Download MGI-2A Is Interleukin-6

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REPORT
CONCISE
The
Myeloid
Blood
Cell
Differentiation-Inducing
MGI-2A
By Yosef
Shabo,
Joseph
Lotem,
Menachem
Rubinstein,
Robert
The
mouse
tein,
myeloid
blood
macrophage
and
2A). was purified.
cleavage
acid
(22
sequence
73
94
of
mouse
IL-6
myeloid
leukemic
by
which
cells
and
HE
NORMAL
includes
normal
that
sequence
found
(IL-6).
are
induced
also
to
differentiation
neutralizes
hematopoietic
of different
four different
proteins
that induce
myeloid
precursor
cells
to form
interleukin-3
system
is
multiplication
of
colonies.
These
factors
(CSFs)
type 1 (MGI-i),
(IL-3).’5
The genes
or
and
for these
growth-inducing
proteins
have been cloned and do not show
apparent
similarities
in their nucleotide
sequence.5
There
is
also another
type of protein
that
normal
myeloid
precursor
cells6’7
this
induces
differentiation
of
and some clones
of mouse
leukemic
cells”3’8”0
but does not
factor
(CSF or IL-3) activity”3’8”0
myeloid
growth
myeloid
differentiation-inducing
different
cell
growth-inducing
have
We
protein,
surface
receptors
from
proteins,’
‘ macrophage
myeloid
cell
have termed
which
also
has
the
four
myeloid
and
granulocyte
inducer
type
differentiation
2 (MGI-2),’3’9
and others
factor
(DF).’#{176}We now show
type of mouse
interleukin-6’2
MGI-2
(MGI-2A)
(IL-6)
are very similar
have
that
called
it
the major
and the protein
and most likely
called
identi-
cal proteins.
MATERIALS
AND
METHODS
Cells.
cell cultures,
and assay for MGI-2.
Clone I 1 myeloid
cells were derived ‘ from the M 1 myeloid leukemic
cell
line,’4 and clone 7-M 12 was derived from an x-ray-induced
myeloid
leukemia
in an SJL/J
mouse.’5 Clone
1 1 cells are inducible
to
differentiate
by MGI-2
and clone 7-M12
by GM-CSF
on IL-3 but
not by MGI-2.’6
These leukemic
cells were cultured
in Dulbecco’s
leukemic
From
the Departments
Institute
bridge,
ofScience,
of Genetics
Rehovot,
Israel,
Submitted
August
by the Ebner Foundation
the Jerome
A. and
Address
reprint
payment.
“advertisement”
indicate
23, 1988;
Estelle
Weizmann
Institute,
to
Sachs,
Leo
ofScience,
costs ofthis
article
article
in accordance
must
with
this fact.
& Stratton,
0006-4971/88/7206-0033$3.00/0
Inc.
August
Cam-
25, 1988.
of Leukemia
Assistance
Institute
This
© I 988 by Grune
2070
accepted
R. Newman
requests
Weizmann
The publication
charge
Virology,
Genetics
MA.
Supported
Genetics,
and
and
Research
and
Fund.
PhD,
Department
of
Israel.
were defrayed in part by page
Rehovot
therefore
18 U.S.C.
76100,
be hereby
section
1734
marked
solely
tein
to
of mouse
(MGI-2A)
C. Clark,
Stanley
These
myeloid
F. Wolf,
and
IL-6
a 41 % similarity
acid
residues
and
also
out
are
very
to mouse
of the
induces
22
results
show
that
differentiation-inducing
similar
proteins.
Recombinant
or B-cell differentiation
only
a 1988
this
Steven
differentiation.
type
kemic
One class
proteins.
proteins
are called
colony-stimulating
macrophage
and granulocyte
inducers,
mouse
antibody,
completely
MYELOID
by a family
one is also called
in posiof
Revel,
identical
interferon-2
amino
Recombinant
antimouse-MGI-2
MGI-2,
regulated
This
differentiation
monoclonal
neutralizes
T
the
major
(MGI-
of a CNBr
determined.
interleukin-6
induces
2A
Michel
and Leo Sachs
IL-6-induced
pro-
type
acid sequence
was
to
mouse
protein
MGI-2,
inducer.
residues)
is identical
to
Kamen,
differentiation-inducing
and the amino
peptide
tions
cell
granulocyte
Protein
Is Interleukin-6
differentiation
and
most
likely
human
IL-6 (also called
factor),
which
shows
IL-6.
in the
the
pro-
has
i i identical
mouse
MGI-2A
of the
same
amino
peptide
myeloid
leu-
cells.
by Grune
& Stratton.
Inc.
modified
Eagle’s medium
(H-21, GIBCO,
Grand Island, NY) and
10% horse serum (GIBCO).
Krebs II carcinoma
cells grown as an
ascites tumor in mice were used as the source of MGI-2.9
MGI-2
activity was assayed by using MGI-2-susceptible,
clone 1 1 myeloid
leukemic cells seeded at 7.5 x i0 cells/mL
in a volume of 2 mL and
measuring
the amount of lysozome produced
by these cells after four
days’ incubation
as described.’7
Cells were analyzed
for morphologic
differentiation
by analysis of 300 cells stained with May-GntinwaldGiemsa
and scoring
the percentage
of cells with monocyte
and
mature
macrophage
morphology.
Polymyxin
B (5 g/mL)
was
added to the cultures
to block any possible effect of contaminating
bacterial
lipopolysaccharide.’6
The presence of polymyxin
B does not
affect induction
of differentiation
by MGI-2.’6 The SE in these
assays was up to 20% of the average values.
Purification
of MGI-2A.
Knebs II ascites carcinoma
cells produce and secrete MGI-2 that is physically
separable
into a major
(MGI-2A)
and minor (MGI-2B)
form that differ in their net
charge.9
MGI-2A
from Krebs
II cell-conditioned
medium
was
purified
by chromatography
on diethyl
aminoethyl
(DEAE)-Sepha-
cel at pH 79 followed by removal of the residual
MGI-2B
form on
heparin-agarose,
further
purification
on phenyl-Sepharose
and
hydroxyapatite,7
and then immunoaffinity
purification
with a monoclonal antimouse
MGI-2
antibody
coupled
to Sepharose.’8
The
affinity-purified
MGI-2A was applied to a 3.9 x 300-mm zBondapak C 18 reverse-phase
high-performance
liquid chromatography
(HPLC)
column
(Waters
Associates,
Milford,
MA)
equilibrated
in
0.3% tnifluoroacetic
acid (Sigma Chemical Co. St Louis) in water,
and the column was developed with a 20-minute
linear gradient of
0.35% acetonitrile
(Merck,
Darmstadt,
W Germany),
a 90-minute
linear gradient to 50% acetontnile,
a 30-minute
linear gradient to
80% acetonitnile,
and then a ten-minute
wash with 80% acetonitrile.
The flow rate was maintained
at 0.5 mL/min,
and fractions of 0.5
mL were collected into polypropylene
tubes containing
polyethylene
glycol 6000 to a final concentration
of0.01%
and assayed for MGI-2
bioactivity.
HPLC
fractions
(15 jL)
containing
MGI-2
activity
were
subjected
to electrophoresis
on a 13.5% polyacrylamide
gel containing 0.1% sodium dodecyl sulfate
(SDS) and the protein
bands
visualized
by silver staining
according
to Sammons
et al.’9
CNBr cleavage and amino acid sequence of an MGI-2A
peptide.
The purified MGI-2A
was cleaved by incubating,
in a volume
of 380 L, 50 zg lyophilized
protein with 10 mg CNBn
in 70% formic
acid (overnight
at room temperature
in the dank). The cleaved
material
was separated
on a 30 x 4.6-mm
Aquapore
RP-300
reverse-phase
HPLC column
(Braunlee,
Foster City, CA), and a
major peptide eluting at 36% acetonitnile
and a minor peptide
eluting at 40% acetonitnile
were subjected to amino acid sequence
analysis using Edman degradation
in a model 470 gas-phase
micro-
Blood,
Vol 72, No 6 (December),
1988: pp 2070-2073
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
MYELOID
DIFFERENTIATION-INDUCER
sequencer with an on-line
Inc. Foster City, CA).
MGI-2A
PTH-AA
IS IL-6
detector
2071
(Applied
Biosystems
,-MCI-2A
9
N N
F) )) A
LA
EN
I
hi
N C
h-IL-A
7)
NKSNMCESSKFALAF.NNLNLPKMAEKD);CFQSGF
N S
1) F; N N N
I
N
I
.-IL-6
F) F) A I.
I
A E
L
K
L
I I
N N
I
I
I
Q
N N DC
P
1
Q
R
P
I
K F
1111111
P
N D
II
C C A (3 T G N
III
0))
II
F))b
RESULTS
Fig 2.
Krebs
that
II mouse
is physically
minor
(MGI-2B)
MGI-2A
from
ascites
carcinoma
separable
into
and
by
eluting
MGI-2
(MGI-2A)
and
HPLC,
reverse-phase
43%
at
23 to 28 kilodaltons
to
46%
MGI-2
antibody
MGI-2A
was
and
activity,
display
the
acetonitrile
active
showed
fracmajor
on an analytic
(Kd)
SDS-polyacrylamide
gel and
1). The 23- to 28-Kd
bands,
silver-stained
15 Kd (Fig
MGI-2
by heparin-agarose,
and immunoaffinity
antimouse
affinity-purified
at
bands
protein
a major
at pH 79 followed
hydroxyapatite,7
purification
with a monoclonal
coupled
to Sepharose.’8
The
tions
produce
form that differ in their net charge.9
The
these cells was purified
by chromatography
on DEAE-Sephacel
phenyl-sepharose
fractionated
cells
a minor
which
band at
all have
of the MGI-
the microheterogeneity
2A protein,7
and the
l5-Kd
band did not have
activity.
The fractions
with the 23- to 28-Kd
proteins,
contained
a small amount
of the I 5-Kd protein
(Fig
MGI-2
which
1), were
purified
cleaved
with
Amino
the amino
human
acid
signals.
CNBr
and
the
The
material
amino
acid
was therefore
sequence
of
cleaved
three
sequence
acid sequence
vertical
lL-6 (bottom
bars.
of CNBr-cleaved
protein
produced
line).’2
of mouse
Matching
peptide
by the cloned
leukemic
to differentiate
by MGI-2.
The
COS-l
monkey
in the
vector
cells
pXM
IL-6
cells
results
transfected
show
acids
mouse
of myeloid
a strong
line)
and
are indicated
by
gene
that
the mouse
HPLC
determined.
The sequence
peptide
eluting
at 40% acetonitrile
was
the fl-major
chain of mouse hemoglobin
the lS-Kd
contaminating
with the major peptide
over
22 Edman
Analysis
the major
2-susceptible
myeloid
leukemic
cells
(Fig
3A)
even at a final
concentration
of 0.1% (Fig 3B). As with MGI-2A,
were also induced
by the recombinant
mouse IL-6
!
I
interleukin-6
MAb
.
40
therefore
20
MAb
of the minor
01
of
to
0.25
I
.!
Mouse
0.5
MGI-2Aadded
0.75
(‘I.)
-NAb
12
4,
‘F
80
-MAb
I
0
cycles.
We
80
with
acid residues
that eluted
of
at
showed
a perfect
match
(Fig
2) with a
stretch
located
in positions
73 to 94 of mouse
(IL-6).’2
the cells
to adhere
00
T
I
individual
found to be part
that corresponds
of the sequence
of the 22 amino
CNBr-cleaved
peptide
of MGI-2A
36% acetonitrile
22-amino
acid
gene’2
differentiation-inducing
12
band (Fig 1). The three fractions
gave an identical
amino acid sequence
degradation
from
IL-6
activity
as measured
by the production
of lysozyme
and
differentiation
to mature
nondividing
macrophages
on MGI-
CNBr-cleaved
peptides
eluting
at 36% acetonitrile
(major
peptide)
and the minor peptide eluting
at 40% acetonitrile
on
reverse-phase
can induce
can be induced
supernatant
with
with
from
of the CNBrto be matched
IL-6 (middle
amino
differentiation
pooled
for amino acid sequence
analysis.
N-terminal
Edman
degradation
of the purified
MGI-2A
did not give any specific
amino
acid
MGI-2A.
The 22-amino
acid sequence
mouse MGI-2A
peptide
(top line) is shown
tested
whether
the
60
UN
B
>
40
O’4
a)
U
4,
20
ON
a)
a
F)
E
015
>‘
N
05
Recombinant
1
mouse
IL-6
2
added
(1.)
C
a)
4,
0
UN
>‘
.-J
I
11
N
12
,oo 0
80
60
40
NAP.NF
20
1
2.5
Recombinant
Fig
1.
molecular
Silver
weight
staining
standards;
with
very weak
(fractions
through
m) MGI-2A
activity
46% acetonitrile.
of purified
MGI-2A.
Lane A. protein
lanes B through
M. fractions
(1 5 giL)
b through
d) or strong
(fractions
a
eluted
from a HPLC column at 43% to
5
10
human
IL-6
20
added
(nWmt)
Fig 3.
Induction
of differentiation
by recombinant
mouse and
human IL-6 protein
in MGI-2-susceptible
mouse myeloid
leukemic
cells.
Material
tested for induction
of differentiation
was added
to MGI-2-susceptible
clone
1 1 mouse
myeloid
leukemic
cells
without
or with 10 gig/mL antimouse
MGI-2
monoclonal
antibody
(MoAb).
(A) MGI-2A
from
mouse
Krebs
II ascites
cells (360
U/mL).
(B) Supernatant
of COS-i
cells transfected
with
the
mouse
IL-6 gene.’2
(C) Recombinant
pure human
lL-6 protein.’2
O-O
and L-&
production
of lysozyme;
and A-A.
percentage
of differentiated
cells (monocytes
and mature
macrophages).
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
ET AL
SHASO
2072
to the tissue
culture
nant
11-6
mouse
clonal
MGI-2A
(Fig
or
antibody
MGI-2
antitnouse
IL-3,
Pctri dish. These
activities
were completely
neutralized
3A)
IL-l.’8
and
does
(Fig
3B)
that
likely
identical
any
did not induce
differentiation
even at a final concentration
of 10%.
that mouse
most likely
MGI-2A
identical
Comparison
and mouse
proteins.
of the CNBr-cleaved
of mouse
MGI-2A
called
interferon-fi2,-2’
protein,23
are closely
IL-6
shows
dues 6 through
to 90, 92, and
22-amino
to the sequence
B-cell
DF
that
nine
related
of human
(BSF-2)21’22
of ten amino
15) of the peptide
93 of the human
acid
acid
leukemic
myeloid
human
recombinant
IL-6
entiation-inducing
(Fig
inducing
cells
cells.
The
protein’2
activity
results
(resi-
to residues 84
and that two
that
pure
also had a strong
on the
mouse
myeloid
the biologic
myeloid
sents
that
both
key
activity
to act
and myeloid
different
target
monoclonal
another
factor
ascites
IL-6
renders
activity
cytokine,
induced
as a DF
cells;
cells
antibody
protein
M 1 myeloid
inhibitory
the
This
the
repre-
previously
in fibroblasts
for cytolytic
T
as a growth
implicate
to IL-6
analysis
both in vivo and in vitro
this multifunctional
cytokine.
Recently,
differ-
Finally,
to block
factor
IL-6
as a
that may serve to integrate
the responses of the
and immune
systems
to different
stimuli
or
availability
of the recombinant
protein
and a
hematopoietic
stresses. The
mouse
murine
certain.
differentiation
is known
sup-
and
of terminal
murine
IL-6.
most
and plasmacytomas;
and as an activation
T cells and hepatocytes.’2-#{176}23 These varied
with
regulator
neutralizing
and
further
human
inducers
cell
of
B cells,
for hybridomas
factor for helper
interactions
the
virtually
antiviral
now
related
was
myeloid
leukemic
cells.
anti-MGI-2
antibody
IL-6
property
activated
closely
of recombinant
and
an
are
conclusion
effective
leukemic
a novel
cells,
differleukemic
3C), but unlike the mouse IL-6, the differentiationactivity
of human
IL-6
was not inhibited
by
activity
of MGI-2A
described
as
(interferon-fl2),
IL-6,’2
also
or 26-Kd
show
finding
IL-6s
are
of the mouse Ml
the ability of our monoclonal
The
and
other
amino
acids (residues
21 and 22) of the peptide
are
identical
to residues
99 and 100 of the human
IL-6 protein
(Fig 2). We, therefore,
tested recombinant
human
IL-6 for
its ability
to induce differentiation
in the MGI-2-susceptible
mouse
IL-6
This
the
by
identity
peptide
residues
are identical
IL-6 protein
and
proteins.
entiation
COS-l
in these leukemic
These results
show
gene
MGI-2A
recombinant
of the CSFs,
mock-transfected
from
ported
neutralized
cells or from
COS-i
cellS transfected
with
the
IL-S gene by using the same vector as for the mouse
monkey
human
IL-6
cells
strated
that
not neutralize
Supernatant
of rccombi-
by a mono-
cells
The
(LIF),
and
that
leukemia
the
induces
cells,
corresponding
of
of
from
leukemiamouse
cDNA
MGI-2’6
were also not induced
to differentiate
by up to 5% of
the COS-l
supernatant
containing
recombinant
mouse IL-6
or by 100 ng/mL
pure recombinant
human IL-6.
tion of M I leukemic
distinct
cytokines
cells.25 This shows that there are several
capable
of inducing
Mi
leukemic
cell
differentiation
entiation-inducing
indicates
proteins
and
sequence
Krebs
molecularly
cloned.24
any regions
Recombinant
acid
the
roles
differentiation
designated
has been isolated
amino
facilitate
monoclonal
antimouse
MGI-2
antibody
(Fig 3C). Mouse
myeloid
leukemic
clone 7-M12
cells that can be induced
to
differentiate
with mouse GM-CSF
and IL-3 but not with
does not include
IL-6 sequence.
predicted
will
of the regulatory
of murine
LIF
with significant
similarity
to the
IL-i
also induces
differentia-
that there is a family
of differfor the myeloid
cell lineages.
DISCUSSION
Our
finding
that
peptide
CNBr-generated
to a sequence
a 22-amino
predicted
acid
from
from
the
sequence
natural
murine
derived
MGI-2A
IL-6
from
cDNA
ACKNOWLEDGMENT
a
is identical
demon-
We thank Rachel Kama,
skillful technical
assistance.
Nunit
Dorevitch,
and Nathan
Tal for
REFERENCES
1 . Sachs L: Normal
developmental
programmes
kemia. Regulatory
proteins in the control of growth
tion.
Cancer
Surveys
1:321,
[Biol] 231:289,
3. Sachs
L: The
leu-
1982
2. Sachs L: The molecular
regulators
blood cells. The Wellcome
Foundation
Lond
in myeloid
and differentia-
of normal and leukaemic
Lecture 1986. Proc R Soc
1987
molecular
control
of blood
cell
development.
238:1374,
1987
4. Metcalf
D: The gnanulocyte-macrophage
colony-stimulating
factors. Science 229:16, 1985
5. Clank SC, Kamen
R: The
human
hematopoietic
colonystimulating
factors. Science 236:1229,
1987
6. Liebermann
D, Hoffman-Lieberman
B, Sachs L: Regulation
and role of different macrophage and granulocyte
inducing proteins
in normal
and leukemic
myeloid cells. Int J Cancer 29:159, 1982
7. Shabo Y, Lotem J, Sachs L: Target cell specificity of hematopoietic regulatory
proteins for different clones of myeloid leukemic
cells: Two regulators
secreted by Krebs carcinoma cells. Int J Cancer
41:622, 1988
8. Lotem J, Lipton JH, Sachs L: Separation of different molecuScience
lan forms
normal
of macrophage
and leukemic
myeloid
and granulocyte-inducing
cells.
Int J Cancer
proteins
25:763,
for
1980
9. Lipton
JH, Sachs L: Characterization
of macrophage
and
granulocyte
inducing proteins for normal and leukemic myeloid cells
produced
by the Krebs ascites tumor.
Biochim
Biophys Acta
673:552, 1981
10. Tomida M, Yamamoto-Yamaguchi
Y, Hozumi
M: Punification of a factor inducing differentiation
of mouse myeloid leukemic
Ml cells from conditioned
medium of mouse fibroblast L929 cells. J
Biol Chem 259:10978,
1984
1 1 . Lotem
J, Sachs L: Regulation
of cell surface
receptors
for
hematopoietic
differentiation-inducing
protein
MGI-2
on normal
and leukemic myeloid cells. Int J Cancer 40:532, 1987
12. Wong GG, Clark SC: Multiple actions of interleukin
6 within
a cytokine network.
Immunol Today 9:137, 1988
13. Fibach E, Hayashi
M, Sachs L: Control of normal differentiation of myeloid leukemic cells to macrophages
and granulocytes.
Proc Natl Acad Sci USA 70:343, 1973
14. Ichikawa
Y: Differentiation
of a cell line of myeloid
leukemia. J Cell Physiol 74:223, 1969
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
MVELOID
DIFFERENTIATION-INDUCER
MGI-2A
IS lL-6
2073
I 5. Lotem
J, Sachs L: Genetic dissection of the control of normal
differentiation
in myeloid
leukemic
cells. Proc NatI Acad Sci USA
74:5554, 1977
16. Lotem J, Sachs L: In vivo control ofdifferentiation
of myeloid
leukemic
cells by recombinant
granulocyte-macrophage
colony
stimulating
factor and interleukin
3. Blood 71:375, 1988
I 7. Krystosek
A, Sachs L: Control of lysozyme induction
in the
differentiation
of myeloid leukemic
cells. Cell 9:675, 1976
18.
Shabo
purification
Y,
with
tiation-inducing
Sachs
L: Inhibition
of differentiation
a monoclonal
antibody
protein.
(in press)
Blood
to a myeloid
and
affinity
cell diffenen-
Sammons
DW, Adams
LD, Nishizawa
EE: Ultrasensitive
silver-based
color staining of polypeptides
in polyacrylamide
gels.
19.
Electrophonesis
2:135,
1981
A, Ruggeri R, Korn JH, Revel M: Structure
and
expression of cDNA
and genes for human interferon
beta-2, a
distinct species inducible
by growth stimulatory
cytokines. EMBO J
5:2529, 1986
21 . Sehgal PB, May LT, Tamm I, Vilcek J: Human fl2 interferon
20.
Zilberstein
B cell differentiation
factor BSF-2 are identical.
Science
235:731, 1987
22. Hirano T, Yasukawa
K, Harada H, Taga T, Watanabe
Y,
Matsuda T, Kashiwamura
5, Nakajima
K, Koyama K, Iwamatsu A,
and
Tsunasawa
S, Sakiyama
F, Matsui
Kishimoto
T: Complementary
DNA
(BSF-2)
that induces B lymphocytes
Nature324:73,
H, Takahara
Y,
Taniguchi
T,
for a novel human intenleukin
to produce
immunoglobulin.
1986
G, Content J, Volckaent
G, Derynck
R, Tavernier
W: Structural
analysis
of the sequence
coding
for an
26-kDa
protein
in human
fibroblasts.
Eur J Biochem
1986
24. Gearing
DP, Gough
NM,
King JA, Hilton
Di, Nicola NA,
Simpson
Ri, Nice EC, Kelso A, Metcalf
D: Molecular
cloning and
expression of cDNA encoding
a murine
myeloid leukaemia
inhibitory factor (LIF). EMBO J 6:3995, 1987
25. Lotem
J, Sachs L: In vivo control of differentiation
of myeloid
leukemic
cells by cyclosponine
A and recombinant
interleukin-I
alpha. Blood (in press)
23.
Haegeman
J, Fiers
inducible
159:625,
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1988 72: 2070-2073
The myeloid blood cell differentiation-inducing protein MGI-2A is
interleukin-6
Y Shabo, J Lotem, M Rubinstein, M Revel, SC Clark, SF Wolf, R Kamen and L Sachs
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