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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 7 (October 1), 1999:
pp. 2333-2342
Demonstration That Human Mast Cells Arise From a Progenitor Cell
Population That Is CD34+, c-kit+,
and Expresses Aminopeptidase N (CD13)
By
Arnold S. Kirshenbaum,
Julie P. Goff,
Tekli Semere,
Barbara Foster,
Linda M. Scott, and
Dean D. Metcalfe
From Laboratory of Allergic Diseases, National Institute of Allergy
and Infectious Diseases, National Institutes of Health, Bethesda, MD;
and Department of Radiation Oncology, University of Pittsburgh Medical
Center, Pittsburgh, PA.
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ABSTRACT |
Human mast cells are known to arise from a
CD34+/c-kit+ progenitor cell
population that also gives rise to neutrophils, eosinophils, basophils,
and monocytes. To further characterize cells within the
CD34+/c-kit+ population that yield
mast cells, this progenitor was additionally sorted for CD13, a myeloid
marker known to appear early on rodent mast cells and cultured human
mast cells, but not expressed or expressed at low levels on human
tissue mast cells; and cultured in recombinant human (rh) stem cell
factor (rhSCF), rh interleukin-3 (rhIL-3; first week only), and rhIL-6.
Initial sorts revealed that although the majority of cells in culture
arose from the CD34+/c-kit+/CD13
cell population, mast cells arose from a
CD34+/c-kit+/CD13+
progenitor cell that also gave rise to a population of monocytes. Sequential sorting confirmed that
CD34+/c-kit+/CD13+
cells in
CD34+/c-kit+/CD13
sorts gave rise to the few mast cells observed in CD13
sorted cells.
CD34+/c-kit+/CD13+
cells plated as single cells in the presence of various cytokine combinations gave rise to pure mast cell, monocyte, or mixed mast cell/monocyte progeny. Addition of either rh
granulocyte-macrophage colony-stimulating factor
(rhGM-CSF) or rhIL-5 to the
CD34+/c-kit+/CD13+
progenitor cell population cultured in rhSCF, rhIL-3, and rhIL-6 did
increase the number of total cells cultured and in the case of rhIL-5,
did increase total mast cell numbers. Neither rhGM-CSF or rhIL-5 led to
additional cell populations, ie, even with the addition of rhGM-CSF or
rhIL-5, only mast cells and monocytes grew from
CD34+/c-kit+/CD13+
cells. Thus, human mast cells and a population of monocytes arise from
precursor cells that express CD34, c-kit, and CD13; and within which, are mast cell, monocyte, and mast/monocyte (bipotential) precursors.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
HUMAN MAST cells originate from
CD34+ progenitor cells when these cultures are maintained
in stem cell factor (SCF), thus defining the mast cell
precursor as
CD34+/c-kit+.1-5 However,
other cells including monocytes, basophils, eosinophils, and
neutrophils may also be cultured from this cell population. To further
define the mast cell precursor, we noted that CD34+ myeloid
progenitor cells have been reported to variably express CD13, CD33,
CD44, CD45, or CD117 (c-kit).6-10 Of particular
interest is CD13, detected on rodent mast cells11 and human
mast cells cultured from liver,12 but which is not
expressed or expressed at low levels by mast cells digested from human
tissues.13-16 CD13, a broadly distributed or myeloid marker
additionally known as aminopeptidase N and gp150, is a type II integral
membrane protein composed of 967 amino acids, and is expressed on the
cell surface as a homodimer.17 This membrane-bound
zinc-binding metalloprotease is known to be expressed during
hematopoiesis at several different stages of myeloid
differentiation.18
Because of its expression on cultured human mast cells and on murine
mast cells, we hypothesized that CD13 might help define an early human
mast cell progenitor. To explore this question, we sorted cells
expressing various combinations of CD34, c-kit, and CD13;
cultured them in selected growth factors; and examined the resulting
cell cultures over 8 weeks. As will be shown,
CD34+/c-kit+/CD13+ cells
preferentially gave rise to mast cells. The remaining cells were
monocytes.
CD34+/c-kit+/CD13
and
CD34+/c-kit/CD13+
cells in culture produced monocytes, eosinophils, basophils, and
neutrophils, but not mast cells. It thus seems that when CD13 is
expressed on CD34+/c-kit+ cells, it
becomes a marker of a progenitor cell population that includes mast
cell, monocyte, and mast cell/monocyte (bipotential) precursors.
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MATERIALS AND METHODS |
CD34+ immunoselection.
Human bone marrow (BM) from mastocytosis patients and peripheral blood
(PB) mononuclear cells from normal volunteers (collected by
leukapheresis) were obtained and processed, after informed consent was
given.19 Twenty milliliters of BM aspirates was collected
for study. Progenitor cells were purified by positive immunomagnetic
selection19 or using commercially available affinity columns.20 CD34+ cells were 95% to 99% pure
and used immediately or were aliquoted and frozen in liquid nitrogen
until ready for use.
Cell selection and fluorescence-activated cell sorting
(FACS) analysis.
CD34+/c-kit+/CD13+
progenitor cells were further sorted from BM or PB CD34+
cells and analyzed for surface antigens.19 For cell
sorting, 1 to 10 × 106 CD34+ cells were
first incubated in 1X phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (BSA) and 1% milk
for 1 hour at 37°C. Cells were then incubated with 1 µg/mL
R-phycoerythrin (PE)-conjugated mouse antihuman c-kit (1 mg/mL; Ancell, Bayport, MN), 1 µg/mL PE Cyanine 5 (PECy5)-conjugated
mouse antihuman CD13 (50 µg/mL; Immunotech, Westbrook, ME), and 10 µL of fluorescein isothiocyanate (FITC)-conjugated
mouse antihuman CD34 (anti-HPCA2; Becton Dickinson, San Jose, CA) for 1 hour at 37°C. Cells examined for Fc RI were incubated
overnight at 37°C with 2 µg/mL FITC-conjugated human
IgEPS (see below). Cells were then washed and resuspended in cold PBS containing 0.1% BSA. Control cells were unstained or
stained with an irrelevant mouse IgG1. Selection of
CD34+ subpopulations and cell analysis were performed using
a FACStarPlus (Becton Dickinson) and CellQuest software
(Becton Dickinson). Monocytes present in mast cell cultures were
characterized for surface expression of CD11b, CD14, CD15 (Becton
Dickinson), c-kit, and FcRI. For lysozyme
staining, paraformaldehyde-fixed slides were blocked for endogenous
peroxidase, incubated for 30 minutes in Tris-buffered saline containing
3% goat serum, rinsed, and then incubated for 2 hours with rabbit
antihuman lysozyme (DAKO, Carpinteria, CA). Secondary staining and
color development were performed on an automated immunostainer (Ventana
Medical Systems, Tucson, AZ).
Cell culture.
To assess hematopoietic potential, BM and PB
CD34+/c-kit+/CD13+,
CD34+/c-kit+/CD13,
CD34+/c-kit/CD13+,
and
CD34+/c-kit/CD13
cells were placed at a concentration of 5 × 104
cells/mL in serum-free media (StemPro-34 SFM; Life Technologies, Grand
Island, NY) supplemented with 2 mmol/L L-glutamine, 100 IU/mL
penicillin, 50 µg/mL streptomycin, 100 ng/mL recombinant human stem
cell factor (rhSCF), 100 ng/mL recombinant human interleukin-6 (rhIL-6), and 30 ng/mL rhIL-3 (first week only; Peprotech, Rocky Hill,
NJ). Hemidepletions were performed weekly with media containing 100 ng/mL rhSCF and 100 ng/mL rhIL-6.19 In some experiments, cultures were supplemented with 10 ng/mL recombinant human
granulocyte-macrophage colony-stimulating factor (rhGM-CSF) or 10 ng/mL
rhIL-5, with hemidepletions performed as above. Cell aliquots were
taken weekly for determination of total cell and mast cell numbers, and
for histochemical and immunohistochemical staining. Acidic toluidine blue (pH 1.0) was used to stain mast cell cytopreparations (Cytospin 3;
Shandon, Pittsburgh, PA) or sections fixed in a mixture of 2%
paraformaldehyde and 1.5% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer, pH 7.3, after sectioning for electron microscopy. Wright-Giemsa staining and qualitative tryptase enzyme determinations on cytocentrifuged cell preparations were performed as
described.1,19 For clonogenic cultures, cells in each well
were gently pipetted and placed on slides treated with
3-aminopropylethoxysilane (Digene, Beltsville, MD). The slides were
incubated in 5% CO2 in air at 37°C in a humidified
incubator for 30 to 40 minutes, centrifuged at 100g for 15 minutes, and stained with Wright-Giemsa.
Clonogenic cultures.
Single-cell liquid cultures were prepared as
described.21,22 Briefly, sequentially sorted and enriched
CD34+/c-kit+/CD13+ cells
were individually seeded into 96-well plates using the automated cell
deposition unit (ACDU; Becton Dickinson), which permits single-cell
sorting with an accuracy of greater than 99%. To allow for the cloning
of most cell phenotypes, 3 liquid culture cytokine mixture systems were
prepared using triplicate 96-well plates: rhIL-3 30 ng/mL, rhSCF 100 ng/mL, and rhIL-6 100 ng/mL; rhIL-3 30 ng/mL, rhSCF 100 ng/mL, rhIL-6
100 ng/mL, rh erythropoietin (rhEpo) 2 U/mL, and rh thrombopoietin
(rhTPO) 50 ng/mL (Peprotech)21; and rhIL-3 30 ng/mL, rhSCF
100 ng/mL, rhIL-6 100 ng/mL, rhEpo 2.5 U/mL, rhGM-CSF 10 ng/mL, rh
insulin-like growth factor-1 (rhIGF-1) 10 ng/mL, and rh basic
fibroblast growth factor (rhbFGF) 2.5 ng/mL (Peprotech).22
All cultures were incubated in 5% CO2 in air at 37°C.
Cell growth was checked on days 3, 5, 7, and 10. On day 14, cells were
scored for the presence of nonadherent cells, adherent cells, or a
mixture of both, and harvested.
FITC conjugation of IgEPS.
IgEPS was conjugated with FITC.23 Protein
concentration (mg/mL) was calculated using the formula:
Moles of FITC per mole of IgE was calculated using the formula:
Conjugated IgEPS was 2.66 mg/mL with 7.3 mol FITC per
mole IgEPS.
Immunomagnetic selection of FcRI+ cells.
2 × 106 cells were removed from
CD34+/c-kit+/CD13+ 2-week
cultures and incubated overnight at 37°C with 2 µg/mL
biotinylated human IgEPS. Cells were washed and resuspended
in 1 mL of PBS with 0.1% BSA and 100 ng/mL rhSCF, to which was added
25 µL (107 beads) of washed superparamagnetic,
polystyrene beads coated with recombinant streptavidin (CELLection Kit;
Dynal, Lake Success, NY). Cells were gently tilted and rotated for 15 minutes at 4°C, recovered with a magnet (Dynal MPC),
and washed 3 times. Cell counts and flow cytometric analysis of
rosetted cells revealed 50% to 60% recovery of >97%
FcRI+ cells. Cells were cultured in
serum-free media, as above, containing either 30 ng/mL rhIL-3 alone or
100 ng/mL rhSCF and 100 ng/mL rhIL-6.
Histamine release and analysis.
Four-week-old
c-kit+/CD13+/FcRI+
mast cells were harvested, incubated (5,000 cells/0.5 mL) with 2 µg/mL human IgEPS overnight at 37°C, washed, and
incubated with 10 µg/mL affinity-purified antihuman IgE (1 mg/mL;
Kirkegaard and Perry, Gaithersburg, MD) for 30 minutes at 37°C.
After incubation with 2 µg/mL human IgEPS, control cells
were incubated with either media alone or 1 µmol/L A23187. In all
cases, supernatants and cell pellets were examined for histamine using
an enzyme-linked immunosorbent assay (ELISA; Immunotech).19 Percentage histamine release was expressed
as the histamine measured in the supernatant divided by the sum of the
histamine in the supernatant and cell pellet.
Electron microscopy.
Mast cells cultured in rhSCF and rhIL-6 were harvested and fixed at
various times for electron microscopy and
immunohistochemistry.1,19 Sections were examined using a
Hitachi 7100 electron microscope (Hitachi, Japan).
Intracytoplasmic staining for tryptase and chymase.
Two-and 8-week-old
c-kit+/CD13+/FcRI+
mast cells were permeabilized and stained for tryptase and chymase as
reported.24 Briefly, 50 to 100 × 103
cells were fixed with 4% paraformaldehyde, washed, and incubated with
a blocking solution of 1X PBS-Saponin (PBS-S) containing 0.1% BSA and 1% milk for 1 hour at 37°C. For tryptase staining only, cells were incubated with 3 µg/mL mouse antihuman tryptase (1.14 mg/mL; Chemicon, Temecula, CA) for 1 hour at 37°C, washed, and incubated with 30 µg/mL PE-conjugated goat antimouse IgG (1 mg/mL; Southern Biotechnology, Birmingham, AL) for 30 minutes at
37°C. For chymase staining, cells first stained for tryptase were
washed and incubated with 3 µg/mL biotinylated mouse antihuman chymase (3 mg/mL; Chemicon) for 1 hour at 37°C, washed, and
incubated with 2 µg/mL allophycocyanine (APC)-conjugated streptavidin
for 30 minutes at 37°C. After staining, all cells were washed and resuspended in cold 1X PBS containing 0.1% BSA. Control cells were
unstained or stained with an irrelevant mouse IgG1. Cell analysis was performed using a FACScan and CellQuest software.
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RESULTS |
It has been documented that mast cells arise from an SCF-dependent
(c-kit+) CD34+ population of cells in
human BM and PB.1-5,19,25 Based on the reports that
CD13+ is expressed early in hematopoiesis as well as on
rodent mast cells, we hypothesized that this marker might help define
the subset of cells within the CD34+ population that is
committed to the mast cell lineage. As a first step, we separated
CD34+ cells and labeled these cells with antibodies to
CD34, c-kit, and CD13. We then analyzed these cells by forward
(FSC) and side (SSC) scatter using flow cytometry
(Fig 1A), and examined gated subpopulations
of cells for their expression of CD34, c-kit, and CD13. As can
be seen in Fig 1B, a subpopulation of cells was observed that was
CD34+, c-kit+, and CD13+ in
gates 1 and 2, whereas other CD34+ cells expressed various
combinations of CD34, c-kit, and CD13 in gates 3, 4, and 5. Nonviable or cell fragments (lowest forward and side scatter
parameters) were gated and eliminated before sorting, and therefore,
were not cultured. The remaining viable CD34+ population
was 95% to 99% pure. A series of cell subpopulations could thus be
identified within the CD34+ population that variously
expressed c-kit, CD13, or both.
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| Fig 1.
FSC, SSC, and surface antigen expression of BM-and
PB-derived CD34+ cells. (A) FSC and SSC display with 5 populations gated for further study. (B) Expression of CD13 versus
c-kit and CD34 in each gate.1-5 The population of
cells in gate 1 is
CD34+/c-kit+/CD13+;
gate 2 is mixed
CD34+/c-kit+/c-kit/CD13+/CD13;
gate 3 is mixed
CD34+/CD34/c-kit/CD13;
gate 4 is
CD34/c-kit+/CD13+;
and gate 5 is
CD34/c-kit/CD13.
Results shown are representative of 6 experiments separately performed,
using either BM (n = 3) or PB (n = 3) cells, and in which the FSC
by SSC patterns resemble one another.
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To determine which of these subpopulations included committed mast cell
progenitors, we sorted CD34+ cells
(Fig 2A) by flow cytometry into 4 distinct
populations of cells that were
CD34+/c-kit+/CD13+ (Fig
2B),
CD34+/c-kit+/CD13(Fig
2C),
CD34+/c-kit/CD13+
(Fig 2D), and
CD34+/c-kit/CD13
(Fig 2E). In 6 CD34+ sorts, the purity of
c-kit+/CD13+ cells averaged 85% ± 5%, c-kit+/CD13 cells 89% ± 3%, c-kit/CD13+ cells
68% ± 4%, and
c-kit/CD13 cells 86% ± 4%. Sorted cells were then placed in culture, and total and mast
cell numbers were determined over 8 weeks.
CD34+/c-kit/CD13+
and
CD34+/c-kit/CD13
cells did not appreciably expand in culture, and through 8 weeks, few,
if any, mast cells developed from these subpopulations
(Fig 3A and B). The most marked cell
expansion was seen from the
CD34+/c-kit+/CD13
subpopulation. Cell numbers increased 60-fold by 2 weeks and declined
in number thereafter. At all time points, mast cells constituted <3%
of the cells cultured from the CD13 subpopulation.
Basophils were detected at 2 weeks in cultures derived from
CD34+/c-kit+/CD13
and
CD34+/c-kit/CD13+,
but not
CD34+/c-kit+/CD13+- sorted
cells. The
CD34+/c-kit+/CD13+ cell
culture expanded 12-fold, peaked at 3 weeks, and declined thereafter.
In contrast with other subpopulations at 2 weeks, approximately 15% of
these cells could be identified as mast cells by morphologic criteria.
By 3 weeks, approximately 60% had the morphologic appearance of mast
cells, and by 4 weeks, approximately 85% were mast cells. Thus, the
CD34+/c-kit+/CD13+
subpopulation contained the mast cell progenitors that gave rise to
approximately 90% of the mast cells cultured from the 4 CD34+ subpopulations. Because of the starting purity of
these cell populations, the data supports the hypothesis that mast cell
progenitors are
CD34+/c-kit+/CD13+.
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| Fig 2.
CD34+ cells sorted into CD13+
and CD13 progenitor cells. (A) Presorted
CD34+ cells.
CD34+/c-kit+/CD13+
cells averaged 10% to 15%. (B) Postsorted
CD34+/c-kit+/CD13+
cells. (C) Postsorted
CD34+/c-kit+/CD13
cells. (D) Postsorted
CD34+/c-kit/CD13+
cells. (E) Postsorted
CD34+/c-kit/CD13
cells. Similar results were obtained in 6 experiments separately
performed, using either BM (n = 3) or PB (n = 3) cells in which the
percentage of cells in quadrants of interest did not significantly
differ between BM and PB.
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| Fig 3.
Total and mast cell numbers after 8 weeks in culture. (A)
Total cell numbers, and (B) mast cell numbers derived from
CD34+/c-kit+/CD13+
cells,
CD34+/c-kit+/CD13
cells,
CD34+/c-kit/CD13+
cells, and
CD34+/c-kit/CD13
cells. Results are shown as the mean ± SEM of 3 separate experiments.
PB cells were used and are representative of data from BM.
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The morphologic appearance of the initial
CD34+/c-kit+/CD13+ cell
population is shown in Fig 4, as well as
mast cells at 4 weeks. Sorted
CD34+/c-kit+/CD13+ cells at
day 0 were agranular and lymphoid in appearance with a large
nucleus/cytoplasm ratio and basophilic-staining cytoplasm, consistent
with immature hematopoietic progenitors (Fig 4A). The appearance of
mast cells containing metachromatic granules is shown for comparison at
4 weeks (Fig 4B).
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| Fig 4.
Wright-Giemsa staining and metachromasia of
CD34+/ c-kit+/CD13+
cells at 0 weeks (A), as well as mast cells at 4 weeks (B). Left panels
show light microscopy of Wright-Giemsa-stained cells. Right panels
show acid toluidine blue-positive cells (original magnification × 1,000).
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Ultrastructural examination of cultured
CD34+/c-kit+/CD13+ progeny
revealed cells with characteristics typical of cultured human mast
cells and monocytes, but not basophils.1,19 Four-week-old mast cells had numerous surface projections and contained cytoplasm with electron-dense lipid bodies and cytoplasmic granules with partial
scrolls, particles, dense cores, and homogeneously dense material
consistent with more mature cultured mast cells. Granules stained
strongly positive for tryptase. Monocytes were larger cells with
abundant cytoplasm and few granules with nonspecific electron-dense material.
Human mast cells variably express chymase, with most if not all mast
cells expressing tryptase.26 We first verified that the
majority of tryptase-containing cells were present within the
CD34+/c-kit+/CD13+-cultured
cell subpopulation. As can be seen in Fig
5, at 2 weeks approximately 59% of the total
CD34+/c-kit+/CD13+ progeny
in culture were tryptase-positive, whereas approximately 2% of cells
derived from
CD34+/c-kit+/CD13
cells in culture were tryptase-positive. This data is consistent with
previous observations that tryptase expression is observed during mast
cell differentiation.1,2 Eight-week-old mast cells were
further characterized by flow cytometry as having tryptase without
chymase (MCT), or both enzymes (MCTC). In these
cultures, 85% of the mast cells were found to exhibit both enzymes
(MCTC); 15% were only positive for tryptase
(MCT).
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| Fig 5.
FACS analysis of intracytoplasmic tryptase expression by
2-week-old c-kit+/CD13+ (A) and
c-kit+/CD13 (B) progeny. Right
panels show tryptase expression. Isotype controls are shown in left
panels. Results shown are representative of 4 experiments separately
performed, using either BM (n = 2) or PB (n = 2) cells.
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We next examined the cells cultured from
CD34+/c-kit+/CD13+
(Fig 6A) and the
CD34+/c-kit+/CD13
(Fig 6B) cell subpopulations at 2 weeks for the expression of FcRI, c-kit, and CD13. As can be seen, greater
than 60% of cell progeny in the
CD34+/c-kit+/CD13+ sort,
and greater than 40% of the cell progeny in the
CD34+/c-kit+/CD13
sort simultaneously expressed FcRI, c-kit, and
CD13. Because there are substantially more cells in Fig 6A, the
majority of mast cells were thus cultured from
CD34+/c-kit+/CD13+ cells
and, as expected, expressed FcRI and c-kit and
remained CD13+. FcRI+ progeny
derived from
CD34+/c-kit+/CD13+ cells at
2 weeks also behaved as mast cells rather than basophils when purified
and recultured. When immunomagnetically purified (>97%)
FcRI+ cells at 2 weeks were recultured in
either rhIL-3 alone or rhIL-6 and rhSCF for an additional 4 weeks, only
those FcRI+ cells cultured in rhIL-6 plus
rhSCF survived, as would be predicted for mast cells. No basophils were
observed.
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| Fig 6.
FACS analysis of 2-week-old
c-kit+/CD13+ (A) and
c-kit+/CD13 (B) progeny for CD13,
c-kit, and Fc RI expression; or c-kit and
FcRI expression on the cell population gated by FSC and
SSC to identify mast cells. Results shown are representative of 4 experiments separately performed, using either BM (n = 2) or PB (n
= 2) cells.
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Histamine was not detected in
CD34+/c-kit+/CD13+ cells on
day 0, but was approximately 2 to 3 pg/cell at 4 weeks of cell culture. Cells cultured from
CD34+/c-kit+/CD13+ cells
were functionally competent, as shown by the capacity to release
histamine. Using 4-week-old mast cells (Fig 4B), histamine release at
30 minutes after addition of 1 µmol/L A23187 averaged 90%. Histamine
release at 30 minutes from mast cells sensitized with IgEPS
and incubated with goat antihuman IgE averaged 23%.
The
CD34+/c-kit+/CD13+-sorted
cells, which in culture gave rise to mast cells, initially expressed
HLA-DR (80%), CD33 (90%), and CD38 (98%). By 2 to 3 weeks in
culture, the
c-kit+/CD13+/FcRI+
mast cells observed within the progeny of the
CD34+/c-kit+/CD13+ cells no
longer expressed CD34 and HLA-DR; none of the mast cell progeny of
CD34+/c-kit+/CD13+ cells
expressed CD3, CD11b, CD15, CD16, CD19, CD36, or erythrocyte markers at
2, 4, and 8 weeks. Monocytes expressed CD14 and CD15, but were negative
for detectable CD11b, FcRI and c-kit. Monocytes present in cultures also stained positive for lysozyme.
CD34+/c-kit+/CD13
cultures were consistently noted to give rise to small percentages
( 5%) of mast cells by 2 to 4 weeks. Because cell selection by
fluorescent cell sorting has technical limitations and therefore did
not allow for 100% pure
CD34+/c-kit+/CD13
cell sorting, we next determined if mast cells arising in
CD34+/c-kit+/CD13 cell
cultures might be derived from
CD34+/c-kit+/CD13+ cells
that contaminated these sorts.
CD34+/c-kit+/CD13+ cells
were therefore first sorted into
CD34+/c-kit+/CD13+ and
CD34+/c-kit+/CD13
cell populations.
CD34+/c-kit+/CD13
cells then underwent a second sort for CD13, yielding double-sorted CD34+/c-kit+/CD13+- and
CD34+/c-kit+/CD13-sorted
cells. These double-sorted cells were also then cultured. As shown in
Fig 7, the majority of mast cells observed
at 2 weeks (15%) and 4 weeks (85%) were again noted in cell cultures
derived from
CD34+/c-kit+/CD13+ (+/+)
sorts. Mast cells (5%) observed in
CD34+/c-kit+/CD13
(+/ ) cultures were reduced by greater than 50% after sequential sorting, confirming that mast cells within the first +/ sort originated from
CD34+/c-kit+/CD13+
progenitor cells.
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| Fig 7.
Effect of sequential sorting of
CD34+/c-kit+/CD13
progenitor cells on the appearance of mast cells at 2 (A) and 4 (B)
weeks of culture. Equal numbers (1.5 × 105 cells) of
c-kit+/CD13+ (+/+) and
c-kit+/CD13 (+/) cells from
the first sort were placed in culture (1st). A portion of
CD34+/c-kit+/CD13
cells underwent a second sort (2nd) into
c-kit+/CD13+ (+/+) and
c-kit+/CD13 (+/) cells that
were also placed in culture. Equal numbers (1.5 × 105
cells) were again plated. Results are presented as percentage of mast
cells and are representative of 2 experiments separately performed,
using PB cells.
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The CD34+/c-kit+/CD13+
progenitor cell population thus gives rise to the mast cells that
develop in culture. This
CD34+/c-kit+/CD13+
progenitor cell population also gives rise to monocytes. To determine whether mast cells and monocytes were derived from the same or different progenitors, single cell clonal assays were performed and
evaluated at 2 weeks. As shown in Table 1,
single CD34+/c-kit+/CD13+
cells seeded in wells using 3 different cytokine mixtures produced similar results and gave rise to either pure mast cells (11-18 clones/480 seeded wells), pure monocytes (2-6 clones/480 seeded wells),
or mixtures of mast cells and monocytes (10-16 clones/480 seeded
wells). No basophils or other cell phenotypes were seen. Mast cells
tended to be rounded (Fig 8A and B),
whereas monocytes adhered to the well bottom (Fig 8B and C).
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| Fig 8.
Phenotypic appearance of single cell clonal assays at 2 weeks by inverted scope. (A) Mast cell colony. (B) Mixed mast
cell/monocyte colony. (C) Monocyte colony.
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Mast cells and monocytes were again the only cell types present in
liquid suspension cultures examined later than 2 weeks. In 4-week
liquid suspension cultures containing only rhSCF and rhIL-6
(single-sorted
CD34+/c-kit+/CD13+ cells;
Table 2), mast cells constituted 1.50 of
1.82 or 82.4% of the cells in culture, and monocytes constituted 0.32 of 1.82 or 17.6%. Similarly, the double-sorted
CD34+/c-kit+/CD13+ cells
gave rise again only to mast cells at 4 weeks (1.00 of 1.21 or 83.3%)
and monocytes (0.21 of 1.21 or 17.4%). Thus, the CD34+/c-kit+/CD13+
progenitor cell population that gives rise to mast cells also gives
rise to a population of monocytes.
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Table 2.
Growth of Mast Cells From Single- and Double-Sorted
CD34+/c-kit+/CD13+
(+/+) and
CD34+/c-kit+/CD13
(+/) Cells
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To further explore the characteristics of the starting
CD34+/c-kit+/CD13+ and
CD34+/c-kit+/CD13
cell subpopulations, we next cultured these cells for 4 weeks in the
usual conditions of rhSCF, rhIL-3 (first week only), and rhIL-6, but
with and without rhGM-CSF or rhIL-5. The
CD34+/c-kit+/CD13+ cells
cultured in rhSCF, rhIL-3, and rhIL-6 at 4 weeks consisted of 80% mast
cells and 20% monocytes (Fig 9A), in
agreement with previous data. In contrast,
CD34+/c-kit+/CD13
cells cultured under these conditions consisted largely of monocytes. The addition of rhGM-CSF to the culture of
CD34+/c-kit+/CD13+ cells
increased the overall cell number, but resulted in fewer mast cells
being observed, the remainder being monocytes (Fig 9A). This inhibition
of mast cell growth by GM-CSF has been reported in murine27
and human28 systems.
CD34+/c-kit+/CD13
cells cultured with the addition of rhGM-CSF gave rise to a variety of
cell types in addition to monocytes, including basophils, eosinophils, and neutrophils (Fig 9B). Mast cells consisted of <5% of cells.
View larger version (17K):
[in this window]
[in a new window]
| Fig 9.
Effect of rhGM-CSF on the differentiation of
CD34+/c-kit+/CD13+
(A) and
CD34+/c-kit+/CD13
(B) cells cultured in rhSCF/rhIL-3/rhIL-6 over 4 weeks. Results shown
are the average of 3 experiments separately performed, using PB cells.
( ), Monocytes; ( ), mast cells; ( ), neutrophils; ( ),
basophils; ( ), eosinophils.
|
|
The addition of rhIL-5 to cultures of
CD34+/c-kit+/CD13+ cells
increased both the total number of cells and the mast cell number approximately 2-fold to 4-fold. Approximately 70% of cells cultured were mast cell and 30% were monocytes under both conditions
(Fig 10A). The addition of rhIL-5 to
CD34+/c-kit+/CD13
cells, as expected, increased the eosinophil number (Fig 10B). Mast
cells consisted of <5% of cells under both conditions. Thus, the
single-sorted
CD34+/c-kit+/CD13+ cell
subpopulation again gave rise to greater than 95% of mast cells
cultured, and this result was not altered by the addition of rhIL-5 or
rhGM-CSF. In addition, the data clearly show that rhIL-5 increases the
mast cell number in rhSCF/rhIL-3/rhIL-6-dependent cell cultures.
View larger version (16K):
[in this window]
[in a new window]
| Fig 10.
Effect of rhIL-5 on the differentiation of
CD34+/c-kit+/CD13+
(A) and
CD34+/c-kit+/CD13
(B) cells cultured in rhSCF/rhIL-3/rhIL-6 over 4 weeks. Results shown
are the average of 3 experiments separately performed, using PB cells.
(), Monocytes; (), mast cells; (), neutrophils; (),
basophils; (), eosinophils.
|
|
| |
DISCUSSION |
A progenitor cell population, capable of giving rise only to mast cells
and monocytes, has not been described. Such progenitor cells were
identified and sorted using FACS analysis for CD34, c-kit, and
CD13 surface antigen expression (Fig 1B), in addition to size (FSC) and
density (SSC) (Fig 1A). Cell-sorting parameters used to isolate
c-kit+/CD13+ progenitors from other
c-kit- or
CD13-committed progenitors were relatively easy to
establish once the larger, more dense BM and PB mast cell progenitor
cells with the
CD34+/c-kit+/CD13+ surface
antigen phenotype were identified (Fig 2B). The most marked cell
expansion (60-fold) was seen from the
CD34+/c-kit+/CD13
subpopulation (Fig 3A), in which mast cells constituted <3% of the
cells cultured (Fig 3B). In contrast,
CD34+/c-kit+/CD13+
cells expanded 12-fold, but were 85% mast cells by 4 weeks (Figs 3B
and 4). The remainder of the cells were monocytes (Table 2). Some mast
cells had an indented or segmented nucleus (Fig 4B). Similar cells have
been observed in human marrow,29 and rodent mast
cells have exhibited lobed nuclei.30
In the present work, cultured human mast cells were shown to express
FcRI at 2 weeks, similar to FcRI
expression on cultured human basophils and rodent mast
cells.31 Flow cytometric analysis of intracytoplasmic
tryptase with 2-week-old
c-kit+/CD13+/FcRI+
cell progeny identified a greater number of probable mast cells than
could be identified by acidic toluidine blue staining and tryptase
immunohistochemistry (Fig 5). Of interest, cultured mast cells at 8 weeks by flow cytometry were approximately 85% MCTC and
15% MCT, documenting that the
CD34+/c-kit+/CD13+
progenitor cell population gives rise to both MCT and
MCTC. The discrepancy between the larger number of tryptase
and chymase-positive (MCTC) mast cells observed with flow
cytometry rather than with Carnoy's-fixed cytopreparations and
immunohistochemistry may be a result of the negative effect on
chymase-positive mast cells by Carnoy's fixative32 and the
increased sensitivity of flow cytometry. BM and PB
c-kit+/CD13+/FcRI+
mast cell progeny at 8 weeks, derived from sorted progenitors, also had
phenotypic FACS profiles closely resembling mast cell cultures derived
from unsorted CD34+ progenitors that were allowed to mature
in culture over 8 weeks (data not shown).
We speculated that, because of the limitations of fluorescent cell
sorting, mast cells noted at 4 weeks in the
CD34+/c-kit+/CD13
cell cultures were a result of
CD34+/c-kit+/CD13+ cells
contaminating the starting cell population (Fig 3B). To prove this
hypothesis, we performed sequential cell sorting of CD34+/c-kit+/CD13
cells before cell culture to remove additional
CD34+/c-kit+/CD13+ (+/+)
cells from the
CD34+/c-kit+/CD13
(+/) subpopulation. This reduced by greater than 50% the number of mast cells arising in CD13 sorts at 4 weeks (Fig
7A and B and Table 2).
A close lineage relationship between mast cells and monocytes is
suggested by clinical findings in mastocytosis. In this disease of mast
cell neoplasia, monocytosis is frequently observed as the disease
progresses,33 and chronic myelogenous leukemia is observed
to arise in some patients.34,35 Thus, the observation that
the CD34+/c-kit+/CD13+
precursor cell population gives rise both to mast cells and monocytes (Tables 1 and 2; Figs 8, 9A, and 10A) under similar conditions is
consistent with these clinical observations of a close lineage relationship between mast cells and monocytes. Note that basophil neoplasia, for instance, is not observed in
mastocytosis.33,35
To verify the potential of
CD34+/c-kit+/CD13+ cells
and confirm the absence of other cell types at 2 weeks, single cell
clonal assays were performed. In addition, both rhGM-CSF and rhIL-5
were added to some liquid suspension cultures to see if other cell lineages might be observed from these starting cells at 4 weeks. Single
CD34+/c-kit+/CD13+ cells
cultured for 2 weeks with combinations of rhIL-3, rhIL-6, rhSCF, rhTPO,
rhEpo, rhGM-CSF, rhIGF-1, and rhbFGF gave rise only to pure mast cell,
monocyte, or mixed mast cell/monocyte clones (Table 1). No erythroid
precursors, myeloid cells, lymphocytes, or megakaryocytes were noted.
In liquid suspension cultures, both rhGM-CSF and rhIL-5 increased the
total number of cells cultured (Figs 9 and 10). As expected, rhGM-CSF
inhibited mast cell outgrowth (Fig 9A), whereas monocytes increased in
number. In contrast, rhIL-5 increased mast cell numbers, but again,
few, if any, mast cells originated from
CD34+/c-kit+/CD13
cells (Fig 10B). These observations using liquid suspension cultures would suggest that rhIL-5 may act as a growth and maturation and/or survival factor for mast cells, an observation especially relevant in
inflammatory conditions known to be associated with increased IL-5
levels, such as in asthma. Taken together, the data confirms that the
CD34+/c-kit+/CD13+ cell
population gives rise to only mast cells and monocytes, even in the
presence of rhIL-3, rhTPO, rhEpo, rhGM-CSF, rhIL-5, rhIGF-1, and
rhbFGF. It should be noted that monocytes are also observed to grow
from
CD34+/c-kit+/CD13
cells, suggesting that an earlier cell population that is
CD13 may be the ultimate monocyte precursor and that
it is possible that the bipotential cell and monocyte precursor cell
herein described arise from this earlier precursor. Most available data
point to the expression of CD13 on immature rather than mature human
mast cells. If CD13 is expressed during mast cell, monocyte, and
myeloid cell differentiation in the bone marrow,18 when
progenitors retain or lose their c-kit positivity, it is
possible to speculate that the mast cell/monocyte progenitor would
retain the c-kit+/CD13+ phenotype,
whereas other myeloid (ie, basophil) and certain monocyte progenitor
cells would lose c-kit expression but express CD13 as these
cells differentiate from earlier
c-kit+/CD13 cells.
Recently, it has been published that human mast cell progenitors are
both CD34+ and CD38+, and often lack
HLA-DR.36 When 1,200 single CD34+
CD38+ cells were cultured in rhSCF, rhIL-6, and rhIL-3 for
7 days, followed by 3 weeks in rhIL-3 or 7 weeks in rhSCF and rhIL-6, they obtained 13 mast cell colonies, 1 colony with mast cells and
another type, and 42 colonies of other cell types, including basophils,
macrophages, and eosinophils. They also reported mast cells in
CD34+,CD38, and HLA-DR+
sorts. The authors concluded that mast cells originate from progenitors different from myeloid progenitors. Although we agree with this conclusion, by using a sorting strategy based on 2 known mast cell
progenitor markers (CD34, c-kit) plus CD13, we were able to
obtain all mast cell precursors, with only one other cell type (monocyte) arising from this progenitor. Furthermore, no matter what
growth factors were added, 40% to 50% of all colonies were pure mast
colonies, and most of the other colonies were mixed mast cell/monocyte
colonies. It should be noted that our
CD34+/c-kit+/CD13+
progenitor cells are HLA-DR+ and express CD38. Thus, the
combination of CD34, c-kit, and CD13 more clearly defines the
human mast cell progenitor population.
Taken together, these results show that the
CD34+/c-kit+/CD13+ cell
population contains the precursors for all human mast cells, and a
subset of monocytes. Within the
CD34+/c-kit+/CD13+ cell
population are mast cell, monocyte, and mast cell/monocyte (bipotential) precursors. The proportion of mast cells to monocytes arising from
CD34+/c-kit+/CD13+ cells
depends on the growth factors added, but in no combination of cytokines
added were any other cell types noted.
| |
ACKNOWLEDGMENT |
The authors thank Stefania Pittaluga, Hemopathology Section, National
Cancer Institute (Bethesda, MD), for her excellent
technical assistance, and Dr Calman Prussin, Laboratory of Allergic
Diseases, NIAID (Bethesda, MD), for his advice.
| |
FOOTNOTES |
Submitted October 28, 1998; accepted June 4, 1999.
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.
Address reprint requests to Arnold S. Kirshenbaum, MD,
NIH/NIAID/Laboratory of Allergic Diseases, Building 10, Room 11C208, 10 Center Dr MSC 1881, Bethesda, MD 20892-1881; e-mail:
Akirshenba{at}atlas.niaid.nih.gov.
| |
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T. R. Hundley, A. M. Gilfillan, C. Tkaczyk, M. V. Andrade, D. D. Metcalfe, and M. A. Beaven
Kit and Fc{epsilon}RI mediate unique and convergent signals for release of inflammatory mediators from human mast cells
Blood,
October 15, 2004;
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[Abstract]
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J. K. Brown, P. A. Knight, A. D. Pemberton, S. H. Wright, J. A. Pate, E. M. Thornton, and H. R. P. Miller
Expression of Integrin-{alpha}E by Mucosal Mast Cells in the Intestinal Epithelium and Its Absence in Nematode-Infected Mice Lacking the Transforming Growth Factor-{beta}1-Activating Integrin {alpha}v{beta}6
Am. J. Pathol.,
July 1, 2004;
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[Abstract]
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C. Tkaczyk, V. Horejsi, S. Iwaki, P. Draber, L. E. Samelson, A. B. Satterthwaite, D.-H. Nahm, D. D. Metcalfe, and A. M. Gilfillan
NTAL phosphorylation is a pivotal link between the signaling cascades leading to human mast cell degranulation following Kit activation and Fc{epsilon}RI aggregation
Blood,
July 1, 2004;
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207 - 214.
[Abstract]
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M. Babina, S. Guhl, A. Starke, L. Kirchhof, T. Zuberbier, and B. M. Henz
Comparative cytokine profile of human skin mast cells from two compartments--strong resemblance with monocytes at baseline but induction of IL-5 by IL-4 priming
J. Leukoc. Biol.,
February 1, 2004;
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[Abstract]
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C. Tkaczyk, M. A. Beaven, S. M. Brachman, D. D. Metcalfe, and A. M. Gilfillan
The Phospholipase C{gamma}1-dependent Pathway of Fc{epsilon}RI-mediated Mast Cell Activation Is Regulated Independently of Phosphatidylinositol 3-Kinase
J. Biol. Chem.,
November 28, 2003;
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[Abstract]
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J. C. Qi, L. Li, Y. Li, K. Moore, M. C. Madigan, G. Katsoulotos, and S. A. Krilis
An Antibody Raised Against In Vitro-derived Human Mast Cells Identifies Mature Mast Cells and a Population of Cells that are Fc{varepsilon}RI+, Tryptase-, and Chymase- in a Variety of Human Tissues
J. Histochem. Cytochem.,
May 1, 2003;
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[Abstract]
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A. R. Migliaccio, R. A. Rana, M. Sanchez, R. Lorenzini, L. Centurione, L. Bianchi, A. M. Vannucchi, G. Migliaccio, and S. H. Orkin
GATA-1 as a Regulator of Mast Cell Differentiation Revealed by the Phenotype of the GATA-1low Mouse Mutant
J. Exp. Med.,
February 3, 2003;
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[Abstract]
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A. Pardanani, J.-Y. Baek, C.-Y. Li, J. H. Butterfield, and A. Tefferi
Systemic Mast Cell Disease Without Associated Hematologic Disorder: A Combined Retrospective and Prospective Study
Mayo Clin. Proc.,
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[Abstract]
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A. Maekawa, K. F. Austen, and Y. Kanaoka
Targeted Gene Disruption Reveals the Role of Cysteinyl Leukotriene 1 Receptor in the Enhanced Vascular Permeability of Mice Undergoing Acute Inflammatory Responses
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J. A. Boyce, E. A. Mellor, B. Perkins, Y.-C. Lim, and F. W. Luscinskas
Human mast cell progenitors use alpha 4-integrin, VCAM-1, and PSGL-1 E-selectin for adhesive interactions with human vascular endothelium under flow conditions
Blood,
April 15, 2002;
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[Abstract]
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M. Arock, E. Schneider, M. Boissan, V. Tricottet, and M. Dy
Differentiation of human basophils: an overview of recent advances and pending questions
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D. D. Hagaman, Y. Okayama, C. D'Ambrosio, C. Prussin, A. M. Gilfillan, and D. D. Metcalfe
Secretion of Interleukin-1 Receptor Antagonist from Human Mast Cells after Immunoglobulin E-Mediated Activation and after Segmental Antigen Challenge
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N. Bannert, M. Farzan, D. S. Friend, H. Ochi, K. S. Price, J. Sodroski, and J. A. Boyce
Human Mast Cell Progenitors Can Be Infected by Macrophagetropic Human Immunodeficiency Virus Type 1 and Retain Virus with Maturation In Vitro
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M. L. Taylor, J. Dastych, D. Sehgal, M. Sundstrom, G. Nilsson, C. Akin, R. G. Mage, and D. D. Metcalfe
The Kit-activating mutation D816V enhances stem cell factor-dependent chemotaxis
Blood,
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[Abstract]
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M. F. Gurish and K. F. Austen
The Diverse Roles of Mast Cells
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C. Chaves-Dias, T. R. Hundley, A. M. Gilfillan, A. S. Kirshenbaum, J. R. Cunha-Melo, D. D. Metcalfe, and M. A. Beaven
Induction of Telomerase Activity During Development of Human Mast Cells from Peripheral Blood CD34+ Cells: Comparisons with Tumor Mast-Cell Lines
J. Immunol.,
June 1, 2001;
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H. H. Mwamtemi, K. Koike, T. Kinoshita, S. Ito, S. Ishida, Y. Nakazawa, Y. Kurokawa, K. Shinozaki, K. Sakashita, K. Takeuchi, et al.
An Increase in Circulating Mast Cell Colony-Forming Cells in Asthma
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April 1, 2001;
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Y. Okayama, D. D. Hagaman, and D. D. Metcalfe
A Comparison of Mediators Released or Generated by IFN-{{gamma}}-Treated Human Mast Cells Following Aggregation of Fc{{gamma}}RI or Fc{{epsilon}}RI
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M. Iida, K. Matsumoto, H. Tomita, T. Nakajima, A. Akasawa, N. Y. Ohtani, N. L. Yoshida, K. Matsui, A. Nakada, Y. Sugita, et al.
Selective down-regulation of high-affinity IgE receptor (Fc{epsilon}RI) {alpha}-chain messenger RNA among transcriptome in cord blood-derived versus adult peripheral blood-derived cultured human mast cells
Blood,
February 15, 2001;
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[Abstract]
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A. Tefferi, C.-Y. Li, J. H. Butterfield, and H. C. Hoagland
Treatment of Systemic Mast-Cell Disease with Cladribine
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F. H. Falcone, H. Haas, and B. F. Gibbs
The human basophil: a new appreciation of its role in immune responses
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December 15, 2000;
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Y. Okayama, A. S. Kirshenbaum, and D. D. Metcalfe
Expression of a Functional High-Affinity IgG Receptor, Fc{gamma}RI, on Human Mast Cells: Up-Regulation by IFN-{gamma}
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April 15, 2000;
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H. Ochi, N. H. De Jesus, F. H. Hsieh, K. F. Austen, and J. A. Boyce
IL-4 and -5 prime human mast cells for different profiles of IgE-dependent cytokine production
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TOP
ABSTRACT
INTRODUCTION
