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Characterization of Cell Phenotype by a Novel cDNA Library Subtraction
System: Expression of CDSa in a Mast Cell-Derived
Interleukin-4-Dependent Cell Line
By Takahiko Hara, Nobuyuki Harada, Hideki Mitsui, Toru Miura, Teruko Ishizaka, and Atsushi Miyajima
We have established a unique variant cell line, MC/9.IL-4,
which continuously proliferates in the presence of interleukin-4 (IL-4). from a murine interleukin-3 (IL-3)-dependent
mast cell line, MC/9 (referred t o as MC/9.IL-3). Compared
with MC/9.IL-3 cells, MC/9.IL-4 cells are smaller, lack cytoplasmic granules and metachromasia, carry a very small
amount of histamine, and express fewer high-affinity IgE
receptors (IgERs) and IL-3 receptors. To further characterize
MC/9.IL-4, w e developed a novel method t o enrich cell typespecific cDNAs by cDNA library subtraction and applied it
for MC/9.IL-3 versus MC19.IL-4. Sequence analysis of cDNA
clones isolated by this technique showed that MC/9.IL-4
cells specifically express CD& and expression of mast cellspecific proteases andmajorhistocompatibility
complex
class II (MHCII) is considerably decreased. It was also noted
that responsiveness t o the IL-3-agonistic antibody F9 and
expression of the transcription factor GATA-2 is diminished
in MC/9.IL-4, indicating that MC/9.IL-4 have lost major characteristics of the bone marrow-derived cultured mastcells.
Because other T-cell marker antigens, CDW, CD4, Thy-l,
were not detected on MC/9.IL-4 cells, MC19.IL-4 cells may
represent an unknown class of hematopoietic cells that express CD&. This cell line will be useful in studies of IL4-mediated signal transduction, as well as transcriptional
regulation of mast cell characteristic genes. This study also
demonstrates the effective use of the cDNA library subtraction strategy t o characterize unknown types of hematopoietic cells at themolecular level.
0 1994 by The American Society of Hematology.
M
long-term growth of MCY9.L-3 cells. Interestingly, neither
IL-4 nor IL-10 alone sustains long-term growth of MC/9.IL3; a combination of these two cytokines supports long-term
growth.’*
During the studies of the IL-4 receptor and signal transduction, we have established an MC/9 variant cell line, which
proliferates permanently in response to L - 4 without L-3.
This IL-4-dependent MC/9 variant cell line (MU9.K-4)
has lost responsiveness to IL-3 due to the loss of the L - 3
receptor (IL-3R) expression (T.H. and A.M., submitted).
What phenotype does MC/9.IL-4 exhibit? To characterize
the cells at the gene expression level, we have developed a
novel method to enrich for cell type-specific cDNAs by
subtracting a cDNA library from another cDNA library, and
we isolated and analyzed several cDNAs that are induced
or downregulated in MC/9.IL-4 cells.
AST CELLS ARE derived from pluripotent hematopoietic stem cells in bone marrow, and are highly
differentiated in tissues such as peritoneum and intestinal
mucosa.’.* They express the high-affinity IgE receptor (IgER,
Fc,RI) on their cell surface3 and contain secretory granules
in their cytoplasm where a carboxypeptidase, several distinct
mast cell serine proteases, and histamine are to red.',^,^ Mast
cells play a role in the immune and inflammatory reactions,
particularly in the allergic reaction and defense against parasitic infection by secreting various kinds of mediators including cytokine~.~~’
Because rapid proliferation of mast cells in
the intestinal mucosa during parasitic infection does not occur in genetically T-cell-deficient n d n u mice,’ T-cell-derived cytokines appear to be involved in the proliferation of
mast cells. Until now four such cytokines, interleukin-3 (IL3), interleukin-4 (IL-4), interleukin-9 (IL-9), and interleukin10 (K-lo), have been molecularly cloned as growth/differentiation factors for mast
IL-3 is a pleiotropic cytokine that stimulates the proliferation and differentiation of hematopoietic stem cells, as well
as various lineage-committed hematopoietic progenitors, including pre-B cells, erythroid progenitors, megakaryocytes,
macrophages, eosinophils, and mast cell^.'^,'^ L - 4 also exhibits diverse activities on a variety of cell types. It induces
B-cell proliferation in the presence of anti-IgM antibodies,
induces lipopolysaccharide (LPS)-activated B cells to express CD23 and produce IgGl, stimulates T cells, monocytes, macrophages, mast cells, fibroblasts, and endothelial
IL-3-dependent immature mast cell lines can be established from murine bone marrow cells in the presence of IL3. MC/9I7(MC/9.IL-3 in this study) is one such cell line, and
has been widely used to study cytokines, cytokine receptors,
cytokine-mediated signal transduction, and mast cell maturation,12.18.19 Besides IL-3, various cytokines including IL-4,
IL-9, and IL-10 are known to stimulate the proliferation
of MC/9.IL-3 cells. While IL-3 alone supports long-term
proliferation of MCl9.L-3, other cytokines augment the proliferation, and none of these cytokines alone can support
Blood, Vol 84. No 1 (July l), 1994: pp 189-199
MATERIALSANDMETHODS
Cell lines, media, andcytokines. A murine IL-3(mIL-3)-dependent mast cell line, MC/9,” and its IL-4-dependent variant cell line,
MC/9.IL-4, were grown in RPM1 1640 medium supplemented with
10% fetal calf serum (FCS), 50 pmol/L 2-mercaptoethanol (2“E),
and either &L-3 (10 ng/mL), or murine L-4( a - 4 ) (10n@mL),
From the Departments of Molecular Biology and Immunology,
DNAX Research Institute of Molecular and Cellular Biology, Palo
Alto, CA; and La Jolla Institute for Allergy and Immunology, La
Jolla, CA.
Submitted August 19, 1993; accepted March 7, 1994.
DNAX Research Institute of Molecular and Cellular Biology is
supported by Schering-Plough Corp.
Address reprint requests to Atsushi Miyajima, PhD, Department
of Molecular Biology, DNAX Research Institute of Molecular and
Cellular Biology, 901 California Ave, Palo Alto, CA 94304.
The publication costs ofthis article were defiayed 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.
0 1994 by The American Sociery of Hematology.
wO6-4971/94/8401-0010$3.00/0
189
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190
respectively. Recombinant mIL-3 and mIL-4 were described previous~y.*o~2'
Cell proliferation assay. To detect a short-term response of cells
to mIL-4 and F9 antibody, a colorimetric assay using 3-(4,5-dimethyl-thiasol-2-yl)-2,5-diphenyltetrmlium bromide (m)
(Sigma,
St Louis, MO) was performed as described by Mosmann.z*Briefly,
IO4 cells (50 pL) were mixed with 50 pL of various concentrations
of mIL-4 or F9 hybridoma supernatant in 96-well plates. After a 2day culture, 10 pL of MTT (5 mg/mL phosphate-buffered saline
[PBS] was added per well and incubated at 37°C for 4 hours before
the colorimetric analysis. The F9 hybridoma supernatantz3was generously provided by Dr M. Furuichi (Nippon Roche, Kanagawa,
Japan).
Analyses of mast cell-characteristicproperties. Morphological
properties of MC/9.IL-4 cells were characterized by staining with
May-Griinwald-Giemsa, Alcian blue (pH 0.4), and Safranin. The
number of Fc,RI expressed on the cells was estimated by direct
binding of 'Z51-labeledmouse IgE.24Histamine content in the cells
was determined by the automated technique of Siraganian2'
Antibodies and jlow-cytometric analysis. Phycoerythrin (PE)conjugated anti-CD8cu, anti-CD80, and anti-CD4 monoclonal antibodies (MoAbs), Fluorescein isothiocyanate (F1TC)-conjugatedantiCD25 and anti-Thy-l MoAbs were provided by DrA. Zlotnik
(DNAX, Palo Alto, CA). FITC-conjugated murine IgE was provided
by S. Hudak (DNAX). Anti-B220/CD45R MoAb (RA3-6B2),*' antiCD24/heat-stable antigen MoAb (J1 Id)'' and isotype control MoAb
were obtained from Pharmingen (San Diego, CA). MoAb against
murine major histocompatibility complex class I1 (MHCII) (M5/
114)'' was obtained from Boehringer Mannheim (Indianapolis, IN).
FITC-conjugated anti-rat IgG or IgM antibodies were purchased
from Pierce (Rockford, IL). Cells (IO5 to 10') were incubated in 50
pL of PBS pH 7.4, containing 5% FCS and PE- or FITC-conjugated
MoAb (10 pg/mL) or FITC-conjugated IgE (40 pg/mL), and analyzed by using a FACScan (Becton Dickinson, San Jose, CA). For
unconjugated MoAbs, FITC-conjugated corresponding second antibodies ( I O pg/mL) were used.
Construction of directional cDNA libraries. Poly(A)' RNA
from MC/9.IL-3 cells or MC/9.IL-4 cells was used to synthesize
first-strand cDNA by using the NotYOligo-dT,, primer (Pharmacia,
Piscataway, NJ). Double-stranded cDNA was synthesized, ligated
with EcoRI adaptors, digested with NotI, size fractionated (> 1.0
kilobase pairs [kbp]) and ligated into EcoRIINotI sites of pME18S
vector, a derivative vector ofpCEV4.''
Electro-competent Escherichia coli WM1100 cells (BioRad, Hercules, CA) were used for
transformation. Total number of independent clones of the cDNA
libraries were 6 X lo5 for MC/9.IL-3 and 1.2 X lo6 for MC19.L
4, respectively.
Library subtraction. The polymerase chain reaction (PCR)based subtraction system developed by Wang and Brown'" was modified to apply to plasmid cDNA libraries. An MC19.IL-4-specific
subtracted library was generated as follows: 40 pg of MC/9.IL-3
cDNA library DNA was digested with EcoRI, NotI, and ScaI (ScaI
cleaves the vector) followed by the filling-in reaction with DNA
polymerase Klenow fragment. After ethanol precipitation, the DNA
was dissolved in 40 pL of HzO, heat-denatured, mixed with 40 pL
(40 pg)of Photoprobe biotin (Vector Laboratories, Burlingame, CA).
According to the manufacturer's manual, it was irradiated with a
270-W sunlamp on ice for20 minutes. Additional Photoprobe biotin
(20 pL)was added and the biotinylation reaction was repeated. After
buthanol extraction, the photobiotinylated DNA (Driver"3)
was
ethanol-precipitated and dissolved in 32 pL of 10 mmoVL Tris-HC11
1 mmol/L EDTA, pH 8 (TE). As a tracer DNA, I pg of MC19.IL4 cDNA was digested with EcoRI and NotI, ethanol-precipitated,
and dissolved in 4 pL of TE (Tracer"4).Tracer"4
was mixed
HARA ET AL
with 16 p L ofDriver"3,
1 pL (10 pg) of E coli tRNA (Sigma),
and 20 pL of 2 X hybridization buffer (1.5 m o m NaCVlO mmol/
L EDTA/5O mmol/L HEPES pH 7.5/0.2% sodium dodecyl sulfate
[SDS]), overlaid with mineral oil, and heat-denatured completely.
The sample tube was immediately transferred into a 68°C water bath
and incubated for 20 hours (long hybridization [LH]). The reaction
mixture was then subjected to the streptavidin treatment followed
by phenol/chroloform extraction exactly as described by Wang and
Brown." Subtracted DNA was precipitated, dissolved in 12 pL of
and 20 pL of 2 X hybridization
TE, mixed with 8 pL of Driver"3
buffer, and subjected to a hybridization at 68°C for 2 hours (short
hybridization [SH]). After removal of biotinylated double-stranded
DNAs, residual DNA was ligated with 250 ng of a purified fragment
(3.0 kbp) of pME18S digested with EcoRI and NotI, and used for
transformation of the electro-competent E coli cells to generate first
MC/9.IL-4-specific subtracted library (M4-l). The same procedure
was performed by using the MC/9.IL-3 library as a tracer and the
MC/9.IL-4 library as a driver to produce first MC/9.IL-3-specific
subtracted library (M3-1). Second MC/9.IL-4-subtracted library
(M4-2) was made from M4-l after LH with M3-I as a driver followed by SH withDriver"3.
Likewise. second MC/9.IL-3-subtracted library (M3-2) was also made from M3-1 byLHwith M41 as a driver followed by SH with the MC/9.IL-4 library as a driver.
Plasmid DNAs were prepared from 100 independent clones, which
were randomly picked from each second subtracted cDNA library,
and grouped based on the insert size and restriction sites. Representative cDNA clones were further characterized by RNA blot analysis
and DNA sequencing.
RNA blot analysisand
reverse transcriptase-PCR analysis.
Poly(A)+ RNA was prepared from MC/9.IL-3 and MC/9.IL-4 cells
by using QuickPrep mRNA purification kit (Pharmacia). RNA blot
and hybridization was performed according to the standard method.
The cDNA fragments isolated by digestion with EcoRI andNot1
were labeled with 32Pby using T7 Quickprimer labeling kit (Pharmacia) and used for probes. As a probe for the detection of 0-actin
transcript, a 1.2-kbp PstI fragment of the murine p-actin cDNA,
kindly provided by Dr R. De Waal Malefyt (DNAX), was used.
Probes for GATA-I and GATA-2 were the 1.8-kbp XhoI fragment
of the mouse GATA- 1 cDNA and the 2.2-kbp EcoRI fragment of the
1.o
0.8
0.6
0.4
4
P
1
I
1
.OOOI
.OOI
.OI
.l
I
IO
loo
loo0
IL-4 nglml
Fig 1. Prolieration response of MC19.11-4 cells to IL-4. MC/9.IL-4
cells (0)
and its parental MC/9.IL-3 cells (0)were incubated for 2
days in the presence of various concentrations of mouse 11-4, and
cell growth was measured by the M l l assay.
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Fig 2. Morphologic appearance of MC/9.IL-4 cells.
Cytospin of MC/9.IL-4 cells (A) and its parental MC/
9.IL-3 cells (B) were stained with May-GriinwaldGiemsa. (Original magnification x 1,000.)
human GATA-2 cDNA (generous gifts from Dr S. Orkin, Harvard
RESULTS
Medical School), respectively. Poly(A)+ RNA was used for reverse
Establishment of an IL-4-dependent MC/9 variant cell
transcriptase (RT)-PCR analysis with First-Strand cDNA synthesis
line. MC/9.IL-3 is an L-3-dependent mouse mast cell
kit (Pharmacia) and AmpliTaq DNA polymerase (Perkin Elmer, Norline, and also proliferates in response to L - 4 for a short
walk, CT) according to the manufacturers' manuals. Primers to deperiod of time (Fig 1). Continuous culture of MC/9.L-3 in
tect mRNA expression of mouse mast cell protease-2 (MMCP-2),
mouse mast cell protease-6 (MMCP-6), and mouse GATA-3 were
medium containing L - 4 without IL-3 resulted in death of
5"AACGGTTdesigned
based on the published sequence
most of the cells in several days. However, an adapted variCGAAGGAGAGGTGT-3'
and
5"CCAGGGCAGGTAATAGGant cell population started to grow after a l-month culture.
AGAT-3' for MMCP-2; 5'-CTGCGGAGGCTCTCTCATCCA-3' These cells responded to L - 4 much more stronglythan MC/
and 5'-GGAATGCTCAGGGACATAGCG-3'for MMCP-6; 5'9.L-3 (Fig 1) and have grown continuously in the presence
CAGCTGCCAGATAGCATGAAG-3' and5"GCTCTTGGGGAof L - 4 for over 2 years. They express approximately twice
AGTCCTCCAG-3' for GATA-3, respectively.
as many L - 4 receptors as the parental cells based on staining
DNA sequencing. PlasmidDNA was denatured,primedwith
with the anti-IL-4 receptor MoAb (data not shown). We
two oligonucleotides, M E - I and ME-4, complementary to sequences
designated
the variant cell line as MU9.L-4. Interestingly,
located 50 bases upstream and downstream from the cloning site of
MC/9.IL-4 cells no longer responded to L - 3 due to the loss
pME18S, respectively. The samples were subjected to the dideoxyof L - 3 receptor expression (T.H. and A.M., submitted).
nucleotide chain termination reaction by using Sequenase kit (US
Morphologic properties of MC/9.IL-4 cells. The bone
Biochemical, Cleveland,OH). Sequences of 150 to 250 bp from the
marrow-derived mast cells cultured in medium containing
5' terminal of each insert cDNA fragment weredeterminedand
searched against GenBank and Eh4BL nucleotide data bases.
L - 3 exhibit immature mast cell ~ h e n o t y p e : ' ~ ,they
~ ' ~ ~are
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HARA ET AL
192
Alcianblue'/Safranin-, contain small amounts of granule
and histamine, contain mast cell-specific secretary granule
serine proteases, and exhibit the high-affinity IgER. To examine if MC/9.IL-4 cells possess such characteristics of the
cultured mast cells, we compared the morphologic appearance and the histochemical profile between MC/9.IL-3 and
MC/9.IL-4 cells. As shown in Fig 2, MC/9.IL-4 cells were
smaller in size and did not contain cytoplasmic granules as
seen in MC/9.IL-3 cells, although we observed fine granules
in a small percentage of MC/9.IL-4 cells. MC/9.IL-4 cells
did not show metachromasia that is characteristic to mast
cells. Expression of the high-affinity IgER, as well as production of histamine, was barely detectable in MC/9.IL-4 cells
(Table l). These results suggested that MC/9.IL-4 cells appeared to have lost characteristic properties of the bone marrow-derived mast cells.
Isolation of cell type-specific genes by cDNA library subtraction. To further characterize MC/9.IL-4 cells, we attemptedto isolate genes that are specifically expressed in
MC/9.IL-4 cells, but not in MC/9.IL-3 cells, and also genes
that are downregulated or diminished in MC/9.IL-4 cells.
For this purpose, we developed a cDNA library subtraction
system (Fig 3A) and applied it for MC/9.IL-3 cells versus
MC/9.IL-4 cells. Directional cDNA libraries were generated
by using a plasmid vector from poly(A)' RNA prepared
from both cells. The cDNA inserts from one library were
released from the vector by restriction enzyme digestion.
The mixture of the DNA fragments were photobiotinylated.
and hybridized with the other library DNA, which was also
digested with restriction enzymes to release the cDNA inserts. After removal of common cDNA species by streptavidin treatment followed by phenol extraction, the remaining
DNA fragments were reinserted into the vector and used for
producing a subtracted library. As shown in Fig 3B, several
enriched cDNA insert bands were visible in the first subtracted libraries and second subtraction resulted in fewer and
more prominent bands. We, therefore, isolated individual
clones from the second subtracted libraries (M3-2; MC/9.1L3 cDNA library subtracted twice with MC/9.IL-4 cDNAs,
and M4-2; MC/9.IL-4 cDNA library subtracted twice with
MC/9.IL-3 cDNAs, see Materials and Methods, and examined their specific expression in either MC/9.IL-3 cells or
MC/9.IL-4 cells byNorthernblot
analysis. Eight of nine
genes isolated from M3-2 were MC/9.IL-3-specific, while
five of six M4-2-derived genes were specifically expressed
in MC/9.IL-4 cells (Fig 4 and data not shown).
Table 1. Loss of Mast Cell-Characteristic Properties
in MC/9.11-4 Cells
Cytoplasmic granules"
Proportion of Alcian Blue positive
cells (%P
Histamine content (ng/lOEcellslt
IgE binding (molecules/cell)t
MCB.IL-3 Cells
MC/9.IL-4Cells
+
-
l00
2
10
2.9 x 10'
1.1
<l02
Based on the microscopic observation of cells.
t The results are shown as the mean for two similar experiments.
A
MC/S.iL? mRNA
MC19.iLi3 mRNA
DirecHo!ml pMEl8S cDNA library
Dirsctio~al
pME18S cDNA library
EwRVNoti digestion
Fragmentalion by E c o R i i N o I ~ V K I e n o w
enzyme
I
I
I
I
'
Pholobiotinylation
I
Driver-M3 (20pg)
Trnmr"4 (1 pg)
Long hybridization (W)
I
/
Stnptavidin treatment
I
Short hybridizallon (W)wlth Driver-M3 (10 pg)
I
Ligationinto EcoRIINotisltes of pME18SMCtor
I
Bacterial transformation
I
F l n t subtracted ilbrary (W-l)
I
Another cycle of U( wilh M51 as II drlvsr
lollowed by Y( wilh Driver-M)
I
Sownd sublmcted library (M4-2)
kbP
VectorB.O+
1.8-
5
d"11
4
d
1.01
0.9
0.4
-
Fig 3. Library subtraction for isolation of genes that are specifically expressed in MC/9.IL-4 cells or MC19.IL-3 cells. (A) Flow diagram
of Library subtraction. EcoRIINot l-digested plasmid DNA (1 p g ) of
MC/S.IL-4-derived cDNA library was hybridized
with fragmented and
photobiotinylated plasmid DNA (20pg) of MC/S.IL-3-derived cDNA
library. Afterremoval of common cDNA species bystreptavidin
treatment, recovered DNA was religated in the vector and used for
generatingasubtracted cDNA library(M4-l). Inversely, MW9.11cDNA-enriched subtracted library(M3-l) was made in the same way.
M4-2 andM 3 2 represent second subtracted cDNA libraries for MC/
9.IL-4-specific genes and MC/9.IL-3-specific genes, respectively. (B)
Restriction enzyme digestion pattern of the subtractedcDNA libraries. Plasmid DNAs from the starting cDNA libraries and the subtracted cDNA libraries derived from
either MC19.IL-4 cells or MC/9.113 cells were digested with EcoRl and Not I t o separate inserts from
the vector DNA (pME18S) and analyzed by 1% agarose gel electrophoresis. Ethidium bromide stain.
We next analyzed the DNA sequences of these 13 cDNA
inserts and searched for homologous sequences in the databases. The results are summarized in Table 2. Nucleotide
sequences (150 to 200 bp) of the 5'-end of MC/9.IL-3specific cDNAs, 3H,M32c1, M32c10, M32c18, M32c47,
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193
CELLCHARACTERIZATIONBYLIBRARYSUBTRACTION
Table 2. 1dent.W of Isolated Genes That Are Specifically Expressed
in Either MC/9.1L-3 or MC19.L-4 Cells
Gene Name
mRNA
(kb)
MC/9.11-3specific
genes
3H
1.5
M32cl
M32c10
M32c18
1.1
1.0
1.o
M32c37
1.2
M32c47
M32c63
MC/9.IL-4specific
genes
4c
4A
1.1
1.7
3.0,1.8
1.4
M42c60
M42c80
M42c63
4.0, 2.4
2.0
3.0
Identity
Reference
Mast cell carboxy peptidase
A (MC-CPA)
MHCll EB
MHCll Am
Mast cell protease-5 (MMCP5) or chymase-l
Related to human monocyte/
neutrophil elastase
inhibitor
MHCll AB
Tryptophan hydroxylase
38
CD&
Cytotoxic cell protein-l
(CCPI) or CTLA-1,
Granzyme B
Unknown
Unknown
Related to human myristoyl
CoA: protein Nmyristoyltransferase
39
40
41, 42
45
43
44
46
47-49
50
and M32c63, were identical to published sequences of mast
cell carboxy peptidase A (MC-CPA)?' MHCII
MHCII
A c Y , mouse
~
mast cell protease-5 (MMCP-5),'""* MHCII
AD': and tryptophan hydroxylase," respectively. One gene,
M32c37, was not completely matched to any known sequence. However, it has significant homology with the hu-
Fig 4.RNA blot analyses of MC-CPA and CD&
mRNAs in MW9.11-3 and MC/9.IL-4 cells. Poly(A1'
RNA samples (2 p g each1 from MC/9.1L-3 cells and
MW9.11-4 cells were electrophoresed on a 1.3% agarose gel, transferred t o a nitrocellulose membrane,
and hybridized with 32P-labeledMC-CPA cDNA 11.5
kbp full length) (A) or CD& cDNA (0.6 kbp €coRl/
N o t I fragment) (B). The membranes were reprobed
with 3ZP-labeledp-actin cDNA t o verify amounts of
RNA loaded.
man monocyte/neutrophil elastase inhibitor gene (71%),45
and the horse serapin gene (68%) (Kordula T, et al, unpublished results. M91 161 in Genbank), suggesting that M32c37
is a mouse counterpart of the elastase inhibitor gene. One
cDNA, M32c64 was a shorter clone of the MC-CPA gene.
Likewise, we identified the MC/9.IL-4-specific cDNA inserts as follows: 4C and 4A were identical to CD8aM and
the cytotoxic cell protein4 (CCPI; also known as CTLA-1
or granzyme B):749 respectively. M42c63 showed homology
with the human myristoyl CoA:protein N-myristoyltransferase gene (86%);' suggesting M42c63 as its mouse homolog.
The other two cDNAs, M42c60 and M42c80, were derived
from novel genes, because entire DNA sequences of the
two cDNAs and their protein sequences deduced from the
putative open reading frames did not show significant homology to any published sequences and known motif sequences
(T.H. and A.M., unpublished results).
Loss of mast cell characteristics in MCD.IL-4 cells. The
library subtraction experiment showed that MC-CPA,as well
as MMCPJ, which are localized in the cytoplasmic granules
of mast cells, are decreased substantially in MC/9.IL-4 cells
(Fig 4 and data not shown). This result is consistent with
the histochemical analysis of the cells. Expression of another
mast cell serine protease, MMCP-2, was also diminished in
MC/9.IL-4 cells based on the RT-PCR analysis. However,
the MMCP-6 transcript was detected by this method in both
cells (data not shown). Absence of the high-affinity IgER
was again confirmed by flowcytometry by using FITC-labeled murine IgE (Fig 5B). As indicated by the library subtraction, MC/9.IL-3 cells were found to express MHCII, and
its expression was considerably decreased in MU9.IL-4
cells. This was also proved by the FACS staining with antiMHCII complex MoAb, MY1 14 (Fig 5B).
As described elsewhere'' (T.H. and A.M., submitted) the
high-affinity &3R, which is composed of the CY and fl subunits,'* and known to be expressed in murine mast cells,
MGCPA+
U
CD8a
c
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HARA ET AL
194
A
MC/9.1L-3
MC/9. IL-4
B
M W9.1
L-3
MC/9.1L-4
lgER
MHCll
l E!
B220
Q)
.->
-m
c
a I
Q)
ld
CD24
Thy-l
Fluorescence Intensity
-
"-
Fluorescence Intensity
-
Fig 5. Expression of hematopoietic cell surface antigens in MC/9.IL-4 cells. Cell surface expression of various hematopoietic antigens in
MC19.IL-4 cells and its parental MW9.11-3 cells was examined by flow cytometry by using (A) PE-conjugated anti-CDh, PE-conjugated antiCDSp, FITC-conjugated anti-CD25 (IL-~RcY),
FITC-conjugated anti-Thy-l monoclonal antibodies, (B) FITC-conjugated murine IgE, and MoAbs
against MHCII, B220 (CD45R). and CD24 (heat-stable antigen). For detection of MHCII, 8220. and CD24, FITC-conjugated anti-rat IgG or IgM
antibody was used as a second antibody. Shaded areas show staining profiles with specific antibodies and blank areas show staining
profiles
with an isotype control MoAb (CDSn, CDSp, 0 2 5 , Thy-l, and IgER) or the second antibody alone (MHCII, 8220. CD24).
were also absent in MC/9.IL-4 cells. Because many IL-3responsive hematopoietic cell lines, including mast cells,
have been shown to respond to the agonistic F9 antibody
for
we examined the responsiveness of MC/
9.IL-4 cells to the F9 antibody. The F9 antibody seems to
recognize a distinct molecule from the cloned IL-3R subunits
because the CTLL-2 transfectant expressing two types of the
high-affinity IL-3R (CTLWABS C32) cannot proliferate in
the presence of F9 antibody (Fig 6). The MC/9.IL-4 cells
were unable to respond to the IL-3 and F9 antibody (Fig 6).
Taken together, our results indicate that MC/9.IL-4 cells
have lost many mast cell-related characteristics.
CD8a expression in MC/9..IL-4 cells. To confirm the specific expression of CD80 in MU9.IL-4 cells atthe protein
level, the cells were stained with anti-CD8a MoAb, as well
as MoAbs against other T-cell surface antigens, CDSP, Thy1 (Fig 5A), and CD4 (data not shown). Interestingly,MC/
9.1L-4 cells expressed CD8a butnot other T-cell antigens.
CD25 and B220, which were expressed
in MC/9.IL-3 cells,
were retained in MU9.IL-4 cells as well (Fig 5, A and B).
indicating that MC/9.IL-4 still possesses
a part of the parental
phenotype. This agrees with the expression of a small amount
of MC-CPA,MMCP-5,andMMCP-6
in MU9.IL-4 cells.
Another antigen found to be differentially expressed in MC/
9.IL-4 cells was the heat-stable antigen,
CD24 (Fig 5B), which
is known to be present in immature thymocytes, pre-B cells,
and erythroid cells, but not in mature lymphocytes."
To examine whether the phenotypic change of MC/9.IL3 to MC/9.IL-4 is reversible, we cultured MC/9.IL-4 cells
in the presence of IL-3 without IL-4. Only a few cells survived under this condition. and IL-3-responsive cells were
obtained after a 1 -month culture. FACS analysis showed that
these IL-3-responsive cells expressed the a and P subunits
of IL-3R. but not CD8a and IgER (Fig 7). This result suggested that the phenotype of MC/9.IL-4can be partially
converted to that of MC/9.IL-3.
Expression of hematopoietic transcriptionfactors. Transcriptional activity of theMC-CPA gene isknowntobe
ERIZATION
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
195
CELL
2 (3.0-kb mRNA) was only detectable in MC/9.IL-3 cells.
Transcript of
the other
member of
the GATA family, GATA3, was detected in both cells by RT-PCR assay and Northern
analysis
(data not shown).
These results
suggested
that re-
MCB.11-3
"-t
CTLUABS C32
markable change of the phenotype in MC/9.IL-4 cells could
be correlated with downregulation of the hematopoietictranscriution factor GATA-2.
0.4
\
1
10
100
DISCUSSION
Oo0
Ooo0
F9 dilution (x)
Fig 6. Loss of proliferation response t o the agonistic MoAbF9 of
MC/9.1L-4 cells. MC/9.IL-3 cells (01,MC19.IL-4 ( C ) ,and CTLL-2 stable
transfectant cells,CTLL/ABSC32 (AI,which expresses the a and
p subunits of IL-3R. were incubated with serial dilutions of the F9
hybridoma supernatant for 2 days. Proliferation response of each cell
line was determined by theMlT assay.
positively regulated by the transcription factor, GATA-2.s3
The GATA-2 binding DNAmotif sequence, (A/T)GATA(G/
A), was found in the 5' upstream regions of the transcriptional initiationsitesof
MMCP-5," rat Fc,RI," andthe
mouse IL-3R /? genes.5' All of these genes were found to
be diminished in MC/9.IL-4 cells. Hence, we examined expression of GATA-I and GATA-2 genes in MC/9.IL-3 and
MC/9.IL-4 cells. Intriguingly, as shown in Fig 8, GATA-I
(1.8-kb mRNA) was expressed in both cells, while GATA-
We have established theIL-4-dependent mast cell-derived cell line, MC/9.IL-4, histochemical analyses. and molecular characterization of this cell line have demonstrated
that MC/9.IL-4 cells have lost mast cell-related properties.
They, instead, express CD8a and CCPI. both of which are
cytotoxic T-cell markers. In themouse, CD8a and CD8P
form the CD8complex, which is expressed
mainly
on
cytotoxic T cells
and
is also involved in interaction with MHC
class I on antigen presenting cells. The CD8 expression is
induced during T-cell developmentin the thymus.5hSofar. it
has been reported that not only T cells. but also lymphokineactivated killer cells and dendritic cells express CD8a."'59
However, MC/9.IL-4 cells do not exhibit exactly the same
phenotype of such cells. Clearly, it is not a typical T-cell
line. because ( I ) MC/9.IL-4 cells do not express CDS/?. as
well as Thy-l(FigSA);(2)
rearrangement of theT-cell
receptor P locus has not occurred in MC/9.IL-4 cells, and
cytokine production (IL-2 and granulocyte-macrophage colony-stimulating factor[GM-CSF]) was not induced upon
stimulation with phorbol ester (Kennedy J. Zlotnik A, Hara
T, et al, unpublished results); (3) small amounts of the mast
cellproducts such as MC-CPA, MMCP-S, and MMCP-6
were still detected in MC/9.IL-4 (Table 3); and (4) expression of GATA- l , absent in T cells, was found at the level
equal to that of MC/9.IL-3 (Fig 8).
IL-3R p
CD8a
lgER
1
MC19.IL-3
Fig 7. Phenotype of IL-3-responsive revertants of MCI9.IL4 cells. Surface expression of IL3Ra.lL-3RP.IgER. and CD& in
MC/9.1L-3, MC/9.IL-4, and revertant cell mixture of
MC/9.IL-4
(MC/g.IL-4 -t IL-31was examined
by
flow
cytometry
by
using
MoAbs against IL-3Ra (Gorman,
D, Hara T, Miyajima A, unpublished) and IL-3Rp (9D3):'
FITCconjugated IgE, and PE-conjugated anti-CD8a antibody. Blank
areas show
staining
profiles
with FITC-conjugated goat-antirat IgG antibody.
MC19.IL-4
L
MC19.IL-4
IL-3
-+
Intensity
Fluorescence
b
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
196
HARA
%
GATA-2 +
downregulation of MC-CPA, MMCP-S, FceR, and IL-3RP
in MC/9.IL-4 may be correlated with the loss of GATA-2.
However, it is unlikely that GATA-2 alone accounts for the
cell type-specific expression of the MC-CPA and the other
genes because GATA-2 is expressed in a wide variety of
cells including embryonic stem cells, fibroblasts, and many
tissues." Presumably, multiple genes, which govern the transcription in mast cells, must have been deregulated in MC/
9.IL-4 cells. As MC/9.IL-4 cell line was established without
any mutagenesis, it is unlikely thatthe drastic change of
the cell phenotype is due to multiple mutations in various
transcription factors. It is more likely that deregulation of a
critical gene caused alteration of other transcription factors
that led to the phenotypic change of MC/9. The phenotype of
MC/9.IL-4 may be aberrant and nonphysiologic. However,
another possibility is that MC/9.IL-4 represents a certain
stage of primitive mast cell precursors. Morphologic appearance and expression of small amounts of the mast cell proteases may support this hypothesis. However, CCPI, expressed
in MC/9.IL-4 but not in MC/9.IL-3, was originally found in
cytotoxic T cells stimulated with mitogens. The recent report
suggested that CCPI is abundantly present in the IL-3-dependent multipotential myeloid progenitor cell line, FDCPmix, andits expression is downmodulated along granulocytic
differentiation." Likewise, CD24, expressed in MC/9.IL-4,
Table 3. Summary of Phenotypic Comparison Between MC/9.11-3
Cells and MCl9.11-4 Cells
Expression Gene
Fig 8. Expression of GATA-1 and GATA-2 in MC/9.IL-3 and MC/
9.11-4 cells. PolyIA)' RNA samples 12 p g each) from MC/9.11-3 cells
and MCl9.IL-4 cells were electrophoresed on a 1.0 46 agarose gel and
transferred to a nylon membrane. Triplicated filters were hybridized
with '*P-labeled mouse GATA-1 cDNA, human GATA-2 cDNA, and pactin cDNA under the stringent condition, respectively.
As shown by Zon et a!,"' GATA-2 transactivates the transcription of the MC-CPA gene. In addition, the GATA binding motif isfound in the S' upstream regions of the transcription initiation sites of the MMCP-S, the rat FER, and the
murine IL-3RB genes,'"~s4~s5
all of which weredownregulated
in MC19.IL-4 cells. In MC/9.IL-3 cells, three members of
the GATA gene family, GATA- I , GATA-2, and GATA-3,
were expressed (Table 3). Among them, GATA-2 expression
was diminished in MC/9.IL-4 cells (Fig 8), suggesting that
Mast cell-related
lgER
MC-CPA
MMCP-2*
MMCP-5
MMCP-6*
IL-3R-related
IL-3R a and 0
Response to F9 antibody
T-cell-related
CD&
CD84
CD4
Thy-l
CCPI
Transcription factors
GATA-1
GATA-2
GATA-3
Cell surface antigens
MHCll Aa, A0, and €0
CD24
CD25
B220
Others
Monocytolneutrophil elastase inhibitor-
MCD.IL-3
+
+
+
+
+
MC19.IL-4
+I+I-
+
+
-
-
+
+
+
+
+
+
-
+
-
+
+
-
+
+I-
+
+
+
+
related gene
Tryptophan hydroxylase
+
+
-
N-myristoyl transferase-related gene
-
+
The results were based on RT-PCR analysis using specific primers.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
CELL CHARACTERIZATION BY LIBRARY SUBTRACTION
197
man for his help with analysis of the sequences, and Dr K. Moore
but not in MC/9.IL-3, is known tobe expressed in immature
for critical reading of this manuscript.
thymocytes, pre-B cells, and erythroid cells, but not in mature lymphocytes. While these findings are in favor of the
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1994 84: 189-199
Characterization of cell phenotype by a novel cDNA library
subtraction system: expression of CD8 alpha in a mast cell-derived
interleukin-4- dependent cell line
T Hara, N Harada, H Mitsui, T Miura, T Ishizaka and A Miyajima
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