T. Regalia

Telocytes in Human Term Placenta: Morphology and Phenotype

In the last few years, a new cell type – interstitial Cajal-like cell (ICLC) – has been described in digestive and extra-digestive organs. The name has recently been changed to telocytes (TC) and their typical thin, long processes have been named telopodes (TP). To support the hypothesis that TC may also be present in human placenta and add to the information already available, we provide evidence on the ultrastructure, immunophenotype, distribution, and interactions with the surrounding stromal cells of TC in the villous core of human term placenta. We used phase-contrast microscopy, light microscopy of semithin sections, transmission electron microscopy, immunohistochemistry, and immunofluorescence of tissue sections or cell cultures, following a pre-established diagnostic algorithm. Transmission electron microscopy showed cells resembling TC, most (∼76%) having 2–3 very thin, longprocesses (tens to hundreds of micrometers), with an uneven calibre(≤0.5 µm thick) and typical branching pattern. The dilations of processes accommodate caveolae, endoplasmic reticulum cisternae, and mitochondria. These TC have close contacts with perivascular SMC in stem villi. In situ, similar cells are positive for c-kit, CD34, vimentin, caveolin-1, vascular endothelial growth factor (VEGF), and inducible nitric oxide synathase (iNOS). The c-kit-positive cells inconsistently co-express CD34, CD44, αSMA, S100, neuron-specific enolase, and nestin. Among cells with a morphologic TC profile in cell cultures, about 13% co-express c-kit, vimentin, and caveolin-1; 70% of the c-kit-positive cells co-express CD34 and 12% co-express iNOS or VEGF. In conclusion, this study confirms the presence of TC in human term placenta and provides their ultrastructural and immunophenotypic characterization.

C-kit immunopositive interstitial cells (Cajal-type) in human myometrium.

Previous reports describing Cajal‐like interstitial cells in human uterus are contradictory in terms of c‐kit immunoreactivity: either negative (but vimentin‐positive) in pregnant myometrium, or positive, presumably in the endometrium. The aim of this study was to verify the existence of human myometrial Cajal‐like interstitial cells (m‐CLIC). Six different, complementary approaches were used: 1) methylene‐blue supravital staining of tissue samples (cryosections), 2) methylene blue and Janus green B vital staining (m‐CLIC and mitochondrial markers, respectively), and 3) extracellular single‐unit electrophysiological recordings in cell cultures, 4) non‐conventional light microscopy on glutaraldehyde/osmium fixed, Epon‐embedded semi‐thin sections (less than 1μm) stained with toluidine blue (TSM), 5) transmission electron microscopy (TEM), and 6) immunofluorescence (IF). We found m‐CLIC in myometrial cryosections and in cell cultures. In vitro, m‐CLIC represented ∼7% of the total cell number. m‐CLIC had 2–3 characteristic processes which were very long (∼ 60 μm), very thin (±0.5μm) and moniliform. The dilated portions of processes usually accomodated mitochondria. In vitro, m‐CLIC exhibited spontaneous electrical activity (62.4 ± 7.22 mV field potentials, short duration: 1.197 ± 0.04ms). Moreover, m‐CLIC fulfilled the usual TEM criteria, the so‐called ‘gold’ or ‘platinum’ standards (e.g. the presence of discontinuos basal lamina, caveolae, endoplasmic reticulum, and close contacts between each other, with myocytes, nerve fibers and/or capillaries etc.). IF showed that m‐CLIC express CD117/c‐kit, sometimes associated with CD34 and with vimentin along their processes.

Insights into the interstitium of ventricular myocardium: interstitial Cajal-like cells (ICLC).

We have previously described interstitial Cajal‐like cells (ICLC) in human atrial myocardium. Several complementary approaches were used to verify the existence of ICLC in the interstitium of rat or human ventricular myocardium: primary cell cultures, vital stainings (e.g.: methylene blue), traditional stainings (including silver impregnation), phase contrast and non‐conventional light microscopy (Epon‐embedded semithin sections), transmission electron microscopy (TEM) (serial ultrathin sections), stereology, immunohistochemistry (IHC) and immunofluorescence (IF) with molecular probes. Cardiomyocytes occupy about 75% of rat ventricular myocardium volume. ICLC represent ∼32% of the number of interstitial cells and the ratio cardiomyocytes/ICLC is about 70/1. In the interstitium, ICLC establish close contacts with nerve fibers, myocytes, blood capillaries and with immunoreactive cells (stromal synapses). ICLC show characteristic cytoplasmic processes, frequently two or three, which are very long (tens up to hundreds of μm), very thin (0.1‐0.5μm thick), with uneven caliber, having dilations, resulting in a moniliform aspect. Gap junctions between such processes can be found. Usually, the dilations are occupied by mitochondria (as revealed by Janus green B and Mito Tracker Green FM) and elements of endoplasmic reticulum. Characteristically, some prolongations are flat, with a veil‐like appearance, forming a labyrinthic system. ICLC display caveolae (about 1 caveola/1μm cell membrane length, or 2‐4% of the relative cytoplasmic volume, Mitochondria and endoplasmic reticulum (rough and smooth) occupy 5‐10% and 1‐2% of cytoplasmic volume, respectively. IHC revealed positive staining for CD34, EGFR and vimentin and, only in a few cases for CD117. IHC was negative for: desmin, CD57, tau, chymase, tryptase and CD13. IF showed that ventricular ICLC expressed connexin 43. We may speculate that possible ICLC roles might be: intercellular signaling (neurons, myocytes, capillaries etc.) and/or chemomechanical sensors. For pathology, it seems attractive to think that ICLC might participate in the process of cardiac repair/remodeling, arrhythmogenesis and, eventually, sudden death.

Cajal-type cells from human mammary gland stroma: phenotype characteristics in cell culture.

We report here the in vitro isolation of Cajal‐like interstitial cells from human inactive mammary‐gland Stroma. Primary cell cultures examined in phase‐contrast microscopy or after vital methylene‐blue staining revealed a cell population with characteristic morphological phenotype: fusiforms, triangular or polygonal cell body and the corresponding (very) long, slender, moniliform cytoplasmic processes. Giremsa staining pointed out the typical knobbed aspect of cell prolongations. Immunofluorescence (IF) showed, like in situ immunohistochemistry, that Cajal‐type cells in vitro (primary culltures), expressed c‐kit/CD117 and vimentin. In conclusion, the images presented here reinforce our previous hypothesis that human mammary glands have a distinct population of Cajal‐like cells in non‐epithelial tissue compartments.

Epicardium: interstitial Cajal-like cells (ICLC) highlighted by immunofluorescence

During the last few years, there is an increasing interest in the role of the epicardium in cardiac development, myocardial remodelling or repair and regeneration. Several types of cells were described in the subepicardial loose connective tissue, beneath the epicardial mesothe‐lium. We showed previously (repeatedly) the existence of interstitial Cajal‐like cells (ICLCs) in human and mammalian myocardium, either in atria or in ventricles. Here, we describe ICLCs in adult mice epicardium and primary culture as well as in situ using frozen sections. The identification of ICLCs was based on phase contrast microscopy and immunophenotyping. We found cells with characteristic morphologic aspects: spindle‐shaped, triangular or polygonal cell body and typical very long (tens to hundreds micrometres) and very thin cyto‐plasmic processes, with a distinctive ‘beads‐on‐a‐string’ appearance. The dilations contain mitochondria, as demonstrated by MitoTracker Green FM labelling of living cells. Epicardial ICLCs were found positive for c‐kit/CD117 and/or CD34. However, we also observed ICLCs positive for c‐kit and vimentin. In conclusion, ICLCs represent a distinct cell type in the subendocardium, presumably comprising at least two subpopulations: (i) c‐kit/CD34‐positive and (ii) only c‐kit‐positive. ICLCs might be essential as progenitor (or promoter) cells for developing cardiomyocyte lineages in normal and/or injured heart.

Apoptosis in human embryo development: 3. Fas‐induced apoptosis in brian primary cultures

Fas (APO‐1/CD95) is an important apoptotic mediator for both immune and nervous systems. In the present study, we have investigated the expression and function of Fas in human embryonic/fetal brain primary cultures from 12 human embryos and fetuses with gestational ages between 5 to 22 weeks. Anti‐Fas fluorescent antibody was used for labeling of Fas positive cells and for quantitation of Fas expression in brain cultures. To demonstrate that Fas receptor is functional in human embryonic/fetal brain cells, anti‐Human‐Fas monoclonal antibody (0.5 μg/ml) was used to induce apoptosis in brain primary cultures. Apoptosis was investigated by flow‐cytometry and fluorescent microscopy using TUNEL and annexin V labeling. Fas was found to be expressed in the embryonic/fetal human primary brain cultures, on neuronal and glial cells or their precursors, varying with gestational ages. Cross‐linking of Fas induced apoptosis in brain cultures indicating that Fas receptor functions as a death receptor. We also showed that cell death triggered through Fas receptor was caspase dependent, hence it was blocked by a selective caspase‐8 inhibitor (IETD‐fmk).These results suggest that Fas is involved in neuronal apoptosis in the developing human brain.

Co-stimulatory and adhesion molecules of dendritic cells in rheumatoid arthritis

Dendritic cells (DCs) in the rheumatoid arthritis (RA) joint mediate the immunopathological process and act as a potent antigen presenting cell. We compared the expression of co‐stimulatory and adhesion molecules on DCs in RA patients versus controls with traumatic joint lesions and evalulated the correlation between the immunophenotypical presentation of DCs and the clinical status of the disease. Samples of peripheral venous blood, synovial fluid (SF) and synovial tissue (ST) were obtained from 10 patients with RA at the time of hip or knee replacement and from 9 control patients with knee arthroscopy for traumatic lesions. Clinical status was appreciated using the DAS28 score. Blood, SF and dissociated ST cell populations were separated by centrifugation and analyzed by flow cytometry. Cells phenotypes were identified using three‐color flow cytometry analysis for the following receptors HLA‐DR, CD80, CD83, CD86, CD11c, CD18, CD54, CD58, CD3, CD4, CD8, CD19, CD20, CD14, CD16, CD56. HLA‐DR molecules, co‐stimulatory receptors CD80, CD86, CD83 and adhesion molecules CD18, CD11c, CD54, CD58, were analyzed by two‐color immunofluorescence microscopy on ST serial sections. In patients with active RA (DAS28>5.1) we found a highly differentiated subpopulation of DCs in the ST and SF that expressed an activated phenotype (HLA‐DR, CD86+, CD80+, CD83+, CD11c+, CD54+, CD58+). No differences were found between circulating DCs from RA patients and control patients. Our data suggest an interrelationship between clinical outcome and the immunophenotypical presentation of DCs. Clinical active RA (DAS28>5.1) is associated with high incidence of activated DCs population in the ST and SF as demonstrated by expression of adhesion and co‐stimulatory molecules.

Apoptosis in the immune system: 1. Fas-induced apoptosis in monocytes-derived human dendritic cells.

Dendritic cells (DC) are cells of the hematopoietic system specialized in capturing antigens and initiating T cell‐mediated immune responses. We show here that human DC generated from adherent peripheral blood mononuclear cells (PBMC) after in vitro stimulation with granulocyte macrophage colony stimulating factor (GM‐CSF) and interleukin‐4 (IL‐4) express Fas antigen (APO‐1, CD95) and can undergo apoptosis upon triggering of Fas by monoclonal antibodies. Immature monocytes‐derived dendritic cells (MDDC) upregulate CD86 and HLA‐DR expression and develop dendrites and veiled processes. Flow cytometry analysis revealed CD95 expression in approx. 40% of these MDDC and incubation with anti‐CD95 mAb (0.5μg/ml) induced apoptosis when compared to untreated controls. The extent of apoptosis induced by the agonist anti‐Fas antibody strongly related to the percentage of cells expressing CD 95. Upon tumor necrosis factor α (TNF‐α) additional stimulation, MDDC assumed a characteristic mature dendritic cells morphology showing prolonged veils, CD83 expression, and high levels of HLA‐DR. These cells have downregulated their Fas receptors (to approx. 20%) and undergo apoptosis to a lesser extent when treated with anti‐CD 95, as demonstrated by the hardly noticeable effect of this antibody on the viability of cultured cells as compared to controls. Thus, upon TNF‐α induced maturation, MDDC became resistant to Fas‐induced apoptosis. The apoptotic episodes surrounding the earlier stage of DC differentiation appeared to be mediated by Fas. In contrast, a Fas independent pathway is probably responsible for the apoptotic events associated with terminally differentiated DC.

MHC Class II Vesicles (CIIV) within immature dendritic cells

Human peripheral blood monocytes can be induced to differentiate in vitro into immature dendritic cells when incubated for up to 7 days with granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) [1]. These myeloid dendritic cells (MDC) have various