Thrombopoietin enhances neutrophil production by bone marrow

Thrombopoietin enhances neutrophil production by bone marrow hematopoietic progenitors with the aid of stem cell factor in congenital neutropenia Nobukuni Sawai* Kenichi Koike,* Hadija Hemed Mwamtemi,* Susumu Ito,† Yumi Kurokawa,* Kazuo Sakashita,* Tatsuya Kinoshita,* Tsukasa Higuchi,* Kouichi Takeuchi,* Masaaki Shiohara,* Takehiko Kamijo,* Yumiko Higuchi,‡ Hiroshi Miyazaki,§ Takashi Kato,§ Masao Kobayashi,储 Munenori Miyake,** Kozo Yasui,* and Atsushi Komiyama* *Department of Pediatrics, Shinshu University School of Medicine, Matsumoto, †Blood Transfusion Service and ‡ Central Clinical Laboratories, Shinshu University Hospital, Matsumoto; §Pharmaceutical Research Laboratory, Kirin Brewery Co. Ltd., Takasaki; 储Department of Pediatrics, Hiroshima University School of Medicine, Hiroshima; and **Department of Pediatrics, Osaka Medical College, Takatsuki, Japan

Abstract: We examined the effects of granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF), and thrombopoietin (TPO), alone or in combination, on the generation of neutrophils by bone marrow (BM) cells from three patients with severe congenital neutropenia (SCN) through the use of a serum-deprived liquid culture system. Synergistic effects of G-CSF and SCF on the neutrophil production by BM CD34ⴙCD38ⴙc-kitⴙ cells were observed in SCN patients as well as in normal controls. The addition of TPO to the culture containing G-CSF and SCF further augmented the growth of neutrophils in the two groups. Single-cell culture experiments revealed that the three-factor combination caused increases in both the number and size of neutrophil colonies compared with G-CSF ⴙ SCF in normal BM cells, whereas only a significant increment in the colony size was observed in SCN patients. Even in the presence of SCF or SCF ⴙ TPO, the concentrations of G-CSF necessary for the substantial production of neutrophils by CD34ⴙCD38ⴙc-kitⴙ cells were higher in two patients compared with the levels obtained by normal control cells. In addition, TPO did not accelerate the maturation of neutrophilic cells supported by G-CSF ⴙ SCF. When BM CD34ⴙCD38ⴚc-kitⴙ cells were targeted, the addition of TPO to the culture containing G-CSF and SCF was required for significant neutrophil colony growth in the two groups. These results suggest that TPO enhances the G-CSF-dependent neutrophil production with the aid of SCF in this disorder. J. Leukoc. Biol. 68: 137–143; 2000.

ration arrest at the stage of promyelocyte and myelocyte in the bone marrow. Treatment with granulocyte colony-stimulating factor (G-CSF) tremendously diminished the number and severity of infections in SCN patients [1]. It is well known that the therapeutic doses of G-CSF necessary to improve neutropenia are higher in most SCN patients than in cancer patients with neutropenia caused by chemotherapy. Based on the evidence obtained by clonal cell culture studies [2, 3], an altered responsiveness of hematopoietic progenitors to growth factors has been proposed as the most likely pathogenesis of SCN. Thrombopoietin (TPO) was initially considered to be a selective stimulator in megakaryocytopoiesis and platelet production. However, Grossmann et al. [4] reported that treatment with TPO accelerates platelet, red blood cell, and neutrophil recovery in myelosuppressed mice, indicating in vivo effects of TPO on multiple cell lineages. They also showed that the combined use of TPO and G-CSF further improves neutropenia associated with intensive chemotherapy in mice [5]. We recently described that neutrophilic cell production was induced by a combination of stem cell factor (SCF) and TPO from CD34⫹ cord blood cells in a long-term serum-deprived liquid culture [6]. Furthermore, Brizzi et al. [7] reported that human polymorphonuclear cells (PMN) express c-Mpl, and that TPO stimulates the activation of PMN by inducing interleukin-8 (IL-8) release and by priming these cells to oxygen metabolite production. Taken together, treatment of SCN patients with TPO may be an alternative therapeutic approach to enhance myeloid differentiation. Therefore, we attempted to elucidate the effects of TPO on the neutrophil production from SCN hematopoietic progenitors in a serum-deprived culture system. Our results provide fundamental insight for the clinical application of TPO in SCN.

Key Words: granulocyte colony-stimulating factor 䡠 CD34⫹CD38⫹ c-kit⫹ cells

INTRODUCTION Severe congenital neutropenia (SCN) is characterized by decreased numbers of circulating neutrophils, and often a matu-

Correspondence: Kenichi Koike, M.D., Department of Pediatrics, Shinshu University School of Medicine, 3-1-1, Asahi, Matsumoto, 390-8621, Japan. E-mail: [email protected] Received October 11, 1999; revised February 15, 2000; accepted February 16, 2000.

Journal of Leukocyte Biology Volume 68, July 2000 137

TABLE 1.

Patient 1

Sex Age (years) At diagnosis Age (months) WBC (⫻109/L) Neutrophil (%) Monocyte (%) Eosinophil (%) Basophil (%) Lymphocyte (%) Hb (g/dL) Platelet (⫻109/L) Episodes of infection

Serum-deprived suspension culture

Summary of Clinical Data

male 9 12 5.5 0 0 1 0 99 10.7 788 Otitis media Gingivitis

Patient 2

Patient 3

female 1.5

female 4

6 6.04 1 3 4 1 91 10.5 334 Otitis media Bacteremia

13 8.94 0 24 7 2 67 10.8 459 Otitis media Bacteremia Enterocolitis Pneumonia

MATERIALS AND METHODS Patients Bone marrow cells were obtained from three SCN patients. They had had severe neutropenia and frequent bacterial infections, as shown in Table 1. Bone marrow examination showed normal neutrophil development up to the promyelocyte or myelocyte stage with a marked depletion of mature neutrophils. At the time of the study, the absolute neutrophil count was less than 200/mL in all of the cases. There was no family history of an increased susceptibility to infection.

Factors and antibodies Human recombinant TPO and SCF were provided by Kirin Brewery (Takasaki, Japan). Human recombinant G-CSF was a gift from Chugai Pharmaceutical (Tokyo, Japan). For the flow cytometric analysis and cell sorting, monoclonal antibodies (mAbs) for CD34 (8G-12, fluorescein isothiocyanate, FITC), CD38 (HB7, allophycocyanin, APC), and c-kit (104D2, phycoerythrin, PE) were purchased from Becton Dickinson Immunocytometry Systems (Mountain View, CA). Recombinant FITC-labeled Annexin V and 7-amino-actinomycin D (7-AAD) were obtained from PharMingen (San Diego, CA). For the immunocytochemical analysis, purified mAbs for human myeloperoxidase (MPO) and CD41 (SZ22) were purchased from Immunotech (Marseilles, France).

Isolation of CD34⫹CD38⫹c-kit⫹ cells and CD34⫹CD38⫺c-kit⫹ cells from bone marrow mononuclear cells (BM MNCs). BM cells from three SCN patients and three healthy volunteers were aspirated in heparinized plastic syringes after informed consent was obtained. BM MNCs were separated by density centrifugation over Ficoll-Paque (Pharmacia, Piscataway, NJ), washed twice, and suspended in Ca2⫹- and Mg2⫹-free phosphate-buffered saline (PBS) containing 1 mmol/L EDTA 2-Na, and 2.5% fetal bovine serum (FBS, Hyclone, Logan, UT). The cells (2 ⫻ 106) were incubated with 20 mL of FITC-conjugated anti-CD34 mAb, 5 mL of APC-conjugated anti-CD38 mAb, and 20 mL of PE-conjugated anti-c-kit mAb for 30 min at 4°C. As negative controls, the cells were stained with FITC-, APC-, and PE-conjugated mouse IgG1 (Becton Dickinson). CD34⫹CD38⫹c-kit⫹ cells and CD34⫹CD38⫺c-kit⫹ cells were individually sorted in 5-mL tubes by the FACStarplus flow cytometer (Becton Dickinson), as described previously [6]. The purity of each subpopulation after sorting was higher than 90%, compared with that before sorting. In the preliminary experiments, in contrast with the plating efficiency of the two subpopulations, CD34⫹CD38⫹c-kit⫺ cells and CD34⫹CD38⫺c-kit⫺ cells showed no or very low proliferative potential under stimulation with G-CSF, SCF, and TPO in SCN patients and normal controls.

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Serum-deprived liquid cultures were carried out in 24-well culture plates (no. 3047; Becton Dickinson) using a technique described previously [6, 8]. One thousand BM CD34⫹CD38⫹ c-kit⫹ cells were cultured in individual wells containing 2 mL of ␣-medium (Flow Laboratories, Rockville, MD) supplemented with 1% deionized bovine serum albumin (Sigma Chemical, St. Louis, MO), 600 ␮g/mL fully iron-saturated human transferrin (⬃98% pure, Sigma), 16 ␮g/mL soybean lecithin (Sigma), and 9.6 ␮g/mL cholesterol (Nakalai Tesque, Kyoto, Japan) in the presence of 10 ng/mL of G-CSF, 10 ng/mL of SCF, or 10 ng/mL of TPO, alone or in combination. The plates were incubated at 37°C in a humidified atmosphere flushed with a mixture of 5% CO2, 5% O2, and 90% N2. Half of the cell-free supernatant was replaced with fresh medium containing growth factor(s) every 7 days. The number of viable cells was determined by a trypan blue exclusion test using hemocytometers. The cells were then processed for the cytochemical and immunological stainings, and for flow cytometric analysis.

Serum-deprived single-cell culture Single-cell sorting was performed by two-step sorting. BM CD34⫹CD38⫹ckit⫹ cells or CD34⫹CD38⫺c-kit⫹ cells were collected in 5-mL tubes, and were re-sorted into individual wells of a 96-well U-bottomed tissue culture plate (no. 3077; Becton Dickinson) containing 100 ␮L of the serum-deprived culture medium supplemented with G-CSF, SCF, or TPO, alone or in combination, using the FACStarplus flow cytometer equipped with an automatic cell deposition unit, as described previously [6, 9]. Ninety-nine percent of the wells contained a single cell on the first day of culture. The plates were incubated at 37°C in a humidified atmosphere flushed with a mixture of 5% CO2, 5% O2, and 90% N2. Colonies consisting of more than 30 cells were scored in situ on an inverted microscope. At 3 weeks of culture, the size of the small colonies (consisting of ⬍300 cells) was determined by counting individual cells in situ. Colonies consisting of ⬎300 cells were individually lifted by an Eppendorf micropipette, and made into single cell suspensions. Colony size was estimated by using a counting chamber. Then, the constituent cells of the colonies were stained with peroxidase (POX).

Cytochemical staining Cultured cells were spread on glass slides with the use of a Cytospin II (Shandon Southern, Sewickly, PA), and stained with May-Gru¨nwald-Giemsa, Biebrich scarlet, or toluidine blue. Cytochemical reactions with POX, ␣-naphthyl butyrate esterase, and alkaline phosphatase (ALP) were performed, as described previously [6, 10].

Immunocytochemical staining The reaction with mouse mAb against MPO or CD41 was detected using the alkaline phosphatase-anti-alkaline phosphatase method (Dako APAAP Kit System, Dako, Carpinteria, CA), as described previously [10]. The isotype mouse mAb was used as a control.

Nitroblue tetrazolium (NBT) assay The NBT assay was performed on cultured neutrophils generated with G-CSF, G-CSF ⫹ SCF, or G-CSF ⫹ SCF ⫹ TPO from BM CD34⫹CD38⫹c-kit⫹ cells according to the procedure described [11]. Briefly, 1 ⫻ 105 cells were suspended in 0.5 mL of 0.2% NBT reaction medium, and stimulated with phorbol myristate acetate (PMA, Sigma) at the final concentration of 20 ng/mL. After incubation for 15 min at 37°C, the cells were spread on glass slides. The percentage of NBT-positive cells (containing blue-purple formazan deposits from reduction of NBT) was determined by evaluating 200 cells with light microscopy. When peripheral blood neutrophils were used as target cells, almost all of the cells were NBT-positive.

Detection of cellular apoptosis Detection of apoptotic cells was performed according to a modification of the procedure described by Koopman et al. [12]. The cultured cells generated by G-CSF ⫹ SCF or G-CSF ⫹ SCF ⫹ TPO from BM CD34⫹CD38⫹c-kit⫹ cells were washed with PBS, and resuspended at a concentration of 1 ⫻ 106 cells/mL in binding buffer (10 mM HEPES/NaOH, pH 7.4, 0.14 mM NaCl, 2.5

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Fig. 1. Sorting of CD34⫹CD38⫹c-kit⫹ cells and CD34⫹CD38⫺c-kit⫹ cells from BM MNCs. BM MNCs were stained with FITC-conjugated anti-CD34 mAb, APCconjugated anti-CD38 mAb, and PE-conjugated anti-c-kit mAb. As negative controls, FITC-, APC-, and PE-conjugated mouse IgG1 were used. The gate (R1) was set on CD34⫹ cells according to FITC fluorescence and side scatter characteristics (SSC). The cells in the R2 and R3 region were sorted as CD34⫹CD38⫹c-kit⫹ cells and CD34⫹CD38⫺c-kit⫹ cells, respectively, as described in Materials and Methods. BM MNCs of normal control 1 were stained with mouse IgG1 (A) or with anti-CD34 mAb (B). BM MNCs of SCN patient 1 were stained with anti-CD34 mAb (C). BM CD34⫹ cells of normal control 1 were stained with APCand PE-conjugated mouse IgG1 (D) or with anti-CD38 mAb and anti-c-kit mAb (E). BM CD34⫹ cells of SCN patient 1 were stained with the mAbs (F).

mM CaCl2). Cells (1 ⫻ 105 in 100 ␮L) were incubated with 5 ␮L of FITC-labeled Annexin V and 5 ␮L of 7-AAD for 15 min at room temperature in the dark. After an addition of 400 ␮L of the binding buffer to the tube, the cells were analyzed with the FACScan flow cytometer (Becton Dickinson) using the Lysis 2 software program. To set up the compensation and quadrants, we prepared unstained cells, cells stained with Annexin V, and cells stained with 7-AAD. The percentages of Annexin V⫹ 7-AAD⫺ cells (cells undergoing apoptosis) and Annexin V⫹ 7-AAD⫹ cells (cells in the end stage of apoptosis, undergoing necrosis or already dead) were estimated.

Statistical analysis Values are expressed as means ⫾ SD. To determine the significance of difference between two independent groups, we used the unpaired t test or Mann-Whitney U test if the data were not normally distributed. One-way analysis of variance, followed by post hoc contrasts with the Bonferroni limitation, was used for more than three independent groups. The paired t test was used to assess the significance of difference in the neutrophilic cell production or the neutrophil colony growth (see Fig. 2 and Tables 3 and 4). To compare the number of neutrophilic cells or the size of neutrophil colonies grown by BM CD34⫹CD38⫹c-kit⫹ cells, the statistical analysis was performed on logarithms of cell numbers.

the FACStarplus flow cytometer, and plated at 1,000 cells/well containing serum-deprived liquid culture medium supplemented with 10 ng/mL of G-CSF, 10 ng/mL of SCF, or 10 ng/mL of TPO, alone or in combination. Half of the culture medium was replaced weekly with fresh medium containing the growth factor(s) for 3 weeks. The results are presented in Figure 2. In the absence of the growth factors, almost all of the cells degenerated within 2 weeks. G-CSF alone supported the growth of 3.7 ⫾ 2.0 ⫻ 103 POX⫹ cells from SCN CD34⫹CD38⫹c-kit⫹ cells at 3 weeks. Neither SCF alone nor TPO alone supported the production of significant numbers of POX⫹ cells in any of the SCN patients, although a small number of mast cells grew in the presence of SCF and a few megakaryocytes were generated by stimulation with TPO. In agreement with the evidence reported previously [3], the com-

RESULTS Effects of TPO on neutrophil production by BM CD34⫹CD38⫹c-kit⫹ cells under stimulation with G-CSF plus SCF To examine the effects of G-CSF, SCF, and TPO, alone or in combination, on the generation of neutrophils by BM cells from SCN patients and normal controls, we used CD34⫹CD38⫹ckit⫹ cells and CD34⫹CD38⫺c-kit⫹ cells as the target cells. The percentage of CD34⫹ cells among total BM MNCs was 1.1 ⫾ 0.2% (mean ⫾ SD) in three SCN patients, being similar to the value in the three normal controls (1.2 ⫾ 0.3%), as shown in Figure 1. The percentages of CD34⫹CD38⫹c-kit⫹ cells and CD34⫹CD38⫺c-kit⫹ cells among BM CD34⫹ cells were 66.8 ⫾ 5.7 and 4.5 ⫾ 3.8%, respectively, in SCN patients; and 54.0 ⫾ 3.3 and 4.7 ⫾ 1.9%, respectively, in the controls. First, BM CD34⫹CD38⫹c-kit⫹ cells were sorted by

Fig. 2. Neutrophil production by BM CD34⫹CD38⫹c-kit⫹ cells under stimulation with G-CSF, SCF, and TPO, alone or in combination. One thousand BM CD34⫹CD38⫹c-kit⫹ cells were plated in a well containing serum-deprived liquid culture medium supplemented with 10 ng/mL of G-CSF, 10 ng/mL of SCF, or 10 ng/mL of TPO, alone or in combination. Half of the cell-free supernatant was replaced with fresh medium containing the growth factor(s) every week. The viable cells were enumerated at 3 weeks. Then the cells were processed for staining with POX. Results shown are the mean ⫾ SD of POX⫹ cells generated by BM CD34⫹CD38⫹c-kit⫹ cells from three SCN patients or three normal controls. G, G-CSF; S, SCF; T, TPO. Significant difference from G-CSF ⫹ SCF (*P ⬍ 0.05, #P ⬍ 0.02, ¶P ⬍ 0.0001).

Sawai et al. TPO enhances neutrophil growth in congenital neutropenia

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bination of G-CSF and SCF supported the production of a greater number of POX⫹ cells by SCN progenitors than G-CSF alone. There was no substantial difference in the number of POX⫹ cells between G-CSF ⫹ TPO and G-CSF alone. The ability of SCF ⫹ TPO to generate POX⫹ cells was one-ninth to one-fourth of that of G-CSF alone. It is of particular interest that the three-factor combination caused a significant increase in the growth of POX⫹ cells by SCN CD34⫹CD38⫹c-kit⫹ cells, compared with the value obtained by G-CSF ⫹ SCF. SCF-mediated amplification of the G-CSF-dependent POX⫹ cell generation and TPO-mediated amplification of the G-CSF ⫹ SCF-dependent POX⫹ cell generation were also observed in normal controls. The numbers of POX⫹ cells grown with G-CSF alone, G-CSF ⫹ SCF, or G-CSF ⫹ SCF ⫹ TPO were substantially higher in normal controls than in SCN patients (P ⬍ 0.05). Percentages of POX-positive cells among the cultured cells were ⬎92%, irrespective of the type of cytokine stimulation, in SCN patients and normal controls. Staining with anti-MPO mAb, alkaline phosphatase, Biebrich scarlet, toluidine blue, and ␣-naphthyl butyrate esterase showed that the POX⫹ cells were of the neutrophilic lineage. In the presence of G-CSF, SCF, and TPO, the cells expressing other lineage markers, including CD41 were not significantly expanded. Consistent with the results described by Hestdal et al. [3], the mean percentage of mature neutrophils (band cells and segmented cells) grown under stimulation with G-CSF alone was lower in SCN patients than in normal controls (29.7 ⫾ 12.3 vs. 82.2 ⫾ 5.3%). In cases 1 and 2, the addition of SCF to the culture with G-CSF induced a twofold increase in the frequency of mature cells, which was not influenced by the further addition of TPO. On the other hand, in case 3 and normal controls, the percentage of mature neutrophils was not different between G-CSF

TABLE 2.

alone and G-CSF ⫹ SCF, and the three-factor combination reduced the relative number of mature cells by ⬃50%. We then compared the ability of neutrophils grown with G-CSF alone, G-CSF ⫹ SCF, or G-CSF ⫹ SCF ⫹ TPO to generate superoxide by the respiratory burst oxidase in cases 1 and 3. When PMA was used as a respiratory burst agonist, there was no significant difference in the percentages of NBTpositive cells among three types of cytokine stimulation in SCN patients and normal controls (⬃20 –30% in the two groups).

Dose response to G-CSF of neutrophil production by BM CD34⫹CD38⫹c-kit⫹ cells We then examined whether the addition of SCF or SCF ⫹ TPO could reduce the requirement for G-CSF in the neutrophil production. The results are presented in Table 2. In all of the normal controls, when stimulated with G-CSF alone, the optimal doses of G-CSF were higher than 1 ng/mL. In the presence of SCF, G-CSF at 0.1–10 ng/mL generated more neutrophils than did G-CSF at 0.01 ng/mL. When TPO was further added to the culture with more than 0.1 ng/mL of G-CSF and SCF, all of the normal control CD34⫹CD38⫹c-kit⫹ cells maximally and equivalently generated neutrophils. In SCN patients, under stimulation with G-CSF alone, the ability of 1 ng/mL of G-CSF was significantly lower than that of 10 ng/mL of G-CSF. The addition of SCF or SCF ⫹ TPO stimulated the production of neutrophils supported by 10 ng/mL of G-CSF in all patients. However, even in the three-factor combination, the numbers of neutrophils obtained with 0.1 and 1 ng/mL of G-CSF were markedly lower than those of neutrophils obtained with 10 ng/mL of G-CSF in cases 1 and 3.

Dose Response to G-CSF of Neutrophil Production by BM CD34⫹CD38⫹c kit⫹ Cells Numbers of POX⫹ cells (⫻103) generated with G-CSF, SCF, or TPO, alone or in combination

G-CSF Patient 1 (⫹) (⫹) (⫹) Patient 2 (⫹) (⫹) (⫹) Patient 3 (⫹) (⫹) (⫹) Normal 1 (⫹) (⫹) (⫹) Normal 2 (⫹) (⫹) (⫹) Normal 3 (⫹) (⫹) (⫹)

None SCF SCF ⫹ None SCF SCF ⫹ None SCF SCF ⫹ None SCF SCF ⫹ None SCF SCF ⫹ None SCF SCF ⫹

TPO

TPO

TPO

TPO

TPO

TPO

0 ng/mL

0.001 ng/mL

0.01 ng/mL

0.1 ng/mL

1 ng/mL

10 ng/mL

0 (0) 0 (0) 0.5 ⫾ 0.2 (0.1) 0 (0) 0 (0) 0.7 ⫾ 0.1 (0.1) 0 (0) 0 (0) 0.3 ⫾ 0.3 (0.2) 0 (0) 0 (0) 2.8 ⫾ 0.9 (0.3) 0 (0) 0 (0) 2.1 ⫾ 0.5 (0.2) 0 (0) 0 (0) 1.8 ⫾ 0.9 (0.2)

0 (0) 0 (0) 0.5 ⫾ 0.2 (0.1) 0 (0) 0 (0) 0.7 ⫾ 0.1 (0.1) 0 (0) 0 (0) 0.2 ⫾ 0.1 (0.2) 0 (0) 1.3 ⫾ 0.5 (0.1) 10.2 ⫾ 0.9 (1.1) 0 (0) 1.2 ⫾ 0.5 (0.1) 9.8 ⫾ 4.3 (1.0) 0 (0) 1.3 ⫾ 0.4 (0.1) 10.2 ⫾ 0.5 (1.1)

0 (0) 0.4 ⫾ 0.4 (0.1) 0.5 ⫾ 0.2 (0.1) 0 (0) 0.7 ⫾ 0.5 (0.1) 7.7 ⫾ 0.9 (1.2) 0 (0) 0.2 ⫾ 0.1 (0.2) 0.7 ⫾ 0.1 (0.5) 0 (0) 11.2 ⫾ 0.9 (1.2) 26.0 ⫾ 1.9 (2.7) 0 (0) 11.0 ⫾ 2.4 (1.2) 26.5 ⫾ 1.6 (2.8) 0 (0) 10.8 ⫾ 2.8 (1.2) 24.5 ⫾ 1.8 (2.7)

0.5 ⫾ 0.4 (0.1) 0.6 ⫾ 0.6 (0.2) 0.7 ⫾ 0.4 (0.2) 1.3 ⫾ 0.2 (0.2) 1.9 ⫾ 0.3 (0.3) 43.4 ⫾ 1.2 (7.0) 0.4 ⫾ 0.1 (0.3) 0.4 ⫾ 0.1 (0.3) 3.2 ⫾ 0.5 (2.5) 2.8 ⫾ 0.9 (0.3) 80.0 ⫾ 4.9* (8.3) 228.5 ⫾ 16.0* (23.8) 2.8 ⫾ 0.9 (0.3) 78.7 ⫾ 4.8* (8.3) 234.9 ⫾ 19.0* (24.7) 2.0 ⫾ 0.5 (0.2) 78.2 ⫾ 8.8* (8.7) 239.2 ⫾ 13.4* (26.6)

0.9 ⫾ 0.6 (0.2) 6.7 ⫾ 2.6 (1.8) 8.2 ⫾ 2.9 (2.2) 0.8 ⫾ 0.4 (0.1) 28.1 ⫾ 3.1* (4.5) 58.9 ⫾ 9.3* (9.5) 0.2 ⫾ 0.1 (0.2) 6.0 ⫾ 0.3 (4.6) 5.8 ⫾ 0.3 (4.5) 8.1 ⫾ 1.4* (0.8) 89.9 ⫾ 5.7* (9.4) 230.0 ⫾ 9.4* (24.0) 7.9 ⫾ 1.4* (0.8) 97.6 ⫾ 5.3* (10.3) 241.0 ⫾ 28.0* (25.4) 7.3 ⫾ 1.3* (0.8) 84.6 ⫾ 5.3* (9.4) 214.7 ⫾ 9.6* (23.9)

3.7 ⫾ 0.6 (1) 59.2 ⫾ 5.6 (16.0) 102.7 ⫾ 3.8 (27.8) 6.2 ⫾ 0.6 (1) 28.1 ⫾ 3.1 (4.5) 61.2 ⫾ 6.4 (9.9) 1.3 ⫾ 0.2 (1) 17.1 ⫾ 1.0 (13.2) 37.7 ⫾ 2.6 (29.0) 9.6 ⫾ 1.4 (1) 94.9 ⫾ 9.8 (9.9) 242.4 ⫾ 17.9 (25.3) 9.5 ⫾ 1.4 (1) 93.3 ⫾ 9.7 (9.8) 241.0 ⫾ 34.6 (25.4) 9.0 ⫾ 1.3 (1) 90.4 ⫾ 5.1 (10.0) 228.1 ⫾ 16.8 (25.3)

One thousand BM CD34⫹CD38⫹c-kit⫹ cells were incubated in a well containing serum-deprived liquid culture medium supplemented with different concentrations of G-CSF and/or the other cytokine(s). Both SCF and TPO were used at 10 ng/mL. The viable cells were enumerated at 3 weeks. Data represent the means ⫾ SD of triplicate wells. The values in parentheses are the mean fold increase relative to the number of POX⫹ cells generated by 10 ng/mL of G-CSF. * No significant difference from the results obtained by the culture containing 10 ng/mL of G-CSF.

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Effects of TPO on neutrophil colony growth supported by G-CSF plus SCF from BM CD34⫹CD38⫹c-kit⫹ cells To analyze the stimulatory effect of TPO on the G-CSF ⫹ SCF-dependent neutrophil production at the hematopoietic progenitor level, we performed a single-cell culture study of BM CD34⫹CD38⫹c-kit⫹ cells. As shown in Table 3, under stimulation with G-CSF alone, approximately 7% of normal CD34⫹CD38⫹c-kit⫹ cells proliferated and differentiated into the neutrophilic lineage. The addition of SCF to the culture containing G-CSF caused an increase in the number of neutrophil colonies. TPO further augmented the growth of neutrophil colonies supported by G-CSF ⫹ SCF. In SCN patients, significantly lower numbers of neutrophilic colonies were formed by G-CSF alone compared with the value obtained by normal control cells (P ⬍ 0.01). The addition of SCF substantially increased the number of G-CSF-dependent neutrophil colonies, as in normal controls. However, there was no difference in the number of neutrophil colonies between the twofactor combination and the three-factor combination. Next, we compared the size of neutrophil colonies generated by three types of cytokine stimulation in two SCN patients and two normal controls. As shown in Figure 3, SCF significantly enlarged neutrophil colonies supported by G-CSF in both of the patients. The addition of TPO to the culture containing G-CSF and SCF resulted in a further increase in the colony size compared with the value obtained by G-CSF ⫹ SCF. Similar results were obtained in the culture containing normal control BM cells.

Effects of TPO on survival of the cultured cells generated by G-CSF ⫹ SCF in SCN patients Because Brizzi et al. [7] reported the functional expression of c-Mpl on human polymorphonuclear cells, we examined whether the TPO-mediated increase in the numbers of neutrophils grown by G-CSF ⫹ SCF from BM CD34⫹CD38⫹c-kit⫹

TABLE 3.

Neutrophil Colony Growth by BM CD34⫹CD38⫹c-kit⫹ Cells in Serum-Deprived Single-Cell Cultures G

Patient 1 Patient 2 Patient 3 mean ⫾

SD

Normal 1 Normal 2 Normal 3 mean ⫾

SD

4 4 3 3.7 ⫾ 0.6 8 7 6 7.0 ⫾ 1.0

G⫹S

15 11 10 12.0 ⫾ 2.6* 13 12 14 13.0 ⫾ 1.0*

G⫹S⫹T

11 14 9 11.3 ⫾ 2.5 18 17 17 17.3 ⫾ 0.6#

The single BM CD34⫹CD38⫹c-kit⫹ cells were sorted into the individual wells of a 96-well culture plate containing the cytokine(s), as described in Materials and Methods. After 3 weeks, colonies containing more than 30 cells were scored on an inverted microscope. More than 95% of the constituent cells of pooled colonies grown by G-CSF alone, G-CSF ⫹ SCF, or G-CSF ⫹ SCF ⫹ TPO were positive for POX in SCN patients and normal controls. G, G-CSF at 10 ng/mL; S, SCF at 10 ng/mL; T, TPO at 10 ng/mL. Significant difference from G-CSF alone (* P ⬍ 0.05) and from G-CSF ⫹ SCF (# P ⬍ 0.05).

Fig. 3. Size of neutrophil colonies grown by BM CD34⫹CD38⫹c-kit⫹ cells. The sizes of neutrophil colonies generated by G-CSF alone, G-CSF ⫹ SCF, or G-CSF ⫹ SCF ⫹ TPO were determined in two SCN patients (case 1 and case 3) and two normal controls (normal 1 and normal 2), as described in Materials and Methods. Mean values (filled circles) and SD for neutrophil colonies are also presented in each group. G, G-CSF at 10 ng/mL; S, SCF at 10 ng/mL; T, TPO at 10 ng/mL. *Significant difference from G-CSF ⫹ SCF.

cells in SCN patients resulted from the prolongation of neutrophil survival. There was no difference in the percentages of Annexin V⫹7-AAD⫺ cells plus Annexin V⫹7-AAD⫹ cells between stimulation with G-CSF ⫹ SCF and stimulation with G-CSF ⫹ SCF ⫹ TPO in the two patients (48.5% for stimulation with G-CSF ⫹ SCF vs. 40.4% for stimulation with G-CSF ⫹ SCF ⫹ TPO in case 1, and 28.5% for stimulation with G-CSF ⫹ SCF vs. 29.7% for stimulation with G-CSF ⫹ SCF ⫹ TPO in case 2).

Effects of TPO on neutrophil colony growth supported by G-CSF plus SCF from BM CD34⫹CD38⫺c-kit⫹ cells We then examined whether TPO exerted action on the neutrophil colony growth by SCN CD34⫹CD38⫺c-kit⫹ cells. The results are presented in Table 4. In all of the SCN patients, neither G-CSF nor G-CSF ⫹ SCF stimulated the formation of colonies. The addition of TPO to the culture containing G-CSF and SCF was required for the colony growth. Similar results were observed in normal control cells except for the data obtained under stimulation with G-CSF ⫹ SCF. There was no difference in the incidence of hematopoietic progenitors responsive to the combination of G-CSF, SCF, and TPO between the two groups. The great majority of colonies emerged after 3 weeks, especially at 4 weeks, in both the SCN patients and normal controls. At 6 weeks, colonies grown by the three factor-combination contained 152 to 50,000 cells in case 1, 310 to 41,000 cells in case 2, and 40 to 8,200 cells in case 3, whereas the values obtained from normal controls were 250 to 12,000 cells per colony. Ninety-three to ninety-nine percent of the constituent cells of colonies were positive for POX, and most of them were positive for ALP. The remaining cells were POX-negative blastic cells.


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TABLE 4.

Neutrophil Colony Growth by BM CD34⫹CD38⫺c-kit⫹ Cells in Serum-Deprived Single-Cell Cultures G

G⫹S

G⫹S⫹T

Patient 1 Patient 2 Patient 3

0 0 0

0 0 0

11 5 9

mean ⫾

0

0

8.3 ⫾ 3.1*

0 0 0

1 2 1

4 9 7

1.3 ⫾ 0.6

6.7 ⫾ 2.5*

SD

Normal 1 Normal 2 Normal 3 mean ⫾

SD

0 ⫹

⫺

⫹

The single BM CD34 CD38 c-kit cells were sorted into the individual wells of a 96-well culture plate containing the cytokine(s), as described in Materials and Methods. Colonies containing more than 30 cells were scored on an inverted microscope. G, G-CSF at 10 ng/mL; S, SCF at 10 ng/mL; T, TPO at 10 ng/mL. Significant difference from G-CSF ⫹ SCF (* P ⬍ 0.05).

DISCUSSION There has been controversy regarding the incidence of G-CSFresponsive progenitors in SCN BM cells under serum-containing culture conditions [2, 3]. Although this discrepancy can be explained by the heterogeneity of SCN, another possibility is that the culture conditions influence the results. FBS is a potential endogenous source of growth factors such as SCF [13], which is demonstrated to improve the response of hematopoietic progenitors to G-CSF in SCN patients [3]. Therefore, in the present study, we used a serum-deprived culture system to better understand the effects of the cytokines on the growth of SCN hematopoietic progenitors. In the culture with BM CD34⫹CD38⫹c-kit⫹ cells, TPO enhanced the neutrophil production supported by G-CSF and SCF in SCN patients as well as normal controls. The single-cell culture study revealed that in normal BM cells, the addition of TPO to the culture containing G-CSF and SCF resulted in increases in both the number and size of neutrophil colonies. On the other hand, TPO did not increase the number of neutrophil colonies supported by G-CSF ⫹ SCF, but significantly enlarged neutrophil colonies in two SCN patients. Morphological analysis showed that TPO did not hasten the G-CSF ⫹ SCF-induced neutrophilic maturation. These results suggest that the combined effect of G-CSF ⫹ SCF is optimal for the initial stage of the proliferation of neutrophilic progenitors in SCN CD34⫹CD38⫹c-kit⫹ cells. Alternatively, TPO may not stimulate the entry of G-CSF ⫹ SCF-dependent neutrophilic progenitors to the proliferative process, but it may be able to enhance their subsequent growth with no acceleration of differentiation into the neutrophilic lineage. G-CSF has been demonstrated to prolong the neutrophil survival by suppressing apoptosis [14, 15]. In the G-CSF receptor-deficient mouse, a model of congenital neutropenia, Gr-1-positive cells have increased susceptibility to apoptosis [16]. Because the functional expression of c-Mpl on human polymorphonuclear cells was demonstrated [7], we examined the effects of TPO on the frequency of cells in apoptosis/ necrosis among the cultured cells generated by G-CSF ⫹ SCF. 142


There was no difference in the percentage of Annexin V⫹7AAD⫺ cells plus Annexin V⫹7-AAD⫹ cells between stimulation with G-CSF ⫹ SCF and stimulation with G-CSF ⫹ SCF ⫹ TPO in the two patients. Therefore, it is unlikely that the TPO-mediated increase in the G-CSF ⫹ SCF-induced neutrophil production results from the prolongation of neutrophil survival. In the majority of SCN patients, the G-CSF receptor appears to be normal [17]. However, three cases have been found to have mutations that truncate the carboxy-terminal cytoplasmic portion of the G-CSF receptor [18, 19]. The mutant receptors can interfere in a dominant-negative manner with the function of wild-type G-CSF receptors and thereby block neutrophil maturation. These findings suggest a receptor problem or a possible intracellular signaling defect in response to G-CSF in this disorder. This study showed that numbers of neutrophils grown by BM CD38⫹c-kit⫹ cells of SCN patients in response to G-CSF ⫹ SCF or G-CSF ⫹ SCF ⫹ TPO as well as G-CSF alone were significantly lower than the values obtained from normal controls (Fig. 2). The dose-response study showed that the capability of 1 ng/mL of G-CSF to yield neutrophils was significantly lower than that of 10 ng/mL of G-CSF in SCN patients, but not in normal controls. Furthermore, even in the presence of SCF or SCF ⫹ TPO, the concentrations of G-CSF necessary for the substantial production of neutrophils by CD34⫹CD38⫹c-kit⫹ cells were higher in two patients. These results support a defect of the G-CSF-dependent signaling pathway as the major pathogenesis in the disorder. It is generally held that the CD34⫹CD38⫺ immunophenotype defines a primitive subpopulation of BM progenitors [20]. The present study showed that, in contrast with BM CD34⫹CD38⫹c-kit⫹ cells, SCN CD34⫹CD38⫺c-kit⫹ cells were unable to form neutrophil colonies in response to G-CSF plus SCF. The addition of SCF plus TPO to the culture containing G-CSF was required for significant colony growth by this subpopulation. There was no difference in the incidence of hematopoietic progenitors responsive to the three-factor combination in CD34⫹CD38⫺c-kit⫹ cells between SCN patients and normal controls. On the other hand, numbers of G-CSFresponsive neutrophil progenitors in BM CD34⫹CD38⫹c-kit⫹ cells were significantly lower in SCN patients than in normal controls. These results suggest that the combination of SCF and TPO is a requisite for G-CSF-dependent generation of neutrophils from SCN primitive hematopoietic progenitors. In addition, a possible pathogenesis of SCN may be an impaired commitment of primitive progenitors to the neutrophilic lineage. Although G-CSF markedly decreased the number and severity of infections in SCN patients, the development of myelodysplastic syndrome or acute myeloblastic leukemia (AML) has been reported in patients receiving G-CSF therapy [21– 23]. Acquired mutations in the G-CSF receptor gene were detected in some patients with SCN progressing to AML [19]. This study elucidated the stimulatory effects of TPO on the neutrophil production supported by G-CSF ⫹ SCF, providing fundamental insight regarding the clinical application of TPO in this disease. However, TPO has been shown to stimulate the proliferation of AML cells [24] and juvenile chronic myelogenous leukemic cells [9]. Thus, further studies are required to http://www.jleukbio.org

examine the safety of treatment with early-acting cytokines in patients with bone marrow stem cell disorders.

ACKNOWLEDGMENTS This work was supported by Grants-in-Aid Nos. 09041178 and 11670753 from the Ministry of Education of Japan.

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