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in Response to Bacterial Pathogens1
*
Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom;
Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand; and
School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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response is essential for
host survival. To identify the cellular sources of IFN-, spleen
cells from uninfected mice were stimulated with B.
pseudomallei in vitro and assayed by ELISA and flow cytometry.
Costaining for intracellular IFN- vs cell surface markers
demonstrated that NK cells and, more surprisingly, CD8+ T
cells were the dominant sources of IFN-. IFN-+ NK
cells were detectable after 5 h and IFN-+
CD8+ T cells within 15 h after addition of bacteria.
IFN- production by both cell populations was inhibited by
coincubation with neutralizing mAb to IL-12 or IL-18, while a mAb to
TNF had much less effect. Three-color flow cytometry showed that
IFN--producing CD8+ T cells were of the
CD44high phenotype. The preferential activation of NK cells
and CD8+ T cells, rather than CD4+ T cells, was
also observed in response to Listeria monocytogenes or a
combination of IL-12 and IL-18 both in vitro and in vivo. This
rapid mechanism of CD8+ T cell activation may be an
important component of innate immunity to intracellular
pathogens. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-activated murine
macrophage cell lines, and the bacteria were susceptible to the killing
effects of chemically generated NO and superoxide anion in a
macrophage-free system (3). However, neither the sites of
latency nor the mechanism whereby the bacterium evades the host immune
system or induces protective immunity have been identified.
Our previous studies have established murine models of both acute and
chronic melioidosis, which mimic many features of the human pathology
and provide the first evidence of host resistance mechanisms against
B. pseudomallei in vivo (4). Infection with
high doses of B. pseudomallei results in acute disease and
death within 2 days. In contrast, at lower doses mice were able to
clear the inoculum over a 3- to 4-wk period, but persistence of the
organism resulted in a chronic infection. Resistance was absolutely
dependent upon the production of IFN- in vivo within the first
24 h after infection, as administration of neutralizing mAbs
against IFN- lowered the LD50 by
100,000-fold. These results demonstrated the obligatory role of
early production of IFN- to host survival against B.
pseudomallei infection.
For many intracellular pathogens such as Listeria monocytogenes,
Salmonella typhimurium, and Toxoplasma gondii
(5), the rapid production of proinflammatory and
phagocyte-activating cytokines by NK cells is a key factor of early
host resistance against infection (6). The triggering of
NK cells for IFN- production involves the synergistic action of
TNF-
and IL-12 (7). IL-18 is also a potent inducer of
IFN-, acting in synergy with IL-12 (8, 9, 10, 11). In vivo
functions of IL-18 have been studied recently in many intracellular
infections, such as Yersinia enterocolitica,
Mycobacterium spp, Cryptococcus neoformans, and
Salmonella typhimurium (12, 13, 14), and reveal a
key role for IL-18 in early host resistance to infection.
The purpose of this study was to identify the cellular sources of
IFN-, which is rapidly produced in response to B.
pseudomallei in the naive host. As predicted from our previous
studies with other intracellular pathogens (6), B.
pseudomallei was a potent inducer of IFN- production from
splenic NK cells in vitro. More surprisingly,
CD8+ TCR
+ cells from
previously uninfected mice were also rapidly activated for IFN-
production via a cytokine-dependent bystander mechanism involving the
synergistic action of IL-12 and IL-18. We propose that this novel
pathway of CD8+ T cell activation may be an
important component of the innate IFN- response against
intracellular pathogens.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B. pseudomallei strain 708A is a gentamicin-sensitive
isolate, originally obtained from the splenic abscess of a melioidosis
patient, and has been used in the previous studies of animal models and
cell biology in our laboratory (4, 15, 16). For this
project, bacteria were subcultured onto Columbia agar (Oxoid, U.K.) for
24 h at 37°C, then scraped and resuspended in sterile
pyrogen-free saline. The number of viable bacteria was determined by
colony-forming counts before irradiation with 5 x
104 rad for 2 h (Gammacell 1000 ELITE,
Wizard, U.K.). -Irradiated B. pseudomallei were aliquoted
and stored in PBS plus 10% v/v glycerol at -70°C. Before use, an
aliquot of the bacteria was thawed, washed twice in RPMI at 10,000
x g for 5 min, and added to cells as described below.
Mice
Female mice at 8–10 wk of age were used throughout this study. C57BL/6 mice were obtained from Charles River (Margate, U.K.). µMT mice (n = 6 to C57BL/6) were originally obtained from Bantin & Kingman (Hull, U.K.) and bred under aseptic conditions in filter top cages at the London School of Hygiene and Tropical Medicine (London, U.K.).
Cytokines, anti-cytokine Abs, and other reagents
The following recombinant murine cytokines were used: IL-2
(PharMingen, San Diego, CA), IL-12 (1.67 x
106 U/mg; Genzyme, Cambridge, MA), IL-18
(PeproTech, London, U.K.), and TNF- (2.7 x
108 U/mg; Genzyme).
The following mAbs against mouse cytokines were used for neutralization: anti-IL-12 (C17.8, rat IgG2a; a kind gift from G.Trinchieri, Wistar Institute, Philadelphia, PA), anti-TNF (TN3.19.12, hamster Ig) and H22 (hamster Ig) (kind gifts from R. D. Schreiber, St. Louis, MO), and isotype-matched control Abs, GL117.4 and L2, respectively. Anti-mouse IL-18 (39-3F, rat IgG1; MBL, Nagoya, Japan) and rat IgG1 isotype control (PharMingen) were also used for neutralization studies.
Preparation and stimulation of murine spleen cells in vitro
Naive mice were killed by cervical dislocation, the spleens removed aseptically, single cell suspensions made by passing through sterile meshes, and erythrocytes lysed using Tris-buffered ammonium chloride. The resulting cell suspensions were washed twice in RPMI, counted by trypan blue exclusion staining, and adjusted to 6 x 106 cells/ml. The cells were suspended in RPMI 1640 medium (Life Technologies, U.K.) supplemented with 10% FCS, 10 mM L-glutamine, 25 mM HEPES, 200 U/ml penicillin, 200 µg/ml streptomycin, 50 µM 2-ME, and 10 µg/ml indomethacin. Cells were plated at 100 µl/well in 96-well flat-bottom cultured plates (Life Technologies, Paisley, U.K.) and incubated with 100 µl/well stimulus or medium control in 5% CO2 at 37°C for 24–48 h; the supernatants and/or cells were harvested for analysis of cytokine production.
ELISA for detection of cytokine production
Spleen cell culture supernatants were assayed for IFN-
production by double Ab sandwich ELISA. Plastic 96-well microtitration
plates (Immulon 2; Dynatech, Sussex, U.K.) were coated with a mAb to
IFN- (H22) at 5 µg/ml in 0.1 M carbonate-bicarbonate buffer (pH
9.6) overnight at 4°C. The excess reagent was removed by washing the
plates three times with 0.05% Tween 20 in PBS, and this was repeated
between each of the following reagent layers. Test supernatants and a
murine IFN- standard (from 1.2 to 20 ng/ml; Life Technologies) were
added and incubated for 1 h at 37°C. Then, rabbit
anti-IFN- (1:1,000) was added and incubated for 45 min at
37°C. Goat anti-rabbit Ig conjugated to HRP (1:10,000; Kirkegaard
& Perry Laboratories, Gaithersburg, MD) was added and incubated for 45
min at 37°C, followed by tetramethylbenzidine substrate (Boehringer
Mannheim, Mannheim, Germany), and read at 450 nm with a Dynatech MR5000
plate reader.
A similar protocol was performed for detection of TNF, except in this
case using 5 µg/ml monoclonal rat anti-mouse TNF mAb (TN3.19.12)
for the coating step, rabbit anti-TNF (1:1000) for the second Ab
step, and a mouse rTNF- standard (Genzyme) ranging from 1.6 to 18.5
ng/ml.
For the detection of IL-12p40, rat monoclonal anti-mouse IL-12
(C15.6; Cambridge Bioscience, Cambridge, U.K.) was used at 2.5 µg/ml
for coating onto plastic 96-well microtitration plates (Immulon2;
Dynatech) overnight at 4°C. The excess reagent was washed
out three times with 0.05% Tween-20 in PBS, and this was repeated
between each of the following steps. This was followed by a 0.5%
BSA-blocking step for 60 min at room temperature, then mouse rIL-12
standard (Genzyme) was added at concentrations ranging from 1.7 to 17.3
ng/ml and incubated for 1 h at 37°C. Then biotinylated rat
anti-mouse IL-12 mAb (C17.5; Cambridge Bioscience) was used for the
second Ab step for 45 min at 37°C and followed by streptavidin
peroxidase (Serotec, Oxford, U.K.) at 2.5 µg/ml for 45 min at 37°C.
Substrate addition and analysis of OD were as described for the IFN-
ELISA. ELISA detection of murine IL-18 was performed as previously
described, with a sensitivity of >300 pg/ml (17).
Flow cytometry for intracellular IFN- and cell surface marker
staining
Spleen cell cultures stimulated with B. pseudomallei
or other reagents were established as described above. Cytokine
secretion was then blocked by the addition of brefeldin A
(BFA;3 Sigma, St. Louis, MO) for 3 h at a
final concentration of 10 µg/ml. Brefeldin A was prepared as a stock
solution of 10 mg/ml in DMSO. Spleen cells were washed once with 1%
FCS-PBS adjusted to 0.5 x 106 cells/tube
and stained for cell surface markers. The following FITC-labeled mAb
were used for cell surface staining at 1–2 µg/tube: anti-CD4
(H129.19, rat IgG2a), anti-CD8a (53-6.7, rat IgG2a),
anti-TCR (H57-597, hamster Ig) (Sigma), anti-TCR
(GL3, hamster Ig), anti-pan-NK cells (DX5,
rat IgM), anti-NK1.1 (PK136, rat IgG2a), and anti-CD44 (IM7,
rat IgG2b) (PharMingen). Isotype-matched control Abs were included in
each analysis. Staining of cell surface markers was performed for 30
min at 4°C; the cells were washed twice, then fixed with 2%
paraformaldehyde-PBS for 20 min at 4°C, and permeabilized by the
addition of 0.1% saponin-1% FCS-PBS. Cells were then incubated with 1
µg/tube PE anti-IFN- (XMG1.2, rat IgG1; PharMingen) for 45 min
at 4°C in 0.1% saponin-1% FCS-PBS, washed twice with 0.1%
saponin-1% FCS-PBS, and kept in PBS. Staining was analyzed for a total
of 50,000 events gated on viable cells by forward/side scatter using a
FACScan flow cytometer with CellQuest software (Becton Dickinson,
Mountain View, CA).
CD4+ or CD8+ T cell purification by immunomagnetic beads
CD4+ or CD8+ cells
were positively selected from naive spleen cells by using
CD4+ (L3T4) or CD8+ (Ly-2)
microbeads and MS+/RS+
columns, as described in the manufacturer’s instructions (Miltenyi
Biotec, Bergisch Gladbach, Germany). Purified cells were then incubated
with various combinations of cytokines for 18 h, and cells were
collected for analysis of intracellular IFN- vs T cell markers.
In vivo infection with L. monocytogenes
Female µMT mice at 10 wk of age
were i.v. injected either with 1 x 107 CFU
L. monocytogenes or 0.2 µg IL-12 plus 1 µg
IL-18 or pyrogen-free saline. To assess bacterial burdens, spleen cells
were collected 45 min and 16 h postinfection, prepared, and plated
onto brain heart infusion agar (Difco, Detroit, MI) for bacterial
colony counts. For intracellular IFN- vs cell marker analysis,
spleen cells were cultured in 10 µg/ml BFA for 3 h, as
previously described, in the presence of 50 ng/ml PMA and 100 ng/ml
ionomycin before staining.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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by murine spleen cells in vitro
IFN- plays an essential protective role within the first 24–48
h after infection with B. pseudomallei in vivo
(4). To develop an in vitro culture system to identify the
cellular sources of this rapid IFN- response, spleen cells from
uninfected mice were incubated with -irradiated B.
pseudomallei and culture supernatants collected and assayed for
IFN- levels by ELISA at 24 or 48 h. Our initial experiments
demonstrated that spleen cells from µMT mice consistently produced 4-
to 5-fold higher levels of IFN- than C57BL/6 mice at all doses of
bacteria tested; therefore, µMT mice were used for all subsequent
experiments. As shown in this study, IFN- production was induced in
a dose-response manner with maximal values observed with 1 x
106–107 B.
pseudomallei/ml (Fig. 1
A). Dose-dependent induction
of IFN- was also observed following incubation of B.
pseudomallei with C57BL/6 rag-/-
spleen cells, which possess functional NK cells but no T or B cells;
however, the levels of IFN- produced were consistently 2- to 3-fold
lower than with C57BL/6 cultures. In contrast, no IFN- was obtained
using cells from CD3
Tg26 transgenic mice that are deficient in both
NK cells and T cells (data not shown). These results indicate that
maximal induction of IFN- was observed in the presence of both NK
cells and T cells.
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response, spleen cells were
incubated with B. pseudomallei, and IFN- production
assayed after 2, 5, 24, and 48 h. IFN- was not detectable by
ELISA until 24 h and reached maximal levels within 48 h (Fig. 1
B). However, analysis of IFN- production by
intracytoplasmic staining and flow cytometry demonstrated an increase
in IFN-+ cells at 5 h, which peaked at
24 h.
Cellular sources of IFN- induced by B.
pseudomallei in vitro
To directly examine the cellular source of IFN-, spleen cells
were stimulated with B. pseudomallei for 18 h, and
stained for intracellular IFN- vs cell surface expression of NK cell
(DX5) or T cell (TCR, CD4, and CD8) markers (Fig. 2). Isotype-matched Ig controls showed
background staining of less than 0.2% in all experiments. The
percentage of IFN-+ cells increased from a
medium control of <0.5% to 10% following stimulation with
B. pseudomallei. Of the IFN-+
cells, 60% were DX5+ NK cells and 40%
TCR+ cells. Of all NK cells, 28–30%
was induced to secrete IFN- by B. pseudomallei, whereas
up to 10% of all CD8+ T cells were IFN-
secretors. In other experiments, three-color flow cytometry
demonstrated that less than 1% of IFN--producing
CD8+ cells expressed NK1.1, and that essentially
all IFN-+ NK1.1+ cells
were also DX5+, arguing against the involvement
of NK T cells under these conditions (data not shown). In all
experiments performed, the frequency of IFN-+
CD4+ T cells was consistently lower than for
CD8+ T cells (Fig. 2).
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production by NK cells vs
CD8+ T cells was then compared. In this
experiment, IFN-+ NK cells were first detected
at 5 h, followed by CD8+ T cells at 24
h (Fig. 3). By 48 h, the frequency
of IFN-+ NK cells had declined, while the
production from T cells remained elevated. Together, these results
identify two major IFN--positive cell populations, NK cells and
CD8+ T cells, which are rapidly induced by
B. pseudomallei in vitro.
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in response to B.
pseudomallei in vitro
Previous studies by Sprent and colleagues (18, 19)
have shown that bacterial components such as LPS can preferentially
activate memory (CD44high)
CD8+ T cells rather than naive
(CD44low/int) cells for proliferation in vitro
and in vivo. To compare the response of naive vs memory
CD8+ T cells to B. pseudomallei,
spleen cells from uninfected mice were incubated with B.
pseudomallei for 15 h and stained for intracellular IFN-
vs CD8 and CD44 markers by three-color flow cytometry. As shown in Fig. 4, under these conditions essentially all
highly IFN-+ CD8+ T
cells also expressed high levels of CD44
(CD44high), suggesting an activated/memory
phenotype.
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by B.
pseudomallei
In other pathogen models, TNF- and IL-12 are two major
stimulators of IFN- production during the innate immune response
(6). To investigate the role of these cytokines in
response to B. pseudomallei, spleen cells were stimulated
with bacteria and cell culture supernatants assayed for levels of
IL-12, TNF, and IFN- by ELISA after 24 h. Addition of B.
pseudomallei induced secretion of both IL-12 and TNF in a
dose-dependent manner (Fig. 5A), which correlated well
with the induction of IFN- (Fig. 1A).
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, neutralizing mAbs to these cytokines were
added at the time of addition of B. pseudomallei. Addition
of 0.1–1 µg/ml Ab to IL-12 inhibited IFN- production by up to
80%, as assayed by ELISA (Fig. 5B). In contrast,
anti-TNF- was less effective, giving 30% inhibition even at
10 µg/ml (Fig. 5C). These results were confirmed using
flow cytometry and showed the effects of neutralizing IL-12 on the
production of IFN- by both NK cells and CD8+ T
cells, whereas neutralizing TNF showed partial effects on NK cells and
even less on CD8+ T cells (data not shown). These
results suggest that IFN- production by both NK cells and
CD8+ T cells requires IL-12, but that TNF- is
less important.
The role of IL-18 in the induction of IFN- in response to
B. pseudomallei
IL-18 is an important inducer of IFN- secretion in response to
LPS and has been implicated in resistance against several intracellular
pathogens (20, 21). To evaluate the role of IL-18 in the
IFN- response seen in this study, supernatants from spleen cells
stimulated with B. pseudomallei were collected after 24
h and assayed for IL-18 by ELISA. High levels of IL-18 were induced in
a dose-dependent manner by B. pseudomallei, which correlated
with the induction of IFN- measured at 48 h (Fig. 6A). Addition of 0.1–10
µg/ml monoclonal rat anti-IL-18 into spleen cell cultures
stimulated with the optimal dose of B. pseudomallei
progressively reduced IFN- production. Complete inhibition was
observed with 
10 µg/ml anti-IL-18, while the same concentration
of a rat IgG1 isotype-matched control had no effect (Fig. 6B). Taken together, the above results indicate that
B. pseudomallei is a potent inducer of both IL-12 and IL-18,
and that these two cytokines are obligatory for the subsequent
production of IFN- by both NK cells and CD8+ T
cells.
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production by NK cells and CD8+ T cells
To ask whether the preferential activation of both NK cells
and CD8+ T cells also occurs with other
intracellular bacterial pathogens, spleen cells were incubated with
-irradiated L. monocytogenes, and IFN- production was
assayed by ELISA and flow cytometry. L. monocytogenes
induced the dose-dependent secretion of IFN- that peaked at between
3 x 105 and 1 x
106 bacteria/ml (data not shown). Intracellular
IFN- vs cell surface marker analysis demonstrated that both
IFN--producing NK cells and CD8+ T cells had
responded to L. monocytogenes, again showing a
CD8+ rather than CD4+ T
cell preference as observed with B. pseudomallei (Fig. 7A). Furthermore, direct
addition of rIL-12 plus IL-18 mimicked the IFN- responses observed
with intact bacteria. Addition of either IL-12 or IL-18 alone had
minimal effects on IFN- synthesis, consistent with the known
synergistic actions of these two cytokines in other systems
(10). The results suggest that cytokine-mediated
activation of CD8+ T cells (and NK cells) is not
unique to B. pseudomallei and point to the key role of IL-12
and IL-18 in this response.
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. The total frequency of IFN-+ cells
was lower in C57BL/6 than µMT spleen cells, consistent with
measurement of IFN- by ELISA (data not shown). However, analysis of
surface phenotypes revealed preferential activation of
CD8+ and NK cells in both mouse strains, with the
frequency of IFN-+ CD8+
T cells being essentially identical (Fig. 7B).
Interestingly, the increased capacity for total IFN- production by
µMT spleen cells (Fig. 7B, inset) appeared to
be mediated by the NK cell population. Effects of proinflammatory cytokines on purified CD4+ and CD8+ cells
To directly compare IFN- production by these two T cell
subsets, CD4+ or CD8+ T
cells were purified from the spleens of uninfected mice by positive
selection using immunomagnetic beads and stimulated with either
bacteria or combinations of cytokines for 18 h. Addition of
B. pseudomallei to purified CD8+ or
CD4+ T cells did not induce IFN- production
above unstimulated controls (Fig. 8). In
contrast, CD8+ rather than
CD4+ T cells responded efficiently to the
combination of exogenous IL-12, IL-18, and TNF, which is found in
unfractionated spleen cell cultures in the presence of B.
pseudomallei, as described above. This cytokine combination
resulted in similar frequencies of IFN-+
CD8+ T cells as unfractionated spleen cells
stimulated with B. pseudomallei. Stimulation with IL-12 and
IL-18 plus IL-2 was shown to induce the greatest response with 18–20%
of all CD8+ T cells becoming IFN- positive vs
2–3% of CD4+ T cells. In summary, these
observations suggest that the IFN- response to B.
pseudomallei is triggered indirectly via the action of IL-12 and
IL-18 and that CD8+ T cells are intrinsically
more responsive to these signals than CD4+ T
cells.
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production by NK cells and CD8+ T cells
also occurs in vivo after L. monocytogenes infection
Finally, we investigated whether the rapid and preferential
activation of CD8+ T cells and NK cells also
occurs in response to bacterial infection in vivo. Mice were infected
i.v. with viable L. monocytogenes and assessed for IFN-
expression ex vivo after 16 h. Compared with saline-treated
controls, mice infected with viable L. monocytogenes showed
a preferential induction of IFN-+
CD8+ and NK cells, but not
CD4+ T cells (Fig. 9). Bacterial burdens immediately after
infection were 2 x 104, and at the time of
spleen cell harvest were 3.5 x 104
bacterial CFU/spleen. A similar but reduced induction of
IFN-+ CD8+ and NK cells
was seen following IL-12 + IL-18 injection in the absence of bacteria.
The results indicate that the rapid activation of
CD8+ T cells (and NK cells) to produce IFN-
also occurs in response to bacterial infection in vivo.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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response
within 24–48 h after primary infection in mice (4). In
this study, we describe an in vitro model using spleen cells from
previously uninfected mice to investigate the cellular sources and
regulation of this response. Our data show that B.
pseudomallei is a potent and rapid inducer of IFN- production
by murine spleen cells. This response could be detected intracellularly
in NK cells within 5 h and in CD44high
activated/memory phenotype CD8+ T cells by
15 h after contact with the bacterium. However, there was little
production by CD4+ T cells at any time point
examined. Neutralization of IL-12 or IL-18 abolished IFN- production
by both responding cell types. Furthermore, recombinant IL-12 plus
IL-18 induced IFN- production with similar magnitude and T cell
subset preference as intact bacteria. Together, these data suggest that
cytokine-mediated bystander activation of CD8+ T
cells contributes to the initial IFN- response to bacterial
pathogens.
Previous studies with a diverse range of pathogens, including bacteria,
protozoan parasites, and some viruses, have demonstrated the role of NK
cells in IFN- production during the innate immune response (6, 22, 23). In this study, addition of B. pseudomallei
to naive spleen cells resulted in the rapid activation of
DX5+/NK1.1+ cells for
IFN- production. Recent evidence suggests that several distinct T
cell populations also have the potential to make IFN- with an
intermediate kinetics between the rapid NK cell response and the
acquisition of acquired immunity (24, 25). These include
NK T cells (26, 27), T cells (11, 28),
CD4- CD8-
(29), or CD8+ T cells responding to
microbial products presented via the CD1 system (30, 31)
and other CD8+ T cells triggered via recognition
of N-formylated bacterial peptides via the H2-M3 Ag
presentation system (32). Several other cell types,
including macrophages, B cells, and dendritic cells, have also been
reported to secrete IFN- (33, 34, 35). However, we do not
believe that these cell types contribute substantially to the IFN-
responses described in this work. Thus, NK cells plus
CD8+ TCR+ cells
together account for greater than 90% of all IFN--positive cells
under these conditions. We have used µMT mice for many of these
experiments, because they produce greater levels of IFN- in response
to B. pseudomallei, presumably due to the relative
enrichment of T cells and NK cells in culture. However, the relative
proportion of IFN-+ CD8+
vs IFN-+ CD4+ T cells
described above was also observed in immunocompetent C57BL/6 spleen
cells.
Several lines of evidence suggest that the activation of
CD8+ T cells seen in this study is a consequence
of cytokine-mediated bystander events rather than through engagement of
the TCR. First, these responses were observed in spleen cell cultures
from naive animals and were maximal within 15–18 h after addition of
bacteria in vitro. Second, up to 10% of all CD8+
T cells are driven into IFN- production within this period. Third,
the responses to intact bacteria were abolished by neutralization of
IL-12 or IL-18 in vitro, and could be mimicked by addition of the
recombinant cytokines in the absence of bacteria. Finally, Ag-specific
activation is unlikely to show such pronounced bias in favor of
CD8+ T cells given that dead bacteria are most
likely to favor the exogenous Ag presentation pathway and activation of
class II-restricted CD4+ T cells
(36).
Our data show a consistent preference for the generation of
IFN--secreting CD8+ rather than
CD4+ T cells in response to either intact
bacteria or IL-12 plus IL-18. Studies by Tough and Sprent et al.
(37, 38) have shown that memory CD8+
T cells are preferentially responsive to bacterial LPS, CpG DNA, and
downstream cytokines such as type I IFNs and IL-15 for incorporation of
5-bromo-2'-deoxyuridine and cell proliferation. This has been suggested
to underlie the Ag-independent maintenance of memory
CD8+ T cells in vivo via bystander activation by
cytokines generated from exposure to antigenically unrelated organisms.
Analysis of CD44 expression profiles in our experiments showed that
CD44high rather than
CD44low/int CD8+ T cells
preferentially expressed IFN- following activation by B.
pseudomallei. Thus, activated/memory CD8+ T
cells exhibited preferential IFN- expression compared with naive
cells by these criteria. The patterns of basal or induced expression of
receptors for IL-12 and IL-18 may explain both the distinct kinetics of
the NK cell vs CD8+ T cell responses as well as
the pronounced bias in CD8 vs CD4 activation seen in this study. NK
cells, which become IFN- positive within 5 h after addition of
bacteria, are reported to constitutively express receptors for both
cytokines (39, 40). In contrast, splenic
CD8+ T cells from uninfected mice express low
levels of IL-18R, but show increased receptor transcripts and
functional responsiveness to IL-18 within several hours after
activation with IL-12 (35). Furthermore, induction of
IL-18R expression is significantly greater in
CD8+ than CD4+ T cells
(41), consistent with the T cell subset preferences we
observe. Studies are in progress to test whether changes in IL-18R
underlie the distinct kinetic and phenotypic characteristics of the
IFN- response to B. pseudomallei.
Our results support the growing evidence that resistance to B.
pseudomallei is strongly influenced by the cell-mediated immune
response and, in particular, the production of IFN-. Mice depleted
of IFN- by either mAbs or gene deletion showed markedly increased
susceptibility to lethal infection in vivo (4). B.
pseudomallei grows intracellularly in both human and murine
macrophages or macrophage cell lines, but can be killed by both NO- and
oxygen-dependent mechanisms following activation of the cells with IFN-
in vitro (3). Furthermore, genetically determined
resistance of different mouse strains to B. pseudomallei
correlates with the induction of type 1 rather than type 2 immune
responses, as measured by the preferential induction of IgG2a vs IgG1
(42). We now demonstrate directly that IL-12 and IL-18,
two cytokines that are key determinants in the generation of type 1 T
cell responses, are rapidly produced in response to B.
pseudomallei in the naive host and serve to generate an
IFN--enriched environment within the first few hours after contact
with the bacterium. These results are also consistent with our previous
demonstration that neutralization of IL-12 increases the susceptibility
of mice to infection with B. pseudomallei in vivo
(4).
The mechanisms of immunity to melioidosis in humans are less well
understood. Incubation of B. pseudomallei with PBMC of
healthy individuals results in the rapid secretion of IFN- , IL-12,
and IL-18, although the nature of the responding cells is not known
(43). However, high levels of IFN- , IL-18, IL-12p40,
and IL-15 have been reported in the serum of patients presenting with
acute septicemic melioidosis, and in the case of IL-18 and IFN- were
higher in nonsurvivors, although these differences did not reach
statistical significance (43). Plasma concentrations of
IL-6, IL-8, and TNF are also elevated in septicemic melioidosis and
correlate with disease severity and clinical outcome (44, 45). It therefore seems that B. pseudomallei induces
a similar spectrum of proinflammatory cytokines in both humans and
mice. From these studies and data from other pathogen models, we
predict that these cytokines will contribute to resistance in the
majority of the population living in endemic areas, which show no signs
of clinical disease, but can nevertheless also contribute to the
pathology of septic shock when immunity fails due to underlying factors
such as diabetes.
In conclusion, the results presented in this work reaffirm the
prominent role of NK cells as rapid and effective producers of IFN-
in response to bacterial pathogens. In addition, we highlight a
substantial contribution from CD8+
TCR+ T cells responding to cytokines in an
Ag-independent manner. Yoshimoto et al. (35), using
recombinant cytokines as stimuli, first suggested the potential
importance of Ag-independent T cell activation via IL-12/IL-18 in
innate immunity. To our knowledge, the data presented in this study
provide the first direct evidence that pathogens can actually trigger
this pathway, supporting the possibility that bystander
CD8+ T cell activation may contribute to
IFN--mediated resistance to infection in vivo. Our findings that
CD8+ T cells (as well as NK cells) are induced to
express IFN- within 16 h after challenge with viable L.
monocytogenes in vivo support this concept. Together, these data
suggest that bystander cytokine activation of
CD8+ T cells may operate at several levels in
contributing to host immunity: first, by providing an additional source
of IFN- during the early phase of response to infection; second, in
expansion of effector T cells (46); and third, by
nonspecifically maintaining the memory T cell pool (47).
These results imply that prior antigenic experience provides the host
not only with specific memory T cells, but a pool of cells, which
collectively can contribute to innate immunity against subsequent
unrelated infection. Thus, we believe that both NK cells and
CD44high CD8+ T cells
respond to cytokines produced by phagocytic cells following contact
with intracellular pathogens and may provide the initial sources of
IFN- following infection.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Gregory J. Bancroft, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, U.K.
3 Abbreviation used in this paper: BFA, brefeldin A.
Received for publication June 19, 2000. Accepted for publication October 23, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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