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. 2012:2012:715049.
doi: 10.1155/2012/715049. Epub 2012 Jun 26.

Impairment and Differential Expression of PR3 and MPO on Peripheral Myelomonocytic Cells with Endothelial Properties in Granulomatosis with Polyangiitis

Susann Patschan  1 , Daniel Patschan, Elvira Henze, Sabine Blaschke, Johannes T Wessels, Gerhard Anton Müller
Affiliations

Impairment and Differential Expression of PR3 and MPO on Peripheral Myelomonocytic Cells with Endothelial Properties in Granulomatosis with Polyangiitis

Susann Patschan et al. Int J Nephrol. 2012.

Abstract

Background. Granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA) are autoimmune-mediated diseases characterized by vasculitic inflammation of respiratory tract and kidneys. Clinical observations indicated a strong association between disease activity and serum levels of certain types of autoantibodies (antineutrophil cytoplasm antibodies with cytoplasmic [cANCA in GPA] or perinuclear [pAN CA in MPA] immunofluorescence). Pathologically, both diseases are characterized by severe microvascular endothelial cell damage. Early endothelial outgrowth cells (eEOCs) have been shown to be critically involved in neovascularization under both physiological and pathological condition. Objectives. The principal aims of our study were (i) to analyze the regenerative activity of the eEOC system and (ii) to determine mPR3 and MPO expression in myelo monocytic cells with endothelial characteristics in GPA and MPA patients. Methods. In 27 GPA and 10 MPA patients, regenerative activity blood-derived eEOCs were analyzed using a culture-forming assay. Flk-1(+), CD133(+)/Flk-1(+), mPR3(+), and Flk-1(+)/mPR3(+) myelomonocytic cells were quantified by FACS analysis. Serum levels of Angiopoietin-1 and TNF-α were measured by ELISA. Results. We found reduced eEOC regeneration, accompanied by lower serum levels of Angiopoietin-1 in GPA patients as compared to healthy controls. In addition, the total numbers of Flk-1(+) myelomonocytic cells in the peripheral circulation were decreased. Membrane PR3 expression was significantly higher in total as well as in Flk-1(+) myelomonocytic cells. Expression of MPO was not different between the groups. Conclusions. These data suggest impairment of the eEOC system and a possible role for PR3 in this process in patients suffering from GPA.

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Figures

Figure 1
Figure 1
Flk-1+ and Flk-1+/CD133+ myelomonocytic cells (eEOCs) in patients with GPA, MPA, and SLE as compared to healthy controls. The percentages of circulating Flk-1+ cells (defined as all cells displaying endothelial characteristics) were lower in all patient groups (GPA, MPA, SLE) as compared to healthy controls. In contrast, total peripheral circulating eEOCs (Flk-1+/CD133+ cells) were not different between the four groups (bars show median).
Figure 2
Figure 2
Proliferative activity of peripheral circulating eEOCs in GPA, MPA, and SLE. Patients with either GPA or MPA showed significantly less eEOC proliferation (defined as smaller numbers of colony unit endothelial cells [CFU-ECs]) than controls (bars show median). SLE patients were not different from controls. In GPA the numbers of colonies did not correlate with clinical activity of the disease as represented by BVAS (not shown).
Figure 3
Figure 3
Percentages of all PR3+ myelomonocytic cells. GPA patients displayed higher percentages of cells expressing Proteinase 3. MPA and SLE patients were not different from controls ((a), bars show median). Images in (b) show representative FACS plots of a patient with GPA. The upper image in (b) displays the gating strategy for analyzing myelomonocytic cells, the lower image indicates increased percentages of mPR3 positive cells.
Figure 4
Figure 4
Percentages of PR3+/Flk-1+ cells. In order to evaluate PR3 expression on cells belonging to the endothelial lineage, peripheral myelomonocytic cells were stained for both, PR3 and Flk-1. As for the total myelomonocytic cell population, GPA patients showed higher percentages of PR3+/Flk-1+ cells than controls. Such differences were not detected between MPA or SLE and controls (bars show median).
Figure 5
Figure 5
(a) Immunoblot analysis of eEOC plasma membrane preparations for mPR3 expression. All but one out of five GPA patients displayed significant mPR3 expression. The one individual with intense mPR3 signal was a patient that refused therapy from the beginning of the disease. (b)–(d) Laser scanning analysis of cultures eEOCs. The cells were detected by UE lectin binding (green immunofluorescence in (c)) and Dil-acLDL uptake lectin binding (red immunofluorescence in (c)). All double-positive cells were analyzed for mPR3 expression (long red in (d)). Only a minority of the cells was mPR3 positive. (e)–(i) Representative images of PR3 expression in cultured peripheral eEOCs. Cells were stained for UE lectin ((f)—green), Dil-acLDL uptake ((g)—orange), and PR3 ((h)—red). Image e merges images (e)–(h) ((e): nuclear staining with DAPI solution, magnification in (e)–(i) × 100).
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