Key adhesion molecules are present on long podia extended by hematopoietic cells.

BACKGROUND:We recently reported that CD34(+) hematopoietic cells and the KG1a cell line extend long, thin podia. These podia can dynamically extend and retract, often adhere to the substrate, and appear to connect cells up to 300 microm apart. The surface receptors found on these podia have not been described. METHODS:By using time-lapse fluorescent microscoscopy and immunostaining techniques, we describe a method for detecting surface receptors on these podia. This includes an in situ antibody staining procedure without fixing cells. RESULTS:We demonstrate, using CD34 selected mobilized peripheral blood cells and KG1a cells, that adhesion molecules known to play important roles in blood-cell migration and adhesion are present on these podia. These include: CD11a, CD18, CD29, CD34, CD45, CD49d, CD49e, and CD62L. Additionally, CD54 and CD44 were present on the podia extended by KG1a cells, but were not detectable on the primary CD34(+) cells. The integrin CD49d localized at the base of these podia in a time-dependent manner in KG1a cells. The frequency and morphology of these long podia on three myeloid leukemia-cell lines (KG1a, MV4-11, and AML-193) and a CD34-negative T-cell line (CEM) are also compared. KG1a and CEM cell lines extend long, dynamic podia that are similar to the podia on primary CD34(+) cells in morphology and adhesion molecule expression. The AML-193 and MV4-11 cell lines, however, did not extend these long podia. CONCLUSIONS:We describe a technique that provides a method of detecting surface receptors on thin cell membrane projections. These results support the likely role of these podia in cell migration and cell-cell communication.     

Intercellular adhesion can be visualized using fluorescently labeled fibrosarcoma HT1080 cells cocultured with hematopoietic cell lines or CD34(+) enriched human mobilized peripheral blood cells

BACKGROUND: Intercellular contacts between adjacent cells migrating over each other are important in many cellular processes. However, it has been difficult to visualize and identify dynamic intercellular adhesions between migrating cells in situ. METHODS: Two fluorescent membrane dyes, PKH2 and PKH26 for staining HT1080 and hematopoietic cells and cell lines, and an automated fluorescence microscopy system were used to monitor intercellular adhesion. RESULTS: Cellular extensions connecting two or more adjacent cells were visualized, showing the intercellular adhesion between migrating cells for minutes and up to hours. After cells adhered to each other, followed by cell migration in different directions, cellular extensions were dragged from the pivotal contact points in different focal planes. CD34(+)-enriched mobilized peripheral blood cells and six hematopoietic cell lines showed intercellular connections in cocultures with HT1080. However, the frequency of intercellular connections was variable in different cocultures. A cell density of about 3.1 x 10(4) cells/cm(2) for both cell lines in cocultures provided an adequate number of cells in each field of view, showing up to four intercellular connections per 100 total cells plated. DISCUSSION: The tools derived from this study will open new areas of investigation for understanding the mechanism of the intercellular adhesion process.     

The effects of three-dimensional cell culture on single myoblasts

Identification of cellular and morphological changes in myoblasts during three-dimensional (3D) culture may provide novel insight into skeletal muscle morphogenesis. One particular morphological change that occurs during the transition from monolayer culture to the 3D environment is the appearance of cytoplasmic projections (podia). The purpose of these studies was to determine if: (1) 3D culture increased podia formation in single cells, and (2) podia were F-actin dependent. C2C12 cells were grown in 3D conditions using a rotary cell culture system (RCCS) for 3, 6, and 9 h, fixed, and stained. Analysis of confocal images revealed that podia were significantly more numerous on RCCS cultured cells than those on suspension controls. Further, the podia of RCCS cultured cells decreased in number and increased in length during the time intervals examined. RCCS cultured cells showed no significant changes in viability, Annexin V staining, and activated Caspase 3 expression over time. In contrast, significant decreases in viability of suspension controls occurred. The application of 2 μM Latrunculin A (Lat A), an actin depolymerizing agent, significantly reduced the number of cells with podia. The number of cells with podia recovered with Lat A removal. Changes in viability and apoptosis markers were not significant during Lat A application or washout experiments. These observations reveal that: (1) culture conditions in the RCCS increase the quantity of podia formation; (2) these podia increase in length with time; and (3) F-actin plays a role in podia formation.     

A novel ganglioside expressed by mouse hematopoietic cell lines.

Mouse progenitor T cell-derived cell lines were established by fusion of cells of hematopoietic organs such as bone marrow and fetal liver with T lymphoma (BW5147) to determine their characteristic cell-surface components. The hybridomas with the phenotype of Thy-1+, CD3-, CD4-, CD8- and expression of T cell receptor gene mRNA (BM216 and FL339) were selected for progenitor T cell-derived cell lines, and their ganglioside compositions were studied. A ganglioside component with a mobility slightly faster than that of bovine brain GD1a on high-performance thin-layer chromatography was found in the cell extracts of these cell lines as one of the most abundant components and was absent in the extract of the parental cell line (BW5147). The structure of the ganglioside was determined to be: NeuAc alpha-Gal beta-Gal beta-Gal alpha-Gal beta-Glc beta-ceramide. Gangliosides with such a sequence have never been found before, suggesting the possibility that the ganglioside is expressed as a surface marker of the cells in hematopoietic organs committed to a specific cell lineage, presumably to T cell lineage. cells in hematopoietic organs committed to a specific     

Expression of CD34 in hematopoietic cancer cell lines reflects tightly regulated stem/progenitor-like state

Hematopoietic cancer stem cells preserve cellular hierarchy in a manner similar to normal stem cells, yet the underlying regulatory mechanisms are poorly understood. It is known that both normal and malignant stem/progenitor cells express CD34. Here, we demonstrate that several cell lines (HL-60, U266) derived from hematopoietic malignancies contain not only CD34(-) but also CD34(+) subpopulations. The CD34(+) cells displayed a stem/progenitor-like phenotype since, in contrast to CD34(-) cells, they frequently underwent cellular division and rapidly formed colonies in methylcellulose-based medium. Strikingly, a constant fraction of the CD34(+) and CD34(-) cell subpopulations, when separated, rapidly switched their phenotype. Consequently, both separated fractions could generate tumors in immunocompromised NOD/LtSz-scid/scid mice. Cultures in vitro showed that the proportion of CD34(+) stem/progenitor-like cells in the population was decreased by cell-cell contact and increased by soluble factors secreted by the cells. Using cytokine arrays, we identified some of these factors, notably thymopoietin that was able to increase the proportion of CD34(+) cells and overall colony-forming capacity in tested cell lines. This action of thymopoietin was conserved in mononuclear cells from bone marrow. Therefore, we propose that hematopoietic cancer cell lines containing subpopulations of CD34(+) cells can provide an in vitro model for studies of cancer stem/progenitor cells.     

Phototoxicity of the fluorescent membrane dyes PKH2 and PKH26 on the human hematopoietic KG1a progenitor cell line.

BACKGROUND: The phototoxic effects of the well-known fluorescent membrane dyes PKH2 and PKH26 have been unknown, although their use in cell tracking experiments has increased dramatically. To eliminate the phototoxicity-induced alteration in cell function and morphology, it is essential to examine the suspicious phototoxicity of these dyes. METHODS: Chemical and phototoxic effects of PKH dyes on the human hematopoietic KG1a cell line were examined. To minimize phototoxicity in long-term cell tracking experiments lasting up to 18 h with a fluorescence microscope system, time-lapse monitoring with different time intervals and exposure times was introduced. RESULTS: There were no significant effects of the two PKH dyes on cell viability and growth when using dye concentrations up to 5 microM. However, when stained cells were exposed to excitation light, cell viability decreased dramatically, showing the phototoxicity of the PKH dyes. More than 60% of cells stained with 5 microM PKH26 died after 5 min of continuous light exposure. The phototoxic effect was more extensive in cells stained with higher concentrations of the dyes. CONCLUSIONS: We present guidelines for the optimal use of these dyes by using a defined hardware configuration.     

Characterization and functionality of cell surface molecules on human mesenchymal stem cells

We have characterized adhesion molecules on the surface of multipotential human mesenchymal stem cells (hMSCs) and identified molecules whose ligands are present on mature hematopoietic cells. Flow cytometric analysis of hMSCs identified the expression of integrins: alpha1, alpha2, alpha3, alpha5, alpha6, alphav, beta1, beta3, and beta4, in addition to ICAM-1, ICAM-2, VCAM-1, CD72, and LFA-3. Exposure of hMSCs to IL-1alpha, TNFalpha or IFNgamma up-modulated ICAM-1 surface expression, whereas only IFNgamma increased both HLA-class I and -class II molecules on the cell surface. Whole cell-binding assays between the hMSCs and hematopoietic cell lines showed that T lymphocytic lines bound hMSCs with higher affinity than lines of either B lymphocytes or those of myeloid lineage. Experiments using autologous T lymphocytes isolated from peripheral blood mononuclear cells showed that hMSCs exhibited increased affinity for activated T-lymphocytes compared to resting T cells by quantitative whole cell binding and rosetting assays. Flow cytometric analysis of rosetted cells demonstrated that both CD4+ and CD8+ cells bound to hMSCs. To determine the functional significance of these findings, we tested the ability of hMSCs to present antigen to T lymphocytes. hMSCs pulsed with tetanus toxoid stimulated proliferation and cytokine production (IL-4, IL-10, and IFNgamma) in a tetanus-toxoid-specific T cell line. Maximal cytokine production correlated with maximal antigen-dependent proliferation. These data demonstrate physiological outcome as a consequence of interactions between hMSCs and human hematopoietic lineage cells, suggesting a role for hMSCs in vivo to influence both hematopoietic and immune function(s).     

Characterization and efficacy of PKH26 as a probe to study the replication history of the human hematopoietic KG1a progenitor cell line

The PKH26 dye can, in principle, be used for the study of asymmetric cell divisions (ASDs). A requirement for the identification of ASDs based on fluorescence intensity is that the PKH26 dye is distributed equally between daughter cells at each division, but this has not been demonstrated at a single-cell level. The efficacy of PKH26 as a probe for the study of ASDs was examined using the human hematopoietic KG1a cell. An automated time-lapse fluorescent microscope system was used to determine changes in cell size and fluorescence intensity during culture, and track cell divisions. The images of daughter cells were analyzed using the Isee software to determine the distribution of PKH26 dye between daughter cells. Ratios of cell size, mean fluorescence intensity, and total fluorescence intensity were calculated by dividing the values for one daughter cell by the value of the other daughter cell. The ratios for cell size, mean intensity, and total intensity were 1.13 +/- 0.12, 1.08 +/- 0.07, and 1.15 +/- 0.14 (mean +/- SD), respectively. Thus, PKH26 is not distributed equally to both daughter cells upon cell division. However, the replication history of individual KG1a cells can be reliably deduced for up to three divisions based solely on the mean and total fluorescence intensity of the PKH26 dye, using PKH26 concentrations below the chemical and phototoxic limits (2 microM).     

Expression of surface antigen and mRNA for the CD11c (alpha X, p150) subunit of the human leukocyte adherence receptor family in hematopoietic cells

We characterized the surface antigen and mRNA expression for the CD11c (alpha X, p150) subunit of the human leukocyte adherence receptor family during hematopoietic cell differentiation. The CD11c subunit antigen and mRNA are constitutively expressed in undifferentiated HL-60 promyelocytic leukemia cells, and levels increase markedly with differentiation along the monocyte/macrophage pathway using phorbol myristate acetate. Human monocyte-derived macrophages and human alveolar macrophages express elevated levels of the CD11c subunit antigen and mRNA, indicating that the changes observed in vitro are present in vivo. Dot blot analysis of immature and mature lymphoid and myeloid cells and cell lines demonstrate equivalent levels of CD11c mRNA expression. We conclude that CD11c gene expression is selectively increased during hematopoietic cell differentiation along the monocyte/macrophage pathway.     

Usefulness of PKHs for studying cell proliferation

Classical methods for proliferative assessment (such as tritiated thymidine or bromodeoxyuridine (BrdUrd) incorporation) need sample fixation. As an alternative, we have evaluated the use of a dye dilution method using PKH26 to determine the rate and extent of proliferation in cell lines. Flow cytometric analysis associated with modelling software makes it possible to estimate the number of cells having undergone different numbers of cell divisions by sampling the cell population at varying times post-labelling. Two major questions were addressed in these studies, (i) Does PKH26 give a stable and reproducible labelling? (ii) Does labelling with PKH26 alter cellular proliferation characteristics? We conclude that the methods developed here provide a simpler, more complete means for assessment of cell proliferation in patients with hematological malignancies.     

γδ T cell homing to skin and migration to skin-draining lymph nodes is CCR7 independent

In most species, γδ T cells preferentially reside in epithelial tissues like the skin. Lymph duct cannulation experiments in cattle revealed that bovine dermal γδ T cells are able to migrate from the skin to the draining lymph nodes via the afferent lymph. For αβ T cells, it is generally accepted that epithelial and mucosal tissue egress is regulated by expression of the CCR7 chemokine receptor. In this study, we tracked the migratory route of bovine lymph-derived γδ T cells and examined their CCR7 cell surface expression in several compartments along this route. Total lymph cells from afferent and efferent origin were labeled with PKH fluorescent dyes and injected into the bloodstream. PKH(+) cells already reappeared in the afferent lymph after 4 h. The vast majority of the PKH(+) cells retrieved from the afferent lymph were of the WC1(+) γδ T cell phenotype, proving that this PKH(+) γδ T cell subset is able to home to and subsequently exit the skin. PKH(+) γδ T cells from afferent and efferent lymph lack CCR7 surface expression and display high levels of CD62L compared with CD4 T cells, which do express CCR7. Skin homing receptors CCR4 and CCR10 in contrast were transcribed by both CD4 and γδ T cells. Our findings suggest that γδ T cell skin egress and migration into the peripheral lymphatics is CCR7-independent and possibly mediated by CD62L expression.     

Expression of CD41 on hematopoietic progenitors derived from embryonic hematopoietic cells

In this study, we have characterized the early steps of hematopoiesis during embryonic stem cell differentiation. The immunophenotype of hematopoietic progenitor cells derived from murine embryonic stem cells was determined using a panel of monoclonal antibodies specific for hematopoietic differentiation antigens. Surprisingly, the CD41 antigen (alphaIIb integrin, platelet GPIIb), essentially considered to be restricted to megakaryocytes, was found on a large proportion of cells within embryoid bodies although very few megakaryocytes were detected. In clonogenic assays, more than 80% of all progenitors (megakaryocytic, granulo-macrophagic, erythroid and pluripotent) derived from embryoid bodies expressed the CD41 antigen. CD41 was the most reliable marker of early steps of hematopoiesis. However, CD41 remained a differentiation marker because some CD41(-) cells from embryoid bodies converted to CD41(+) hematopoietic progenitors, whereas the inverse switch was not observed. Immunoprecipitation and western blot analysis confirmed that CD41 was present in cells from embryoid bodies associated with CD61 (beta3 integrin, platelet GPIIIa) in a complex. Analysis of CD41 expression during ontogeny revealed that most yolk sac and aorta-gonad-mesonephros hematopoietic progenitor cells were also CD41(+), whereas only a minority of bone marrow and fetal liver hematopoietic progenitors expressed this antigen. Differences in CD34 expression were also observed: hematopoietic progenitor cells from embryoid bodies, yolk sac and aorta-gonad-mesonephros displayed variable levels of CD34, whereas more than 90% of fetal liver and bone marrow progenitor cells were CD34(+). Thus, these results demonstrate that expression of CD41 is associated with early stages of hematopoiesis and is highly regulated during hematopoietic development. Further studies concerning the adhesive properties of hematopoietic cells are required to assess the biological significance of these developmental changes.     

血源细胞系中CD39的表达和功能分析

细胞外ATP通过激活细胞膜上核苷酸受体介导细胞间通讯。CD39是一个钙,镁离子依赖的ATP双磷酸酶,其对ATP信号的调节机制尚未完全阐明。采用半定量RT．PCR、ABC免疫酶标和流式细胞术以及荧光素／荧光素酶法,研究了七个血源细胞系中CD39的表达和功能。结果表明,不同细胞中CD39的表达水平差异显著：在J6-1和LCL-H中高水平表达,在HL60中低水平表达,而在Namalva、Jurkat,U937细胞中极低水平表达．CD39的表达与这些细胞膜上ATP酶活性、胞外ATP的基础水平结果一致,提示CD39是这些细胞膜上ATP酶活性的主要来源,可能在调节P2受体介导的细胞间通讯中起重要作用。     

Cellular specificity for the activation of fibroblast growth factor-2 by heparan sulfate proteoglycan

Heparan sulfate proteoglycans (HSPGs) promote cellular proliferation through interaction with FGF-2. To examine the role of cellular specificity of HSPG in FGF-2 function, a recombinant soluble isoform of CD44 (rsCD44v3,8-10) was expressed in various cell types; 293 T fibroblasts, the epithelial carcinoma cell lines A431 and HOTZ, the myelomonocytic cell line THP-1, and the Ig-secreting B lymphoblast IM9. The capacity of the recombinant HSPGs expressed in these cell lines to bind and present FGF-2 to the high-affinity receptor FGFR1 was addressed. This novel approach showed a minor difference in the binding and in the FGF-2 stimulating activity of rsCD44v3,8-10 HSPGs from fibroblasts and epithelial cells. However, FGF-2 binding of rsCD44v3,8-10 from IM9 and THP-1 cells was significantly lower, and stimulation of FGF-2 by rsCD44v3,8-10 from these two cell types could not be detected. We tested the possibility that the differences among cell types were related to the functional profile of endogenous HSPGs. The initial survey of a wider panel of cell types revealed high levels of HSPGs synthesis on the surface of 293 T, epithelial and IM9 cells, but low levels on the surface of other cells of hematopoietic origin. Surprisingly, native HSPGs from fibroblasts and epithelial cell lines promoted FGF-2 biological activity to vastly different extents, and cell surface HSPGs from IM9 cells induced an FGF-2 response. Altogether, the results suggested a role for cell-specific HS modification in addition to synthesis as regulatory mechanisms for the cellular specificity of proteoglycan function.     

Epstein-Barr virus latent membrane protein (LMP1) and nuclear proteins 2 and 3C are effectors of phenotypic changes in B lymphocytes: EBNA-2 and LMP1 cooperatively induce CD23.

Latent Epstein-Barr virus (EBV) infection and growth transformation of B lymphocytes is characterized by EBV nuclear and membrane protein expression (EBV nuclear antigen [EBNA] and latent membrane protein [LMP], respectively). LMP1 is known to be an oncogene in rodent fibroblasts and to induce B-lymphocyte activation and cellular adhesion molecules in the EBV-negative Burkitt's lymphoma cell line Louckes. EBNA-2 is required for EBV-induced growth transformation; it lowers rodent fibroblast serum dependence and specifically induces the B-lymphocyte activation antigen CD23 in Louckes cells. These initial observations are now extended through an expanded study of EBNA- and LMP1-induced phenotypic effects in a different EBV-negative B-lymphoma cell line, BJAB. LMP1 effects were also evaluated in the EBV-negative B-lymphoma cell line BL41 and the EBV-positive Burkitt's lymphoma cell line, Daudi (Daudi is deleted for EBNA-2 and does not express LMP). Previously described EBNA-2- and LMP1-transfected Louckes cells were studied in parallel. EBNA-2, from EBV-1 strains but not EBV-2, induced CD23 and CD21 expression in transfected BJAB cells. In contrast, EBNA-3C induced CD21 but not CD23, while no changes were evident in vector control-, EBNA-1-, or EBNA-LP-transfected clones. EBNAs did not affect CD10, CD30, CD39, CD40, CD44, or cellular adhesion molecules. LMP1 expression in all cell lines induced growth in large clumps and expression of the cellular adhesion molecules ICAM-1, LFA-1, and LFA-3 in those cell lines which constitutively express low levels. LMP1 expression induced marked homotypic adhesion in the BJAB cell line, despite the fact that there was no significant increase in the high constitutive BJAB LFA-1 and ICAM-1 levels, suggesting that LMP1 also induces an associated functional change in these molecules. LMP1 induction of these cellular adhesion molecules was also associated with increased heterotypic adhesion to T lymphocytes. The Burkitt's lymphoma marker, CALLA (CD10), was uniformly down regulated by LMP1 in all cell lines. In contrast, LMP1 induced unique profiles of B-lymphocyte activation antigens in the various cell lines. LMP1 induced CD23 and CD39 in BJAB; CD23 in Louckes; CD39 and CD40 in BL41; and CD21, CD40, and CD44 in Daudi. In BJAB, CD23 surface and mRNA expression were markedly increased by EBNA-2 and LMP1 coexpression, compared with EBNA-2 or LMP1 alone. This cooperative effect was CD23 specific, since no such effect was observed on another marker, CD21.(ABSTRACT TRUNCATED AT 400 WORDS)     

Profiling blood lymphocyte interactions with cancer cells uncovers the innate reactivity of human gamma delta T cells to anaplastic large cell lymphoma

Quantifying the contacts that circulating lymphocytes have with cancer cells is useful, because their deficit favors malignancy progression. All normal lymphocytes contact, scan, and acquire membrane fragments (trogocytosis) from foreign cells for their immunosurveillance. So in this study, we used the in vitro trogocytosis of PKH67-stained cancer cell lines as a measure of their interactions with bulks of PBMC freshly isolated from healthy donors. Allogeneic PBMC mixed and coincubated in vitro for 1 h did not trogocytosis, whereas in the same conditions CD20(+), CD4(+), CD8(+), gammadelta T, and CD16(+) PBMC interacted strongly with the cancer cells. Although most unprimed lymphoid effectors of innate (NK) and adaptive (B and T) immunity from healthy donors spontaneously trogocytosed different tumoral cell lines, some carcinoma cell lines could escape them in the coculture. This also uncovered the strong interactions of circulating Vgamma9/Vdelta2(+) central memory gammadelta T cells with anaplastic large cell lymphoma. These interaction profiles were stable upon time for healthy blood donors but were different with other tumors and blood donors. This profiling provides interaction signatures for the immunomonitoring of cancer.     

The Moloney murine leukemia virus repressor binding site represses expression in murine and human hematopoietic stem cells

The Moloney murine leukemia virus (MLV) repressor binding site (RBS) is a major determinant of restricted expression of MLV in undifferentiated mouse embryonic stem (ES) cells and mouse embryonal carcinoma (EC) lines. We show here that the RBS repressed expression when placed outside of its normal MLV genome context in a self-inactivating (SIN) lentiviral vector. In the lentiviral vector genome context, the RBS repressed expression of a modified MLV long terminal repeat (MNDU3) promoter, a simian virus 40 promoter, and three cellular promoters: ubiquitin C, mPGK, and hEF-1a. In addition to repressing expression in undifferentiated ES and EC cell lines, we show that the RBS substantially repressed expression in primary mouse embryonic fibroblasts, primary mouse bone marrow stromal cells, whole mouse bone marrow and its differentiated progeny after bone marrow transplant, and several mouse hematopoietic cell lines. Using an electrophoretic mobility shift assay, we show that binding factor A, the trans-acting factor proposed to convey repression by its interaction with the RBS, is present in the nuclear extracts of all mouse cells we analyzed where expression was repressed by the RBS. In addition, we show that the RBS partially repressed expression in the human hematopoietic cell line DU.528 and primary human CD34(+) CD38(-) hematopoietic cells isolated from umbilical cord blood. These findings suggest that retroviral vectors carrying the RBS are subjected to high rates of repression in murine and human cells and that MLV vectors with primer binding site substitutions that remove the RBS may yield more-effective gene expression.