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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 2 (January 15), 2000:
pp. 610-618
NEOPLASIA
Expression of functional interleukin-15 receptor and autocrine
production of interleukin-15 as mechanisms of tumor propagation in
multiple myeloma
Inge Tinhofer,
Ingrid Marschitz,
Traudl Henn,
Alexander Egle, and
Richard Greil
From the Laboratory of Molecular Cytology, Department of Internal
Medicine, University of Innsbruck, Innsbruck, Austria.
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Abstract |
Interleukin-15 (IL-15) induces proliferation and promotes cell
survival of human T and B lymphocytes, natural killer cells, and
neutrophils. Here we report the constitutive expression of a functional
IL-15 receptor (IL-15R) in 6 of 6 myeloma cell lines and in
CD38high/CD45low plasma cells belonging to 14 of 14 patients with multiple myeloma. Furthermore, we detected IL-15
transcripts in all 6 myeloma cell lines, and IL-15 protein in 4/6 cell
lines and also in the primary plasma cells of 8/14 multiple myeloma
patients. Our observations confirm the existence of an autocrine IL-15
loop and point to the potential paracrine stimulation of myeloma cells
by IL-15 released from the cellular microenvironment. Blocking
autocrine IL-15 in cell lines increased the rate of spontaneous
apoptosis, and the degree of this effect was comparable to the
pro-apoptotic effect of depleting autocrine IL-6 by antibody targeting.
IL-15 was also capable of substituting for autocrine IL-6 in order to promote cell survival and vice versa. In short-term cultures of primary
myeloma cells, the addition of IL-15 reduced the percentage of tumor
cells spontaneously undergoing apoptosis. Furthermore, IL-15 lowered
the responsiveness to Fas-induced apoptosis and to cytotoxic treatment
with vincristine and doxorubicin but not with dexamethasone. These data
add IL-15 to the list of important factors promoting survival of
multiple myeloma cells and demonstrate that it can be produced and be
functionally active in an autocrine manner.
(Blood. 2000;95:610-618)
© 2000 by The American Society of Hematology.
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Introduction |
Multiple myeloma is characterized by the accumulation
of malignant plasma cells in the bone marrow. Because the myeloma cells proliferate and accumulate in close proximity to stromal and
hematopoietic cells, a great deal of effort has focused on identifying
factors responsible for the survival and growth of the neoplastic clone in the stromal matrix. Some of these soluble factors are produced and
secreted by cells of the microenvironment,1,2 while others are produced by the myeloma cells themselves. These include
interleukin-1 ,3,4 interleukin-6 (IL-6),4,5
transforming growth factor ,4 tumor necrosis factor
,6 granulocyte colony-stimulating factor,4 and macrophage colony-stimulating factor.7 IL-6, a potent
growth factor for murine hybridomas and plasmacytomas, is at present considered the most relevant growth and survival factor for human multiple myeloma as well.5,8,9 The function of IL-6 as survival factor is demonstrated by its ability to inhibit apoptosis induced by growth factor withdrawal,10,11
dexamethasone,12 and triggering of the
cell-death receptor Fas.13 However, malignant plasma cells
lose their IL-6 dependence during in vitro culturing,1 which raises the question as to whether unknown cytokines might have
the potential to replace IL-6 signaling in promoting survival and
growth of myeloma cells.
IL-15, a recently identified cytokine,14 was found to share
many biological activities with IL-2, which led to a series of studies
on its effect on T cells and on natural killer (NK) cells. These
studies revealed that IL-15 stimulated proliferation of cytotoxic T
cells14 and regulated survival of NK cells.15 Furthermore, a proliferation and differentiation promoting function of
IL-15 has also been demonstrated in preactivated human B
cells.16 Its inhibitory effects on apoptosis induced by
anti-Fas, anti-CD3, anti-immunoglobulin M (anti-IgM) antibodies, or
dexamethasone in activated human T and B cells have recently been
demonstrated.17 IL-15 not only shares biological activities
with IL-2 but also uses the same -receptor and  -receptor
subunits.18 These subunits are indispensable for signal
transmission whereas the  -receptor subunit is responsible for the
high-affinity binding of IL-15 to the receptor.18,19 While
much attention has been paid to the expression patterns of the IL-15
receptor (IL-15R) components in T cells, there is no information
available on IL-15R expression in normal and neoplastic B cells
during maturation and differentiation. In the present study, we
examined whether all components of the IL-15R are expressed in both
myeloma cell lines and in native neoplastic plasma cells. Furthermore,
the effects of IL-15 on myeloma cell viability and the potential
contribution of an autocrine IL-15 loop to the propagation of the
neoplastic clone were investigated.
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Patients and methods |
After the acquisition of informed consent, tumor cells were
collected during routine examinations from the bone marrow of 14 patients suffering from multiple myeloma. The patients'
characteristics are given in Table 1. The
samples were treated with a erythrocyte lysis buffer
(NH4Cl, 155 mmol/L; KHCO3, 100 mmol/L; and
ethylene diamine tetra-acetic acid [EDTA], 1 mmol/L) to remove
erythrocytes. The samples were then washed in RPMI 1640 medium (10% fetal calf serum [FCS]) and immediately analyzed. During
flow cytometric analysis, plasma cells were characterized by high
expression of CD38 and dim or absent expression of CD45. This 2-color
analysis has been described as a reliable identification of plasma
cells in peripheral blood and bone marrow.20
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Table 1.
Patients' characteristics and expression levels of
surface IL-15R , IL-2R, and intracellular IL-15 in the
malignant plasma cell fraction
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Cell lines and culture conditions
The following neoplastic plasma cell lines were used in this
investigation: ARH-77; LP-1; MC-Car, the plasmacytoma cell line (The
American Type Culture Collection, Rockville, MD); OPM-2, the myeloma
cell line, and IM-9, the myeloma/lymphoblastoid cell line (German
Collection of Microorganisms and Cell Cultures, Braunschweig, Germany);
and RPMI 8226, the myeloma cell line (Dr T Otani, Fujisaki Cell Center,
Okayama, Japan). All cell lines and native plasma cells were cultured
in RPMI 1640 media (Seromed, Berlin, Germany) at 37°C
in a humidified atmosphere containing 5% CO2. The culture was supplemented with heat-inactivated FCS, 10% (Biological
Industries, Beth Haemek, Israel); L-glutamine, 2 mmol/L (Seromed); and
gentamycin, 100 µg/mL (GIBCO, Grand Island, NY).
Reagents
The following antibodies were used to determine the
protein expression levels by flow cytometry: monoclonal mouse antihuman IL-2R, IL-2R, and IL-2R antibodies (PharMingen, San Diego, CA); polyclonal goat antihuman IL-15R antibody (Santa Cruz
Biotechnology, Santa Cruz, CA); murine antihuman Fas (clone UB2;
Immunotech, Marseilles, France); antihuman FasL (clone
H11; Alexis, Laeufelfingen, Switzerland); antihuman Bcl-2 mAb (Dako,
Vienna, Austria); and goat antihuman Bax (Santa Cruz Biotechnology).
Murine antihuman IL-2R monoclonal antibody (mAb) (clone Mik-2,
PharMingen) was used to block IL-15 signaling.21
Monoclonal mouse antihuman IL-15 antibody (clone M112;
Genzyme Diagnostics, Cambridge, MA) was used in inhibitory experiments
and for detection of intracellular IL-15 protein in myeloma cells.
Monoclonal mouse antihuman IL-6 antibody (R&D Systems, Minneapolis, MN)
was used to block IL-6 signaling. The murine mAbs anti-CD38
(Pharmingen) and anti-CD45 (Becton Dickinson, San Jose,
CA) were used for the detection of neoplastic plasma cells
in bone marrow aspirates of patients. We also used recombinant IL-15
and IL-6 (PBH; Eubio, Vienna, Austria) and IL-15 enzyme
immunosorbent assay (ELISA) (Genzyme Diagnostics), with a detection
limit of 10 pg/mL, for detection of IL-15 in culture supernatants.
RNA extraction, cDNA synthesis, and reverse transcription-polymerase
chain reaction
Total RNA from approximately 1 × 106 cells was
extracted according to the guanidinium thiocyanate-phenol-chloroform
protocol described by Chomczynski and Sacchi.22
In distilled water, we diluted 1 µg RNA with 250 ng of
oligo (dT)15 primer to a final volume of 14 µL. The
solution was denatured at 70°C for 5 minutes and immediately
chilled on ice. We added the following reagents (Promega, Madison, WI)
to each reaction: reverse transcription (RT) mixture (6 µL)
containing 4 µL 5 × buffer; dATP, dCTP, dGTP, and dTTP (2 pmol each); and Moloney-murine leukemia virus RT (200 units). For complementary DNA (cDNA) synthesis, all samples were incubated at 37°C for 60 minutes, and the reaction was then stopped by heating the sample to 80°C for 2 minutes; 100 ng of cDNA
obtained was amplified by 36 cycles for IL-15, IL-15R, bcl-2, and
bax-; 28 cycles for Fas; and 50 cycles for FasL with 1 unit
polymerase (Taq, Promega). The reaction conditions were: for denaturing
60 seconds at 95°C, for annealing 60 seconds each at 63°C (cycle 1-3), 59°C (cycle 4-6), and 56°C (cycle 7-50) and for extension 45 seconds at 72°C.
The oligonucleotide primers used were as follows: for IL-15
23: TAA AAC AGA AGC CAA CTG (sense) and CAA GAA GTG TTG ATG AAC AT (antisense); for IL-15R 23: GTC AAG AGC TAC AGC
TTG TAC (sense) and GGT GAG CTT GCT CCT GGA G (antisense); for bcl-2 24: GGT GCC ACC TGT GGT CCA CCT G (sense) and CTT CAC TTG
TGG CCC AGA TAG G (antisense); for bax- 24: ATG GAC GGG
TCC GGG GAG CAG C (sense) and CCC CAG TTG AAG TTG CCG TCA G
(antisense); for FasL 25: TTC TTC CCT GTC CAA CCT CTG TGC
(sense) and TCA TCT TCC CCT CCA TCA TCA CCA (antisense); and for Fas:
TTC TGC CAT AAG CCC TGT CC (sense) and GGT GTT GCT GGT GAG TGT GC
(antisense). The expected amplification product sizes were: IL-15 (357 base pair [bp]); IL-15R (778 bp); bcl-2 (459 bp); bax- (323 bp); FasL (603 bp); and Fas (318 bp). GAPDH (987 bp)
served as internal control for the reverse transcriptase-polymerase
chain reaction (RT-PCR).
Detection of IL-15R components and IL-15 by flow cytometry
For the detection of IL-15R/IL-2R proteins,
0.5 × 106 cells were treated with the specific
antibody (1 µg per each sample) or with a nonreactive
(isotype-matched) negative control antibody. After 30 minutes
incubation at 4°C in PBS containing 0.3% bovine serum albumin
(BSA), the cells were washed and a secondary FITC-labeled antibody (dilution 1:10) was added for a further 30 minutes. Cells were
subsequently washed, resuspended in PBS/2% paraformaldehyde, and
analyzed within 1 hour (FACScan, Becton Dickinson). A minimum of
10 000 viable cells, gated according to their forward scatter/side scatter profile, were analyzed for their fluorescence intensity. For
analysis of native plasma cells of multiple myeloma patients, R-phycoerythrin-labeled anti-CD38 (Pharmingen) and peridinin
chlorophyll protein-labeled anti-CD45 (Becton Dickinson) antibodies
were also used. At least 5000 primary plasma cells were analyzed for
their fluorescence signals.
For intracellular IL-15 staining, 0.5 × 106 cells
were fixed and permeabilized (Fix & Perm; An-der-Grub Bio Research,
Kaumberg, Austria) according to manufacturer's
instructions. Permeabilized cells were incubated with murine
anti-IL-15 mAb (1 µg per each sample) or an isotype-matched negative
control antibody for 15 minutes at RT. Subsequently, secondary staining
using a rabbit antimouse antibody (Dako) and a tertiary staining using
a FITC-labeled goat antirabbit antibody (Dako) (both at a 1:10
dilution) were performed.
Detection of Fas, FasL, Bcl-2, and Bax by flow cytometry
For detection of surface Fas and FasL expression,
0.5 × 106 cells were stained with either a specific
mAb (1 µg) or an isotype-matched negative control mAb for 30 minutes
at 4°C, washed, and immediately analyzed by flow cytometry. For
intracellular staining of Bcl-2 and Bax, 0.5 × 106
cells were fixed and permeabilized (An-der-Grub Bio Research) according
to manufacturer's instructions. Permeabilized cells were stained with
specific monoclonal antibodies (mAbs) (1 µg) or the
relevant control mAbs for 20 minutes at RT. For Bax detection, a
secondary rabbit antigoat mAb was used for 20 minutes at RT. Cells were
washed and immediately analyzed by flow cytometry for their specific
fluorescence signals.
Detection of apoptotic cells
Staining of cells with the combination of annexinV/FITC and
propidium iodide (PI) was performed according to manufacturer's instructions for detection of early
(annexinV/FITC+/PI ) and late
(annexinV/FITC+/PI+) apoptotic
cells26; both subpopulations were considered to represent
the total fraction of apoptotic cells. Briefly,
2.5 × 105 cells were incubated with saturating
concentrations of annexinV/FITC (Alexis) and PI (Sigma, Vienna
Austria) for 15-30 minutes at RT and immediately analyzed
by flow cytometry.
The breakdown of the mitochondrial transmembrane potential
(  ) was followed by staining the cells with the
fluorescent dye 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazol-carbo-cyanine iodide (10 µmol/L) (JC-1; Molecular Probes, Leiden,
Netherlands) for 20 minutes at RT. This fluorochrome has been
demonstrated to be a reliable probe for assessing changes during
apoptosis.27 Immediately after washing with PBS, the
fluorescence signal intensity of FL-2 (representing cells
with high mitochondrial transmembrane potential) and of FL-1 (low
potential) were analyzed by flow cytometry.
Statistical analysis
For statistical analysis of data, P values were assessed
using a Fisher PLSD test in the analysis of variance
(ANOVA) program or using the paired Student t test program
(Statview 5.1; Abacus Concepts, Berkeley, CA) as appropriate.
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Results |
IL-15R subunits expressed in myeloma cells
It has been demonstrated that IL-15 and IL-2 share the
receptor subunits, but the high-affinity binding of IL-15
(kd = 10-80 pmol/L) depends on the expression of the
IL-15R chain.18,28 To analyze IL-15R expression in
the myeloma cell lines ARH-77, LP-1, MC-Car, OPM-2, and RPMI 8226 and
the myeloma/lymphoblastoid cell line IM-9, a RT-PCR analysis of
IL-15R messenger RNA (mRNA) was performed. PBMC of healthy donors
were used as a positive control,23 and erythrocytes of
these donors were used as a negative control. All cell lines had
detectable amounts of the IL-15R mRNA in 2 molecular forms (Figure
1A), which have been reported to be
generated by alternative splicing.28 Additionally, using flow cytometry, we detected constitutive expression of IL-15R protein in all myeloma cell lines (Table
2). Representative result for expression in
RPMI 8226 cells is depicted in Figure 1B. Since functional studies have
shown that the and chains are necessary for signaling by IL-15,
we subsequently analyzed the myeloma cell lines for these subunits. We
found low but significant expression of IL-2R and medium expression
levels of the chain (Table 2). Representative result in RPMI 8226 cells is depicted in Figure 1B. To investigate the expression of
IL-15R/IL-2R on native malignant plasma cells, we used flow
cytometry to examine CD38high/CD45low cells
from the bone marrow aspirates of 14 patients with multiple myeloma. In
all cases, the myeloma cell fraction expressed all components of the
IL-15R (Table 1).
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| Fig 1.
RT-PCR and flow cytometric analyses of
IL-15R, IL-2R, and control GAPDH.
(A) RT-PCR analysis of expression of IL-15R (upper) and control
GAPDH (lower) in myeloma cell lines. The results indicated that all
myeloma cell lines expressed the mRNA of IL-15R (778 bp) in 2 alternatively spliced isoforms. Lane 1: PBMC from a healthy donor as
positive control; lane 2: erythrocytes from a healthy donor as negative
control; lane 3: dH2O as technical negative control; lane
4: RPMI 8226; lane 5: OPM-2; lane 6: MC-Car; lane 7: LP-1; lane 8:
IM-9; lane 9: ARH-77; and lane 10: the molecular weight marker
pGEM (Promega). (B) Flow cytometric analysis of all
constituents of the IL-15R and the IL-2R in RPMI 8226 cells. Cells were
stained with antibodies specific for the relevant subunits of the
receptors or the respective isotype-matched control mAb. Fluorescence
intensities (FI) were determined by flow cytometry. Each histogram
represents 3 independent measurements.
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Regulation of IL-15R expression by IL-15
It has been reported that stimulation by IL-15 leads to a rapid
down-regulation of its own high-affinity binding site IL-15R, which can be observed as early as 4 hours after
incubation and can last no less than 72 hours.29 At the
same time, IL-15 and IL-2 up-regulate the expression of IL-2R in
human T and B cells. We determined the expression levels for both
IL-15R and IL-2R after an incubation period of 4 hours. As can be
seen in Table 2, stimulation by IL-15 did not down-regulate IL-15R
expression in cells lines, but rather led to a slight increase of the
expression in all cell lines. Even continuous stimulation of myeloma
cell lines with IL-15 for up to 72 hours did not change IL-15R
expression (data not shown). None of the myeloma cell lines expressed
the IL-2R subunit constitutively (Table 2). Representative results of the RPMI 8226 cell line are depicted in Figure 1B. Expression levels
did not appear to increase after IL-15 treatment (Table 2).
Myeloma cells express IL-15 mRNA and protein
IL-15 mRNA has been found in various tissues and cell lines but not
in T cells or B cells under physiological conditions, although
neoplastic T cells may express the cytokine.23,30 We found
that all myeloma cells tested in the present study expressed IL-15 mRNA
(Figure 2A). In addition, we tested whether
intracellular IL-15 protein is present in the myeloma cell lines by
flow cytometry. The ARH-77, IM-9, LP-1, and RPMI 8226 cell lines had a
distinct signal specific for IL-15 protein, whereas the MC-Car and
OPM-2 cell lines had no detectable IL-15 protein (Figure 2B). To
evaluate the significance of IL-15 protein expression in myeloma cell
lines, we analyzed bone marrow aspirates of patients and detected IL-15 protein in the plasma cell fraction of 8 out of 14 patients (Table 1).
Representative examples are given in Figure 2B. The concentration of
IL-15 in culture supernatants of the myeloma cell lines and in sera of
30 successive patients was determined using an ELISA specific for IL-15
(lower detection limit, 10 pg/mL). None of these samples contained
detectable amounts of IL-15 (data not shown).
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| Fig 2.
RT-PCR analysis of IL-15 and control GAPDH, with
expression in myeloma cell lines.
(A) RT-PCR analysis of expression of IL-15 (upper) and control GAPDH
(lower) in myeloma cell lines. All myeloma cell lines expressed the
mRNA of IL-15 (357 bp). Lane 1: PBMC from a healthy donor as positive
control; lane 2: erythrocytes from a healthy donor as negative control;
lane 3: dH2O as technical negative control; lane 4: RPMI
8226; lane 5: OPM-2; lane 6: MC-Car; lane 7: LP-1; lane 8: IM-9; lane
9: ARH-77; and lane 10: the molecular weight marker pGEM (Promega). (B)
Expression of intracellular IL-15 protein in myeloma cell lines and in
the plasma cell fraction of myeloma patients. Cells were stained with a
specific anti-IL-15 mAb (solid line) or the respective isotype-matched
control mAb (dashed line), and FI were determined by flow cytometry.
Representative histograms of the cell lines for 1 of 3 independent
measurements are presented. Staining profiles representative for IL-15
protein-negative (Pat. #4) and for IL-15 protein-positive primary
myeloma cells (Pat. #13) are shown.
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Blocking of autocrine IL-15 or IL-6 and rescuing with the alternate
cytokine
Intracellular IL-15 protein was found in 4 of 6 myeloma cell lines and in 8 of 14 primary cell populations in the
absence of detectable IL-15 in the culture supernatants of cell lines or in the sera of patients. This suggests that small amounts of IL-15
secreted by these myeloma cells could be trapped by the surface IL-15R
and could exert a signal via an autocrine or juxtacrine loop. To test
this hypothesis, we analyzed the effects of neutralization of
endogenous IL-15 on cell survival by saturating concentrations of
blocking mAbs; the effects were compared to those of blocking autocrine
IL-6. ARH-77, LP-1, MC-Car, and RPMI 8226 cells were cultured in low
serum culture medium (0.5% FCS) to reduce the levels of exogenous
growth-promoting and survival factors. These suboptimal culture
conditions did not increase the apoptotic cell fraction in the myeloma
cell lines within the 3-day time frame analyzed
(.07 < P < .66 for the respective cell
lines, Figures 3A, B).
We then added either neutralizing anti-IL-15 mAb (5 µg/mL),
anti-IL-6 mAb (5 µg/mL), or a combination of both and cultivated the cells for 72 hours. According to the manufacturer's
protocol, the concentration of the anti-IL-15 mAb applied is able
to neutralize 5 ng/mL IL-15, which is 500 times the lower
detection limit of the IL-15 analyzed by ELISA. The concentration of
the anti-IL-6 mAb used is about 30 times the one described to inhibit
50% of the biological activity of 2500 pg/mL IL-6. The range of IL-6 detected in the supernatants of the cell lines investigated was 0-26 pg/mL.31 To exclude nonspecific effects of the neutralizing antibodies, all cell lines were treated with a nonreactive
isotype control mAb under identical experimental conditions in each
experiment (Figure 3A, B).
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| Fig 3.
Effects of blocking IL-15 or IL-6 in myeloma cell lines.
(A) Increase in apoptosis by blocking IL-15 or IL-6 in myeloma cell
lines producing IL-15. ARH-77, LP-1, and RPMI 8226 cells were cultured
in RPMI-1640 supplemented with 10% FCS or 0.5% FCS. Neutralizing
agents anti-IL-15 (5 µg/mL) or anti-IL-6 (5 µg/mL) mAbs were
added alone or in combination with recombinant IL-15 (10 ng/mL) or IL-6
(100 ng/mL), respectively. Bars indicate the mean percentages of
apoptotic cells ± SEM (standard error of mean), which was
determined after a 3-day cultivation in 4 independent experiments. (B)
Effects of blocking IL-15 in the MC-Car cell line without detectable
intracellular IL-15 protein. MC-Car cells were treated as described for
part A. Bars indicate the mean percentages of apoptotic cells ± SEM
of 4 independent experiments. Statistical analysis revealed no
significant effects of blocking either autocrine IL-15
(P = 0.7) or IL-6 (P =0.3).
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Blocking endogenous IL-15 led to an increase in the apoptotic fraction
in cell lines with detectable intracellular IL-15 (ARH-77, P = 0.01; LP-1, P = 0.001; RPMI 8226, P = 0.008) (Figure 3A). The effect of neutralizing IL-15 was
comparable to that of blocking autocrine IL-6 in ARH-77, LP-1, and RPMI
8226 cell lines (ARH-77, P = 0.02; LP-1, P = 0.03;
and RPMI 8226, P = 0.005). Autocrine IL-6 production has
previously been reported in the RPMI 8226 cell line, and antisense
experiments confirmed its functional role.32 However,
subclones may exist without detectable IL-6 production.33
The combination of anti-IL-15 and anti-IL-6 mAbs yielded no
synergistic or additive effect on these cell lines (ARH-77,
P = 0.29; LP-1, P = 0.39; and RPMI 8226, P = 0.4). Recombinant IL-15 (10 ng/mL) was able to rescue
cells from apoptosis induced by IL-6 depletion (ARH-77,
P = 0.003; LP-1, P = 0.04; and RPMI 8226, P = 0.01). In turn, exogenous IL-6 abrogated the reduction of
cell survival resulting from anti-IL-15 mAb incubation (ARH77, P = 0.03; LP-1, P = 0.004; and RPMI 8226 cells,
P = 0.04) (Figure 3A). In the MC-Car cell line without
detectable IL-15, blocking experiments with the cytokine-specific
antibody and rescue experiments with the alternative cytokine showed no
significant effects on cell survival (Figure 3B).
IL-15 represents an anti-apoptotic factor for native neoplastic
plasma cells
Next we tried to determine whether stimulation by IL-15 also
functions as a survival signal in native neoplastic plasma cells. The
mononuclear fractions of bone marrow samples of multiple myeloma patients (n = 14) were cultured in the absence or presence of IL-15
(10 ng/mL) for 24 hours, and the percentages of apoptotic cells in the
CD38high/CD45low subpopulations were determined
by the annexinV/FITC/PI assay. Culturing with IL-15 led to a
significant reduction in the percentage of apoptotic cells in the
plasma cell fraction (Figure 4, P = 0.001).
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| Fig 4.
Decrease of spontaneous apoptosis in primary myeloma
cells by IL-15 treatment.
The mononuclear fractions of bone marrow samples of 14 myeloma patients
were cultured without or with IL-15 (10 ng/mL) for 24 hours. The
percentages of apoptotic cells in the
CD38high/CD45low subpopulations were determined
by the annexinV/FITC/PI assay. The significance of IL-15 treatment was
analyzed using the paired Student t test.
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IL-15 and Fas-induced apoptosis in myeloma cell lines
There is accumulating evidence in various cell types that IL-15 is a
potent inhibitor of distinct apoptosis pathways including cell death
induced by Fas.17,30,34 In order to test whether IL-15 also
protects neoplastic plasma cells from Fas-induced apoptosis, the
myeloma cell lines were incubated in cell culture medium. Sixteen hours
prior to induction of apoptosis with the agonistic anti-Fas mAb
CH11 (50 ng/mL), the cell lines were either treated with
IL-15 (10 ng/mL) or left untreated. The kinetics of phosphatidylserine exposure and loss of plasma membrane integrity were monitored from 30 minutes to 48 hours after apoptosis induction. Furthermore, in order to
have a second readout system and to confirm the sensitivity of the
annexinV/FITC/PI test, the breakdown of mitochondrial transmembrane potential , which is also a consistent feature of Fas-induced apoptosis in myeloma cells (I.T., unpublished
observation, 1999), was detected by flow cytometry using
the potential-sensitive dye JC-1. As shown for the RPMI 8226 cell line
in Figure 5A, apoptosis started 2 hours after the CH11
mAb addition, and the apoptotic cell fraction increased in a
time-dependent manner.
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| Fig 5.
Kinetics of apoptosis induction by CH11 mAb and effects
of IL-15 pretreatment.
(A) RPMI 8226 cells were either treated with IL-15 (10 ng/mL) or left
untreated for 16 hours prior to addition of the agonistic anti-Fas mAb
CH11 (50 ng/mL). The percentages of early and late apoptotic cells were
determined 30 minutes to 48 hours after CH11 mAb addition. Results from
4 independent experiments ± SEM are given. Specificity control of
IL-15 signaling via IL-2R. Cells were pretreated with an
anti-IL-2R mAb (2.5 µg/mL;  ) or an isotype-matched control mAb
() 30 minutes prior to stimulation with IL-15 and CH11 mAb. The
extent of apoptosis induction at 7, 24, and 48 hours was compared to
that of cells treated with either CH11 mAb alone ( ) or the
combination of IL-15 and CH11 mAbs ( ). Mean values ± SEM of 3 independent experiments are given. (B) Dose-dependence of IL-15
prestimulation to inhibit Fas-induced cell death. IM-9, MC-Car, and
RPMI 8226 cells were stimulated with increasing concentrations of IL-15
(0-100 ng/mL) 16 hours prior to addition of CH11 mAb (50 ng/mL). The
percentages of apoptotic cells were determined 7 hours after
Fas-triggering by CH11 mAb (50 ng/mL). Results from 3 independent
experiments ± SEM are given. (C) Dose-dependence of CH11
mAb-induced apoptosis in RPMI 8226 cells. Cells were treated for 24 hours with increasing concentrations of CH11 mAb, and the percentages
of apoptotic cells were determined. Statistical analysis revealed a
significant increase in the apoptotic cell fraction following CH11 mAb
treatment compared with untreated cells (control versus 50 ng/mL:
P = .02; control versus 100 ng/mL: P = .002;
control versus 250 ng/mL: P = .001; control versus 500 ng/mL:
P = .0007). No significant difference was observed when
comparing the effects of 50 ng/mL with those at higher concentrations
of CH11 mA: 50 ng/mL versus 100 ng/mL: P = .2; 50 ng/mL
versus 250 ng/mL: P = .1; and 50 ng/mL versus 500 ng/mL:
P = .07. (D) Interdependence of the IL-15 and Fas-pathways in
RPMI 8226 cells. RPMI 8226 cells were pretreated with IL-15 (10 ng/mL)
16 hours prior to the addition of increasing concentrations of CH11 mAb
(10-250 ng/mL). The percentages of apoptotic cells were determined 24 hours after CH11 mAb addition. Results of 3 independent experiments ± SEM are given, and P values were as follows: 10 ng ± IL-15: P = .0009; 20 ng ± IL-15:
P = .0009; 50 ng ± IL-15: P = .001; 100 ng ± IL-15: P = .003; and 250 ng ± IL-15:
P = .08. Apoptotic cells were detected by the
annexinV/FITC/PI assay, and the sum of the percentages of early and
late apoptotic cells was calculated as the percentage of the apoptotic
cell fraction. Statistical analyses were done using a Fisher's PLSD
test, and statistically significant different results are marked.
*P < .05; **P < .01; ***P < .005;
and **** P < .001.
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Prestimulation with IL-15 significantly reduced the sensitivity of RPMI
8226 cells toward Fas-triggering (Figure 5A), and similar results were
obtained with all other myeloma cell lines. The respective percentages
of apoptotic cells at the time points 0 and 24 hours are depicted in
Table 3. Identical results were observed
using the mitochondrial transmembrane potential as a readout system
(data not shown). Since the expression levels of the signal-transducing
subunit IL-2R were low and the specificity of IL-15
signaling via its receptor needed to be confirmed, we performed
experiments with the antagonistic antihuman IL-2R mAb (2.5 µg/mL).21 When added 30 minutes prior to the cytokine
addition, the IL-2R mAb abolished the effect of IL-15 prestimulation
on CH11-induced apoptosis of RPMI 8226 cells (Figure 5A insert). The
signals triggered by IL-15 were transduced rapidly because even
simultaneous addition of IL-15 and CH11 mAbs proved to be as effective
in protecting myeloma cells from Fas-induced apoptosis (data not shown)
as a preincubation period of 16 hours. Furthermore, the effect of IL-15
was dose-dependent in the cell lines RPMI 8226, IM-9, and MC-Car
(Figure 5B). Maximal inhibition was observed at a concentration of 10 ng/mL IL-15. All subsequent experiments were performed using this
concentration.
Testing the dose-dependence of CH11 mAb-induced apoptosis in the RPMI
8226 cell line, a plateau in cell death induction was observed at 50 ng/mL, with only a slight and statistically insignificant increase in
apoptotic cell death at higher concentrations up to 500 ng/mL (Figure
5C). The interdependence of the IL-15 and Fas-pathways in RPMI 8226 cells was demonstrated by: (1) the above-mentioned decrease in the
responsiveness to CH11 mAb by increasing concentrations of the cytokine
(Figure 5B) and (2) the increase in the pro-apoptotic Fas-stimulus to
the maximal effective dose in RPMI 8226 cells, which lessened the
protective effect of IL-15 pretreatment (Figure 5C, D).
IL-15 does not modify expression patterns
Since IL-15 had a significant effect on the regulation
of spontaneous as well as Fas-induced apoptosis in myeloma cells, we investigated IL-15 as a cause of alterations in the expression pattern
of Fas or its physiological ligand. mRNA and protein analyses by RT-PCR
and flow cytometry, respectively, revealed that stimulation of myeloma
cells with IL-15 (10 ng/mL) for 4-24 hours did not decrease the
expression of Fas or FasL (data not shown). Using native plasma cells,
which express Fas at low levels, no significant effect of IL-15
treatment on Fas expression could be observed (data not shown). To
determine whether IL-15 acts as a survival factor for myeloma cells via
regulation of Bcl-2 or Bax, we analyzed the expression of both antigens
at the mRNA as well as protein levels after incubation with IL-15 (10 ng/mL) for 4-24 hours. We found no significant changes in Bcl-2 or Bax
expression levels in any of the cell lines (data not shown).
Effect of blocking IL-15 on induced apoptosis by Fas, dexamethasone,
or cytotoxic treatment
We found that autocrine IL-15 preserves myeloma cells
from spontaneous cell death. This raised several questions: Does
autocrine IL-15 also contribute to the reported low sensitivity to
apoptosis induced by anti-Fas antibodies in a fraction of myeloma cell
lines and native malignant plasma cells?10,35-37 Or does it
contribute to the development of resistance toward treatment with
dexamethasone or cytotoxic drugs such as doxorubicin or vincristine? As
measured by the degree of apoptosis induced, our long-term in vitro
propagated cell lines were relatively insensitive to CH11 mAb
concentrations of 10 ng/mL; vincristine (VCR), 2 µg/mL; doxorubicin
(Doxo), 2 µg/mL; or dexamethasone (DEX), 1 µmol/L. (See Figure
6.)
View larger version (21K):
[in this window]
[in a new window]
| Fig 6.
Blocking autocrine IL-15 increases cell death of myeloma
cells induced by Fas-triggering, vincristine, or doxorubicin treatment
but not that induced by dexamethasone.
RPMI 8226 and MC-Car cells were cultured in the absence or presence of
anti-IL-15 mAb (5 µg/mL) for 48 hours and treated with CH11 mAb (10 ng/mL), vincristine (VCR, 2 µg/mL), doxorubicin (Doxo, 2 µg/mL), or
dexamethasone (DEX, 1 µmol/L) for another 16 hours. The bars indicate
the mean percentages of apoptotic cells ± SEM determined in 3 independent experiments.
|
|
In the IL-15-producing cell line RPMI 8226, prior to
incubation with anti-Fas mAb, vincristine, or doxorubicin for 24 hours, we blocked autocrine IL-15 for 48 hours. This led to a significant increase in the proportion of apoptotic cells (CH11 mAb,
P = .0002; vincristine, P = .0015; and doxorubicin,
P = .0001), but it did not influence the sensitivity of
myeloma cells toward incubation with dexamethasone (P > .3)
(Figure 6). In the MC-Car cell line with no detectable intracellular
IL-15 protein, treatment with neutralizing anti-IL-15 mAb did not
increase the sensitivity of these cells to Fas-triggering
(P = .9); to treatment with cytotoxic drugs (vincristine,
P = .75; doxorubicin, P = .6); or to dexamethasone (P = .75) (Figure 6).
| |
Discussion |
The present study reports the expression of all functional IL-15R
components including the specific -chain in 6 of 6 myeloma cell
lines and in the neoplastic plasma cell fraction from all 14 myeloma
patients investigated. In normal B cells, a rapid down-regulation of
IL-15R by IL-15 was reported.29 The down-regulation has been found to attenuate the responsiveness of B cells to this cytokine,
while the concomitant up-regulation of IL-2R sensitizes them to IL-2
ligation.29 Thus, IL-15 may be important for initial T- and
B-cell responses, but subsequently it might favor their responses to
IL-2. In contrast, treatment of myeloma cell lines with IL-15 for up to
72 hours did not decrease the expression levels of IL-15R (Table 2
and data not shown). Furthermore, we detected neither constitutive
IL-2R expression in myeloma cell lines nor up-regulation following
IL-15 stimulation (Table 2, Figure 1B). This altered regulation of
IL-15R seems to be a prerequisite in order for the cytokine to
provide a continuous signal to myeloma cells independent of IL-2.
The expression of all IL-15R constituents in all myeloma cell samples
investigated raises the question of a potential autocrine IL-15 loop in
this tumor. In fact, some neoplastic cell types that produce IL-15
mRNA, namely lung carcinoma,38 adult T-cell leukemia,23 cutaneous T-cell lymphoma,30 and
melanoma cells,39 have recently been identified. We
observed constitutive expression of IL-15 mRNA in 6 of 6 myeloma cell
lines (Figure 2A) and the intracellular presence of IL-15 protein in 4 of 6 myeloma cell lines (Figure 2B). IL-15 mRNA production in the
absence of measurable IL-15 protein is frequently found in several cell
systems.18,38,40 It is thought to be caused by a low
efficiency of mRNA translation40,41 and the low
intracellular levels of the protein, which require sophisticated
techniques like confocal laser scanning microscopy for
detection.39 This might explain why, in the plasma cell fraction of 6 of 14 patients, intracellular IL-15 protein was never
found. However, the finding that 2 cell lines produced the transcript
but lacked detectable protein of the cytokine might also be caused by a
tight control of the translation process under an as-yet undefined
stimulus. Alternatively, the production of detectable amounts of IL-15
protein may differ in various phases of the disease. The fact that all
myeloma cell lines as well as the primary neoplastic cells of all
patients investigated expressed the IL-15R/IL-2R chains but
only 8 of 14 detectable amounts of its physiological ligand may
also point to the coexistence of both autocrine and paracrine
stimulation of myeloma cells. IL-15 mRNA and protein have been detected
in various cell types found in the microenvironment of plasma cells,
such as primary human bone marrow stroma cells,42
endothelial cells,43 and fibroblasts, but not in normal B
cells.14,44 The potential impact of autocrine IL-15
production, its regulation during the transformation process, the
course of disease, and the relative contribution of autocrine or
paracrine stimulation loops in the bone marrow of myeloma patients will
need to be addressed in future studies.
Investigating the biological consequences of either autocrine or
paracrine IL-15R stimulation in myeloma cell lines, we focused on
apoptosis control, which has previously been demonstrated to be under
the influence of the cytokine in several cell
types.17,30,34 As demonstrated by the blocking experiments
shown in Figure 3A, autocrine IL-15 protected these cells from
spontaneous apoptosis. Using exogenous cytokine as a surrogate for
paracrine IL-15, we observed a decrease in the percentage of primary
myeloma cells undergoing spontaneous apoptosis (Figure 4). These data
point to a role of IL-15 in the propagation of a neoplastic plasma cell clone and raise the question concerning the extent of its
biological activity and its role as a survival factor in the
context of other cytokines.
It has been demonstrated that both autocrine45 and
paracrine IL-646 represent important anti-apoptotic factors
for myeloma cells by protecting them from spontaneous apoptosis or
death by serum starvation.11 We found that the degree of
cell survival reduction resulting from neutralizing endogenous IL-15
loops is comparable with the reduction resulting from neutralizing
autocrine IL-6 loops (Figure 3A). The extent of protection observed for both cytokines (range for all cell lines tested: IL-6, 25%-48%; IL-15, 38%-48%) is in agreement with the 40% mean reduction in apoptosis following paracrine IL-6 stimulation in the IL-6-dependent ANBL6 cell line.46 Although the cytokines did
not prove additive or synergistic in protecting against spontaneous
apoptosis, each of them was able to compensate for the neutralization
of the other respective cytokine loop (Figure 3A). Several cytokines
may contribute to the longevity of neoplastic plasma cells, but to our
knowledge none of them has been shown to substitute for IL-6, although
the data on GM-CSF remains controversial.47,48
Our data show that IL-15 not only protects against spontaneous
apoptosis but also against a broader range of death-inducing signals
including Fas-triggering. The expression of functional Fas in myeloma
cell lines as well as in native plasma cells from myeloma patients has
been reported.10,36,37 The responsiveness of myeloma cells
to Fas-induced apoptosis proved to be variable and was suggested to be
regulated by a soluble factor10; subsequently, IL-6 was the
identified factor.13,49 In the present study, exogenous
IL-15 persistently decreased the percentage of cells succumbing to
Fas-induced apoptosis in a dose-dependent fashion in all 6 myeloma cell
lines tested (Figures 5A and B, Table 3). However, it has to be
mentioned that increasing the concentration of CH11 mAb to the maximal
effective dose in RPMI 8226 cells (Figure 5C) reduced the protective
effect of the maximal inhibitory concentration of IL-15 (Figure 5D).
This might indicate that the balance between the degree of recruitable
IL-15 and the extent of Fas-stimulation influences the fate of the
neoplastic clone and that the cytokine might be operative over a
limited range of concentration of molecules available for cross-linking Fas.
There are two unknowns for myeloma cells in their natural
bone marrow microenvironment: the median number of FasL molecules meeting their receptors in a given period of time and the degree of
juxtacrine IL-15 stimulation. Despite these facts, inhibition of
endogenous IL-15 increased the sensitivity of RPMI 8226 cells to CH11
mAb (Figure 6). In addition, exogenous IL-15 lowered the sensitivity of
myeloma cells to Fas-triggering over a broad range of CH11 mAb
concentrations. The pro-apoptotic efficacy of CH11 mAb, even at the
maximally lytic concentration, was reduced by IL-15, although this
effect was not statistically significant (Figure 5D). These data
suggest that IL-15 might provide a survival advantage for the
neoplastic clone during the immune attack of FasL+
Fas-sensitive cytotoxic T cells. As we demonstrated in a recent publication,25 when exposed to FasL+ effector
cells, the myeloma cells were completely resistant to Fas-induced
apoptosis, whereas they proved to be potent effectors and killed
T-ALL cells using their own FasL.25
These data imply a highly effective intrinsic control mechanism
governing sensitivity as well as direction of FasL/Fas signaling in
myeloma cells. Our demonstration of functional IL-15R expression on
myeloma cells and of its anti-apoptotic capacity indicates that beside
IL-6,13,49 IL-15 might also be a signal leading to the
protection of these cells from the attack of FasL+ effector
cells in tumor surveillance.
The exact cellular mechanisms of action of IL-15 are still to be
elucidated because the cytokine did not influence the expression levels of either Fas or FasL (data not shown). In this study, previous investigations of the myeloma cell lines tested have demonstrated that the expression levels of both Fas and Bax, but no
other Bcl-2 family member, were positively correlated with the
sensitivity to CH11 mAb.36 Although in NK cells, IL-15
augmented survival during serum-free cultivation by maintaining the
endogenous Bcl-2 protein levels,15 the results of our
present study exclude the possibility of IL-15 exerting its
anti-apoptotic effects on myeloma cells by modulating the
expression of Bcl-2 or Bax. Similarly, anti-apoptotic activity of IL-6
was not mediated via regulation of Bcl-2 or Bax,11,13 but
recent results suggest that Bcl-XL might be a target for
IL-6.50 Whether this holds true also for IL-15 will have to
be tested in future studies.
To further analyze the role of autocrine IL-15 in the regulation of
apoptosis in myeloma cells, we also investigated cell death following
treatment with chemotherapeutic drugs. While autocrine IL-15 protected
RPMI 8226 cells from cell death induced by Fas-triggering, vincristine,
or doxorubicin treatment (Figure 6), it had no protective effect on
dexamethasone-induced cell death (Figure 6). Our data on the effects of
IL-15 and IL-6, either alone or as a rescue factor when 1 of the
cytokines is depleted, in conjunction with data previously reported on
IL-6,11,13,49 suggest redundancy for these cytokines
in protecting myeloma cells from spontaneous and Fas-induced apoptosis.
However, their anti-apoptotic activities may differ when a broader
spectrum of death inducers is considered. In fact, in previous data,
IL-6 protected from corticosteroid-induced apoptosis12 but
not from doxorubicin-induced apoptosis,11 while in our
study IL-15 behaved in the opposite way. This might have
implications on the therapeutic exploitation of cytokine targeting by mAbs, superantagonists, or antisense strategies. A
cocktail of inhibitors might be required for antagonizing myeloma cell
survival in its microenvironment, while deprivation of a single cytokine might be sufficient for sensitization toward
cytotoxic therapies.
| |
Footnotes |
Submitted February 1, 1999; accepted August 31, 1999.
Supported by a grant from the Österreichische
Krebshilfe-Krebsgesellschaft Tirol.
Reprints: Richard Greil, Laboratory of Molecular Cytology,
Department of Internal Medicine, University of Innsbruck, Anichstrasse
35, A-6020 Innsbruck, Austria; e-mail: richard.greil{at}uibk.ac.at.
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