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

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

doi:10.3892/br.2022.1527

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

Authors:
- Vedrana Drvar
- Božena Ćurko‑Cofek
- Ljerka Karleuša
- Merica Aralica
- Marija Rogoznica
- Tatjana Kehler
- Dalen Legović
- Daniel Rukavina
- Gordana Laskarin
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Affiliations: Clinical Department of Laboratory Diagnostics, University Hospital Centre Rijeka, 51000 Rijeka, Croatia, Department of Physiology and Immunology, Faculty of Medicine, University of Rijeka, 51000 Rijeka, Croatia, Hospital for Medical Rehabilitation of Health and Lung Diseases and Rheumatism ‘Thalassotherapia‑Opatija’, 51410 Opatija, Croatia, Clinic for Orthopaedic Surgery Lovran, 51415 Lovran, Croatia
Published online on: March 24, 2022 https://doi.org/10.3892/br.2022.1527
Article Number: 44
Copyright: © Drvar et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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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.

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1	Koh SM, Chan CK, Teo SH, Singh S, Merican A, Ng WM, Abbas A and Kamarul T: Elevated plasma and synovial fluid interleukin-8 and interleukin-18 may be associated with the pathogenesis of knee osteoarthritis. Knee. 27:26–35. 2020.PubMed/NCBI View Article : Google Scholar
2	Woodell-May JE and Sommerfeld SD: Role of inflammation and the immune system in the progression of osteoarthritis. J Orthop Res. 38:253–257. 2020.PubMed/NCBI View Article : Google Scholar
3	Kehler T, Laskarin G, Massari D, Dominovic M, Persic V, Rosovic I, Laginja J and Rukavina D: Possible role of granulysin in pathogenesis of osteoarthritis. Med Hypotheses. 85:850–853. 2015.PubMed/NCBI View Article : Google Scholar
4	Chenoweth MJ, Mian MF, Barra NG, Alain T, Sonenberg N, Bramson J, Lichty BD, Richards CD, Ma A and Ashkar AA: IL-15 can signal via IL-15Rα, JNK, and NF-κB to drive RANTES production by myeloid cells. J Immunol. 188:4149–4157. 2012.PubMed/NCBI View Article : Google Scholar
5	Zeng R, Spolski R, Casas E, Zhu W, Levy DE and Leonard WJ: The molecular basis of IL-21-mediated proliferation. Blood. 109:4135–4142. 2007.PubMed/NCBI View Article : Google Scholar
6	Hu X, Chen Q, Sowrirajan B, Bosche M, Imamichi T and Sherman BT: Genome-wide analyses of MicroRNA profiling in interleukin-27 treated monocyte-derived human dendritic cells using deep sequencing: A pilot study. Int J Mol Sci. 18(925)2017.PubMed/NCBI View Article : Google Scholar
7	Gotthardt D, Trifinopoulos J, Sexl V and Putz EM: JAK/STAT cytokine signaling at the crossroad of NK cell development and maturation. Front Immunol. 10(2590)2019.PubMed/NCBI View Article : Google Scholar
8	Wu Y, Lin Z, Yan Z, Wang Z, Fu X and Yu K: Sinomenine contributes to the inhibition of the inflammatory response and the improvement of osteoarthritis in mouse-cartilage cells by acting on the Nrf2/HO-1 and NF-κB signaling pathways. Int Immunopharmacol. 75(105715)2019.PubMed/NCBI View Article : Google Scholar
9	Lettau M, Dietz M, Dohmen K, Leippe M, Kabelitz D and Janssen O: Granulysin species segregate to different lysosome-related effector vesicles (LREV) and get mobilized by either classical or non-classical degranulation. Mol Immunol. 107:44–53. 2019.PubMed/NCBI View Article : Google Scholar
10	Liu X and Lieberman J: Knocking ‘em Dead: Pore-forming proteins in immune defense. Annu Rev Immunol. 38:455–485. 2020.PubMed/NCBI View Article : Google Scholar
11	Krawczyk PA, Laub M and Kozik P: To kill but not be killed: Controlling the activity of mammalian pore-forming proteins. Front Immunol. 11(601405)2020.PubMed/NCBI View Article : Google Scholar
12	Tewary P, Yang D, de la Rosa G, Li Y, Finn MW, Krensky AM, Clayberger C and Oppenheim JJ: Granulysin activates antigen-presenting cells through TLR4 and acts as an immune alarmin. Blood. 116:3465–3474. 2010.PubMed/NCBI View Article : Google Scholar
13	Clayberger C, Finn MW, Wang T, Saini R, Wilson C, Barr VA, Sabatino M, Castiello L, Stroncek D and Krensky AM: 15 kDa granulysin causes differentiation of monocytes to dendritic cells but lacks cytotoxic activity. J Immunol. 188:6119–6126. 2012.PubMed/NCBI View Article : Google Scholar
14	Robinson WH, Lepus CM, Wang Q, Raghu H, Mao R, Lindstrom TM and Sokolove J: Low-grade inflammation as a key mediator of the pathogenesis of osteoarthritis. Nat Rev Rheumatol. 12:580–592. 2016.PubMed/NCBI View Article : Google Scholar
15	Griffin TM and Scanzello CR: Innate inflammation and synovial macrophages in osteoarthritis pathophysiology. Clin Exp Rheumatol. 37 (Suppl 120):S57–S63. 2019.PubMed/NCBI
16	Berenbaum F and Walker C: Osteoarthritis and inflammation: A serious disease with overlapping phenotypic patterns. Postgrad Med. 132:377–384. 2020.PubMed/NCBI View Article : Google Scholar
17	World Medical Association. World medical association declaration of Helsinki: Ethical principles for medical research involving human subjects. JAMA. 310:2191–2194. 2013.PubMed/NCBI View Article : Google Scholar
18	Altman R, Asch E, Bloch D, Bole G, Borenstein D, Brandt K, Christy W, Cooke TD, Greenwald R, Hochberg M, et al: Development of criteria for the classification and reporting of osteoarthritis. Classification of osteoarthritis of the knee. Diagnostic and therapeutic criteria committee of the American rheumatism association. Arthritis Rheum. 29:1039–1049. 1986.PubMed/NCBI View Article : Google Scholar
19	American Heart Association: Classes of heart failure. American Heart Association, Dallas, TX, 2017. https://www.heart.org/en/health-topics/heart-failure/what-is-heart-failure/classes-of-heart-failure. Accessed May 31, 2017.
20	Menkes CJ: Radiographic criteria for classification of osteoarthritis. J Rheumatol Suppl. 27:13–15. 1991.PubMed/NCBI
21	Persic V, Ruzic A, Miletic B, Samsa DT, Rakic M, Raljevic D, Pejcinovic VP, Eminovic S, Zaputovic L and Laskarin G: Granulysin expression in lymphocytes that populate the peripheral blood and the myocardium after an acute coronary event. Scand J Immunol. 75:231–242. 2012.PubMed/NCBI View Article : Google Scholar
22	Donaldson JG: Immunofluorescence staining. Curr Protoc Cell Biol. 69:4.3.1–4.3.7. 2015.PubMed/NCBI View Article : Google Scholar
23	Kabakov AE and Gabai VL: Cell death and survival assays. Methods Mol Biol. 1709:107–127. 2018.PubMed/NCBI View Article : Google Scholar
24	Ogawa K, Takamori Y, Suzuki K, Nagasawa M, Takano S, Kasahara Y, Nakamura Y, Kondo S, Sugamura K, Nakamura M and Nagata K: Granulysin in human serum as a marker of cell-mediated immunity. Eur J Immunol. 33:1925–1933. 2003.PubMed/NCBI View Article : Google Scholar
25	Krensky AM and Clayberger C: Biology and clinical relevance of granulysin. Tissue Antigens. 73:193–198. 2009.PubMed/NCBI View Article : Google Scholar
26	Belizário JE, Neyra JM and Setúbal Destro Rodrigues MF: When and how NK cell-induced programmed cell death benefits immunological protection against intracellular pathogen infection. Innate Immun. 24:452–465. 2018.PubMed/NCBI View Article : Google Scholar
27	Saini RV, Wilson C, Finn MW, Wang T, Krensky AM and Clayberger C: Granulysin delivered by cytotoxic cells damages endoplasmic reticulum and activates caspase-7 in targeT cells. J Immunol. 186:3497–3504. 2011.PubMed/NCBI View Article : Google Scholar
28	Biassoni R, Ugolotti E and De Maria A: NK cell receptors and their interactions with MHC. Curr Pharm Des. 15:3301–3310. 2009.PubMed/NCBI View Article : Google Scholar
29	Laskarin G, Persic V, Kukic SR, Massari D, Legovic A, Boban M, Miskulin R, Rogoznica M and Kehler T: Can pain intensity in osteoarthritis joint be indicator of the impairment of endothelial function? Med Hypotheses. 94:15–19. 2016.PubMed/NCBI View Article : Google Scholar
30	Zakaria ZF, Bakar AA, Hasmoni HM, Rani FA and Kadir SA: Health-related quality of life in patients with knee osteoarthritis attending two primary care clinics in Malaysia: A cross-sectional study. Asia Pac Fam Med. 8(10)2009.PubMed/NCBI View Article : Google Scholar
31	Courties A, Sellam J and Berenbaum F: Metabolic syndrome-associated osteoarthritis. Curr Opin Rheumatol. 29:214–222. 2017.PubMed/NCBI View Article : Google Scholar
32	Coskun Benlidayi I: Diet in osteoarthritis. Rheumatol Int. 41:1699–1700. 2021.PubMed/NCBI View Article : Google Scholar
33	Villalvilla A, Larrañaga-Vera A, Lamuedra A, Pérez-Baos S, López-Reyes AG, Herrero-Beaumont G and Largo R: Modulation of the inflammatory process by hypercholesterolemia in osteoarthritis. Front Med (Lausanne). 7(566250)2020.PubMed/NCBI View Article : Google Scholar
34	Deveza LA, Melo L, Yamato TP, Mills K, Ravi V and Hunter DJ: Knee osteoarthritis phenotypes and their relevance for outcomes: A systematic review. Osteoarthritis Cartilage. 25:1926–1941. 2017.PubMed/NCBI View Article : Google Scholar
35	Laitner MH, Erickson LC and Ortman E: Understanding the impact of sex and gender in osteoarthritis: Assessing research gaps and unmet needs. J Womens Health (Larchmt). 30:634–641. 2021.PubMed/NCBI View Article : Google Scholar
36	Hughes SM, Pandey U, Johnston C, Marrazzo J, Hladik F and Micks E: Impact of the menstrual cycle and ethinyl estradiol/etonogestrel contraceptive vaginal ring on granulysin and other mucosal immune mediators. Am J Reprod Immunol. 86(e13412)2021.PubMed/NCBI View Article : Google Scholar
37	Alter G, Malenfant JM and Altfeld M: CD107a as a functional marker for the identification of natural killer cell activity. J Immunol Methods. 294:15–22. 2004.PubMed/NCBI View Article : Google Scholar