Development and Validation of Human Psoriatic Skin Equivalents
2008; Elsevier BV; Volume: 173; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2008.080173
ISSN1525-2191
AutoresGeuranne S. Tjabringa, Mieke Bergers, Desiree van Rens, Roelie de Boer, Evert N. Lamme, Joost Schalkwijk,
Tópico(s)Cytokine Signaling Pathways and Interactions
ResumoPsoriasis is an inflammatory skin disease driven by aberrant interactions between the epithelium and the immune system. Anti-psoriatic drugs can therefore target either the keratinocytes or the immunocytes. Here we sought to develop an in vitro reconstructed skin model that would display the molecular characteristics of psoriatic epidermis in a controlled manner, allowing the screening of anti-psoriatic drugs and providing a model in which to study the biology of this disease. Human skin equivalents generated from normal human adult keratinocytes after air exposure and stimulation by keratinocyte growth factor and epidermal growth factor displayed the correct morphological and molecular characteristics of normal human epidermis whereas the psoriasis-associated proteins, hBD-2, SKALP/elafin, and CK16, were absent. Skin equivalents generated from foreskin keratinocytes were clearly abnormal both morphologically and with respect to gene expression. When normal skin equivalents derived from adult keratinocytes were stimulated with psoriasis-associated cytokines [tumor necrosis factor-α, interleukin (IL)-1α, IL-6, and IL-22] or combinations thereof, strong expression of hBD-2, SKALP/elafin, CK16, IL-8, and tumor necrosis factor-α was induced as shown by quantitative polymerase chain reaction and immunohistochemistry. Retinoic acid but not cyclosporin A was found to inhibit cytokine-induced gene expression at both the mRNA and protein levels. These results illustrate the potential of this disease model to study the molecular pathology and pharmacological intervention in vitro. Psoriasis is an inflammatory skin disease driven by aberrant interactions between the epithelium and the immune system. Anti-psoriatic drugs can therefore target either the keratinocytes or the immunocytes. Here we sought to develop an in vitro reconstructed skin model that would display the molecular characteristics of psoriatic epidermis in a controlled manner, allowing the screening of anti-psoriatic drugs and providing a model in which to study the biology of this disease. Human skin equivalents generated from normal human adult keratinocytes after air exposure and stimulation by keratinocyte growth factor and epidermal growth factor displayed the correct morphological and molecular characteristics of normal human epidermis whereas the psoriasis-associated proteins, hBD-2, SKALP/elafin, and CK16, were absent. Skin equivalents generated from foreskin keratinocytes were clearly abnormal both morphologically and with respect to gene expression. When normal skin equivalents derived from adult keratinocytes were stimulated with psoriasis-associated cytokines [tumor necrosis factor-α, interleukin (IL)-1α, IL-6, and IL-22] or combinations thereof, strong expression of hBD-2, SKALP/elafin, CK16, IL-8, and tumor necrosis factor-α was induced as shown by quantitative polymerase chain reaction and immunohistochemistry. Retinoic acid but not cyclosporin A was found to inhibit cytokine-induced gene expression at both the mRNA and protein levels. These results illustrate the potential of this disease model to study the molecular pathology and pharmacological intervention in vitro. Psoriasis is a highly prevalent inflammatory skin disease that has both environmental and genetic components to its etiology.1Bowcock AM Krueger JG Getting under the skin: the immunogenetics of psoriasis.Nat Rev Immunol. 2005; 5: 699-711Crossref PubMed Scopus (396) Google Scholar Linkage analysis has been used to identify multiple loci and alleles that confer risk of the disease, with the strongest genetic effect found at chromosome 6p21.3, where haplotypes carrying the HLA-Cw6 allele are associated with an increase in risk.2Nair RP Stuart PE Nistor I Hiremagalore R Chia NV Jenisch S Weichenthal M Abecasis GR Lim HW Christophers E Voorhees JJ Elder JT Sequence and haplotype analysis supports HLA-C as the psoriasis susceptibility 1 gene.Am J Hum Genet. 2006; 78: 827-851Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar Recently we have found that increased β-defensin copy numbers are associated with psoriasis, suggesting that both the adaptive immune system and the epidermal innate immune system are causally involved in the disease.3Hollox EJ Huffmeier U Zeeuwen PL Palla R Lascorz J Rodijk-Olthuis D van de Kerkhof PC Traupe H de Jongh G Heijer MD Reis A Armour JA Schalkwijk J Psoriasis is associated with increased beta-defensin genomic copy number.Nat Genet. 2008; 40: 23-25Crossref PubMed Scopus (548) Google Scholar Psoriasis is characterized by erythro-squamous plaques, and histological examination of psoriatic lesions shows inflammation, increased proliferation, and disturbed epidermal differentiation.4Schön MP Boehncke WH Psoriasis.N Engl J Med. 2005; 352: 1899-1912Crossref PubMed Scopus (1010) Google Scholar At the molecular level, a regenerative epidermal differentiation program is induced that includes expression of psoriasis-associated genes such as cytokeratin 16 (CK16), SKALP/elafin, psoriasin, and β-defensin-2 (hBD-2).5de Jongh GJ Zeeuwen PL Kucharekova M Pfundt R van der Valk PG Blokx W Dogan A Hiemstra PS van de Kerkhof PC Schalkwijk J High expression levels of keratinocyte antimicrobial proteins in psoriasis compared with atopic dermatitis.J Invest Dermatol. 2005; 125: 1163-1173Crossref PubMed Scopus (242) Google Scholar Furthermore, high levels of proinflammatory cytokines and chemokines have been demonstrated, including interferon-γ, interleukin (IL)-1, tumor necrosis factor (TNF)-α, IL-6, and IL-22, which are produced by multiple cell types.6Uyemura K Yamamura M Fivenson DF Modlin RL Nickoloff BJ The cytokine network in lesional and lesion-free psoriatic skin is characterized by a T-helper type 1 cell-mediated response.J Invest Dermatol. 1993; 101: 701-705Abstract Full Text PDF PubMed Google Scholar, 7Ettehadi P Greaves MW Wallach D Aderka D Camp RD Elevated tumour necrosis factor-alpha (TNF-alpha) biological activity in psoriatic skin lesions.Clin Exp Immunol. 1994; 96: 146-151Crossref PubMed Scopus (446) Google Scholar, 8Grossman RM Krueger J Yourish D Granelli-Piperno A Murphy DP May LT Kupper TS Sehgal PB Gottlieb AB Interleukin 6 is expressed in high levels in psoriatic skin and stimulates proliferation of cultured human keratinocytes.Proc Natl Acad Sci USA. 1989; 86: 6367-6371Crossref PubMed Scopus (747) Google Scholar, 9Bonifati C Ameglio F Cytokines in psoriasis.Int J Dermatol. 1999; 38: 241-251Crossref PubMed Scopus (169) Google Scholar, 10Wolk K Kunz S Witte E Friedrich M Asadullah K Sabat R IL-22 increases the innate immunity of tissues.Immunity. 2004; 21: 241-254Abstract Full Text Full Text PDF PubMed Scopus (1155) Google Scholar A wide array of mechanistically distinct anti-psoriatic therapies is available, including agents that presumably target the adaptive immune system (corticosteroids, UVB, PUVA, calcineurin inhibitors), agents that are thought to be directed to the keratinocyte (retinoids, vitamin D3 derivatives, dithranol) and agents that possibly target multiple cell types (methotrexate, anti-TNF).4Schön MP Boehncke WH Psoriasis.N Engl J Med. 2005; 352: 1899-1912Crossref PubMed Scopus (1010) Google Scholar, 11Lowes MA Bowcock AM Krueger JG Pathogenesis and therapy of psoriasis.Nature. 2007; 445: 866-873Crossref PubMed Scopus (1409) Google Scholar, 12Gottlieb AB Psoriasis: emerging therapeutic strategies.Nat Rev Drug Discov. 2005; 4: 19-34Crossref PubMed Scopus (133) Google Scholar, 13Krueger G Ellis CN Psoriasis—recent advances in understanding its pathogenesis and treatment.J Am Acad Dermatol. 2005; 53: S94-S100Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar Although the anti-psoriatic armamentarium has been expanded throughout the last years there is still room for improvement with regard to efficacy and side effects. The development of relevant, high-content in vitro models would greatly enhance the evaluation of novel therapeutic agents. Submerged keratinocyte culture systems have been widely used for biological and pharmacological studies and some of these have been developed for high-throughput screening of anti-psoriatic drugs.14Pol A Bergers M Van Ruissen F Pfundt R Schalkwijk J A simple technique for high-throughput screening of drugs that modulate normal and psoriasis-like differentiation in cultured human keratinocytes.Skin Pharmacol Appl Skin Physiol. 2002; 15: 252-261Crossref PubMed Scopus (18) Google Scholar, 15Pol A Bergers M Schalkwijk J Comparison of antiproliferative effects of experimental and established antipsoriatic drugs on human keratinocytes, using a simple 96-well-plate assay.In Vitro Cell Dev Biol Anim. 2003; 39: 36-42Crossref PubMed Scopus (49) Google Scholar, 16Amigó M Schalkwijk J Olthuis D De Rosa S Paya M Terencio MC Lamme E Identification of avarol derivatives as potential antipsoriatic drugs using an in vitro model for keratinocyte growth and differentiation.Life Sci. 2006; 79: 2395-2404Crossref PubMed Scopus (24) Google Scholar Recent studies introduced more advanced systems such as three-dimensional tissue-engineered human skin equivalents.17MacNeil S Progress and opportunities for tissue-engineered skin.Nature. 2007; 445: 874-880Crossref PubMed Scopus (843) Google Scholar Tissue-engineered skin equivalents were initially developed for treatment of skin defects such as burn wounds and ulcers, but have also made a great impact on basic and applied research.17MacNeil S Progress and opportunities for tissue-engineered skin.Nature. 2007; 445: 874-880Crossref PubMed Scopus (843) Google Scholar Commercially available skin equivalents, mostly derived from foreskin keratinocytes, mimic normal skin to a large extent and are used in a wide range of biological studies, skin corrosion testing, and irritation studies.18Netzlaff F Lehr CM Wertz PW Schaefer UF The human epidermis models EpiSkin, SkinEthic and EpiDerm: an evaluation of morphology and their suitability for testing phototoxicity, irritancy, corrosivity, and substance transport.Eur J Pharm Biopharm. 2005; 60: 167-178Crossref PubMed Scopus (271) Google Scholar el-Ghalbzouri and colleagues19el-Ghalbzouri A Gibbs S Lamme E Van Blitterswijk CA Ponec M Effect of fibroblasts on epidermal regeneration.Br J Dermatol. 2002; 147: 230-243Crossref PubMed Scopus (231) Google Scholar have described the development of skin equivalents using de-epidermized dermis (DED) and adult keratinocytes. The addition of fibroblasts or defined growth factors to the equivalents stimulated the development of a good morphology of the epithelium. However, in contrast to normal skin, the psoriasis-associated markers SKALP/elafin and cytokeratin 6 were still present in this model. More recent studies have introduced skin equivalent models for diseased skin, by using keratinocytes from psoriasis patients,20Barker CL McHale MT Gillies AK Waller J Pearce DM Osborne J Hutchinson PE Smith GM Pringle JH The development and characterization of an in vitro model of psoriasis.J Invest Dermatol. 2004; 123: 892-901Crossref PubMed Scopus (82) Google Scholar induction of a psoriatic phenotype by inhibition of transglutaminases,21Harrison CA Layton CM Hau Z Bullock AJ Johnson TS Macneil S Transglutaminase inhibitors induce hyperproliferation and parakeratosis in tissue-engineered skin.Br J Dermatol. 2007; 156: 247-257Crossref PubMed Scopus (41) Google Scholar or by the addition of lymphocytes.22Engelhart K El Hindi T Biesalski HK Pfitzner I In vitro reproduction of clinical hallmarks of eczematous dermatitis in organotypic skin models.Arch Dermatol Res. 2005; 297: 1-9Crossref PubMed Scopus (38) Google Scholar All these models exhibit features of psoriatic epidermis and some of them were validated by anti-psoriatic agents. Here we aimed to generate a reconstructed skin model from normal adult human keratinocytes, that would allow controlled induction of psoriasis-associated features and gene expression by the addition of relevant pro-inflammatory cytokines. The system should allow quantitative measurement of established psoriasis markers in the epidermal keratinocytes. Preferably, the starting point should be reconstructed skin that would mimic normal skin both morphologically and with respect to gene expression, as defined by the expression of established marker genes (CK10, involucrin, loricrin) and the absence of psoriasis-associated markers (hBD-2, SKALP/elafin, CK16) and low expression levels of psoriasis-associated pro-inflammatory cytokines such as IL-8 and TNF-α. Here we describe a model system that fulfills these criteria and that is potentially useful for studying biology of the disease and screening of anti-psoriatic drugs. Cells from the mouse fibroblast cell line 3T3 were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc., Grand Island, NY) supplemented with penicillin/streptomycin (50 IU/ml; ICN Biomedicals, Zoetermeer, The Netherlands) and 10% calf serum with iron (Hyclone, Logan, UT). Keratinocytes were obtained from human abdominal skin derived from donors who underwent surgery for abdominal wall correction. After isolation by trypsin treatment for 16 to 20 hours at 4°C, keratinocytes were cultured in the presence of irradiated (3295 cGy for 4.10 minutes) cells from the 3T3 cell line. 3T3 cells were seeded at a concentration of 3 × 104 cells per cm2 in Greens medium, which consisted of two parts Dulbecco's modified Eagle's medium (Life Technologies, Inc.) and one part of Ham's F12 medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Hyclone), l-glutamine (4 mmol/L; Life Technologies, Inc.), penicillin/streptomycin (50 IU/ml; Life Technologies, Inc.), adenine (24.3 μg/ml; Calbiochem, San Diego, CA), insulin (5 μg/ml; Sigma, St. Louis, MO), hydrocortisone (0.4 μg/ml; Merck, Darmstadt, Germany), triiodothyronine (1.36 ng/ml, Sigma) and cholera toxin (10−10 mol/L, Sigma). The next day keratinocytes were added at a concentration of 5 × 104 cells per cm2. After 3 days, medium was replaced by Greens medium containing epidermal growth factor (EGF, 10 ng/ml; Sigma). The cells were then refreshed every 2 to 3 days, and when wells were almost confluent, cells were trypsinized and stored in the liquid nitrogen. DED was generated using abdominal skin from donors who underwent surgery for abdominal wall correction. After incubation of the skin for 5 to 10 minutes in phosphate-buffered saline (PBS) at 56°C, the epidermis was separated from the dermis. The dermis was then incubated for 1 month in PBS containing gentamicin (0.5 mg/ml; Life Technologies, Inc.) and antibiotic/antimycotic (Life Technologies, Inc.) at 37°C. Punches were prepared from this DED using an 8-mm biopter. DED still contained basal membrane, as demonstrated by expression of heparin sulfate and collagen type IV. A hollow metal ring with a diameter of 1 cm was placed on the DED, and 2 × 105 keratinocytes were seeded in the ring in medium containing 5% serum, consisting of two parts Dulbecco's modified Eagle's medium (Life Technologies, Inc.) and one part Ham's F12 medium (Life Technologies, Inc.) supplemented with 5% calf serum (Hyclone), l-glutamine (4 mmol/L, Life Technologies, Inc.), penicillin/streptomycin (50 IU/ml, Life Technologies, Inc.), adenine (24.3 μg/ml, Calbiochem), ascorbic acid (50 μg/ml, Sigma), insulin (0.2 μmol/L, Sigma), hydrocortisone (1 μmol/L, Merck), triiodothyronine (1.36 ng/ml, Sigma), and cholera toxin (10−10 mol/L, Sigma). After 1 day, the ring was removed, and the construct was cultured submerged for 3 days. Then, the skin equivalents were placed on a grid, and cultured for 10 days at the air-liquid interface in medium without serum, consisting of two parts Dulbecco's modified Eagle's medium (Life Technologies, Inc.) and one part Ham's F12 medium (Life Technologies, Inc.) supplemented with l-glutamine (4 mmol/L, Life Technologies, Inc.), penicillin/streptomycin (50 IU/ml, Life Technologies, Inc.), adenine (24.3 μg/ml, Calbiochem), l-serine (1 mg/ml, Sigma), l-carnitine (2 μg/ml, Sigma), BSA lipid mix (palmitic acid (25 μmol/L, Sigma), arachidonic acid (7 μmol/L, Sigma), linoleic acid (15 μmol/L, Sigma), vitamin E (0.4 μg/ml, Sigma), ascorbic acid (50 μg/ml, Sigma), insulin (0.1 μmol/L, Sigma), hydrocortisone (1 μmol/L, Merck), triiodothyronine (1.36 ng/ml, Sigma), cholera toxin (10−10 mol/L, Sigma), keratinocyte growth factor (KGF) (5 ng/ml, Sigma) and epidermal growth factor (2 ng/ml, Sigma). Psoriatic skin equivalents were obtained by incubating normal skin equivalents the last 4 days of the air-liquid interface culture with various combinations of cytokines, including IL-1α (Preprotech, Rocky Hill, NJ), TNF-α (Preprotech), IL-6 (2 × 108 IU/ml; Gentaur, Brussels, Belgium), and IL-22 (Preprotech). Epidermis was separated for the skin equivalents by dispase (Roche Diagnostics, Mannheim, Germany) treatment for 4 hours at 4°C, and total RNA was isolated from the epidermis using Trizol reagent (Life Technologies, Gaithersburg, MD). Generation of first strand cDNA was performed as described previously.23Zeeuwen PL van Vlijmen-Willems IM Jansen BJ Sotiropoulou G Curfs JH Meis JF Janssen JJ Van Ruissen F Schalkwijk J Cystatin M/E expression is restricted to differentiated epidermal keratinocytes and sweat glands: a new skin-specific proteinase inhibitor that is a target for cross-linking by transglutaminase.J Invest Dermatol. 2001; 116: 693-701Crossref PubMed Scopus (91) Google Scholar The reverse transcriptase reaction products were used for quantitative real-time PCR amplification, which was performed with MyiQ single-color real-time detection system for quantification with SYBR Green and melting curve analysis (Bio-Rad, Richmond, CA). Primers for hBD-2, SKALP/elafin, TNF-α, CXCL-8, and the housekeeping gene human acidic ribosomal protein P0 (hARP) were obtained from Biolegio (Malden, The Netherlands). DNA was PCR-amplified using iQ SYBR Green Supermix (Bio-Rad, Hercules, CA) under the following conditions: 2 minutes at 50°C and 10 minutes at 95°C followed by 40 cycles of 15 minutes at 95°C and 1 minute at 60°C, with data collection in the last 30 seconds. All primer concentrations were 300 nmol/L in a total reaction volume of 25 μl. The amount of each mRNA was normalized to the amount of hARP in the same sample. Relative mRNA expression levels of all examined genes were measured using the comparative 2−δδCT method.24Livak KJ Schmittgen TD Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (124899) Google Scholar Human skin equivalents were fixed in buffered 4% formalin for 4 hours, and processed for routine histology. Skin equivalents were embedded in paraffin, and 6-μm sections were cut. Sections were stained with hematoxylin and eosin (H&E) or processed for immunohistochemical staining using an indirect immunoperoxidase technique with avidin-biotin complex enhancement. To study epidermal proliferation, an antibody directed against Ki-67 (MIB-1; Immunotech, SA, Marseilles, France) was used whereas epidermal differentiation was studied using an antibodies directed against cytokeratin 10 (clone RKSE60; Sanbio, Uden, The Netherlands) and cytokeratin 16 (Novacastra, Newcastle-On Tyne, UK). SKALP/elafin was stained using polyclonal antibodies as described previously25Wingens M van Bergen BH Hiemstra PS Meis JF van Vlijmen-Willems IM Zeeuwen PL Mulder J Kramps HA Van Ruissen F Schalkwijk J Induction of SLPI (ALP/HUSI-I) in epidermal keratinocytes.J Invest Dermatol. 1998; 111: 996-1002Crossref PubMed Scopus (106) Google Scholar and hBD-2 was stained using goat anti-hBD-2 polyclonal serum derived from Peprotech (London, UK). Statistical analysis was performed using the Statistica software package (Statsoft Inc., Tulsa, OK). Analysis of qPCR data were done on the δCt values, using a paired t-test or analysis of variance (repeated design), followed by posthoc testing (Duncan's multiple range test). Adult abdominal keratinocytes were seeded on DED, and cultured for 4 days submerged followed by 10 days culturing at the air-liquid interface. To obtain a well-differentiated epithelium that would lack expression of psoriasis-associated genes, the effect of a number of relevant growth factors in a chemically defined medium that lacked serum or bovine pituitary extract, was examined. This was done in various concentrations of these factors, either alone or in combination, and the effect on morphology and expression of SKALP/elafin, hBD-2, and CK16 by the keratinocytes was examined by quantitative real-time PCR (qPCR) and histology. We found that a combination of 5 ng/ml of KGF and 2 ng/ml of EGF in the medium during the air-liquid interface culture was optimal to obtain a good morphology of the epithelium of the skin equivalent, as demonstrated by H&E staining (Figure 1). The cells were shown to attach to the DED in the submerged phase (Figure 1A), followed by formation of a multilayered epithelium during the air-liquid interface culture (Figure 1, B–D). At day 7 of the air-liquid interface culture, differentiation of the epithelium was shown as demonstrated by the development of a granular and a cornified cell layer (Figure 1C). After 10 days culturing at the air-liquid interface, a well-differentiated fully stratified epithelium was formed (Figure 1D). As shown by immunohistochemistry, normal expression of CK10 was noted (Figure 2B) whereas no expression of SKALP/elafin, hBD-2, or CK16 was detected (Figure 2, C–E). This pattern of protein expression, which is also found in normal skin in vivo, was consistently noted in primary cells derived from more than 10 different donors. Because most commercially available skin equivalents use foreskin keratinocytes, we generated skin constructs from neonatal foreskins, for comparison. Under the conditions used here, the constructs containing foreskin keratinocytes displayed parakeratosis (Figure 2F) and consistently expressed the psoriasis-associated marker SKALP/elafin (Figure 2J). Expression of the other marker genes was similar to that found in abdominal keratinocytes (Figure 2, G–I). These results show that adult normal human keratinocytes cultured at an air-liquid interface with defined medium and two defined growth factors (EGF and KGF) exhibit a normal stratification and a protein expression profile found in normal human epidermis.Figure 2Comparison of adult and neonatal keratinocytes in the development of normal human skin equivalents. Abdominal (A–E) or foreskin (F–J) human keratinocytes were seeded on DED, and cultured for 4 days submerged, followed by 10 days of culture at the air-liquid interface. Morphology of the skin constructs was studied by H&E staining (A and F), whereas protein expression of CK10 (B and G), CK16 (C and H), hBD-2 (D and I), and SKALP/elafin (E and J) was determined by immunohistochemistry. Note the expression of SKALP/elafin and the presence of parakeratosis in the constructs derived from neonatal foreskin keratinocytes.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To induce a psoriatic phenotype in the normal human skin equivalents, the constructs were stimulated during the last 4 days of the air-liquid interface culture with various combinations of IL-1α, TNF-α, and IL-6. mRNA was isolated, and gene expression of SKALP/elafin and hBD-2 was determined by qPCR, see Figure 3, A and B, respectively. Treatment of the skin constructs with any of the stimuli induced an increase in expression of SKALP/elafin and/or hBD-2 compared to the control medium. The combination of IL-1α, TNF-α, and IL-6 was found to induce significantly higher expression levels of psoriasis-associated genes than any of the other treatments. TNF-α alone variably induced hBD-2 and SKALP/elafin expression, and high concentrations of TNF-α had a deleterious effect on epidermal morphology (not shown). IL-6 alone moderately affected hBD-2 and SKALP/elafin expression (not shown). The effect of cytokine stimulation of the normal skin constructs was further investigated at the protein level by immunohistochemical analysis of SKALP/elafin and hBD-2 (Figure 4). In the control skin equivalents, no expression of SKALP/elafin (Figure 4A) and hBD-2 (Figure 4E) could be demonstrated. Stimulation with IL-α alone (Figure 4, B and F), or a combination of IL-1α and TNF-α (Figure 4, C and G) induced weak to moderate expression of SKALP/elafin and hBD-2 protein in the upper layers of the epidermis. Stimulation of the skin construct with the combination of IL-1α, TNF-α, and IL-6 (Figure 4, D and H) induced high expression levels of both SKALP/elafin and hBD-2 protein in the stratum granulosum and stratum spinosum, a pattern also found in lesional psoriatic epidermis. In skin equivalents with high epidermal hBD-2 expression, hBD-2 staining was also found in the acellular dermal compartment (Figure 4H), presumably by the interaction of secreted hBD-2, which is a highly cationic protein, with negatively charged dermal structures such as basal membranes. Cytokine stimulation minimally affected morphology of the skin constructs. To study the effects of pro-inflammatory cytokines on epidermal differentiation expression of the normal differentiation marker CK10 and the regeneration-associated marker CK16 was determined by immunohistochemistry (Figure 5). As shown before, control skin constructs showed high CK10 expression in the suprabasal layers (Figure 5A), whereas CK16 was almost absent in the skin constructs (Figure 5E). Stimulation of the constructs with IL-1α, or a combination of IL-1α and TNF-α did not affect CK10 expression (Figure 5, B and C), whereas CK16 expression was slightly increased (Figure 5, F and G). The combination of IL-1α, TNF-α, and IL-6 slightly decreased CK10 expression (Figure 5D), whereas CK16 expression was markedly increased (Figure 5H), showing that cytokine stimulation affects differentiation of the skin constructs in a way that is very similar to that found in lesional psoriatic epidermis. Another hallmark of lesional psoriatic skin are high levels of inflammatory mediators produced both by infiltrate cells and the keratinocytes. We therefore measured the expression of IL-8 and TNF-α in cytokine-stimulated normal human skin equivalents, using qPCR. The mixture of cytokines that induced strong expression of SKALP/elafin, hBD-2, and CK16 was also shown to increase gene expression of both TNF-α (Figure 6A) and IL-8 (Figure 6B), as compared to the control skin construct. Because recent studies have suggested a role for Th17 cell-derived cytokines in psoriasis we studied the effect of IL-22 on the skin equivalents. Figure 7 demonstrates that IL-22 induces a dose-dependent induction of hBD-2 protein expression as determined by immunohistochemistry. No effect was noted on epidermal morphology or cellular proliferation (not shown). We performed a limited validation of the psoriasis skin equivalent model by testing the effect of selected anti-psoriatic drugs. The prediction would be that drugs known to target the epidermal compartment would show a therapeutic effect, whereas drugs that are mainly active against T cells (absent in our model) would be without effect. Normal skin constructs were stimulated the last 4 days of the air-liquid interface culture with a mixture of IL-1α, TNF-α, and IL-6 in the presence or absence of all-trans retinoic acid (ATRA) or cyclosporine A. Gene expression of SKALP/elafin and hBD-2 in the epithelium was determined by qPCR, and protein expression was determined by immunohistochemistry. ATRA was shown to inhibit cytokine-induced SKALP/elafin (Figure 8A) and hBD-2 (Figure 8B) gene expression as determined by qPCR. Also cytokine-induced SKALP/elafin (Figure 9, A–C) and hBD-2 (Figure 9, D–F) protein expression were inhibited by ATRA, suggesting a therapeutic effect of ATRA, and illustrating the potential use of the model for drug screening. ATRA also inhibited the expression of the normal differentiation marker CK10, which is a known side effect of retinoid therapy in vivo (data not shown). In contrast, no inhibition of cytokine-induced TNF-α and IL-8 expression by ATRA was shown (data not shown). No significant effect of cyclosporine A on hBD-2 and SKALP/elafin expression was noted in concentrations up to 10−6 mol/L (data not shown).Figure 9Effect of all-trans retinoic acid (ATRA) on cytokine-induced SKALP/elafin and hBD-2 protein expression in skin equivalents. Human skin equivalents were stimulated for 4 days of culture with a mixture of 10 ng/ml of IL-1α, 5 ng/ml of TNF-α, and 5 ng/ml of IL-6 in the presence or absence of ATRA (10−6 or 10−7 mol/L). Protein expression of SKALP/elafin (A: no cytokines; B: cytokines; C: cytokines and ATRA) and hBD-2 (D: no cytokines; E: cytokines; F: cytokines and ATRA) was determined by immunohistochemistry. Note the reduction of SKALP/elafin and hBD-2-positive cell layers in the ATRA-treated constructs (C and F) compared to the untreated cytokine-stimulated constructs (B and E).View Large Image Figure ViewerDownload Hi-res image Download (PPT) In the present study we sought to develop skin model systems, using a minimal set of relevant growth factors and cytokines, that would reproducibly display morphological and molecular features of normal and psoriatic epidermis. A chemically defined medium with EGF and KGF was found to stimulate development of normal epidermis lacking psoriasis markers, whereas a combination of IL-1α, TNF-α, and IL-6 induced expression of the psoriasis-associated prot
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