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32: 994–1002
The production of IL-12 by dendritic cells (DC) early in an immune response is considered
critical for the polarization of CD4+ T lymphocyte response towards a Th1 pattern, a key pro-
cess in the clearance of intracellular pathogens. Infection of bone marrow-derived DC with
Mycobacterium bovis Bacillus Calmette Guérin (BCG) induced a concurrent and dose-
dependent release of IL-10 and IL-12. Here we examined whether the production of IL-10 by
DC affected their IL-12 response to mycobacterial infection and the generation of protective
immune responses in vivo. Compared to wild-type (WT) DC, DC deficient for IL-10 synthesis
(IL-10–/–) showed increased IL-12 production in response to BCG infection and CD40 stimuli
in vitro. Moreover, when transferred into mice, infected IL-10–/– DC were more efficient than
WT DC at inducing IFN- + production to mycobacterial antigens in the draining lymph nodes
(DLN). This effect was associated with increased trafficking of IL-10–/– DC to the DLN and
enhanced IL-12 production by DC within the DLN. These data show that autocrine IL-10
exerts a dual inhibitory effect on the induction of primary immune responses by DC: first, by
down-regulating the migration of infected DC to the DLN and second, by modulating the
IL-12 production by DC in the DLN. Received 5/11/01
Revised 20/12/01
Accepted 21/1/02
Key words: Dendritic cell / Mycobacteria / Cell trafficking / IL-12 / IL-10
If endogenous IL-10 inhibits the IL-12 response of DC to 2.5 IL-10–/– and WT DC differ by their ability to
microbial stimuli and to CD40 stimulation, IL-10–/– DC reach draining lymph nodes following
should be more effective than WT DC at priming IFN- + - transfer
secreting T cells against mycobacterial antigens in vivo.
WT and IL-10–/– DC were infected with BCG in vitro and We thus compared WT and IL-10–/– DC for their migratory
compared for their ability to activate naive T cells follow- capacities following transfer in vivo. Following infection
ing transfer in mice. Seven days following subcutaneous with BCG and prior to s.c. injection, DC were stain-
(s.c.) immunization, cells from pooled draining lymph ed with the fluorescent cell tracker 5-chloromethyl-
Fig. 3. Endogenous IL-10 regulates the IL-12 response of DC to CD40 stimulation. (A) IL-12 production by BCG-infected DC
derived from WT or IL-10–/– mice, in the presence of agonistic anti-CD40 ( § CD40 Ab) or irrelevant (ctrl Ab) antibody, is shown.
Data are mean and SE of triplicate measurements, and are representative of two independent experiments. (B) The percentage
of DC producing IL-12 in response to BCG infection in the presence ( § CD40 Ab), or absence (ctrl Ab), of agonistic anti-CD40
antibody is shown.
Eur. J. Immunol. 2002. 32: 994–1002 Autocrine IL-10 suppresses DC-derived immune responses 997
mote alloantigen-specific T cell responses [10]. However, is an important regulator of DC migration in vivo in
the expression of CD86 and MHC class II molecules was response to microbial stimuli.
comparable in IL-10–/– DC and WT DC (Fig. 5), and was
not modified in splenic DC by treatment with IL-10 [21], Enhanced IL-12 production was observed in the DLN of
suggesting that the effect of IL-10 on DC maturation mice immunized with IL-10–/– DC, as compared to WT
might vary with the type of DC. Moreover, WT and IL- DC. Intriguingly, the CMFDA+ subset of DC in the DLN
10–/– DC did not differ in the percentage of apoptotic or was consistently negative for IL-12 production, indicat-
necrotic cells. Therefore, the differences in the immune ing that the IL-12-producing cells were of recipient ori-
responses driven by IL-10–/– and WT DC following trans- gin. This raises the question as to how the information
fer in mice were essentially due to differential production transfer between the different DC populations occurs. It
of IL-12 and migratory capacities in this model. is possible that although most injected DC do not
migrate, they deliver a cytokine message and/or anti-
Tissue damage, presence of stressed or necrotic cells genic peptides to surrounding uninfected DC, which
and microbial products are danger signals triggering the induces these to migrate to secondary lymphoid organs.
release of locally important concentrations of inflamma- Alternatively, one can speculate that the few migrating
tory mediators of DC migration [34, 35]. The capacity of DC of donor origin are able to activate local DC to
IL-10–/– or WT DC to migrate to the DLN was examined secrete IL-12 when they reach the lymph nodes, result-
24 h after inoculation. Previous studies have shown that ing in a local amplification of the initial response. This
the number of splenic DC trafficking to the DLN after s.c. could be mediated by cytokine release or cell/cell con-
injection reached a maximum within 24 h [26, 36]. tact with resident DC or by recruiting other circulating DC
Accordingly, migrating DC could be detected in DLN fol- in the nodes. Taken together, our results support the
lowing loading of splenic [37] or bone marrow-derived emerging concept (Maraskovsky et al., unpublished) that
[38] DC in the trachea within 24 h and were no longer in physiological situations of infection, only a small sub-
detectable 48 h later, supporting the rapid migration of set of the DC population activated by microbial agents in
DC. However, the proportion of DC migrating to the DLN peripheral tissues migrates effectively to the DLN. In
always constituted a small percentage of the injected contrast, the vast majority of infected DC do not leave
pool (0.1–1.0%). This proportion of migratory DC is con- the site of infection. Our experiments suggest that this
sistent with other studies [26, 36], and questions the fate retention may be IL-10 dependent. Further, IL-10
of the remaining cells. It is likely that some of the injected appears to be an important factor regulating the genera-
DC die at the site of inoculation, and released antigens tion of the appropriate type of immune responses
are recaptured and processed by resident DC. Interest- against microbial infections. Blocking DC-derived IL-10
ingly, T cell priming in the DLN occurs with viable but not appears to be a potent way to stimulate enhanced Th1
with killed antigen-pulsed DC following transfer [39]. immune responses and may contribute to more efficient
Therefore, despite the fact that the proportion of traffick- anti-microbial vaccine strategies.
ing cells is relatively small, the direct migration of
antigen-bearing DC appear to be essential for the initia-
tion of specific T cell responses. If so, then the manipula- 4 Materials and methods
tion of DC to increase their migration properties before
transfer may be a way to improve the efficacy of DC- 4.1 Mice
based therapies.
Six- to eight-week-old C57BL/6 mice were obtained
The trafficking of DC from inflammatory sites to lymphoid from the Animal Resources Centre (Perth, Australia). IL-
tissues has been associated with a chemokine receptor 10–/– mice on a C57BL/6 background [43] were kindly
switch on DC surface, with down-regulation of inflamma- provided by Dr. Donna Rennick (DNAX Research Insti-
tory receptors and induction of CCR7 [40]. Recently, tute, Palo Alto, CA) and kept under specific pathogen-
it was demonstrated that concomitant exposure of free conditions at the Centenary Institute animal facility.
monocyte-derived DC with LPS and IL-10 blocks this
chemokine receptor switch on DC and impairs their abil-
ity to respond to inflammatory chemokines [41], sug- 4.2 DC cultures
gesting that IL-10 can suppress this migration process.
Consistently, FITC-induced epidermal Langerhans cell Bone marrow-derived DC were generated by a modifica-
migration from skin to DLN was enhanced in IL-10–/– tion of the method previously described by Inaba et al.
mice [42]. In the present study, BCG-infected IL-10–/– DC [44]. Briefly, murine bone marrow cell suspensions were
migrated to the DLN more efficiently than WT DC follow- incubated with a mixture of M5.114 (anti-MHC class II),
ing transfer into mice, suggesting that autocrine IL-10 RA3-6B2 (anti-B220), 53-6.7 (anti-CD8), GK1.5 (anti-
1000 C. Demangel et al. Eur. J. Immunol. 2002. 32: 994–1002
CD4) and RB6-8C5 (anti-Ly-6G) mAb. Cells bound to Diego, CA). For DC migration assays, CMFDA fluores-
antibodies were eliminated using Dynabeads M-450 cence was used to distinguish donor-derived DC from
coated with sheep anti-rat IgG. Remaining cells were recipient DC. For intracytoplasmic staining, DC puri-
cultured in complete medium (CM) of RPMI 1640 con- fied from the lymph nodes were fixed with 4% (w/v)
taining 5% FCS, 50 ? M 2-ME, 2 mM glutamine supple- paraformaldehyde 20 min at RT and permeabilized
mented with 2.5 ng/ml recombinant murine granulocyte/ in 0.1% BSA, 0.5% saponin PBS. Cells were then stain-
macrophage colony-stimulating factor and 5 ng/ml ed with PE-conjugated rat anti-mouse IL-12 (C15.6,
recombinant murine IL-4 (both from Peprotech, Rocky PharMingen), and with biotinylated anti-CD11c (HL3,
Hill, NJ). To test the effect of CD40 ligation on DC, a rat PharMingen) followed by streptavidin coupled to PE-
anti-CD40 IgG (clone 1C10) [15, 45] or an irrelevant rat Cy5. Analysis was then performed on CD11c+-gated
IgG (GL113, ATCC HB11679) was added to the CM. For cells. Pre-incubation of the fixed/permeabilized cells
BCG infection, day 4 DC cultures were incubated with with unlabeled C15.6 antibody (20 ? g/ml) prior to stain-
live BCG (Tokyo strain, ATCC 35737) at various MOI ing with PE-C15.6 abolished the detection of intracyto-
(from 1 to 10). Cells were washed after 16 h to eliminate plasmic IL-12, confirming the specificity of the staining
free mycobacteria, and cultured in fresh medium for an (data not shown). Samples were analyzed on a FACScali-
additional 48 h before harvesting culture supernatants bur (Becton Dickinson, San Jose, CA).
for cytokine assays. For transfer experiments, DC were
harvested 4 h after infection with BCG at a MOI of 10,
and washed twice in PBS before inoculation. 4.5 Cytokine production assays
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