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The kinase Btk negatively regulates the production of reactive oxygen species and stimulation-induced apoptosis in human neutrophils

Nature Immunology volume 13, pages 369–378 (2012)Cite this article

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Abstract

The function of the kinase Btk in neutrophil activation is largely unexplored. Here we found that Btk-deficient neutrophils had more production of reactive oxygen species (ROS) after engagement of Toll-like receptors (TLRs) or receptors for tumor-necrosis factor (TNF), which was associated with more apoptosis and was reversed by transduction of recombinant Btk. Btk-deficient neutrophils in the resting state showed hyperphosphorylation and activation of phosphatidylinositol-3-OH kinase (PI(3)K) and protein tyrosine kinases (PTKs) and were in a 'primed' state with plasma membrane–associated GTPase Rac2. In the absence of Btk, the adaptor Mal was associated with PI(3)K and PTKs at the plasma membrane, whereas in control resting neutrophils, Btk interacted with and confined Mal in the cytoplasm. Our data identify Btk as a critical gatekeeper of neutrophil responses.

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Figure 1: Btk-deficient neutrophils show enhanced production of ROS.
Figure 2: Btk-deficient neutrophils show augmented apoptosis due to excessive production of ROS.
Figure 3: Excessive production of ROS and apoptosis in neutrophils from patients with XLA are abrogated by transduction of Hph-1–tagged full-length recombinant Btk but not by Hph-1–tagged Btk with deletion of the kinase or PH domain.
Figure 4: Btk-deficient neutrophils show targeting of Rac2 to the plasma membrane, colocalization of Rac2 with gp91phox and higher membrane expression of gp91phox.
Figure 5: Btk-deficient neutrophils have higher baseline activity of PTKs and PI(3)K, which is reversed by transduction of recombinant Btk protein.
Figure 6: Mal in neutrophils from healthy controls associates with Btk in the resting state and translocates to the plasma membrane after stimulation, whereas Mal associates with PI(3)K at the plasma membrane in Btk-deficient neutrophils.
Figure 7: Btk associates with Mal at the PH and kinase domains.
Figure 8: SFKs and Syk are involved in PI(3)K activation and the augmented production of ROS, whereas SFKs are involved in the membrane localization of Mal, in Btk-deficient neutrophils.

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References

  1. Flannagan, R.S., Cosio, G. & Grinstein, S. Antimicrobial mechanisms of phagocytes and bacterial evasion strategies. Nat. Rev. Microbiol. 7, 355–366 (2009).

    Article  CAS  PubMed  Google Scholar 

  2. Nauseef, W.M. How human neutrophils kill and degrade microbes: an integrated view. Immunol. Rev. 219, 88–102 (2007).

    Article  CAS  PubMed  Google Scholar 

  3. Lambeth, J.D. NOX enzymes and the biology of reactive oxygen. Nat. Rev. Immunol. 4, 181–189 (2004).

    Article  CAS  PubMed  Google Scholar 

  4. Babior, B.M. NADPH oxidase. Curr. Opin. Immunol. 16, 42–47 (2004).

    Article  CAS  PubMed  Google Scholar 

  5. Sumimoto, H. Structure, regulation and evolution of Nox-family NADPH oxidases that produce reactive oxygen species. FEBS J. 275, 3249–3277 (2008).

    Article  CAS  PubMed  Google Scholar 

  6. Fang, F.C. Antimicrobial reactive oxygen and nitrogen species: concepts and controversies. Nat. Rev. Microbiol. 2, 820–832 (2004).

    Article  CAS  PubMed  Google Scholar 

  7. Singh, A., Zarember, K.A., Kuhns, D.B. & Gallin, J.I. Impaired priming and activation of the neutrophil NADPH oxidase in patients with IRAK4 or NEMO deficiency. J. Immunol. 182, 6410–6417 (2009).

    Article  CAS  PubMed  Google Scholar 

  8. Woollard, K.J. & Geissmann, F. Monocytes in atherosclerosis: subsets and functions. Nat. Rev. Cardiol. 7, 77–86 (2009).

    Article  Google Scholar 

  9. Finkel, T. Radical medicine: treating ageing to cure disease. Nat. Rev. Mol. Cell Biol. 6, 971–976 (2005).

    Article  CAS  PubMed  Google Scholar 

  10. Conley, M.E. et al. Genetic analysis of patients with defects in early B-cell development. Immunol. Rev. 203, 216–234 (2005).

    Article  CAS  PubMed  Google Scholar 

  11. Winkelstein, J.A. et al. X-linked agammaglobulinemia: report on a United States registry of 201 patients. Medicine (Baltimore) 85, 193–202 (2006).

    Article  Google Scholar 

  12. Mohamed, A.J. et al. Bruton's tyrosine kinase (Btk): function, regulation, and transformation with special emphasis on the PH domain. Immunol. Rev. 228, 58–73 (2009).

    Article  CAS  PubMed  Google Scholar 

  13. Gray, P. et al. MyD88 adapter-like (Mal) is phosphorylated by Bruton's tyrosine kinase during TLR2 and TLR4 signal transduction. J. Biol. Chem. 281, 10489–10495 (2006).

    Article  CAS  PubMed  Google Scholar 

  14. Doyle, S.L., Jefferies, C.A., Feighery, C. & O'Neill, L.A. Signaling by Toll-like receptors 8 and 9 requires Bruton's tyrosine kinase. J. Biol. Chem. 282, 36953–36960 (2007).

    Article  CAS  PubMed  Google Scholar 

  15. Mangla, A. et al. Pleiotropic consequences of Bruton tyrosine kinase deficiency in myeloid lineages lead to poor inflammatory responses. Blood 104, 1191–1197 (2004).

    Article  CAS  PubMed  Google Scholar 

  16. Fiedler, K. et al. Neutrophil development and function critically depend on Bruton tyrosine kinase in a mouse model of X-linked agammaglobulinemia. Blood 117, 1329–1339 (2011).

    Article  CAS  PubMed  Google Scholar 

  17. Conley, M.E. et al. Primary B cell immunodeficiencies: comparisons and contrasts. Annu. Rev. Immunol. 27, 199–227 (2009).

    Article  CAS  PubMed  Google Scholar 

  18. Kerner, J.D. et al. Impaired expansion of mouse B cell progenitors lacking Btk. Immunity 3, 301–312 (1995).

    Article  CAS  PubMed  Google Scholar 

  19. Khan, W.N. et al. Defective B cell development and function in Btk-deficient mice. Immunity 3, 283–299 (1995).

    Article  CAS  PubMed  Google Scholar 

  20. O'Neill, L.A.J. & Bowie, A.G. The family of five: TIR-domain-containing adaptors in Toll-like receptor signalling. Nat. Rev. Immunol. 7, 353–364 (2007).

    Article  CAS  PubMed  Google Scholar 

  21. Piao, W. et al. Tyrosine phosphorylation of MyD88 adapter-like (Mal) is critical for signal transduction and blocked in endotoxin tolerance. J. Biol. Chem. 283, 3109–3119 (2008).

    Article  CAS  PubMed  Google Scholar 

  22. Jenkins, K.A. & Mansell, A. TIR-containing adaptors in Toll-like receptor signalling. Cytokine 49, 237–244 (2010).

    Article  CAS  PubMed  Google Scholar 

  23. Taneichi, H. et al. Toll-like receptor signaling is impaired in dendritic cells from patients with X-linked agammaglobulinemia. Clin. Immunol. 126, 148–154 (2008).

    Article  CAS  PubMed  Google Scholar 

  24. Pérez de Diego, R. et al. Bruton's tyrosine kinase is not essential for LPS-induced activation of human monocytes. J. Allergy Clin. Immunol. 117, 1462–1469 (2006).

    Article  PubMed  Google Scholar 

  25. Horwood, N.J. et al. Bruton's tyrosine kinase is required for TLR2 and TLR4-induced TNF, but not IL-6, production. J. Immunol. 176, 3635–3641 (2006).

    Article  CAS  PubMed  Google Scholar 

  26. Marron, T.U., Rohr, K., Martinez-Gallo, M., Yu, J. & Cunningham-Rundles, C. TLR signaling and effector functions are intact in XLA neutrophils. Clin. Immunol. 137, 74–80 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Honda, F. et al. Transducible form of p47phox and p67phox compensate for defective NADPH oxidase activity in neutrophils of patients with chronic granulomatous disease. Biochem. Biophys. Res. Commun. 417, 162–168 (2012).

    Article  CAS  PubMed  Google Scholar 

  28. Dang, P.M. et al. A specific p47phox -serine phosphorylated by convergent MAPKs mediates neutrophil NADPH oxidase priming at inflammatory sites. J. Clin. Invest. 116, 2033–2043 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Scapini, P., Pereira, S., Zhang, H. & Lowell, C.A. Multiple roles of Lyn kinase in myeloid cell signaling and function. Immunol. Rev. 228, 23–40 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Zhu, Q.S. et al. G-CSF induced reactive oxygen species involves Lyn-PI3-kinase-Akt and contributes to myeloid cell growth. Blood 107, 1847–1856 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Pereira, S. & Lowell, C. The Lyn tyrosine kinase negatively regulates neutrophil integrin signaling. J. Immunol. 171, 1319–1327 (2003).

    Article  CAS  PubMed  Google Scholar 

  32. Vlahos, C.J., Matter, W.F., Hui, K.Y. & Brown, R.F. A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H–1-benzopyran-4-one (LY294002). J. Biol. Chem. 269, 5241–5248 (1994).

    CAS  PubMed  Google Scholar 

  33. Sadhu, C., Masinovsky, B., Dick, K., Sowell, C.G. & Staunton, D.E. Essential role of phosphoinositide 3-kinase δ in neutrophil directional movement. J. Immunol. 170, 2647–2654 (2003).

    Article  CAS  PubMed  Google Scholar 

  34. Morris, A.C. et al. C5a-mediated neutrophil dysfunction is RhoA-dependent and predicts infection in critically ill patients. Blood 117, 5178–5188 (2011).

    Article  CAS  PubMed  Google Scholar 

  35. Santos-Sierra, S. et al. Mal connects TLR2 to PI3Kinase activation and phagocyte polarization. EMBO J. 28, 2018–2027 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Nam, S. et al. Action of the Src family kinase inhibitor, dasatinib (BMS-354825), on human prostate cancer cells. Cancer Res. 65, 9185–9189 (2005).

    Article  CAS  PubMed  Google Scholar 

  37. Lai, J.Y. et al. Potent small molecule inhibitors of spleen tyrosine kinase (Syk). Bioorg. Med. Chem. Lett. 13, 3111–3114 (2003).

    Article  CAS  PubMed  Google Scholar 

  38. Slack-Davis, J.K. et al. Cellular characterization of a novel focal adhesion kinase inhibitor. J. Biol. Chem. 282, 14845–14852 (2007).

    Article  CAS  PubMed  Google Scholar 

  39. Korade-Mirnics, Z. & Corey, S.J. Src kinase-mediated signaling in leukocytes. J. Leukoc. Biol. 68, 603–613 (2000).

    CAS  PubMed  Google Scholar 

  40. Bradshaw, J.M. The Src, Syk, and Tec family kinases: distinct types of molecular switches. Cell. Signal. 22, 1175–1184 (2010).

    Article  CAS  PubMed  Google Scholar 

  41. Vassilev, A., Ozer, Z., Navara, C., Mahajan, S. & Uckun, F.M. Bruton's tyrosine kinase as an inhibitor of the Fas/CD95 death-inducing signaling complex. J. Biol. Chem. 274, 1646–1656 (1999).

    Article  CAS  PubMed  Google Scholar 

  42. Wang, A.V., Scholl, P.R. & Geha, R.S. Physical and functional association of the high affinity immunoglobulin G receptor (FcγRI) with the kinases Hck and Lyn. J. Exp. Med. 180, 1165–1170 (1994).

    Article  CAS  PubMed  Google Scholar 

  43. Boyle, K.B. et al. Class IA phosphoinositide 3-kinase β and δ regulate neutrophil oxidase activation in response to Aspergillus fumigatus hyphae. J. Immunol. 186, 2978–2989 (2011).

    Article  CAS  PubMed  Google Scholar 

  44. Kawabuchi, M. et al. Transmembrane phosphoprotein Cbp regulates the activities of Src-family tyrosine kinases. Nature 404, 999–1003 (2000).

    Article  CAS  PubMed  Google Scholar 

  45. Saito, K. et al. BTK regulates PtdIns-4,5–P2 synthesis: importance for calcium signaling and PI3K activity. Immunity 19, 669–678 (2003).

    Article  CAS  PubMed  Google Scholar 

  46. Condliffe, A.M. et al. Sequential activation of class IB and class IA PI3K is important for the primed respiratory burst of human but not murine neutrophils. Blood 106, 1432–1440 (2005).

    Article  CAS  PubMed  Google Scholar 

  47. Uckun, F.M. et al. Anti-breast cancer activity of LFM-A13, a potent inhibitor of Polo-like kinase (PLK). Bioorg. Med. Chem. 15, 800–814 (2007).

    Article  CAS  PubMed  Google Scholar 

  48. Mahajan, S. et al. Rational design and synthesis of a novel anti-leukemic agent targeting Bruton's tyrosine kinase (BTK), LFM-A13 [alpha-cyano-β-hydroxy-β-methyl-N-(2,5-dibromophenyl)propenamide]. J. Biol. Chem. 274, 9587–9599 (1999).

    Article  CAS  PubMed  Google Scholar 

  49. Futatani, T. et al. Deficient expression of Bruton's tyrosine kinase in monocytes from X–linked agammaglobulinemia as evaluated by a flow cytometric analysis and Its clinical application to carrier detection. Blood 91, 595–602 (1998).

    CAS  PubMed  Google Scholar 

  50. Takahashi, N. et al. Impaired CD4 and CD8 effector function and decreased memory T cell populations in ICOS-deficient patients. J. Immunol. 182, 5515–5527 (2009).

    Article  CAS  PubMed  Google Scholar 

  51. Morio, T. et al. Ku in the cytoplasm associates with CD40 in human B cells and translocates into the nucleus following incubation with IL-4 and anti-CD40 mAb. Immunity 11, 339–348 (1999).

    Article  CAS  PubMed  Google Scholar 

  52. Choi, J.M. et al. Intranasal delivery of the cytoplasmic domain of CTLA-4 using a novel protein transduction domain prevents allergic inflammation. Nat. Med. 12, 574–579 (2006).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank E. Tsitsikov, E. Rachlin, K. Imai and J. Yata for discussions; all patients who participated in this study; S. Goo Rhee (Ewha Womans University) for antibody to Prx1 phosphorylated at Tyr194; and J.A. Lindquist (Otto-von-Guericke University) for antibody to Cbp (PAG) phosphorylated at Tyr317. Supported by the Ministry of Health, Labour and Welfare of Japan (H. Kane, S.N. and T.M.), the Ministry of Education, Culture, Sports, Science and Technology of Japan (S.M. and T.M.) and by the National Research Foundation of Korea (National Creative Research Initiatives grant to S.-K.L.).

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Authors and Affiliations

  1. Department of Pediatrics and Developmental Biology, Tokyo Medical and Dental University Graduate School of Medical and Dental Sciences, Tokyo, Japan

    Fumiko Honda, Masatoshi Takagi, Shuki Mizutani & Tomohiro Morio

  2. Department of Pediatrics, Teikyo University School of Medicine Hospital, Mizonokuchi, Kawasaki, Japan

    Hirotsugu Kano

  3. Department of Pediatrics, Toyama University School of Medicine, Toyama, Japan

    Hirokazu Kanegane

  4. Department of Pediatrics, National Defense Medical College, Tokorozawa, Japan

    Shigeaki Nonoyama

  5. Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, Republic of Korea

    Eun-Sung Kim & Sang-Kyou Lee

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F.H. did experiments; E.-S.K. and S.-K.L. contributed to protein-delivery experiments and provided some technical support; H. Kano and H. Kane made suggestions on data analysis and interpretation; S.N. and S.M. provided advice on project planning and data interpretation; M.T. provided advice on project plan and edited the manuscript; T.M. directed the project, designed research and wrote the manuscript; and all authors reviewed and approved the manuscript.

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Correspondence to Tomohiro Morio.

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Honda, F., Kano, H., Kanegane, H. et al. The kinase Btk negatively regulates the production of reactive oxygen species and stimulation-induced apoptosis in human neutrophils. Nat Immunol 13, 369–378 (2012). https://doi.org/10.1038/ni.2234

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