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HEMATOPOIESIS
Human skin–derived mast cells can proliferate while retaining their
characteristic functional and protease phenotypes
Naotomo Kambe, Michiyo Kambe, Jarema P. Kochan, and Lawrence B. Schwartz
Human mast cells in adult tissues have
been thought to have limited, if any, proliferative potential. The current study examined mast cells obtained from adult skin
and cultured in serum-free medium with
recombinant human stem cell factor. During the first 4 weeks of culture, the percentages of mast cells increased from 10
to almost 100. After 8 weeks, a 150-fold
increase in the number of mast cells was
observed. When freshly dispersed mast
cells were individually sorted onto human
fibroblast monolayers and cultured for 3
weeks, one or more mast cells were detected in about two thirds of the wells,
and in about two thirds of these wells the
surviving mast cells showed evidence of
proliferation, indicating most mast cells
in skin can proliferate. Such mast cells all
expressed high surface levels of Kit and
Fc⑀RI, each of which were functional,
indicating IgE was not required for Fc⑀RI
expression on mast cells. Such mast cells
also retained the MCTC protease phenotype of mast cells that normally reside in
the dermis. After 4 to 8 weeks of culture
these mast cells degranulated in response
to substance P and compound 48/80, characteristics of skin-derived mast cells that
persist outside of the cutaneous microenvironment. (Blood. 2001;97:2045-2052)
© 2001 by The American Society of Hematology
Introduction
Multipotential progenitor cells, capable of becoming mast cells,
leave the bone marrow and enter the circulation, but they complete
their differentiation into mature mast cells only after arriving in
peripheral tissues such as lung, bowel, skin, and nasal and
conjunctival mucosa. Mature human mast cells can be distinguished from other cell types by expression of high levels of
surface Fc⑀RI and Kit, and of granule tryptase and heparin. Two
types of human mast cells have been identified based on their
composition of neutral proteases.1-6 MCTC cells contain tryptase,
chymase, mast cell carboxypeptidase, and cathepsin G in their
secretory granules and are the predominant type of mast cells in
normal skin and in intestinal submucosa. MCT cells also contain
tryptase in their granules, but lack these other proteases, and are the
predominant type of mast cell in normal alveolar wall and in small
intestinal mucosa.
In a number of human diseases such as asthma,7,8 allergic
rhinitis,9,10 rheumatoid arthritis,11-13 and vernal keratoconjunctivitis,14 significant increases in mast cell density at the local affected
sites have been described and mast cells were estimated to play a
central role in the pathophysiology of the associated inflammation.
The principal mechanisms involved in mast cell hyperplasia were
thought to be either the recruitment of progenitor cells and their
differentiation to mast cells or the migration of mast cells from
other regions of the tissue. Proliferation has been considered less
likely in part because cells were considered to be terminally
differentiated and also because mitotic mast cells are rarely
appreciated at these sites.
The proliferative potential of most myelomonocytic multipotential hematopoietic cells diminishes as they differentiate. However,
mature murine mast cells have been reported to proliferate both in
vivo and in vitro. About 6% of mouse peritoneal mast cells
proliferate after being injected into skin of W/Wv mast cell-deficient
mice.15,16 Purified peritoneal mast cells17,18 or dispersed skin mast
cells,19 when plated in methylcellulose containing pokeweed
mitogen-stimulated spleen cell-conditioned medium, produced
mast cell colonies. However, in humans, isolated tissue-derived
mast cells have shown little capacity for proliferation. For example,
dispersed newborn foreskin or adult skin mast cells placed
into culture with lymphocyte-conditioned media and various
cytokines showed bromodeoxyuridine incorporation in up to 15%
mast cells.20
The current study shows that a substantial portion of mast cells
dispersed from adult human skin can proliferate under serum-free
culture conditions with recombinant human stem cell factor
(rhuSCF), while retaining expression of chymase and tryptase in
secretory granules, Kit, and Fc⑀RI on their surface and the ability to
degranulate in response to IgG anti-Fc⑀RI␣, compound 48/80 and
substance P.
From the Department of Internal Medicine, Division of Rheumatology, Allergy
and Immunology, Virginia Commonwealth University, Richmond, Virginia, and
the Department of Metabolic Diseases, Hoffman-La Roche, Nutley, New
Jersey.
Reprints: Lawrence B. Schwartz, Virginia Commonwealth University, PO Box
980263, Richmond, VA 23298-0263; e-mail: [email protected].
Submitted October 27, 2000; accepted December 7, 2000.
Supported by National Institutes of Health grants AI27517 and AI20487
(to L.B.S.).
BLOOD, 1 APRIL 2001 䡠 VOLUME 97, NUMBER 7
Materials and methods
Antibodies and reagents
Biotin-conjugated mouse IgG monoclonal antibody (mAb) antichymase,
B7-biotin, and IgG mAb antitryptase, G3, were obtained and prepared as
described.21 The mAb anti-Kit, YB5.B8, was a gift from Dr L. K. Ashman
(Institute of Medical and Veterinary Science, Adelaide, Australia); IgG
mAb anti-Fc⑀RI␣, 22E7, was obtained and used as described22,23; IgG mAb
R24 against disialoganglioside (GD3),24 was a gift from Dr P. O. Livingston
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2001 by The American Society of Hematology
2045
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2046
BLOOD, 1 APRIL 2001 䡠 VOLUME 97, NUMBER 7
KAMBE et al
(Memorial Sloan-Kettering Cancer Center, New York, NY); and rhuSCF
was provided by Amgen (Thousand Oaks, CA).
Dispersal purification and culture of human skin mast cells
Mast cells were dispersed from human skin tissue and enriched essentially
as described.25-27 Fresh samples of skin were obtained after breast reduction
or mastectomy for breast cancer, or from abdominoplasties, at the Medical
College of Virginia Hospital or through the National Disease Research
Interchange (Philadelphia, PA) and Cooperative Human Tissue Network of
the National Cancer Institute (Columbus, OH) as approved by the Human
Studies Internal Review Board at Virginia Commonwealth University.
Subcutaneous fat was removed by blunt dissection and residual tissue was
cut into 1- to 2-mm fragments, which were incubated in a solution of Hanks
balanced salt solution (HBSS) containing 1.5 mg/mL type 2 collagenase,
0.7 mg/mL hyaluronidase (Worthington Biochemical, Lakewood, NJ), 0.3
mg/mL type 1 DNase (Sigma Chemical, St Louis, MO), 1% fetal calf serum
(FCS), and 1 mM CaCl2 for 2 hours at 37°C with constant stirring. The
dispersed cells were separated from residual tissue by filtration through a
no. 80 mesh sieve and suspended in HBSS containing 1% FCS and 10 mM
4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (Hepes) (washing
buffer). The remaining tissue was subjected to an additional digestion as
above, and combined with the cells from first digestion. Isotonic Percoll
was prepared by mixing 9 parts Percoll (Sigma) and 1 part of 10 ⫻ HBSS,
and further diluted with HBSS to give final concentration of 75%. Cells
were resuspend in washing buffer, layered over Percoll cushion, and
centrifuged at 700g at room temperature for 20 minutes. Nucleated cells
were collected from the buffer/Percoll interface.
Percoll gradient-enriched cells were suspended at the concentration of
1 ⫻ 106 cells/mL in serum-free AIM-V medium (Life Technologies,
Rockville, MD). Some cells were cultured at the same concentration in
complete RPMI 1640 medium supplemented with 10% controlled process
serum replacement-3 (CPSR-3; Sigma), 2 mM L-glutamine, 0.1 mM
nonessential amino acids, 10 mM Hepes, 50 ␮M 2-mercaptoethanol, 200
U/mL penicillin, and 100 ␮g/mL streptomycin. In both cases, 100 ng/mL
rhuSCF was included, and half the culture medium was changed every
week. When the cell number reached 2 ⫻ 106 cells/mL, half the cells were
split to another well. Cell numbers per sample were adjusted to account for
the cells that were split. Total cell number and viability were assessed by
trypan blue staining. The percentages of mast cells were assessed cytochemically by metachromatic staining with toluidine blue, and by flow cytometry
with anti-Kit and anti-Fc⑀RI mAbs. The protease phenotype of cultured
cells was examined by immunochemistry with antitryptase and antichymase mAbs. For each sample of cytocentrifuged cells, 100 cells or more per
field, 4 fields per slide, and 3 slides were examined.
phosphatidylserine, a phospholipid that transfers from the inner to the outer
side of the lipid bilayer of the plasma membrane during an early stage of
apoptosis, and with propidium iodide (PI) for 15 minutes at room
temperature to detect dead cells. Samples were directly analyzed by flow
cytometry. To estimate the relative DNA content of cultured mast cells, cells
were washed with PBS, incubated for 10 minutes on ice in hypotonic
fluorochrome solution, which contained 50 ␮g/mL PI and 0.1% TritonX100 in 0.1% sodium citrate, and then directly analyzed by flow cytometry.
Immunochemistry
Mast cells were assessed in cytocentrifuge preparations subjected to
indirect immunocytochemical labeling as described.23 For chymase detection, B7-biotin–labeled cells were incubated with peroxidase-conjugated
streptavidin and visualized with 3-amino-9-ethylcarbazole in 0.01% H2O2
to stain positive cells reddish brown. For tryptase detection, alkaline
phosphatase-conjugated goat antimouse IgG (Sigma) was added to G3labeled cells and visualized with naphthol AS-BI/new fuchsin to stain
positive cells red. Cells were lightly counterstained with hematoxylin.
Immunoaffinity magnetic enrichment and individual sorting
Enzymatically dispersed skin cells were further enriched with mAb R24
against GD3, which was recently reported to be selectively expressed on the
surface of mast cells in normal skin.28 Briefly, cells were incubated in PBS
containing 10% human AB serum for 30 minutes, washed with PBS, and
incubated with R24 (3 ␮g/1 ⫻ 107 cells) at 4°C for 30 minutes. Cells were
washed with PBS to remove unbound mAb, incubated with microbeads
conjugated to antimouse IgG (Miltenyi Biotec, Auburn, CA) at 4°C for 15
minutes, washed, and applied to the separation column. The column was
exposed to a magnetic field in the magnetic cell separation system (Miltenyi
Biotec) and washed to remove cells without attached microbeads. Retained
cells were then eluted after the column was withdrawn from the magnetic
field. Eluted cells were incubated with normal mouse IgG to saturate the
antimouse antibody, and then with PE-conjugated mAb anti-Kit or control
antibody for 1 hour at 4°C. Cells were then washed, and resuspended in
PBS containing 20% AIM-V medium. Kit⫹ cells were individually seeded
onto confluent 1059SK human fibroblasts (American Type Culture Collection, CRL-2072, Manassas, VA) in 96-well plates using a Coulter Epics Cell
Sorting System (Coulter, Miami, FL) at Virginia Commonwealth University’s Flow and Imaging Cytometry Facility, which permits single-cell sorting
with an accuracy of more than 99%. Sorted cells were then cultured in
AIM-V medium supplemented with 100 ng/mL rhuSCF. Half the medium
was changed every week. After 3 weeks mast cell numbers were checked by
immunocytochemistry for tryptase.
Flow cytometry
Surface Kit and Fc⑀RI␣ expression were assessed by flow cytometry with
mAbs YB5.B8 and 22E7, respectively. Cells were incubated in 10% human
AB serum in phosphate-buffered saline (PBS). After washing in PBS
containing 1% FCS and 0.1% sodium azide, cells then were incubated with
anti-Fc⑀RI␣ mAb or control mouse IgG for 30 minutes at 4°C. After
washing as above, cells were incubated with fluorescein isothiocyanate
(FITC)-conjugated goat antimouse immunoglobulins (BD Biosciences, San
Jose, CA) for 15 minutes at 4°C, and washed as above. After incubation
with normal mouse IgG to saturate antimouse antibody binding sites, cells
were incubated with phycoerythrin (PE)-conjugated anti-Kit mAb (BD
Pharmingen) or control IgG for 30 minutes at 4°C, resuspended in sheath
solution, and analyzed with a FACScan flow cytometer (BD Biosciences).
The percentage of positive cells was calculated by subtracting the percentage of cells labeled with a control antibody (⬍ 3% positive cells) from that
of cells with experimental mAb. HMC-1 cells, a human mast cell line, and
KU812 cells, a human basophil leukemia cell line, were used as positive
controls for surface Kit and Fc⑀RI␣ expression, respectively.
For the detection of cells undergoing apoptosis after withdrawal of
rhuSCF, cells were washed in PBS and incubated in the binding buffer as
provided and recommended by the manufacturer (R & D Systems,
Minneapolis, MN) with FITC-conjugated annexin-V, which binds to
Activation of mast cells and tryptase immunoassay
Cultured mast cells were washed in Tyrode buffer containing 10 mM Hepes,
pH 7.4, 0.1% gelatin, and 100 ng/mL DNase (TGD⫺/⫺), then suspended in
TGD⫺/⫺ buffer containing 1 mM MgCl2 and 2.5 mM CaCl2 (TGD⫹/⫹) and
preincubated for 5 minutes at 37°C. For stimulation, 5 ␮L mAb 22E7 or
control mouse IgG, or nonimmune reagents such as substance P or
compound 48/80 (Sigma) were added to 25 ␮L cell suspension (1 ⫻ 106
cells/mL) in a 96-well plate, and incubated for a further 15 minutes at 37°C.
The reaction was stopped by adding 200 ␮L ice-cold TGD⫺/⫺ buffer. The
cells were separated by centrifugation at 300g for 7 minutes at 4°C, and
supernatant was carefully removed and adjusted to 1.2 M NaCl by adding
40 ␮L 5 M NaCl. The cell pellet was resuspended in 200 ␮L ice-cold
extraction buffer (10 mM MES, pH 6.5, with 2 M NaCl), quickly frozen in
liquid nitrogen, and thawed 4 times to release all cellular tryptase. After
centrifugation at 12 000g for 15 minutes at 4°C, the soluble extract was
collected and stored at ⫺70°C until ready to be assayed. Total immunoreactive tryptase levels were measured by an enzyme-linked immunosorbent
assay (ELISA) using mAb B12 to capture tryptase and biotinylated mAb
G5 to detect captured tryptase as described29; purified human lung-derived
tryptase was used as standard.
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BLOOD, 1 APRIL 2001 䡠 VOLUME 97, NUMBER 7
PROLIFERATION OF HUMAN SKIN–DERIVED MAST CELLS
2047
Results
Proliferation of human skin–derived mast cells
in a serum-free culture
Because serum may contain both beneficial and detrimental factors
for mast cell growth in vitro, synthetic media are potentially
advantageous. Serum-free AIM-V media permitted an earlier, more
selective and greater expansion of fetal liver-derived mast cells
than serum-containing media in the presence of rhuSCF.23 However, most tissue-derived human mast cells, though probably
long-lived, are thought to be terminally differentiated with limited,
if any, proliferative capacity. The growth of skin-derived mast cells
in the presence of rhuSCF (100 ng/mL) was assessed with this
serum-free media compared to serum-containing RPMI 1640
(Figure 1). As expected with RPMI 1640 medium with serum, after
an 8-week culture period, total cell numbers diminished by 90%
(Figure 1A), mast cell percentage by about 80% (Figure 1B), and
mast cell number by over 90% (Figure 1C). At 1 week there was a
slight increase in the percentage of mast cells due to a more rapid
loss of other cell types, but there was no increase in mast cell
number. In contrast to serum-containing media, results with AIM-V
media were dramatically different. Total cell numbers diminished
slightly after the first week, but then increased 16-fold by 8 weeks
(Figure 1A). Under these serum-free culture conditions the mast
cell percentage increased from about 10% at the initiation of
culture to nearly 100% by 4 weeks, and remained so for the
remaining 4 weeks (Figure 1B). Mast cell numbers increased
progressively from 0.1 ⫻ 106 cells on day zero to 15.9 ⫻ 106 after
8 weeks. A similar growth pattern was recognized in another 5
independent specimens. Of the 4 specimens kept in culture under
serum-free conditions beyond 8 weeks, one stopped proliferating at
8 to 9 weeks, 2 stopped at 9 to 10 weeks and one continued until the
culture was harvested at 12 weeks.
Skin-derived mast cells were examined by light microscopy
(Figure 2). A cytospin of freshly dispersed skin cells, stained with
toluidine blue, shows about 10% of the cells to be metachromatic
with oval, nonsegmented nuclei (Figure 2A). After 4 weeks of
culture in AIM-V medium, monolayers of tightly adherent cells
were not observed. Instead, almost all of the cells were metachromatic (Figure 2B), although the staining intensity appeared to be
less than for newly dispersed mast cells. In contrast, after 4 weeks
of culture in RPMI 1640 with CPSR-3, the culture well was
completely covered by fibroblast-like cells. Toluidine blue staining
of cytocentrifuged cell preparations revealed few metachromatic
cells, many fibroblast-like cells, and occasional metachromatic
bodies in fibroblast- or macrophage-like cells, suggesting that
ingestion of mast cells or mast cell granules may have occurred
(Figure 2C). Also, morphometric measurements (mean ⫾ SE,
n ⫽ 4 separate skin samples, 30 mast cells examined per preparation) of mast cell diameter revealed cells cultured for 4 to 8 weeks
in AIM-V media (25.7 ⫾ 0.2 ␮m,) to be larger (P ⬍ 0.001, t test)
than those prior to culture (13.2 ⫾ 0.2 ␮m). Although most nuclei
were oval and nonsegmented, 4.6% ⫾ 2.1% (n ⫽ 5 specimens) of
the cultured mast cells were binucleated or showed mitotic figures
(Figure 2B), features not observed before culture. Almost no
fibroblast-like cells were observed. Essentially all mast cells in
normal adult skin contain both tryptase and chymase (MCTC type of
mast cell).21 To test whether skin-derived mast cells retained this
protease phenotype after 4 to 8 weeks of culture in serum-free
AIM-V media containing rhuSCF, cytospins of these cells were
Figure 1. Expansion of skin-derived mast cell numbers in serum-free media.
Dispersed skin cells, enriched with mast cells by Percoll density-dependent sedimentation, were placed into culture with rhuSCF (100 ng/mL) in either RPMI 1640
supplemented with CPSR-3 (E) or AIM-V (F) as described. Total viable cell numbers
(A), percentages of mast cells (metachromatic staining) (B), and numbers of mast
cells (product of total cell number and mast cell percentage) (C) are depicted. Each
data point is the mean value from 3 separate experiments, each performed in
triplicate. Error bars indicate the SD. When the total cell concentration in a well
exceeded 2 ⫻ 106 cells/mL, half of the cells were either transferred to another well or
discarded; cell numbers were adjusted to account for the cells that were discarded as
well as those that were retained when cultures were split.
stained with antichymase (Figure 2 D) and antitryptase (Figure 2 E)
mAbs, and counterstained with hematoxylin. Essentially all cells
were labeled with each mAb, indicating the MCTC phenotype was
preserved under these culture conditions.
To further evaluate the proliferation activity of 4-week-old
cultured mast cells, DNA content was assessed by flow cytometry
with PI. In a DNA fluorescence histogram, the cells in S, G2, and M
phases are shown in the M1 region of Figure 3A, whereas those in
G0 and G1 phases reside in the peak that precedes M1. When
4-week-old mast cells grown in AIM-V media were examined
before (left panel, 7 days after prior feeding) and 24 hours after
rhuSCF feeding (right panel), 3.7% ⫾ 1.8% and 9.4% ⫾ 3.3% of
the cells, respectively, were distributed in M1 (n ⫽ 3, mean ⫾ SD).
Thus proliferation rates appeared to be somewhat greater 24 hours
than 7 days after treatment with rhuSCF. Also, to confirm that these
cells were mast cells, surface Kit (Figure 3B) and Fc⑀RI (Figure
3C) were evaluated. As shown, after 6 weeks of culture in AIM-V
media (right panels), essentially all cells were strongly Kit⫹ (96%)
and Fc⑀RI⫹ (96%). Similar results were obtained at 4- and 8-week
time points. The fluorescence intensity of the Kit⫹ population
within 24 hours after their dispersion from skin (Figure 3B, left
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2048
KAMBE et al
BLOOD, 1 APRIL 2001 䡠 VOLUME 97, NUMBER 7
Figure 2. Light microscopic appearance of cytospins
of skin-derived mast cells. Toluidine blue staining
dispersed skin cells at the initiation of culture (A) and after
4 weeks of culture in AIM-V (B) and in RPMI 1640 with
10% CPSR-3 (C) as described. In panel B, arrows show 2
cells with dual nuclei; an arrowhead points out a mitotic
cell. In panel C, arrows point to mast cells (17% of total
cells counted on this slide); the arrowhead shows a
metachromatic body inside a nonmast cell. Cells grown
for 4 weeks in AIM-V medium (98% mast cells) were
stained for chymase (D) and tryptase (E). Essentially all
cells contained both chymase and tryptase. The original
magnification of all photomicrographs was ⫻ 200.
panel) was similar to or slightly higher than that of the mast cells
after 4 to 8 weeks of culture in AIM-V media. In contrast, Fc⑀RI
levels on Kit⫹ cells appeared to be lower just after dispersion of
skin cells (Figure 3C, left panel) than after 4 to 8 weeks of culture.
Skin-derived mast cells cultured in AIM-V media are
dependent on exogenous SCF for survival
Survival of human mast cells is acknowledged to be primarily
dependent on SCF. To test whether proliferating skin-derived mast
cells were dependent on this growth factor, 3 separate preparations
of 6- to 8-week cultures of mast cells in AIM-V media were washed
and placed into culture with or without rhuSCF (100 ng/mL). As
shown in Figure 4A, viability (trypan blue) remained near 100% in
the presence of rhuSCF, but steadily decreased in the absence of
rhuSCF. By day 1 and beyond the percent viabilities of cells
cultured with rhuSCF were significantly different from those
cultured without rhuSCF; by day 3 and beyond the percent
viabilities of cells cultured in the absence of rhuSCF were
significantly lower than the day 0 cells. The percentages of
apoptotic cells on day 2 were evaluated by annexin-V surface and
PI labeling. Without rhuSCF 49% of the cells were labeled with
annexin V, whereas less than 20% (as with trypan blue) were also
labeled with PI. All of the annexin V⫹ cells were smaller than the
viable unlabeled cells (forward side scatter by flow cytometry),
consistent with apoptosis being their death pathway. With rhuSCF
only about 6% of the cells were labeled with annexin V and about
5% with PI. As shown in Figure 4B, with rhuSCF the number of
viable cells increased significantly by day 3, whereas without
rhuSCF the cell numbers had significantly declined by day 3 when
compared to day 0 cells. Also, viable cell numbers in the presence
and absence of rhuSCF were significantly different by day 3.
Figure 3. Cell cycle and surface phenotype of skin-derived mast cells. (A)
Relative DNA content of skin-derived mast cells after about 4 weeks of culture in
AIM-V media. Cells were incubated with 50 ␮g/mL PI for 10 minutes on ice just before
(7 days after last rhuSCF feeding, left panel) and 24 hours (right panel) after a
rhuSCF feeding, and analyzed by flow cytometry. (B) Surface Kit⫹ mast cells 1 day
after their initial dispersion (left panel) and 7 days after rhuSCF feeding of cells grown
in AIM-V suspension culture for 6 weeks (right panel). (C) Fc⑀RI surface expression of
Kit⫹ cells 1 day after their initial dispersion (left panel) and 7 days after rhuSCF
feeding of cells grown in AIM-V suspension culture for 6 weeks (right panel). Shaded
areas in panels B and C show anti-Fc⑀RI and anti-Kit labeling, respectively; bold lines
indicate negative IgG controls. Each panel is representative of 3 separate experiments.
Proliferative potential of human skin–derived mast cells
From the experiments described thus far, it was not clear whether
the proliferating mast cells in this serum-free culture condition
were derived from mature mast cells or from progenitors that might
reside in preparations of dispersed skin cells. To address this, mast
cells were immunoaffinity purified with anti-GD3 mAb to at least
95% purity as described,28 and then subjected to cell sorting after
labeling the cells with PE-conjugated anti-Kit mAb. A portion of
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BLOOD, 1 APRIL 2001 䡠 VOLUME 97, NUMBER 7
PROLIFERATION OF HUMAN SKIN–DERIVED MAST CELLS
2049
skin-derived mast cells are capable of proliferating in serum-free
media containing rhuSCF.
Proliferating skin-derived mast cells retain their
characteristic functional phenotype
Experiments were performed to determine whether cultured skinderived mast cells degranulate in response to Fc⑀RI cross-linking
and to agents known to stimulate skin but not lung-derived mast
cells. As shown in Figure 6A, tryptase release occurred in a
dose-response fashion to anti-Fc⑀RI␣ mAb, maximal release being
observed at 0.3 to 3 ␮g/mL. Also, the amount of tryptase per mast
cell was calculated to be 22 ⫾ 4 pg/mast cell (n ⫽ 5), slightly less
than the amount measured per mast cell from skin tissue shortly
after their dispersion and density-dependent enrichment, which
was 29 ⫾ 3 pg/mast cell (n ⫽ 3). Degranulation also was observed
in a dose-dependent manner to substance P (Figure 6B) and to
compound 48/80 (Figure 6 C).
Discussion
The current study shows that skin-derived mast cells are capable of
proliferation, which could contribute to mast cell hyperplasia in
tissues. However, human mast cells that reside in peripheral tissues
Figure 4. Proliferating skin-derived mast cells require SCF to survive. After 6 to
8 weeks in culture with rhuSCF (100 ng/mL) in AIM-V medium, cells (95%-99% mast
cells) were washed with PBS and placed into culture with (closed circles and square)
or without (open circles and square) rhuSCF (100 ng/mL) in AIM-V medium. Cell
numbers and viabilities were measured daily for 6 days. (A) Percent viability (circles).
Also shown are the annexin V⫹ cells measured on day 2 (squares). (B) Viable cell
numbers. Each data point in panels A and B is the mean ⫾ SD for 3 independent
experiments, each performed in duplicate. *, P ⬍ .05 for a difference compared to the
day 0 value using a one-way repeated measures ANOVA and the Dunnet method for
multiple comparisons versus a control. #, P ⬍ .05 for a difference between the plus
and minus rhuSCF values using a paired t test.
these cells was placed directly onto glass slides that then were
stained with toluidine blue. All exhibited metachromasia, indicating this population contained virtually 100% mature mast cells
(Figure 5). Another portion of the cells was individually sorted into
96-well culture plates, each well containing a confluent monolayer
of human fibroblasts to act as feeder cells. Fibroblasts together with
rhuSCF were necessary for cell survival; in the absence of either
fibroblasts or exogenous rhuSCF, no cells were recovered after 3
weeks. These confluent fibroblasts survived but did not proliferate
under the serum-free culture condition used. After 3 weeks of
culture with AIM-V medium containing 100 ng/mL rhuSCF, the
number of mast cells per well was evaluated by immunocytochemistry with antitryptase mAb. Results are shown in Figure 5B and
Table 1. Mean data taken from 4 separate experiments indicated
only one tryptase-positive cell in 19% of the wells, and 2 or more
tryptase-positive cells per well in 44% of the wells (11 cells/well on
average). Thus, one or more mast cells were detected in 63% of the
wells, and in about two thirds of the mast cell-containing wells
there was evidence for mast cell proliferation. In 2 control
experiments single Kit-sorted HMC-1 cells yielded detectable cells
in 65% (58% and 71%) of the wells, the remainder presumably not
surviving the sorting or the culture conditions. The percentages of
wells with surviving mast cells are similar for skin-derived mast
cells and HMC-1 cells. These results indicate that most mature
Figure 5. Human skin-derived mast cells after GD3- and Kit-dependent immunoaffinity purification and coculture with fibroblasts. Dispersed and densitydependent sedimentation-enriched skin-derived mast cells were purified using
anti-GD3 mAb and were then labeled with PE-anti-Kit mAb. (A) A portion of these
cells was placed on a slide, air dried, and stained with toluidine blue. Mast cells exhibit
metachromatic purple cytoplasm and blue nuclei. (B) Another portion of these cells
was subjected to sorting of individual cells onto fibroblast monolayers, which were
then cultured with rhuSCF (100 ng/mL) for 3 weeks and stained with anti-tryptase
mAb (naphthol AS-BI/new fuchsin). Positive cells stain red. Panel B shows a well
containing multiple tryptase-positive cells. Original magnifications at the time of
photomicroscopy were ⫻ 400 for panel A, and ⫻ 200 for panel B (inverted microscope).
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2050
BLOOD, 1 APRIL 2001 䡠 VOLUME 97, NUMBER 7
KAMBE et al
Table 1. Mast cell proliferation of individually sorted skin mast cells
No. wells per plate
Plate no.
1 mast cell
At least 2
mast cells
Mast cells per well in wells
with at least 2 mast cells
(mean ⫾ SD, range)
1
16
39
14 ⫾ 12, 2-47
2
9
33
8.7 ⫾ 6.0, 2-25
3
33
47
11 ⫾ 11, 2-56
4
15
51
12 ⫾ 13, 2-56
19 ⫾ 11
44 ⫾ 8
Mean ⫾ SD
11 ⫾ 11
Enzymatically dispersed skin cells were immunoaffinity purified with the R24
mAb against GD3, and then sorted with the anti-Kit YB5.B8 mAb using a Coulter
Epics Cell Sorting System. Individual mast cells were sorted into each well of 96-well
plates containing human fibroblast monolayers, and were then cultured in AIM-V
medium containing 100 ng/mL rhuSCF. After 3 weeks of culture, mast cell numbers
were determined by immunochemistry for tryptase with the G3 mAb. Results from 4
separate sorts, one plate per sort, are shown.
of adult hosts have been considered to be long-lived cells with
limited, if any, proliferative capacity. At sites where mast cell
hyperplasia occurs, such as urticaria pigmentosa in skin,30 rheumatoid synovium,11 bronchopulmonary dysplasia and fibrotic lung
disease,31 and atopic keratoconjunctivitis,14,32 the rare presence of
mitotic mast cells has been used to argue that proliferation of
mature mast cells does not account for their increased presence.
Even when mast cell hyperplasia is iatrogenically induced by
administration of rhuSCF, mitotic mast cells in skin biopsies were
not reported.33 Consequently, expansion of mast cell populations at
specific tissue sites in humans has been predicted to occur by
diverting a greater portion of the pool of hematopoietic progenitor
cells to a mast cell lineage, by stimulating proliferation of mast cell
progenitors, by increasing recruitment of mast cells and their
precursors into tissues, or by increasing survival of mast cells.
Recruitment of mast cell from one region of a tissue to another or of
precursors from the circulation to tissue sites may be driven by
various chemokines,34-37 transforming growth factor-␤ (TGF-␤),38
SCF,39 and complement anaphylatoxins.40 Proliferation of mast cell
progenitors may occur before these cells acquire the characteristic
phenotypic features of the granules and cell surface of mature mast
cells, even in cases of systemic mastocytosis associated with
activating somatic mutations of c-kit.41 However, mitosis should be
a relatively brief event in the life of a long-lived cell such as a mast
cell. Thus, mitotic figures as a sign of mature mast cell proliferation
may lack adequate sensitivity.
The current study demonstrates that most mast cells derived
from adult skin can proliferate in serum-free media containing
rhuSCF. This suggests that SCF is a proliferation factor as well as a
survival42,43 and priming or activation44,45 factor for mature human
mast cells. Mast cell numbers in suspension cultures increased
150-fold by 8 weeks. At 4 weeks, mitotic or binucleated cells
accounted for 4.6% of the mast cells. A DNA fluorescence
histogram with PI revealed that 9.4% of the 4-week cells were
distributed in the S, G2, and M phases of the cell cycle. To rule out
the possibility that this represented expansion of a small progenitor
cell population, mature mast cells were purified by densitydependent sedimentation and immunoaffinity purification with
anti-GD3 mAb,28 and then were individually sorted using PElabeled anti-Kit mAb onto fibroblast monolayers in wells of
microtiter plates. At least two thirds of the mature mast cells that
survived such Kit-dependent sorting had the ability to proliferate.
Why this was observed in serum-free but not serum-containing
media is not known, but is consistent with previous observations
showing survival but not proliferation of mast cells in serumcontaining media.26,27 With serum, a substantial increase in fibro-
blast-like cells and metachromatic bodies in nonmast cells were
observed in our experiments. Consequently, the attenuating effects
of serum on mast cell growth, in part, could be indirect by
stimulating other cell types to grow and to phagocytose mast cells.
However, when serum was added to AIM-V–cultured mast cells at
4 weeks, when mast cells predominated and few if any fibroblastlike cells were observed, proliferation of mast cells was curtailed,
suggesting direct as well as indirect effects of serum on mast cell
growth. Thus, mature mast cells in human skin have the capacity to
proliferate when exposed to SCF, but factor(s) in serum or plasma
may regulate this response. Whether the extracellular fluid to which
tissue mast cells are exposed in vivo acts similarly to serum in vitro
remains to be determined.
Proliferating skin-derived mast cells appear to retain several
key phenotypic features of freshly dispersed cells. For example,
nearly all expressed high levels of cell-surface Kit and Fc⑀RI after
4 weeks of culture. That both receptors were functional can be
inferred because survival of these cells was dependent on rhuSCF,
and degranulation was induced by cross-linking Fc⑀RI with
anti-Fc⑀RI␣ mAb. Also, given that a substantial amount of
proliferation had occurred between day 0 and 4 and 8 weeks of
Figure 6. Skin-derived mast cells retain their capacity to degranulate in
response to several agonists after proliferating in vitro. Anti-Fc⑀RI␣ mAb (A),
substance P (B), and compound 48/80 (C) were assessed as degranulating agents
on skin-derived mast cells cultured for 4 to 8 weeks in AIM-V media containing 100
ng/mL rhuSCF. Tryptase release was determined after each agonist was incubated
with the mast cells for 15 minutes at 37°C. In each case data are presented as net
percent tryptase release. Spontaneous tryptase release values were always less
than 5%. Cell viabilities were determined at the high dose of each agonist at the end
of the experiment by trypan blue staining, and were observed to be more than 90% in
all cases. Bar graphs display mean ⫾ SD values from 3 independent experiments.
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BLOOD, 1 APRIL 2001 䡠 VOLUME 97, NUMBER 7
PROLIFERATION OF HUMAN SKIN–DERIVED MAST CELLS
culture, new receptors must have been synthesized. For Kit, similar
fluorescence intensities were observed between freshly dispersed
mast cells and those cultured for 4 to 8 weeks. In contrast, for
Fc⑀RI, higher intensities of surface labeling were observed on mast
cells after 4 to 8 weeks of culture than those freshly dispersed from
skin. Regardless as to whether the low Fc⑀RI levels on day 1 reflect
actual levels in vivo or removal of surface Fc⑀RI during the
proteolytic digestion of skin, new receptors must have been
synthesized in the absence of IgE to account for the high levels
after 4 to 8 weeks of culture. Such persistently high surface levels
of Fc⑀RI on skin-derived mast cells in vitro in the absence of IgE
contrasts with reports that Fc⑀RI levels dramatically fall in the
absence of IgE on human peripheral blood-derived basophils46-49
and in vitro-derived mast cells.50,51 However, Fc⑀RI levels on
peripheral blood-derived monocytes and eosinophils do not correlate with serum IgE levels,52 indicating factors other than IgE levels
may influence Fc⑀RI levels in some cell types. The current in vitro
observation suggests that mast cells in tissues might retain high
Fc⑀RI levels after IgE is neutralized with anti-IgE antibody, which
in turn may help to explain the persistence of immediate hypersensitivity responses in the skin and airway to allergens in sensitive
subjects treated with anti-IgE mAb at a time when basophil Fc⑀RI
levels are very low.46,53
The typical protease phenotype of skin-derived mast cells,
namely, both tryptase and chymase expression by immunocytochemistry, also was maintained. After 4 to 8 weeks of culture, the
tryptase content measured in cell extracts by an ELISA averaged 22
pg/mast cell, slightly less than the 29 pg/mast cell measured after
their initial dispersion. These values are comparable to what has
been reported for freshly dispersed skin-derived mast cells (35
2051
pg/mast cell),25 and greater than the content of lung-derived mast
cells (11 pg/mast cell), and fetal liver-derived mast cells (3.7 ⫾ 0.1
pg/mast cell, n ⫽ 323). As for tryptase, essentially all skin-derived
mast cells also express chymase when they are dispersed from skin,
and then retain their chymase-positive phenotype during culture in
serum-free AIM-V medium. In contrast, suspension cultures of
fetal liver-derived cells with rhuSCF produce mast cells with
abundant chymase messenger RNA, but few mast cells with
detectable chymase protein when serum-containing media is used.50
About one third of the mast cells express chymase protein
when they develop in suspension cultures with serum-free AIMV medium.23
Although human mast cells from all tissues degranulate in
response to Fc⑀RI cross-linking and to calcium ionophores,
heterogeneity has been reported for the response to substance P and
to compound 48/80. For example, freshly dispersed skin-derived
mast cells respond to both agonists; mast cells from most other
tissues do not, regardless of their protease phenotype.54-56 The
current study showed that proliferating skin-derived mast cells still
respond to these agonists after 4 to 8 weeks of culture in serum-free
medium. This result suggests that once responsiveness to substance
P and compound 48/80 develops, the cutaneous microenvironment
is not needed to maintain this functional phenotype.
Acknowledgment
We are grateful to Frances K. White (Flow and Imaging Cytometry
Facility, Virginia Commonwealth University, Richmond, VA) for
technical support with cell sorting.
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2001 97: 2045-2052
doi:10.1182/blood.V97.7.2045
Human skin−derived mast cells can proliferate while retaining their
characteristic functional and protease phenotypes
Naotomo Kambe, Michiyo Kambe, Jarema P. Kochan and Lawrence B. Schwartz
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