Interaction of cells of Helicobacter pylori with human polymorphonuclear leucocytes: Possible role of haemagglutinins

Abstract The interaction of fluorescein isothiocynate (FITC)-labelled cells of Helicobacter pylori with human polymorphonuclear leucocytes (PMNs) was studied. Two strains with surface haemagglutinins expressing different receptor specificity were used in order to decide if cell surface haemagglutinins of H. pylori may play a role in lectin-mediated binding to/uptake by phagocytes: (1) strain 17874 (NCTC 11637) which expresses sialic acid-specific haemagglutin; and (2) strain 17875 (NCTC 11638) which expresses a sialic acid-independent haemagglutinin. Cells of strain 17874 were poorly attached to/ingested by PMNs compared to cells of strain 17875. Pre-treatment of bacteria with fetuin or rabbit antibodies against partly purified sialic acid-specific haemagglutinin enhanced interaction of cells of strain 17874 with PMNs. The enhancement did not occur in the case of strain 17875. Phagocytosis of H. pylori 17874 bacteria was slightly increased by fresh human sera positive for anti- H. pylori antibodies. The results suggest that the sialic-acid-specific haemagglutinin complex of 17874 bacteria might disturb their uptake by human PMNs.     

Interaction of extracellular vesicles of Bacteroides gingivalis W50 with human polymorphonuclear leucocytes

The effects of B. gingivalis W50 extracellular vesicles (ECV) on neutrophil chemotaxis and viability were assessed and compared with those of whole cells and the extracellular non-dialysable soluble protein (EP) fraction. None of the fractions tested, including soluble fractions derived from cells and ECV by sonication, induced neutrophil chemotaxis. Only ECV and cells inhibited f-MLP-stimulated chemotaxis. ECV and cells were cytotoxic towards neutrophils. The cytotoxic response was time dependent. The soluble EP fraction did not influence cell viability.     

Interaction of lactoferrin and lysozyme with casein micelles

On addition of lactoferrin (LF) to skim milk, the turbidity decreases. The basic protein binds to the caseins in the casein micelles, which is then followed by a (partial) disintegration of the casein micelles. The amount of LF initially binding to casein micelles follows a Langmuir adsorption isotherm. The kinetics of the binding of LF could be described by first-order kinetics and similarly the disintegration kinetics. The disintegration was, however, about 10 times slower than the initial adsorption, which allowed investigating both phenomena. Kinetic data were also obtained from turbidity measurements, and all data could be described with one equation. The disintegration of the casein micelles was further characterized by an activation energy of 52 kJ/mol. The initial increase in hydrodynamic size of the casein micelles could be accounted for by assuming that it would go as the cube root of the mass using the adsorption and disintegration kinetics as determined from gel electrophoresis. The results show that LF binds to casein micelles and that subsequently the casein micelles partly disintegrate. All micelles behave in a similar manner as average particle size decreases. Lysozyme also bound to the casein micelles, and this binding followed a Langmuir adsorption isotherm. However, lysozyme did not cause the disintegration of the casein micelles.     

Interaction of rotavirus with human myeloid dendritic cells

We have previously shown that very few rotavirus (RV)-specific T cells that secrete gamma interferon circulate in recently infected and seropositive adults and children. Here, we have studied the interaction of RV with myeloid immature (IDC) and mature dendritic cells (MDC) in vitro. RV did not induce cell death of IDC or MDC and induced maturation of between 12 and 48% of IDC. Nonetheless, RV did not inhibit the maturation of IDC or change the expression of maturation markers on MDC. After treatment with RV, few IDC expressed the nonstructural viral protein NSP4. In contrast, a discrete productive viral infection was shown in MDC of a subset of volunteers, and between 3 and 46% of these cells expressed NSP4. RV-treated IDC secreted interleukin 6 (IL-6) (but not IL-1beta, IL-8, IL-10, IL-12, tumor necrosis factor alpha, or transforming growth factor beta), and MDC released IL-6 and small amounts of IL-10 and IL-12p70. The patterns of cytokines secreted by T cells stimulated by staphylococcal enterotoxin B presented by MDC infected with RV or uninfected were comparable. The frequencies and patterns of cytokines secreted by memory RV-specific T cells evidenced after stimulation of peripheral blood mononuclear cells (PBMC) with RV were similar to those evidenced after stimulation of PBMC with RV-infected MDC. Finally, IDC treated with RV strongly stimulated naive allogeneic CD4+ T cells to secrete Th1 cytokines. Thus, although RV does not seem to be a strong maturing stimulus for DC, it promotes their capacity to prime Th1 cells.     

Interaction of human mesenhymal stromal with immune cells

Multipotential mesenchymal stromal cells (MMSCs) are the subject of increasing scientific interest due to the key role of these cells in physiological renewal and repair. Allogeneic MMSC interaction with other components of tissue processes in the environment, in particular, with immune cells is one of the most intriguing questions of modern cell physiology. MMSCs possess pronounced immunomodulatory capabilities based on their immmunoprivilege properties and the ability to suppress an immune response. This review considers the state-of-the-art in the field of MMSC immunomodulatory effects and their mechanisms. The interaction between MMSCs and immune cells is a complex, multidirectional process governed by both direct cell-to-cell interactions and soluble factors (interferon-γ, tumor necrosis factor, prostaglandin E₂, hepatocyte growth factor, interleukins, etc.). The importance of physical environmental factors, primarily oxygen tension, for the characteristics of the interaction between MMSCs and immune cells is discussed.     

Interaction of Eglin c with polymorphonuclear cells: evidence for binding to the cell surface

The interaction of Eglin c with human polymorphonuclear cells was investigated in order to explain the effect of this (and other) proteinase inhibitor(s) on the biological activities of neutrophils. We have identified binding sites on human neutrophils by using [3H]Eglin. Binding is rapid and reversible at 5 degrees C. There are approximately 100,000 binding sites per cell, with an equilibrium dissociation constant of 0.2microM. Eglin binding was not inhibited by other proteinase inhibitors (alpha 1-PI, PhCH2SO2F, Tos-Phe-CH2Cl), and was enhanced four-fold by the chemotactic peptide fMet-Leu-Phe. The results indicate that Eglin c, a peptide proteinase inhibitor, is able to bind to human PMN cells and that this initial interaction does not involve a known proteinase such as cathepsin G or elastase.     

Interaction of polymorphonuclear leukocytes and viruses in humans: adherence of polymorphonuclear leukocytes to respiratory syncytial virus-infected cells

The nature of neutrophil-respiratory syncytial virus (RSV) interaction was investigated by assessing factors that influence neutrophil adherence to RSV-infected tissue culture monolayers. The adherence of neutrophils to infected cells was directly proportional to the degree of RSV replication as evidenced by infectious virus production, cytopathological changes, or viral antigen appearance. Sixty-one percent of the neutrophils adhered to the RSV-infected cells as compared with 52.7% on noninfected monolayers (P less than 0.05). The addition of RSV-specific antibody markedly increased polymorphonuclear leukocyte adherence to 88.5% (P less than 0.001). Complement in the absence of antibody augmented polymorphonuclear leukocyte adherence, but to a lesser degree, 69.0% (P less than 0.025). Arachidonic acid metabolism appeared to play a critical role in the adherence process; thromboxane was the single most important arachidonic acid metabolite. Inhibition of thromboxane synthesis reduced antibody-dependent polymorphonuclear leukocyte adherence on RSV-infected cells to 52.3% (P less than 0.025). These observations suggest a role for neutrophils in RSV infection. It is proposed that neutrophils may participate in RSV infection at the site of viral replication through the attachment to infected cells and the subsequent release of mediators of inflammation.     

Interaction of chemotactic factors with human polymorphonuclear leukocytes: Studies using a membrane potential-sensitive cyanine dye

Summary Changes in the fluorescence intensity of the dye 3-3 dipentyloxacarbocyanine were measured in suspensions of purified human peripheral blood polymorphonuclear leukocytes (PMNs) during exposure to the chemotactic factors N-formyl-methionylleucyl-phenylalanine (f-met-leu-phe) and partially purified C5a. Incubation of PMNs with dye resulted in a stable fluorescence reflecting the resting membrane potential of the cell. Exposure of PMNs to dye did not affect stimulated chemotaxis or secretion. The mechanism of cell-associated dye fluorescence involved solvent effects from partitioning of the dye between the aqueous incubation medium and the cell and not dye aggregation, Chemotactically active concentrations of f-met-leu-phe (5×10^–9 m or greater) produced a biphasic response characterized as a decrease followed by an increase in fluorescence. No fluorescence response was seen in lysed PMNs, and no response was elicited by an inhibitor of f-met-leu-phe binding (carbobenzoxy-phenylalanyl-methionine). The ability of several other synthetic peptides to elicit a fluorescence response corresponded to their effectiveness as chemotactic agents. Although the first component of the response suggested a depolarization, it was not influenced by variation in the external concentration of sodium, potassium, chloride, or calcium, and could not be characterized as a membrane potential change. The second component of the response, which was inhibited by both Mg²⁺ (10mm)-EGTA (10mm) and high external potassium, was compatible with a membrane hyperpolarization. The data indicate that chemotactic factors produce changes in dye fluorescence which can, at least in part, be attributed to a hyperpolarizing membrane potential change occurring across the plasma membrane.Presented in part at the 17th Annual Cell Biology Meeting.Cell Biol. 75:103a, 1977.     

Comparison of casein of cynomolgus monkey (Macaca fascicularis) with human casein

Casein of cynomolgus monkey was compared with those from human and bovine milk. Cynomolgus monkey casein showed similar electrophoretical patterns to those of human casein on Disc- and SDS-electrophoresis. It consisted of beta- and kappa-casein-like components. The component corresponding to bovine alpha s1-casein was not detected. The beta-casein-like fraction of cynomolgus monkey showed 9 bands on Disc-PAGE. These were suggested to be the same protein binding different levels of phosphorus by dephosphorylation experiment using an acid phosphatase. The kappa-casein-like component of cynomolgus monkey was highly glycosylated (about 50% carbohydrate) similarly as human kappa-casein and the constituent carbohydrates were same as those detected in human kappa-casein (galactose, fucose, N-acetylgalactosamine, N-acetylglucosamine, and sialic acid). Amino acid composition of cynomolgus monkey kappa-casein bore a resemblance to those of both human and bovine kappa-caseins. Amino acid composition of cynomolgus monkey beta-casein was also similar to those of human and bovine beta-caseins.     

Interaction of fibronectin with polymorphonuclear leukocytes normally and in anaphylaxis

Fibronectin is known as an opsonic glycoprotein possessing a wide range of specificity. Fibronectin interaction with neutrophils (normal ones and those obtained at peak of anaphylactic shock) was studied. Neutrophils were brought in contact with purified homologous fibronectin previously bound to gelatin-Sepharose granules. The presence of fibronectin-binding proteins on the surface of human and rabbit neutrophils was demonstrated, the binding being strongly dependent on Ca2+ and especially Mg2+ ions. Anaphylactic neutrophils, in contrast to normal ones, did not interact with fibronectin, which may be important for the pathogenesis of the immediate type hypersensitivity reactions.     

Biochemical characterisation of porcine polymorphonuclear leucocytes: comparison with human polymorphonuclear leucocytes

Polymorphonuclear leucocytes were isolated from pig blood relatively free from other cells and were characterised biochemically and morphologically and compared with human PMNLs. The activities of 16 enzymes of porcine and human PMNLs were measured and compared. Alkaline phosphatase, acid phosphatase, phosphodiesterase, gamma-glutamyl transpeptidase, NADH-cytochrome c oxidoreductase, malate dehydrogenase and acetylcholinesterase had higher specific activities in procine than in human cells. Alkaline phosphatase has an 87-fold higher specific activity in porcine than in human cells. beta-glucuronidase, lysozyme, beta-galactosidase, N-acetyl-glucosaminidase, beta-glucosidase, myeloperoxidase and catalase had higher specific activities in human than in porcine cells. beta-glucuronidase and myeloperoxidase showed over a 1000- and a 13-fold higher specific activity, respectively, in human than in porcine cells. Porcine PMNLs are readily available in large numbers and are recommended for studies of phagocytosis, chemotaxis and membrane biochemistry.     

Interaction of single-chain urokinase-type plasminogen activator with human endothelial cells

The interaction of urokinase-type plasminogen activators with receptors on the surface of endothelial cells may play an important role in the regulation of fibrinolysis and cell migration. Therefore, we investigated whether human umbilical vein endothelial cells (HUVEC) express receptors for single-chain urokinase (scu-PA) on the cell surface and examined the effect of such binding on plasminogen activator activity. Binding of 125I-labeled scu-PA to HUVEC, performed at 4 degrees C, was saturable, reversible, and specific (k+1 4 +/- 1 X 10(6) min-1 M-1, k-1 6.2 +/- 1.4 X 10(-3) min-1, Kd 2.8 +/- 0.1 nM; Bmax 2.2 +/- 0.1 X 10(5) sites/cell; mean +/- S.E.). Binding of radiolabeled scu-PA was inhibited by both natural and recombinant wild-type scu-PA, high molecular weight two-chain u-PA (tcu-PA), catalytic site-inactivated tcu-PA, an amino-terminal fragment of u-PA (amino acids 1-143), and a smaller peptide (amino acids 4-42) corresponding primarily to the epidermal growth factor-like domain. Binding was not inhibited by low molecular weight urokinase or by a recombinant scu-PA missing amino acids 9-45. Cell-bound scu-PA migrated at its native molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of plasminogen, scu-PA bound to endothelial cells generated greater plasmin activity than did scu-PA in the absence of cells. In contrast, when tcu-PA was added directly to HUVEC, sodium dodecyl sulfate-stable complexes formed with cell or matrix-associated plasminogen activator inhibitors with a loss of plasminogen activator activity. These studies suggest that endothelial cells in culture express high affinity binding sites for the epidermal growth factor domain of scu-PA. Interaction of scu-PA with these receptors may permit plasminogen activator activity to be expressed at discrete sites on the endothelial cell membrane.     

Cyclic AMP-independent casein/glycogen synthase kinases from pig polymorphonuclear leucocytes

1. Two cyclic AMP-independent casein/glycogen synthase kinases were purified from pig polymorphonuclear leucocytes by chromatography on phosphocellulose followed by affinity chromatography on casein-Sepharose 4B or gel filtration on Bio-Gel A-1.5m. When the affinity step was used, the specific activities were 86 and 43units/mg of protein for casein kinase 1 and 2, respectively, whereas these values were 94 and 90units/mg of protein when the gel-filtration step was used. 2. These kinases differ as follows: (a) the molecular weight of casein kinase 1 (38000) is very much lower than that of casein kinase 2 (185000); (b) the K(m) for casein (0.46mg/ml) and K(a) for Mg(2+) (0.3mm) of casein kinase 1 are lower than those of casein kinase 2 (0.90mg/ml and 1.7mm respectively); (c) KCl stimulates the phosphorylation of casein by casein kinase 1, whereas it inhibits phosvitin phosphorylation by this enzyme; on the contrary, the effect of KCl on casein kinase 2 is very similar with either casein or phosvitin as substrate; (d) although both kinases phosphorylate rabbit muscle glycogen synthase I, the ratio of glycogen synthase to casein phosphorylation by casein kinase 1 is about 4-fold greater than that by casein kinase 2. Furthermore, (32)P incorporation into glycogen synthase promoted by casein kinase 1 (3.6mol of (32)P/mol of 85000-dalton subunit) is twice that observed with casein kinase 2 (1.8mol of (32)P/mol of 85000-dalton subunit). Such a phosphorylation results in a decrease in the glucose 6-phosphate-independence ratio of glycogen synthase to 10-15 with casein kinase 1 and to 35-45 with casein kinase 2. 3. The activity of both kinases is neither stimulated by cyclic AMP, Ca(2+) and calmodulin nor inhibited by cyclic AMP-dependent protein kinase inhibitor protein. 4. No phosphorylation kinase activity was observed with casein kinase 1 and 2 at either pH6.8 or 8.2 in the presence of Ca(2+). 5. Activities of both kinases on casein and glycogen synthase decreased in parallel when incubated at 50 degrees C.     

Interaction of polyriboinosinic acid. Polyribocytidylic acid with human lymphoblastoid cells

The double-stranded RNA, poly(I).poly(C), failed to induce interferon in Namalva lymphoblastoid cells even when tested under varying conditions. Striking differences were observed between lymphoblastoid cells and human diploid fibroblasts in the binding, release and degradation of radiolabelled poly(I).poly(C). The cells were able to take up radiolabelled poly(I).poly(C) for only a short time. Cell-associated radioactivity was immediately released into the supernatant fluid. Although the released material was still TCA-precipitable, partial or complete degradation could not be excluded. Pretreatment of the cells with DEAE-dextran enabled the cells to take up a much larger amount of radiolabelled poly(I).poly(C) and this material was not being released. However, this procedure did not lead to any detectable interferon production.     

Interaction of human peripheral blood mononuclear cells with Coccidioides immitis arthroconidia

We explored the in vitro interaction of human peripheral blood mononuclear cells with the arthroconidial stage of the fungus Coccidioides immitis. Fresh peripheral blood monocytes in an adherent monolayer were capable of ingesting C. immitis. Further, peripheral blood monocytes from either skin-test-positive or skin-test-negative donors significantly decreased the in vitro growth of C. immitis when coccidioidal arthroconidia were incubated with monocytes. Peripheral blood mononuclear cells also reduced fungal incorporation of the chitin precursor N-acetyl glucosamine. Cell fractions consisting predominantly of monocytes were significantly more active in this regard than fractions containing predominantly lymphocytes. Moreover, this activity was independent of the coccidioidal skin-test status of the donor. We conclude that human fresh peripheral blood mononuclear cells are able to phagocytize C. immitis arthroconidia and have the ability to inhibit its growth in vitro. That these abilities are independent of the immune status of the donor supports the possibility that the peripheral blood monocyte may contribute to the early defense against initial coccidioidal infection.     

Interaction of casein kinase II with ribosomal protein L22 of Drosophila melanogaster

The ubiquitous eukaryotic protein kinase CKII (casein kinase II) has been found to interact with a number of cellular proteins, either through the catalytic subunit or the regulatory subunit. Using the yeast two-hybrid screening method, we found that the catalytic subunit of Drosophila melanogaster CKII (DmCKII) interacts with Drosophila ribosomal protein L22 (rpL22). This interaction was also observed in vitro with a glutathione-S-transferase (GST)-rpL22 fusion protein. The predicted full-length Drosophila rpL22 protein has an N-terminal extension rich in alanine, lysine, and proline that appears to be unique to Drosophila. Deletion mapping revealed that the conserved core of rpL22 is responsible for the interaction with CKII. Moreover, purified DmCKII can phosphorylate a GST-L22 fusion protein at the C-terminal end, suggesting that this protein may be a substrate of CKII in Drosophila.     

Interaction between casein and the oppositely charged surfactant

The interactions between the classical cationic surfactant dodecyltrimethylammonium bromide (DTAB) and 2.0 mg/mL casein were investigated using isothermal titration calorimetry (ITC), turbidity, dynamic light scattering (DLS), and fluorescence spectra measurements. The results suggest that the cationic headgroup of the surfactant individually binds to the negatively charged amino acid sites on the casein chains because of the electrostatic attraction upon the addition of DTAB. When the surfactant concentration reaches a critical value c1, DTAB forms micelle-like aggregates on the casein chain, resulting in the formation of insoluble casein/DTAB complexes. Further addition of DTAB leads to the redissolution of casein/DTAB complexes because of the net positive charge on casein/DTAB complexes and the formation of DTAB free micelles. The addition of salt screens the repulsion between the surfactant headgroups and the attraction between casein and surfactant molecules, which weakens the binding of surfactant onto the casein chain, favoring the formation of free surfactant micelles.