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1
*
Department of Pathobiology, University of Pennsylvania, and
The Wistar Institute, Philadelphia, PA 19104; and
Department of Molecular Biology, DNAX Research Institute, Palo Alto, CA 94304
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. This study compares the abilities of the cytokine IL-18 and
the costimulatory cell surface molecule CD28 to enhance IL-12-driven
IFN- production by NK cells. Studies with other cytokines (IL-1ß,
IL-6, TNF-
, IL-15) showed that IL-18 or anti-CD28 treatments
were the most efficient inducers of IFN- when combined with IL-12.
The ability of IL-18 to enhance IFN- was shown to be dependent on
the presence of IL-12. Similarly, although anti-CD28 stimulation
alone could enhance IFN- synthesis, this effect was significantly
increased in the presence of IL-12. Although neither method of
costimulation required de novo protein synthesis for their effects on
IFN- mRNA expression, these molecules used distinct mechanisms.
Specifically, nuclear run-on analysis revealed that IL-18 in
combination with IL-12 enhanced the rate of transcription of the
IFN- gene. Conversely, treatment with anti-CD28 plus IL-12 did
not significantly up-regulate the rate of transcription of the IFN-
gene, but stabilized IFN- mRNA expression within NK cells. These
findings illustrate costimulatory pathways that result in potent
IFN- responses by NK cells and show that although IL-18 and
anti-CD28 can enhance the synthesis of IL-12-driven IFN-, they
employ molecular mechanisms that are distinct from one
another. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, which subsequently activates macrophage
killing of organisms (4, 5). Furthermore, the production of IFN- by
NK cells during the innate phase of the immune response influences
ensuing Th cell differentiation by promoting development of a Th1
phenotype (6), illustrating the importance of this pathway in both
innate and adaptive immunity. IFN- is secreted by NK cells primarily
in response to IL-12, a heterodimeric cytokine that is secreted by
cells such as macrophages and dendritic cells early during infection
(7). However, recent evidence has demonstrated a role for other
cytokines and costimulatory molecules in enhancing the activation and
function of NK cells. In particular, cytokines such as IL-1ß and
TNF- (8, 9) have been shown to augment both NK cell proliferation
and IFN- production. Similarly, the type I IFNs have been shown to
enhance IL-12-driven IFN- production in vitro (10). Another cytokine
with particular relevance to innate immunity is IL-18 (IFN--inducing
factor) (11), which has been characterized as a potent enhancer of
IL-12-induced IFN- production by Th1 cells (12, 13) and has been
shown recently to enhance NK cell proliferation and IFN- synthesis
(14). Previous studies in our laboratory have shown that IL-18 enhances
IFN- production by IL-12-stimulated murine NK cells more efficiently
than either IL-1 or IL-1ß (15), and a recent study with
IL-18-deficient mice showed that these animals have defective NK cell
activity in terms of decreased cytotoxic killing ability (16). These
findings suggest that IL-18 has an important role in NK cell responses.
However, the molecular mechanisms responsible for the effects of IL-18
on NK cells, particularly its ability to induce IFN- production,
remain poorly characterized.
Similar to IL-18, the effects of costimulation via the CD28 cell
surface molecule on T cell responsiveness have been well described (17, 18); however, the role of this molecule in influencing NK cell
responses remains less well defined. CD28 costimulation has been shown
to induce optimal proliferation of murine NK cells and enhance IFN-
secretion (19), and the CD28 pathway has been shown to be involved in
cytotoxic killing of tumor cells (20, 21). However, although it has
been demonstrated that the natural ligand for CD28 (B7-1) is involved
in triggering of NK cell cytotoxicity, it was also shown in the same
study that this activity was independent of CD28 (22). Furthermore, a
recent study demonstrated that human NK cells could not be induced to
express CD28, and cytotoxic responses could not be elicited by
stimulation with B7-1 transfected tumor cell lines (23). Therefore, the
role of the CD28 costimulatory pathway in influencing the nature of NK
cell responses remains controversial, and although previous studies
have shown that IFN- production by NK cells can be enhanced by
anti-CD28 in the presence of IL-12 (24), the molecular events of
CD28 costimulation in NK cell activation and IFN- synthesis remain
to be determined.
This study was designed to elucidate the molecular basis of CD28 and
IL-18 costimulation in modulation of IL-12-driven IFN- synthesis by
NK cells. To determine the relative importance of IL-18 and CD28 we
compared the effects of these molecules with those of other cytokines
known to be capable in vitro of enhancing IL-12-induced IFN-
synthesis. We show by analyzing IFN- protein synthesis, mRNA
induction, and gene transcription that although IL-18 and CD28 both use
pathways that do not require de novo protein synthesis, they use
distinct molecular mechanisms to enhance the production of IFN- in
the presence of IL-12. These findings demonstrate that molecules such
as IL-18 and CD28 play an important role in the induction of IFN-
synthesis by NK cells and identify the distinct molecular mechanisms
responsible for these effects.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Homogeneous populations of NK cells were prepared by culturing bone marrow cells from 4- to 6-wk-old C57BL/6 recombination-activating gene-1-deficient mice (The Jackson Laboratory, Bar Harbor, ME) in rIL-2 as previously described (25). Briefly, bone marrow cells were plated in petri dishes at an initial density of 1 x 106 cells/ml in a volume of 10 ml of RPMI 1640 (containing 10% FCS, 2 mM L-glutamine, 100 U/ml of penicillin, 10 mg/ml of streptomycin, 1 mM sodium pyruvate, 10 mM HEPES, and 1% nonessential amino acids) with 4000 U of recombinant human IL-2 (Chiron, Emeryville, CA). The IL-2 was replenished every 2 days, and after 7 days NK cells were harvested from dishes by washing with cold RPMI 1640. These NK preparations consisted of cells that were morphologically large granular lymphocytes and were negative for the expression of CD4 or CD8 as determined by FACS analysis. Staining with the murine NK surface marker NK1.1 revealed that >98% of the harvested population expressed this phenotype. These IL-2-activated NK cells also expressed the CD28 marker as previously described (19). In all experiments NK cells were plated at a final density of 1 x 106/ml either in 24-well tissue culture plates (1 ml/well) or, in the case of larger cultures (5–10 ml), in 25-cm2 tissue culture flasks (Corning, New York, NY).
Stimulation of NK cells
In all experiments, unless otherwise indicated, NK cells were
cultured with various murine cytokines at a final concentration of 1
ng/ml. IL-12 was supplied by Genetics Institute (Cambridge, MA). IL-18
was supplied by DNAX (Palo Alto, CA). IL-1ß, TNF-, and IL-6 were
supplied by Genzyme (Cambridge, MA). IL-15 was supplied by Immunex
(Seattle, WA). Anti-CD28 stimulation of NK cells was performed by
precoating the wells of tissue culture plates with anti-CD28 mAb
(PharMingen, San Diego, CA) before addition of cells. Plates were
coated overnight (4°C) with anti-CD28 (hamster IgG, clone 37.51)
diluted to a final concentration of 1.0 µg/ml in PBS, pH 7.2. Before
addition of NK cells, excess Ab was removed from culture plates by
washing with sterile PBS. In IL-2 neutralization experiments, all Abs
were purchased from PharMingen, were low endotoxin screened, and
contained no sodium azide. Anti-human IL-2 (MQ1-17H12), anti-murine
IL-2 (S4B6), or nonspecific rat IgG (R3-34) was added to cultures 30
min before addition of inducers at a final concentration of 10 µg/ml.
In experiments analyzing the rate of mRNA decay (half-life),
actinomycin D (Calbiochem-Behring, La Jolla, CA) was added to cultures
at a final concentration of 5 µg/ml, 4 h after stimulation with
appropriate inducers. In experiments testing the requirements for de
novo protein synthesis, cycloheximide
(CHX;3 Sigma, St. Louis, MO) was
added at a final concentration of 10 µg/ml, 2 h before and
during induction with appropriate stimuli.
Measurement of IFN- synthesis by NK cells
IFN- levels in culture supernatants were measured using a
standard capture cytokine ELISA protocol. Briefly, the wells of
Immunlon microtiter plates (Dynex Technologies, Chantilly, VA) were
coated with detecting Ab (R4-6A2) in PBS, pH 9.0. After addition of
samples, bound cytokine was detected with biotinylated anti-IFN-
(XMG1.2) followed by streptavidin-horseradish peroxidase conjugate (The
Jackson Laboratory). Bound peroxidase was visualized with
2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (Sigma). Unknown
values were extrapolated from a standard curve that was constructed
using recombinant mouse IFN- (Genzyme) of known concentration.
Total RNA was extracted from cultured NK cells by the guanidine
isothiocyanate method and was assayed for cytokine mRNA content using
the Riboquant MultiProbe RNase Protection Assay System (PharMingen).
Briefly, 10 µg of RNA from each sample was hybridized in solution
with the appropriate radiolabeled antisense RNA probe set. mCK-1 (IL-4,
IL-5, IL-10, IL-13, IL-15, IL-9, IL-2, IL-6, IFN-) or mCK-3
(TNF-ß, LTß, TNF-, IL-6, IFN-, IFN-ß, TGFß1, TGFß2) was
employed for detection of cytokine mRNA as recommended by the
manufacturers. Following hybridization, free probe and remaining ssRNA
were digested with RNases, and the protected probes were purified and
resolved on 5% denaturing polyacrylamide gels using Ultra Pure
Sequagel reagents (National Diagnostics, Atlanta, GA). Dried gels were
then exposed to phosphorimaging screens, and protected fragments
visualized using a PhosphorImager 445S1 (Molecular Dynamics, Sunnyvale,
CA).
Nuclear gene transcription analysis in NK cells (run-on assay)
Isolation of nuclei and in vitro transcription in the presence
of [32P]UTP (300 Ci/mmol; DuPont, Boston, MA) were
performed essentially as previously described (26, 27). Nuclear RNA was
then isolated after DNase I and proteinase K (both from Boehringer
Mannheim, Mannheim, Germany) treatment followed by four
phenol/chloroform/isoamyl alcohol extractions and ethanol precipitation
at -70°C for 2 h. Unincorporated [32P]UTP was
removed using Sephadex G-50 columns (Boehringer Mannheim). Nuclear RNA
was partially degraded by treatment with 0.2 N NaOH for 10 min at 4°C
and was hybridized for 2 days at 60°C to prehybridized nylon filters
(Schleicher & Schuell, Keene, NH) on which 500 ng of denatured
PCR-amplified cDNAs corresponding to the coding regions of murine
IFN- and ß-actin genes had been immobilized using a slot-blot
apparatus (Hoeffer Scientific, San Francisco, CA). After hybridization
filters were washed at room temperature with 2x SSC, and ssRNA was
digested with the same solution containing 10 µg/ml RNase A (37°C
for 30 min). Filters were then washed twice in 2 x SSC/0.1% SDS
for 15 min at 50°C and once in 0.1x SSC/0.1% SDS for 30 min at
50°C. The extent of hybridization was quantified using ImageQuant
software on a PhosphorImager 445S1 (Molecular Dynamics).
Statistical analysis
All data are expressed as the mean ± 1 SD. IFN- levels
were compared by Student’s two-tailed t test. Fold
induction was calculated according to the formula: densitometric values
for IFN- bands (normalized against L32/GAPDH for RNase protection
assay (RPA) or ß-actin for run-on)/densitometric values
(unstimulated). Fold induction values were compared by Mann-Whitney
U test. p < 0.05 was considered
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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synthesis by NK
cells
To determine the relative contribution of IL-18 to the production
of IFN- by NK cells we compared the stimulatory ability of this
cytokine with those of other proinflammatory cytokines (IL-1ß, IL-6,
IL-15, TNF-) at the same concentration. NK cells were stimulated
with these cytokines for 4 h, either alone or in combination with
IL-12, and their effects were determined by measuring IFN- protein
synthesis (Fig. 1
A) and mRNA
induction (Fig. 1B). Stimulation of NK cells in the presence
of IL-18, IL-1ß, TNF-, IL-15, or IL-6 (1 ng/ml) alone did not
result in significant production of IFN- protein or IFN- mRNA
synthesis above background levels. However, in the presence of IL-12,
IL-18 was able to significantly up-regulate the synthesis of IFN-
compared with the effect of IL-12 alone. IL-1ß, TNF-, and IL-15
were consistently observed to have small enhancing effects on IFN-
synthesis when combined with IL-12, although at the concentrations
employed (1 ng/ml) these effects were small and not significantly
higher than those found in cultures stimulated with IL-12 alone.
Indeed, although not tested at higher concentrations in this study,
previous studies from our laboratory have demonstrated that cytokines
such as IL-1ß can enhance IL-12-driven IFN- synthesis by
IL-2-activated NK cells but requires higher stimulatory concentrations
(10 ng/ml) to induce levels of IFN- protein synthesis comparable
with those in IL-18-stimulated cultures (15). IL-6 alone or in
combination with IL-12 did not have any significant enhancing
effect on NK cell IFN- production. These results demonstrate that
IL-18 is a potent enhancer of IL-12-driven IFN- synthesis by NK
cells, and in comparison, other proinflammatory cytokines, such as
IL-1ß and TNF-, are inefficient in this process.
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synthesis by NK cells
To further characterize the ability of IL-18 to enhance
IL-12-driven IFN- production we compared IL-18 with stimulation via
CD28, a costimulatory surface molecule known to enhance IFN-
production in T cells. NK cells were cultured in the presence of IL-12
plus IL-18 or in the presence of plate-bound anti-CD28 plus IL-12
for 4 h. Similar to IL-18, anti-CD28 costimulation of NK cells
in the presence of IL-12 resulted in significant enhancement of IFN-
protein production (Fig. 2A)
and mRNA synthesis (Fig. 2B) compared with the effects of
IL-12 alone. Interestingly, in additional experiments (data not shown),
costimulation of NK cells with the combination of IL-12, IL-18, and
anti-CD28 resulted in a further small enhancement of both IFN-
mRNA and protein synthesis (10% increase), suggesting that these
different stimuli together have an additive effect on IL-12-driven
IFN- production. It should be noted that under these various
stimulatory conditions we failed to detect the expression of other
cytokines (including IL-2, IL-4, IL-10, IL-13, IL-15, TNF-, and
IFN-ß) using the multi- probe RPA, with the exception of low
level constitutive expression of TGF-ß2 (data not shown). Cells
cultured with only anti-CD28 produced levels of IFN- comparable
to those in cultures stimulated with IL-12 alone (Fig. 2A).
Also, NK cells stimulated with IL-12 and anti-CD28 produced higher
quantities of IFN- than NK cells stimulated with IL-12 and IL-18,
although this was not statistically significant at the protein level.
It should be noted that the amount of anti-CD28 Ab used to coat the
tissue culture plates was relatively high (1 µg/ml) compared with the
amount of IL-12 or IL-18 present (1 ng/ml), making it difficult to
directly compare their effectiveness. To eliminate any residual effects
of IL-2, cultures were pretreated with a mixture consisting of
neutralizing human IL-2 Ab (to remove any possible contaminating human
IL-2 from stock cultures) and neutralizing murine IL-2 Ab (to inhibit
any possible endogenous IL-2). In the presence of this neutralizing Ab
mixture, IL-18 and anti-CD28 were still able to enhance
IL-12-driven IFN- production, although the total amount of induced
IFN- mRNA and protein in each condition was slightly reduced
compared with that in rat IgG-treated control cultures (not shown).
This illustrates that IL-18 and CD28 can enhance the production of
IL-12-driven IFN- production in an IL-2-independent manner.
Furthermore, even after anti-CD3 stimulation of our cultures, IL-2
mRNA was not detectable by RPA in any experiments, confirming the
purity of our cultures from contaminating T cells.
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production does not
require de novo protein synthesis
To further characterize the nature of the enhancing effects of
IL-18 and CD28 on IFN- synthesis we tested whether these molecules
require de novo protein synthesis for their effects. The requirements
for newly synthesized intracellular proteins on IFN- mRNA expression
were determined by culturing NK cells in the presence of the protein
synthesis inhibitor CHX for 2 h before and during stimulation (4
h) with the appropriate cytokines. The concentration of CHX used in
these experiments was highly effective at blocking cellular protein
synthesis, as no IFN- protein could be detected in the supernatants
from any of the cultures by ELISA (data not shown). It should be noted
that the background levels of IFN- mRNA were enhanced in the
presence of CHX. Control levels of IFN- mRNA in the presence of CHX
were, on the average, 10 times higher than those in control cultures
without CHX. This effect of CHX on IFN- mRNA is consistent with
nonspecific stabilization of mRNA degradation, an attribute previously
assigned to CHX (28). NK cells stimulated with IL-12 plus IL-18 in the
presence of CHX still exhibited enhanced IFN- mRNA expression
compared with that after stimulation by IL-12 alone (Fig. 3). Similarly, anti-CD28
costimulation enhanced IL-12-driven IFN- mRNA expression even in the
presence of CHX, demonstrating that IL-18 or anti-CD28
costimulation does not require de novo protein synthesis for its
enhancing effects on IFN- mRNA expression in NK cells. In summary,
neither IL-18 nor CD28 costimulation requires newly synthesized
proteins for its enhancing effect on IFN- mRNA expression,
suggesting that these molecules signal through intracellular pathways
in NK cells that use preformed protein components.
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gene transcription in NK cells
Having established that IL-18- or CD28-mediated costimulation of
NK cells can potently enhance the synthesis of IFN- in a de novo
protein synthesis-independent manner, we further investigated the
molecular basis for these effects by using nuclear run-on analysis to
determine the rate of IFN- gene transcription in appropriately
stimulated NK cells. The combination of IL-12 plus IL-18 resulted in
markedly elevated levels of IFN- gene transcription within 4 h
of culture compared with those in NK cells either left unstimulated or
stimulated with IL-12 alone (Fig. 4).
IL-18 alone was not able to enhance the rate of transcription of the
IFN- gene above that found in unstimulated control cultures,
consistent with its inability to stimulate IFN- protein synthesis.
When NK cells were stimulated in the presence of IL-12 or IL-12 plus
anti-CD28 there was a small enhancement in the amount of IFN-
gene transcription over unstimulated control levels. However, this was
low compared with the level of transcription observed in IL-12- plus
IL-18-treated cultures and appeared to be dependent on the presence of
IL-12, as anti-CD28 alone did not enhance transcription above basal
levels. In some experiments (as shown in the example run-on in Fig. 3
)
the combination of CD28 plus IL-12 did result in some transcriptional
activity above that detected in NK cells cultured with IL-12 alone,
suggesting that CD28 may have some small transcriptional activity in
the presence of IL-12. However, this difference was not significant
when analyzed in multiple experiments. Therefore, despite the ability
of IL-18 or anti-CD28 treatment to enhance IFN- mRNA and protein
synthesis in the presence of IL-12, these molecules have different
effects on the rate of transcription of the IFN- gene. These results
illustrate that even though both methods of costimulation elicit high
levels of IFN- protein synthesis, this appears to be achieved
through distinct intracellular mechanisms.
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mRNA in NK
cells
CD28 costimulation of T cells has been shown to directly enhance
the stability of certain cytokine mRNAs, including IFN- (28).
Therefore, to determine whether CD28 treatment was enhancing IFN-
protein synthesis by stabilizing cytokine mRNA in NK cells, we analyzed
the rate of decay of IFN- mRNA after blocking gene transcription by
treatment with the transcriptional inhibitor actinomycin D. Culture of
NK cells with the appropriate inducers for 4 h followed by the
addition of actinomycin D resulted in a steady decay of IFN- mRNA
over time (Fig. 5). However, notable
differences in the rate of decay were apparent depending on the nature
of the initial stimulus. NK cells stimulated with either IL-12 or IL-12
plus IL-18 had similar rates of decay of IFN- mRNA; both these
conditions produced an mRNA half-life of 2 h. In contrast, when NK
cells were stimulated with anti-CD28 in the presence of IL-12, the
rate of decay of IFN- mRNA was significantly longer with a half-life
in excess of 4 h. Therefore, in contrast to IL-18, which directly
increases the rate of transcription of the IFN- gene in the presence
of IL-12, the enhancing effects of CD28 costimulation on IFN-
protein synthesis appear to be due primarily to the enhanced stability
of IFN- mRNA within activated NK cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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synthesis by T
cells and its role in Th1 development (11, 12, 13, 29), with few studies
addressing in detail the role of this cytokine in NK cell function. In
reporting the initial cloning of IL-18 it was noted that this cytokine
augmented NK activity in spleen cells (11), and recent studies have
shown that IL-18 is important in the induction of NK cell activation
(14) and cytotoxicity (30). However, the role of IL-18 in modulating
IFN- production by NK cells has not been extensively studied.
Similarly, despite a few notable exceptions concerning NK cell
proliferation (19) and activation of NK cell cytotoxicity (20, 21, 22), the
role of the costimulatory molecule CD28 in NK cell function has
received less attention, particularly with regard to IFN- synthesis.
Therefore, to address this deficiency and given previous findings from
our laboratory showing that costimulation of NK cells can play an
important role in enhancing IFN- production during infection (24),
this study further elucidates the role of these costimulatory molecules
in IFN- production by NK cells at the molecular level. We show that
IL-18 and anti-CD28 can enhance IL-12-driven IFN- synthesis by
NK cells, and despite these similar effects, they use distinct
intracellular mechanisms to achieve this. IL-18 increases the synthesis
of IFN- mRNA by directly up-regulating the rate of transcription of
the IFN- gene, whereas costimulation via CD28 results in little
transcriptional activity but marked stabilization of presynthesized
IFN- mRNA. These findings illustrate that in addition to influencing
T cell function, these costimulatory molecules play an important role
in the synthesis of IFN- by activated NK cells.
Although the induction of IFN- by costimulation via IL-18 and CD28
has been described for T cells, our results suggest important
differences in the mechanisms used by NK cells. For example, it was
recently reported that T cells require stimulation via CD3/CD28 in the
presence of IL-12 before acquiring responsiveness to IL-18 (31).
Similarly, in a previous study by the same group using T cell clones it
was observed that the synergy observed between IL-12 and IL-18 was due
to up-regulation of the IL-18R by IL-12 (32). Our findings demonstrate
that NK cells do not require any further prestimulation and respond
rapidly to the combination of IL-12 plus IL-18, suggesting that in
addition to expressing the IL-12R, these cells may also constitutively
express the IL-18R. However, it is also possible that in the short
stimulation period (4 h) IL-12 is able to sufficiently up-regulate
expression of the IL-18R. Receptor binding studies or analysis of
receptor mRNA expression during stimulation will resolve this issue.
Our findings are in agreement with these previously mentioned studies
using T cells, in that IL-18 requires the presence of IL-12 for its
enhancing effects on IFN- synthesis. Furthermore, the kinetics of
this response suggested a direct cooperation between IL-12 and IL-18 at
the level of IFN- gene transcription. In contrast to IL-18,
anti-CD28 costimulation of NK cells is influenced by IL-2
preactivation, as murine NK cells have been shown to up-regulate CD28
expression upon stimulation with IL-2 (19). CD28 costimulation of T
cells results not only in IFN- synthesis but in up-regulation of
other cytokines including IL-2 and TNF- (33), and it has been
suggested that CD28 costimulation of NK cells may enhance IFN-
synthesis via indirect stimulation with these cytokines (19). However,
in contrast to T cells the only major effect that we observed upon CD28
costimulation was increased stability of IFN- mRNA, and mRNA for
other cytokines, such as TNF- and IL-2, were not detectable by RPA.
Our studies also show that IL-18 and CD28 signaling uses intracellular
processes that do not require de novo protein synthesis. Interestingly,
in contrast to our findings with IFN- synthesis by NK cells, the
CD28-mediated post-transcriptional stability of the IL-2 gene in T
cells has been shown to require de novo protein synthesis (34).
Recently, CD28-mediated induction of CTLA-4 expression in the T cell
line EL4 was shown to involve both increased transcription and
stability of mRNA, and expression of this mRNA was shown to be
dependent on new protein synthesis (35). Also, binding of the nuclear
factor NF-MATp35 to the CD28 response element of the IL-2 promoter has
been shown to require de novo protein synthesis (36), illustrating that
many of the effects mediated by CD28 require the synthesis of new
proteins, such as specific transcription factors, or that pre-existing
intracellular proteins require modification or activation by newly
induced proteins. This does not appear to be the case for CD28-mediated
effects on IFN- mRNA expression, and it is possible that our
findings illustrate unique properties of NK cells. With regard to
IL-18, our study is novel in that it demonstrates that this cytokine
does not require de novo protein synthesis for its ability to mediate
its IL-12-dependent enhancing effects on NK cells. Interestingly, a
recent study by Barbulescu et al. showed that IL-18 induces AP-1
activity in CD4+ T cells (37), and AP-1 induction in T
cells has been shown to be sensitive to inhibition of protein synthesis
(38). In preliminary studies we have found that IL-18 also induces AP-1
in NK cells. Since our data indicate that the effects of IL-18 are not
sensitive to CHX treatment, this transcription factor either is not
required for IL-18 to mediate its effects or does not require de novo
protein synthesis for its effects on IFN- synthesis by NK cells.
Barbulescu et al. also showed that IL-18 in combination with IL-12
induces high IFN- promoter activity in T cells and concluded that
both AP-1 and STAT4 are required for IL-12-dependent IFN- promoter
activity (37). However, although we observed a synergism between IL-12
and IL-18, IL-12 alone induced IFN- protein synthesis without
additional costimulation, and IL-12 induced STAT4 in nuclear extracts
of NK cells (unpublished observations). This may reflect signaling
differences between NK cells and T cells. In preliminary studies we
have also found that IL-18 induces NF-
B activation in NK cells,
consistent with previous studies showing that IL-18 (29, 39) can induce
this transcription factor in T cells. The IL-18R has now been
identified as the previously cloned IL-1R-related protein (40), and the
induction of NF-B in NK cells is consistent with signaling via an
IL-1R pathway and with findings that IL-18 induces IL-1R-associated
kinase activity (29). NF-B (41), AP-1 (42), and STAT4 (43) binding
sites have been demonstrated in the IFN- promoter and its proximal
regions, so it is probable that these transcription factors act
directly at these sites to enhance IL-12-driven IFN- transcription
in NK cells, as observed with IL-18 costimulation. Whether
IL-18-induced AP-1 and NF-B are both required for up-regulation of
IFN- gene expression or whether they mediate distinct functions in
NK cells remains to be determined and is the subject of ongoing studies
in our laboratories.
CD28 is known to stabilize certain cytokine mRNAs in T cells, including
IFN- (28), but the mechanism remains poorly defined. Indeed, as
previously discussed by others (28), the ability of CD28 to stabilize
IFN- and other cytokine mRNAs appears to be related to the
structural characteristics of these mRNAs, in that they possess
homologous AU-rich sequences in their 3' untranslated regions. Control
of mRNA degradation by AU-rich elements (ARE) has become a paradigm for
post-transcriptional regulation (44). However, little is known about
which trans-acting factors participate in or regulate
ARE-mediated mRNA decay. Different proteins have been identified that
are capable of binding ARE sequences, and a recent report provided in
vivo evidence that an ARE binding protein, termed HuR, participates in
the regulation of mRNA turnover by inhibiting c-fos-mediated
mRNA decay (45). Redistribution of HuR expression from the nucleus to
the cytoplasm was associated with enhanced mRNA stability, and it is
possible that CD28 mediates its effects on IFN- mRNA in a similar
manner using this or other related proteins. Further investigations of
CD28-induced expression of these proteins in NK cells and T cells will
elucidate the precise role of CD28 in mediating mRNA stability. Also,
the relation of these ARE binding proteins to CD28-induced
transcription factor expression requires further investigation.
In summary, this study demonstrates an important role for IL-18 and
CD28 in costimulation of IFN- synthesis by NK cells. In comparison
with other costimulatory cytokines these molecules are extremely
efficient at up-regulating IL-12-driven IFN- synthesis, yet achieve
this via distinct effects on IFN- gene expression. IL-18 synergizes
with IL-12 in directly up-regulating transcription of the IFN- gene.
In contrast, CD28 uses post-transcriptional mechanisms that are still
poorly defined to stabilize IL-12-induced IFN- mRNA in NK cells. The
importance of these findings to infectious disease are aptly
demonstrated by recent in vivo studies showing that IL-18 promotes
resolution of bacterial infections by stimulating IFN- production
(46) and that fungicidal activity mediated by NK cell-derived IFN-
is dependent on the synergistic action of IL-12 and IL-18 (47). Our
findings are also of relevance to inflammatory diseases such as
rheumatoid arthritis, in which blockade of CD28 reduces IFN-
production, leading to amelioration of symptoms (48), and tumor
immunology, where IL-12-activated CD28-deficient NK cells were shown to
have markedly reduced ability to lyse syngeneic tumor cells (21). Our
findings clarify at the cellular and molecular levels how expression of
these costimulatory molecules can influence IFN- production by NK
cells and suggest that these molecules, by enhancing IFN- synthesis,
will have a profound effect on innate immunity, Th1 development, and
disease outcome.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Christopher A. Hunter, Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce Street, Philadelphia, PA 19104. E-mail address:
3 Abbreviations used in this paper: CHX, cycloheximide; RPA, RNase protection assay; ARE, AU-rich element.
Received for publication November 19, 1998. Accepted for publication March 2, 1999.
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