IL-21 Promotes Skin Recruitment of CD4+ Cells and Drives IFN-γ–Dependent Epidermal Hyperplasia

Massimiliano Sarra, Roberta Caruso, Maria Laura Cupi, Ivan Monteleone, Carmine Stolfi, Elena Campione, Laura Diluvio, Annamaria Mazzotta, Elisabetta Botti, Sergio Chimenti, Antonio Costanzo, Thomas T. MacDonald, Francesco Pallone and Giovanni Monteleone
J Immunol May 1, 2011, 186 (9) 5435-5442; DOI: https://doi.org/10.4049/jimmunol.1003326

Abstract

Psoriasis is a chronic inflammatory disorder of the skin characterized by epidermal hyperplasia and infiltration of leukocytes into the dermis and epidermis. T cell-derived cytokines, such as IFN-γ and IL-17A, play a major role in the psoriasis-associated epidermal hyperplasia, even though factors/mechanisms that regulate the production of these cytokines are not fully understood. We have recently shown that IL-21 is synthesized in excess in psoriatic skin lesions and causes epidermal hyperplasia when injected intradermally in mice. Moreover, in the human psoriasis SCID mouse model, neutralization of IL-21 reduces both skin thickening and expression of inflammatory molecules, thus supporting the pathogenic role of IL-21 in psoriasis. However, the basic mechanism by which IL-21 promotes skin pathology remains unknown. In this study, we show that CD4+ cells accumulate early in the dermis of IL-21–treated mice and mediate the development of epidermal hyperplasia. Indeed, IL-21 fails to induce skin damage in RAG1-deficient mice and CD4+ cell-depleted wild-type mice. The majority of CD4+ cells infiltrating the dermis of IL-21–treated mice express IFN-γ and, to a lesser extent, IL-17A. Studies in cytokine knockout mice show that IFN-γ, but not IL-17A, is necessary for IL-21–induced epidermal hyperplasia. Finally, we demonstrate that IFN-γ–producing CD4+ cells infiltrating the human psoriatic plaque express IL-21R, and abrogation of IL-21 signals reduces IFN-γ expression in cultures of psoriatic CD4+ cells. Data indicate that IL-21 induces an IFN-γ–dependent pathogenic response in vivo, thus contributing to elucidate a mechanism by which IL-21 sustains skin-damaging inflammation.

Psoriasis is one of the most common immune-inflammatory disorders in the world, affecting 1–2% of white individuals (1). Histological features of psoriatic skin lesions include epidermal hyperplasia with acanthosis, caused by aberrant terminal differentiation and hyperproliferation of epidermal keratinocytes, and a marked infiltration of mononuclear leukocytes into the dermis and epidermis (1). The exact sequence of events leading to the development of psoriatic plaque is not fully understood, but accumulating evidence suggests that, following innate immunity activation, T cells are recruited into the skin, where they release huge amounts of cytokines that sustain the excessive growth of keratinocytes (1, 2). Specifically investigation on the immunophenotype and cytokine secretion patterns of T cells in psoriatic patients has indicated that Th1 (i.e., IFN-γ) and Th17 cell-derived cytokines (i.e., IL-17A and IL-22) are involved in the development of psoriatic plaque (3). Although various factors, including IL-23, are supposed to play a decisive role in expanding Th17 cell responses (4–6), the mechanism by which Th1 cells are induced and maintained within the psoriatic environment remains unclear.

We have recently shown that in psoriatic skin there is an exaggerated production of IL-21 (7), a cytokine produced by activated CD4+ T cells, NK T cells, and T follicular cells (8). Injection of IL-21 into the skin of wild-type mice induces epidermal hyperplasia, parakeratosis, and accumulation of T cells into the skin in an IL-22–independent manner (7). Consistently, in the human psoriasis SCID mouse model, neutralization of IL-21 reduces epidermal hyperplasia (7), thus supporting the pathogenic role of IL-21 in psoriasis. Nonetheless, the mechanism whereby IL-21 promotes skin pathology remains unknown.

In this study, we examined whether T cells are involved in the IL-21–driven epidermal hyperplasia.

Materials and Methods

Animals

Six- to 8-wk-old wild-type (Harlan, Udine, Italy), Rag1 knockout (KO) (The Jackson Laboratory Bar Harbor, ME), IFN-γ KO (Charles River Laboratories, Wilmington, MA), and IL-17A KO (Regeneron, Tarrytown, NY) mice, all on C57BL/6 background, were used for the in vivo experiments. Wild-type, IFN-γ KO, and IL-17A KO mice were maintained in standard animal cages under specific pathogen-free conditions in the animal facility at the University Tor Vergata (Rome, Italy), whereas RAG1 KO mice were hosted in specific pathogen-free conditions at the Fondazione Parco Biomedico San Raffaele (Rome, Italy). Experiments were approved by the local ethics committee.

In vivo mouse studies

All reagents were from Sigma-Aldrich (Milan, Italy) unless specified. Coupling of IL-21 to 500-nm silica microparticles (Kisker-Biotech, Steinfurt, Germany) was performed according to the manufacturer’s instructions. Briefly, silica microparticles (50 mg/ml) were incubated with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (31 mM) and N-hydroxysuccimide (104 mM) for 1 h at room temperature. Afterward, 25 μl microparticle suspension was washed with MES buffer 0.1 M (pH 6.3) and incubated with 500 ng IL-21 (R&D Systems, Minneapolis, MN) at room temperature in continuous mixing. After 3 h, glycine (final concentration, 2.5 mM) was added to the IL-21–coated microparticle suspension and incubated for an additional 30 min. At the end, IL-21–coupled silica microparticles were washed and resuspended in PBS and given intradermally to the mice (500 ng/50 μl/mouse). Control mice were intradermally injected with uncoated silica microparticles (vehicle, 50 μl/mouse). Mice were epilated at day −3 and daily disinfected during the treatment. In some experiments, wild-type mice were treated i.p. with a depleting anti-mouse CD4 Ab (100 μg/mouse; R&D Systems) 2 d before and 2 d after IL-21 injection or with control IgG (100 μg/mouse; R&D Systems). In addition, wild-type mice were given i.p. a neutralizing mouse IFN-γ Ab or control IgG (300 μg/mouse; R&D Systems) 2 h before and 3 d after IL-21 administration. In another set of studies, wild-type mice were i.p. administered with a neutralizing anti-mouse CCL27 Ab or control IgG (500 μg/mouse; R&D Systems) 2 h before and 2 d after IL-21 administration. CD4+ cells were isolated from splenocytes of wild-type and IFN-γ KO mice by using a CD4+ cell isolation kit (Miltenyi Biotec, Bologna, Italy) and i.v. administrated (10 × 106/mouse) into RAG1 KO mice, and the animals were then injected with IL-21.

Histopathological analysis and immunofluorescence

Frozen sections were stained with H&E- (Sigma-Aldrich), anti-mouse CD4-, CD3 (both from BD Biosicences)-, and CD8-specific Ab (BioLegend, San Diego, CA), followed by incubation with a highly sensitive biotinylated secondary Ab (DakoCytomation, Glostrup, Denmark) and a Tyramide Signal Amplification Kit (PerkinElmer, Waltham, MA). Epidermal thickness and skin T cell infiltrate were analyzed as described previously (7).

Flow cytometry

The following anti-mouse Abs were used: CD4-PerCP, CD4-allophycocyanin, CD4-FITC, IL-10–allophycocyanin (BD Biosciences, Milan, Italy), IFN-γ-PE, IL-17A-Alexa Fluor 647, CCR7-PerCP, Foxp3-Alexa Fluor 647 (eBioscience, San Diego, CA), CCR5-PE, CD25-PE (BioLegend), and isotype control IgGs (BD Biosciences). Anti-human Abs included IL-21R-PE (R&D Systems), IFN-γ-FITC, CD4-PerCP, CD161-allophycocyanin (BD Biosciences), CD4-allophycocyanin (Miltenyi Biotec), and isotype control IgGs (BD Biosciences). Annexin V (AV) and propidium iodide (PI)-positive cells were examined by a commercially available kit (Beckman Coulter, Brea, CA).

Mononuclear cell culture and analysis

Mononuclear cells were isolated from the skin of wild-type mice injected with IL-21 or vehicle as described previously (9). Freshly isolated cells from IL-21–treated mice were analyzed for CD4, CCR5, CCR7, CD25, and Foxp3 by flow cytometry or alternatively stimulated with PMA (80 pM; Sigma-Aldrich), ionomycin (1 mg/ml; Sigma-Aldrich), and monensin (2 μM; eBioscience) for 5 h and then analyzed by flow cytometry for the expression of IFN-γ, IL-17A, and IL-10.

Patients and controls

Biopsies were collected from the lesional skin of seven patients with psoriasis taking no drug and six normal controls and used for isolating mononuclear cells as described previously (10). Dermal cells from psoriatic patients were stimulated with PMA, ionomycin, and monensin for 5 h and then analyzed for CD4, IFN-γ, and IL-21R or cultured with a neutralizing human IL-21 or control Ab (10 μg/ml) for 72 h and then analyzed for AV/PI. PMA, ionomycin, and monensin were added in the last 5 h of culture with the anti–IL-21 or control IgG, and cells were then analyzed for CD4 and IFN-γ expression by flow cytometry. Dermal cells isolated from normal skin were stimulated with IL-21 (25–100 ng/ml; Biosource International, Camarillo, CA) for 24 and 48 h and then analyzed for the content of IFN-γ RNA transcripts and protein by real-time PCR and flow cytometry, respectively.

Real-time PCR

The following conditions were used: denaturation 1 min at 95°C, annealing 30 s at 58°C for CCL20, at 60°C for CCL5 and at 62°C for CCL27, followed by 30 s of extension at 72°C. Primers sequence was as follows: CCL5 FWD, 5′-TTCTACACCAGCAGCAAGTG-3′; CCL5 REV, 5′-AGCAAGCAATGACAGGGAAG-3′; CCL20 FWD, 5′-GTCTGCTCTTCCTTGCTTTG-3′; CCL20 REV, 5′-GCCATCTGTCTTGTGAAACC-3′; CCL27 FWD, 5′-TCCTGAAGCAGCCTTGCCTC-3′; and CCL27 REV: 5′-GGGGATGAACACAGACACTGC-3′. Primers for human IFN-γ, mouse IFN-γ, mouse IL-17A, mouse and human β-actin, and real-time PCR conditions were described elsewhere (7).

IL-21 ELISA

Total proteins were extracted from skin samples of wild-type mice intradermally injected with silica microparticles coated or not with IL-21 and used to analyze IL-21 by ELISA using a commercially available kit (R&D Systems).

Cultures of skin draining lymph node cells

Mononuclear cells, isolated from skin draining lymph nodes of untreated wild-type mice, were either stimulated with PMA, ionomycin, and monensin for 5 h or cultured in the presence or absence of activating anti-CD3 with or without exogenous IL-21 (50 ng/ml) for 24 h. Cells were analyzed by flow cytometry using the following Abs: CD4-PerCP, IFN-γ-FITC (BD Biosciences), IL-21R-PE (BioLegend), or relative control IgGs (BD Biosciences).

Proliferation assay

Mononuclear cells isolated from the skin draining lymph nodes were labeled with CFSE (Molecular Probes, Eugene, OR), as previously described (7), and cultured in the presence or absence of activating anti-CD3 with or without exogenous IL-21 (50 ng/ml) for 72 h. Cells were then harvested and analyzed for the content of CFSE and CD4 by flow cytometry.

Data analysis

Differences between groups were compared using the Student t test or the Mann-Whitney U test.

Results

CD4+ cells are necessary for the IL-21–driven epidermal hyperplasia

In initial studies, we characterized the skin T cell infiltrate in C57BL/6 mice injected intradermally with IL-21. To prevent skin damage caused by multiple injections, IL-21 was coated to silica particles and administered once (day 0). Enhanced levels of IL-21 protein were seen in the skin of IL-21–treated mice at day 2; there was then a decline in such a production at days 4 and 6 (Supplemental Fig. 1A). Time-course studies revealed also that CD4+, but not CD8+, cells accumulated into the dermis as early as 2 d following IL-21 injection, whereas a significant skin hyperplasia was seen only at day 6 (Fig. 1A). At this later time point, some CD4+ cells were also seen in the epidermal compartment of IL-21–treated mice (Fig. 1A). Because accumulation of CD4+ cells into the dermis of IL-21–injected mice preceded the development of epidermal hyperplasia, we next examined whether IL-21–driven epidermal hyperplasia was mediated by CD4+ cells. First, we injected RAG1-deficient mice with IL-21. No epidermal thickening was seen in these animals following IL-21 administration (Fig. 1B). Second, wild-type mice were depleted of CD4+ cells before IL-21 injection. Depletion of CD4+ cells (Fig. 1C) largely protected mice against IL-21–induced epidermal damage (Fig. 1D).

FIGURE 1.
FIGURE 1.

IL-21–induced epidermal hyperplasia is mediated by CD4+ cells. A, Photomicrographs of CD4/DAPI-stained (upper panels), CD8/DAPI-stained (middle panels), and H&E-stained (lower panels) frozen sections of skin samples taken from mice injected intradermally with 500 ng IL-21–coated silica particles (IL-21) or silica particles (vehicle). The mice were sacrificed at days 2, 4, and 6 after IL-21 injection. The photomicrographs are representative of three separate experiments in which 12 mice/group were analyzed. Original magnification ×100. In the right panels corresponding to CD4 and CD8 staining, insets show staining at higher magnification (×200). B, Representative H&E-stained sections of skin biopsies taken from RAG1-deficient mice (n = 14) injected intradermally with 500 ng IL-21 or vehicle. Mice were sacrificed at day 6, and skin samples were stained with H&E. Right inset shows the epidermal thickness measured at day 6. Data indicate mean ± SD of all experiments. C, Representative dot plots of CD4 staining in cells isolated from the spleen of wild-type mice injected i.p. with 100 μg depleting CD4 Ab (aCD4) or control IgG at day −2 and day 2. Mice were killed at day 6, and splenic cells were analyzed by flow cytometry. Numbers above boxes indicate the percentages of CD4-positive cells. Staining with a nonrelevant isotype IgG is also shown. D, Representative H&E-stained sections of skin biopsies taken from wild-type mice treated as indicated in C and injected intradermally with 500 ng IL-21 or vehicle at day 0. Mice were killed at day 6, and skin section was stained with H&E. Right inset shows the epidermal thickness measured at day 6. Data indicate mean ± SD of all experiments (n = 10 mice/group). **p = 0.01, *p = 0.03.

IFN-γ mediates the development of epidermal hyperplasia in IL-21–treated mice

We previously showed that, in vivo in mice, intradermal injection of IL-21 is followed by enhanced gene expression of IFN-γ and IL-17A (7). To show that these cytokines were induced in CD4+ cells, we collected mononuclear cells from the skin of IL-21–treated mice and analyzed IFN-γ and IL-17A at a single-cell level by flow cytometry. The majority of dermal CD4+ cells expressed IFN-γ and, to a lesser extent, IL-17A; moreover, ∼10% of cells coexpressed IFN-γ and IL-17A (Fig. 2A). By contrast, <5% of these cells were positive for IL-10 or Foxp3 (Fig. 2A; data not shown). Consistently, more than two-thirds of dermal CD4+ cells were positive for CCR5, a chemokine receptor that is predominantly expressed on Th1 cells (Fig. 2B) (11). Moreover, ∼40% of CCR5+ cells coexpressed CCR7, a secondary lymphoid tissue homing receptor that is also induced in Th17 cells (Fig. 2B) (12). To explore the role of IFN-γ and IL-17A in IL-21–driven skin thickening, IL-21 was injected into the skin of IFN-γ or IL-17A KO mice. IFN-γ KO mice did not develop epidermal hyperplasia (Fig. 2C), although they exhibited a marked infiltration of CD4+ cells into the dermis (Fig. 2D) and the high IL-17A expression (Fig. 2E). By contrast, IL-21–injected IL-17A KO mice showed a marked epidermal thickening (Fig. 2F), accumulation of CD4+ cells into the dermis (Fig. 2G), and increased expression of IFN-γ (Fig. 2H).

FIGURE 2.
FIGURE 2.

IL-21–induced epidermal hyperplasia is mediated by IFN-γ. A, Percentage of CD4+ cells expressing IFN-γ and/or IL-17A or IL-10 as assessed by flow cytometry. Mononuclear cells were isolated from the skin of wild-type mice injected intradermally with IL-21. On day 4, mice were sacrificed, and dermal cells were stained with CD4 and cytokine Abs and analyzed by flow cytometry. Data indicate mean ± SD of four separate experiments. B, Representative dot plots showing the percentage of CD4+ cells expressing CCR5 and CCR7 in dermal cells isolated from the skin of mice treated as indicated in A. Numbers in the quadrants indicate the percentages of CD4+ cells expressing CCR5/CCR7. Staining with nonrelevant isotype IgGs is also shown. One of four representative experiments is shown. C, Representative H&E-stained sections of skin biopsies taken from IFN-γ KO (IFN-γ−/−) mice injected intradermally with 500 ng IL-21 or vehicle. On day 6, mice were sacrificed, and skin samples were stained with H&E. Right insets show the epidermal thickness measured at day 6 (n = 6). D, Skin sections were also stained with CD4 Ab/DAPI, and the number of CD4+ cells for field was calculated and shown as mean ± SD of all experiments (n = 6; *p = 0.03). E, Real-time PCR for IL-17A RNA transcripts in samples prepared from the skin of IFN-γ−/− mice injected as above. Levels are normalized to β-actin. Values are mean ± SD of all experiments (n = 6; *p = 0.04). F, Representative H&E-stained sections of skin biopsies taken from IL-17A KO (IL-17A−/−) mice injected intradermally with 500 ng IL-21 or vehicle. On day 6, mice were sacrificed, and skin samples were stained with H&E. Right insets show the epidermal thickness measured mice at day 6 (n = 7; **p = 0.01). G, Skin sections were also stained with CD4 Ab/DAPI, and the number of CD4 T cells for field was calculated and shown as mean ± SD of all experiments (n = 6; *p = 0.02). H, Real-time PCR for IFN-γ RNA transcripts in samples prepared from the skin of IL-17A−/− mice injected as indicated in F. Levels are normalized to β-actin. Values are mean ± SD of all experiments (n = 7; **p = 0.01). I, Representative H&E-stained sections of skin biopsies taken from wild-type (WT) mice, which received administrations of a neutralizing anti–IFN-γ Ab (aIFN-γ) or control (IgG) Ab and injected intradermally with 500 ng IL-21 or vehicle. The anti–IFN-γ and control Abs were injected i.p. 2 h before and 3 d after IL-21 injection. Mice were killed at day 6, and skin sections were stained with H&E. Right inset shows the epidermal thickness measured at day 6. Data indicate mean ± SD of all experiments (n = 6). **p = 0.01, *p = 0.03.

The lack of epidermal damage we observed in IFN-γ KO mice after injection of IL-21 was not due to any developmental defect in this strain, because a significant decrease in epidermal thickness was observed in wild-type mice receiving a neutralizing IFN-γ Ab and exposed to IL-21 in comparison with those receiving an isotype control Ab (Fig. 2I).

IL-21 promotes CD4+ cell recruitment into the skin through an IFN-γ–independent mechanism and stimulates dermal CD4+ cells to produce IFN-γ

To confirm that IL-21 promotes skin homing of CD4+ cells, IL-21– or vehicle-treated RAG1-deficient mice were injected i.v. with purified splenic wild-type or IFN-γ–deficient CD4+ cells, and 6 d later, skin sections were stained with anti-CD3 Ab. Both wild-type and IFN-γ–deficient CD4+ cells migrated equally in the dermis of RAG1-deficient mice in response to IL-21 (Fig. 3A). However, IL-21–treated RAG1 mice injected with wild-type but not IFN-γ–deficient CD4+ cells developed epidermal hyperplasia (Fig. 3B), thus confirming that IFN-γ is necessary for IL-21–driven epidermal hyperproliferation but not for skin recruitment of CD4+ cells. Skin samples taken from IL-21–treated wild-type mice expressed high levels of transcripts for CCL20 and CCL27, two chemokines involved in the recruitment of T cells (13, 14), whereas CCL5 remained unchanged (Fig. 4A). Because interactions between CCL27 and CCR10 play a key role in T cell-mediated skin pathology (13), we next assessed the role of CCL27 in the IL-21–mediated skin infiltration of T cells. In vivo in mice, blockade of CCL27 with a neutralizing Ab largely reduced T cell migration as well as epidermal hyperplasia seen in IL-21–treated mice (Fig. 4B, 4C).

FIGURE 3.
FIGURE 3.

IL-21 promotes skin homing of CD4+ cells. A, Representative CD3/DAPI-stained sections of skin biopsies taken from RAG1-deficient mice injected intradermally with 500 ng IL-21 or vehicle and then i.v. with splenic (10 ×106 cells/mouse) CD4+ T cells purified from wild-type (WT) mice and IFN-γ−/− mice. On day 6, mice were sacrificed, and skin samples were stained with CD3/DAPI. Original magnification ×100. In the left upper panel corresponding to CD4/DAPI staining in mice injected with IFN-γ–deficient cells, inset shows staining at higher magnification (×200). Right inset shows the number of CD3+ T cells per field measured at day 6. Data indicate mean ± SD of all experiments (n = 9). B, Mice were treated as in A, and skin sections were stained with H&E. Right inset shows the epidermal thickness measured at day 6. Data indicate mean ± SD of all experiments. *p < 0.04.

FIGURE 4.
FIGURE 4.

Involvement of CCL20 in the IL-21–induced skin homing of T cells. A, Real-time PCR for CCL5, CCL27, and CCL20 RNA transcripts in samples prepared from the skin of wild-type mice injected intradermally with IL-21 or vehicle. Mice were killed 12 h after IL-21 injection. Levels are normalized to β-actin. Values are mean ± SD of all experiments (n = 7; *p < 0.05). B, Mice were injected intradermally with 500 ng IL-21 or vehicle and i.p. treated with anti-mouse CCL27 neutralizing Ab (aCCL27) or isotype control (IgG). Mice were killed 6 d after IL-21 administration. Skin samples were stained for CD4/DAPI. Original magnification ×100. In the middle panel, right inset shows CD4 staining at higher magnification (×200). Right inset shows the number of CD4+ cells per field measured at day 6. Data indicate mean ± SD of all experiments (n = 9). *p < 0.02, **p < 0.005. C, Mice were treated as in B and skin samples were stained for H&E. Original magnification ×100. Right inset shows the epidermal thickness measured at day 6. Data indicate mean ± SD of all experiments (n = 9). *p = 0.04, **p < 0.001.

The fact that CD4+ cells migrate into the skin of IL-21–treated mice independently of IFN-γ and that the expression of this cytokine is upregulated in IL-21–treated skin suggests that IL-21 can stimulate CD4+ cells to synthesize IFN-γ. Indeed, skin draining lymph node cells isolated from untreated wild-type mice express IL-21R and respond in vitro to IL-21 with enhanced expression of IFN-γ and proliferation (Supplemental Fig. 2).

IL-21 positively regulates IFN-γ production in human psoriasis

In a final set of experiments, we determined whether IL-21 controls IFN-γ production in human skin. In psoriatic patients, more than two-thirds of IFN-γ–producing CD4+ cells expressed IL-21R, and these percentages were significantly greater than in normal controls (Fig. 5A). Because IL-21 is spontaneously produced by psoriatic CD4+ T cells (7), we next evaluated the effect of a neutralizing IL-21 Ab on IFN-γ synthesis in cultures of dermal mononuclear cells isolated from psoriatic lesions. In all experiments, neutralization of IL-21 reduced the fraction of IFN-γ–expressing CD4+ cells (Fig. 5B) without affecting cell survival (Fig. 5C). In both anti–IL-21 and IgG-treated cell cultures, <5% of the of IFN-γ–expressing CD4+ cells were positive for the NKT cell marker CD161 (data not shown), thus indicating that the majority of IFN-γ–expressing CD4+ cells in these cultures are T cells.

FIGURE 5.
FIGURE 5.

IL-21 positively regulates IFN-γ expression in the human skin. A, Percentage of IFN-γ–expressing CD4+ cells positive for IL-21R in four healthy controls (HC) and four psoriatic patients (Pso). Dermal mononuclear cells were cultured in vitro with PMA, ionomycin, and monensin for 5 h and then assessed for the expression of CD4, IFN-γ, and IL-21R by flow cytometry. Data indicate mean ± SD of all experiments. B, Cells isolated from the dermis of psoriatic patients were cultured in vitro with a neutralizing IL-21 or control Ab for 72 h and then assessed for the expression of CD4 and IFN-γ by flow cytometry. Each point represents the percentage of CD4+ cells expressing IFN-γ in cultures treated with anti–IL-21 or control IgG. Horizontal bar indicates median. C, Cells cultured as indicated in B were evaluated for the content of AV and/or PI by flow cytometry. Results are mean ± SD of four experiments. D and E, Cells isolated from the dermis of normal subjects were cultured in vitro with or without increasing doses of IL-21 for 24 h (D) or 48 h (E). At the end, cells were used either to extract RNA (D) or to analyze CD4 and IFN-γ by flow cytometry (E). One of two separate experiments in which cells from two healthy subjects were analyzed is shown. **p = 0.01.

To confirm that IL-21 positively regulates IFN-γ production in the skin, we cultured normal dermal T cells with exogenous IL-21 and then assessed IFN-γ expression by real-time PCR and flow cytometry. IL-21 dose-dependently enhanced IFN-γ production (Fig. 5D, 5E).

Discussion

The present study was undertaken to investigate the mechanism by which IL-21 sustains the excessive growth of keratinocyte in vivo in mice. Our previous work showed that the pathological changes seen in the epidermal compartment of IL-21–treated mice were not mediated by IL-22 (7), a cytokine known to exert proliferative effects on keratinocytes (15, 16). Because the epidermal hyperplasia induced by IL-21 in mice was associated with a marked accumulation of T cells into the skin (7), we hypothesized that these cells could be involved in the IL-21–driven epidermal pathology. Indeed, a considerable amount of experimental and clinical work has been produced to show that T cells are key regulators of keratinocyte growth and survival (2, 17, 18). Immunohistochemical analysis of skin sections taken from mice treated with IL-21 showed a marked accumulation of CD4+ but not CD8+ cells into the dermis, which preceded the development of epidermal hyperplasia. Interestingly, no significant epidermal change was seen in RAG1-deficient mice, which lack both lymphocytes and NK cells, and CD4+ cell-depleted wild-type mice following IL-21 injection, clearly indicating that CD4+ cells are mediators of IL-21–induced epidermal pathology. The lack of epidermal thickening we observed in IL-21–treated RAG1-deficient mice apparently conflicts with our previous demonstration that IL-21 is growth stimulatory for human keratinocytes in vitro (7). In this context, however, it is noteworthy that other studies have previously shown that keratinocytes can respond differently to recombinant cytokines in terms of proliferation and survival depending on whether they are stimulated in vitro or in vivo (19, 20).

CD4+ T cells cloned from psoriatic lesions stimulate keratinocyte growth in vitro through a mechanism, which is supposed to be largely mediated by Th1/Th17-related cytokines (3, 21, 22). The lack of IFN-γ but not IL-17A protected mice against the IL-21–mediated skin pathology. This observation together with the demonstration that CD4+ cells infiltrated normally the skin of IFN-γ null mice following IL-21 injection strongly suggest that IFN-γ is crucial in the IL-21–mediated epidermal changes. In line with this is the demonstration that neutralization of IFN-γ with a blocking Ab largely prevented the development of epidermal hyperplasia in IL-21–treated wild-type mice. These findings are consistent with results of previous studies showing that IFN-γ administration in vivo to psoriatic uninvolved skin results in epidermal hyperproliferation (19). By contrast, IFN-γ was reported to exert inhibitory effects on the growth of keratinocytes in vitro in primary cultures (20). A plausible explanation for these discrepancies is that IFN-γ is critical but not sufficient to drive keratinocytes proliferation and that, in vivo in the epidermis, other factors provide the necessary costimulatory signals for IFN-γ to become growth stimulatory.

Although IL-21 has been reported to exert suppressive effects on IFN-γ expression in particular experimental settings (23), the above data clearly show that IL-21 is a positive regulator of IFN-γ synthesis in the skin and are in line with studies in other systems showing that IL-21 assists the in vitro induction of Th1-related molecules (24, 25). Support to this notion derives also from the demonstration that stimulation of T cells isolated from skin draining lymph nodes of wild-type mice with IL-21 enhanced IFN-γ expression. Moreover, our study with human skin samples revealed that IL-21R is highly expressed by IFN-γ–producing CD4+ cells in psoriatic plaque and that neutralization of IL-21 reduced IFN-γ synthesis in cultures of dermal CD4+ cells isolated from psoriatic lesions.

The demonstration that IL-21 enhances cutaneous expression of IL-17A well fits with recent reports showing the involvement of IL-21 in the positive regulation of Th17 cell differentiation and IL-17A production (26–28). IL-17A KO mice were not protected against IL-21–driven skin hyperplasia, thus suggesting that IL-17A is not necessary for driving the epidermal pathology in our model. We cannot, however, exclude the possibility that IL-17A can cooperate with IFN-γ in promoting epidermal hyperplasia in wild-type mice, because IL-17A has been reported to induce inflammatory genes in human keratinocytes, particularly in combination with IFN-γ (29).

The fact that IFN-γ null mice did not develop epidermal hyperplasia, even though they exhibited a marked infiltration of CD4+ cells into the dermis, indicates that IL-21 promotes skin homing of CD4+ cells through an IFN-γ–independent mechanism. This is also supported by the demonstration that CD4+ cells injected systemically into RAG1-deficient mice migrate to the skin in response to IL-21 independently of their ability to make IFN-γ. Although the exact mechanism whereby IL-21 promotes skin trafficking remains to be ascertained, we show in this study that IL-21–induced skin reaction causes upregulation of CD4+ cell chemoattractants such as CCL20 and CCL27, two chemokines that are supposed to play a pivotal role in T cell-mediated skin inflammation (13, 30). Consistently, blockade of CCL27 with a neutralizing Ab impaired T cell recruitment to the skin of IL-21–treated mice. The anti-CCL27 Ab also attenuated the IL-21–induced skin damage, thus supporting the role of recruited T cells in the IL-21–driven epidermal hyperplasia. The fact that anti-CCL27 did not abrogate T cell skin infiltration could stem from our technical difficulty to completely neutralize CCL27. Another possibility is that additional chemokines can cooperate with CCL27 in promoting T cell migration to the skin of IL-21–treated mice.

The skin changes seen in IL-21–treated mice do not model human psoriasis. However, some immune-morphological features documented in our model, such as infiltration of CD4+ cells into the dermis and epidermis, induction of IFN-γ and epidermal hyperplasia are similar to features present in psoriasis (1, 2). These findings together with the demonstration that IL-21 stimulates the growth of primary keratinocytes isolated from nonlesional skin of psoriatic patients (7) suggest a multiple-step mechanism whereby IL-21, produced within the psoriatic plaque, can amplify the skin-damaging Th1 cell inflammation. A critical question remains as to what induces IL-21 in the psoriatic plaque. An intriguing hypothesis is that IL-21 production is positively regulated by plasmacytoid dendritic cells-derived IFN-α, because studies in other systems have shown that IFN-α stimulates T cells to produce IL-21 (31).

In conclusion, these data show that IL-21 promotes skin trafficking of CD4+ T cells and elicits an abnormal IFN-γ–dependent epidermal reaction in vivo.

Disclosures

G.M. has filed a patent entitled “Interleukin-21 (IL-21) binding proteins and methods of making and using same” (European Patent Application No. 08425294.9).

Footnotes

  • This work was supported by the Fondazione Umberto di Mario and Giuliani SpA (Milan, Italy).

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    AV
    annexin V
    KO
    knockout
    PI
    propidium iodide.

  • Received October 6, 2010.
  • Accepted February 23, 2011.

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