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IMMUNOBIOLOGY
From the Department of Biomedical Sciences and Human
Oncology, Section of Internal Medicine and Clinical Oncology,
University of Bari Medical School, Bari, Italy.
In this study, flow cytometry was used to evaluate interleukin-6
(IL-6) production by bone marrow mononuclear cells from 47 patients
with multiple myeloma (MM) in different clinical stages and 15 patients
with monoclonal gammopathy of undetermined significance. In patients
with MM, autocrine IL-6 production paralleled the clinical disease
stage. The largest proportion of
syndecan-1+/IL-6+ cells was detected in
patients with resistant relapse or primary refractory disease,
suggesting that tumor progression involves expansion of myeloma cells
producing IL-6. The authors assessed autocrine IL-6 production and in
vitro proliferation and apoptosis of myeloma cells in 6 myeloma cell
clones (MCCs) and in 2 myeloma cell lines, namely IM-9 and U-266-1970,
which showed different sensitivities to the addition of exogenous IL-6.
Autocrine IL-6 production was observed in IL-6-independent MCC-2,
MCC-3, and MCC-5 cloned from patients with aggressive disease and in
the IM-9 cell line. In contrast, IL-6-dependent MCC-1, MCC-4, and MCC-6 were syndecan-1+ and IL-6 Interleukin 6 (IL-6), a pleiotropic cytokine
produced by a variety of cells, is the most important growth factor for
human multiple myeloma (MM).1-3 Several findings support
in vivo and in vitro roles for IL-6 in the disease: specifically, (1)
serum IL-6 and IL-6R levels were found to correlate with disease
activity4-6; (2) therapy with anti-IL-6 monoclonal
antibody (mAb) transiently reversed disease
manifestations7; (3) in vitro proliferation of myeloma
cells was suppressed by neutralizing mAbs to either IL-6 or its
cellular receptors8-9; and (4) inactivation of IL-6 messenger RNA by antisense oligonucleotides inhibited proliferation of
plasma cells.10 Furthermore, other cytokines, such as
IL-1, IL-3, and granulocyte-macrophage colony-stimulating factor,
regulate myeloma cell proliferation in synergy with IL-611
or by inducing IL-6 production in myeloma cells or the tumor
environment.12-13
The cellular origin of IL-6 is controversial. Several
authors13-16 showed that it is produced by the myeloma
cells themselves (autocrine hypothesis). Other
studies,17-19 however, point to its paracrine production
by cells in the bone marrow (BM) and suggest that proliferation of
myeloma cells depends on close contact with stromal
cells.20-21 The major criticism of the autocrine
hypothesis is based on the facts that marrow IL-6-producing
cells other than myeloma cells may occur in the enriched myeloma cell
population1 and that sorting of malignant plasma cells
using CD38 or CD45 antigen expression can activate IL-6
production.1
More recent studies22-23 demonstrated that IL-6 is an
antiapoptotic factor for myeloma cells in that it prevents
spontaneous,22,24 drug-induced,22-23,25 and
Fas-induced26 apoptosis and thereby modulates MM cell
growth and survival. Experimental evidence suggests that these 2 effects are dissociable and mediated by distinct signaling
pathways.27 Interaction of IL-6 with its Dexamethasone (Dex) alone or in combination with cytotoxic drugs is
frequently used in the treatment of MM.32 Its mechanism of
action is unclear: it has been shown that the binding of
glucocorticoids to specific receptors alters the gene transcription in
a positive or negative manner. In particular, Dex
(10 Because of the central role of IL-6 in MM, it is important to define
its cellular source precisely. Therefore, in this study, we evaluated
autocrine and paracrine IL-6 production in bone marrow mononuclear
cells (BMMC) from patients with MM by using flow cytometry to identify
directly the phenotype of cells producing IL-6. We also analyzed the
relation between autocrine IL-6 production and susceptibility of
myeloma cells to spontaneous and drug-induced apoptosis.
Patients
BMMC and cell cultures
Flow cytometry identification of myeloma cells Flow cytometry was used to phenotype fresh plasma cells and established MCCs by comparing their cytoplasmic immunoglobulin (Ig) content with their membrane expression of CD38 and syndecan-1 (CD138) antigens. Briefly, 1 × 106 cells were incubated with the unconjugated mAb to CD38 (Becton Dickinson, Mountain View, CA) and with the second antibody conjugated to peridinin chlorophyll protein (Becton Dickinson). Cells were then treated with a fluorescein isothiocyanate (FITC)-conjugated mAb to syndecan-1 (Serotec Limited Ltd, Oxford, United Kingdom), fixed, permeabilized, and incubated with a polyclonal phycoercythrin (PE)-conjugated antihuman -chain or  -chain
antiserum (Jackson Immunoresearch, West Grove, PA). Cytometric analysis
was done with the Cell Quest program in a fluorescence-activated
cell-sorter scanner (FACScan; Becton Dickinson).
Intracellular IL-6 production Immunofluorescence staining of intracellular IL-6 was done according to the method described by Prussin and Metcalfe,39 with slight modifications. Briefly, in accordance with the procedure suggested by the manufacturer (Biosource, Camarillo, CA), cell samples (1 × 106) were stimulated for 6 hours with 1 µg/mL lipopolysaccharide (LPS; Sigma, St Louis, MO) in round-bottomed tissue culture tubes and in the presence of 2 µM monensin (Sigma) to prevent extracellular transport of the cytokine. LPS is a nonspecific immunostimulatory treatment used to elicit cytokine production.40 Cultures without monensin or exogenous stimuli were included as controls. In preliminary experiments, we found that the optimal incubation time for cells to express IL-6 in short-term cultures was 6 hours. After incubation, cells were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 15 minutes at room temperature. After 2 additional washings in PBS, samples were resuspended in permeabilization buffer (PBS containing 0.5% bovine serum albumin and 0.5% saponin) and incubated for 30 minutes at 4°C with PE-conjugated anti-IL-6 mAb (Biosource). To ensure the specificity of IL-6 staining, the binding of anti-IL-6 mAb was blocked with an excess of cytokine (500 U/mL) before staining. As a last step, myeloma cells were identified by using an FITC-conjugated mAb specific to syndecan-1. The negative controls included isotype-matched irrelevant antibodies. Samples were analyzed with a FACScan.Proliferation assay The proliferative rate of each MCC and myeloma cell line was evaluated by assessing tritium-thymidine uptake. Briefly, cells (104/well) were incubated overnight in culture medium or the presence of 500 U/ml IL-6 (Genzyme) and pulsed overnight with tritium-thymidine (7400 Bq/well; Du Pont NEN, Bad Homburg, Germany). Uptake was measured in a -counter (Beckman, Palo Alto, CA). The
sensitivity of MCCs to exogenous IL-6 was evaluated on the basis of
their tritium-thymidine uptake and expressed as a stimulation index (SI) calculated as follows: counts per million per sample plus IL-6
divided by counts per million per sample. SI values below 3 and greater
than 3 indicated IL-6-independent and IL-6-dependent cell
proliferation, respectively.
Inhibition assay To verify the effect of IL-6 on myeloma cell growth, an inhibition assay of the proliferative rate of MCCs was done by using an anti-IL-6 mAb (clone AH65; Immunotech, Marseilles, France). Preliminary experiments showed that this mAb inhibited the in vitro growth of IL-6-dependent MCCs in the presence of 25 pg/mL exogenous IL-6. Complete blocking was obtained with a mAb concentration of 10 ng/mL. The inhibitory effect of the same mAb on IL-6-independent MCCs was evaluated by using bromodeoxyuridine (BrdU)-propidium iodide (PI) staining.41 Briefly, washed MCCs were cultured in 96-well plates at a density of 1 × 106 cells/mL for 20 hours at 37°C in culture medium or the presence of anti-IL-6 mAb (100 ng/mL). Cells were then incubated for 3 hours in the presence of 15 µM BrdU (Sigma), fixed with 70% cold ethanol, and treated with 2N hydrochloric acid to partly denature the DNA. After washing with 0.1 M sodium borate to neutralize the acid, the cell suspensions were incubated with FITC-conjugated mAb to BrdU (Becton Dickinson), resuspended in 5 µg/mL PI solution (Sigma), and analyzed by using flow cytometry. The uptake of BrdU (a thymidine analog) into DNA identifies cells undergoing DNA synthesis. The inhibitory effect was calculated as follows: (percentage of BrdU+ cells in the presence of anti-IL-6 mAb divided by the percentage of BrdU+ cells in culture medium) times 100.Assessment of apoptosis The susceptibility of MCCs and myeloma cell lines to spontaneous and Dex-induced apoptosis was evaluated by using cytofluorometric analysis of PI cell staining.42 Cells (1 × 106) incubated in complete medium or with 10 6 M Dex (Sigma) for 24 hours at 37°C were fixed with
70% cold ethanol for 3 hours at 4°C, incubated overnight with PI
isotonic solution (50 µg/mL), and evaluated by using flow cytometry.
The extent of the subdiploid DNA peak reflected the percentage of
apoptotic cells.
Statistical analysis The Student t test and, in several instances, the Wilcoxon nonparametric method were used to compare the mean values of specific phenotype expression and in vitro variables.
Autocrine and paracrine IL-6 production Detection of cytoplasmic IL-6 in BMMC by double-fluorescence staining directly identifies the phenotype of IL-6-producing cells and consequently distinguishes autocrine from paracrine cytokine secretion. Myeloma cells were identified using an FITC-conjugated mAb to syndecan-1, which is specifically expressed by myeloma tumor cells among BMMC (Figure 1). Usually, myeloma cells are identified by the phenotype CD38bright (Figure 1B, R2 region). However, CD38 is an activation antigen that is also expressed by other cells and is sometimes not expressed on myeloma cells (Figure 1A, R1 region). Gating of BMMC on syndecan-1+ populations (R1 and R2 regions) demonstrated expression of monoclonal cytoplasmic Ig on 95% of syndecan-1+ cells (Figure 1C and 1D) and suggested that syndecan-1+ populations were indeed myeloma cells.43 Therefore, the presence of syndecan-1+/IL-6+ cells and syndecan-1/IL-6+ cells indicates autocrine
and paracrine cytokine production, respectively (Figure
2). In particular, IL-6 staining was
detected only in the saponin-permeabilized samples (Figure 2C compared with Figure 2B and Figure 2I compared with Figure 2H) and was blocked
by the addition of an excess of IL-6 (Figure 2D and 2L), indicating
that cell staining was due to intracellular cytokine, not false
staining. In addition, because the percentages of
syndecan-1+/IL-6+ cells in short-term cultures
of LPS-stimulated BMMC (Figure 2E,M) were similar to those in
unstimulated cells (Figure 2F,N), they indicated the in vivo capacity
of cells to produce the cytokine.44 LPS stimulation
increased the fluorescence intensity of positive cells (Figure 2M
compared with Figure 2N and Figure 2E compared with Figure 2F) and
allowed a clear definition of the cut-off point at which a
cytokine-specific signal was considered positive.
Cytofluorometric analysis of cytoplasmic IL-6 in BMMC showed the
presence of both autocrine and paracrine IL-6 production in patients
with MM and MGUS (Table 1). However,
whereas elevated percentages of
syndecan-1
Heterogeneous IL-6 production by myeloma cells Flow cytometry analysis showed that IL-6 production by malignant plasma cells was heterogeneous (Figure 3). The proportion of syndecan-1+ cells expressing cytoplasmic IL-6 ranged from 16% to 99% of total syndecan-1+ cells and was higher in patients with relapse (70.1% ± 12.2%) or aggressive disease (resistant relapse and primary refractory disease, 88.0% ± 7.8%) than in patients with a recent diagnosis (41.3% ± 16.2%) or patients in remission (30.9% ± 10.1%; Figure 3).
Figure 4 shows the heterogeneous IL-6
production and its parallelism to clinical stage in 4 representative
patients. In particular, patients with a recent diagnosis (Figure 4B,C)
had a wide distribution of ratio values (21%-65%), whereas in
patients with aggressive disease (Figure 4A), the entire myeloma cell
population produced IL-6. These data suggest that selection and
expansion of syndecan-1+/IL-6+ cells occur
during tumor progression.
Autocrine IL-6 production and IL-6-independent cell growth Because the in vitro proliferation of some myeloma cell lines is independent of IL-6, the relation between IL-6 insensitivity and autocrine IL-6 secretion was investigated by determining cytoplasmic cytokine production in 6 MCCs and 2 myeloma cell lines. In vitro growth of the cell line U-266-1970 is dependent on exogenous IL-6, whereas that of IM-9 is independent of exogenous IL-6.8The in vitro sensitivity to IL-6 of the MCCs paralleled their
heterogeneous IL-6 production (Table 2).
IL-6 expression was minimal in MCC-1, MCC-4, and MCC-6, which showed
the highest proliferative response to exogenous IL-6, whereas MCC-2,
MCC-3, and MCC-5, the proliferative rates of which were insensitive to
IL-6, expressed cytoplasmic IL-6 in 77% to 100% of cells. Two groups
of myeloma cell clones were thus distinguished. IL-6+ MCCs
(MCC-2, MCC-3, and MCC-5) and the IM-9 myeloma cell line showed great
spontaneous proliferation not influenced by exogenous IL-6 (SI < 3), whereas in vitro growth of IL-6
Furthermore, blocking experiments using anti-IL-6 mAb, which inhibits
IL-6/IL-6R
Specific results obtained with 3 representative MCCs are shown in
Figure 6. The elevated percentages of
spontaneously proliferating cells observed in the IL-6-insensitive
MCC-2 and MCC-3 (BrdU+ cells, 61.3% and 30.9%,
respectively; Figure 6A) paralleled the positivity of the clones for
cytoplasmic IL-6 (Figure 6B). Addition of anti-IL-6 mAb inhibited
proliferation of IL-6+ MCCs (BrdU+ cells,
27.2% and 9.9%, respectively), but it had no effective on
IL-6
Autocrine production of IL-6 and susceptibility of MCCs to apoptosis Because IL-6 rescues cells from spontaneous22,24 and drug-induced apoptosis,22-23,25 our next experiments compared the apoptotic susceptibility of MCCs to IL-6 production by using cytofluorometric analysis of DNA content after PI staining. As expected, 2 profiles of response were obtained (Table 3). Spontaneous and Dex-induced apoptosis was observed only in the IL-6 MCCs, the growth of which,
in the absence of exogenous IL-6, was characterized by a high
susceptibility to spontaneous apoptosis. This also was increased
significantly by the addition of Dex (106 M). In
contrast, IL-6+ MCC-2, MCC-3, and MCC-5 were completely
insusceptible to apoptosis. Addition of exogenous IL-6 promptly rescued
IL-6/IL-6-dependent MCC-1, MCC-4, and MCC-6 from
apoptosis and stimulated their proliferation (SI > 3), whereas it
had no effect on IL-6+/IL-6-independent MCCs.
These data demonstrate the close relation between IL-6 production and apoptotic susceptibility of MCCs. They also imply that autocrine IL-6 production offers protection against spontaneous and drug-induced apoptosis.
In this study, flow cytometric detection of intracellular IL-6 and the correlation of its expression with cell-surface phenotype in fresh BMMC, without sorting of cell subpopulations, revealed that autocrine IL-6 production occurs in MM and parallels its clinical stages. Although several studies support the idea that IL-6 is produced by myeloma cells,14-16,45 its origin remains controversial. The major criticism of these studies is based on the methods used to evaluate autocrine IL-6 production.1 In the current study, primary myeloma cells were identified by assessing the expression of syndecan-1 on viable cells.43,46-48 We showed that 95% of these syndecan-1+ cells secreted monoclonal Ig, but could not rule out the possibility that a small percentage of them were not tumor cells. However, compared with other techniques, which are based on sorting of myeloma cells from BMMC, cytoplasmic cytokine staining provides a simple method for directly evaluating IL-6 production by tumor cells. We found that autocrine IL-6 production was heterogeneous and that the highest proportion of IL-6-producing cells among syndecan-1+ cells was observed in samples from patients with active disease. These findings probably reflect the heterogeneity of myeloma cells49 related to differences in their degree of maturation.15-16 We speculate that the IL-6+ phenotype could be a feature of immature cells15-16 (an idea supported by the presence of the relative defect of CD38 expression24) and may reflect clinically aggressive disease. In this study, patients with primary refractory disease and resistant relapse had the largest proportions (almost 100%) of IL-6-producing syndecan-1+ myeloma cells, a result suggesting that aggressive MM involves in vivo expansion of IL-6-producing malignant plasma cells. In vitro functional studies revealed a close correlation between
autocrine IL-6 production and IL-6-independent cell proliferation. The
proliferative rate (SI Our investigation of defective apoptosis observed in myeloma
cells24 in relation to autocrine IL-6 production and the
proliferative response to IL-6 showed that IL-6+ MCCs
resist both spontaneous and Dex-mediated apoptosis,53 whereas IL-6 Urashima et al31 demonstrated that changes in the myeloma cell cycle triggered by Dex and IL-6 are related to changes in p21 protein expression. Increased p21 protein expression inhibited cyclin D-CDK4, cyclin D-CDK6, and cyclin E-CDK2 complexes and increased dephosphorylation of pRb, resulting in growth suppression.54 p21 was constitutively expressed in IL-6-responsive myeloma cell lines and its expression was down-regulated and up-regulated by IL-6 and Dex, respectively.34 IL-6 prevented Dex-induced G1 growth arrest by overcoming p21 up-regulation. In contrast, Dex and IL-6 had no effect on p21 expression of myeloma cell lines not responsive to IL-6, whereas p21 was not detected in Dex-resistant MM-derived cell lines.34 It can thus be speculated that autocrine IL-6 prevents Dex-mediated apoptosis and drives cell proliferation of IL-6+/IL-6-insensitive myeloma cells by down-modulating p21 expression. Another intriguing hypothesis is related to Ras oncogene mutations. Ras-dependent mitogenic signals regulate cell-cycle progression by pRb. It was shown that Ras inactivation induces dephosphorylation of pRb and G1 cell arrest.55 Several studies56 found that N-Ras and K-Ras mutations are common in MM and that their frequency increases with disease progression. The association of Ras mutations with the disease stages described by Durie and Salmon suggests that they are progression events.56 On the other hand, transfection of activated Ras complementary DNA into the IL-6-dependent ANBL6 myeloma cell line resulted in IL-6-independent cell growth and cell protection from glucocorticoid-induced apoptosis, similar to that observed with the addition of IL-6.57 Activating Ras mutations may thus be supposed to induce myeloma cell growth independent of paracrine IL-6 and resistance to glucocorticoid apoptosis. Studies assessing whether Ras mutations are related to autocrine IL-6 production are currently being conducted in our laboratory. In conclusion, our results indicate that autocrine IL-6 production reflects a highly malignant phenotype of myeloma cells. MM is a multistep transformation process56 in which several oncogenic events result in the selection and malignant expansion of a single IL-6+ clone. IL-6+ myeloma cells have a high proliferative capacity and are refractory to drug-induced apoptosis. Because Dex is used to treat MM, our results may have clinical implications. Selection and expansion of IL-6+ Dex-resistant myeloma cells could explain Dex resistance and MM progression.
We thank Mr Vito Iacovizzi and Miss Simona Squicciarini for secretarial and technical assistance.
Submitted January 24, 2000; accepted September 17, 2000.
Supported by grants from Associazione Italiana della Ricerca sul Cancro, Milan, Italy; and the Ministry of University and Scientific and Technological Research, Rome, Italy.
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.
Reprints: Franco Dammacco, DIMO
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Top
Abstract
Introduction
complexes, prevented cell proliferation of
IL-6+ MCCs. Flow cytometry evaluations after propidium
iodide staining revealed different susceptibilities of MCCs to
cell death. IL-6-producing MCCs showed minimal spontaneous and
dexamethasone-induced apoptosis, whereas a regular amplitude of
apoptosis occurred in the IL-6
3 or > 3) of each MCC paralleled the IL-6+ phenotype, and proliferation was specifically
inhibited by an anti-IL-6 mAb. In addition, the spontaneous
proliferative capacity of IL-6+ MCCs was higher than that
of IL-6
Section of Internal
Medicine and Clinical Oncology, 11 Plaza G Cesare, 70124, Bari,
Italy; e-mail: 
in multiple myeloma cells.
Blood.
1997;90:279-289