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- MATERIALS AND METHODS
Adult-onset Still's disease (AOSD) is a multisystem inflammatory disorder of unknown etiology and pathogenesis (1, 2). A major pathologic finding of Still's rash in patients with AOSD is perivascular inflammation of the superficial dermis, with prominent lymphocytes and histiocytes associated with dermal edema (3). Synovial biopsy specimens also show moderate vascular engorgement and moderate infiltrates with mononuclear cells in AOSD patients with active synovitis (3). In inflammatory and immune responses, adhesion molecules play a major role in the recruitment of leukocytes and immunocompetent cells to the inflamed tissues (4–6). Intercellular adhesion molecule 1 (ICAM-1; CD54), which is an important adhesion molecule in cell–cell interactions and leukocyte extravasation at inflammatory sites, exerts its influence by binding to 2 integrins, CD11a/CD18 (leukocyte function–associated antigen 1) and CD11b/CD18 (7, 8). It is expressed on various cell types in many tissues and is considered to play a vital role in some autoimmune diseases (5, 6, 9).
A soluble form of ICAM-1 (sICAM-1) that is found in the circulation as well as in body fluids is thought to be cleaved from the surface of ICAM-1–expressing cells (10, 11). Moreover, sICAM-1 has been recognized as a marker for the presence of inflammatory mediators in vivo, such as cytokines (12). High levels of sICAM-1 have been reported in a variety of inflammatory diseases (13–15). However, most of the ICAM-1 is produced near sites of inflammation. Quantification of ICAM-1 gene expression at inflammatory sites would provide a better reflection of in vivo ICAM-1. To our knowledge, there has been no report on the serum levels of sICAM-1 and the levels of ICAM-1 gene expression in biopsy specimens obtained from the skin lesions and synovial membrane of patients with AOSD.
The findings of previous reports, as well as those of our previous study, showed that serum interleukin-18 (IL-18) levels were markedly elevated and significantly correlated with disease activity in AOSD, indicating that IL-18 is likely involved in the pathogenesis of this disease (16–18). ICAM-1 expression can be induced in vitro and in vivo by IL-18 (19–21), suggesting that it may be an important mechanism for regulating the inflammatory response. A recent study demonstrated that IL-18 up-regulated the expression of ICAM-1 on rheumatoid arthritis (RA) synovial fibroblasts (22). However, the influence of IL-18 on ICAM-1 expression in peripheral blood mononuclear cells (PBMCs) from patients with AOSD has not yet been reported.
The aim of this study was to determine the serum levels of sICAM-1 and to investigate their associations with clinical features in 50 patients with active AOSD. To clarify the in vivo involvement of ICAM-1 in these patients, we examined the levels of ICAM-1 messenger RNA (mRNA) expression in biopsy specimens obtained from the skin lesions and synovial membranes of 8 patients with active untreated AOSD. We chose patients with RA as disease controls because several studies have documented ICAM-1 involvement in this disease (14, 23, 24). In addition, the articular manifestations of RA and chronic articular AOSD are similar. Furthermore, in patients with AOSD we extended our previous study regarding serum levels of IL-18 (18) and investigated whether a correlation exists between levels of IL-18 and ICAM-1.
- Top of page
- MATERIALS AND METHODS
ICAM-1 (CD54) is a cytokine-inducible member of the immunoglobulin superfamily and plays a central role in the cell-to-cell–mediated immune response (4, 7, 8). Circulating sICAM-1 is thought to be cleaved from the surface of ICAM-1–expressing cells (10, 11). The pathophysiologic role of sICAM-1 has not been well elucidated. It has been suggested that sICAM-1 might regulate the activation of leukocytes in the interaction with endothelium, might compete in ICAM-1–mediated cell-to-cell adhesion, or might simply reflect activation of endothelial cells and immunocompetent cells (29–31). Previous studies have shown high sICAM-1 levels in serum from patients with autoimmune diseases (12–15). This is the first study to demonstrate that serum sICAM-1 levels were significantly higher in patients with active AOSD than in healthy controls (Figure 1). Furthermore, serum levels of sICAM in patients with AOSD correlated well with the clinical activity score (r = 0.565, P < 0.001) and the serum ferritin value (r = 0.462, P < 0.005), which is considered to be a marker for adult Still's disease activity (32). Our results indicate that sICAM-1 may play an important role in the pathogenesis of the acute inflammatory process in AOSD, although no significant correlation between sICAM-1 levels and the peripheral blood leukocyte counts were observed. This suggests that elevated levels of sICAM-1 in sera from patients with AOSD simply did not reflect raised levels of leukocytes.
The disease course and prognosis of AOSD may vary considerably. In contrast to the distribution of patterns of disease course reported by Cush et al (33), our study revealed that the polycyclic systemic disease course was the most common (46%) in patients with AOSD, followed by the monocyclic systemic course (28%); only 26% of patients had the chronic articular course. In this study, we investigated whether the pretreatment levels of serum sICAM-1 differed among patients with distinct courses of this disease. We found that serum levels of sICAM-1 in the active stage were higher in patients who had the polycyclic systemic pattern compared with those who had the monocyclic systemic pattern or the chronic articular pattern (Figure 2). This suggests that sICAM-1 levels may be related to the intensity of the systemic inflammatory response in AOSD.
Numerous studies have investigated cellular adhesion molecules in sera, synovial fluid, and synovial membranes in patients with RA and found that ICAM-1 participates in mechanisms involved in synovial inflammation (6, 14, 23, 34). The clinical benefits seen in patients with RA treated with monoclonal antibody to ICAM-1 (24, 35) may support this hypothesis. In the present study, serum levels of sICAM-1 were significantly higher in patients with active RA than in healthy controls (Figure 1). However, serum levels of sICAM-1 were significantly higher in patients with active untreated AOSD than in patients with active RA (Figure 1). The results of assessment of sICAM-1 levels in the sera of our patients with AOSD or RA were similar to those reported for patients with juvenile rheumatoid arthritis, in whom sICAM-1 levels were significantly higher in those with systemic-onset disease than in those with polyarticular-onset disease (13). Another study also showed the serum levels of sICAM-1 were significantly higher in RA patients with systemic vasculitis than in patients with uncomplicated disease (36). The higher levels of serum sICAM-1 in patients with active AOSD relative to patients with active RA may be an indicator of the higher systemic involvement and may account for the associated intense IL-18 release observed in the former group (16, 18). However, bias may have been introduced in this study, because corticosteroid treatment used in patients with RA may suppress expression of ICAM-1 in human monocytic cell lines (37).
The notion of a central role for IL-18 in the pathogenesis of AOSD is supported by previous reports and our previous results, which demonstrated that serum IL-18 levels were markedly elevated and significantly correlated with disease severity in AOSD (16–18). IL-18 plays pivotal roles in the modulation of inflammation through stimulation of ICAM-1 expression in a wide variety of tissues (19, 22). In this study, we showed that serum sICAM-1 levels correlated well with serum IL-18 levels (r = 0.443, P < 0.005) in patients with AOSD. Furthermore, we demonstrated that IL-18 up-regulated ICAM-1 mRNA expression by cultured PBMCs from patients with AOSD in a dose-dependent manner (Figure 3). This observation is consistent with the results of previous studies showing up-regulated expression of ICAM-1 by IL-18 in the human myelomonocytic cell line KG-1 (20) and in freshly isolated human monocytes from healthy volunteers (21). Our data suggest that a crucial pathogenic step in the progression of inflammation in AOSD may be related to IL-18–up-regulated expression of ICAM-1, which mediates recruitment of immunocompetent cells to inflammatory sites. The mechanism of the effect on ICAM-1 by IL-18 remains to be elucidated in AOSD.
ICAM-1 is expressed on various cell types, including keratinocytes, fibroblasts, vascular endothelial cells, leukocytes, and synoviocytes (38, 39). The up-regulated expression of ICAM-1 on target cells is observed in a variety of diseases, including RA and dermatitis (34, 40–42). To determine the in vivo involvement of ICAM-1 in patients with AOSD, we investigated the expression of ICAM-1 mRNA in biopsy specimens obtained from 8 patients with Still's rash or synovitis with active untreated AOSD using real-time quantitative PCR technique. This is the first study to demonstrate that ICAM-1 mRNA expression was significantly higher in the biopsy specimens obtained from AOSD patients with Still's rash than in normal skin specimens (Figure 4A). Our results were similar to those of previous studies, which showed increased ICAM-1 expression on basal keratinocytes in biopsy specimens from lesional skin of patients with subacute cutaneous lupus erythematosus (42). We demonstrated that the levels of ICAM-1 transcripts were significantly higher in synovial membranes from patients with AOSD than those from OA controls (Figure 4B), which was consistent with previous findings that patients with RA had higher ICAM-1 mRNA expression on synovial membranes even in the early stages of disease compared with controls (34, 40, 41). Our previous results also showed significantly higher levels of IL-18 mRNA expression in the biopsy tissues obtained from patients with Still's rash or synovitis with AOSD compared with those in normal skin and OA controls (18). A recent study showed that IL-18 significantly enhanced expression of ICAM-1 on vascular endothelial cells and RA synovial fibroblasts (22). Based on the above findings, we speculate that raised expression of ICAM-1 in the pathologic tissues from patients with active AOSD may be related to local release of IL-18. However, this hypothesis needs to be confirmed by further investigations. Although corticosteroid treatment used in patients with RA may suppress expression of ICAM-1 (43), the levels of ICAM-1 mRNA expression in synovial membranes were still significantly lower in AOSD than in RA (Figure 4B). A previous study has shown that faintly positive ICAM-1 molecules were expressed in synovial tissue of patients with RA at one month from onset, while intensive expression of ICAM-1 appeared 3 months after onset (44). The results of ICAM-1 mRNA expression in synovial membranes of our patients with AOSD and RA may account for the difference in persistence of synovitis between both diseases. A prospective study with a large number of patients including those with early untreated RA should be conducted to support these findings.
AOSD is a multisystem inflammatory disorder characterized by heterogeneous clinical manifestations (1–3). Although the functional role of sICAM-1 remains unclear, Rothlein et al suggested that the presence of circulating molecule reflects inflammation and tissue damage (10). We constructed a logistic regression model to evaluate the effects of serum sICAM-1 on the presence of clinical features in patients with AOSD. We found that the serum sICAM-1 level was a significant predictor for the occurrence of hepatic dysfunction (P < 0.05). Previous studies showed that normal human hepatocytes express low or undetectable levels of ICAM-1, as determined by immunohistochemistry (45, 46). Up-regulation of ICAM-1 expression on human hepatocytes has been reported in autoimmune liver diseases (45) and induction of ICAM-1 gene expression and protein secretion has been demonstrated in cultured hepatocytes stimulated with IL-18, tumor necrosis factor α, and interferon-γ (46). Thomson et al reported that serum sICAM-1 levels were elevated in patients with autoimmune liver diseases and suggested that hepatocytes may be an important source of sICAM-1 in inflammatory liver disease (47). In this study, we showed that serum sICAM-1 levels correlated well with hepatic enzyme (ALT) values (r = 0.466, P < 0.005) and serum IL-18 levels (r = 0.443, P < 0.005) in patients with AOSD. We speculate that the raised levels of serum sICAM-1 in these patients with hepatic dysfunction may be partly the result of IL-18–stimulated synthesis followed by shedding of ICAM-1 by hepatocytes. Further studies should include an investigation of the effects of IL-18 on up-regulation of ICAM-1 gene expression directly in primary hepatocyte cultures.
DIC is a rare but fatal complication of AOSD in the absence of infection or salicylates therapy (48). Several predisposing factors of DIC in AOSD have been proposed and include infection, medications, hepatic dysfunction, altered hemostasis, and vasculitis (48). A recent study showed that serum sICAM-1 levels were more elevated in patients after severe trauma complicated by DIC than in those without DIC, and were correlated with maximum DIC scores (49). Our study included 5 patients with AOSD complicated by DIC in the absence of inciting infection or previous medications. All patients had thrombocytopenia, elevated hepatic enzyme levels, hyperferritinemia, and an unexpectedly low ESR. Among them, one patient had pancytopenia, marked hyperferritinemia (91,600 ng/ml), hemophagocytosis in bone marrow, and elevated levels of serum IL-18 (1,500 pg/ml). Data for this patient were consistent with a recent report showing the presence of macrophage activation syndrome and increased IL-18 expression in bone marrow from a patient with Still's disease (50). In this study, we found that the serum sICAM-1 level was a significant predictor of occurrence of DIC (OR 1.013, 95% CI 1.002–1.025; P < 0.05). In AOSD patients with DIC, elevated sICAM-1 levels may be an indicator of endothelial cell activation.
In conclusion, our results showed significantly higher levels of sICAM-1 in sera and enhanced expression of ICAM-1 mRNA in pathologic tissues from patients with active AOSD. The levels of sICAM-1 convey additional information about the clinical features of AOSD. We also demonstrated that IL-18–up-regulated gene expression of ICAM-1 may mediate the recruitment of immunocompetent cells to the inflammatory sites. The investigation of ICAM-1 in AOSD not only improves our understanding of its immunopathogenesis but also offers a foundation for the development of a novel therapeutic strategy.