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Accumulating evidence indicates that apoptosis plays an important role in immunologic tolerance by halting the unwanted expansion of activated T and B cell clones beyond the course of inflammation (1, 2). The Fas (APO-1, CD95) is a cell-surface receptor, which mediates apoptosis when engaged by its ligand (FasL) via its cytoplasmic death domain (3, 4). Therefore, Fas-dependent apoptosis may be involved in the pathogenesis of autoimmune diseases (1, 5). In addition, Fas-mediated apoptosis could be modulated by Bcl-2 family members and p53 phosphoprotein (6, 7).
Adult-onset Still's disease (AOSD) is a systemic inflammatory disorder characterized by high fever, evanescent rash, arthritis, lymphadenopathy, and hepatosplenomegaly (8, 9). Our previous study demonstrated a predominance of Th1 cytokine in peripheral blood and pathologic tissues of patients with active AOSD (10). Zhang et al demonstrated that Th1 cells undergo activation-induced cell death via the Fas/FasL pathway more readily than Th2 cells (11). Furthermore, we and other investigators have shown that patients with active AOSD often have high levels of serum interleukin-18 (IL-18) (12, 13). Dao et al indicated that IL-18 might play a potential role in immunoregulation by enhancing FasL-mediated cytotoxicity of murine Th1 cells (14). Marked elevation of serum IL-18 levels and Th1 predominance in AOSD lead us to hypothesize that apoptosis may play a role in the pathogenesis of this disease.
The goal of this study was to determine spontaneous and IL-18–stimulated apoptosis of peripheral blood lymphocytes (PBLs) from patients with active untreated AOSD. We chose patients with systemic lupus erythematosus (SLE) as disease controls because increased IL-18 and the occurrence of apoptosis have been documented in SLE (15–17), and because of the similarity of some manifestations between SLE and AOSD. The changes in apoptotic PBLs and serum IL-18 levels during longitudinal followup of AOSD patients were also evaluated. To explore the role of IL-18 in the pathogenesis of apoptosis, we further analyzed the change in transcripts of apoptosis-regulating genes in recombinant human IL-18 (rHuIL-18)–treated peripheral blood mononuclear cells (PBMCs) from patients with AOSD and patients with SLE.
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- PATIENTS AND METHODS
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This study is the first attempt to investigate in vitro apoptosis, both spontaneous and IL-18–stimulated, of PBLs from patients with active untreated AOSD and patients with active untreated SLE. The apoptotic assay used in this study, by combining usage of FITC-labeled annexin V and dye exclusion of PI, discriminates intact cells (FITC−/PI−), early apoptotic cells (FITC+/PI−), and late apoptotic cells mixed with necrotic cells (FITC+/PI+) (22). To avoid the contamination of necrotic cells, only early apoptotic cells were gated in accordance with previous studies in the assessment of apoptotic lymphocytes from SLE patients (26). The results of apoptotic assay, which was used in this study, correlate with the results of DNA-flow cytometry and DNA electrophoresis (27, 28). Our data showed that accelerated in vitro apoptosis, both spontaneous and IL-18–stimulated, occurred in PBLs from patients with active AOSD. This observation was supported by RQ-PCR analysis showing an increase in transcript levels of caspase 3, which is thought to be responsible for the actual demolition of the cell during apoptosis (29). Our results were consistent with recent reports demonstrating an increase in the mean apoptotic index of PBLs and in the activation-induced apoptotic PBMCs from patients with juvenile-onset Still's disease (30, 31). In addition, we demonstrated that the percentages of peripheral blood apoptotic lymphocytes significantly correlated with the disease activity scores and declined markedly after effective therapy in AOSD patients. Our results suggest that an increase in apoptosis may play an important role in the elimination of autoreactive lymphocytes in AOSD.
The notion of a central role for IL-18 in the pathogenesis of AOSD is supported by our and other investigators' reports (12, 13). To examine the effects of IL-18 on the in vitro apoptosis in AOSD patients, we investigated the frequency of apoptotic lymphocytes after 24-hour incubation with rHuIL-18. Because a growth factor deficiency may contribute to apoptosis of autoreactive lymphocytes in vitro and apoptotic cell preparations may contain increasing numbers of secondary necrotic cells during prolonged incubation, we used higher concentrations of IL-18 (100 ng/ml) in the shorter culture period (24-hour time point) in accordance with previous studies (23). Our data showed that increased percentages of IL-18–stimulated apoptotic lymphocytes correlated positively with disease activity scores and serum IL-18 levels in AOSD patients. The effect of IL-18 on the apoptosis is further supported by our results, which showed the up-regulation of mRNA expression of caspase 3 on IL-18–treated PBMCs from AOSD patients in a dose-dependent manner (Figure 4), and by a recent report demonstrating that IL-18 up-regulated caspase 3 expression in human cardiac endothelial cells (23). Our results suggest that IL-18 plays an important role in the pathogenesis of apoptosis associated with AOSD. There are conflicting findings concerning the role of IL-18 in the pathogenesis of inflammation and apoptosis in AOSD. However, accumulating evidence supports the concept of a dual role of interferon-γ that can be induced by IL-18 (32) in inflammation and in the regulatory pathways including activation of apoptosis (33). Therefore, we speculated that IL-18 might act as a proinflammatory and proapoptotic cytokine, and enhance apoptosis of autoreactive lymphocytes in patients with AOSD. Because the local concentrations of IL-18 in tissues are not known, it is impossible to indicate whether the elevated serum concentrations observed are sufficient to cause the apoptosis found in this study.
Similar to AOSD patients, augmented apoptosis in IL-18–stimulated lymphocytes and up-regulated caspase 3 transcripts on IL-18–treated PBMCs were observed in our SLE patients. Amerio et al also demonstrated that increased levels of serum IL-18 correlated positively with disease activity in SLE (34). Our results suggest that accelerated in vitro apoptosis and up-regulation of caspase 3 transcripts by IL-18 may be a general characteristic of systemic inflammation. It may be speculated that IL-18 priming can render PBLs susceptible to activation-induced cell death in AOSD and SLE. However, this hypothesis needs to be confirmed by future studies. Although other proapoptotic cytokines were not investigated in the present study, tumor necrosis factor α and IL-10 have been shown to promote lymphocyte apoptosis through the Fas-dependent pathway in patients with active SLE (35, 36). To test whether the enhanced in vitro apoptosis is unique to IL-18 in systemic inflammatory diseases, further investigation of other apoptosis inducers are needed.
It remains unclear whether increased apoptosis is a contributing factor to the development of AOSD, or is just a secondary phenomenon common to systemic inflammation. Several reports have shown that mutations in Fas and FasL are associated with peripheral lymphoid expansion, indicating that defective apoptosis of autoreactive lymphocytes may be involved in the pathogenesis of SLE and other autoimmune diseases (1, 37, 38). We also demonstrated that the increased frequency of apoptotic lymphocytes correlated positively with both disease activity and levels of proinflammatory cytokine IL-18 in AOSD patients and SLE patients. Accumulating evidence showed that activation-induced apoptosis plays an important role in deletion of autoreactive T cells (39), and apoptotic cells can inhibit the production of proinflammatory cytokines and induce the production of immunosuppressive cytokines from PBMCs and monocytes (40, 41). Our results indicated that enhanced apoptosis of lymphocytes might be a compensatory mechanism designed to eliminate excess autoreactive cells in AOSD and SLE.
To clarify the role of IL-18 in the accelerated apoptosis of PBLs from patients with active AOSD, we used RQ-PCR analysis to investigate the mRNA expression of apoptosis-regulating genes on rHuIL-18–stimulated PBMCs. The 45-kd membrane protein Fas is a major apoptogenic cell-surface receptor, which mediates apoptosis when engaged by its ligand (FasL) via cytoplasmic death domain (3, 4). The p53 gene is a potent transcriptional factor that promotes apoptosis in response to a variety of stresses (7), whereas Bcl-2 belongs to apoptosis-inhibitory members that can inhibit the release of mitochondrial factors (6). Our results demonstrated that the transcript levels of FasL and p53 were up-regulated on PBMCs treated with IL-18 in a dose-dependent manner for AOSD patients and SLE patients (Figure 5). Our observation is similar to recent reports showing that IL-18 can up-regulate FasL in human cardiac endothelial cells (23), and FasL as well as p53 in myelomonocytic KG-1 cells (42). Based upon these findings, we postulate that IL-18 may play a role in the pathogenesis of apoptosis through up-regulation of FasL and p53 transcripts in AOSD and SLE. However, further investigation to demonstrate the real involvement of FasL and p53 in the pathogenesis of apoptosis is needed.
During apoptosis, surface-exposed phosphatidylserine (PS) offers a recognition signal for clearance of apoptotic cells by macrophages and other scavenger cells (43). This overexpression of surface PS is supported by our finding of a considerable increase in PBLs with positive FITC-labeled annexin V staining in patients with active AOSD and patients with SLE. Increased numbers of apoptotic lymphocytes in patients with active AOSD may occur as a result of increased apoptosis of circulating cells, or because of defective clearance. Although there are no data concerning the clearance of apoptotic cells in AOSD compared with SLE, impaired clearance of apoptotic cells is already known to be a hallmark of SLE (44, 45). Increased apoptosis of lymphocytes together with impaired engulfment of early apoptotic cells may cause secondary necrosis and release of intracellular autoantigens, and then trigger autoimmune reactions in SLE (46, 47). Moreover, the presence of apoptotic cells may shift the helper T cells' response toward Th2 cells that characterize the immune response in SLE (48), while a predominance of Th1 cells was observed in AOSD (10). Additionally, elevation of C-reactive protein, which is a common finding in AOSD patients, enhanced opsonization and phagocytosis of apoptotic cells by macrophage (49) and hyperferritinemia, which is a hallmark of AOSD patients (50), may be a marker of excessive macrophage activation (51). Our data suggest that the increased apoptotic lymphocytes in peripheral blood from patients with active AOSD are not due to defective clearance. This hypothesis may be supported by the absence of antinuclear antibodies (18) and the self-limited course in a majority of AOSD patients (12, 52).
In conclusion, accelerated in vitro apoptosis, both spontaneous and IL-18–stimulated, was found in patients with active AOSD and patients with SLE, and may be a common characteristic of systemic inflammatory diseases. IL-18 may play an important role in apoptosis by augmenting FasL and p53 expression on PBMCs from AOSD patients and SLE patients. Although the present study does not directly address the causative role of IL-18 and apoptosis in the pathogenesis of AOSD, our work may provide a new insight into the effects of IL-18 on the regulation of apoptosis-related genes in this disease.