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*Psoriasis
The Journal of Immunology, 2007, 178: 2229-2240.
Copyright © 2007 by The American Association of Immunologists, Inc.

The Effects of IL-20 Subfamily Cytokines on Reconstituted Human Epidermis Suggest Potential Roles in Cutaneous Innate Defense and Pathogenic Adaptive Immunity in Psoriasis

Susan M. Sa*, Patricia A. Valdez{dagger}, Jianfeng Wu{dagger}, Kenneth Jung{ddagger}, Fiona Zhong*, Linda Hall*, Ian Kasman*, Jane Winer§, Zora Modrusan§, Dimitry M. Danilenko1,* and Wenjun Ouyang1,{dagger}

* Department of Pathology, {dagger} Department of Immunology, {ddagger} Department of Bioinformatics, and § Department of Molecular Biology, Genentech, South San Francisco, CA 94080


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
IL-19, IL-20, IL-22, IL-24, and IL-26 are members of the IL-10 family of cytokines that have been shown to be up-regulated in psoriatic skin. Contrary to IL-10, these cytokines signal using receptor complex R1 subunits that are preferentially expressed on cells of epithelial origin; thus, we henceforth refer to them as the IL-20 subfamily cytokines. In this study, we show that primary human keratinocytes (KCs) express receptors for these cytokines and that IL-19, IL-20, IL-22, and IL-24 induce acanthosis in reconstituted human epidermis (RHE) in a dose-dependent manner. These cytokines also induce expression of the psoriasis-associated protein S100A7 and keratin 16 in RHE and cause persistent activation of Stat3 with nuclear localization. IL-22 had the most pronounced effects on KC proliferation and on the differentiation of KCs in RHE, inducing a decrease in the granular cell layer (hypogranulosis). Furthermore, gene expression analysis performed on cultured RHE treated with these cytokines showed that IL-19, IL-20, IL-22, and IL-24 regulate many of these same genes to variable degrees, inducing a gene expression profile consistent with inflammatory responses, wound healing re-epithelialization, and altered differentiation. Many of these genes have also been found to be up-regulated in psoriatic skin, including several chemokines, beta-defensins, S100 family proteins, and kallikreins. These results confirm that IL-20 subfamily cytokines are important regulators of epidermal KC biology with potentially pivotal roles in the immunopathology of psoriasis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Psoriasis is a chronic inflammatory skin disease characterized by excessive proliferation and abnormal differentiation of epidermal keratinocytes (KCs),2 the growth and dilation of blood vessels, and the infiltration of leukocytes into the dermis and epidermis. Analyses of infiltrates in psoriatic lesions have most consistently identified increases in myeloid-derived CD11c+ dendritic cells and CD4+ and CD8+ T cells, with plasmacytoid dendritic cells (CD11c–), monocytes/macrophages, neutrophils, and mast cells also frequently found in increased numbers (1, 2). The role of T cells in the pathogenesis of psoriasis has been supported by the recent development of therapeutics for psoriasis that target T cells, such as efalizumab and alefacept, which bind CD11a and CD2, respectively (1, 2). Infiltrating CD4+ T cells in psoriatic skin have thus far been characterized as displaying an inflammatory Th1 phenotype due to elevated levels of IFN-{gamma}, TNF-{alpha}, and IL-12. (3, 4). Consistent with this hypothesis, an Ab anti-blocking the p40 subunit of IL-12, a cytokine essential for Th1 development, has demonstrated early clinical efficacy in the treatment of psoriasis (5). However, p40 is a common subunit for two cytokines, IL-12 and IL-23. Recently, IL-23 has been found to be the dominant p40-containing cytokine identified in psoriatic lesions (6).

Despite the fact that significant progress has recently been made in the treatment of psoriasis by targeting the immune system, not all psoriatic patients respond to these targeted therapies (1, 2). One explanation for the lack of complete efficacy by these therapies is that resident skin cell populations and the disregulation of normal cutaneous defense mechanisms are also playing important roles in the pathogenesis of psoriasis (7, 8). Moreover, the mechanisms underlying abnormal KC hyperproliferation and differentiation in psoriasis are not completely understood, although a number of epidermal growth factor (EGF) family growth factors have been proposed to play an important role (9). Another group of soluble factors that have been implicated in the pathophysiology of psoriasis are the recently described IL-20 subfamily cytokines, which include IL-19, IL-20, IL-22, IL-24, and IL-26. Of these cytokines, IL-19, IL-20, IL-22, and IL-24 have been shown to be up-regulated in psoriatic skin (10, 11, 12, 13). In addition, IL-20 and IL-22 promoted hyperproliferation and abnormal differentiation of KCs both in vitro and in vivo (11, 13, 14, 15). IL-20 overexpression triggered abnormal epidermal proliferation and differentiation in transgenic mice when it was expressed in the epidermis (11), and IL-22 induced hyperplasia and inhibited differentiation in a cultured reconstituted human epidermis (RHE) (14). Finally, all of these cytokines cause Stat3 activation. Sano et al. (8) recently found Stat3 to be activated in lesional psoriatic skin. Furthermore, overexpression of a constitutively active form of Stat3 in epidermal KCs led to the development of psoriatic lesions in K5.Stat3C transgenic mice. Although this model still required T cells for disease development, blocking Stat3 activation led to alleviation of lesions. Therefore, targeting upstream signals that activate Stat3 in lesional skin could be a potential new therapy for psoriasis.

IL-19 and IL-20 are mainly produced by activated monocytes (16, 17, 18, 19), IL-24 is produced by Th2 cells, monocytes, and melanocytic cells (20, 21, 22), and IL-22 and IL-26 are primarily produced by activated T cells (16, 17, 18, 19). Like IL-10, these class II cytokines signal through heterodimeric receptors. Class II cytokine receptors (CRF2) are comprised of a long R1 type subunit with a long cytoplasmic tail paired with a shorter R2 type subunit with a short cytoplasmic tail. IL-19 signals exclusively through the IL-20R1/IL-20R2 heterodimer, whereas IL-20 and IL-24 can signal through IL-20R1/IL-20R2 as well as the IL-20R2 chain paired with IL-22R1 (11, 23, 24, 25). IL-22 signals through IL-22R1 paired with IL-10R2 (26, 27), and IL-26 signals through a heterodimer composed of IL-20R1 and IL-10R2 (28, 29). IL-20R1 and IL-20R2 are expressed in skin, lung, testis, and other tissues (11, 17, 28, 29). IL-22R1 is expressed by KCs, hepatocytes, and pancreatic acinar cells, as well as in the colon and the small intestine (13, 17, 26, 27, 30). The expression profiles of these receptors indicate that, unlike IL-10, the IL-20 subfamily cytokines mainly target epithelial tissues. Although there are a few reports of these cytokines having activities on hemopoietic cell types (31, 32, 33, 34), the expression of their receptors on immune cells has not been clearly demonstrated.

Given the immune origins of IL-20 subfamily cytokines, they are prime candidates for consideration as upstream targets that might directly mediate the cross-talk between infiltrating T cells, monocytes, and resident skin cells. However, the functions of IL-20 subfamily cytokines on KCs have not been systematically analyzed and compared. In this study, we evaluated the effects of IL-19, IL-20, IL-22, IL-24, and IL-26 on cultured human KC monolayers and in a reconstituted human epidermal culture system. As part of our analysis, we evaluated the biological consequences of IL-20 subfamily cytokine signaling in KCs using gene microarray analysis. Our data demonstrate that the members of this cytokine subfamily not only affect the growth and differentiation of KCs but that its members also induce a number of chemokines, proteases, S100 family proteins, beta-defensins, and growth factors, including vascular endothelial growth factor (VEGF), suggesting that the IL-20 subfamily plays a prominent role in epidermal immunity and wound repair. In addition, our findings support the hypothesis that these cytokines may form important links between leukocytic infiltration, inflammatory leukocyte responses, and abnormal KC proliferation and differentiation in the pathogenesis of psoriasis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell culture, cytokines, and other reagents

Normal human epidermal KCs (NHEKs) from a neonatal foreskin were cultured in serum-free EpiLife medium with human KC growth supplement (0.2% bovine pituitary extract (v/v), 5 µg/ml bovine insulin, 5 µg/ml bovine transferrin, 0.2 ng/ml human EGF, and 0.18 µg/ml hydrocortisone), all purchased from Cascade Biologics. Three different single donor NHEK lots were used. Recombinant human cytokines and growth factors were purchased from R&D Systems.

Blocking activity of Abs specific for receptor subunits

Abs targeting human IL-20R1, human IL-20R2, and human IL-22R2 (5C9, 1B4, and 7E9, respectively) were generated by immunizing mice with the ectodomain of these proteins. The blocking activities of the Abs were determined using 293 cells stably expressing IL-20R1 with IL-20R2, IL-22R1 with IL-20R2, or IL-22R1 with IL-10R2. Cells were plated at 0.2 x 106/well in a 24-well plate and the following day the receptor-expressing cell lines were transfected with a Stat3 Luciferase reporter and a Renilla luciferase control using Lipofectamine 2000 (Invitrogen Life Technologies). The next day IL-19, IL-20, IL-22, or IL-24 (R&D Systems) were added at the indicated concentrations (0.5–3 nM) along with a dose range of Ab. Sixteen hours later the cells were lysed and samples read on a luminometer. Dose curves were plotted to determine the IC50 for the blocking activity of the 5C9, 1B4, and 7E9 Abs.

Flow cytometry

To assess cell surface expression of IL-20 subfamily cytokine receptor subunits on primary human KCs in monolayer and RHE tissue culture, the following mAbs were used: anti-IL-20R1 (Genentech; clone 5C9), anti-IL-20R2 (Genentech; clone 1B4), anti-IL-22R1 (Genentech; clone 7E9), anti-IL-10R2 (Genentech; clone 3213), and PE-conjugated anti-IL-10R2 (R&D Systems; clone 90220). Isotype controls were mouse IgG1 (BD Biosciences; clone X40) and PE-conjugated mouse IgG1 (BD Biosciences; clone MOPC-21). Secondary mAb was R-PE-conjugated F(ab')2 goat anti-mouse IgG (Jackson ImmunoResearch Laboratories; catalog no. 115-116-071). NHEK monolayer cultures were trypsinized briefly at ambient temperature (20–23°C) followed by neutralization using a defined trypsin inhibitor (Cascade Biologics) to lift cells and prepare single-cell suspensions. The primary Ab (0.5 µg) was used for 3–6 x 105 cells per sample with propidium iodide added just before analysis to exclude dead cells. To disaggregate RHE tissues into KC cell suspensions, RHE tissues were first trypsinized for 2–3 h at ambient temperature in 0.83% trypsin (Invitrogen Life Technologies). Subcorneal layer cells were collected and pelleted at 180 x g at 4°C after neutralization of trypsin with the defined trypsin inhibitor. Cells were collected on a FACScan cytometer (BD Biosciences) and analysis was performed with Flow Jo software (Tree Star).

Proliferation assay

NHEK proliferation was assessed using a CyQUANT cell proliferation kit (Invitrogen Life Technologies). NHEK were seeded at 1550 cells/well in 96-well plates in EpiLife medium with complete human KC growth supplement. Fifteen to 24 h later, the medium was changed to EpiLife supplemented with only bovine insulin and transferrin, with the addition of either anti-EGF receptor (EGFR) mAb (Calbiochem; clone 225) or an isotype control Ab (anti-gp120; Genentech; clone 10E7) at a concentration of 0.5 µg/ml. Twelve to 20 h later, test factors were added. The plates were harvested 2–4 days later, decanted of medium, and frozen at –70°C until they were assayed according to the manufacturer’s protocol (Invitrogen Life Technologies).

RHE studies

EpiDerm RHE tissue models and EPI-100-NMM medium were purchased from MatTek. All experiments using RHE were started after first equilibrating tissues with medium at 37°C with 5% CO2 overnight to recover from shipping. Three tissues per treatment condition were cultured at the air-liquid interface for 4 days in a total volume of 5 ml in 6-well plates. The medium was changed every other day, with fresh cytokines/growth factors applied. For receptor blocking studies, the following mAbs were used at a final concentration of 20 µg/ml added 1 h before cytokines: anti-IL-20R1 (Genentech; clone 5C9), anti-IL-20R2 (Genentech; clone 1B4), anti-IL-22R1 (Genentech; clone 7E9), and anti-gp120 (Genentech; clone 10E7). All mAbs were mouse anti-human IgG1 isotype.

For routine histologic analysis, 5-µm sections were stained with H&E. Tissues were collected and fixed in 10% neutral-buffered formalin and embedded in paraffin. For immunohistochemistry (IHC), 5-µm sections on glass slides were deparaffinized and hydrated to distilled water. Slides were incubated for 20 min in Dako target retrieval solution (DakoCytomation) at 99°C. Endogenous peroxidase, avidin, and biotin were quenched using KPL blocking buffer (Kirkegaard & Perry Laboratories) and a Vector avidin/biotin kit (Vector Laboratories), respectively. Slides were incubated for 30 min in 10% normal horse serum in 3% BSA/PBS and then incubated with either 1 µg/ml anti-cytokeratin 16 (CK16) (Serotec; clone LL025), 1 µg/ml anti-S100A7 (Imgenex; clone 47C1068), or 5 µg/ml anti-phospho-Stat3 (Tyr705) (Cell Signaling Technology; clone 3E) for 60 min at room temperature. After washes in TBST, slides were incubated with 2.5 µg/ml biotinylated horse anti-mouse secondary Ab (Vector Laboratories) for 30 min, Vectastain ABC Elite reagents (Vector Laboratories) for 30 min, and metal enhanced diaminobenzidine (Pierce) for 4 min. Slides were then counterstained with Mayer’s hematoxylin, dehydrated, mounted, and coverslipped for bright field viewing.

For measurements of epidermal thickness and IHC staining intensity, MetaMorph (Molecular Devices) image analysis software was used. Thickness measurements in micrometers were made by calibrating the scale bar to pixels using captured x20 (original magnification) images of H&E-stained tissues and taking four measurements for each tissue across the viable (subcorneal) cell layers. For CK16 and S100A7 staining intensity, four equal area regions across the viable cell layers were measured for average staining intensity on a scale of 0–255 in each captured x20 image. Mean epidermal thickness and mean intensity values were calculated per tissue and for each treatment group.

Statistics

Statistical significance was determined by one-way Student’s t test using JMP software (SAS).

Microarray analysis and real-time RT-PCR

RHE tissues were cultured for 4, 24, 48, or 96 h with or without cytokines (concentration used for all IL-20 subfamily members was 20 ng/ml; for IL-1beta, TNF-{alpha}, IFN-{gamma}, and KC growth factor (KGF) the concentration was 10 ng/ml, and for EGF it was 6 ng/ml). Tissues were snap frozen in liquid nitrogen, and total RNA was prepared using RNeasy mini kit (Qiagen) after homogenization (Omni H-01 with saw-tooth blade). cRNA was hybridized to Affymetrix U133 Plus gene chips containing 54675 probe sets. Signal intensity values from the Affymetrix microarray analysis suite version 5 were normalized according to the procedure described by Choe et al. (35). Hierarchical clustering using the Pearson correlation as the similarity metric was performed using R software from the R Project for Statistical Computing (Department of Statistics and Mathematics, Vienna University of Economics and Business Administration, Vienna, Austria; www.r-project.org/). Statistical tests for differential expression were also performed using the R software. False discovery rates were estimated using the q value package (36). For differential expression contrasting IL-20 subfamily treated cultures vs untreated controls, a t statistic with Bayesian regularization (37) was used with false discovery rate cutoff values of 0.05 and 0.005, yielding 1955 and 266 probe sets, respectively. For ANOVA, a false discovery rate of 0.005 was used, yielding 7936 probe sets with significant F statistics. To compare the fold induction in RHE by different cytokines, we calculated a Pearson correlation of the fold changes for all probes that showed a fold change ≥2 for either treatment group relative to the untreated control samples. The correlation of the gene list from our differential expression analysis with that of Zhou et al. (38) was evaluated by using NetAffx mapping (available from Affymetrix) of probe sets on the U133 Plus and U95 chips to reference sequence (RefSeq) identifiers. This resulted in 16621 genes in common between the two chips, with 661 differentially expressed in Zhou et al. (38) and 1333 in our study. The overlap between the two sets of genes was then assessed by a {chi}2 test. For real-time RT-PCR, 50 ng of total RNA was run using primers and FAM/TAMRA probes directed against the exon boundaries of targeted genes. One-step RT-PCR master mix (Applied Biosystems) was used and reactions were run on the ABI Prism 7700 sequence detector.

Primer/probe pairs used were as follows: CXCL1, 5'-CGTGAAGTCCCCCGGAC-3' (forward), 5'-GCCCATTCTTGAGTGTGGCT-3' (reverse), and 5'-CCACTGCGCCCAAACCGAAGTC-3' (probe); CXCL7, 5'-TGATCGGGAAAGGAACCCA-3' (forward), 5'-GGCAGATTTTCCTCCCATCC-3' (reverse), and 5'-TGCAACCAAGTCGAAGTGATAGCCACACT-3' (probe); CXCL8/IL-8, 5'-AAGGAAAACTGGGTGCAGAGG-3' (forward), 5'-GATACCACAGAGAATGAATTTTTTTATGA-3' (reverse), and 5'-TTGTGGAGAAGTTTTTGAAGAGGGCTGAGAA-3' (probe); Stat3, 5'-TGAACCCTCAGCAGGAGGG-3' (forward), 5'-AGGTAGCGCACTCCGAGGT-3' (reverse), and 5'-AGTTTGAGTCCCTCACCTTTGACATGGAGTT-3' (probe); SOCS3, 5'-TGGGACGATAGCAACCACAA-3' (forward), 5'-GTCCCCTGTTTGGAGGCAG-3' (reverse), and 5'-TGGATTCTCCTTCAATTCCTCAGCTTCCC-3' (probe); BD02, 5'-GCCATGAGGGTCTTGTATCTCC-3' (forward), 5'-CCTATACCACCAAAAACACCTGG-3' (reverse), and 5'-CTTCTCGTTCCTCTTCATATTCCTGATGCCTC-3' (probe); S100A7, 5'-CCTGCTGACGATGATGAAGGA-3' (forward), 5'-GCGAGGTAATTTGTGCCCTTT-3' (reverse), and 5'-ACTTCCCCAACTTCCTTAGTGCCTGTGACA-3' (probe); S100A12, 5'-GCAGCTGCTTACAAAGGAGCTT-3' (forward), 5'-TCCAGGCCTTGGAATATTTCA-3' (reverse), and 5'-CAAACACCATCAAGAATATCAAAGATAAAGCTGTCATTG-3' (probe); S100A15, 5'-TGCTGACGATGATGAAGGAGAA-3' (forward), 5'-GCGAGGTAATGTATGCCCTTTT-3' (reverse), and 5'-TTCCCCAATTTCCTCAGTGCCTGTGAC-3' (probe); heparin-binding (HB)-EGF, 5'-GAAAGACTTCCATCTAGTCACAAAGA-3' (forward), 5'-GGG AGG CCC AAT CCT AGA-3' (reverse), and 5'-TCCTTCGTCCCCAGTTGCCG-3' (probe); and IL-20, 5'-GCCAGATTCTGAGTCACTTTGAAA-3' (forward), 5'-AGAATGTCTAGTTCCCCCAAAGC-3' (reverse), and 5'-CTGGAACCTCAGGCAGCAGTTGTGAA-3' (probe).

ELISA

IL-8 and MIP-3{alpha} ELISAs were performed using the DuoSet ELISA development kit from R&D Systems following the manufacturer’s protocol. Samples tested were supernatants from RHE treated with 20 ng/ml IL-20 family cytokines for 48 h in triplicate. Briefly, the capture Ab was coated overnight in PBS at room temperature. After washing the next morning, the plates were blocked with 1% BSA in PBS for 1 h at room temperature. After washing, 100 µl of undiluted samples was added to each well for 2 h at room temperature. After washing, 100 µl of streptavidin-HRP was added to each well for 20 min at room temperature followed by washes and the addition of substrate and stop solution. The concentration of IL-8 and MIP-3{alpha} was determined based on the standard curve. ELISAs were run using samples from experiments performed on three separate occasions, all of which yielded similar results.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Expression of receptor subunits for novel IL-20 subfamily cytokines on primary human KCs

Although the receptor subunits IL-20R1, IL-20R2, and IL-22R1 have previously been shown to be present in psoriatic lesions and in KCs at the mRNA level (10, 11, 12, 13), the cell surface expression of these proteins has not been unequivocally demonstrated. To address this, we generated a panel of Abs recognizing cell surface expression of human IL-20R1, IL-20R2, and IL-22R1. All of the Abs were specific for the Ag they were raised against as demonstrated by their ability to block signaling in reporter assays using cell lines expressing these receptor subunits (J. Wu and W. Ouyang, unpublished data). Clones 5C9 and 1B4 recognize the human IL-20R1 chain and the IL-20R2 chain, respectively. Both of these Abs were able to completely block IL-19- and IL-20-induced Stat3 activation in a Stat3-driven luciferase assay in which 293 cells stably overexpressed IL-20R1 and IL-20R2 chains (Table I). Clone 1B4 also inhibited IL-20 signaling through the IL-20R2 chain paired with the IL-22R1 chain. However, IL-24 induced activity could not be blocked by 5C9 and could only be blocked by 1B4 at much higher concentration in similar assays, although both receptor chains are absolutely required for IL-24 activity in these cell lines (Table I). Clone 7E9 binds to IL-22R1 and blocked both IL-22 signaling through IL-22R1 paired with IL-10R2, as well as the activity induced by IL-20 through IL-22R1/IL-20R2. In contrast, 7E9 failed to block IL-24-induced Stat3 activation through IL-22R1/IL-20R2 (Table I).


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  		Table I. Blocking activity of receptor targeted Absa

 
We assessed the expression of IL-20R1, IL-20R2, IL-22R1, and IL-10R2 on primary human KCs using flow cytometry and found that NHEK cells express the receptor subunits in both a monolayer culture and in stratified RHE. IL-20R2 and IL-10R2 were consistently expressed on the surface of NHEKs regardless of confluence, passage number, or calcium levels in the medium (Fig. 1A). In contrast, the surface expression of both IL-20R1 and IL-22R1 on NHEK varied from donor to donor and was always at a relatively low but detectable level (Fig. 1A and our unpublished data). Compared with expression levels in monolayer NHEK, IL-20R1 and IL-22R1 were expressed at much higher levels on KCs isolated from RHE (Fig. 1B). The reasons for this difference and the factors affecting the expression level of these two chains are unknown. We also surveyed the expression of these receptors on human immune cells by FACS analysis. To date, we have not detected any surface expression of these receptors by our Abs on the cell types examined, which included T cells, B cells, NK cells, and monocytes (our unpublished data).


Figure 1
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  		FIGURE 1. Flow cytometric analysis of primary human KCs for cell surface expression of IL-20 subfamily cytokine receptor subunits. Open peaks are isotype control Abs. Shaded peaks are the indicated receptor subunits. A, NHEK monolayer culture. Three single donor lots were assessed through six passages with differing culture conditions in regard to the degree of confluence and medium Ca2+ levels. Histogram overlays from one donor are shown to represent consistent expression patterns. B, RHE. Tissues were disaggregated to collect NHEK from viable cell layers by trypsinization after 1–4 days of culture at the air-liquid interface. Histogram overlays from one representative experiment of five are shown.

 
Differential effects of IL-20 subfamily cytokines on KC proliferation and differentiation

We first evaluated whether the IL-20 subfamily members could induce KC proliferation in monolayer cultures. We found that to detect effects on KC proliferation, even for KGF, it was necessary to block signaling by EGF family ligands through the EGFR (Fig. 2A, left). Using a neutralizing anti-EGFR Ab to block EGFR-mediated autocrine growth, we observed modest but significant NHEK proliferation induced by IL-20, IL-22, and IL-24 (Fig. 2A, right). Thus, the proliferation of NHEK induced by IL-20 subfamily cytokines was unlikely to be mediated indirectly by the EGFR pathway. Among the IL-20 subfamily cytokines, IL-22 induced the highest level of proliferation, followed by IL-24 and IL-20. IL-19 and IL-26 both failed to induce significant proliferation even at a concentration of 100 ng/ml (Fig. 2A and our unpublished data). In addition to failing to induce proliferation in NHEK, IL-26 also had no effects in any of the RHE studies, even when used at 100 ng/ml. This was despite the ability of IL-26 to induce Stat3 activation in Colo205 cells (our unpublished data), a result that confirmed a previous report (29). Thus, IL-26 was not further analyzed.


Figure 2
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  		FIGURE 2. Effects of IL-20 subfamily cytokines on primary KC proliferation. A, NHEK proliferation in monolayer culture was assessed using a fluorescent DNA-binding dye to quantitate cell number as detailed in Materials and Methods. Detection of the effects on proliferation by test factors required treatment with anti-EGFR to block autocrine growth. Results on the left show proliferation in the absence of Ab, with an isotype control Ab (anti-gp120), or with an anti-EGFR neutralizing Ab. Data on the right show results for IL-20 subfamily cytokines with anti-EGFR used in all samples. Results shown are from one assay representative of four experiments using three single donor lots. RFU, relative fluorescence units (excitation wavelength of 485 nm). Concentrations used were 50 ng/ml for IL-19 and IL-20, 25 ng/ml for IL-22 and IL-24, 5 ng/ml for KGF, and 0.5 µg/ml Abs. Data are shown as mean ± SD (error bars); n = 3 for test factors, n = 6 for medium control. **, p = 0.0005; ***, p < 0.0001. B, IL-20 subfamily members induce epidermal hyperplasia in RHE. H&E-stained sections of RHE were treated for 4 days in culture with the indicated cytokines. Images are representative of results from triplicate tissues from one study of six. EGF was used as a positive control at 6 ng/ml, and IL-19, IL-20, IL-22, and IL-24 were used at 20 ng/ml. Bars, 50 µm. C, Dose response of RHE cultured for 4 days with IL-20 subfamily cytokines. Epidermal thickness was quantified by taking measurements across viable cell layers (excluding the stratum corneum) on x20 images (original magnification) of H&E-stained sections as detailed in Materials and Methods. Data shown as mean + SD (error bars); n = 2 for IL-19 and IL-20 at 2.5 and 10 ng/ml and IL-22 and IL-24 at 50 ng/ml; n = 3 for medium control, KGF, and IL-19 and IL-20 at 50 ng/ml and IL-22 and IL-24 at 2.5 and 10 ng/ml. p < 0.05 for all treatments compared with the medium control group except for IL-19 at 10 ng/ml.

 
Next, to evaluate the effects of IL-20 subfamily cytokines on KCs in a stratified epidermal culture system, RHE tissues were cultured for 4 days at the air-liquid interface with IL-20 subfamily cytokines added to the culture medium. Histologic examination showed that IL-19, IL-20, IL-22, and IL-24 all induced acanthosis or hyperplasia of the viable, noncornified epidermis (Fig. 2B), with IL-22 and IL-24 again having the greatest hyperplastic effects. To further assess hyperplastic effects, epidermal thickening was quantified in H&E-stained sections. IL-19, IL-20, IL-22, and IL-24 all induced epidermal thickening in viable cell layers in a dose-dependent manner (Fig. 2C), with IL-22 increasing epidermal thickness by >50% compared with control tissues in all studies. Although the effect of IL-22 peaked at 25 ng/ml, the activity of IL-24 plateaued at 25–50 ng/ml, IL-20 at 50 ng/ml, and IL-19 at 100 ng/ml. These effects were statistically significant (p < 0.05) compared with untreated medium control tissues at doses as low as 2.5 ng/ml for IL-19, IL-20, IL-22, and IL-24. To evaluate whether this epidermal thickening was due to KC hyperplasia, we counted the number of epidermal KC cell layers. All IL-20 subfamily members, as well as KGF, caused a similar, statistically significant increase in the average number of RHE epidermal cell layers compared with medium control tissues (our unpublished data). These results verified that the IL-20 subfamily members induced hyperplasia of epidermal KCs, although we could not entirely rule out the possibility that some KC hypertrophy might also have occurred. Interestingly, although we saw an increase in epidermal hyperplasia, we could not demonstrate any significant differences between IL-20 subfamily cytokine-treated and medium control tissues using Ki-67 staining as a proliferation marker (our unpublished data), suggesting that the proliferation effects that led to hyperplasia occurred earlier than we were able to measure. These results are in agreement with those found in a previous report (14).

In addition to inducing the greatest degree of epidermal hyperplasia, IL-22 was the only cytokine that also induced hypogranulosis (Fig. 3A) or a decrease in the granular cell layer, a finding that was consistent with a previous report (14). We also observed that IL-22 induced the formation of a prominent zone of acellular hyalinized keratin above the hypogranular zone of KCs and the lower stratum corneum (Fig. 3A, asterisks). Furthermore, in a full thickness model that included dermal fibroblasts, IL-22 induced parakeratosis in tissues cultured for 7 days (our unpublished data). Hypogranulosis and parakeratosis are both frequently observed histologic features of psoriatic epidermis (39). In contrast to these IL-22-induced changes, EGF induced hypergranulosis with compacting of the KCs within the stratum granulosum while the other three IL-20 subfamily cytokines, IL-19, IL-20, and IL-24, had little to no apparent effect on the epidermal granular cell layer in RHE.


Figure 3
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  		FIGURE 3. Effects of IL-20 subfamily cytokines on epidermal differentiation. The RHE was cultured for 4 days and treated with various cytokines as indicated (20 ng/ml). Images are representative of results from treatments on tissues run in triplicate in six studies. A, IL-22 alters normal epidermal differentiation. Shown are H&E-stained sections. All cytokines induced hyperplasia of the viable nucleated epidermis as denoted by the increased length of the double-headed arrows compared with the medium control. In addition, IL-22 induced hypogranulosis (arrowheads) as well as hyalinization of the lower stratum corneum (asterisks). In contrast, the other three IL-20 subfamily cytokines, IL-19, IL-20, and IL-24, induced only epidermal hyperplasia with little to no apparent effect on either the granular cell layer or the stratum corneum. EGF induced epidermal hyperplasia with hypergranulosis and compacting of the KCs within the stratum granulosum (arrows). Bars, 25 µm. B, CK16 IHC. CK16 was increased in all cytokine treated cultures compared with the medium control, most markedly by IL-22 and IL-24. Bars, 50 µm. C, Psoriasin (S100A7) IHC. Psoriasin was up-regulated by all members of the IL-20 subfamily to a variable degree, but most markedly by IL-22. EGF did not up-regulate psoriasin. Bars, 50 µm. D, pY (705)-Stat3 IHC. Stat3 was activated by all members of the IL-20 subfamily as shown by nuclear staining of KCs using a pY(705)-Stat3 specific Ab. In contrast, Stat3 was not activated in medium control or KGF-treated RHE. Bars, 25 µm. E and F, Quantification of IHC staining intensity in 4-day RHE cultures. Average intensity was measured by defining regions in viable cell layers (excluding stratum corneum) on x20 (original magnification) images of stained sections as detailed in Materials and Methods. Data are shown as group means (lines) with individual tissue values (points); n = 3 per group. E, CK16 average intensity; p < 0.005 for IL-20, IL-22, IL-24, and EGF compared with medium control. F, S100A7 average intensity; p < 0.0005 for IL-19, IL-20, IL-22, and IL-24 compared with medium control.

 
IHC was performed on RHE to assess the expression of KC differentiation markers as well as other features that have been associated with the psoriatic phenotype. As shown in Fig. 3B, we found that CK16, a marker of epidermal hyperproliferation, was increased in all cytokine-treated cultures compared with the control tissues. The extent of CK16 expression induced by each cytokine varied, with IL-19 and IL-20 only inducing an increase in CK16 expression in the basal zone while IL-22, IL-24, and EGF all induced an increase in CK16 expression throughout the noncornified epidermis. S100A7 (psoriasin), one of several S100 family proteins that has been found to be up-regulated in certain hyperproliferative and inflammatory skin conditions, including psoriasis, was induced in the suprabasal epidermis by all members of the IL-20 subfamily to a variable degree (Fig. 3C), but, again, most extensively by IL-22 followed by IL-24. Intense S100A7 staining was observed in the nuclei and cytoplasm of the KCs, with some protein also appearing to be extracellular. In contrast, despite inducing epidermal hyperplasia, EGF did not up-regulate S100A7. Quantification of staining intensity (Fig. 3F) revealed that induction of S100A7 by IL-19, IL-20, IL-22, and IL-24 was statistically significant (p < 0.05) compared with untreated control tissues. Quantification of CK16 (Fig. 3E) yielded similar results.

Activated Stat3 has been shown to be elevated in psoriatic lesional skin (8). We found that IL-20 subfamily cytokines induced persistent Stat3 activation that was detected using phosphotyrosine (pY)-Stat3 IHC in RHE. IL-19, IL-20, IL-22, and IL-24 all induced pY-Stat3 nuclear staining in KCs of all viable cell layers (Fig. 3D). Although EGF and TGF-{alpha} have been reported to activate Stat3 (40, 41), no pY-Stat3 nuclear staining was observed in RHE treated with these two growth factors. Furthermore, real-time RT-PCR analysis of the RNA harvested from RHE in these experiments indicated that IL-20 subfamily cytokines also augmented Stat3 expression at the mRNA level (see Fig. 6A).


Figure 6
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  		FIGURE 6. IL-20 subfamily up-regulation of expression for selected genes verifies microarray results. A, Real-time RT-PCR was performed as detailed in Materials and Methods to determine relative mRNA levels for selected genes as indicated. Results are expressed as mean fold change normalized to human RPL19 for triplicate samples prepared from separate RHE tissues with the same treatment conditions. Fold induction was calculated relative to expression in time 0 samples. Error bars, SD. B, Media collected from RHE cultures treated with 20 ng/ml cytokines as indicated were assayed by ELISA for the presence of IL-8 and MIP-3{alpha}. Data shown are mean concentration for triplicate samples taken at 48 h from separate RHE in 5-ml total well volume per tissue. Error bars, SD.

 
IL-20 uses both receptor pairs for its signaling in RHE

IL-20 signals through two receptor pairs, IL-20R1/IL-20R2 and IL-22R1/IL-20R2 (11, 23, 25). Because both of these receptors are expressed on primary KCs derived from RHE, it is unclear whether IL-20 preferentially uses one receptor pair over the other. To address this question, mAbs specific to individual IL-20 subfamily receptor subunits were used to determine whether the observed effects of IL-19, IL-20, and IL-22 on cultured RHE could be blocked (Fig. 4). Consistent with previous data, Abs against either IL-20R1 or IL-20R2 could completely block IL-19-induced S100A7 expression and Abs against IL-22R1 blocked IL-22-induced S100A7 expression. Although induction of S100A7 by IL-20 was completely blocked using anti-IL-20R2, Abs against IL-20R1 or IL-22R1 could block IL-20-induced S100A7 expression only if they were applied in combination (our unpublished data), findings that suggest that IL-20R1 and IL-22R1 might have complementary roles in IL-20 signaling in human KCs (Fig. 4).


Figure 4
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  		FIGURE 4. Abs specific for IL-20 subfamily cytokine receptor subunits block induction of S100A7 by IL-19, IL-20, and IL-22. Shown is S100A7 IHC of 4-day RHE cultures treated with IL-20 subfamily cytokines with or without Abs directed to IL-20 subfamily receptor subunits, as indicated. Induction by IL-19, which uses IL-20R1/IL-20R2 for signaling, was blocked by both anti ({alpha})-IL-20R1 and anti-IL-20R2, while induction by IL-20, which can use either IL-22R1/IL-20R2 or IL-20R1/IL-20R2, was blocked only by anti-IL-20R2. Induction by IL-22, which signals through IL-22R1/IL-10R2, was reduced by anti-IL-22R1. Cytokines were used at a concentration of 20 ng/ml and Abs at 20 µg/ml. Anti-gp120 was used as an isotype control. Images are representative of results from treatment groups run in triplicate in three studies. Bars, 50 µm.

 
Unique gene signatures induced by IL-20 subfamily cytokines in RHE suggest potential roles of these cytokines in tissue repair and defense of infection

To further unveil the biological effects of this group of cytokines on KCs, we performed microarray experiments. RHE tissues were treated with IL-20 subfamily cytokines for 4 days as described above. RNA was prepared and cRNA was hybridized to Affymetrix U133 Plus gene chips containing 54675 probe sets. Image data were processed into signal intensities using the Microarray Analysis Suite version 5 and normalized according to the procedure described by Choe et al. (35). The data were analyzed as described in Materials and Methods; each treatment condition was run in duplicate or triplicate. Hierarchical clustering was performed to analyze genes based on their expression pattern. The most differentially expressed genes based on ANOVA analysis are shown in Fig. 5A. RHE that were treated under the same conditions were clearly clustered together. Consistent with results in previous experiments, IL-26 treated samples had an expression pattern that was very similar to that of the untreated control samples (our unpublished data). RHE samples treated with IL-22 and IL-24 and, to a lesser degree, IL-20 and IL-19 shared similar gene signatures. IFN-{gamma}-, IL-1beta-, and KGF-treated samples were also included as controls. As illustrated by Fig. 5A, these factors had gene signatures that were clearly different from those induced by IL-20 subfamily cytokines, indicating that these factors induce different biological functions. Although KGF induced significant epidermal hyperplasia in RHE, the downstream mechanisms induced by KGF were clearly different from those of the IL-20 subfamily cytokines, as illustrated by their distinct gene clustering (Fig. 5A). Similarly, although IFN-{gamma} and IL-1beta have been reported to be up-regulated in psoriatic skin (42, 43), their downstream targets were also clearly different from those of the IL-20 subfamily cytokines.


Figure 5
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  		FIGURE 5. Microarray gene expression analysis of 4-day cultured RHE treated with IL-20 subfamily cytokines. A, Hierarchical clustering of differentially expressed genes, based on ANOVA analysis, with a false discovery rate of 0.005, yielding 7936 probe sets with significant F statistics. B, The top 20 genes most induced or repressed by IL-20, IL-22, and IL-24 were selected based on their fold of change when compared with untreated control samples. C, Significant correlation of gene profiles induced by IL-22 and IL-24 in cultured RHE. Pearson correlation coefficient (R2) was calculated for fold changes relative to untreated control samples.

 
Because we observed that each of the IL-20 subfamily of cytokines had quite similar biological effects on KCs, we asked whether they induced similar gene profiles. We compared the fold induction of each gene by each different cytokine by calculating the Pearson correlation coefficient of the fold changes relative to the untreated RHE. Unexpectedly, although IL-22 and IL-24 use different receptors to signal, the gene profiles induced by these two cytokines were significantly correlated, as demonstrated in Fig. 5C. Similar correlations were also evident when we compared genes induced by IL-22 vs IL-20 and IL-22 vs IL-19, although the degree of correlation was less than that observed between IL-22 and IL-24 in both these cases (our unpublished data). It is unclear whether these similarities in gene induction that we observed were due to IL-22, IL-20, and IL-24 all signaling through IL-22R or whether it was due to shared downstream pathways of this group of cytokines. IL-22 did induce several unique genes, including trichohyalin and fibrinogen (our unpublished data). Fibrinogen is part of the provisional matrix formed in the tissue repair process that provides a scaffold for cell migration (44), and trichohyalin is a keratin-associated protein that is expressed in hair follicles, foreskin, and tongue and in pathological epithelia, including the psoriatic epidermis (45, 46). Trichohyalin has an S100-like calcium-binding domain and is in a gene family with profilaggrin whose members are thought to be fused genes of cell envelope precursor protein genes and S100 family protein genes (47). These genes are located in the epidermal differentiation complex along with those for S100A proteins and small proline-rich proteins (SPRRs). Expression of this protein may thus be related to the band of hyalinized keratin noted above the hypogranular zone of KCs and the lower stratum corneum in IL-22-treated RHE and is further evidence of the altered differentiation pattern induced by IL-22.

Given the similarity of the gene profiles induced by IL-20, IL-22, and IL-24 and their common usage of the IL-22 receptor, we decided to focus on genes that were commonly regulated by them when compared with control samples. At false discover rates of <0.005 and 0.05 there were 266 (supplemental Table I)3 and 1955 (our unpublished data) total probe sets identified, respectively. The top 20 most induced or repressed genes are shown in Fig. 5B. Among the induced genes previously reported to be associated with psoriasis were S100A7, S100A12, SCCA2, SERPINB4, CCL20, CD36, and Stat3 (38, 48, 49, 50). To further assess potential correlation between genes regulated by IL-20 subfamily cytokines and genes regulated in psoriasis, we compared our study with a previous microarray study of psoriatic skin (38). Because the studies were on different chips, we only compared unique RefSeqs in common from both studies. We found our 1955 gene list was highly correlated with the list of genes regulated in involved psoriatic skin vs normal skin ({chi}2 = 231.55, p < 2.73E-52; details in Materials and Methods). Of 457 common RefSeqs that were significantly up-regulated in psoriatic skin (39), 355 RefSeqs also were induced and 188 of them were significant (p < 0.05) in our study. Of 165 common RefSeqs that were down-regulated in psoriatic skin (39), 138 RefSeqs also were repressed in our study and 71 were significant. These genes are shown in supplemental Table II.

One of the most important biological functions of the IL-20 subfamily of cytokines appears to be the induction of antimicrobial defense mechanisms in KCs. Chemokines play key roles in recruiting leukocytes to sites of infection. We identified CXCL8/IL-8, CXCL1/GRO-{alpha}, DMC, CXCL7/NAP-2, CCL20/MIP-3{alpha}, and CCL2/MCP-1 as chemokines induced by IL-20 subfamily cytokines (supplemental Table I and Fig. 6). S100 family members have also been demonstrated to be involved in the response to infection (51, 52, 53). In addition to S100A7, S100A8, and S100A9, which were previously reported to be up-regulated by IL-22 (13, 14), we found that S100A12 and S100A15 were strongly induced by the IL-20 subfamily of cytokines (supplemental Table I and Fig. 6A). Other antimicrobial genes such as beta-defensins were also up-regulated. To further validate the induction of these genes, real- time RT-PCR was performed on total RNA prepared from RHE treated with IL-20 subfamily cytokines for 4, 24, 48, and 96 h. As demonstrated in Fig. 6A, IL-22 was the most potent cytokine in inducing chemokines as well as many other genes, including IL-20, from RHE. The secretion of two chemokines, IL-8 and MIP-3{alpha}, after 48 h of cytokines treatment was further confirmed by ELISA (Fig. 6B).

Other families of genes regulated by IL-20 subfamily cytokines included the kallikrein subgroup of serine proteases and a large of group of protease inhibitors (supplemental Table I). Among the kallikrein genes up-regulated were kallikrein 6, 10, 12, 13, and 14. Several serpin family protease inhibitors were also up-regulated in our experiments, including clade A serpin 1 and 6, clade B serpin 1, 4, 6, 9, and 13, and clade G serpin 1. Additionally, a number of keratins were regulated by IL-20 subfamily cytokines, confirming that this subfamily plays a role in KC differentiation. Expression of the EGF family ligands amphiregulin and HB-EGF were augmented, whereas betacellulin expression was down-regulated. Significantly, we also discovered that the proangiogenic factors VEGF and endothelial cell growth factor 1 were induced by IL-20 subfamily cytokines (supplemental Table I).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we systemically compared the biology of IL-20 subfamily cytokines on cultured KCs. Our data suggest that the IL-20 subfamily cytokines are messengers linking immune cells to KCs, as these cytokines are primarily produced by immune cells while their receptors are expressed on KCs but not on T cells, B cells, or monocytes (13, 17, 54, 55). Though these cytokines signal through different receptors, they had very similar biological activities. Histologic and microarray analyses support the hypothesis that these cytokines play an important role in cutaneous innate immunity, skin repair, and remodeling. It is, moreover, likely that they contribute to the disregulation of normal differentiation and immune homeostasis of the skin that is evident in psoriasis.

We first demonstrate that KCs express all of the receptors for this subfamily of cytokines on the cell surface. Other inflammatory cytokines such as TNF-{alpha}, IFN-{gamma}, and IL-1beta are considered important in the pathophysiology of psoriasis due to their biological effects on both KCs and immune cells. On KCs, they up-regulate the expression of MHC, costimulatory ligands for T cells, chemokines, and adhesion molecules (43, 56, 57, 58). In contrast, IL-20 subfamily cytokines specifically target KCs, provoking epidermal thickening in RHE and inducing a number of other features that are characteristic to psoriatic epidermis, including activated Stat3, expression of CK16, S100A7 and other S100 family members, and up-regulation of many proteases involved in skin mobility and remodeling. It has been previously hypothesized that growth factors in both the EGF and fibroblast growth factor families might play a critical role in the pathogenesis of psoriasis, in particular contributing to hyperplasia of the epidermis (9, 59, 60, 61, 62). EGF and TGF-{alpha}, both members of the EGF family, and KGF, a member of the fibroblast growth factor family, are all potent stimulators of KC proliferation. We found that while these three growth factors all induced significant KC hyperplasia in RHE, none of them induced hypogranulosis, persistent Stat3 activation, or S100A7 expression. In addition, the gene expression profiles induced by each of these three growth factors in RHE did not correlate well with the gene profile of psoriatic skin (our unpublished data). IL-20 subfamily cytokines, except IL-26, all induced proliferation of monolayer KCs that was independent of EGFR pathways, because blocking of EGFR in the monolayer culture system did not abolish KC proliferation. However, KC proliferation might be further enhanced in vivo through the up-regulation of the EGF family ligands amphiregulin and HB-EGF by the IL-20 subfamily cytokines. Both amphiregulin and HB-EGF bind to EGFR, have been determined to be the primary growth factors driving autocrine KC proliferation (9), and are overexpressed by KCs in psoriatic lesions (61). The potential role of amphiregulin in psoriasis and psoriatic arthritis has also been supported by preclinical animal studies (59, 63, 64).

Although IL-20 subfamily cytokines have similar biological functions on KCs, there are differences among family members. First, they use different receptors to signal as demonstrated by our studies with neutralizing Abs against IL-20R1, IL-20R2, or IL-22R1. In contrast to the activity of IL-20, IL-24 activity could only be blocked by anti-IL-20R2 at very high concentrations (our unpublished data). It is unclear what properties of IL-20 and IL-24 caused this difference. One potential explanation might be that IL-20 and IL-24 have differing affinities for the IL-20R2 chain, although these have been reported to be similar (11, 24, 25). Comparison of the relative potency of the five IL-20 subfamily members in stimulating proliferation and inducing genes in KCs demonstrate that IL-22 consistently produced responses of the greatest magnitude followed by IL-24, then IL-20, and finally IL-19. IL-22 was also the only cytokine that caused hypogranulosis in the RHE culture system. The finding of less discrete epidermal cell layers with a lack of prominent keratohyalin granules in the upper layers of the epidermis is commonly seen in psoriatic skin and distinguishes psoriasis from other dermatological diseases such as lichen simplex chronicus in which hypergranulosis, an expansion of the granular cell layer, is the predominant feature (39). Given the almost identical gene signatures of IL-22 and IL-24, the exact molecular mechanisms responsible for the histologic differences in epidermal differentiation patterns induced by these two cytokines are still unknown. Another interesting result was that IL-26 did not induce any observable biological activities in the RHE system, although it was able to induce Stat3 activation in the Colo205 cell line as previously reported (29). The reason for this lack of activity in our system is unclear but may have to do with structural properties of IL-26 that it does not share with the other IL-20 subfamily members, such as its ability to bind cell surface proteoglycans and the possibility of IL-26 forming dimers in solution (65). Colon cell lines and intestinal tissues express the IL-20R1 chain at relatively high levels, and IL-26 is the only IL-20 subfamily cytokine that binds this subunit with high affinity (28, 29). It is possible that IL-26 is primarily active on gut epithelial cells due to higher IL-20R1 receptor levels and differences in cell surface proteoglycans.

A recent report from Wolk et al. (15) demonstrated that IL-22 regulates genes involved in antimicrobial defense, cellular differentiation, and KC mobility by using gene chip analysis of RNA samples from monolayer KCs treated with IL-22. In addition, Wang et al. (55) evaluated the effects of IL-20 on HaCaT cells and compared the gene expression profile to that induced by IFN-{gamma}. Although we did find similar induction of genes by IL-22 as those found in the Wolk study (15), we have identified a number of additional up-regulated genes such as chemokines, EGF family members, and VEGF that were not identified in that report. However, our initial results using HaCaT cells and primary KCs in monolayer culture identified limitations in these monolayer systems, including inconsistent Stat3 activation and lower expression levels of IL-20R1 and IL-22R1. Moreover, the monolayer culture system does not recapitulate the processes of KC stratification, differentiation, and cornification that take place during normal epidermal growth and homeostasis. Therefore, we believe that the RHE system is much more representative of epidermal KC biology than is the monolayer system. In further support of our findings, several other studies have reported that IL-20 subfamily cytokines induced chemokine production from various cell types (66, 67, 68).

IL-19, IL-20, IL-22, and IL-24 are all elevated in psoriatic skin (10, 11, 12, 13, 32, 55). We recently discovered that IL-22 is augmented by IL-23 in CD4+, CD8+, and {gamma}{delta} T cells (69). IL-23 has recently come into prominence as a cytokine that may have a crucial role in the development and maintenance of autoimmune inflammation in peripheral tissues due to its potential polarization effects on T cells (70). These data suggest that IL-22 might be one of the primary soluble factors that link the adaptive immune response with KCs and innate immunity in the pathophysiology of psoriasis. Expression studies of psoriatic vs normal or uninvolved skin have identified altered expression of >1300 genes in psoriatic lesions (38, 48, 49). We demonstrated here that the genes regulated by IL-20 subfamily cytokines were highly correlated with genes modified in psoriatic skin, further supporting their potential roles in the pathogenesis of psoriasis. S100 family proteins were some of the most highly up-regulated genes in our microarrays and as determined by quantitative RT-PCR. Proteins of this family, whose genes are found in the epidermal differentiation complex, are calcium-binding proteins that are associated with inflammation, some of which have been shown to have chemotactic and direct antimicrobial activities (51, 52, 53). Other genes induced by the IL-20 subfamily cytokines and also in psoriatic skin include chemokines, angiogenic factors, tissue kallikreins, and beta-defensins (38, 48, 49, 71). Chemokines and angiogenic factors might further enhance proinflammatory responses through a positive feedback loop by recruiting additional leukocytes and promoting angiogenesis in the skin. Some kallikreins have been shown to be important in the process of desquamation, the shedding of corneocytes from the stratum corneum (72, 73). beta-Defensins are one of several families of antimicrobial peptides produced by KCs that have the ability to kill bacteria, fungi, and viruses. Psoriatic plaques are known to be highly resistant to bacterial, viral, and fungal infection (38).

In summary, our results demonstrate that the IL-20 subfamily cytokines all induce epidermal KCs to proliferate and to express inflammatory and immunomodulatory mediators. In addition, these cytokines all cause persistent Stat3 activation and alter the expression of many genes involved in epidermal terminal differentiation. The gene expression profile induced by the IL-20 subfamily is consistent with that found during the re-epithelialization phase in wound healing, a process referred to as regenerative maturation. In this process, increased proliferation and altered differentiation of KCs as part of the tissue repair process is demonstrated by the expression of the hyperproliferative keratins CK6 and CK16, a number of chemokines, and several of the S100 proteins, including S100A7 (74). This pattern is similar to that observed in KCs in psoriatic lesions and indicates that these cytokines are important in cutaneous innate immunity and response to injury. Furthermore, our findings suggest that the IL-20 subfamily may form an important link between adaptive and innate immunity of the skin by exacerbating the inappropriate adaptive immune response and disturbed epidermal homeostasis that is evident in psoriasis through numerous potential feedback loops. These findings support the hypothesis that disregulation of the normal epidermal response to injury, which is an essential element of the skin’s innate homeostatic immune surveillance, may result in an inappropriate adaptive immune response that, if sustained, might lead to a chronic inflammatory condition such as psoriasis.


   Acknowledgments
 
We acknowledge the Histology Laboratory and the Immunohistochemistry Laboratory in the Pathology Department for contributions and assistance in optimizing tissue staining. We also acknowledge Chae Reed, Terence Wong, and Anan Chuntharapai for their technical assistance for generation of the anti-IL-20R1, anti-IL-20R2, and anti-IL-22R1 Abs. We also thank the Protein Chemistry Department in Genentech for its contribution in the generation and purification of the Fc fusion proteins of the ectodomains of IL-20R1, IL-20R2, and IL-22R1, which were used for Ab generation.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors are all current or former employees of Genentech.


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

1 Address correspondence and reprint requests to Dr. Wenjun Ouyang, Department of Immunology, Genentech, 1 DNA Way, MS 34, South San Francisco, CA 94080; E-mail address: ouyang{at}gene.com or Dr. Dimitry M. Danilenko, Genentech, 1 DNA Way, MS 72B, South San Francisco, CA 94080; E-mail address: danilenko.dimitry{at}gene.com Back

2 Abbreviations used in this paper: KC, keratinocyte; CK16, cytokeratin 16; EGF, epidermal growth factor; EGFR, EGF receptor; HB, heparin binding; IHC, immunohistochemistry; KGF, KC growth factor; NHEK, normal human epidermal KC; pY, phosphotyrosine; RefSeq, reference sequence; RHE, reconstituted human epidermis; VEGF, vascular endothelial growth factor. Back

3 The online version of this article contains supplemental material. Back

Received for publication August 29, 2006. Accepted for publication November 24, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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