PR3-ANCA in Wegener's granulomatosis prime human mononuclear cells for enhanced activation via TLRs and NOD1/2

Abstract

Background: Anti-neutrophil cytoplasmic antibodies (ANCA) is autoantibodies characteristic of vasculitis diseases. A connection between ANCA and Wegener's granulomatosis was well established. The interaction of both ANCA phenotypes (PR3-ANCA and MPO-ANCA) with leukocytes provoked cell activation, which might be involved in the pathogenesis of ANCA-related Wegener's granulomatosis.

Methods: In this study, we examined whether PR3-ANCA sera and purified immunoglobulins from patients with Wegener's granulomatosis prime human monocytic cells for enhanced responses to microbial components in terms of production of proinflammatory cytokines.

Results: Flow cytometry demonstrated that stimulation with antibodies to proteinase 3 enhanced the expression of TLR2, 3, 4, 7, and 9, NOD1, and NOD2 in human mononuclear cells. The sera and purified immunoglobulins significantly primed human mononuclear cells to secrete interleukin-8 in response to microbial components via TLRs and NODs. Priming effects were also observed for the production of interleukin-6, monocyte chemoattractant protein-1, and tumor necrosis factor-alpha. On the other hand, PR3-ANCA-negative sera from patients with polyarteritis nodosa which possibly related to MPO-ANCA and aortitis syndrome as well as control sera from a healthy volunteer did not have any priming effects on PBMCs.

Conclusion: In conclusion, PR3-ANCA prime human mononuclear cells to produce cytokines upon stimulation with various microbial components by up-regulating the TLR and NOD signaling pathway, and these mechanisms may partially participate in the inflammatory process in Wegener's granulomatosis.

Figure 1

Up-regulation of the expression of TLR2, TLR3, TLR4, TLR7, TLR9, NOD1, and NOD2 in human PBMCs in response to anti-PR3 Abs. PBMCs were stimulated with murine anti-human PR3 Abs (1 μg/ml, filled graphs) or an isotype-matched control IgG (1 μg/ml, bold line). After 6 h of incubation, the expression of cell-surface TLR2 and TLR4 and intracellular TLR3, TLR7, TLR9, NOD1, and NOD2, was assessed by flow cytometry. The thin lined curve is the staining with a control Ab. The results presented are representative of four different experiments.

Figure 2

Enhancement of TLR and NOD ligand-induced IL-8 production in human monocytic THP-1 cells preincubated with PR3-ANCA sera. THP-1 cells (2 × 10⁵ per ml) were preincubated for 6 h with 1:100 dilutions of PR3-ANCA sera from WG patients (Q9-40, R9-10, S9-31, R9-7, S9-29, S9-32, and D10-41) (closed bar) or with equal amounts of normal serum (sham incubation, open bar). Subsequently, THP-1 cells were challenged with FSL-1 (1 nM), poly I:C (1 μg/ml), lipid A (10 ng/ml), ssPoly U (10 μg/ml), CpG DNA (1 μM), FK156 (100 μg/ml), or MDP (100 μg/ml) for 18 h. IL-8 levels in the culture supernatants were determined by ELISA, and expressed as means ± S.D. *^,# Significantly different from sham-incubated THP-1 cells and from respective cultures stimulated with the respective ligands alone. The results are representative of three different experiments.

Figure 3

Enhancement of TLR and NOD ligand-induced production of IL-8, MCP-1, IL-6, and TNF-α in human PBMCs preincubated with PR3-ANCA sera. PBMCs (donor 1 and 2, 2 × 10⁵ cells per ml) were preincubated for 6 h with a 1:20 dilution of PR3-ANCA sera (Q9-40 or R9-10 from WG patients or ANCA-negative vasculitis seum (M10-24, M10-29, H10-51, or G10-37) (closed bar), or with equal amounts of normal serum (sham incubation, open bar). Subsequently, PBMCs were challenged with FSL-1 (1 nM), poly I:C (1 μg/ml), lipid A (10 ng/ml), ssPoly U (10 μg/ml), FK156 (100 μg/ml), or MDP (100 μg/ml) for 18 h. The levels of IL-6, IL-8, MCP-1, and TNF-α in the culture supernatants were determined by ELISA, and expressed as means ± S.D. *^,# Significantly different from sham-incubated PBMCs and from respective cultures stimulated with the respective ligands alone. The results are representative of four different experiments.

Figure 4

Enhancement of TLR and NOD ligand-induced production of IL-8 and MCP-1 in human PBMCs preincubated with purified IgGs from PR3-ANCA sera. PBMCs (donor 1 and 2, 2 × 10⁵ cells per ml) were preincubated for 6 h with PR3-ANCA IgG (50 μg/ml, Q9-40 or R9-10) (closed bar) or with an equal amount of normal IgG (sham incubation, open bar). Subsequently, PBMCs were challenged with FSL-1 (1 nM), poly I:C (1 μg/ml), lipid A (10 ng/ml), ssPoly U (10 μg/ml), FK156 (100 μg/ml) or MDP (100 μg/ml) for 18 h. The levels of IL-8 and MCP-1 in the culture supernatants were determined by ELISA, and expressed as means ± S.D. *^,# Significantly different from sham-incubated PBMCs and from respective cultures stimulated with the respective ligands alone. The results are representative of four different experiments.