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Mast Cells Cultured From the Peripheral Blood of Normal Donors and Patients With Mastocytosis Originate From a CD34+/Fc6RI- Cell Population By Menachem Rottem, Tadashi Okada, Julie P. Goff, and Dean D. Metcalfe Mast cells may be cultured from humapne ripheral blood in the presence of recombinant human stemc ell factor( rhSCF). The characteristics of the cells in peripheral blood that give rise t o mast cells are unknown. Because mast cellp recursors in human marrow are CD34+, human peripheral blood mononuclear cells from patientsw ith 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 (rhlL-31, or both for 6 weeks. Cell cultures were examined every 2 weeks for total and mascte ll numbera nd cell differential using Wright Giemsa and acid toluidine bluest ains and antibodies to mast cell tryptase and chymase, cell-associated histamine, and expression of CD34, c-&it, Fc,RI, and Fc,.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 rhlL-3 gave rise to cell cultures consistingo f greater than 80% mast cells by 6 weeks. CD34+ cells cultured in rhlL-3 alone did not give rise to mast cells, whereas rhlL-3 plus rhSCF increased the final mast celln umber eightfold when compared with cells cultured in rhSCF alone. Mast cells increased concomitantly M AST CELLS ORIGINATE from pluripotential hematopoietic cells within the marrow',2 and reach their mature phenotype within t i s s~e sI.t~ is believed that the mast cell progenitors arising within bone marrow (BM) reach peripheral tissues after distribution via the peripheral circulation. Indeed, mast cells cannot be identified in peripheral blood (PB) under normal conditions: and a murine mononuclear cell population separated by density gradients and giving rise to mast cells has been de~cribed.T~h e concept is further supported by the demonstration that human PB mononuclear cells (PBMC) cultured in conditioned medium derived from human placenta, supernatants from a human lymphocyte leukemic cell line, and phytohemagglutininstimulated normal human peripheral blood lymphocytes give rise to histamine-containing colonies resembling mast cells/ basophik6 More recently, it has been shown that human cord blood and blood from adult donors cultured in the presence of c-kif ligand (stem cell factor [SCF]) give rise to human mast cells defined by traditional criteria.'.* In an attempt to identify the mast cell progenitor within human PB, we hypothesized that, as is true for human marrowt,h is cell would be CD34+, and might or might not express Fc,RI. As will be shown, a CD34' Fc," cell population within PB does give rise to mast cells when culturined S CF. Interleukin-3 (IL-3) enhances this SCFdependent mast cell proliferation. The CD34- population does not give rise to mast cells. The kinetics of the appearance of mast cells; their morphologic features; expression of c-kif, Fc,RI, and Fc,RII, and cell-associated histamine are also examined. The number of mast cells arising from CD34+ cell populations obtained from normal subjects and patients with indolent or aggressive mastocytosis9 is compared. MATERIALS AND METHODS Cell separation. PB was obtained from 8 patients with indolent systemic mastocytosis, 2 patients with aggressive mastocytosis, and Blood, Vol 84, No 8 (October 151, 1994: pp 2489-2496 with a decrease in large undifferentiated mononuclear cells. CD34- cells did not give rise to mast cells. Histamine content per cell a6t 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 (66%) and Fc,RII (95%). but not Fc,RI, by fluorescence-activated cell sorter analysis. At 6 weeks, CD34+-derived mast cells exhibited Fc,RI in addition to Kit and FcJtII, 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+/FceRl- and gives rise to mast cells in the presence of rhSCF with or without rhlL-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. This is a US government work. There are no restrictions on its use. 3 normal controls according to an Institutional Review Board approved protocol and after obtaining informed consent. BM was similarly obtained from 3 patients with indolent systemic mastocytosis after obtaining informed consent. PBMC and BM-derived mononuclear cells (BMMC) were obtained using Ficoll Hypaque (density, 1.077 to 1.080 g/mL; Organon Teknika CO, Durham, NC). CD34+ cells were separated from PBMC and BMMC by an avidin column using a Ceprate LC CD34 kit (CellPro, Inc, Bothell, WA), as described."," Briefly, PBMC or BMMC were centrifuged at 8OOg for 10 minutes and resuspended in phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) twice at a density of 1 X 10' cellslml. PBMC or BMMC were then incubated at room temperature for 25 minutes in the presence of mouse antihuman CD34 IgM monoclonal antibody (CD34 MoAb, 40 pg/mL). Cells were washed twice, resuspended to 100 x lo6 cellslmL in PBS with 1% BSA, and incubated at room temperature for an additional 25 minutes in the presence of biotinylated rat antimouse IgM (0.5 pgl mL). Cells were next washed once, resuspended in PBS containing 5% BSA, and placed over an avidin column. The column was washed with 6 mL PBS to collect the CD34- cell population, after which the column was agitated to release the CD34+ cell population. The From the Allergic Diseases Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD; and theD ivision of Molecular Virology and Immunology, Georgetown Universiy Medical Center, Rockville, MD. Submitted August 16, 1993; accepted June 13, 1994. Address reprint requests to Dean D. Metcalfe, MD, National Institutes of Health, Bldg IO, Room IIC-205, 9000 Rockville Pike, Bethesda, MD 20892. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact. This is a US government work. There are M restrictions on its use. 0006-4971/94/8408-0002$0.00/0 2489
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