CD45 expression is modulated and essential for cell mobilization. (A–D) CD45 expression. WT mice were untreated (untr.), or were treated with G-CSF for 5 consecutive days (A–C) or with a single injection of LPS (D). MNCs from BM and PB samples were examined for their CD45 expression by FACS. A representative histogram of CD45 levels is shown in A. Isotype control staining (dotted lines), untreated cells (black line), and cells after 5 d of G-CSF stimulations (gray line) are shown. A summary of four independent experiments is shown for G-CSF (B and C) and LPS (D). CD45 expression (presented as a geometric mean value) on different sorted subpopulations after G-CSF stimulations for 5 d is shown in C. (E) In vivo mobilization. WT and CD45KO mice were injected with G-CSF for 3 and 5 d or were left untreated. Total numbers of viable circulating WBCs were evaluated. (F) Frequency of CFCs in the PB of 3 d of G-CSF–treated mice. The data summarize three independent experiments (±SD). (G) Representative FACS analysis of MNCs derived from the PB after 3 d of G-CSF stimulations. Indicated values represent the percentage of SKL primitive cells in PB MNCs. One representative figure out of five independent experiments is shown. (H) Kinetics of the increased CFC numbers in the BM of G-CSF–treated WT and CD45KO mice. Numbers of colonies were evaluated in untreated mice or after G-CSF administration for 3 and 5 d. Bars represent at least three mice in each group, showing mean ± SD. *, P < 0.01; **, P < 0.05. (I) Table summarizing the percentage of SKL cells out of BM and PB MNCs in WT or CD45KO mice after G-CSF treatment for 3 d.

CD45 deficiency reduces MMP-9 levels and increases cell adhesion via activation of Src kinase. (A and B) BM or PB MNCs were isolated from CD45KO or WT mice, either untreated or treated with G-CSF for 3 d. Cells were cultured and the activity of MMP-9 in the conditioned media was detected by the gelatin zymography assay. A summary of three independent experiments and representative gel images of BM cells (A) and PB cells (B) is shown (±SD). (C) FACS analyses of the β1-activated epitope 9EG7 in untreated WT or CD45KO BM MNCs. (C, i) Representative histogram of background staining and 9EG7 expression in WT (black line) and CD45KO (gray line). (C, ii) Summary of five independent experiments (±SD). (D) Adhesion assay of WT and CD45KO BM MNCs to FN-coated wells. Shown is a summary of four independent experiments (mean ± SD). (E and F) Western blot analysis of Src phosphorylation or Src activity levels (F) in whole-cell lysates that were prepared from BM MNCs of WT or CD45KO mice, either untreated or after G-CSF injections. Histogram represents a summary of four independent experiments. Values represent the fold change of the control. (G) Western blot analysis for Erk phosphorylation. Lysates were prepared as described in E. (H) SDF-1–induced migration of WT and CD45KO BM MNCs pretreated with 1 μM of the Src inhibitor PP2 or DMSO as a control. Data represent mean ± SD values of three independent experiments and are shown as the fold increase from DMSO-treated cells. *, P < 0.01; **, P < 0.05.

Defective maturation of CD45KO osteoclasts in vitro involving impaired expression of MMPs, DC-STAMP, and Src kinase. (A) BM-derived osteoclasts from WT (top) and CD45KO (bottom) mice immunolabeled for CD45 expression (green), and stained for polymerized actin (red) and nuclear DNA (blue). Bars, 20 μm. (B) TRAP staining (purple) of BM-derived WT (top) and CD45KO (bottom) osteoclasts. Bars, 200 μm. (C and D) Semiquantitative PCR analysis for DC-STAMP mRNA levels in WT and CD45KO BM-derived cells. (C) Representative PCR image. (D) Summary of four independent experiments showing the ratio between DC-STAMP and GAPDH mRNA expression (±SE; *, P < 0.01). (E) Conditioned medium of WT versus CD45KO BM-derived osteoclasts was tested for the activity of secreted MMP-9 in a gelatin zymography assay. (F) WT and CD45KO BM-derived osteoclasts cultured with or without G-CSF were immunolabeled for expression of MT1-MMP (green), and stained for polymerized actin (red) and nuclear DNA (blue). Bar, 10 μm. (G) TRAP staining (purple) of BM-derived WT (top) and CD45KO (bottom) osteoclasts cultured in vitro in the presence of DMSO vehicle (left) or the Src inhibitor PP2 (right). Bars, 200 μm. (H) Src activity assay for osteoclast precursors incubated with 1 μM PP2 or DMSO (ctrl) for 5 d. Values indicate the fold changes of WT control mice ± SE (indicated by a horizontal line), showing a representative experiment.

Defective engraftment and reduced rapid mobilization in CD45KO chimeras. (A) Percentage of donor-derived WT cells in the BM of WT and CD45KO recipients. Shown is a summary of seven mice in each group (±SE; *, P < 0.01). Values demonstrate reduced engraftment levels in CD45KO hosts. (B) AMD3100-induced mobilization of donor WBCs in the PB of WT and CD45KO hosts. Values indicate fold change of mobilization index compared with PBS control mice (±SE). **, P < 0.05. (C) Rapid mobilization of WT and CD45KO donor cells. WT chimeric hosts were injected with PBS or AMD3100 and were tested for the presence of donor-derived cells in the PB. The mobilization index of specific lineages was tested, as indicated in the figure legend. Values indicate the fold change compared with PBS control mice, which are marked as a dashed line (mean ± SE). *, P < 0.01; ***, P = 0.07.

Impaired migration and reduced response to SDF-1 by CD45KO progenitors. (A and B) In vitro migration of MNCs from the BM of CD45KO or WT mice either untreated or treated with G-CSF for 3 or 5 d. Cells were allowed to migrate without (spont.) or with addition of SDF-1 to the lower well. (B) Migration of WT and CD45KO BM MNCs of untreated mice through bare or FN-coated filters toward SDF-1. (C) Homing of BM MNCs derived from untreated WT and CD45KO mice to the BM and spleens of recipient NOD/SCID mice 3 h after transplantation. (D and E). In vitro migration (D) or homing assay (E) of BM CD11b⁺ cells sorted from the BM of untreated or G-CSF–treated WT or CD45KO mice. (F) c-Kit⁺ cells were sorted from the BM of WT or CD45KO mice, untreated or treated with G-CSF for 5 d and stimulated in vitro with SDF-1. Representative images demonstrate cell polarization (indicated by black arrows). Bar, 20 μm. (G) Homing of c-Kit⁺ cells sorted from the BM of WT or CD45KO mice to the BM of NOD/SCID recipient mice 3 h after transplantation. (H) Homing of spleen progenitor cells isolated from WT or CD45KO mice to the BM and spleens of lethally irradiated NOD/SCID/β2 recipients. (I) Percentages of engraftment in WT-recipient BM and PB transplanted with spleen-derived cells from WT and CD45KO mice. Data represent the levels of specific lineages, as indicated. Shown are the means of four recipients in each group ± SE. *, P < 0.01; **, P < 0.05.

CD45KO mice show impaired progenitor mobilization and endosteal modulation induced by RANKL. (A) Colony-forming progenitor cells in the BM of WT and CD45KO mice after RANKL treatment for 3 d. Values indicate fold changes compared with control mice ± SE (*, P < 0.01). (B) The percentage of SKL primitive cells in the BM of WT versus CD45KO mice after treatment with RANKL for 3 or 5 d. Values indicate the fold changes compared with control mice ± SE (*, P < 0.02; **, P < 0.05). (C) Colony-forming progenitor cells in the spleens of WT and CD45KO mice after RANKL treatment. Values indicate fold changes compared with control mice ± SE (*, P < 0.01). (D) Numbers of colony-forming progenitor cells in the PB of WT and CD45KO mice after RANKL treatment for 5 d (±SE). (E) Soluble SCF levels in the plasma of WT and CD45KO, control, or RANKL-treated mice. Values indicate the fold change in plasma SCF compared with control mice ± SE (*, P < 0.05). (F) Immunoreactivity (brown) of osteopontin in femoral metaphyseal trabecules of WT and CD45KO, control, and RANKL-stimulated mice for 3 and 5 d. Black arrowheads indicate endosteal bone-lining osteoblasts. Bars, 20 μm. (G) Western blot analysis of an osteopontin-degraded product (32 kD) in BM superannuates of WT and CD45KO mice after RANKL treatments for 3 and 5 d.

Reduced osteoclast function in CD45KO mice is associated with elongated trabecular zone and irregular localization of progenitors. (A) Plasma levels of PYD indicating bone resorption in WT and CD45KO. (B) Plasma levels of osteocalcin in WT and CD45KO mice. Values represent the mean ± SE (*, P < 0.05). (C) Three-dimensional constructions of the distal femurs of WT and CD45KO mice. The red frame indicates the metaphyseal region, near the bone growing plates, and the yellow frame indicates a region 1 mm ahead toward the diaphysis (see Materials and methods). Bars, 2 mm. (D) TRAP staining of femoral bone sections. Osteoclasts stained in red (arrowheads) are shown along the metaphyseal trabecules (Tb). (E) The ratio between osteoclast surface and bone surface (Ocl.S/B.S). Data are presented as the percentage of WT cells ± SE (*, P < 0.05). (F–H) Flow cytometry analyses of BM immature Lin⁻/c-Kit⁺ progenitors (F), presented as the percentage of WT (mean ± SE), and the more primitive SKL stem cells. A representative FACS plot (G) and summary (H) indicate lower levels of these primitive cells in the BM of CD45KO mice. *, P < 0.01. (I) Flow cytometry analyses of spleen-derived immature Lin⁻/c-Kit⁺ progenitors, presented as the percentage of WT (mean ± SE). Numbers indicate higher levels of these primitive cells in the spleen of CD45KO mice, *, P < 0.01.