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Department of Molecular Microbiology and Immunology, Division of Biology and Medicine, Brown University, Providence, RI 02912
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production and downstream antiviral defenses
during innate immune responses. Even though lymphocytic
choriomeningitis virus (LCMV) can be sensitive to IFN--mediated
antiviral effects, infections with this agent do not elicit IL-12 or
early IFN- in immunocompetent hosts. Studies presented here
demonstrate that LCMV infections of mice not only fail to induce IL-12,
but also modify responsiveness to exogenous IL-12 for IFN-
production. IFN- responses induced by IL-12 administration were
greatly diminished in splenic populations, but significantly increased
in serum and hepatic leukocytes, during the early course of LCMV
infections. The IFN- production was NK cell dependent, and the
compartmental dichotomy between spleen and liver was also demonstrated
in response to in vitro IL-12 stimulation. Although infections did
increase proportions and numbers of liver NK cells, changes in
responsiveness for IFN- expression could not be explained by cell
redistribution. Corroborating changes in proportions of NK cells
induced to express intracellular IFN- protein within the
compartments were observed. The reduction in ability of splenic
populations to produce IL-12-induced IFN- after infection by LCMV
was associated with decreased efficacy of administered IL-12 for
promoting IFN--dependent antiviral effects in the spleen.
Concomitantly, the maintenance of hepatic population IFN- production
was associated with preserved efficacy of administered IL-12 to elicit
IFN--dependent antiviral effects in the liver. Taken together, these
results demonstrate modifications of compartmental responses to IL-12
by viral infections and the consequences of these changes for efficacy
of cytokine therapy. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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synthesis, and endogenous
expression of IL-12 is critical for NK cell IFN- production during
innate responses to a variety of agents (1, 2, 3, 4, 5, 6). However,
early IL-12 and IFN- are induced in some, but not all, viral
infections. In particular, the cytokine proteins are not readily
detectable during infections of immunocompetent mice with lymphocytic
choriomeningitis virus
(LCMV)3 (4, 5, 7, 8), even though this virus can be sensitive to
IFN--dependent antiviral effects (9). The lack of early
IFN- expression is associated with a lack of demonstrable role for
NK cells in defense against this virus (4, 5, 7, 8). It is
not clear whether the absence of NK cell IFN- is simply a result of
IL-12 absence and/or a consequence of other virus-induced changes. If
the lack of a response is exclusively the result of poor IC-12
induction, administration of the cytokine may provide a therapeutic
modality for inducing NK cell IFN- and promoting resistance at early
times of infection.
The studies presented here were undertaken to evaluate efficacy of
administered IL-12 for promoting NK cell IFN- responses and
downstream antiviral effects during LCMV infections. The results
demonstrate compartmental differences between splenic and hepatic
leukocytes for IFN- induction by IL-12, with an infection-induced
loss of splenic, but not hepatic, leukocyte IL-12 responsiveness for
IFN- production. The decrease in splenic cell responsiveness was
associated with diminished efficacy of IL-12 intervention to induce
antiviral states, whereas maintenance of hepatic cell responsiveness
was associated with preserved antiviral efficacy. This striking
dichotomy in compartmental IFN- production and antiviral efficacy in
response to exogenous IL-12 suggests differential pathways regulating
NK cells in specific microenvironments and has implications for
cytokine therapy for eliciting effects at particular sites.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male C57BL/6 (Taconic Laboratory Animals and Services,
Germantown, NY) and T and B cell-deficient C57BL/6J-SCID and C57BL/6J
immunocompetent mice (The Jackson Laboratory, Bar Harbor, ME) were
purchased for use in these studies. Also used were E26 mutant mice,
containing high copy numbers of human CD3
transgenes and deficient
in NK and T cells (10), and
C57BL/6J-IFN--/- mice (11) (a
generous gift from Herbert W. Virgin, IV, Washington University, St.
Louis, MO) maintained through brother x sister matings in
breeding facilities at Brown University (Providence, RI). All mice were
used between 5 and 9 wk of age. Experiments were conducted in
accordance with institutional guidelines for animal care and
use.
In vivo treatment protocols
Infections were established by i.p. injection with 2 x
104 PFU of LCMV Armstrong strain, clone E350.
This isolate of LCMV replicates to high titers in spleen, but is
relatively nonhepatotropic. Some studies used 2 x
102 PFU of the Armstrong or the hepatotropic LCMV
isolate, WE, for establishing infections. Murine recombinant IL-12
(12) (sp. act. 4.9 x 106
U/mg), a gift from Genetics Institute (Andover, MA), was diluted in
vehicle (PBS supplemented with 5% heat-inactivated C57BL/6 mouse serum
from Taconic Laboratory Animals and Services) and administered i.p. for
in vivo treatments. Effects of IL-12 administration on viral titers
were investigated by initiating IL-12 injections at different times
relative to day of infection. Some experiments used daily IL-12
treatments beginning either 1 day before (day -1) or 1 day after (day
+1) LCMV infection. In these studies, mice received daily IL-12
treatments up to, and including, day 2 after infection. In other
experiments, only a single dose of IL-12 was given. In all treatment
studies, the spleens were harvested on day 3. In vivo effects of IL-12
for induction of IFN- were examined using a single injection of
IL-12 given to uninfected or day 0, 1, or 2 LCMV-infected mice. One day
after IL-12 administration, mice were anesthetized with methoxyflurane
(Pitttman-Moore, Mundelein, IL), and whole blood was collected into
heparinized tubes for preparation of serum and/or PBMCs. Spleens were
harvested from killed mice into ice-cold RPMI 1640 (RPMI 1640 media
supplemented with NaHCO3, 10 mM HEPES, 2 mM
L-glutamine, 100 U/ml penicillin, and 100 µg/ml
streptomycin from Life Technologies, Grand Island, NY), followed by
perfusion of the hepatic portal vein with 5 ml PBS and harvesting of
livers for isolation of leukocytes.
Isolation of leukocytes
Whole blood, collected into 2 ml RPMI 1640 with heparin, was overlaid on 3 ml Histopaque-1083 (Sigma, St. Louis, MO) and centrifuged according to manufacturer’s directions (800 x g for 15 min at room temperature). PBMCs were harvested at the interface. To obtain splenic leukocytes, spleens were minced, passed through nylon mesh (Tetko, Kansas City, MO), and treated with ammonium chloride to osmotically lyse erythrocytes. Hepatic leukocytes were prepared using published methods (13) with modifications. Briefly, livers were forced through steel mesh and digested with 0.05% collagenase IV and 0.004% DNase I (Sigma) in RPMI 1640 at 37°C for 30 min. Digested slurry was passed through nylon mesh and treated with ammonium chloride. Cell suspensions were then layered on two-step discontinuous Percoll gradients (Pharmacia Fine Chemicals, Piscataway, NJ) in D-PBS (Life Technologies) for density separation. Hepatic leukocytes were harvested at the 35/80% Percoll interface after centrifugation at room temperature for 30 min at 900 x g. Cell yields and viabilities were determined by trypan blue exclusion (Life Technologies).
Generation of conditioned media (CM) and cytokine measurements
To investigate induction of IFN- production after in vivo
IL-12 administration, leukocytes were plated in flat-bottom microtiter
plates at 107 cells/ml in RPMI 1640 supplemented
with 10% heat-inactivated FBS (HyClone Laboratories, Logan, UT).
Plates were incubated for 24 h at 37°C in a 5%
CO2 incubator before collection of supernatants
and storage at -80°C. To determine in vitro sensitivity of
leukocytes to IL-12, plated leukocytes were supplemented with 100
µl/well of indicated concentrations of IL-12 prepared in 10%
FBS-RPMI 1640 to a final volume of 200 µl/well. Cytokine levels in CM
and serum were determined by sandwich ELISA as previously described
(7, 14, 15).
Flow cytometric analysis
Cells were blocked with 20% FBS-PBS supplemented with 100
µg/ml mouse IgG (Sigma), stained using R-PE-conjugated anti-NK1.1
(PK136) and CyChrome-conjugated anti-TCR-
(H57-597) or isotype
control Abs (PharMingen, San Diego, CA), and fixed with 4%
paraformaldehyde in PBS. More than 25,000 events were collected within
a leukocyte acquisition gate using a FACScalibur (Becton Dickinson, San
Jose, CA). Argon laser output was operating at 15 mW at 488 nm. Data
were acquired and analyzed using CellQuest software (Becton Dickinson).
Intracellular staining of IFN- protein was conducted as described
(16). Briefly, cells were first incubated for 5 h at
37°C in 10% FBS-RPMI 1640 containing 10 µg/ml brefeldin A (Sigma),
harvested, and blocked with 20% FBS-PBS containing mouse IgG.
Biotinylated anti-NK1.1 (PK136) and CyChrome-conjugated
anti-TCR- (H57-597), followed by streptavidin-conjugated APC
(PharMingen), were used for surface staining. Cells were fixed with 4%
formaldehyde in PBS, then treated with permeabilization buffer (1%
saponin in 0.5% BSA-PBS with 0.006% NaN3;
Sigma). Samples were blocked in permeabilization buffer containing 300
µg/ml rat IgG (Sigma) followed by staining with R-PE-conjugated
anti-mouse-IFN- (XMG1.2) or isotype control Ab (PharMingen).
Specificity of intracellular IFN- staining was demonstrated by
blocking with unlabeled soluble IFN- or unconjugated XMG1.2. Samples
were acquired using a FACScalibur with the first laser operating as
described above and a second laser operating at a wavelength of 635 nm.
More than 100,000 events were collected within a leukocyte
acquisition gate.
Plaque assays
LCMV titers were quantitated as previously described (17). Briefly, monolayers of Vero cells were grown until confluent. Frozen organs were homogenized, and debris was removed by centrifugation. Serially diluted supernatants were incubated in duplicate on cell monolayers for 1.5 h at 37°C. Cell monolayers were overlaid with 10% FBS-2x Media 199 (Life Technologies) mixed with equal part 1% SeaKem ME agarose (FMC Bioproducts, Rockland, ME). Plates were incubated for 4 days at 37°C and overlaid with 10% FBS-2x Media 199/1% ME agarose containing a 6.7% neutral red solution (Life Technologies) to visualize plaques. LCMV standards and negative controls were included in each assay.
Statistical analyses
Results are given as means ± SEM. Statistical analyses using 2-tailed homoscedastic Student’s t tests were run on Microsoft Excel 98 (Microsoft Corporation, Redmond, WA). ANOVA tests were run on StatView 4.5 (Abacus Concepts, Berkeley, CA) where indicated.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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responses in
spleen and serum
To characterize the effects of viral infection on the efficacy of
IL-12 administration for IFN- induction, uninfected and
LCMV-infected C57BL/6 mice were vehicle treated or treated with a
single 1-µg dose of the cytokine. One day after injection, splenic
leukocytes and serum samples were harvested. IFN- production by
leukocytes was assessed in culture media conditioned for 24 h
without further ex vivo stimulation (CM). Cells isolated from all mice
treated with vehicle injections failed to produce detectable IFN- in
culture (Table I
). Cells isolated from
uninfected mice treated with IL-12 for 1 day produced 47.9
pg/106 cells of IFN- (Table I; C57BL/6,
uninfected). Those isolated from mice treated with IL-12 on day 2 and
harvested on day 3 of infection produced only 6.3
pg/106 cells of IFN- (Table I; C57BL/6, LCMV
infection day 3), representing an infection-induced inhibition of
>80%. This inhibition was reproducibly observed in over seven
independent experiments, comprising an aggregate of 18 samples that
demonstrated an average IFN- production of 80.9 ± 24.3
pg/106 cells by splenic leukocytes from
IL-12-treated uninfected but only 24.5 ± 5.9
pg/106 cells by those from IL-12-treated infected
mice (significantly different; p 
0.02). The IFN-
was primarily produced by NK cells because it was present in T and B
cell-deficient SCID but absent in NK and T cell-deficient E26 mice
(Table I). In contrast to the infection-induced inhibition of splenic
leukocyte production, IL-12-induced IFN- levels were significantly
augmented in serum during the infection. In this compartment, IL-12
treatment of uninfected mice induced 218.5 ± 45.2, and of
LCMV-infected mice induced 2655.6 ± 330.3, pg/ml serum IFN-
(p < 0.001; Table II). The serum responses were also shown
to be NK cell dependent because they were observed in SCID but not E26
mice (Tables II). Thus, LCMV infection results in dramatically
inhibited IL-12-elicited IFN- production by splenic leukocytes but
augmented serum IFN-, and these effects appear to be at the level of
NK cell responsiveness to the cytokine. As the enhancement in serum
IFN- levels occurs concomitantly with diminished splenic production,
the results suggest that other compartments are contributing to
IL-12-induced circulating levels of IFN-.
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To expand characterization of responses in spleen and serum, as
well as to extend studies to cells in other compartments known to
harbor significant proportions of NK cells, i.e., blood (circulating
PBMCs) and liver, time course experiments were conducted with
populations isolated from these different sites. Uninfected mice and
mice infected with LCMV for 0, 1, or 2 days were treated with a single
dose of vehicle control or 1 µg IL-12. One day following treatment,
i.e., 0 (uninfected), 1, 2, or 3 days after infection, sera were
collected, and cells were isolated to prepare CM. IL-12 induced
significant levels of serum IFN-, and these levels were
progressively increased during the course of LCMV infection. Mice
treated for 1 day with IL-12, on the day of infection (0) or day 1 or
day 2 after infection, respectively, exhibited 2-, 6-, and 10-fold
greater serum IFN- levels as compared with treated uninfected mice
(-1) (Fig. 1A). In contrast,
the ability of splenic leukocytes to produce IFN- was severely
attenuated as infection progressed such that administration of IL-12 1
day or later after infection resulted in up to 80% decreases in
IFN- production (Fig. 1B). Interestingly, PBMCs from
IL-12-treated mice failed to produce detectable levels of IFN- at
any time point examined, suggesting that circulating leukocytes were
not significant contributors to serum IFN- (Fig. 1C). In
contrast, hepatic leukocytes were a rich source of IFN- under these
conditions, with cells from mice receiving IL-12 on day 2 and isolated
on day 3 of infection producing >5-fold more IFN-, reaching >2000
pg/106 cells (Fig. 1D). Estimated
total production based on cell yields from specific organs indicated
that IFN- production by cells from IL-12-treated uninfected or day
2-infected mice was, respectively, 3,322 (±234) and 355 (±53)
pg/total cells isolated from spleen, but 2,477 (±443) and 10,278
(±970) pg/total cells isolated from livers. Thus during infection,
cells in the liver sustain their ability to respond to IL-12 and
synthesize IFN-, and the compartment is a significant source of the
factor.
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As production of IFN- was primarily dependent upon NK cells
(Tables I and II), experiments were conducted to determine whether
changes in NK cell subset distributions accounted for the compartmental
differences. Flow cytometric analyses were conducted to quantitate
classical NK cells
(NK1.1+TCR--). For
these studies, cell populations were prepared from uninfected and
LCMV-infected C57BL/6 mice that had been untreated, vehicle treated,
and/or IL-12 treated for 1 day. Although infections did induce
significant increases in proportions and numbers of liver NK cells,
differences in subset distribution could not account for the observed
decreases in splenic, or increases in liver, cell IFN- production.
As compared with the average of 1.7% (±0.2) in IL-12-treated,
uninfected mice, NK cell proportion in the spleens of mice administered
a single 1 µg dose of IL-12 on day 2 and harvested on day 3 of
infection averaged 2.1% (±0.3) (Table III). The modest change in NK cell
proportions was neither significant nor congruent with the >80%
infection-induced inhibition of splenic leukocyte IFN- production
(Fig. 1
). Similarly, differences in hepatic NK cell percentages (Table III) could not fully account for the >5-fold enhancement in hepatic
leukocyte IFN- production after IL-12 administration during
infection (Fig. 1). Taken together, these studies demonstrate that
compartmental changes in NK cell proportions following LCMV infection
fail to satisfactorily account for infection-induced changes in IFN-
induction within each particular compartment or across
compartments.
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To directly evaluate intrinsic cell populations as opposed to
potential secondary effects in vivo, IL-12 responsiveness was also
evaluated in culture. For these experiments, leukocytes were isolated
from C57BL/6 mice uninfected or LCMV infected for various periods, and
IFN- production was examined by 24-h culture with the cytokine.
Splenic leukocytes from uninfected animals produced high levels of
IFN- upon in vitro exposure to IL-12, reaching 39,399.0 ±
10,895.5 pg/ml (at a final cell concentration of 5 x
106 cells/ml). However, those prepared during the
course of the infection were progressively attenuated such that on day
2 their IFN- production was reduced by >95% to only 924.5 ±
427 pg/ml (Fig. 2). In contrast, hepatic
leukocytes from uninfected animals produced 1,518 pg/ml of IFN- and
from day 2-infected mice produced >8-fold higher levels, reaching
13,492 pg/ml. Thus, in vitro reflected in vivo responses of the
populations to IL-12. Flow cytometric analyses comparing cells from day
3 LCMV-infected and uninfected mice (Table III) indicated that changes
in IFN- production following in vitro IL-12 stimulation could not be
fully explained by virus-induced alterations in NK cell proportions.
Hence, compartmental differences in IL-12 responsiveness induced
during viral infection are maintained by isolated cell populations
stimulated with the cytokine in vitro.
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-expressing cell subsets
To directly quantitate proportions of NK cells induced to express
IFN- protein, normal or SCID C57BL/6 mice were treated with vehicle
or IL-12 without infection or on day 2 of infection. One day later,
splenic and hepatic cells were isolated, stained for intracellular
IFN- protein expression, without further stimulation in culture and
analyzed by flow cytometry. Experiments examining total
IFN--positive populations demonstrated that specific staining for
IFN- under these conditions occurred almost exclusively in the
classical NK cell subset, i.e.,
NK1.1+TCR--
populations, in both spleens and livers (data not shown). These results
confirmed and extended the studies presented above indicating that NK
cells were the primary source of IFN- (Tables I and II). Evaluation
of the percentages of positive splenic cells demonstrated that IL-12
treatment induced 16% (±2.0) of the NK cells in uninfected mice to
express IFN-. In contrast, only 10% (±1.0) of the splenic NK cells
in infected mice were IFN- positive after treatment, representing a
38% decrease in the proportion of responders (Fig. 3A). SCID mice, naturally
enriched in NK cells, also demonstrated similar reductions after
infection (Fig. 3A). Thus, both the percentages and numbers
of splenic NK cells induced to express IFN- by administered IL-12
were decreased as a result of LCMV infection. Moreover, the level of
expression within individual positive cells was decreased as determined
by mean channel of fluorescence intensity (data not shown).
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expression during
infection. Similar proportions of NK cells isolated from
immunocompetent C57BL/6 mice were induced by IL-12 administration to
express IFN-, comprising 24% and 23% of the NK cell subset in
uninfected and infected mice, respectively (Fig. 3B). The
trends were also observed in liver populations from IL-12-treated SCID
mice, where percentages of IFN--expressing NK cells increased from
25% to 32% after infection (Fig. 3B). Thus, in agreement
with patterns of IFN- responses observed in CM, these flow
cytometric results demonstrate that NK cells are a primary source for
IFN- after IL-12 treatment, and that relative proportions of
IFN--expressing NK cells are decreased in spleen, but maintained or
enhanced in liver, after LCMV infection. Requirements for antiviral IL-12 intervention in spleen
LCMV is reported to be sensitive to IFN- antiviral effects, but
the infection itself does not induce IL-12 and/or early IFN- protein
in immunocompetent hosts. The results presented above suggest that
administered IL-12 may be used therapeutically to induce early IFN-
expression and downstream antiviral effects, but that these effects may
be limited to particular compartments due to infection-induced changes
in NK cell responsiveness to IL-12. Studies were initiated to determine
whether IL-12 administration could be used therapeutically during LCMV
infection. The primary site of replication for the LCMV Armstrong
strain used here is the spleen. In immunocompetent C57BL/6 mice, daily
IL-12 treatments initiated 1 day after establishment of infection (day
+1) resulted in no significant decreases in splenic viral titers, i.e.,
6.7–6.9 logs of virus were detected in both vehicle control and
IL-12-treated groups (Fig. 4A). In contrast, daily IL-12
treatments initiated 1 day before infection (day -1) resulted in
significant decreases of up to 1 log virus over vehicle control
treatments (Fig. 4B). These results could not be explained
by total aggregate dosage of cytokine received as statistically
significant inhibitory effects on viral replication were observed with
low-dose (1 ng/day) IL-12 initiated on day -1, whereas marginal
effects at 1000-fold higher doses were detected when initiated on day
+1. Furthermore, studies using only a single 1-µg dose of IL-12 on
different days relative to infection demonstrated significant
reductions in day 3 splenic viral titers when administered once on day
-1, but not when administered any time thereafter (Fig. 4C). As these results directly mirrored the reductions in
IL-12-induced splenic IFN- production after LCMV infection (Fig. 1),
additional studies to demonstrate the IFN--dependency of antiviral
effects were performed. The antiviral effects of daily IL-12 treatment
initiated on day -1 were indeed found to be dependent upon the
induction of IFN- as treatments failed to reduce viral burden in
C57BL/6 mice genetically deficient in IFN- (Fig. 4D).
Taken together, these results suggest that the reduced ability of
splenic cells to produce IL-12-induced IFN- after infection is
correlated with biological consequences that narrow the window of
opportunity for cytokine therapy to exert antiviral effects locally in
the spleen.
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The experiments thus far have definitively shown a dichotomy
between spleen and liver in IL-12 responsiveness for IFN- induction
over the course of LCMV infection. As IL-12 responsiveness was
preserved in the liver, additional experiments were performed to
determine whether antiviral effects in this compartment can be accessed
with IL-12 therapy even though efficacy is lost in the spleen. Because
the Armstrong strain of LCMV does not replicate to detectable titers in
the liver under the conditions examined, a hepatotropic isolate of
LCMV, WE, was used for these studies. The WE strain is more aggressive
and replicates to higher titers than Armstrong, thus 100-fold lower
infecting doses (2 x 102 PFU) of either
Armstrong or WE were used. Consistent with previous results (Fig. 4),
immunocompetent C57BL/6 mice administered 100 ng IL-12 per day
demonstrated a significant 2 log drop in splenic viral titers if
treatments were initiated on day -1, but not if initiated on day +1,
with either strain of virus (Fig. 5,
A and B). As expected, no virus was detected in
livers of mice infected with the Armstrong strain regardless of
treatment (Fig. 5C). In contrast, there was high replication
of the WE virus in livers and significant, 2 log reductions in viral
titers in this compartment resulting from IL-12 treatments initiated on
either day -1 or day +1 of infection (Fig. 5D). The
antiviral effects elicited in liver with IL-12 administration starting
1 day after WE infection were IFN- dependent, as such treatments did
not reduce viral burden in mice genetically deficient in IFN- (Fig. 6). Taken together, the data reveal that
the previously demonstrated compartmental dichotomy in IL-12
responsiveness for IFN- induction has biological consequences
distinctly affecting IFN--dependent antiviral efficacy of IL-12
therapy in the spleen and liver after viral infections.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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responses in different host compartments during the
early, innate immune response. After LCMV infection, a diminished
capacity for IL-12-induced IFN- production was found in splenic cell
populations. In striking contrast, IFN- induction in serum was
dramatically increased, and hepatic leukocytes maintained and/or
increased their ability to produce IFN-, in response to IL-12
stimulation. The synthesis of IFN- was largely NK cell dependent
under these conditions, and the virus-induced decreases in splenic
IFN- responses were also detected as reductions in proportions of
splenic NK cells expressing IFN-. The changes were associated with
affected abilities to elicit IFN--dependent antiviral effects in a
therapeutic setting: IL-12 treatments initiated after infections with
either LCMV Armstrong or hepatotropic WE resulted in minimal reductions
in splenic viral titers, whereas they significantly inhibited LCMV WE
replication in the liver. Taken together, these results demonstrate
differential regulation of IL-12-induced NK cell IFN- responses in
particular compartments during viral infection. Furthermore, they
suggest that these putative local regulatory mechanisms influence the
effectiveness of cytokine treatments in particular compartments.
Although it is not yet known why LCMV infections do not induce
endogenous IL-12 and NK cell IFN- responses in immunocompetent
hosts, it is becoming clear that the lack of such responses is not
exclusively a result of an absence of the ability to elicit IL-12. This
laboratory has previously demonstrated infection-induced mechanisms in
place to actively inhibit endogenous IL-12 expression (8).
The studies presented here demonstrate that bypassing this inhibition
by adding exogenous cytokine reveals another tier of regulation at the
level of ability for IL-12 to induce IFN- in splenic NK cells. This
repression of IL-12-induced IFN- production is reminiscent of human
NK cell studies demonstrating particular conditions driving in vitro
differentiation of NK1 cells to preferentially produce IFN-, as
compared with conditions driving NK2 cells to produce IL-5/IL-13
(18, 19, 20). However, the dichotomy in NK cell responsiveness
between organ compartments is more similar to demonstrated
hyporesponsiveness after sequential augmentation of splenic, but not
hepatic, NK cells following treatments with biological response
modifiers, such as maleic anhydride divinyl ether or heat-killed
Propionibacterium acnes (13). Taken together
with these other studies, our results suggest that localized responses
are a result of unidentified microenvironmental influences in place to
either negatively or positively regulate endogenous cytokine
responses.
This report adds to characterization of how ongoing immune responses to
infections may modify responses to administered cytokines, i.e.,
cytokine therapy. Other studies from our group, examining administered
IL-12 during peak adaptive immune responses to LCMV, have shown
dramatic synergistic effects between these and endogenous T cell
responses in enhancing proinflammatory cytokine expression and
cytokine-mediated toxicities (17, 21). This synergism
limits tolerated doses of IL-12. The work presented here extends
knowledge to the context of ongoing endogenous innate immune responses
and has important implications for therapeutic use of IL-12 under these
conditions. Our results indicate that, depending upon the nature of
infection-induced modulation of innate immune responses within
different compartments, achieving optimal antiviral defenses at
particular sites may be limited to prophylactic regimens. This is
consistent with observations from others that IL-12 treatments
initiated before vesicular stomatitis virus infections significantly
decrease viral titers in the central nervous system (22),
but can also be effective postinfection (23); and that
prophylactic is more effective than therapeutic IL-12 for enhancing
host survival during lethal herpes simplex virus infections
(24). Thus, different viral systems indicate that it is
important to treat with IL-12 before infection to elicit maximal
antiviral defenses at some sites. Our results indicate that LCMV
infections can enhance IL-12 IFN- induction at sites other than the
spleen, i.e., serum and liver. Therefore, IL-12 administration
initiated during an established infection may be beneficial in
particular organs if the cellular targets of a virus are localized in
compartments with either maintained or enhanced IL-12 responsiveness,
such as the liver. In this regard, IL-12 therapy may prove to be
protective against infections of humans with hepatitis viruses
sensitive to IFN- (25, 26, 27, 28).
Surprisingly, the protective effects of IL-12 treatments in the spleen
did not correlate with serum IFN- levels but with local IFN-
production. This dissociation of circulating IFN- responses from
IFN--mediated protective effects in spleens suggests that the
cytokine must be locally produced to mediate antiviral effects at
target sites. Other work from our group has been characterizing the
requirements for NK cell-mediated defense during endogenous responses
to murine cytomegalovirus (MCMV) infections in liver. Those studies
have similar implications. During MCMV infections, 1) NK cells mediate
antiviral effects in liver through an IFN--dependent pathway, 2) NK
cells traffic to focal sites of viral Ag expression in this tissue, and
3) optimal antiviral defenses in liver require chemokine-dependent
accumulation of NK cells (14, 29, 30). Taken together with
the MCMV studies, the experiments presented here indicate that local NK
cell responses must be regulated to deliver IFN- within particular
tissues and that there is a requirement for local delivery even in the
face of high serum IFN-.
In summary, the findings in this report demonstrate differential
modification of compartmental responses to IL-12-induced IFN-
production over the course of viral infection and highlight
requirements for effective IL-12-mediated antiviral therapy resulting
from these modifications. Furthermore, the data reveal intriguing
differences between spleen and liver for regulation of NK cell IFN-
responses during viral infection and suggest the existence of local
conditions regulating cytokine responses.
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Acknowledgments |
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sera, Dr. Cox
Terhorst for providing original breeding pairs of E26, and Dr. Herbert
W. Virgin, IV for breeding pairs of IFN--deficient
mice. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Christine A. Biron, Department of Molecular Microbiology and Immunology, Division of Biology and Medicine, Box G-B629, Brown University, Providence, RI 02912. E-mail address:
3 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; MCMV, murine cytomegalovirus; CM, conditioned media.
Received for publication April 9, 1999. Accepted for publication November 5, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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by an intracellular parasite and induces resistance in T-cell-deficient hosts. Proc. Natl. Acad. Sci. USA 90:6115. production and antiviral defense. J. Immunol. 156:1138.[Abstract]
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production. J. Immunol. 162:5238.. J. Immunol. 162:51. and interferon- inhibit synergistically viral replication in hepatitis B virus-replicating cells. J. Med. Virol. 47:272.[Medline]
inhibits duck hepatitis B virus replication in primary hepatocytes. J. Virol. 73:3162. (MIP-1)-dependent pathways. J. Exp. Med. 187:1.This article has been cited by other articles:
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, Values below the limits of detection (serum, 20 pg/ml;
splenic and PBMC CM, 2 pg/106 cells; hepatic CM, 10
pg/106 cells). Significant differences (*,
p < 0.05) were observed with IL-12 treatment of
LCMV-infected vs uninfected controls in serum (A) and
splenic CM (B).
, p 
