Intercellular Adhesion Molecule-1 Expression in Human Endometrium
Neutrophils infiltrate the endometrium pre-menstrually and after long-term progestin only-contraceptive (LTPOC) treatment. Trafficking of neutrophils involves endothelial cell-expressed intercellular adhesion molecule (ICAM-1).
An
Published 30 January 2006
Background
Neutrophils infiltrate the endometrium pre-menstrually and after long-term progestin only-contraceptive (LTPOC) treatment. Trafficking of neutrophils involves endothelial cell-expressed intercellular adhesion molecule (ICAM-1). Previous studies observed that ICAM-1 was immunolocalized to the endothelium of endometrial specimens across the menstrual cycle, but disagreed as to whether extra-endothelial cell types express ICAM-1 and whether ICAM-1 expression varies across the menstrual cycle.
Methods
Endometrial biopsies were obtained from women across the menstrual cycle and from those on LTPOC treatment (either Mirena or Norplant). The biopsies were formalin-fixed and paraffin-embedded with subsequent immunohistochemical staining for ICAM-1.
Results
The current study found prominent ICAM-1 staining in the endometrial endothelium that was of equivalent intensity in different blood vessel types irrespective of the steroidal or inflammatory endometrial milieu across the menstrual cycle and during LTPOC therapy. Unlike the endothelial cells, the glands were negative and the stromal cells were weakly positive for ICAM immunostaining.
Conclusion
The results of the current study suggest that altered expression of ICAM-1 by endothelial cells does not account for the influx of neutrophils into the premenstrual and LTPOC-derived endometrium. Such neutrophil infiltration may depend on altered expression of neutrophil chemoattractants.
Background
The premenstrual human endometrium displays increased prostaglandin-generating capacity, elevated levels of inflammatory cytokines [1,2] and a leukocyte infiltrate that comprises nearly one-half of the cell population [3-5]. Among endometrial leukocyte subtypes, neutrophils are virtually absent until the mid-luteal phase, but comprise a significant portion of the leukocytes in the menstrual phase. During long-term progestin-only contraceptive (LTPOC) administration, the endometrium also experiences enhanced prostaglandin-generating capacity and increased inflammatory cytokine levels [6,7]. Administration of Norplant, which releases levonorgestrel (LNG) from subdermal rods, and Mirena, which releases LNG from an intrauterine system, leads to endometrial infiltration of matrix metalloproteinase-9 (MMP-9) positive neutrophils [8], and macrophages [9], respectively.
Endothelial cell-expressed cellular adhesion molecules mediate leukocyte trafficking [10]. In this regard, particular attention has been directed at the physiological and pathological roles played by intercellular adhesion molecule (ICAM-1), a 76-114-kDa surface glycoprotein that has five extracellular immunoglobulin-like domains [11-14].Transmigration of leukocytes involves high-affinity binding of LFA-1 or Mac-1 on their surface to ICAM-1 expressed on the endothelium [10]. ICAM-1 deficient mice experience numerous inflammatory response abnormalities including impaired neutrophil trafficking [15,16]. Although ICAM-1 has been immunolocalized to the endothelium of various blood vessel types in specimens of cycling endometrium, there are conflicting reports as to whether extra-endothelial cell types also express ICAM-1, and whether ICAM-1 expression varies across the menstrual cycle [17-19]. In view of this lack of consensus, the current study reassessed immunohistochemical (IHC) staining for ICAM-1 in endometrial biopsies across the menstrual cycle, and extended the use of IHC staining of ICAM-1 to include endometrial tissues exposed to subdermal (Norplant) and intra-uterine (Mirena, Schering) exogenous progestogens. Both LTPOC types provide safe and effective contraception for several years. Norplant is particularly well suited for use in underdeveloped countries where access to trained medical personnel is limited. They are discontinued primarily because of inflammation-associated abnormal uterine bleeding (AUB) as a source of personal annoyance and discomfort as well as cultural and religious taboo [20,21]. The levonorgestrel-releasing intra-uterine system (LNG-IUS, Mirena) is now increasingly used as an effective contraceptive and for its associated health benefits, including reduction in menstrual blood loss [22].
Prior to menstruation and during progestin-only contraception (Norplant, Mirena), secretion of MMPs by endometrial leukocytes as well as cytokines that can act as autocrine/paracrine modulators of MMP expression [5], are thought to enhance degradation of the vascular support structure leading to stromal collapse and bleeding [23-26]. The current study sought to determine whether altered expression of ICAM-1 could account for infiltration of neutrophils into the menstrual and LTPOC-derived endometrium.
Methods
Tissues
After receiving written informed consent and approval from the Institutional Research Board (IRB) of New York University Medical Center and Bellevue Hospital, specimens of endometrium were obtained across the menstrual cycle (four each from the follicular and luteal phases and five from the menstrual phase) from hysterectomies for benign conditions (e.g. myomas without abnormal uterine bleeding), and histologically dated by the criteria of Noyes et al [27]. For studies on LTPOC-derived endometrium, institutional ethical review and approval was obtained from the New York University IRB and the Lothian Research Ethical Committee, Scotland and written informed consent was obtained for biopsy collection.
The subjects had regular menstrual cycles and had not used hormonal or intrauterine contraception in the six months prior to insertion of Norplant or Mirena. Patients did not exhibit symptoms characteristic of endometriosis such as pelvic pain, dysmennorhea, dysparunia, or infertility. The only way to confirm a diagnosis of endometriosis is through exploratory surgery. Such surgery would be prompted by symptoms that would have ruled out the use of those patients for our study. For the cycling endometrium patients were pre-menopausal between 32 and 43 years of age who were not receiving any hormonal treatments. For the LTPOC endometrium patients were premenopausal, between 28 and 45 years of age, had regular menstrual cycles and had not used any hormonal or intrauterine contraception in the six months prior to receiving the LTPOC treatment.
Norplant specimens
Prior to insertion of Norplant biopsies were collected from four women (two in the follicular and two in the luteal phase) by Pipelle suction curette (Laboratoire CCD, Paris, France). Only patients who experienced bleeding while on the Norplant treatment were used. Biopsies were collected using an operative hysteroscope connected to a video camera to facilitate separate sampling of bleeding and non-bleeding sites as previously described [25]. These samples were taken after 3 and 12 months post Norplant insertion
Mirena specimens
Endometrial biopsies were also obtained from four women (two in the follicular and two in the luteal phase) prior to and at 1, 3, 6, and 12 months after intrauterine insertion of the LNG-intrauterine system by Pipelle suction biopsy.
Immunohistochemistry (IHC)
Specimens of endometrium obtained across the menstrual cycle as well as from control, and levonorgestrel treated (Norplant, Mirena) subjects were fixed in 4% paraformaldehyde and embedded in paraffin. Four μm sections (4 μm) were deparaffinized, rehydrated and washed in Tris-buffered saline [TBS: 20 mmol/l Tris-HCl, 150 mmol/l NaCl (pH 7.6)], which was used for all washes and for dilution of the antibody. Antigen retrieval was carried-out by incubating sections in sodium citrate buffer (10 mM, pH 6.0) in a microwave oven at 750 Watts for 5 minutes. The sections were then rinsed in 3% hydrogen peroxide to block endogenous peroxidase and incubated for 1 hour at room temperature with either of the following primary antibodies: a goat polycolonal ICAM-1 (CD54) antibody from R&D Systems (R&D Systems, Inc., Minneapolis, MN) or a monoclonal antibody against the Platelet Adhesion Molecule (PECAM) (CD31) from Dako (DakoCytomation California, Inc., Carpinteria, CA). Staining was visualized using the avidin-biotin peroxidase complex (Vectastain ABC kit, Vector Laboratories, Burlingame, CA) and the 3,3'-diaminobenzidine tetrahydrochloride (Sigma-Aldrich, St. Louis, MI) chromogen substrate. Light hematoxylin stain was used for nuclear counterstaining. Negative controls for each tissue section consisted of substituting the corresponding pre-immune serum for the primary antibody.
Assessment of immunohistochemical (IHC) staining and statistical analysis
Intensity of ICAM-1 staining was evaluated using a semi-quantitative 4-point rating method with the following scoring system: 0, absence of staining; 1, light staining; 2 moderate staining; and 3, strong staining. Each of these possible scores was established in advance of rating the fields via reference to external stained specimens unrelated to this study. In order to determine inter-rater reliability of this scale, two independent judges scored a series of 35 separate fields on slides from 4 separate patient samples. The degree of concordance was then assessed by use of Cohen's kappa statistic, which yielded a value of 0.67, indicating a high degree of agreement between the judges.
Non-parametric statistical analysis was performed by the Mann-Whitney Rank Sum Test with p < 0.05 considered significant.