Journal: Respiratory Research

Article Title: Oxidative stress augments toll-like receptor 8 mediated neutrophilic responses in healthy subjects

doi: 10.1186/1465-9921-10-50

Figure Lengend Snippet: Detection of toll-like receptor (TLR) 8 in human polymorphonuclear cells (PMNs), and the effects of TLR 7/8 ligand R848 on interleukin(IL)-8 release . (A) TLR8 in PMN was detected by immunocytochemistry. Left panel indicates isotype control. Right panel shows TLR8 immunoreactivity in PMN. (Original magnification: × 400, Scale bars = 10 μm). (B) TLR8 expression was analyzed by flow-cytometry. PMNs were stained by anti-human TLR8 (solid lines) or the isotype control (gray histograms) in the permeabilized (left panel) and unpermeabilized condition (right panel). Left panel indicates both intercellular and cell surface expression of TLR8. Right panel shows cell surface expression alone. (C-F) Effect of R848 on the release of IL-8, and effect of bafilomycin or dexamethasone on the R848-induced IL-8 release from PMN. (C) PMNs were treated with 10 μM R848. The media were harvested at various time points and assayed for IL-8 by ELISA. (D) PMNs were treated for 24 hrs with R837, a ligand of TLR7, or various concentrations of R848, a ligand of TLR 7/8. Media were assayed for IL-8 by ELISA. (E, F) PMNs were treated with 10 μM R848 or vehicle in the presence of various concentrations of bafilomycin, an inhibitor of endosomal acidification (E), or dexamethasone (F). Media were assayed for IL-8 by ELISA. All values are mean values ± SEM of three to four separate experiments. *p < 0.05, **p < 0.01, compared with the values of control; +p < 0.05, ++p < 0.01, compared with the values of the vehicle-pretreated and 10 μM R848-treated group.

Article Snippet: Commercially available reagents were obtained as follows: Mono-Poly Resolving Medium was from Dainippon Pharmaceutical Co. Ltd. (Osaka, Japan); fetal calf serum (FCS) and RPMI medium 1640 (RPMI 1640) were from Invitrogen (Carlsbad, California, USA); R848 (resiquimod: 4-amino-2-etoxymethyl-α,α-dimethyl-1 H -imidazo [4,5- c ]quinolin-1-ethanol), bafilomycin and 12-o-tetradecanoylphorbol 13-acetate were from Alexis Biochemicals (San Diego, California, USA); R837 (Imiquimod: 1-isobutyl-1 H -imidazo [4,5- c ]quinolin-4-amine) was from Biomol (Plymouth Meeting, Pennsylvania, USA); N-acethyl- L -cysteine, MG-132, dexamethasone and anti-β-actin antibody were from Sigma (St. Louis, Missouri, USA); anti-TLR8 rabbit polyclonal antibody was from Abgent (San Diego, California, USA); Cellfix solution was from Becton Dickinson (San Jose, California, USA); phycoerythrin (PE)- conjugated anti-TLR8 antibody solution was from Imgenex (San Diego, California, USA); dihydro-rhodamine-123 (DHR-123) was from Cayman Chemical (Ann Arbor, Michigan, USA); human recombinant IL-8 was from Acris antibodies (Hiddenhausen, Germany); anti-human MyD88 antibody, anti-human TRAF6, and anti-human IkBα were from Santa Cruz (San Diego, California, USA); peroxidase-conjugated secondary antibodies were from Rockland Immunochemicals (Gilbertsville, Pennsylvania, USA)

Techniques: Immunocytochemistry, Expressing, Flow Cytometry, Staining, Enzyme-linked Immunosorbent Assay