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Department of Immunology and
Department of Virology, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is therefore of great interest to determine how GC have
opposite immunoregulatory effects on the Th1 and Th2 responses. In
contrast to their suppressive effect on the production of such Th1-type
cytokines as IL-2, IFN-
, and TNF-
(10, 11), GC have been reported
to augment the production of such Th2-type cytokines as IL-4, IL-10,
and IL-13 both in vitro and in vivo (12, 13, 14, 15). The most implicative
model of this regulation of Th1/Th2 responses by GC appears to be EAE
in Lewis rats, which is a cell-mediated autoimmune disease induced by
Th1-type CD4+ T cells in response to myelin basic protein
(16). In these rats, the spontaneous recovery from EAE is accompanied
by an elevation in the plasma levels of GC, and adrenalectomy leads to
progressive disease with a fatal outcome instead of remission (17, 18).
In addition, the replacement of GC in adrenalectomized rats is known to
induce a recovery from EAE (17). Since EAE in Lewis rats is caused by
Th1-type CD4+ T cells that produce IL-2 and IFN-, these
lines of evidence suggest that GC may thus induce a shift from the Th1
to Th2 responses in the recovery phase of EAE. However, the mechanism
by which GC preferentially induce Th2-type responses remains to be
elucidated.
NK1.1+ T cells are a recently identified cell
population that coexpresses TCR and NK1.1, a member of the NKR-P1
receptor family, and they have been reported to exist in the bone
marrow (19), thymus (20), liver (21), and, to a lesser degree, spleen
(22). Such NK1.1+ T cells are reported to be either
CD4+ CD8- or CD4-
CD8-, and to use a single TCR V-chain and skewed
Vß-chains (20, 23). Although the functions of NK1.1+ T
cells have not been fully elucidated, they are suggested to play some
immunoregulatory roles in the thymic selection (24), the suppression of
hemopoiesis or CTL generation (25, 26), and the regulation of
autoimmune responses (27, 28). More interestingly, NK1.1+ T
cells are considered to influence the Th1/Th2 lineage commitment
because they promptly produce IL-4 after stimulation with anti-CD3
mAb both in vivo and in vitro (22, 29). Evidence that certain mouse
strains, such as SJL or ß2m-deficient mice lacking
NK1.1+ T cells, fail to produce IL-4 and IgE in response to
anti-IgD Ab injection (30, 31) also implies that NK1.1+
T cells contribute to the development of Th2-type responses. However,
there are also opposite reports that IL-4 and IgE production are
normally induced in ß2m-deficient mice (32, 33) or
CD1-deficient mice (34, 35), which also lack NK1.1+ T
cells, suggesting that the contribution of NK1.1+ T cells
remains to be determined.
In this study, we investigated the participation of NK1.1+ T cells in the GC-induced IL-4 production and thus found a preferential survival of IL-4-producing NK1.1+ T cells in the spleen and liver of GC-treated mice. In addition, NK1.1+ T cells were revealed to be resistant to GC and radiation-induced apoptosis probably due to higher expression of intracellular Bcl-2 than conventional T cells. Our findings thus suggest that NK1.1+ T cells play a role in the regulation of Th1/Th2 balance by GC and in the immune responses under apoptotic stimuli.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female C57BL/6 mice and C57BL/6-lpr/lpr mice were purchased from Japan SLC (Shizuoka, Japan), and ß2m-deficient mice (H-2b) were originally supplied by The Jackson Laboratory (Bar Harbor, ME). These mice were maintained under specific pathogen-free conditions and were used for experiments at 8 to 10 wk of age.
Reagents and Abs
Dexamethasone (DEX) was purchased from Sigma (St. Louis, MO).
Anti-CD3 mAb (hamster IgG) and anti-TCRß mAb (hamster IgG)
were obtained from the supernatants of anti-CD3 mAb-producing
hybridoma 145-2C11 (provided by Dr. J. A. Bluestone, Department of
Pathology, University of Chicago, Chicago, IL) and anti-TCRß
mAb-producing hybridoma H57-597 (provided by Dr. R. T. Kubo,
Cytel, San Diego, CA), respectively. These Abs were purified by
collecting the supernatants of the hybridoma cells growing in a
serum-free medium (101, Nissui Pharmaceutical, Tokyo, Japan).
Anti-murine IL-4 mAb (11B11; rat IgG1) was provided by Dr. S. Hamano
(Department of Parasitology, Kyushu University, Fukuoka, Japan), and
anti-murine IL-4R mAb was purchased from Genzyme (Cambridge, MA).
The mAbs used in flow cytometric analysis were as follows: FITC- or
PE-conjugated anti-TCRß mAb, PE- or biotin-conjugated
anti-NK1.1 mAb, FITC-conjugated anti-IL-4 mAb, FITC-conjugated
rat IgG2b, PE-conjugated anti-Fas mAb, anti-murine Bcl-2 mAb
(hamster IgG), biotin-conjugated mouse anti-hamster IgG mAb, and
FITC-conjugated anti-bromodeoxyuridine (BrdUrd) mAb. These mAbs
were all purchased from PharMingen (San Diego, CA). Anti-CD3 mAb (rat
IgG2a), purified from the supernatants of anti-CD3 mAb-producing
hybridoma KT3.2 and conjugated with FITC in our laboratory, were also
used in some experiments.
Preparation of liver mononuclear cells (MNC)
Isolation of liver MNC was conducted as previously reported with minor modifications (36). Briefly, the liver was pressed through a 100-gauge stainless steel mesh in a complete medium, which consisted of RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 10% heat-inactivated FCS (HyClone, Logan, UT), 5 x 10-5 M 2-ME, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 30 µg/ml gentacin (Schering, Kenilworth, NJ), and 0.2% sodium bicarbonate. The cells were resuspended in 5 ml of 45% Percoll (Sigma) and layered on 5 ml of 67.5% Percoll solution. The gradient was centrifuged at 2500 rpm at 20°C for 25 min. The cells at the interface were harvested, washed with the complete culture medium, and then used as liver MNC.
In vitro cytokine production
The mice were injected i.p. with either 500 µl of PBS or the
indicated doses of DEX dissolved in 500 µl of PBS. After 18 h,
the spleen cells and liver MNC were harvested and cultured at a dose of
1 x 106 cells/ml in flat-bottom 96-well microtiter
plates (Corning, Corning, NY) precoated with 10 µg/ml of
anti-TCRß mAb. After 2 days, the culture supernatants were
harvested, and the amounts of IFN- and IL-4 were determined using a
sandwich ELISA, as previously reported (37).
Flow cytometric analysis
The spleen cells and liver MNC prepared from either DEX-treated
or untreated mice were stained with mAbs and analyzed by a FACS Calibur
flow cytometer with CellQuest software (Becton Dickinson, Mountain
View, CA). The supernatants of anti-FcR II/III mAb-producing
hybridoma (2.4G2: rat IgG2b) were used to block nonspecific binding in
all experiments. In some experiments the prepared cells were stained
with 2 µg/ml propidium iodide (Sigma) to exclude any dead cells.
Analysis of intracellular IL-4 synthesis
The mice were injected i.p. with either PBS or 200 µg of DEX,
and 18 h later they were injected i.v. with 2.5 µg of
anti-CD3 mAb. After 90 min, the spleen cells and liver MNC were
prepared, washed, and suspended at 1 x 106 cells/ml
in the complete culture medium, and then incubated for 4 h at
37°C in the presence of 10 µg/ml brefeldin A (Wako, Osaka, Japan).
These cells were harvested, washed, and incubated for 30 min at 4°C
with both PE-conjugated anti-TCRß mAb and biotin-conjugated
anti-NK1.1 mAb, followed by Red 670-conjugated streptavidin (Life
Technologies, Gaithersburg, MD). The cells were washed and fixed in 100
µl of solution A (Cell Perm & Fix, Caltag, South San Francisco, CA)
for 30 min at room temperature, and then washed again and resuspended
in 50 µl of solution B (permeabilization buffer) containing 1 µg of
FITC-conjugated anti-IL-4 mAb. The cells were incubated for 30 min
at 4°C and washed, and then cell fluorescence was analyzed by a flow
cytometer using the gates set by FL2 and FL3 to determine the
proportion of IL-4-producing cells in TCRß+
NK1.1- and TCRß+ NK1.1+ cell
subsets. As negative controls, the samples were also incubated with
isotype-matched Ab (FITC-conjugated rat IgG2b) instead of
FITC-conjugated anti-IL-4 mAb.
Detection of apoptotic cells
For the measurement of apoptosis, spleen cells and liver MNC
were prepared from either the untreated mice or the mice injected i.p.
with 200 µg of DEX either 3, 6, or 12 h previously. To analyze
the apoptotic cells in conjunction with cell surface staining, we used
the terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine
triphosphate-biotin nick-end labeling (TUNEL) method, as
previously reported (38). The prepared cells (1 x 106
cells) were briefly incubated with both FITC-conjugated
anti-TCRß mAb and PE-conjugated anti-NK1.1 mAb for 30 min
at 4°C. For fixation and permeabilization, the cells were washed and
suspended with 250 µl of 1% paraformaldehyde in PBS for 60 min at
4°C, and then stored in 200 µl of 75% ethanol at -20°C. For
analysis, these cells were incubated in 50 µl of buffer containing
250 U/ml TdT and 10 µM biotin-16-dUTP (both from Boehringer Mannheim,
Indianapolis, IN) for 60 min at 37°C, followed by Red
670-streptavidin staining for 30 min. Cell fluorescence was analyzed by
a flow cytometer using the gates of the TCRß+
NK1.1- and TCRß+ NK1.1+ cell
subsets, and the negative controls were determined by the samples
incubated without TdT.
In vitro assay of cell survival
The spleen cells or liver MNC (1 x 106
cells/ml) prepared from the untreated mice were incubated at 37°C in
24-well tissue culture plates (Corning) in a volume of 1 ml/well of the
complete medium in the absence or the presence of the indicated doses
of DEX. In some wells, either anti-IL-4 mAb (25 µg/ml) or
anti-IL-4R mAb (5 µg/ml) was added to neutralize the endogenous
IL-4 production. After 8 h, these cells were harvested and stained
with both FITC-conjugated anti-TCRß mAb and PE-conjugated
anti-NK1.1 mAb, and then with 2 µg/ml propidium iodide. The
surviving cells in TCRß+ NK1.1- and
TCRß+ NK1.1+ cell subsets were analyzed by
a flow cytometer with the gate excluding dead cells that were stained
with propidium iodide. The percentage of survival in each subset was
calculated according to the following formula: % survival =
(number of surviving cells in the presence of DEX, DEX and
anti-IL-4 mAb, or DEX and anti-IL-4R mAb)/(number of surviving
cells in the absence of DEX) x 100. As for radiation-induced
apoptosis, the spleen cells or the liver MNC prepared from the
untreated mice were irradiated with or without 150 rad by
137Cs irradiator (Gammacell 40, Atomic Energy of Canada,
Ottawa, Canada) and then incubated in 24-well tissue culture plates
(Corning) at 1 x 106 cells/well at 37°C. After 0,
4, 8, 12, and 20 h, the number of surviving cells of
TCRß+ NK1.1- and TCRß+
NK1.1+ cell subsets were analyzed in each well by a flow
cytometer.
Analysis of intracellular Bcl-2 expression
The spleen cells or liver MNC (1 x 106 cells/ml) prepared from the untreated mice were analyzed for their Bcl-2 expression either before or after incubation in the presence of 10-7 M DEX for 2 or 4 h. These cells were stained with both FITC-conjugated anti-CD3 mAb (KT3.2; rat IgG2a) and PE-conjugated anti-NK1.1 mAb (mouse IgG2a) for 30 min at 4°C. The cells were washed and fixed in 100 µl of solution A (Cell Perm & Fix, Caltag) for 30 min at room temperature, and then washed and incubated with 50 µl of solution B containing 10 µg/ml of hamster anti-murine Bcl-2 mAb for 30 min at 4°C. The cells were washed again and incubated with biotin-conjugated anti-hamster IgG mAb followed by Red 670-conjugated streptavidin. The expression level of Bcl-2 was analyzed by a flow cytometer using the gates of either the CD3+ NK1.1- or the CD3+ NK1.1+ cell subset, and the negative controls were determined by the addition of normal hamster IgG instead of anti-Bcl-2 mAb.
BrdUrd incorporation analysis
The mice were injected i.p. with 500 µl of 2 mg/ml BrdUrd
(Sigma) every 12 h for 3 days and analyzed 12 h after the
last injection. In a group, the mice were also injected i.p. with 200
µg of DEX 18 h before the analysis. The liver MNC prepared from
these mice were stained with both PE-conjugated anti-TCRß mAb
and biotin-conjugated anti-NK1.1 mAb, followed by Red
670-conjugated streptavidin. After surface staining, BrdUrd
incorporation was analyzed as previously reported (39). The cells were
washed, resuspended in cold 0.15 M NaCl, and fixed by dropwise addition
of cold 75% ethanol. The cells were incubated for 30 min at 4°C,
washed with PBS, then incubated with PBS containing 1%
paraformaldehyde and 0.01% Tween-20 for 1 h. The cells were
pelleted, then incubated with 50 Kunitz units of DNase I (Boehringer
Mannheim) in 0.15 M NaCl and 4.2 mM MgCl2, pH 5, for 10
min. After washing, the cells were incubated with FITC-conjugated
anti-BrdUrd mAb and analyzed by a flow cytometer with the gates of
TCRß+ NK1.1- and TCRß+
NK1.1+ cell subsets. As a negative control, the mice not
injected with BrdUrd were also analyzed.
Statistics
The statistical significance of the data was determined using Student’s t test. p < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been reported that GC promote the production of Th2-type
cytokines such as IL-4, IL-10, and IL-13 both in vivo and in vitro
(12, 13, 14, 15). To investigate the contribution of NK1.1+ T cells
to the increased production of Th2-type cytokines by GC, we at first
examined the effect of the in vivo administration of DEX, a synthetic
GC, on the potential of the spleen cells and liver MNC to produce
IFN- and IL-4 in wild-type mice and ß2m-deficient mice
that lack NK1.1+ T cells. As shown in Figure 1
, the in vivo treatment of wild-type
mice with DEX inhibited IFN- production of both spleen cells and
liver MNC in a dose-dependent manner. On the other hand, the IL-4
production of both the spleen cells and liver MNC in wild-type mice was
augmented by the administration of DEX, and the ability of DEX to
induce IL-4 production was most remarkable at a dose of 200 µg of
DEX. In contrast to wild-type mice, IL-4 production was down-regulated
in ß2m-deficient mice, and more importantly, no
enhancement of IL-4 production was induced in
ß2m-deficient mice even when they were injected with 200
µg of DEX. These results indicate that the DEX treatment promotes
IL-4 production in both spleen cells and liver MNC and that
NK1.1+ T cells would thus be necessary for such increased
IL-4 production by DEX. In addition, we selected 200 µg of DEX as an
administration dose in the following experiments.
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ß+ NK1.1+ cells in spleen and liver
To further investigate the contribution of NK1.1+ T
cells to the induction of Th2-type cytokines by GC, we next examined
the phenotypic changes in spleen cells and liver MNC in DEX-treated
mice. Figure 2 shows that DEX treatment
increased the proportion of TCRß+ NK1.1+
cells in both spleen and liver and reduced that of
TCRß+ NK1.1- cells in liver MNC. No
definite effect on TCRß- NK1.1+ cells was
observed. Since GC are reported to induce apoptosis in several cell
types, including mature T cells (40), we further examined the absolute
cell number of these subsets in the spleen and liver of DEX-treated
mice. As shown in Table I, the
administration of DEX decreased the cellularity in the spleen but not
that in the liver and also decreased the cell number of
TCRß+ NK1.1- cells in both organs 18
h after the injection. Interestingly, the cell number of
TCRß+ NK1.1+ cells did not decrease in the
spleen and even increased in the liver. The cell number of
TCRß- NK1.1+ cells decreased in the
spleen after DEX treatment. These changes in cellularity in each subset
were similar 36 h after the injection of DEX, except for a
recovery of TCRß+ NK1.1- cells in the
liver. These results suggest that TCRß+
NK1.1+ cells preferentially survive the in vivo treatment
with DEX.
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ß+ NK1.1+ cells in spleen and liver
To directly confirm the participation of TCRß+
NK1.1+ cells in GC-induced IL-4 production, we next
examined the intracellular IL-4 synthesis in NK1.1+ T cells
of DEX-treated mice. Since it has been reported that the i.v. injection
of a low dose of anti-CD3 mAb induces IL-4 production in
NK1.1+ T cells (22), we used this protocol and found that
the proportion of IL-4-producing TCRß+
NK1.1+ cells in TCRß+ cells increased in
both spleen and liver of DEX-treated mice (Fig. 3). We also confirmed that these
IL-4-positive cells were not detected by the staining with
isotype-matched control Ab. These results thus indicate that IL-4
production is enhanced by DEX through the increased proportion of
IL-4-producing TCRß+ NK1.1+ cells in
DEX-treated mice.
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ß+ NK1.1+ cells by DEX
To investigate the underlying mechanism by which
TCRß+ NK1.1+ cells preferentially survived
in the DEX-treated mice, we at first examined the possible role of
Fas/Fas ligand interaction, because NK1.1+ T cells possess
an ability to kill lymphocytes through Fas-mediated cytolysis (24). As
shown in Figure 4A, the
expression level of Fas on TCRß+ NK1.1-
cells and TCRß+ NK1.1+ cells was
comparable in untreated and DEX-treated mice in both spleen cells and
liver MNC. In addition, Figure 4B shows that DEX
treatment increased the proportion of TCRß+
NK1.1+ cells and reduced that of TCRß+
NK1.1- cells in the liver of genetically Fas-deficient
C57BL/6-lpr/lpr mice. We also examined
the absolute cell number of each subset in
C57BL/6-lpr/lpr mice and found that the
preferential survival of TCRß+ NK1.1+
cells, similar to that shown in Table I
, was also observed after
treatment with DEX (data not shown). These results thus suggest that
the different survival rates of TCRß+
NK1.1- cells and TCRß+ NK1.1+
cells after DEX treatment are independent of Fas-mediated
apoptosis.
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ß+ NK1.1+ cells are resistant to
DEX-induced apoptosis
We next compared the sensitivity to DEX-induced apoptosis between
NK1.1+ T cells and conventional T cells, since GC have been
reported to induce apoptotic cell death in mature lymphocytes as well
as immature thymocytes (40). As shown in Figure 5A, the proportion of
apoptotic cells in TCRß+ NK1.1+ cells was
significantly lower than that in TCRß+
NK1.1- cells after the injection of DEX. The differences
were apparent in the liver, and there were small but significant
differences at 3 and 6 h after the injection of DEX in the spleen
cells. We further showed the representative data of the TUNEL analysis
in Figure 5B, which demonstrated that the percentage of
apoptotic cells in hepatic TCRß+ NK1.1+
cells was approximately 10 times lower than that in hepatic
TCRß+ NK1.1- cells at 6 h after the
injection of DEX. These results suggest that the preferential survival
of TCRß+ NK1.1+ cells in the DEX-treated
mice is due to their resistance to DEX-induced apoptosis.
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ß+ NK1.1+ cells to
DEX-induced apoptosis is independent of their IL-4 production
We further tried to clarify the reason why TCRß+
NK1.1+ cells were resistant to DEX-induced apoptosis, and
at first examined whether the potential of NK1.1+ T cells
to produce IL-4 was involved in their resistance to GC, since IL-4
protects T cells from GC-induced apoptosis (41, 42). Figure 6A shows that in vivo
administration of anti-IL-4 mAb before treatment with DEX resulted
in changes in cellularity similar to those in the mice injected with
DEX alone. We also examined the in vitro sensitivity of conventional T
cells and NK1.1+ T cells to the DEX-induced apoptosis, and
the influence of IL-4 on the survival of these cells in vitro. As shown
in Figure 6B, the percentage of survival in
TCRß+ NK1.1+ cells was significantly
higher than that of TCRß+ NK1.1- cells
with 10-7 M DEX in spleen cells and 10-8 and
10-7 M DEX in liver MNC. Moreover, the addition of neither
anti-IL-4 mAb nor anti-IL-4R mAb to the culture abrogated such
preferential survival of TCRß+ NK1.1+
cells in the presence of DEX. These results suggest that the resistance
of TCRß+ NK1.1+ cells to DEX-induced
apoptosis is not ascribed to their potential to produce IL-4.
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We further investigated the expression level of intracellular
Bcl-2 in conventional T cells and NK1.1+ T cells, since
overexpression of Bcl-2 has been reported to prevent GC-induced
apoptosis in T cell hybridomas (43). As shown in Figure 7, the expression level of Bcl-2 in
CD3+ NK1.1+ cells was higher than that in
conventional CD3+ NK1.1- cells, especially in
the liver MNC. In addition, the higher expression of Bcl-2 in
CD3+ NK1.1+ cells than in CD3+
NK1.1- cells was also exhibited after the in vitro culture
with DEX, although the DEX treatment elevated Bcl-2 expression in both
subsets. These results suggest that the resistance of
NK1.1+ T cells to GC-induced apoptosis could depend on
their high expression of intracellular Bcl-2.
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ß+ NK1.1+ cells are resistant to
radiation-induced apoptosis
We next determined whether TCRß+
NK1.1+ cells could also exhibit resistance to
radiation-induced apoptosis, since a high expression of Bcl-2 is known
to protect lymphocytes from radiation-induced apoptosis as well as
GC-induced apoptosis (44). As shown in Figure 8, the in vitro exposure to radiation
significantly reduced the number of surviving TCRß+
NK1.1- cells in both spleen cells and liver MNC from 8 to
20 h after radiation. In contrast, the numbers of surviving
splenic and hepatic TCRß+ NK1.1+ cells
after radiation were almost equal to those without radiation, although
the number of hepatic TCRß+ NK1.1+ cells
spontaneously decreased even without radiation. These results suggest
that TCRß+ NK1.1+ cells are resistant not
only to DEX-induced apoptosis but also to radiation-induced apoptosis,
probably due to their high expression of intracellular Bcl-2.
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ß+ NK1.1+ cells in liver
We lastly examined whether the in vivo treatment of DEX induced
the proliferative responses of TCRß+
NK1.1+ cells in the liver, since the absolute cell number
of hepatic TCRß+ NK1.1+ cells increased
after DEX treatment (Table I). As shown in Figure 9, the incorporation of BrdUrd in hepatic
TCRß+ NK1.1+ cells did not increase after
in vivo treatment with DEX and was almost comparable to that of
TCRß+ NK1.1- cells. These results suggest
that the increased number of hepatic TCRß+
NK1.1+ cells after DEX treatment is independent of their
proliferative activation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß+ NK1.1+ cells in
spleen and liver. More interestingly, such preferential survival of
TCRß+ NK1.1+ cells was ascribed to their
resistance to DEX-induced apoptosis, probably through a higher
expression of intracellular Bcl-2 than that in conventional T cells. In
addition, the splenic and hepatic TCRß+
NK1.1+ cells showed resistance to radiation-induced cell
death in vitro. These findings therefore suggest that
NK1.1+ T cells play an important role in Th1/Th2 regulation
by GC and their possible functions under the conditions of various
apoptotic stimuli.
Although several researchers have reported that GC promote the
production of Th2-type cytokines in contrast to their inhibitory effect
on Th1-type cytokines (10, 11, 12, 13, 14, 15), the underlying mechanisms of the
effects have yet to be fully elucidated. In general, there appear to be
at least two possible explanations. One is that GC preferentially
induce differentiation in Th2-type T cells from naive CD4+
T cells, and the other is that GC enhance the proliferation and
cytokine production of already differentiated Th2-type T cells. The
latter possibility is supported by the reports that IL-4-dependent
proliferation of Th2-type clone is augmented by GC (45) and that IL-4
rescues Th2 cells from GC-induced apoptosis (41, 42). However, this
idea alone is not sufficient to explain the finding that GC increase
the production of Th2-type cytokines from naive spleen and lymph node
cells as well as primed T cells (13). In this study we also
demonstrated that IL-4 production could be augmented by DEX in naive
spleen cells and liver MNC (Fig. 1). In this regard, we propose another
possibility: that GC promote the development of Th2-type T cells
through the preferential survival of IL-4-producing NK1.1+
T cells. We demonstrated the survival of NK1.1+ T cells
after treatment with DEX both in vivo (Fig. 2 and Table I) and in vitro
(Fig. 6B). Moreover, the importance of NK1.1+ T
cells in DEX-induced IL-4 production was also confirmed by the
experiments using ß2m-deficient mice (Fig. 1) and by an
analysis of intracellular IL-4 synthesis (Fig. 3). Although it remains
controversial that IL-4 production by NK1.1+ T cells is
necessary for the development of Th2-type T cells (30, 31, 32, 33, 34, 35), our
findings suggest a novel mechanism concerning the regulation of Th1/Th2
balance by GC.
To elucidate the underlying mechanism for the preferential survival of
NK1.1+ T cells by DEX, we at first examined the involvement
of Fas/Fas ligand interaction because NK1.1+ T cells were
reported to kill lymphocytes through Fas-mediated cytolysis (24).
However, this possibility could be ruled out by the comparable
expression of Fas between NK1.1+ T cells and conventional T
cells (Fig. 4A) and the increased proportion of
NK1.1+ T cells by DEX even in Fas-mutated lpr
mice (Fig. 4B). Furthermore, these findings also suggest
that GC-induced apoptosis may be independent of the interaction of
Fas/Fas ligand, in contrast to a possible role of this interaction in
radiation-induced apoptosis (46). Next, we examined the sensitivity of
NK1.1+ T cells to DEX-induced apoptosis and found that they
were more resistant to DEX-induced apoptosis than conventional T cells
in both spleen and liver (Fig. 5). Based on this finding, we concluded
that the preferential survival of NK1.1+ T cells would be
ascribed to their resistance to GC-induced apoptosis. This explanation
is plausible but not sufficient, because we cannot yet explain the
result that the cellularity of hepatic TCRß+
NK1.1+ cells increased after treatment with DEX (Table I).
To explore this question, we also examined the in vivo proliferative
activity of NK1.1+ T cells, but detected no increased
proliferation by the treatment with DEX (Fig. 9). We could not clarify
the reason for the increase in hepatic NK1.1+ T cells by
DEX in this study, but it is possible that the activity of migration in
NK1.1+ T cells would be altered by DEX, since GC have been
reported to regulate the production of several chemoattractants for
leukocytes (47, 48).
The precise mechanism by which GC induce apoptosis still remains
unclear, but several means to protect T cells from GC-induced apoptosis
have been reported. In considering such reports, we investigated the
reason why NK1.1+ T cells are resistant to GC-induced
apoptosis. We first examined a possibility that IL-4 production by
NK1.1+ T cells contributed to their resistance to
GC-induced apoptosis, since IL-4 protects T cells from GC-induced
apoptosis (41, 42) and the binding affinity of GC receptors is reduced
by IL-4 and IL-2 (49). However, neither in vivo nor in vitro
neutralization of IL-4 abrogated the resistance of
NK1.1+ T cells to apoptosis by DEX (Fig. 6), thus ruling
out this possibility. Other cytokines, such as IL-6 and IL-9, and some
chemokines are also reported to protect lymphocytes from GC-induced
apoptosis (50, 51, 52), but we consider these factors to have only minor
effects, since NK1.1+ T cells have not been shown to
produce these cytokines. We next examined the expression of
intracellular Bcl-2, since the transfection of this proto-oncogene into
T cell hybridoma inhibits GC-induced apoptosis (43) and peripheral T
cells in Bcl-2 knockout animals are sensitive to GC-induced apoptosis
(53). The expression of Bcl-2 in NK1.1+ T cells was higher
than that in conventional T cells regardless of culture with DEX (Fig. 7), suggesting a possible contribution of Bcl-2 to the resistance of
NK1.1+ T cells to GC-induced apoptosis. The expression
level of Bcl-2 was less high in the splenic NK1.1+ T cells,
but it may be compatible with the finding that the resistance of
splenic NK1.1+ T cells to DEX-induced apoptosis was less
apparent both in vivo (Fig. 5A) and in vitro (Fig. 6B). Interestingly, the level of Bcl-2 in both conventional
T cells and NK1.1+ T cells increased after culture with
DEX. With regard to the mechanism for such an increase in Bcl-2, we
consider the possibility that the cells expressing high levels of Bcl-2
selectively survived the treatment with DEX or that DEX directly
increased the expression level of Bcl-2, as previously reported (54).
We further demonstrated the radioresistance of NK1.1+ T
cells in vitro (Fig. 8), which is consistent with a previous report
that hepatic IL-2Rß+ intermediate TCR cells are resistant
to in vivo radiation (55). These findings may support our idea, since
Bcl-2 can also inhibit radiation-induced apoptosis (44). Although the
cell number of hepatic NK1.1+ T cells spontaneously
decreased even without radiation, this result would depend on
mechanisms irrelevant to Bcl-2, such as Bcl-2-independent apoptosis
(43) or down-regulation of NK1.1 expression by the in vitro culture
(56).
In this study we demonstrated the increase in IL-4 production and the survival of NK1.1+ T cells after the exogenous administration of DEX, a synthetic GC. To confirm that these findings could also be applied to physiologic conditions, we conducted experiments in which mice were burdened with repeated restraint stress. As a result, the concentration of GC in the serum was elevated in the stressed mice, and their spleen cells and liver MNC exhibited both an increase in IL-4 production and a preferential survival of IL-4-producing NK1.1+ T cells (K. Tamada, unpublished observation). These findings thus suggest that endogenously secreted GC may also potentially induce Th2-type cytokines through the survival of IL-4-producing NK1.1+ T cells.
In conclusion, we herein revealed a novel mechanism regarding GC-induced IL-4 production in which IL-4-producing NK1.1+ T cells were resistant to GC-induced apoptosis through their high expression of Bcl-2 and thereby preferentially survived in GC-treated mice. Our findings thus suggest that NK1.1+ T cells play an important role in the immune responses during therapy with GC, stressful events, and various types of apoptotic stimuli.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to the current address of Dr. Koji Tamada, Department of Immunology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905.
3 Abbreviations used in this paper: GC, glucocorticoids; EAE, experimental allergic encephalomyelitis; DEX, dexamethasone; PE, phycoerythrin; BrdUrd, bromodeoxyuridine; MNC, mononuclear cells; TdT, terminal deoxynucleotidyl transferase; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling.
Received for publication November 3, 1997. Accepted for publication April 3, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ßTCR+ NK1.1+ cells. J. Immunol. 145:3209.[Abstract]
/ß+ cells in the liver of mice. J. Exp. Med. 180:699./ß+ thymocytes against a CD4+ 8+ thymocyte population associated with intact Fas antigen expression on the target. J. Exp. Med. 180:423.24JQ antigen receptor in patients with systemic sclerosis. J. Exp. Med. 182:1163.14+ NK T cells associated with disease development in autoimmune-prone mice. J. Immunol. 156:4035.[Abstract]
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