Final Maturation of Dendritic Cells Is Associated with Impaired Responsiveness to IFN-γ and to Bacterial IL-12 Inducers: Decreased Ability of Mature Dendritic Cells to Produce IL-12 During the Interaction with Th Cells

Paweł Kaliński, Joost H. N. Schuitemaker, Catharien M. U. Hilkens, Eddy A. Wierenga and Martien L. Kapsenberg
J Immunol March 15, 1999, 162 (6) 3231-3236;

Abstract

Activation of immature CD83− dendritic cells (DC) in peripheral tissues induces their maturation and migration to lymph nodes. Activated DC become potent stimulators of Th cells and efficient inducers of Th1- and Th2-type cytokine production. This study analyzes the ability of human monocyte-derived CD1a+ DC at different stages of IL-1β and TNF-α-induced maturation to produce the major Th1-driving factor IL-12. DC at the early stages of maturation (2 and 4 h) produced elevated amounts of IL-12 p70 during interaction with CD40 ligand-bearing Th cells or, after stimulation with the T cell-replacing factors, soluble CD40 ligand and IFN-γ. The ability to produce IL-12 was strongly down-regulated at later time points, 12 h after the induction of DC maturation, and in fully mature CD83+ cells, at 48 h. In contrast, the ability of mature DC to produce IL-6 was preserved or even enhanced, indicating their intact responsiveness to CD40 triggering. A reduced IL-12-producing capacity of mature DC resulted mainly from their impaired responsiveness to IFN-γ, a cofactor in CD40-induced IL-12 p70 production. This correlated with reduced expression of IFN-γR (CD119) by mature DC. In addition, while immature DC produced IL-12 and IL-6 after stimulation with LPS or Staphylococcus aureusCowan I strain, mature DC became unresponsive to these bacterial stimuli. Together with the previously described ability of IL-10 and PGE2 to stably down-regulate the ability to produce IL-12 in maturing, but not in fully mature, DC, the current data indicate a general resistance of mature DC to IL-12-modulating factors.

Interleukin-12 is a major Th1-promoting factor. IL-12 is essential in generation of Th1-biased cells from naive precursors 1 . In addition, IL-12 plays an important role in the effector phase of immune responses, providing costimulatory and anti-apoptotic signals that regulate the activity of committed effector/memory CTL, Th, and NK cells 1 . Several in vivo studies, using IL-12 neutralizing Abs or IL-12- or STAT-4-deficient animals, documented an important role for IL-12 in the development of functional Th1 responses and in the protection against intracellular pathogens and tumors 1, 2, 3 . Two recent reports 4, 5 on IL-12R-deficient individuals, who show impaired IFN-γ production and increased susceptibility to intracellular pathogens, documented a similar role for IL-12 in humans.

IL-12 is a product of several types of APC, including dendritic cells (DC)3, 1, 6, 7, 8 . DC produce IL-12 in response to exogenous stimuli, e.g., bacteria and their products, such as Staphylococcus aureus Cowan I strain (SAC) and LPS 9, 10, 11, 12 . In addition, IL-12 production can be induced by CD40 ligation 7, 8, 9, 10, 13 . While CD40 triggering alone is sufficient to induce the production of the p40 subunit of IL-12 13 , the optimal induction of bioactive IL-12 p70 heterodimer requires either very high levels of CD40 cross-linking, e.g., provided by CD40 ligand (CD40L)-transfected J558 cells 7 , or additional signals, which may be provided by IFN-γ or bacterial LPS 9, 10, 13 . The combination of CD40L and IFN-γ represents an endogenous pathway of IL-12 induction that operates during the interaction of CD40-bearing DC with CD40L-expressing Th cells 9, 13 .

Depending on their maturation stage and their localization, DC perform different functions within the immune system. Immature CD83− DC residing in peripheral tissues are poor stimulators of primary T cell responses. Instead, they act as immune sentinels by virtue of their ability to collect information about invading pathogens and carry it to the draining lymph nodes. Activation of immature DC directly by a pathogen or indirectly by the pathogen-induced cytokines, such as IL-1β and TNF-α 14, 15, 16 , results in the up-regulation of their stimulatory capacity and the migration to lymph nodes. Depending on the type of tissue in which they reside and the character of the pathogen-related migration-inducing signals, DC reach the lymph nodes within 4–48 h 17, 18, 19, 20, 21 . Here, mature CD83+ DC act as effective inducers of primary responses of Ag-specific naive Th cells 14, 15, 16 .

In addition to this role of DC as the initiators of immune responses, tissue-type DC perform an important function during the effector phase of immune responses, acting as APC for peripheral tissue-homing memory T cells 14, 22 .

Since in either of these two situations the induction of IL-12 occurs in different conditions and can serve different populations of responding cells (naive Th cells in lymph nodes vs effector cells in peripheral nonlymphoid tissues, such as primed Th cells, NK cells, and CD8+ CTLs), we analyzed the requirements for the effective IL-12 induction in immature and mature monocyte-derived human CD1+ DC 23 and compared the levels of IL-12 production in either DC population.

Materials and Methods

Culture media and mAb used

All cultures were performed in Iscove’s modified Dulbecco’s medium with 10% FCS (HyClone, Logan, UT). The following mAb were used: CD1a (OKT6; Ortho, Beerse, Belgium), HLA-DR (L243; Becton Dickinson, San Jose, CA), CD83 (HB15; Immunotech, Marseille, France), CD115 (GR12; Oncogene, Cambridge, MA), CD80 and CD86 (B7-24 and 1G10; both provided by Innogenetics (Ghent, Belgium), CD40 (EA-5; a gift from Dr. T. LeBien, University of Minnesota, Minneapolis, MN), and CD119 (IFN-γR α-chain, BB1E2; provided by InstruChemie, Hilversum, The Netherlands). The following isotype-matched control mAbs were used: MOPC-21, UPC-10, and MOPC-141 (respectively, IgG1, IgG2a, and IgG2b; all obtained from Sigma, St.Louis, MO). The mAb-labeled cells were analyzed using FACScan (Becton Dickinson), after application of FITC-coupled goat F(ab′)2 anti-mouse IgG and IgM (Jackson ImmunoResearch Laboratories, West Grove, PA) as a secondary reagent.

Isolation of monocytes and CD4+ Th cells

Monocytes and lymphocytes were isolated from peripheral blood of healthy volunteers as described previously 10 . CD4+ Th cells were isolated from the lymphocyte fraction using CD4-specific Dynabeads/Detatchabead system (Dynal, Oslo, Norway), accordingly to the manufacturer’s instructions.

Generation of immature CD1a+CD83− DC and induction of their final maturation

A procedure of Sallusto 23 was used. Monocytes were cultured in the presence of recombinant human granulocyte-macrophage CSF (500 U/ml; a gift from Schering-Plough, Uden, The Netherlands) and recombinant human IL-4 (250 U/ml; PBH, Hannover, Germany). On day 6 the cultures consisted of uniformly HLA-DR+, CD83−CD115+ immature DC, without any detectable CD3+ cells. Over 90% of the cells expressed high levels of CD1a. To induce final maturation, recombinant human IL-1β (10 ng/ml, corresponding to 5 × 102 U/ml; Boehringer Mannheim, Germany) and recombinant human TNF-α (50 ng/ml, corresponding to 5 × 103 U/ml; PBH) or LPS (100 ng/ml; Difco, Detroit, MI) were added at the indicated time points, 2–48 h before harvesting the cells. On day 8 DC were harvested; washed four times (each time in 10 ml of fresh medium) to remove IL-1β, TNF-α, LPS, granulocyte-macrophage CSF, and IL-4; and subsequently used for cocultures with Th cells or stimulated as indicated.

Cocultures of DC with CD4+ Th cells and DC stimulation

Freshly isolated CD4+ T cells (6 × 104) were cocultured with DC (2 × 104) at different stages of maturation in a final volume of 200 μl in the presence of superantigen (1 ng/ml staphylococcal enterotoxin B (SEB); Serva, Heidelberg, Germany). Supernatants were harvested after 24 h of coculture. Alternatively, DC at different time points after the induction of their maturation (2 × 104 cells in 200 μl) were stimulated for 24 h with one of the following stimuli: soluble recombinant CD40L 24 (1 μg/ml; a gift from Immunex, Seattle, WA) in combination with recombinant human IFN-γ (1000 U/ml; a gift from Dr. P. H. van der Meide, Biomedical Primate Research Center, Rijswijk, The Netherlands); CD40L-transfected J558 plasmocytoma cells (J558-CD40L; 5 × 104 cells/well; a gift from Dr. P. Lane, Birmingham, U.K.), which were previously shown to induce IL-12 p70 in an IFN-γ-independent manner 7 ; SAC (75 μg/ml; Calbiochem, San Diego, CA); or LPS (100 ng/ml; Difco). The concentrations of cytokines in 24-h supernatants were analyzed with specific solid phase sandwich ELISAs.

Cytokine measurements

An IFN-γ ELISA (sensitivity, 100 pg/ml) 25 was performed using a pair of specific mAb, MD2 and MD1, both gifts from Dr. P. van der Meide (Biomedical Primate Research Center). The IL-4 ELISA (sensitivity, 50 pg/ml) 26 was performed using a pair of specific mAb, CLB-IL-4/5 and CLB-IL-4/1 (both obtained from CLB, Amsterdam, The Netherlands). The IL-5 ELISA (sensitivity, 10 pg/ml) was performed using a specific TRFK5 mAb (a gift from Dr. H. Savelkoul, Erasmus University, Rotterdam, The Netherlands) and JES1-5A10 mAb (PharMingen, San Diego CA). The IL-12 p70 ELISA (sensitivity, 3 pg/ml) 27 was performed using p70-specific mAb 20C2 (a gift from Dr. M. K. Gately, Hoffmann-La Roche, Nutley, NJ) and p40-specific C8.6 mAb (a gift from Dr. G. Trinchieri, The Wistar Institute, Philadelphia, PA). Concentrations of IL-6 in 24-h supernatants were measured by specific IL-6 ELISA (sensitivity, 20 pg/ml), using a pair of anti-IL-6 mAb, 58.126.08 and biotinylated 58.126.02, both obtained from Medgenix (Fleurus, Belgium).

Results

The interaction of CD4+ Th cells with mature DC results in reduced amounts of IL-12 despite the presence of elevated amounts of IFN-γ

The exposure of monocyte-derived CD1a+CD83− immature DC 10, 28 to the inflammatory cytokines IL-1β (5 × 102 U/ml) and TNF-α (5 × 103 U/ml) resulted in the initiation of their final maturation. This was evidenced by a rapid appearance of CD83 and the up-regulation of surface expression of CD40, CD80 (B7.1), and particularly CD86 (B7.2; Fig. 1⇓). To test whether DC at different stages of maturation produce different levels of IL-12 p70 heterodimer and induce different patterns of Th1- and Th2-type cytokine production in Th cells, DC harvested at different time points after the initiation of their maturation were cocultured with freshly isolated CD4+ Th cells in the presence of superantigen (SEB).

FIGURE 1.
FIGURE 1.

The kinetics of changes in the surface phenotype of DC undergoing IL-1β- and TNF-α-induced final maturation. IL-1β (5 × 102 U/ml) and TNF-α (5 × 103 U/ml) were added to the cultures of immature DC at the indicated time points before harvesting of the cells on day 8. The cells were analyzed by FACScan after labeling with the indicated Abs. The levels of nonspecific fluorescence, obtained with isotype-matched irrelevant mAbs, were set in each case at 0.3 × 101.

Consistent with the enhanced expression of costimulatory molecules, maturing DC induced increasingly higher levels of the Th1-type cytokine IFN-γ compared with immature DC (Fig. 2⇓A). This increased IFN-γ induction by more mature DC was paralleled by a similar increase in the induction of the Th2-type cytokine, IL-5. IL-4 was undetectable in these cultures of SEB-stimulated CD4+ Th cells.

FIGURE 2.
FIGURE 2.

The interaction of CD4+ Th cells with mature DC results in reduced amounts of IL-12 despite the presence of elevated amounts of IFN-γ. A, Production of the Th cell cytokines IFN-γ and IL-5 (top) and of DC-derived IL-12 p70 (bottom) during 24-h cocultures of CD4+ Th cells with DC induced to mature by IL-1β (5 × 102 U/ml) and TNF-α (5 × 103 U/ml) for the indicated periods of time (see Materials and Methods). Data are shown as the mean (±SD) of triplicate cultures from a single donor and represent one experiment of four, which all gave similar results. B, Decreased production of IL-12 is an intrinsic property of maturing DC. DC at different stages of IL-1β- and TNF-α-induced maturation were stimulated with sCD40L and IFN-γ, and the concentration of IL-12 p70 was analyzed in 24-h supernatants. Data are shown as the mean (±SD) of triplicate cultures of DC from a single donor and represent one experiment of four, which all gave similar results.

Although IFN-γ is a cofactor in the induction of IL-12 p70 by CD40L-expressing Th cells 9, 13 , the increasing levels of IFN-γ induced in Th cells by more mature DC did not correlate with the changes in IL-12 p70 production. The induction of final DC maturation resulted in a transiently elevated ability to produce IL-12 at early time points (2 and 4 h) of maturation, followed by the decline of this ability, that was evident in DC matured for 12 h and to an even greater extent in fully mature DC at 48 h (Fig. 2⇑A). As an average of four different donors, during cocultures with Th cells mature DC produced only 19% (range, 7–32%) of the IL-12 produced by immature DC despite the fact that mature DC displayed higher levels of CD40 expression (Fig. 1⇑) and induced higher IFN-γ production in Th cells.

The diminished IL-12 levels in the cultures containing mature DC could result from the decreased ability of mature DC to produce this cytokine or, alternatively, could result from its consumption by more strongly stimulated Th cells or from their production of some putative inhibitory factors. To distinguish among these possibilities, DC at different stages of maturation were stimulated with a soluble CD40L trimer (sCD40L) plus IFN-γ, a mode of stimulation that mimics the physiological IL-12-induction by CD4+ T cells 9, 13 . As shown in Fig. 2⇑B, the maturation-dependent changes in the ability to produce IL-12 were observed after this mode of DC stimulation, indicating that the decreased ability to produce IL-12 is indeed an intrinsic property of mature DC. Interestingly, after stimulation with CD40L plus IFN-γ, the eventual down-regulation of IL-12 production in fully mature DC was preceded by an initial transient up-regulation of this ability at 4 h after the induction of final maturation of DC.

Mature DC show a decreased production of IL-12, but not of IL-6, in response to CD40 ligation and IFN-γ

To test whether the reduced ability of mature DC to produce IL-12 is not due to impaired CD40 signaling or to a general decrease in their metabolic activity, we compared the production of IL-12 and another DC cytokine IL-6 11, 29, 30 after the stimulation of mature and immature DC with a combination of sCD40L plus IFN-γ or with CD40L-transfected J558 cells (J558-CD40L). As reported previously, stimulation of immature DC with sCD40L requires the presence of IFN-γ 9, 10, 13 , while J558-CD40L can induce IL-12 p70 in the absence of IFN-γ 7 , which may result from the very high levels of their CD40L expression.

Consistent with the previous set of experiments, CD40-mediated stimulation of mature DC resulted in reproducibly lower production of IL-12 p70 (Fig. 3⇓, A and B). This was especially evident after the stimulation with CD40L plus IFN-γ, but, interestingly, it was much less pronounced after the IFN-γ-independent stimulation with J558-CD40L. These functional changes appear to be independent from the mode of induction of final DC maturation, since similar observations were made using either DC matured in response to IL-1β and TNF-α or DC matured in response to LPS (Fig. 3⇓B).

FIGURE 3.
FIGURE 3.

Mature DC are resistant to exogenous stimuli, while they respond to CD40 triggering with selectively diminished IL-12 production without a decrease in IL-6 production. Immature DC and DC matured for 48 h in response to IL-1β (5 × 102 U/ml) and TNF-α (5 × 103 U/ml; eight donors tested) or, when specified, in response to LPS (100 ng/ml; three donors tested) were stimulated with the indicated factors or their combinations, and the levels of IL-12 p70 and IL-6 were analyzed in 24-h supernatants. A, IL-12 p70 production by immature DC and mature DC. To avoid a donor-to-donor variation, the results (mean ± SEM) obtained with eight different donors are expressed as a percentage of IL-12 p70 production by immature DC (the black bar (100%) represents the production of IL-12 by immature DC after each mode of stimulation). B, IL-12 p70 production by immature DC and mature DC. Data are shown as the mean (±SD) of triplicate cultures from a representative donor. C, IL-6 production by immature and mature DC. The data are shown as the mean (±SEM) of the results obtained with eight different donors, expressed as a percentage of IL-6 production by immature DC after each mode of stimulation. D, IL-6 production by immature and mature DC. Data shown are the mean (±SD) of triplicate cultures from a representative donor.

In contrast to their reduced IL-12 production, mature DC produced similar or even elevated levels of IL-6 in response to either manner of CD40-mediated stimulation (Fig. 3⇑, C and D), demonstrating that their CD40-mediated signaling pathway is undisturbed.

Mature DC are resistant to the bacterial cytokine inducers, LPS and SAC

Several previous studies demonstrated the induction of IL-12 p70 production in immature DC by bacterial products, LPS and SAC 9, 10, 11 . Both these factors are known to induce low level IL-12 p70 production in the absence of IFN-γ, and their combination with IFN-γ results in high IL-12 p70 production 9, 10, 13 . In contrast to IL-12, the production of IL-6 is relatively IFN-γ independent 11, 27, 31 .

In accord with the previous reports 9, 10, 11 , SAC and LPS alone induced low, but clearly detectable, production of IL-12 p70 in immature DC (Fig. 3⇑B). Their effectiveness was strongly up-regulated in the presence of IFN-γ. The ability to produce IL-12 in response to SAC or LPS, alone or in combination with IFN-γ, was abolished in fully mature DC. Surprisingly, although mature DC were good producers of IL-6 (Fig. 3⇑, C and D; as well as of IL-8; data from three donors; not shown) after CD40 triggering, they did not produce these cytokines after exposure to SAC or LPS, suggesting a general loss of responsiveness of mature DC to these bacterial stimuli.

Mature DC show reduced responsiveness to IFN-γ and express reduced levels of IFN-γR

The differential pattern of regulation of the ability of maturing DC to produce IL-12 p70 and IL-6 suggested a modulation of IFN-γ responsiveness in the course of final DC maturation. Therefore, taking advantage of the ability of CD40L-transfected J558 cells to induce substantial amounts of IL-12 p70 in either DC type in the absence of IFN-γ (Fig. 3⇑, A and B), we tested whether the production of IL-12, in response to this mode of stimulation, could be enhanced by IFN-γ to a similar extent in mature and immature DC.

As shown in Fig. 4⇓A, at the concentration of 10 U/ml, IFN-γ strongly up-regulated IL-12 p70 production in immature DC. This IFN-γ effect was dose dependent up to 1000 U/ml. In contrast, although mature DC did respond to IFN-γ, the effect of this cytokine was much less pronounced. In view of the previously reported requirement for IFN-γ in the induction of the p35 subunit of IL-12 and the production of biologically active IL-12 p70 heterodimer 13, 27, 32 , these observations suggest that the decreased production of IL-12 by mature DC interacting with Th results mainly from their reduced ability to receive IFN-γ-mediated signals. To gain insight into the mechanism of the decreased responsiveness to IFN-γ, we analyzed the expression of surface IFN-γR (α-chain IFN-γR; CD119) on DC at different stages of maturation. Consistent with their decreased responsiveness to IFN-γ, maturing DC displayed progressively reduced levels of surface expression of CD119 (Fig. 4⇓B).

FIGURE 4.
FIGURE 4.

Mature DC show reduced responsiveness to IFN-γ and reduced expression of IFN-γR (CD119). A, Production of IL-12 p70 by immature DC and by DC matured for 48 h in response to IL-1β (5 × 102 U/ml) and TNF-α (5 × 103 U/ml) after the stimulation with CD40L-transfected J558 cells in the presence of increasing doses of IFN-γ. Data are shown as the mean (±SD) of triplicate stimulation cultures from a representative donor. Similar data were obtained in three additional donors. B, The levels of expression of IFN-γR (CD119) on immature DC and DC exposed to IL-1β (5 × 102 U/ml) and TNF-α (5 × 103 U/ml) for 24 or 48 h before harvesting the cells on day 8. The levels of nonspecific fluorescence, obtained with isotype-matched (IgG2a), irrelevant Ab are shown in the inset. The bold line represents the binding of nonspecific Ab to mature DC and was used to demonstrate the residual expression of CD119 on fully mature DC. Data are from a representative donor of four.

Discussion

The present results show that DC undergoing final maturation become unresponsive to stimulation with exogenous bacterial products, such as LPS and SAC, while they maintain responsiveness to CD40-mediated stimulation, as demonstrated by the efficient induction of IL-6. The process of final DC maturation is associated with the initial elevation of the ability of DC to produce the bioactive IL-12 p70 heterodimer during the interaction with CD4+ Th cells followed by a gradual decrease in IL-12-producing ability at later stages of DC maturation. This is associated with reduced expression of IFN-γR on mature DC and their impaired responsiveness to IFN-γ. In contrast to the reduced IL-12 production, the ability of mature DC to produce IL-6 in response to CD40 triggering is preserved or even enhanced. This indicates that in contrast to the lost susceptibility of mature DC to exogenous cytokine inducers, such as bacterial LPS or SAC, mature DC preserve an intact CD40-mediated signaling pathway involved in the communication within the immune system. Taking into account the previous observations that IFN-γ is an important cofactor in the CD40-induced high level IL-12 p70 production 9, 13 , the current data suggest that the selectively reduced ability of maturing DC to produce bioactive IL-12 results from their reduced responsiveness to IFN-γ. The preserved ability of mature DC to produce IL-6, a factor known to costimulate the proliferation of naive Th cells 33 , may contribute to their high efficiency as the initiators of primary responses. This issue is currently being studied.

The issue of a changing ability to produce IL-12 in DC at different stages of development and maturation has been addressed previously. A study in a murine model demonstrated that mature DC produce higher levels of IL-12 than their early proliferating precursors 34 . However, in that study an intermediate population of nonproliferating immature DC was not available for comparison. This may explain the different results from the current report, which compares mature DC with nonproliferating immature DC, functionally corresponding to tissue-type DC 15 .

The reduced ability of mature DC to produce IL-12 and their impaired responsiveness to bacterial stimuli and to IFN-γ are consistent with different functions of DC, performed at different stages of their maturation. At the spot of infection, immature DC are likely to be activated by the pathogen itself, which requires their responsiveness to bacterial products. Recently, it was shown that LPS or Toxoplasma gondii extract induces migration of DC from nonlymphoid tissues and their in vivo production of the p40 subunit of IL-12 12 . Such a direct induction of IL-12 by pathogens will be especially important for the activation of effector mechanisms in the initial phase of immune response in infected tissues, when only few, if any, Ag-specific memory/effector T cells are available to induce the production of IL-12. Since such an early local response is usually induced by a single pathogen, the fact that pathogen-induced or Th cell-induced IL-12 is secreted in high amounts and will also reach all the effector cells in the vicinity does not carry with it the risk of dysregulating other responses to unrelated Ags. On the contrary, this may be beneficial, since high levels of IL-12 may be required for the optimal activation of other than Th types of responding cells, such as class I-restricted CTLs or NK cells, which can recognize infected or transformed cells but not necessarily be able to specifically interact with DC themselves.

This is in sharp contrast to the situation in the lymph nodes. In a recent study Sangster et al. showed that the responses characterized by different Th-dependent isotypes (IgG2a and IgA dominated) to different viruses do not affect each other, even when they are induced simultaneously in the same lymph nodes 35 . An independent regulation of the simultaneously induced responses with different characters against different Ags in a single lymph node requires their functional isolation. This indicates that DC-derived signals should only affect these T cells, which can directly interact with Ag-loaded DC. The risk of affecting other Th cells that have different Ag specificities can be reduced by reducing the amounts of IL-12 released by mature DC.

On the other hand, the resistance of mature DC to IL-12-modulating factors may help the cells to avoid influences from the responses to unrelated Ags occurring in the same lymph node. Previously, we have reported that the levels of IL-12 produced by DC during the presentation of Ag to Th cells are strongly affected by the previous experience of mature DC, i.e., by the conditions in which they develop 10 and are induced to mature 36 . While DC matured under the influence of IL-1β and TNF-α produce intermediate levels of IL-12 and induce the development of Th0/Th1-like cytokine profiles in naive Th cells, final DC maturation in the additional presence of a common inflammatory mediator, PGE2 37 , leads to the development of mature CD1a+CD83+ DC that show a reduced ability to produce IL-12 and induce Th2-biased differentiation of naive Th cells 36 . The ability of DC to produce IL-12 is affected to a similar extent by another inflammatory mediator, IL-10. However, IL-10, in contrast to PGE2, inhibits the process of final DC maturation 36 , which may lead to the development of a suppressive DC type 38, 39 . Despite their strong IL-12 inhibitory activity on immature and maturing DC, neither PGE2 nor IL-10 can inhibit IL-12 production in DC that have completed final maturation 36 . Together with the current data, these observations suggest a general resistance of mature DC to IL-12-modulating factors. These maturation-associated changes in susceptibility to modulatory factors may help DC to acquire the signals affecting the initial polarization of primary immune response (signal 3), to the relevant site of the infected tissue and to avoid further modification of this message within the lymph nodes. This aim may be additionally facilitated by the resistance of mature DC to soluble pathogen-derived IL-12 inducers, such as LPS, which can reach all the DC within the lymph node regardless of their source of origin, and the antigenic message they carry. These maturation-associated changes in the susceptibility of DC to IL-12-modulating factors resemble the changing ability of DC to take up Ag, which is up-regulated at the onset of final DC maturation and is subsequently shut down 23, 28 .

While the interaction of naive Th cells with DC results in only limited amounts of IL-12 production 6, 9, 13, 40 , the functional significance of such low doses of IL-12 was demonstrated in the mouse by showing that neutralization of IL-12 activity in the cultures of TCR transgenic naive Th cells with spleen-isolated DC results in the reduced IFN-γ production 6 . Although we could not quantitate the amounts of IL-12 induced in maturing DC by naive Th cells due to their low levels (2–10% the amounts induced by bulk CD4+ Th cells) 9, 13 , the current study suggests that the differences in the kinetics of DC migration may result in different IL-12 levels in the lymph nodes and may affect the character of the Ag-specific response subsequently induced.

The observation of strong maturation stage-dependent differences in the IL-12-producing capacity of DC generated in FCS-supplemented cultures raises the question of whether similar maturation-related differences are also present in DC obtained in the presence of autologous serum or in serum-free conditions that are used in immunotherapeutic protocols. The demonstration of similar differences may help in designing optimal strategies for the therapeutic induction of Th1-type responses with the use of DC.

Footnotes

  • ↵1 This work was supported in part by grants from The Netherlands Asthma Foundation (no. NAF 94.60 to C.M.U.H.) and the Royal Netherlands Academy of Arts and Sciences (to E.A.W.).

  • ↵2 Address correspondence and reprint requests to Dr. Paweł Kaliński, Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. E-mail address: p.kalinski{at}amc.uva.nl

  • ↵3 Abbreviations used in this paper: DC, dendritic cells; SAC, Staphylococcus aureus Cowan I strain; sCD40L, soluble CD40 ligand trimer; SEB, staphylococcal enterotoxin B; J558-CD40L, CD40 ligand-transfected J558 cells.

  • Received August 11, 1998.
  • Accepted December 11, 1998.

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The Journal of Immunology: 162 (6)
The Journal of Immunology
Vol. 162, Issue 6
15 Mar 1999
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