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Prepublished online as a Blood First Edition Paper on June 21, 2002; DOI 10.1182/blood-2001-12-0360.
IMMUNOBIOLOGY
From The Melbourne Tumour Biology Branch, The Ludwig
Institute for Cancer Research, Austin and Repatriation Medical Centre,
Heidelberg, Victoria, Australia; Medizinische Klinik und Poliklinik V,
University of Heidelberg, Heidelberg, Germany; Institut für
Experimentelle Hämatologie und Transfusionsmedizin Uniklinik,
Bonn, Germany; The Walter and Eliza Hall Institute of Medical Research,
Melbourne, Australia; Immunex Corporation, Seattle, WA.
Migration of antigen (Ag)-loaded dendritic cells (DCs) from sites
of infection into draining lymphoid tissues is fundamental to the
priming of T-cell immune responses. We evaluated monocyte-derived DCs (MoDCs) and peripheral blood DCs (PBDCs) to respond to
proinflammatory mediators, CD40L, and intact bacteria. All classes of
stimuli induced DC phenotypic maturation. However, for MoDCs, only
prostaglandin E2 (PGE2)-containing stimuli
induced migratory-type DCs. Thus, immature MoDCs that encountered
proinflammatory cytokines or CD40L or intact bacteria in the presence
of PGE2 acquired migratory capacity but secreted low levels
of cytokines. Conversely, MoDCs that encountered pathogens or CD40L
alone become nonmigratory cytokine-secreting cells (proinflammatory
type). Interestingly, both migratory- and proinflammatory-type DCs
expressed equivalent levels of chemokine receptors, suggesting that the
role of PGE2 was to switch on migratory function. We
demonstrate that PGE2 induces migration via the
E-prostanoid 2/E-prostanoid 4 (EP2/EP4) receptors and the cAMP pathway. Finally, migratory-type MoDCs stimulated T-cell proliferation and predominantly IL-2 secretion, whereas proinflammatory-type MoDCs induced IFN- Dendritic cells (DCs) represent a heterogeneous
family of leukocytes that establishes sentinel networks throughout body
tissues.1 DCs sample the microenvironment for evidence of
barrier breakage, tissue damage, and pathogen entry. Such perturbations
of the local microenvironment induce DC maturation.1
The induction of adaptive immune responses is initiated once
antigen (Ag)-bearing DC traffic from peripheral sites of inflammation
into draining lymph nodes.1,2 One functional consequence
of DC maturation is the up-regulation of the lymph node-directing
chemokine receptor, CCR7, and acquisition of migratory capacity toward
lymph node-directing chemokines CCL21 (6Ckine) and CCL19
(MIP-3 DCs are being evaluated as cellular vaccine adjuvants in the
immunotherapy of cancer.1 In humans, DCs can be directly
isolated from peripheral blood dendritic cells (PBDCs), and at least 2 subsets of immature PBDCs can be identified on the basis of HLA-DR and
CD1b/c or IL-3R expression.1 However, PBDCs are not widely used as vaccine adjuvants in clinical trials due to their rarity in
blood and the feasibility of procuring sufficient numbers for multiple
vaccinations. The recent discovery that the systemic administration of
the hematopoietic growth factor Flt-3 ligand (FL) can dramatically
increase the numbers of PBDCs in humans may substantially overcome
these limitations.21,22
More commonly, DC-like cells (resembling dermal or interstitial DCs of
skin and lymphoid organs) can be generated in vitro from blood
monocyte-derived DCs (MoDCs) when cultured in granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 for 5 to 7 days.1 Although it is unclear whether MoDCs represent
physiologic DC subsets in vivo or the fate of monocytes under highly
selective conditions, Randolph et al23 have shown that a
subset of monocytes can rapidly (< 24 h) transform into MoDCs in
the absence of GM-CSF and IL-4 simply by migrating through an
endothelial monolayer and ingesting particulates in the collagen matrix
below.23 However, regardless of whether MoDCs are
physiologically relevant cells in vivo, they are, nonetheless,
clinically relevant given that the majority of human DC-based trials
use MoDCs for vaccine delivery.1 Therefore, understanding
the mechanisms that regulate MoDC function and thus their ability to
stimulate appropriate T-cell effector function will ultimately benefit
their clinical use.
Proinflammatory mediators found at sites of infection and inflammation
(eg, IL-1 In this study we investigated how proinflammatory factors (TNF- Media
Monoclonal antibodies, enzyme-linked immunosorbent assay (ELISA)
kits, and cytokines
MoDCs Peripheral blood mononuclear cells (PBMCs) were obtained from buffy coat preparations from healthy donors from the Red Cross Blood Bank (Melbourne, Australia) and used to produce MoDCs. CD14+ monocytes (5 × 105) were affinity purified using the MACS CD14 isolation kit (Miltenyi Biotech, Sunnyvale, CA) and cultured in 1 mL RPMI, 10% FCS, GM-CSF (40 ng/mL), and IL-4 (500 U/mL) in 24-well plates. By day 7, MoDCs represented more than 90% of cultured cells. On day 7, all wells were pooled and readjusted to a concentration of 1 × 105 DCs per mL. Maturation-inducing factors were added on day 7, and cells and supernatants were harvested on day 10 for functional assessment. All cytokines and stimuli examined for their ability to stimulate DC functional maturation in the present study (eg, IFN- 2a, IFN-2a,
CD40L, PGE2, IL-1 , and intact E coli) were thoroughly tested in dose titration analyses, and the concentrations used in the figures represent those found to be optimal.
Enrichment of PBDCs from FL-treated patients PBDCs were enriched from frozen PBMC samples obtained from a Phase 1 randomized study performed in HLA-A2+ patients with minimal residual disease stages III and IV malignant melanoma receiving 14 consecutive days of Flt-3 ligand, FL (25 µg/kg/d) (Immunex), with or without peptide vaccines (LUD-97-012). Blood for PBDCs was taken at day 15 of FL treatment. The protocol was approved by the Ludwig Institute's Investigators Review Board and the Ethics Committee at the Austin and Repatriation Medical Centre, and informed consent was obtained from all patients. Alternatively, PBDCs were isolated from buffy packs obtained from healthy donors from the Australian Red Cross Blood Bank. After thawing, CD14+ monocytes were depleted using immunomagnetic beads (MACS; Miltenyi Biotech) according to the manufacturer's instructions. These CD14-depleted PBMCs underwent a second round of depletion using MACS beads coupled to anti-CD3, anti-CD14, and anti-CD19 (Miltenyi Biotech) in combination with rat-anti-mouse IgG MACS beads (Miltenyi Biotech). This depletion procedure yielded more than 60% CD1b/c+ CD14 HLA-DR+ PBDCs as assessed by
fluorescence-activated cell-sorting scanner (FACS). The
enriched PBDCs were then sorted on the basis of CD1b/c and HLA-DR
expression on a MoFlo cell sorter (Cytomation, Fort Collins, CO). These
immature PBDCs were then cultured in 96-well plates
(1 × 105/well) in RPMI-10% FCS for 3 days with various
combinations of stimuli prior to examination of function.
Measurement of Ag uptake MoDCs were harvested after culture in maturation-inducing conditions. Following incubation with 1 mg/mL FITC-dextran (44 kd and 260 kd) (Sigma) for 30 to 60 minutes at 0°C or 37°C, cells were washed 3 times in phosphate-buffered saline (PBS) 5% FCS and then incubated with PE-anti-CD11c. FITC-dextran uptake was quantified as mean fluorescence intensity (MFI) on gated CD11c+ cells. Nonspecific FITC signal was assessed by incubating MoDCs with FITC-dextran at 0°C. Phagocytosis was assessed by incubating cells with 1 mg/mL PE-latex beads (Sigma) for 90 minutes at 37°C. In some conditions, cells were pretreated with 10 uM cytochalasin D (Sigma) for 30 minutes at 37°C to depolymerize actin. To verify that the flow cytometry-based FITC signal represented internalized dextran or beads, cells were analyzed by epifluorescence and phase-contrast microscopy.RNA isolation and cDNA synthesis Total RNA was isolated from MoDCs using a Rneasy Mini Kit (Qiagen, Hilden, Germany) according to manufacturer's instructions. In brief, cells were lysed and homogenized in lysis buffer containing guanidine isothiocyanate (GITC) and-mercaptoethanol. Ethanol (70%)
was added to the samples, and the RNA was immobilized on spin columns
and eluted in RNase-free water. Total RNA (0.16 µg) was used to
synthesize cDNA using 1 µg random hexamers (Promega, Madison, WI), 1 mM deoxynucleoside triphosphate (dNTP) (Amersham Pharmacia Biotech,
Piscataway, NJ), 2 U RNase inhibitor (Promega), 5 mM MgCl2
(Applied Biosystems, Foster City, CA), 1x polymerase chain
reaction (PCR) buffer (Applied Biosystems), and 2 U
Moloney-murine leukemia virus (M-MLV) reverse transcriptase (Life
Technologies, Rockville, MD) in a 20 µL reaction for 60 minutes at
42°C. The enzyme was inactivated at 95°C for 5 minutes. Of the
resulting 20µLs, 1 µL cDNA was used for real-time polymerase chain
reaction (PCR) quantitation.
Quantitative real-time PCR Gene expression levels were quantitated using ABI Prism 7700 Sequence Detection System (Applied Biosystems). Predeveloped assay reagents (PDARs) for CCR7 were obtained from Applied Biosystems and used in multiplex reactions with 18S rRNA PDAR (Applied Biosystems) for normalization. PCR reactions were set up in 96-well plates (25 µL/reaction) according to the manufacturer's instructions and analyzed using the SDS program version v1.7. Relative expression was calculated using the Ct method and is expressed
relative to a calibrator, in this case the GM-CSF/IL-4 DC
control:
Cytokine ELISAs Cytokine secretion by stimulated MoDCs or by allogeneic T cells was measured by cytokine ELISAs. IL-1, IL-6, IL-10, and IL-12p70 ELISAs were performed on supernatants (SNs) of MoDCs and
IFN- , IL-2, and IL-5 ELISAs were performed on SNs of MLRs
according to the manufacturer's instructions using Maxisorp plates
(Nunc, Melbourne, Australia). The HRP-substrate was
tetramethylbenzidine (TMB) peroxidase (KPL, Gaithersburg, MD);
the color reaction was terminated by adding 100 µL orthophosphoric
acid (1 M). Wells were developed using a substrate solution of 548 mg/mL ABTS (2,2'azino-bis13-ethylbenz-thiazoline-6-sulfonic acid)
(Sigma Aldrich Pty Ltd, Castle Hill, Australia) with 0.001% hydrogen
peroxide (Ajax Chemicals, Auburn, Australia) in 0.1M citric acid,
pH4.2. Plates were read in a Thermomax microplate reader (BioMediq,
Melbourne, Australia).
Migration assays MoDCs matured with the indicated stimuli for 24 to 48 hours were harvested from their wells, washed 3 times, and tested for migration toward chemokines using the transwell assay. Briefly, lower chambers of transwell plates (8.0 µm pore size; Costar, Corning, NY) were filled with 500 µL IMDM/5% HS with or without chemokines. Chemokines tested included CCL19 (MIP-3; 3-300 ng/mL); CCL21 (6Ckine; 5-250 ng/mL);
CXCL12 (SDF-1; 100 ng/mL); CCL3 (MIP-1; 50 ng/mL); CXCL9 (Mig; 50 ng/mL); and CCL7 (MCP-3, 50 ng/mL; PeproTech, Rocky Hill, NJ). Briefly,
1-2 × 104 DCs were added in 50 µL IMDM/5% HS into the
upper chamber, and cells were incubated at 37°C for 2 hours. Cells in
the lower chambers were harvested, concentrated to 50 µL volumes in
Eppendorf tubes, and counted with a hemocytometer. Migration for all
stimulation conditions was performed in triplicate wells.
T-cell purification and MLR Allogeneic CD2+ T lymphocytes were obtained by rosetting PBMCs with aminoethylisothiouronium (AET)-treated sheep red blood cells (SRBCs). T cells were further fractionated using anti-CD4 and anti-CD8 MACS beads (Miltenyi Biotech) and were between 88% and 95% pure. Negatively selected CD4 and
CD8 cells were further separated into CD45RA+
and CD45RA cells using MACS beads (Miltenyi Biotech). DCs
were cultured in round-bottomed 96-well plates in triplicate at various
cell numbers with 105 allogeneic PBMCs for 5 days in RPMI
with 10% HS. After 5 days, 200 µL of SNs were harvested, and fresh
medium containing 1 µCi (0.037 MBq)/well
[3H]thymidine (DuPont, Sydney, MA) was added for 8 hours.
Cells were transferred onto a glass fiber filter (Wallac, Turku,
Finland), and [3H]thymidine incorporation was measured
using an NXT TopCount Betaplate scintillation counter (Packard,
Meriden, CT).
Phenotypic changes of MoDCs in response to soluble proinflammatory factors MoDCs were generated from sorted CD14+ monocytes. Phenotypic maturation of MoDCs can be induced by pathogen signals7-10 or CD40L.11-20 Alternatively, maturation also can be induced by proinflammatory cytokines.24-31 To determine the role of individual proinflammatory mediators, immature MoDCs were exposed to combinations of TNF- (10 ng/mL), IFN- (1000 IU/mL), and PGE2 (1 µM) for 3 days. The optimal concentrations of each of these factors
in combination were previously determined by cross-titration of each
soluble mediator. Figure 1A shows the
fold increase in the MFI of CD83 expression above that expressed by
immature MoDCs following stimulation with either TNF- or IFN- or
PGE2 or combinations thereof. TNF- alone weakly
up-regulated CD83 expression on MoDCs. The effects of dual combinations
(ie, TNF- and PGE2 or TNF- and IFN-) and, in
particular, the triple combination of TNF-, IFN-, and
PGE2 substantially increased the level of expression of
CD83. Similar effects were seen when examining CD80, CD86, HLA-ABC, and
HLA-DR (data not shown). Up-regulation of the above surface markers was induced as early as 18 hours, reaching maximal levels by 48 hours (data
not shown). These results demonstrate that phenotypic maturation of
MoDCs is a multistep process that can be enhanced by combining several
proinflammatory mediators.
We next compared how maturation induced by the combination of
proinflammatory mediators compares to other known inducers of MoDC
maturation such as soluble CD40 ligand (CD40L) or intact E
coli. Proinflammatory mediators (TNF- Migration of immature and mature MoDCs toward CXCR4 or CCR7 ligands Maturation of MoDCs results not only in loss of Ag uptake capacity but also in substantial changes in the expression of chemokine receptors and the ability to migrate toward chemokine gradients. The capacity of the 3 classes of stimuli to induce MoDC migratory capacity toward chemokines was therefore evaluated, and several important points were identified. First, MoDCs matured with proinflammatory mediators (TNF-, IFN-, PGE2) were highly sensitive to the
CCR7-ligands CCL19 (MIP-3) (Figure
2A,C) and CCL21 (6Ckine) (data not
shown), migrating toward 3 to 10 ng/mL of either chemokine. Second, in contrast to previous reports,34,35 immature MoDCs did not
migrate substantially to the CXCR4-binding proinflammatory chemokine
CXCL12 (SDF-1) (Figure 2C). Third, although immature MoDCs matured
with either CD40L or intact E coli were phenotypically
identical to those matured with TNF-, IFN-, PGE2,
they were poor migratory cells (Figure 2B). Fourth, immature MoDCs
matured with TNF-, IFN-, PGE2 not only migrated
toward the CCR7-ligands CCL19 and CCL21 but also to proinflammatory
chemokines such as CXCL12 (Figure 2C) and CCL3 (MIP-1), CXCL9 (Mig),
and CCL7 (MCP-3) (data not shown). Finally, simultaneous
stimulation of immature MoDCs with either CD40L and PGE2 or
intact E coli and PGE2 induced mature MoDCs that
migrated toward chemokines (Figure 2C,D). These results demonstrate
that in our cultures, migration of MoDCs was induced by all 3 classes
of physiologic stimuli, provided PGE2 was present.
Analysis of chemokine receptor expression on mature MoDCs DC maturation involves the coordinated expression of specific chemokine receptors.34,35 In this way, the presence or absence of chemokine receptors has been used to predict migratory capacity toward specific chemokines. To evaluate the levels of chemokine receptor expression on DCs before and after maturation, analysis of surface expression for CXCR4 (Figure 3A) and CCR7 (Figure 3B) were performed by flow cytometry. Figure 3A demonstrates that CXCR4 protein can be constitutively expressed on the surface of nonmigratory immature DCs and that this level of expression was not considerably increased following stimulation. Figure 3B demonstrates that CCR7 expression was expressed on a minority of immature MoDCs and up-regulated on most MoDCs following maturation with either proinflammatory mediators (TNF-, IFN-, PGE2) or CD40L. Molecular analysis of
CCR7 mRNA by quantitative real-time PCR (qRT-PCR) shows that CCR7 mRNA
was induced in immature MoDCs following stimulation with all 3 classes
of stimuli (Figure 3C). In addition, analysis of 3 separate experiments
revealed that the up-regulation of CCR7 mRNA expression between
migratory-type MoDCs (ie, those matured with
PGE2-containing conditions) and nonmigratory-type MoDCs
(ie, CD40L or E coli alone) varied by no more than 2-fold
(Figure 3C). These data highlight the fact that chemokine
receptor expression is not necessarily predictive of migratory
capacity.
Actions of PGE2 involve cAMP pathway via EP2/EP4 receptor signaling The effects of PGE2 have been shown to be mediated via the elevation of intracellular cAMP.36 The mobilization of cAMP can be synthetically induced using an activator of adenylate cyclase, such as forskolin. Figure 4A shows the means of migration assays generated from 5 separate experiments. The addition of forskolin (50 µM) to TNF- and IFN-
or CD40L or intact E coli could significantly replace the
requirement for PGE2 in the induction of MoDC migration toward CCL19 (Figure 4A). This demonstrates that a major mechanism used
by PGE2 during the induction of migratory capacity is via cAMP mobilization. We next examined whether cAMP analogs, such as
db-cAMP or 8-bromo-cAMP (8-b-cAMP), could also induce migratory function in MoDCs. Figure 4B demonstrates that both of these cAMP analogs, when combined with TNF- and IFN-, could mimic
PGE2 at inducing MoDC migration toward CCL19, confirming
the effects of forskolin.
PGE2 can bind to 4 separate receptors for signaling:
EP1-4.36,37 The EP2 and
EP4 receptors signal via cAMP mobilization, while the
EP1/EP3 receptors do not. We examined which of
the EP receptors PGE2 acts through to induce MoDC migratory
function by using the EP receptor agonists 11-deoxy-PGE1
(which mediates signaling via the EP2/EP4
receptors) or Saprostone (which signals via the
EP1/EP3 receptors).36,37 Figure 4B
shows that only the EP2/EP4 receptor agonist
11-deoxy-PGE1 mimics PGE2 in inducing MoDC
migratory function when combined with TNF- DC cytokine secretion is induced by CD40L or intact E coli and inhibited by PGE2 DCs secrete an array of cytokines, including IL-12, when stimulated with either infectious organisms such as intact bacteria, viruses, and protozoa7-10 or with CD40L.11-20 In this way, DCs may be central to the development of IFN--secreting T-cell effectors.1 Although cytokines
such as TNF-, IL-1, and IFN- are potent inducers of DC
maturation, they are not sufficient to induce secretion of
IL-12p7026-29,37 and can even inhibit IL-12 secretion in
response to IL-12-inducing stimuli.26-29 Optimal
CD40L-mediated IL-12p70 production requires the presence of cofactors
such as IFN-, IL-4, and IL-1 or IFN-.13-16,18-20
Figure 5A shows that immature MoDCs stimulated with either CD40L or intact bacteria in the presence of
their own conditioned media secrete high levels of IL-12p70 (Figure
5A), and this was further potentiated by addition of IFN- (Table
1). Table 1 also shows that MoDCs were
induced to secrete high levels of IL-6, IL-10, and IL-12p70 in response
to CD40L, CD40L + IFN-, or E coli. In contrast,
MoDCs stimulated with the proinflammatory mediators TNF-, IFN-,
PGE2, were poor cytokine-secreting cells (Figure 5A).
Furthermore, the addition of PGE2 to CD40L or intact
E coli inhibited the induction of IL-12p70 secretion by MoDCs (Figure 5B). Finally, the inhibitory effect of
PGE2 could be replaced by the cAMP agonist forskolin
(Figure 5B) and the cAMP analogs db-cAMP and 8-b-cAMP (data not shown),
indicating that mobilization of cAMP was, in part, responsible for
down-regulation of IL-12p70 production. Finally, Table
2 shows that mature, migratory-type MoDCs
produce substantially lower levels of cytokines following stimulation
with either CD40L or intact E coli as compared with immature
MoDCs, suggesting a differential developmental commitment of the 2 cell
populations. This is in accordance with the reports by others showing
that cytokine production by MoDCs is optimal when the stimulus is given
at the onset of maturation.13-16,38
Effect of mature MoDCs on T-cell proliferation and cytokine secretion Because DCs are the most potent stimulators of naive T cells, we investigated the ability of differentially matured MoDCs to stimulate cytokine secretion in alloreactive, naive, and memory CD4+ T cells. Figure 6 shows that the differential capacity of MoDCs matured with proinflammatory factors or CD40L to secrete IL-12p70 correlated with their capacity to induce IFN- and inhibit IL-5 secretion by naive
CD4+CD45RA+ T cells. Only CD40L- or CD40L + IFN--activated DCs induced the highest levels of IFN- in both
naive (Figure 6A) and memory T cells (data not shown) and significantly
reduced IL-5 secretion in these cultures (Figure 6C). In contrast, both
IFN- and IL-5 were secreted in low but significant quantities by
naive (and memory) T cells stimulated with either immature DCs or DCs
matured with TNF-, IFN-, PGE2 (Figure 6A,C). This
indicates immature MoDCs matured with PGE2-containing
stimuli will induce T cells to secrete both IL-5 and IFN-.
Surprisingly, migratory-type MoDCs (ie, matured with
PGE2-containing stimuli) induced significant IL-2 secretion
in naive (and memory) CD4+CD45RA+ T cells
(Figure 6B). These increased IL-2 levels also correlated with
significantly higher proliferation of T cells in the MLR (Figure 6D).
These differences between migratory-type and IL-12-producing nonmigratory-type MoDCs (proinflammatory DCs) were most prominent at
the lower DC:T cell ratios (102 and 103 DCs per
105 T cells) and was less significant at higher DC
concentrations (104 DCs per 105 T cells, data
not shown). These results demonstrate that the different functional
states of MoDCs (ie, migratory-type versus nonmigratory
proinflammatory-type) stimulate different T-cell cytokine profiles.
While CD40L-activated DCs induced higher IFN- and decreased IL-5
secretion by T cells, migratory-type DCs stimulated IL-2, IL-5, and
IFN- secretion and augmented T-cell proliferation.
Fate of irreversibly differentiated MoDCs and role of CD40L as a survival factor Maturation of MoDCs is a graded process involving several intermediate stages, which maintain the potential for reversion to a macrophage phenotype upon removal of cytokines.28,30,31 To further elucidate these findings, we investigated the stability of immature and mature DCs upon withdrawal of cytokines and exposure to human serum. MoDC cultures were washed on day 7 and resuspended in fresh medium with or without cytokines. After 3 days, cells were washed again and resuspended in medium containing 5% heat-inactivated human serum. Immature MoDCs reverted to adherent macrophages in the absence of cytokines and presence of HS (data not shown). This confirmed that immature MoDCs represent a transient and reversible state of development, analogous to a bipotential precursor of macrophages and MoDCs.30,31 In contrast, MoDCs matured with the proinflammatory factors TNF-, IFN-, and PGE2 did not
revert to adherent macrophages but rather underwent cell death.
Interestingly, the addition of CD40L prevented migratory-type MoDCs
from undergoing cell death, inducing them instead to vigorously form
cell clusters (data not shown).
To address whether CD40L prevented migratory-type DCs from
undergoing apoptosis, we assessed annexin V (apoptotic cells) versus propidium iodide (dead and necrotic cells) uptake. Figure
7 demonstrates Annexin V binding to
migratory-type MoDCs that were washed and exposed to HS for 3 days
(Figure 7A) or exposed to HS and CD40L (Figure 7B). The presence of
CD40L significantly decreased the proportion of Annexin V+,
apoptotic MoDCs (5.0% vs 34.3%), or PI+, dead/dying MoDCs
(5.2% vs 22.7%). This suggests that the productive interaction with
antigen-specific T cells (via CD40L) may be necessary for the prolonged
survival of mature migratory-type DCs once they enter the T-cell areas
of lymphoid tissues. It also demonstrates the importance of the
sequence of cytokine signals on the recipient cells.
PGE2 is not essential for the induction of migratory function of PBDCs To evaluate whether the effect of PGE2 on the regulation of DC migratory function was specific to only MoDCs or necessary for other DC subsets, we examined how PGE2 affects the migratory functions of PBDCs. Here we sorted to high purity CD1b/c+ PBDCs (> 98%) from healthy individuals and patients receiving the hematopoietic growth factor FL. We have previously shown that FL-expanded PBDCs are phenotypically immature but mature rapidly following culture in vitro.19 Figure 8 compares MoDCs and autologous CD1b/c+ PBDCs generated from 4 separate donors. Unlike MoDCs, which required PGE2 to acquire migratory capacity when stimulated with CD40L or intact E coli (Figure 8A), sorted CD1b/c+ PBDCs were efficient migratory cells following stimulation with these 2 classes of stimuli even in the absence of PGE2 (Figure 8B). Although the overall trends suggested that the addition of PGE2 could enhance, to some degree, the migratory function of CD1b/c+ PBDCs, this varied substantially from donor to donor. In addition, unlike MoDCs, which required at least 24 hours for the acquisition of migratory capacity, CD1b/c+ PBDCs acquired migratory capacity within 8 hours of culture (M.J., manuscript in preparation). This suggests that CD1b/c+ PBDCs may represent a presensitized population of antigen-presenting cells (APC) that migrates following encounter with all 3 classes of stimuli, whereas immature MoDCs are temporally delayed in acquiring migratory function following stimulation and require the presence of PGE2 to do so. Finally, unlike MoDCs, which secrete high levels of IL-12p70 following stimulation with either CD40L or intact E coli (Figure 8C), CD1b/c+ PBDCs were poor producers of IL-12p70 (Figure 8D). In this regard, CD1b/c+ PBDCs produced less than 150 pg/mL of IL-12p70 even when stimulated with combinations known to induce IL-12p70 in MoDCs (eg, GM-CSF + IL-4 + CD40L + IL-1 + IFN- + intact E coli), suggesting
that CD1b/c+ PBDCs are not major producers of this
cytokine.19
During bacterial and viral infections, high levels of TNF- Several novel findings have been made in the present study. First,
MoDCs differentiate into mature migratory-type DCs in response to
PGE2-containing stimuli (eg, TNF- Second, immature MoDCs may have an alternate functional fate.
Proinflammatory-type MoDCs were generated when immature MoDCs were
stimulated with CD40L-trimers or intact E coli in the
absence of PGE2. These cells secreted high levels of
cytokines, including IL-10 and IL-12p70, and induced naive T cells to
secrete high amounts of IFN- PGE2 has previously been shown to induce migration in a variety of different cell types, such as endothelial cells, mesangial cells, Lewis lung carcinoma cells, mammary tumor cells, and even Oesophagostomum dentatum larvae.48-52 This suggests a more universal mechanism of action of PGE2 that may go beyond our focus on DC maturation and activation. We show that mobilization of cytoplasmic cAMP is likely one of the main mediators for PGE2 activity since forskolin and the cAMP analogs, db-cAMP and 8-bromo-cAMP, could mimic the effect of PGE2. In addition, only the EP2/EP4 receptor agonist 11-deoxy-PGE1 could mimic the effects seen with PGE2, suggesting that these 2 receptors (which signal via the cAMP cascade) mediate the effects of PGE2 upon MoDC migratory capacity. This latter finding confirms the findings of Scandella and colleagues, who recently demonstrated by quantitative real-time PCR that exposure of immature MoDCs to PGE2-containing stimuli results in the down-regulation of EP2 and EP4 mRNA and induces MoDC migration (Scandella et al, personal written communications, November 15, 2001). Interestingly, cAMP induction decreases lipopolysaccharide
(LPS)- and TNF-mediated NFkB-activation in a variety of experimental systems, resulting in reduced secretion of cytokines, including IL-12.53-59 It is therefore tempting to speculate on the
involvement of these 2 antagonistic pathways (both stress induced) in
the regulation of DC differentiation. However, it is intriguing that the same PGE2 signal that enhances TNF- Interestingly, purified PBDCs resembled migratory-type MoDCs rather
than immature MoDCs in their response to the 3 classes of stimuli
tested. This was also the case with respect to the types of T-cell
cytokines induced by PBDCs (ie, higher levels of IL-2 regardless of
stimulus class, with no clear bias toward either IL-5 or IFN- Finally, migratory-type MoDCs did not revert to adherent macrophages when removed from their maturation stimuli but underwent cell death within 5 days. In contrast, immature MoDCs differentiated into macrophages as previously reported.28,30,31 Interestingly, CD40L prolonged the survival of migratory-type DCs, emphasizing the importance of the sequence of cytokine signals on recipient cells. Similar enhancement of in vitro survival was seen when CD1b/c+ PBDCs were stimulated with CD40L (data not shown). This suggests that the productive interaction with antigen-specific T cells (via induction of CD40L expression) is a positive signal for maintaining DC survival and Ag presentation, whereas absence of Ag-specific T cells will result in the death of the immigrant DC. This may provide a critical selective mechanism against inappropriate immune-response induction. Our data suggest that DCs that acquire migratory capacity are capable
of efficiently stimulating the expansion of antigen-specific T cells in
lymphatic organs. Indeed, several clinical groups have now demonstrated
that MoDCs matured with PGE2-containing stimuli (eg,
TNF- The data are also consistent with the proposal that the type of T-cell
immune response may be initiated and antigen-specific T cells
preferentially expanded by migratory-type DCs within lymph nodes,
while T-cell cytokine bias and effector function may be terminally
coordinated at the effector site by proinflammatory-type DCs or APC
(site high in IL-12p70 and other IFN-
Submitted December 31, 2001; accepted May 29, 2002.
Prepublished online as Blood First Edition Paper, June 21, 2002; DOI 10.1182/blood-2001-12-0360.
Supported by the Sylvia and Charles Viertel Foundation. Dr T. Luft is supported by a fellowship from The Anti Cancer Council of Victoria, Australia; Dr P. Leutjens and Dr H. Hochrein are supported by a Deutche Krebshilfe fellowship; and Dr M. Jefford is supported by the Stewardson Family Trust.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Eugene Maraskovsky, Ludwig Institute Oncology Unit, Austin and Repatriation Medical Centre, Studley Road, Heidelberg, Victoria 3084, Australia; e-mail: eugene.maraskovsky{at}ludwig.edu.au.
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T. Luft, E. Rodionova, E. Maraskovsky, M. Kirsch, M. Hess, C. Buchholtz, M. Goerner, M. Schnurr, R. Skoda, and A. D. Ho Adaptive functional differentiation of dendritic cells: integrating the network of extra- and intracellular signals Blood, June 15, 2006; 107(12): 4763 - 4769. [Abstract] [Full Text] [PDF]
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C. Jakubzick, F. Tacke, J. Llodra, N. van Rooijen, and G. J. Randolph Modulation of Dendritic Cell Trafficking to and from the Airways J. Immunol., March 15, 2006; 176(6): 3578 - 3584. [Abstract] [Full Text] [PDF]
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D. Giordano, D. M. Magaletti, and E. A. Clark Nitric oxide and cGMP protein kinase (cGK) regulate dendritic-cell migration toward the lymph-node-directing chemokine CCL19 Blood, February 15, 2006; 107(4): 1537 - 1545. [Abstract] [Full Text] [PDF]
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D. F. Legler, P. Krause, E. Scandella, E. Singer, and M. Groettrup Prostaglandin E2 Is Generally Required for Human Dendritic Cell Migration and Exerts Its Effect via EP2 and EP4 Receptors J. Immunol., January 15, 2006; 176(2): 966 - 973. [Abstract] [Full Text] [PDF]
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 M. T. Rubio, T. K. Means, R. Chakraverty, J. Shaffer, Y. Fudaba, M. Chittenden, A. D. Luster, and M. Sykes Maturation of human monocyte-derived dendritic cells (MoDCs) in the presence of prostaglandin E2 optimizes CD4 and CD8 T cell-mediated responses to protein antigens: role of PGE2 in chemokine and cytokine expression by MoDCs Int. Immunol., December 1, 2005; 17(12): 1561 - 1572. [Abstract] [Full Text] [PDF]
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 R. B. Mailliard, S. M. Alber, H. Shen, S. C. Watkins, J. M. Kirkwood, R. B. Herberman, and P. Kalinski IL-18-induced CD83+CCR7+ NK helper cells J. Exp. Med., October 3, 2005; 202(7): 941 - 953. [Abstract] [Full Text] [PDF]
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T. Luft, E. Maraskovsky, and M. Schnurr IDO production, adaptive immunity, and CTL killing Blood, October 1, 2005; 106(7): 2228 - 2229. [Full Text] [PDF]
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D. Braun, R. S. Longman, and M. L. Albert A two-step induction of indoleamine 2,3 dioxygenase (IDO) activity during dendritic-cell maturation Blood, October 1, 2005; 106(7): 2375 - 2381. [Abstract] [Full Text] [PDF]
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N. Bertho, H. Adamski, L. Toujas, M. Debove, J. Davoust, and V. Quillien Efficient migration of dendritic cells toward lymph node chemokines and induction of TH1 responses require maturation stimulus and apoptotic cell interaction Blood, September 1, 2005; 106(5): 1734 - 1741. [Abstract] [Full Text] [PDF]
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M. Rossi and J. W. Young Human Dendritic Cells: Potent Antigen-Presenting Cells at the Crossroads of Innate and Adaptive Immunity J. Immunol., August 1, 2005; 175(3): 1373 - 1381. [Abstract] [Full Text] [PDF]
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 A. Smed-Sorensen, K. Lore, J. Vasudevan, M. K. Louder, J. Andersson, J. R. Mascola, A.-L. Spetz, and R. A. Koup Differential Susceptibility to Human Immunodeficiency Virus Type 1 Infection of Myeloid and Plasmacytoid Dendritic Cells J. Virol., July 15, 2005; 79(14): 8861 - 8869. [Abstract] [Full Text] [PDF]
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J. Dannull, S. Nair, Z. Su, D. Boczkowski, C. DeBeck, B. Yang, E. Gilboa, and J. Vieweg Enhancing the immunostimulatory function of dendritic cells by transfection with mRNA encoding OX40 ligand Blood, April 15, 2005; 105(8): 3206 - 3213. [Abstract] [Full Text] [PDF]
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M. Schnurr, Q. Chen, A. Shin, W. Chen, T. Toy, C. Jenderek, S. Green, L. Miloradovic, D. Drane, I. D. Davis, et al. Tumor antigen processing and presentation depend critically on dendritic cell type and the mode of antigen delivery Blood, March 15, 2005; 105(6): 2465 - 2472. [Abstract] [Full Text] [PDF]
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M. Schnurr, T. Toy, A. Shin, M. Wagner, J. Cebon, and E. Maraskovsky Extracellular nucleotide signaling by P2 receptors inhibits IL-12 and enhances IL-23 expression in human dendritic cells: a novel role for the cAMP pathway Blood, February 15, 2005; 105(4): 1582 - 1589. [Abstract] [Full Text] [PDF]
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E. Vassiliou, V. Sharma, H. Jing, F. Sheibanie, and D. Ganea Prostaglandin E2 Promotes the Survival of Bone Marrow-Derived Dendritic Cells J. Immunol., December 1, 2004; 173(11): 6955 - 6964. [Abstract] [Full Text] [PDF]
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T. D. Shellenberger, M. Wang, M. Gujrati, A. Jayakumar, R. M. Strieter, M. D. Burdick, C. G. Ioannides, C. L. Efferson, A. K. El-Naggar, D. Roberts, et al. BRAK/CXCL14 Is a Potent Inhibitor of Angiogenesis and a Chemotactic Factor for Immature Dendritic Cells Cancer Res., November 15, 2004; 64(22): 8262 - 8270. [Abstract] [Full Text] [PDF]
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F. E. Baratelli, N. Heuze-Vourc'h, K. Krysan, M. Dohadwala, K. Riedl, S. Sharma, and S. M. Dubinett Prostaglandin E2-Dependent Enhancement of Tissue Inhibitors of Metalloproteinases-1 Production Limits Dendritic Cell Migration through Extracellular Matrix J. Immunol., November 1, 2004; 173(9): 5458 - 5466. [Abstract] [Full Text] [PDF]
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R. B. Mailliard, A. Wankowicz-Kalinska, Q. Cai, A. Wesa, C. M. Hilkens, M. L. Kapsenberg, J. M. Kirkwood, W. J. Storkus, and P. Kalinski {alpha}-Type-1 Polarized Dendritic Cells: A Novel Immunization Tool with Optimized CTL-inducing Activity Cancer Res., September 1, 2004; 64(17): 5934 - 5937. [Abstract] [Full Text] [PDF]
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 J. A. Long, M. Fogel-Petrovic, D. A. Knight, P. J. Thompson, and J. W. Upham Higher Prostaglandin E2 Production by Dendritic Cells from Subjects with Asthma Compared with Normal Subjects Am. J. Respir. Crit. Care Med., September 1, 2004; 170(5): 485 - 491. [Abstract] [Full Text] [PDF]
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A. Rivas-Carvalho, M. A. Meraz-Rios, L. Santos-Argumedo, S. Bajana, G. Soldevila, M. E. Moreno-Garcia, and C. Sanchez-Torres CD16+ human monocyte-derived dendritic cells matured with different and unrelated stimuli promote similar allogeneic Th2 responses: regulation by pro- and anti-inflammatory cytokines Int. Immunol., September 1, 2004; 16(9): 1251 - 1263. [Abstract] [Full Text] [PDF]
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A. K. Bengtsson, E. J. Ryan, D. Giordano, D. M. Magaletti, and E. A. Clark 17{beta}-Estradiol (E2) modulates cytokine and chemokine expression in human monocyte-derived dendritic cells Blood, September 1, 2004; 104(5): 1404 - 1410. [Abstract] [Full Text] [PDF]
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C. Caux Signal strength and persistence control DC fate Blood, August 15, 2004; 104(4): 913 - 914. [Full Text] [PDF]
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T. Luft, E. Maraskovsky, M. Schnurr, K. Knebel, M. Kirsch, M. Gorner, R. Skoda, A. D. Ho, P. Nawroth, and A. Bierhaus Tuning the volume of the immune response: strength and persistence of stimulation determine migration and cytokine secretion of dendritic cells Blood, August 15, 2004; 104(4): 1066 - 1074. [Abstract] [Full Text] [PDF]
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 S. Kubo, H. K. Takahashi, M. Takei, H. Iwagaki, T. Yoshino, N. Tanaka, S. Mori, and M. Nishibori E-Prostanoid (EP)2/EP4 Receptor-Dependent Maturation of Human Monocyte-Derived Dendritic Cells and Induction of Helper T2 Polarization J. Pharmacol. Exp. Ther., June 1, 2004; 309(3): 1213 - 1220. [Abstract] [Full Text] [PDF]
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E. Maraskovsky, S. Sjolander, D. P. Drane, M. Schnurr, T. T. T. Le, L. Mateo, T. Luft, K.-A. Masterman, T.-Y. Tai, Q. Chen, et al. NY-ESO-1 Protein Formulated in ISCOMATRIX Adjuvant Is a Potent Anticancer Vaccine Inducing Both Humoral and CD8+ T-Cell-Mediated Immunity and Protection against NY-ESO-1+ Tumors Clin. Cancer Res., April 15, 2004; 10(8): 2879 - 2890. [Abstract] [Full Text] [PDF]
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E. Scandella, Y. Men, D. F. Legler, S. Gillessen, L. Prikler, B. Ludewig, and M. Groettrup CCL19/CCL21-triggered signal transduction and migration of dendritic cells requires prostaglandin E2 Blood, March 1, 2004; 103(5): 1595 - 1601. [Abstract] [Full Text] [PDF]
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C. C. Bowman and K. L. Bost Cyclooxygenase-2-Mediated Prostaglandin E2 Production in Mesenteric Lymph Nodes and in Cultured Macrophages and Dendritic Cells after Infection with Salmonella J. Immunol., February 15, 2004; 172(4): 2469 - 2475. [Abstract] [Full Text] [PDF]
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T. Chen, J. Guo, M. Yang, C. Han, M. Zhang, W. Chen, Q. Liu, J. Wang, and X. Cao Cyclosporin A impairs dendritic cell migration by regulating chemokine receptor expression and inhibiting cyclooxygenase-2 expression Blood, January 15, 2004; 103(2): 413 - 421. [Abstract] [Full Text] [PDF]
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D. Giordano, D. M. Magaletti, E. A. Clark, and J. A. Beavo Cyclic Nucleotides Promote Monocyte Differentiation Toward a DC-SIGN+ (CD209) Intermediate Cell and Impair Differentiation into Dendritic Cells J. Immunol., December 15, 2003; 171(12): 6421 - 6430. [Abstract] [Full Text] [PDF]
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I. J. M. de Vries, W. J. Lesterhuis, N. M. Scharenborg, L. P. H. Engelen, D. J. Ruiter, M.-J. P. Gerritsen, S. Croockewit, C. M. Britten, R. Torensma, G. J. Adema, et al. Maturation of Dendritic Cells Is a Prerequisite for Inducing Immune Responses in Advanced Melanoma Patients Clin. Cancer Res., November 1, 2003; 9(14): 5091 - 5100. [Abstract] [Full Text] [PDF]
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A. Soruri, J. Riggert, T. Schlott, Z. Kiafard, C. Dettmer, and J. Zwirner Anaphylatoxin C5a Induces Monocyte Recruitment and Differentiation into Dendritic Cells by TNF-{alpha} and Prostaglandin E2-Dependent Mechanisms J. Immunol., September 1, 2003; 171(5): 2631 - 2636. [Abstract] [Full Text] [PDF]
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M. Jefford, M. Schnurr, T. Toy, K.-A. Masterman, A. Shin, T. Beecroft, T. Y. Tai, K. Shortman, M. Shackleton, I. D. Davis, et al. Functional comparison of DCs generated in vivo with Flt3 ligand or in vitro from blood monocytes: differential regulation of function by specific classes of physiologic stimuli Blood, September 1, 2003; 102(5): 1753 - 1763. [Abstract] [Full Text] [PDF]
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A. Martin-Fontecha, S. Sebastiani, U. E. Hopken, M. Uguccioni, M. Lipp, A. Lanzavecchia, and F. Sallusto Regulation of Dendritic Cell Migration to the Draining Lymph Node: Impact on T Lymphocyte Traffic and Priming J. Exp. Med., August 18, 2003; 198(4): 615 - 621. [Abstract] [Full Text] [PDF]
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M. Schnurr, T. Toy, P. Stoitzner, P. Cameron, A. Shin, T. Beecroft, I. D. Davis, J. Cebon, and E. Maraskovsky ATP gradients inhibit the migratory capacity of specific human dendritic cell types: implications for P2Y11 receptor signaling Blood, July 15, 2003; 102(2): 613 - 620. [Abstract] [Full Text] [PDF]
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H. Harizi, C. Grosset, and N. Gualde Prostaglandin E2 modulates dendritic cell function via EP2 and EP4 receptor subtypes J. Leukoc. Biol., June 1, 2003; 73(6): 756 - 763. [Abstract] [Full Text] [PDF]
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M. Dauer, B. Obermaier, J. Herten, C. Haerle, K. Pohl, S. Rothenfusser, M. Schnurr, S. Endres, and A. Eigler Mature Dendritic Cells Derived from Human Monocytes Within 48 Hours: A Novel Strategy for Dendritic Cell Differentiation from Blood Precursors J. Immunol., April 15, 2003; 170(8): 4069 - 4076. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
