All chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated. Recombinant human IL-17A and IL-22 were purchased from PeproTech (Rocky Hill, NJ, USA). The following monoclonal antibodies (mAbs) were used: anti-BrdU antibody (BU-33; B2531) was obtained from Sigma-Aldrich, anti-BrdU antibody conjugated with FITC (ab74545) was purchased from Abcam (Cambridge, MA, USA), antibodies against differentiation-specific markers, namely K15 (ab52816; Abcam), K10 (SC31770; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), integrin β1/FITC (ab46920), filaggrin (ab24584), and Ki67 (ab16667) were all purchased from Abcam. Secondary antibodies conjugated with Alexa Fluor 488 (green; A11001) or 555 (red; A21432) were purchased from Molecular Probes (Eugene, OR, USA). Mowiol 4–88 anti-fade mounting solution was obtained from Polyscience, Inc. (Warrington, PA, USA).

Eleven patients suffering from psoriasis vulgaris and ten healthy controls were enrolled in this study. The clinical and demographic characteristics of the subjects included in this study were summarized. Certain patients had a family history of the disease (data not shown). At the time that skin biopsies were taken, medications had been discontinued for several months. Samples were obtained under local anesthesia from psoriasis skin plaques using a 6-mm biopsy punch. All patients were recruited from the Department of Dermatology (Renmin Hospital of Wuhan University, Wuhan, China), and normal skin samples were collected from patients who underwent breast reconstruction in the Department of Plastic Surgery (Renmin Hospital of Wuhan University). This study was conducted in accordance with the Declaration of Helsinki Principles, and was approved by the Ethics Committee of Renmin Hospital of Wuhan University.

BALB/c male mice (8–11 weeks old) were purchased from the animal facility of Hubei Provincial Center for Disease Control and Prevention (CDC; Wuhan, China), raised in pathogen-free conditions and provided with food and water ad libitum. All experiments were approved by the Animal Ethics Committee according to Chinese legislation on animal experiments. Mice were treated daily with commercially available IMQ cream (5%) (Aldara; 3M Pharmaceuticals, Minneapolis, MN, USA), which induced psoriasiform dermatitis, on shaved dorsal skin, or treated with a control cream on shaved dorsal skin for 6 consecutive days as previously reported (6). BrdU incorporation was performed to monitor the mitotic activity of basal cells in the inflamed skin. A single dose of BrdU at 50 µg/gram body weight was administered to mice by intraperitoneal injection 2 h prior to mice being sacrificed by carbon dioxide inhalation. Dorsal skin was biopsied and fixed in buffered formalin and embedded in paraffin. Five-micrometer sections were cut on a microtome and stained with mouse-anti-BrdU (Sigma-Aldrich) and a commercially available streptavidin-peroxidase (SP) staining kit (Maixin-Bio, Fuzhou, China) in order to visualize BrdU immunostaining in the paraffin-embedded sections.

As previously described (17), the skin tissue was trimmed to remove subcutaneous adipose tissue and cut into small strips. These skin strips were soaked in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Carlsbad, CA, USA) containing 0.25% dispase (Sigma-Aldrich) and digested overnight at 4°C. The following day, the epidermis was peeled off and further dissociated after simple digestion in 0.25% trypsin. Single cell cultures were obtained by passing the cell suspension through a nylon cell strainer with 70-µm pore size (Becton-Dickinson, Franklin Lakes, NJ, USA) and then washing with PBS. Primary cells were maintained in EpiLife keratinocyte medium with 1% (v/v) human keratinocyte growth supplement (both from Invitrogen, Camarillo, CA, USA) and 60 µM calcium. Cells at an early passage (≤5) were used for all experiments. To analyze the expression pattern of differentiation-related proteins, the calcium concentration was increased to 1.3 mM in cultures. The primary cells were also stimulated with recombinant IL-17A (10 ng/ml) and IL-22 (10 ng/ml) for 24 h before protein extraction (17,18).

For labeling of cells in culture, the cells were plated at a low density (~10–20 cells/mm²) in 6-well tissue culture plates which contained a collagen (Sigma-Aldrich)-coated coverslip in each well, as previously described (19,20). After cells had attached, 10 µM BrdU (B9285; Sigma-Aldrich) was added for 30 h of labeling. Subsequently, cell cultures were washed with PBS twice and replaced with fresh medium containing 5 µM cytochalasin D (C8273; Sigma-Aldrich) to arrest cytokinesis. After a chase period of 30 h, cells on the coverslips were fixed with 4% paraformaldehyde solution for subsequent BrdU immunostaining.

Passage one epidermal cell sheets were prepared following a previously reported protocol (16). Briefly, skin tissue samples were trimmed to remove subcutaneous tissue and were further cut into small strips using ophthalmologic scissors. Skin strips were placed on glass coverslips at the bottom of 6-well plates with the epidermal side facing upwards. After allowing the skin pieces to firmly attach to the coverslips, DMEM containing 20% fetal bovine serum (FBS) and antibiotics (0.625 µg/ml amphotericin B, 100 IU/ml penicillin and 100 µg/ml streptomycin) were carefully added to the plates, ensuring no tissue pieces floated upwards. After 3–5 days of cultivation, passage one epidermal cells had grown out from the edge of each skin explant to form a tongue-like epidermal sheet, as visualized in Fig. 6A. The epidermal sheet cultures were treated with recombinant IL-17A (10 ng/ml) and IL-22 (10 ng/ml) for 24 h to further analyze the expression pattern of differentiation-related proteins. Finally, the skin pieces were removed gently using an ophthalmologic forceps and fixed with 4% paraformaldehyde solution for subsequent immunofluorescence staining.

In the present study, formalin-fixed paraffin sections (5 µM) were dewaxed by melting for 30–60 min at 60°C, and they were cleared in xylene 3 times for 5 min each and then rehydrated in water bath solutions containing decreasing percentages of ethanol. The sections were then treated to inhibit endogenous peroxidases, blocked and incubated overnight at 4°C with specific primary antibodies against proliferation and/or differentiation-related protein markers: anti-BrdU (dilution 1:100), anti-K15 (1:100), anti-K10 (1:100), anti-integrin β1 (1:50), anti-filaggrin (1:100) and anti-Ki67 (1:100). After incubation with the primary antibodies, sections were rinsed with Tris-buffered solution (TBS) and were then incubated with a biotinylated secondary antibody (dilution 1:2,000; Maixin-Bio, Fuzhou, China) for 1 h. The immune signals were detected using a commercially available SP staining kit (Maixin-Bio). Sections were counterstained with hematoxylin and eosin and mounted. The number of positively immunostained cells in the basal layers (aside from K10 and Ki67 in the suprabasal layer) was counted from at least 6 randomly selected fields under a magnification of x400 and expressed as a percentage of the total cells counted. For BrdU immunofluorescence staining, cell cultures were fixed in 70% ethanol, washed with PBS, and incubated with 4 M HCl for 10 min at room temperature, and then washed 3 times with PBS containing 1% FBS and 0.01% sodium azide. They were then incubated with anti-BrdU/FITC antibody for 45 min at 37°C. The sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen) or propidium iodide (Sigma-Aldrich) to identify the nuclei. The routine protocol was also carried out for the detection of intracellular and extracellular epitopes, as detailed in the figure legends. Double immunofluorescence was performed with anti-BrdU/FITC (dilution 1:100) and anti-K15 (1:50). Slides were mounted with Mowiol 4–88 anti-fade mounting solution. Immunostaining was subsequently observed using an Olympus IX71 fluorescence microscope.

Cells were washed in PBS and lysed in extraction buffer containing 1% Nonidet P-40, 0.01% SDS and a protease inhibitor cocktail (Roche, Indianapolis, IN, USA). Protein content was determined with a BCA assay kit (Pierce, Rockford, IL, USA). Equal amounts of each protein extract (30 µg/lane) were then resolved by using 6% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Following blotting onto Immobilon-P membranes (Millipore, Bedford, MA, USA) and blocking with 5% non-fat milk in saline buffer, the membranes were incubated with anti-K15, anti-K10, anti-integrin, anti-filaggrin, and anti-Ki67, each at a 1:2,000 dilution, or with an anti-GAPDH antibody (Santa Cruz Biotechnology, Inc.) at a dilution of 1:1,000, for 1 h at room temperature. The membranes were then washed and incubated with horseradish-peroxidase-conjugated anti-rabbit IgG (Pierce) at a dilution of 1:2,000 for 1 h at room temperature. Membranes were then washed again, and specific bands were visualized using enhanced chemiluminescence (ECL) reaction (Amersham, Piscataway, NJ, USA).

The Student's t-test was used to compare the average band intensities resulting from western blotting, the average epidermal thickness and the average cell-pair number of asymmetric cell divisions. Epidermal thickness of the dorsal skin was quantifed using ImageJ densitometry software (NIH, Bethesda, MD, USA) (21). Groups were compared using ANOVA. In the present study, a P-value <0.05 (5%) was considered to indicate a statistically significant difference and was denoted with asterisks. SPSS software for Windows version 11.0 was used to analyze the results.

To fully characterize the histological alterations of psoriatic TA cell expansion, we first analyzed the expression of phenotypic markers relevant to proliferation and differentiation in psoriatic and normal skin samples. We used immunohistochemical analysis and a panel of specific antibodies against putative stem cells (K15 and integrin β1), TA cells (integrin β1) and PM cells (K10) as well as a cellular proliferative marker (Ki67) (Fig. 1). The immunostaining results listed in Table I indicate the percentage of positive cells in the psoriatic lesions and the normal epidermis. K15 expression was significantly decreased in the psoriatic epidermis (11.7±3.2%) compared with normal skin (43.3±12.6%, P<0.05). Immunostaining for Ki67 revealed a markedly increased proportion of stained cells in the suprabasal layer, but the emergence of K10-positive keratinocytes was postponed until they reached the granular layer. By contrast to the immunostaining pattern of K10 in the psoriatic epidermis, intensive staining for K10 was clear in the entire suprabasal layer in the normal epidermis, aside from the single basal cell layer that did not express K10. These findings suggest that excessive expansion of the TA cell population existed in the psoriatic epidermis, which may have exhausted the stem cell pool to some extent since K15-positive cells were barely detectable in the psoriatic basal layer. Considering that in vivo lineage tracing to directly monitor changes of the stem cell pool is not feasible in humans (12), we thus measured the number of mitotic basal cells using BrdU labeling in the mouse model of IMQ-induced dermatitis (Fig. 2), which is an animal model simulating some clinical features of human psoriasis (6). Representative stained images of BrdU-labeled basal cells are shown in Fig. 3. A proportion (6%) of BrdU-labeled mitotic basal cells was easily detected in the inflamed skin of the mice, but those cells were negligible in the control mice. Interestingly, two types of asymmetric cell division, perpendicular and parallel (17), were clearly discerned in BrdU-labeled basal cells (Fig. 3). These data indicate that the quiescent basal cells become activated to undergo cell division, which may serve as a prelude to epidermal hyperplasia in this model of psoriasis.

Based on the above observation that K15 immunostaining was significantly reduced in psoriatic basal cells, we further examined whether there was a reduction of the stem cell pool in cases of psoriasis. It should be noted, however, that one may speculate that decreased K15 expression contributes to the active states of stem cells (22,23). First, the BrdU pulse-chase labeling protocol was employed to identify template-DNA strand segregation during mitosis in primary cultures of psoriatic epidermal cells. In general, daughter cells inheriting the older template DNA are considered stem cells according to the immortal strand hypothesis (19,20). The data demonstrated that an increased proportion (~10%) of asymmetric segregation of BrdU was noted in the cell pairs of psoriatic epidermal cells, whereas only 3% was noted in normal epidermal cells (P<0.001) (Fig. 4). This suggests that hyperactive asymmetric stem cell division exists in cultured psoriatic keratinocytes. Secondly, the correlation between asymmetric cell division and the transition from stem cells to TA cells was also investigated by examination of BrdU pulse-chased anaphase cells using dual immunostaining for both BrdU segregation and K15 expression. The results showed that primary keratinocytes underwent asymmetric division in which the template DNA (unlabeled) always segregated to the daughter cell that retained K15, as shown in Fig. 5A. The percentage (~13%) of cells expressing K15 that were asymmetrically labeled with BrdU (BrdU⁻/K15⁺; BrdU⁺/K15⁻; putative asymmetric stem cell division in the second round) was increased in psoriatic keratinocytes compared with normal cells (P<0.05), as shown in Fig. 5B (bottom row). The percentage (~32%) of cells expressing K15 that were symmetrically labeled with BrdU (BrdU⁺/K15⁺; BrdU^+/+; putative stem cell self-renewal in the first round) was also increased in psoriatic keratinocytes compared with normal cells (P<0.01), as shown in Fig. 5B (top row). It seems safe to conclude that the increased proportion of both symmetric and asymmetric cell division may provide a mechanism by which the psoriatic epidermis maintains persistent hyperplastic plaques.

To explore the effects of IL-17A and IL-22 on basal cells expressing stem cell markers characteristic for K15 and the integrin β1, we first established a short-term culture system that allowed passage-one epidermal cells migrating from skin explants to form an epidermal cell sheet, as shown in Fig. 6A. We then treated these tongue-like epithelial sheets with 10 ng/ml recombinant IL-17A plus 10 ng/ml IL-22 for 24 h in vitro. The pattern of K15 and integrin β1 expression was then examined in those epithelial sheets by immunofluorescence staining. The results showed that cells expressing K15 and/or integrin β1 were restricted to the leadingedge zone of the epithelial sheet in the absence of cytokine stimulation. However, following exposure to IL-17A and IL-22, both markers were expressed by cells throughout the epithelial tongue, and integrin β1 was very brightly labeled in the inner cells close to the explant (Fig. 6C). Likewise, western blotting revealed that the expression of K15 and integrin β1 was significantly upregulated in cells stimulated with both cytokines under low (0.06 mM) and high (1.3 mM) calcium conditions compared with untreated controls, and those 2 cytokines also exhibited a mild synergistic action (Fig. 7). These findings demonstrate that IL-17A and IL-22 play critical roles in maintaining an undifferentiated stem-cell like phenotype in basal keratinocytes.