The OHSU Institutional Review Board approved all of the described studies, and the study was conducted according to the Declaration of Helsinki Principles. For archival tissue, written informed consent was waived as the tissue specimens were anonymized. Formalin-fixed, paraffin-embedded lesional skin obtained from seven individuals with psoriasis and three healthy individuals were retrieved from the OHSU dermatopathology archives maintained by Dr Clifton White. Sections were de-paraffinized and hydrated through heating and washing sections in a graded alcohol series. The sections were incubated in antigen-unmasking solution (Vector Laboratories, Burlingame, CA) at 95 °C for 20 minutes, and then exposed to either goat anti-human IL-17A polyclonal Abs (R&D Systems, Minneapolis, MN) or rabbit anti-human CD3 (Thermo Fisher Scientific, Waltham, MA) monoclonal Abs overnight at 4 °C. Samples were washed and incubated for 1 hour with biotinylated anti-goat or anti-rabbit secondary Abs (Vector Laboratories), followed by 1 hour incubation with streptavidin-conjugated horseradish peroxidase (Vectastain Elite ABC kit, Vector Laboratories), developed using Vector VIP substrate kit for peroxidases (Vector Laboratories), and counter-stained with hematoxylin (Vector Laboratories).
For fresh tissue, patients first gave their written informed consent. Four-mm punch biopsies of lesional skin from six individuals with psoriasis were obtained and frozen immediately in OCT compound. Frozen sections were cut, fixed with methanol for 10 minutes, and then incubated with either rabbit anti-human IL-22 polyclonal Abs (Capralogics, Hardwick, MA) or CD3 mAbs overnight at 4 °C. Samples were washed and incubated for 1 hour with biotinylated anti-rabbit secondary Abs (Vector Laboratories) and developed and counter-stained as above.
Frozen mouse skin sections were cut and fixed in methanol for 10 minutes and incubated overnight with rat anti-mouse CD3 monoclonal Abs (Biolegend, San Diego, CA). Sections were then incubated for 1 hour with biotinylated goat anti-rat secondary Abs (Jackson ImmunoResearch Laboratories, West Grove, PA), for 45 minutes with streptavidin-conjugated horseradish peroxidase (Vectastain Elite ABC kit, Vector Laboratories), and developed and counter-stained as above.
All IHC images were captured using a microscope equipped with Imagepro software. For each antibody, the number of positive cells was counted in 10 separate high-powered (×40) fields and then divided by 10 to obtain the average number of positive cells per high-powered field.
Normal human KC cultures
Primary normal human KC (Cascade Biologics, Portland, OR) were cultured in Keratinocyte Growth Media containing bovine pituitary extract, human epidermal growth factor, insulin, hydrocortisone, gentamicin, and amphotericin B. Cytokine stimulation experiments were initiated on 60% confluent KC cultured between passages 2–5 in 6 cm plastic dishes at 37 °C in the presence of 0.05% heat-inactivated bovine serum albumin and in the absence of hydrocortisone. KC were cultured either alone or with one of the following cytokines: 1, 10, 100, or 1,000 ng ml−1 of IL-17A; 0.1, 1, 10, or 100 ng ml−1 of IL-22, TNF-α, or IFN-γ; or 0.002, 0.02, 0.2, or 2 ng ml−1 of TGF-β1 (R&D Systems). At indicated time points following cytokine exposure, conditioned culture media and cells were collected for CCL20 protein and mRNA quantification, respectively.
HEKn-E6/E7 cells were established and propagated as described (Iordanov et al., 2002
). HEKn-E6/E7 were propagated in EpiLife basal KC medium (BKM) (Cascade Biologics) supplemented with a semi-defined human keratinocyte growth supplement (HKGS) (Cascade Biologics), which included bovine pituitary extract, bovine insulin, hydrocortisone, bovine transferrin, and human epidermal growth factor. RHE were established as described (Poumay and Coquette, 2007
). Briefly, HEKn-E6/E7 cells were plated at 5 × 105
cells per ml on 0.4 μm, 12 mm-diameter polycarbonate membranes (Millipore, Billerica, MA) and allowed to reach confluence in BKM + HKGS. Cells were then exposed to the air–liquid interface by removing the medium above the confluent monolayer. Simultaneously, the cells were exposed from underneath the monolayer to a differentiation medium (BKM + HKGSF supplemented with 1.5mM CaCl2
, 50 μg ml−1
ascorbic acid, and 10 ng ml−1
rhKGF (Peprotech, Rocky Hill, NJ)). The differentiation medium was changed every 48 hours for 14 days. RHE cultures were treated with 10 or 100 ng ml−1
of IL-17A, IL-22, or TNF-α, and incubated at 37 °C. At indicated time points following cytokine exposure, conditioned culture media and cells were collected for CCL20 protein and mRNA quantification, respectively.
Cells from normal human KC monolayer and RHE cultures were lysed directly in TRIzol, whereas mouse ear tissue was minced first and then placed in TRIzol and total RNA was extracted according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). Integrity of RNA was determined by the appearance of distinct 28S and 18S rRNA bands when analyzed by electrophoresis on 1% agarose gels. The iScript kit (Bio-Rad, Hercules, CA) was employed for cDNA synthesis and resulting cDNA was analyzed to determine CCL20 mRNA levels relative to GAPDH mRNA levels. cDNA was amplified using SYBR green (Bio-Rad) and the following primers: human CCL20, forward TACTCCACCTCTGCGGCGAATCAGAA, and reverse GTGAAACCTCCAACCCCAGCAAGGTT; human GAPDH, forward GAGTCAACGGATTTGGTCGT, and reverse TTGATTTTG GAGGGATCTCG. Primers were annealed at 60 °C and amplified using My IQ single color RT-PCR detection system (Bio-Rad) for a total of 40 cycles.
Secreted CCL20 protein levels in conditioned human KC culture media were quantified using the Quantikine ELISA kit according to the manufacturer’s protocol (R&D Systems). For the murine studies, total protein was extracted from ears by snap-freezing tissue in liquid nitrogen, pulverizing tissue while immersed in liquid nitrogen, and subsequently re-suspending in cell extraction buffer (Invitrogen). Samples underwent three freeze-thaw cycles and were centrifuged at full speed for 15 minutes to fractionate samples and remove protein. Mouse CCL20 ELISA (R&D Systems) was performed according to the manufacturer’s instructions using 150 μg total protein.
The Portland VA Medical Center Institutional Animal Care and Use Committee approved all of the described studies using mice. Wild type Balb/c mice were injected in each ear with either 500 ng of IL-17A, IL-22, or TNF-α in 25 μl of PBS/0.1% of BSA, or with 25 μl of PBS/0.1% BSA alone. Ten animals per cytokine treatment group were injected daily for 5 days. All animals were killed at day 5 and mRNA was extracted from ears using a standard Trizol protocol, followed by further purification using an RNeasy kit (Qiagen, Valencia, CA). cDNA was synthesized using an iScript cDNA synthesis kit (Bio-Rad) and amplified using Taqman primers and fluorescent probes for GAPDH, CCL20, and CCR6 using My IQ single color RT-PCR detection system for a total of 40 cycles.