Mast cells cultured from the peripheral blood of normal donors and patients with mastocytosis originate from a CD34+/Fc epsilon RI- cell population.
Mast cells may be cultured from human peripheral blood in the presence of recombinant human stem cell factor (rhSCF). The characteristics of the cells in peripheral blood that give rise to mast cells are unknown. Because mast cell precursors in human marrow are CD34+, human peripheral blood mononuclear cells from patients with mastocytosis and normal controls were sorted on the basis of CD34 expression and the positive and negative cell populations were cultured in rhSCF, recombinant human interleukin-3 (rhIL-3), or both for 6 weeks. Cell cultures were examined every 2 weeks for total and mast cell number and cell differential using Wright Giemsa and acid toluidine blue stains and antibodies to mast cell tryptase and chymase, cell-associated histamine, and expression of CD34, c-kit, Fc epsilon RI, and Fc gamma RII using flow cytometric analysis. The ultrastructural anatomy of mast cells was examined by electron microscopy. Peripheral blood CD34+ cells cultured in rhSCF with or without rhIL-3 gave rise to cell cultures consisting of greater than 80% mast cells by 6 weeks. CD34+ cells cultured in rhIL-3 alone did not give rise to mast cells, whereas rhIL-3 plus rhSCF increased the final mast cell number eightfold when compared with cells cultured in rhSCF alone. Mast cells increased concomitantly with a decrease in large undifferentiated mononuclear cells. CD34- cells did not give rise to mast cells. Histamine content per cell at 6 weeks was approximately 5 pg. Electron microscopy of 4-week cultures showed immature mast cells containing predominantly tryptase-positive granules that were either homogeneous or contained lattice structures, partial scroll patterns, or central dense cores and mixtures of vesicles, fine granular material, and particles. The CD34+ population at day 0 expressed Kit (65%) and Fc gamma RII (95%), but not Fc epsilon RI, by fluorescence-activated cell sorter analysis. At 6 weeks, CD34+-derived mast cells exhibited Fc epsilon RI in addition to Kit and Fc gamma RII, and were negative for CD34 antigen. Patients with mastocytosis showed a higher number of mast cells per CD34+ cell cultured compared with normal controls. Thus, the mast cell precursor in human peripheral blood is CD34+/Fc epsilon RI- and gives rise to mast cells in the presence of rhSCF with or without rhIL-3, and the number of mast cells arising per CD34+ cell in culture is greater when the CD34+ cells are obtained from patients with mastocytosis compared with normal subjects.
Rottem
M
M
Allergic Diseases Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892.
Okada
T
T
Goff
J P
JP
Metcalfe
D D
DD
D015703:Antigens, CD / Q000032:analysis*; D018952:Antigens, CD34; D002454:Cell Differentiation; D002469:Cell Separation; D002478:Cells, Cultured; D053818:Chymases; D003594:Cytoplasmic Granules / Q000201:enzymology / Q000648:ultrastructure; D005434:Flow Cytometry; D016298:Hematopoietic Cell Growth Factors / Q000494:pharmacology; D006632:Histamine / Q000032:analysis; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology; D007700:Kinetics; D018655:Lymphocyte Count; D008407:Mast Cells / Q000166:cytology* / Q000276:immunology; D008415:Mastocytosis / Q000473:pathology*; D008854:Microscopy, Electron; D011518:Proto-Oncogene Proteins / Q000032:analysis; D019009:Proto-Oncogene Proteins c-kit; D020794:Receptor Protein-Tyrosine Kinases / Q000032:analysis; D016184:Receptors, Colony-Stimulating Factor / Q000032:analysis; D017455:Receptors, IgE / Q000032:analysis*; D011994:Recombinant Proteins / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000032:analysis; D019089:Stem Cell Factor; D053802:Tryptases
D015703:Antigens, CD; D018952:Antigens, CD34; D016298:Hematopoietic Cell Growth Factors; D007377:Interleukin-3; D011518:Proto-Oncogene Proteins; D016184:Receptors, Colony-Stimulating Factor; D017455:Receptors, IgE; D011994:Recombinant Proteins; D019089:Stem Cell Factor; D006632:Histamine; D019009:Proto-Oncogene Proteins c-kit; D020794:Receptor Protein-Tyrosine Kinases; D012697:Serine Endopeptidases; C083294:chymase 2; D053818:Chymases; D053802:Tryptases