1 Departments of Pathology and 2 Immunology, University of Newcastle, Royal Victoria Infirmary, Newcastle upon TyneNE1 4LP, UK
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Abstract |
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Key words: activation/decidual lymphocytes/immunohistochemistry/pregnancy/spontaneous abortion
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Introduction |
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The activation molecules that are expressed on leukocytes can be classified as early activation markers, such as CD69 and CD25, and late activation markers, such as human leukocyte antigen (HLA) DR and very late antigen (VLA)1. CD69, a cell surface glycoprotein, is expressed by activated T and B lymphocytes and natural killer (NK) cells (Testi et al., 1994) and has been identified in both normal pregnant and non-pregnant human endometrium (King et al., 1991
; Nishikawa et al., 1991
; Saito et al., 1992
; Chen et al., 1995
). Single and double immunostaining of tissue sections has indicated that most of these CD69-positive cells in endometrial stroma are T cells rather than the CD56-positive eGL (Vassiliadou and Bulmer, 1998c
). Although intracellular signalling occurs following CD69 crosslinking, the function of CD69 is still unknown (Testi et al., 1994
). The expression of both interleukin (IL)-2 receptor subunits, IL-2R
(CD25) and IL-2Rß in apparently normal first trimester human decidua, has also been investigated (Bulmer and Johnson, 1986
; Sato et al., 1990
; Nishikawa et al., 1991
; Starkey, 1991
; King et al., 1992
; Saito et al., 1993
). Although IL-2Rß is expressed by decidual lymphocytes, IL-2R
is virtually absent and it is generally accepted that IL-2 itself is not normally present in early human pregnancy decidua (Jokhi et al., 1997
). HLA DR, a class II MHC molecule, is expressed by macrophages in normal pregnant human endometrium (Bulmer et al., 1988
) and T lymphocytes in both non-pregnant and normal pregnant human endometrium (Nishikawa et al., 1991
; Saito et al., 1992
; Chen et al., 1995
). VLA1 functions as a collagen and/or laminin receptor, facilitating cellular interactions with the extracellular matrix (Hemler, 1990
). Studies of endometrial VLA1 expression have primarily focused on the eGL subpopulation as well as on the role of VLA1 in trophoblast invasion of uterine decidua, and have generally been limited to normal human pregnancy (Dietl et al., 1992
; Damsky et al., 1994
; Geiselhart et al., 1995
).
Systematic investigation of the activation marker profile of decidual leukocytes in spontaneous early pregnancy loss could provide valuable information concerning the functional status of decidual leukocytes in this pathological situation. The aim of the present study was to investigate and compare the expression of CD69, CD25, HLA DR and VLA1 activation markers in sporadic spontaneous abortion and apparently normal first trimester human pregnancy.
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Materials and methods |
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Monoclonal antibodies (mAb)
Eight murine mAb were employed to stain cryostat sections. Leukocyte common antigen (LCA) (CD45; Dako, High Wycombe, UK), which is reactive with the majority of human leukocytes, was diluted 1:10. N-CAM (CD56; Novocastra, Newcastle upon Tyne, UK), which reacts with the isoform of neural adhesion molecule (N-CAM) expressed by NK cells and some T cells, was used at 1:100 dilution. UCHT1 (Dako), reactive with the T cell-associated CD3 antigen, was diluted 1:400. HLA DR (Dako) reacts with the ß chain of all products of the DP, DQ and DR subregions and in human decidua principally labels macrophages, although activated T cells will also react with this antibody; it was used at a 1:100 dilution. CD69 (Serotec, Oxford, UK) reacts with a heterodimer known as activation inducer molecule (AIM) which is rapidly expressed by activated T and B lymphocytes and was used at a 1:200 dilution. CD25 (Dako) reacts with the subunit of the IL-2 receptor and is strongly expressed on activated T cells; it was diluted 1:200. CD49a-VLA
1 (Immunotech, Bournbrook, UK) reacts with the
1ß1 integrin (VLA1) known to be a receptor for laminin and collagen and expressed primarily by activated T and B cells, as well as monocytes, melanoma cells, smooth muscle cells and fibroblasts (Hemler et al., 1987
); it was used at a 1:100 dilution. VLA-1 (T cell Diagnostics, Cambridge, MA, USA), reactive with the
1 chain of the ß1 integrin heterodimer, was also used and was diluted 1:10. The optimal dilution for each mAb was determined in positive control tissue (frozen sections of tonsil).
Single immunohistochemical labelling
Acetone-fixed 7 µm cryostat sections were labelled by the avidinbiotin complex (ABC) method using the Vectastain Elite kit (Vector Laboratories, Peterborough, UK) as detailed previously (Vassiliadou and Bulmer, 1996b). Briefly, rehydrated sections were sequentially incubated with diluted normal blocking serum, appropriately diluted primary mAb in blocking serum, biotinylated immunoglobulins (Ig) and the Vectastain avidinbiotin complex (ABC)peroxidase reagent. Endogenous peroxidase activity was blocked by incubating the sections with a 0.5% solution of hydrogen peroxide in methanol; this treatment took place after incubation with biotinylated Ig to ensure that the antigen was not damaged by treatment with hydrogen peroxide. The reaction was developed with diaminobenzidine (DAB) (Sigma Chemical Co., Poole, UK) to give a brown reaction product. Sections were counterstained with Mayer's haematoxylin and mounted in DPX (Raymond Lamb, London, UK). Positive (frozen sections of tonsil) and negative (test sections in which the primary mAb was replaced with normal mouse serum) controls were included in each immunohistochemical run and for each mAb.
Double immunohistochemical labelling
Eight selected spontaneous abortion samples were subjected to double immunohistochemical labelling in order to characterise further those cells which expressed activation markers. The double immunostaining procedure has been described in detail previously (Stewart et al., 1998). Briefly, sections were first labelled for CD3 or CD56 using the ABCperoxidase method described above. The reaction was developed with DAB. The slides were then washed in 0.05 M Tris0.15 M saline pH 7.6 (TBS) for 10 min, overlain with blocking serum for 30 min and then incubated for 60 min with the second primary antibody, anti-CD25 or anti-CD69. After washing in TBS, sections were then incubated sequentially with biotinylated anti-mouse Ig (30 min) and ABC alkaline phosphatase (30 min) (Vector Laboratories). The reaction was developed at room temperature using alkaline phosphatase substrate III (Vecta Blue) (Vector Laboratories). The reaction was stopped by excess water and slides were mounted in Supermount (BioGenex, San Ramon, CA, USA). Double-labelled sections were not counterstained.
The antibody combinations used were CD3/CD25, CD3/CD69, CD56/CD25 and CD56/CD69. Negative controls were performed at all levels of the reaction and included replacement of either the first or second primary antibodies with normal serum. Single- and double-labelled sections were also compared in order to confirm that there was no spurious double labelling.
Quantification and analysis of results
For single immunohistochemical labelling, cells positive for CD45, CD56, CD3, HLA DR and CD69 were counted in eight high power (x400) fields using a 10x10 mm graticule. Counting was performed in equivalent fields in serial sections for each case and each mAb. CD25-positive cells were scanty and were therefore counted in the whole section; all sections were comparable in size. Positive cells were quantified independently by two investigators who were blinded as to which group each case was designated. The distribution of VLA1-positive cells was assessed qualitatively. The proportion that CD56-positive, CD3-positive, HLA DR-positive and CD69-positive cells formed in relation to the total CD45-positive cell population was calculated and the results were analysed by calculating the mean, the standard error of the mean (SEM) and the median for each positive cell population. The MannWhitney statistical test was employed to assess whether there were significant differences in the absolute numbers or proportions of the decidual leukocyte subpopulations between apparently normal pregnancy and spontaneous abortion. Two-sided P values were calculated on all occasions and the conventional level of P < 0.05 was considered as the limit of significance.
Double-labelled sections were assessed qualitatively. CD3 or CD56 single-labelled cells were brown, whereas cells also expressing CD25 or CD69 were double-labelled with both blue and brown reaction products.
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Results |
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CD69-positive cells were detected in all apparently normal and pathological pregnancy samples and were mainly scattered within the stroma, although sometimes they formed aggregates close to endometrial glands. Examination of serial cryostat sections indicated that distribution of CD69-positive cells was very similar to that of CD3-positive cells in both therapeutic abortion and spontaneous abortion cases. Significantly more CD69-positive cells were detected in decidua associated with spontaneous abortion (therapeutic abortion: mean ± SEM: 20 ± 2.26, median: 18; spontaneous abortion: 36 ± 5.04, median: 31; P = 0.02) (Figure 1a); the proportion that CD69-positive cells formed in relation to the total CD45-positive leukocyte population was also significantly greater in spontaneous abortion (therapeutic abortion: 11% ± 1.20; median: 9; spontaneous abortion: 19% ± 2.87; median: 15; P = 0.02).
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Double immunohistochemical labelling
As suggested by examination of single-labelled sections, double labelling of spontaneous abortion decidua for CD3 and CD69 indicated that the increased numbers of CD69-positive cells were also CD3-positive (Figure 1d). In sections double-labelled for CD3 and CD69, single-labelled CD3-positive and double-labelled CD3/CD69-positive cells were identified but single-labelled CD69-positive cells were not seen. In contrast, in sections labelled for CD56 and CD69, single-labelled CD56-positive and CD69-positive cells were easily identified but there were no double-labelled cells (Figure 1e
). Similarly, although CD25-positive cells were scanty, they appeared to be double labelled for CD3 (Figure 1f
). These double labelling studies indicated that the CD69-positive and CD25-positive cells in spontaneous abortion decidua were CD3-positive T cells rather than CD56-positive eGL.
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Discussion |
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Although none of the major decidual leukocyte populations differs significantly in number or proportion in spontaneous abortion (Vassiliadou and Bulmer, 1996a), considerably elevated numbers of CD69-positive cells were detected in decidua from all the spontaneous abortion cases compared with the apparently normal pregnancy controls. Double immunohistochemical labelling studies indicated that these CD69-positive cells in spontaneous abortion decidua are CD3-positive T cells. In normal first trimester decidua it has been reported that the vast majority of T cells [58% of CD4-positive and 73% of CD8-positive cells, and up to 54% of eGL] express CD69 (King et al., 1991
; Nishikawa et al., 1991
; Saito et al., 1992
). However, immunohistochemical studies of first trimester sections and CD56-positive eGL after purification have suggested that CD69 expression by eGL may be induced by cell purification and culture (Vassiliadou and Bulmer, 1998c
).
CD25-positive cells were rarely detected in the vast majority (78%) of apparently normal early pregnancy decidua, in agreement with previous immunohistochemical and flow cytometry reports (Bulmer and Johnson, 1986; Sato et al., 1990
; Starkey, 1991
; King et al., 1992
). The frequency of CD25-bearing cells was significantly higher within pathological decidua when compared with the therapeutic abortion cases. Double labelling studies demonstrated that CD25-positive cells were CD3-positive T cells; this altered phenotype in the spontaneous abortion samples may be indicative of an abnormal maternal immunological response in a proportion of spontaneous abortion cases.
Those cases with increased CD25 cell numbers had significantly increased HLA DR-positive cells when compared with low CD25-positive cell samples. HLA DR was not investigated in double immunohistochemical labelling studies since the high expression of HLA DR by non-lymphoid cells in decidua rendered interpretation very difficult. Macrophages are unlikely to be responsible for the elevated HLA DR expression since studies in paraffin sections have shown no difference in their number and proportion between therapeutic abortion and spontaneous abortion samples (Vassiliadou and Bulmer, 1996a). Studies of apparently normal pregnant decidua have indicated that a proportion of T cells express HLA DR (Saito et al., 1992
); and it has been suggested albeit in a limited flow cytometry study that the number of decidual T cells expressing HLA DR is higher in spontaneous abortion than in apparently normal pregnancy (Maruyama et al., 1992
). Thus, it appears that T lymphocytes may be the cell population responsible for the phenotypic differences detected in spontaneous abortion.
Spontaneous abortion cases could be separated into two categories according to the differential expression of the CD25 activation marker and this classification was further enhanced by comparing expression of the CD69 early activation marker and the HLA DR late activation marker. Separation of spontaneous abortion cases into two groups was also proposed when investigation of a `classical' NK cell marker was performed (Vassiliadou and Bulmer, 1996a). Investigation of the cytotoxic activity of decidual CD56-positive lymphocytes has also identified two distinct subject groups (Vassiliadou and Bulmer, 1998b
). It has already been established that women suffering spontaneous abortion form a heterogeneous group and a number of factors may be implicated in the etiology of this pathological situation. Hence, a common cause for all cases of spontaneous miscarriage would not be expected. It is interesting, however, that approximately half of the women could be classified into each category. Approximately 46% of sporadic spontaneous abortions are assumed to be due to identifiable karyotypic abnormalities (Hassold et al., 1980
) and in such cases it is possible that a lethal combination of chromosomes affects fetal survival. Furthermore, it is unlikely that fetal genetic abnormalities influence maternal immune function. Although anembryonic pregnancies were excluded from the present study, all the patients were sporadic aborters and it is possible that those women who did not differ from the control group may have experienced a spontaneous abortion due to chromosomal abnormalities; in view of the altered activation marker expression, it is possible that an immunopathological mechanism may be implicated in the remaining cases. The fact that the present study, as well as a previous immunohistochemical study (Vassiliadou and Bulmer, 1996a
) and a functional study (Vassiliadou and Bulmer, 1998b
), employing different groups of sporadic spontaneous aborters, allowed separation of patients into two distinct subgroups supports this. The same selection criteria were employed in both studies, the primary aim being to investigate the phenotype and function of leukocytes in sporadic spontaneous abortion decidua that had an apparently normal histological appearance. It is not possible to subject all sporadic spontaneous abortion samples to karyotypic analysis in Newcastle; it would be interesting to perform both immunohistochemical and functional studies of sporadic spontaneous abortion decidua in association with karyotypic investigation.
In-situ immunohistochemical analysis of tissue sections has the advantage that decidual lymphocytes have not been subjected to disaggregation, purification and culture procedures. Analysis of CD69 expression has suggested that this antigen may be induced during tissue and cell manipulation (Vassiliadou and Bulmer, 1998c) and this may be the case for other early activation molecules. Nevertheless, there are certain limitations in studies of this nature which cannot be controlled. First, the changes noted in the spontaneous abortion group may be the consequence rather than the cause of the pregnancy failure; substances secreted within endometrium following fetal rejection and before the patient's manifested genital bleeding may have influenced residual or migrated immune cell function. Second, although women undergoing elective terminations are considered to be the best available `control' group, it is not certain whether they can be considered as truly normal because the outcome of the pregnancy, had there been no intervention, is unknown. Hence, it is uncertain whether those women who, although they were undergoing apparently normal pregnancies, had increased CD25-positive cell numbers were destined for a different pregnancy outcome from the remainder. Third, there is the possibility that the spontaneously aborted fetuses may have died several days before the ultrasonograph documentation of fetal death. Hence the decidual tissue in the spontaneous abortion group may not have reached the same level of development as the decidua of the terminated pregnancies. The site of spontaneous abortion decidua sampled could also be an important factor, as sites closer to endometrial breakdown may be more immunologically affected.
In conclusion, increased T cell expression of activation markers occurs in a proportion of spontaneous abortion cases. Further studies should attempt to correlate the altered decidual immune function in spontaneous abortion with fetal chromosomal data as well as with maternal clinical features such as ultrasonography and endocrine data.
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Acknowledgments |
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Notes |
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References |
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Submitted on September 9, 1998; accepted on January 28, 1999.