The Psoriatic Transcriptome Closely Resembles That Induced by Interleukin-1 in Cultured Keratinocytes
2007; Elsevier BV; Volume: 171; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2007.061067
ISSN1525-2191
AutoresJohn Mee, Claire M. Johnson, Nilesh Morar, Frank Burslem, Richard Groves,
Tópico(s)T-cell and B-cell Immunology
ResumoPsoriasis has been considered an autoimmune, T cell-mediated disorder in which adaptive immune responses predominate over those of non-antigen-specific innate immunity. To test this hypothesis, we profiled the transcriptome of psoriatic tissue and compared the data with that from cultured human keratinocytes exposed to the proinflammatory cytokine interleukin (IL)-1α and the Th1 cytokine interferon-γ. When compared with patient-matched, nonlesional skin biopsies, psoriatic samples exhibited regulation of 90 transcripts including several members of the epidermal differentiation complex, molecules with antimicrobial activity, and hyperproliferation-associated keratins. Stimulation of keratinocytes with interferon-γ resulted in regulation of 252 transcripts, with particularly strong expression of the CXCR3-binding ligands CXCL9, -10, and -11 and class II major histocompatibility complex genes, primarily those of the HLA-DR and -DP families. In contrast, the transcriptome resulting from exposure of keratinocytes to IL-1α elicited differences in just 19 transcripts, particularly genes within the epidermal differentiation complex and antimicrobial molecules, including PI3 and DEFB4. Major differences between the two keratinocyte transcriptomes were exhibited with only five induced IL-1α transcripts also regulated in the interferon-γ set. Unexpectedly, there was a high correlation between psoriatic lesional tissue and the IL-1α transcriptome. These findings suggest that the inflammatory milieu in the epidermal microenvironment in psoriasis is more likely dependent on evolutionarily ancient cytokines such as IL-1, rather than those of the adaptive immune response. Psoriasis has been considered an autoimmune, T cell-mediated disorder in which adaptive immune responses predominate over those of non-antigen-specific innate immunity. To test this hypothesis, we profiled the transcriptome of psoriatic tissue and compared the data with that from cultured human keratinocytes exposed to the proinflammatory cytokine interleukin (IL)-1α and the Th1 cytokine interferon-γ. When compared with patient-matched, nonlesional skin biopsies, psoriatic samples exhibited regulation of 90 transcripts including several members of the epidermal differentiation complex, molecules with antimicrobial activity, and hyperproliferation-associated keratins. Stimulation of keratinocytes with interferon-γ resulted in regulation of 252 transcripts, with particularly strong expression of the CXCR3-binding ligands CXCL9, -10, and -11 and class II major histocompatibility complex genes, primarily those of the HLA-DR and -DP families. In contrast, the transcriptome resulting from exposure of keratinocytes to IL-1α elicited differences in just 19 transcripts, particularly genes within the epidermal differentiation complex and antimicrobial molecules, including PI3 and DEFB4. Major differences between the two keratinocyte transcriptomes were exhibited with only five induced IL-1α transcripts also regulated in the interferon-γ set. Unexpectedly, there was a high correlation between psoriatic lesional tissue and the IL-1α transcriptome. These findings suggest that the inflammatory milieu in the epidermal microenvironment in psoriasis is more likely dependent on evolutionarily ancient cytokines such as IL-1, rather than those of the adaptive immune response. Psoriasis affects approximately 2% of Western populations and is characterized by epidermal inflammation and hyperplasia, chronic neutrophilic and T-lymphocytic infiltrates, and abnormal keratinocyte differentiation.1Schön MP Boehncke WH Psoriasis.N Engl J Med. 2005; 352: 1899-1912Crossref PubMed Scopus (1010) Google Scholar Previous studies have suggested a predominance of type 1 (Th1 and Tc1 T cell subset)-associated cytokines, such as interferon (IFN)-γ and interleukin (IL)-2, within psoriatic lesions.2Austin LM Ozawa M Kikuchi T Walters IB Krueger JG The majority of epidermal T cells in psoriasis vulgaris lesions can produce type 1 cytokines, interferon-γ, interleukin-2, and tumor necrosis factor-α, defining TC1 (cytotoxic T lymphocyte) and TH1 effector populations: a type 1 differentiation bias is also measured in circulating blood T cells in psoriatic patients.J Invest Dermatol. 1999; 113: 752-759Crossref PubMed Scopus (434) Google Scholar, 3Uyemura 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 Xenotransplantation experiments involving grafting of nonlesional skin from psoriatic patients to immunodeficient mice and the subsequent introduction of autologous, stimulated immunocytes to induce plaque formation4Wrone-Smith T Nickoloff BJ Dermal injection of immunocytes induces psoriasis.J Clin Invest. 1996; 98: 1878-1887Crossref PubMed Scopus (391) Google Scholar have demonstrated the obligatory role of these cells in the pathology. Further, many effective antipsoriatic therapies, including cyclosporin A, alefacept, and efalizumab, directly target T cells as their major mode of action.5Griffiths CE T-cell-targeted biologicals for psoriasis.Curr Drug Targets Inflamm Allergy. 2004; 3: 157-161Crossref PubMed Scopus (11) Google Scholar As the predominant cell type within the epidermis, keratinocytes have been demonstrated to be pivotal in the initiation, maintenance, and regulation of immune responses in the skin.6Kupper TS Fuhlbrigge RC Immune surveillance in the skin: mechanisms and clinical consequences.Nat Rev Immunol. 2004; 4: 211-222Crossref PubMed Scopus (601) Google Scholar They can synthesize a diverse array of immunomodulatory molecules, including a number of primary cytokines [eg, IL-1 and tumor necrosis factor (TNF)-α] and chemokines such as IL-8 required to induce vascular endothelial molecules and recruit T cells to the epidermis. In addition, stimulation of keratinocytes with T-cell-derived molecules such as IFN-γ up-regulates a number of major histocompatibility complex and costimulatory molecules inducing amplification of cytokine cascades essential in the potentiation of cutaneous inflammation.7Barker JN Mitra RS Griffiths CE Dixit VM Nickoloff BJ Keratinocytes as initiators of inflammation.Lancet. 1991; 337: 211-214Abstract PubMed Scopus (640) Google Scholar A key difference between the immunological responses induced in T cells and keratinocytes is that of antigen specificity. Keratinocytes react to infection or other trauma with an evolutionarily ancient, non-antigen-specific set of responses (innate immunity), whereas T cells use somatically recombined receptor genes to mediate an antigen-specific response (adaptive immunity). Effective host defense activities within the skin require interactions between the innate and adaptive immune systems. For example, innate proinflammatory mediators such as IL-1α released from keratinocytes following trauma are a prerequisite for the extravasation of memory T cells to the epidermis and subsequent antigen-specific responses.8Kupper TS Groves RW The interleukin-1 axis and cutaneous inflammation.J Invest Dermatol. 1995; 105: 62S-66SCrossref PubMed Scopus (160) Google Scholar Such interactions suggest that keratinocyte-mediated (innate) responses may have a fundamental role to play in psoriatic pathology.9Bos JD de Rie MA Teunissen MB Piskin G Psoriasis: dysregulation of innate immunity.Br J Dermatol. 2005; 152: 1098-1107Crossref PubMed Scopus (195) Google Scholar, 10Nickoloff BJ Skin innate immune system in psoriasis: friend or foe?.J Clin Invest. 1999; 104: 1161-1164Crossref PubMed Scopus (109) Google Scholar A number of transgenic mice exhibiting psoriasiform phenotypes, in which keratinocyte molecules were either overexpressed11Carroll JM Romero MR Watt FM Suprabasal integrin expression in the epidermis of transgenic mice results in developmental defects and a phenotype resembling psoriasis.Cell. 1995; 83: 957-968Abstract Full Text PDF PubMed Scopus (286) Google Scholar, 12Sano S Chan KS Carbajal S Clifford J Peavey M Kiguchi K Itami S Nickoloff BJ DiGiovanni J Stat3 links activated keratinocytes and immunocytes required for development of psoriasis in a novel transgenic mouse model.Nat Med. 2005; 11: 43-49Crossref PubMed Scopus (597) Google Scholar or deleted,13Pasparakis M Courtois G Hafner M Schmidt-Supprian M Nenci A Toksoy A Krampert M Goebeler M Gillitzer R Israel A Krieg T Rajewsky K Haase I TNF-mediated inflammatory skin disease in mice with epidermis-specific deletion of IKK2.Nature. 2002; 417: 861-866Crossref PubMed Scopus (403) Google Scholar, 14Zenz R Eferl R Kenner L Florin L Hummerich L Mehic D Scheuch H Angel P Tschachler E Wagner EF Psoriasis-like skin disease and arthritis caused by inducible epidermal deletion of Jun proteins.Nature. 2005; 437: 369-375Crossref PubMed Scopus (486) Google Scholar support this hypothesis, and recent data indicating an essential role for resident plasmacytoid predendritic cells in psoriatic pathology15Nestle FO Conrad C Tun-Kyi A Homey B Gombert M Boyman O Burg G Liu YJ Gilliet M Plasmacytoid predendritic cells initiate psoriasis through interferon-α production.J Exp Med. 2005; 202: 135-143Crossref PubMed Scopus (879) Google Scholar further underpin the concept that innate immune mechanisms are central to this disease. Novel insights into the molecular mechanisms driving psoriatic plaques have been provided by a number of recent studies using high-density cDNA microarrays to perform global comparisons of gene expression in lesional skin with biopsies from clinically normal skin.16Bowcock AM Shannon W Du F Duncan J Cao K Aftergut K Catier J Fernandez-Vina MA Menter A Insights into psoriasis and other inflammatory diseases from large-scale gene expression studies.Hum Mol Genet. 2001; 10: 1793-1805Crossref PubMed Scopus (217) Google Scholar, 17Nomura I Gao B Boguniewicz M Darst MA Travers JB Leung DY Distinct patterns of gene expression in the skin lesions of atopic dermatitis and psoriasis: a gene microarray analysis.J Allergy Clin Immunol. 2003; 112: 1195-1202Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar, 18Quekenborn-Trinquet V Fogel P Aldana-Jammayrac O Ancian P Demarchez M Rossio P Richards HL Kirby B Nguyen C Voegel JJ Griffiths CE Gene expression profiles in psoriasis: analysis of impact of body site location and clinical severity.Br J Dermatol. 2005; 152: 489-504Crossref PubMed Scopus (32) Google Scholar, 19Zhou X Krueger JG Kao MC Lee E Du F Menter A Wong WH Bowcock AM Novel mechanisms of T-cell and dendritic cell activation revealed by profiling of psoriasis on the 63,100-element oligonucleotide array.Physiol Genomics. 2003; 13: 69-78Crossref PubMed Scopus (258) Google Scholar Thus, the psoriatic transcriptome may be broadly defined as comprising molecules found on the epidermal differentiation complex on chromosome 1q (eg, S100A7, SPRR1B), molecules with antimicrobial activity (eg, DEFB4, LCN2), hyperproliferation-associated molecules (eg, KRT6A, KRT16), and molecules involved in the regulation of proteolysis (eg, SERPINB3, PI3). In addition, numerous cytokines or cytokine-induced molecules associated with both keratinocytes (eg, IL1B, CXCL8/IL-8) and T cells (eg, IFNG, STAT1) were commonly induced in these studies. To explore whether psoriasis is associated with the innate or adaptive immune response at the molecular level, we used microarray techniques to analyze psoriatic skin and compared this with the transcriptomes generated from primary human keratinocytes exposed to either IL-1α or IFN-γ, as simple models of innate and adaptive immunity, respectively. Six unrelated patients with chronic plaque psoriasis were recruited from a local dermatology outpatient clinic. None had received any systemic or topical therapy (except emollients) for at least 6 weeks before biopsy. Following local ethical committee approval and informed consent, a pair of shave biopsies was excised from each patient using a keratome, one intralesionally and the other from clinically normal skin, at least 20 mm from any plaque. All biopsies were snap-frozen in liquid nitrogen to maximize ex vivo transcriptome accuracy before RNA extraction. In two additional patients, 2-mm punch biopsies were removed from the center of the keratomes subsequent to excision, fixed, and processed according to standard techniques for histological examination. Following local ethical committee approval and informed consent, primary human keratinocyte cultures were established as described previously20Mee JB Alam Y Groves RW Human keratinocytes constitutively produce but do not process interleukin-18.Br J Dermatol. 2000; 143: 330-336Crossref PubMed Scopus (74) Google Scholar from breast reduction tissue derived from six unrelated female donors. Second-passage cells were grown to approximately 90% confluence in 25-cm2 flasks and quiesced in supplement-free keratinocyte growth medium (EpiLife, Cascade Biologics, Mansfield, UK) for 24 hours before the addition of either recombinant human IL-1α (100 ng/ml final concentration), recombinant human IFN-γ (20 ng/ml final concentration; specific activity = 1 × 107 IU/mg) (R&D Systems, Abingdon, UK), or vehicle alone (equivalent dilution of 1% bovine serum albumin in phosphate-buffered saline). Cultures were incubated for a further 24 hours at 37°C, 5% CO2, before RNA extraction. Total RNA was isolated from all samples using TRIzol reagent (Invitrogen, Paisley, UK) according to the manufacturer's instructions. Skin biopsies were processed in 2 ml of TRIzol using a Polytron PT 1200CL homogenizer (Kinematica, Lucerne, Switzerland) for 30 seconds to commence RNA extraction. First-strand cDNA was derived from 10 μg of total RNA using a T7-(dT)24 primer and Superscript II reverse transcriptase (Invitrogen) (42°C, 1 hour). Second-strand cDNA synthesis was performed using Escherichia coli DNA ligase, E. coli DNA polymerase I, and RNase H (Invitrogen) (16°C, 2 hours). Biotin-labeled antisense cRNA was synthesized from purified double-stranded cDNA using the BioArray HighYield RNA transcript labeling kit (Enzo Diagnostics, Farmingdale, NY) according to the manufacturer's instructions (37°C, 5 hours). Two Affymetrix microarrays were used in the present studies. For the psoriatic biopsies, the HuGeneFL array, representing approximately 5600 full-length human genes, was used, whereas the HG-U133A array, containing approximately 18,000 characterized genes, was used in the analysis of the cytokine-exposed keratinocytes. For the HuGeneFL arrays, fragmentation was performed according to the Affymetrix protocol (20 μg of biotinylated cRNA in 40 mmol/L Tris-acetate, pH 8.1, 100 mmol/L potassium acetate, and 150 mmol/L magnesium acetate at 94°C for 35 minutes). Ten micrograms of fragmented cRNA were hybridized to HuGeneFL arrays according to standard Affymetrix protocols in 100 mmol/L MES buffer, 1 mol/L [Na+], 20 mmol/L ethylenediamine tetraacetic acid, 0.01% Tween 20, 0.1 mg/ml herring sperm DNA, 0.5 mg/ml acetylated bovine serum albumin, and control oligos B2, BioB, BioC, BioD, and cre at 45°C for 16 hours. The fragmentation of antisense cRNA and hybridization to HG-U133A arrays was performed at the Clinical Sciences Centre/Imperial College Microarray Centre using in-house protocols based on those provided by Affymetrix. Further details are available online (http://microarray.csc.mrc.ac.uk, accessed March 2006). For both sets of microarrays, washing and subsequent staining with streptavidin phycoerythrin (two rounds at 10 μg/ml) were performed according to standard Affymetrix protocols before scanning (Affymetrix GeneChip scanner 3000). Data from Affymetrix Microarray Suite 5.0 scanning software (CEL files) were imported into GeneSpring (version 7.2, Agilent Technologies, Palo Alto, CA) for analysis. For each experimental set of chips, array normalization was performed using the Robust Multichip Average preprocessing module of GeneSpring 7.2, followed by normalization of each probe set relative to the mean intensity value calculated on the control array. For each experiment, probe sets were initially filtered to exclude all those called as “absent” on all arrays, then for those exhibiting a twofold minimum difference in normalized expression levels. The resulting probe lists were exported to a custom-written database that was used to filter the probe sets further to include only those regulated in at least four of the six replicate experiments for all three comparisons, ie, lesional versus nonlesional skin, IL-1α versus no treatment, and IFN-γ versus no treatment, to minimize false-positive data and provide final transcript lists. Biological Process description data from the Gene Ontology Consortium, supplied for each probe set by Affymetrix (Q4 2005 update), were used for transcript annotation. To better understand molecular dysregulation within psoriatic tissue, we were first interested in defining the transcriptional changes occurring in lesional skin compared with adjacent clinically normal tissue. Two shave biopsies were taken from each of six patients with chronic plaque psoriasis. The use of keratome sections reduced the dermal content of these biopsies relative to standard punch or ellipse methodologies (Figure 1). RNA was extracted from all biopsies and subsequently processed for microarray analysis using the Affymetrix HuGeneFL array, comprising approximately 5600 well-characterized human transcripts. Analysis of these data revealed 95 probe sets (representing 90 separate transcripts) regulated by a factor of two or more in at least four of the six patients examined (Table 1 and Supplemental Table S1 at http://ajp.amjpathol.org). Seventy-five transcripts were induced and 15 were repressed. The serine protease inhibitor (SERPIN) B4 was the most strongly up-regulated transcript, with a mean induction of 64-fold. In accordance with previous studies,18Quekenborn-Trinquet V Fogel P Aldana-Jammayrac O Ancian P Demarchez M Rossio P Richards HL Kirby B Nguyen C Voegel JJ Griffiths CE Gene expression profiles in psoriasis: analysis of impact of body site location and clinical severity.Br J Dermatol. 2005; 152: 489-504Crossref PubMed Scopus (32) Google Scholar, 19Zhou X Krueger JG Kao MC Lee E Du F Menter A Wong WH Bowcock AM Novel mechanisms of T-cell and dendritic cell activation revealed by profiling of psoriasis on the 63,100-element oligonucleotide array.Physiol Genomics. 2003; 13: 69-78Crossref PubMed Scopus (258) Google Scholar, 21Hardas BD Zhao X Zhang J Longqing X Stoll S Elder JT Assignment of psoriasin to human chromosomal band 1q21: coordinate overexpression of clustered genes in psoriasis.J Invest Dermatol. 1996; 106: 753-758Crossref PubMed Scopus (80) Google Scholar we found marked enrichment for epidermal differentiation complex genes on chromosome 1q21, including S100A7/psoriasin, S100A9/calgranulin B, S100A12/calgranulin C, and the small proline-rich proteins (SPRR) 1A, 1B, 2A, 2B, 2D, and 2E. In addition to SERPINB4, a number of other protease inhibitors were induced (PI3, SERPINB3, and cystatin A), along with the antimicrobial genes β4 defensin and lipocalin 2. Marked enhancements in the transcription of the hyperproliferative keratins K6, K16, and K17 were also observed as demonstrated previously.22Leigh IM Navsaria H Purkis PE McKay IA Bowden PE Riddle PN Keratins (K16 and K17) as markers of keratinocyte hyperproliferation in psoriasis in vivo and in vitro.Br J Dermatol. 1995; 133: 501-511Crossref PubMed Scopus (258) Google Scholar Functional classification of transcripts revealed clusters of genes involved in immune responses, cellular and electron transport, and epidermal differentiation all markedly up-regulated. Surprisingly, no T cell-specific transcripts were consistently up-regulated. No functional groupings could be discerned among the 15 down-regulated transcripts, with the marked reduction of hemoglobin subunit mRNA likely indicative of greater reticulocytic contamination of the nonlesional biopsies.Table 1Differentially Expressed Transcripts between Lesional and Nonlesional Psoriatic SkinProbe IDPt 1Pt 2Pt 3Pt 4Pt 5Pt 6Fold ChangeGeneDefinitionChromosomeU19557_s_at86.2151.2394.4061.5162.2030.0664.27SERPINB4Serpin peptidase inhibitor, clade B (ovalbumin), member 418q21L10343_at36.4427.6531.6427.3828.7126.0729.65PI3Peptidase inhibitor 3, skin-derived (SKALP)20q12-q13M26311_s_at28.4940.8525.3038.1217.3723.5128.94S100A9S100 calcium binding protein A9 (calgranulin B)1q21M86757_s_at19.1029.3714.6540.077.8433.0324.01S100A7S100 calcium binding protein A7 (psoriasin 1)1q21Z71389_at36.6918.2733.5815.6625.778.4923.08DEFB4Defensin, β48p23-p22V01516_f_at7.6226.3639.0522.3026.049.2021.76KRT6AKeratin 6A12q12-q13L42583_f_at8.1424.0032.5225.6524.689.4420.74KRT6AKeratin 6A12q12-q13L42601_f_at7.6924.1834.3021.5024.5311.3120.58KRT6AKeratin 6A12q12-q13S66896_at34.1621.4414.4316.6216.1911.9219.13SERPINB3Serpin peptidase inhibitor, clade B (ovalbumin), member 318q21L05188_f_at16.6814.4213.2833.668.0412.7016.46SPRR2BSmall proline-rich protein 2B1q21-q22Z19574_rna1_at4.2018.5816.2924.2318.647.0014.82KRT17Keratin 1717q12-q21S72493_s_at10.8220.4617.5616.9510.386.7813.82KRT16Keratin 16 (focal nonepidermolytic palmoplantar keratoderma)17q12-q21X53065_f_at12.598.899.5526.147.1810.9412.55SPRR2ASmall proline-rich protein 2A1q21-q22M21302_at15.4112.1115.9814.009.766.1212.23SPRR2DSmall proline-rich protein 2D1q21-q22M86849_at4.916.1314.807.6017.325.899.44GJB2Gap junction protein, β2, 26 kd (connexin 26)13q11-q12L42611_f_at12.6913.2112.985.387.242.889.06KRT6EKeratin 6E12q13L00205_at8.8019.908.194.328.832.368.73KRT6BKeratin 6B12q12-q13D88422_at26.613.382.812.802.683.616.98CSTACystatin A (stefin A)3q21M19888_at3.514.1913.6712.265.652.446.95SPRR1BSmall proline-rich protein 1B (cornifin)1q21-q22J05068_at14.074.506.264.506.915.476.95TCN1Transcobalamin I (vitamin B12 binding protein, R binder family)11q11-q12L05187_at5.369.319.437.356.723.506.95SPRR1ASmall proline-rich protein 1A1q21-q22L33930_s_at11.742.366.405.136.653.635.99CD24CD24 antigen (small cell lung carcinoma cluster 4 antigen)6q21S75256_s_at7.897.119.673.465.412.255.96LCN2Lipocalin 2 (oncogene 24p3)9q34M20030_f_at4.612.453.9814.802.915.775.75SPRR2ESmall proline-rich protein 2E1q21-q22M94856_at7.734.205.315.195.045.655.52FABP5Fatty acid binding protein 5 (psoriasis-associated)8q21HG880-HT880_at1.030.710.450.390.350.350.55MUC6Mucin 6, gastric11p15M57710_at0.590.290.420.500.431.030.54LGALS3Lectin, galactoside-binding, soluble, 3 (galectin 3)14q21-q22HG3115-HT3291_at1.430.790.330.190.180.150.51MBPMyelin basic protein18q23S73591_at0.360.340.400.460.550.880.50TXNIPThioredoxin interacting protein1q21U62800_at0.210.290.440.590.490.960.50CST6Cystatin E/M11q13HG3925-HT4195_at0.790.360.420.720.380.200.48SFTPA2Surfactant, pulmonary-associated protein A210q22-q23X13839_at0.190.220.390.860.600.490.46ACTA2Actin, α2, smooth muscle, aorta10q23M94077_at0.380.240.090.580.371.080.46LORLoricrin1q21X58072_at0.430.120.580.390.380.820.45GATA3GATA binding protein 310p15X76717_at0.400.210.660.390.470.540.44MT1XMetallothionein 1X16q13HG2149-HT2219_at1.111.040.140.130.120.110.44MUC5BMucin 5, subtype B, tracheobronchial11p15L11672_r_at0.470.170.460.530.410.510.42ZNF91Zinc finger protein 91 (HPF7, HTF10)19p13-p12M25079_s_at0.220.140.401.050.090.580.41HBBHemoglobin, β (aka β-globin)11p15S43646_at0.280.200.200.580.230.850.39KRT2AKeratin 2A (epidermal ichthyosis bullosa of Siemens)12q11-q13Z84721_cds2_at0.100.040.310.960.090.680.36HBA2Hemoglobin, α216p13HG1428-HT1428_s_at0.060.030.191.220.070.500.35HBBHemoglobin, β11p15Data represent the fold increases in expression between the two tissue types in six unrelated psoriatic patients (Pt 1–6), ranked by mean fold change. Probe IDs refer to Affymetrix HuGeneFL microarrays. Values in dark shading represent increases, light shading denotes decreases, and no shading indicates a fold change below the twofold threshold used. Note that only the top 25 of 79 induced probe sets are displayed (see Supplemental Table S1 at http://ajp.amjpathol.org for full data set). Open table in a new tab Data represent the fold increases in expression between the two tissue types in six unrelated psoriatic patients (Pt 1–6), ranked by mean fold change. Probe IDs refer to Affymetrix HuGeneFL microarrays. Values in dark shading represent increases, light shading denotes decreases, and no shading indicates a fold change below the twofold threshold used. Note that only the top 25 of 79 induced probe sets are displayed (see Supplemental Table S1 at http://ajp.amjpathol.org for full data set). We were interested in dissecting out the influence of individual cytokines on the chronic inflammatory microenvironment observed in psoriatic skin through in vitro stimulation of keratinocytes with either IL-1α or IFN-γ. Following exposure of second-passage normal human epidermal keratinocytes to either 100 ng/ml IL-1α or 20 ng/ml IFN-γ cytokine for 24 hours, cRNA was generated for microarray analysis using the Affymetrix HG-U133A microarray. Keratinocytes expressed approximately 10,000 of the 22,283 probe sets present on the arrays. Many genes are represented by more than one probe set on the U133A array, hence 266 separate transcripts were regulated consistently in these experiments (247 by IFN-γ alone, 14 by IL-1α alone, and five by both cytokines), according to the filtering criteria used. The small set of 19 transcripts induced by IL-1α exposure in keratinocytes contained a number of genes found in the epidermal differentiation complex, namely S100A7, S100A9, S100A12, and SPRR2B (Table 2). In addition, a group of proteases (MMP1 and MMP10) and protease inhibitors (SERPINB4 and PI3) were identified, along with the antimicrobial transcripts β4 defensin and lipocalin 2 and a number of genes involved in regulation of the key proinflammatory nuclear factor κB (NF-κB) signaling pathway (IL-32, IL-1F9, IκB, and TNF-α-induced protein 3). Bioactivity of the IL-1α used in the present study was confirmed by induction of IL-8 release from keratinocytes, observed from 1 ng/ml IL-1α (data not shown).Table 2Differentially Expressed Transcripts between IL-1α−Exposed and Vehicle-Treated KeratinocytesProbe IDD1D2D3D4D5D6Fold changeGeneDefinitionChromosome205916_at122.4030.2015.8610.4210.178.1732.87S100A7S100 calcium binding protein A7 (psoriasin 1)1q21211906_s_at91.469.213.121.771.546.0318.85SERPINB4Serpin peptidase inhibitor, clade B (ovalbumin), member 418q21219554_at55.009.927.092.993.613.5113.69RHCGRhesus blood group, C glycoprotein15q25207356_at45.4911.935.621.551.554.7311.81DEFB4Defensin, β48p23-p22220322_at20.947.834.473.405.354.967.83IL1F9IL-1 family, member 92q12-q21212531_at16.076.542.802.562.252.785.50LCN2Lipocalin 2 (oncogene 24p3)9q34203535_at7.788.523.383.403.554.565.19S100A9S100 calcium binding protein A9 (calgranulin B)1q2141469_at13.677.382.812.412.302.585.19PI3Peptidase inhibitor 3, skin-derived (SKALP)20q12-q13203691_at12.837.213.302.432.322.855.16PI3Peptidase inhibitor 3, skin-derived (SKALP)20q12-q13210413_x_at14.645.072.081.311.342.704.52SERPINB4Serpin peptidase inhibitor, clade B (ovalbumin), member 418q21219630_at5.026.464.122.723.244.984.42PDZK1IP1PDZK1 interacting protein 11p33215223_s_at10.214.362.881.502.623.194.13SOD2Superoxide dismutase 2, mitochondrial6q25204475_at4.793.861.882.944.943.573.66MMP1Matrix metallopeptidase 1 (interstitial collagenase)11q22208539_x_at4.406.432.732.391.764.043.62SPRR2BSmall proline-rich protein 2B1q21-q22205863_at9.393.392.271.101.343.533.50S100A12S100 calcium binding protein A12 (calgranulin C)1q21205680_at1.745.383.472.632.105.253.43MMP10Matrix metallopeptidase 10 (stromelysin 2)11q22218810_at4.112.612.061.962.132.112.50ZC3H12AZinc finger CCCH-type containing 12A1p34203180_at3.523.281.992.111.722.252.48ALDH1A3Aldehyde dehydrogenase 1 family, member A315q26202643_s_at4.992.341.472.232.061.742.47TNFAIP3TNF-α-induced protein 36q23203828_s_at2.301.672.162.712.503.022.39IL32IL-3216p13202644_s_at4.492.101.472.042.101.732.32TNFAIP3TNF-α-induced protein 36q23201502_s_at3.432.492.001.762.421.492.27NFKBIANF-κ light polypeptide gene enhancer in B-cell inhibitor α14q13Data represent the fold increases in expression between the two treatment types in second-passage cultured keratinocytes derived from six unrelated donors (D1–6), ranked by mean fold change. Probe IDs refer to Affymetrix HG-U133A microarrays. Shading is as in Table 1. Open table in a new tab Data represent the fold increases in expression between the two treatment types in second-passage cultured keratinocytes derived from six unrelated donors (D1–6), ranked by mean fold change. Probe IDs refer to Affymetrix HG-U133A microarrays. Shading is as in Table 1. By contrast, 252 transcripts (156 increased, 96 decreased) were regulated by IFN-γ exposure (Table 3 and Supplemental Table S2 at http://ajp.amjpathol.org). Particularly strong induction (mean >100-fold) of CXCL9, -10, and -11, all ligands of the Th1 cell chemokine receptor CXCR3, and a large number of genes located in the major histocompatibility complex on chromosome 6p, particularly those of the class II family including HLA-DR and -DP, were observed. In addition to the immune response genes, a number of proteolysis-associated transcripts were induced, whereas groups of genes involved in cell cycle regulation and DNA replication and repair were markedly down-regulated following IFN-γ exposure.Table 3Differentially Expressed Transcripts between IFN-γ-Exposed and Vehicle-Treated KeratinocytesProbe IDD1D2D3D4D5D6Fold changeGeneDefinitionChromosome203915_at141.80209.0086.1396.8679.2687.41116.74CXCL9Chemokine (C-X-C motif) ligand 94q21204533_at92.3380.28117.50132.9072.28184.20113.25CXCL10Chemokine (C-X-C motif) li
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