Disruption of the CXCR4/CXCL12 chemotactic interaction during hematopoietic stem cell mobilization induced by GCSF or cyclophosphamide

Abstract

Hematopoietic progenitor cells (HPCs) normally reside in the bone marrow (BM) but can be mobilized into the peripheral blood (PB) after treatment with GCSF or chemotherapy. In previous studies, we showed that granulocyte precursors accumulate in the BM during mobilization induced by either GCSF or cyclophosphamide (CY), leading to the accumulation of active neutrophil proteases in this tissue. We now report that mobilization of HPCs by GCSF coincides in vivo with the cleavage of the N-terminus of the chemokine receptor CXCR4 on HPCs resident in the BM and mobilized into the PB. This cleavage of CXCR4 on mobilized HPCs results in the loss of chemotaxis in response to the CXCR4 ligand, the chemokine stromal cell-derived factor-1 (SDF-1/CXCL12). Furthermore, the concentration of SDF-1 decreased in vivo in the BM of mobilized mice, and this decrease coincided with the accumulation of serine proteases able to directly cleave and inactivate SDF-1. Since both SDF-1 and its receptor, CXCR4, are essential for the homing and retention of HPCs in the BM, the proteolytic degradation of SDF-1, together with that of CXCR4, could represent a critical step leading to the mobilization of HPCs into the PB in response to GCSF or CY.

Figure 1

Mobilized CD34⁺ PBPCs express a CXCR4 molecule truncated in the first extracellular domain. (a) Nalm-6 cells were incubated for 2 hours at 37°C in the presence of indicated concentrations of purified NE (circles) or CG (squares) and were stained with the mAbs 6H8 or 12G5. After analysis by flow cytometry, results are expressed as a percentage of 6H8/12G5 binding of nontreated cells. Data represent means ± SD of three independent experiments. (b) Binding of 6H8 and 12G5 mAbs to steady-state CD34⁺ BM cells, GCSF–mobilized CD34⁺ PBPCs, and GCSF–mobilized CD34⁺ PBPCs after overnight culture. The flow cytometry analysis was gated on CD34⁺ cells. Representative data from two experiments are shown. (c) CD34⁺ cells isolated from normal steady-state BM were treated with 100 μg/ml of NE or CG. Control cells were pretreated in an identical manner with PBS in the absence of protease. The chemotactic response of cells was assessed in the presence (black bars) or absence (white bars) of 200 ng/ml of CXCL12 in the lower chamber. Representative data from two experiments in triplicate are shown. P, PBS. (d) Freshly isolated GCSF–mobilized CD34⁺ PBPCs or CD34⁺ cells derived from steady-state BM were compared for their chemotactic response in the absence (white bars) and presence (black bars) of 200 ng/ml CXCL12. Representative data from two experiments are shown.

Figure 2

BM loses reactivity for anti-human CXCR4 mAb 6H8 during mobilization with GCSF. BM sections taken before mobilization were stained with a nonimmune IgG1 (a) and mAb 6H8 (b and c). Positive staining appears in purple (magnification, ×400). 6H8 staining on BM on day 4 of GCSF administration was very dim (c). The scale bar represents 50 μm.

Figure 3

CXCL12 concentration in the BM decreases when HPCs are mobilized in the PB. (a) BM extracellular fluids were extracted at the indicated time points from mice injected with either saline (open circles), CY alone (filled circles), GCSF alone (filled triangles), or CY in combination with GCSF (filled squares). CXCL12 concentrations were quantified by ELISA. (b) PB from mice injected with either CY alone (filled circles), GCSF alone (filled triangles), CY in combination with GCSF (filled squares), or saline (open circles) was taken at the indicated time points and plated in triplicate in clonogenic assays. The numbers of CFCs were determined after 14 days of incubation at 37°C. Data are means ± SD of three to six mice per group, with each sample analyzed in triplicate. Statistically significant differences with noninjected animals are indicated (*P < 0.05, **P < 0.01, as determined by Student’s t test).

Figure 4

BM extracellular fluids from mobilized mice contain proteases cleaving exogenous human synthetic CXCL12α. (a–c) Aliquots of exogenous synthetic human CXCL12α were incubated overnight at 37°C in the presence of an equal volume of BM extracellular fluids taken from mice mobilized with either GCSF alone (a), CY alone (b), or CY in combination with GCSF (c). The remaining chemotactic activity of exogenous CXCL12α was measured by performing transmigration assays with CD34⁺ cells freshly isolated from normal human BM. Nil indicates that PBS was added instead of BM extracellular extracts. In b and c, Sal represents the BM extracellular fluid from mice injected with saline for 6 days. Black bars show transmigration in the presence of digested CXCL12α, whereas white bars show controls in which exogenous CXCL12α was omitted. Data represent means ± SD of duplicates. Representative data from three independent experiments are shown. (d) The same samples of synthetic human CXCL12α incubated with BM extracellular fluids (as in a) were electrophoresed on a 20% polyacrylamide Tris-Trycine-SDS gel and analyzed by Western blotting with a goat anti-human CXCL12α antibody. A representative experiment from three performed is shown.

Figure 5

Cleavage and inactivation of CXCL12 in mobilized BM is due to serine proteases. Aliquots of synthetic human CXCL12α were incubated overnight at 37°C in the presence of PBS (lane 1), BM extracellular fluids isolated on day 4 of GCSF mobilization (lanes 2–5) after preincubation in the absence of protease inhibitor (lane 2) or in the presence of human α1-antitrypsin (lane 3), PMSF (lane 4), or BB-94 (lane 5). In the top panel, samples (black bars) together with controls without exogenous CXCL12α were analyzed for chemotactic activity on Nalm-6 cells as described in Figure 4a. A representative experiment from two performed is shown. In the bottom panel, the same samples were analyzed by Western blot with a goat anti-human CXCL12 antibody. A representative experiment from two performed is shown.

Figure 6

Neutrophil proteases NE and CG cleave and inactivate CXCL12α. Aliquots of synthetic human CXCL12α were incubated overnight at 37°C in the presence of medium conditioned by either human BM CD34^– cells, PB neutrophils, or nonconditioned medium. In parallel, CXCL12α was also incubated with purified human NE, CG, or proteinase-3. In the top panel, the remaining chemotactic activity of exogenous human CXCL12α was measured on purified BM CD34⁺ cells in transmigration assays as described in Figure 4a. A representative experiment from three performed in triplicate is shown. The bottom panel shows Western blot analysis of the same samples with a goat anti-human CXCL12 antibody. BM, BM CD34^– cells; Neut, PB neutrophils; NC, nonconditioned medium; P3, proteinase-3.

Figure 7

Pretreatment of BM extracellular fluids from mobilized mice with a specific NE inhibitor together with a specific CG inhibitor prevents degradation and inactivation of CXCL12. Aliquots of synthetic human CXCL12α were incubated overnight at 37°C in the presence of BM extracellular fluids on day 4 of GCSF-induced mobilization and day 6 of CY-induced mobilization that were pretreated with 1 mM PMSF, 10 μM specific NE inhibitor MetOSuc-Ala-Ala-Pro-Val-CMK, or 10 μM specific CG inhibitor MetOSuc-Ala-Ala-Phe-PO(Phe)₂ alone or in combination. In the top panel, the remaining chemotactic activity of exogenous human CXCL12α was measured on purified Nalm-6 cells in transmigration assays as described in Figure 4a. A representative experiment from two performed in triplicate is shown. The bottom panel shows Western blot analysis of the same samples with a goat anti-human CXCL12 antibody. G4, day 4 of GCSF-induced mobilization; CY6, day 6 of CY-induced mobilization.