Interleukin-10 is produced by human uterine natural killer cells but does not affect their production of interferon-gamma.

A predominance of T helper (Th)2-type cytokines and a weakening of Th1 responses seem to be critical for the maintenance of a successful gestation. Among Th2-type cytokines, interleukin (IL)-10 is produced by human cytotrophoblasts and defects in this production result in specific pathological conditions of pregnancy. The current opinion is that IL-10 serves to protect the fetus from a harmful maternal immune response. However, production of the cytokine and its direct effect on uterine natural killer (uNK) cells, which represent the predominant lymphocyte population infiltrating the pregnant endometrium, are largely unknown. Thus, to shed light on the cytokine network at the maternal-fetal barrier during early pregnancy, we investigated the IL-10 system in uNK cells. We showed that uNK cells express the mRNA transcripts for IL-10 and IL-10 receptor. Production of IL-10 by the uNK cells was enhanced by both IL-2 and IL-12. Treatment with IL-10 alone enhanced uNK cell cytotoxic activity. In contrast, the cytokine did not modify the basal or stimulated production of interferon (IFN)-gamma by uNK. Thus, IL-10 does not act as a direct antagonist of uNK cell function and activation. However, IL-10 produced by uNK cells in response to IL-12 and IL-2 may still have a feedback inhibitory effect on the production of deleterious cytokines within the uterine microenvironment.

Effect of a single dose of ibuprofen lysinate before embryo transfer on pregnancy rates in cows.

Embryo implantation is a critical step in both cows and humans. The use of ibuprofen lysinate to enhance implantation has been investigated in cattle with the specific aim of improving pregnancy rates after embryo transfer. In this study, heifers (n = 100) were assigned randomly to one of two groups: one group was treated i.m. with 5 mg ibuprofen lysinate kg(-1) body weight 1 h before embryo transfer and a control group received vehicle only. A single embryo was transferred into each recipient cow. There was a significant difference in the number of pregnancies after embryo transfer between cows in the treated (41 of 50; 82%) and control (28 of 50; 56%) groups (P < 0.05). These data indicate that ibuprofen lysinate may be an effective adjunctive treatment for assisted reproduction in cattle. Further studies are needed to clarify whether this effect is associated with the reduction of cyclooxygenase enzyme isoforms during embryo transfer or whether other mechanisms are involved.

Unexplained habitual abortion is associated with a reduced endometrial release of soluble intercellular adhesion molecule-1 in the luteal phase of the cycle.

Although the mechanisms causing recurrent spontaneous abortion (RSA) remain frequently speculative, recent evidence indicates that a specific uterine immune-endocrine network plays a pivotal role in the continuation of pregnancy. We have recently demonstrated that an adhesion molecule of the immune system, named intercellular adhesion molecule (ICAM)-1, is markedly expressed at both protein and mRNA levels in endometrial stromal cells and is able to mediate their interaction with lymphoid cells. Moreover, we have shown that the soluble form of ICAM-1 (sICAM-1) can be released by the endometrium in a hormone-dependent manner. The present study was designed to determine whether surface and/or sICAM-1 expression by cultured endometrial stromal cells could be related to early pregnancy loss in patients with a history of unexplained RSA. Luteal-phase endometrial biopsies were obtained from eight patients who had experienced three or more consecutive unexplained RSAs in the first trimester and 12 control fertile women. Surface ICAM-1 was similarly expressed on luteal-phase endometrial cells obtained from women with and without a history of unexplained RSA. In contrast, the endometrial release of sICAM-1 was significantly lower in abortion-prone patients than in control women. sICAM-1 is a cytokine-inducible molecule able to interfere with several immunological responses and the reduced levels of the protein shed by the endometrium in patients who have suffered from unexplained RSAs may reflect the presence of an altered immunological environment during the early phases of pregnancy.

Expression of GnRH receptor gene in human ectopic endometrial cells and inhibition of their proliferation by leuprolide acetate.

The present study was conducted to investigate whether GnRH-receptor (GnRH-R) gene is expressed in endometriosis ovarian implants and whether a GnRH-analogue (GnRH-a) may exert an effect on endometriosis cell proliferation in vitro. The presence of GnRH-R transcripts in ovarian endometriosis cells was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and further confirmed by Southern blot analysis. GnRH-R mRNA was detected in all the 13 samples examined. In contrast, GnRH-R transcripts were not detectable in endometriosis-free peritoneal tissue. In the second part of the study, endometriosis cells were cultured for 9 days with different doses of leuprolide acetate (ranging from 0 to 10(-5) M). In 4 out of 13 cases, a significant anti-proliferative effect was observed at doses of leuprolide acetate ranging from 10(-9) to 10(-5) M. In one case, a significant inhibition of cell proliferation was observed only at 10(-5) M leuprolide acetate concentration. In contrast, the GnRH-a did not affect cell growth, regardless of the expression of GnRH-R transcripts and the given doses, in the remaining 8 experiments. To date, this is the first evidence indicating that GnRH-R mRNA is expressed in human ovarian endometriomas. Moreover, the inhibition of endometriosis cell proliferation induced by the GnRH-a in vitro suggests that, at least in some cases, this compound might exert a direct effect on endometriosis lesions.

Endometrial release of soluble intercellular adhesion molecule 1 and endometriosis: relationship to the extent of the disease.

OBJECTIVE: To relate endometrial release of the soluble form of intercellular adhesion molecule 1 with extent of endometriosis. 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.

Soluble intercellular adhesion molecule-1 in ovarian follicles: production by granulosa luteal cells and levels in follicular fluid.

OBJECTIVE: To determine the concentration of the soluble form of intercellular adhesion molecule (ICAM)-1 in granulosa luteal cell-conditioned media and in follicular fluid (FF). DESIGN: Granulosa cells and FF samples were obtained at the time of oocyte retrieval for IVF. In 10 women, a total of 33 fluids were obtained from individual follicles, whereas in 70 women, the follicular aspirates were pooled. SETTING: Clinica "L. Mangiagalli" and Reproductive Center, San Raffaele Hospital, Milan, Italy. PATIENT(S): Eighty women referred for IVF for tubal factor or male factor infertility. INTERVENTION(S): Women underwent ovarian hyperstimulation. MAIN OUTCOME MEASURE(S): Soluble ICAM-1 was measured by an ELISA, and its levels were correlated with follicular size, the number of retrieved oocytes, and the number of follicles with a diameter of >15 mm. RESULT(S): The concentration of soluble ICAM-1 in granulosa luteal cell-conditioned media was 17.8 +/- 1.8 ng/5 x 10(5) cells. Interleukin-1beta can stimulate soluble ICAM-1 release in a dose-dependent manner. A significant positive correlation was demonstrated between levels of soluble ICAM-1 in pooled FF and the number of retrieved oocytes or the number of follicles with a diameter of >15 mm. CONCLUSION(S): Soluble ICAM-1 can be released by granulosa luteal cells and can be detected in FF after ovarian hyperstimulation. Levels of soluble ICAM-1 in FF correlate directly with some indices of ovarian function.

Expression of intercellular adhesion molecule-1 messenger ribonucleic acid and protein in human term placental cells and its modulation by pro-inflammatory cytokines (interleukin-1beta and tumor necrosis factor alpha).

Intercellular adhesion molecule-1 (ICAM-1) is a ligand for the integrins lymphocyte function-associated antigen-1 (LFA-1) and complement receptor-3 (Mac-1), making it an important participant in many immune and inflammatory processes. Previous studies suggested that lack or reduced expression of ICAM-1 on trophoblast might partially explain its resistance to lysis by cytotoxic effectors. However, whether or not the adhesion molecule is expressed on placental cells is still a matter of debate. In this study, we determined ICAM-1 expression at mRNA, surface, and soluble protein levels on human trophoblasts throughout their functional differentiation in culture from cytotrophoblasts into syncytiotrophoblasts. Placental cells were obtained from 6 term placentas derived from normal pregnancies. ICAM-1 mRNA was detected by reverse transcription-polymerase chain reaction using two oligonucleotide primers specific for the human ICAM-1 gene. A single major DNA band of the expected size (943 base pairs) was obtained in both cytotrophoblasts and syncytiotrophoblasts. Flow cytometric analysis demonstrated expression of surface ICAM-1 protein on 45.5+/-3.5% of cytotrophoblasts. No changes were observed during differentiation in culture. Levels of the soluble form of ICAM-1 (sICAM-1) released by placental cells were undetectable when assessed by a specific ELISA. Finally, we investigated the effect of pro-inflammatory cytokines on placental ICAM-1 expression. Treatment of cultured trophoblasts for 24 h with interleukin-1beta (1 ng/ml) or tumor necrosis factor alpha (1 ng/ml) increased surface expression of ICAM-1 without inducing sICAM-1 shedding. However, on placental cells, the two cytokines exerted stimulatory effects lower than those detected on endometrial cells used as positive control. These observations document that the ICAM-1 gene is expressed in both cytotrophoblasts and syncytiotrophoblasts, suggesting that the molecule may be of value for some immune-mediated processes. On the other hand, the low sensitivity of trophoblasts to cytokine-mediated induction of ICAM-1 expression might represent a functional mechanism contributing to maternal tolerance for fetal graft.

Soluble intercellular adhesion molecule-1 serum profile in physiologic and preeclamptic pregnancy.

PROBLEM: The aim of this study was to determine serum levels of soluble intercellular adhesion molecule (sICAM)-1, an adhesion receptor that mediates interactions with the immune system, in physiologic and preeclamptic pregnancies. Moreover, we evaluated whether the release of sICAM-1 during pregnancy correlated to plasma fibronectin concentrations. METHOD OF STUDY: Serum was collected from 18 nonpregnant, control women, from 58 normal pregnant women during the first (n = 13), second (n = 15), and third (n = 30) trimesters, and from 25 preeclamptic patients at 27-39 weeks' gestation. All samples were assayed for sICAM-1 by a specific enzyme-linked immunoassay and for fibronectin by a nephelometric system. Serum sICAM-1 levels in preeclamptic patients were compared to those obtained from gestational-matched normal pregnant women. RESULTS: Levels of sICAM-1 were significantly elevated (P < 0.001) in each of the three trimesters of normal pregnancy (I trimester: 390.4 +/- 25.7 ng/ml; II trimester: 386.3 +/- 15.4 ng/ml; and III trimester: 367.3 +/- 15.8 ng/ml) when compared to those of healthy nonpregnant women (263.3 +/- 11.6 ng/ml). No significant difference in sICAM-1 concentrations was observed among the three trimesters. Preeclampsia was associated to a significant decrease (P < 0.01) of sICAM-1 levels (309.8 +/- 11.6 ng/ml) relative to those observed in gestational-matched pregnant women (367.3 +/- 15.8 ng/ml). Fibronectin and sICAM-1 levels did not correlate. CONCLUSION: The increased levels of sICAM-1 found in physiologic pregnancies and its reduction in preeclampsia may account for some of the immunologic alterations demonstrated to be associated with pregnancy.

Expression of intercellular adhesion molecule (ICAM)-1 mRNA and protein is enhanced in endometriosis versus endometrial stromal cells in culture.

The relationship between the immune and the endometrial systems has been recently suggested to be critical to the development of endometriosis. We previously showed that one of the molecules involved in the complex events that allow this interaction is the adhesion molecule intercellular adhesion molecule (ICAM)-1. This study was designed to evaluate whether differences in ICAM-1 mRNA and protein expression might exist between eutopic endometrial cells and ectopic cells derived from endometriomas. Stromal cells were dispersed from samples of endometrium and ovarian endometriomas biopsied synchronously from 24 patients with endometriosis. We established that the relative expression of ICAM transcript was significantly higher in ectopic cells than that found in cultures derived from endometrial samples. Moreover, ectopic cells demonstrated a significant overexpression of ICAM-1 protein in both its cell-bound and soluble form (sICAM-1) (P < 0.05). Interestingly, endometrial secretion of sICAM-1 was shown to vary during the menstrual cycle as proliferative phase samples released significantly higher concentrations of the soluble protein compared to the secretory phase. In contrast, this cycle-dependent pattern was absent in stromal cells derived from endometriomas. Moreover, interleukin (IL-1beta) was able to increase sICAM-1 shedding from endometrial cells in a concentration-dependent manner and this IL-1beta-mediated induction could be slightly enhanced by oestradiol. As sICAM-1 is able to interfere with ICAM-1-mediated immune functions, the release of higher concentrations from ectopic samples may be the mechanism by which ectopic endometrial cells escape immunosurveillance.

Comparative effect of the calcium antagonist verapamil and the synthetic steroids gestrinone and danazol on human monocyte phagocytosis in vitro.

Recent evidence suggested that periovulatory treatment with an immunomodulatory agent such as verapamil might be an effective alternative to conventional treatment for endometriosis-associated subfertility. In particular, it has been reported that the drug might reduce the accentuated macrophage peritoneal activation demonstrated in patients with endometriosis. In this study, we compared the effect of the calcium antagonist verapamil with those of gestrinone, danazol and testosterone on human monocyte phagocytosis in an attempt to evaluate any significant differences in their ability to influence a parameter of cell inflammatory activation. Peripheral blood monocytes were isolated from 37 healthy women. Monocyte function was determined by phagocytosis of fluorescent microspheres after an overnight incubation in the presence or absence of the various agents. This study indicates that verapamil at the pharmacological concentration of 0.4 micrograms/ml, the systemic level in patients taking 40-80 mg/8 h p.o., significantly inhibits monocyte function. A lower immunosuppressive but still significant effect was achieved in this assay system with gestrinone at a concentration of 3 x 10(-8) M). The pharmacological concentration of danazol (10(-6) M) and the physiologic concentration of testosterone (10(-8) M) did not significantly affect this immunologic test system. These results provide evidence that verapamil is able to exert a slightly greater immunosuppressive effect than steroidal drugs on monocyte phagocytosis. However, due to the small differences observed, further studies on the biological mechanism of the drug seem to be necessary to completely elucidate its potential role in endometriosis-associated subfertility.

Intercellular adhesion molecule-1 is expressed on human granulosa cells and mediates their binding to lymphoid cells.

Some immune cellular components have been recently demonstrated to play a critical role in ovarian physiology. Resident ovarian white blood cells are known to produce cytokines that modulate granulosa cell (GC) functions and differentiation. Moreover, it has been postulated that, during the formation of the corpus luteum and luteolysis, human luteal cells are able to interact with lymphocytes and macrophages through some adhesion molecules. This study was designed to examine, at messenger RNA and protein levels, whether intercellular adhesion molecule (ICAM)-1, known to be involved in leukocyte-cell binding, is expressed by human GCs. Furthermore, we also investigated whether this molecule could be involved in the complex events that allow the interaction between the ovary and the immune system. GCs, obtained from women undergoing in vitro fertilization procedures, were enzymatically dispersed with collagenase and cultured for different time periods. To assess the presence of ICAM-1 messenger RNA, total RNA obtained from freshly aspirated GCs and GCs luteinized in culture was reverse transcribed and then amplified using two oligonucleotide primers specific for the human ICAM-1 gene. A single major DNA band of the expected size (943 bp) was obtained. The identity of this material with the human ICAM-1 sequence was further confirmed by restriction enzyme analysis. Surface ICAM-1 protein was detected by flow cytometric analysis on luteinized GCs cultured for 7 and 15 days. Finally, to evaluate a possible functional activity of ICAM-1, a 51Cr-release-binding assay between peripheral blood lymphocytes and luteinized GCs was performed in the presence and absence of a monoclonal antibody against ICAM-1. As a result, lymphocyte adhesion to GC monolayers was significantly, but not completely, inhibited by the anti-ICAM-1 monoclonal antibody. These findings demonstrate that intercellular interactions between GCs and the immune system are, at least in part, mediated by the adhesion molecule ICAM-1. Based on this data, we might speculate that this molecule could participate in the remodeling processes of the ovarian endocrine compartment.

Modulation of NK cell lytic function by endometrial secretory factors: potential role in endometriosis.

PROBLEM: Development and progression of endometriosis have been suggested to be related to a defect of NK cells in the ability to eliminate endometrial fragments regurgitated with menstrual debris in ectopic sites. However, it has not been clarified yet whether the origin of this defect has to be attributed to an intrinsic alteration of NK cell population or to a consequence of the environmental system. The present study was designed to evaluate whether soluble factors of endometrial origin might directly interfere with NK cell lytic function. METHOD: Media conditioned by 20 endometrial stromal cultures (10 from endometriosis patients and 10 from women without the disease) were examined to test their effects on NK cell-mediated cytotoxicity directed against endometrial stromal targets. NK cells were purified from peripheral blood samples by a two steps panning technique and incubated overnight with and without endometrial supernatants. Thereafter, the percentage of lysis against endometrial cells was detected in a 51Cr cytotoxicity assay. RESULTS: Media conditioned by the overall endometrial stromal cultures exerted a significant suppressive effect (P < 0.001) on NK cell function when compared to control NK fractions (mean percentage of cytotoxicity +/- SEM 17.46 +/- 2.8 and 29.41 +/- 1.49, respectively). No inhibitory effect was detect with conditioned media obtained from peritoneal cells and keratinocytes. Moreover, NK cell-mediated cytotoxicity resulted significantly more inhibited by the addition of endometrial supernatants obtained from endometriosis patients (mean percentage cytotoxicity +/- SEM 13.55 +/- 2.3, P < 0.05) when compared to those from women without the disease (mean percentage cytotoxicity +/- SEM 21.38 +/- 4.9). CONCLUSION: The results of this study indicate the existence of soluble non-specific factors(s) released by human endometrial cells able to interfere with NK cell killing. These factors could participate in the local regulation of the immunological mechanisms suggested to be involved in the development of endometriosis.

Alteration in the pulsatility index values of the internal carotid and middle cerebral arteries after suspension of postmenopausal hormone replacement therapy: a randomized crossover study.

OBJECTIVE: The aim of the study was to investigate the effect of the suspension of hormone replacement therapy on blood flow in the internal carotid and the middle cerebral arteries. STUDY DESIGN: Doppler ultrasonography was used to measure the pulsatility index of the internal carotid and middle cerebral arteries of 23 women. The patients were all receiving continuous transdermal estradiol replacement therapy (50 micrograms/day) with a cyclic supplementation of medroxyprogesterone acetate every second month (10 mg/day for 12 days). The duration of the study was 12 months. The patients were randomly assigned to one of two groups. The first group (11 subjects) continued therapy for the first 6 months and then suspended it for the following 6 months; the second group (12 subjects) interrupted hormone replacement therapy for the first 6 months and then resumed it for the following 6 months. The internal carotid and middle cerebral artery pulsatility index was measured at the start of the 12-month period and then every 3 months. Serum estradiol levels were measured to check compliance. RESULTS: A statistically significant difference was found between the internal carotid and middle cerebral artery pulsatility index values of the two groups at each of the measurements after the first one. Over the first 6 months the pulsatility index values rapidly increased in the patients kept off hormone replacement therapy and remained stable in those receiving hormone replacement therapy. After the crossover at 8 months, the pulsatility index rapidly dropped to values similar to those at baseline in the patients who resumed hormone replacement therapy and increased in those who suspended therapy. CONCLUSIONS: Resistance to blood flow in cerebral vessels of postmenopausal women rapidly changes after hormone replacement therapy suspension. In postmenopausal women estrogen administration should be continued to maintain the favourable variations of vascular reactivity induced by hormone replacement therapy.

Human endometrial stromal cells as a source of soluble intercellular adhesion molecule (ICAM)-1 molecules.

Intercellular adhesion molecule (ICAM)-1-mediated cell-cell adhesion is essential for various immunological functions, including natural killer (NK) cell-mediated cytotoxicity against endometrium. The present study was designed to establish whether shedding of ICAM-1 from cultured endometrial stromal cells occurred and to characterize its potential functional significance in endometrial physiology and pathology. The shed sICAM-1 molecule was detected and quantified in supernatants from endometrial stromal cultures and in peritoneal fluid by a specific enzyme-linked immunosorbent assay. The results of this study indicate that cultured endometrial stromal cells constitutively shed ICAM-1 from their surface. This ability is regulated during the menstrual cycle, as it appears to be higher in the proliferative than in the secretory phase of the cycle (16.93 +/- 2.2 and 7.7 +/- 1.76 ng/ml respectively). In order to evaluate whether the release of sICAM-1 could interfere with cell-mediated lysis of endometrium we compared the determinations of sICAM-1 in endometrial supernatants with the ability of such supernatants to suppress NK cell-mediated cytotoxicity toward endometrial targets. A significant correlation (r = 0.6, P < 0.05) was found between the sICAM-1 concentration in endometrial supernatants and the percentage of inhibition of NK cell-mediated lysis exerted by the same supernatant samples. Finally, endometrial stromal shedding of sICAM-1 appears to be related to endometriosis since endometrial stromal cultures obtained from patients with advanced stages of the disease released significantly higher amounts of the soluble protein compared to the control group (P < 0.05). sICAM-1 is a soluble molecule which can interfere with immunological functions, and its shedding may be one of the mechanisms by which refluxed endometrial cells escape immunosurveillance.