Close Intercellular Contacts Research Articles

CSF1R Inhibition Combined with GM-CSF Reprograms Macrophages and Disrupts Protumoral Interplays with AML Cells.

Simple SummaryAcute myeloid leukemia is a blood disease whose long-term treatment is not satisfactory due to frequent (>50%) relapse despite complete initial remission. By adopting protumoral properties, macrophages in the leukemia cell microenvironment may promote resistance to treatment leading to relapse. Our goal was to assess whether macrophages in contact with leukemia cells have an impact on blast survival and sensitivity to chemotherapy. We observed that leukemia cells can educate macrophages to support their survival and proliferation and reduce their sensitivity to therapy, as long as the CSF1 receptor remains active and they are in close contact. By inhibiting the activity of the CSF1 receptor, in the presence of GM-CSF, we could modify the macrophage phenotype and increase blast apoptosis and sensitivity to treatment. Our results indicate that leukemia therapies should not only target blasts but also their microenvironment and specifically macrophages and their receptor for CSF1.Relapse is a major issue in acute myeloid leukemia (AML) and while the contribution of gene mutations in developing drug resistance is well established, little is known on the role of macrophages (MΦs) in an AML cell microenvironment. We examined whether myeloblasts could educate MΦs to adopt a protumoral orientation supporting myeloblast survival and resistance to therapy. Flow cytometry analyses demonstrated that M2-like CD163+ MΦs are abundantly present, at diagnosis, in the bone marrow of AML patients. We showed that myeloblasts, or their conditioned medium, polarize monocytes to M2-like CD163+ MΦs, induce the secretion of many protumoral factors, and promote myeloblast survival and proliferation as long as close intercellular contacts are maintained. Importantly, pharmacologic inhibition of the CSF1 receptor (CSF1R), in the presence of GM-CSF, reprogrammed MΦ polarization to an M1-like orientation, induced the secretion of soluble factors with antitumoral activities, reduced protumoral agonists, and promoted the apoptosis of myeloblasts interacting with MΦs. Furthermore, myeloblasts, which became resistant to venetoclax or midostaurin during their interplay with protumoral CD163+ MΦs, regained sensitivity to these targeted therapies following CSF1R inhibition in the presence of GM-CSF. These data reveal a crucial role of CD163+ MΦ interactions with myeloblasts that promote myeloblast survival and identify CSF1R inhibition as a novel target for AML therapy.

The influence of mesenchymal stromal cells on B-cell line growth and immunoglobulin synthesis

A number of publications contain contradictory data about influence of mesenchymal stromal cells (MSC) on B-lymphocyte growth, differentiation and production of immunoglobulins (Ig). The aim of the study was to investigate the influence of MSC derived from adipose tissue of healthy donors and cancer patients on the proliferation and Ig synthesis of lymphoblastoid cell line Namalva and myeloma cell line U266. Co-cultivation of Namalva cells with MSC stimulated their proliferation, decreased the doubling time and the minimal effective seeding dose and therefore made cloning of these lymphoblastoid cells possible. The presence of MSC supported the survival and proliferation of Namalva cells cultivated in growth factor deficient medium. MSC also stimulated proliferation of U266 myeloma cells. Both MSC derived from adipose tissue from the healthy donors and from patients with breast cancer effectively stimulated B-cell lines proliferation. Presence of MSC in mixed cultures had no influence on the production of IgM or IgE by Namalva or U266 cells respectively. Co-cultivation of Namalva or U266 with MSC resulted in the formation of close intercellular contacts between cells of both types.

Germ layer differentiation during early hindgut and cloaca formation in rabbit and pig embryos

Relative to recent advances in understanding molecular requirements for endoderm differentiation, the dynamics of germ layer morphology and the topographical distribution of molecular factors involved in endoderm formation at the caudal pole of the embryonic disc are still poorly defined. To discover common principles of mammalian germ layer development, pig and rabbit embryos at late gastrulation and early neurulation stages were analysed as species with a human-like embryonic disc morphology, using correlative light and electron microscopy. Close intercellular contact but no direct structural evidence of endoderm formation such as mesenchymal-epithelial transition between posterior primitive streak mesoderm and the emerging posterior endoderm were found. However, a two-step process closely related to posterior germ layer differentiation emerged for the formation of the cloacal membrane: (i) a continuous mesoderm layer and numerous patches of electron-dense flocculent extracellular matrix mark the prospective region of cloacal membrane formation; and (ii) mesoderm cells and all extracellular matrix including the basement membrane are lost locally and close intercellular contact between the endoderm and ectoderm is established. The latter process involves single cells at first and then gradually spreads to form a longitudinally oriented seam-like cloacal membrane. These gradual changes were found from gastrulation to early somite stages in the pig, whereas they were found from early somite to mid-somite stages in the rabbit; in both species cloacal membrane formation is complete prior to secondary neurulation. The results highlight the structural requirements for endoderm formation during development of the hindgut and suggest new mechanisms for the pathogenesis of common urogenital and anorectal malformations.

Barrière hématoencéphalique Partie III : approche thérapeutique pour franchir la barrière hématoencéphalique

Over the last few years, the blood-brain barrier has come to be considered as the main limitation for the treatment of neurological diseases caused by inflammatory, tumor or neurodegenerative disorders. In the blood-brain barrier, the close intercellular contact between cerebral endothelial cells due to tight junctions prevents the passive diffusion of hydrophilic components from the bloodstream into the brain. Several specific transport systems (via transporters expressed on cerebral endothelial cells) are implicated in the delivery of nutriments, ions and vitamins to the brain; other transporters expressed on cerebral endothelial cells extrude endogenous substances or xenobiotics, which have crossed the cerebral endothelium, out of the brain and into the bloodstream. Recently, several strategies have been proposed to target the brain, (i) by by-passing the blood-brain barrier by central drug administration, (ii) by increasing permeability of the blood-brain barrier, (iii) by modulating the expression and/or the activity of efflux transporters, (iv) by using the physiological receptor-dependent blood-brain barrier transport, and (v) by creating new viral or chemical vectors to cross the blood-brain barrier. This review focuses on the illustration of these different approaches.

DDAVP enhances the ability of blood monocytes to form rosettes with activated platelets by increasing the expression of P-selectin sialylated ligands on the monocyte surface.

The mechanism through which DDAVP (1-deamino-8-d-arginine vasopressin) promotes blood coagulation is not completely understood. As blood monocytes have been identified as a target for DDAVP, we investigated whether this drug increased monocyte adhesion to activated platelets, which would result in the close intercellular contact that is necessary for a juxtacrine effect on platelets and/or endothelium at sites of vascular injury. Monolayers of non-confluent monocytes adhered to glass slides were incubated with thrombin-activated, formaldehyde-fixed platelets before and after the adherent monocytes were stimulated with DDAVP or n-formyl-methyl-leucyl-phenylalanine (fMLP). The number of platelets involved in rosettes with monocytes was quantified, and the effect of DDAVP or fMLP on the monocyte surface expression of P-selectin ligands and CD11b/CD18 was assessed. DDAVP or fMLP increased the number of activated platelets involved in rosettes with monocytes by 2.8- and 4.9-fold respectively. EDTA and inhibitors of the P-selectin/counter-receptor interaction decreased the platelet numbers in rosettes by 80-90%, whereas inhibitors of the integrin-mediated adhesion reduced rosettes by 40-50%. Blocking the P-selectin glycoprotein ligand-1 (PSGL-1) with the monoclonal antibody, Pl-1, decreased the platelet numbers in rosettes by only 50%. In contrast, surface expression of the sialylated ligands of P-selectin and, to a lesser extent, of CD11b/CD18 increased upon monocyte activation with DDAVP or fMLP, whereas it decreased slightly with PSGL-1. These results indicate that DDAVP enhanced the ability of blood monocytes to bind activated platelets, mainly by increasing the expression of P-selectin sialylated ligands on the monocyte surface. A similar effect was achieved with fMLP.

Cell Hybridization by Electrofusion on Filters

Electric field pulses induce permeabilization and associated fusogenicity in cell membranes. Electrofusion of cells is usually performed in two steps: the first is the creation of close intercellular contacts; the second is an application of electric pulses that induces membrane fusion. Very large cell contacts can be obtained by a filter aspiration method. A cell monolayer is created by controlled suction on biocompatible filter. No spontaneous fusion results. Just after filtration, electrofusion is obtained by field pulses applied parallel to the filter. Cell viability is not strongly affected and cells recover their spherical shape in the minute time range after filtration. The electrical parameters, the cell density, and the flow rate control fusion. Fusion is obtained with cells of different origins with very different adhesion properties. Hybrid cells are easily formed. This approach appears to be a very efficient method for cell hybridization with an easy-to-use protocol.

Mitogen stimulation favours replication of equine herpesvirus-1 in equine blood mononuclear cells by inducing cell proliferation and formation of close intercellular contacts.

In the present study, equine herpesvirus-1 (EHV-1)-infected cells were identified in ionomycin/phorbol dibutyrate (IONO/PDB)-stimulated peripheral blood mononuclear cells (PBMC) and the mechanism by which stimulation increases the percentage of infected cells was examined. In the population of viral antigen-positive PBMC, 38.4+/-4.5% were CD5(+) T-lymphocytes (18.1+/-3.2% CD4(+) 13.6+/-1.8% CD8(+)), 18.1+/-5.4% were B-lymphocytes, 8.5+/-3.9% were monocytes and 35% remained unidentified. The role of the cell cycle in the increased susceptibility to EHV-1 upon stimulation was examined by stimulating PBMC for 0, 12, 24 or 36 h prior to inoculation. A high correlation was found between the increase of cells in the S- (r=0.974) and G(2)/M-phase (r=0.927) at the moment of inoculation and the increase of infected cells at 12 h post-inoculation (p.i.). This suggests that a specific stage of the S-phase or S- and G(2)/M-phase facilitates virus replication. At 24 h p.i. lower correlations were found, suggesting that other effects are involved. From 12 h after addition of IONO/PDB, formation of clusters of PBMC became manifest. We examined whether close intercellular contacts in these clusters facilitated cell-to-cell transmission of EHV-1. Between 8 and 17 h p.i., the percentage of clusters containing adjacent infected cells increased from 1.6 to 13.4% and the maximal number of adjacent infected cells increased from two to four. Confocal microscopy visualized close intercellular contacts between adjacent infected cells. It can be concluded that mitogen stimulation favours EHV-1 infection of PBMC (i) by initiating specific cell cycle events and (ii) by inducing formation of clusters, thereby facilitating transmission of virus between cells.

Tyrosine phosphorylation and src family kinases control keratinocyte cell-cell adhesion.

In their progression from the basal to upper differentiated layers of the epidermis, keratinocytes undergo significant structural changes, including establishment of close intercellular contacts. An important but so far unexplored question is how these early structural events are related to the biochemical pathways that trigger differentiation. We show here that beta-catenin, gamma-catenin/plakoglobin, and p120-Cas are all significantly tyrosine phosphorylated in primary mouse keratinocytes induced to differentiate by calcium, with a time course similar to that of cell junction formation. Together with these changes, there is an increased association of alpha-catenin and p120-Cas with E-cadherin, which is prevented by tyrosine kinase inhibition. Treatment of E-cadherin complexes with tyrosine-specific phosphatase reveals that the strength of alpha-catenin association is directly dependent on tyrosine phosphorylation. In parallel with the biochemical effects, tyrosine kinase inhibition suppresses formation of cell adhesive structures, and causes a significant reduction in adhesive strength of differentiating keratinocytes. The Fyn tyrosine kinase colocalizes with E-cadherin at the cell membrane in calcium-treated keratinocytes. Consistent with an involvement of this kinase, fyn-deficient keratinocytes have strongly decreased tyrosine phosphorylation levels of beta- and gamma-catenins and p120-Cas, and structural and functional abnormalities in cell adhesion similar to those caused by tyrosine kinase inhibitors. Whereas skin of fyn-/- mice appears normal, skin of mice with a disruption in both the fyn and src genes shows intrinsically reduced tyrosine phosphorylation of beta-catenin, strongly decreased p120-Cas levels, and important structural changes consistent with impaired keratinocyte cell adhesion. Thus, unlike what has been proposed for oncogene-transformed or mitogenically stimulated cells, in differentiating keratinocytes tyrosine phosphorylation plays a positive role in control of cell adhesion, and this regulatory function appears to be important both in vitro and in vivo.

Perforin, Fas/Fas ligand, and TNF-alpha pathways as specific and bystander killing mechanisms of hepatitis C virus-specific human CTL.

In chronic hepatitis C, Fas expression is up-regulated in the hepatocytes, especially near liver-infiltrating lymphocytes, and Fas ligand is expressed on the lymphocytes. The presence of hepatitis C virus (HCV)-specific CTLs has been demonstrated both in peripheral blood and among liver-infiltrating lymphocytes of patients with chronic hepatitis C. We studied the killing mechanisms of HCV-specific human CTLs using target cells that were sensitive or resistant to agonistic anti-Fas Abs and TNF-alpha. We show that HCV-specific CTL clones kill non-Ag-bearing bystander cells as well as Ag-bearing cells, although the bystander killing is less efficient than the specific target cell killing, and the efficacy of the bystander killing of anti-Fas- and soluble TNF-alpha-sensitive cells is greater than that of resistant cells. We also show that the killing of Ag-presenting, sensitive cells is mediated by Fas ligand and TNF-alpha as well as perforin, although the latter plays a major role in the killing at a low E:T ratio, and that the killing of sensitive bystander cells is primarily mediated by Fas ligand and TNF-alpha on CTLs expressed upon specific Ag stimulation, which may be relevant to the bystander lysis by HCV-specific CTLs of uninfected hepatocytes, in which Fas expression is up-regulated. Activated CTLs also kill bystander cells by the perforin-based mechanism, although it requires a high E:T ratio. The effective bystander killing requires a close intercellular contact between CTLs and target cells, although TNF-alpha released from the CTLs mediates lysis of the bystander cells without a close cell-cell contact.

Tenascin mediates human glioma cell migration and modulates cell migration on fibronectin.

The role of tenascin in mediating tumor cell migration was studied using two cell migration models. In migration/invasion Transwell assays U251.3 glioma cells rapidly migrated through the 8 mu m pore size membranes onto tenascin- and fibronectin-coated surfaces. In this assay the number of cells migrating onto tenascin was 52.2 +/- 9.6% greater than on fibronectin within 4 hours. To assess cell migration rates and cell morphology, U251.3 migration was examined in a two-dimension spheroid outgrowth assay. The radial distance migrated by U251.3 cells from tumor spheroids was found to be 53.8 +/- 4.9% greater on tenascin than on fibronectin. Cells migrating on tenascin display a very motile appearance, while cells migrating on fibronectin spread and maintain close intercellular contacts. Cell migration in the presence of integrin blocking antibodies demonstrated that migration on tenascin and fibronectin is mediated by distinct integrins, alpha2beta1 and alphavbeta5/alphavbeta3, respectively. Since tenascin is coexpressed in malignant tumor matrices with fibronectin, we assessed the effects of tenascin on U251.3 cell migration mediated by fibronectin. Tenascin was found to provide a positive effect on fibronectin-mediated migration by altering cell morphology and enhancing cell motility. These effects of tenascin on fibronectin-mediated cell migration were inhibited by blocking beta1 and alpha2beta1 integrins. The results suggest that tenascin may play a significant role in promoting tumor cell migration and invasiveness by modulating cell responses to normal matrix components.

Enhanced Human Kupffer Cell-Mediated Cytotoxicity after Activation of the Effector Cells and Modulation of the Target Cells by Interferon-γ: A Mechanistic Study at the Cellular Level

In this study we demonstrate enhanced Kupffer cell (KC) cytotoxicity against several colorectal cell lines by activation of KC and by modulation of the targets (SW948, WiDR, HT29, and SW620) with IFN-γ. We demonstrated that soluble TNF-α had no effect on these tumor cells, while cytotoxicity against SW948 and WiDR was blocked by anti-TNF-α. Experiments using a transwell system stressed the importance of close intercellular contact for this process. Anti-IL-1 did not inhibit cytotoxicity against SW948. Modulation of HT29, WiDR, and SW948 by IFN-γ (500 U/ml) induced a significant increase in cytotoxicity. We conclude that cell-associated TNF-α may be responsible for KC cytotoxicity against SW948, a process requiring close intercellular contact. WiDR is only partly lysed by a TNF-α-dependent mechanism, whereas HT29 is not. Furthermore, IFN-γ is involved in the regulation of tumor susceptibility.

Local modulation of neurofilament phosphorylation, axonal caliber, and slow axonal transport by myelinating Schwann cells

Studies in Trembler and control mice demonstrated that myelinating Schwann cells exert a profound influence on axons. Extensive contacts between myelin and axons have been considered structural. However, demyelination decreases neurofilament phosphorylation, slow axonal transport, and axonal diameter, as well as significantly increasing neurofilament density. In control sciatic nerves with grafted Trembler nerve segments, these changes were spatially restricted: they were confined to axon segments without normal myelination. Adjacent regions of the same axons had normal diameters, neurofilament phosphorylation, cytoskeletal organization, and axonal transport rates. Close intercellular contacts between myelinating Schwann cells and axons modulate a kinase-phosphatase system acting on neurofilaments and possibly other substrates. Myelination by Schwann cells sculpts the axon-altering functional architecture, electrical properties, and neuronal morphologies.

Orthogonal arrays of intramembrane particles in the supporting cells of the guinea-pig vestibular sensory epithelium.

Membrane specializations of the contact region between afferent nerve endings and supporting cells of the sensory epithelia of guinea-pig vestibular endorgans were examined by thin-section and freeze-fracture electron microscopy. The calyx-type nerve endings (C-endings) are separated from supporting cells (SC) by a 25-30 nm space. At irregular intervals along the upper lateral surface of supporting cells, the intercellular space narrows markedly to form special close contacts between the C-ending and SC plasma membranes. Freeze-fracture replicas reveal membrane specializations--orthogonal arrays of particulate units--in the region where the close intercellular contacts were found in sections. Orthogonal arrays consisting of from 5 to 20 units were observed on the cytoplasmic (P) fracture face of the lateral SC plasma membrane. These particulate units from a 12 x 12-nm square, and each unit is composed of four 6-nm subunits. Possible roles of the orthogonal arrays are discussed.

Absence of Contact Effects in Irradiated EMT6-Rw Tumors

Some cells have been reported to show greater resistance to drugs or radiation when growing with close intercellular contacts in spheroids or in solid tumors than when growing with few intercellular contacts in sparse cultures. In some cases this increased resistance reflects an increased capacity of cells in close contact to repair cytotoxic damage. However, not all tumors show contact effects, and in some tumors and spheroids the increased resistance appears to be produced by environmental factors, such as hypoxia, rather than by changes in the repair capacity of the cells. To assess whether EMT6-Rw cells showed increased intrinsic radioresistance when grown as solid tumors, we compared survival curves for cells in exponentially growing monolayers and in solid tumors in BALB/c mice. To avoid complications arising from regional heterogeneity in oxygenation within solid tumors, these irradiations were performed under conditions of uniform, maximal hypoxia. The two survival curves were indistinguishable. Moreover, survival curves for cells suspended from solid tumors, plated at low densities and irradiated immediately, after 5 h of incubation or after 24 h of incubation, were indistinguishable from one another and were indistinguishable from survival curves for cells suspended from exponentially growing monolayers and irradiated immediately using an identical protocol. It therefore appears that contact effects are insignificant for irradiated EMT6-Rw tumors and that the intrinsic radiosensitivity of these cells is similar in culture and in solid tumors.

Histochemical and ultrastructural analysis of developing adipocytes in the fetal pig.

Histochemical and ultrastructural aspects of adipocyte differentiation in subcutaneous tissue of fetal pigs were analyzed in a longitudinal study. A matrix of collagen fibers surrounding adipocytes developed after the establishment of a distinct and continuous PAS-positive basement membrane. The degree of plasma membrane invagination and specialization was positively correlated with the extent of basement membrane and collagen matrix formation. Close spatial relationships between narrow, smooth endoplasmic reticulum, plasma membrane invaginations, the surface of lipid droplets and mitochondria were observed in differentiating adipocytes. Histochemical and ultrastructural criteria for the identification of preadipocytes are: (1) perivascular location; (2) mitochondria localized in the Golgi zone; (3) cytosolic glycogen; (4) rough endoplasmic reticulum with cisternae uniformly and approximately 600 A wide; (5) free ribosomes and few polysomes, and (6) lipid droplets encased by microfilaments. These criteria permitted clear distinction from obvious fibroblasts and macrophages. Other stromal cells were morphologically abnormal. Occasionally, adipocytes and perivascular cells exhibited close intercellular contacts that were morphologically distinct from intercellular contacts between contiguous endothelial cells.

Fine structural studies on white adipocyte differentiation.

AbstractThe differentiation of mammalian white adipocytes from prenatal through early postnatal periods was studied by light and electron microscopy in C57BL mice. Anatomical regions chosen for this study were the epididymal, mesometrial, mesenteric and inguinal fat pads. In each of these regions, adipocytes differentiated from fibroblast‐like cells (preadipocytes) characterized by an ovoid nucleus, profiles of rough endoplasmic reticulum, microtubules, microfilaments, spherical mitochondria, and small multiple lipid inclusions. Preadipocytes of the inguinal fat pad were first observed prior to birth (17–19 days), whereas, in the other anatomical sites, these cells were not observed until one to three days postnatally. As differentiation proceeded, and as the adipocytes assumed a spherical shape, there was a progressive decrease in the amount of rough endoplasmic reticulum and microfilaments concomitant with transient glycogen storage and an increase in the size of lipid droplets. Mature unilocular adipocytes were observed in the inguinal fat pads at three days of age. On the other hand, these cells did not appear until seven days after birth in the epididymal fat pad, mesometrium and mesentery. Regardless of the anatomical region studied, the differentiation of preadipocytes to adipocytes proceeded similarly. Preadipocytes could not be distinguished from fibroblasts morphologically within the fat depots studied. Adipocytes at the mid‐stages of differentiation and in all regions studied occasionally exhibited close intercellular contacts of varying morphology.

Cell cycle-dependent surface changes in Chinese hamster cells grown in suspension culture

The cell surface appears to play an important part in the control of cell replication. It has been demonstrated that the cell membrane undergoes cyclic changes in appearance which bear a relation to the cell cycle phase, irrespective of close intercellular contact. The surface of Chinese hamster (CHO) cells was investigated using the scanning electron microscope (SEM). The cells were synchronized in suspension culture and were sampled at frequent intervals during the cell cycle. During mitosis, the cells showed microvilli and few blebs. In early G 1 phase, profuse microvilli were seen. In late G 1 phase, blebs appeared and persisted in great numbers. During the synthesis of DNA in the S phase, blebs were observed in the early stages and then declined in number; in G 2 phase, the blebs appeared to be larger (1–2 μm) and more sparsely distributed than in late S phase. Some of these blebs were pedunculated and, in some instances, the diameter of the pedicles approximated the diameter of microvilli. Since the reasons for these changes are not understood, our long-range goal is to correlate the observed surface changes with internal biochemical events during the cell cycle.

Application of freeze-etching to the study of myogenesis in cell culture

A method is described for in situ freeze-etching of cells grown in monolayer culture, with preservation of delicate surface structures such as filopodia. The close intercellular contact which is a prerequisite for fusion of myogenic cells appears to depend on and be mediated by filopodial contacts: cells grown in standard fusion-permitting medium show frequent filopodial contacts, whereas cells in medium depleted of Ca 2 ions by the addition of EGTA lack filopodia and fail to fuse.

Effect of phytohaemagglutinin on the surface charge of human and murine lymphocytes.

NON-IMMUNE murine lymphocytes are cytotoxic to allogeneic target cells in the presence of phytohaemagglutinin (PHA)1. In a similar system using cells of human origin this “allogeneic inhibition” occurs without PHA (ref. 2). The role of PHA in the mouse experiments is unknown, but it has been suggested that it acts by promoting intercellular contact, thus permitting the close apposition of dissimilar groups of histocompatibility antigens3. This would imply that there is some factor in the mouse experiments which tends to inhibit close intercellular contact but which does not operate in experiments using human cells.