Transcription of proteinase 3 and related myelopoiesis genes in peripheral blood mononuclear cells of patients with active Wegener's granulomatosis

Abstract

Objective: Wegener's granulomatosis (WG) is a systemic inflammatory disease that is associated with substantial morbidity. The aim of this study was to understand the biology underlying WG and to discover markers of disease activity that would be useful for prognosis and treatment guidance.

Methods: Gene expression profiling was performed using total RNA from peripheral blood mononuclear cells (PBMCs) and granulocyte fractions from 41 patients with WG and 23 healthy control subjects. Gene set enrichment analysis (GSEA) was performed to search for candidate WG-associated molecular pathways and disease activity biomarkers. Principal components analysis was used to visualize relationships between subgroups of WG patients and controls. Longitudinal changes in proteinase 3 (PR3) gene expression were evaluated using reverse transcription-polymerase chain reaction, and clinical outcomes, including remission status and disease activity, were determined using the Birmingham Vasculitis Activity Score for WG (BVAS-WG).

Results: Eighty-six genes in WG PBMCs and 40 in WG polymorphonuclear neutrophils (PMNs) were significantly up-regulated relative to controls. Genes up-regulated in WG PBMCs were involved in myeloid differentiation, and these included the WG autoantigen PR3. The coordinated regulation of myeloid differentiation genes was confirmed by GSEA. The median expression values of the 86 up-regulated genes in WG PBMCs were associated with disease activity (P = 1.3 x 10(-4)), and WG patients with low-level expression of the WG signature genes showed expression profiles that were only modestly different from that in healthy controls (P = 0.07). PR3 transcription was significantly up-regulated in WG PBMCs (P = 1.3 x 10(-5), false discovery rate [FDR] 0.002), but not in WG PMNs (P = 0.03, FDR 0.28), and a preliminary longitudinal analysis showed that the fold change in PR3 RNA levels in WG PBMCs corresponded to changes in the BVAS-WG score over time.

Conclusion: Transcription of PR3 and related myeloid differentiation genes in PBMCs may represent novel markers of disease activity in WG.

Figure 1

Identification of discrete sets of up-regulated genes in Wegener's PBMCs and PMNs. Heat maps of the expression levels of the genes significantly differentially expressed between WG patients and normal controls in PBMCs (n=86) (A), and in PMNs (n=40) (B).

Figure 2

Association of the WG signature with disease activity. (A), Bar charts of the median expression levels of the significantly up-regulated genes in WG vs. control PBMCs, and (B), WG vs. control PMNs. The samples in (B) are in the same order as in (A). The short vertical lines below the x-axis in (A) indicate the BVAS score for each sample; cyan for BVAS=0, lavender for BVAS=1, and blue for BVAS>1. The single gap in the WG Sig- section in (B) represents a PMN sample that did not pass stringent quality control. (C), Medians of the expression levels in PBMCs of the 86 signature genes, grouped as indicated. The mean and the mean ± the standard error of the mean for each group are designated by black lines (D), PCA visualization of all samples using the top 400 most significantly regulated genes (up or down) in WG vs. control PBMCs, ranked by p-value, viewed using the top three PCA axes. The fraction of the total variance captured in the top three PCA axes was 76%. Controls are in green, and the WG samples are colored by positive (red) and negative (gold) signature status.

Figure 3

PR3 transcription occurs primarily in PBMCs and not in PMNs in WG. (A), Bar chart of PR3 expression levels in WG and control PBMCs and (B), PMNs. Samples are displayed in the same order as in Figure 2A. (C), Comparison of PR3 expression by RT-PCR and microarray for all 41 Wegener's PBMC samples. For RT-PCR, fold changes are relative to a pool of RNA from healthy controls. The points are color coded by patient BVAS score as in Figure 2A; cyan for BVAS=0, lavender for BVAS=1, and blue for BVAS>1.

Figure 4

Longitudinal expression of PR3 and other WG signature genes in WG patients. RT-PCR results for selected signature genes including PR3 were performed on paired samples (PBMCs) taken from patients at two time points at least 3 months apart. Fold change values are presented as average fold change = 2^{-(average ΔΔCt)} for genes in the patient samples relative to a pool of control samples. Error bars for fold changes from PCR correspond to 2^{-(average ΔΔCt ± sem)} where sem is the standard error of the mean for each gene.