1 Department of OB/GYN, Julius-Maximilians University Würzburg and 2 Charité, Campus Virchow Klinikum, Humboldt University Berlin, Germany
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Abstract |
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Key words: apoptosis/ectopic pregnancy/leukocytes/progesterone receptor
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Introduction |
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In ectopic pregnancies, the fertilized ovum implants into the Fallopian tube (Breen, 1970). Immunohistochemical characterizations have suggested that ectopic pregnancy represent abnormal implantation of a morphologically normal blastocyst, giving rise to immunologically normal and hormonally active trophoblast cells (Vassiliadou and Bulmer, 1998
). On the other hand, decidualization cannot occur normally in the epithelium of the Fallopian tube (Earl et al., 1987
; Maruyama et al., 1992
; Stewart-Akers et al., 1997
). Therefore, this pathological situation provides an excellent model in helping us to understand immunological reactions at the eutopic feto-maternal interface during early gestation.
Decidualized endometrium is more complex than just a collection of enlarged glycogen-filled stromal cells. There are immunocompetent cells in direct contact with trophoblast cells expressing paternal antigens (Clark, 1991; King et al., 1997
). The decidua contains intraepithelial lymphocytes, and in the stroma, T cells, B cells, CD56+ natural killer (NK) cells, macrophages and mast cells are present (Bulmer et al., 1988
; King and Loke, 1991
; Hunt 1994
; Clark 1995
; Marx et al., 1999
). There are changes in the composition of decidual tissue as pregnancy progresses, differences between species, and differences depending on the nature of the stimulus initiating decidualization, like that given in ectopic pregnancies (Pace et al., 1991
; Chernyshov et al., 1993
; Clark, 1995
).
Programmed cell death by apoptosis has been accepted as a mechanism for maintaining tolerance in the immune system. Expression of BCL-2 and Fas ligand (FasL) by human trophoblast cells has been proposed as a mechanism providing protection against the lytic action of decidual immune cells (Hunt et al., 1997; Lea et al., 1997a
; McLaren et al., 1997
; Jones et al., 1998
; Quenby et al., 1998
). However, very little is known about the protection mechanism of decidual cells for FasL-mediated apoptosis. Therefore, it was an aim of the present study to investigate decidual cells for the presence of BCL-2. The proto-oncogene product BCL-2 is associated with the inhibition of apoptosis (Nunez et al., 1994
). BCL-2 immunopositive cells have been identified in non-pregnant endometrial stroma and glandular epithelium (Koh et al., 1995
; Rodger et al., 1995
; Tabibzadeh et al., 1995
). Lea et al. (1997a) have shown that during first trimester healthy pregnancies, a majority of stromal BCL-2+ cells were CD56+ large granular lymphocytes. We performed double staining immunohistochemistry for various decidual and tubal leukocytes and examined their expression of BCL-2. In addition, we examined the tissue for apoptotic cell bodies by in-situ detection of fragmented DNA. The following experiments were designed to investigate if the distribution of immunocompetent decidual cells, hormone receptor expression and apoptosis show similarities between intrauterine decidua, reflecting normal invasion of trophoblast, and ectopic decidua. This may help us to improve our understanding of the mechanism underlying trophoblast invasion in normal or failing pregnancies.
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Materials and methods |
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Immunolocalization of single staining for cytokeratin (CK), CD56, CD3, CD8, CD68, BCL-2, ER, PR
Tissue sections were dewaxed in xylene and rehydrated through a descending ethanol series. Non-specific endogenous peroxidase activity was blocked by treatment with 3% hydrogen peroxide in methanol for 30 min at room temperature. All stainings required microwave boiling in citrate buffer pH 6.0 for 15 min. Tissue sections were then washed with Tris-buffered saline (TBS; pH 7.4) for 2x5 min and exposed to a non-immune, serum-free protein block (Dako). Monoclonal mouse anti-human cytokeratin antibody was applied to the different sections at a dilution of 1 in 100 for 60 min at room temperature; on different sections anti-CD3 was used at 1:75, anti-CD8 at 1:25, anti-CD68 at 1:75, anti-BCL-2 at 1:40, and anti-PR and anti-ER at 1:50. This was followed by biotinylated multilink anti-mouse antibody for 15 min at room temperature, and an avidinbiotinperoxidase detection system (LSAB-Kit; Dako) for a further 15 min at room temperature. Finally, the sections were developed with diaminobenzidine (DAB; Sigma, Munich, Germany) for 5 min to generate a brown-coloured product and lightly counterstained with haemalaim (Roth, Munich, Germany) prior to mounting. Negative controls were performed by replacing the primary antibody with a serum-free protein block (Dako) at the same concentration as the primary antibody.
Immunolocalization of double staining for BCL-2, CD56, CD3
Staining with mouse monoclonal antibody to CD56 or CD3 was performed as described earlier in this paper and followed by multi-link antibody. The same avidinbiotin detection kit as in the single staining was used, and developed with DAB to generate a brown-coloured product. Mouse monoclonal antibody to BCL-2 was incubated overnight at 4°C at a dilution of 1:40, followed by biotinylated multi-link antibody. Then the avidinbiotin peroxidase system was applied and developed with Vector VIP substrate kit (Linaris, Bettingen, Germany), which generated a purple-coloured product. For the CD3/CD56 double staining the same protocol was used except that anti-CD56 was used instead of anti-BCL-2. Negative controls were carried out by replacing either BCL-2 antibody or CD56 and CD3 respectively by normal mouse IgG, or by replacing both primary antibodies with normal mouse IgG.
In-situ cell death detection
The in-situ detection of fragmented DNA was performed on one slide of all samples used. We used a commercially available kit (TUNEL test kit; Boehringer Mannheim, Germany) and performed the assay according to the guidelines of the distributor with the following modification: after step 8, we washed in PBS/Triton 100 twice for 5 min at 60° C and incubated in PBS plus 10% fetal calf serum for 15 min at room temperature, instead of washing in PBS, as recommended by the distributor.
Evaluation
The evaluation of immunohistochemistry, double immunostaining and detection of apoptotic cell bodies was performed by two independent observers using a light microscope with scaled eye pieces. The number of positive cells/mm2 tissue was counted at a magnification of x25.
Statistics
Mean scores and SD of all samples were calculated for absolute and relative cell numbers within the same groups. Statistical analysis was performed on relative cell numbers using Student's t-test, and significance was set at P 0.05.
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Results |
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Discussion |
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In ectopic tubal pregnancy, fetal trophoblast cells show an identical reaction pattern with regard to their MHC antigen expression in comparison to that seen in intrauterine pregnancy, suggesting that ectopic implantation is not related to an inherent immunological abnormality of fetal trophoblast (Earl et al., 1985). Therefore we suggest that the increase in CD3 and CD68 positive cells is a sign of inflammation due to the mechanical irritation of the Fallopian tube through the implanted and growing embryo. However, the increased proportion of CD8+ cells among the total number of T cells is striking. CD8+ T cells are thought to be pregnancy-protective in humans, as well as in mice (Arck et al., 1996
; Szekeres-Bartho and Wegmann, 1996
). Recent data have shown that peripheral CD8+ lymphocytes with PR of women during early pregnancy also bear the g
T cell receptor. These cells produce progesterone-induced blocking factor (PIBF) which contributes to the success of gestation via cytokine-mediated inhibition of NK activity (Szekeres-Bartho et al., 1997
; Barakonyi et al., 1998
).
It has been suggested that decidual CD56+ NK cells may control key events in trophoblast migration and placentation (King and Loke, 1991). However, CD56+ NK cells are absent from the tubal implantation site, as demonstrated by the present study and others (Maruyama et al., 1992
; Vassiliadou and Bulmer, 1998
) These results suggest that sufficient placentation and invasion may also occur in the absence of CD56+ cells, since in ectopic pregnancy we observe a lack of CD56+ cells but progressing pregnancy until the mechanical rupture of the Fallopian tube.
The immunomodulation of CD56+ cells is most likely not induced by the local presence of trophoblast due to the lack of CD56+ cells in the tubal epithelium in the case of ectopic pregnancy. The data published by others and presented in this study allow us to postulate that CD56+ cells are relatively inactive in the endometrium. A stimulus, other than invading trophoblast cells, might induce immunomodulation. This certainly requires receptor(s) which are present in the intrauterine decidua and absent in the tubal epithelium. It is quite possible that hormones may induce stimulation of CD56+ immunocompetent cells in the decidua. It has been shown that the number of stromal leukocytes in human endometrium varies during the menstrual cycle and, in particular, CD56+ cell numbers increase in the late secretory phase (Jones et al., 1995). The stimulus responsible for these cyclic changes is unknown, but it is likely that steroid hormones such as oestrogen and particularly progesterone play a role. The investigation of steroid hormone receptors on leukocytes in non-pregnant and pregnant human endometrium demonstrated that, despite the prominence of CD56+ endometrial granulated lymphocytes in late secretory phase endometrium and early pregnancy decidua, there was no expression of either PR or ER by these cells or other endometrial leukocyte populations. Rather than acting directly, steroid hormones are more likely to influence endometrial leukocyte populations indirectly via products of endometrial stromal or epithelial cells that express steroid hormone receptors (Stewart et al., 1998
).
Progesterone is secreted by trophoblast cells; the production can be doubled in vitro in the presence of pure HCG (Yagel et al., 1989). Assuming that both ectopic and eutopic trophoblast produces progesterone, and keeping in mind that CD56+ NK cells do not express PR, we evaluated the distribution of PR in ectopic pregnancy and uterine pregnancy tissues and observed a high density of PR positive cells in both types of tissue. ER and PR expression in Fallopian tubes had been investigated by Land and Arends (1992) using ectopic pregnancy tissue; no PR expression was seen in glandular epithelium or stroma of the Fallopian tube and it was postulated that the absence of PR is correlated to poor decidualization. We believe that the improved availability of more specific antibodies to PR led to the results we observed with regard to PR expression in ectopic pregnancy and uterine pregnancy.
Cell death by apoptosis is regarded as an important feature in reproductive biology. Recent reports have suggested that BCL-2, a proto-oncogene responsible for the suppression of apoptosis, is expressed in the endometrium and may be involved in the regulation of menstruation (Koh et al., 1995). On the other hand, the presence of BCL-2 on decidual CD56+ cells has recently been described, resulting in the hypothesis that the CD56+ population has been selected for survival during early pregnancy (Lea et al., 1997a
). In the present study it was therefore our aim to identify the phenotype of the remaining BCL-2+ cells in the decidua. Our data suggest that BCL-2 expression among the decidual leukocyte population is not restricted to CD56+ cells since we observed comparable percentages of BCL-2/CD3 double positive cells. The co-expression of BCL-2 with CD56 or CD3 may be a reflection of a normal cell turnover. If BCL-2 expression would only be detectable on `selected cells', further studies are needed to identify the mechanism of selection for the various BCL-2+ cells in the human decidua.
Only very few data are currently available on apoptosis in reproduction (Hunt et al., 1997). We observed apoptotic cell bodies in the stroma of eutopic early pregnancy decidua. The presence of fragmented DNA as a sign of apoptosis might be attributed to the presence of tumour necrosis factor (TNF)-
in the decidua (Tabibzadeh et al., 1995
). Induction of apoptosis in decidual epithelium may also be related to the amount of TNF-
present at the feto-maternal interface (Lea et al., 1997b
). In ectopic pregnancy we observed fewer apoptotic cell bodies, which supports recently published data (Kokawa et al., 1998
). These apoptotic differences between ectopic pregnancy and uterine pregnancy may be related to the increase in CD8+ cells producing Th2 type cytokines in ectopic pregnancy.
The results discussed in the present paper suggest that reproductive biologists should refrain from the hypothesis that CD56+ confers privilege on foreign embryonic tissue. Normal placentation appears to be possible in the absence of these cells, as demonstrated by the use of tissue from ectopic pregnancies lacking CD56+ cells. In addition, the expression of BCL-2, which had previously been thought to be present only on `selected' CD56 cells, was also detectable on CD3 T cells. Rather, the results presented in this paper allow us to conclude that CD8+ cells may be of particular importance during early pregnancy whereby further experiments are neccessary to identify their Th1/Th2 and TcR ß or
phenotypes. In addition, the use of tissue obtained from ectopic pregnancies has been demonstrated to be an excellent model to identify the mechanism of trophoblast invasion in normal pregnancies.
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Acknowledgments |
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Notes |
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References |
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Submitted on September 2, 1998; accepted on December 11, 1998.