Archive for the ‘Neurokinin Receptors’ Category

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

Wednesday, October 27th, 2021

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.

Supplementary Materials Supplemental Materials supp_26_4_622__index

Tuesday, July 13th, 2021

Supplementary Materials Supplemental Materials supp_26_4_622__index. play a key role in cell interactions with extracellular matrix (Gieger 0.001 compared with WT, c 0.001 compared SGI-110 (Guadecitabine) with DKO. (C) Expression of arrestins in DKO and WT cells was detected by Western blot. Purified bovine arrestin-2 and arrestin-3 (0.2 ng/lane) were run for comparison. (D, E) DKO cells were retrovirally infected with Ha-tagged arrestin-2 (Arr2), arrestin-2-7 (Arr27), arrestin-3 (Arr3), arrestin-3-7 (Arr37), or GFP as a control (DKO and WT). Cells were plated on FN and PDL. Arrestin-expressing cells were stained for actin and HA (E), and control cells were stained for actin and GFP (D). Level bar, 10 m. (F) Western blots showing the expression of HA-arrestins and GFP. GAPDH is used as a loading control. (G) Cell size was measured on FN and analyzed as explained for B. # 0.001 DKO from all other conditions, * 0.001, ** 0.01, * 0.05 to WT. Data are from 37C82 cells/condition from three or four experiments. (H) Cell size was measured on PDL from 29C54 cells in three experiments and analyzed as in B. # 0.001 for DKO from all other conditions, * 0.001 from WT. To confirm that the absence of arrestin-2/3 is responsible for the morphological phenotype of DKO cells, we tested whether retroviral expression of arrestin-2 or arrestin-3 rescues them. To ensure that infection did not impact cell morphology, we used cells infected with green fluorescent protein (GFP) as controls (Physique 1D). Cells plated on FN or PDL were SGI-110 (Guadecitabine) stained for hemagglutinin (HA)-tagged arrestins and actin filaments (Physique 1E). The expression of either of the nonvisual arrestins (Physique 1F) reduces DKO cell size nearly back to WT on SGI-110 (Guadecitabine) FN and PDL. Cells expressing arrestin-3 are closer to WT, whereas the rescue by arrestin-2 is usually partial (Physique 1, G and H). Thus each nonvisual arrestin significantly affects cell distributing. Single- Rabbit polyclonal to annexinA5 arrestin-2 or -3Cknockout cells do not reach the size of SGI-110 (Guadecitabine) DKO MEFs and behave like WT MEFs on PDL, further supporting this notion (Physique 1, ACC). The best-characterized function of arrestins is usually their high-affinity binding to active phosphorylated GPCRs (Gurevich and Gurevich, 2006b ). To test whether arrestin interactions with GPCRs play a role in cell distributing, we used receptor bindingCdeficient arrestin mutants with a 7-residue deletion in the interdomain hinge (7; Hanson 0.001, ** 0.01. Means SD from three experiments. (C) Adhesion of DKO cells expressing arrestin-2 + GFP, arrestin-3 SGI-110 (Guadecitabine) + GFP, or GFP alone (controls). Cells were plated on 0.32 g/ml FN. Means SD from 24 data points in three experiments. *** 0.001 compared with DKO. (D) Cells were plated in Transwell chambers coated with 0.32 g/ml FN and allowed to migrate for 4 h. Cells were counted in six fields/chamber in each of four impartial experiments. The data were analyzed by one-way ANOVA with cell type as the main factor, *** 0.001. Insets, representative membranes postmigration. (E) Migration of DKO cells expressing arrestin-2 and GFP or arrestin-3 and GFP, or cells expressing GFP only (DKO and WT). Means SD from 5 fields/chamber from three impartial experiments performed in duplicate analyzed by one-way ANOVA with cell type as the main factor. *** 0.001 compared with WT. DKO-Arr2, ## 0.01, and DKO-Arr3, # 0.05, compared with DKO. (F) Arrestin expression in DKO cells was decided using arrestin-2C or arrestin-3Cspecific antibodies, with corresponding purified bovine arrestins (0.1 ng/lane) run as standards. To determine whether the reversal of DKO morphology by arrestin-2 or -3 rescues enhanced adhesion and motility deficit, were infected DKO cells with arrestin-2 or -3 in constructs that drive GFP coexpression, with controls expressing only GFP. Cells were sorted for GFP expression (Physique 2F) and used in adhesion and Transwell migration assays. Of interest, arrestin-3 but not arrestin-2 reduces the adhesion of DKO cells, although not to WT level (Physique 2C). Similarly,.