Active RA patients
were defined as those presenting DAS 28 scores of above 3.2 and inactive patients were those defined as presenting DAS 28 scores of less BMN 673 research buy than 2.6. Patients were subdivided into three groups according to their treatment: therapy with DMARDs (DMARD, most patients were also in treatment with methotrexate, MTX = 7.5–25 mg/week), anti-TNF-α therapy (AB; 3 mg/kg Infliximab with/without MTX; intravenous infusions every 8 weeks) and a non-treated group, not treated with drugs specific for RA (NT). To be included in the study, patients must have been on treatment regimens for at least 3 months, without co morbidities and without excessive bone destruction. Healthy individuals were used as controls (CON). The ages of individuals ranged between 21–75 years and informed written consent was obtained from all patients and controls. The study was approved by the Ethics Committee of the University of Campinas, Brazil. Neutrophil isolation. Peripheral blood samples from controls and patients were collected in sodium citrate learn more (3.13% w/v). Neutrophils were isolated by centrifuging whole
blood over two layers of Ficoll-Paque of densities of 1.077 and 1.119 g/l . After lysis of contaminating erythrocytes by resuspension of the cell pellet in lysis buffer (155 mm NH4Cl, 10 mm KHCO3, 4 °C, 10 min), cells were washed in phosphate-buffered saline (PBS) before resuspending in RPMI medium for immediate use in assays. Histological and morphological analyses of isolated neutrophil populations indicated them to demonstrate over 95% purity and over 98% viability with no significant differences in morphology. Neutrophil adhesion assays. Neutrophil static adhesion assays were performed as previously described
. Briefly, neutrophils (2 × 106 cells/ml in RPMI medium) were seeded onto 96-well plates previously coated with 20 μg/ml FN; cells were allowed to adhere for 30 min at 37 °C, 5% CO2. Following incubation, PAK5 non-adhered cells were discarded and wells washed thrice with PBS. RPMI (50 μl) was added to each well and varying concentrations of the original cell suspension were added to empty wells to form a standard curve. Percentage cell adhesion was calculated by measuring the myeloperoxidase (MPO) content  of each well and comparing with the standard curve. For IL-8 stimulation, cells were co-incubated with IL-8 (500 ng/ml) during the assay. In vitro neutrophil chemotaxis. Cell migration assays were performed using a 96-well chemotaxis chamber (Chemo Tx; Neuro Probe, Gaithersburg, MD, USA). Twenty-five microlitres of cell suspension (4 × 106 cells/ml in RPMI) were added to the upper compartment of the chamber and separated from the lower chamber, which contained 29 μl of RPMI (unstimulated) or IL-8 (100 ng/ml), by a polycarbonate filter (5-μm pore). Chambers were incubated (37 °C, 5% CO2) for 120 min.