Cathepsin S, a new serum biomarker of sarcoidosis discovered by transcriptome analysis of alveolar macrophages

Abstract

Background: Development of reliable new biomarkers remains crucial to improve diagnosis and assessing disease activity in sarcoidosis. The objective of this study was to seek such markers from the gene expression signature of alveolar macrophages by transcriptome analysis.

Methods: Pooled RNA extracted from alveolar macrophages from patients with active sarcoidosis and control patients was subjected to transcriptome analysis using microarrays. Expressed gene intensity in sarcoidosis relative to that in control was calculated. We measured serum cathepsin S (CTSS) concentrations in 89 healthy volunteers, 107 patients with sarcoidosis, 26 with interstitial pneumonia, 150 with pneumoconiosis, and 76 with pulmonary mycobacteriosis by the enzyme-linked immunosorbent assay.

Results: Among 12 genes with ratios higher than that of a housekeeping gene, we selected CTSS for scrutinizing protein expression in serum because of the feasibility of the protein assay. CTSS concentrations were significantly increased in sarcoidosis compared with not only controls but also all the other lung diseases. Receiver operating characteristics curve for sarcoidosis and parenchymal lung diseases revealed an area under the curve of 0.800 (95% confidence interval, 0.751-0.850; p=1.4 x 10^-18) with 70% sensitivity and 78% specificity at a CTSS concentration of 15.5 ng/ml. A significant trend was identified between CTSS concentrations and the number of affected organs. Serum CTSS concentrations varied in parallel with clinical courses both spontaneously and in response to corticosteroid therapy. Epithelioid cells in granulomas were positive for immunohistochemical staining with CTSS.

Conclusions: CTSS has the potential to be a useful biomarker in sarcoidosis.

Keywords: biomarkers; cathepsins; macrophages; sarcoidosis; transcriptome.

Fig. 1.

Serum CTSS concentrations. Data are presented in box-and-whisker plots with ends of the whiskers representing the lowest datum still within the 1.5 interquartile range (IQR) under the lower quartile and the highest datum still within 1.5 IQR above the upper quartile. Circles represent outliers between 1.5 and 3.0 IQR under the lower quartile or above the upper quartile. Asterisks represent extreme values below 3.0 IQR under the lower quartile or above 3.0 IQR over the upper quartile. The horizontal dotted line indicates the 95th percentile value of controls. * Significantly higher than controls. P values for SA, IP, and PC were 1.3X10^-30, 8.6X10^-8, and 4.0X10^-31, respectively. † Significantly higher than the other pulmonary diseases. P values for IP, PC, and PM were 6.7X10^-6, 7.8X10^-8, and 2.1X10^-9, respectively. Definitions for abbreviations: CO, control; SA, sarcoidosis; IP, interstitial pneumonia; PC, pneumoconiosis; PM, pulmonary mycobacteriosis.

Fig. 2.

Receiver operating characteristics analysis for differentiation between sarcoidosis and some of parenchymal lung diseases. Receiver operating characteristics curve was generated as the actual state and higher values of CTSS as positive. Definitions of abbreviations: AUC, area under the curve; CI, confidence interval

Fig. 3.

Changes in CTSS concentrations and clinical course of sarcoidosis. CTSS concentrations in serum were measured twice and compared in patients with unchanged clinical activity (A), spontaneous improvement (B), improvement by systemic corticosteroids (C), and disease progression (D)

Fig. 4.

Representative immunohistochemical staining with anti-CTSS antibody for a lymph node from a patient with sarcoidosis. Positive cells for CTSS are stained brown. A hematoxylin-eosin stained picture of the same area is shown in the upper right corner.