Volume 63, Issue 1, Pages 96-106 (January 2003) Extracellular signal-regulated kinase inhibition by statins inhibits neutrophil activation by ANCA  Mira Choi, Susanne Rolle, Madhavi Rane, Hermann Haller, Friedrich C. Luft, Ralph Kettritz  Kidney International  Volume 63, Issue 1, Pages 96-106 (January 2003) DOI: 10.1046/j.1523-1755.2003.00718.x Copyright © 2003 International Society of Nephrology Terms and Conditions

Figure 1 Concentration-dependent effect of cerivastatin (CV) and simvastatin (SV) on tumor necrosis factor-α (TNF-α)-primed neutrophils stimulated with a monoclonal antibody (mAb) to myeloperoxidase (MPO). In parallel experiments, increasing doses of CV (A) or SV (B) were added 30 minutes prior to priming with 2 ng/mL TNF-α. Symbols are: (□) control; (○) 1 μmol/L; (⋄) 5 μmol/L; (▵) 10 μmol/L; (*) 25 μmol/L. Seven (CV) or six (SV) independent experiments were done and superoxide was measured by the continuous ferricytochrome C assay up to 45 minutes. Kidney International 2003 63, 96-106DOI: (10.1046/j.1523-1755.2003.00718.x) Copyright © 2003 International Society of Nephrology Terms and Conditions

Figure 2 Respiratory burst activity in response to a mAb to MPO by CV and SV using nitro blue tetrazolium (NBT). Neutrophils were incubated for 45 minutes in buffer control (A), PMA (B), or stimulated with the mAb to MPO after TNF-α priming (C). TNF-α primed cells stimulated with the mAb to MPO were pretreated with 10 μmol/L CV (D) or 10 μmol/L SV (E). Representative pictures from 5 independent experiments are shown. (F) The corresponding statistical analysis. Respiratory burst activity results in a reduction from yellow NBT to blue-black formazan. This response was inhibited by CV and SV (**P < 0.01). Kidney International 2003 63, 96-106DOI: (10.1046/j.1523-1755.2003.00718.x) Copyright © 2003 International Society of Nephrology Terms and Conditions

Figure 3 Effects of 10 μmol/L cerivastatin (CV) and 10 μmol/L simvastatin (SV) (dotted bars) on superoxide generation of TNF-α–primed neutrophils stimulated with human PR3-ANCA (open bars) and MPO-ANCA (gray bars) was tested. For CV, a total of 18 experiments with six different PR3-ANCA preparations and a total of 10 experiments with two different MPO-ANCA preparations were performed. For SV, 12 experiments with five different PR3-ANCA preparations and 5 experiments with one MPO-ANCA were carried out. Superoxide formation was measured using the continuous ferricytochrome C assay, and results are shown for the 45-minute time point (*P < 0.05; **P < 0.01). Kidney International 2003 63, 96-106DOI: (10.1046/j.1523-1755.2003.00718.x) Copyright © 2003 International Society of Nephrology Terms and Conditions

Figure 4 Effects of mevalonic acid (MVA) on CV and SV mediated inhibition of respiratory burst activity were tested in parallel. TNF-α–primed neutrophils were stimulated with a mAb to MPO without statin preincubation (▪, N = 7 independent experiments), with 10 μmol/L CV preincubation (, N = 7) or with 10 μmol/L SV preincubation (□, N = 6) in the presence or absence of 500 μmol/L MVA. Superoxide formation was measured using the continuous ferricytochrome C assay, and results are shown for the 45-minute time point. These results show that both statins inhibit the respiratory burst in a mevalonic acid independent manner (**P < 0.01 compared to TNF-α–primed cells activated with the mAb to MPO). Kidney International 2003 63, 96-106DOI: (10.1046/j.1523-1755.2003.00718.x) Copyright © 2003 International Society of Nephrology Terms and Conditions

Figure 5 Effects of statins on ANCA antigen expression studied by FACS. A typical flow cytometry experiment of the effect of 10 μmol/L cerivastatin (A) and 10 μmol/L simvastatin (B) on TNF-α–induced PR3 translocation is depicted. In these experiments, cells were incubated either with buffer (▪), or stimulated with TNF-α (), or TNF-α stimulated cells were preincubated with increasing concentrations of either cerivastatin (CV; □) or simvastatin (SV; ) prior to TNF-α stimulation, respectively. Three independent experiments show a dose dependent inhibition of membrane translocation of PR3 (C, D) and MPO (E, F) by both statins. Kidney International 2003 63, 96-106DOI: (10.1046/j.1523-1755.2003.00718.x) Copyright © 2003 International Society of Nephrology Terms and Conditions

Figure 6 Effects of 10 μmol/L CV and SV on TNF-α–induced PR3 and MPO membrane expression studied by flow cytometry. Cells incubated with buffer control () or without TNF-α stimulation (▪) were preincubated with CV (□) or SV (). PR3 (A) and MPO (B) membrane expression was measured in 6 independent experiments (**P < 0.01). These results indicate a small but significant inhibition of ANCA antigen expression by both statins. Kidney International 2003 63, 96-106DOI: (10.1046/j.1523-1755.2003.00718.x) Copyright © 2003 International Society of Nephrology Terms and Conditions

Figure 7 Effects of simvastatin (SV) and cerivastatin (CV) on ERK phosphorylation during TNF-α priming assessed by Western blotting. Cells were either left untreated (Ctrl) or treated with 2 ng/mL tumor necrosis factor-α (TNF). TNF-α–treated cells were preincubated with buffer control, 25 μmol/L SV or 50 μmol/L PD (PD), respectively (A, N = 5); a representative experiment for simvastatin is shown. Preincubation also was carried out with 25 or 50 μmol/L cerivastatin; a typical experiment is given in panel B (N = 5). The corresponding statistical analysis of phosphorylated ERK for (C) simvastatin and for (D) cerivastatin (*P < 0.05; **P < 0.01). Kidney International 2003 63, 96-106DOI: (10.1046/j.1523-1755.2003.00718.x) Copyright © 2003 International Society of Nephrology Terms and Conditions

Figure 8 Effects of simvastatin (SV) on p38 phosphorylation during TNF-α priming assessed by Western blotting. Cells were either left untreated (Control) or treated with 2 ng/L TNF-α (TNF). TNF-α–treated cells were preincubated with buffer control, 25 μmol/L SV or 25 μmol/L SB (SB) (N = 5). A representative experiment is shown. Kidney International 2003 63, 96-106DOI: (10.1046/j.1523-1755.2003.00718.x) Copyright © 2003 International Society of Nephrology Terms and Conditions