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CUTTING EDGE |
Production In Vivo1
*
Department of Internal Medicine, Division of Hematology/Oncology, and
Department of Molecular Virology, Immunology, and Medical Genetics, Division of Human Cancer Genetics, and the Comprehensive Cancer Center, Ohio State University, Columbus, OH 43210
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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. Such IFN- may then in turn sensitize macrophages to elicit
the Shwartzman reaction following a subsequent LPS challenge.
Supporting this, IL-12 and IL-15 synergized to induce murine NK cell
IFN- production in vitro. LPS stimulation of SCID mouse splenocytes
resulted in measurable IFN- production, which was reduced when IL-15
was neutralized or IL-2/15Rß was blocked. Pretreatment with either
anti-IL-2/15Rß or anti-IL-15 Abs reduced serum IFN-
protein following LPS administration to SCID mice. Collectively, these
data provide the first in vivo evidence that IL-15 participates in
LPS-induced innate immune IFN- production and significantly
contributes to the lethal Shwartzman reaction. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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-chain for binding and signaling (2, 3, 4, 5).
However, the IL-2 and IL-15 receptor complexes each contain unique
-chains that confer specific high-affinity binding (6).
As IL-15 is produced by macrophages following stimulation with
bacterial components in vitro, it has been proposed to have a role in
the early innate proinflammatory response to infection (7, 8).
The generalized Shwartzman reaction is a lethal cytokine-induced shock
response elicited by sequential priming and challenge with bacteria or
bacterial components (e.g., LPS) originally identified in rabbits and
later in mice and humans (9, 10). IL-12-induced IFN- is
critical for sensitization of macrophages during LPS priming
administered s.c. in the footpad (11, 12). After initial
IFN--dependent priming, a subsequent i.v. LPS challenge 18–24 h
later results in cytokine-induced shock and mortality, largely due to
the release of TNF- and IL-1 (11, 12, 13). The monokines
that may act synergistically with IL-12 to induce the IFN- important
for LPS priming of the Shwartzman reaction have yet to be fully
characterized.
NK cells are a critical component of the innate immune response to
infection, commonly through their elaboration of IFN- before
development of an effective adaptive immune response (14, 15). IL-15, acting through the IL-15Rß, induces human NK
cell proliferation, cytotoxicity, and synergizes with IL-12 to
stimulate production of IFN-, TNF-, and macrophage inflammatory
protein-1 in vitro (5, 16). Coculture of LPS-activated
human macrophages and NK cells results in abundant IFN- production
that is partially dependent upon IL-15 (7). In this innate
immune cytokine loop, LPS-stimulated IL-15 acts in concert with other
monokines (e.g., IL-12) to stimulate IFN- production by human NK
cells in vitro. NK cells have been implicated as important contributors
of IFN- during priming of the generalized Shwartzman reaction in
mice (17) and contribute to IFN- production and
lethality in lymphocytic choriomeningitis virus-infected mice
challenged with LPS (18).
In the current study, we demonstrate that neutralization of IL-15
during the priming phase of the generalized Shwartzman reaction
provides protection from this lethal process. We also show that
endogenous IL-15 is critical for optimal IFN- production by
LPS-challenged SCID mice in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Purified cytokines were provided: recombinant murine IL-15
(Immunex, Seattle, WA); recombinant human IL-2 (Hoffmann-LaRoche,
Nutley, NJ); and recombinant murine IL-12 (Genetics Institute,
Cambridge, MA). Anti-mouse IL-2/15Rß (TM-ß1) Ab (19)
and rat IgG2b control were purified from hybridoma supernatants.
Additional reagents include sheep anti-mouse IL-12 Ab and sheep IgG
control (Genetics Institute), rabbit anti-mouse IL-15 antiserum
(Immunex), and normal rabbit serum control, LPS from Escherichia
coli (Difco, Detroit, MI). Rat anti-mouse IL-2 Ab and isotype
control, anti-mouse IFN--FITC, anti-DX5 (PAN-NK)-PE, and
PE/FITC-conjugated isotype control Abs were purchased from PharMingen
(San Diego, CA). C.B.-17 SCID mice (Taconic, Germantown, NY) and
IL-2-/- mice (The Jackson Laboratory, Bar
Harbor, ME) were housed in specific pathogen-free animal facilities.
All experiments were performed under approved Ohio State University
University Laboratory Animal Resources protocols.
Induction of the generalized Shwartzman reaction
SCID mice (6-wk-old females) were given a s.c. 5-µg priming dose of LPS, and 24 h later were given an i.v. 50-µg LPS challenge dose. These doses were established to induce 80–100% mortality within 24 h following the LPS challenge (data not shown). Mice (n = 11/group) were pretreated (i.p.) with the anti-mouse IL-15 antiserum or control 1 h before the priming dose of LPS. The specificity of the anti-mouse IL-15 antiserum was confirmed through neutralization of CTLL-2 proliferation stimulated by rIL-15, but not by rIL-2 (data not shown). All injections were performed in a blinded fashion.
Isolation and stimulation of SCID splenocytes in vitro
SCID mouse spleens were harvested, processed into a single-cell
suspension, and stimulated with medium (RPMI 1640 plus 10% FCS) alone,
recombinant murine IL-15 (1 ng/ml), recombinant murine IL-12 (10
ng/ml), or IL-15 plus IL-12 (5 x 105/well).
These concentrations were chosen based upon their ability to
synergistically induce IFN- production by SCID mouse splenocytes,
while inducing little or no IFN- when used alone in repeated
experiments. Some splenocyte preparations were stimulated with LPS (10
µg/ml) or PBS (control). After 48 h, supernatants were harvested
and assayed for murine IFN- by ELISA (BioSource International,
Camarillo, CA; sensitivity 10 pg/ml). Some preparations were
preincubated with anti-mouse IL-2 Ab or control (50 µg/ml),
anti-mouse IL-2/15Rß or control (50 µg/ml), anti-mouse
IL-12 or control (12 µg/ml), or anti-mouse IL-15 or control
(1:100 dilution), before stimulation.
Inhibition of LPS-induced IFN- in vivo
SCID or IL-2-/- mice (n
= 5–6) were injected i.v. with LPS (400 µg/SCID mouse and 600
µg/IL-2-/- mouse), blood was collected 6
h after challenge, and serum was assayed for IFN- by ELISA. In some
experiments, groups of mice were injected i.p. with
anti-IL-2/15Rß or control Ab (100 µg/mouse) and anti-IL-15
antiserum or control 1 h before LPS challenge. Splenocytes were
obtained 6 h after LPS injection, cultured in brefeldin-A (10
µg/ml; Sigma, St. Louis, MO) and LPS (10 µg/ml) for 4 h to
allow IFN- protein to accumulate in the golgi, harvested, stained,
and analyzed for intracellular IFN- as described previously
(20). No IFN- was detectable by flow cytometry in
splenocytes from PBS-treated mice cultured in brefeldin-A and LPS for
4–24 h (data not shown), suggesting that differences in IFN-
observed in LPS-injected mice were due to in vivo activation.
Real-time PCR quantitation of cytokine transcripts in vivo
Real-time PCR (PE Applied Biosystems, Foster City, CA; TaqMan technology) is a novel method that allows an accurate and precise quantitation of gene transcripts through measurement of target amplification during (i.e., in real time) the reaction using fluorochrome-labeled probes (21). Groups of SCID mice (n = 5/time point) were injected i.v. with LPS (400 µg/mouse), and spleens were snap-frozen at the indicated time points after LPS challenge. RNA isolation, RT, and real-time PCR assays were performed as described (20) with modifications to specifically quantitate murine cytokine transcripts (21). Final quantitation is reported as the fold difference relative to a calibrator cDNA (untreated SCID splenocytes) prepared in parallel with the experimental cDNAs.
Statistical analysis
Experimental groups were compared by the Student’s t test with p < 0.05 considered significant. Survival (Kaplan-Meier) significance was determined by the log rank test.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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We hypothesized that IL-15 may contribute to IL-12-induced IFN-
in the Shwartzman reaction and therefore tested whether administration
of an anti-IL-15 antiserum affected mortality during this lethal
response. Pretreatment of SCID mice with anti-IL-15 antiserum
before LPS priming provided significant protection from lethality after
the subsequent i.v. LPS challenge, compared with the control (Fig. 1
, p = 0.0172). We next
performed a series of experiments to determine whether LPS-induced
IL-15 costimulated IFN- production.
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production
by SCID mouse splenocytes in vitro
Stimulation of resting SCID mouse splenocytes (macrophages and NK
cells) with IL-15 or IL-12 alone induced little or no IFN-
production, while costimulation with IL-15 plus IL-12 resulted in
abundant IFN- protein (Fig. 2A). We next cultured resting
SCID splenocytes in the presence or absence of LPS to examine whether
endogenous IL-15 could costimulate innate immune IFN- production.
Resting SCID splenocytes produced no IFN-, while identical
LPS-stimulated cultures produced IFN- protein (Fig. 2B).
To determine whether endogenous production of IL-15 contributed to the
in vitro IFN-, splenocytes were preincubated with
anti-IL-2/15Rß, anti-IL-15, anti-IL-12, or anti-IL-2
Abs or appropriate controls and then stimulated for 48 h with LPS
(Fig. 2B). Preincubation with controls or the anti-IL-2
Ab had no effect on LPS-induced IFN- (n = 5). In
contrast, preincubation with anti-IL-2/15Rß Ab (50.7 ±
6.2% decrease, p < 0.04, n = 5) or
anti-IL-15 antiserum (64.4 ± 9.6% decrease,
p < 0.02, n = 3) resulted in
significantly lower amounts of IFN-. The anti-IL-12 Ab also
abrogated LPS-induced IFN- in vitro (61.3 ± 4.1% decrease,
p < 0.01, n = 5), confirming previous
results demonstrating a role for IL-12 in NK cell IFN- production
(22, 23). Preincubation with both anti-IL-15 and
anti-IL-12 Abs reduced IFN- to nearly undetectable levels
(p < 0.03, n = 3). These data
suggest that endogenous IL-15 protein, in combination with IL-12, is
produced in vitro by LPS-activated macrophages and contributes to
IFN- production.
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The expression of IL-15, IL-12 p40, IL-2, and IFN- mRNA was
quantitated in vivo following LPS injection of SCID mice by real-time
RT-PCR (20, 21). While there was no change in cytokine
gene expression following PBS administration to SCID mice, LPS-induced
IL-15 transcript in the spleen increased 
6-fold with a peak at
3 h. This induction of IL-15 partially overlapped with that of
IL-12, which increased 60-fold peaking at 1 h. Importantly, the
first increase in IFN- mRNA (3 h) follows simultaneous elevation of
IL-12 and IL-15 gene expression. IL-2 gene expression was unchanged at
all time points (data not shown). Thus, the time course of IL-12 and
IL-15 expression in vivo supports a role for these monokines during
LPS-induced IFN- production.
Blockade of the IL-2/15Rß reduces LPS-induced IFN- in vivo
As IL-15 was important for optimal LPS-induced IFN- production
by SCID mouse splenocytes in vitro, and LPS increased IL-15 transcript
in vivo, we next tested whether endogenous IL-15 was important for
IFN- production in vivo. We first targeted the IL-2/15Rß, one
signaling component of the IL-15R complex (2, 4), with an
anti-IL-2/15Rß Ab that blocks ligand binding (19).
Injection of SCID mice with LPS resulted in measurable serum IFN-
production that peaked 6 h postinjection and was unaffected by
pretreatment with PBS (data not shown). However, pretreatment with
anti-IL-2/15Rß Ab significantly decreased serum IFN- measured
in response to LPS (54.8 ± 9.4% decrease, p <
0.02), compared with control Ab (Fig. 3A). As IL-15 shares the
IL-2/15Rß with IL-2, we also examined IL-2-/-
mice pretreated with the anti-IL-2/15Rß Ab for an effect upon
LPS-induced IFN-. A similar decrease in LPS-induced IFN- was
observed in IL-2-/- mice (Fig. 3B;
53.4 ± 8.3% decrease, p < 0.02), indicating
that the cytokine binding to the IL-2/15Rß-chain in this system was
not IL-2. Importantly, during the time course of this experiment,
administration of the anti-IL-2/15Rß Ab did not significantly
change the percentage of DX5+ NK cells present in
the spleen, as assessed by flow cytometry (data not shown). The data
presented in Fig. 3 suggest that an LPS-induced factor that requires
the IL-2/15Rß for signaling, but not IL-2, contributes to LPS-induced
IFN- in vivo. We next confirmed that this factor was IL-15.
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production in vivo
We tested whether LPS-induced IL-15 contributed to the IFN-
response in vivo by pretreating SCID mice with the anti-IL-15
antiserum or control before challenge with LPS. Pretreatment with
anti-IL-15 significantly decreased the serum IFN- measured
following LPS challenge in vivo (66.3 ± 7.0% decrease,
p < 0.004, n = 5) compared with
pretreatment with control (Fig. 4A). We also examined SCID
mouse DX5+ splenocytes after in vivo LPS
administration for production of intracellular IFN-. Pretreatment
with anti-IL-15 antiserum (n = 5) significantly
decreased the percentage of DX5+ splenocytes
producing IFN- (41.2 ± 8.5% decrease, p <
0.008) and the DX5+ IFN- mean fluorescence
intensity (54.2 ± 8.3% decrease, p < 0.004)
induced by LPS in vivo, compared with mice pretreated with control
(Fig. 4B and data not shown).
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production in vivo following
certain infectious insults. As IFN- is the prototypic
macrophage-activating factor (28), such NK-derived IFN-
may feedback to further activate the LPS-stimulated macrophage in vivo.
These results clarify how neutralization of IL-15 during LPS priming of
the Shwartzman reaction may reduce IFN- production and ultimately
enhance survival to subsequent LPS challenge.
While our data show that IL-15 is required for optimal IFN-
production in response to LPS in vivo, it is likely that other
LPS-induced monokines (e.g., IL-18 and TNF-) also contribute to
generate IFN- in vivo (20, 22, 23, 29). We also
examined whether IL-15 may be operating through the induction of IL-18
and found that while LPS challenge induced measurable IL-18 protein in
the serum of SCID mice, there was not a significant difference
comparing groups of mice pretreated with the anti-IL-15 antiserum
(2449 ± 231 pg/ml) or control (2607 ± 490 pg/ml). One
possible explanation for these observations is that IL-15 may be
required for IL-12 to efficiently induce IFN-, regardless of other
operative costimuli (e.g., IL-18), a hypothesis currently being
evaluated. In addition, our data suggest that the host immune response
to infection may be augmented through the supply of exogenous IL-15,
especially for those pathogens that require IFN- for effective
clearance. This is supported by a report showing that rIL-15 augments
IFN- production by SCID splenocytes stimulated with T.
gondii in vitro (30).
In conclusion, we provide in vivo evidence that IL-15 participates in
the innate, proinflammatory response leading to IFN- production in
SCID mice. Consistent with these results, neutralization of IL-15
during LPS priming of the generalized Shwartzman reaction protected
mice against mortality. Our results suggest that IL-15 may be
considered a proinflammatory cytokine produced by macrophages to
activate NK cells. Therefore, IL-15 may be a therapeutic target to
manipulate the innate immune response, to either augment host defense
or diminish excessive immune activation.
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Acknowledgments |
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Footnotes |
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2 T.A.F. and H.Y. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Michael A. Caligiuri, Ohio State University, 458A Starling-Loving Hall, 320 West 10th Avenue, Columbus, OH 43210. E-mail address:
Received for publication July 23, 1999. Accepted for publication December 13, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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chains of the IL-2 receptor by the novel cytokine IL-15. EMBO J. 13:2822.[Medline]
chain of the IL-2 receptor. EMBO J. 14:3654.[Medline]
by natural killer cells in vitro. J. Clin. Invest. 96:2578.
, and tumor necrosis factor are the key cytokines of the generalized Shwartzman reaction. J. Exp. Med. 180:907. production and lethality in lipopolysaccharide-induced shock in mice. Eur. J. Immunol. 25:672.[Medline]
). Curr. Opin. Immunol. 9:17.[Medline]
in response to monocyte-derived cytokines. J. Clin. Invest. 97:2722.[Medline]
production. J. Immunol. 162:5238. are costimulators of interferon production by natural killer cells in severe combined immunodeficiency mice with listeriosis, and interleukin 10 is a physiologic antagonist. Proc. Natl. Acad. Sci. USA 90:3725. by an intracellular parasite and induces resistance in T-cell-deficient hosts. Proc. Natl. Acad. Sci. USA 90:6115.-chain administration prevents murine collagen-induced arthritis: a role for IL-15 in development of antigen-induced immunopathology. J. Immunol. 160:5654.2a protein blocks delayed-type hypersensitivity. J. Immunol. 160:5742.This article has been cited by other articles:
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