1. No category

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Interleukin-4 Inhibits Growth of Multiple Myelomas by Suppressing
In t erleu kin - 6 Ex pre s s ion
By F. Herrmann, M. Andreeff, H.-J. Gruss, M.A. Brach, M. Lubbert, and R. Mertelsmann
Unfractionated bone marrow (BM) cells obtained form patients with multiple myeloma (MM) exhibit high levels of
interleukin (IL)-6. Secretion of IL-6 by these cells as well as
spontaneous plasma cell proliferation can be abrogated by
neutralizing anti-IL-6 monoclonal antibody (MoAb). Treatment of BM cells with recombinant human (rh)lL4 at doses
of 50 to 250 U/mL blocked endogenous IL-6 synthesis in a
dose-dependent fashion and was associated with significant
reduction of plasma cell growth that could be reversed by
exogenous rhlL-6. Enrichment of BM cells from MM patients
for plasma cells and adherent cells and analysis of IL-6 mRNA
in these subpopulations by means of quantitative polymerase chain reaction (PCR) showed that adherent BM cells
accounted for most of the synthesis of IL-6 transcripts,
whereas plasma cells displayed negligible levels of IL-6
mRNA only. These results suggest therapeutic evaluation of
rhlL-4 in patients with plasma cell neoplasms.
o 1991 by The American Society of Hematology.
I
Bence-Jones protein only. All patients exhibited BM plasmacytosis
greater than 35% by morphologic inspection of BM smears. Seven
patients had stage 111 disease, and 2 patients stage I1 disease
according to the system of Durie and Sa1m0n.I~
Fractionation of EM cells. The method of fractionation of
various subpopulations from BM of MM patients has been previously reportedI4; involving the use of a panel of monoclonal
antibodies (MoAbs) and a two-step immune rosette technique to
select positively or negatively for MM BM subsets. MoAbs used are
directed against the following antigens: CD2, CD3 (T cells), CD10,
CD19 (B cells), CD16 (NK cell and neutrophils), CD17 (myeloid
cells), CD33, CD34 (early myeloid cells and hematopoietic progenitor cells), HLA-DR, glycophorin A (nucleated RBC), PC1 and
PCAl (plasma cells). To prepare plasma cell-enriched fractions,
BM cells were depleted from CD2, 3, 10, 16, 17, 19, 33, 34,
HLA-DR, and glycophorin A-positive cells. The remaining (rosette negative) fraction was further enriched for plasma cells
( > 95% pure by morphology) using a second rosetting step (rosette
positive fraction) to select positively for PC1- and PCAl-expressing
cells. The CD2, 3, 10, 16, 17, 19, 33, 34, HLA-DR, glycophorin
A-positive PClIPCA1 negative cell fraction was enriched for
monocyteslmacrophages by repeated adherence to plastic surfaces
( > 88% cells positively stained with nonspecific esterase and free
of morphologically detectable plasma cells). Cells positively enriched for PClIPCA1 cells and adherent PClIPCA1 negative cells
were then either directly subjected to polymerase chain reaction
(PCR) analysis or were cultured for a period of 12 hours (37”C, 5%
CO, in air) with or without rhIL-4 (250 UlmL) before PCR analysis
was performed. Student’s t-test was used for statistical analysis.
Recombinant human IL-4 (rhlL-4). rhIL-4 was obtained as a
purified protein from supernatants of Chinese hamster ovary cells
transfected with the cDNA clone encoding IL-4 (specific activity
lo* Ulmg) and was obtained through Dr P. Trotta, ScheringPlough, Bloomfield, NJ). One unit IL-4 was defined as the
concentration of IL-4 resulting in half-maximal proliferation of
phytohemagglutinin-stimulated T cells.
IL-6 antibody. A neutralyzing MoAb to rhIL-6 (B-E8) was
provided by Dr U. Schwulera (Biotest GmbH, Dreieich, German~).’~
cDNAs. cDNAs used in this study were a cloned IL-6lBSF-2
cDNA (pBSF2-38.1)16and a cDNA encoding human IL-6 receptor
(pBSF2R.236).I7 Both probes were provided by Dr T. Hirano
(Osaka University, Osaka, Japan). A cDNA clone coding for the
housekeeping protein glyceraldehyde phosphate dehydrogenase
(GAPD) was obtained through Dr J. Bauer, University of Freiburg,
Germany.I8
Cell culture. BM cells previously subjected to density gradient
separation through Ficoll-Hypaque (Pharmacia, Uppsala, Sweden)
were cultured at 1 x lo6 cells1mL under serum-free conditions as
previously described.” Serum-free culture medium was prepared
from powdered Iscove’s modified Dulbecco’s medium (IMDM)
NTERLEUKIN-4 (IL-4) acts on many cell types by
displaying either agonistic or antagonistic effects.’ Perhaps more important, IL-4 exhibits different effects on cells
of a single lineage at different stages of differentiation. IL-4
costimulates with anti-IgM antibody the proliferation of
murine B-cells’ and induces secretion of IgGl by polysaccharide (LPS)-activated B lymphocytes,’ but inhibits growth of
malignant plasma cells in vitro: IL-4 either induces or
inhibits expression of certain cytokines by various cell types.
rhIL-4 stimulates secretion of granulocyte CSF and macrophage CSF by monocytes and fibroblast^^.^ but inhibits,
however, gene expression of IL-1, tumor necrosis
IL-6,8,9 IL-8,’” and prostaglandin E; by monocytes, consistent with the effects of an antiinflammatory agent. Endogenous IL-6 has been shown to be a major growth factor of
MM cells by establishing either autocrine” or paracrine
loops.” Because of the inhibitory effects of IL-4 and
anti-IL-6 on MM growth in vitro,4,”,’’ we investigated
whether the effects of IL-4 on this cell type involves an
interruption of growth stimulation mediated by endogenous IL-6.
MATERIALS AND METHODS
Patients and tumor specimens. After patients gave their informed consent, heparinized bone marrow (BM) aspirations were
collected from 9 patients with MM and 6 healthy individuals. All
biopsy procedures were part of the routine diagnostic workup.
Four plasmacytoma patients were previously untreated, 3 patients
had received melphalan and prednisone, 1 patient had received
cyclophosphamide only, and 1patient was treated with vincristine,
adriamycin, and dexamethasone. At time of study, all patients had
been untreated for at least 2 months. Seven patients had an IgG
myeloma, 1 patient had an IgA myeloma, and 1 patient had
From the Department of Hematology and Oncology, University of
Freiburg Medical Center, Freibue, Germany; and the University of
Texas,MD Anderson Cancer Center, Houston, TX.
Submitted January 24,1991; accepted May 21,1991.
Supported by Grant No. W 19/86/0sfromthe Deutsche fiebshiye.
Address reprint requests to Friedhelm Hemnann, MD, PhD, Department of Hematology and Oncology, University of Freiburg Medical
Center, Hugstetter Str. 55, 0-7800 Freiburg i.Br., 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 1991 by TheAmerican Society of Hematology.
0006-4971191 I 7808-0128$3.00/0
2070
Blood, Vol78, No 8 (October 15). 1991: pp 2070-2074
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207 1
EFFECT OF IL-4 ON MYELOMA GROWTH
(GIBCO, Grand Island, NY).To 900 mL reconstituted IMDM
triple distilled water (40.5 mL), sodium bicarbonate (3.0 mg/mL),
3-mercapto-1-2-propanediol
(8 pg/mL), penicillin (100 U/mL),
and streptomycin (100 pg/ml; Sigma Chemicals, Munich, FRG)
were added. Five milliliters of 10% fatty acid-free deionized bovine
serum albumin was added to 44.35 mL prepared IMDM. To this
mixture was added 0.65 mL transferrin (90 mg/mL) saturated with
7.0 mmol/L ferric chloride in 1 mmol/L HCR (Sigma). In experiments indicated, the cultures received rhIL-4 at 50 to 250 U/mL, or
anti-IL-6 MoAb at 150 final dilution.
IL-6 bioassay. IL-6 biologic activity present in cell-free culture
supernatants was assessed by its proliferative action on the murine
hybridoma cell line B9 with a calorimetric assay as previously
detailed.20In these experiments, a preparation of rhIL-6 (lot no.
703) was used as internal standard (provided by Dr Hirano).
RNA extraction and Northem blot analysis. Total cellular RNA
was isolated by lysing low-densityBM cells in guanidium isothiocyanate followed by recovery of RNA by centrifugation through
cesium chloride?’ After denaturation at 6WC, RNA was electrophoresed in an agarose formaldyhydegel (1.2%) and transferred to
synthetic membranes (Schleicher and Schuell, Dassel, Germany).
Filters were hybridized with labeled probe for 12 to 24 hours at
42°C in 50% formamide 2 x SSC, 5 x Denhardts, 0.1% sodium
dodecyl sulfate (SDS), 10% dextran sulfate, and 100 pg/mL
salmon sperm DNA.*’ Filters were washed to a stringency of 0.1%
SSC, 65°C for 12 minutes and used to expose Kodak Xomat films
with intensifying screens. To exclude incomplete RNA transfer in
single lanes, all filters were reprobed with GAPD cDNA.
Analysis of mRNA expression by the PCR. RNA was prepared as
described above. Avian reverse transcriptase (Promega, Heidelberg, FRG) was used for cDNA synthesis with the 3’ IL-6 and the
3’ p,-microglobulin (&M) primer. After addition of the 5’ IL-6 or
5’ P,-M primer, 35 PCR cycles were performed using Taq polymerase (Pharmacia) and a thermocycler (Perkin Elmer Cetus,
Emeryville, CA). Reaction conditions were used as previously
described.u Deoxyribonucleotides were synthesized on an automated solid phase synthesizer (model 381A, Applied Biosystems).
The nucleotide sequences of the direct and reverse primers used
were as follows: IL-6; direct primer 5’-GGACTGCAGGAACTCClT-3’, reverse primer 5’-GTACCCCCA GGAGAAGAT-3’.
p2-M; direct primer 5’-CTCGCGCTACTCTCTCTCT-3‘, reverse primer 5’-TCCATTClTCAGTAAGTCAACT-3’.
PCR products [525 base pairs (bp) for IL-6 and 160 bp for pZ-M]were
separated on 1.2% agarose gels.
Proliferation assay ofplasmacytoma cells. Low-density BM cells
of MM patients were cultured in serum-free medium in the
presence or absence of rhIL-4 (50, 125, 250 U/mL), anti-IL-6
MoAb (1:50 final dilution) or rhIL-6 (250 ng/mL) for the indicated
periods (37”C,5% CO, in humidified air). At day 5 , the percentage
of plasmacytomacells was determined by intracytoplasmic immunofluorescence using anti-lc or anti-A light chain antibodies conjungated to fluorescein (Behringwerke, Marburg, Germany). The
percentage of myeloma cells in the S-phase of cell cycle was
assessed as a measure of the proliferative capacity of myeloma cells
by means of two-color immunofluorescence using a MoAb to
bromodeoxyuridine (BrdUrd) and a rhodamine-labeled goat antimouse immunoglobulin (Cappel, Malvern, PA).”
RESULTS
Secretion of IL-6 and growth requirements for ZL-6 in
plasmacytoma cell-containing BM cultures. As shown in
Table 1, culture supernatants of BM cells sampled from
nine M M patients and cultured for 36 hours displayed
significant levels of IL-6 (94 to 1,009 U/mL). Cultures of
Table 1. Effect of rhlL-4 on IL-6 Secretion by BM Cells From Patients
With Plasmacytoma and From Healthy Individuals
IL-6 W m L )
IL-4 (U/mL)
Treatment
Patient
1
2
3
4
5
6
7
a
9
Healthy individual
1
2
3
4
5
6
Culture
Medium
50
125
493
974
349
904
1,009
248
94
332
617
104
77
37
114
345
43
14
72
a5
17
ND
ND
39
102
ND
ND
9
ND
32
ND
11
ND
ND
ND
7
ND
ND
ND
ND
ND
ND
ND
ND
ai
ND
39
250
<5
<5
<5
14
17
Culture
Medium
+ Anti-IL-6
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
15
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
Cells were cultured for 36 hours at lo6 cellslmL in serum-free culture
medium. rh-IL-4 and anti-IL-6 MoAb (1:50) were added at the initiation
of culture. IL-6 biologic activity was measured by means of the E9
proliferation assay in cell-free culture supernatants. One unit per
milliliter IL-6 is defined as the concentration of IL-6 leading to halfmaximal stimulation of E9 cells and equals 1 pg/mL. Values are means
of triplicate determinations.
BM cells from four of six healthy individuals also contained
measurable IL-6 biologic activity (11 to 81 U/mL). Treatment of cultures with excess concentrations of MoAb to
rhIL-6 completely abrogated levels of IL-6 activity detectable in culture supernatant of BM cells of both patients and
healthy individuals. Endogenous synthesis of IL-6 led to a
spontaneous growth of myeloma cells in short-term culture
for 5 days that could be prevented when anti-IL-6 MoAb
was present in the cultures during the cultivation period
(Fig 1).This spontaneous growth was detectable in six of
the nine patients investigated. All six patients had a
myeloma cell labeling index (LI; percentage of plasmacytoma cells in S-phase) of greater than 1% while the
remaining three had LI < 0.7%.
Effectof rhIL-4 on IL-6synthesis and spontaneousplasmacytoma growth. As shown in Fig 2, treatment of BM cell
cultures obtained from these six patients with rhIL-4
resulted in significant growth inhibition of plasmacytoma
cells. The effects of rhIL-4 were dose-dependent, could b e
duplicated by anti-IL-6 MoAb, and reversed by addition of
excess (250 ng/mL) exogenous rhIL-6 to the cultures
(Table 2). Treatment of cultures with rhIL-4 also resulted
in dose-dependent downregulation of IL-6 secretion (Table
1) that was almost complete when cultures had been
exposed to 250 U / m L of IL-4 and occurred in cultures of
both MM patients and healthy individuals. Analyses of the
effects of IL-4 on IL-6 expression in low-density BM cells
containing spontaneously proliferating plasma cells were
also made at the mRNA level. As indicated in Fig 3,
cytoplasmic R N A (20 kg per lane) harvested from BM cells
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HERRMANN ET AL
2072
Table 2. Reversalof Growth Inhibitory Action of rhlL-4 on M M Cells
by Exogenous rhlL-6
M M Cells in S-Phase'
Day 0 (Medium)
Day 5 (Medium)
Day 5 (anti-IL-6)
0
a
4
12
16
Percent of Plasmacytoma cells in Sqhase
Fig 1. Spontaneous proliferation of MM cells in serum-free medium. Cells were cultured at 1 x lO'cells/mL. Before and 5 days after
initiation of culture, plasmacytoma cells in S-phase were determined
by two-color immunofluorescence as described in the Materials and
Methods section. In additional experiments, the culture was performed with anti-IL-6 MoAb at a final dilution of 1:50(shown in pilot
experiments t o neutralize >3,000 U/mL). Results are the mean and
SE of six patients with LI greater than 1%.
of all six patients disclosed transcripts of IL-6 and IL-6
receptors detectable by Northern blot analysis. On exposure of cells to rhIL-4 (250 U/mL), IL-6 transcripts were
almost undetectable whereas expression of IL-6 receptor
mRNA remained almost unchanged. To determine the
source of IL-6 production in MM, BM cells of the patient
who had produced the highest amount of IL-6 in culture
were fractionated into various subpopulations by an immune-rosette technique and were assessed (with or without
rhIL-4) for synthesis of IL-6 transcripts by quantitative
PCR. As shown in Fig 4, BM cells highly enriched for
plasma cells disclosed only low levels of IL-6 transcripts,
barely detectable by PCR analysis, whereas IL-6 transcripts
were easily detectable in RNA harvested from adherent
BM cells. Culture of these cells with rhIL-4 (250 U/mL) for
12 hours, but not with medium only, completely abrogated
their ability to synthesize IL-6 transcripts (Fig 4).
Experiment
Experiment
Experiment
Treatment
1
2
3
rhlL-4
rhlL-4 + rhlL-6
24
107
22
98
25
92
Cells (lOa/mL)were cultured in serum-free medium with or without
rhlL-4 (250 U/mL) or rhlL-4 (250 U/mL) plus rhlL-6 (250 ng/mL). After 5
days, MM cells in S-phase were determined by two-color immunofluorescence analysis as described in the Materials and Methods section.
Results are the percentageof S-phase cells in control cultures (medium
treatment only), which were assumed to be 100%. Experiments were
performed with cells from three different donors of MM-derived BM
(experiments 1 through 3) and were repeated twice with SE < 10%.
*Percentage of S-phase cells in medium-treated control cultures.
DISCUSSION
IL-6 has been shown to act as an endogenous growth
promoter of various tumor types, including rcnai cell
carcinomaz4 and hematopoietic neoplasms such as acute
myelogenous leukemia," megakaryoblastic leukemia" and
MM.I
l,12,26,27
Either the tumor cells themselves or the tumoral environment may account for the IL-6 activity
detectable in these disease states. Particularly in plasmacytoma, the source of endogenous IL-6 has been controversial. Kawano et all' and Fiedler et aIz7 suggested that
myeloma cells may secrete IL-6 in an autocrine fashion to
escape growth restrictions, whereas Klein et allzfavored the
hypothesis of paracrine growth stimulation by demonstrating high levels of IL-6 expression in plasma cell-depleted
BM cells of plasmacytoma patients. In the present study, we
confirm previous findings to show that MM cells spontaneously proliferate in short-term liquid cultures, that high
levels of IL-6 can be found in BM cells of MM, and finally
that anti-IL-6 MoAb can effectively suppress growth of
plasmacytoma cells in vitro. As estimated by quantitative
PCR analysis, adherent cells appeared to be the major
I
I
100 -
75
Medium
IL-4 (50 U/mL)
-
IL-4 (125 U/mL)
IL-4 (250 WmL)
50
-
25
-
anti-IL-6
01
2
3
4
Par, No.
5
6
Fig 2. Effect of rhlL-4 on proliferation of M M cells (six patients with LI > l%].
Cells were
cultured at 1 x lo" cellslmL in
serum-free culture medium. After 5 days, MM cells in S-phase
were determined by two-color
immunofluorescence as described in the Materials and
Methods section. Results are the
percentage of S-phase cells in
control cultures (medium treatment only) which were assumed
t o be 100%.
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2073
EFFECT OF I L 4 ON MYELOMA GROWTH
1 2 3 4
IL-6
5 6 7 8 9 1 0 1 1 12 13
t
Fig 3. Effect of rhlL-4 o n
mRNA accumulation of 11-6 and
its receptor (IL-6R) in low-density BM cells of six patients
(LI > 1%) with plasmacytoma.
Low-density BM cells of patients
were cultured for 12 hours without (lanes 1through 6) and with
(lanes 7 through 12) r h l L 4 (250
U/mL). The control lane” contains RNA from peripheral blood
monocytes activated with rhlL-16
(20 U/mL, provided by Dr D.
Krumwieh, Behringwerke AG,
Marburg, Germany) and LPS (10
pg/mL) for 2 hours. Hybridization with the GAPD cDNA controls for identical RNA loading in
single lanes.
source of IL-6 in MM BM, whereas plasma cells themselves
produced negligible amounts of IL-6 mRNA only.
Various studies have indicated that certain cytokines may
downregulate growth factor-dependent proliferation of tumor cells by interrupting autocrine or paracrine loops.
Cordingley et aIB showed that interferon (IFN)-a
inhibits
.
.
Fig 4. PCR analysis of expresd o n of 11-6 and &-microglobulin
(&M) mRNA in subsets of MMderived BM cells: Analysis of amplified DNA sequence by evaluation of migration in agarose gel
and ethidium bromide staining.
MM-derived BM cells were enriched for Plasma cells (PCA1In
PC1~expressin~ce~~s)and
d
adherent cells as described in the
Materials and Methods
The various subsets (as indicated) were subjected t o PCR
analysis either
after
enrichment procedures or were first
cultured for ,2 hours
[medium) or with rhlL-4 (250
UlmL).
growth of hairy cell leukemia cells mediated by endogenous
TNF-a. IFN-y was shown to suppress G-CSF-dependent
terminal divisions in chronic myelogeneous leukemia cells.”
IL-4 is a potent inhibitor of gene expression of various
inflammatory cytokines including ILl,TNF, IL-8, and also
IL-6.7” Because of previous findings by other investigators4
demonstrating that-IL-4 downregulates clonogenic growth
of myeloma cells, we investigated whether IL-4 acts in this
disease by antagonizing the growth stimulatory effects of
endogeneous IL-6. We showed at the mRNA and protein
level that rhIL-4 dose-dependently inhibits IL-6 synthesis in
the BM of plasmacytoma patients which was associated
with a significant decrease of plasmacytoma growth. There
was a strong suggestion that monocytes are the major
source of IL-6 in MM BM and thus are target cells for the
IL-&suppressing action of rhIL-4.
The mechanism of action of rhIL-4 that interferes with
IL-6 expression is unclear, but preliminary transcriptional
runoff analysis (not shown) suggests that IL4may decrease
transcriptional activity of the I L 6 gene in BM cells of
plasmacytoma. Our results also have chica! ramifications
____
in that they supmrt
.. therapeutic evaluation of IL-4 in this
patient group. Use of I L 4 in patients with plasma cell
neoplasms might prove beneficial, analogous to use of
endocrine hormone derivatives in treatment of breast or
prostate cancer.
I
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1991 78: 2070-2074
Interleukin-4 inhibits growth of multiple myelomas by suppressing
interleukin-6 expression
F Herrmann, M Andreeff, HJ Gruss, MA Brach, M Lubbert and R Mertelsmann
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