1 Deartment of Obstetrics and Gynecology, and 3 Department of Public Heath, Hokkaido University Graduate School of Medicine and 2 Division of Immunobiology, Research Section of Pathophysiology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan
4 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Graduate School of Medicine, Hokkaido University, Kita-ku N15 W7, Sapporo 060-8638, Japan. e-mail: yhideto{at}med.hokudai.ac.jp
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Abstract |
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Key words: immunodystrophism/natural killer cell/natural killer T cell/recurrent miscarriage/Th cytokine
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Introduction |
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An increasing number of unique functions of natural killer T (NKT) cells have been reported ever since this new lymphocyte was first reported in 1987 (Joyce, 2001). In the field of reproductive medicine, it has been found that NKT cells increase in the uterine decidua during early pregnancy (Dang and Heyborne, 2001
), and might control the T-helper (Th) cell function by producing interferon (IFN)-
and interleukin (IL)-4 at the materno-fetal interface (Tsuda et al., 2001
). However, no abnormality of the NKT cell population has yet been demonstrated in relation to the aetiology of RM. We previously found no difference in the NKT cell population of peripheral lymphocytes in the mid-luteal phase between RM women and fertile control women (Shimada et al., 2003
).
The Th cell responses following activation are functionally characterized according to cytokine production; type-1 Th (Th1) and type-2 Th (Th2) cells (Mosmann and Coffman, 1989). The Th1 cells mainly secrete IL-2, IFN-
and tumour necrosis factor (TNF), whereas the Th2 cells synthesize IL-4, IL-5, IL-10 and IL-13. This Th1/Th2 balance model has been expected to provide a framework to explain materno-fetal immune reactions. Murine studies have demonstrated that the predominant Th1 immunity is related to implantation failure and fetal resorptions (Krishnan et al., 1996
). The Th2 cytokines produced at the materno-fetal interface are thought to be beneficial for the maintenance of pregnancy because these suppress cellular cytotoxicity (Lin et al., 1993
; Wegmann et al., 1993
).
An abnormal Th1/Th2 balance with Th1 predominance of peripheral mononuclear cells in response to trophoblast antigens has been found to be associated with the cause of RM (Hill et al., 1995; Raghupathy et al., 1999
). Recently, by using flow cytometry, one study found higher ratios of IFN-
+/IL-4+, TNF-
+/IL-4+ and TNF-
+/IL-10+ cells in CD3+CD8 peripheral lymphocytes in non-pregnant RM women (Kwak-Kim et al., 2003
). However, the same study also found no differences in the percentages of IFN-
+, TNF-
+ or IL-4+ cells. In contrast, we demonstrated Th2 and Tc2 predominance of CD4+ and CD8+ peripheral lymphocytes in non-pregnant RM women (Shimada et al., 2003
). Another study, using stimulated peripheral lymphocytes, found that TNF-
production levels at 610 weeks of gestation in RM women with subsequent miscarriages were lower than those in RM women with a successful reproductive outcome. The authors also found that IL-4 and IL-10 production levels at 610 weeks of gestation in RM women were higher than those in normal pregnant women (Bates et al., 2002
). Thus, there still are many controversies concerning peripheral Th1/Th2 balance as the underlying pathophysiology and predictive values for miscarriage in RM women.
In the present study, we aimed to investigate the NK cell and NKT cell populations, the cytokine expression of lymphocyte, and the Th1/Th2 balances in the endometrium. The concept of immunodystrophism as the underlying pathophysiology of RM was proposed.
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Materials and methods |
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During the study period, of 41 consecutive RM women, nine had endocrine abnormalities, four had haematological abnormalities, three had uterine anomaly or myoma, three had antiphospholipid antibody syndrome or definite autoimmune disease, and two had chromosome translocation. The remaining 20 women without plausible causes were classified as having unexplained aetiology (48.8%). Among these 20 women with unexplained aetiology, only two showed a positive test for ANA without any definite diagnosis of autoimmune disease or the presence of antiphospholipid antibody. The other 18 women carried no autoantibody.
The control group consisted of 17 non-pregnant women (34.9 ± 6.0 years old, range 2644) who had experienced one or more normal live births without a history of miscarriage or ectopic pregnancy. All control women had regular menstrual periods.
Endometrial samples were obtained from the RM and control women by using an endometrial cell sampler, ENDOCELL (WALLACH Surgical Devices, Inc., Orange, CT), with informed consent during the mid-luteal phases of their menstrual cycle. The mid-luteal phases (5th9th day of a high phase) in RM women were confirmed by basal body temperature.
This study has been approved by the ethics committee of the Graduate School of Medicine, Hokkaido University.
Flow cytometric analysis
The endometria were suspended in phosphate-buffered saline (PBS) containing 0.2% bovine serum albumin (BSA) and 0.1% sodium azide, and were minced by surgical scissors and strained through a nylon mesh (59 µm). A lysing solution containing NH4Cl and EDTA was added for 10 min at room temperature to lyse the erythrocytes. The cells were washed twice with PBS and resuspended with 1 ml of PBS before flow cytometric analysis. For the NK cell analyses, the cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD16 (Becton Dickinson & Co., San Jose, CA) and phycoerythrin (PE)-conjugated anti-CD56 (Beckman Coulter, Inc., Fullerton, CA). For the NKT cell analyses, the cells were stained with phycoerythrin-cyanine 5 (PC5)-conjugated anti-CD3, allophycocyanin (APC)-conjugated anti-CD4 and anti-CD8, PE-conjugated anti-V24 and FITC-conjugated anti-V
11 monoclonal antibodies (mAbs), all purchased from Immunotech. Two- and four-colour flow cytometric analyses were carried out using FACS Calibur flow cytometry (Becton Dickinson & Co.) and CellQuest Software (Figure 1).
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Results |
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NKT cell percentages in V24+V
11+ cells/CD3+CD4CD8 cells, V
24+ cells/CD3+CD4CD8 cells, V
24+V
11+ CD3+CD4CD8 cells/lymphocytes or V
24+CD3+CD4CD8 cells/lymphocytes showed no difference between the two groups (Table I).
Table II shows IFN--, TNF-
- and IL-4-expressing cell populations and ratios in CD4+ cells. In RM women compared with controls, CD4+IFN-
+ cell percentages (28.4 ± 13.4% versus 39.5 ± 12.1%, respectively) and CD4+TNF-
+ cell percentages (32.9 ± 17.6% versus 45.8 ± 12.3%, respectively) were significantly lower than those in control women. CD4+IFN-
IL-4 cell percentages in RM women were significantly higher than those in controls (71.0 ± 13.6 versus 59.6 ± 11.7%). The CD4+IL-4+/CD4+IFN-
+ (Th2 + Th0/Th1 + Th0) cell ratio, CD4+IL-4+IFN-
/CD4+IFN-
+IL-4 (Th2/Th1) cell ratio and the CD4+IL-4+/CD4+TNF-
+ cell ratio were not different between the two groups.
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Discussion |
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Recently, CD1d antigen has been found to be expressed on trophoblasts and to interact with NKT cells in the decidua. The decidual NKT cells produce much more granulocytemacrophage colony-stimulating factor (GM-CSF) and IFN- than do peripheral NKT cells (Boyson et al., 2002
). Murine studies have demonstrated that fetal growth and viability are jeopardized in the absence of maternal GM-CSF (Robertson et al., 1999
) and that IFN-
plays a crucial role in the murine implantation phenomenon (Ashkar and Croy, 1999
; Ashkar et al., 2000
). Therefore, NKT cells seem to be beneficial to maintaining the normal reproduction process. In contrast, it has been demonstrated in a murine study that NKT cells can provoke fetal resorption if overstimulated by a specific ligand for V
14+NKT cells,
-galactosylceramide (
GalCer) (Ito et al., 2000
). In the previous study, we found no difference in V
24+V
11+ NKT cell percentages among CD3+CD4CD8 cells in peripheral blood between RM women (mean ± SD; 0.61 ± 0.51%) and fertile controls (0.46 ± 0.47%). In the present study, we assessed the NKT cell population in the endometrium and also found no difference in the V
24+V
11+ NKT cell population between RM women (1.90 ± 1.78%) and fertile control women (2.34 ± 1.76%). However, NKT cell accumulation in the endometrium compared with that in peripheral blood was noted; this phenomenon was in agreement with the results of another study (Tsuda et al., 2001
). Although NKT cells were not found to be involved in RM aetiologies, these cells might play some roles in the reproduction process.
In the present study, we demonstrated that the percentage of CD3+ cells in endometrial lymphocytes was decreased in comparison with controls, while the percentage of IFN-IL-4 cells in CD4+ cells was increased. The percentages of IFN-
+ cells and TNF-
+ cells in CD4+ cells were also decreased in RM women. These findings suggest the diminution of T-cell activity rather than accentuation in the endometrium of RM women. One study has demonstrated lower IFN-
expression in the peripheral CD4+ and CD8+ lymphocytes of RM women than in those of controls (Rein et al., 2002
). We have also found Th2 and Tc2 predominance of peripheral lymphocytes in non-pregnant RM women compared with fertile women (Shimada et al., 2003
). The IFN-
+/IL-4+cell ratio and TNF-
+/IL-4+cell ratio in CD4+ and CD8+ peripheral lymphocytes have been observed to be decreased in RM women (Shimada et al. 2003
). Thus, we confirmed the diminution of IFN-
+ and TNF-
+ cells in peripheral lymphocytes as well as in the endometrium in RM.
Murine studies have demonstrated that the predominant Th1 immunity is related to implantation failure and fetal resorptions (Krishnan et al., 1996). The Th2 cytokines produced at the materno-fetal interface have been thought to be beneficial to the maintenance of pregnancy because these suppress cellular cytotoxicity (Lin et al., 1993
; Wegmann et al., 1993
). However, a recent murine study has demonstrated that Th2 cytokines, IL-4 and IL-10, were not crucial to the successful completion of allogeneic pregnancies (Svensson et al., 2001
). In humans, no differences in the Th2 cell marker in the endometrium during the peri-implantation period have been observed between RM women and controls, and this marker has also failed to predict subsequent pregnancy outcome (Michimata et al., 2002
). One recent study has demonstrated that the IFN-
concentration in the uterine cavity fluid is decreased in women with recurent implantation failure (Inagaki et al., 2003
). Another study has found decreases in the percentages of the Th1-inducing cytokine IL-12 in lymphocytes, monocytes and granulocytes derived from peripheral blood and the decidua of RM women when compared with those of normal pregnant women (Zenclussen et al., 2002
). Using stimulated peripheral lymphocytes, it has been found that TNF-
production levels at 610 weeks of gestation in RM women with subsequent miscarriages are lower than those in RM women with successful reproductive outcomes (Bates et al., 2002
). There are still many controversies concerning peripheral Th1/Th2 balance as the underlying pathophysiology and predictive values for miscarriage in RM women.
TNF- is a primarily pro-inflammatory cytokine produced preferentially, but not uniquely, by Th1 cells like GM-CSF, and the main source of this cytokine is assumed to be macrophages/monocytes (Cherwinski et al., 1987
). Embryo-protective effects of TNF-
against teratogenic stress have been discovered recently (Torchinsky et al., 2003
). IFN-
has been known to contribute essentially to implantation, decidual integrity and placental growth (Ashkar et al., 2000
); therefore, decreased production of TNF-
and IFN-
at the materno-fetal interface could adversely affect implantation and fetal development. Our results in the present and previous studies in addition to the recent findings of others suggest that the diminution of cytokine production early in gestation rather than the Th1 dominance is related to the aetiology of RM and poor reproductive prognosis. The insemination and the implantation normally induce remarkable changes of Th cytokine expression in the endometrium and decidua (Krasnow et al., 1996
). It is hypothesized that diminution of Th1 cytokine expression in the endometrium disturbs the induction of immune tolerance during the processes of insemination and implantation and during subsequent embryonic development.
In conclusion, we found diminution of CD3+ cells, CD4+IFN-+ cells and CD4+TNF-
+ cells but not Th1 predominance, NK cell or NKT cell accentuation in the endometrium of RM women. The immunodystrophism, i.e. reduced Th1 cell function, in the endometrium may be the underlying pathophysiology of RM.
However, the number of subjects in this study population was rather small. The differences of cytokine expression in the endometrium between RM women and controls were not sufficient to conclude that these changes would be essential to cause RM. Further immunological characterization of T cells in endometrium as well as in the decidua would promote our understanding of the underlying pathology of RM. Further studies should be performed in order to determine the causeeffect relationship, especially in miscarriage with normal fetal chromosome karyotype.
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Acknowledgments |
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References |
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Submitted on July 28, 2003; accepted on January 5, 2004.