|
|
Blood, 1 August 2007, Vol. 110, No. 3, pp. 860-869.
Prepublished online as a Blood First Edition Paper on April 6, 2007; DOI 10.1182/blood-2006-06-031401.
 Previous Article | Table of Contents | Next Article 
HEMATOPOIESIS
The chemokine GROß mobilizes early hematopoietic stem cells characterized by enhanced homing and engraftment
Seiji Fukuda1,
Huimin Bian1,
Andrew G. King2, and
Louis M. Pelus1
1 Department of Microbiology and Immunology, the Walther Oncology Center, Indiana University School of Medicine, and the Walther Cancer Institute, Indianapolis; and
2 Microbial, Musculoskeletal, and Proliferative Diseases Centers of Excellence in Drug Discovery (CEDD), GlaxoSmithKline, Collegeville, PA
 |
Abstract
|
|---|
Mobilized peripheral blood hematopoietic stem cells (PBSCs) demonstrate accelerated engraftment compared with bone marrow; however, mechanisms responsible for enhanced engraftment remain unknown. PBSCs mobilized by GROß (GROß 4/CXCL24) or the combination of GROß4 plus granulocyte colony-stimulating factor (G-CSF) restore neutrophil and platelet recovery faster than G-CSF–mobilized PBSCs. To determine mechanisms responsible for faster hematopoietic recovery, we characterized immunophenotype and function of the GROß-mobilized grafts. PBSCs mobilized by GROß4 alone or with G-CSF contained significantly more Sca-1+-c-kit+-lineage– (SKL) cells and more primitive CD34–-SKL cells compared with cells mobilized by G-CSF and demonstrated superior competitive long-term repopulation activity, which continued to increase in secondary and tertiary recipients. GROß4-mobilized SKL cells adhered better to VCAM-1+ endothelial cells compared with G-CSF–mobilized cells. GROß4-mobilized PBSCs did not migrate well to the chemokine stromal derived factor (SDF)-1 in vitro that was associated with higher CD26 expression. However, GROß4-mobilized SKL and c-Kit+ lineage– (KL) cells homed more efficiently to marrow in vivo, which was not affected by selective CXCR4 and CD26 antagonists. These data suggest that GROß4-mobilized PBSCs are superior in reconstituting long-term hematopoiesis, which results from differential mobilization of early stem cells with enhanced homing and long-term repopulating capacity. In addition, homing and engraftment of GROß4-mobilized cells is less dependent on the SDF-1/CXCR4 axis.
|
Introduction
|
|---|
Granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood hematopoietic stem cells (PBSCs) are widely used for transplantation1,2 and restore platelet and neutrophil levels faster than bone marrow or cord blood cells.2–4 However, 10% to 30% of patients and volunteers mobilize poorly to G-CSF, resulting in collection of suboptimal numbers of PBSCs for transplantation,5,6 highlighting the need for more efficacious mobilizing regimens/agents. Single administration of the chemokines GROß (CXCL2) or GROß4 (CXCL24) rapidly mobilize hematopoietic stem and progenitor cells (HSPCs), including long-term repopulating cells (LTRC), to an equivalent degree as G-CSF when used alone and there is synergy when combined with G-CSF.7,8 Neutrophil and platelet recoveries in mice receiving GROß4-mobilized PBSCs are significantly accelerated compared with mice receiving G-CSF–mobilized cells,7 suggesting that GROß4-mobilized PBSCs engraft more efficiently
Differences in engraftment using HSPCs from different sources may reflect stem cell number, cell cycle status,9,10 presence of facilitating cells,11,12 or intrinsic differences in homing properties or proliferative capacity.13,14 Hematopoietic stem cells (HSCs) require an appropriate marrow niche to differentiate and self renew15,16 and only HSCs homing to marrow contribute to long-term repopulation.17,18 The SDF-1/CXCR4 axis is involved in HSPC trafficking.13,14,19–21 SDF-1 can attract HSCs that express CXCR4 to the marrow microenvironment,14,20 and enhanced in vitro transmigration of G-CSF–mobilized CD34+ cells to SDF-1 is associated with hematopoietic recovery.22 However, blocking Gi-coupled receptors, which includes CXCR4, does not affect homing of Sca-1+-lin–-Rhlow cells23 and marrow engraftment of CXCR4–/– Sca-1+-c-kit+-lineage– (SKL) cells is equivalent to CXCR4+/+ SKL cells,24 suggesting that the SDF-1/CXCR4 axis is not absolutely required for HSPC homing and engraftment. Furthermore, marrow HPCs rendered unresponsive to SDF-1 home efficiently, provided that the 4-integrin/VCAM-1 pathway is intact.25 In the absence of 4-integrin or VCAM-1, blocking CXCR4 reduces homing, indicating a dominant role for the 4-integrin/VCAM-1 pathways in progenitor cell homing.
To determine mechanisms responsible for enhanced hematopoietic repopulation by GROß4-mobilized HSC, we examined in vitro migration to SDF-1, adhesion, static expression of adhesion molecules, expression of the cell surface exopeptidase CD26, CXCR4 expression, apoptosis, cell cycle, in vivo marrow homing capacity, and competitive LTRC capacity of the mobilized graft. Our results indicate that compared with G-CSF, GROß4 mobilizes more primitive HSCs characterized by enhanced adhesion to endothelial cells, more competitive repopulating capacity, and more efficient marrow homing that is less dependent on the SDF1/CXCR4 axis.
|
Materials and methods
|
|---|
Animals
SPF female BALB/c and C57Bl/6 mice, 6-8 weeks of age, were purchased from Harlan Sprague-Dawley, Indianapolis, IN. B6.SJL-PtrcAPep3B/BoyJ (B6.BoyJ) mice were bred at Indiana University. Indiana University School of Medicine Institutional Animal Care and Use Committee (IACUC) approved all experimental procedures.
Reagents
Endotoxin-free human GROß was purchased from R&D Systems (Minneapolis, MN). Recombinant human GROß4 was produced and purified as previously described.7 G-CSF was from Amgen (Thousand Oaks, CA). Recombinant murine stem cell factor was a gift from Dr Karl Nocka, UCB Research, Cambridge, MA. Recombinant murine stem cell factor was purchased from BioVision, Palo Alto, CA. MegaCult-C reagents were obtained from Stem Cell Technologies (Vancouver, BC, Canada).
Peripheral blood cell mobilization
Mobilized peripheral blood was collected 15 minutes after subcutaneous injection of 2.5 mg/kg GROß or GROß4 or 16 hours after 4 days of subcutaneous administration (50 µg/kg per day, twice daily) of G-CSF. In the combination setting, G-CSF was administered for 4 days, GROß or GROß4 was administered 16 hours after the last dose of G-CSF, and peripheral blood was collected 15 minutes later. These regimens induce optimal HSPC mobilization.7,8 Mononuclear cells (PBMCs) were isolated as previously described.7,8 Total colony forming units (CFUs)/mL blood were calculated as follows: (%CFU in PBMC) x (PBMC/mL blood) x 100–1.
In vitro migration
Mobilized PBMCs were blocked with antimouse CD16/CD32(Fc III/II) (BD Biosciences, San Diego, CA), stained with phycoerythrin (PE)-conjugated antimouse Ly-6G (Gr-1) and Ly-6C (RB6-8C5), CD45R/B220 (RA-6B2), CD3e (145-2C11), TER119 (TER-119), and CD11b (M1/70; BD Biosciences), and incubated with anti-PE magnetic beads (Miltenyi Biotech, Auburn, CA). Cells passed through a magnetic column were collected and stained with FITC-anti-c-kit antibody (2B8; BD Biosciences). PE– KL cells were isolated using fluorescence-activated cell sorter (FACS) (Figure 6A) and KL cells subjected to in vitro migration to rmSDF-1 (R&D Systems) for 4 hours. Input and migrated cells were collected, washed, and CFU enumerated in 1% methylcellulose/IMDM containing 30% fetal bovine serum, 1 U/mL rhEpo, 10 ng/mL recombinant murine granulocyte-macrophage colony-stimulating factor, and 50 ng/mL recombinant murine stem cell factor. Percent migration was calculated as (total CFU/lower chamber ÷ total input CFU x 100). SKL cell migration was determined using lin– cells and input cells and migrated cells stained with anti-c-kit and anti-Sca-1 (E13-161.7; BD Biosciences) antibodies. Migration was calculated as (migrated SKL cells ÷ SKL input cells x 100). Total granulocyte macrophage colonies (CFU-GM) and granulocyte-erythrocyte-megakaryocyte-macrophage (GEMM)/mL in mobilized blood migrating to SDF-1 was calculated as: (CFU/mL blood) x (percent migration to SDF-1) x 1/100. In some experiments, linneg cells were pre-incubated with 10 µM AMD3100 (AnorMED, Langley, BC, Canada).
PBSC transplantation in mice
BALB/c mice received 875 cGy total body irradiation (lethal dose [LD]100 [radiation dose lethal to 100% of mice]) from a Gammacel-40 irradiator (Nordon International, Kanata, ON, Canada) in 2 sessions, 6 hours apart. Irradiated mice received mobilized PBMCs via the tail vein in 0.2 mL endotoxin-free saline (Baxter Healthcare, Deerfield, IL). Neutrophil (ANC) and platelet (PLT) counts were monitored using a Hemavet Mascot hematology analyzer (CDC Technologies, Oxford, CT).
Competitive transplants
Peripheral blood mononuclear cells from C57BL/6 (CD45.2) mice mobilized by GROß4 or G-CSF were mixed with 5 x 105 competitor marrow cells from Boy J mice (CD45.1) at ratios of 4:1, 3:1, or 2:1 and transplanted into lethally irradiated C57BL/6 mice (1050-Gy split dose the day before transplantation). Peripheral blood was sampled monthly and the proportion of CD45.1 and CD45.2 cells determined by flow cytometry.
Flow cytometry
Lin–-PBMCs were blocked with anti-FcIII/II and stained with antibodies against CXCR4 (2B11; BD Biosciences) or CD11a (2D7), CD62L (MEL-14), CD49d (9C10), CD49e (5H10-27; BD Biosciences) with anti-c-kit (2B8, BD Biosciences) with or without anti-Sca-1. Total CD34–-SKL cells/mL blood was determined by staining lin– cells with anti-CD34 (RAM34; BD Biosciences), Sca-1, and c-kit and calculated using the following formula: (percent CD34– SKL cells in lin– fraction) x (percent lin– cells in PBMC) x (PBMC/mL blood) x 10–4. Total SKL or KL cells/mL blood was similarly calculated. Mobilized PBMC were stained with CD8a-PE (53-6.7; BD Biosciences) and TCR-FITC (H57-597; BD Biosciences) for determination of facilitating cells. Lin– cells prestained with APC-c-kit and PE-Cy7-Streptavidin-biotin-Sca-1 were stained with FITC–Annexin-V (BD Bioscience). Intracellular CXCR4 in mobilized KL or SKL cells was determined by staining lin– cells with FITC-Sca-1, APC-c-kit, and a saturating amount of nonconjugated antimouse CXCR4 sufficient to block surface CXCR4 (2B11; BD Biosciences). Cells were fixed with 1% paraformaldehyde, permeabilized by 0.25% Triton-X/0.5% bovine serum albumin/phosphate-buffered saline (BSA/PBS), and stained with PE-CXCR4 (2B11, BD Biosciences) to examine intracellular CXCR4.
Adhesion assays
Mouse endothelial C166 cells (3 x 104; ATCC, Manassas, VA), which express VCAM-126 and support proliferation of primitive hematopoietic cells,27 were seeded on replicate plates precoated with 5 µg/mL human fibronectin (BD Biosciences) 48 hours before adhesion assays. Mobilized PBMCs in 0.5% bovine serum albumin/RPMI-1640 (4 x 104 cells/well) were seeded into wells coated with C166 cells. Cells were briefly centrifuged to facilitate adhesion, incubated for 30 minutes at 37°C, washed, and nonadherent cells collected. Input cells and nonadherent KL and SKL cells were quantitated and percent adhesion determined by subtracting percent nonadherent cells from 100.
Statistics
Data are expressed as mean plus or minus a standard error of the mean (SEM) and statistical significance was evaluated using the 2-tailed Student t test in Microsoft Excel (Microsoft, Seattle WA). Synergy in response to G-CSF plus GROß/GROß4 was compared with G-CSF or GROß/GROß4 used alone by ANOVA with Bonferroni correction for multiple comparisons using GraphPad InStat3 (Graphpad Software, San Diego, CA). Hematologic recovery for mice that underwent transplantation with PBMCs mobilized by G-CSF, GROß, and GROß4 alone and with G-CSF were compared by nonlinear regression analysis as described.7
|
Results
|
|---|
Accelerated hematologic recovery in mice receiving GROß or GROß4-mobilized PBMCs
We previously reported that GROß4-mobilized PBMCs restore neutrophil and platelet recovery faster in mice than G-CSF–mobilized PBMCs.7 In separate studies, we reported that GROß mobilized fewer total WBCs but a greater number of early HSPCs into peripheral blood than G-CSF, and that compared with G-CSF–mobilized PBSCs, the PBSCs mobilized by GROß showed a stronger competitive trend at least when used at a 2:1 ratio.28 To more precisely understand the faster engraftment observed with the more clinically relevant GROß4 ligand, we analyzed in depth the phenotypic and functional composition of the chemokine-mobilized graft that produced faster engraftment and directly compared PBSCs mobilized by full-length GROß to evaluate the role of N-terminal truncation. Comparison of PBMCs mobilized by full-length and truncated GROß showed similar engraftment and accelerated neutrophil and platelet recovery compared with G-CSF (Table 1), demonstrating that the effect of GROß4 is not a consequence of N-terminal truncation. Neutrophil recovery was significantly improved further using PBMCs mobilized by either chemokine in combination with G-CSF (13 days, P < .05 vs 15 and 17 days for GROß/GROß4 and G-CSF used alone, respectively), whereas, 80% platelet recovery using combination-mobilized PBMCs (29 days, P < .05) was slower than observed with cells mobilized by GROß or GROß4 alone (23 days) but still faster than by G-CSF (39 days). The reasons for slower platelet recovery with G-CSF or GROß/GROß4 with G-CSF are not clear, but may reflect graft composition. It has been shown that G-CSF mobilizes CD14+ monocytes with suppressive activity, at least for T-cell proliferation.29
While total HSPCs in hematopoietic grafts influence recovery,30 GROß, GROß4, and G-CSF–mobilized grafts contained equivalent total CFU-GM, CFU-GEMM, and CFU-megakaryocyte (Meg) (Figure 1A), as well as immunophenotypically defined KL cells (Figure 1B),7 making it unlikely that accelerated recoveries were due to the number of transplanted HPCs. PBMCs mobilized by GROß/GROß4 plus G-CSF contained 6 to 8-fold more CFU-GM, 5- to 7-fold more CFU-GEMM, 8 to 12-fold more CFU-Meg, and 3- to 4-fold more KL cells (Figures 1A,B), which probably contributed to accelerated recovery.
View larger version (17K):
[in this window]
[in a new window]
|
Figure 1. Mobilization of HPC in mice treated with GROß or GROß4 alone or in combination with G-CSF. (A) CFU-GM, CFU-GEMM, and CFU-Meg/2 x 106 PBMC graft mobilized with G-CSF, GROß, GROß4, or the combination of G-CSF plus either GROß or GROß4. Data are an average of 5 experiments for CFU-GM and 2 to 4 experiments for CFU-GEMM and CFU-Meg from 3 mice/group, each assayed individually.  Synergy compared with G-CSF or GROß/GROß4 (P < .05), as determined using ANOVA with Bonferroni multiple comparison test. (B) C-kit+, lineage– (KL) cells/2 x 106 PBMC graft mobilized by G-CSF, GROß4 or the combination of G-CSF plus GROß4. Data are expressed as mean (± SEM) from 6 replicates of 10 mice per group in 2 experiments. Synergy compared with G-CSF or GROß4, P < .05, determined using ANOVA with Bonferroni multiple comparison test.
|
|
GROß4-mobilized LTRCs are more competitive than G-CSF–mobilized LTRCs
HPCs provide short-term protection; however, HSCs are required for long-term repopulation. Because hematopoietic recovery occurs faster with GROß4-mobilized PBMCs, we assessed their long-term repopulating ability using limiting dilution competitive repopulation. Lethally irradiated C57BL/6 mice underwent transplantation with 0.5 x 106 congenic B6.BoyJ (CD45.1) marrow competitor cells plus 2 x 106 (4:1), 1.5 x 106 (3:1), and 1 x 106 (2:1) CD45.2 PBMCs mobilized by GROß4 and/or G-CSF. All ratios of mobilized donor/recipient competitor cells showed significant chimerism (> 10-fold) compared with cells from mice mobilized by saline. Little difference in chimerism was seen between mice receiving GROß4 or G-CSF–mobilized PBMCs at the highest ratio, whereas enhanced chimerism was observed using PBMCs mobilized by the combination of both compounds (Figure 2A). Because this graft contains more HPCs, enhanced chimerism is also likely because of more LTRCs. At ratios of 3:1 and 2:1, PBMCs mobilized by GROß4 alone or with G-CSF produced significantly higher chimerism than G-CSF–mobilized PBMCs, suggesting that these PBMCs contain more LTRCs or LTRCs with enhanced repopulating ability/self-renewal.
To evaluate if GROß-mobilized HSCs have greater intrinsic repopulating/self-renewal activity, at 6 months after transplantation marrow from competitively engrafted mice was transplanted into secondary recipients in noncompetitive fashion (Figure 2B). All secondary mice survived less than 6 months, with significant levels of chimerism, demonstrating durable engraftment of primary mice. Secondary mice engrafted with marrow from mice receiving G-CSF–mobilized PBMCs showed the same level of chimerism in peripheral blood as the donor mice. In contrast, mice engrafted with HSCs from mice that underwent transplantation with GROß4 or GROß4 plus G-CSF–mobilized cells showed significantly higher chimerism than observed in the primary mice at all dilutions and significantly higher than mice that received marrow from mice that originally underwent transplantation with G-CSF–mobilized PBMCs. When marrow cells from the secondary recipients were transplanted into the tertiary recipients, chimerism was again significantly higher in mice receiving marrow reconstituted from GROß4 or GROß4 plus G-CSF–mobilized cells compared with G-CSF–mobilized cells (Figure 2C). The reasons for enhanced competitiveness in secondary transplants and the continued enhanced competitiveness on tertiary transplantation displayed by HSCs from mice receiving GROß4-mobilized grafts is not clear, but probably represents mobilization of earlier and more competitive HSCs, which is consistent with the enhanced hematopoietic recovery in mice that received GROß4–mobilized PBMCs.
Mobilization of SKL cells and CD34– SKL cells by GROß4 and G-CSF
To further define HSCs mobilized by GROß alone or with G-CSF, we quantitated and compared the SKL cell population (Figure 3A) that contains mouse LTRCs.31,32 We have previously shown that G-CSF and GROß4 each mobilized significant numbers of SKL and CD34–SKL cells.28 However, although no significant difference in total SKL cells/mL blood was observed between GROß4 or G-CSF, fewer PBMCs are obtained from GROß4-mobilized blood than G-CSF–mobilized blood; therefore, the GROß4-mobilized PBMCs contained 30% more SKL cells, which is numerically meaningful (1.3-fold ± 0.1-fold; P < .05; Figure 3B). GROß4 also mobilized significantly more CD34–SKL cells/mL blood (1.5-fold ± 0.1-fold; P < .05; not shown), and the GROß4-mobilized PBMC graft used for transplantation contained significantly more CD34–SKL cells (2.0-fold ± 0.2-fold; P < .05; Figure 3C), which contains one of the most primitive HSC populations in vivo.31,32 GROß4 plus G-CSF synergistically mobilized SKL cells, similar to what is observed for CFU-GM8,28 (not shown), and the G-CSF plus GROß4-mobilized graft contained significantly more SKL and CD34–SKL cells compared with G-CSF (Figure 3C). These results suggest that GROß-mobilized grafts, either alone or with G-CSF, contain significantly more primitive long-term repopulating HSCs than G-CSF–mobilized grafts. CD8+, TCR–, or CD8+ TCR+-facilitating cells can accelerate HSC engraftment in the allogeneic settings.12 There was no significant change in CD8a+ TCR– cells (0.5% ± 0.02%, 0.5% ± 0.03%, and 0.5% ± 0.03%) and CD8a+ TCR+ cells (7.9% ± 0.4%, 8.2% ± 1.2%, and 6.3% ± 2.8%) between G-CSF, GROß4, and G-CSF plus GROß4-mobilized cells.
Reduced apoptosis in GROß4 plus G-CSF–mobilized SKL cells
Stem cell survival contributes to homing and engraftment efficiency.33,34 G-CSF–mobilized CD34+ cells express higher levels of caspase-3, -4, and -8 with elevated Annexin-V binding, compared with marrow CD34+ cells.35 We therefore quantitated apoptosis in the KL and SKL cell populations mobilized by GROß4 or G-CSF to determine whether differences in survival contribute to engraftment. Annexin-V staining was equivalent in GROß4 and G-CSF–mobilized KL and SKL cells (Figure 4A,B), suggesting that enhanced engraftment of GROß4-mobilized PBMCs is unrelated to apoptosis. However, Annexin-V staining was significantly reduced in SKL cells mobilized by GROß4 plus G-CSF (Figure 4A,B), suggesting that enhanced survival probably contributes to accelerated hematopoietic recovery.
View larger version (24K):
[in this window]
[in a new window]
|
Figure 4. Annexin-V on SKL cells mobilized by G-CSF or GRO. (A) Representative Annexin-V staining on mobilized SKL cells. (B) Fold change Annexin-V mean fluorescence intensity on KL or SKL cells, expressed as mean (± SEM) from 2 independent experiments performed in triplicate using 10 mice per group per experiment. *P < .01 compared with G-CSF and GROß4.
|
|
GROß4-mobilized cells adhere better to VCAM-1+ endothelial cells
HSC trafficking is regulated by adhesion molecules, including leukocyte function associated antigen 1 (LFA-1),36 very late antigen-4 (VLA-4),36–40 and VLA-5.36 L-selectin expression in CD34+ cells predicts rapid platelet recovery in patients undergoing transplantation with G-CSF–mobilized cells.41 Firm adhesion to the vascular endothelial cells is the first step in homing, followed by transendothelial migration.42 We compared adhesion of G-CSF- and GROß4-mobilized KL and SKL cells to VCAM-1+ C166 endothelial cells that support proliferation of HSPCs26,27 under static conditions to determine whether adhesion differences are involved in enhanced engraftment of GROß4-mobilized HSCs. Adhesion of GROß4-mobilized KL and SKL cells to C166 cells was greater than G-CSF–mobilized cells (Figure 5A, left panel). GROß4-mobilized HSPC adhesion was 2- to 4-fold greater than cells mobilized by G-CSF (Figure 5A, right panel), consistent with enhanced adhesion to immobilized recombinant VCAM-1, as we reported previously.28 Cells mobilized by GROß4 plus G-CSF also demonstrated enhanced adhesion (not shown). However, despite enhanced adhesion to C166 cells, static expression of CD11a (L-integrin, LFA-1), CD49d (4-integrin), CD49e (5-integrin), and CD62L (L-selectin, LCECAM-1) was equivalent in SKL cells mobilized by GROß4 or G-CSF (Figure 5B), suggesting that adhesion molecule activation rather than expression is probably responsible for enhanced adhesion of GROß4-mobilized cells.
Although cell cycle state can influence engraftment and homing of HSCs,9,10 cell cycle distribution determined by DNA staining was equivalent in KL cells mobilized by either GROß4 or G-CSF, with less than 90% of both cell populations falling in G0/G1 (Figure 5C), which is similar to nonhuman primate CD34+ cells mobilized by AMD3100.43 Similarly, cell cycle distribution of cells mobilized by GROß4 plus G-CSF was identical to cells mobilized by either agent alone (not shown).
In vitro migration of CFU and SKL cells mobilized by GROß4 to SDF-1 is lower than that of G-CSF–mobilized cells
The SDF-1/CXCR4 migration axis is believed to play a major role in HSPC homing.13,14,20 Enhanced in vitro migration to SDF-1 by G-CSF–mobilized CD34+ cells is associated with hematopoietic recovery.22 We quantitated transmigration of mobilized HPCs to SDF-1 in vitro to determine whether the enhanced engraftment of GROß4-mobilized PBMCs is attributable to enhanced migration capability. Migration of CFU-GM and CFU-GEMM in the sorted KL cell fraction mobilized by GROß/GROß4 alone or with G-CSF to recombinant murine SDF-1 (Figure 6B) was significantly lower compared with G-CSF. Migration of GROß4-mobilized CD34–-SKL cells was also significantly lower (Figure 6C). Despite reduced migration, surface CXCR4 expression on GROß4-mobilized SKL (Figure 6D, top) and KL cells (not shown) were equivalent. Similarly, surface CXCR4 on G-CSF plus GROß4-mobilized SKL or KL cells was not different from cells mobilized by G-CSF or GROß4 alone (Figure 6D). We examined intracellular CXCR4 level because human CD34+CXCR4– cells harbor intracellular CXCR4 that can restore NOD/SCID repopulation potential44 and found that intracellular CXCR4 expression in SKL (Figure 6D, bottom) and KL cells (not shown) mobilized by GROß4 were not statistically different from expression in G-CSF–mobilized cells.
Enhanced homing of GROß4-mobilized SKL cells to marrow
HSC homing can be regulated by a hierarchy of pathways including the SDF-1/CXCR4 and 4-integrin/VCAM-1 pathways.25 Because neutrophil and platelet recovery is faster in recipients that underwent transplantation with GROß4-mobilized PBMCs, whereas in vitro migration of mobilized HPCs to SDF-1 was significantly lower but their adhesion to endothelial cells was higher, we evaluated the in vivo marrow homing capacity of CFSE-labeled mobilized KL and SKL cells (Figure 7 and Table 2). Although GROß4-mobilized SKL and KL cells showed reduced migration to SDF-1 in vitro, their marrow homing in vivo was significantly higher than G-CSF–mobilized SKL cells (Table 2). Moreover, the total number of SKL cells homing to recipient marrow in vivo in the 2 x 106 GROß4-mobilized PBMC graft was significantly higher than in an equivalent G-CSF–mobilized PBMC graft (Figure 7C). Homing of SKL cells mobilized by G-CSF or GROß4 was significantly higher than KL cells (Table 2). Despite superior homing of SKL cells, there was no significant difference in surface CXCR4 expression between mobilized KL and SKL cells (mean fluorescent intensity [MFI] = 4.7 ± 0.5 and 4.8 ± 1, respectively, for G-CSF and MFI = 5.4 ± 0.8 and 5.9 ± 0.7 for GROß4, respectively).
View larger version (17K):
[in this window]
[in a new window]
|
Figure 7. In vivo marrow homing of mobilized KL and SKL cells. (A) The top histogram shows lineage markers (PE) and isotype staining of donor PBMNCs before transplantation. The bottom 2 plots show Sca-1 (PE-Cy7) vs c-kit (APC; right) and their isotype staining (left) in lin– cells. M1: lin– cells; R1: Sca-1+, c-kit+ cells in lin– cells. (B) PBMCs mobilized by G-CSF or GROß4 were stained with CFSE and 4 x 107 cells were transplanted into lethally irradiated syngeneic recipients. Recipient marrow cells were harvested 16 hours after transplantation. The top plots show CFSE staining in nontransplanted recipient (left) and transplanted recipient marrow (right) stained with lineage markers (PE), anti-Sca-1 (PE-Cy7), and anti-c-kit (APC) antibodies. The R1 gate represents CFSE+ cells. The middle histogram represents lineage markers (PE) and isotype staining of CFSE+ cells. The bottom plots shows Sca-1 (PE-Cy7) vs c-kit (APC) staining and its isotype staining in the CFSE+ lin– population. Gate R2 are Sca-1+ c-kit+ cells in CFSE+ lin– cells. (C) Total KL or SKL cells in 2 x 106 PBMCs homing to recipient marrow. Data are expressed as mean (± SEM) from 3 experiments using 30 donors and 3 recipients per group per experiment. *P < .05 compared with G-CSF.
|
|
The dissociation between in vitro migration to SDF-1 and in vivo homing of SKL cells mobilized with GROß4 suggested that their homing may be less dependent on the SDF-1/CXCR4 axis. To directly address this possibility, we used the selective CXCR4 antagonist AMD3100, which inhibits homing and migration of mouse HSPC and Diprotin A, a selective inhibitor for the cell surface peptidase CD26 that increases marrow stem cell homing and engraftment by blocking CD26-mediated cleavage of SDF1.13 Consistent with published findings, treatment of normal bone marrow cells and G-CSF–mobilized SKL cells with AMD3100 significantly inhibited in vitro migration to SDF-1 and in vivo marrow homing of SKL cells; however, in vivo homing of GROß4-mobilized SKL cells was unaffected by pretreatment with AMD3100, despite significant reduction in migration to SDF-1 in vitro (Table 3). The lack of inhibition of homing of GROß4-mobilized SKL by AMD3100 was not caused by altered CXCR4 expression. In fact, CXCR4 expression on GROß4-mobilized SKL cells was higher than on normal marrow SKL cells (MFI: 30.7 ± 8.8 vs 13.1 ± 0.4, respectively). Similarly, the CD26 inhibitor Diprotin A significantly enhanced homing of G-CSF–mobilized SKL cells as reported,13 but had little effect on homing of GROß4-mobilized cells (Table 4). However, CD26 expression on GROß4-mobilized SKL cells was significantly higher than on G-CSF–mobilized cells and the number of CD26+SKL cells in the GROß4-moblized graft was significantly higher than G-CSF (Table 4). While the higher CD26 expression on GROß4-mobilized HSCs relative to G-CSF is consistent with their reduced migration to SDF1 in vitro, it is counterintuitive to enhanced in vivo homing and provides further evidence for the fact that in vivo homing of GROß4-moblized SKL cells may not be primarily dependent on the SDF1/CXCR4 axis. Mobilization of CFU-GM by G-CSF was inhibited by Diprotin A as reported;13 however, mobilization by GROß4 was unaffected (Table 4). Identical findings were observed in CD26-deficient mice. These data indicate that CD26 is not involved in the release of stem cells by GROß4 (Table 4).
View this table:
[in this window]
[in a new window]
|
Table 4. CD26 expression on mobilized SKL cells and the effect of CD26 inhibition on HSPC mobilization and homing
|
|
|
Discussion
|
|---|
GROß4 mobilizes HSPCs when used alone and synergizes with G-CSF when used in combination. In mice, GROß4-mobilized PBMCs restore neutrophil and platelet counts more rapidly and demonstrate more competitive LTRC ability than G-CSF–mobilized PBMCs. Compared with G-CSF, the GROß4-mobilized PBSC graft contains significantly more SKL and primitive CD34–- SKL cells, demonstrates superior adhesion to VCAM-1+ endothelial cells, and shows enhanced marrow homing capacity that is less dependent on CXCR4. The combination of GROß and G-CSF produces highly synergistic mobilization and further enhanced engraftment. Our findings provide functional evidence supporting the utility of GROß4 as a rapid and effective HSC mobilizer and evidence that PBSCs mobilized by GROß4, either used alone or with G-CSF, are a superior hematopoietic graft, at least in mice.
The absolute number of transplanted HSCs is critical to hematopoietic recovery.30 Although total CFU-GM and KL cells in GROß4 and G-CSF–mobilized blood were nearly identical, the GROß4-mobilized graft contained significantly more primitive LTR-SKL cells and CD34–-SKL cells31,32 and contributed to hematopoiesis at a significantly higher degree than G-CSF–mobilized HSC. One might expect that mature HSPCs rather than immature primitive HSCs would contribute to accelerated short-term hematopoietic recovery. GROß4 may mobilize short-term repopulating cells with greater intrinsic proliferative capacity, although clonogenic assays show no obvious differences in colony size between GROß4 and G-CSF–mobilized colony-forming cells (CFCs). Alternatively, GROß4 may mobilize a population of cells that are not identified by our clonogenic or phenotypic analyses, such as high proliferative potential-colony forming cells (HPP-CFCs) that facilitate accelerated white blood cell recovery. However, our results are consistent with previous reports that primitive stem cells alone mediate rapid marrow recovery after transplantation17 and that the early phase of engraftment after transplantation is mediated by stem cells.30 PBSCs mobilized by GROß4 plus G-CSF demonstrated greater engraftment and repopulating capacity and contained significantly more CFU-GM, KL, SKL, and CD34–-SKL cells, suggesting that both mature and immature HSPCs contribute to their faster engraftment and enhanced chimerism. Lower apoptosis of SKL cells mobilized by GROß4 plus G-CSF probably also contributes to accelerated engraftment. While CD8+, TCR–, or CD8+ TCR+-facilitating cells accelerate HSC engraftment in the allogeneic settings12 and ours is a syngeneic transplant model, involvement of these facilitating cells in the enhanced engraftment is unlikely because we observed no significant differences between G-CSF and GROß4-mobilized cells.
Osteoblasts are implicated in HSC maintenance,45–47 and imaging studies show an interaction of primitive HSPCs with endosteal bone,16,46,48 leading to the hypothesis that primitive HSCs are found in the endosteum niche, whereas HPCs and mature cells migrate to the vascular niche and egress marrow. The capacity of chemokines such as GROß/GROß47,8 and IL-849 to mobilize HSPCs within 15 minutes is kinetically inconsistent with mobilization from the endosteal niche and suggests either that these chemokines do not mobilize long-term repopulating-hematopoietic stem cells (LTR-HSCs), which is clearly not the case, or that a more readily accessible pool of HSPCs can be mobilized. Using expression of SLAM (signaling lymphocytic activation molecule) family receptors that distinguish HSCs and hematopoietic progenitor cells (HPCs), it has been shown that a relatively large pool of HSCs is associated with sinusoidal endothelium.50 This may explain the rapid mobilization of primitive and mature HSCs by GROß4 or IL-8. In addition, synergistic mobilization of large numbers of primitive HSCs by GROß4 plus G-CSF suggests that a relatively large pool of primitive HSCs inhabit the vascular niche or are mobilized to the vascular niche from the osteoblast niche by G-CSF, but not released in response to G-CSF alone.
Numerous studies implicate the SDF-1/CXCR4 axis in HSPC trafficking and chemoattraction to the marrow microenvironment.13,14,19–21 Migration of GROß4-mobilized CFU-GM, KL, and SKL cells to SDF-1 in vitro was significantly lower than G-CSF–mobilized cells, which was not caused by differences in CXCR4 expression. Higher CD26 levels on GROß4-mobilized SKL cells that can inactivate SDF-1 is consistent with reduced migration to SDF-1. In addition, plasma and marrow SDF-1 levels in mice mobilized with GROß4 are significantly higher than in mice mobilized by G-CSF,8 raising the possibility that CXCR4 desensitization on GROß4-mobilized HSPCs could also account for their reduced migration in vitro. Alternatively, administration of GROß4 may inhibit HSPC CXCR4 responsiveness, given that KC, the mouse homologue of GROß, can desensitize CXCR4 on mouse polymorphonuclear neutrophils (PMNs).51 However, there is no clear evidence that HSPCs express CXCR2.52
Despite reduced migration to SDF-1 in vitro, homing of GROß4-mobilized SKL cells to marrow was significantly enhanced compared with G-CSF–mobilized SKL cells, indicating a lack of correlation between in vivo homing and in vitro migration to SDF-1. Lack of correlation between in vitro migration to SDF-1 and marrow homing of CD34+ cells mobilized by G-CSF plus AMD3100 has also been reported.53 Pretreatment of nonmobilized SKL cells with the CXCR4 antagonist AMD3100 significantly blocked their homing as described;13 however, although AMD3100 inhibited in vitro migration of SKL cells from control and GROß4-mobilized blood to SDF-1, it failed to block homing of GROß4-mobilized SKL cells, even at doses 10-fold higher than required to block homing of resident marrow HPCs.13 In addition, the CD26 inhibitor Diprotin A, while enhancing homing of G-CSF–mobilized cells, did not enhance homing of GROß |
Top
Abstract
Introduction
Synergy compared with G-CSF or GROß
