INTRODUCTION

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Sarcoidosis is a systemic granulomatous disease of unknown origin, which is characterized by noncaseating epithelioid cell granulomas with dominant infiltration of CD4⁺ T cells and macrophages (1). It has been revealed that many cytokines and chemokines are concerned with the immunopathogenesis of sarcoidosis (2). Modern immunology has confirmed a Th1 and Th2 paradigm, which has been applied to human disorders (3). Recent immunological studies on sarcoidosis suggest a Th1 response in this disease (2).

Interleukin-18 (IL-18) is a novel cytokine that was found to be an interferon- (IFN-)-inducing factor (4) and has the ability to induce IFN- from Th1 cells with IL-12, and enhances the cytotoxicity of NK cells and T cells (5). Furthermore, IL-18 promotes apoptosis through enhancing expression of the Fas ligand (6). Pathological roles of IL-18 have been found in experimental animal models (5), however, there are few reports concerning human disorders. To determine the pathological roles of IL-18 in pulmonary sarcoidosis, we measured the level of IL-18 in sera and bronchoalveolar lavage (BAL) fluids, and investigated its clinical usefulness

	METHODS

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Study Population

The diagnosis of sarcoidosis was established in 37 individuals (14 men, mean age 26 yr, 23 women, mean age 46 yr). Nineteen patients had never smoked, and 18 patients were smokers. They had histological evidence consistent with sarcoidosis of the lung (showing noncaseating epithelioid cell granuloma) without any evidence of mycobacterial, fungal, or parasitic infection. None had a history of exposure to organic or inorganic materials known to cause granulomatous lung diseases. No patient had received corticosteroid therapy at the time of the study. The assessment of disease included clinical features, chest X-ray, high-resolution computed tomography, lung function tests, gallim-67 (⁶⁷Ga) scan, BAL examinations, and routine blood studies. BAL examinations were performed as described previously (7). Twenty-nine patients had abnormal chest X-ray findings; 14 patients demonstrated hilar lymphadenopathy alone (stage I), 10 patients had hilar lymphadenopathy and interstitial infiltrates of the lung field (stage II), 3 patients had interstitial infiltrates of the lung field alone (stage III), and 2 patients demonstrated fibrotic lesions of the lung field (stage IV). Eight patients with normal chest X-ray findings (stage 0), however, had positive biopsy findings of lung and uveitis and thus were diagnosed as having sarcoidosis. Blood samples were collected at the time of diagnosis and BAL fluids were concentrated 10-fold by membrane dialysis using CENTRIPLUS (Amicon), and samples were cryopreserved at 80° C until use.

Serum samples of healthy control subjects were obtained from 25 healthy volunteers (aged 22 to 62). BAL fluid samples were obtained from 9 healthy all lifelong nonsmokers. They had no history or symptoms of respiratory diseases, and their chest X-ray and pulmonary function tests showed no evidence of respiratory diseases.

IL-18 Assay

Enzyme-linked immunosorbent assay (ELISA) for determination of serum IL-18 was performed according to a previously described method (7). Briefly, a maxisorp plate was coated with monoclonal antibody (MoAb) #125-2H to human IL-18 (20 µg/ml in phosphate-buffered saline [PBS]) at room temperature (RT) for 3 h and blocked with PBS containing 1% bovine serum albumin (BSA) at 4° C overnight. After washing with PBS containing 0.05% Tween 20, 50 µl of the assay buffer (PBS containing 1% BSA, 5% fetal calf serum [FCS], and 1 M NaCl) was dispensed and 50 µl of 5-fold diluted serum, 10-fold concentrated BAL fluid samples, or standard human IL-18 were added to the assay buffer and the plate was incubated at RT for 2 h. After washing, peroxidase-conjugated MoAb #159-12B to human IL-18 (0.5 µg/ml in PBS containing 1% BSA, 5% FCS, 0.1% CHAPS, and 0.3 M NaCl) was added and incubated at RT for 2 h. After washing, the substrate solution (100 µl of 0.1 M sodium phosphate-citrate buffer containing 0.5 mg/ml o-phenylenediamine and 0.003% H₂O₂, pH 5.0) was added. The reaction was stopped with 100 µl of 1 M H₂SO₄ and the absorbance at 490 nm was measured. All assays were performed in duplicate. The detectable range of this ELISA was between 10 and 1,000 pg/ml.

IFN- Assay

IFN- in sera and 10-fold concentrated BAL fluid samples was measured with an ELISA (high sensitivity interferon-gamma human ELISA system, Amersham Life Science, Buckinghamshire, UK) according to the manufacturer's instructions. The lower detection limit of this assay was 0.10 pg/ml.

Statistical Analysis

Data were expressed as mean ± SEM. The Mann-Whitney U test was used to test for differences between the groups. Pearson's product- moment correlation coefficient analysis was used. (Spearman rank correlation coefficient analysis was used between serum IL-18 and IFN-.) The level of critical significance was assigned at p < 0.05.

	RESULTS

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The levels of serum IL-18 of patients with sarcoidosis (419.4 ± 36.6 pg/ml) were significantly higher than those of healthy control subjects (169.0 ± 19.2 pg/ml [n = 19], 6 controls < 50 pg/ml, p < 0.0001) (Figure 1). In the comparison between each radiographic stage of chest X-ray and healthy control subjects, all radiographic stages had significantly elevated serum IL-18 levels (stage 0, 249.9 ± 48.2 pg/ml [p = 0.0255]; stage I, 477.9 ± 61.2 pg/ml [p < 0.0001]; stage II, 482.9 ± 77.6 pg/ml [p < 0.0001]; and stage III + IV, 399.9 ± 44.1 pg/ml [p = 0.0009]). In comparison between the different radiographic stages, stage I (p = 0.0169) and stage II (p = 0.0456) cases had concentrations of serum IL-18 significantly increased over stage 0 cases.

View larger version (11K): [in this window] [in a new window]   Figure 1.   Levels of serum interleukin-18 (IL-18) of patients with sarcoidosis and healthy control subjects. The dashed line indicates the level of sensitivity of this assay (50 pg/ml). Stage I (p = 0.0169 and p < 0.0001) and stage II (p = 0.0456 and p < 0.0001) sarcoid cases had significantly higher levels of serum IL-18 than those of stage 0 sarcoid cases and healthy control subjects, respectively. Stage III + IV sarcoid cases had significantly elevated levels over those of healthy control subjects (p = 0.0009).

The levels of IFN- in 21 of 25 healthy control subjects were very low (< 0.10 pg/ml), and the levels of the remaining four healthy controls were 0.66 ± 0.15 pg/ml (Figure 2). In stage 0 sarcoid cases, the levels of seven cases were below the lower limit, that of the remaining patient was 1.10 pg/ml, and the levels of stage 0 cases were not significantly elevated as compared with those of healthy control subjects (p = 0.2822). In stage I cases, the levels of five patients were below the lower limit and the average of the remaining nine patients was 2.59 ± 0.58 pg/ml. The average serum IFN- level of nine stage II cases was 2.29 ± 0.49 pg/ml except one patient below the lower limit, and that of stage III + IV cases was 4.21 ± 1.92 pg/ml. The levels of stage I and stage II cases were significantly increased as compared with those of healthy control subjects (p = 0.0001 and p < 0.0001, respectively) and stage 0 cases (p = 0.0230 and p = 0.0044, respectively), and the levels of stage III + IV cases were significantly elevated as compared with those of healthy control subjects (p = 0.0031). Then we examined the correlation between levels of serum IL-18 and IFN-. The serum IL-18 levels of patients with sarcoidosis significantly correlated with those of serum IFN- levels (rs = 0.436, p = 0.0084).

View larger version (10K): [in this window] [in a new window]   Figure 2.   Levels of serum interferon- (IFN-) of patients with sarcoidosis and healthy control subjects. The dashed line indicates the level of sensitivity of this assay (0.1 pg/ml). Stage I (p = 0.0230 and p = 0.0001) and stage II (p = 0.0044 and p < 0.0001) sarcoid cases had significantly higher levels of serum IFN- than those of stage 0 sarcoid cases and healthy control subjects, respectively. Stage III + IV sarcoid cases had significantly elevated levels over those of healthy control subjects (p = 0.0031).

Next, we measured IL-18 and IFN- levels in BAL fluid of patients with sarcoidosis and healthy control subjects. Because it is well known that cigarette smoking has a influence on cytokine concentration in BAL fluid, only levels in nonsmokers were measured. The levels of IL-18 in 19 sarcoid BAL fluids (19.3 ± 2.3 pg/ml) were significantly increased as compared with those of nine healthy controls (7.6 ± 2.0 pg/ml [n = 5], four control subjects < 1 pg/ml, p = 0.0004) (Figure 3). The levels of IL-18 in sarcoid BAL fluids significantly correlated with lymphocyte proportions in BAL fluid (r = 0.493, p = 0.0319) and the IL-18 levels in sera (r = 0.559, p = 0.0128), whereas they did not correlate with BAL fluid CD4/CD8 ratios (r = 0.034, p < 0.8928). IFN- was not detectable in 10-fold concentrated BAL fluid samples of the patients or healthy control subjects.

View larger version (9K): [in this window] [in a new window]   Figure 3.   Levels of bronchoalveolar lavage (BAL) fluid IL-18 of patients with sarcoidosis and healthy control subjects. The dashed line indicates the level of sensitivity of this assay (1.0 pg/ml). Concentrations of IL-18 in BAL fluids of sarcoid cases were significantly increased as compared with those of healthy control subjects (p = 0.0004).

Finally, we investigated the clinical usefulness of serum IL-18 as a marker of disease activity of sarcoidosis. The levels of serum IL-18 significantly correlated with the activities of serum lysozyme (r = 0.604, p < 0.0001), whereas no correlation of serum angiotensin-converting enzyme (ACE) activity was found (r = 0.194, p = 0.2497). The levels of serum and BAL fluid IL-18 did not significantly correlate with the results in pulmonary function tests (%VC, %FEV_1.0 or DL_CO/VA). Furthermore, we compared the serum IL-18 levels between the ⁶⁷Ga scan positive (positive uptake for intrathoracic lymph nodes and/or lung fields) and negative groups (negative uptake for both intrathoracic lymph nodes and lung fields). The group having positive ⁶⁷Ga scan had significantly elevated serum IL-18 levels (465.1 ± 39.5 pg/ml, n = 30) as compared with those of the negative group (223.7 ± 46.7 pg/ml, n = 7, p = 0.0046). It was demonstrated that serum IL-18 had potential as a useful clinical marker for disease activity of sarcoidosis.

	DISCUSSION

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Recent studies concerning the immunopathogenesis of sarcoidosis have demonstrated a Th1 dominant response of this disease, especially under conditions of disease activity. IL-12 is an important cytokine that differentiates naive T cells into Th1 cells, and induces IFN- production by Th1 cells (9). IL-18 is also a potent cytokine that synergistically induces IFN- production by differentiated Th1 cells with IL-12, and augments the cytotoxicity of NK cells and T cells.

In this study, we demonstrated that the concentrations of serum IL-18 and IFN- were elevated, and there was a significant correlation between both concentrations in pulmonary sarcoidosis. In addition, we demonstrated increased IL-18 levels in sarcoid BAL fluids. These levels correlated with serum IL-18 levels, suggesting that elevated local production of IL-18 reflects circulating IL-18. We also indicated that the serum IL-18 levels of patients with sarcoidosis closely correlated with the clinical markers of disease activity (⁶⁷Ga scan and serum lysozyme activity) and, further, that BAL fluid IL-18 levels of patients correlated to BAL fluid lymphocyte proportions. Up-regulation of IFN- was found in the sarcoid lesions (10) and serum (12). Serum IL-12p70 was not detected in patients with juvenile chronic arthritis (13) and we also failed to detect IL-12p70 in sera and BAL fluids of patients with sarcoidosis (data not shown). However, our recent observation demonstrated that IL-12p70 and IL-18 were produced by alveolar macrophages in sarcoid lung and IL-12p70 and IL-18 synergistically induced IFN- production (14). Thus, IL-18 probably plays important roles in the immunopathogenesis of sarcoidosis. In this study, we could not detect IFN- in BAL fluids. Walker and coworkers (11) reported detecting concentrations of IFN- in BAL fluid using 20-fold membrane dialysis concentration. Although there were differences between study populations, further concentrations of greater than 10-fold may be needed to detect IFN- in BAL fluids. We showed that the levels of serum IL-18 did not correlate with the serum ACE activity. ACE and lysozyme are secreted by epithelioid cells and their serum levels indicated the total body granuloma burden (15). However, because of an insertion/deletion polymorphism of the ACE gene, serum ACE levels are not necessarily a good marker of disease activity in single measurement (16).

In this study, we documented increased levels of BAL fluid IL-18 in pulmonary sarcoidosis and a significant correlation of the serum IL-18 levels. To our knowledge, this is the first report to document increased levels of serum IL-18 in patients with sarcoidosis. Up-regulated IL-18 expression has been found in Th1-mediated chronic inflammatory diseases such as Crohn's disease and rheumatoid arthritis (17, 18) and acute Epstein-Barr virus-induced infectious mononucleosis (19). Elevated levels of IL-18 have found in sera of hemophagocytic lymphohistiocytosis (20). In patients with sarcoidosis, IL-18 is speculated to induce IFN- production and to reflect disease activity. To clarify the pathophysiological roles of IL-18 in sarcoidosis, further examinations will be needed.