Background The role of the cell-mediated immune response is recently recognized in osteoarthritis (OA) (1). Granulysin (GNLY) is mediator of cellular immunity and cytotoxic molecule expressed in T and NK cells in regulatory (15 kDa) and cytotoxic (9 kDa) forms (2). Cytotoxic 9 kDa form of GNLY mediates apoptosis of eukaryotic cells (3) and might be responsible for silent unscheduled apoptosis of joint tissue cells in patients with OA without clinically recognized systematic immune reaction, as measured by erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) (4). We investigate GNLY distribution and intracellular setting in peripheral blood lymphocytes of patients with OA.
Objectives Women with knee OA (17), and age and sex appropriated control (17) were tested. All of them signed informed consent before sampling of peripheral blood (PB).
Methods Medical history, clinical examination, X-ray and routine laboratory testing (ESR, CRP) were used for diagnosing OA. Peripheral blood mononuclear cells (PBMC) were isolated by gradient density centrifugation and used for multiple, simultaneous intracellular [total GNLY, 9 kDa GNLY, 15 kDa GNLY and Lysosomal-associated membrane protein-1 (LAMP-1)] and surface antigens (CD3 and CD56) detection by immunofluorescence. Data were analyzed by flow cytometry or confocal microscopy.
Results In OA and control samples the percentage of total GNLY+ cells and GNLY expressing NK and T cells did not significantly differ and correlate with ESR and CRP. The frequency of GNLY+ cells was always higher in natural killer (NK) then in T cells and 9 kDa GNLY dominated over 15 kDa GNLY. However, 9 kDa GNLY co-localized more with the marker of cell degranulation, LAMP-1 in polarized granules of OA patients when compared to the control or to the 15 kDa GNLY.
Conclusions The increase in the expression of cytotoxic (9 kDa) over regulatory (15 kDa) GNLY form in PBMC and its intracellular setting in polarized exocytic granules suggests the involvement of activated GNLY+ lymphocytes in the immunopathogenesis of knee OA, indispensable of ESR and CRP.
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Acknowledgements The experiments were financed by the University of Rijeka (No.13.06.1.1.06) and “Thalassotherapia- Opatija”, Opatija, Croatia.
Disclosure of Interest None declared