Tatjana Kehler

Analysis of granulysin-mediated cytotoxicity in peripheral blood of patients with psoriatic arthritis.

The objective of the present study was to investigate possible changes in granulysin (GNLY)-mediated cytotoxicity of peripheral blood lymphocytes in psoriatic arthritis (PsA) patients with respect to different phases of the disease. We prospectively enrolled 25 PsA patients in the active phase, 26 PsA patients in remission and 24 healthy controls. The simultaneous detection of intracellular GNLY and cell surface antigens (CD3 and CD56) was performed with flow cytometry. GNLY apoptotic protein was visualised by immunocytochemistry. Natural killer (NK) cell cytotoxicity was analysed with a cytotoxicity assay against human erythroleukaemia K-562 cells. The percentage of GNLY+ cells did not differ significantly between PsA patients in the acute phase and those in remission; however, it was always higher than in healthy examinees due to the increased percentage of GNLY+ cells within T cells, NKT cells, and both, and in the CD56+dim and CD56+bright NK subsets. The mean fluorescence intensity for GNLY was higher in all lymphocyte subpopulations in the acute phase than in remission and in healthy controls. Accordingly, GNLY-mediated NK cell cytotoxicity against K-562 cells of active phase PsA patients was significantly higher than that in patients in remission or in healthy controls. These findings demonstrated the involvement of GNLY in the worsening of PsA and suggested that GNLY mediated the development of joint lesions.

Can pain intensity in osteoarthritis joint be indicator of the impairment of endothelial function

We propose that pathological remodeling in joint tissues of osteoarthritis (OA) patients persistently stimulates local secretion of pro-inflammatory mediators, which overflow into the blood, activating leukocytes that impair endothelial function and accelerate the atherosclerotic process. During periods of pain, endothelial dysfunction progresses more aggressively due to elevated secretion of these pro-inflammatory mediators, which are involved in both atherosclerosis and the sensation of pain. Concentrations of pro-inflammatory cytokines and their antagonists, activating and decoy receptors of the broad interleukin (IL)-1 and IL-17 families, IL-15, and monocyte chemotactic protein-1 should be measured in peripheral blood samples of OA patients and compared with (I) OA clinical severity; (II) subclinical parameters of atherosclerosis; (III) ischemic heart disease risk factors; (IV) soluble factors indicating endothelial dysfunction; (V) degree of bone destruction; and (VI) results of a six-minute walk test. Arthroscopy and joint replacement surgery provide an opportunity to estimate mRNA and protein expression of inflammatory mediators in specimens of synovial fluid, synovial membrane, cartilage, and/or subarticular bone. A range of methods, including questionnaires, X-ray, computed tomography, ultrasound, enzyme-linked immunosorbent assay, immunohistology, immunofluorescence, and reverse transcription and in situ polymerase chain reaction are available. Understanding the inflammatory and immune mechanisms underlying OA may allow the early identification of patients at high risk of cardiovascular disease, independently of classical coronary risk factors. Pain may constitute an extrinsic indicator of currently worsening endothelial function.

Possible role of granulysin in pathogenesis of osteoarthritis

Increased presence of immune mediator and cytotoxic/apoptotic molecule granulysin was noticed in different tissues during pathological processes with the domination of Th1 over Th2 mediated immunity. Beside granulysin expression in T and NKT cells, activated NK cells are thought to be the major source of chemotactic 15 kDa and cytotoxic 9 kDa granulysin in vivo. As NK cells are the principal joints tissue-infiltrating lymphocyte subset, we hypothesized that granulysin mediated human cell death (apoptosis) could be responsible for the relatively silent damage of the joints tissue without clinically notable signs of systemic inflammation in the patients with osteoarthritis (OA). The analyzes of the presence and frequency of granulysin expressing lymphocytes at protein and gene levels in peripheral blood and synovial samples and/or the samples of joints tissue after the joint replacement therapy in patients with OA could give the initial insight to evaluate our hypothesis. It would be of the particular interest to differentiate the expression of 9 kDa and 15 kDa granulysin forms in the effector cells, since only the shorter form exhibits cytotoxic properties. The measurement of granulysin mediated early apoptosis in human NK sensitive K562 cells could be suitable in vitro model for evaluating granulysin activity. Furthermore, disturbed balance of pro-inflammatory and anti-inflammatory cytokines in OA patients, could influence the level of the granulysin expression. Having in mind that the granulysin and its regulation is still unknown in the pathogenesis of OA, it could be worth to explore this important pro-inflammatory, cytotoxic/apoptotic mediator.

SAT0009 Distribution and intracellular settitng of granulysin in women with knee osteoarthritis

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. References Yamada H, Nakashima Y, Okazaki K, et al. Preferential accumulation of activated Th1 cells not only in rheumatoid arthritis but also in osteoarthritis joints. J Rheumatol 2011;38:1569–75. Clayberger C, Finn MW, Wang T, et al. 15 kDa granulysin causes differentiation of monocytes to dendritic cells but lacks cytotoxic activity. J Immunol 2012;188:6119–26. Pardo J, Pérez-Galán P, Gamen S, et al. A role of the mitochondrial apoptosis-inducing factor in granulysin-induced apoptosis. J Immunol 2004;167:1222–9. Kehler T, Laskarin G, Massari D, et al. Possible role of granulysin in pathogenesis of osteoarthritis. Med Hypotheses 2015;85:850–3. 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