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Expression and localization of cell adhesion molecules in human fetal membranes during parturition
Journal of Reproductive Immunology 63 (2004) 11–21
Expression and localization of cell adhesion molecules in human fetal membranes during parturition Inass Osman∗ , Marshia Crawford, Fiona Jordan, Anne Young, Jane Norman, Andrew Thomson Reproductive and Maternal Medicine, Division of Developmental Medicine, Glasgow Royal Infirmary, 10 Alexandra Parade, Glasgow G31 2ER, UK Received in revised form 16 March 2004; accepted 19 April 2004
Abstract There is increasing evidence to support the view that human parturition represents an inflammatory process. We have previously demonstrated that parturition is associated with leukocyte invasion and pro-inflammatory cytokine production in the cervix and myometrium. Furthermore, we have shown that several cell adhesion molecules are upregulated in these tissues during labor. In fetal membranes, previous studies have shown intercellular adhesion molecule-1 (ICAM-1) upregulation in association with labor. The role of other adhesion molecules has not been explored. The aims of this study were, therefore, to determine the expression of ICAM-1, platelet endothelial cell adhesion molecule (PECAM), vascular cell adhesion molecule (VCAM) and E-selectin in pre- and post-laboring amnion and choriodecidua and to identify cell types responsible for their expression. Biopsies of fetal membranes were obtained from pregnant women delivered by caesarean section before the onset of labor (n = 8) and following spontaneous vaginal delivery (n = 8). Cell adhesion molecules were identified using immunohistochemistry and messenger RNA expression quantified using Northern analysis. We found that following labor, ICAM-1 mRNA expression was significantly upregulated in amnion and choriodecidua (P < 0.05). PECAM mRNA expression was also increased in choriodecidua (P < 0.05). The main cell types responsible for adhesion molecule expression were leukocytes, amniotic epithelial cells and endothelial cells.
∗ Corresponding author. Tel.: +44 141 211 4702; fax: +44 141 552 0873. E-mail address: [email protected] (I. Osman).
0165-0378/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jri.2004.04.003
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The upregulation of ICAM-1 and PECAM mRNA expression in fetal membranes following labor provides further evidence that fetal membranes play an important role in the inflammatory process of parturition. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Labor; Cell adhesion molecules; Fetal membranes
1. Introduction There is mounting evidence supporting the role of inflammation during human parturition (Bowen et al., 2002; Kelly, 2002; Yellon et al., 2003). A hallmark of inflammation is the extravasation of leukocytes into tissues, which requires the expression of cell adhesion molecules. In general, cell adhesion molecules aid recruitment, adhesion and transendothelial migration of leukocytes into tissues (Smith et al., 1989; Springer, 1994; Van de Stolpe and Van der Saag, 1996). We and others have reported the infiltration of leukocytes into uterine tissues and increased expression of pro-inflammatory cytokines co-incident with parturition (Junqueira et al., 1980; Thomson et al., 1999; Osman et al., 2003). In addition, we have reported an increase in the expression of the cell adhesion molecules intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule (VCAM) and platelet endothelial cell adhesion molecule (PECAM) messenger RNA in cervix and myometrium (Ledingham et al., 2001). The fetal membranes play a key role in the inflammatory response of term labor through increased production of pro-inflammatory cytokines, matrix metalloproteinases (MMPs) and prostaglandins via inducible cyclo-oxygenase 2 (Bryant-Greenwood and Yamamoto, 1995; Vadillo-Ortega et al., 1995; Dudley et al., 1996; Keelan et al., 1999; Sawdy et al., 2000). In association with these inflammatory events, we speculate that there is a greater expression of cell adhesion molecules by fetal membranes during parturition. The role of only one cell adhesion molecule, ICAM-1, has previously been studied in relation to labor in fetal membranes. ICAM-1 was found to be upregulated in choriodecidua during preterm labor, and amnion during preterm and term labor (Marvin et al., 1999b, 2000). At present, no studies have determined which cells are responsible for this increased expression of ICAM-1 in fetal membranes during term and preterm labor. Our aims were, therefore, (i) to determine the expression of ICAM-1, PECAM, E-selectin and VCAM in amnion and choriodecidua during parturition at term using Northern blotting and (ii) to determine which cells are responsible for the expression of cell adhesion molecules in fetal membranes using immunohistochemistry.
2. Materials and methods 2.1. Subjects Biopsies of fetal membranes were obtained from pregnant women delivered at term (>37 weeks gestation) (i) by caesarean section before the onset of labor (n = 8) and (ii) following
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spontaneous vaginal delivery (n = 8). Fetal membranes were sampled in accordance with techniques described by McLaren et al., 2000; Malak and Bell, 1994. Briefly, this involved sampling from the zone of altered morphology, which is the area in which membrane rupture arises. The zone of altered morphology was assessed since previous studies state that this is the site where inflammatory changes and extracellular matrix re-modelling occurs. This region is characterized by thinning of the cytotrophoblast layer and decidua, and thickening of connective tissue. In group (i), membranes were obtained from the portion of membranes overlying the uterine cervix following delivery of the baby. This region was identified by application of a Babcock’s forcep, and an area of approximately 5 cm2 was then removed from around the forcep. For group (ii), tissue was removed from along the line of fetal membrane rupture. Women with clinical evidence of infection, multiple pregnancy, dysfunctional labor and those who had received prostaglandin or artificial oxytocin were excluded. There were no differences in maternal age, parity or gestational age of the patients recruited. Written informed consent was obtained from each woman prior to recruitment and the study was approved by the Local Research Ethics Committee. In each biopsy, amnion was separated from choriodecidua by dissection. All samples were either fixed in 10% buffered formalin (BDH, Poole, UK) and embedded in paraffin or snap-frozen in liquid nitrogen and stored at −70 ◦ C for total RNA extraction. 2.2. Northern analysis Total RNA was extracted from amnion and choriodecidua using the RNAzol B method (Biogenesis, Bournemouth, UK) and quantified using Northern blotting. Isolated RNA was further dissolved in distilled water treated with diethylpyrocarbonate and quantified using ultraviolet spectrophotometry. Ten microliters of RNA sample loading buffer was added to 10 g of total RNA and the resulting solution separated onto 1.2% agarose gels. Gels were electrophoresed at 60 V for 2.5 h. RNA was transferred and fixed by ultraviolet irradiation onto Hybond-N nylon membranes in 20× sodium saline citrate overnight. Membranes were prehybridized for 2–3 h at 42 ◦ C in 12 ml of UltrahybTM (AMS Biotecnology, Oxon, UK) and then hybridized in the same buffer overnight with the appropriate 32 P-labelled cDNA probes (Oligolabelling kit, Pharmacia, Biotech) 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). The nylon filters were washed to a final stringency of 0.5× sodium saline citrate, 0.1% sodium dodecyl sulphate at 65 ◦ C and autoradiographs for each cell adhesion molecule were compared with GAPDH and the ratio determined. For a positive control of ICAM-1, VCAM, PECAM and E-selectin expression, primary cultures of human umbilical vein endothelial cells were isolated with collagen digestion (0.1% collagenase type II, Sigma, UK) and then confluent cultures were incubated with interleukin-1 (10 IU/ml) for 4 h. Total RNA was extracted and loaded at 5 g per lane. 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 (pregnant not in labor
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versus pregnant in labor) to investigate changes in mRNA expression during pregnancy and parturition. 2.3. Immunohistochemistry Cell adhesion molecules were identified using primary antibodies directed against ICAM-1, VCAM, PECAM and E-selectin using the ABC-peroxidase method on biopsies of paraffin-embedded amnion and choriodecidua before and after labor (n = 8 in each group). Leukocytes were immunolocalized in each tissue using an antibody directed against CD45, the common leukocyte antigen. In order to identify decidua and trophoblast, respectively, immunohistochemistry was undertaken using antibodies directed against vimentin and cytokeratins, thereby facilitating the localization of cell adhesion molecule staining (Table 1). PECAM (CD 31) immunostaining identified blood vessels (Parums et al., 1990) within fetal membranes. Paraffin-embedded tissue sections (5 m thick) were mounted on silane-coated slides and heated to 60 ◦ C for 35 min. They were then deparaffinized using xylene and gradually rehydrated with ethanol. Some sections required pretreatment by microwaving at full power for 4 × 5 min in citrate buffer to retrieve the antigen (Table 1). Preincubation was carried out with 1.5% (w/v) normal horse serum in phosphate-buffered solution (PBS: 10 mM sodium phosphate, pH 7.5, 120 mM sodium chloride) for 30 min at room temperature. Thereafter, slides were incubated for 60 min with the primary antibody diluted in 1.5% horse serum and then washed with PBS. Further incubation was carried out with biotinylated anti-mouse immunoglobulin (Vector Laboratories, Peterborough, UK) diluted in 1.5% horse serum and 1.5% normal human serum. Sections were washed in PBS and then incubated with avidin DH/biotinylated horseradish peroxidase H reagent (Vector Laboratories, Peterborough, UK) in PBS for 30 min before final washing. The antigen was localized using 1 mg/ml diaminobenzidene tetrahydrochloride (Sigma, UK), 0.02% H2 O2 in 50 mM Tris–HCl, pH 7.6, which appeared as a brown end product. Sections were then counterstained with Harris haematoxylin (Sigma, UK). For negative controls, some slides were incubated without the primary antibody and others with mouse monoclonal antibody against IgG1 Aspergillus niger glucose oxidase (Dako), an enzyme that is neither present nor inducible in mammalian tissues. The positive tissue controls for each of the antibodies used are listed in Table 1. The immunohistochemistry slides were examined by observers who were blinded to the clinical condition of the patients. Table 1 Primary antibodies used for immunohistochemistry Antibody
Clone
Pretreatment
Dilution
Source
Positive control tissue
E-selectin ICAM-1 PECAM VCAM CD 45 Vimentin Cytokeratin
Polyclonal Polyclonal Clone JC/70A Polyclonal 2B11 + PD7/26 Polyclonal Clone MNF116
No Microwave Microwave Microwave Microwave No Trypsin
1:500 1:1000 1:500 1:500 1:100 1:150 1:150
R + D, Abingdon, UK R + D, Abingdon, UK Dako, Cambridge, UK R + D, Abingdon, UK Dako, Cambridge, UK Euro-path Cornwall, UK Dako, Cambridge, UK
Kidney Placenta Placenta Arthritic joint Tonsil Decidua Placenta
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2.4. Statistical analysis Statistical analysis of autoradiograph band intensity for Northern analysis was performed using Kruskal–Wallis with Mann–Whitney-U as a post hoc test. A P-value of less than 0.05 was considered significant. SPSS version 8.0 was the statistics package employed for data analysis. Minitab version 13.1 was used to produce the boxplot graphs. 3. Results 3.1. Histology Histological examination confirmed that tissue specimens were of amnion and choriodecidua. The amnion consists of a layer of amniotic epithelial cells and its supporting connective tissue. The chorion is composed of several layers of epithelial cells known as the trophoblast, a basement membrane and its connective tissue, called the reticular layer, and stromal cells of the maternal decidua lying adjacent to the chorionic trophoblast (Malak and Bell, 1994). 3.2. ICAM-1 expression and localization Messenger RNA for the cell adhesion molecule, ICAM-1, was identified in pre- and post-laboring amnion and choriodecidua. ICAM-1 mRNA expression was significantly greater in amnion (P < 0.05) and choriodecidua (P < 0.01) in tissues obtained following spontaneous labor compared with non-laboring tissues (Figs. 1, 2 and 3a,b). In the amnion, ICAM-1 was identified using immunohistochemistry in amniotic epithelial cells and leukocytes. ICAM-1 was also identified in the vascular endothelium, extravillous trophoblast and stromal cells of the decidua (Fig. 4a–d). However, the immunostaining was stronger and more consistent in tissues collected following labor than in those collected before labor.
Fig. 1. Northern blot hybridization of total RNA (5 g per lane) from human amnion samples (n = 8) collected (a) before labor and (b) after spontaneous labor at term. Intercellular adhesion molecule-1 (ICAM-1) hybridization was compared with the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The expression of ICAM-1 mRNA was upregulated in amnion following labor.
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Fig. 2. Northern blot hybridization of total RNA (5 g per lane) from human choriodecidua samples (n = 8) collected (a) before labor and (b) after spontaneous labor at term. Intercellular adhesion molecule (ICAM)-1 hybridization was compared with the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The expression of ICAM-1 mRNA was upregulated in choriodecidua following labor.
3.3. PECAM expression and localization The messenger RNA for PECAM was expressed in amnion and choriodecidua both before and after labor. There was a significant increase in PECAM mRNA expression (P < 0.05) after the onset of labor in choriodecidua (Figs. 3b and 5). In the amnion, labor did not alter PECAM mRNA expression. PECAM protein localized to leukocytes in amnion (Fig. 4e,f), but was absent from the amniotic epithelial cells. In choriodecidua, PECAM was identified in vascular endothelium (Fig. 4g) and in leukocytes (Fig. 4h). 3.4. VCAM expression and localization Messenger RNA for VCAM was not detected in either pre- or post-laboring amnion. Bands for VCAM mRNA were detected in choriodecidua (Fig. 3b). VCAM immunolocalized to cells within the chorionic connective tissue. These were PECAM (CD 31)- and CD 45-negative, implying they are neither endothelial cells nor leukocytes (Fig. 4k,l). We speculate, therefore, that they are fibroblasts or myofibroblasts. 3.5. E-selectin expression and localization E-selectin was not identified in amnion or choriodecidua before or after labor. Additionally, messenger RNA for E-selectin was not detected in amnion or choriodecidua regardless of labor status.
4. Discussion This report describes the change in expression of several key cell adhesion molecules within term fetal membranes. We have also identified specific cell types responsible for
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Fig. 3. Boxplot graphs demonstrating the median and interquartile ranges of the densitometric ratios of the cell adhesion molecules identified (a) in the amnion before and after labor and (b) in the choriodecidua before and after labor. ICAM-1 mRNA expression was significantly greater in amnion (P < 0.05) and choriodecidua (P < 0.01) in tissues obtained after labor than before. PECAM mRNA expression was significantly greater in laboring compared to non-laboring choriodecidua (P < 0.05).
the expression of cell adhesion molecules. During term parturition, there is a significant increase in ICAM-1 mRNA expression in amnion and choriodecidua and PECAM mRNA expression in choriodecidua. There were no significant changes in VCAM expression in either amnion or choriodecidua. Neither E-selectin mRNA nor antigen was detected in amnion or choriodecidua. Using immunohistochemistry, ICAM-1 immunolocalized to amniotic epithelial cells and leukocytes, suggesting that increased ICAM-1 mRNA expression is attributable to these cell types. In a recent study, we have shown no increase in leukocyte density in amnion
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Fig. 4. Immunolocalization of cell adhesion molecules in human fetal membranes (amnion and choriodecidua). In amnion, intercellular adhesion molecule (ICAM)-1 localized to amniotic epithelial cells and leukocytes in samples obtained (a) before and (b) after labor. In choriodecidua obtained (c) before and (d) after labor, ICAM-1 localized to the vascular endothelium, extravillous trophoblast and stromal cells of the decidua. In amnion, platelet endothelial cell adhesion molecule (PECAM) antigen was identified on leukocytes, which were sparse (e) before and (f) after labor. In choriodecidua, PECAM was identified on vascular endothelium (g) and in leukocytes (h). Vascular cell adhesion molecule (VCAM) was not identified in amnion collected (i) before or (j) after labor. In samples of choriodecidua collected (k) before and (l) after labor, VCAM was identified within cells of the chorionic connective tissue (indicated in (k) and (l) by arrows). (Scale bars = 50 m. The arrows in (e), (f) and (h) indicate leukocytes. The light blue background is haematoxylin counterstain. D indicates decidual stromal cells and EVT indicates extravillous trophoblast). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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Fig. 5. Northern blot hybridization of total RNA (5 g per lane) from samples of choriodecidua (n = 8) collected (a) before labor and (b) after spontaneous labor. Platelet endothelial cell adhesion molecule (PECAM) hybridization was compared with the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The expression of PECAM mRNA was upregulated in choriodecidua during labor.
or choriodecidua during labor (Osman et al., 2003). These data suggest that the change in ICAM-1 mRNA expression is a result of increased expression by existing cells and, consistent with this, previous studies have shown that ICAM-1 expression is inducible in amniotic epithelial cells (Marvin et al., 1999a). Previous studies have focused on quantification of ICAM-1 expression in fetal membranes during term and preterm labor using Northern analysis and ELISA (Marvin et al., 1999b, 2000). In these reports, ICAM-1 mRNA in amnion was upregulated following term and preterm labor. Following preterm labor, ICAM-1 mRNA expression was again increased in choridecidua, but there was no significant difference in expression following term labor. This is in contrast to our findings of ICAM-1 mRNA upregulation in choriodecidua during this time. Subject selection may explain this discrepancy; we did not include patients who received induction or augmentation agents during labor. It may also be related to sampling technique. Our post-laboring biopsies were removed from the zone of altered morphology (ZAM), the structurally weakened area in which membrane rupture arises (Malak and Bell, 1994). The region of fetal membranes sampled in the studies by Marvin et al. (1999b, 2000) was not stated. If ICAM-1 increases within the zone of altered morphology during parturition but not elsewhere in fetal membranes, it may account for the apparent discrepancy between our results and those of Marvin et al. (1999b, 2000). In other tissue types, numerous factors are known to control ICAM-1 expression, including pro-inflammatory cytokines such as IL-1, TNF-␣ and interferon-␥ (IFN-␥), hormones and viral infections (Roebuck and Finnegan, 1999). Cell-culture studies have shown that IL-1 and TNF-␣ regulate ICAM-1 expression in amnion-derived WISH cells in the absence of leukocytes (Marvin et al., 1999a). These cytokines stimulate inducible promoter regions on the ICAM-1 gene, increasing its expression. Furthermore, nuclear factor-kappa B is considered one of the most important transcription factors mediating the induction of ICAM-1 (Roebuck and Finnegan, 1999). PECAM is considered to be constitutively ex-
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pressed; however, expression can be stimulated by IFN-␥ (Tang and Hendricks, 1996). As with ICAM-1, endothelial VCAM requires induction by cytokines such as TNF-␣, IL-1 and IL-4. The primary function of cell adhesion molecules is to mediate transendothelial migration of leukocytes during inflammation and, as we have shown, ICAM-1 and PECAM mRNA are upregulated within fetal membranes during labor. There is however no associated increase in leukocyte density suggesting that, in fetal membranes with labor, adhesion molecules have additional roles within this tissue (Osman et al., 2003). Acting through physical and biochemical signalling, cell adhesion molecules are known to mediate other physiological processes such as gene transcription and apoptosis (Bischoff, 1997; Freemont, 1998). These mechanisms, perhaps influenced by cell adhesion molecules, are pertinent to changes in the ZAM prior to and during membrane rupture. In summary, we have demonstrated that the inflammatory process of spontaneous term labor is associated with increased cell adhesion molecule expression by fetal membranes. The control of expression of these molecules may represent new targets in the control of labor.
Acknowledgements This work was supported by a grant from the Glasgow Royal Infirmary Research and Development Endowment fund for which we are grateful. Reference number 0501728 (02REF0030-Osman).
References Bischoff, J., 1997. Cell adhesion and angiogenesis. J. Clin. Invest. 100, S37–S39. Bowen, J.M., Chamley, L., Keelan, J.A., Mitchell, M.D., 2002. Cytokines of the placenta and extra-placental membranes: roles and regulation during human pregnancy and parturition. Placenta 23, 257–273. Bryant-Greenwood, G.D., Yamamoto, S.Y., 1995. Control of peripartal collagenolysis in the human chorion-decidua. Am. J. Obstet. Gynecol. 172, 63–70. Dudley, D.J., Collmer, D., Mitchell, M.D., Trautman, M.S., 1996. Inflammatory cytokine mRNA in human gestational tissues: implications for term and preterm labor. J. Soc. Gynecol. Investig. 3, 328–335. Freemont, A.J., 1998. Demystified . . . adhesion molecules. Mol. Pathol. 51, 175–184. Junqueira, L.C., Zugaib, M., Montes, G.S., Toledo, O.M., Krisztan, R.M., Shigihara, K.M., 1980. Morphologic and histochemical evidence for the occurrence of collagenolysis and for the role of neutrophilic polymorphonuclear leukocytes during cervical dilation. Am. J. Obstet. Gynecol. 138, 273–281. Keelan, J.A., Marvin, K.W., Sato, T.A., Coleman, M., McCowan, L.M., Mitchell, M.D., 1999. Cytokine abundance in placental tissues: evidence of inflammatory activation in gestational membranes with term and preterm parturition. Am. J. Obstet. Gynecol. 181, 1530–1536. Kelly, R.W., 2002. Inflammatory mediators and cervical ripening. J. Reprod. Immunol. 57, 217–224. Ledingham, M.A., Thomson, A.J., Jordan, F., Young, A., Crawford, M., Norman, J.E., 2001. Cell adhesion molecule expression in the cervix and myometrium during pregnancy and parturition. Obstet. Gynecol. 97, 235–242. Malak, T.M., Bell, S.C., 1994. Structural characteristics of term human fetal membranes: a novel zone of extreme morphological alteration within the rupture site. Br. J. Obstet. Gynaecol. 101, 375–386.
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Marvin, K.W., Hansen, W.R., Miller, H.C., Eykholt, R.L., Mitchell, M.D., 1999a. Amnion-derived cells express intercellular adhesion molecule-1: Regulation by cytokines. J. Mol. Endocrinol. 22, 193–205. Marvin, K.W., Keelan, J.A., Sato, T.A., Coleman, M.A.G., McCowan, L.M.E., Mitchell, M.D., 1999b. Expression of intercellular adhesion molecule-1 (ICAM-1) in choriodecidua with labour and delivery at term and preterm. Reprod. Fertil. Dev. 11, 255–262. Marvin, K.W., Keelan, J.A., Sato, T.A., Coleman, M.A.G., McCowan, L.M.E., Miller, H.C., Mitchell, M.D., 2000. Enhanced expression of intercellular adhesion molecule-1 (ICAM-1) in amnion with term and preterm labour. Placenta 21, 115–121. McLaren, J., Taylor, D.J., Bell, S.C., 2000. Prostaglandin E2-dependent production of latent matrix metalloproteinase-9 in cultures of human fetal membranes. Mol. Hum.Reprod. 6, 1033–1040. Osman, I., Young, A., Ledingham, M.A., Thomson, A.J., Jordan, F., Greer, I.A., Norman, J.E., 2003. Leukocyte density and pro-inflammatory cytokine expression in human fetal membranes, decidua, cervix and myometrium before and during labour at term. Mol. Hum. Reprod. 9, 41–45. Parums, D.V., Cordell, J.L., Micklem, K., Heryet, A.R., Gatter, K.C., Mason, D.Y., 1990. JC70: A new monoclonal antibody that detects vascular endothelium associated antigen on routinely processed tissue sections. J. Clin. Pathol. 43, 752–757. Roebuck, K.A., Finnegan, A., 1999. Regulation of intercellular adhesion molecule-1 (CD54) gene expression. J. Leukoc. Biol. 66, 876–888. Sawdy, R.J., Slater, D., Dennes, W.J., Sullivan, M.H., Bennett, P.R., 2000. The roles of the cyclo-oxygenases types one and two in prostaglandin synthesis in human fetal membranes at term. Placenta 21, 54–57. Smith, C.W., Marlin, S.D., Rothlein, R., Toman, C., Anderson, D.C., 1989. Cooperative interactions of LFA-1 and Mac-1 with intercellular adhesion molecule-1 in facilitating adherence and transendothelial migration of human neutrophils in vitro. J. Clin. Invest. 83, 2008–2017. Springer, T.A., 1994. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 76, 301–314. Tang, Q., Hendricks, R.L., 1996. Interferon gamma regulates platelet endothelial cell adhesion molecule 1 expression and neutrophil infiltration into herpes simplex virus- infected mouse corneas. J. Exp. Med. 184, 1435–1447. Thomson, A.J., Telfer, J.F., Young, A., Campbell, S., Stewart, C.J., Cameron, I.T., Greer, I.A., Norman, J.E., 1999. Leukocytes infiltrate the myometrium during human parturition: further evidence that labour is an inflammatory process. Hum. Reprod. 14, 229–236. Vadillo-Ortega, F., Gonzalez-Avila, G., Furth, E.E., Lei, H., Muschel, R.J., Stetler-Stevenson, W.G., Strauss 3rd, J.F., 1995. 92kd type IV collagenase (matrix metalloproteinase-9) activity in human amniochorion increases with labor. Am. J. Pathol. 146, 148–156. Van de Stolpe, A., Van der Saag, P.T., 1996. Intercellular adhesion molecule-1*. J. Mol. Med. 74, 13–33. Yellon, S.M., Mackler, A.M., Kirby, M.A., 2003. The role of leukocyte traffic and activation in parturition. J. Soc. Gynecol. Investig. 10, 323–338.
Expression and localization of cell adhesion molecules in human fetal membranes during parturition Inass Osman∗ , Marshia Crawford, Fiona Jordan, Anne Young, Jane Norman, Andrew Thomson Reproductive and Maternal Medicine, Division of Developmental Medicine, Glasgow Royal Infirmary, 10 Alexandra Parade, Glasgow G31 2ER, UK Received in revised form 16 March 2004; accepted 19 April 2004
Abstract There is increasing evidence to support the view that human parturition represents an inflammatory process. We have previously demonstrated that parturition is associated with leukocyte invasion and pro-inflammatory cytokine production in the cervix and myometrium. Furthermore, we have shown that several cell adhesion molecules are upregulated in these tissues during labor. In fetal membranes, previous studies have shown intercellular adhesion molecule-1 (ICAM-1) upregulation in association with labor. The role of other adhesion molecules has not been explored. The aims of this study were, therefore, to determine the expression of ICAM-1, platelet endothelial cell adhesion molecule (PECAM), vascular cell adhesion molecule (VCAM) and E-selectin in pre- and post-laboring amnion and choriodecidua and to identify cell types responsible for their expression. Biopsies of fetal membranes were obtained from pregnant women delivered by caesarean section before the onset of labor (n = 8) and following spontaneous vaginal delivery (n = 8). Cell adhesion molecules were identified using immunohistochemistry and messenger RNA expression quantified using Northern analysis. We found that following labor, ICAM-1 mRNA expression was significantly upregulated in amnion and choriodecidua (P < 0.05). PECAM mRNA expression was also increased in choriodecidua (P < 0.05). The main cell types responsible for adhesion molecule expression were leukocytes, amniotic epithelial cells and endothelial cells.
∗ Corresponding author. Tel.: +44 141 211 4702; fax: +44 141 552 0873. E-mail address: [email protected] (I. Osman).
0165-0378/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jri.2004.04.003
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The upregulation of ICAM-1 and PECAM mRNA expression in fetal membranes following labor provides further evidence that fetal membranes play an important role in the inflammatory process of parturition. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Labor; Cell adhesion molecules; Fetal membranes
1. Introduction There is mounting evidence supporting the role of inflammation during human parturition (Bowen et al., 2002; Kelly, 2002; Yellon et al., 2003). A hallmark of inflammation is the extravasation of leukocytes into tissues, which requires the expression of cell adhesion molecules. In general, cell adhesion molecules aid recruitment, adhesion and transendothelial migration of leukocytes into tissues (Smith et al., 1989; Springer, 1994; Van de Stolpe and Van der Saag, 1996). We and others have reported the infiltration of leukocytes into uterine tissues and increased expression of pro-inflammatory cytokines co-incident with parturition (Junqueira et al., 1980; Thomson et al., 1999; Osman et al., 2003). In addition, we have reported an increase in the expression of the cell adhesion molecules intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule (VCAM) and platelet endothelial cell adhesion molecule (PECAM) messenger RNA in cervix and myometrium (Ledingham et al., 2001). The fetal membranes play a key role in the inflammatory response of term labor through increased production of pro-inflammatory cytokines, matrix metalloproteinases (MMPs) and prostaglandins via inducible cyclo-oxygenase 2 (Bryant-Greenwood and Yamamoto, 1995; Vadillo-Ortega et al., 1995; Dudley et al., 1996; Keelan et al., 1999; Sawdy et al., 2000). In association with these inflammatory events, we speculate that there is a greater expression of cell adhesion molecules by fetal membranes during parturition. The role of only one cell adhesion molecule, ICAM-1, has previously been studied in relation to labor in fetal membranes. ICAM-1 was found to be upregulated in choriodecidua during preterm labor, and amnion during preterm and term labor (Marvin et al., 1999b, 2000). At present, no studies have determined which cells are responsible for this increased expression of ICAM-1 in fetal membranes during term and preterm labor. Our aims were, therefore, (i) to determine the expression of ICAM-1, PECAM, E-selectin and VCAM in amnion and choriodecidua during parturition at term using Northern blotting and (ii) to determine which cells are responsible for the expression of cell adhesion molecules in fetal membranes using immunohistochemistry.
2. Materials and methods 2.1. Subjects Biopsies of fetal membranes were obtained from pregnant women delivered at term (>37 weeks gestation) (i) by caesarean section before the onset of labor (n = 8) and (ii) following
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spontaneous vaginal delivery (n = 8). Fetal membranes were sampled in accordance with techniques described by McLaren et al., 2000; Malak and Bell, 1994. Briefly, this involved sampling from the zone of altered morphology, which is the area in which membrane rupture arises. The zone of altered morphology was assessed since previous studies state that this is the site where inflammatory changes and extracellular matrix re-modelling occurs. This region is characterized by thinning of the cytotrophoblast layer and decidua, and thickening of connective tissue. In group (i), membranes were obtained from the portion of membranes overlying the uterine cervix following delivery of the baby. This region was identified by application of a Babcock’s forcep, and an area of approximately 5 cm2 was then removed from around the forcep. For group (ii), tissue was removed from along the line of fetal membrane rupture. Women with clinical evidence of infection, multiple pregnancy, dysfunctional labor and those who had received prostaglandin or artificial oxytocin were excluded. There were no differences in maternal age, parity or gestational age of the patients recruited. Written informed consent was obtained from each woman prior to recruitment and the study was approved by the Local Research Ethics Committee. In each biopsy, amnion was separated from choriodecidua by dissection. All samples were either fixed in 10% buffered formalin (BDH, Poole, UK) and embedded in paraffin or snap-frozen in liquid nitrogen and stored at −70 ◦ C for total RNA extraction. 2.2. Northern analysis Total RNA was extracted from amnion and choriodecidua using the RNAzol B method (Biogenesis, Bournemouth, UK) and quantified using Northern blotting. Isolated RNA was further dissolved in distilled water treated with diethylpyrocarbonate and quantified using ultraviolet spectrophotometry. Ten microliters of RNA sample loading buffer was added to 10 g of total RNA and the resulting solution separated onto 1.2% agarose gels. Gels were electrophoresed at 60 V for 2.5 h. RNA was transferred and fixed by ultraviolet irradiation onto Hybond-N nylon membranes in 20× sodium saline citrate overnight. Membranes were prehybridized for 2–3 h at 42 ◦ C in 12 ml of UltrahybTM (AMS Biotecnology, Oxon, UK) and then hybridized in the same buffer overnight with the appropriate 32 P-labelled cDNA probes (Oligolabelling kit, Pharmacia, Biotech) 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). The nylon filters were washed to a final stringency of 0.5× sodium saline citrate, 0.1% sodium dodecyl sulphate at 65 ◦ C and autoradiographs for each cell adhesion molecule were compared with GAPDH and the ratio determined. For a positive control of ICAM-1, VCAM, PECAM and E-selectin expression, primary cultures of human umbilical vein endothelial cells were isolated with collagen digestion (0.1% collagenase type II, Sigma, UK) and then confluent cultures were incubated with interleukin-1 (10 IU/ml) for 4 h. Total RNA was extracted and loaded at 5 g per lane. 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 (pregnant not in labor
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versus pregnant in labor) to investigate changes in mRNA expression during pregnancy and parturition. 2.3. Immunohistochemistry Cell adhesion molecules were identified using primary antibodies directed against ICAM-1, VCAM, PECAM and E-selectin using the ABC-peroxidase method on biopsies of paraffin-embedded amnion and choriodecidua before and after labor (n = 8 in each group). Leukocytes were immunolocalized in each tissue using an antibody directed against CD45, the common leukocyte antigen. In order to identify decidua and trophoblast, respectively, immunohistochemistry was undertaken using antibodies directed against vimentin and cytokeratins, thereby facilitating the localization of cell adhesion molecule staining (Table 1). PECAM (CD 31) immunostaining identified blood vessels (Parums et al., 1990) within fetal membranes. Paraffin-embedded tissue sections (5 m thick) were mounted on silane-coated slides and heated to 60 ◦ C for 35 min. They were then deparaffinized using xylene and gradually rehydrated with ethanol. Some sections required pretreatment by microwaving at full power for 4 × 5 min in citrate buffer to retrieve the antigen (Table 1). Preincubation was carried out with 1.5% (w/v) normal horse serum in phosphate-buffered solution (PBS: 10 mM sodium phosphate, pH 7.5, 120 mM sodium chloride) for 30 min at room temperature. Thereafter, slides were incubated for 60 min with the primary antibody diluted in 1.5% horse serum and then washed with PBS. Further incubation was carried out with biotinylated anti-mouse immunoglobulin (Vector Laboratories, Peterborough, UK) diluted in 1.5% horse serum and 1.5% normal human serum. Sections were washed in PBS and then incubated with avidin DH/biotinylated horseradish peroxidase H reagent (Vector Laboratories, Peterborough, UK) in PBS for 30 min before final washing. The antigen was localized using 1 mg/ml diaminobenzidene tetrahydrochloride (Sigma, UK), 0.02% H2 O2 in 50 mM Tris–HCl, pH 7.6, which appeared as a brown end product. Sections were then counterstained with Harris haematoxylin (Sigma, UK). For negative controls, some slides were incubated without the primary antibody and others with mouse monoclonal antibody against IgG1 Aspergillus niger glucose oxidase (Dako), an enzyme that is neither present nor inducible in mammalian tissues. The positive tissue controls for each of the antibodies used are listed in Table 1. The immunohistochemistry slides were examined by observers who were blinded to the clinical condition of the patients. Table 1 Primary antibodies used for immunohistochemistry Antibody
Clone
Pretreatment
Dilution
Source
Positive control tissue
E-selectin ICAM-1 PECAM VCAM CD 45 Vimentin Cytokeratin
Polyclonal Polyclonal Clone JC/70A Polyclonal 2B11 + PD7/26 Polyclonal Clone MNF116
No Microwave Microwave Microwave Microwave No Trypsin
1:500 1:1000 1:500 1:500 1:100 1:150 1:150
R + D, Abingdon, UK R + D, Abingdon, UK Dako, Cambridge, UK R + D, Abingdon, UK Dako, Cambridge, UK Euro-path Cornwall, UK Dako, Cambridge, UK
Kidney Placenta Placenta Arthritic joint Tonsil Decidua Placenta
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2.4. Statistical analysis Statistical analysis of autoradiograph band intensity for Northern analysis was performed using Kruskal–Wallis with Mann–Whitney-U as a post hoc test. A P-value of less than 0.05 was considered significant. SPSS version 8.0 was the statistics package employed for data analysis. Minitab version 13.1 was used to produce the boxplot graphs. 3. Results 3.1. Histology Histological examination confirmed that tissue specimens were of amnion and choriodecidua. The amnion consists of a layer of amniotic epithelial cells and its supporting connective tissue. The chorion is composed of several layers of epithelial cells known as the trophoblast, a basement membrane and its connective tissue, called the reticular layer, and stromal cells of the maternal decidua lying adjacent to the chorionic trophoblast (Malak and Bell, 1994). 3.2. ICAM-1 expression and localization Messenger RNA for the cell adhesion molecule, ICAM-1, was identified in pre- and post-laboring amnion and choriodecidua. ICAM-1 mRNA expression was significantly greater in amnion (P < 0.05) and choriodecidua (P < 0.01) in tissues obtained following spontaneous labor compared with non-laboring tissues (Figs. 1, 2 and 3a,b). In the amnion, ICAM-1 was identified using immunohistochemistry in amniotic epithelial cells and leukocytes. ICAM-1 was also identified in the vascular endothelium, extravillous trophoblast and stromal cells of the decidua (Fig. 4a–d). However, the immunostaining was stronger and more consistent in tissues collected following labor than in those collected before labor.
Fig. 1. Northern blot hybridization of total RNA (5 g per lane) from human amnion samples (n = 8) collected (a) before labor and (b) after spontaneous labor at term. Intercellular adhesion molecule-1 (ICAM-1) hybridization was compared with the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The expression of ICAM-1 mRNA was upregulated in amnion following labor.
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Fig. 2. Northern blot hybridization of total RNA (5 g per lane) from human choriodecidua samples (n = 8) collected (a) before labor and (b) after spontaneous labor at term. Intercellular adhesion molecule (ICAM)-1 hybridization was compared with the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The expression of ICAM-1 mRNA was upregulated in choriodecidua following labor.
3.3. PECAM expression and localization The messenger RNA for PECAM was expressed in amnion and choriodecidua both before and after labor. There was a significant increase in PECAM mRNA expression (P < 0.05) after the onset of labor in choriodecidua (Figs. 3b and 5). In the amnion, labor did not alter PECAM mRNA expression. PECAM protein localized to leukocytes in amnion (Fig. 4e,f), but was absent from the amniotic epithelial cells. In choriodecidua, PECAM was identified in vascular endothelium (Fig. 4g) and in leukocytes (Fig. 4h). 3.4. VCAM expression and localization Messenger RNA for VCAM was not detected in either pre- or post-laboring amnion. Bands for VCAM mRNA were detected in choriodecidua (Fig. 3b). VCAM immunolocalized to cells within the chorionic connective tissue. These were PECAM (CD 31)- and CD 45-negative, implying they are neither endothelial cells nor leukocytes (Fig. 4k,l). We speculate, therefore, that they are fibroblasts or myofibroblasts. 3.5. E-selectin expression and localization E-selectin was not identified in amnion or choriodecidua before or after labor. Additionally, messenger RNA for E-selectin was not detected in amnion or choriodecidua regardless of labor status.
4. Discussion This report describes the change in expression of several key cell adhesion molecules within term fetal membranes. We have also identified specific cell types responsible for
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Fig. 3. Boxplot graphs demonstrating the median and interquartile ranges of the densitometric ratios of the cell adhesion molecules identified (a) in the amnion before and after labor and (b) in the choriodecidua before and after labor. ICAM-1 mRNA expression was significantly greater in amnion (P < 0.05) and choriodecidua (P < 0.01) in tissues obtained after labor than before. PECAM mRNA expression was significantly greater in laboring compared to non-laboring choriodecidua (P < 0.05).
the expression of cell adhesion molecules. During term parturition, there is a significant increase in ICAM-1 mRNA expression in amnion and choriodecidua and PECAM mRNA expression in choriodecidua. There were no significant changes in VCAM expression in either amnion or choriodecidua. Neither E-selectin mRNA nor antigen was detected in amnion or choriodecidua. Using immunohistochemistry, ICAM-1 immunolocalized to amniotic epithelial cells and leukocytes, suggesting that increased ICAM-1 mRNA expression is attributable to these cell types. In a recent study, we have shown no increase in leukocyte density in amnion
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Fig. 4. Immunolocalization of cell adhesion molecules in human fetal membranes (amnion and choriodecidua). In amnion, intercellular adhesion molecule (ICAM)-1 localized to amniotic epithelial cells and leukocytes in samples obtained (a) before and (b) after labor. In choriodecidua obtained (c) before and (d) after labor, ICAM-1 localized to the vascular endothelium, extravillous trophoblast and stromal cells of the decidua. In amnion, platelet endothelial cell adhesion molecule (PECAM) antigen was identified on leukocytes, which were sparse (e) before and (f) after labor. In choriodecidua, PECAM was identified on vascular endothelium (g) and in leukocytes (h). Vascular cell adhesion molecule (VCAM) was not identified in amnion collected (i) before or (j) after labor. In samples of choriodecidua collected (k) before and (l) after labor, VCAM was identified within cells of the chorionic connective tissue (indicated in (k) and (l) by arrows). (Scale bars = 50 m. The arrows in (e), (f) and (h) indicate leukocytes. The light blue background is haematoxylin counterstain. D indicates decidual stromal cells and EVT indicates extravillous trophoblast). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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Fig. 5. Northern blot hybridization of total RNA (5 g per lane) from samples of choriodecidua (n = 8) collected (a) before labor and (b) after spontaneous labor. Platelet endothelial cell adhesion molecule (PECAM) hybridization was compared with the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The expression of PECAM mRNA was upregulated in choriodecidua during labor.
or choriodecidua during labor (Osman et al., 2003). These data suggest that the change in ICAM-1 mRNA expression is a result of increased expression by existing cells and, consistent with this, previous studies have shown that ICAM-1 expression is inducible in amniotic epithelial cells (Marvin et al., 1999a). Previous studies have focused on quantification of ICAM-1 expression in fetal membranes during term and preterm labor using Northern analysis and ELISA (Marvin et al., 1999b, 2000). In these reports, ICAM-1 mRNA in amnion was upregulated following term and preterm labor. Following preterm labor, ICAM-1 mRNA expression was again increased in choridecidua, but there was no significant difference in expression following term labor. This is in contrast to our findings of ICAM-1 mRNA upregulation in choriodecidua during this time. Subject selection may explain this discrepancy; we did not include patients who received induction or augmentation agents during labor. It may also be related to sampling technique. Our post-laboring biopsies were removed from the zone of altered morphology (ZAM), the structurally weakened area in which membrane rupture arises (Malak and Bell, 1994). The region of fetal membranes sampled in the studies by Marvin et al. (1999b, 2000) was not stated. If ICAM-1 increases within the zone of altered morphology during parturition but not elsewhere in fetal membranes, it may account for the apparent discrepancy between our results and those of Marvin et al. (1999b, 2000). In other tissue types, numerous factors are known to control ICAM-1 expression, including pro-inflammatory cytokines such as IL-1, TNF-␣ and interferon-␥ (IFN-␥), hormones and viral infections (Roebuck and Finnegan, 1999). Cell-culture studies have shown that IL-1 and TNF-␣ regulate ICAM-1 expression in amnion-derived WISH cells in the absence of leukocytes (Marvin et al., 1999a). These cytokines stimulate inducible promoter regions on the ICAM-1 gene, increasing its expression. Furthermore, nuclear factor-kappa B is considered one of the most important transcription factors mediating the induction of ICAM-1 (Roebuck and Finnegan, 1999). PECAM is considered to be constitutively ex-
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pressed; however, expression can be stimulated by IFN-␥ (Tang and Hendricks, 1996). As with ICAM-1, endothelial VCAM requires induction by cytokines such as TNF-␣, IL-1 and IL-4. The primary function of cell adhesion molecules is to mediate transendothelial migration of leukocytes during inflammation and, as we have shown, ICAM-1 and PECAM mRNA are upregulated within fetal membranes during labor. There is however no associated increase in leukocyte density suggesting that, in fetal membranes with labor, adhesion molecules have additional roles within this tissue (Osman et al., 2003). Acting through physical and biochemical signalling, cell adhesion molecules are known to mediate other physiological processes such as gene transcription and apoptosis (Bischoff, 1997; Freemont, 1998). These mechanisms, perhaps influenced by cell adhesion molecules, are pertinent to changes in the ZAM prior to and during membrane rupture. In summary, we have demonstrated that the inflammatory process of spontaneous term labor is associated with increased cell adhesion molecule expression by fetal membranes. The control of expression of these molecules may represent new targets in the control of labor.
Acknowledgements This work was supported by a grant from the Glasgow Royal Infirmary Research and Development Endowment fund for which we are grateful. Reference number 0501728 (02REF0030-Osman).
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