The discriminative capacity of soluble Toll-like receptor (sTLR)2 and sTLR4 in inflammatory diseases

ten Oever, Jaap; Kox, Matthijs; van de Veerdonk, Frank L; Mothapo, Khutso M; Slavcovici, Adriana; Jansen, Tim L; Tweehuysen, Lieke; Giamarellos-Bourboulis, Evangelos J; Schneeberger, Peter M; Wever, Peter C; Stoffels, Monique; Simon, Anna; van der Meer, Jos WM; Johnson, Melissa D; Kullberg, Bart-Jan; Pickkers, Peter; Pachot, Alexandre; Joosten, Leo AB; Netea, Mihai G

In order to determine expression of TLR2 and TLR4, blood was collected in EDTA-containing vacutainers. The following directly conjugated mouse anti-human antibodies were used: TLR2: CD282 PE (mouse IgG2a, TLR 2.1 clone, eBioscience, San Diego, CA), TLR4: CD284 PE-Cy7 (mouse IgG2a, HTA125 clone eBioscience, San Diego, CA), and CD14 ECD (mouse IgG2a, RMO52 clone Immunotech, Beckman Coulter, Marseille, France). Isotype and fluorochrome matched controls from Beckman Coulter were used. Cell buffer solution was used containing 0.5% Bovine Serum Albumin in Phosphate Buffered Saline and 0.1% sodium azide. Rabbit serum (Invitrogen, Carlsbad, CA) for blocking was diluted to 20% with cell buffer solution. Red blood cell lysis was performed using 0.075 M ammonium chloride (NH4Cl, pH7.4), freshly prepared. 1 ml of blood was mixed with 20 ml of NH4Cl lysing solution and was left at room temperature for 10 minutes. After centrifuging for 5 minutes at 500 g the supernatant was discarded. The cell pellet was resuspended in 50 ml of PBS and centrifuged again. After this washing step the cell pellet was resuspended in 0.5 ml cell buffer solution. 0.1 ml of this cell suspension was mixed with 0.1 ml 20% rabbit serum and left at room temperature for 10 min. Subsequently, cells were incubated with the appropriate antibody concentration mixture for 15 min in the dark at room temperature. After washing, samples were resuspended in 0.5 ml cell buffer solution and analyzed on a Beckman Coulter FC500 flow cytometer (Beckman Coulter, Miami, FL). Monocytes were gated in a Side Scatter vs. CD14 plot. Fluorochrome matched isotype controls, non-stained samples, as well as cells incubated with only a secondary antibody, were used to set the photo multiplier detectors. The TLR2 and TLR4 expression was analyzed within CD14 + monocytes.