1 Department of Obstetrics and Gynaecology and 2 Institute of Clinical Chemistry and Pathobiochemistry, University Hospital, Technical University (RWTH), 52057 Aachen, 3 Institute of Pathology, University of Tübingen, 72076 Tübingen and 4 Institute of Biochemistry, University of Bielefeld, 33615 Bielefeld, Germany
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Abstract |
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Key words: cervix uteri/interleukin-8/matrix metalloproteinases/neutrophils/parturition
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Introduction |
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Interleukin (IL)-8 is a cytokine that has a chemotactic effect on neutrophils (Peveri et al., 1988) and also stimulates activation and degranulation of these cells, thus provoking the release of proteolytic enzymes (Willems et al., 1989
). Human cervical tissue has been shown to produce IL-8 (Barclay et al., 1993
).
This study was undertaken to determine the nature and number of leukocytes infiltrating the stroma of the lower uterine segment during cervical dilatation, and to look for a relationship with the tissue concentrations of IL-8, matrix metalloproteinase (MMP)-8 and MMP-9.
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Materials and methods |
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Protein extraction
Specimens of frozen tissue (~100200 mg wet weight) were homogenized for 1 min (Dismembranator; Braun, Melsungen, Germany) and extracted overnight at 4°C in Tris/NaCl (0.02 mol/l TrisHCl, pH 8.5, with 0.125 mol/l NaCl) containing protease inhibitors [1 mmol/l diisopropyl flurophosphate, 10 mmol/l EDTA (disodium salt), 5 µmol/l pepstatin, and 50 µmol/l E64]. All chemicals were obtained from Sigma (Munich, Germany).
IL-8, MMP-8 and MMP-9 assays
The IL-8, MMP-8 and MMP-9 concentrations were determined in aliquots of the clarified extract (100 000 g, 45 min, 4°C). The IL-8 concentration was determined by an enzyme-linked immunosorbent assay (ELISA; human IL-8 Quantikine, R&D Systems, Abingdon, UK) using a monoclonal antibody specific for both natural and recombinant human IL-8, as described elsewhere (Osmers et al., 1995). The inter- and intra-assay coefficients of variation (CV) were between 5% and 10%. The IL-8 ELISA was performed using a zero standard and seven standard concentrations (31.52000 pg/ml), each analysis being performed in triplicate. MMP-8 and MMP-9 concentrations were determined by a sandwich ELISA described by Bergmann et al. (1989). The inter- and intra-assay CV were between 2% and 5%. The MMP-8 and MMP-9 assays were performed using standard concentrations of the latent proenzymes. The sensitivities of the MMP-8 and MMP-9 assays were 0.9425.00 ng/ml and 0.3825.00 ng/ml respectively. Each analysis was performed in triplicate. The total protein (total protein) concentration was determined by the pyrogallol method using a Dimension assay (Dade, Munich, Germany).
Investigation of leukocytes
Paraffin sections were cut at 5 µm, rehydrated and stained with Leder's stain (naphthol AS-D chloroacetate esterase reaction; Leder, 1964) for neutrophils and mast cells and immunostained by the avidinbiotinperoxidase complex method (Hsu et al., 1981
) for mast cells (tryptase), macrophages (CD68), plasma cells (VS38c) and various lymphocyte subsets (CD3, CD20 and CD57). Details of the antibodies applied are given in Table I
. The number of each of the various cell types infiltrating the stroma of the lower uterine segment was determined light microscopically by two independent observers (M.W. and T.M.), who evaluated the number of stained cells in the stroma in each of five randomly selected fields using an eyepiece with a counting grid at a magnification of x250. In cases where the number of these cells was very small, the whole section was evaluated. The number of cells per mm2 was then calculated, and the average of the values obtained by the two observers was recorded. Interobserver variability was between 10% and 150%; it was especially high in cases with very low density of neutrophils (<20/mm2).
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Results |
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A good correlation was found between the concentrations of MMP-8 and MMP-9 (Pearson's correlation coefficient: 0.63), and IL-8 and MMP-9 (Pearson's correlation coefficient: 0.63), a weaker correlation between the concentrations of IL-8 and MMP-8 (Pearson's correlation coefficient: 0.33), and no correlation between the number of stromal neutrophils and the concentrations of IL-8, MMP-8 or MMP-9 (Pearson's correlation coefficients <0.2).
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Discussion |
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The concentration of IL-8 in the lower uterine segment rose significantly up to the stage of 46 cm dilatation. The finding that there is a significant increase in the concentration of this cytokine in amniotic fluid during term labour (Romero et al., 1991) also suggests that it plays an important role in the process of parturition. Cells of the human trophoblast, decidua, amnion and chorion have been shown to release IL-8 in vitro (Saito et al., 1993
; Fortunato et al., 1995
), and synthesis by amnion and chorion cells has been demonstrated with reverse transcriptasepolymerase chain reaction (Fortunato et al., 1995
). In addition to these findings, which suggest a relationship between the rise in IL-8 concentration and the onset of labour, there is other evidence that this cytokine plays a decisive role in cervical dilatation. Local administration of IL-8 in the rabbit and guinea-pig has been found to lead to cervical ripening, with a significant increase in extensibility (Chwalisz et al., 1994
; type="bib">El Maradny et al., 1995), a decrease in collagen content and an increase in water and glycosaminoglycan content of the cervix, and increased infiltration of the cervical stroma by neutrophils (type="bib">El Maradny et al., 1994). The findings of this study are consistent with those of Osmers et al. (Osmers et al., 1995
), who found a significant increase in IL-8 concentration in the myometrium of the lower uterine segment during labour, and suggest that IL-8 is also involved in the process of cervical dilatation during parturition. Recently, Sennström et al. (1997) described an increase in IL-8 concentration in specimens obtained transvaginally from the anterior lip of the cervix of pregnant women at term before the onset of labour, compared with the concentrations in non-pregnant women, and a further increase after vaginal delivery. The finding in this study that the significant increase in IL-8 concentration continues only up to the stage of 46 cm dilatation is new. IL-8 concentrations at >6 cm dilatation were the same as at 46 cm, or slightly lower. Together with the morphological findings described here, this suggests that IL-8-induced leukocyte chemotaxis ceases to increase during the later stages of cervical dilatation.
The number of neutrophils invading the stroma of the human lower uterine segment was evaluated for the first time in this study and compared with changes in the concentrations of IL-8, MMP-8 and MMP-9. The greatest numbers of neutrophils were found at >6 cm dilatation. As long ago as 1980, Junqueira et al. reported increased infiltration of the cervical stroma by neutrophils during parturition. The presence of a fibre-free `halo' and increased amounts of amorphous ground substance in the vicinity of these granulocytes, and the positive correlation between the number of infiltrating cells and the degree of collagenolysis seen in many cases were taken as evidence of the release of proteolytic enzymes by these cells. The immunohistochemical results in this study are consistent with these findings. There was also increasing degranulation of the leukocytes up to 6 cm dilatation (Osmers et al., 1992). These observations underline the crucial role of neutrophils, not only in normal and prostaglandin-induced cervical ripening (Greer et al., 1992
), but also in cervical dilatation during parturition at term.
Animal experiments using immunohistochemical techniques have revealed an increase in mast cell numbers in the cervix during parturition (Spanggaard et al., 1997). In this study, the number of mast cells and other leukocytes was not found to be related to the degree of cervical dilatation, so it is probable that these cells play only a minor role, if any, in this process in humans.
Also increases were observed in the concentrations of the granulocyte proteases MMP-8 and MMP-9 with cervical dilatation, which is consistent with other published findings (Rath et al., 1987; Rajabi et al., 1988
; Osmers et al., 1992
; Rechberger and Woessner, 1993
). These increases occurred parallel to the invasion of the lower uterine segment stroma by granulocytes. Unlike the IL-8 concentration, these parameters continued to increase after the stage of 46 cm dilatation. The levelling off of the IL-8 concentration may suggest that there is strict temporal limitation of the degradative processes in the cervix to prevent more profound tissue damage and enable rapid regeneration of cervical structure after parturition.
The specimens investigated in this study were obtained from the lower border of the uterine incision at Caesarean section. They therefore derived from the lower uterine segment, and not the cervix. Even without the ethical problems that make transvaginal biopsy of the vaginal part of the cervix in pregnancy unjustifiable, it would be difficult to obtain enough tissue by this route to investigate all the parameters of interest here. By measuring collagenase activity, Rajabi et al. showed that the same changes occur in both the lower uterine segment and the cervix during parturition (Rajabi et al., 1988). The composition of the lower uterine segment (i.e. the cranial part of the cervix) is not, of course, the same as that of the more caudal parts of the cervix (there is, for example, proportionately less muscle), but we believe that the changes occurring in the lower uterine segment during parturition can, with some reservation, be taken to reflect those occurring in the cervix.
The findings of this study can therefore be considered to support the hypothesis that the increase in IL-8 concentration and the invasion of the stroma by neutrophils, with the subsequent release of proteolytic enzymes, play a significant role in the process of cervical dilatation during parturition.
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Acknowledgments |
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Notes |
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References |
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Submitted on June 4, 1998; accepted on December 21, 1998.