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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 8 (April 15), 2000:
pp. 2630-2636
NEOPLASIA
Vascular endothelial growth factor and interleukin-6 in
paracrine tumor-stromal cell interactions in multiple myeloma
Berno Dankbar,
Teresa Padró,
Regine Leo,
Birgit Feldmann,
Martin Kropff,
Rolf M. Mesters,
Hubert Serve,
Wolfgang E. Berdel, and
Joachim Kienast
From the Department of Medicine/Hematology and Oncology, University
of Muenster, Muenster, Germany
 |
Abstract |
Vascular endothelial growth factor (VEGF), a multifunctional
cytokine, potently stimulates angiogenesis including tumor
neovascularization. Although well established in solid tumors, the role
of VEGF in bone marrow neoangiogenesis and paracrine tumor-stromal cell
interactions in lymphohematopoietic malignancies has not been fully
elucidated. In multiple myeloma (MM), marrow neovascularization
parallels disease progression. This parallel prompted us
to investigate the expression and secretion of VEGF by myeloma cells
and its potential effects in myeloma-marrow stroma interactions. The
biologically active splice variants VEGF165 and VEGF121 were expressed
and secreted by myeloma cell lines and plasma cells isolated from the
marrow of patients with MM. As shown by immunocytochemistry or
RT-PCR, myeloma cells did not express or weakly expressed the VEGF receptors FLT-1 and FLK-1/KDR, indicating that autocrine stimulation is unlikely. In contrast, FLK-1/KDR was abundantly expressed by marrow stromal cells. Therefore, we studied the effects of
VEGF on marrow stroma, focusing on the secretion of interleukin-6 (IL-6), a potent growth factor for myeloma cells and an inhibitor of
plasma cell apoptosis. Exposure of stromal and microvascular endothelial cells to recombinant human (rh) VEGF165 or VEGF121 induced
a time- and dose-dependent increase in IL-6 secretion (14- to 27-fold
at 50 ng/mL after 24 hours, P < .001). Conversely, rhIL-6
stimulated VEGF expression and secretion in myeloma cell lines
(40%-60%; P < .05) and to a variable degree (up to
5.3-fold; P < .005) in plasma cells purified from the
marrow of patients with MM. This mutual stimulation suggests paracrine
interactions between myeloma and marrow stromal cells triggered by VEGF
and IL-6.
(Blood. 2000;95:2630-2636)
© 2000 by The American Society of Hematology.
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Introduction |
Multiple myeloma (MM) is a human B-cell neoplasm
characterized by the clonal expansion of malignant plasma cells in the
bone marrow. Interleukin-6 (IL-6) has been identified as a major
cytokine involved in the emergence of the tumor clone and in
tumor-associated toxicities in patients with MM.1-6 Indeed,
high levels of IL-6 have been found in the serum of patients with
active MM,7-9 and a reduction of the tumor cell mass has
been reported by inhibition of myeloma cell proliferation with
anti-IL-6 monoclonal antibodies.6,10 In addition, IL-6 is
known to inhibit fas- and dexamethasone-induced plasma cell apoptosis
in vitro.11-14 Thus, IL-6 is considered a most important
cytokine involved in myeloma cell growth, both in vitro and in vivo.
Interactions between myeloma cells and the bone marrow stroma, a
heterogeneous compartment of various cell types and the main producer
of IL-6, are well described.15-21 Although various
cytokines have been shown to mediate IL-6 secretion by myeloma-derived
marrow stromal cells,21-24 the involvement of angiogenic
factors has not been investigated. Vascular endothelial growth factor
(VEGF) is considered a potent stimulator of angiogenesis in
vivo.25 In solid tumors, VEGF expression is closely
associated with the induction of neovascularization and correlates with
tumor growth and metastatic potential.26-32 In MM, marrow
neoangiogenesis parallels tumor progression and correlates with poor
prognosis, suggesting an angiogenesis-dependent regulation of disease
activity.33-35 In addition, VEGF
expression36,37 and angiogenic activity38 of
myeloma cells have recently been described.
In the present study, we have demonstrated that VEGF165 and VEGF121 are
expressed and secreted by myeloma cells and that both isoforms
stimulate the expression of IL-6 by microvascular endothelial cells
(MVECs) and bone marrow stromal cells (BMSCs). In turn, IL-6 stimulated
the expression of both VEGF splice variants by myeloma cells,
suggesting a paracrine role for VEGF in tumor-stroma interactions in MM.
| |
Materials and methods |
Patients and control subjects
Bone marrow samples were obtained from 17 patients aged 38 to 69 years with stage III MM.39 Monoclonal isotype was
immunoglobulin G (IgG) in 9 patients, IgA in 6, and light chains only
in 2 patients. Marrow sampling was performed at diagnosis (n = 3), at
relapse off therapy (n = 4), in refractory progressive disease
(n = 5), or in chemotherapy responsive or stable disease (n = 5).
In addition, mononuclear cells (MNCs) were isolated from nonaffected
bone marrow obtained from patients with solid tumors (n = 1) or
various skin diseases (n = 4) who served as control subjects. B
lymphocytes were isolated from the peripheral blood of healthy
volunteers. Marrow aspirates from three patients with active MM and
three patients with non-Hodgkin's lymphoma or solid tumors without
bone marrow involvement were used for the preparation of bone marrow stromal cell cultures (MM-BMSC and BMSC, respectively). Patients and
volunteers gave informed written consent prior to the
sampling procedure.
Immunofluorescence and cell sorting
Anti-CD38-phycoerythrin/cyanin5 and anti-CD19-fluorescein
isothiocyanate were purchased from Coulter-Immunotech (Hamburg, Germany). Anti-CD56 and anti-CD14 conjugated to phycoerythrin were
obtained from Becton Dickinson (Heidelberg, Germany), and anti-CD138-fluorescein isothiocyanate was obtained from Serotec (Oxford, UK).
Bone marrow MNCs were separated by density gradient centrifugation
using Ficoll-Paque (Pharmacia, Upsala, Sweden).
CD38high/CD138+ or
CD38high/CD56+ plasma cells were isolated from
the marrow MNC fraction of patients with MM by fluorescence-activated
cell sorting using a FacsVantage (Becton Dickinson). The remaining
cells that were negative for the myeloma phenotype were defined as the
nontumor cell fraction. CD19+/CD14- B
lymphocytes were sorted from peripheral blood MNCs of healthy volunteers. On reanalysis, sorted populations had a purity of at least
96% for the defining phenotype.
Cell cultures
The human myeloma-derived cell lines RPMI-8226, U-266, OPM-2, and
IM-9 were obtained from the German Collection of Microorganisms and
Cell Cultures (DSMZ, Braunschweig, Germany). Cell lines, marrow MNC,
and sorted cells from individual patients were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS; GIBCO-BRL,
Eggenstein, Germany) and 2 mmol/L L-glutamine (GIBCO-BRL) at 37°C and 5% CO2. Human MVECs isolated from skin
biopsies (pool: 20 samples; Technoclone, Austria) were cultured at
37°C and 5% CO2 in MCDB 131 medium (GIBCO-BRL)
supplemented with 20% FCS. BMSC and MM-BMSC cultures were established
from the MNC fraction of nonaffected or MM bone marrow, respectively,
according to the method of Lagneaux et al40 with minor
modifications. In brief, 5 × 105 cells/mL were
cultured in  -minimal essential medium (GIBCO-BRL) supplemented with
10% FCS at 37°C and 5% CO2 in 75-cm2
flasks. The culture medium was replaced at weekly intervals until a
confluent monolayer had developed (usually after 4-8 weeks). All
culture media were supplemented with 100 U/mL penicillin and 100 µg/mL streptomycin. For stimulation experiments, MVECs and BMSCs in
passages 7 and 3, respectively, were grown in 24-well plates. Prior to
stimulation, confluent cells were starved for 24 hours (MVEC: 5% FCS,
BMSC: 1% FCS).
Cell extracts
Extracts from myeloma cell lines were generated by lysis of cells at
4°C, using a Triton-based extraction buffer (100 mmol/L Tris-HCl,
pH 8.0, 0.5% Triton X-100, 10 mmol/L EDTA). Supernatants were
collected and centrifuged for 5 minutes at 12 000 rpm at 4°C.
Extracts were frozen in liquid nitrogen and stored at -80°C until
analysis. The protein content in cell extracts was determined by the bicinchoninic acid method using a commercial assay (Pierce Chemical, Rockford, IL).
Stimulation of cell cultures and cytokine measurements
MVECs and BMSCs were stimulated with different concentrations of
recombinant human VEGF165 or VEGF121 (R&D Systems, Wiesbaden, Germany)
for 6, 24, and 48 hours in their respective starvation medium. In
addition, MM-BMSCs were stimulated with different concentrations of
both VEGF isoforms for 48 hours. Thereafter, cells were pelleted and
the supernatants were analyzed for IL-6. From BMSC pellets, total RNA
was isolated for analysis of IL-6 transcripts. IL-6 concentrations in
culture supernatants were determined by a commercial enzyme-linked
immunosorbent assay (ELISA; Genzyme, Cambridge, MA) with a lower
detection limit of 2 pg/mL. Concentrations of IL-6 are presented as
pg/mL corrected for 105 cells. Inhibition experiments were
performed, using a polyclonal goat anti-human VEGF antibody (R&D Systems).
Sorted marrow myeloma cells from patients were cultured for 24 hours
prior to stimulation. Myeloma cells at a concentration of
1 × 106 cells/mL were stimulated with recombinant
human IL-6 (PeproTech, Rocky Hill, NJ) at concentrations up to 10 ng/mL. After 72 hours of stimulation, cells were harvested and
centrifuged, and the supernatants were analyzed for VEGF. From pelleted
cells, either total RNA was isolated or proteins were extracted and
subjected to quantitative analyses. VEGF levels in culture supernatants and in cell extracts were determined by a commercial ELISA (Cytimmune Science, MD) that detects both isoforms, VEGF121 and VEGF165. The lower
detection limit of the assay is 12.5 pg/mL. Calibration curves were
prepared by dilution of the VEGF standard provided by the manufacturer
either in culture medium or extraction buffer as required. VEGF
concentrations in supernatants are presented as pg/mL corrected for
106 cells. For inhibition experiments, a polyclonal goat
anti-human IL-6 neutralizing antibody was used (R&D Systems).
RT-PCR analyses
Total RNA was prepared, using the guanidine isothiocyanate/phenol
method.41 Complementary DNA (cDNA) was synthesized for 2 hours at 37°C, using 1 µg total RNA, random hexamers, and M-MLV reverse transcriptase (Promega, Heidelberg, Germany). VEGF transcripts were amplified, using Taq polymerase (Promega) on a Hybaid thermocycler (MWG-Biotech, Ebersberg, Germany) for 35 cycles as follows: at 94°C
for 1.5 minutes, at 60°C for 3 minutes, and at 72°C for 4 minutes. VEGF primers 5'-TCGGGCCTCCGAAACCATGA-3' (sense)
and 5'-CCTGGTGAGAGATCTGGTTC-3' (antisense) corresponding to
sequences in the 3' and 5' untranslated regions were used,
allowing the amplification of the known splice variants (516 base pair
[bp], 648 bp, 720 bp, and 771 bp).42 PCR products were
separated on a 1.2% low-melting agarose gel, and DNA fragments of the
expected size were cloned in the pCR II-TOPO vector using the TOPO TA
cloning kit (Invitrogen, San Diego, CA). Specificity of the PCR was
verified by sequencing the cloned fragments, using an automated DNA
sequencer (ABI PRISM 310; Perkin Elmer, Weiterstadt, Germany).
Detection of IL-6 receptor (IL-6R) transcripts was performed by
amplification for 35 cycles at 94°C for 1 minute, at 65°C for
30 seconds, and at 72°C for 30 seconds, using a human IL-6R
amplimer set (Clontech Laboratories, Heidelberg, Germany). Expression
of the VEGF receptors FLK-1/KDR and FLT-1 by myeloma cells isolated
from patients was analyzed, using specific primer pairs as described
previously.43 Amplification was performed for 35 cycles
composed of 45 seconds at 94°C, 45 seconds at 58°C, 1 minute at
72°C, and, finally, 5 minutes at 72°C.
For semiquantitative VEGF messenger RNA (mRNA) analyses in patient
samples, 300 ng total RNA was reversely transcribed, and cDNA was
amplified as described above with a reduced cycle number (28-32 cycles). IL-6 mRNA expression was determined by the amplification of
cDNA generated from 300 ng total RNA for 28 cycles at 94°C for 45 seconds, 60°C for 45 seconds, and 72°C for 2 minutes, followed by a 7-minute final extension step, using a human IL-6 amplimer set
(Clontech). In all PCR amplifications, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was co-amplified as an internal control for RNA
integrity and quantification, using the GAPDH primers 5'-CCCTCCAAAATCAAGTGGGG-3' (sense) and
5'-CGCCACAGTTTCCCGGAGGG-3' (antisense). All PCR-amplified
products were separated on 6% polyacrylamide gels and stained with
ethidium bromide. For the estimation of VEGF and IL-6 expression, the
corresponding signals were normalized against GAPDH, using a
fluorometric analysis system (Gel Doc 1000; Bio-Rad Laboratories,
München, Germany).
Ribonuclease protection assay
VEGF mRNA expression in myeloma cell lines was quantitated by
ribonuclease protection assays. Radioactive full-length antisense cRNA
probes were generated in vitro from a 648 bp VEGF165 cDNA cloned in the
PCR II-TOPO vector and from a 316 bp GAPDH cDNA (pTRI-GAPDH; Ambion,
Austin, TX) in the presence of -[32P]CTP or
[35S]CTPS (Amersham Buchler, Freiburg, Germany), using
the Riboprobe Transcription System (Promega, Madison, WI). Probes and
sample RNAs were denatured in hybridization buffer (80% formamide; 0.5 mol/L NaCl; 40 mmol/L Pipes, pH 6.4; 0.2 mmol/L EDTA, pH 8.0) and
hybridized at 53°C for at least 16 hours. Samples were co-assayed for GAPDH mRNA as an internal quantification standard. Protected RNA
fragments were electrophoresed through a 5% denaturating
polyacrylamide gel. After exposure of the dried gel to an x-ray film
(Hyperfilm; Amersham, Heidelberg, Germany), VEGF signals were
quantitated by densitometric scanning (GelScan XL; Pharmacia LKB,
Freiburg, Germany) and normalized against GAPDH.
Immunocytochemistry
BMSC cultures grown to semiconfluence on 8-well chamber slides and
cytospin preparations of the above myeloma cell lines were processed
for immunocytochemical detection of the VEGF receptors FLT-1 and
FLK-1/KDR. The cells were dried and fixed for 4 minutes with cold
acetone (-20°C). Unspecific binding of antibodies was blocked by
TBS/0.5% BSA for 30 minutes at 4°C. Immunocytochemical analyses
were performed, using polyclonal rabbit anti-human FLT-1 and FLK-1/KDR
antibodies (Santa Cruz Biotechnology, Santa Cruz, CA; working dilution
1:200) for 30 minutes at 4°C. Bound receptor antibodies were
detected by the alkaline phosphatase/anti-alkaline phosphatase method according to the manufacturer's
instructions, using Fast Red as a substrate (DAKO Diagnostica,
Hamburg, Germany). After counterstaining with hematoxylin, cells were
evaluated for receptor expression by light microscopy.
Murine monoclonal antibodies against human CD54, CD68, CD31, and
thrombomodulin were purchased from DAKO and used for phenotypic characterization of BMSCs.
Statistics
Data are presented as individual data plots or as medians and
interquartile ranges (IQRs). Statistical significance of overall differences between multiple groups was analyzed by the Kruskal-Wallis one-way analysis of variance. If the test was significant, pairwise comparisons were done by the multiple-comparisons' criterion. Differences between two independent groups were analyzed by the Mann-Whitney rank sum test. The Wilcoxon matched-pair signed rank test
was used for comparison of differences within pairs.44 A
P value of .05 or less was considered significant.
| |
Results |
Expression and secretion of VEGF by myeloma cells
The expression of VEGF transcripts was determined by RT-PCR in
myeloma-derived cell lines (RPMI-8226, OPM-2, U-266, and IM-9) and in
purified marrow myeloma cells from 12 patients. Peripheral blood B
lymphocytes from healthy individuals served as controls. Myeloma cells
consistently expressed VEGF transcripts of the two lower molecular
weight isoforms, VEGF165 and VEGF121. In contrast, VEGF mRNA was not
detected in normal peripheral blood B lymphocytes (Figure
1).
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| Fig 1.
Vascular endothelial growth factor (VEGF) expression in
myeloma cells.
VEGF transcripts in human myeloma cell lines (lanes 2-5) and in
CD38high/CD138+ or
CD38high/CD56+ sorted myeloma cells from the
bone marrow of patients with multiple myeloma (MM 1-4) were analyzed by
RT-PCR. The primers used allowed the amplification of the known splice
variants of VEGF. Only the two lower molecular weight isoforms VEGF165
(648 base pairs [bp]) and VEGF121 (516 bp) were detected. Peripheral
blood B lymphocytes from healthy volunteers served as controls (PBB-L
1-4). Glyceraldehyde-3-phosphate dehydrogenase (347 bp) was used as a
control for RNA integrity. M = size marker; C = PCR control.
|
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Basal secretion of VEGF protein was similar in the myeloma cell lines
RPMI-8226, OPM-2, and U266. Median concentrations in culture
supernatants after 72 hours were 239.8 pg/mL (IQR, 235.8-260.6 pg/mL),
264.5 pg/mL (IQR, 247.1-291.5 pg/mL), and 368.3 pg/mL (IQR, 342.9-406.0 pg/mL) per 106 cells, respectively. By comparison, VEGF
levels in IM-9 cells were significantly lower (median 19.8 pg/mL; IQR,
18.8-20.5 pg/mL; P < .001, Mann-Whitney test). After 72 hours of culture, VEGF concentrations in the media of sorted marrow
myeloma cells from patients ranged from 16.0 to 321.5 pg/mL (median
115.3 pg/mL; IQR, 34.3-172.7 pg/mL) (Figure
2).
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| Fig 2.
Vascular endothelial growth factor (VEGF) secretion by
myeloma cells.
Basal VEGF concentrations were determined in supernatants of myeloma
cell lines (n = 5 independent experiments per cell line performed in
triplicates) and of sorted marrow myeloma cells from patients with
multiple myeloma (MM; n = 12) after 72 hours of culture. Data are
presented as medians and interquartile ranges.
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As determined in a subsequent series of experiments, VEGF levels in the
culture media of marrow MNCs from patients with MM (n = 5; median
67.1 pg/mL; IQR, 43.6-88.2 pg/mL) were found to be significantly higher
than in the media of marrow MNCs from control subjects (n = 5, see
"Materials and methods" section; median 22.9 pg/mL; IQR,
10.3-28.1 pg/mL; P < .0025, Mann-Whitney test). In
addition, a highly significant difference between sorted marrow myeloma
cells (median 199.2 pg/mL; IQR, 132.6-253.4 pg/mL) and the
corresponding nontumor cell fractions (median 10.9 pg/mL; IQR, 5.0-20.1 pg/mL) was observed (P < .001, Mann-Whitney)(Figure 3).
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| Fig 3.
Secretion of vascular endothelial growth factor (VEGF) by
marrow mononuclear cells (MNCs) as well as tumor and nontumor cells.
VEGF concentrations were determined in supernatants of marrow MNCs from
control subjects (n = 5), marrow MNCs from patients with multiple
myeloma (MM; n = 5), and of the corresponding sorted tumor and
nontumor cells of the MM marrows after 72 hours of culture. Data are
presented as medians and interquartile ranges. The Mann-Whitney test
was employed to identify differences between control MNCs and MM-MNCs
(P < .0025) and between tumor and nontumor cells
(P < .001).
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VEGF receptors in myeloma cells and BMSCs
To examine whether VEGF might have an autocrine regulatory effect on
myeloma cells, expression of the VEGF receptors FLT-1 and FLK-1/KDR was
analyzed by immunocytochemistry. None of the four myeloma
cell lines showed positive signals for either receptor. In
contrast, BMSCs stained strongly positive for FLK-1/KDR, whereas expression of the FLT-1 receptor was consistently low (data not shown).
In addition, expression of the two VEGF receptors by sorted myeloma
cells from patients was analyzed by RT-PCR. Ten of 12 patients
expressed neither the FLK-1/KDR nor the FLT-1 receptor. One patient
expressed exclusively transcripts for FLK-1/KDR, whereas another
patient showed a weak signal for FLT-1 (data not shown).
Effects of VEGF on IL-6 secretion by BMSCs, MM-BMSCs, and MVECs
BMSC cultures were established from bone marrow aspirates of three
patients with malignant disorders who had no evidence of marrow
involvement. Adherent cells in third passage showed mainly CD54+ fibroblast-like cells with few CD68+
macrophages. No endothelial cells were detected by CD31 and
thrombomodulin immunoreactivity. Stimulation of BMSCs with VEGF165 or
VEGF121 for up to 48 hours induced a time- and dose-dependent increase in IL-6 secretion (17- and 19-fold, respectively, at 50 ng/mL after 24 hours; P < .001 for overall group differences) (Figure 4). This increase in IL-6
secretion was inhibited in the presence of an anti-human
VEGF antibody (working concentration 100 µg/mL) (data not shown).
Stimulation of MM-BMSC cultures for 48 hours with VEGF165 or VEGF121
revealed a similar dose-dependent increase in IL-6 levels as observed
in BMSC cultures. Notably, basal IL-6 concentrations in MM-BMSCs tended
to be higher than in BMSCs (median 1729.2 pg/mL; IQR, 1580.4-3165.2 pg/ml vs 624.7 pg/mL; IQR, 488.1-1244.1 pg/mL; P < .05,
Mann-Whitney test) (Figure 5).
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| Fig 4.
Effect of vascular endothelial growth factor (VEGF) on
interleukin-6 (IL-6) secretion by bone marrow stromal cells (BMSCs).
IL-6 concentrations were determined in culture supernatants of BMSCs
after stimulation with 2 ( ), 10 ( ), or 50 ( ) ng/mL of VEGF165
(A) or VEGF121 (B) for 6, 24, and 48 hours, respectively; ( ),
unstimulated controls. Data represent median values and interquartile
ranges of stimulation experiments performed in triplicates in three
different BMSC cultures. Analysis of significance for overall group
differences was performed by the Kruskal-Wallis test
(P < .001). The multiple comparisons' criterion was
employed to identify differences between groups (10 ng/mL vs controls,
P < .01; 50 ng/mL vs controls, P < .001; 50 ng/mL vs 2 ng/mL, P < .001 for all time periods).
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| Fig 5.
Comparison of interleukin-6 (IL-6) secretion by bone
marrow stromal cells (BMSCs) and multiple myeloma (MM)-BMSCs.
IL-6 levels in culture supernatants of BMSCs (A, C) and MM-BMSCs (B, D)
were measured after stimulation with 0, 10, or 50 ng/mL of VEGF165 or
VEGF121 for 48 hours, respectively. Data represent median values and
interquartile ranges of stimulation experiments performed in
triplicates using three different BMSC and MM-BMSC cultures.
P < .05 for differences between basal IL-6 secretion of
BMSCs and MM-BMSCs (Mann-Whitney test).
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A time- and dose-dependent increase in IL-6 secretion was also observed
in MVEC cultures stimulated with VEGF165 and VEGF121, respectively,
although IL-6 levels in supernatants were consistently lower than in
BMSCs. At higher concentrations, VEGF121 tended to be more potent than
VEGF165 in stimulating IL-6 secretion by MVECs (median fold increase
over controls, × 27 vs × 14 at 50 ng/mL after 24 hours;
P < .05, Mann-Whitney test) (Figure
6).
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| Fig 6.
Effect of vascular endothelial growth factor (VEGF) on
interleukin-6 (IL-6) secretion by microvascular endothelial cells
(MVECs).
IL-6 concentrations were determined in culture supernatants of MVECs
after stimulation with 2 (), 10 (), or 50 () ng/mL of VEGF165
(A) or VEGF121 (B) for 6, 24, and 48 hours, respectively; (),
unstimulated controls. Data represent median values and interquartile
ranges of three independent experiments performed in triplicates.
Analysis of significance for overall group differences was performed by
the Kruskal-Wallis test (P < .001). The multiple
comparisons' criterion was employed to identify differences between
groups (10 ng/mL vs controls, P < .05; 50 ng/mL vs
controls, P < .001; 50 ng/mL vs 2 ng/mL,
P < .001 for all time periods).
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Semiquantitative RT-PCR showed an increase in IL-6 transcripts in both
BMSCs and MVECs after exposure to 50 ng/mL VEGF165 or VEGF121 for 6 and
24 hours, respectively, indicating that VEGF-induced stimulation of
IL-6 occurred at the mRNA level (Figure 7).
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| Fig 7.
Interleukin-6 (IL-6) transcripts in bone marrow stromal
cells (BMSCs) and microvascular endothelial cells (MVECs) after
exposure to vascular endothelial growth factor (VEGF).
Adherent cells were incubated with 50 ng/mL of VEGF165 or VEGF121 for 6 and 24 hours, respectively, and analyzed for IL-6 expression by RT-PCR.
Levels of IL-6 messenger RNA (628 base pairs) were estimated by
normalization against co-amplified glyceraldehyde-3-phosphate
dehydrogenase. C = unstimulated controls; M = size marker.
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IL-6 stimulates VEGF expression in myeloma cells
To study whether IL-6 would in turn stimulate the expression and
secretion of VEGF by myeloma cells, the four myeloma cell lines were
cultured for 72 hours with and without the addition of exogenous IL-6.
Except for IM-9 cells, a dose-dependent increase in VEGF concentrations
was observed in culture supernatants on stimulation with IL-6 (Figure
8A). In the presence of 10 ng/mL IL-6,
median VEGF concentrations increased by 62% over controls in U-266
(P < .005), by 39% in OPM-2 (P < .025), and by
38% in RPMI-8226 (P < .005). The effect of IL-6 on VEGF
secretion was completely abrogated in the presence of an
IL-6-neutralizing antibody (data not shown). Consistent with these
results, RNase protection assays revealed an IL-6-induced increase of
VEGF mRNA transcripts in the IL-6-sensitive cell lines, whereas no
effect of IL-6 on VEGF mRNA levels was evident in IM-9 cells.
Representative experiments in U-266 and IM-9 are shown in Figure 8B.
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| Fig 8.
Effect of interleukin-6 (IL-6) on vascular endothelial
growth factor (VEGF) expression in myeloma cell lines.
(A) VEGF concentrations in culture supernatants. The myeloma cell lines
U-266 (), OPM-2 (), RPMI-8226 (), and IM-9 () were exposed
to 0, 1, and 10 ng/mL IL-6, respectively, for 72 hours. Data represent
median values and interquartile ranges of five independent experiments
performed in triplicates. Stars denote significant differences versus
unstimulated controls (U-266, P < .005; OPM-2,
P < .025; RPMI-8226, P < .005;
multiple-comparisons' criterion). (B) RNase protection assays showing
VEGF messenger RNA (mRNA) levels in U-266 and IM-9 cells after exposure
to 0, 1, and 10 ng/mL IL-6, respectively, for 72 hours. Total RNA was
hybridized against radioactively labeled complementary RNA probes for
VEGF and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Sizes of
protected fragments were 648 nucleotides for VEGF165, 438 nt for VEGF121, and 316/258 nt for GAPDH. Signals were analyzed by
densitometric scanning and normalized against GAPDH. In contrast to
U-266, VEGF mRNA levels were not upregulated in IM-9 cells lacking IL-6
receptor.
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Determinations of intracellular VEGF levels in protein extracts of
U-266, OPM-2, and RPMI-8226 cells demonstrated an increase on
stimulation with 10 ng/mL IL-6 for 72 hours (median fold increase over
controls × 1.54; range: × 1.21 to × 1.73; n = 3
independent experiments per cell line; P < .01 for
IL-6-stimulated vs unstimulated myeloma cells, Wilcoxon test). In
contrast, VEGF protein concentrations in IM-9 extracts remained below
the detection limit of the ELISA, even when the cells were stimulated
with IL-6. The IL-6-sensitive cell lines U-266, OPM-2, and RPMI-8226
expressed IL-6R mRNA as revealed by RT-PCR, whereas no IL-6R
transcripts were detected in IM-9 cells (not shown).
In a subsequent series of experiments, myeloma cells sorted from bone
marrow aspirates of 12 patients were cultured in the presence or
absence of IL-6 (10 ng/mL) for 72 hours. Although to a variable degree,
IL-6 stimulated VEGF secretion into culture supernatants up to 5.3-fold
(P < .005, Wilcoxon test) (Figure 9A). In line with these results, an
increase in VEGF mRNA levels was observed in IL-6-stimulated marrow
myeloma cells using semiquantitative RT-PCR analysis (Figure 9B).
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| Fig 9.
Effect of interleukin-6 (IL-6) on vascular endothelial
growth factor (VEGF) expression in sorted marrow myeloma cells from
patients (n = 12).
(A) VEGF concentrations in supernatants of myeloma cell cultures after
exposure to 0 and 10 ng/mL IL-6 for 72 hours, presented as pg/mL
corrected for 106 cells. P < .005 for
difference between unstimulated and stimulated myeloma cells (Wilcoxon
test). (B) VEGF transcription in IL-6-stimulated myeloma cells from
patients. RT-PCR analysis of VEGF transcripts after exposure to 0 or 10 ng/mL IL-6 for 72 hours. Representative examples of three patients with
increased VEGF messenger RNA levels after IL-6 stimulation are shown.
Corresponding patient numbers (UPN) are indicated in panels A and B.
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| |
Discussion |
In MM, three lines of evidence suggest a role for angiogenic
factors, such as VEGF, in the regulation of tumor cell growth and
disease activity. First, bone marrow neoangiogenesis parallels disease
progression and predicts poor outcome.33,35,38 Second, the
marrow microenvironment supports both the growth of myeloma cells and
the neovascularization of areas with myeloma cell
infiltration.45 Third, myeloma cells express both
angiogenic activity and angiogenic cytokines.36-38,46 On
the basis of these premises, we studied the role of VEGF in
myeloma-marrow stroma interactions.
In accordance with a recent report,37 we found consistent
expression of the splice variants VEGF165 and VEGF121 in purified myeloma cells from patients and in human myeloma-derived cell lines.
Basal VEGF secretion was variable in cultured marrow myeloma cells and
particularly low in IM-9 cells. Purified myeloma cell fractions of MM
marrow were found to secrete significantly more VEGF than the
corresponding nontumor fractions. This finding suggests that the presence of myeloma cells indeed accounts for the higher amounts of VEGF secretion by marrow MNCs from patients with MM as
compared with marrow MNCs from control subjects.
In general, basal VEGF secretion rates appeared to be low in comparison
with other types of tumor cells.37,47 However, VEGF
concentrations in culture supernatants were within its range of
biological activity. Native VEGF at concentrations of 150 to 300 pg/mL
has been reported to stimulate endothelial cells in vitro.48,49 Thus, VEGF concentrations within the tumor
microenvironment should also suffice to exert potential biological
effects on marrow stromal cells. Because of the limited number of
samples from patients with MM that were available for the study, we
were unable to relate VEGF secretion by cultured myeloma cells to the
actual stage and activity of the disease.
The activity of VEGF is mediated by the tyrosine kinase receptors FLT1
(VEGFR-1) and FLK1/KDR (VEGFR-2).50-52 However, none of the
cell lines studied (U-266, RPMI-8226, OPM-2, and IM-9) expressed
VEGFR-1 or VEGFR-2. In addition, myeloma cells of only 1 of 12 patients
expressed VEGFR-2, and, in another patient, weak expression of VEGFR-1
by myeloma cells was observed. This finding indicates that
relevant autocrine stimulation of myeloma cells by VEGF is unlikely. In
contrast, FLK1/KDR (VEGFR-2) was abundantly expressed by BMSCs,
suggesting a potential paracrine role for VEGF in MM.
Paracrine interactions between myeloma cells and the stromal cell
compartment of the bone marrow have been extensively
studied.15-24 IL-6, a key cytokine of myeloma cell
growth,2,3,6,10 is expressed in BMSC cultures and its
release is stimulated by adhesion of myeloma cells.17-20 On
the one hand, bone marrow cultures mainly consisted of fibroblast-like
cells, which is in agreement with previously reported
data.53 On the other hand, MVECs, another functional
component of the bone marrow stroma in vivo, have been shown to release
cytokines relevant to the regulation of hematopoiesis.54 Likewise, stimulation of human umbilical vein endothelial cells with
VEGF leads to increased secretion of granulocyte-macrophage colony-stimulating factor and an up-regulation of several cytokine and
growth factor transcripts.37,42 Thus, it is conceivable that the stromal cell compartment may, in response to angiogenic factors, release cytokines capable of sustaining tumor growth. Our
results demonstrate that the VEGF isoforms 165 and 121 induce a time-
and dose-dependent increase in IL-6 secretion by BMSCs and MVECs.
Furthermore, a similar increase in IL-6 levels was found in MM-BMSCs
and BMSCs from nonaffected marrow. The findings suggest that VEGF is a
paracrine mediator supporting myeloma cell growth through the induction
of IL-6 release by bone marrow stroma. In MM, stimulation of IL-6
secretion by marrow stromal cells was previously shown for tumor
necrosis factor- (TNF-), interleukin-1 (IL-1), and
transforming growth factor- (TGF-)21-24 but had not
been described for angiogenic cytokines. Because plasma cells can
produce various cytokines able to induce the expression of IL-6 (eg,
IL-1, TNF-, TGF-, and VEGF),21,22,24 it is likely that the close proximity of myeloma and stromal cells creates favorable
conditions for myeloma cell growth in vivo.
We also demonstrated that IL-6 in turn stimulates VEGF secretion by
IL-6R-expressing myeloma cell lines and sorted marrow myeloma cells
from patients. This observation is in line with recent reports on VEGF
induction by both cellular and viral IL-6 in epidermoid carcinoma and
NIH3T3 cells, respectively, further supporting the notion that IL-6 can
indirectly promote angiogenesis.55,56 However, in 5 of 12 cases studied, we found no or only minor effects of IL-6 on VEGF
secretion by myeloma cells. This finding may reflect an
insensitivity to IL-6 caused by altered expression of or impaired signaling through the IL-6R. Thus, IL-6-induced VEGF secretion may only
occur in a subset of myeloma cells, eg, more mature myeloma cells. This
notion would be in line with reports on a variable response pattern of myeloma cells to exogenous IL-6, particularly in
freshly isolated tumor cells.5,57 In this context, it is again tempting to speculate that the IL-6 sensitivity of VEGF secretion
by myeloma cells may be related to the actual activity of the disease,
a hypothesis that we were unable to confirm or refute in the present study.
Along with IL-6, other cytokines and growth factors, such as IL-1,
platelet-derived growth factor, insulin-like growth factor, TGF-,
fibroblast growth factor, TNF-, and keratinocyte growth factor, have
been reported to stimulate VEGF expression, whereas IL-10 and IL-13
inhibit the release of VEGF.58 This plethora of regulating
factors suggests that cytokines other than IL-6 may also be involved in
the control of VEGF production by myeloma cells.
In conclusion, our data provide strong evidence for a mutual
stimulation of myeloma and marrow stromal cells triggered by VEGF and
IL-6. What appears to represent a perpetual paracrine cooperation
between VEGF and IL-6 may be part of a complex regulatory mechanism
sustaining tumor growth and protecting myeloma cells from
treatment-induced apoptosis. In addition, directly or indirectly, both
cytokines are likely to be involved in the neovascularization of bone
marrow infiltrated by myeloma cells. The data add to current evidence
providing a rationale for anti-angiogenic therapy in MM.
| |
Footnotes |
Submitted July 21, 1999; accepted December 13, 1999.
Supported in part by a grant (ZE 378/1NW) from the
Stifterverband für die Deutsche Wissenschaft, Essen, Germany.
Preliminary results presented as oral communication at the
40th (1998) Annual Meeting of the American Society of Hematology, Miami
Beach, FL.
Reprints: Joachim Kienast, Department of Medicine/Hematology
and Oncology, University of Muenster, Albert-Schweitzer-Str 33, D-48129
Muenster, Germany; e-mail: kienast{at}uni-muenster.de.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Top
Abstract
Introduction