Activation of Naive NK Cells in Response to
Listeria monocytogenes Requires IL-18 and
Contact with Infected Dendritic Cells
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J Immunol 2010; 184:5172-5178; Prepublished online 29
March 2010;
doi: 10.4049/jimmunol.0903759
http://www.jimmunol.org/content/184/9/5172
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References
Jessica Humann and Laurel L. Lenz
The Journal of Immunology
Activation of Naive NK Cells in Response to Listeria
monocytogenes Requires IL-18 and Contact with Infected
Dendritic Cells
Jessica Humann and Laurel L. Lenz
N
atural killer cells play an important role in innate immune
responses to tumors, viruses, and bacteria. Host cells
lacking MHC I, such as tumor cells, are targeted by NK
cells because of missing self-recognition (1). NK cells also recognize upregulated self and nonself molecules induced in response
to stress or infection, such as the NKG2D ligands RAE1 and
MULT1 in mice (2). Certain viral proteins also activate NK cells.
For example, the MCMV viral protein m157 is presented on the
surface of virally infected cells and is recognized by the NK receptor Ly49H (2). In addition to lysis of infected and tumor cells,
NK cells produce IFN-g. IFN-g promotes inflammatory and antibacterial responses by inducing other inflammatory chemokines
and cytokines and by eliciting NO and reactive oxygen species in
IFN-g–responsive cells (3).
Several cytokines are known to promote NK cell effector
mechanisms. For example, viral induction of type I IFNs (IFN-a
and -b) promotes NK cell cytotoxicity (4). Dendritic cell transpresentation of IL-15 has also been implicated recently in the
priming of NK cells to become fully activated for lysis and IFN-g
secretion (5). In addition, IL-12 promotes NK and Th1 type
T cells to produce IFN-g, in part via induction of the transcription
factor Tbet (6). TNF-a and TLR signaling through IRFs and NFkB enhances NK cell production of IFN-g in concert with IL-12 to
National Jewish Health and Integrated Department of Immunology, University of
Colorado, Denver, CO 80206
Received for publication November 25, 2009. Accepted for publication February 18,
2010.
This work was supported by National Institutes of Health Grant AI065638 (to L.L.L)
and National Institutes of Health Training Grants AI07505 and AI5206606 (to J.H.).
Address correspondence and reprint requests to Dr. Laurel L. Lenz, National Jewish
Health, 1400 Jackson Street, Room K-510, Denver, CO 80206. E-mail address:
[email protected]
Abbreviations used in this paper: BMDC, bone marrow-derived dendritic cell; MOI,
multiplicity of infection; NWNA, nylon wool nonadherent; wt, wild type.
Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903759
enhance NK cell activation (7, 8). NF-kB is also activated by
IL-1b and IL-18 through an MyD88-dependent pathway (9, 10).
IL-1b and IL-18 are synthesized as procytokines that are processed into their active forms by caspases 1 and 11 (11). Caspase1–deficient mice exhibit decreased IFN-g levels in response to
infection with the bacterial pathogen Listeria monocytogenes
(12), further implicating IL-1b, IL-18, or possibly other recently
identified caspase-1 substrates in NK cell responses to L. monocytogenes infection (13).
L. monocytogenes is a facultative intracellular bacterium that
replicates within the host cell cytosol and uses host actin machinery
to spread from cell to cell (14). L. monocytogenes requires the
hemolysin LLO to escape from phagosomes and enter the host cell
cytosol. Mice infected with LLO-deficient L. monocytogenes (Dhly
L. monocytogenes) do not produce IFN-g (15). Mice lacking the
ability to produce or respond to IFN-g fail to control L. monocytogenes expansion and succumb to normally sublethal doses as
early as 4 d after systemic inoculation (16, 17). Early production of
IFN-g is thought to promote a Th1 response required for efficient
clearance of the pathogen (18). Cells involved in the early production of IFN-g include NK and T cells (19), with the NK cells
being the major source of this cytokine during the first 2 d of
infection in C57BL/6 mice (20). However, previous data from
our laboratory and others suggest that activation of NK cells by
L. monocytogenes can promote virulence (20, 21). It is thus important to determine how NK cells are activated during infection
with live wild type (wt) L. monocytogenes.
One challenge in dissecting the requirements for NK cell activation during in vivo infection is defining whether various cytokines
primarily act on NK cells or other specific cell types. Although cell
culture models can be used to shed light on this issue, previous
in vitro studies have largely used NK cells expanded or cultured with
IL-15 or IL-2 prior to stimulation with killed infectious agents (8, 22,
23). To determine the factors necessary for activation of naive NK
cells during infection with live L. monocytogenes and to characterize the respective effects of such factors on NK cells or other cell
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The mechanisms for NK cell activation during infection by intracellular bacterial pathogens are not clearly defined. To dissect how
Listeria monocytogenes infection elicits NK cell activation, we evaluated the requirements for activation of naive splenic NK cells
by infected bone marrow-derived dendritic cells (BMDCs). We found that NK cell activation in this setting required infection of
BMDCs by live wild type bacteria. NK cells were not activated when BMDCs were infected with a live hemolysin deficient (Dhly)
strain. Neutralization of IL-12, TNF-a, or caspase-1 each dramatically reduced NK cell IFN-g production in response to live wt
L. monocytogenes infection. Addition of recombinant IL-18, but not IL-1b, reversed the effects of caspase-1 inhibition. Recombinant IL-18 also restored NK cell activation by BMDCs infected with Dhly L. monocytogenes, which produced IL-12 but not IL-18.
IL-18 acted on NK cells because MyD88 expression was required in responding NK cells, but not infected BMDC. However,
secreted cytokines were not sufficient for activation of naive NK cells by infected BMDCs. Rather, NK cell activation additionally
required contact between infected BMDCs and NK cells. These data suggest that the activation of NK cells during L. monocytogenes infection requires both secreted cytokines and ligation of NK activating receptors during direct contact with infected
DCs. The Journal of Immunology, 2010, 184: 5172–5178.
The Journal of Immunology
types, we developed a novel in vitro NK cell activation assay. Using
fresh NK cells isolated from naive mouse spleens and infected
bone marrow-derived dendritic cells (BMDCs), we were able to
reproducibly induce activation of a large fraction of NK cells. With
this assay system we confirmed that cytokines such as IL-12 and
IL-18 are essential for potent NK cell activation by L. monocytogenes infection. We further demonstrated that LLO expression
by L. monocytogenes is required to trigger NK cell activation via
activation of caspase-1 and the subsequent production of IL-18.
However, our findings also revealed that cytokines alone are not
sufficient to drive NK cell activation by live L. monocytogenes
infection. Rather, we found that cell contact between naive NK
cells and infected BMDCs was essential for efficient L. monocytogenes-induced NK cell production of IFN-g. This requirement
for NK cell contact with infected DCs is not explained by MHC I
downregulation or IL-15 trans-presentation by BMDCs, and instead appears to reflect the involvement of an infection-induced
NK cell activating ligand(s).
Mice
Rag12/2IL-15Ra2/2 and MyD882/2 mice crossed onto the B6 background for at least 10 generations were used as sources for bone marrow
stem cells to cultivate BMDCs, to isolate spleen NK cells, and for in vivo
mouse infections. C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Rag12/2IL-15Ra2/2 mice were obtained from
Dr. Steve Jameson (University of Minnesota). MyD882/2 mice were obtained from Dr. Christina Leslie (National Jewish Health). Bone marrow
and spleens from IL-12R2/2 mice were obtained from Dr. Ross Kedl
(University of Colorado). Mice were housed in the National Jewish Health
Biological Resource Center, and all animal studies were approved by the
National Jewish Health Institutional Animal Care and Use Committee.
Mouse infections
Female C57BL/6 and MyD882/2 mice between 8 and 10 wk of age were
used for all in vivo experiments. Mice were infected with 3 3 104 CFU of
log-phase mouse-passaged L. monocytogenes strain 10403S via intraperitoneal injection. At the indicated times postinfection, sera and
spleens were harvested for analysis. Spleens were processed into singlecell suspensions for staining and flow cytometry.
Adoptive cell transfers
NK cells were isolated from C57BL/6 mouse spleens via nylon-wool
column enrichment followed by depletion of CD3, CD19, CD11c, and
CD11b cells using PE-conjugated Abs (BD Biosciences, San Jose, CA) and
anti-PE magnetic beads (Miltenyi Biotech, Auburn, CA). The resulting NK
cell population was ∼85% pure; 1.5 3 106 purified NK cells were injected
into recipient MyD882/2 mice at the indicated time before infection.
Cell culture
For BMDCs, bone marrow cells were flushed from both femurs of one
mouse and cultured for 7 d in RPMI 1640 media supplemented with 10%
FBS, 1% sodium pyruvate, 1% L-glutamine, 1% penicillin/streptomycin, 2ME, and ∼2% GM-CSF (B78hi hybridoma supernatants). During culture,
media were changed at days 2 and 4. Nonadherent cells were plated on day
7 for use in experiments in vitro.
Infection and stimulation of cultured BMDCs
Following overnight culture in antibiotic-free media, BMDCs were infected
with L. monocytogenes strain 10403S or an isogenic L. monocytogenes
strain with an in-frame deletion of the hly gene coding for LLO (Dhly).
Strains were originally obtained from Dr. Daniel Portnoy (University of
California–Berkeley). BMDCs were infected with wt L. monocytogenes at
a multiplicity of infection (MOI) of 1 and Dhly L. monocytogenes at an
MOI of 20, unless otherwise indicated. LPS was used in some experiments
at 10 ng/ml. At 1 h postinfection, fresh media and 10 mg/ml gentamicin
were added to each well to kill extracellular bacteria.
Isolation of NK cells
Spleens were harvested from uninfected naive mice and processed into
single cell suspensions. Cell suspensions were added to nylon wool columns
and incubated for 1 h at 37˚C. Column elutions were collected, and cells
were stained for NK 1.1 (PK136) and CD3 (145-2C11). NK cells composed ∼6% of the cell population collected from the columns, compared
with 2% from total spleen preparations. For pure NK cells, nylon wool
nonadherent (NWNA) cells were sorted using NK1.1 (PK136) and CD3
(145-2C11) Abs on a MoFlo XDP sorter (Beckman Coulter, Brea, CA) to
achieve .97% NK cell purity. All Abs were obtained from BD Biosciences and eBioscience.
Coculture of NK cells and BMDCs
BMDCs were infected with the indicated L. monocytogenes strains. At 2 h
postinfection, NK cells were added to the infected BMDCs at a ratio of
0.1:1 and allowed to incubate in coculture for up to 19 h. At the indicated
time points, cells and/or supernatants were collected for analysis. For cell
staining, NK1.1 (PK136), CD3 (145-2C11), and IFN-g (XMG1.2) Abs
were used. Cells were fixed and permeabilized using saponin and paraformaldehyde buffers. ELISAs were performed using kits for murine IL-1b,
TNF-a, IL-12p70, and IFN-g (BD Biosciences), and IL-18 (MBL International, Woburn, MA). Anti–IL-12 (C17.8), anti–TNF-a (XT.11), rIL18 (MBL International), rIL-1b (R&D Systems, Minneapolis, MN), control
mouse IgG2a (BD Biosciences), control caspase inhibitor FA-FMK (R&D
Systems), caspase-1–specific inhibitor Z-WHED-FMK (R&D Systems),
and pan-caspase inhibitor ZVAD-FMK (R&D Systems) were used at the
indicated concentrations. Anti-mouse IL-18 (93-10C) (MBL International)
and anti-mouse ICAM (R&D Systems) were also used at the indicated
concentrations.
Statistics
All conditions were tested in triplicate in each experiment, and each experiment was repeated three times. Data shown are a representative experiment. Statistics were done for each experiment using a Student t test.
Significance was determined to be p # 0.05.
Results
Coculture of naive NK cells and BMDCs infected with live,
cytosolic L. monocytogenes elicits IFN-g production
We previously showed that up to 60% of splenic NK1.1+CD32 NK
cells produce IFN-g within 24 h of wt L. monocytogenes infection
in C57BL/6 (20). We sought to evaluate the mechanism for this
bulk activation of NK cells. We used freshly isolated naive splenic
lymphocytes (NWNA) as our source of NK cells. CD11b+CD11c+
BMDCs were used as the infected cell population (Fig. 1A).
BMDCs were infected with wt L. monocytogenes at an MOI of 1.
Postinfection, NWNA cells were added into the culture. At 19 h
postinfection, the cultures were analyzed for IFN-g production by
both intracellular cytokine staining and ELISA of cell supernatants.
BMDCs infected with wt L. monocytogenes triggered .40% of the
NK cells in the NWNA population to produce IFN-g (Fig. 1B, 1C).
However, heat-killed wt L. monocytogenes (data not shown) and
LLO-deficient L. monocytogenes (Dhly L. monocytogenes) used at
an MOI of 20 failed to induce significant IFN-g in parallel cocultures. IFN-g production by the NK cells peaked by 12 h of coculture with infected BMDCs (Fig. 1D). These data indicate that
cytosolic L. monocytogenes infection of BMDCs is both necessary
and sufficient to activate naive splenic NK cells to produce IFN-g
during in vitro cocultures.
Cell staining revealed that NWNA cell preparations contained
NK cells (∼6%), T cells (∼70%), a few contaminating B cells, and
monocytes (Fig. 1A and data not shown). Depletion of CD3+ cells
from NWNA using magnetic bead separation did not affect IFN-g
production in cocultures with infected BMDC (data not shown),
suggesting that T cells did not significantly contribute to the IFN-g
produced in response to infected BMDCs. To confirm that only
NK cells within the NWNA populations produced IFN-g in response to live wt L. monocytogenes infection, we sorted NWNA
cells into three populations: NK1.1+CD32 (NK cells), CD3+
(T cells), and CD32NK1.12 (other cells). All sorted populations
were .97% pure for their respective cell type (Fig. 2A and data
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Materials and Methods
5173
5174
IL-18 AND CELL CONTACT ACTIVATE NAIVE NK CELLS
FIGURE 1. NK cells produce IFN-g
in vitro in response to coculture with L.
monocytogenes-infected BMDCs. A,
CD11c+CD11b+ BMDCs were derived
from bone marrow cells cultured in GMCSF. Spleen cells were processed via
nylon wool columns to enrich for lymphocytes. These NWNA cells were cocultured with wt L. monocytogenes or
Dhly L. monocytogenes-infected BMDC
for 19 h. B and C, NK cells produced
IFN-g only in response to wt L. monocytogenes infection as measured by intracellular staining and ELISA. D,
Kinetics of IFN-g production by NWNA
responding to wt L. monocytogenes in
infected BMDCs as measured by ELISA.
Proinflammatory cytokines produced by infected BMDCs
contribute to NK cell activation
Several proinflammatory cytokines previously implicated in
the induction of IFN-g by NK cells were also produced by L.
monocytogenes-infected cocultures. As measured by ELISA, wt L.
monocytogenes infection of BMDCs induced secretion of nanogram concentrations of IL-12p70, TNF-a, and IL-1b, and picogram concentrations of IL-18 by 19 h of coculture with NWNA
cells (Fig. 3A–D). In contrast, Dhly L. monocytogenes infection of
BMDCs, which failed to induce IFN-g (Fig. 1C), induced high
levels of IL-12p70 and TNF-a, but less IL-1b and no IL-18
(Fig. 3A–D).
LPS stimulation of BMDCs induces a similar proinflammatory
cytokine profile to Dhly L. monocytogenes infection of BMDCs
(24). We thus compared LPS stimulation of BMDCs with L.
monocytogenes infection of BMDCs to determine which cytokines
were necessary for NK activation. After 19 h of stimulation, LPS
induced high levels of IL-12p70, but TNF-a and IL-1b concentrations were low when compared with wt L. monocytogenes infection (Fig. 3A–D). However, there was a substantial level of
TNF-a (nanogram concentrations) after 6 h of LPS treatment
(data not shown). LPS stimulation of BMDCs also poorly induced
IFN-g (Fig. 3E). These data indicate that neither LPS nor a noncytosolic L. monocytogenes infection were sufficient to induce
naive NK cells to make IFN-g.
To further determine the relative importance of BMDC-derived
cytokines for naive NK cell activation, we inhibited IL-12 and
TNF-a using neutralizing mAbs at a concentration of 10 mg/ml.
We blocked caspase activation with the pan-caspase inhibitor
Z-VAD-FMK. In each case, IFN-g production was significantly
attenuated (Fig. 4). Neutralization of IL-12 did not affect the
levels of TNF-a produced by infected BMDCs, and vice versa
(data not shown). These results suggest that IL-12, TNF-a, and
caspase-1 activation are all involved in naive NK cells production
of IFN-g. However, eliminating individual cytokines or caspases
failed to prevent NK cell activation.
Role of Il-18 in L. monocytogenes-induced NK cell activation
Given that IL-18 was produced only during wt L. monocytogenes
infection of BMDCs, and only wt L. monocytogenes potently induced IFN-g production by naive NK cells, we hypothesized that
caspase-1 activation and release of IL-18 might act as a limiting
factor in naive NK cell activation. To test this, we pretreated wt
L. monocytogenes-infected BMDCs with the irreversible caspase-1
specific inhibitor Z-WHED-FMK. Z-WHED-FMK treatment of
infected BMDCs reduced IL-1b levels in cell culture supernatants
as measured by ELISA (data not shown). The BMDCs were
washed after inhibitor treatment and incubated with NWNA
cells. Inhibition of caspase-1 activity in the BMDCs prior to coculture reduced NWNA IFN-g production to basal levels (Fig.
5A). Furthermore, addition of recombinant IL-18 reversed the
effects of Z-WHED-FMK on IFN-g production, whereas addition
FIGURE 2. Purified NK cells are solely responsible for IFN-g production in response to L.
monocytogenes-infected BMDCs. A, NWNA cells
were isolated as in Fig. 1 and sorted into three populations: NK1.1+CD32 (NK), CD3+NK1.12 (T), and
CD32NK1.12 (other), each population .90% pure.
B, Sorted NK cells produce IFN-g in response to coculture with wt L. monocytogenes-infected BMDCs.
T cells or other cells alone do not produce IFN-g.
Addition of either of the other sorted populations does
not enhance IFN-g production in cocultures.
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not shown). Each sorted cell type was then cocultured with wt
L. monocytogenes-infected BMDCs for 19 h. Neither CD3+ nor
CD32NK1.12 cells produced IFN-g in coculture. In contrast,
sorted NK1.1+CD32 cell populations produced significant amounts
of IFN-g (Fig. 2B). Furthermore, addition of purified CD3+ or
CD32NK1.12 cells to the sorted NK cells did not affect the ability
of NK cells to produce IFN-g (Fig. 2B). Because NK cell activation
did not require help from T cells or other cell populations present in
the NWNA preparations, we used this in vitro system to further
study the requirements for activation of naive NK cells during live
L. monocytogenes infection.
The Journal of Immunology
5175
of Dhly L. monocytogenes to induce NK cell IFN-g. Indeed, when
recombinant IL-18 was added to Dhly L. monocytogenes infected
cocultures, IFN-g secretion by NWNA cells was restored to levels
seen with wt L. monocytogenes infected BMDC cocultures (Fig.
5B). Hence, caspase-1 dependent release of IL-18 is crucial for
NK cell IFN-g production and the failure of Dhly L. monocytogenes to induce IL-18 secretion explains why infection with
this attenuated L. monocytogenes strain fails to activate naive
NK cells.
MyD88 signaling in NK cell activation
of recombinant IL-1b had no effect (Fig. 5A). Specific inhibition
of IL-18 with a neutralizing mAb also dramatically reduced IFN-g
production in cocultures (Fig. 5A). In the context of these results
and our above findings that Dhly L. monocytogenes infection induces IL-12p70 and TNF-a comparable to wt L. monocytogenes,
but reduced IL-1b and no measurable IL-18 (Fig. 3), we hypothesized that lack of IL-18 might be responsible for the failure
NK-DC contact is required for IFN-g production in response to
infection
FIGURE 4. Effects of cytokine inhibition on IFN-g production in response
to wt L. monocytogenes-infected BMDCs. Control IgG, anti–IL-12, and anti–
TNF-a were used at 10 mg, and the caspase inhibitor ZVAD-FMK and control
FA-FMK were used at 10 mM. Inhibition of IL-12, TNF-a, and caspases reduced the level of IFN-g produced in response to wt L. monocytogenesinfected BMDCs, indicating that each of these cytokines is necessary but not
sufficient for the activation of naive NK cells.
The data above indicate roles for IL-12, TNF-a, IL-1b, and IL-18 in
the activation of naive NK cells by wt L. monocytogenes infection.
To test whether these secreted cytokines were sufficient for L.
monocytogenes-induced NK cell activation, we transferred conditioned media from infected BMDCs onto uninfected BMDCs cultured with NWNA cells. Surprisingly, the transfer of conditioned
media from infected BMDCs failed to induce IFN-g production and
secretion (Fig. 7A). Furthermore, when a 0.5-mM membrane was
used to separate infected BMDCs from responding NWNA cells for
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FIGURE 3. Inflammatory cytokines produced during in vitro coculture
of BMDCs and NWNA cells in response to wt L. monocytogenes, Dhly L.
monocytogenes, or LPS. A–D, BMDCs were infected with wt L. monocytogenes or Dhly L. monocytogenes or were treated with 10 ng/ml LPS.
IL-12p70, TNF-a, IL-1b, and IL-18 were measured in supernatants after
19 h. Both Dhly L. monocytogenes and LPS failed to induce IL-18, but
they did induce varying levels of each of the other cytokines. E, Compared
with wt L. monocytogenes infection, Dhly L. monocytogenes and LPS fail
to induce significant levels of IFN-g in cocultures.
MyD88 is a well-known adaptor molecule for many TLRs. In
addition, IL-1R and IL-18R use MyD88 as an adaptor molecule for
downstream signaling (8). MyD882/2 mice exhibit reduced levels
of IL-12 and TNF-a and fail to make IFN-g in response to
L. monocytogenes infection (10). We sought to directly test
whether the reduced IFN-g production in MyD882/2 mice was
due to a requirement for TLR or IL-18 signaling in DCs, naive NK
cells, or both. We thus infected C57BL/6 and MyD882/2 BMDCs
with wt L. monocytogenes and cocultured them with either
C57BL/6 or MyD882/2 NWNA. Wt L. monocytogenes infection
induced similar IFN-g production by NWNA cells regardless of
MyD88 expression by the infected BMDCs (Fig. 5C). Conversely,
NWNA cells lacking MyD88 did not produce IFN-g in response to
wt L. monocytogenes-infected BMDCs, even when BMDCs expressed MyD88 (Fig. 5C). These data reveal that signaling via
MyD88 is necessary only in responding NK cells during infection,
likely because of its role in the NK cell response to IL-18 produced by infected dendritic cells.
To further examine the requirement and sufficiency of MyD88
signaling in NK cells during in vivo L. monocytogenes infection,
we established an adoptive transfer system using MyD882/2 mice.
On day 0, we isolated NK cells from the spleens of C57BL/6 mice
by negative selection, labeled them with CFSE, and transferred
them into recipient MyD882/2 mice. The purity of the transferred
NK cell population was ∼84% (data not shown). Recipient and
control mice were infected with L. monocytogenes on day 1, and
all mice were analyzed on day 2. At the time of harvest, we detected a significant population of CFSE-labeled NK cells in the
spleens of infected mice that had received the adoptive transfer
(Fig. 6A), indicating that the transferred cells were able to populate this organ. Analysis of the CFSE staining levels in the
transferred NK cells suggested that these cells failed to proliferate
within the first day of the L. monocytogenes infection (Fig. 6B).
However, the sera of infected MyD882/2 mice that had received
C57BL/6 NK cells exhibited a significantly increased IFN-g concentration when compared with control MyD882/2 mice (Fig.
6C). These findings confirm that expression of MyD88 by naive
NK cells is both necessary and sufficient to permit their activation
during L. monocytogenes infection. Furthermore, our data suggest
the requirement for MyD88 is due to its role in the response to
IL-18 rather than IL-1b or TLR agonists.
5176
IL-18 AND CELL CONTACT ACTIVATE NAIVE NK CELLS
FIGURE 5. IL-18 is necessary for IFN-g production by naive NK cells. A, Wt L. monocytogenes-infected BMDC cocultures were treated with the
caspase-1 specific inhibitor Z-WEHD-FMK at 100 mM. Caspase-1 inhibition abrogated IFN-g induction. Addition of 500 pg/ml exogenous IL-18 restored
IFN-g induction, but 2.5 ng/ml of exogenous IL-1b did not. Specific inhibition of IL-18 (1 mg/ml of aIL-18) significantly reduced IFN-g induction in
cocultures infected with wt L. monocytogenes. B, Addition of recombinant IL-18 to Dhly L. monocytogenes-infected cocultures restored IFN-g induction to
levels seen with wt L. monocytogenes infection. C, Wt L. monocytogenes induction of IFN-g requires MyD88 expression on NWNA. C57BL/6 and
MyD882/2 BMDC were infected with wt L. monocytogenes and cocultured with either C57BL/6 or MyD882/2 NWNA. Only cocultures with MyD882/2
NWNA failed to induce IFN-g in response to L. monocytogenes infection.
FIGURE 6. C57BL/6 NK cells can restore IFN-g production in infected
MyD882/2 mice. 1.5 3 106 CFSE-labeled C57BL/6 NK cells were
adoptively transferred into MyD882/2 mice before wt L. monocytogenes
infection. A, At 24 h postinfection, the transferred CFSE-labeled NK cells
were clearly visible in the spleens of L. monocytogenes-infected mice. B,
Transferred NK cells did not show evidence of proliferation with the first
day postinfection. In the total NK cell population, infected MyD882/2
mice that received C57BL/6 NK cells (black line) did not exhibit NK cell
proliferation compared with uninfected MyD882/2 mice that received
C57BL/6 NK cells (shaded). This finding was apparent in both the total
NK cell population and the population of NK cells gated as CFSE+. C,
IFN-g was undetectable in MyD882/2 and significantly higher in the sera
of MyD882/2 mice that received the transferred C57BL/6 NK cells, as
determined by the t test (p = 0.04).
obtained during cocultures with direct contact of infected BMDCs
and NWNA cells (data not shown). Thus, we concluded that both
cytokines and cell contact were necessary for NK cell IFN-g production in response to wt L. monocytogenes infection.
Given a previous report suggesting that cell contact was required
in DC priming of NK cells by IL-15 (25), we asked whether IL-15
trans-presentation might account for the contact-dependent interaction in our infection system. Consistent with the previous
work, we found that IL-15 trans-presentation was important for
NK cell priming, because infected IL-15Ra2/2 BMDC induced
lower amounts of NK cell IFN-g production than did control
C57BL/6 BMDC. However, wt L. monocytogenes infection of the
BMDCs still activated NK cell IFN-g production well above the
background level seen with the control DHly L. monocytogenes
infection (Fig. 8A). Thus, IL-15Ra expression by BMDCs contributed to priming of naive NK cells in our system, but failed to
fully account for the contact-dependent induction of IFN-g. To
examine cell contact requirements further, we asked whether
blocking cell–cell interactions using an mAb against ICAM reduced IFN-g production in the cocultures. We found that blocking
ICAM reduced NK cell activation and IFN-g production to a low
level. However, such blockade failed to fully abrogate production
of IFN-g, which was still modestly but significantly higher in the
anti-ICAM treated wt L. monocytogenes-infected cultures than
that seen in the DHly L. monocytogenes infected cultures for this
experiment (Fig. 8B). These data suggest that either ICAMspecific dendritic cell–NK cell interactions are directly involved in
the activation of naive NK cells or that aICAM interferes with the
ability of naive NK cells to interact with the infected BMDCs in
a manner that permits NK cell receptors to bind ligands induced
by the wt L. monocytogenes infection.
Discussion
Our results help to clarify the mechanisms for activation of naive
NK cells early after L. monocytogenes infection. We show that
infection of BMDCs with live wt L. monocytogenes potently activates naive NK cells to release IFN-g. The mechanism for such
activation involves the activation of caspase-1 and the release of
IL-18 by infected BMDCs. Caspase-1 activation in response to
L. monocytogenes infection is known to require the pore-forming
L. monocytogenes LLO protein (26). Our data show that Dhly L.
monocytogenes-infection of BMDCs is not sufficient to trigger
activation of naive NK cells. However, the Dhly L. monocytogenes
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the 19-h coculture, the NWNA cells also failed to produce IFN-g by
both intracellular staining and ELISA (Fig. 7B, 7C). ELISAs confirmed that proinflammatory cytokines such as IL-12 and IL-18
were present in these cultures at concentrations similar to those
The Journal of Immunology
5177
FIGURE 7. IFN-g induction by naive NK cells requires cell contact between NWNA cells and BMDCs.
A, BMDCs were infected with wt and Dhly L. monocytogenes for 8 h. Media were collected from the infected BMDCs and added to cultures of uninfected
BMDCs and NWNA cells. IFN-g production was
measured after 19 h by ELISA and intracellular staining. Infected BMDCs directly cocultured with NWNA
cells induced IFN-g. Infected BMDCs separated from
NWNA cells by a 0.4 mM trans-well did not induce
significant levels of IFN-g, either by (B) intracellular
staining or by (C) ELISA.
FIGURE 8. Inhibition of receptor-ligand binding impairs NK cell activation. A, C57BL/6 or Rag12/2/IL-15Ra2/2 BMDCs were infected with
wt or Dhly L. monocytogenes and cocultured with C57BL/6 NWNA for
19 h. IFN-g in supernatants was measured by ELISA. B, C57BL/6 BMDC
were infected with wt or Dhly L. monocytogenes and cocultured with
NWNA. In parallel, BMDCs infected with wt L. monocytogenes were also
treated with anti-mouse ICAM1 at 1 mg/ml prior to addition of NWNA
cells. Inhibition of ICAM1 severely attenuated the IFN-g response to infection as measured by ELISA.
IFN-g production (10), we show that transfer of C57BL/6 NK
cells is sufficient to partially restore IFN-g production. MyD88
expression by NK cells was also necessary and sufficient for their
IFN-g production in our coculture system. In contrast to MyD88,
our in vitro data reveal that IL-12R expression is required on both
DCs and NK cells for efficient induction of IFN-g (data not
shown). We speculate that IL-12 may likewise have roles on both
infected and responding cells during the activation of naive NK
cells during in vivo L. monocytogenes infection.
An additional conclusion from our studies is that soluble proinflammatory cytokines are not sufficient for activation of naive NK
cells in response to wt L. monocytogenes infection. Although naive
NK cells secrete IFN-g in our hands after extended culture in
recombinant IL-12 and IL-18 (data not shown), the rapid NK cell
IFN-g production seen in response to live wt L. monocytogenes
additionally requires direct cell contact between the infected
BMDCs and responding NK cells. Cell contact has also recently
been implicated as a requirement for activation of human NK cells
by macrophages infected with Salmonella (22) and RBCs infected
with Plasmodium (29). In the case of in vivo infection, cell contact
may explain why only 30–60% of splenic NK cells are activated in
response to wt L. monocytogenes (20). Cell contact likely occurs
within the lymphoid follicles of the spleen, because recent work
suggests that NK cells cluster near DCs and L. monocytogenes
organisms in these areas prior to producing IFN-g (30). The requirement for cell contact might be a way for the immune system
to localize NK cell activation within the lymphoid follicles. Such
localization might help reduce immunotoxicity and damage to
neighboring uninfected cells and could indicate an important role
for NK cells in regulating the function of other lymphocytes.
The specific mechanisms of cell contact in infection-induced NK
cell activation are still unclear. Some previous studies have suggested that IL-12 is secreted across a synapse between NK cells and
DCs (31). It has also been published that NK/DC interactions can
result in the directed release of IL-18 to NK cells (32). Such
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infection is sufficient when exogenous IL-18 is added. We also
show that TNF-a and caspase-1 activity contribute to NK cell
activation in this system. Although inhibition of either of these
molecules impairs IFN-g production, neither is essential for wt
L. monocytogenes-induced NK cell activation.
The precise roles of IL-12 and IL-18 in activation of naive NK
cells are not clear. Mice deficient in both IL-12 and IL-18 have
significantly impaired NK cell IFN-g activity compared with mice
deficient in either cytokine alone (27). Furthermore, previous
in vitro studies have indicated that IL-18 can act in synergy with
IL-12 to induce NK cell IFN-g production by L. monocytogenes
and other stimuli (9, 23, 28). One study suggested that IL-12 induces IFN-g transcription while IL-18 stabilizes these transcripts
and promotes translation of IFN-g (9). Our results reveal that
MyD88 signaling is essential in naive NK cells, likely owing to its
role in the response of these cells to IL-18. Although L. monocytogenes infection of MyD882/2 mice fails to induce NK cell
5178
Acknowledgments
We thank Rebecca Schmidt for critical reading of the manuscript and the
National Jewish Cytometry Core for help with NK cell sorting experiments.
Disclosures
The authors have no financial conflicts of interest.
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directed release of cytokines might increase their effective concentrations in cultures in which NK cells and DCs are allowed to
intimately associate with one another. Thus, one possible mechanistic explanation for the cell contact requirement to activate
NK cells is that the low level of IL-18 produced by wt L. monocytogenes infection (,500 pg/ml in vitro) is more efficiently presented to NK cells that are in close proximity to the infected DCs.
Numerous receptor–ligand interactions have been implicated in
the activation of NK cells. NKG2D is expressed by almost all NK
cells responding to L. monocytogenes infection (data not shown)
and is associated with cytokine production and cytotoxicity in
NK cells (33–37). However, our studies argue against a role for
NKG2D in NK cell activation, because blocking NKG2D had no
effect on IFN-g production in response to wt L. monocytogenes
infection (data not shown). Adhesion molecules such as ICAM
have been associated previously with NK cell activation during
Mycobacterium tuberculosis infection (38), and we also found that
blocking cell contact with an Ab to ICAM1 significantly reduced
NK cell IFN-g production in our coculture system. These findings
suggest that proximity between infected and responding cells is
not sufficient for NK cell activation. Rather, NK cell activation in
response to wt L. monocytogenes infection requires an association
between the NK cell and the infected BMDCs that can be disrupted by blockade of ICAM1. Whether the actual activating
stimulus received by the NK cell is due to ligation of ICAM1 or
other specific receptor-ligand pairs remains to be determined.
Our findings reveal that regulation of the NK cell response
involves a complex combination of priming, proinflammatory cytokine stimulation, and contact-dependent firing in response to
contact with L. monocytogenes infected DCs. Our in vitro assay
system can be used as a tool to further examine the precise mechanisms of NK cell activation by this, and possibly other, intracellular pathogens.
IL-18 AND CELL CONTACT ACTIVATE NAIVE NK CELLS