Medical Hypotheses 85 (2015) 850–853
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Medical Hypotheses journal homepage: www.elsevier.com/locate/mehy
Possible role of granulysin in pathogenesis of osteoarthritis Tatjana Kehler a,b, Gordana Laskarin a,d,⇑, Drazen Massari a,b, Marin Dominovic d, Viktor Persic b,c, Ivan Rosovic c, Josip Laginja f, Daniel Rukavina d,e a Department of Rheumatology, Rehabilitation and Physical Medicine, Hospital for Medical Rehabilitation of Heart and Lung Diseases and Rheumatism ‘‘Thalassotherapia-Opatija”, M. Tita 188, 51410 Opatija, Croatia b Department of Medical Rehabilitation, Medical Faculty, University of Rijeka, B. Branchetta 20, 51000 Rijeka, Croatia c Division of Cardiology, Hospital for Medical Rehabilitation of the Heart and Lung Diseases and Rheumatism ‘‘Thalassotherapia-Opatija”, M. Tita 188, 51410 Opatija, Croatia d Department of Physiology and Immunology, Medical Faculty, University of Rijeka, B. Branchetta 20, 51000 Rijeka, Croatia e Department of Clinical and Transplantation Immunology and Molecular Medicine in Rijeka, Croatian Academy of Sciences and Arts, Radmile Matejcic 2, Rijeka, Croatia f Hospital for Medical Rehabilitation of Heart and Lung Diseases and Rheumatism ‘‘Thalassotherapia-Opatija”, M. Tita 188, 51410 Opatija, Croatia
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Article history: Received 20 April 2015 Accepted 22 September 2015
a b s t r a c t 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 joint’s tissue-infiltrating lymphocyte subset, we hypothesized that granulysin mediated human cell death (apoptosis) could be responsible for the relatively silent damage of the joint’s 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 joint’s 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 proinflammatory, cytotoxic/apoptotic mediator. Ó 2015 Elsevier Ltd. All rights reserved.
Introduction It has been considered that OA is induced by a mechanical stress, which causes cartilage destruction without significant participation of the immune response, particularly when compared with rheumatoid arthritis (RA) [1,2]. However, there were no statistical differences between the OA and RA patients regarding the percentages of T cells (T helper and T cytotoxic), B cells and natural
⇑ Corresponding author at: Department of Physiology and Immunology, Medical Faculty, University of Rijeka, B. Branchetta 20, 51000 Rijeka, Croatia. Tel.: +385 51 65 11 85; fax: +385 51 67 56 99. E-mail addresses: [email protected] (T. Kehler), gordana.laskarin@ medri.uniri.hr (G. Laskarin), [email protected] (D. Massari), marin.dominovic@ gmail.com (M. Dominovic), [email protected] (V. Persic), ivan_rosovic@ yahoo.com (I. Rosovic), [email protected] (J. Laginja), [email protected] (D. Rukavina). http://dx.doi.org/10.1016/j.mehy.2015.09.025 0306-9877/Ó 2015 Elsevier Ltd. All rights reserved.
killer (NK) cells in the peripheral blood, and the peripheral CD4/ CD8 ratio, reflecting the similarities in the immune cell profiles of RA and OA patients [2,3]. Immune cells were also found in the synovial fluids and synovial membrane. T cells and their Th1oriented CD4+ subset were much less numerous in the joints of patients with OA than in the RA joints, but they showed a similar expression of activation HLA-DR and CD69 markers and profile of IFN-gamma cytokine secretion [3]. The predominance of Th1 cells were confirmed in both OA and RA joints [4]. Only the frequency of Th17 cells tended to be lower in the joint of patients with OA, than with RA [4]. Locally present CD4+ T lymphocytes increased concentration of macrophage inflammatory protein-1 gamma in homogenized synovial tissue in an anterior cruciate ligament-transection model of OA in wild-type B6 mice [5]. The macrophage inflammatory protein-1 gamma, subsequently supported osteoclast formation, enhanced macrophage infiltration and matrix metalloproteinase-9 expression, which are all responsible for tis-
T. Kehler et al. / Medical Hypotheses 85 (2015) 850–853
sue destruction in mice model of OA [5]. Similarly, CD8+ cells in the synovial membrane in OA patients correlated positively with increased matrix metalloproteinase-1 [6]. Several more studies in animal models [7] and human tissue [8] illustrate the potential importance of cytokine balance in local tissue changes and the development of OA. Thus the counteraction of the main proinflammatory interleukin (IL)-1 cytokine can prevent the progression of joint’s structural changes in mice with OA [7]. The concentration of interferon-c inducible protein-10 is low in the plasma and synovial fluid of OA patients and it inversely correlates with radiographic severity of the disease [8]. Conversely, IL-15 is elevated in the synovial fluid and the synovial membranes in early knee OA and positively correlated with IL-6 concentration [6], suggesting that these cytokines are the local factors responsible for the differentiation and the proliferation of T cells [9] and B cells [10] in situ, and activation of an innate immune response, mediated primarily by NK cells [6]. IL-15 is an effective chemoattractant for resting and activated T and NK cells [11]. It regulates the cytokine production and the cytotoxic potential of NK cells [12], as well as the mRNA expressions of perforin, Fas ligand [13], and granulysin [14]. However, little is known about the role of the cell-mediated immune response and its cytotoxic/apoptotic mechanisms, particularly perforin and granulysin in the pathogenesis of OA at the local and systemic levels. The presence of cytotoxic protein – perforin within the cytoplasm granules of the CD4+, CD8+, CD56+, CD16+, and CD25+ lymphocyte subsets from the synovial fluid and the synovial membrane of OA patients, was identified using flow cytometry [3]. Perforin expression suggests the role of activated cytotoxic immune T and NK cells in the pathogenesis of OA [3], as perforin correlates with cell mediated cytotoxic potential of T cell and NK cells in different tissues [15–17]. In the activated NK effector cells, perforin is stored in dense vesicles together with pro-apoptotic mediators, such as granzymes, Fas ligand, and cytotoxic 9 kDa granulysin form [17,18]. Longer, 15 kDa form of granulysin is stored in distinct granules, situated directly beneath the cell membrane and it can be processed to the shorter, cytotoxic 9 kDa granulysin form [18]. The longer 15 kDa granulysin shows predominantly regulatory properties as alarmin, due to the recruitment of immune cells to the site of inflammation [17,19]. It strongly chemoattracts monocytes, CD4+ and CD8+ memory T cells, NK cells and mature dendritic cells [20] and induce differentiation of monocyte into dendritic cells [18]. Both granulysin containing granules are released from the effector cells on demand, in accordance with cell stimulation [17]. However, 9 kDa form is released after the receptor mediated granule exocytosis pathway, depending of current expression of activation and inhibitory NK cell receptors and their ligands on target cells [18]. Longer 15 kDa form of granulysin is released constantly by IL-15 activated NK cells mostly [18]. Granulysin uses multiple mechanisms to enter the cells and kill targets. As granulysin is kationic molecule of saposin-like family of proteins, it possesses structural features allowing him to associate with lipids [21]. 9 kDa granulysin is endocytosed by the infected eukaryotic cells via lipid rafts [21]. In the cells 9 kDa granulysin kills bacteria efficiently within minutes [20,22], because it forms defects in negatively charged, cholesterol-free membranes of prokaryotic bacterial cells, but leave the eukaryotic cell intact [21]. It is believed that cytotoxic activity of granulysin against eukaryotic cells is due to binding of granulysin for negatively charged mitochondrial membrane [23], after the quick cytoplasmic access by perforin pores [24,25]. Mitochondrial cell damage supports the quick release of apoptosis-inducing factors and mitochondrial cytochrome C, which then induces DNA fragmentation in a caspase-dependent or caspase-independent manner [23,26,27]. Granulysin can also mediate apoptosis by nuclear accumulation [28]. A slower mechanism of granulysin-mediated apoptosis seems to be conducted by
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ceramide generation in the target cell membrane, likely without perforin support [27]. Hypothesis We hypothesized that cytolytic immune mediator granulysin participates in cartilage destruction in patients with OA. In OA patients, granulysin could increase lymphocyte infiltration in synovial tissue and synovial fluid, as well as in the plasma and peripheral blood lymphocyte subpopulations. NK cells might be the main source of granulysin in OA patients, since activated NK cells has been shown as the major source of serum granulysin in vivo [18]. Moreover, NK cells are the principal tissue-infiltrating lymphocyte subset in patients with OA, comprising nearly 30% of the CD45+ mononuclear cell infiltrates in the synovial tissue obtained from patients undergoing total joint replacement surgery [29]. Increased presence of granulysin has been noticed in different tissues during pathological processes with the domination of Th1 over Th2 mediated immunity. For example, the frequency of granulysin expressing NK cells is enhanced in peripheral blood of patients with preeclampsia [30] and psoriatic arthritis [31]. Skeletal muscles and the hearth are shown to be infiltrated with granulysin positive cells during polymyosytis [32] or acute infarction [33], respectively. Granulysin concentration in human serum and broad cytotoxic activity of granulysin against virally infected cells, tumors, and transplanted cells [28] indeed classify this mediator as a human marker of the Th1 immune response [20,26,30,34]. Thus the dynamic changes in the expression of granulysin in patients with OA could be the reflection of disturbed balance of humoral pro-inflammatory and anti-inflammatory factors, at least at the local level. And finally, granulysin mediated cell death supporting apoptosis could be responsible for the relatively silent damage of the joint’s tissue without clinically notable, eclatant systemic inflammatory reaction. Evaluation of the hypothesis To estimate the granulysin-mediated cytotoxic potential of lymphocytes in patients with OA, it is essential to form homogenous group of patients with OA and healthy control. The inclusive criteria could be set up according to the recommendations of the American College of Rheumatology for diagnosis of knee [35], hand [36] or hip [37] OA. The exclusion criteria for the group of patients with OA and control group should also be defined precisely, particularly in terms of other possible systemic immune diseases. The therapy of participants deserves particular attention and should not interfere with the immunological examinations. The estimation of the presence and frequency of granulysin expressing lymphocyte subpopulations in freshly isolated peripheral blood samples of patients with OA at protein and gene levels will give the first approach to evaluate our hypothesis. Our preliminary data show that granulysin positive cells in peripheral blood samples of OA patients counts about 30% and it was approximately twice higher, when compared with healthy control. It was due to higher granulysin expressing in NK, T and NKT leukocyte subsets of OA patients, as measured by flow cytometry. The second approach to test this hypothesis could be the analyzes of granulysin-mediated NK cell cytotoxicity in different time points using major histocompatibility complex (MHC) devoid, NK sensitive K562 cells of human erythroleukaemia cell-line, as targets in classical in vitro model for the assessment of NK cell activity [38]. 51Cr release assay measures necrosis/lysis [39] very precisely [40], but the measurement of NK cell mediated target cell apoptosis could be more suitable for evaluating granulysin activity. Late
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apoptosis evaluated by flow cytometry assay as frequency of propidium iodide positive and annexin V positive target cells cannot be distinguished well from the total necrosis [41]. But the early apoptosis, expressed as the percentage of propidium iodide negative and annexin V positive cells is better indicator for apoptotic activity of cells according to data of Vermes et al. [41]. It would be of the particular interest to differentiate the expression of shorter (9 kDa) and longer (15 kDa) granulysin forms in the effector cell, since their immuno-biological roles have been distinguished. Only the shorter form possesses cytolytic activity [18], whereas longer form attracts, activates and differentiates monocytes into dendritic cells [17], as well as exhibits potent in vivo immunoactivating properties [18,20]. Production of cytokines in lymphocyte subpopulations and cytokine concentration in the plasma by flow cytometry or enzyme linked immunoassay, respectively, could help to evaluate c1ytokine support, at the time of granulysin estimation. The analysis of the synovial tissue and synovial fluid obtained from patients undergoing total joint replacement surgery could provide better insight in locally acting pathophysiological mechanisms during late OA, while early pathophysiological events might be analyzed in synovial fluids obtained by diagnostic puncture of the joint or eventually by arthroscopy. Since the clinical studies may be limited, the experiments in animal models for example with granulysin transgenic mice could be of particular importance, since there has not been homologous molecule to granulysin in the mice [25].
Consequences of the hypothesis and discussion There is increasing body of evidence that cell-mediated immunity supported by pro-inflammatory cytokines and chemokines has a critical role in the pathogenesis of OA [4–7,29]. However, the role of pro-inflammatory, cytotoxic mediator granulysin in the pathogenesis of OA is still unexplored, although it has been shown to reflect the activation status of cell-mediated immunity [42]. Granulysin mediates unscheduled human cell death by apoptosis induction and nuclear accumulation [34]. The apoptosis is the important biological phenomenon, which occurs in the human adult approximately in 50 and 70 billion of cells daily [43]. The quick removal of apoptotic bodies by neighboring phagocytic cells, before the contents of dying cells could spill out onto surrounding cells and cause damage, does not elicit an inflammatory response [43]. This mechanism could be responsible for the silent damage of the joint’s tissue without clinically notable, eclatant systemic inflammatory reaction, in spite of the presence of immune cells and a chronic, progressive immune response, directed toward unknown antigen(s) in the fluid and synovial membrane of osteoarthritic joints [4,6,29]. Granulysin exists in the effector T, NKT and NK cells [44,45] in the longer 15 kDa form, which is produced rapidly and shaded from IL-15 stimulated NK cells, exerting regulatory immune functions [18]. The shorter 9 kDa form with cytolytic properties, appears in the cell after the cleavage of the precursor 15 kDa form [44]. Since functions of granulysin forms have been different, it would be valuable to determine the frequency of cells that express the particular forms. As far as we know there is no clear data showing difference in the frequency of 9 and 15 kDa expressing subsets by flow cytometry, although it was recently attempted by other investigators [18] and by our group [preliminary, unpublish data]. To distinguish forms of granulysin is not very convenient at the moment, although more monoclonal and polyclonal antibodies, which recognize different epitopes, and do not compete with each other, are available. Polyclonal rabbit anti-9 kDa granulysin antibodies, designed by the group of Clayberger et al. [18] seem to rec-
ognize the epitope of 9 kDa granulysin, independently whether it is cleaved or not from the precursor 15 kDa granulysin form. However, only under denaturing conditions in Western blot analysis the distinction of 15 kDa and 9 kDa was possible [18]. Granulysin isoforms share a common core, whose epitope is likely to be recognized in the proteins of both forms by the same antibody [18] depending also of the biological properties of the antibody and the laboratory method implemented. Our group has shown previously the higher co-localisation of granulysin and perforin by immunofluorescence using RC8 anti-granulysin mAb and deltaG9 anti-perforin mAb, respectively, in freshly isolated early human pregnancy decidual NK cells, which were in vivo activated by IL15, when compared with peripheral blood NK cells of the same pregnant women [19]. RC8 mAb recognizes an core epitope contained in both, 9 kDa and 15 kDa forms of granulysin [42], because RC8 anti-granulysin antibody was purified from RC8 hybridoma, which had been established by fusion of mouse myeloid cell Sp2/0-Ag8 with Balb/c mouse splenocyte immunized with fulllength human granulysin expression plasmid [18]. Accordingly, our above mentioned results only suggest, but does not prove the presence of lytic 9 kDa granulysin in activated decidual NK cells, as positivity could appear due to the staining of 15 kDa granulysin form, in spite of the facts that 9 kDa granulysin co-localizes with perforin in the granules of highly stimulated NK cells much more than in cells at lower level of activation [19]. Testing of granulysin induced apoptosis in the presence and absence of blocking anti-granulysin antibody, could be valuable proof for the release of the cytotoxic mature 9 kDa granulysin form from the effector cell, assuming that the activity of probably coexcreted 15 kDa granulysin from does not exhibit cytotoxic properties [18] and therefore cannot be measured by cytotoxicity assay. RC8 mAb might be useful in blocking granulysin mediated cytotoxicity (apoptosis), if it is present in vitro in the medium surrounding effector and target cells, where cytolytic 9 kDa granulysin could be secreted by receptor mediated pathway [18]. It could recognize secreted cytolytic 9 kDa granulysin, unless the specific epitope becomes inaccessible in the tertiary structure of secreted 9 kDa granulysin form. The results of proposed study should be compared with the effects of other anti-granulysin antibodies that are also capable to recognize 9 kDa granulysin with the aim of seeking for the antibody with the most effective blocking properties. All these data suggest that further investigation of the granulysin-induced apoptosis is needed, in particular due to its possible involvement in the pathogenesis of OA. We hope that our hypothesis will help to generate novel ideas that would encourage investigations of such important biological phenomenon. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements This work was supported by the Special Hospital for Medical Rehabilitation of Heart and Lung Diseases and Rheumatism ‘‘Thalas sotherapia-Opatija”, Opatija, Croatia and a grant from the University of Rijeka (Science Support No. 13.06.1.1.06 to Dr. D. Rukavina). References [1] Klit J, Gosvig K, Jacobsen S, Sonne-Holm S, Troelsen A. The prevalence of predisposing deformity in osteoarthritic hip joints. Hip Int 2011;21:537–41. [2] Leheita O, Abed Elrazek NY, Younes S, Mahmoud AZ. Lymphocytes subsets in osteoarthritis versus rheumatoid arthritis. Egypt J Immunol 2005;12:113–24.
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Contents lists available at ScienceDirect
Medical Hypotheses journal homepage: www.elsevier.com/locate/mehy
Possible role of granulysin in pathogenesis of osteoarthritis Tatjana Kehler a,b, Gordana Laskarin a,d,⇑, Drazen Massari a,b, Marin Dominovic d, Viktor Persic b,c, Ivan Rosovic c, Josip Laginja f, Daniel Rukavina d,e a Department of Rheumatology, Rehabilitation and Physical Medicine, Hospital for Medical Rehabilitation of Heart and Lung Diseases and Rheumatism ‘‘Thalassotherapia-Opatija”, M. Tita 188, 51410 Opatija, Croatia b Department of Medical Rehabilitation, Medical Faculty, University of Rijeka, B. Branchetta 20, 51000 Rijeka, Croatia c Division of Cardiology, Hospital for Medical Rehabilitation of the Heart and Lung Diseases and Rheumatism ‘‘Thalassotherapia-Opatija”, M. Tita 188, 51410 Opatija, Croatia d Department of Physiology and Immunology, Medical Faculty, University of Rijeka, B. Branchetta 20, 51000 Rijeka, Croatia e Department of Clinical and Transplantation Immunology and Molecular Medicine in Rijeka, Croatian Academy of Sciences and Arts, Radmile Matejcic 2, Rijeka, Croatia f Hospital for Medical Rehabilitation of Heart and Lung Diseases and Rheumatism ‘‘Thalassotherapia-Opatija”, M. Tita 188, 51410 Opatija, Croatia
a r t i c l e
i n f o
Article history: Received 20 April 2015 Accepted 22 September 2015
a b s t r a c t 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 joint’s tissue-infiltrating lymphocyte subset, we hypothesized that granulysin mediated human cell death (apoptosis) could be responsible for the relatively silent damage of the joint’s 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 joint’s 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 proinflammatory, cytotoxic/apoptotic mediator. Ó 2015 Elsevier Ltd. All rights reserved.
Introduction It has been considered that OA is induced by a mechanical stress, which causes cartilage destruction without significant participation of the immune response, particularly when compared with rheumatoid arthritis (RA) [1,2]. However, there were no statistical differences between the OA and RA patients regarding the percentages of T cells (T helper and T cytotoxic), B cells and natural
⇑ Corresponding author at: Department of Physiology and Immunology, Medical Faculty, University of Rijeka, B. Branchetta 20, 51000 Rijeka, Croatia. Tel.: +385 51 65 11 85; fax: +385 51 67 56 99. E-mail addresses: [email protected] (T. Kehler), gordana.laskarin@ medri.uniri.hr (G. Laskarin), [email protected] (D. Massari), marin.dominovic@ gmail.com (M. Dominovic), [email protected] (V. Persic), ivan_rosovic@ yahoo.com (I. Rosovic), [email protected] (J. Laginja), [email protected] (D. Rukavina). http://dx.doi.org/10.1016/j.mehy.2015.09.025 0306-9877/Ó 2015 Elsevier Ltd. All rights reserved.
killer (NK) cells in the peripheral blood, and the peripheral CD4/ CD8 ratio, reflecting the similarities in the immune cell profiles of RA and OA patients [2,3]. Immune cells were also found in the synovial fluids and synovial membrane. T cells and their Th1oriented CD4+ subset were much less numerous in the joints of patients with OA than in the RA joints, but they showed a similar expression of activation HLA-DR and CD69 markers and profile of IFN-gamma cytokine secretion [3]. The predominance of Th1 cells were confirmed in both OA and RA joints [4]. Only the frequency of Th17 cells tended to be lower in the joint of patients with OA, than with RA [4]. Locally present CD4+ T lymphocytes increased concentration of macrophage inflammatory protein-1 gamma in homogenized synovial tissue in an anterior cruciate ligament-transection model of OA in wild-type B6 mice [5]. The macrophage inflammatory protein-1 gamma, subsequently supported osteoclast formation, enhanced macrophage infiltration and matrix metalloproteinase-9 expression, which are all responsible for tis-
T. Kehler et al. / Medical Hypotheses 85 (2015) 850–853
sue destruction in mice model of OA [5]. Similarly, CD8+ cells in the synovial membrane in OA patients correlated positively with increased matrix metalloproteinase-1 [6]. Several more studies in animal models [7] and human tissue [8] illustrate the potential importance of cytokine balance in local tissue changes and the development of OA. Thus the counteraction of the main proinflammatory interleukin (IL)-1 cytokine can prevent the progression of joint’s structural changes in mice with OA [7]. The concentration of interferon-c inducible protein-10 is low in the plasma and synovial fluid of OA patients and it inversely correlates with radiographic severity of the disease [8]. Conversely, IL-15 is elevated in the synovial fluid and the synovial membranes in early knee OA and positively correlated with IL-6 concentration [6], suggesting that these cytokines are the local factors responsible for the differentiation and the proliferation of T cells [9] and B cells [10] in situ, and activation of an innate immune response, mediated primarily by NK cells [6]. IL-15 is an effective chemoattractant for resting and activated T and NK cells [11]. It regulates the cytokine production and the cytotoxic potential of NK cells [12], as well as the mRNA expressions of perforin, Fas ligand [13], and granulysin [14]. However, little is known about the role of the cell-mediated immune response and its cytotoxic/apoptotic mechanisms, particularly perforin and granulysin in the pathogenesis of OA at the local and systemic levels. The presence of cytotoxic protein – perforin within the cytoplasm granules of the CD4+, CD8+, CD56+, CD16+, and CD25+ lymphocyte subsets from the synovial fluid and the synovial membrane of OA patients, was identified using flow cytometry [3]. Perforin expression suggests the role of activated cytotoxic immune T and NK cells in the pathogenesis of OA [3], as perforin correlates with cell mediated cytotoxic potential of T cell and NK cells in different tissues [15–17]. In the activated NK effector cells, perforin is stored in dense vesicles together with pro-apoptotic mediators, such as granzymes, Fas ligand, and cytotoxic 9 kDa granulysin form [17,18]. Longer, 15 kDa form of granulysin is stored in distinct granules, situated directly beneath the cell membrane and it can be processed to the shorter, cytotoxic 9 kDa granulysin form [18]. The longer 15 kDa granulysin shows predominantly regulatory properties as alarmin, due to the recruitment of immune cells to the site of inflammation [17,19]. It strongly chemoattracts monocytes, CD4+ and CD8+ memory T cells, NK cells and mature dendritic cells [20] and induce differentiation of monocyte into dendritic cells [18]. Both granulysin containing granules are released from the effector cells on demand, in accordance with cell stimulation [17]. However, 9 kDa form is released after the receptor mediated granule exocytosis pathway, depending of current expression of activation and inhibitory NK cell receptors and their ligands on target cells [18]. Longer 15 kDa form of granulysin is released constantly by IL-15 activated NK cells mostly [18]. Granulysin uses multiple mechanisms to enter the cells and kill targets. As granulysin is kationic molecule of saposin-like family of proteins, it possesses structural features allowing him to associate with lipids [21]. 9 kDa granulysin is endocytosed by the infected eukaryotic cells via lipid rafts [21]. In the cells 9 kDa granulysin kills bacteria efficiently within minutes [20,22], because it forms defects in negatively charged, cholesterol-free membranes of prokaryotic bacterial cells, but leave the eukaryotic cell intact [21]. It is believed that cytotoxic activity of granulysin against eukaryotic cells is due to binding of granulysin for negatively charged mitochondrial membrane [23], after the quick cytoplasmic access by perforin pores [24,25]. Mitochondrial cell damage supports the quick release of apoptosis-inducing factors and mitochondrial cytochrome C, which then induces DNA fragmentation in a caspase-dependent or caspase-independent manner [23,26,27]. Granulysin can also mediate apoptosis by nuclear accumulation [28]. A slower mechanism of granulysin-mediated apoptosis seems to be conducted by
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ceramide generation in the target cell membrane, likely without perforin support [27]. Hypothesis We hypothesized that cytolytic immune mediator granulysin participates in cartilage destruction in patients with OA. In OA patients, granulysin could increase lymphocyte infiltration in synovial tissue and synovial fluid, as well as in the plasma and peripheral blood lymphocyte subpopulations. NK cells might be the main source of granulysin in OA patients, since activated NK cells has been shown as the major source of serum granulysin in vivo [18]. Moreover, NK cells are the principal tissue-infiltrating lymphocyte subset in patients with OA, comprising nearly 30% of the CD45+ mononuclear cell infiltrates in the synovial tissue obtained from patients undergoing total joint replacement surgery [29]. Increased presence of granulysin has been noticed in different tissues during pathological processes with the domination of Th1 over Th2 mediated immunity. For example, the frequency of granulysin expressing NK cells is enhanced in peripheral blood of patients with preeclampsia [30] and psoriatic arthritis [31]. Skeletal muscles and the hearth are shown to be infiltrated with granulysin positive cells during polymyosytis [32] or acute infarction [33], respectively. Granulysin concentration in human serum and broad cytotoxic activity of granulysin against virally infected cells, tumors, and transplanted cells [28] indeed classify this mediator as a human marker of the Th1 immune response [20,26,30,34]. Thus the dynamic changes in the expression of granulysin in patients with OA could be the reflection of disturbed balance of humoral pro-inflammatory and anti-inflammatory factors, at least at the local level. And finally, granulysin mediated cell death supporting apoptosis could be responsible for the relatively silent damage of the joint’s tissue without clinically notable, eclatant systemic inflammatory reaction. Evaluation of the hypothesis To estimate the granulysin-mediated cytotoxic potential of lymphocytes in patients with OA, it is essential to form homogenous group of patients with OA and healthy control. The inclusive criteria could be set up according to the recommendations of the American College of Rheumatology for diagnosis of knee [35], hand [36] or hip [37] OA. The exclusion criteria for the group of patients with OA and control group should also be defined precisely, particularly in terms of other possible systemic immune diseases. The therapy of participants deserves particular attention and should not interfere with the immunological examinations. The estimation of the presence and frequency of granulysin expressing lymphocyte subpopulations in freshly isolated peripheral blood samples of patients with OA at protein and gene levels will give the first approach to evaluate our hypothesis. Our preliminary data show that granulysin positive cells in peripheral blood samples of OA patients counts about 30% and it was approximately twice higher, when compared with healthy control. It was due to higher granulysin expressing in NK, T and NKT leukocyte subsets of OA patients, as measured by flow cytometry. The second approach to test this hypothesis could be the analyzes of granulysin-mediated NK cell cytotoxicity in different time points using major histocompatibility complex (MHC) devoid, NK sensitive K562 cells of human erythroleukaemia cell-line, as targets in classical in vitro model for the assessment of NK cell activity [38]. 51Cr release assay measures necrosis/lysis [39] very precisely [40], but the measurement of NK cell mediated target cell apoptosis could be more suitable for evaluating granulysin activity. Late
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apoptosis evaluated by flow cytometry assay as frequency of propidium iodide positive and annexin V positive target cells cannot be distinguished well from the total necrosis [41]. But the early apoptosis, expressed as the percentage of propidium iodide negative and annexin V positive cells is better indicator for apoptotic activity of cells according to data of Vermes et al. [41]. It would be of the particular interest to differentiate the expression of shorter (9 kDa) and longer (15 kDa) granulysin forms in the effector cell, since their immuno-biological roles have been distinguished. Only the shorter form possesses cytolytic activity [18], whereas longer form attracts, activates and differentiates monocytes into dendritic cells [17], as well as exhibits potent in vivo immunoactivating properties [18,20]. Production of cytokines in lymphocyte subpopulations and cytokine concentration in the plasma by flow cytometry or enzyme linked immunoassay, respectively, could help to evaluate c1ytokine support, at the time of granulysin estimation. The analysis of the synovial tissue and synovial fluid obtained from patients undergoing total joint replacement surgery could provide better insight in locally acting pathophysiological mechanisms during late OA, while early pathophysiological events might be analyzed in synovial fluids obtained by diagnostic puncture of the joint or eventually by arthroscopy. Since the clinical studies may be limited, the experiments in animal models for example with granulysin transgenic mice could be of particular importance, since there has not been homologous molecule to granulysin in the mice [25].
Consequences of the hypothesis and discussion There is increasing body of evidence that cell-mediated immunity supported by pro-inflammatory cytokines and chemokines has a critical role in the pathogenesis of OA [4–7,29]. However, the role of pro-inflammatory, cytotoxic mediator granulysin in the pathogenesis of OA is still unexplored, although it has been shown to reflect the activation status of cell-mediated immunity [42]. Granulysin mediates unscheduled human cell death by apoptosis induction and nuclear accumulation [34]. The apoptosis is the important biological phenomenon, which occurs in the human adult approximately in 50 and 70 billion of cells daily [43]. The quick removal of apoptotic bodies by neighboring phagocytic cells, before the contents of dying cells could spill out onto surrounding cells and cause damage, does not elicit an inflammatory response [43]. This mechanism could be responsible for the silent damage of the joint’s tissue without clinically notable, eclatant systemic inflammatory reaction, in spite of the presence of immune cells and a chronic, progressive immune response, directed toward unknown antigen(s) in the fluid and synovial membrane of osteoarthritic joints [4,6,29]. Granulysin exists in the effector T, NKT and NK cells [44,45] in the longer 15 kDa form, which is produced rapidly and shaded from IL-15 stimulated NK cells, exerting regulatory immune functions [18]. The shorter 9 kDa form with cytolytic properties, appears in the cell after the cleavage of the precursor 15 kDa form [44]. Since functions of granulysin forms have been different, it would be valuable to determine the frequency of cells that express the particular forms. As far as we know there is no clear data showing difference in the frequency of 9 and 15 kDa expressing subsets by flow cytometry, although it was recently attempted by other investigators [18] and by our group [preliminary, unpublish data]. To distinguish forms of granulysin is not very convenient at the moment, although more monoclonal and polyclonal antibodies, which recognize different epitopes, and do not compete with each other, are available. Polyclonal rabbit anti-9 kDa granulysin antibodies, designed by the group of Clayberger et al. [18] seem to rec-
ognize the epitope of 9 kDa granulysin, independently whether it is cleaved or not from the precursor 15 kDa granulysin form. However, only under denaturing conditions in Western blot analysis the distinction of 15 kDa and 9 kDa was possible [18]. Granulysin isoforms share a common core, whose epitope is likely to be recognized in the proteins of both forms by the same antibody [18] depending also of the biological properties of the antibody and the laboratory method implemented. Our group has shown previously the higher co-localisation of granulysin and perforin by immunofluorescence using RC8 anti-granulysin mAb and deltaG9 anti-perforin mAb, respectively, in freshly isolated early human pregnancy decidual NK cells, which were in vivo activated by IL15, when compared with peripheral blood NK cells of the same pregnant women [19]. RC8 mAb recognizes an core epitope contained in both, 9 kDa and 15 kDa forms of granulysin [42], because RC8 anti-granulysin antibody was purified from RC8 hybridoma, which had been established by fusion of mouse myeloid cell Sp2/0-Ag8 with Balb/c mouse splenocyte immunized with fulllength human granulysin expression plasmid [18]. Accordingly, our above mentioned results only suggest, but does not prove the presence of lytic 9 kDa granulysin in activated decidual NK cells, as positivity could appear due to the staining of 15 kDa granulysin form, in spite of the facts that 9 kDa granulysin co-localizes with perforin in the granules of highly stimulated NK cells much more than in cells at lower level of activation [19]. Testing of granulysin induced apoptosis in the presence and absence of blocking anti-granulysin antibody, could be valuable proof for the release of the cytotoxic mature 9 kDa granulysin form from the effector cell, assuming that the activity of probably coexcreted 15 kDa granulysin from does not exhibit cytotoxic properties [18] and therefore cannot be measured by cytotoxicity assay. RC8 mAb might be useful in blocking granulysin mediated cytotoxicity (apoptosis), if it is present in vitro in the medium surrounding effector and target cells, where cytolytic 9 kDa granulysin could be secreted by receptor mediated pathway [18]. It could recognize secreted cytolytic 9 kDa granulysin, unless the specific epitope becomes inaccessible in the tertiary structure of secreted 9 kDa granulysin form. The results of proposed study should be compared with the effects of other anti-granulysin antibodies that are also capable to recognize 9 kDa granulysin with the aim of seeking for the antibody with the most effective blocking properties. All these data suggest that further investigation of the granulysin-induced apoptosis is needed, in particular due to its possible involvement in the pathogenesis of OA. We hope that our hypothesis will help to generate novel ideas that would encourage investigations of such important biological phenomenon. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements This work was supported by the Special Hospital for Medical Rehabilitation of Heart and Lung Diseases and Rheumatism ‘‘Thalas sotherapia-Opatija”, Opatija, Croatia and a grant from the University of Rijeka (Science Support No. 13.06.1.1.06 to Dr. D. Rukavina). References [1] Klit J, Gosvig K, Jacobsen S, Sonne-Holm S, Troelsen A. The prevalence of predisposing deformity in osteoarthritic hip joints. Hip Int 2011;21:537–41. [2] Leheita O, Abed Elrazek NY, Younes S, Mahmoud AZ. Lymphocytes subsets in osteoarthritis versus rheumatoid arthritis. Egypt J Immunol 2005;12:113–24.
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