lfm-a13 and Agammaglobulinemia

The role of the Bruton tyrosine kinase (Btk) protein in neutrophil function has been evaluated using neutrophils from healthy volunteers after incubation with a Btk inhibitor, leflunomide metabolite analog (LFM-A13), suggesting an important role for Btk in neutrophil function. We sought to determine the role of Btk protein on neutrophil superoxide generation and chemotaxis stimulated by N-formyl-methionine-leucine-phenylalanine (fMLP).. Chemotaxis was assayed on agarose gel and superoxide generation by cytochrome C reduction. The affects of LFM-A13 on chemotaxis and superoxide generation in unstimulated and fMLP stimulated neutrophils were studied in Btk deficient neutrophils from XLA patients compared with matched controls analyzed simultaneously.. Chemotaxis and stimulated superoxide production were similar in the normal and Btk deficient neutrophils and were similarly inhibited by LFM-A13. In one patient, LFMA13 had no effect on superoxide generation in Btk deficient neutrophils up to a concentration of 25 microM, while inhibited superoxide production by control neutrophils.. Our results suggest that Btk does not have a specific role in neutrophil fMLP-stimulated superoxide generation and chemotaxis since these activities were similarly inhibited by LFM-A13 in Btk deficient and normal neutrophils. The lack of superoxide generation following Btk inhibition by LFM-A13 in Btk deficient neutrophils from one patient may suggest some heterogeneity in the role of Btk in fMLP induced neutrophil superoxide generation.

Topics: Adolescent; Agammaglobulinaemia Tyrosine Kinase; Agammaglobulinemia; Amides; Case-Control Studies; Chemotactic Factors; Chemotaxis, Leukocyte; Child; Child, Preschool; Gene Expression; Genetic Diseases, X-Linked; Humans; Infant; Male; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Nitriles; Primary Cell Culture; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Superoxides; Young Adult

Bruton tyrosine kinase (BTK) is a component of signaling pathways downstream from Toll-like receptors (TLRs) 2, 4, 7, 8, and 9. Previous work in BTK-deficient mice, cell lines, and cultured cells from patients with X-linked agammaglobulinemia (XLA) suggested defective TLR-driven cytokine production.. We sought to compare TLR-4-, TLR-7-, and TLR-8-induced cytokine production of primary cells from patients with XLA with that seen in control cells.. PBMCs from patients with XLA, freshly isolated plasmacytoid dendritic cells, monocytes, and monocytoid dendritic cells were activated with TLR-4, TLR-7, and TLR-8 agonists. Signaling intermediates and intracellular and secreted cytokine levels were compared with those seen in control cells.. Although TLR-4, TLR-7, and TLR-8 activation of nuclear factor κB and mitogen-activated protein kinase pathways in cells from patients with XLA and control cells were comparable, TLR-activated freshly isolated monocytes and monocytoid dendritic cells from patients with XLA produced significantly more TNF-α, IL-6, and IL-10 than control cells. TLR-7/8-activated plasmacytoid dendritic cells produced normal amounts of IFN-α. In murine models BTK regulates the degradation of Toll-IL-1 receptor domain-containing adaptor protein, terminating TLR-4-induced cytokine production. Although this might explain the heightened TLR-4-driven cytokine production we observed, Toll-IL-1 receptor domain-containing adaptor protein degradation is intact in cells from patients with XLA, excluding this explanation.. In contrast to previous studies with BTK-deficient mice, cell lines, and cultured cells from patients with XLA suggesting impaired TLR-driven cytokine production, these data suggest that BTK inhibits TLR-induced cytokine production in primary human cells.

Topics: Adolescent; Adult; Agammaglobulinaemia Tyrosine Kinase; Agammaglobulinemia; Amides; Child; Child, Preschool; Cytokines; Dendritic Cells; Genetic Diseases, X-Linked; Humans; Infant; Inflammation Mediators; Male; Membrane Glycoproteins; Mitogen-Activated Protein Kinases; Monocytes; Myeloid Cells; NF-kappa B; Nitriles; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Receptors, Interleukin-1; Signal Transduction; Toll-Like Receptor 4; Toll-Like Receptor 7; Toll-Like Receptor 8; Toll-Like Receptors; Young Adult

Heme oxygenase (HO)-1 is the inducible isoform of the rate-limiting enzyme of heme degradation and provides cytoprotection against oxidative stress by its products carbon monoxide and biliverdin. More recently, HO-1 has also been shown to exert immunomodulatory functions via cell type-specific anti-inflammatory effects in myeloid/macrophage cells. In the current study, it is demonstrated that Bruton's tyrosine kinase (Btk), the gene of which is mutated in the human immunodeficiency X-linked agammaglobulinemia, is involved in the upregulation of HO-1 gene expression via TLR signaling in macrophages. The specific Btk inhibitor LFM-A13 blocked HO-1 induction by the classical TLR4 ligand LPS in cell cultures of RAW264.7 monocytic cells and primary mouse alveolar macrophages. Moreover, upregulation of HO-1 gene expression was abrogated in LPS-stimulated alveolar macrophages from Btk(-/-) mice. Transfection studies with luciferase reporter gene constructs demonstrated that LPS-dependent induction of HO-1 promoter activity was attenuated by pharmacological Btk inhibition and by an overexpressed dominant-negative mutant of Btk. This induction was mediated by the transcription factor Nrf2, which is a master regulator of the antioxidant cellular defense. Accordingly, nuclear translocation of Nrf2 in LPS-treated macrophages was reduced by Btk inhibition. The generation of reactive oxygen species, but not that of NO, was involved in this regulatory pathway. Btk-dependent induction of HO-1 gene expression was also observed upon macrophage stimulation with ligands of TLR2, TLR6, TLR7, and TLR9, suggesting that Btk is required for HO-1 gene activation by major TLR pathways.

Topics: Agammaglobulinaemia Tyrosine Kinase; Agammaglobulinemia; Amides; Animals; Cell Line; Cells, Cultured; Gene Expression Regulation, Enzymologic; Genetic Diseases, X-Linked; Heme Oxygenase-1; Lipopolysaccharides; Macrophages, Alveolar; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Mutation; NF-E2-Related Factor 2; Nitriles; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Signal Transduction; Toll-Like Receptors; Transcriptional Activation