engleski

Inflammatory characteristics of peripheral blood lymphocytes from patients with osteoarthritis

Osteoarthritis (OA) is a chronic joint disease caused by mechanical damage and metabolic factors that support the development of low grade inflammation. Biomechanical forces constantly damage chondrocytes and change their phenotype with intensive production of pro-inflammatory cytokines which may support granulysin (GNLY) mediated cytotoxicity. GNLY is a constitutive cytotoxic mediator in natural killer (NK) cells. After direct stimulation of effector NK cells in close contact with targets, the cytotoxic GNLY form is released into the immunological synapse, enters the target cells through the perforin pores, and kills human cells by DNA fragmentation. According to this, we investigated the inflammatory status of peripheral blood lymphocytes (PBL) in patients with OA and compared it with controls. 40 non-obese women aged 64 (56 ; 67) years, median (25th, 75th percentiles) with knee OA, and 40 controls were recruited in the investigation. Interferon-γ (IFN-γ) and interleukin-4 (IL-4) were labelled intracellularly and acquired by flow cytometry. GNLY and lysosomal-associated membrane protein-1 (LAMP-1), a marker of lysosome exocytosis were co-labelled in PBL and analysed using confocal microscopy. Serum GNLY concentration were measured using ELISA. GNLY mediated apoptotic activity of peripheral NK cells were measured against K-562 targets in 18 hour- assay using PKH26 cytotoxicity assay. Different ratios of NK effectors and K-562 targets were labelled with FITC-conjugated annexin V (5 μg/105 cells). Propidium iodide (PI), at a final concentration of 5 μg/ml, was added to the samples 15-20 minutes before analyses using FACS Calibur. The subset of PKH26 labelled K-562 cells, detected as PI-negative and FITC-annexin V-positive, were considered for the examination of early apoptosis. The serum GNLY concentration was below 0.3 ng/ml and GNLY localization inside LAMP-1+ granules was approximately 40% in both groups. Early apoptosis did not differ significantly between the OA patients and controls cultured in medium only. However, the analysis revealed GNLY-mediated early apoptosis only in the OA patients. RC8 anti-GNLY mAb in the combination with δG9 anti-perforin mAb significantly reduced NK-mediated early apoptosis of K-562 targets in the OA patients in effector target ratios 50:1 (P=0.01), 25:1 (P=0.006), 12.5:1 (P=0.01), whereas they were inefficient in the controls. IFN-γ dominated over IL-4 in NK and T cells of OA patients (P=0.03 and P=0.01, respectively), while they were codominant in NKT cells. In the controls, IL-4 levels were higher than IFN-γ levels in NK and NKT cells (P=0.004), and it was only marginally increased in T cells (P=0.07). Based on the results, we can conclude that PBL pro-inflammatory polarization in OA patients supports GNLY-mediated apoptosis through NK effectors against K-562 targets in 18 hour- cytotoxicity assay in vitro.

apoptosis ; granulysin ; interferon gamma ; interleukin-4 ; NK cells ; osteoarthritis

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