1. No category

From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
1996 87: 1928-1938
Transforming growth factor-beta1: differential effects on multiple
myeloma versus normal B cells
M Urashima, A Ogata, D Chauhan, M Hatziyanni, MB Vidriales, DA Dedera, RL Schlossman and
KC Anderson
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Transforming Growth Factor-P 1: Differential Effects on Multiple
Myeloma Versus Normal B Cells
By Mitsuyoshi Urashima, Atsushi Ogata, Dharminder Chauhan, Maria Hatziyanni, Maria
Douglas A. Dedera, Robert L. Schlossman, and Kenneth C. Anderson
B. Vidriales,
Interleukin-6 (IL-6). a product of bone marrow stromal cells
(BMSCs),is a growth factor for multiple myeloma (MM)cells.
Transforming growth factor-pl (TGF-PI) is also produced by
BMSCs and can regulate IL-6 secretion by several tissues,
including BMSCs. The present study was designed t o characterize in vitro tumor growth regulation by TGF-p1 in MM.
Sorted CD38+CD45RA- MM cells secreted significantly more
TGF-p1 (8.2 f 2.0 ng/mL) than peripheral blood mononuclear cells ( P < .001), splenic B cells (P< .001), and CD40
ligand (CD40L) pretreated B cells ( P < .05). TGF-/31secretion
by MM-BMMCs (3.8 k 0.9 ng/mL) was significantly greater
than by N-BMMCs (1.2 f 0.1 ng/mL, P < .001). MM-BMSCs
also secreted significantly more TGF-PI (6.6 f 2.5 ng/mL, n
= 11) than N-BMSCs (4.4 f 0.6 ng/mL, P < .02, n = I O ) and
N-BMSC lines (3.9 f 0.2 ng/mL, P < .02, n = 6). TGF-p1
secretion was correlated with IL-6 secretion in MM-BMSCs.
Anti-TGF-pl monoclonal antibody both blocked IL-6 secretion by BMSCs and inhibited the increments in IL-6 secretion
by BMSCs induced by MM cell adhesion. Moreover, exogenous TGF-PI upregulated IL-6 secretion by MM-BMSCs, normal BMSCs, and CD38+ CD45RA- MM cells, as well as tumor
cell proliferation. This is in contrast t o the inhibitory effect
of TGF-/?I on proliferation and Ig secretion of normal splenic
B cells. Finally, retinoblastoma proteins (pRB) are constitutively phosphorylated in MM cells; TGF-/31 either did not
alter or increased pRB phosphorylation. pRB are dephosphorylated in splenic B cells and phosphorylated in CD40L
triggered B cells; in contrast t o its effects on MM cells, TGFp l decreased phosphorylation of pRB in CD40L treated B
cells. These results suggest that TGF-p1 is produced in MM
by both tumor cells and BMSCs and can trigger IL-6 secretion
by both MM cells and BMSCs, with related tumor cell
growth. Moreover, MM cell growth may be enhanced by
resistance of tumor cells t o the inhibitory effects of TGF-p1
on normal B-cell proliferation and l g secretion.
0 1996 by The American Society of Hematology.
I
locyte macrophage-colony stimulating factor (GM-CSF),12
and most recently IL-l0,l3 have been suggested to augment
the proliferation of MM cells and derived cell lines. Our
preliminary studies show that IL-1 and tumor necrosis factora (TNF) do not mediate the MM cell adhesion-related upregulation of IL-6 in BMSCs. However, to date the role of
other cytokines triggering MM cell growth either directly,
or indirectly by regulating IL-6, has not been characterized.
Transforming growth factor-p1 (TGF-P1) was originally
purified from platelets and found to mediate morphologic
transformation as well as anchorage-independent growth of
fibroblasts.I4It has pleitropic biologic effects, including suppression of hematopoiesis by an action antagonistic to stem
cell factor,I5 as well as inhibition of wound healing and the
immune response. The BM failure that occurs in B-chronic
lymphocytic leukemia (B-CLL), for example, has been attributed to excessive production of TGF Pl by BMSCS.’~
In
wound healing, Battegay et all’ have postulated a bimodal
response to TGF Pl, because TGF-P1 at low concentrations
upregulates and at high concentrations downregulates proliferation of fibroblasts. Acute limited injury may therefore be
accompanied by only a transient increase in TGF-P 1, without sustained increases and related fibrosis. TGF-P1 also
inhibits the normal immune response: it suppresses normal
B-cell proliferation and immunoglobulin (Ig) producti~n.’~.’~
Finally, TGF-P1 may play a role in the pathophysiology of
malignancies because many kinds of cancer cells produce
TGF ,B 1 ,*’ which may regulate tumor progression by stimulating stroma formation and angiogenesis. For example, excessive production of TGF-PI may serve as an endogenous
growth inhibitor for B-CLL cells and account for the slow
progression of the malignant process in vivo.” Morever.
lack of response to TGF-P1 may also contribute to disease
pathology. For example, loss or alteration of TGF-P 1 high
affinity receptors results in continuous proliferation of Epstein-Barr virus (EBV) transformed B cells.”.23 In some
cases, TGF-P 1 does not significantly inhibit the proliferation
NTERLEUKIN-6 (IL-6) has been proposed as an autocrine and/or paracrine growth factor for multiple myeloma (MM).’ Reports that freshly isolated MM cells and
derived cell lines express IL-6 receptors and specifically
proliferate in response to IL-6, express IL-6 mRNA, are
intracytoplasmic IL-6 positive, and secrete IL-6 suggest an
autocrine growth mechani~m.’.~Recent observations that
MM cells and cell lines can be triggered via CD40 ligand
(CD40L) to secrete IL-6 and proliferate also support this
vie^.^,^ However, many studies have also suggested that
bone marrow stromal cells (BMSCs) are the major source
of IL-6, consistent with a paracrine growth mechani~m.~
Most recently, several studies confirm upregulation of IL-6
gene transcription and secretion by BMSCs related either to
MM cell adhesions%9
or triggering via CD4OL.’ MM cell
adhesion-induced IL-6 expression in BMSCs, for example,
involves activation of NF-KB.loAlthough further studies of
IL-6 gene regulation in both MM cells and BMSCs are ongoing, it is unlikely that IL-6 is the sole growth factor for MM,
and the role of other cytokines produced in the marrow
microenvironment in the pathophysiology of MM has been
examined in preliminary studies. For example, IL- 1,I’ granu-
From the Division of Hematologic Malignancies, Dana-Farber
Cancer Institute; and the Department of Medicine, Harvard Medical
School, Boston, MA.
Submitted June 12, 1995; accepted October 4, 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, DanaFarber Cancer Institute, 44 Binney St, Boston, MA 02215.
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.
0006-4971/96/%705-0001$3.00/0
1928
Blood, Vol 87, No 5 (March I ) , 1996:pp 1928-1938
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
TGF-01 IN MULTIPLE MYELOMA
of malignant cell lines, ie, HL-60 or squamous cell carci-
noma line^.^^-*^ This decreased responsiveness to the inhibitory effects of TGF 01 facilitates their long-term proliferation and malignant transformation; once transformed, cells
lose any inhibitory response to TGF-P1 permitting their unregulated growth.
In the present report, we characterized in vitro tumor
growth regulation by TGF Pl in MM. TGF-P1 was secreted
in greater amounts by CD38TD45RK MM cells than normal peripheral blood mononuclear cells (PBMCs), splenic
B cells, or CD40 ligand (CD4OL) pretreated B cells. Similarly MM-BMMCs and BMSCs secreted more TGF-P1 than
normal (N) BMMCs and BMSCs. TGF-P1 secretion correlated with IL-6 secretion by MM; anti-TGF-P monoclonal
antibody (MoAb) both blocked IL-6 secretion by BMSCs
and inhibited the increments in IL-6 secretion by BMSCs
induced by MM cell adhesion. Moreover, exogenous TGFPl increased IL-6 secretion by MM-BMSCs, N-BMSCs, and
M M cells, and related tumor cell proliferation; in contrast,
TGF-P1 inhibited proliferation and Ig secretion of normal
B cells. Finally, TGF-P1 did not affect or upregulated the
constitutive phosphorylation of retinoblastoma protein
(pRB) in M M cells, but decreased phosphorylation of pRB
in CD40L-triggered normal B cells. These results suggest
that TGF-01 secretion by MM cells and BMSCs can trigger
IL-6 secretion by MM cells and BMSCs, with resultant autocrine and paracrine IL-6-mediated tumor cell growth, respectively. Moreover, the resistance of M M cells to the downregulatory effects of TGF-P1 observed on normal B cells
may facilitate autocrine and paracrine tumor cell growth.
Our data, coupled with reports that plasmacytoma-bearing
mice develop marked impairment of B-cell function attributable to up to 30-fold increments in serum levels of TGFP1,27 suggest that TGF-P1 may also contribute to the impairment of humoral immunity characteristic of MM.28
MATERIALS AND METHODS
M M cells and MM-derived cell lines. Fresh BM was obtained
from seven patients with MM, and mononuclear (MCs) were isolated
by Ficoll-Hypaque (FH; Pharmacia Biotech, Uppsala, Sweden) density gradient centrifugation, washed, and resuspended in media.
CD38+CD45RK MM cells within BMMCs were isolated using an
Epics C Cell Sorter (Coulter Electronics, Hialeah, FL) after incubation with HB7 (anti-CD38) MoAb-biotin-streptavidin and 2H4 (antiCD45RA) MoAb-fluorescein isothiocyanate on ice. The ARH-77,
IM-9, RPMI-8226, and U-266 human MM-derived cell lines were
obtained from American Type Culture Collection (Rockville, MD).
Our previous studies showed that ARH-77 and IM-9 cells were
infected with EBV, in contrast to RPMI-8226 and U-266 cells that
were uninfected.2CD38TD45RK MM cells and MM derived cell
lines were cultured in RPMI-1640 media (Sigma Chemical CO,St
Louis, MO) containing 10% fetal bovine serum (FBS),L-glutamine
(L-glu) (GIBCO, Grand Island, NY), 100 U/mL penicillin (pen),
and 100 U/mL streptomycin (strep) (GIBCO). The JKB (acute
lymphoblastic leukemia: ALL),29 Ramos (non-Hodgkin’s
lymphoma), U937 (acute myelomonocytic leukemia), and CMK
(acute megakaryocytic leukemia) cell lines were cultured as described for the MM cells/MM-derived cell lines.
M M BMSCs and normal BMSCs/BMSC-derived cell lines. BM
specimens were obtained from patients with MM (n = 11) and from
1929
healthy donors (n = 10). MCs separated by FH density sedimentation
were either used fresh or were used to establish long-term BM
cultures, as described by Gartner and Kaplan” with a slight modification.’ BMMCs were suspended in growth medium containing Iscove’s modified Dulbecco medium (IMDM) (Sigma), 20% FBS,Lglu, and penlstrep in 162-cm2flasks (Costar, Cambridge, MA). Cells
were incubated at 37°C in a CO2 incubator, with fresh media added
weekly. After culture for 2 to 3 months, the cells (predominantly of
fibroblast morphology) were procured in Hanks’ Buffered Saline
Solution containing 0.25% trypsin and 0.02% EDTA (GIBCO),
washed, collected by centrifugation, and used as MM and noma1
(N) BMSCs. The normal human BMSC-derived cell lines LPlOl
and LS501 were kindly provided by Dr Shin Aizawa (Tokyo Medical
College, Tokyo, Japan)3’ and cultured in IMDM (Sigma) containing
10% FBS, L-glu, and penlstrep.
Preparation of PBMCs, splenic B cells, and CD40 ligand pretreated B cells. PBMCs (n = 5) were collected by centrifugation
and FH density sedimentation of heparinized blood from normal
donors. Normal spleen (n = 7) was obtained from operative specimens of patients not known to have any systemic or malignant
diseases. Single cell suspensions from spleen were prepared by extrusion through sterile stainless steel mesh. Splenic MCs were isolated by centrifugation on FH density sedimentation, and adherent
cells removed from MCs by double adherence to plastic petri dishes
for 1 hour at 37”C, as previously described.32Further enrichment
for B cells in spleen was done by rosetting with sheep red blood
cells to deplete T cells. B cells were cultured in RPMI-1640 containing 10% FBS, L-glu, and penlstrep. Splenic B cells [Bl:
>90%(+ +), B2: >85%(++), CD38: negative, PCA-1: negative] (n
= 3) were also cultured with supematant from CD40 ligand (CD40L)
transfected COS cells (titer of 1:4) for 10 days to trigger their differentiation [Bl: s%(+),B2: negative, CD38: 85%(++), PCA-1:
47%(+ +)I, as in prior studies33;these CWOL pretreated cells were
washed 3 times with HBSS before use in experiments.
M M cell to BMSC adhesion assay. MM cell to BMSC adhesion
assays were performed as previously de~cribed.~
In brief, BMSCs
(1 X 10s cells/mL) (n = 3) were cultured in 6 well plates (Falcon,
Lincoln, NJ) for 2 2 4 hours. After these cells formed >80% adherent
layer, assessed by phase-contrast microscopy, nonadherent cells
were removed by washing. MM derived cell lines (2 x 106/2 mL)
were then directly added to the BMSCs for 24 hours. In some experiments, anti-TGF p1.2.3 MoAb (100 pglmL) (Genzyme, Cambridge,
MA) was added to neutralize the effects of endogenous TGF p;
unreactive murine IgGl MoAb (Coulter) served as a control. In
addition, in some experiments either MM cells or BMSCs were fixed
with 1% paraformaldehyde before adhesion assays to delineate the
source of IL-6 secretion into culture supematants.
Measurement of ZL-6 secretion. IL-6 levels in culture supematants were measured using an Enzyme linked immunosorbent assay
(ELISA), as previously described5.I0: ( I ) 96-well plates (Costar,
Cambridge, MA) were coated with IG61 anti-IL-6 MoAb (murine
IgG1, Toray, Ohtsu, Japan); (2) wells were saturated with calfskin
gelatin (BioRad)-phosphate buffered-saline (PBS) for 1 hour; (3)
serial dilutions (100 pL) of test sample supematants were added in
duplicate to plates; and (4) biotinylated detector anti-IL-6 MoAb
(Genetics Institute, Cambridge, MA) was next added and developed
with avidin-peroxidase (Amersham, Arlington Heights, IL), tetramethylbenzidine (Sigma), and 30% peroxide (Sigma). IL-6 levels in
each supematant were determined by comparison with a standard
curve. The level of detection of IL-6 was linear in the range of 10
pg/mL to 100 ng/mL. These MoAbs can detect only human IL-6.
Measurement of TGF-PI secretion. TGF-p1 concentration was
measured using a TGF-PI ELISA kit (Genzyme, Cambridge, MA).
To activate TGF-01, samples and standards were diluted and acidi-
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
1930
URASHIMA ET A t
fied using hydrochloride (HCL) for 1 hour before assay. To test
whether there was any preactivated TGF-PI in cell culture supematants, 10 samples were assayed with and without acidification. The
standard curve was linear in the range of 0.10 to 4.0 ng/mL.
Solid phase ELISA for IgC. The quantitative ELISA used to
measure IgG secreted into culture supernatants was done in a manner
similar to that for IL-6 described above. Goat-antihuman polyvalent
Ig Ab (Sigma) was used as the coating Ab, and biotin-conjugated
goat-antihuman IgG Abs (Sigma) were used as detection Abs. The
level of detection of IgG was 1 ng/mL.
Assays of DNA synthesis. DNA synthesis was measured as previously described.' Cells (2 X 10'/well) in RPMI-1640 media supplemented with 10% FBS, L-glu, and streplpen were incubated in 96
well plates, and DNA synthesis was measured at day 4 of culture
using 'H-thymidine ['H-TdR] uptake. Cells were pulsed with 'HTdR during the last 18 hours of incubation (1 pCi/well), procured
onto glass filters with the aid of an automatic cell harvester (Cambridge Technology, Cambridge, MA), and counted on a liquid scintillation counter (Packard Tri-Carb 4530, Downers Grove, IL). Proliferation was defined by the stimulation index (SI): 'H-TdR uptake
of sample with both CD40L and TGF-PlI'H-TdR uptake of control
sample with CD40L (1:4 dilution) alone (see Fig 7); or as 'H-TdR
uptake of sample with TGF-PI (20 nglmL)/'H-TdR uptake of sample
in media alone (see Fig 8).
Immunoprecipitation. RPMI-8226 MM cells, patient MM cells,
normal splenic B cells, CD4OL-triggered B cells, and JKB-1 ALL
cells (1 x IO' cells/mL) were cultured in the presence or absence of
TGF-PI (20 ng/mL) for 2 hours in RPMI-1640 media. CD4OL-triggered B cells were prepared by culture of splenic B cells with CD40L
supematants for 3 days. All cells were washed three times with PBS
including NasVOl and lysed for 30 minutes at 4°C in buffer: 10 mmoV
L Tris-HCL buffer (pH 7.6), 150 mmoW NaCI, 0.5% Noidet P-40,
5 mmoW EDTA, 1 mmoVL phenyl methyl sulfonyl fluoride, 200
pmollL Na'VO,, aprotinin, and 1 mmoVL NaF. Anti-pRB Ab (Oncogene Science, Uniondale, NY) was added for an overnight incubation
at 4°C. RB protein (pRB) was collected using rabbit antimouse IgG
pretreated protein A sepharose. Aliquots of each lysate were analyzed
by 5.O% sodium dodecyl sulfate-polyacrylamide gel electrophoresis
under reducing conditions. Protein was transferred ovemight onto
nitrocellulose membrane, and nonspecific binding was blocked by
incubation of the membrane with 5% skim milk. The membrane was
probed with MoAb against pRB and detected using the enhanced
chemiluminescence system (Amersham).
Reagents. Serial dilutions of TGF pl (Kirin-Brewrey CO Ltd,
Tokyo, Japan) were added to cultures of B cells, CD40L pretreated
B cells or MM cells, and the effects on IL-6 secretion were assayed.
Soluble CD40L and soluble CD72 were kindly provided by Dr Diane
Hollenbaugh (Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, WA). COS cells were transfected with CD40L or CD72,
as previously de~cribed.",'~One day after transfection, cell culture
medium was changed to Dulbecco's modified Eagle's medium containing 2% FBS, and supematants were procured 8 days later. Supernatants from CD4OL COS transfectants were used at a titer of 1.4,
which triggered a 9.0 ? 0.9-fold increment in proliferation of normal
splenic B cells. Supematants from CD72 COS transfectants were
used as a control and had no effect on DNA synthesis or IgG secretion by B cells, MM cells, or MM derived cell lines. IL-6 was
not detectable by ELISA in supematants of CD4OL or CD72 COS
transfectants, or in FBS. IL-10 (Genzyme) and CD40L were used
to stimulate Ig secretion by splenic B cells, CD40L pretreated B
cells, CD38'CD45RA- MM cells, and MM derived cell lines.
RESULTS
Secretion of TGF-PI by CD38+CD45Rt- MM cells, Blineage malignant cell lines, B cells, N-BMMCs, MM-
12
-
10
0
-
0:
0
8'
6'
0
42'
-&
@-
to
Fig 1. Secretion of TGF-p1 by PBMCs, splenic B cells, CD40 ligand
pretreated B cells, MM cells, N-BMMCs, and MM-BMMCs. PBMCs
were isolated from healthy voluntmn (n = 5) and B d l s 1>90%
CD20(++), >85% CD21(++), CD38-. PCAl-I were isolated from
spleen (n = 7). Splenic B cells (n = 3) were cultured with C W L (1:4
dilution) for 10 days to induce differentiation I8% CD20(+), CD21-,
85% CD38(++), 47% PCAl(++)l. These CD40L pretreated B cells were
thoroughly washed before subsequent cultures. CD38+CD45RA- MM
cells were separated by duel fluorescence cell sorting (n = 61. MMBMMCs (n = 6) and N-BMMCs In = 6 ) were isolated by FH density
centrifugation. All cells were cultured at 1 x 10'lmL in 10% FBS RPMI1640, and supernatants at day 7 were assayed for TGF-pl by ELISA.
Two sample ttests were used to compare TGF 8 1 secretion by these
cell populations.
BMMCs, MM-BMSCs, and N-BMSCs/derived cell lines.
Normal PBMCs (n = 5 ) , splenic B cells (n = 7), CD40L
pretreated B cells (n = 3), CD38+CD45RA- MM cells (n
= 6), N-BMMCs (n = 6), and MM-BMMCs (n = 6 ) were
cultured for 7 days, and TGF-P1 concentrations in culture
supematants were quantitated by ELISA (Fig 1). Normal
PBMCs secreted 0.9 t 0.2 ng/mL of TGF-P1. Splenic B
cells secreted 3.7 5 0.2 ng/mL TGF PI, significantly more
than PBMCs (P< ,001). CD40L pretreated B cells secreted
more TGF-P1 (4.7 % 0.5 ng/mL) than splenic B cells (P <
.05). Sorted CD38TD45RA- MM cells secreted significantly more TGF 01 (8.2 2 2.0 ng/mL) than CD40L pretreated B cells ( P < .05), splenic B cells ( P < .001), and
PBMCs (P < .001). TGF-Dl secretion by MM-BMMCs
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
I
TGF-B1 IN MULTIPLE MYELOMA
1931
:
:
10
4
8
:
00
ob
0
0
0
0
02
I
2E
I
I
.-3
QE
I
9
I
z
z
Fig 2. Secretion of TGF p l by MM-BMSCs, N-BMSCs, and N-BMSC
lines. BMSCs from healthy donors (n = 10) and patients with MM (n
= 11). as well as the LPlOl (n = 3) and LS501 (n = 3) BMSC lines,
were cultured at 1 x ld cellslml in IMDM, and supernatants at 24
hours were assayed for TGF-p1 by ELISA. Two sample t-tests were
used to compare TGF-/31 secretion by these cell types.
(3.8 t 0.9 ng/mL) was significantly greater than that of NBMMCs (1.2 t 0.1 ng/mL, P < .001).
TGF-Pl secretion in supernatants from 24-hour cultures
of tumor cell lines derived from MM and other hematologic
malignancies was also measured using ELISA. TGF-01 secretion by MM cell lines was as follows: ARH-77 cells: 6.3
t 1.2 ng/mL; IM-9 cells: 5.9 t 1.8 ng/mL; RPMI-8226
cells: 6.3 t 1.5 ng/mL; and U-266 cells: 4.3 +. 1.4 ng/mL).
The mean TGF Pl secretion (5.7 t 1.6 ng/mL) was not
significantly different from the JKB ALL (5.8 t 0.8 ng/mL)
or Ramos B-cell lymphoma (5.5 t 0.7 ng/mL) cell lines.
However, TGF Pl secretion by the U-937 myelomonocytic
leukemia (0.9 t 0.3 ng/mL) and the CMK megakaryocytic
leukemia (1.7 t 0.6 ng/mL) cell lines was significantly lower
than that by malignant cell lines of B lineage ( P < .001).
MM-BMSCs, N-BMSCs, and N-BMSC derived cell lines
(LP101 and LS501) were cultured for 24 hours, and TGFPl concentration in supematants also quantitated by ELISA
(Fig 2). There was 'a broad range of TGF-PI secretion by
MM-BMSCs; however, MM-BMSCs secreted significantly
more TGF-P1 (6.6 ? 2.5 ng/mL, n = 11) than N-BMSCs
(4.4 2 0.6 ng/mL, P < .02, n = IO) and N-BMSC lines (3.9
t 0.2 ng/mL, P < .02, n = 6). There was no significant
difference in TGF-01 secretion by N-BMSCs and N-BMSC
derived cell lines.
In these experiments, all normal and MM cells, as well
as N-BMMCs, MM-BMMCs, N-BMSCs, and MM-BMSCs
were cultured in media with 10% FBS. This media contained
1.7 t 0.2 ng/mL (n = 3) of immunoreactive TGF-P1, which
is significantly greater than measured in supematants of
PBMCs ( P < .02), N-BMMCs ( P < .02), and U-937 cells
(P < .OS). Sample measurements were therefore not adjusted
for the presence of TGF-P1 in FBS. To determine whether
preactivated TGF-Dl was present in culture supematants, 10
samples were assayed with and without acidification. TGFPl (0.2 to 0.4 ng/mL) was detected in nonacidfied samples
only when the TGF-01 was 23.8 ng/mL in corresponding
acidified samples. In supernatants from CD40L or CD72
COS transfectants, 3.5 t 0.2 ng/mL of TGF-PI was measured after acidification, but no immunoreactive TGF-Pl
was detected without acidification.
Relationship between TGF-PI and IL-6 secretion by NBMSCs, MM-BMSCs, CD38'CD45Rt- MM cells, and B
cells. The relationship between TGF-D1 and IL-6 secretion
in supernatants of 24-hour cultures of MM-BMSCs and NBMSCs was next examined (Fig 3). TGF-P1 secretion by
N-BMSCs (4.4 +. 0.6 ng/mL) was significantly lower than
by MM-BMSCs (6.6 t 2.5 ng/mL) ( P < .02). IL-6 secretion
by MM-BMSCs (24.5 ? 25.6 ng/mL, n = 11) was significantly greater (P < .01) than that of N-BMSCs (2.0 t 1.2
ng/mL, n = 10). There was a correlation between TGF-81
secretion and IL-6 secretion by MM-BMSCs: Y = 8.8X 31.3 ng/mL, in which X = TGF-01 concentration, Y =
IL-6 concentration, and coefficient = 0.85, P < .02. No
correlation in secretion of these cytokines by N-BMSCs was
observed.
To determine whether TGF-0 1 stimulates IL-6 secretion
from BMSCs, IL-6 concentration was measured in 24-hour
cultures of N-BMSCs in the presence or absence of anti-
so-
=
8
2
60-
v
0
e
0
0
2
TGF p1
4
Secretion
6
8
10
(ng/ml)
Fig 3. Relationship between TGF-p1 and 11-6 secretion by NBMSCs and MM-BMSCs. N-BMSCs (n = 10) (01and MM-BMSCs (n
= 11) (0)were cultured at 1 x lo6 cellslmL in 20% IMDM, and supernatants at 24 hours assayed for TGF-Bl and IL-6 by EUSA. Pearson's
correlation coefficient was used to define the relationship between
TGF-pl and IL-6 secretion by MM-BMSCs.
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
URASHIMA ET AL
Fig 4. Effects of anti-TGF-p MoAb on 11-6 secretion triggered by
M M cell t o BMSC adhesion. N-BMSCs (1 x lo5 cellslmL) were cultured in media (m) or withARH-77 (B),IM-9 (m), RPMI-8226 (B),and
U-266 (0)M M cell lines (2 x lo6 cellslml) for 24 hours. Anti-TGF-p
MoAb (100 pg/mL) or control unreactive isomatched murine lgGl
MoAb were added t o some cultures. In addition, either BMSCs or
M M cells were fixed with1% paraformaldehyde before adhesion cultures t o delineate the source of IL-6 secretion into culture supernatants. 11-6 concentration in culture supernatants was analyzed by
ELISA. Data shown are mean standard deviation (SDI from three
independent experiments. Two sample t-tests wereused t o compare
IL-6 secretion by these celltypes.
*
TGF-P1 MoAb (100 pg/mL) (Fig 4). Anti-TGF-P MoAb,
but notcontrol isotype identical unreactive MoAb, decreased
IL-6 secretion to 47% of levels secreted by N-BMSCs in
media alone ( P < .005, n = 3). Adhesion of ARH-77, IM9, RPMI-8226, and U-266 MM cell lines to BMSCs increased IL-6 secretion 3.0-fold, 3.2-fold, 2.8-fold, and 1.8fold, respectively. The addition of anti-TGF-P MoAb during
MM cellto BMSC adhesion assays abrogated IL-6 secretion:
61 % decrease for ARH-77 cells, P < .05; 81% decrease for
IM-9 cells, P < .05; 87% decrease for RPMI-8226 cells, P
< .05; and 66% decrease for U-266 cells, P < .02. Control
MoAb did not affect IL-6 secretion induced by MM cell to
BMSC adhesion. Fixation of BMSCs withparaformaldehyde
before adhesion of MM cell lines abrogated IL-6 secretion
( P < .001). In contrast, paraformaldehyde fixation of MM
cell lines before their adhesion to BMSCs inhibited IL-6
secretion (55% decrease for ARH-77, P < .05; 59% decrease
for IM-9, P < .05;59% decrease for RPMI-8226, P < .005;
and 59% decrease for U-266, P < .05) to levels secreted by
BMSCs in media alone. IL-6 secretion was not detectable
in cultures of MM cell lines in media alone.
TGF-P1 was next added to N-BMSCs, N-BMSC derived
cell lines, and MM-BMSCs,and effect on IL-6 secretion
was determined (Fig 5). N-BMSCs, N-BMSC derived cell
lines (LPIOI and LS501), andMM-BMSCs(n = 3) were
cultured for 24hours with serial dilutions of exogenous TGFPI, and IL-6 concentration in the culture supernatants was
analyzed by ELISA. TGF-81 upregulated IL-6 secretion by
N-BMSCs, LP101 BMSCs, LS501 BMSCs, and
MMBMSCs in geometrical progression up to 11.6-fold ( P <
.Ol), 4.0-fold ( P < .Ol), and 6.1-fold ( P < .Ol), and 10.1fold ( P < .001), respectively, at 80 ng/mL.
Effects of serial dilutions of TGF-P1 on IL-6 secretion by
CD38'45RA- MM cells and splenic B cells were investigated in a similar fashion (Fig 6). TGF P1 at concentrations
of >5 ng/mL significantly increased IL-6 secretion by MM
cells ( P < .05, n = 3). In contrast, TGF-81 decreased IL-6
secretion by splenic B cells ( P < .05, n = 3). The decrease
in IL-6 secretion was temporally associated with inhibitory
effects of TGF-PI on B cell proliferation. TGF-PI had no
significant effects on IL-6 secretion by MM cell lines (data
not shown).
Effects of TGF-PI on proliferation of B cells, MM cells,
and MM-derived cell lines. Effects of TGF-01 on CD40Ltriggered DNA synthesis of B cells (n = 5). MM cells (n =
5), and MM derived cell lines were analyzed by'H-TdR
uptake (Fig 7). CD40L induced proliferation of B cells (SI
9.0 ? 0.9) and CD38TD45"M
cells (SI 5.8 2 3.8), but
did not significantly alter proliferation ofARH-77,IM-9,
RPMI-8226, and U-266 MM cell lines. Serial dilutions of
TGF-PI were added with CD40L, and effect on 'H-TdR
uptake by B cells was measured. B-cell proliferation stimulated by CD40L was blocked by 50% and 76% at 0.1 ng/
mL and 20 ng/mL of TGF-P1, respectively.
Proliferation of CD38TD45RA- tumor cells from patients MM1 and MM2 cultured with 20 ng/mL of TGF-01
was 62% and 54%, respectively, of proliferation notedin
the presence of CD4OL alone. In contrast, DNA synthesis
-v-
r.
_"..""""""
\"W
"",
Fig 5. Effects of TGF-p1 on 11-6 secretion by N-BMSCslderived
cell lines andMM-BMSCs. Direct effects ofexogenous TGF-p1 on IL6 secretion by N-BMSCs (01,LP101 ( 0 1 and LS501 ( 0 )N-BMSC lines,
as well as MM-BMSCs (AI were investigated. Serial dilutions of TGF
p1 (0 t o 80 ng/mL) were added t o cultures of BMSCs I1 x 10' cells/
mL), and supernatants at 24 hours were assayed for IL-6 by ELISA.
Data shown are mean
SD from three independent experiments
and werecompared using two sample t-tests.
*
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
TGF-01 IN MULTIPLE MYELOMA
1933
R
0
.I
1
TGF p 1 Concentration
10
100
(ng/ml)
Fig 6. Effects of TGF-pl on IL-6 secretion by MM cells and B cells.
The direct effect of exogenous TGF-p1 on IL-6 secretion by splenic B
cells In = 3) (0)and CD38+CD45RA-MM cells (n = 3) (0)was investigated. Serial dilutions of TGF p1(0 to 80 ng/mL) were added to either
MM or B cell cultures (1 x 10' cells/mL), and supernatants at 7 days
were assayed for IL-6 by EUSA. Data shown are mean 2 SD from
three independent experiments and were compared using two sample t-tests.
of MM cells in the presence of 20 ng/mL of TGF-P1 was
either unaffected (MM3) or increased (SI 2.6 for MM4 and
SI 1.9 for MM5). Proliferation of MM cell lines was unaffected even at high (>20 ng/mL) concentrations of TGFPl. Supernatants from CD72 COS tranfectants were used as
controls and did not alter proliferation of B cells, MM cells,
or MM-derived cell lines.
Effects of TGF-PI on IgG secretion by B cells, CDIOLtriggered B cells, M M cells, and MM-derived cell lines. Effect of TGF-P1 on IgG secretion by splenic B cells, CD40L
pretreated B cells, MM cells (patients MMl-MM4), and MM
derived cell lines (ARH-77 and IM-9) was analyzed by
ELISA (Table 1). To enhance Ig secretion, these cells were
stimulated with CD4OL + E-10. As previously
IL-10 CD4OL significantly increased IgG secretion by B
cells (87 +. 6-fold, P < .001, n = 5). CD4OL pretreated B
cells increased IgG secretion (8 ? 0.7-fold, P < .001, n =
3) in the presence of CD4OL + IL-10. In contrast, CD40L
+ IL-10 did not stimulate significant increments in IgG secretion by MM cells (1.8 +. 1.2-fold, n = 4) or MM cell
lines (1.2 ? 0.2-fold for ARH-77 and IM-9).
Fifty percent inhibition of IgG secretion by B cells or
CD40L pretreated B cells cultured in the presence of CD4OL
+ IL-10 was noted at concentrations between 0.08 ng/mL
and 0.31 ng/mL of TGF-P1. IgG secretion by ARH-77 and
IM-9 cells was 50% inhibited at concentrations of 1.25 to
20.0 ng/mL of TGF-P1. Inhibition of IgG secretion of MM
cell lines was obtained at higher TGF-P1 concentrations
than those that downregulated normal B cells or CD40L
pretreated B cells. Specifically, IgG secretion by tumor cells
from patients MM1, MM2, MM3, and MM4 cultured with
20 ng/mL TGF-P1 were decreased to U%,82%, 69%, and
65%, respectively, of levels noted in cultures with CD40L
+ IL10.
Effects of TGF-PI on proliferation and phosphorylation
state of pRB in RPMI-8226 M M cells, patient M M cells,
JKB ALL cells, and n o m 1 B cells. Effects of TGF-P1 on
proliferation and phosphorylation state of pRB in RPMI8226 MM cells, patient MM cells, JKB ALL cells, as well
as splenic B cells and CD4OL triggered B cells were investigated with immunoprecipitation followed by immunoblotting (Fig 8). TGF-P1 did not alter proliferation of RPMI8226 MM cells; pRB were constitutively phosphorylated,
and TGF-01 did not affect their phosphorylation state. Patient MM cells proliferated in response to TGF-P1 (SI 2.2,
P < .02); pRB were again constitutively phosphorylated
with two bands of strong (upper) and weak (lower) intensity,
and the latter was absent in the presence of TGF-P1. In
contrast, proliferation of JKB-1 ALL cells was significantly
inhibited by TGF-P1 (SI 0.1, P < ,001); these cells again
31
+
TGF pl
Concentration
(ng/ml)
Fig 7. Effects of TGF-pl on proliferation of MM cells and MMderived cell lines. CD38+CD45RA- MM cells (n = 5) (patients MM1:
--U-; MM2 -M-;
MM3: -A-;
MM4 -A-; MM5 -+-I;
MM
derived cell lines IARH-77, IM-9, RPMl-8226, U-2661(-0-1 or splenic
B cells In = 7) (-0-1 were suspended at 1 x 10' cells/mL in 10%
FBS RPMI-16M media. CD4OL (titer of 1:4) and serial dilutions of TGFp l IO to 20 ng/mLl were added. Two hundred-microliter aliquots
were dispensed into 96-well round-bottomed tissue culture plates.
Cells were pulsed during the last 18 hours of 72-hour cultures with
1 pCi *H-TdR/well. procured onto glass filters, and counted on a
scintillation counter. SI = 'H-TdR uptake of cells with C W L + TGF
p1I'H-TdR uptake of cells with C W L alone.
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
URASHIMA ET AL
1934
Table 1. Effects of TGF-p1 on IgG Secretion
Splenic B Cells
Treatment.
Media alone
+ IL-10
+ CD40L
+ CD72
TGF-bl
+ IL-10 + CD40L
+ IL-10 + CD40L + TGF-Dl
(0.005 ng/mL)
(0.02 nglmL)
(0.08 nglmL)
(0.31 ng/mL)
(1.25 ng/mL)
(5.0 nglmL)
(20.0 ng/mL)
+
MM Cells From Patients*
Normal Splenic
B Cellst
CD40L Pretreated
B Cellst
MM1
105 f 17
122 f 18
166 c 20
108 f 19
26 2 4
9,128 c 102
250 c 12
535 f 46
342 2 71
238 f 41
1 8 2 2 20
2,005 f 139
207 c 21
224 I
18
488 t 36
213 c 20
2 0 8 2 19
496 f 56
9,036
7.987
4,536
1,204
931
593
493
2,105
1,804
1,624
741
561
501
461
485
467
473
448
434
371
218
f 87
t 74
f 52
f 110
2 75
f 34
c 30
f 163
f 205
2 144
f 80
f 63
I
72
c 55
MM3
MM2
c 50
f 44
2 38
t 32
f 37
c 33
c 12
2 74
c 38
t 42
f 55
167 c 14
135 f 12
384 t 35
161 f 21
1 5 5 2 12
332 f 35
608 c 70
583 f 41
556 2 64
560 t 58
551 f 39
559 f 35
514 c 32
299 I
22
288 f 24
273 2 23
252 f 22
262 f 23
269 I
21
229 c 19
556
432
1,040
436
448
627
c 43
f 40
MM Cell Linet
ARH 77
IM-9
f 32
246
f 65
132 f 12
143 f 13
145 I
15
140 f 14
28 Z 5
185 c 11
1,496 f 121
1,520 t 133
1.487 f 148
1,503 t 106
468f 58
1,533 f 107
955 f 71
916 f 87
875 t 49
799 f 45
753 c 45
707 f 36
672 c 57
168 f 13
159 c 12
172 f 14
175 2 14
163 f 17
93 f 16
21 f 7
1,548 t 104
1,502 f 136
1.487 f 117
1,366 c 108
996 f 121
486 t 77
423 f 60
MM4
554
345
1,207
560
426
1,035
c 51
f 30
2 63
~
Supernatants were collected at 7 days of culture and IgG levels (ng/mL) quantitated by ELISA. IL-10 was used at 10 ng/mL, CD4OL at 1:4
titer, CD72 at 1:4 titer, and TGF-bl at 20 nglmL.
t Data shown are mean c SD from three independent experiments.
Data from duplicate experiments.
*
showed constitutively phosphorylated pRB, with dephosphorylation of pRB in response to TGF-PI . In normal splenic
B cells, proliferation was inhibited by TGF-PI (SI 0.28, P
< .OOl); in this case, no phosphorylated pRB was detectable
before or after treatment with TGF PI. CD40L triggering of
B cells increased both proliferation (SI 9.0) and increased
pRB phosphorylation. TGF-P 1 treatment of these CD40L
triggered B cells both inhibited their proliferation (SI 2.2)
and decreased phosphorylation of pRB.
DISCUSSION
A broad spectrum of normal and malignant cells secrete
TGF-PI and has specific high-affinity receptors for this pep-
tide,2h suggesting a role for TGF-01 in both normal and
malignant processes. In MM, earlier studies have shown
TGF-PI mRNA in tumor cells and derived cell lines,3hbut
the role of TGF PI in the pathophysiology of MM has not
previously been characterized. In the present studies, MM
cells were found to secrete more TGF-Dl than splenic B
cells or CD40L pretreated B cells, and MM BMMCs to
produce more TGF-PI than N-BMMCs. These data are consistent with studies of TGF-Dl mRNA reported by Matthes
et a],37.38 and suggest that TGF-PI secretion increases with
stage of B-cell differentiation, ie, TGF-PI secretion by B
cells <CD40L pretreated B cells <malignant plasma cells.
Proliferation of normal B cells decreases as they differenti-
*
TGFP
-
+
-
+
-
+
-
+
- +
Phosphorylated
87kd
Fig 8. Effects of TGF-p1 on
phosphorylation of retinoblastoma protein in MM cells, ALL
cells, and B cells. RPMI-8226 MM
cells, patient MM cells, JKB-1
ALL cells, normal splenic B cells,
and CD40L triggered B cells (B
cells cultured for 3 days with
CD40L)were cultured for 2 hours
in the presence or absence of
TGF-pl (20ng/mL). Cell lysates
were immunoprecipitated with
anti-pRB MoAb, transferred to
nitrocellulose membranes and
immunoblotted with anti-pRB
antibody, followed by detection
using chemiluminescence. Dephosphorylated pRB was recognized at 105 kD and phosphorylated pRB at 110 kD.
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
TGF-81 IN MULTIPLE MYELOMA
ate, related to autoinhibitory effects of TGF-61, and is terminated in the BM microenvironment where the concentration
of TGF-01 is greater than in PB. In contrast to its effects
on normal B cells, TGF-01 does not decrease proliferation
of MM cells and, at high concentrations, may augment their
IL-6 secretion and related proliferation. This pattern of MM
cell response to TGF-PI may facilitate tumor cell proliferation and accumulation in the BM. Furthermore, localization
of tumor cells in BM accounts for the higher amounts of
TGF-PI secretion by MM BM than by normal BM. Therefore, TGF Pl may trigger autocrine IL-6-mediated MM cell
growth in the BM.
The present studies showed that exogenous TGF-P1
upregulated IL-6 secretion by MM cells as well as BMSCs
and derived cell lines. Previous reports showed that TGF01 stimulated IL-6 production by lung fibroblasts, and that
TGF-PI could either augment or inhibit IL-6 production by
IL-1-stimulated fibroblasts." Upregulation of IL-6 triggered
by TGF-P 1 has also been reported in murine B M S C S ,intes~
tinal epithelial
astrocytoma
and rat hepatoma cells.44In contrast, Shalaby et a145reported that TGFPl inhibited IL-6 production by endothelial cells induced by
IL-10 and TNF-a, suggesting that the effect of TGF Pl on
IL-6 secretion may in some cases depend on interactions
with other cytokines. For example, although IL-6 secretion
by N-BMSCs and CLL-BMSCs is equivalent, Lagneaux et
all" showed higher TGF-01 production by B-CLL BMSCs
and related suppression of blast-colony forming cell growth
in the presence of IL- 1 or lipopolysaccharide; this inhibitory
effect of TGF-P1 could be partially overcome by exogenous
IL-6. In our studies, TGF-01 upregulated IL-6 secretion by
some freshly isolated CD38TD45RA- MM cells, but did
not increase IL-6 secretion by B cells, as in previous reports.& Excess TGF-P1 secretion by MM cells and MM
BMSCs increased their IL-6 secretion, because anti-TGFP MoAb inhibited and exogenous TGF-01 stimulated IL-6
secretion by BMSCs. Moreover, TGF-P1 secretion by MMBMSCs was correlated with IL-6 secretion. Therefore, accumulation of MM cells in BM increases TGF-P1 concentration within the BM microenvironment, which may cause
further upregulation of IL-6 and related tumor cell proliferation. Thus, our studies suggest that TGF-01 can affect MM
cell growth not only via an autocrine mechanism, ie, via
induction of IL-6 in tumor cells, but also via a paracrine
mechanism, ie, induction of IL-6 in BMSCs. Concomitant
increases in both IL-6 and TGF-P have also been observed
in other disease states, ie, plasmacytoma bearing micez7and
cardiac allografting in h~mans.4~
We have previously reported that MM cells express cell
surface adhesion molecules (CD29/CDw49d: very late antigen 4; CD 18/CD11a: lymphocyte associated antigen- 1, and
CD44) and may localize to BM via specific adherence to
both BM extracellular matrix proteins and BMSCS.~'We
have also shown that MM cell adherence triggers JL-6 secretion by BMSCS.'*'-'~However, our previous attempts to block
tumor cell adhesion induced IL-6 secretion by BMSCs using
MoAbs to adhesion molecules had minimal effects. In the
present studies, anti-TGF-P MoAb inhibited IL-6 secretion
1935
by BMSCs; moreover, the increments in IL-6 secretion induced by MM cell adhesion were also blocked by anti-TGFP MoAb. In these studies, paraformaldehyde fixation of
BMSCs before MM cell adhesion abrogated IL-6 secretion,
suggesting that the major source of IL-6 was BMSCs. Moreover, fixation of MM cells reduced IL-6 secretion, suggesting
that TGF-/3 produced by MM cells can induce IL-6 secretion
by BMSCs. These studies confirm the role of TGF-P1 in
triggering IL-6 secretion during the interaction between MM
cells and BMSCs. As is true in our previous studies triggering IL-6 secretion via either MM to BMSC adhesion or
CD40L,s.'-'0 these results also suggest that the major source
of IL-6 in MM is BMSCs and that MM cell growth is primarily via an IL-6-mediated paracrine mechanism.
Both DNA synthesis and Ig secretion by normal splenic
B cells were downregulated by TGF-PI; in contrast, MM
cells and derived cell lines did not similarly decrease their
proliferation or Ig secretion in response to TGF-01. TGFPl acts via dephosphorylation of pRB, which suppresses
E2F transcription and causes subsequent cell growth arrest
in late G1 p h a ~ e . ~In~ our
. ~ ' studies, TGF-P1 could not inhibit
phosphorylation of pRB or DNA synthesis in MM cells/
derived cell lines, but did both trigger dephosphorylation of
pRB and block proliferation of the JKB ALL cell line and
normal splenic B cells cultured with CD40L. Mutations in
RB gene have been reported in many types of malignancies?
35% to 52% of MM patients have abnormalities or deletions
in RB protein or gene.53,54
Moreover, p53 mutation and ras
mutations have also been recognized in MM.s3 The lack of
response of MM cells to TGF-P1 suggests that abnormalities
in signaling may occur in MM cells between the level of the
TGF-P receptor and dephosphorylation of pRB, involving
other cell cycle associated proteins, which may facilitate
disease progression.
TGF-P1 has been shown to inhibit in vitro proliferation
and Ig secretion of B cells,20,2'IL-2-dependent T-cell prolife r a t i ~ n development
,~~
of cytotoxic T ~ e l l s , ~ ' .as
' ~ well as
natural killer (NK) cell5* and lymphokine activated killer
cell f~nction.'~
In addition, TGF-0 1 can suppress cytotoxic
effector function of activated macrophages by downregulating IL-2 receptor mRNA
Clinical immunodeficiency has also been attributed to increases in TGF-81 in
vivo. For example, increases in TGF-PI mediate immunosuppression during either chronic graft versus host disease
post allogeneic BMT or cardiac allograft reje~tion.'~.'~
In
patients infected with human immunodeficiency virus and
those with adult T-cell leukemia, high levels of TGF-01
released by PBMCs lead to defects in both cellular and humoral immunity.61,65Moreover, it is of note that TGF-01
knock-out mice succumb to a wasting syndrome characterized by a multifocal, mixed inflammatory cell response and
tissue necrosis, leading to organ failure and death at 20 days
after
These immunomodulatory effects of TGF-P 1
suggest that it may also contribute to the immunosuppression
characteristic of MM. The immunodeficiencyin MM is characterized by a nonspecific polyclonal B-cell defect and a
profound impairment of primary antibody responsiveness,
and is a major cause of morbidity and
Our
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
1936
URASHIMA ET AL
studies showed high levels of TGF-P1 secretion by human
MM cells and derived cell lines, consistent with previous
reports of high serum levels of TGF-P1 in plasmacytomabearing mice.*' In addition, both DNA synthesis and Ig secretion of normal B cells were inhibited by TGF-P1. Finally,
anti-TGF-P Abs have been reported to inhibit tumorigenicity
of breast cancer cells by increasing NK cell activity:' suggesting that TGF-P1 may facilitate progression of disease
by suppressing host immune surveillance. Future studies will
determine whether high levels of TGF-P1 secretion may also
contribute to immunosuppression and disease progression in
patients with MM, and if so, whether treatment strategies
could be targeted to decreasing TGF-P to both interrupt IL6-mediated tumor cell growth and decrease immunodeficiency in patients with MM.
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