Yoneyama, Yoshio MD; Sawa, Rintaro MD; Suzuki, Shunji MD; Yoneyama, Koichi MD; Doi, Daisuke MD; Araki, Tsutomu MD
Department of Obstetrics and Gynecology, Nippon Medical School, Tokyo, Japan.
Address reprint requests to: Yoshio Yoneyama, MD, Nippon Medical School, Department of Obstetrics and Gynecology, 1–1‐5, Sendagi, Bunkyo‐ku, Tokyo, 113–8603, Japan; E‐mail: yoshi‐email@example.com.
Supported by a Grant‐in‐Aid (No. 12470349) from the Ministry of Education, Culture, Sports, Science and Technology.
Received January 30, 2002. Received in revised form April 11, 2002. Accepted May 2, 2002.
OBJECTIVE: To evaluate the relationship between plasma adenosine deaminase activity and the proportion of cytokine‐secreting T cells as causes of changes in adenosine deaminase activity in normal pregnancy.
METHODS: Plasma adenosine deaminase activity and the proportions of cytokine‐secreting T cells were measured in the peripheral blood of 26 nonpregnant and normal pregnant women in the third trimester. The proportion of CD4‐positive T cells secreting interferon‐γ derived from T helper 1 cells, and interleukin‐4 derived from T helper 2 cells, were analyzed by flow cytometry. The ratio of interferon‐γ–secreting cells to interleukin‐4–secreting cells was taken as the T helper 1/T helper 2 ratio.
RESULTS: Mean plasma adenosine deaminase activity in normal pregnant women, averaged, was significantly lower than that in nonpregnant women (10.3 ± 0.6 U/L versus 13.8 ± 0.5 U/L, P < .05). In normal pregnant women, the proportion of interferon‐γ–secreting cells was significantly lower than that in nonpregnant women (20.5% ± 1.0% versus 24.7% ± 1.2%, P < .05), but the proportion of interleukin‐4–secreting cells did not differ from that of nonpregnant women. Consequently, the T helper 1/T helper 2 ratios were significantly decreased during normal pregnancy. A significant correlation was found between adenosine deaminase activity and the proportion of interferon‐γ–secreting cells (r = .54, P < .05).
CONCLUSION: Decreased plasma adenosine deaminase activity in normal pregnant women may be in part associated with changes in the immunological status, especially the decrease of the proportion of interferon‐γ–secreting cells.
Adenosine deaminase catalyzes the hydrolytic deamination of adenosine and 2′‐deoxyadenosine to inosine and 2′‐deoxyinosine, respectively. Adenosine deaminase is ubiquitously distributed in human tissues and cells, which may contribute to the proliferation, maturation, and function of lymphoid cells.1 Plasma adenosine deaminase activity is changed in several autoimmune and inflammatory diseases in which the immunological status is changed.2,3 The major sources of plasma adenosine deaminase may be lymphocytes or the monocyte–macrophage cell system,3,4 and other immunological factors are also involved in the regulation of plasma adenosine deaminase activity; however, the exact mechanisms of the regulation of plasma adenosine deaminase activity have not been elucidated.
Cytokine‐secreting T cells play a central role in the immune response and have been classified into subsets based on their type of cytokine production. T helper 1 cells synthesize mainly interleukin‐2 and interferon‐γ, which induce cellular immunity. T helper 2 cells produce predominantly interleukin‐4, ‐5, ‐6, and ‐10, which promote humoral immunity.5 The shift of T helper 1/T helper 2 balance to T helper 2 predominance occurs in normal pregnancy and appears to protect the fetus and placenta from being rejected and to aid in the maintenance of normal pregnancy.6 Because normal pregnancy is characterized by depressed cell‐mediated immunity in conjunction with enhanced humoral immunity, plasma adenosine deaminase activity may be altered. Only a few studies have been performed regarding plasma adenosine deaminase activity during pregnancy; these demonstrated plasma adenosine deaminase activity as increased7 or decreased8 during normal pregnancy. There has not been, however, any study evaluating the relationship of plasma adenosine deaminase activity to immunological status, especially changes in the proportions of cytokine‐secreting T cells in normal pregnancy. Furthermore, the clinical significance of changes in adenosine deaminase activity during normal pregnancy has not been elucidated.
To address these questions, we measured plasma adenosine deaminase activity and the proportion of CD4‐positive T cells secreting interferon‐γ (as an index of T helper 1 cells) and interleukin‐4 (as an index of T helper 2 cells9) in the peripheral blood of women with normal pregnancies.
MATERIALS AND METHODS
Subjects were recruited from the Obstetrics Outpatient Department of Nippon Medical School Hospital, Tokyo, Japan, from January 2000 until December 2001. This study was approved by the Institutional Review Board at the Nippon Medical School. The inclusion criteria were as follows: a woman in the third trimester of a normal pregnancy, well‐established gestational age corroborated by ultrasonography, a singleton fetus, nonsmoker, no evidence of recent infection, no medical complications. Women who developed complications during pregnancy were excluded from the study.
Twenty‐seven women with normal pregnancies meeting the inclusion criteria volunteered for the study, and 26 gave informed consent after being advised they would not benefit directly from this study. In this study, we also recruited 26 healthy nonpregnant women, who were matched for age (within 0.9 years) and parity.
The measurements were taken after an overnight fast. An hour before the study, subjects were placed at bed rest in a semi‐recumbent position in a quiet room and an indwelling 18‐gauge catheter (Medikit Co., Tokyo, Japan) was placed into an antecubital vein. After a 1‐hour period of accommodation, blood was taken through the indwelling catheter for the measurement of plasma adenosine deaminase activity. Obtained plasma was stored at −80C until analyzed. Plasma adenosine deaminase activity was measured by a specific colorimetric method using adenosine as the substrate.10 The detection limit of the assay was 0.5 U/L, with intra‐ and interassay coefficients of variation of 6.2% and 8.3%, respectively.
Flow cytometric determination of interferon‐γ and interleukin‐4 in the cytoplasm of peripheral CD4‐positive T cells was performed using the Fastimmune cytokine detection system (Becton Dickinson Immunocytometry Systems, San Jose, CA). Briefly, whole‐blood samples (500 μL) were cultured in tubes for 4 hours at 37C under 7% CO2 in Rosewell Park Memorial Institute #1640 medium. Cells were activated with 50 ng/mL phorbol 12‐myristate 13‐acetate (Sigma‐Aldrich Japan K.K., Tokyo, Japan) and 1 μg/mL calcium ionomycin (Sigma‐Aldrich Japan K.K.) in the presence of 10 μmol/L brefeldin A (Sigma‐Aldrich Japan K.K.), which inhibits intracellular transport processes. Cultured cells were stained with 20 μL of peridin chlorophyll protein‐conjugated monoclonal anti‐CD4 monoclonal antibody (Becton Dickinson Immunocytometry Systems) for 15 minutes at room temperature, then washed twice with phosphate‐buffered saline. Red blood cells were lysed by adding 1 mL of fluorescence‐activated cell sorter lysing solution (Becton Dickinson Immunocytometry Systems) for 10 minutes. After washing with phosphate‐buffered saline, fluorescence‐activated cell sorter permeabilizing solution (5 mL) (Becton Dickinson Immunocytometry Systems) was added, and the mixture was incubated for 10 minutes at room temperature to render the cells permeable.
The cells were washed twice with phosphate‐buffered saline. The treated cells were then stained with fluorescein‐isothiocyanate‐conjugated anti‐human interferon‐γ monoclonal antibody (Becton Dickinson Immunocytometry Systems), and phycoerythrin‐conjugated anti‐human interleukin‐4 monoclonal antibody (Becton Dickinson Immunocytometry Systems) for 30 minutes at room temperature in the dark. Fluorescein‐isothiocyanate‐conjugated, isotype‐matched immunoglobulin G (IgG) 2a and phycoerythrin‐conjugated IgG 1 were used as controls for detecting nonspecific binding. After two washes with phosphate‐buffered saline, the cells were resuspended in 1% paraformaldehyde in phosphate‐buffered saline. Flow cytometry was performed using a FACSCalibur (Becton Dickinson Immunocytometry Systems).
The forward and side scatter gates for lymphocytes and the CD4‐positive gate (logical gate) were set to exclude contaminating monocytes and dead cells from the analysis. The sorted cells were collected in sterile tubes that had been precoated with fetal calf serum to prevent T cell adhesion. The cells were counted, and the purity was verified. The purity of the sorted CD4‐positive cells uniformly exceeded 99%. Fifty thousand cells were acquired in the list mode and analyzed with CELL Quest software (Becton Dickinson Immunocytometry Systems). The results were expressed as the percentage of cytokine‐secreting cells in the total CD4‐positive cell population.
Data are presented as mean ± standard error of the mean. Statistic analyses were performed using Student t test for normally distributed data and the Mann–Whitney U test as appropriate. Normality of the data was evaluated by Kolmogorov–Smirnov test. Linear regression analysis was performed by the least‐squares method. Differences were considered significant at P < .05.
Demographic data are given in Table 1. All subjects were married Japanese women of middle socioeconomic status, and all were nonsmokers. All deliveries resulted in live infants with 5‐minute Apgar scores > 7. Each infant's weight was appropriate for gestational age.
In normal pregnant women, mean adenosine deaminase activity was significantly lower than that of non‐pregnant women (Table 2) (P < .05). The proportion of CD4‐positive T cells secreting cytokines and the T helper 1/T helper 2 ratio are shown in Table 2. The proportion of interferon‐γ–secreting cells in normal pregnant women was significantly lower than that in nonpregnant women (P < .05). The proportion of interleukin‐4–secreting cells in normal pregnant women was rather high but not significantly higher than that in nonpregnant women. Consequently, the T helper 1/T helper 2 ratio in normal pregnant women was significantly lower than that in nonpregnant women (P < .05). A significant correlation was found between plasma adenosine deaminase activity and the proportion of interferon‐γ–secreting cells in normal pregnant women (r = .54, P < .05). There were, however, no significant correlations between plasma adenosine deaminase activity and the proportion of interleukin‐4–secreting cells (r = .23, P = .45) or the T helper 1/T helper 2 ratios (r = .01, P = .95) in normal pregnant women. Representative data from flow cytometry of peripheral blood T cells in normal pregnant women that were CD4‐positive are shown in Figure 1. The spontaneous production of cytokines was minimal in this study, and the percentages of interferon‐γ‐ and interleukin‐4‐negative cells were 99.89% and 99.92% in normal pregnant and healthy nonpregnant women, respectively.
In the present study, a significant decrease in plasma adenosine deaminase activity was found, which was accompanied by a decrease in the proportion of interferon‐γ–secreting cells and the T helper 1/T helper 2 ratio in normal pregnant women. There was a significant relationship between plasma adenosine deaminase activity and the proportion of interferon‐γ–secreting cells. These results suggest that reduced plasma adenosine deaminase activity in normal pregnancy may be in part related to the decrease in the proportion of interferon‐γ–secreting cells, which eventually causes the T helper 1/T helper 2 balance to shift toward a T helper 2 bias.
Plasma adenosine deaminase activity in normal pregnant women was significantly lower than that in non‐pregnant women, which is in agreement with a previous study.8 In contrast, another study demonstrated an increase of plasma adenosine deaminase activity during pregnancy7; however, that study did not use a control, and the study population differed from that of the present study, which may account for the discrepancy in the results.
The proportion of interferon‐γ–secreting cells significantly decreased, which eventually caused a shift in T helper 1/T helper 2 balance to the T helper 2 predominance, in normal pregnant women. These results were in accordance with those of a previous study.11 Various cytokines produced by T helper cells play an important regulatory role in the immune response during pregnancy.6,12 T helper 1 and T helper 2 cells secrete many cytokines that regulate membrane adenosine deaminase on human lymphocytes.13 T helper 1 cytokines stimulate and T helper 2 cytokines inhibit ecto‐adenosine deaminase expression on lymphocytes.13 Because lymphocytes are one of the major sources of plasma adenosine deaminase activity,2,3 a decrease of the proportion of interferon‐γ–secreting cells as an index of T helper 1 cells and the shift of T helper 1/T helper 2 balance toward T helper 2 predominance observed in this study may be in part related to a decrease in plasma adenosine deaminase activity. The T helper 1 cytokine, interferon‐γ, activates the monocyte–macrophage cell system,14 which is also assumed to contribute to the regulation of plasma adenosine activity.3,4 The proportion of interferon‐γ–secreting cells was decreased in the present study, and a decrease in serum interferon‐γ levels was also demonstrated in normal pregnancy.11 Taken together, these mechanisms may also contribute to a decrease in plasma adenosine activity in normal pregnancy.
The clinical significance of a decrease in plasma adenosine deaminase activity in normal pregnancy is not clear. Because 5′‐nucleotidase is increased in normal pregnancy,15 and a decrease in adenosine deaminase was found in the present study, those kinetic factors may tend to increase plasma adenosine levels. In normal pregnancy, the increased adenosine may modulate platelet activation.16 Because adenosine also regulates cytokine production and may shift the T helper 1/T helper 2 ratio toward T helper 2 dominance,17 decreased adenosine deaminase may play a role in the maintenance of normal pregnancy via increasing adenosine levels.
A number of limitations in the present study should be noted. First, circulating T cells secreting cytokines in the peripheral blood do not necessarily reflect, for instance, the more important local immunologic environments, such as the cell surface of lymphocytes in the regulation of plasma adenosine deaminase activity. Second, in addition to T helper cells, other elements including monocytes, natural killer cells, and other immunologic factors are involved in the regulation of plasma adenosine deaminase activity.2,13 Further, T helper 1 and T helper 2 cells and their secreting cytokines influence each other,12 and crosstalk between them could affect plasma adenosine deaminase activity. Thus, changes in plasma adenosine deaminase activity are likely to reflect, at least in part, changes in the immune status during pregnancy. Overall, the mechanisms by which immunological changes may influence and possibly regulate plasma adenosine deaminase activity in normal pregnancy are not fully understood, and further study is needed to clarify them.
1. Adams A, Harkness RA. Adenosine deaminase activity in thymus and human tissues. Clin Exp Immunol 1976;26:647–9.
2. Koehler LH, Benz EJ. Serum adenosine deaminase: Methodology and clinical applications. Clin Chem 1962;8:133–140.
3. Ungerer JPJ, Oosthuizen HM, Bissbort SH, Vermaak WJH. Serum adenosine deaminase: Isoenzyme and diagnostic application. Clin Chem 1992;38:1322–6.
4. Zuckerman SH, Olson JM, Douglas SD. Adenosine deaminase activity during in vitro culture of human peripheral blood monocytes and pulmonary alveolar macrophages. Exp Cell Res 1980;129:281–7.
5. Mosmann TR, Sad S. The expanding universe of T-cell subsets: Th1, Th and more. Immunol Today 1996;17:138–46.
6. Wegmann TG, Lin H, Guilbert L, Mosmann TR. Bidirectional cytokine interactions in the maternal-fetal relationship: Is successful pregnancy a TH2 phenomenon? Immunol Today 1993;14:353–6.
7. Henkiewicz J, Michalski J. Adenosine deaminase in pregnancy and in some gynecological diseases. Enzymologia 1971;41:261–77.
8. Jaqueti D, M-H, Hernandez-Garcia R, Navarro-Gallar F. Adenosine deaminase in pregnancy serum. Clin Chem 1990;36:2144.
9. Picker LJ, Singh MK, Zdraveski Z, Treer JR, Waldrop SL, Bergstresser PR, et al. Direct demonstration of cytokine synthesis heterogenity among human memory/effector T cells by flow cytometry. Blood 1995;86:1408–19.
10. Giusti G, Galanti B. Colorimetric method. In: Bergmeyer HU, ed. Methods of enzymatic analysis. Weinheim, Germany: Verlag Chemie, 1984:315–23.
11. Saito S, Sakai M, Sasaki Y, Tanabe K, Tsuda H, Michihama T. Quantitative analysis of peripheral blood Th0, Th1, Th2 and the Th1:Th2 cell ratio during normal human pregnancy and preeclampsia. Clin Exp Immunol 1999;117:550–5.
12. Saito S. Cytokine network at the feto-placental interface. J Reprod Immunol 2000;47:87–103.
13. Cordero OJ, Salgado FJ, Fernandez-Alonso CM, Herrera C, Lluis C, Franco R, Nogueira M. Cytokines regulate membrane adenosine deaminase on human activated lymphocytes. J Leukoc Biol 2001;70:920–30.
14. Wilson CB, Westall J. Activation of neonatal and adult human macrophages by alpha, beta, and gamma interferons. Infect Immunol 1985;49:351–6.
15. Yoneyama Y, Suzuki S, Sawa R, Otsubo Y, Power GG, Araki T. Plasma adenosine levels increase in women with normal pregnancies. Am J Obstet Gynecol 2000;182:1200–3.
16. Yoneyama Y, Suzuki S, Sawa R, Kiyokawa Y, Power GG, Araki T. Plasma adenosine levels and P-selectin expression on platelets in preeclampsia. Obstet Gynecol 2001;97:366–70.
17. Zidek Z. Adenosine-cyclic AMP pathways and cytokine expression. Eur Cytokine Netw 1999;10:319–28.