We compared a potential to generate mast cells among various sources of CD34+ peripheral blood (PB) cells in the presence of stem cell factor (SCF) with or without thrombopoietin (TPO), using a serum-deprived liquid culture system. From the time course of relative numbers of tryptase-positive and chymase-positive cells in the cultured cells grown by CD34+ PB cells of nonasthmatic healthy individuals treated with G-CSF, TPO appears to potentiate the SCF-dependent growth of mast cells without influencing the differentiation into mast cell lineage. CD34+ PB cells from asthmatic patients in a stable condition generated significantly more mast cells under stimulation with SCF alone or SCF+TPO at 6 wk of culture than did steady-state CD34+ PB cells of normal controls. Single-cell culture studies showed a substantial difference in the number of SCF-responsive or SCF+TPO-responsive mast cell progenitors in CD34+ PB cells between the two groups. In the presence of TPO, CD34+ PB cells from asthmatic children could respond to a suboptimal concentration of SCF to a greater extent, compared with the values obtained by those of normal controls. Six-week cultured mast cells of asthmatic subjects had maturation properties (intracellular histamine content and tryptase/chymase enzymatic activities) similar to those derived from mobilized CD34+ PB cells of nonasthmatic subjects. An increase in a potential of circulating hemopoietic progenitors to differentiate into mast cell lineage may contribute to the recruitment of mast cells toward sites of asthmatic mucosal inflammation.

Asthma is an inflammatory disease, characterized by the infiltration of eosinophils, neutrophils, basophils, and lymphocytes in the airway wall and the surrounding parenchyma. Recently, it is proposed that hemopoietic myeloid progenitors contribute to the ongoing recruitment of inflammatory cells such as eosinophils to sites of allergen challenge in this disorder (1, 2, 3, 4, 5). It is well known that this inflammatory process is caused by mast cell activation through cross-linking of the high affinity IgE receptor (FcεRΙ). Mast cells originate from pluripotent hemopoietic cells within the marrow. Mast cell progenitors depart from the bone marrow (BM)3 and migrate into the connective or mucous tissues, where they differentiate into the mature form. In the human system, mast cell progenitors are positive for CD34, c-kit, CD13, and CD38, but lack HLA-DR (6, 7, 8, 9). Stem cell factor (SCF) has been reported to act as a major growth and differentiation factor for the human mast cell development from cord blood mononuclear cells (10), BM cells (11, 12), and fetal liver cells (13). In contrast, our recent study showed that the addition of thrombopoietin (TPO) to culture containing SCF is a requisite for the significant production of mast cells from CD34+ BM cells (14).

It is demonstrated that PBMCs or CD34+ cells generate mast cells in the presence of SCF with or without IL-3 (7, 12). In addition, Rottem et al. (7) reported that the number of mast cells arising per CD34+ cell is greater in patients with aggressive mastocytosis than normal subjects. However, little is known about the kinetics of mast cell progenitors in allergic disorders. In this study, we compared the production of mast cells from CD34+ peripheral blood (PB) cells between asthmatic patients and normal controls, using a serum-deprived culture system.

Six normal donors for allogeneic PB stem cell transplantation aged 7.5 ± 4.7 (range, 3–15) years were enrolled in this study. They had no known diseases including allergic disorders, and took no medications. All donors and/or their parents provided written informed consent. Donors received G-CSF (Chugai Pharmaceutical, Tokyo, Japan) s.c. at a dose of 10 μg/kg for 5 consecutive days, and blood sampling was performed on day 5. The protocol was approved by the ethics committee of Shinshu University School of Medicine.

PB samples (10 ml) were harvested by venous puncture from a total of 10 males and three females with bronchial asthma aged 6.5 ± 4.0 (2–15) years after obtaining the fully informed consent of each patient and/or the parents. The asthma was defined according to the criteria of the American Thoracic Society. Twelve aged-matched healthy subjects and three nonallergic patients with lower respiratory tract infection were used as the control group. Based on the guidelines for the diagnosis and management of asthma established by the National Heart, Lung, and Blood Institute (15), five cases were classified as mild and the rest as moderate. None of the patients had had an asthmatic attack within 1 month before the study. In moderate cases, oral theophylline with or without a β-adrenergic agonist was given. All medications were withdrawn 12 h before blood sampling. The mean PB eosinophil count was 0.83 ± 0.27 × 109/L (0.56–1.36 × 109/L), and the total serum IgE concentration was 1448 ± 1340 IU/ml (256–4711 IU/ml). All of the patients had positive immediate skin reactions to several Ags including house dust mite. Specific IgE against mite Ag was scored as 4 or more in all of the patients according to the radioimmunosorbent assay. PB samples were also collected from two infants with atopic dermatitis, and from a 6-year-old boy with allergic rhinitis after informed consent.

Human recombinant SCF, TPO, and IL-3 were provided by Kirin Brewery (Takasaki, Japan). Human recombinant IL-6 was a gift from Ajinomoto (Kawasaki, Japan).

For immunocytochemical staining, purified mAbs for tryptase (MAB1222) and chymase (3D5) were purchased from Chemicon International (Temecula, CA) and Biogenesis (Sandown, NH), respectively. Anti-CD2 mAb (T11) and anti-CD41 mAb (SZ.22) were obtained from Immunotech (Marseilles, France). Anti-CD11b mAb (2LPM19c), anti-CD15 mAb (C3D-1), anti-CD19 mAb (HD37), and anti-glycophorin A (anti-GPA) mAb (JC159) were obtained from Dako (Glostrup, Denmark).

For the flow cytometric analysis and cell sorting, mAbs for CD34 (8G12, FITC) and c-kit (104D2, PE) were purchased from Becton Dickinson Immunocytometry Systems (Mountain View, CA). The mAb for CD13 (Immu103.44, PE-cyanin 5.1, PC5) was obtained from Immunotech.

PB samples were aspirated in heparinized plastic syringes. PBMCs were separated by density centrifugation over Ficoll-Paque (Pharmacia, Piscataway, NJ), washed twice, and suspended in Ca2+- and Mg2+-free PBS containing 1 mmol/L EDTA-2Na and 2.5% FBS (HyClone, Logan, UT). After treatment with Silica (Immuno-Biological Laboratories, Fujioka, Japan) for 30 min at 37°C, CD34-positive cells were enriched using a Dynal CD34 Progenitor Cell Selection System (Dynal, Oslo, Norway). Briefly, 1–4 × 107 cells were mixed with the same number of polystyrene beads coated with mAb specific for CD34 (Dynabeads M-450 CD34) and incubated for 30 min at 4°C. Bead-rosetted cells were separated by a magnet. For the detachment of the beads from the cells, affinity-purified polyclonal Abs against the Fab portion of anti-CD34 Ab (Detach-a-Bead CD34) were added, and incubation was conducted for 45 min at room temperature. The detached beads were removed by the magnet, and the cells were collected as CD34+ cells. Approximately 90% of the isolated cells were CD34-positive, as determined by FACScan flow cytometry (Becton Dickinson).

Unless otherwise specified, serum-deprived liquid cultures were conducted in 24-well culture plates (no. 3047; Becton Dickinson) using a modification of the technique described previously (14, 16, 17, 18). CD34+ cells (2 × 104/well) were cultured in 2 ml of α-medium supplemented with 1% deionized BSA; 300 μg/ml fully iron-saturated human transferrin (∼98% pure; Sigma); 16 μg/ml soybean lecithin (Sigma); 9.6 μg/ml cholesterol (Nacalai Tesque, Kyoto, Japan); and 10 ng/ml of SCF, 10 ng/ml of TPO, 100 U/ml of IL-3, or 50 ng/ml of IL-6, alone or in combination. In dose-response studies, CD34+ cells were plated at 1 × 104 cells in a well containing 200 μl of the serum-free culture medium. 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 culture medium was replaced weekly with fresh medium containing the factor(s). The number of viable cells was determined by a trypan-blue exclusion test using a hemocytometer. We presented the actual counts of progeny in the results. Preliminary experiments showed that SCF at 10 ng/ml or higher was required for maximal cell growth by mobilized CD34+ cells in the presence of 10 ng/ml of TPO.

Single-cell sorting was performed by a two-step process, as described previously (14, 16, 17). PBMCs (1–4 × 107) were incubated with 20 μl of FITC-conjugated anti-CD34 mAb for 30 min at 4°C. As negative controls, the cells were stained with FITC-conjugated mouse IgG1 (Becton Dickinson). After two washes, CD34+ cells were individually sorted in 5-ml tubes by a FACStarPlus flow cytometer. The percentages of CD34+ cells in PBMCs were 0.13 ± 0.10% (0.03–0.21%) in asthmatic children and 0.26 ± 0.26% (0.09–0.64%) in normal controls. The CD34+ cells were then resorted into individual wells of a 96-well U-bottom tissue culture plate (no. 3077; Becton Dickinson) containing 100 μl of serum-deprived culture medium supplemented with 10 ng/ml of SCF with or without 10 ng/ml of TPO, using the FACStarPlus flow cytometer equipped with an automatic cell deposition unit (Becton Dickinson). 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. If the constituent cells numbered 20 or more at 4 wk under direct microscopic visualization, aggregates were scored as colonies. Then, colonies were picked up with a 3-μl Eppendorf micropipette and pooled. The constituent cells were stained with anti-tryptase mAb.

For the analysis of surface markers on CD34+ PB cells, 1–2 × 107 PBMCs were incubated with 20 μl FITC-conjugated anti-CD34 mAb, 20 μl PE-conjugated anti-c-kit mAb, and 10 μl PC5-conjugated anti-CD13 mAb for 30 min at 4°C, as described previously (14). The cells were washed twice, after which their surface markers were analyzed with the FACScan flow cytometer, using the Lysis 2 software program. The lymphoblastic region was gated on the basis of their forward light and side scatter characteristics. Then, the second gate was set on CD34+ cells. The expressions of c-kit and CD13 on CD34+ cells were examined. The proportion of positive cells was determined by comparison to cells stained with FITC-, PE-, or PC5-conjugated mouse isotype-matched Ig.

The cultured cells were spread on glass slides using a Cytospin II (Shandon Southern, Sewickly, PA) and stained with May-Grünwald-Giemsa or peroxidase. Reactions with mouse mAbs against tryptase, chymase, CD2, CD11b, CD15, CD19, CD41, and GPA were detected using the alkaline phosphatase-anti-alkaline phosphatase (APAAP) method (Dako APAAP Kit System; Dako, Carpinteria, CA), as described previously (19). The isotype mouse mAb was also used as a control. Briefly, cytocentrifuged samples were fixed with Carnoy’s fluid, washed with PBS, and preincubated with normal rabbit serum to saturate the Fc receptors on the cell surface. After being washed with PBS three times, the samples were reacted with each of the mouse mAbs for 30 min at room temperature in a humidified chamber. After three more washes with PBS, the samples were incubated with rabbit anti-mouse IgG Ab, washed three times, and successively reacted with the calf intestinal alkaline phosphatase-mouse monoclonal anti-alkaline phosphatase complex. Finally, alkaline phosphatase activity was detected with naphthol AS-MX phosphate, Fast Red TR, and levamisole to inhibit nonspecific alkaline phosphatase activity. The specimens were counterstained with hematoxylin. Three hundred cells were examined.

Histamine concentrations in the cell lysates obtained by the treatment of 6-wk cultured cells (5 × 104) grown with SCF or SCF+TPO with 0.5 ml of 1% Triton X-100 containing 0.5 M KCl were measured with the Histamine Enzyme Immunoassay Kit (Immunotech). The detection limit was 1 nM. All assays were conducted in triplicate.

Tryptase and chymase enzymatic activities were measured according to the procedure described by Xia et al. (20). The cultured cells (5 × 104) grown with SCF or SCF+TPO were lysed with 0.5 ml of 1% Triton X-100 containing 0.5 M KCl, and sonicated. Aliquots (20 μl) of the samples were incubated with 0.2 mM tosyl-Gly-Pro-Lys-p-nitroanilide (Sigma), 50 mM HEPES (pH 7.6) containing 0.12 M NaCl, 100 μg/ml of a soybean trypsin inhibitor (Sigma), and 1 μg/ml of heparin sulfate proteoglycan (Sigma) in a total volume of 180 μl at 37°C for 1 h. The release of p-nitroaniline was determined spectrophotometrically. As a control, we used p-nitroaniline purchased from Sigma. The addition of PMSF (Nacalai Tesque) at 1 × 10−3 M reduced the levels of tryptase enzymatic activity by 87.3%. For the assay of chymase enzymatic activity, aliquots of the samples (20 μl) were incubated with 0.2 mM succinyl-Ala-Ala-Pro-Phe-MCA (Peptide Institute, Osaka, Japan), 100 mM Tris (pH 7.8) containing 2 M NaCl, and 150 μg/ml of aprotinin (Sigma) in a total volume of 180 μl at 37°C for 1 h. Reactions were stopped by the addition of 200 μl of 10% SDS (Sigma) and 2 ml of 100 mM Tris (pH 9.0). The release of MCA was measured spectrofluorometrically. As a control, we used MCA obtained from Peptide Institute. The addition of chymostatin (Sigma) at 3.3 × 10−8 M to 3.3 × 10−4 M almost completely suppressed the release of MCA.

The results 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 when or if the data were not normally distributed. To compare the size of mast cell colonies, the unpaired t test was performed on logarithms of the cell numbers of individual colonies. One-way ANOVA, followed by post hoc contrasts with the Bonferroni limitation, was used for more than three independent groups.

We examined the effects of SCF (10 ng/ml), TPO (10 ng/ml), IL-3 (100 U/ml), and IL-6 (50 ng/ml), alone or in combination, on the generation of mast cells from CD34+ PB cells mobilized with G-CSF in serum-deprived liquid cultures. Half of the culture medium was replaced weekly with fresh medium containing the factor(s). As presented in Fig. 1, SCF alone induced the production of significant numbers of progeny from CD34+ PB cells, with a peak of ∼3 times the input quantity at 6 wk. The number of viable cells decreased at 8 wk. In the presence of TPO, IL-3, and IL-6 alone, the total cell number in a well estimated at 2 wk was 6400 ± 800, 3500 ± 900, and 1200 ± 200, respectively. Subsequently, the cell numbers were not measurable. The addition of IL-6 significantly reduced the number of progeny grown with SCF, consistent with the previous result (17). In the presence of SCF and IL-3, the total cell number was maintained from 2 to 6 wk. Two-thirds of 6-wk cultured cells reacted with anti-tryptase mAb, and some of the remaining cells were positive for peroxidase. In contrast, the addition of TPO caused a significant enhancement of the SCF-dependent cell generation from CD34+ PB cells. The number of viable cells reached maximal at 6 wk, and was 5- to 6-fold that at the beginning of the culture. There was a decline in the number of viable cells at 8 wk.

FIGURE 1.

Mast cell production by CD34+ PB cells mobilized with G-CSF under stimulation with SCF, TPO, IL-3, or IL-6, alone or in combination. CD34+ PB cells mobilized with G-CSF (2 × 104) were cultured in wells containing 2 ml of serum-deprived liquid culture medium supplemented with 10 ng/ml of SCF, 10 ng/ml of TPO, 100 U/ml of IL-3, or 50 ng/ml of IL-6, alone or in combination. The number of viable cells was serially counted, and the actual counts of progeny are presented. Values are expressed as the mean ± SD. ∗, Significantly higher than the values with SCF alone (p < 0.0001).

FIGURE 1.

Mast cell production by CD34+ PB cells mobilized with G-CSF under stimulation with SCF, TPO, IL-3, or IL-6, alone or in combination. CD34+ PB cells mobilized with G-CSF (2 × 104) were cultured in wells containing 2 ml of serum-deprived liquid culture medium supplemented with 10 ng/ml of SCF, 10 ng/ml of TPO, 100 U/ml of IL-3, or 50 ng/ml of IL-6, alone or in combination. The number of viable cells was serially counted, and the actual counts of progeny are presented. Values are expressed as the mean ± SD. ∗, Significantly higher than the values with SCF alone (p < 0.0001).

Close modal

Under stimulation with SCF alone, ∼70% of the cultured cells became positive for tryptase at 2 wk. A large portion of the cultured cells reacted with anti-tryptase mAb after 4 wk. Although the frequency of chymase+ cells was at a negligible or very low level at 2 wk, the percentage of the cells positive for chymase increased to ∼80–90% at 4 wk. It is of interest that the relative numbers of both tryptase+ cells and chymase+ cells in the cultured cells generated by stimulation with SCF+TPO increased in parallel with the values in the cells grown with SCF alone during 8 wk. At 6 wk of the culture with SCF alone or SCF+TPO, the cells with other lineage-specific markers (CD2, CD19, CD11b, CD15, CD41, or GPA) were at negligible levels.

The combination of SCF and TPO was the most favorable stimulus for mast cell growth from CD34+ PB cells of asthmatic patients as well as from those mobilized with G-CSF. The numbers of tryptase+ cells grown at 4 wk by 1 × 104 CD34+ PB cells were 20,700 ± 2,000 in SCF alone; 61,400 ± 9,000 in SCF+TPO; 17,700 ± 2,100 in SCF+IL-6; and 23,800 ± 3,100 in SCF+IL-3. Then, we compared the ability of CD34+ PB cells to generate mast cells between asthmatic children in a stable condition and controls. The results are presented in Fig. 2. CD34+ PB cells (2 × 104) from healthy children generated 12,500 ± 6,300 cells at 6 wk of the culture with SCF+TPO. The numbers of the cultured cells from nonallergic patients with lower respiratory tract infection were 8300 ± 8100, being similar to the values obtained by healthy subjects. In contrast, CD34+ PB cells of asthmatic patients had a significantly higher potential to generate the progeny than did those of nonallergic controls (p < 0.01). A substantial difference was also observed in the culture containing SCF alone (84,000 ± 6,500 cells from 2 × 104 CD34+ PB cells of three asthmatic children, and 3500 ± 1600 cells in three healthy subjects). Furthermore, in the presence of TPO, CD34+ PB cells from asthmatic children responded to a suboptimal concentration of SCF (1 ng/ml) to a greater extent than those from normal controls (Fig. 3). In both asthmatic individuals and nonallergic control subjects, >99% of 6-wk cultured cells were positive for tryptase, and >95% of them positive for chymase. However, an increase in the generation of mast cells from CD34+ PB cells was found in a part of patients with other allergic disorders (Fig. 2).

FIGURE 2.

Comparison of mast cell production by CD34+ PB cells under stimulation with stem cell factor + thrombopoietin between patients with allergic diseases and nonallergic controls. CD34+ PB cells (2 × 104) from patients with allergic diseases and nonallergic controls were cultured in a well containing 2 ml of serum-deprived liquid culture medium supplemented with 10 ng/ml of SCF plus 10 ng/ml of TPO. After 6 wk, the viable cells were enumerated. Patients 1–6, asthmatic children; patients 7 and 8, infants with atopic dermatitis; patient 9, child with allergic rhinitis. Controls 1–5, healthy children; controls 6–8, patients with lower respiratory tract infection.

FIGURE 2.

Comparison of mast cell production by CD34+ PB cells under stimulation with stem cell factor + thrombopoietin between patients with allergic diseases and nonallergic controls. CD34+ PB cells (2 × 104) from patients with allergic diseases and nonallergic controls were cultured in a well containing 2 ml of serum-deprived liquid culture medium supplemented with 10 ng/ml of SCF plus 10 ng/ml of TPO. After 6 wk, the viable cells were enumerated. Patients 1–6, asthmatic children; patients 7 and 8, infants with atopic dermatitis; patient 9, child with allergic rhinitis. Controls 1–5, healthy children; controls 6–8, patients with lower respiratory tract infection.

Close modal
FIGURE 3.

Dose response of mast cell generation by CD34+ PB cells to SCF. CD34+ PB cells (1 × 104) were plated with SCF in concentrations ranging from 1 to 100 ng/ml and TPO at 10 ng/ml. At 6 wk, the numbers of progeny were counted. The results shown (mean ± SD) were derived from three samples in each group. ▪, Asthmatic children; ○, normal controls. ∗, Significantly different from the values obtained by normal controls (p < 0.02).

FIGURE 3.

Dose response of mast cell generation by CD34+ PB cells to SCF. CD34+ PB cells (1 × 104) were plated with SCF in concentrations ranging from 1 to 100 ng/ml and TPO at 10 ng/ml. At 6 wk, the numbers of progeny were counted. The results shown (mean ± SD) were derived from three samples in each group. ▪, Asthmatic children; ○, normal controls. ∗, Significantly different from the values obtained by normal controls (p < 0.02).

Close modal

To elucidate why CD34+ PB cells from patients with asthma had the superior capability to yield mast cells, we compared the number of progenitors that gave rise to mast cell colonies by stimulation with SCF or SCF+TPO in CD34+ PB cells between the two groups, using single-cell cultures. The results are presented in Table I. In the presence of SCF+TPO, significantly greater numbers of colonies were formed in the cultures containing CD34+ PB cells from patients with asthma, as compared with the values obtained by normal controls. A prominent difference was also noted in the culture with SCF alone. A majority of the constituent cells of pooled colonies were positive for tryptase under stimulation with SCF alone or SCF+TPO in children with or without asthma. There was no significant difference in the size of mast cell colonies formed in the presence of SCF+TPO between asthmatic children and normal controls. The mean number of constituent cells in mast cell colonies was 70 ± 84 (20–312) in patient 1, 89 ± 129 (20–500) in patient 2, 28 ± 7 (23–48) in patient 3, 110 ± 258 (20–1300) in patient 4, and 66 ± 89 (20–400) in the controls. As it has been demonstrated that most of the human mast cells originate from CD34+c-kit+ cells or from CD34+c-kit+CD13+ cells (6, 9), we investigated whether relative numbers of these particular CD34+ cell subsets were also increased in CD34+ PB cells of patients with asthma. The results are presented in Table II. There was no significant difference in the proportion of c-kit+ cells or c-kit+CD13+ cells in CD34+ cells between asthmatic children and normal controls.

Table I.

An increase in mast cell colony-forming cells in CD34+ PB cells from asthmatic patientsa

Number of Mast Cell Colonies
SCFSCF + TPO
Asthmatic patients   
Patient 1 14 
Patient 2 13 
Patient 3 11 
Patient 4 22 
Mean± SD 6.8 ± 1.7b 15.0 ± 4.8b 
Normal controls   
Control 1 
Control 2 
Control 3 
Control 4 
Mean± SD 1.5 ± 1.7 3.0 ± 1.4 
Number of Mast Cell Colonies
SCFSCF + TPO
Asthmatic patients   
Patient 1 14 
Patient 2 13 
Patient 3 11 
Patient 4 22 
Mean± SD 6.8 ± 1.7b 15.0 ± 4.8b 
Normal controls   
Control 1 
Control 2 
Control 3 
Control 4 
Mean± SD 1.5 ± 1.7 3.0 ± 1.4 
a

CD34+ cells from PBMCs of asthmatic patients or normal controls were sorted as a single cell into the individual wells of a 96-well culture plate containing 10 ng/ml of SCF with or without 10 ng/ml of TPO. Aggregates were scored as colonies, if the constituent cells numbered 20 or more at 4 wk.

b

, Significantly different from normal controls (p < 0.005).

Table II.

Surface marker expression on CD34+ PB cells of asthmatic patientsa

CD34+ PB Cells
% of c-kit+ cells% of c-kit+CD13+ cells
Asthmatic Patients   
Patient 1 61 14 
Patient 2 65 25 
Patient 3 66 16 
Mean± SD 64.0 ± 2.6 18.3 ± 5.9 
Normal Controls   
Control 1 63 30 
Control 2 71 11 
Control 3 58 22 
Mean± SD 64.0 ± 6.6 21.0 ± 9.5 
CD34+ PB Cells
% of c-kit+ cells% of c-kit+CD13+ cells
Asthmatic Patients   
Patient 1 61 14 
Patient 2 65 25 
Patient 3 66 16 
Mean± SD 64.0 ± 2.6 18.3 ± 5.9 
Normal Controls   
Control 1 63 30 
Control 2 71 11 
Control 3 58 22 
Mean± SD 64.0 ± 6.6 21.0 ± 9.5 
a

Expression of c-kit and CD13 on CD34+ PB cells was analyzed by flow cytometry using FITC-conjugated anti-CD34 mAb, PE-conjugated anti-c-kit mAb, and PC5-conjugated anti-CD13 mAb.

Finally, we compared intracellular levels of histamine and proteases of 6-wk-old cultured cells between asthmatic subjects and normal individuals. To estimate the cellular amounts of tryptase and chymase protein, we measured protease enzymatic activity in a total of 5 × 104 cells, as described by Xia et al. (20). In addition, we used the cultured mast cells derived from G-CSF-mobilized CD34+ PB cells of nonasthmatic individuals as controls, because of the paucity of mast cells grown from normal steady-state CD34+ PB cells. There were no significant differences in intracellular histamine content and tryptase/chymase enzymatic activities between the two groups. The histamine concentration of 5 × 104 6-wk cultured cells grown with SCF (10 ng/ml) + TPO (10 ng/ml) from CD34+ PB cells was 1508 ± 532 nM (826–2197 nM) in asthmatic patients, and 2097 ± 1079 nM (739–3667 nM) in controls. Tryptase enzymatic activity of them was 5284 ± 2124 μM (3434–9336 μM) in asthmatic patients, and 3519 ± 1439 μM (1634–5631 μM) in controls. Chymase enzymatic activity of them was 16.0 ± 8.8 μM (7.5–28.8 μM) in asthmatic patients, and 28.5 ± 23.8 μM (3.7–62.1 μM) in controls.

In the culture containing CD34+ cord blood cells and SCF at 10 ng/ml, a progressive, steady increase in mast cell production was achieved during 50 wk (17). When G-CSF-mobilized CD34+ PB cells were target cells, SCF alone induced the generation of significant numbers of mast cells for up to 6 wk. However, the number of cultured cells decreased at 8 wk of culture. A large portion of 4-wk progeny grown from CD34+ cord blood cells reacted with anti-tryptase mAb, but were negative for chymase (17). At 36 wk, a vast majority of the cord blood-derived cultured cells became positive for chymase. In contrast, immunoreactivity for chymase appeared markedly earlier in mast cells derived from mobilized CD34+ PB cells. A great part of the cultured cells generated from steady-state CD34+ PB cells of normal controls or from CD34+ PB cells of asthmatic patients were also positive for two types of protease at 6 wk. These lines of evidence suggest the age-related advance in mast cell maturation.

Rottem et al. (7) demonstrated that IL-3 substantially increases the numbers of mast cells grown with SCF from CD34+ PB cells. In contrast, Valent et al. (12) found IL-3-mediated down-regulation of SCF-dependent mast cell formation in long-term cultures. In this study, the addition of IL-3 to the culture containing SCF failed to augment the generation of mast cells both from G-CSF-mobilized CD34+ PB cells and from CD34+ PB cells obtained without G-CSF. In contrast, a combination of SCF and TPO exerted a prominent synergism on the production of mast cells from CD34+ PB cells obtained with or without G-CSF treatment. The time course study of the culture containing mobilized CD34+ PB cells showed that relative numbers of both tryptase+ cells and chymase+ cells in the cultured cells grown under stimulation with SCF+TPO increased in parallel with the values in the cells grown with SCF alone. These results suggest that TPO can expand the SCF-dependent growth of mast cells from mobilized CD34+ PB cells without influencing the differentiation into the mast cell lineage.

It is of interest that CD34+ PB cells from stable asthmatic children generated substantially greater numbers of mast cells in response to SCF alone or SCF+TPO than did steady-state CD34+ PB cells from controls. However, such an increase in the generation of mast cells from CD34+ PB cells is unlikely observed in all types of allergic disorders. Based on the results of the flow cytometric and immunocytochemical analyses, the discrepancy does not appear to result from the difference in the percentages of the particular subsets in CD34+ PB cells and from the distinct maturation stage of the progeny between asthmatic patients and normal subjects. The single-cell culture experiments clearly demonstrated that significantly greater numbers of mast cell colonies were formed by SCF alone in the cultures containing CD34+ PB cells from patients with asthma, as compared with the values obtained by normal controls. A prominent discrepancy was also noted in the SCF+TPO-responsive mast cell progenitors. Additionally, in the presence of TPO, CD34+ PB cells of asthmatic children generated apparently higher numbers of progeny under stimulation with a suboptimal concentration of SCF than did those of normal subjects. Thus, in patients with allergic asthma, greater numbers of CD34+ PB cells appear committed to the mast cell lineage. Moreover, it is suggested that mast cell progenitors have a hypersensitivity to SCF in this disorder.

Denburg and coworkers (1, 2, 3, 4, 5) have proposed that activation of specific hemopoietic pathways in the BM contribute to the allergic diathesis through increased production and traffic of lineage-committed inflammatory progenitors such as those of eosinophils. Moreover, significant changes are observed in the expression of hemopoietic cytokine receptors on CD34+ cells. In particular, increased expression of IL-5 receptor α on CD34+ cells favors eosinophilopoiesis, and may thus contribute to the subsequent development of blood and tissue eosinophilia. In vivo allergen-stimulated products of cytokines may account for the increases in CFU for eosinophils and/or basophils. Actually, detectable serum IL-5 concentrations are found in a proportion of patients with acute severe asthma, but not in the same patients following oral glucocorticoid therapy or in normal controls (21). Hence, there were significant falls in circulating eosinophil/basophil progenitor counts with resolution of the asthma exacerbation on beclomethasone therapy (22). In contrast, an increase in the number of mast cell progenitors in CD34+ PB cells was observed in stable asthmatic patients. Additionally, the concentrations of SCF and TPO in venous plasma were not elevated in asthmatic children compared with the values in normal subjects (the values of SCF and TPO were 1271 ± 233 pg/ml and <0.20–0.39 fmol/ml, respectively, in asthmatic children; 1047 ± 277 pg/ml and 0.26–0.74 fmol/ml, respectively, in normal controls). Therefore, it is likely that a raised level of mast cell progenitors in CD34+ PB cells from asthmatic children is mediated through a mechanism different from hemopoietic progenitors differentiating into eosinophils or basophils.

It is demonstrated that mast cell numbers are increased in bronchoalveolar lavage fluid in relatively stable asthmatic patients (23). The active recruitment of mast cell progenitors from the circulation into the tissue may contribute to ongoing airway inflammation during asymptomatic periods.

We are grateful to T. Shinbo (Department of Pediatrics, Mizonokuchi Hospital, Teikyo University School of Medicine) for supplying plasma of asthmatic patients and normal controls.

1

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

3

Abbreviations used in this paper: BM, bone marrow; PB, peripheral blood; SCF, stem cell factor; TPO, thrombopoietin; GPA, glycophorin A.

1
Sehmi, R., L. J. Wood, R. Watson, R. Foley, Q. Hamid, P. M. O’Byrne, J. A. Denburg.
1997
. Allergen-induced increases in IL-5 receptor α-subunit expression on bone marrow-derived CD34+ cells from asthmatic subjects: a novel marker of progenitor cell commitment towards eosinophilic differentiation.
J. Clin. Invest.
100
:
2466
2
Wood, L. J., M. D. Inman, R. M. Watson, R. Foley, J. A. Denburg, P.M. O’Byrne.
1998
. Changes in bone marrow inflammatory cell progenitors after inhaled allergen in asthmatic subjects.
Am. J. Respir. Crit. Care Med.
157
:
99
3
Wood, L. J., M. D. Inman, J. A. Denburg, P. M. O’Byrne.
1998
. Allergen challenge increases cell traffic between bone marrow and lung.
Am. J. Respir. Cell. Mol. Biol.
18
:
759
4
Wood, L. J., R. Sehmi, G. M. Gauvreau, R. M. Watson, R. Foley, J. A. Denburg, P. M. O’Byrne.
1999
. An inhaled corticosteroid, budesonide, reduces baseline but not allergen-induced increases in bone marrow inflammatory cell progenitors in asthmatic subjects.
Am. J. Respir. Crit. Care. Med.
159
:
1457
5
Upham, J. W., L. M. Hayes, J. Lundahl, R. Sehmi, J. A. Denburg.
1999
. Reduced expression of hemopoietic cytokine receptors on cord blood progenitor cells in neonates at risk for atopy.
J. Allergy Clin. Immunol.
104
:
370
6
Agis, H., M. Willheim, W. R. Sperr, A. Wilfing, E. Kromer, E. Kabrna, E. Spanblochl, H. Strobl, K. Geissler, A. Spittler, et al
1993
. Monocytes do not make mast cells when cultured in the presence of SCF: characterization of the circulating mast cell progenitor as a c-kit+, CD34+, Ly, CD14, CD17, colony-forming cell.
J. Immunol.
151
:
4221
7
Rottem, M., T. Okada, J. P. Goff, D. D. Metcalfe.
1994
. Mast cells cultured from the peripheral blood of normal donors and patients with mastocytosis originate from a CD34+/FcεRI cell population.
Blood
84
:
2489
8
Kempuraj, D. H., A. Saito, K. Kaneko, M. Fukagawa, H. Nakayama, M. Toru, H. Tomikawa, M. Tachimoto, A. Akasawa Ebisawa, et al
1999
. Characterization of mast cell-committed progenitors present in human umbilical cord blood.
Blood
93
:
3338
9
Kirshenbaum, A. S., J. P. Goff, T. Semere, B. Foster, L. M. Scott, D. D. Metcalfe.
1999
. Demonstration that human mast cells arise from a progenitor cell population that is CD34+, c-kit+, and expresses aminopeptidase N (CD13).
Blood
94
:
2333
10
Mitsui, H., T. Furitsu, A. M. Dvorak, A. M. Irani, L. B. Schwartz, N. Inagaki, M. Takei, K. Ishizaka, K. M. Zsebo, S. Gillis, T. Ishizaka.
1993
. Development of human mast cells from umbilical cord blood cells by recombinant human and murine c-kit ligand.
Proc. Natl. Acad. Sci. USA
90
:
735
11
Kirshenbaum, A. S., S. W. Kessler, J. P. Goff, D. D. Metcalfe.
1991
. Demonstration of the origin of human mast cells from CD34+ bone marrow progenitor cells.
J. Immunol.
146
:
1410
12
Valent, P., E. Spanblochl, W. R. Sperr, C. Sillaber, K. M. Zsebo, H. Agis, H. Strobl, K. Geissler, P. Bettelheim, K. Lechner.
1992
. Induction of differentiation of human mast cells from bone marrow and peripheral blood mononuclear cells by recombinant human stem cell factor/kit-ligand in long-term culture.
Blood
80
:
2237
13
Irani, A. M., G. Nilsson, U. Miettinen, S. S. Craig, L. K. Ashman, T. Ishizaka, K. M. Zsebo, L. B. Schwartz.
1992
. Recombinant human stem cell factor stimulates differentiation of mast cells from dispersed human fetal liver cells.
Blood
80
:
3009
14
Sawai, N., K. Koike, H. H. Mwamtemi, T. Kinoshita, Y. Kurokawa, K. Sakashita, T. Higuchi, K. Takeuchi, M. Shiohara, T. Kamijo, et al
1999
. Thrombopoietin augments stem cell factor-dependent growth of human mast cells from bone marrow multipotential hematopoietic progenitors.
Blood
93
:
3703
15
O’Connell, E. J., D. C. Heilman.
1996
. Asthma. F. D. Burg, and J. R. Ingelfinger, and E. R. Wald, and R. A. Polin, eds.
Gellis & Kagan’s Current Pediatric Therapy 15
708
Saunders, Philadelphia.
16
Sawai, N., K. Koike, S. Ito, H. H. Mwamtemi, Y. Kurokawa, T. Kinoshita, K. Sakashita, T. Higuchi, K. Takeuchi, M. Shiohara, et al
1999
. Neutrophilic cell production by combination of stem cell factor and thrombopoietin from CD34+ cord blood cells in long-term serum-deprived liquid culture.
Blood
93
:
509
17
Kinoshita, T. N., E. Sawai, T. Yamashita Hidaka, K. Koike.
1999
. Interleukin-6 directly modulates stem cell factor-dependent development of human mast cells derived from CD34+ cord blood cells.
Blood
94
:
496
18
Kinoshita, T., K. Koike, H. H. Mwamtemi, S. Ito, S. Ishida, Y. Nakazawa, Y. Kurokawa, K. Sakashita, T. Higuchi, K. Takeuchi, et al
2000
. Retinoic acid is a negative regulator for the differentiation of cord blood-derived human mast cell progenitors.
Blood
95
:
2821
19
Ma, F., K. Koike, T. Higuchi, T. Kinoshita, K. Takeuchi, H. H. Mwamtemi, N. Sawai, T. Kamijo, M. Shiohara, S. Horie, et al
1998
. Establishment of a GM-CSF-dependent megakaryoblastic cell line with the potential to differentiate into an eosinophilic lineage in response to retinoic acids.
Br. J. Haematol.
100
:
427
20
Xia, H. Z., Z. Du, S. Craig, G. Klisch, N. Noben-Trauth, J. P. Kochan, T. H. Huff, A. M. Irani, L. B. Schwartz.
1997
. Effect of recombinant human IL-4 on tryptase, chymase, and Fcε receptor type I expression in recombinant human stem cell factor-dependent fetal liver-derived human mast cells.
J. Immunol.
159
:
2911
21
Alexander, A. G., J. Barkans, R. Moqbel, N. C. Barnes, A. B. Kay, C. J. Corrigan.
1994
. Serum interleukin 5 concentrations in atopic and non-atopic patients with glucocorticoid-dependent chronic severe asthma.
Thorax
49
:
1231
22
Gibson, P. G., J. Dolovich, A. Girgis-Gabardo, M. M. Morris, M. Anderson, F. E. Hargreave, J. A. Denburg.
1990
. The inflammatory response in asthma exacerbation: changes in circulating eosinophils, basophils and their progenitors.
Clin. Exp. Allergy
20
:
661
23
Stevenson, E. C., G. Turner, L. G. Heaney, B. C. Schock, R. Taylor, T. Gallagher, M. Ennis, M. D. Shields.
1997
. Bronchoalveolar lavage findings suggest two different forms of childhood asthma.
Clin. Exp. Allergy
27
:
1027