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Department of Adult Oncology, Dana-Farber Cancer Institute, Department of Medicine, Harvard Medical School, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. This activation of STAT4 in
response to IL-2 is not due to the autocrine production of IL-12 or
IFN-. STAT4 activated in response to IL-2 is able to bind to a
STAT-binding DNA sequence, suggesting that in NK cells IL-2 is capable
of activating target genes through phosphorylation of STAT4. IL-2
induces the activation of Jak2 uniquely in NK cells, which may underlie
the ability of IL-2 to activate STAT4 only in these cells. Although the
activation of STAT4 in response to IL-2 occurs in primary resting and
activated NK cells, it does not occur in primary resting T cells or
mitogen-activated T cells. The unique activation of the STAT4-signaling
pathway in NK cells may underlie the distinct functional effect of IL-2
on this cell population. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-2 mediates its effects through interaction with a multichain cell surface receptor and activation of the Janus kinase (Jak)3/STAT pathway (11). Upon binding to its receptor, IL-2 induces tyrosine phosphorylation of two members of the Jak family, Jak1 and Jak3 (12, 13). In T cells, NK cell lines, and activated NK cells, the phosphorylation of Jak1 and Jak3 leads to the recruitment and activation of STAT1, STAT3, and STAT5, which then translocate to the nucleus and activate target genes (14, 15, 16). Since these signaling events are identical in NK cells and T cells, it is likely that these pathways mediate effects of IL-2 that are common to both cell populations, such as proliferation. That IL-2 enhances cytotoxicity in NK cells, but not in T cells suggests that an alternative IL-2 signaling pathway may exist that is unique to NK cells.
To explore this hypothesis, we investigated alternative pathways of
IL-2 signaling in primary NK cells and in two NK cell lines. In
addition to activating STAT1 and STAT5, we found that IL-2 activates
STAT4, a STAT that was previously reported to be activated only in
response to IL-12 and IFN-. Although IL-2 can activate STAT1 and
STAT5 in T cells, it does not activate STAT4 in these cells. IL-2 also
leads to the activation of Jak2 only in NK cells. Thus, the selective
activation of Jak2 and STAT4 in response to IL-2 in NK cells may
explain the enhancement of cytolytic activity of these cells by IL-2.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human rIL-2 (specific activity, 6 x 106 U/ml)
was provided by Amgen (Thousand Oaks, CA). Human rIL-12 (specific
activity, 1.7 x 107 U/mg) was kindly provided by
Genetics Institute (Cambridge, MA). Human rIFN- was obtained from
Hoffman-La Roche (Nutley, NJ). Purified IL-4 (specific activity, 1
x 107 U/mg) was purchased from Genzyme (Cambridge, MA).
Purified unconjugated and phycoerythrin (PE)-conjugated murine mAbs,
including B1 (CD20, IgG2a), My4 (CD14, IgG2b), NKH1 (CD56, IgG1), T1
(CD5, IgG2a), T3 (CD3, IgG1), and MsIgG1, were obtained from Coulter
Immunology (Miami, FL). Anti-phosphotyrosine Ab PY99, and STAT4, Tyk2,
Jak1, Jak2, and Jak3 Abs were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). The generation of Abs to tyrosine-phosphorylated
STAT1 (14) (which also cross-reacts with tyrosine-phosphorylated STAT5
(17, 18)), and the IL-12 receptor ß1 subunit, 12Rß44 (19), were
described previously. Anti-IFN- neutralizing Ab was generously
provided by Endogen (Woburn, MA).
Reagents
PMA and FCS were obtained from Sigma (St. Louis, MO). Ionomycin
was purchased from Calbiochem (La Jolla, CA).
[
-32P]ATP was purchased from New England Nuclear Life
Science Products (Boston, MA). Lymphocult was obtained from Biotest
Diagnostics (Denville, NJ).
Purification and culture of NK cells and T cells
PBMCs were isolated from heparinized blood obtained from normal donors by Ficoll-Hypaque (Pharmacia LKB, Piscataway, NJ) gradient centrifugation. Primary NK cells (>96% purity) were obtained by first depleting monocytes, B cells, and T cells from PBMCs with B1 (CD20, IgG2a), My4 (CD14, IgG2b), T1 (CD5, IgG2a), and T3 (CD3, IgG1) Abs and immunomagnetic anti-IgG beads (Advanced Magnetics, Cambridge, MA). The remaining cells were incubated with PE-conjugated CD56 and sorted on an EPICS Elite ESP flow cytometer (Coulter). Activated NK cells were obtained by culturing primary NK cells for 4 days in RPMI 1640 medium containing 15% FCS and 10% Lymphocult, with 750 ng/ml ionomycin included for the first 16 h of culture.
Primary T cells (>98% purity) were obtained by incubating PBMCs with PE-conjugated CD5 and sorting by flow cytometry. PHA-activated T cells were prepared by culturing purified primary T cells for 4 days in RPMI 1640 medium containing 15% FCS and 2.5 µg/ml PHA (Murex, Dartford, U.K.). PHA-activated T cells used for immunoprecipitation experiments (>94% purity) were purified by depleting monocytes, B cells, and NK cells from PBMCs with My4 (CD14, IgG2b), B1 (CD20, IgG2a), and NKH1 (CD56, IgG1) Abs and immunomagnetic anti-IgG beads.
NK3.3 cells (20) were cultured in RPMI 1640 medium containing 15% FCS, 10% Lymphocult, and 50 U/ml IL-2. NKL cells (21) were cultured in RPMI 1640 medium containing 15% FCS and 50 U/ml IL-2.
Whole cell extracts and Western blotting
Following stimulation, cells were washed once with PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 (pH 7.4)) and extracted with EBC 250 buffer containing 0.5% Nonidet P-40, 250 mM NaCl, 10 mM Tris (pH 8), 10 mM sodium orthovanadate, 100 µM PMSF, 1 µg/ml pepstatin, and 1 µg/ml aprotinin. Insoluble material was removed by centrifugation at 12,000 x g for 5 min. Protein was separated on a 9% gel by SDS-PAGE and subsequently electrotransferred to nitrocellulose. The membrane was blocked with either 5% BSA (for phosphotyrosine blots) or 5% dry milk in TBST (100 mM Tris-HCl (pH 8), 150 mM NaCl, and 0.05% Tween 20), and probed with primary Ab diluted 1/10,000. After incubation with horseradish peroxidase-conjugated secondary Ab, specific protein was detected by chemiluminescence (ECL; New England Nuclear Life Science Products).
Immunoprecipitation
Whole cell extracts were precleared with rabbit anti-mouse Ig-coupled protein A beads, followed by a 1-h incubation at 4°C with anti-STAT4, anti-Jak2, or anti-Tyk2 Ab and 1 h with rabbit anti-mouse-coupled protein A beads. After extensive washing, beads were boiled in sample buffer containing 2-ME; precipitated protein was resolved by SDS-PAGE.
Nuclear extracts and electrophoretic mobility shift assay (EMSA)
Nuclear extracts were prepared as described previously (22).
Nuclear extract (2 µl) was mixed with 1 ng of 32P-labeled
oligonucleotide in 10 µl of binding buffer (25 mM HEPES (pH 7.9), 100
µM EDTA, 200 µM MgCl2, 500 µM DTT, 1 mg/ml BSA, 0.4
mg/ml double-stranded poly(dI:dC), 30% glycerol, and 0.1 mg/ml salmon
sperm DNA) and incubated at room temperature for 15 min. The
STAT-binding oligonucleotide used,
5'-GAGCCTGATTTCCCCGAAATGATGAGC-3' and its complement, is
derived from the IFN--responsive factor 1 gene promoter (23). After
incubation at room temperature, 1 µl of anti-STAT4 Ab was added
where indicated for supershift analysis, and samples were incubated on
ice for an additional 15 min. The protein-DNA complexes were separated
on a 4% acrylamide gel in 0.5x TBE (45 mM Tris-borate, 1 mM EDTA) and
visualized by autoradiography.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To explore alternative pathways for IL-2 signaling in NK cells, we
examined the activation of different STATs in response to IL-2 in two
human NK cell lines, NKL and NK3.3. Both of these cell lines are IL-2
responsive and require IL-2 to maintain growth in vitro. While IL-2
treatment induced STAT1 and STAT5 activation, as reported previously
(14, 16) (data not shown), it also induced forms of STAT4 with slow
migration in both NK cell lines (Fig. 1
,
A and B, lane 2). IFN-, which was
shown to activate tyrosine phosphorylation of STAT4 in NK3.3 cells
(24), also produced slow migration STAT4 bands in both NK cell lines
(Fig. 1A, lane 4, and 1B, lane
5). IL-12, which is known to induce tyrosine phosphorylation of
STAT4 in NK3.3 cells, also led to the formation of slow migration forms
of STAT4 in these cells (Fig. 1B, lane 4). NKL
cells lack expression of the ß2 subunit of the IL-12 receptor, as
assessed by reverse-transcriptase PCR, and do not respond to IL-12
(data not shown). No slow migration forms of STAT4 were observed in
untreated cells (Fig. 1, A and B, lane
1) or in IL-4-treated cells (Fig. 1, A and
B, lane 3).
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A, lane 1), IL-2,
IL-12, and IFN- all induced tyrosine phosphorylation of STAT4. The
degree of STAT4 tyrosine phosphorylation induced by IL-2 was greater
than that of IL-12 and was comparable with that induced by IFN-.
Reprobing with an Ab to STAT4 revealed that tyrosine-phosphorylated
STAT4 (Fig. 2A) induced by IL-2, IL-12, or IFN-
migrated as the band with slowest mobility (Fig. 2B).
The intermediate bands most likely represent STAT4 phosphorylated on
serine residues ((24) and data not shown).
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STAT4 has been reported to be activated only in response to IL-12
in NK cells and T cells (24, 25, 26), and IFN- in NK3.3 cells (24).
Although IL-2 treatment of NK cells is not known to lead to production
of IL-12 or IFN-, we considered the possibility that autocrine
production of one of these factors might lead to STAT4 phosphorylation.
NK3.3 cells were treated with cytokines in the presence or absence of
Abs that block IL-12 or IFN- signaling. The presence of an
IFN--neutralizing Ab, which abolished the activation of STAT4 in
response to exogenous IFN- (1000 U/ml) (Fig. 3A, lane 5), did
not affect the activation of STAT4 in response to IL-2 (Fig. 3A, lane 3). Similarly, STAT4 activation in
response to IL-2 was not affected by the presence of an Ab against the
ß1 subunit of the IL-12R (12Rß44), which could block the ability of
IL-12 to activate STAT4 phosphorylation (Fig. 3B). These
results indicate that the STAT4 activation in response to IL-2 is not
due to exogenous stimulation with IL-12 or IFN-.
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Although the NK cell lines NKL and NK3.3 exhibit many properties
of NK cells, through adaptation to in vitro growth, they may display
differences from primary NK cells. To test whether IL-2 activates STAT4
in primary human NK cells, NK cells were purified from PBMCs of healthy
donors by FACS. NK cells were activated by ionomycin for 16 h, and
IL-2 plus Lymphocult for 3 days, then cells were rested overnight and
treated with specific cytokines. Whole cell extracts were prepared and
Western blotting was performed. As indicated by the appearance of the
slow migrating STAT4 band, IL-2 could activate STAT4 in these activated
NK cells (Fig. 4A, lane
2). To investigate whether IL-2 could also activate STAT4 in
activated T cells, T cells were isolated from PBMCs of healthy donors
and activated for 4 days with PHA, then washed and rested overnight. In
these activated T cells, IL-12 and IFN- induced slow migration forms
of STAT4 (Fig. 4B, lanes 4 and 5),
consistent with tyrosine phosphorylation of this protein. By contrast,
IL-2 did not induce any change in the mobility of STAT4 in these cells
(Fig. 4B, lane 2). That these activated T cells
could respond to IL-2 was indicated by the tyrosine phosphorylation of
STAT1 and STAT5 induced by this cytokine (Fig. 4C,
lane 2). When extracts from cytokine-treated activated T
cells were analyzed by immunoprecipitation with Abs to STAT4 and probed
with PY99, it was confirmed that IL-12 or IFN-, but not IL-2,
induced tyrosine phosphorylation of STAT4 (Fig. 5). These results suggest that IL-2
activates STAT4 in activated NK cells, but not in activated T cells. To
test whether similar signaling occurs in resting primary NK cells and T
cells, IL-2-induced phosphorylation of STAT4 was compared in NK cells
and T cells that were purified from the same healthy donor. In cells
from four individuals, IL-2 induced STAT4 activation in primary NK
cells (Fig. 6A, upper
panel, lane 2), but not in the primary T cells from the
same donor (Fig. 6B, upper panel, lane
2). Reprobing the membrane with anti-phosphotyrosine-STAT1 Ab
showed that IL-2 activates STAT1 in both primary NK cells and primary T
cells (Fig. 6, A and B, lower panels,
lane 2), indicating that these cells can respond to this
cytokine. Activation of STAT5 in response to IL-2 also occurred in
primary T cells (Fig. 6B, lower panel, lane
2). These results suggest that the lack of STAT4 activation in
response to IL-2 in primary resting T cells or in activated T cells is
not due to an absence of IL-2 responsiveness in these cells. From these
data, we conclude that, as in NK cell lines, IL-2 activates STAT4 in
primary resting and activated NK cells. Moreover, the activation of
STAT4 by IL-2 is restricted to NK cells, and is not found in either
primary or activated T cells.
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The number of primary NK cells required for immunoprecipitation
exceeds that which can be obtained by cell sorting. Thus, to confirm
the activation of STAT4 in primary NK cells, DNA-binding assays were
performed. Once activated by tyrosine phosphorylation, STATs dimerize,
translocate to the nucleus, bind to specific DNA sequences in promoter
regions, and activate the transcription of their target genes.
Therefore, if STAT4 phosphorylated in response to IL-2 is functionally
active, it must translocate to the nucleus and bind to cognate DNA
sequences. To investigate whether STAT4 activated by IL-2 in NK cells
is in the nucleus and capable of binding DNA, nuclear extracts were
prepared from untreated or cytokine-treated activated NK cells, and
EMSA was performed. The oligonucleotide used in the EMSA is derived
from the IFN--responsive factor-1 gene promoter, and can bind
multiple STATs, including STAT1, STAT3, STAT4, and STAT5 (14, 23, 27).
As indicated in Fig. 7A, IL-2
induced three protein-DNA complexes, A, B, and C. By contrast, IL-12
induced only complex B, and IFN- induced complexes A and C. It has
been shown previously that in NK cells, IL-12 activates STAT4, and
IFN- activates STAT4 and STAT1. Given our finding that IL-2
activates STAT1, STAT5, and STAT4, the common complex induced by all
three cytokines, complex A, would be most likely to include STAT4.
Indeed when Ab to STAT4 was introduced into the binding mixture,
complex A disappeared. Instead, a supershifted complex, complex S,
appeared in all three cytokine conditions. Complex C, which was induced
by IL-2 and IFN-, has been found to contain STAT1 (data not shown).
Complex B, which was formed only in response to IL-2, most likely
contains STAT5. These data indicate that STAT4 activated in response to
IL-2, like STAT4 activated by other cytokines, is capable of
translocating to the nucleus and binding to target DNA sequences. To
verify further that IL-2 does not induce STAT4 activation in T cells,
EMSA was performed with nuclear extracts from activated T cells that
were untreated or treated with cytokines. While IL-12 and IFN-
induced complexes in activated T cells similar to those in activated NK
cells, IL-2 did not induce formation of complex A, which contains
activated STAT4 (Fig. 7B). This indicates that in contrast
to activated NK cells, IL-2 does not induce DNA-binding activity of
STAT4 in activated T cells.
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Upon binding to their receptors, cytokines induce phosphorylation
and activation of members of the Jak family, which leads to the
recruitment and phosphorylation of STATs. It has been shown that Jak2
and/or Tyk2 are involved in the IL-12 and IFN- signaling events that
activate STAT4, in contrast to the kinases reported to be activated by
IL-2, Jak1, and Jak3. To investigate whether Jak2 or Tyk2 is involved
in the activation of STAT4 in response to IL-2 in NK cells, Jak2 and
Tyk2 were immunoprecipitated from NKL, NK3.3, and activated T cells
that had been untreated or treated with cytokines. The phosphorylation
of Jak2 and Tyk2 was assessed by performing a Western blot with
anti-phosphotyrosine Ab PY99. IL-2 did not induce an increase in
the basal phosphorylation of Tyk2 in any of these cells (Fig. 8A, upper panel).
In contrast, IFN- led to the activation of Tyk2 in NKL and NK3.3
cells, and both IL-12 and IFN- caused activation of Tyk2 in
activated T cells. However, IL-2 did induce phosphorylation of Jak2 in
both NK cell lines, but not in activated T cells (Fig. 8B,
upper panel). These data suggest that Jak2, but not Tyk2,
may be involved in the activation of STAT4 in response to IL-2 in NK
cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This finding is significant in that it suggests that one mechanism by which a single cytokine can induce unique effects in different cell types is to activate distinct signaling pathways in each. Cell-restricted signaling activation would allow a relatively small group of cytokines to exert distinct effects on various cell types. Cells of clearly disparate lineages may have other mechanisms for modulating the subset of genes activated by a given cytokine. For example, gene silencing through DNA methylation or other mechanisms may restrict the potential target genes activated by a cytokine. In more closely derived cell types, such as T cells and NK cells, such a mechanism may not be practical, as similar genes may ultimately need to be activated at some time in each. Thus, restricting the transcription factors that can be activated in a specific cell type would allow an easily modified mechanism to alter gene activation in response to a given cytokine.
The STAT family of transcription factors appears to play an important role in mediating gene activation by most hemopoietic, and many nonhemopoietic cytokines, growth factors, and neurotrophic factors (28). Some members, such as STAT1, STAT3, and STAT5, are activated in numerous cell types by a wide variety of cytokines. These proteins may be involved in mediating more general types of signals, such as those for cell growth and survival. Others, including STAT4, are more restricted in their expression (29), and are activated in response to a very small number of factors. This may reflect a more specialized role for these STATs. The loss of STAT4 appears to have a major impact on the differentiation of Th subsets (30, 31), indicating that one of these roles most likely involves T cell differentiation. The findings presented in this study would suggest that STAT4 is also important in mediating the effects of IL-2 on NK cells, perhaps in the activation of cytotoxicity. This cell type restriction of STAT activation is an attractive model for increasing the diversity of cellular responses induced by a given cytokine.
To determine the mechanism by which IL-2 induces STAT4 activation only
in NK cells, we examined activation of Jak family members in response
to IL-2 in these cells. While IL-2 is known to induce the activation of
Jak1 and Jak3, we considered whether IL-2 might activate another Jak
family member only in NK cells. Although specificity in STAT signaling
is not thought to be restricted by the kinase that is activated, it is
conceivable that in this system a kinase might influence the choice of
substrate. In fact, we found that IL-2 induced the activation of Jak2
in NK cells, but not in T cells. This unique signaling event in NK
cells might underlie the ability of IL-2 to activate STAT4 only in this
population, although the mechanism by which this occurs is unclear. One
hypothesis is that an additional receptor chain is found associated
with the IL-2R in NK cells, but not in T cells, and this mediates
activation of Jak2 and STAT4. For example, recruitment of one or more
chains of the IL-12R by the IL-2R in NK cells might allow Jak2 to be
activated and STAT4 to become phosphorylated uniquely in NK cells. Such
a common receptor chain would be similar to the ßc chain
shared by the receptors for granulocyte-macrophage CSF, IL-3, and IL-5.
These cytokines can compete for this signal-transducing chain on the
cell surface, and the ßc chain confers signaling
competency to the cognate ligand of the receptor -chain with which
it associates. Such a common receptor chain would allow IL-2 to
replicate at least some of the signaling events induced by IL-12 in NK
cells.
There are other mechanisms for recruitment of Jak2 and/or STAT4 to IL-2R complexes. For example, a novel docking protein whose expression is restricted to NK cells might allow the recruitment of these molecules to the IL-2R in NK cells, but not in T cells. STAT proteins are believed to interact with tyrosine-phosphorylated receptor-kinase complexes via their SH2 domain. Thus, the presence of a protein in NK cells that associates with the IL-2R and can become tyrosine phosphorylated at a site that is recognized by the STAT4 SH2 domain would allow STAT4 to become phosphorylated in response to IL-2 uniquely in NK cells.
While the stimulation of cytotoxicity is a known effect of IL-2 on NK cells, it is possible that IL-2 also affects NK cells in other ways. For example, IL-2 may alter the expression of cell surface molecules or the survival of NK cells. These effects of IL-2 may all be mediated by STAT4 activation, although it is possible that other signaling pathways might also be regulated by IL-2 in NK cells. While the principal substrates for the Jak family of kinases are believed to be STAT transcription factors, it is possible that Jak2 may phosphorylate other proteins in NK cells. Thus, in addition to STAT4, the activation of Jak2 might lead to signaling events through other pathways that might further modulate NK cell function.
In conclusion, the restriction of STAT4 activation to NK cells sheds light on the differential response of NK cells and T cells to IL-2. Importantly, this finding provides a potential mechanism by which a single cytokine can have overlapping but distinct effects on two critical effector cells. Furthermore, it suggests that the recruitment and phosphorylation of individual STAT proteins following the binding of a cytokine to its receptor are regulated in a cell type-restricted fashion. The finding that Jak2 is activated in response to IL-2 uniquely in NK cells suggests that this specificity is mediated at the level of the receptor-kinase complex. Investigation into how these events are regulated will help define the mechanism by which cytokines exert distinct functional effects in different cell types.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David A. Frank, Department of Adult Oncology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115. E-mail address:
3 Abbreviations used in this paper: Jak, Janus kinase; EMSA, electrophoretic mobility shift assay; PE, phycoerythrin.
Received for publication April 20, 1998. Accepted for publication September 2, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and interleukin-4 regulate T cell interleukin-12 responsiveness through the differential modulation of high-affinity interleukin-12 receptor expression. Eur. J. Immunol. 27:647.[Medline]
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interferon activation site-binding protein expressed in early myeloid differentiation. Mol. Cell. Biol. 14:4342.This article has been cited by other articles:
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