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Schering-Plough, Laboratory for Immunological Research, Dardilly, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Molecularly, we confirmed the phenotypic differences discriminating the
two subsets: E-cadherin mRNA was only detected in
CD1a+-derived DC, whereas CD68 and factor XIIIa mRNAs were
observed exclusively in CD14+-derived DC. Semiquantitative
reverse-transcriptase PCR analysis revealed that both DC subpopulations
spontaneously expressed IL-1, IL-1ß, IL-6, IL-7, IL-12 (p35 and
p40), IL-15, IL-18, TNF-, TGF-ß, macrophage CSF, and
granulocyte-macrophage CSF, but not IL-2, IL-3, IL-4, IL-5, IL-9, and
IFN-
transcripts. Both subpopulations were shown to secrete IL-12
after CD40 triggering. Interestingly, only the
CD14+-derived DC secreted IL-10 after CD40 activation,
strengthening the notion that the two DC subpopulations indeed
represent two independent pathways of DC development. Furthermore, both
DC subpopulations expressed IL-13 mRNA and protein following activation
with PMA-ionomycin, but not with CD40 ligand, in contrast to IL-12 and
IL-10, revealing the existence of different pathways for DC activation.
Finally, we confirmed the expression of IL-7, IL-10, and IL-13 mRNA by
CD4+CD11c+CD3- DC isolated ex vivo
from tonsillar germinal centers. Thus, CD14+-derived DC
expressing IL-10 and factor XIIIa seemed more closely related to
germinal center dendritic cellsGCDC than to Langerhans
cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Difficulty to isolate DC ex vivo has limited our current knowledge
regarding the cytokines produced by different DC subsets. After
exposure to contact allergens in vivo, Langerhans cells up-regulate
IL-1ß mRNA expression, suggesting an important role for this cytokine
in the initiation of primary immune responses in the skin (10). IL-12
production by DC, which has been documented by several authors
(11, 12, 13, 14), favors the differentiation of Th0 cells into Th1 cells (11, 15). Scheicher et al. (16) have reported that uptake of
particle-adsorbed Ag by DC up-regulates the transcription of both
IL-1 and IL-12 (p35 and p40). Finally, blood-derived DC were shown
to express mRNA for numerous cytokines (17, 18) and to secrete
functional IL-15 (19).
Study of DC has recently been facilitated by the development of
in vitro culture systems, allowing the generation of large number of
highly pure DC (20, 21, 22, 23). In this context, human cord blood
CD34+ hemopoietic progenitors cultured in presence of
GM-CSF and TNF- were shown recently to differentiate along two
independent DC pathways (24, 25). Thus, CD1a+-derived DC,
related to epidermal Langerhans cells, are characterized by the
expression of Birbeck granules, Langerhans-associated granule Ag, and
E-cadherin (26, 27). In contrast, CD14+-derived
DC, expressing CD68 and factor XIIIa (two dermal DC markers), are more
closely related to interstitial DC and/or peripheral blood DC. Whereas
both DC subpopulations are equally potent in stimulating naive T cell
proliferation, CD14+-derived DC are more efficient in Ag
uptake and have the unique capacity to induce naive B cells to
differentiate into IgM-secreting cells (28). To understand the
functional differences between these two DC subpopulations generated in
vitro, their pattern of cytokine expression was analyzed by
semiquantitative RT-PCR. Both cell types express a large array of
cytokine mRNAs, including IL-7, and secrete IL-13, but not IL-4, upon
PMA-ionomycin activation. Of particular interest, the production of
IL-10 mRNA and protein is restricted exclusively to
CD14+-derived DC. This represents a major difference
between the two DC subsets, given the strong effect of IL-10 on T cell
priming (29, 30). Different activation signals up-regulate different
cytokines: CD40 engagement induces IL-12 and IL-10 secretion, while
PMA-ionomycin activation turns on IL-13 production. Finally, IL-7,
IL-10, and IL-13 were also expressed by GCDC isolated ex vivo (31),
suggesting close relationship with CD14+-derived DC. Taken
together, these results indicate that the outcome of a primary immune
response will depend on which subset of DC is involved, as well as on
the activation signal delivered to the DC during the early phase of the
response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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rhGM-CSF (sp. act., 2 x 106 U/mg;
Schering-Plough Research Institute, Kenilworth, NJ) was used at a
saturating concentration of 100 ng/ml (200 U/ml). rhTNF- (sp. act.,
2 x 107 U/mg; Genzyme Corp., Boston, MA) was used at
an optimal concentration of 2.5 ng/ml (50 U/ml). rhSCF (sp. act.,
4 x 105 U/mg; R&D, Abington, U.K.) was used at an
optimal concentration of 25 ng/ml.
PBMC were obtained from healthy donors after Ficoll-Hypaque gradient centrifugation (d = 1.077; Eurobio, Paris, France). Cells were activated by PMA-ionomycin for 6 h (PMA, 1 ng/ml; Sigma Chemical Co., St. Louis, MO) (ionomycin, 1 µg/ml; Calbiochem, La Jolla, CA). MT9 is a CD4 T cell clone obtained in the laboratory; the cells were stimulated by PMA-ionomycin for 6 h. Murine fibroblasts transfected with human CD40 ligand (CD40L L cells) were produced in the laboratory (32). All cell types were cultured in RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with 10% (v/v) heat-inactivated FBS (Flow Laboratories, Irvine, U.K.), 10 mM HEPES, 2 mM L-glutamine, 5 x 10-5 M 2-ME, penicillin (100 U/ml), and streptomycin (100 µg/ml) (hereafter referred to as complete medium).
Purification of cord blood CD34+ hemopoietic progenitor cells
Umbilical cord blood samples were obtained according to institutional guidelines. Cells bearing CD34 Ag were isolated from mononuclear fractions through positive selection, using anti-CD34 mAb (Immu-133.3; Immunotech, Marseille, France) and goat anti-mouse IgG-coated microbeads (Miltenyi Biotec GmbH, Bergish Gladbach, Germany). Isolation of CD34+ progenitors was achieved using Minimacs separation columns (Miltenyi Biotec GmbH), as described (24). In all experiments, the isolated cells were 80 to 99% CD34+, as judged by staining with anti-CD34 mAb. After purification, CD34+ cells were cryopreserved in 10% DMSO.
Generation of DC from CD34+ cell culture
Cultures were established in the presence of SCF, GM-CSF, and
TNF-, as described, in complete medium (20, 24). After thawing,
CD34+ cells were seeded for expansion in 25- to
75-cm2 flasks (Corning, New York, NY) at 1 to 2 x
104 cells/cm2. Optimal conditions were
maintained by splitting these cultures at day 4 with medium containing
fresh GM-CSF and TNF- (cell concentration, 1–3 x
104 cells/cm2). For most experiments, cells
were routinely collected after 5 to 6 days of culture for FACS sorting
(24). Culture medium was supplemented with 5% AB+ pooled
human serum at initiation of the cultures, and by day 5 to 6, human
serum was washed away. Unsorted cells were cultured in presence of
GM-CSF and TNF- until day 12.
CD1a+ and CD14+ cell FACS sorting
After 5 to 6 days of culture in presence of SCF, GM-CSF, and
TNF-, cells were collected and labeled with FITC-conjugated OKT6
(CD1a; Ortho Diagnostic Systems, Raritan, NJ) and PE-conjugated Leu-M3
(CD14; Becton Dickinson, Mountain View, CA). Cells were separated
according to CD1a and CD14 expression into
CD14+CD1a- and
CD14-CD1a+, using a FACStarPlus
(Becton Dickinson) (laser setting: power, 250 mW; excitation
wavelength, 488 nm). To obtain highly purified populations of
CD1a+- and CD14+-derived cells, DC precursors
were sorted twice. Reanalysis of the sorted populations showed a purity
higher than 99% (T cells could never be detected, even by PCR). Sorted
cells were seeded in the presence of GM-CSF and TNF- (0.5–1 x
105 cells/cm2) for 6 to 7 additional days, a
last medium change being performed at day 10. At day 12 of the culture,
cells were divided into three fractions: one was untreated, whereas the
two others were stimulated either by PMA-ionomycin for 6 h, or by
irradiated CD40L L cells for 24 h (1 CD40L L cell for 5
DC).
Purification of GCDC from tonsils
Germinal center dendritic cells (GCDC) were prepared as previously described (31). In brief, tonsils obtained from children undergoing tonsillectomy were finely minced and digested with collagenase IV and DNase. The collected cells were centrifuged through Ficoll-Hypaque for 15 min at 500 rpm, then for 30 min at 2000 rpm. CD3+ T cells, CD19+ B cells, and CD14+ monocytes were removed from the resulting low density cells by magnetic beads (anti-mouse Ig-coated Dynabeads; Dynal, Oslo, Norway). Anti-CD3 (OKT3), anti-CD19 (4G7), and anti-CD14 (MOP9) mAb were purified from ascites. A second depletion was performed with anti-NKH1 (Coulter Corp., Hialeah, FL), anti-glycophorin A (Immunotech), and anti-CD20 (purified from ascites). The remaining cells were stained with the following mAbs: anti-CD1a FITC (Ortho Diagnostic Systems); anti-CD14 FITC, anti-CD57 FITC, anti-CD16 FITC, anti-CD7 FITC, anti-CD20 FITC, and anti-CD3 FITC (Becton Dickinson); and anti-CD4 PE-Cy5 (Immunotech) and anti-CD11c PE (Becton Dickinson). CD4+CD11c+CD3-CD20-CD1a- GCDC were isolated by cell sorting using a FACStarPlus (Becton Dickinson). A quantity amounting to 2 to 5 x 109 tonsil cell suspension was required to purify 2 x 105 to 2 x 106 GCDC with a purity higher than 97%.
GCDC were stimulated by either PMA-ionomycin for 3 h or an anti-CD40 mAb (10 µg/ml of G28-5 mAb kindly provided by Ed. Clark, University of Washington, Seattle, WA).
RNA extraction
Total RNA was extracted from the cells following established procedures (33). Briefly, the cells (1–10.106) were washed twice with PBS; lysed in 4 M guanidine isothiocyanate (100 µl/1.106 cells), 25 mM sodium acetate (pH = 7), 0.5% N-lauroylsarcosine, and 100 mM 2-ME; vortexed; and frozen at -20°C until used. Cell lysates were extracted once with acidic phenol (pH 4.8) and once with chloroform-isoamyl alcohol (24:1). Precipitation was performed with 2 vol of absolute ethanol, and lysates were centrifuged at 13,000 rpm for 30 min. The pellet was washed with 70% ethanol, and redissolved in diethylpyrocarbonate 0.1%, water.
Reverse transcriptase-PCR
Total RNA obtained following the procedure described above was reverse transcribed using a random hexamer pN6 and Superscript RNase-H reverse transcriptase (Life Technologies, Bethesda, MD). PCR was conducted in a 100 µl vol using 1 µl cDNA, 10 µl 10x PCR reaction buffer (Perkin-Elmer Corp., Norwalk, CT), 2.5 U Taq polymerase (Gene Amp PCR reagents kit; Perkin-Elmer), 200 mM dNTPs, and 500 nM of the 5' and 3' amplification primers. The PCR was performed in a DNA thermal cycler (Perkin-Elmer) for 40 cycles (1-min denaturation at 94°C, 2-min annealing at 60°C, and 3-min elongation at 72°C). ß actin mRNA amplification was performed on the cDNA as positive control of reaction efficiency. To evaluate mRNA expression semiquantitatively, in addition to the PCR product from the 40 cycles, 15 µl of the PCR product from the 28 cycles and the 35 cycles was run simultaneously on 1% agarose gels and transferred to nylon membranes. Negative controls were performed by omitting cDNA. As positive controls, RNA from cells known to abundantly express the respective mRNA was used: PMA-ionomycin-stimulated normal human PBMC or elutriated monocytes from PBMC.
Oligonucleotide primers and the expected sizes of PCR products from
cytokines are listed in Table I
. Sense
and antisense primers for ß actin, IL-2, IL-3, IL-5, and IL-12 p35
were obtained from Stratagene (La Jolla, CA).
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All PCR products obtained were hybridized with a specific
internal digoxygenin (DIG)-labeled probe (Boehringer Mannheim Corp.,
Mannheim, Germany) (Table I
). Revelation of DIG-labeled cDNA on nylon
membranes was performed as decribed by the manufacturer (Boehringer
Mannheim Corp.).
Intracellular IL-10 production studies by flow cytometry
Intracellular IL-10 was detected by flow cytometry using the method of Andersson et al. (34) with modifications (35). DC cultured for 10 days were stimulated by irradiated CD40L L cells for 24 h (1 CD40L L cell for 5 DC), and submitted to Brefeldin A (10 µg/ml) for 5 h. Then cells were washed in PBS, fixed 15 min at room temperature in Fixation Medium A (Fix and Perm cells permeabilization kit; Caltag Laboratories, Burlingame, CA), washed twice with PBS, and incubated with FITC-labeled CD1a (Ortho Diagnostic Systems) and PE-labeled anti-IL-10 mAb (PharMingen, San Diego, CA) in the presence of permeabilization Medium B (Caltag Laboratories) for 15 min at 4°C. To compete IL-10 production, PE-labeled anti-IL-10 mAb was incubated previously with an excess of exogenous hIL-10 (100 µg/ml) (Schering-Plough Research Institute) for 30 min. Then cells were washed twice in PBS, resuspended in 1% formaldehyde (Sigma Chemical Co.), and analyzed using a FACScan flow cytometer (Becton Dickinson).
Cytokine assays
DC cultured for 12 days were stimulated by PMA-ionomycin for 6 h either by irradiated CD40L or control L cells for 24 h (1 CD40L L cell for 5 DC). Supernatants from 1.106 DC were assayed for cytokine production. The production of IL-10 was measured in culture supernatants by IL-10-specific ELISA (sensitivity, 100 pg/ml) using two rat mAbs kindly provided by Dr. J. S. Abrams (DNAX, Palo Alto, CA) (36). IL-8 production was measured by IL-8-specific immunoenzymetric assay (Medgenix Diagnostics S.A., Fleurus, Belgium) (sensitivity, 0.7 pg/ml). IL-13 production was measured by IL-13-specific ELISA (BioSource International, Camarillo, CA) (sensitivity, <12 pg/ml).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human cord blood CD34+ hemopoietic progenitors
cultured in the presence of GM-CSF and TNF- differentiate into DC
along two distinct pathways (24). Mutually exclusive expression of CD1a
or CD14 at day 6 of culture allowed the isolation, after two rounds of
FACS sorting, of pure populations of CD1a+ and
CD14+ DC precursor cells. These DC precursor cells were
then recultured in the presence of GM-CSF and TNF- for 6 additional
days. cDNA generated from the different samples were normalized
according to the results of semiquantitative PCR amplification of
ß-actin (Fig. 1). At day 12,
differentiation of both precursor subsets into mature DC with typical
morphology and phenotype (CD1a+, CD80+,
CD86+, high HLA class II) was confirmed by PCR
amplification of CD1a, CD83, and CD86 mRNA. Only the CD1a+
subset differentiates into Langerhans cells characterized by the
presence of Birbeck granules and the expression of
E-cadherin. Semiquantitative analysis of
E-cadherin by PCR showed a strong signal after 28 cycles of
amplification in CD1a+-purified DC, while a weak band was
detected only after 35 and 40 cycles in CD14+-purified DC
(Fig. 1). The CD14+ subset has been characterized
previously by the distinctive expression of intracytoplasmic CD68 and
coagulation factor XIIIa, which are present in dermal DC, but not in
epidermal Langerhans cells. Both factor XIIIa and CD68 mRNA expression
were found to be restricted to purified CD14+ DC: a strong
signal was observed after 35 cycles of PCR-derived amplification using
CD14+-derived DC cDNA as template. In contrast, no message
could be detected in purified CD1a+-derived DC even after
hybridizing a 40-cycle PCR product with factor XIIIa-specific (Fig. 1)
or CD68-specific (data not shown) probes. Semiquantitative RT-PCR
analysis therefore confirmed the differential expression of specific
markers by the two subsets of DC and established the lack of
cross-contaminating cells.
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The presence of cytokine transcripts in the two purified DC
subsets was compared by semiquantitative RT-PCR, using both resting
cells and cells activated by either PMA-ionomycin (6 h) or
CD40L-transfected L cells (24 h). For each cytokine, a positive control
was amplified from cDNA of PMA-ionomycin-activated PBMC, except for
IL-15, which was amplified from elutriated monocytes. Before studying
the DC subsets, we analyzed the pattern of cytokines in the bulk DC
population. None of the samples contained mRNA for IL-2, IL-3, IL-4,
IL-5, IL-9, and IFN-, neither constitutively nor after activation.
The absence of contaminating T cells was established further by the
lack of PCR amplification of CD3 transcripts in any DC sample (data not
shown). The numerous cytokine mRNAs detected in unsorted DC are listed
in Table II.
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, IL-1ß, IL-6, IL-15, TNF-, TGF-ß, and macrophage CSF
were amplified from unstimulated DC from both subsets (Table II).
However, IL-1 and IL-1ß signals were consistently lower in
CD1a+DC than in CD14+-derived DC. GM-CSF
mRNA was expressed in both DC subsets, but only after PMA-ionomycin
activation.
IL-7 mRNA was detected in both DC subsets, albeit at a different level:
a specific PCR product was detected after 40 and 28 cycles of
amplification in CD1a+ and CD14+-derived DC,
respectively (Fig. 2). Specificity of
IL-7 amplification was confirmed by sequencing the PCR product (data
not shown). However, secretion of the protein could not be observed
using an IL-7-specific ELISA, a possible consequence of IL-7 release
below detection levels.
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DC and Langerhans cells were shown previously to secrete IL-12, a
heterodimeric cytokine composed of two covalently linked (p40 and p35)
chains (11, 14, 37), and this secretion is enhanced by CD40 ligation
(12, 13). Consistent with these reports, IL-12 p40 and IL-12 p35 mRNA
were constitutively present in both DC subsets (Fig. 3A), and their
expression was up-regulated after either CD40L or PMA-ionomycin
activation. Moreover, a p70 IL-12-specific ELISA detected the protein
both in CD1a+ and CD14+ subset supernatants,
but only after CD40L activation (CD1a+, 0.1 ± 0.08
ng/ml, mean of n 
10, range 0.03–0.29;
CD14+, 0.11 ± 0.06 ng/ml, mean of n
10, range 0.03–0.19). Interestingly, both DC subpopulations also
expressed mRNA for IL-18 (or IFN--inducing factor), a recently
described cytokine produced by monocyte and macrophage cell lines (38),
and which shares some biologic activities with IL-12 (Fig. 3B).
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IL-10 is a cytokine produced by numerous cell types, including T
cells, activated B cells, monocytes/macrophages, basophils/mast cells,
and keratinocytes, but to date, no production of IL-10 protein by DC
has been reported (39). Using semiquantitative RT-PCR analysis, total
DC were found to transcribe IL-10 mRNA (Table II). Remarkably, a
specific IL-10 PCR product was detected in unactivated
CD14+-derived DC after 40 cycles, but not in
unactivated CD1a+-derived DC, even after Southern blot
hybridization (Fig. 4). The exclusive
expression of IL-10 mRNA by CD14+-derived DC was confirmed
after activation with either CD40L or PMA-ionomycin, which both
up-regulated IL-10 mRNA expression in CD14+-derived DC,
whereas IL-10 transcripts remained undetectable in
CD1a+-derived DC. IL-10 protein production was next
measured by ELISA. As shown in Figure 5A, low levels of IL-10 were
produced constitutively by unsorted DC (0.16 ± 0.3 ng/ml, mean of
n 10, range <0.05–0.7). Up to 0.367 ng/ml of IL-10
was produced spontaneously by CD14+-purified DC, while no
IL-10 was detected in the supernatant of purified
CD1a+-derived DC (n 10). IL-10
production was also analyzed after PMA-ionomycin or CD40L activation,
which induced a strong secretion of IL-8 in the supernatants (24
± 8.2 ng/ml, mean of n 10, range 13.1–43.4, and
11.6 ± 7.6 ng/ml, mean of n 10, range
4.4–25.3, respectively). In line with RNA data, spontaneous secretion
of IL-10 by unpurified DC was enhanced significantly following CD40L
activation (0.6 ± 0.5 ng/ml, mean of n 10,
range 0.2–1.7) and was restricted strictly to the
CD14+-derived cells that secreted up to 2.14 ng/ml
(0.8 ± 0.6 ng/ml, mean of n 10, range 0.2–2).
However, PMA-ionomycin activation did not up-regulate, but rather
decreased the production of IL-10.
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B, CD40-activated
CD1a+ DC produced IL-10, and an excess of exogenous hIL-10
completely prevented IL-10 detection. At that stage of differentiation,
CD1a stained both CD1a+- and CD14+-derived DC.
Day 10 DC were used to optimize intracytoplasmic IL-10 detection, as
IL-10 is best visualized at immature stage. Taken together, these results demonstrate that CD14+-derived DC (expressing CD1a) produce IL-10. This restriction of IL-10 secretion to the CD14+-derived DC subpopulation is likely to result in functional differences between the two subsets.
In contrast to IL-10 and IL-12, PMA-ionomycin, but not CD40 activation, induces IL-13 production
Unlike many other cytokines, human IL-13 and IL-4 are produced by
relatively few cell types, including activated T cells, mast cells, and
basophils (40, 41, 42, 43, 44, 45). Surprisingly, high amounts of IL-13 transcripts
were found in both DC subsets following PMA-ionomycin activation (Table II and Fig. 6), but not after CD40L
activation. In contrast, no IL-4 transcript could be detected even
after activation. The absence of CD3, IL-3, IL-4, and IL-5 mRNA in
the highly purified CD1a+ and CD14+ DC
subsets (data not shown) argues against contaminating T cells or
basophils being the source of the IL-13 signal. No IL-13 secretion was
detected by ELISA in supernatants of either resting DC subsets (Fig. 7). Consistent with PCR results,
activation of total DC by PMA-ionomycin resulted in detectable
secretion of IL-13 (0.12 ± 0.09 ng/ml, mean of n
10, range 0.03–0.36). Both CD1a+- and
CD14+-derived cells produced significant amounts of IL-13
(0.46 ± 0.54 ng/ml, mean of n 10, range
0.04–1.38, and 0.8 ± 0.32 ng/ml, mean of n
10, range 0.08–1.25, respectively), in response to PMA-ionomycin, but
failed to respond to CD40L activation, contrasting with IL-10 and IL-12
productions. Taken together, these data suggest that an activation
signal distinct from CD40/CD40L triggers both DC subsets to secrete
IL-13, but not IL-4.
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The physiologic relevance of IL-7, IL-10, and IL-13 expression by
DC was evaluated on DC isolated ex vivo.
CD4+CD11c+CD3- DC (GCDC) that
strongly express class II Ags, but are CD1a- and do not
contain Birbeck granules (31), were purified from tonsillar germinal
centers. The absence of T and B cell contaminations in purified GCDC
was established by the absence of CD3 and CD19 PCR products (Fig. 8, lanes 3, 4,
7, and 8). Similar levels of IL-7
transcription were detected in GCDC with or without PMA-ionomycin
activation (lanes 11 and 12). IL-10
mRNA, which was detected in unactivated GCDC (lane
15), was up-regulated following PMA-ionomycin
(lane 16) or CD40L activation (data not
shown). Similar to in vitro generated DC, GCDC expressed IL-13 mRNA
only following PMA-ionomycin activation (lane
20), and not after CD40L triggering (data not shown).
Therefore, production of IL-7, IL-10, and IL-13, which is not limited
to DC generated in vitro, may have physiologic consequences on in vivo
immune response. In addition, similar cytokine production patterns
raise the possibility that GCDC are related to the
CD14+-derived DC generated in vitro.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The results demonstrate that 1)
both populations of DC produce a large array of cytokines known to
contribute to T cell priming (IL-1, IL-6, IL-15, TNF-) or to T
cell maturation (IL-12, IL-18, IL-7); 2) different signals lead to a
different regulation of the production of immunologically relevant
cytokines (IL-12, IL-13); 3) only CD14+-derived DC produce
IL-10, a key immunomodulator; 4) finally, DC isolated ex vivo (GCDC)
express IL-7, IL-10, and IL-13 and thus may represent derivatives of
the CD14+ interstitial lineage.
Among the various cytokines tested, both subsets express transcripts
for IL-1, IL-1ß, IL-6, IL-15, TNF-, and TGF-ß, cytokines that
potentially regulate naive T cell activation. Both CD1a+-
and CD14+-derived cells expressed IL-1 and IL-1ß
transcripts, although transcription levels were lower in the
CD1a+-derived DC. This is in agreement with the reported
expression of IL-1 and IL-1ß mRNA within Langerhans cells and
peripheral blood DC (17, 46, 47, 48). The secretion of IL-1ß by DC, and
in particular by Langerhans cells, seems to be essential for induction
of primary immune responses in skin (10). IL-15, a recently identified
cytokine with IL-2-like properties, was shown to increase Ag-specific T
cell activity (49). The expression of IL-15 mRNA in both DC subsets is
in line with description of this cytokine in Langerhans cells and in
human blood-derived DC (50). Furthermore, human blood DC were reported
to produce functional IL-15 protein with chemotactic activity for T
cells (19). Both DC subsets were found to express IL-6, TNF-,
TGF-ß, macrophage CSF, and GM-CSF (only following activation)
transcripts that may regulate the capacity of DC to initiate an immune
response.
IL-12 is a heterodimeric molecule produced by APCs that appears to be
central in promoting Th1 differentiation through induction of IFN-
production (51). It is not yet clear whether IL-12 p35 and p40 chains
are expressed constitutively in APCs, but both chains need to be
assembled to form biologically active IL-12 protein (37). In our hands,
the two constitutively expressed chains were up-regulated following
CD40L activation, and IL-12 protein was detectable in both DC subsets,
although only after CD40L stimulation. Our results are in agreement
with a previous study reporting constitutive IL-12 p40 and p35 mRNA
expression in DC, and secretion of an active IL-12 p70, up-regulated by
Staphylococcus aureus Cowan I stimulation (15) or CD40
triggering (12, 13). Furthermore, both DC subpopulations were found to
express IL-18 (or IGIF) transcript. IL-18 is a recently identified
cytokine that synergizes with IL-12 to induce the production of IFN-
by Th cells (38, 52) and to block IgE production from B cells (53). It
is therefore not unlikely that the large amounts of IFN- secreted by
T cells activated by DC result from the cooperation between DC-derived
IL-12 and IL-18.
In contrast to the results of a previous study (17) using mature blood DC, we report in this work constitutive expression of IL-7 mRNA in both DC subsets analyzed. IL-7 was shown originally as a bone marrow stromal cell-derived cytokine supporting growth of B cell (54, 55) and T cell precursors (56). It also has been detected in human follicular DC (57), but was never observed in cells of hemopoietic origin. The finding that DC express IL-7 is of interest in regard to the recently identified role of IL-7 in naive CD4+ T cell activation, early IL-4 secretion, and commitment toward Th2 development (58). Furthermore, in mice, IL-7 appears to regulate the functional development of an MHC class I-like restricted NK.1.1 T cell subset (59) that is involved in Th2 commitment through production of high levels of IL-4 (60, 61, 62).
Of interest, both DC subpopulations presently analyzed secreted IL-13,
but not IL-4, upon PMA-ionomycin stimulation. Both cytokines are known
to be secreted mostly by the same cell types, including activated T
cells (40, 41), mast cells, and basophils (42, 43, 44, 45). However,
differences in cellular sources have been reported inasmuch as IL-13 is
produced by CD4+ and CD8+ T cell clones
belonging to Th0, Th1, and Th2 subsets (de Waal Malefyt, (63)), while
IL-4 is secreted mostly by Th0, Th2-like, and
NK1.1+CD4+ T cells (60, 64). Moreover, contrary
to eosinophils producing IL-4, but not IL-13, malignant and
EBV-transformed B cells only produce IL-13 (63, 65, 66). DC-derived
IL-13 might participate in several aspects of DC functions: 1) as it
can substitute for IL-4, IL-13 might have an autocrine function during
differentiation of CD14+ precursors into DC; 2) by strongly
inhibiting the production of proinflammatory cytokines, such as
IL-1, IL-1ß, IL-6, IL-8, IL-10, GM-CSF, TNF- (67), and IL-12,
IL-13 could indirectly favor the commitment of naive T cells toward the
Th2 pathway; 3) DC-derived IL-13 is likely to contribute to the
regulation of B cell proliferation and differentiation by DC
(68, 69, 70).
Of importance, only PMA-ionomycin activation up-regulates IL-13, GM-CSF secretion, and lymphotactin mRNA expression (data not shown), while only CD40 triggering induces IL-10 and IL-12 production. This differential response suggests the existence of DC activation signal(s) distinct from CD40/CD40L, the surface triggers of which remain to be identified.
Of particular interest, we found that only CD14+-derived DC
cells can produce IL-10, either constitutively at a low level, or in
larger amounts after CD40L activation. In this context,
monocyte-derived DC (data not shown) as well as blood DC (17) express
IL-10, while Langerhans cells fail to express IL-10 mRNA (71). The
unique capacity of CD14+-derived cells to produce IL-10
strongly supports the concept that these two populations represent two
independent pathways of DC development (24, 28), and suggests a
particular role of this DC subset in the priming of naive T cells.
Thus, IL-10 might be involved in controlling the levels of T cell
activation induced by DC (72). Furthermore, IL-10 has been shown
recently to directly act on T cells to induce a state of anergy (73).
Thus, CD14+-derived DC might have a specific role in the
induction of T cell tolerance through the production of IL-10.
Moreover, by analogy to IL-10 effects on monocytes (74), endogenous
IL-10 may down-regulate the production of IL-1, IL-1ß, IL-6, IL-8,
TNF-, and GM-CSF by DC.
Another important aspect of this study is the correlation between cytokine expression of DC generated in vitro and DC isolated ex vivo. Thus, IL-7, IL-10, and IL-13 mRNA expression was observed on a recently identified ex vivo purified CD4+CD11c+CD3- DC population (GCDC) (31). GCDC are in close contact with T cells and B cells in germinal centers, suggesting an important role of these cytokines during B cell responses in vivo. Like CD14+-derived DC generated in vitro, GCDC express IL-10 and factor XIIIa mRNA (data not shown), but lack Birbeck granules, CD1a, CD40, CD80, CD83, and CD86 molecules. In this context, CD14+-derived DC may be related to GCDC, and thus represent an interesting model to study in vitro DC functions.
In conclusion, human DC secrete a large array of soluble factors, including several cytokines and growth factors that are immunologically relevant. IL-7, IL-10, and IL-13 were found unexpectedly to be expressed by DC, and the role of these cytokines during DC-T and DC-B cell interactions remains to be established. Taken together, our findings indicate that, depending on the subset of cells considered and on the condition of activation, human DC can produce different sets of cytokines. It is therefore likely that the outcome of a primary immune response will be affected not only by the subset of DC involved, but also by the activation signal engaged during the initiation phase of the response.
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Acknowledgments |
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Footnotes |
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2 Current address: Baylor Institute for Immunology Research, 3500 Gaston Avenue, P.O. Box 710699, Dallas, TX 75246.
3 Address correspondence and reprint requests to Dr. Christophe Caux, Schering-Plough, 27 chemin des peupliers, BP 11, 69571, Dardilly, France. E-mail address:
4 Abbreviations used in this paper: DC, dendritic cell; CD40L, CD40 ligand; DIG, digoxygenin; GCDC, germinal center dendritic cell; GM-CSF, granulocyte-macrophage colony-stimulating factor; h, human; PE, phycoerythrin; RT-PCR, reverse-transcriptase-polymerase chain reaction; SCF, stem cell factor.
Received for publication August 4, 1997. Accepted for publication October 29, 1997.
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S. Corinti, C. Albanesi, A. la Sala, S. Pastore, and G. Girolomoni Regulatory Activity of Autocrine IL-10 on Dendritic Cell Functions J. Immunol., April 1, 2001; 166(7): 4312 - 4318. [Abstract] [Full Text] [PDF]
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M. Ulanova, A. Tarkowski, M. Hahn-Zoric, and L. A. Hanson The Common Vaccine Adjuvant Aluminum Hydroxide Up-Regulates Accessory Properties of Human Monocytes via an Interleukin-4-Dependent Mechanism Infect. Immun., February 1, 2001; 69(2): 1151 - 1159. [Abstract] [Full Text] [PDF]
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Y. Yu, M. Hagihara, K. Ando, B. Gansuvd, H. Matsuzawa, T. Tsuchiya, Y. Ueda, H. Inoue, T. Hotta, and S. Kato Enhancement of Human Cord Blood CD34+ Cell-Derived NK Cell Cytotoxicity by Dendritic Cells J. Immunol., February 1, 2001; 166(3): 1590 - 1600. [Abstract] [Full Text] [PDF]
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C. Schmitt, H. Fohrer, S. Beaudet, P. Palmer, M.-J. Alpha, B. Canque, J. C. Gluckman, and A. H. Dalloul Identification of mature and immature human thymic dendritic cells that differentially express HLA-DR and interleukin-3 receptor in vivo J. Leukoc. Biol., December 1, 2000; 68(6): 836 - 844. [Abstract] [Full Text]
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B. Canque, S. Camus, A. Dalloul, E. Kahn, M. Yagello, C. Dezutter-Dambuyant, D. Schmitt, C. Schmitt, and J. C. Gluckman Characterization of dendritic cell differentiation pathways from cord blood CD34+CD7+CD45RA+ hematopoietic progenitor cells Blood, December 1, 2000; 96(12): 3748 - 3756. [Abstract] [Full Text] [PDF]
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S. Thoma-Uszynski, S. M. Kiertscher, M. T. Ochoa, D. A. Bouis, M. V. Norgard, K. Miyake, P. J. Godowski, M. D. Roth, and R. L. Modlin Activation of Toll-Like Receptor 2 on Human Dendritic Cells Triggers Induction of IL-12, But Not IL-10 J. Immunol., October 1, 2000; 165(7): 3804 - 3810. [Abstract] [Full Text] [PDF]
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C. Soulas, T. Baussant, J.-P. Aubry, Y. Delneste, N. Barillat, G. Caron, T. Renno, J.-Y. Bonnefoy, and P. Jeannin Cutting Edge: Outer Membrane Protein A (OmpA) Binds to and Activates Human Macrophages J. Immunol., September 1, 2000; 165(5): 2335 - 2340. [Abstract] [Full Text] [PDF]
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 P. Brossart, A. Zobywalski, F. Grünebach, L. Behnke, G. Stuhler, V. L. Reichardt, L. Kanz, and W. Brugger Tumor Necrosis Factor {{alpha}} and CD40 Ligand Antagonize the Inhibitory Effects of Interleukin 10 on T-Cell Stimulatory Capacity of Dendritic Cells Cancer Res., August 1, 2000; 60(16): 4485 - 4492. [Abstract] [Full Text]
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E. Riedl, J. Stockl, O. Majdic, C. Scheinecker, K. Rappersberger, W. Knapp, and H. Strobl Functional Involvement of E-Cadherin in TGF-{beta}1-Induced Cell Cluster Formation of In Vitro Developing Human Langerhans-Type Dendritic Cells J. Immunol., August 1, 2000; 165(3): 1381 - 1386. [Abstract] [Full Text] [PDF]
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P. Jourdan, J.-P. Vendrell, M.-F. Huguet, M. Segondy, J. Bousquet, J. Pene, and H. Yssel Cytokines and Cell Surface Molecules Independently Induce CXCR4 Expression on CD4+ CCR7+ Human Memory T Cells J. Immunol., July 15, 2000; 165(2): 716 - 724. [Abstract] [Full Text] [PDF]
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H. Nagayama, K. Sato, H. Kawasaki, M. Enomoto, C. Morimoto, K. Tadokoro, T. Juji, S. Asano, and T. A. Takahashi IL-12 Responsiveness and Expression of IL-12 Receptor in Human Peripheral Blood Monocyte-Derived Dendritic Cells J. Immunol., July 1, 2000; 165(1): 59 - 66. [Abstract] [Full Text] [PDF]
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D. Rea, C. van Kooten, K. E. van Meijgaarden, T. H. M. Ottenhoff, C. J. M. Melief, and R. Offringa Glucocorticoids transform CD40-triggering of dendritic cells into an alternative activation pathway resulting in antigen-presenting cells that secrete IL-10 Blood, May 15, 2000; 95(10): 3162 - 3167. [Abstract] [Full Text] [PDF]
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C. G. Feng, W. J. Britton, U. Palendira, N. L. Groat, H. Briscoe, and A. G. D. Bean Up-Regulation of VCAM-1 and Differential Expansion of {beta} Integrin-Expressing T Lymphocytes Are Associated with Immunity to Pulmonary Mycobacterium tuberculosis Infection J. Immunol., May 1, 2000; 164(9): 4853 - 4860. [Abstract] [Full Text] [PDF]
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A. Matsukawa, C. M. Hogaboam, N. W. Lukacs, P. M. Lincoln, H. L. Evanoff, R. M. Strieter, and S. L. Kunkel Expression and Contribution of Endogenous IL-13 in an Experimental Model of Sepsis J. Immunol., March 1, 2000; 164(5): 2738 - 2744. [Abstract] [Full Text] [PDF]
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B. Johansson, S. Ingvarsson, P. Bjorck, and C. A. K. Borrebaeck Human Interdigitating Dendritic Cells Induce Isotype Switching and IL-13-Dependent IgM Production in CD40-Activated Naive B Cells J. Immunol., February 15, 2000; 164(4): 1847 - 1854. [Abstract] [Full Text] [PDF]
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A. Khanna, A. E. Morelli, C. Zhong, T. Takayama, L. Lu, and A. W. Thomson Effects of Liver-Derived Dendritic Cell Progenitors on Th1- and Th2-Like Cytokine Responses In Vitro and In Vivo J. Immunol., February 1, 2000; 164(3): 1346 - 1354. [Abstract] [Full Text] [PDF]
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 M. Brenner, C. Rossig, U. Sili, J. W. Young, and E. Goulmy Transfusion Medicine: New Clinical Applications of Cellular Immunotherapy Hematology, January 1, 2000; 2000(1): 356 - 375. [Abstract] [Full Text] [PDF]
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A. Galy, I. Christopherson, G. Ferlazzo, G. Liu, H. Spits, and K. Georgopoulos Distinct signals control the hematopoiesis of lymphoid-related dendritic cells Blood, January 1, 2000; 95(1): 128 - 137. [Abstract] [Full Text] [PDF]
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 D. Rea, F. H. E. Schagen, R. C. Hoeben, M. Mehtali, M. J. E. Havenga, R. E. M. Toes, C. J. M. Melief, and R. Offringa Adenoviruses Activate Human Dendritic Cells without Polarization toward a T-Helper Type 1-Inducing Subset J. Virol., December 1, 1999; 73(12): 10245 - 10253. [Abstract] [Full Text]
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A. Aicher, G. L. Shu, D. Magaletti, T. Mulvania, A. Pezzutto, A. Craxton, and E. A. Clark Differential Role for p38 Mitogen-Activated Protein Kinase in Regulating CD40-Induced Gene Expression in Dendritic Cells and B Cells J. Immunol., December 1, 1999; 163(11): 5786 - 5795. [Abstract] [Full Text] [PDF]
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S. Nakagawa, C. W. Koomen, J. D. Bos, and M. B. M. Teunissen Differential Modulation of Human Epidermal Langerhans Cell Maturation by Ultraviolet B Radiation J. Immunol., November 15, 1999; 163(10): 5192 - 5200. [Abstract] [Full Text] [PDF]
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 E. L. NELSON, S. STROBL, J. SUBLESKI, D. PRIETO, W. C. KOPP, and P. J. NELSON Cycling of human dendritic cell effector phenotypes in response to TNF-{alpha}: modification of the current `maturation' paradigm and implications for in vivo immunoregulation FASEB J, November 1, 1999; 13(14): 2021 - 2030. [Abstract] [Full Text]
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S. Jaksits, E. Kriehuber, A. S. Charbonnier, K. Rappersberger, G. Stingl, and D. Maurer CD34+ Cell-Derived CD14+ Precursor Cells Develop into Langerhans Cells in a TGF-{beta}1-Dependent Manner J. Immunol., November 1, 1999; 163(9): 4869 - 4877. [Abstract] [Full Text] [PDF]
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S. Corinti, D. Medaglini, A. Cavani, M. Rescigno, G. Pozzi, P. Ricciardi-Castagnoli, and G. Girolomoni Human Dendritic Cells Very Efficiently Present a Heterologous Antigen Expressed on the Surface of Recombinant Gram-Positive Bacteria to CD4+ T Lymphocytes J. Immunol., September 15, 1999; 163(6): 3029 - 3036. [Abstract] [Full Text] [PDF]
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Y. Delneste, N. Herbault, B. Galea, G. Magistrelli, I. Bazin, J.-Y. Bonnefoy, and P. Jeannin Vasoactive Intestinal Peptide Synergizes with TNF-{alpha} in Inducing Human Dendritic Cell Maturation J. Immunol., September 15, 1999; 163(6): 3071 - 3075. [Abstract] [Full Text] [PDF]
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R. Sacedon, A. Vicente, A. Varas, E. Jimenez, J. J. Munoz, and A. G. Zapata Glucocorticoid-mediated regulation of thymic dendritic cell function Int. Immunol., August 1, 1999; 11(8): 1217 - 1224. [Abstract] [Full Text] [PDF]
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C. Chougnet, S. S. Cohen, T. Kawamura, A. L. Landay, H. A. Kessler, E. Thomas, A. Blauvelt, and G. M. Shearer Normal Immune Function of Monocyte-Derived Dendritic Cells from HIV-Infected Individuals: Implications for Immunotherapy J. Immunol., August 1, 1999; 163(3): 1666 - 1673. [Abstract] [Full Text] [PDF]
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S. Corinti, E. Fanales-Belasio, C. Albanesi, A. Cavani, P. Angelisova, and G. Girolomoni Cross-Linking of Membrane CD43 Mediates Dendritic Cell Maturation J. Immunol., June 1, 1999; 162(11): 6331 - 6336. [Abstract] [Full Text] [PDF]
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B. Canque, Y. Bakri, S. Camus, M. Yagello, A. Benjouad, and J. C. Gluckman The Susceptibility to X4 and R5 Human Immunodeficiency Virus-1 Strains of Dendritic Cells Derived In Vitro From CD34+ Hematopoietic Progenitor Cells Is Primarily Determined by Their Maturation Stage Blood, June 1, 1999; 93(11): 3866 - 3875. [Abstract] [Full Text] [PDF]
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F. Geissmann, P. Revy, A. Regnault, Y. Lepelletier, M. Dy, N. Brousse, S. Amigorena, O. Hermine, and A. Durandy TGF-{beta}1 Prevents the Noncognate Maturation of Human Dendritic Langerhans Cells J. Immunol., April 15, 1999; 162(8): 4567 - 4575. [Abstract] [Full Text] [PDF]
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P. Kalinski, J. H. N. Schuitemaker, C. M. U. Hilkens, E. A. Wierenga, and M. L. Kapsenberg Final Maturation of Dendritic Cells Is Associated with Impaired Responsiveness to IFN-{gamma} and to Bacterial IL-12 Inducers: Decreased Ability of Mature Dendritic Cells to Produce IL-12 During the Interaction with Th Cells J. Immunol., March 15, 1999; 162(6): 3231 - 3236. [Abstract] [Full Text] [PDF]
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B. Dubois, C. Barthelemy, I. Durand, Y.-J. Liu, C. Caux, and F. Briere Toward a Role of Dendritic Cells in the Germinal Center Reaction: Triggering of B Cell Proliferation and Isotype Switching J. Immunol., March 15, 1999; 162(6): 3428 - 3436. [Abstract] [Full Text] [PDF]
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R. Josien, B. R. Wong, H.-L. Li, R. M. Steinman, and Y. Choi TRANCE, a TNF Family Member, Is Differentially Expressed on T Cell Subsets and Induces Cytokine Production in Dendritic Cells J. Immunol., March 1, 1999; 162(5): 2562 - 2568. [Abstract] [Full Text] [PDF]
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P. A. Stumbles, J. A. Thomas, C. L. Pimm, P. T. Lee, T. J. Venaille, S. Proksch, and P. G. Holt Resting Respiratory Tract Dendritic Cells Preferentially Stimulate T Helper Cell Type 2 (Th2) Responses and Require Obligatory Cytokine Signals for Induction of Th1 Immunity J. Exp. Med., December 7, 1998; 188(11): 2019 - 2031. [Abstract] [Full Text] [PDF]
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B. Dubois, C. Massacrier, B. Vanbervliet, J. Fayette, F. Briere, J. Banchereau, and C. Caux Critical Role of IL-12 in Dendritic Cell-Induced Differentiation of Naive B Lymphocytes J. Immunol., September 1, 1998; 161(5): 2223 - 2231. [Abstract] [Full Text] [PDF]
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M.-C. Dieu, B. Vanbervliet, A. Vicari, J.-M. Bridon, E. Oldham, S. Ait-Yahia, F. Briere, A. Zlotnik, S. Lebecque, and C. Caux Selective Recruitment of Immature and Mature Dendritic Cells by Distinct Chemokines Expressed in Different Anatomic Sites J. Exp. Med., July 20, 1998; 188(2): 373 - 386. [Abstract] [Full Text] [PDF]
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) by unsorted DC, and by CD1a+- or
CD14+-derived DC was measured at day 12 of the culture
using a specific ELISA, with supernatants from unactivated cells,
PMA-ionomycin, or CD40L-activated cells. To control the efficiency of
activation, comparatively to IL-10 production, IL-8 secretion () was
measured by a specific ELISA in the samples tested. Results, expressed
in ng/ml, represent one experiment, representative of 10. In the
experiment shown, unactivated or CD40L-activated DC produced an average
0.278 and 0.475 ng/ml of IL-10, respectively, whereas unactivated or
CD40L-activated CD14+-derived cells produced 0.223 and 1.57
ng/ml, respectively. B, The production of IL-10 by unsorted
DC was analyzed by intracellular flow cytometry at day 10 of the
culture after 24-h stimulation by irradiated CD40L L cells. CD1a
expression was used as a DC marker recognizing both CD1a+-
and CD14+-derived cells. To compete IL-10 production,
PE-labeled anti-IL-10 mAb was incubated previously with an excess
of exogenous hIL-10 (100 µg/ml).
