Analysis of the impact of Bruton tyrosine kinase on myeloid cell development and function

Abstract

In the present study, it could be demonstrated that Bruton tyrosine kinase is not only necessary for the proper hematopoietic differentiation of B cells, but also of granulocytes and monocytes downstream of the granulocyte-macrophage progenitor (GMP) in the bone marrow. We could show that GMP isolated from the bone marrow of Btk-deficient mice preferentially developed into granulocytes at the expense of monocytes or undifferentiated cells when cultivated in the presence of granulocyte-monocyte-colony stimulating factor (GM-CSF) or TLR-ligands as differentiation cues. In addition, the enhanced granulopoiesis could be confirmed by the evaluation of the bone marrow compartment of Btk-mutant mice. With respect to the terminal differentiation of granulocytes in the bone marrow we also found a substantial maturation defect in granulocytes obtained from Btk-deficient mice, although the granulopoiesis was enhanced in these mice. This immature phenotype of granulocytes was associated with an inefficient development of granules as well as a reduced expression of granule proteins, like myeloperoxidase, neutrophil elastase, lactoferrin or gelatinase. Moreover, analyzes of neutrophil function in the reverse passive Arthus reaction demonstrated a significant impairment of Btk-deficient neutrophils in tissue migration at the site of immune complex deposition. The examination of the molecular mechanism revealed a decreased phosphorylation of the regulatory subunit p85 of the phosphatidylinositol-3-kinase, Akt and glycogen synthase kinase-3ß in Btk-deficient myeloid cells after GM-CSF engagement, where Btk is activated in wild type cells. In addition, we could show that the expression of the lineage-determining transcription factors CAAT-enhancer binding protein alpha (C/EBPalpha) and PU.1 is diminished in Btk-deficient bone marrow cells.