Figure 4.

TIMP-1 triggers maturation of neutrophils in the bone marrow (BM). (A–D) DBA/2 mice (n=5) were adenovirally transduced and bone marrow (BM) was analyzed three days later. (A) Gating (left) and relative quantification (right) of pre-mature neutrophils (CD11b⁺ Ly6G^int) by flow cytometry. (B) Expression of neutrophil maturation markers and granulopoiesis-inducing transcription factors in BM as analyzed by qRT-PCR. (C) CFU assay of BM isolates from AdCtrl or AdTIMP-1-transduced mice ex vivo. (D) A single BrdU pulse was injected 1 h before BM isolation. The proportion of BrdU-positive neutrophil progenitors (BM gated for CD11b⁺ Ly6G^int) was analyzed by flow cytometry. Columns: mean; Bars: SEM; Student t-test. ns: not significant; *P<0.05; **P<0.01. (E and F) G-CSF-induced differentiation of 32Dcl3 cells. Cells were stimulated with G-CSF for up to seven days in the absence or presence of rTIMP-1. (D) CD11b surface expression was analyzed by flow cytometry and (E) cells with segmented nuclei were counted on cytospins stained with DiffQuick. Representative image (left; arrows indicate cells with segmented nuclei; bar: 50 μm) and quantification of three independent experiments (right). Columns: mean; Bars: SEM; Student’s t-test. *P<0.05; **P<0.01.