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Binding of proteinase 3 and myeloperoxidase to endothelial cells: ANCA-mediated endothelial damage through ADCC?
B E Ballieux1, K T Zondervan, P Kievit
1Department of Nephrology, University Hospital, Leiden, The Netherlands.
Abstract:
Binding of both proteinase 3 (PR3) and myeloperoxidase (MPO) to endothelial cells (EC) has been suggested to be involved in the vascular damage seen in patients with Wegener's granulomatosis or microscopic polyangiitis. In the present study we investigated in detail the interaction of MPO and PR3 with cultured human umbilical vein endothelial cells (HUVEC) and its matrix products. In addition, we investigated whether interaction of PR3 or MPO with HUVEC monolayers also resulted in antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by anti-neutrophil cytoplasmic antibody (ANCA)-positive patient sera or rabbit IgG anti-PR3 or anti-MPO. Preincubation of HUVEC monolayers with PR3 or MPO resulted in a dose-dependent binding of both PR3 and MPO. However, HUVEC, preincubated with PR3 or MPO, followed by ANCA or by rabbit anti-PR3 or anti-MPO, were not susceptible to ADCC. Detailed analysis of the binding of PR3 to HUVEC monolayers showed that PR3 binds primarily to the extracellular matrix of endothelial cells, and to a very limited extent to the cells themselves. For MPO it was shown that it binds both to the extracellular matrix and to the endothelial cells themselves. However, after binding to HUVEC cultures, MPO was not detectable by polyclonal rabbit or human antibodies specific for MPO, probably because MPO is bound to sites not accessible for immunoglobulins. Binding of PR3 to HUVEC cultures (cells + matrix) was inhibited by fetal calf serum and by alpha 1-antitrypsin, but inactivation of enzymatic activity of PR3 by PMSF did not influence binding of PR3 to HUVEC cultures. Binding of MPO to HUVEC cultures was not influenced by fetal calf serum.
Insights
Proteinase 3 (PR3) and myeloperoxidase (MPO) bind to endothelial cells and their matrix. However, this binding does not lead to antibody-dependent cell-mediated cytotoxicity in vasculitis models.
Area of Science:
- Immunology
- Cell Biology
- Vascular Biology
Background:
- Proteinase 3 (PR3) and myeloperoxidase (MPO) binding to endothelial cells (EC) is implicated in vascular damage in Wegener's granulomatosis and microscopic polyangiitis.
- Understanding the precise interactions of PR3 and MPO with EC is crucial for elucidating mechanisms of vasculitis.
Purpose of the Study:
- To investigate the detailed interaction of MPO and PR3 with cultured human umbilical vein endothelial cells (HUVEC) and their matrix.
- To determine if PR3 or MPO interaction with HUVEC induces antibody-dependent cell-mediated cytotoxicity (ADCC).
Main Methods:
- Dose-dependent binding assays of PR3 and MPO to HUVEC monolayers.
- Assessment of ADCC using ANCA-positive patient sera or specific rabbit IgG.
- Analysis of PR3 and MPO binding localization (cell vs. extracellular matrix).
Main Results:
- Both PR3 and MPO demonstrated dose-dependent binding to HUVEC cultures.
- HUVEC pre-incubated with PR3 or MPO were not susceptible to ADCC.
- PR3 primarily bound to the extracellular matrix, with limited cell binding; MPO bound to both cells and matrix.
- MPO binding to HUVEC rendered it undetectable by specific antibodies.
Conclusions:
- PR3 and MPO interact with HUVEC and their extracellular matrix, but this interaction does not trigger ADCC.
- The binding characteristics of PR3 and MPO suggest distinct interactions with endothelial cells and their matrix.
- Further research is needed to understand the functional consequences of MPO's altered accessibility after binding.