-
Selective Growth of Human Mast Cells Induced by Steel Factor,
11-6, and Prostaglandin E, from Cord Blood Mononuclear Cells'
Hirohisa Saito,** Motohiro Ebisawa,* Hiroshi Tachimoto,*
Michitaka Shichijo,* Kazumi Fukagawa,* Kenji Matsumoto,* Yoji
likura,* Takeo Awaji,+ Gozo Tsujimoto,+ Makoto Yanagida,* Hiroya
Uzumaki,* Gen Takahashi,§ Koichiro Tsuji,' and Tatsutoshi
Nakahata'
To establish the method for generating a large number of mature
human mast cells, we cultured cord blood mononuclear cells (CBMC)
in several conditions in the presence of Steel factor (SF). Among
several cytokines tested, 11-6 enhanced SF-dependent mast cell
growth from purified CD34+ cells for more than 8 wk in culture.
When CBMC were cultured instead of CD34+ cells, IL-6 enhanced the
mast cell development in the presence but not in the absence of
PGE,. PGE, enhanced the SF- and IL-6- dependent development of mast
cells from CBMC probably by blocking granulocyte-macrophage CSF
(GM-CSF) secretion from accessory cells, because 1 ) PGE, or
anti-GM-CSF enhanced the mast cell development induced by SF and
11-6 from CBMC, but not from CD34+ cells; 2) CM-CSF inhibited the
enhancing effect of 11-6 on the mast cell development from CD34+
cells; and 3) PGE, inhibited CM-CSF secretion from CBMC. The mast
cells cultured in the presence of SF, 11-6, and PGE, for >10 wk
were 99% pure, and seemed to be functionally mature, because 1)
they contained 5.62 p g of histamine and 3.46 p g of tryptase per
lo6 cells; and 2) when sensitized with human IgE and then
challenged with anti-human IgE, the cells released a variety of
mediators such as histamine, and an increase in intracellular Caz+
was found in advance of the activation of membrane movement by
using a confocal laser-scanning microscope. Electron-microscopic
analysis revealed that some of the cultured mast cells are
morphologically mature since they filled with scroll granules and
contained crystal granules. The journal of Immunology, 1996,157:
343-350.
M ast cells play a central role in allergic inflammation by
releasing various kinds of cytokines as well as vaso- active
mediators (1, 2). Since mast cell heterogeneity has been found in
different species and tissues (3,4), it is expected to use human
cells for investigating the role of mast cells in al- lergic
disorders. And, since the number of cells that can be ob- tained
from human tissues is limited, numerous attempts to estab- lish
human mast cell culture had been made for a decade after discovery
of the method for culturing a large number of mouse mast cells in
the presence of mouse IL-3 (5). The development of human mast cells
was found in the coculture system of hemopoi- etic cells with 3T3
fibroblast cell line (6).
One of the major factors supporting the development of human
mast cells has later been found to be stem cell factor (7-lo),
which
*Division of Allergy and 'Department of Pediatric Pharmacology,
National Chil- dren's Medical Research Center, Tokyo, Japan;
*Pharmaceutical Development Laboratory, Kirin Brewery Co.,
Maebashi, Gunrna, Japan; §Department of Anat- omy, Hirosaki
University School of Medicine, Hirosaki, Aomori, Japan; and
¶Department of Clinical Oncology, The Institute of Medical Science,
University of Tokyo, Tokyo, Japan
Received for publication November 15, 1995. Accepted for
publication April 22, 1996.
The costs of publication of this article were defrayed in part
by the payment of page charges. This article must therefore be
hereby marked advertisement in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
' This work was supported in part by Pediatric Research Grant
6-04 from the ences Foundation, 1995. Ministry of Health and
Welfare, and by Grant 51 14 from the Japan Health Sci-
Allergy, National Children's Medical Research Center, 3-35-31,
Taishido, Seta- ' Address correspondence and reprint requests to
Dr. Hirohisa Saito, Division of gaya-ku, Tokyo 154, Japan.
Copyright 0 1996 by The American Association of
Immunologists
is derived from the Steel locus gene and has a ligand activity
for the c-kit proto-oncogene product (1 1-13). Stem cell factor,
which is also called c-kit ligand or Steel factor (SF),3 is known
to promote colony growth of crude hemopoietic cells (14-16). SF
alone, how- ever, induces only minimum proliferation of hemopoietic
colonies from purified CD34" cells. In combination with other
cytokines, SF strongly stimulates hemopoietic colonies from CD34+
cells (1 7-1 9). The synergistic effect of SF with other cytokines
is hardly detected when crude cell populations are cultured, since
accessory cells spontaneously secrete a variety of cytokines (20,
21). Simi- larly, SF induces development of a substantial number of
mast cells from crude hemopoietic cell preparations (8-10).
Although human mast cells have been proven to be derived from CD34'
cells, SF alone induces only a minimum proliferation of the cells
from purified CD34+ cells (7). These reports suggest that addi-
tional growth factors for mast cell development may be released
from accessory cells.
We have recently found in a preliminary experiment (22) that
IL-6 enhances the SF-induced development of human mast cells from
purified CD34+ cells. We have also previously reported that PGE
enhances the growth of mast cell colonies and inhibits the growth
of granulocyte-macrophage (GM) colonies in the murine system (23).
In the present study, therefore, we tried to confirm the effect of
IL-6 in several culture conditions, and tested the effect of PGE on
the SF- and IL-6-dependent development of human mast cells from
crude cord blood mononuclear cells (CBMC) to establish a simple
method for generating a large number of human mast cells.
' Abbreviations used in this paper: SF, Steel factor; CBMC, cord
blood mono- nuclear cells: CM, granulocyte-macrophage.
0022-1 767/96/$02.00
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344 CULTURED HUMAN MAST CELLS
Materials and Methods Cell preparation
Heparin-treated umbilical cord blood was obtained under informed
consent based on guidance from the hospital, diluted, and layered
over lymphocyte separation medium (LSM; Organon Teknika Corp.,
Durham, NC) at room temperature within 12 h after delivery.
Mononuclear cell fractions, which contained lymphocytes (60-80%),
monocytes (20-40%), and other cell types (
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The Journal of Immunology 345
FIGURE 1. Effect of cytokines on SF-induced proliferation of
tryptase-positive or -negative cells. Purified CD34+ cells were
cultured at 10’ to 104/ml in the presence of cytokines, as
indicated. After 4 wk in culture, the number of cultured cells was
counted, the smears were stained for tryptase, and the percentages
of cells having tryptase-pos- itive granules (B) or
tryptase-negative cells (0) were calculated. Each column and bar
represents the mean and SE of four separate experi- ments, and p
values between the number of tryptase-positive cells in cultures
with SF and that in cultures with SF plus cytokines were 95% of the
tryptase- negative cells were judged to be macrophages by their
prominent phagosomes) developed in the presence of SF. IL-6
enhanced the SF-induced development of tryptase-positive mast cells
with the highest proportion. Further addition of GM-CSF at 100
pg/ml to the cultures containing SF and IL-6 enhanced the
proliferation of tryptase-negative cells, and inhibited the
enhancing effect of IL-6 on the SF-induced growth of
tryptase-positive mast cells (Fig. 1). In the absence of SF, the
growth of tryptase-positive cells was not detected even in the
presence of these three cytokines (data not shown). Other than the
experiments shown in Figure 1 , we have obtained similar results
that IL-6, IL-3, and GM-CSF in combina- tion with SF stimulated the
growth of tryptase-positive cells in three separate experiments,
although the cell number was not suf- ficient enough for counting
tryptase-positive cells in the cultures
FIGURE 2. Effect of PGE, on mast cell development from crude
CBMC induced by SF and IL-6. CBMC were cultured at 105/ml in the
presence of SF at 80 ng/ml and IL-6 at 50 ng/rnl for 4 wk. After 4
wk in culture, the number of cultured cells was counted, the smears
were stained for tryptase, and the percentages of tryptase-positive
cells were calculated. Each column and bar represents the mean and
SE of eight separate experiments, and p values were
-
346 CULTURED HUMAN MAST CELLS
Table II. Effect of PGE, on the increase in cellular histamine
and cellular tryptase concentrations in mast cells cultured in the
presence of SF and IL-6 for 4 wk
Control PCE, lo-’ M PGE, 10-7 M PCE, 1 0-6 M
Histamine (nglwell)” 51.4 t 35.2‘ 75.2 t 49.9‘ 11 9.6 t 75.2‘ 1
10.0 ? 84.8 Tryptase (nglwell)” 81.2 t 53.2 220.6 ? 189.2 286.4 t
182.8‘ 242.2 t 105.1‘
a Each value indicates the concentration of mediators in the
total cultured cells per each well where 1 X lo5 mononuclear cord
blood cells were plated.
c p < 0.05 by a double-tailed paired Student’s t test. Each
value represents an average 2 SEM of five separate experiments.
FIGURE 3. Effect of PGE, on the SF- and IL-6-induced mast cell
de- velopment from purified CD34+ cells. Cord blood-derived CD34+
cells were cultured at 1 O3 to 104/ml in a well in the presence of
SF at 80 nglml and IL-6 at 50 nglml for 4 wk. After cell count, the
smears were stained for tryptase, and then percentages of
tryptase-positive cells were calculated. Each column represents the
mean of three sep- arate experiments. Tryptase-positive cells were
developed at 2.98 t 1, 3.05 t 0.82,2.62 t 0.8, and 2.89 t 0. A
quantity amounting to 98 X 1 Os per well for 4 wk in culture was in
the presence of PGE, at 0 , l O-’ M, 10” M, and M, respectively
(not significant).
lo4 tryptase-positive cells from lo5 mononuclear cord blood
cells after 4 wk in the medium supplemented with IL-6 at 0, 2, 10,
and 50 ng/ml, respectively (mean ? SEM; n = 3; p < 0.05 by Stu-
dent’s paired t test between groups cultured with IL-6 at 10-50
ng/ml and 0 ng/ml). In the absence of PGE, and the presence of SF,
the addition of IL-6 did not affect the number of tryptase-
positive cells that arose from 10’ mononuclear cells (3.99 X io4
cells for IL-6 at 50 ng/ml and 3.82 X lo4 cells for control in
three separate experiments).
When lo5 CBMC were cultured for 4 wk in 1 ml of medium provided
with SF and IL-6, but not PGE,, the addition of anti- GM-CSF (10
pg/ml) enhanced the growth of tryptase-positive cells ((8.03 ?
2.01) X lo4 for anti-GM-CSF and (1.54 ? 2.01) X io4 for control, in
four separate experiments. The value represents the mean ? SEM; p
< 0.05 by paired t test.).
Morphologic features
In the presence of SF at 80 ng/ml, IL-6 at 50 ng/ml, and 300 nM
of PGE,, the cultured mast cells increased in size as well as in
number until day 100 in culture (Fig. 4). Ultrastructural analysis
revealed that most granules of the cultured mast cells were filled
with scroll structures, although irregular periodicity was seen in
the structures (Fig. 5). We were also able to demonstrate a typical
crystal granule in the mast cells (Fig. 6). We used IL-6 at 50
ng/ml, because the cytokine at 100 ng/ml induced atypical
characteristics, i.e., the cells had >10 pg of cellular
histamine per cell and mul- tilobed nuclei. As has been reported
elsewhere (28), the cultured mast cells were stained for tryptase
at 98.6 ? 0.4%, and chymase for 18.4 ? 4.4% (n = 7).
Functional features
When CBMC were cultured in the presence of SF at 80 ng/ml, IL-6
at 50 ng/ml, and 300 nM of PGE, for >10 wk, the mast cells
FIGURE 4. Typical mast cells developed in the presence of SF,
IL-6, and PGE, for 12 wk. May-Grunwald Giemsa stain. Note the
prominent microvilli-like processes. X1099.
contained 5.62 ? 1.88 pg of histamine (n = 6) and 3.46 ? 0.89 pg
of tryptase (n = 6; each value represents mean ? SEM) per lo6
cells. Cells sensitized with 1 pglml of human IgE released 52.9%
histamine at 30 min, and 1.76 ng of TNF-a per lo6 cells at 6 h
after challenge with 1.5 pg/ml of anti-human IgE, whereas the
control cells spontaneously released 3.7% histamine and 0.18 ng of
TNF-a (Fig. 7). Release of TNF-a reached a plateau at 6 h (data not
shown), while the increase in intracellular Ca2+ (Fig. 8) and his-
tamine release (data not shown) reached a plateau before 10 min. An
increase in intracellular Ca” could be detected in advance of
partial swelling of the plasma membrane by using a confocal laser-
scanning microscope (Fig. 9). Some parts of the membrane had
ballooned outward and were soon shrinking. The ballooning was
repeated from 196 to 476 s after anti-IgE challenge. We could not
observe the dynamic movement of the plasma membrane after 8 min.
Mast cells also released 3, 85, 98, and 58 pg of IL-5 per lo6 cells
at 6 h after challenge with anti-human IgE at 0,0.15, 1.5, and 15
pg/ml, respectively (n = 2). The protein levels of IL-3 and IL-4
were
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The Journal of Immunology 347
FIGURE 5. Mast cell developed in the presence of SF, IL-6, and
PGE, for 16 wk. The photograph shows that the cell filled with
dense scroll-type granules (A) . Higher magnification of the
granules was seen in 6. A, X4,200. 6, X21,OOO.
absence of accessory cells. Several cytokines such as IL-3,
IL-6, and GM-CSF have been shown to synergize with SF for prolifer-
ation of hemopoietic cells (17-19, 27). Therefore, it was not sur-
prising that these cytokines enhanced the SF-induced proliferation
of mast cells probably by stimulating the expansion of primitive
hemopoietic cells in the early stage of cell proliferation, as
shown in this study. Among these cytokines, only IL-6 stimulated
the expansion of hemopoietic cells without inducing marked
prolifer- ation of macrophages, which are known to secrete a
variety of cytokines such as GM-CSF (20, 21, 30). Subsequent
secretion of GM-CSF, which may occur in the presence of exogenous
GM-CSF or IL-3, would therefore accelerate the differentiation of
primitive hemopoietic cells toward the GM lineage, thereby reducing
the differentiation capacity of the cells toward the mast cell
lineage in a prolonged period. IL-6 was also effective for
proliferation of differentiated mast cells after 4 wk in culture,
as well as for the expansion of undifferentiated progenitors.
Indeed, we have ob- served recently that IL-3, IL-4, and IL-5 as
well as IL-6 prevent apoptosis of the mast cells cultured for >
I O wk (31).
FIGURE 6. Mast cell developed in the presence of SF. IL-6, and
PGE, for 14 wk, showing immature granules and microvilli-like mem-
brane processes (A). A crystal granule was seen in 6. A, x5,400. 6,
X83,OOO.
anti-lgE ( Fglml)
FIGURE 7. IgEdependent release of histamine and TNF-a from CUI-
tured mast cells. The mast cells cultured >10 wk were sensitized
with 1 pg of myeloma IgE, and were challenged with either control
buffer or 0.1 5 to 15 pg of anti-human IgE for 30 min (0)
(histamine release) and for 6 h (0) (TNF-a release). Each point
represents mean t SEM of five (TNF-o! release) or six (histamine
release) separate experiments. The purity of mast cells used in
this experiment was 98.6% 2 0.4% (mean 2 SEM).
It has been reported by Metcalfe et al. (7,32) that IL-3
markedly enhanced the SF-dependent mast cell growth from bone
marrow- derived CD34+ cells in the early culture period. We had
similar
-
348 CULTURED HUMAN MAST CELLS
t - 1 min
Anti-lgE (1.5 ,ug/ml)
FIGURE 8. Increase in the intracellular concentrations of Ca2+
in the cultured mast cells. The mast cells with 99.5% purity at 1 2
wk in culture were sensitized with 1 pg of myeloma IgE, and were
labeled with 1 p M of fura-2 AM and suspended at 1 X 1 Ob per 1 mi
of Tyrode‘s solution in a cuvette of a Ca2+ analyzer, CAF-100. The
cells were then challenged with either 1.5 pg (left side) or 15 pg
(right side) of anti- human IgE in the analyzer. The two
fluorescent intensities were mea- sured at excitation wavelengths
of 340 nm and 380 nm, and their ratio was calculated. Similar
results were obtained in two other separate experiments.
results in the present study, especially in three of the seven
exper- iments showing SF alone failed to induce a substantial
amount of mast cells from purified CD34+ cells. In those three
experiments, E - 3 seemed to be a powerful synergistic factor for
the SF-induced proliferation of mast cells. In one of the seven
purified CD34+ samples and in two of ten crude samples with PGE,
addition, we failed to obtain a substantial number (>lo6 per
sample) of mast cells even in the presence of SF and IL-6, whereas
IL-3 alone ‘always induced a substantial amount of basophils (33)
from the same cord blood samples (data not shown). It may be
related to the number of cytokine receptors present on cord
blood-derived CD34+ cells, which may be individually different. In
most cases, however, the combination of L - 6 and SF would be the
best way for generating highly purified mast cells in a prolonged
period.
We have reported previously that PGE enhances the colony for-
mation of mast cells and inhibits GM colony formation in the
presence of IL-3-containing medium in murine system (23). In the
present study, PGE, inhibited proliferation of adherent macro-
phages by blocking GM-CSF secretion, as has been reported pre-
FIGURE 9. Membrane ballooning of the cultured mast cells
following an increase in intracellular Ca*+. The mast cells at 12
wk in culture were sen- sitized with 1 pg of myeloma IgE overnight
on a fi- broblast layer to which the mast cells were fixed, and
weie labeled with 1 p M of fluo-3, and sus- pended in a
temperature-controlled dish. The cells were then challenged with 15
pg of anti-human IgE. A single cell was scanned every 7 s by using
a con- focal laser microscope, Olympus GB-200. The mast cell at 0 s
( A ) , 21 s (61, and 399 s (C) after challenge with anti-lgE was
respectively shown. Relative Ca’+ concentrations were shown in the
upperpartby col- oring the fluorescent intensities in the following
rank order, i.e., red > orange > yellow > green > blue.
In the lower part, the same cell was examined by using a
phase-contrast technique. Similar results were obtained in three
separate experiments, al- l though intracellular Ca*+ started to
increase at var- ious intervals, from 35 s to >1 min after
anti-lgE challenge. The fading of fluo-3 fluorescence was clearly
found in the control cells within 5 min.
267 bp. I p-actln IL-3 IL-4 IL-5
FIGURE 10. IgE-dependent mRNA expression of cytokines. A half-
million mast cells at day 150 in culture with >99.9% purity were
challenged with 1.5 pgml of anti-lgE. After 60 min of incubation at
37C, the cells were treated with RNAzol B, as shown in Materials
and Methods.
viously by others (29), and it enhanced the development of human
mast cells. Since PGE, failed to affect mast cell development from
purified CD34+ cells, and GM-CSF was never detected until day 4 in
culture with CD34+ cells in the presence of SF and E-6, the
enhancing effect of the lipid mediator is probably brought on by
modulating cytokine secretion, especially by blocking GM-CSF
secretion from accessory cells. Indeed, exogenous addition of anti-
GM-CSF Ab enhanced the proliferation of mast cells in the pres-
ence of SF and IL-6. IL-6 and granulocyte CSF were released from
the 2 X 10’ mononuclear cord blood cells at 181 pg and 150 pg in
the present study (data not shown in the results) and previous re-
ports (20, 21, 29), and the addition of PGE, slightly enhanced the
secretion of the cytokines. However, their concentrations were not
sufficient for modulating the proliferation of mast cells.
Thus, we established a method for obtaining a large number 01
human mast cells. We were able to generate >lo7 mast cells in 2
of IO samples and 10‘ to IO’ mast cells in 6 of 10 samples with
>95% purity when 3 to 8 X lo7 crude CBMC were cultured in the
presence of SF at 80 ng/ml, IL-6 at 50 ng/ml, and 300 nM of PGE,
for > I O wk. Similar inconstant results were obtained even by
cul- turing purified CD34+ cells depending on samples. Since the
num- bers of CD34’ cells, GM colony-forming cells, monocytes, and
lymphocytes present in the CBMC were almost constant depend- ing on
samples (data not shown), we may have to examine the
A C
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The Journal of Immunology
expression of c-kit and ILdR on the CD34+ cells to define dif-
ferentiation capacity toward the mast cell lineage of the cells.
Hu- man mast cells developed in the presence of SF alone often
exert somewhat immature functional properties (9, 10). The lung
mast cells are known to possess 2 to 3.9 p g of histamine and 11 p
g of tryptase per 10" cells (34, 35). The mast cells cultured in
the present method seemed to be functionally mature, because 1)
they contained 5.62 pg of histamine and 3.46 pg of tryptase per lo6
cells; 2) the cultured mast cells had IgE receptors (36), and the
cells sensitized with 1 pglml of human IgE released 52.9% of the
histamine content, 693 ng LTC, (28), 1.76 ng TNF-a, and 98 pg IL-5
per 10" cells when challenged with 1.5 pg/ml anti-human IgE,
whereas the control cells spontaneously released 3.7% hista- mine,
9 ng LTC,, 0.18 ng TNF-a, and 3 pg IL-5; and 3) the re- actions
were accompanied by an increase in intracellular Ca2+. By using a
confocal laser-scanning microscope, some parts of the plasma
membrane were found to have ballooned outward follow- ing the
IgE-dependent increase in intracellular Ca2+. The mem- brane
ballooning was started from 3 min and finished until 8 min after
anti-IgE challenge, and its time course was similar with that of
histamine release (34), suggesting that the partial swelling of the
membrane reflects IgE-dependent degranulation of human mast
cells.
The cultured mast cells were positively stained for
anti-tryptase and anti-chymase Abs at 99 and 18% (28),
respectively. In an electron-microscopic analysis, some of the
cells nearly filled with scroll-type granules that are seen
frequently in lung mast cells (37). Crystal granules, which are
seen frequently in human skin mast cells and in some of the
cultured mast cells developed in coculture system with 3T3
fibroblasts (6), were also detectable. These results suggest that
the cultured mast cells developed in SF plus IL-6 are
morphologically more mature than those developed in SF alone, which
are reported to have only incomplete condensation of gran- ule
materials (lo), and are a mixture of tryptase- and chymase-
positive skin-type mast cells and tryptase-positive lung-type mast
cells (35).
Since it is hard to obtain lo7 purified mast cells from human
tissues, analyses requiring a large number of pure cells, such as
intracellular mechanisms or cytokine production, have not been
intensively examined. Contaminated monocytes may produce a va-
riety of cytokines when they adhere to plastic flasks (21, 29), and
basophils may produce IL-4, as has been reported (38). In the
present experiment for cytokine production, cells consisting of
>99.9% mast cells and 1 ng of TNF-a per lo6 cells, which amount
is enough for eosinophil transendothelial migration (41). A
substantial amount of IL-5 was also detected. We were able to show
that the cultured mast cells expressed IL-5 mRNA after challenge
with anti-IgE. In the same samples, however, mRNA expression of
IL-3 and IL-4 was not detected. As has been reported elsewhere
(28), the cultured mast cells expressed a series of surface CD
molecules in keeping with human mast cells in vivo (42,43), except
CD13, CD14, and CD38 present on the cultured cells. Taken together,
the present method for generating human mast cells should greatly
facilitate investi- gation of the role of mast cells in human
allergic disorders, espe- cially when such studies require large
numbers of pure mast cells.
349
Acknowledgments We thank Drs. Kimishige Ishizaka and Teruko
Ishizaka (La Jolla Institute for Allergy and Immunology, L a Jolla,
CA) for their critical reviewing of the manuscript. We are grateful
to Dr. Shigenobu Shoda (Department of Obstetrics, Gyoda Chuo
Hospital, Saitama, Japan) for his continuous sup- port by
generously providing the umbilical cord blood. We also thank Dr.
Naoya Sakaguchi, Dr. Katsushi Miura. Dr. Akira Akasawa, Mr. Takashi
Numazaki, and Mr. Masahiro Kimata (Division of Allergy, National
Chil- dren's Medical Research Center, Tokyo, Japan) for their
advice on the manuscript and for excellent technical suppon for the
experiments.
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