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Blood, Vol. 94 No. 8 (October 15), 1999:
pp. 2871-2879
Involvement of Interleukin-10 (IL-10) and Viral IL-6 in the Spontaneous
Growth of Kaposi's Sarcoma Herpesvirus-Associated Infected Primary
Effusion Lymphoma Cells
By
Karen D. Jones*,
Yoshiyasu Aoki*,
Yuan Chang,
Patrick S. Moore,
Robert Yarchoan, and
Giovanna Tosato
From the Division of Hematologic Products, Center for Biologics
Evaluation and Research, Food and Drug Administration, Bethesda, MD;
the Department of Pathology, School of Public Health, Columbia
University, New York, NY; and the HIV and AIDS Malignancy Branch,
National Cancer Institute, National Institutes of Health, Bethesda, MD.
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ABSTRACT |
Primary effusion lymphoma (PEL) is a distinct type of lymphoma
associated with Kaposi's sarcoma-associated herpesvirus (KSHV) infection. To determine the factors responsible for the unrestrained proliferation of PEL, we have studied the growth factor requirements of
the PEL-derived BCBL-1 and BC-1 cell lines. Both cell lines were found
to be autocrine growth factor dependent and to release human
interleukin-6 (IL-6), viral IL-6 (vIL-6), and human IL-10 in the
culture supernatant. To establish whether these cytokines contribute to
autocrine growth, neutralizing antibodies against human IL-6, vIL-6,
human IL-10, and soluble IL-10 receptor were used. These experiments
showed that human IL-10 and, to a lesser degree, vIL-6 serve as
autocrine growth factors for BCBL-1 and BC-1 cells. Thus, human IL-10
and vIL-6 are growth factors released and used by PEL cells for
autonomous proliferation and may be critical to the development and
progression of PEL.
This is a US government work. There are no restrictions on its use.
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INTRODUCTION |
BODY CAVITY-BASED/PRIMARY effusion
lymphoma (PEL) is a rare and distinct type of non-Hodgkin's B-cell
lymphoma occurring in patients with acquired immunodeficiency syndrome
(AIDS) and some human immunodeficiency virus (HIV)-negative individuals
that is typically associated with Kaposi's sarcoma-associated
herpesvirus (KSHV; also known as herpes virus 8 [HHV-8])
infection.1-6 Characteristic features of this lymphoma
include involvement of the pleural, pericardial, and abdominal cavities
as lymphomatous effusions in the absence of a solid tumor mass, B-cell
lineage of the lymphoma cells based on Ig gene rearrangement, and, in
most cases, coinfection with Epstein-Barr virus (EBV).5,7,8
The lymphoma cells display immunoblastic morphology, express the cell
activation CD38 marker, and are generally monoclonal. They also
generally fail to express B-cell-associated surface antigens, lack
rearrangements of the c-myc gene, and do not display alterations of the
bcl-2, ras, and p53 genes.3,8,9
The presence of KSHV in PEL has raised questions on the virus'
potential pathogenetic role. In addition to its association with PEL,
KSHV is regularly found in all types of Kaposi's sarcoma and is
believed to infect asymptomatically a proportion of normal individuals.
Unlike 2 other gammaherpesviruses, EBV and Herpesvirus Saimiri, KSHV
has not been previously shown to immortalize or reproducibly infect
normal cells in vitro.10,11 However, many spontaneous cell
lines have been derived from PEL that grow autonomously in
vitro.6,12-14 Reflecting the infectious status of the
original lymphomas, some of these cell lines are coinfected with KSHV
and EBV, whereas others are infected with KSHV alone.
Previous studies on EBV-infected B cell lines have shown that growth
and survival of these cells is dependent on autocrine and/or paracrine
growth factors.15,16 Human interleukin-6 (IL-6) and IL-10
are present in the culture supernatants of EBV-infected cells and play
an important role in promoting the continuous proliferation of
EBV-immortalized cells in vitro.16-18 Patients with
posttransplant lymphoproliferative syndrome, an illness attributed to
the unbridled expansion of B cells latently infected with EBV, often
display abnormally high levels of circulating IL-6 and
IL-10.19,20 In addition, IL-10 was reported to serve as an
autocrine growth factor for AIDS-related B-cell lymphoma.21
Efforts aimed at identifying factors responsible for PEL autonomous
growth in vitro have suggested a role for human IL-6 based on
experiments with antisense oligonucleotides.22 However,
antisense oligonucleotides containing 4 contiguous guanosine residues,
such as the human IL-6 antisense oligonucleotide used in these
experiments, can inhibit cell proliferation via a
hybridization-independent mechanism.23 In addition, because
PEL cell growth was not inhibited by neutralizing antibodies against
human IL-6 or stimulated by IL-6 protein added to the cells, the role
of human IL-6 as a growth or survival factor for PEL is
uncertain.22 In particular, KSHV is known to code for viral
IL-6 (vIL-6), a cytokine that shares 24.7% amino acid identity with
human IL-6.24,25 Like human IL-6, vIL-6 can support the
growth of the murine hybridoma B9 cell line and the human myeloma INA-6
cell line, albeit at concentrations approximately 1,000-fold greater
than human IL-6.24
In this study, we have examined the potential contribution of vIL-6 and
other growth factors to the growth and survival of PEL cells.
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MATERIALS AND METHODS |
Cell lines.
The PEL-derived BCBL-16,11 and BC-111 cell
lines and the murine Ad5mE526 B-cell line were maintained
in RPMI 1640 medium (BioWhittaker, Walkersville, MD) supplemented with
2 mmol/L L-glutamine (Life Technologies, Grand Island, NY), 10% fetal
bovine serum (BioWhittaker), and 5 µg/mL gentamicin (Life Technologies).
Reagents.
The human cytokines IL-1 , tumor necrosis factor  (TNF), IL-4,
transforming growth factor (TGF), and IL-10 were purchased from
R&D Systems, Inc (Minneapolis, MN); IL-6 was either a gift from Sandoz
Pharmaceutical Co (Basel, Switzerland) or was purchased from R&D
Systems, Inc. Purified recombinant vIL-6 (MBP-vIL-6) was obtained from
Escherichia coli-expressing a fusion protein of vIL-6 (amino
acids 22-204) and a maltose binding protein (MBP), as
described.27 Neutralizing rat monoclonal antibodies (MoAbs) against human IL-10 (antibody 19F1) and an isotype control rat MoAb
(R35.95) were purchased from Pharmingen (San Diego, CA). A neutralizing
rabbit antibody against vIL-6 was obtained by immunizations with
recombinant purified vIL-6 (MBP-vIL-6; 1 mg/injection) and subsequent
serum purification over protein G column (Amersham Pharmacia Biotech,
Piscataway, NJ), following the manufacturer's instructions. A
neutralizing mouse MoAb against human IL-6 (6709.111) was purchased
from R&D Systems, Inc. Soluble human IL-10 receptor was obtained from
R&D Systems, Inc.
Cytokine measurements.
Conditioned media from the BCBL-1 and BC-1 cell lines were tested by
enzyme-linked immunosorbent assay (ELISA) for the human cytokines human
IL-1, IL-6, leukemia inhibitory factor (LIF), and IL-10 using
commercially available kits from R&D Systems, Inc. The sensitivity of
detection for IL-1, IL-6, and IL-10 ranged between 20 and 40 pg/mL.
Viral IL-10 was measured as previously described28 using
the rat anti-vIL-10 6B11 MoAb that recognizes viral but not human
IL-10. The sensitivity of vIL-10 detection was estimated to be 20 to 25 pg/mL. IL-6 bioactivity was measured by the B9 cell proliferation
assay, as described previously.29 The lower limit of
sensitivity of the B9 assay was estimated to correspond to 15 pg/mL
human IL-6. One unit of B9 activity, defined as the activity
stimulating 1 half-maximal cell proliferation, corresponds to
approximately 20 pg/mL human IL-6. Viral IL-6 was detected by Western
blotting, using a rabbit antiserum raised against recombinant vIL-6.
Flow cytometric analysis.
Biotinylated human IL-10 (1.5 mg/mL), human IL-6 (1.5 mg/mL), vIL-6
(MBP-vIL-6, 1.5 mg/mL), MBP (1.5 mg/mL), or biotinylated control
protein (soybean trypsin inhibitor, 1.5 mg/mL) was added to 1 × 105 IgG-treated cells and incubated for 60 minutes at
4°C. After incubation, streptavidin was added to the cells (10 µg/mL), and incubation was continued at 4°C for 30 minutes in the
dark. After washing, the cells were analyzed by flow cytometric
analysis (FACScan flow cytometer). Biotinylation of vIL-6 (MBP-vIL-6),
human IL-6, and MBP was performed using EZ-Link
Sulfo-NHS-LC-Biotinylation Kit (Pierce, Rockford, IL). Reagents for
human IL-10 receptor detection were purchased from R&D Biosystems, Inc.
Preparation of autologous conditioned media.
BCBL-1 and BC-1 cells obtained 48 to 72 hours after subculture were
washed free of serum (6 washes in RPMI 1640 medium), suspended at 2 × 106 cells/mL in culture medium (consisting of RMPI
1640 medium [BioWhittaker] supplemented with 0.25 mg/mL bovine serum
albumin [Boehringer Mannheim, Indianapolis, IN], 2.5 mg/mL
transferrin [Sigma Chemical Co, St Louis, MO], and 5 mg/mL gentamicin
[Life Technologies]), and incubated for 48 hours in
25-cm2 culture flasks (Corning, Corning, NY).
At the end of incubation, culture supernatants were harvested by
centrifugation, filtered, and frozen at  30°C until use.
Western blot analysis.
Aliquots (0.1 to 1 mL) of conditioned media, prepared in serum-free
conditions as described above and then subjected to deoxycholate trichloroacetic acid (DOC-TCA) precipitation, cell lysates from 1 × 105 cells, or purified proteins (vIL-6, human IL-6,
human IL-1, human TGF, and human TNF) were solubilized in
tricine sodium dodecyl sulfate (SDS) sample buffer (Novex,
San Diego, CA), boiled, and run through 10% to 20% tricine gels
(Novex). Protein was then transferred onto Immobilon-P membranes
(Millipore Corp, Bedford, MA). Membranes were reacted with a rabbit
anti-vIL-6 antiserum (1:1,000 dilution); bound antibody was detected
with an affinity-purified, peroxidase-linked, donkey antirabbit IgG
antibody (Amersham Pharmacia Biotech) and a chemiluminescence detection
system (ECL kit; Amersham Pharmacia Biotech). For detection of human
IL-6 (recombinant human IL-6, 100 ng), the membranes were reacted with
a biotinylated, affinity-purified goat antihuman IL-6 antiserum (R&D
Systems), followed by a streptavidin-horseradish-peroxidase-conjugated
antigoat IgG antibody (Life Technology) and a chemiluminescence
detection system (ECL kit).
Cell proliferation assays.
Exponentially growing BCBL-1 and BC-1 cells were first washed free of
serum, suspended in RMPI 1640 medium (BioWhittaker) supplemented with
0.25 mg/mL bovine serum albumin (Boehringer Mannheim), 2.5 µg/mL
transferrin (Sigma Chemical Co), and 5 µg/mL gentamicin (Life
Technologies) and then incubated (5 to 10 × 103
cells/well) alone or with additives in microtiter culture plates (Costar, Cambridge, MA) for 3 days. DNA synthesis was measured by
3H thymidine deoxyribose uptake (0.5 mCi/well, 6.7 Ci/mmol;
New England Nuclear, Boston, MA) during the last 18 to 20 hours of culture. The results of proliferation assays were expressed as the mean
radioactivity (± standard deviation) of triplicate cultures.
Cytokine neutralization assays.
Exponentially growing BCBL-1 and BC-1 cells that had been washed free
of serum were incubated (5 to 10 × 103 cells/well for
3 days) in microtiter culture wells (Costar) in RMPI 1640 medium
(BioWhittaker) supplemented with 0.25 mg/mL bovine serum albumin
(Boehringer Mannheim), 2.5 µg/mL transferrin (Sigma Chemical Co), and
5 µg/mL gentamicin (Life Technologies) alone or with the
addition of autologous conditioned medium (prepared as described above)
with or without the addition of purified neutralizing antibodies (1 to
10 µg/mL) against various cytokines or soluble human IL-10 receptor
(0.1 to 0.2 µg/mL). The specificity of reactions was tested by
addition of the appropriate cytokines (1 to 10 ng/mL) to the
neutralized cultures. Cell proliferation was measured by 3H
thymidine deoxyribose uptake, as described above.
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RESULTS |
Dependency of body cavity lymphoma cell lines on autocrine growth
factors.
Body cavity-based/PEL-derived cell lines are either infected with HHV-8
alone or with HHV-8 plus EBV. We therefore selected the HHV-8-infected
BCBL-111 and the HHV-8 plus EBV-infected BC-114
cell lines. To test for autocrine growth factor dependency, PEL cells
were cultured under conditions that minimize the contribution of
autocrine growth factors, including low cell densities (2.2 to 10 × 104 cells/mL) and restricted culture medium (RPMI
1640 culture medium supplemented with 0.25 mg/mL bovine serum albumin
and 2.5 µg/mL transferrin). Conditioned medium was prepared by
incubation of PEL cell lines and a control murine B-cell line (Ad5mE5)
at 2 × 106 cells/mL in RPMI 1640 culture medium
supplemented with 0.25 mg/mL bovine serum albumin and 2.5 µg/mL
transferrin for 2 days. When incubated in medium alone (RPMI 1640 medium supplemented with 0.25 mg/mL bovine serum albumin and 2.5 µg/mL transferrin) for 3 days at low cell densities, BCBL-1 and BC-1
cells showed minimal spontaneous proliferation
(Fig 1). However, when incubated with autologous conditioned medium under otherwise identical conditions, BCBL-1 and BC-1 cells showed a dose-dependent increase in proliferation (Fig 1). Conditioned medium from a control murine B-cell line prepared
under identical conditions had variable stimulatory effects on BCBL-1
and BC-1 cells (Fig 1). These experiments demonstrate that BCBL-1 and
BC-1 cells are dependent on autocrine growth factors for sustained
growth.
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| Fig 1.
Enhanced proliferation of BCBL-1 and BC-1 cell lines in
response to autologous conditioned medium. The cell lines BCBL-1 and
BC-1 in exponential growth phase (5 × 103 and 10 × 103/0.2 mL flat-bottom microwell, respectively) were
cultured for 3 days in RPMI 1640 medium supplemented with either fresh
medium alone or medium that had previously been conditioned for 24 hours by either the autologous (BCBL-1 or BC-1;  ) or control (murine
AdmEm5;  ) cells seeded at 2 × 106 cells/mL in tissue
culture flasks. 3H thymidine was added during the final 18 hours of culture. The results represent the mean radioactivity (±SD)
of triplicate cultures. Shown is a representative experiment of 9 performed.
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Expression of human IL-10, human IL-6, and vIL-6 by PEL cells.
To identify potential compounds responsible for autocrine growth factor
activity, we first measured by ELISA the levels of selected cytokines
in the conditioned media from BCBL-1 and BC-1 cell lines. In
representative determinations (Table 1),
human IL-10 was detected at the concentration of 20.8 ng/mL in BCBL-1 and at 3.3 ng/mL in BC-1 conditioned media; and human IL-6 was detected
at 116.0 pg/mL in BCBL-1 and at 12.7 ng/mL in BC-1 conditioned media.
However, vIL-10 (assay sensitivity, 10 × 10 3
U/mL; corresponding to ~20 to 25 pg/mL), IL-1 (assay sensitivity, ~35 pg/mL), and LIF (assay sensitivity, ~50 pg/mL) were not
detectable in BCBL-1 and in BC-1 conditioned media. Because of a lack
of suitable reagents, vIL-6 cannot currently be measured by ELISA. Instead, we used a rabbit antiserum raised against recombinant vIL-6
that recognizes vIL-6 but not human IL-6
(Fig 2A), IL-1, IL-10, TGF, or TNF
(not shown) in immunoblotting. Using this method, vIL-6 was detected in
the culture supernatants of BCBL-1 and BC-1 cells (Fig 2B). Using
various concentrations of recombinant purified vIL-6 in Western
blotting (Fig 2C), we estimated the content of vIL-6 in culture
supernatants of BC-1 and BCBL-1 cell lines to range between 5 and 20 ng/mL. Consistent with these results, BCBL-1 and BC-1 culture
supernatants contained IL-6 bioactivity (42 and >100 IL-6 units/mL,
respectively), as measured by the B9 cell proliferation assay that can
detect both cellular and vIL-6. These results demonstrate that
conditioned media from BCBL-1 and BC-1 cells contain human IL-10, human
IL-6, and vIL-6.
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| Fig 2.
Viral IL-6 in the culture supernatants of BCBL-1 and BC-1
cells detected by immunoblotting with a specific rabbit antiserum. (A)
A rabbit antiserum to vIL-6 was used in immunoblotting of viral and
human IL-6 protein. (B) Conditioned medium from the BCBL-1 and BC-1
cell lines (1 mL) was TCA-precipitated and then analyzed by
immunoblotting with a rabbit antiserum to vIL-6. Cell lysates from
NIH3T3 cells (1 × 103 cells) transfected with a control
vector or with the vIL-6 gene were used as controls. (C) Detection of
purified recombinant vIL-6 (0.32 to 200 ng) in immunoblotting using a
rabbit antiserum to vIL-6.
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Effect of IL-10, IL-6, and vIL-6 neutralizing antibodies on PEL cell
growth.
To assess the potential role for cytokines as autocrine growth factors,
neutralizing antibodies specific for human IL-10, IL-6, and vIL-6 or
control antibodies were added to PEL cell cultures, and their effects
on cell proliferation were measured. Neutralizing antibodies against
human IL-10 (19F1 MoAb, 5 µg/mL) reduced autocrine growth
factor-induced proliferation of BCBL-1 cells by approximately 88% and
of BC-1 cells by 70% (Fig 3). An
isotype-matched control rat MoAb (R35-95) had a minimal effect (Fig 3).
Addition of IL-10 (25 ng/mL) to these cultures reduced the level of
antibody neutralization by 33% to 60%. Titration experiments showed
that, at the concentration of 2.5 µg/mL, the antihuman IL-10 antibody
neutralized 42% and 51% of the BCBL-1 and BC-1 autocrine growth
factor activity, respectively, whereas at the concentration of 1.25 µg/mL, the antihuman IL-10 antibody had minimal neutralizing effect
with either cell line. However, IL-10 (25 ng/mL) added alone to BCBL-1
and BC-1 cultured under conditions that minimize the contribution of
autocrine growth factors stimulated little cell proliferation (Fig 3).
Because these results suggested that IL-10 is a critical component of the autocrine growth factor activity in BCBL-1 and BC-1 cells, we
tested the effects of soluble IL-10 receptor. The addition of soluble
IL-10 receptor (100 ng/mL) to BCBL-1 and BC-1 cells substantially
reduced cell proliferation in the presence of autologous conditioned
medium (Fig 4). Thus, soluble IL-10
receptor and antibodies against human IL-10, reagents that specifically
bind and neutralize IL-10, reduced autocrine growth factor-dependent
PEL proliferation, substantiating a role for IL-10 in PEL cell growth.
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| Fig 3.
Effects of a neutralizing antibody to human IL-10 on the
proliferation of BCBL-1 and BC-1 cells. Cells (5 × 103
cells/microwell) from the BCBL-1 and BC-1 cell lines were cultured for
3 days with or without the addition of 25% autologous conditioned
medium either alone or in the presence of a neutralizing MoAb (19F1; 5 µg/mL) to human IL-10 or an isotype-matched control MoAb (R35-95; 5 µg/mL). Sets of cultures were supplemented with recombinant human
IL-10 (25 ng/mL). 3H thymidine was added during the final
20 hours of culture. The results represent the mean (±SD)
radioactivity of triplicate cultures. Shown is a representative
experiment of 4 performed.
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| Fig 4.
Effects of soluble IL-10 receptor on the proliferation of
BCBL-1 and BC-1 cells. Cells (5 × 103 cells/microwell)
from the BCBL-1 and BC-1 cell lines were cultured for 3 days with or
without the addition of 25% autologous conditioned medium with medium
alone or in medium supplemented with soluble IL-10 receptor (100 ng/mL). 3H thymidine was added during the final 20 hours of
culture. The results represent the mean (±SD) radioactivity of
triplicate cultures. Shown is a representative experiment of 5 performed.
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To test for a potential contribution of human IL-6 to PEL autocrine
cell growth, we selected a MoAb against human IL-6 that can neutralize
human, but not viral, IL-6 bioactivity detected by the B9 assay and can
recognize human but not viral IL-6 in immunoblotting (not shown). This
antihuman IL-6 antibody consistently failed to inhibit BCBL-1 and BC-1
cell proliferation induced by autologous conditioned media (not shown).
Also, human recombinant purified IL-6 (100 pg/mL to 1 mg/mL)
consistently failed to stimulate the proliferation of BCBL-1 and BC-1
cells grown under restricted culture conditions (not shown). Similarly,
the addition of the cytokines TGF, IL-4, and TNF (used at
concentrations ranging between 1 and 100 ng/mL) consistently failed to
stimulate the proliferation of BCBL-1 and BC-1 cells grown under
restricted culture conditions (not shown). Thus, human IL-6, which is
detected in the culture supernatant of BCBL-1 and BC-1 cells, appears
not to function as an autocrine growth factor for these cells.
To assess the potential role of vIL-6 to autocrine growth factor
activity, we tested the effects of a purified rabbit antiserum against
vIL-6. This reagent can specifically neutralize vIL-6-induced B9 cell
proliferation, but has minimal neutralizing effect on human
IL-6-induced B9 cell proliferation (Fig
5). When tested for its ability to neutralize PEL autocrine growth
factor activity (Fig 6), anti-vIL-6
antibody (5 µg/mL) reduced autocrine growth factor activity by 23%
in BCBL-1 cells and by 28% in BC-1 cells. A dose-response study showed
that, at the reduced concentration of 1 µg/mL, this anti-vIL-6
antibody neutralized 0% and 4% of BCBL-1 and BC-1 autocrine growth
factor activity, respectively. A control rabbit IgG preparation did not
reduce the autocrine growth factor activity in BCBL-1 and BC-1 cells
(Fig 6). The specificity of the neutralizing effect was documented by
the addition of recombinant purified vIL-6, which reversed the
neutralizing effect of the anti-vIL-6 antibody, whereas human IL-10
did not. We compared the neutralizing effect of anti-vIL-6 and
antihuman IL-10 antibodies used at the same concentration (5 µg/mL).
In parallel cultures (Fig 6), antihuman IL-10 antibodies were more
effective at neutralizing autocrine growth factor activity than
anti-vIL-6 antibodies (75% v 23% in BCBL-1 cells; and 32%
v 28% in BC-1 cells). When added together in culture,
antihuman IL-10 and anti-vIL-6 antibodies neutralized 89% and 35% of
the autocrine growth factor activity in BCBL-1 and BC-1 cells,
respectively (Fig 6). The addition of recombinant purified vIL-6 and
human IL-10 to these cultures reversed the combined neutralizing effect
of the anti-vIL-6 and antihuman IL-10 antibodies (Fig 6). However,
when added together in the absence of conditioned medium, IL-10 and
vIL-6 displayed little growth stimulation of BCBL-1 and BC-1 cells (Fig
6). These experiments document that human IL-10 and vIL-6 contribute to
the autocrine growth factor activity in the BCBL-1 and BC-1 cell lines
and suggest that other growth factors yet to be defined are important
contributors to autocrine growth in these cells.
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| Fig 5.
Effects of anti-vIL-6 antibody on B9 cell proliferation
induced by viral and human IL-6. Exponentially growing B9 cells (2 × 103 cells/microwell) were cultured in medium alone or
medium supplemented with vIL-6 (MBP-vIL-6; 1 to 100 ng/mL) or human
IL-6 (1 to 100 pg/mL), with () or without () anti-vIL-6 antibody
(10 µg/mL). 3H thymidine was added during the final 6 hours of culture. The results represent the mean radioactivity of
triplicate cultures; SDs were within 5% of the mean.
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| Fig 6.
Effects of a neutralizing antibody to vIL-6 on the
proliferation of BCBL-1 and BC-1 cells. Cells (5 × 103
cells/microwell) from the BCBL-1 and BC-1 cell lines were cultured
for 3 days with or without the addition of 25% autologous conditioned
medium either alone or in the presence of a rabbit neutralizing
antibody (5 µg/mL) against vIL-6, control rabbit IgG (5 µg/mL), a
MoAb against human IL-10 (19F1; 5 µg/mL), or anti-vIL-6 plus
antihuman IL-10 antibodies (each at 5 µg/mL). Selected cultures were
supplemented with recombinant purified vIL-6 (MBP-vIL-6; 50 ng/mL) or
human IL-10 (25 ng/mL). 3H thymidine was added during the
final 20 hours of culture. The results represent the mean (±SD)
radioactivity of triplicate cultures.
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IL-10 and vIL-6 receptor expression by BCBL-1 and BC-1 cells.
To define further the roles of IL-10 and vIL-6 as growth factors for
PEL cells, we evaluated the expression of the IL-10, vIL-6, and human
IL-6 receptors using biotinylated IL-10, vIL-6, or human IL-6 and
flow-cytometric analysis. BCBL-1 and BC-1 cells exhibited significant
binding of IL-10, vIL-6, and human IL-6 (Fig 7). The specificity of cytokine
binding was confirmed by use of a control biotinylated protein, soybean
trypsin inhibitor, whereas the specificity of vIL-6 (MBP-vIL-6) binding
was confirmed by use of control biotinylated MBP (Fig 7). These results
are consistent with BCBL-1 and BC-1 cell expression of human IL-10, human IL-6, and vIL-6 receptors.
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| Fig 7.
Human IL-10, human IL-6, and vIL-6 binding to BCBL-1 and
BC-1 cells. Biotinylated human IL-10 (1.5 µg/mL), human IL-6 (1.5 µg/mL), soybean trypsin inhibitor (1.5 µg/mL), vIL-6 (MBP-vIL-6;
1.5 µg/mL), or MBP (1.5 µg/mL) was incubated with BCBL-1 and BC-1
cells. Cell-bound protein was shown by the addition of
avidin-fluorescein (10 µg/mL). After washing, surface fluorescence
was evaluated by FACScan analysis. Unshaded histograms reflect binding
from control biotinylated control proteins (soybean trypsin inhibitor
or MBP).
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| |
DISCUSSION |
In this study, we show that KSHV-infected spontaneous cell lines
derived from PEL are dependent on autocrine growth factors for
continuous proliferation in vitro and demonstrate that human IL-10 and,
to a lesser degree, vIL-6 account for some of the autocrine growth
factor activity. When cultured at low cell densities in serum-free
medium, the BCBL-1 and BC-1 cell lines showed little spontaneous
proliferation. However, the addition of autologous supernatant prepared
in serum-free medium at optimal cell densities induced a high rate of
cell proliferation. Human IL-10, human IL-6, and vIL-6, but not other
cytokines, were detected in BCBL-1 and BC-1 culture supernatants.
Neutralizing antibodies to human IL-10 and to vIL-6 added individually
to BCBL-1 and BC-1 cell cultures reduced significantly cell
proliferation induced by autologous conditioned media. When added
together, these neutralizing antibodies removed a substantial amount of
the autocrine growth factor activity in BCBL-1 and BC-1 culture
supernatants. The neutralizing effect was specific, because human IL-10
and vIL-6 proteins reversed the growth-neutralizing effect of the
respective antibodies. However, when added together to BCBL-1 and BC-1
cells grown at low cell densities in serum-free medium, human IL-10 and
vIL-6 failed to stimulate cell growth. Neutralizing antibodies to human
IL-6, oncostatin M, LIF, ciliary neurotropic factor
(CNTF), and IL-11 had minimal effect on the growth of
BCBL-1 or BC-1 cells (not shown). Thus, we show that 2 PEL-derived cell
lines are dependent, in part, on the cellular cytokine IL-10 and, to a
lesser extent, the viral cytokine IL-6 for autonomous proliferation in
vitro. Other growth factors yet to be defined may contribute to the
autocrine growth of PEL cells.
The cell lines used in the current studies are representative of PEL
that is either infected with KSHV alone or is coinfected with EBV and
KSHV.23 Previous studies have demonstrated that EBV-infected cell lines derived from the spontaneous outgrowth of
either normal B cells from EBV-seropositive individuals or posttransplant lymphoproliferative disease tissues are largely dependent on autocrine growth factors, particularly IL-6 and IL-10, for
continuous proliferation.16-18 By contrast, vIL-10, a
product of the EBV gene BCRF-1, and a variety of other cytokines and
chemokines are not active as a growth-stimulatory factor for these cell
lines.30 LMP-1, an EBV latency protein that is regularly
expressed in EBV-immortalized cell lines, is known to mediate the
activation of NF-kB transcription factor and, by this mechanism,
stimulate expression of IL-10, IL-6, and other cellular genes that
contain kB elements.31-33 Previous studies have documented
that LMP-1 is not expressed in the PEL-derived cell line BC-1 that is
coinfected with KHSV and EBV, suggesting that EBV-infected PEL may
display a limited expression of EBV latency genes similar to certain
EBV-infected Burkitt cells.34,35 Thus, IL-6 and IL-10
expression in BC-1 cells may not be attributable to EBV infection and
expression of the LMP-1 protein. Rather, because the pattern of
autocrine cytokine requirement is quite similar in BCBL-1 and BC-1 cell
lines, which differ with respect to their EBV status while both being
KSHV-infected, KSHV could play an important and unifying role in the
regulation of autonomous cell growth in these cell lines. KSHV open
reading frame K2 encodes vIL-6; thus, the virus plays a very direct
role in the production of this cytokine.24,25,36 By
contrast, the mechanism by which human IL-10 is expressed in PEL cell
lines is currently undefined.
In addition to being expressed in PEL and the cell lines derived from
PEL, vIL-6 is detected in some forms of KSHV-positive multicentric
Castleman's disease, whereas it is generally not expressed in
KSHV-positive Kaposi's sarcoma tissues.24,37 Because vIL-6
exhibits 24.7% amino acid identity to human IL-6, it was suggested
that KSHV may have captured this cellular gene during evolution for its
own advantage.24,25,36 The results presented here
demonstrate that vIL-6 contributes some of the autocrine growth factor
activity in PEL cell lines. By promoting the expansion of KSHV-infected
cells, vIL-6 may help KSHV persist and spread in the human host.
It is worth noting that, whereas both viral and human IL-6 are detected
in PEL culture supernatants, our results suggest that human IL-6 and
vIL-6 differ with respect to growth stimulation of PEL cells. The
reasons for this difference, particularly the failure of human IL-6 to
promote PEL cell growth, are currently not clear. We know that the
human IL-6 present in the culture supernatant of PEL cell lines is
biologically active, because it promotes the growth of the
IL-6-dependent indicator B9 cells. We also know that the failure of
human IL-6 to stimulate PEL cell growth is not due to insufficient
cytokine amounts, because even microgram quantities of human IL-6 added
to culture were inactive. Previous studies with the Hep-G2 cell line, a
human hepatoma cell line that expresses cellular IL-6 receptors,
demonstrated potential differences in IL-6 receptor use by human and
vIL-6.38 Other studies using the human myeloma cell line
INA-6 provided evidence that the IL-6 receptor chain as well as
GP130 are involved in viral as well as human IL-6 signaling, albeit
with different affinities, perhaps due to amino acid differences at
human IL-6 positions Phe74 and Arg182.25,39 In addition,
Western blot analysis has demonstrated that the IL-6 receptor chain
is expressed in several PEL cell lines.22 We showed here
that PEL cells bind human IL-6. Thus, current information on IL-6
receptor structure and signaling does not explain the biological
differences of human and vIL-6 noted here, and additional studies will
be needed.
Human IL-10, a cytokine normally produced by activated T lymphocytes,
monocytes, B lymphocytes, and other cells, can promote the growth of
primary B lymphocytes that are costimulated by anti-IgM or anti-CD40
antibodies and can profoundly inhibit T-cell immunity.40 In
conjunction with IL-4, IL-10 promoted the expansion and increased substantially the survival of primary B lymphocytes in
vitro.41-43 When added to B lymphocytes in conjunction with
EBV, IL-10 potentiated virus-induced cell
proliferation.44,45 IL-10 is expressed by PEL cells in
vitro and in vivo.6,10 The results presented here suggest
that, in addition to other previously reported stimulatory effects on B
lymphocytes, IL-10 may cooperate with KSHV to potentiate virus-induced
proliferation of cells of B-cell lineage.
Currently, there are no effective treatments available for PEL, and
patients usually succumb of their illness within months from diagnosis.
The observation that PEL cells are dependent on IL-10 and vIL-6 for
expansion, suggests that rational treatments could be designed to
impair production or cell responsiveness to these cytokines.
| |
ACKNOWLEDGMENT |
The authors thank Sandy Pike, Lei Yao, Barry Cherney, Fonda Newcomb,
and Andy Lewis.
| |
FOOTNOTES |
*
K.D.J. and Y.A. contributed equally to this report.
Submitted January 27, 1999; accepted June 15, 1999.
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 Giovanna Tosato, MD, Division of
Hematologic Products, Center for Biologics Evaluation and Research,
Bldg 29A, Room 2D16, 8800 Rockville Pike, Bethesda, MD 20892.
| |
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[Full Text]
[PDF]
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Y. Aoki, G. M. Feldman, and G. Tosato
Inhibition of STAT3 signaling induces apoptosis and decreases survivin expression in primary effusion lymphoma
Blood,
February 15, 2003;
101(4):
1535 - 1542.
[Abstract]
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[PDF]
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M. Cannon, N. J. Philpott, and E. Cesarman
The Kaposi's Sarcoma-Associated Herpesvirus G Protein-Coupled Receptor Has Broad Signaling Effects in Primary Effusion Lymphoma Cells
J. Virol.,
December 6, 2002;
77(1):
57 - 67.
[Abstract]
[Full Text]
[PDF]
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M. Chatterjee, J. Osborne, G. Bestetti, Y. Chang, and P. S. Moore
Viral IL-6-Induced Cell Proliferation and Immune Evasion of Interferon Activity
Science,
November 15, 2002;
298(5597):
1432 - 1435.
[Abstract]
[Full Text]
[PDF]
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M.-Q. Du, T. C. Diss, H. Liu, H. Ye, R. A. Hamoudi, J. Cabecadas, H. Y. Dong, N. L. Harris, J. K. C. Chan, J. W. Rees, et al.
KSHV- and EBV-associated germinotropic lymphoproliferative disorder
Blood,
October 16, 2002;
100(9):
3415 - 3418.
[Abstract]
[Full Text]
[PDF]
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H. Li and J. Nicholas
Identification of Amino Acid Residues of gp130 Signal Transducer and gp80 {alpha} Receptor Subunit That Are Involved in Ligand Binding and Signaling by Human Herpesvirus 8-Encoded Interleukin-6
J. Virol.,
May 3, 2002;
76(11):
5627 - 5636.
[Abstract]
[Full Text]
[PDF]
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E. Oksenhendler, E. Boulanger, L. Galicier, M.-Q. Du, N. Dupin, T. C. Diss, R. Hamoudi, M.-T. Daniel, F. Agbalika, C. Boshoff, et al.
High incidence of Kaposi sarcoma-associated herpesvirus-related non-Hodgkin lymphoma in patients with HIV infection and multicentric Castleman disease
Blood,
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99(7):
2331 - 2336.
[Abstract]
[Full Text]
[PDF]
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C. Liu, Y. Okruzhnov, H. Li, and J. Nicholas
Human Herpesvirus 8 (HHV-8)-Encoded Cytokines Induce Expression of and Autocrine Signaling by Vascular Endothelial Growth Factor (VEGF) in HHV-8-Infected Primary-Effusion Lymphoma Cell Lines and Mediate VEGF-Independent Antiapoptotic Effects
J. Virol.,
November 15, 2001;
75(22):
10933 - 10940.
[Abstract]
[Full Text]
[PDF]
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Y. Aoki, M. Narazaki, T. Kishimoto, and G. Tosato
Receptor engagement by viral interleukin-6 encoded by Kaposi sarcoma-associated herpesvirus
Blood,
November 15, 2001;
98(10):
3042 - 3049.
[Abstract]
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T. Portis, J. C. Harding, and L. Ratner
The contribution of NF-{kappa}B activity to spontaneous proliferation and resistance to apoptosis in human T-cell leukemia virus type 1 Tax-induced tumors
Blood,
August 15, 2001;
98(4):
1200 - 1208.
[Abstract]
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[PDF]
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Y. Aoki, G. Tosato, T. W. Fonville, and S. Pittaluga
Serum viral interleukin-6 in AIDS-related multicentric Castleman disease
Blood,
April 15, 2001;
97(8):
2526 - 2527.
[Full Text]
[PDF]
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H. Li, H. Wang, and J. Nicholas
Detection of Direct Binding of Human Herpesvirus 8-Encoded Interleukin-6 (vIL-6) to both gp130 and IL-6 Receptor (IL-6R) and Identification of Amino Acid Residues of vIL-6 Important for IL-6R-Dependent and -Independent Signaling
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April 1, 2001;
75(7):
3325 - 3334.
[Abstract]
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M.-Q. Du, H. Liu, T. C. Diss, H. Ye, R. A. Hamoudi, N. Dupin, V. Meignin, E. Oksenhendler, C. Boshoff, and P. G. Isaacson
Kaposi sarcoma-associated herpesvirus infects monotypic (IgM{lambda}) but polyclonal naive B cells in Castleman disease and associated lymphoproliferative disorders
Blood,
April 1, 2001;
97(7):
2130 - 2136.
[Abstract]
[Full Text]
[PDF]
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Y. Aoki, R. Yarchoan, K. Wyvill, S.-i. Okamoto, R. F. Little, and G. Tosato
Detection of viral interleukin-6 in Kaposi sarcoma-associated herpesvirus-linked disorders
Blood,
April 1, 2001;
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2173 - 2176.
[Abstract]
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J Nicholas
Evolutionary aspects of oncogenic herpesviruses
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[Abstract]
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E. Oksenhendler, G. Carcelain, Y. Aoki, E. Boulanger, A. Maillard, J.-P. Clauvel, and F. Agbalika
High levels of human herpesvirus 8 viral load, human interleukin-6, interleukin-10, and C reactive protein correlate with exacerbation of multicentric Castleman disease in HIV-infected patients
Blood,
September 15, 2000;
96(6):
2069 - 2073.
[Abstract]
[Full Text]
[PDF]
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Y. Aoki, R. Yarchoan, J. Braun, A. Iwamoto, and G. Tosato
Viral and cellular cytokines in AIDS-related malignant lymphomatous effusions
Blood,
August 15, 2000;
96(4):
1599 - 1601.
[Abstract]
[Full Text]
[PDF]
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TOP
ABSTRACT
INTRODUCTION