[CANCER RESEARCH
45, 9-13, January 1985]
Distinct Activities of lnterferon-% Lymphokine and Cytokine Differentiationinducing Factors Acting on the Human Monoblastic Leukemia Cell Line
U9371
Paul E. Harris,2 Peter Ralph,3 Patricia Litcof sky, and Malcolm A. S. Moore
Department of Developmental Hematopolesis, Memorial Sloan-Kettering Cancer Center, New York, New York 10021
ABSTRACT
INTRODUCTION
The human monoblast leukemia line U937 is growth inhibited
and induced to express various characteristics of mature
monocytes by lymphokines (LK) and other cytokines. Previous
experiments have shown that interferon-7 (IFN-7) is responsible
for some but not all of the differentiation-inducing effects on
The recent discovery of the maturation-inducing activity of
IFN-74 on U937 cells (7, 17) has raised several new questions
regarding the role of IFN-7 and other factors in the regulatory
biology of normal and leukemic monoblasts. Such factors have
therapeutic potential not only as leukemia-specific growth inhib
itors but as potential immunomodulators. Both LK and IFN-7
U937. To determine the variety and specificity of activity, the
following factors were studied: phytohemagglutinin-induced LK
that contained IFN-7 (100 units/ml); purified IFN-7; human col
ony-stimulating factor 1 (CSF-1); and conditioned medium(a)
(CM) from the human bladder carcinoma cell line 5637 and the
hepatoma cell line SK-HEP. LK preparations contained no col
ony-stimulating activity, whereas CM from 5637 and SK-HEP
both contained granulocyte-macrophage
CSF (3000 to 4000
units/ml) but no IFN activity. IFN-7 ¡sthe major immunoglobulin
G Fc receptor-inducing species within lymphokine, since antiinterferon-7 antibody inhibited most of this activity. Other
sources of Fc receptor-inducing activity were CM from SK-HEP
and 5637 cell lines. Human CSF-1 when tested up to 800 units/
ml was inactive for Fc receptor induction. LK induced the
chemotactic peptide receptor, but this induction was due to
factors other than IFN-7 as anti-IFN-7 antibody did not inhibit
the induction, and purified IFN-7 at a dose equivalent to that
found in LK (100 units/ml) had no activity in the assay. SK-HEP
and 5637 CM had strong chemotactic peptide receptor-inducing
activity, but human CSF-1 was inactive up to 800 units/ml.
Peroxide production after stimulation with phorbol myristic acid
could be induced by LK, LK with anti-IFN-7 antibody, 5637, and
SK-HEP treatment. IFN-7 (100 units/ml) and CSF-1 (800 units/
ml) were ineffective. Peroxide production was induced by IFN-7
have been shown to block cell proliferation and induce immuno
globulin FcRs and antibody-dependent cellular cytotoxicity in
U937 (9, 25). IFN-7, however, is not a "global" inducing agent,
as there are monocyte characteristics which are not inducible by
IFN-7 treatment. This fact prompted us to search for other
activities that could induce a functional terminal differentiation in
U937 cells. LK was found to possess the capactiy to induce
certain markers of mature monocytes after constituent IFN-7
had been neutralized. Such non-IFN-7 inducing activities appear
related, at least functionally, to inducing activities contained
within the conditioned media of the human bladder carcinoma
cell line 5637 and hepatoma cell line SK-HEP. This report ex
amines the activities of purified IFN-7, LK, LK with neutralizing
anti-IFN-7 antibody, human CSF-1, and conditioned media from
5637 and SK-HEP in the induction of several aspects of monocyte-macrophage function.
MATERIALS AND METHODS
Cell Lines and Biochemicals. The human monoblast cell line U937
has been described elsewhere (28). U937 was grown in RPMI 1640
medium containing 10% fetal bovine serum. U937 cells were cultured for
5 days in preparations containing media and the various factors in
question at an initial concentration of 2.0 x 10s cells/ml. Human urinary
at concentrations above 1000 units/ml. The inducibility of several
enzymatic activities was determined as additional measures of
maturation. /V-Acetylglucuronidase was induced, for example, by
LK, IFN-7, 5637 CM, and phorbol myristic acid. Alkaline phosphatase was induced by LK, IFN-7, dexamethasone, and phorbol
myristic acid. 1,25-Dihydroxycholecalciferol
was also examined
and could induce most of the maturational markers examined.
The results demonstrate that non-IFN cytokines from several
sources have profound differentiation-inducing
effects on
monoblast leukemia cells in a pattern different from that of IFN-
bladder carcinoma line 5637 (19) and hepatoma line SK-HEP (3) were
used as sources of inducing factors. PHA, PMA, dexamethasone (Sigma
Chemical Co., St. Louis, MO), VD3 (Hoffman LaRoche, Nutley, NJ) were
dissolved in growth media.
LK and Cytokine Preparations. LK was prepared from peripheral
blood mononuclear cells (1 x 106 cells/ml) by culturing 2 days with 1%
PHA-M (Difco Laboratories, Detroit, Ml) in growth medium. Cell-free
supematants were harvested and frozen for subsequent use. Supernatants of the human bladder carcinoma cell line 5637 were obtained as
follows. Cells were cultured in growth medium until confluent. This
medium was then replaced with RPM11640 plus 2% PCS, and the cells
were cultured for an additional 3 days, after which the supematants were
harvested and frozen until subsequent use. SK-HEP 1 cells were cultured
in Iscove's medium (Grand Island Biological Co., Grand Island, NY) with
7. The inducing factors in LK and nonlymphoid cytokines have
similar biological activity on U937 cells, and the LK factor is not
associated with CSF-1.
1Supported in part by Grant CH-3F from the American Cancer Society and a
4 The abbreviations used are: IFN-y, Interferon-^; CSA, colony-stimulating activ
ity; FcR, IgG receptor; PCS, fetal calf serum; FMLP, formyl-methionyl-leucyl-phenylalanine; GM-CSF, granulocyte-macrophage colony-stimulating factor; CSF-1, hu
man urinary colony-stimulating factor; LK, lymphokine from phytohemagglutinintreated peripheral blood leukocytes; PHA, phytohemagglutinin; PMA, phorbol myr
istic acid; VD3.1,25-dihydrocholecalciferol.
grant from the Gar Reichman Foundation.
2 To whom requests for reprints should be addressed, at S1101, Department of
Developmental Hematopoiesis, Memorial Sloan-Kettering Cancer Center, 1275
York Avenue, New York, NY 10021.
3 Present address: Cetus Corporation, 1400-53 Street, Emeryville, CA 94608.
Received July 17,1984; accepted September 12,1984.
CANCER
RESEARCH
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45 JANUARY
1985
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CYTOKINES
IN MONOCYTIC
LEUKEMIA
10% PCS for 24 hr, after which the medium was replaced with serumfree Iscove's medium. Following a 4-day incubation, the medium was
nm of a suspension of Micmcoccus lysodeikticus cells at pH 6.24 at 25°
in a 2.6-ml reaction mixture and 1-cm light path (18). A unit of lysozyme
harvested and frozen. LK and the conditioned media from SK-HEP and
Lymphokine contained no CSA (19). The 10% lymphokine preparation
had an IFN--) titer of 100 units/ml (17). In a 2-week human bone marrow
activity is described as 0.001 A units/min. Growth medium and inducers
did not contain significant amounts of lysozyme activity. Measurements
were made with a Gilford Model 1500 spectrophotometer and Syva
CP5000 clinical processor.
Alkaline, Acid Phosphatase, 0-Glucuronidase, and N-Acetylglu-
assay, the 10% SK-HEP and the 10% 5637 preparation each contained
GM-CSF activity (approximately 400 and 300 units/ml, respectively).5
curonidase. Cell extracts were prepared by suspending cells in 0.5 ml
PBS:1% Nonidet P-40, incubating 5 min at room temperature, and then
Both conditioned media were free of IFN-7. Partially purified human
urinary CSF-1 was kindly supplied by R. Stanley (2.5 x 106 units/mg)
centrifuging for 10 min at 5000 rpm. Extracts were assayed for alkaline
and acid phosphatase, iS-glucuronidase, and W-acetylglucuronidase, as
(27) and was active in a 2-week
colony-
described elsewhere (6, 8, 23, 24). Activity of extracts is expressed as
change in absorbance per hr per 106 cells. Measurements were made in
Human immune IFN was obtained from B.
a Beckman ACTA-CV spectrophotometer.
Statistical Analysis. Student's f test to compare means was carried
5637 were diluted to a final concentration of 10% (v/v) in growth medium.
These preparations were then assayed for their CSA and IFN-7 content.
forming cell assay.
IFN, Anti-iFN-7 Antibody.
bone marrow macrophage
Rubin (17) and Cellular Products (Buffalo, NY) with a specific activity of
1.0 to 2.6 x 10" units/mg. A murine IgG monoclonal antibody that
neutralizes the antiviral and anticelluiar effects of IFN--,, but not of IFNa or IFN-/3, was kindly supplied by B. Rubin (20). IFN content of LK
preparations was assayed in vesicular stomatitis virus-infected WISH
cells by inhibition of the cytopathic effect as compared to a laboratory
IFN-7 standard (21).
out using the significance limits of a 2-taiied test.
RESULTS
Induction of FcR and FMLP Receptors: The FcR was inducible on the surface of U937 cells by various cytokine and LK
preparations (Table 1). The strongest inducers were LK and
purified IFN-? (100 units/ml). An excess of anti-IFN-r antibody
blocked most of the FcR-inducing activity of LK. However, a
residual and significant level of FcR-positive cells were induced
Rosette Assays for FcR, OKM1, and Leu M2 Antigens. Cell recep
tors for immunoglobulin Fc were assayed by resetting with IgG- (Cordis
Laboratories, Coral Gables, FL) coated erythrocytes (17). Anti-OKMl
(Ortho Diagnostic Systems, Inc., Raritan, NJ) or anti-Leu M2 (BectonDickmson Monoclonal Center, Inc., Mountain View, CA) was used to
detect surface antigens by incubating 10* cells in 0.1 ml phosphate-
in the presence of the neutralizing antibody, suggesting the
presence of active LK factors other than IFN-7 active in the
induction of FcR. IFN--, -neutralizing antibody completely blocked
the FcR-inducing activity of purified IFN-7. Tne conditioned media
from the human hepatoma cell lines SK-HEP and 5637 also
possessed significant FcR-inducing capacity despite the lack of
IFN-7 activity. Purified CSF-1 had no FcR-inducing capacity at
buffered saline with monoclonal antibody (1 ^g/ml) for 20 min at room
temperature, washing, and incubating for 20 min at room temperature
with 1:100 dilution of rabbit anti-rat (Leu M2) or anti-mouse (OKM1 ) IgG
serum (Cappel Laboratories, Cochranviiie, PA), washing, and resetting
with protein A-coated erythrocytes, as described previously (29).
Assay for FMLP Receptor. Two million U937 cells were incubated in
15 HM [3H]FMLP (New England Nuclear, Boston, MA) in a total volume
100 and 800 units/ml. Vitamin D3and PMA were used as positive
standards, and they induced high levels of FcR-positive cells.
Dexamethasone induced FcR-positive cells weakly.
The induction of chemotactic peptide receptor, FMLP, was
measured in parallel with FcR. LK- and IFN--,-neutralized LK
were strong inducers of FMLP-receptor (Chart 1). IFN-y-neutral-
of 0.2 ml in the presence or absence of 10 MM unlabeled FMLP (Sigma).
After 3 hr at 4°,the cell suspensions were rapidly filtered onto glass
fiber discs (Whatman GF/C; Whatman, Inc., Clifton, NJ) and washed with
30 ml of 4°phosphate-buffered saline. Radioactivity on the discs was
measured by liquid scintillation spectroscopy. Specific binding was de
fined as the total amount of labeled FMLP bound minus the amount
bound in the presence of an excess of unlabeled cold FMLP. Specific
binding as cpm was converted to sites per cell (14).
Phagocytosis Assay. Fifty pi of latex beads (1.1 pm diameter; 109/
ml) were added to 2.0 ml of cells (5 x 105/ml) in growth medium and
incubated at 37°for 2 hr with light agitation every 30 min. After incuba
ized LK consistently induced the receptor to higher level than LK
Table1
Inductionof FcRs in 1/937cells
U937cells were incubated 5 days with final concentrations of the agents shown
and assayed for FcR as described in 'Materials and Methods.'
tion, the cells were washed through FCS, cytocentrifuged, and stained.
Cells with 6 or more beads were scored as phagocytosis positive with
at least 100 cells counted.
Assay for H2O2. The production of H2O2 in response to PMA stimu
lation was assayed by horseradish peroxidase (Sigma)-mediated hfeOr
InducerMediumLK
(5%)6LK(10%)"LK(10%)
dependent oxidation of homovanillic acid (Sigma), as described else
where (22). Briefly, cells (1 x 10°)were suspended in 2 ml solution
A7AU'CIFN-7
+
units/ml)IFN-y
(100
ASK-HEP
(100 units/ml) + A-y
CM (10%)"
(10%)"CSF-1
5637 CM
units/ml)CSF-1
(100
units/ml)Dexamethasone
(800
M)PMA
(10"*
containing 100 UM homovanillic acid and horseradish peroxidase; (5
units/ml) in the absence or presence of PMA (0.030 /«g/ml).Following a
90-min incubation at 37°, the incubation product was centrifuged, and
0.25 ml of a 25 mM EDTA:0.1 M glycine-NaOH, pH 12, solution was
added to the supematants. A 30% stock solution of H2O2 (Sigma) was
used to prepare H2O2standards (0.001 to 50 nmol/assay) for contruction
of a standard curve. The homovanillic acid oxidation product was mea
sured on a Perkins-Elmer Model MPF-44A fluorescence spectrofluoro-
341010156030108
ng/ml)VD3(10-*M)PHA-M(0.1%,v/v)AfA
(3
(1200 nanounits/ml)%
* From a representative experiment.
* LK contained IFN-y (1000 units/ml); neutralizingATA was added to LK or IFN-
meter. Excitation and emission were measured at 312 and 420 nm,
respectively.
Lysozyme Assay. Lysozyme activity in cell supematants was deter
mined spectrophotometrically
by following the change in turbidity at 450
y at 1200 nanounits/ml before testing for induction.
c ATA, anti-IFN-Tantibody; CM, conditioned medium.
" Final concentrations of CSA (300 units/ml) (SK-HEP)and of 5637 CM (400
5J. Gabnloveand E. Platzer, personal communication.
CANCER
of positive
cells"10357624801024
units/ml).
RESEARCH
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1985
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CYTOKINES
Inducen
IN MONOCYTIC
Inductionof IMLPReceptorIn U937Cells*
concentration tested. The release of lysozyme into growth me
dium was induced by LK, IFN-7, SK-HEP, 5637, PMA, VD3, and,
to a lesser extent, IFN-7-neutralized LK. Low doses of LK and
CSF-1 were ineffective.
Media
LK5%"
LK 10%"
LK 10% + AGA"
Production of Lysosomal Enzymes. U937 cells contain small
amounts of alkaline phosphatase, acid phosphatase, /3-glucuronidase, and A/-acetylglucuronidase (Table 3). The inducibility of
these lysosomal enzymes was examined. Intracellular alkaline
phosphatase activity was increased by incubation with LK, IFN7, PMA, dexamethasone, and VD3. SK-HEP and 5637 condi
tioned media and CSF-1 were inactive at the concentrations
tested. Acid phosphatase levels increased moderately in re
sponse to treatment with LK, IFN-7, 5637 conditioned medium,
PMA, and VD3. Incubation with SK-HEP conditioned medium
and CSF-1 preparations did not significantly alter the expression
of acid phosphatase from control level. /8-Glucuronidase levels
were not significantly altered by the agents tested. /V-acetylglucuronidase was inducible by LK, IFN-7, 5637 conditioned me
ylFN 100 u/ml
ylFN 1000 u/ml
SK-Hep CM 10%
5637 CM 10%
CSF-1 100 U/ml
CSF-1 800 U/ml
Oexamethasone
52
VD3
PHA-M
AGA
I
10
20
30
Sites/Cell
40
50
LEUKEMIA
60
Chart 1. U937 cells were induced as described in Table 1 and assayed for
chemotactic peptide (FMLP) receptor. ", average value from 3 or more experiments;
", LK containing IFN-r (1000 units/ml). Neutralizing anti-IFN--y antibody (AGA) was
added to LK or IFN-y at 1200 nunits/ml before testing for induction. U, units; CM,
conditioned media.
dium, VD3, and PMA. Extracellular levels of these enzymes were
not appreciably affected by the induction protocols (data not
shown).
Induction of Antigens Identifiable by OKM1 and Leu M2.
The monocyte-related antigens, OKM1 and Leu M2, defined by
Table 2
Induction of H¿02and tysozyme production
U937 cells were induced as described in Table 1 and assayed for PMA-stimulated
production of H2O2and lysozyme secretion.
monoclonal antibodies, can be induced on the surface of U937
(units/10*cells)50-60"75-80490-525125-160190-200NT260-300390-41040-5070-8050-60220-230500-60050-6050-70"
cells (Table 4). The induction of these markers was readily seen
after incubation with LK and IFN-7. Purified IFN-7 alone in
creased OKM1- and Leu M2-positive cells at a concentration
InducerMediumLK
(5%)LK(10%)LK
(100 units/ml) similar to that in the LK preparation and may be
the major inducing species contained within LK. SK-HEP and
CSF-1 (100 units/ml) did not increase the number of reactive
AfA6\FN-y
+
units/ml)IFN-7
(100
units/ml)SK-HEP
(1000
(10%)5637
CM
(10%)CSF-1
CM
units/ml)CSF-1
(100
units/ml)Dexamethasone
(800
M)PMA
(10~*
cells at the concentrations tested. The conditioned medium of
5637 cells was moderately active in the 5-day assay. Dexameth
asone did not induce these antigens at the concentration tested,
while PMA and VD3 were both active.
ng/ml)VD3(1Q-"M)PHA-M
(3
Induction of Phagocytosis and Growth Inhibition. The ca
v/v)A-yAHjCMnmol/10«cells/hr)0.07-0.32"0.34-0.481.31-0.631.31-0.630.07-0.111.51-1.321.37-1.550.49-1.570.07-0.100.14-0.070.14-0.07NT0.08-0.250.08-0.320.08-0.32Lysozyme
(0.1%,
pacity to phagocytize latex beads was induced by LK, LK with
neutralized IFN-7, SK-HEP- and 5637 conditioned media, VD3,
experiments.6
Ranges from 3
PMA, and weakly by IFN-7 (100 units/ml). Human CSF-1, dexa
A-*A 9nti.IPN.-v antihnrtu- NT not toQterl- C.M ronrlitinnort merlii im
methasone, PHA, and anti-IFN-7-antibody were inactive (Table
5). Growth inhibition was observable after treatment with LK,
alone, suggesting that IFN-7 may interfere in the expression of
IFN-7-neutralized LK, IFN-7, SK-HEP and 5637 conditioned methis marker. IFN-7 at 100 units/ml (the concentration of IFN-7 in
the induction cultures containing LK) and 1000 units/ml did not
TabteS
include additional receptor sites. The conditioned media from
Induction of acid phosphatase, alkaline phosphatase, ß-glucuronidase, and NSK-HEP and 5637 were strong inducers of FMLP receptor.
acetylglucuronidase
U937 cells were induced as described in Table 1 and assayed for their intracelHuman CSF-1, however, was inactive at 100 and 800 units/ml.
Dexamethasone (10~6 M) was a strong inducer, while 10~8 M lular enzyme levels. Values are expressed as control and/or treatment and are the
mean of 3 or more experiments.
VD3 showed intermediate effects.
Production of H2O2and Lysozyme. The capacity to produce
H2O2 in response to PMA stimulation constitutes another inducible characteristic of U937 cells (Table 2). Uninduced but PMAstimulated U937 cells produced very small amounts of peroxide,
perhaps the product of a small subpopulation of spontaneously
matured cells. LK and IFN-7-neutralized LK both induced the
ability of U937 cells to produce H202. IFN-7 at 100 units/ml was
inactive. A 10-fold increase in the IFN-7 concentration (1000
units/ml), however, induced U937 cells to produce H2O2 after
PMA stimulation. Both 5637- and SK-HEP-conditioned media
induced peroxide response capacity, while CSF-1 did not. Vita
min D3 had little activity in inducing peroxide production at the
CANCER
RESEARCH
Alkaline
phosphatase
Inducer
Acid phosphatase
0-glucuronidase
N-acetylglucuronidase
MediumLK
(5%)LK
100units/ml)IFN-7
(10%; IFN-7,
units/ml)SK-HEP
(100
(10%)5637
CM
(20%)CSF-1
CM
units/ml)CSF-1
(100
units/ml)Dexamethasone
(800
M)PMA
(10"*
(3 ng/ml)
VD3(10-*M)1.05.2"3.1"1.81.51.12.6"2.7"
2.8"1.03.0"1.8"1.22.7"1.01.11.9"
2.2"1.01.71.61.21.41.01.31.21.51.04.4"2.5"1.53.2"1.11.62.6"
2.6"
Significantlydifferent from 1.00 at p < 0.05.
VOL. 45 JANUARY
1985
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CYTOKINES
IN MONOCYTIC
Table 4
increase. These cell lines produce a GM-CSF (300 to 400 units/
ml final concentration in growth medium). However, the related
human urinary CSF-1 (11) tested at doses (100 to 800 units/ml)
exceeding the comparable GM-CSF concentration in the cell lineconditioned medium was inactive in FcR induction.
IFN-7 appears to be an incomplete inducer as it is inactive in
Induction of OKM1 and Leu M2 markers
U937 cells were induced as described in Table 1 and assayed for their expression
of surface antigens.
Inducer
OKM1
(% positive)
LEUKEMIA
Leu M2
(% positive)
MediumLK
(5%)LK(10%)IFN-7
the induction of chemotactic peptide receptor at 100 to 1000
units/ml. LK induced a dramatic increase in the number of
receptors for chemotactic peptide. Anti-IFN-7 antibody did not
(10%)5637
CM
block this effect showing that the LK activity is not IFN-7. Purified
(20%)CSF-1
CM
human CSF-1 was inactive. The conditioned media from 5637
units/ml)CSF-1
(100
and SK-HEP demonstrated strong activity in the induction of the
units/ml)Dexamethasone
(800
M)PMA
(10"6
FMLP receptor in distinct contrast to CSF-1.
ng/ml)VD3(10-*M)9a33"4527"820620"14161259"476834"5737"1114"14"410660"27*
(3
Results similar to the induction of FMLP receptor were ob
tained in the production of H202. LK and LK with neutralized IFN8 Mean of 3 or more experiments.
6 Significantly different from control at p < 0.05.
7 induced the capacity to respond to PMA stimulation by the
c CM, conditioned medium.
elaboration of H2O2. IFN-7, at the same dose found within LK
preparation, was inactive. Only when the IFN concentration was
Table 5
raised to 1000 units/ml did U937 cells acquire the capacity to
Induction of growth inhibition and phagocytosis
produce H202. These results suggest that there are other factors
U937 cells were induced as described in Table 1 and assayed for growth
acting alone or in synergy with IFN-7 in the induction of peroxide
inhibition and latex bead phagocytosis.
production. Human lymphotoxin is reported to be one such factor
of growth in
of positive
(2). The ability of SK-HEP and 5637 conditioned media to induce
phagocytosis2104338113639222526855
InducerMediumLK
hibitiono*,»2580307060300.o"307070015%
units/ml)SK-HEP
(100
(10%)5637
CMC
this same response lend additional support to the idea that there
are factors other than IFN-7 capable of inducing the production
of H202, as these conditioned media are free of IFN-7. The
possibility that CSA-like activity is responsible for the acquisition
of peroxide production was tested directly by using purified CSF1; however, this did not induce secretion of this microbiocidal
product at the concentrations tested. The production and release
of lysozyme is another maturationally acquired characteristic. LK
and the conditioned media of SK-HEL and 5637 were strong
inducers of lysozyme in U937 cells. IFN-7 was a moderate
(5%)LK(10%)LK(10%)
A-yA0\FN-y+
units/ml)SK-HEPCM(10%)5637
(100
(10%)CSF-1
CM
units/ml)CSF-1
(100
units/ml)Dexamethasone
(800
M)PMA
(10"*
ng/ml)VD3(10-8M)PHA-M(0.1%,
(3
v/v)AT-A%
inducer of lysozyme production and release, shown by purified
IFN-7 and by the fact that much of the induction activity of LK
was removed when neutralizing antibody was added. Human
CSF-1 was inactive in the induction of this bacteriolytic enzyme.
a Mean of 3 or more experiments.
"Average increase in cell number in the medium control was 4.1-fold; CSF-1
(800 units/ml) reproducibly increased cell number about 4.3-fold.
c A-yA, anti-IFN-7 antibody; CM, conditioned medium.
Induction of lysosomal enzymes examined was most readily
accomplished by the use of LK and IFN-7, although the increase
dia, dexamethasone, VD3, and PMA. Anti-IFN-7 antibody greatly
reduced the toxicity of LK and alone caused a slight reduction in
cell growth. PHA did not inhibit growth, and human CSF-1
in enzyme levels was generally smaller than that for the markers
described previously. IFN-7 accounted for much of the observed
activity in LK and probably represents the major inducing spe
cies. Of the conditioned media tested, 5637 was active in stim
ulating acid phosphatase, and A/-acetylglucuronidase and SKHEP was inactive. Human CSF-1 at 100 units/ml consistently
increased the final cell concentration 5% above that of controls.
DISCUSSION
failed to increase any of the activities examined.
Leu M2 and OKM1 are monoclonal antibodies which recognize
monocyte-related determinants (1,16). LK-mediated induction of
LKM1 antigen (12) and of a different epitope Mac-1 (17) on the
Our approach to defining the biological activities of complex
mixtures such as LK and other conditioned media was dependent
on the use of a wide phenotypic panel and partially purified
known constituents of the biological supernatants in question. In
side-by-side comparisons of inducible characteristics, it has been
possible to identify the activity of known and new LK-cytokine
species. The induction of FcR and FMLP receptors signal monocyte maturation. It is well known that LK preparations are able
to effect such a transformation (7, 14). IFN-7 is the major FcR-
same molecule has been reported for U937, but the participation
of IFN-7 in this effect has not been measured. Our results
suggest that LK constituents responsible for the induction of this
marker may be in whole or in part IFN-7 (Table 4). Further
experiments are planned to ascertain this fact. The ability of
5637 conditioned medium to increase the expression of this
antigen suggests the existence of a non-IFN-7 inducer. Signifi
inducing moiety of LK preparations (17). However, it is not the
only inducing species found within LK, as these results show.
LK preparations with anti-IFN-7 antibody added in 5-fold excess
can still modulate a weak induction of the FcR. Additionally, the
conditioned media from SK-HEP and 5637 cell lines that lacked
any IFN activity were able to induce a moderate amount of FcR
CANCER
RESEARCH
cant growth inhibition was observed during the acquisition of the
maturational markers examined, suggesting that terminal differ
entiation had occurred. LK, IFN-7, SK-HEP, 5637, VD3, dexa
methasone, and PMA all yielded significant growth inhibitions at
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1985
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CYTOKINES
IN MONOCYTIC
the levels tested. The action of human CSF-1 on U937 cells was
growth stimulatory. Normal monocyte progenitors respond to
the dual action of CSA by proliferation and maturation (26). A
dissociation of CSA activity appears to occur in the U937 re
sponse to CSF-1, as the cells did not show any evidence of
maturation by the markers tested. The inability to respond to
CSA maturational activity appears to be a general feature of
explanted myeloid leukemic cells (10).
The induction of latex bead phagocytosis may in part be
mediated by IFN-7, but there appears to be other active species
contained within LK. The activities contained within 5637 and
SK-HEP conditioned media are strong inducers of phagocytosis.
The similarity of residual LK activity after IFN-7 neutralization
5.
6.
7.
8.
9.
and the conditioned media tested suggest functional identity in
U937 induction potential. There have been reports of similar
activity. Gately ef al. (5) report a factor obtained from supernatants of the HUT 102 T-cell line apparently distinct from IFN-7
10.
that is able to induce peroxide production without PMA stimula
tion. Small amounts of H2O2 were spontaneously liberated after
maturation induction in our experiments and perhaps represent
a similar maturational event.
Olsson ef a/. (13) likewise report a U937 differentiation factor
able to induce mature morphology and increase the percentage
of nitroblue tetrazolium-positive cells, but only after retinoic acid
priming (13). The activities reported here have no /3-retinoic acid
12.
dependency for their actions and represent distinct molecular
species or assay differences. The conditioned media of 5637
has been reported to induce antibody-dependent cellular cyto-
15.
11.
13.
14.
16.
toxicity in U937 cells (6). Gabrilove ef al. (4) and Platzer ef al.
(15) find that conditioned media from SK-HEP and 5637 contain
a leukemia differentation-inducing activity copurifying with a GMCSF and active on the human promyelocytic leukemia cell line
HL-60 on the basis of a colony morphology assay (3).6 Results
presented herein lend support to the notion that SK-HEP and
5637 contain activities that modulate the maturation of myelo-
17.
18.
19.
monocytic leukemias. The relative inactivity of purified human
CSF-1 provides evidence, albeit circumstantial, that the action
of 5637 and SK-HEP are not due to CSA-like species. LK, SK-
20.
HEP, and 5637 conditioned media are currently being purified in
the attempt to isolate the leukemia-active moieties.
21.
ACKNOWLEDGMENTS
22.
The authors thank Rose M. Vecchiolla for preparation of the manuscript.
23.
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45 JANUARY
1985
13
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1985 American Association for Cancer Research.
Distinct Activities of Interferon-γ, Lymphokine and Cytokine
Differentiation-inducing Factors Acting on the Human
Monoblastic Leukemia Cell Line U937
Paul E. Harris, Peter Ralph, Patricia Litcofsky, et al.
Cancer Res 1985;45:9-13.
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