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• W2010043145 abstract "Natural killer (NK) cells are innate immune effectors that produce various immunoregulatory cytokines. Recent studies have shown that NK cells are involved in the initiation of autoimmunity. In this study, we determined abnormalities of NK cells in systemic sclerosis (SSc), an autoimmune connective tissue disease, by assessing the frequency and absolute number, activation marker expression, cytokine production, and killing activity. The frequency and absolute number of NK cells increased in diffuse cutaneous SSc (dcSSc), whereas they were normal in limited cutaneous SSc (lcSSc). NK cells from both dcSSc and lcSSc patients exhibited activated phenotypes characterized by up-regulated CD16 and CD69 expression and downregulated CD62L expression. Interferon (IFN)-γ production by non-stimulated NK cells from both dcSSc and lcSSc patients was increased compared to the normal control, whereas on stimulation, a reduced amount of IFN-γ was produced. Interleukin (IL)-5 and IL-10 production by non-stimulated NK cells and IL-6 production by stimulated NK cells were augmented in dcSSc patients, but not in lcSSc patients. Despite the augmented cytokine production by non-stimulated NK cells, natural cytotoxicity activity and granzyme B secretion was reduced in NK cells from dcSSc and lcSSc patients. These results suggested that altered NK cell function contributes to immunological abnormalities in SSc. Natural killer (NK) cells are innate immune effectors that produce various immunoregulatory cytokines. Recent studies have shown that NK cells are involved in the initiation of autoimmunity. In this study, we determined abnormalities of NK cells in systemic sclerosis (SSc), an autoimmune connective tissue disease, by assessing the frequency and absolute number, activation marker expression, cytokine production, and killing activity. The frequency and absolute number of NK cells increased in diffuse cutaneous SSc (dcSSc), whereas they were normal in limited cutaneous SSc (lcSSc). NK cells from both dcSSc and lcSSc patients exhibited activated phenotypes characterized by up-regulated CD16 and CD69 expression and downregulated CD62L expression. Interferon (IFN)-γ production by non-stimulated NK cells from both dcSSc and lcSSc patients was increased compared to the normal control, whereas on stimulation, a reduced amount of IFN-γ was produced. Interleukin (IL)-5 and IL-10 production by non-stimulated NK cells and IL-6 production by stimulated NK cells were augmented in dcSSc patients, but not in lcSSc patients. Despite the augmented cytokine production by non-stimulated NK cells, natural cytotoxicity activity and granzyme B secretion was reduced in NK cells from dcSSc and lcSSc patients. These results suggested that altered NK cell function contributes to immunological abnormalities in SSc. diffuse cutaneous systemic sclerosis fluorescein isothiocyanate interferon interleukin limited cutaneous systemic sclerosis natural killer peripheral blood mononuclear cells phycoerythrin phorbol 12-myristate 13-acetate systemic sclerosis transforming growth factor T helper type 1 tumor necrosis factor Systemic sclerosis (SSc) is a connective tissue disease characterized by increased collagen production by fibroblasts, resulting in cutaneous and internal organ fibrosis, with an autoimmune background. One of the central features in SSc is the presence of a variety of immunological abnormalities, such as in vivo T lymphocyte activation, elevated serum levels of various cytokines and growth factors, chronic activation of memory B lymphocytes, the frequent occurrence of autoantibodies, and hyper-γ-globulinemia (White, 1996White B. Immunopathogenesis of systemic sclerosis.Rheum Dis Clin North Am. 1996; 32: 695-708Google Scholar; Sato et al., 2004Sato S. Fujimoto M. Hasegawa M. Takehara K. Altered blood B lymphocyte homeostasis in systemic sclerosis: Expanded naive B cells and diminished but activated memory B cells.Arthritis Rheum. 2004; 50: 1918-19127Google Scholar). Although the pathogenesis of SSc remains unknown, these immunological abnormalities play an important role in the initiation and development of this disease. Natural killer (NK) cells are characterized by their ability to lyse transformed cells and virally infected cells without antigen sensitization, thereby contributing to the early host defense of innate immunity (Biron et al., 1999Biron C.A. Nguyen K.B. Pien G.C. Cousens L.P. Salazar-Mather T.P. Natural killer cells in antiviral defense: Function and regulation by innate cytokines.Annu Rev Immunol. 1999; 17: 189-220Google Scholar; Seaman, 2000Seaman W.E. Natural killer cells and natural killer T cells.Arthritis Rheum. 2000; 43: 1204-1217Google Scholar). In recent years, other functions of NK cells have been identified, particularly their capacity to produce various cytokines (Biron et al., 1999Biron C.A. Nguyen K.B. Pien G.C. Cousens L.P. Salazar-Mather T.P. Natural killer cells in antiviral defense: Function and regulation by innate cytokines.Annu Rev Immunol. 1999; 17: 189-220Google Scholar; Seaman, 2000Seaman W.E. Natural killer cells and natural killer T cells.Arthritis Rheum. 2000; 43: 1204-1217Google Scholar). NK cells produce not only T helper type 1 (Th1) cytokines such as interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and granulocyte-macrophage colony stimulating factor, but also Th2 cytokines such as interleukin (IL)-5, IL-10, and IL-13 and other cytokines such as transforming growth factor (TGF)-β (Peritt et al., 1998Peritt D. Robertson S. Gri G. Showe L. Aste-Amezaga M. Trinchieri G. Differentiation of human NK cells into NK1 and NK2 subsets.J Immunol. 1998; 161: 5821-5824Google Scholar; Biron et al., 1999Biron C.A. Nguyen K.B. Pien G.C. Cousens L.P. Salazar-Mather T.P. Natural killer cells in antiviral defense: Function and regulation by innate cytokines.Annu Rev Immunol. 1999; 17: 189-220Google Scholar; Seaman, 2000Seaman W.E. Natural killer cells and natural killer T cells.Arthritis Rheum. 2000; 43: 1204-1217Google Scholar; Shi et al., 2001Shi F. Ljunggren H.G. Sarvetnick N. Innate immunity and autoimmunity: From self-protection to self-destruction.Trends Immunol. 2001; 22: 97-101Google Scholar). Thus, NK cells are able to regulate immune responses either directly or indirectly through cytokine secretion (Peritt et al., 1998Peritt D. Robertson S. Gri G. Showe L. Aste-Amezaga M. Trinchieri G. Differentiation of human NK cells into NK1 and NK2 subsets.J Immunol. 1998; 161: 5821-5824Google Scholar; Biron et al., 1999Biron C.A. Nguyen K.B. Pien G.C. Cousens L.P. Salazar-Mather T.P. Natural killer cells in antiviral defense: Function and regulation by innate cytokines.Annu Rev Immunol. 1999; 17: 189-220Google Scholar; Seaman, 2000Seaman W.E. Natural killer cells and natural killer T cells.Arthritis Rheum. 2000; 43: 1204-1217Google Scholar; Shi et al., 2001Shi F. Ljunggren H.G. Sarvetnick N. Innate immunity and autoimmunity: From self-protection to self-destruction.Trends Immunol. 2001; 22: 97-101Google Scholar). It has been revealed that NK or NK1.1+ T (NKT) cells are involved in various experimental autoimmune diseases (Maruyama et al., 1991Maruyama T. Watanabe K. Takei I. et al.Anti-asialo GM1 antibody suppression of cyclophosphamide-induced diabetes in NOD mice.Diabetes Res. 1991; 17: 37-41Google Scholar; Zhang et al., 1997Zhang K. Gharaee-Kermani M. McGarry B. Remick D. Phan S.H. TNF-α-mediated lung cytokine networking and eosinophil recruitment in pulmonary fibrosis.J Immunol. 1997; 158: 954-959Google Scholar; Fort et al., 1998Fort M.M. Leach M.W. Rennick D.M. A role for NK cells as regulators of CD4+ T cells in a transfer model of colitis.J Immunol. 1998; 161: 3256-3261Google Scholar). Although the effects of NK cells were not distinguished from those of NKT cells in these studies (Shi et al., 2001Shi F. Ljunggren H.G. Sarvetnick N. Innate immunity and autoimmunity: From self-protection to self-destruction.Trends Immunol. 2001; 22: 97-101Google Scholar), a recent study has shown that NK cells, but not NKT cells, promote the development of murine myasthenia gravis, a T cell-dependent, B cell- and autoantibody-mediated autoimmune disease (Shi et al., 2000Shi F.D. Wang H.B. Li H. et al.Natural killer cells determine the outcome of B cell-mediated autoimmunity.Nat Immunol. 2000; 1: 245-251Google Scholar). In this model, the requirement for NK cells is reflected by the lack of a Th1 response and autoantibodies, suggesting that NK cells regulate auto-reactive T and B cells (Shi et al., 2000Shi F.D. Wang H.B. Li H. et al.Natural killer cells determine the outcome of B cell-mediated autoimmunity.Nat Immunol. 2000; 1: 245-251Google Scholar). NK cell abnormalities have been reported in SSc with conflicting results (Wright et al., 1982Wright J.K. Hughes P. Rowell N.R. Spontaneous lymphocyte-mediated (NK cell) cytotoxicity in systemic sclerosis: A comparison with antibody-dependent lymphocyte (K cell) cytotoxicity.Ann Rheum Dis. 1982; 41: 409-413Google Scholar; Majewski et al., 1987Majewski S. Blaszczyk M. Wasik M. Jablonska S. Natural killer cell activity of peripheral blood mononuclear cells from patients with various forms of systemic scleroderma.Br J Dermatol. 1987; 116: 1-8Google Scholar; Miller et al., 1988Miller E.B. Hiserodt J.C. Hunt L.E. Steen V.D. Medsger Jr, T.A. Reduced natural killer cell activity in patients with systemic sclerosis. Correlation with clinical disease type.Arthritis Rheum. 1988; 31: 1515-1523Google Scholar; Grazia Cifone et al., 1990Grazia Cifone M. Giacomelli R. Famularo G. et al.Natural killer activity and antibody-dependent cellular cytotoxicity in progressive systemic sclerosis.Clin Exp Immunol. 1990; 80: 360-365Google Scholar; Kantor et al., 1992Kantor T.V. Whiteside T.L. Friberg D. Buckingham R.B. Medsger Jr, T.A. Lymphokine-activated killer cell and natural killer cell activities in patients with systemic sclerosis.Arthritis Rheum. 1992; 35: 694-699Google Scholar). Specifically, the killing activity of NK cells from SSc patients was decreased in three previous studies (Wright et al., 1982Wright J.K. Hughes P. Rowell N.R. Spontaneous lymphocyte-mediated (NK cell) cytotoxicity in systemic sclerosis: A comparison with antibody-dependent lymphocyte (K cell) cytotoxicity.Ann Rheum Dis. 1982; 41: 409-413Google Scholar; Majewski et al., 1987Majewski S. Blaszczyk M. Wasik M. Jablonska S. Natural killer cell activity of peripheral blood mononuclear cells from patients with various forms of systemic scleroderma.Br J Dermatol. 1987; 116: 1-8Google Scholar; Miller et al., 1988Miller E.B. Hiserodt J.C. Hunt L.E. Steen V.D. Medsger Jr, T.A. Reduced natural killer cell activity in patients with systemic sclerosis. Correlation with clinical disease type.Arthritis Rheum. 1988; 31: 1515-1523Google Scholar), whereas it was enhanced in two studies (Grazia Cifone et al., 1990Grazia Cifone M. Giacomelli R. Famularo G. et al.Natural killer activity and antibody-dependent cellular cytotoxicity in progressive systemic sclerosis.Clin Exp Immunol. 1990; 80: 360-365Google Scholar; Wanchu et al., 1995Wanchu A. Singh V.K. Yadav V.S. Biswas S. Misra R. Agarwal S.S. Lack of natural killer cell augmentation in vitro by human interferon gamma in a subset of patients with systemic sclerosis.Pathobiology. 1995; 63: 288-292Google Scholar). One study showed normal killing activity in SSc (Kantor et al., 1992Kantor T.V. Whiteside T.L. Friberg D. Buckingham R.B. Medsger Jr, T.A. Lymphokine-activated killer cell and natural killer cell activities in patients with systemic sclerosis.Arthritis Rheum. 1992; 35: 694-699Google Scholar). Two studies showed the normal frequency and absolute number of circulating NK cells in SSc (Miller et al., 1988Miller E.B. Hiserodt J.C. Hunt L.E. Steen V.D. Medsger Jr, T.A. Reduced natural killer cell activity in patients with systemic sclerosis. Correlation with clinical disease type.Arthritis Rheum. 1988; 31: 1515-1523Google Scholar; Kantor et al., 1992Kantor T.V. Whiteside T.L. Friberg D. Buckingham R.B. Medsger Jr, T.A. Lymphokine-activated killer cell and natural killer cell activities in patients with systemic sclerosis.Arthritis Rheum. 1992; 35: 694-699Google Scholar), whereas one study showed increased NK cell frequency (Grazia Cifone et al., 1990Grazia Cifone M. Giacomelli R. Famularo G. et al.Natural killer activity and antibody-dependent cellular cytotoxicity in progressive systemic sclerosis.Clin Exp Immunol. 1990; 80: 360-365Google Scholar). In all these studies, the NK cell killing activity was assessed with total peripheral blood mononuclear cells (PBMC), but not with isolated NK cells. Since NK activity is proportional to the percentage of NK cells (Ichikawa et al., 1985Ichikawa Y. Yoshida M. Takaya M. Uchiyama M. Shimizu H. Arimori S. Circulating natural killer cells in Sjogren's syndrome.Arthritis Rheum. 1985; 28: 182-187Google Scholar), the discrepancy among killing activity results may be due to different NK cell numbers. In addition, the presence of other lymphocytes in PBMC might affect NK cell killing. Furthermore, the analysis of cell surface activation marker expression on NK cells and cytokine production by NK cells from SSc patients were not analyzed in any of the previous studies. In this study, to determine NK cell dysfunction in SSc, we assessed the percentage and number of blood NK cells, activation marker expression, and production of cytokine and granzyme B, and natural cytotoxicity by isolated NK cells from SSc patients. The frequency and absolute number of NK cells in blood from SSc patients and normal individuals were examined by flow cytometry. The NK cell frequency in dcSSc patients was significantly increased relative to lcSSc patients (p<0.0001) and normal controls (p<0.001; Figure 1a). Similarly, the absolute number of NK cells in dcSSc patients was significantly increased compared to normal controls (p<0.05; Figure 1b). By contrast, there was no significant difference in the NK cell frequency and number between lcSSc patients and normal controls, although some patients with lcSSc showed relatively high numbers of NK cells. To assess the NK cell activation state, the expression of CD16, CD25, CD54, CD62L, CD69, and CD95 was analysed with flow cytometry (Figure 2 and 3). CD56+ NK cells in dcSSc and lcSSc patients exhibited significantly higher CD16 expression levels than normal controls (p<0.05). The frequency of cells expressing CD69, an early activation marker, in CD56+ NK cells was significantly increased in dcSSc and lcSSc patients compared with normal controls (p<0.001 and p<0.005, respectively). The frequency of CD62L expression on NK cells was significantly decreased in dcSSc and lcSSc patients than in normal controls (p<0.001 and p<0.005, respectively), indicating that NK cells from SSc patients are activated since NK cell activation results in the loss of CD62L (L-selectin) expression (Frey et al., 1998Frey M. Packianathan N.B. Fehniger T.A. et al.Differential expression and function of L-selectin on CD56bright and CD56dim natural killer cell subsets.J Immunol. 1998; 161: 400-408Google Scholar). There was no significant difference in the frequency of NK cells expressing CD25, CD54, or CD95 and the expression levels of these molecules between SSc patients and normal controls (data not shown). Therefore, NK cells from SSc patients exhibited an activated phenotype characterized by up-regulated CD16 and CD69 expression and down-regulated CD62L expression.Figure 3CD16 expression level, CD69, and CD62L frequency on natural killer (NK) cells from diffuse cutaneous systemic sclerosis patients, limited cutaneous systemic sclerosis patients, and normal controls (CTL). CD16 expression levels on CD56+ NK cells (A) and frequency of CD69+ cells (B) and CD62L (C) in CD56+ NK cells were determined by flow cytometric analysis as described in Figure 2. The horizontal bars represent mean values with statistically significant differences between groups indicated.View Large Image Figure ViewerDownload (PPT) The production of IFN-γ, IL-5, IL-6, IL-10, and IL-13 by NK cells stimulated with or without phorbol 12-myristate 13-acetate (PMA) and ionomycin was examined by ELISA (Figure 4). IFN-γ production by non-stimulated NK cells form dcSSc and lcSSc patients was significantly increased 3–5-fold compared to normal controls (p<0.005). By contrast, on stimulation, NK cells from dcSSc and lcSSc patients produced a significantly, ∼40%, lower amount of IFN-γ than from normal controls (p<0.05 and p<0.01, respectively). IFN-γ production by non-stimulated or stimulated NK cells did not significantly differ between dcSSc and lcSSc patients. Non-stimulated NK cells from dcSSc patients produced a significantly, 3–4-fold, larger amount of IL-5 than from lcSSc patients (p<0.005) or normal controls (p<0.005); however, no such significance difference was observed after stimulation. Non-stimulated NK cells from dcSSc and lcSSc patients produced a normal IL-6 amount, whereas stimulated NK cells from dcSSc patients released a significantly, 2.6-fold, larger amount of IL-6 relative to normal controls (p<0.005). IL-10 production by non-stimulated NK cells was detected in seven of ten dcSSc patients, whereas it was detected in only one of nine lcSSc patients and none of the normal controls. After stimulation, IL-10 production was detected in four of ten dcSSc patients and three of nine lcSSc patients, whereas it could not be detected in the normal controls. IL-13 production could not be detected from either non-stimulated or activated NK cells from SSc patients and healthy controls. Thus, dcSSc patients generally exhibited enhanced production of IL-5, IL-6, and IL-10 by NK cells compared with lcSSc patients, except that altered IFN-γ production was similarly detected in both dcSSc and lcSSc patients. To further investigate the functional abnormalities of SSc NK cells, we examined NK cell killing activity by performing a cell-mediated cytotoxicity assay with flow cytometric analysis, which has been widely used to determine cytotoxicity instead of the standard 51Cr release assay (Chang et al., 1993Chang L. Gusewitch G.A. Chritton D.B. Folz J.C. Lebeck L.K. Nehlsen-Cannarella S.L. Rapid flow cytometric assay for the assessment of natural killer cell activity.J Immunol Methods. 1993; 166: 45-54Google Scholar; Hoppner et al., 2002Hoppner M. Luhm J. Schlenke P. Koritke P. Frohn C. A flow-cytometry based cytotoxicity assay using stained effector cells in combination with native target cells.J Immunol Methods. 2002; 267: 157-163Google Scholar). This two-color fluorescence assay allows direct assessment of cell-mediated cytotoxicity measurements that correlate well with those obtained using conventional 51Cr release assays (Chang et al., 1993Chang L. Gusewitch G.A. Chritton D.B. Folz J.C. Lebeck L.K. Nehlsen-Cannarella S.L. Rapid flow cytometric assay for the assessment of natural killer cell activity.J Immunol Methods. 1993; 166: 45-54Google Scholar). Isolated NK cells from SSc patients had 10%–20% reduction in their ability to lyse K562 target cells compared with normal controls at all E:T ratios (p<0.05; Figure 5). There was no significant difference in the killing activity between dcSSc and lcSSc patients. Granzyme B, a serine protease, is present in cytoplasmic granules of NK cells and is involved in natural cytotoxicity by NK cells (Seaman, 2000Seaman W.E. Natural killer cells and natural killer T cells.Arthritis Rheum. 2000; 43: 1204-1217Google Scholar). Therefore, granzyme B released by isolated NK cells was assessed by ELISA (Figure 6). Granzyme B released by unstimulated NK cells was ∼40% significantly lower in lcSSc or dcSSc patients than in normal controls (p<0.05). Furthermore, NK cells stimulated by PMA and ionomycin produced a significant reduction in granzyme B release in lcSSc patients compared to normal controls (p<0.05), whereas granzyme B production by stimulated NK cells was similar between dcSSc patients and normal controls. In this study, an activated phenotype was observed in circulating NK cells from both dcSSc and lcSSc patients, with a more activated phenotype in dcSSc NK cells. CD69 expression on SSc NK cells was upregulated, whereas CD25 expression was normal. This may be explained by the finding that CD69 expression is increased in very early phase of activation, whereas CD25 expression is up-regulated in late phase (Marzio et al., 1999Marzio R. Mauel J. Betz-Corradin S. CD69 and regulation of the immune function.Immunopharmacol Immunotoxicol. 1999; 21: 565-582Google Scholar; Reddy et al., 2004Reddy M. Eirikis E. Davis C. Davis H.M. Prabhakar U. Comparative analysis of lymphocyte activation marker expression and cytokine secretion profile in stimulated human peripheral blood mononuclear cell cultures: An in vitro model to monitor cellular immune function.J Immunol Methods. 2004; 293: 127-142Google Scholar). Indeed, CD25 and CD69 expression on NK cells is inversely correlated (Clausen et al., 2003Clausen J. Vergeiner B. Enk M. Petzer A.L. Gastl G. Gunsilius E. Functional significance of the activation-associated receptors CD25 and CD69 on human NK-cells and NK-like T-cells.Immunobiology. 2003; 207: 85-93Google Scholar). Both dcSSc and lcSSc patients exhibited abnormal function of NK cells characterized by altered cytokine production, diminished natural cytotoxicity, and decreased granzyme B release. The frequency and absolute number of NK cells were increased in dcSSc compared to normal controls. This NK cell expansion may be related to increased serum levels of IL-2 and IL-15, cytokines that can induce NK cell proliferation, in SSc patients (Needleman et al., 1992Needleman B.W. Wigley F.M. Stair R.W. Interleukin-1, interleukin-2, interleukin-4, interleukin-6, tumor necrosis factor-α, and interferon-g levels in sera from patients with scleroderma.Arthritis Rheum. 1992; 35: 67-72Google Scholar; Suzuki et al., 2001Suzuki J. Morimoto S. Amano H. Tokano Y. Takasaki Y. Hashimoto H. Serum levels of interleukin 15 in patients with rheumatic diseases.J Rheumatol. 2001; 28: 2389-2391Google Scholar), Furthermore, augmented IL-5 by non-stimulated NK cells and IL-6 production by stimulated NK cells were increased in dcSSc, but not in lcSSc. IL-5 promotes the proliferation and differentiation of eosinophils that stimulate matrix production in dermal fibroblasts (Birkland et al., 1994Birkland T.P. Cheavens M.D. Pincus S.H. Human eosinophils stimulate DNA synthesis and matrix production in dermal fibroblasts.Arch Dermatol Res. 1994; 286: 312-318Google Scholar). SSc patients with active lung fibrosis exhibit a higher frequency of eosinophils in their bronchoalveolar lavage fluid, and bronchoalveolar lavage cells express higher IL-5 levels (Atamas et al., 1999Atamas S.P. Yurovsky V.V. Wise R. et al.Production of type 2 cytokines by CD8+ lung cells is associated with greater decline in pulmonary function in patients with systemic sclerosis.Arthritis Rheum. 1999; 42: 1168-1178Google Scholar), suggesting that increased IL-5 production by NK cells in dcSSc may be related to lung inflammation. IL-6 production by PMBC and serum IL-6 levels are increased in dcSSc patients, especially those with lung involvement (Bolster et al., 1997Bolster M.B. Ludwicka A. Sutherland S.E. Strange C. Silver R.M. Cytokine concentrations in bronchoalveolar lavage fluid of patients with systemic sclerosis.Arthritis Rheum. 1997; 40: 746-751Google Scholar; Hasegawa et al., 1998Hasegawa M. Sato S. Fujimoto M. Ihn H. Kikuchi K. Takehara K. Serum levels of interleukin 6 (IL-6), oncostatin M, soluble IL-6 receptor, and soluble gp130 in patients with systemic sclerosis.J Rheumatol. 1998; 25: 308-313Google Scholar; Scala et al., 2004Scala E. Pallotta S. Frezzolini A. et al.Cytokine and chemokine levels in systemic sclerosis: Relationship with cutaneous and internal organ involvement.Clin Exp Immunol. 2004; 138: 540-546Google Scholar). Consistently, in our study, eight of ten dcSSc patients but no lcSSc patients examined had lung fibrosis. Thus, the increased IL-5 and IL-6 production by dcSSc NK cells might contribute to the more severe clinical manifestations. The results of previous studies regarding natural cytotoxicity abnormalities in SSc were conflicted, probably due to the use of total PBMC as the NK cell source (Wright et al., 1982Wright J.K. Hughes P. Rowell N.R. Spontaneous lymphocyte-mediated (NK cell) cytotoxicity in systemic sclerosis: A comparison with antibody-dependent lymphocyte (K cell) cytotoxicity.Ann Rheum Dis. 1982; 41: 409-413Google Scholar; Majewski et al., 1987Majewski S. Blaszczyk M. Wasik M. Jablonska S. Natural killer cell activity of peripheral blood mononuclear cells from patients with various forms of systemic scleroderma.Br J Dermatol. 1987; 116: 1-8Google Scholar; Miller et al., 1988Miller E.B. Hiserodt J.C. Hunt L.E. Steen V.D. Medsger Jr, T.A. Reduced natural killer cell activity in patients with systemic sclerosis. Correlation with clinical disease type.Arthritis Rheum. 1988; 31: 1515-1523Google Scholar; Grazia Cifone et al., 1990Grazia Cifone M. Giacomelli R. Famularo G. et al.Natural killer activity and antibody-dependent cellular cytotoxicity in progressive systemic sclerosis.Clin Exp Immunol. 1990; 80: 360-365Google Scholar; Kantor et al., 1992Kantor T.V. Whiteside T.L. Friberg D. Buckingham R.B. Medsger Jr, T.A. Lymphokine-activated killer cell and natural killer cell activities in patients with systemic sclerosis.Arthritis Rheum. 1992; 35: 694-699Google Scholar; Wanchu et al., 1995Wanchu A. Singh V.K. Yadav V.S. Biswas S. Misra R. Agarwal S.S. Lack of natural killer cell augmentation in vitro by human interferon gamma in a subset of patients with systemic sclerosis.Pathobiology. 1995; 63: 288-292Google Scholar). This study using isolated NK cells showed that cytotoxicity activity by SSc NK cells was constitutively reduced, excluding the effects of different NK cell numbers and other lymphocytes in PBMC. This diminished cytotoxicity activity by SSc NK cells may be explained in part by reduced granzyme B release, since granzyme B secretion correlates with cytotoxicity activity evaluated by the standard 51Cr-release cytotoxicity assay (Rininsland et al., 2000Rininsland F.H. Helms T. Asaad R.J. Boehm B.O. Tary-Lehmann M. Granzyme B ELISPOT assay for ex vivo measurements of T cell immunity.J Immunol Methods. 2000; 240: 143-155Google Scholar; Shafer-Weaver et al., 2003Shafer-Weaver K. Sayers T. Strobl S. Derby E. Ulderich T. Baseler M. Malyguine A. The Granzyme B ELISPOT assay: An alternative to the 51Cr-release assay for monitoring cell-mediated cytotoxicity.J Transl Med. 2003; 1: 14-22Google Scholar). Alternatively, chronic NK cell activation in SSc may prevent efficient activation when active killing is required. A recent study has shown that human NK cells are able to polarize in vitro into two functionally distinct subsets, NK type 1 (NK1) and NK2, analogous to T-cell subsets Th1 and Th2 (Peritt et al., 1998Peritt D. Robertson S. Gri G. Showe L. Aste-Amezaga M. Trinchieri G. Differentiation of human NK cells into NK1 and NK2 subsets.J Immunol. 1998; 161: 5821-5824Google Scholar). NK1 cells mainly produce IFN-γ and IL-10, whereas NK2 cells predominantly produce IL-5 and IL-13 (Peritt et al., 1998Peritt D. Robertson S. Gri G. Showe L. Aste-Amezaga M. Trinchieri G. Differentiation of human NK cells into NK1 and NK2 subsets.J Immunol. 1998; 161: 5821-5824Google Scholar). In SSc, it has been reported that serum levels and production levels by stimulated PBMC of Th2 cytokines, such as IL-4, IL-6, and IL-13, are increased, suggesting that SSc may be a Th2-predominant disease (Needleman et al., 1992Needleman B.W. Wigley F.M. Stair R.W. Interleukin-1, interleukin-2, interleukin-4, interleukin-6, tumor necrosis factor-α, and interferon-g levels in sera from patients with scleroderma.Arthritis Rheum. 1992; 35: 67-72Google Scholar; Hasegawa et al., 1997Hasegawa M. Fujimoto M. Kikuchi K. Takehara K. Elevated serum levels of interleukin 4 (IL-4), IL-10, and IL-13 in patients with systemic sclerosis.J Rheumatol. 1997; 24: 328-332Google Scholar). Recent studies, however, have shown that Th1 cytokine production is also increased in SSc. Serum levels of IL-12, a potent inducer of Th1 cells, are elevated in SSc patients (Sato et al., 2000Sato S. Hanakawa H. Hasegawa M. et al.Levels of interleukin 12, a cytokine of type 1 helper T cells, are elevated in sera from patients with systemic sclerosis. [In Process Citation].J Rheumatol. 2000; 27: 2838-2842Google Scholar). The finding that NK cells produced a larger amount of Th2 cytokines (IL-5, IL-6, and IL-10) in dcSSc suggests NK2 response activation. In contrast, IFN-γ production by non-stimulated NK cells in dcSSc and lcSSc was significantly increased, which suggests NK1 response activation. NK1 cells produced high IFN-γ levels when cocultured with IL-12 (Peritt et al., 1998Peritt D. Robertson S. Gri G. Showe L. Aste-Amezaga M. Trinchieri G. Differentiation of human NK cells into NK1 and NK2 subsets.J Immunol. 1998; 161: 5821-5824Google Scholar). This elevated IFN-γ production by non-stimulated NK cells may result from increased serum IL-12 levels in SSc (Sato et al., 2000Sato S. Hanakawa H. Hasegawa M. et al.Levels of interleukin 12, a cytokine of type 1 helper T cells, are elevated in sera from patients with systemic sclerosis. [In Process Citation].J Rheumatol. 2000; 27: 2838-2842Google Scholar). Our results suggest that both NK1 and NK2 responses occur in dcSSc. Thus, similar to Th1/Th2 responses, NK1/NK2 responses appear to be complicated, and activation of each response may vary according to various factors, including NK cell activation status and disease subsets. Although a possible link between NK cell abnormalities and SSc has long been suggested, research has focus on NK cell cytotoxic activity. This study reveals distinct abnormalities in various aspects of NK cell functions in SSc. Therefore, the analysis of NK cells from this viewpoint would provide an important clue for further understanding immunological abnormalities and their relevance to clinical manifestations in SSc. Blood samples were obtained from 46 Japanese patients with SSc (44 females and two males). All patients fulfilled the criteria by the American College of Rheumatology (Subcommittee, 1980Subcommittee for Scleroderma Criteria of the American Rheumatism Association Diagnostic and Therapeutic Criteria Committee: Preliminary criteria for the classification of systemic sclerosis (scleroderma).Arthritis Rheum. 1980; 23: 581-590Google Scholar). Localized scleroderma and morphea patients were excluded in this study. These SSc patients were grouped according to the classification system proposed byLeRoy et al., 1988LeRoy E.C. Black C. Fleischmajer R. et al.Scleroderma (systemic sclerosis): Classification, subsets and pathogenesis.J Rheumatol. 1988; 15: 202-205Google Scholar: 31 patients (all females) had lcSSc and 15 patients (13 females and two males) had dcSSc. According to theBarnett et al., 1988Barnett A.J. Miller M. Littlejohn G.O. The diagnosis and classification of scleroderma (systemic sclerosis).Postgrad Med J. 1988; 64: 121-125Google Scholar classification, SSc patients were classified into 13 patients with type 1 (sclerodactyly only), 25 with type 2 (sclerosis proximal to the metacarpophalangeal joints but excluding the trunk), and eight with type 3 (diffuse skin sclerosis including the trunk). All type 1 patients were lcSSc, whereas all type 3 patients belong to dcSSc. Type 2 patients consisted of 18 lcSSc and seven dcSSc patients. The age of patients with lcSSc and dcSSc (mean±SD) was 54±11 and 49±16 y old, respectively. The disease duration of patients with lcSSc and dcSSc was 10.4±8.2 and 6.1±5.3 y, respectively. None of the SSc patients was treated with steroids, D-penicillamine, or other immunosuppressive therapy. The antinuclear antibody (Ab) was determined by indirect immunofluorescence using HEp-2 cells as the substrate, and autoantibody specificities were further assessed by ELISA and immunoprecipitation. Anticentromere Ab was positive in 24 patients, anti-topoisomerase I Ab in ten, anti-U1RNP Ab infour, anti-mitochondria Ab in one, anti-RNA polymerases I and III Ab in two, and other unknown antinuclear Ab in two. The remaining three patients were negative for autoantibody. Twenty age- and sex-matched healthy Japanese individuals (19 females and one male aged 49±10 y) were used as normal controls. The protocol was approved by the Kanazawa University Graduate School of Medical Science, and informed consent was obtained from all patients. The study was conducted according to the Declaration of Helsinki Principles. Two-color analysis for NK cell percentage was performed using fluorescein isothiocyanate (FITC)-conjugated anti-CD16 (Beckman Coulter, Miami, Florida) and phycoerythrin (PE)-conjugated anti-CD56 (Exalpha Biologicals, Boston, Massachusetts) monoclonal Abs (mAb). For activation marker expression, two-color analysis was conducted using a combination of FITC-conjugated anti-CD56 (Exalpha Biologicals) and PE-conjugated mAb, and anti-CD25 (Beckman Coulter), anti-CD54 (Beckman Coulter), anti-CD62L (Beckman Coulter), anti-CD69 (Beckman Coulter), or anti-CD95 (Beckman Coulter). Fresh heparinized whole blood samples were collected and immediately placed on ice. Blood samples (50 μL) were stained at 4°C using predetermined saturating concentrations of the test mAb for 20 min as previously described (Sato et al., 2004Sato S. Fujimoto M. Hasegawa M. Takehara K. Altered blood B lymphocyte homeostasis in systemic sclerosis: Expanded naive B cells and diminished but activated memory B cells.Arthritis Rheum. 2004; 50: 1918-19127Google Scholar). Blood erythrocytes were lysed after staining using a Coulter Whole Blood Immuno-Lyse kit as detailed by the manufacturer (Beckman Coulter). Cells were analyzed on a FACScan flow cytometer (BD PharMingen, San Diego, California). Absolute numbers of cells were calculated from the relative frequency of NK cells and the absolute lymphocyte counts. PBMC were separated using Ficoll–Paque (Pharmacia Biotech, Uppsala, Sweden) after centrifugation. NK cells were isolated from PBMC using a magnetic cell sorting NK-cell-isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer's recommendations. Briefly, non-NK cells (T cells, B cells, monocytes, basophils, dendritic cells, platelets, and early erythroid cells) were magnetically labeled using a cocktail of biotin-conjugated mAbs to CD3, CD4, CD14, CD15, CD19, CD36, CD123 and glycophorin A in combination with magnetic bead-conjugated anti-biotin mAb. The magnetically labeled cells were depleted by retaining on a magnetic column. After isolation, >95% of cells were CD56+ by flow cytometric analysis. The patients analyzed for cytokine production by NK cells were different from those described above: 21 SSc patients (17 females and 4 males aged 57.6±10.1 y; 11 lcSSc patients and ten dcSSc patients) and nine healthy individuals (six females and three males aged 41.3±12.5 y) for cytokine production and 16 healthy individuals (12 females and four males aged 40.1±12.1 y) for granzyme B production were examined. None of these patients was treated with steroids, D-penicillamine, or other immunosuppressive therapy. Isolated NK cells (1 × 105) were suspended in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum and incubated in a 5% CO2 incubator with or without 25 ng per mL of PMA and 1 μg per mL of ionomycin at 37°C for 24 h. The culture supernatant was collected, and levels of cytokines and granzyme B were determined by ELISA specific for IFN-γ (R&D Systems, Minneapolis, Minnesota), IL-5 (R&D System), IL-6 (BD PharMingen), IL-10 (BD PharMingen), IL-13 (R&D System), and granzyme B (Euroclone Life Sciences, Pero, Italy), according to the manufacturer's instructions. The patients examined for analysis of cytotoxicity activity were different from those described above: 6 SSc patients (6 females aged 52.6±11.0 y; 4 lcSSc and 2 dcSSc patients) and 3 healthy individuals (3 females aged 35±7.2 y) were examined. None of these patients was treated with steroids, D-penicillamine, or other immunosuppressive therapy. To assess the cytotoxic activity of NK cells, K562, a human erythroleukemic cell line, was used as the target cell. Cultures of K562 cells were split to a concentration of 3 × 105 cells per mL 3 d prior to testing, to ensure that cells were in the log phase. A cytotoxicity assay was performed using a cell-mediated cytotoxicity kit (Molecular Probes, Eugene, Oregon), according to the manufacturer's instructions. The target cells were stained with DiOC18 (Molecular Probes) for 20 min at 37°C. The stained target cells (1 × 106 cells per mL) were resuspended in culture medium and then mixed with isolated NK cells to yield the desired effector:target (E:T) ratios of 40:1, 20:1, 10:1, and 5:1. A counterstaining solution containing propidium iodide, which detects dead cells, was added to the mixture and incubated at 37°C for 2 h. Two-color flow cytometric analysis was performed to assess NK lytic activity. Percent lysis was calculated by the frequency of dead target cells divided by the total target cells. Spontaneous cell lysis as determined in the absence of effector cells was subtracted from total lysis to calculate the amount of specific lysis. Statistical analysis was performed using the Mann–Whitney U test to determine the level of significance of differences between the sample means, and Bonferroni's test was used for multiple comparisons. A p value less than 0.05 considered statistically significant." @default.
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• W2010043145 title "Abnormal Natural Killer Cell Function in Systemic Sclerosis: Altered Cytokine Production and Defective Killing Activity" @default.
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• W2010043145 cites W1966108522 @default.
• W2010043145 cites W1974717741 @default.
• W2010043145 cites W1994145503 @default.
• W2010043145 cites W1995485547 @default.
• W2010043145 cites W1996665823 @default.
• W2010043145 cites W2000939080 @default.
• W2010043145 cites W2001320879 @default.
• W2010043145 cites W2002269996 @default.
• W2010043145 cites W2006448899 @default.
• W2010043145 cites W2010789559 @default.
• W2010043145 cites W2011036671 @default.
• W2010043145 cites W2022270470 @default.
• W2010043145 cites W2024852458 @default.
• W2010043145 cites W2027009572 @default.
• W2010043145 cites W2027949435 @default.
• W2010043145 cites W2028921539 @default.
• W2010043145 cites W2052232839 @default.
• W2010043145 cites W2056149800 @default.
• W2010043145 cites W2060120946 @default.
• W2010043145 cites W2061422374 @default.
• W2010043145 cites W2079828804 @default.
• W2010043145 cites W2085339199 @default.
• W2010043145 cites W2098795862 @default.
• W2010043145 cites W2099019634 @default.
• W2010043145 cites W2106054874 @default.
• W2010043145 cites W2109534516 @default.
• W2010043145 cites W2132503390 @default.
• W2010043145 cites W2145076333 @default.
• W2010043145 cites W4313343422 @default.
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