1 Department of Obstetrics and Gynaecology and 2 Department of Pathology, University of Glasgow, 10 Alexandra Parade, Glasgow, G31 2ER, Scotland, UK
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Abstract |
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Key words: adhesion molecules/labour/macrophages/neutrophils/uterus
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Introduction |
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There are no data on whether inflammatory cells infiltrate myometrium during labour. However, indirect evidence suggests that inflammatory cells may infiltrate the myometrium at the time of parturition. Rechberger and Woessner (1993) demonstrated that collagenase activity is increased in lower segment myometrium during labour and proposed that neutrophils may be responsible for much of the increase. Furthermore, Osmers et al. (1995) demonstrated a significant increase in interleukin-8 concentration in lower segment myometrium during labour, and proposed that this increase could result in an influx of neutrophils. The presence of these leukocytes in myometrium has been examined prior to the onset of labour only. Butterworth et al. (1991) found no neutrophils in the myometrium of both normal and pre-eclamptic women before the onset of labour.
The attachment and extravasation of circulating leukocytes is controlled by the expression of cell surface adhesion molecules on both the circulating cells and the vascular endothelium (Akyama et al., 1989; Bevilacqua, 1993
). An increased expression of adhesion molecules on the endothelium occurs in inflammatory conditions and supports the recruitment and aggregation of leukocytes (Bevilacqua, 1993
) The major endothelial adhesion molecules involved in leukocyte attachment and transendothelial migration include E-selectin, intercellular adhesion molecule-1 and 2 (ICAM-1 and 2), platelet endothelial cell adhesion molecule (PECAM) and vascular cell adhesion molecule-1 (VCAM-1).
The purpose of the present study was to investigate inflammatory cell subpopulations in the human myometrium during pregnancy and labour. Specifically, we aimed to determine whether myometrium in each of the lower and upper uterine segments, like cervical stroma, maternal decidua and the fetal membranes, is infiltrated by leukocytes during parturition. Furthermore, the expression and distribution of the cell adhesion molecules, E-selectin, ICAM-1 and 2, PECAM and VCAM-1, were assessed in myometrium during labour to investigate possible mechanisms of leukocyte accumulation.
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Materials and methods |
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Myometrial biopsies were collected from three groups of women: (i) Eighteen pregnant women who were delivered before the onset of labour at term (>37 weeks gestation); (ii) Eighteen pregnant women who were delivered during active labour at term (cervical dilatation >4 cm and <9 cm). Women were excluded from the study if they had a multiple pregnancy, evidence of active infection, or following induction of labour; (iii) Thirteen non-pregnant, pre-menopausal women with regular menstrual cycles undergoing hysterectomy for benign disease.
In groups (i) and (ii), a myometrial biopsy was obtained from the upper margin of the lower uterine segment incision during the Caesarean section. Additionally, in seven of the women in group (i), and five of the women in group (ii), a myometrial biopsy was obtained from the upper uterine segment by dissecting a strip of myometrium from the inner aspect of the posterior uterine wall and inserting a haemostatic suture when required. In group (iii), myometrial biopsies were taken from the anterior wall of the lower uterine body, immediately following hysterectomy.
Each of the 12 upper segment and the 13 non-pregnant myometrial biopsies was fixed in 10% neutral buffered formalin (BDH, Poole, UK) and embedded in paraffin. The 36 lower segment myometrial biopsies were divided; one half was fixed in 10% neutral buffered formalin and embedded in paraffin, and the other half was snap frozen in liquid nitrogen and stored at 70°C.
Identification of inflammatory cells
Inflammatory cells were detected using immunocytochemistry, with a panel of antibodies as shown in Table I. Serial sections were stained for naphthol AS-D chloroacetate esterase activity, an enzyme considered specific for cells of granulocytic lineage (neutrophils, eosinophils and basophils).
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The sections were pre-incubated with 1.5% (w/v) horse serum in PBS (10 mM sodium phosphate, pH 7.5, 120 mM sodium chloride) for 30 min at room temperature. They were then incubated for 1 h with the primary antibody diluted in 1.5% horse serum. Next, sections were washed in PBS before incubation with biotinylated anti-mouse immunoglobulin. This antibody, from a Vectastain Elite ABC kit (Vector, Peterborough, UK) was first diluted in 1.5% horse serum and 1.5% normal human serum. After washing as before, sections were placed in 3% hydrogen peroxide in methanol for 10 min at room temperature. The sections were thoroughly washed again, then incubated for 30 min with avidin DH/biotinylated horseradish peroxidase H reagent (Vectastain Elite ABC kit) in PBS before final washing. The antigen was localized using 1 mg/ml diaminobenzidine tetrahydrochloride (DAB), 0.02% H2O2 in 50 mM Tris.Cl, pH 7.6, and appeared as a brown end-product.
Negative controls included sections incubated without the primary antibody and sections incubated with a mouse monoclonal antibody against IgG1 Aspergillus niger glucose oxidase (Dako Ltd, High Wycombe, UK), an enzyme which is not expressed in mammalian cell systems. Tonsillar tissue was used as positive controls for CD 3, CD 20 and CD 68.
Mast cells
Mast cells were localized in non-pregnant and pregnant myometrial biopsies. The paraffin embedded sections were prepared as before and digested in a trypsin solution to retrieve the antigen (Table I). The sections were then pre-incubated with 20% normal goat serum (SAPU, Carluke, UK) in PBS (10 mM sodium phosphate, pH 7.5, 120 mM sodium chloride) for 30 min at room temperature. They were then incubated for 90 min with a monoclonal antibody raised against mast cell tryptase (Dako Ltd) diluted 1/300 in 2% normal goat serum. The primary antibody was omitted from the negative control slides. Next, sections were washed in PBS before incubation with goat anti-mouse IgG alkaline phosphatase (Sigma) diluted 1/200 in 2% normal goat serum and 5% normal human serum. The sections were thoroughly washed again and immunoreactive tryptase was localized using Fast Red Substrate (Sigma). Finally, the sections were counter-stained with Harris haematoxylin.
Cell adhesion molecules
Sections 5 µm thick were cut from the frozen tissue and mounted on silane-coated slides. The sections were fixed in acetone for 10 min, washed in TBS, and placed in 0.5% hydrogen peroxide in methanol for 30 min at room temperature. The sections were then washed as before and pre-incubated with 20% (w/v) normal goat serum (SAPU) in TBS for 30 min at room temperature. The sections were then incubated for 16 h at 4°C with the primary antibody diluted in 1.5% horse serum. Table II shows the characteristics of the primary antibodies. The primary antibody was omitted from the negative control slides. Next, sections were washed in TBS before incubation for one h with biotinylated goat anti-mouse immunoglobulin (Dako Ltd), diluted in 2% normal goat serum and 1.5% normal human serum. The sections were thoroughly washed again, then incubated for 30 min with streptavidin horseradish peroxidase (Dako Ltd) in TBS before final washing. The antigen was localized as previously described using DAB.
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Quantification of inflammatory cells and statistical analysis
The inflammatory infiltrate was quantified (i) to determine whether inflammatory cells infiltrate the myometrium during labour at term (ii) to compare the density of the inflammatory cell infiltrate in upper and lower uterine segment myometrium and (iii) to compare mast cell density in non-pregnant and pregnant myometrium. Inflammatory cells were identified by either brown staining (neutrophils, macrophages, T cells and B cells) or red staining (mast cells). In each section of myometrium, the number of positive cells was counted in a high powered field (x400 magnification within parts of a lined grid covering an area of 0.02 mm2). Six different fields were counted by two observers who were blinded to the specimen details. Areas containing blood vessels were avoided and leukocytes within vessels were not included. The average number (arithmetic mean) of positive cells recorded per field by each observer was calculated, and then a mean of these two values obtained. Statistical comparisons of the means were performed using three-factor analysis of variance (ANOVA) with Scheffé's S as a post hoc test. Significant differences between groups were explored using the MannWhitney U-test. The sites of expression of each of the cell adhesion molecules were recorded by the observers who were blinded to the specimen details. Differences in the expression of these molecules before and after labour were analysed using the 2 test.
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Results |
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The leukocyte subpopulations were characterized as follows:
Neutrophils
Neutrophils were sparse in myometrium obtained before the onset of labour and abundant in biopsies obtained during labour (Figure 2). A significant increase in myometrial neutrophil density occurred following the onset of labour in both the lower (Table III
), and the upper uterine segments (Table IV
). During labour, the neutrophil density was significantly greater in the lower than in the upper uterine segment (P < 0.02). Within the labouring biopsies, the elastase antigen was localized both within the neutrophil cytoplasm and extracellularly in the vicinity of the leukocytes, suggesting that a proportion of these cells had degranulated.
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Mast cells
There was no significant difference in mast cell density in myometrium before and after the onset of labour at term (Table III). Furthermore, mast cells were sparse in both upper and lower uterine myometrial biopsies (Table IV
), with no significant differences in mast cell density between the biopsy sites. We assessed mast cell density in non-pregnant myometrium [group (iii)], and found that this was significantly greater than in biopsies obtained from each of the pregnant groups, term not in labour [group (i)], and term in labour [group (ii)] (Figure 4
). These results are summarized in Table V
.
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Discussion |
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In this study, we have examined both lower and upper uterine segment myometrium for the presence of inflammatory cells. Whether lower segment tissue reflects the state of the cervix or that of the uterine fundus, has aroused considerable interest over the last half century (Calder, 1994). Some workers contest that lower uterine segment biopsies provide an alternative source of tissue that closely resembles cervix (Rajabi et al., 1988
). Because of the difficulties in obtaining cervical tissue for study, lower segment biopsies have been used in place of cervical tissue to investigate collagenase activity during cervical dilatation (Rechberger and Woessner, 1993
). Our results indicate that lower uterine segment myometrium behaves quite differently from reported behaviour in cervix, and has similarities to upper segment myometrium. Bokstrom et al. (1997) demonstrated an abundance of neutrophils and macrophages in cervical biopsies in late pregnancy before the onset of labour with no significant increase in their densities during labour. In contrast, we have shown that neutrophils and macrophages are sparse in lower and upper segment myometrium before, and abundant during labour. Although the upper and lower uterine segments behave in a similar manner, they are not identical. During labour, the inflammatory infiltrate is more dense in lower segment myometrium than in upper segment myometrium. Consistent with this histological finding, functional studies have demonstrated that in normal labour the upper uterine segment contracts more strongly than the lower, a situation which is reversed in abnormal labour (Caldeyro-Barcia and Poseiro, 1960
; Margono et al., 1993
).
Activated neutrophils and macrophages are a rich source of inflammatory mediators. These include plasminogen activators, eicosanoids, collagenase and elastase, and proinflammatory cytokines, including interleukin-1 and tumour necrosis factor- (Nathan, 1987
; Osmers et al., 1992
; Casatella, 1995
). Since these mediators have many diverse functions, the inflammatory infiltrate could have different roles in different regions of the uterus. Within the lower segment it could be involved in tissue remodelling and thereby facilitate cervical dilatation and passage of the fetus. In the upper segment, leukocyte products, including eicosanoids, interleukins and tumour necrosis factor-
, may stimulate uterine contractions directly, or indirectly by facilitating the production of uterotonic prostaglandins (Casey et al., 1990
). Furthermore, inflammatory mediators may also initiate tissue remodelling in the uterine body. Granstrom et al. (1989) demonstrated that the connective tissue of the uterine isthmus (lower segment) and the uterine body undergoes a biochemical ripening process similar to that found in the cervix, with an increase in collagenolytic activity following the onset of labour. A breakdown of the connective tissue within the myometrium may facilitate the co-ordination of uterine contractions by allowing the formation of gap junctions (Garfield and Hayashi, 1981
).
We found no change in myometrial mast cell density before and after the onset of labour at term. The function of mast cells in the pregnant uterus remains unclear, although it has been proposed that myometrial mast cells regulate uterine contractility during labour (Rudolph et al., 1993). Mast cells produce mediators (histamine and serotonin) and prostaglandins which can induce strong contractions in human myometrium in vitro (Cruz et al., 1989
; Rudolph et al., 1990
, 1993
). Further, these cells are considered to play a pivotal role in wound healing, fibrosis and tissue remodelling (Galli, 1993
), and might be involved in promoting collagen degradation and uterine involution in the postnatal period (Jeffrey et al., 1991
). We were surprised at the low density of mast cells in the pregnant myometrial biopsies since this is in contrast with the known distribution of mast cells in non-pregnant myometrium (Mori et al., 1997
). Since mast cell mediators are capable of stimulating uterine contractions, the low density of mast cells in pregnant myometrium may be involved in the maintenance of myometrial quiescence as the uterus expands during pregnancy. Whilst we could demonstrate no change in myometrial mast cell density following the onset of labour, mast cell mediators might be capable of stimulating uterine contractions at term since the sensitivity of human myometrium to histamine and serotonin is upregulated at the end of pregnancy (Cruz et al., 1989
). This means that myometrial smooth muscle cells might be stimulated by mast cell mediators even when the mast cell density is reduced. In non-pregnant myometrium, the high density of mast cells has been proposed to have a role in implantation (Brandon and Evans, 1983
; Hore and Mehrotra, 1988
), or in remodelling uterine smooth muscle and extracellular matrix during the menstrual cycle (Mori et al., 1997
).
The mechanisms involved in the accumulation, extravasation and degranulation of inflammatory cells in uterine tissues during parturition are poorly understood. Chemotactic cytokines, including interleukin-1, tumour necrosis factor- and interleukin-8, seem to play a role (Barclay et al., 1993
; Chwalisz et al., 1994
; Osmers et al., 1995
), as well as other chemotactic agents, such as C5a (El Maradny et al., 1995
). E-selectin is involved in the infiltration of leukocytes to the maternal decidua and fetal membranes during labour (Rosenberg et al., 1996
).
Since the leukocytes in our myometrial biopsies were concentrated in and around blood vessels, we postulated that an up-regulation in the expression of vascular cell adhesion molecules in lower segment myometrium was involved in the accumulation of leukocytes in this tissue. ICAM-1, ICAM-2, PECAM and VCAM are members of the immunoglobulin superfamily (Frenette and Wagner, 1996). ICAM-1 is important in the adhesion of monocytes, lymphocytes and neutrophils to activated endothelium, whilst VCAM binds to leukocyte integrins on many cells including eosinophils and activated T lymphocytes. E-selectin, a member of the selectin family of adhesion molecules, is expressed by cytokine activated endothelial cells and has a major role in attracting neutrophils, monocytes, eosinophils and some lymphocytes (Lasky 1992
; Bevilacqua and Nelson, 1993
).
The expression of cell adhesion molecules in the endometrium of the non-pregnant uterus is well described (Tawia et al., 1993; Tabibzadeh et al., 1994
). A recent report has identified ICAM-1, VCAM, PECAM and E-selectin in pregnant human myometrium (Winkler et al., 1998
). We have shown that ICAM-1, ICAM-2, PECAM and VCAM are expressed on the vascular endothelium in myometrium obtained before the onset of labour at term and we propose that these molecules play a role in regulating leukocyte trafficking into this tissue. We found no change in the localization and intensity of staining of ICAM-1, ICAM-2, PECAM and VCAM in the biopsies obtained during labour compared with those obtained before the onset of labour. In contrast, E-selectin expression was absent in all of the biopsies collected before labour, but was expressed in three of the six biopsies obtained during labour, suggesting a role for this molecule in the recruitment of leukocytes in at least some of the tissues. These results are in broad agreement with Winkler et al. (1998), who also found that E-selectin expression was up-regulated during labour. Both our own study and that of Winkler et al. (1998) have employed immunocytochemistry, a qualitative technique. In order to confirm the changes in cell adhesion molecule expression during parturition, further studies are required using quantitative techniques.
Factors responsible for the initiation of parturition remain obscure. We have demonstrated that leukocytes infiltrate both the upper and lower uterine segments of the myometrium during spontaneous labour at term, and we propose that these cells play a fundamental role in normal parturition. A better understanding of the mechanisms involved in the initiation of labour both at term and preterm would allow the development of novel strategies to prevent premature delivery. Our results suggest that strategies aimed at preventing the influx of inflammatory cells into the myometrium could be crucial in averting preterm delivery, and thus in reducing the excess perinatal mortality and morbidity associated with this condition.
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Acknowledgments |
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Notes |
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References |
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Submitted on May 7, 1998; accepted on September 24, 1998.