Lipopolysaccharide lps from pseudomonas aeruginosa

Lipopolysaccharide (LPS; from Pseudomonas aeruginosa), Lipoteichoic acid (LTA; from Staphylococcus aureus), 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), propidium iodide (PI), n-phenyl-1-naphthylamine (NPN), HEPES, and 3,3′-dipropylthiadicarbocyanine iodide (DisC₃-5) were obtained from Sigma-Aldrich (St Louis, MO, USA).
Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 25923 were obtained from the ATCC (American Type Culture Collection, Manassas, VA, USA), and Acinetobacter baumannii KCTC 2508 and Bacillus subtilis KCTC 2217 were obtained from the KCTC (Korean Collection for Type Cultures, Jeongeup-si, Jeollabuk-do, Korea). Human skin epithelial cells (HaCaT cells) were obtained from ATCC.

Monocytes were isolated from buffy coats of healthy blood donors (Sanquin Blood Bank, Leiden, The Netherlands) using anti-CD14 microbeads (Miltenyi Biotec, Auburn, CA, USA) according to the manufacturer’s protocol. MΦ1 and MΦ2 were derived as described previously [23 (link), 24 (link)]. Briefly, cells were cultured for six days in RPMI 1640 medium in 48 well plates (Invitrogen, Life Technologies, Bleiswijk, The Netherlands) containing 10 % fetal calf serum (FCS, Invitrogen), 2 mM L-glutamine, 100U/ml penicillin and 100 μg/ml streptomycin (all Bio Whittaker, Walkersville, MD, USA) at 37 °C in 5 % CO₂ atmosphere with either GM-CSF (5 ng/ml, Invitrogen) or M-CSF (50 ng/ml, R&D Systems, Minneapolis, MN, USA) to obtain MΦ1 and MΦ2, respectively [13 (link), 14 (link)]. Differentiated macrophages were stimulated with the pro-inflammatory stimuli lipopolysaccharide (LPS, from Pseudomonas aeruginosa, 100 ng/ml, Sigma-Aldrich, St. Louis, MO, USA), TNF-α (10 ng/ml, Peprotech, Rocky Hill, NJ, USA) or oncostatin M (OSM, 100 ng/ml, R&D Systems) for 24 h. Dexamethasone (0.1, 0.3 and 1nM, Sigma) was added during differentiation at day 0, 3 and/or day 7. The demethylating agent 5-AZA-2’-deoxycytidine (5-AZA, 0.1, 1 and 10 μM, Sigma) was added during differentiation. Every day 100 μl per well was removed and replaced by fresh medium containing growth factors and 5-AZA.

Purified blood neutrophils or sputum cells were resuspended in RPMI medium without phenol red (Gibco, Waltham, MA, USA) at a concentration of 1.5 × 10⁶ cells/ml or 3 × 10⁶ cells/ml, respectively. Prior to stimulation, the cells were incubated for 10 min at 37 °C in the presence of 50 ng/ml tumor necrosis factor-α (TNF-α; Peprotech, Rocky Hill, NJ, USA). Subsequently, the cells were added to a white clear-bottom 96-well plate (Perkin-Elmer, Waltham, MA, USA) and supplemented with luminol (5 mM; Sigma-Aldrich) and one of the following compounds: lipopolysaccharide (LPS) from Pseudomonas aeruginosa (10 µg/ml; Sigma-Aldrich); peptidoglycan (PGN) from Staphylococcus aureus (10 µg/ml; Sigma-Aldrich); N-formyl-methionyl-leucyl-phenylalanine (fMLF) (10^–8 M; Sigma-Aldrich). Phorbol 12-myristate 13-acetate (PMA; 150 ng/ml; Sigma-Aldrich) was used as a positive control. An additional condition without luminol was included to determine the background luminescence. The light signal was measured every minute for a period of 3 h by a Clariostar Monochromator microplate reader (BMG Labtech, Orthenberg, Germany). Analysis was performed by subtracting the background luminescence and determining the maximal luminescence as a measure for maximal ROS production. The maximal ROS production of cells stimulated by one of the compounds was normalized to the ROS production of unstimulated cells.