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Blood, Vol. 93 No. 1 (January 1), 1999:
pp. 208-216
Mechanism of Interleukin-10 Inhibition of T-Helper Cell
Activation by Superantigen at the Level of the Cell Cycle
By
George Q. Perrin,
Howard M. Johnson, and
Prem S. Subramaniam
From the Department of Microbiology and Cell Science, University of
Florida, Gainesville, FL.
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ABSTRACT |
We have analyzed the effects of interleukin-10 (IL-10) on the entry
of quiescent CD4+ T cells into the cell cycle upon
stimulation with the superantigen staphylococcal enterotoxin B (SEB).
IL-10 arrested cells at G0/G1. IL-10 treatment
prevented the downregulation of p27Kip1, an inhibitory
protein that controls progression out of the G0 phase of
the cell cycle. IL-10 also prevented the upregulation of the
G1 cyclins D2 and D3, proteins necessary for entry and progression through the G1 phase of the cell cycle.
Associated with the inhibition of the cell cycle, IL-10 suppressed SEB
induction of interleukin-2 (IL-2). Addition of exogenous IL-2 to
IL-10-treated cells significantly reversed the antiproliferative
effects of IL-10. Moreover, IL-10 effects on the early G1
proteins p27Kip1 and cyclin D2 were similarly reversed by
exogenous IL-2. Although this reversal by IL-2 was pronounced, it was
not complete, suggesting that IL-10 may have some effects not directly
related to the suppression of IL-2 production. Cell separation
experiments suggest that IL-10 can effect purified CD4+ T
cells directly, providing functional evidence for the presence of IL-10
receptors on CD4+ T cells. IL-10 also inhibited
expression of IL-2 transcriptional regulators c-fos and c-jun, which
also inhibit other cell functions. Our studies show that the mechanism
of IL-10 regulation of quiescent CD4+ T-cell activation
is mainly by blocking induction of IL-2 that is critical to
downregulation of p27Kip1 and upregulation of D cyclins in
T-cell activation and entry into the cell cycle.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
INTERLEUKIN-10 (IL-10) is a
multifunctional cytokine produced by B cells, stimulated macrophages,
and subsets of CD4+ T cells. It was originally termed
cytokine synthesis inhibition factor due to its ability to inhibit
production of some cytokines such as interferon  and interleukin-2
(IL-2).1 It has been shown to have both immunostimulatory
and immunosuppressive properties, depending on the target cell type.
IL-10 exerts immunostimulatory properties on B cells and mast cells and
is a growth factor for immature thymocytes.2 It exerts
immunosuppressive effects on cells of the monocyte/macrophage
lineage3,4 and also on subsets of activated helper
CD4+ T cells,5,6 which are normally found in a
quiescent or resting G0 state before activation. However,
little is known of the molecular effects of IL-10 on the progression of
the target cells through the cell cycle. The IL-10 receptor has been
found on macrophages and B cells as well as T cells, including murine
CD4+ T-cell clones.7-9 Significant levels of
IL-10 receptor mRNA has also been detected in human CD4+
T-cell clones.8 The IL-10 receptor is a member of the class II (interferon-like) subgroup of the cytokine receptor family and has
been found to have a high affinity for the IL-10
molecule.8,10 In this study, we have specifically examined
the effects of IL-10 on the molecular events controlling the cell cycle
of CD4+ T cells in peripheral blood after superantigen
stimulation.
In the entry of quiescent T cells from the G0 phase into
the G1 phase of the cell cycle, two proteins act as mitogenic sensors. These are the cyclin-dependent kinase (cdk) inhibitor
p27Kip1 and the D-type cyclins.10 These
proteins act in a reciprocal fashion. Mitogenic stimulation causes the
destruction of the inhibitor p27Kip1 and the upregulation
of cyclins D2 and D3. Cyclin D2 expression occurs early in the
G1 phase and is followed later in G1 by the expression of cyclin D3.11 The inhibitor
p27Kip1 itself inhibits the kinase activity of complexes
formed between the D cyclins and select cdks. These cyclin-cdk
complexes are responsible for the phosphorylation of key substrates
required for continued G1 progression, an important one
being the tumor-suppressor retinoblastoma protein pRB.10 In
general, the expression profiles of p27Kip1 and the D-type
cyclins are faithful markers of the activation state of T cells. In
CD4+ T cells, the downregulation of p27Kip1 and
the upregulation of D-type cyclins upon mitogenic stimulation are
dependent on the binding of IL-2 to its receptor.12 The importance of p27Kip1 is underscored by the fact that
immunosuppressive drugs such as rapamycin that prevent the activation
of T cells do so by preventing the downregulation of
p27Kip1 that occurs upon mitogenic
stimulation.13
In this study, we have used the superantigen staphylococcal enterotoxin
B (SEB) as the mitogen. The superantigen SEB is a potent
T-cell-specific mitogen that activates the T-cell receptor in a major
histocompatibility complex (MHC) class II-restricted manner.14 Thus, SEB more closely resembles conventional
antigens in terms of T-cell activation when compared with mitogens such as Concanavalin A (ConA) or phytohemagglutinin (PHA).
Activation of T cells by mitogens such as superantigens induces the
production of IL-2.15,16
Our data show that IL-10 exerts its immunosuppressive effects on
SEB-stimulated CD4+ T cells very early in the cell cycle.
IL-10 prevents the activation of quiescent CD4+ T cells
from the normal resting G0 state and their entry into the
cell cycle. This quiescent state is characterized by high levels of the
cell cycle inhibitory protein p27Kip1 and low levels of the
early D-type cyclin proteins that are associated with progression into
the G1 phase of the cell cycle. These effects occur in good
part as a consequence of the inhibition of IL-2 production
but may also involve other mechanisms affecting IL-2 receptor
signaling.
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MATERIALS AND METHODS |
Isolation of peripheral blood mononuclear cells (PBMCs) and cell
culture.
Human cells were collected from the whole blood of normal healthy
volunteers or from leukocyte source packs (Civitan Regional Blood
Center, Gainesville, FL). PBMCs were isolated by Histopaque (Sigma, St
Louis, MO) density gradient centrifugation17 and viability
was determined to be greater than 95% by trypan blue exclusion. For
isolation of CD4+ T cells, PBMCs were loaded onto CD4 T
Cellect columns (Biotex, Edmonton, Alberta, Canada) as per the
manufacturer's recommendation and cell purity was assessed by flow
cytometry. Cells were maintained in RPMI 1640 supplemented with 10%
(vol:vol) heat-inactivated fetal bovine serum (FBS), 200 U/mL
penicillin, and 200 µg streptomycin in a 5% CO2
atmosphere at 37°C and used immediately.
Proliferation assay.
The proliferative response of PBMCs to SEB (Toxin Technology, Sarasota,
FL) was determined by the incorporation of [3H]thymidine
into DNA. Specifically, PBMCs were added to wells in a 96-well plate at
a concentration of 3 × 105 cells/mL and were treated
with either IL-10 (Intergen, Purchase, NY) at 10 U/mL or media alone
and incubated for 48 hours. Media or SEB was then added at the
indicated concentrations and the cultures were incubated for another 48 hours in a final volume of 200 µL/well. Next, 1 µCi of
[3H]thymidine (Amersham, Arlington Heights, IL) in media
was added per well and the plates were incubated for 8 hours before
harvest. All experiments were performed in quadruplicate. The
proliferative response of purified CD4+ T cells to SEB was
performed in a similar manner, except that cells were added to the
plates at a concentration of 2.5 × 106 cells/mL. This
was necessary to obtain substantial proliferation, as determined by
experimentation. These cells were only incubated with IL-10 for 2 hours
before the addition of SEB.
Flow cytometry.
PBMCs were added to 6-well plates at a concentration of 3 × 106 cells/mL and treated with IL-10, SEB, and/or
IL-2 (Intergen) at the indicated concentrations in a final volume of 5 mL. Plates were incubated for the time indicated before harvest for
flow cytometry analysis. At harvest, cells were gently mixed and cell counts were performed. Cells (1 × 106) were added to
a 15-mL tube and washed twice with 12 mL cold standard azide buffer
[phosphate-buffered saline (PBS) containing 5% (vol:vol) FBS and
0.1% (wt:vol) sodium azide], centrifuging each time at 500 RPM for 10 minutes at 4°C. After carefully removing the supernatant, the cells
were adjusted to 1 × 107 cells/mL by resuspending the
pellet in 80 µL cold standard azide buffer and adding 20 µL
fluorescein isothiocyanate (FITC)-labeled anti-CD4 antibody
(Pharmingen, San Diego, CA) and incubating on ice for 30 minutes in the
dark. Cells were then washed twice as described before. For propidium
iodide staining, the cells were then resuspended in 1 mL of a 0.112%
sodium-citrate buffer containing 50 µg/mL propidium iodide (Sigma)
and 100 U/mL RNase A (Sigma) and allowed to stain for up to 1 hour at
room temperature.
For intracellular staining of c-fos and c-jun proteins, the cells were
harvested as before, washed with cold staining buffer [PBS containing
1% (vol:vol) FBS and 0.1% (wt:vol) sodium azide], and stained with
FITC-labeled anti-CD4 antibody in staining buffer as described before.
After washing twice with cold staining buffer and once with cold PBS,
the cells were fixed with 100 µL of 4% (wt:vol) paraformaldehyde in
PBS for 20 minutes at 4°C. The cells were then washed once with
cold PBS and twice with cold permeabilization buffer [staining buffer
with 0.1% (wt:vol) saponin]. The cells were then resuspended in 100 µL of permeabilization buffer containing 0.7 µg of either c-fos or
c-jun specific rabbit antibody (Santa Cruz Biotechnology, Santa Cruz,
CA) on ice for 30 minutes. The cells were then washed twice with
permeabilization buffer and resuspended in permeabilization buffer
containing 0.7 µg of phycoerythrin-labeled antirabbit antibody
(Sigma) on ice for 30 minutes. The cells were again washed twice with
permeabilization buffer and washed once with staining buffer. Finally,
the cells were resuspended in 1 mL of staining buffer.
For all flow cytometry analysis, samples were filtered through 44-µm
nylon mesh and data from 30,000 events were acquired with a FACSort
(Becton Dickinson Immunocytometry Systems, San Jose, CA) using the
LYSIS II software system. Analysis of the cell cycle was performed
using CellFIT software (Becton Dickinson, San Jose, CA).
Immunoblotting.
PBMCs were cultured in 6-well plates and harvested as described above.
CD4+ T cells were then isolated by passing PBMCs over CD4 T
Cellect columns (Biotex, Edmonton, Alberta, Canada) as per the
manufacturer's recommendations. The resulting CD4+ T cells
were counted and stored at  70°C.
Cells were lysed at 4°C for 20 minutes in ice-cold lysis buffer
consisting of 50 mmol/L Tris-HCl (pH 7.5), 250 mmol/L NaCl, 1%
(vol:vol) Triton X-100, 2 mmol/L EDTA, 2 mmol/L EGTA, 50 mmol/L NaF, 20 mmol/L  -glycerophosphate, 2 mmol/L Na-orthovanadate, aprotinin (10 µg/mL), leupeptin (10 µg/mL), pepstatin (10 µg/mL), benzamidine
(5 µg/mL), and 1 mmol/L phenylmethanesulfonyl fluoride. Samples were
centrifuged at 13,000 RPM for 10 minutes and a BCA protein assay
(Pierce, Rockford, IL) was performed on the supernatants. Equal amounts
of protein from cell lysates (30 to 70 µg/lane) were subjected to
sodium dodecyl sulfate (SDS) gel electrophoresis. After
Western transfer, membranes were probed with 1.2 µg of
anti-p27Kip1, anti-cyclin D2, or anti-cyclin D3 antibodies
(Santa Cruz Biotechnology) and developed using an enhanced
chemiluminescence (ECL) detection kit (Amersham). Densitometric
analysis of radiographic film using IA-1000 Digital Image Analysis
Software (Alpha Innotech Corp, San Leandro, CA) was used
to determine fold increase or decrease between band intensities based
on total pixel value.
IL-2 assay.
IL-2 levels were determined from cell-free culture supernatants stored
at 70°C using IL-2 enzyme-linked immunosorbent assay (ELISA)
kits (Intergen). Assay protocol was conducted as described by the
manufacturer. All supernatant samples were tested in duplicate.
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RESULTS |
IL-10 blocks SEB-induced proliferation in PBMCs.
IL-10 was first tested for its ability to inhibit superantigen-induced
proliferation of human lymphocytes. Human PBMCs were treated with IL-10
for up to 2 days before the addition of SEB. These cultures were then
incubated for up to 5 days, which was the time of optimal proliferation
as reflected by [3H]thymidine incorporation. IL-10
significantly inhibited SEB-induced proliferation in a dose-dependent
manner (data not shown), and an optimal concentration of 10 U/mL of
IL-10 was determined. Furthermore, as shown in
Fig 1, 10 U/mL of IL-10 inhibited PBMC
proliferation over a wide range of concentrations of SEB. Thus, IL-10
blocked SEB-induced PBMC proliferation.
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| Fig 1.
IL-10 suppression of SEB stimulation: SEB dose-response.
PBMCs were added to 96-well plates at 3 × 105 cells/mL
along with IL-10 at 10 U/mL or media only. After 2 days of incubation,
media alone or SEB at varying concentrations was added and plates were
further incubated for another 2 days. Proliferation was measured by
[3H]thymidine incorporation. Different PBMC donors are
represented in each experiment. Data are expressed as the mean CPM ± SD. Results were determined to be statistically significant by the
Student's t-test (P < .002). Numbers between curves
indicate the percentage of suppression by IL-10.
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IL-10 inhibits SEB-stimulated CD4+ T cells from
progressing through the cell cycle.
We next determined the stage of the cell cycle in which IL-10 blocked
SEB-induced proliferation, looking specifically at CD4+ T
cells, which are one of the primary targets of superantigen stimulation. After stimulation for up to 5 days, CD4+ T
cells were examined for cell cycle progression using FITC-labeled anti-CD4 antibodies and propidium iodide staining. Preliminary experiments using SEB showed that CD4+ T cells were
entering the S phase between days 4 and 5, although there was some
variation among donors with respect to the percentage of cells in S
phase at these times. As shown in Table 1,
untreated cells were all essentially quiescent (>98%
G0/G1). Treatment with SEB induced cells to
progress through the G1 phase and into the S phase of the
cell cycle. However, IL-10 blocked SEB-induced progression of
CD4+ T cells through the G1 phase of the cell
cycle. A similar pattern of inhibition was observed with 3 and 30 U/mL
of IL-10, with a resulting dose response
(Table 2). These data suggest that IL-10 inhibits the early SEB-mediated activation of CD4+ T cells
and prevents their progression through the G1 phase of the
cell cycle and their subsequent entry into the cell cycle.
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Table 2.
Flow Cytometry Analysis of the Effect of Increasing
Concentrations of IL-10 on SEB-Stimulated CD4+ T
Cells
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IL-10 inhibition of cell cycle progression in CD4+ T
cells stimulated with SEB is dependent on their activation state.
We then examined the inhibitory effect of IL-10 on SEB stimulation of
PBMCs when added to cultures at various times relative to superantigen
addition. As seen in Table 3, the addition
of IL-10 (10 U/mL) 2 days before, 1 day before, or at the time of SEB
addition inhibited cell proliferation in the
G0/G1 phase of the cell cycle to essentially
the same extent. The addition of IL-10 at 1 to 4 days after SEB
resulted in progressively less inhibition of cell cycling. The addition
of IL-10 4 days after SEB allowed essentially the same percentage of
cells to leave the G1 phase and enter the cell cycle as did
cells treated with SEB and no IL-10. These data suggest that, once
CD4+ T cells have been activated by mitogen and enter the
cell cycle, IL-10 has no further effect on these cells. Thus, it
appears that IL-10 blocks the activation of quiescent CD4+
T cells but not the subsequent proliferation of these cells.
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Table 3.
Flow Cytometry Analysis of the Effect of
IL-10 Addition at Various Times Relative to SEB Incubation on
SEB-Stimulated CD4+ T Cells
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IL-10 prevents the elimination of the tumor suppressor protein
p27Kip1 in SEB-stimulated CD4+ T cells.
To gain insight into how IL-10 blocked superantigen-induced activation
in the G0/G1 phase of the cell cycle, we
determined the effects of IL-10 on the tumor suppressor gene protein
p27Kip1 and on the cyclins D2 and D3 in CD4+ T
cells. p27Kip1 levels are high in quiescent G0
cells and are rapidly reduced in cells induced to enter the cell cycle
by mitogens,18 whereas cyclin D levels are low in quiescent
cells and increase upon mitogenic stimulation.19 In human T
cells, cyclin D2 migrates as a doublet, with the faster migrating
species representing the activated form.20 PBMCs were
incubated with IL-10 (10 U/mL) for 2 days before the addition of SEB.
After incubating for the specified number of days, the CD4+
T cells were isolated using negative selection chromatography columns
and the cell extracts were subjected to Western analysis for
p27Kip1 and cyclins D2 and D3. As shown in
Fig 2, untreated quiescent cells showed
high levels of p27Kip1. Treatment with SEB dramatically
reduced the levels of p27Kip1 by day 3.5. By comparison,
the p27Kip1 level remained relatively high in cultures
treated with IL-10 even in the presence of SEB. The effect of IL-10 on
the expression of p27Kip1 in SEB-treated cultures supports
the conclusion that IL-10 inhibits cellular proliferation in the early
G0/G1 phase of the cell cycle.
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| Fig 2.
IL-10 inhibits the SEB reduction of p27Kip1
and prevents SEB-induced upregulation of cyclins D2 and D3 in
CD4+ T cells. (A) PBMCs were incubated in 6-well plates
at 6 × 105 cells/mL along with IL-10 at 10 U/mL or media
only. After 2 days of incubation, media alone or SEB at 100 pg/mL was
added. The cells were further incubated until the indicated harvest
day. Cells were harvested and CD4+ T cells were separated
and collected by negative selection affinity chromatography. Lysates
from these cells were subjected to SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) and Western analysis using antibodies
specific for p27Kip1, cyclin D2, and cyclin D3. The lower
band in the cyclin D2 doublet represents the faster-migrating activated
form of cyclin D2. (B) Quantitation was performed by densitometric
analysis of bands from day 3.5. The differences between SEB treatment
and treatment with IL-10 and SEB were found to be statistically
significant for all immunoblots (P < .001) by the
Student's t-test.
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IL-10 inhibits the upregulation of cyclins D2 and D3 in
SEB-stimulated CD4+ T cells.
The expression of cyclins D2 and D3 were also strongly affected by
IL-10 treatment of SEB-stimulated CD4+ T cells. Figure 2
shows that SEB treatment of quiescent CD4+ T cells
increased the levels of cyclin D3 and the activated faster migrating
form of cyclin D2 by day 3.5. In contrast, IL-10 prevented the
expression of comparable amounts of both cyclin D3 and the faster
migrating form of cyclin D2 over the same time period. As mentioned
earlier, p27Kip1 and the D-type cyclins behave as sensors
for mitogenic stimuli and play a crucial role in the decision of cells
to become activated, enter the cell cycle, and subsequently
proliferate. Taken together with the effects on p27Kip1,
these data demonstrate that IL-10 affects CD4+ T cells very
early in the cell cycle by antagonizing the ability of mitogens such as
SEB to activate these cells to enter the cell cycle.
IL-10 inhibits SEB-induced IL-2 production.
The potent mitogenic activity of SEB requires the induction and
autocrine effects of IL-2 for the activation of CD4+ T
cells. IL-2 is the primary cytokine secreted by CD4+ T
cells that is responsible for the activation of competent T cells and
their subsequent progression through the G1 phase of the
cell cycle. IL-10 has also been reported to block induction of
IL-2.1 Thus, we next examined the role of IL-2 in this
system and its effects on the cell cycle events affected by IL-10. As seen in Table 4, treatment with IL-10
dramatically inhibited the amount of IL-2 produced during the time
period of SEB stimulation. These data represent IL-2 levels in
supernatants taken from the same cell cultures analyzed in Fig 2. The
changes in IL-2 levels are seen to precede changes in the cell cycle
proteins. We then measured the amount of IL-2 produced during the first
24 hours after SEB stimulation (Table 5) to
study the effect IL-10 has on IL-2 production during the time of
initial T-cell activation. IL-2 production was induced as early as 12 hours after the addition of SEB, and IL-10-treated cells showed
significantly less IL-2 production than did cells incubated with SEB
only. In both cases, similar values were obtained when IL-10 was added
2 days before the addition of SEB and when IL-10 and SEB were added
simultaneously (data not shown). Thus, IL-10 blocks induction of IL-2
in the SEB-stimulated cultures.
The addition of IL-2 reverses IL-10's effects on cell cycle
progression in SEB-stimulated CD4+ T cells.
Next, we determined the effect of the addition of IL-2 on the
IL-10-induced suppression of SEB stimulation. IL-2 at varying concentrations was added to PBMCs along with IL-10 and SEB. The cells
were then incubated for 5 days and the CD4+ T cells were
examined for cell cycle progression using FITC-labeled anti-CD4
antibodies and propidium iodide staining.
Table 6 shows that the addition of IL-2
overcame the suppressive effect of IL-10 in these cells and allowed
them to become activated and progress from the
G0/G1 phase into S and G2/M phases
of the cell cycle. This reversal of the IL-10 effect by IL-2 occurred
in a dose-dependent manner. A similar experiment was performed in which
cells were incubated with IL-10 for 2 days before the addition of IL-2
and SEB (Table 7). Results very similar to
those in Table 6 were seen in which the addition of IL-2 reversed the
G0/G1 block imposed by IL-10 in a
dose-dependent manner. In either case, the concentration of IL-2
required to produce an approximately 50% reversal of inhibition was
similar to the concentration of IL-2 induced in cultures stimulated by
SEB alone (see Table 4). These IL-2 levels probably represent steady-state levels of IL-2 in culture supernatants. These data strongly support the conclusion that one important mechanism by which
IL-10 exerts its effects is by downregulating the production of IL-2,
especially since pretreatment with IL-10 had no greater effect on the
ability of IL-2 to reverse the cell cycle block. In a few donors
tested, as shown in Table 7, IL-10 also inhibited cell cycle
progression in cells stimulated with exogenous IL-2, indicating that
IL-10 may also interfere with the response of CD4+ T cells
to IL-2 to varying extents.
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Table 6.
Flow Cytometry Analysis of the Effect of IL-2
Addition on Reversing the Effect of IL-10 on SEB-Stimulated
CD4+ T Cells: No IL-10 Pretreatment
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Table 7.
Flow Cytometry Analysis of the Effect IL-2 Addition
on Reversing the Effect of IL-10 on SEB-Stimulated CD4+ T
Cells: Two Day IL-10 Pretreatment
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The addition of IL-2 partially reversed IL-10's effects on cell
cycle proteins p27Kip1 and cyclin D2 in SEB-stimulated
CD4+ T cells.
We next examined the effect of adding exogenous IL-2 on the levels of
the cell cycle proteins p27Kip1 and cyclin D2 in
CD4+ T cells. We concentrated on p27Kip1 and
cyclin D2, because changes in p27Kip1 and cyclin D2 occur
earlier in G1 as compared with cyclin D3. PBMCs were
treated simultaneously with IL-10, SEB, and IL-2. The CD4+
T cells were then isolated and the cell extracts were subjected to
Western analysis as described before. As seen in
Fig 3, p27Kip1 levels again
decreased in SEB-treated cultures by day 3.5 and remained high in
cultures treated with IL-10, as described earlier (see Fig 2). The
addition of IL-2 along with the IL-10 and SEB caused the
p27Kip1 levels to decrease to nearly those seen in cells
treated with SEB only. Figure 3 also shows the effects of adding IL-2
on the level of cyclins D2. The addition of IL-2 to cells treated with SEB and IL-10 significantly restored the expression of cyclin D2. These
data thus demonstrate that one mechanism by which IL-10 inhibits entry
into the G1 phase of the cell cycle is by the blockage of
IL-2 production that is critical to the downregulation of
p27Kip1 and the upregulation of early D-type cyclins in
T-cell activation. Absence of a complete block of p27Kip1
induction and restoration of the active form of cyclin D2, as per Fig
3, would suggest other possible IL-2-independent effects of IL-10 on
CD4+ T cells.
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| Fig 3.
Addition of IL-2 reverses the effects of IL-10 on the SEB
reduction of p27Kip1 and SEB-induced upregulation of cyclin
D2 in CD4+ T cells. (A) PBMCs were incubated in 6-well
plates at 6 × 105 cells/mL along with IL-10 at 10 U/mL or
media only. After 2 days of incubation, media alone, SEB at 100 pg/mL,
or SEB and IL-2 (100 U/mL) were added. The cells were further incubated
until the indicated harvest day. Cells were harvested and
CD4+ T cells were separated and collected by negative
selection affinity chromatography. Lysates from these cells were
subjected to SDS-PAGE and Western analysis using antibodies specific
for p27Kip1 and cyclin D2. The lower band in the cyclin D2
doublet represents the faster-migrating activated form of cyclin D2.
(B) Quantitation was performed by densitometric analysis of bands from
day 3.5. The differences between SEB treatment and treatment with IL-10
and SEB were found to be statistically significant for both immunoblots
(P < .001) by the Student's t-test. Differences were
also statistically significant when comparing IL-10 and SEB treatments
with those in which IL-2 was also added: P < .001 for the p27Kip1 blot and P < .002 for
the activated form in the cyclin D2.
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IL-10 reduced the levels of the IL-2 transcriptional regulators c-fos
and c-jun in SEB-stimulated CD4+ T cells.
To determine the effects of IL-10 on the production of IL-2 at the
molecular level, we performed flow cytometry analysis looking specifically at the transcriptional regulatory proteins for IL-2. AP-1
is an important transcriptional regulator for IL-2 and is a heterodimer
composed of the proteins c-fos and c-jun.21 As seen in
Fig 4, flow cytometry showed an increase in
the expression of both c-fos and c-jun proteins in CD4+ T
cells induced by treatment with SEB, consistent with the cells becoming
activated. We further show that the addition of IL-10 to these cultures
along with SEB caused a decrease in the levels of the c-fos and c-jun
proteins. There appeared to be a stronger effect of IL-10 on the
expression of c-fos than c-jun, but the reason for this is not known at
this time. These results are consistent with our data showing that
IL-10 can significantly decrease the amount of IL-2 produced by cells
stimulated with SEB and prevents CD4+ T cells from becoming
activated and entering the cell cycle.
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| Fig 4.
IL-10 reduces SEB induction of c-fos and c-jun proteins
in CD4+ T cells. PBMCs were incubated in 6-well plates at
6 × 105 cells/mL with media alone, SEB (100 pg/mL), or
IL-10 (10 U/mL) and SEB. Cells were incubated for 45 minutes,
harvested, and subjected to flow cytometry analysis. Data were gated to
analyze CD4+ T cells. Results were determined to be
statistically significant by the Student's t-test (P < .01 for c-fos and P < .0005 for c-jun). These values
represent net effects after the subtraction of relative fluorescent
intensity for untreated cells.
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IL-10 blocks proliferation of CD4+ T cells.
To investigate the effects of IL-10 on CD4+ T cells in the
absence of antigen-presenting cells (APCs), we studied the
antiproliferative effects of IL-10 on CD4+ T cells in a
system depleted of APCs. CD4+ T cells were isolated from
PBMCs and their purity was verified by flow cytometry. These
CD4+ T cells were stimulated either with SEB or a mixture
of phorbol 12-myristate 13-acetate (PMA) and ionomycin and their
proliferation was measured by [3H]thymidine
incorporation. SEB is a unique superantigen in that it has been shown
to directly activate T cells in the absence of APCs.22
Figure 5A shows that SEB did induce
CD4+ T cells to proliferate in the absence of APCs and that
IL-10 was able to significantly block this proliferation. PHA, whose effects are known to be APC dependent,23 had a negligible
effect on proliferation of the CD4+ T-cell population used
while also stimulating a population that contained APCs. We also
stimulated CD4+ T cells with varying mixtures of PMA and
ionomycin, which can also directly activate CD4+ T cells.
IL-10 significantly inhibited the proliferation of CD4+ T
cells at all of the PMA concentrations tested (Fig 5B) and also at
various ionomycin concentrations tested (data not shown). These data
show that IL-10 had a direct antiproliferative effect on
CD4+ T cells and that these antiproliferative effects are
independent of IL-10's effects on APCs, such as downregulation of MHC
class II molecules and downregulation of CD80/86 molecules.
Furthermore, these data provide functional evidence for the presence of
IL-10 receptors on CD4+ T cells.
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| Fig 5.
IL-10 blocks proliferation of CD4+ T cells.
(A) CD4+ T cells were isolated from PBMCs by negative
selection chromatography and were added to 96-well plates at 2.5 × 106 cells/mL along with IL-10 at 10 U/mL or media only.
After incubating for 2 hours, SEB at varying concentrations was added
and plates were incubated for 4 days. Proliferation was measured by
[3H]thymidine incorporation. Data are expressed as the
mean CPM ± SD. The percentage of suppression by IL-10 and statistical
significance, as determined by the Student's t-test, for each
treatment are indicated above the graphs. (B) CD4+ T
cells were incubated as indicated above and stimulated with a mixture
of ionomycin (100 ng/mL) and varying concentrations of PMA.
Proliferation was measured by [3H]thymidine
incorporation. Data are expressed as the mean CPM ± SD. The
percentage of suppression by IL-10 and statistical significance, as
determined by the Student's t-test, for each treatment are
indicated above the graphs. In both (A) and (B), the proliferation of
CD4+ T cells incubated with PHA at 5 µg/mL is compared
with that of whole PBMCs incubated with 3 µg/mL.
|
|
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DISCUSSION |
The immunosuppressive effects of IL-10 have been well documented in a
number of different systems both in vitro and in vivo.2 However, the mechanism of these effects at the level of cell cycle events is currently not known. We showed here that IL-10 blocks SEB-stimulated CD4+ T cells from entering the cell cycle.
Specifically, IL-10 treatment of cells prevented the downregulation of
p27Kip1, a cell cycle inhibitory protein. Resting cells are
characterized by relatively high levels of p27Kip1, which
binds to cyclin-cdk complexes, inhibiting phosphorylations that are
required for moving cells out of G0 and through the
G1 phase of the cell cycle.11,24 IL-10 also
prevented upregulation of the G1 cyclins D2 and D3 in
SEB-stimulated CD4+ T cells, which are necessary for the
cells to enter the cell cycle. Cyclin D2 has been shown to be present
at low levels in resting T cells, whereas cyclin D3 is only detected
after T-cell activation.19 Thus, IL-10 blocked
downregulation of p27Kip1 and upregulation of cyclins D2
and D3, which provides a mechanism for its inhibition of lymphocyte
activation.
Related to the inhibitory effects of IL-10 on CD4+ T cells,
IL-10 blocked SEB induction of IL-2. It has previously been shown that
IL-10 blocked IL-2 induction, but the consequences of this in terms of
cell cycle events have not been determined. Addition of IL-2 to
cultures treated with SEB in the presence of IL-10 reversed IL-10
inhibition of CD4+ T-cell cycling. This correlated with a
downregulation of p27Kip1 and upregulation of cyclin D2.
Furthermore, because pretreatment with IL-10 did not significantly
affect responses to IL-2, it suggests that events affecting the binding
of IL-2 to its receptor are probably not involved in the immediate
effects of IL-10. However, the reversal by exogenous IL-2 of the
effects on the cell cycle proteins p27Kip1 and cyclin D2
was not complete. Thus, it appears that IL-10 acts by IL-2-independent
mechanisms in addition to suppressing induction of IL-2. We have
observed in some instances that IL-2 alone can induce a proliferative
response, which varied between donors. However, even in these instances
the ability of these cells to respond to IL-2 can be suppressed by
IL-10. This would suggest that IL-10 could possibly affect signals that
do not directly stem from IL-2 but are nonetheless required in
conjunction with IL-2-induced signals in mediating a mitogenic
response.
IL-10 was examined for its effect on inhibiting upregulation of the
AP-1 transcription factors c-fos and c-jun. These proteins are involved
in activation of the IL-2 gene as well as a number of other cytokines
involved in cell growth.21 Treatment with IL-10 caused a
decrease in the amounts of both c-fos and c-jun proteins in
SEB-stimulated CD4+ T cells. Thus, the effects of IL-10 on
c-fos and c-jun could contribute to the inhibition of IL-2 induction.
Given the broad gene-target specificity of c-fos and c-jun, it is
probable that IL-10 may also have effects that go beyond the early
blockage of IL-2 production.
IL-10 receptors have been shown to be present on murine
CD4+ T cells and CD4+ T-cell clones of the
TH1 subtype.9 In addition, significant levels
of IL-10 receptor mRNA have been detected in human CD4+
T-cell clones.8 Consistent with these data, we have
demonstrated that IL-10 directly affects the proliferation of purified
human CD4+ T cells in response to both the superantigens
such as SEB and T-cell mitogens such as PMA and ionomycin. Our data
provide further functional evidence for the presence of IL-10 receptors
on human CD4+ T cells.
Recent studies have shown that IL-10 induced an anergic state in human
CD4+ T cells stimulated with alloantigens or cross-linked
with anti-CD3 antibodies.25,26 These studies involved the
long-term treatment of cells with IL-10 in which the cells become
unresponsive to IL-2. Although we have not directly studied the effect
of long-term treatment with IL-10, we found that pretreatment of cells
for up to 2 days with IL-10 did not affect the ability of IL-2 to reverse the cell cycle block. Thus, our data, along with those of
others,25 would suggest that the effects of IL-10 are
twofold. Short exposures to IL-10 produce an immediate inhibition of
the activation of CD4+ T-cell entry into the cell cycle
that is IL-2 dependent and is reflected by high levels of
p27Kip1 and low levels of cyclin D2. This phase is
reversible by IL-2. However, continued treatment with IL-10 apparently
produces permanent changes in these cells that ultimately makes them
unresponsive to IL-2 and renders the cells anergic. The mechanism of
this shift to a permanent state of IL-2 unresponsiveness remains
unclear.
IL-10 is produced relatively late in the immune response and is thought
to be important in the shift from a cell-mediated response to an
antibody-mediated response. In this regard, IL-10 functions in a
paracrine fashion, affecting neighboring cells. Because IL-10
effectively blocked cell cycling in the presence of SEB, our data
suggest that IL-10 can prevent naive CD4+ T cells from
being activated even when they are subsequently presented with antigen.
This highlights an important use for IL-10 as an immunosuppressive
drug. Furthermore, when compared in this fashion, immunosuppressive
drugs such as cyclosporin A and FK506 would be expected to resemble
IL-10 in their effects on the cell cycle. These drugs act to prevent
T-cell activation primarily by blocking IL-2 synthesis27
and, thereby, presumably also preventing the cell cycling of target
cells in a manner analogous to IL-10. This is in contrast to drugs such
as rapamycin that act by blocking signaling after the binding of IL-2
to its receptor.13 Rapamycin, like IL-10, also prevents the
downregulation of p27Kip1, but this effect cannot be
reversed by exogenous IL-2.13
Overall, our results suggest the importance of IL-10 in regulating the
cell cycle proteins necessary for stimulated CD4+ T cells
to enter the cell cycle and implicate IL-2 as a key cytokine that
mediates the early short-term immunosuppressive effects of IL-10 on the
cell cycle. However, failure of IL-2 to completely reverse the IL-10
effects would suggest some direct effects of IL-10 on CD4+
T cells.
| |
ACKNOWLEDGMENT |
The authors gratefully acknowledge Melissa Chen and Neal Benson at the
Flow Cytometry Core Facility, University of Florida, for expert
assistance with flow cytometry data acquisition and analysis. This
manuscript is Florida Agricultural Experiment Station, Journal Series
Number R-06558.
| |
FOOTNOTES |
Submitted March 11, 1998;
accepted August 23, 1998.
Supported by National Institues of Health Grant No. AI 25904-08 to
H.M.J.
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 George Q. Perrin, Department of
Microbiology and Cell Science, Room 1019, Bldg 981, No Name and Museum
Roads, University of Florida, Gainesville, FL 32611; e-mail:
gperrin{at}micro.ifas.ufl.edu.
| |
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Top
Abstract
Introduction