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Abstract: TH-OR054

Proteinase 3 Promotes Giant Cell Formation and Granulomata In Vitro and In Vivo Implicating a Key Role in GPA Pathogenesis

Session Information

Category: Glomerular Diseases

  • 1202 Glomerular Diseases: Immunology and Inflammation


  • Henderson, Scott R., University College London, London, United Kingdom
  • Khosravi, Maryam, University College London, London, United Kingdom
  • Greig, Alan J., University College London, London, United Kingdom
  • Frankel, Paul, University College London, London, United Kingdom
  • Salama, Alan D., University College London, London, United Kingdom

Granulomatosis with polyangiitis (GPA) is characterised by ANCA reactivity towards proteinase 3 (PR3), PR3 deposition in granulomatous lesions and increased neutrophil membrane PR3 expression, inhibiting apoptotic cell phagocytosis by macrophages. We investigated whether persistent and excessive PR3 expression in GPA may underlie giant cell and granuloma formation.


PBMCs were isolated from healthy controls(HC)(n=10), GPA(n=10) and microscopic polyangiitis(MPA)(n=10) patients and monocytes isolated. Cells were stimulated with either enzymatically active (aPR3) or inactive PR3(iPR3) or control auto-antigen myeloperoxidase (MPO). Light, epifluorescence and confocal microscopy was used to confirm cell fusion at different time points. Mpeg:mcherry macrophage zebrafish at 24 hours post-fertilisation were injected with aPR3 or iPR3 or albumin. Fish were imaged by lightsheet microscopy 6 days later.


Monocyte aggregation and giant cell formation occurred following stimulation with both aPR3 and iPR3 with a greater number (p<0.01) and size of aggregates (p<0.001) in GPA patients compared to MPA patients and HC. Typical granuloma were observed using PBMC preparations with a greater number (p<0.001) and size (p<0.001) of aggregates seen in GPA patients. There was no significant difference between aPR3 or iPR3. No effect was seen with MPO. Supernatant profiling implicated specific roles for pro-inflammatory cytokines. In zebrafish (n=9/group), both aPR3 and iPR3 were associated with a significant increase in cell fusion and aggregate volume (p<0.001) when compared to albumin injected controls (figure).


These models support a role for PR3 in promoting monocyte and macrophage fusion and granuloma formation in vitro and in vivo, and provide an opportunity to test specific therapeutics.

Macrophage aggregation in zebrafish (volume=purple min;red max)