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*
Institute for Medical Microbiology, Immunology and Hygiene, Klinikum Rechts der Isar, Technical University of Munich, Munich, Germany; and
Institute for Experimental Hematology, GSF National Research Center for Environment and Health, Munich, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. In
fact, either CpG-DNA or dsRNA (poly(I:C)) induced IFN- from purified
ppDC. Surprisingly, only CpG-DNA triggered purified ppDC survival,
maturation, and production of TNF, GM-CSF, IL-6, and IL-8, but not
IL-10 or IL-12. Known DC activators such as CD40 ligation triggered
ppDC maturation, but only IL-8 production, while bacterial LPS was
negative for all activation criteria. An additional finding was that
only CpG-DNA could counteract IL-4-induced apoptosis in ppDC.
Therefore, CpG-DNA represents a pathogen-associated molecular pattern
for ppDC. In contrast to these finding, CpG-DNA, like LPS, caused TNF,
IL-6, and IL-12 release from PBMC and purified monocytes; however,
differentiation of monocytes into DCs with GM-CSF and IL-4 unexpectedly
resulted in refractoriness to CpG-DNA, but not LPS. Taken together,
these results suggest that within a DC subset a multiplicity of
responses can be generated by distinct environmental stimuli and that
responses to a given stimulus may be dissimilar between DC
subsets. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DCs have been subdivided into lineage subsets based on surface marker
phenotype. Functional characterization of human DCs has established
myeloid-like DCs as Th1-inducing precursor DC type 1 (pDC1) and
lymphoid-like DCs as Th2-inducing precursor DC type 2 (pDC2)
(8). pDC1 are generated from peripheral blood monocytes by
treatment with GM-CSF and IL-4 and are also known as monocyte-derived
DCs (MDDCs). These DCs express CD11c, CD13, CD33, and GM-CSF-R
(CD116), but not CD4, and become mature after stimulation with CD40L or
PAMP. pDC1 production of IL-12 upon stimulation is a likely explanation
for Th1 polarization. pDC2 are plasmacytoid cells isolated from the
tonsil, termed here plasmacytoid precursor DC (ppDC) (8).
These cells are CD4+
CD11c- CD13-,
CD33-, CD45RA+,
IL-3R+ (CD123+) and use
IL-3 as a survival factor (9, 10, 11). DCs of this phenotype
can also be found circulating in the peripheral blood or resident in
lymphoid organs (9, 10, 12, 13, 14, 15, 16).
CD4+/CD11- DCs from the
blood have also been termed plasmacytoid cells, IFN-producing cells,
natural IFN-
-producing cells, IL-3Rhigh
DCs, or pDC2 (8, 9, 10, 17, 18). CD40 ligation matures ppDC,
but does not induce IL-12; however, they do produce type I IFNs if
stimulated with UV-irradiated HSV (8, 18). Type I IFNs
(IFN- and IFN-) are involved in antiviral defense, cell growth
regulation, immune activation, and Th1 polarization. ppDC have been
implicated as the major source of type I IFNs after viral or bacterial
stimulation (17, 19).
Viruses and bacteria probably activate MDDC and ppDC through engagement
of pattern recognition receptors (e.g., Toll-like receptor (TLR) or
dsRNA-responsive protein kinase). Well-documented PAMP are endotoxins
(LPS), dsRNA, and immunostimulatory bacterial CpG-DNA sequences
(CpG-DNA) (7). LPS, a prototypic PAMP, matures and induces
cytokine production from murine bone marrow-derived DCs and human MDDC
(2). A LPS binding and signaling complex assembles when
TLR4 interacts with LPS bound to CD14, thus initiating the IL-1R/TLR
receptor transduction pathway (20, 21, 22). CpG-DNA-driven
activation of APCs also acts through the IL-1R/TLR-like signal
transduction pathway; however, cellular uptake and translocation into
early endosomes are required (23, 24, 25). It has been
recently determined that CpG-DNA signals via TLR9 (26).
TNF-associated factor-6 is a critical element in the IL-1R/TLR as well
as CD40 signaling pathways (27). Subsequent to
TNF-associated factor-6 both I
B kinase and Jun kinase are activated.
Interestingly, dsRNA activation of dsRNA-responsive protein kinase also
results in IB kinase and Jun kinase activation (28).
The convergence of these multiple stimuli may explain how they are all
able to activate and mature DCs.
Bacteria and virus stimulate the release of IFNs from plasmacytoid
cells; however, the PAMPs involved remain unidentified with the
possible exception of dsRNA. Bacterial CpG-DNA was originally
recognized for its ability to induce IFNs from both murine spleen cells
and human peripheral blood cells. Given that bacterial stimuli activate
DCs (29, 30, 31), we attempted to characterize the effects of
CpG-DNA and other stimuli on human MDDC and ppDC. We describe that in
contrast to LPS, bacterial CpG-DNA activates human lymphoid
CD4+, CD11c-, ppDC cells
to produce IFN- and subsequently to mature into phenotypic DCs that
display dendritic morphology, express high levels of costimulatory
molecules, and produce cytokines. Conversely, LPS, but not CpG-DNA,
activated myeloid MDDC/pDC1. Additionally, the effects of dsRNA
and CD40 ligation were examined.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Escherichia coli DNA and poly(I:C) (Sigma, Deisenhofen, Germany) were used at a concentration of 50 µg/ml (32). When needed as a control, E. coli DNA was digested by DNase I (Roche, Mannheim, Germany) overnight and checked for complete digestion by gel electrophoresis. LPS (Sigma, Germany) was used at a concentration of 100 ng/ml. All cytokines were purchased from PharMingen (San Diego, CA). The following oligodeoxynucleotides (ODNs) were used in their phosphorothioate form: CpG-ODN 2006, 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3'; the nonstimulatory oligonucleotide GpC-ODN, 5'-TGCTGCTTTTGTGCTTTTGTGCTT-3'; and the control oligonucleotide C-ODN, 5'-GCTTGATGACTCAGCCGGAA-3' (32). All ODN were used at 2 µM, which yielded maximal activity (data not shown) (32).
Cell culture
Cells were cultivated in RPMI 1640 supplemented with 50 µM 2-ME, 2 mM L-glutamine, 100 U/ml penicillin G, 100 µg/ml streptomycin, 10 mM HEPES, and 10% FCS (Seromed, Berlin, Germany). IL-3 was used at a concentration of 500 U/ml, GM-CSF at 1000 U/ml, and IL-4 at 800 U/ml. The fibroblast cell line 3T3 stably transfected with human CD40L was described previously (33).
Cell preparation and purification
PBMC were isolated from citrate-stabilized buffy coats by centrifugation over Ficoll-Hypaque gradient. Briefly, 15 ml of the buffy coat was diluted 1/1 with PBS, underlayed with 15 ml of Ficoll-Hypaque solution, 1.077 g/l (Biochrom, Berlin, Germany), and centrifuged for 30 min at 1000 x g. Cells at the interface were harvested and washed four times with HBSS. Depletion of CD123+ DCs from PBMC was performed by MACS separation (Miltenyi Biotec, Bergisch Gladbach, Germany) on the basis of CD123 expression.
Monocytes were purified by plastic adherence. Briefly, PBMC (10 x 106/well) were incubated in RPMI 1640/10% FCS in six-well plates (Falcon, Heidelberg, Germany) for 2 h at 37°C. After decanting the medium, the cells were washed with PBS/2% FCS and incubated for an additional hour. The washing step was repeated, and the adherent cells were harvested with a cell scraper. The monocytes were >90% pure as analyzed by flow cytometry.
Myeloid DC were generated from monocytes (MDDC/pDC1). Monocytes were isolated from PBMC by positive selection using a MACS separation kit according to the manufacturer’s instruction (Miltenyi Biotec). The purity was determined by FACS analysis using a Coulter EPICS XL (Coulter, Krefeld, Germany) and was >95%. The monocytes were cultured for 7 days in 800 U/ml GM-CSF and 500 U/ml IL-4. Every second day 500 U/ml GM-CSF and 300 U/ml IL-4 were added.
ppDC/pDC2 were isolated from tonsils by a combination of elutriation and FACS sorting or MACS separation followed by FACS sorting. Fresh tonsils were cut into small fragments and pushed through a mesh. The cell suspension was fractionated by countercurrent centrifugation in an elutriation rotor (JE-6B with Sandersonchamber; Beckman, Krefeld, Germany) at constant speed (1800 rpm) with increasing flow rate (8–24 ml/min). Fractions enriched for CD123+ cells, as accessed by FACS analysis, were pooled. For further purification, the enriched CD123+ cell fractions were sorted by FACS according to CD123+, HLA-DR+ expression. The purity was determined by flow cytometry and was >98%. All steps were performed with the addition of DNase I (Roche) to prevent clumping of the cells. The anti-CD123 mAb, clone 9F5, is a nonblocking Ab, so signaling by IL-3 via the receptor was possible after sorting. For MACS separation, a tonsilar single-cell suspension was stained with PE-conjugated mAb against CD123 (PharMingen) and counterstained with anti-PE microbeads. CD123+ cells were positively selected on a column. Further purification was performed by FACS as stated above.
Cytology analysis
Cells were cytocentrifuged onto slides and fixed with methanol for 5 min. Dried slides were stained for 20 min with May-Giemsa solution (Merck, Darmstadt, Germany) and rinsed with distilled water. The slides were analyzed by confocal microscopy on an LSM 510 (Zeiss, Heidelberg, Germany) and imaged digitally.
Determination of cell survival and proliferation
ppDC were cultured at a concentration of 20 x 103/well at 37°C in 96-well U-bottom plates with different stimuli. After 24 h cells were harvested and incubated with propidium iodide at an end concentration of 1 µg/ml for 10 min. Staining of the DNA in dead cells was determined by FACS and presented as the percentage of living cells. In parallel experiments proliferation was determined by seeding ppDC (20 x 103/well) in triplicate 96-well U-bottom plates (Falcon) followed by culture for 3 days at 37°C with or without stimulation. For the last 16 h cells were pulsed with 1 µCi of [3H]thymidine (6.7 Ci/mM). Lysed cells were harvested onto filter papers, the filters were washed, and the [3H]thymidine incorporation into the DNA was measured and expressed as counts per minute. When CD40L transgenic 3T3 cells were used as stimulus, they were irradiated with a dose of 4000 rad.
Measurement of cytokine release
PBMC (5 x 106/ml) or MDDC/pDC1
(1 x 106/ml) were incubated at 37°C in
24-well plates in the presence or the absence of stimuli. The
supernatants were harvested after 12 h for TNF- or after
24 h for all other cytokines. ppDC were cultured at a
concentration of 0.2 x 106/ml at 37°C in
24-well plates, and supernatants were taken after 12 h for TNF-
or after 36 h for all other cytokines. The samples were analyzed
in duplicate, and the ELISAs were performed according to the
manufacturer’s instruction. ELISA kits for IL-6, IL-10, and TNF-
were purchased from PharMingen; those for IL-8, GM-CSF, and total IL-12
were obtained from R&D Systems (Minneapolis, MN); and those for IFN-
were obtained from PBL Biomedical Laboratories (New Brunswick, ME).
Streptavidin-peroxidase conjugate, as enzyme (Sigma), and
o-phenylenediamine, as substrate (Sigma), were used for
development when not included in the kits.
Expression of surface markers
PBMC (5 x 106/ml) and MDDC/pDC1 (1 x 106/ml) were cultured for 24 h at 37°C in 24-well plates in the presence or the absence of stimuli. ppDC (0.2 x 106/ml) were cultured for 36 h. Cells were harvested, washed, and preincubated with human IgG (Miltenyi) for FcR blockade. Abs used for specific staining or isotype controls were FITC- or PE-conjugated anti-CD1a, anti-CD3, anti-CD4, anti-CD11c, anti-CD13, anti-CD14, anti-CD16, anti-CD19, anti-CD33, anti-CD40, anti-CD56, anti-CD80, anti-CD86, anti-CD116, anti-CD123, anti-HLA-DR, and anti-HLA-ABC (PharMingen). The staining was performed on ice for 30 min; cells were washed twice and then fixed with 1% paraformaldehyde solution (Sigma). FACS analysis was performed on a Coulter EPICS XL (Coulter).
Th1 vs Th2 induction
ppDC (2 x 105) were cultured in
24-well plates in the presence or the absence of the stimuli at 37°C.
After 24-h incubation 106 allogenic naive T cells
were added. Naive T cells were obtained from PBMC depleted of
CD11b+, CD16+,
CD19+, CD36+, and
CD56+ (Pan T Cell Isolation Kit) and additionally
of CD8+ and CD 45RO+ cells
on a MACS column (Miltenyi). Naive T cells and ppDC were cocultured in
supplemented RPMI 1640 with added IL-3 (500 U/ml) and sodium pyruvate
(1 mM) for 6 days. The T cells were then washed, counted, and
restimulated at a concentration of 5 x 105
cells/ml with PMA (50 ng/ml) and ionomycin (0.5 µg/ml) for 24 h.
The secretion of IL-4, IL-5, and IFN-
was determined by ELISA (R&D
Systems, Minneapolis, MN) according to the manufacturer’s
instructions.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In studies designed to determine APC reactivity to PAMPs, we found
that bacterial CpG-DNA and LPS stimulated human monocytes to produce
cytokines, such as IL-12, IL-6, and TNF- (Fig. 1
, A and B)
(32). Monocyte activation through E. coli DNA
was DNA dependent, because DNase digestion destroyed cytokine release.
CpG-DNA also caused IFN- release from PBMCs in a CpG-dependent
manner; in contrast, however, LPS did not induce IFN- (Fig. 1C). Poly(I:C), a dsRNA known to induce IFN- from PBMCs,
was used as a positive control (Fig. 1C). Interestingly,
purified monocytes did not produce IFN- upon CpG-DNA stimulation,
although poly(I:C) remained effective (Fig. 1D). These data
implied that IFN-producing cells within PBMCs were lost during the
monocyte preparation. Additionally, CpG-DNA, LPS, and poly(I:C) were at
variance with regard to IFN production pattern.
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DC are a known source of various cytokines and IFNs
(34, 35, 36). Most contemporary studies rely on MDDC as a
source of DCs. MDDC generated by culturing monocytes in GM-CSF and IL-4
are myeloid in origin and have been termed pDC1 based on functional
analysis (8). We tested MDDC/pDC1 for their responsiveness
toward CpG-DNA or LPS. In agreement with others (2, 37, 38), LPS induced human MDDC to up-regulate the expression of
costimulatory molecules such as CD40 and CD86 and to produce cytokines
such as TNF-, IL-12, IL-6, and IL-8 (Fig. 2 and Table I). MHC class I and II molecules were
also up-regulated 2-fold (data not shown). However, CpG-DNA was
negative for up-regulation of activation markers and cytokine
production (Fig. 2 and Table I). Additionally, LPS and CpG-DNA were
both negative for the production of IFN- (Table I), although these
cells have been shown to release type I IFNs upon poly(I:C) stimulation
(34, 35). Given that ex vivo-prepared human monocytes are
sensitive to immunostimulatory CpG-DNA, (Fig. 1
A)
(32), these results implied that the conversion of
monocytes to MDDC is associated with a CpG-DNA refractory state.
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Plamacytoid CD4+,
CD11c-, CD123+
(IL-3R+) ppDC, functionally described as pDC2,
acutely respond to microbial stimuli with the production of type I IFN
(18, 19). Upon depletion of CD123+
cells from PBMCs, bacterial CpG-DNA failed to trigger IFN- from the
remaining PBMCs, yet poly(I:C) was still effective (Table II). In contrast, IFN- producer
cells within PBMCs were nonresponsive to LPS (Table II). This
contrasted with IL-12 production, which was induced in PBMCs or
CD123-depleted PBMCs by LPS, CpG-ODN, and E. coli DNA, but
not by poly(I:C) (Table II). Furthermore, CpG-DNA, but not LPS,
up-regulated the expression of CD86 and CD40 costimulatory molecules on
CD123+, CD4+ cells within
PBMCs (Fig. 3). These data implied that
CD123+, CD4+ cells are
responsive to bacterial DNA and thus focussed our attention on the
purification and characterization of ppDC.
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The tonsil is a rich source of ppDC (9). Upon
purification (>98%; see Materials and Methods) the
phenotype of these cells was lin-,
CD1a-, CD11c-,
CD13-, CD33-,
CD45RO-, CD45RA+,
CD123+, CD4+ (data not
shown), as demonstrated previously (9, 10). Consistent
with previous reports, nearly all freshly isolated ppDC apoptosed
during overnight culture in medium alone; however, 70% survived if
cultured with IL-3 (Fig. 4A).
CD40 ligation in the absence of IL-3 promoted some survival over a 24-h
period (Fig. 4A); however, over extended periods cells
continued to die (data not shown). In our hands, GM-CSF also promoted
survival (Fig. 4A), consistent with data derived from
blood-borne CD123+ DCs (11). In
agreement with others TNF- alone did not promote survival (data not
shown) (11). The most effective survival stimuli were
E. coli DNA and CpG-ODN, which supported 80% survival of
ppDC (Fig. 4A). Control DNAs were ineffective, with the
possible exception of a minimal effect from the GpC-ODN. LPS was an
ineffective anti-apoptotic agent (Fig. 4A), consistent
with CD123+, CD4+ cell
nonresponsiveness to LPS (Fig. 3). Poly(I:C) was also ineffective in
blocking ppDC apoptosis (Fig. 4A).
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B). Activation via CD40 cross-linking plus IL-3 was
also inefficient in promoting survival when IL-4 was added
simultaneously (Fig. 4B). In contrast, CpG-DNA stimulation
was able to overcome the negative effects of IL-4 (Fig. 4B).
Additionally, ppDC proliferated upon culture with IL-3 (Fig. 4C). In contrast, neither CD40 cross-linking nor CpG-DNA
promoted proliferation, implying that maturation/differentiation (see
below) in these DCs curtailed proliferation (11). The ppDC display activated DC morphology upon CpG-DNA stimulation
The cell morphology of ppDC was consistent with published
descriptions of plasmacytoid cells that reside in T cell-rich areas of
the tonsil and lymph nodes, and CD11c-,
CD4+ DCs from the blood (Fig. 5A) (11, 18).
Although IL-3 was anti-apoptotic, the overall plasmacytoid
morphology was maintained for 36 h with perhaps the appearance of
a few small dendrites and a subtle increase in size (Fig. 5E). In contrast, CpG-DNA promoted long dendrite formation,
a strong increase in size, an alteration in nuclear structure, and
increased vacuolization, seemingly clustered around a dense focal point
(Fig. 5, B–D). This was similar in nature to IL-3/CD40
activation (Fig. 5F), which has been documented previously
(10). IL-3/LPS or IL-3/poly(I:C) treatment did not differ
from the mild maturational effects of IL-3 alone (compare Fig. 5, G and H, with Fig. 5E). These data
implied that CpG-DNA is a strong activating agent for ppDC and
confirmed the lack of activation by LPS.
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Up-regulation of costimulatory molecules such as CD86 and CD40 are
a hallmark of DC activation (1). IL-3 was inefficient in
activating ppDC (Fig. 6). In contrast,
CpG-DNA induced strong CD86 and CD40 expression in a CpG-dependent
fashion, although some activation was seen with the GpC-ODN (Fig. 6).
CD40 ligation induced CD86 up-regulation, but not that of CD40,
presumably due to CD40 ligation-driven receptor down-regulation (Fig. 6). CD80 and MHC classes I and II were also up-modulated by CpG-DNA and
CD40 ligation (data not shown). In contrast, poly(I:C) and LPS were
ineffective (Fig. 6). Thus, CpG-DNA activates/matures ppDC, but LPS and
poly(I:C) are ineffective.
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IFN-producing cells/pDC2 produce IL-8 upon CD40 cross-linking, but
in acute response to enveloped viruses and bacteria they produce type I
IFNs (8, 18, 19, 36, 39). Given the strong stimulatory
effects of bacterial CpG-DNA, we tested the cytokine release pattern of
ppDC after CpG-DNA exposure. Confirming previous reports, CD40 ligation
induced only IL-8 release (Fig. 7)
(8). Poly(I:C) induced only IFN-, while LPS was
nonstimulatory (Fig. 7). However, CpG-DNA sequence specifically induced
not only IFN- and IL-8, but also TNF and GM-CSF (Fig. 7). IL-6 was
weakly induced, but, interestingly, neither IL-10 nor IL-12 was induced
(data not shown). The control GpC-ODN induced significant TNF and
GM-CSF; however, this was not true for the control unrelated sequence,
C-ODN. This implies that the activation of ppDC by GpC-ODN was not due
to phosphorothioate modification, but perhaps was due to flanking
sequences outside the GpC held in common with the CpG-ODN. Overall,
these data implied that the type of stimuli used dictates the response
pattern triggered in purified ppDC. For example, poly(I:C) triggered
only IFN- production, but not DC maturation. CD40 ligation induced
maturation, but only IL-8 production. In contrast, CpG-DNA triggered
phenotypic maturation into DCs and the production of a variety of
cytokines.
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Although ppDC did not produce IL-12, both CpG-DNA and poly(I:C)
induced IFN type I production, which may influence T cell
differentiation. We stimulated ppDC with either IL-3 or IL-3 plus
CpG-DNA, LPS, CD40L, or poly(I:C) and used these DC to differentiate
naive CD4+ T cells (Fig. 8). IL-3-cultured ppDC differentiated T
cells toward a mixed response, producing both Th1 and Th2 cytokines,
IFN-, or IL-4 and IL-5, respectively (similar to published data)
(36). LPS or CD40L did not significantly alter this
response; however, LPS depressed IL-4 production, while CD40L depressed
both IL-4 and IL-5. Poly(I:C) behaved similarly to viral stimulation,
that is, it enhanced IFN- production and significantly reduced IL-4
and IL-5 production. CpG-DNA, although strongly trending toward a
poly(I:C)-like induced response, did not prove to be significantly
different from IL-3 alone.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human monocytes responded to CpG-DNA and LPS, as determined by
activation marker up-regulation and cytokine production (Fig. 1, A and B) (32). Unexpectedly, MDDC
were refractory to CpG-DNA (Table I), but responsive to LPS, confirming
previous reports (2, 43). The protocol used to compel in
vitro transformation of human monocytes into immature MDDC cells uses
culture in GM-CSF and IL-4 (44). During the culture period
the cells loose their expression of CD14, an LPS coreceptor
(44). However, the resultant MDDC remain responsive to
LPS, because the signaling complex is reconstituted by serum-borne
soluble CD14 (43). Interestingly, culture of murine
macrophages or primary bone marrow-derived DCs in IL-4 also renders
these cells refractory to CpG-DNA (our unpublished
observations). Human DCs grown from CD34+ bone
marrow progenitors in the absence of IL-4, in contrast, are CpG-DNA
responsive (C. Meyer zum Büschenfelde, unpublished observations).
Whether IL-4 suppresses the response of MDDC to CpG-DNA via mechanisms
such as receptor loss or signal interference needs to be analyzed.
However, CpG-DNA uses a nearly identical signal transduction pathway to
LPS, namely, the IL-1R/TLR pathway (25). This signaling
pathway was apparently fully active, as judged by LPS
activation/maturation of MDDC, implying that the CpG-DNA receptor may
be absent in MDDC. Very recently, Hemmi et al. reported that murine
TLR9-deficient mice do not respond to CpG-DNA (26). We
determined that MDDC do not express human TLR9, but do express the LPS
receptor TLR4 by semiquantitative PCR. Conversely, ppDC express TLR9,
but not TLR4 or TLR2.4
Type I IFNs produced by a variety of cells in response to viruses,
bacteria, and mycoplasma confer cellular resistance to virus, affect
cell growth and differentiation, and modulate the immune system. It has
been observed that PBMC produce type I IFNs in response to bacteria,
but not to bacterial LPS (45). We document here that PBMC
produce type I IFNs in response to bacterial CpG-DNA and not LPS (Fig. 1 and Table II), extending earlier studies that used CpG-DNA
transfection with lipofectin (46, 47). Within PBMC,
plasmacytoid cells (lin-
CD123+ CD11C-
CD4+) that bear characteristic DC surface markers
were recently identified as the major source of type I IFN
(18). These precursor DCs were stimulated to produce
IFN- by both live and UV-irradiated enveloped viruses and
Gram-negative and Gram-positive bacteria (8, 18, 19, 39).
We show here that ppDC produce IFN- in response to CpG-DNA and
poly(I:C), but not LPS. Nucleic acid-based PAMPs could thus be the
common link between viral and bacterial pathogen-derived stimuli and
induction of IFN responses. Furthermore, nucleic acid structures should
be considered a potential immunostimulatory component within crude
pathogen mixtures.
Here we show that the nature of the stimulus dictates the quality of
ppDC activation and maturation. For example, poly(I:C), a mimic of
viral infection, triggered IFN- production, but no obvious cellular
activation, as defined by DC morphology, up-regulation of costimulatory
molecules, and production of proinflammatory cytokines such as TNF-.
In contrast, bacterial CpG-DNA not only caused IFN- production, but
acted as a survival factor and also effectively brought about
maturation into DCs expressing high amounts of costimulatory molecules.
Furthermore, the maturing DCs, in addition to IFN-, produced
TNF-, GM-CSF, IL-6, and IL-8, but no IL-10 or IL-12. CD40 ligation,
used as a mimic for DC-activating T cells, seemed intermediate to
poly(I:C) or CpG-DNA, in that DCs matured, but only produced IL-8.
Thus, bacterial CpG-DNA induced in ppDC a breath of responses not seen
by other stimuli, including CD40 ligation (8, 11).
Therefore, given the proper stimulus, these cells demonstrate a
repertoire of responses commonly ascribed to myeloid DCs, with the
exception of IL-12 production. The induction of Th polarization by
CpG-DNA was noninformative, although consistent enhancement of IFN-
and diminished IL-4 and IL-5 were observed (Fig. 8). In allogenic mixed
lymphocyte reactions none of the tested stimuli enhanced allogenic T
cell proliferation over IL-3 only (data not shown), confirming
observations made with HSV- vs IL-3-stimulated ppDC (36).
Overall, these results demonstrate that ppDC act as environmental
sentinels, converting to effector DCs in response to inflammatory
pathogen stimuli. This implies that ppDC are not simply intermediate
stage DCs in transition to more differentiated forms (16)
or that they are only responsible for peripheral T cell tolerance
(1).
Purified human ppDC succumbed within 24 h to apoptosis unless
cultured together with IL-3 (Fig. 4A) (10). The
survival factor IL-3 promoted long term proliferation of phenotypically
immature ppDC (Figs. 4C and 5E), and the Th2
cytokine IL-4 abolished IL-3-driven survival (Fig. 4B). It
has been postulated that IL-4 may self-limit, via apoptosis, any
potential recruitment of immature pDC2 within repopulating Th2 biased
lymph nodes or sites of infection (8). Interestingly,
CpG-DNA acted as a survival factor for ppDC (Fig. 4A) and
was anti-apoptotic in the presence of IL-4 (Fig. 4B).
Anti-apoptotic effects of CpG-DNA have been observed in B cells due to
activation of NF-B and prevention of mitochondrial membrane
potential disruption via a chloroquine-sensitive pathway
(48). However, cross-linking of CD40 on ppDC failed to
counteract the apoptotic effect of IL-4, suggesting that CpG-DNA
induces additional signaling events compared with CD40 (Fig. 4B). This is in agreement with the findings of others and
implies that activation/maturation alone is insufficient to promote
survival (11). Rissoan et al. demonstrated that IFN-
was able to rescue ppDC from IL-4-induced apoptosis (8).
They concluded that the induction of Th1 differentiation by pDC1 cells
would promote the survival of Th2, supporting pDC2 as a reciprocal
control mechanism. Although not formally demonstrated in the human,
CpG-DNA is a strong Th1-inducing adjuvant in murine models (47, 49, 50). Therefore, it is intriguing that only CpG-DNA
counteracted IL-4-induced apoptosis of purified ppDC (see below).
It has been postulated that DC lineage may determine the type of Th
cell differentiation (1, 51, 52), although the potential
roles of various DC culture conditions and activation protocols have
not been considered. DC-like cells have been expanded in vitro from
various sources, but whether in vitro-generated DCs are equivalent to
their in vivo counterparts is a question of debate (44, 53, 54, 55, 56). Here we focused on ex vivo-purified ppDC. These cells
produced IFN-, but not IL-12, when stimulated with CpG-DNA (Fig. 7).
During productive T cell responses, IFN- functions in vitro as a T
cell survival factor and in humans as a Th1-polarizing cytokine
(57, 58, 59). We have previously noted that CpG-DNA induced
Th1-polarized responses in PBMC via the induction of IFN- and IL-12
(60). Here we show that CD123+ cells
produce IFN-, while IL-12 originates from a
CD123- cell population (Table II). Others have
reported that ppDC induced Th2 responses after CD40 cross-linking
(8), a stimulus unable to trigger Th1 promoting IFN-
production (Fig. 7). Additional studies challenged the classification
of plamacytoid/pDC-2 cells as Th2-polarizing DCs because blood-borne
lin-/ILT3+/ILT1-
DCs with a plasmacytoid DC phenotype
(CD4+/CD123+/CD11c-),
which responded to virus, CD40 ligation, and LPS, induced a
nonpolarized Th1 and Th2 differentiation (38). Here we
show that CpG-DNA stimulation of ppDC trended toward enhanced Th1
polarization, although it was not statistically significant (Fig. 8).
In our hands, while CD40L and LPS were not neutral, poly(I:C)
was a significant inducer of Th1 polarization (Fig. 8). Stimulating
ppDC with HSV can drive a very strong type I IFN-dependent Th1
differentiation (36). The apparent difference in Th1
promotion among CpG-DNA, poly(I:C), and HSV seems partly related to
type I IFN output by ppDC. Although CpG-DNA induces full maturation of
ppDC, the type I IFN production was low relative to HSV stimulation
(Fig. 7) (36). Overall, these data suggest that within a
given DC subset it is the quality of the stimulus encountered that
dictates the respective response pattern and that the respective DC
stimulus needs to be considered when accessing T cell
differentiation.
Currently it is believed that infection is sensed through pattern recognition receptors driving Th cell-independent DC activation to professional APCs. The idea of DCs directing either Th1 or Th2 responses has been expressed in the context of different DC lineage, such as myeloid vs lymphoid or pDC1 vs pDC2, but not via integration of signal within one subset of DCs. Our data show that ppDC responded to LPS, dsRNA, CpG-DNA, or CD40 ligation with a broad range of distinct responses. This implies that DCs interpret environmental stimuli, rendering the appropriate response, and that the nature of environmental stimuli determines the DC response pattern. It follows that the propensity of ex vivo DCs to direct Th1 or Th2 responses needs to be evaluated in the context of various activation schemes.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Grayson B. Lipford or Prof. Hermann Wagner, Institute for Medical Microbiology, Immunology, and Hygiene, Technical University of Munich, Trogerstrasse 9, 81675 Munich, Germany.
3 Abbreviations used in this paper: DC, dendritic cell; MDDC, monocyte-derived DC; CD40L, CD40 ligand; PAMP, pathogen-associated molecular patterns; ppDC, plasmacytoid precursor DC; pDC1, precursor DC type 1; pDC2, precursor DC type 2; CpG-DNA, immunostimulatory bacterial CpG-DNA sequences; TLR, Toll-like receptor; ODN, oligodeoxynucleotide.
4 S. Bauer, C. Kirschning, V. Redecke, H. Hacker, S. Akira, H. Wagner, and G. B. Lipford. Human TLR9 expression correlates with responsiveness to bacterial DNA. Submitted for publication.
Received for publication March 29, 2000. Accepted for publication February 13, 2001.
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M. O'Keeffe, H. Hochrein, D. Vremec, B. Scott, P. Hertzog, L. Tatarczuch, and K. Shortman Dendritic cell precursor populations of mouse blood: identification of the murine homologues of human blood plasmacytoid pre-DC2 and CD11c+ DC1 precursors Blood, February 15, 2003; 101(4): 1453 - 1459. [Abstract] [Full Text] [PDF]
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M. O'Keeffe, H. Hochrein, D. Vremec, I. Caminschi, J. L. Miller, E. M. Anders, L. Wu, M. H. Lahoud, S. Henri, B. Scott, et al. Mouse Plasmacytoid Cells: Long-lived Cells, Heterogeneous in Surface Phenotype and Function, that Differentiate Into CD8+ Dendritic Cells Only after Microbial Stimulus J. Exp. Med., November 18, 2002; 196(10): 1307 - 1319. [Abstract] [Full Text] [PDF]
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R. Takauji, S. Iho, H. Takatsuka, S. Yamamoto, T. Takahashi, H. Kitagawa, H. Iwasaki, R. Iida, T. Yokochi, and T. Matsuki CpG-DNA-induced IFN-{alpha} production involves p38 MAPK-dependent STAT1 phosphorylation in human plasmacytoid dendritic cell precursors J. Leukoc. Biol., November 1, 2002; 72(5): 1011 - 1019. [Abstract] [Full Text] [PDF]
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M.-C. Rissoan, T. Duhen, J.-M. Bridon, N. Bendriss-Vermare, C. Peronne, B. d. S. Vis, F. Briere, and E. E. M. Bates Subtractive hybridization reveals the expression of immunoglobulinlike transcript 7, Eph-B1, granzyme B, and 3 novel transcripts in human plasmacytoid dendritic cells Blood, October 16, 2002; 100(9): 3295 - 3303. [Abstract] [Full Text] [PDF]
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K. Heckelsmiller, K. Rall, S. Beck, A. Schlamp, J. Seiderer, B. Jahrsdorfer, A. Krug, S. Rothenfusser, S. Endres, and G. Hartmann Peritumoral CpG DNA Elicits a Coordinated Response of CD8 T Cells and Innate Effectors to Cure Established Tumors in a Murine Colon Carcinoma Model J. Immunol., October 1, 2002; 169(7): 3892 - 3899. [Abstract] [Full Text] [PDF]
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H. T. Ichikawa, L. P. Williams, and B. M. Segal Activation of APCs Through CD40 or Toll-Like Receptor 9 Overcomes Tolerance and Precipitates Autoimmune Disease J. Immunol., September 1, 2002; 169(5): 2781 - 2787. [Abstract] [Full Text] [PDF]
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M. Gierynska, U. Kumaraguru, S.-K. Eo, S. Lee, A. Krieg, and B. T. Rouse Induction of CD8 T-Cell-Specific Systemic and Mucosal Immunity against Herpes Simplex Virus with CpG-Peptide Complexes J. Virol., June 5, 2002; 76(13): 6568 - 6576. [Abstract] [Full Text] [PDF]
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T. Ito, R. Amakawa, T. Kaisho, H. Hemmi, K. Tajima, K. Uehira, Y. Ozaki, H. Tomizawa, S. Akira, and S. Fukuhara Interferon-{alpha} and Interleukin-12 Are Induced Differentially by Toll-like Receptor 7 Ligands in Human Blood Dendritic Cell Subsets J. Exp. Med., June 3, 2002; 195(11): 1507 - 1512. [Abstract] [Full Text] [PDF]
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V. Hornung, S. Rothenfusser, S. Britsch, A. Krug, B. Jahrsdorfer, T. Giese, S. Endres, and G. Hartmann Quantitative Expression of Toll-Like Receptor 1-10 mRNA in Cellular Subsets of Human Peripheral Blood Mononuclear Cells and Sensitivity to CpG Oligodeoxynucleotides J. Immunol., May 1, 2002; 168(9): 4531 - 4537. [Abstract] [Full Text] [PDF]
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W. Barchet, M. Cella, B. Odermatt, C. Asselin-Paturel, M. Colonna, and U. Kalinke Virus-induced Interferon {alpha} Production by a Dendritic Cell Subset in the Absence of Feedback Signaling In Vivo J. Exp. Med., February 19, 2002; 195(4): 507 - 516. [Abstract] [Full Text] [PDF]
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I. Miconnet, S. Koenig, D. Speiser, A. Krieg, P. Guillaume, J.-C. Cerottini, and P. Romero CpG Are Efficient Adjuvants for Specific CTL Induction Against Tumor Antigen-Derived Peptide J. Immunol., February 1, 2002; 168(3): 1212 - 1218. [Abstract] [Full Text] [PDF]
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 M. F. Lipscomb and B. J. Masten Dendritic Cells: Immune Regulators in Health and Disease Physiol Rev, January 1, 2002; 82(1): 97 - 130. [Abstract] [Full Text] [PDF]
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A. Dzionek, Y. Sohma, J. Nagafune, M. Cella, M. Colonna, F. Facchetti, G. Gunther, I. Johnston, A. Lanzavecchia, T. Nagasaka, et al. BDCA-2, a Novel Plasmacytoid Dendritic Cell-specific Type II C-type Lectin, Mediates Antigen Capture and Is a Potent Inhibitor of Interferon {alpha}/{beta} Induction J. Exp. Med., December 17, 2001; 194(12): 1823 - 1834. [Abstract] [Full Text] [PDF]
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H. Nakano, M. Yanagita, and M. D. Gunn Cd11c+B220+Gr-1+ Cells in Mouse Lymph Nodes and Spleen Display Characteristics of Plasmacytoid Dendritic Cells J. Exp. Med., October 15, 2001; 194(8): 1171 - 1178. [Abstract] [Full Text] [PDF]
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N. Kadowaki, S. Ho, S. Antonenko, R. de Waal Malefyt, R. A. Kastelein, F. Bazan, and Y.-J. Liu Subsets of Human Dendritic Cell Precursors Express Different Toll-like Receptors and Respond to Different Microbial Antigens J. Exp. Med., September 17, 2001; 194(6): 863 - 870. [Abstract] [Full Text] [PDF]
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M. Gilliet, A. Boonstra, C. Paturel, S. Antonenko, X.-L. Xu, G. Trinchieri, A. O'Garra, and Y.-J. Liu The Development of Murine Plasmacytoid Dendritic Cell Precursors Is Differentially Regulated by FLT3-ligand and Granulocyte/Macrophage Colony-Stimulating Factor J. Exp. Med., April 1, 2002; 195(7): 953 - 958. [Abstract] [Full Text] [PDF]
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) or DNase-digested 50
µg/ml EC DNA (
), and cytokine release was measured by ELISA.
B, Human monocytes were cultured with 1 µg/ml LPS
(
0.05).