The potential pathogenic roles of S100A8/A9 and S100A12 in patients with MPO-ANCA-positive vasculitis

Abstract

Background: The significance of S100A8/A9 and S100A12 in anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) has not been clarified. This study was dedicated to exploring the potential pathogenic roles of S100A8/A9 and S100A12 in patients with myeloperoxidase (MPO)-ANCA-positive vasculitis.

Methods: Serum and urine concentrations of S100A8/A9 and S100A12 of forty-two AAV patients were evaluated. The influence of S100A8/A9 and S100A12 on the chemotaxis, the apoptosis, the release of IL-1β, the complement activation, the respiratory burst, as well as the neutrophil extracellular traps (NETs) formation of MPO-ANCA-activated neutrophils was investigated.

Results: The serum and urine S100A8/A9 and S100A12 of active MPO-AAV significantly increased (compared with inactive AAV and healthy controls, p < 0.001) and were correlated with the severity of the disease. In vitro study showed that S100A8/A9 and S100A12 activated the p38 MAPK/NF-κB p65 pathway, increased the chemotaxis index (CI) and the release of IL-1β, extended the life span, and enhanced the complement activation ability of MPO-ANCA-activated neutrophils. The Blockade of TLR4 and RAGE inhibited the effects of S100A8/A9 and S100A12. All above-mentioned effects of S100A8/A9 and S100A12 were ROS-independent because neither S100A8/A9 nor S100A12 enhanced the ROS formation and NETs formation of MPO-ANCA-activated neutrophils.

Conclusion: S100A8/A9 and S100A12 serve as markers for assessing the disease severity, and they may also play a role in MPO-AAV pathogenesis.

Keywords: AAV; ANCA; MPO; S100A12; S100A8/A9.

Fig. 1

Levels of serum and urinary S100A8/A9 and S100A12 in MPO-AAV patients and normal controls. Comparison of concentrations of serum S100A8/A9 (A), serum S100A12 (B), urinary S100A8/A9 (C), and urinary S100A12 (D) between MPO-AAV patients in the active period or remission and NC. E The relationship between serum S100A8/A9 and serum S100A12 in active MPO-AAV. F The relationship between urinary S100A8/A9 and urinary S100A12 in active MPO-AAV. NC: normal controls. ns: not significant

Fig. 2

The correlations between serum/urinary S100A8/A9 and S100A12 and clinical parameters in 34 active MPO-AAV. A–G showed the correlations of serum S100A8/A9 and the level of MPO-ANCA (A), serum ferritin (B), CRP (C), D-Dimer (D), erythrocyte sedimentation rate (E), rheumatoid factor (F), serum albumin (G). H and I showed the correlations of serum S100A12 and the level of MPO-ANCA (H) and serum ferritin (I). J–N showed the correlations of urinary S100A8/A9 and MPO-ANCA (J), BVAS (K), serum creatinine (L), hematuria (M), and urinary NGAL (N). O Showed the correlation between urinary S100A12 and serum creatinine. BVAS: Birmingham vasculitis activity score. NGAL: neutrophil gelatinase-associated lipocalin

Fig. 3

ANCA IgG stimulated neutrophils to release S100A8/A9 (A) and S100A12 (B) concentration-dependently. *p < 0.05, **p < 0.01

Fig. 4

Influence of different concentrations of S100A8/A9, and S100A12 on the chemotaxis and cell death of neutrophils. A The chemotaxis index of ANCA-activated neutrophils treated with different concentrations of S100A8/A9 and S100A12. B Effects of S100A8/A9 and S100A12 on the cell death of ANCA-activated neutrophils. C–K Representative images of the flow cytometry analysis. C Neutrophils were incubated with PBS. D Neutrophils were incubated with normal-IgG. E Neutrophils were incubated with ANCA-IgG alone. F–H Neutrophils were incubated with ANCA combined with 1, 5, and 10 μg/ml S100A8/A9, respectively. I–K Neutrophils were incubated with ANCA combined with 1, 5, and 10 μg/ml S100A12, respectively. *p < 0.05, **p < 0.01, ns: not significant

Fig. 5

S100A8/A9 and S100A12 enhanced the release of IL-1β and complement activation of ANCA-stimulated neutrophils through TLR4/RAGE. A Effects of S100A8/A9 and S100A12 on the release of IL-1β from ANCA-activated neutrophils. B–D Effects of S100A8/A9 and S100A12 on the supernatant concentrations of C5a, CBb, and sC5b-9 of ANCA-activated neutrophils. *p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant

Fig. 6

S100A8/A9 and S100A12 played their pro-inflammatory effects through the p38 MAPK/NF-κB p65 pathway. The full-length blot of GAPDH in A was absent for the limited exposure space. A S100A8/A9 and S100A12 induced p38 MAPK phosphorylation and NF-κB p65 expression. B The phosphorylation ratios of p38 after neutrophils were stimulated with a combination of ANCA and S100A8/A9 or S100A12. C The activation of NF-κB p65 after neutrophils were stimulated with a combination of ANCA and S100A8/A9 or S100A12. D The phosphorylation of p38 MAPK and the activation of NF-κB p65 after the blockade of TLR4 and RAGE. E The phosphorylation ratio of p38 after blocking TLR4 and RAGE. F The expression of NF-κB p65 after blocking TLR4 and RAGE. *p < 0.05, **p < 0.01, ns: not significant

Fig. 7

Effects of S100A8/A9 and S100A12 on the production of ROS and NETs in AAV. A–I Representative images of influences of S100A8/A9 and S100A12 on ANCA-induced production of ROS. Neutrophils were incubated with different stimulators and analyzed using flow cytometry. A Neutrophils were incubated with PBS. B Neutrophils were incubated with normal-IgG. C Neutrophils were incubated with ANCA. D–F Neutrophils were incubated with ANCA combined with 1, 5, and 10 μg/ml S100A8/A9, respectively. G–I Neutrophils were incubated with ANCA combined with 1, 5, and 10 μg/ml S100A12, respectively. J The mean fluorescence intensity of ROS after neutrophils were incubated with different stimulators. K Effects of S100A8/A9 and S100A12 on ANCA-induced formation of NETs. NE: neutrophil elastase