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Production1
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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levels after LPS administration. All
three cytokines contributed to lethality during dual challenge, because
neutralization of any one of the factors protected from death.
Production of TNF was not dependent on either NK or T cells. In
contrast, these populations were the predominant sources of IFN-, as
determined by lack of detectable IFN- production in NK and T
cell-deficient mice and by intracellular cytokine expression in the
cell subsets. Concordant with the demonstrations that both cell
populations produced IFN- and that this factor was critical for
lethality, removal of either subset alone was not sufficient to protect
mice from death resulting from dual challenges. Increased resistance
required absence of both cell subsets. Taken together, the data show
that during viral infections, the normally protective immune responses
can profoundly modify reactions to secondary heterologous challenges,
to result in dysregulated cytokine expression and consequent heightened
detrimental effects. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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are rapidly
induced following LPS administrations (3, 4, 5, 6), and these factors all
contribute to endotoxin-induced lethality (1, 2, 4, 6, 7, 8, 9, 10, 11, 12, 13). TNF is
the primary mediator of mortality (7, 8, 14, 15, 16). IFN- appears to
act by promoting increased responsiveness to TNF, in part by enhancing
synthesis of both TNF and its receptors (10, 11, 17, 18). IL-12 also
can play a critical role during endotoxic shock (4, 6), primarily
through its function as an inducer of IFN- (4, 6). Cells of
monocyte/macrophage and dendritic lineages are major sources of IL-12
and TNF (19, 20, 21, 22). NK and T cells are not required for production of
IL-12 and TNF, but they are primary producers of IFN- (5, 23, 24).
However, after endotoxin challenge, NK cells are the major contributors
to the IFN- response and are required for both production of the
cytokine and lethality (23, 25). In contrast, T cells, and consequently
T cell-produced IFN-, appear to have more peripheral roles, given
that LPS-induced lethality in uninfected mice is unabated in the
absence of T lymphocytes (23, 25). Thus, during bacterial
endotoxin-initiated shock reactions, cells of the innate immune system
are the critical producers of excess cytokines, there is a requirement
for NK cells in IFN- responses, and these cells and factors are key
mediators of disease and lethality.
Viral infections also can induce cytokine responses, including certain
of those found during endotoxic shock reactions, i.e., IL-12, TNF, and
IFN- (26, 27, 28, 29, 30), and cytokine-mediated diseases have been demonstrated
under particular conditions of infections with these agents (31, 32).
Such responses are generally tightly regulated and do not result in
cytokine-mediated death. However, in both humans and experimental
animals, disease and clinical status during viral infections can be
aggravated by concurrent or secondary bacterial challenges. In humans,
the best documented example is that of influenza virus. Subsequent to
infections with this pathogen, infections with bacteria such as
Streptococcus pneumoniae, Staphylococcus aureus, or
Haemophilus influenzae can lead to sudden and abrupt
worsening of disease symptoms (33, 34). In mice, a number of viruses,
including coxsackie, influenza, lymphocytic choriomeningitis
(LCMV),3 murine cytomegalovirus
(MCMV), and Theiler’s murine encephalomyelitis, increase
susceptibility to lethality and disease following secondary challenges
with either bacteria or bacterial products (35, 36, 37, 38, 39, 40, 41, 42, 43). Although
heightened sensitivity has been observed in a number of dual challenge
settings in both humans and mice, the mechanisms promoting deleterious
effects under these conditions are not well understood. Because both
viruses and bacteria are inducers of cytokines, and as viral infections
have been demonstrated to increase sensitivity to toxic effects
resulting from cytokine exposures under particular conditions (44, 45),
the responses elicited from dual challenge conditions could result from
synergisms promoting elevated expression of the proinflammatory
cytokines and/or potentiating cytokine-mediated toxicities.
The studies presented here characterized parameters of increased
sensitivity to challenge with bacterial products during viral
infections further and defined pathways responsible for induction of
disease and lethality under these conditions. The viral/bacterial
immune interactions resulting from LCMV infections followed by LPS
challenges were examined. During infections with LCMV, NK cells undergo
limited proliferation but are not induced to produce IFN- (46, 47).
In contrast, CD8 T lymphocytes are induced to undergo extensive
proliferation and become prominent producers of IFN- (48, 49, 50, 51, 52, 53). To
address potential contributions of the respective cell subsets to
pathologic processes, experiments were conducted at earlier times as
well as at the later peak times of T cell responses to the viral
infections. They demonstrated that endotoxin-elicited systemic cytokine
responses were dramatically elevated in virus-infected mice, with TNF,
IL-12, and IFN- all playing critical roles in enhanced LPS-induced
lethality under the dual challenge conditions. As a result of producing
IFN-, both NK and T cells contributed to disease under these
conditions. The role of T cells for IFN- production was shown to be
dependent upon priming during the viral infection. The results
conclusively establish that the cytokine responses induced during dual
challenge are critical mediators of toxicities. Furthermore, they
define a previously unappreciated role for T, in addition to NK, cells
in mediating such cytokine-dependent immunopathology.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Specific pathogen-free male and female C57BL/6 (C57BL/6NTacfBR)
mice were purchased from Taconic Laboratory Animals and Services
(Germantown, NY). Male T and B cell-deficient C57BL/6-SCID, male
C57BL/6 athymic nude T cell-deficient, and male T and B cell-deficient
C57BL/6-RAG-1 mutant (RAG-1-/-) (54) mice were
obtained from The Jackson Laboratory (Bar Harbor, ME). C57BL/6
IFN--deficient (55) mice, originally purchased from The Jackson
Laboratory, were generously provided by Dr. Herbert Virgin (Washington
University, St. Louis, MO), and a colony was established and maintained
at Brown University. E26 mice, deficient in NK and T cells, were
established with CBA x C57BL/6 backgrounds as described (56),
bred in strict isolation under pathogen-free conditions by
brother-to-sister mating in the animal care facility at Brown
University, and maintained on sterile food, water, and caging. Some
RAG-1-/- mice also were maintained
and bred in the animal care facility. All mice used in experiments were
6 to 14 wk old. Animals obtained from sources outside of Brown
University were housed in our facility for at least 1 wk before use.
Handling of mice and experimental procedures were conducted in
accordance with institutional guidelines for animal care and use.
Virus, infections, and in vivo treatment protocols
Experiments were initiated on day 0, with mice either not
infected or infected i.p. with 2.0 x 104 PFU of LCMV
Armstrong strain clone E350, as previously described (44, 57). At 4 or
7 days after infection, mice were injected i.p. either with PBS or with
various doses of LPS from Escherichia coli strain O111:B4
(Difco, Detroit, MI). Experiments evaluating the roles for the
cytokines IL-12, IFN-, and TNF were conducted by neutralizing
cytokine functions in vivo. To neutralize IL-12, mice were given a
single 1.0-mg i.p. injection of the rat mAb C17.8 (a generous gift from
Dr. Giorgio Trinchieri, Wistar Institute of Anatomy and Biology,
Philadelphia, PA). To neutralize IFN-, mice were injected i.p. with
1.0 mg of the rat monoclonal XMG1.2 (kindly provided by Dr. Robert
Coffman, DNAX Research Institute, Palo Alto, CA). In vivo
neutralization of TNF was conducted by i.p. injection of mice with 0.5
mg of a chimeric hamster/murine anti-TNF mAb having the F(ab) of
TN3.19-12 and a murine IgG1 F(c) region (58) (gift of Celltech, Slough,
U.K.). Mice were treated with 500 µg of purified Ab in PBS on day 4
relative to infection. Control animals received equivalent amounts of
rat IgG (for IL-12 and IFN- neutralizations) or mouse IgG (for TNF
neutralization). Previous studies from our laboratory have demonstrated
that these treatment protocols are efficient at neutralizing endogenous
cytokine functions (45, 46, 59). For in vivo cell depletions,
anti-CD8 rat mAb 2.43 was used to eliminate CD8 T cells, and
anti-NK1.1 mouse mAb PK136 was used to eliminate NK cells. Mice
were treated, relative to infection, on days -1, 2, and 5 with 2.43
mAb to a cumulative total of 1.0 mg of Ab, and 1 day before LPS
challenge with 0.25 mg of PK136 Ab, per animal. Control animals were
given equivalent amounts of rat IgG or mouse IgG. These treatment
protocols are 
90% efficient at removing the respective cell subsets
(53, 57, 59) and performed at this level for the elimination of splenic
populations in the studies reported here. With exception of the
anti-TNF, all Abs for in vivo studies were purified in our
laboratory from ascites fluid preparations. In survival studies, mice
were monitored for their condition and viability twice daily for at
least 18 days.
Serum preparation and organ collection
On day 7 following initiation of experiments, animals were first anesthetized with methoxyflurane (Pittman Moore, Mundelein, IL), and blood was collected via the retroorbital route into microcentrifuge tubes containing 25 U of heparin. Because there was always clotting in blood samples, collected fluids were identified as sera. Mice were then sacrificed and spleens were harvested and maintained at 4°C in polystyrene tubes containing RPMI media with 10% FBS.
Preparation of cells
Splenocytes were obtained by teasing apart spleen and by passing single-cell suspensions through a nylon mesh. Erythrocytes were osmotically lysed by brief ammonium chloride treatment. Viable cell yields were determined by trypan blue exclusion.
Flow cytometric analyses
Studies evaluating intracellular expression of IFN- were
performed using previously described protocols (53, 60) with specific
modifications to examine expression in NK cell subsets and evaluate
spontaneous as compared with ex vivo stimulation-enhanced expression.
Cells were resuspended at 106 cells/ml in RPMI media
containing 10% FBS. Unstimulated analyses were conducted by
immediately exposing cells to brefeldin A (Sigma Chemical, St. Louis,
MO), at a final concentration of 100 ng/ml to block cytokine secretion,
and then incubated for 4 h at 37°C. For analyses after
stimulation, cells were ex vivo activated with plate-bound anti-CD3
mAb clone 145-2C11 for a total of 6 h at 37°C, with brefeldin A
added during the last 4 h of culture. Cells were then collected,
washed with ice-cold PBS, and used at 106 cells/test. For
NK cell analyses, cells were incubated for 30 min at 4°C with
biotinylated mouse anti-NK1.1 mAb clone PK136 and anti-CD3
CyChrome-conjugated hamster mAb clone 145-2C11, washed, and incubated
for a further 30 min at 4°C with streptavidin-FITC (PharMingen, San
Diego, CA), washed, and fixed with 4% formaldehyde in PBS. For T cell
analyses, cells were incubated for 30 min at 4°C with biotinylated
rat anti-mouse CD4 mAb clone RM4–5 and FITC-conjugated
anti-mouse CD8
mAb clone 53-6.7, followed by washing,
streptavidin-PerCP (Becton Dickinson, San Jose, CA) incubation, and
fixation. Fixed cells were permeabilized with a 1% saponin solution
(Sigma) in staining buffer, resuspended in permeabilization buffer
containing 300 µg/ml rat IgG, incubated for 10 min at room
temperature, and further incubated for 20 min after addition of
anti-mouse IFN- mAb XMG1.2 conjugated to R-PE. Demonstration of
specificity for intracellular visualization of IFN- was performed by
preincubating R-PE conjugated anti-IFN- with either recombinant
murine IFN- (PharMingen) or purified unconjugated XMG1.2. The
blocking experiments established the limit of detection for proportions
of cells expressing cytoplasmic IFN- as 2%. All directly conjugated
mAbs were purchased from PharMingen. Samples were acquired using a
FACSCalibur (Becton Dickinson), with the CellQuest version
3.1 software package. Laser outputs were 15 mW at 488 and 635 nm
wavelengths. At least 50,000 events were collected for analysis.
Proportions of cells within a subset expressing IFN- were determined
by using cell surface markers to gate on the population for 100% and
determining the proportion within the gate expressing the cytokine.
Relative contributions to IFN--expressing cells were determined by
using splenic cell yields with flow cytometric analyses to determine
numbers of NK, CD8, and CD4 cells expressing the cytokine per spleen,
and the relative contribution of each subset to their total.
Cytokines and cytokine measurement
Serum TNF, IL-12 p40, and IFN- levels were determined by
standard sandwich ELISA as previously described (46, 61). The TNF assay
was specific for detection of TNF-. Limits of detection for diluted
serum samples in the assays were 0.6 ng/ml for TNF, 0.1 ng/ml for IL-12
p40, and 0.6 ng/ml for IFN-. Colorimetric changes of enzyme
substrates were detected at 405 nm wavelength using a SpectraMax 250
reader (Molecular Devices, Sunnyvale, CA).
Statistical analysis
For survival studies, p values were obtained by comparing groups using the nonparametric Mantel-Cox test provided by the statistics software package Statview 4.0 (Abacus Concepts, Berkeley, CA). In all other studies, Student’s two-tailed t tests were performed, using Microsoft Excel 5.0 (Microsoft, Redmond, WA). Unless otherwise indicated, means ± SE are shown.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The effects of LCMV infection on susceptibility to endotoxin were
examined by secondary administration of LPS. To evaluate changes at
times of prominent T cell responses, initial studies contrasted
responses in day 7 LCMV-infected to those in uninfected mice.
Infections with LCMV i.p. did not result in death of mice (Fig. 1
A), whereas injection of a
high dose (100 µg) of LPS resulted in 100% lethality within 2 days
after challenge in both uninfected and 7 day-infected mice (Fig. 1B). At this dose, however, LPS administration to the
infected mice resulted in accelerated kinetics of lethality, with all
animals succumbing by 18 h after endotoxin injection (Fig. 1B). Moreover, in contrast to the resistance of uninfected
mice to lower doses, 100% mortality was induced in day 7-infected mice
given 4- or 10-fold less LPS (Fig. 1, C and D).
Both uninfected and infected mice tolerated lower doses of 2 µg (Fig. 1E). Experiments conducted to examine whether the increased
susceptibility to endotoxin could be observed in mice at earlier times
after LCMV infection demonstrated that enhanced sensitivity to LPS also
occurred at other times during the infection. In particular, at 4 days
after infection, 25 µg of LPS induced 80 to 100% mortality in
virus-infected but no death in uninfected mice (see below).
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were detected systemically during infections with LCMV (data
not shown and samples at 0 h following LPS administration in Fig. 2). All three cytokines were induced
following LPS administration, and systemic levels of these factors were
greatly elevated in dual-challenged mice (Fig. 2). TNF levels peaked in
serum samples from uninfected mice at 90 min after LPS injections,
reaching values of 26.0 ± 17.6 ng/ml and subsided quickly to near
undetectable levels by 6 h. In contrast, dual-challenged mice had
136.7 ± 19.5 ng/ml of serum TNF at the height of factor
production, and the cytokine was still apparent (8.9 ± 1.6 ng/ml)
at 6 h after LPS challenge (Fig. 2A). Similar effects
were observed for IL-12 p40 and IFN-. At peak times of production,
infected mice given LPS had, respectively, 43- and 28-fold more of the
cytokines than uninfected challenged mice (Fig. 2, B and
C). Serum IL-12 p40 levels peaked at 4 h after LPS
exposure, with 7.6 ± 2.8 as compared with 0.2 ± 0.0 ng/ml,
respectively, in virus-infected and uninfected mice. Serum IFN-
levels were below detection at this time after LPS challenge of
uninfected mice but were at 24.7 ± 10.3 ng/ml in virus-infected
mice. Systemic IFN- in virus-infected as compared with uninfected
mice, respectively, reached 83.5 ± 25.6 and 2.9 ± 1.1 ng/ml
at 6 h after LPS challenge. Thus, during infections with LCMV,
inoculated mice are extremely sensitive to the lethal effects of
endotoxin, and the exacerbated susceptibility correlates with
dramatically elevated systemic levels of the proinflammatory mediators.
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to lethality during dual
challenge
To evaluate the contributions of the cytokines to death, their
biological functions were neutralized in vivo. Experiments blocking
TNF, IL-12, and IFN- were conducted because all of these have been
shown to be required for lethality during endotoxemia (1, 2, 4, 6, 7, 8, 9, 10, 11, 12, 13). LCMV-infected animals were given a single dose of neutralizing
anti-IL-12, anti-IFN-, or anti-TNF Abs before challenge
with LPS as described in Materials and Methods. Greater than
or equal to 50% of day 7 LCMV-infected mice having had any one of the
cytokine functions blocked were protected from LPS-induced death (Fig. 3A). To further examine the
role for IFN- under conditions of complete absence of the factor,
studies were conducted in mice rendered genetically factor deficient as
a result of gene mutation (IFN--/-). The IFN-
deficiency completely protected day 7 LCMV-infected mice from LPS
challenge (Fig. 3B). An enhanced sensitivity to LPS-induced
lethality observable on day 4 of LCMV infection also was IFN-
dependent because the effect at this time was completely ablated by the
genetic factor deficiency (Fig. 3C). Hence, during
challenges of LPS subsequent to viral infections, mice are induced to
produce significantly elevated levels of TNF, IL-12, and IFN-, and
all three factors are required to promote lethality.
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Because IFN- was contributing to the LPS-induced lethality in
LCMV-infected mice and because NK and T populations are the known major
producers of this cytokine, roles for the cell subsets in promoting
death were evaluated. For these studies, mice were rendered deficient
in various lymphocyte subsets either by in vivo depletions resulting
from Ab treatment or by genetic mutation. Ab-mediated depletion of
NK1.1+ cells alone did not protect LCMV-infected animals
challenged with endotoxin on either day 4 (Fig. 4A) or day 7 (Fig. 4B) after LCMV infection. Even though CD8 T cells have been
demonstrated to be the predominant producers of IFN- at later times
after LCMV infection, Ab-mediated depletion of these T cell populations
before LPS challenge did not protect infected mice from death (Fig. 4C). Moreover, LPS-induced death was not ablated in T
cell-deficient athymic nude mice (Fig. 4D), or in T and B
cell-deficient RAG-1-/- mice (Fig. 4, E and F). However, dramatically increased
resistance was observed in the absence of both NK and T cell subsets;
the transgenic E26 mice, genetically deficient in these populations,
had profoundly and significantly enhanced resistance to LPS challenge,
respectively, on day 4 (Fig. 4G) and day 7 (Fig. 4H) of infection. Taken together, the results demonstrate
that during dual LCMV-LPS challenges, both NK and T cells are critical
players in promoting lethality, and each lymphocyte population by
itself is capable of promoting these adverse responses in the host.
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Because both NK and T cell populations contributed to pathology
during dual challenges and because these lymphocyte subsets can be
sources of two of the cytokines required for lethality, i.e., TNF and
IFN-, NK and T cell contributions to production of these cytokines
were evaluated. Immunocompetent or lymphocyte subset deficient animals
were infected with LCMV and subsequently injected with LPS 7 days after
viral inoculation. Because high levels of both TNF and IFN- were
detectable at 4 h after endotoxin challenge (Fig. 2
), cytokines
were measured in sera of mice at this time. The elevated serum TNF
levels in virus-infected and LPS-challenged mice, as compared with
LPS-challenged alone mice, were not significantly reduced either in T
and B cell-deficient SCID and RAG-1-/- mice or
in T and NK cell-deficient E26 mice (Table I). Examination of the response at the
earlier time of 1.5 h after challenge indicated that peak TNF
levels were reduced in E26 as compared with immunocompetent mice.
However, the significant levels of 69.5 ± 14.9 ng/ml serum TNF
were still achieved in the T and NK cell-deficient mice. Thus, NK, T,
and B cells were not required for the virus-induced enhanced TNF
response. In contrast, NK and T cells both contributed to the
virus-induced enhanced IFN- production. The primary requirement was
for T cells in infected mice because serum IFN- levels were >90%
reduced in LPS-challenged NK cell-competent, but T and B
cell-deficient, SCID or RAG-1-/- mice as
compared with immunocompetent mice (Table I). Even though the lack of T
cells resulted in dramatic IFN- reductions, there was still a modest
synergism between virus infection and LPS challenge for induction of
this cytokine. Absence of NK cells, along with T cells, in E26 mice
resulted in complete abrogation of detectable IFN- production (Table I). These results indicate that during LPS challenge of virus-infected
mice, TNF production is not dependent on T, B, or NK cells, but NK and
T cells are required for IFN- production.
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protein were modified to
examine spontaneous expression by both NK and T cells, and conducted
without ex vivo stimulation as described in Materials and
Methods. No intracellular IFN- protein was detectable in any
splenic leukocyte population isolated from uninfected and untreated
mice, including NK cells defined as NK1.1+CD3-
subsets (Fig. 5A), CD4 T cells
defined as CD4+CD8- subsets (Fig. 5B), and CD8 T cells defined as
CD8+CD4- subsets (Fig. 5B). After
in vivo stimulation with LPS for 4 (Fig. 5, C and
D) or 6 h (Fig. 5, E and F), NK,
but not T, cells were induced to express the factor. At 4 h,
IFN- was detectable in NK cells from some but not all mice, but by
6 h, the cytokine was detectable in NK cells from all LPS-treated
mice (Fig. 5, C and E; Table II). More than one-half of the NK cells,
i.e., 61.7% (±4.0), were induced to express IFN- (Table II). In
contrast, no detectable CD4 or CD8 T cells, i.e., below the 2% limit
of detection for specific cytoplasmic IFN--expressing proportions,
were stimulated to spontaneously express the factor at these times
(Fig. 5, D and F; Table II). Overall, the NK
cells represented virtually 100% of the cells expressing detectable
IFN- after LPS challenge in the evaluated NK and T cell subsets
(Table III). Factor expression also was
observed within the "nonclassical" NK cells, e.g., NK1 T cells
defined as NK1.1+CD3+ (data not shown).
However, given that these populations represented low frequencies of
cells in the spleen, it was difficult to quantitate their contribution.
Ex vivo stimulation with anti-CD3 did not significantly modify the
percentages and/or proportions of T cells responding to express IFN-
(data not shown). Thus, under these conditions, the induced NK cell
expression of IFN- can be detected without, and T cells are not
primed to respond with IFN- expression to, purposeful additional
stimulation ex vivo. The results indicate that after endotoxin
challenge in uninfected mice, NK cells are the predominant, whereas CD4
and CD8 T cells represent minor, populations of IFN--expressing
cells.
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was not detectable in either NK or
T cells on day 7 of LCMV infection (Fig. 5, G and
H). However, LPS challenge induced both cell subsets to
express detectable IFN- under these conditions (Fig. 5, I-L, and Table II). At 4 h after endotoxin
injection, averages of 18.6% (±3.9) NK cells (Fig. 5I,
Table II), and 12.4% (±3.1) and 30.6% (±8.0) CD4 and CD8 T cells,
respectively, were positive for IFN- (Fig. 5J, Table II).
Similar observations were found at 6 h after LPS challenge of
virus-infected mice (Fig. 5, K and L, and Table II). Overall, the CD8 T cells represented >80%, whereas NK cells were
<5%, of the cells expressing IFN- in the evaluated splenic NK and
T cell subsets at either 4 or 6 h after LPS challenge of
LCMV-infected mice (Table III). Ex vivo stimulation with anti-CD3
did enhance the proportions of T cells expressing IFN- (data not
shown). Consistent with other studies from this laboratory (53), T
cells from day 7 LCMV-infected mice, not challenged with LPS, were
primed to express IFN- such that 
42% of CD8 and 12% of CD4 T
cells were factor positive after ex vivo stimulation with anti-CD3.
Ex vivo stimulation of cells from LPS-treated mice modestly enhanced
the proportions of positive CD8 T cells by 15% and did not increase
the proportions of positive CD4 T cells over that observed without
stimulation. Hence, by a number of criteria, including direct
intracellular visualization of IFN-, NK and T cells are the
demonstrated predominant sources of IFN- after LPS challenge of
LCMV-infected mice, but T cells comprise the major subsets expressing
the cytokine. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, as compared with uninfected, LPS-injected, mice. The increased
production of the proinflammatory factors correlated with enhanced
susceptibility of LCMV-infected animals to LPS-induced death, and these
factors were definitively shown to be required for lethality.
Furthermore, the contributions by NK and T cells to death of
dual-challenged mice were defined. These cell populations facilitated
the adverse consequences largely by producing IFN-. Although T
cells, CD8 lymphocytes in particular, were the major producers of the
cytokine, IFN- production by NK cells alone was sufficient to induce
mortality in infected animals, and protection required absence of both
NK and T cell subsets. Taken together, these results define previously
unappreciated mechanisms elicited during viral infections to profoundly
alter responses to secondary heterologous challenges and to potentiate
detrimental consequences. Moreover, they identify the critical soluble
and cellular factors promoting these adverse effects.
The demonstration of a role for T, in addition to NK, cell-produced
IFN- in contributing to lethal reactions following LPS challenge of
virus-infected animals sharply contrasts studies of endotoxin-induced
death in uninfected mice (23, 25). Under those conditions, NK cells are
the predominant contributors to IFN- production (23). In addition,
NK cells are the critical mediators of lethality whereas T cell
contributions appear marginal (23, 25). Here we demonstrate by flow
cytometric analyses that although NK cells comprise most of the total
splenic IFN- producers following LPS injection of uninfected mice,
they represent only low proportions of these populations after LPS
challenge of infected mice. Under the conditions of infection, T cells
are the major IFN- producers, making up >90% of the
IFN--expressing populations (Fig. 5; Tables II and III). Moreover, T
cells are required for most, but not all, of the heightened LPS-induced
serum IFN- response in infected mice (Table I). Thus, unlike LPS
injections of uninfected animals, during dual viral/LPS challenge, T
cells along with NK cells are induced to synthesize IFN-, and the T
cells are the major producers of the cytokine.
However, absence of T cells alone is not sufficient to protect from the
increased sensitivity to LPS-induced lethality during infection (Fig. 4). NK cells also contribute to promoting death. On day 7 following
LCMV infection of immunocompetent mice, the T cell responses are at
high levels and NK cell activities are waning (62, 63). Nevertheless,
exposure to LPS results in NK cell IFN- production. Moreover, a
synergy for IFN- production between the viral infection and the LPS
stimulation, albeit diminished, occurs in the absence of T lymphocytes.
NK cells are the principal players under these conditions. Their
production levels of the factor are sufficient to play a
physiologically critical role, because virus-infected, T cell-deficient
mice succumb to LPS challenge, but mice rendered completely deficient
in IFN- functions are protected from lethality.
The focus of these studies has been on the classical non-T NK cells,
i.e., the NK1.1+CD3- subset, and conventional
T cells, i.e., the CD8+CD4- and
CD4+CD8- subsets. Another nonclassical NK cell
population has been identified based on its expression of both NK1.1
and CD3 (24), and both the CD3- and CD3+ NK
cells subsets can be induced by LPS to express IFN- in
immunocompetent mice (23). Because the NK1 T cell is found only in very
low frequencies in the spleen, it is not a major contributor to the
IFN--expressing cells in this compartment. It is found at high
frequencies in certain other compartments including the liver and may
contribute to the IFN- responses at such sites and/or in the serum.
This has not been examined in the experiments reported here. The flow
cytometric analyses of non-T NK cells clearly demonstrate that
classical NK cells are responding to LPS challenge with IFN-
expression (Fig. 5), and the experiments in infected SCID and
RAG-1-/- mice, genetically lacking all T cells
including NK1 T cells, suggest that they are primed to do so during
viral infection (Table I).
To our knowledge, the experiments presented here are the first
carefully examining NK cell IFN- production by flow cytometric
analysis of intracellular protein. Remarkably, it was possible to
demonstrate this after LPS treatment of both uninfected and
LCMV-infected mice without further ex vivo stimulation (Fig. 5).
Ongoing studies in our laboratory indicate that NK cell IFN-
expression also can be detected spontaneously during infections with a
virus eliciting an early NK cell IFN- response, MCMV (K. B.
Nguyen and C. A. Biron, unpublished observation). This is in
contrast to results from others examining T cell IFN- expression in
nonviral systems (60) and those from both our own and other
laboratories examining T cell IFN- expression during LCMV infections
(this report) (52, 53). Generally, T cells appear to require sustained
stimulation through their Ag receptors to express significant
cytoplasmic cytokine. Clearly, this is not the case for the
aforementioned NK cell cytokine expression. Interestingly, both of the
NK cell IFN- responses examined are downstream to IL-12 induction
and dependent on this cytokine. The demonstration of T cell IFN-
expression without ex vivo stimulation in infected mice challenged with
LPS (Fig. 5) suggests that the general T cell requirement for sustained
stimulation is not a difference in T as compared with NK cell
responses, but rather a difference in the conditions and/or signals
supporting IFN- responses. Thus, certain pathways, such as LPS or
MCMV induction of NK cell IFN- production in normal mice or LPS
induction of T cell IFN- production in LCMV-infected mice, may be
regulated through fundamentally different mechanisms than Ag-driven T
cell IFN- expression. Alternatively, sustained presence of another
inducing cytokine during the labeling procedure may substitute for Ag
stimulation. However, this is unlikely to explain the results presented
here, because the cells were immediately exposed to brefeldin A to
allow accumulation of intracellular cytokines, and this agent should
block secretion of all cytokines. Whatever the mechanism, the
LPS-induced spontaneous T cell IFN- expression still has a
requirement for endogenous activation, most likely through the Ag
receptor, because it is observed only in infected mice and apparent at
frequencies approaching those of infection-induced T cells primed for
IFN- expression in response to stimulation through the Ag receptor.
Thus, the results of our studies not only identify the contribution of
T cells to IFN- expression under the conditions of LPS challenge
during viral infections but also suggest the existence of different
conditions and requirements for prominent IFN- production by
activated T cells.
The observations of synergy between viral infection and endotoxin
challenge for lethality and proinflammatory cytokine expression extend
previous work from this laboratory demonstrating enhanced sensitivity
to IL-12 during LCMV infections (44, 45). Those studies demonstrated
that IL-12 administration to LCMV-infected mice resulted in profound
induction of serum TNF and IFN- as well as cytokine-mediated
pathologies. Thus, virus-infected animals are primed for elevated
cytokine responses and resulting disease induced by either LPS or
IL-12. However, the infection-enhanced TNF production appears to depend
on endogenous IL-2-dependent T cell responses under conditions of IL-12
administration (45) but is clearly T cell independent in response to
LPS (Table I). This may be a result of the fact that LPS is a potent
inducer of systemic TNF, whereas IL-12 is not. Hence, the IL-12-induced
TNF is likely to be more dependent on synergism with T cell responses.
Taken together, the two systems clearly demonstrate the importance of
endogenous cytokine milieu and cellular activation state in outcome of
exposure to additional challenges. They provide mechanistic
explanations for certain reported complications resulting from
concurrent viral and bacterial infections in humans (33, 34), and
toxicities observed during dual challenges of mice with these agents
(35, 36, 41, 42). Moreover, the results suggest parameters to be
considered in development of therapeutic interventions with cytokine
treatment and/or vaccination in the context on going viral infections.
In summary, these studies define mechanisms whereby the immune response to an underlying viral infection could modify outcome of a secondary challenge. They demonstrate that viral infections can potently synergize with components from bacteria to hyperstimulate the immune system, leading to exaggerated expression of proinflammatory cytokines and pathological consequences. The data highlight the contributions by both NK and T cells and emphasize the pathology-inducing potential of NK cells, especially in the context of T cell immunodeficiency. In addition, they demonstrate a virus-induced priming of T cells for cytokine expression in response to secondary challenges. These results help define the complex interactions in place during dual or multiple infectious challenges and have major implications for consequences of therapeutic intervention in the context of infection.
|
Acknowledgments |
|---|
sera; and Dr. Steve Opal for his
gift of anti-TNF Abs. |
Footnotes |
|---|
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; RAG-1, recombination activation gene 1.
Received for publication December 11, 1998. Accepted for publication February 17, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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),
anti-IL12 (
), or anti-IFN-
). Mice for these experiments were used at 12
to 13 wk of age. B, C57BL/6 (
