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Standardised assessment of membrane proteinase 3 expression. Analysis in ANCA-associated vasculitis and controls
André P van Rossum1, Minke G Huitema, Coen A Stegeman
1Department of Rheumatology and Clinical Immunology, Groningen University Medical Centre, University of Groningen, The Netherlands. c.g.m.kallenberg@int.umcg.nl
Objectives:
Increased numbers of neutrophils expressing proteinase 3 on their membrane (mPR3) have been reported in anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitis (AAV) and are suggested to be involved in AAV immunopathogenesis. In most studies, neutrophils were analysed for mPR3 expression without priming with TNFalpha, suggesting that mPR3 expression on neutrophils is dependent on other priming events, such as isolation procedures . These priming events can be variable. Therefore, we analysed mPR3 expression on neutrophils before and after priming with TNFalpha to assess whether standardised assessment of mPR3 expression requires priming. Using neutrophils before and after priming with TNFalpha, we assessed percentages of mPR3(+) neutrophils in patients with AAV and in disease and healthy controls.
Methods:
Neutrophils from patients with PR3-AAV and MPO-AAV, systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), and from healthy controls were analysed before and after priming with TNFalpha for mPR3 expression.
Results:
42% of all individuals analysed showed minimal expression for mPR3 on all neutrophils before priming with TNFalpha, whereas after priming a clear mPR3(+) subset was observed next to mPR3(-) neutrophils, corresponding to bimodal mPR3 expression. In patients with PR3-AAV or MPO-AAV, the percentage of mPR3(+) neutrophils after priming with TNFalpha was significantly increased (p<0.01 and p<0.05, respectively) compared with healthy controls. Percentages of mPR3(+) PMN were also increased in patients with SLE (p<0.01) but not in RA.
Conclusion:
Standardised assessment of proteinase 3 on the membrane of neutrophils requires priming with TNFalpha. Percentages of mPR3(+) PMN are increased in AAV and SLE, but not in RA.
Insights
Standardized assessment of membrane proteinase 3 (mPR3) on neutrophils requires TNFalpha priming. Percentages of mPR3-expressing neutrophils are elevated in anti-neutrophil cytoplasm antibody-associated vasculitis (AAV) and systemic lupus erythematosus (SLE), but not rheumatoid arthritis (RA).
Area of Science:
- Immunology
- Rheumatology
- Cell Biology
Background:
- Elevated membrane proteinase 3 (mPR3) on neutrophils is implicated in anti-neutrophil cytoplasm antibody-associated vasculitis (AAV) pathogenesis.
- Previous studies on mPR3 expression lacked standardization, as neutrophils were often analyzed without priming, potentially influenced by isolation procedures.
- TNFalpha priming is crucial for revealing distinct mPR3 expression patterns on neutrophils.
Purpose of the Study:
- To determine if TNFalpha priming is necessary for standardized assessment of membrane proteinase 3 (mPR3) expression on neutrophils.
- To compare mPR3 expression levels in neutrophils from patients with AAV, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and healthy controls.
- To investigate the role of TNFalpha priming in revealing bimodal mPR3 expression.
Main Methods:
- Neutrophils were isolated from patients with PR3-AAV, MPO-AAV, SLE, RA, and from healthy controls.
- Membrane proteinase 3 (mPR3) expression was analyzed before and after in vitro priming with TNFalpha.
- Flow cytometry was used to quantify the percentages of mPR3-positive neutrophils.
Main Results:
- Priming neutrophils with TNFalpha induced a bimodal mPR3 expression pattern, with a distinct mPR3-positive subset.
- Significantly increased percentages of mPR3-positive neutrophils were observed in patients with PR3-AAV (p<0.01) and MPO-AAV (p<0.05) after TNFalpha priming compared to controls.
- Elevated mPR3-positive neutrophil percentages were also found in SLE patients (p<0.01), but not in RA patients.
Conclusions:
- Standardized assessment of membrane proteinase 3 (mPR3) on neutrophils necessitates TNFalpha priming.
- Increased percentages of mPR3-positive neutrophils are characteristic of AAV and SLE.
- This finding suggests a potential role for mPR3 in the immunopathogenesis of AAV and SLE, but not RA.
