Disruption of the CXCR4/CXCL12 chemotactic interaction during hematopoietic stem cell mobilization induced by GCSF or cyclophosphamide (original) (raw)
Mobilized human PB CD34+ cells express a truncated form of CXCR4. Previous reports have shown that neutrophil proteases NE and CG accumulate in the BM of humans and mice mobilized with GCSF (13, 14) and that human CXCR4 is cleaved in vitro by purified NE (24). This prompted us to determine whether CXCR4 expressed by HPCs could be cleaved in vivo during the process of mobilization in response to GCSF administration. In a first set of experiments, we analyzed the binding of the mAb 6H8, which is specific for residues 22–25 of human CXCR4 (23, 24) — a site located within the first N-terminal extracellular domain of CXCR4 — on acute B lymphoid leukemia cell line Nalm-6 after treatment with increasing concentrations of purified NE and CG. As shown in Figure 1a, both proteases induced a dose-dependent reduction of 6H8 binding to Nalm-6 cells, whereas the binding of mAb 12G5, which recognizes an epitope located within the second extracellular domain of CXCR4 (23, 24), was increased by 20% after treatment with NE and decreased by 40% after treatment with CG. These data confirm that both NE and CG can cleave between the 6H8 epitope and the first transmembrane domain of CXCR4, a cleavage known to inactivate the chemotactic properties of CXCR4 (23, 24).
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.
In a second set of experiments, we followed the binding of 6H8 and 12G5 mAbs to CD34+ cells isolated from steady-state BM and GCSF–mobilized PB (Figure 1b). Although all CD34+ cells isolated from steady-state BM were stained brightly by either 6H8 or 12G5 mAbs, CD34+ cells from GCSF–mobilized PB were characterized by a complete lack of 6H8 binding despite lower but still positive staining with 12G5. Overnight culture of purified CD34+ mobilized PBPCs resulted in re-expression of the 6H8 epitope (Figure 1b). These data do not rule out the possibility that the partial decrease of 12G5 binding may be due to reduced transcription of the CXCR4 mRNA. However, the fact the mAb 6H8 failed to bind to mobilized CD34+ PBPCs that were still positive for 12G5 demonstrates that, like Nalm-6 cells treated in vitro with purified NE and CG, CD34+ PBPCs mobilized in vivo with GCSF express a truncated form of CXCR4 containing the second transmembrane domain but lacking at least the 25 N-terminal residues in the first extracellular domain.
Loss of CXCR4 N-terminus in GCSF–mobilized BM. Immunohistochemical stains were performed with mAb 6H8 on human BM sections taken before and on day 4 of GCSF administration (Figure 2). Before GCSF administration, 6H8 binding was particularly strong in the endosteal region in the vicinity of trabecular bone, where the most primitive HPCs reside. On day 4 of GCSF administration, staining for 6H8 was greatly reduced, showing that the truncation of CXCR4 is not only seen on CD34+ cells once they are mobilized into the PB but also occurs in the BM when GCSF is administered.
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.
GCSF–mobilized CD34+ PBPCs do not migrate toward CXCL12. The cleavage of the N-terminal CXCR4 by NE has previously been reported to reduce binding of CXCL12 to Jurkat T cells to undetectable levels and to abolish the chemotactic response to CXCL12 gradient in vitro (24). We therefore examined the effect of exposure to NE or CG on the chemotactic response of steady-state BM CD34+ cells that expressed 6H8 epitope before in vitro exposure to proteases. Both NE and CG treatments resulted in the complete inhibition of CXCL12-driven chemotaxis of CD34+ progenitors (Figure 1c), in good accord with our finding that treatment with either protease resulted in the complete loss of 6H8 epitope (Figure 1a). Since GCSF–mobilized CD34+ PBPCs lack the 6H8 epitope (Figure 1b), we then investigated the chemotactic response of freshly isolated CD34+ PBPCs to CXCL12. In contrast to the robust chemotactic response observed with CD34+ cells isolated from steady-state BM, GCSF–mobilized CD34+ PBPCs failed to respond to CXCL12 (Figure 1d).
Decrease of CXCL12 concentration in mouse BM extracellular fluids during mobilization. We next investigated whether the CXCR4 ligand CXCL12 may be also be degraded in mobilized BM using the murine model in which we demonstrated accumulation of active NE and CG during mobilization of HPCs induced by GCSF or CY (13, 14). For this purpose, balb/c mice were mobilized after injection of either GCSF alone, CY alone, or CY in combination with GCSF. At various time points, BM extracellular fluids were extracted, and the concentration of endogenous murine CXCL12 was determined by ELISA. Concentrations of CXCL12 in these BM extracellular extracts were significantly decreased on day 6 after administration of CY alone or CY plus GCSF (Figure 3a). This corresponded precisely to the time at which HPCs were mobilized into the PB (Figure 3b). Similarly, in animals receiving GCSF alone, CXCL12 concentration in the BM was significantly decreased between days 2 and 6 of cytokine administration, again corresponding to the time of maximal HPC numbers in the PB. On day 10, CXCL12 concentrations in the BM returned to levels seen before the initiation of mobilization, and there was a concordant decrease in the number of circulating HPCs. It is interesting to note that on day 3 after injection of CY alone or CY plus GCSF, when mice are neutropenic and HPCs are not mobilized into the PB (14), CXCL12 concentration in the BM was significantly increased as compared with steady-state BM. Thus, CXCL12 levels in the BM are inversely related to the numbers of PBPCs in mice receiving three different mobilization regimens involving either a cytokine alone (GCSF), chemotherapy alone, or the combination of both.
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).
BM fluids from mobilized mice contain proteases inactivating CXCL12. We have previously reported that active neutrophil proteases accumulate in the BM extracellular fluid during mobilization and that these proteases cleave VCAM-1, which is essential to the retention of HPCs in the BM (13, 14). On the basis of this observation, we hypothesized that the decrease of endogenous CXCL12 concentration in the BM of mobilized mice could be due to proteolytic degradation. Since the BM from a 8- to 11-week-old mouse femur represents a total volume of 10 μl (approximately 90–95% cells and 5–10% fluid) and is flushed into 1 ml of PBS, our BM extracellular fluids were consequently diluted between 500 and 1,000 times during the extraction process. Since a concentration of at least 10 ng/ml CXCL12 is required to promote chemotaxis in CD34+ cells (16), 400 pg of endogenous mouse CXCL12 contained within the BM of one femur and diluted into 1 ml of PBS represented a concentration too low to induce chemotaxis in vitro. Therefore, to assess the possibility that proteases cleaving and inactivating CXCL12 were released in mobilized BM, synthetic human CXCL12α (which is identical to mouse CXCL12α except for a Val to Ile substitution in position 18) was incubated at 37°C with the BM extracellular fluids extracted from mice at different time points of mobilization. The residual bioactivity of the exogenous synthetic human CXCL12α was subsequently evaluat-ed in transmigration assays on CD34+ cells isolated from steady-state human BM and on Nalm-6 cells. BM extracellular fluids without exogenous CXCL12α were used to control basal transmigration. Synthetic CXCL12α preincubated in the presence of PBS was used as a positive control.
As anticipated, BM extracellular fluids in the absence of exogenous synthetic CXCL12α were unable to promote chemotaxis due to the 500- to 1,000-fold dilution of endogenous murine CXCL12 (Figure 4a, white bars), whereas exogenous synthetic human CXCL12α preincubated with either PBS or BM extracellular fluids from uninjected mice induced chemotaxis of human steady-state CD34+ BM cells (Figure 4a, black bars). However, preincubation of synthetic CXCL12α with BM extracts isolated between days 2 and 6 of GCSF mobilization completely inactivated the chemotactic activity of exogenous CXCL12α on CD34+ cells isolated from steady-state BM. Similarly, incubation of synthetic CXCL12α with BM extracts isolated on day 6 after injection of either CY or CY plus GCSF, when HPC mobilization peaked, completely inactivated the chemotactic activity of exogenous CXCL12α (Figure 4a). Results on Nalm-6 cells showed the same result with a total inactivation of CXCL12α chemotactic activity after incubation with BM extracts taken between days 2 and 6 of GCSF–induced mobilization or on day 6 of CY-induced or CY plus GCSF–induced mobilization (data not shown).
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.
When these digests were analyzed by immunoblotting, exogenous CXCL12α was no longer detectable after incubation with BM extracellular fluids from mice during HPC mobilization — that is, between days 2 and 6 of GCSF–induced mobilization (Figure 4a) — and on day 6 of mobilization with CY alone (Figure 4b) or in combination with GCSF (Figures 4c). On day 10, when mobilization had ceased (Figure 3b), none of the BM extracellular fluids inactivated or degraded the exogenous CXCL12α (Figure 4).
To assess whether this inactivation of exogenous CXCL12α was due to proteases present in the BM extracellular fluids, BM extracellular fluids extracted on day 4 of mobilization with GCSF were preincubated with human α1-antitrypsin (tissue inhibitor of serine proteases), PMSF (a serine protease inhibitor) or BB-94 (a broad specificity inhibitor of matrix metalloproteinases) (28, 29) before addition to synthetic CXCL12α. As shown in Figure 5, pretreatment of GCSF–mobilized BM extracellular fluids with human α1-antitrypsin or PMSF completely abolished inactivation and degradation of exogenous synthetic CXCL12α in both transmigration assays and Western blots, whereas pretreatment with BB-94 had no effect. This result demonstrates that proteases released in mobilized BM and responsible for CXCL12α degradation and inactivation are serine proteases.
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.
Neutrophils release in the BM proteases that inactivate CXCL12. To determine which cell population within the BM was responsible for the release of these proteases, synthetic human CXCL12α was incubated with media conditioned either by BM CD34– mononucleated cells or PB neutrophils and further analyzed in transmigration assays and by Western blotting (Figure 6). Incubation with either PB neutrophil-conditioned or BM CD34– cell–conditioned media abolished the chemotactic activity of exogenous synthetic CXCL12α. This result was confirmed by immunoblotting, which showed that these two conditioned media completely degraded CXCL12α. In contrast, neither BM stromal cell–conditioned nor bone cell–conditioned media degraded CXCL12α (data not shown). This result demonstrates that neutrophils are a major source of proteases with the ability to degrade and inactivate CXCL12α.
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.
Active NE and CG cleave and inactivate CXCL12. Since NE, CG, and proteinase-3 are the three major serine proteases produced and released by neutrophils (30), we tested whether these enzymes were capable of inactivating the chemotactic activity of exogenous human CXCL12α as assessed using the CD34+ cell transmigration assay. These experiments demonstrated that both NE and CG inactivated CXCL12α, whereas proteinase-3, a neutrophil serine protease closely related to NE (30), did not (Figure 6). In accord with this finding, CXCL12α was no longer detected by Western blot analysis after digestion with NE, suggesting that it was digested into small fragments. In contrast, after digestion with CG, CXCL12α exhibited a slightly faster electrophoretic mobility when overrun on a 20% SDS-PAGE (data not shown). These findings are consistent with the previous observation that CG cleaves CXCL12α between the fifth and sixth residues from the N-terminus, resulting in its complete inactivation (31).
Combination of NE- and CG-specific inhibitors prevents inactivation of CXCL12 by mobilized BM extracellular fluids. Proof that the proteases released in BM extracellular fluids during mobilization are NE and CG was provided by preincubating BM extracellular fluids on day 4 of GCSF–induced mobilization and day 6 of CY-induced mobilization with the specific NE inhibitor MetOSuc-Ala-Ala-Pro-Val-CMK and the specific CG inhibitor MetOSuc-Ala-Ala-Phe-PO(Phe)2. A preliminary experiment using NE and CG purified from human sputum and the chromogenic substrates MetOSuc-Ala-Ala-Pro-Val paranitroanilide (pNA) and Suc-Ala-Ala-Pro-Phe-pNA, which are specific for NE and CG, respectively (13, 14), demonstrated unambiguously that MetOSuc-Ala-Ala-Pro-Val-CMK inhibits NE but not CG, whereas MetOSuc-Ala-Ala-Phe-PO(Phe)2 inhibits CG but not NE (data not shown). Pretreatment of the BM extracellular fluids with MetOSuc-Ala-Ala-Pro-Val-CMK alone significantly reduced the degradation of CXCL12α, as observed by Western blotting (Figure 7b), but had a marginal effect on the inactivation of the CXCL12α chemotactic activity (Figure 7a), suggesting that a protease different from NE was present and, like CG, could inactivate CXCL12 activity with a minor shortening of the molecule. MetOSuc-Ala-Ala-Phe-PO(Phe)2 alone did not prevent inactivation of the chemotactic activity nor the degradation of CXCL12. However, preincubation of BM extracellular fluids with both MetOSuc-Ala-Ala-Pro-Val-CMK and MetOSuc-Ala-Ala-Phe-PO(Phe)2 prevented the inactivation and proteolytic degradation of CXCL12α by either CY-mobilized or GCSF–mobilized BM extracellular fluids.
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.