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• W2011238768 abstract "In order to characterize the phenotype of human mast cell precursors in the peripheral blood mononuclear fraction and its alterations during in vivo mast cell differentiation, cells were studied before and during culture with stem cell factor or stem cell factor-containing cell supernatants. Prior to culture, 86% of cells were immunoreactive for the monocytic marker CD14, slightly fewer for CD11b and CD64, <10% expressed FcεRIα, rare cells were CD34+ (<0,1%), and none stained for CD1, CD33, c-Kit, and tryptase. After 2 wk of culture, there was de novo expression of c-Kit (14%-43% positive cells), tryptase (26%-79%), CD33 (57%), and CD64 (64%), an upregulation of FcεRIα (23%-52%), CD11b (93%), and CD68 (95%), but no expression of CD34. Levels of mRNA for FcεRIα and c-Kit were detectable prior to culture and increased during culture, together with de novo expression of tryptase. Double staining after 2 wk of culture showed that FcεRIα-positive cells were mostly CD14+ (90%), CD64+ (82%), and CD68+ (52%) on flow cytometry. Intracellular tryptase activity was first detectable after 1 wk of culture, increased FcεRIα expression was only detectable by week 2. Cultured cells acquired the ability to release histamine during IgE-dependent stimulation, and culture with the c-Kit antibody YB5.B8 resulted in a downregulation of tryptase and FcεRIα, but not of c-Kit. These data show that human mast cells develop from c-Kit- and tryptase-negative precursors in the myelomonocytic fraction of peripheral blood and that they upregulate, maintain, and share many phenotypic characteristics of cells from the monocyte/macrophage lineage during early phases of in vitro differentiation. In order to characterize the phenotype of human mast cell precursors in the peripheral blood mononuclear fraction and its alterations during in vivo mast cell differentiation, cells were studied before and during culture with stem cell factor or stem cell factor-containing cell supernatants. Prior to culture, 86% of cells were immunoreactive for the monocytic marker CD14, slightly fewer for CD11b and CD64, <10% expressed FcεRIα, rare cells were CD34+ (<0,1%), and none stained for CD1, CD33, c-Kit, and tryptase. After 2 wk of culture, there was de novo expression of c-Kit (14%-43% positive cells), tryptase (26%-79%), CD33 (57%), and CD64 (64%), an upregulation of FcεRIα (23%-52%), CD11b (93%), and CD68 (95%), but no expression of CD34. Levels of mRNA for FcεRIα and c-Kit were detectable prior to culture and increased during culture, together with de novo expression of tryptase. Double staining after 2 wk of culture showed that FcεRIα-positive cells were mostly CD14+ (90%), CD64+ (82%), and CD68+ (52%) on flow cytometry. Intracellular tryptase activity was first detectable after 1 wk of culture, increased FcεRIα expression was only detectable by week 2. Cultured cells acquired the ability to release histamine during IgE-dependent stimulation, and culture with the c-Kit antibody YB5.B8 resulted in a downregulation of tryptase and FcεRIα, but not of c-Kit. These data show that human mast cells develop from c-Kit- and tryptase-negative precursors in the myelomonocytic fraction of peripheral blood and that they upregulate, maintain, and share many phenotypic characteristics of cells from the monocyte/macrophage lineage during early phases of in vitro differentiation. alkaline phosphatase anti-alkaline phosphatase high affinity IgE receptor high performance liquid chromatography human fibroblast supernatants human keratinocyte supernatants immunoglobulin L-cell fibroblast supernatant nerve growth factor peripheral blood monocytic cells stem cell factor Mast cells are bone marrow-derived, tissue resident cells whose numbers are known to increase in a wide variety of inflammatory and neoplastic conditions (Weber et al., 1995Weber S. Krüger-Krasagakes S. Grabbe J. Zuberbier T. Czarnetzki B.M. Mast cells.Int J Dermatol. 1995; 34: 1-10Crossref PubMed Scopus (61) Google Scholar;Metcalfe et al., 1997Metcalfe D.D. Baram D. Mekori Y.A. Mast cells.Physiol Rev. 1997; 77: 1033-1079Crossref PubMed Scopus (1705) Google Scholar). Mechanisms underlying these processes, particularly in the human system, are however largely unclear (Denburg, 1995Denburg J.A. Differentiation of human basophils and mast cells.in: G. Marone Human Basophils and Mast Cells: Biological Aspects, Vol. 61: Chem Immunol. Basel, Karger1995: 49-71Crossref Google Scholar;Czarnetzki et al., 1996Czarnetzki B.M. Grabbe J. Kolde G. Krüger-Krasagakes S. Zuberbier T. Mast cells in the cytokine network – the what, where from and what for.Exp Dermatol. 1996; 4: 221-226Crossref Scopus (15) Google Scholar). One possible way for mast cells to increase at tissue sites might involve the influx of precursors from the peripheral blood into the tissue, with subsequent differentiation of the cells under the influence of locally secreted growth factors. Stem cell factor (SCF), also known as mast cell growth factor, Steel factor, or c-Kit ligand, has been identified as a potent mast cell growth factor (Zsebo et al., 1990Zsebo K.M. Williams D.A. Geissler E.N. Stem cell factor is encoded at the S1 locus of the mouse and is the ligand for the c-kit tyrosine kinase receptor.Cell. 1990; 63: 213-224Abstract Full Text PDF PubMed Scopus (1187) Google Scholar;Williams et al., 1990Williams D.E. Eisenmann J. Baird A. et al.Identification of a ligand for the c-kit protooncogen.Cell. 1990; 63: 167-174Abstract Full Text PDF PubMed Scopus (750) Google Scholar;Takagi et al., 1990Takagi M. Nokahata T. Kubo T. et al.Stimulation of mouse connective tissue-type mast cells by hematopoietic stem cell factor, a ligand for c-kit receptor.J Immunol. 1990; 148: 3446-3453Google Scholar) and appears to be the main mast cell differentiation factor identified so far in humans (reviewed inGrabbe et al., 1994aGrabbe J. Welker P. Dippel E. Czarnetzki B.M. Stem cell factor, a novel cutaneous growth factor for mast cells and melanocytes.Arch Dermatol Res. 1994; 287: 78-84Crossref PubMed Scopus (113) Google Scholar). SCF is produced by a number of tissue resident cells including fibroblasts, keratinocytes, endothelial, and bone marrow stroma cells, supporting the concept that mast cell precursors can differentiate in situ in diverse organs including the skin. A potential role of SCF in the pathogenesis of mastocytosis has also been discussed by different groups (Longley et al., 1993Longley B.J. Morganroth G.S. Tyrrell L. Ding T.G. Anderson D.M. Williams D.E. Halaban R. Altered metabolism of mast-cell growth factor (c-kit ligand) in cutaneous mastocytosis.N Engl J Med. 1993; 328: 1302-1307Crossref PubMed Scopus (244) Google Scholar;Castells et al., 1996Castells M.C. Friend D.S. Bunnell C.A. Hu X. Fraus M. Osteen R.T. Austen K.F. The presence of membrane-bound stem cell factor on highly immature nonmetachromatic mast cells in the peripheral blood of a patient with aggressive systemic mastocytosis.J Allergy Clin Immunol. 1996; 98: 831-840Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar;Henz, 1998Henz B.M. SCF and c-Kit in mastocytosis – a pandora’s box holding more theories than proven facts.J Invest Dermatol. 1998; 110: 186Crossref PubMed Scopus (9) Google Scholar). As suggested by its name, SCF is however not mast cell specific, as it also affects a number of other cells expressing its c-Kit receptor, including hematopoietic stem cells, melanocytes, and germ cells (Grabbe et al., 1994aGrabbe J. Welker P. Dippel E. Czarnetzki B.M. Stem cell factor, a novel cutaneous growth factor for mast cells and melanocytes.Arch Dermatol Res. 1994; 287: 78-84Crossref PubMed Scopus (113) Google Scholar). We and colleagues have shown in the past that cells expressing all major features of mast cells can be induced in cultures of the monocytic fraction from human peripheral blood (PBMC) under the influence of SCF or SCF-containing fibroblast- and keratinocyte-derived mast cell conditioning medium (Czarnetzki et al., 1983Czarnetzki B.M. Krüger G. Sterry W. In vitro generation of mast cell-like cells from human peripheral mononuclear phagocytes.Int Arch Allergy Appl Immunol. 1983; 71: 161-167Crossref PubMed Scopus (21) Google Scholar,Czarnetzki et al., 1984Czarnetzki B.M. Figdor C.G. Kolde G. Vroom T. Aalberse R. de Vries J. Development of human connective tissue mast cells from purified blood monocytes.Clin Exp Immunol. 1984; 51: 549-554Google Scholar;Valent et al., 1992Valent P. Spanblöchl E. Sparr W.R. et al.Induction of differentiation of human mast cells from bone marrow and peripheral blood mononuclear cells by rhSCF/c-kit-ligand (KL) in long time culture.Blood. 1992; 80: 2237-2245Crossref PubMed Google Scholar;Agis et al., 1993Agis H. Willheim M. Sperr W.R. Monocytes do not make mast cells when cultured in the presence of SCF. Characterization of the circulating mast cell progenitors as a c-kit+, LY–, CD14–, CD17–, colony-forming cell.J Immunol. 1993; 151: 4221-4227PubMed Google Scholar;Grabbe et al., 1994bGrabbe J. Welker P. Möller A. Dippel E. Ashman L.F. Czarnetzki B.M. Comparative cytokine release from human monocytes, monocyte-derived immature mast cells and a human mast cell line (HMC1).J Invest Dermatol. 1994; 103: 504-508Crossref PubMed Scopus (59) Google Scholar;Welker et al., 1995Welker P. Grabbe J. Czarnetzki B.M. Human keratinocytes release mast cell differentiation factors other than stem cell factor.Int Arch Allergy Immunol. 1995; 107: 139-141Crossref PubMed Scopus (19) Google Scholar). In the bone marrow and in cord blood, mast cells have been shown to develop from CD34+, SCF-responsive, multipotent hematopoietic progenitor cells (Kirshenbaum et al., 1992Kirshenbaum A.S. Goff J.P. Kessler S.W. Mican J.M. Zsebo K.M. Metcalfe D.D. Effect of IL3 and stem cell factor on the appearance of human basophils and mast cells from CD34+ and pluripotent progenitor cells.J Immunol. 1992; 148: 772-777PubMed Google Scholar;Agis et al., 1993Agis H. Willheim M. Sperr W.R. Monocytes do not make mast cells when cultured in the presence of SCF. Characterization of the circulating mast cell progenitors as a c-kit+, LY–, CD14–, CD17–, colony-forming cell.J Immunol. 1993; 151: 4221-4227PubMed Google Scholar;Rottem et al., 1994Rottem M. Okada T. Goff J.P. Metcalfe D.D. Mast cells cultured from peripheral blood of normal donors and patients with mastocytosis originate from a CD34+/FcεRI– cell population.Blood. 1994; 84: 2489-2496PubMed Google Scholar;Huang and Terstappen, 1995Huang S. Terstappen L.W. Lymphoid and myeloid differentiation of single human CD34+, HLA-DR–, CD38– hematopoietic stem cells.Blood. 1995; 83: 1515-1526Google Scholar). The phenotype of the committed, specific mast cell precursor remains elusive however. In peripheral blood that serves as a direct source of mast cell precursors for diverse organs, including the skin, CD34+ cells are extremely rare and yield only a minor fraction of mast cells during in vitro culture, whereas CD14– cells have been described to provide the main proportion of these cells (Valent, 1994Valent P. The riddle of the mast cell: kit (CD117) -ligand as the missing link?.Im Today. 1994; 15: 111-114Abstract Full Text PDF PubMed Scopus (109) Google Scholar). In nasal mucosa, a c-Kit+, tryptase–, FcεRI– cell has recently been identified and proposed to serve as a committed mast cell precursor (Kawabori et al., 1997Kawabori S. Kanai N. Tosho T. Adachi T. Existence of c-kit receptor-positive, tryptase-negative, IgE-negative cells in human allergic mucosa: a candidate for mast cell progenitor.Int Arch Allergy Immunol. 1997; 112: 36-43Crossref PubMed Scopus (14) Google Scholar). In order to further delineate the phenotype of the mast cell precursor in peripheral blood, which would serve as source of mast cells in diverse organs including the skin, this study was designed to focus on the identification of several typical mast cell markers and of monocytic markers on PBMC prior to and during culture in the presence of SCF or mast cell growth factor-containing conditioning media. The data confirm the previously suggested close relationship between mast cells and peripheral blood myelomonocytic cells (Czarnetzki et al. 1992,Czarnetzki et al., 1983Czarnetzki B.M. Krüger G. Sterry W. In vitro generation of mast cell-like cells from human peripheral mononuclear phagocytes.Int Arch Allergy Appl Immunol. 1983; 71: 161-167Crossref PubMed Scopus (21) Google Scholar,Czarnetzki et al., 1984Czarnetzki B.M. Figdor C.G. Kolde G. Vroom T. Aalberse R. de Vries J. Development of human connective tissue mast cells from purified blood monocytes.Clin Exp Immunol. 1984; 51: 549-554Google Scholar;Valent et al., 1989Valent P. Ashman L.K. Hinterberger W. Eckersberger F. Majdic O. Lechner K. Bettelheim P. Mast cell typing: demonstration of a distinct hematopoietic cell type and evidence for immunophenotypic relationship to mononuclear phagocytes.Blood. 1989; 73: 1178-1784Google Scholar). They furthermore underline the central role of SCF compared with possible additional mast cell growth factors in this process. PBMC were prepared from peripheral blood of different healthy donors by a sequence of differential centrifugation on Ficoll-Hypaque, adherence to polystyrene culture flasks for 2 h, and subsequent washing with RPMI containing 10% fetal calf serum (both from Seromed, Berlin, Germany) to remove nonadherent cells (Grabbe et al., 1994bGrabbe J. Welker P. Möller A. Dippel E. Ashman L.F. Czarnetzki B.M. Comparative cytokine release from human monocytes, monocyte-derived immature mast cells and a human mast cell line (HMC1).J Invest Dermatol. 1994; 103: 504-508Crossref PubMed Scopus (59) Google Scholar). Human leukemic mast cells (HMC-1) were kindly provided by J.H. Butterfield, Minneapolis, U.S.A. (Butterfield et al., 1988Butterfield J.H. Weiler D. Dewald G. Gleich G.J. Establishment of an immature mast cell line from a patient with mast cell leukemia.Leukemia Res. 1988; 12: 345-355Abstract Full Text PDF PubMed Scopus (618) Google Scholar) and human basophilic leukemic cells (KU 812 cells) (Kishi, 1985Kishi K. A new leukemic cell line with Philadelphia chromosome characterized as basophil precursor.Leukemia Res. 1985; 9: 381-390Abstract Full Text PDF PubMed Scopus (215) Google Scholar) were from the Research Institute in Borstel, Germany. Cells were kept routinely in mast cell growth medium consisting of Iscove’s medium (GIBCO, Eggenstein, Germany) and 30% horse serum (Seromed) because this basic medium has been proven in the past to be optimal for supporting the activity of mast cell differentiation factors (Czarnetzki et al., 1983Czarnetzki B.M. Krüger G. Sterry W. In vitro generation of mast cell-like cells from human peripheral mononuclear phagocytes.Int Arch Allergy Appl Immunol. 1983; 71: 161-167Crossref PubMed Scopus (21) Google Scholar). For mast cell differentiation, L-cell fibroblasts (LCS), human fibroblast (HFS), or HaCaT keratinocyte supernatants (HKS), obtained from confluent culture of cells after a 5 d culture in Dulbecco’s minimal essential medium (DMEM, GIBCO) with 5% newborn calf serum or 10% fetal calf serum (both GIBCO), were added to the basic mast cell growth medium. In addition to SCF, LCS contained 250 pg NGF per ml and 500 pg TGFβ per ml, HKS 125 pg NGF per ml and 1250 pg TGFβ per ml, as measured by commercial ELISA (Biermann, Bad Nauheim, Germany). These conditioning media were added at a 30% concentration throughout, as lower concentrations had proven in the past to be less effective for the induction of mast cell differentiation from PBMC (Czarnetzki et al., 1983Czarnetzki B.M. Krüger G. Sterry W. In vitro generation of mast cell-like cells from human peripheral mononuclear phagocytes.Int Arch Allergy Appl Immunol. 1983; 71: 161-167Crossref PubMed Scopus (21) Google Scholar;Welker et al., 1995Welker P. Grabbe J. Czarnetzki B.M. Human keratinocytes release mast cell differentiation factors other than stem cell factor.Int Arch Allergy Immunol. 1995; 107: 139-141Crossref PubMed Scopus (19) Google Scholar). rh SCF (Peptro Tech, London, U.K.) was added to the basic culture medium at 100 ng per ml because this concentration has been shown to be equal or superior to other concentrations during mast cell differentiation studies (Grabbe et al., 1994aGrabbe J. Welker P. Dippel E. Czarnetzki B.M. Stem cell factor, a novel cutaneous growth factor for mast cells and melanocytes.Arch Dermatol Res. 1994; 287: 78-84Crossref PubMed Scopus (113) Google Scholar), and because this concentration approximated the SCF concentration in HKS (Grabbe et al., 1996Grabbe J. Welker P. Rosenbach T. et al.Release of stem cell factor from a human keratinocyte line, HaCaT, is increased in differentiating versus proliferating cells.J Invest Dermatol. 1996; 107: 1-6Crossref Google Scholar). Cells were seeded into the different culture media at 1 × 105 cells per ml and kept under standard conditions, as described (Czarnetzki et al., 1983Czarnetzki B.M. Krüger G. Sterry W. In vitro generation of mast cell-like cells from human peripheral mononuclear phagocytes.Int Arch Allergy Appl Immunol. 1983; 71: 161-167Crossref PubMed Scopus (21) Google Scholar;Grabbe et al., 1994bGrabbe J. Welker P. Möller A. Dippel E. Ashman L.F. Czarnetzki B.M. Comparative cytokine release from human monocytes, monocyte-derived immature mast cells and a human mast cell line (HMC1).J Invest Dermatol. 1994; 103: 504-508Crossref PubMed Scopus (59) Google Scholar). In another set of experiments, cells were cultured with LCS in the presence of the SCF-receptor antibody YB5.B8 or an irrelevant antibody of the same isotype (anti-desmin, see below). Both antibodies had been purified by HPLC using ion exchange columns (Pharmacia, Freiburg, Germany), with subsequent dialysis. The antibodies were added together with LCS to culture media from days 0–14 at 2 μg per ml. The antibody against mast cell tryptase (AA1) (Walls et al., 1990Walls A.F. Jones D.B. Williams J.H. Church M.K. Holgate S.T. Immunohistochemical identification of mast cells in formaldehyde-fixed tissue using monoclonal antibodies specific for tryptase.J Pathol. 1990; 162: 119-126Crossref PubMed Scopus (153) Google Scholar) was kindly provided by A. Walls, Southampton, U.K., a competitively binding antibody against the α-chain of the FcεRI (29C6) (Riske et al., 1991Riske F. Hakimi J. Mallamaci M. High affinity human IgE receptor (FcεRI). Analysis of functional domains of the α-subunit with monoclonal antibodies.J Biol Chem. 1991; 266: 11245-11251Abstract Full Text PDF PubMed Google Scholar) by J. Hakimi, Nutley, U.S.A., a partially competitively binding antibody against c-Kit (YB5.B8) (Lerner et al., 1991Lerner N.B. Nocka K.H. Cole S.R. Qui F. Strife A. Ashman L.K. Besmer P. Monoclonal antibody YB5.B8 identifies the human c-kit protein product.Blood. 1991; 77: 1876-1883Crossref PubMed Google Scholar;Ashman et al., 1994Ashman L.K. Buhring R.J. Aylett G.W. Broudy V.C. Muller C. Epitope mapping and functional studies with three monoclonal antibodies to the c-kit receptor tyrosine kinase.J Cell Physiol. 1994; 158: 545-554Crossref PubMed Scopus (28) Google Scholar) by L. Ashman, Adelaide, Australia, and an HLA-DR antibody (Tü36) by A. Ziegler, Berlin Germany (Ziegler et al., 1986Ziegler A. Heinig J. Müller C. Götz H. Thinnes F.P. Uchanska-Ziegler B. Wernet P. Analysis by sequential immunoprecipitations of the specificities of monoclonal antibodies TÜ22, 34, 35, 36, 37, 39, 43, 58 and YD1/63.HLK directed against human HLA Class II antigens.Immunobiol. 1986; 171: 77-90Crossref PubMed Scopus (89) Google Scholar). Antibodies against CD1, CD3, CD11b, CD14, CD26, CD33, CD34, CD64, and CD68 as well as isotype matched control antibodies against desmin and laminin were all purchased from Dianova (Hamburg, Germany). Immunocytochemistry using the APAAP technique was performed on cytocentrifuge preparations of cells at days 0 and 14 of in vitro culture. Antibody reactivity was evaluated by two independent observers, counting at least 100 cells, recording distinctly stained cells only, as described before (Schadendorf et al., 1991Schadendorf D. Tiedemann K-.H. Haas N. Czarnetzki B.M. Detection of human papilloma viruses in paraffin-embedded condylomata accuminata – comparison of immunohistochemistry, in situ hybridization, and polymerase chain reaction.J Invest Dermatol. 1991; 97: 549-554Abstract Full Text PDF PubMed Google Scholar;Hamann et al., 1994Hamann K. Grabbe J. Welker P. Haas N. Algermissen B. Czarnetzki B.M. Phenotypic evaluation of cultured human mast and basophilic cells and of normal human skin mast cells.Arch Dermatol Res. 1994; 286: 380-385Crossref PubMed Scopus (54) Google Scholar). For flow cytometric analysis, 5 × 105 cells were incubated for 60 min at 4°C with the monoclonal receptor antibodies in 50 μl of phosphate-buffered saline (PBS), containing human AB serum (Seromed) (0.1%). After two washes with PBS, cells were incubated for 45 min at 4°C with a 1:20 dilution of fluorescein isocyanate (FITC)-conjugated anti-mouse IgG (DAKO, Denmark) or phycoerythrine (PE)-conjugated anti-mouse IgG (Dianova, Hamburg, Germany). Following two washes, cells were suspended in 400 μl PBS containing 0.1% NaN3, fixed with 50 μl of a 37% formaldehyde solution, and analysed on an EPICES Profile flow cytometer (Coulter, Krefeld, Germany). Negative controls were done in the absence of antibody, with IgG and isotype-matched desmin antibody D33 (DAKO). For flow cytometric analysis, the elite workstation software (Coulter) was used. The enzyme activity of mast cell tryptase was detected by cleavage of the peptide Z-Gly-Pro-Arg-pNA (4 mM) in the presence of heparin (5 mg per ml) and α-1-antitrypsin (2 mg per ml) (all from Sigma, St Louis, MO), as described before (Harvima et al., 1988Harvima I.T. Schechter N.M. Harvima R.J. Fraeki J.E. Human skin tryptase: purification, partial characterisation and comparison with human lung tryptase.Biochem Biophys Acta. 1988; 957: 71-80Crossref PubMed Scopus (82) Google Scholar). Cells were lysed by three cycles of freezing and thawing for determination of intracellular tryptase. Activity was expressed as mU per 106 cells. In order to analyse tryptase activities of FcεRI-positive and negative cells, PBMC cultured for 14 d were separated using the FcεRI antibody 29C6 and magnetic cell sorting with microbeads (MACS) (Miltenyi Biotec, Bergisch Gladbach, Germany), as described before (Zuberbier et al., 1999Zuberbier T. Guhl S. Handtke T. Handtke C. Welker P. Grabbe J. Henz B.M. Role of gangliosides during the in vitro human mast cell differentiation.Exp Dermatol. 1999; 8: 380-385Crossref PubMed Scopus (11) Google Scholar), yielding >95% purity of either cell population. Histamine was quantified in perchloric acid lysed cells and in cell supernatants, using a modified automated fluorometric method as described (Zuberbier et al., 1995Zuberbier T. Pfrommer C. Beinhölzl J. Hartmann K. Czarnetzki B.M. Gangliosides enhance IgE-receptor dependent histamine and LTC4 release from human mast cells.Biochem Biophys Acta. 1995; 1269: 79-84Crossref PubMed Scopus (17) Google Scholar). For studies of histamine release, cells were preincubated with IgE (1 μg per ml, Calbiochem, Bad Soden, Germany) for 30 min at 37°C, followed by addition of anti-IgE (4000 U per ml, Behring, Marburg, Germany) and another 30 min incubation. Samples were kept at -20°C until analysis. Cells from days 0 and 14 were lysed with 3 M lithium chloride and 6 M urea, centrifuged at 20 000 rpm for 60 min and extracted with phenol-chloroform, according toSambrock et al., 1989aSambrock J. Fritsch E.F. Maniatis T. Analysis of genomic DNA.in: Ford N. Nolan C. Ferguson M. Molecular Cloning. Cold Spring Harbor Laboratory Press, New York1989: 7.37-7.79Google Scholar). cDNA was synthesized by reverse transcription of 5 μl total RNA, using a cDNA synthesis kit (InVitrogen, Stade). The following sets of oligonucleotide primers were used to amplify cDNA (expected fragment lengths are given in parentheses): tryptase, 5′ GGA GCT GGA GGA GCC CGT GA and 5′ ACC TGG GTA AGG AAG CAG TGG TG (531 bp) (Miller et al., 1989Miller J.S. Westin E.H. Schwartz L.B. Cloning and characterisation of complementary DNA for human tryptase.J Clin Invest. 1989; 84: 1188-1195Crossref PubMed Scopus (174) Google Scholar); FcεRIα, 5′ CTG TTC TTC GCT CCA GAT GGC GT and 5′ TAC AGT AAT GTT GAG GGG CTG AG (536 bp); FcεRIβ, 5′ GGA CAC AGA AAG TAA TAG GAG AG and 5′ GAT CAG GAT GGT AAT TCC CGT T (446 bp) (Robertson et al., 1991Robertson M.W. Mehl V.S. Richards M.L. Fu-Tong L. m-RNA variants encoding multiple forms of the high-affinity IgE receptor alpha subunit in transformed and nontransformed mast cells.Int Arch Allergy Appl Immunol. 1991; 96: 289-295Crossref PubMed Scopus (8) Google Scholar;Schöneich et al., 1992Schöneich J.T. Victoria V.L. Kado-Fong H. Presky D.H. Kochan J.P. Association of the human FcεRI subunit with novel cell surface polypeptides.J Immunol. 1992; 148: 2181-2185PubMed Google Scholar); GAPDH, 5′ GAT GAC ATC AAG AAG GTG GTG and 5′ GCT GTA GCC AAA TTC GTT GTC (197 bp) (Togumaga et al., 1987Togumaga K. Nakamura Y. Sakata K. Fujimori F. Ohkubo M. Sawada K. Sakiyama S. Enhanced expression of glyceraldehyde-3-phosphate.Cancer Res. 1987; 47: 5616-5619PubMed Google Scholar); c-Kit, 5′ CGT TGA CTA TCA GTT CAG CG AG and 5′ CTA GGA ATG TGT AAG TGC CTC C (369 bp) (Ratajczak et al., 1992Ratajczak M.Z. Luger S.M. De Riel K. Abraham J. Calabretta B. Gewirtz A.M. Role of the KIT protooncogene in normal and malignant human hematopoiesis.Proc Natl Acad Sci USA. 1992; 89: 1710-1718Crossref PubMed Scopus (128) Google Scholar). Amplification was done using taq polymerase (GIBCO) over 35 cycles with an automated thermal cycler (Perkin Elmer, Germany). Each cycle consisted of the following steps: denaturation at 94°C, annealing at 55°C, and extension at 72°C for 1 min each. PCR products were analysed by agarose gel electrophoresis and enzymatic digestion, using standard techniques (Sambrook et al., 1989bSambrook J. Fritsch E.F. Maniatis T. Analysis of RNA.in: Ford N. Nolan C. Ferguson M. Molecular Cloning. Cold Spring Harbor Laboratory Press, New York1989: 9.31-9.57Google Scholar). Data are expressed as means ± 1 SD, and significant differences between values were calculated, using the unpaired two-tailed Student t-test. Prior to culture, <10% of adherent PBMC reacted with the FcεRIα antibody 29C6 on immunocytochemistry (Figure 1), and none were positive for mast cell specific tryptase and for c-Kit. The majority of cells stained with markers of the monocyte/macrophage lineage, namely CD11b, CD14, CD68, and HLA-DR (Table 1). No or hardly any CD1, CD3, CD16, CD33, or CD26-positive cells were detected and CD34+ cells were found at a frequency of <1:10,000 (100,000 cells counted).Table 1Immunoreactivity of adherent PBMC prior to and after 14 d of culture with LCS, as determined by the APAAP methodMaReactivity with human leukemic mast cells (HMC-1) and basophils (KU812) is shown for comparison. Data are expressed as means ± SD of the percentage of positively staining cells (N=3)% stained cellsAntibody (specificity)bMarkers coexpressed on mast cells are underlined.Known expression oncListing of relevant cell types only.PBMC day 0PBMC day 14HMC-1KU-812CD1Langerhans cells, B cells, dendritic cells0 ± 00 ± 00 ± 00 ± 0CD3T cells, dendritic cells0 ± 033 ± 15 *0 ± 00 ± 0CD11b (C3b)Monocytes, granulocytes, macrophages, basophils, mast cells77 ± 1593 ± 5*p<0.05.15 ± 565 ± 15CD14Monocytes, granulocytes, macrophages86 ± 1280 ± 80 ± 00 ± 0CD16 (FcγRIII)Monocytes, granulocytes, macrophages0 ± 037 ± 15*p<0.05.0 ± 00 ± 0CD33Monocytes, myeloic cells, mast cells0 ± 057 ± 9*p<0.05.n.d.n.d.dPreviously described to be positive (Hiroshi et al. 1995).CD34Hematopoietic stem cells≤0.10 ± 00 ± 00 ± 0CD64(FcγRI)Monocytes, macrophages0 ± 064 ± 11*p<0.05.0 ± 00 ± 0CD68Macrophages77 ± 2195 ± 5100 ± 0100 ± 0Tü36 (HLA-DR)Langerhans cells, monocytes, macrophages, mast cells65 ± 1895 ± 5*p<0.05.0 ± 00 ± 0CD26Activated T cells, B cells, macrophages<1<10 ± 00 ± 0Desmin(IgG1control)0 ± 00 ± 00 ± 00 ± 0LamininIgG2control)0 ± 00 ± 00 ± 00 ± 0a Reactivity with human leukemic mast cells (HMC-1) and basophils (KU812) is shown for comparison. Data are expressed as means ± SD of the percentage of positively staining cells (N=3)* p<0.05.b Markers coexpressed on mast cells are underlined.c Listing of relevant cell types only.d Previously described to be positive (Hiroshi et al., 1995Hiroshi S. Katsui M. Takahashi G. et al.Development of tryptase-positive KU812 cells cultured in the presence of Steel factor.Int Arch Allergy Immunol. 1995; 107: 330-332Crossref PubMed Scopus (8) Google Scholar). Open table in a new tab Double staining of the FcεRIα-positive cells with markers for monocytes and/or macrophages showed that 87% were also CD14+, CD68+, 45% stained weakly for CD64. None were reactive for c-Kit or CD34 (Table 2).Table 2Double staining of adherent PBMC prior to and after 14 d of culture with LCS, as determined by flow cytometryaData are expressed as means ± SD of the percentage of positively staining cells (N=3).% positive cellsDay 0Day 14FcεRIα (single staining) FcεRIα+ cells (100%) also positive for:7 ± 334 ± 12 CD1487 ± 590 ± 4 CD6816 ± 652 ± 10 CD6445 ± 12bFaint staining only.82 ± 15 c-KitnegativenegativecNote that intracellular staining was detected with the APAAP technique (Figure 1). CD34negativenegativea Data are expressed as means ± SD of the percentage of positively staining cells (N=3).b Faint staining only.c Note that intracellular staining was detected with the APAAP technique (Figure 1). Open table in a new tab Quantitative assessment of mRNA of selected molecules typically expressed on mast cells was done by serial dilution, w" @default.
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• W2011238768 date "2000-01-01" @default.
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• W2011238768 title "Mast Cell and Myeloid Marker Expression During Early In Vitro Mast Cell Differentiation from Human Peripheral Blood Mononuclear Cells" @default.
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• W2011238768 doi "https://doi.org/10.1046/j.1523-1747.2000.00827.x" @default.
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