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Journal of Virology, October 1999, p. 8427-8434, Vol. 73, No. 10
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Regulation of Bovine Leukemia Virus tax
and pol mRNA Levels by Interleukin-2 and -10
Dohun
Pyeon
and
Gary A.
Splitter*
Department of Animal Health and Biomedical
Sciences, University of Wisconsin
Madison, Madison, Wisconsin
53706
Received 11 March 1999/Accepted 2 July 1999
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ABSTRACT |
Recently, particular cytokines have been identified to affect
progression of a variety of diseases and retrovirus infections. Previously, we demonstrated that interleukin-2 (IL-2), IL-12, and gamma
interferon increased in peripheral blood mononuclear cells (PBMCs) from
animals with early disease and decreased in PBMCs from animals with
late disease stages of bovine leukemia virus (BLV) infection. In
contrast, IL-10 increased with disease progression. To examine the
effects of these cytokines on BLV expression, BLV tax and
pol mRNA and p24 protein were quantified by competitive PCR
and immunoblotting, respectively. IL-10 inhibited BLV tax
and pol mRNA levels in BLV-infected PBMCs; however, the inhibitory effect of IL-10 was prevented in PBMCs depleted of monocytes
and/or macrophages (monocyte/macrophages). To determine whether these
factors were secreted or monocyte/macrophage associated, monocyte/macrophage-depleted PBMCs were cultured with isolated monocyte/macrophages in transwells where contact between
monocyte/macrophages and nonadherent PBMCs was blocked. BLV
tax and pol mRNA levels increased in transwell
cultures similar to cultures containing nonseparated cells, and IL-10
addition inhibited the increase of BLV tax and
pol mRNA. These results suggest that monocyte/macrophages secrete soluble factor(s) that increases BLV mRNA levels and that secretion of these soluble factor(s) could be inhibited by IL-10. In
contrast, IL-2 increased BLV tax and pol mRNA
and p24 protein production. Thus, IL-10 production by BLV-infected
animals with late stage disease may serve to control BLV mRNA levels,
while IL-2 may increase BLV mRNA in the early disease stage. To
determine a correlation between cell proliferation and BLV expression,
the effect of IL-2 and IL-10 on PBMC proliferation was tested. As anticipated, IL-2 stimulated while IL-10 suppressed antigen-specific PBMC proliferation. The present study, combined with our previous findings, suggests that increased IL-10 production in late disease stages suppresses BLV mRNA levels, while IL-2-activated immune responses stimulate BLV expression by BLV-infected B cells.
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INTRODUCTION |
Bovine leukemia virus (BLV), which
is closely related to human T-cell leukemia virus type 1 (HTLV-1), is a
type C retrovirus that infects bovine B cells and leads to development
of enzootic bovine leukosis (13). Less than 5% of infected
animals develop malignant lymphosarcoma (8), while 30% of
infected animals progress to persistent lymphocytosis. In persistent
lymphocytosis, nonneoplastic B cells proliferate, and leukocyte counts
may exceed 10,000 cells/mm3 (16). However, most
infected animals remain in the alymphocytotic (AL) stage. Despite the
often long duration for disease progression, the mechanism for
progression is unknown. Previously, we determined that cytokine
profiles of BLV-infected animals differ depending on the stage of
disease (24, 27). Interleukin-2 (IL-2), IL-12, and gamma
interferon (IFN-
) were expressed in high amounts in AL animals. In
contrast, interleukin 10 (IL-10) was increased in persistently
lymphocytotic (PL) animals. While IL-2, IL-12, and IFN- trigger
cellular immune responses that activate macrophages, NK, Th1, and
cytotoxic T cells to remove virus from the host, IL-10 suppresses these
cytokine-activated immune responses (17). Increased IL-10
production in BLV infection could be deleterious for clearing viral
infection from the host. Lundberg et al. reported that cytotoxic 
T-cell (CTL) activity is crippled in PL animals, while CTLs from
AL animals efficiently lysed BLV Env and Tax presenting cells
(18). Cytokine imbalance may also contribute to disease
progression in human immunodeficiency virus (HIV) infection (6), autoimmune disease, and cancer (17).
Alternatively, there may be beneficial effects of IL-10 for virus
clearance. In HIV infection, IL-10 suppresses immune activation
(20, 30), and reduced immune surveillance may permit a
suitable environment for virus replication. Interestingly, whereas HIV
replication was significantly reduced in experiments with macrophage
cell lines and primary macrophages, this inhibitory effect was
not observed in experiments with T-cell lines and primary T cells alone (29, 30, 36). These reports suggest that monocytes and/or macrophages (monocyte/macrophages) have an important role in
regulating virus replication in T cells, as well as
monocyte/macrophages responding to IL-10. To examine the influence of
cytokines on BLV mRNA levels, BLV tax and pol
mRNA were quantified from peripheral blood mononuclear cells (PBMCs)
cultured with IL-2, IL-10, and IL-12. Here, we demonstrate that IL-10
inhibits detection of BLV tax and pol mRNA, while
IL-2 activates BLV tax and pol mRNA and p24
protein levels. The inhibitory effect of IL-10 on BLV tax and pol mRNA was eliminated in monocyte/macrophage-depleted PBMCs.
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MATERIALS AND METHODS |
Animals and cell preparation.
Adult female Holstein cattle,
2 to 12 years of age, were assigned to two groups according to their
disease stage. Three AL and three PL animals were used. Each experiment
was performed with three different animals and at least one from each
disease stage except the immunoblotting assay with two PL animals.
Heparinized or EDTA-treated blood was obtained from the jugular vein,
and PBMCs were isolated by density gradient centrifugation
(5).
Cell cultures and monocyte/macrophage separation.
Isolated
PBMCs were cultured at 5 × 106 to 10 × 106 cells/ml for BLV quantification and 5 × 105 cells/ml for cell proliferation. The cells were treated
with human recombinant IL-2 (hrIL-2; 100 U/ml; PharMingen, San Diego, Calif.), hrIL-10 (10 ng/ml; R&D systems, Minneapolis, Minn.), hrIL-12
(5 ng/ml; R&D systems), anti-hrIL10 neutralizing antibody (10 µg/ml;
R&D systems), concanavalin A (ConA; 10 µg/ml; Sigma, St. Louis, Mo.),
and BLV (10 µg/ml). BLV was purified from the culture supernatant of
the BL3 cell line (ATCC CRL-8037, Rockville, Md.) by using metrizamide
(Sigma) gradient centrifugation and confirmed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
immunoblotting. After incubation of the PBMCs for 3 to 5 days, the
cells were harvested for further experiments. In cell proliferation
assays, [3H]thymidine was added 8 to 12 h before
cell harvest, and the radioactivity of the harvested cells was measured
with a 
-scintillation counter (MATRIX 9600; Packard, Meriden,
Conn.).
Monocyte/macrophages were isolated from PBMCs by exploiting their
ability to adhere to plastic (34). After PBMCs were
incubated for 4 to 6 h, nonadherent cells were removed and
transferred to other wells. Isolated monocyte/macrophages were cultured
with nonadherent cells separated by a transwell filter (0.4 µm pore size; Costar, Acton, Mass.). The purity of separated
monocyte/macrophages was confirmed by flow cytometry with mouse
anti-bovine CD14 immunoglobulin G (IgG; CAM36A; VMRD, Pullman, Wash.)
and fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Jackson
ImmunoResearch, West Grove, Pa.) by using an EPICS profile analyzer.
Adherent cells were also stained for esterase as previously performed
(24, 27).
Primers and reverse transcriptase PCR (RT-PCR).
Based on
GenBank sequence information, primers for tax and
pol were designed with the Oligo 5.0 software (National
Bioscience, Plymouth, Minn.). To produce plasmids containing standard
tax and pol fragments, PCR products from
BLV-infected PBMCs were cloned into the TA cloning vector pCR 2.1 (Invitrogen, San Diego, Calif.) as recommended by the manufacturer.
Plasmids were purified from transformed Escherichia coli by
using a Wizard miniprep system (Promega, Madison, Wis.) and then
screened for the insert by EcoRI digestion. The amplified
PCR products were assessed by agarose gel electrophoresis. Plasmid
concentration was determined by spectrophotometry at 260 nm. Inserts
were confirmed by automated DNA sequencing at the University of
Wisconsin Biotech Center.
Cytoplasmic lysates from freshly isolated PBMCs were obtained by adding
200 µl of chloroform, and total RNA was prepared by
using TRI reagent
according to the manufacturer's protocol (MRC,
Cincinnati, Ohio) with
DNase treatment. The concentration of purified
total RNA was determined
by spectrophotometry. Then, the RT reaction
was performed with purified
total RNA. The reaction mixture, including
400 U of Moloney murine
leukemia virus (MMLV) RT, 10 mg of bovine
serum albumin BSA, 40 units
of RNasin, 1 µg of oligo(dT), 0.5
mM deoxynucleoside triphosphates
(dNTP), and a 5× reaction buffer
(250 mM Tris-HCl, pH 8.3; 375 mM KCl;
15 mM MgCl
2; 50 mM dithiothreitol)
was incubated with 1 to
10 µg of total RNA for 2 h at 37°C. PCR
was performed in a DNA
thermocycler (Perkin-Elmer, Norwalk, Conn.)
for 35 cycles consisting of
1 min at 94°C for denaturation, 1
min at 62 (for
pol
amplification) or 65°C (for
tax amplification)
for
annealing, and 1 min at 72°C for polymerization. Each PCR
reaction
contained 1.25 U of
Taq polymerase, 1.5 mM
MgCl
2, 0.8
mM dNTP, 1 µM primers, template, and 10×
thermobuffer (500 mM
KCl; 100 mM Tris-HCl, pH 9.0; 1% Triton X-100).
tax (5'-CAGCATTTGGGCCGCCTTTTCTAAC,
3'-ACAGCCGGAGGGGGTCCACAAGGAG, 691-bp product) and
pol (5'-GCCGCCCCGCCTGAACCTGT,
3'-CCCACGCTTCGCCGAGGCATGAGTAG, 530-bp product) were
used as amplification
primers (Table
1).
Amplified products were analyzed by 1% agarose
gel electrophoresis.
Samples from RT reactions without MMLV-RT
and mixtures without cDNA
template were used in PCR assays as
controls for amplification of
contaminated DNA fragments.
Generation of mimics and quantitative-competitive PCR
(QC-PCR).
Mimics of tax and pol were
generated by using PCR primers and an internal nonspecific stuffer
region (24). The nonspecific fragment was produced by PCR by
using a DNA thermocycler for five cycles consisting of 1 min at 94°C
for denaturation, 1 min at 42°C for annealing, and 1 min at 72°C
for polymerization, followed by 35 cycles consisting of 1 min at 94°C
for denaturation, 1 min at 62°C or 65°C for annealing, and 1 min at
72°C for polymerization. The mimic band was excised, and the fragment
was purified by using a GenElute agarose spin column (Supelco,
Bellefonte, Pa.). The purified mimic fragment was amplified again by
PCR with tax and pol primers, and the PCR
reaction mixture was removed by using a QIAquick PCR purification kit
(Qiagen, Chatsworth, Calif.). The mimic fragment was then cloned by
using the TA cloning kit as described above. The concentration of
tax and pol mimic-ligated plasmids were
determined by spectrophotometry.
To quantify the
tax and
pol mRNA produced by
PBMCs from BLV-infected animals, QC-PCR was performed (
32).
For standardization,
PCR was performed by using this serially
twofold-diluted standard
plasmid with concentrations ranging from 8,192 to 16 fg/µl for
tax and from 2,048 to 4 fg/µl for
pol and a fixed amount of mimic
(10 fg/µl). Synthesized
cDNA from each sample and fixed amounts
of mimic were added into the
same tube and amplified simultaneously
with tubes for standard
reaction. Gel photographs were scanned,
and the amplified DNA bands
were analyzed by densitometry by using
NIH Image program version 1.61 with standard curves constructed
with Cricket Graph. Amplified products
were analyzed by the methods
previously described. The amount of
cytokine produced was determined
by comparing the density ratios of
sample reactions and standard
reaction.
Immunoblotting.
To detect BLV p24, PBMCs from PL animals
were cultured with IL-2 and ConA in 135-cm3 flasks
(Costar). The harvested cell pellet was resuspended in Triton X-100
lysis buffer (300 mM NaCl, 50 mM Tris-Cl, 0.5% Triton X-100, 10 µg
of leupeptin per ml, 10 µg of aprotinin per ml) at 108
cells/ml, kept for 30 to 45 min on ice, lightly vortexed, and centrifuged for 15 min at 12,000 × g. The supernatant
was removed from the nuclear pellet. SDS-PAGE was performed with the
supernatant, and the separated protein bands were transferred to
nitrocellulose filter paper (Bio-Rad Laboratories, Hercules, Calif.).
Immunoblotting was performed with mouse anti-BLV p24 IgG (BLV3; VMRD),
anti-BLV gp51 (BLV2; VMRD), and alkaline phosphatase-conjugated
anti-mouse IgG (Bio-Rad). The bands were scanned and then analyzed by
densitometry as described above. The relative densities of bands from
IL-2- and ConA-stimulated PBMCs were calculated and compared with those from PBMCs cultured with medium only.
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RESULTS |
IL-10 inhibits BLV tax and pol mRNA
expression.
To verify differences in the level of BLV expression
by PBMC cultures, tax and pol mRNA were
quantified by using mimics. Tax (450 bp) and pol
(700 bp) mimics were generated by nonspecific PCR amplification.
According to band ratios in standard reactions, standardization graphs
were drawn (Fig. 1).
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FIG. 1.
Standardization curve for QC-PCR of tax (A
and B) and pol (C and D) mRNA assays. Plasmids containing
mimic for tax (450 bp) or pol (700 bp) were
generated by using primers and an internal nonspecific stuffer region,
followed by cloning. Standard fragment-ligated plasmids for
tax (691 bp) and pol (539 bp) were also generated
by using the primers and BLV-infected cells. Serial twofold dilution of
standard plasmid and a fixed amount of mimic were amplified
simultaneously in the same tube. Standard DNA (8,192 fg to 16 fg for
tax [panel A, lanes 1 to 10] or 2,048 fg to 4 fg for
pol [panel c, lanes 1 to 10]) was amplified with 10 fg of
mimic DNA for both. The standard reactions for tax and
pol are shown in panels A and C, respectively. Amplified DNA
bands were analyzed by densitometry by using NIH Image program version
1.61 to generate standard curves (panels B and D). Each sample reaction
was performed simultaneously with a standard reaction, and the amount
of tax and pol level by PBMCs was calculated from
the representative standard curve.
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Interestingly, BLV
tax and
pol mRNA levels
decreased in the PBMCs cultured with hrIL-10 (Fig.
2), whereas
-
actin mRNA
level
was independent of IL-10 addition. This experiment was conducted
for 1, 3, 5, 7, and 9 days, and
tax mRNA level peaked in
PBMCs
cultured for 3 days, while
pol mRNA peaked in PBMCs
cultured for
5 to 7 days (data not shown). To confirm the inhibitory
effect
of IL-10 on BLV mRNA, anti-hrIL10 neutralizing antibody was
added
to the PBMCs cultured with IL-10. While hrIL-10 alone inhibited
BLV
tax mRNA, the addition of anti-hrIL-10 neutralizing
antibody
reversed BLV
tax mRNA levels inhibited by IL-10
(Fig.
3). These
results suggest that
IL-10 decreases BLV
tax and
pol mRNA levels
in
BLV-infected PBMCs.
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FIG. 2.
IL-10 inhibits BLV tax and pol
mRNA levels. PBMCs from the AL animal S201 were cultured with different
concentrations of human recombinant IL-10 (0.1 to 100 ng/ml) for 5 days. (A) QC-PCR analysis. The bands are as follows: 890 bp, -actin;
691 bp, tax standard; 450 bp, tax mimic; 700 bp,
pol mimic; and 530 bp, pol standard. All products
were separated on a 1% agarose gel stained by ethidium bromide. (B)
The bands were analyzed by densitometry in NIH image version 1.61, and
the amount of BLV tax and pol mRNA is shown. The
data are representative of experiments from three different animals.
Standard error bars are shown from at least three experiments.
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FIG. 3.
Inhibition of BLV tax mRNA level is blocked
by anti-hrIL-10 neutralizing antibody. PBMCs from the AL animal S17
were cultured with hrIL-10 with or without 10 µg of anti-IL-10
neutralizing antibody per ml for 5 days. Isotype antibody was used as a
negative control. The amounts of BLV tax mRNA level are
shown with standard error bars from at least three different assays.
The data are representative of experiments from three different
animals.
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Monocyte/macrophage-depleted PBMCs expressed reduced BLV
tax and pol mRNA levels.
Previous reports
indicate that monocyte/macrophages have a critical role in regulating
retrovirus expression (29, 36). To examine whether
monocyte/macrophages regulate BLV mRNA levels, monocyte/macrophages
were depleted by adherence to plastic. PBMCs, 2 × 107
to 3 × 107, were used for separation, and 2 to 5 × 106 cells were isolated by adherence. Flow cytometry
confirmed that more than 85% of the isolated cells were
CD14+ and were more than 95% esterase positive, a result
similar to our previous findings (24, 27).
Monocyte/macrophage-depleted cells contained fewer than 5%
CD14+ cells (data not shown). Monocyte/macrophage-depleted
PBMCs had dramatically reduced BLV tax and pol
mRNA compared to nonseparated PBMCs (Fig.
4). When hrIL-10 was added to
monocyte/macrophage-depleted PBMCs, the inhibitory effect of IL-10 on
BLV tax and pol mRNA level was not observed. To
determine whether monocyte/macrophages regulate BLV levels directly or
via soluble product(s), isolated monocyte/macrophages were cultured
with nonadherent cells separated by a 0.4-µm (pore-size) filter in
transwell plates. After culture for 3 to 5 days with or without
hrIL-10, nonadherent cells were harvested, and BLV tax and
pol mRNA levels were assessed. The nonadherent PBMCs, when
cultured with monocyte/macrophages in transwells expressed
approximately 10 times more BLV tax and twice as much
pol mRNA as nonadherent PBMCs cultured alone (Fig. 4). In
addition, hrIL-10 inhibited BLV tax and pol mRNA
levels in nonadherent PBMCs cultured with monocyte/macrophages (Fig.
4). These results suggest that factor(s) secreted by
monocyte/macrophages affect BLV mRNA levels and that IL-10 can regulate
these factor(s).
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FIG. 4.
The levels of BLV tax (A) and pol
(B) are reduced in monocyte/macrophage-depleted PBMCs and are not
affected by IL-10. Monocyte/macrophages were depleted by using plastic
adherence. Nonseparated PBMCs and monocyte/macrophage-depleted PBMCs
(M -depleted) were cultured with or without hrIL-10. Isolated
monocyte/macrophages were also cultured with
monocyte/macrophage-depleted PBMCs in transwell plates (Transwell),
where contact was blocked by a 0.4-µm (pore-size) membrane. After 5 days, the cells were harvested and QC-PCR was performed. Standard error
bars indicate variation of at least three different assays, and the
data are representative of experiments from three different animals. In
panel B, the medium and medium-macrophage-depleted columns have a
P  0.05 by using the Student's t test.
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IL-2 increased BLV tax and pol mRNA levels
and the level of BLV p24 protein.
To determine the effect of IL-2
and IL-12 on BLV detection, hrIL-2 and hrIL-12 were added to PBMC
cultures for 3 to 5 days, and the BLV tax and pol
mRNA levels were quantified by QC-PCR. BLV tax and
pol mRNA levels were increased more than five times in PBMCs
cultured with hrIL-2 and ConA compared to PBMCs cultured with medium
only, while the -actin level was unchanged (Fig. 5). In contrast, although the addition of
hrIL-12 enhanced antigen-specific PBMC proliferation, hrIL-12 did not
affect BLV tax and pol mRNA levels. To examine
the effect of IL-2 on BLV translation, 1.5 × 108
PBMCs were cultured with hrIL-2 and ConA. After 3 days, cellular proteins were isolated from cultured PBMCs, and semiquantitative immunoblotting was performed. Increased BLV p24 protein production was
detected by semiquantitative immunoblotting with anti-p24 antibody
(Fig. 6). BLV p24 protein amount
paralleled BLV tax and pol mRNA levels when PBMCs
were cultured in the presence of IL-2. Purified BLV by metrizamide
gradient centrifugation was used for a positive control. As predicted,
p24 protein was not detected in PBMCs cultured with hrIL-10, and gp51
production was not detected in PBMCs cultured with hrIL-2 and hrIL-10
(data not shown).
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FIG. 5.
IL-2 and ConA stimulate BLV tax and
pol mRNA levels. PBMCs from the PL animal P191 were cultured
with recombinant human IL-2 (100 U/ml) or ConA (10 µg/ml) for 5 days.
QC-PCR was performed (A), and the amounts of BLV tax and
pol mRNA are shown (B), with standard error bars from at
least three assays, and the data are representative of experiments with
three different animals. The bands are as follows: 691 bp,
tax standard; 450 bp, tax mimic; 700 bp
pol mimic; 530 bp pol standard; and 890 bp,
-actin. All products were analyzed on a 1% agarose gel stained with
ethidium bromide.
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FIG. 6.
IL-2 and ConA stimulate BLV p24 protein production.
PBMCs from PL animal P49 were cultured with hrIL-2 (100 U/ml) or ConA
(10 µg/ml) for 3 days. After protein purification, an equal amount of
protein from each sample was analyzed by SDS-10% PAGE. (A)
Immunoblotting was performed with mouse anti-BLV p24 antibody and
alkaline phosphatase-conjugated goat anti-mouse IgG antibody. (B) Each
band was analyzed by densitometry by using the NIH Image program
version 1.61 to measure relative band density. Purified BLV was used as
a positive control. The data are representative data of experiments
with two different PL animals. Standard error bars are shown from at
least four separate experiments.
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IL-10 reduces antigen-specific PBMC proliferation, while IL-2
enhances antigen-specific PBMC proliferation.
To examine whether
BLV expression correlates with PBMC proliferation, increasing
concentrations of hrIL-10 were added to PBMC cultures with 25% of BL3
supernatant as an antigen source (23). Although low
concentrations of hrIL-10 slightly activated PBMC proliferation, 10 times more than the 50% effective dose (1 ng/ml) of hrIL-10
dramatically reduced antigen-specific PBMC proliferation (Fig.
7A). Anti-hrIL-10 neutralizing antibody
restored antigen-specific PBMC proliferation, confirming that PBMC
proliferation is specifically inhibited by IL-10 (Fig. 7B). As
expected, hrIL-2 enhanced antigen-specific PBMC proliferation (Fig.
7B). These results indicated a correlation between BLV expression and
antigen-specific PBMC proliferation and suggest that cell proliferation
may provide a suitable environment for BLV expression by BLV-infected B
cells.
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FIG. 7.
IL-10 inhibits PBMC proliferation to BLV. PBMCs from AL
animal S201 were cultured with BLV containing supernatant from the BL3*
cell line or with medium alone for 5 days. Different concentrations of
hrIL-10 (0.1 to 100 ng/ml) (A) or of hrIL-10 (10 ng/ml), hrIL-2 (100 U/ml), anti-IL-10 antibody (10 µg/ml), and/or isotype antibody (10 µg/ml) were added (B). Proliferation was assessed by measuring
[3H]thymidine incorporation with a -scintillation
counter. The data are representative of experiments with three
different animals. Standard error bars are from three assays.
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DISCUSSION |
The results presented here demonstrate that IL-10 inhibits BLV
tax and pol mRNA levels, while IL-2 stimulates
BLV tax and pol mRNA and the level of p24
protein. Interestingly, IL-10 inhibition of BLV mRNA is removed when
adherent monocyte/macrophages were depleted from PBMCs. Also, the
background level of tax and pol mRNA was
dramatically decreased in monocyte/macrophage-depeleted PBMCs without
IL-10 addition. These findings suggest that IL-2 and IL-10 regulate the
amount of BLV and that monocyte/macrophages have an important role in
regulating BLV expression.
Previously, we showed that IL-2, IL-12, and IFN- were increased in
AL animals, while decreased in PL and tumor-bearing animals (24,
27). In contrast, IL-10 was increased in PL and tumor-bearing animals. These results suggest that disease progression in BLV infection is associated with specific cytokine expression. Retroviruses have a common immunosuppressive domain in their transmembrane protein.
A retroviral envelope peptide, termed CKS-17, caused a shift in the
cytokine balance, suppressing cell-mediated immunity by upregulating
IL-10 and downregulating IL-2, IL-12, and tumor necrosis factor alpha
production (10). Thus, previously we quantified IL-10 mRNA
expression of PBMCs infected by tax- and
env-recombinant vaccinia virus and also treated PBMCs with
the comparable oligopeptide of BLV CKS-17; however, a difference in
IL-10 expression was not detected (25). IL-10 can be
produced by a variety of cells, including macrophages. IL-10 production
also could be a result of antigen-specific T-cell apoptosis
(33). T helper (Th) 1 cells express Fas and Fas ligand upon
activation (33), and these cells are selectively removed by
programmed cell death, while Th2 cells are not affected.
High levels of IL-10 could have deleterious or beneficial effects on
the host immune defense against virus infections. Numerous studies have
reported that type 2 cytokines, as well as IL-10, inhibit cell-mediated
immunity and impair clearance of viral infections by cytotoxic T cells
and NK cells (17, 31). In BLV infection, PL animals have
impaired T-cell-mediated immunity to BLV infection. Although
CTLs from AL animals efficiently lysed cells presenting BLV Env
and Tax, CTLs isolated from PL animals could not respond to BLV Env and
Tax (18). In addition, lymphocytes from PL and tumor-bearing
animals failed to proliferate when cultured with BLV recombinant
proteins, while lymphocytes from AL animals proliferated to BLV
recombinant Gag and Env proteins (23). In addition to type 2 cytokines, IL-10 has a role in B-cell activation and proliferation. Excessive activation and proliferation may cause B-cell lymphocytosis and transformation by a chromosomal translocation of the oncogene c-myc in the immunoglobulin genes (9, 11, 12).
Alternatively, IL-10 may have a beneficial role in viral clearance. The
inhibitory effect of IL-10 on virus expression observed in the present
study has also been reported in HIV infection (20, 29, 30).
Interestingly, IL-10 can inhibit HIV replication in the
monocyte/macrophage lineage cells and in PBMCs but not in T cells
(29). These results implicate that the
IL-10-monocyte/macrophage interplay may have an important role in
regulating HIV expression. Thus, we depleted monocyte/macrophages from
PBMCs to examine whether monocyte/macrophages affect regulation of BLV
expression. The inhibitory effect of IL-10 on BLV tax and
pol mRNA levels was removed, and the background levels of
tax and pol mRNA was dramatically decreased
without addition of IL-10. When monocyte/macrophage-depleted PBMCs were
cultured with isolated monocyte/macrophages in transwells, monocyte/macrophage-depleted PBMCs expressed high amounts of BLV tax and pol mRNA. The addition of IL-10
dramatically reduced this level. These results suggest that
monocyte/macrophages secrete soluble factor(s) that activate BLV
expression and whose action is inhibited by IL-10. Recently, we found
that IL-10 inhibited and IL-2 enhanced detection of COX-2 mRNA in PBMCs
(26). Also, prostaglandin E2 (PGE2)
reversed IL-10 inhibition of BLV tax and pol mRNA
levels. Therefore, PGE2 might be one secretory factor from
monocyte/macrophages that increases BLV expression by BLV-infected B
cells. Signal transduction by PGE2 receptors mediates
increased cyclic AMP production (2). BLV long terminal
repeats contain a cyclic AMP response element that facilitates BLV gene
transcription. Although macrophages may be infected by BLV and produce
low levels of BLV mRNA (37), this idea is controversial
(19), and our data indicate that nonadherent cells produce
significant amount of BLV mRNA that is apparently regulated by the
presence of monocyte/macrophages.
IL-2 has been used therapeutically to boost the host immune response
against several infectious diseases and in cancer therapy (7,
15). Although IL-2 stimulates PBMC proliferation and CTL activity
in virus infections, IL-2 also activates BLV (Fig. 5, 6) and HIV
expression (1, 14). In addition, opportunistic infections
can stimulate HIV replication and disease progression (21,
35). Our preliminary data also show that bovine herpesvirus, one
of the most prevalent opportunistic infections in cattle, increased the
detection of BLV tax and pol mRNA
(25). These results suggest that cellular activation may
provide a suitable environment for virus replication.
To examine the level of BLV, BLV tax and pol mRNA
levels were quantified in the fentagram range by QC-PCR. Anti-p24
antibody also detected expression of p24, a part of gag, in
PBMCs cultured with IL-2 and ConA. However, anti-p24 antibody was not
sensitive enough to detect a difference in the amount of p24 between
untreated and IL-10-treated PBMCs. In addition, anti-gp51 antibody
could not detect a gp51 signal from cultured cells, while gp51 was
clearly detected in purified BLV. Changes in BLV tax and
pol transcription paralleled the translation of BLV p24 in
PBMC stimulated with IL-2 and ConA. Therefore, measuring the
transcription of BLV by QC-PCR is a reliable method for examining
differences in the levels of BLV. The level of pol can be
detected only from genomic mRNA (28). Thus, the replication
rate of BLV genomic RNA can be measured by amplification of
pol mRNA. Tax is an early gene product of BLV expression and
regulates both cellular and viral transcription. Although Tax protein
is translated only from the shortest mRNA, all four different mRNAs
expressed in BLV transcription can contain the tax sequence.
Thus, amplification of the tax region can be detected in all
BLV mRNAs, providing an explanation for greater tax mRNA
detection than pol in the present study. Since bovine cytokines are not commercially available, hrIL-2 (22),
hrIL-10 (3), and hrIL-12 (4) were used. Previous
studies (3, 4, 22) and the data in the present experiments
indicate that these cytokines have reactivity with bovine PBMCs.
In conclusion, the findings that IL-10 inhibits BLV tax and
pol mRNA levels suggest that increased IL-10 production by
monocyte/macrophages could be serve as a host immune defense mechanism
during late-stage BLV infection to limit the amount of virus
production. In addition, the observation that IL-2 stimulates BLV
production implicates that IL-2 treatment, to boost the immune
response, may contribute a deleterious effect stimulating virus
multiplication in the host.
| |
ACKNOWLEDGMENTS |
This work was supported by the National Cancer Institute grant
RO1 CA59127, BARD 95-34339-2556, and the College of Agricultural and
Life Sciences.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Animal Health and Biomedical Sciences, University of
WisconsinMadison, 1656 Linden Dr., Madison, WI 53706. Phone: (608)
262-1837. Fax: (608) 262-7420. E-mail:
splitter{at}ahabs.wisc.edu.
Present address: Beth Israel Deaconess Medical Center, Harvard
Medical School, Boston, MA 02115.
| |
REFERENCES |
| 1.
|
Al-Harthi, L.,
K. A. Roebuck, and A. Landay.
1998.
Induction of HIV-1 replication by type 1-like cytokines, interleukin (IL)-12 and IL-15: effect on viral transcriptional activation, cellular proliferation, and endogenous cytokine production.
J. Clin. Immunol.
18:124-131[Medline].
|
| 2.
|
An, S.,
J. Yang,
S. W. So,
L. Zeng, and E. J. Goetzl.
1994.
Isoforms of the EP3 subtype of human prostaglandin E2 receptor transduce both intracellular calcium and cAMP signals.
Biochemistry
33:14496-14502[Medline].
|
| 3.
|
Brown, W. C.,
V. M. Woods,
C. G. Chitko-McKown,
S. M. Hash, and A. C. Rice-Ficht.
1994.
Interleukin-10 is expressed by bovine type 1 helper, type 2 helper, and unrestricted parasite-specific T-cell clones and inhibits proliferation of all three subsets in an accessory-cell-dependent manner.
Infect. Immun.
62:4697-4708[Abstract/Free Full Text].
|
| 4.
|
Brown, W. C.,
W. C. Davis, and W. Tuo.
1996.
Human interleukin-12 upregulates proliferation and interferon-gamma production by parasite antigen-stimulated Th cell clones and gamma/delta T cells of cattle.
Ann. N. Y. Acad. Sci.
795:321-324[Medline].
|
| 5.
|
Choi, S. H., and G. A. Splitter.
1994.
Induction of MHC-unrestricted cytolytic CD4+ T cells against virally infected target cells by cross-linking CD4 molecules.
J. Immunol.
153:3874-3881[Abstract].
|
| 6.
|
Clerici, M., and G. M. Shearer.
1994.
The Th1-Th2 hypothesis of HIV infection: new insights.
Immunol. Today
15:575-581[Medline].
|
| 7.
|
Davey, R. T., Jr.,
D. G. Chaitt,
S. C. Piscitelli,
M. Wells,
J. A. Kovacs,
R. E. Walker,
J. Falloon,
M. A. Polis,
J. A. Metcalf,
H. Masur,
G. Fyfe, and H. C. Lane.
1997.
Subcutaneous administration of interleukin-2 in human immunodeficiency virus type 1-infected persons.
J. Infect. Dis.
175:781-789[Medline].
|
| 8.
|
Esteban, E. N.,
R. M. Thorn, and J. F. Ferrer.
1985.
Characterization of the blood lymphocyte population in cattle infected with the bovine leukemia virus.
Cancer Res.
45:3225-3230[Abstract/Free Full Text].
|
| 9.
|
Gupta, P.,
S. V. Kashmiri,
M. D. Erisman,
P. G. Rothberg,
S. M. Astrin, and J. F. Ferrer.
1986.
Enhanced expression of the c-myc gene in bovine leukemia virus-induced bovine tumors.
Cancer Res.
46:6295-6298[Abstract/Free Full Text].
|
| 10.
|
Haraguchi, S.,
R. A. Good,
M. James-Yarish,
G. J. Cianciolo, and N. K. Day.
1995.
Differential modulation of Th1- and Th2-related cytokine mRNA expression by a synthetic peptide homologous to a conserved domain within retroviral envelope protein.
Proc. Natl. Acad. Sci. USA
92:3611-3615[Abstract/Free Full Text].
|
| 11.
|
Holland, G., and A. Zlotnik.
1993.
Interleukin-10 and cancer.
Cancer Invest.
11:751-758[Medline].
|
| 12.
|
Ishiguro, N.,
T. Shinagawa,
T. Matsui, and M. Shinagawa.
1994.
Putative bovine B cell lineage tumor in sporadic bovine leukosis.
Vet. Immunol. Immunopathol.
42:185-197[Medline].
|
| 13.
|
Kettmann, R.,
D. Portetelle,
M. Mammerickx,
Y. Cleuter,
D. Dekegel,
M. Galoux,
J. Ghysdael,
A. Burny, and H. Chantrenne.
1976.
Bovine leukemia virus: an exogenous RNA oncogenic virus.
Proc. Natl. Acad. Sci. USA
73:1014-1018[Abstract/Free Full Text].
|
| 14.
|
Kinter, A. L.,
G. Poli,
L. Fox,
L, E. Hardy, and A. S. Fauci.
1995.
HIV replication in IL-2-stimulated peripheral blood mononuclear cells is driven in an autocrine/paracrine manner by endogenous cytokines.
J. Immunol.
154:2448-2459[Abstract].
|
| 15.
|
Kovacs, J. A.,
S. Vogel,
J. M. Albert,
J. Falloon,
R. T. Davey, Jr.,
R. E. Walker,
M. A. Polis,
K. Spooner,
J. A. Metcalf,
M. Baseler,
G. Fyfe, and H. C. Lane.
1996.
Controlled trial of interleukin-2 infusions in patients infected with the human immunodeficiency.
New Engl. J. Med.
335:1350-1356[Abstract/Free Full Text].
|
| 16.
|
Koyama, H.,
H. Nakanishi,
O. Kajikawa,
H. Yoshikawa,
S. Tsubaki,
T. Yoshikawa, and H. Saito.
1983.
T and B lymphocytes in persistently lymphocytotic and leukemic cattle.
Jpn. J. Vet. Sci.
45:471-475.
|
| 17.
|
Lucey, D. R.,
M. Clerici, and G. M. Shearer.
1996.
Type 1 and type 2 cytokine dysregulation in human infectious, neoplastic, and inflammatory diseases.
Clin. Microbiol. Rev.
9:532-562[Abstract].
|
| 18.
| Lundberg, P. S., and G. A. Splitter.
+ T-lymphocyte cytotoxicity against
envelope-expressing target cells is unique to the non-progressed
alymphocytotic stage of bovine leukemia virus-infection in the natural
host. Submitted for publication.
|
| 19.
|
Mirsky, M. L.,
C. A. Olmstead,
Y. Da, and H. A. Lewin.
1996.
The prevalence of proviral bovine leukemia virus in peripheral blood mononuclear cells at two subclinical stages of infection.
J. Virol.
70:2178-2183[Abstract].
|
| 20.
|
Naif, H. M.,
J. Chang,
M. Ho-Shon,
S. Li, and A. L. Cunningham.
1996.
Inhibition of human immunodeficiency virus replication in differentiating monocytes by interleukin 10 occurs in parallel with inhibition of cellular RNA expression.
AIDS Res. Human Retroviruses
12:1237-1245[Medline].
|
| 21.
|
Orenstein, J. M.,
C. Fox, and S. M. Wahl.
1997.
Macrophages as a source of HIV during opportunistic infections.
Science
276:1857-1861[Abstract/Free Full Text].
|
| 22.
|
Orlik, O., and G. A. Splitter.
1996.
Optimization of lymphocyte proliferation assay for cells with high spontaneous proliferation in vitro: CD4+ T cell proliferation in bovine leukemia virus-infected animals with persistent lymphocytosis.
J. Immunol. Methods
199:159-165[Medline].
|
| 23.
|
Orlik, O., and G. A. Splitter.
1996.
Progression to persistent lymphocytosis and tumor development in bovine leukemia virus (BLV)-infected cattle correlates with impaired proliferation of CD4+ T cells in response to gag- and env-encoded BLV proteins.
J. Virol.
70:7584-7593[Abstract].
|
| 24.
|
Pyeon, D., and G. A. Splitter.
1998.
Interleukin-12 p40 mRNA expression in bovine leukemia virus-infected animals: increase in alymphocytosis but decrease in persistent lymphocytosis.
J. Virol.
72:6917-6921[Abstract/Free Full Text].
|
| 25.
| Pyeon, D., and G. A. Splitter. Unpublished
data.
|
| 26.
| Pyeon, D., F. J. Diaz, and G. A. Splitter. Prostaglandin E2 increases bovine leukemia
virus tax and pol mRNA expression with
cyclooxygenase-2 regulation by interleukin-2, -10, and bovine leukemia
virus. Submitted for publication.
|
| 27.
|
Pyeon, D.,
K. L. O'Reilly, and G. A. Splitter.
1996.
Increased interleukin-10 mRNA expression in tumor-bearing or persistently lymphocytotic animals infected with bovine leukemia virus.
J. Virol.
70:5706-5710[Abstract/Free Full Text].
|
| 28.
|
Radke, K.
1994.
Bovine leukemia virus, p. 166-175.
In
R. G. Webster, and A. Granoff (ed.), Encyclopedia of Virology. Academic Press, Inc., New York, N.Y.
|
| 29.
|
Saville, M. W.,
K. Taga,
A. Foli,
S. Broder,
G. Tosato, and R. Yarchoan.
1994.
Interleukin-10 suppresses human immunodeficiency virus-1 replication in vitro in cells of the monocyte/macrophage lineage.
Blood
83:3591-3599[Abstract/Free Full Text].
|
| 30.
|
Schuitemaker, H.
1994.
IL4 and IL10 as potent inhibitors of HIV1 replication in macrophages in vitro: a role for cytokines in the in vivo virus host range?
Res. Immunol.
145:588-592[Medline].
|
| 31.
|
Shearer, G. M., and M. Clerici.
1996.
Protective immunity against HIV infection: has nature done the experiment for us?
Immunol. Today
17:21-24[Medline].
|
| 32.
|
Tsai, S. J., and M. C. Wiltbank.
1996.
Quantification of mRNA using competitive RT-PCR with standard-curve methodology.
BioTechniques
21:862-866[Medline].
|
| 33.
|
Van Parijs, L., and A. K. Abbas.
1998.
Homeostasis and self-tolerance in the immune systemturning lymphocytes off.
Science
280:243-248[Abstract/Free Full Text].
|
| 34.
|
Wahl, L. M., and P. D. Smith.
1995.
Isolation of monocyte/macrophage populations, p. 7.6.1-7.6.8.
In
J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober (ed.), Current protocols in immunology. John Wiley & Sons, Inc., New York, N.Y.
|
| 35.
|
Wahl, S. M., and J. M. Orenstein.
1997.
Immune stimulation and HIV-1 viral replication.
J. Leukoc. Biol.
62:67-71[Abstract].
|
| 36.
|
Weissman, D.,
G. Poli, and A. S. Fauci.
1994.
Interleukin 10 blocks HIV replication in macrophages by inhibiting the autocrine loop of tumor necrosis factor alpha and interleukin 6 induction of virus.
AIDS Res. Human Retroviruses
10:1199-1206[Medline].
|
| 37.
|
Werling, D.,
C. J. Howard,
E. Niederer,
O. C. Straub,
A. Saalmuller, and W. Langhans.
1998.
Analysis of the phenotype and phagocytic activity of monocytes/macrophages from cattle infected with the bovine leukaemia virus.
Vet. Immunol. Immunopathol.
62:185-195[Medline].
|
Journal of Virology, October 1999, p. 8427-8434, Vol. 73, No. 10
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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Top
Abstract
Introduction
