Substances that penetrate the skin surface can act as allergens and induce a T cell–mediated inflammatory skin disease called contact hypersensitivity (CHS). IL-17 is a key cytokine in CHS and was originally thought to be produced solely by CD4+ T cells. However, it is now known that several cell types, including γδ T cells, can produce IL-17. In this study, we determine the role of γδ T cells, especially dendritic epidermal T cells (DETCs), in CHS. Using a well-established model for CHS in which 2,4-dinitrofluorobenzene (DNFB) is used as allergen, we found that γδ T cells are important players in CHS. Thus, more IL-17–producing DETCs appear in the skin following exposure to DNFB in wild-type mice, and DNFB-induced ear swelling is reduced by ∼50% in TCRδ−/− mice compared with wild-type mice. In accordance, DNFB-induced ear swelling was reduced by ∼50% in IL-17−/− mice. We show that DNFB triggers DETC activation and IL-1β production in the skin and that keratinocytes produce IL-1β when stimulated with DNFB. We find that DETCs activated in vitro by incubation with anti-CD3 and IL-1β produce IL-17. Importantly, we demonstrate that the IL-1R antagonist anakinra significantly reduces CHS responses, as measured by decreased ear swelling, inhibition of local DETC activation, and a reduction in the number of IL-17+ γδ T cells and DETCs in the draining lymph nodes. Taken together, we show that DETCs become activated and produce IL-17 in an IL-1β–dependent manner during CHS, suggesting a key role for DETCs in CHS.

Contact hypersensitivity (CHS), which manifests as redness, swelling, and itching of the skin, is a T cell–mediated inflammatory disease induced by exposure of the skin to contact allergens. IL-17 is an important cytokine in the pathogenesis of various autoimmune and allergic diseases, and IL-17−/− mice show a strong reduction in ear swelling compared with wild-type (WT) mice in experimental models of CHS (1). In line with this, several studies (25) suggested the involvement of IL-17 in CHS based on the presence of Th17- and IL-17–producing CD8+ T cells in allergic reactions in both humans and mice. However, recent studies (6, 7) found that Th17 and CD8+ T cells are not the only sources of IL-17. Among the newly identified important sources of IL-17 during early inflammatory responses are γδ T cells and NKT cells.

Splenic γδ T cells can produce IL-17 following stimulation with IL-1β and IL-23, independently of TCR stimulation (7). This suggests that these IL-17–producing γδ T cells play an innate role in the immune response (7). Interestingly, both IL-1β and IL-23 are upregulated in the skin following exposure to contact allergens (3, 5, 8). Recently, it was shown that dermal γδ T cells can be activated in a similar fashion as splenic γδ T cells by IL-1β and IL-23, and it was suggested that dermal γδ T cells are the major source of IL-17 during psoriatic skin inflammation (9). However, dermal γδ T cells are not the only subtype of γδ T cells found in the skin. Murine epidermis contains a large number of a special subpopulation of Vγ3+ γδ T cells (Garman nomenclature) (10) called “dendritic epidermal T cells” (DETCs) because of their dendritic morphology (11). The role of DETCs as IL-17–producing cells is still debated (7, 9, 1215). Interestingly, it was shown recently that a subset of DETCs can produce IL-17 (14). This DETC subset was shown to be critical for epidermal barrier function and for effective wound healing (14).

Several studies (1623) found that γδ T cells can play both pro- and anti-inflammatory roles in CHS. In adoptive-transfer experiments, γδ T cells were found to assist αβ T cells in mediating the CHS response in a nonallergen- and non-MHC–specific manner (21). The role of γδ T cell help in the CHS response was investigated further, and DETCs were identified as γδ T cells assisting αβ T cells (17, 18). Furthermore, in vitro studies found that keratinocytes could induce DETC proliferation in a non–MHC-restricted manner following exposure to various contact allergens, whereas exposure to irritants could not (20). Thus, these studies indicated that DETCs play an inflammatory role in the immune response to contact allergens. However, other studies (16, 19, 22, 23) found that DETCs have a regulatory role in CHS by downregulating the response in an allergen-specific, MHC-independent pathway. Thus, the role of DETCs in CHS is unclear and seems to depend on the experimental setting, including the presence or absence of keratinocytes.

The aims of this study were to investigate the role of γδ T cells, especially DETCs, in CHS and to determine the ability of DETCs to produce IL-17. Using a well-established model for CHS in which 2,4-dinitrofluorobenzene (DNFB) is used as allergen, we found that γδ T cells, as well as IL-17, are required in CHS, as reflected by reduced ear swelling in TCRδ−/− mice and IL-17−/− mice compared with WT mice following DNFB exposure (24). We found that the numbers of IL-17–producing DETCs were increased significantly in the skin of WT mice following exposure to DNFB. Furthermore, analysis of epidermal ear sheets demonstrated that DETCs became activated following exposure to 2,4-dinitrobenzene sulfonic acid (DNSB). However, purified DETCs did not become activated when stimulated with the allergen, which indicated that a route of activation other than direct allergen recognition is required for DETC activation. We found that IL-1β is produced in the skin following exposure to DNFB and that keratinocytes upregulate IL-1β mRNA following DNSB treatment. We show that TCR stimulation and IL-1β act in synergy in mediating DETC activation and IL-17 production. Furthermore, intradermal (i.d.) injection with anti-CD3 in combination with IL-1β induced increased ear thickness compared with mice treated with PBS. Interestingly, DETCs activated in vitro with anti-CD3 and IL-1β induced increased ear thickness following i.d. injection compared with mice injected with unstimulated DETCs. Finally, treatment with the IL-1R antagonist anakinra inhibited ear swelling, local DETC activation, and the numbers of DETCs and IL-17+ γδ T cells in the draining lymph nodes (dLNs) after DNFB exposure. We suggest a model in which allergen exposure induces IL-1β production and expression of the still-unknown TCR ligand for DETCs in keratinocytes. Together, these molecules induce DETC activation and IL-17 production required for the CHS reaction.

Female C57BL/6 (WT) mice were purchased from Taconic (Ry, Denmark). B6.129P2-Tcrdtm1Mom/J (TCRδKO) mice were purchased from The Jackson Laboratory (San Diego, CA). IL-17−/− mice were provided by Y. Iwakura (University of Tokyo, Tokyo, Japan) (1) and K. Ley (La Jolla Institute for Allergy and Immunology). Mice were housed in specific pathogen–free facilities at either the Department of Experimental Medicine, Panum Institute, The University of Copenhagen, in accordance with national animal-protection guidelines (license no. 2007/561-1357) or at The Scripps Research Institute, according to The Scripps Research Institute Institutional Animal Care and Use Committee guidelines.

DNFB, DNSB, PMA, Con A, indomethacin, ionomycin, and monensin were purchased from Sigma-Aldrich (Brøndby, Denmark). Recombinant murine (rm)IL-1β, rmIL-23, IFN-γ, IL-10, and TGF-β ELISA kits, Fixable Viability Dye eFluor 780, and intracellular staining kits were purchased from eBioscience (San Diego, CA). Mouse TH1/TH2 9-Plex and Mouse IL-17A Ultrasensitive kits were purchased from Meso Scale Discovery. Anti-CD16/CD32 (2.4G2), anti-γδ (GL3), anti-Vγ3 (536), anti-CD3ε (145-2C11), anti–IL-17 (TC11-18H10), anti–IFN-γ (XMG1.2), and isotype control (R3-34) were all purchased from BD Pharmingen. Anti-CD25 (PC61) was purchased from BioLegend (San Diego, CA). TaqMan RevertAid First Strand cDNA synthesis Kit (K1622), TaqMan Universal PCR Master Mix (4326708), and primers GAPDH (Mm999999_g1) and IL-1β (Mm01336189) were purchased from Applied Biosystems (Foster City, CA).

The murine keratinocyte cell line PAM2.12 was cultured in DMEM at 37°C, 5% CO2 (Sigma-Aldrich). DMEM was supplemented with 10% FBS, 0.5 IU/l penicillin, 500 mg/l streptomycin, 1% l-glutamine, 2-ME, 0.63 mM HEPES (Life Technologies, San Diego, CA), and 1 mM Na pyruvate (Life Technologies). Medium was changed every other day, and cells were split when 80% confluent. Freshly isolated DETCs were cultured in flat-bottom 96-well plates in RPMI 1640 at 37°C, 5% CO2. RPMI 1640 was supplemented with 10% FBS, 0.5 IU/l penicillin, 500 mg/l streptomycin, 1% l-glutamine, 2-ME, 0.63 mM HEPES, 1 mM Na pyruvate, 1 μM nonessential amino acids (Life Technologies), and 5 U/ml IL-2 (Invitrogen, Naerum, Denmark). Every other day half of the medium was gently removed, and fresh medium was added.

To induce CHS, mice were painted with 25 μl 0.15% DNFB in a 1:4 olive oil:acetone (OOA) mixture on the dorsal side of both ears for three consecutive days (days 0–2). On day 23, mice were challenged on the dorsal side of both ears with 25 μl 0.15% DNFB in OOA. Control mice were exposed to the vehicle on day 23. Mice were euthanized 24 h after challenge, and ear thickness was measured on both ears using an engineer’s micrometer (Mitutoyo, Tokyo, Japan). dLNs were harvested, and single-cell suspensions were prepared for further analysis.

C57BL/6 mice were sensitized for three consecutive days (days 0–2) and challenged on day 23. Anakinra (200 μl 150 μg/μl) or PBS was administered i.p. 12 h before and immediately before challenge. Ear thickness was measured 24 h postchallenge, and dLNs were collected for FACS analysis.

To produce epidermal cell suspensions, the hair was removed from euthanized naive C57BL/6 mice. Skin pieces were floated with the dermis side down in 0.3% trypsin/GNK at 37°C overnight. The next day, the epidermis was peeled from the dermis and treated with trypsin/GNK with 0.1% DNase (Sigma-Aldrich). Lymphocytes were purified using Lympholyte-M (Life Technologies) and plated in complete DETC medium containing 2 μg/ml Con A and 2 μg/ml indomethacin. Every second day, half of the medium was removed, and fresh medium was added. Cells were expanded in culture for 3 wk prior to the experiments. Cultured epidermal cells were collected, and nonspecific binding was blocked by anti-CD16/CD32 (2.4G2). Cells were stained with anti-Vγ3 (536), anti-CD3 (145-2C11), and anti-γδ (GL3). DETCs (CD3+γδ+Vγ3+) were sorted on a FACSAria to a purity > 98%.

Single-cell suspensions from dLNs were adjusted and plated at 1 × 106 cells/well and restimulated with PMA (1.25 μg/ml) and ionomycin (625 ng/ml) in complete RPMI 1640 including monensin (2.08 μg/ml) for 5 h at 37°C. Subsequently, cells were treated with anti-CD16/CD32 (2.4G2) to block nonspecific binding, washed, and stained with anti-γδ (GL3) and anti-Vγ3 (536). Cells were fixed and permeabilized with eBioscience intracellular staining kit. Cells were subsequently stained with anti–IL-17 (TC11-18H10) or anti–IFN-γ (XMG1.2). A single-cell suspension from epidermis was obtained as described in “Cell sorting.” Cells were rested overnight to allow re-expression of trypsin-sensitive surface markers. The following day, cells were incubated in the presence of PMA (1.25 μg/ml) and ionomycin (625 ng/ml) in complete RPMI 1640, including monensin (2.08 μg/ml), for 5 h at 37°C. Subsequently, cells were treated with anti-CD16/CD32 (2.4G2) to block nonspecific binding, washed, and stained with anti-CD3 (145-2C11), anti-γδ (GL3), anti-Vγ3 (536), and Fixable Viability Dye eFluor 780. Cells were fixed and permeabilized with an intracellular staining kit (eBioscience). Subsequently, cells were stained with anti–IL-17 (TC11-18H10), anti–IFN-γ (XMG1.2), or anti–IL-10 (JE55-16E3). Cell samples were analyzed on a FACSCalibur or Fortessa (BD, Brøndby, Denmark) with CellQuest Pro or FACSDiva software. Data were analyzed using FlowJo software.

Ear sheets were prepared by separating the dorsal and ventral sides of the ears. Subsequently, ear pieces were floated dermis side down for 20 h (DNSB stimulation) or 24 h (cytokine or anti-CD3 stimulation) at 37°C in DMEM containing DMSO, 0.1% DNSB, 10 ng/ml IL-1β, or 1 μg/ml anti-CD3. Subsequently, epidermal sheets were prepared as previously described (25). Sheets were stained with anti-γδ TCR (GL3) for 1 h at room temperature, washed, and mounted on slides using DAKO fluorescent mounting medium (Carpinteria, CA). Samples were imaged directly after staining with a Nikon Eclipse E800 microscope. Digital images were collected with an AxioCam HRc camera and AxioCam software (Zeiss, Oberkochen, Germany), and image processing was performed using Adobe Photoshop and InDesign. Changes in DETC morphology after anti-CD3 and cytokine treatment were assessed by counting the number of dendrites on each cell (more than two dendrites = resting; one or two dendrites = partially activated; no dendrites = activated). A minimum of 200 cells from at least three experiments was counted for each treatment.

Mice were exposed once to 0.15% DNFB in OOA or, as a control, to OOA. At the indicated times postallergen exposure, mice were euthanized, and 8-mm punch biopsies were immediately taken from both ears. Biopsies were split in half, snap-frozen, and stored at −80°C for later protein or RNA purification. Biopsies were crushed individually and lysed simultaneously using Precellys technology (Bertin Technologies, Montigny-le-Bretonneux, France). For mRNA analysis, RNA was purified using the mirVana mRNA purification kit (Life Technologies, Naerum, Denmark) and reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The samples were amplified by real-time RT-PCR using Applied Biosystems validated gene-expression assays and PRISM 7900HT sequence detection system (SDS 2.3). Fold changes in mRNA expression were calculated by the comparative Ct method and normalized to GAPDH using RealTime StatMiner software (Integromics, Granada, Spain). For protein analysis, total amounts of protein in each sample were quantified by the Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL), and the total protein concentration of each sample was standardized before assessment for cytokine expression. Cytokines from homogenized ear biopsies were measured by Mouse TH1/TH2 9-Plex Ultra-Sensitive Kits on a Meso Scale Discovery platform. PAM2.12 cells were plated at 2.5 × 105 cells/well in six-well plates and incubated overnight at 37°C. The following day, cells were stimulated for the indicated time and subsequently harvested using 0.5% trypsin/EDTA (Life Technologies). RNA extraction was performed using the standard protocol RNeasy Mini Kit (QIAGEN, Copenhagen, Denmark) and reverse transcribed as described for tissue lysates.

Following cell sorting, DETCs were rested overnight and restimulated to determine IL-17 production. In brief, 1.0 × 105 DETCs/well were stimulated with rmIL-1β, rmIL-23 (10 ng/ml), anti-CD3 (1 μg/ml), or combinations of these. For IL-17A detection, cells were incubated at 37°C, and supernatant was collected at 12, 24, and 48 h and analyzed using Mouse IL-17A Ultra-Sensitive Kits on a Meso Scale Discovery platform. For detection of IFN-γ, IL-10, and TGF-β, supernatant was collected at 24 h and analyzed by ELISA. For FACS analysis of sorted DETCs, cells were restimulated for 24 h and stained with anti-Vγ3 (536), anti-γδ (GL3), anti-CD25 (PC61), and anti–IL-17 (TC11-18H10), as described in “Intracellular cytokine staining.”

C57BL/6 mice were injected i.d. in the ears with 20 μl 1 μg/ml anti-CD3, isotype control, or 10 ng/ml IL-1β, alone or in combination. Ear thickness was measured 24 h after the injections to evaluate the degree of inflammation. For cell-transfer experiments, sorted DETCs were either left untreated or stimulated with 1 μg/ml anti-CD3 and 10 ng/ml IL-1β for 24 h prior to i.d. injection. A total of 20 μl fresh medium containing 1 × 104 DETCs was injected i.d. in the ears, and the ear thickness was measured 24 h later.

To investigate the role of γδ T cells and IL-17 in CHS, we compared CHS responses to DNFB in WT, TCRδ−/−, and IL-17−/− mice, as measured by ear thickness. We found that ear swelling was significantly reduced in both TCRδ−/− mice and IL-17−/− mice compared with WT mice (Fig. 1A, 1B). Interestingly, suppression of the CHS response was similar in IL-17−/− and TCRδ−/− mice. The important role for IL-17 in CHS is in good agreement with a previous study (1); however, it is still unknown whether skin-derived γδ T cells contribute to the production of IL-17 during CHS. To investigate this, we isolated and restimulated cells from the dLNs and skin from DNFB-exposed and control WT mice with PMA and ionomycin and determined intracellular IL-17. A significantly increased number of γδ T cells and IL-17+ γδ T cells was found in the dLNs of DNFB-exposed mice compared with control mice (Fig. 1C, 1D). To analyze whether skin-derived γδ T cells contributed to the increased number of γδ T cells in the dLNs, we determined the number of DETCs in the dLNs. A 10-fold increase in the number of DETCs was found in DNFB-exposed mice compared with control mice (Fig. 1E). However, DETCs did not seem to produce IL-17A and IFN-γ in the dLNs (Fig. 1F). In contrast, when analyzing IL-17A and IFN-γ production by DETCs isolated from the skin, more IL-17A+ and IFN-γ+ DETCs were found in mice exposed to DNFB compared with control mice (Fig. 1G, 1H). Taken together, these results show that both γδ T cells and IL-17 are involved in the pathogenesis of CHS and that more DETCs that produce IL-17A and IFN-γ are found in skin exposed to DNFB. Furthermore, the numbers of γδ T cells, including DETCs and IL-17+ γδ T cells, increase in the dLNs during CHS. However, DETCs do not seem to produce IL-17A and IFN-γ in the dLNs.

FIGURE 1.

γδ T cells are involved in the pathogenesis of CHS. (A) C57BL/6, IL-17−/−, and TCRδ−/− mice were split into two groups, with four or five mice/group. Mice were exposed to vehicle or DNFB on the dorsal side of the ears for three consecutive days (days 0–2) and then re-exposed to DNFB on day 23. Ear thickness was measured 24 h after the final exposure and is shown relative to vehicle-treated mice. (B) Suppression of ear inflammation in DNFB-treated IL-17−/− and TCRδ−/− mice compared with WT mice. Suppression was calculated as ([ear thickness(IL-17−/−, DNFB)] − [ear thickness(IL-17−/−, vehicle)]/[ear thickness(WT, DNFB)] − [ear thickness(WT, vehicle)]) × 100%. Total number of γδ T cells (GL3+ cells) (C), IL-17–producing γδ T cells (GL3+IL-17+ cells) (D), and DETCs (GL3+Vγ3+ cells) (E) in dLNs of C57BL/6 mice exposed to vehicle or DNFB. (F) Representative FACS plots of Vγ3+IL-17A+ cells and Vγ3+IFN-γ+ cells in the dLNs. (G) Percentages of Vγ3+IL-17A+ cells and Vγ3+IFN-γ+ cells in epidermis from C57BL/6 mice exposed to vehicle or DNFB. (H) Representative FACS plots of Vγ3+IL-17A+ and Vγ3+IFN-γ+ cells in epidermis from C57BL/6 mice exposed to vehicle or DNFB. Data are mean + SEM of two independent experiments consisting of four or five mice/group. **p < 0.01, ***p < 0.001, ****p < 0.0001, Student t test.

FIGURE 1.

γδ T cells are involved in the pathogenesis of CHS. (A) C57BL/6, IL-17−/−, and TCRδ−/− mice were split into two groups, with four or five mice/group. Mice were exposed to vehicle or DNFB on the dorsal side of the ears for three consecutive days (days 0–2) and then re-exposed to DNFB on day 23. Ear thickness was measured 24 h after the final exposure and is shown relative to vehicle-treated mice. (B) Suppression of ear inflammation in DNFB-treated IL-17−/− and TCRδ−/− mice compared with WT mice. Suppression was calculated as ([ear thickness(IL-17−/−, DNFB)] − [ear thickness(IL-17−/−, vehicle)]/[ear thickness(WT, DNFB)] − [ear thickness(WT, vehicle)]) × 100%. Total number of γδ T cells (GL3+ cells) (C), IL-17–producing γδ T cells (GL3+IL-17+ cells) (D), and DETCs (GL3+Vγ3+ cells) (E) in dLNs of C57BL/6 mice exposed to vehicle or DNFB. (F) Representative FACS plots of Vγ3+IL-17A+ cells and Vγ3+IFN-γ+ cells in the dLNs. (G) Percentages of Vγ3+IL-17A+ cells and Vγ3+IFN-γ+ cells in epidermis from C57BL/6 mice exposed to vehicle or DNFB. (H) Representative FACS plots of Vγ3+IL-17A+ and Vγ3+IFN-γ+ cells in epidermis from C57BL/6 mice exposed to vehicle or DNFB. Data are mean + SEM of two independent experiments consisting of four or five mice/group. **p < 0.01, ***p < 0.001, ****p < 0.0001, Student t test.

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Because we found that DETCs from the skin produce IL-17A and IFN-γ following exposure of the skin to DNFB, we wanted to study whether exposure of the skin to DNFB induced DETC activation in situ. It was shown that DETCs change their morphology and round up during their activation in the epidermis (11). To investigate whether exposure of the skin to allergens activates DETCs in situ, we treated epidermal ear sheets with 0.1% DNSB or vehicle for 20 h and subsequently determined the morphology of the DETCs. We found that DETCs retained their classical dendritic morphology in epidermal ear sheets treated with vehicle, whereas DNSB treatment induced rounding up (Fig. 2A). To investigate whether DETC activation was caused by direct recognition of the allergen, we purified DETCs and treated them for 24 h with nonsupplemented medium or medium supplemented with 0.1% DNSB, anti-CD3, or anti-CD3 plus IL-1β. DETC activation was assessed by CD25 upregulation (Fig. 2B, 2C). DNSB did not activate DETCs, whereas stimulation through the TCR with anti-CD3 did (Fig. 2B, 2C). Interestingly, addition of IL-1β seemed to increase CD25 expression on the DETCs, although this was not significant. Taken together, these results indicate that DETCs are activated in the epidermis when allergens penetrate the skin; furthermore, they are not activated directly, rather they are activated indirectly by the allergens via allergen-induced activation of other cells in the skin.

FIGURE 2.

Exposure of the skin to allergens leads to DETC activation in the epidermis. (A) Ears sheets from untreated mice were treated for 20 h with vehicle or 0.1% DNSB. The epidermal ear sheets were isolated and stained with anti-γδ TCR (green) (original magnification ×20). Data are representative of five separate experiments. (B and C) DETCs were isolated by sorting CD3+GL3+Vγ3+ cells and rested overnight. DETCs were stimulated for 24 h, as indicated, and CD25 expression was determined by FACS. (B) FACS graph of CD25 expression on untreated DETCs and DETCs treated with anti-CD3, anti-CD3 + IL-1β, or DNSB. (C) CD25 expression on DETCs treated with anti-CD3, anti-CD3 + IL-1β, or DNSB normalized to CD25 expression on untreated DETCs. Data are mean + SEM of four individual experiments.

FIGURE 2.

Exposure of the skin to allergens leads to DETC activation in the epidermis. (A) Ears sheets from untreated mice were treated for 20 h with vehicle or 0.1% DNSB. The epidermal ear sheets were isolated and stained with anti-γδ TCR (green) (original magnification ×20). Data are representative of five separate experiments. (B and C) DETCs were isolated by sorting CD3+GL3+Vγ3+ cells and rested overnight. DETCs were stimulated for 24 h, as indicated, and CD25 expression was determined by FACS. (B) FACS graph of CD25 expression on untreated DETCs and DETCs treated with anti-CD3, anti-CD3 + IL-1β, or DNSB. (C) CD25 expression on DETCs treated with anti-CD3, anti-CD3 + IL-1β, or DNSB normalized to CD25 expression on untreated DETCs. Data are mean + SEM of four individual experiments.

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It was reported that, although spleen and dermal γδ T cells have the ability to produce IL-17A independently of TCR stimulation following treatment with IL-1β and IL-23, DETCs do not have this ability (7, 9). To investigate this further, we purified DETCs from the epidermis (Fig. 3A) and treated them for the indicated periods with IL-1β, IL-23, and anti-CD3, either alone or in combination. We found that DETCs treated with IL-1β, IL-23, or anti-CD3 alone produce low amounts of IL-17A (Fig. 3B). However, IL-23 and anti-CD3 in combination resulted in a 50% increase in IL-17A production compared with cells treated with anti-CD3 or IL-23 alone. Even more impressive, combined IL-1β and anti-CD3 treatment resulted in a 3-fold increase in IL-17A secretion compared with cells stimulated with anti-CD3 or IL-1β alone (Fig. 3B). No additional effect was seen when IL-23 was added with IL-1β. To further validate that DETCs have the capacity to produce IL-17A, we purified DETCs, stimulated them for 24 h with anti-CD3, alone or in combination with IL-1β, and measured intracellular IL-17A (Fig. 3C). We found that DETCs can produce IL-17A following TCR stimulation. Again, when IL-1β was added in combination with anti-CD3, a marked increase in IL-17A+ DETCs was observed (Fig. 3C). Because we found more IL-17A+ and IFN-γ+ DETCs in the skin following exposure of the skin to DNFB, we wanted to investigate the effect of anti-CD3 and IL-1β on IFN-γ production by DETCs. Furthermore, because the role of γδ T cells during CHS has been suggested to be anti-inflammatory, we investigated whether treatment with anti-CD3 and IL-1β led to induction of anti-inflammatory cytokines IL-10 and/or TGF-β (17, 18, 22, 23). Treatment with anti-CD3 induced the production of both IFN-γ and IL-10 (Fig. 3D). Addition of IL-1β increased the production of IFN-γ, whereas it did not seem to have an effect on IL-10 production (Fig. 3D). Interestingly, anti-CD3, alone or in combination with IL-1β, did not lead to production of TGF-β (Fig. 3D). Treatment with DNSB did not induce production of any of the cytokines (Fig. 3D). Taken together, these results indicate that DETCs have the capacity to produce IL-17A and that TCR and IL-1β signaling in combination is highly potent in inducing IL-17A production in DETCs. The same was seen for IFN-γ production, whereas IL-10 production depended only on TCR signaling. Finally, DNSB treatment did not induce cytokine production by DETCs, further supporting that DNFB-induced activation of DETCs in situ is mediated by an indirect pathway.

FIGURE 3.

DETCs produce IL-17A when stimulated with IL-1β, IL-23, and anti-CD3. (A) Representative FACS plot of DETC purity after cell sorting on CD3+GL3+Vγ3+ cells from short-term epidermal cultures. (B) DETCs were sorted, rested overnight, and treated with IL-1β, IL-23, anti-CD3, or combinations of these for the time indicated. Subsequently, supernatants were collected, and the concentration of IL-17A was determined. Data are mean + SEM and are representative of two independent experiments. (C) IL-17A profile of sorted DETCs cultivated for 24 h in medium alone, medium with anti-CD3, or medium with both anti-CD3 and IL-1β. Data are representative of two separate experiments. (D) DETCs were sorted, rested overnight, and treated with anti-CD3, anti-CD3 + IL-1β, or DNSB for 24 h. Subsequently, the supernatants were collected, and the concentration of IFN-γ, IL-10, or TGF-β was determined. Data are mean + SEM of four individual experiments. **p < 0.01, ***p < 0.001, Student t test.

FIGURE 3.

DETCs produce IL-17A when stimulated with IL-1β, IL-23, and anti-CD3. (A) Representative FACS plot of DETC purity after cell sorting on CD3+GL3+Vγ3+ cells from short-term epidermal cultures. (B) DETCs were sorted, rested overnight, and treated with IL-1β, IL-23, anti-CD3, or combinations of these for the time indicated. Subsequently, supernatants were collected, and the concentration of IL-17A was determined. Data are mean + SEM and are representative of two independent experiments. (C) IL-17A profile of sorted DETCs cultivated for 24 h in medium alone, medium with anti-CD3, or medium with both anti-CD3 and IL-1β. Data are representative of two separate experiments. (D) DETCs were sorted, rested overnight, and treated with anti-CD3, anti-CD3 + IL-1β, or DNSB for 24 h. Subsequently, the supernatants were collected, and the concentration of IFN-γ, IL-10, or TGF-β was determined. Data are mean + SEM of four individual experiments. **p < 0.01, ***p < 0.001, Student t test.

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It was shown that IL-1β is produced in the skin following allergen exposure (8). Based on this observation and our findings described above, we hypothesized that activation of DETCs in the epidermis following allergen exposure is mediated by IL-1β derived from keratinocytes activated by the allergen. To determine whether IL-1β was produced in the skin in our experimental setup, we treated mice with DNFB on the dorsal side of the ears. IL-1β mRNA levels in the ears were determined by real-time RT-PCR after 6, 24, and 48 h. IL-1β was rapidly upregulated after exposure to DNFB (Fig. 4A). The protein levels of IL-1β in ear tissue extracts were in good agreement with the mRNA results (Fig. 4B). To determine whether allergens directly induce IL-1β in keratinocytes, we stimulated the PAM2.12 keratinocyte cell line for various periods of time with DNSB. We found that exposure to DNSB induced a significant upregulation of IL-1β mRNA (Fig. 4C). IL-1β mRNA peaked after 24 h of DNSB treatment, with an ∼25-fold upregulation compared with cells treated with vehicle (Fig. 4C). To determine the role of IL-1β in DETC activation, epidermal ear sheets were treated with IL-1β or anti-CD3 for 24 h, and DETC morphology was determined. Nontreated ear sheets contained mostly highly dendritic DETCs (more than two dendrites), whereas the majority of DETCs had a partial retraction of dendrites following treatment of the ear sheets with IL-1β, suggesting that IL-1β induced partial activation of the DETCs (Fig. 4D). Full DETC activation, as indicated by a rounded morphology, was seen in ∼80% of the DETCs following treatment with anti-CD3 (Fig. 4D). These results suggested that DETCs become activated in situ when stimulated with IL-1β or anti-CD3. To investigate this further, we injected mice i.d. in the ears with anti-CD3, alone or in combination with IL-1β, and measured ear thickness 24 h later. We found that anti-CD3, alone and in combination with IL-1β, induced significant ear swelling (Fig. 4E). To analyze whether DETCs might contribute to this anti-CD3–induced ear swelling, we injected purified DETCs, which had been activated with anti-CD3 and IL-1β in vitro or were left untreated, i.d. in the ears of untreated mice. We found that injection of activated DETCs induced increased ear swelling compared with injections of untreated DETCs (Fig. 4F). Taken together, these experiments suggested that keratinocytes are activated directly by DNFB and start to produce IL-1β, which, together with the still-unidentified DETC TCR ligand, induces DETC activation, IL-17 production, and skin inflammation.

FIGURE 4.

Allergens induce keratinocytes to produce IL-1β that activates DETCs. (A) Fold change in IL-1β mRNA in ear biopsies from mice exposed to DNFB for 6, 24, and 48 h compared with mice exposed to vehicle. The mRNA level of IL-1β was determined by real-time RT-PCR. The relative expression was determined compared with GAPDH. **p < 0.01, ΔCT values obtained from real-time RT-PCR (vehicle treated versus DNFB treated), Student t test. (B) Protein levels of IL-1β in ear biopsies from mice exposed to vehicle or DNFB for 6, 24, and 48 h. (C) Fold change in IL-1β mRNA in PAM2.12 cells treated with 0.1% DNSB for the indicated time compared with PAM2.12 cells treated with vehicle. The relative expression was determined compared with GAPDH. Data are mean ± SEM for two separate experiments (n = 4). (D) Epidermal ear sheets were treated with IL-1β or anti-CD3 for 24 h, and the morphology of DETCs was determined by fluorescence microscopy. Inset shows examples of fluorescence microscopy images of DETCs stained with anti-γδ TCR Abs (red). A minimum of 200 cells from at least three experiments was counted and characterized based on their number of dendrites (more than two dendrites = resting DETC; one or two dendrites = partially activated DETC; no dendrites = activated DETC). Ear thickness of C57BL/6 mice 24 h after i.d. injection with anti-CD3, anti-CD3 + IL-1β, PBS, or isotype control (E) or with untreated DETCs or DETCs preactivated with anti-CD3 and IL-1β (F). Data are mean + SEM of two individual experiments (n = 4). *p < 0.05, **p < 0.01, Student t test.

FIGURE 4.

Allergens induce keratinocytes to produce IL-1β that activates DETCs. (A) Fold change in IL-1β mRNA in ear biopsies from mice exposed to DNFB for 6, 24, and 48 h compared with mice exposed to vehicle. The mRNA level of IL-1β was determined by real-time RT-PCR. The relative expression was determined compared with GAPDH. **p < 0.01, ΔCT values obtained from real-time RT-PCR (vehicle treated versus DNFB treated), Student t test. (B) Protein levels of IL-1β in ear biopsies from mice exposed to vehicle or DNFB for 6, 24, and 48 h. (C) Fold change in IL-1β mRNA in PAM2.12 cells treated with 0.1% DNSB for the indicated time compared with PAM2.12 cells treated with vehicle. The relative expression was determined compared with GAPDH. Data are mean ± SEM for two separate experiments (n = 4). (D) Epidermal ear sheets were treated with IL-1β or anti-CD3 for 24 h, and the morphology of DETCs was determined by fluorescence microscopy. Inset shows examples of fluorescence microscopy images of DETCs stained with anti-γδ TCR Abs (red). A minimum of 200 cells from at least three experiments was counted and characterized based on their number of dendrites (more than two dendrites = resting DETC; one or two dendrites = partially activated DETC; no dendrites = activated DETC). Ear thickness of C57BL/6 mice 24 h after i.d. injection with anti-CD3, anti-CD3 + IL-1β, PBS, or isotype control (E) or with untreated DETCs or DETCs preactivated with anti-CD3 and IL-1β (F). Data are mean + SEM of two individual experiments (n = 4). *p < 0.05, **p < 0.01, Student t test.

Close modal

To determine the role of IL-1β in CHS and DETC activation in vivo, we investigated CHS responses in WT mice treated with the IL-1R antagonist anakinra or PBS. We found that mice treated with anakinra had significantly reduced ear swelling compared with mice treated with PBS (Fig. 5A, 5B). Furthermore, anakinra treatment led to reduced numbers of total γδ T cells, IL-17+γδ T cells, and DETCs in the dLNs (Fig. 5C–E). To investigate whether the reduced number of DETCs in the dLNs from mice treated with anakinra could be explained by inhibition of DETC activation in the epidermis, we determined DETC activation in situ, as measured by DETC morphology in mice treated with either anakinra or PBS. DETCs were clearly activated in the epidermis from PBS-treated mice, as judged by their round shape (Fig. 5F). In contrast, DETCs retained their dendritic morphology in the epidermis from anakinra-treated mice, indicating that DETCs are in a no/low-activation state (Fig. 5F). Taken together, these results indicate that IL-1β is one of the key cytokines for a full-blown CHS response and for the activation and migration of DETCs to the dLNs during CHS.

FIGURE 5.

IL-1β is required for DNFB-induced DETC activation in vivo. Mice were sensitized and challenged with DNFB, as described in 2Materials and Methods. Before challenge, mice were given anakinra or PBS i.p. (A) Ear thickness measured 24 h after DNFB exposure. (B) Percentage suppression of inflammation in mice treated with anakinra compared with mice treated with PBS. Total number of γδ T cells (GL3+) (C), IL-17A–producing γδ T cells (GL3+IL-17+) (D), and DETCs (GL3+Vγ3+) (E) in the dLNs of mice treated with PBS or anakinra before challenge with DNFB. Data represent two individual experiments with four mice/group. (F) Epidermal ear sheets from mice treated with PBS or anakinra before challenge with DNFB were isolated and stained with anti-γδ TCR (green) (original magnification ×20). Data are representative of two separate experiments. *p < 0.05, Student t test.

FIGURE 5.

IL-1β is required for DNFB-induced DETC activation in vivo. Mice were sensitized and challenged with DNFB, as described in 2Materials and Methods. Before challenge, mice were given anakinra or PBS i.p. (A) Ear thickness measured 24 h after DNFB exposure. (B) Percentage suppression of inflammation in mice treated with anakinra compared with mice treated with PBS. Total number of γδ T cells (GL3+) (C), IL-17A–producing γδ T cells (GL3+IL-17+) (D), and DETCs (GL3+Vγ3+) (E) in the dLNs of mice treated with PBS or anakinra before challenge with DNFB. Data represent two individual experiments with four mice/group. (F) Epidermal ear sheets from mice treated with PBS or anakinra before challenge with DNFB were isolated and stained with anti-γδ TCR (green) (original magnification ×20). Data are representative of two separate experiments. *p < 0.05, Student t test.

Close modal

In this study, we show that both γδ T cells and IL-17 are involved in the pathogenesis of CHS and that γδ T cells, IL-17+γδ T cells, and DETCs accumulate in the dLNs following exposure of the skin to allergens. We found that exposure of the skin to DNFB leads to local production of IL-1β and activation of DETCs in the epidermis, as seen by the rounding of the DETCs and increased numbers of DETCs producing IL-17A and IFN-γ. We demonstrate that DETCs have the ability to produce IL-17A, IFN-γ, and IL-10 following TCR stimulation and that TCR-induced IL-17A and IFN-γ production is significantly enhanced by IL-1β. We show that anti-CD3 and IL-1β injected i.d. into the ears induce significant ear swelling and, furthermore, that DETCs activated in vitro induce an increased ear swelling following i.d. injection into the ears. Finally, we found that blocking of IL-1R in vivo resulted in decreased ear swelling and DETC activation and significantly lowered the numbers of γδ T cells, IL-17+ γδ T cells, and DETCs in the dLNs.

In agreement with Nakae et al. (1), we found that IL-17 is important for the CHS response, because mice lacking IL-17 had an ∼50% reduced response to DNFB, as measured by changes in ear thickness, compared with WT mice. Furthermore, we found a strong reduction in ear swelling following allergen exposure in TCRδ−/− mice compared with WT mice on the C57BL/6 background. In contrast, Girardi et al. (26) found that γδ T cells did not have an effect on the CHS response in mice on the C57BL/6 background. However, the protocols used in the two studies differ significantly. Girardi et al. (26) sensitized the mice on the abdominal skin and challenged on the ears 5 d after sensitization. We sensitized for three consecutive days on the ears and challenged after a minimum of 21 d on the ears. It is well known that primary adaptive responses during CHS induction and infectious diseases peak between days 5 and 10, depending on the presence of allergen and type of infection (27, 28). We (24) showed previously that the primary response is ongoing in the dLNs for ≥10 d when using our CHS protocol. Thus, the primary response is most likely ongoing at day 5 in the protocol used by Girardi et al. (26), and a challenge at this time would primarily be driven by the activated CD4+ and CD8+ effector T cells. In contrast, our model is likely to be more dependent on early responders, like DETCs. Although γδ T cells are thought to be more innate than αβ T cells, it was shown that they can develop a memory-like phenotype and that they become more efficient during secondary Ag exposure (29, 30). Furthermore, existence of memory-like γδ T cells in CHS is further supported by previous studies (18), which found that DETCs are required for adaptive transfer of CHS.

Conflicting results have been reported concerning the role of γδ T cells as pro- or anti-inflammatory in CHS. Adaptive-transfer experiments indicated that γδ T cells are required for optimal transfer of CHS from sensitized to allergen-inexperienced mice (17, 18, 21). Interestingly, DETCs were shown to be the γδ T cell subset required for the full transfer of CHS (17, 18). In contrast, transfer of DETCs primed with allergen in vitro induced tolerance toward the specific allergen (17, 18, 22, 23). The divergent results obtained in these experiments might be explained by the different conditions during priming of DETCs in vivo and in vitro. Based on the changes in DETC morphology, we found that DETCs become activated following exposure of ears/ear sheets to DNFB/DNSB and that their number increases in the dLNs following DNFB exposure of the skin. We found that highly purified DETCs from naive mice produce IL-17, IFN-γ, and IL-10 when stimulated with anti-CD3. This TCR-induced production of IL-17 and IFN-γ was increased further by IL-1β, whereas IL-10 production was unaffected. From these data, it might be suggested that DETCs play a proinflammatory role in the allergic response as long as IL-1β is present in the skin; however, when the concentration of IL-1β wanes, they can convert to playing an anti-inflammatory role.

The ability of DETCs to produce IL-17 is in agreement with previous studies (12, 14) that found that DETCs produce IL-17 following skin infection with Staphylococcus aureus, when stimulated with PMA and ionomycin in the presence of IL-1β, IL-23, or a combination of the two, and following skin wounding. In contrast, other studies (7, 9, 13, 15), using either Mycobacterium or a psoriasis model, found that DETCs were unable to produce IL-17 and that dermal γδ T cells are the primary source of IL-17 in the skin. The different results obtained concerning the ability of DETCs to produce IL-17 might be explained by the requirement for TCR stimulation. Thus, DETCs might require TCR triggering, whereas dermal γδ T cells seem to be able to produce IL-17 by a cytokine-dependent, TCR-independent pathway, as reported for spleen γδ T cells (7, 13). Cai et al. (9) used an IL-23–induced psoriasis model to investigate the main IL-17–producing cell types within the skin; it is likely that this model did not induce any stress to the keratinocytes because IL-23 was injected i.d. A recent study (31) using this model characterized a subtype of γδ T cells in mouse epidermis that was distinct from DETCs, because they did not express Vγ3. This γδ T cell subtype was suggested to be the main IL-17– and IL-22–producing cell type in the skin when the IL-23–induced psoriasis model was used (31). Interestingly, in a CHS model using DNFB as allergen, the same group (31) was unable to detect this γδ T cell subtype in the epidermis. This indicated that different γδ T cell subtypes exist in the epidermis. It is also likely that different types of skin infections have different effects on the keratinocytes (12, 32). Interestingly, DETCs have higher levels of TCR expression at their surface than do dermal γδ T cells, which might indicate that DETCs are more dependent on TCR signaling than are dermal γδ T cells (9, 32).

Taken together, our data show that γδ T cells play a proinflammatory role during CHS. We believe that our data support the hypothesis that DETCs produce IL-17 when allergens penetrate the skin and that this is important for the immune response to allergens in the skin. Based on our study and a previous study (33) that demonstrated upregulation of a still-uncharacterized ligand for the DETC TCR at the cell surface of stressed keratinocytes, we propose the following model for cross-talk between DETCs and keratinocytes: exposure of the skin to allergens leads to keratinocyte stress, with rapid production of IL-1β and upregulation of the still-unidentified TCR ligand on the keratinocyte cell surface. Together, IL-1β and the TCR ligand provide the necessary signals required for full activation and IL-17 production by DETCs and the development of CHS. Interestingly, IL-1β is upregulated rapidly in the skin of allergic patients after exposure to contact allergens (34). Although DETCs are a unique cell population in murine skin, another subset of γδ T cells seems to play a role as IL-17–producing cells in human inflammatory skin diseases, like psoriasis and contact allergy (35, 36). Finally, our study suggests that anakinra might be efficient in the treatment of patients with severe contact dermatitis, in part by inhibiting IL-17 production from γδ T cells. However, this needs further investigation.

We thank Drs. Morten Alhede and Thomas Bjarnsholt for expert advice on fluorescent microscopy.

This work was supported by the LEO Pharma Research Foundation, the A.P. Møller Foundation for the Advancement of Medical Sciences, the Kongelig Hofbuntmager Aage Bangs Fond, the Danish Medical Research Council, the Novo Nordisk Foundation, the Lundbeck Foundation, and National Institutes of Health Grants AI36964 (to W.L.H.), AI64811 (to W.L.H.), and T32AI004244 (to A.S.M.).

Abbreviations used in this article:

CHS

contact hypersensitivity

DETC

dendritic epidermal T cell

dLN

draining lymph node

DNFB

2,4-dinitrofluorobenzene

DNSB

2,4-dinitrobenzene sulfonic acid

i.d.

intradermal(ly)

OOA

olive oil:acetone

rm

recombinant murine

WT

wild-type.

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M.M.N. is partly employed by LEO Pharma A/S and P.L. is employed by LEO Pharma A/S, which funded part of this research.