Single-chain variable fragment intrabody impairs LPS-induced inflammatory responses by interfering with the interaction between the WASP N-terminal domain and Btk in macrophages.
Wiskott-Aldrich syndrome protein (WASP) plays important roles in both acquired and innate immune responses. We recently uncovered that the WASP N-terminal domain specifically binds the Src homology (SH) 3 domain of Bruton's tyrosine kinase (Btk) in macrophages. Over-expression of the WASP N-terminal domain impairs LPS-induced inflammatory responses. To evaluate the significance of this interaction in LPS signaling, we established bone marrow-derived macrophage (BMDM) cell lines from transgenic (Tg) mice expressing anti-WASP N-terminal domain single-chain variable fragment (scFv) intrabody. Anti-WASP scFv specifically bound endogenous WASP and inhibited its specific binding to the SH3 domain of Btk in the Tg BMDMs. Tyrosine phosphorylation in WASP was inhibited after LPS stimulation. As a result, TNF-α, IL-6, and IL-1β gene transcription and NF-κB phosphorylation were impaired. These observations strongly suggest that the phosphorylation of WASP by Btk plays a pivotal role in transducing the LPS signaling pathway in macrophages.
Animal Immune and Cell Biology Research Unit, National Institute of Agrobiological Sciences, 1-2 Ohwashi, Tsukuba, Ibaraki 305-8634, Japan.
Chisato
C
Sakuma
Mitsuru
M
Sato
Takato
T
Takenouchi
Joe
J
Chiba
Hiroshi
H
Kitani
D000077329:Agammaglobulinaemia Tyrosine Kinase; D000818:Animals; D002460:Cell Line; D007249:Inflammation / Q000235:genetics / Q000276:immunology*; D053583:Interleukin-1beta / Q000235:genetics; D015850:Interleukin-6 / Q000235:genetics; D008070:Lipopolysaccharides / Q000276:immunology; D008264:Macrophages / Q000276:immunology*; D051379:Mice; D008822:Mice, Transgenic; D016328:NF-kappa B / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000276:immunology*; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology*; D014158:Transcription, Genetic / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics; D014443:Tyrosine / Q000378:metabolism; D051316:Wiskott-Aldrich Syndrome Protein / Q000235:genetics / Q000276:immunology*; D018909:src Homology Domains / Q000235:genetics / Q000276:immunology*
D053583:Interleukin-1beta; D015850:Interleukin-6; D008070:Lipopolysaccharides; D016328:NF-kappa B; D057127:Single-Chain Antibodies; D014409:Tumor Necrosis Factor-alpha; D051316:Wiskott-Aldrich Syndrome Protein; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase; C000625951:Btk protein, mouse