Summary
We investigated the interaction between GM-CSF and its receptor on human granulocytes and on several human tumor cell lines. Specific high-affinity binding for GM-CSF was characterized by Scatchard plot analysis. The specific radioactivity of the 125I-labeled derivative of rH. GM-CSF was determined by self-displacement analysis and calculated to be 30 μCi/μg. The maximum concentration of binding sites (B max) in granulocytes was 40 fmol/mg protein (2,200 molecules GM-CSF bound/cell) and the dissociation constant (KD) was 0.42 nM. No binding sites for GM-CSF were found in two lung cancer cell lines, SCLC-16HV and NCI-N417 or in the urinary bladder carcinoma cell line 5637, whereas the promyelocytic leukemia cell line HL60 was positive for GM-CSF binding. Time course experiments showed maximum binding of GM-CSF in granulocytes after an incubation period of 60 min and a decrease in binding after an incubation period of 2 h. In parallel, we found a maximum biological signal when granulocytes were preincubated for 90 min with GM-CSF, and a decrease after an incubation time of 120 min. Preincubation of the cells with rH. GM-CSF induced an enhancement of the production of activated oxygen species by the cells in response to PMA.
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Abbreviations
- CSF :
-
colony stimulating factor
- rH.GM-CSF :
-
recombinant human granulocyte-macrophage colony-stimulating factor
- PMNs :
-
polymorphonuclears
- ADCC :
-
antibody dependent cell mediated cytotoxicity
- PMA :
-
phorbol myristate acetate
- SCLC :
-
small cell lung cancer
- DAG :
-
diacylglycerol
- PIP 2 :
-
phosphatidylinositol-biphosphate
References
Bell RM (1986) Protein kinase C activation by diacylglycerol second messengers. Cell 45: 631–632
Bepler G, Jaques G, Neuman K, Aumüller G, Gropp C, Havemann K (1987) Establishment, growth properties, and morphological characteristics of permanent human small cell lung cancer cell lines. J Cancer Res Clin Oncol 113: 31–40
Calvo JC, Radicella JP, Charreau EH (1983) Measurement of specific radioactivities in labelled hormones by self-displacement analysis. Biochem J 212: 259–264
Cantrell MA, Anderson D, Cerretti DP, Price V, Mc Kereghan K, Tushinsky R, Mochizuki DJ, Larsen A, Grabstein K, Gillis S, Cosman D (1985) Cloning, sequence and expression of a human granulocyte-macrophage colony-stimulating factor. Proc Natl Acad Sci USA 82: 6250–6254
Carney DN, Gazdar AF, Bepler G, Guccion JG, Marangos PJ, Moody TW, Zweig MH, Minua JD (1985) Establishment and identification of small cell lung cancer cell lines having classic and variant features. Cancer Res 45: 2913–2923
Collins SJ, Gallo RC, Gallagher RE (1977) Continuous growth and differentiation of human myeloid leukaemic cells in suspension culture. Nature 270: 347–348
Fleischmann J, Golde DW, Weisbart RH, Gasson JC (1986) Granulocyte-macrophage colony-stimulating factor enhances phagocytosis of bacteria by human neutrophils. Blood 68: 708–711
Fogh J (1978) Cultivation, characterization and identification of human tumor cells with emphasis on kidney, testis, and bladder tumors. Natl Cancer Inst Monogr 49: 5–9
Gabrilove JL, Welte K, Harris P, Platzer E, Lu I, Levi E, Mertelsmann R, Moore MAS (1986) Pluripoietin: a second human hematopoietic CSF produced by the human bladder carcinoma cell line 5637. Proc Natl Acad Sci USA 83: 2478–2482
Gasson JC, Kaufmann SE, Weisbart RH, Tomonoga M, Golde DW (1986) High-affinity binding of granulocyte-macrophage colony-stimulating factor to normal and leukemic human myeloid cells. Proc Natl Sci USA 83: 669–673
Gasson E, Weisbart RH, Kaufmann SE, Clark SC, Hewick RM, Wong GG, Golde DW (1984) Purified human granulocyte-macrophage colony-stimulating-factor: direct action on neutrophils. Science 226: 1339–1342
Gough NM, Gough J, Metcalf D, Kelso A, Grail D, Nicola NA, Burgess AW, Dunn AR (1984) Molecular cloning of cDNA encoding a murine haematopoietic growth regulator, granulocyte-macrophage colony-stimulating factor. Nature 309: 763–767
Grabstein KH, Urdal DL, Tushinski RJ, Mochizuki DY, Price VL, Cantrell MA, Gillis S, Conlon PJ (1986) Induction of macrophage tumoricidal activity by granulocyte-macrophage colony-stimulating factor. Science 232: 506–508
Greenberger JS, Eckner RJ, Ostertag W, Colletta G, Boshetti S, Nagasawa H, Karpas A, Weichselbaum R, Moloney WC, Sakakeeny MA (1980) Release of spleen focus-forming virus (SFFV) in differentiation inducible granulocytic leukemia cell lines transformed in vitro by Friend leukemia virus. Virology 105: 425–435
Klausmann M, Pflüger KH, Krumwieh D, Seiler FR, Havemann K (1987) Stimulation of oxidative metabolism of granulocytes by recombinant granulocyte-macrophage colony-stimulating factor and a conditioned medium of a urinary bladder carcinoma cell line. Blut 54: 307–31
Lee F, Yokota T, Otsuka T, Gemmel L, Larsan N, Luh J, Arai KI, Rennick D (1985) Isolation of a cDNA for a human GM-CSF by functional expression in mammalian cells. Proc Natl Acad Sci USA 82: 4360–4364
Lopez AF, Williamson DJ, Gamble JR, Glenn BC, Harian JM, Klebanoff SJ, Waltersdorph A, Wong G, Clark SC, Vadas MA (1986) Recombinant human granulocyte-macrophage colony-stimulating factor stimulates in vitro mature human neutrophil and eosinophil function, surface receptor expression, and survival. J Clin Invest 78: 1220–1228
Metcalf D (1985) The granulocyte-macrophage colony-stimulating factors. Science 229: 16–22
Metcalf D (1985) The granulocyte-macrophage colony-stimulating factors. Cell 43: 5–6
Morrison DC, Jacobs DM (1976) Inhibition of lipopolysaccharide-initiated activation of serum complement by polymyxin B. Infect Immunol 13: 298–301
Park LS, Friend D, Gillis S, Urdal DL (1986) Characterization of the cell surface receptor for human granulocyte-macrophage colony-stimulating factor. J Exp Med 164: 251–262
Sharkey NA, Leach KL, Blumberg PM (1984) Competitive inhibition by diacylglycerol of specific phorbol ester binding. Proc Natl Sci USA 81: 607–610
Suzuki Y, Lehrer RI (1980) NADPH oxidase activity in human neutrophils stimulated by phorbol myristate acetate. J Clin Invest 66: 1409–1418
Tejedor F, Ballesta JPG (1982) Iodination of biological samples without loss of functional activity. Anal Biochem 127: 143–149
Tomonoga M, Golde DW, Gasson JC (1986) Biosynthetic (recombinant) human granulocyte-macrophage colony-stimulating factor: effect on normal bone marrow and leukemia cell lines. Blood 67: 31–36
Vadas MA, Nicola NA, Metcalf D (1983) Activation of antibody-dependent-cell-mediated cytotoxicity of human neutrophils and eosinophils by separate colony stimulating factors. J Immunol 130: 795–799
Weisbart RH, Golde DW, Clark SC, Wong GG, Gasson JC (1985) Human granulocyte-macrophage colony-stimulating factor is a neutrophil activator. Nature 314: 361–363
Wong GG, Witek J, Temple PA, Wilkens KM, Leary AC, Luxenberg DP, Jones SS, Brown EC, Kay RM, Orr EC, Shoemaker C, Golde DW, Kaufmann RJ, Hewick RM, Wang EA, Clark RC (1985) Human GM-CSF: molecular cloning of the complementary DNA and purification of the natural and recombinant proteins. Science 228: 810–815
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Häder, M., Klausmann, M., Pflüger, K.H. et al. Granulocyte-macrophage colony-stimulating factor binding sites and oxidative metabolism in human granulocytes. Blut 59, 486–492 (1989). https://doi.org/10.1007/BF00329493
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DOI: https://doi.org/10.1007/BF00329493