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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 10 (November 15), 1998:
pp. 3887-3897
Induction of CD45 Expression and Proliferation in U-266 Myeloma
Cell Line by Interleukin-6
By
Maged S. Mahmoud,
Hideaki Ishikawa,
Ryuichi Fujii, and
Michio M. Kawano
From the Department of Immuno-hematology, Yamaguchi University School
of Medicine, Ube, Japan.
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ABSTRACT |
Recently, there has been an increasing interest in the expression
pattern and biological significance of the CD45 molecule in myeloma
cells. In this study, we have further defined the phenotypic pattern of
CD45 expression on myeloma cells. Using a panel of myeloma cell lines,
we showed that CD45 showed a remarkably heterogeneous pattern of
expression. Whereas some cell lines were CD45+ and others
were CD45 , the U-266 cell line, although predominantly
CD45, still had a considerable subpopulation of
CD45+ cells. Among the myeloma cell lines examined, there
was a direct correlation between interleukin-6 (IL-6) dependency and
CD45 positivity. Moreover, we showed that IL-6 stimulation led to the
induction of expression of CD45 and cellular proliferation. Using
independent experimental approaches, we could show that the
IL-6-induced effects were closely linked to CD45 expression. First,
sorting out CD45+ and CD45 subsets of
U-266 cell line followed by IL-6 stimulation, only the
CD45+ cells showed a proliferative advantage after IL-6
stimulation. Second, IL-6 stimulation of sorted CD45
cells was gradually followed by phenotypic conversion to
CD45+ cells that started after 2 days as judged by the
detection of CD45 mRNA by reverse transcription polymerase chain
reaction (RT-PCR) and immunophenotypic analysis by flow
cytometry. Withdrawal of IL-6 from the medium led to gradual loss of
CD45 expression in CD45+ flow-sorted U-266 cells. Third,
the use of vanadate, a potent inhibitor of protein tyrosine phosphatase
(PTP), abrogated the IL-6-induced proliferation in the
CD45+ myeloma cells. On the other hand, cellular
proliferation induced by IL-6 was not affected by the serine-threonine
phosphatase inhibitor okadaic acid. Our data show that the expression
pattern of CD45 in myeloma cell lines is heterogeneous and show for the
first time that CD45 expression can be induced by IL-6 stimulation. Finally, these data shed some light on the biological role of CD45 in
myeloma by determining the proliferative population among myeloma
cells.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
THE CD45 MOLECULE IS A transmembrane
glycoprotein expressed on the surface of all hematopoietic cells and
their precursors except for mature erythrocytes and
platelets.1 Differential usage of three variable exons
tentatively called A-, B-, and C-encoding amino acids near the
N-terminus of the molecule results in the generation of at least 5 alternatively spliced isoforms. CD45 isoforms reactive with exon 4-, 5-, and 6-specific monoclonal antibodies are referred to as CD45RA,
CD45RB, and CD45RC, respectively.2 In humans, the lowest
molecular weight isoform of CD45 (CD45RO) was found to be selectively
expressed on CD34+ progenitor cells with functional
erythroid colony-forming activity.3 The cytoplasmic
portion of CD45 molecule contains two tyrosine phosphatase domains in
which activity has been shown to be crucial for both T- and B-cell
activation through the corresponding antigen receptor.4
Mice homozygous for CD45-exon 6 mutation had a block in thymocyte
maturation at the transitional stage from double-positive to
single-positive stage and a functional B-cell defect in the form of
abrogated Ig µ-induced proliferation.5
The initial signal transduction events essential for eliciting an
immune response are controlled by concerted action of protein tyrosine
kinases and protein tyrosine phosphatases (PTPs). Distinct enzymes can
be positive or negative regulators of a molecular reaction, and in
certain circumstances a single enzyme may have both functions. The
biological functions of two PTPs, the src homology region
2-containing protein tyrosine phosphatase and CD45, are considered to
serve to illustrate the dichotomy by which such class of enzymes
regulate immune responses.6 Another role for CD45 in
regulation of B-cell-negative and -positive selection has been shown
by experiments crossing CD45-deficient mice with mice carrying Ig
transgenes specific for hen egg lysozyme.7
The expression of CD45 molecule in primary myeloma cells and cell lines
is quite variable, where in some studies myeloma cells were
described as CD45/dim mature cells,8-12
whereas in others they were described as CD45+/++ immature
cells.13-16 The biological functions of CD45 in myeloma are
still largely unknown.
Interleukin-6 (IL-6) had been originally characterized as a B-cell
differentiation factor, but it also has a variety of biological functions in various cells and tissues, including the hematopoietic cells, hepatocytes, and nerve cells.17 In myeloma cells,
IL-6 had been shown to play a role as an autocrine growth
factor.18 IL-6 is the original member of a family of
cytokines that share gp130 as a critical component for signal
transduction. The ubiquitous expression of gp130 explains the basis for
the extensive function of IL-6 family in hematopoietic, cardiovascular,
and nervous tissues. After IL-6 stimulation, gp130 homodimers or
heterodimers activate associated cytoplasmic tyrosine kinases and
subsequently modify downstream transcription factors.19 In
this work, we examined the expression pattern and the biological
significance of CD45 in a panel of myeloma cell lines. The observation
of concordant correlation between IL-6 dependency and CD45 positivity
led us to proceed to investigate the possible correlation between IL-6 and CD45 expression.
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MATERIALS AND METHODS |
Cell culture.
The IL-6-independent KMS-5,20 U-266, and IL-6-dependent
(ILKM-2 and ILKM-3) human myeloma cell lines (kindly provided by Dr S. Shimizu, Shimane Prefectural Hospital, Matsue, Japan) were maintained
in RPMI 1640 medium (Nissui, Tokyo, Japan) supplemented with 10% fetal
calf serum (M.A. Bioproducts, Walkersville, MD). IL-6 was added to the
culture of IL-6-dependent cell lines and in other experiments as
appropriately mentioned in a final concentration of 2 ng/mL. Cell
counts were performed both manually on a hemocytometer and
automatically by harvesting the whole culture into 0.5 mL and counting
cells at a constant flow rate for 1 minute using the cell sorter (Epics
Elite ESP; Coulter, Hialeah, FL).
Monoclonal antibodies and flow cytometry.
Labeled monoclonal antibodies fluorescein isothiocyanate (FITC)-CD38,
phyroerythrin (PE)-CD45, PE-Cy5-CD45, PE-CD45RO, and PE-CD45RA were
purchased from Immunotech (Marseille, France), and R-PE-CD45B was
purchased from Pharmingen (San Diego, CA). Cells were incubated with 20 µg of each appropriate antibody, left to react for 30 minutes on ice,
and washed twice in phosphate-buffered saline (pH 7.4)
with 200 µg/mL bovine serum albumin and 0.01% sodium azide. Cells
were then analyzed for surface immunofluorescence, cell count, and
viability by the cell sorter.
Cell sorting.
For cell sorting, the cells were stained with PE-CD45 and FITC-CD38 as
described above under sterile conditions. Either CD45+ or
CD45U-266 subpopulations were isolated via the cell
sorter (Epics Elite ESP; Coulter) for subsequent in vitro culture.
Single-cell assay.
U-266 cells were extensively washed and cultured without exogenous IL-6
to minimize the number of CD45+ cells. The
CD45 subpopulation was sorted out and plated at 1 cell
per well in a 96-well plate and cultured for 28 days. Forty-eight wells
received exogenous IL-6 (2 ng/mL), and the other 48 wells were
maintained without exogenous IL-6. The number of wells that received
one cell were verified microscopically on the second day of plating. Wells with significant proliferating cells were analyzed by flow cytometry after staining with PE-CD45 monoclonal antibody, as described
above.
Semiquantitative RT-PCR.
Total cellular RNA was collected by guanidine thiocyanate and
phenol/chloroform extraction and reverse transcribed by Superscript II
(GIBCO-BRL, Gaithersburg, MD) at 37°C for 60 minutes as recommended by the manufacturer. The primers used were as follows:  -actin forward, ATC TGG CAC CAC ACC TTC TAC A AT GAG CTG CG; -actin reverse, CGT CAT ACT CCT GCT TGC TGA TCC ACA TCT GC; IL-6 forward, ATG
AAC TCC TTC TCC ACA AGC GC; IL-6 reverse, GAA GAG CCC TCA GGC TGG ACT
G; CD45 forward, AAC AGT GGAGAA AGG ACA CA; and CD45 reverse, TGT GTC
CAG AAA GGC AAA GC. Thermal cycling was performed for 30 cycles:
denaturation at 94°C for 1 minute, primer extension at 72°C for 1 minute, and primer annealing for 1 minute at 65°C for -actin and
IL-6 gene and at 55°C for CD45 gene. For the semiquantitative polymerase chain reaction (PCR), complementary DNA load was
standardized in preliminary experiments by making serial dilution and
amplifying -actin for 20 cycles to produce just detectable signal,
thus keeping the PCR condition within the linear range of amplification before reaching saturation.
CD45 phosphatase blocking experiments.
CD45+ subpopulations of U-266 cells were extensively washed
in medium and subsequently plated in 24-well plates at 5 × 104/well. The cells were incubated for 2 days in medium
alone: rIL-6 at a concentration of 2 ng/mL; IL-6 and
sodium orthovanadate (Na3VO4; Sigma, St Louis,
MO); IL-6 and okadaic acid (Wako Pure Chemicals, Osaka, Japan); or IL-6
and dimethyl sulfoxide (DMSO). The PTP inhibitor
Na3VO4 was dissolved in sterile water just
before use and added to the culture at 50 µmol/L final concentration.
Preliminary experiments were performed using a range of 12.5 µmol/L
to 100 µmol/L to determine the optimum concentration for blocking
CD45 phosphatase. Okadaic acid was dissolved in DMSO to 0.25 mmol/L and
stocked at  80°C; for control experiments, okadaic acid was diluted
in Tris-HCl buffer and used at a concentration of 10 ng/mL.
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RESULTS |
Expression of CD45 was heterogeneous and possibly correlated with IL-6
dependency.
To study the role and biological importance of CD45 expression in
myeloma cells, we first examined its pattern of expression by flow
cytometry on a panel of myeloma cell lines. The expression pattern of
CD45 showed a remarkable heterogeneity between the various myeloma cell
lines (Fig 1). Whereas some cell lines were strongly
positive (ILKM-2 and ILKM-3; Fig 1A), others, such as KMS-5, were
almost completely CD45 (Fig 1B). U-266 cells showed a
peculiar pattern of CD45 expression, in which a relatively high
percentage of cells (>25%) were CD45+ (Fig 1B). ILKM-2
and ILKM-3 were strictly IL-6-dependent cell lines, and they could not
be grown in culture without the addition of exogenous IL-6 (2 ng/mL).
They had a duplication time of 4 and 3 days, respectively (Fig 1A,
bottom). On the other hand, KMS-5 was completely IL-6-independent and
had a duplication time of less than 2 days (Fig 1B, bottom). Again,
U-266 cell line showed a peculiar pattern in which it grew
satisfactorily in cultures without exogenous IL-6, yet the addition of
IL-6 to the culture enhanced its growth by about 30% (Fig 1B, bottom).
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| Fig 1.
CD45 was heterogeneously expressed on myeloma cells and
correlated with IL-6 dependency. (A) IL-6-dependent myeloma cell lines
ILKM-2 and ILKM-3 were stained for 30 minutes by PE-Cy5-CD45 (forward
scatter) and FITC-38 (side scatter) and analyzed by cell sorter (top
panels), and growth curves are shown of the cells cultured in medium
alone (solid lines) or in the presence of IL-6 (2 ng/mL; dotted lines)
for the indicated period of time (bottom panels). (B) CD45 expression (top) and
growth curves (bottom) of the IL-6-independent cell lines KMS-5 and
U-266.
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Next, we have extended our analyses to delineate the expression pattern
of CD45 isoforms in myeloma cells. Within the CD45+ lines,
all of them expressed the CD45RO isoform and all were CD45RA and CD45RB+ (data not shown). These
data showed that the expression pattern of CD45 was heterogeneous in
myeloma cells, not only in the context of line-to-line variation, but
also within a given cell line. It also showed a concordant correlation
between CD45 expression and IL-6 dependency and raised the possibility
that the CD45+ populations were those that responded to
IL-6 stimulation in the form of proliferation.
CD45 expression was induced by IL-6 and was lost again upon its
withdrawal.
To further analyze the relationship between IL-6 dependency and the
expression of CD45, we have sorted out both CD45+ and
CD45 subpopulations from U-266 and KMS-5 cell lines.
Sorted cells were maintained in culture either in the presence or
absence of IL-6 for about 20 days and were analyzed at different time
points by flow cytometry and reverse transcription polymerase chain
reaction (RT-PCR) for the expression of CD45. Surprisingly, more than
35% of the CD45 U-266 cells underwent phenotypic
conversion to CD45+ after 8 days, and more than 60%
underwent phenotypic conversion after 20 days of culture with IL-6 (2 ng/mL) compared with a minor fraction of 15% of cells kept in medium
without IL-6 (Fig
2A). Conversely, the sorted CD45 fraction from the
IL-6-independent KMS-5 line did not show significant phenotypic
conversion, even after culture for 20 days with IL-6 (Fig 2B). Using
RT-PCR to detect CD45 mRNA in U-266 cells, we observed that although it
was hardly detectable after 2 days (Fig 2C, lane 3), it was clearly
detectable after 4 days (Fig 2C, lane 4) of IL-6 stimulation. PCR
analysis showed that the level of endogenous IL-6 mRNA did not change
during the course of IL-6 treatment and that both CD45
and CD45+ cells produced endogenous IL-6 of comparatively
the same level. In the culture supernatant of U-266 cell line,
endogenous IL-6 was produced at a concentration of 10 pg/mL/1 × 106 cells, as previously reported.21
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| Fig 2.
CD45 expression was induced by IL-6 and lost on its
withdrawal. (A) Flow-sorted CD45 U-266 cells were
cultured in medium alone (left) or in medium + IL-6 (2 ng/mL; right),
and cells were analyzed by flow cytometry after staining with
PE-Cy5-CD45 and FITC-CD38. The percentage of CD45+ cells
is indicated on each flow cytogram. (B) CD45
IL-6-independent KMS-5 cells were cultured with or without exogenous
IL-6 (2 ng/mL) and analyzed after staining by PE-Cy5-CD45 and
FITC-CD38. (C) RT-PCR analysis of
-actin, CD45, and IL-6 of CD45 U-266 cells cultured
with IL-6. Lane M,  X174/Hae III digest DNA size marker;
lane 1, negative control without RT product; lanes 2, 3, 4, 5, and 6, IL-6-treated CD45 cells at day 0, 2, 4, 6, and 8, respectively; lane 7, CD45+ U-266 cells. (D) Flow-sorted
CD45+ U-266 cells were incubated in medium without IL-6.
Cells were stained with PE-CD45 and FITC-CD38 and subjected to flow
cytometric analysis as described in Materials and Methods. The
percentage of CD45+ cells is indicated on each flow
cytogram.
(E) Flow-sorted
CD45+ U-266 cells were cultured without exogenous IL-6
for 2 weeks (left), and only CD45+ fraction was sorted
out and incubated with or without IL-6 (2 ng/mL) for another 2 weeks
(middle). Both CD45+ and CD45 fractions
were separately sorted out and cultured for 5 days with and without
IL-6 (right). The percentages of both CD45+ and
CD45 fractions are indicated on each flow cytogram.
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To further confirm the effect of IL-6 on CD45 expression, we have
cultured CD45+ U-266 in the absence of exogenous IL-6.
Withdrawal of IL-6 led to the loss of CD45 expression, with gradual
conversion of the CD45+ cells into CD45.
More than 65% of the cells lost CD45 after 7 days, and more than 90%
lost CD45 after 16 days of culture in IL-6 free medium (Fig 2D). To
further confirm that U-266 cells can freely change from the
CD45+ to the CD45 phenotype and vice versa
depending on IL-6 and that this phenotypic conversion is not a one-way
process, we have performed the following experiment. Flow-sorted
CD45+ cells were maintained in culture without exogenous
IL-6 for approximately 2 weeks. After this period of time, more than
40% of the cells had a CD45 phenotype (Fig 2E, left).
The CD45+ fraction of these cells was sequentially sorted
and then cultured with and without exogenous IL-6 (2 ng/mL; Fig 2E,
middle). After culture for another 2 weeks, both the CD45+
and the CD45 fractions that appeared in culture without
IL-6 were again sorted and then cultured with and without IL-6 for 5 days. These cultures (Fig 2E, right) had a strikingly heterogeneous
pattern of CD45 expression despite all originating from flow-sorted
CD45+ cells. These data showed that CD45 expression could
be induced by IL-6 stimulation at least in the U-266 cell line and that
CD45 mRNA is apparently upregulated by IL-6 and precedes the surface expression of CD45. Phenotypic conversion from CD45 to
CD45+ and vice versa occurred freely in both directions and
was highly dependent on the IL-6 level to which the cells were exposed.
Single-cell assay confirmed that induction CD45 are IL-6-induced
effects and not due to contaminating CD45+ cells.
To address the question of a possible sorting defect that could result
in a small number of contaminating CD45+ cells, which could
proliferate and result in the observed phenotypic conversion, we have
performed a single-cell assay by limiting dilution of flow-sorted
CD45 cells. Of 45 wells cultured in the presence of
exogenous IL-6 (2 ng/mL), 35 wells (77.7%) had proliferating cells
after 28 days of culture. We have analyzed 14 (40%) of those clones
for the expression of CD45. All of the analyzed clones without
exception showed CD45+ cells with varying percentages
ranging from 29.9% to 86% (Fig 3 and Table
1). On the
other hand, the number of wells containing proliferating cells cultured
without exogenous IL-6 were considerably less than those cultured with
IL-6. Most of those wells had a considerably lower cell number (<100
cells/well), and only 4 wells had about 200 cells. When those wells
were analyzed, they showed low percentages of CD45+ cells
(0.9% to 8.9%), accounting for their ability to proliferate depending
only on endogenous IL-6 production (Table 1). The results of this
experiment confirm that phenotypic conversion of CD45 to
CD45+ in U-266 cells is an IL-6-induced effect rather than
caused by contamination by CD45+ cells during cell sorting.
It also shows that the proliferative capacity is confined to the
CD45+ population and much enhanced by IL-6.
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| Fig 3.
Single-cell assays confirmed that CD45 expression and
proliferation are IL-6-induced effects. CD45 U-266 were
flow-sorted, diluted, and plated at 1 cell per well in 96-well plate.
Cells were cultured for 28 days with or without IL-6 (2 ng/mL). At the
end of the culture period, cells were harvested from wells containing
enough cells for flow cytometric analysis, stained with PE-CD45
monoclonal antibody, and analyzed. Representative flow cytograms from
cells cultured with IL-6 are shown. The percentage of
CD45+ cells is shown on each flow cytogram.
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Myeloma cells expressing CD45 proliferated in response to IL-6
stimulation.
To assess the proliferative activity of U-266 CD45+ and
CD45 subpopulations in response to IL-6
stimulation, flow-sorted CD45+ and
CD45 cells were cultured separately either in
the presence or absence of IL-6 (2 ng/mL), and cell counts were
performed at different time points. As shown in Fig
4, CD45+ cells showed a rapid
response that led to a 2.4-fold increase in cell number after 4 days
compared with a 1.4-fold increase in the absence of exogenous IL-6.
This increase scaled up to 5.1- and 7.9-fold after 6 and 8 days of
culture with IL-6, respectively, compared with only 2.5- and 3.9-fold
increases in the absence of IL-6. On the other hand, sorted
CD45 cells did not show any increase after 4 days of
culture with IL-6, but after 6 and 8 days, 1.9- and 3.0-fold increases,
respectively, were observed. This proliferative activity is probably
attributed to emergence of CD45+ cells during culture with
IL-6. The CD45 fraction cultured without IL-6 remained
consistently unchanged during the whole culture period as the medium
was frequently changed to remove any endogenously produced IL-6. These
results showed that the CD45+ fraction of U-266 constituted
the continuously proliferating fraction in response to IL-6
stimulation, and this response was largely dependent on the level of
IL-6 available in culture.
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| Fig 4.
CD45+ cells proliferated in response to
IL-6 stimulation. Growth curves of flow-sorted CD45
cells (dotted lines) cultured in medium alone ( ) or in the presence
of IL-6 (2 ng/mL;  ), and CD45+ cells (solid lines)
cultured in medium alone ( ) or in the presence of IL-6 ( ). Cells
were cultured for the indicated period of time, and cell counts were
performed as described in Materials and Methods.
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IL-6-induced effects were abolished by the PTP inhibitor sodium
vanadate.
To verify whether the IL-6-induced proliferation could be mediated
through CD45, we have used Na3VO4, which is
known to be a potent PTP inhibitor, to block the action of CD45
phosphatase. Okadaic acid, a serine-threonine phosphatase inhibitor,
was used as a control, and because it was dissolved in DMSO, another
experiment using DMSO at the same concentration was the control. After
1 day of treatment, no remarkable difference was observed, but on day 2 cells treated with medium alone and those treated with vanadate and
IL-6 (2 ng/mL) had a similar proliferative pattern that was clearly
less active compared with that of cells grown in medium with IL-6.
Conversely, cells treated with IL-6 together with either okadaic acid
or DMSO had an almost identical proliferative pattern to that of cells
treated with IL-6 alone (Fig 5). These data
suggested that the IL-6-induced cellular proliferation could be
mediated through the CD45 molecule, because this effect could only be
abolished by PTP inhibitor (vanadate), but not by serine-threonine
phosphatase inhibitor (okadaic acid). However, because we have used
vanadate in this experiment, the possibility of involvement of other
PTPs cannot be ruled out.
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| Fig 5.
IL-6-induced proliferation was abrogated by vanadate.
Flow-sorted CD45+ U-266 cells were cultured in medium
alone; in medium +IL-6 (2 ng/mL); or in medium + IL-6 + either 50 µmol/L Na3VO4; 10 ng/mL okadaic acid; or
DMSO. Cell counts were performed after 1 and 2 days after treatment.
Only vanadate could abrogate IL-6-induced proliferation after 2 days.
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| |
DISCUSSION |
In this study, we showed that CD45 expression in myeloma cell lines was
quite heterogeneous, not only regarding the presence of positive and
negative lines, but also within a given cell line such as U-266, in
which both positive and negative subpopulations did exist (Fig 1A and
B). This is in agreement with the existence of different phenotypes for
myeloma cells regarding CD45 expression, in which it is both reported
as CD45/dim,8-12 and as CD45+/++
in other studies.13-16 Our finding that CD45 expression
could be induced by IL-6 can explain the different patterns of
expression that appear in the literature. Different microenvironmental
levels of IL-6, either endogenously produced by myeloma cells
(autocrine loop) or exogenously supplied by the bone marrow stroma
cells, may modulate the pattern of CD45 expression in different cases. Alterations in the level of IL-6 in a given case may result in different CD45 phenotypes when analyzed at different time points. Indeed, different myeloma cell lines produce variable levels of endogenous IL-6, and it is quite high in some and barely detectable in
others (unpublished data).
The isoform expression pattern in CD45+ myeloma cells is
much more uniform in that almost all CD45+ myeloma cell
lines predominantly express the CD45RO and CD45RB isoforms (data not
shown). The biological significance of isoform expression is still ill
understood, but it could be speculated that RO and RB isoforms are
involved in positive regulation of cellular proliferation in myeloma
cells.
We also showed that CD45 expression could be induced by exogenous IL-6
(2 ng/mL) stimulation in sorted CD45U-266 cells and that
this phenotypic conversion occurred spontaneously but at a much lower
rate in the absence of exogenous IL-6 (Fig 2A). This spontaneous
conversion might be caused by the effect of endogenously produced IL-6
(10 pg/mL; Fig 2C). Because immature CD45+ cells are
generally considered to differentiate into mature CD45,
it would be argued that a contamination with a small fraction of
CD45+ cells during sorting could be responsible for those
observations. Given that the sorting purity was greater than 97% and
that the doubling time for U-266 was definitely more than 2 days, if
contamination with CD45+ cells was the cause, we should not
have seen such a high percentage of CD45+ cells (3.2% to
36%) within 8 days (Fig 2A). This result instead can be explained if
some CD45 convert to CD45+ that continue to
proliferate. Single-cell assays from flow-sorted CD45
cells unequivocally confirmed that IL-6 induces phenotypic conversion (Fig 3 and Table 1). Single-cell assays also showed clear difference in
the rate of emergence of CD45+ cells depending on the
availability of IL-6 in culture (0.9% to 8.9% in response to
endogenous IL-6 compared with 29.9% to 86.0% with exogenous IL-6) and
show the proliferative advantage of cells expressing CD45. The limited
proliferation of cells incubated without exogenous IL-6 (Table 1) is
not surprising, because we observed spontaneous conversion to
CD45+ phenotype depending only on endogenous IL-6
production (Fig 2A). Moreover, withdrawal of IL-6 from the medium led
to gradual loss of the CD45 from sorted CD45+ cells (Fig
2D).
Sequential culture and sorting of CD45+ cells further
confirms the ability to modify CD45 expression by IL-6 (Fig 2E). This indicates that induction of expression and loss of the CD45 is an
exogenous IL-6 effect and that this effect is a dose-dependent one,
because both CD45 and CD45+ subpopulations
produce similar levels of endogenous IL-6 (10 pg/mL; Fig 2C).
Also, the CD45 fraction is a nonproliferative one, and
it starts to proliferate only after expressing CD45 in response to
IL-6. Conversely, the CD45+ cells are continuously
proliferating, and their proliferation is much enhanced in response to
exogenously added IL-6 (Fig 4).
The underlying mechanism by which IL-6 modulates CD45 expression is
still not fully known, but apparently IL-6 has a positive regulatory
role on CD45 gene transcription as determined by RT-PCR (Fig 2C).
Despite the characterization of upstream22 and
intragenic23 promoters of CD45, nothing is known about
cis- and trans-acting elements. Examination of the
upstream promoter region shows a putative STAT consensus sequence, but
it is still unknown whether IL-6 stimulation is followed by binding of
gp130 heterodimers with a STAT member to that site. Because we observe
a lag time between IL-6 stimulation and the appearance of CD45 mRNA, we
speculate that an indirect mechanism is more likely in IL-6-induced
CD45 transcriptional activation. Work is going on in our laboratory to
delineate downstream signaling events of IL-6 using CD45+
and CD45 U-266 cells.
Finally, to verify that IL-6-induced proliferation could be mediated
through CD45, we have performed a blocking experiment in which we used
a potent PTP inhibitor (Na3VO4). As shown in Fig 5, vanadate could abolish the IL-6 enhancement of proliferation; on
the other hand, okadaic acid, which is an unrelated phosphatase inhibitor that acts specifically on serine-threonine phosphatases, had
almost no effect.
In conclusion, in this study we showed that CD45 had a remarkably
heterogeneous pattern of expression in myeloma cell lines. We show that
CD45 expression could be acquired or lost depending on the availability
of IL-6 in culture. Our observations indicate that CD45 may act as a
cellular growth regulatory molecule rather than solely as a
differentiation molecule. Finally, we also showed that the
IL-6-induced cellular proliferation in U-266 cells was attributed to
the CD45+ population of cells in an IL-6 dose-dependent
manner, and it seems plausible that this effect could be mediated
through the CD45 molecule.
| |
ACKNOWLEDGMENT |
The authors thank Dr S. Shimizu for his generous gift of the
IL-6-dependent myeloma cell lines, Dr H. Asaoku (Hiroshima Red Cross
Hospital, Hiroshima, Japan) for providing bone marrow samples, and N. Aoki for her secretarial help in preparing the manuscript.
| |
FOOTNOTES |
Submitted December 30, 1997;
accepted July 16, 1998.
Supported in part by grants from the Japanese Ministry of Education,
Science, and Culture.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Michio M. Kawano, MD, Department of
Immuno-hematology, Yamaguchi University School of Medicine, 1144 Kogushi, Ube, Yamaguchi 755-8505, Japan.
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Abstract
Introduction