Microanatomy of the rat diaphragm with special reference to the lymphatics and mesothelial stomata

The three-dimensional microstructure of the rat diaphragm was studied in order to reveal morphological bases which permit peritoneal fluids to pass across the diaphragm to enter the pleural cavity. The methods used include scanning electron microscopy of either intact or alkali-treated tissues, enzyme-histochemistry, and confocal laser scanning microscopy (CLSM). The peritoneal and pleural surfaces of the diaphragm are covered with mesothelial cells studded with numerous microvilli. There are many round gaps between mesothelial cells on the peritoneal side of the diaphragm. The subperitoneal connective tissue contains voluminous, irregularly shaped lymphatics which extended many funnel-shaped projections of the endothelia towards the pored region of the mesothelium. On coming into contact with the mesothelium, many of the lymphatic projections are perforated at their ends, thus giving rise to stomata connecting the peritoneal cavity and lymphatic lumen. Some projections ended blindly while plugging the mesothelial pores, thereby making visible some intercellular gaps in this contact. The subperitoneal sheet of collagen fiber network possesses clusters of pores which tightly fit the passage of the lymphatic projections. CLSM of the diaphragm after intraperitoneal injection of FITC-dextran has demonstrated the tracer both in the lymphatic lumen and in the connective tissue spaces. The tracer has also been detected in the lymphatics located in the subpleural connective tissue space. These results indicate that peritoneal fluid is allowed to flow into the lymphatics directly through the stomata and indirectly through the intercellular gaps between endothelia and mesothelial cells, and then drain into the subpleural lymphatics. Discussions were made on the probable mechanisms by which a hydrothorax may occur during continuous ambulatory peritoneal dialysis.     

The Mesothelium, Teflon or Velcro? Mesothelium in endometriosis pathogenesis

Sampson's transplantation theory for the pathogenesis of peritoneal endometriosis is widely accepted. The events that take place, however, on the cellular and subcellular level during the transition of endometrial tissue in the abdominal cavity into peritoneal endometriosis remain controversial. The mesothelium plays a central role in the debate on this subject.The interaction between endometrium and peritoneum has been studied in an in-vitro model using amnion, peritoneum and mesothelial cells in culture on the one hand and cyclic and menstrual endometrium on the other hand. The results of these studies indicate that (i) an intact mesothelial lining prevents adhesion of shed endometrial tissue, (ii) shed endometrial tissue adheres to the peritoneal extracellular matrix and (iii) menstrual effluent creates its own adhesion sites by damaging the mesothelial lining thus exposing the extracellular matrix. Therefore we conclude that the mesothelium has the properties of Teflon, while the extracellular matrix resembles Velcro.     

Feasibility of mesothelial transplantation during experimental peritoneal dialysis and peritonitis

The mesothelial cell layer lining the peritoneum orchestrates peritoneal homeostasis. Continuous exposure to peritoneal dialysis fluids and episodes of peritonitis may damage the monolayer irreversibly, eventually leading to adhesion formation and fibrosis/sclerosis of the peritoneum. Autologous mesothelial cell transplantation is thought to be one of the options to reduce dysfunction of the peritoneal membrane. In this article we will review the mesothelial cell transplantation experiments performed in the field of peritoneal dialysis and peritonitis. In addition we will focus on the trouble shooting using cultured autologous mesothelial cells for transplantation.     

Transplantation of an autologous mesothelial cell sheet prepared from tunica vaginalis prevents post-operative adhesions in a canine model

Post-operative adhesions often cause severe complications such as bowel obstruction and abdominopelvic pain. Previously, we reported that transplantation of a mesothelial cell sheet is effective for preventing adhesion in rat model. We also proposed a new technique for harvesting autologous mesothelial cells from tunica vaginalis without intra-abdominal maneuvers. In this study, we examined whether an autologous mesothelial cell sheet can prevent post-operative peritoneal adhesions in a canine adhesion model. Mesothelial cells were isolated from the tunica vaginalis of male beagles. Isolated cells were cultured on fibrin gel. We named this construct the "mesothelial cell sheet." Animals underwent surgery to induce peritoneal adhesion formation and were then transplanted with the mesothelial cell sheets (sheet group, n = 4), fibrin gel (fibrin group, n = 4), or no materials (sham group, n = 4). Four weeks after the transplantation, we evaluated adhesion formation and scored adhesion levels. The abdominal wall transplanted with the mesothelial cell sheet was covered with mesothelium. The total adhesion score of the sheet group was significantly lower than that of the fibrin group and the sham group. These results indicated that transplantation of an autologous mesothelial cell sheet is effective for preventing post-operative adhesion formation in the canine adhesion model. Our mesothelial cell sheet has the potential to be a powerful adhesion prophylactic material in surgery.     

腹透液增强腹膜间皮细胞CD40表达及其意义

目的:研究腹膜透析液对大鼠腹膜间皮细胞CD40表达的影响及CD40活化后与细胞间粘附分子-1(ICAM-1)分泌的相关性, 以揭示腹膜透析时腹腔局部炎症的发生机制。方法: 分离、培养大鼠腹膜间皮细胞, 分别用IFN-γ、4.25%腹透液、4.25%腹透液+IFN-γ作为刺激因子, 通过逆转录-多聚酶链反应(RT-PCR)及流式细胞仪(FACS)检测间皮细胞CD40表达;并通过CD40单克隆抗体(CD40mAb)活化高表达的间皮细胞CD40, 用FACS检测间皮细胞ICAM-1的表达。结果:腹膜间皮细胞结构性表达低水平CD40mRNA及蛋白。用4.25%腹透液刺激后, 间皮细胞CD40mRNA及蛋白的表达显著高于对照组。用4.25%腹透液+IFN-γ刺激后, 间皮细胞表面CD40mRNA及蛋白的表达进一步增加, 且明显高于单纯腹透液刺激组。活化CD40受体可显著增强间皮细胞ICAM-1的表达。结论:腹膜间皮细胞可表达CD40, 4.25%透析液及IFN-γ均能显著增加腹膜间皮细胞CD40的表达。间皮细胞CD40的上调, 可能是腹透过程中腹腔局部炎症启动、放大的重要机制之一。     

Implant of autologous mesothelial cells in animals and a peritoneal dialysis patient

Success in culturing human and animal peritoneal mesothelial cells for the purpose of study, led us to determine whether these cells could be autoimplanted in animals and man during peritoneal dialysis in cases of acute and extensive loss of mesothelial surface area. Using an original biopsy technique, we were able to cultivate and characterize from the structural and caryological point of view, human and rabbit peritoneal mesothelial cells. Staphylococcal peritonitis was provoked in 12 rabbits with in-dwelling peritoneal catheters and after 4 days of antibiotic therapy, 6 of them were autoimplanted with cultured mesothelial cells. In the animals sacrificed on the third and sixth days, direct morphological observation and autoradiographic techniques showed that the transplanted cells had taken and revealed a different picture from that in the non-transplanted rabbits. In a 56 year old female diabetic patient, upon insertion of the first peritoneal catheter, a specimen of mesothelial cells was cultured and then frozen. Seven months later after an episode of peritonitis from candida which dictated removal of the peritoneal catheter, since there was a sufficient number of cultured mesothelial cells and the patient consented, the implant was performed. Peritoneal biopsy by laparoscopy three and six days later showed that the cells had taken. The purpose of the study was merely to show that autoimplant of mesothelium in man and animals is possible.     

Carcinoma‐associated fibroblasts derive from mesothelial cells via mesothelial‐to‐mesenchymal transition in peritoneal metastasis

Peritoneal dissemination is a frequent metastatic route for cancers of the ovary and gastrointestinal tract. Tumour cells metastasize by attaching to and invading through the mesothelial cell (MC) monolayer that lines the peritoneal cavity. Metastases are influenced by carcinoma‐associated fibroblasts (CAFs), a cell population that derives from different sources. Hence, we investigated whether MCs, through mesothelial–mesenchymal transition (MMT), were a source of CAFs during peritoneal carcinomatosis and whether MMT affected the adhesion and invasion of tumour cells. Biopsies from patients with peritoneal dissemination revealed the presence of myofibroblasts expressing mesothelial markers in the proximity of carcinoma implants. Prominent new vessel formation was observed in the peritoneal areas harbouring tumour cells when compared with tumour‐free regions. The use of a mouse model of peritoneal dissemination confirmed the myofibroblast conversion of MCs and the increase in angiogenesis at places of tumour implants. Treatment of omentum MCs with conditioned media from carcinoma cell cultures resulted in phenotype changes reminiscent of MMT. Adhesion experiments demonstrated that MMT enhanced the binding of cancer cells to MCs in a β1‐integrin‐dependent manner. Scanning electron microscopy imaging showed that the enhanced adhesion was mostly due to increased cell–cell interaction and not to a mere matrix exposure. Invasion assays suggested a reciprocal stimulation of the invasive capacity of tumour cells and MCs. Our results demonstrate that CAFs can derive from mesothelial cells during peritoneal metastasis. We suggest that MMT renders the peritoneum more receptive for tumour cell attachment/invasion and contributes to secondary tumour growth by promoting its vascularization. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Polarized secretion of interleukin-8 by human mesothelial cells: a role in neutrophil migration.

We investigated the role of human mesothelium in an in vitro model of peritonitis with emphasis on the secretion of the neutrophil chemoattractant interleukin-8 (IL-8) and the migration of polymorphonuclear leucocytes (PMN) across monolayers of peritoneal mesothelial cells. PMN showed minimal migration across non-activated mesothelial monolayers (< 2%). However, migration was induced after mesothelial cell activation by IL-1 beta (24%) and this induced migration was significantly blocked by antibodies against IL-8 (63% inhibition; P < or = 0.01). IL-1 beta-activated mesothelial monolayers were shown to secrete IL-8 in a polarized way, which was preferentially oriented towards the apical side of the monolayer. Our results indicate that the influx of PMN into the peritoneal cavity is, at least in part, controlled by the mesothelial cell layer of the peritoneal membrane.     

Effects of rapamycin on the epithelial-to-mesenchymal transition of human peritoneal mesothelial cells

The preservation of the peritoneal membrane is crucial for long-term survival in peritoneal dialysis. Epithelial-to-mesenchymal transition (EMT) is a process demonstrated in mesothelial cells (MC), responsible for negative peritoneal changes and directly related to PD. EMT enables neovascularization and fibrogenic capabilities in MC. Vascular endothelial growth factor (VEGF) is the mediator for neo-vascularization. Rapamycin is a potent immunosuppressor with antifibrotic action in renal allografts and has a demonstrated anti-VEGF effect. We performed this study with the hypothesis that rapamycin may regulate the EMT of MC. MC from human omentum were cultured. When mesothelial cells reached confluence, some of them were stimulated with r-TGF-beta (1 ng/mL) to induce EMT, co-administered with rapamycin (0.2, 2, 4, 20 and 40 nM). Other groups of cells received similar doses of rapamycin or r-TGF-beta, separately. Cells were analyzed at 6, 24, 48 hours and 7 days. As markers of EMT we included alfa -SMA, E-cadherin and snail nuclear factor by quantitative RT-PCR. EMT markers and regulators demonstrated the following changes with rapamycin: E-cadherin (a protective gene for EMT) increased 2.5-fold relative to controls under 40 nM, at 24h. Importantly, rapamycin inhibited snail expression induced by TGF-beta at 6h, whereas TGF-beta increased snail 10-fold. At day 7, rapamycin showed no anti-EMT properties. An important decrease in alfa -SMA expression by MC after rapamycin addition was observed. In conclusion, rapamycin shows a mild protective effect on EMT, as it increases E-cadherin and decreases alfa -SMA expression. Consequently, rapamycin might partially regulate the epithelial-to-mesenchymal transition of mesothelial cells.     

Hyaluronic acid secreted by mesothelial cells: a natural barrier to ovarian cancer cell adhesion

The adhesion to mesothelial monolayers of eight cultured ovarian tumour cell lines was studied in multiwell plates as a model for some of the interactions of ovarian cancer in the peritoneal cavity. When only the upper half of the conditioned medium (CM) from a confluent mesothelial cell culture was aspirated, the adhesion of the tumour cells was low (3.5%–36%). When the medium was removed completely the adhesion increased. The tumour cell lines showing the greatest enhancement of adhesion were those which had previously been shown to express the highest amounts of CD44. By adding erythrocyte suspensions to mesothelial cells it was shown that there was a pericellular coat around the mesothelial cells that could be destroyed by aspirating the medium, or by treating the medium with hyaluronidase (Hase). Treatment of the CM with Hase also considerably increased tumour cell adhesion. Furthermore, CM was shown to contain high amounts of hyaluronic acid (HA). HA blocked adhesion in the absence of CM, but the effect was not as large as that produced by the pericellular coat. It is proposed that pericellular HA produced by mesothelial cells has an important role in the invasion of ovarian tumour cells in the peritoneal cavity.     

人腹膜间皮细胞的改良分离及其鉴

背景：腹膜间皮细胞作为腹膜的重要组分,分泌多种细胞因子,在参与抗炎、免疫调节、腹膜纤维化等方面起着重要作用,如何获得优质、均一的腹膜间皮细胞成为解决这些问题的关键。 目的：拟建立改良的人腹膜间皮细胞消化培养法。 方法：0.1%胶原酶Ⅰ消化腹部大网膜组织,去除红细胞后用含体积分数为10%胎牛血清的1640培养基培养。在培养过程中以倒置显微镜观察细胞形态变化,CCK-8观察培养液对间皮细胞生长的促进作用,透射电子显微镜观察细胞超微结构,激光共聚焦免疫荧光鉴定细胞。 结果与结论：分离培养的细胞为多角形,汇合时呈铺路石样排列,培养细胞的纯度达90%以上;生长良好,迅速,可传代至四五代。透射电子显微镜下见细胞表面大量的微绒毛,免疫荧光显示角蛋白、波形蛋白抗原阳性,白细胞CD45、第Ⅷ因子相关抗原阴性;所有被鉴定特征均符合间皮细胞的特点。结果提示胶原酶Ⅰ消化法是一种简单、高效、重复性高的分离腹膜间皮细胞的方法。     

Stem cells for mesothelial repair: an understudied modality

Adhesions are bands of fibrous tissue that form between opposing organs and the peritoneum, restricting vital intrapleural and abdominal movement. They remain a major problem in abdominal surgery, occurring in more than three fourths of patients following laparotomy. Adhesions result when injury to the mesothelium is not repaired by mesothelial cells and can be viewed as scar tissue formation. The mechanism of mesothelial healing suggested the involvement of stem cells in the process. It has long been known that peritoneal wounds heal in the same amount of time regardless of size. Therefore, the mesothelium could not regenerate solely by proliferation and centripetal migration of cells at the wound edge as occurs in the healing of skin epithelium. Several studies suggest the presence of i) mesothelial stem cells that can differentiate into mesothelial cells and a few other phenotypes and/or ii) that mesothelial cells are themselves stem cells. Other studies have suggested that adult stem cells in the muscle underlying the peritoneum can differentiate into mesothelial cells and contribute to healing. Prevention of abdominal adhesions have been accomplished by delivery of autologous mesothelial cells and multipotent adult stem cells isolated from skeletal muscle. Adult stem cells from sources other than the serosal tissue offer an alternative treatment modality to prevent the formation of abdominal adhesions.     

Antigen-presenting function of human peritoneum mesothelial cells

Mesothelial cells (MC) from human peritoneal omentum fragments obtained during surgical insertion of peritoneal catheters for continuous peritoneal dialysis in end stage renal failure (ESRF) patients were cultured in vitro. MC exhibited a phenotype different from macrophages, but MHC class II molecules were well expressed. Therefore MC lines were tested for antigen-presenting capacity by pulsing with soluble antigens (tetanus toxoid and purified protein derivative (PPD)) or with a corpusculate antigen (Candida albicans bodies). Autologous peripheral blood mononuclear cells (PBMC) depleted of adherent monocytes and cloned T cells generated from an individual matched for the MHC class II antigen DR2 were used to test antigen-presenting function. MC effectively presented the soluble and corpusculate antigens to autologous and MHC-compatible allogeneic lymphocytes, indicating that they are endowed with both endocytic/phagocytic activity and with processing/presenting capacity. Preincubation of MC with human recombinant interferon-gamma (IFN-γ) up-regulated MHC class II and intercellular adhesion molecule-I (ICAM-I) expression, but the effect on antigen-presenting function was not consistent. Since MC are an important component of the peritoneal environment, they may participate, along with macrophages, in activation of specific T cells and in the generation of local cell-mediated immunity to various pathogens.     

Expression of adhesion molecules relevant to leukocyte migration on the microvilli of liver peritoneal mesothelial cells

To help assess the immunological functions of the liver peritoneum, expression and 3D-microlocalization of adhesion molecules were studied by immuno-SEM and -TEM. The peritoneal tissues of the liver obtained from lipopolysaccharide (LPS, 1.5 microg/g BW for 24 hr)-stimulated (n = 18 including nine controls) and non-stimulated mice (n = 6 including three controls) were analyzed by immunolabeling with 15 nm gold particle single-labeling analysis of ICAM-1, ICAM-2, VCAM-1, MAdCAM-1, PECAM-1, ELAM-1, and CD105 expression. In addition, 10 and 20 nm gold particle double-labeling analysis of ICAM-1 and VCAM-1 was carried out with conventional TEM and BSE (backscatter electron) imaging. Gold particles detected in the peritoneal mesothelial cells were quantified using a computer analyzer, LUZEX III. Only ICAM-1 in non-stimulated mice and both ICAM-1 and VCAM-1 in LPS-stimulated mice were expressed on the mesothelium, but no other adhesion molecules were detected in either condition. Expression of ICAM-1 was consistently about four times greater than that of VCAM-1. Each adhesion molecule was restricted to the microvilli. ICAM-1 was expressed on all microvilli and tended to form clusters of three or four molecules. On the other hand, about 24% of the microvilli expressed VCAM-1 and less clustering was seen. Double-labeling techniques disclosed that VCAM-1 and ICAM-1 were rarely closely associated, usually spaced by about 40 nm. These results suggest that microvilli of the mesothelial cell play a significant role in leukocyte migration in the peritoneal cavity, by providing the important substrates for adhesion, ICAM-1 and VCAM-1.     

The potential of mesothelial cells in tissue engineering and regenerative medicine applications

Injury to the serosa through injurious agents such as radiation, surgery, infection and disease results in the loss of the protective surface mesothelium and often leads to fibrous adhesion formation. Mechanisms that increase the rate of mesothialisation are therefore actively being investigated in order to reduce the formation of adhesions. These include intraperitoneal delivery of cultured mesothelial cells as well as administration of factors that are known to increase mesothelial proliferation and migration. An exciting alternative that has only recently received attention, is the possible role of mesothelial progenitor cells in the repair and regeneration of denuded serosal areas. Accumulating evidence suggests that such a population exists and under certain conditions is able to form a number of defined cell types indicating a degree of plasticity. Such properties may explain the extensive use of mesothelial cells in various tissue engineering applications including the development of vascular conduits and peripheral nerve replacements. It is likely that with the rapid explosion in the fields of tissue engineering and regenerative medicine, a greater understanding of the potential of mesothelial progenitor cells to repair, replace and possibly regenerate damaged or defective tissue will be uncovered.     

Changes in peritoneal mesothelial cells phenotype after chronic exposure to glucose or N-acetylglucosamine

Glucose is commonly used as an osmotic solute in peritoneal dialysis fluids despite vast knowledge about deleterious peritoneal and systemic effects of that solute. N-acetylglucosamine (NAG) is a solute of the comparable size to glucose, with strong anti-inflammatory properties. We compared the chronic in vitro effect of both solutes on phenotype of peritoneal mesothelial cells. Experiments were performed of primary cultures of human peritoneal mesothelial cells, which were cultured over 4 weeks in medium supplemented either with glucose 45 mmol/L (GLU) or with NAG 45 mmol/L (NAG). Generation of reactive oxygen species (ROS) in cells was studied, as well as their ability to proliferate, synthesis of cytokines, fibronectin, and factors regulating peritoneal fibrinolysis. Cells cultured in the presence of glucose 45 mmol/L generated more ROS (+73% vs control, P < 0.01), whereas NAG did not stimulate generation of ROS. GLU caused hypertrophy of mesothelial cells (+53% vs control, P < 0.001) and prolonged their population doubling time (+16% vs control, P < 0.01); NAG did not cause significant changes in these parameters. Healing of mesothelial monolayer after mechanical injury was impaired in GLU treated cells: (-48% vs control, P < 0.001 and -40% vs NAG, P < 0.05). Synthesis of Il-6, vascular endothelial growth factor (VEGF), transforming growth factor beta (TGFbeta), and fibronectin was higher in GLU group as compared with control: + 86%, P < 0.001, +38%, P < 0.05, +51%, P < 0.001, +38%, P < 0.05, respectively. In the presence of NAG, these parameters were comparable with the control group, but at the same time NAG stimulated synthesis of hyaluronan: +116% versus control, P < 0.001 and + 96% versus GLU, P < 0.01. Treatment with GLU resulted in decline of tissue plasminogen activator/plasminogen activator inhibitor-1 (t-PA/PAI-1) ratio by 23% versus control, P < 0.001, whereas NAG increased that parameter by 43%, P < 0.01 versus control. Glucose, contrary to NAG, induces oxidative stress and proinflammatory and profibrotic changes in mesothelial cells. NAG seems to be more biocompatible osmotic solute than glucose.     

Culturing mouse peritoneal mesothelial cells

Obtaining normal cells has become increasingly important for use in comparative genetic analytical techniques to examine alterations in gene expression during transformation and progression into malignancy. Normal mesothelial cells are not currently available in cell banks and are essential for comparison of genetic expression analysis in current mouse mesothelioma models. The purpose of this investigation was to extract normal mouse peritoneal mesothelial cells using minimal culture techniques to obtain sufficient cells for gene expression analysis. Mesothelial cells were collected from the mouse peritoneal cavity in vivo with minimal contamination of lymphocytes and macrophages. The cells were cultured for approximately eight days until they were just confluent and retained normal mesothelial phenotype and cytokeratin immunoperoxidase staining.     

CD40在人腹膜间皮细胞的表达

目的:对人腹膜间皮细胞CD40的表达及其调节因素进行初步探讨。方法:从CAPD患者的透出液中分离、培养腹膜间皮细胞,用IFN-γ、TNF-α、IL-1、LPS刺激24h,通过流式细胞仪(FACS)检测分析腹膜间皮细胞CD40、CD40L及ICAM-1的表达。结果:腹膜间皮细胞结构性表达少量的CD40;IFN-γ可显著增加腹膜间皮细胞表面CD40蛋白的表达,而TNF-α、IL-1、LPS对腹膜间皮细胞表面CD40蛋白的表达无显著影响。未见间皮细胞表达CD40L。IFN-γ、TNF-α、IL-1、LPS对间皮细胞ICAM-1表达均有显著增强作用。IFN-γ增强ICAM-1表达作用显著高于TNF-α、IL-1、LPS,间皮细胞CD40表达强度与ICAM-1呈显著正相关。结论:人腹膜间皮细胞可功能性表达CD40。     

The mesothelium: a cytochemical study of "activated" mesothelial cells

The cytochemical profile of activated mesothelial cells differs from resting cells. The reaction products of enzymes associated with oxidative mechanisms of cell respiration were easily displayed in activated mesothelial cells; in resting mesothelial cells, only enzymes of the pentose pathway were readily demonstrable. Acid hydrolases were detected in greater quantity than in resting cells, possibly reflecting an increased potential for endocytosis. In addition, the cytochemical assays indicated increased Golgi activity was reflected by the demonstration of thiamine pyrophosphatase, while the content of ribonucleic acid was also increased. These cytochemical features compare well with the ultrastructure of active mesothelial cells which possess abundant ergastoplasm and a well-developed Golgi apparatus. In comparison with peritoneal macrophages, activated mesothelial cells differ mainly in the quantity of reaction product, there being more in macrophages. The results were significantly different only in the demonstration of lipids which were never found in mesothelial cells, but were invariably present in macrophages. It is still unclear whether the acid mucopolysaccharide hyaluronic acid is produced by or merely transported through the mesothelium from the subserosal site.     

Tumour necrosis factor-alpha, interleukin-6 and interleukin-8 do not promote adhesion of human endometrial epithelial cells to mesothelial cells in a quantitative in vitro model

BACKGROUND: A key factor in the pathogenesis of endometriosis is the endometrial-peritoneal adhesion. To study the pathogenesis of endometriosis, a quantitative in vitro assay (QIVA) was developed to measure in vitro adhesion between human endometrial epithelial cells and mesothelial cells using commercially available cell lines. Using the QIVA, the hypothesis was tested that tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and interleukin-8 (IL-8) promote adhesion of endometrial epithelial cells to mesothelial cells. METHODS: Mesothelial cells were pre-treated with TNF-alpha, IL-6 or IL-8 in various concentrations (ranging from 0 to 1000 IU/ml) for 24 h. Confluent endometrial epithelial cells were labelled with [35S]methionine, added to the confluent mesothelial cells and incubated for 1 h. After incubation, non-adhering cells were removed and adherent cells were solubilized and their [35S]methionine radioactivity was counted to quantify the adherence of endometrial epithelial cells to mesothelial cells. RESULTS: The in vitro adhesion of human endometrial epithelial cells to human mesothelial cells was inhibited in a dose-dependent manner by TNF-alpha (P=0.0007), IL-6 (P<0.0001) and IL-8 (P=0.0004). CONCLUSIONS: Using a quantitative in vitro adhesion assay, we were unable to confirm our hypothesis that TNF-alpha, IL-6 and IL-8 promote the in vitro adhesion between endometrial epithelial cells and mesothelial cells.