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Interleukin-6 Gene Expression in Multiple Myeloma:
A Characteristic of Immature Tumor Cells
By Hiroyuki Hata, Huiqing Xiao, Maria Teresa Petrucci, Jeff Woodliff, Ruixin Chang, and Joshua Epstein
Interleukin-6 (IL-6) has been suggestedto play a major role
in multiple myeloma. To investigate the source and target
cells of IL-6 activity in multiple myeloma, expression of
the cytokine and its receptor genes by myeloma plasma
cells was studied. Tumor cells were sorted from bone
marrow aspirates of myeloma patients using 4-parameter gating. Myeloma cells were identified as CD38high
CD45negnive-inte""edirts
and by their light-scatter characteristics. Sorted cells contained only myeloma plasma cells. No
contaminating cells were present as determined morphologically, by monoclonal cytoplasmic lg analysis, and by
polymerase chain reaction (PCR) amplification of marker
genes. Myeloma cells from 45% of patients expressed IL6; IL-6 receptor transcripts were found in 68% of the specimens. IL-6 gene expression correlated with expression of
the IL-6 receptor gene (P < .005). Correlations observed
between the expression of CD45, a protein tyrosine phosphatase expressed by B lymphocytes but not by plasma
cells, and the expression of the IL-6 and IL-6-receptor
genes (P < .0002 and P < .005, respectively) suggest
that an autocrine IL-6 loop is functioning in myeloma in
preplasma cells.
0 1993 by The American Society of Hematology.
T
some surface antigens, coexisted in most patients. Expression of IL-6 and its receptor genes by highly purified myeloma cells from 22 patients was determined.
HE MATURATION STAGE of B-cell neoplasias vary
from pre-B-cell leukemia to multiple myeloma, a hematologic malignancy characteristically associated with
mature plasma cell morphology and function. Whereas the
recognizable tumor cells in myeloma are the most mature B
cells, early lymphoid cells are involved in the disease and
probably represent the proliferative, preplasma cells compartment.'-' The apparent involvement of a common early
progenitor in the B-cell neoplasias and possibly in all hematologic malignancies6underscores the importance of defining the factors that determine the phenotypic presentation
of the neoplasms.
Recently, studies have shown a role of lymphokines in the
regulation of proliferation and differentiation ofthe hematopoietic system cells. The multifunctional interleukin-6 (IL6) has been proposed as the major myeloma cell growth
factor.' Whereas some studies have suggested that IL-6
functions as an autocrine growth f a ~ t o r ,others
~ , ~ have favored a paracrine growth stimulation mechanism." IL-6
and IL-3 function in concert to promote proliferation and
differentiation of early B cells in the bone marrow (BM) and
blood from myeloma patients into myeloma plasma
cells. ',I2 Apparently, granulocyte-macrophagecolony-stimulating factor (GM-CSF) and IL- l a modulate the proliferative response of myeloma cells to IL-6.'3,'4 Tumor necrosis
factor a (TNF-a), which is produced by accessory cells,''-"
stimulates the production of IL-6 and other lymphokines by
a variety of hematopoietic and other cells.
Whether IL-6 stimulation of myeloma is autocrine or
paracrine is important clinically. For instance, neutralizing
anti-IL-6 antibody therapy could present an attractive option for a paracrine-stimulated IL-6-dependent tumor.
However, such therapy may not be effective against an autocrine IL-6-dependent tumor, especially if IL-6 does not
need to exit the cell to be active." In such case, IL-6 receptor
~ ~ ~ be an
targeting therapy, such as recently r e p ~ r t e d , "could
attractive alternative.
Investigations aimed at clarifying the sources and activities of IL-6 in myeloma have been impeded by the difficulty
of obtaining purified tumor cells from myeloma patients in
adequate numbers. A modification to a recently developed
method2' was used to purify myeloma cells from BM aspirates of patients with plasma cell myeloma. Two myeloma
plasma cell populations, differing only in the expression of
Blood, Vol81, No 12 (June 15),1993:pp 3357-3364
MATERIALS AND METHODS
Patients. Twenty-two patients with multiple myeloma, 6
women and 16 men, 36 to 67 years of age (median 52 years), at all
treatment stages were studied. Their relevant clinical information is
summarized in Table 1. All had a single monoclonal myeloma cell
population as determined by heavy chain Ig gene rearrangements.
The only selection criteria for study were marow aspirate plasmacytosis of greater than 5% and a sufficiently high recovery of purified
myeloma cells.
Myeloma cell preparation. Heparinized BM aspirates were obtained during routine visits to the clinic, as required by treatment
protocols. Signed consent forms were obtained before study. Myeloma cells were purified using a modification of a recently described method." After density separation (Histopaque; Sigma, St
Louis, MO), light-density cells were reacted for 30 minutes at 4°C
with a mixture of phycoerythrin-conjugated anti-CD38 and fluorescein isothiocyanate-conjugated (FITC) anti-CD45 monoclonal antibodies (MoAbs) (Becton Dickinson Immunocytometry, San Jose,
CA) and washed twice with phosphate-buffered saline (PBS). Cells
showing high-intensity CD38 and low-to-intermediate CD45 fluorescence with negative-to-intermediate orthogonal and intermediate-to-high forward light-scatter profile were sorted using a FACStar Plus flow cytometer/cell sorter (Becton Dickinson) at a flow
rate of 1,000 to 1,200 events per second. Cytospin slides were pre-
From the Department of Medicine, Division of Hematology/Oncology, University of Arkansas for Medical Sciences, Arkansas
Cancer Research Center; and the John L. McClellan Memorial Veteran's Hospital, Little Rock, AR.
Submitted September 29, 1992; accepted January 27, 1993
Supported in part by Grant Nos. CA37161 and CA28771 of the
National Institutes of Health, Bethesda, MD, and a grant from the
International Exchange Program on Cancer Research.
Address reprint requests to Joshua Epstein, D Sc, Division of Hematology/Oncology, Arkansas Cancer Research Center, 4301 W
Markham St, Slot 508, Little Rock, AR 72205.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section I734 solely to
indicate this fact.
0 I993 by The American Society of Hematology.
0006-4971/93/8112-0015$3.00/0
3357
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3358
HATA ET AL
Table 1. Patient Characteristics
Variable
n
Stage*
111
II
I
11
9
2
tion was used for amplification with IL-6 primers, and the total
amplification product was analyzed by ethidiun bromide gel electrophoresis. In several experiments the amplification products were
transferred to a membrane, hybridized to the appropriate "P-labeled probe, and processed autoradiographically, to further confirm their identity and the specificity of the PCR reaction.
lg isotype
IgA
IgG
BJP
K
h
NS
Prior treatment
Statust
Prim resist
Resis rel§
6
10
3
12
9
3
16
12
3
Abbreviations: BJP, Bence-Jones protein only; NS, non-secreting patients.
* Stage according to the Durie-Salmon staging.
t Disease status at time of study.
t Primary unresponsive to therapy.
§ Therapy-resistant relapse.
pared and stained with May-Grunwald Giemsa stain for morphologic evaluation. Ethanol-fixed slides were reacted with F(ab), fragments of antihuman Ig light-chains antibodies for evaluation of
monotypic cIg content. Nonmyeloma cells (lymphocytes, monocytes, and myeloid cells) were sorted into a separate tube and were
used as controls.
h4onocyte.x Mononuclear cells separated from I O mL of blood
by density centrifugation as described above, were layered over 5
mL Percoll (49.2%,Sigma) and centrifuged at 1,130g for 25 minutes. The cells from the upper band were recovered and washed
with PBS. The proportion of monocytes was determined by morphology using May-Griinwald Giemsa-stained cytospin slides and
by flow cytometry using MoAb to CD14 (Becton Dickinson).
Gene expression. To overcome the obstacles presented by the
relatively small numbers of purified cells as well as the anticipated
low levels of IL-6 gene expression by myeloma cells,22the polymerase chain reaction (PCR) technique for detection of specific mRNA
was used. Total cellular RNA was isolated using a microadaptation
of the guanidium thiocyanate/cesium chloride pr~cedure.'~
Briefly,
cell pellets containing 1 to 4 X IO4 purified cells were resuspended
in 100 pL of 4 mol/L guanidium isothiocyanate, vortexed for 1 to 2
minutes, layered over a 100 pL cushion of 5.7 mol/L cesium chloride in a polycarbonate tube and centrifuged at 80,000 rpm for 2
hours at 20°C in a refrigerated tabletop ultracentrifuge (TL- 100;
Beckman, Fullerton, CA). The RNA pellet was resuspended in 50
pL diethyl pyrocarbonate (DEPC) water, precipitated with ethanol,
dried, and resuspended in I O pL cDNA-synthesizing solution.
First-strand cDNA was synthesized using Moloney murine leukemia virus (MMLV) reverse transcriptase and oligo dT (Boehringer
Mannheim, Indianapolis, IN) for 1 hour at 42°C. After inactivation
ofthe reverse transcriptase at 95°C for 10 minutes, 20 pL water was
added to a final volume of 30 pL, and a 1- to 5-pL aliquot was used
as template for 35-cycle amplification by PCR with cytokine-specific primers (Table 2). Temperature cycling was 1 minute at 94"C, 2
minutes at 6 0 T , followed by 3 minutes at 72°C. Aliquots of I O pL
of the PCR product were analyzed by electrophoresis on 2%agarose
gel containing 0.5 pg/mL ethidium bromide. In single-cell PCR
experiments, the total amount of cDNA left after &actin amplifica-
RESULTS
Some samples contained as few as 5% myeloma cells as
determined by morphology or by monotypic cytoplasmic Ig
content analyzed by flow cytometry. Nevertheless, the myeloma cell preparations consisted of pure myeloma plasma
cells (Fig IA). CD38 and CD45 profiles of two representative samples are presented in Figs I , B and C. The monocyte-enriched preparations contained greater than 96%
monocytes by morphology and CD14 expression, with occasional lymphoid cells.
Cytokine gene expression was studied by PCR. Figure 2
shows examples of PCR analysis using primers for IL-6,
IL-6-receptor (IL-6-R), IL-18, and TNF-a (Table 2).
Primers for the P-actin gene were used as control for cDNA
integrity, for the presence of cells in single-cell PCR experiments, and for PCR. IL-6 mRNA was detected in purified
myeloma cells from 10 of 22 patients (45%). IL-6-R mRNA
was detected in the 10 patients in whom IL-6 gene expression was found, and in five additional patients in whom
IL-6 transcripts were not detected. Myeloma cells from
seven patients did not express the IL-6-R gene. Thus, the
receptor mRNA was detected in every instance where IL-6
mRNA was expressed.
TNF-a mRNA was detected in 8 of 21 patients tested.
IL- 1/3 transcripts were not detected in myeloma cell preparations from any of the patients studied. TNF-a gene expression did not correlate with expression of the IL-6 and IL-6R genes. The results for IL-6, IL-6-R, TNF-a, and IL-ID
expression by myeloma cells are summarized in Table 3.
PCR is capable of detecting the presence of even one contaminating cell among 1O5 cells. Although morphologically
100%of the cells were myeloma plasma cells, this high sensitivity of the PCR mandated the use of controls to assure that
the results obtained using this procedure represented gene
expression by myeloma cells and not the phenotype of rare
contaminating cells. Because neither immunocytochemical
staining,24enzyme-linked immunospot (ELISPOT),25or insitu hybridization (S.M. Hsu, personal communication,
May 1992;and M.T.P. and J.E., data not shown) techniques
Table 2. Primers for PCR
Product
Size
Gene
IL-6
IL-lP
0-Actin
IL-6-R
TNF-a
(bp)
627
802
548
435
43 1
SourceISequence
Clontech
Clontech
CIont e ch
'485-GTAGAGCCGGAAGACAATGCCACT-3'
58355'-CGACGCACATGGACACTATGTAGA-3
"5-TGTTCCTCAGCCTCTTCTCCTT-3
5235-TCTTGATGGCAGAGAGGAGGTT-3
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3359
AUTOCRINE IL-6 IN IMMATURE MYELOMA CELLS
-
Fig 1. (A) Sorted myeloma cells. Myeloma cells were identified as
CD38NO"CD45--*
and by light-scatter properties. Sorted cells
contained only myeloma cells by morphology, monoclonal clg content and as determined by PCR (see Results). (B and C) CD38/CD45
expression by myeloma cells. CD38h10hCD45- (B) and CD38'Oh
CD45"-"
myeloma cells (C) are boxed. Coordinates represent
fluorescence intensities (channel number) on a logarithmic scale.
Note the presence of a small number of C D 3 8 m CD451m""d'R"cells
in (B).
104
104
J
. . . _ :... . .
.
.I
..
, ..
:.,., . . .. .,
.. . . .
.. .. ..'
103
..
.
'
..
1 .
. '.-
.
.
. .
IC
.
:
103
. ..
co
g
102
102
0
10'
10'
100
10 0
100
10'
102
103
1
CD45
were capable of detecting the cytokine or IL-6 gene transcripts in myeloma cells, a different approach had to be used
to ascertain purity of the myeloma cell preparations. The
patterns of cytokine gene expression by purified myeloma
cells were compared with those ofmonocyte-enriched preparations and of sorted nonmyeloma cells from the same patients, studied simultaneously. Results from several such
experiments are presented in Fig 2 and in Table 4. Whereas
all control cell preparations expressed IL-lp and TNF-a
genes, none of the myeloma cell samples contained IL-10
gene transcripts, and only eight expressed TNF-a. In addition, while control cells from patients 2, 5 , and 6 in Table 4
expressed IL-6, no IL-6 mRNA was detected in the purified
myeloma cells; the opposite was observed for IL-6-R expression for patients 1 through 4. These clear and consistent
100
10'
102
103
104
CD45
differences in IL-lp gene expression between the myeloma
and control cell populations, as well as additional differences in expression of the IL-6, IL-bR, and TNF-a genes,
prove the high degree of purity of the myeloma cell preparations.
Expression ofthe IL-6 gene by myeloma cells is suggestive
of an autocrine IL-6 loop. Unless one accepts the possible
existence of exclusively autocrine and paracrine forms of
the disease, the variation in IL-6 expression by myeloma
cells among different patients probably reflects tumor-cell
hetrogeneity. In the normal immune system, IL-6 functions
as a differentiation-promoting factor, inducing maturation
of B cells into Ig-producing cells?"28 If IL-6 plays a similar
role in myeloma, it would be plausible to assume an autocrine IL-6 loop to be functional in less differentiated tumor
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HATA ET AL
3360
Myeloma Cells
Control
'1 2 3 4 5 '
' 1 2 3 4 5'6
Table 4. Cytokine Gene Expression
bv Mveloma and Nonmveloma Cells
Patient
Cells
1
Plasma cells
Monocytes'
Plasma cells
Monocytes
Plasma cells
Monocytes
Plasma cells
Nonplasma cellst
Plasma cells
2
3
4
1 - p-actin
4 - TNF-a
5
2 - 11-6
5 - IL-lP
6
3 - IL-6-R
6 - Size Markers
Nonplasma cells
Plasma cells
Nonplasma cells
11-6
IL-6-R
IL-lg
TNF-a
+
+
+
-
+
-
+
+
+
-
+
+
+
+
+
+
+
-
-
+
+
-
-
+
+
+
+
-
+
-
+
+
+
+
-
+
+
-
-
+
-
-
+
+
+
+
* Cell preparations containing >95% monocytes.
Fig 2. Cytokine gene expression by purified myeloma and control cells. Total RNA was extracted from 2 X 1O4 sorted myeloma
cells and from monocytes or sorted nonmyeloma cells (controls),
cDNA synthesized and presence of @-actin,11-6, IL-6-receptor,
TNF-a, and IL-la gene transcripts determined by PCR. Size
markers (sizes in basepairs are given) were run in lane 6.
cells. Thus, the observed heterogeneity in IL-6 expression
among patients could reflect differences in myeloma cell
maturation.
Expression of CD45 by malignant B cells and shifts in
CD45 isoform expression are indicators ofthe level of maturation of neoplastic B cell^.^^-^' Therefore, expression of the
IL-6 and IL-6-R genes was compared with the expression of
CD45, also heterogeneously expressed on myeloma cells
(Figs 1A, 1B, 3, and 4). CD45 expression data were collected
at the time of cell sorting. Myeloma plasma cells from I O
patients expressed CD45; of these, 9 also expressed the IL-6
gene. In contrast, only I of 12 CD45- cell preparations
showed IL-6 gene expression. The correlation between
CD45 and IL-6 expression was highly significant (P<.0002,
Table 5).
The observed association between CD45 and IL-6 expression was further investigated in four patients who had both
CD45-expressing as well as CD45- myeloma-cell subpopulations (Fig 5). Both cell populations consisted exclusively
of monoclonal clg-containing plasma cells. Only purified
CD45+myeloma cells from these patients expressed the IL6 gene, confirming the relationship between IL-6 and CD45
expression. These results also add support to the punty of
the sorted myeloma cell preparations. Expression of CD45
t Cells sorted with the sort windows set to include all cells except
plasma cells.
also correlated with IL-6-R expression (Pc .005, Table 5).
No relationship was apparent between CD45, IL-6 or IL-6R expression and clinical parameters such as stage and
marrow plasmacytosis.
To further investigate the association between CD45 and
IL-6 expression, individual CD45- and CD45+ myeloma
cells were sorted using the Automatic Cell Deposition Unit
(Becton Dickinson) attachment, RNA extracted, and IL-6
gene expression studied. 0-Actin was used as control for cell
presence, cDNA integrity, and for PCR. 0-Actin mRNA
was detected in 16 of the 20 cell preparations. Of the 16,
IL-6 transcripts were detected by ethidum bromide fluorescence only in CD45+ cells (Table 6).
--a
100
b
C
I
Table 3. Expression of IL-6, IL-6-R, IL-lB, and TNF-a
by Myeloma Plasma Cells
IL-6
IL-6-R
IL-lB
TNF-a
N
CD45
Fig 3. Between-patient heterogeneity of CD45 expression by
myeloma cells. Histograms from two patients are shown. The myeloma cells from one patient were CD45- (histogram a), from the
other CD45"-'""
(histogram b). Histograms c represent nonmyeloma cells expressing high levels of CD45 from these patients,
for comparison. Abscissa: intensity of CD45-associated fluorescence (log scale). Ordinate: relative cell number.
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336 1
AUTOCRINE IL-6 IN IMMATURE MYELOMA CELLS
CD45+
10 4
’1 2 3 4 5 ‘
CD45
1 2 3 4 5’6
103
i
~
- 1353
- 1078
-072
-603
- 310
10’
100
103
102
104
CD45
Fig 4. Coexistence of CD45- (box on left) and CD45(box on right) myeloma cells in the BM of a myeloma patient. The
t w o myeloma cell subpopulations were sorted from this and three
other patients and gene expressionby each cell subpopulationsdetermined. Both cell populations consisted entirely of myeloma
plasma cells containing the same monotypic cytoplasmic immunoglobulin.
A bioassay was used to test whether myeloma cells that
express the IL-6 gene also produce IL-6 protein product.
Purified myeloma cells, 2 X los, from nine patients were
cultured in 100 pL media for 48 to 72 hours, after which the
medium was filtered through a 0.22-pm pore-size membrane and the concentration of IL-6 determined using the
IL-6-dependent B9 murine plasmacytoma cell^.'^^" IL-6
for standard was a kind gift from Interpharm Laboratories
(Israel). Only the five myeloma cell preparations with IL-6
mRNA expression produced IL-6, detected at concentrations of0.5 to 12.8 pg/mL (median = 1.16 pg/mL).
DISCUSSION
The controversy surrounding the mechanism of IL-6 activity in myeloma reflects, in part, the difficulty in obtaining
myeloma cells in sufficient quantities and of an adequate
degree of purity to allow application of sensitive analytical
methods. While enrichment of myeloma cells could be accomplished by phy~ical,’~
immun~logic,’~
and immunomagnetic b e a d - b a ~ e d ’ ~ .and
’ ~ . ~panning
~
methods, only cell
Table 5. Relationship Between CD45 Expression and
the Expression of IL-6 and IL-6-R Genes by Myeloma Cells
CD45
11-6
N
CD45
+
-+
19
+
-
+
1
-
11
(P < .0002)
IL-6-R
N
+
10
-
+
11-6
+
0
5
7
(P < ,005)
-
1 - p-actin
4 - IL-lP
2 - IL-6
5 - TNF-a
3 - IL-6-R
6 - Size Markers
Fig 5. Cytokine gene expression by CD45- and CD45’ myeloma plasma cells, studied by PCR. Both cell populations were
purified from the same patient. IL-6 transcripts (lane 2) were found
only in the CD45-expressingtumor cells, whereas bothcell populations expressed IL-6-R (lane 3).
sorting provided the high degree of selectivity needed for
obtaining myeloma cells of a sufficiently high level of purity. The absence of contaminating cells was sought by adjusting instrument settings and sort windows to exclude all
cells with marginal properties. As a result, collected cells
represented only 20% to 50% of those selected, thus limiting
the quantity of cellsavailable for study and imposing the use
of PCR as the most feasible method for studying cytokine
and receptor gene expression.
Use of PCR in turn necessitated using strict controls to
guarantee the validity of the results. The high sensitivity of
PCR mandated negation ofthe possibility that the observed
phenotype resulted from cosorted myeloma and nonmyeloma cells. The inability of less sensitive techniques to detect IL-6 gene expression by individual myeloma cells necessitated a different approach to be taken. This was
accomplished by comparing expression of IL-6, IL-6-R, IL18, and TNF-a genes by myeloma cells with their expression by monocyte-enriched and by sorted nonmyeloma cell
preparations. If one was to attribute the PCR results to rare
contaminatingcells. the followingwould imply: ( 1) contamination by IL-6-expressing nonmyeloma cells occurred only
in the 45% of patients in whose myeloma cells IL-6 transcripts were detected (Table 3); (2) contamination with IL6’ cells consistently occurred in patients with CD45-ex-
Table 6. IL-6 Expression by Individual Myeloma Cells
CD45
Sorted.
8-Actint
1L-6t
-
IL-6-R
N
-
5
+
5
0
10
+
15
11
96
-
0
+
5
7
(P < ,005)
~~
* Number of single cells sorted.
t Number of preparations with 8-actin transcripts.
$ 8-actin’ samples with 11-6 expression.
5 Difference significant at P < ,005.
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3362
pressing myeloma cells but only rarely in CD45- myeloma
cells (9 of 10 v 1 of 1 1, respectively, Table 5);(3) contamination in all patients was only by IL-lp-, never by IL-lp+
nonmyeloma cells; (4) even in patients with coexisting
CD45- and CD45+ myeloma cell subpopulations, only the
sorted CD45+ subpopulations were contaminated with ILIp- IL-6' nonmyeloma cells; ( 5 ) the majority of CD45+
myeloma cells studied were, in fact, nonmyeloma cells (Table 6); and this is not compatible with morphologic and cIg
content evaluation of the sorted myeloma cells (Fig 1); and
(6) sorted myeloma cells were preferentially contaminated
by nonmyeloma cells while the reverse did not occur, and in
Table 4, nonmyeloma cells of patients 1 through 4 were not
contaminated with IL-6-R-expressing myeloma cells.
Thus, it was concluded that the observed differences between these cell preparations in the patterns of cytokine and
receptor-gene expression were proof of the purity of the myeloma cell preparations and attested to the validity of the
PCR-derived data.
IL-6 expression by myeloma plasma cells correlated
strongly with a CD45+ phenotype. Expression of CD45 on
the surface of malignant B cells and shifts in CD45 isoform
expression are indicators of the level of maturation of neoplastic B cell^.*^-^' Heterogeneity in CD45 expression by myeloma cells among different patients, observed in this study,
likely reflects differences in the degree of maturation of the
tumor cells. Indeed, CD45' but not CD45- myeloma cells
coexpressed the B-cell antigen CD19 (data not shown). In
the majority (88%)of patients with CD45- myeloma, small
numbers of CD4Sintemediate
cIg-containing myeloma cells
coexisted (eg, Fig 1B). In these, the levels of CD19 expression by the myeloma cells declined with the decrease in the
level of CD45 expression, indicating a maturation process
or a phenotypic shift to a more immature, possibly more
aggressive disease. In four of these patients, sufficient numbers of cells from each myeloma cell subpopulation were
purified for study; only the tumor cells displaying the less
mature CD45' phenotype expressed IL-6, underscoring the
correlation between the two parameters.
A central role for IL-6 in myeloma has been proposed by
several investigator^.^-'^ The current finding of IL-6 expression by freshly isolated myeloma cells displaying a less mature phenotype, although not evidence, strongly supports
the possibility of an autocnne IL-6 loop in multiple mye10ma.~Further support for this can be found in the close
association between expression of IL-6 and IL-6-R genes ( P
< .005, Fisher's exact test). This association as well as production of IL-6 by purified CD45-expressing myeloma cells
tends to discount the possibility that regulatory processes at
the posttranscriptional level prevent translation of IL-6 as
well as IL-6-R mRNA into the active IL-6 molecule and its
receptor. It would appear that only minuscule amounts of
IL-6 are required for maintaining an autocrine loop, as was
reported for the myeloma cell line U266.22This low level of
expression could account for the difficulty in detecting IL-6
production in myeloma cells using even sensitive immunologic and immunocytochemical methods.24 The reported
inhibition of proliferation of myeloma cell lines in which
IL-6 production could not be detected by antisense to IL-6'*
HATA ET AL
underscores the likelihood of an autocrine IL-6 stimulation
loop in multiple myeloma even when IL-6 expression cannot be detected with available methods other than PCR and
a sensitive bioassay.
Both the CD45- and CD45' myeloma cells used in this
study were morphologically and phenotypically myeloma
plasma cells. Both cell populations were nonproliferative,
did not respond to exogenous IL-6,24,37
and likely are not the
target cells for IL-6 activity. Rather, CD45+ myeloma
plasma cells represent an intermediate stage in B-cell maturation. As such, while expressing plasma cell morphology
and function (Ig secretion), they are still in the process of
losing the characteristics of B cells, such as CD19 and
CD45, and also IL-6 and IL-6-R expression. This would
imply that an autocrine IL-6 loop is functional in an even
earlier, as yet unrecognized, tumor cell of B-cell morphology and phenotype (preplasma cell). Circulating monoclo.~'
nal B cells found in patients with multiple m y e l ~ m a ~and
Waldenstrom's macroglob~linemia~~
could represent such
preplasma cells. It is significant that these circulating cells
have normal, albeit polyclonal counterparts, because this
lends support to the hypothesis that the central role of IL-6
in myeloma is not different from the cytokine's role in normal B-cell development.2628
IL-6 could provide a proliferative stimulus to myeloma
progenitor cells, as has been suggested. Alternatively, akin
to its role in normal B-cell development,26-28the cytokine
could act as a differentiation factor, facilitating maturation
of premyeloma B cells into Ig-secreting myeloma plasma
cells, without direct growth-stimulating a~tivity.~'
As a third
possibility, IL-6 could affect both proliferation and differentiation. Existence of an autocnne IL-6 loop in premyeloma
B cells, as the data suggest, would imply that these cells are
constantly stimulated. The fact that a largely expanded preplasma cell compartment in myeloma patients has not been
identified may suggest that the rate of preplasma cell proliferation is matched or probably exceeded by the rate of differentiation, or that the proliferative, preplasma cell population is maintained at a constant size. Under the latter
scenario, cell proliferation could be regulated, possibly
through a "feedback" loop, so as to compensate for the
"loss" of cells to IL-6-mediated differentiation, without expansion of the proliferative pool. In this case, IL-6 would
directly affect differentiation and indirectly, by allowing expansion to take place, also proliferation.
CD45 is a tyrosine p h o ~ p h a t a s eIt. ~functions
~
in receptor-mediated transmembrane signal transduction associated with tyrosine phosph~rylation.~~
Inhibition of CD45
activity by mutation or with MoAbs inhibits, among other
pathways, B-cell activation, proliferation, and differentiatiom4IA3One could speculate that loss of CD45 expression
with normal or malignant B-cell maturation into normal or
myeloma plasma cell^^.*^ is compatible with the loss of response to activating signals that accompanies terminal differentiation. In this context, the close association seen between IL-6, IL-6-R, and CD45 expression is particularly
interesting. In addition to indicating the possible operation
ofan autocrine IL-6 loop in preplasma cells, it may suggest a
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AUTOCRINE IL-6 IN IMMATURE MYELOMA CELLS
role for CD45 in IL-6/IG6-R complex-mediated signal
transduction.
In contrast to a previous reportt4 IL-IP expression was
not detected in myeloma cells of the 22 patients studied,
whereas IL-ID transcripts were present in all control cell
preparations. This discrepancy likely reflects differences in
myeloma cell purity, which was greater than 95% using a
physical/immunologic methodu compared with 100% reported here.
The data presented here strongly suggest the existence of
an autocrine IL-6 loop in multiple myeloma; that the autocrine IG6 loop is functional in preplasma cells; and that the
recognizable myeloma cells, of plasma cell morphology and
function, are not the target for IL-6 activity. Ongoing clinical trials with IL-6-neutralizing antibodies and work with
IL-6-dependent and -independent cell lines as well as with
agents such as dexamethasone that regulate IL-6 and IL-6R gene expression, could potentially help solve the IL-6
enigma.
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1993 81: 3357-3364
Interleukin-6 gene expression in multiple myeloma: a characteristic of
immature tumor cells
H Hata, H Xiao, MT Petrucci, J Woodliff, R Chang and J Epstein
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