Role of Mitogen-Activated Protein Kinases in Activation of Human Neutrophils by Antineutrophil Cytoplasmic Antibodies

J Am Soc Nephrol 12: 37–46, 2001 Role of Mitogen-Activated Protein Kinases in Activation of Human Neutrophils by Antineutrophil Cytoplasmic Antibodies

RALPH KETTRITZ, ADRIAN SCHREIBER, FRIEDRICH C. LUFT, and HERMANN HALLER Franz Volhard Clinic and Max Delbrück Center for Molecular Medicine, Medical Faculty of the Charité, Humboldt University of Berlin, Berlin, Germany.

Abstract. Antineutrophil cytoplasmic antibodies (ANCA) may to 3.3 Ϯ 1.3 for MPO-ANCA). The p38-MAPK inhibitor be important in the pathophysiology of necrotizing vasculitis. (SB202190) and the ERK inhibitor (PD98059) diminished ANCA activate human neutrophils primed with tumor necrosis PR3-ANCA–mediated superoxide production dose depen- ␣ ␣ ␣ Ϯ – Ϯ ␮ factor- (TNF- ) in vitro. TNF- priming results in translo- dently (11.6 1.7 nmol O2 to 1.9 0.6 with 50 M cation of ANCA antigens to the cell surface, where they are SB202190 and 4.0 Ϯ 0.6 with 50 ␮M PD098059, respective- recognized by the antibodies. The signaling mechanisms in- ly). For MPO-ANCA, the results were similar (16.0 Ϯ 2.8 volved in TNF-␣ priming and subsequent ANCA-induced ac- nmol to 0.9 Ϯ 1.0 nmol with SB202190 and 6.4 Ϯ 2.4 nmol tivation were investigated. TNF-␣–primed neutrophils were with PD98059, respectively). Western blot showed phosphor- stimulated with monoclonal antibodies (MAb) to human my- ylation of both p38-MAPK and ERK during TNF-␣ priming. eloperoxidase (MPO) and proteinase 3 (PR3), and with prep- The p38-MAPK inhibitor and the ERK inhibitor showed the arations of human ANCA (three patients with PR3-ANCA and strongest effect on respiratory burst when added before TNF-␣ two patients with MPO-ANCA). Respiratory burst was mea- priming, further supporting an important role for both signaling sured with superoxide dismutase-inhibitable ferricytochrome C pathways in the priming process. Flow cytometry showed that reduction and using dihydro-rhodamine-1,2,3. Phosphorylation p38-MAPK inhibition decreased the translocation of PR3 (by of p38 mitogen-activated protein kinase (p38-MAPK) and the 93 Ϯ 2%) and of MPO (by 64 Ϯ 2%). In contrast, no such extracellular signal-regulated kinase (ERK) were assessed by effect was seen when ERK was inhibited. Thus, p38-MAPK immunoblotting. ANCA-antigen translocation was studied by and ERK are important for the TNF-␣–mediated priming of flow cytometry. The tyrosine phosphorylation inhibitor neutrophils enabling subsequent ANCA-induced respiratory genistein, but not calphostin or staurosporin, resulted in a burst. However, both pathways show differential effects, significant dose-dependent superoxide generation inhibition whereby p38-MAPK controls the translocation of ANCA an- (11.6 Ϯ 1.7 nmol to 2.1 Ϯ 0.5 for PR3-ANCA, and 16.0 Ϯ 2.8 tigens to the cell surface.

Antineutrophil cytoplasmic antibodies (ANCA) occur in pa- TNF-␣–treated neutrophils in vitro, triggering the release of tients with microscopic polyangiitis, Wegener’s granulomato- reactive oxygen species, toxic granule components, cytokines, sis, Churg-Strauss syndrome, and pauci-immune necrotizing and leukotrienes (7,10–16). ANCA-activated neutrophils ad- crescentic glomerulonephritis (NCGN) (1–6). The two major here to and damage endothelial cells (17–19). This cytotoxic target antigens for ANCA are proteinase 3 (PR3) and myelo- effect is at least in part mediated by PR3 and elastase (20) and peroxidase (MPO), which are stored in intracellular granules of can initiate endothelial cell apoptosis (21,22). The early pres- neutrophils and in the lysosomes of monocytes (reviewed in ence of neutrophils within vasculitic lesions has been shown in references 5,6). Neutrophil treatment with subactivating con- patients (23). The importance of neutrophils for the develop- centrations of tumor necrosis factor-␣ (TNF-␣) (priming) re- ment of early vasculitic changes has been demonstrated in sults in the translocation of target antigens from the cytoplasm animals (24). However, little is known about the intracellular to the extracellular membrane, where the antigens become events that control ANCA-mediated activation of TNF-␣– accessible to circulating antibodies (7,8). Circulating neutro- primed neutrophils. Several soluble and particulate stimuli phils that express PR3 were detected in patients with active result in increased tyrosine phosphorylation of cellular pro- Wegener’s granulomatosis (9). Human ANCA can activate teins, and the importance of this signaling event to neutrophil functions, including generation of reactive oxygen species, has been demonstrated (25–27). Radford et al. (28) reported that Received December 16, 1999. Accepted June 15, 2000. activation of cytokine-primed neutrophils by ANCA IgG re- Correspondence to Dr. Ralph Kettritz, Division of Nephrology, Franz Volhard sults in tyrosine phosphorylation of multiple proteins, includ- Clinic, Wiltbergstrasse 50, 13122 Berlin, Germany. Phone: 49-30-9417-2202; Fax: 49-30-9417-2206; E-mail: [email protected] ing p39, p41, and p46 kD bands, respectively. The data that we present here indicate a functional role for tyrosine phosphory- 1046-6673/1201-0037 Journal of the American Society of Nephrology lation in ANCA activation of neutrophils. We explored this Copyright © 2001 by the American Society of Nephrology issue in more detail and focused on mitogen-activated protein 38 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 37–46, 2001

kinases (MAPK). MAPK are activated via phosphorylation of purified by protein G column chromatography. A mouse monoclonal threonine and tyrosine residues by upstream dual-specificity to MPO (MPO-7, IgG1k) and an isotype-matched control (IgG1␬) kinases and provide potent inflammatory signaling pathways were purchased from Dako. (29,30). Furthermore, the activation of MAPK activity in neutrophils can be inhibited by genistein (31–33). Therefore, we Superoxide Generation Assay tested the hypothesis that the p38-MAPK and the extracellular Superoxide was measured using the assay of SOD-inhibitable signal-regulated kinase (ERK) are intimately involved in acti- reduction of ferricytochrome C as described by Pick and Mizel (34). ␮ vation of human neutrophils by ANCA. Briefly, neutrophils were pretreated with 5 g/ml cytochalasin B for 15 min at 4°C. Cells (1 ϫ 106) were primed with 2 ng/ml TNF-␣ for 15 min at 37°C before stimuli or buffer control was added. The final Materials and Methods concentrations were 5 ␮g/ml for the MAb to MPO, 15 ␮g/ml for the Materials MAb to PR3, 75 ␮g/ml for purified IgG preparations, 1 ␮M FMLP, Plasmagel was obtained from Cellular Products Inc. (Buffalo, NY), and 50 ng/ml PMA. All experiments were set up in duplicates. When and Ficoll-Hypaque was obtained from Sigma (Deisenhofen, Germa- indicated, cells were preincubated with inhibitory compounds or ny). Hanks balanced salt solution (HBSS), trypan blue, and phos- buffer control for 30 min on ice before cells were primed with 2 ng/ml phate-buffered saline (PBS) were obtained from Seromed (Berlin, TNF-␣. The mixture was incubated in 96-well plates at 37°C for up to Germany). Recombinant TNF-␣ was obtained from Genzyme (Rüs- 120 min, and the absorption of samples with and without 300 U/ml selsheim, Germany). Genistein, SB202190, PD98059, staurosporin, SOD was scanned repetitively at 550 nm using a Microplate Auto- and calphostin C were purchased from Calbiochem (Bad Soden, reader. No activating effect was seen when human and mouse control Germany). Formyl-methionyl-leucyl-phenylalanine (FMLP), daid- antibodies were used or when cells were primed with 2 ng/ml TNF-␣. zein, bovine erythrocyte superoxide dismutase (SOD; 2500 to 7000 The baseline activity of TNF-␣–treated neutrophils was determined in U/mg protein), ferricytochrome C, cytochalasin B, and phorbol my- every experiment and was factored for each condition. In each inde- ristate acetate (PMA) were from Sigma. Dihydro-rhodamine-1,2,3 pendent experiment using human ANCA-IgG preparations, three dif- (DHR) was from Molecular Probes (Eugene, OR). The polyclonal ferent PR3-ANCA and two different MPO-ANCA preparations were rabbit antibodies to tyrosine phosphorylated p38-MAPK and ERK tested. were acquired from New England Biolabs, Inc. (Beverly, MA), horseradish peroxidase-labeled donkey anti-rabbit IgG was from Amer- Measurement of Respiratory Burst by Oxidation of sham (Braunschweig, Germany), and FITC-conjugated F(ab) -frag- 2 DHR to Rhodamine ment of goat anti-mouse IgG was from Dako (Hamburg, Germany). The generation of reactive oxygen radicals was additionally as- The 96-well microtiter plates were from TPP-Company (Munich, sessed using DHR. This method is based on the fact that reactive Germany). Endotoxin-free reagents and plastic disposables were used oxygen radicals cause an oxidation of the nonfluorescence DHR to the in all experiments. green fluorescence rhodamine. In brief, neutrophils (1 ϫ 107/ml HBSS) were placed in polypropylene tubes and kept for 5 min at 37°C Isolation of Human Neutrophils in a water bath. Cells were loaded with DHR (1 ␮M) for 10 min at Polymorphonuclear leukocytes (PMN) of healthy human volun- 37°C. After 15 min of priming with 2 ng/ml TNF-␣, cells were teers were isolated from heparinized whole blood by red blood cell divided and 5 ϫ 105 cells were incubated with the stimuli in a total sedimentation with plasma gel, followed by Ficoll-Hypaque density assay volume of 100 ␮l. When indicated, cells were preincubated with gradient centrifugation. Erythrocytes were lysed by incubation with inhibitory compounds or buffer control for 30 min on ice before the hypotonic saline for 15 s. PMN were spun down (200 g, 7 min) and priming. After 45 min, the reactions were stopped by adding 900 ␮l reconstituted in HBSS with calcium and magnesium (HBSSϩϩ). ice-cold PBS/1% bovine serum albumin. Samples were kept on ice PMN (10 ␮l) in suspension were incubated with 40 ␮l of trypan blue and analyzed using a FACScan (Becton Dickinson, Heidelberg, Ger- for 5 min at room temperature. Cells were counted in duplicate using many). Neutrophils were identified in the scatter diagram, and data a hemocytometer and considered viable if able to exclude trypan blue. were collected from 10,000 cells per sample. The shift of green The cell viability was detected in every cell preparation and found to fluorescence in the FL-1 mode was determined. For each condition, be greater than 99%. The percentage of PMN in the suspension was the mean fluorescence intensity (MFI, representing the amount of greater than 95% by a Wright-Giemsa staining and by light generated reactive oxygen radicals) is reported. microscopy. Western Blot Analysis for Phosphorylated p38-MAPK Preparation of Immunoglobulins and ERK Human IgG was prepared from Wegener’s granulomatosis (three PMN were stimulated with TNF-␣ 2 ng/ml or buffer for 15 min PR3-ANCA) and microscopic polyangiitis (two MPO-ANCA) pa- followed by stimulation with the MAb to MPO, isotype control, and tients and from three healthy controls. Samples were obtained from buffer control, respectively. Samples were harvested at the indicated freshly drawn blood and kept at Ϫ20°C. Plasma was filtered through time points, and cell lysates were prepared by resuspending 2 ϫ 106 a 0.2-␮m syringe filter (Gelman Sciences, Ann Arbor, MI) and cells in 20 ␮l of ice-cold lysing solution (20 mM Tris-Hcl [pH 8.0] applied to a protein G affinity column (Pharmacia, Uppsala, Sweden). containing 138 mM NaCl, 1% Triton X-100, 2 mM ethylenediami- Bound immunoglobulins were eluted with 0.1 M glycine-HCl buffer, netetraacetate, 10% glycerol, 0.2 mM sodium orthovanadate, 1 mM pH 2.75 (elution buffer). After the antibodies emerged the column, the PMSF, 10 ␮g/ml aprotinin, 10 ␮g/ml leupeptin, 0.1 mM quercetin, 5 pH was immediately adjusted to pH 7.0 using 1 M Tris-HCl, pH 9.0. mM Iodoacetamide). Samples were stored for 20 min on ice and A mouse monoclonal anti-PR3 antibody (IgG1␬) previously used in centrifuged at 12,000 ϫ g for 5 min at 4°C. Supernatant was recov- our laboratory (13) was concentrated from tissue culture supernatant ered and protein concentration was estimated by bicinchoninic acid by using dia-flo ultrafilter YM 100 (Amicon, Beverly, MA) and protein assay (Pierce, Munich, Germany). Samples were incubated for J Am Soc Nephrol 12: 37–46, 2001 MAPK in Activation of Human Neutrophils by ANCA 39

5 min at 95°C in loading buffer (250 mM Tris-Hcl [pH 6.8] with 4% sodium dodecyl sulfate, 20% glycerol, 0.01% bromphenol blue, 6% ␤-mercaptoethanol) and 25 ␮g of protein per lane were loaded on 10% sodium dodecyl sulfate-polyacrylamide gel, electrophoresed, and blotted onto polyvinylidene difluoride membrane by semidry tech- nique. Membrane was blocked in 5% skim milk/0.05% Tween/PBS overnight at 4°C. Phosphotyrosine was detected using specific polyclonal rabbit antibodies to phospho-p38 and phospho-ERK1/2, respectively (1:1,000 dilution) and a horseradish peroxidase-labeled donkey anti-rabbit IgG (1:1,000). Blot was developed by incubation in a chemiluminescence substrate (ECL, Amersham) and exposed to a x-ray film. Densitometry of phospho-p38 and phospho-ERK1/2 was performed with scanned x-ray films and the NIH image program.

Assessment of ANCA-Antigen Expression by Flow Cytometry Flow cytometry was used to evaluate the effect of genistein, the p38-MAPK inhibitor (SB202190), and the ERK-inhibitor (PD98059) on PR3 and MPO expression on neutrophils. Immunostaining was performed as described previously (13). Cells were preincubated with 50 ␮M of the inhibitors or buffer control for 30 min on ice, followed by treatment with 2 ng/ml TNF-␣ or buffer control for 15 min at 37°C. Cells were pelleted at 200 ϫ g for 5 min at 4°C and resuspended in 1 ml ice-cold PBS/0.5% paraformaldehyde. After the cells were washed in HBSS without Ca2ϩ/Mg2ϩ, they were incubated with dilutions of MAb to PR3, MPO, or an isotype control followed by a secondary FITC-conjugated F(ab)2-fragment of goat anti-mouse IgG. Flow cytometry was performed on the same day using a FACScan, and 10,000 events per sample were collected.

Statistical Analysis Results are given as mean Ϯ SEM. Comparisons were made by t test or ANOVA as appropriate. Figure 1. The effect of 50 ␮M genistein, 100 nM calphostin, and 100 nM staurosporin on superoxide release of tumor necrosis factor-␣ Results (TNF-␣)-primed neutrophils (2 ng/ml) activated with a monoclonal Signal Transduction Inhibitors and Respiratory Burst antibody (MAb) to MPO (5 ␮g/ml) was studied in three parallel We first studied the effect of the classical signal transduction experiments (A). The inhibitors were added before TNF-␣ priming. inhibitors genistein, staurosporin, and calphostin on the super- Superoxide release was measured using a continuous assay of superoxide generation of human neutrophils primed with TNF-␣ and oxide dismutase (SOD)-inhibitable reduction of ferricytochrome C. A activated by ANCA. Neutrophils were preincubated with sig- dose-response curve for genistein was titrated over a range of 10–7 to nal transduction inhibitors before the priming with TNF-␣ and 10–4 M. The results for the 60-min time point are shown in B. the subsequent stimulation with ANCA. Using the assay of SOD-inhibitable reduction of ferricytochrome C, our results demonstrate that genistein decreased superoxide production by nmol. Preincubation with genistein resulted in a release of 3.3 30 min, whereas staurosporin and calphostin did not (Figure Ϯ 1.3 nmol (P Ͻ 0.001). To exclude the possibility that 1A, n ϭ 3). Increasing concentrations of genistein (10–7 to genistein resulted in toxic effects that compromised the respi- 10–4 M) had a progressively greater effect (Figure 1B). We ratory burst, we determined the generation of superoxide in next tested the effect of the inhibitors in experiments using PMA-treated cells in the absence and in the presence of 50 ␮M human ANCA as stimulators. Figure 2 indicates that 50 ␮M genistein. Neutrophils were incubated with 50 ␮M genistein or genistein also inhibited the respiratory burst when neutrophils buffer control for 30 min on ice before the stimulation with 50 were activated with human PR3- and MPO-ANCA, respec- ng/ml PMA. At 60 min, cells that were pretreated with buffer Ϯ – 6 tively. In each of the five experiments using human PR3 control generated 47.7 6.0 nmol O2 /10 cells, and pretreat- ANCA, ANCA preparations from three different patients were ment with genistein resulted in 45.7 Ϯ 5.0 nmol (n ϭ 3). These tested, and in each experiment using human MPO-ANCA, data indicate that genistein used at 50 ␮M did not affect the ANCA preparations from two different patients were tested. functional activity to generate superoxide. In addition, we Cells activated with PR3-ANCA released 11.6 Ϯ 1.7 nmol excluded that the observed effect of genistein on ANCA- – 6 O2 /10 cells per 60 min. Cells that were preincubated with 50 stimulated respiratory burst was nonspecific by showing that ␮M genistein released only 2.1 Ϯ 0.5 nmol (P Ͻ 0.001). These daidzein, an inactive analog of genistein, did not inhibit respi- values for MPO-ANCA without genistein were 16.0 Ϯ 2.8 ratory burst. After 60 min, cells that were pretreated with 40 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 37–46, 2001

Figure 2. The inhibitory effect of genistein on superoxide release was Figure 3. The concentration dependence of the effect of SB202190 studied in experiments using human proteinase 3–antineutrophil cy- and PD98059 on the generation of superoxide was studied in three ϭ Ͻ toplasmic antibodies (PR3-ANCA; n 5; P 0.001), and human experiments. Neutrophils were preincubated for 30 min on ice with ϭ Ͻ myeloperoxidase (MPO)-ANCA (n 5; P 0.001), in addition to increasing doses of SB202190 (z) and PD98059 (1), primed with 2 ϭ Ͻ ϭ the murine MAb to MPO (n 8, P 0.001) and to PR3 (n 4; P ng/ml TNF-␣, and activated with 5 ␮g/ml MAb to MPO. Superoxide Ͻ f e ␮ 0.05). , controls; , samples that were pretreated with 50 M release was measured using a continuous assay of SOD-inhibitable genistein. In each of the five independent experiments using human reduction of ferricytochrome C, and the results are shown for the PR3-ANCA, ANCA preparations from three different patients were representative 60-min time point. tested, and in each experiment using human MPO-ANCA, ANCA preparations from two different patients were tested. Superoxide release was measured using a continuous assay of SOD-inhibitable reduction of ferricytochrome C. Immunoglobulins from healthy hu- and the MAb to MPO and PR3, respectively. In each of the six ␬ man controls (human Control) and a murine IgG1 isotype control experiments using human PR3-ANCA, ANCA preparations (murine Control) did not induce a respiratory burst (n ϭ 5). *, P Ͻ from three different patients were tested, and in each of the six 0.05; **, P Ͻ 0.001. experiments using human MPO-ANCA, ANCA preparations from two different patients were tested. For clarity, although tested in a continuous superoxide assay, the data are given for buffer control and stimulated with the MAb to MPO (5 ␮g/ml) the representative 60-min time point of activation (Figure 4). Ϯ – generated 27.8 2.2 nmol O2 . Pretreatment with genistein Both components resulted in a significant inhibition of the decreased this amount to 11.3 Ϯ 3.2 nmol, whereas daidzein respiratory burst. Activating neutrophils with MPO-ANCA resulted in 30.5 Ϯ 2.5 nmol (n ϭ 3). As demonstrated for the Ϯ – 6 resulted in 16.0 2.8 nmol O2 /10 cells, whereas preincuba- activation with the MAb to MPO, staurosporin and calphostin tion with SB202190 decreased this amount to 0.9 Ϯ 1.0 nmol also had no effect on neutrophil activation by human ANCA (P Ͻ 0.001) and preincubation with PD98059 to 6.4 Ϯ 2.4 (data not shown), whereas staurosporin inhibited the superox- nmol (P Ͻ 0.01). PR3-ANCA stimulated 11.6 Ϯ 1.7 nmol O –, Ϯ 2 ide generation in response to PMA (at 60 min from 41.6 5.6 and this amount was decreased by SB202190 to 1.9 Ϯ 0.6 nmol – 6 Ϯ ϭ nmol O2 /10 cells to 4.5 3.0 nmol; n 3), and calphostin – Ͻ Ϯ – Ͻ O2 (P 0.001) and by PD98059 to 4.0 0.6 nmol O2 (P decreased superoxide release in neutrophils stimulated with 0.01). These results indicate that p38-MAPK and ERK are FMLP (at 60 min from 18.1 Ϯ 4.6 nmol to 5.9 Ϯ 2.8 nmol; important for the respiratory burst of human neutrophils n ϭ 3), demonstrating that both inhibitors were functioning. primed with TNF-␣ and activated by ANCA. These data indicate that tyrosine kinase activation plays a role Using the dihydrorhodamine oxidation test, we studied the in the respiratory burst of human neutrophils primed with effect of genistein, SB202190, and PD98059 on ANCA-medi- TNF-␣ and activated by ANCA. ated respiratory burst in human neutrophils by a second inde- Because tyrosine kinase activation can result in stimulation Ϯ of MAPK-dependent pathways, we examined the role of p38- pendent method. The MFI was 11 1 in untreated cells and 20 Ϯ ␣ MAPK and ERK in ANCA-mediated superoxide release of 1 in cells primed with 2 ng/ml TNF- . The MFI value Ϯ ␣ human neutrophils. Using the MAb to MPO, we titrated a increased to 578 139 in TNF- –primed neutrophils activated Ϯ dose-response curve for the effect of pretreatment of neutro- with the MAb to MPO. This number was decreased to 18 4 ␮ Ϯ ␮ phils with the specific p38-MAPK inhibitor (SB202190) and by 50 M genistein, to 18 5by50 M SB202190, and to the ERK inhibitor (PD98059). Figure 3 demonstrates that 267 Ϯ 89 by 50 ␮M PD98059 (n ϭ 3). A typical experiment SB202190 inhibits already at lower concentrations of 1 ␮M, is shown in Figure 5. These results confirm the assay of whereas the inhibitory effect of PD98059 required higher con- SOD-inhibitable reduction of ferricytochrome C, showing that centrations of 50 ␮M. Both compounds strongly inhibit at 50 all three inhibitors decreased the respiratory burst in response ␮M and were selected for the experiments with human ANCA to ANCA. J Am Soc Nephrol 12: 37–46, 2001 MAPK in Activation of Human Neutrophils by ANCA 41

TNF-␣ and after 15 min activated with 5 ␮g/ml of the MAb to MPO. On the basis of the kinetics of tyrosine phosphorylation seen in Figure 6, samples were analyzed after 5 min of priming with TNF-␣ and after 5 min of stimulation with the MAb to MPO, respectively. Our data indicate that genistein and PD98059 but not daidzein inhibited phosphorylation of ERK, whereas none of the inhibitors diminished the phosphorylation of p38-MAPK.

Time-Dependent Inhibition of Superoxide by Genistein, the p38-MAPK Inhibitor, and the ERK Inhibitor To analyze further the role of p38-MAPK and ERK during TNF-␣ priming and during the subsequent ANCA stimulation, we studied the effect of the p38-MAPK inhibitor, the ERK inhibitor, and genistein when added at different time points. Figure 4. The effect of the p38-MAPK inhibitor (50 ␮M SB202190, z) and the extracellular signal-regulated kinase (ERK) inhibitor (50 Figure 8 shows that the effect of all three inhibitory compounds ␮M PD98059, 1) on superoxide release was determined in experi- is time dependent. Adding the p38-MAPK inhibitor SB202190 ments using the MAb to MPO (n ϭ 6), the MAb to PR3 (n ϭ 4), and genistein 15 min before TNF-␣ and within in the 15-min human PR3-ANCA (n ϭ 6), and human MPO-ANCA (n ϭ 6), time period of TNF-␣ priming resulted in a strong decrease of respectively. Cells were preincubated for 30 min on ice with superoxide release. Both substances demonstrated a similar SB202190 and PD98059, respectively. Superoxide release was mea- degree of inhibition. The inhibiting effect of the ERK inhibitor sured using a continuous assay of SOD-inhibitable reduction of fer- PD98059 was observed only when the component was added ricytochrome C. f, controls. In each of the experiments using human before TNF-␣. All three inhibitors did not decrease superoxide PR3-ANCA, ANCA preparations from three different patients were release significantly when added 15 min after the MAb to tested, and in each experiment using human MPO-ANCA, ANCA MPO. These experiments provide further support for an im- preparations from two different patients were tested. *, P Ͻ 0.05; **, Ͻ portant role of tyrosine kinases and particularly for the p38- P 0.01 compared with cells that were preincubated only with ␣ control buffer. SB202190 inhibited significantly stronger than MAPK and ERK pathway during TNF- priming. PD98059 when neutrophils were activated with the MAb to MPO (†, P Ͻ 0.01). Effect of the p38-MAPK Inhibitor and the ERK Inhibitor on Translocation of ANCA Antigens We finally investigated a possible mechanism by which the p38-MAPK and the ERK pathway may control ANCA-stimu- Phosphorylation of p38-MAPK and ERK in TNF-␣– lated respiratory burst in TNF-␣–primed neutrophils. We ex- Primed Neutrophils Activated by ANCA plored the hypothesis that p38-MAPK and ERK control the We performed Western blot analysis to study tyrosine phos- TNF-␣–mediated translocation of ANCA antigens from the phorylation of p38-MAPK and ERK, respectively. Using an- intracellular granules to the cell surface. Using flow cytometry, tibodies that detect only the phosphorylated forms of both we showed in eight parallel experiments that inhibiting p38- kinases, we analyzed the effect of TNF-␣ priming as well as MAPK with SB202190 (50 ␮M) resulted in a decreased TNF- the effect of the subsequent ANCA stimulation. Figure 6 shows ␣–induced translocation of PR3. Untreated cells showed an representative immunoblots of phosphorylated p38-MAPK and MFI for PR3 membrane expression of 31.4 Ϯ 4.4. That number phosphorylated ERK (Figure 6A) and the corresponding den- was increased to 66.7 Ϯ 8.6 by 2 ng/ml TNF-␣. The TNF-␣ sitometric analysis (Figure 6, B through E). Minimal phosphor- effect was almost abrogated by SB202190 (34.1 Ϯ 6.4; P Ͻ ylation was seen at 0 time, whereas a significantly increased 0.01). MPO membrane expression increased from 4.8 Ϯ 1.1 in phosphorylation was observed after priming with TNF-␣, untreated cells to 14.3 Ϯ 2.8 after TNF-␣ treatment and was reaching a maximum at 5 min. The data show no significant diminished to 8.2 Ϯ 2.2 by SB202190 (P Ͻ 0.05). In contrast, additional effect of the MAb to MPO on the phosphorylation of no such inhibitory effect was seen when the ERK pathway was neither p38-MAPK nor ERK. With the use of antibodies that inhibited (50 ␮M PD98059). A representative FACS analysis recognize the nonphosphorylated forms of both kinases, no for PR3 membrane expression is shown in Figure 9A with the change in expression was observed (data not shown). These corresponding statistics in Figure 9B. In addition, we assessed data suggest an important role for both the p38-MAPK and the effect of 50 ␮M genistein on the TNF-␣–induced increase ERK pathways, during TNF-␣–mediated priming of human in expression of PR3 and MPO (n ϭ 5). The PR3 expression in neutrophils. untreated cells was 27.5 Ϯ 6.1 MFI, and genistein decreased We determined the effect of genistein and the specific in- the TNF-␣–induced increase of PR3 expression from 69.2 Ϯ hibitors of ERK and p38-MAPK on the tyrosine phosphoryla- 14.0 MFI to 46.4Ϯ 9.5 MFI (not significant). The MPO ex- tion of ERK and p38-MAPK (Figure 7). Daidzein, an inactive pression was 4.2 Ϯ 0.8 MFI for untreated cells, and genistein analog of genistein, was used as control. PMN were preincu- decreased the TNF-␣–induced MPO expression from 11.3 Ϯ bated with 50 ␮M of the inhibitors, primed with 2 ng/ml 2.4 to 7.3 Ϯ 1.9 (P Ͻ 0.05). Together, these experiments 42 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 37–46, 2001

indicate that tyrosine phosphorylation and p38-MAPK control the TNF-␣–mediated translocation of ANCA antigens to the cell surface.

Discussion TNF-␣–pretreated human neutrophils can be activated by ANCA in vitro. The results of this study show that tyrosine phosphorylation plays an important role in this process. Ty- rosine kinase activation can result in stimulation of mitogenic signal transduction pathways, including p38-MAPK and ERK. Our data indicate that inhibiting either of these two pathways results in decreased respiratory burst. Furthermore, our data demonstrate that both signaling pathways are crucial for ANCA activation by controlling TNF-␣–mediated priming. We show that both kinases act by different mechanisms. P38- MAPK controls the translocation of ANCA antigens to the cell surface. ERK does not seem to be specifically involved in this translocation. Several groups have shown that MAb to human ANCA antigens and human ANCA itself can activate TNF-␣–primed neutrophils and cause the release of reactive oxygen species, toxic-granule components, cytokines, and leukotrienes (7,10– 16,35,36). These effector functions may play an important role in inducing inflammation in ANCA-associated vasculitis. Sev- eral investigations were performed to determine which part of the ANCA molecule is important for the neutrophil activation, Ј e.g., F(ab )2 fragments versus Fc part (10–13). However, the underlying intracellular signaling events in ANCA-induced activation have not been elucidated in detail. An earlier report indicated that activation of cytokine-primed neutrophils by ANCA IgG results in tyrosine phosphorylation of multiple proteins, including p39, p41, and p46 kD bands, respectively (28). Our observation that genistein abrogates superoxide generation in ANCA-activated TNF-␣–primed neutrophils is in agreement with these findings. In addition, our results indicate a functional significance of tyrosine phosphorylation in this activation process. Neutrophils that are challenged with various stimuli including ultraviolet irradiation, growth factors, and cytokines re- spond with an activation of the MAPK cascade. MAPK are activated via phosphorylation of threonine and tyrosine residues by upstream dual-specificity kinases and phosphorylate substrates on serine or threonine adjacent to proline residues (29,30). Three MAPK pathways have been described; however, only p38-MAPK and ERK play a role in human neutrophils. No stimulus resulting in activation of Jun N-terminal

MAb to MPO (anti-MPO) after preincubation with buffer, 50 ␮M genistein (Gen), 50 ␮M SB202190 (SB), and 50 ␮M PD98059 (PD), respectively. The increase in fluorescence intensity represents the Figure 5. Using the dihydrorhodamine oxidation test, the effect of oxidation of the nonfluorescent dihydrorhodamine to the green fluo- genistein (A), the p38-MAPK inhibitor SB202190 (B), and the ERK rescent rhodamine reflecting the amount of generated reactive oxygen inhibitor PD98059 (C) on the respiratory burst of TNF-␣–primed radicals. A typical of three independent experiments is shown. Prein- neutrophils activated with 5 ␮g/ml MAb to MPO was assessed in cubation with all three inhibitory compounds decreased the respira- parallel. Each graph shows the results from unstimulated cells (cells), tory burst in TNF-␣–primed neutrophils that were challenged with the TNF-␣–primed cells (TNF), and TNF-␣–primed cells treated with MAb to MPO. J Am Soc Nephrol 12: 37–46, 2001 MAPK in Activation of Human Neutrophils by ANCA 43

Figure 6. Phosphorylation of p38-MAPK and ERK in response to TNF-␣ priming and to the subsequent ANCA stimulation was studied by immunoblotting. Cell aliquots were obtained after 0, 2, 5, and 15 min of TNF-␣ priming (T) with 2 ng/ml and after 2, 5, and 15 min of subsequent treatment with buffer control (C), isotype control (I), and 5 ␮g/ml MAb to MPO (M), respectively. At each time point, phosphorylated p38-MAPK and ERK was determined by immunoblotting, and a representative example is shown (A). The corresponding densitometric analysis is shown in B through E (n ϭ 5). (B) The effect of TNF-␣ priming on ERK phosphorylation with the effect of the subsequent antibody treatment in C. (D) The effect of TNF-␣ priming on p38-MAPK phosphorylation with the effect of the subsequent antibody treatment in E. These data show that p38-MAPK and ERK activation is predominantly a feature of TNF-␣ priming. (Co) -x-, untreated controls; (T) –⅙–, TNF-␣ priming; (M) –▫–, MAb to MPO; (I) --‚--, isotype control buffer; (C) –⅙–, control.

kinases/stress-activated protein kinase in neutrophils has been PD98059 and SB202190 show a good dose-response curve reported. With the use of in vitro kinase activity assays, it was from 0.1 ␮Mupto200␮M with a maximal effect observed at shown that bacterial phagocytosis, granulocyte-macrophage approximately 50 to 100 ␮M (38–41). Here, we demonstrate colony-forming unit, and TNF-␣ stimulate ERK and p38- that genistein and, more important, the specific ERK and MAPK activation in human neutrophils. This effect was inhib- p38-MAPK inhibitors PD98059 and SB202190 decreased ited by the specific inhibitors PD98059 and SB202190 and by ANCA-induced respiratory burst in TNF-␣–primed neutro- the tyrosine phosphorylation inhibitor genistein (31–33). In phils. These findings indicate an important role of both MAPK contrast, cross-linking of the Fc␥II-receptor resulted in a pathways for the activation of neutrophils by ANCA. Conceiv- genistein-independent activation of MAPK (37). Carefully es- ably, the p39, p41, and p46 kD bands observed by Radford et tablished dose-response experiments for the specific inhibitors al. (28) in fact reflect p38-MAPK and ERK (p42/44). 44 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 37–46, 2001

Figure 8. The time dependence of the inhibitory effect of genistein, the p38-MAPK inhibitor, and the ERK inhibitor on superoxide generation was studied using the MAb to MPO. Fifty ␮M genistein, the Figure 7. The effect of genistein (G) and daidzein (D) an inactive p38-MAPK inhibitor SB202190, or the ERK inhibitor PD98059 was analog of genistein on the phosphorylation of ERK and p38 MAPK added 30 min before TNF-␣ (2 ng/ml), 5 min after TNF-␣, and 5 and was studied by immunoblotting. The ERK inhibitor PD98059 (PD) 15 min after the subsequent stimulation with the MAb to MPO and the p38-MAPK inhibitor SB202190 (SB) were tested for com- (␣MPO). Superoxide release was assayed continuously for up to 120 parison. Neutrophils were preincubated with 50 ␮M of the inhibitors, min, and the data are given for the representative 60-min time point primed with 2 ng/ml TNF-␣, and after 15 min activated with 5 ␮g/ml (n ϭ 3; z, SB202190; 1, PD98059; □, genistein). These data show MAb to MPO. Cells were analyzed after isolation (0), after 5 min of that the maximum effect of all three inhibitors is observed if they are treatment with TNF-␣ (T), and after 5 min of stimulation with the applied before TNF-␣ priming. MAb to MPO (M), respectively. Phosphorylation of ERK and p38- MAPK was assessed by Western blotting. Genistein and PD98059 but not daidzein inhibited phosphorylation of ERK. None of the inhibitors (41,43,45). It is interesting that our data also indicate that affected the phosphorylation of p38-MAPK. A typical of two inde- genistein prevented phosphorylation of ERK but not of pendent experiments is shown. p38-MAPK. We demonstrated that p38-MAPK and ERK inhibit TNF-␣– mediated neutrophil priming via different mechanisms. P38- We found a rapid tyrosine phosphorylation of ERK and MAPK inhibition but not ERK inhibition abrogated TNF-␣– p38-MAPK in response to small priming concentrations of mediated translocation of ANCA antigens from cytoplasmic TNF-␣ peaking at 5 min and an additional, although not granules to the cell surface. Several investigators showed that significant, increase during the subsequent ANCA stimulation. treatment of resting neutrophils with TNF-␣ results in in- The strongest effect of the p38-MAPK and ERK inhibitors, as creased expression of PR3 and MPO. This process enables an well as of the tyrosine kinase inhibitor genistein, on superoxide interaction of ANCA with the expressed target antigens, re- generation occurred when the inhibitory compounds were sulting in a full-blown neutrophil activation. Witko-Sarsat et added before TNF-␣ priming. These data indicate the signifi- al. (46) showed that within the normal population, the percent- cance for tyrosine kinases and, in particular, both MAPK age of neutrophils that express PR3 is variable and that a high pathways in TNF-␣–mediated neutrophil priming rather than in PR3 expression is significantly more frequent in patients with the consecutive ANCA stimulation. Other investigators also ANCA-associated vasculitis. Additional activation resulted in reported that TNF-␣ phosphorylates p38-MAPK and ERK a further increase in the amount of PR3 expressed. Conceiv- observing the highest phosphorylation or kinase activities after ably, high membrane PR3-expressing neutrophils could favor 10 min of stimulation (33,42). ERK activation by TNF-␣ was the occurrence or the progression of vasculitic inflammation. also demonstrated by Rafiee et al. (32). However, there is some Thus, understanding the mechanisms that control PR3 and controversy regarding this issue. Other investigators reported MPO expression is important. To our knowledge, our report is that TNF-␣ activates only p38-MAPK (43,44). We showed that the first indicating that the TNF-␣–induced translocation of PD98059, the specific inhibitor of the ERK pathway, blocked ANCA antigens from cytoplasmic granules to the cell surface tyrosine phosphorylation of ERK, whereas the specific inhib- is controlled by p38-MAPK. itor of the p38-MAPK pathway, SB202190, did not prevent Our data demonstrate a similar effect of genistein and the tyrosine phosphorylation of p38-MAPK. This finding is in p38-MAPK inhibitor SB202190 on the respiratory burst to agreement with reports from the literature; others demonstrated ANCA. The strongest inhibition occurred when given before that PD98059 inhibits the upstream MAPK/ERK kinase pre- and during TNF-␣ priming, whereas the ERK inhibitor venting the phosphorylation and therefore the activation of PD98059 affected only superoxide generation when given be- ERK, whereas SB202190 inhibits the p38-MAPK pathway by fore TNF-␣. We showed that genistein and SB202190 reduced competing with ATP for the ATP-binding site on p38-MAPK respiratory burst activity in the entire population of cells, without affecting phosphorylation of p38-MAPK itself whereas PD98059 decreased the activity in only a portion of J Am Soc Nephrol 12: 37–46, 2001 MAPK in Activation of Human Neutrophils by ANCA 45

ferent mechanisms, whereby p38-MAPK but not ERK controls the translocation of ANCA antigens to the cell surface. Phar- macologic blockade of p38-MAPK and ERK may limit inflammatory damage caused by ANCA-activated PMN.

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