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Keywords:

  • fetal bovine serum;
  • human serum;
  • RARα antagonist;
  • retinol;
  • stem cell factor;
  • TGF-β

Abstract

  1. Top of page
  2. Abstract
  3. Methods
  4. Serum samples
  5. Cytokines, reagents and antibodies
  6. Cell preparation
  7. Suspension cultures
  8. Flow cytometric analysis
  9. Clonal cell cultures
  10. Cytochemical and immunologic staining
  11. Assay of histamine, tryptase, cytokine and retinol levels
  12. Statistical analysis
  13. Results
  14. Effects of human serum on SCF-dependent human mast cell development
  15. Contribution of retinol to human serum-mediated inhibition of mast cell growth
  16. Differential regulation by human serum during hematopoiesis
  17. RARα antagonist decreases the FBS-induced suppression of human mast cell growth supported by SCF
  18. Discussion
  19. Acknowledgments
  20. References

Background:In vitro culture systems have been used to study the physiological and pathological characteristics of human mast cells. However, there are some differences in proliferation and maturation of mast cells between fetal bovine serum (FBS)-containing and serum-deprived cultures. Accordingly, we attempted to identify circulating factor(s) affecting the development of human mast cells.

Methods: We measured the serum levels of retinol and several cytokines. To elucidate the antiproliferative effects of the serum, a retinoic acid receptor (RARα) antagonist and neutralizing antibodies against cytokines were used.

Results: Similar to FBS, human serum dose-dependently suppressed the growth of tryptase+ cells from CD34+ cord blood cells or 20-week cultured mast cells under stimulation with stem cell factor (SCF). The serum-mediated inhibition might be based on a decline in proliferation rate. Among inhibitors for mast cell growth, retinol and transforming growth factor (TGF)-β1 were present at high levels in human serum. In contrast with anti-TGF-β1 antibody, an RARα antagonist counteracted the serum-induced suppression of human mast cell proliferation.

Conclusions: Our results suggest that retinol and its derivatives act as a circulating regulator for human mast cell growth. The RARα antagonist may be a useful tool to obtain higher numbers of mast cells in FBS-containing cultures.

Mast cells are distributed throughout the body, and play a pivotal role in immediate-type hypersensitivity reactions. Stem cell factor (SCF) is a major growth and differentiation factor for cells of the human mast cell lineage, and produced by stromal cells. There are two types of SCF, i.e., a membrane-bound form and a soluble form. Soluble SCF circulates in the blood at detectable levels (1, 2). Because only limited numbers of mast cells can be obtained from human tissues, attempts have been made to establish human mast cell cultures using SCF. However, the purity of cultured mast cells grown with SCF alone has ranged from approximately 40 to 85% (3–5). Nakahata et al. (6) reported the growth of large, almost 100%-pure, mast cell populations from human cord blood cells in a serum-containing liquid culture medium supplemented with SCF plus interleukin (IL)-6. Therefore, this culture method has been widely adopted to elucidate the physiological and pathological characteristics of human mast cells.

We have reported a serum-deprived culture system that supports the selective growth of human mast cells from CD34+ cord blood cells under stimulation with SCF alone (7, 8). When we compared the plating efficiency obtained with SCF between the two different culture conditions, an inferior proliferation of mast cells was observed in fetal bovine serum (FBS)-containing culture (7). Dahl et al. (9) also reported different development of human mast cells between these two culture systems. Based on these results, we attempted to identify potent inhibitors for human mast cell growth in human adult serum (human serum) and FBS.

Serum samples

  1. Top of page
  2. Abstract
  3. Methods
  4. Serum samples
  5. Cytokines, reagents and antibodies
  6. Cell preparation
  7. Suspension cultures
  8. Flow cytometric analysis
  9. Clonal cell cultures
  10. Cytochemical and immunologic staining
  11. Assay of histamine, tryptase, cytokine and retinol levels
  12. Statistical analysis
  13. Results
  14. Effects of human serum on SCF-dependent human mast cell development
  15. Contribution of retinol to human serum-mediated inhibition of mast cell growth
  16. Differential regulation by human serum during hematopoiesis
  17. RARα antagonist decreases the FBS-induced suppression of human mast cell growth supported by SCF
  18. Discussion
  19. Acknowledgments
  20. References

Peripheral blood or cord blood samples were harvested after obtaining fully informed consent from adult volunteers or the mothers of all neonates. Fetal bovine serum was obtained from Hyclone (2 batches, Logan, UT), Gibco BRL (2 batches, Grand Island, NY), ICN Biomedicals Inc. (1 batch, Aurora, OH), Sigma (1 batch, St Louis, MO), and Stem Cell Technologies (1 batch, Vancouver, Canada). All sera were heat inactivated by placement in a water bath for 30 min at 56°C. Aliquots were frozen at −80°C until use.

Cytokines, reagents and antibodies

  1. Top of page
  2. Abstract
  3. Methods
  4. Serum samples
  5. Cytokines, reagents and antibodies
  6. Cell preparation
  7. Suspension cultures
  8. Flow cytometric analysis
  9. Clonal cell cultures
  10. Cytochemical and immunologic staining
  11. Assay of histamine, tryptase, cytokine and retinol levels
  12. Statistical analysis
  13. Results
  14. Effects of human serum on SCF-dependent human mast cell development
  15. Contribution of retinol to human serum-mediated inhibition of mast cell growth
  16. Differential regulation by human serum during hematopoiesis
  17. RARα antagonist decreases the FBS-induced suppression of human mast cell growth supported by SCF
  18. Discussion
  19. Acknowledgments
  20. References

Human recombinant SCF, granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoietin (EPO) and thrombopoietin (TPO) were generously provided by Kirin Brewery Co. Ltd. (Takasaki, Japan). Human recombinant transforming growth factor (TGF)-β1 was purchased from R & D Systems (Minneapolis, MN).

Retinol (vitamin A) and all-trans retinoic acid (ATRA) were obtained from Sigma. Ro 41-5253, an retinoic acid receptor (RARα) antagonist (10, 11), was kindly provided by F. Hoffman-La Roche, Ltd (Basel, Switzerland).

The polyclonal rabbit anti-human IL-6 antibody (Ab, 1.2 mg/ml) was a gift from Ajinomoto Co. (7). One microgram of this Ab neutralized the activity of 3 ng of IL-6, as determined with the cell line, SKW6-CL4. A polyclonal sheep anti-human IL-4 Ab was purchased from Genzyme Co. (Cambridge, MA). The Ab at 0.1–1 μg/ml neutralized the bioactivity of a 0.25 ng/ml solution of IL-4. The mouse monoclonal antibody (mAb) against human GM-CSF was purchased from Oncogene Science Inc. (Uniondale, NY). This azide-free Ab at 2 μg/ml reduced the growth of granulocyte-macrophage colonies supported by 10 ng/ml of GM-CSF to 37% (12). The neutralizing anti-human TGF-β1 Ab was obtained from R & D Systems. The ND50 of the Ab was determined to be 0.2–0.6 μg/ml in the presence of 0.25 ng/ml of TGF-β1, using TGF-β-responsive HT-2 cells.

For immunocytochemical staining, a purified mAb for tryptase (MAB1222) was purchased from Chemicon International Inc (Temecula, CA).

For flow cytometric analysis, a mAb for CD34 (HPCA-2, fluorescein isothiocyanate, FITC) was purchased from Becton Dickinson Immunocytometry Systems (Mountain View, CA). The mAbs for glycophorin A (GPA; JC159, FITC) and CD41b (TP80, FITC) were from Dako (Glostrup, Denmark) and Nichirei (Tokyo, Japan), respectively.

Cell preparation

  1. Top of page
  2. Abstract
  3. Methods
  4. Serum samples
  5. Cytokines, reagents and antibodies
  6. Cell preparation
  7. Suspension cultures
  8. Flow cytometric analysis
  9. Clonal cell cultures
  10. Cytochemical and immunologic staining
  11. Assay of histamine, tryptase, cytokine and retinol levels
  12. Statistical analysis
  13. Results
  14. Effects of human serum on SCF-dependent human mast cell development
  15. Contribution of retinol to human serum-mediated inhibition of mast cell growth
  16. Differential regulation by human serum during hematopoiesis
  17. RARα antagonist decreases the FBS-induced suppression of human mast cell growth supported by SCF
  18. Discussion
  19. Acknowledgments
  20. References

Cord blood samples were aspirated in heparinized plastic syringes from the umbilical vein at normal delivery after obtaining the informed consent from the mothers of all neonates. Mononuclear cells (MNCs) were separated by density centrifugation over Ficoll-Paque (Pharmacia Fine Chemicals, Piscataway, NJ), washed twice, and suspended in Ca2+- and Mg2+-free phosphate-buffered saline containing 1 mM ethylenediaminetetraacetic acid-2Na and 2.5% FBS. CD34-positive cells were enriched using the Direct CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions. After treatment with 100 μl of Fc-receptor blocking reagent, 0.5–1 × 108 MNCs were mixed with 100 μl of colloidal super-paramagnetic MicroBeads conjugated to a mouse mAb specific for CD34 (QBEND/10), and incubated for 30 min at 4°C. The magnetically labeled cells were separated with a MS+/RS+ column in the magnetic field of the MACS separator (VarioMACS). More than 90% of the isolated cells were CD34-positive, as determined by FACScan flow cytometry (Becton Dickinson).

Suspension cultures

  1. Top of page
  2. Abstract
  3. Methods
  4. Serum samples
  5. Cytokines, reagents and antibodies
  6. Cell preparation
  7. Suspension cultures
  8. Flow cytometric analysis
  9. Clonal cell cultures
  10. Cytochemical and immunologic staining
  11. Assay of histamine, tryptase, cytokine and retinol levels
  12. Statistical analysis
  13. Results
  14. Effects of human serum on SCF-dependent human mast cell development
  15. Contribution of retinol to human serum-mediated inhibition of mast cell growth
  16. Differential regulation by human serum during hematopoiesis
  17. RARα antagonist decreases the FBS-induced suppression of human mast cell growth supported by SCF
  18. Discussion
  19. Acknowledgments
  20. References

Suspension cultures were carried out in 24-well culture plates (no. 3047; Becton Dickinson) using a modification of the technique described previously (7, 8, 13). CD34+ cells or 20-week cultured mast cells grown with 10 ng/ml of SCF from CD34+ cord blood cells were cultured in each well with 2 ml of α-medium supplemented with 1% bovine serum albumin, 300 μg/ml of fully iron-saturated human transferrin (approximately 98% pure, Sigma), 16 μg/ml of soybean lecithin (Sigma), 9.6 μg/ml of cholesterol (Nakalai Chemicals Ltd., Tokyo, Japan) and 100 ng/ml of SCF, human serum, FBS, retinol or TGF-β1, alone or in combination. The GM-CSF, EPO, and TPO were used at 10 ng/ml, 2 U/ml, and 10 ng/ml, respectively. 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 cells and 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.

Assay of histamine, tryptase, cytokine and retinol levels

  1. Top of page
  2. Abstract
  3. Methods
  4. Serum samples
  5. Cytokines, reagents and antibodies
  6. Cell preparation
  7. Suspension cultures
  8. Flow cytometric analysis
  9. Clonal cell cultures
  10. Cytochemical and immunologic staining
  11. Assay of histamine, tryptase, cytokine and retinol levels
  12. Statistical analysis
  13. Results
  14. Effects of human serum on SCF-dependent human mast cell development
  15. Contribution of retinol to human serum-mediated inhibition of mast cell growth
  16. Differential regulation by human serum during hematopoiesis
  17. RARα antagonist decreases the FBS-induced suppression of human mast cell growth supported by SCF
  18. Discussion
  19. Acknowledgments
  20. References

Histamine concentrations in cell lysates obtained by the treatment of the cultured cells with 0.5% Nonidet P-40 were measured with a Histamine Radioimmunoassay Kit (Immunotech, S.A.), as described previously (7). The tryptase concentrations in the cell lysates were measured with a fluoroenzymeimmunoassay (UniCAP Tryptase, Pharmacia & Upjohn Diagnostics AB, Uppsala, Sweden). The concentrations of GM-CSF, IL-4, and IL-6 in human serum were measured by an enzyme-linked immunosorbent assay (Amersham International, Buckinghamshire, UK). We also determined circulating TGF-β1 levels using the quantitative sandwich enzyme immunoassay technique (Quantikine, R & D Systems) after the treatment of the samples with acetic acid. The serum concentration of retinol was measured with high-performance liquid chromatography (HPLC). All assays were conducted in triplicate.

Effects of human serum on SCF-dependent human mast cell development

  1. Top of page
  2. Abstract
  3. Methods
  4. Serum samples
  5. Cytokines, reagents and antibodies
  6. Cell preparation
  7. Suspension cultures
  8. Flow cytometric analysis
  9. Clonal cell cultures
  10. Cytochemical and immunologic staining
  11. Assay of histamine, tryptase, cytokine and retinol levels
  12. Statistical analysis
  13. Results
  14. Effects of human serum on SCF-dependent human mast cell development
  15. Contribution of retinol to human serum-mediated inhibition of mast cell growth
  16. Differential regulation by human serum during hematopoiesis
  17. RARα antagonist decreases the FBS-induced suppression of human mast cell growth supported by SCF
  18. Discussion
  19. Acknowledgments
  20. References

Because Abs against multiple bovine cytokines were unavailable, we used human serum to analyze circulating inhibitors for the growth of human mast cells. One × 104 20-week cultured cells (>99% of the cells were positive for tryptase) grown with 10 ng/ml of SCF from CD34+ cord blood cells were incubated in wells containing SCF at 100 ng/ml with or without human serum at concentrations ranging from 0.3 to 20%. As shown in Fig. 1A, in the presence of SCF alone, the total viable cell number increased to approximately nine times the input quantity after 2 weeks. The addition of human serum to the culture with SCF resulted in a dose-dependent decrease in the numbers of progeny. When four serum samples from healthy adults were compared at the concentration of 10%, almost equal levels of inhibition were obtained (Fig. 1B). Furthermore, there was no significant difference in the serum-induced suppression of mast cell growth between asthmatic patients and normal controls. In clonal cell cultures, 22.3 ± 3.4 mast cell colonies and 50.8 ± 8.7 clusters were formed in the presence of SCF at 100 ng/ml from 4 × 103 20-week cultured mast cells. The addition of 10% human serum caused a significant inhibition of the SCF-dependent growth of mast cell colonies (no colonies and 9.0 ± 2.3 clusters). We then evaluated whether human serum exerted its inhibitory effects at an earlier phase of mast cell development. As shown in Fig. 1C, the addition of human serum at 5–20% apparently suppressed the SCF-dependent growth of tryptase+ cells from CD34+ cord blood cells at 4 weeks of culture. Interestingly, numbers of POD+ cells were significantly higher in the presence than absence of serum.

image

Figure 1. Effects of human serum on mast cell growth supported by stem cell factor (SCF). (A) The 20-week cultured mast cells (1 × 104) were incubated in wells containing SCF (100 ng/mL) and different concentrations of human serum. After 2 weeks, the viable cells were enumerated. (B) Inhibitory effects of four serum samples on the growth of 20-week cultured mast cells were compared at a final concentration of 10%. HS, human serum. (C) CD34+ cord blood cells (2 × 104) were cultured in wells containing SCF (100 ng/ml) with or without human serum. Numbers of tryptase+ cells and peroxidase+ cells were determined after 4 weeks. The results are the mean ± SD of three independent experiments. *P < 0.0001 compared with the values obtained with SCF alone.

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To clarify whether the serum-induced inhibition resulted from a decrease in either the proliferation rate or survival, we examined the cell cycle status of 20-week cultured mast cells exposed to SCF alone or SCF + human serum. As shown in Fig. 2, flow cytometric analysis showed that the addition of 10% human serum to the culture with SCF caused a decline in the percentage of S plus G2/M cells on day 2 (17.3 ± 3.8% in SCF alone vs 7.2 ± 2.7% in SCF + human serum, n = 3, P < 0.05). However, the serum did not increase the frequency of a sub-G1 peak of the cultured cells grown with SCF.

image

Figure 2. Human serum reduces the proliferation rate of mast cells under stimulation with stem cell factor (SCF). The 20-week cultured mast cells (1 × 105) were incubated with 100 ng/ml of SCF, 10% human serum, 1 × 10−7 M retinol and 10 ng/ml of TGF-β1, alone or in combination. The DNA distribution was examined by labeling the cells with propidium iodide on day 2.

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On the contrary, human serum substantially increased the histamine content of 1 × 105 20-week cultured mast cells (140.7 ± 23.4 ng in 100 ng/ml of SCF vs 279.0 ± 22.1 ng in 100 ng/ml of SCF + 10% serum, P < 0.0001).

Contribution of retinol to human serum-mediated inhibition of mast cell growth

  1. Top of page
  2. Abstract
  3. Methods
  4. Serum samples
  5. Cytokines, reagents and antibodies
  6. Cell preparation
  7. Suspension cultures
  8. Flow cytometric analysis
  9. Clonal cell cultures
  10. Cytochemical and immunologic staining
  11. Assay of histamine, tryptase, cytokine and retinol levels
  12. Statistical analysis
  13. Results
  14. Effects of human serum on SCF-dependent human mast cell development
  15. Contribution of retinol to human serum-mediated inhibition of mast cell growth
  16. Differential regulation by human serum during hematopoiesis
  17. RARα antagonist decreases the FBS-induced suppression of human mast cell growth supported by SCF
  18. Discussion
  19. Acknowledgments
  20. References

It has been reported that various factors inhibit the proliferation of human mast cells or the HMC-1 mast cell line (8, 17–21). To identify the negative regulator(s) for mast cell growth in human serum, we measured the concentrations of IL-4, IL-6, GM-CSF, retinol (vitamin A), and TGF-β1, using the conventional enzyme immunoassay and HPLC. The levels of IL-4, IL-6, and GM-CSF in 11 serum samples were <5, <0.15–0.365 and <2 pg/ml, respectively. Neither neutralizing anti-IL-4 Ab at 10 μg/ml, anti-IL-6 Ab at 10 μg/ml nor anti-GM-CSF Ab at 2 μg/ml affected the progeny generation from 20-week cultured mast cells under stimulation with 100 ng/ml of SCF plus 1% human serum (data not shown).

On the contrary, the mean concentration of retinol was 1.89 ± 0.51 × 10−6 M and the mean concentration of TGF-β1 was 42.4 ± 18.5 ng/ml. We then examined whether retinol and TGF-β1 inhibited the mast cell growth supported by SCF. As shown in Fig. 3A and C, both factors decreased the SCF-dependent generation of progeny by 20-week cultured mast cells in a dose-dependent manner. A significant inhibition of mast cell production was also found in the culture containing retinol or TGF-β1, when CD34+ cord blood cells were target cells (Fig. 3B and D). In the culture with retinol at 10−8 to 10−7 M, CD34+ cord blood cells yielded substantially greater numbers of POD+ cells, compared with the value obtained by SCF alone. The addition of either retinol or TGF-β1 to the culture with SCF decreased the percentage of S plus G2/M cells, but did not increase the sub-G1 peak of the cultured mast cells (Fig. 2). The intracellular histamine level was increased 1.7-fold by TGF-β1 (10 ng/ml), whereas retinol (1 × 10−7 M) did not affect the mediator content.

image

Figure 3. Effects of retinol or transforming growth factor (TGF)-β1 on stem cell factor (SCF)-dependent mast cell growth. The 2 × 104 20-week cultured mast cells (A, C) were incubated in wells containing SCF (100 ng/ml) and different concentrations of retinol or TGF-β1 for 2 weeks. In the culture with 2 × 104 CD34+ cord blood cells (B, D), cell numbers were determined at 4 weeks. The results are the mean ± SD of three independent experiments. *P < 0.0001, **P < 0.001, ***P < 0.005 compared with the values obtained with SCF alone.

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Retinol is converted to RA by the action of retinol dehydrogenases and retinal dehydrogenases. The action of RA is thought to be mediated by two types of nuclear retinoid receptors, RARs and RXRs (22). Each class of the receptors comprises three subtypes designated α, β and γ. Our previous study indicated that both ATRA and 9-cis RA inhibited human mast cell development through RARα (8). To analyze whether retinol and TGF-β1 accounted for the inhibitory effects of human serum, we used Ro 41-5253 (an RARα antagonist) and anti-TGF-β1 Ab, respectively. As shown in Fig. 4A, the addition of Ro 41-5253 at 10−6 M or higher markedly counteracted the 1% human serum-induced suppression of mast cell growth. When the antagonist was used at 10−5 M, the 5% serum-mediated inhibition was also reduced (data not shown). However, anti-TGF-β1 Ab at 10 μg/ml failed to increase the number of mast cells grown with SCF + 1% human serum (Fig. 4B).

image

Figure 4. Effects of an RARα-selective antagonist or anti-transforming growth factor (TGF)-β1 Ab on mast cell growth under stimulation with stem cell factor (SCF) + human serum. (A) The effects of Ro 41-5253 on the progeny generation from 20-week cultured mast cells (2 × 104) in the presence of SCF (100 ng/ml) and human serum (1%) were examined. The results are the mean ± SD of three independent experiments. (B) The effects of anti-TGF-β1 Ab on the progeny generation from 20-week cultured mast cells (2 × 104) in the presence of SCF (100 ng/ml) plus human serum (1%) were examined. Anti-TGF-β1 Ab and Ro 41-5253 were used at 10 μg/ml and 1 × 10−5 M, respectively. The results for human sera were derived from three samples. Significantly different from no Ro 41-5253 or no anti-TGF-β1 Ab (*P < 0.0001).

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Differential regulation by human serum during hematopoiesis

  1. Top of page
  2. Abstract
  3. Methods
  4. Serum samples
  5. Cytokines, reagents and antibodies
  6. Cell preparation
  7. Suspension cultures
  8. Flow cytometric analysis
  9. Clonal cell cultures
  10. Cytochemical and immunologic staining
  11. Assay of histamine, tryptase, cytokine and retinol levels
  12. Statistical analysis
  13. Results
  14. Effects of human serum on SCF-dependent human mast cell development
  15. Contribution of retinol to human serum-mediated inhibition of mast cell growth
  16. Differential regulation by human serum during hematopoiesis
  17. RARα antagonist decreases the FBS-induced suppression of human mast cell growth supported by SCF
  18. Discussion
  19. Acknowledgments
  20. References

As shown in Fig. 1C, the addition of human serum to the culture with SCF decreased mast cell growth, but increased POD+ cell production from CD34+ cord blood cells, implying a differential effect of human serum on hematopoiesis. We therefore compared the effects of human serum on cell growth among stimulation with SCF, GM-CSF, EPO and TPO. The results are shown in Fig. 5. The addition of human serum to the culture with EPO decreased numbers of GPA+ cells in a dose-dependent fashion. In contrast, GM-CSF-dependent myeloid cell production was enhanced by the addition of human serum. Numbers of megakaryocytic cells grown with TPO plus human serum were similar to or higher than the value obtained with TPO alone.

image

Figure 5. Effects of human serum on the cell production supported by stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoietin (EPO) or thrombopoietin (TPO) from CD34+ cord blood cells. CD34+ cord blood cells (2 × 104) were plated in wells containing SCF (100 ng/ml), GM-CSF (10 ng/ml), EPO (2 U/ml), or TPO (10 ng/ml) with or without human serum. The viable cells were enumerated at 2 weeks in the culture with GM-CSF, EPO or TPO, and at 4 weeks in the culture with SCF. The results are the mean ± SD of three independent experiments. Statistical analysis was performed on the numbers of tryptase+ cells (▪), POD+ cells (bsl00039), glycophorin A+ cells (bsl00004), and CD41+ cells (bsl00052) in the culture with SCF, GM-CSF, EPO, and TPO, respectively. Significantly different from no serum (*P < 0.0001). White bars show lineage-negative cells.

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RARα antagonist decreases the FBS-induced suppression of human mast cell growth supported by SCF

  1. Top of page
  2. Abstract
  3. Methods
  4. Serum samples
  5. Cytokines, reagents and antibodies
  6. Cell preparation
  7. Suspension cultures
  8. Flow cytometric analysis
  9. Clonal cell cultures
  10. Cytochemical and immunologic staining
  11. Assay of histamine, tryptase, cytokine and retinol levels
  12. Statistical analysis
  13. Results
  14. Effects of human serum on SCF-dependent human mast cell development
  15. Contribution of retinol to human serum-mediated inhibition of mast cell growth
  16. Differential regulation by human serum during hematopoiesis
  17. RARα antagonist decreases the FBS-induced suppression of human mast cell growth supported by SCF
  18. Discussion
  19. Acknowledgments
  20. References

Retinol is an essential nutrient common to various animal species and humans, all having lost the capacity to synthesize the molecule. It is also known that retinoids can be transferred across the placenta into the fetus (23). The mean concentrations of retinol in FBS were 0.51 ± 0.06 × 10−6 M (n = 7), and equivalent to the values in human cord blood serum (0.60 ± 0.10 × 10−6 M, n = 5). Similar to human serum, FBS at 20% apparently inhibited the SCF-dependent generation of progeny from human cultured mast cells, but increased the intracellular levels of histamine and tryptase (Fig. 6). We then examined whether Ro41-5253 could attenuate the FBS-induced suppression of the growth of human mast cells under stimulation with SCF. The addition of Ro41-5253 to the culture containing SCF + FBS resulted in a significant increase in numbers of progeny. However, the treatment with the RARα antagonist exerted no substantial effects on the concentrations of histamine and tryptase in 1 × 105 cultured mast cells grown in the presence of SCF and FBS.

image

Figure 6. Effects of retinoic acid receptor α antagonist on the growth and maturation of human mast cells in fetal bovine serum (FBS)-containing cultures with stem cell factor (SCF). The 20-week cultured mast cells (2 × 104) were incubated in serum-deprived culture medium containing 100 ng/ml of SCF, 20% FBS, and 1 × 10−5 M Ro 41-5253, alone or in combination. (A) After 2 weeks, numbers of tryptase+ cells were determined. At the same time, the intracellular concentrations of histamine (B) and tryptase (C) were measured. The results are the mean ± SD of three independent experiments. Significantly different from no Ro 41-5253 (*P < 0.001).

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Discussion

  1. Top of page
  2. Abstract
  3. Methods
  4. Serum samples
  5. Cytokines, reagents and antibodies
  6. Cell preparation
  7. Suspension cultures
  8. Flow cytometric analysis
  9. Clonal cell cultures
  10. Cytochemical and immunologic staining
  11. Assay of histamine, tryptase, cytokine and retinol levels
  12. Statistical analysis
  13. Results
  14. Effects of human serum on SCF-dependent human mast cell development
  15. Contribution of retinol to human serum-mediated inhibition of mast cell growth
  16. Differential regulation by human serum during hematopoiesis
  17. RARα antagonist decreases the FBS-induced suppression of human mast cell growth supported by SCF
  18. Discussion
  19. Acknowledgments
  20. References

The present study showed that the addition of human serum apparently suppressed the SCF-dependent generation of tryptase+ cells from CD34+ cord blood cells, and that of progeny from 20-week cultured mast cells. We and other investigators (8, 17–21) reported that IL-4, IL-6, GM-CSF, RA and TGF-β1 suppressed the proliferation of human mast cells or the HMC-1 mast cell line. However, concentrations of IL-4, IL-6 and GM-CSF in human serum were very low or below detectable limits. None of the neutralizing Abs against IL-4, IL-6 and GM-CSF could increase the numbers of progeny grown in the presence of SCF + human serum. On the contrary, TGF-β1 and retinol were present at high concentrations in human serum. Both factors inhibited the SCF-dependent growth of CD34+ cord blood cells and 20-week cultured mast cells. According to the flow cytometric analysis, each of the serum-, retinol-, and TGF-β1-mediated inhibitions of mast cell growth appear to be based on a decline in the proliferation rate rather than an increase in apoptotic death. The addition of Ro 41-5253 profoundly counteracted the human serum-induced suppression of mast cell growth, whereas no significant effects of anti-TGF-β1 Ab were found. Considering that the plasma concentration of ATRA in normal subjects was 0.65 ± 0.18 × 10−8 M (24), it is likely that the antiproliferative effects of human serum depend largely on retinol and its derivatives. However, we cannot exclude the presence of other negative regulator(s) in the serum.

Mast cells originate from pluripotent hematopoietic cells within the bone marrow. Mast cell progenitors depart from the marrow and enter blood circulation. Then, the progenitors migrate into the connective or mucous tissues, where they differentiate into the mature form. Thus, mast cells cannot be detected in peripheral blood of normal individuals, although soluble SCF circulates in the blood. Given the present and previous observations (7) that retinol and their derivatives exerted their effects on the formation of mast cell colonies and clusters, retinoid appears to protect against the SCF-mediated activation of mast cell progenitors in blood vessels. Redlich et al. (25) reported that human lung tissue contains retinol at 0.15 μg ± 0.06 μg/g. Coupled with 1.5-fold higher numbers of peritoneal mast cells in vitamin A-deficient rats (26), it is possible that retinoids function as a physiological regulator for mast cell production in the human body.

Mastocytosis is a rare disease caused by an excessive accumulation of human mast cells in various tissues. Manifestations of the disorder largely reflect the local and systemic consequences of mediator release from tissue mast cells. Because of no curative treatment, STI571 (imatinib mesilate, a competitive inhibitor for a few tyrosine kinases including c-kit) was expected as a novel therapeutic agent in the disease. The most frequent c-kit mutation identified in patients with mastocytosis occurs in the catalytic pocket coding region with substitutions at codon 816, and the other in the intracellular juxtamembrane coding region. Zermati et al. (27) demonstrated that STI571 inhibits juxtamembrane mutant c-kit kinase activity, but has no effect on the activity of the D816 V mutant. From these results, they concluded that the compound is not a good candidate to treat the disease. Recently, Alexandrakis et al. (28) reported that ATRA significantly decreased both the growth and intracellular tryptase levels of HMC-1 cells. Taken together with our results, retinol and their derivatives may be a possible agent in the treatment of mastocytosis.

The bioactive forms of TGF-β are composed of two mature peptide chains linked by disulfide bonds. Once synthesized and processed intracellularly, TGF-β is secreted as two types of latent complexes. The small complex consists of TGF-β and latency-associated protein (LAP). The large complex contains TGF-β, LAP and latent TGF-β-binding proteins. TGF-β is activated by proteases such as plasmin. For the measurement of TGF-β1 in human serum and plasma by immunoassay, acidification is required to disrupt the interaction between TGF-β1 and LAP. Thus, the failure of anti-TGF-β1 Ab to antagonize the serum-mediated inhibition of mast cell growth may be due to a latent form of the cytokine present in human serum.

Serum-containing and serum-deprived culture methods have been used to elucidate mechanisms for the development and function of human mast cells. Compared with serum-deprived cultures, serum-containing cultures were superior in mast cell maturation, but had demerits such as decreased growth of mast cells and concomitant growth of myeloid cells. For this reason, Dahl et al. (9) reported a new culture system combining serum-deprived culture for 8 weeks, followed by serum-containing culture, using SCF plus IL-6 as a stimulus. The present study clearly showed that retinol and its derivatives acted as the main inhibitors for human mast cell growth in FBS-containing cultures. The RARα antagonist may be a useful tool to obtain higher numbers of mast cells in this culture system.

References

  1. Top of page
  2. Abstract
  3. Methods
  4. Serum samples
  5. Cytokines, reagents and antibodies
  6. Cell preparation
  7. Suspension cultures
  8. Flow cytometric analysis
  9. Clonal cell cultures
  10. Cytochemical and immunologic staining
  11. Assay of histamine, tryptase, cytokine and retinol levels
  12. Statistical analysis
  13. Results
  14. Effects of human serum on SCF-dependent human mast cell development
  15. Contribution of retinol to human serum-mediated inhibition of mast cell growth
  16. Differential regulation by human serum during hematopoiesis
  17. RARα antagonist decreases the FBS-induced suppression of human mast cell growth supported by SCF
  18. Discussion
  19. Acknowledgments
  20. References
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