The maturation and functional defects of Btk-deficient neutrophils are neutrophil-intrinsic. Chimeric mice were generated by transplanting either CD45.2⁺ wild-type or Btk-deficient bone marrow into CD45.1⁺ lethally irradiated mice. Alternatively, CD45.1⁺ lethally irradiated mice received a mixture of CD45.1⁺ wild-type and CD45.2⁺ Btk-deficient bone marrow in different ratios. (A-C) Blood analyses were performed 4 weeks after transplantation. The bone marrow of CD45.1/CD45.2 chimeras was analyzed 6 weeks after transplantation for (D) the reconstitution of the neutrophil compartment and (E) the maturation status of the CD11b/Gr-1 population. CD45.1⁺ wild-type or CD45.2⁺ Btk-deficient granulocytes were sorted out of the bone marrow of chimeric mice and analyzed for the expression of Btk as a control and for C/EBPα (Cebpa), gelatinase (Mmp9), elastase (Elane), and myeloperoxidase (Mpo) by quantitative PCR (F). Granulocytes obtained from CD45.1⁺ lethally irradiated mice that received bone marrow from CD45.2⁺ wild-type (wt) or Btk-deficient (Btk-ko) mice were analyzed for (G) the maturation status of the CD11b/Gr-1 population, as well as the release of elastase (H) or gelatinase (I) in an immune-complex/Fc-mediated degranulation assay. CD45.1⁺ lethally irradiated mice that received bone marrow from CD45.2⁺ wild-type (wt) or Btk-deficient mice (Btk-ko) were investigated in the IC-mediated Arthus reaction (J) for ear swelling as a sign of tissue damage and edema formation. Treated ears were harvested 8 hours after eliciting the immune response and analyzed by immunofluorescence (K); red = CD31; green = Gr-1, arrowheads; blue = DAPI. (L) In parallel, one-half of the treated ears were analyzed by toluidin-staining for the presence of mast cells. (M) To determine the origin of mast cells, ear slides were analyzed by anti-CD45.2 (green; corresponds to donor cells), anti-Kit (red), and DAPI (blue). A representative image taken from an ear of a lethally irradiated CD45.1⁺ mouse that received CD45.2⁺ Btk-deficient bone marrow cells is presented. n represents the number of biological replicates.