Department of Dermatology, Faculty of Medicine, University of Tokyo, Tokyo, Japan
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Abstract |
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Methods. Serum SP-D levels were assayed using a sensitive enzyme-linked immunosorbent assay in 59 patients with PM/DM and in 29 healthy controls.
Results. The serum level of SP-D was significantly higher in patients with PM/DM than in healthy controls (mean±S.D. 61.7±122.6 vs 31.0±12.4 ng/ml, P < 0.01). The serum SP-D level in patients with interstitial lung disease (ILD) was significantly higher than in those without ILD (118.7±220.2 vs 38.7±21.0 ng/ml, P < 0.001). Serum level of SP-D was correlated with the presence of ILD. The incidences of decreased vital capacity (%VC) and of decreased diffusing capacity of carbon monoxidase (%DLCO) were also significantly greater in patients with an elevated SP-D level than in those with a normal level (64 vs 7%, P < 0.02; 73 vs 27%, P < 0.01). Moreover, the serum SP-D level was inversely correlated with %VC (r=-0.452, P < 0.01) and %DLCO (r=-0.349, P < 0.05).
Conclusion. The serum SP-D level may be a useful marker for ILD in patients with PM/DM.
KEY WORDS: Interstitial lung disease, Enzyme-linked immunosorbent assay, Vital capacity.
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Introduction |
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Pulmonary surfactant is composed of phospholipids and associated proteins. It covers the alveolar surface and maintains alveolar gas exchange during expiration. There are four distinct proteins in pulmonary surfactant, which are designated surfactant proteins: SP-A, SP-B, SP-C and SP-D [3]. These surfactant proteins play various important roles in the lung, such as maintaining the biophysical activity of the surfactant and regulating surfactant homeostasis in the alveoli [4]. SP-D is a hydrophilic glycoprotein with a reduced molecular mass of 43 kDa. SP-D is produced and secreted by alveolar type II epithelial cells and Clara cells [3].
Recently, the SP-D levels in bronchoalveolar lavage (BAL) fluids and the systemic circulation have been measured in several lung diseases, including idiopathic pulmonary fibrosis [57]. It was reported that the SP-D level in BAL fluids was decreased in patients with pulmonary fibrosis, while the serum SP-D level was significantly elevated. This may be because the increased permeability of SP-D results in an increase in alveolar-to-vascular leakage of SP-D in these patients [6]. Increased serum SP-D levels were detected in patients with idiopathic pulmonary fibrosis, interstitial pneumonia with collagen diseases and pulmonary alveolar proteinosis [6, 7]. In contrast, the serum SP-D levels in patients with bacterial pneumonia were not elevated [6]. Moreover, serum SP-D levels reflected the disease activity of pulmonary fibrosis. However, previous studies have included only small numbers of PM/DM patients [6, 7]. The mechanism of serum SP-D elevation is unknown, but probably involves the combination of type II cell hyperplasia with a concomitant increase in the synthesis of surfactant proteins and spillover into the circulation.
SP-B and SP-C are extremely hydrophobic proteins that are rarely detected in serum. SP-D gets into the bloodstream from the alveoli more easily than SP-A, resulting in higher levels of SP-A. Because SP-D exhibited a wider range of serum concentration and was detected in patients with pulmonary fibrosis more than SP-A, SP-D is thought to be a better diagnostic marker than SP-A [6].
In this study, we investigated the serum SP-D levels in patients with PM/DM and assessed the relationships among serum SP-D level and clinical features in these patients.
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Patients and methods |
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Enzyme-linked immunosorbent assay (ELISA) for SP-D
Aliquots of serum were frozen at -20°C until assayed. Specific ELISA kits were used for the measurement of SP-D according to the manufacturer's instructions (Yamasa, Tokyo, Japan) [5]. In brief, polystyrene cups coated with SP-D antibodies were incubated with 20 µl of 11-fold diluted serum at 4°C for 24 h. The cups were then washed and incubated at 25°C for 2 h with 100 µl of 111-fold diluted horseradish peroxidase conjugated anti-SP-D antibody. The cups were washed again and 100 µl of tetramethylbenzene was added. Incubation was then performed at 25°C for 15 min. Finally, 2 mmol/l NaN3 was added to terminate the peroxidase reaction and the absorbance at 450 nm was measured. Serum SP-D levels more than 2 S.D. greater than the mean level in the normal controls were regarded as elevated.
Antinuclear or anticytoplasmic antibodies
Antinuclear or anticytoplasmic antibodies were detected by the indirect immunofluorescence method using HEp-2 cells as the substrate [9].
Statistical analysis
Statistical analysis was carried out with the MannWhitney test for the comparison of means and Fisher's exact probability test for the analysis of frequency. Correlations with clinical data were assessed using Spearman's rank correlation coefficient. Two-tailed P values less than 0.05 were considered significant.
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Results |
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Correlation of serum SP-D level with clinical and serological features of patients with PM/DM
The clinical and serological features of the patients with elevated or normal levels of SP-D are shown in Table 1. There was no significant difference between these groups in terms of sex or age. The percentage of individuals with Gottron's papules was significantly lower among patients with an elevated SP-D level than among those with a normal level (18 vs 63%, P < 0.01). The percentage of individuals with ILD was significantly greater among patients with an elevated SP-D level than among those with a normal level (73 vs 24%, P < 0.01). The incidences of decreased %DLCO and decreased vital capacity (%VC) were also significantly higher in patients with an elevated SP-D level than in those with a normal level (73 vs 27%, P < 0.01; 64 vs 7%, P < 0.02). The patients with an elevated SP-D level had significantly lower %DLCO values than those with a normal level (mean±S.D., 55.5±30.1 vs 83.4±20.9%, P < 0.02). The serum SP-D level was inversely correlated with %VC (r=-0.452, P < 0.01) and %DLCO (r=-0.349, P < 0.05). The percentage of individuals with decreased oxygen saturation in the blood was significantly higher among patients with an elevated SP-D level than among those with a normal level (55 vs 15%, P < 0.01).
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Longitudinal measurements of serum SP-D levels
In 15 patients, the serum SP-D level was examined longitudinally over a period of 12 yr. The activity of ILD was evaluated on the basis of subjective symptoms (e.g. cough) and objective signs, including chest radiographs, computed tomography and pulmonary function tests. The cases were divided into four groups as described [9, 11]: those in whom ILD became exacerbated, those in whom ILD improved, those with unchanged ILD and those without ILD (Fig. 2).
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Discussion |
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In the present study, the serum SP-D level of PM/DM patients with ILD was significantly elevated compared with those without ILD. The incidences of decreased %DLCO and decreased %VC were significantly greater in patients with an elevated SP-D level than in those with a normal level. Furthermore, the serum SP-D level was inversely correlated with %VC and %DLCO. These results suggest that the serum SP-D level is a serum marker for ILD and reflects the severity of ILD in patients with PM/DM. In this study, the diagnosis of ILD was made on the basis of the findings of chest radiography, computed tomography of the chest, lung function tests and %DLCO. However, there may be patients with minor respiratory crepitations, probably due to fibrosis, who do not have abnormal values in these examinations. Further longitudinal studies of the serum SP-D level in these patients are needed to clarify the clinical significance of the serum SP-D level.
Longitudinal measurements of the serum SP-D level showed that the exacerbation of ILD was accompanied by definite increases in serum SP-D. In a patient whose ILD improved, the serum SP-D level decreased from elevated to normal. In two patients with unchanged ILD, the serum SP-D level was elevated at both points. In seven of the eight patients without ILD, the serum SP-D level was within normal limits at both points. All of these patients were treated with oral prednisolone. In particular, a patient whose ILD improved received high doses of prednisolone. Glucocorticoid treatment increased the concentrations of SP-D mRNA and protein in cultured lung fibroblasts [13]. This indicates that the serum SP-D level reflects the severity of ILD rather than the effect of treatment with glucocorticoid in patients with PM/DM.
Significantly fewer individuals had Gottron's papules among the patients with an elevated SP-D level than in those with a normal level. This may be due to the fact that PM patients had ILD more frequently than DM patients in our study. In this study, four of the six PM patients (67%) and 17 of the 53 DM patients (32%) had ILD.
Recently, we showed that the serum level of KL-6, a MUC 1 mucin expressed on alveolar type II cells and also on epithelial cells in other organs, was significantly elevated in PM/DM patients with ILD compared with those without ILD, and was inversely correlated with %DLCO and %VC [13]. The sensitivity of serum KL-6 levels for ILD in patients with PM/DM was 100%. However, the specificity of serum KL-6 levels for ILD in PM/DM patients was 56%, because some patients in whom DM was associated with neoplasia had an elevated serum SP-D level. In this study, none of the PM/DM patients with neoplasia had an elevated serum SP-D level and the specificity of serum SP-D level was 93%. These results, including those of our previous study, showed that SP-D may serve as a specific marker for evaluating ILD in patients with PM/DM.
In conclusion, we have demonstrated the elevation of serum SP-D level in PM/DM patients with ILD. SP-D may be a useful marker for evaluating ILD in patients with PM/DM. However, this study was undertaken with a small patient population. Further studies are needed to clarify the usefulness of SP-D in PM/DM patients.
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Acknowledgments |
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Notes |
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References |
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