Phosphoinositol 3-kinase-γ mediates antineutrophil cytoplasmic autoantibody-induced glomerulonephritis
2009; Elsevier BV; Volume: 77; Issue: 2 Linguagem: Inglês
10.1038/ki.2009.420
ISSN1523-1755
AutoresAdrian Schreiber, Susanne Rolle, Ludmilla Peripelittchenko, Jörg Rademann, Wolfgang Schneider, Friedrich C. Luft, Ralph Kettritz,
Tópico(s)Coagulation, Bradykinin, Polyphosphates, and Angioedema
ResumoAntineutrophil cytoplasmic autoantibodies (ANCA) are associated with necrotizing crescentic glomerulonephritis (NCGN) and systemic vasculitis. We examined the role of phosphoinositol 3 kinase-γ isoform (PI3Kγ) in ANCA-activated neutrophil functions. Further, we tested whether its inhibition protects a mouse model of ANCA NCGN from developing NCGN. We transplanted bone marrow from wild-type mice or PI3Kγ-deficient mice into myeloperoxidase-deficient mice immunized with myeloperoxidase. Bone marrow from PI3Kγ−/− mice protected against development of the disease. Similarly, bone marrow transplanted from wild-type mice followed by treatment with the specific PI3Kγ inhibitor AS605240 also protected these mice against NCGN in this model. AS605240 significantly abrogated myeloperoxidase- or proteinase 3-ANCA-stimulated superoxide production in vitro. Furthermore, ANCA-induced degranulation and GM-CSF-stimulated migration in a transwell assay of isolated human neutrophils were also abrogated by the drug. We found that PI3Kγ plays a pivotal role in ANCA-induced NCGN and suggest that its specific inhibition may provide a novel treatment target. Antineutrophil cytoplasmic autoantibodies (ANCA) are associated with necrotizing crescentic glomerulonephritis (NCGN) and systemic vasculitis. We examined the role of phosphoinositol 3 kinase-γ isoform (PI3Kγ) in ANCA-activated neutrophil functions. Further, we tested whether its inhibition protects a mouse model of ANCA NCGN from developing NCGN. We transplanted bone marrow from wild-type mice or PI3Kγ-deficient mice into myeloperoxidase-deficient mice immunized with myeloperoxidase. Bone marrow from PI3Kγ−/− mice protected against development of the disease. Similarly, bone marrow transplanted from wild-type mice followed by treatment with the specific PI3Kγ inhibitor AS605240 also protected these mice against NCGN in this model. AS605240 significantly abrogated myeloperoxidase- or proteinase 3-ANCA-stimulated superoxide production in vitro. Furthermore, ANCA-induced degranulation and GM-CSF-stimulated migration in a transwell assay of isolated human neutrophils were also abrogated by the drug. We found that PI3Kγ plays a pivotal role in ANCA-induced NCGN and suggest that its specific inhibition may provide a novel treatment target. Patients with necrotizing crescentic glomerulonephritis (NCGN) and systemic small-vessel vasculitis feature antineutrophil cytoplasmic autoantibodies (ANCAs).1.van der Woude F.J. Rasmussen N. 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Kimberly W.T. et al.Anti-neutrophil cytoplasmic antibodies engage and activate human neutrophils via Fc gamma RIIa.J Immunol. 1994; 153: 1271-1280PubMed Google Scholar The phosphoinositol 3-kinase (PI3K)/Akt pathway is pivotal for ANCA-induced respiratory burst activity.13.Ben-Smith A. Dove S.K. Martin A. et al.Antineutrophil cytoplasm autoantibodies from patients with systemic vasculitis activate neutrophils through distinct signaling cascades: comparison with conventional Fcgamma receptor ligation.Blood. 2001; 98: 1448-1455Crossref PubMed Scopus (105) Google Scholar, 15.Kettritz R. Choi M. Butt W. et al.Phosphatidylinositol 3-kinase controls antineutrophil cytoplasmic antibodies-induced respiratory burst in human neutrophils.J Am Soc Nephrol. 2002; 13: 1740-1749Crossref PubMed Scopus (61) Google Scholar, 21.Williams J.M. Savage C.O. 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Ferguson G.J. et al.Gbetagammas and the Ras binding domain of p110gamma are both important regulators of PI(3)Kgamma signalling in neutrophils.Nat Cell Biol. 2006; 8: 1303-1309Crossref PubMed Scopus (133) Google Scholar, 30.Ferrandi C. Ardissone V. Ferro P. et al.Phosphoinositide 3-kinase gamma inhibition plays a crucial role in early steps of inflammation by blocking neutrophil recruitment.J Pharmacol Exp Ther. 2007; 322: 923-930Crossref PubMed Scopus (53) Google Scholar implicated the γ-isoform also in ANCA-mediated neutrophil activation. All of these functions participate in ANCA-mediated organ damage. Recently, the small-molecule PI3Kγ inhibitor AS605240 with more than 30-fold selectivity over PI3Kδ and PI3Kβ and 7.5-fold selectivity over PI3Kα was generated. AS605240 (1 μmol/l) blocked PI3Kγ-dependent Akt phosphorylation in primary monocytes, whereas PI3Kγ-independent Akt phosphorylation was not affected.28.Camps M. Ruckle T. Ji H. et al.Blockade of PI3Kgamma suppresses joint inflammation and damage in mouse models of rheumatoid arthritis.Nat Med. 2005; 11: 936-943Crossref PubMed Scopus (644) Google Scholar Furthermore, specific PI3Kγ inhibition blocked glomerulonephritis and improved survival in a lupus erythematosus mouse model.31.Barber D.F. Bartolome A. Hernandez C. et al.PI3Kgamma inhibition blocks glomerulonephritis and extends lifespan in a mouse model of systemic lupus.Nat Med. 2005; 11: 933-935PubMed Google Scholar AS605240 is orally active and 50 mg/kg twice a day suppressed rheumatoid arthritis in another mouse model.28.Camps M. Ruckle T. Ji H. et al.Blockade of PI3Kgamma suppresses joint inflammation and damage in mouse models of rheumatoid arthritis.Nat Med. 2005; 11: 936-943Crossref PubMed Scopus (644) Google Scholar We tested whether the PI3Kγ isoform controls neutrophil functions that mediate ANCA-induced NCGN. We found that PI3Kγ could be a treatment target for ANCA-induced NCGN. We first tested the hypothesis that PI3Kγ has an important function in an ANCA-glomerulonephritis mouse model. Anti-myeloperoxidase (MPO)-mediated NCGN was induced by the previously described bone marrow (BM) transplantation approach.32.Schreiber A. Xiao H. Jennette J.C. et al.C5a receptor mediates neutrophil activation and ANCA-induced glomerulonephritis.J Am Soc Nephrol. 2009; 20: 289-298Crossref PubMed Scopus (255) Google Scholar,33.Schreiber A. Xiao H. Falk R.J. et al.Bone marrow-derived cells are sufficient and necessary targets to mediate glomerulonephritis and vasculitis induced by anti-myeloperoxidase antibodies.J Am Soc Nephrol. 2006; 17: 3355-3364Crossref PubMed Scopus (82) Google Scholar MPO-deficient animals were immunized with MPO, irradiated, and transplanted with BM cells from either wild-type (WT) mice or PI3Kγ gene-deficient (PI3Kγ−/−) animals (n=8 in each group). At 8 weeks after transplantation, mice were killed. All mice (100%) transplanted with WT BM developed hematuria and proteinuria, whereas mice transplanted with PI3Kγ−/− BM did not develop significant urine abnormalities. These results were confirmed by a higher urinary albumin excretion in the mice transplanted with WT BM compared with the PI3Kγ−/− BM group (1047.0±783.5 μg/ml albumin in the WT mice versus 203.9±67.4 μg/ml in the PI3Kγ−/− mice). The values did not reach statistical significance because of high interindividual variability. All mice transplanted with WT BM developed NCGN on histology (100% disease induction) whereas in the PI3Kγ−/− BM group mice developed only weak glomerular abnormalities (six of the eight mice got weak glomerular disease, two did not develop any glomerular abnormalities). When these findings were quantitatively assessed, mice in the WT BM group showed on average 22.3±7.5% crescents and 9.7±2.8% necrosis whereas mice in the PI3Kγ−/− BM group developed 3.1±1.1% crescents and 2.5±0.3% necrosis (P<0.05 for both) (Figure 1). Immunohistology showed weak IgG, IgA, IgM, and C3 deposition that did not differ between both groups (data not shown). By enzyme-linked immunosorbent assay we found similar anti-MPO titer in both experimental groups (WT group: d0 1.25±0.38 arbitrary units (AUs), d56 0.69±0.44 AU; PI3Kγ−/− group: d0 1.22±0.26 AU, d56 0.77±0.22 AU) excluding the possibility that differences in anti-MPO titers were responsible for the protection from NCGN in the PI3Kγ−/− mice. Furthermore, mice transplanted with WT BM showed slightly higher circulating white blood counts and neutrophil counts at d56, probably reflecting disease activity similar to what is seen in patients with active ANCA vasculitis (Table 1). Finally, both groups showed similar engraftment rates with MPO-positive circulating neutrophils (data not shown). These data establish that neutrophil PI3Kγ is important for NCGN induction by anti-MPO antibodies without affecting BM engraftment, anti-MPO titers, or the deposition of immunoglobulins and complement.Table 1Peripheral blood cell analysis at the day of killing in MPO-deficient mice transplanted with WT or PI3Kγ−/− BM (Exp 1) and in VC-treated mice versus AS-treated mice (Exp 2) (data are means±s.e.m.)WBC (Gpt/l)HGB (mg/dl)Lym (Gpt/l)Mo (Gpt/l)Gra (Gpt/l)Exp 1 WT17.2±10.013.4±1.19.7±5.21.9±1.95.6±3.9 PI3Kγ−/−11.7±2.714.4±1.07.0±2.31.0±0.43.5±1.2Exp 2 VC5.9±1.212.3±0.52.7±0.70.3±0.12.4±0.6 AS5.1±0.313.6±0.42.5±6.00.5±0.12.1±0.3Abbreviations: AS, AS605240; BM, bone marrow; MPO, Gpt/l, giga-particles/liter; Gra, granulocyte; HGB, hemoglobin; Lym, lymphocyte; Mo, monocyte; myeloperoxidase; PI3K, phosphoinositol 3-kinase; VC, vehicle control; WBC, white blood cell count; WT, wild type.None of the comparisons showed significant differences. Open table in a new tab Abbreviations: AS, AS605240; BM, bone marrow; MPO, Gpt/l, giga-particles/liter; Gra, granulocyte; HGB, hemoglobin; Lym, lymphocyte; Mo, monocyte; myeloperoxidase; PI3K, phosphoinositol 3-kinase; VC, vehicle control; WBC, white blood cell count; WT, wild type. None of the comparisons showed significant differences. To test whether PI3Kγ has an important function in leukocyte migration in vivo, we assessed glomerular neutrophil and monocyte/macrophage accumulation. The data show a reduced neutrophil and monocyte/macrophage influx in glomeruli from mice transplanted with PI3Kγ−/− BM (Table 2). These findings indicate that PI3Kγ is needed for circulating leukocytes to accumulate in the glomeruli and induce NCGN.Table 2Quantitated immunohistochemical microscopy for glomerular neutrophil and macrophage infiltration in MPO-deficient mice transplanted with WT or PI3Kγ−/− BM (Exp 1) and in VC-treated mice versus AS-treated mice (Exp 2)NeutrophilsMacrophagesGroup% Positive GlomCells/+GlomCells/Glom% Positive GlomCells/+GlomCells/GlomExp 1 WT36.9±6.6*P<0.05.1.4±0.10.5±0.1*P<0.05.48.5±8.0*P<0.05.1.4±0.10.7±0.1 PI3Kγ−/−14.5±4.01.4±0.20.2±0.122.5±5.31.4±0.10.3±0.1Exp 2 VC35.2±11.0*P<0.05.1.5±0.10.5±0.235.3±12.42.4±0.51.0±0.5 AS7.2±2.01.3±0.20.1±0.012.1±2.41.3±0.20.2±0.1Abbreviations: AS, AS605240; BM, bone marrow; MPO, myeloperoxidase; PI3K, phosphoinositol 3-kinase; VC, vehicle control; WT, wild type.Numbers represent mean numbers±s.e.m. of positive glomeruli (% Positive Glom), mean numbers±s.e.m. of positive cells per positive glomeruli (Cells/+Glom), mean numbers±s.e.m. of positive cells per glomerular cross section (Cells/+Glom).* P<0.05. Open table in a new tab Abbreviations: AS, AS605240; BM, bone marrow; MPO, myeloperoxidase; PI3K, phosphoinositol 3-kinase; VC, vehicle control; WT, wild type. Numbers represent mean numbers±s.e.m. of positive glomeruli (% Positive Glom), mean numbers±s.e.m. of positive cells per positive glomeruli (Cells/+Glom), mean numbers±s.e.m. of positive cells per glomerular cross section (Cells/+Glom). We showed earlier that ANCA stimulation results in a second Akt phosphorylation wave in cytokine-primed neutrophils.15.Kettritz R. Choi M. Butt W. et al.Phosphatidylinositol 3-kinase controls antineutrophil cytoplasmic antibodies-induced respiratory burst in human neutrophils.J Am Soc Nephrol. 2002; 13: 1740-1749Crossref PubMed Scopus (61) Google Scholar We used two priming cytokines that differ in their ability to induce PI3Kγ. Tumor necrosis factor-α (TNFα) acts through G-protein-coupled-receptor-mediated PI3Kγ activation and granulocyte macrophage colony-stimulating factor (GM-CSF) activates RTK that does not induce PI3Kγ.34.al-Shami A. Bourgoin S.G. Naccache P.H. Granulocyte-macrophage colony-stimulating factor-activated signaling pathways in human neutrophils. I. Tyrosine phosphorylation-dependent stimulation of phosphatidylinositol 3-kinase and inhibition by phorbol esters.Blood. 1997; 89: 1035-1044Crossref PubMed Google Scholar, 35.Cadwallader K.A. Condliffe A.M. McGregor A. et al.Regulation of phosphatidylinositol 3-kinase activity and phosphatidylinositol 3,4,5-trisphosphate accumulation by neutrophil priming agents.J Immunol. 2002; 169: 3336-3344Crossref PubMed Scopus (59) Google Scholar, 36.Yasui K. Sekiguchi Y. Ichikawa M. et al.Granulocyte macrophage-colony stimulating factor delays neutrophil apoptosis and primes its function through Ia-type phosphoinositide 3-kinase.J Leukoc Biol. 2002; 72: 1020-1026PubMed Google Scholar Accordingly, we observed significant inhibition of TNFα-induced Akt phosphorylation with 10 μmol/l LY294002 as a nonspecific PI3K inhibitor and with 0.5 μmol/l AS605240 as a PI3Kγ-specific inhibitor (Figure 2a). In contrast, LY294002 prevented Akt phosphorylation in GM-CSF-treated neutrophils whereas AS605240 did not. Western blot experiments indicate that 0.5 μmol/l AS605240 does not block p38 and extracellular signal-regulated kinase (ERK) phosphorylation underscoring the specificity for PI3Kγ inhibition (Figure 2b and c). Stimulation with human ANCA after cytokine priming caused a second strong Akt phosphorylation wave that was not observed with control IgG. Pretreatment with either LY294002 or AS605240 blocked Akt phosphorylation in both GM-CSF- and TNFα-primed neutrophils subsequently activated by human ANCA IgG (Figure 3). These findings show that ANCA-activated PI3K and that PI3Kγ isoform inhibition blocked Akt phosphorylation.Figure 3Western blot analysis for phospho-Akt is shown. Neutrophils were preincubated with 10 μmol/l LY294002 (LY), 0.5 μmol/l AS605240 (AS), or dimethyl sulfoxide as a control (-). Cells were subsequently primed with buffer (B) or tumor necrosis factor α (TNF) (a) or buffer or granulocyte macrophage colony-stimulating factor (GM-CSF) (b) for 45 min, followed by stimulation with control IgG (ctrl), myeloperoxidase-antineutrophil cytoplasmic autoantibodies (MPO-ANCA), or PR3-ANCA. After 10 min, samples were harvested, S473 phosphorylated Akt was determined by immunoblotting, and a representative example of four independent experiments is shown in panels a and b. Total Akt is shown as loading control. The corresponding densitometric analysis is given in panel c (n=4). These data show that LY294002 and AS605240 block ANCA-induced phosphoinositol 3-kinase (PI3K)/Akt activation.View Large Image Figure ViewerDownload (PPT) We next tested whether PI3Kγ inhibition prevents respiratory burst by ANCA. We first titrated a dose-response study where cells were preincubated over an AS605240 range from 2 × 10−6 to 1 × 10−9 mol/l for 30 min followed by GM-CSF priming and treatment with a monoclonal antibody (mAb) to MPO (Figure 4a). On the basis of these data, neutrophils were then pretreated with 0.5 μmol/l AS605240, primed with GM-CSF or TNFα, and subsequently activated with human ANCA IgG. Incubation of TNFα- or GM-CSF-primed neutrophils with control IgG did not result in generation of superoxide, whereas stimulation with MPO-ANCA IgG or proteinase 3 (PR3)-ANCA IgG induced a strong respiratory burst (Figure 4b). Pretreatment with LY294002 or with AS605240 abrogated the ANCA-stimulated superoxide production in either TNFα- or GM-CSF-primed neutrophils (n=4, P<0.05). The difference between both inhibitors was not statistically significant. These data indicate that the PI3Kγ isoform is necessary for superoxide production by ANCA. To investigate whether the reduction in superoxide generation was merely a consequence of reduced ANCA antigen expression, we performed flow cytometry experiments. mPR3 expression of resting neutrophils was 47.9±4.9 mean fluorescence intensity that was upregulated with 2 ng/ml TNFα to 90.6±12.8 mean fluorescence intensity. Treatment with 0.5 μmol/l AS605240 did not significantly change the membrane expression. We observed 79.6±14.6 mean fluorescence intensity for TNFα-treated neutrophils (n=5, n.s.). Expectantly, treatment with 10 μmol/l SB202190 significantly abrogated TNFα-induced membrane PR3 expression as showed in parallel experiments (90.6±12.8 mean fluorescence intensity to 59.0±11.2; P<0.05; n=5). We next tested whether PI3Kγ inhibition abrogates neutrophil degranulation after stimulation with ANCA. Neutrophils were pretreated with LY294002 or AS605240, followed by priming with GM-CSF or TNFα, and subsequently activated with human ANCA IgG. Degranulation of primary granules was assayed by β-glucuronidase release. As shown in Figure 4c, degranulation of TNFα- and GM-CSF-primed neutrophils stimulated with ANCA IgG was significantly inhibited by pretreatment with both PI3K inhibitors (n=4, P<0.05). These data show that activation of PI3Kγ isoform by ANCA IgG is essential for ANCA-induced neutrophil degranulation. Glomerular neutrophil recruitment is a hallmark of ANCA-induced glomerulonephritis. When we tested whether specific inhibition of PI3Kγ blocks migration toward GM-CSF, we observed that neutrophil migration increased within the first 2 h reaching a plateau thereafter. Furthermore, a strong reduction of migration occurred with both LY294002 and AS605240 pretreatment (Figure 5). These data show that PI3Kγ controls neutrophil migration toward GM-CSF in vitro. In a second set of experiments, the effect of LY294002 and AS605240 on migration in the presence of human ANCA IgG was assessed. Compared with control IgG, GM-CSF-mediated migration was slightly decreased by MPO and PR3 ANCA (n=6, n.s.). However, LY294002 and AS605240 showed a similar inhibitory effect on migration in the presence of ANCA. For clarity, migration data after 2 h are shown in Figure 5b. Other important neutrophil functions involved in the multistep process of glomerular neutrophil influx in ANCA-induced NCGN are adhesion to and spreading on extracellular matrices. After 120 min, we observed an increase in neutrophil adhesion to fibronectin with GM-CSF treatment from 1.6±0.1 to 2.4±0.5 × 104 cells (P<0.05). The GM-CSF effect was not inhibited by LY294002 (2.1±0.5 × 104) or AS605240 (2.3±0.7 × 104, n=5, n.s.). We then assessed whether mAb to PR3 or MPO further increased GM-CSF-mediated adhesion. GM-CSF treatment in the presence of isotype control for 120 min resulted in 2.3±0.3 × 104, with anti-MPO mAb in 2.6±1.2 × 104 and with anti-PR3 mAb in 2.4±0.6 × 104 adherent cells. This increase was small and not statistically significant (n=5). Furthermore, we found that GM-CSF treatment increased neutrophil spreading on fibronectin (from 8±3 to 45%±12, n=5, P<0.05). The GM-CSF effect was not affected by LY294002 (37%±14) or AS605240 (39%±14, n=5, n.s.) pretreatment. GM-CSF treatment in the presence of isotype control resulted in 43%±12 with a variable effect by anti-MPO mAb (57%±10) and anti-PR3 mAb (72%±8) that was not statistically significant (n=5, n.s.). We finally tested whether specific PI3Kγ inhibition by orally administered AS605250 provides a novel treatment option for ANCA-induced NCGN. Immunized MPO-deficient mice were irradiated and subsequently transplanted with WT BM cells. At day 21 after transplantation treatment with oral gavage twice daily containing 30 mg/kg body weight AS605250 or vehicle control for the next 5 weeks was started. We chose day 21 as a starting point to avoid interference with BM engraftment. At this time point, we already found engraftment with MPO-positive neutrophils in the circulation and the beginning of urine abnormalities. At 8 weeks after BM transplantation, all mice transplanted with WT BM and treated with vehicle control showed hematuria and proteinuria, whereas AS605240-treated mice did not develop significant urine abnormalities. These results were confirmed by a higher urinary albumin excretion in the mice treated with vehicle control in comparison to the AS605240-treated group: 118.2±67.1 μg/ml in the control mice compared with 32.8±15.1 μg/ml in the AS605240-treated group. The difference did not reach statistically significance. All vehicle-control-treated mice developed NCGN on histology (100% disease induction) whereas AS605240-treated mice developed only weak glomerular abnormalities (three of the six mice developed weak glomerular disease, three did not develop any glomerular abnormalities). Mice in the control group showed on average 15.6±3.9% crescents and 6.0±1.5% necrosis, whereas mice in the AS605240 group developed 1.4±0.6% crescents and 0.5±0.4% necrosis (P<0.01 for both; Figure 6). Both groups showed similar anti-MPO titers by enzyme-linked immunosorbent assay (vehicle-control group: d0 1.25±0.41 AU, d56 1.10±0.64 AU; AS group: d0 1.39±0.52 AU, d56 0.99±0.83 AU), similar engraftment with MPO-positive cells (data not shown), and no difference in blood cell counts. These data establish that AS605240 did not exert toxic effects that interfered with BM engraftment (Table 1). The glomerular neutrophil and macrophage accumulation in anti-MPO NCGN was diminished in mice treated with AS605240 as shown in Table 2, indicating that the blockade of PI3Kγ reduces in vivo glomerular neutrophil and monocyte/macrophage accumulation. We used in vivo models to show that PI3Kγ controls neutrophil functions that participate in ANCA-induced glomerular damage. Our data suggest PI3Kγ inhibition is a new therapeutic approach in ANCA vasculitis. ANCAs induce NCGN that resembles the human disease in several animal models.8.Xiao H. Heeringa P. Hu P. et al.Antineutrophil cytoplasmic autoantibodies specific for myeloperoxidase cause glomerulonephritis and vasculitis in mice.J Clin Invest. 2002; 110: 955-963Crossref PubMed Scopus (918) Google Scholar, 33.Schreiber A. Xiao H. Falk R.J. et al.Bone marrow-derived cells are suf
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