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, IL-12, and IL-15 Regulation of NK Cell Responses to Viral Infection1
* Deparment of Molecular Microbiology and Immunology, Brown University, Providence, RI 02912;
Department of Internal Medicine, Ohio State University, Columbus, OH 43210;
Department of Immunology, University of Glasgow, Glasgow, United Kingdom; and
Department of Pathology, Children’s Hospital and Research Institute, Columbus, OH 43205
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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expression, proliferation, and
accumulation are rapidly induced after murine CMV infections.
Under these conditions, the responses were shown to be elicited in
overlapping populations. Nevertheless, there were distinct signaling
molecule requirements for induction of functions within the subsets.
IL-12/STAT4 was critical for NK cell IFN- expression, whereas
IFN-
/STAT1 were required for induction of cytotoxicity. The
accumulation/survival of proliferating NK cells was STAT4-independent
but required IFN-/STAT1 induction of IL-15. Taken together, the
results define the coordinated interactions between the cytokines
IFN-, IL-12, and IL-15 for activation of protective NK cell
responses during viral infections, and emphasize these factors’
nonredundant functions under in vivo physiological
conditions. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, proliferate, and accumulate
(1, 2, 3, 4). Under these conditions, the innate cytokines
IFN-, i.e., type 1 IFNs, and IL-12 are induced early after
infection and are critical for stimulation of particular NK cell
functions, with IFN- promoting cytotoxicity and proliferation,
and IL-12 eliciting IFN- production (1, 2, 3). Hence, at
early times after MCMV infection, NK cell responses are controlled by
innate cytokines and the factors IFN- and IL-12 are nonredundant
endogenous regulators of these.
Although the critical roles for the cytokines in activating NK cell
responses have been defined, several important questions remain
unanswered. First, the lack of functional overlap between these factors
for activation of NK cells raises questions about the endogenous
regulation pathways. IFN- and IL-12 may be accessing different NK
cell subsets, or activating the same subset of cells through
nonoverlapping intracellular signals. Available biochemical data
suggest potential overlapping spectra of transcription factors.
IFN- and IL-12 both can activate STAT1 (5, 6, 7, 8, 9). STAT4
is activated by IL-12 functions in both humans and mice
(5, 6, 7, 8), and by IFN- in humans (5, 10, 11). Hence, under certain conditions, both STAT1 and STAT4 can
be stimulated by type 1 IFNs and IL-12. In addition, recent data
indicated that both cytokine classes may have STAT-independent
signaling pathways (12, 13). Thus, while it is clear that
IFN- and IL-12 are crucial for induction of NK cell responses
during MCMV infection, mechanistic details regarding: 1) whether the
factors act on different or the same cell NK cell populations, and 2)
the intracellular signaling mechanisms by which these cytokines exert
their effects in vivo, remain poorly characterized.
In addition to the undefined signaling mechanisms for cytokine-mediated
effects, it is uncertain how IFN- promote cell proliferation in
vivo. These cytokines are not likely to stimulate NK cell proliferation
directly, as they fail to elicit NK cell proliferation in vitro and
exert anti-proliferative effects at high concentrations (14, 15). Thus, IFN--mediated promotion of this response may
depend on stimulation of secondary cytokine(s) in vivo. One candidate
is IL-15 (16, 17). This cytokine can be a potent regulator
of NK cell differentiation, proliferation, and survival, and an
accessory factor for NK cell IFN- (16, 17, 18, 19, 20, 21, 22, 23). However,
little is known about the expression and function of IL-15 in viral
infections. The nonlymphoid cell-derived factor shares many
characteristics with the T cell-produced IL-2, and type 1 IFNs can
induce IL-15 mRNA expression in vitro (24). IL-15 binds to
a receptor complex consisting of the common -chain (CD132), the
IL-2/IL-15R subunit (CD122), and a unique high affinity chain
designated IL-15R (16, 17). The IFN- to IL-15
axis for induction of cell proliferation has been hypothesized as the
mechanism for IFN-dependent stimulation of memory CD4 and CD8 T cell
proliferation (24). However, definitive demonstration for
such a pathway remains elusive. Thus, it is unclear how IFN- can
elicit NK cell proliferation in vivo but not in vitro, and although
IL-15 is a possible intermediary, its expression and function during
viral infections are largely uncharacterized.
These studies defined the IFN- and IL-12-mediated
immunoregulatory effects on NK cells during viral infections, and
delineated the signaling molecules requisite for transduction of these
responses. In addition, the role of IL-15 in promoting NK cell
responses during viral infections was defined. STAT1, but not STAT4,
was required for IFN--mediated activation of NK cell
cytotoxicity. In contrast, STAT4, but not STAT1, was essential for
IL-12-dependent NK cell IFN- production. STAT1, but not STAT4, was
required for the accumulation of proliferating NK cells. IL-15 mRNA
expression was induced under conditions of IFN- exposure in vivo,
and the lack of NK cell accumulation in the absence of STAT1 was
associated with failure to induce IL-15. Finally, NK cell accumulation
in response to type 1 IFNs was dramatically reduced in the absence of
endogenous IL-15 function. Taken together, these results delineate the
in vivo STAT-mediated pathways requisite for activation of specific NK
cell functions and reveal unexpected mechanisms for IFN--induced
functions in vivo. In addition, these studies definitively establish
for the first time a distinct mechanism for IFN- support of NK
cell proliferation in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Specific pathogen-free male immunocompetent C57BL/6 and 129SvEv
mice were purchased from Taconic Farms (Germantown, NY). Male C57BL/6J,
129 x C57BL/6 F2 (129B6
F2), endotoxin-resistant C3H/HeJ, and IL-12
p40- (25) mice were purchased from
The Jackson Laboratory (Bar Harbor, ME). STAT1-
(26) and IL-12 p35-
(27) animals were backcrossed for at least five
generations onto the C57BL/6 background before use in experiments.
STAT4- breeding pairs (28),
maintained on a mixed 129 x C57BL/6 background, were from Dr. J.
Ihle (St. Jude’s Children’s Hospital, Memphis, TN).
129Sv-IFN-R- (29) breeding
pairs were purchased from B&K Universal (North Humberside, U.K.). All
mice used in experiments were 5–12 wk in age. Animals obtained outside
of Brown University were housed for at least 1 wk before use. Handling
of mice and experimental procedures were in accordance with
institutional guidelines for animal care and use.
Viral infections and in vivo cytokine treatments
All experiments were initiated on day 0. For viral infections,
mice were either not infected or infected i.p. as described (3, 4, 27, 30, 31, 32) with 1.0 x 104 PFU
MCMV Smith strain. There were no appreciable effects on the
characteristics of NK cell responses induced following infection with
different batches of virus, but there were differences in the levels of
serum cytokines induced. IL-12 and IFN- effects on MCMV
replication in the spleen have been reported previously
(3). After 1.5 days of MCMV infection,
STAT1- spleens had 4.8 ± 0.0 log PFU/g
virus, compared with 4.1 ± 0.1 log PFU/g in
STAT1+ mice. There were, respectively, 4.7
± 0.3 and 4.9 ± 0.1 log PFU/g virus in
STAT4+ and STAT4- mice.
For in vivo cytokine stimulation, mice were i.p. administered
recombinant murine IL-12 (Genetics Institute, Andover, MA), recombinant
simian IL-15 (Immunex, Seattle, WA), or recombinant universal IFN-
(Hoffman-LaRoche, Nutley, NJ). Mice received a total of 2 µg of
rIL-12 (sp. act. 4.6 x 106 U/mg; 1 µg/day
for 2 days), 12 µg of rIL-15 (sp. act. 3.5 x
108 U/mg; 4 µg/day for 3 days), or 3 x
105 U of rIFN- (sp. act. 6 x 106 U/mg;
105/day for 3 days). Control vehicle treatments
were conducted similarly. All experiments were evaluated at 1.5 days
after MCMV infection, 2 days following IL-12, and 3 days following
rIFN- or IL-15 administration.
In vivo neutralization of IL-15 functions
IL-15 functions were neutralized in vivo by either blocking the
IL-2/IL-15R subunit or by treatment with soluble IL-15
(sIL-15)R (33, 34). F(ab)' of anti-IL-2/IL-15R
mAb clone TM-1 were used and F(ab)' of clone CRL-8164 were used as
controls. F(ab)' preparation from hybridoma-purified mAb was performed
as previously described (35, 36). Briefly, Abs were
enzymatically cleaved with mercuripapain (Sigma-Aldrich, St. Louis,
MO) in a buffered solution containing 20 mM sodium phosphate, 10
mM cysteine, and 2 mM EDTA. Reactions were stopped by addition of
iodoacetamide (Sigma-Aldrich), followed by dialysis, and sterile
filtration. For MCMV infection, mice were given 0.5-mg doses F(ab)' at
2 h before, and 18 h after, viral inoculation. For
experiments with rIFN- administration, F(ab)' was given to mice on
days 0 (1.0 mg), 1 (0.5 mg), and 2 (0.5 mg) relative to the initial
injection of recombinant cytokine. Under conditions of treatment, NK
cells were still present in spleens and livers of
anti-IL-2/IL-15R-treated mice, and anti-receptor F(ab)'
appeared to still bind to NK cells (data not shown). Recombinant
sIL-15R (34), binding with high affinity to IL-15 but
not to IL-2, and a mutated recombinant sIL-15R, designated M4
(33), no longer binding to IL-15, were purified and
analyzed by SDS-PAGE and Western blot as described. LPS was not
detectable by the Amebocyte Limulus test. These reagents
were administered with schedules similar to the F(ab)' treatments but
at 400 µg per injection. This dose was established in preliminary
experiments as the minimum necessary to observe IL-15 blockade effects
following MCMV infection.
Serum preparation and organ collection
Animals were anesthetized at the indicated times, and blood was collected via the retroorbital route into low amounts of heparin. Following centrifugation, supernatants were collected and identified as sera. Mice were sacrificed, and spleens and livers were harvested and maintained at 4°C.
Preparation of cells
Splenic and liver leukocytes were obtained using previously
described protocols (3, 4, 27, 30, 31, 32, 37, 38). Viable
cell yields were determined by trypan blue exclusion. In certain
studies, NK cells were enriched by negative magnetic selection as per
manufacturer protocol (StemCell Technologies, Vancouver, British
Columbia, Canada). Enrichments yielded preparations containing 
50%
NK cells, as determined by flow cytometric analyses.
Flow cytometric analyses
Studies evaluating spontaneous ex vivo intracellular expression
of IFN- were performed using previously described protocols
(4, 27, 30, 38). Briefly, cells were fluorescently labeled
with the surface markers NK1.1 or DX5 and CyChrome-conjugated
anti-CD3
, washed, fixed, permeabilized, and incubated with
allophycocyanin-conjugated anti-IFN- mAb clone XMG1.2.
Specificity for IFN- staining was demonstrated by lack of labeling
with isotype-matched Ab or by coincubating samples with ascite-purified
unconjugated XMG1.2. All directly conjugated mAb and
streptavidin-allophycocyanin were purchased from BD PharMingen
(San Diego, CA). Intracellular perforin expression was evaluated using
protocols similar to those for IFN-, using purified
anti-perforin mAb clone KM585 (Kamiya Biomedical, Seattle, WA),
followed by PE-conjugated polyclonal rabbit anti-rat Abs (BD
PharMingen). A rat mAb with the same isotype as the perforin mAb was
used for controls. To determine levels of perforin expression, NK cells
were gated and evaluated for proportions of cells expressing the
molecule. Under these conditions, there was no staining with
isotype-control mAbs. Staining conditions were established based upon
published protocols (39). Studies evaluating
5-bromo-2'-deoxyuridine (BrdU) incorporation by NK cells after
in vivo labeling were adapted from previously described methods
(24). Briefly, mice were given drinking water containing
BrdU (Sigma-Aldrich) at 0.8 mg/ml starting on day 0 relative to viral
infection or cytokine treatment and sacrificed on the indicated days
thereafter for preparation of splenic leukocytes. Isolated cells were
labeled with NK1.1 or DX5 and CD3, washed, fixed with ethyl
alcohol and paraformaldehyde containing Tween 20, and stained
with anti-BrdU mAb clone 3D4 or isotype-matched control Abs (BD
PharMingen). Samples were acquired using a FACSCalibur (BD Biosciences,
Mountain View, CA), with the CellQuest version 3.1 software package.
Laser outputs were 15 mW at 488- and 635-nm wavelengths. At least
100,000 events were collected for analyses. In experiments where NK
cells were quantified, proportions of NK cells, which were identified
as NK1.1+CD3- in C57BL/6
strains and DX5+CD3- in
129 and C3H strains of mice, were first calculated. This proportion was
then used to calculate the total number of NK cells in the spleen based
on total cell yields. Numbers of live spleen cells were quantitated
after trypan blue exclusion of dead cells.
Serum cytokine measurements
Serum IFN- and IL-12 p40 levels were determined by standard
sandwich ELISA as was previously described (3, 4, 27, 30, 31, 32, 37, 38). IL-20 p70 ELISA was done according to manufacturer protocol
(R&D Systems, Minneapolis, MN). Sandwich ELISA for IL-15 was conducted
using recombinant sIL-15R as the primary and polyclonal rabbit
anti-murine IL-15 as the secondary Ab. Recombinant murine IL-15 was
used for cytokine standards. IL-15 and polyclonal anti-IL-15 were
provided by Immunex. For ELISAs, limits of detection varied between
experiments but always were <20 pg/ml. Colorimetric changes of enzyme
substrates were detected at 405- or 450-nm wavelengths using a
SpectraMax 250 reader (Molecular Devices, Sunnyvale, CA). IFN-
bioassays were conducted as described (27).
Cytotoxicity assays
Cytotoxicity was measured as release of radioactive isotope from YAC-1 target cells labeled with sodium chromate (51Cr) following incubation with unlabeled effector splenic leukocytes, as described (3, 14). Spontaneous release was <15% of maximum release. Percent specific lysis was calculated as 100 x (cpm test sample - cpm spontaneous release)/(cpm total release - cpm spontaneous release). Consistent with previous studies demonstrating the role of IL-15 for NK cell development (22, 23), treatments to block endogenous IL-15 resulted in decreases in NK cell proportions, both in infected and uninfected mice. Hence, cytotoxicity was evaluated after specific lysis was normalized to the proportions of NK cells. Under these conditions, the 100:1 E:T ratio data were used to calculate lytic units, and the lytic units per spleen were compared between different treatment groups.
Semiquantitative RT-PCR analysis for IL-15 expression
Total spleen and bone marrow RNA samples were prepared using the RNAWiz isolation kit (Ambion, Austin, TX). Relative quantitative RT-PCR was performed as reported (32). Briefly, 1 µg samples of total RNA were reversed-transcribed into cDNA, and cDNA samples were then used as templates for PCR amplification using primers specific for IL-15 (24). To control for sample preparation, gel loading, and random variations in RT-PCR, 18S rRNA primers, and 18S rRNA Competimers (Ambion), used to modify 18S cDNA amplification efficiency, were also included in each PCR. Amplifications were conducted in the programmable thermocycler PTC-100 (MJ Research, Waltham, MA) using previously reported parameters (24). IL-15 primer sequences were synthesized by Operon Technologies (Alameda, CA). Products were separated on 1.8% agarose gels and visualized by ethidium bromide staining. IL-15 sequence specificity was confirmed following RT-PCR amplification by hybridizing with an internal oligonucleotide probe.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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expression, and
proliferation
After 1.5 days of MCMV infection, NK cells were induced to mediate
cytotoxicity, synthesize IFN-, and proliferate (Fig. 1
, Tables I and II)(1, 2, 3). To examine whether the functions could
occur within the same NK cell,
intracellular flow cytometric analyses were performed for expression of
perforin, a cytotoxic effector molecule, IFN-, and incorporation of
BrdU, a synthetic nucleic analog used to evaluate DNA synthesis, in
uninfected mice or those infected for 1.5 days with MCMV. BrdU was
supplied continuously in the drinking water during the experimental
period. As shown in Fig. 1, very low proportions of splenic NK cells
from uninfected mice were expressing perforin or incorporating BrdU,
and IFN- was undetectable. After 1.5 days of infection, 60% of
NK cells were induced to express perforin, 30–60% to express IFN-,
and 25% had incorporated BrdU. Simultaneous perforin vs IFN-
detection revealed that virtually all IFN--expressing cells also
were positive for perforin (Fig. 1A). Similarly, 75% of
BrdU incorporating NK cells were synthesizing IFN- (Fig. 1B). Hence, there was large overlap for induction of
responses within NK cells following MCMV infection, and at least 20%
of NK cells are estimated to have all three functions after infection.
Thus, individual NK cells were responding in multiple ways.
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expression
Confirming earlier studies with Ab neutralization
(3), mice blocked in IFN- function by genetic
mutation of IFN-R and infected for 1.5 days with MCMV were
profoundly inhibited in NK cell cytotoxicity (Table I
) but not IFN-
production (Table II). In contrast, infected mice blocked in IL-12
function as a result of genetic mutation of the p35 (Table II) or the
p40 (data not shown) subunit of IL-12 were inhibited in IFN-
production (Table II) but not cytotoxicity (Table I). Differences in
responses could not be accounted for by changes in proportions or total
numbers of NK cells.
To further define the molecular bases for IFN-- and
IL-12-mediated immunoregulatory functions, STAT requirements for
IFN-- or IL-12-dependent induction of specific NK cell responses
were evaluated. NK cell cytotoxicity was comparable in MCMV-infected,
STAT4-, compared with immunocompetent, mice
after 1.5 days of MCMV infection (Table I). In contrast, cytotoxicity
from infected STAT1- mice was >70% reduced
(Table I). The reductions could not be accounted for by differences in
the proportions of NK cells in STAT1- mice (data
not shown). Likewise, decreased cytotoxicity was not due to reduced
IFN- expression; both mouse strains had similar levels of serum
IFN- at this time after infection (data not shown). However,
IL-12 induction of NK cell IFN- required STAT4 but not STAT1.
Systemic IFN- levels in STAT4+ mice were
3000 pg/ml, whereas the factor was undetectable in sera of infected
STAT4- animals (Table II). Proportions and
numbers of spleen NK cells expressing intracellular IFN- in infected
STAT4- mice decreased >60% compared with
infected STAT4+ animals (Table II). Under these
conditions, STAT4- had equivalent levels of
IL-12 p40 and p70 to STAT4+ mice (data not
shown). In contrast, systemic IFN- levels were dramatically elevated
in infected STAT1- mice; infected
STAT1- mice had at least 6-fold higher serum
IFN- compared with STAT1+ animals (Table II).
After infection, >50% of NK cells from
STAT1- animals expressed intracellular IFN-,
compared with 30% of those from STAT1+ mice
(Table II). The elevated IFN- production under these conditions was
associated with increased IL-12 production;
STAT1- animals had more than five times more
systemic IL-12 p40 levels compared with STAT1+
mice (data not shown). Taken together, the results demonstrate that NK
cell cytotoxicity is dependent on the IFN-/STAT1 pathway, whereas
IFN- expression requires IL-12/STAT4.
Cytokine and STAT requirements for NK cell proliferation and accumulation
Parameters examined were extended to include requirements for
induction of NK cell proliferation by incorporation of BrdU during in
vivo labeling. MCMV infection induced dramatic accumulation of NK cells
labeled with BrdU (Fig. 2). Under these
conditions, biologically active IL-12 was not required for BrdU
incorporation (Fig. 2A). By comparison, MCMV-infected
IFN-R- animals were significantly reduced
in the proportions of BrdU+ cells (Fig. 2B). Although percentages and yields of total NK cells were
either not reduced or reduced by no more than 50% (yields of 410
x 103 (±50) vs 240 x
103 (±20) per spleen), numbers of
BrdU+ NK cells were decreased by 60–70% as a
result of blocking IFN- functions during infection. There were
90 x 103 (±10) NK cells incorporating BrdU
in IFN-R+ but only 30 x
103 (±10) in IFN-R-
mice. Because activated NK cells are induced to migrate to livers
(31, 32), yields from this tissue also were examined.
Similar to spleen, liver NK cell numbers were higher in infected
IFN-R+ as compared with
IFN-R- mice (230 x
103 vs 154 x 103 per
liver). Thus, during MCMV infections, biologically active IL-12 is
dispensable, but IFN-R functions are necessary for accumulation
and induction of NK cell proliferation.
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-dependent stimulation of
proliferating NK cell accumulation. STAT4+ and
STAT4- mice had similar proportions and numbers
of BrdU+ splenic NK cells (Fig. 2C).
In contrast, BrdU+ NK cell accumulation was
severely impaired in STAT1-deficient mice (Fig. 2D). In
several experiments totaling >12 mice per group, proportions of
BrdU+ NK cells in MCMV-infected
STAT1- mice consistently did not rise above the
basal accumulation seen in uninfected mice, in contrast to the >300%
increase in the proportions and numbers of BrdU+
NK cells seen in infected STAT1+ animals (Fig. 2D and data not shown). Thus, IL-12- and STAT4-independent
accumulation of BrdU+, i.e., proliferating NK
cells after MCMV infection, requires IFN- and STAT1 functions.
Taken together, the experiments showed that under conditions of a mixed
cytokine milieu, IFN--dependent accumulation of proliferating NK
cells requires STAT1 but not STAT4. STAT requirements for induction of NK cell responses after IL-12 or type 1 IFN administration
To directly examine the in vivo pathways activated by individual
cytokines, as well as to move to nonviral conditions and thus limit
potential secondary effects resulting from changes in inducing cytokine
levels or viral burdens, studies were conducted examining in vivo
responses to administered recombinant cytokine. The IL-12 induction of
NK cell IFN- expression also required only STAT4 and not STAT1, and
rIFN- effects on NK cells in vivo did not require STAT4 (data not
shown). However, similar to MCMV infection, STAT1 was necessary for
rIFN--mediated induction of NK cell cytotoxicity (Fig. 3A). There were also
reductions in the accumulation of BrdU+ NK cells
following rIFN- administration in STAT1- mice
(Fig. 3B). Total spleen and liver NK cell yields in
IFN-treated STAT1- mice reflected the impaired
ability of the proliferating NK cells to accumulate; there were 2- and
6-fold increases in NK cells, respectively, in spleens and livers after
rIFN- treatment of immunocompetent mice, contrasted to more modest
increases in spleens and no dramatic increases in livers of
STAT1- mice (Fig. 3, C and
D). Thus, STAT1 is requisite for IFN--mediated
induction of NK cell cytotoxicity and accumulation of
proliferating cells.
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-mediated, STAT1-dependent, induction of IL-15 expression
in vivo
The impaired ability of proliferating NK cells from
STAT1- mice to accumulate in vivo may be due to
absence of factors that can promote NK cell survival, such as IL-15. To
evaluate whether IL-15 was induced during MCMV infection or after
rIFN- exposure and to define requirements for expression, sera as
well as spleen and bone marrow leukocyte-conditioned media were
evaluated for IL-15 expression by ELISA. IL-15 protein was not
detectable in any of the samples. Total RNA samples also were prepared
from spleen and bone marrow cells and evaluated in semiquantitative
RT-PCRs for IL-15 induction. The conditions of infection as well as
rIFN- treatment both induced elevated levels of IL-15 mRNA (Fig. 4). Increases were more readily
detectable in spleen samples from infected, and in bone marrow samples
from rIFN--treated, mice. Endogenous IFN- function was
critical for induction of IL-15 mRNA during MCMV infection because it
was not detected in samples from IFN-R-
mice (Fig. 4A). Samples from STAT1-
mice demonstrated that STAT1 also was required for induced IL-15
expression after viral infection (Fig. 4B) and after
rIFN- treatment (Fig. 4C). Thus, IFN-, through
STAT1, mediates enhanced expression of the NK cell survival cytokine
IL-15.
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IL-15 effects and requirements for NK cell responses were
evaluated in vivo. Under the conditions examined, NK cell cytotoxicity
and serum IFN- production were not elicited to detectable levels
following rIL-15 administration (data not shown), but the factor
readily induced splenic NK cells to incorporate BrdU (Table III). The responses were observed across
several different strains (Table III and data not shown). Concordantly,
total NK cell numbers increased in both spleens (Table III) and livers
(data not shown) of rIL15-treated mice. The effects on NK cells did not
require IFN-, as IL-15 stimulated NK cells in
IFN-R- mice to incorporate BrdU and to
accumulate to levels similar to those from
IFN-R+ animals (Table III). IL-15
treatments of STAT1-deficient mice also induced NK cell proliferation
(data not shown). Thus, IL-15, independent of IFN- and STAT1, had
potent effects in inducing NK cell proliferation and accumulation.
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administration, endogenous IL-15
functions were neutralized by treatment with IL-2/IL-15R mAb
F(ab)' to block the receptor or by treatments with recombinant
sIL-15R to block the factor. Blocking endogenous IL-15 functions
with either IL-2/IL-15R F(ab)' or sIL-15R did not inhibit the
IFN--dependent induction of NK cell cytotoxicity or the
IL-12-driven NK cell IFN- expression (data not shown). In contrast,
IL-2/IL-15R blockade had dramatic effects on the accumulation of
proliferating NK cells, both in proportions (Fig. 5A) and numbers (Fig. 5B). Because IL-2/IL-15R F(ab)' treatment could block the
functions of other cytokines, sIL-15R was a more specific inhibitor
of endogenous IL-15. Similar to IL-2/IL-15R, sIL-15R treatment
dramatically reduced both proportions (Fig. 5C) and numbers
(Fig. 5D) of NK cells incorporating BrdU. Administration of
rIFN- yielded responses similar to those observed after MCMV
infection (Fig. 5, E and F), except that NK cell
IFN- production was not observed (data not shown).
Anti-IL-2/IL-15R treatment during rIFN- treatment also blocked
accumulation of proliferating NK cells (Fig. 5, E and F).
Interestingly, IL-15 blockade, especially after treatment with
anti-IL-2/IL-15R, also decreased NK cell proportions in
uninfected mice (data not shown). Taken together, the data demonstrated
that IL-15 and its downstream effects are induced by type 1 IFNs, and
that although it is not critical for induction of NK cell cytotoxicity
or IFN- expression, the cytokine is required for the maintenance
and/or accumulation of proliferating NK cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-induced endogenous IL-15 expression in regulating NK cells
during viral infections. Under these conditions, the type I IFNs
promote NK cell cytotoxicity and proliferation, whereas IL-12 elicits
IFN- production. IFN- production, cytotoxic activity, and
proliferation were shown in this study to occur in overlapping
populations of NK cells. STAT1, but not STAT4, was required for the
IFN--dependent induction of cytotoxicity. In contrast, the
IL-12-dependent NK cell IFN- response was dependent on STAT4, but
not STAT1. STAT1, but not STAT4, was required for accumulation of the
cells. Impaired NK cell expansion in STAT1- mice
was due to failure to induce IL-15 in response to IFN-. Taken
together, the studies delineate the STAT pathways required for
induction of specific NK cell functions in vivo. They suggest that the
distinct immunoregulatory effects mediated by IFN- and IL-12 are
due at least in part to nonoverlapping signaling pathway requirements
for induction. They demonstrate the nonredundant but critical functions
for the innate cytokines IFN-, IL-12, and IL-15 in regulating NK
cell responses during viral infections. Lastly, they establish an
IFN--STAT1-IL-15 axis critical for maintenance of NK.
By differentiating between signals for induction of cells to
proliferate, to acquire effector functions, and to maintain these
populations in vivo, the complexity of NK cell regulation during an
infectious process is revealed (Fig. 6).
In the endogenous milieu of multiple cytokine stimulations of the same
cell subsets, individual cytokines nevertheless retain unique
functions. During MCMV infections, even though IFN- and IL-12 are
accessing overlapping NK cell populations, IL-12, through STAT4,
exclusively regulates IFN-. Likewise, the IFN- cytokines,
through STAT1-dependent signaling, uniquely regulate NK cell
cytotoxicity and proliferation. NK cell activation by IFN- does
not appear to confer intrinsic survival capabilities. Survival signals,
conferred by IL-15 elicited in response to IFN-, are required to
sustain the NK cell response following activation. The observation that
STAT1 is critical for induction of cytotoxicity is consistent with
another recent report demonstrating that the signaling molecule is
required for basal and dsRNA-induced NK cell killing (40).
STAT1 is also required for a variety of IFN--dependent effects
(41). At 1.5 days after MCMV infection, IFN- is not a
major regulator of NK cell cytotoxicity, because this cytokine is just
being produced by NK cells. In the absence of IFN- functions, a
modest contribution by IFN- to NK cell cytotoxicity can be observed
at later times after infection. However, in the presence of IFN-
functions, IFN- contribution to cytotoxicity is not readily observed
(K. B. Nguyen and C. A. Biron, unpublished observations).
Thus, during immune responses to viral infections, multiple cytokines
are induced, and each of these has unique roles in regulating distinct
aspects of the NK cell responses with IFN--inducting
cytotoxicity, IL-12-inducing IFN- production, and IFN--induced
IL-15 supporting expansion.
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Although certain studies have suggested the role for IL-15 in
maintaining memory T cell responses and for maintaining long-term
protection to viruses (16, 42, 43), these are the first
studies to our knowledge to define and document the expression and
function of IL-15 during acute viral infections and to critically
define this factor’s role in regulation of NK cell functions. The
importance for endogenous IL-15 in NK cell development (16, 17, 22, 23), pathogenesis during collagen-induced arthritis
(34), and NK cell IFN- production following endotoxin
challenge (20) have been demonstrated. One of the
challenges for identifying specific IL-15 functions in vivo has been
the lack of reagents selectively neutralizing this cytokine without
eliminating NK cells. As mice genetically deficient in either the
IL-15R -chain or IL-15 do not develop NK cells (22, 23), they would not have been informative for the questions
being addressed in this study, i.e., effects of induced IL-15 on NK
cell responses to viral infections. Thus, for our studies, in vivo
IL-15 functions were evaluated by two complementary, short-term
approaches (Fig. 5). First, the shared IL-2/IL-15R -chain was
blocked by administering F(ab)'' before infection or rIFN-
administration. Second, the soluble rIL-15R was given to neutralize
endogenous factor. The sIL-15R used was generated by recombinant
technology and expression in Gram-negative bacteria (33, 34). It was purified and had no detectable endotoxin, i.e., LPS,
using standard assays. However, because previous work from our
laboratory has demonstrated that sensitivity to endotoxin can be
increased 10- to 100-fold during viral infections (37, 38), and because treatment of both mutated and functional
sIL-15R had stimulatory effects on NK cells in C57BL/6 mice,
experiments with this reagent were performed in the endotoxin-resistant
C3H/HeJ mice. Under these conditions, this very specific blocker of
IL-15 functions inhibited the accumulation of NK cells.
We also demonstrated that administration of rIL-15 induced NK cell
accumulation (Table III). In contrast, IL-15 was not crucial for either
NK cell cytotoxicity or IFN- expression under the conditions of
viral infections. This is in contrast to other works demonstrating that
IL-15 can stimulate NK cell cytotoxicity in vitro and synergize with
IL-12 for induction of NK cell IFN- production
(16, 17, 18, 19, 20, 21, 22, 23). Given differences in in vitro vs in vivo
settings, the experimental conditions, and the amounts of IL-15 used in
our studies and others, the results presented in this study do not
necessarily contradict published work on the role of this cytokine in
promoting NK cell cytotoxicity. Rather, we show that under conditions
of short-term exposure in vivo, IL-15 effects on proliferation and
cytotoxicity readily can be separated. Taken together, the results
conclusively demonstrate the critical role for endogenous IL-15,
induced during MCMV infection, for NK cell survival, and suggest that
IL-15 functions are not static and may have different functions
depending on the in vivo stimulus.
The observation that NK cells from MCMV-infected STAT1-deficient mice
express higher levels of IFN- (Table II), is consistent with our
previous report demonstrating that STAT1 acts to dampen and/or inhibit
the NK cell IFN- response (30). STAT1-mediated
inhibition of IFN- is downstream of IFN-. Type 1 IFNs under
normal conditions do not elicit IFN- production by NK cells.
However, when STAT1 is absent or somehow negatively regulated, type 1
IFNs now can induce IFN- expression. Thus, in the absence of STAT1,
the IFN- cytokines may have roles in the enhanced IFN-
production during MCMV infection. In other studies, IL-12
administration to STAT1- mice also resulted in
increased NK cell and serum IFN- as compared with
STAT1+ animals (K. B. Nguyen and C. A.
Biron, unpublished observations). These results indicate that STAT1 has
a role in dampening the IFN- responses to IL-12 in the absence of
virus-induced IFN- production and in modulating IL-12 levels
during viral infections. Such effects may be critical to the balance of
beneficial vs detrimental effects of IL-12. Thus, STAT1 may be a
general inhibitor of IFN- production, and the increased IFN-
production by NK cells in STAT1-deficient MCMV-infected mice may be the
result of heightened sensitivity of NK cells to IFN- production in
response to cytokines, including IFN- and IL-12.
In summary, these studies characterize key cytokines and STAT molecules
required for induction of NK cell effector functions during immune
responses in vivo. They reveal IFN--dependent, STAT1-dependent
mechanisms for induction of biological effects, separate signaling
pathways for IL-12- as compared with IFN--mediated effects, and
identify an IFN- to STAT1 to IL-15 cascade critical for NK cell
responses in vivo. Given that particular NK cell responses may be more
important in defense against certain infectious agents, and that NK
cells have the potential to mediate detrimental effects if not
appropriately regulated, the unique effects mediated by, and signaling
pathways accessed by, IFN-, IL-12, and IL-15, provide elegant
mechanisms to precisely control these cells. Moreover, although in
vitro and certain in vivo studies suggest a strong degree of overlap
for immunoregulation between these factors, our studies show the
nonredundancy of these cytokines for regulation of protective responses
under physiologic conditions. These results emphasize the need for
characterization of cytokine effects and interactions under conditions
of mixed milieus.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Christine A. Biron, Molecular Microbiology and Immunology, Brown University Box G-B6, Providence, RI 02912. E-mail address: Christine_Biron{at}brown.edu
3 Abbreviations used in this paper: MCMV, murine CMV; BrdU, 5-bromo-2'-deoxyuridine; LU, lytic unit.
Received for publication June 25, 2002. Accepted for publication August 16, 2002.
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N. Dikopoulos, A. Bertoletti, A. Kroger, H. Hauser, R. Schirmbeck, and J. Reimann Type I IFN Negatively Regulates CD8+ T Cell Responses through IL-10-Producing CD4+ T Regulatory 1 Cells J. Immunol., January 1, 2005; 174(1): 99 - 109. [Abstract] [Full Text] [PDF]
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G. Ferlazzo, M. Pack, D. Thomas, C. Paludan, D. Schmid, T. Strowig, G. Bougras, W. A. Muller, L. Moretta, and C. Munz Distinct roles of IL-12 and IL-15 in human natural killer cell activation by dendritic cells from secondary lymphoid organs PNAS, November 23, 2004; 101(47): 16606 - 16611. [Abstract] [Full Text] [PDF]
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G. Alter, J. M. Malenfant, R. M. Delabre, N. C. Burgett, X. G. Yu, M. Lichterfeld, J. Zaunders, and M. Altfeld Increased Natural Killer Cell Activity in Viremic HIV-1 Infection J. Immunol., October 15, 2004; 173(8): 5305 - 5311. [Abstract] [Full Text] [PDF]
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A. Ahmad and F. Alvarez Role of NK and NKT cells in the immunopathogenesis of HCV-induced hepatitis J. Leukoc. Biol., October 1, 2004; 76(4): 743 - 759. [Abstract] [Full Text] [PDF]
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F. Granucci, I. Zanoni, N. Pavelka, S. L.H. van Dommelen, C. E. Andoniou, F. Belardelli, M. A. Degli Esposti, and P. Ricciardi-Castagnoli A Contribution of Mouse Dendritic Cell-Derived IL-2 for NK Cell Activation J. Exp. Med., August 2, 2004; 200(3): 287 - 295. [Abstract] [Full Text] [PDF]
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J. Siren, T. Sareneva, J. Pirhonen, M. Strengell, V. Veckman, I. Julkunen, and S. Matikainen Cytokine and contact-dependent activation of natural killer cells by influenza A or Sendai virus-infected macrophages J. Gen. Virol., August 1, 2004; 85(8): 2357 - 2364. [Abstract] [Full Text] [PDF]
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M. Strengell, I. Julkunen, and S. Matikainen IFN-{alpha} regulates IL-21 and IL-21R expression in human NK and T cells J. Leukoc. Biol., August 1, 2004; 76(2): 416 - 422. [Abstract] [Full Text] [PDF]
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S. H. Robbins, M. S. Tessmer, T. Mikayama, and L. Brossay Expansion and Contraction of the NK Cell Compartment in Response to Murine Cytomegalovirus Infection J. Immunol., July 1, 2004; 173(1): 259 - 266. [Abstract] [Full Text] [PDF]
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M. Franchini, H. Hefti, S. Vollstedt, B. Glanzmann, M. Riesen, M. Ackermann, P. Chaplin, K. Shortman, and M. Suter Dendritic Cells from Mice Neonatally Vaccinated with Modified Vaccinia Virus Ankara Transfer Resistance against Herpes Simplex Virus Type I to Naive One-Week-Old Mice J. Immunol., May 15, 2004; 172(10): 6304 - 6312. [Abstract] [Full Text] [PDF]
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D. Tosi, R. Valenti, A. Cova, G. Sovena, V. Huber, L. Pilla, F. Arienti, F. Belardelli, G. Parmiani, and L. Rivoltini Role of Cross-Talk between IFN-{alpha}-Induced Monocyte-Derived Dendritic Cells and NK Cells in Priming CD8+ T Cell Responses against Human Tumor Antigens J. Immunol., May 1, 2004; 172(9): 5363 - 5370. [Abstract] [Full Text] [PDF]
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P. Bjorck Dendritic Cells Exposed to Herpes Simplex Virus In Vivo Do Not Produce IFN-{alpha} after Rechallenge with Virus In Vitro and Exhibit Decreased T Cell Alloreactivity J. Immunol., May 1, 2004; 172(9): 5396 - 5404. [Abstract] [Full Text] [PDF]
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B. G. Dorner, H. R.C. Smith, A. R. French, S. Kim, J. Poursine-Laurent, D. L. Beckman, J. T. Pingel, R. A. Kroczek, and W. M. Yokoyama Coordinate Expression of Cytokines and Chemokines by NK Cells during Murine Cytomegalovirus Infection J. Immunol., March 1, 2004; 172(5): 3119 - 3131. [Abstract] [Full Text] [PDF]
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J. Brady, Y. Hayakawa, M. J. Smyth, and S. L. Nutt IL-21 Induces the Functional Maturation of Murine NK Cells J. Immunol., February 15, 2004; 172(4): 2048 - 2058. [Abstract] [Full Text] [PDF]
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G. Ferlazzo and C. Munz NK Cell Compartments and Their Activation by Dendritic Cells J. Immunol., February 1, 2004; 172(3): 1333 - 1339. [Full Text] [PDF]
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 M. J. Loza and B. Perussia Differential regulation of NK cell proliferation by type I and type II IFN Int. Immunol., January 1, 2004; 16(1): 23 - 32. [Abstract] [Full Text] [PDF]
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M. Jinushi, T. Takehara, T. Tatsumi, T. Kanto, V. Groh, T. Spies, T. Suzuki, T. Miyagi, and N. Hayashi Autocrine/Paracrine IL-15 That Is Required for Type I IFN-Mediated Dendritic Cell Expression of MHC Class I-Related Chain A and B Is Impaired in Hepatitis C Virus Infection J. Immunol., November 15, 2003; 171(10): 5423 - 5429. [Abstract] [Full Text] [PDF]
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T. Ranson, C. A. J. Vosshenrich, E. Corcuff, O. Richard, W. Muller, and J. P. Di Santo IL-15 is an essential mediator of peripheral NK-cell homeostasis Blood, June 15, 2003; 101(12): 4887 - 4893. [Abstract] [Full Text] [PDF]
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M. Dalod, T. Hamilton, R. Salomon, T. P. Salazar-Mather, S. C. Henry, J. D. Hamilton, and C. A. Biron Dendritic Cell Responses to Early Murine Cytomegalovirus Infection: Subset Functional Specialization and Differential Regulation by Interferon {alpha}/{beta} J. Exp. Med., April 7, 2003; 197(7): 885 - 898. [Abstract] [Full Text] [PDF]
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K. Abel, L. Compton, T. Rourke, D. Montefiori, D. Lu, K. Rothaeusler, L. Fritts, K. Bost, and C. J. Miller Simian-Human Immunodeficiency Virus SHIV89.6-Induced Protection against Intravaginal Challenge with Pathogenic SIVmac239 Is Independent of the Route of Immunization and Is Associated with a Combination of Cytotoxic T-Lymphocyte and Alpha Interferon Responses J. Virol., March 1, 2003; 77(5): 3099 - 3118. [Abstract] [Full Text] [PDF]
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