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Department of Cell and Molecular Biology, Section for Immunology, Lund University, Lund, Sweden
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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+,
CD8-CD4-, and
CD8-CD4+ DC, from these organs in response
to Salmonella. Infection of purified splenic DC with
S. typhimiurium expressing green fluorescent protein
(GFP) and OVA revealed that all three splenic DC subsets internalize
bacteria, and splenic as well as MLN DC process
Salmonella for peptide presentation. Furthermore,
presentation of Salmonella Ags on MHC-I and MHC-II was
evident in both CD8+ and CD8- splenic DC
subsets. Direct ex vivo analysis of splenic DC from mice infected with
GFP-expressing Salmonella showed that all three subsets
harbored bacteria, and splenic DC purified from mice given
Salmonella-expressing OVA presented OVA-derived peptides
on MHC-I and MHC-II. Cytokine production analyzed by intracellular
staining of splenic DC infected with GFP-expressing
Salmonella revealed that TNF- was produced by a large
percentage of CD8- DC, while only a minor proportion of
CD8+ DC produced this cytokine following bacterial
exposure. In contrast, the greatest number of IL-12p40-producing DC
were among CD8+ DC. Experiments inhibiting bacterial
uptake by cytochalasin D as well as use of a Transwell system revealed
that bacterial contact, but not internalization, was required for
cytokine production. Thus, DC in sites of Salmonella
replication and T cell activation, spleen and MLN, respond to bacterial
encounter by Ag presentation and produce cytokines in a subset-specific
fashion. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Murine splenic DC were originally divided into two subsets based on
surface expression of CD8 (12). The observation that
CD8- splenic DC could be further divided into
CD4+ and CD4- populations
resulted in the definition of three major murine splenic DC subsets,
CD8+,
CD8-CD4+
(CD4+), and
CD8-CD4-
double-negative (DN) subsets (7, 13, 14), which appear to
develop along independent pathways (7). The DC subsets
have a differential capacity to secrete cytokines upon microbial
stimulation (15, 16, 17, 18, 19, 20, 21). Recent studies have also suggested
that the splenic DC subsets may differ in their capacity to take up and
present soluble or particulate Ags (5, 6, 7, 8).
A critical feature of DC is their capacity to activate naive T cells (1). Despite this, the role of DC, particularly DC subsets, in triggering T cells during bacterial infection has not been established. Using a murine infection model with the Gram-negative bacterium Salmonella enterica serovar Typhimurium (Salmonella typhimurium) it has been shown, however, that both splenic (22) and Peyer’s patch (23) DC harbor Salmonella during infection. Furthermore, splenic DC are activated in Salmonella-infected mice (22), and bone marrow-derived DC loaded with S. typhimurium can elicit bacteria-specific CD4+ and CD8+ T cells after transfer into naive animals (22). In addition, the three splenic DC subsets are differentially modulated with respect to distribution, number, and cytokine production in response to oral Salmonella infection (16). Despite these observations, and the finding that immature bone marrow-derived DC can process Salmonella for peptide presentation on MHC-I and MHC-II (24, 25, 26), the capacity of DC from lymphoid organs to process bacteria for peptide presentation is not known.
Thus, the present study examined the capacity of DC from spleen and MLN, sites of Salmonella replication and T cell activation, to present bacterial Ags. In addition, the capacity of splenic DC subsets to internalize bacteria and produce cytokines upon infection as well as the relationship between bacterial internalization and cytokine production were examined.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c, C57BL/6, C3H/HeN, C3H/HeJ, and OT-I (27) mice were bred at the animal facilities of Lund University (Lund, Sweden) or purchased from Charles River Laboratories (Sulzfeld, Germany). DO11.10 mice (28) were provided by Active Biotech (Lund, Sweden).
Bacterial strains and infection of mice
S. typhimurium 14028r harboring pJLP-2H-Kan
(25), pJLP-1E-Kan (25), or pOVA
(29) were used for in vitro studies. S.
typhimurium 
4550 harboring pYA3259rOVA or pYA3259rOVA-GFP were
used in vivo (22). pJLP-2H-Kan encodes the fusion protein
Crl-OVA, which contains residues 257–277 of OVA fused near the
carboxyl terminus of the cytoplasmic bacterial protein Crl. pJLP-1E-Kan
encodes the Crl-HEL fusion protein, which contains residues 45–61 of
hen egg white lysozyme (HEL) fused to Crl. pOVA and pYA3259rOVA encode
full-length OVA. pYA3259rOVA-GFP encodes green fluorescent protein
(GFP) fused to the carboxyl terminus of OVA. S. typhimurium
14028r containing pJLP-2H-Kan or pJLP-1E-Kan were grown in
Luria-Bertoni (LB) broth or on LB agar plates supplemented with 50
µg/ml kanamycin overnight at 37°C. When pOVA was used,
carbenicillin was substituted for kanamycin. 4550 pYA3259rOVA
(called 4550 rOVA) and 4550 pYA3259rOVA-GFP (called 4550
rOVAGFP), which contain the cloned inserts in a balanced lethal vector
system to allow plasmid maintenance in the absence of antibiotic
selection (30), were grown in LB broth without
antibiotics.
Bacterial suspensions were prepared by removing colonies from agar plates into PBS, pH 7.4 (Life Technologies, Paisley, U.K.). Suspensions were centrifuged at 1700 x g, resuspended in IMDM (without antibiotics; Life Technologies) or PBS, and quantitated spectrophotometrically by determining the OD at 600 nm. Alternatively, liquid bacterial cultures were centrifuged and resuspended in IMDM without antibiotics. Bacterial suspensions were then diluted in IMDM without antibiotics to obtain the desired amount of bacteria to be added to the Ag processing assay or in PBS for inoculation of mice.
Mice were inoculated i.v. in the lateral tail vein with 150 µl bacterial suspension and were sacrificed 4 h later. The quantity of live bacteria actually administered as well as the number of CFU recovered in the spleen upon sacrifice were determined by viable plate counts from the suspension used for infection or from a splenocyte suspension, respectively.
Flow cytometry
Flow cytometric analysis was performed using a FACSCalibur flow
cytometer (BD Biosciences, Mountain View, CA). Abs from hybridomas
2.4.G2 (anti-Fc
II/III), M5/114 (anti-MHC-II), N418
(anti-CD11c), GK1.5 (anti-CD4), YTS.169 (anti-CD8), and
C17.8 (anti-IL-12p40) were used. Abs were purified from
supernatants using -Bind Plus columns (Pharmacia Biotech, Uppsala,
Sweden) and were labeled with biotin (Sigma-Aldrich, St. Louis, MO),
Cy5 (Amersham Pharmacia Biotech, Uppsala, Sweden), or FITC (Sigma).
FITC-labeled anti-CD11c; PE-labeled anti-CD4, CD8, CD11c,
and MHC-II; PerCP-labeled anti-CD8; allophycocyanin-labeled
anti-CD4; biotinylated anti-CD11b; and biotinylated
anti-IFN- Abs were purchased from BD PharMingen (San Diego, CA).
Biotinylated anti-TNF- Ab was purchased from Caltag Laboratories
(Burlingame, CA). Streptavidin-allophycocyanin or streptavidin-PE (both
from BD PharMingen) were used as second-step reagents. In stainings
where PerCP-labeled anti-CD8 was absent, 7-amino-actinomycin D
(Sigma-Aldrich) was used to exclude dead cells. Incubations with Abs or
reagents for surface staining were performed for 15 min at 4°C in
FACS buffer. FACS buffer is HBSS (Life Technologies, Paisley, U.K.)
containing 3% FCS, 2 mM EDTA, and 0.01% sodium azide.
Isolation of splenic DC
Splenic DC were purified from naive mice or mice immunized i.p. for 9 consecutive days with 10 µg recombinant human Flt3 ligand (Flt3L; Immunex, Seattle, WA) as indicated in individual experiments. Spleens were digested with 1.6 mg/ml collagenase type IV (Worthington Biochemicals, Freehold, NJ) and 1 mg/ml DNase 1 (Worthington Biochemicals) in IMDM for 45 min at 37°C. The cells were washed once, and CD11c-expressing cells were enriched using N418 magnetic beads and MidiMACS columns or AutoMACS (Miltenyi Biotec, Bergisch Gladbach, Germany) following the manufacturer’s protocol.
The purity of splenic and mesenteric lymph node (MLN) DC used in
processing and presentation experiments was enhanced by two sequential
passages of splenocytes through an AutoMACS. The first passage over the
column was performed without any addition of beads. This resulted in
cells containing a large proportion of autofluorescent cells (gate R2
in Fig. 1
a) binding to the
column. The negative fraction from this passage was collected,
coincubated with N418 beads, and run over the column again. This
reduced the number of autofluorescent cells in the CD11c-positive
fraction that bound to the column (gate R2 in Fig. 4a). The
enriched population consisted of 
80–90%
CD11c+ and MHC-II+ cells as
determined by flow cytometric analysis. Similar results were obtained
using spleens and MLN from naive and Flt3L-treated mice.
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+ and
CD11c+CD8- cells from
Flt3L-treated mice was performed by FACS sorting of splenocytes stained
with CD11c-FITC and CD8-PE. Alternatively, these cells were purified
using a Multisort anti-FITC kit (Miltenyi Biotec), followed by
anti-PE beads (Miltenyi Biotec). These purification procedures
resulted in 85–90% purity of each CD11c+ subset
as determined by subsequent analysis of CD8 expression by flow
cytometry. In experiments in which splenic DC were purified from mice given Salmonella, all steps were conducted on ice where possible. The spleens from immunized animals were homogenized, and cell suspensions were washed once in HBSS. CD11c-expressing cells were enriched using N418 magnetic beads as described above. This resulted in 85–90% pure CD11c+ cells as determined by flow cytometry.
Intracellular cytokine staining and detection of GFP+ cells
Splenic DC (0.5–1 x 106) were
seeded in Ultra-Low Cluster 24-well tissue culture plates (Costar
Corning, Cambridge, MA) or in the bottom of a Transwell plate
(pore size, 0.1 µm; Costar Corning). S. typhimurium
4550rOVA or 4550rOVAGFP were then added either directly to the DC
or to the top chamber of Transwells at a bacteria to DC ratio of 10:1,
25:1, 50:1, or 250:1 as indicated in individual experiments. Plates
were centrifuged at 270 x g for 4 min and were
incubated for 2 h at 37°C. In some experiments DC were
pretreated with 10 µg/ml cytochalasin D (CCD; Sigma-Aldrich) for 45
min before addition of bacteria, and CCD was also present during the
2-h bacterial coculture at this concentration.
After 2 h of coincubation the cells were washed three times,
resuspended in FACS buffer, and stained on ice as described above.
Alternatively, cells were resuspended in IMDM containing 5% FCS, 50
µg/ml gentamicin, and 5 µg/ml brefeldin A (BFA; Sigma-Aldrich) and
incubated for 5 h at 37°C. The BFA-treated cells were then
labeled with anti-CD11c and anti-CD8. If the DC were
infected with 4550rOVA, they were stained with anti-CD4 as well.
The cells were then fixed in 2% paraformaldehyde (PFA) for 20 min at
room temperature. Fixed cells were permeabilized in HBSS containing 3%
FCS (Sigma-Aldrich), 0.5% saponin (Sigma-Aldrich), and 0.05% azide
for 20 min at room temperature. Intracellular cytokines were detected
by addition of biotinylated Abs followed by
streptavidin-allophycocyanin in permeabilization buffer. Each step was
for 30 min at room temperature. The cells were then analyzed by
four-color flow cytometry.
Ag processing assays
Splenic DC (2 x 105) isolated from
Flt3L-treated mice were seeded in 96-well plates in IMDM containing 5%
FCS. Titrated numbers of 14028r pJLP-2H, 14028r pJLP-1E, or 14028r pOVA
were then added to triplicate wells. As a positive control, the
Kb-binding OVA257–264
peptide or the I-Ad-binding
OVA323–339 peptide was added at the indicated
concentrations. After 2 h the cells were washed three times in
HBSS and were either left in IMDM containing 5% FCS and 50 µg/ml
gentamicin or were fixed in 0.5% PFA for 15 min at room temperature
before addition of IMDM containing 5% FCS and 50 µg/ml gentamicin.
Alternatively, the DC were incubated for an additional 4 h in IMDM
containing 5% FCS and 50 µg/ml gentamicin before fixation and
washing. In some experiments DC were pretreated with 10 µg/ml CCD for
45 min before addition of bacteria and then with 5 µg/ml CCD during
the coincubation with bacteria. Finally, either 2 x
105
Kb/OVA257–264-specific
CD8+ OT-I T cells or 1 x
105
I-Ad/OVA323–339-specific
CD4+ DO11.10 T cells were added. Before addition
to DC, OT-I and DO11.10 T cells were MACS-purified from spleen or MLN
using anti-CD8 or anti-CD4 magnetic beads, respectively. The
T cells were 90–95% pure as determined by flow cytometry. After
64 h of incubation at 37°C, cultures were pulsed with
[3H]thymidine for 8 h, and incorporation
into cellular DNA was subsequently determined.
Experiments using DC from naive mice were performed as described above with the following modifications. DC were seeded in Ultra-Low Cluster 24-well tissue culture plates (Costar Corning) and were infected at a 10:1 bacteria to DC ratio. After washing in HBSS, the cells were resuspended in IMDM containing 5% FCS and 50 µg/ml gentamicin. DC were then seeded in triplicate or duplicate wells of 96-well plates and were diluted 2-fold into IMDM containing 5% FCS and 50 µg/ml gentamicin. The titrated DC were then coincubated with 1 x 105 OT-I or DO11.10 T cells. Proliferation was measured as described above.
When DC were isolated from Salmonella-infected mice the cells were seeded in triplicate or duplicate wells of 96-well plates. Serial 2-fold dilutions of DC were performed in IMDM supplemented with 5% FCS and 50 µg/ml gentamicin. Transgenic T cells were added, and proliferation was measured as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To assess the capacity of the three major splenic DC subsets to
internalize S. typhimurium, CD11c+
cells were purified from spleens of naive mice. Flow cytometric
analysis of CD11c, CD4, CD8, and MHC-II expression by the purified
cells revealed autofluorescent cells previously described as
contaminating macrophages (Fig. 1
a, gate R2) (5, 13). However, the autofluorescence did not interfere with
the fluorochrome used in the third channel, making it possible to gate
out CD11c+ that were CD4+,
CD8-CD4- (DN), or
CD8+ (Fig. 1a). These three
subpopulations of the splenic DC all expressed high levels of MHC-II
(Fig. 1a).
Flow cytometric analysis following coincubation of purified
CD11c+MHC-II+ cells with
S. typhimurium expressing GFP was used to assess bacterial
uptake by the DC subsets. For these analyses, MHC-II staining was
omitted to allow detection of GFP. The data revealed that all three
splenic DC subsets internalize Salmonella (Fig. 1b). Compared with the other two subsets, a slightly higher
percentage of GFP+ cells among
CD8+ DC was consistently observed following
bacterial coculture. Active uptake of bacteria by DC was distinguished
from adherence to the cell surface by performing parallel experiments
in the presence of CCD. A drastically reduced percentage of
GFP+ cells was apparent when DC were pretreated
with CCD (Fig. 1b), which inhibits actin polymerization and
therefore phagocytosis. In addition, uptake was not induced by the
bacteria, as heat-killed Salmonella were also internalized
by all three DC subsets (data not shown). Together these data
demonstrate that CD8+,
CD4+, and DN splenic DC subsets phagocytose
Salmonella following brief coculture in vitro.
Cytokine production by splenic DC after bacterial encounter
We previously showed that CD4+ and
CD8+ splenic DC subsets have a differential
capacity to produce cytokines in mice orally infected with
Salmonella (16). However, cytokine production
by the DN DC subset was not analyzed in these studies. In addition,
these studies did not allow us to ascertain whether the DC producing
cytokines did so because they contained internalized bacteria, because
they had contacted bacteria/bacterial products but did not contain
phagocytosed bacteria, or because they were influenced by cytokines
produced in the microenvironment. To address these issues and ascertain
the requirements for bacterial contact or internalization for cytokine
production, purified splenic DC were coincubated with S.
typhimurium in vitro, and cytokine production was measured by
intracellular staining after a 2-h bacterial pulse.
TNF- was produced by CD4+ and DN DC after
coculture with S. typhimurium (Fig. 2a). In contrast, only a minor
proportion of CD8+ DC produced this cytokine
following bacterial exposure. The production of IL-12p40 measured in
parallel showed that 25–30% of CD8+ DC
produced this cytokine in response to Salmonella, whereas
relatively few CD4+ and DN DC did so (Fig. 2a). To investigate whether bacterial contact with DC was
sufficient to induce cytokine production, and whether the LPS of
Salmonella was responsible for inducing TNF- or IL-12p40
production, several types of experiments were performed. First, the
number of cytokine-producing DC from C3H/HeN and C3H/HeJ mice, the
latter having a point mutation in Toll-like receptor 4, a receptor
critical for LPS-induced cellular responses (31),
following coculture with Salmonella was measured. S.
typhimurium infection of splenic DC from LPS-hyporesponsive
C3H/HeJ mice resulted in a low percentage of TNF--producing DC,
while TNF-+ DC from C3H/HeN mice were 4 times
as abundant (Fig. 2b). Coculture of Salmonella
with C3H/HeN DC also resulted in 25–30%
IL-12p40+ cells, approximately twice as many as
C3H/HeJ IL-12p40-producing DC (Fig. 2b).
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a). Experiments performed in a Transwell, where DC
and bacteria were physically separated by a filter with 1-µm pores,
resulted in markedly reduced numbers of TNF-+
DC. In addition, induction of IL-12p40-producing DC was abrogated in
the Transwell system (Fig. 2a). Thus, a diffusable bacterial
product present in the cultures is not by itself sufficient to elicit
the observed cytokine response.
A final set of experiments was performed to determine the fraction of
DC-producing cytokines when bacteria coculture was performed in the
presence of CCD. These conditions led to partial inhibition of the
number of DC producing IL-12p40 and TNF-, suggesting that contact
between Salmonella and DC was sufficient to induce cytokine
production in a fraction of the cells (Fig. 2a). To directly
assess the relationship between bacterial internalization and cytokine
production, DC were cocultured with GFP-expressing bacteria, and the
number of DC producing cytokines was determined (Fig. 3a). These studies showed that
a major fraction of cytokine-producing cells were GFP negative, i.e.,
they did not contain internalized bacteria (Fig. 3a).
Examining GFP+ CD8- and
CD8+ DC revealed that among
GFP+ CD8- DC roughly
50% produce TNF- (Fig. 3b). In contrast, in the
GFP+ CD8+ DC population
only 10% produce TNF-. A similar analysis of IL-12p40-producing
cells revealed that 25 and 15% of
GFP+CD8+ and
GFP+CD8- DC,
respectively, produced this cytokine (Fig. 3b).
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The capacity of splenic and MLN DC to process bacteria confined to vacuolar compartments such as Salmonella for peptide presentation on MHC-I and MHC-II is presently not known. To address this, freshly isolated DC from these organs from either naive or Flt3L-treated mice were cocultured with S. typhimurium expressing the Kb-binding 257–277 epitope of OVA as the Crl-OVA fusion protein or full-length OVA, the latter also containing the I-Ad-binding OVA323–339 epitope.
Splenic DC purified from Flt3L-treated C57BL/6 mice coincubated with
S. typhimurium-expressing Crl-OVA resulted in proliferation
of OVA257–264/Kb-specific
OT-I T cells (Fig. 4b). The
observed proliferation of OT-I T cells was peptide specific. This was
demonstrated by the lack of proliferation when bacteria expressing an
epitope irrelevant for OT-I cells, the
I-Ak-binding HEL46–61
epitope contained in Crl-HEL, were used (Fig. 4b). That the
OT-I response was abrogated when the DC were pretreated with CCD showed
that active Salmonella uptake was required to observe
peptide presentation, and that peptides present in the culture could
not account for T cell stimulation (Fig. 4b). The processing
of Salmonella-expressing Crl-OVA occurred rapidly, as
significant presentation of OVA257–264 was
apparent even when the DC were fixed after only 2 h of coculture
with bacteria (Fig. 4b).
Similar experiments using Flt3L-treated BALB/c mice showed that freshly
isolated splenic DC could also process S. typhimurium
expressing rOVA for presentation of OVA323–339
on I-Ad (Fig. 4c). More than 2 h
of bacteria coculture with splenic DC was required to detect
presentation of OVA323–339 on
I-Ad, as no DO11.10 proliferation was detected if
the DC were fixed at this time point (data not shown). However, if the
2-h bacteria coincubation was chased 4 h before fixing the DC,
DO11.10 proliferation was detected (Fig. 4c). As for
MHC-I presentation of a Salmonella-derived Ag,
MHC-II presentation was peptide specific, since DC that engulfed
bacteria expressing Crl-OVA, which lacks the
OVA323–339 epitope, resulted in no proliferation
of DO11.10 T cells. CCD inhibition experiments revealed that
presentation of the MHC-II epitope by DC required phagocytosis of the
bacteria, and that DO11.10 stimulation was not due to peptides present
in the culture (Fig. 4c).
To ensure that the capacity of splenic DC to process and present
Salmonella-encoded Ags was not influenced by Flt3L
treatment, splenic DC were purified from naive mice using the same
procedure. Flow cytometric analysis showed a similar level of CD86 and
MHC-II expression on splenic DC from naive and Flt3L-treated mice (Fig. 5a). In parallel stainings,
MACS-purified splenic DC from naive and Flt3L-treated mice were stained
with CD8-FITC and CD11b-bio, followed by streptavidin-PE. Analysis
revealed that the expression of these markers was mutually exclusive
within CD11c+ cells from naive and Flt3L-treated
mice (data not shown).
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b). Likewise, splenic DC
from naive BALB/c mice processed S. typhimurium expressing
rOVA for OVA323–339 presentation on
I-Ad (Fig. 5c). As with DC from
Flt3L-treated mice, presentation by splenic DC from naive mice was
epitope specific (Fig. 5, b and c).
The surface phenotype and Ag presentation capacity of DC purified from
MLN of naive mice was also analyzed. Flow cytometric analysis revealed
that, relative to splenic DC purified in parallel from the same
animals, DC from MLN expressed similar surface levels of CD86. However,
some MLN DC expressing slightly higher levels of MHC-II were apparent
(Fig. 5d). Like splenic DC, purified MLN DC also possessed
the capacity to process S. typhimurium expressing Crl-OVA
for OVA257–264 presentation on
Kb (Fig. 5e).
Together these data show that freshly isolated splenic and MLN DC from either naive or Flt3L-treated mice are capable of phagocytic processing of Salmonella for peptide presentation to primary T cells.
CD8+ and CD8-splenic DC process S. typhimurium for
peptide presentation on MHC-I and MHC-II
Data from in vivo studies suggest that
CD8+ and CD8- DC
have a differential capacity to present soluble (8) or
particulate (5) Ags to CD8+ T cells.
However, the ability of splenic DC subsets to process live bacteria for
peptide presentation on MHC-I and MHC-II has not been determined. To
address this, CD8+ and
CD8- DC purified from the spleen of
Flt3L-treated mice (Fig. 6, a
and d) were used in Ag presentation assays. Both
CD8+ and CD8- DC
processed and presented a Salmonella-derived Ag on MHC-I to
CD8+ OT-I T cells (Fig. 6b) and on
MHC-II to CD4+ DO11.10 T cells (Fig. 6e). As observed using total splenic
CD11c+ DC (Fig. 4), presentation of the
bacteria-derived OVA257–264 and
OVA323–339 epitopes on MHC-I and MHC-II,
respectively, by the purified DC subsets was epitope specific (Fig. 6, b and e).
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Splenic DC internalize S. typhimurium in infected mice
(22), but the capacity of splenic
CD8+, CD4+, and DN DC
subsets to take up Salmonella in vivo is not known. In pilot
experiments analysis of splenic DC at 2, 4, 12, and 24 h
postinfection showed increased numbers of DC associated with
GFP-expressing Salmonella (data not shown). Splenic DC were
therefore purified from mice given GFP-expressing bacteria 4 h
earlier and stained with anti-CD8 and anti-CD4. Analysis of
GFP fluorescence among CD11c+ cells gated into
CD8+, CD4+, and DN
populations showed that all three DC subsets associated with
Salmonella (Fig. 7a). At the lower two doses of
bacteria administered, a higher percentage of
GFP+ cells was detected in the
CD8+ DC subset relative to the
CD4+ and DN populations, while DN DC contained
the highest percentage of GFP+ cells at the
highest dose administered (Fig. 7a). Analysis of the mean
value of GFP+ cells in three independent
experiments, however, revealed a similar percentage of
GFP+ cells in each subset at the highest dose of
bacteria administered (Fig. 7b). The percentage of
GFP+ cells in each DC subset was consistent in
independent experiments with increasing bacterial dose and bacterial
load in the spleen (Fig. 7, b and c).
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We next investigated whether splenic DC isolated from
Salmonella-infected mice could present bacterial
Ags upon coculture with specific T cells directly ex vivo. Indeed,
OVA257–264/Kb-specific
OT-I T cells proliferated following coincubation with DC purified from
mice that received Salmonella-expressing Crl-OVA (Fig. 8a). In contrast, no
proliferation was apparent when OT-I cells were coincubated with DC
purified from mice receiving bacteria expressing the irrelevant epitope
in Crl-HEL. Likewise, DC purified from mice that received S.
typhimurium-expressing OVA induced proliferation of
CD4+ DO11.10 T cells (Fig. 8b). In
contrast, little DO11.10 proliferation was apparent upon coculture with
DC purified from mice that received Salmonella-expressing
Crl-OVA, which lacks the OVA323–339 epitope
recognized by DO11.10 T cells. These data demonstrate that DC isolated
from the spleen of Salmonella-infected mice present
bacterial Ags to CD4+ and
CD8+ T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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+,
CD4+, and DN splenic DC subsets are beginning to
be elucidated, little is known about the outcome of interaction between
the DC subsets and live bacteria. Furthermore, data showing a
differential response of DC subsets in mice orally infected with
S. typhimurium (16) and in mice given endotoxin
(32, 33) or a soluble parasite extract (17, 20) suggest that the DC subsets may have a different function as
APC upon microbial encounter in vivo. The present study was thus aimed
at addressing bacterial uptake, cytokine production, and Ag
presentation capacity of purified splenic DC subsets upon encounter
with S. typhimurium.
The data show that all three splenic DC subsets internalize
Salmonella using a mechanism requiring cytoskeletal
rearrangements. CD8+ DC were found to be
somewhat more capable of internalizing bacteria compared with
CD4+ and DN DC at several bacteria to DC ratios.
Phagocytosis of Salmonella by splenic DC also resulted in
presentation of bacterial Ags to primary T cells. That is, freshly
isolated splenic DC, both total CD11c+
MHC-II+ splenocytes and purified
CD8- and CD8+
subsets, processed Salmonella for peptide presentation on
MHC-I and MHC-II following bacterial uptake. Although immature, bone
marrow-derived DC have previously been shown to have this capacity
(24, 25, 26, 34), this is the first time that splenic DC have
been shown to be capable of phagocytic processing of
Salmonella for MHC-I and MHC-II presentation. In addition,
we show that DC purified from MLN have the capacity to present
bacterial Ags to CD8+ T cells. Thus, DC in sites
of Salmonella replication (spleen and MLN) are capable of
bacterial phagocytosis and presentation of bacterial Ags.
Salmonella encounter with splenic DC also resulted in
subset-specific cytokine production. Furthermore, infecting purified
splenic DC with GFP-expressing Salmonella and measuring
cytokine production by intracellular staining allowed us to elucidate
the relationship between bacterial internalization and cytokine
production. Consistent with previous reports showing that
CD8+ DC are the dominant producers of IL-12 in
response to other microbial stimuli (17, 18, 20, 21, 35),
the largest fraction of IL-12p40-producing cells following
Salmonella encounter is within the
CD8+ DC subset. In contrast, few
CD8+ DC produced TNF- following
coincubation with Salmonella, while numerous cells within
the CD4+ and DN populations produced this
cytokine. Thus, different splenic DC subsets are the predominant
producers of TNF- and IL-12p40 upon Salmonella
encounter.
Interestingly, the major population of TNF--producing cells was
among those that had not engulfed GFP-expressing bacteria. This was
true for both the major TNF--producing DC subset,
CD8-, as well as for the
CD8+ subset. Among
GFP+CD8- DC, however,
50% were TNF-+. In contrast, only 10% of
GFP+CD8+ DC produced
TNF-. Thus, although bacterial internalization was not required for
TNF- production, as shown by the CCD inhibition studies and by the
capacity of bystander DC to produce this cytokine, uptake of bacteria
increased the likelihood that CD8- DC
produced TNF-. The bacterial component(s) important in stimulating
TNF- production by splenic DC is not known. However, the paucity of
TNF- producers among DC from LPS-hyporesponsive C3H/HeJ mice
suggests that LPS and Toll-like receptor 4 may be important.
Similar to TNF- production by CD4+ and DN DC,
IL-12p40 production by CD8+ as well as
CD8- DC did not require bacterial
internalization. However, data from Transwell and CCD inhibition
experiments suggest that the bacteria must either be in contact with
the DC or in close proximity to them so bacterial components in the
microenvironment can induce cytokine production by the DC. Furthermore,
the major population of cells producing IL-12p40, like those making
TNF-, had not engulfed the GFP-expressing bacteria. Infection of DC
from C3H/HeJ mice with Salmonella resulted in
IL-12p40-producing DC. Although the frequency of these cells was
50% of that detected following Salmonella coculture with
C3H/HeN DC, the data suggest that splenic DC with a mutant Toll-like
receptor 4 can produce IL-12p40 upon Salmonella
encounter.
Although Salmonella encounter with splenic DC results in
IL-12p40 production, only negligible levels of IL-12p70 are detected.
This is true in supernatants of bone marrow-derived DC
(24) or splenic DC cocultured with Salmonella
even in the presence of anti-CD40 mAb (data not shown). This is
consistent with a recent report showing that DC isolated from M.
bovis bacillus Calmette-Guérin-infected mice and cultured ex
vivo produce IL-12p40, but not IL-12p70 (36). Thus, even
if Salmonella contact with DC induces IL-12p40 production,
additional signals are needed to maximize the number of DC producing
IL-12p70. Such signals may come in the form of soluble products such as
IFN- (18, 21) and/or CD40 engagement (19).
The latter would occur following activation of specific T cells, which
could then engage CD40 via CD40 ligand. If IFN- is needed, the
source is probably cells other than DC, as IFN- production by DC
after coincubation with Salmonella is not detected (data not
shown) (16).
Multiple forms of IL-12 have been identified. For example,
heterodimerization of IL-12p40 with IL-12p19 forms IL-23
(37). A homodimer of the p40 subunit,
IL-12(p40)2, is also made (38).
IL-23 has been shown to induce T cell proliferation, while
IL-12(p40)2 has been suggested to be an
antagonist of IL-12p70 by competing for binding to IL-12R
1
(38). A recent report, however, has shown that
IL12-p70-independent, but IL-12p40-dependent, mechanisms contribute to
protective immunity to Salmonella (39). This
study showed that IL-12p35-/- mice survived
better and had higher serum levels of TNF- and IFN- than
IL-12p40-/- mice. The induction of IL-12p40
mRNA, but not IL-12p19, in Salmonella-infected mice suggests
that IL-12p40 as a monomer or homodimer rather than IL-23 is involved
in the immune response to Salmonella (39).
Deciphering the roles of the different forms of IL-12 to
anti-Salmonella immunity as well as the cellular sources
requires further investigation.
In addition to demonstrating that splenic DC could internalize and process bacteria in vitro, we showed that splenic DC could perform these functions in vivo. GFP fluorescence was apparent in all three splenic DC subsets in mice infected with Salmonella expressing this protein. Thus, not only can splenic DC subsets take up particulate and soluble Ag in vivo (5, 7, 8), but they have the capacity to internalize live Salmonella early during the course of infection.
The dose required to detect DC associated with fluorescent bacteria in
lymphoid organs is very high, as shown here and previously (22, 36). Given the need to administer unphysiological quantities of
bacteria to overcome technical constraints of detecting bacteria
associated with (rare) splenic DC and to diminish the likelihood of
downstream effects caused by i.v. bacterial administration, analyses
were performed 4 h after infection. Administration of different
doses of bacteria within the range where GFP+ DC
were clearly detected revealed fine differences in the capacity of the
splenic DC subsets to internalize bacteria. At the lower doses of
bacteria administered, more cells within the
CD8+ DC subset internalized
Salmonella. At the highest dose given, however, somewhat
more GFP+ cells were detected among DN DC. These
results suggest that the bacterial load may influence the relative
capacities of the different splenic DC subsets to internalize bacteria
in vivo. Finally, we also showed that splenic DC could stimulate both
CD4+ and CD8+ TCR
transgenic T cells specific for bacteria-encoded Ags directly ex vivo
following administration of Salmonella to mice. Together
these data show that DC in organs targeted by Salmonella
during infection (MLN and spleen) harbor bacteria, present
Salmonella-encoded Ags, and produce cytokines in a
subset-specific fashion. These functions underscore the importance of
DC in anti-Salmonella immunity.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Mary Jo Wick at the current address: Department of Clinical Immunology, University of Goteborg, Guldhedsgatan 10A, SE-413 46 Goteborg, Sweden. E-mail address: mary-jo.wick{at}immuno.gu.se
3 Abbreviations used in this paper: DC, dendritic cell; BFA, brefeldin A; CCD, cytochalasin D; DN, double negative; Flt3L, Flt3 ligand; GFP, green fluorescent protein; HEL, hen egg white lysozyme; LB, Luria-Bertani; MLN, mesenteric lymph node; PFA, paraformaldehyde.
Received for publication December 13, 2001. Accepted for publication April 29, 2002.
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)
or the presence (
) of CCD. Alternatively, as a control for epitope
specificity, 14028r expressing Crl-HEL (
) was used. After 2 h
of incubation cells were washed to remove extracellular bacteria and
were either left untreated (left panel) or were fixed in
PFA and washed (middle panel). The cells were
resuspended in medium containing 50 µg/ml gentamicin and were
coincubated with MACS-purified OT-I T cells. Proliferation of OT-I
cells was measured by quantitating [3H]thymidine
incorporation at 72 h. The proliferative response of OT-I cells to
OVA257–264 peptide added to either untreated (open bars)
or PFA-fixed (filled bars) splenic DC is shown (right
panel). One representative experiment of six is shown.
c, Splenic DC from Flt3L-treated BALB/c mice were
coincubated with increasing numbers of 14028r expressing rOVA in either
the absence (
) or were incubated in medium
alone (
). After 2 h of
bacteria coincubation followed by washing, the cells were treated with
BFA for 5 h. Cells were then stained for surface expression of
CD11c, CD4, and CD8
) DC were loaded with 0.1 nM OVA257–264 peptide for
2 h. For both e and f, the cells
were then washed, serially diluted 2-fold, and coincubated with
purified OT-I cells. Proliferative responses was measured as described
in Fig. 4
