Utility of HBME-1 immunostaining in serous effusions

Utility of HBME-1 immunostaining in serous effusions
HBME-1 is an anti-mesothelial cell monoclonal antibody derived from human mesothelioma cells. We investigated 227 body cavity effusions to test its utility in differentiating mesothelioma from adenocarcinoma. HBME-1 outlined cell membranes in non-neoplastic mesothelial cells. Thick surface staining was observed on all mesotheliomas. HBME-1 reactivity was also detected in 24% of metastatic carcinomatous effusions. Most ovarian carcinomas (83%) reacted with this antibody, showing surface staining. Cytoplasmic HBME-1 immunoreactivity was observed in a small proportion of non-ovarian adenocarcinomas (14%). Despite its limited specificity, HBME-1 might be added to the battery of other markers of epithelial and/or mesothelial differentiation to be used in cases of suspected mesothelioma. Evaluation of suspicious cells should include careful study of the pattern of immunostaining.     

A scanning electron microscope study of mouse peritoneal mesothelium.

As seen in the scanning electron microscope, peritoneal mesothelial cells of the mouse diaphragm, anterior abdominal wall and intestinal serosa carry numerous microvilli. These microvilli are absent over certain areas of the cell surface and are sometimes, interlocked in meshwork patterns or coronal formation. The apical cell membranes of the mesothelium at the base of the microvilli, are invaginated by many plasmalemmal vesicles and vacuoles and carry a number of protruding spherical structures. Deep circular craters, giving the impression of stomata, are also visible.     

Surface configuration of mesothelial cells in effusions. A comparative light microscopic and scanning electron microscopic study.

Surface configuration of mesothelial cells identified by light microscopy (LM) has been studied by scanning electron microscopy (SEM). It has been shown that mesothelial cells may have a variable SEM appearance. The surfaces of a small proportion of mesothelial cells are covered by regular microvilli (MV) and show openings of the pinocytotic vesicles. The surfaces of the majority of these cells are covered by vesicles or blebs. An intermediate population of mesothelial cells, i.e., cells displaying side-by-side blebs and MV, has also been observed. The latter cells no longer display pinocytotic vesicles. Occasional mesothelial cells have smooth surfaces. It has been shown by LM and transmission electron microscopy that cells with blebs are viable and capable of mitotic activity. It is concluded that mesothelial cells, detached from their epithelial setting, lose microvilli and pinocytotic vesicles and acquire surface blebs. The possible relationship between mesothelial cells and macrophages based on surface features has been discussed.     

Mesothelial primary cilia of peritoneal and other serosal surfaces

The conspicuous presence of primary cilia, a small immotile cilium present on most cell types, left researchers with little doubt of their functional relevance. Recently mechanosensitive functional significance was established and a link with the pathogenesis of polycystic kidney disease. Together these discoveries have raised the profile of this, previously considered "vestigial", organelle. Primary cilia are expressed on the apical surface of serosal mesothelium and display regional variation but are more abundant on biosynthetically active cells. Adult mesothelial cells are highly biosynthetic producing a phospholipid rich surfactant that lubricates and protects the visceral organs. The mesothelium is utilized as a semipermeable membrane during peritoneal dialysis for patients with end stage renal failure. However, little is known about the functional role of primary cilia on this highly specialized cell type. The present review, examines the significance of the primary cilium in serosal mesothelial cell biology with an emphasis on ciliary location, structure, form and function. Future research is identified and discussed in view of the emerging role cilia have in other cells and the established function of the serosal mesothelium in development, normal function, peritoneal dialysis and pathology of the serosal membranes.     

Light and electron microscope observations of the lymphatic drainage units of the peritoneal cavity of rodents

Fluid, particles, and cells are taken up from the peritoneal cavity by lymphatic drainage units, which, in the mouse and rat, are located along the peritoneal surface of the muscular portion of the diaphragm. The drainage units are composed of three specifically differentiated components: a lymphatic lacuna, a covering of lacunar mesothelium, and intervening submesothelial connective tissue. The units are drained by connecting lymphatic vessels that cross the diaphragm to empty into collecting lymphatic vessels running along the pleural surface of the diaphragm. The collecting lymphatics empty into parasternal lymphatic trunks. In this report, we briefly review critical features of the drainage apparatus and describe new observations, summarized below, about their structure. Around the rim of stomata, the mesothelial openings that lead into the lymphatic lacunae, plasma membranes of lacunar mesothelial cells and of lacunar endothelial cells abut but are not linked to one another by recognizable junctional specializations. Lacunar endothelial cells often extend valve-like processes that bridge the distal end of the channel beneath the stoma. The configuration of the endothelial processes may be complex. Occasionally, processes from fibroblasts in the submesothelial connective tissue adjacent to stomata make contact with the interstitial surface of lacunar endothelial cells. A discontinuous elastic layer in the submesothelial connective tissue spans the roof of each lacuna. Connecting and collecting lymphatics, which drain lymphatic lacunae, possess endothelial valves. Possible functions for each of these newly described structural features are discussed.     

Autophagy promotes fibrosis and apoptosis in the peritoneum during long‐term peritoneal dialysis

Long‐term peritoneal dialysis is accompanied by functional and histopathological alterations in the peritoneal membrane. In the long process of peritoneal dialysis, high‐glucose peritoneal dialysis solution (HGPDS) will aggravate the peritoneal fibrosis, leading to decreased effectiveness of peritoneal dialysis and ultrafiltration failure. In this study, we found that the coincidence of elevated TGF‐β1 expression, autophagy, apoptosis and fibrosis in peritoneal membrane from patients with peritoneal dialysis. The peritoneal membranes from patients were performed with immunocytochemistry and transmission electron microscopy. Human peritoneal mesothelial cells were treated with 1.5%, 2.5% and 4.25% HGPDS for 24 hrs; Human peritoneal mesothelial cells pre‐treated with TGF‐β1 (10 ng/ml) or transfected with siRNA Beclin1 were treated with 4.25% HGPDS or vehicle for 24 hrs. We further detected the production of TGF‐β1, activation of TGF‐β1/Smad2/3 signalling, induction of autophagy, EMT, fibrosis and apoptosis. We also explored whether autophagy inhibition by siRNA targeting Beclin 1 reduces EMT, fibrosis and apoptosis in human peritoneal mesothelial cells. HGPDS increased TGF‐β1 production, activated TGF‐β1/Smad2/3 signalling and induced autophagy, fibrosis and apoptosis hallmarks in human peritoneal mesothelial cells; HGPDS‐induced Beclin 1‐dependent autophagy in human peritoneal mesothelial cells; Autophagy inhibition by siRNA Beclin 1 reduced EMT, fibrosis and apoptosis in human peritoneal mesothelial cells. Taken all together, these studies are expected to open a new avenue in the understanding of peritoneal fibrosis, which may guide us to explore the compounds targeting autophagy and achieve the therapeutic improvement of PD.     

Ultrastructural aspects of and observations on the permeability of the rat mesentery to colloidal iron

In works already published, it was made clear that many researches were interested in the absorption phenomena, permeability and structure of the visceral mesothelial tissue. Attention was concentrated on the mesentery and observations were made using the application of lanthanum nitrate and osmium-amine. The penetration of lanthanum nitrate is impeded by the basement membrane situated between the connective and mesothelial tissues. The heavy salt moves through and not between the mesothelial cells by passive diffusion. No reaction was observed in general with osmium-amine, with the exception of a few cases. In those instances, the osmium-amine reacted not only in the outer surface of the mesentery, but also penetrated with no visible reaction all the way to the connective tissue where it was detected in the elastic layer. In this paper, the colloidal iron was employed using different techniques, and depositions were detected in the surface of the mesentery, in the mesothelial cells and also in the connective tissue. A final conclusion that the permeability of different layers of tissues is of great variety and has a definite capacity for selectivity is suggested.     

Identification of a novel developmental mechanism in the generation of mesothelia

Mesothelium is the surface layer of all coelomic organs and is crucial for the generation of their vasculature. Still, our understanding of the genesis of this essential cell type is restricted to the heart where a localized exogenous population of cells, the proepicardium, migrates to and envelops the myocardium supplying mesothelial, vascular and stromal cell lineages. Currently it is not known whether this pattern of development is specific to the heart or applies broadly to other coelomic organs. Using two independent long-term lineage-tracing studies, we demonstrate that mesothelial progenitors of the intestine are intrinsic to the gut tube anlage. Furthermore, a novel chick-quail chimera model of gut morphogenesis reveals these mesothelial progenitors are broadly distributed throughout the gut primordium and are not derived from a localized and exogenous proepicardium-like source of cells. These data demonstrate an intrinsic origin of mesothelial cells to a coelomic organ and provide a novel mechanism for the generation of mesothelial cells.     

HGF/SF Induces Mesothelial Cell Migration and Proliferation by Autocrine and Paracrine Pathways

Mesothelial repair differs from that of other epithelial-like surfaces as healing does not occur solely by centripetal in-growth of cells as a sheet from the wound margins. Mesothelial cells lose their cell-cell junctions, divide, and adopt a fibroblast-like morphology while scattering across and covering the wound surface. These features are consistent with a cellular response to hepatocyte growth factor/scatter factor (HGF/SF). In this study, we examined the ability of mesothelial cells to secrete HGF/SF and investigated its possible role as an autocrine regulator of mesothelial cell motility and proliferation. We found that human primary mesothelial cells expressed HGF/SF mRNA and secreted active HGF/SF into conditioned medium as determined by ELISA and in a scattering bioassay. These cells also expressed the HGF/SF receptor, Met, as shown by RT-PCR and by Western blot analysis and immunofluorescence. Incubation of mesothelial cells with neutralizing antibodies to HGF/SF decreased cell migration to 25% of controls, whereas addition of HGF/SF disrupted cell-cell junctions and induced scattering and enhanced mesothelial cell migration. Furthermore, HGF/SF showed a small but significant mitogenic effect on all mesothelial cell lines examined. In conclusion, HGF/SF is produced by mesothelial cells and induces both motility and proliferation of these cells. These data are consistent with HGF/SF playing an autocrine role in mesothelial healing.     

Immunocytochemical analysis of ascitic fluid due to cirrhosis. A contribution to understanding the origin of markedly atypical cells.

In some cases of ascitic fluid due to cirrhosis, benign mesothelial clusters may be observed, accompanied by markedly atypical cells that have been proposed to be abnormal macrophages, mesothelial cells or necrotic cells of hepatic origin. The aim of this study was to determine the origin of these cells with the use of a panel of monoclonal antibodies (MAbs) against cell surface antigens. Furthermore, the lymphocyte subpopulations were analyzed for a possible correlation with the presence of abnormal cells. Markedly atypical cells were found in 4 of 12 cases. They showed no phagocytosis of latex particles and were negative for MAbs My4 (CD14), HLE-1 (CD45), Leu M1 (CD15), CEA 3-13 and HEA-125. They reacted positively with BMA-120 and HLA-1. This staining pattern demonstrated the mesothelial origin of the markedly atypical cells. The profile of the lymphocyte subpopulations in the cases with markedly atypical cells was not different from the other cases. We propose that these cells are abortive cluster formations of mesothelial cells.     

Vitronectin adsorption to chrysotile asbestos increases fiber phagocytosis and toxicity for mesothelial cells

Biological modification of asbestos fibers can alter their interaction with target cells. We have shown that vitronectin (VN), a major adhesive protein in serum, adsorbs to crocidolite asbestos and increases fiber phagocytosis by mesothelial cells via integrins. Because chrysotile asbestos differs significantly from crocidolite in charge and shape, we asked whether VN would also adsorb to chrysotile asbestos and increase its toxicity for mesothelial cells. We found that VN, either from purified solutions or from serum, adsorbed to chrysotile but at a lower amount per surface area than to crocidolite. Nevertheless, VN coating increased the phagocytosis of chrysotile as well as of crocidolite asbestos. VN coating of both chrysotile and crocidolite, but not of glass beads, increased intracellular oxidation and apoptosis of mesothelial cells. The additional apoptosis could be blocked by integrin-ligand blockade with arginine-glycine-aspartic acid peptides, confirming a role for integrins in the fiber-induced toxicity. We conclude that VN increases the phagocytosis of chrysotile as well as of crocidolite asbestos and that phagocytosis is important in fiber-induced toxicity for mesothelial cells.     

Nod1/RICK and TLR signaling regulate chemokine and antimicrobial innate immune responses in mesothelial cells

Mesothelial cells that line the serous cavities and outer surface of internal organs are involved in inflammatory responses induced by microbial stimuli and bacterial infection. Upon exposure to bacterial products, mesothelial cells secrete chemokines, but the signaling pathways by which these cells recognize bacteria to mediate innate immune responses remain largely unknown. We report that stimulation of primary peritoneal mesothelial cells via nucleotide-binding oligomerization domain (Nod)1, a member of the intracytoplasmic Nod-like receptor family, induced potent secretion of the chemokines CXCL1 and CCL2 as well as expression of inducible NO synthase and such responses required the kinase RICK. Mesothelial cells also produced chemokines in response to TLR2, TLR3, TLR4, and TLR5 agonists, but unlike that induced by Nod1 stimulation, the TLR-mediated responses were independent of RICK. Yet, Nod1 stimulation of mesothelial cells via RICK enhanced chemokine secretion induced by LPS or IFN-gamma and cooperated with IFN-gamma in the production of NO. The i.p. administration of KF1B, a synthetic Nod1 agonist, elicited chemokine production in the serum and peritoneal fluid as well as the recruitment of neutrophils into the peritoneal cavity of wild-type mice, but not RICK-deficient mice. Finally, infection of mesothelial cells with Listeria monocytogenes induced production of CXCL1 and this response was significantly reduced in Nod1- or RICK-deficient cells. These results define mesothelial cells as microbial sensors through TLRs and Nod-like receptors and identify Nod1 and RICK as important mediators of chemokine and antimicrobial responses in mesothelial cells.     

Lubrication regimes in mesothelial sliding

To function normally, the lungs, heart, and other organs must undergo changes in shape and size, sliding against surrounding body walls. It is not known whether the delicate mesothelial surfaces covering these organs and body wall are in contact during sliding, or if hydrodynamic pressure in the lubricating liquid increases separation between their surfaces. To address this question, we measured the coefficient of friction (mu) of the mesothelial surface of nine rat-abdominal walls sliding in saline on a smooth glass surface. Sliding at physiological velocities of 0.0123-6.14 cm/s with normal stresses of 50-200 Pa, mu varied with velocity (P<0.001). On average, mu was relatively high at low speeds (0.078 at 0.041 cm/s), decreased to a minimum at intermediate speeds (0.034 at 1.23 cm/s), and increased slightly again at higher speeds (0.045 at 6.14 cm/s), consistent with a mixed lubrication regime in which there is at least partial hydrodynamic separation of surfaces. We conclude that mesothelial surfaces, sliding under physiological conditions, are protected from excessive shear by hydrodynamic pressures that increase separation of surfaces.     

Malignant ascites of serous papillary ovarian adenocarcinoma. An immunocytochemical study of the tumor cells

In 17 malignant peritoneal effusions due to papillary serous adenocarcinoma of the ovary, the reaction patterns of the tumor cells to monoclonal antibodies (MAbs) against surface antigens were studied and compared with the reaction patterns of mesothelial cells in the same effusions. The following surface markers were used with the adhesive slide method: epithelial membrane antigen (EMA), human epithelium-specific cell surface antigen (HEA-125), human endothelial antigen (BMA-120), carcinoembryonic antigen (CEA 3-13), an antibody against natural killer cells and cytotoxic cells (BMA-070), granulocyte antigen (Leu M1) and leukocyte antigen of class I (HLA-1). In all cases, from 30% to 95% of the tumor cells reacted with EMA and HEA-125. Tumor cells showed a positive staining with CEA 3-13 in only five cases. In all cases, from 75% to 95% of the tumor cells reacted positively with BMA-120. The reactivity of a few mesothelial cells with EMA and of all mesothelial cells with BMA-120 did not interfere with the identification of positive tumor cells since the reaction patterns were different. Interestingly, our study demonstrated that BMA-070, an MAb identifying natural killer cells and cytotoxic cells, is also a most useful tumor marker. The same was found to be true for Leu M1, an MAb originally thought to react only with granulocytes. The tumor cells showed a partial or total loss of the expression of HLA-1 reactivity. Since all cases were immunocytochemically positive for tumor cells while conventional cytology was positive in only 13 of the cases, the immunocytochemical analysis of malignant peritoneal effusions due to papillary serous adenocarcinoma of the ovary seems able to improve the cytologic diagnosis of the fluids.     

Quantitative immunocytochemical studies of endogenous albumin in rat aortic endothelial and mesothelial cells

Endogenous albumin was revealed over cellular structures of rat ascendent aorta endothelia and mesothelium, with high resolution and specificity, by applying the protein A-gold immunocytochemical approach. This approach allows albumin distribution to be studied under steady-state conditions. The cellular layers evaluated were the aortic endothelium, the capillary endothelium (vasa vasorum), and the mesothelium externally lining the aorta at this level. Gold particles, revealing albumin antigenic sites, were preferentially located over plasmalemmal vesicles and intercellular clefts of endothelial and mesothelial cells, though with different labeling intensities. The interstitial space was also labeled. Morphometrical evaluation of plasmalemmal vesicles demonstrated a higher surface density for these structures in capillary endothelial cells (12%) compared with those in aortic endothelial (5%) and mesothelial cells (2%). Quantitation of gold labeling intensities over these structures revealed a higher labeling over plasmalemmal vesicles of capillary endothelium than over those of aortic endothelium and mesothelium. This result, together with the higher surface density of plasmalemmal vesicles found in capillary endothelium, suggest an important role of these structures in the transendothelial passage of endogenous albumin, particularly for capillary endothelium. On the other hand, labeling densities over mesothelial clefts were found to be higher than those of capillary and aortic endothelia. Results from this study concur with the proposal of a differential passage of albumin according to the cell lining considered, and suggest to a role for mesothelial intercellular clefts in contributing to the presence of albumin in interstitial spaces.     

Binding of ovarian cancer cells to immobilized hyaluronic acid

Ovarian cancer has the highest mortality rate of any gynaecological malignancy. This is caused by metastatic deposits obstructing the intestinal tract. Very little is known about the molecules involved in the initial attachment of the metastatic tumour cells to the peritoneal mesothelial lining. Previously, we showed that many ovarian tumour lines express the adhesion molecule, CD44, on their cell surface. The major ligand for CD44 is the extracellular matrix glycosaminoglycan, hyaluronic acid (HA). Because mesothelial cells have a pericellular cost that contains large amounts of HA, it was postulated that the CD44/HA interaction is an important stage in ovarian cancer spread. However, it was difficult to demonstrate this interaction in an in vitro adhesion assay with mesothelial cells as most of the HA, and presumably the bound tumour cells, were lost from the mesothelial cells during the washing steps of the assay. In order to try and clarify the situation, the adhesion of six ovarian tumour lines to immobilized HA was measured. Four lines expressed high levels of CD44 and two lines expressed negligible amounts. Preliminary experiments were carried out with one of the CD44-expressing lines. After coating a plate overnight with 3 mg ml−1 HA, the 5 min adhesion of this line varied between 2% and 73% according to the type of plate that was used. Falcon Micro Test III flexible plates gave the highest adhesion and was used for further experiments. Plates were coated with concentrations of HA between 0.001 mg ml−1 and 3 mg ml−1. All CD44 expressing lines adhered to HA, but the maximum adhesion and the adhesion strength varied with the line studied and was not closely related to the total CD44 expression. These results suggest that CD44 on ovarian tumour cells binds to HA on mesothelial cells. As much of the HA can be very easily lost from the mesothelial cell surface, additional factors such as the strength of the CD44/HA interaction, and the formation of bonds by the tumour cells with other membrane adhesion molecules, such as integrins, are also important in promoting tumour spread. This revised version was published online in November 2006 with corrections to the Cover Date.     

Cytochemical investigation of cathepsin B in rheumatoid synovial membrane and fluid

Cathepsin B was cytochemically investigated in the cells of synovial membranes and in the cell pellet of synovial fluids obtained from 50 patients with rheumatoid arthritis and eight patients with various nonrheumatoid arthropathies. The activity of Cathepsin B was estimated by using the substrate N-alpha-benzoyl-DL-arginine-naphthylamide HCl and diazoic dye Fast Corinth V in phosphate buffer pH 6.0 in the presence of EDTA and cysteine. A significant activity of cathepsin B was shown in lining mesothelial cells, in macrophages of the submesothelial infiltrations, as well as in fibroblasts prominent in the deep areas of rheumatoid synovial membranes. In the cell pellets of synovial fluids the highest activity of cathepsin B was found in the macrophages and polymorphonuclear leukocytes, accompanied by a variable activity in lymphocytes. The considerable activity of cathepsin B, an enzyme with degradative action upon collagen and proteoglycans, in the main cellular populations of rheumatoid synovial membranes and fluids, suggests its involvement in the genesis and maintenance of rheumatoid lesions.     

Biodefense function of omental milky spots through cell adhesion molecules and leukocyte proliferation

To evaluate the immunological functions of the greater omentum in the peritoneal cavity, the localization of cell adhesion molecules (CAMs) on mesothelial cells and leukocytes in the omental milky spots were studied in normal and lipopolysaccharide (LPS)-stimulated mice by means of immunoelectron microscopy. The milky spots featured numerous leukocytes among the dome-shaped mesothelial cells, even in the normal stable state. Leukocyte integrins LFA-1, Mac-1, and VLA-4 were preferentially localized to microvilli and ruffles of macrophages and lymphocytes. The mesothelial cells of the milky spots showed higher ICAM-1 levels than did those of other omental regions, and fibronectin was detected in the stomata. The number of leukocytes markedly increased following an increase in proliferating cell nuclear antigen (PCNA)-positive cells in the milky spots after LPS stimulation. The mesothelial cells contained VCAM-1 newly restricted to the microvilli and increasing amounts of ICAM-1. These results show that the omental milky spots are active sites for leukocyte migration and peritoneal leukocyte supply because of the presence of adhesion molecules and active cell proliferation. Proliferative active leukocytes and those that have migrated from vessels pass through the stomata via an interaction of VLA-4 and fibronectin, adhere to the microvilli of the activated mesothelial cell surface as the result of an interaction between ICAM-1/VCAM-1 and integrins, and exude into the peritoneal cavity. Much of the exudation and adhesion of leukocytes seen in the milky spots of LPS-stimulated mice may be attributable to an increase in cell proliferation and in the amounts of ICAM-1 and VCAM-1.     

Analysis of number, size and distribution patterns of lipid vacuoles in benign and malignant mesothelial cells

A quantitative analysis of the lipid vacuoles in benign hypertrophic and neoplastic mesothelial cells, using a size-independent distribution index, showed that computer-assisted image analysis for distribution patterns of cytoplasmic components can aid in distinguishing benign from malignant cells. Benign mesothelial cells had fewer lipid vacuoles, which were smaller and predominantly found around the nuclei. It is argued that, due to the high surface tension in the lipid vacuoles, the largest vacuoles are found in the center of the cells, which is the least flattened part of the air-dried mesothelial cells. It seems likely that the distribution pattern of rigid substructures, such as lipid vacuoles, varies between histologic and cytologic material as well as between cells processed by different cytologic methods with various cell-flattening artifacts. The study of the distribution of cytoplasmic components that differ in size was enhanced by using the defined size-independent distribution index, which incorporates the radius of the cell, the radius of the vacuoles and their respective centers of gravity.