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ORIGINAL RESEARCH |
From the II Department of Obstetrics and Gynecology, University of Milan and Istituto Auxologico Italiano, Milan, Italy.
Address reprint requests to: Paola Viganò, DSc, II Department of Obstetrics and Gynecology, Clinica "L. Mangiagalli", Via Commenda 12, 20122 Milan, Italy
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Abstract |
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Methods: Samples of endometrium were collected from 23 women with endometriosis. Soluble intercellular adhesion molecule 1 was quantified in conditioned medium from 48-hour endometrial stromal cell cultures with use of a specific enzyme-linked immunosorbent assay. Levels were correlated with revised American Society for Reproductive Medicine classification score for adhesions, implants, and cysts and total score; number of endometriotic implants; cyst diameter; and presence or absence of pelvic pain symptoms and previous surgical procedures for endometriosis.
Results: Endometrial release of soluble intercellular adhesion molecule 1 directly correlated with number of implants (r = .64, P < .005) and score for implants (r = .61, P < .005). There was no significant correlation between levels of the soluble molecule and score for adhesions or total score. Soluble intercellular adhesion molecule 1 shed by endometrium did not correlate with the score for ovarian cysts, although an inverse relationship was found with ovarian cyst diameter (r = -0.52, P < .05). No differences were detected between women who had pelvic pain and those who did not and between those who had previous surgery for endometriosis and those who had not.
Conclusion: The association between endometrial release of soluble intercellular adhesion molecule 1 and the number and score of endometriotic implants suggests that the molecule might be of value in evaluating spread potential of refluxed endometrium.
Cellular adhesion molecules have been implicated in homotypic and heterotypic cell interactions. Intercellular adhesion molecule 1, a member of the immunoglobulin (Ig) supergene family of adhesion molecules, has been found in various cell types, including vascular endothelial cells and leukocytes, and its expression can be modulated by cytokines.1 Intercellular adhesion molecule 1 affects inflammatory and immune responses and has been implicated in migration of tumor and normal cells.1–3 The soluble form of intercellular adhesion molecule 1 is the shedding domain of the surface molecule and has been described in serum and medium conditioned by different cell types.4–6 Serum levels of the molecule fluctuate, appearing to correlate with distinct diseases such as malignant melanoma, neutropenic pneumonia, multiple sclerosis, and insulin-dependent diabetes mellitus, making it possible that its detection might be of diagnostic value.1,7 Increased serum levels also have been noted in women with endometriosis.8,9 We showed that eutopic and ectopic endometrial cells release soluble intercellular adhesion molecule 1 and that shedding of the protein by eutopic endometrium was increased significantly in women with endometriosis.10,11 However, despite expanding knowledge regarding serum levels of soluble intercellular adhesion molecule 1 in many clinical settings, little is known about the basic physiology of this soluble protein. Shedding of the molecule from the cell surface is believed to interfere with immune surveillance, promoting cellular spreading potential.2,12,13 To gain insight into soluble intercellular adhesion molecule 1–dependent mechanisms in endometriosis, we correlated levels of the molecule released by endometrial stromal cells collected from women with endometriosis, with specific characteristics pertaining to the severity of the disease.
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Materials and Methods |
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At the time of pathologic examinations, ovarian endometriotic cysts were confirmed histologically and their diameters were recorded. Single ovarian endometriotic cysts were present in 15 women; bilateral ovarian endometriotic cysts were identified in four. In seven women with ovarian endometriomas, no peritoneal implants were seen. All types of lesions (deep, red, black, and white) were considered equally for data analysis. Lesions were considered different when clearly separated at the time of macroscopic evaluation. Single deep endometriotic peritoneal implants were found in three women. Before laparoscopy, information was obtained on pain symptoms (dyspareunia, dysmenorrhea, pelvic pain) considered present when the visual analogue score exceeded 5; parity; previous surgery for endometriosis; medical treatment; and menstrual cycles. Subjects who had had any surgery or medical therapy within 3 months were excluded from the study. All women had regular menstrual cycles. Eleven (48%) were in the proliferative phase and 12 (52%) were in the secretory phase of the cycle. All characteristics evaluated were represented similarly in the two phases. Three women were fertile, eight were infertile, and in 12, fertility could not be assessed because the women had not attempted to conceive. Endometriosis was classified as stage I-II in four women (17%), stage III in 15 (66%), and stage IV in four (17%). Subjects were voluntary participants and gave informed consent according to the protocol of our institution’s committee on human experimentation.
Samples of uterine endometrium were collected at the time of laparoscopy with the use of endometrial biopsy curet. Specimens were transported to the laboratory in sterile vials containing Ham’s F-10 medium (EuroClone, Celbio, Milan, Italy). We used a previously described procedure to establish a stromal cell monolayer from normal endometrial samples,15,16 so diffuse and strong cytoplasmic immunostaining for vimentin occurred in nearly all cultured stromal cells (90%). Cytofluorimetric analysis showed that macrophage contamination of our cultures was less than 2%.
Endometrial stromal cultures for supernatant collection were allowed to proliferate to subconfluence in Ham’s F-10 medium supplemented with 10% fetal calf serum (EuroClone, Celbio) and antibiotics in a humidified atmosphere of 95% air and 5% CO2 at 37C. Afterward, cells were harvested, counted, resuspended in Ham’s F-10 medium plus 10% fetal calf serum, and plated at a density of 5 x 105/mL in Petri dishes (1 mL per dish, 35 mm in diameter) for an additional 48 hours. The medium was collected, centrifuged for 5 minutes at 400g, and stored at -20C until analysis.
The quantitative detection of soluble intercellular adhesion molecule 1 in cell-free supernatants was done with use of a commercially available enzyme-linked immunosorbent assay kit provided by Bender MedSystem (Vienna, Austria). A dose of standard culture medium also was evaluated, to exclude the possibility of the presence of soluble intercellular adhesion molecule 1 in the culture medium. Levels of soluble intercellular adhesion molecule 1 were expressed as nanograms per milliliter and the interassay and intra-assay coefficients of variation were 8% and 4%, respectively.
Spearman correlation coefficients for nonnormally distributed parameters were calculated to assess associations between variables. The Mann-Whitney non-parametric test was used to assess statistical significance of differences between groups when pelvic pain and previous surgery for endometriosis were being considered. P < .05 was considered statistically significant.
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). That strong correlation was still present when numbers of ovarian and peritoneal implants were considered separately (r = .70, P < .002; and r = .46, P < .05, respectively). A positive significant association also was noted between soluble intercellular adhesion molecule 1 levels and the revised American Fertility Society for Reproductive Medicine classification score for implants (Table 1). No significant correlations were detected when subject age, score for adhesions, and total score were considered (Table 1).
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). When bilateral endometriotic cysts were present, we decided to use the diameter of the largest cyst for statistical analysis. However, that correlation also was noted when we considered the diameter of the smallest cyst (r = -.51, P < .05), the sum of the diameters of both cysts (r = -.54, P < .05), and the diameters of all cysts except the bilateral ones (r = -.58, P < .05). However, that association was not confirmed when the score for cysts was considered instead of cyst diameter (Table 1).
Soluble intercellular adhesion molecule 1 shed by endometrium did not seem to be related to clinical characteristics of the disease (Table 2). Levels were similar between women who had had previous surgery for endometriosis and those who had not and between women with pelvic pain and those without. There were deep endometriotic peritoneal implants in only three women. Fertility was established definitively in only three women. Therefore, the effect of the molecule on frequency of deep endometriosis and fertility could not be assessed.
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Discussion |
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Intercellular adhesion molecule 1 is a cytokine-inducible adhesion molecule of the Ig family that is the natural ligand for the leukocyte integrin lymphocyte function–associated antigen 1. The interaction between lymphocyte function–associated antigen 1 and intercellular adhesion molecule 1 is essential for cell-mediated cytotoxicity, the interaction of T and B cells, homotypic adhesion of monocytes, and B cell–B cell homotypic adhesion. Intercellular adhesion molecule 1 is necessary for lymphocyte–endothelial cell adhesion and is believed to facilitate lymphocyte migration to sites of inflammation along endothelial cells.1 Like other leukocyte receptors, this adhesion molecule also exists in a soluble form, which is believed to act as antagonist of intercellular adhesion molecule 1 and lymphocyte function–associated antigen 1–mediated cellular events and related cellular immune functions.5,12,13 Many investigators have examined shedding of soluble intercellular adhesion molecule 1 in normal and abnormal tissues and its relationship with many conditions in which the molecule was proposed as an indicator of disease progression.2,5–7 Elevated serum levels have been associated with liver metastasis in stomach, colon, gallbladder, and pancreatic cancer and with reduced survival in malignant melanoma.1,7 Among cellular sources are normal and malignant epithelial tissues, including cells from melanoma, liver, and kidney.5–7
We found that the endometrial release of soluble intercellular adhesion molecule 1 correlated with number and score of endometriotic implants, findings in line with reports that suggested that the soluble molecule might be of biologic relevance in cellular spreading.2 However, the precise mechanism underlying shedding and function of soluble adhesion molecules is still a matter of speculation. The release of the molecule from the cell surface might interfere directly with the attachment of natural killer or cytotoxic T cells, because lymphocyte function–associated antigen 1 could be blocked by soluble intercellular adhesion molecule 1.5,12 Recognition and susceptibility to killing by immune cells consequently would be reduced. On the other hand, because expression and release of intercellular adhesion molecule 1 can be stimulated by cytokines,1 levels of the molecule might show indirectly the action of endometrium-derived cytokines or of those derived from resident lymphoid cells. In both cases, the direct correlation between soluble intercellular adhesion molecule 1 shed by endometrium and the spreading potential of endometriosis suggests that eutopic endometrium could be a source of the elevated serum levels seen in women with the disease. The fact that a disregulation of endometrial factors might contribute to endometriosis strengthens our hypothesis.
As we expected, endometrial release of soluble inter-cellular adhesion molecule 1 did not correlate with adhesion score and results were similar in women with and in those without pelvic pain. There is no evidence that the molecule could be involved in processes of proliferation, repair, or prostaglandin production. The correlation with number and score of implants is not mirrored by the association with the total score because pelvic endometriotic implants were accorded only slight importance in the classification system. Scores for adhesions and cysts have greater effects on total scores.
We were surprised that levels of the molecule were associated negatively with ovarian cyst diameter, but there are potential explanations for that. From a more accurate evaluation of the data, it was evident that the inverse association was due predominantly to the inclusion of the largest ovarian cysts in the analysis. That negative correlation was not present when cysts with diameters of 6 cm or more were excluded (data not shown). It has been hypothesized that large endometriomas might develop from secondary involvement of functional ovarian cysts by the endometriotic process18; therefore, extent of endometriosis can be overestimated in those cases. Ovarian cysts, especially larger ones, are likely to affect ovarian function and influence endometrial cycles. Endometrial release of soluble intercellular adhesion molecule 1 was shown to be menstrual cycle–dependent; proliferative-phase samples released significantly higher levels of the soluble protein compared with samples obtained in the secretory phase.10,11 Thus, reduced secretion by endometrium might be the consequence of subclinical ovarian dysfunction. Another possible explanation is that ovarian endometriomas and peritoneal lesions might be different entities with different pathogeneses.19 If that is the case, a different role of soluble intercellular adhesion molecule 1 in the development of the two entities should be sought. The etiology of the increased secretion by the endometrium in women with endometriosis remains unknown.
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Footnotes |
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Received March 25, 1999. Received in revised form June 1, 1999. Accepted June 17, 1999.
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References |
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2. Giavazzi R, Nicoletti MI, Chirivi RGS, Hemingway I, Bernasconi SP, Allavena P, et al. Soluble intercellular adhesion molecule-1 (ICAM-1) is released into the serum and ascites of human ovarian carcinoma patients and in nude mice bearing tumor xenografts. Eur J Cancer 1994;30:1865–70.
3. Viganò P, Pardi R, Magri B, Busacca M, Di Blasio AM, Vignali M. Expression of intercellular adhesion molecule-1 (ICAM-1) on cultured human endometrial stromal cells and its role in the interaction with natural killers. Am J Reprod Immunol 1994;32:139–45.
4. Rothlein R, Mainolfi EA, Czajkowski M, Marlin SD. A form of circulating ICAM-1 in human serum. J Immunol 1991;11:3788–93.
5. Becker JC, Dummer R, Hartmann AA, Burg G, Schmidt RE. Shedding of ICAM-1 from human melanoma cell lines induced by INF-
and tumor necrosis factor-
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6. Thomson AW, Satoh S, Nussler AK, Tamura K, Woo J, Gavaler J, et al. Circulating intercellular adhesion molecule-1 (ICAM-1) in autoimmune liver disease and evidence for the production of ICAM-1 by cytokine-stimulated human hepatocytes. Clin Exp Immunol 1994;95:83–90.[Medline]
7. Gearing AJH, Newman W. Circulating adhesion molecules in disease. Immunol Today 1993;14:506–12.[Medline]
8. Wu M-H, Yang B-C, Hsu C-C, Lee Y-C, Huang K-E. The expression of soluble intercellular adhesion molecule-1 in endometriosis. Fertil Steril 1998;70:1139–42.[Medline]
9. De Placido G, Alviggi C, Di Palma G, Carravetta C, Matarese G, Landino G, et al. Serum concentration of soluble human leukocyte class I antigens and of the soluble intercellular adhesion molecule-1 in endometriosis: Relationship with stage and non-pigmented peritoneal lesions. Hum Reprod 1998;13:3206–10.
10. Somigliana E, Viganò P, Gaffuri B, Guarneri D, Busacca M, Vignali M. Human endometrial stromal cells as a source of intercellular adhesion molecule (ICAM)-1 molecules. Hum Reprod 1996;11:1190–4.
11. Viganò P, Gaffuri B, Somigliana E, Busacca M, Di Blasio AM, Vignali M. Expression of intercellular adhesion molecule (ICAM)-1 mRNA and protein is enhanced in endometriosis versus endometrial stromal cells in culture. Mol Hum Reprod 1998;4:1150–6.
12. Becker JC, Termeer C, Schmidt RE, Brocker EB. Soluble intercellular adhesion molecule-1 inhibits MHC-restricted specific T cell/tumor interaction. J Immunol 1993;151:7224–32.[Abstract]
13. Meyer DM, Dustin ML, Carron CP. Characterization of intercellular adhesion molecule-1 ectodomain (sICAM-1) as an inhibitor of lymphocyte function-associated molecule-1 interaction with ICAM-1. J Immunol 1995;155:3578–84.[Abstract]
14. American Society for Reproductive Medicine. Revised American Society for Reproductive Medicine classification of endometriosis: 1996. Fertil Steril 1997;67:817–21.[Medline]
15. Viganò P, Di Blasio AM, Dell’Antonio G, Vignali M. Culture of endometrial cells: A new simple technique to completely separate epithelial glands. Acta Obstet Gynecol Scand 1993;72:87–92.[Medline]
16. Di Blasio AM, Pecori-Giraldi F, Viganò P, Petraglia F, Vignali M, Cavagnini F. Expression of corticotropin-releasing hormone and its R1 receptor in human endometrial stromal cells. J Clin Endocrinol Metab 1997;82:1594–7.
17. Kupker W, Schultze-Mosgau A, Diedrich K. Paracrine changes in the peritoneal environment of women with endometriosis. Hum Reprod Update 1998;4:719–23.
18. Nezhat F, Nezhat C, Allan CJ, Metzger DA, Sears DL. Clinical and histologic classification of endometriomas. J Reprod Med 1992; 3709:771–6.
19. Nisolle M, Donnez J. Peritoneal endometriosis, ovarian endometriosis, and adenomyotic nodules of the rectovaginal septum are three different entities. Fertil Steril 1997;68:585–96.[Medline]
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