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Departments of
*
Dermatology and
Internal Medicine, University of Innsbruck, Innsbruck, Austria; and
Robert-Koch-Institut, Federal Health Administration, Berlin, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-secreting
Th1 cells. Dendritic cells have repeatedly been shown to produce IL-12 both in an unstimulated state (4) and, in much larger amounts, when stimulated by either bacteria or bacterial products (5, 6), virus (7), or by ligation of their CD40 and/or MHC class II molecules (5, 8, 9). In the human system, this may only be true for dendritic cells of the myeloid lineage, so-called dendritic cells type 1 (10). Dendritic cell-derived IL-12 was functional in that it skewed primary T cell responses toward a Th1 pattern (4, 11). Most studies hitherto, except one recent report (12), have not specifically defined the maturational status of dendritic cells analyzed. This aspect is important, though, because it is the mature, T cell-activating dendritic cell in which IL-12 would presumably be most relevant for the generation of specific Th1 immunity. This prompted us to systematically study IL-12 production of dendritic cells as a function of their maturational state as well as of different maturation stimuli. In the light of recently reported feedback loops on IL-12 by Th2 cytokines (10), we have specifically investigated the influence of IL-4 and IL-10 on dendritic cell-derived IL-12. We emphasized the study of monocyte-derived dendritic cells, a population that is preferably used for immunotherapeutic approaches.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Culture medium used throughout was RPMI 1640 supplemented with 10% FCS (endotoxin <0.06 ng/ml), gentamicin (all obtained from PAA, Linz, Austria), and 2-ME (Sigma, St. Louis, MO). Alternatively, dendritic cell cultures were also set up in 1% autologous plasma, as described (13, 14).
Generation of dendritic cells
Dendritic cells were generated from adherent mononuclear cells in human blood according to established standard procedures (13, 14). Blood cells were from freshly drawn blood or from buffy coats that were obtained from the local blood center. Briefly, an initial 7-day priming culture in the presence of GM-CSF (800 U/ml) and IL-4 (1000 U/ml) was followed by a 3-day differentiation culture in the additional presence of monocyte-conditioned medium (MCM).3 GM-CSF and IL-4 were still present during this period. In the majority of experiments, populations of immature dendritic cells were split in half on day 7 of culture. They were cultured for 3 more days in the presence or absence of MCM (13, 14). On day 10, cells were collected, and immature (i.e., those without MCM) and mature (i.e., those with MCM) dendritic cells were analyzed for IL-12 production in parallel. Alternatively, in a few experiments, immature dendritic cells on day 7 of culture were compared with mature dendritic cells on day 10 of culture, after maturation in the presence of MCM. Both types of immature dendritic cells were identical with regard to phenotype and IL-12 production. GM-CSF was obtained from Novartis (Basel, Switzerland; Leukomax, sp. act., 1.1 x 106 U/mg), and IL-4 was purchased from Genzyme (Cambridge, MA; sp. act., 5 x 107 U/mg). Alternatively, we used culture supernatant (5% v/v) of a cell line transfected with human IL-4 (IL-4-62) that was provided by Dr. A. Lanzavecchia (Basel, Switzerland).
Stimuli to induce and modulate IL-12 production in dendritic cells
Fixed Staphylococcus aureus Cowan I strain (SACS, 10 µg/ml Ig-binding capacity, Pansorbin cells, catalogue number 507861) was obtained from Calbiochem (La Jolla, CA). Murine myeloma cells transfected with the human CD154/CD40 ligand molecule (P3 x TBA7 cells) were used to ligate the CD40 molecule on the surface of dendritic cells (15). Wild-type cells served as negative control (P3 x 63Ag8.653-WT). Alternatively, we cross-linked CD40 with anti-CD40 mAbs G28-5 (gift of Dr. E. Clark, Seattle, WA (16)) and MAB089 (Immunotech-Coulter, Marseille, France) as well as with total and ultracentrifuged culture supernatants of CD40 ligand-transfected cells containing CD40 ligand bound to membrane fragments and soluble CD40 ligand, respectively (17). IL-10 (sp. act., 1 x 107 U/mg) was a gift of Dr. Ann O’Garra (DNAX Research Institute, Seattle, WA). IL-4 was from Genzyme (see above).
Determination of IL-12 production
Immature or mature dendritic cells were washed out (3x) of cytokine-containing culture media. They were counted under the hemocytometer and analyzed for CD83 expression by flow cytometry, and 1 x 106 dendritic cells/ml were plated into 24-well or 48-well multiwell tissue culture plates in total volumes of 1 and 0.5 ml of culture medium, respectively. (mAb HB-15a, anti-CD83 was a gift of Dr. Thomas F. Tedder, Durham, NC; FITC-conjugated anti-CD83 was from Coulter-Immunotech, Marseille, France.) Supernatants were taken at 48 and 72 h and stored at -80°C until analysis by ELISA. For most experiments, we used a sandwich ELISA, which was generously provided by Drs. D. H. Presky and M. K. Gately from Hoffmann-LaRoche (Nutley, NJ; capture mAb, 20C2; detection mAb, peroxidase-conjugated 4D6). The exact protocol has been described previously (18, 19). Few experiments were analyzed by means of a commercial IL-12 ELISA (Quantikine; R&D Systems, Minneapolis, MN). The capture Abs used in both tests specifically recognize the p70 heterodimer, but not the free p40 chains. Detection limits were 20 pg/ml of IL-12.
Flow cytometric detection of IL-12 production
PE-conjugated mouse mAb C11.5, directed against the p40 subunit of human IL-12, was used to stain saponin-permeabilized cell populations that had been stimulated for 30 h with CD40 ligand-expressing cells in the presence (last 5 h) of brefeldin A to achieve some accumulation of cytokine within the cell (20). All reagents and the staining protocol were from BD PharMingen (San Diego, CA).
Other methods
Human IL-12 p40 and p35 mRNA was detected by PCR and liquid
hybridization, as described previously (21). IFN- was
measured with a commercial ELISA (BioSource-Medgenix, Fleurus,
Belgium). Binding of mAb dendritic cell-lysosome-associated membrane
glycoprotein (DC-LAMP) (mouse IgG1) (22) on acetone-fixed
cytospins was visualized by a biotinylated anti-mouse Ig
(Amersham-Pharmacia, Amersham, U.K.), followed by Texas
Red-conjugated streptavidin (Amersham); after blocking of residual
binding sites with an excess of mouse -globulin (100 µg/ml),
dendritic cells were counterstained with an FITC-conjugated
anti-HLA-DR mAb (clone L243; BD PharMingen, San Jose, CA). DC-LAMP
was a gift of Dr. Serge Lebecque, Laboratory for Immunological
Research, Schering-Plough (Dardilly, France). Neutralizing mAbs against
human IFN- (clone B27) and isotype-matched control Abs were
purchased from BD Phar-Mingen and used at final concentrations of 20
µg/ml.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). This
contrasted with the expression of CD83 that clearly distinguished the
two maturational states (Figs. 1 and 2A). In addition, the
up-regulated expression of CD86, the lack of CD115 expression, and a
pronounced veiled morphology under phase contrast were used as markers
for mature dendritic cells (data not shown). It should be emphasized in
this work that we use "immature dendritic cell" as the term to
describe a monocyte differentiated for 6–7 days in the presence of
GM-CSF and IL-4 (23, 24). It is clearly more advanced than
the early (25) immature dendritic cell such as a
Langerhans cell in situ, in that it has already sizeable levels of
surface MHC class II molecules and it expresses high levels of CD40.
Yet, it still requires maturation stimuli to further differentiate and
to acquire those features that characterize the terminally mature
dendritic cell (13, 14): 1) no reversion back to a
macrophage, 2) greatly augmented T cell-stimulatory capacity for MLR
and CTL, and 3) de novo expression of markers such as CD83 or DC-LAMP
(22). These features are similar to those of dendritic
cells that matured spontaneously from blood after 2 days of culture
(26) or of cultured epidermal Langerhans cells (27, 28).
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In 11 independent experiments, IL-12 values from
immature dendritic cells ranged between 2.1 and 0.02 ng/ml; values from
corresponding populations of mature dendritic cells ranged from 0.5 to
0 (i.e., below detection threshold) ng/ml. However, in all experiments,
immature dendritic cells produced higher levels than mature dendritic
cells (Fig. 3). It is of note that in 7
of 11 populations of mature dendritic cells, IL-12 in the supernatants
was below the level of detection, i.e., <20 pg/ml. Analysis of data by
means of the two-sample t test showed that the differences
between immature and mature dendritic cells were statistically
significant (p < 0.05).
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A). Ligation of CD40 induces more IL-12 in immature than in mature monocyte-derived dendritic cells
Initially, we tested the conditions for cross-linking CD40 by means of CD40 ligand-expressing cells (TBA7 cells). CD40 ligand expression of TBA7 cells was high; it was clearly more than the levels reported for activated T cells (data not shown). Maximal IL-12 release by dendritic cells was achieved at a ratio of one TBA7 cell to two dendritic cells. This ratio was kept for all additional experiments. When proliferation of TBA7 cells was stopped by irradiation with 15–30 Gy from a Cs source, they elicited considerably lower amounts of IL-12 from dendritic cells (data not shown). Therefore, viable TBA7 cells were used in all assays.
In 11 independent experiments, IL-12 values from immature dendritic
cells ranged from >100 to 1.26 ng/ml; values from corresponding
populations of mature dendritic cells were between 29 and 0.11 ng/ml.
Like with SACS stimulation, immature dendritic cells produced higher
levels than mature dendritic cells in all experiments (Fig. 4). However, in contrast to SACS
stimulation, populations of mature dendritic cells did elaborate
substantial amounts of IL-12 in all experiments, the lowest
concentration measured being 110 pg/ml. Analysis of data by means of
the two-sample t test showed that the differences between
immature and mature dendritic cells were statistically significant
(p < 0.05). These data were confirmed with
dendritic cells that had been cultured in the presence of 1%
autologous human plasma (n = 22) rather than 10% FCS,
as in the experiments reported above (Fig. 4). IL-12 production in
plasma-supplemented cultures was lower than in FCS-containing media,
though. The down-regulation of IL-12 production upon maturation of
dendritic cells did not only occur when MCM was used as a maturation
stimulus, but also when a defined cytokine cocktail consisting of
IL-1
, IL-6, TNF-
, and PGE2
(29) was employed: Experiment 1, 19 ng/ml in immature vs
1.3 ng/ml in mature dendritic cells; experiment 2, 1 vs 0.5 ng/ml.
Stimulation of dendritic cells with control wild-type cells was
consistently negative. Addition of a mAb against the CD40 ligand
completely abrogated IL-12 induction (data not shown).
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A) as well as of
intracellular DC-LAMP (22) (Fig. 2B). It is
also noteworthy that CD83 of already mature dendritic cells (on day 10)
remained stably on the surface during the 2-day duration of the
stimulation culture, even in controls in which no stimulus was added
(Fig. 2A). Ligation of CD40 by anti-CD40 mAbs yielded inconsistent results. mAb G28-5 did not induce IL-12 in some experiments; in some it did. In those experiments, the same phenomenon was noted: immature dendritic cells made more IL-12 than mature dendritic cells in response to the Ab (immature vs mature dendritic cells, IL-12 p70 in pg/ml: Expt. 1, >500 vs 52; Expt. 2, 456 vs 46). MAB089 never induced IL-12. Likewise, total and ultracentrifuged culture supernatants of CD40 ligand-transfected cells, containing CD40 ligand bound to membrane fragments and soluble CD40 ligand, respectively (17), were inactive in our assays.
Finally, we wished to test the possibility that immature and mature dendritic cells made similar amounts of IL-12, but that populations of mature dendritic cells contained proteases that would efficiently degrade the cytokine. Therefore, rIL-12 was added to immature and mature dendritic cells and incubated for 48 h. Supernatants analyzed by ELISA showed no substantial degradation of IL-12 in either population (data not shown).
Fully mature skin-derived dendritic cells make no detectable IL-12
Dendritic cells emigrated from whole skin explants were also tested for their ability to produce bioactive IL-12. These cells are a mixture consisting of epidermal Langerhans cells and dermal dendritic cells. All dendritic cells within these populations were fully mature, as previously shown (30, 31), and as monitored by morphology under phase contrast and by CD83 and CD86 expression (data not shown). In three independent experiments, SACS did not induce any measurable IL-12 p70. In two different experiments using CD40 ligand-transfected cells as stimulus, IL-12 p70 production was also below the threshold of detection.
MCM induces maturation without IL-12 production
Because the stimuli that brought about IL-12 production (CD40
ligation and SACS) also induced maturation, we wondered whether MCM,
the classical stimulus for maturation (13, 14, 32), would
also do so. Immature dendritic cells on day 7 or 10 were assayed for
IL-12 in the presence or absence of MCM. In six independent
experiments, virtually no IL-12 was induced by MCM; however, dendritic
cells from parallel cultures that were stimulated with CD40 ligand or
SACS did elaborate the cytokine (Fig. 5).
FACS analyses (Fig. 2A) proved that MCM rendered dendritic
cells stably mature also under the specific culture conditions used for
collecting supernatants for ELISA (i.e., 1 x
106 cells/ml; 48 h).
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Comparison of IL-12-inducing stimuli
From Figs. 1 and 4 it can be read that CD40 ligation is the
strongest of the three stimuli tested. This becomes more apparent when
the same data are plotted as side-by-side comparisons within different
individual experiments (Fig. 5). When CD40-induced IL-12 production of
immature dendritic cells is set equal to 100%, SACS elicits on average
about one-fifth of this amount (19.9 ± 37%; range 0.1–139%;
n = 16). MCM induce only very little IL-12 (4.4 ±
6, 9%; range 0.1–17, 9%; n = 6).
IL-12 production at the single cell level
Intracellular FACS staining of CD40 ligand-stimulated dendritic
cell populations using a mAb against the p40 subunit of IL-12 showed
unequivocally that IL-12 had accumulated in CD83+
cells (Fig. 6). This indicated that IL-12
synthesis and maturation had proceeded simultaneously. It also means
that the high levels of IL-12 are not produced by immature, but rather
by maturing dendritic cells. FACS analyses also confirmed that already
mature dendritic cells made less IL-12 than maturing dendritic
cells.
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Next we examined whether the differential secretion of IL-12
heterodimer protein in immature and mature dendritic cell was due to de
novo synthesis. To this end, the expression of mRNA for the p35 and p40
subunits of IL-12 was investigated. A semiquantitative PCR analysis
revealed that, in response to ligation of CD40, immature dendritic
cells expressed more mRNA for both p35 and p40 than mature dendritic
cells (Fig. 7). This was not as
pronounced when SACS was used as a stimulus for IL-12 production.
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IFN- has been described as necessary costimulus for IL-12
production by dendritic cells (33). Therefore, this
cytokine was measured in parallel with the IL-12 assays. In a series of
13 independent experiments (in medium containing autologous plasma), a
significant (r = 0.81) positive correlation between the
values of IL-12 and IFN- became apparent (data not shown): Most
cultures of CD40-stimulated immature dendritic cells contained more
IFN- (maximum, >60 ng/ml IFN-) than cultures of CD40-stimulated
mature cells (maximum, 9.2 ng/ml IFN-). Few experiments with
SACS-stimulated dendritic cells revealed the same correlation (data not
shown). It was not further investigated whether this cytokine was
produced by dendritic cells or by few contaminating T cells. NK cells
were ruled out as producers of IFN-: In five independent FACS
analyses, we detected virtually no CD56+ cells,
i.e., NK cells.
IL-4 enhances IL-12 production by monocyte-derived dendritic cells
When IL-4 was present during the stimulation of dendritic cells
with CD40 ligand-expressing cells, a marked increase of IL-12 secretion
was observed. Up to almost the 10-fold amount of IL-12 was induced by
IL-4. This observation was made both in FCS-containing cultures (Fig. 8) and in cultures with 1% autologous
plasma: 698, 108, 995, 961, 139, 546, and 356% for immature dendritic
cells in seven experiments, and 125, 438, 183, and 243% for mature
dendritic cells in four experiments; IL-12 production in the absence of
IL-4 was set equal to 100%. Thus, IL-4 enhanced IL-12 production
irrespective of the state of dendritic cell maturation. IL-4 did not
alter the degree of maturation of dendritic cells, as determined by
morphology under phase contrast, CD83 expression by FACS (data not
shown), and DC-LAMP expression on cytospins (Fig. 2B). In a
series of five independent experiments, we found that IL-4 did not lead
to increased amounts of IFN- in the cultures, but to clearly
enhanced levels of IL-12 (698, 108, 995, 961, and 139%). Conversely,
the neutralization of IFN- with a mAb did not prevent the
IL-4-induced augmentation of IL-12 (Table I).
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IL-10 was shown to inhibit IL-12 synthesis in murine dendritic
cells (8). In this study, we investigated the effects of
IL-10 on CD40-induced IL-12 production in human dendritic cells. In six
independent experiments, a concentration of 10 U/ml (i.e., 1 ng/ml)
IL-10 did not consistently inhibit IL-12 production. When 100 U/ml
(i.e., 10 ng/ml) IL-10 was present during the 48-h stimulation with
CD40 ligand, a clear-cut reduction of IL-12 secretion was observed
(Fig. 8). The mean reduction with 100 U/ml IL-10 was 61% for
populations of immature dendritic cells, and 66% for mature dendritic
cells. Thus, dendritic cells at both states of maturation were
inhibited to a similar degree by the high dose of IL-10.
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Discussion |
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IL-12 p70 production is not a general feature of dendritic cell maturation
We observed that CD40 ligation and (less though) bacteria induced
massive IL-12 p70 production in immature dendritic cells. In contrast,
MCM, the classical maturation stimulus (13, 14), did not
induce substantial IL-12 production when applied in an identical
experimental setting as the other stimuli (nor did the combination of
TNF- and PGE2 in two experiments). This latter
combination was reported to induce IL-12 p40 secretion, though
(35). Thus, of the three stimuli tested in this study, two
induced maturation and IL-12 secretion (CD40 ligation and bacteria),
whereas one (MCM) led to maturation without concomitant IL-12
secretion. This is similar to the findings of Cella et al.
(5), who noted IL-12 p70 induction in dendritic cells only
with CD40 ligation and to some degree with viral infection
(7), but not with other stimuli such as LPS or TNF-.
The discrepancy with regard to bacterially induced IL-12 (in this
study, good IL-12 induction; Cella et al. (5), no IL-12
induction) may be due to different reagents (staphylococci, FCS). In
vivo it would be advantageous if a dendritic cell made the potent
cytokine IL-12 only if threatened by microbes or if in physical touch
with T cells, rather than in response to any inflammatory cytokine
milieu.
Relevance of maturation-linked IL-12 production capacity: immature dendritic cells
In vivo (e.g., in the epidermis), dendritic cells receive maturation and migration stimuli by inflammatory cytokines (36), often in the absence of microbes. In that case, IL-12 is needed only when dendritic cells have arrived in the lymph nodes and interact with T cells. Maturation in the presence of MCM may be regarded as an in vitro equivalent for this case. In a scenario in which microbes are present, high levels of bacterially induced dendritic cell-derived IL-12 could be beneficial in that they would contribute to the inflammation (activation of NK cells, maintenance and enhancement of Th1 state of infiltrating Th cells, and, as a consequence, macrophage activation) and thus help with the clearance of microorganisms. Inflammation (e.g., in the skin) might be further fueled by high levels of IL-12 derived from the interaction of CD40-expressing migrating dendritic cells that encounter effector or memory T cells expressing CD40 ligand. In vivo examples underscore our in vitro data: Dendritic cells in the spleen of mice exposed in vivo to Toxoplasma Ags respond with vigorous IL-12 production (6). The initial strong IL-12 immunostaining was predominantly found at the edge of the T cell area, but also in the periarteriolar region, implying that these dendritic cells were not fully mature in situ. Similarly, Leishmania-infected dendritic cells in the spleen produce IL-12 in situ (37).
Relevance of maturation-linked IL-12 production capacity: mature dendritic cells
Upon encounter with Ag-specific T cells in the T cell areas of lymphoid organs, IL-12 is crucial for the establishment of a Th1 response. When a dendritic cell that arrives in the lymph node via the afferent lymphatics finds and binds an Ag-specific T cell in the T areas (38), it signals to the resting naive T cell via its MHC/peptide complexes (signal 1) and costimulatory molecules (signal 2). T cell activation ensues and within a period of few hours, activated T cells up-regulate CD154 (CD40 ligand) expression. CD154, in turn, engages with CD40 on the surface of the dendritic cell and now signals flow in the inverse direction: The T cell induces terminal maturation (i.e., further up-regulation of costimulatory molecules) and IL-12 production in the dendritic cells (39, 40). This determines the default pathway of dendritic cells to induce Th1 responses. Probably in the microenvironment of the intracellular spaces in lymph nodes or spleen, small quantities of IL-12 might suffice to reach biologically active concentrations. It should be emphasized that IL-12 production was found to be markedly down-regulated in mature dendritic cells; it did not completely disappear, though. Substantial quantities (up to some hundred pg/ml) were still made by mature dendritic cells in most experiments. Higher concentrations of IL-12 may even be harmful.
Augmenting effects of IL-4: mechanism
The presence of IL-4 during the stimulation period strongly
increased the levels of IL-12 in response to CD40 ligation. The reason
for this somewhat unexpected finding may be a previous conditioning of
dendritic cells during the 7-day culture in the continuous presence of
IL-4, perhaps similar, but clearly not identical with what was
described by D’Andrea et al. (41) as priming: These
authors had observed an increased IL-12 production by
IL-4-pretreated PBMCs in response to bacteria. We observed an increased
IL-12 production by IL-4-pretreated dendritic cells in response to CD40
ligation plus IL-4. Thus, the otherwise IL-12-inhibiting cytokine IL-4
turned out to be IL-12 enhancing when the cells had been pretreated
(conditioned) with IL-4. From very recent data by Hochrein et al.
(42), who used dendritic cells that had been generated in
the absence of IL-4, it appears that the IL-12-enhancing effect of IL-4
does not depend on a prior exposure to the same cytokine. A similar
observation was made with CD40 ligand-stimulated murine dendritic
cell-containing populations by Takenaka et al. (43).
IFN- appears not to mediate the IL-4 effect, because its production
was not induced by IL-4 in our hands, and moreover, IL-4 has typically
been described to inhibit IFN- rather than enhancing it (44, 45). In addition, neutralization of IFN- in the stimulation
assays did not prevent the IL-4-induced increase in IL-12 production.
This is in line with a recent report by Kalinski et al.
(46), who observed the same phenomenon induced by IL-4
derived from a Th2 clone that was deficient in IFN- production. IL-4
also does not act by influencing the maturation status of dendritic
cells: maturation markers CD83 and DC-LAMP were up-regulated in
response to CD40 ligation, irrespective of IL-4 treatment. Although
IL-4 can augment IL-12 production in such a potent way, it is not a
prerequisite for the large amounts of IL-12 made by dendritic cells.
This was first concluded by Cella et al. (5), who showed
high levels of CD40-induced IL-12 in freshly isolated dendritic cells
that had never encountered IL-4 in vitro. It is underscored by the data
of Koch et al. (8): murine spleen dendritic cells were
induced to make large amounts of IL-12 p70 by cross-linking with
anti-CD40 mAb. These dendritic cells also never had contact with
IL-4 during their generation.
Augmenting effects of IL-4: relevance
When exogenous IL-4 is added to cocultures of APCs and T cells, a
Th2 response (i.e., IL-4-producing T cells) is the consequence
(47). If a Th2 response, e.g., to fungal hyphae
(48) or to helminthic parasites (49), occurs
in the environment of a lymph node, one might expect that the resulting
T cell-derived IL-4 would skew all other ongoing or beginning immune
responses toward a Th2 pattern. Our data would indicate that there may
be some balancing mechanism ensuring that Th1 responses are not
necessarily suppressed in an IL-4-rich milieu. This finding seems
important for immunotherapy (see below). While our work was in the
final stages of review, Hochrein et al. (42) reported that
IL-4, and even more so IL-4 plus IFN-, enhance the IL-12 p70
production of murine and human dendritic cells, and Kalínski et
al. (46) found that IL-4 secreted by Th2 cells mediates
high level IL-12 p70 production by immature human dendritic cells. We
confirm these data and extend them in that we show the effect of IL-4
in an FCS-free culture system relevant for adoptive immunotherapy and
for populations of mature monocyte-derived dendritic cells, i.e., those
dendritic cells that are able to prime naive T cells and are
preferentially used in immunotherapy (50, 51).
Inhibitory effects of IL-10
The inhibition of IL-12 synthesis by IL-10 in mature dendritic
cells was also somewhat unexpected. Steinbrink et al. (52)
and Thurner et al. (53) have demonstrated that mature
dendritic cells are resistant to the effects of IL-10 in the MLR. Three
explanations are conceivable. First, it is possible that the dose of
100 U/ml (i.e., 10 ng/ml) of IL-10 is unphysiologically high and
already toxic. We have not further explored this possibility, except
for simple trypan blue staining of cell populations at the end of the
48-h IL-12 assay. However, by this criterium, no IL-10-induced toxicity
was detected. The low dose of IL-10 (1 ng/ml) did not consistently
inhibit mature dendritic cells, the average inhibition being -22%.
Second, one may assume that the amounts of IL-12 that were measured in
populations of mature dendritic cells were derived from few, still
immature or maturing dendritic cells that were still susceptible to
inhibition by IL-10. Third, IL-10 may have differential effects on
mature dendritic cells. We observed in this study that the same dose of
100 U/ml of IL-10 did not affect the phenotypical (CD83, CD86
expression) and morphological (nonadherence, veils) characteristics of
mature dendritic cells, whereas in parallel cultures it inhibited IL-12
secretion, as described. This might shift the Th1/Th2 balance in
ensuing T cell responses toward Th2. Neither IL-12 nor the IFN-/IL-4
balance of resulting T cell responders was measured in previous work
pinpointing the stability of mature dendritic cells (52, 53). This hypothesis is underscored by our previous finding with
a population of classically mature dendritic cells, namely mouse spleen
dendritic cells (8): IL-10 totally blocked IL-12 p70
secretion. When mature spleen dendritic cells were used to repetitively
stimulate allogeneic T cells, the presence of IL-10 led to the
development of a Th2 pattern of T cell cytokines (F. Koch, personal
communication).
Significance for clinical immunotherapy
Dendritic cells have been widely used and (successfully) tested in animal models of tumor therapy, and a number of clinical trials are currently running (e.g., 51, 54, 55). Monocyte-derived mature dendritic cells are often used as a convenient source of large numbers of human dendritic cells (53). Three sets of data from our experiments may be of relevance in a clinical setting: 1) Our finding that mature dendritic cells were less responsive to CD40 ligation (i.e., T cell interaction) in terms of IL-12 production seems counterproductive at first glance. Yet, it is likely that the small amounts of IL-12 still produced by mature dendritic cells will suffice for Th1 skewing within the microenvironment of the lymph nodes. 2) Our observation that IL-10 inhibits CD40-induced IL-12 production in dendritic cells should alert us that under circumstances of high IL-10 levels in the body, e.g., in tumor situations (56), dendritic cell therapies might be impaired (57) and might need adjuvant treatment such as cytokines. Induction of anergy in melanoma-specific CTL by IL-10-pretreated immature (i.e., during the maturation culture) dendritic cells was recently demonstrated (58). 3) Finally and most importantly, the fact that dendritic cells make much more IL-12 when IL-4 is present seems encouraging for strategies in which a predominant Th1 response is desired, for example, therapy of tumors or microbial infections. It seems conceivable that Ag-pulsed dendritic cells that arrive in a lymph node with an IL-4-rich milieu (e.g., atopic state) would still be able to skew a T cell response toward a Th1 pattern, perhaps even better. Additionally, IL-4 may allow for the development of Th2 mechanisms that also appear to be critically involved in tumor immunity (59, 60), without inhibiting therapeutically administered dendritic cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Nikolaus Romani, Department of Dermatology, University of Innsbruck, Anichstrasse 35, A-6020–Innsbruck, Austria.
3 Abbreviations used in this paper: MCM, monocyte-conditioned medium; DC-LAMP, dendritic cell-lysosome-associated membrane glycoprotein; SACS, fixed Staphylococcus aureus Cowan I strain.
Received for publication May 11, 2000. Accepted for publication September 27, 2000.
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References |
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  A. R. Mathers, B. M. Janelsins, J. P. Rubin, O. A. Tkacheva, W. J. Shufesky, S. C. Watkins, A. E. Morelli, and A. T. Larregina Differential Capability of Human Cutaneous Dendritic Cell Subsets to Initiate Th17 Responses J. Immunol., January 15, 2009; 182(2): 921 - 933. [Abstract] [Full Text] [PDF]
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S. J. A. M. Santegoets, H. J. Bontkes, A. G. M. Stam, F. Bhoelan, J. J. Ruizendaal, A. J. M. van den Eertwegh, E. Hooijberg, R. J. Scheper, and T. D. de Gruijl Inducing Antitumor T Cell Immunity: Comparative Functional Analysis of Interstitial Versus Langerhans Dendritic Cells in a Human Cell Line Model J. Immunol., April 1, 2008; 180(7): 4540 - 4549. [Abstract] [Full Text] [PDF]
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A. T. Prechtel, N. M. Turza, A. A. Theodoridis, and A. Steinkasserer CD83 Knockdown in Monocyte-Derived Dendritic Cells by Small Interfering RNA Leads to a Diminished T Cell Stimulation J. Immunol., May 1, 2007; 178(9): 5454 - 5464. [Abstract] [Full Text] [PDF]
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 M. Wittmann, C. Killig, M. Bruder, R. Gutzmer, and T. Werfel Critical involvement of IL-12 in IFN-{gamma} induction by calcineurin antagonists in activated human lymphocytes J. Leukoc. Biol., July 1, 2006; 80(1): 75 - 86. [Abstract] [Full Text] [PDF]
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A. E. Morelli, J. P. Rubin, G. Erdos, O. A. Tkacheva, A. R. Mathers, A. F. Zahorchak, A. W. Thomson, L. D. Falo Jr., and A. T. Larregina CD4+ T Cell Responses Elicited by Different Subsets of Human Skin Migratory Dendritic Cells J. Immunol., December 15, 2005; 175(12): 7905 - 7915. [Abstract] [Full Text] [PDF]
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 C. F. Narvaez, J. Angel, and M. A. Franco Interaction of Rotavirus with Human Myeloid Dendritic Cells J. Virol., December 1, 2005; 79(23): 14526 - 14535. [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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 P. A. Efron, H. Tsujimoto, F. R. Bahjat, R. Ungaro, J. Debernardis, C. Tannahill, H. V. Baker, C. K. Edwards, and L. L. Moldawer Differential maturation of murine bone-marrow derived dendritic cells with lipopolysaccharide and tumor necrosis factor-{alpha} Innate Immunity, June 1, 2005; 11(3): 145 - 160. [Abstract] [PDF]
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 C. Munz, T. Dao, G. Ferlazzo, M. A. de Cos, K. Goodman, and J. W. Young Mature myeloid dendritic cell subsets have distinct roles for activation and viability of circulating human natural killer cells Blood, January 1, 2005; 105(1): 266 - 273. [Abstract] [Full Text] [PDF]
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A. Smed-Sorensen, K. Lore, L. Walther-Jallow, J. Andersson, and A.-L. Spetz HIV-1-infected dendritic cells up-regulate cell surface markers but fail to produce IL-12 p70 in response to CD40 ligand stimulation Blood, November 1, 2004; 104(9): 2810 - 2817. [Abstract] [Full Text] [PDF]
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G. Guruli, B. R. Pflug, S. Pecher, V. Makarenkova, M. R. Shurin, and J. B. Nelson Function and survival of dendritic cells depend on endothelin-1 and endothelin receptor autocrine loops Blood, October 1, 2004; 104(7): 2107 - 2115. [Abstract] [Full Text] [PDF]
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G. Ratzinger, J. Baggers, M. A. de Cos, J. Yuan, T. Dao, J. L. Reagan, C. Munz, G. Heller, and J. W. Young Mature Human Langerhans Cells Derived from CD34+ Hematopoietic Progenitors Stimulate Greater Cytolytic T Lymphocyte Activity in the Absence of Bioactive IL-12p70, by Either Single Peptide Presentation or Cross-Priming, Than Do Dermal-Interstitial or Monocyte-Derived Dendritic Cells J. Immunol., August 15, 2004; 173(4): 2780 - 2791. [Abstract] [Full Text] [PDF]
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 D. Avigan, B. Vasir, J. Gong, V. Borges, Z. Wu, L. Uhl, M. Atkins, J. Mier, D. McDermott, T. Smith, et al. Fusion Cell Vaccination of Patients with Metastatic Breast and Renal Cancer Induces Immunological and Clinical Responses Clin. Cancer Res., July 15, 2004; 10(14): 4699 - 4708. [Abstract] [Full Text] [PDF]
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M. A. P. Oliveira, G. M. A. C. Lima, M. T. Shio, P. J. M. Leenen, and I. A. Abrahamsohn Immature macrophages derived from mouse bone marrow produce large amounts of IL-12p40 after LPS stimulation J. Leukoc. Biol., November 1, 2003; 74(5): 857 - 867. [Abstract] [Full Text] [PDF]
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J. Xie, J. Qian, S. Wang, M. E. Freeman III, J. Epstein, and Q. Yi Novel and Detrimental Effects of Lipopolysaccharide on In Vitro Generation of Immature Dendritic Cells: Involvement of Mitogen-Activated Protein Kinase p38 J. Immunol., November 1, 2003; 171(9): 4792 - 4800. [Abstract] [Full Text] [PDF]
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S. Miyazaki, H. Tsuda, M. Sakai, S. Hori, Y. Sasaki, T. Futatani, T. Miyawaki, and S. Saito Predominance of Th2-promoting dendritic cells in early human pregnancy decidua J. Leukoc. Biol., October 1, 2003; 74(4): 514 - 522. [Abstract] [Full Text] [PDF]
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L. Skelton, M. Cooper, M. Murphy, and A. Platt Human Immature Monocyte-Derived Dendritic Cells Express the G Protein-Coupled Receptor GPR105 (KIAA0001, P2Y14) and Increase Intracellular Calcium in Response to its Agonist, Uridine Diphosphoglucose J. Immunol., August 15, 2003; 171(4): 1941 - 1949. [Abstract] [Full Text] [PDF]
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M. Gilliet, M. Kleinhans, E. Lantelme, D. Schadendorf, G. Burg, and F. O. Nestle Intranodal injection of semimature monocyte-derived dendritic cells induces T helper type 1 responses to protein neoantigen Blood, July 1, 2003; 102(1): 36 - 42. [Abstract] [Full Text] [PDF]
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J. H. Bream, R. E. Curiel, C.-R. Yu, C. E. Egwuagu, M. J. Grusby, T. M. Aune, and H. A. Young IL-4 synergistically enhances both IL-2- and IL-12-induced IFN-{gamma} expression in murine NK cells Blood, July 1, 2003; 102(1): 207 - 214. [Abstract] [Full Text] [PDF]
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T. Luft, M. Jefford, P. Luetjens, T. Toy, H. Hochrein, K.-A. Masterman, C. Maliszewski, K. Shortman, J. Cebon, and E. Maraskovsky Functionally distinct dendritic cell (DC) populations induced by physiologic stimuli: prostaglandin E2 regulates the migratory capacity of specific DC subsets Blood, July 30, 2002; 100(4): 1362 - 1372. [Abstract] [Full Text] [PDF]
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S. Ebner, S. Hofer, V. A. Nguyen, C. Furhapter, M. Herold, P. Fritsch, C. Heufler, and N. Romani A Novel Role for IL-3: Human Monocytes Cultured in the Presence of IL-3 and IL-4 Differentiate into Dendritic Cells That Produce Less IL-12 and Shift Th Cell Responses Toward a Th2 Cytokine Pattern J. Immunol., June 15, 2002; 168(12): 6199 - 6207. [Abstract] [Full Text] [PDF]
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 B. Schuler-Thurner, E. S. Schultz, T. G. Berger, G. Weinlich, S. Ebner, P. Woerl, A. Bender, B. Feuerstein, P. O. Fritsch, N. Romani, et al. Rapid Induction of Tumor-specific Type 1 T Helper Cells in Metastatic Melanoma Patients by Vaccination with Mature, Cryopreserved, Peptide-loaded Monocyte-derived Dendritic Cells J. Exp. Med., May 20, 2002; 195(10): 1279 - 1288. [Abstract] [Full Text] [PDF]
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T. Luft, M. Jefford, P. Luetjens, H. Hochrein, K.-A. Masterman, C. Maliszewski, K. Shortman, J. Cebon, and E. Maraskovsky IL-1{beta} Enhances CD40 Ligand-Mediated Cytokine Secretion by Human Dendritic Cells (DC): A Mechanism for T Cell-Independent DC Activation J. Immunol., January 15, 2002; 168(2): 713 - 722. [Abstract] [Full Text] [PDF]
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A. E. Morelli, A. F. Zahorchak, A. T. Larregina, B. L. Colvin, A. J. Logar, T. Takayama, L. D. Falo, and A. W. Thomson Cytokine production by mouse myeloid dendritic cells in relation to differentiation and terminal maturation induced by lipopolysaccharide or CD40 ligation Blood, September 1, 2001; 98(5): 1512 - 1523. [Abstract] [Full Text] [PDF]
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 C. W. Cutler, R. Jotwani, and B. Pulendran Dendritic Cells: Immune Saviors or Achilles' Heel? Infect. Immun., August 1, 2001; 69(8): 4703 - 4708. [Full Text] [PDF]
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H. Hackstein, A. E. Morelli, A. T. Larregina, R. W. Ganster, G. D. Papworth, A. J. Logar, S. C. Watkins, L. D. Falo, and A. W. Thomson Aspirin Inhibits In Vitro Maturation and In Vivo Immunostimulatory Function of Murine Myeloid Dendritic Cells J. Immunol., June 15, 2001; 166(12): 7053 - 7062. [Abstract] [Full Text] [PDF]
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 R. M. Steinman and M. C. Nussenzweig Inaugural Article: Avoiding horror autotoxicus: The importance of dendritic cells in peripheral T cell tolerance PNAS, January 8, 2002; 99(1): 351 - 358. [Abstract] [Full Text] [PDF]
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