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ORIGINAL RESEARCH |
From the Department of Obstetrics and Gynaecology, University of Glasgow, Glasgow, Scotland, United Kingdom.
Address reprint requests to: Marie-Anne Ledingham, MRCOG Specialist Registrar Department of Obstetrics and Gynaecology University of Glasgow 10 Alexandra Parade Glasgow, Scotland G31 2ER United Kingdom E-mail: ml50y{at}udcf.gla.ac.uk
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Abstract |
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Methods: Biopsies of myometrium and cervix were obtained from non-pregnant women and from pregnant women before and after onset of spontaneous labor at term. Cell adhesion molecule mRNA expression was quantified using Northern blotting and cell adhesion molecule protein was localized using immunohistochemistry.
Results: ICAM-1 mRNA was upregulated in the cervix (10-fold increase, P < .01) and myometrium (10.5-fold increase, P < .01) during labor. ICAM-1 was localized in the vascular endothelium and in leukocytes in the cervix and myometrium from all three groups of women. VCAM mRNA was upregulated in the cervix (2.5-fold increase, P < .01) during pregnancy and there was no further change during labor. VCAM localized weakly to the vascular endothelium in cervical and myometrial biopsies from pregnant and non-pregnant women. PECAM mRNA was significantly upregulated in myometrium during pregnancy (ninefold increase, P < .01) and did not change with the onset of labor. PECAM localized to the vascular endothelium in all cervical and myometrial biopsies and was identified on leukocytes. There were no significant changes in E-selectin mRNA expression in either tissue with pregnancy or parturition.
Conclusion: Cell adhesion molecule expression changes in human cervix and myometrium during pregnancy and parturition. At least part of these changes are attributable to expression by leukocytes infiltrating these tissues.
Accumulating evidence suggests that parturition in the human represents an inflammatory process involving a wide variety of mediators, with leukocytes now recognized to play a crucial role.1–3 The factors controlling leukocyte infiltration into the uterus and cervix during parturition are incompletely understood. In other tissues, leukocyte infiltration from the vasculature is controlled by the expression of a number of cell adhesion molecules present on the leukocyte cell surface and the vascular endothelium.4 Recent evidence suggests that cell adhesion molecules have a variety of other biochemical and physiologic functions including the regulation of cellular differentiation, gene transcription, angiogenesis, apoptosis, and cell signalling.5–7
Improved understanding of the mechanisms involved in controlling the inflammatory events in the cervix and myometrium is crucial to understanding the control of parturition. We8 and others9,10 previously reported studies describing changes in cell adhesion molecule expression in the vascular endothelium of human lower segment myometrium. Although cell adhesion molecules have been reported in non-pregnant human cervix,11,12 their expression in the cervix during pregnancy and parturition has not been determined.
We hypothesize that cell adhesion molecules are involved in the control of leukocyte infiltration in the human cervix and myometrium during late pregnancy and labor onset. Further, we hypothesize that changes occurring in the cervix are fundamentally different from those occurring in the myometrium, reflecting the functional and structural differences between these tissues.1,13 We wanted to 1) examine the expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule (VCAM), platelet–endothelial cell adhesion molecule (PECAM), and E-selectin in the cervix during pregnancy and parturition using qualitative (immunohistochemistry) and quantitative (Northern blotting) methods, 2) determine whether expression of these adhesion molecules in the cervix and myometrium is influenced by pregnancy (by analyzing samples from non-pregnant and pregnant women), and 3) identify whether cells within the myometrium and cervix other than the vascular endothelium express adhesion molecules during pregnancy and labor.
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Materials and Methods |
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We calculated that our study had a power of 90% at the 5% significance level to detect a 2.5-fold increase in expression of ICAM-1 in laboring compared with non-laboring samples. This calculation was based on previous data obtained in our laboratory on mean (±SD) expression of ICAM-1 in non-laboring tissues.
Cervical biopsies in non-pregnant women were taken from anterior cervical lips immediately after hysterectomy. In pregnant women not in labor, biopsies were obtained from the lower parts of the cervixes per vaginam before cesarean delivery. Cervical biopsies in women in labor were taken after they bore infants, with cervixes approached through uterine incisions. Myometrial biopsies in non-pregnant women were obtained from the anterior walls of lower uterine bodies after removal of uteri at the time of hysterectomy. In pregnant women, myometrial biopsies were obtained from the upper margins of lower uterine incisions at cesarean deliveries. In all myometrial biopsies, myometria were separated from surrounding structures, ie, endometrium and decidua, by sharp dissection.
Half of the cervical and myometrial samples were snap frozen in liquid nitrogen and stored at -70C for Northern blotting and half were fixed in 10% neutral buffered formalin and paraffin embedded for immunohistochemistry. We confirmed tissue origin by immunohistochemistry using antimyosin smooth muscle antibody to quantify the smooth muscle content of paraffin-embedded sections of cervical and myometrial biopsies (Table 1
). Leukocytes in each tissue were examined using immunohistochemistry with an antibody directed against CD45, the common leukocyte antigen1 (Table 1).
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We hybridized nylon filters in the same buffer overnight with the appropriate 32P-labeled (Oligolabelling kit, Pharmacia Biotech) cDNA added to the prehybridization buffer. ICAM-1 (1.8-kb insert), VCAM (1.9 kb), and PECAM (2.5 kb) cDNA were a gift from Dr. Simmons (Oxford, UK). E-selectin (1.2 kb) cDNA was a gift from Dr. M. Bevilacqua (San Diego, CA). The cDNA probe for the housekeeping gene human glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1.1 kb) was purchased from Clontech Laboratories UK Ltd. (Basingstoke, UK). We washed the nylon filters to a final stringency of 0.5x sodium saline citrate, 0.1% sodium dodecyl sulphate at 65C and autoradiography was done with Fuji x-ray film at -70C for 3 days. The intensity of the bands on the autoradiographs for each cell adhesion molecule was compared with GAPDH and the ratio was determined.
Human umbilical vein endothelial cells were isolated by collagenase digestion. Once confluent, we incubated the cells in medium containing interleukin (IL)–1ß (10 IU/mL) for 4 hours and used those as a positive control for the expression of ICAM-1, VCAM, PECAM, and E-selectin. Total RNA was extracted and loaded at 5 µg per lane.
We extracted total RNA from cervical samples (n = 6 in each group) using the Trizol method. We quantified RNA by ultraviolet spectrophotometry and confirmed its integrity. We added RNA sample loading buffer to 10 µg of total RNA and separated it on 1.2% agarose gels as for myometrial samples. We electrophoresed gels at 60 V for 2.5 hours. We transferred RNA onto Hybond-N nylon membranes overnight in 20x sodium saline citrate and fixed it to the membrane by ultraviolet irradiation. We prehybridized membranes for 1–2 hours at 42C in 12 mL of Ultrahyb (AMS Biotechnology, Oxon, UK) and then hybridized these with the appropriate 32P-labeled cDNA added to the prehybridization buffer. We washed the nylon filters and autoradiography was done at -70C for 2–7 days.
Human umbilical vein endothelial cells were used as a positive control for the expression of each cell adhesion molecule.
We used immunohistochemistry on paraffin-embedded cervical and myometrial biopsies (n = 8 in each group) using antibodies against ICAM-1, VCAM, PECAM, E-selectin, smooth muscle myosin, and CD45.1 Table 1
shows the antibodies used. We localized antigen using 1 mg/mL diaminobenzidene tetrahydrochloride (Sigma), 0.02% H2O2 in 50 mM Tris-Cl, pH 7.6, and the antigen appeared as a brown endproduct that was counterstained with Harris hematoxylin. As negative controls, we used slides incubated without the primary antibody and sections incubated with mouse monoclonal antibody against immunoglobulin G1 Aspergillus niger glucose oxidase (Dako Ltd., Buckinghamshire, UK), an enzyme that is neither present nor inducible in mammalian tissues. We used placentas as positive controls for ICAM-1 and PECAM. As positive controls, we used arthritic joint for VCAM14 and kidney for E-selectin.15
The intensity of the bands on the autoradiographs for each cell adhesion molecule undergoing Northern analysis was compared with GAPDH and the ratio was determined using Bio-Rad Multi-Analyst/PC 1.1. We compared ratios between groups (non-pregnant versus pregnant not in labor versus pregnant in labor) to investigate changes in mRNA expression during pregnancy and parturition. Statistical analysis of band intensity for Northern analysis was done using the Kruskal–Wallis test with the Mann–Whitney U test as a post hoc test. P less than .05 was considered significant.
For immunohistochemical analysis, two observers (M.A.L. and A.J.T.) blinded to the source of the biopsy analyzed each section and the localization of staining was recorded for each antibody used. Staining for CD45 was used to colocalize leukocytes in the tissues.
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Results |
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Northern blotting confirmed the presence of mRNA for ICAM-1 in myometrial and cervical samples (Figure 1). There were significant changes in the expression of ICAM-1 across the three groups in the cervix and myometrium (P = .009 and .03, respectively, Kruskal–Wallis test). The expression of ICAM-1 mRNA was 10-fold greater in laboring cervix than in non-laboring cervix (P < .01; Figure 2) and 10.5-fold greater in laboring myometrium than in non-laboring myometrium (P < .01). There was no significant change in the expression of ICAM-1 between the non-laboring and non-pregnant groups. Immunohistochemistry localized ICAM-1 strongly to the vascular endothelium in both tissues (Figure 3a and b). In pregnant samples, ICAM-1 was present in leukocytes that had infiltrated the tissues, as confirmed by CD45 staining (Figure 3c). Substantially greater numbers of these cells were visualized in the laboring samples from both tissues. Staining was absent in the negative control slides.
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). There were significant changes in the cervical expression of VCAM mRNA in the three groups (P = .003, Kruskal–Wallis test). Expression was increased 2.5-fold in the cervix during pregnancy (P < .01) with no further increase with labor onset (Figure 5). The expression of VCAM in the myometrium was not altered during pregnancy. VCAM localized weakly to the vascular endothelium in the cervix and myometrium from all groups of women. Staining was also identified in leukocytes (Figure 3d). We did not observe staining in the negative control slides.
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). Expression of PECAM mRNA increased ninefold in pregnant myometria compared with non-pregnant samples (P < .01), with no further change with labor onset (Figure 7). There was no corresponding change in cervical tissue. Immunohistochemistry localized PECAM strongly to the vascular endothelium in both tissues and to leukocytes (Figure 3e).
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Discussion |
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In view of the technical difficulties involved in obtaining cervical biopsies for research purposes, several investigators suggested that the lower uterine segment might be representative of the cervix.9,16,17 However, the cervix and myometrium are structurally and functionally different during pregnancy and labor.13 In the cervix at term, a significantly greater number of neutrophils and macrophages are present compared with the first trimester, with no increase after labor onset.3 In contrast, in the myometrium, neutrophils and macrophages are sparse in the lower segment myometrium before labor and abundant during labor.3 In this study, we characterized the structure of each myometrial and cervical biopsy by determining the smooth muscle content and confirmed that lower segment myometrium is composed of substantially more smooth muscle during pregnancy and labor in comparison with the cervix. Our results showing different changes in VCAM and PECAM mRNA in cervixes and lower segment myometria provide further evidence that lower segment myometrium cannot be considered representative of the cervix for the purpose of research in this field.
We found that infiltrating leukocytes in the laboring cervix and myometrium expressed ICAM-1, VCAM, and PECAM. In light of these immunohistochemical findings, we conclude that the increase in ICAM-1 mRNA expression within the lower segment myometrium and cervix with the onset of labor seems to be attributable, at least in part, to the leukocytic infiltration occurring in these tissues with the onset of labor. Leukocytic influx is a characteristic feature in the cervix and myometrium before and at labor onset. Leukocytes expressing ICAM-1 might modulate cell signaling pathways, activation of cytokines and metalloproteinases, and apoptosis, events that occur in the cervix and myometrium around parturition onset.
We showed an increase in VCAM expression in the cervix during pregnancy. VCAM is principally expressed on endothelial cells but is also found on macrophages, dendritic cells, and bone marrow fibroblasts and plays a significant role in the migration of leukocytes that express VLA-4, ie, lymphocytes, monocytes eosinophils, and basophils. VCAM is not expressed on endothelial cells in the resting state but, like ICAM-1, is upregulated by inflammatory mediators such as IL-1ß, IL-4, tumor necrosis factor-
, and IL-13. Prostaglandins also might regulate VCAM expression.18 Because prostaglandins, IL-1ß, and tumor necrosis factor- are also recognized mediators of cervical ripening,19–21 the increased expression of VCAM in the cervix during pregnancy might reflect one of the mechanisms of action of these agents.
PECAM is constitutively found on platelets, leukocytes, and endothelial cells and mediates leukocyte–endothelial and platelet–endothelial interactions. It is essential for the transendothelial migration of leukocytes through intracellular junctions of vascular endothelial cells. Its transcription is not affected by cytokine release. PECAM mRNA increased in the myometrium during pregnancy with no further increase with labor onset. The reason for the increase in PECAM expression within the myometrium during pregnancy is unclear but might reflect the involvement of this cell adhesion molecule in cell signaling and angiogenesis.
E-selectin is expressed by cytokine-activated endothelial cells and mediates neutrophil, monocyte, and lymphocyte recruitment.22 In this study, we found no change in E-selectin expression in either tissue during pregnancy or labor. These results might reflect the small sample size studied and further investigations are required to determine whether E-selectin expression is altered during pregnancy.
Recent research suggested that cell adhesion molecules are potential targets for therapeutic intervention.23 Antibodies directed against cell adhesion molecules might have a major functional role in the treatment of such inflammatory conditions as rheumatoid arthritis,24 allergic airways disease and asthma,25 inflammatory bowel disease,26 stroke,27 and ischemia reperfusion injury.28 The mechanisms controlling the onset of human parturition remain unclear. However, we demonstrated that the processes of cervical ripening and the initiation of labor constitute an inflammatory response with increased cell adhesion molecule expression in the cervix and myometrium. Improved understanding of the mechanisms involved in these events will ultimately lead to new therapeutic strategies for the treatment of preterm labor, which remains a leading cause of neonatal morbidity and mortality despite improvements in neonatal intensive care facilities. In the future, antibodies directed against cell adhesion molecules in the cervix and myometrium might be beneficial in reducing the morbidity and mortality associated with preterm labor.
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Footnotes |
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Received June 7, 2000. Received in revised form September 27, 2000. Accepted October 12, 2000.
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8. Thomson A, Greer M, Young A, et al. Expression of intercellular adhesion molecules ICAM-1 and ICAM-2 in human endometrium: Regulation by interferon-gamma. Mol Hum Reprod 1999;5:64–70.
9. Winkler M, Ruck P, Horny HP, et al. Expression of cell adhesion molecules by endothelium in the human lower uterine segment during parturition at term. Am J Obstet Gynecol 1998;178:557–61.[Medline]
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