Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Binding of leucocytes to endothelial cells (EC) is essential as an initial step in inflammatory responses. We present a rapid, non-radioactive method to measure adhesion of human lymphoid cells to EC using flow cytometry. Freshly isolated peripheral blood mononuclear cells (PBMC) were allowed to adhere to EC grown in 24-well plates. Non-adhering cells were removed, after which adhering cells and EC were dissociated using trypsin/EDTA. These samples were subsequently analysed by flow cytometry, using scatter properties to distinguish between adhering cells and EC. The ratio of the number of adhering leucocytes and EC was calculated to quantify adhesion. Results of the flow cytometric adhesion assay were comparable to those obtained with a conventional adhesion assay using chromium-labelled cells. We additionally show that by using the flow cytometric adhesion assay, adhesion of lymphocytes and monocytes present within the adhering PBMC can be quantified simultaneously. As a model, the contribution of LFA-1 (CD11a/CD18) and ICAM-1 (CD54) in adhesion of PBMC to EC was studied. It was found that adhesion of lymphocytes and monocytes is regulated differently by phorbol ester and that the relative contribution of LFA-1 and ICAM-1 differs for both cell types.
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PMID:Adhesion of subsets of human blood mononuclear cells to endothelial cells in vitro, as quantified by flow cytometry. 136 Oct 77

Neutrophils and mononuclear cells have been associated with the lower respiratory tract inflammation observed in both acute and chronic bronchitis. In order to transit into and remain within the airways, neutrophils and mononuclear cells would likely need to adhere to bronchial epithelium. To test this hypothesis, bovine bronchial epithelial cells (BBECs) were isolated and cultured on a round coverslip. After 7 to 10 days, 51Cr-labeled neutrophils and mononuclear cells were evaluated for their capacity to adhere to the BBEC monolayer. Both neutrophils and mononuclear cells readily bound to the BBEC monolayer (10.8 +/- 1.2% bound neutrophils; 40.5 +/- 2.8% bound mononuclear cells). Stimulation of the neutrophils and mononuclear cells with phorbol 12-myristate 13-acetate (PMA) increased the adherence (45.8 +/- 10.6% bound neutrophils, P less than 0.01 compared with unstimulated cells; 58.7 +/- 6.2% bound mononuclear cells, P less than 0.01 compared with unstimulated cells). Importantly, stimulating the BBEC monolayer with PMA, bacterial lipopolysaccharide, or a cigarette smoke extract for 4 to 72 h also increased the adherence of both cell types (P less than 0.01, all comparisons at 24 h). The adherence was not decreased by exposure of either the BBEC monolayer, the neutrophils, or the mononuclear cells to cycloheximide or to the anti-CD11/CD18 monoclonal antibody 60.3 (P greater than 0.05). However, exposure of the BBEC monolayer to trypsin before addition of the neutrophils significantly decreased adherence (P less than 0.05). Because neutrophils and mononuclear cells are thought to mediate cell cytotoxicity by adhering to the target cells, BBECs were labeled with 51Cr, and 51Cr release was measured as an index of cytotoxicity. There was a modest increase in 51Cr release by the addition of unstimulated neutrophils and mononuclear cells, and culturing the BBEC monolayer with PMA before the addition of the neutrophils or mononuclear cells resulted in a further modest enhancement of 51Cr release (P less than 0.05). Similar results were obtained using lactate dehydrogenase release as a measure of cytotoxicity. These results demonstrate that inflammatory cells can adhere to BBECs and may be capable of mediating cytotoxicity and adherence and cytotoxicity can be increased by stimulating BBECs.
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PMID:Modulation of neutrophil and mononuclear cell adherence to bronchial epithelial cells. 162 34

Adhesive interactions between CD34+ myeloid progenitors, cytomatrix components, and marrow fibroblast and stromal monolayers are described and compared to the binding interactions of the CD34+ myeloid leukemic cell lines KG1a and KG1. Both normal precursors and their leukemic counterparts showed adhesion to marrow stroma and fibroblasts. CD34+ myeloid progenitors bound to the extracellular matrices of marrow stromal cell and fibroblast monolayers and to laminin and fibronectin to a lesser extent than to cellular stromal layers. These adhesive interactions were not inhibited by polyclonal antibodies to laminin or fibronectin, nor by 1 mM Arg-Gly-Asp-Ser (RGDS)-containing peptides. Also, although both normal and leukemic cells expressed the CD18 antigen, binding of these cells to stroma was not inhibited by blocking anti-CD18 monoclonal antibodies. Finally, KG1a adhesion was not blocked in the presence of anti-CD54 (ICAM) antibody, nor was it blocked when galactosyl or mannosyl pyranosides were added. KG1a binding was trypsin sensitive and enhanced in the presence of neuraminidase. These studies serve to characterize adhesive properties of normal and leukemic myeloid progenitors and begin to establish interactions important for the lodgement of early progenitor cells in human marrow.
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PMID:Adhesive interactions of normal and leukemic human CD34+ myeloid progenitors: role of marrow stromal, fibroblast, and cytomatrix components. 170 95

The nature of the binding sites for lipopolysaccharide (LPS) on human monocytes was investigated using fluorescein isothiocyanate (FITC)-labelled LPS from Salmonella minnesota R595 (ReLPS). In the absence of serum, ReLPS bound to monocytes and this interaction was trypsin sensitive. A concentration of 0.1 mg/ml resulted in a 90% loss of LPS binding, while low concentrations increased this binding. Trypsin-treated monocytes recovered FITC-ReLPS binding after 20 hr culture, which was abrogated in the presence of cycloheximide and actinomycin D. This showed that de novo protein and mRNA synthesis were essential. A number of different proteins have been implicated in cellular binding of LPS to monocytes. In this paper we show that CD14 is not involved in direct binding of FITC-ReLPS to monocytes, since anti-CD14 monoclonal antibody (mAb) (3C10) and removal of most of cell-surface CD14 by phosphatidylinositol-specific phospholipase C did not prevent FITC-ReLPS binding. Furthermore, LPS also bound to CD14-deficient cells from a patient with paroxysmal nocturnal haemoglobinuria (PNH). FITC-ReLPS binding was not mediated by the CD11/CD18 complex since mAb to the alpha and beta chains of the CD11/CD18 complex did not alter the binding of FITC-ReLPS to cells. These observations indicate that ReLPS may interact with monocyte membrane protein(s) in the absence of serum. This binding site(s) for LPS might be different from those previously described by others.
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PMID:Serum-independent binding of lipopolysaccharide to human monocytes is trypsin sensitive and does not involve CD14. 750 49

Three different intercellular adhesion molecules (ICAMs) have been identified acting as ligand for counter-receptor leukocyte-function-associated antigen-1 (LFA-1) (CD11a/CD18). We have recently shown that ICAM-1 (CD54) is present on cultured human epidermal Langerhans cells but not on freshly isolated Langerhans cells, and that this molecule participates in the generation of an antigen-specific T-cell response. ICAM-2 (CD102) was not involved because this molecule is expressed by neither fresh nor cultured Langerhans cells. In this study, the presence of ICAM-3 (CD50) on Langerhans cells was examined. Flow cytofluorometric analysis demonstrated that ICAM-3 is strongly displayed by fresh Langerhans cells, and daily determinations showed that the level of this trypsin-resistant molecule remained nearly unchanged during in vitro culture for up to 4 d, indicating that Langerhans cells constitutively express this molecule. Analysis of RNA extracted from purified cultured Langerhans cells by means of reverse transcriptase-polymerase chain reaction demonstrated the presence of mRNA specific for ICAM-3. Antigen-specific T-cell responses triggered by Langerhans cells were dose-dependently inhibited by anti-ICAM-3 if the antibody was added within the first 16 h of T-cell stimulation. Simultaneous addition of anti-ICAM-1 and anti-ICAM-3 synergistically inhibited T-cell responses, although a total block was never achieved. Pretreatment of Langerhans cells with anti-ICAM-3 resulted in a reduced T-cell response, whereas pretreatment of T cells did not. These results suggest that ICAM-3 on Langerhans cells, like ICAM-1, is functionally involved in the initiation of antigen-specific activation of T cells, but the expression of these two ICAMs on Langerhans cells is differently regulated.
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PMID:Intercellular adhesion molecule-3 (CD50) on human epidermal Langerhans cells participates in T-cell activation. 753 71

Human fetal livers contain progenitor cells that become mast cells after 4 weeks of culture with recombinant human stem cell factor. Expression of cell surface CD29 (beta 1), CD18 (beta 2), CD61 (beta 3), and beta 5 integrins was investigated on such cells by flow cytometry and adhesion measurements. High surface expression of CD49e, CD51, and CD61 along with kit was apparent by 4 weeks of culture, whereas expression of each at day 0 was low to undetectable. CD29 and CD49d were detected on cells from day 0 to 4 weeks of culture; CD49b, CD49c, CD49f, CD18, and CD54 expression was negligible. The fetal liver-derived mast cells spontaneously adhered to vitronectin. No evidence for degranulation was found during vitronectin-dependent adhesion. Adhesion occurred in part through the CD61/CD51 receptor. No evidence for adhesion to vitronectin through CD29 and beta 5 integrins was obtained. Almost all of the vitronectin-adherent cells expressed CD51, CD61, kit, and tryptase, and exhibited metachromasia with toluidine blue. Thus, among the fetal liver-derived cells, developing mast cells were selectively adherent to vitronectin. These mast cells and the other cell types present also adhere spontaneously to fibronectin and to laminin, this adhesion being partially inhibited by antibodies against CD61 and CD29 integrins. In conclusion, human mast cells acquire functional vitronectin receptors as they develop from fetal liver progenitors under the influence of rhSCF. This may be important for the recruitment, localization, and retention of developing mast cells.
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PMID:Human mast cells derived from fetal liver cells cultured with stem cell factor express a functional CD51/CD61 (alpha v beta 3) integrin. 754 4

Complement-dependent activation of immune cells is regulated by cell surface membrane receptors. In this study, expression of complement receptors (CR) on human blood basophils (n = 11), tissue mast cells (lung, n = 7; skin, n = 10; uterus, n = 4; tonsil, n = 3; heart, n = 10), and on respective human cell lines (basophil line KU-812, mast cell line HMC-1) was analyzed by the use of mAbs and indirect immunofluorescence. Normal blood basophils and KU-812 cells were found to express C5aR (CD88), membrane cofactor protein (CD46), decay-accelerating factor (CD55), and membrane attack complex inhibitory factor (CD59), as well as the previously recognized CR1 (CD35), CR3 alpha (CD11b), CR4 alpha (CD11c), and CR3/4 beta (CD18). Mast cells from all organs as well as HMC-1 cells expressed CD46, CD55, and CD59, but not CD11b, CD21, or CD35. The C5aR (CD88) was detectable on skin mast cells, a subset (5 to 15%) of cardiac mast cells, and on HMC-1 cells, but not on lung, uterus, or tonsillar mast cells (< 5%). Moreover, double immunoperoxidase staining (tryptase vs C5aR/CD88) revealed in situ expression of C5aR on skin, but not lung mast cells. Recombinant human (rh) C5a, at 10(-10) to 10(-7) M, induced secretion of histamine from basophils (rhC5a, 10(-8) M: 53.4 +/- 3.1% vs control < 5%) and from skin mast cells (rhC5a, 10(-8) M: 25.8 +/- 16.1% vs control < 10% histamine release), but not from other mast cells (rhC5a or control: < 10%, p > 0.05). The rhC5a-induced secretion of histamine from basophils and skin mast cells was inhibited by S5/1, a blocking Ab against CD88 (basophils: 37.2% to 75.1%; skin mast cells: 39.2% to 83.9% inhibition, p < 0.05). Together, this study shows that a) basophils and mast cells express a different profile of complement receptors, b) C5a-dependent mediator release in skin mast cells and basophils is mediated via CD88, and c) mast cells constitute a heterogeneous lineage in terms of expression of the C5a binding site CD88.
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PMID:Differential expression of complement receptors on human basophils and mast cells. Evidence for mast cell heterogeneity and CD88/C5aR expression on skin mast cells. 767 28

The molecular basis for direct bacteria-macrophage interactions that distinguishes nontypeable (NT) Haemophilus influenzae from type b organisms is not known. Because of similarities between filamentous hemagglutinin (FHA) adhesin of Bordetella pertussis and high-molecular-weight (HMW) proteins commonly expressed by NT H. influenzae, the role that HMW proteins play in determining NT H. influenzae-macrophage interactions was assessed. In tests with genetically engineered organisms, HMW protein-expressing bacteria bound significantly better than isogenic HMW protein-deficient bacteria to macrophages. HMW protein-dependent binding to macrophages is trypsin-sensitive, is independent of divalent cations, does not occur via the leukocyte integrin CD11b/CD18, and is not affected by galactose-containing carbohydrates. Organisms bound via HMW proteins remain largely extracellular and viable. Like FHA of Bordetella organisms, HMW proteins mediate binding of NT H. influenzae to macrophages. However, unlike the interaction determined by FHA, this interaction is characteristically one of adhesion and requires additional serum opsonization for efficient killing of bacteria by macrophages.
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PMID:High-molecular-weight surface-exposed proteins of Haemophilus influenzae mediate binding to macrophages. 810 76

Two hamster pancreatic cancer cell lines, PC-1 and PC1.0, established from N-nitrosobis(2-oxopropyl)amine-induced pancreatic ductal/ductular adenocarcinomas exhibit different growth patterns. PC-1 cells, which produce well differentiated adenocarcinomas in vitro after allogeneic inoculation, form cell aggregates and characteristic island-like structures in vitro. PC1.0 cells, which produce poorly differentiated tumors in vivo, form dispersed colonies in vitro. Conditioned medium prepared from PC1.0 cells inhibits PC-1 cells from forming island-like colonies. The conditioned medium also prevents several human pancreatic carcinoma cell lines, HPAF, CD11 and CD18, from forming compact colonies. These properties are similar to those described previously as scatter factors. The scatter factor-like activity is heat-labile, acid-stable, non-dialyzable, trypsin sensitive and unaffected by reducing agents. The activity is not suppressed by addition of heparin, and it does not bind to heparin. In addition, the scatter phenomenon is not reproduced by acidic or basic fibroblast growth factor, epidermal growth factor or transforming growth factor-beta 1. Based on these findings, it appears that the scattering activity produced by PC1.0 cells differs from the scatter factors that have been identified in other systems.
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PMID:Production of scatter factor-like activity by a nitrosamine-induced pancreatic cancer cell line. 838 14

The primary objective of this study was to test the hypothesis that human neutrophil elastase (HNE) affects neutrophil infiltration (adhesion and emigration) into inflamed vessels. To determine whether HNE contributes to neutrophil adhesion in vivo, intravital microscopy was used to study neutrophil-endothelial cell interactions in single inflamed postcapillary venules. Superfusion of platelet-activating factor (PAF) (100 nmol/L) onto the mesentery caused an increase in neutrophil-neutrophil interactions, neutrophil adhesion to postcapillary venules, and cellular emigration out of the vasculature. Both L658 758 (an elastase-specific inhibitor), and Eglin C (an elastase and cathepsin G inhibitor) significantly attenuated all of these parameters in vivo. To further characterize the mechanism(s) involved, various in vitro parameters were assessed. HNE, but not trypsin, caused a dose-dependent (0.01 to 1.0 microgram/mL) increase in the expression of the beta subunit (CD18) of the CD11/CD18 adhesive glycoprotein complex on neutrophils. An HNE-dependent increase in CD11b expression was also observed; however, HNE did not affect the expression of other neutrophil adhesion molecules (L-selectin), superoxide production, or degranulation. PAF-enhanced CD18 expression on neutrophils and neutrophil migration were both abolished by L658 758 but PAF-induced neutrophil adhesion to endothelial monolayers was not affected by the antiproteinase. The in vitro data suggest that the antiproteinases do not directly prevent neutrophil adhesion in vivo but may be important in other CD18-dependent events such as neutrophil-neutrophil interaction or neutrophil infiltration (chemotaxis). These results translate into an important, rate-limiting role for elastase in the process of leukocyte infiltration and accumulation in inflamed microvessels.
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PMID:Effects of human neutrophil elastase (HNE) on neutrophil function in vitro and in inflamed microvessels. 840 Feb 69


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