Mobilization Kinetics of CD34+ Cells in Association with Modulation of CD44 and CD31 Expression during Continuous Intravenous Administration of G-CSF in Normal Donors SEOK LEE,a SEOCK-AH IM,a EUN-SUN YOO,b EUN-MI NAM,a MI-AE LEE,c JEE-YOUNG AHN,a JUNG-WON HUH,c DO-YON KIM,a SOON-NAM LEE,a MI-JUNG KIM,d SEUNG-JU LEE,b WHA-SOON CHUNG,c CHU-MYONG SEONGa a
Departments of Hematology-Oncology, bPediatrics and cClinical Pathology, College of Medicine, Ewha Woman’s University, Seoul, Korea; dDept. of Cell Biology & Immunology, Asan Institute for Life Science, Seoul, Korea Key Words. Continuous i.v. G-CSF · Mobilization kinetics · CD34+ cells · CD31 · CD44 · Normal donors
A BSTRACT The aim of the present study is to evaluate the kinetics of CD34+ cells and investigate the potential modulation of CD44 and CD31 expression on CD34+ cells during continuous i.v. administration of G-CSF, thus to elucidate the possible mechanism of peripheral blood progenitor cell (PBPC) mobilization. Fifteen healthy donors were enrolled in this study. G-CSF (10 µg/kg/day) was administered for four consecutive days through continuous 24-h i.v. infusion. For measurement of complete blood counts, CD34+ cell levels and their expression of CD44 and CD31, PB sampling was performed immediately before the administration of G-CSF (steady-state) and after 4, 8, 24, 48, 72, 96, and 120 h of G-CSF administration. The percentage and absolute number of CD34+ cells significantly
increased at day 3 (0.55 ± 0.09%, 51.12 ± 24.83 × 103/ml) and day 4 (0.47 ± 0.09%, 46.66 ± 24.93 × 103/ml), compared to the steady-state level (0.06 ± 0.09%, 2.03 ± 5.69 × 103/ml). At day 3 to day 5 following the onset of G-CSF administration, a strong decrease of CD44 and CD31 expression was observed on mobilized CD34+ cells compared to controls: the relative fluorescence intensity of CD44 and CD31 was, respectively, 50%-70% and 40%90% lower than that of controls. We conclude that continuous i.v. administration of G-CSF apparently results in more rapid mobilization of CD34+ cells, and downregulation of CD44 and CD31 on CD34+ cells is likely to be involved in the mobilization of PBPC after treatment with G-CSF. Stem Cells 2000;18:281-286
INTRODUCTION Allogeneic peripheral blood progenitor cell (PBPC) transplantation from normal donors who undergo mobilization with G-CSF is an increasingly frequent procedure resulting in durable engraftment and rapid hematopoietic reconstitution [1-3]. Although allogeneic transplantation using G-CSFmobilized PBPC has been well studied in clinical practice, there are still uncertainties about the most efficient method and the mechanism of PBPC mobilization. We and several investigators have studied the kinetics of mobilization of PBPC in normal donors [4-7]. After daily s.c. administration of G-CSF at a dose of 10-16 µg/kg/day, the peak level of
circulating CD34+ cells is usually reached on days 4-5, with a subsequent decline thereafter despite continued G-CSF administration. However, there are no reports about the kinetics of PBPC release induced by continuous i.v. administration of G-CSF. Since the most efficient method of PBPC mobilization remains to be defined, we hypothesized that continuous i.v. administration of G-CSF might influence the kinetics of mobilization of PBPC. The mechanisms of hematopoietic progenitor cell (HPC) mobilization from the bone marrow (BM) into the circulation are still poorly understood. Whether HPC circulate or remain within the BM may depend on the presence and function of
Correspondence: Chu-Myong Seong, M.D., Department of Hematology-Oncology, Ewha Woman’s University Mock-Dong Hospital, Yangchun-Ku, Mock-6-Dong 911-1 Seoul, Korea 158-710. Telephone:82-2-650-5015; Fax: 82-2-650-5062; e-mail:
[email protected] Received May 15, 2000; accepted for publication May 17, 2000. ©AlphaMed Press 1066-5099/2000/$5.00/0
STEM CELLS 2000;18:281-286
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Lee, Im, Yoo et al. adhesion molecules. Adhesion molecules mediate the adhesion of HPC to the marrow stromal and endothelial cells and play a pivotal role in HPC mobilization and homing [8]. CD34+ cells have been shown to express a wide repertoire of adhesion molecules including integrins (very late antigen-4 [VLA-4]; CD49d/CD29), VLA-5 (CD49e/CD29), leukocyte function antigen-1 ([LFA-1]; CD11a/CD18), selectins (Lselectin [CD62L], sialyl LewisX), immunoglobulin superfamily (intercellular adhesion molecule-1 [ICAM-1; CD54]), LFA-3 (CD58), platelet/endothelial cell adhesion molecule-1 (PECAM-1; CD31), and homing-associated cell adhesion molecule (H-CAM; CD44). Several studies have suggested that among the many families of adhesion molecules, the β1 integrin VLA-4 and L-selectin are probably the most involved in the mobilization of PBPC [9-12]. However, the expression of other adhesion molecules on mobilized PBPC has shown substantial variations and especially, the phenotypic modulation of CD44 or CD31 during G-CSF-induced PBPC mobilization has not been adequately studied. The aim of the present study is to evaluate the kinetics of CD34+ cells and investigate the potential modulation of CD44 and CD31 expression on CD34+ cells during continuous i.v. G-CSF administration, thus to elucidate possible mechanisms of PBPC mobilization. MATERIALS AND METHODS Normal Donors Fifteen healthy donors (5 men and 10 women) were enrolled in this study. The median age was 38 years (range, 2056). Written informed consent was obtained from all donors approved by the institutional review board of Ewha Woman’s University Medical Center. All were normal on physical examination and routine hematological and biochemical screening. Mobilization and Collection of PBPC G-CSF (Filgrastim, Kirin Brewery Co; Tokyo, Japan; http://www1.Kirin.co.jp/english/r_d/pha/index.html), 10 µg/kg/day was administered for four consecutive days through continuous 24-h i.v. infusion. We collected PBPC on the day following the fourth dose of G-CSF using a blood cell separator (COBE Spectra; Lakewood, CO; http://cobebct.com). The total blood volume processed per apheresis was three times the donor’s blood volume at flow rates of 70-80 ml/min. The whole blood:anticoagulant ratio was programmed, and anticoagulant citrate dextrose solution was used. The total collected CD34+ cells were counted according to the International Society of Hematotherapy and Graft Engineering guideline [13]. In 12 of 15 subjects, the target dose of ≥3 × 106 CD34+ cells/kg was collected with a single
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apheresis. The remaining three subjects required two collections on consecutive days. All donors successfully completed mobilizing treatment and PBPC collection without any significant adverse effects. Immunophenotyping For measurement of complete blood counts, CD34+ cell levels and their expression of CD31 and CD44, venous peripheral blood (PB) sampling was performed immediately before the administration of G-CSF (baseline) and 4, 8, 24, 48, 72, 96, and 120 h thereafter. PB mononuclear cells (MNC) were isolated by standard Ficoll-Hypaque (Pharmacia; Uppsala, Sweden; http://www.pnu.com; density, 1.077 g/ml) density gradient centrifugation. Freshly isolated cell preparations (1 × 106 MNC) were incubated for 30 min at 4°C in the dark with fluorescein isothiocyanate-conjugated anti-CD34 (HPCA-2; Becton Dickinson; San Jose, CA; http://www.bd.com), phycoerythrin (PE)-conjugated anti-CD31 (PharMingen, San Diego; CA; http://www.pharmingen.com) and PE-conjugated antiCD44 (clone F10-44-2, Serotec; Oxford, UK). Fluorescenceactivated cell sorter lysing solution (Becton Dickinson) was used to lyse red cells after staining. As isotype controls for the staining, we used FITC-conjugated mouse immunoglobulin (Ig) G1 and PE-conjugated mouse IgG1 or IgG2a (Becton Dickinson). Phenotypic analysis of CD34+ cells was performed with a FACScan flow cytometer (Becton Dickinson) equipped with a 15-mW air-cooled argon-ion laser tuned at 488 nm. LYSIS II software was used for data acquisition and analysis. To calculate the total CD34+ cells, the number of MNC per µl of PB was multiplied by the percentage of CD34+ cells. The expression of CD44 and CD31 molecules on CD34+ cells was shown in relative fluorescence intensity ([RFI] = mean fluorescence intensity of CD34+ cells stained with antiCD44 or anti-CD31/mean fluorescence intensity of CD34+ cells stained with isotype-matched control antibody). Statistical Analysis Data are presented as mean × standard error (SE). For the statistical comparisons of values before and after G-CSF administration, repeated measure analysis was performed using mixed procedures of SAS for Windows (version 6.12). A significant difference was defined as a p value < 0.05. RESULTS Kinetics of CD34+ Cell Mobilization Serial changes of total WBC, granulocyte and lymphocyte concentrations relative to baseline values are shown in Table 1. The baseline numbers of WBC, granulocytes and lymphocytes were 5.88 ± 2.85, 3.34 ± 2.37 and 1.77 ± 0.35
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Table 1. Absolute numbers of WBC, granulocytes, and lymphocytes in donor peripheral blood before and during continuous i.v. G-CSF administration Baseline
4h
8h
d+1
d+2
d+3
d+4
d+5
WBC (× 109/l) Difference
5.88 ± 2.85
4.35 ± 3.26
7.99 ± 3.56
18.41 ± 3.16
28.61 ± 3.03
36.03 ± 3.04
44.40 ± 3.04
49.14 ± 3.03
p = 0.0002
p = 0.0001
p < 0.0001
p < 0.0001
p < 0.0001
Granulocytes (× 109/l) Difference
3.34 ± 2.37
14.61 ± 2.53
23.48 ± 2.42
28.13 ± 2.43
36.58 ± 2.42
38.35 ± 2.43
p = 0.0001
p < 0.0001
p < 0.0001
p < 0.0001
p < 0.0001
Lymphocytes (× 109/l) Difference
1.77 ± 0.35
2.45 ± 0.43
3.66 ± 0.41
4.82 ± 0.41
4.60 ± 0.40
3.56 ± 0.42
p = 0.1187
p < 0.0001
p < 0.0001
p < 0.0001
p = 0.0001
2.07 ± 2.53
1.66 ± 0.43
5.90 ± 2.85
1.24 ± 0.48
Data represent mean ± SE.
Table 2. CD34+ cell concentrations in donor peripheral blood before and during continuous i.v. G-CSF administration Baseline
4h
8h
d+1
d+2
d+3
d+4
d+5
CD34 (%) Difference
0.06 ± 0.09
0.04 ± 0.10
0.04 ± 0.09
0.10 ± 0.09
0.29 ± 0.10 p = 0.0216
0.55 ± 0.09 p = 0.0020
0.47 ± 0.09 p = 0.0697
0.33 ± 0.13 p = 0.2341
(× 103/l) Difference
2.03 ± 5.69
1.48 ± 5.06
3.85 ± 5.17
5.50 ± 6.23
15.56 ± 8.49 p = 0.1031
51.12 ± 24.83 p < 0.0001
46.66 ± 24.93 p < 0.0001
23.40 ± 16.62 p = 0.0030
+
Data represent mean ± SE.
Table 3. Modulation of CD44 and CD31 expression on CD34+ cells during continuous i.v. G-CSF administration Baseline
4h
8h
CD31 (RFI) 145.86 ± 30.52 143.25 ± 39.65 138.20 ± 39.52 Difference
d+1
d+2
d+3
d+4
d+5
121.48 ± 41.45
134.91 ± 42.83
85.63 ± 38.86 p = 0.1255
34.47 ± 38.87 p = 0.0055
17.33 ± 50.68 p = 0.0134
CD44 (RFI) 579.59 ± 133.69 501.60 ± 119.61 535.35 ± 109.95 478.75 ± 119.62 435.80 ± 123.89 281.02 ± 105.15 164.76 ± 107.44 210.69 ± 129.25 Difference p = 0.0059 p = 0.0002 p = 0.0056 Data represent mean ± SE. RFI = mean fluorescence intensity (MFI) of CD34+ cells stained with anti-CD44/CD31 ⫼ MFI of CD34+ cells with isotype-matched control antibody.
× 109/l, respectively. After continuous i.v. administration of G-CSF, the WBC counts increased up to day 5 and reached approximately 8.4-fold above the baseline. Changes in the granulocyte count were similar to those in WBC counts. The number of lymphocytes increased slightly up to day 4 (2.7-fold above the baseline), but no further increase occurred on day 5. Although there were considerable variations among the healthy donors, the statistical peaks of CD34+ cell levels were consistently observed on day 3 or day 4. Up to day 4 of G-CSF treatment, the circulating CD34+ cell population expanded by 25-fold. The percentage and absolute number of CD34+ cells significantly increased on day 3 (0.55 ± 0.09%, 51.12 ± 24.83 × 103/ml) and day 4 (0.47 ± 0.09%, 46.66 ± 24.93 × 103/ml), compared to baseline values (0.06 ± 0.09%, 2.03 ± 5.69 × 103/ml) (Table 2).
Modulation of CD44 and CD31 Expression on CD34+ Cells Table 3 shows the modulation of CD44 and CD31 RFI on CD34+ cells before and after G-CSF administration. The RFI of CD44 molecules on CD34+ cells, used as a parameter for antigen density, gradually decreased during G-CSF administration and reached the lowest level on day 4, compared to baseline levels (164.8 ± 107.4 versus 579.6 ± 133.7, p = 0.0002) and then slightly increased on day 5. The expression of CD31 on CD34+ cells also significantly decreased from the steady-state level after day 4 of G-CSF treatment (34.5 ± 38.9 versus 145.7 ± 30.5, p = 0.0055), confirming the data found in double fluorescence analysis (Table 3). The number of CD34+ cells in association with the modulation of CD44 and CD31 expression from steadystate to day 5 of G-CSF treatment is indicated in Figure 1.
Lee, Im, Yoo et al.
284
1,000
100
10 CD34+ (× 103/ml) CD44 (RFI) CD31 (RFI)
1 Baseline 4 h
8h
d+1
d+2
d+3
d+4 d+5
Figure 1. Effect of continuous i.v. G-CSF administration on the mean number of CD34+ cells and their expression of CD44 and CD31.
DISCUSSION As the number of allogeneic PBPC transplantations increases, progenitor cell mobilization by G-CSF in normal donors is a more widely used procedure and will increase further since PBPC has been used in unrelated donor transplants. Although it is considered to be a relatively safe procedure, there are still uncertainties about the most efficient method of progenitor cell mobilization. On the basis of currently available data, G-CSF appears to have reproducible biologic activity and an acceptable safety profile in normal subjects [4, 5, 14, 15]. The pharmacological profile of G-CSF in normal donors shows maximum serum concentrations after s.c. administration within 2-8 h [16]. Due to the short elimination half-life of G-CSF of about 3-4 h, we considered that the administration of G-CSF once or twice daily might be suboptimal. Therefore we investigated the efficacy in mobilizing PBPC with a continuous i.v. administration schedule and compared it with a once-or-twice-daily schedule suggested from previous studies [14]. Several investigators have found that after daily administration of G-CSF, the peak level of circulating CD34+ progenitor cells (ordinarily a 15- to 35-fold increase over baseline values) is usually reached around day 5, with a subsequent decline thereafter despite continued administration of G-CSF [4-6]. We also previously found that the CD34+/Thy-1dim subset of CD34+ progenitors was mobilized with similar kinetics during recombinant human G-CSF treatment, increasing in frequency by 42.1-fold [7]. Our data indicated that the peak level of CD34+ cells was reached after three days of continuous i.v. G-CSF. A 25-fold increase in the number of PB CD34+ cells was seen on day 3 of G-CSF administration compared to steady-state. This was confounded by the broad interindivid-
ual variation in the capacity of normal subjects to mobilize PBPC. Within the limitations imposed by this biologic heterogeneity and group size, our results showed that the peak time of mobilization of CD34+ cells could be shortened about 24-48 h by continuous i.v. administration of G-CSF. Although we did not check the serial alternation of serum G-CSF level, we can postulate that the maintenance of the serum G-CSF level through continuous i.v. administration may be responsible for shortening the time of PBPC mobilization. Recently, CD44 and CD31 expression has been demonstrated on CD34+ cells, both of which can bind to extracellular matrix components in the BM [17, 18]. CD44 is thought to play an important role in myelopoiesis. CD44 mediates the adhesion of CD34+ cells to hyaluronan, its best-known ligand, which is present in the hematopoietic extracellular matrix. In addition, CD44 may also be involved in the regulation of CD34+ cell proliferation and development, because the addition of anti-CD44 monoclonal antibodies was reported to either inhibit or enhance stromal cell-dependent hematopoiesis [18]. Recently, we found that the patterns of apoptosis during ex vivo expansion of human cord blood CD34+ cells were distinctively associated with downregulated CD44 appearing from the fourth day up to the second week according to the type of cytokines used (i.e., G-CSF or thrombopoietin) [19]. Meanwhile, CD31 has been shown to be involved in mediating the penetration of HPC through the endothelial layer. However it was unknown whether PBPC egress may result from downregulation of CD44 or CD31 adhesion molecule expression. Our data showed that there were significant decreases of CD44 and CD31 expression on CD34+ cells after three days of G-CSF treatment. Several
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groups have shown differences in adhesion molecule expression between mobilized PB CD34+ cells and BM CD34+ cells, in particular, downregulation of VLA-4, VLA-5, and LFA-3. But there were no significant changes of CD44 or CD31 expression on mobilized CD34+ cells in PB [9-12, 20]. Although we did not evaluate the expression of VLA-4, VLA-5, and LFA-3 at the same time, the reason for the different observation between the results by others shown above and ours is that downregulation of CD44 and CD31 may be due to the different subjects and mobilization methods. However, how this change in expression is brought about and whether it determines the release of CD34+ cells into the PB is unknown. Recently, Papayannopoulou and Nakamoto [9] have proposed the predominant role of VLA-4 in the adhesion of hematopoietic progenitors to the BM stroma by preincubation of transplanted marrow cells with antibodies to VLA-4 or in vivo administration of an anti-VLA-4, which led to a dramatic mobilization of clonogenic cells into the PB. Bellucci et al. [12] have suggested that the CD34+ cell compartment, exposed in vivo to a five-day administration of G-CSF, showed a decreased expression of VLA-4 and L-selectin and a reduced adherence to stromal cells, compared to the steady-state PB. Taken together, one may postulate that the decrease in the adhesive properties of CD34+ cells with downregulation of CD44 and CD31 might facilitate the selective mobilization of CD34+ cells and the retention of these cells in the PB during G-CSF administration. The bulk of CD34+ cells are CD38+ and comprise many proliferating cells, whereas CD34+CD38– cells are mostly in G0. These phenotypic differences can be also explained by changes in the cell cycle
Mobilization of CD34+ Cells During Continuous i.v. Administration of G-CSF status, and downregulation of CD44 and CD31 expression may occur as CD34+ cells enter the cell cycle. For a better understanding of the significance of CD44 and CD31 modulation on G-CSF-mobilized CD34+ cells, functional experiments investigating the cell cycle status and stem cell adhesion capacity before and after G-CSF administration are necessary. Interestingly, the fact that stimulation with G-CSF does not induce an immediate increase of CD34+ cells suggests a more complex interaction of other adhesion molecules or cytokines in the BM microenvironment. Papayannopoulou et al. [21] reported that anti-VLA-4- or anti-VCAM-1-induced mobilization requires signaling which is likely accomplished through the kit/kit ligand pathway. Therefore, further characterization of the molecular mechanisms relating to the role of adhesion molecules, including CD44/CD31 and cytokines of the BM microenvironment involved in the PBPC mobilization, is required. We conclude that continuous i.v. administration of GCSF apparently results in more rapid mobilization of CD34+ cells, at least 24-48 h, and downregulation of CD44 and CD31 on CD34+ cells is likely to be involved in the mobilization of PBPC after treatment of continuous i.v. G-CSF. ACKNOWLEDGMENT We thank Ms. Jee-Young Ahn (Department of Hematology-Oncology, Ewha Woman’s University Cancer Research Center, Seoul, Korea) for her excellent technical assistance. This work was supported in part by grants from the Kirin Research Fund and the Good Health R & D Project (HMP-98-D-3-0024) R.O.K. (to C-M Seong).
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