated. cytokines studied. In the course of this analysis we detected statistically significant (P < 0.05 to P < 0.001) differences betw een the non-pregnant state and pregnancy for IL-1a (drop in second trimester by 13%), IL-2 (decrease mainly at labour by 11%), IL-3 (decrease at second and third trimester by 14%), IL-10 (increase at labour by 28%), GM-CSF (increase at third trimester and labour by 16% and 30% respectively) and IFN-g (increase at third trimester and labour by 25% and 26% respectively). By the same token, significant differences w ithin the different pregnancy stages are obtained by IL-1a (minimal production in the second trimester), IL-2 (decrease in third trimester and labour), IL-3 (decrease after the first trimester), IL-6 (increase after the first trimester), IL-10 (increase during labour), GM-CSF and IFN-g (steady increase after the first trimester).
TNF-a , accused for pregnancy failure, 5-7 presents a stable production profile in all stages. The same is also shown for IL-12, a factor that has been reported as an immunomodulator in various mechanisms during pregnancy. 10 -12 Finally, IL-10 and IL-4, named as protective agents during pregnancy, 3,13 show a constant presence at the first tw o trimesters with IL-10 showing a peak of production during labour (as stated above). These four cytokines show a minimal presence in the serum of pregnant or non-pregnant women at levels ranging from 0.88 to 0.14 ng/ml, implying a possible maintenance role during the gestation period.

Assessment of cytokine levels in the first trimester abortions
The serum from women who aborted during their first trimester was also examined for the same cytokine content and the corresponding values were determined. Table 2 shows not only the cytokine values of aborted women but also a statistical comparison w ith the actual values (taken from Table 1) of women in their first trimester of continuing pregnancy. It is shown that although there are no differences for IL-4, IL-12 and TNF-a (we consider P £ 0.025 as not significant, as stated in the Methods), the situation with the remaining cytokines is totally different. Whilst only the IL-1a and IL-3 levels drop during abortion (IL-1a not significantly by 8% or P < 0.3 but w ith a considerable 17% decrease in IL-3), on the contrary, IL-2, IL-6, IL-10, GM-CSF and IFN-g levels increase when abortion occurs with IFN-g being more obvious in magnitude (53% of increase). Although it is not clear yet whether increase in cytokine production is responsible for the abortion process or it is the abortion itself that modulates cytokine levels, this latter observation gives an insight to and supports the Th1 versus Th2 situation in pregnancy. It is seen here that Th1 cytokine levels are augmented during abortion whereas, Th2 are not significantly different or dropping. Another point that merits attention is the IFN-g increased levels during abortion. This piece of data confirms previous reports and strengthens the correlation between this agent and fetal rejection. 7 The results presented here do not differ from the recently published report by Tranchot-Diallo et al. 14 where modulation of cytokine gene expression (TNFa , IFN-g , GM-CSF, IL-1b , IL-2, IL-4, IL-6 and IL-10) by RT-PCR in pregnancy was evaluated.