Increased monocyte transcription of the proteinase 3 gene in small vessel vasculitis

Abstract

Proteinase 3 (PR3) is a pleiotropic and destructive serine protease and it is also a major target for autoantibodies in systemic small vessel vasculitis. We have shown recently that patients in stable remission have increased circulating levels of PR3, independent of autoantibody titre, inflammation, neutrophil degranulation and renal function. Here we explore the possibility of increased PR3 gene transcription. RNA was purified from peripheral blood monocytes from vasculitis patients and controls. Specific mRNA was measured by TaqMan real-time polymerase chain reaction (PCR). The monocyte-like cell lines THP-1 and U937 and human peripheral blod monocytes from healthy controls were stimulated with cytokines and lipopolysaccharide (LPS) for different time periods. PR3 protein was measured in plasma with enzyme-linked immunosorbent assay (ELISA). The median result for PR3 mRNA was 9.6 (1.8-680) for 22 patients, compared to 1 (0.1-2.8) for the 15 healthy controls. Elastase expression was also significantly increased, whereas myeloperoxidase and interleukin-8 were not. Stimulation of monocytes with tumour necrosis factor (TNF)-alpha, interferon (IFN)-gamma or LPS did not result in any increase of PR3 or elastase transcription, whereas interleukin (IL)-8 transcription was increased 10-fold. Circulating monocytes from patients with systemic vasculitis display increased PR3 gene transcription compared to healthy controls and patients with sytemic lupus erythematosus (SLE). This may be important for the development of vasculitis. Our results do not favour a role for cytokines, antineutrophil cytoplasmic antibodies (ANCA) or immunosuppressive medication in the upregulation of PR3 transcription in vasculitis.

Fig. 1

Monocyte proteinase 3 (PR3) mRNA levels among 22 patients with antineutrophil cytoplasmic antibodies (ANCA)-associated small vessel vasculitis. Patient enumeration is the same as in Table 1. Patients 1–9 are myeloperoxidase (MPO)-ANCA positive, 10–21 are PR3-ANCA-positive and patient 22 is seronegative. Levels are normalized using β-actin as housekeeping gene and the median value of our 15 healthy controls was set as 1·0 (range 0·1–2·8). The difference between ANCA-positive patients and healthy controls is highly significant, while the difference between MPO-ANCA and PR3-ANCA is not significant. X = outlier reaching 680.

Fig. 2

Correlation between plasma proteinase 3 (PR3) concentrations and monocyte PR3 mRNA levels among 18 of 22 patients with antineutrophil cytoplasmic antibodies (ANCA)-associated small vessel vasculitis. Plasma levels are measured in µg/l using enzyme-linked immunosorbent assay (ELISA). mRNA levels are normalized using β-actin as housekeeping gene and the median value of the healthy controls is set to 1. The correlation coefficient is 0·3 (not significant) when using the Spearman's rank correlation test.