Scales were formerly removed from the index lesions by a 3-day treatment with 5% salicylic acid in petrolatum

Scales were formerly removed from the index lesions by a 3-day treatment with 5% salicylic acid in petrolatum. different leukocyte subsets in the skin in these diseases. 8,9 Keratinocytes exposed to IFN-, TNF-, and IL-17 also express membrane ICAM-1, which plays a relevant role in the adhesion of lymphocytes to keratinocytes, and in the COG 133 regulation of lymphocyte effector functions. 3,10,11 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 COG 133 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. 12-15 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-, and endothelial cells express iNOS after activation with IL-1. 14-16 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. 17 A novel function of NO is its ability to modulate chemokine expression, as already assessed in leukocytes and glomerular cells. 18,19 In particular, IFN– and TNF–induced IP-10 and Mig (CXCL9) expression decreased in resident glomerular cells of kidneys on NO treatment through inhibition of nuclear factor (NF)-B activity. 19 Moreover, the production of MCP-1 and RANTES by the human keratinocyte cell line HaCaT could be reduced by NO donors. 20,21 Finally, NO donors down-regulated endothelial cell expression COG 133 of various adhesion molecules including ICAM-1, VCAM-1, and E-selectin. 22,23 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. Materials and Methods Chemicals Glutathione (GS-H), = 3, two females and one male; ages 28 to 37 years) and normal-appearing skin of patients with psoriasis vulgaris (= 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. 24 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 5 days before performing experiments. Keratinocytes were stimulated with 100 U/ml of IFN- and 50 ng/ml of TNF- (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. 1-4,24 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). Enzyme-Linked Immunosorbent Assay (ELISA) Cell-free supernatants from resting or stimulated keratinocyte cultures Rabbit polyclonal to AIPL1 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 manufacturers 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. RNase Protection Assay and Northern Blot Analysis 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. [32P]-labeled anti-sense riboprobes were generated from DNA corresponding to RANTES, IP-10, MIP-1 (CCL3), MIP-1 (CCL4), MCP-1, IL-8, and I-309 (CCL1), as well as the housekeeping genes, L32 and GAPDH. Ten.

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