Nitric Oxide Donors Suppress Chemokine Production by Keratinocytes in Vitro and in Vivo
2002; Elsevier BV; Volume: 161; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)64416-1
ISSN1525-2191
AutoresMaria Laura Giustizieri, Cristina Albanesi, Claudia Scarponi, Ornella De Pitá, Giampiero Girolomoni,
Tópico(s)Immune Response and Inflammation
ResumoNitric oxide (NO) is involved in the modulation of inflammatory responses. In psoriatic skin, NO is highly produced by epidermal keratinocytes in response to interferon-γ and tumor necrosis factor-α. In this study, we investigated whether the NO donors, S-nitrosoglutathione (GS-NO) and NOR-1, could regulate chemokine production by human keratinocytes activated with interferon-γ and tumor necrosis factor-α. In addition, we studied the effects of the topical application of a GS-NO ointment on chemokine expression in lesional psoriatic skin. NO donors diminished in a dose-dependent manner and at both mRNA and protein levels the IP-10, RANTES, and MCP-1 expression in keratinocytes cultured from healthy patients and psoriatic patients. In contrast, constitutive and induced interleukin-8 production was unchanged. GS-NO-treated psoriatic skin showed reduction of IP-10, RANTES, and MCP-1, but not interleukin-8 expression by keratinocytes. Moreover, the number of CD14+ and CD3+ cells infiltrating the epidermis and papillary dermis diminished significantly. NO donors also down-regulated ICAM-1 protein expression without affecting mRNA accumulation in vitro, and suppressed keratinocyte ICAM-1 in vivo. Finally, NO donors inhibited nuclear factor-κB and STAT-1, but not AP-1 activities in transiently transfected keratinocytes. These results define NO donors as negative regulators of chemokine production by keratinocytes. Nitric oxide (NO) is involved in the modulation of inflammatory responses. In psoriatic skin, NO is highly produced by epidermal keratinocytes in response to interferon-γ and tumor necrosis factor-α. In this study, we investigated whether the NO donors, S-nitrosoglutathione (GS-NO) and NOR-1, could regulate chemokine production by human keratinocytes activated with interferon-γ and tumor necrosis factor-α. In addition, we studied the effects of the topical application of a GS-NO ointment on chemokine expression in lesional psoriatic skin. NO donors diminished in a dose-dependent manner and at both mRNA and protein levels the IP-10, RANTES, and MCP-1 expression in keratinocytes cultured from healthy patients and psoriatic patients. In contrast, constitutive and induced interleukin-8 production was unchanged. GS-NO-treated psoriatic skin showed reduction of IP-10, RANTES, and MCP-1, but not interleukin-8 expression by keratinocytes. Moreover, the number of CD14+ and CD3+ cells infiltrating the epidermis and papillary dermis diminished significantly. NO donors also down-regulated ICAM-1 protein expression without affecting mRNA accumulation in vitro, and suppressed keratinocyte ICAM-1 in vivo. Finally, NO donors inhibited nuclear factor-κB and STAT-1, but not AP-1 activities in transiently transfected keratinocytes. These results define NO donors as negative regulators of chemokine production by keratinocytes. The skin is a frequent site of T-cell-mediated diseases, such as psoriasis, atopic dermatitis, and allergic contact dermatitis. In these disorders, infiltrating T cells release lymphokines that influence the immune functions of keratinocytes. In particular, cytokine-activated keratinocytes become an important source of chemokines, which direct the recruitment of specific leukocyte populations in the skin.1Albanesi C Scarponi C Sebastiani S Cavani A Federici M Sozzani S Girolomoni G A cytokine-to-chemokine axis between T lymphocytes and keratinocytes can favor Th1 cell accumulation in chronic inflammatory skin diseases.J Leukoc Biol. 2001; 70: 617-623PubMed Google Scholar, 2Sebastiani S Albanesi C De Pità O Puddu P Cavani A Girolomoni G The role of chemokines in allergic contact dermatitis.Arch Dermatol Res. 2002; 293: 552-559Crossref PubMed Scopus (131) Google Scholar Several in vitro and in vivo studies have documented that keratinocytes produce a variety of chemokines in a coordinated manner and with distinct expression profiles according to the inducing stimulus. Interferon (IFN)-γ and tumor necrosis factor (TNF)-α are the cytokines most effective in eliciting chemokine synthesis in keratinocytes, with interleukin (IL)-1, IL-4, and IL-17 also having a modulatory activity.3Albanesi C Cavani A Girolomoni G IL-17 is produced by nickel-specific T lymphocytes and regulates ICAM-1 expression and chemokines production in human keratinocytes: synergistic or antagonistic effects with IFN-γ and TNF-α.J Immunol. 1999; 162: 494-502PubMed Google Scholar, 4Albanesi C Scarponi C Sebastiani S Cavani A Federici M De Pità O Puddu P Girolomoni G IL-4 enhances keratinocytes expression of CXCR3 agonistic chemokines.J Immunol. 2000; 165: 1395-1402PubMed Google Scholar Moreover, keratinocytes from patients with psoriasis or atopic dermatitis may have intrinsic defects in chemokine gene expression. In particular, psoriatic keratinocytes produce exaggerated amounts of IL-8 (CXCL8), IP-10 (CXCL10), and MCP-1 (CCL2),5Nickoloff BJ Mitra RS Varani J Dixit VM Polverini PJ Aberrant production of interleukin-8 and thrombospondin-1 by psoriatic keratinocytes mediates angiogenesis.Am J Pathol. 1994; 144: 820-828PubMed Google Scholar, 6Gillitzer R Wolff K Tong D Müller C Yoshimura T Hartmann AA Stingl G Berger R MCP-1 mRNA expression in basal keratinocytes of psoriatic lesions.J Invest Dermatol. 1993; 101: 127-131Abstract Full Text PDF PubMed Google Scholar, 7Gottlieb AB Luster AD Posnett DN Carter DM Detection of a γ interferon-induced protein IP-10 in psoriatic plaques.J Exp Med. 1998; 168: 941-948Crossref Scopus (213) Google Scholar, 8Giustizieri ML Mascia F Frezzolini A De Pità O Chinni LM Giannetti A Girolomoni G Pastore S Keratinocytes from patients with atopic dermatitis and psoriasis show a distinct chemokines production profile in response to T cell-derived cytokines.J Allergy Clin Immunol. 2001; 107: 871-877Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar whereas keratinocytes from patients with atopic dermatitis synthesize higher levels of RANTES (CCL5).8Giustizieri ML Mascia F Frezzolini A De Pità O Chinni LM Giannetti A Girolomoni G Pastore S Keratinocytes from patients with atopic dermatitis and psoriasis show a distinct chemokines production profile in response to T cell-derived cytokines.J Allergy Clin Immunol. 2001; 107: 871-877Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar These alterations can contribute to the accumulation of different leukocyte subsets in the skin in these diseases.8Giustizieri ML Mascia F Frezzolini A De Pità O Chinni LM Giannetti A Girolomoni G Pastore S Keratinocytes from patients with atopic dermatitis and psoriasis show a distinct chemokines production profile in response to T cell-derived cytokines.J Allergy Clin Immunol. 2001; 107: 871-877Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 9Sallusto F Mackay CR Lanzavecchia A The role of chemokine receptors in primary, effector, and memory immune response.Annu Rev Immunol. 2000; 18: 593-620Crossref PubMed Scopus (938) Google Scholar Keratinocytes exposed to IFN-γ, TNF-α, and IL-17 also express membrane ICAM-1, which plays a relevant role in the adhesion of lymphocytes to keratinocytes, and in the regulation of lymphocyte effector functions.3Albanesi C Cavani A Girolomoni G IL-17 is produced by nickel-specific T lymphocytes and regulates ICAM-1 expression and chemokines production in human keratinocytes: synergistic or antagonistic effects with IFN-γ and TNF-α.J Immunol. 1999; 162: 494-502PubMed Google Scholar, 10Barker JN Sarma V Mitra RS Dixit VM Nickoloff BJ Marked synergism between tumor necrosis factor factor-alpha and interferon-gamma in regulation of keratinocyte-derived adhesion molecules and chemotactic factors.J Clin Invest. 1990; 85: 605-608Crossref PubMed Scopus (231) Google Scholar, 11Traidl C Sebastiani S Albanesi C Merk HF Puddu P Girolomoni G Cavani A Disparate cytotoxic activity of nickel-specific CD8+ and CD4+ T cell subset against keratinocytes.J Immunol. 2000; 165: 3058-3064PubMed Google Scholar Nitric oxide (NO) is a short-lived radical produced from the l-arginine pathway by different isoforms of NO synthase (NOS) that are expressed by various cell types residing in the skin. Increasing evidence indicates that NO is involved in the maintenance of skin homeostasis as well as in the modulation of inflammatory reactions. High levels of NO have been measured in the skin affected with psoriasis, atopic dermatitis, or allergic contact dermatitis.12Ormerod AD Weller R Copeland P Benjamin N Ralston SH Grabowksi P Herriot R Detection of nitric oxide and nitric oxide synthases in psoriasis.Arch Dermatol Res. 1998; 290: 3-8Crossref PubMed Scopus (118) Google Scholar, 13Ormerod AD Dwyer CM Reid CM Copeland P Thomson WD Inducible nitric oxide synthase demonstrated in irritant and allergic contact dermatitis.Acta Derm Venereol (Stockh). 1997; 77: 436-440PubMed Google Scholar, 14Rowe A Farrel AM Bunker CB Constitutive endothelial and inducible nitric oxide synthase in inflammatory dermatoses.Br J Dermatol. 1997; 136: 18-23Crossref PubMed Scopus (97) Google Scholar, 15Sirsjo A Karlsson M Gidlof A Rollman O Torma H Increased expression of inducibile nitric oxide synthase in psoriatic skin and cytokine-stimulated cultured keratinocytes.Br J Dermatol. 1996; 134: 643-648Crossref PubMed Scopus (119) Google Scholar In these conditions, proinflammatory cytokines stimulate keratinocytes to express inducible NOS (iNOS), which in turn catalyzes NO production. Fibroblasts and dendritic cells also become iNOS-positive after exposure to bacterial endotoxin and IFN-γ, and endothelial cells express iNOS after activation with IL-1β.14Rowe A Farrel AM Bunker CB Constitutive endothelial and inducible nitric oxide synthase in inflammatory dermatoses.Br J Dermatol. 1997; 136: 18-23Crossref PubMed Scopus (97) Google Scholar, 15Sirsjo A Karlsson M Gidlof A Rollman O Torma H Increased expression of inducibile nitric oxide synthase in psoriatic skin and cytokine-stimulated cultured keratinocytes.Br J Dermatol. 1996; 134: 643-648Crossref PubMed Scopus (119) Google Scholar, 16Qureshi AA Hosoi J Xu S Takashima A Granstein RD Lerner EA Langerhans cells express inducible nitric oxide synthase and produce nitric oxide.J Invest Dermatol. 1996; 107: 815-821Crossref PubMed Scopus (77) Google Scholar The role of NO in the regulation of inflammatory responses has been extensively investigated. Depending on the concentration, the cell type, and its state of activation, as well as the presence of other inflammatory mediators, NO can either block or stimulate inflammatory responses.17Bodgan C Nitric oxide and the immune response.Nat Immunol. 2001; 2: 907-916Crossref PubMed Scopus (2710) Google Scholar A novel function of NO is its ability to modulate chemokine expression, as already assessed in leukocytes and glomerular cells.18Zouki C Jozsef L Ouellet S Paquette Y Filep JG Peroxynitrite mediates cytokine-induced IL-8 gene expression and production by human leukocytes.J Leukoc Biol. 2001; 69: 815-824PubMed Google Scholar, 19Romagnani P Lazzeri E Lasagni L Mavilia C Beltrame C Francalanci M Rotondi M Annunziato F Maurenzig L Coami L Galli G Salvadori M Maggi E Seri M IP-10 and Mig production by glomerular cells in human proliferative glomerulonephritis and regulation by nitric oxide.J Am Soc Nephrol. 2002; 13: 53-64Crossref PubMed Google Scholar In particular, IFN-γ- and TNF-α-induced IP-10 and Mig (CXCL9) expression decreased in resident glomerular cells of kidneys on NO treatment through inhibition of nuclear factor (NF)-κB activity.19Romagnani P Lazzeri E Lasagni L Mavilia C Beltrame C Francalanci M Rotondi M Annunziato F Maurenzig L Coami L Galli G Salvadori M Maggi E Seri M IP-10 and Mig production by glomerular cells in human proliferative glomerulonephritis and regulation by nitric oxide.J Am Soc Nephrol. 2002; 13: 53-64Crossref PubMed Google Scholar Moreover, the production of MCP-1 and RANTES by the human keratinocyte cell line HaCaT could be reduced by NO donors.20Wetzler C Kämpfer H Pfeilschifter J Frank S Keratinocyte-derived chemotactic cytokines: expressional modulation by nitric oxide in vitro and during cutaneous wound repair in vivo.Biochem Biophys Res Commun. 2000; 274: 689-696Crossref PubMed Scopus (44) Google Scholar, 21Frank S Kämpfer H Wetzler C Stallmeyer B Pfeilschifter J Large induction of the chemotactic cytokine RANTES during cutaneous wound repair: a regulatory role for nitric oxide in keratinocyte-derived RANTES expression.Biochem J. 2000; 347: 265-273Crossref PubMed Scopus (77) Google Scholar Finally, NO donors down-regulated endothelial cell expression of various adhesion molecules including ICAM-1, VCAM-1, and E-selectin.22Spiecker M Darius H Kaboth K Hübner F Liao JK Differential regulation of endothelial cell adhesion molecule expression by nitric oxide donors and antioxidants.J Leukoc Biol. 1998; 63: 732-739Crossref PubMed Scopus (188) Google Scholar, 23Grisham MB Granger DN Lefer DJ Modulation of leukocyte-endothelial interactions by reactive metabolites of oxygen and nitrogen: relevance to ischemic heart disease.Free Radic Biol Med. 1998; 25: 404-433Crossref PubMed Scopus (252) Google Scholar In this study we tested whether synthetic NO donors could modulate the expression of chemokines and ICAM-1 in keratinocyte primary cultures established from healthy patients and patients with psoriasis. In addition, the expression of chemokines and ICAM-1 on keratinocytes as well as the amount and quality of inflammatory infiltrate were investigated in psoriatic skin before and after application of a NO-releasing cream. Glutathione (GS-H), S-nitrosoglutathione (GS-NO) and (±)-(E)-methyl-2-((E)-hydroxyimino)-5-nitro-6-methoxy-3-hexenamide (NOR-1) were purchased from Calbiochem (Darmstadt, Germany). Keratinocyte cultures were prepared from skin biopsies taken from healthy patients (n = 3, two females and one male; ages 28 to 37 years) and normal-appearing skin of patients with psoriasis vulgaris (n = 3, two males and one female; ages 25 to 42 years). Biopsies were disaggregated to single-cell suspensions using 0.25% trypsin (Biochrom, Berlin, Germany). Primary cultures were prepared by seeding cell suspensions on a feeder layer of irradiated 3T3/J2 mouse fibroblasts, and cultured according to an optimized Rheinwald and Green culture technique.24Pastore S Fanales-Belasio E Albanesi C Chinni LM Giannetti A Girolomoni G Granulocyte macrophage colony-stimulating factor is overproduced by keratinocytes in atopic dermatitis. Implications for sustained dendritic cell activation in the skin.J Clin Invest. 1997; 99: 3009-3017Crossref PubMed Scopus (187) Google Scholar Second or third passage keratinocytes were used in all experiments, with cells cultured in six-well plates in serum-free medium (Keratinocyte Growth Medium; Clonetics, San Diego, CA) for at least 3 to 5 days before performing experiments. Keratinocytes were stimulated with 100 U/ml of IFN-γ and 50 ng/ml of TNF-α (R&D Systems, Abingdon, Oxon, UK) for 16 or 24 hours. These time points were chosen because they were optimal for studying the expression of most inflammatory genes in keratinocytes in response to cytokines.1Albanesi C Scarponi C Sebastiani S Cavani A Federici M Sozzani S Girolomoni G A cytokine-to-chemokine axis between T lymphocytes and keratinocytes can favor Th1 cell accumulation in chronic inflammatory skin diseases.J Leukoc Biol. 2001; 70: 617-623PubMed Google Scholar, 2Sebastiani S Albanesi C De Pità O Puddu P Cavani A Girolomoni G The role of chemokines in allergic contact dermatitis.Arch Dermatol Res. 2002; 293: 552-559Crossref PubMed Scopus (131) Google Scholar, 3Albanesi C Cavani A Girolomoni G IL-17 is produced by nickel-specific T lymphocytes and regulates ICAM-1 expression and chemokines production in human keratinocytes: synergistic or antagonistic effects with IFN-γ and TNF-α.J Immunol. 1999; 162: 494-502PubMed Google Scholar, 4Albanesi C Scarponi C Sebastiani S Cavani A Federici M De Pità O Puddu P Girolomoni G IL-4 enhances keratinocytes expression of CXCR3 agonistic chemokines.J Immunol. 2000; 165: 1395-1402PubMed Google Scholar, 24Pastore S Fanales-Belasio E Albanesi C Chinni LM Giannetti A Girolomoni G Granulocyte macrophage colony-stimulating factor is overproduced by keratinocytes in atopic dermatitis. Implications for sustained dendritic cell activation in the skin.J Clin Invest. 1997; 99: 3009-3017Crossref PubMed Scopus (187) Google Scholar Treatments with NO donors and/or cytokines were performed in medium devoid of hydrocortisone and bovine pituitary extract, but supplemented with 0.1% bovine serum albumin (Sigma-Aldrich, Milan, Italy). Cell-free supernatants from resting or stimulated keratinocyte cultures were tested for RANTES content using the antibody (Ab) pair, rabbit polyclonal 20581D for coating and 20582D for detection (BD PharMingen, San Diego, CA). IP-10 was assayed using the purified 4D5/A7/C5 and the biotinylated 6D4/D6/G2 anti-human IP-10 monoclonal antibodies (mAbs) (BD PharMingen). IL-8 and MCP-1 were measured with OptEIA kits (BD PharMingen), as per the manufacturer's protocol. Soluble ICAM-1 was detected with an ELISA kit from Bender MedSystems (Vienna, Austria). The plates were analyzed in an ELISA reader (model 3550 UV; Bio-Rad, Hercules, CA). Keratinocyte cultures were performed in triplicate for each condition. Results are given as mean ng/106 cells ± SD. Total RNA was extracted from cultured keratinocytes using the Trizol reagent (Invitrogen Italia, Milan, Italy). The multiprobe template set hCK5 and the complete kit for RNase protection assay were purchased from BD PharMingen. [α32P]-labeled anti-sense riboprobes were generated from DNA corresponding to RANTES, IP-10, MIP-1α (CCL3), MIP-1β (CCL4), MCP-1, IL-8, and I-309 (CCL1), as well as the housekeeping genes, L32 and GAPDH. Ten μg of each RNA sample were used in the assays and processed as per the manufacturer's protocol. Two separate experiments with keratinocytes from different donors were performed with similar results. For Northern blot experiments, 5 μg of total RNA were fractionated on 1% formaldehyde-agarose gel, blotted to nylon membranes (Amersham-Pharmacia-Biotech, Milan, Italy), and fixed by UV irradiation. The ICAM-1 probe (accession no. M83071) was obtained by reverse transcriptase-polymerase chain reaction performed on RNA isolated from keratinocyte cultures stimulated with IFN-γ plus TNF-α. ICAM-1 probe was labeled with [32P] dCTP, and used for hybridization performed for 1 hour at 68°C in Quickhyb solution (Stratagene, La Jolla, CA). Blots were washed under highly stringent conditions and subjected to autoradiography. Equal loading and integrity of RNA were assessed either by ethidium bromide staining of the gels or hybridizing the membrane with a probe specific for 28S rRNA. Keratinocyte expression of membrane ICAM-1 and HLA-DR was evaluated using fluorescein isothiocyanate-conjugated anti-CD54 (clone 84H10; Immunotech, Marseille, France) and anti-HLA-DR (clone L243, BD PharMingen) mAbs. In control samples, staining was performed using isotype-matched control Abs. Apoptosis and necrosis of keratinocytes were evaluated using the Genzyme TACS Annexin V apoptosis detection kit (R&D Systems). Cells were analyzed with a FACScan equipped with Cell Quest software (Becton Dickinson, Mountain View, CA). Results are expressed as net mean fluorescence intensity, which represents the mean fluorescence intensity subtracted of the fluorescence of isotype-matched control Ab. Three patients (two females and one male, ages 29 to 39 years) with chronic plaque psoriasis underwent treatment with an ointment containing 1% GS-NO or vehicle alone applied for 2 weeks (two applications/day) on two similar lesions. Scales were formerly removed from the index lesions by a 3-day treatment with 5% salicylic acid in petrolatum. Patients were not receiving any systemic or topical therapy for at least 2 weeks before testing. Informed consent was obtained from the patients, and the local ethical committee approved the study. Four-mm punch biopsies were taken from both vehicle- and GS-NO-treated psoriatic skin and snap-frozen in OCT compound. Cryostat sections were fixed with 4% paraformaldehyde, treated with 0.3% hydrogen peroxide, and normal horse serum, and finally permeabilized with 0.1% Triton X-100. Staining was performed using the following Abs: goat polyclonal anti-RANTES (2 μg/ml), anti-MCP-1 (2 μg/ml), and anti-IL-8 (5 μg/ml) (R&D Systems), mouse mAbs anti-IP-10 (1:20) (kindly provided by M. G. Uguccioni, Institute for Research in Biomedicine, Bellinzona, Switzerland), anti-ICAM-1 (1:20), anti-CD14 (1:10), anti-CD3 (1:10) (BD PharMingen), and anti-Ki67 (1:40) (DAKO, Glostrup, Denmark). Immunoreactivity was revealed using avidin-biotin-peroxidase system and 3-amino-9-ethylcarbazole as chromogen. Sections were counterstained with Mayer's hematoxylin. As negative controls, primary Abs were omitted or replaced with isotype-matched Ig. Slides were analyzed blind by two observers. CD14+ and CD3+ cells as well as Ki67+ keratinocytes were counted on two different sections with an eyepiece graticule at a magnification of 200 in 10 adjacent fields. Keratinocytes from healthy patients and patients with psoriasis were transiently transfected in duplicate using Lipofectin reagent (Invitrogen). Typically, 2 to 2.5 × 105 cells were seeded in six-well plates 24 to 48 hours before transfection (60 to 80% confluence), and co-transfected with 1.0 μg of pCMV.SPORT-β-gal plasmid (Invitrogen) and 1.0 μg of pNF-κB-Luc, pGAS-Luc or pAP-1-Luc vectors (Stratagene). The latter plasmids contain the luciferase reporter gene driven by a basic promoter element (TATA box) joined to tandem repeats of prototypical NF-κB-, STAT1-, and AP-1-binding sites. After a 6-hour transfection, culture medium was removed, and keratinocytes were stimulated for 24 hours with IFN-γ plus TNF-α in the presence or the absence of 2.5 mmol/L of GS-H, GS-NO, or NOR-1. β-Galactosidase and luciferase activities were then measured in keratinocyte lysates using the β-Gal ELISA kit (Boehringer Mannheim, Mannheim, Germany) and the luciferase assay system (Promega, Madison, WI), respectively. The luciferase activity of each sample was normalized to the β-galactosidase activity. Wilcoxon's signed rank test was used (SigmaStat; Jandel, San Rafael, CA) to compare differences in chemokine release, cell apoptosis/necrosis, luciferase activities of transiently transfected keratinocytes, and CD14+, CD3+, and Ki67+ cells in psoriatic skin sections. P values ≤0.05 were considered significant. In the first set of experiments we sought to determine whether NO donors could regulate the expression of chemokines in activated keratinocyte cultures prepared from healthy patients and patients with psoriasis. Untreated keratinocyte cultures released spontaneously only moderate amounts of IL-8, with psoriasis keratinocytes producing more chemokine than healthy cells (data not shown).5Nickoloff BJ Mitra RS Varani J Dixit VM Polverini PJ Aberrant production of interleukin-8 and thrombospondin-1 by psoriatic keratinocytes mediates angiogenesis.Am J Pathol. 1994; 144: 820-828PubMed Google Scholar, 8Giustizieri ML Mascia F Frezzolini A De Pità O Chinni LM Giannetti A Girolomoni G Pastore S Keratinocytes from patients with atopic dermatitis and psoriasis show a distinct chemokines production profile in response to T cell-derived cytokines.J Allergy Clin Immunol. 2001; 107: 871-877Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar Keratinocytes treated with IFN-γ and TNF-α secreted high levels of chemokines with psoriasis keratinocytes releasing much higher amounts of IP-10, MCP-1, and IL-8 compared to keratinocytes from healthy donors (Figure 1). Keratinocytes stimulated with IFN-γ and TNF-α in the presence of GS-NO showed a markedly inhibited production of IP-10, MCP-1, and RANTES. This effect was dose-dependent and was not shared by GS-H. Moreover, NOR-1, which releases NO with a more rapid kinetics compared to GS-NO,25Percival MD Ouellet M Campagnolo C Claveau D Li C Inhibition of cathepsin K by nitric oxide donors: evidence for the formation of mixed disulfides and a sulfenic acid.Biochemistry. 1999; 38: 13574-13583Crossref PubMed Scopus (126) Google Scholar resulted more efficacious than GS-NO in blocking IP-10, MCP-1, and RANTES release. In contrast, both NOR-1 and GS-NO were ineffective in reducing IL-8 secretion (Figure 1). The effects of NO donors on chemokine expression were also examined at the mRNA level (Figure 2). IP-10, MCP-1, and IL-8 mRNA signals induced by IFN-γ plus TNF-α were more represented in keratinocytes from patients with psoriasis than in keratinocytes from control patients, as previously described.8Giustizieri ML Mascia F Frezzolini A De Pità O Chinni LM Giannetti A Girolomoni G Pastore S Keratinocytes from patients with atopic dermatitis and psoriasis show a distinct chemokines production profile in response to T cell-derived cytokines.J Allergy Clin Immunol. 2001; 107: 871-877Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar On the other hand, RANTES and I-309 (CCL1) mRNA signals were similar in the two keratinocyte types. GS-NO or NOR-1 decreased IFN-γ/TNF-α-induced RANTES, IP-10, MCP-1, and I-309 mRNA expression in both healthy and psoriasis keratinocytes, whereas IL-8 mRNA was not affected (Figure 2). IFN-γ and TNF-α induced a modest increase in keratinocyte apoptosis, as assessed by fluorescence-activated cell-sorting analysis after staining with propidium iodide and anti-annexin V antibody (Table 1). GS-NO or NOR-1 did not significantly elicit apoptosis nor necrosis of keratinocytes, and did not augment IFN-γ/TNF-α-induced apoptosis.Figure 2IFN-γ/TNF-α-induced IP-10, MCP-1, and RANTES mRNA expression in keratinocytes is down-regulated by NO donors. Normal and psoriatic keratinocytes were stimulated with IFN-γ and TNF-α in the presence or not of GS-NO (2.5 mmol/L) or NOR-1 (2.5 mmol/L). After 16 hours, total RNA was extracted and subjected to RNase protection assay using a human CK5 multiprobe template. Films were exposed for 8 hours.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table 1NO Donors Do Not Induce Keratinocyte Apoptosis or Necrosis in VitroTreatment*Keratinocyte cultures were left untreated or stimulated with 100 U/ml of IFN-γ and 50 ng/ml of TNF-α and/or 2.5 mmol/L of GS-NO or NOR-1. After 24 hours, cells were stained with propidium iodide and anti-annexin V antibody, and then analyzed by flow cytometry. Results are expressed as mean percentage ± SD of positive cells from three independent experiments.% Annexin V+ cells% PI+ cells% Annexin V+/PI+ cellsNone0.6 ± 0.46.1 ± 2.50.9 ± 0.3IFN-γ/TNF-α2.6 ± 0.8†P < 0.05 compared to untreated keratinocytes.6.6 ± 1.45.1 ± 0.4†P < 0.05 compared to untreated keratinocytes.GS-NO0.8 ± 0.37.6 ± 0.91.6 ± 0.5NOR-11.1 ± 0.47.3 ± 0.62.6 ± 0.6IFN-γ/TNF-α + GS-NO3.6 ± 1.4†P < 0.05 compared to untreated keratinocytes.8.6 ± 1.26.8 ± 1.1†P < 0.05 compared to untreated keratinocytes.IFN-γ/TNF-α + NOR-13.8 ± 0.7†P < 0.05 compared to untreated keratinocytes.8.2 ± 0.46.6 ± 0.5†P < 0.05 compared to untreated keratinocytes.* Keratinocyte cultures were left untreated or stimulated with 100 U/ml of IFN-γ and 50 ng/ml of TNF-α and/or 2.5 mmol/L of GS-NO or NOR-1. After 24 hours, cells were stained with propidium iodide and anti-annexin V antibody, and then analyzed by flow cytometry. Results are expressed as mean percentage ± SD of positive cells from three independent experiments.† P < 0.05 compared to untreated keratinocytes. Open table in a new tab Resting keratinocytes do not express ICAM-1 or MHC class II molecules, but they do so after activation with IFN-γ and/or TNF-α.3Albanesi C Cavani A Girolomoni G IL-17 is produced by nickel-specific T lymphocytes and regulates ICAM-1 expression and chemokines production in human keratinocytes: synergistic or antagonistic effects with IFN-γ and TNF-α.J Immunol. 1999; 162: 494-502PubMed Google Scholar ICAM-1 provides a major adhesion pathway for the retention of T lymphocytes in the epidermis. Moreover, ICAM-1 serves as an important co-stimulatory molecule for the cytotoxic activity of CD4+ and some CD8+ T lymphocytes against keratinocytes.11Traidl C Sebastiani S Albanesi C Merk HF Puddu P Girolomoni G Cavani A Disparate cytotoxic activity of nickel-specific CD8+ and CD4+ T cell subset against keratinocytes.J Immunol. 2000; 165: 3058-3064PubMed Google Scholar Therefore, we next examined whether NO donors could modulate ICAM-1 and HLA-DR expression on activated keratinocytes. Keratinocytes exposed to IFN-γ plus TNF-α showed high ICAM-1 expression, with psoriatic keratinocytes expressing higher levels of ICAM-1 compared to healthy cells (Figure 3). When activation was performed in the presence of GS-NO or NOR-1, but not GS-H, a markedly and dose-dependent reduction of membrane ICAM-1 was observed (Figure 3). In contrast, the IFN-γ/TNF-α-induced expression HLA-DR did not vary in keratinocytes co-treated with NO donors (data not shown). In the following experiments, we tested whether the decreased membrane ICAM-1 promoted by NO donors was associated with changes in the release of sICAM-1. As shown in Figure 4, A and B, addition of GS-NO or NOR-1, but not GS-H, strongly and dose dependently reduced the IFN-γ/TNF-α-induced ICAM-1 content in supernatants from both healthy and psoriasis keratinocyte cultures. Northern blot analysis revealed that psoriatic keratinocytes treated with IFN-γ and TNF-α had ICAM-1 mRNA levels fourfold higher compared to normal keratinocytes. Surprisingly, GS-NO or NOR-1 did not alter ICAM-1 mRNA accumulation (Fig
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