49 results on '"S. Krüger-Krasagakes"'
Search Results
2. MAST CELLS
- Author
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S, Weber, S, Krüger-Krasagakes, J, Grabbe, T, Zuberbier, and B M, Czarnetzki
- Subjects
Animals ,Humans ,Receptors, Cell Surface ,Mast Cells ,Dermatology ,Immunoglobulin E ,Receptors, Immunologic ,Hematopoietic Stem Cells ,Skin Diseases ,Cell Division ,Mastocytosis - Published
- 1995
3. Expression of interleukin 10 in human melanoma
- Author
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Tibor Diamantstein, Christoph Hüls, S. Krüger-Krasagakes, Claus Garbe, Thomas Blankenstein, Konstantin Krasagakis, and Edgar Schmitt
- Subjects
Keratinocytes ,Cancer Research ,Pathology ,medicine.medical_specialty ,medicine.medical_treatment ,Gene Expression ,Biology ,Immune system ,Gene expression ,Tumor Cells, Cultured ,medicine ,Humans ,RNA, Messenger ,RNA, Neoplasm ,Neoplasm Metastasis ,Melanoma ,neoplasms ,Skin ,Messenger RNA ,Biological activity ,medicine.disease ,Reverse transcriptase ,Interleukin-10 ,Interleukin 10 ,Cytokine ,Oncology ,Cancer research ,Research Article - Abstract
The expression of interleukin 10 (IL-10) mRNA in human malignant melanoma was investigated by reverse transcriptase polymerase chain reaction analysis. Selective expression of IL-10 mRNA in tissues of primary melanomas and melanoma metastases was found in comparison with normal skin. In addition, strong expression of IL-10 mRNA and of biologically active IL-10 was detected in 3 out of 13 melanoma cell lines. Normal melanocytes consistently expressed low levels of IL-10 mRNA but did not produce detectable IL-10 protein, nor did keratinocytes or fibroblasts. The production of biologically active IL-10 by melanoma cell lines suggests that IL-10 mRNA in melanoma lesions may derive at least in part from the tumour cells themselves. Tumour-infiltrating cells, however, could also be a source of IL-10 in melanoma tissues. The presence of IL-10 in melanoma lesions may contribute to the postulated 'paralysis' of an anti-melanoma immune response. Images Figure 1 Figure 2
- Published
- 1994
4. Expression of tumor necrosis factor by different tumor cell lines results either in tumor suppression or augmented metastasis
- Author
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S. Krüger-Krasagakes, Ulrich Kunzendorf, Zhihai Qin, Tibor Diamantstein, Hanno Hock, and Thomas Blankenstein
- Subjects
Pathology ,medicine.medical_specialty ,Cancer Research ,CD30 ,medicine.medical_treatment ,Immunology ,Molecular Sequence Data ,Biology ,Transfection ,Metastasis ,Mice ,medicine ,Tumor Cells, Cultured ,Immunology and Allergy ,Animals ,Neoplasm Metastasis ,Base Sequence ,Tumor Necrosis Factor-alpha ,Lymphoblast ,Immunotherapy ,Articles ,Neoplasms, Experimental ,medicine.disease ,Cytokine ,Cell culture ,Mice, Inbred DBA ,Cancer research ,Tumor necrosis factor alpha ,Cell Adhesion Molecules - Abstract
Tumor necrosis factor (TNF) produced by tumor cells after gene transfer can effectively suppress the growth of locally growing tumors. We wanted to test the effects of "local" TNF on the growth of a highly metastatic cell line. Therefore, a recombinant retrovirus allowing expression of the TNF gene by the beta-actin promotor has been constructed and used to infect the two tumor cell lines EB and ESB, which grow as solid tumor or metastasize, respectively. Expression of TNF by EB cells resulted in their rapid and dose-dependent rejection. In sharp contrast, mice injected with ESB cells producing similar amounts of TNF showed no signs of tumor suppression, but rather had reduced survival rates that correlated with enhanced hepatic metastases. The accelerated formation of liver metastases by ESB TNF cells could be reversed by an anti-TNF mAb. These results demonstrate the opposite effects TNF may have on tumor growth: suppression of a locally growing tumor and promotion of metastasis formation.
- Published
- 1993
5. Supplement II: Abstracts of the international symposium on Skin Carcinogenesis in man and in experimental models. Heidelberg, 29–31 October 1991 (pp S61–S88)
- Author
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C. J. Kemp, G. Stingl, C. Caulín, E. G. Jung, H. Tanooka, J. Lassus, E. F. Griffin, Douglas R. Lowy, J. L. Jorcano, J. C. Wang, L. Weber, R. Kato, Paul Janiaud, S. Ohno, A. Schwaaf, R. Gollhausen, N. Sönnichsen, H. Hug, Toshio Kuroki, M. Yaar, J. R. Schlehofer, K. Krasagakis, PE Purkis, Monika M. Gross, H. Heine, H. Mukhtar, J. A. Newton, G. Reisbach, C. Bauer, A. Winter, K. M. Niemi, S. Yamamoto, Bernd L. Sorg, V. A. DeLeo, S. Bruvers, P. Navarro, A. Ootsuyama, G. Tadini, Bert J. Vermeer, D. English, A. B. Bianchi, S. Feil, A. Lehmus, H. Winter, P. T. Strickland, C. Proby, J. M. Foidart, R. Eckert, R. E. Albert, N. E. Fusenig, E. Lee, R. D. Granstein, P. Bums, E. Berti, J. Jürgensmeier, H. Roeser, J. Nährig, A. Anders, F. R. de Gruijl, C. S. Baxter, R. Mailhammer, H. van Weelden, Y. Fujiwara, E. Filvaroff, E. Weber, S. Froschermaier, G. Graf, J. C. Barrett, J. Weiss, H. Weber, B. Hennig, M. Miller, F. Urbach, K. Yamamura, E. Pâques, A. Hülsen, Seymour Garte, B. A. Gilchrest, S. Neill, K. Thalmeier, C. Zechel, Jan P. Vandenbroucke, B. Epe, P. Höfler, B. Przybilla, A. Markey, C. Gilles, C. Bauluz, I. B. Weinstein, U. Van der Piepen, Fokko J. Van Der Woude, T. Jimbo, A. Cano, P. Tomakidi, M. Quintanilla, A. Real, T. Grande, G. T. Bowden, H. Friesel, Y. Mishima, Jan N. Bouwes Bavinck, D. Breitkreutz, Stanley J. Miller, M. Piette, E. Wagner, M. Buček, A. Kopp-Schneider, C. A. Afshari, A. Ranki, M. Garmyn, Margaret L. Kripke, C. Baxter, E. Hecker, Hiroshi Tanooka, F. Harks, E. Lopez-Bran, P. A. Futreal, H. Wei, M. B. Abdel-Naser, A. Diugosz, S. Altmeier, J. Macejewski, Uwe Wollina, J. Römisch, B. Eberlein, E. B. Broecker, Y. Funasaka, M. Glover, M. Haas, S. Gruner, T. Bishop, J. Leers, G. Picht, A. Gilani, W. Diezel, D. S. Silvers, A. Glick, R. Krauß, H. Harris, Anne Østerlind, J. Levy, A. Cerri, E. Danen, K. Schiess, E. Viesel, H. Gröger, B. C. Bastian, K. Hayashibe, K. H. Richter, K. Frenkel, Odilia Popanda, M. Gómez, I. Moll, U. Schleenbecker, M. Ueda, Fritz Anders, H. D. Volk, K. Möller, M. Ichihashi, M. Martín, G. Krauter, S. Krüger-Krasagakes, D. J. Ruiter, J. C. van der Leun, M. Götschl, R. Niedner, Sylvia A. Sedman, T. M. Rünqer, Akira Ootsuyama, Judith P. Johnson, A. Montes, A. G. Ushmorov, G. Bauer, R. Schnapke, S. Kahn, B. Kempkes, C. Garbe, B. Steinbauer, B. K. Armstrong, P. Plein, T. Schneider, C. Missero, B. Schlatterer, M. Schara, P. J. Heenan, M. Stephan, B. A. Burkhart, A. J. P. Klein-Szanto, Eva-B. Bröcker, R. Halaban, S. Grabbe, G. N. P. van Muijen, E. Azizi, D. Schaefer, A. A. Hartmann, C. Ballestin, P. Klein-Bauernschmitt, R. Shukla, G. Kelfkens, M. Nelson, Friedrich Rippmann, M. Kaszkin, S. G. Zubova, Bruce D. Cohen, T. Cody, A. Kricker, V. B. Okulov, P. Fuchs, V. Kinzel, S. Osada, A. Balmain, A. B. Stoler, T. T. Sun, J. Svetek, W. D. Lehmann, F. Larcher, P. Krieg, Jürgen Schweizer, M. Hergenhahn, A. Faissner, G. P. Dotto, C. J. Conti, U. Burcin, L. Hültner, V. Bataille, G. Fürstenberger, EB Lane, A. Smith, D. Jahrens, K. Elgjo, Walter Troll, A. Gandarillas, M. Schön, R. D. Owen, S. Ramón y Cajal, Heinz Walter Thielmann, A. O. Danilov, S. H. Yuspa, J. Cuzick, P. L. Randell, Sylvia Unger, J. A. Boyd, C. Sutter, N. M. Navone, IM Leigh, H. J. Stark, L. A. Annab, R. Gitto, James M. Spencer, C. E. Orfanos, R. M. Lavker, W. Tilgen, R. Albert, H. L. Moses, Eric J. Stanbridge, R. Kosters, Rainer Schmidt, P. Boukamp, E. Schöpf, U. Pascheberg, Yuichi Hashimoto, A. Robledo, F. Marks, J. Sherman, J. Richards, C. E. Klein, Frans H.J. Claas, S. Pečar, Bernard M. Mechler, Doris Rueß, B. Fiebich, Lutz Edler, John T. Schiller, and H. Fujiki
- Subjects
Oncology ,Cancer Research ,medicine.medical_specialty ,business.industry ,Internal medicine ,Medicine ,General Medicine ,business ,Carcinogenesis ,medicine.disease_cause - Published
- 1991
6. Adhesion of human mast cells to extracellular matrix provides a co-stimulatory signal for cytokine production
- Author
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S, Krüger-Krasagakes, A, Grützkau, K, Krasagakis, S, Hoffmann, and B M, Henz
- Subjects
Extracellular Matrix Proteins ,Ionophores ,Interleukin-6 ,Reverse Transcriptase Polymerase Chain Reaction ,Interleukins ,Interleukin-8 ,Granulocyte-Macrophage Colony-Stimulating Factor ,Enzyme-Linked Immunosorbent Assay ,Cell Line ,Fibronectins ,Cell Adhesion ,Humans ,Tetradecanoylphorbol Acetate ,Original Article ,Mast Cells ,RNA, Messenger ,Vitronectin ,Calcimycin - Abstract
Engagement of integrin receptors during cell adhesion leads to changes in the morphology and the state of activation of cells. We therefore examined whether mast cell adhesion to extracellular matrix proteins affects the synthesis and release of various proinflammatory cytokines. Cells of the human mast cell line HMC-1 were added to fibronectin (FN)-, vitronectin (VN)- or, as a control, bovine serum albumin (BSA)-coated wells and were stimulated with phorbol 12-myristate 13-acetate (PMA) and/or calcium ionophore A23187 (ionophore). Cytokine production was evaluated using semiquantitative reverse transcription–polymerase chain reaction (RT–PCR) analysis of cell extracts and enzyme-linked immunosorbent assay (ELISA) analysis of cell supernatants. After a 4-hr incubation, mRNA expression of interleukin (IL)-8 (and weakly of IL-6) was up-regulated in matrix-adherent cells, with further increase in the presence of PMA and/or ionophore, compared with unstimulated cells. High-level de novo expression of IL-3 and of granulocyte–macrophage colony-stimulating factor (GM-CSF) was observed mainly in matrix-adherent cells. These changes were paralleled by the secretory pattern of HMC-1 cells after a 24-hr stimulation. Unstimulated cells adherent to FN or VN had already released small amounts of IL-8, and both VN- and FN-adherent cells produced, almost invariably, a higher level of cytokines than BSA-exposed cells after additional stimulation. These results show that mast cell adhesion to matrix proteins by itself has only selected and minor effects, but additional activation of mast cells by secretory stimuli causes significantly enhanced cytokine gene expression and secretion, suggesting that mast cells are far more active in their natural tissue environment than hitherto suggested from data in suspension cultures.
- Published
- 1999
7. Desensitization of melanoma cells to autocrine TGF-beta isoforms
- Author
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K, Krasagakis, S, Krüger-Krasagakes, S, Fimmel, J, Eberle, D, Thölke, M, von der Ohe, U, Mansmann, and C E, Orfanos
- Subjects
Gene Expression ,DNA ,DNA, Neoplasm ,Growth Inhibitors ,Recombinant Proteins ,Cell Transformation, Neoplastic ,Transforming Growth Factor beta ,Tumor Cells, Cultured ,Humans ,Melanocytes ,RNA, Messenger ,RNA, Neoplasm ,Neoplasm Metastasis ,Melanoma ,Cell Division ,Cells, Cultured - Abstract
Previous studies have suggested that transforming growth factor-beta 1 (TGF-beta1) acts as an autocrine growth inhibitor on normal human melanocytes, while melanoma cells may not respond to this stimulus. The role of other TGF-beta isoforms such as TGF-beta2 and TGF-beta3 remained less well characterized. In the present study, the mRNA and protein levels of all three isoforms of TGF-beta were analyzed in a panel of human melanoma cell lines and in cultures of normal human melanocytes in vitro. Northern analysis showed that the degree of TGF-beta1, -beta2, -beta3 mRNA expression varied considerably in melanoma cells, whereas TGF-beta expression was very low in melanocytes. In melanoma cells, secreted amounts of TGF-beta1 and TGF-beta3 were found increased in comparison to normal melanocytes: 615 pg/ml vs. 118 pg/ml and 193 pg/ml vs. 30 pg/ml (mean values). In addition, low levels of TGF-beta2 were detected (mean value: 28 pg/ml). Although TGF-beta secretion increased, the proliferation of melanoma cells was found to be only moderately inhibited by TGF-beta isoforms, in contrast to its strong antiproliferative effect on normal human melanocytes: - 15%, -11%, and -18% vs. -52%, -46%, and -50% average inhibition at 0.5 ng/ml TGF-beta1, -beta2, and -beta3, respectively. The different efficacy of TGF-beta on melanocyte and melanoma cells was highly significant (P0.0001); in addition, TGF-beta-dependent growth inhibition of melanoma cells from primary tumors vs. cells from metastases showed a trend for further decreased response for the metastatic populations (Por = 0.075). Measurements of DNA synthesis revealed even more pronounced differences between melanocytes (-86%, -78%, and -80% inhibition, respectively, for TGF-beta1, -beta2, and -beta3) and melanoma cells (no inhibition). Our data show loss of responsiveness of melanoma cells to the growth-inhibitory function of TGF-beta isoforms but not of melanocytes. Although melanoma cells are not growth-inhibited by all three TGF-beta isoforms, they secrete significantly higher levels of TGF-beta, as compared to melanocytes. The reduced response indicates their escape from TGF-beta surveillance with ongoing tumor progression.
- Published
- 1999
8. Comparative cytokine gene expression: regulation and release by human mast cells
- Author
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A, Möller, B M, Henz, A, Grützkau, U, Lippert, Y, Aragane, T, Schwarz, and S, Krüger-Krasagakes
- Subjects
Gene Expression Regulation ,Interleukin-6 ,Interleukin-8 ,Tumor Cells, Cultured ,Cytokines ,Humans ,Tetradecanoylphorbol Acetate ,Mast Cells ,RNA, Messenger ,Polymerase Chain Reaction ,Calcimycin ,Research Article - Abstract
Since data on the ability of human mast cells to produce various cytokines are scanty, we examined the mRNA expression, its modulation and the resulting protein expression of a number of well-characterized cytokines, using semi-quantitative reverse transcription-polymerase chain reaction of cell extracts and enzyme-linked immunosorbent assays for analysis of cell supernatants. One million cells/ml of the human mast cell line HMC-1 were stimulated with 25 ng/ml phorbol myristate acetate (PMA), 5 x 10(-7) M calcium ionophore A 23187 (ionophore) or both stimuli combined for various time periods. Constitutive expression in unstimulated cells was found for interleukin-1 beta (IL-1 beta) -3, -4, -8, tumour necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta). Maximal mRNA up-regulation was observed by 2-4 hr, with a second peak for TNF-alpha at 24 hr. After a 4-hr stimulation, IL-13 expression was detectable as well, whereas for IL-12, only the p35 but not the p40 chain was found, and IL-2, -5, -7 and interferon-gamma (IFN-gamma) were not expressed at all. Large quantities of IL-8, TNF-alpha, granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-3 were secreted time-dependently over a 72-hr period, with lower levels of IL-1 beta, -6, -10 and TGF-beta and no detectable IL-2, -4 and IFN-gamma protein. When IL-6 and IL-8 expression was compared in more detail, IL-6 mRNA was found to be up-regulated only with ionophore but not PMA, whereas both stimuli alone or combined increased IL-8 mRNA expression. Preincubation with cycloheximide inhibited IL-6 but not IL-8 transcription, and incubation of stimulated cells with actinomycin D stabilized IL-8 and also IL-6 mRNA. These data suggest a selective regulation of distinct cytokines in human mast cells at the transcriptional and post-transcriptional levels. Furthermore, the spectrum of cytokines produced by HMC-1 cells supports the well-recognized role of mast cells in immediate-type hypersensitivity reactions as well as their potential colony-stimulating and tissue-remodelling abilities.
- Published
- 1998
9. C3a and C5a stimulate chemotaxis of human mast cells
- Author
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K, Hartmann, B M, Henz, S, Krüger-Krasagakes, J, Köhl, R, Burger, S, Guhl, I, Haase, U, Lippert, and T, Zuberbier
- Subjects
Dose-Response Relationship, Drug ,Chemotaxis ,Complement C5a ,Fetal Blood ,Cell Line ,Pertussis Toxin ,Cell Adhesion ,Complement C3a ,Cytokines ,Humans ,Calcium ,Laminin ,Mast Cells ,Virulence Factors, Bordetella ,Complement Activation ,Signal Transduction - Abstract
The factors that control migration of mast cells to sites of inflammation and tissue repair remain largely undefined. Whereas several recent studies have described chemotactic factors that induce migration of murine mast cells, only stem cell factor (SCF) is known to induce migration of human mast cells. We report here that the anaphylatoxins C3a and C5a are chemotactic factors for the human mast cell line HMC-1, human cord blood-derived mast cells (CBMC) and cutaneous mast cells in vitro. The presence of an extracellular matrix protein, laminin, was required for chemotaxis in response to complement peptides. Migration of mast cells towards C3a and C5a was dose-dependent, peaking at 1 microg/mL (100 nmol/L), and was inhibited by specific antibodies. Pretreatment with pertussis toxin inhibited the anaphylatoxin-mediated migration of HMC-1 cells, indicating that Gi proteins are involved in complement-activated signal transduction pathways in human mast cells. Both C3a and C5a also induced a rapid and transient mobilization of intracellular free calcium ([Ca2+]i) in HMC-1 cells. Besides SCF, other chemotactic factors tested, such as interleukin-3, nerve growth factor, transforming growth factor beta, RANTES (regulated upon activation, normal Tcell expressed and secreted), monocyte chemotactic protein-1 (MCP-1), MCP-2, MCP-3, macrophage inflammatory protein-1alpha (MIP-1alpha), and MIP-1beta, failed to stimulate migration of human mast cells. In summary, these findings indicate that C3a and C5a serve as chemotaxins for human mast cells. Anaphylatoxin-mediated recruitment of mast cells might play an important role in hypersensitivity and inflammatory processes.
- Published
- 1997
10. Production of cytokines by human melanoma cells and melanocytes
- Author
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S, Krüger-Krasagakes, K, Krasagakis, C, Garbe, and T, Diamantstein
- Subjects
Gene Expression Regulation, Neoplastic ,Skin Neoplasms ,Immune Tolerance ,Tumor Cells, Cultured ,Cytokines ,Humans ,Melanocytes ,RNA, Messenger ,RNA, Neoplasm ,Melanoma ,Polymerase Chain Reaction ,Cells, Cultured ,Neoplasm Proteins - Abstract
Experimental animal models have shown that various cytokines, depending of their specific properties, may support growth and metastasis of tumor cells or even lead to tumor rejection. The analysis of expression of cytokine genes by melanoma cell lines indicated that melanoma cells constitutively produce both autostimulatory and inhibitory cytokines. Using reverse transcriptase polymerase chain reaction analysis, simultaneous expression of several cytokines, including interleukin-1 beta (IL-1 beta), IL-6, IL-8, tumor necrosis factor-alpha, and granulocyte-macrophage colony-stimulating factor, by melanoma cells was found. The same cytokine transcripts were detected in melanocytes, suggesting that cells of the melanocytic lineage express a specific pattern of cytokines in vitro. All these cytokines are known to be able to stimulate effector cells of the host. Additionally, production of mRNA for IL-10, a cytokine with potential immunosuppressive properties, was detected in melanoma cells and melanocytes. These and other cytokines are likely to be involved in the immune response to cancer and at this time it is unknown what the net effects of multiple cytokines are on the outcome of the host response to tumor.
- Published
- 1995
11. Production of Cytokines by Human Melanoma Cells and Melanocytes
- Author
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S. Krüger-Krasagakes, Claus Garbe, T. Diamantstein, and Konstantin Krasagakis
- Subjects
Cytokine ,Immune system ,Effector ,Melanoma ,medicine.medical_treatment ,medicine ,Cancer research ,Tumor necrosis factor alpha ,Biology ,medicine.disease ,In vitro ,Reverse transcriptase ,Metastasis - Abstract
Experimental animal models have shown that various cytokines, depending on their specific properties, may support growth and metastasis of tumor cells or even lead to tumor rejection. The analysis of expression of cytokine genes by melanoma cell lines indicated that melanoma cells constitutively produce both autostimulatory and inhibitory cytokines. Using reverse transcriptase polymerase chain reaction analysis, simultaneous expression of several cytokines, including interleukin-1 beta (IL-1β), IL-6, IL-8, tumor necrosis factor-α, and granulocyte-macrophage colony-stimulating factor, by melanoma cells was found. The same cytokine transcripts were detected in melanocytes, suggesting that cells of the melanocytic lineage express a specific pattern of cytokines in vitro. All these cytokines are known to be able to stimulate effector cells of the host. Additionally, production of mRNA for IL-10, a cytokine with potential immunosuppressive properties, was detected in melanoma cells and melanocytes. These and other cytokines are likely to be involved in the immune response to cancer and at this time it is unknown what the net effects of multiple cytokines are on the outcome of the host response to tumor.
- Published
- 1995
12. Tumor-cell-targeted cytokine gene transfer in experimental models for cancer therapy
- Author
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H, Hock, M, Dorsch, G, Richter, U, Kunzendorf, S, Krüger-Krasagakes, T, Blankenstein, Z, Qin, and T, Diamantstein
- Subjects
Mice ,Macrophages ,Gene Transfer Techniques ,Leukocytes ,Animals ,Cytokines ,Genetic Therapy ,Neoplasms, Experimental ,Lymphocyte Subsets - Abstract
The genetic manipulation of tumor cells to express immunostimulatory molecules provides a current approach for the analysis of immune reactions against tumor cells in vivo. Experiments with multiple cytokines have demonstrated that an array of different host effector cells can be recruited by different cytokines in vivo, but more studies are necessary to distinguish cytokine-specific effects from as yet uncharacterized influences of different tumor models. A technically feasible clinical application of this approach is to be seen in the generation of vaccines by introducing such immunostimulatory genes into cancer cells and boosting systemic immune reactions against the unmodified cells. The experimental basis of these vaccination studies is critically discussed.
- Published
- 1994
13. A sialyl-Le(x)-negative melanoma cell line binds to E-selectin but not to P-selectin
- Author
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U, Kunzendorf, S, Krüger-Krasagakes, M, Notter, H, Hock, G, Walz, and T, Diamantstein
- Subjects
Base Sequence ,Molecular Sequence Data ,Lewis X Antigen ,Platelet Membrane Glycoproteins ,Flow Cytometry ,Fucosyltransferases ,Polymerase Chain Reaction ,P-Selectin ,Cell Adhesion ,Tumor Cells, Cultured ,Humans ,RNA, Messenger ,E-Selectin ,Cell Adhesion Molecules ,Melanoma - Abstract
The adhesion molecules E-selectin (ELAM-1) and P-selectin (GMP-140/CD62) recognize the carbohydrate motives sialyl-Le(x), sialyl-diLe(x), or sialyl-Lea, though with different affinity. We found that the melanoma cell line NKI-4 bound to E-selectin, but not to P-selectin. This melanoma cell line did not express sialyl-Le(x), but was positive for sialyl-diLe(x) and sialyl-Le(a). In contrast, 2 other melanoma cell lines, MeWo and SK-MEL-28, expressing either sialyl-diLe(x) or sialyl-Le(a) on the cell surface, bound neither E-selectin nor P-selectin. Transfection of the fucosyltransferases Fuc-TIII, Fuc-TIV, and Fuc-TV mediates cell surface expression of sialyl-Le(x) in many cell lines. We detected transcripts of the fucosyltransferases Fuc-TIII and Fuc-TV in 4 melanoma cell lines despite the absence of cell surface sialyl-Le(x). Our observations indicate that expression of fucosyltransferases (Fuc-TIII and -TV) and generation of cell-surface sialyl-diLe(x) are not sufficient to permit adherence to E-selectin or P-selectin. Furthermore, it seems possible that a yet undefined ligand different from sialyl-Le(x), sialyl-diLe(x), or sialyl-Le(a) enables melanoma cells to adhere to E-selectin.
- Published
- 1994
14. Interleukin 10 transfected into Chinese hamster ovary cells prevents tumor growth and macrophage infiltration
- Author
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G, Richter, S, Krüger-Krasagakes, G, Hein, C, Hüls, E, Schmitt, T, Diamantstein, and T, Blankenstein
- Subjects
Mice ,Mice, Inbred BALB C ,Base Sequence ,Cricetinae ,Macrophages ,Molecular Sequence Data ,Animals ,Female ,CHO Cells ,Genetic Therapy ,Neoplasms, Experimental ,Transfection ,Interleukin-10 - Abstract
Expression of cytokines in tumor cells provides a sensitive modality to analyze the consequences of local cytokines in vivo on tumor infiltrating cells and tumorigenicity. We have transfected Chinese hamster ovary (CHO) cells with an interleukin 10 (IL-10) expression vector. CHO-IL10 cells although unaltered with respect to their in vitro growth lost tumorigenicity, both in nude and in SCID mice and in an IL-10 dose dependent manner. In addition, CHO-IL10 cells suppressed the growth of equal numbers of coinjected but not of contralaterally injected CHO cells. Immunohistology with anti-CR3/Mac-1 and anti-Mac-3 monoclonal antibodies revealed that CHO tumors were substantially infiltrated by macrophages. However, in CHO-IL10 tumors macrophages were virtually absent within the tumor tissue. Our results suggest that IL-10 indirectly suppresses tumor growth of certain tumors by inhibiting infiltration of macrophages which may provide tumor growth promoting activity.
- Published
- 1993
15. Eosinophils infiltrating interleukin-5 gene-transfected tumors do not suppress tumor growth
- Author
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Günther Richter, Weiqun Li, Thomas Blankenstein, Tibor Diamantstein, and S. Krüger-Krasagakes
- Subjects
Adoptive cell transfer ,medicine.medical_treatment ,Immunology ,Molecular Sequence Data ,Biology ,medicine.disease_cause ,Transfection ,Polymerase Chain Reaction ,Mice ,Immune system ,medicine ,Immunology and Allergy ,Animals ,Interleukin 5 ,Immunity, Cellular ,Mice, Inbred BALB C ,Base Sequence ,Neoplasms, Experimental ,Eosinophil ,medicine.disease ,Eosinophils ,medicine.anatomical_structure ,Cytokine ,Oligodeoxyribonucleotides ,Cancer research ,Plasmacytoma ,Female ,Interleukin-5 ,Carcinogenesis - Abstract
Tumor-associated eosinophils have been observed in human tumors and in experimental tumor models, but their function is poorly understood. To study the role of eosinophils during tumor growth, the plasmacytoma J558L and the mammary adenocarcinoma TS/A were transfected with an expression vector encoding the murine gene for interleukin-5 (IL-5), a cytokine inducing proliferation and activation of eosinophils. Injection of parental cells, mock-transfectants and IL-5-producing cells into syngeneic mice showed that local IL-5 secretion induced rapid tumor infiltration by eosinophils, as evidenced by immunohistochemical staining, but nevertheless did not alter the tumor growth kinetics of IL-5 transfectants. Therefore, the mere presence of IL-5 and eosinophils was not sufficient to induce a protective host immune response.
- Published
- 1993
16. 46 Cytokine network regulating melanoma cell growth in vitro
- Author
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Ch.C. Zouboulis, C. E. Orfanos, Konstantin Krasagakis, and S. Krüger-Krasagakes
- Subjects
Cancer Research ,Oncology ,Cell growth ,Cytokine Network ,Melanoma ,medicine ,Cancer research ,Dermatology ,Biology ,medicine.disease ,In vitro - Published
- 1997
17. Production of cytokines by melanoma cells
- Author
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C. Garbe, T. Diamanstein, Konstantin Krasagakis, and S. Krüger-Krasagakes
- Subjects
Cancer Research ,Oncology ,Melanoma ,medicine ,Cancer research ,Production (economics) ,Dermatology ,Biology ,medicine.disease - Published
- 1993
18. Human mast cells and extracellular matrix: immunohistochemical studies of normal skin and inflammatory dermatoses.
- Author
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Krüger-Krasagakes S, Kelm I, and Henz BM
- Subjects
- Humans, Extracellular Matrix metabolism, Extracellular Matrix Proteins metabolism, Mast Cells metabolism, Skin metabolism, Skin Diseases metabolism
- Published
- 2004
- Full Text
- View/download PDF
19. Adhesion of human mast cells to extracellular matrix provides a co-stimulatory signal for cytokine production.
- Author
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Krüger-Krasagakes S, Grützkau A, Krasagakis K, Hoffmann S, and Henz BM
- Subjects
- Calcimycin pharmacology, Cell Adhesion, Cell Line, Enzyme-Linked Immunosorbent Assay, Fibronectins metabolism, Granulocyte-Macrophage Colony-Stimulating Factor analysis, Granulocyte-Macrophage Colony-Stimulating Factor genetics, Humans, Interleukin-6 analysis, Interleukin-6 genetics, Interleukin-8 analysis, Interleukin-8 genetics, Ionophores pharmacology, RNA, Messenger analysis, Reverse Transcriptase Polymerase Chain Reaction, Tetradecanoylphorbol Acetate pharmacology, Vitronectin metabolism, Extracellular Matrix Proteins metabolism, Interleukins metabolism, Mast Cells metabolism
- Abstract
Engagement of integrin receptors during cell adhesion leads to changes in the morphology and the state of activation of cells. We therefore examined whether mast cell adhesion to extracellular matrix proteins affects the synthesis and release of various proinflammatory cytokines. Cells of the human mast cell line HMC-1 were added to fibronectin (FN)-, vitronectin (VN)- or, as a control, bovine serum albumin (BSA)-coated wells and were stimulated with phorbol 12-myristate 13-acetate (PMA) and/or calcium ionophore A23187 (ionophore). Cytokine production was evaluated using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis of cell extracts and enzyme-linked immunosorbent assay (ELISA) analysis of cell supernatants. After a 4-hr incubation, mRNA expression of interleukin (IL)-8 (and weakly of IL-6) was up-regulated in matrix-adherent cells, with further increase in the presence of PMA and/or ionophore, compared with unstimulated cells. High-level de novo expression of IL-3 and of granulocyte-macrophage colony-stimulating factor (GM-CSF) was observed mainly in matrix-adherent cells. These changes were paralleled by the secretory pattern of HMC-1 cells after a 24-hr stimulation. Unstimulated cells adherent to FN or VN had already released small amounts of IL-8, and both VN- and FN-adherent cells produced, almost invariably, a higher level of cytokines than BSA-exposed cells after additional stimulation. These results show that mast cell adhesion to matrix proteins by itself has only selected and minor effects, but additional activation of mast cells by secretory stimuli causes significantly enhanced cytokine gene expression and secretion, suggesting that mast cells are far more active in their natural tissue environment than hitherto suggested from data in suspension cultures.
- Published
- 1999
- Full Text
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20. Desensitization of melanoma cells to autocrine TGF-beta isoforms.
- Author
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Krasagakis K, Krüger-Krasagakes S, Fimmel S, Eberle J, Thölke D, von der Ohe M, Mansmann U, and Orfanos CE
- Subjects
- Cell Division drug effects, Cell Transformation, Neoplastic, Cells, Cultured, DNA biosynthesis, DNA, Neoplasm biosynthesis, Gene Expression, Growth Inhibitors pharmacology, Humans, Melanocytes cytology, Melanocytes drug effects, Melanocytes metabolism, Melanoma pathology, Neoplasm Metastasis, RNA, Messenger genetics, RNA, Messenger metabolism, RNA, Neoplasm genetics, RNA, Neoplasm metabolism, Recombinant Proteins pharmacology, Transforming Growth Factor beta pharmacology, Tumor Cells, Cultured, Melanoma genetics, Melanoma metabolism, Transforming Growth Factor beta genetics, Transforming Growth Factor beta metabolism
- Abstract
Previous studies have suggested that transforming growth factor-beta 1 (TGF-beta1) acts as an autocrine growth inhibitor on normal human melanocytes, while melanoma cells may not respond to this stimulus. The role of other TGF-beta isoforms such as TGF-beta2 and TGF-beta3 remained less well characterized. In the present study, the mRNA and protein levels of all three isoforms of TGF-beta were analyzed in a panel of human melanoma cell lines and in cultures of normal human melanocytes in vitro. Northern analysis showed that the degree of TGF-beta1, -beta2, -beta3 mRNA expression varied considerably in melanoma cells, whereas TGF-beta expression was very low in melanocytes. In melanoma cells, secreted amounts of TGF-beta1 and TGF-beta3 were found increased in comparison to normal melanocytes: 615 pg/ml vs. 118 pg/ml and 193 pg/ml vs. 30 pg/ml (mean values). In addition, low levels of TGF-beta2 were detected (mean value: 28 pg/ml). Although TGF-beta secretion increased, the proliferation of melanoma cells was found to be only moderately inhibited by TGF-beta isoforms, in contrast to its strong antiproliferative effect on normal human melanocytes: - 15%, -11%, and -18% vs. -52%, -46%, and -50% average inhibition at 0.5 ng/ml TGF-beta1, -beta2, and -beta3, respectively. The different efficacy of TGF-beta on melanocyte and melanoma cells was highly significant (P<0.0001); in addition, TGF-beta-dependent growth inhibition of melanoma cells from primary tumors vs. cells from metastases showed a trend for further decreased response for the metastatic populations (P< or = 0.075). Measurements of DNA synthesis revealed even more pronounced differences between melanocytes (-86%, -78%, and -80% inhibition, respectively, for TGF-beta1, -beta2, and -beta3) and melanoma cells (no inhibition). Our data show loss of responsiveness of melanoma cells to the growth-inhibitory function of TGF-beta isoforms but not of melanocytes. Although melanoma cells are not growth-inhibited by all three TGF-beta isoforms, they secrete significantly higher levels of TGF-beta, as compared to melanocytes. The reduced response indicates their escape from TGF-beta surveillance with ongoing tumor progression.
- Published
- 1999
- Full Text
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21. Expression and functional activity of the IL-8 receptor type CXCR1 and CXCR2 on human mast cells.
- Author
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Lippert U, Artuc M, Grützkau A, Möller A, Kenderessy-Szabo A, Schadendorf D, Norgauer J, Hartmann K, Schweitzer-Stenner R, Zuberbier T, Henz BM, and Krüger-Krasagakes S
- Subjects
- Actins metabolism, Antigens, CD genetics, Antigens, CD physiology, Antigens, CD ultrastructure, Binding, Competitive, Calcium metabolism, Cell Movement drug effects, Chemokine CXCL1, Chemotactic Factors metabolism, Chemotactic Factors pharmacology, Flow Cytometry, Growth Substances metabolism, Growth Substances pharmacology, HL-60 Cells, Humans, Interleukin-8 pharmacology, Iodine Radioisotopes, Mast Cells physiology, Mast Cells ultrastructure, Peptides pharmacology, Polymerase Chain Reaction, Protein Binding, RNA, Messenger biosynthesis, Receptors, Chemokine genetics, Receptors, Chemokine physiology, Receptors, Interleukin genetics, Receptors, Interleukin physiology, Receptors, Interleukin ultrastructure, Receptors, Interleukin-8A, Receptors, Interleukin-8B, Skin metabolism, Skin ultrastructure, Tumor Cells, Cultured, beta-Thromboglobulin, Antigens, CD biosynthesis, Chemokines, CXC, Intercellular Signaling Peptides and Proteins, Interleukin-8 metabolism, Mast Cells metabolism, Receptors, Chemokine biosynthesis, Receptors, Interleukin biosynthesis
- Abstract
To further elucidate mechanisms involved in mast cell accumulation at sites of cutaneous inflammation, we have studied the ability of human leukemic mast cells (HMC-1 cells) to express functionally active IL-8 receptors. Expression of mRNA for both types of IL-8 receptors (CXCR1 and CXCR2) was demonstrated by PCR and of both proteins by flow cytometry. Binding and competition studies with 125I-labeled IL-8 and its homologue melanoma growth stimulating activity (125I-labeled MGSA) revealed two specific binding sites for IL-8, K1 = 1.1 x 10(11) M(-1) and K2 = 5 x 10(7) M(-1); and for MGSA, K1 = 2.8 x 10(10) M(-1) and K2 = 5 x 10(7) M(-1). This finding was supported by a dose-dependent rise of cytosolic free calcium concentration ([Ca2+]i) induced by both chemokines and to a lesser extent by the homologue neutrophil-activating peptide-2 (NAP-2). A significant migratory response of human leukemic mast cells (HMC-1) was observed with all three chemokines at a range from 10(-8) M to 10(-9) M. Moreover, the formation of cellular F-actin was induced in a rapid, dose-dependent fashion, with a maximally 1.7-fold increase at 10(-7) M. Using postembedding immunoelectron microscopy, we could show the expression of CXCRI on the cytoplasmatic membrane of isolated human skin mast cells whereas CXCR2 was located in mast cell-specific granules. These findings demonstrate for the first time the functional expression of both types of IL-8 receptors on human mast cells, suggesting a role for their ligands during mast cell activation and recruitment.
- Published
- 1998
22. Synthesis, storage, and release of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) by human mast cells: implications for the biological significance of VEGF206.
- Author
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Grützkau A, Krüger-Krasagakes S, Baumeister H, Schwarz C, Kögel H, Welker P, Lippert U, Henz BM, and Möller A
- Subjects
- Blotting, Western, Calcimycin pharmacology, Culture Media, Conditioned, Endothelial Growth Factors analysis, Enzyme-Linked Immunosorbent Assay, Flow Cytometry, Humans, Leukemia pathology, Lymphokines analysis, Lymphokines drug effects, Mast Cells drug effects, Mast Cells ultrastructure, Microscopy, Immunoelectron, Polymerase Chain Reaction methods, Skin cytology, Tetradecanoylphorbol Acetate pharmacology, Tumor Cells, Cultured, Vascular Endothelial Growth Factor A, Vascular Endothelial Growth Factors, Endothelial Growth Factors genetics, Endothelial Growth Factors metabolism, Lymphokines genetics, Lymphokines metabolism, Mast Cells metabolism
- Abstract
Mast cells have been implicated in various diseases that are accompanied by neovascularization. The exact mechanisms by which mast cells might mediate an angiogenic response, however, are unclear and therefore, we have investigated the possible expression of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) in the human mast cell line HMC-1 and in human skin mast cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that mast cells constitutively express VEGF121, VEGF165, and VEGF189. After a prolonged stimulation of cells for 24 h with phorbol 12-myristate 13-acetate (PMA) and the ionophore A23187, an additional transcript representing VEGF206 was detectable, as could be verified by sequence analysis. These results were confirmed at the protein level by Western blot analysis. When the amounts of VEGF released under unstimulated and stimulated conditions were compared, a significant increase was detectable after stimulation of cells. Human microvascular endothelial cells (HMVEC) responded to the supernatant of unstimulated HMC-1 cells with a dose-dependent mitogenic effect, neutralizable up to 90% in the presence of a VEGF-specific monoclonal antibody. Flow cytometry and postembedding immunoelectron microscopy were used to detect VEGF in its cell-associated form. VEGF was exclusively detectable in the secretory granules of isolated human skin mast cells. These results show that both normal and leukemic human mast cells constitutively express bioactive VEGF. Furthermore, this study contributes to the understanding of the physiological role of the strongly heparin-binding VEGF isoforms, since these were found for the first time to be expressed in an activation-dependent manner in HMC-1 cells.
- Published
- 1998
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23. Comparative cytokine gene expression: regulation and release by human mast cells.
- Author
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Möller A, Henz BM, Grützkau A, Lippert U, Aragane Y, Schwarz T, and Krüger-Krasagakes S
- Subjects
- Calcimycin immunology, Cytokines genetics, Humans, Interleukin-6 genetics, Interleukin-6 metabolism, Interleukin-8 genetics, Interleukin-8 metabolism, Polymerase Chain Reaction, RNA, Messenger genetics, Tetradecanoylphorbol Acetate immunology, Tumor Cells, Cultured immunology, Cytokines metabolism, Gene Expression Regulation immunology, Mast Cells immunology
- Abstract
Since data on the ability of human mast cells to produce various cytokines are scanty, we examined the mRNA expression, its modulation and the resulting protein expression of a number of well-characterized cytokines, using semi-quantitative reverse transcription-polymerase chain reaction of cell extracts and enzyme-linked immunosorbent assays for analysis of cell supernatants. One million cells/ml of the human mast cell line HMC-1 were stimulated with 25 ng/ml phorbol myristate acetate (PMA), 5 x 10(-7) M calcium ionophore A 23187 (ionophore) or both stimuli combined for various time periods. Constitutive expression in unstimulated cells was found for interleukin-1 beta (IL-1 beta) -3, -4, -8, tumour necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta). Maximal mRNA up-regulation was observed by 2-4 hr, with a second peak for TNF-alpha at 24 hr. After a 4-hr stimulation, IL-13 expression was detectable as well, whereas for IL-12, only the p35 but not the p40 chain was found, and IL-2, -5, -7 and interferon-gamma (IFN-gamma) were not expressed at all. Large quantities of IL-8, TNF-alpha, granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-3 were secreted time-dependently over a 72-hr period, with lower levels of IL-1 beta, -6, -10 and TGF-beta and no detectable IL-2, -4 and IFN-gamma protein. When IL-6 and IL-8 expression was compared in more detail, IL-6 mRNA was found to be up-regulated only with ionophore but not PMA, whereas both stimuli alone or combined increased IL-8 mRNA expression. Preincubation with cycloheximide inhibited IL-6 but not IL-8 transcription, and incubation of stimulated cells with actinomycin D stabilized IL-8 and also IL-6 mRNA. These data suggest a selective regulation of distinct cytokines in human mast cells at the transcriptional and post-transcriptional levels. Furthermore, the spectrum of cytokines produced by HMC-1 cells supports the well-recognized role of mast cells in immediate-type hypersensitivity reactions as well as their potential colony-stimulating and tissue-remodelling abilities.
- Published
- 1998
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24. Detection of intracellular interleukin-8 in human mast cells: flow cytometry as a guide for immunoelectron microscopy.
- Author
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Grützkau A, Krüger-Krasagakes S, Kögel H, Möller A, Lippert U, and Henz BM
- Subjects
- Antibodies, Monoclonal, Cell Compartmentation, Cell Membrane Permeability, Cytoplasmic Granules chemistry, Cytoplasmic Granules ultrastructure, Fluorescent Antibody Technique, Humans, Interleukin-8 immunology, Mast Cells ultrastructure, Tissue Fixation, Flow Cytometry methods, Interleukin-8 isolation & purification, Mast Cells chemistry, Microscopy, Immunoelectron methods
- Abstract
The chemokine interleukin-8 (IL-8) mediates infiltration and adhesion of neutrophils during inflammatory processes. We have previously shown that this cytokine can be produced and released by normal and leukemic human mast cells (HMC-1 cells). To assess whether and to what extent this cytokine is stored intracellularly, we investigated production and localization of IL-8 at the single-cell level by combined use of flow cytometry (FACS) and immunoelectron microscopy. Conditions necessary for optimal fixation and permeabilization of HMC-1 cells were determined by measuring changes in cell-specific light scatter parameters and by estimating cellular uptake of propidiumiodide (PI). In this way, we were able to detect IL-8 with a monoclonal antibody in stimulated cells that were microwave-fixed with a combination of paraformaldehyde (4%) and glutaraldehyde (0.1%), followed by permeabilization with saponin (0.025%). FACS analysis revealed time-dependent synthesis of IL-8 with at most 50% positively stained cells at 8-12 hr after stimulation. For pre-embedding immunogold electron microscopy, cells were treated according to the protocol established by flow cytometry. IL-8 was found to be located in specific cytoplasmic, electron-dense granules of stimulated HMC-1 cells. These results confirm and extend our previous findings by demonstrating IL-8 expression in HMC-1 cells at the single-cell level. In addition, we propose that quantitative FACS can be reliably used in a timesaving manner to establish appropriate conditions for pre-embedding immunoelectron microscopy of intracellular antigens.
- Published
- 1997
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25. C3a and C5a stimulate chemotaxis of human mast cells.
- Author
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Hartmann K, Henz BM, Krüger-Krasagakes S, Köhl J, Burger R, Guhl S, Haase I, Lippert U, and Zuberbier T
- Subjects
- Calcium metabolism, Cell Adhesion, Cell Line, Complement Activation, Cytokines pharmacology, Dose-Response Relationship, Drug, Fetal Blood cytology, Humans, Laminin physiology, Pertussis Toxin, Signal Transduction, Virulence Factors, Bordetella pharmacology, Chemotaxis drug effects, Complement C3a pharmacology, Complement C5a pharmacology, Mast Cells drug effects
- Abstract
The factors that control migration of mast cells to sites of inflammation and tissue repair remain largely undefined. Whereas several recent studies have described chemotactic factors that induce migration of murine mast cells, only stem cell factor (SCF) is known to induce migration of human mast cells. We report here that the anaphylatoxins C3a and C5a are chemotactic factors for the human mast cell line HMC-1, human cord blood-derived mast cells (CBMC) and cutaneous mast cells in vitro. The presence of an extracellular matrix protein, laminin, was required for chemotaxis in response to complement peptides. Migration of mast cells towards C3a and C5a was dose-dependent, peaking at 1 microg/mL (100 nmol/L), and was inhibited by specific antibodies. Pretreatment with pertussis toxin inhibited the anaphylatoxin-mediated migration of HMC-1 cells, indicating that Gi proteins are involved in complement-activated signal transduction pathways in human mast cells. Both C3a and C5a also induced a rapid and transient mobilization of intracellular free calcium ([Ca2+]i) in HMC-1 cells. Besides SCF, other chemotactic factors tested, such as interleukin-3, nerve growth factor, transforming growth factor beta, RANTES (regulated upon activation, normal Tcell expressed and secreted), monocyte chemotactic protein-1 (MCP-1), MCP-2, MCP-3, macrophage inflammatory protein-1alpha (MIP-1alpha), and MIP-1beta, failed to stimulate migration of human mast cells. In summary, these findings indicate that C3a and C5a serve as chemotaxins for human mast cells. Anaphylatoxin-mediated recruitment of mast cells might play an important role in hypersensitivity and inflammatory processes.
- Published
- 1997
26. A subclone (5C6) of the human mast cell line HMC-1 represents a more differentiated phenotype than the original cell line.
- Author
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Weber S, Babina M, Krüger-Krasagakes S, Grützkau A, and Henz BM
- Subjects
- Cell Adhesion Molecules metabolism, Cell Differentiation physiology, Chymases, Clone Cells immunology, Clone Cells metabolism, Histamine Release, Humans, Mast Cells immunology, Mast Cells metabolism, Phenotype, Proto-Oncogene Proteins c-kit metabolism, Receptors, IgE metabolism, Serine Endopeptidases metabolism, Tryptases, Tumor Cells, Cultured, Antigens, Surface analysis, Clone Cells cytology, Mast Cells cytology
- Published
- 1996
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27. Fucosyltransferase III and sialyl-Le(x) expression correlate in cultured colon carcinoma cells but not in colon carcinoma tissue.
- Author
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Hanski C, Klussmann E, Wang J, Böhm C, Ogorek D, Hanski ML, Krüger-Krasagakes S, Eberle J, Schmitt-Gräff A, and Riecken EO
- Subjects
- Antibodies, Monoclonal immunology, Antibodies, Monoclonal metabolism, Blotting, Northern, Butyrates pharmacology, Butyric Acid, Colonic Neoplasms enzymology, Colonic Neoplasms metabolism, Dimethyl Sulfoxide pharmacology, Humans, Lewis X Antigen biosynthesis, Polymerase Chain Reaction, RNA, Messenger metabolism, Tumor Cells, Cultured, Fucosyltransferases metabolism, Gene Expression Regulation, Neoplastic genetics, Lewis X Antigen metabolism
- Abstract
The potential contribution of fucosyltransferases to the overexpression of sialyl-Le(x) antigen was investigated in the colon carcinoma cell line HT-29 and in human colon carcinoma tissue. In HT-29 cells as well as in normal or malignant colonic tissues Fuc-TIII, Fuc-TIV, Fuc-TVI but not Fuc-TV nor Fuc-TVII were detectable after RT-PCR. Sodium butyrate treatment of HT-29 cells increased (to about 200%) and DMSO treatment decreased (to about 20%) the expression of sialyl-Le(x). This modulation of sialyl-Le(x) was concomitant with the analogous increase/decrease of mRNA of Fuc-TIII but not Fuc-TIV. Fuc-TVI was not detectable by Northern blotting in HT-29 cells. In six human colon carcinomas which exhibited strong overexpression of sialyl-Le(x), the expression of Fuc-TIII-mRNA was the same or lower than in the corresponding normal colonic tissue. Thus Fuc-TIII expression may be affecting the expression of the sialyl-Le(x) moiety in HT-29 cells but not in human colon carcinoma tissue.
- Published
- 1996
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28. Release of stem cell factor from a human keratinocyte line, HaCaT, is increased in differentiating versus proliferating cells.
- Author
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Grabbe J, Welker P, Rosenbach T, Nürnberg W, Krüger-Krasagakes S, Artuc M, Fiebiger E, and Henz BM
- Subjects
- Base Sequence, Cell Differentiation, Cell Division, Cell Line, Humans, Immunohistochemistry, Molecular Probes genetics, Molecular Sequence Data, RNA Splicing, RNA, Messenger genetics, RNA, Messenger metabolism, Stem Cell Factor genetics, Keratinocytes metabolism, Stem Cell Factor metabolism
- Abstract
Stem cell factor, a recently discovered growth factor for hematopoietic stem cells, mast cells, and melanocytes, was initially reported to be produced by fibroblasts. In this study, we investigated the secretion of this factor from human HaCaT cells during in vitro culture and compared it to synthesis by cells in the skin. Release of stem cell factor from freshly cultured keratinocytes was comparable to that of HaCaT cells and was nearly half that produced by fibroblasts and umbilical vein endothelial cells. No stem cell factor was detectable in culture supernatants of melanocytes. HaCaT cells underwent spontaneous differentiation after a period of proliferation until confluency. Depending on duration of culture, they released increasing amounts of stem cell factor (approximately 150 pg/10(6) cells on day 3 (proliferating cells) vs approximately 450 pg/10(6) cells on day 14 (differentiating cells) measured by enzyme-linked immunosorbent assay. Stimulation for 24 h with the calcium ionophore A 23187 (10(-6) to 10(-8) M) further enhanced release. Western blot analysis of HaCaT cell lysates with a stem cell factor antibody revealed two proteins with the known molecular weights of membrane-bound and soluble stem cell factor. By semiquantitative reverse transcriptase polymerase chain reaction, full-length as well as spliced type stem cell factor mRNA was found to be increased in differentiating versus proliferating HaCaT cells. Keratinocytes are thus potentially important sources of stem cell factor in human skin, and HaCaT cells provide a useful model for further studies of stem cell factor from keratinocytes.
- Published
- 1996
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29. Interactions of immature human mast cells with extracellular matrix: expression of specific adhesion receptors and their role in cell binding to matrix proteins.
- Author
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Krüger-Krasagakes S, Grützkau A, Baghramian R, and Henz BM
- Subjects
- Cell Adhesion drug effects, Cell Differentiation, Cell Line, Collagen metabolism, Extracellular Matrix Proteins metabolism, Fibronectins metabolism, Humans, Laminin metabolism, Mast Cells cytology, Mast Cells drug effects, Microscopy, Electron, Scanning, Tetradecanoylphorbol Acetate pharmacology, Vitronectin metabolism, Extracellular Matrix metabolism, Integrins metabolism, Mast Cells metabolism
- Abstract
Interactions of cells with their extracellular matrix (ECM) are central to tissue-specific migration, localization, and function of migratory cells. Since mast cells circulate as immature precursor cells and home to tissues in a characteristic distribution, with increases in various disease states, we used the immature human mast cell line HMC-1 as a model to investigate the poorly understood mast cell-ECM interactions in humans. Functional adhesion studies showed that HMC-1 cells spontaneously adhere to fibronectin and laminin (80% at 6 and 12 microgram/ml, respectively) and to collagen type I and III (50% at 20 microgram/ml), whereas binding to vitronectin and collagen type IV required cell activation by phorbol myristate acetate. HMC-1 cells did not adhere to hyaluronic acid. Moreover, both fibronectin and laminin supported pronounced cytoplasmatic spreading with formation of isolated lamellipodia, whereas these cells exhibited a round cell shape on collagen and vitronectin, as shown by scanning electron microscopy. On flow cytometric analysis, HMC-1 cells expressed several adhesion molecules including the integrins beta 1, alpha 2 through alpha 6, alpha v, and alpha v beta 5, as well as CD44. Adhesion to fibronectin and vitronectin was found to be divalent cation- and arginine-glycine-aspartic acid-dependent, and could be blocked by antibodies to beta 1 or alpha 5, and alpha v or alpha v beta 5, respectively. In contrast, binding to laminin and collagen could not be blocked by monoclonal antibodies to any of the cell surface adhesion receptors expressed. Our results show that immature mast cells are able to modify their adhesive behavior in response to various ECM proteins and activating stimuli, and that this phenomenon is partly integrin mediated. These findings may be important for our understanding of the mechanisms leading to tissue-specific localization of mast cells.
- Published
- 1996
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30. Glucocorticoid-induced modulation of cytokine secretion from normal and leukemic human myelomonocytic cells.
- Author
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Welker P, Lippert U, Nürnberg W, Krüger-Krasagakes S, Möller A, and Czarnetzki BM
- Subjects
- Administration, Topical, Anti-Inflammatory Agents pharmacology, Cytokines blood, Dose-Response Relationship, Drug, Humans, Leukocytes, Mononuclear metabolism, Lipopolysaccharides pharmacology, Methylprednisolone analogs & derivatives, Methylprednisolone pharmacology, Tumor Cells, Cultured drug effects, Cytokines metabolism, Glucocorticoids pharmacology, Leukemia, Myelomonocytic, Acute metabolism, Leukemia, Myelomonocytic, Acute pathology, Leukemia, Myelomonocytic, Chronic metabolism, Leukemia, Myelomonocytic, Chronic pathology
- Abstract
Since glucocorticoid effects on inflammatory processes may be mediated via modulation of cytokine release, different types of myelomonocytic cells were stimulated in vitro with lipopolysaccharide (50 ng/ml) or phorbol myristate acetate (25 ng/ml) plus the ionophore A23187, 2 x 10(-7) M, and release of interleukin (IL)-1 beta, IL-8 and tumor necrosis factor (TNF)-alpha was measured after 24 h by ELISA. Peripheral blood mononuclear cells from two allergic and two normal human donors released similarly large quantities of IL-8 and lower amounts of IL-1 beta and TNF-alpha. This also held for myelomonocytic cell lines, with THP-1 cells being most active, followed by U-937 and HL-60 cells. All potent glucocorticoids studied caused a dose-dependent inhibition of cytokine release from donor cells, being most marked for IL-1 beta and lowest for IL-8. Inhibition of cytokine release was also noted with U-937 cells, with clear differences in potency between the glucocorticoids, whereas release was enhanced in all experiments with THP-1 cells. These results were confirmed with Northern blot analysis. Modulating effects of glucocorticoids on cytokine release are thus complex, and are particularly dependent on the cell type studied.
- Published
- 1996
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31. Modulation of in vitro cytokine release from human leukemic mast cells (HMC-1) by glucocorticoids.
- Author
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Lippert U, Welker P, Krüger-Krasagakes S, Möller A, and Henz BM
- Subjects
- Administration, Topical, Dose-Response Relationship, Drug, Glucocorticoids, Humans, Mast Cells metabolism, Tumor Cells, Cultured drug effects, Tumor Cells, Cultured metabolism, Anti-Inflammatory Agents pharmacology, Interleukins metabolism, Mast Cells drug effects, Tumor Necrosis Factor-alpha metabolism
- Abstract
Mast cells are well known effector cells not only in allergic but also in diverse acute and chronic inflammatory diseases. We have shown previously that these cells produce a broad spectrum of cytokines which might contribute to mast cell-dependent pathology. In the present study, we have investigated the influence of four potent glucocorticoids, methylprednisolone-aceponate, methylprednisolone-17-propionate, prednicarbate, and betametasone valerate (10(-5) M-10(-9) M), on the IL-1 beta, IL-3, IL-8, and tumor necrosis factor alpha secretion of the HMC-1 mast cell line as measured by ELISA. All four glucocorticoids caused a comparable dose- and time-dependent inhibition of cytokine release from HMC-1 cells stimulated for 24 h with phorbol 12-myristate 13-acetate 25 ng/ml and calcium ionophore 2 x 10(-7) M. These results shed further light on the mechanisms involved in antiinflammatory effects of glucocorticoids in allergic inflammation.
- Published
- 1996
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32. Production of interleukin-6 by human mast cells and basophilic cells.
- Author
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Krüger-Krasagakes S, Möller A, Kolde G, Lippert U, Weber M, and Henz BM
- Subjects
- Biological Assay, Biopsy, Cells, Cultured, Enzyme-Linked Immunosorbent Assay, Humans, Immunoblotting, Interleukin-6 blood, Microscopy, Immunoelectron, Polymerase Chain Reaction, Skin pathology, Transcription, Genetic, Basophils metabolism, Interleukin-6 biosynthesis, Mast Cells metabolism, Skin metabolism
- Abstract
Since mast cells and basophils are thought to play a central role in several types of cutaneous inflammatory and allergic reactions, and since interleukin-6 (IL-6) is an important mediator in these processes, we have studied the ability of the human mast cell line HMC-1, the human basophilic cell line KU812, and human skin mast cells to produce IL-6. All three cell types proved to be potent sources of this cytokine after appropriate stimulation. Transcription of IL-6 mRNA was first detectable 2 h after stimulation with the ester phorbol myristate acetate (PMA) and the calcium ionophore A23187 in both cell lines, as evidenced by semiquantitative reverse transcriptase polymerase chain reaction analysis. Whereas resting cells did not produce IL-6 protein, PMA/A23187-stimulated cells released immunoreactive and biologically active IL-6, as demonstrated and quantitated by enzyme-linked immunosorbent assay and by the use of TEPC 1033 cells, an IL-6-dependent murine plasmacytoma cell line. Stimulated KU812 cells secreted sevenfold more IL-6 (up to 15 ng/ml) than HMC-1 cells (up to 2.4 ng/ml). Immunoblotting of HMC-1- and KU812 cell-derived IL-6 revealed several IL-6 forms in the molecular weight range of 21 to 30 kDa. Immunoelectron microscopic studies of human skin biopsies provided evidence that unstimulated mast cells do not contain preformed IL-6 but accumulate IL-6 in cytoplasmic and extruded granules after IgE-dependent stimulation. These findings suggest that IL-6 secreted by human mast cells and basophils potentially contributes to allergic, other immunologically mediated and nonspecific inflammatory responses.
- Published
- 1996
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33. Mast cells in the cytokine network: the what, where from and what for.
- Author
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Czarnetzki BM, Grabbe J, Kolde G, Krüger-Krasagakes S, Welker P, and Zuberbier T
- Subjects
- Animals, Humans, Mast Cells ultrastructure, Cytokines physiology, Mast Cells physiology
- Abstract
The basic understanding of mast cell ontogeny and function has been fundamentally changed in recent years with observations that the cells produce and respond to a broad range of cytokines. These rapidly accruing data and their potential significance were discussed at the recent symposium "Mast Cells in the Cytokine Network", and the overview lectures of most speakers are summarized in this special journal issue. In the present introductory manuscript, the organizers of the meeting discuss data fundamental to an understanding of the topic and highlight aspects of special interest. They consider mast cells to be defined most reliably by their unique ultrastructure since the cells are highly heterogeneous in dependence of the species studied, their tissue location, their stage of development and probably also in relation to cytokines. Most other characteristics of mast cells are shared with diverse other cell types. Murine mast cell development is induced by several cytokines. These factors are mostly ineffective in human cells except for stem cell factor which causes mast cell development from CD34+/c-kit+ progenitors. There is however recent evidence that fibroblasts and keratinocytes produce additional growth factors for human mast cells. Regarding cytokine secretion, most molecules known so far are produced by both murine and human mast cells. The cells furthermore bear receptors for several cytokines, enabling them to respond in an autocrine and paracrine fashion. Mast cells may thus function within a complex cytokine network, affecting physiological as well as immunological and inflammatory processes.
- Published
- 1995
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34. Cytokine secretion by human mast cells.
- Author
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Krüger-Krasagakes S and Czarnetzki BM
- Subjects
- Humans, Inflammation pathology, Mast Cells pathology, Cytokines metabolism, Inflammation physiopathology, Mast Cells metabolism
- Abstract
Mast cells have been traditionally viewed as effector cells of immediate-type hypersensitivity reactions. Besides this, mast cell activation and degranulation have been associated with various biologically and clinically important functions. Results of the past few years suggest that mast cells are involved in the development of late-phase reactions and influence other chronic inflammatory responses through the generation and secretion of various multipotential cytokines.
- Published
- 1995
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35. Pharmacological modulation of IL-6 and IL-8 secretion by the H1-antagonist decarboethoxy-loratadine and dexamethasone by human mast and basophilic cell lines.
- Author
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Lippert U, Krüger-Krasagakes S, Möller A, Kiessling U, and Czarnetzki BM
- Subjects
- Basophils metabolism, Cell Line, Enzyme-Linked Immunosorbent Assay, Humans, Mast Cells metabolism, Basophils drug effects, Dexamethasone pharmacology, Histamine H1 Antagonists pharmacology, Interleukin-6 metabolism, Interleukin-8 metabolism, Loratadine pharmacology, Mast Cells drug effects
- Abstract
Mast cells and basophils are central effector cells of allergic reactions and are involved in inflammatory diseases. These cell types produce an array of mediators including a broad spectrum of cytokines. In order to examine whether antiallergic drugs modulate the release of these mediators, we have investigated the influence of dexamethasone and decarboethoxy-loratadine (DEL), the active metabolite of the H1-blocking agent loratadine, on the release of IL-6 and IL-8 by the human mast cell line HMC-1 and the human basophilic cell line KU812 by ELISA. Dexamethasone (10(-6)-10(-11) M) or Del (10(-5)-10(-14) M) were added to the cells either 1 h prior to or simultaneously with PMA and Ca-ionophore A23187. When preincubated with the cells, DEL dose-dependently suppressed IL-6 release by up to 40% and IL-8 release by up to 50%. Dexamethasone potently suppressed secretion of both cytokines if simultaneously added to the cells with the stimuli by up to 60% and after preincubation by up to 80%. Since both antihistamines and glucocorticoids are used for treatment of allergic diseases, the findings reported here indicate that these drugs may modulate allergic reactions via inhibition of cytokine release from mast cells and basophils.
- Published
- 1995
- Full Text
- View/download PDF
36. Mast cells.
- Author
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Weber S, Krüger-Krasagakes S, Grabbe J, Zuberbier T, and Czarnetzki BM
- Subjects
- Animals, Cell Division, Hematopoietic Stem Cells cytology, Humans, Immunoglobulin E immunology, Mast Cells cytology, Mast Cells immunology, Mastocytosis pathology, Receptors, Cell Surface physiology, Receptors, Immunologic immunology, Skin Diseases pathology, Mast Cells physiology
- Published
- 1995
- Full Text
- View/download PDF
37. Possible aggravation of hepatitis A by acitretin.
- Author
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Krüger-Krasagakes S, Grabbe J, and Czarnetzki BM
- Subjects
- Acitretin administration & dosage, Hepatitis, Chronic physiopathology, Humans, Male, Middle Aged, Mycosis Fungoides drug therapy, Photochemotherapy adverse effects, Skin Neoplasms drug therapy, Acitretin adverse effects, Hepatitis A physiopathology
- Published
- 1995
- Full Text
- View/download PDF
38. Production of cytokines by human melanoma cells and melanocytes.
- Author
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Krüger-Krasagakes S, Krasagakis K, Garbe C, and Diamantstein T
- Subjects
- Cells, Cultured, Cytokines genetics, Humans, Immune Tolerance, Melanoma genetics, Melanoma immunology, Melanoma pathology, Neoplasm Proteins genetics, Polymerase Chain Reaction, RNA, Messenger biosynthesis, RNA, Messenger genetics, RNA, Neoplasm biosynthesis, RNA, Neoplasm genetics, Skin Neoplasms genetics, Skin Neoplasms immunology, Skin Neoplasms pathology, Tumor Cells, Cultured, Cytokines biosynthesis, Gene Expression Regulation, Neoplastic, Melanocytes metabolism, Melanoma metabolism, Neoplasm Proteins biosynthesis, Skin Neoplasms metabolism
- Abstract
Experimental animal models have shown that various cytokines, depending of their specific properties, may support growth and metastasis of tumor cells or even lead to tumor rejection. The analysis of expression of cytokine genes by melanoma cell lines indicated that melanoma cells constitutively produce both autostimulatory and inhibitory cytokines. Using reverse transcriptase polymerase chain reaction analysis, simultaneous expression of several cytokines, including interleukin-1 beta (IL-1 beta), IL-6, IL-8, tumor necrosis factor-alpha, and granulocyte-macrophage colony-stimulating factor, by melanoma cells was found. The same cytokine transcripts were detected in melanocytes, suggesting that cells of the melanocytic lineage express a specific pattern of cytokines in vitro. All these cytokines are known to be able to stimulate effector cells of the host. Additionally, production of mRNA for IL-10, a cytokine with potential immunosuppressive properties, was detected in melanoma cells and melanocytes. These and other cytokines are likely to be involved in the immune response to cancer and at this time it is unknown what the net effects of multiple cytokines are on the outcome of the host response to tumor.
- Published
- 1995
- Full Text
- View/download PDF
39. Adhesion molecules on the human mast cell line HMC-1 are upregulated during cell activation.
- Author
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Weber S, Ruh B, Krüger-Krasagakes S, and Czarnetzki BM
- Subjects
- Binding Sites, Cell Line, Humans, Up-Regulation, Intercellular Adhesion Molecule-1 metabolism, Mast Cells metabolism
- Published
- 1995
- Full Text
- View/download PDF
40. Expression of interleukin 10 in human melanoma.
- Author
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Krüger-Krasagakes S, Krasagakis K, Garbe C, Schmitt E, Hüls C, Blankenstein T, and Diamantstein T
- Subjects
- Gene Expression, Humans, Keratinocytes metabolism, Melanoma genetics, Neoplasm Metastasis, RNA, Messenger genetics, RNA, Neoplasm genetics, Skin metabolism, Tumor Cells, Cultured, Interleukin-10 genetics, Melanoma metabolism
- Abstract
The expression of interleukin 10 (IL-10) mRNA in human malignant melanoma was investigated by reverse transcriptase polymerase chain reaction analysis. Selective expression of IL-10 mRNA in tissues of primary melanomas and melanoma metastases was found in comparison with normal skin. In addition, strong expression of IL-10 mRNA and of biologically active IL-10 was detected in 3 out of 13 melanoma cell lines. Normal melanocytes consistently expressed low levels of IL-10 mRNA but did not produce detectable IL-10 protein, nor did keratinocytes or fibroblasts. The production of biologically active IL-10 by melanoma cell lines suggests that IL-10 mRNA in melanoma lesions may derive at least in part from the tumour cells themselves. Tumour-infiltrating cells, however, could also be a source of IL-10 in melanoma tissues. The presence of IL-10 in melanoma lesions may contribute to the postulated 'paralysis' of an anti-melanoma immune response.
- Published
- 1994
- Full Text
- View/download PDF
41. Expression of MUC2-mucin in colorectal adenomas and carcinomas of different histological types.
- Author
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Blank M, Klussmann E, Krüger-Krasagakes S, Schmitt-Gräff A, Stolte M, Bornhoeft G, Stein H, Xing PX, McKenzie IF, and Verstijnen CP
- Subjects
- Adenocarcinoma, Mucinous pathology, Adenoma pathology, Adenoma, Villous metabolism, Adenoma, Villous pathology, Adenomatous Polyps metabolism, Adenomatous Polyps pathology, Base Sequence, Colorectal Neoplasms pathology, DNA Primers, Diagnosis, Differential, Gene Expression, Humans, Immunoenzyme Techniques, Molecular Sequence Data, Mucin-2, Mucins genetics, Polymerase Chain Reaction, Precancerous Conditions metabolism, Precancerous Conditions pathology, RNA, Messenger analysis, Adenocarcinoma, Mucinous metabolism, Adenoma metabolism, Biomarkers, Tumor metabolism, Colorectal Neoplasms metabolism, Mucins metabolism, Neoplasm Proteins metabolism
- Abstract
The expression of mucin MUC2 was investigated in normal colonic tissue, in colonic adenomas and in carcinomas of the mucinous and non-mucinous type. The latter were subdivided into carcinomas originating from the adenoma-carcinoma sequence (ACS) and de novo (DN) carcinomas. The expression was assayed by immunohistochemistry with the monoclonal anti-MUC2 antibody CCP58 and by mRNA semiquantitation. MUC2 protein epitope CCP58 was strongly expressed in 21% of normal colonic tissues, in 40% of villous and in 48% of tubular adenomas. Mucinous carcinomas exhibited strong expression in 72%, ACS carcinomas in 21% and DN adenocarcinomas in none of the tumors investigated. Compared with the adjacent non-malignant tissue (transitional mucosa), CCP58 epitope expression in the tumor was higher in 74% of mucinous carcinomas, but equal or lower in 69% of ACS carcinomas and in 100% of de novo carcinomas. The alterations of MUC2 expression detected by immunohistochemistry in adenocarcinomas were confirmed on mRNA level. These data indicate that the MUC2 expression pattern is different in the 3 carcinoma types investigated. MUC2 over-expression occurs in the adenomatous tissue. It is always maintained in mucinous carcinomas, but frequently decreased in non-mucinous ACS carcinomas. DN carcinomas are most frequently associated with decreased expression of MUC2.
- Published
- 1994
- Full Text
- View/download PDF
42. Tumor-cell-targeted cytokine gene transfer in experimental models for cancer therapy.
- Author
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Hock H, Dorsch M, Richter G, Kunzendorf U, Krüger-Krasagakes S, Blankenstein T, Qin Z, and Diamantstein T
- Subjects
- Animals, Gene Transfer Techniques, Leukocytes physiology, Lymphocyte Subsets physiology, Macrophages physiology, Mice, Neoplasms, Experimental immunology, Cytokines genetics, Genetic Therapy methods, Neoplasms, Experimental therapy
- Abstract
The genetic manipulation of tumor cells to express immunostimulatory molecules provides a current approach for the analysis of immune reactions against tumor cells in vivo. Experiments with multiple cytokines have demonstrated that an array of different host effector cells can be recruited by different cytokines in vivo, but more studies are necessary to distinguish cytokine-specific effects from as yet uncharacterized influences of different tumor models. A technically feasible clinical application of this approach is to be seen in the generation of vaccines by introducing such immunostimulatory genes into cancer cells and boosting systemic immune reactions against the unmodified cells. The experimental basis of these vaccination studies is critically discussed.
- Published
- 1994
43. A sialyl-Le(x)-negative melanoma cell line binds to E-selectin but not to P-selectin.
- Author
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Kunzendorf U, Krüger-Krasagakes S, Notter M, Hock H, Walz G, and Diamantstein T
- Subjects
- Base Sequence, Cell Adhesion, E-Selectin, Flow Cytometry, Fucosyltransferases genetics, Humans, Melanoma pathology, Molecular Sequence Data, P-Selectin, Polymerase Chain Reaction, RNA, Messenger analysis, Tumor Cells, Cultured, Cell Adhesion Molecules metabolism, Lewis X Antigen analysis, Melanoma metabolism, Platelet Membrane Glycoproteins metabolism
- Abstract
The adhesion molecules E-selectin (ELAM-1) and P-selectin (GMP-140/CD62) recognize the carbohydrate motives sialyl-Le(x), sialyl-diLe(x), or sialyl-Lea, though with different affinity. We found that the melanoma cell line NKI-4 bound to E-selectin, but not to P-selectin. This melanoma cell line did not express sialyl-Le(x), but was positive for sialyl-diLe(x) and sialyl-Le(a). In contrast, 2 other melanoma cell lines, MeWo and SK-MEL-28, expressing either sialyl-diLe(x) or sialyl-Le(a) on the cell surface, bound neither E-selectin nor P-selectin. Transfection of the fucosyltransferases Fuc-TIII, Fuc-TIV, and Fuc-TV mediates cell surface expression of sialyl-Le(x) in many cell lines. We detected transcripts of the fucosyltransferases Fuc-TIII and Fuc-TV in 4 melanoma cell lines despite the absence of cell surface sialyl-Le(x). Our observations indicate that expression of fucosyltransferases (Fuc-TIII and -TV) and generation of cell-surface sialyl-diLe(x) are not sufficient to permit adherence to E-selectin or P-selectin. Furthermore, it seems possible that a yet undefined ligand different from sialyl-Le(x), sialyl-diLe(x), or sialyl-Le(a) enables melanoma cells to adhere to E-selectin.
- Published
- 1994
44. Further evidence for involvement of both cell mediated and humoral immunity in generalized vitiligo.
- Author
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Abdel-Naser MB, Krüger-Krasagakes S, Krasagakis K, Gollnick H, Abdel-Fattah A, and Orfanos CE
- Subjects
- Adolescent, Adult, Aged, Antibodies, Monoclonal immunology, Antibody Formation, Antibody-Dependent Cell Cytotoxicity, Antigens, CD analysis, Biomarkers, Biopsy, Blood immunology, Cells, Cultured, Complement System Proteins immunology, Complement System Proteins pharmacology, Female, Humans, Immunity, Cellular, Infant, Newborn, Lymphocyte Activation, Male, Melanocytes immunology, Melanocytes pathology, Middle Aged, Receptors, Antigen, B-Cell analysis, Receptors, Interferon analysis, Receptors, Interleukin-2 analysis, Vitiligo blood, Vitiligo pathology, Interferon gamma Receptor, Vitiligo immunology
- Abstract
Immunohistochemical and immunoserological evidence supports the involvement of both cell-mediated and humoral mechanisms in the pathogenesis of melanocyte destruction in vitiligo. Punch biopsies from depigmented vitiliginous skin (VS), normal-looking pigmented skin (PS), and marginal skin (MS) from patients with generalized vitiligo (n = 15) were labeled with K 1.2.58, OKM1 (CD11b), Leu 11b (CD16), Leu 19 (CD56), IFN-gamma receptor, IL-2 receptor (CD25), IgG, IgM, C3c, and C3d MoAbs. In addition, in vitro effects of vitiligo sera (n = 13) on human newborn melanocytes (HMel) under different culture conditions were studied. The immunohistochemical findings showed absence of K 1.2.58+ epidermal melanocytes in VS and abnormal morphology in MS. In these areas, a few CD11b+ cells in the dermis and epidermis could be detected but no significant numbers of CD16+ or CD56+ cells were seen among the mononuclear cellular infiltrate. IL-2 and IFN-gamma receptors were clearly expressed by the cellular infiltrate. No significant deposition of complement or immunoglobulin was seen. The addition of vitiligo sera to HMel cultures induced a significant cellular proliferation. The stimulation of cell proliferation occurred regardless whether the sera were added alone or when preheated (56 degrees C for 1 hr) and then supplemented with a complement source (P < 0.01 at 2%, P < 0.001 at 10%, and P < 0.01 at 20% for sera alone) (P > 0.05 at 2%, P < 0.05 at 10%, and P < 0.01 at 20% for decomplemented sera plus complement).(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1994
- Full Text
- View/download PDF
45. Interleukin 10 transfected into Chinese hamster ovary cells prevents tumor growth and macrophage infiltration.
- Author
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Richter G, Krüger-Krasagakes S, Hein G, Hüls C, Schmitt E, Diamantstein T, and Blankenstein T
- Subjects
- Animals, Base Sequence, CHO Cells, Cricetinae, Female, Genetic Therapy, Interleukin-10 physiology, Mice, Mice, Inbred BALB C, Molecular Sequence Data, Neoplasms, Experimental therapy, Interleukin-10 genetics, Macrophages pathology, Neoplasms, Experimental pathology, Transfection
- Abstract
Expression of cytokines in tumor cells provides a sensitive modality to analyze the consequences of local cytokines in vivo on tumor infiltrating cells and tumorigenicity. We have transfected Chinese hamster ovary (CHO) cells with an interleukin 10 (IL-10) expression vector. CHO-IL10 cells although unaltered with respect to their in vitro growth lost tumorigenicity, both in nude and in SCID mice and in an IL-10 dose dependent manner. In addition, CHO-IL10 cells suppressed the growth of equal numbers of coinjected but not of contralaterally injected CHO cells. Immunohistology with anti-CR3/Mac-1 and anti-Mac-3 monoclonal antibodies revealed that CHO tumors were substantially infiltrated by macrophages. However, in CHO-IL10 tumors macrophages were virtually absent within the tumor tissue. Our results suggest that IL-10 indirectly suppresses tumor growth of certain tumors by inhibiting infiltration of macrophages which may provide tumor growth promoting activity.
- Published
- 1993
46. Expression of tumor necrosis factor by different tumor cell lines results either in tumor suppression or augmented metastasis.
- Author
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Qin Z, Krüger-Krasagakes S, Kunzendorf U, Hock H, Diamantstein T, and Blankenstein T
- Subjects
- Animals, Base Sequence, Cell Adhesion Molecules physiology, Immunotherapy, Mice, Mice, Inbred DBA, Molecular Sequence Data, Neoplasms, Experimental metabolism, Transfection, Tumor Cells, Cultured, Tumor Necrosis Factor-alpha genetics, Neoplasm Metastasis, Neoplasms, Experimental pathology, Tumor Necrosis Factor-alpha biosynthesis
- Abstract
Tumor necrosis factor (TNF) produced by tumor cells after gene transfer can effectively suppress the growth of locally growing tumors. We wanted to test the effects of "local" TNF on the growth of a highly metastatic cell line. Therefore, a recombinant retrovirus allowing expression of the TNF gene by the beta-actin promotor has been constructed and used to infect the two tumor cell lines EB and ESB, which grow as solid tumor or metastasize, respectively. Expression of TNF by EB cells resulted in their rapid and dose-dependent rejection. In sharp contrast, mice injected with ESB cells producing similar amounts of TNF showed no signs of tumor suppression, but rather had reduced survival rates that correlated with enhanced hepatic metastases. The accelerated formation of liver metastases by ESB TNF cells could be reversed by an anti-TNF mAb. These results demonstrate the opposite effects TNF may have on tumor growth: suppression of a locally growing tumor and promotion of metastasis formation.
- Published
- 1993
- Full Text
- View/download PDF
47. 12-O-tetradecanoylphorbol-13-acetate not only modulates proliferation rates, but also alters antigen expression and LAK-cell susceptibility of normal human melanocytes in vitro.
- Author
-
Krasagakis K, Garbe C, Krüger-Krasagakes S, and Orfanos CE
- Subjects
- Alkaloids pharmacology, Cell Division drug effects, Cytotoxicity, Immunologic, Humans, Killer Cells, Natural physiology, Protein Kinase C antagonists & inhibitors, Reference Values, Staurosporine, Antigens immunology, Killer Cells, Lymphokine-Activated physiology, Melanocytes immunology, Melanocytes physiology, Tetradecanoylphorbol Acetate pharmacology
- Abstract
For serial cultivation of normal human melanocytes media supplemented with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) are largely employed. By using a culture medium that permits cultivation of melanocytes without TPA, the effects of TPA on melanocyte proliferation, phenotype, and susceptibility to lymphokine-activated killer cells were studied. Addition of 50 ng/ml TPA to the medium induced rapid dendrite formation and increased the cell proliferation rate by 16-63% in mitogen-rich media (four of seven cultures, p < 0.01), and by 237% in mitogen-reduced media (p < 0.001). Furthermore, several phenotypic changes indicating early stages of melanocyte transformation were induced by 50 ng/ml TPA. These included increased expression of melanoma progression-associated antigens such as A.1.43 and A.10.33, upregulation of nerve-growth factor receptor as well as of the melanocyte-activation marker HMB-45 and of histocompatibility class I antigens. In contrast, the expression of the differentiation marker K.1.2 and of intercellular adhesion molecule-1 was decreased in TPA-treated cultures. Most of these changes persisted even after removal of TPA from the culture medium (> or = 2 weeks). Staurosporine, a protein kinase C inhibitor, modulated melanocyte-antigen expression similar to TPA, suggesting that protein kinase C downmodulation rather than activation by TPA is involved. In addition to the antigenic alterations, the susceptibility of TPA-treated melanocytes to lymphokine-activated killer cell cytotoxicity decreased by 40% (p < 0.01), possibly due to their altered surface antigen expression. The presented data reveal that the tumor promoter TPA hitherto used as a supplement of melanocyte culture media induces profound phenotypic and functional changes of the cultured cells, indicating incipient transformation of normal human melanocytes in vitro.
- Published
- 1993
- Full Text
- View/download PDF
48. Eosinophils infiltrating interleukin-5 gene-transfected tumors do not suppress tumor growth.
- Author
-
Krüger-Krasagakes S, Li W, Richter G, Diamantstein T, and Blankenstein T
- Subjects
- Animals, Base Sequence, Female, Immunity, Cellular, Mice, Mice, Inbred BALB C, Molecular Sequence Data, Neoplasms, Experimental immunology, Oligodeoxyribonucleotides chemistry, Polymerase Chain Reaction, Transfection, Eosinophils physiology, Interleukin-5 physiology, Neoplasms, Experimental pathology
- Abstract
Tumor-associated eosinophils have been observed in human tumors and in experimental tumor models, but their function is poorly understood. To study the role of eosinophils during tumor growth, the plasmacytoma J558L and the mammary adenocarcinoma TS/A were transfected with an expression vector encoding the murine gene for interleukin-5 (IL-5), a cytokine inducing proliferation and activation of eosinophils. Injection of parental cells, mock-transfectants and IL-5-producing cells into syngeneic mice showed that local IL-5 secretion induced rapid tumor infiltration by eosinophils, as evidenced by immunohistochemical staining, but nevertheless did not alter the tumor growth kinetics of IL-5 transfectants. Therefore, the mere presence of IL-5 and eosinophils was not sufficient to induce a protective host immune response.
- Published
- 1993
- Full Text
- View/download PDF
49. A rapid and sensitive fluorometric microassay for determining cell mediated cytotoxicity to adherent growing cell lines.
- Author
-
Krüger-Krasagakes S, Garbe C, Kossman P, and Orfanos CE
- Subjects
- Cell Adhesion, Chromium Radioisotopes, Humans, Hymecromone analogs & derivatives, Hymecromone chemistry, In Vitro Techniques, Melanoma immunology, Tumor Cells, Cultured, Cytotoxicity, Immunologic, Fluoroimmunoassay methods, Immunity, Cellular, Killer Cells, Lymphokine-Activated immunology
- Abstract
In order to measure cell mediated cytotoxicity to adherent growing cell lines in vitro more rapidly and conveniently, a fluorometric microassay was developed and results were compared with those obtained by the 51Cr release assay. The fluorometric method is based on the hydrolysis of the fluorochrome 4-methylumbelliferyl heptanoate (MUH) by intracellular esterases of viable cells. Melanoma cell monolayers were incubated with lymphokine activated killer (LAK) cells for 4 h at various effector: target (E:T) cell ratios (E:T = 16, 8, 4, 2:1). Thereafter surviving adherent melanoma cells were stained with MUH for 30 min and fluorescence was measured directly in a 96 well plate reader. For the calculation of LAK cell cytotoxicity fluorescence values were corrected for the number of nonspecifically detached tumor cells during the washes and the number of nonspecifically adherent LAK cells. Using identical target and effector cell preparations both assays showed a nearly proportional increase of percentage cytotoxicity with rising numbers of lymphocytes. Compared with the 51Cr release assay, however, higher cytotoxicity values were obtained with the fluorometric MUH microassay: 57% with MUH versus 26% with 51Cr and 39% versus 14% for cell lines StML-11 and SKMel-28, respectively (E:T ratio = 16:1). The higher cytotoxicity rates obtained with the fluorometric MUH microassay were not due to the additional 30 min staining with MUH or due to nonspecific hydrolysis of MUH by extracellular esterases released from damaged cells, as could be shown by a series of experiments. In conclusion, a simple and rapid fluorometric microassay has been developed showing reliable reproducibility and a higher sensitivity compared with the 51Cr release assay for the determination of cellular cytotoxicity to adherent growing cell lines, avoiding hazardous radioactive labels.
- Published
- 1992
- Full Text
- View/download PDF
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