1 Department of Obstetrics and Gynaecology, University of Würzburg, Josef-Schneider-Strasse 4, D-97080 Würzburg and 2 Department of Pathology University of Tübingen, D-72076 Tübingen, Germany
3 To whom correspondence should be addressed. e-mail: frak057{at}mail.uni-wuerzburg.de
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Abstract |
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Key words: development/fetal uterus/immunocompetent cells/immunohistochemistry/LGL
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Introduction |
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The development of the human uterus during fetal life has only been described in a few reports, which have focused on morphological features. Huber and colleagues demonstrated that the formation of glands can be observed in the fetal human endometrium from the 20th week of gestation (Huber et al., 1971). Proliferation and differentiation of epithelial and stromal cells occur subsequently, and from the 33rd week of gestation secretory changes can be seen in the endometrium. Because of the withdrawal of maternal estrogens, regressive changes occur immediately after birth and a transitional type of endometrium without glandular activity appears (Huber et al., 1971
). The only information about immunocompetent cells in the developing uterus pertains to mice. It has been shown that in the mouse, large granular lymphocyte (LGL)-1+ NK cells appear in the uterus 23 weeks after birth, unrelated to puberty or the microbial environment (Kiso et al., 1992
). There are very few T cells in the murine uterus at birth, but their number reaches adult levels at
3 weeks of age. As in humans, the majority of murine uterine CD3+ T cells are CD8+ (Croy et al., 1993
).
As very little is known about the development of the immune system of the human uterus, this study was undertaken to investigate the distribution of the various different leukocyte subpopulations in the endometrium of human fetuses and children between 17 weeks gestation and 5 years of age.
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Materials and methods |
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Blocking immunohistochemistry
As staining of endothelial cells for HLA-DR and neural cells for CD56 would make it difficult to identify leukocytes reactive for these antigens, a blocking technique in which these cells were specifically stained first by antibodies against CD34 and S100 respectively, was employed. For this technique, the primary antibodies were used at 2x the normal concentrations and the chromogen (DAB) developed to a very high concentration of precipitate. This precipitate then completely covers the antigenic structures with the bound specific antibody, thus protecting this cellular structure from other antibodies. Subsequent staining with the second primary antibody will then produce no staining of the blocked structures.
Evaluation of results
To determine the average density of specific leukocyte subpopulations, fields containing only endometrium were randomly selected. The number of positive cells in three to 15 single fields (depending on the size of the uterus investigated) of 0.31 mm2 was determined at 250x magnification for each of the sections by two independent observers (L.R. and U.K.).
Potential differences in the findings of the two observers were investigated with Students t-test (significance set at P < 0.05); no statistically significant differences between their findings were noted (P > 0.9). The mean values for the various cell types in the two groups (fetuses, n = 11; neonates/children, n = 9) were analysed using the non-parametric MannWhitney U-test (significance set at P < 0.05).
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Results |
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The median number of endometrial CD45+ leukocytes was 18.6 cells/mm2 in fetal specimens and 30.6 cells/mm2 after birth (Figure 1A).
Lymphocytes
In most tissue specimens, moderate numbers of CD3+ cells were scattered irregularly throughout the endometrial stroma and epithelium (Figure 1B). The median number of CD3+ lymphocytes before and after birth was the same (3.2 cells/mm2).
A few CD4+ T-helper cells were seen, but only in the fetal uteri (median 1.3 cells/mm2; Figure 1C). The median number of CD7+ cells before and after birth was comparable (1.6 and 2.6 cells/mm2 respectively; Figure 1D). Only a few cytotoxic T cells (CD8+, median 0.6 cells/mm2 in both groups; Figure 1E) and B lymphocytes (CD20+, median 1.0 and 0.6 cells/mm2, respectively; Figure 1H) were found in the endometrial stroma in the uteri investigated.
Monocytes and mast cells
Endothelial cells were HLA-DR positive in all cases (Figure 1L), but HLA-DR-immunoreactive leukocytes were found only in neonates and children. Their median density was 19.5 cells/mm2, and they were often seen in clusters close to the surface epithelium (Figure 1N). In a double immunostaining procedure using the blocking technique with the antibody against CD34, no staining for HLA-DR was seen in the fetal endometrium, thus confirming the absence of HLA-DR+ leukocytes (Figure 1M). CD68+ cells could be detected scattered throughout the endometrial stroma in all cases (median 2.3 cells/mm2 in fetuses and 8.8 cells/mm2 after birth; Figure 1K), except one fetus at the 17th week of gestation, in which no endometrial CD68+ cells were found. A few mast cells were detected in some of the specimens (not shown). Owing to the limited number of sections per uterus in which endometrium was present, the evaluation of CD14+ cells was possible only in 14 of the 20 specimens (six fetuses, eight neonates/children). The median number of endometrial CD14+ cells was 5.85 cells/mm2 in fetuses, increasing to 28.4 cells/mm2 after birth (Figure 1F). Thus, CD14-immunoreactive cells represented the largest leukocyte subpopulation in both groups investigated.
CD56+ uterine NK cells
Single staining revealed marked reactivity for CD56 in most specimens. However, staining was restricted mainly to smooth muscle cells in the myometrium and nerve fibres (Figure 1I), and only in neonatal/childhood endometrium were some lymphocytes with annular staining detected (Figure 1J). After staining of the nerve fibres had been blocked by the antibody against S100 protein, no CD56 reactivity was observed in the fetal specimens (Figure 1O), indicating the absence of CD56+ NK cells. In contrast, a small number of CD56+ leukocytes was found scattered throughout the endometrium of neonates and children, with a median density of 3.4 cells/mm2 (Figure 1P). The density of CD56+ cells did not correlate with age (Figure 3). A very few CD16+ cells were found in the endometrium of neonates and children (median density 0.2 cells/mm2) and those cells were clearly located in small vessels (Figure 1G). Investigation of fetal endometrium for CD16+ cells was not possible due to the limited amount of tissue available.
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Discussion |
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The largest endometrial leukocyte populations between the 19th week of gestation and term were found to be CD14+ monocytes and CD68+ macrophages. Their proportions in relation to the total number of CD45+ bone marrow derived leukocytes were similar to those described in the adult endometrium (Bulmer et al., 1988). However, in contrast to the situation in neonates and children, staining for HLA-DR in the fetal endometrium was confined to vascular endothelium. Thus, at this stage of development of the fetal uterine immune system, CD68+ and CD14+ cells are negative for HLA-DR. These cells may be identical to the RFD7+/UCHM-1+/HLA-DR majority of tissue macrophages, described by Janossy and colleagues, in the yolk sac and mesenchymal tissue of fetuses (Janossy et al., 1986
). Since RFD7 is a marker for mature tissue macrophages (Collings et al., 1985
), it is likely that this marker detects the same macrophage subpopulation as the CD68 antibody used in our study. However, in the endometrium of neonates and children a large number of HLA-DR+ cells with dendritic/macrophage-like morphology was found. The appearance of HLA-DR+ cells in the endometrium after birth may be due either to the induction of HLA-DR expression on the fetal monocytes/macrophages by mediators in the microenvironment or to the selective infiltration of the endometrium by HLA-DR+ cells. It is possible that interferon-
induces HLA-DR expression here, as this cytokine induces the expression of HLA-DR on fetal monocytes (Kelley et al., 1984
) and is also known to play a key role in modulating host defences during delivery (Buonocore et al., 1995
). On the other hand, foreign antigens or the changes in hormone levels might also induce infiltration of the endometrium by HLA-DR+ cells after birth.
CD3+ T cells accounted for 17.2% of the CD45+ endometrial cells before birth, and 10.5% after birth, the latter figure resembling that in adults (Starkey et al., 1991). In contrast to the situation in adults, only one-third of the CD3+ cells in the endometrium of fetuses and children were CD8+. CD4+ cells were found only in fetal endometrium and not after birth. The highest proportion of T cells were CD7+, consistent with the fact that CD7 is the earliest marker antigen expressed in T cell development (Haynes, 1990
).
In contrast to the endometrium after the onset of puberty, where CD56+ uterine NK cells are the dominant lymphocyte population (Starkey et al., 1991), fetal endometrium was totally devoid of CD56+ uterine NK cells in all cases investigated, and neonatal/childhood endometrium contained only small numbers of these cells. This finding is surprising because CD56+ NK cells are found very early in the development of the immune system, and membrane CD3 CD56+ NK cells are identifiable in the fetal liver at around the 6th week of gestation (Phillips et al., 1992
). Because only a limited amount of tissue was available in each case, there were not enough sections to perform double labelling procedures for CD56 and CD16. However, as the number of CD56+ cells was substantially higher than that of CD16+ cells, we conclude that the CD56+ cells correspond to the uterine CD56+ CD16 NK cells found in adult endometrium. In the mouse model, LGL-1+ NK cells seed the uterus during the first weeks of post-natal life, independent of onset of puberty and environmental factors (Kiso et al., 1992
), and this study shows that this is also the case in humans. What triggers the first appearance of CD56+ uterine NK cells in human endometrium cannot be determined on the basis of the results of this study. However, it seems likely that these cells, which exhibit the same phenotype as a small proportion of NK cells in the peripheral blood, migrate into the endometrium at the onset of puberty, probably due to changes in the endometrial microenvironment induced by rising levels of steroid hormones. A recent study indicated that the high density of CD56+ NK in the uterus is not only due to homing mechanisms but, at least in pregnancy, also results from in situ expansion of these cells (Kammerer et al., 1999
).
In summary, our data show that the leukocyte population in the endometrium of fetuses and small children is different to that in the endometrium of adult women. We demonstrated that the seeding of the endometrium with antigen-presenting HLA-DR+ cells and uterine NK cells is a post-natal event and may be induced by the changes in hormone levels occurring at this time and/or reflect the adaptation of the local immune system in the uterine mucosa to the change in circumstances from the almost sterile environment of the fetus to the normal environment of the newborn.
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Acknowledgements |
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References |
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Submitted on June 6, 2002; resubmitted on November 14, 2002; accepted on January 30, 2003.