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*
Laboratory of Experimental Immunology,
Laboratory of Molecular Immunoregulation, Division of Basic Sciences;
Intramural Research Support Program, Science Applications International Corporation-Frederick; National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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secretion. When NK cells were costimulated with IL-13 and IL-2, IL-13
generally resulted in two types of reactivity: IL-13 synergized with
IL-2 to stimulate IFN- production or it modestly inhibited
IL-2-mediated IFN- production. In both types of donors, the effect
of IL-13 on IL-2-induced IFN- production was in marked contrast to
the strong inhibition seen with IL-4 in NK cells. Additionally, IL-13
suppresses IL-2-induced NK cytolytic and proliferative activities
although less efficiently than IL-4. In T cells, IL-13 inhibits
anti-CD3 mAb/IL-2- or PHA-mediated IFN- production and enhances
cytolytic potential. Furthermore, we demonstrate that IL-13, like IL-4,
induces distinct STAT6-DNA binding complexes and tyrosine
phosphorylation of STAT6 and Janus kinase 3 (JAK3) in NK and T cells.
We observed that Abs directed against unique domains of STAT6 have
differential effects on complexes in T cells but not in NK cells,
suggesting different STAT6 isoforms. These findings show that IL-13 and
IL-4 have the ability to regulate NK and T cell activation and that
IL-13 is a potent regulator of STAT6 and JAK3 in these cell types. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In human NK cells, while it has been demonstrated that IL-13, unlike
IL-4, increases IL-2-induced IFN- production (4, 5) and cytolytic
activity (4), the IL-13 signaling mechanism in these cells has not been
investigated. In human T cells, the biologic effects of IL-13 have not
been fully defined although it has been demonstrated that IL-13 is
incapable of promoting T cell proliferation (5, 6, 13). In addition, it
also has been reported that T cells (5, 13, 14, 15), as well as NK and
LAK2 cells (14, 15), lack a
functional IL-13R. In contrast, it also has been reported that IL-13,
like IL-4, induces IL-4 responsive element DNA protein complexes in
human T cells (16) and FcRI GAS promoter DNA protein binding
complexes in fresh human PBL (17). Nevertheless, this limited
information suggests that the biologic effects of IL-13 might be
distinct when NK and T cells are compared. Therefore, we investigated
the signaling and biologic effects of IL-13 on NK and T cells to define
the possible different effects of IL-13 on these cell types and to
determine whether these effects are distinct from those of
IL-4.
The molecular basis for cytokine functional redundancy is thought to
occur through shared receptor chains or signaling molecules (18).
According to binding assays and functional analysis, it has been
demonstrated that IL-13 and IL-4 share common signaling molecule(s) (6, 12, 19, 20, 21, 22, 23, 24). In T cells, IL-4R
conjugates with the common -chain
(c) (25) to form a high affinity IL-4R complex (26, 27). Recently,
human and murine IL-13R chains, responsible for binding IL-13, were
cloned and found capable of associating with the IL-4R to form a
high affinity IL-13 receptor complex (28, 29, 30). Thus, these data suggest
that IL-13 might share some biologic effects with IL-4.
The Janus kinase (JAK) STAT pathway is an important cytokine-induced
signal transduction pathway that directly transfers signals from cell
surface cytokine receptors to the nucleus (31, 32, 33). In the case of
IL-4, IL-4R and c are believed to be associated with JAK1 and
JAK3, respectively (27, 34). The activation of JAKs leads to
phosphorylation of IL-4R and recruitment and activation of STAT6
(21, 35, 36, 37, 38, 39, 40). STAT6 has been demonstrated to play a critical role in
regulating the expression of MHC II, CD23 molecules, and production of
IgE in B cells, as well as secretion of IL-4 and IFN- in T cells in
the STAT6-deficient mouse model (41, 42, 43), suggesting that the
activation of STAT6 is a key event for IL-4-mediated biologic effects.
Therefore, we further investigated whether or not STAT6 and JAK3 were
involved in the IL-13-induced signal transduction in NK and T
cells.
In this study, we demonstrate that IL-13 is able to differentially
modulate specific biologic properties of NK and T cells, including
IFN- production, cytotoxic activity, and NK cell proliferation, and
that these effects are distinctive from those mediated by IL-4. In
addition, we found that IL-13 induces distinct activation of STAT6 when
primary human NK and T cells are compared. These findings indicate that
IL-13 may play an important role in regulating the differentiation and
activation of NK and T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cells were cultured in RPMI 1640 (BioWhittaker, Walkersville,
MD) and supplemented with 10% FCS (Atlanta Biologic, Atlanta, GA), 100
U/ml penicillin and 100 µg/ml streptomycin (Biofluids, Rockville,
MD). Recombinant human IL-2 was provided by Chiron (Emeryville, Ca),
and IL-4, IL-6, and IL-13 were purchased from Pepro Tech (Rocky Hill,
NJ). IL-12 was provided by Hoffmann-La Roche (Nutley, NJ). Antisera
against STAT1, STAT3, STAT5a, and STAT5b were kindly provided by Dr.
Andrew C. Larner (Food and Drug Administration, Center for Biologics
Evaluation and Research (CBER), Division of Cytokine Biology, Bethesda,
MD). Antisera to STAT4, human STAT6 (residues 828–847, Cat: S-20,
denoted as STAT6h1), and mouse STAT6 (residues 280–480, Cat: M-200,
designated as STAT6m) were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). Antiserum to hSTAT6 (residues 787–804, designated as
STATh2) was kindly provided by Dr. William J. LaRochelle (Bethesda, MD)
(44). Abs against JAK1 and JAK3 were generated as previously described
(21, 22). mAb against phosphotyrosine, 4G10, and JAK1 were purchased
from Upstate Biotechnology (Lake Placid, NY). A hybridoma producing Ab
against CD3 (OKT3) was purchased from American Type Culture Collection
(ATCC) (Manassas, VA). Phytohemagglutinin (PHA) was purchased from
Murex Diagnostics (Darfort, England). Animal care was provided in
accordance with the procedures outlined in the Guide for the Care
and Use of Laboratory Animals (National Institutes of Health
Publication No. 86–23, 1985).
Purification of NK and T cells and cell stimulation
PBMC were separated on Ficoll-Hypaque gradients by leukophoresis packages obtained from healthy donors through the National Institutes of Health blood bank as previously described (45). Leukocyte suspensions were washed in HBSS and resuspended in RPMI 1640 containing 1% heat-inactivated FCS. Adherent cells (monocytes and B cells) were removed by adherence to plastic dishes to nylon wool and via elutriation. Highly enriched populations of CD3+ CD56- T cells (95–99%) (<2% LGL; large granular lymphocytes) and CD3-CD56+CD16+ LGL (>90% pure) (<10% CD3+ T cells, <2% monocytes) were obtained from PBMC by centrifugation of nylon wool-passed and elutriated cells on discontinuous density gradients of percoll (Pharmacia Fine Chemicals, Uppsala, Sweden). The purity of T and NK cells was analyzed by flow cytometry. Activation was monitored by surface activation Ag expression (46). For cytotoxicity and cytokine secretion assays, the cell cultures were treated with 100 U/ml of IL-2, 1 µg of mAb against CD3/106 T cells, or 5 µg/ml of PHA.
For EMSA, freshly isolated human NK cells and T cells were immediately washed three times with RPMI 1640 medium to remove contaminating percoll. Next, aliquoted cells were resuspended in RPMI 1640 medium, incubated at 37°C for 30 min in the absence of FCS, and then immediately stimulated with various cytokines at times and concentrations described in the figure legends. Preactivated T cells were obtained by treating freshly isolated T cells with 100 U/ml IL-2 for 72 h. For EMSA, before restimulation, cytokines were removed from the cell culture by washing, and rested in RPMI 1640 medium containing 1% FCS for 24 h at a concentration of 2 x 106 cells/ml.
Cytotoxicity assays and cytokine measurement
Freshly isolated NK and T cells were cultured in the absence or presence of cytokines. The cells harvested from the cultures were used for cytotoxic assays while their supernatants were collected for cytokine analysis. In the cytotoxicity assay, target cells, K562, which were derived from a patient with chronic myelogenous leukemia in blast crisis (47), were labeled with 100 µCi of 51Cr (New England Nuclear, Boston, MA) for 1 h at 37°C and washed. Labeled target cells (5 x 103) and effector cells were cultured together in 200 µl of medium in microtiter plates (Linbro, Hamden, CT) at several effect-to-target ratios (25:1, 12:1, 6:1, 3:1). After 4 h of incubation at 37°C, the supernatant was harvested and counted in a gamma scintillation counter. Cytotoxicity was determined by the amount of 51Cr released from lysed target cells. Three replicates were used per experimental group, and the percentage of specific lysis was calculated according to the formula: lysis = 100 x [(cpm in experimental wells) - (cpm in wells with target cells alone)]/(cpm incorporated in target cells).
IFN- production was measured using IFN- Elisa Kits (R&D Systems,
Minneapolis, MN). All samples were measured in duplicate and plotted
against the standard curve and reported as pg/ml. In all assays, the SD
of cytokine measurement was less than 25 pg/ml.
Proliferation assay
Freshly isolated human NK or T cells (2 x 105) were cultured in flat-bottom 96-well plates. Each sample was tested in triplicate in 200:l total volume. After 2 to 3 days culture with or without cytokines, wells were pulsed with 1 µCi of methyl[3H]thymidine ([3H]TdR; DuPont NEN, Boston, MA; 6.7 Ci/mmol, ICN Radiochemicals, Costa Mesa, CA) and harvested 4 h later on a Skatron Cell Harvester (Skatron, Sterling, VA). Thymidine incorporation was assessed by liquid scintillation counting.
Flow cytometry analysis
Cells were stained as previously described (48). Cell sorting was performed on either an Epics 750 (Coulter Electronics, Hialeah, FL) or a FACStar (Becton Dickinson, Mountain View, CA). Cells were directly stained using PE or FITC-labeled primary Abs against CD25 or HLA-Dr (Becton Dickinson) or indirectly stained using a primary Ab followed by an isotype-specific FITC-labeled or PE-conjugated secondary or biotinylated primary Ab followed by Streptaviden PE (Becton Dickinson) or Avidium red 670 (Becton Dickinson).
Preparation of nuclear extracts
Nuclear extracts were prepared as described (49). Cells were washed once with cold PBS and once with buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM DTT, 1 mM Na-vanadate, 25 mM NaF, 10 mM Na-pyrophosphate, and 25 mM p-nitrophenyl guanidinobenzoate), and lysed in buffer A containing 0.05% NP40. The lysate was placed on ice for 10 min and centrifuged at 4,000 rpm and 4°C for 4 min to remove cytoplasmic proteins. The nuclear proteins in the pellet were extracted in high salt buffer (410 mM KCl, 25% glycerol, and 0.2 mM EDTA in buffer A). Insoluble material was removed by centrifugation at 15,000 rpm for 10 min. Protein concentration was measured with a Bio-Rad (Hercules, CA) protein assay, and samples were stored at -70°C until use.
Electrophoretic mobility shift assays
EMSA was performed as previously described (49).
Oligonucleotides were synthesized in DNA/RNA Synthesizer (PE Applied
Biosystems, Foster, CA). The double-stranded oligonucleotides
containing GAS-like motifs from the human C
regulatory region
(5'-AGTCAAGACCTTTTCCCAAGAAATCTATC-3') (42, 50), and the
c-fos promoter sis-inducible element m67 SIE
(5'-AGCTTGTCGACATTTCCCGTAAATCGTCGG-3') (51, 52) were end-labeled with
the Klenow fragment DNA polymerase (New England BioLabs, Beverly, MA)
with [-32P]dATP (3000 Ci/mmol; DuPont) as previously
described (49). Labeled DNA probe was further purified from a 12%
polyacrylamide gel by excising the corresponding radioactive band after
electrophoresis. The purified DNA probe was aliquoted at 15,000 cpm per
µl and stored at -20°C until use. For supershift analysis,
extracts were incubated with 1 µl antisera to STAT1, STAT3,
STAT5a, STAT5b, and STAT6, while 1 µg anti-STAT4, and 1–2 µl
normal rabbit serum were incubated with samples for 2 h on ice,
before addition of 32P-labeled DNA probe. Cold competition
experiments were performed with unlabeled wild-type probes or C GAS
motif-mutated probes (GTCCCAAGAC). Results were visualized by
autoradiography after 1 to 4 days exposure at -70°C.
Immunoprecipitation and Western blotting
Immunoprecipitation and Western blotting were performed as described previously (22). The classified cell lysates were incubated with anti-STAT6 antiserum for 2 h at 4°C. Abs were captured by incubation for 30 min with protein A-Sepharose beads (Pharmacia). Immunoprecipitated protein was washed and eluted by boiling in SDS-sample buffer for 4 min, and then subjected to 7.5% SDS-PAGE under reducing conditions. Proteins were transferred to the polyvinylidene difluoride (PVDF) membrane (Immobilon; Millipore, Bedford, MA; Cat No. 1PVH 00010). The membrane was blotted with 4G10 and then stripped and reblotted with anti-STAT6 antiserum. Proteins were detected with an ECL kit (Amersham, Arlington Heights, IL), according to the manufacturer’s instructions.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production in primary NK and T cells
To examine whether IL-13 and IL-4 differentially regulate IFN-
production in primary NK and T cells, fresh primary NK cells were
stimulated with medium, 10, 100, and 1,000 ng/ml of IL-13, or 10, 100,
and 1,000 ng/ml of IL-4 in the presence or absence of 50 U/ml of IL-2
for 18 h. Fresh primary T cells were cultured with 100 ng/ml of
IL-4 or 100 ng/ml of IL-13 in presence or absence of anti-CD3 mAb
(1 µg/106 cells)/IL-2 (100 U/ml) or PHA (5 µg/ml)
for 48 h. Supernatants from these cultures were harvested for the
measurement of IFN- by ELISA whereas the cells were collected for
cytotoxicity assays (discussed below).
In primary NK cells, IL-13, but not IL-4, could induce low levels of
IFN- secretion in both representative donors (Table I
, donors 1 and 2). When NK cells were
costimulated with IL-13 and IL-2, IL-13 generally resulted in two types
of reactivity from nine donors tested. Donor 1 is representatives of
five of nine donors, in which IL-13 synergized with IL-2 to stimulate
IFN- production (3- to 27-fold increase) (Table I) and is consistent
with previous reports (4, 5). However, donor 2 represents four of nine
donors where IL-13 modestly inhibited IFN- production (about a
twofold reduction) (Table I). Currently, the reasons for variability in
IFN- production, probably resulting from the differences between NK
cell populations in response to IL-13 stimulation, are unknown (i.e.,
IL-13R or IL-2R expression, signaling molecules, and differentiation
states). Nevertheless, in both types of donors, the effect of IL-13 on
IFN- production was in marked contrast to the strong inhibition seen
with IL-4 in NK cells (Table I) since 10 ng/ml of IL-4 was able to
effectively inhibit IL-2-induced IFN- production.
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production (Table II).
However, different levels of IFN- production were detected in the
cultures in which T cells were stimulated with either anti-CD3
mAb/IL-2 or PHA (Table II). IL-13 inhibited both anti-CD3 mAb/IL-2-
and PHA-mediated IFN- production (approximately 50% reduction)
while IL-4 inhibited only PHA-induced IFN- production. This
inhibition was not seen in anti-CD3/IL-2-treated cells, suggesting
that the inhibition might vary under certain T cell activation
conditions. However, these data suggest that NK and T cells might
express a functional IL-13R, and IL-13 and IL-4 may have different
roles in regulating IFN- production in these cell types.
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The NK and T cells from the above-mentioned cultures were utilized
for cytotoxicity assays. IL-13 itself induced low NK cytolytic
activity, while IL-4 had no effect (Table I
, donors 1 and 2). In
addition, IL-13 had a lower (less than 50%) inhibitory effect on the
IL-2-induced NK cytolytic activity (Table I, donors 1 and 2), which was
different from the previous report that demonstrated that IL-13 could
increase the IL-2-induced LAK activity (4). This difference is probably
due to donor-to-donor variations or possibly different sources of
IL-13. In contrast, as previously reported (53), IL-4 strongly
suppressed IL-2-mediated LAK potential (>90% reduction). These data
suggest that both IL-13 and IL-4 have an inhibitory effect on
IL-2-induced NK cytolytic activity, but IL-4 is a more potent
inhibitor, which is consistent with previous reports that IL-13 and
IL-4 have different roles in the regulation of LAK activity (4, 5).
In T cells, IL-13 or IL-4 alone did not induce T cell cytolytic
activity (Table II). If T cells were stimulated with anti-CD3
mAb/IL-2 for 48 h, a low level of T cell cytolytic activity was
detected. However, when T cells were costimulated with anti-CD3
mAb/IL-2 and either IL-13 or IL-4, both IL-4 and IL-13 increased the
cytolytic potential of T cells although IL-4 is much more potent (Table II). These results are consistent with the previous report that IL-4
augmented LAK activity with other T cell mitogens in the absence or
presence of IL-2 (54). In contrast, in NK cells, IL-4 and IL-13
inhibited IL-2-mediated LAK activity (Table I). These data suggest that
NK and T cells are responsive to IL-13 stimulation and that IL-13, like
IL-4, can differentially regulate cytolytic activity in T and NK
cells.
IL-13 inhibits IL-2-induced NK cell proliferation, but has no significant proliferative activity in primary T and NK cells
Previous reports have demonstrated that IL-4 can inhibit
IL-2-dependent proliferation of human NK and T cells (53, 55), but the
effect of IL-13 on NK cell proliferation has not been determined.
Freshly isolated NK and T cells were cultured for 60 h in the
absence or presence of 10 and 100 U/ml of IL-2, 10 and 100 ng/ml of
IL-13, or 10 and 100/ml of IL-4 alone or in combinations of IL-2 with
IL-13 or IL-4 as indicated in Figure 1.
In NK cells, the results of [3H]thymidine
incorporation experiments showed that 10 or 100 U/ml of IL-2
significantly induced NK cell proliferation while 10 or 100 ng/ml of
IL-13 or IL-4 did not (Fig. 1A), suggesting that
IL-13, like IL-4, might not be a NK cell mitogen. However, when IL-13
was combined with IL-2, IL-13 inhibited IL-2-induced NK cell
proliferation (Fig. 1A), similar to that observed
with IL-4 (53). These data demonstrate that the inhibitory effect of
IL-13 on NK cell proliferation is similar to that of IL-4.
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B). In contrast, IL-4 itself or
in combination with IL-2, had a stimulatory (or additive) effect on
primary T cell proliferation, consistent with the previous report that
IL-4 stimulates T cell growth by an IL-2-independent mechanism (56).
The inhibitory effects of IL-4 on IL-2-dependent proliferation of T
cells occurred when T cells also were costimulated with anti-CD3 Ab
(57). In addition, IL-13 or IL-4 alone did not induce the expression of
CD25 and HLA-Dr molecules on the cell surface, while the stimulation of
T cells with anti-CD3 mAb/IL-2 or PHA significantly induced CD25
and HLA-Dr expression (Table II). IL-13, but not IL-4, when used in
combination with anti-CD3 mAb/IL-2, increased the number of T cells
expressing CD25 (from 35% to 50%) and HLA-Dr (from 14% to 24%). In
addition, PHA is more effective than anti-CD3-Ab/IL-2 in the
induction of CD25 and HLA-Dr expression in T cells, indicating that the
PHA-stimulated T cells potentially express more high affinity IL-2R
than anti-CD3 Ab/IL-2 stimulated ones, suggesting that IL-13 might
expand the pool of IL-2 responding cells (Table II).
IL-13, like IL-4, induces distinct C DNA-protein binding
complexes in fresh primary human NK and T cells
Transcription factor STAT6 is one of the key elements in the
transduction of IL-4-mediated signaling (41, 42, 43). To determine whether
NK and T cells were responsive to IL-13 stimulation resulting in STAT6
activation, nuclear extracts were prepared from fresh primary T and NK
cells stimulated with IL-13, IL-4 (positive control), IL-6, and IL-12
(negative control) for 15 min at 37°C. Using a C STAT6 DNA-binding
element as an oligonucleotide probe (42, 50), EMSA analysis revealed
that IL-13 and IL-4 induced two similar C DNA-protein complexes in
fresh primary NK cells, which were designated as complexes B and C,
according to their electrophoretic mobility (Fig. 2A, lanes 3and 4). We also observed a slow migrating band
(designated as complex A) induced by IL-13 and IL-4 in fresh primary T
cells (lanes 8 and 9) that was not
seen in NK cells. In addition, the complex C appeared constitutively at
low levels in primary T cells in some donors (Fig. 2A, and
data not shown). In contrast, IL-6 and IL-12 did not induce any
specific DNA protein complex with this oligonucleotide probe
(lanes 2, 5, 7, and 10). These
results revealed several interesting findings. First, the ability of
IL-13 to induce C GAS DNA-protein complexes further suggests that
primary human NK and T cells express functional IL-13R. Second, IL-13
and IL-4 induce similar binding complexes in NK or T cells. Third,
distinct DNA-protein binding complexes are induced by IL-13 and IL-4,
when comparing both cell types.
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, STAT3, and STAT4 (induced by IL-6 and IL-12, respectively)
(49, 52, 59, 60). IL-13 and IL-4 did not induce DNA-protein complex
formation with its oligonucleotide probe (Fig. 2B,
lanes 3, 4, 7, and 8). In contrast, IL-6
induced three SIE GAS DNA-protein-binding complexes with similar
mobility (lanes 2 and 7) when
comparing NK and T cells, while IL-12 induces two unique bands upon
prolonged exposure (data not shown). These results indicate that
different GAS-like element DNA probes (ie, C or SIE) can selectively
bind activated STAT proteins as evidenced by the unique binding
patterns. Moreover, these data also suggest that IL-13 and IL-4 might
not significantly induce the activation of STAT1 and STAT3 proteins
in primary NK and T cells.
Dose and time-dependence of IL-13-induced C DNA-protein-binding
complexes
To further characterize IL-13 activation of STAT6 in NK and T
cells, time-course and dose-dependence assays were performed. Cells
were stimulated with 1, 10, 100, 500, or 1,000 ng/ml of IL-13 for 15
min or with 100 ng/ml of IL-13 for 1, 5, 15, 30, and 90 min for EMSA
assay, or with 100 ng/ml of IL-4 as a positive control (Fig. 3). IL-13 (10 ng/ml) very weakly induced
C DNA-protein-binding complexes while 100 (8 nM), 500, and 1000
ng/ml of IL-13 induced two C DNA-protein-binding complexes
(designated as complexes A and B) (Fig. 3, lanes 4, 5,and 6). Peak induction of C
DNA-protein-binding complexes was found to occur between 15 and 30 min
(lanes 10 and 11). These results
are similar to those observed with IL-4 induction of IL-4NAF-binding
complexes (35). Lastly, 100 ng/ml of IL-4 induced the same complexes
that were induced by IL-13 (Fig. 3, lane
7).
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DNA-protein complexes contain STAT6
protein(s)
To identify the protein components in IL-13- and IL-4-induced C
DNA-binding complexes, supershift analysis with STAT6-specific Abs was
performed (Fig. 4, A and
B). Three anti-STAT6 antisera were tested, which
recognize distinct STAT6 sites (as described in Materials and
Methods). In the IL-13-treated nuclear extracts, Ab
prepared against amino acids 827–847 (designated STAT6h1) effectively
blocked and supershifted both complexes A and B (Fig. 4A,
lane 3). The other Ab recognizing amino acids
787–804 (designated STATh2) blocked and supershifted only complex A
(Fig. 4A, lane 5). In contrast, antisera
against STAT3 and STAT6m (lanes 2 and
4) had no effect on these DNA-protein complexes.
These results indicated that complexes A and B contain STAT6
protein(s). Whether Ab prepared against amino acids 787–804 is less
efficient or incapable of recognizing possible STAT6 isoforms or other
associated protein involvement is unknown. Similar supershift results
also were obtained in nuclear extracts prepared from IL-4-treated T
cells (Fig. 4B). Complexes A and B induced by IL-4
contained STAT6 proteins, but not STAT3 and STAT4 (Fig. 4B,
lanes 2, 3, and 5), or STAT5a and
STAT5b (data not shown). Western STAT6 supershift results were obtained
with NK cells (data not shown). These data demonstrated that IL-13 is
able to directly activate STAT6 in fresh primary T and NK cells,
analogous to IL-4.
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DNA-protein-binding
complexes in preactivated primary T cells vs fresh primary T cells
The activation of primary T cells for several days by various
stimuli, such as IL-2, anti-CD3 Ab, or PHA, leads to morphologic
and functional cell changes. To determine whether preactivated T cells,
stimulated with PHA/IL-2 for 3 days (described in Materials and
Methods), are distinct from the freshly isolated primary T
cells stimulated with IL-4 or IL-13, STAT6 activation was measured by
EMSA, and supershift analysis was performed (Fig. 5). In both primary (lane
1–3) and preactivated T cells (lanes
4–12) obtained from the same donor, IL-4 and IL-13 induced
complex A and B in primary T cells (Fig. 5, lanes 2 and
3) (see also Figs. 2 and 3) while significantly
inducing three C DNA-protein-binding complexes (designated as A, B,
and C) in the preactivated T cells (Fig. 5, lanes 5 and
6). Similar results were observed in three other
donors (data not shown). IL-2 did not induce any DNA-protein complex in
this donor’s cells, but a single band, more slowly migrating than the
bands induced by IL-13 or IL-4, was seen with cells obtained from other
donors (data not shown).
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, lane
7) while only complex A was blocked by anti-STAT6h2
antiserum for IL-13- and IL-4-treated cells (data not shown). This
result is consistent with results described in Figure 4. However,
complex C was neither blocked nor supershifted by anti-STAT1,
-STAT3, -STAT4, or -STAT5a/b (Fig. 5, lanes 8, 9, and data
not shown). To elucidate the identity of complex C, specific and
nonspecific cold oligo competition assays within EMSA were performed.
Complex C, as well as complexes A and B, was completely competed with 1
ng of specific cold C oligonucleotides but not with 1 ng SIE or
mutated oligos, suggesting that complex C is a specific one. Therefore,
complex C may be specific for preactivated primary T cells. These data
suggest that IL-13 and IL-4 might induce different patterns of STAT6
activation when comparing preactivated and fresh primary T cells and
further suggest that STAT6 might be differentially utilized in various
T cell types. IL-13 induces activation of STAT6 via tyrosine phosphorylation of STAT6 and JAK3
To determine whether tyrosine phosphorylation of STAT6 occurs
following IL-13-treatment in NK and T cells, we analyzed lysates of
stimulated cells with anti-STAT6 antiserum via immunoprecipitation
(Fig. 6). Anti-phosphotyrosine Western
blotting revealed that STAT6 was phosphorylated following IL-13
stimulation in NK cells (lane 3) and T cells
(lane 6) in a manner analogous to IL-4-treated
cells (lanes 2 and 5). This blot
was then stripped and reprobed with anti-STAT6 antiserum to verify
equal amounts of loaded protein (Fig. 6B). A small
fragment migrating with an apparent molecular mass of 100 kDa also was
recognized by the anti-STAT6 antiserum although it was not detected
with anti-phosphotyrosine Ab (Fig. 6B). Whether
this protein represents an isoform of STAT6 or a degraded product is
currently being investigated. In any case, these results demonstrate
that IL-13, as well as IL-4, induce STAT6 tyrosine phosphorylation in
both cell types.
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). However, in comparison with JAK3,
IL-13 very weakly induced the tyrosine phosphorylation of JAK1 (data
not shown). However, these results suggest that IL-13 induced the
activation of STAT6, due to the activation of JAK3.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DNA-protein-binding
complexes when comparing primary NK cells and T cells. These results
suggest that STAT6 may be differentially utilized in a cell-dependent
fashion and that IL-13 may play an important role in the regulation of
NK and T cells.
We have observed that IL-13, but not IL-4, weakly induced IFN-
production in NK cells and either synergized with IL-2 or weakly
inhibited (with 30–50% reduction) IL-2-mediated IFN- production.
This result likely reflects the variability in NK cells seen from donor
to donor. However, in both situations, the IL-13 effect was in marked
contrast to the strong inhibition (with more than 90% reduction) seen
with IL-4 in NK cells (Table I). The different effects of IL-13 and
IL-4 on IFN- production in NK cells also were confirmed by analyzing
the effects of IL-4 and IL-13 pretreatment for the first 24 h on
IL-2-mediated IFN- production (data not shown). These results
demonstrate that IL-13 and IL-4 might have a different role in
regulating IFN- production in NK cells. However, in T cells, IL-13
and IL-4 similarly inhibit PHA-induced IFN- secretion. This is in
contrast to the effect of IL-4 on NK cells where it acts as a strong
inhibitor of IFN- production. Furthermore, in NK cells, IL-13 weakly
and IL-4 strongly inhibit the IL-2-induced cytolytic activities
whereas, in T cells, both IL-13 and IL-4 enhance CD3/IL-2-mediated LAK
potential. These data also suggest that T cells express functional
IL-13R and IL-13 might share similar biologic effects with IL-4 in NK
and T cells. A previous report has demonstrated that IL-13 was able to
increase IL-2-mediated LAK activity, while our data demonstrated IL-13
weakly inhibits this activity. The discrepancy in these findings may be
due to donor differences or the different sources of IL-13 used in
these studies. However, it is consistent with a previous report that
IL-13 and IL-4 have a different role in the regulation of LAK activity.
Collectively, this evidence further elucidates that NK and T cells are
functionally regulated by IL-13 and that its effects on NK and T cells
are unique and overlapping compared with IL-4.
Previous studies have suggested that the functional IL-13R are not
expressed on T cells (5, 13, 14, 15), LAK (15), and NK cells (14). However,
IL-13, like IL-4, has been demonstrated to induce IL-4 responsive
element DNA-protein complexes in primary human T cells (16) or
FcRI-binding DNA-protein complexes in fresh human PBL (17). Thus, a
potential question arises as to whether or not the cells utilized in
the experiments were contaminated by monocytes or B cells. However, in
the data presented in this manuscript, the primary NK and T cells did
not contain a significant level of contaminating monocytes and B cells,
based on fluorescent staining for phenotype determination. In addition,
we have observed different patterns of STAT6 activation between NK and
T cells, which also could not be explained by the possibility of
monocyte and B cell contamination in these cell preparations. Moreover,
IL-13 is able to modulate the NK and T cell biologic activities as
described above. Thus, our data provide evidence that IL-13 may
regulate primary T cells and thereby substitute for IL-4, perhaps
explaining why impairment in Th2 development seen in
STAT6-/- mice is more pronounced than observed in
IL-4-deficient mice (43). Therefore, in this manuscript, we have
provided important observations that offer insight into the biology of
IL-13 and on the expression and function of IL-13R on human T and NK
cells.
Data from Table I would suggest that IL-13 is not a significant inducer
of IFN- production or LAK activity for NK cells, but perhaps acts as
a co-regulatory agent when combined with IL-2. Indeed, many factors
regarding NK cell responsiveness to IL-2, such as levels and types of
IL-2R expression, may all determine differential biologic outcomes
following IL-13 stimulation. In the same donor, what appears to be
clear is that IL-13 or IL-4 can have different or opposing effects on
IL-2-induced IFN- production. Whether these effects are due to
differences in unique signal transduction pathways rather than being
dependent on the levels of IL-2R expression alone is not readily
apparent and requires future study.
It is known that the primary NK and T cells express different IL-2R
complexes (61). Therefore, these differences may determine the type of
IL-13 effect seen in NK cells. In addition, the receptor components for
IL-13R complex and its state of expression on NK and T cells is not
clear. On the other hand, IL-13 and IL-4 have overlapping and distinct
biologic effects on both NK and T cells. Here, we have demonstrated
that IL-13 and IL-4 are similarly able to induce distinct C GAS
DNA-protein-binding complexes between NK and T cells, but they only
partially share similar biologic effects on IL-2-induced proliferation
and LAK activity, but not IFN- production in NK cells. These data
suggest that the differential effects of IL-13 on NK and T cells might
result from cell-dependent signaling pathways, differentiation status,
and unique cytokine-signaling pathways.
While the signaling pathway responsible for mediating IL-13 responses
is not well established in NK and T cells, the IL-4R probably serves
as the signaling transducing subunit responsible for activation of
STAT6, 4PS, and JAK1, as previously demonstrated with genetic and
molecular approaches (21, 37, 38, 39, 62). In NK and T cells, IL-13 induces
the activation of STAT6, probably via IL-4R, that is also consistent
with the models where IL-4R is able to associate with the newly
discovered IL-13R (28, 29, 30). However, it would be interesting to
determine whether or not these IL-13R(s) are differentially
distributed between NK and T cells in the future study. A role for
IL-2R (or c) in IL-13 signaling also has been suggested based
upon IL-13-inducible tyrosine phosphorylation of JAK3 (21) and its
association with IL-4R (63). In this study, we have demonstrated
that IL-13 induced the tyrosine phosphorylation of JAK3 in primary
human T cells, although less effectively than IL-4 (Fig. 7), suggesting
that common might be one of the IL-13R components in T cells,
probably in NK cells. In contrast, we did not observe a detectable
level of JAK1 tyrosine phosphorylation although all proteins are
expressed in equivalent amounts (data not shown). These findings are in
contrast to a previous report that showed IL-13 induced JAK1 tyrosine
phosphorylation, but not JAK3, in human B cells (62). Thus, distinct
patterns of IL-13 and IL-4 signaling may be observed within different
cell types although they may share only the common signaling chain,
IL-4R. In support of this hypothesis, we demonstrate that IL-13, as
well as IL-4, distinctively induces cell-specific patterns of C GAS
DNA-protein-binding complexes between NK and T cells.
Previous studies have reported the existence of alternatively spliced
forms of STAT proteins such as STAT1 and STAT18 (64), STAT3 (65) and
STAT5 (66). Since our results derived from anti-STAT6 supershift
analysis suggest unique STAT6 DNA-protein complexes, we hypothesize
that these distinct complexes may represent either homodimeric and
homotetrimeric forms of STAT6 or different spliced isoforms of STAT6
protein(s). This hypothesis is consistent with the previous observation
that the multiple of STAT6 mRNA species were found in spleen, thymus,
and peripheral blood lymphocytes (36, 67). Thereby, the activation of
distinct splice variants of STAT6 also may explain a putative mechanism
by which IL-13 and IL-4 can differentially signal in various type
cells, such as NK and T cells in our study, with different biologic
effects. However, it is unclear why IL-13 and IL-4 induce similar STAT6
DNA-binding complexes in NK cells, but have distinctive effects on
IFN- production. Nevertheless, our data suggest that other signal
transduction pathway(s) induced by IL-13 or IL-4 also might be involved
in regulating IFN- production in this cell type.
In summary, we have demonstrated that IL-13, like IL-4, can directly
and differentially induce STAT6 activation in freshly isolated primary
human T and NK cells, providing new evidence that these cytokines may
exert cell-dependent effects on primary lymphocyte populations. In
addition, we have established that IL-13 has a unique biologic effect
on the modulation of IFN- secretion, cytolytic potential, and
proliferation of primary human NK and T cells. Therefore, IL-13 may
play an important role in the regulation of NK and T cell biologic
activities.
|
Acknowledgments |
|---|
, STAT3, STAT5a, and STAT5b, and for
helpful discussions, and Dr. William J. LaRochelle (Laboratory of
Cellular and Molecular Biology, NCI, Bethesda, MD 20892) for antiserum
to STAT6. We thank Dr.Daniel McVicar for critical review of this
manuscript. We also are grateful to Joyce Vincent and Susan Charbonneau
for editorial assistance and manuscript preparation. |
Footnotes |
|---|
2 Abbreviations used in this paper: LAK, lymphokine activated killer; GAS, IFN- activated sequence; JAK, Janus kinase; EMSA, electrophoretic mobility shift assays; PHA, phytohemagglutinin; PE, phycoerythrin; SIE, sis-inducible element.
Received for publication September 9, 1997. Accepted for publication March 3, 1998.
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