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The Journal of Immunology, 2005, 175: 5866-5874.
Copyright © 2005 by The American Association of Immunologists

IL-12 Breaks Dinitrothiocyanobenzene (DNTB)-Mediated Tolerance and Converts the Tolerogen DNTB into an Immunogen1

Helge Riemann2,*, Karin Loser2,{dagger}, Stefan Beissert{dagger}, Mayumi Fujita*, Agatha Schwarz{ddagger}, Thomas Schwarz{ddagger} and Stephan Grabbe3,{dagger},§

* Department of Dermatology, UCHSC at Fitzsimons, Aurora, CO 80010; {dagger} Ludwig Boltzmann Institute for Cell Biology and Immunobiology of the Skin, Department of Dermatology, University of Münster, Germany; {ddagger} Department of Dermatology, University of Kiel, Germany; § Department of Dermatology, University of Essen, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Epicutaneous application of dinitrothiocyanobenzene (DNTB) induces tolerance against its related compound dinitrofluorobenzene (DNFB), because DNTB-pretreated mice cannot be sensitized against the potent hapten DNFB. This tolerance is hapten-specific and transferable. In this study, we demonstrate that IL-12 can break DNTB-mediated tolerance. Furthermore, naive mice treated with IL-12 before DNTB application responded to DNFB challenge with a pronounced ear swelling response without previous sensitization to DNFB, showing that IL-12 can convert the tolerogen DNTB into an immunogen. No differences in numbers or regulatory activity were observed between CD4+CD25+ regulatory T cells isolated from mice treated with DNFB, DNTB, or IL-12 followed by DNTB. However, the number of CD207+ Langerhans cells in regional lymph nodes of DNTB-treated mice was significantly lower than in animals treated with DNFB or IL-12 plus DNTB. Additionally, CD11c+ dendritic cells (DC) isolated from regional lymph nodes of DNTB-treated mice had a significantly lower ability to stimulate T cell proliferation and produced reduced amounts of inflammatory cytokines. Application of both DNFB and DNTB induced apoptotic cell death of DC in the epidermis and the regional lymph nodes. However, the number of apoptotic DC in regional lymph nodes was significantly higher in DNTB-treated animals compared with mice treated with DNFB or IL-12 plus DNTB. Therefore, we conclude that DNTB-mediated tolerance is secondary to inefficient Ag presentation as a result of apoptotic cell death of DC and that IL-12 converts the tolerogen DNTB into an immunogen by preventing DNTB-induced apoptosis of DC.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The heterodimeric cytokine IL-12 is composed of covalently linked 35- and 40-kDa subunits (1). Besides stimulatory effects on both, NK cells and CD8+ cytotoxic T lymphocytes, IL-12 has costimulatory and regulatory effects on CD4+ Th cells and favors the differentiation of Th1 cells (2, 3, 4). Although contact hypersensitivity (CHS)4 (4) is unaltered in IL-12-deficient mice, exogenous IL-12 can enhance the CHS response by amplifying the development of hapten-specific CD8+ T cells and by inhibiting the induction of Ag-specific CD4+ regulatory cells (5, 6). Furthermore, neutralization of IL-12 by injection of blocking Abs inhibits the induction of CHS in wild-type mice (7) and even induces hapten-specific tolerance (8). In contrast, administration of exogenous IL-12 can overcome UV-induced immune tolerance (9, 10). Epicutaneous application of haptens onto mice that have been exposed to UV radiation does not result in sensitization but induces tolerance (11). This tolerance is hapten-specific and can be adoptively transferred by injecting bulk T cells obtained from animals tolerized in this manner (12). We and others have reported that IL-12 is able to prevent UV-induced immunosuppression when injected i.p. into mice between UV exposure and hapten sensitization (7, 9, 10). In addition, tolerance did not develop in these animals (9). Even more importantly, mice tolerized by hapten application on UV-irradiated skin can be fully sensitized with the same hapten when IL-12 is injected before re-sensitization, demonstrating that IL-12 cannot only prevent but also break established UV-mediated tolerance (9, 10). Although IL-12 seems to act on regulatory T cells (13), the detailed mechanism by which IL-12 breaks established tolerance remains to be determined.

Epicutaneous application of dinitrothiocyanobenzene (DNTB) has been reported to induce tolerance against its related compound dinitrofluorobenzene (DNFB) because DNTB-pretreated mice cannot be sensitized against the potent hapten DNFB (14, 15). Specific immune tolerance is induced by topical application of DNTB 7 days before sensitization to DNFB. Tolerance can be abrogated by cyclophosphamide, indicating that DNTB-induced suppressor/regulatory T cells may be involved, and adoptive transfer studies showed that DNTB induces hapten-specific regulatory T cells (15). In addition, lymph node cells from DNTB-treated mice are defective in proliferation and produce significantly lower amounts of IL-1, IL-2, and IL-4 compared with DNFB-treated animals, suggesting that DNTB-induced tolerance results in deficient Th1 as well as deficient Th2 responses to hapten re-exposure (16).

Here, we show that injection of IL-12 into DNTB-treated mice before sensitization with DNFB restores CHS responses and that injection of IL-12 before DNTB treatment enables subsequent sensitization against DNFB. In addition, when naive mice are injected with IL-12 followed by DNTB treatment, primary contact with DNFB results in a specific ear swelling response, indicating that IL-12 converts the tolerogen DNTB into an immunogen. Furthermore, we show that treatment with DNTB leads to increased numbers of apoptotic dendritic cells (DC) both in epidermis and regional lymph nodes and that this effect can be mitigated by IL-12.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

BALB/c mice between 8 and 12 wk of age were purchased from Charles River Laboratories. Animals were housed under specific pathogen-free conditions and treated according to institutional guidelines.

Contact hypersensitivity

Mice were sensitized by painting 25 µl of 2,4-dinitrofluorobenzene solution (Sigma-Aldrich; 0.5% in acetone/olive oil 4:1) on the shaved back on day 0. On day 5, the left ear was challenged by applying 20 µl of 0.3% DNFB, and the right ear was treated with acetone/olive oil alone. Ear swelling was measured in a blinded fashion with a spring-loaded micrometer (Mitutoyo) 24 h after challenge. Contact hypersensitivity was determined as the amount of swelling of the hapten-challenged ear compared with the thickness of the vehicle-treated ear and was expressed in centimeters x 10–3 (mean ± SD). Mice that were ear challenged without previous sensitization served as negative controls. Sensitization against oxazolone was performed by applying 100 µl of a 2% oxazolone solution on day 0, and ears were challenged using 20 µl of 0.5% oxazolone on day 5. Each group consisted of at least 5 mice, experiments were performed at least three times.

DNTB treatment

Mice were painted with 100 µl of DNTB (Lancaster Synthesis) solution (1% in acetone/olive oil 4:1) on the shaved abdomen. On day 7, mice were sensitized with 0.5% DNFB, and DNFB challenge was performed 5 days later.

Injection of rIL-12

For in vivo injection, 250 ng of recombinant murine IL-12 (Genetics Institute) diluted in sterile endotoxin-free saline (PAA) were used. The cytokine was injected i.p. 24 and 3 h before treatment with DNTB or DNFB. Control mice were treated i.p. with equal volumes of saline, which had no effect on the outcome of the sensitization procedure or on the suppressive effect of DNTB.

Adoptive transfer of immune response

Donor mice were treated with DNTB, draining lymph nodes and spleens were removed 7 days later, and single cell suspensions were prepared. Cell number was adjusted to 5 x 108 cells/ml, and 200 µl (1 x 108 cells) were injected i.v. into naive recipients. Recipients were sensitized 24 h after injection by painting 0.5% DNFB on the shaved back. Recipient mice were challenged on the left ear 5 days later, and ear swelling was evaluated.

Immunofluorescence and in situ TUNEL analysis

Immunofluorescence stainings were performed on cryostat sections of mouse ears according to standard methods (17). Epidermal Langerhans cells (LC) were identified using anti-CD207 (clone 929F3, diluted 1/50 in PBS; kindly provided by Dr. S. Saeland, Schering-Plough, Dardilly, France) and OregonGreen-coupled secondary Ab (Molecular Probes). Apoptosis of Langerhans cells was detected using TUNEL staining and the Texas Red in situ cell death detection kit (Roche) according to the manufacturer’s instructions. Slides were examined using an Olympus BX61 microscope and the MetaMorph software (Visitron Systems).

Cell preparation and flow cytometry

Single cell suspensions of regional lymph nodes were prepared as described before (18). DC were isolated from lymph node cell suspensions by MACS (Miltenyi Biotec) using anti-CD11c-coupled microbeads according to the manufacturer’s instructions. Expression of cell surface and intracellular markers was analyzed by standard four-color flow cytometry with a FACSCalibur flow cytometer and CellQuest software (BD Pharmingen). Cells were stained for FACS analysis in PBS containing 1% FCS with the following mAbs: anti-neuropilin-1 (clone H-286; Santa Cruz Biotechnology), FITC-conjugated anti-CD103 (clone 2E7), anti-CD80 (clone 16-10A1), goat anti-rabbit Ig (Dianova), PE-conjugated anti-CD25 (clone PC61), anti-CTLA-4 (clone UC10-4F10-11), anti-CD86 (clone GL1), peridinin chlorophyll protein-conjugated anti-CD3 (clone 145-2C11), anti-CD4 (clone RM4-5), allophycocyanin-conjugated anti-CD4 (clone RM4-5), anti-CD25 (clone PC61), anti-CD11c (clone HL3), Cy5-conjugated anti-CD207 (Langerin, clone 929F3; kindly provided by Dr. S. Saeland, Schering-Plough, Dardilly, France). CD207 staining was performed after cell permeabilization. Isotype-matched control Abs were included in each staining. All Abs as well as isotype-matched controls were obtained from BD Pharmingen unless otherwise noted. Apoptotic and necrotic cells were identified using the Annexin V apoptosis detection kit (BD Pharmingen) according to the manufacturer’s instructions.

Proliferation assays

Naive CD4+CD25– and CD4+CD25+ cells were sorted by MACS as described (18). Proliferation assays were performed in triplicate, and T cell proliferation was assessed by [3H]thymidine incorporation. CD4+CD25– and CD4+CD25+ T cells (1 x 106/ml alone or mixed at indicated ratios) were cultured in 96-well round-bottom plates, and cells were stimulated with 1 µg/ml anti-CD3 (clone 145-2C11) and 1 µg/ml anti-CD28 (clone 37.51). Proliferation assays were done for 96 h in a final volume of 200 µl; 1 µCi/well [3H]thymidine was added for the last 12 h of the experiment, and thymidine incorporation was measured by liquid scintillation counting.

Mixed lymphocyte reactions

Proliferation of CD4+CD25– T cells isolated from lymph nodes of naive BALB/c mice was assessed by [3H]thymidine incorporation. Cells (1 x 106/ml) were cultured in triplicate in 96-well round-bottom plates in the presence of 1 x 104 DC isolated from lymph nodes of naive BALB/c mice or mice treated with DNFB, DNTB, or IL-12 followed by DNTB. Mixed lymphocyte reactions were done for 72 h in a final volume of 200 µl. T cell proliferation was evaluated by adding 1 µCi/well [3H]thymidine for the last 12 h of the experiment, and thymidine incorporation was measured by liquid scintillation counting.

Cytometric bead array (CBA)

The cytokine activity in culture supernatants of CD11c+ cells from skin draining lymph nodes of DNFB-sensitized, DNTB-sensitized, and IL-12-treated, and DNTB-sensitized mice was assayed by CBA (BD Pharmingen) according to the manufacturer’s instructions. Cells (2 x 106/ml) were incubated for 3 days without any further stimulation at 37°C and 5% CO2 in 96-well round-bottom plates (BD Falcon) in a volume of 200 µl of RPMI 1640 containing 10% FCS. Supernatants were collected and subjected to cytokine quantification using CBA kits.

Statistical analysis

Data were analyzed by Student’s t test and differences were considered significant at p < 0.05. Each experiment was performed at least three times.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Induction of hapten-specific tolerance against DNFB by epicutaneous application of DNTB

DNTB (1%) was painted on the shaved abdomen of BALB/c mice. A sensitizing dose of DNFB was applied 7 days later epicutaneously on the shaved back and challenge with DNFB was performed 5 days thereafter. Although animals sensitized with DNFB showed a significant ear swelling response upon challenge, animals pretreated with DNTB could not be sensitized with DNFB, as indicated by the absent ear swelling response. Likewise, animals challenged with DNFB without prior sensitization showed no ear swelling response (Fig. 1A). The tolerizing effect of DNTB appeared to be dose dependent, because both lower and higher concentrations of DNTB failed to induce tolerance (data not shown).



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  		FIGURE 1. A, DNTB induces tolerance against DNFB. Mice were tolerized by epicutaneous application of 100 µl of 1% DNTB on the abdomen, followed by sensitization against DNFB (25 µl of 0.5% painted on the back) 7 days later. Ears were challenged with DNFB (20 µl 0.3%) 5 days after sensitization with DNFB. B, DNTB-mediated tolerance is transferable. Donor mice were painted with DNTB or left untreated, and single cell suspensions were prepared from draining lymph nodes and spleens 7 days later. Cells (1 x 108) were injected i.v. into naive recipients followed by DNFB sensitization 1 day after transfer. Recipient mice were ear challenged with DNFB 5 days after sensitization. C, DNTB-induced tolerance is hapten-specific; IL-12 followed by DNTB does not allow for an unspecifc challenge. Mice were treated with DNTB, and a sensitizing dose of oxazolone was applied 7 days later. One group of mice was injected with IL-12 followed by DNTB application but not sensitized against oxazolone. All mice were challenged with oxazolone 5 days after sensitization with oxazolone. Ear swelling response is expressed as the difference (centimeters x 10–3, mean ± SD) between the thickness of the challenged ear and the vehicle-treated ear. *, p < 0.001 versus positive control; n.s., nonsignificant.

 
DNTB-mediated tolerance against DNFB is transferable

Next we analyzed whether DNTB-mediated tolerance was transferable. Therefore, 1% DNTB was painted on the shaved abdomen of BALB/c mice. Animals were sacrificed 7 days later, and regional lymph nodes and spleens were removed. Single cell suspensions were prepared as described and injected i.v. into naive recipient BALB/c. Recipients were sensitized with DNFB 1 day after transfer, and challenge was performed with DNFB 5 days after sensitization. Whereas control animals that had received cells from untreated donors showed a significant CHS response upon challenge with DNFB, no ear swelling response was observed in recipients of cells from DNTB-treated donors, indicating that these animals could not be sensitized against DNFB (Fig. 1B).

DNTB-mediated tolerance is hapten-specific

To exclude that DNTB induces tolerance against an unrelated hapten, mice were treated by epicutaneous application of DNTB onto the shaved abdomen. Sensitization with oxazolone was performed on the shaved back 7 days later, and ear challenge with oxazolone was performed 5 days later. Upon challenge, mice pretreated with DNTB and sensitized with oxazolone had ear swelling reponses similar to positive control animals sensitized with oxazolone alone, showing that DNTB-mediated tolerance is specific for DNFB (Fig. 1C).

IL-12 prevents DNTB-mediated tolerance

To evaluate whether IL-12 can prevent DNTB-mediated tolerance, IL-12 was injected 24 and 3 h before DNTB application. Seven days later, sensitization with DNFB was performed, and ears were challenged with DNFB 5 days thereafter. CHS response in DNTB-pretreated mice was significantly suppressed compared with positive control animals. In contrast, when IL-12 was injected 24 and 3 h before DNTB application, the tolerizing effect of DNTB was not observed because animals responded with a marked ear swelling response to sensitization with DNFB (Fig. 2A).



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  		FIGURE 2. A, IL-12 prevents DNTB-mediated tolerance toward DNFB. Mice were treated with DNTB on the shaved abdomen and sensitized against DNFB on the back 7 days later. All groups were ear challenged with DNFB 5 days thereafter. One group received IL-12 injected i.p. 24 and 3 h before DNTB application. B, IL-12 breaks DNTB-mediated tolerance. Mice were treated with DNTB on the shaved abdomen and sensitized against DNFB on the back 7 days later. All groups were ear challenged with DNFB 5 days later. One group received IL-12 injected i.p. 24 and 3 h before DNFB sensitization. Ear swelling response is expressed as the difference (centimeters x 10–3, mean ± SD) between the thickness of the challenged ear and the vehicle-treated ear. *, p < 0.01; **, p < 0.05, n.s., nonsignificant.

 
IL-12 breaks DNTB-mediated tolerance

To study whether IL-12 can break established DNTB-mediated tolerance, BALB/c mice were treated by epicutaneous application of DNTB. Animals were sensitized with 0.5% DNFB 7 days after application of DNTB, and challenge with DNFB was performed 5 days later. One group of BALB/c mice received i.p. injections of IL-12, 24 and 3 h before sensitization with DNFB. Control animals sensitized with DNFB showed a significant ear swelling response upon challenge, whereas animals pretreated with DNTB could not be sensitized against DNFB. However, when IL-12 was injected into DNTB-pretreated mice before application of DNFB, a significant CHS response was observed (Fig. 2B).

IL-12 converts the tolerogen DNTB into an immunogen

To study whether injection of IL-12 before application of DNTB enables a CHS response to DNFB challenge without prior DNFB sensitization, mice received IL-12 i.p. 24 and 3 h before DNTB application. Challenge with DNFB was performed 7 days later. Although animals treated with DNTB alone showed no ear swelling response upon challenge with DNFB, mice treated with the combination of IL-12 injection followed by DNTB application showed a significant CHS response upon challenge with DNFB (Fig. 3). This ear swelling response appeared hapten-specific, because the combination of IL-12 injection followed by DNTB application did not allow for a CHS response upon challenge with the unrelated hapten oxazolone (Fig. 1C).



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  		FIGURE 3. IL-12 converts the tolerogen DNTB into an immunogen. Mice were treated with DNTB, and ear challenge with DNFB was performed 5 days later. One group received IL-12, 24 and 3 h before DNTB application. As a positive control, mice were sensitized against DNFB. All groups were ear challenged with DNFB. Ear swelling response is expressed as the difference (centimeters x 10–3, mean ± SD) between the thickness of the challenged ear and the vehicle-treated ear. *, p < 0.005.

 
Concomitant application of DNTB and an irrelevant hapten does not reconstitute DNFB sensitization

Because DNFB sensitizes whereas DNTB induces tolerance against the same hapten, we reasoned that DNTB might fail to generate the immunostimulatory signal(s) necessary for induction of CHS. To substitute for these signal(s), animals were sensitized by epicutaneous application of DNTB in conjunction with the non-related hapten oxazolone in the conventional sensitizing dose onto the same area. Ears were challenged with DNFB 5 days later. Upon challenge with DNFB, no CHS response was observed in animals sensitized with DNTB alone or in combination with oxazolone (Fig. 4, lines 3 and 4). Thus, unlike IL-12 treatment, concomitant application of an immunologically unrelated hapten could not convert the tolerogen DNTB into an immunogen, suggesting that the tolerogenic effects of DNTB are not due to the lack of an immunostimulatory effect.



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  		FIGURE 4. The combination of DNTB and oxazolone fails to induce CHS against DNFB but induces ear challenge in DNFB-sensitized mice. Mice were sensitized by epicutaneous application of DNFB, or by application of DNTB in combination with oxazolone (2%). Ear challenge was performed 5 days later by applying either DNFB (0.3%), DNTB (0.5, 1, or 2%), or DNTB (0.5, 1, or 2%) in combination with oxazolone (0.5%). Ear swelling response was measured 24 h later and is expressed as the difference (centimeters x 10–3, mean ± SD) between the thickness of the challenged ear and the vehicle-treated ear. *, p < 0.005 versus line 1, n.s., nonsignificant versus line 1; #, nonsignificant versus line 5.

 
DNTB fails to elicit a CHS response in DNFB-sensitized animals, but CHS reactivity is restored by simultaneous application of an irrelevant hapten

It has been shown earlier that an inherent feature of contact allergens is their capacity to induce a nonspecific inflammatory response necessary for elicitation of CHS (19). Thus, to investigate whether DNTB, in addition to its tolerogenic effects during hapten sensitization, might also have an altered capacity to elicit CHS responses in DNFB-sensitized mice, animals were DNFB sensitized and subsequently challenged with DNTB. Indeed, application of DNTB also failed to elicit a CHS response in DNFB-sensitized mice, suggesting that it not only lacks sensitizing capacity but also signal(s) necessary for elicitation of a CHS response (Fig. 4, lines 8–10).

We were interested whether concomitant application of a non-related hapten could substitute for these signals. Thus, DNFB-sensitized mice were ear challenged with either DNTB alone or with DNTB in combination with a conventional challenging dose of the irrelevant hapten oxazolone. Although DNFB-sensitized animals challenged with DNTB alone failed to develop a CHS response, a significant ear swelling could be observed in animals simultaneously challenged with DNTB plus oxazolone. Moreover, the ear swelling response was clearly dependent on the dose of DNTB applied (Fig. 4, lines 11–13).

IL-12 does not affect the induction of CD25+CD4+ regulatory T cells

We next investigated the mechanism by which DNTB might exert its tolerogenic effects during primary hapten exposure. As shown previously, CD4+CD25+ regulatory T cells can control CHS responses (20). To study the role of CD4+CD25+ regulatory T cells in DNTB-induced tolerance, mice were left untreated or were treated with DNFB, DNTB, or IL-12 plus DNTB. Lymph nodes and spleens were removed 7 days later, and single cell suspensions were prepared. CD4+CD25– and CD4+CD25+ cells were separated. CD4+CD25– T cells from naive mice were stimulated with anti-CD3 and anti-CD28 Ab in the absence or presence of CD4+CD25+ T cells from animals treated with DNFB, DNTB, or IL-12 plus DNTB. Proliferation was measured by the amount of [3H]thymidine incorporation (Fig. 5A). Whereas CD4+CD25– cells from naive mice alone showed vigorous proliferation, proliferation was significantly inhibited by addition of CD4+CD25+ cells from mice treated with DNFB, DNTB, or IL-12 plus DNTB. CD4+CD25+ cells from different groups exhibited similar inhibitory capacities. Flow cytometric analyses of CD4+ T cells isolated from lymph nodes of naive, DNFB-sensitized, DNTB-treated, and IL-12 plus DNTB-treated mice showed no significant differences in the numbers of CD4+CD25+ T cells (Fig. 5B). Moreover, the expression of surface markers typically associated with regulatory T cells like CTLA-4, CD103, or neuropilin-1 was not changed (data not shown). These findings suggest that the generation of CD4+CD25+ cells is not the primary mechanism of tolerance induction by DNTB and that generation of CD4+CD25+ T cells is not affected by IL-12.



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  		FIGURE 5. A, CD4+CD25+ T cells are suppressive in vitro. CD4+CD25– and CD4+CD25+ T cells were separated by MACS, and proliferation assays were perfomed by stimulating CD4+CD25– T cells from naive BALB/c mice (1 x 105) with anti-CD3 and anti-CD28 Ab in the absence or presence of 7.5 x 104 CD4+CD25+ T cells from DNFB-sensitized, DNTB-sensitized, or IL-12-treated and DNTB-sensitized mice. *, p < 0.05 versus CD4+CD25–. B, Flow cytometric analyses of CD4+ lymph node T cells from naive BALB/c, DNFB-sensitized, DNTB-sensitized, and IL-12-treated plus DNTB-sensitized mice.

 
DC from DNTB-treated animals fail to prime naive T cells

Mice were treated with DNFB, DNTB, or IL-12 plus DNTB, and DC were isolated from regional lymph nodes. DC were coincubated with CD4+CD25– T cells from naive BALB/c mice for 3 days, and T cell proliferation was measured. Whereas T cells incubated with DC from DNFB-treated animals proliferated vigorously, T cells incubated with DC from DNTB-treated mice showed only a weak proliferative response, suggesting that DNTB fails to activate DC for efficient priming of naive T cells. This effect could partially be reversed by treatment with IL-12 (Fig. 6A).



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  		FIGURE 6. Functional and flow cytometric analysis of regional lymph node cells. A, DC (5 x 104) were isolated from skin draining lymph nodes of DNFB-sensitized, DNTB-sensitized, or IL-12-treated plus DNTB-sensitized mice. DC were incubated for 3 days with 1 x 105 CD4+ T cells from naive BALB/c mice, and proliferation assays were performed. *, p < 0.05 versus DNTB treatment. B, Lymph node cells from mice treated with DNFB, DNTB, and IL-12 plus DNTB were stained for CD11c or Langerin (CD207) and analyzed by flow cytometry. CD207 staining was performed after cell permeabilization. Percentage of positive cells in total lymph nodes from five independent experiments are shown. *, p < 0.05 versus DNFB and IL-12 plus DNTB. C, Lymph node cells from DNFB-sensitized, DNTB-sensitized mice, or IL-12 plus DNTB-treated mice were stained for CD80 and CD86 and analyzed by flow cytometry. Cells were gated for CD11c+ and percentage of CD80+CD86+ cells in lymph nodes from five independent experiments are shown. *, p < 0.05 versus DNFB and IL-12 plus DNTB. D, Cytokine production of CD11c+ DC isolated from skin draining lymph nodes of DNFB-sensitized, DNTB-sensitized, and IL-12 plus DNTB-treated mice was measured by CBA. Data are shown as one of three different experiments with similar results.

 
IL-12 prevents DNTB-induced apoptosis of DC in epidermis and regional lymph nodes

Mice were treated with DNFB, DNTB, or IL-12 plus DNTB, and single cell suspensions were prepared from regional lymph nodes 3 days later. Lymph node cells were stained for CD11c and the Langerhans cell-specific intracellular marker Langerin (CD207) and analyzed by flow cytometry. Although the number of CD11c+ cells was slightly increased, the number of Langerin+ cells was considerably reduced in the skin draining lymph nodes of DNTB-treated compared with DNFB-treated mice (Fig. 6B). Injection of IL-12 into mice before application of DNTB raised the number of Langerin+ lymph node cells to an amount similar to that observed in DNFB-treated animals (Fig. 6B). Interestingly, CD11c+ cells isolated from skin draining lymph nodes of DNTB-sensitized mice were less activated than DC isolated from the lymph nodes of DNFB-sensitized or IL-12 plus DNTB-treated mice as demonstrated by the reduced expression of CD80 and CD86 (Fig. 6C). Moreover, lymph node DC from DNTB-sensitized mice produced reduced amounts of inflammatory cytokines like TNF-{alpha} and IFN-{gamma} compared with CD11c+ cells isolated from skin draining lymph nodes of DNFB-sensitized or IL-12 plus DNTB-treated mice (Fig. 6D).

Staining with propidium iodide and annexin V showed that the number of apoptotic CD11c+ cells was increased in lymph nodes from DNTB-sensitized mice compared with control, DNFB, and IL-12 plus DNTB-treated animals (Fig. 7A). To evaluate whether DNTB induces apoptosis of DC not only in regional lymph nodes but also in the skin, mice were treated with DNFB, DNTB, or IL-12 plus DNTB. One day later, skin samples were taken from the area of DNTB, DNFB, or vehicle application. Biopsies were stained for Langerin and for apoptotic cells using the TUNEL assay. Although application of DNFB slightly increased the number of apoptotic cells compared with vehicle-treated skin, the total number of apoptotic cells and especially of apoptotic Langerhans cells was significantly higher after DNTB treatment. However, injection of IL-12 before DNTB application reduced the number of apoptotic cells to background levels (Fig. 7B). Therefore, induction of apoptosis in Langerin+ CD11c+ DC correlates with tolerance induction in the DNFB/DNTB model, and IL-12 can prevent DNTB-induced apoptosis.



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  		FIGURE 7. Detection of apoptotic CD11c+ and Langerhans cells in skin draining lymph nodes and in the skin of sensitized mice. A, Mice were treated with DNFB, DNTB, or IL-12 plus DNTB. Lymph node cells were prepared 3 days later, stained for annexin V and propidium iodide (PI), and subjected to flow cytometry analysis. Cells were gated for CD11c+. Statistical evaluation of apoptotic lymph node DC from five independent experiments is shown. *, p<0.05. B, Immunofluorescence analysis of (i) BALB/c mice as well as (ii) DNFB sensitized, (iii) DNTB-sensitized, or (iv) IL-12 plus DNTB-treated mice. One day after sensitization cryosections of treated skin areas were stained for CD207 (green) and for apoptotic cells (TUNEL assay, red). Apoptotic Langerhans cells are marked with white arrows. Original magnification, x400. Statistical evaluation of apoptotic Langerhans cells from five independent experiments is shown. In each experiment, numbers of apoptotic LC were determined by two independent investigators in a blinded fashion from 15 randomly selected slide sections. *, p<0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
DNTB has initially been described to induce tolerance to subsequent treatment with DNFB (14). Because later reports could not confirm the tolerizing capacity of DNTB (21, 22), we first determined whether DNTB in our hands acts as a tolerogen. DNTB at a concentration of 1% applied to the abdomen clearly suppressed subsequent sensitization with DNFB, whereas lower and higher concentrations failed to exert this effect (data not shown). Therefore, we speculate that the different properties attributed to DNTB are due to different concentrations used and different sites of application (ear vs abdomen). We were able to further consolidate this tolerance model by confirming previous reports that DNTB-mediated tolerance is transferable and that this is due to the development of hapten-specific regulatory/suppressor cells (15).

Once we had established the model of DNTB-mediated tolerance we evaluated the effects of IL-12 at different time points, i.e., after and before tolerance had developed. IL-12 injected after DNTB treatment and just before DNFB application clearly enabled DNFB sensitization, indicating that IL-12 can break DNTB-mediated tolerance. It is important to mention that IL-12 was injected at a time point when regulatory T cells had already developed as demonstrated by adoptive transfer of suppression. These findings are similar to those observed in UV-mediated tolerance (9, 13). We also found that IL-12 is able to prevent DNTB-mediated tolerance, because animals that received IL-12 before DNTB application could be sensitized to DNFB. Furthermore, although animals treated with DNTB alone did not react with ear swelling to DNFB challenge, mice which received IL-12 before DNTB application showed a significant ear swelling response following DNFB application on the ear. Because in these mice, ear challenge with DNFB represents the primary contact with DNFB in a concentration that does not cause ear swelling by itself as shown by the negative controls, the combination of IL-12 plus DNTB obviously sensitizes the animals against DNFB.

Our data suggest that IL-12 specifically prevents immune tolerance, but does not enhance immunity in a general fashion, because injection of IL-12 before DNTB application induced CHS against DNFB, whereas IL-12 in the amounts used throughout this study did not further enhance the induction of CHS if injected before sensitization with DNFB (data not shown). Although this is similar to results we previously obtained in a different mouse strain (9), IL-12 applied before sensitization did exaggerate CHS responses in other studies, albeit in significantly higher doses (5, 6).

In the model of UV-induced tolerance, the immune suppression seems to be induced by the release of keratinocyte-derived cytokines, especially IL-10 (23). However, the mechanisms by which chemical tolerogens, including DNTB, exert their effects, are poorly understood. Enk and Katz (24) showed that tolerizers in contrast to immunogens do not induce IL-1, MIP-2, IL-10, or class II mRNA. Because we demonstrated that IL-12 can prevent DNTB-mediated tolerance we hypothesized that DNTB might fail to induce IL-12 and thereby induces hapten-specific tolerance. In the human system, induction of IL-12 has been observed upon application of haptens (25). However, we were not able to identify either p35 or p40 transcripts in murine skin after hapten application (data not shown). Because IL-12 can prevent DNTB-mediated tolerance, we studied whether DNTB fails to induce IL-12 in the regional lymph nodes and thereby induces tolerance. Both p35 and p40 transcripts could be detected in regional lymph nodes of both DNFB- and DNTB-treated animals (data not shown). Thus, there is little evidence that a failure to induce IL-12 might account for the differences between DNTB and DNFB.

CD4+CD25+ regulatory T cells provide an important pathway for immune tolerance as shown in different models (20, 26, 27, 28, 29). CD4+CD25+ T cells are classically anergic, unable to proliferate in vitro in response to mitogenic stimulation or stimulation through the TCR, and inhibit proliferation of CD4+CD25– T cells by inhibiting production of IL-2 (30). It was tempting to hypothesize that CD4+CD25+ T cells have a crucial function in DNTB-mediated tolerance and that IL-12 either interferes with their function or impairs their development. However, we were not able to detect any differences in the suppressive activity of CD4+CD25+ regulatory T cells in mice treated with DNFB, DNTB, or IL-12 plus DNTB. Furthermore, these CD4+CD25+ regulatory T cells did not differ in their expression of surface markers like CTLA-4, CD103, or neuropilin-1 (data not shown). It therefore seems unlikely that modulation of development or function of CD4+CD25+ T cells is the main mechanism of IL-12 in reversing DNTB-mediated tolerance.

Instead, however, a major reduction in the relative numbers of CD11c+ Langerin+ DC in regional lymph nodes was observed after application of DNTB when compared with DNFB. Interestingly, injection of IL-12 before DNTB application could raise the number of CD11c+ Langerin+ cells to levels in the range observed after DNFB treatment. Initially, we speculated that the reduced number of CD11c+ Langerin+ cells after DNTB treatment could be due to the inability of DNTB to sufficiently activate Langerhans cells for migration to regional lymph nodes. The hapten then would be presented less efficiently, which might lead to tolerance (11, 31). If DNTB failed to activate Langerhans cells to migrate to the lymph nodes, one would expect that simultaneous application of the unrelated hapten oxazolone would substitute for this deficiency as this potent allergen is obviously able to activate DC for migration and efficient Ag presentation. However, concomitant application of oxazolone and DNTB did not lead to induction of CHS. We then analyzed the possibility that DNTB application induces LC damage or even death, thereby decreasing the number of cells that reached the regional lymph node. Indeed, we found that DNTB consistently induced enhanced apoptotic cell death of DC both in epidermis and regional lymph nodes. This toxic, apoptosis-inducing effect of DNTB on APCs is able to explain the dose dependency we observed for the tolerogenic effects of DNTB. Thus, at low concentrations, the damaging effect of DNTB on DC is absent, but the amount of Ag is too small to induce CHS. At high concentrations, apoptotic death of DC will be so extensive that sufficient numbers of viable DC do not reach the regional lymph nodes and thus fail to prime for tolerance. In our study, administration of IL-12 was able to prevent DNTB-induced cell death in vivo. It has recently been shown that IL-12 has anti-apoptotic properties, because it is able to protect keratinocytes from UV-induced apoptosis by inducing DNA repair (32) and to prevent activation-induced cell death in T cells by down-regulating Fas ligand and inhibiting caspase activation (33, 34, 35). Now, our findings indicate that IL-12 also protects LC against hapten-mediated apoptosis. Likewise, we demonstrated earlier that UV-mediated tolerance is also dependent on apoptosis of DC and can be reversed by IL-12 (36). Thus, similar mechanisms are responsible for both UV-mediated and hapten-induced tolerance.

In addition to its tolerogenic effects during hapten sensitization, DNTB also failed to elicit a CHS response even in mice that had been sensitized to the relevant hapten by using DNFB. This effect is due to a different mechanism and cannot be explained by induction of LC apoptosis. We have previously shown that for the elicitation of hapten-specific CHS responses, two signals are required: 1) the specific Ag, and 2) a nonspecific proinflammatory signal (19). This conclusion was based on the observation that coadministration of an irrelevant hapten with a low dose of the specific hapten in sensitized mice yielded a full CHS response, whereas application of either substances alone, or the combination of haptens applied to nonsensitized animals, failed to elicit a significant reaction. Although the nature of the proinflammatory signal remains to be characterized, there is strong evidence that this signal is specifically provided by haptens because it could not be substituted by application of irritants (19). To determine whether DNTB differs from haptens in its ability to provide this second signal, mice were either treated by epicutaneous application of DNTB alone or in combination with oxazolone in the induction or effector phase. While in the effector phase a vigorous ear swelling response could be observed in DNFB-sensitized animals challenged with the combination of DNTB and oxazolone, no sensitization against DNFB was observed in animals treated with the combination of DNTB and oxazolone in the induction phase of CHS (Fig. 4). These findings suggest that an absent irritative effect of DNTB could be responsible for the inability of DNTB to elicit a CHS response in DNFB-sensitized animals. In contrast, application of an irrelevant hapten during sensitization did not restore the sensitizing potential of DNTB.

In summary, the present study shows that DNTB acts as a tolerogen upon primary hapten exposure and also fails to elicit CHS responses during secondary hapten application. IL-12 can both prevent and overcome DNTB-mediated tolerance and even convert the tolerogen DNTB into an immunogen. Our data also link the tolerogenic effect of DNTB to induction of apoptosis in LC and demonstrate an anti-apoptotic effect of IL-12 on LC.


   Acknowledgments
 
The excellent technical assistance of Joachim Windau and Maik Voskort is gratefully acknowledged.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the German Research Foundation [DFG Schw 1177/1-1 (to A.S.), SFB 415-A16 (to T.S.), SFB 293-B1 and SFB492-B2 (to S.G.), DFG 1580/6-2 and SFB 293-B8 (to S.B.), and IZKF Lo2/065/04 (to K.L. and S.B.)]. Back

2 H.R. and K.L. contributed equally to this study. Back

3 Address correspondence and reprint requests to Dr. Stephan Grabbe, Department of Dermatology, University of Essen, Hufelandstrasse 55, D-45122 Essen, Germany. E-mail address: grabbe{at}med.uni-essen.de Back

4 Abbreviations used in this paper: CHS, contact hypersensitivity; CBA, cytometric bead array; DC, dendritic cell; DNFB, 2,4-dinitrofluorobenzene; DNTB, 2,4,-dinitrothiocyanobenzene; LC, Langerhans cell. Back

Received for publication January 1, 2005. Accepted for publication August 18, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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