Cell-to-cell binding induced by different lectins

The cell-to-cell binding induced by concanavalin A (Con A) and the lectins from wheatgerm, soybean, and waxbean has been analyzed by measuring the ability of single cells to bind to lectin-coated cells immobilized on nylon fibers. The cells used were lymphoma, myeloid leukemia, and normal fibroblast cells. With all lectins, cell-to-cell binding was inhibited if both cells were prefixed with glutaraldehyde. However, in most cases cell-to-cell binding was enhanced when only the lectin-coated cell was prefixed. With normal fibroblasts, treatment of either one or both cells with trypsin enhanced the cell-to-cell binding induced by Con A and the wheatgerm lectin. Neuraminidase, which increases the number of receptors for soybean agglutinin, increased cell-to-cell binding only if both cells were treated. Although cell-to- cell binding induced by the lectins from soybean and wheatgerm could be partially reversed by the appropriate competitive saccharide inhibitor, binding induced by Con A could not be reversed. The experiments indicate that cell-to-cell binding induced by a lectin can be prevented by an insufficient density of receptors for the lectin, insufficient receptor mobility, or induced clustering of receptors. These effects can explain the differences in cell-to-cell binding and agglutination observed with different cell types and lectins. They also suggest that cell-to-cell binding induced by different lectins with a variety of cell types is initiated by a mechanism involving the alignment of complementary receptors on the colliding cells for the formation of multiple cell-to-lectin-to-cell bridges.     

Differential apoptotic response to ionizing radiation in subpopulations of human white blood cells.

The purpose of this paper is to characterize the apoptotic response of various subpopulations of human white blood cells after in vitro exposure to ionizing radiation using the modified neutral comet assay (MNCA). White blood cells, isolated from human whole blood, were fractionated into granulocytes and mononuclear cells which were further separated into B-cells, natural killer (NK) cells, and CD4(+) and CD8(+) T-cells. The separated fractions were exposed to low doses of X-rays and then MNCA was used to measure the apoptotic fraction (AF) at different time points in irradiated and unirradiated aliquots of sorted cultures. The spontaneous AF in unirradiated control cells was the most critical determinant of whether an apoptotic response could be detected in irradiated cells. When cultured in isolation granulocytes and B-cells had the highest background AF, with NK cells having the next highest. CD4(+) and CD8(+) T-cells had a low, stable, spontaneous AF which gave them the highest signal-to-noise ratio. Although B-cells demonstrated the highest radiation-induced apoptotic response to 1Gy of X-rays, CD8(+) T-cells were the most radiation-responsive lymphocytes due to their low spontaneous AF. By generating dose response curves for CD4(+) and CD8(+) T-cells, the sensitivity of the MNCA for detecting apoptosis in these two cell types was also examined.     

Determination of the LDL receptor binding capacity of human lymphocytes by immunocytofluorimetric assay

The determination of the LDL receptor binding capacity of human blood lymphocytes was assessed by indirect immunocytofluorimetric assay. To produce the maximal synthesis of the LDL receptor, the cholesterol efflux was enhanced by incubation of lymphocytes with HDL3 subfractions. The binding capacity of the LDL receptor was measured by incubation at 4 degrees C either with LDL and rabbit anti-LDL immunoglobulins or with peptide receptor antibody (ARP-Ig) raised against the NH2-terminal sequence of the LDL receptor. Thereafter complexes were incubated with fluorescein-labelled anti-rabbit immunoglobulin (FITC-Ig). Fluorescence flow cytometry was used to quantify the number of fluorescent lymphocytes and results were expressed as the percentage of lymphocytes with a fluorescent intensity above the threshold. Using preimmune rabbit immunoglobulin and then FITC-Ig, only 5-10% of cells were fluorescent. Neither LDL nor ARP-Ig could bind to homozygous familial hypercholesterolemia (FH) lymphocytes. Normal lymphocytes preincubated with HDL3 could bind LDL or ARP-Ig, the number of fluorescent cells being 59 and 39.2% respectively. Subjects with confirmed or suspected heterozygous FH demonstrated cell fluorescence at about half the normal level.     

Differential DNA damage induced by H2O2 and bleomycin in subpopulations of human white blood cells

The purpose of this study was to characterize the differential sensitivities of various subpopulations of human white blood cells after exposure to H2O2 (an oxidant agent) and bleomycin (a radiomimetic glycopeptide), in vitro, using single-cell gel electrophoresis (SCGE). Human peripheral blood was fractionated into mononuclear cells, which were further separated into monocytes, CD4+ T-cells, CD8+ T-cells, B-cells and natural killer cells (NK cells). The separated fractions were exposed to different doses of H2O2 and bleomycin, and then used to measure levels of induced and basal DNA damage. There was a significant increase in the amount of DNA damage in CD4+ T-cells, CD8+ T-cells, NK cells and B-cells when treated with H2O2 and bleomycin, whereas monocytes had the lowest sensitivity to H2O2 compared with the other cell fractions, but no lower sensitivity to bleomycin. Furthermore, CD4+ T-cells and CD8+ T-cells had the highest levels of basal DNA damage. When basal DNA damage was taken into account, NK cells tended to show a higher sensitivity to H2O2 than CD4+ T-cells, CD8+ T-cells and monocytes. In addition, B-cells, which showed lower sensitivity to H2O2 than CD4+ T-cells, CD8+ T-cells and NK cells when exposed to lower doses of H2O2 (<10 microM), showed higher sensitivity to H2O2 at higher doses (>20 microM). On the other hand, B-cells showed the highest sensitivity to bleomycin.     

The cell surface of Phytomonas davidi

Carbohydrates were located on the surface of Phytomonas davidi using ultrastructural cytochemistry, and agglutination induced by lectins which bind to residues of mannose, N-acetylglucosamine, galactose, N-acetylgalactosamine, fucose and sialic acid. The surface charge of the cells was analysed by the binding of cationic particles (colloidal iron and cationized ferritin) to the cell surface and by cell electrophoretic mobility (EPM). Based on observations of binding of cationic particles to the cell surface; a decrease in the binding of these particles to the cell surface; a decrease in the mean EPM of the cells after their incubation in the presence of neuraminidase; and detection of N-acetylneuraminic acid by paper and gas-liquid chromatography, it was concluded that sialic acid residues are exposed on the surface of P. davidi. These residues may be glycolipids or are masked on the cell surface since only after brief trypsinization were the cells agglutinated by the lectin from Limulus polyphemus.     

Differential apoptotic response to ionizing radiation in subpopulations of human white blood cells

The purpose of this paper is to characterize the apoptotic response of various subpopulations of human white blood cells after in vitro exposure to ionizing radiation using the modified neutral comet assay (MNCA). White blood cells, isolated from human whole blood, were fractionated into granulocytes and mononuclear cells which were further separated into B-cells, natural killer (NK) cells, and CD4⁺ and CD8⁺ T-cells. The separated fractions were exposed to low doses of X-rays and then MNCA was used to measure the apoptotic fraction (AF) at different time points in irradiated and unirradiated aliquots of sorted cultures. The spontaneous AF in unirradiated control cells was the most critical determinant of whether an apoptotic response could be detected in irradiated cells. When cultured in isolation granulocytes and B-cells had the highest background AF, with NK cells having the next highest. CD4⁺ and CD8⁺ T-cells had a low, stable, spontaneous AF which gave them the highest signal-to-noise ratio. Although B-cells demonstrated the highest radiation-induced apoptotic response to 1 Gy of X-rays, CD8⁺ T-cells were the most radiation-responsive lymphocytes due to their low spontaneous AF. By generating dose response curves for CD4⁺ and CD8⁺ T-cells, the sensitivity of the MNCA for detecting apoptosis in these two cell types was also examined.     

Nonspecific human lymphocyte-mediated cytotoxicity induced by immobilized IgG aggregate

Normal human lymphocytes were induced to lyse nonsensitized erythrocytes when concomitantly incubated on immobilized IgG aggregate with various erythrocyte target cells. These included ox, sheep, chicken, and human red blood cells. Only immobilized aggregate would initiate cytolysis. The IgG aggregate was prepared from normal, healthy adult donors and did not possess target cell specificity (e.g., human erythrocyte lysis was initiated by autologous IgG). Normal human lymphocytes could be induced to lyse the red blood cell targets only after a preincubation with adherent mononuclear cells; however, freshly prepared lymphocytes depleted of IgG-FcR^? cells were cytolytic. Cytolysis induced by immobilized IgG-aggregate can be distinguished from NCMC and ADCC by its requirement for immobilized IgG aggregate and the absence of target cell specificity in the IgG-aggregate preparation.     

Cooperative binding of concanavalin A to thymocytes at 4 degrees C and micro-redistribution of concanavalin A receptors.

The mode of binding of 125I-labelled concanavalin A and succinyl-concanavalin A to rat thymocytes at 4 degrees C was investigated. Simultaneously, the free binding sites of the cell-bound lectin molecules were quantified by horseradish peroxidase binding. Concanavalin A showed cooperative binding while succinyl-concanavalin A did not. The number of molecules of concanavalin A bound to the cell surface when it was saturated was twice the number of molecules of succinyl-concanavalin A. We interpret these results as showing that the binding of native concanavalin A to thymocytes at 4 degrees C brings about a cooperative modification of the membrane which leads to appearance of new receptors. Divalent succinyl-concanavalin A has no such effect. Horseradish peroxidase binding to cell-bound lectin was shown to be related to the immobilization of membrane receptors; the more they are immobilized, the more receptor-associated lectin can bind horseradish peroxidase. This allowed us to establish that post-binding events, which we called micro-redistribution, occurred at 4 degrees C when either concanavalin A or succinyl-concanavalin A binds to cells. A cooperative restriction of the micromobility of cell receptors is produced by increasing concentrations of concanavalin A. Succinyl-concanavalin A does not restrict cell receptor mobility at any concentration tested. The results are discussed in terms of cell stimulation and cell agglutination.     

Evidence for the presence of specific binding sites for transcortin in human liver plasma membranes

Binding sites which recognize and bind specifically asialotranscortin and the native transcortin-cortisol complex have been found in plasma membranes of human liver cells. The native conformation of transcortin is an absolute requirement for the binding reaction of the transcortin-hormone complex. Sex-hormone-binding globulin and thyroxine-binding globulin from human serum do not bind to this binding sites.     

Equilibrium binding of cytotoxic T lymphocytes to class I antigen

Cloned cytotoxic T lymphocytes specifically bind to purified alloantigen that has been immobilized on a surface. When the time course was examined, it was found that binding reached a plateau level within about 1 h at 37 degrees C, at which time about 30% of the CTL were tightly adhered to the surface. Analysis of the properties of binding demonstrated that this does not simply result because only a fraction of the cells in the clonal population are capable of binding. Instead, the binding is shown to result from an equilibrium involving tightly bound and unbound (or weakly bound) cells. Thus, the cells cycle between a tightly bound and unbound state, despite continuous contact with the Ag-bearing surface. The results suggest that dissociation of the bound cells may be an actively signaled event. A model that could account for these results based on activated CD8 binding is discussed.     

WGA binding to the surface of two autologous human melanoma cell lines: different expression of sialyl and N-acetylglucosaminyl residues

Two autologous human melanoma cell lines were studied to determine their capacities to bind wheat germ agglutinin (WGA). Both cell lines were derived from the same patient, the first, IGR 39, originated from the primary tumor, the second, IGR 37, was established from a metastatic lymph node. WGA binding sites on the surface of these cell lines were compared before and after sialidase and/or tunicamycin treatments. IGR 39 cells exhibited two classes of WGA binding sites with high and low affinities, whereas IGR 37 cells had only one class of high affinity binding sites. After tunicamycin treatment, the capacity of IGR 39 cells to bind WGA was markedly altered, since only one class of WGA binding sites with high affinity was observed under these conditions, whereas tunicamycin did not induce significant changes in the lectin binding of IGR 37 cells. The low affinity WGA binding sites, which were only found on IGR 39 cells, corresponded to sialyl residues present in N-linked glycoproteins. The high affinity binding sites present on both cell lines probably involved sialyl and N-acetyl-glucosaminyl residues associated with O-linked glycoproteins and/or glycolipids. No direct correlation could be drawn between the number of WGA binding sites and the overall sialic acid levels exposed to sialidase treatment. The 3-fold increase in the amount of cell surface glycopeptides obtained after pronase digestion and specifically binding to WGA-Sepharose was in good agreement with the overall higher number of WGA binding sites on IGR 39 compared to IGR 37 cells. Thus, subtle carbohydrate changes of cell surface glycoconjugates might account for the differences between the biological properties of human melanoma cell lines of low and high tumorigenicity.     

Structural studies of T lymphocyte Fc receptors. Association of Gc protein with IgG binding to Fc gamma

To obtain further information concerning the structure of Fc IgG-binding sites on human peripheral blood lymphocytes, enriched T-cells were surface-radioiodinated and treated with nonionic detergent, and the soluble supernatant was submitted to affinity chromatography selecting for components binding complexed IgG. Analysis of eluted material by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and two-dimensional electrophoresis demonstrated the major proteins to be of Mr 56,000, pI 4.8-5.1 and Mr 60,000, pI 5.0-5.6, and these were radiolabeled, indicating an origin in part from the T-cell membrane. While the Mr 56,000 band gave positive reactions upon transblotting with antisera to Gc protein, the identity of the Mr 60,000 protein remains unknown. Three other components were detected, although with less consistency; Mr 78,000, 42,000, and 20,000-25,000, respectively. Immunocytochemical experiments showed that less than 5% freshly isolated native T-cells were positive with antiserum to human Gc, but, after IgG antibody-coated erythrocyte rosetting, the number of positive cells increased to 15-25%, in close agreement with the percentage of IgG antibody-coated erythrocyte rosette-positive T-cells. These findings therefore indicate that, in addition to interaction with components of Mr 60,000, 42,000, and 20,000-25,000, complexed IgG binding to Fc gamma of human peripheral blood T-cells also becomes spatially associated with Gc protein.     

Immune islet killing mechanisms associated with insulin-dependent diabetes: in vitro expression of cellular and antibody-mediated islet cell cytotoxicity in humans

In previous work we have shown that some bacteria can bind to human lymphocytes and can be used to identify lymphocyte subpopulations in conventionally stained blood smears. These bacteria are of different species or genera, which makes it difficult to study the binding mechanism. Also, the main marker for B cells, Brucella melitensis, is of very small size and highly pathogenic. Here we show that B cells as well as some of the T cell subpopulations can be identified by different mutants obtained from a strain of an Escherichia coli. Two procedures were used to generate mutants. First, E. coli-YS57 (pro-his-trp-) was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine and the binding to mouse spleen cells was used as a selective pressure. Second, phage-resistant mutants of E. coli-YS57 were obtained and tested for the ability to bind to lymphocytes. Out of 10 strains selected by the former procedure, 5 bound to a significant number of human lymphocytes. All four phage-resistant mutants bound to human lymphocytes. Out of the total of nine mutants that bound to lymphocytes, six bound consistently, i.e., they bound to similar percentages of peripheral blood lymphocytes from different normal donors. One phage-resistant mutant, E. coli USC-106, bound only to B cells. The subpopulations of lymphocytes identified by the mutants were essentially the same as those identified by different species or genera of bacteria. We concluded that E. coli mutants can be obtained that identify human lymphocyte subpopulations and that one of these mutants recognizes B cells; these mutants may be used to study the nature of the receptors for bacteria on lymphocytes, which appear to have a lectin-like nature.     

Susceptibility to duck hepatitis B virus infection is associated with the presence of cell surface receptor sites that efficiently bind viral particles.

To test the hypothesis that susceptibility of hepatocytes to duck hepatitis B virus (DHBV) infection requires cell surface receptors that bind the virus in a specific manner, we developed an assay for the binding of DHBV particles to monolayers of intact cells, using radiolabeled immunoglobulin G specific for DHBV envelope protein. Both noninfectious DHBV surface antigen particles and infectious virions bound to a susceptible fraction (approximately 60%) of Pekin duck hepatocytes. In contrast, binding did not occur to cells that were not susceptible to DHBV infection, including Pekin duck fibroblasts and chicken hepatocytes, and binding to Muscovy duck hepatocytes, which are only weakly susceptible (approximately 1% of cells) to DHBV infection, was virtually undetectable. Within a monolayer, individual Pekin duck hepatocytes appeared to differ markedly in the capacity to bind DHBV, which may explain difficulties that have been encountered in infecting 100% of cells in culture. We have also found that the loss of susceptibility to infection with DHBV that occurs when Pekin duck hepatocytes are maintained for more than a few days in culture correlates with a decline in the number of cells that bind virus particles efficiently. All of these results support the interpretation that the binding event detected by our assay is associated with the interaction between DHBV and specific cell surface receptors that are required for initiation of infection. Our assay may facilitate isolation and identification of hepatocyte receptors for this virus.     

Endocytosis of cationized ferritin in human resting peripheral blood by T-lymphocytes

Summary We have examined the binding and internalization of cationized ferritin in T-lymphocytes of human peripheral blood, as a model for resting cells. After 30 min of incubation only 8% of endocytotic vesicles contain cationized ferritin. T-cells internalie the equivalent of their entire surface area in approximately 54 h, a longer time than is required by non-resting cells such as PHA-stimulated human lymphocytes. These tracer experiments suggest that the endocytosis of cationized ferritin by T-lymphocytes follows a lysosome pathway similar to that described for other cell types.     

Cytological observations on the cytotoxic interaction between lymphocytes and antibody-coated monolayer cells

Chang cells in monolayers were killed when exposed to highly purified human peripheral blood lymphocytes in the absence of complement and in the presence of a concentration of heat inactivated rabbit anti-Chang cell serum (ACS) chosen for inability by itself to cause cytolysis. As evident by plaque formation, the cytotoxic reaction was confined to areas of the cultures covered with lymphocytes. In the presence of ACS, lymphocytes attached to and infiltrated the monolayers. Uropod formation indicated activation of the lymphocytes. Close contact was established between effector and target cells. The cytotoxic reaction was not accompanied by protracted cytopathological changes but led to detachment and rapid lysis of the target cells. Horse anti-thymocyte globulin, added to the cultures, completely inhibited plaque formation and lymphocyte infiltration.     

The viral sigma1 protein and glycoconjugates containing alpha2-3-linked sialic acid are involved in type 1 reovirus adherence to M cell apical surfaces

Type 1 reoviruses invade the intestinal mucosa of mice by adhering selectively to M cells in the follicle-associated epithelium and then exploiting M cell transport activity. The purpose of this study was to identify the apical cell membrane component and viral protein that mediate the M cell adherence of these viruses. Virions and infectious subviral particles of reovirus type 1 Lang (T1L) adhered to rabbit M cells in Peyer's patch mucosal explants and to tissue sections in an overlay assay. Viral adherence was abolished by pretreatment of sections with periodate and in the presence of excess sialic acid or lectins MAL-I and MAL-II (which recognize complex oligosaccharides containing sialic acid linked alpha2-3 to galactose). The binding of T1L particles to polarized human intestinal (Caco-2(BBe)) cell monolayers was correlated with the presence of MAL-I and MAL-II binding sites, blocked by excess MAL-I and -II, and abolished by neuraminidase treatment. Other type 1 reovirus isolates exhibited MAL-II-sensitive binding to rabbit M cells and polarized Caco-2(BBe) cells, but type 2 or type 3 isolates including type 3 Dearing (T3D) did not. In assays using T1L-T3D reassortants and recoated viral cores containing T1L, T3D, or no sigma1 protein, MAL-II-sensitive binding to rabbit M cells and polarized Caco-2(BBe) cells was consistently associated with the T1L sigma1. MAL-II-recognized oligosaccharide epitopes are not restricted to M cells in vivo, but MAL-II immobilized on virus-sized microparticles bound only to the follicle-associated epithelium and M cells. The results suggest that selective binding of type 1 reoviruses to M cells in vivo involves interaction of the type 1 sigma1 protein with glycoconjugates containing alpha2-3-linked sialic acid that are accessible to viral particles only on M cell apical surfaces.     

Affinity binding of cells to cryogel adsorbents with immobilized specific ligands: effect of ligand coupling and matrix architecture

The capture of human acute myeloid leukemia KG-1 cells expressing the CD34 surface antigen and the fractionation of human blood lymphocytes were evaluated on polyvinyl alcohol (PVA)-cryogel beads and dimethyl acrylamide (DMAAm) monolithic cryogel with immobilized protein A. The affinity ligand (protein A) was chemically coupled to the reactive PVA-cryogel beads and epoxy-derivatized monolithic cryogels through different immobilization techniques and the binding efficiency of the cell surface receptors specific antibody-labeled cells to the gels/beads was determined. The binding of cells to monolithic cryogel was higher (90-95%) compared with cryogel beads (76%). B-lymphocytes, which bound to the protein A-cryogel beads, were separated from T-lymphocytes with yields for the two cell types 74 and 85%, respectively. About 91% of the bound B-cells could be recovered without significantly impairing their viability. Our results show differences in the percentage of cell-binding to the immunosorbents caused by ligand density, flow shear forces and bond strength between the cells and the affinity surface once distinct chemical coupling of protein A, size of beads, sequence of antibody binding to protein A adsorbents, morphology and geometry of surface matrices were compared.     

A kinetic model of the agglutination process.

We propose a kinetic model of the aggregation process in a system consisting of two different types of particles. Aggregating particles (cells) are polyvalent and bear on the surface a huge number of binding sites for the other type of particles, ligands. The ligand is bivalent and has two identical active sites for binding to cells. The cross-linking of the cells by the ligands causes the aggregation phenomenon called agglutination. We obtained the analytical solution of this model task describing the time dependence of the aggregate mean size versus the composition of the system. The comparison of the analytical solution with the experimental data for the agglutination of bacterial cells by bivalent antibodies shows that the main factors affecting agglutination were correctly taken into account.     

A large subpopulation of lymphocytes with T helper phenotype (Leu-3/T4+) exhibits the property of binding to NK cell targets and granular lymphocyte morphology

A discrete subpopulation of lymphocytes sharing several phenotypic characteristics with natural killer (NK) cells was identified within the circulating pool of human lymphocytes that bear the T helper marker Leu-3. This Leu-3+ subpopulation of cells formed cell conjugates with the NK target cell lines K562 and MOLT-4, but did not bind to mouse myeloma and hybridoma cell lines that are insensitive to NK cells. The Leu-3+ lymphocytes binding to NK cell targets contained cytoplasmic granules similar in ultrastructure and cytochemistry to those previously defined in granular lymphocytes with NK function, except that the granules in Leu-3+ cells were smaller and fewer in number. Unlike classical NK cells, however, the granular Leu-3+ cells did not kill the target cells to which they bound, even after treatment with interferon. The proportion of granular Leu-3+ cells with the capacity to bind to NK cell targets was approximately 7% at birth and increased to approximately 21% of the Leu-3+ cells in adults. These observations suggest the possibility of a lineal relationship between the granular Leu-3+ cells and granular Leu-3- cells with NK capability.