Multiple Myeloma Cell Adhesion-Induced Interleukin-6 Expression in Bone
Marrow Stromal Cells Involves Activation of NF-KB
By Dharminder Chauhan, Hiroshi Uchiyama, Yasmin Akbarali, Mitsuyoshi Urashima, Ken-lchi Yamamoto,
Towia A. Libermann, and Kenneth C. Anderson
Adhesion of multiple myeloma (MM) cells to bone marrow
stromal cells (BMSCsl not only localizes MM cells in the
marrow microenvironment, but also triggers interleukin-6
(IL-6) secretion by BMSCs and related MM cell proliferation.
In the present study, we characterized the regulation of IL-
6 gene expression in BMSCs during MM cell adhesion. Adhe-
sion of ARH-77, HS-Sultan, IM-9, and U266 MM cell lines to
BMSCs and BMSC lines (LP 101 and AA 101) triggered 5-
through 15-fold and 2- through 4-fold increases in IL-6 secre-
tion, respectively. IL-6 mRNA transcripts were undetectable
by Northern blotting in IM-9 MM cells or LP 101 BMSCs
cultured alone; however, adherence of IM-9 cells to LP 101
cells induced a transient increase in IL-6 transcripts at 6
hours, followed by peak IL-6 secretion at 24 hours. To con-
firm increased IL-6 transcription and characterize its regula-
tion, LPlOl BMSCs were transiently transfected with full
length and deletion fragments of the IL-6 promoter linked to
the chloramphenicol acetyltransferase (CAT) reporter gene.
Transient transfection of LPlOl BMSCs with plasmid con-
E HAVE used B-cell antigens and immunofluores-
cence techniques to characterize secretory B-cell
malignancies and to define the role of recombinant B-cell
growth factors in their gr0~th.I.~ Interleukin-4 (IL-4) has
been noted to inhibit the in vitro growth of tumor samples
from patients with multiple myeloma In contrast,
IL-6 is proposed to be either an autocrine or paracrine growth
factor for MM.”.h.10”4 Specifically, Kawano et alls postulate
an autocrine growth mechanism because (I) IL-6 induces in
vitro growth of freshly isolated MM cells; (2) MM cells
express the IL-6 receptor (IL-6R); (3) purified MM cells
produce IL-6; and (4) in vitro growth of MM cells is inhib-
ited by anti-IL-6 antibody. In support of an autocrine theory,
Freeman et all6 have shown that MM and plasma cell leuke-
mia cells express IL-6 mRNA. Hata et all7 found that tumor
cells from 45% of patients expressed IL-6 mRNA; IL-6R
transcripts were found in 68% of tumor specimens, also
consistent with an autocrine growth mechanism in some pa-
tients. In addition, some cell line data supports this view:
W
Frvm the Division of Hematologic Malignancies, Dana-Farber
Cancer Institute, Department of Medicine, Harvard Medical School,
Boston, MA: the Department of Medicine, Beth Israel Hospital, and
Harvard Medical School, Boston, MA; and the Department of Molec-
ular Pathology, Cancer Research Institute, Kanazawa University,
Japan.
Submitted July 5, 1995; accepted September 7, 1995.
Supported by National Institutes of Health Grant No. CA 50947
and the Kraft Family Research Fund.
Address reprint requests to Kenneth C. Anderson, MD, Division
of Hematologic Malignancies, Dana-Farber Cancer Institute. 44
Binney St, Boston, MA 02115.
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 oj Hematology.
0006-4971/96/8703-0032$3.00/0
1 l04
taining an intact NF-KB site showed a 6.8 ? 0.4-fold increase
in CAT activity triggered by IM-9 MM cell adhesion (n = 3,
P< .05). Transfection of LP 101 cells with plasmid containing
a single base pair deletion from the NF-KB binding motif
abolished the MM adhesion-induced increase in CAT activ-
ity, whereas transfection with plasmid containing three cop-
ies of synthetic NF-KB sequence resulted in an 8.1 f 0.7-fold
increase in CAT activity related to MM adhesion (n = 3, P <
.05). These data suggest that the NF-KB site is one of the
essential regulatory elements for MM cell adhesion-induced
IL-6 transcription in BMSCs. Electrophoretic mobility shift
assays confirmed the involvement of NF-KB activation in
regulating MM adhesion-induced IL-6 transcription i
BMSCs. Further characterization of the upstream events in
the signalling cascade regulating IL-6 may not only delineate
mechanisms of IL-6 regulation during paracrine MM cell
growth, but also provide new therapeutic strategies based
on interruption of IL-6 mediated tumor cell growth.
0 1996 by The American Society of Hematology.
in particular, MM cell lines have been described that are
responsive to and produce IL-6; their growth can be inhibited
by an antibody to IL-6 and/or IL-6 antisense oligonucleo-
tides.lX-22 Most recently, an IL-6 dependent human MM-
derived cell line as well as freshly isolated MM cells have
been triggered via their cell surface CD 40 to both secrete
IL-6 and to proliferate, suggesting the potential for induction
of IL-6-mediated autocrine growth in
Despite the above studies, an IL-6-mediated autocrine
growth mechanism in MM remains controversial, because
many studies have shown that bone marrow stromal cells
(BMSCs) are the major source of IL-6.”.12,’4 Furthermore,
although all human MM-derived cell lines express IL-6R
mRNA, only a subset express IL-6 mRNA. Our studies have
shown that freshly isolated MM cells or MM-derived cell
lines express functional IL-6Rs and proliferate in response
to exogenous IL-6; however, only a fraction of cells also
synthesize IL-6, compatible with an autocrine growth pat-
tern.’.‘ Multiple groups have shown that MM cells express
cell surface adhesion molecules, ie, CD29KDw49d (VLA-
4), VLA-5, CDl8KDlla (LFA-l), CD44, CD 21, MPC-I,
syndecan, which localize MM cells in marrow via specific
adherence to both marrow extracellular matrix proteins and
to BMSCS.’~”~ Most importantly, MM cell adhesion triggers
IL-6 secretion by normal or MM BMSCs and related IL-6-
mediated tumor cellThis process requires cell
to cell contact between tumor and BMSCs. Moreover, para-
formaldehyde fixation of the BMSCs before tumor cell adhe-
sion abrogates IL-6 secretion, suggesting that BMSCs are
the major source of IL-6-mediating paracrine tumor cell
growth.” To date, however, the mechanism whereby tumor
cell adhesion induced IL-6 expression in BMSCs is regulated
remains undefined.
In the present report, we studied the molecular mechanism
involved in the transcriptional activation of IL-6 gene in
BMSCs triggered by MM cell adhesion. Adhesion of MM
cell lines to BMSCs and BMSC lines resulted in significant
Blood, Vol 87, No 3 (February l), 1996: pp 1104-1112

INTERLEUKIN-6 REGULATION IN MULTIPLE MYELOMA 105
increases in IL-6 secretion by BMSCs. Paraformaldehyde
fixation of BMSCs before MM adhesion abrogated this re-
sponse, suggesting that BMSCs are the major source of IL-
6. Adherence of IM-9 MM cells to LP 101 or AA 101
BMSCs triggered a transient increase in IL-6 transcripts that
preceded maximal IL-6 secretion. Full-length or deletion
fragments of the IL-6 promoter linked to the chlorampheni-
col acetyltransferase (CAT) reporter gene were transfected
into BMSCs before MM cell adhesion: these experiments
indicated involvement of the NF-KB binding site in the in-
duction of IL-6 gene transcription. Finally, electrophoretic
mobility shift assays (EMSAs) showed increased NF-KB
binding activity in BMSCs after MM cell adhesion, and
antibodies against members of the reVNF-KB family revealed
the presence of p50, c-rel, and smaller quantities of p65 in
the complex induced by IM-9 MM cell adherence to LPlOl
BMSCs. These studies confirm a role for NF-KB activation
in regulation of IL-6 transcription triggered in BMSCs after
MM cell adhesion.
MATERIALS AND METHODS
Cells and cell culrure. The IM-9, ARH-77, HS Sultan, and U-
266 human MM-derived cell lines6 were obtained from American
Type Culture Collection (Rockville, MD). Cells were cultured in
RPMI-1640 containing 10% to 15% fetal bovine serum (FBS), 100
U/mL penicillin (pen), and 100 mg/mL streptomycin (strep)
(GIBCO, Grand Island, NY). The normal human BMSC lines LP101
and AAlOl were kindly provided by Dr Shin Aizawa (Tokyo Medi-
cal College) and cultured in Iscove’s Modified Dulbecco Medium
(IMDM), containing 10% FBS, and pen/strep. Assays of adhesion
of MM cell lines to BMSCs were done as previously described.’”.’”
Briefly, BMSCs were cultured (1 X lo5 cells/mL) for 24 hours to
obtain a confluent monolayer. After BMSCs formed a confluent
adherent layer, remaining nonadherent cells were washed three times
with Hanks’ Buffered Saline Solution. Myeloma cells (5 X lo5 cell/
mL) were then added directly to BMSCs. After incubation at 37°C
for 24 hours, the supernatants were collected, and remaining bound
cells were procured for either Northern blot analysis or CAT assays.
Measurernenr of IL-6 secretion. IL-6 levels in the supernatants
of BMSCs cultured in media or with adherent MM cell lines were
measured in a bioassay using the B9 IL-6 dependent hybridoma
cells, as previously described.’ Test samples were heat-inactivated
(56°C for 30 minutes) and sterile filtered before use. Triplicate test
samples were serially diluted in 96-well microtitre plates (100 pW
well) and 100 pL of B9 cells (5 X IO’ cellslwell) suspended in
IMDM containing 5% FBS, 5 X mercaptoethanol, and pen/
strep. Dilutions of recombinant IL-6 were included as a standard.
B9 cells were then cultured for 72 hours, with the addition of tritiated
thymidine (7.4 kBq/well) for the last 4 hours of culture. Thymidine
uptake by the B9 cells was determined as described7; the lower limit
of detection of IL-6 was 0.5 pg/mL.
IL-6 was also measured using an enzyme-linked immunosorbent
assay (ELISA; Endogen, Cambridge, MA), as previously de-
scribed.’6 Serial dilutions (100 pL) of test sample supernatants were
added in duplicate to 96 well plates (Costar, Cambridge, MA) coated
with IgGl anti-IL-6 antibody (murine IgGI; TORAY, Kanagawa,
Japan). Biotinylated detector anti-IL-6 monoclonal antibody (MoAb)
(TORAY) was next added and developed with streptavidin (Amer-
sham, Arlington Heights, IL). IL-6 level in each supernatant was
determined by comparison with a standard curve. The level of detec-
tion of IL-6 was 1 ng/mL.
Measurement of IL-I and IL-8 secretion. The IL-I and IL-8
levels in culture supernatants were measured using ELISA kits (En-
dogen). The minimal detection limits were 50 pg/mL and 1 pg/mL
for IL-I and IL-8, respectively.
RNA isolarion and Norrhern blotring. Total cellular RNA was
isolated by a modification of the guanidine-isothiocyanate technique,
as previously described.’6 Total cellular RNA (20 pg/lane) was sub-
jected to electrophoresis in a 1% agarose12.2 mom formaldehyde
gel, transferred to nitrocellulose paper, and hybridized to one of the
following ’*P-labeled DNA probes: (l) the pAl plasmid containing
a 2.0-kb Psr I insert of the chicken &actin gene’7; and (2) the BanII-
Taq I fragment containing nucleotides 215 to 657 of a full-length
human IL-6 cDNA.‘ The hybridizations were performed for 16 to
24 hours at 42°C in 50% (voUvol) formamide, 2 X SSC (SSC: 0.15
moVL sodium chloride, 0.015 mol/L sodium citrate), 1 X Denhardt’s
solution, 0.1% (wthol) sodium dodecyl sulfate, and 200 pg/mL
salmon sperm DNA. Filters were washed and exposed to Kodak
X-Omat XAR film (Eastman Kodak, Rochester, NY) using an inten-
sifying screen. The autoradiograms were scanned using an LKB
produkter (Bromma, Sweden) Ultrascan XL laser densitometer and
analysed with the Gelscan XL software package (LKB). Signal inten-
sity was determined in a linear range and normalized to that for
actin.
CAT assays. The various IL-6 CAT constructs were prepared as
previously described.’x Briefly, the parental BAMHIIXhoI fragment
of IL-6 promoter was subjected to restriction enzyme digestion with
Mva I (-630) and Hho I (-225). 5’ deletional constructs were
prepared from the pIL-6 CAT 225. The 5’ deletions were constructed
by BAL 31 resection of pIL-6 CAT 225 from the Xba I site and
addition of BAMHI linkers. The resulting IL-6 BarnH1-Xho I frag-
ments were ligated to pIL-6 CAT 3 digested with BarnHI and Xho
I to generate 5’ deletions (see Fig 3). For cloning, complementary
oligonucleotides were treated with kinase, annealed, and inserted at
the BamHI site of the plasmid pIL-6 CAT 59, creating pIL-6 CAT
The indicated plasmids (30 kg) (see Fig 3) were transfected into
LP101 or AA101 BMSCs using electroporation, as previously de-
scribed.’“ Briefly, l .5 X IO7 BMSCs were procured and washed with
phosphate-buffered saline. The cells were resuspended in 800 pL of
media without serum, and incubated on ice for 10 minutes. The cells
were pulsed with 240 V, 960 mF (Biorad Gene Pulser, Richmond,
CA) using the standard vendor’s protocol; incubated further on ice
for 10 minutes; resuspended in complete media; and then incubated
at 37°C. Twenty-four hours after transfection, IM-9 MM cells were
added directly to the transfected confluent LPlOl or AAlOl BMSCs.
The transfected BMSCs were also cultured alone as a control. After
24 hours, either BMSCs alone or BMSCs and IM-9 MM cells were
procured and lysed by three cycles of freezing and thawing in 0.25
mom Tris-HC1 (pH 7.8) and 1 mmol/L phenylmethylsulfonyl fluo-
ride. To assay for CAT activity, equal amounts of the cell extracts
were incubated with 0.025 pCi [I4C] chloramphenicol (Amersham;
57 uCi/mmol), 0.15 mom Tris-HC1 (pH 7.8), and 0.4 mmol/L ace-
tyl-coA (Sigma, St Louis, MO) for 4 hours at 37°C. The enzyme
assay was terminated by the addition of ethyl acetate. The organic
layer containing the acetylated [I4C] chloramphenicol was separated
by thin layer chromatography using chlorofonmnethanol (95%/5%;
voUvol). Following autoradiography, both acetylated and unace-
tylated forms of [‘JC]chloramphenicol were cut from the plates,
and the conversion of chloramphenicol to the acetylated form was
calculated by measurement of radioactivity in a p-scintillation
counter, as previously described.”
The IM-9 MM cells were also transiently transfected with pIL-
6CAT 630 and pIL-6CAT ~~/NF-KB X 3 constructs. After 24 hours
of culture, the transfected IM-9 MM cells were adhered to LP101
BMSCs, and the extracts analysed for CAT activity.
Cotransfections were also performed with a luciferase gene re-
porter construct (pGL2) as an internal control plasmid, as in previous
~~/NF-KB X 3.