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BIBTEX RIS

Phenotypic Characterization of Human Skin Mast Cells by Combined Staining with Toluidine Blue and CD Antibodies

1998; Elsevier BV; Volume: 111; Issue: 4 Linguagem: Inglês

10.1046/j.1523-1747.1998.00359.x

ISSN

1523-1747

Autores

Minoo Ghannadan, Mehrdad Baghestanian, Friedrich Wimazal, Michael Eisenmenger, D Latal, Gülriz Kargül, Sabine Walchshofer, Christian Sillaber, Klaus Lechner, Peter Valent,

Tópico(s)

Monoclonal and Polyclonal Antibodies Research

Resumo

Mast cells (MC) are important cellular components of the immune network in diverse organs. The skin MC has likewise been implicated in IgE- and complement-mediated cutaneous reactions. Such reactions supposedly involve specific cell surface membrane receptors. In this study, the cell surface marker profile of human skin MC was established using monoclonal antibodies (MoAb) against defined CD antigens. MC were isolated from juvenile foreskin (n = 55) and adult mammary skin (n = 5). The reactivity of MC with MoAb was assessed by a combined toluidine blue/immunofluorescence staining technique. Confirming our previous analyses on lung MC, foreskin MC reacted with MoAb against CD9, CD29, CD33, CD43, CD44, CD45, CD46, CD51, CD54, CD55, CD58, CD59, CD61, and CD117 (c-kit). Foreskin MC were also recognized by MoAb to CD47, CD48, CD49d, CD53, CD60, CD63, CD81, CD82, CD84, CD87, CD92, CD97, CD98, and CD99. Recently clustered CD antigens detectable on foreskin MC were CD147 (neurothelin), CD149 (MEM133), CD151 (PETA-3), and CD157 (BST-1). In contrast to lung MC and MC from adult skin, foreskin MC were found to express CD88 (C5aR). Also, cutaneous MC (from both juvenile foreskin and adult mammary skin), but not lung MC, were found to bind the CD32 MoAb IV.3, 2E1, and FLI8.26 (FcγRII). The CD50 antigen (ICAM-3) was detectable on lung MC, but not on foreskin MC or MC of adult mammary skin. In summary, our data show that cutaneous MC and lung MC express an almost identical phenotype; however, in contrast to lung MC, cutaneous MC appear to express substantial amounts of CD32 and to lack CD50. In addition, foreskin MC, unlike MC from adult skin or lung, express CD88. Mast cells (MC) are important cellular components of the immune network in diverse organs. The skin MC has likewise been implicated in IgE- and complement-mediated cutaneous reactions. Such reactions supposedly involve specific cell surface membrane receptors. In this study, the cell surface marker profile of human skin MC was established using monoclonal antibodies (MoAb) against defined CD antigens. MC were isolated from juvenile foreskin (n = 55) and adult mammary skin (n = 5). The reactivity of MC with MoAb was assessed by a combined toluidine blue/immunofluorescence staining technique. Confirming our previous analyses on lung MC, foreskin MC reacted with MoAb against CD9, CD29, CD33, CD43, CD44, CD45, CD46, CD51, CD54, CD55, CD58, CD59, CD61, and CD117 (c-kit). Foreskin MC were also recognized by MoAb to CD47, CD48, CD49d, CD53, CD60, CD63, CD81, CD82, CD84, CD87, CD92, CD97, CD98, and CD99. Recently clustered CD antigens detectable on foreskin MC were CD147 (neurothelin), CD149 (MEM133), CD151 (PETA-3), and CD157 (BST-1). In contrast to lung MC and MC from adult skin, foreskin MC were found to express CD88 (C5aR). Also, cutaneous MC (from both juvenile foreskin and adult mammary skin), but not lung MC, were found to bind the CD32 MoAb IV.3, 2E1, and FLI8.26 (FcγRII). The CD50 antigen (ICAM-3) was detectable on lung MC, but not on foreskin MC or MC of adult mammary skin. In summary, our data show that cutaneous MC and lung MC express an almost identical phenotype; however, in contrast to lung MC, cutaneous MC appear to express substantial amounts of CD32 and to lack CD50. In addition, foreskin MC, unlike MC from adult skin or lung, express CD88. recombinant human mast cells mast cell growth factor stem cell factor Mast cells (MC) are proinflammatory effector cells of the immune system (Kaplan and Schwartz, 1985Schwartz L.B. The mast cell.in: Kaplan A.P. Allergy. Vol. 1. Churchill Livingston, Edinburgh1985: 53-92Google Scholar;Galli, 1993Galli S.J. New concepts about the mast cell.New Eng J Med. 1993; 328: 257-265Crossref PubMed Scopus (897) Google Scholar). These cells produce several vasoactive and other biologically relevant molecules (Lewis and Austen, 1981Lewis R.A. Austen K.F. Mediation of local homeostasis and inflammation by leukotrienes and other mast cell dependent compounds.Nature. 1981; 293: 103-108Crossref PubMed Scopus (366) Google Scholar;Serafin and Austen, 1987Serafin W.E. Austen K.F. Mediators of immediate hypersensitivity reactions.N Engl J Med. 1987; 317: 30-34Crossref PubMed Scopus (227) Google Scholar;Gordon et al., 1990Gordon J.R. Burd P.R. Galli S.J. Mast cells as a source of multifunctional cytokines.Immunol Today. 1990; 11: 458-464Abstract Full Text PDF PubMed Scopus (162) Google Scholar). Under certain conditions and in response to diverse stimuli, MC release their products and thereby can participate in a number of pathophysiologic processes (Kaplan and Siraganian, 1985Siraganian R.P. Biochemical events in basophil/mast cell activation and mediator secretion.in: Kaplan A.P. Allergy. Vol. 1. Churchill Livingston, Edinburgh1985: 31-51Google Scholar;Galli, 1990Galli S.J. New insights into "the riddle of the mast cells" microenvironmental regulation of mast cell development and phenotypic heterogeneity.Lab Invest. 1990; 62: 5-33PubMed Google Scholar,Galli, 1993Galli S.J. New concepts about the mast cell.New Eng J Med. 1993; 328: 257-265Crossref PubMed Scopus (897) Google Scholar;Dvorak and Harris, 1991Dvorak A.M. Basophil and mast cell degranulation and recovery.in: Harris J.R. Blood Cell Biochemistry. New York, Plenum Press1991Crossref Google Scholar;Walsh et al., 1991Walsh L.J. Trinchieri G. Waldorf H. Whitaker D. Murphy G.F. Human dermal mast cells contain and release tumor necrosis factor alpha, which induces endothelial leukocyte adhesion molecule-1.Proc Natl Acad Sci USA. 1991; 88: 4220-4224Crossref PubMed Scopus (535) Google Scholar). The various functional properties of MC are associated with expression of distinct cell surface membrane receptors (Valent and Bettelheim, 1992Valent P. Bettelheim P. Cell surface structures on human basophils and mast cells: Biochemical and functional characterization.Adv Immunol. 1992; 52: 333-423Crossref PubMed Scopus (179) Google Scholar). The mast cell growth factor receptor (= c-kit) is expressed on mature MC as well as on MC progenitors (Galli, 1993Galli S.J. New concepts about the mast cell.New Eng J Med. 1993; 328: 257-265Crossref PubMed Scopus (897) Google Scholar;Valent, 1994Valent P. The riddle of the mast cell: c-kit ligand as missing link?.Immunol Today. 1994; 15: 111-114Abstract Full Text PDF PubMed Scopus (109) Google Scholar;Rodewald et al., 1996Rodewald D.R. Dessing M. Dvorak A.M. Galli S.J. Identification of a committed precursor for the mast cell lineage.Science. 1996; 271: 818-822Crossref PubMed Scopus (292) Google Scholar). The ligand of c-kit, mast cell growth factor (MGF), also termed stem cell factor (SCF), induces differentiation, mediator secretion, and chemotaxis in human (and murine) MC (Irani et al., 1992Irani A.M. Nilsson G. Miettinen U. et al.Recombinant human stem cell factor stimulates differentiation of human mast cells from dispersed fetal liver cells.Blood. 1992; 80: 3009-3016Crossref PubMed Google Scholar;Bischoff and Dahinden, 1992Bischoff S.C. Dahinden C.A. c-kit Ligand: a unique potentiator of mediator release from human lung mast cells.J Exp Med. 1992; 175: 237-244Crossref PubMed Scopus (233) Google Scholar;Valent et al., 1992Valent P. Spanblöchl E. Sperr W.R. et al.Induction of differentiation of human mast cells from bone marrow and peripheral blood mononuclear cells by recombinant human stem cell factor (SCF) / kit ligand (KL) in long term culture.Blood. 1992; 80: 2237-2245Crossref PubMed Google Scholar;Mitsui et al., 1993Mitsui H. Furitsu T. Dvorak A.M. et al.Development of human mast cells from umbilical cord blood cells by recombinant human and murine c-kit ligand.Proc Natl Acad Sci USA. 1993; 90: 735-739Crossref PubMed Scopus (233) Google Scholar;Nilsson et al., 1994Nilsson G. Butterfield J.H. Nilsson K. Siegbahn A. Stem cell factor is a chemotactic factor for human mast cells.J Immunol. 1994; 153: 3717-3723PubMed Google Scholar). The mast cell high affinity receptor for IgE (immunoglobulin E) is involved in allergen-induced mediator release (Ishizaka and Ishizaka, 1984Ishizaka T. Ishizaka K. Activation of mast cells for mediator release through IgE receptors.Prog Allergy. 1984; 34: 188-235PubMed Google Scholar). MC also express a number of adhesion receptors and recognition molecules, as well as cell surface receptors for complement activation products (Valent and Bettelheim, 1992Valent P. Bettelheim P. Cell surface structures on human basophils and mast cells: Biochemical and functional characterization.Adv Immunol. 1992; 52: 333-423Crossref PubMed Scopus (179) Google Scholar). During the past few decades substantial evidence for the existence of subpopulations of MC has been accumulated (Befus et al., 1985Befus A.D. Bienenstock J. Denburg J.A. Mast cell differentiation and heterogeneity.Immunol Today. 1985; 6: 281-284Abstract Full Text PDF PubMed Scopus (18) Google Scholar;Irani et al., 1986Irani A.M. Schechter N.M. Craig S.S. Deblois G. Schwartz L.B. Two types of human mast cells that have distinct neutral protease compositions.Proc Natl Acad Sci USA. 1986; 83: 4464-4468Crossref PubMed Scopus (760) Google Scholar;Enerbäck, 1987Enerbäck L. Mucosal mast cells in rat and man.Int Arch Allergy Appl Immunol. 1987; 82: 249-255Crossref PubMed Scopus (70) Google Scholar;Kitamura, 1989Kitamura Y. Heterogeneity of mast cells and phenotypic changes between subpopulations.Ann Rev Immunol. 1989; 7: 59-76Crossref PubMed Google Scholar). Human MC can be subclassified based on expression of the two proteolytic MC enzymes, tryptase and chymase (Irani et al., 1986Irani A.M. Schechter N.M. Craig S.S. Deblois G. Schwartz L.B. Two types of human mast cells that have distinct neutral protease compositions.Proc Natl Acad Sci USA. 1986; 83: 4464-4468Crossref PubMed Scopus (760) Google Scholar), expression of cell surface membrane receptors (Füreder et al., 1995Füreder W. Agis H. Willheim M. et al.Differential expression of complement receptors on human basophils and mast cells.J Immunol. 1995; 155: 3152-3160PubMed Google Scholar), response to activating ligands, and distinct ultrastructural properties (Dvorak and Harris, 1991Dvorak A.M. Basophil and mast cell degranulation and recovery.in: Harris J.R. Blood Cell Biochemistry. New York, Plenum Press1991Crossref Google Scholar). A number of studies suggest that MC heterogeneity is an organ-specific phenomenon. In particular, major differences were found when phenotypic and functional properties were analyzed and compared in human cutaneous and "noncutaneous" MC. Specifically, cutaneous MC, but not lung MC, express large amounts of the mast cell specific enzyme chymase (Irani et al., 1986Irani A.M. Schechter N.M. Craig S.S. Deblois G. Schwartz L.B. Two types of human mast cells that have distinct neutral protease compositions.Proc Natl Acad Sci USA. 1986; 83: 4464-4468Crossref PubMed Scopus (760) Google Scholar). Furthermore, juvenile foreskin MC release their mediators in response to C5a, morphine, and substance-P, whereas lung MC were found to be unresponsive against these agonists (Benyon et al., 1987Benyon R.C. Lowman M.A. Church M.K. Human skin mast cells: their dispersion, purification, and secretory characterization.J Immunol. 1987; 138: 861-867PubMed Google Scholar;Lawrence et al., 1987Lawrence I.D. Warner J.A. Cohan V.L. Hubbard W.C. Kagey-Sobotka A. Lichtenstein L.M. Purification and characterization of human skin mast cells.J Immunol. 1987; 139: 3062-3069PubMed Google Scholar;Füreder et al., 1995Füreder W. Agis H. Willheim M. et al.Differential expression of complement receptors on human basophils and mast cells.J Immunol. 1995; 155: 3152-3160PubMed Google Scholar). We have recently described that MC derived from human foreskin, as opposed to lung or other visceral organs, express CD88, the receptor for the complement activation product C5a (Füreder et al., 1995Füreder W. Agis H. Willheim M. et al.Differential expression of complement receptors on human basophils and mast cells.J Immunol. 1995; 155: 3152-3160PubMed Google Scholar). These observations prompted us to look for additional biologic and antigenic differences between cutaneous and "noncutaneous" MC. For this purpose, we isolated human MC from juvenile foreskin, adult mammary skin, and lung tissue specimens, and analyzed the immunophenotypic and functional properties of these cells. A number of MoAb were used to immunophenotype MC. One part of MoAb were obtained from the IVth (Vienna 1989), Vth (Boston 1993), or VIth (Kobe 1996) International Workshops and Conferences on Human Leukocyte Differentiation Antigens. A specification of MoAb (CD, Ig subclass) is provided in Table 1 , Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, Table 8. The following MoAb were purchased: A2MRα-2 and A2MRβ-1 (both CD91) from American Diagnostica (Greenwich, CT); S-HCL3 (CD11c), L138 (CD13), and L60 (CD43) from Becton Dickinson (San Jose, CA); and HD39 (CD22) from Behring (Marburg, Germany). The MoAb VNR147 (CD51), W-CAM-1 (CD54), G3 (anti-tryptase), B7 (anti-chymase), and human myeloma IgE were from Chemicon (Temecula, CA); F10–44–2 (CD44) was from Cymbus Bioscience (Southampton, U.K.); and MoAb 366 (CD13) was from Coulter Clone (Hialeah, FL). The MoAb JC/70A (CD31), PD7/26 + 2B11 (CD45), PG-M1 (CD68R), and F8/86 (against von Willebrand factor, vWF) were purchased from Dako (Glostrup, Denmark); MoAb M10 (CD121a) and 1H7 (CD121b) were from Genzyme (Cambridge, MA); and BL4 (CD4), BEAR 1 (CD11b), K20 (CD29), 2E1 (CD32), QBEND10 (CD34), SZ2 (CD42b), J4–48 (CD46), J4.57 (CD48), HP2B6 (CD49a), HP2/1 (CD49d), AMF7 (CD51), 84H10 (CD54), and E124.2.8 (anti-IgE) were from Immunotech (Marseilles, France). The MoAb MEM-102 (CD48) was from Monosan (Uden, Netherlands), and WM15 (CD13), 2F3 (CD15s), 2H5 (CD15s), HIB22 (CD22), FLI8.26 (CD32), M-B371 (CD37), HIT2 (CD38), GoH3 (CD49f), IA10 (CD55), H19 (CD59), 68–5H11 (CD62E), DREG-56 (CD62L), JS-81 (CD81), A59 (CD89), and 9F5 (anti-IL-3Rα/CD123) were from Pharmingen (San Diego, CA). A MoAb against GM-CSFRα (CD116) was from Santa Cruz Biotechnology (Santa Cruz, CA), the anti-C5aR MoAb S5/1 and W17/1 (CD88) were from Serotec (Oxford, U.K.), and MoAb RFB4 (CD22) was from Southern Biotechnology (Birmingham, AL). The MoAb LAM-89 (anti-laminin) and BA-4 (anti-elastin) were purchased from Sigma (St. Louis, MO). The anti-c-kit MoAb YB5.B8 (CD117) was kindly provided by Dr. L.K. Ashman (University of Adelaide, Australia), and MoAb 1A2.C5 (CD117) was kindly sent by Dr. H.J. Bühring (University of Tübingen, Germany).Table 1Reactivity of mast cells with MoAb to cytokine receptorsaForeskin and lung MC were purified and prepared as described in the text. A combined toluidine blue/immunofluorescence staining technique was applied to determine the reactivity of primary MC with MoAb. At least three MC preparations (three different donors) were analyzed for each individual MoAb. HMC-1 cells were analyzed by flow cytometry. The results are expressed as percentage reactive cells using a score system: ++, more than 90% of cells reactive; +, 50%–90% of cells reactive; +/–, 20%–49% of cells reactive;–/+ , 10%–19% of the cells reactive;–, less than 10% of cells reactive. Results obtained with lung MC and HMC-1 (CD25–130) were found to correspond to our previous data (Agis et al. 1996).CDRMoAbIg-classReactivity of MoAb withSkin MCLung MCHMC-1025IL-2RαBC96IgG1–––/+040CD40LRG28–5IgG1––++095APO1/FAS7C11IgM––+105TGFβRF430C-5IgG1–––114G-CSFR129IgG1–––116GM-CSFRαhGM-CSFR-M1IgG1––+117SCFR/c-kitYB5.B8IgG1++++++118IFNRα/βIFNαR3IgG1–––/+121aIL-1RIhIL-1R1-M1IgG1–––121aIL-1RIM10IgG1–––121bIL-1RII1H7IgG2b–––/+122IL-2RβMikβ1IgG2a–––/+123IL-3Rα9F5IgG1–––126IL-6RαB-C22IgG1–––127IL-7RαIL-7RIgG–––w128IL-8RB-F25IgM–––130gp130AM64IgG––+w131β to IL-3/5R3D7IgG1–––132γ to IL-2/4/7/9RAG43CIgG1–––134OX40L106IgG1–––135FLT34G8IgG1–––w136MSP-RID1IgG2a––/+–w1374–1BB4B4IgG1–––140aPDGF-RAPR292IgG1–––140bPDGF-RBPR7212IgG1–––a Foreskin and lung MC were purified and prepared as described in the text. A combined toluidine blue/immunofluorescence staining technique was applied to determine the reactivity of primary MC with MoAb. At least three MC preparations (three different donors) were analyzed for each individual MoAb. HMC-1 cells were analyzed by flow cytometry. The results are expressed as percentage reactive cells using a score system: ++, more than 90% of cells reactive; +, 50%–90% of cells reactive; +/–, 20%–49% of cells reactive;–/+ , 10%–19% of the cells reactive;–, less than 10% of cells reactive. Results obtained with lung MC and HMC-1 (CD25–130) were found to correspond to our previous data (Agis et al., 1996Agis H. Füreder W. Bankl H.C. et al.Comparative immunophenotypic analysis of human mast cells, blood basophils and monocytes.Immunology. 1996; 87: 535-543Crossref PubMed Scopus (121) Google Scholar). Open table in a new tab Table 2Reactivity of mast cells with MoAb to integrinsaForeskin MC and lung MC were enriched and analyzed as described in the text. For the score of antibody reactivity see the footnote to Table 1. Results obtained with lung MC and HMC-1 were found to correspond to our previous data (Agis et al. 1996). VNR, vitronectin receptor.CDintegrinMoAbIg-classReactivity of MoAb withSkin MCLung MCHMC-1β129β chainK20IgG2a++++++49aVLA-1αHP2B6IgG1––+/–49bVLA-2α6F1IgG1–––/+49dVLA-4αHP2/1IgG1+/–++49fVLA-6αGoH3IgG2aκ–––β211aLFA-1α25–3-1IgG1–––11aLFA-1αMHM24IgG1–––11bC3biRVIM-12IgG1–––11bC3biRBEAR 1IgG1κ–––11cCR43.9IgG1–––11cCR4S-HCL3IgG2b–––18β chainMHM23IgG1–––β341αIIbPL1.64IgG1–––51VNRα13C2IgG1++–/+51VNRαAMF7IgG1+/–++/–61VNRβSZ.21IgG1+/–+/–+/–61VNRβY – 2/51IgG1κ+/––/+–/+β4104β4-chain439–9BIgG2b–––β7n.c.β7-chainF1B21IgG2a–––n.c.β7-chainF1B30IgG2a–––n.c.β7-chainF1B504IgG2a–––103HML-1αHML-1IgG2a–––a Foreskin MC and lung MC were enriched and analyzed as described in the text. For the score of antibody reactivity see the footnote to Table 1. Results obtained with lung MC and HMC-1 were found to correspond to our previous data (Agis et al., 1996Agis H. Füreder W. Bankl H.C. et al.Comparative immunophenotypic analysis of human mast cells, blood basophils and monocytes.Immunology. 1996; 87: 535-543Crossref PubMed Scopus (121) Google Scholar). VNR, vitronectin receptor. Open table in a new tab Table 3Reactivity of mast cells with MoAb to Ig-superfamily type recognition receptorsaFor score and specifications see the footnotes to Table 1 and Table 2. Results obtained with lung MC and HMC-1 were found to correspond to our previous data (Agis et al. 1996).CDantigenMoAbIg-classReactivity of MoAb withSkin MCLung MCHMC-102LFA-2O275IgG1––+/–28B7-ligandKOLT2IgG1–––31PECAM-1SG134IgG–––31PECAM-15 A2.G5IgG1–––54ICAM-1RR1/1IgG1++/–+54ICAM-184H10IgG1+/–+++56N-CAMLeu19IgG1–––58LFA-3TS2/9IgG1+++++80BB1/B7BB1IgM–––102ICAM-2CBR-IC2/1IgG2aκ––/+–102ICAM-2CBR-IC2/2IgG2aκ+/––/+–106VCAM-12G7IgG1–––a For score and specifications see the footnotes to Table 1 and Table 2. Results obtained with lung MC and HMC-1 were found to correspond to our previous data (Agis et al., 1996Agis H. Füreder W. Bankl H.C. et al.Comparative immunophenotypic analysis of human mast cells, blood basophils and monocytes.Immunology. 1996; 87: 535-543Crossref PubMed Scopus (121) Google Scholar). Open table in a new tab Table 4CD50 (ICAM-3) expression on MC derived from juvenile foreskin and other tissuesaForeskin MC, adult mammary skin MC, and lung MC were enriched and analyzed by MoAb as described in the text. HMC-1 cells were analyzed by flow cytometry. For definitions and score of antibody reactivity see the footnote to Table 1.CDRMoAbIg-classReactivity of MoAb withSkin MC(foreskin)Skin MC(adult skin)Lung MCHMC-150ICAM-3CBR-IC3/1IgG1κ–n.t.+++50ICAM-3BU68IgG1––+++50ICAM-3186–2G9IgG2b––+++50ICAM-3B-N2IgG––+++50ICAM-3B-P12IgG––+++50ICAM-3B-R1IgG––+/–++50ICAM-3AZN-ICM3.1IgG1κ–––/+++a Foreskin MC, adult mammary skin MC, and lung MC were enriched and analyzed by MoAb as described in the text. HMC-1 cells were analyzed by flow cytometry. For definitions and score of antibody reactivity see the footnote to Table 1. Open table in a new tab Table 5Reactivity of mast cells with MoAb to other adhesion receptors and related moleclusaFor definitions and score of antibody reactivity see the footnote to Table 1.CDRMoAbIg-classReactivity of MoAb withSkin MCLung MCHMC-104T4/Cl-IIRBL4IgG2a–––08T8/Cl-IRBB29IgM–––09p24MM2/57IgG2b++++++153-FALPM-81IgM–––15ssLex2F3IgMκ–––15ssLex2H5IgMκ–––30Ki-1AC10IgG2b–––36TSP-RESIVC7IgG1–––42aGPIX/gp53Beb1IgG1–––43LeukosialinL10IgG1++++++44Pgp-1A3D8IgG1++++++62EE-selectin68–5H11IgG1κ–––62LL-selectinDREG-56IgG1κ–––62PP-selectinG1IgG1–––86B7–2FUN-1IgG1–––138syndecan 1MI15IgG1–––144VE-cadherin55–7H1IgG1κ–––146MUC-18OJ79IgG1–––147NeurothelinUM-8D6IgG3+/––/+++147NeurothelinH84IgG2b++/–++147NeurothelinHI197IgG1–/+–/+++147NeurothelinHIM6IgG1–/+–/+++151PETA-311B1.G4IgG2a++++++151PETA-314A2.H1IgG1++++153CD30LM81IgG2b–––154CD40L5C8IgG2a–––162PSGL-1PL1IgG1κ––+/–164MGC-24105A5IgG3––+165gp37AD2IgG1––+166ALCAM-13A6IgG1–––a For definitions and score of antibody reactivity see the footnote to Table 1. Open table in a new tab Table 6Reactivity of mast cells with MoAb against immunoglobulin receptorsaFor definitions and score of antibody reactivity see the footnote to Table 1. Results obtained with lung MC and HMC-1 were found to correspond to our previous data (Agis et al. 1996).CDFc-receptorMoAbIg classReactivity of MoAb withSkin MCLung MCHMC-1CD89FcαRA59IgG1–––CD23FcεRIIMHM6IgG1–––CD64FcγRI22.2IgG1–––/+CD32FcγRIIIV.3IgG2b+–++CD32FcγRIIFLI8.26IgG2bκ+–++CD32FcγRII2E1IgG2a+–++CD16FcγRIII3G8IgG1–––a For definitions and score of antibody reactivity see the footnote to Table 1. Results obtained with lung MC and HMC-1 were found to correspond to our previous data (Agis et al., 1996Agis H. Füreder W. Bankl H.C. et al.Comparative immunophenotypic analysis of human mast cells, blood basophils and monocytes.Immunology. 1996; 87: 535-543Crossref PubMed Scopus (121) Google Scholar). Open table in a new tab Table 7Overview of reactivity of foreskin MC with MoAb: comparison with other MCaFor definitions and score of antibody reactivity see the footnote to Table 1. Results obtained with lung MC and HMC-1 correspond to our previous dataFüreder et al. 1995,Agis et al. 1996Skin MC++009, 029, 043, 044, 045RB, 046, 047, 055, 059, 060, 063, 082, 092, 097, 099, 117, w149, 151+032, 033, 045, 051, 053, 054, 058, 081, 084, 088, 098, 157+/–048, 049d, 061, 102, 147–/+083, 087–01a, 01b, 01c, 002, 003, 004, 005, 006, 007, 008, 010, 011a, 011b, 011c, 012, 014, 015, 015s, 016, 017, 018, 019, 020, 021, 023, 024, 025, 027, 028, 030, 031, 034, 035, 036, 037, 038, 039, 040, 041, 042a, 045RO, 049a, 049b, 049f, 050, 052, 056, 062E, 062L, 062P, 064, 065, 066, 067, 068, 069, 070, 071, 072, 073, 074, 075, 076, 077, 078, 079, 080, 085, 086, 089, 090, 091, 093, 094, 095, 096, 101, 103, 104, 105, 106, 108, 114, 115, 116, 118, 121a, 121b, 122, 123, 126, 127, w128, 130, 131, 132, 134, 135, w136, w137, 138, 139, 140a, 140b, 141, 142, 143, 144, w145, 146, 148, w150, 152, 153, 154, 155, 156, 162, 164, 165, 166Lung MC++009, 029, 043, 044, 046, 047, 048, 053, 055, 058, 059, 063, 081, 082, 084, 092, 097, 098, 099, 117, w149, 151+033, 049d, 050, 051, 054, 157, 163+/–060, 061, 069, 087, 090, 107b, 147–/+052, 093, 102, 108, 124, w136–001a, 001b, 001c, 002, 003, 004, 005, 006, 007, 008, 010, 011a, 011b, 011c, 014, 015, 015s, 016, 017, 018, 019, 020, 021, 023, 024, 025, 027, 028, 030, 031, 032, 034, 035, 036, 037, 038, 039, 040, 041, 042a, 049a, 049b, 049f, 056, 057, 062E, 062L, 062P, 064, 065, 066, 066b, 068, 070, 071, 072, 073, 074, 075, 076, 077, 078, 079b, 080, 083, 085, 086, 088, 089, 091, 094, 095, 096, 101, 103, 104, 105, 106, 114, 116, 118, 120a, 120b, 121a, 121b, 122, 123, 126, 127, w128, 130, 131, 132, 134, 135, w137, 138, 139, 140a, 140b, 141, 142, 143, 144, w145, 146, 148, w150, 152, 153, 154, 155, 156, 162, 164, 165, 166HMC-1++009, 013, 029, 032, 037, 040, 043, 044, 046, 047, 048, 050, 053, 054, 055, 058, 059, 063, 066, 081, 082, 084, 087, 092, 097, 098, 099, 117, 147, w149, 151, 155+033, 049d, 072, 088, 095, 116, 130, 164, 165+/–002, 049a, 051, 052, 061, 071, 101, 108, 118, 157, 162–/+025, 049b, 060, 064, 118, 120a, 120b, 121b, 122, 152-001a, 001b, 001c, 002R, 003, 004, 005, 006, 007, 008, 010, 011a, 011b, 011c, 012, 014, 015, 015s, 016, 017, 018, 019, 020, 021, 023, 024, 027, 028, 030, 031, 034, 035, 036, 038, 039, 041, 042a, 049f, 056, 062E, 062L, 062P, 065, 066b, 068, 069, 070, 073, 074, 075, 076, 077, 078, 079b, 080, 083, 085, 086, 089, 090, 091, 093, 094, 096, 102, 103, 104, 105, 106, 114, 121a, 123, 124, 126, 127, w128, 131, 132, 134, 135, w136, w137, 138, 139, 140a, 140b, 141, 142, 143, 144, w145, 146, 148, w150, 153, 154, 156, 163, 166a For definitions and score of antibody reactivity see the footnote to Table 1. Results obtained with lung MC and HMC-1 correspond to our previous dataFüreder et al., 1995Füreder W. Agis H. Willheim M. et al.Differential expression of complement receptors on human basophils and mast cells.J Immunol. 1995; 155: 3152-3160PubMed Google Scholar,Agis et al., 1996Agis H. Füreder W. Bankl H.C. et al.Comparative immunophenotypic analysis of human mast cells, blood basophils and monocytes.Immunology. 1996; 87: 535-543Crossref PubMed Scopus (121) Google Scholar Open table in a new tab Table 8Reactivity of antibodies on paraffin embedded juvenile foreskinaIn situ detection of antigens in sections of paraffin embedded foreskin. The applied immunohistochemistry technique is described in the text. The applicability in paraffin embedded sections and the specificity of the MoAb were tested by evaluating their reactivity with reference cells known to express the respective antigen.CDAntigenCloneIg-classReactivity of MCReference cells31PECAM-1JC/70AIgG1–endothelial cells34HPCA-1QBEND10IgG1–endothelial cells43leukosialinL60IgG1+lymphocytes44HCAMF10–44–2IgG2a+lymphocyte45LCAPD7/26 + 2B11IgG1+lymphocytes51integrin αVVNR147IgG1+osteoclastsbIn the case of CD51, a bone marrow section was used to control reactivity with reference cells.54ICAM-1W-CAM-1IgG+keratinocytes68RmacrosialinPG-M1IgG3+macrophages117SCF-R1 A2.C5IgG3+mast cellsn.c.α-vWFF8/86IgG1–endothelial cellsn.c.MC-tryptaseG3IgG1+mast cellsn.c.MC-chymaseB7IgG1+mast cellsn.c.lamininLAM-89IgG1–vessel wall cellsn.c.elastinBA-4IgG1–vessel wall cellsa In situ detection of antigens in sections of paraffin embedded foreskin. The applied immunohistochemistry technique is described in the text. The applicability in paraffin embedded sections and the specificity of the MoAb were tested by evaluating their reactivity with reference cells known to express the respective antigen.b In the case of CD51, a bone marrow section was used to control reactivity with reference cells. Open table in a new tab Collagenase type II was purchased from Sebak (Suben, Austria), fetal calf serum was from Hyclone (Logan, Utah), and L-glutamine, penicillin/streptomycin, gentamycin, and fungizone were from Gibco Life Technologies (Gaithersburg, MD). Recombinant human (rh) SCF, as well as rhIL-1, rhIL-2, rhIL-4 through rhIL-11, rhIL-13, and rhIL-15 were from Peprotech (Rocky Hill, NY), rhIL-3 was from Sandoz (Vienna, Austria), rhIL-12 was from R&D Systems (Minneapolis, MN), rhC5a, toluidine blue, protease type XXIV, and 3-amino-9-ethylcarbazole were from Sigma, and tris-(hydroxymethyl)-aminomethan was from Biomol (Hamburg, Germany). Fluorescein isothiocyanate-goat F(ab′)2 anti-mouse IgG and fluorescein isothiocyanate-goat anti-rat IgG were from Caltag Laboratories (San Francisco, CA) and human AB-serum from Sera Lab (Crawley Down, U.K.). Biotinylated horse anti-mouse IgG and avidin-biotin-peroxidase complex (ABC Elite kit) were from Vector Laboratories (Burlingame, CA). Biotinylated goat anti-mouse immunoglobulin and peroxidase conjugated streptavidin were purchased from Biogenex Laboratories (San Ramon, CA). Histamine release buffer (Immunotech) contained 20 mM PIPES, glucose (1 g per liter), and 1 mM CaCl2. One litre of Mg/Ca-free Tyrode's buffer contained 0.2 g KCl, 0.05 g NaH2 PO4.H2O, 8.0 g NaCl, and 1 g glucose. Iscove's modified Dulbecco's medium was purchased from Gibco Life Technologies and RPMI 1640 medium from PAA Laboratories (Linz, Austria). Foreskin was obtained from 55 children (age 2 mo to 16 y; median, 4 y; mean, 5 y) undergoing circumcision. Informed consent was obtained from parents in each case. Normal mammary skin was obtained from five adult females who underwent reduction surgery after informed consent was obtained. Resected skin specimens were transported to the laboratory immediately after resection. Cutaneous MC were isolated according to standard procedures (Füreder et al., 1995Füreder W. Agis H. Willheim M. et al.Differential expression of complement receptors on human basophils and mast cells.J Immunol. 1995; 155: 3152-3160PubMed Google Scholar). In brief, tissue was cut into small pieces and washed in Tyrode's buffer. Tissue fragments were incubated in collagenase type II (2 mg per ml) for 90–180 min at 37°C. After digestion, cells were recovered, washed, and examined for MC by toluidine blue staining. Isolated foreskin MC were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum at 37°C for at least 12 h before being used in immunostaining or release experiments. Human lung MC were enriched from lung tissue obtained from patients with bronchiogenic carcinoma (n = 24, after informed consent) using collagenase type II as described (Schulman et al., 1982Schul

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