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
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