1. Art and Diseño

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Transcript Synthesis and Surface Expression of the Interleukin-2 Receptor
(a-,
p-, and ?-Chain) by Normal and Malignant Myeloid Cells
By Ralf R. Schumann, Takayuki Nakarai, Hans-Jurgen Gruss, Marion A. Brach, Ute von Arnim, Carsten Kirschning,
Leonid Karawajew, Wolf-Dieter Ludwig, Jean-Christophe Renauld, Jerome Ritz, and Friedhelm Herrmann
Expression of the interleukin-2 receptor a- (IL-2Ra-1, IL-ZRP-,
and the recently identified IL-2Ry-chainwas examined on a
wide range of cells of myeloid origin including neutrophils,
monocytes, normal bone marrow-derived myeloid progenitors enriched for CD34+ cells, bone marrow blasts obtained
from acute myelogenous leukemia (AML) patients, and permanent myeloid leukemia cell lines by reverse transcriptasepolymerase chain reaction and surface membrane analysis
using receptor chain-specific monoclonal antibodies and
flow cytometry. Expression of the p75 IL-PRP- and the p64
IL-2Rychain was a common finding in most of the myeloid
cell samples investigated, whereas IL-2Ra-chain was less
frequently expressed. Although the high-affinity IL-2R form
(ie, the a+, p+, y+ IL-2R form) was detectable in a small
minority of primary AML samples as well as the KG-l cell
line and 11-2 binding t o these cells wassufficient to initiate
signal transduction as evidenced by an increase in overall
protein tyrosine phosphorylation and more specifically in
tyrosine phosphorylation of the Janus kinase (JAK) 3, in
none of these cell types did exposure t o IL-2 affect cell
growth kinetics. These results suggest that,in myeloid cells,
the IL-2R may not stimulate mitogenic responses orthat its
components may be expressed in a combinational association withreceptorsfor other cytokines and that IL-2Ry may
play a regulatory role in normal and malignant myelopoiesis
possibly independent from IL-2. Because recent studies by
others have indicated that theIL-ZRy- chain may be shared
by the I L 4 , the IL-7R. and most likely the lL-9R. expression
of mRNA of these receptor types was also investigated in
these cell samples. Surprisingly, in a substantial part of the
myeloid lineage cells examined, an IL-2Ry+, IL-4R-, IL-7Rconfiguration was noted that was, however, frequently associated with expression of IL-9R. Sharing of IL-9R/IL-2R
components was furthermore suggested by inhibition of
'251-lL-2 binding t o primary AML cells with excess of unlabeled 11-9.
0 1996 by The American Society of Hematology.
T
Such an unexplained discrepancy between deficient ligand
and deficit of a single receptor chain has invited basic research to examine whether IL-2Ry functions exclusively as
a part of the IL-2R or acts by forming part of other cytokine
receptors. This mystery was recently solved whenit was
shown that the IL-2Ry-chain also served as a functional
component of the receptors for both IL-4 and IL-7.23-25
The recent discovery of IL-2Ry chains displayed on the
membrane surface of human neutrophils" has prompted us
to examine expression of the IL-2R components and their
functionality on a broader range of normaland leukemic
myeloid cells.
HOUGH HUMAN interleukin-2 (IL-2) plays a central
role in growth and function of T lymphocytes, it also
boosts the activity of other lymphoid cells such as B
lymphoctes, natural killer cells, and lymphokine-activated
killer cells.'*2More recently, it has been shown that IL-2 also
affects effector functions of nonlymphoid cells including
fibroblasts:"
normal and malignant epithelial cells,6.' myeloid cells including polymorphonuclear and monomorphonuclear phagocytes,8"2 and normal and malignant myelopoietic progenitor cell^.'^,^^"^ The action of IL-2 is mediated
through binding to a specific surface IL-2 receptor (R) consisting of a low-affinity IL-2 binding subunit, the p55 IL2Ra-chain, and an effector unit, which is made up of at least
two distinct components, the p75 IL-2RO-chain and the p64
IL-2Ry-chai11.'~"' Expression of different combinations of
these three components gives rise to the generation of various forms of the IL-2R with high-affinity IL-2Rs containing
all three chains. IL-2RP possesses the largest cytoplasmic
domain and expression studies with IL-2Rp deletion mutant
cDNAs have indicated that a serine-rich cytoplasmic region
confers the transduction of the IL-2 signal."
Analysis of the role of IL-2Ry has just begun. Although
it has been shown that IL-2Ry is part of the intermediateand high-affinity receptor" and thereby essential for ligand
internalization," its role in signaling of IL-2 is not completely understood. An IL-2Ry-defective mutant T-cell line
that expresses normal IL-2Ra and IL-2Rp has been shown
to be also deficient in IL-2 signaling:' whereas, in human
embryonic fibroblasts expressing IL-2Ra- and IL-2Rpchains, the IL-2R was shown to be functional in the absence
of the p64 IL-2Ry-chai11.~Nevertheless, evidence for a role
of IL-2Ry in IL-2 signaling also comes from the clinical
level. Patients with X-linked severe combined immunodeficiency (X-SCD), characterized by highly reduced numbers
of mature and immature T cells, have been shown to cany
nonsense mutations within their IL-2Ry-genes." Humans
and mice that lack IL-2 entirely suffer far milder symptoms.
Blood, Vol 87, No 6 (March 15). 1996: pp 2419-2427
MATERIALSANDMETHODS
Source, purification, and culture of cells. The hematopoietic cell
lines K562, HEL,KG-l, HL-60, U937, and M07; the lymphoid cell
lines Daudi and HUT 102B2; and the ACHN renal cell carcinoma
From the Department of Medical Oncology and Applied Molecular Biology, Virchow Klinikum der Humboldt Universitiit Berlin,
Robert-Rossle Cancer Center, and Max-Delbriick Center for Molecular Medicine, Berlin, Germany: the Ludwig Institute for Cancer
Research, Brussels, Belgium; and the Division of Hematologic Malignancies, Dana-Farber Cancer Institute, Harvard Medical School,
Boston, MA.
Submitted February 14, 1995; accepted November 2, 19%.
Supported by the Deutsche Krebshilfe Grant No. W 27-91 (to
F.H.) and National Institutes of Health Grant No. CA 41619 (to
J.R.).
Address reprint requests to Friedhelm Hernnann, MD, PhD,
Humboldt Universitat Berlin, Robert-Rossle Cancer Center, Lindenberger Weg 80, 0-13122 Berlin, Germany.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1996 by The American Society of Hematology.
OOW-4971#6/S706-0$3.00/0
2419
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2420
line were obtained through the American Type Culture Collection
(Bethesda, MD). The cell line GF-D8 was kindly provided by Dr
A. Rambaldi (University of Bergamo, Bergamo, Italy). Mono Mac
6 cells were provided by Dr L. Ziegler-Heitbrock (University of
Munich, Munich, Germany). Both cell lines also represent myeloid
leukemia-derived cell types. CTLL-2 cells were a gift of Dr K.
Welte (Hannover Medical School, Hannover, Germany). Cells were
cultured in RPM1 1640 medium (Seromed Biochrom, Berlin, Germany) supplemented with 10%low-endotoxin fetal calf serum (Hazelton, Vienna, UT) in the presence of 2 mmoVL L-glutamine and
1% penicillidstreptomycin (standard culture medium) in 5% C02in
air at 37°C. M07 and GF-D8 cells received in addition 5 ng/mL of
each recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and rhIL-3 (kindly provided by Dr S. Gillis,
Immunex Corp. Seattle, WA). Monocytes and neutrophils were isolated from b u Q coats of blood donated by consenting healthy volunIn brief, interphase cells, obtained
teers as previously
after centrifugation of buffy coat cells over a Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) cushion, were collected and first subjected
to two subsequent rosetting steps with aminoethylisothiouronium
bromide (AET)-treated sheep red blood cells (SRBC; Flow Laboratories, Meckenheim, Germany) for depletion of T cells. Monocytes
were further purified by two adherence steps of the E-rosette-negative cell fraction. Monocytes obtained in this way were greater than
97% pure by a-naphtyl acetate esterase staining and morphology.
Neutrophils were isolated by dextran sedimentation and centrifugation on a first density cut using Ficoll-Hypaque followed by a second
density centrifugation using a Percoll (Pharmacia) gradient." Neutrophils obtained in this way were greater than 98% pure by myeloperoxydase staining and morphologic inspection, Primary leukemic
blast cells were obtained from a series of acute myelogenous leukemia (AML) samples previously depleted from contaminating T lymphocytes and adherent cells by the above methods (>97% pure blast
cell populations by morphology). AML samples were selected from
the leukemia tissue bank of our institute based on their phenotypes
according to the various subsets of the French-American-British
(FAB) leukemia classification system. CD34+ bone marrow mononuclear cells (BMMC) were obtained from bone marrow aspirates
of consenting healthy donors and purified as detailed before.33To this
end, BMMC were separated by centrifugation over Ficoll-Hypaque.
Ficolled cells were first depleted from monocytes by repeated plastic
adherence and from T cells by rosetting with AET-treated SRBC.
Nonadherent E-rosette-negative cells were then exposed to an antiCD34 monoclonal antibody (MoAb; Becton Dickinson, Heidelberg,
Germany) at a concentration of 15 pg per lo7 cells for 60 minutes
followed by 30 minutes of incubation (rocking) with magnetic beadIg (Dynabeads; Dynal, Oslo, Norway)
coated goat antimouse (GAM)
at a target cell to bead ratio of 1 to 3. CD34+ cells were harvested
using a magnetic particle concentrator (Dynal). An additional incubation step over 16 hours at 37°C and a subsequent density gradient
centrifugation was performed to allow for detachment of magnetic
beads. To determine purity of the cell population to be studied, cells
were re-exposed to anti-CD34 MoAbs followed by incubation with
biotinylated GAM Ig and streptavidin-conjugated phycoerythnn
(Becton Dickinson). Preparations showed positive staining for CD34
in 91% to 98% of cells by flow cytometry.
Polymerase chain reaction (PCR), primer design, and molecular
probes. The presence of transcripts for IL-2Ra. IL-2Rp, IL-2Ry,
IL-4R. L 7 R , and IL-BR in'the above cells was examined by reverse
transcriptase-PCR (RT-PCR), as previously described." Briefly, 0.2
p g of total RNA from cells to be studied was first reversely transcribed (RT) using poly-(d)T primers (Phannacia) and the Moloney's
murine leukemia virus (M-MLV) reverse transcriptase (GIBCO
BRL, Heidelberg, Germany). PCR was performed in a solution containing 50 mmoVL KCl, 10 m o V L Tris-HC1, pH 8.3, 1.5 ~ o V L
SCHUMANN ET AL
Table 1. PCR Primers Used for Detection of Cytokine
Receptor Transcripts
Receotor
ll-2Ra:
lL-2Rp:
IL-2Ry:
IL-4R:
IL-7R
IL-9R
GAPDH:
Primer
Upstream
Downstream
Upstream
Downstream
Upstream
Downstream
Upstream
Downstream
Upstream
Downstream
Upstream
Downstream
Upstream
Downstream
5"GGGACTGAGCTGGCATAGAG-3'
5"GGCTCTACACAGAGGTCCTG-3'
5"GCTGCAGGCllTGTCCTGAA-3'
5"CCCGTGAGTCAAGCATCCTG-3'
5"CCAGGACCCACGGGAACCCA-3'
5'-GGTGGGAAlTCGGGGCATCG-3'
5"CACCAGTGGCTATCAGGAG-3'
5"CGAGGACCCGTCTCCACAGC-3'
5"ATGATGTAGClTACCGCCAG-3'
3"CTAClTGAATGTCATCCACC-3'
5"ATGTGGTAGAGGAGGAGCGT-3'
3"TGAACAGGACGTAGGTCGG-3'
5"GGTGAAGGTCGGAGTCAACGGA-3'
5"GAGGGATCTCGCTCCTGGAAGA-3'
Abbreviation: GAPHD, glyceraldehyde-3-phosphate dehydrogenase.
MgCIZ,0.01% gelatine, 2 pmoVL primers, 200 pmoVL of each
deoxynucleotide, 50 ng of reversely transcribed cDNA, and 2.5 U
of Amplitaq polymerase (Cetus, Emeryville, CA). The 35 PCR cycles were for 1 minute of denaturation at 95°C 2 minutes of annealing at 55°C to 60°C. and 3 minutes of extension at 72°C. Primer
oligonucleotides were synthesized using a Gene Assembler Plus
synthesizer (Pharmacia) as detailed in Table 1. All primers were
complementary to sequences contained in the coding sequence of
each gene and designed to attain comparable T, and PCR products
of similar size. PCR products were also subcloned into pBS followed
by sequencing of 5 independent clones that showed the identity of
the PCR product to the published sequence of the respective cDNA.
These probes were then also used as probes for Southern blot analysis. In addition, Southern blot results were confirmed by rehybridization with cDNA-specific oligonucleotides. Labeled probes were separated from nonincorporated nucleotides on a G-50 column
(Pharmacia). RT-PCR reaction products were electrophoresed on
agarose gels and transfered to nitrocellulose membranes (Hybond N;
Amersham-Buchler, Braunschwig, Germany) by capillary blotting
overnight. Filters were baked for 2 hours at 80°C and prehybridized
for 16 hours followed by hybridization with the labeled probe for 2
hours. Filters were washed several times and exposed to x-ray films
for 10 to 20 hours using intensifying screens at -70°C.
Immunojfuorescence analysis by jfow cytometry. Surface expression of the IL-2Ra-and p- and y-chain by myeloid cell lines, monocytes, neutrophils, CD34+ BMMC, and primary AML cells was
assessed by immunofluorescence analysis and flow cytomeay as
previously described"*33using anti-IL-2Ra MoAb (anti-CD25; Becton Dickinson), anti-IL-2RP MoAb (Mik-p1; Nichirei, Tokyo, Japan), and anti-IL-2Ry MoAb (3G11).35 In selected experiments,
receptors for IL-9 were determined in the same manner using antiIL-9R MoAb AH9R1.
CeNprolijerarion assay. To quantitate the proliferative response
to rhIL-2, M'IT rapid colorimetric assay (Sigma Chemicals, Munich,
Germany) was performed. To this end, triplicate aliquots of cells
(2.5 x l@/mL) were suspended in standard culture medium (see
above) in 96-well flat-bottomed microtiter plates at 37°Cinthe
presence or absence of rhIL-2 ( E m e t u s , Amsterdam, The Netherlands) in concentrations ranging from 0.1 to 10,OOO standard units/
mL. "T (5 mg/mL) was added for the final 4 hours of culture.
After a total culture period of 72 hours, 100 pL of acid isopropanol
was added and absorbance was assessed on a microelisa plate reader
at 550 nm. With this assay, more reproducible and reliable results
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242 t
IL-2 RECEPTOR COMPLEXES EXPRESSION BY MYELOID CELLS
than with [3H] thymidine incorporation assays have been described"
(own unpublished observations). As controls, cells were also stimulated with a combination of rhG-CSF, rhGM-SCF, and rhIL-3 (25
ng/mL each) or rhIL-9 (100 ng/mL) for 72 hours.
Binding of 1Z51-labeled
IL-2. L - 2 binding assays were performed
with AML-4, AML-5, AML-10, KG-l, HUT 102B2 (positive control), and ACHN cells (negative control), as described elsewhere.5
To this end cells were washed and treated with acid medium to
remove prebound growth factors (1.5 X 10' cells in 5 mL of cold
[4"C] PBS [pH 3.0]), followed by immediate washing in 50 mL cold
PBS. Aliquots of 5 X 106 cells were then cultured at 4°C for 4 hours
or at 37°C for 90 minutes in 100 pL of binding buffer consisting of
RPM1 1640 with bovine serum albumin (2 mg/mL), 25 pmoW 4(2-hydroxyethyl)-l-piperazineethanesulfonic acid, pH 7.5,and 0.1%
sodium azide with serial dilutions of '=I-IL-2 (Amersham-Buchler;
specific activity, 30 to 50 mCi/mg) in the presence or absence of
450-fold excess of cold ligand to assess specifically bound counts.
Under these conditions, nonspecific binding was typically less than
20%. After incubation, cells were separated from the medium by
placing each cell suspension on a 1:4 mixture of olive oil and diN-butyl phthalate in a microfuge tube and centrifugating at 10,OOOg
for 2 minutes. The tube was cut, and cell-bound and free IzI-labeled
factor was counted in a gamma counter. Numbers and kdwere examined by Scatchard plot analysis using mean data from triplicate
samples. The SEM was less than 15%. For competition experiments,
A M L - ~cells were mixed with a single concentration of '=I-L-2
in the presence of unlabeled rhIL-2, rhIL-6 (kindly provided by Dr
T. Hirano, Osaka University, Osaka, Japan), or rhIL-9 (kindly provided by Dr S. Clark, Genetics Institute, Cambridge, MA) at different
concentrations. Binding of '%labeled L-9 to A M L J cells was
assessed in the same fashion.
Antiphosphotyrosine enzyme-linked imnwwsorbeni assay (EWSA).
Fifteen minutes after exposure of cells to rhIL-2 (1,000 U/mL), cell
lysates were prepared and assessed for protein content by the Bradford assay. To assay for overall protein tyrosine phosphorylation,
the Tyroscan-ELISA kit (Laboserv, Heidelberg, Germany) was used
according to the manufacturer's guidelines. Briefly, 20 and 150 ng,
respectively, of lysate-derived protein was added to each well of a
96-well flat-bottom plate precoated with tyrosine residues poly (glu,
tyr) as substrate for total phospho-tyrosine kinase activity. Detection
of tyrosine phosphorylation was performed with specific MoAb,
followed by a peroxidase-coupled antibody and calorimetric detection. The color reaction was quantitate by Milenia Kinetic Analyzer
(Diagnostic Products Corp. Los Angeles, CA) at a wave length of
450 m.
Analysis of JAK3 protein levels and tyrosine phosphorylation.
For analysis of JAK3 protein levels, representative cell samples
(neutrophils, KG-l, CD34+ BMMC, HUT 102 B2) were lysed in
lysis buffer containing 1% Triton X-100.After removal of insoluble
materials by centrifugation, the protein concentration was determined (Bradford assay) and equal amounts of protein for each sample
were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoretical transfer to Immobilon (Millipore, Bedford, MA) filters were probed with a polyclonal
antihuman JAK3 serum (Santa Cruz Biotechnology, Santa Cruz,
CA). For analysis of JAK3 tyrosine phosphorylation, JAK3 was
immunoprecipitated from cell lysates by incubation with anti-JAK3
prebound to protein A-Sepharose beads (Pharmacia). The beads were
washed with buffer containing 0.1% SDS-PAGE, and t r a n s f e d to
Immobilon. Antiphosphotyrosine immunoblotting was performed as
previously described?' The blots were washed with TBS/0.5%
Tween 20 and then incubated with sheep antimouse antibody conjugated to horseradish peroxidase. Bound antibodies were visualized
by the enhanced chemiluminescence method. Enhanced chemilumi-
nescence reagents for immunoblotting were from AmershamBuchler.
RESULTS
Expression of transcripts for IL-2R components by myeloid cells. Given previous findings by others and ourse1ves8-15.26-29
showing the presence of the IL-2Ra- and/or
IL-2RP-chain on the surface of cells of myelopoietic origin,
along with the recent discovery of the y-chain of this receptor:'
we were interested in studying the simultaneous presence of these three L-2R components on a wide range of
myeloid cells. IL-2Ra, -P, and - y expression was examined
on the mRNA and protein level. Because of the low number
of pure cells available in some samples, RNA studies were
generally performed by RT-PCR analysis. To control the
quality of the RNA preparation and PCR analysis, in all
reactions GAPDH was coamplified from reversely transcribed RNA. Specificity of the RT-PCR reaction was assessed by using RNA of cells known to abundantly express
the respective gene or not that were included as positive or
negative controls, respectively. Moreover, RT-PCR was also
performed in reactions containing water instead of reversely
transcribed RNA. In addition, PCR was performed in samples containing RNA not reversely transcribed to exclude
amplification of contaminating DNA. Identity of the resulting PCR product was shown by Southern blotting using
specific radiolabeled probes or oligonucleotides.
As shown in Table 2andFig
1, IL-2Ry mRNA was
ubiquitously expressed in all samples of myeloid origin investigated, which included 8 different myeloid leukemia cell
lines, 14 different primary AML samples of all subtypes
according to the FAB classification, and normal neutrophils,
monocytes, and CD34+ BMMC. Variations of the L-2Ry
to GAPDH ratio were obvious but did not appear to be
related to the differentiation stage of the respective cell type,
except for a single sample with a megakaryocytic leukemia
(AML-14), in which the highest IL-2Ry mRNA levels were
detected.
IL-2Ra mRNA was also expressed in 8 of 8 myeloid
leukemia cell lines, 8 of 14 primary AML samples, and
in neutrophils, monocytes, and CD34+ BMMC. Similarly,
transcripts for IL-2RP were found in 8 myeloid leukemia
cell lines, 12 of 14 primary AML specimens, neutrophils,
monocytes, and CD34+ BMMC as well. The order of magnitude of mRNA expression of the various L 2 R components
in leukemia-derived samples was heterogenous in that transcript levels of IL-2Ry mostly exceeded those for IL-2Rp,
which themselves exceeded those for IL-2Ra.
Surface expression of the IL-2Ra-, IL-2RP-, and IL-2Rychain by myeloid cells. Ina second set of experiments
we sought to establish the relationship between mRNA and
protein levels of the various chains of the IL-2R in myeloid
cells. Table 3 shows that surface expression of IL-2Ra was
detectable by immunofluorescence and flow cytometry using
an anti-CD25 MoAb in the KG-l cell line, as well as in 2
primary AML samples (AML-5 and AML-10) only. Inconsistency of the expression data as shown by RT-PCR and
flow cytometry analysis was striking particularly regarding
IL-2Ra expression, which was most likely related to the low
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SCHUMANN ET AL
2422
Table 2. Expression of Transcripts of the Receptors for 11-2,114.
IL-7, and 11-9 by MyeloidCells
Cell Type
Cell lines
K562
HEL
KG-l
HL-60
U937
Mono Mac 6
M07
GF-D8
Daudi
HUT 10282
ACH N
Primary AML
AML-1 (FAB "l)
AML-2 (FAB M1)
AML-3 (FAB "I)
AML-4 (FAB M2)
AML-5 (FAB M2)
AML-6 (FAB M2)
AML-7 (FAB M3)
AML-8 (FAB M4)
AML-9 (FAB M4)
AML-10 (FAB M5)
AML-11 (FAB M5)
AML-12 (FAB M5)
AML-13 (FAB M6)
AML-14 (FAB M7)
Normal myeloid cells
Neutrophils
Monocytes
CD34' BMMC
IL-PRO
IL-PRO
(+)
+
++
+
++
+
++
++
+
+
++
+
++
(+)
IL-4R
IL-7R
IL-9R
+
+
+
+
+
+
++
++
++
ND
ND
++
++
ND
ND
-
-
++
++
++
+
+
++
+
++
++
+
+
+
++
+
+
+
++
+
+
+
+
+
+
+
-
+
+
+
++
+
+
+
+++
+
+
+
+
+
-
(+)
2
MlHZO1
3
4
5
6
+
-
++
(+l
IL-PRy
though surface analysis of the IL-2R components has shown
an a+,p+, y + configuration in 3 of the 25 samples, and at
least a D+, y + configuration in additional 9 samples, this
result does not prove high- or intermediate-affinity IL-2
binding and does not offer proof for the presence of an
1
2
4
3
5
HPOM2
6
+
+
+
+
+
+
+
11-7R
ND
+
+
H201
2
3
4
5
6
All cell lines and primary AMLsamples were analyzed at least twice.
Normal myeloid cells of three different donors were examined and
gave comparable results.
Abbreviations: +,transcript levels in the same order of magnitude
as GAPDH mRNA; ++, transcript levels up to twofold greater than
GAPDH mRNA; +++,transcript levels more than twofold greaterthan
GAPDH mRNA; (+l, transcripts below levels of GAPDH mRNA controls, but detectable; -, no transcripts detectable; ND, not determined.
abundance of surface expression of this receptor chain not
detectable with the antibody used. However, in this regard,
it should be noted that previous reports have suggested inducibility of the a-receptor component in myeloid leukemia
cells as well as blood-derived phagocytes by interferon-y
and L - 2 i t ~ e l f . " . ' ~ . ~ ~Surface
.~'
expression of the IL-2Rp
chain was detectable by the above means in all of six myeloid
leukemia lines and in 9 of 14 primary AML samples investigated. In line with previous reports,s." significant IL-2RP
levels were also detected on the surface of neutrophils and
monocytes that constitutively failed to display IL-2Ra. IL2Ry chains were detectable on the surface of 5 of 7 myeloid
leukemia lines and 7 of 13 primary AML samples. Bloodderived polymorphonuclear and mononuclear phagocytes as
well as CD34+ BMMC displayed surface L-2Ry as well.
Representative flow cytometry data are shown in Fig 2.
Lack of proliferative response of myeloid cells to IL-2
inspite of tyrosine phosphorylation of target proteins. Al-
M2
l
H,OM1
1
2
3
4
5
11-9R
6
Fig 1. RT-PCR analysis of transcript levels of IL-ZRy (320 bp), 1L7R
(456 bp), IL-9R (174 bp), and GAPDH (371 bp) in myeloid cells. Shown
are representative cell samples as follows: lane 1, ACHN cells; lane
2, HL-60 cells; lane 3, KG-l cells; lane 4, AML-l2 cells; lane 5, U937
cells; lane 6, monocytes. Molecular weight standards are as follows:
M1, l - k b DNA ladder (Boehringer Mannheim, Mannheim, Germany;
201. 220. 298, 314, 396, 506/517, 1,018, 1,636, 2,036,
and 3,054 bp);
M2, 100-bp DNA ladder (Boehringer Mannheim; 100, 200,300, 400,
500, and 600 bp). RT-PCR was also performed in reactions containing
HzO instead of reversely transcribed RNA.
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IL-2RECEPTORCOMPLEXESEXPRESSION
BY MYELOID CELLS
Table 3. Sudaco Expression of the a-,p-. and yChain of the Il-2R
by Myeloid Cells
Percentaaa of Cells Stained With MoAb to
Call Type
~
IL-2RB
lL-2Ry
3
NO
2
41
79
77
45
43
ND
91
13
15
53
11
90
1
13
ll-2Ra
~~~~
Cell lines
K562
HEL
KG-l
HL-60
U937
Mono Mac 6
M07
GF-D8
HUT 10282
ACHN
Primary AML
AML-1 (FAB MI)
AML-2 (FAB MI)
AML-3 (FAB "l)
AML-4 (FAB M2)
AML-5 (FAB M2)
AML-6 (FAB M2)
A M L J (FAB M3)
AML-8 (FAB M4)
AML-9 (FAB M41
AML-10 (FAB M5)
AML-11 (FAB M5)
AML-l2 (FAB M5)
AML-13 (FAB M6)
AML-14 (FAB M7)
Normal myeloid cells
Neutrophils
Monocytes
CD34' BMMC
2
0
21
0
0
ND
0
ND
95
78
0
0
0
1
3
2
3
22
2
1
20
ND
8
12
0
0
0
68
10
16
0
0
0
0
14
17
0
0
1
22
64
23
13
1
0
1
0
0
20
2
44
8
47
3
16
0
41
37
ND
12-79
51
11-52
1
ND
All cell lines and primary AML samples were analyzed at least twice.
Results represent means with SD always ~ 5 % Normal
.
myeloid cells
of four different donors per sample were examined and gave comparable results exceptfor neutrophilsand CD34+ BMMC, for which some
differences in the levels of expression were observed because of donor-to-donor variations.
appropriate signaling machinery that would allow delivery
of mitogenic stimulation. Analysis of KG-l cells and of
blasts of one of the samples obtained from patients with
primary AML (AMLJ), which both also exhibited IL-2Rat
IL-2RP-, and IL-2Ry-protein, showed intermediate- to highaffinity IL-2 binding by Scatchard analysis (Table 4 and Fig
3). In these cells, L-2-mediated signaling was estimated by
a recently developed Tyroscan-ELISA detecting the overall
protein tyrosine phosphorylation. As shown in Table 5 , IL2 treatment of KG-l and AMLJ cells was associated with
tyrosine phosphorylation in a comparable fashion as seen in
IL-2-treated HUT 102B2 cells, whereas ACHN cells, devoid of IL-2R, failed to exhibit tyrosine phosphorylation in
response to this factor. The Tyroscan-ELISA was performed
with two concentrations of lysate-derived protein (20 and
150 ng per well) and gave concordant results, thus confirming the reproducibility of this assay and corroborating our
previous results on L-2 signaling in myeloid cells."
Recently, the nonreceptor tyrosine kinases belonging to
2423
the Janus (JAK) family have emerged as key elements in
the signaling pathway elicited by several c y t o k i n e ~and
~ ~it
was shown that one member of this family, ie, JAK3, is
coupled functionally and physically to the IL-2Ry-~hain.~~
Therefore, we examined whether JAK3 is involved in signaling of IL-2 in myeloid cells (neutrophils, KG-l, and CD34+
BMMC). To this end, immunoprecipitation experiments
were performed with anti-JAK3 antibody followed by immunoblotting with antiphosphotyrosine antibody with cell lysates obtained from various types of myeloid cells. As shown
in Fig 4, JAK3 is constitutively expressed in myeloid cells
and tyrosine phosphorylated after IL-2 stimulation of these
cells in a way comparable to JAK3 phosphorylation seen in
HUT 102B2 T-lymphoblasts.
Given these findings and the mitogenic potential that IL2 exerts in lymphoid cells displaying high-affinity IL-2 binding, and based on a previous study of IL-2-inducible blast
cell proliferation in some cases of AML,I4 we also aimed at
studying the possible role of IL-2 in regulating proliferation
of immature myeloid cells. To this end, all 8 myeloid leukemia lines, 10 primary AML samples (including AML-5),
and CD34' BMMC were exposed to rhIL-2 in a concentration range of up to 10,000 U/mL and their proliferative response was determined in a 72-hour liquid culture. Except
for M 0 7 cells, in which a concentration of L - 2 of greater
than 1,OOO U/mL spurred some minor proliferative responses
(1.%fold greater than levels seen in cultures stimulated with
GM-CSF and IL-3 only), in none of these samples was a
significant stimulatory effect of IL-2 on growth kinetics of
these cells noted (data not shown). Controls were also performed in all experments in which cells were exposed to a
combination of rhG-CSF, rhGM-CSF, and rhIL-3. Myeloid
cell lines (except U937 and KG-l cells), 7 of 8 primary
AML samples, and CD34' BMMCT responded to this factor
combination at least 10-fold above levels seen in cultures
stimulated with medium only (data not shown).
Taken together, these results suggest that at least in early
myeloid cells of normal and leukemic origin IL-2R triggering may lead to cell activation without proliferation. In
leukemia cells, the lack of IL-2-induced proliferation may
thus not be a reflection ofthe abnormal nature of these cells
but may represent the presence of anintact signaling pathway
through this receptor.
Expression of transcripts for IL-4R, IL-7R, and IL-9R by
myeloid cells. Several previous reports have indicated (see
Kishimoto et also for review) that IL-2Ry may be expressed
in a combinational association with receptors for other cytokines. The recent unraveling of the multimeric nature of
many cytokine receptors has led to the view that ligandinduced association of multiple receptor chains is needed
for efficient signal recognition and transmission to occur.
Meanwhile several receptors have been shown to share subunits (see Kishimoto et a130 for review). For instance, a common P-chain is shared by receptors for L-3, IL-5, and GMCSF.30Another molecule, gp 130, is shared as an effector
unit by the receptors for IL-6, IL-l 1, leukemia inhibitory
factor, oncostatin M, and ciliary neurotrophic factor.% Recently, it has been shown that the IL-2Ry chain represents
a subunit also being shared by the IL4R and IL-7R.23-25
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
2424
SCHUMANN ET AL
B
A
Log Fluorescence
"
+
Fig 2. Flow cytomatry analysis of membrane surface expression of the lL-2Ra-, ILaRg-, and IWRychain in myeloid colla. Shown are
representative cell samples as follows: (A) monocytes, (B) neutrophils, (C) A M - 5 calls, (D} KG-1 calk, (E) C m +B-,
and (F) AWN dls.
There have also been some hints to suggest a possible participation of the IL-9R in this scenario as well.30 Therefore,
we decided to supplement our studies on IL-2Ry mRNA
expression in myeloidcells by examining transcript synthesis
of the IL-4R, IL-7R, and IL-9R in these cells to also gain
Table 4. Presence of Intermediate- and High-Affinity 11-2 Binding
Sites on KG-l, AML-5, End AML-10 Cells
Intermediate-Affinity
High-Affinity
Cell
Binding
SitesKell
KG-l
40-58
AML4
AML-5
62-91
AML-10
52
147-1
HUT 10282
20-29
4,400-5,100
ACH N
~
kd
(pmol/L)
Binding
Sites/Cell
(nmolR)
78-88
709-903
1.4-1.5
76-112
83-90
1,166-1,412
587-599
1.5-1.8
2.1-2.2
-
-
-
kd
-
-
-
-
~
Results indicate the average number of IL-2R per cell (range of
4 independent experiments) and the apparent dissociation constant
calculated by Scatchard plot analysis of equilibrium binding data.
Dashes indicate an absence of measurable receptors of the respective
affinity class.
first insights into a role for myeloid cell IL-2Ry expression
outside the IL-2 system.In 4 samples of primary AMLderived blasts that simultaneoulsy displayed transcripts for
the IL-~Rcu-,
IL-2R$, and E-2Ry-chain and in 1 additional
patient whose leukemic cells synthesized IL-2Rp and IL2Ry transcripts, neither IL-4R mRNA nor IL-7R mRNA
was detectable. The same IL-~Rcu', IL-2RP+, IL-2Ry+,
IL4R-, IL-7R- phenotype was also noted in KG-l cells.
IL-BR transcripts were detectable in all myeloid leukemia
cell lines and primary AML samples examined, as well as
in monocytes and CD34+ BMMC. However, interestingly,
in all of the IL-2Ry mRNA expressing samples that failed
to exhibit IL-4R- and IL-7R transcripts, IL-9R mRNA was
detectable by RT-PCR analysis (Table 2 and Fig 1) and
surface IL-9R protein by Row cytometry using AH9Ri
MoAb (data not shown). In Ah4L-5cells displaying intermediate- to high-affinity IL-2 binding, unlabeled IL-2 inhibited
I25
I-IL-2 binding in adose-dependent fashion andcompletely relieved "'I-IL-2 binding with a 450-fold excess.
Similarly, a 450-fold excess of unlabeled rhIL-9 competed
with 65% of 'z51-IL-2 for binding to IL-2R (Fig 5 ) . Though
further studies will be necessary to clarify the issue of a
possible sharing of the y-component of the IL-2R with the
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
IL-2 RECEPTOR COMPLEXES EXPRESSION BY MYELOID CELLS
2425
500
400
300
200
100
100200300400500
R (moleculedcell)
1
Fig 3. Scatchard plot and isotherm curve examining IL-2 binding to AML-10 cells. Forprotocol see the
Materials and Methods and Table 4.
DISCUSSION
In the study presented here, synthesis of the IL-2Ry-chain
was examined on the mRNA and protein level in a variety of
myeloid cells incuding myeloid leukemia cell- lines, primary
AML cells, normal CD34+ myeloid progenitor cells, and
mature neutrophils and monocytes. Synthesis of the IL-2Rychain by these cells was correlated with mRNA and protein
expression of the IL-2Ra and IL-2Rp subunits and mRNA
Table 5. Stimulation of Overall Protein Tyrosine Phosphorylation
by 11-2 in Myeloid Cells
Overall Tyrosine Phosphorylation(amount of cell
lysate-derived protein loaded)
Cell
and
Treatment
+
KG-l
IL-2$
KG-l + medium
11-2
AML-5
AML-5
medium
CTLL-2 IL-2
CTLL-2 + medium
ACHN IL-2
medium
ACHN 0.04
+
+
+
+
+
Phosphorylation
Phosphorylation
OD* Stimulation Factort
0.14
0.06
0.15
0.06
0.18
0.06
0.04
2.3
2.5
3
1
150 ng/Well
-
OD
Stimulation Factor
0.61
0.26
0.69
0.27
0.73
0.24
0.20
0.20
2.4
2.5
-
3
-
1
-
OD was examined at 450 nm.
t Fold tyrosine phosphorylation-induction of IL-2 treated versus untreated cells.
Cells (5 x 105/mL) were cultured for a period of 15 minutes in
the presence or absence (medium) of rhlL-2 (1.000 U/mL) before cell
lysates were prepared and subjected to overall protein phosphorylation analysis by Tyroscan-ELISA. CTLL-2 cells (kept overnight in CUIture under serum- and IL-2-free conditions before assaying) and
ACHN cells served as positive or negative controls, respectively. The
experiment was repeated once and gave almost identical (SD <2%)
results.
x
*
3
4
5
6
c (XlO10)
TL-9R, the studies shown heremay be to some extent in
favor of this hypothesis.
20 ngMlell
2
expression of IL-4R and IL-7R. Given some evidence for a
possible participation of the IL-2Ry-chain in formation of
the IL-9R complex:’
we also investigated IL-9R mRNA
and, in selected experiments, IL-9R surface expression in
these cells.
In contrast to lymphoid cells, the function of IL-2R in
cells of myeloid origin is largely unknown. In postmitotic
myeloid cells such as monocytes that constitutively exhibit
IL-2R@and inducibly I L - ~ R c Y , ~
IL-2
’ . ~ induces
~
secretion of
secondary ~ytokines,~.”
whereas in neutrophils treatment
with IL-2 was found to prevent apoptotic cell death.’ Also,
in myeloid leukemia cell lines, such as KG-l, U937, and
M07, expression of at least one chain of the IL-2R complex
has been reported,’032s
whereas in primary blast cells from
patients with AML IL-2R expression appears to be controversial. Hoshino et a129failed to detect both IL-2Ra and IL2Rp in 18 of 18 AML samples examined, whereas other
investigators, including Pizzolo et all3 and Rosolen et al,14
found IL-2Rp to be expressed in almost all AML and IL2Ra in a small minority of these samples. However, IL-2Ry
chain expression in myeloid cells (neutrophils excepted) has
not yet been under detailed investigation.
In the study presented here, additional evidence for the
expression of the CY- and @-componentof the IL-2R is presented in cells of myelopoietic origin. Expression of the IL2Ry-chain is also a consistent finding in myeloid cells, which
seems to be independent from the differentiation state (immature v mature) and lineage commitment (granulocytic v
monocytic v erythroid v megakaryocytic). Despite possible
high- and intermediate-affinity IL-2 binding in some myeloid
cell samples, IL-2 is unlikely to transmit mitogenic signals
in these cells. Moreover, the presence of IL-2R-y may also
point to a role of this chain in normal and malignant myelopoiesis independent from IL-2, as suggested by the coexpression of IL-2Ry and L-4R and/or IL-7R by these cells.
Nevertheless, myeloid samples may also exhibit the IL-2Rychain in the absence of detectable IL-4R and IL-7R. On a
speculative basis, coexpression of the IL-9R in these cells
may result from formation ofan IL-2Ry/IL-9R complex.
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
SCHUMANN ET AL
2426
1
2
3
4
5
6
7
8
200 kDa-
4 JAK3
97 kDa69 kDa-
200 kDa97 kDa-
-- -
___
--
Thus, the expression of the IL-9R or other receptors that
also use the common IL-2Ry chain may influence the availability of this receptor component to participate or effectively signal through IL-2. In particular, in myeloid cells IL2RylIL-9R complexing willneed further attention, which
T
4 JAK~
Fig 4. Tyrosine phosphorylation of JAK3 in neutrophils (lanes 1 and 21, KG-l cells (lanes 3 and 4).
HUT 10262 cells (lanes 5 and 61, and CD34+ BMMC
(lanes 7 and 8). Cells were either left untreated (lanes
1, 3,5, and 71 or were stimulated with rhlL-2 (1,000
U/mL) for 10 minutes (lanes 2,4,6, and 8 ) and lysed
and JAK3 was immunoprecipitated. The resulting
filters
were
immunoblotted
with
antiphosphotyrosine MoAb (upper panel) and then with anti-JAK3
antibody (lower panel).
implies analysis of whether the myeloid cell response to IL9 requires participation of the L - 2 R y chain. These studies
are currently in progress. First experiments (Fig 5 ) using
AML-5 cells that exhibited intermediate- to high-affinity IL2 binding sites indicated inhibition of '*'I-IL-2 binding with
excess unlabeled rhIL-9 for binding to IL-2R and thus support this hypothesis. Finally, the number of high-affinity
binding sites we observed in KG-l, A M L J , and AML-10
cells was unexpectedly low despite the abundant surface
expression of the IL-2Rp and IL-2Ry protein. However, the
formation of a high-affinity IL-2R requires the expression
of all three components in a complexed fashion. In this regard, the low IL-2Ra expression levels in KG1 and AML5 cells have to be considered. Perhaps more importantly, it
should be noted that lower functional expression of the ychain (eg, due to engagement with other cytokine receptors)
would also limit the number of high-affinity binding sites
present. However, more detailed analysis is needed to clearly
prove that, in this cell type, the IL-2Ry-chain is not entirely
accessible for complexing with other IL-2R components.
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1996 87: 2419-2427
Transcript synthesis and surface expression of the interleukin-2
receptor (alpha-, beta-, and gamma-chain) by normal and malignant
myeloid cells
RR Schumann, T Nakarai, HJ Gruss, MA Brach, U von Arnim, C Kirschning, L Karawajew, WD
Ludwig, JC Renauld, J Ritz and F Herrmann
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