Granulysin expression and granulysin-mediated apoptosis in the peripheral blood of osteoarthritis patients

Vedrana Drvar; Božena Ćurko-Cofek; Ljerka Karleuša; Merica Aralica; Marija Rogoznica; Tatjana Kehler; Dalen Legović; Daniel Rukavina; Gordana Laskarin

doi:10.3892/br.2022.1527

Granulysin expression and granulysin-mediated apoptosis in the peripheral blood of osteoarthritis patients

Biomed Rep. 2022 May;16(5):44. doi: 10.3892/br.2022.1527. Epub 2022 Mar 24.

Authors

Vedrana Drvar¹, Božena Ćurko-Cofek², Ljerka Karleuša², Merica Aralica¹, Marija Rogoznica³, Tatjana Kehler^{3

4}, Dalen Legović⁵, Daniel Rukavina^{2

6}, Gordana Laskarin^{2

3}

Affiliations

¹ Clinical Department of Laboratory Diagnostics, University Hospital Centre Rijeka, 51000 Rijeka, Croatia.
² Department of Physiology and Immunology, Faculty of Medicine, University of Rijeka, 51000 Rijeka, Croatia.
³ Hospital for Medical Rehabilitation of Health and Lung Diseases and Rheumatism 'Thalassotherapia-Opatija', 51410 Opatija, Croatia.
⁴ Department of Medical Rehabilitation, Faculty of Medicine, University of Rijeka, 51000 Rijeka, Croatia.
⁵ Clinic for Orthopaedic Surgery Lovran, 51415 Lovran, Croatia.
⁶ Department of Biomedical Sciences in Rijeka, Croatian Academy of Sciences and Arts, 51000 Rijeka, Croatia.

Abstract

Osteoarthritis (OA) is a chronic joint disease caused by mechanical damage and metabolic factors that support the development of low-grade inflammation. Increased levels of T helper 1 pro-inflammatory cytokines in the serum of OA patients may support granulysin (GNLY) mediated cytotoxicity, which in-turn may contribute to the pathogenesis of OA. In the present study, GNLY expression and cytotoxic/apoptotic mechanisms mediated by GNLY in the peripheral blood of OA patients were assessed. A total of 40 non-obese women (median age of 64 years old) with knee OA, and 40 controls (median age 62 years old) were enrolled in the study. GNLY, IFN-γ and IL-4 expression levels were investigated in peripheral blood lymphocytes (PBLs) using flow cytometry, immunocytochemistry and/or confocal microscopy. Natural killer (NK) GNLY-mediated apoptosis through NK effectors against K-562 targets was analyzed using the PKH-26 18-h cytotoxicity assay. Serum GNLY levels were assessed using ELISA. The percentage of GNLY⁺PBLs was higher in the OA patients than that in the controls due to the increase in the proportions of GNLY⁺ cells in the natural killer (NK), T and natural killer T (NKT) subsets. GNLY localization inside exocytotic lysosomal-associated membrane protein-1⁺ granules was ~40% in both groups. However, the intensity of GNLY labeling in PBLs was higher in OA patients than in the controls, and it was supported by the increased expression of IFN-γ relative to IL-4 in NK and T cells from OA patients. The serum GNLY concentration was <0.3 ng/ml in both groups. RC8 anti-GNLY mAb by itself was unable to significantly alter early apoptosis, whereas RC8 anti-GNLY mAb combined with anti-perforin mAb significantly reduced NK-mediated early apoptosis of K-562 targets in the OA patients, whilst not exerting a notable effect in the controls. Anti-perforin mAb by itself did not affect apoptosis significantly. These results suggest that in women with knee OA, GNLY expression in the PBL subsets and GNLY-mediated early apoptosis of K-562 targets are increased compared with the controls and accompanied by intracellular dominance of IFN-γ over IL-4 in NK cells.

Keywords: apoptosis; cytotoxicity; granulysin; lymphocytes; osteoarthritis.

Grants and funding

Funding: This work was supported by funding from the University of Rijeka, Croatia (grant nos. 13.06.1.1.06, Uni-ri-biomed-18-110, and Uni-ri-biomed-18-160).