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EARLY PREGNANCY: Biology and Medicine Editor-in-Chief: Eytan R. Barnea MD, FACOG

July 2001
Volume V, Number 3
ISSN: 1537-6583
Pages: 164-175

Immunohistochemical Detection Of CD45+, CD56+, And CD14+ Cells In Human Decidua During Early Pregnancy

Çiler Çelik Özenci(1)*, Emin Türkay Korgun (1)**, Ramazan Demir (1)

(1) Department of Histology and Embryology, Faculty of Medicine, Akdeniz University, Antalya, Turkey
(*) Obstetrics and Gynaecology, School of Medicine, Yale University, New Haven, CT, USA
(**) Institute of Histology and Embryology, Karl-Franzens University, Graz, Austria

Short title: Immunohistochemical Detection of Leukocyte Cells Population in Human Decidua

Key words: Immunohistochemistry/NK cell/macrophage/human decidua/early pregnancy

Correspondence address: Prof. Dr. Ramazan Demir Department of Histology and Embryology Faculty of Medicine, Akdeniz University 07070, Campus-Antalya-Turkey,
Telefax: + 90 242 227 44 86, Email: demir@med.akdeniz.edu.tr

Summary

The densities and distribution of innate immune system cells, NK cells and macrophages, was investigated between days 22-30 post coitus of very early pregnancy. Paraffin sections were labelled by the avidin-biotin complex-peroxidase method using monoclonal antibodies specific for CD45, CD56 and CD14. Positive cells were quantified and the results were analysed using ANOVA tests. It was found that ~40% of all cells were leukocytes and ~30% of the leukocytes were NK cells and ~22% were macrophages. The number of CD56+ cells increased as pregnancy progressed. CD56+ were observed cells close to the luminal epithelium and were especially noticeable around glands where they often occurred in small clusters. Decidual macrophages (CD14+) were distributed throughout the decidual stroma of early pregnancy and were observed in greatest numbers around the vessel walls. Their numbers have also increased as pregnancy progressed. The great abundance of these leukocytes, which are known as members of the innate immune system, in early pregnancy decidua suggests that these cells are important for fetal survival and pregnancy success. The relevance of the distribution of CD45+, CD56+ and CD14+ in decidua at the fourth week of pregnancy to the immunological barrier between mother and fetus is discussed.

1. Introduction

Pregnancy starts with decidualization of endometrial stromal cells just before implantation of the blastocyst (Abrahamson, 1993). The functions of the decidua include protection of maternal tissues from invasion by the trophoblast, nutrition of the developing embryo (Finn, 1971), immunological protection of the embryo and/or the mother (Bernard and Rachman, 1980) and isolation of individual embryos (DeFeo, 1967). It is known that, like other mucosal surfaces, the uterus must be able to respond to antigenic challenge in order to accept this foreign body to be developed within it. According to our knowledge of immune system cell behaviour in response to a foreign body, we might naturally expect that this invasion of semi-allogeneic cells into a host tissue would be reflected by different types of leukocytes. The main hypothesis which have been advanced to explain the immunological status of the "fetal semi-allograft" is the unusual distribution of maternal leukocytes within the pregnant uterus (King et al., 1989; Bulmer et al., 1991; Loke and King., 1995).

In early pregnancy, the establishment of the human placenta is characterised by the presence of large numbers of uNK cells within the maternal decidua (Chao et al., 1999). The dominant NK population in blood is CD16++CD56+CD3-, in contrast the decidua is infiltrated almost exclusively by CD16+-CD56+CD3- NK cells (uNK) which seem that decidua is an immunepriviliged tissue (Moller et al., 1998). Histological studies have shown that the number of uNK cells is low in proliferative phase endometrium but increases in the mid-luteal phase, reaching a maximum in the late secretory phase of the cycle (King et al., 1998, Stewart-Akers et al., 1997). The stimulus for the in-vivo proliferation of uNK cells is still unknown.

The Uterus contains a large number of macrophages in the endometrium (Hunt, 1994). Uterine endometrial, epithelial and stromal cells with oestrogen receptors respond to steroid hormones and the oestrogen-stimulated uterine growth factors attract macrophages (Hunt and Robertson, 1996). Their densities and distribution patterns in cycling and pregnant uterus changes with the circulating estrogens and progesterone levels (Arck et al., 2000). Although these cells are also abundant at the implantation site, there are not many studies concerning their functions at implantation.

Besides uNK cells and uterine macrophages, the next abundant population of leukocytes in decidua are classic T lymphocytes (Stewart-Akers et al., 1997, Vince and Johnson., 2000). Relative T-cell numbers decrease in first trimester decidua; T cells are therefore unlikely to have an important role in the immunological maintenance of pregnancy but could be more important in implantation, when their relative numbers are greater (Vince and Johnson., 2000). Because of the fact that in the uterus the major leukocyte population is uNK cells and uterine macrophages, and not T and B cells, the idea that implantation is likely to involve an innate immune system becomes more important (Loke and King., 2000). Due to a study that didn’t support the idea that spontaneous abortion could be a result of atypical expression of complement regulatory proteins by villous trophoblast, implantation appears to be influenced by uNK cell allorecognition system rather than by a T cell system (Hill et al., 1995).

Although there have been many previous studies of leukocyte populations in human decidua in the first trimester of pregnancy, this study is unique in the sense that it describes densities and distribution of uNK cells and uterine macrophages in the decidua at a very early stage of pregnancy. Information about decidual leukocytes early in gestation is scarce and that’s why we think that this sample group is potentially important. So, the objective of the present study was to investigate the identification and distribution of leukocytes, uNK cells and decidual macrophages fourth fifth between days 22-30 post coitus of pregnancy, in very early human decidua by immunohistochemical methods.

2. Material and Methods

2.1. Immunohistochemistry
Human decidual tissue samples (n=3, 22 days post conception (p.c.); n=2, 27 days p.c.; n=2, 29 days p.c.; n=3, 30 days p.c.) were obtained by curettage from ten healthy women undergoing abortion of unwanted healthy pregnancies between days 22-30 post conception. Age of pregnancy was determined from women’s histories, according to the first day of the last menstruation period. All the women (age range =29-37 years) included in the study were multiparous (less than 4) and none were using medications including oral contraceptives. Specimens were dissected and washed several times in phosphate buffered saline (PBS) immediately after curettage. The decidual tissue was then cut with a scalpel into tissue blocks 1 cm³ for fixation in 10% formaldehyde for 4 h. After dehydration, the samples were embedded in paraffin wax. Serial sections 6 - 8 m m thick were collected on Poly-L-Lysin coated slides (Sigma, St. Louis, MO, USA) and incubated overnight at 40°C. After dehydrating, the samples were placed in citrate buffer (pH= 6; 1l distilled water; 2.1g citric acid, 15ml NaOH) and antigen retrieval was performed, by treating the samples in a microwave oven at 750 W for 5 min twice. After cooling for 20 min at room temperature, the sections were washed in PBS. To remove endogenous peroxidase activity, sections were kept in 3% hydrogen peroxidase (Dako A/S, Glostrup, Denmark) for 30 min and afterwards washed with PBS three times. We have identified decidua basalis and the parietalis via trophoblast marker (CK 7). Using this antibody to CK 7 (Dako, Glostrup, Denmark) the decidua was classified as decidua basalis (trophoblast present) or decidua parietalis (no trophoblast). Only decidua basalis was used for further experiments The following, primary antibodies were used: CD45 (Dako, mouse anti-human, prediluted), CD56 (Dako, mouse anti-human, 1/300), CD14 (Zymed Laboratories Inc., San Francisco, CA, USA, mouse anti-human, prediluted). Immunohistochemistry was performed using a horseradish-peroxidase-labelled streptavidin biotin (HRP-LSAB) kit (Dako) according to the manufacturer’s instructions. The resulting signal was developed with diaminobenzidine (DAB) (Dako). All incubations were performed in a moist chamber at room temperature, using PBS for washes between the incubation steps. Briefly, after a 30-min incubation with blocking solution, sections were incubated for 60 min with the primary antibodies, followed by sequential 30-min incubations with a biotinylated link antibody and peroxidase labelled streptavidin. The sections were counterstained with Mayers’s haematoxylin and mounted with glycerol gelatine (Dako). Negative controls were performed by using normal mouse Ig control antibodies at the same concentration as the primary antibodies, provided in the kit. Normal mouse IgG for CD45 and CD14, mouse IgG1 for CD56 primary antibodies were used at a concentration of 5µg/ml as negative controls. Photomicrographs were taken with an Axioplan microscope (Zeiss, Oberkochen, Germany).

2.2 Histomorphometric analysis
Morphometry was performed using light microscopy and positioning an unbiased counting frame of known area (modified from Gundersen, 1977). A random section was selected for each gestational day and 25 randomly selected areas were scored on the same slide for each CD marker in the series. The morphometric accounting was done as previously described (Gundersen et al., 1988), based on the original physical dissector counting rules (Sterio, 1984). A total of 25 randomly selected areas were scored on the same slide for each day of pregnancy (22, 27, 29, 30 p.c.). The counts obtained from each field were averaged for each single patient as a mean. And the average of the means were used to come up with the grand mean for each day (22, 27, 29, 30 p.c.) individually which is seen as plotted on fig 4 with their +/-2SE’s. All sections were scored blindly by one observer (Ç.Ç.Ö) first and then a random selection of the same number of slides was scored blindly by a second observer (E.T.K.). Similar numbers of positively staining cells were found. Results scored by the two observers were strongly correlated (r = 0.989, P < 0.05). The number of positively staining cells for each marker was counted at X20 in the 25 fields. The number of positive cells included only that found in stroma, that all the quantitative analyses were done in decidua basalis.

2.3 Statistical analysis
The statistical difference according to days and immunostainings of specific antibodies for CD45, CD56 and CD14 were analysed with ANOVA tests and appropriate post hoc tests were done. The means for counts obtained from each pregnancy day were plotted with their +/-2SE’s as seen in figure 4. On the other hand, to compare the difference between CD56+ and CD14+ cell numbers according to pregnancy days, T-tests were done. The appropriate P values for each statistical test were calculated by the computer with Sigma Stat Version 2.0.

3. Results

3.1 Immunohistochemistry
Decidual leukocytes (CD45+) were numerous on all pregnancy days (22, 27, 29, 30 p.c.; Table 1) in decidual stroma . The decidual stroma was also positive for CD56+ uNK cells and CD14+ macrophages. It was found that ~40% of the total cells were leukocytes and ~30% of these leukocytes were NK cells and ~22% were macrophages. The number of CD56+ cells tended to increase as pregnancy progressed. The intensity of staining for CD45, CD56 and CD14 positive cells is indicated according to the gestational age (Table1). The localisation patterns of the CD45+ leukocytes and leukocyte subgroups are summarised semi-quantitively in Table 2. Typical clusters of CD45+ leukocytes were observed (Fig. 1a) as well as their deep infiltration of the area around the glands (Fig. 2a). They were located close to the luminal epithelium, around the vessel walls and even in the vicinity of the endothelium (Fig. 3a).

CD56+ cells were found scattered throughout the stroma at the fourth week of pregnancy (Fig. 1b). They were especially noticeable around the glands (Table 2), where they often occurred in small clusters (Fig. 2b). CD56+ cells were also observed close to the luminal epithelium. uNK cells were particularly located around spiral arteries (Fig. 3b). The peri-arterial localisation of CD56+ cells was not dense as for CD14+ cells (Fig. 3).

Decidual macrophages (CD14+) were the least numerous types of leukocytes when the serial sections were stained against monoclonal antibodies to CD14. They were distributed throughout the stroma but only in less numbers (Fig. 1c) when compared to uNK cells. These macrophages were more commonly located close to the vessel walls compared to glands (Table 2; Fig. 2c, 3c).

3.2 Quantitative analysis
When the positive cells were quantified and the results were analysed using ANOVA tests, it was observed that, while the numbers of CD45+ leukocytes decreased (p<0.001) the numbers of uNK cells and decidual macrophages increased as the pregnancy progressed (22, 27, 29, 30 p.c.). All pairwise multiple comparisons (Tukey) for CD45+ cells showed that the decrease is statistically significant except between days 29 and 30 p.c. (p<0.05). There was a statistically different (p<0.001) increase of uNK cells through the pregnancy days When post hoc tests (Tukey) was done, the difference between all pairwise multiple comparisons was statistically significant (p<0.05), except between days 22 and 27 p.c. In the meanwhile, the increase of macrophages in numbers were statistically different (p<0.001) utilising ANOVA test and the appropriate post hoc test (Tukey) has indicated that the increase was statistically different after day 29 p.c. (p<0.05). On the other hand, when we compared the difference between CD56+ and CD14 + cells according to pregnancy days, we have observed that the difference was significant between all days except day 22 p.c. (p<0.05).

4. Discussion

The present study confirmed that by the fourth week and the beginning of fifth week of early pregnancy, decidua is infiltrated by CD45+, CD56+ and CD14+ cells; it also showed, however, that there were differences in the proportions of infiltrating. The distribution in this study was ~40% of total cells were CD45+ leukocytes, ~30% of these leukocytes were CD56+ cells and ~22% of them were CD14+ cells at 4-5^th week of early pregnancy. These percentages are important because, very early stage of pregnancy was observed in our study compared to previous studies (Bulmer et al., 1988, 1991; Beer et al., 1998) in which the whole first trimester was included as early pregnancy. In our study, we have observed a rapid increase of uNK cells after day 27 p.c. and uterine macrophages after day 29 p.c. in numbers at the 4-5^th week of early pregnancy. It may suggest that, these uterine uNK cells and macrophages may be involved in the maintenance of normal pregnancy. The increase of uNK cells was more significant than the increase of macrophages (p<0.05) utilising T-tests. In the present study, uNK cells were found scattered throughout the stroma; they were also located around spiral arteries but not so frequently, and were particularly located around glands, where they were often seen creating small cell clusters. To our previous knowledge, the periglandular distribution of CD56+ cells is remarkable in decidua and is more marked than in luteal phase endometrium (Mincheva-Nilsson et al., 1994).

During pregnancy, macrophages are present at the fetomaternal interface, suggesting an involvement in both the response to infection and in the immune interaction between fetal and maternal tissues (Hunt and Robertson, 1996). In the present study, 22% of the leukocytes were macrophages in the fourth week of pregnancy and their numbers were also seemed to be increase after day 29 p.c. As they occur in the placental bed throughout gestation (Kabawat et al., 1985), the key to their presence in the decidua may be the distribution pattern in this immune-privileged tissue. In this study, the macrophages were observed especially around the vessel walls when compared to decidual glands semi-quantitively.

In our study, while the number of CD56+ cells and CD14+ cells have tend to increase, the numbers of CD45+ cells seemed to be decreasing. The decrease between pregnancy days 29 and 30 is not significant (p<0.05). Given both that CD56+ and CD14+ cell numbers increased through the pregnancy days one would expect that the numbers of CD45+ cell to increase also. This numerical discrepancy may be a technical limitation of immunohistochemical analysis. There are also other groups who have reported that the total numbers of uterine leukocyte subpopulations are greater than the number obtained by a pan leukocyte marker (Bulmer et al., 1996, Stewart-Akers et al., 1997). However, as the numbers of CD56+ and CD14+ cells do not add up to the number of CD45+ cells, it would implicate that there is a sizeable number of CD45+ cells which are not CD56+ or CD14+. According to these results, when we consider the missing cell population as classical T cells (CD3+), then the percentage of CD3+ cells might be around fifties. So, as the number of CD45+ cells were decreasing and the numbers of CD56+ and CD14+ cells increased; the number of CD3+ cells might be decreasing rapidly, leaving decidua to the innate immune system cells. It has been suggested that the number of CD3+ cells decrease after implantation (Vince and Johnson, 2000). Our study suggests that the rapid increase of innate immune system cells alongside with the rapid decrease of specific immune system cells might be significant around 4-5^th week of early pregnancy.

In conclusion, the significant abundance of leukocytes in the early pregnancy suggests that these cells are important for fetal survival and pregnancy success, indicating that the immunological barrier between mother and fetus is established very early in pregnancy. On the other hand, as NK cells are abundant throughout the decidua at the fourth week of pregnancy, it is clear that these cells are mainly responsible for the immunological barrier between mother and fetus. Because of the fact that, the major leukocyte population in the decidua seems to be the uNK cells and macrophages and distinct changes in distribution and localisation of these cells occur during the differentiation of decidual components; it supports the idea that implantation is likely involve an innate immune system. It is necessary to further investigate the innate immune system and its relations with various cytokines in early pregnancy.

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Table 1

Intensity of staining for CD45, CD56 and CD14 at different stages of pregnancy.

Intensity is indicated as +: weak, ++: moderate, +++: strong.

Table 2

Density of distribution of the leukocytes and leukocyte subgroups are indicated semi-quantitively as (+):rare, (++):moderate, (+++): frequent, (++++):dense.

Figure 1

Immunostaining in decidual stroma of early pregnancy on day 22 p.c. Typical clusters of CD45+ cells are seen in the stroma (a; double arrows). The scattered distribution of CD56+ cells is seen throughout the stroma (b; single arrows). CD14+ cells are rarely observed (c; arrows). Pregnancy day is 22 p.c. Original Magnification = 100

Figure 2

Immunostaining in decidual stroma of early pregnancy on day 29 p.c. CD45+ cells are seen deeply infiltrating the glandular epithelium (a; single arrows). CD56+ cells are seen as small cell clusters in stroma around the glandular epithelium (b; double arrows). CD14+ cells are observed as subglandular lines and in the stroma (c; single arrows). Original Magnification = 100.

Figure 3

Immunostaining in decidual stroma of early pregnancy on day 30 p.c. CD45+ cells are closely associated with the vessel wall and even infiltrated the endothelial cells (a; single arrow). A few CD56+ cells are seen around vessel walls (b; single arrow). CD14 cells are especially seen in large numbers around vessel walls (c; single arrow). Original Magnification = 100

Figure 4

The mean numbers of CD45+, CD56+, CD14+ cells at the days between 22-30 p.c. of pregnancy.