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Keywords:

  • Intercellular adhesion molecule-1 (ICAM-1);
  • Interleukin-18 (IL-18);
  • Adult-onset Still's disease (AOSD);
  • Rheumatoid arthritis (RA);
  • Real-time PCR

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Objective

To investigate the association of intercellular adhesion molecule 1 (ICAM-1) with clinical manifestations and interleukin-18 (IL-18) levels in patients with active untreated adult-onset Still's disease (AOSD).

Methods

We determined serum soluble ICAM-1 (sICAM-1) levels by enzyme-linked immunosorbent assay in 50 patients with active untreated AOSD, 20 patients with active rheumatoid arthritis (RA), and 20 healthy controls. The levels of ICAM-1 messenger RNA expression in IL-18–stimulated peripheral blood mononuclear cells (PBMCs) and in biopsy specimens obtained from AOSD patients with Still's rash or synovitis were investigated using real-time quantitative polymerase chain reaction.

Results

Significantly higher serum levels of sICAM-1 were observed in patients with active untreated AOSD compared with those with active RA and healthy controls. Serum sICAM-1 levels were significantly correlated with the clinical activity score (r = 0.565, P < 0.001), ferritin values (r = 0.462, P < 0.005), and IL-18 levels (r = 0.462, P < 0.005) in patients with AOSD. The serum sICAM-1 level was identified as a predictor of hepatic dysfunction (odds ratio [OR] 1.016, P = 0.011) and disseminated intravascular coagulation (DIC) (OR 1.013, P = 0.023). Up-regulation of ICAM-1 gene expression was demonstrated in IL-18–stimulated PBMCs from patients with AOSD. Increased levels of ICAM-1 transcripts were observed in the biopsy specimens obtained from AOSD patients with Still's rash or synovitis compared with healthy skin and patients with osteoarthritis.

Conclusion

The serum sICAM-1 level may be used as a clinical marker to assess disease activity and may predict the occurrence of hepatic dysfunction and DIC in AOSD. IL-18–up-regulated gene expression of ICAM-1 may contribute to the inflammatory response in AOSD.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

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.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Patients and healthy controls.

The study group comprised 50 patients (33 women and 17 men, mean ± SEM age 35.2 ± 14.7 years) all of whom fulfilled the criteria for AOSD described by Yamaguchi et al (25). Patients with infection, malignancy, or other rheumatic diseases were excluded. According to the criteria described by Pouchot et al in 1991 (3), the clinical activity score (range 0–12) for each patient with AOSD was assessed by assigning and adding 1 point for each of the following manifestations: fever, evanescent rash, sore throat, arthritis, myalgias, pleuritis, pericarditis, pneumonitis, lymphadenopathy, hepatomegaly or abnormal liver function test results, abdominal pain, and leukocytosis ≥15,000/mm3. After diagnosis of AOSD and initial assessment of ICAM-1 and IL-18 levels, all patients were treated with nonsteroidal antiinflammatory drugs (NSAIDs) and corticosteroids with or without disease-modifying antirheumatic drugs (DMARDs) including hydroxychloroquine, sulfasalazine, and/or methotrexate. The mean ± SEM duration of followup was 65.4 ± 39.1 months (range 24–178 months). Three patterns of disease course were determined and defined as follows: monocyclic systemic (only 1 episode of systemic manifestations, followed by complete remission within 1 year after disease onset); polycyclic systemic (>1 episode of systemic manifestations, followed by partial or complete remission after onset of the initial or the subsequent attack); and chronic articular (persistent arthritis involving at least 1 joint area, lasting >6 months). Twenty age- and sex-matched healthy volunteers were used as controls (13 women and 7 men, mean ± SEM age 33.5 ± 7.2 years). Twenty patients with active classic RA (18 women and 2 men, mean ± SEM age 45.0 ± 7.6 years) were included in this study as a disease control if they fulfilled the American College of Rheumatology (formerly the American Rheumatism Association) criteria for RA (26), had a disease duration <1 year, and had active arthritis, which was defined as having >6 swollen and 6 tender joints for at least 3 months. All patients with RA received low dosages of oral prednisolone (5–10 mg/day) and NSAIDs, but no DMARDs for at least 1 month before enrolling in this study. This study protocol was approved by the Ethics Committee of Clinical Research, Taichung Veterans General Hospital, and informed consent was obtained from each participant.

Determination of serum levels of sICAM-1, IL-18, and ferritin.

Serum levels of sICAM-1 were determined using an enzyme-linked immunosorbent assay (ELISA) according to the supplier's instructions (Bender MedSystems, Vienna, Austria). In our previous study (18), serum IL-18 levels were determined using an ELISA (Bender MedSystems), and serum ferritin levels were measured by nephelometry (Dade Behring, Newark, NJ).

RNA extraction and TaqMan real-time quantitative polymerase chain reaction (PCR).

Skin biopsy specimens were obtained from lesions of 8 patients with active untreated AOSD with Still's rash and from normal skin of 4 healthy individuals undergoing plastic surgery. Synovial membrane samples were obtained from 6 untreated patients with AOSD with active synovitis, 6 patients with active RA undergoing therapy with NSAIDs and low-dose prednisolone, and 4 patients with osteoarthritis who had been receiving no medication for at least 1 month before enrolling in this study. Total cellular RNA was isolated from biopsy tissues by the guanidinium isothiocyanate method (27). RNA was quantitated by spectrophotometry at 260 nm. A 2.5-μg aliquot was reverse transcribed with 200 units of Moloney murine leukemia virus reverse transcriptase according to standard procedures (Boehringer Mannheim, Mannheim, Germany).

The levels of ICAM-1 mRNA expression were quantified by TaqMan real-time quantitative PCR (28) according to the manufacturer's instructions (Applied Biosystems, Foster City, CA). The TaqMan probe consists of an oligonucleotide with a 5′FAM reporter dye and a 3′nonfluorescent quencher dye. PCR was performed in a total volume of 50.0 μl containing 100 ng of complementary DNA (cDNA), 0.5 μl Taq DNA polymerase (5 units/μl), 5.0 μl TaqMan probe, 5.0 μl (each) oligonucleotide primers, 25.0 μl PCR buffer, and 9.5 μl RNase-free water. PCR conditions were incubation for 2 minutes at 50°C, activation of Taq DNA polymerase for 10 minutes at 95°C, and then 40 cycles at 95°C for 15 seconds, followed by 58°C for 1.5 minutes. To standardize ICAM-1 mRNA concentrations, transcript levels of the housekeeping gene β-actin were determined in parallel for each sample. Final results were expressed as the copy ratio of ICAM-1/β-actin transcripts.

TaqMan real-time quantitative PCR of recombinant human IL-18 (rhIL-18)–-stimulated PBMCs from patients with active AOSD.

PBMCs were immediately isolated using Ficoll-Hypaque (Amersham Biosciences, Uppsala, Sweden) density-gradient centrifugation. After washing 3 times with phosphate buffered saline, cells were resuspended in medium (RPMI 1640 + 2 ng glutamine + 60 μg/ml gentamicin + 5% human pool serum) in a final concentration of 1 × 106 cells/well. Culture conditions included medium alone; recombinant human IL-18 (rhIL-18) (Bender MedSystems) 10 ng/ml; and rhIL-18 100 ng/ml. The culture was then incubated at 37°C in 5% CO2 for 24 hours. TaqMan real-time quantitative PCR of cDNA from rhIL-18–treated PBMCs for quantification of ICAM-1 mRNA expression was performed by the previously described method.

Statistical analysis.

Except where indicated otherwise, values are the mean ± SEM. Differences among groups were determined by the Kruskal-Wallis test for nonparametric analysis of variance. The correlation between serum sICAM-1 levels and clinical activity scores were determined by linear regression, and the correlation between laboratory parameters and clinical activity scores were determined using the nonparametric Spearman's test. For comparison of the levels of ICAM-1 mRNA expression on PBMCs, which were treated with different doses of rhIL-18, Wilcoxon's signed rank test was used. Moreover, we used logistic regression modeling to evaluate the effects of serum sICAM-1 on the occurrence of clinical features in patients with AOSD. P values less than 0.05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Clinical features and laboratory findings in active untreated AOSD.

All patients with AOSD had high fever (>39°C). The typical evanescent rash was present in 44 patients (88%), and synovitis was seen in 36 patients (72%). Forty-seven patients (94%) had hyperferritinemia (>300 μg/liter). Approximately two-thirds of patients had neutrophilic leukocytosis. Hepatic dysfunction (alanine aminotransferase [ALT] ≥ 40 IU/liter) was present in 13 patients (26%). In 5 patients (10%), AOSD was associated with disseminated intravascular coagulation (DIC), which was defined by a prothrombin time of >1.3 times control, a partial thromboplastin time of >1.3 times control, thrombocytopenia (<50,000/mm3), and positive fibrin degradation product. Among patients with AOSD complicated by DIC, all had easy bruising and active polyarthritis, and 3 patients had hepatosplenomegaly and clinical jaundice. Hepatic dysfunction, hypofibrinogenemia (<400 mg/dl), a low erythrocyte sedimentation rate (ESR; <20 mm/hour), and elevated levels of serum IL-18 (mean value 787.7 pg/ml) were observed in all patients with AOSD complicated by DIC. Results of a bone marrow biopsy performed in one patient who had pancytopenia showed a mild degree of hemophagocytosis.

Serum levels of sICAM-1 in patients with AOSD and patients with RA.

As shown in Figure 1, serum levels of sICAM-1 were significantly increased in patients with active untreated AOSD compared with those in controls (mean ± SEM 92.27 ± 8.85 pg/ml versus 17.06 ± 1.08 pg/ml; P < 0.01), and were increased in patients with active RA compared with healthy controls (55.79 ± 12.57 pg/ml versus 17.06 ± 1.08 pg/ml; P < 0.01). Patients with active AOSD had significantly higher serum levels of sICAM-1 than patients with active RA (P < 0.01).

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Figure 1. Serum levels of soluble intercellular adhesion molecule 1 (sICAM-1) in 50 patients with active untreated adult-onset Still's disease (AOSD), 20 patients with active rheumatoid arthritis (RA), and 20 healthy controls (HC). Individual values are plotted, with the mean ± SEM shown for each group. P values are indicated for comparison of the 3 groups.

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Correlation of serum sICAM-1 levels with clinical activity score and laboratory parameters in patients with AOSD.

Serum sICAM-1 levels correlated well with the clinical activity score (r = 0.510, P < 0.001), serum levels of ferritin (r = 0.462, P < 0.005), and hepatic enzyme (ALT) values (r = 0.466, P < 0.005) in patients with AOSD. Using previous data concerning serum IL-18 levels (18), we found that sICAM-1 levels correlated well with IL-18 levels (r = 0.443, P < 0.005) in patients with AOSD. However, there was no significant correlation between serum sICAM-1 levels and peripheral blood leukocyte counts in these patients.

Serum levels of sICAM-1 in 3 patterns of AOSD patients grouped according to disease course.

A followup analysis of 50 patients with AOSD was performed over a mean ± SEM period of 65.4 ± 39.1 months (range 24–178 months). Twenty-three patients (46%) had polycyclic systemic pattern, 14 patients (28%) had monocyclic systemic pattern, and the remaining 13 patients (26%) had the chronic articular pattern. Significantly higher pretreatment levels of serum sICAM-1 in the active stage were demonstrated in patients with AOSD who had the polycyclic systemic pattern compared with those who had the monocyclic systemic pattern or the chronic articular pattern (mean ± SEM 116.08 ± 15.02 pg/ml versus 74.18 ± 16.01 pg/ml or 69.65 ± 6.49 pg/ml; P < 0.05 for both) (Figure 2). No significant difference in pretreatment levels of serum sICAM-1 was demonstrated between patients with AOSD who had the monocyclic systemic pattern and those who had the chronic articular pattern.

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Figure 2. Serum levels of soluble intercellular adhesion molecule 1 (sICAM-1) in patients with active untreated adult-onset Still's disease grouped according to the pattern of disease course. Individual values are plotted, with the mean ± SEM shown for each group. P values are indicated for comparison of the 3 groups.

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Logistic regression analysis of the effect of serum sICAM-1 on the occurrence of clinical features in AOSD.

For patients with AOSD, a logistic regression analysis was performed to evaluate the effect of serum sICAM-1 levels on the occurrence of clinical features, which included Still's rash, synovitis, hepatic dysfunction, and DIC. The serum sICAM-1 level was identified as a predictor of hepatic dysfunction (odds ratio [OR] 1.016, 95% confidence interval [95% CI] 1.004–1.028; P = 0.011) and a predictor of DIC (OR 1.013, 95% CI 1.002–1.025; P = 0.023).

ICAM-1 mRNA expression in rhIL-18–stimulated PBMCs from patients with active AOSD.

We examined whether cultured PBMCs stimulated with rhIL-18 could up-regulate ICAM-1 gene expression in 5 patients with active AOSD and 3 healthy controls. As shown in Figure 3, basal levels of ICAM-1 mRNA expression were significantly higher in PBMCs from patients with active AOSD than in those from healthy controls (P < 0.05). After 24 hours of stimulation of cultured PBMCs with different concentrations of rhIL-18 (10 ng/ml and 100 ng/ml), the levels of ICAM-1 mRNA expression were significantly greater than those of unstimulated cells from patients with active AOSD (P < 0.01), in a dose-dependent manner (Figure 3). A similar trend of up-regulation of ICAM-1 mRNA expression was observed in PBMCs from healthy controls after the addition of rhIL-18 (P = 0.05).

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Figure 3. Levels of intercellular adhesion molecule 1 (ICAM-1) messenger RNA (mRNA) expression in peripheral blood mononuclear cells stimulated with different concentrations (0 ng/ml, 10 ng/ml and 100 ng/ml) of recombinant human interleukin-18 (rhIL-18) in patients with active adult-onset Still's disease (AOSD) (n = 5) and healthy controls (n = 3), were analyzed by real-time TaqMan PCR method. Data are presented with logarithmic scales of mean ± SEM. * P < 0.05 comparison of AOSD patients and healthy controls; ** P < 0.01 comparison of the levels of ICAM-1 mRNA expression in peripheral blood mononuclear cells with and without rhIL-18 stimulation in AOSD patients.

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ICAM-1 mRNA expression in biopsy specimens of Still's rash and synovitis from patients with active untreated AOSD.

Significantly higher levels of ICAM-1 mRNA expression were demonstrated in biopsy specimens of Still's rash from patients with active AOSD compared with skin specimens from normal controls (copy ratio to β-actin: 1.79 ± 0.26 versus 0.25 ± 0.05, P < 0.01) (Figure 4A). Regarding the expression of ICAM-1 transcripts on the synovial membrane, the levels of ICAM-1 mRNA expression were significantly higher in patients with active AOSD compared with OA controls (copy ratio to β-actin: 3.33 ± 0.21 versus 0.38 ± 0.04, P < 0.05) and also higher in patients with active RA compared with OA controls (copy ratio to β-actin: 4.89 ± 0.36 versus 0.38 ± 0.04, P < 0.05) (Figure 4B). The levels of ICAM-1 mRNA expression on synovial membrane were significantly higher in patients with active RA compared with those in patients with active AOSD (copy ratio to β-actin: 4.89 ± 0.36 versus 3.33 ± 0.21, P < 0.01).

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Figure 4. Levels of intercellular adhesion molecule 1 (ICAM-1) messenger RNA expression in A) biopsy specimens obtained from the skin lesions of patients with active adult onset Still's disease (AOSD; n = 8) and from patients with healthy skin (normal controls; n = 4), and B) in the biopsy specimens obtained from the synovial membranes from patients with active AOSD (n = 6), from patients with active rheumatoid arthritis (RA; n = 6) and from patients with osteoarthritis (OA; n = 4), were analyzed by real-time TaqMan PCR method. Data are presented as mean ± SEM. P values are provided for comparison of different groups.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

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.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES