ab17_4

In vitro up-regulation of E-selectin and induction of interleukin-6 in endothelial cells by autoantibodies in Wegener’s granulomatosis and microscopic polyangiitis

A.C. Muller Kobold¹, R.T. van Wijk¹, C.F.M. Franssen², G. Molema¹,³, C.G.M. Kallenberg¹, J.W. Cohen Tervaert¹

Departments of Clinical Immunology¹ and Nephrology², University Hospital Groningen, and Department of Pharmacokinetics and Drug Delivery³, State University Groningen, The Netherlands.

ABSTRACT
Objective
In patients with Wegener’s granulomatosis (WG) or microscopic polyangiitis (MPA) autoantibodies to myeloid granule proteins (ANCA), particularly proteinase 3 (Pr3) and myeloperoxidase (MPO), and to endothelial cells (AECA) are frequently detected. The role of these autoantibodies in the development of vascular injury is incompletely understood. Since the expression of E-selectin and the production of interleukin 6 by endothelial cells is an early step in the sequence of events leading to vascular injury, we examined the capacity of IgG fractions from patients with WG and/or MPA to activate endothelial cells to the expression of E-selectin and the production of IL-6. We related those findings to the presence of ANCA and AECA in the IgG preparations.

Methods
Human umbilical vein endothelial cells (HUVEC) were incubated with immunoglobulin (IgG) preparations from 28 patients (17 positive for anti-Pr3, 10 for anti-MPO, and one for anti-Pr3/MPO) with active vasculitis and from 10 healthy volunteers. The final IgG concentration in the activation assay was 2 mg/ml. TNFa (10 ng/ml) and LPS (10 ng/ml) were used as positive controls for HUVEC activation. The extent of HUVEC activation was assessed by the measurement of E-selectin expression by flow cytometry (after 4 hours of incubation) and the production of interleukin 6 by ELISA (after 24 hours).

Results
We found that 11 of the 28 ANCA positive IgG samples were capable of activating endothelial cells: 6 samples induced IL-6 production alone, 1 sample upregulated E-selectin expression alone, and 4 samples induced both IL-6 production and E-selectin upregulation. Five of 17 anti-Pr3 positive samples (one of which was also positive for AECA) and 6 of 10 anti-MPO positive samples (all simultaneously positive for AECA) induced endothelial cell activation. AECA positive samples that induced endothelial cell activation (n = 7) had higher AECA titres than samples that did not induce endothelial cell activation (n = 6).

Conclusion
Our data suggest that the activation of endothelial cells in patients with WG and MPA can be induced by circulating autoantibodies. Both ANCA and AECA can be responsible for this effect.

Key words
ANCA, AECA, IgG, endothelium, cell-activation, E-selectin, interleukin-6.

This study was financially supported by departmental funds.

Please address correspondence and reprint requests to: Dr. A.C. Muller Kobold, Department of Clinical Immunology, University Hospital Groningen, PO Box 30001, 9700 RB Groningen, The Netherlands.